CHROMENE COMPOUNDS, METHODS AND USES THEREOF - Patent application

Abstract

The present disclosure relates to a compound for use in medicine, or a pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer, or polymorph thereof, comprising the formula: [Formula 1] JPEG2022539146000002.jpg6082 here R 1 , R 2 and R 3 are chosen independently of each other, R 1 is selected from an aryl or heterocyclic ring; R 2 is selected from H, alkyl, aryl, alkoxyl, halogen, hydroxyl, amine, carbonyl, or heterocycle; R 3 is selected from H, or (II).

Description

[Technical field]

[0001] The present disclosure relates to a novel class of chromene anti-cancer compounds. The present disclosure describes the anti-cancer properties of a novel class of chromene anti-cancer compounds. The present disclosure further describes methods for synthesizing and isolating the novel class of chromene anti-cancer compounds. [Background technology]

[0002] Cancer was responsible for approximately 10 million deaths worldwide in 2018, with 18.1 million new cases of cancer diagnosed in the same year. 1 The prevalence of the disease in 5 years is expected to exceed 43 million. This justifies the urgency of finding new and better treatments. The problem must be addressed globally, as there is an overall increase in the number of cases and associated mortality. Breast cancer accounted for 6.6% of cancer deaths in 2018. It is the fifth most deadly type of cancer, with 11.6% of new cases recognized in 2018 (more than 2 million cases). 2 It is the second most common type of cancer in terms of incidence. The type of treatment always depends on the stage of the cancer, but basically consists of surgery, radiation, conventional chemotherapy, immunotherapy or molecular therapy if molecular markers such as ER, PR and HER-2 are detected. However, for triple-negative breast cancer (TNBC), which is a highly aggressive form of breast cancer and accounts for approximately 15%-20% of all breast cancers, conventional treatments have limited effectiveness, and molecular and individualized therapies have not yet been developed. Due to this poor prognosis, this subtype of breast cancer has a high mortality rate. 3

[0003] The chromene skeleton has been an inspiration to medicinal chemists due to its widespread occurrence in nature and diverse biological properties. 4 Research into the synthesis and biological importance of this natural scaffold and its synthetic derivatives has received increasing attention in recent years.

[0004] The document "Tetrahedron" by Costa et al. describes the preparation of 2-aryl-1,9-dihydrochromeno[3,2-d]imidazoles by a one-pot cascade reaction with salicylaldehyde and arylideneaminoacetonitrile. Furthermore, the document describes a novel fused tricyclic system combining chromene and imidazole as an alternative drug candidate with improved pharmacological properties. However, the described system does not allow the generation of molecules incorporating different substituents on the aromatic unit.

[0005] These facts are disclosed to explain the technical problem addressed by the present disclosure. Summary of the Invention

[0006] The present disclosure relates to a novel class of chromene anti-cancer compounds. The present disclosure describes the anti-cancer properties of a novel class of chromene anti-cancer compounds. The present disclosure further describes methods for synthesizing and isolating the novel class of chromene anti-cancer compounds.

[0007] Specifically, the present disclosure describes methods for the synthesis of novel chromene-based compounds that combine at least two different molecules to yield compounds with interesting and related biological profiles.

[0008] The synthesis process of chromene compounds described in the prior art does not allow the isolation of these novel compounds.The method disclosed herein allows the synthesis and isolation of novel chromene compounds that are surprisingly effective when used as anti-cancer compounds in several cancer subtypes, such as breast cancer, renal cell carcinoma, acute leukemia and glioma.These compounds are particularly effective against breast cancer and renal cell carcinoma.

[0009] In one embodiment, the synthesized compounds were tested for their anti-cancer activity by in vitro screening using appropriate cell lines. The activity of the compounds was examined at various levels, including effects on cell viability, proliferation, migration, aggressiveness, cell death and metabolism.

[0010] In one embodiment, the toxic effects of the chromene compounds were also tested in non-neoplastic cell lines.

[0011] In one embodiment, the Caenorhabditis elegans (C. elegans nematode) model was used for early stage in vivo toxicity screening of chromene compounds, and mice were also used as a model to assess in vivo toxicity.

[0012] In one embodiment, the anti-cancer properties of the chromene compounds were also characterized using an in vivo CAM (chick chorioallantoic membrane) assay to evaluate the efficacy and angiogenic properties of the novel chromenes. Additionally, the in vivo efficacy was evaluated using mice as a model.

[0013] The subject compounds exhibit a unique and promising anti-cancer profile.

[0014] One aspect of the disclosure relates to a compound comprising the formula: [ka] (In the formula, R 1 , R 2 and R 3 are selected independently of each other, R 1 is selected from H, alkyl, aryl, alkoxyl, acyl, halogen, nitro, hydroxyl, amine, amide, ketone, ester, heterocycle; R 2is selected from H, alkyl, aryl, alkoxyl, acyl, halogen, nitro, hydroxyl, amine, amide, carbonyl, ketone, ester, heterocycle; R 3 is H, alkyl, aryl, alkoxyl, acyl, halogen, nitro, hydroxyl, amine, amide, ketone, ester, heterocycle or [ka] )

[0015] Another aspect of the disclosure relates to a compound or a pharma- ceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer, or polymorph comprising the formula: R 1 is selected from aryl or heterocycle; R 2 is selected from H, alkyl, aryl, alkoxyl, halogen, hydroxyl, amine, carbonyl or heterocycle; R 3 H or [ka] is selected from. However, preferably, [ka] is excluded.

[0016] In one embodiment, R 1 is selected from aryl, R 2 is selected from H, alkyl, alkoxyl, halogen, hydroxyl, or amine, or a pharma- ceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer, or polymorph thereof.

[0017] In one embodiment, the compound or a pharma- ceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer or polymorph, wherein the dimer is preferably a homodimer.

[0018] In one embodiment, R 1 is substituted aryl or a pharma- ceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer, or polymorph thereof.

[0019] In one embodiment, R 1 is selected from hydroxyphenyl, hydroxy-methoxyphenyl, hydroxy-bromophenyl, hydroxy-chlorophenyl, fluorophenyl, bromophenyl, 24-chlorophenyl, phenyl, methoxyphenyl, difluoro-hydroxyphenyl, ethoxyphenyl, or bromo-hydroxy-methoxyphenyl, or a pharma- ceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer, or polymorph.

[0020] In one embodiment, R 1 is 2-hydroxyphenyl, 2-hydroxy-3-methoxyphenyl, 2-hydroxy-5-methoxyphenyl, 2-hydroxy-5-bromophenyl, 2-hydroxy-5-chlorophenyl, 4-fluorophenyl, 4-bromophenyl, 2-fluorophenyl, 3-fluorophenyl, 4-chlorophenyl, phenyl, 3-hydroxyphenyl, 2-hydroxyphenyl, 2-methoxyphenyl, 3,5-difluoro-2-hydroxyphenyl, 4-ethoxyphenyl, or 2-bromo-3-hydroxy-4-methoxyphenyl, or a pharma- ceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer, or polymorph.

[0021] In one embodiment, R 2is substituted or unsubstituted aryl or a pharma- ceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer, or polymorph thereof.

[0022] In one embodiment, R 2 is selected from H, 5-methoxy, 7-methoxy, 7-bromo, 7-chloro, or 7-fluoro, or a pharma- ceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer, or polymorph thereof.

[0023] In one embodiment, R 3 is a chromene unit or H or a pharma- ceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer, or polymorph thereof.

[0024] In one embodiment, R 3 is selected from H, a 2-(4-fluorophenyl)-5-methoxy-1,9-dihydrochromeno[2,3-d]imidazole unit, a 2-(4-bromophenyl)-5-methoxy-1,9-dihydrochromeno[2,3-d]imidazole unit, or a 2-(2-fluorophenyl)-5-methoxy-1,9-dihydrochromeno[2,3-d]imidazole unit, or a pharma- ceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer, or polymorph.

[0025] In one embodiment, [ka] [ka] [ka] or a pharma- ceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer or polymorph selected from:

[0026] In one aspect of the disclosure, the compounds of the disclosure are for use in human or veterinary medicine.

[0027] In one aspect of the disclosure, the compounds may be used in the treatment, therapy or diagnosis of diseases or cancers characterized by hyperproliferation of benign or malignant cells or characterized by areas of neovascularization or hypervascularization.

[0028] In one aspect of the disclosure, the compounds may be used in the treatment, therapy or diagnosis of hyperproliferative tissue or neoplasms.

[0029] In one aspect of the disclosure, the compounds may be used in the treatment, therapy, or diagnosis of breast cancer, renal cell carcinoma, leukemia, glioma, or glioblastoma.

[0030] In one aspect of the disclosure, the compounds may be used in the treatment, therapy or diagnosis of renal cell carcinoma, leukemia, glioma, glioblastoma, breast cancer.

[0031] In one aspect of the disclosure, the compounds may be used in the treatment, therapy or diagnosis of triple negative breast cancer, acute renal cell carcinoma, luminal breast cancer, basal-like breast cancer, acute leukemia.

[0032] Another aspect of the present disclosure relates to pharmaceutical compositions comprising at least one of the compounds of the present disclosure.

[0033] In one embodiment, the pharmaceutical composition of the present disclosure further comprises a pharma- ceutically acceptable carrier.

[0034] In one embodiment, the pharmaceutical composition of the present disclosure further comprises an antiviral agent, an analgesic agent, an anti-inflammatory agent, a chemotherapeutic agent, a radiotherapeutic agent, an antibiotic, an antifungal agent, an antiparasitic agent, or a diuretic agent, or a mixture thereof.

[0035] In one embodiment, the pharmaceutical composition of the present disclosure further comprises a filler, a binder, a disintegrant, a lubricant, or a mixture thereof.

[0036] In one embodiment, the pharmaceutical composition of the present disclosure is used in intradermal or transdermal therapy or local or systemic or intravenous therapy or a combination thereof.

[0037] Another aspect of the present disclosure relates to a method for obtaining the compounds of the present disclosure, comprising the steps of: adding concentrated HCl (1.1 equiv.) to an orange solution of 2-imino-8-methoxy-2H-chromen-3-amine (0.150 mg, 0.79 mmol) in 1 mL of CH3CN; Stir the mixture at room temperature (20°C) for 10 to 15 minutes (precipitation is immediately observed); filtering the solid, preferably by simple filtration, to obtain 3-amino-8-methoxy-2H-chromene-2-iminium chloride; adding an aldehyde (1.1-1.7 equivalents) to a suspension of 3-amino-8-methoxy-2H-chromene-2-iminium chloride (0.25-0.35 mmol) in CHCN (1-2 mL); stirring the suspension at 60°C for 24 to 48 hours; filtering the resulting solid to separate the pure product, preferably washing with CH3CN; Optionally, if HCl contamination is observed in the 1H NMR spectrum, wash the solid with aqueous NaHCO3 (0.05 M), filter, and wash with water to obtain the pure product 2-(5-methoxy-3,9-dihydrochromeno[2,3-d]imidazole.

[0038] In one embodiment, for the preparation of 5,5'-dimethoxy-2,2'-diphenyl-1,1',9,9'-tetrahydro-9,9'-bichromeno[2,3-d]imidazole, aldehyde (1-1.2 equiv.) was added to a solution of 2-imino-8-methoxy-2H-chromen-3-amine in CH3CN (1-2 mL) and the solution was stirred for 7-24 h at 80° C. A solid product gradually began to precipitate, was filtered, washed with CH3CN and identified as the pure product, 5,5'-dimethoxy-2,2'-diphenyl-1,1'',9,9'-tetrahydro-9,9'-bichromeno[2,3-d]imidazole.

[0039] Another aspect of the present disclosure relates to nanoparticles comprising a compound of the present disclosure and / or a pharmaceutical composition of the present disclosure.

[0040] In one embodiment, the nanoparticles comprising the compounds and / or pharmaceutical compositions of the present disclosure are encapsulated by the nanoparticles.

[0041] Another aspect of the present disclosure relates to kits that include the compounds and / or pharmaceutical compositions of the present disclosure.

[0042] Another aspect of the disclosure relates to the use of the compound or a pharma- ceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer or polymorph for use in human or veterinary medicine. In particular, for use in the treatment or therapy of diseases or cancers characterized by hyperproliferation of benign or malignant cells or by areas of neovascularization. In particular, for use in the treatment, therapy or diagnosis of hyperproliferative tissues, such as those associated with cancer. Furthermore, for use in the treatment, therapy or diagnosis of breast cancer, renal cell carcinoma, acute leukemia and glioma. Furthermore, for use in the treatment, therapy or diagnosis of glioblastoma and triple negative breast cancer.

[0043] According to the definition of the International Union of Pure and Applied Chemistry (IUPAC), an alkyl group is a monovalent group obtained by removing a hydrogen atom from any carbon atom of an alkane. n CH2n+1 By removing a hydrogen atom from the terminal carbon atom of an unbranched alkane, the resulting group is the normal alkyl (n-alkyl) group H(CH2). n The RCH2, R2CH (R≠H) and R3C (R≠H) groups are primary, secondary and tertiary alkyl groups, respectively. Aryl groups are derived by removing hydrogen atoms from ring carbon atoms of arenes (monocyclic and polycyclic aromatic hydrocarbons).

[0044] "Alkyl" includes "lower alkyl" and extends to encompass carbon fragments having up to 30 carbon atoms. Examples of alkyl groups include octyl, nonyl, norbornyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, eicosyl, 3,7-diethyl-2,2-dimethyl-4-propylnonyl, 2-(cyclododecyl)ethyl, adamantyl, and the like.

[0045] The term "lower alkyl" refers to an alkyl group having 1 to 7 carbon atoms. Examples of lower alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec- and tert-butyl, pentyl, hexyl, heptyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, 2-methylcyclopropyl, cyclopropylmethyl, and the like.

[0046] In this disclosure, halogen refers to an element selected from the list consisting of fluorine (F), chlorine (Cl), bromine (Br), iodine (I), and astatine (At).

[0047] In the present disclosure, the term "heterocycle" refers to a ring in which at least one of the atoms forming the ring backbone is not carbon. Unless otherwise specified, the heterocycle may be saturated, partially unsaturated or fully unsaturated. "Saturated heterocycle" refers to a heterocycle containing only single bonds between the ring members. "Partially saturated heterocycle" refers to a non-aromatic heterocycle containing at least one double bond. The term "aromatic heterocycle" refers to a fully unsaturated aromatic ring in which at least one atom forming the ring backbone is not carbon. Typically, an aromatic heterocycle contains up to four nitrogens, up to one oxygen and up to one sulfur. Unless otherwise specified, an aromatic heterocycle can be bonded through any available carbon or nitrogen by replacing a hydrogen atom on said carbon or nitrogen. The term "aromatic heterobicyclic ring system" refers to a ring system consisting of two fused rings in which at least one of the two rings is an aromatic heterocycle as defined above.

[0048] The term "carbocycle" refers to a ring in which the atoms forming the ring backbone are selected only from carbon. Unless otherwise specified, a carbocycle may be saturated, partially unsaturated or fully unsaturated. If a fully unsaturated carbocycle satisfies Hückel's rule, the ring may also be called an "aromatic ring". "Saturated carbocycle" refers to a ring having a backbone made of carbon atoms bonded together by single bonds, and unless otherwise specified, the remaining carbon valences are occupied by hydrogen atoms. [Brief description of the drawings]

[0049] The following figures provide preferred embodiments to illustrate the present disclosure and should not be considered as limiting the scope of the invention. [Figure 1] FIG. 1 shows the effect of chromene compounds on the migration of MCF-7 cells assessed in a wound-healing assay (respective ½IC50 and IC50 values ​​for each compound at 12, 24, 48 and 72 hours of treatment). [Diagram 2] FIG. 1 shows the effect of chromene compounds on migration of Caki-2 cell line assessed in a wound-healing assay (respective IC50 values ​​for each compound at 12, 24, 36, 48 hours of treatment). [Diagram 3] FIG. 1 shows the effect of chromene compounds on the proliferation of 786-O cells after 48 hours of treatment. [Figure 4] Flow cytometric analysis of MCF-7 cell viability assessed by Annexin V / PI assay. Figure 4A shows representative dot plots of MCF-7 cells treated with 0.5% DMSO (control) or IC50 concentrations of compounds for 12 and 24 hours. Figure 4B shows quantification of the percentage of cells in each quadrant of the dot plot. [Diagram 5] Flow cytometric analysis of Hs578t cell viability assessed by Annexin V / PI assay. Figure 5A shows representative dot plots of Hs578t cells treated with 0.5% DMSO (control) or IC50 concentrations of drugs for 12 and 24 hours. Figure 5B shows quantification of the percentage of cells in each quadrant of the dot plot. [Figure 6] Figure 1 shows Western blot analysis of full-length PARP, caspase 3 and 9, BIM and Bcl-xL after treatment of MCF-7 cells with compounds MC409, MC408, MC406 and MC421 for (A) 24 hours and (B) 48 hours with the corresponding IC50 concentrations. Protein levels detected by Western blotting in lysates using 12% polyacrylamide gels. [Figure 7] Figure 1 shows Western blot analysis of full-length PARP, caspase 3 and 9, BIM, Bcl-xL and Bax after treatment of Hs578t cells with compounds MC409, MC408, MC406 and MC421 for (A) 24 hours and (B) 48 hours with the corresponding IC50 concentrations. Protein levels detected by Western blotting in lysates using 12% polyacrylamide gel. [Figure 8]Flow cytometric analysis of DNA content of MCF-7 cells. Figure 8A shows representative histograms showing cell cycle profiles of MCF-7 cells treated with 0.5% DMSO (control) or IC50 concentrations of drugs for 12 and 24 hours. Figure 8B shows quantification of cells in different phases of the cell cycle. [Figure 9] Flow cytometry analysis of DNA content of Hs578t cells. Figure 9A shows representative histograms showing cell cycle profiles of Hs578t cells treated with 0.5% DMSO (control) or IC50 concentrations of drugs for 12 and 24 hours. Figure 9B shows quantification of cells in different phases of the cell cycle. [Figure 10] FIG. 1 shows the effects of compounds MC408 and MC421 on the microtubule network of Hs578t (A, B) and MCF-7 (C, D) cells. [Figure 11] Figure 1. Strategy used to determine toxic effects in C. elegans. Worms utilized heat-inactivated bacteria (E. coli OP50) as food source and were cultured in liquid medium for 7 days in the presence of several concentrations of compounds MC421, MC406, MC369 and MC408, MC409, MC407. Food consumption rate from day 3 to day 5 was used as an indicator of worm development, health and even fecundity, since after day 3 worm progeny contribute to the rapid decrease in food amount. An example is shown for a fictitious compound X. DMSO 1% and 5% were used as negative and positive controls for toxicity. [Figure 12] FIG. 1 shows the lack of in vivo toxicity of compounds in C. elegans. [Figure 13] FIG. 1 shows the effect of compound MC408 on C57BI / 6 mice. [Figure 14] FIG. 1 shows the results of a series of welfare tests carried out on C57BI / 6 mice treated with compound MC408. [Figure 15] FIG. 1 shows the effect of MC408 treatment on enzymatic liver function in C57Bl / 6 mice. [Figure 16]Effect of MC408 treatment on C57Bl / 6 mice - second experiment. Figure 16A is a schematic of the experimental timeline. Figure 16B shows the body weight of the mice. [Figure 17] Figure 17 shows the in vivo therapeutic effects of MC408 and MC421 on breast cancer Hs578t cell line, Figure 17A shows representative photographs of CAM assay, and Figure 17B shows tumor growth rate. [Figure 18] Flow cytometric analysis of 786-O cell viability assessed by Annexin V / PI assay. Figure 18A shows representative dot plots of 786-O cells treated with 0.5% DMSO (control) or IC50 concentrations of drugs for 24 and 48 hours. Figure 18B shows quantification of the percentage of cells in each quadrant of the dot plot. [Figure 19] Western blot analysis of KDM4C, full-length PARP, Hsp90, full-length JNK, full-length p53, Bid and p21 after treatment of 786-O cells with compounds MC408 and MC421 for (A) 24 h and (B) 48 h at the corresponding IC50 concentrations. Protein levels detected by Western blotting in lysates using 12% polyacrylamide gels. [Figure 20] Figure 1 shows Western blot analysis of mTOR, ERK, VEGFR2, EGFR, AKT, PTEN and AMPK intact and phosphorylated forms, PDK1, NF-κB and p18 phosphorylated forms, and c-Myc, Hsp90 and PRAS40 proteins after 2 hours starvation and treatment of 786-O cells with Cediranib, compounds MC350, MC415, MC412, MC408 and MC421 at a specific concentration of 2 μM for 6 hours. Protein levels detected by Western blotting in lysates using 10% polyacrylamide gels. [Figure 21] 21 shows the in vivo therapeutic effects of MC408 and MC421 on renal cancer 786-O cell line, in which Fig. 21A shows representative photographs of CAM assay, and Fig. 21B shows the tumor growth rate. [Figure 22] Flow cytometry analysis of DNA content of 786-O cells. Figure 22A shows representative histograms showing cell cycle profiles of 786-O cells treated with 0.5% DMSO (control) or IC50 concentrations of drugs for 24 and 48 hours. Figure 22B shows quantification of cells in different phases of the cell cycle. [Diagram 23] FIG. 1 shows the effect of chromene compounds on cell proliferation after treatment of A498 resistant and parental cell lines with ½ IC50 or IC50 of selected compounds for 24 and 48 hours. [Figure 24] FIG. 1 shows the effect of chromene compounds on cell proliferation after treatment of Caki-2 resistant and parental cell lines with ½ IC50 or IC50 of selected compounds for 24 and 48 h. [Diagram 25] Figure 1 shows Western blot analysis of intact and phosphorylated ERK, GAPDH, HK2, LDHA, Hif2α, MCT1, PKM, PFKL, Hsp90 and c-Myc proteins after a 48 hour treatment period with IC50 of rapamycin and cediranib, compounds MC350, MC413, MC408 and MC421 in A498 parental (P) and resistant (R) cells. Protein levels detected by Western blotting in lysates using 10% polyacrylamide gels. [Figure 26] Figure 1 shows the in vivo effect of compounds MC408 and MC421 on angiogenesis. Representative images (20x magnification) of in ovo and ex ovo CAM assays on days 13 and 17. [Figure 27] FIG. 1 shows tumor volume and weight in an orthotopic breast cancer NSG mouse xenograft model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] The present disclosure relates to a novel class of chromene anti-cancer compounds. The present disclosure describes the anti-cancer properties of a novel class of chromene anti-cancer compounds. The present disclosure further describes methods for synthesizing and isolating the novel class of chromene anti-cancer compounds.

[0051] Specifically, the present disclosure describes methods for the synthesis of novel chromene-based compounds that combine at least two different molecules to yield compounds with interesting and related biological profiles.

[0052] In one embodiment, 19 chromene imidazole compounds were isolated. Table 1 shows the structure of the chromene scaffold and the 19 chromene imidazole compounds that were synthesized and isolated. [Table 1-1] [Table 1-2]

[0053] In one embodiment, the cell growth inhibition rate and IC of the synthesized and isolated chromene compounds are measured as part of the evaluation of the antiproliferative activity of the isolated agents. 50 The IC values ​​of the compounds were measured. A thorough structure-activity relationship study was carried out for all the compounds synthesized. The antiproliferative activity against MCF-7 breast cancer cell line was analyzed. Table 2 shows the IC values ​​of the compounds against breast cancer cell line MCF-7 and non-neoplastic cell line MCF-10A. 50 The IC values ​​(μM) and selectivity index (SI) are shown. The non-neoplastic cell line MCF-10A was also used to examine the selectivity of the compounds against tumor cells. 50 For compounds with IC values ​​(concentration required to reduce cell viability by 50%) higher than 20 μM, 50 To evaluate the cytotoxicity of the test compounds, the selectivity index (SI) was calculated. [Table 2-1] [Table 2-2]

[0054] The dimeric compound showed superior IC against MCF-7 cell line 50 The monomer compounds with halogen atoms or no substituents on the aromatic rings attached to the imidazole moiety showed the lowest IC 50 Imidazochromenes bearing OH or OCH3 / OCH2CH3 showed IC values ​​(<1 μM) even when halogen atoms were present in the same aromatic position. 50 The value increased significantly.

[0055] Several chromenes also showed promising IC10 activity against the non-neoplastic cell line MCF-10A. 50 values ​​were obtained and high SI values ​​were calculated. Toxicity towards non-neoplastic cells was much lower for the dimeric compounds, giving excellent SI values ​​(higher than 60). The SI values ​​were generally very encouraging.

[0056] In one embodiment, the monomers and their respective dimers were selected for comparative studies and further evaluated in Hs578t and MDA-MB-468 breast cancer cell lines. Table 3 shows the IC values ​​of selected chromene compounds against breast cancer cell lines Hs578t and MDA-MB-468. 50 The therapeutic efficacy values ​​(μM) and selectivity index (SI) are shown. These cell lines are negative for estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) molecular markers, and therefore represent the basal-like subtype of breast cancer, which is included in the known triple-negative subtypes. The clinical behavior of these breast cancer subtypes is highly aggressive and there is still no specific molecular therapy. [Table 3-1] [Table 3-2]

[0057] Chromene compounds have very low IC values ​​of 0.035-0.27 μM against Hs578t. 50 Bromine-substituted chromene compound MC408 and dimeric compound MC421 showed very low IC values ​​against Hs578t and MDA-MB468 cell lines. 50 Compound MC408 is particularly active (IC 50 = 0.027 μM). In general, the compounds showed more interesting antiproliferative potential against these aggressive breast cancer subtypes, with good SI values.

[0058] In one embodiment, the antiproliferative activity of compounds MC408, MC409, MC421 and MC406 was further measured against glioma cell lines (U87, GAMG and GL18) and acute leukemia cell lines (HL-60, KG-1 and Jurkat). This was done to assess whether the compounds show an interesting anticancer profile in other cancer cell models as well. Cell viability was measured using the MTS assay after exposing cells to the respective compounds at a range of appropriate concentrations for 72 hours, and the respective IC 50 Table 4 shows the IC values ​​of compounds MC408, MC409, MC421, and MC406 against glioma and leukemia cancer cell models. 50 Values ​​(μM) are shown. [Table 4]

[0059] These compounds had IC 50 The IC values ​​were in the nanomolar or low micromolar range, indicating excellent antiproliferative potency. Compound MC421 showed very high cell proliferation inhibition against glioma cell lines, especially U87 and GAMG. Compound MC408 had a lower antiproliferative potency compared to the other compounds, but still showed an IC 50The values ​​were still in the low μM range. In the leukemia models, all compounds appeared to be more selective for the Jurkat cell line, but excellent growth inhibition was achieved when other leukemia cell lines were treated with all compounds. These results indicated that these compounds are promising in several cancer cell models and can be considered as candidates for cancer therapy.

[0060] In one embodiment, the antiproliferative activity of the compounds was further measured against renal cancer cell lines and non-neoplastic renal cell lines. Table 5 shows the IC 50 The IC values ​​(μM) and selectivity indices (SI) are shown. Compounds M955, M1220, M1143 and M1221 showed no biological activity in the initial screening, with IC 50 Determination of the selectivity of the agonist was not pursued. Several compounds showed high activity and good selectivity index against renal cell carcinoma (RCC) cell lines. [Table 5]

[0061] In one embodiment, the antiproliferative activity of compounds MC421, MC409, MC408, MC350, MC416, MC410, MC411, MC415 and MC413 was further measured against drug-resistant renal cancer cell lines. Table 6 shows the IC50 values ​​against rapamycin-resistant A498 cell line and cediranib-resistant Caki-2 cell line and non-neoplastic cell line (HK2). 50 Values ​​(μM) and selectivity indices (SI) are shown. [Table 6]

[0062] In one embodiment, the effects of four selected compounds (MC408, MC409, MC406, and MC421) were further evaluated on cell migration (Figure 1). MCF-7 cells were cultured at their respective IC values 50 and the IC 50The wound healing assay was performed after treatment with half the IC value for 72 hours. 50 Half the concentration was used to assess concentration-dependent effects. Figure 1 shows the effect of chromene compounds on MCF-7 cell migration assessed by wound-healing assay (1 / 2 IC for each compound). 50 and IC 50 (Treatment with 0.01% DMSO for 12, 24, 48, and 72 h was performed at 0.05–0.50 mM NaCl). Results are presented as the mean ± SD of at least three independent experiments, and *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 compared to control (DMSO, 0.5%).

[0063] For all compounds, extensive cell death was observed microscopically after only 12 hours of incubation. After 72 hours, the medium was removed from each well before taking pictures because the large amount of floating dead cells prevented accurate measurement of cell migration. Compound MC409 showed an early effect on cell migration, and after 12 hours, this compound increased the percentage of cell migration (compared to the 0.5% DMSO control) by approximately 10% compared to the IC 50 and 1 / 2IC 50 However, the cells appear to recover from the effects of the compound, as a gradual recovery of cell migration is observed. After 72 hours of treatment, the IC 50 Using the IC50 concentration, cell migration was inhibited to approximately 12% of the control. For the other three compounds, lower effects were observed after 24 or 48 hours of treatment. 50 Concentration and 1 / 2 50 Compounds MC408 and MC406 treatment with IC concentrations had similar effects on cell migration, reducing cell migration by 20% compared to controls after 72 hours. Compound MC421 showed a concentration-dependent effect. IC 50 and 1 / 2IC 50 All concentrations had a small effect on cell migration over time, with a reduction in cell migration only observed after 72 hours of treatment (13% reduction from control).

[0064] In one embodiment, the effect of compounds MC408, MC421, MC412 and MC415 was also evaluated on cell migration in the renal cell carcinoma cell line Caki-2 (Figure 2) at four different time points. Cediranib was used as the reference drug and all compounds were evaluated at their respective IC 50 The concentration values ​​were used to test. Figure 2 shows the effect of chromene compounds on migration of Caki-2 cell line (respective IC 50 Treatment with 0.5% DMSO for 12, 24, 36, and 48 hours (values ​​shown) was assessed by wound healing assay. Results were normalized to control (dashed line) and presented as mean ± SEM. *p<0.05, **p<0.005, ***p<0.002, ****p<0.0001 compared to control (0.5% DMSO). The significance of differences between different groups was determined by Student's t-test.

[0065] In one embodiment, the effect of the preferred chromene compounds on cell proliferation was evaluated. 786-O cells were treated with an IC 50 and 1 / 2 50 The cells were treated with IC values ​​for 48 hours. The ability of BrdU incorporation during DNA synthesis was measured, and the results are shown in Figure 3. Figure 3 shows the effect of chromene compounds on the proliferation of 786-O cells treated for 48 hours. The results are expressed as the mean ± SD of at least three independent experiments. *p<0.05, **p<0.01, ***p<0.0005, ****p<0.0001 compared to the control (0.5% DMSO). The significance of the differences between different groups was determined by Student's t-test. In general, chromenes induced a significant decrease in cell proliferation in 786-O cells in a concentration-dependent manner. Exceptions were observed for compounds MC409, MC410, and MC368.

[0066] In one embodiment, the compounds, particularly compounds MC406, MC409 and MC408, showed a clear effect on cell proliferation and cell death, which can be seen by morphological changes (cells viewed under a microscope) and also by the presence of round, floating cells.

[0067] In one embodiment, cells were examined for induction of apoptosis by flow cytometry to understand the mechanisms leading to cell death. MCF-7 and Hs578t cells were examined for 12 and 24 hours, and 786-O cells were examined for 24 and 48 hours, with the respective IC 50 After incubation with the compounds at 1000 μg / ml, the cells were double-stained with Annexin V and propidium iodide (PI) to detect the externalization of phosphatidylserine. The externalization of phosphatidylserine occurs early in apoptosis. Annexin V binds to phosphatidylserine, while PI stains only cells that have lost membrane integrity, making this an experimental procedure that can distinguish between live cells, early apoptotic cells, late apoptotic / necrotic cells, or necrotic cells. Compounds MC408 and MC421 induced apoptotic cell death (early apoptosis) in the three cell lines after 24 hours, as shown in Figures 9 and 10, and demonstrated associated anticancer effects. Compounds MC406 and MC409 also induced apoptotic cell death in the Hs578t cell line.

[0068] Figure 4 shows flow cytometry analysis of MCF-7 cell viability assessed by Annexin V / PI assay. Figure 4A shows MCF-7 cells treated with 0.5% DMSO (control) or IC 50 Figure 4B shows representative dot plots of MCF-7 cells treated with compounds MC408, MC409, MC406 and MC421 at concentrations for 12 and 24 hours. Figure 4B shows quantification of the percentage of cells in each quadrant of the dot plot. The results are obtained using the mean ± SEM of three independent experiments, with DMSO-treated cells as control (100%). Annexin V / PI data were analyzed by two-way ANOVA and Bonferroni post-hoc test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 compared to control (0.5% DMSO). Figure 5 shows flow cytometry analysis of Hs578t cell viability assessed by Annexin V / PI assay. Figure 5A shows the viability of Hs578t cells treated with 0.5% DMSO (control) or IC 50Figure 5B shows representative dot plots of Hs578t cells treated with compounds MC408, MC409, MC406 and MC421 at concentrations for 12 and 24 hours. Figure 5B shows quantification of the percentage of cells in each quadrant of the dot plot. The results are obtained using the mean ± SEM of three independent experiments, with DMSO-treated cells as control (100%). Annexin V / PI data were analyzed by two-way ANOVA and Bonferroni post-hoc test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 compared to control (0.5% DMSO). Figure 18 shows flow cytometry analysis of 786-O cell viability assessed by Annexin V / PI assay. Figure 18A shows the results of 786-O cells treated with 0.5% DMSO (control) or IC 50 Figure 18A shows representative dot plots of 786-O cells treated with compounds MC408 and MC421 at 24 and 48 h. Figure 18B shows quantification of the percentage of cells in each quadrant of the dot plot. Results are obtained using the mean ± SEM of three independent experiments, with cells treated with 0.5% DMSO as control (100%). Annexin V / PI data were analyzed by two-way ANOVA with Bonferroni post-hoc test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 compared to control (0.5% DMSO).

[0069] In one embodiment, induction of cell death was further investigated through examination of apoptotic pathways by Western blot analysis of key apoptotic markers. 50 After 24 and 48 h treatment with compounds, poly(ADP-ribose) polymerase 1 (Parp), caspases 3 and 9 were assessed as markers of induced apoptosis (Figure 6).

[0070] Treatment of cells with the compounds resulted in cleavage of PARP, the final step in caspase activation and considered a hallmark of apoptosis (Figures 6 and 7). Cleaved caspase 3 was also observed at two time points. Apoptosis was induced by all compounds, as observed by the presence of these markers.

[0071] Bim protein was also used and elevated levels were observed with the test compounds, but only in the Hs578t cell line. BH3 domain proteins such as Bim interact with tubulin and it is said that in the early stages, microtubules bind to dynein light chains and sequester Bim, thus preventing the initiation of the apoptosis signaling pathway. After being released from microtubules, Bim translocates to mitochondria, where it interacts with several proteins (e.g., Bax, Bcl-2 and Bcl-xL) and ultimately promotes apoptosis. This suggests that cells treated with the test compounds release apoptotic signals early, thereby affecting the levels of pro- and anti-apoptotic proteins involved in mitochondria-induced apoptosis.

[0072] In one embodiment, cell cycle analysis was performed to determine the ability of compounds MC408, MC409, MC406 and MC421 to inhibit cell proliferation. MCF-7 and Hs578t cells were assayed using their respective IC 50 The 786-O cells were treated with the compounds at the respective IC 50 The cells were treated with the compounds MC408 and MC421 for 24 and 48 hours. Cell cycle analysis was assessed by DNA content using flow cytometry (Figures 8, 9 and 22). Figure 8 shows the flow cytometric analysis of DNA content of MCF-7 cells. Figure 8A shows the flow cytometric analysis of DNA content of MCF-7 cells treated with 0.5% DMSO (control) or IC 50Figure 8A shows representative histograms of cell cycle profiles of MCF-7 cells treated with MC408, MC409, MC406 and MC421 at various concentrations for 12 and 24 hours. Figure 8B shows the quantification of cells in different phases of the cell cycle, excluding compound MC409. Results are presented as mean ± SD of three independent experiments. Data were analyzed by two-way ANOVA with Bonferroni post-hoc test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 compared to control (0.5% DMSO).

[0073] Figure 9 shows flow cytometry analysis of DNA content of Hs578t cells. Figure 9A shows Hs578t cells treated with 0.5% DMSO (control) or IC 50 Figure 9B shows representative histograms showing cell cycle profiles of Hs578t cells treated with MC408, MC409, MC406 and MC421 at various concentrations for 12 and 24 hours. Figure 9B shows the quantification of cells in different phases of the cell cycle, excluding compound MC409. Results are shown as mean ± SD of three independent experiments. Data were analyzed by two-way ANOVA with Bonferroni post-hoc test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 compared to control (0.5% DMSO).

[0074] Figure 22 shows flow cytometry analysis of DNA content of 786-O cells. Figure 22A shows flow cytometry analysis of DNA content of 786-O cells treated with 0.5% DMSO (control) or IC 50 Figure 22B shows representative histograms showing cell cycle profiles of 786-O cells treated with MC408 and MC421 at different concentrations for 24 and 48 hours. Figure 22B shows the quantification of cells in different phases of the cell cycle. Results are shown as mean ± SD of three independent experiments. Data were analyzed by two-way ANOVA with Bonferroni post-hoc test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 compared to control (0.5% DMSO).

[0075] In one embodiment, the effect of chromene-based compounds on microtubule dynamics was analyzed. Figure 10 shows the effect of compounds MC408 and MC421 on the microtubule network of Hs578t (A, B) and MCF-7 (C, D) cells. Untreated (control), paclitaxel (Hs578t 12h: 0.25μM, 24h: 0.01μM; MCF-7 12 / 24h: 1μM) and compounds MC408 and MC421 were treated with IC 50 Cells treated with β-tubulin for 12 h (A, C) or 24 h (B, D) were stained for β-tubulin and counterstained with 4,6-diamino-2-phenylindole (DAPI). Microtubules and unassembled tubulin are shown in green. DAPI-stained DNA is shown in blue.

[0076] In one embodiment, the toxicity of compounds was evaluated in vivo using Caenorhabditis elegans (C. elegans) as a model. A food clearance based assay was performed, in which bacterial consumption over time is a proxy for worm development, health and fecundity (Figure 11). Figure 11 shows the strategy used to determine toxic effects in C. elegans. Worms utilized heat-inactivated bacteria (E. coli OP50) as a food source and were cultured in liquid medium for 7 days in the presence of several concentrations of compounds MC421, MC406, MC369 and MC408, MC409, MC407. The rate of food consumption from day 3 to day 5 was used as an indicator of worm development, health and even fecundity, since after day 3, worm progeny contribute to the rapid decrease in food amount. An example is shown for a hypothetical compound X. DMSO 1% and 5% were used as negative and positive controls for toxicity, respectively. In this experiment, compounds were dissolved in DMSO 1% (vehicle).

[0077] Compounds were tested at several concentrations, up to the highest possible soluble concentration of 150 μM (MC369, 75 μM) (Figure 12). Figure 12 shows the results of the analysis of the in vivo toxicity of MC421, MC406, MC369 and MC408, MC409, MC407 in C. elegans. Food consumption rate on days 3-5 was used as an index of worm development, health and fecundity and was compared to 1% DMSO (vehicle, non-toxic). No statistical differences were found for MC421, MC406, MC369 and MC408, MC409, MC407 (analysis of variance followed by Games-Howell post-hoc test). No statistical differences were found for food consumption rate on days 3-5 for any compound / concentration of compound. Compounds appear to be well tolerated at concentrations significantly higher than those used in in vitro studies, which may be a good predictor of toxicity in mammalian models.

[0078] In one embodiment, the safety profile of the compound was evaluated in rodents. Figure 13 shows the effect of MC408 treatment on C57Bl / 6 mice - first study. Figure 13A is a schematic diagram of the experimental timeline. Five-month-old male and female animals were used (n=3 / group / sex) and were injected daily for 7 days (ip) with either 3mg / Kg MC408 or vehicle (saline, Tween 80 and methylcellulose). A series of welfare tests were performed daily. Figure 13B shows the immediate aftermath of the first injection. In this analysis, animals were videotaped for 10 minutes and the immobility time and vertical movement (REARS) were counted by the experimenter. No differences were found between vehicle and MC408 mice. Figure 13B shows the weight of the mice monitored throughout the experiment and no differences were found between the treatment groups. Figure 13D shows the vertical exploration behavior of the mice. This behavior was measured in an observation jar, counting the number of vertical movements over a 5-minute period. Figure 13E shows the horizontal behavior of the mice. This activity was recorded in an open arena with labeled squares, and the number of squares was counted for 1 min while the animals were freely exploring. No differences were found in the exploratory behavior. Figure 13F shows an indirect measurement of anxiety in the mice. To indirectly measure anxiety, the number of boluses faecalis produced by the animals during the behavioral setting was counted, and no differences were found between the groups. In Figures 13G and H, water and food intake, respectively, were analyzed. Each day, 0.300 g of food and 200 mL of water were placed in each cage. At the end of the study (day 7), food was weighed and water was measured, and no differences were found in both parameters. Data are presented as mean ± SE.

[0079] Figure 14 shows the effect of MC408 treatment on C57Bl / 6 mice. A series of welfare tests were performed to evaluate signs of toxicity of treatment with MC408 compared to vehicle animals. All parameters evaluated were scored as normal or abnormal, present or absent, and all animals had the same score (MC408 and vehicle), suggesting that the compound was not affecting the welfare of the mice.

[0080] In one embodiment, the effect of treatment on liver function in C57Bl / 6 mice was analyzed. Figure 15 shows the effect of MC408 treatment on enzymatic liver function in C57Bl / 6 mice. Aspartate aminotransferase (AST / TGO) and alanine aminotransferase (ALT / TGP) were measured in serum from (Figure 15A) females and (Figure 15B) males using standard techniques. Blood was collected at the end of the experiment (day 7). No statistical differences were found between compound-treated and vehicle-treated animals in each sex. Of note, one male from the MC408-treated group showed higher levels of both AST and ALT compared to the other two animals from the same group. Data are presented as mean ± SE.

[0081] In one embodiment, the effect of compound treatment on C57Bl / 6 mice - second study was analyzed. Figure 16 shows the effect of MC408 treatment on C57Bl / 6 mice - second study. Figure 16A is a schematic diagram of the experimental timeline. Three-month-old male and female animals were used (n=6 / group / sex) and were injected daily for 7 days (ip) with either 3mg / Kg MC408 or vehicle (saline, Tween 80 and methylcellulose). In this study, we considered an experiment with a larger number of animals per group and blood sampling before and after treatment. Figure 16B shows the body weight of the mice. Body weight was monitored throughout the experiment and no differences were found between groups within each sex. Data are shown as mean ± SE.

[0082] In one embodiment, the in vivo therapeutic efficiency test of the compounds was analyzed using the CAM model. Figure 17 shows the in vivo therapeutic effect of MC408 and MC421 on breast cancer Hs578t cell line. Figure 17A shows representative pictures (10x magnification) of the in ovo and ex ovo (days 13 and 17) CAM assays. Figure 14B shows the tumor growth rate. The results are expressed as the mean ± SD of the tumor growth rate from day 13 to day 17 of development. ****p<0.0001. Data were analyzed by one-way ANOVA test. Eggs were treated with control (DMSO 0.5%), MC408 (2xIC50 =0.094μM) or MC421(2xIC 50 =0.070 μM).

[0083] In one embodiment, induction of cell death was further investigated through examination of apoptotic pathways by Western blot analysis of key apoptotic markers. Poly(ADP-ribose) polymerase 1 (Parp), heat shock protein 90 (Hsp90), c-Jun N-terminal kinase (JNK), p53, BH3 interacting domain death agonist (Bid) and p21 were identified as induced apoptotic markers when 786-O cells were treated with IC 50 The results were evaluated after 24 and 48 hours of treatment with (Figure 19).

[0084] In one embodiment, interactions with RTK receptors and mTOR / PI3K / AKT pathways were further explored through evaluation of mammalian target of rapamycin (mTOR), extracellular signal-regulated kinase (ERK), vascular endothelial growth factor receptor 2 (VEGFR2), endothelial growth factor receptor (EGFR), protein kinase B (AKT), phosphatase and tensin homolog (PTEN), 5' adenosine monophosphate-activated protein kinase (AMPK), pyruvate dehydrogenase kinase 1 (PDK1), nuclear factor kappa B (NF-kappa B), p18, c-Myc, Hsp90, and proline-rich Akt substrate of 40 kDa (PRAS40) proteins. These markers were evaluated after 6 hours of treatment with cediranib (a potent inhibitor of vascular endothelial growth factor used in the treatment of renal cell carcinoma) and compounds MC350, MC412, MC415, MC408, and MC421 at a unique dose of 2 μM (Figure 20).

[0085] Treatment of 786-O cells with several compounds reduced phosphorylated mTOR and AKT protein levels. Furthermore, in some cases, both EGFR and VEGFR2 levels were reduced compared to control and cediranib. Several of the compounds tested affect the mTOR / PI3K / AKT pathway and inhibit various protein markers.

[0086] VEGFR and EGFR are tyrosine kinase (RTK) receptors specific for the vascular endothelial growth factor (VEGF) and endothelial (EGF) growth factor families and play an important role in tumor growth and metastasis. Their autophosphorylation stimulates downstream activation and signaling by many other proteins bound to the phosphorylated tyrosines. These downstream signaling proteins initiate several signaling cascades, including the Akt pathway, leading to gene expression, cell proliferation and migration, vasculogenesis and angiogenesis. In addition, mTOR regulates cell growth, proliferation and metabolism. Its activity is controlled by two multiprotein complexes: mTORC1, which is highly sensitive to rapamycin, and mTORC2, which is thought to be resistant or inhibited only when high doses are administered for a long period of time. Second-generation dual mTOR inhibitors have been developed, and the most important advantages of these new drugs are the significant reduction in AKT phosphorylation upon mTORC2 blockade and the better inhibition of mTORC1. Taken together, these results suggest that reduced expression of the RTKs, mTOR and AKT, reduces key regulators of all the functions mentioned above.

[0087] In one embodiment, the CAM model was used to analyze the in vivo therapeutic efficacy of compounds. Figure 21 shows the in vivo therapeutic effects of MC408 and MC421 on renal carcinoma 786-O cell line. Figure 21A shows representative pictures (10x magnification) of in ovo and ex ovo (days 13 and 17) CAM assays. Figure 21B shows the tumor growth rate. Results are expressed as mean ± SD of tumor growth rate from day 13 to day 17 of development, p<0.0001 compared to control (statistical analysis was performed using one-way ANOVA test). Eggs were treated with control (DMSO 0.5%), MC408 (2xIC 50 =0.128μM) or MC421(2xIC 50 =0.154 μM).

[0088] In one embodiment, the effects of cediranib, rapamycin and chromene-based compounds on cell proliferation were evaluated. A498 (parental and rapamycin-resistant) and Caki-2 (parental and cediranib-resistant) cells were treated with IC 50 Value and 1 / 2IC 50 The cells were treated with 100 μg / ml of 10 ...

[0089] Overall, the chromenes induced a significant decrease in cell proliferation in a concentration- and time-dependent manner in either parental or resistant A498 cells, especially for compounds MC408, MC421 and MC413 (Figure 23).

[0090] In parental and drug-resistant Caki-2 cells (Figure 24), the chromenes MC421, MC350 and MC413 were able to reduce cell proliferation in a concentration- and time-dependent manner.

[0091] In one embodiment, interactions with metabolic function were further investigated by assessing the intact and phosphorylated forms of extracellular signal-regulated kinase (ERK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hexokinase 2 (HK2), lactate dehydrogenase A (LDHA), hypoxia-inducible factor 2α (Hif2α), monocarboxylate transporter 1 (MCT1), pyruvate kinase isozyme (PKM), 6-phosphofructokinase (PFKL), heat shock protein 90 (Hsp90), and c-Myc proteins. These markers were associated with increased IC 50The activity of rapamycin and cediranib, and compounds MC350, MC413, MC408 and MC421 was evaluated after 48 hours of treatment of A498 parental (P) and resistant (R) cells. Protein levels were detected by Western blotting of lysates on 10% polyacrylamide gels (Figure 25).

[0092] Treatment of A498 cells with various compounds reduced the levels of several proteins. Indeed, in rapamycin-resistant cells, the protein expression levels of GAPDH, Hif2α, c-Myc and Hsp90 were significantly decreased compared to control and parental cells. Considering the results obtained, chromenes alter the metabolic response of cancer cells, even when they are resistant to currently available RCC therapies.

[0093] In one embodiment, the CAM model was used to analyze the in vivo ability of compounds to inhibit angiogenesis. To assess the effect on angiogenesis, sterile 5 mm diameter filter disks were coated with a fixed concentration (IC 50 The cells were then impregnated with culture medium containing 0.5% chromene MC408 and MC421 (2x the value) or 0.5% DMSO (control group) and placed in the vascular area of ​​the CAM. Figure 26 shows representative pictures (10x magnification) of the in ovo and ex ovo (days 13 and 17) CAM assays.

[0094] After 4 days of treatment with the novel chromenes MC408 and MC421, new blood vessel formation was reduced. Furthermore, destruction of existing blood vessels (black arrows in Figure 26) was observed, suggesting that the compounds inhibit angiogenesis in the CAM model.

[0095] In one embodiment, the compound MC408 was analyzed for its in vivo therapeutic efficacy using a mouse model. Figure 27 shows the in vivo therapeutic efficacy of MC408 in an orthotopic breast cancer mouse xenograft model using the TNBC cell line MDA-MB-231. MDA-MB-231 cells were injected into the mammary fat pad of NSG mice (n=6 or 7 per group). Treatment was performed for 1 week starting from day 3 after implantation. NT=vehicle; dose 1=3 mg / kg; dose 2=10 mg / kg; dose 3=50 mg / kg. *, p<0.05; **, p<0.01; ****, p<0.0001 (comparison with NT group; two-way ANOVA Tukey's post-hoc test or unpaired t-test with Welch's correction).

[0096] Chromene MC408 significantly reduced tumor volume (FIG. 27A) and weight (FIG. 27B) in an orthotopic breast cancer xenograft mouse model in a dose-dependent manner. At a dose of 50 mg / kg, both tumor volume and weight showed a greater than 40% reduction compared to the vehicle group. Importantly, animals did not show weight loss or other side effects throughout the experiment.

[0097] In one embodiment, three human breast cancer cell lines Hs578T, MDA-MB-468, and MCF-7 and a normal breast cell line MCF-10A were obtained from ATCC (American Type Culture Collection). Cancer cell lines were cultured in Dulbecco's Modified Eagle's Medium, 4.5 g / l glucose (DMEM, Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco) and 1% antibiotic solution (Penicillin-Streptomycin, Gibco). Normal cell lines were cultured in Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 (DMEM / F12, Gibco) supplemented with 5% heat-inactivated FBS (Gibco), 1% antibiotic solution (Penicillin-Streptomycin, Gibco), 1% steroid hormone (Hydrocortisone, Sigma-Aldrich), 0.1% peptide hormone (Insulin, Sigma-Aldrich) and 0.01% protein complex (Cholera Toxin, Gibco). Two different human acute myeloid leukemia (AML) cell lines, HL-60 (FAB M2) and KG-1 (Erythroleukemia-FAB M6) and one human lymphoblastic leukemia (ALL) cell line, Jurkat (T-cell type), were also used. These three cell lines were obtained from the German Collection of Microbial Cell Cultures (DMSZ®, in German Deutsche Sammlung von Mikroorganismen und Zellkulturen). Cells were cultured in RPMI1640 medium (Biochrom®-Merck Millipore) supplemented with 10% heat-inactivated FBS (Biochrom®-Merck Millipore) and 1% antibiotic / antimitotic mixture (Invitrogen®). Three human glioblastoma (GBM) cell line models were also used, two established commercial cell lines (U87MG and GAMG, a gift from Rui M. Reis) and one primary GBM culture (GL18). All GBM cells were cultured in DMEM (Biochrom-Merck Millipore) supplemented with 10% FBS. Renal cell carcinoma cell lines A498, 786-O, Caki-2 and HK2 were obtained from ATCC.Cancer cell lines A498 were cultured in MEM medium (Biochrom®-Merck Millipore), 786-O in RPMI1640 medium (Biochrom®-Merck Millipore), Caki-2 in Mc Coys medium (Biochrom®-Merck Millipore), and HK2 in RPMI1640 medium (Biochrom®-Merck Millipore). All media were supplemented with 10% heat-inactivated FBS (Biochrom®-Merck Millipore) and 1% antibiotic / antimitotic mixture (Invitrogen®). All cells were cultured in a humidified incubator at 37°C and 5% CO2. DMSO (dimethyl sulfoxide, Sigma-Aldrich) control was used in all assays.

[0098] In one embodiment, cell viability assays were performed. MCF-7, Hs578T and MCF-10A cells were plated in triplicate (100 μL / well) in 96 multi-well culture plates at 3000 cells / mL or 5000 cells / mL for MDA-MB-468. Cells were then allowed to attach for 18-20 hours in complete medium. Cells were then treated with seven concentrations of compounds (0.1-40 μM or 5-60 μM) or controls in fresh medium. HL-60, KG-1 and Jurkat cell lines were plated at 50.000 cells / 100 μL / well in 96 multi-well culture plates and treated with various concentrations of compounds (or controls) (0.001-2 μM). Cells were plated in triplicate in 96-well plates at initial densities of 2000 cells / well for GAMG, 4000 cells / well for GL18, and 6000 cells / well for U87MG, and allowed to attach for 18–20 h. Cells were then treated with various concentrations of compounds (0.005–100 μM) or controls. A498 and 786-O cells were plated in triplicate at 2000 cells / mL, Caki-2 cells at 3000 cells / mL, and HK2 cells at 2000 cells / mL in 96 multiwell culture plates (100 μL / well) and allowed to attach for 18–20 h in complete medium. Cells were then treated with seven concentrations of compounds (0.1–40 μM or 5–60 μM) or controls in fresh medium. After 72 h of culture, MTS (Promega) reduction assays were performed according to the manufacturer's instructions to indirectly assess the percentage of live cells via metabolic cell viability. After 1-2 h of incubation with MTS in a humidified atmosphere of 5% CO at 37 °C, absorbance was measured at 490 nm. For RCC cell lines, the sulforhodamine B assay was used according to the manufacturer's instructions. Data were log-transformed and the concentration of each compound that reduced the number of viable cells relative to the control by 50% (IC) was calculated using GraphPad Prism 6 software. 50 ) was calculated.

[0099] In one embodiment, the IC of all compounds against MCF-7 and MCF10A cell lines 50 The values ​​were used to calculate the selectivity index (SI) value using the following formula: (Formula) SI=(IC 50 Normal cell line-IC 50 Cancer cell lines) / IC 50 Cancer cell lines

[0100] At SI values ​​>1, cytotoxicity towards cancer cell lines is higher than towards non-neoplastic cell lines.

[0101] In one embodiment, cell migration was evaluated by a wound healing assay that mimics the cell migration process during wound healing in vivo. The method is based on creating a wound, simulating the wound with a cell monolayer, taking images at the beginning and at regular intervals of the cell migration process, closing the wound, and comparing the images to quantify the cell migration rate. MCF-7 and Caki-2 cells were cultured in 6-well plates at 9.0x10 per 2mL, respectively. 5 and 3.0x10 5 The cells were plated at a density of 100 μL per well and grown overnight at 37°C in a humidified atmosphere of 5% CO2. Two wounds were made in the confluent cell layer using a 200 μL pipette tip. The cells were gently washed once with 500 μL PBS. MCF-7 cells were cultured at 100 μL per well with each IC 50 , 1 / 2IC 50 The wounds were treated with 0.5% compound or 0.5% DMSO (control) for 72 hours. Specific wound sites (four sites per wound) were photographed at 0, 12, 24, 48 and 72 hours. For Caki-2 cells, the respective IC 50 Treatment was performed for 48 h with 0.5% DMSO (control) and pictures were taken at 0, 12, 24, 36 and 48 h. Images were taken at 100x magnification using an Olympus IX51 inverted microscope equipped with an Olympus DP20 digital camera system. Evaluation of five migration distances was performed using BeWound 1.7.1 and percentage of cell migration normalized to control was plotted using GraphPad Prism 6 software. Three independent experiments were performed for each compound. The significance of differences between different groups was determined by Student's t-test.

[0102] In one embodiment, a proliferation assay was performed. 786-O cells were plated in 96-well plates at a density of 7000 cells / 10 μl and incubated overnight at 37° C. in a 5% CO2 humidified atmosphere. The adherent cells were then cultured using IC 50 Or 1 / 2IC 50 The cells were treated with 10 μl / well of compounds MC350, MC408, MC412, MC415, MC409, MC410, MC413 and MC369 or 0.5% DMSO (control) for 48 h. After incubation, cells were labeled by adding 10 μl / well of BrdU labeling solution (final concentration: 40 μM BrdU). The cells were then reincubated for 16 h to allow BrdU to be incorporated into the DNA of proliferating cells in place of thymidine. After labeling, the cells were fixed by removing the medium and incubated at room temperature with 200 μl of FixDenat solution to denature the DNA. Denaturation of the DNA is essential for the antibody conjugate to bind to the incorporated BrdU. After removing FixDenat, 100 μl of anti-BrdU-POD antibody was incubated at room temperature for 90 min. The anti-BrdU-POD antibody binds to the BrdU incorporated into newly synthesized cellular DNA. After removing the antibody conjugate, the wells were washed three times with washing solution. Substrate solution (100 μl / well) was added to detect immune complexes, and the plates were incubated at room temperature until color development was achieved for photometric detection (5–10 min). The substrate reaction was stopped by adding 25 μl of 1 M H2SO4 to each well and gently mixing. The reaction products were quantified by measuring the absorbance at 450 nm (reference wavelength: 690 nm) with a microplate reader (Tecan Infinite M200). A blank test was performed at each experimental time point without cells, performing all the above steps. The results of at least three independent experiments (quadruplicates) were evaluated using GraphPad Prism 5 software.

[0103] In one embodiment, protein extraction and Western blot analysis were performed. MCF-7 cells and Hs578t cells were grown in T25 flasks, and 786-O cells in 6-well plates. When the cells reached 70-80% confluency, the cells were analyzed for their respective IC 50Cells were treated with 0.5% DMSO or 0.5% DMSO (control). Cells were treated for 24 and 48 hours. Specifically for RTKs and mTOR pathway analysis, 786-O cells were starved for 2 hours and then treated with 2 μM cediranib or compound at their own dose, or with 0.5% DMSO (control). After treatment, adherent and floating cells were scraped and centrifuged at 2000 rpm, 4°C for 10 minutes. The pellet was resuspended in PBS and centrifuged again (1200 rpm; 5 minutes, 4°C). The supernatant was discarded and the pellet was suspended in lysis buffer (50 mM Tris pH 7.6-8, 150 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, 10 mM NaF, 1% NP-40, 1% Triton-X100 and 1 / 7 protease inhibitor cocktail (Roche Applied Sciences)) and incubated on ice for 20 minutes. The lysate was centrifuged at 14,000 rpm for 15 minutes at 4°C, and the supernatant was collected and the protein concentration was measured using a DC protein assay kit (BioRad).

[0104] Briefly, 30 μg of total protein from each sample was separated on 10%, 12% or 15% polyacrylamide gels (100 V) and then transferred to nitrocellulose membranes (100 V, 30 min). The membrane was blocked with 5% milk in 1XTBS for 60 minutes and then incubated with specific primary antibodies (rabbit anti-PARP (Cell Signaling, #9542), 1:1000 5% milk; mouse anti-caspase 9 (Cell Signaling, #9508), 1:500 5% milk; rabbit anti-caspase 3 (Cell Signaling, #9665), 1:500 5% milk; mouse anti-Bax (Santa Cruz Biotechnology, sc-7480), 1:500 5% milk; mouse anti-Bax (Santa Cruz Biotechnology, sc-8392), 1:500 5% milk; rabbit anti-Bim (Cell Signaling, #2933), 1:1000 5% BSA; rabbit anti-Bid (Cell Signaling, #2002), 1:1000 5% BSA; rabbit anti-KDM4C antibody (Abcam, ab27532), 1:2000 5% BSA; rat anti-Hsp90 antibody (Calbiochem, catalog #386041), 1:500 5% BSA; rabbit anti-c-Myc antibody (Cell Signaling, #5605), 1:1000 5% BSA; rabbit anti-p53 antibody (Cell Signaling, #2527), 1:1000 5% BSA; rabbit anti-phospho-p53 antibody (Cell Signaling, #2521), 1:500 5% BSA; rabbit anti-p21 antibody (Cell Signaling, #2947), 1:1000 5% BSA; rabbit anti-JNK antibody (Cell Signaling, #92525), 1:500 5% BSA; rabbit anti-phospho-JNK antibody (Cell Signaling, #10 ... Signaling, #46685), 1:500 5% BSA; rabbit anti-mTOR antibody (Cell Signaling, #2983), 1:1000 5% BSA; rabbit anti-phospho-mTOR antibody (Cell Signaling, #5536), 1:500 5% BSA; rabbit anti-PRAS40 antibody (Cell Signaling, #2691), 1:1000 5% BSA;Rabbit anti-VEGFR2 antibody (Cell Signaling, #2479), 1:500 5% BSA; rabbit anti-phospho-VEGFR2 (Tyr1175) antibody (Cell Signaling, #2478), 1:500 5% BSA; rabbit anti-EGFR antibody (Cell Signaling, #4267), 1:1500 5% BSA; rabbit anti-phospho-EGFR (Tyr1068) antibody (Cell Signaling, #2234), 1:1500 5% BSA; mouse anti-phospho-NF-κB p65 (Ser536) antibody (Cell Signaling, #3036), 1:1000 5% BSA; rabbit anti-AMPKα antibody (Cell Signaling, #2532), 1:1000 5% BSA; rabbit anti-phospho-AMPKα (Thr172) (Cell Signaling, #2532), 1:1000 5% BSA Rabbit anti-AKT (Cell Signaling, #4691), 1:1000 5% BSA; rabbit anti-phospho-AKT (Thr308) (Cell Signaling, #13038), 1:1000 5% BSA; rabbit anti-PTEN (Cell Signaling, #9559), 1:1000 5% BSA; rabbit anti-phospho-PTEN (Ser380) (Cell Signaling, #9551), 1:1000 5% BSA; rabbit anti-phospho-PDK1 (Ser241) (Cell Signaling, #3438), 1:1000 5% BSA; rabbit anti-phospho-p38 (Thr180 / Tyr182) (Cell Signaling, #4511), 1:1000 5% BSA; rabbit anti-p44 / 42 MAPK (Erk1 / 2) antibody (Cell Signaling, #4695), 1:1000 5% BSA; rabbit anti-phospho-p44 / 42 MAPK (Thr202 / Tyr204) (phospho-Erk1 / 2) antibody (Cell Signaling, #4370), 1:1000 5% BSA; rabbit anti-β-tubulin antibody (Abcam, ab6046), 1:10000 5% BSA; mouse anti-GAPDH antibody (Santa Cruz Biotechnology, sc-32233), 1:1000 5% BSA; mouse anti-HK2 (Abcam, ab104836), 1:1000 5% BSA;The cells were incubated overnight at 4°C with mouse anti-LDHA antibody (Santa Cruz Biotechnology, sc-137243), 1:1000 5% BSA; rabbit anti-Hif2α antibody (Cell Signaling, #36169), 1:1000 5% BSA; rabbit anti-MCT1 antibody (Santa Cruz Biotechnology, sc-365501), 1:1000 5% BSA; rabbit anti-PKM (Abcam, ab38237), 1:1000 5% BSA; rabbit anti-PFKL (Abcam, ab37583), 1:1000 5% BSA and mouse anti-β-actin antibody (Santa Cruz Biotechnology, #E1314), 1:500 5% milk). After washing with 0.1% Tween 20 for 5 min (twice) and for another 15 min (once), the blots were incubated with the respective secondary antibodies for 1 h at room temperature (Apoptosis Antibody Sampler Kit-Cell Signaling (#9915): goat anti-rabbit IgG-HRP (7074) and horse-anti-mouse IgG-HRP (7076) secondary antibodies, 1:2000 in 5% milk, Cell Signaling; and rabbit anti-rat IgG-HRP secondary antibody (Abcam, ab6734), 1:30000, 5% BSA). After washing with TBS / 0.1% Tween 20 for 5 min (twice) and for another 15 min (once), immunoreactive bands were detected using the chemiluminescent WesternBright™ Sirius kit (Advansta) on a ChemiDoc XRS+ system (BioRad). Quantification of immunoblots was performed with Quantity One 4.6.9. ;

[0105] In one embodiment, cell cycle distribution was performed by flow cytometry. MCF-7, Hs578t and 786-O cells were seeded in 6-well culture plates. Cells were allowed to attach for 18-20 hours in complete DMEM (for MCF-7 and Hs578t cells) and complete RPMI (for 786-O cells) medium and IC 50The cells were treated with the test compound or 0.5% DMSO (control) for 12 and 24 hours. Each experiment was performed in triplicate. The floating and adherent cells were collected by centrifugation, combined, and fixed with cold ethanol (70%). The cells were resuspended in PBS. After centrifugation to remove the supernatant, the cells were resuspended in a solution containing PBS, PI (50 μg / mL, P1304MP, Invitrogen), ribonuclease A (20 mg / mL, 12091-021, Invitrogen), and TritonX100. After a final incubation of 1 hour in the dark at 50°C, the PI signal was measured and captured using a FACS LSRII flow cytometer (BD Biosciences®) with an excitation laser of 488 nm, and FACS Diva was used as the acquisition software. The percentage of cells in each phase was analyzed using FlowJo 7.6 (Tree Star®) software. Three independent biological replicates were performed.

[0106] In one embodiment, MCF-7, Hs578t and 786-O cells were seeded in 6-well culture plates and allowed to attach for 18-20 hours in complete DMEM (for MCF-7 and Hs578t cells) and complete RPMI (for 786-O cells) medium, and apoptosis was detected by flow cytometry analysis. 50The cells were treated with test compounds at 1000 μg / mL or 0.5% DMSO (control) for 12 and 24 hours. Each experiment was performed in triplicate. Floating and adherent cells were collected by centrifugation and combined. After removing the supernatant, 1 mL of binding buffer was added. 8 μL of FITC Annexin V (556419, BD Pharmingen) and 30 μL of PI (50 μg / mL, P1304MP, Invitrogen) were added to the cell pellet. The samples were incubated for 15 minutes at room temperature in the dark. An additional 200 μL of binding buffer was added to each sample. The PI signal was measured using a FACS LSRII flow cytometer (BD Biosciences®) with a 488 nm excitation laser. The Annexin V signal was collected through a 488 nm blocking filter, a 550 nm long pass dichroic with a 525 nm band pass. The signal was captured and FACS Diva was used as the acquisition software. The percentage of cells at each stage was analyzed using FlowJo 7.6 (Tree Star®) software. Three independent biological replicates were performed.

[0107] In one embodiment, the Bristol strain N2 (provided by CGC under National Institutes of Health Research Infrastructure Program funding (P40 OD010440)) was used to test the toxicity of each compound based on a food clearance assay using C. elegans. The assay was performed in liquid culture in 96-well plates (Voisine et al. 2007; Teixeira-Castro et al. 2015). Each well in a final volume of 60 μL contained approximately 20 egg-stage worms, OP50 bacteria to a final OD of 0.6-0.8 (595 nm), and the appropriate concentration of each compound. Worms were grown for 7 days with continuous shaking at 180 rpm / 20°C (Shel lab Si series incubator) and the absorbance (OD595) was measured daily with a microplate reader (Tecan Infinite M200). The effect of compounds on C. elegans physiology was monitored by the rate at which E. coli food suspension was consumed (Figure 14). Eggs of worms of the same age were obtained by "egg preparation" as follows: adults were treated with an alkaline hypochlorite solution (20% bleach, 25% 1M NaOH) for 6 min, then centrifuged and washed twice with M9 buffer. The egg pellet was then resuspended in S medium to obtain the appropriate egg concentration, before being transferred to a 96-well plate. OP50 bacteria were prepared by inactivating an overnight culture (37 °C, 180 rpm, Luria broth medium) by four freeze-thaw cycles. Before use, the compounds were resuspended in S medium supplemented with OP50 (cholesterol, streptomycin, penicillin and nystatin). Compounds were prepared in 100% DMSO (Sigma) and diluted to test concentrations in 1% DMSO to prevent solvent toxicity. Each compound was tested at several concentrations (150-10 μM), except for MC369 (75-10 μM) due to solubility issues.

[0108] For each compound, several concentrations were used and the corresponding OD595 consumption rates (slope) on days 3–5 were calculated. Compounds were tested in two independent experiments in worms grown in 96-well plates (PlateLayout.xls, Supplementary Information). For statistical analysis, data from both experiments were pooled together and analysis of variance (Brown-Forsythe robust test for equality of means) followed by Games-Howell post-hoc tests was applied using IBM SPSS to compare the various concentrations used for each compound and 5% DMSO (wt / v) with the control (1% DMSO (wt / v)).

[0109] In one embodiment, C57 / Bl6 male and female mice (n=3 / group / sex) were intraperitoneally administered MC408 (3mg / Kg) or vehicle (saline, Tween80 and methylcellulose) daily for 7 days to investigate the toxicity of the compound in the mouse model. A series of tests to evaluate the welfare of the mice were performed daily. Immediately after the first drug administration, the animals were videotaped to record the immediate effects of MC408. The number of vertical movements and immobility time were recorded. During the 7 days of the experiment, water and food intake was evaluated by adding a certain amount of food and water to the animals' home cage on day 1 and measuring the amount remaining on day 7. Body weight was measured daily using a dynamic mouse scale. Horizontal and vertical movements were analyzed to evaluate the animals' intrinsic exploratory behavior. These were measured for 1 min in an open arena with labeled squares and 5 min in an observation jar, respectively. In addition, the number of boluses faecalis was also counted while the animals were performing the daily behavioral assessment protocol as an index of anxiety. The experimenters applied a series of tests related to animal welfare, measuring (i) body posture and body curvature, (ii) spontaneous activity, (iii) respiratory rate, (iv) eyelid opening and reflexes, (v) skin picking to analyze dehydration, (vi) grooming, (vii) hair erection, (viii) tremors, (ix) paw grasping, and (x) locomotion responses. On day 7, the animals were euthanized. All animals were deeply anesthetized (ketamine hydrochloride (150 mg / kg) and medetomidine (0.3 mg / kg)) and perfused transcardially with saline (NaCl 0.9% (wt / v)). Blood was collected from the vena cava and centrifuged at 13.000 rpm for 10 min, after which the plasma was transferred to new tubes and stored at -80°C until further processing for enzymatic liver function measurements using standard techniques. Organs (kidneys, intestines, stomach, brain, liver, ovaries / testes) were harvested and placed in tubes containing 4% paraformaldehyde and further processed for paraffin embedding and pathological analysis.

[0110] In one embodiment, a CAM assay was performed. Fertilized chicken eggs were incubated at 37° C., and on day 3 of development, a hole was punched in the eggshell after the air cell was punctured. The hole was sealed with BTK tape and returned to the incubator. On day 9 of development, Hs578t cells (2×10 6cells) or 786-O cells (3.5 × 10 6 Cells) were placed on the CAM, tumors were allowed to form, and the eggs were tapped and returned to the incubator. Tumors were photographed on day 14 of development. Treatment groups included 2× IC in complete DMEM (for Hs578t cells) or complete RPMI (for 786-O cells) medium. 50 20 μL of MC408 or MC421 at 100% concentration or 20 μL of 0.5% DMSO in complete DMEM or RPMI was added to the established tumors. 72 hours after treatment (day 17 of development), tumors were photographed again in ovo. Chick embryos were sacrificed at -80°C for 10 min, and the CAM alone was fixed in 4% paraformaldehyde and photographed again ex ovo.

[0111] In one embodiment, NSG (NOD Scid Gamma) female mice (3-6 months) were injected with 0.6 x 10 6 A mixture of MDA-MB-231 breast cancer cells and Matrigel (1:1 ratio) was injected into the mammary fat pad. Mice were randomly assigned to treatment groups (at least 6 mice / group) with three independent doses of compound MC408 (Dose 1=3mg / kg, Dose 2=10mg / kg and Dose 3=50mg / kg) or vehicle (PBS1x and Matrigel, 1:1 ratio) starting 3 days after implantation and administered daily for 7 days. Mice were kept under standard laboratory conditions. Animal tumor size (calculated by measuring the two largest sides, L1 being the largest side and L2 being the other side, applying the formula v=3.14xL1xL2 / 6) was measured and recorded periodically. On day 47 after implantation, animals were sacrificed and tumors were excised. All animal experiments were performed in accordance with the guidelines for the care and use of laboratory animals (European Directive 2010 / 63 / EU).

[0112] In one embodiment, reactions of all chemical compounds were monitored by thin layer chromatography (TLC) using 0.2 mm silica gel 60 plates (Macherey-Nagel) and fluorescent indicators. For their exposure, a UV chamber (CN-6 Vilber Lourmat) equipped with a 254 nm lamp was used. Dry flash chromatography was performed using MN Kieselgel 60 (230 ASTM) silica gel (particle size <0.063 mm). Reactions using temperature were performed at various temperatures according to a specific procedure using a hotplate stirrer IKAMAG RCT with appropriate magnetic stirring. Solvents were evaporated using a Buchi RE 11 rotary evaporator under vacuum and variable bath temperature. NMR spectra were recorded using a Bruker Avance III ( 1 400MHz for H NMR, 13 C NMR was measured at 100 MHz at 25 °C using deuterated dimethyl sulfoxide (DMSO-d6) as the solvent. Chemical shifts are reported in parts per million (ppm) using the residual solvent peak as the internal standard. IR spectra were recorded on an FT-IR Bomem MB 104 using a Nujol mul and NaCl cell. Melting points were determined on a Stuart SMP3 instrument and are uncorrected. Elemental analyses were performed on a LECO CHNS-932 instrument. [ka]

[0113] 2-Imino-8-methoxy-2H-chromen-3-amine (4) and chromene derivatives M1159, M955, M1220, M1221 and M1143 can be synthesized by the methods described above.

[0114] In one embodiment, the synthesis of 3-amino-8-methoxy-2H-chromene-2-iminium chloride (5) was carried out. Concentrated HCl (1.1 equiv.) was added to an orange solution of 2-imino-8-methoxy-2H-chromene-3-amine (4) (0.150 mg; 0.79 mmol) in 1 mL of CH3CN. Immediate precipitation was observed and the reaction mixture was stirred at room temperature for 10-15 min. The orange solid was isolated by simple filtration and identified as 3-amino-8-methoxy-2H-chromene-2-iminium chloride (5). Orange solid; 91% yield; mp>300°C; 1 H NMR (400 MHz, DMSO-d6) δ 3.93 (s, 3H), 7.12-7.15 (m, 2H), 7.18 (dd, J = 8.0, 1.2 Hz, 1H), 7.32 (t, J = 8.0 Hz, 1H), 11.48 (s, 1H); 13 C NMR (75 MHz, DMSO-d6) δ 56.02, 110.13, 113.52, 117.42, 122.21, 126.39, 130.57, 135.33, 146.55, 161.15; IR (Nujolmar) ν 3322, 3192, 1678, 1654, 1600, 1576, 1552, 1460 cm -1 ; C 10 H 11 Analysis for N2O2Cl.0.2H2O: Calculated: C, 52.16; H, 4.96; N, 12.17. Found: C, 52.06; H, 4.77; N, 12.40.

[0115] In one embodiment, chromeno[2,3-d]imidazole was synthesized. Aldehyde (1) (1.1-1.7 equiv.) was added to a suspension of 3-amino-8-methoxy-2H-chromene-2-iminium chloride (5) (0.25-0.35 mmol) in CH3CN (1-2 mL) and the suspension was stirred at 60 °C for 24-48 h. The solid was filtered, washed with CH3CN, and identified as pure product. 1 In cases where HCl contamination was observed in the H NMR spectrum, the solid was washed with aqueous NaHCO3 (0.05 M), filtered, and washed with water to give the pure product.

[0116] In one embodiment, 2-(4-fluorophenyl)-5-methoxy-3,9-dihydrochromeno[2,3-d]imidazole (MC409) was synthesized. 4-Fluorobenzaldehyde (0.0375 mg; 0.30 mmol) was added to a suspension of 3-amino-8-methoxy-2H-chromene-2-iminium chloride (5) (0.047 mg; 0.21 mmol) in CH3CN (1.6 mL) and stirred at 60° C. for 32 hours. The solid was filtered, washed with CH3CN, and identified as pure product. Beige solid; yield 98%; mp 191-192° C.; 1 H NMR (400 MHz, DMSO-d6) δ 3.82 (s, 3H), 4.12 (s, 2H), 6.94 (dd, J = 8.4, 1.8 Hz, 1H), 6.86 (dd, J = 7.4, 1.8 Hz, 1H), 7.00 (t, J = 7.8 Hz, 1H), 7.25-7.32 (m, 2H), 7.86-7.92 (m, 2H), 12.44 (s, 1H); 13 C NMR (75 MHz, DMSO-d6) δ 23.64, 55.75, 103.64, 110.48, 115.77 (J = 21.7 Hz), 119.31, 121.82, 122.59, 126.29 (J = 8.3 Hz), 127.19 (J = 3.2 Hz), 138.88, 141.25, 148.19, 148.38, 161.78 (J = 243.4 Hz); IR (Nujol-Marl) ν 3348, 1700, 1650, 1608, 1578, 1538, 1500, 1461 cm -1 ; C 17 H 13 Analysis of N2O2F Calculated: C, 68.91; H, 4.42; N, 9.45. Found: C, 68.89; H, 4.65; N, 9.56.

[0117] In one embodiment, 2-(4-bromophenyl)-5-methoxy-3,9-dihydrochromeno[2,3-d]imidazole (MC408) was synthesized. 4-Bromobenzaldehyde (0.0737 mg; 0.40 mmol) was added to a suspension of 3-amino-8-methoxy-2H-chromene-2-iminium chloride (5) (0.0758 mg; 0.34 mmol) in CH3CN (2 mL) and stirred at 60° C. for 24 hours. The solid was filtered, washed with CH3CN, and identified as the pure product. 1 In cases where HCl contamination was observed in the H NMR spectrum, the solid was washed with aqueous NaHCO3 (0.05 M; 2 mL), filtered, and washed with water to give the pure product. Yellow solid; yield 99%; mp 215-217 °C; 1 H NMR (400 MHz, DMSO-d6) δ 3.82 (s, 3H), 4.11 (s, 2H), 6.86 (dd, J = 7.6, 1.2 Hz, 1H), 6.94 (dd, J = 7.8, 1.2 Hz, 1H), 7.01 (t, J = 8.0 Hz, 1H), 7.63 (dd, J = 6.8, 2.0 Hz, 2H), 7.80 (dd, J = 6.8, 2.0 Hz, 2H), 12.62 (s, 1H); 13 C NMR (75 MHz, DMSO-d6) δ 23.61, 55.77, 104.40, 110.54, 119.25, 120.87, 121.81, 122.70, 126.15 (2C), 129.53, 131.76 (3C), 138.51, 141.16, 148.36; IR (Nujolmar) ν 3435, 1717, 1639, 1603, 1576, 1536, 1463 cm -1 ; C 17 H 13 Analysis for N2O2Br.1.9H2O Calculated: C, 52.12; H, 4.29; N, 7.15. Found: C, 52.12; H, 3.93; N, 7.37.

[0118] In one embodiment, 2-(2-fluorophenyl)-5-methoxy-3,9-dihydrochromeno[2,3-d]imidazole (MC407) was synthesized. 2-Fluorobenzaldehyde (0.0389 mg; 0.31 mmol) was added to a suspension of 3-amino-8-methoxy-2H-chromene-2-iminium chloride (5) (0.0571 mg; 0.25 mmol) in CH3CN (2 mL) and stirred at 60° C. for 24 hours. The solid was filtered, washed with CH3CN, and identified as pure product. Beige solid; yield 82%; mp 102-103° C.; 1 H NMR (400 MHz, DMSO-d6) δ 3.82 (s, 3H), 4.10 (s, 2H), 6.86 (dd, J = 7.6, 2.4 Hz, 1H), 6.94 (dd, J = 8.0, 2.4 Hz, 1H), 7.00 (t, J = 8.0 Hz, 1H), 7.26-7.40 (m, 3H), 7.95 (td, J = 8.0, 1.6 Hz, 1H), 12.09 (s, 1H); 13 C NMR (75 MHz, DMSO-d6) δ 23.93, 55.79, 104.61, 110.54, 116.22 (J = 16.1 Hz), 118.22 (J = 8.6 Hz), 119.44, 121.85, 122.67, 124.94 (J = 2.3 Hz), 128.20 (J = 2.2 Hz), 129.61 (J = 6.2 Hz), 134.50, 141.24, 148.15, 148.38, 158.53 (J = 184.5 Hz); IR (Nujolmal) ν 3426, 1710, 1631, 1603, 1576, 1536, 1463 cm -1 ; C 17 H 13 Analysis of N2O2F1.1H2O Calculated: C, 64.60; H, 4.46; N, 8.87. Found: C, 64.36; H, 4.46; N, 8.91.

[0119] In one embodiment, 2-(3-fluorophenyl)-5-methoxy-3,9-dihydrochromeno[2,3-d]imidazole (MC349) was synthesized. 3-Fluorobenzaldehyde (0.0482 mg; 0.39 mmol) was added to a suspension of 3-amino-8-methoxy-2H-chromene-2-iminium chloride (5) (0.0712 mg; 0.31 mmol) in CH3CN (1 mL) and the suspension was stirred at 60° C. for 33 h. The solid was filtered, washed with CH3CN and identified as pure product. Beige solid; yield 100%; mp 198-200° C.; 1 H NMR (400 MHz, DMSO-d6) δ 3.82 (s, 3H), 4.12 (s, 2H), 6.86 (dd, J = 7.6, 1.2 Hz, 1H), 6.94 (dd, J = 8.0, 1.2 Hz, 1H), 7.00 (t, J = 8.6 Hz, 1H), 7.13 (td, J = 8.6, 2.8 Hz, 1H), 7.44-7.51 (m, 1H), 7.73 (dt, J = 10.4, 2.8 Hz, 1H), 7.70 (d, J = 7.6 Hz, 1H), 12.58 (s, 1H); 13 C NMR (75 MHz, DMSO-d6) δ 23.60, 55.79, 104.46, 110.56, 110.68 (J = 18.4 Hz), 114.43 (J = 15.8 Hz), 119.24, 120.23 (J = 1.8 Hz), 121.81, 122.68, 130.95 (J = 6.4 Hz), 132.78 (J = 6.4 Hz), 138.39 (J = 2.3 Hz), 141.21, 148.31, 148.38, 162.52 (J = 180.8 Hz); IR (Nujolmal) ν 3356, 1707, 1652, 1628, 1522, 1508, 1461cm -1 ; C 17 H 13 Analysis of N2O2F Calculated: C, 68.91; H, 4.42; N, 9.45. Found: C, 68.98; H, 4.58; N, 9.66.

[0120] In one embodiment, 2-(4-chlorophenyl)-5-methoxy-3,9-dihydrochromeno[2,3-d]imidazole (MC412) was synthesized. To a suspension of 3-amino-8-methoxy-2H-chromene-2-iminium chloride (5) (0.0417 mg; 0.18 mmol) in CH3CN (1 mL) was added 4-chlorobenzaldehyde (0.0291 mg; 0.21 mmol) and the suspension was stirred at 60° C. for 24 hours. The solid was filtered, washed with CH3CN, and identified as pure product. Beige solid; 100% yield; mp 212-214° C.; 1 H NMR (400 MHz, DMSO-d6) δ 3.82 (s, 3H), 4.12 (s, 2H), 6.86 (dd, J = 7.6, 1.6 Hz, 1H), 6.94 (dd, J = 8.2, 1.6 Hz, 1H), 7.00 (t, J = 8.0 Hz, 1H), 7.50 (d, J = 8.4 Hz, 2H), 7.86 (d, J = 8.4 Hz, 2H), 12.53 (s, 1H); 13 C NMR (75 MHz, DMSO-d6) δ 23.62, 55.76, 104.18, 110.52, 119.25, 121.80, 122.62, 125.84 (2C), 128.84 (2C), 129.36, 132.18, 138.55, 141.22, 148.31, 148.36; IR (Nujolmar) ν 3352, 1700, 1656, 1601, 1532, 1489, 1462 cm -1 ; C 17 H 13 Analysis for N2O2Cl: Calculated: C, 65.29; H, 4.19; N, 8.96. Found: C, 65.47; H, 4.23; N, 8.74.

[0121] In one embodiment, 5-methoxy-2-phenyl-3,9-dihydrochromeno[2,3-d]imidazole (MC350) was synthesized. Benzaldehyde (0.0520 mg; 0.49 mmol) was added to a suspension of 3-amino-8-methoxy-2H-chromene-2-iminium chloride (5) (0.0653 mg; 0.29 mmol) in CH3CN (1 mL), and the suspension was stirred at 60° C. for 33 h. The solid was filtered, washed with CH3CN, and identified as the pure product. 1 In cases where HCl contamination was observed in the H NMR spectrum, the solid was washed with aqueous NaHCO3 (0.05 M), filtered, and washed with water to give the pure product. Beige solid; yield 99%; mp 162-162 °C; 1 H NMR (400 MHz, DMSO-d6) δ 3.83 (s, 3H), 4.12 (s, 2H), 6.86 (dd, J = 7.6, 1.6 Hz, 1H), 6.94 (dd, J = 8.0, 1.6 Hz, 1H), 7.00 (t, J = 8.0 Hz, 1H), 7.31 (t, J = 7.6 Hz, 1H), 7.43 (t, J = 8.0 Hz, 2H), 7.86 (d, J = 7.8 Hz, 2H), 12.43 (s, 1H); 13 C NMR (75 MHz, DMSO-d6) δ 23.68, 55.77, 110.50, 119.33, 121.83, 122.56, 124.20 (2C), 127.77, 128.75 (2C), 130.50, 139.62, 141.29, 148.23, 148.38; IR (Nujolmar) ν 3346, 1702, 1648, 1602, 1526, 1496, 1461cm -1 ; C 17 H 14 Analysis of N2O2 calculated: C, 73.37; H, 5.07; N, 10.07. Found: C, 73.58; H, 5.12; N, 10.34.

[0122] In one embodiment, 3-(5-methoxy-3,9-dihydrochromeno[2,3-d]imidazol-2-yl)phenol (MC359) was synthesized. 3-Hydroxybenzaldehyde (0.0427 mg; 0.35 mmol) was added to a suspension of 3-amino-8-methoxy-2H-chromene-2-iminium chloride (5) (0.0679 mg; 0.30 mmol) in CH3CN (1.2 mL), and the suspension was stirred at 60° C. for 28 hours. The solid was filtered, washed with CH3CN, and identified as the pure product. 1 In cases where HCl contamination was observed in the H NMR spectrum, the solid was washed with aqueous NaHCO3 (0.05 M), filtered, and washed with water to give the pure product. Light green solid; yield 94%; mp162-164℃; 1H NMR (400 MHz, DMSO-d6) δ 3.82 (s, 3H), 4.10 (s, 2H), 6.71 (dq, J = 8.2, 1.2 Hz, 1H), 6.85 (dd, J = 7.8, 1.2 Hz, 1H), 6.92 (dd, J = 8.2, 1.2 Hz, 1H), 7.00 (t, J = 7.6 Hz, 1H), 7.21 (t, J = 7.6 Hz, 2H), 7.27-7.30 (m, 2H), 9.55 (s, 1H), 12.33 (s, 1H); 13C NMR (75 MHz, DMSO-d6) δ 23.70, 55.75, 110.47, 111.23, 114.99, 115.07, 119.36, 121.84, 122.54, 129.74, 131.77, 139.76, 141.30, 148.10, 148.37, 157.62; IR (Nujolmar) ν 3351, 3218, 1702, 1659, 1611, 1523, 1507, 1460 cm -1 Calculated for C17H14N2O3: C, 69.38; H, 4.79; N, 9.52. Found: C, 69.42; H, 4.88; N, 9.56.

[0123] In one embodiment, 4-(5-methoxy-3,9-dihydrochromeno[2,3-d]imidazol-2-yl)phenol (MC413) was synthesized. To a suspension of 3-amino-8-methoxy-2H-chromene-2-iminium chloride (5) (0.0579 mg; 0.26 mmol) in CH3CN (2 mL) was added 4-hydroxybenzaldehyde (0.0388 mg; 0.32 mmol) and the suspension was stirred at 60° C. for 48 h. The solid was filtered, washed with CH3CN, and identified as pure product. In cases where HCl contamination was observed in the 1H NMR spectrum, the solid was washed with aqueous NaHCO3 (0.05 M), filtered, and washed with water to give the pure product. Yellow solid; yield 64%; mp245-246℃; 1H NMR (400 MHz, DMSO-d6) δ 3.82 (s, 3H), 4.09 (s, 2H), 6.81 (dd, J = 6.8, 2.0 Hz, 2H), 6.85 (dd, J = 7.6, 1.6 Hz, 1H), 6.92 (dd, J = 8.0, 1.6 Hz, 1H), 6.99 (t, J = 8.0 Hz, 1H), 7.68 (dd, J = 6.8, 2.0 Hz, 2H), 9.63 (s, 1H), 12.11 (s, 1H); 13C NMR (75 MHz, DMSO-d6) δ23.76, 55.75, 102.26, 110.43, 115.48 (2C), 119.45, 121.86 (2C), 122.45, 125.88 (2C), 140.34, 141.36, 147.86, 148.37, 157.44; IR (Nujolmar) ν 3338, 3246, 1706, 1645, 1612, 1548, 1503, 1463 cm -1 Calculated for C17H14N2O3: C, 69.38; H, 4.79; N, 9.52. Found: C, 69.39; H, 4.42; N, 9.54.

[0124] In one embodiment, 5-methoxy-2-(2-methoxyphenyl)-3,9-dihydrochromeno[2,3-d]imidazole (MC411) was synthesized. 2-Methoxybenzaldehyde (0.0632 mg; 0.46 mmol) was added to a suspension of 3-amino-8-methoxy-2H-chromene-2-iminium chloride (5) (0.0780 mg; 0.34 mmol) in CH3CN (1 mL), and the suspension was stirred at 60° C. for 47 h. The solid was filtered, washed with CH3CN, and identified as the pure product. 1 If HCl contamination was observed in the H NMR spectrum, the solid was washed with aqueous NaHCO3 (0.05 M), filtered, and washed with water to give the pure product. Yellow solid; yield 85%; mp 188-190 °C; 1 H NMR (400 MHz, DMSO-d6) δ 3.82 (s, 3H), 3.95 (s, 3H), 4.10 (s, 2H), 6.86 (dd, J = 7.6, 1.6 Hz, 1H), 6.93 (dd, J = 8.0, 1.6 Hz, 1H), 6.97-7.04 (m, 2H), 7.13 (dd, J = 8.0, 0.8 Hz, 1H), 7.30 (td, J = 7.7, 2.0 Hz, 1H), 7.99 (dd, J = 8.0, 2.0 Hz, 1H), 11.63 (s, 1H); 13 C NMR (100 MHz, DMSO-d6) δ 24.30, 55.50, 55.78, 103.05, 110.47, 111.72, 118.44, 119.67, 120.78, 121.91, 122.51, 127.39, 128.95, 137.14, 141.34, 147.76, 148.38, 155.59; IR (Nujolmar) ν 3340, 1706, 1659, 1594, 1530, 1506, 1460 cm -1 ; C 18 H 16 Analysis of N2O3 calculated: C, 70.12; H, 5.23; N, 9.09. Found: C, 70.44; H, 5.20; N, 9.16.

[0125] In one embodiment, 2,4-difluoro-6-(5-methoxy-3,9-dihydrochromeno[2,3-d]imidazol-2-yl)phenol (MC410) was synthesized. 2,4-Difluoro-6-hydroxybenzaldehyde (0.0493 mg; 0.31 mmol) was added to a suspension of 3-amino-8-methoxy-2H-chromene-2-iminium chloride (5) (0.061 mg; 0.27 mmol) in CH3CN (2 mL), and the suspension was stirred at 60° C. for 36 hours. The solid was filtered, washed with CH3CN, and identified as the pure product. 1 In cases where HCl contamination was observed in the H NMR spectrum, the solid was washed with aqueous NaHCO3 (0.05 M), filtered, and washed with water to give the pure product. Yellow solid; yield 49%; mp 260-262 °C; 1 H NMR (400 MHz, DMSO-d6) δ 3.83 (s, 3H), 4.15 (s, 2H), 6.87 (dd, J = 7.6, 2.4 Hz, 1H), 6.96 (dd, J = 8.2, 2.4 Hz, 1H), 7.04 (t, J = 8.0 Hz, 1H), 7.24 (td, J = 10.0, 1.6 Hz, 1H), 7.51 (dt, J = 10.0, 1.6 Hz, 1H), 11.91 (s, 1H), 12.90 (s, 1H); 13 C NMR (75 MHz, DMSO-d6) δ 23.28, 55.70, 104.31 (dd, J = 20.6, 16.5 Hz), 104.39, 105.38 (dd, J = 18.8, 4.5 Hz), 110.59, 115.36 (dd, J = 7.9, 4.5 Hz), 119.03, 121.71, 123.14, 137.71-137.79 (m), 140.30 (J = 10.2, 4.5 Hz), 140.68, 145.62, 148.29, 150.80 (dd, J = 181.9, 10.0), 153.9 (dd, J = 175.9, 8.8); IR (Nujolmar) ν 3348, 3225, 1698, 1651, 1601, 1542, 1461 cm -1 ; C17 H 12 Analysis for N2O3F20.8H2O Calculated: C, 59.23; H, 3.72; N, 8.13. Found: C, 59.13; H, 3.53; N, 8.42.

[0126] In one embodiment, 2-(4-ethoxyphenyl)-5-methoxy-3,9-dihydrochromeno[2,3-d]imidazole (MC415) was synthesized. To a suspension of 3-amino-8-methoxy-2H-chromene-2-iminium chloride (5) (0.0614 mg; 0.27 mmol) in CH3CN (2 mL) was added 4-ethoxybenzaldehyde (0.0403 mg; 0.30 mmol) and the suspension was stirred at 60° C. for 47 h. The solid was filtered, washed with CH3CN, and identified as the pure product. 1 In cases where HCl contamination was observed in the H NMR spectrum, the solid was washed with aqueous NaHCO3 (0.05 M), filtered, and washed with water to give the pure product. Yellow solid; yield 96%; mp 215-217 °C; 1 H NMR (400 MHz, DMSO-d6) δ 1.20 (t, J = 7.6 Hz, 3H), 2.62 (q, J = 7.6 Hz, 2H), 3.84 (s, 3H), 4.12 (s, 2H), 6.87 (dd, J = 7.2, 1.6 Hz, 1H), 6.94 (dd, J = 8.2, 1.2 Hz, 1H), 7.01 (t, J = 7.6 Hz, 1H), 7.28 (d, J = 8.4 Hz, 2H), 7.79 (dd, J = 6.6, 1.6 Hz, 2H), 12.36 (s, 1H); 13 C NMR (75 MHz, DMSO-d6) δ 15.43, 23.73, 27.94, 55.80, 103.30, 110.52, 119.41, 121.87, 122.59, 124.33 (2C), 128.08, 128.16 (2C), 139.87, 141.33, 143.60, 148.40; IR (Nujolmar) ν 3342, 1703, 1656, 1610, 1539, 1502, 1460 cm -1; C 19 H 18 Analysis of N2O3: Calculated: C, 70.81; H, 5.59; N, 8.70. Found: C, 70.74; H, 5.67; N, 8.77.

[0127] In one embodiment, 2-bromo-6-methoxy-3-(5-methoxy-3,9-dihydrochromeno[2,3-d]imidazol-2-yl)phenol (MC416) was synthesized. 2-Bromo-3-hydroxy-4-methoxybenzaldehyde (0.0780 mg; 0.34 mmol) was added to a suspension of 3-amino-8-methoxy-2H-chromene-2-iminium chloride (5) (0.0607 mg; 0.27 mmol) in CH3CN (1 mL) and the suspension was stirred at 60° C. for 46 h. The solid was filtered, washed with CH3CN and identified as the pure product. 1 In cases where HCl contamination was observed in the H NMR spectrum, the solid was washed with aqueous NaHCO3 (0.05 M), filtered, and washed with water to give the pure product. Beige solid; yield 71%; mp 160-161 °C; 1 H NMR (400 MHz, DMSO-d6) δ 3.82 (s, 3H), 3.86 (s, 3H), 4.08 (s, 2H), 6.85 (dd, J = 7.6, 1.2 Hz, 1H), 6.93 (dd, J = 8.0, 1.6 Hz, 1H), 6.99 (t, J = 8.0 Hz, 1H), 7.03-7.08 (m, 2H), 9.55 (s, 1H), 11.97 (s, 1H); 13 C NMR (75 MHz, DMSO-d6) δ 23.82, 55.74, 56.23, 102.75, 109.30, 110.43, 110.54, 119.46, 121.38, 121.84, 122.46, 125.26, 139.04, 141.33, 144.05, 147.52, 148.29, 148.37; IR (Nujolmar) ν 3338, 3212, 1703, 1647, 1535, 1497, 1461 cm -1 ; C 18 H 15Analysis for N2O4Br1.8H2O Calculated: C, 49.61; H, 3.86; N, 6.43. Found: C, 49.68; H, 3.91; N, 6.43.

[0128] In one embodiment, 5,5'-dimethoxy-2,2'-diphenyl-1,1',9,9'-tetrahydro-9,9'-bichromeno[2,3-d]imidazole was synthesized. To a solution of 2-imino-8-methoxy-2H-chromen-3-amine (4) in CH3CN (1-2 mL) was added aldehyde (1) (1-1.2 equiv.) and the solution was stirred at 80°C for 7-24 h. A solid product slowly started to precipitate, was filtered, washed with CH3CN and identified as pure product 8.

[0129] In one embodiment, 2,2'-bis(4-fluorophenyl)-5,5'-dimethoxy-1,1',9,9'-tetrahydro-9,9'-bichromeno[2,3-d]imidazole (MC406) was synthesized. To a solution of 2-imino-8-methoxy-2H-chromen-3-amine (4) (0.0962 mg; 0.51 mmol) in CH3CN (1 mL) was added 4-fluorobenzaldehyde (0.0629 mg; 0.51 mmol) and the solution was stirred at 80°C for 7 hours. The solid product slowly started to precipitate, was filtered, washed with CH3CN and identified as pure product. Beige solid; yield 18%; mp 272-274°C; 1 H NMR (400 MHz, DMSO-d6) δ 3.68 (s, 6H), 4.89 (s, 2H), 5.80 (dd, J = 7.8, 2.0 Hz, 2H), 6.71 (t, J = 7.8 Hz, 2H), 6.80 (dd, J = 8.7, 1.6 Hz, 2H), 7.32-7.49 (m, 4H), 7.98-8.03 (m, 4H), 12.57 (s, 2H); 13C NMR (75 MHz, DMSO-d6) δ 41.21, 55.64, 105.81, 111.00, 115.83 (2C, J = 21.2), 119.62, 120.44, 122.01, 126.80 (2C, J = 8.0), 127.12, 139.98, 141.90, 147.62, 150.12, 162.00 (J = 243.8); IR (Nujolmar) ν 3348, 1700, 1650, 1608, 1538, 1500, 1461 cm -1 ; C 34 H 24 Analysis for N4O4F21.2H2O Calculated: C, 64.23; H, 4.85; N, 8.82. Found: C, 64.21; H, 4.79; N, 8.80.

[0130] In one embodiment, 2,2'-bis(4-bromophenyl)-5,5'-dimethoxy-1,1',9,9'-tetrahydro-9,9'-bichromeno[2,3-d]imidazole (MC421) was synthesized. 4-Bromobenzaldehyde (0.069 mg; 0.37 mmol) was added to a solution of 2-imino-8-methoxy-2H-chromen-3-amine (4) (0.0705 mg; 0.37 mmol) in CH3CN (1.5 mL) and the solution was stirred at 80°C for 8 hours. The solid product slowly started to precipitate, was filtered, washed with CH3CN and identified as pure product. Beige solid; yield 32%; mp 266-268°C; 1 H NMR (400 MHz, DMSO-d6) δ 3.68 (s, 6H), 4.89 (s, 2H), 5.78 (dd, J = 7.8, 1.6 Hz, 2H), 6.71 (t, J = 7.8 Hz, 2H), 6.80 (dd, J = 8.2, 2.0 Hz, 2H), 7.71 (dd, J = 6.6, 1.4 Hz, 2H), 7.91 (dd, J = 6.9, 1.6 Hz, 2H), 12.69 (s, 2H); 13C NMR (75 MHz, DMSO-d6) δ 41.24, 55.65, 106.33, 111.06, 119.52, 120.41, 121.14, 122.06, 126.58, 129.65, 131.80, 139.70, 141.86, 147.62, 150.25; IR (Nujol NMR) ν 3352, 1703, 1654, 1610, 1543, 1508, 1461cm -1 ; C 34 H 24 Analysis for N4O4Br2: Calculated: C, 57.32; H, 3.40; N, 7.86. Found: C, 57.78; H, 3.37; N, 7.88.

[0131] In one embodiment, 2,2'-bis(2-fluorophenyl)-5,5'-dimethoxy-1,1',9,9'-tetrahydro-9,9'-bichromeno[2,3-d]imidazole (MC369) was synthesized. 2-Fluorobenzaldehyde (0.0446 mg; 0.36 mmol) was added to a solution of 2-imino-8-methoxy-2H-chromen-3-amine (4) (0.0590 mg; 0.31 mmol) in CH3CN (2 mL), and the solution was stirred at 80°C for 24 hours. The solid product gradually started to precipitate, was filtered, washed with CH3CN, and identified as pure product. Beige solid; yield 15%; mp 276-278°C; 1 H NMR (400 MHz, DMSO-d6) δ 3.69 (s, 6H), 5.00 (s, 2H), 5.91 (dd, J = 8.0, 1.2 Hz, 2H), 6.75 (t, J = 8.0 Hz, 2H), 6.82 (dd, J = 8.2, 1.6 Hz, 2H), 7.31-7.48 (m, 6H), 7.99 (td, J = 5.7, 1.6 Hz, 2H), 11.88 (s, 2H); 13C NMR (75 MHz, DMSO-d6) δ 41.08, 55.65, 106.63, 110.99, 116.28 (J = 21.3), 118.24 (J = 11.4), 120.02, 120.45, 122.06, 124.98, 128.60 (J = 2.8), 129.98 (J = 8.2), 135.58, 141.90, 147.62, 149.93, 158.73 (J = 246.6); IR (Nujolmar) ν 3438, 3447, 1637, 1575, 1614, 1575, 1530, 1469 cm -1 ; C 34 H 24 Analysis for N4O4F2.0.8H2O Calculated: C, 67.51; H, 4.23; N, 9.26. Found: C, 67.58; H, 4.30; N, 9.11.

[0132] As used herein, the term "comprising" is intended to indicate the presence of stated features, integers, steps or components, but does not exclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0133] Those skilled in the art will appreciate that, unless otherwise indicated, the particular order of steps described is merely exemplary and can be varied without departing from the present disclosure. Thus, unless otherwise indicated, the steps described are unordered, meaning that, where possible, they can be performed in any convenient or desirable order.

[0134] Where a singular form of an element or feature is used in the claims, the plural form is also included, and vice versa, unless specifically excluded. For example, the term "compound" or "said compound" includes the plural form "compound" or "said compound", and vice versa. In the claims, articles such as "a", "an" and "the" may mean one or more, unless indicated to the contrary or clear from the context. A claim or description containing "or" between one or more members of a group is considered to be satisfied if one, more than one or all of the group members are present in, employed in or otherwise relevant to a given product or process, unless indicated to the contrary or clear from the context. The invention includes embodiments in which exactly one member of a group is present in, employed in or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one or all of the members of a group are present in, employed in or otherwise relevant to a given product or process.

[0135] Moreover, it is to be understood that the invention encompasses all variations, combinations, and permutations that introduce into another claim one or more limitations, elements, clauses, descriptive terms, etc. from one or more claims, or from the relevant portions of this specification. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim.

[0136] Furthermore, when a composition is recited in the claims, it is to be understood that unless otherwise indicated or a contradiction or inconsistency would be apparent to one of ordinary skill in the art, methods of using the composition for any purpose disclosed herein are included, and methods of making the composition according to any method of manufacture disclosed herein or other methods known to one of ordinary skill in the art are included.

[0137] When ranges are specified, the endpoints are included. Moreover, unless otherwise indicated or otherwise clear from the context and / or the understanding of one of ordinary skill in the art, it is to be understood that values ​​expressed as ranges can assume any particular value within the stated range, to the tenth of the unit of the lower limit of the range, in different embodiments of the invention, unless otherwise clearly indicated by the context and / or the understanding of one of ordinary skill in the art. It is also to be understood that values ​​expressed as ranges can assume any subrange within the stated range, with the endpoints of the subrange being expressed to the same degree of precision as the tenth of the unit of the lower limit of the range, unless otherwise indicated or otherwise clear from the context and / or the understanding of one of ordinary skill in the art.

[0138] The present disclosure is in no way limited to the described embodiments, and those skilled in the art will envision many possible modifications thereof.

[0139] The above-described embodiments can be combined.

[0140] (reference) (1) https: / / www.uicc.org / new-global-cancer-data-globocan-2018 (02 / 01 / 2019) (2)http: / / gco.iarc.fr (02 / 01 / 2019) (3)Bao, B; Mitrea, C.; Wijesinghe, P.; Marchetti, L.; Girsch, E.; Farr, R.; Boerner, J.; Mohammad, R.; Dyson, G.; 7, 44125. (4) Costa, M.; Dias, T.; Brito, A. And Proenca, F. Biological importance of structurally diversified chromenes. Eur. J. Med. Chem, 2016, 123, 487-507. (5)Costa, M.; Rodrigues, A. I.; Proenca, F. Synthesis of 3-aminochromenes: the Zincke reaction revisited. Tetrahedron 2014, 70 (33), 4869. (6)Costa, M.; Proenca, F. 2-Aryl-1,9-dihydrochromeno[3,2-d]imidazoles: a facile synthesis from salicylaldehydes and arylideneaminoacetonitrile. Tetrahedron 2011, 67 (10), 1799.

Claims

1. 1. An agent for use in human or veterinary medicine, comprising: A pharmaceutical agent comprising a compound or a pharma- ceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereoisomer, enantiomer, or atropisomer of the formula: 【Chemistry 1】 (In the formula, R １ , R ２ and R ３ are selected independently of each other, R １ is selected from aryl; R ２ is selected from H, alkyl, alkoxy, halogen, hydroxyl or amine; R ３ is H or 【Chemistry 2】 is selected from. however, 【Chemistry 3】 are excluded.)

2. R １ The method of claim 1 , wherein is substituted aryl.

3. R １ is selected from hydroxyphenyl, hydroxy-methoxyphenyl, hydroxy-bromophenyl, hydroxy-chlorophenyl, fluorophenyl, bromophenyl, 4-chlorophenyl, phenyl, methoxyphenyl, difluoro-hydroxyphenyl, ethoxyphenyl or bromo-hydroxy-methoxyphenyl.

4. R １ is 2-hydroxyphenyl, 2-hydroxy-3-methoxyphenyl, 2-hydroxy-5-methoxyphenyl, 2-hydroxy-5-bromophenyl, 2-hydroxy-5-chlorophenyl, 4-fluorophenyl, 4-bromophenyl, 2-fluorophenyl, 3-fluorophenyl, 4-chlorophenyl, phenyl, 3-hydroxyphenyl, 2-hydroxyphenyl, 2-methoxyphenyl, 3,5-difluoro-2-hydroxyphenyl, 4-ethoxyphenyl or 2-bromo-3-hydroxy-4-methoxyphenyl.

5. R ２ An agent according to any one of claims 1 to 4, wherein is selected from H, 5-methoxy, 7-methoxy, 7-bromo, 7-chloro or 7-fluoro.

6. R ３ The agent according to any one of claims 1 to 5, wherein is a chromene unit or H.

7. R ３ is selected from H, a 2-(4-fluorophenyl)-5-methoxy-1,9-dihydrochromeno[2,3-d]imidazole unit, a 2-(4-bromophenyl)-5-methoxy-1,9-dihydrochromeno[2,3-d]imidazole unit or a 2-(2-fluorophenyl)-5-methoxy-1,9-dihydrochromeno[2,3-d]imidazole unit.

8. The compound 【Chemistry 4-1】 【Chemistry 4-2】 The drug according to any one of claims 1 to 7, which is selected from the group consisting of

9. An agent according to any one of claims 2 to 8 for use in medicine or veterinary medicine.

10. 10. An agent according to any one of claims 1 to 9 for use in the treatment, therapy or diagnosis of a disease or cancer characterised by hyperproliferation of benign or malignant cells or by areas of neo- or hypervascularisation.

11. Agent according to any one of claims 1 to 10 for use in the treatment, therapy or diagnosis of hyperproliferative tissue or neoplasms.

12. The agent according to any one of claims 1 to 11 for use in the treatment, therapy or diagnosis of breast cancer, renal cell carcinoma, leukemia, glioma or glioblastoma.

13. Agent according to any one of claims 1 to 12 for use in the treatment, therapy or diagnosis of renal cell carcinoma, leukemia, glioma, glioblastoma, breast cancer.

14. Agent according to any one of claims 1 to 13 for use in the treatment, therapy or diagnosis of triple negative breast cancer, renal cell carcinoma, luminal breast cancer, basal-like breast cancer, acute leukemia.

15. A pharmaceutical composition comprising at least one compound according to any one of claims 1 to 14.

16. 16. The pharmaceutical composition of claim 15, further comprising an antiviral agent, an analgesic agent, an anti-inflammatory agent, a chemotherapeutic agent, a radiotherapeutic agent, an antifungal agent, an antiparasitic agent, an antibiotic, a diuretic agent, or a mixture thereof.

17. 17. A pharmaceutical composition according to claim 15 or 16 for use in intradermal or topical or systemic or transdermal or intravenous therapy or a combination thereof.

18. Nanoparticles comprising a compound according to any one of claims 1 to 14 and / or a pharmaceutical composition according to any one of claims 15 to 17.

19. The compound of claim 1 having the structure: 【Chemistry 5】 A process for producing a compound having the structure 【Chemistry 6】 is reacted with concentrated hydrochloric acid in an organic solvent to produce a compound having the following structure: 【Chemistry 7】 which is then reacted with an aldehyde having the structure R1-CHO in an organic solvent to produce a compound having the structure 【Chemistry 8】 a process comprising the step of producing or The compound of claim 1 having the structure: 【Chemistry 9】 A process for producing a compound having the structure 【Chemistry 10】 with an aldehyde having the structure R1-CHO in an organic solvent to produce a compound having the structure 【Chemistry 11】 The process comprising the step of producing

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