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 the treatment of cancer, including reducing the likelihood of and / or preventing tumor recurrence and metastasis. The mitochondrial-specific transcription inhibitor compounds disclosed herein have demonstrated to inhibit tumor spherogenic ability, migration ability, and stemness-related signaling in cancer stem cells. These properties are the result of selective inhibition of mitochondrial RNA polymerase (POLRMT) targeting mitochondrial transcription in cancer cells.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to mitochondrial-specific transcription inhibitors, or MSTI compounds, which inhibit the proliferation of cancer stem cells (CSCs) and senescent cells by selectively inhibiting CSC mitochondrial transcription. BACKGROUND

[0002] The biological process of aging has been a focus of scientific and medical research communities. Physiological aging is related, at least in part, to an increase in the rate of oxidative damage to cellular components, including DNA, lipids, and proteins. The increase in oxidative damage causes an imbalance that disrupts self-regulatory processes at the cellular level. In addition, aging is associated with the accumulation of lipofuscin in the cytoplasm of neuronal cells. Modern research has also shown that aging is the result of naturally occurring DNA damage that leads to abnormal changes in DNA and accumulates over time. Both mitochondrial and nuclear DNA damage can lead to aging, indirectly through an increase in apoptosis and cellular senescence, and directly through an increase in cellular dysfunction. Accumulated DNA damage leads to cell loss and, in surviving cells, to a loss of gene expression and mutations— effects that contribute to signs of aging in cells that do not divide frequently. Cellular senescence is the result of aging cells that stop cell division and is believed to occur after various environmental damaging events, abnormal cell growth, autophagy, and oxidative stress, among other factors. The Senescence Associated Secretory Phenotype (“SASP”) is a characteristic of senescent cells and causes protein toxic damage to healthy cell function, including inflammatory or anti-inflammatory effects and tumor or anti-tumor effects, depending on a variety of factors. The effects of SASP-related chronic inflammation can impair the normal ability of the immune system to clear senescent cells, and cells that provide immune function can be recruited by the SASP to senescent cells. It has been found that biomarkers of cellular senescence accumulate as mammals age and contribute to a range of age-related diseases, including Alzheimer’s disease, lateral sclerosis, and type 2 diabetes. For cells that divide frequently, accumulated DNA damage can be a major 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 or anti-senescence therapies. Traditional cancer therapies (e.g., radiation therapy, alkylating agents such as cyclophosphamide, and antimetabolites such as 5-fluorouracil) attempt to selectively detect and eradicate rapidly growing cancer cells by interfering with cellular mechanisms related to cell growth and DNA replication. Other cancer therapies employ immunotherapy, selectively binding mutated tumor antigens (e.g., monoclonal antibodies) to rapidly growing cancer cells. Unfortunately, tumors often recur in the same or different sites after these therapies, indicating that not all cancer cells are eradicated. Cancer stem cells, in particular, are able to survive for various reasons, leading to treatment failure. Recurrence may be due to insufficient chemotherapy dosage and / or the emergence of cancer cell clones that develop resistance to the therapy. Therefore, new cancer treatment strategies are needed to overcome the shortcomings of traditional therapies.

[0004] Advances in mutation analysis have enabled in-depth research into gene mutations occurring during cancer development. Despite the availability of genomic maps, modern oncology still faces challenges in de-topping major driver mutations across various cancer subtypes. The harsh reality is that each patient's tumor is unique, and a single tumor may contain multiple distinct clonal cells. Therefore, a new approach is needed that emphasizes commonalities across different cancer types. Targeting metabolic differences between tumor cells and normal cells holds promise as a novel cancer treatment strategy. Transcriptional profiling data analysis from human breast cancer samples revealed that elevated levels of over 95 mRNA transcripts are associated with mitochondrial biosynthesis and / or mitochondrial translation. (Sotgia et al., ...) Cell Cycle , 11(23):4390-4401 (2012). Furthermore, over 35 of the 95 upregulated mRNAs encoded mitochondrial ribosomal proteins (MRPs). Proteomic analysis of human breast cancer stem cells also revealed significant overexpression of several mitochondrial ribosomal proteins and other proteins related to mitochondrial biosynthesis. Lamb et al., Oncotarget , 5(22):11029-11037 (2014).

[0005] In recent years, mitochondrial metabolism in cancer cells has been a target of exploratory research aimed at identifying therapeutic targets for both cancer and aging. Mitochondria are highly dynamic organelles that constantly divide, elongate, and interconnect to form tubular networks or fragmented particles to meet cellular needs and adapt to the cellular microenvironment. The balance between mitochondrial fusion and division determines the morphology, number, function, and spatial distribution of mitochondria, thereby influencing numerous important mitochondrial-dependent biological processes, such as adenosine triphosphate (ATP) production, mitophagy, apoptosis, and calcium homeostasis. Conversely, mitochondrial dynamics can be 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, the mitochondrial organelle acts as the cell's "powerhouse." Mitochondria produce large amounts of ATP through the TCA cycle and oxidative phosphorylation (OXPHOS), while glycolysis contributes only a small amount. Conversely, mitochondrial dysfunction induces ATP depletion, leading to mitochondrial-driven apoptosis (programmed cell death) and / or necrosis. Therefore, we propose that ATP-depletion therapy may be a viable strategy for targeting and eradicating even the most "adapted" 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%. Therefore, like normal cells, cancer cells are highly dependent on mitochondrial ATP production. However, whether ATP levels in cancer cells contribute to 3D anchorage-independent growth and cell migration (two hallmarks of metastasis and spread) remains unclear.

[0008] Therefore, what we need are novel anti-aging compositions and methods that treat aging at the cellular level, overcoming accumulated oxidative damage and DNA damage, as well as the many adverse effects of aging.

[0009] Furthermore, therapeutic agents targeting unhealthy senescent cells and SASPs are needed to reduce the accumulation of cellular senescence and counteract chronic aging.

[0010] There is also a need to develop therapeutics that can target a broad range of CSCs, acting through the common characteristics of CSCs, regardless of cancer type, with minimal or negligible impact on normal healthy cells.

[0011] What is needed further are therapeutic agents that can inhibit the proliferation of CSCs, which include circulating tumor cells and tumor-initiating cells that have the potential to cause tumor recurrence and / or metastasis. Summary of the Invention

[0012] In light of the foregoing background, the purpose of this disclosure is to describe therapeutic agents or compounds that can be used to reduce the accumulation of senescent cells and inhibit CSC proliferation. The purpose of this disclosure is to describe therapeutic agents for eradicating CSCs and senescent cells. The purpose of this disclosure is to describe therapeutic agents for preventing and reducing the likelihood of tumor recurrence and metastasis. Another purpose of this disclosure is to describe compositions (such as pharmaceutical compositions) and methods for treating and preventing cancer (including tumor recurrence and / or metastasis). A further purpose of this disclosure is to describe compositions (such as pharmaceutical compositions) and methods for anti-aging therapeutic agents.

[0013] This article describes mitochondria-specific transcription inhibitors (MSTIs), compounds that can be used as therapeutic agents with anticancer activity, pharmaceutical compositions containing such therapeutic agents, methods for synthesizing such compounds, and methods for treating cancer. The implementation schemes of the MSTI therapeutic agents described herein can be compared with the following formula to demonstrate the unexpected advantages of this scheme: , This compound is known to be an orally effective inhibitor of mitochondrial RNA polymerase (POLRMT). It was originally described in Bonekamp, ​​NA, Peter, B., Hillen, HS, et al., Small-molecule inhibitors of human mitochondrial DNA transcription. Nature 588, 712–716 (2020), incorporated herein by reference in its entirety. As a POLRMT inhibitor, this compound inhibits mitochondrial DNA expression. Its IUPAC name is 3-piperidinic acid, 1-[(2R)-2-[[4-(2-chloro-4-fluorophenyl)-2-oxo-2H-1-benzopyran-7-yl]oxy]-1-oxopropyl]-,(3S)-, and its assigned CAS registry number is 2304621-06-3.

[0014] According to this scheme, some embodiments of the MSTI compound can have the general chemical structure of formula [I] as shown below: ,in: R1 is selected independently from: -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 -; R 1a Independently selected from: H, alkyl, alkenyl, ynyl, cycloalkyl, heterocycloalkyl, heteroaryl, or phenyl, R 1a Where chemically permissible, it may be optionally substituted by one to four independently selected groups from the following: oxo, C1-C 18 -alkyl, C2-C 18 -Alynyl group, C2-C 18 -Alkenyl, C1-C 18 - Haloalkyl; OR a NR a R b SR a C(O)OR a C(O)NR a R b Halogen, cyano, nitro, C(O)R a and S(O)2OR a ; Among them, R a Each time it appears, it is independently selected from: H and C1-C 18 -alkyl; and R b Each time it appears, it is independently selected from: H and C1-C 18 -alkyl, C(O)C1-C 18 -alkyl groups and S(O)2-C1-C 18 -alkyl; R2 is independently selected from: halogens, -CF2H, -CF3, -OCF2H, -OCF3, substituted or unsubstituted C5-C18 carboxyl groups, 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 alkyl-amides, monocyclic or polycyclic aromatic hydrocarbons, heteroaromatic hydrocarbons, 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 olefin, 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 alkyl-amide, substituted or unsubstituted phenol, and benzoic acid; R5 is selected from H, -CF3, -OCF3, -OCHF2, -NO2, and -CN; L1 is selected independently from: -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 =CR2a -、-CC- and -CH2R 2a -; R 2a Independently selected from: H, alkyl, alkenyl, ynyl, cycloalkyl, heterocycloalkyl, heteroaryl, or phenyl, R 2a Where chemically permissible, it may be optionally substituted with one to four independently selected groups from the following: oxo, C1-C6-alkyl, C2-C6-ynyl, C2-C6-alkenyl, C1-C6-haloalkyl; OR a NR y R b SR y C(O)OR y C(O)NR y R b Halogen, cyano, nitro, C(O)R y S(O)2OR y ; Among them, R y Each time it appears, it is independently selected from: H and C1-C6-alkyl; and R b Each time it appears, it is independently selected from: H and C1-C6-alkyl, C(O)C1-C6-alkyl and S(O)2-C1-C6-alkyl; And X - Selected from suitable anions to form pharmaceutically acceptable salts.

[0015] It should be recognized that a variety of proven counterions can be used to form pharmaceutically acceptable salts of the disclosed compounds. For example, X - It can be selected from acetate, benzenesulfonate, benzoate, besylate, hydrogen tartrate, bromide, camphorsulfate, chloride, citrate, decanoate, ethylenediaminetetraacetic acid, ethanesulfonate, fumarate, gluconate, hexanoate, iodide, hydroxyethylsulfonate, lactate, malate, maleate, mandelic acid, methanesulfonate, methyl sulfate, mucilage, naphthalenesulfonate, nitrate, caprylate, oleate, dihydroxynaphthalate, pantothenate, phosphate, propionate, salicylate, stearate, succinate, sulfate, tartrate, theocyanate, and p-toluenesulfonate. The embodiments shown below as chloride salts should be considered as disclosed embodiments having any salt identified herein. In the preferred embodiment, X - Selected from Cl - ,Br - I - and MeSO3 -Research on the optimal anion for a specific implementation scheme is underway.

[0016] This regimen can also be used to treat and / or prevent tumor recurrence and / or metastasis. For example, MSTI compounds according to Formula I above can be administered as agents alone or in combination with other therapies to treat and / or prevent tumor recurrence and / or metastasis. Anticancer treatments often fail due to tumor recurrence or metastasis, especially after surgery. CSC mitochondrial activity is understood to be at least partially responsible for these treatment failures. Implementation of this regimen can be used in cases of conventional cancer treatment failure and / or in conjunction with anticancer therapy, or prior to anticancer therapy, to prevent or reduce the likelihood of treatment failure due to tumor recurrence and / or metastasis. Attached Figure Description

[0017] Figures 1A-1C The results of mammary globulin formation, SRB test results of MCF-7 cells, and SRB test results of BJ1-hTERT cells under different concentrations of compound [I] are shown.

[0018] Figure 2A and 2B The effect of compound [I] at different concentrations on MCF-7 colony formation is shown; represented by representative phase images of 2D colonies and colony formation relative to the control group.

[0019] Figure 3A and 3B The effects of compound [IV] at different concentrations on MCF-7 cells in the colony formation assay and hTERT-BJ1 cells in the SRB assay were shown (respectively).

[0020] Figure 4A and 4B The effects of compound [IX] at different concentrations on MCF-7 cells in the colony formation assay and hTERT-BJ1 cells in the SRB assay are shown (respectively).

[0021] Figure 5A and 5B The effects of compound [X] at different concentrations on MCF-7 cells in the colony formation assay and hTERT-BJ1 cells in the SRB assay are shown (respectively).

[0022] Figure 6A and 6B The effects of compound [XI] at different concentrations on MCF-7 cells in the colony formation assay and hTERT-BJ1 cells in the SRB assay are shown (respectively).

[0023] Figure 7A and 7BThe effects of compound [XII] at different concentrations on MCF-7 cells in the colony formation assay and hTERT-BJ1 cells in the SRB assay are shown (respectively). Detailed Implementation

[0024] The following description details embodiments of the method to enable its practice. 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 described herein. Rather, these embodiments are provided to make this disclosure exhaustive and to fully communicate the scope of the method to those skilled in the art.

[0025] This description uses various terms that should be understood by one of ordinary skill in the art. To avoid any ambiguity, the following clarifications are provided.

[0026] The term "cancer" refers to a physiological condition in mammals characterized by the uncontrolled growth of cells. This definition includes both benign and malignant cancers. Examples of cancers include cancer types, lymphomas, blastomas (including medulloblastomas and retinoblastomas), sarcomas (including liposarcomas and synovial sarcomas), neuroendocrine tumors (carcinoid tumors, gastrin-producing tumors, including but not limited to tumors and islet cell carcinomas), sarcomas, schwannomas (including acoustic neuromas), medullary carcinomas, adenocarcinomas, melanomas, and leukemias or lymphocytic tumors. Specific examples of cancers include bladder cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung cancer (including squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, stomach 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 (kidney cancer) or 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.

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

[0028] The term "metastasis" refers to the process by which cancer spreads from its primary site to other parts of the body. Cancer cells can escape from the primary tumor, invade lymphatic vessels and blood vessels, circulate through the bloodstream, and grow or "metastasize" in distant lesions in normal tissues elsewhere in the body. Metastasis can be local or distant. Metastasis is a gradual process that requires tumor cells to escape from the primary tumor, circulate through the bloodstream, and reach distant sites. At this new site, the cells can establish a blood supply and grow to form a life-threatening mass. Stimulating and inhibitory molecular pathways within the tumor cells control this behavior, and long-distance interactions between tumor cells and distant host cells are also important.

[0029] The terms “treat,” “treated,” “treating,” and “treatment” encompass the reduction or alleviation of at least one symptom associated with or caused by the treated condition, symptom, or disease (particularly cancer). In some embodiments, the treatment method comprises reducing and / or alleviating at least one symptom associated with or caused by the treated cancer through the compounds disclosed herein. In embodiments, the treatment method comprises causing the death of a specific type of cell in the host body that is a cancer (such as senescent cells, SASP cells, or CSCs that may be involved in metastasis or recurrence), and may be achieved by preventing further proliferation of senescent cells and / or cancer cells, and / or by, for example, by depriving such cells of mechanisms for energy production. For example, treatment may be the alleviation 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, eradicate (e.g., kill CSCs at a rate higher than their proliferation rate) cancer cells, 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 chemotherapy, sensitize cancer to radiotherapy, and sensitize cancer to phototherapy. As another example, this treatment can reduce the accumulation of senescent cells and / or slow the rate of senescent cell accumulation.

[0030] In the context of tumor recurrence and / or metastasis, "prevention" and "reduction of likelihood" refer to reducing the presence of circulating tumor cells (CSCs), transient tumor cells (TICs), and circulating tumor cells (CTCs) in a subject's body to a level that makes recurrence and / or metastasis from the primary site less likely compared to the control group (i.e., without treatment to prevent or reduce the likelihood of tumor recurrence and / or metastasis). In practice, the treatments described herein for preventing and / or reducing the likelihood of tumor recurrence and / or metastasis target and inhibit or eradicate CSCs and TICs, and suppress circulating tumor cells.

[0031] The terms "cancer stem cells" and "CSCs" refer to a subset of cancer cells within a tumor that possess the ability to self-renew, differentiate, and tumorigenerate when transplanted into an animal host. Compared to "mass" cancer cells, CSCs exhibit increased mitochondrial mass, enhanced mitochondrial biosynthesis, and higher mitochondrial protein translation activation. As used herein, "circulating tumor cells" refer to cancer cells that detach from the primary tumor, enter blood vessels or lymphatic vessels, and are carried throughout the body via the bloodstream. The CellSearch Circulating Tumor Cell Assay can be used to detect circulating tumor cells.

[0032] As used herein, the phrase "pharmaceutically effective amount" refers to the amount required to achieve a therapeutic outcome when administered to a host or its cells, tissues, or organs, such as modulating, adjusting, or inhibiting protein kinase activity, for example, inhibiting protein kinase activity or treating cancer. A physician or veterinarian with ordinary skill in the art can readily determine and prescribe an 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 may start the use of the compound of the invention in a pharmaceutical composition at a level below that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. The determination of a pharmaceutically effective amount is considered to be within the scope of a person with ordinary skill in the art who has read this disclosure.

[0033] As used herein, the term "therapeutic agent" refers to embodiments of the compounds described herein, which may include pharmaceutically acceptable salts or isotopic analogs thereof. It should be understood that therapeutic agents can be administered to subjects by any suitable method, as is well known to those skilled in the art. It should also be recognized that the dosage of a therapeutic agent and the timing of its administration may depend on the individual subject being treated (e.g., factors such as age and weight), the route of administration, the pharmacokinetic properties of the particular therapeutic agent, and the judgment of the prescribing physician. Therefore, due to the variability among subjects, any dosage described herein is intended as an initial guideline, and physicians may titrate the dosage of the therapeutic agent to achieve treatment that the physician deems appropriate for the subject. In considering the desired level of treatment, physicians may weigh various factors such as the subject's age and weight, the presence of pre-existing conditions, and other illnesses. Pharmaceutical formulations can be prepared for any desired route of administration, including but not limited to oral, intravenous, or aerosol administration, as discussed in more detail below.

[0034] The phrase “pharmaceutically acceptable carrier” as used in this article refers to pharmaceutically acceptable materials, compositions, or carriers, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials. Each carrier must be “acceptable,” meaning it is compatible with other components of the formulation and will not cause harm to the patient. Some examples of materials that can 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 carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth gum; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, and corn oil. Rice oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer solution; and (21) other non-toxic compatible substances used in pharmaceutical preparations.

[0035] "Pharmaceutically acceptable salt" refers to a relatively non-toxic inorganic or organic base addition salt of the compound used in this method. Pharmaceutically acceptable salts can be formed, for example, by reacting a compound in its free acid form with a base (such as a hydroxide or carbonate of a pharmaceutically acceptable metal cation), with ammonia, or with a pharmaceutically acceptable amine. Typical alkali metal or alkaline earth metal salts include, for example, sodium, potassium, calcium, magnesium, and aluminum salts. Examples of amines that can be used for base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, and piperazine. It should be understood, of course, that other salts may be used, and those skilled in the art can identify suitable salt forms using methods known in the art without departing from the present method.

[0036] Suitable salts of the MSTI compounds described herein may be selected from acetates, benzenesulfonates, benzoates, benzenesulfonates, tartrates, bromides, camphorsulfonates, chlorides, citrates, decanoates, ethylenediaminetetraacetic acid salts, ethanesulfonates, fumarates, gluconates, hexanoates, iodides, hydroxyethylsulfonates, lactates, malates, maleates, mandelates, methanesulfonates, methyl sulfates, mucilages, naphthalenesulfonates, nitrates, octanoates, oleates, dihydroxynaphthaleneates, pantothenates, phosphates, propionates, salicylates, stearates, succinates, sulfates, tartrates, theochlorophosphates, and p-toluenesulfonates. In a preferred embodiment, X - Selected from Cl - ,Br- I - and MeSO3 - .

[0037] Recent research has identified a mitochondrial transcription inhibitor targeting mitochondrial RNA polymerase (POLRMT), a gene product in the OXPHOS system responsible for mitochondrial gene expression and biosynthesis. The inhibitor has been tested in mice for four weeks. Oral treatment with this compound in mice showed no signs of OXPHOS dysfunction or toxicity, but it demonstrated strong antitumor activity in xenografts of human cancer cells.

[0038] The mitochondrial-specific transcription inhibitor (MSTI) compounds disclosed herein represent a new class of compounds with excellent selectivity and activity against cancer cells. Embodiments of the MSTI compounds disclosed herein can be used as therapeutic agents to selectively eradicate cancer cells (CSCs), thereby treating and / or preventing tumor recurrence and / or metastasis. Data indicate that the MSTI compounds disclosed herein possess enhanced anticancer activity relative to the aforementioned POLRMT inhibitors and are suitable for use as therapeutic agents in anticancer therapy, including treating and / or preventing tumor recurrence and metastasis. The data described herein demonstrate anticancer activity by inhibiting MCF7 cells via mammary globulin formation assays, and cell viability is demonstrated using the sulfonylrhodamine B assay (also referred to in the art and herein as the SRB assay). This assay measures the amount of residual protein adhering to tissue culture dishes and is a surrogate marker of cell viability.

[0039] According to this scheme, some embodiments of the MSTI compound can have the general chemical structure of formula [I] as shown below: ,in: R1 is selected independently from: -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 -、-NR1a C(O)OR 1a -、-NR 1a C(O)NR 1a -、-CR 1a =CR 1a -、-CC- and -CH2R 1a -; R 1a Independently selected from: H, alkyl, alkenyl, ynyl, cycloalkyl, heterocycloalkyl, heteroaryl, or phenyl, R 1a Where chemically permissible, it may be optionally substituted by one to four independently selected groups from the following: oxo, C1-C 18 -alkyl, C2-C 18 -Alynyl group, C2-C 18 -Alkenyl, C1-C 18 - Haloalkyl; OR a NR a R b SR a C(O)OR a C(O)NR a R b Halogen, cyano, nitro, C(O)R a and S(O)2OR a ; Among them, R a Each time it appears, it is independently selected from: H and C1-C 18 -alkyl; and R b Each time it appears, it is independently selected from: H and C1-C 18 -alkyl, C(O)C1-C 18 -alkyl groups and S(O)2-C1-C 18 -alkyl; R2 is independently selected from: halogens, -CF2H, -CF3, -OCF2H, -OCF3, substituted or unsubstituted C5-C18 carboxyl groups, 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 alkyl-amides, monocyclic or polycyclic aromatic hydrocarbons, heteroaromatic hydrocarbons, 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 olefin, 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 alkyl-amide, substituted or unsubstituted phenol, and benzoic acid; R5 is selected from H, -CF3, -OCF3, -OCHF2, -NO2, and -CN; L1 is selected independently from: -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 -; R 2a Independently selected from: H, alkyl, alkenyl, ynyl, cycloalkyl, heterocycloalkyl, heteroaryl, or phenyl, R 2a Where chemically permissible, it may be optionally substituted with one to four independently selected groups from the following: oxo, C1-C6-alkyl, C2-C6-ynyl, C2-C6-alkenyl, C1-C6-haloalkyl; OR a NR y R b SR y C(O)OR y C(O)NRy R b Halogen, cyano, nitro, C(O)R y S(O)2OR y ; Among them, R y Each time it appears, it is independently selected from: H and C1-C6-alkyl; and R b Each time it appears, it is independently selected from: H and C1-C6-alkyl, C(O)C1-C6-alkyl and S(O)2-C1-C6-alkyl; And X - Selected from suitable anions to form pharmaceutically acceptable salts.

[0040] It should be recognized that a variety of proven counterions can be used to form pharmaceutically acceptable salts of the disclosed compounds. For example, X - It can be selected from acetate, benzenesulfonate, benzoate, benzenesulfonate, hydrogen tartrate, bromide, camphorsulfonate, chloride, citrate, decanoate, ethylenediaminetetraacetic acid, ethanesulfonate, fumarate, gluconate, hexanoate, iodide, hydroxyethylsulfonate, lactate, malate, maleate, mandelic acid, methanesulfonate, methyl sulfate, mucoate, naphthalenesulfonate, nitrate, caprylic acid, oleate, dihydroxynaphthalate, pantothenic acid, phosphate, propionate, salicylate, stearate, succinate, sulfate, tartrate, theocyanate, and p-toluenesulfonate. In the preferred embodiment, X - Selected from Cl - ,Br - I - and MeSO3 - Research on the optimal anion for a specific implementation scheme is underway.

[0041] This regimen can also be used to treat and / or prevent tumor recurrence and / or metastasis. For example, MSTI compounds according to Formula I above can be used as pharmaceutical preparations alone or in combination with other therapies to treat and / or prevent tumor recurrence and / or metastasis.

[0042] In an exemplary embodiment of the MSTI compound of this scheme based on formula [I], compound [I] has the following chemical formula: , Or another pharmaceutically acceptable salt thereof. The IUPAC name of this MSTI compound is [6-[4-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chroman-7-yl]oxypropionyl]piperazin-1-yl]-6-oxo-hexyl]-triphenylphosphonium chloride. It should be recognized that other salts may be used without departing from this formulation.

[0043] This document also describes a method for preparing compounds according to this scheme. For example, an embodiment for preparing compound [1] according to the following reaction scheme.

[0044]

[0045] The following paragraphs describe an example of the synthesis of compound [I] according to the above reaction scheme. In step 1, 2-chloro-4-nitrobenzoic acid is used as a starting material to synthesize 2-chloro-4-nitrobenzoyl chloride, as shown below.

[0046]

[0047] Under a nitrogen atmosphere, oxaloyl chloride (10.6 ml, 125 mmol) and DMF (8 drops) were added to a stirred solution of 2-chloro-4-nitrobenzoic acid (10.3 g, 50 mmol) in dry dichloromethane (200 ml) at room temperature. The mixture was stirred for 16 hours, and then the solvent was evaporated under reduced pressure to obtain crude 2-chloro-4-nitrobenzoyl chloride (11.78 g) as a light brown oil.

[0048] In step 2, ethyl 3-(2-chloro-4-nitrophenyl)-3-oxopropionate is prepared from 2-chloro-4-nitrobenzoyl chloride (as shown below).

[0049]

[0050] Under a nitrogen atmosphere, triethylamine (14.6 ml, 105 mmol) and anhydrous magnesium chloride (11.4 g, 120 mmol) were added to a stirred suspension of potassium ethyl malonate (17.0 g, 100 mmol) in 300 ml of dried MeCN at +10 °C, and the mixture was stirred at room temperature for 3 hours. A solution of crude 2-chloro-4-nitrobenzoyl chloride (11.7 g, 50 mmol) in 80 ml of dried acetonitrile was added over 60 minutes, and the mixture was stirred at room temperature for 16 hours under a nitrogen atmosphere. The solvent was evaporated under reduced pressure, and the residue was diluted with aqueous NH4Cl (200 ml), EtOAc (300 ml), and 4 M HCl (120 ml). The organic phase was separated, washed with 0.5 M HCl (170 ml) and brine (100 ml), dried over MgSO4, filtered, and the filtrate was concentrated under reduced pressure to give the crude product (19.8 g). Purification on silica gel (isohexane containing 10-20% EtOAc) yielded ethyl 3-(2-chloro-4-nitrophenyl)-3-oxopropionate (12.3 g) as a light brown solid. LC-MS 272.0 [M+1] + RT 5.56 min. 1 H-NMR (CDCl3) d12.50 (s,1H), 8.32-8.30 (m, 1H), 8.19-8.15 (m, 1H), 5.63 (s, 1H), 4.30 and4.19 (q, 2H), 4.04 (s, 1H), 1.35 and 1.25 (t, 3H).

[0051] In step 3, ethyl 3-(2-chloro-4-nitro-phenyl)-3-oxo-propionate is used to prepare 4-(2-chloro-4-nitro-phenyl)-7-hydroxy-chroman-2-one (as shown below).

[0052]

[0053] Under a nitrogen atmosphere, ethyl 3-(2-chloro-4-nitro-phenyl)-3-oxo-propionic acid (11.6 g, 42.7 mmol) was added in portions to a stirred solution of resorcinol (4.14 g, 41 mmol) in methanesulfonic acid (40 mL) at +45 °C for 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, the solution was poured into water (390 mL), cooled in an ice bath for 1 hour, and then warmed to room temperature. The solvent was decanted, and the precipitate was washed with diethyl ether (2 × 100 mL). The solid was collected by filtration, washed with diethyl ether (2 × 100 mL), DCM (2 × 50 mL), and diethyl ether (1 × 100 mL), and dried under vacuum to give 11.9 g of 4-(2-chloro-4-nitrophenyl)-7-hydroxy-chroman-2-one as a red solid. LC-MS 318.0 [M+1] + RT 5.69 min. 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).

[0054] In step 4, 4-(4-amino-2-chloro-phenyl)-7-hydroxy-chroman-2-one is prepared using the product of step 3 (as shown below).

[0055]

[0056] Under a nitrogen atmosphere, a solution of dry DCM (20 ml) of trichlorosilane (3.60 ml, 35 mmol) was added to a stirred, chilled solution of 4-(2-chloro-4-nitro-phenyl)-7-hydroxy-chroman-2-one (3.18 g, 10 mmol) and DiPEA (8.60 ml, 50 mmol) in 200 ml of stirred, cold DCM, over a period of 90 minutes. The mixture was stirred at +0°C for 1 hour, quenched with water (10 ml) and methanol (10 ml), and the pH was adjusted to 8-9 by adding solid sodium bicarbonate (10 g). The suspension was filtered through a diatomaceous earth pad, the pad was washed with DCM (2 x 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 give a dark reddish-brown solid of 4-(4-amino-2-chlorophenyl)-7-hydroxy-chroman-2-one (3.02 g). LC-MS 288.0 [M+1] +, RT 4.94min. 1 H-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 (width s, 2H).

[0057] In step 5, N-[3-chloro-4-(7-hydroxy-2-oxo-chroman-2-one)phenyl]tetradecanoamide is prepared using 4-(4-amino-2-chlorophenyl)-7-hydroxy-chroman-2-one (as shown below).

[0058]

[0059] Under a nitrogen atmosphere, at room temperature, a solution of dry 2-methyltetrahydrofuran (25 mL) containing tetradecanoyl chloride (0.90 g, 3.63 mmol) was added to a stirred solution of 4-(4-amino-2-chlorophenyl)-7-hydroxy-chroman-2-one (1.0 g, 3.48 mmol) and pyridine (1.1 mL, 4.00 mmol). The mixture was stirred at room temperature under a nitrogen atmosphere for 16 hours, diluted with 2-methyltetrahydrofuran (50 mL), 2M HCl (70 mL), and brine (40 mL), dried over MgSO4, filtered, and the solvent was evaporated under reduced pressure to give the crude product. Purification on silica gel (isohexane containing 5-60% EtOAc) gave N-[3-chloro-4-(7-hydroxy-2-oxo-chroman-4-yl)phenyl]tetradecanoamide (1.25 g) as a light brown solid. LC-MS 498.3 [M+1] + RT 6.47 min. 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).

[0060] The product from step 5 is then used in step 6 to prepare ethyl (2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chroman-7-yl]oxypropionate as shown below.

[0061]

[0062] Under a nitrogen atmosphere, ethyl-S-lactic acid ester (0.23 ml, 1.20 mmol) was added to a stirred, chilled solution of diisopropyl azodicarboxylate (0.24 ml, 1.20 mmol) and triphenylphosphine (0.31 g, 1.20 mmol) in a dry solution of 2-methyltetrahydrofuran (10 ml). The mixture was stirred for 15 minutes, and then a solution of N-[3-chloro-4-(7-hydroxy-2-oxo-chroman-4-yl)phenyl]tetradecanoamide (0.50 g, 1.00 mmol) was added. The mixture was stirred at +0 °C for 0.5 h, then heated to room temperature and stirred for 64 h. The solvent was evaporated under reduced pressure to give the crude product. Purified on silica gel (containing 10-50% MTBE in isohexane) to give ethyl(2R)-2-[4-(4-amino-2-chlorophenyl)-2-oxo-chroman-7-yl]oxypropionate (0.46 g) as a light brown solid.

[0063] LC-MS 598.4 [M+1] + RT 6.93 min. 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).

[0064] In step 7, 2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chroman-7-yl]oxypropionic acid (as shown below) was prepared from the product of step 6.

[0065]

[0066] At room temperature and under a nitrogen atmosphere, a stirred solution of ethyl (2R)-2-[4-(4-amino-2-chloro-phenyl)-2-oxo-chroman-7-yl]oxypropionic acid (0.42 g, 1.0 mmol) in MeTHF (5 ml) and MeOH (5 ml) was added to water (5 ml) containing hydrated LiOH (0.42 g, 10.0 mmol). The mixture was stirred for 2 hours, diluted with MeTHF (30 ml), washed with 2H HCl (20 ml) and brine (20 ml), dried over MgSO4, filtered, and the solvent was evaporated under reduced pressure to give a light brown solid of (2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chroman-7-yl]oxypropionic acid (0.45 g). LC-MS 570.0 [M+1] + RT 6.70 min. 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).

[0067] Next, step 8 involves preparing (6-chloro-6-oxo-hexyl)triphenylphosphonium chloride (as shown below) to prepare the phosphine moiety.

[0068]

[0069] At room temperature, under a nitrogen atmosphere, oxalyl chloride (0.24 ml, 2.8 mmol) and dry DMF (2 drops) were added to a stirred solution of (5-carboxypentyl)triphenylphosphonium bromide (0.46 g, 1.0 mmol) in 20 ml of dry DCM. The mixture was stirred for 90 minutes, and the solvent was removed under reduced pressure to give crude (6-chloro-6-oxo-hexyl)triphenylphosphonium chloride (0.43 g).

[0070] Then, the product from step 8 is used to prepare [6-(4-tert-butyloxycarbonylpiperazin-1-yl)-6-oxo-hexyl]-triphenylphosphonium chloride (as shown below).

[0071]

[0072] Tert-butylpiperazine-1-carboxylate (0.45 g, 2.4 mmol) was added to a stirred solution of crude (6-chloro-6-oxo-hexyl)triphenylphosphonium chloride (0.94 g, 2.2 mmol) in 30 mL of THF and 1 mL of pyridine at room temperature under a nitrogen atmosphere. The mixture was stirred for 80 hours and then 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 give 1.07 g of a colorless solid, [6-(4-tert-butyloxycarbonylpiperazine-1-yl)-6-oxo-hexyl]-triphenylphosphonium chloride. LC-MS 545.4 [M+] + RT 5.26 min. 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.

[0073] Continuing to utilize the phosphonium moiety, the product of step 9 is used in step 10 to prepare 6-oxo-6-piperazin-1-yl-hexyl)-triphenyl-phosphonium chloride hydrochloride (as shown below).

[0074]

[0075] At room temperature, under a nitrogen atmosphere, dioxane (2 ml, 8 mmol) containing 4 M HCl was added to a stirred solution of [6-(4-tert-butyloxycarbonylpiperazin-1-yl)-6-oxo-hexyl]-triphenylphosphonium chloride (0.052 g, 0.09 mmol) in 3 ml of DCM. The mixture was stirred for 3 hours and concentrated under reduced pressure to give a light brown solid (6-oxo-6-piperazin-1-yl-hexyl)-triphenylphosphonium chloride hydrochloride (0.052 g). LC-MS 445.3 [M+] + RT 3.46 min. 1 H-NMR (CDCl3)d9.84 (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.

[0076] In step 11, compound [I], [6-[4-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chroman-7-yl]oxypropionyl]piperazin-1-yl]-6-oxo-hexyl]-triphenylphosphonium chloride is prepared using the products of steps 7 and 10.

[0077] At room temperature, under a nitrogen atmosphere, a 5 mL solution of MeTHF containing (2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chroman-7-yl]oxypropionic acid (0.065 g, 0.11 mmol), EDC (0.033 g, 0.17 mmol), HOBt (0.023 g, 0.17 mmol), and NMM (50 µl, 0.45 mmol) was added to a 3 mL solution of DMF containing (6-oxo-6-piperazin-1-yl-hexyl)-triphenyl-phosphonium chloride hydrochloride (0.052 g, 0.09 mmol). The 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 on silica gel (DCM containing 1-15% MeOH) to give a light brown solid of [6-[4-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chroman-7-yl]oxypropionyl]piperazin-1-yl]-6-oxo-hexyl]-triphenylphosphonium chloride (0.021 g). LC-MS 996.3 [M+] + RT 6.45min. 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 23.8ppm.

[0078] As another exemplary embodiment, compound [II] (shown below) has the IUPAC name [6-[4-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chroman-7-yl]oxypropionyl]piperazin-1-yl]-6-oxo-hexyl]tris[4-trifluoromethyl)phenyl]phosphine chloride.

[0079]

[0080] In step 1, tert-butyl 4-[(2S)-2-hydroxypropionyl]piperazine-1-carboxylate was prepared (as shown below).

[0081]

[0082] At room temperature, under a nitrogen atmosphere, a solution of (S)-lactic acid (2.00 g, 22.2 mmol), tert-butylpiperazine-1-carboxylate (4.11 g, 22.2 mmol), and DiPEA (4.0 mL, 22.2 mmol) in DMF (10 mL) was stirred and added to DMF (3 mL) containing HATU (10.1 g, 26.2 mmol). The mixture was stirred for 24 hours, concentrated, diluted with DCM (220 mL) containing 10% MeOH, 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 give the crude product. Purification was performed on silica gel (MTBE containing 1–5% MeOH) to give a light brown solid of tert-butyl 4-[(2S)-2-hydroxypropionyl]piperazine-1-carboxylate (1.56 g). LC-MS 261.1 [M+23] + RT 3.94 min. 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).

[0083] In step 2, tert-butyl 4-[(2S)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chroman-7-yl]oxypropionyl]piperazine-1-carboxylate was prepared from the product of step 1 (as shown below).

[0084]

[0085] Under a nitrogen atmosphere, diisopropyl azodicarbonate (0.23 ml, 1.08 mmol) was added to a stirred, ice-cold solution of N-[3-chloro-4-(7-hydroxy-2-oxo-chroman-4-yl)phenyl]tetradecanoamide (0.45 g, 0.90 mmol) and 4-[(2S)-2-hydroxypropionyl]piperazine-1-carboxylic acid tert-butyl ester (0.28 g, 1.08 mmol) in 10 ml of dry 2-methyltetrahydrofuran. The mixture was stirred for 10 min, and then triphenylphosphine (0.28 g, 1.08 mmol) was added. The mixture was stirred at +0 °C for 0.5 h, then heated to room temperature and stirred for 16 h. The solvent was evaporated under reduced pressure to give the crude product. Purification on silica gel (isohexane containing 20-100% EtOAc) yielded a light brown solid of 0.51 g of tert-butyl 4-[(2S)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chroman-7-yl]oxypropionyl]piperazine-1-carboxylate. LC-MS 738.3 [M+1] + RT 6.99 min. 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).

[0086] Next, in step 3, N-[3-chloro-4-[7-[(1S)-1-methyl-2-oxo-2-piperazin-1-yl-ethoxy]-2-oxo-chroman-4-yl]phenyl]tetradecanoamide; hydrochloride was prepared.

[0087]

[0088] To a stirred solution of N-[3-chloro-4-[7-[(1S)-1-methyl-2-oxo-2-piperazin-1-yl-ethoxy]-2-oxo-chroman-4-yl]phenyl]tetradecanoamide hydrochloride (0.20 g, 0.27 mmol) in DCM (5 mL), dioxane (5 mL, 20 mmol) containing 4 M HCl was added. The mixture was stirred for 3 hours and concentrated under reduced pressure to give a light brown solid of N-[3-chloro-4-[7-[(1S)-1-methyl-2-oxo-2-piperazin-1-yl-ethoxy]-2-oxo-chroman-4-yl]phenyl]tetradecanoamide hydrochloride (0.16 g). LC-MS 638.5 [M+1] + RT 5.85 min. 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).

[0089] In step 4, 5-carboxypentyl-tris[4-(trifluoromethyl)phenyl]phosphonium bromide (as shown below) was prepared as a precursor for the phosphonium moiety.

[0090]

[0091] 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) in dried MeCN was refluxed for 64 hours. Insoluble residues were removed by filtration, and the filtrate was concentrated under reduced pressure to give the crude product. Purification was performed on silica gel (DCM containing 5–15% MeOH) to give 0.28 g of 5-carboxypentyl-tris[4-(trifluoromethyl)phenyl]phosphonium bromide as a colorless solid. LC-MS 581.2 [M+] + RT 4.86min. 1 H-NMR (DMSO-d6) d 8.15 (m, 16H), 3.82 (m, 2H), 2.13 (t, 2H), 1.51 (m, 6H).

[0092] In step 5, compound [II] is prepared using the products of steps 3 and 4.

[0093] At room temperature, 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-piperazin-1-yl-ethoxy]-2-oxo-chroman-4-yl]phenyl]tetradecanoamide; 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 dried DMF (2 mL). The 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. Purification on silica gel (DCM containing 1-10% MeOH) yielded a light brown solid of [6-[4-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chroman-7-yl]oxypropionyl]piperazin-1-yl]-6-oxo-hexyl]tris[4-trifluoromethyl)phenyl]phosphonium chloride (0.0442 g). LC-MS 1200.5 [M+] + ,RT 6.24min. 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 25.2ppm. 19 F-NMR (MeOD-d4) d 65.1ppm.

[0094] Another exemplary embodiment of this scheme is compound [III] (as shown below), whose IUPAC name is [5-[4-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chroman-7-yl]oxypropionyl]piperazin-1-yl]-5-oxo-pentyl]-triphenylphosphonium chloride. Compound [II] is prepared according to the following scheme.

[0095]

[0096] Compound [III] was prepared from N-[3-chloro-4-[7-[(1S)-1-methyl-2-oxo-2-piperazin-1-yl-ethoxy]-2-oxo-chroman-4-yl]phenyl]tetradecanoamide; its hydrochloride and (4-carboxybut-1-yl)triphenylphosphonium bromide according to step 5 of Example Compound 2, yielding a light brown solid of [5-[4-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chroman-7-yl]oxypropionyl]piperazin-1-yl]-5-oxo-pentyl]triphenyl-phosphonium chloride (0.0351 g). LC-MS 968.3 [M+] + RT 6.44 min. 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 23.7ppm.

[0097] As another example of this scheme, compound [IV] (as shown below), whose IUPAC name is [4-[4-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chroman-7-yl]oxypropionyl]piperazin-1-yl]-4-oxo-butyl]-triphenylphosphonium chloride, is prepared as follows.

[0098]

[0099] Compound [IV] was prepared from N-[3-chloro-4-[7-[(1S)-1-methyl-2-oxo-2-piperazin-1-yl-ethoxy]-2-oxo-chroman-4-yl]phenyl]tetradecanoamide; hydrochloride and (3-carboxypropyl-1-yl)triphenylphosphonium bromide according to step 5 of Example Compound 2, yielding a light brown solid of [4-[4-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chroman-7-yl]oxypropionyl]piperazin-1-yl]-4-oxo-butyl]triphenyl-phosphonium chloride (0.024 g). LC-MS 982.5 [M+] + RT 6.39 min. 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 23.7ppm.

[0100] Figure 3A and 3B The effects of compound [iV] at different concentrations on MCF-7 cells in the colony formation assay and hTERT-BJ1 cells in the SRB assay are shown (respectively). Figure 3A As shown, compound [IV] exhibited anticancer activity at 0.5 µM and eradicated MCF-7 cells at a concentration of 2.5 µM. In contrast, Figure 3B The compound [IV] showed no activity against hTERT-BJ1 cells, a non-cancerous normal cell line, which could be used to demonstrate the lack of toxicity of the drug compound.

[0101] As another example of this scheme, compound [V] (as shown below), whose IUPAC name is [3-[4-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chroman-7-yl]oxypropionyl]piperazin-1-yl]-3-oxo-propyl]-triphenylphosphonium chloride, is prepared as follows.

[0102]

[0103] Compound [V] was prepared from N-[3-chloro-4-[7-[(1S)-1-methyl-2-oxo-2-piperazin-1-yl-ethoxy]-2-oxo-chroman-4-yl]phenyl]tetradecanoamide; hydrochloride and (2-carboxyethyl-1-yl)triphenylphosphonium bromide according to step 5 of compound [II] in Example, yielding a light brown solid of [3-[4-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chroman-7-yl]oxypropionyl]piperazin-1-yl]-3-oxo-propyl]-triphenylphosphonium chloride (0.0243 g). LC-MS 954.5 [M+] + RT 6.39 min. 1 H-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) d24.8ppm.

[0104] As another example of this scheme, compound [VI] (as shown below), whose IUPAC name is [6-[[1-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chroman-7-yl]oxypropionyl]-4-piperidinyl]amino]-6-oxo-hexyl]-triphenylphosphonium chloride, is prepared as follows.

[0105]

[0106] The preparation method of compound [VI] is as follows. In step 1, the intermediate phosphonium compound [6-[(1-tert-butyloxycarbonyl-4-piperidinyl)amino]-6-oxohexyl]-triphenylphosphonium chloride was prepared (as shown below).

[0107]

[0108] At room temperature and under a nitrogen atmosphere, a solution of crude (6-chloro-6-oxohexyl)triphenylphosphonium chloride (0.94 g, 2.2 mmol) in THF (30 mL) and pyridine (1 mL) was added to tert-butyl-4-amino-piperidine-1-carboxylate (0.48 g, 2.4 mmol). The mixture was stirred for 80 hours and then 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 give 0.84 g of a light brown solid, [6-[(1-tert-butyloxycarbonyl-4-piperidinyl)amino]-6-oxohexyl]-triphenylphosphonium chloride. LC-MS 559.5 [M+] + RT 5.24 min. 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 H-NMR (CDCl3) d 24.8 ppm.

[0109] In step 2, the aforementioned product is converted to [6-oxo-6-(4-piperidinylamino)hexyl]-triphenylphosphonium chloride (as shown below).

[0110]

[0111] At room temperature and under a nitrogen atmosphere, a 10 mL solution of [6-[(1-tert-butyloxycarbonyl-4-piperidinyl)amino]-6-oxohexyl]-triphenylphosphonium chloride (0.24 g, 0.09 mmol) in DCM was added to dioxane (5 mL, 20 mmol) containing 4 M HCl. The mixture was stirred for 90 minutes and concentrated under reduced pressure to give [6-oxo-6-(4-piperidinylamino)hexyl]-triphenylphosphonium chloride hydrochloride (0.28 g) as a light brown solid. LC-MS 459.3 [M+] + RT 3.58 min. 1H-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 H-NMR (CDCl3) d 24.0 ppm.

[0112] In step 3, the product of step 2 and (2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chroman-7-yl]oxypropionic acid were used to prepare compound [VI] according to step 11 of Compound 1 in Example, yielding [6-[[1-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chroman-7-yl]oxypropionyl]-4-piperidinyl]amino]-6-oxo-hexyl]-triphenyl-phosphonium chloride (0.0267 g) as a light brown solid. LC-MS 1010.4 [M+] + , RT6.42 min. 1 H-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 H-NMR (CDCl3) d 23.8 ppm.

[0113]

[0114] As another example of this scheme, compound [VII] (as shown below), whose IUPAC name is [6-[4-[[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chroman-7-yl]oxypropionyl]amino]-1-piperidinyl]-6-oxo-hexyl]-triphenylphosphonium chloride, is prepared as follows.

[0115]

[0116] In step 1, the phosphonium intermediate compound [6-[4-(tert-butoxycarbonylamino)-1-piperidinyl]-6-oxohexyl]-triphenylphosphonium chloride was prepared (as shown below).

[0117]

[0118] N-(4-piperidinyl)carbamate tert-butyl ester (0.86 g, 4.3 mmol) was added to a stirred solution of crude (6-chloro-6-oxohexyl)triphenylphosphonium chloride (1.30 g, 3.0 mmol) in THF (35 mL) and pyridine (1 mL) at room temperature under a nitrogen atmosphere. The mixture was stirred for 3 hours and then concentrated under reduced pressure. Purification on silica gel (DCM containing 3-12% MeOH) gave 1.30 g of colorless solid [6-[4-(tert-butoxycarbonylamino)-1-piperidinyl]-6-oxohexyl]-triphenylphosphonium chloride. LC-MS 559 [M+] + RT 5.26 min. 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 H-NMR (CDCl3) d 24.3ppm.

[0119] In step 2, the product of step 1 is converted to [6-(4-amino-1-piperidinyl)-6-oxohexyl]-triphenylphosphonium chloride.

[0120]

[0121] Dioxane (5 ml, 20 mmol) containing 4 M HCl was added to a stirred solution of [6-[4-(tert-butoxycarbonylamino)-1-piperidinyl]-6-oxohexyl]-triphenylphosphonium chloride (0.25 g, 0.42 mmol) in DCM (5 ml) under a nitrogen atmosphere at room temperature. The mixture was stirred for 90 minutes and concentrated under reduced pressure to give a colorless solid of [6-(4-amino-1-piperidinyl)-6-oxohexyl]-triphenylphosphonium chloride (0.29 g). LC-MS 459.3 [M+] + , RT 3.61 min.1 H-NMR (DMSO-d6) d8.43 (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 H-NMR (CDCl3) d 24.1 ppm.

[0122] In step 3, using the product from step 2, compound [VII], [6-[4-[[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromanyl-7-yl]oxypropionyl]amino]-1-piperidinyl]-6-oxo-hexyl]-triphenylphosphonium chloride, was prepared according to step 11 of compound [I], yielding a light brown solid of [6-[4-[[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromanyl-7-yl]oxypropionyl]amino]-1-piperidinyl]-6-oxo-hexyl]-triphenylphosphonium chloride (0.0375 g). LC-MS 1010.5 [M+] + RT 6.41 min. 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 0.89 (t, 3H). 31 P-NMR (MeOD-d4) d 23.8ppm.

[0123] With modifications obvious to those skilled in the art, the synthetic examples can be used to prepare compounds according to formula [I]. It should be recognized that other synthetic methods can be used to obtain the compounds of this scheme. The following structures are examples of other compounds obtained according to this scheme.

[0124] .

[0125] Data generated according to the implementation scheme of this protocol indicate that these compounds have effective anticancer activity in both inhibiting tumor growth and preventing metastasis, but are non-toxic to healthy cells. The following paragraphs summarize the data for compound [I]. It should be understood that preliminary results from other implementation schemes are consistent with those for compound [I], and analyses of compounds [I]–[VII], etc., are ongoing.

[0126] Figures 1A-1C The effects of different concentrations of compound [I] on mammary globulin formation, SRB assay results in MCF-7 cells, and SRB assay results in BJ1-hTERT cells are shown. An asterisk (p-value) indicates the p-value. This indicates that P < 0.01. This indicates that P < 0.001. This indicates that P < 0.0001. For example, from... Figure 1A As can be seen, compound [I] showed inhibition of MCF-7 spheres at all concentrations, with almost complete inhibition at 2.5 µM. Compound [I] achieved more than 50% inhibition at concentrations from 0.5 to 1.0 μM, demonstrating the remarkable potency of the compounds according to this scheme.

[0127] Figure 1B and 1C The SRB test results for (B) MCF-7 cells and (C) hTERT-BJ1 cells are shown separately. The data show that compound [I] begins to show cytotoxicity against MCF-7 cells at levels below 1.0 µM, but has almost no cytotoxicity against hTERT-BJ1 cells up to around 10 µM.

[0128] Figure 2A and 2B The effects of compound [I] at different concentrations on MCF-7 colony formation in petri dishes (A) and (B) relative to the control group were shown. Figure 2A The representative phase diagram of 2D colonies shows that treatment with compound [I] significantly reduced MCF-7 colony formation. MCF-7 colony formation was completely inhibited starting at a concentration of 1.0 µM of compound [I]. The significant inhibitory activity against MCF-7 colony formation is clearly visible in the graphical representation, as shown below. Figure 2BCompound [I] was shown to have an IC50 concentration below 0.5 µM. 50 It exhibited complete inhibition at 1.0 µM. Preliminary results indicate that other compounds according to this regimen possess, if not increased, similar potency and selectivity against cancer stem cells.

[0129] Figure 4A and 4B The effects of compound [IX] at different concentrations on MCF-7 cells in the colony formation assay and on hTERT-BJ1 cells in the SRB assay are shown (separately). Compound [IX] exhibits anticancer activity starting at concentrations below 0.5 µM and is non-toxic to hTERT-BJ1 cells at concentrations up to 10 µM.

[0130]

[0131] Figure 5A and 5B The effects of compound [X] at different concentrations on MCF-7 cells in the colony formation assay and on hTERT-BJ1 cells in the SRB assay were shown (respectively). Compound [X] exhibited anticancer activity starting at concentrations as low as 0.25 µM and was non-toxic to hTERT-BJ1 cells at concentrations up to 10 µM.

[0132]

[0133] Figure 6A and 6B The effects of compound [XI] at different concentrations on MCF-7 cells in the colony formation assay and on hTERT-BJ1 cells in the SRB assay were shown (respectively). Compound [XI] exhibited anticancer activity at 0.5 µM and complete inhibition at 2.5 µM, but showed no toxicity to hTERT-BJ1 cells at all tested concentrations.

[0134]

[0135] Figure 7A and 7B The effects of compound [XII] at different concentrations on MCF-7 cells in the colony formation assay and on hTERT-BJ1 cells in the SRB assay were shown (separately). Compound [XII] exhibited anticancer activity at 0.25 µM, complete inhibition at 0.5 µM, and no toxicity to hTERT-BJ1 cells up to concentrations above 1 µM.

[0136]

[0137] The following abbreviations are used to describe the synthetic methods and reaction schemes: acetonitrile (MeCN), ethyl acetate (EtOAc), methanol (MeOH), hydrochloric acid (HCl), magnesium sulfate (MgSO4), diisopropylethylamine (DiPEA), tert-butyloxycarbonyl (Boc), 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 azodicarbonate (DEAD), dimethylacetamide (DMA), dimethylformamide (DMF), ammonia (NH3), and 4-dimethylaminopyridine (DMAP), trifluoroacetic acid (TFA), 1-hydroxybenzotriazole (HOBt), and ammonia (NH3).

[0138] Liquid chromatography-mass spectrometry (LC-MS) data were prepared using a Waters Sunfire C18 30x4.6 mm column. Gradient eluent: 20-100% acetonitrile / water containing 0.05% formic acid. Time: 0-10 minutes.

[0139] NMR spectra were recorded using a Bruker 500MHz Avance III HD spectrometer equipped with a broadband prodigy cryoProbe (1H, 13C, 15N, 19F, 31P) and TopSpin version 3.6, patch level 4 running on Windows 10.

[0140] The following paragraphs describe the materials and tests used to generate the data described herein. It should be understood that a person skilled in the art can perform the same tests as described herein, and / or can utilize other tests known in the art to evaluate the physical, chemical, and pharmaceutical properties of the compounds described herein.

[0141] Reagents and model cell lines: Obviously, other cell lines can be used without departing from this method. The human breast cancer 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-BJ-1 cells were cultured in DMEM supplemented with 10% fetal bovine serum, GlutaMAX, and 1% penicillin-streptomycin, and incubated at 37°C in a 5% CO2 humidified incubator. The medium was changed 2-3 times per week.

[0142] 3D Anchored Independent Growth Assay: This assay is also known as the microsphere formation assay. A single-cell suspension was prepared using an enzyme and manually disintegrated (using a 25-g needle). Cells were then seeded at a density of 500 cells / cm² in a non-adherent microsphere culture medium (DMEM-F12 + 1X B-27 Plus supplement + 20 ng / ml EGF + penicillin / streptomycin) in culture dishes pre-coated with (2-hydroxyethyl methacrylate) (poly-HEMA, Sigma Aldrich Inc.), termed "microsphere plates". Cells were grown for 5 days and maintained in a humidified incubator at 37°C. After 5 days of culture, 3D microspheres >50 μm were counted using an eyepiece ("grid"), and the percentage of cells forming microspheres was calculated and termed the microsphere formation percentage, normalized to one (1 = 100% MFE). The microsphere formation efficiency (MFE) of low-ATP and high-ATP subpopulations was analyzed. All 3D microsphere experiments were conducted three times, at least three times independently.

[0143] Statistical Analysis: All analyses were performed using GraphPad Prism 6. Data are presented as mean ± standard deviation (or ± SEM, if indicated). All experiments were performed independently at least three times, with >3 technical replicates for each experimental condition (unless otherwise specified, e.g., when representative data are shown). Statistically significant differences were determined using Student's t-test or analysis of variance (ANOVA). One-way ANOVA was used to determine statistical significance for comparisons among multiple groups. p < 0.05 was considered significant.

[0144] This method includes a means of confirming cell viability. Those skilled in the art may choose one or more methods to confirm cell viability suitable for a particular embodiment. The inventors initially used the sulfonylrhodamine (SRB) assay, which is based on the measurement of cellular protein content. After treatment in 96-well plates for 72 hours, cells were fixed in 10% trichloroacetic acid (TCA) in a cold room for 1 hour and dried overnight at room temperature. 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-bound dye was dissolved in a 10 mM Tris solution, pH 8.8, and read at 540 nm using a plate reader. Using the SRB assay, the inventors selected only those 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. Of course, embodiments using other cell viability confirmation methods may select compounds for further analysis based on other considerations known in the art.

[0145] Therapeutic agents can be used in the form of pharmaceutical compositions, which can be prepared using one or more known methods. For example, pharmaceutical compositions can be prepared using diluents or excipients, such as, for example, one or more fillers, compatibilizers, binders, wetting agents, disintegrants, surfactants, lubricants, etc., known in the art. Various types of administration unit forms can be selected according to the therapeutic purpose. Examples of forms of pharmaceutical compositions include, but are not limited to, tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, suppositories, injectable formulations (solutions and suspensions), topical creams, and other forms known in the art. To form pharmaceutical compositions in tablet form, any known excipients can be used, such as carriers, such as lactose, white sugar, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, cyclodextrin, crystalline cellulose, silica, etc.; binders, such as water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, etc. In addition, disintegrants such as dried starch, sodium alginate, agar powder, flake powder, sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitol fatty acid ester, sodium lauryl sulfate, glyceryl monostearate, starch, lactose, etc., can be used. Disintegrant inhibitors such as white sugar, stearic acid, coconut oil, hydrogenated oil, etc., can be used; absorption promoters such as quaternary ammonium bases, sodium lauryl sulfate, etc., can be used. Wetting agents such as glycerin, starch, and other wetting agents known in the art can be used. Adsorbents such as, for example, starch, lactose, kaolin, bentonite, colloidal silica, etc., can be used. Lubricants such as pure talc, stearates, boric acid powder, polyethylene glycol, etc., can be used. If tablets are required, they can be further coated with commonly used coating materials to make sugar-coated tablets, gelatin film-coated tablets, enteric-coated tablets, film-coated tablets, bilayer tablets, and multilayer tablets. Pharmaceutical compositions suitable for topical application can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, foams, sprays, aerosols, or oils. Such pharmaceutical compositions may include conventional additives, including but not limited to preservatives, solvents that aid drug penetration, co-solvents, emollients, propellants, viscosity modifiers (gelling agents), surfactants, and carriers.

[0146] This method can be used to prevent and / or reduce the likelihood of tumor recurrence and metastasis. Anticancer treatments often fail due to tumor recurrence or metastasis, especially after surgery. CSC mitochondrial activity is at least partially responsible for these treatment failures. The implementation of this method can be used in cases where conventional cancer treatments have failed, and / or in combination with anticancer therapy, to prevent or reduce the likelihood of failure due to tumor recurrence and / or metastasis.

[0147] This method provides a way to selectively target cancer cells. The targeted cancer cells can be at least one of CSCs, energetic cancer stem cells (e-CSCs), circulating tumor cells (CTCs, seed cells that lead to the subsequent growth of more tumors in distant organs, a mechanism that leads to a large proportion of cancer-related death), and therapy-resistant cancer cells (TRCCs, cells that have developed resistance to one or more chemotherapy, radiation therapy, and other common cancer treatments).

[0148] As described in co-pending U.S. Provisional Patent Application Nos. 62 / 686,881, filed June 19, 2018, and 62 / 731,561, filed September 14, 2018, and both incorporated herein by reference in their entirety, e-CSC represents a CSC phenotype associated with proliferation. In addition to mass cancer cells and CSCs, it should be understood that this method can be used to target a highly proliferating cell subpopulation referred to by the inventors as e-CSC, which exhibits a gradual increase in stemness markers (ALDH activity and microsphere formation activity), a high increase in mitochondrial mass, and increased glycolysis and mitochondrial activity.

[0149] In view of the above, it should be understood that, depending on the implementation, this method can take many forms. For example, the implementation of this method can take the form of a composition, and in particular, a pharmaceutical composition. The therapeutic compound can be an active ingredient and can be present in an effective pharmaceutical amount.

[0150] Implementations of this method may also take the form of a method for preventing or reducing the likelihood of at least one of tumor recurrence and metastasis. In some embodiments, an effective amount of a composition having the compound of this method may be administered as a therapeutic agent. In some embodiments, an effective amount of a composition having the embodiment of the compound described herein may be administered as its therapeutic agent.

[0151] The terminology used in this disclosure is for the purpose of describing particular embodiments only and is not intended to limit the disclosure. As used in the description of this disclosure 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. This disclosure includes numerous substitutions, modifications, and equivalents, which will become apparent upon consideration of the following detailed description.

[0152] It should be understood that although the terms “first,” “second,” “third,” “a),” “b),” and “c),” etc., are used herein to describe various elements of this disclosure, the claims should not be limited by these terms. These terms are used only to distinguish one element of this disclosure from another. Thus, the first element discussed below may be referred to as an element aspect, and similarly, the third element may be referred to as an element aspect without departing from the teachings of this disclosure. Therefore, the terms “first,” “second,” “third,” “a),” “b),” and “c),” etc., are not intended to convey a sequence or other hierarchical structure to the relevant elements, but are used only for identification purposes. The order of operations (or steps) is not limited to the order presented in the claims.

[0153] Unless otherwise defined, all terms used herein (including technical and scientific terms) shall have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. It will also be understood that terms, such as those defined in commonly used dictionaries, shall be interpreted as having a meaning consistent with their meaning in the context of this application and 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 this disclosure is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict of terminology, this specification is controlling.

[0154] Furthermore, as used herein, “and / or” refers to and includes any and all possible combinations of one or more of the related listed items, as well as combinations that are missing when interpreted in the alternatives (“or”).

[0155] The terms “decrease,” “lower,” “lessen,” and “reduce” generally refer to the ability of a composition according to this method to produce and / or induce a smaller physiological response (i.e., a measurable downstream effect) rather than a response induced by the carrier or control molecule / composition, such as a reduction in tumor volume. A “decrease” or “reduced” response is generally a “statistically significant” response and may include a reduction of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, or more times (e.g., 500, 1000 times) in response produced by normal, untreated, or controlled subjects (including all integers and decimals between and above 1, such as 1.5, 1.6, 1.7, 1.8, etc.).

[0156] The phrase "treatment cycle" refers to a course of treatment, such as a regular or pre-determined schedule of repeated dosing. A treatment cycle can consist of several days of treatment followed by several days of rest. For example, in a 4-week treatment cycle, the medication may be administered daily for two weeks, followed by two weeks of no treatment. It should be understood that treatment cycles can depend on many factors, such as an individual's disease state, age, sex, and weight, as well as the specific medication and / or method used to elicit the desired response in the individual.

[0157] Unless the context otherwise requires, it is specifically intended that the various features of this disclosure described herein can be used in any combination. Furthermore, this disclosure also contemplates that, in some embodiments of this disclosure, any feature or combination of features stated herein can be excluded or omitted. For illustration, if the specification states that a complex comprises components A, B, and C, it is specifically intended that any one or a combination of A, B, or C can be omitted or excluded.

[0158] As used herein, the term “about” refers to a measurable value, such as a quantity or concentration, meaning a variation of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified quantity. The ranges provided herein for measurable values ​​may include any other ranges and / or individual values ​​thereof.

[0159] Having described some embodiments of this disclosure in this way, it should be understood that this disclosure as defined by the appended claims is not limited to the specific details set forth in the foregoing description, as many obvious variations are possible without departing from its spirit or scope, as claimed below.

Claims

1. Compounds with the following chemical structures: ,in: R1 is selected independently from: -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 -; R 1a Independently selected from: H, alkyl, alkenyl, ynyl, cycloalkyl, heterocycloalkyl, heteroaryl, or phenyl, R 1a Where chemically permissible, it may be optionally substituted by one to four independently selected groups from the following: oxo, C1-C 18 -alkyl, C2-C 18 -Alynyl group, C2-C 18 -Alkenyl, C1-C 18 - Haloalkyl; OR a NR a R b SR a C(O)OR a C(O)NR a R b Halogen, cyano, nitro, C(O)R a and S(O)2OR a ; Among them, R a Each time it appears, it is independently selected from: H and C1-C 18 -alkyl; and R b Each time it appears, it is independently selected from: H and C1-C 18 -alkyl, C(O)C1-C 18 -alkyl groups and S(O)2-C1-C 18 -alkyl; R2 is independently selected from: halogens, -CF2H, -CF3, -OCF2H, -OCF3, substituted or unsubstituted C5-C18 carboxyl groups, 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 alkyl-amides, monocyclic or polycyclic aromatic hydrocarbons, heteroaromatic hydrocarbons, 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 olefin, 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 alkyl-amide, substituted or unsubstituted phenol, and benzoic acid; R5 is selected from H, -CF3, -OCF3, -OCHF2, -NO2, and -CN; L1 is selected independently from: -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 -; R 2a Independently selected from: H, alkyl, alkenyl, ynyl, cycloalkyl, heterocycloalkyl, heteroaryl, or phenyl, R 2a Where chemically permissible, it may be optionally substituted with one to four independently selected groups from the following: oxo, C1-C6-alkyl, C2-C6-ynyl, C2-C6-alkenyl, C1-C6-haloalkyl; OR a NR y R b SR y C(O)OR y C(O)NR y R b Halogen, cyano, nitro, C(O)R y S(O)2OR y ; Among them, R y Each time it appears, it is independently selected from: H and C1-C6-alkyl; and R b Each time it appears, it is independently selected from: H and C1-C6-alkyl, C(O)C1-C6-alkyl and S(O)2-C1-C6-alkyl; And X - Selected from suitable anions to form pharmaceutically acceptable salts.

2. The compound according to claim 1, wherein, X - Choose from the following groups: acetate, benzenesulfonate, benzoate, benzenesulfonate, hydrogen tartrate, bromide, camphorsulfonate, chloride, citrate, decanoate, ethylenediaminetetraacetic acid, ethanesulfonate, fumarate, gluconate, hexanoate, iodide, hydroxyethylsulfonate, lactate, malate, maleate, mandelic acid, methanesulfonate, methyl sulfate, mucilage, naphthalenesulfonate, nitrate, caprylic acid, oleate, dihydroxynaphthalate, pantothenic acid, phosphate, propionate, salicylate, stearate, succinate, sulfate, tartrate, theocyanate, and p-toluenesulfonate.

3. The compound according to any one of the preceding claims, wherein, X - Choose from the following groups: Cl - ,Br - I - and MeSO3 - .

4. The compound according to any one of claims 1 to 3, wherein, R5 is CF3.

5. The compound according to claim 1, having a structure selected from the group consisting of: , , , , , and 。 6. The compound according to claim 1, having a structure selected from the group consisting of: , , , , , , , , , , , , , , , , , , , 。 7. The compound according to claim 1, having a structure selected from the group consisting of: , , , and .

8. The compound according to claim 1, wherein, R5 is CF3 each time it appears.

9. The compound according to claim 1, wherein, R5 is H each time it appears.

10. A pharmaceutical composition comprising a compound according to any one of claims 1-9 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 carboxymethyl cellulose, ethyl cellulose, cellulose acetate, tragacanth gum, malt, gelatin, talc, cocoa butter, ethylene glycol, propylene glycol, polyols, glycerol, sorbitol, mannitol, polyethylene glycol, esters, ethyl oleate, ethyl laurate, agar, buffer, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethanol, and phosphate buffer solution.

12. The pharmaceutical composition according to claim 10, wherein, The excipients are selected from the group consisting of: lactose, white sugar, 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, the method comprising administering a pharmaceutically effective amount of a pharmaceutical composition to a patient at risk of tumor recurrence and / or metastasis, the pharmaceutical composition comprising any one of claims 1-9.

14. The method according to claim 13, wherein, The application is performed at least once before cancer treatment, during cancer treatment, or after cancer treatment.

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

16. The method according to claim 15, wherein, The application is performed at least once before cancer treatment, during cancer treatment, or after cancer treatment.

17. A method for treating cancer, the method comprising: Administering a pharmaceutically effective amount of a pharmaceutical composition to a person suffering from cancer, said pharmaceutical composition comprising any one of claims 1-9.

18. The method according to claim 17, wherein, The application is performed at least once before cancer treatment, during cancer treatment, or after cancer treatment.

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