Benzopyranyl compounds, methods and uses thereof

Abstract

The present invention relates to a compound or pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer or polymorph for use in medicine comprising the following chemical formula (I): wherein R 1 , R 2 and R 3 are selected independently of each other; R 1 is selected from aryl or heterocycle; R 2 is selected from H, alkyl, aryl, alkoxy, halogen, hydroxyl, amine, carbonyl or heterocycle; R 3 is selected from H or (II).

Description

TECHNICAL FIELD

[0001] The present invention relates to a new class of benzopyranyl anticancer compounds. The present invention describes the anticancer properties of a new class of benzopyranyl anticancer compounds. The present invention also describes the synthesis and isolation methods of a new class of benzopyranyl anticancer compounds. BACKGROUND

[0002] In 2018, cancer caused nearly 10 million deaths worldwide and 18.1 million new cases of cancer were detected that same year. 1 It is expected that in 5 years the incidence of this disease will exceed 43 million cases. This demonstrates the urgency of finding new and better treatments. The problem must be addressed on a global scale due to the overall increase in the number of cases and associated mortality. In 2018, breast cancer accounted for 6.6% of cancer deaths. Breast cancer is the fifth leading cause of death; in 2018, 11.6% of new cases were detected (more than 200 million cases). 2 In terms of incidence, it is the second most common type of cancer. The type of treatment is always based on the stage of the cancer, but mainly includes surgery, radiotherapy, conventional chemotherapy, immunotherapy or, if molecular markers such as ER, PR and HER-2 are detected, molecular therapy. However, in the case of triple-negative breast cancer (TNBC), which is a very aggressive subtype of breast cancer that accounts for approximately 15-20% of all breast cancers, conventional therapies have limited efficacy and molecular or personalized therapies have not yet been developed. Due to this poor prognosis, high mortality is associated with this subtype of breast cancer. 3

[0003] The benzopyranyl skeleton has inspired medicinal chemists, as these compounds are widely present in nature and have multiple biological properties. 4 In recent years, research on the synthesis and biological importance of this naturally occurring skeleton and its synthetic derivatives has gained attention.

[0004] The document by Costa et al. "Tetrahedron" describes a one-pot cascade reaction involving salicylaldehyde and arylideneaminoacetonitriles for the preparation of 2-aryl-1,9- dihydrobenzopyrano[3,2-d]imidazoles. This document further describes a new type of fused tricyclic system combining benzopyran and imidazole as an alternative drug candidate with improved pharmacological properties. However, the system described does not allow the generation of molecules in which the aromatic unit incorporates different substituents.

[0005] These facts are disclosed to show the technical problem solved by the present invention. SUMMARY

[0006] The present invention relates to a new class of benzopyranyl anticancer compounds. The present invention describes the anticancer properties of a new class of benzopyranyl anticancer compounds. The present invention also describes the methods of synthesis and isolation of a new class of benzopyranyl anticancer compounds.

[0007] In particular, the present invention describes a method of synthesizing novel benzopyranyl compounds by combining at least two different molecules, thereby generating compounds with relevant biological properties of interest.

[0008] The methods of synthesis of benzopyranyl compounds described in the prior art do not allow the isolation of these novel compounds. The methods described in the present invention allow the synthesis and isolation of novel benzopyranyl compounds that are unexpectedly effective when used as anticancer compounds in several cancer subtypes, such as breast cancer, renal cell carcinoma, acute leukemia and glioma. The compounds are particularly effective against breast cancer and renal cell carcinoma.

[0009] In one embodiment, the synthesized compounds are tested for their anticancer potential by in vitro screening using appropriate cell lines. The activity of the compounds is studied at different levels, including the effect on cell viability, proliferation, migration, invasion, cell death and metabolism.

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

[0011] In one embodiment, the Caenorhabditis elegans (C. elegans nematode) model is used for early in vivo toxicity screening of the benzopyranyl compounds. Mice are also used as a model to evaluate in vivo toxicity.

[0012] In one embodiment, the anticancer properties of the benzopyranyl compounds are also identified using the in vivo CAM (chick chorioallantoic membrane) assay, which evaluates the potency and angiogenicity of the novel benzopyrans. Mice are also used as a model to further evaluate in vivo potency.

[0013] The compounds of the subject matter of the present invention show unique and promising anticancer properties.

[0014] One aspect of the present invention relates to a compound comprising the following chemical formula or a pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer or polymorph:

[0015]

[0016] wherein

[0017] R 1 , R 2 , and R 3 are selected independently of one another;

[0018] R 1 is selected from H, alkyl, aryl, alkoxy, acyl, halogen, nitro, hydroxyl, amine, amide, carbonyl, ketone, ester, heterocycle;

[0019] R 2 is selected from H, alkyl, aryl, alkoxy, acyl, halogen, nitro, hydroxyl, amine, amide, carbonyl, ketone, ester, heterocycle;

[0020] R 3 is selected from H, alkyl, aryl, alkoxy, acyl, halogen, nitro, hydroxyl, amine, amide, ketone, ester, heterocycle, or

[0021] Another aspect of the present application relates to a compound or pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer, or polymorph comprising the following formula:

[0022] R 1 is selected from aryl or heterocycle;

[0023] R 2 is selected from H, alkyl, aryl, alkoxy, halogen, hydroxyl, amine, carbonyl, or heterocycle;

[0024] R 3 is selected from H or

[0025] Preferably, the condition is that R

[0026] In one embodiment, the compound or pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer, or polymorph, wherein: R 1 is selected from aryl; and R 2 is selected from H, alkyl, alkoxy, halogen, hydroxyl, or amine.

[0027] In one embodiment, the compound or pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer, or polymorph, wherein the dimer is preferably a homodimer.

[0028] In one embodiment, the compound or pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer, or polymorph thereof, wherein R 1 is substituted aryl.

[0029] In one embodiment, the compound or pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer, or polymorph thereof, wherein R 1 is selected from the list: hydroxyphenyl, hydroxy-methoxyphenyl, hydroxy-bromophenyl, hydroxy-chlorophenyl, fluorophenyl, bromophenyl, 24-chlorophenyl, phenyl, methoxyphenyl, difluoro-hydroxyphenyl, ethoxyphenyl, or bromo-hydroxy-methoxyphenyl.

[0030] In one embodiment, the compound or pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer, or polymorph thereof, wherein 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.

[0031] In one embodiment, the compound or pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer, or polymorph thereof, wherein R 2 is substituted or unsubstituted aryl.

[0032] In one embodiment, the compound or pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer, or polymorph thereof, wherein R 2 is selected from the list: H, 5-methoxy, 7-methoxy, 7-bromo, 7-chloro, or 7-fluoro.

[0033] In one embodiment, the compound or pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer, or polymorph thereof, wherein R 3 is benzopyran unit or H.

[0034] In one embodiment, the compound or pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer, or polymorph thereof, wherein R 3 is selected from the following list: H, 2-(4-fluorophenyl)-5-methoxy-1,9-dihydrobenzopyrano[2,3- d]imidazole unit, 2-(4-bromophenyl)-5-methoxy-1,9-dihydrobenzopyrano[2,3-d]imidazole unit, or 2-(2-fluorophenyl)-5-methoxy-1,9-dihydrobenzopyrano[2,3-d]imidazole unit.

[0035] In one embodiment, the compound or pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer, or polymorph thereof, wherein the compound is selected from the following list:

[0036]

[0037]

[0038]

[0039] In one aspect of the application, the disclosed compounds are used in medicine or veterinary medicine.

[0040] In one aspect of the application, the compounds can be used in the treatment, therapy or diagnosis of a disease characterized by benign or malignant cell proliferation or by areas of neovascularization or hyper- vascularization or cancer.

[0041] In one aspect of the application, the compounds can be used in the treatment, therapy or diagnosis of hyperproliferative tissue or neoplasia.

[0042] In one aspect of the application, the compounds can be used in the treatment, therapy or diagnosis of breast cancer, renal cell carcinoma, leukemia, glioma or glioblastoma.

[0043] In one aspect of the application, the compounds can be used in the treatment, therapy or diagnosis of renal cell carcinoma, leukemia, glioma, glioblastoma, breast cancer.

[0044] In one aspect of the application, the compounds can 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.

[0045] Another aspect of the application relates to a pharmaceutical composition comprising at least one of the disclosed compounds.

[0046] In one embodiment, the disclosed pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0047] In one embodiment, the disclosed pharmaceutical composition further comprises an antiviral agent, an analgesic, an anti-inflammatory agent, a chemotherapeutic agent, a radiotherapy agent, an antibiotic, an antifungal agent, an anthelmintic, or a diuretic, or a mixture thereof.

[0048] In one embodiment, the disclosed pharmaceutical composition further comprises a filler, a binder, a disintegrant, a lubricant, or a mixture thereof.

[0049] In one embodiment, the disclosed pharmaceutical composition is used for intradermal or transdermal therapy, or local or systemic therapy, or intravenous therapy, or a combination thereof.

[0050] Another aspect of the invention relates to a method for obtaining the disclosed compound, comprising the following steps:

[0051] Concentrated HCl (1.1 equivalents) was added to an orange solution of 2-imino-8-methoxy-2H-benzopyran-3-amine (0.150 mg; 0.79 mmol) in 1 mL of CH3CN;

[0052] Stir the mixture at room temperature (20°C) for 10-15 minutes (observe direct precipitation);

[0053] Filtering the solid, preferably by simple filtration, to obtain 3-amino-8-methoxy-2H-benzopyran-2-ammonium chloride;

[0054] The aldehyde (1.1-1.7 equivalents) was added to a suspension of 3-amino-8-methoxy-2H-benzopyran-2-ammonium chloride (0.25-0.35 mmol) in CH3CN (1-2 mL);

[0055] The suspension was stirred at 60°C for 24-48 hours.

[0056] The obtained solid by filtration is used to separate pure product, preferably by washing with CH3CN;

[0057] Optional when in 1 When HCl contamination was observed in the 1H NMR spectrum, the solid was washed with an aqueous solution of NaHCO3 (0.05M), filtered, and washed with water, resulting in the production of the pure product 2-(5-methoxy-3,9-dihydrobenzopyrano[2,3-d]imidazole).

[0058] In one embodiment, for the preparation of 5,5'-dimethoxy-2,2'-diphenyl- 1,1',9,9'-tetrahydro-9,9'-bifuranopyrano[2,3-d]imidazole, the aldehyde (1-1.2 eq) is added to a solution of 2-imino-8-methoxy-2H-benzopyran-3-amine in CH3CN (1-2 mL) and the solution is stirred at 80 °C for 7-24 hours. The solid product starts to precipitate slowly, filtered, washed with CH3CN, and identified as pure product 5,5'-dimethoxy-2,2'-diphenyl- 1,1',9,9'-tetrahydro-9,9'-bifuranopyrano[2,3-d]imidazole.

[0059] Another aspect of the application relates to nanoparticles comprising the disclosed compounds and / or the disclosed pharmaceutical compositions.

[0060] In one embodiment, the nanoparticles comprising the disclosed compounds and / or pharmaceutical compositions are encapsulated by nanoparticles.

[0061] Another aspect of the application relates to kits comprising the disclosed compounds and / or the disclosed pharmaceutical compositions.

[0062] Another aspect of the application relates to the use of the disclosed compounds or pharmaceutically acceptable salts, hydrates, solvates, N-oxides, stereoisomers, diastereomers, enantiomers, atropisomers, dimers or polymorphs thereof for medical or veterinary use. In particular, for use in the treatment or therapy of a disease characterized by benign or malignant cell proliferation or by areas of neovascularization or cancer. In particular, for use in the treatment, therapy or diagnosis of hyperproliferative tissues, such as those associated with cancer. In addition, 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.

[0063] Alkyl is defined as a monovalent group derived from an alkane by removal of a hydrogen atom from any carbon atom - C n H 2n+1 The group derived by removal of a hydrogen atom from a terminal carbon atom of an unbranched alkane forms the n-alkyl (n-alkyl) subcategory H(CH2) n The groups RCH2, R2CH(R≠H) and R3C(R≠H) are primary, secondary and tertiary alkyl groups, respectively. Aryl is derived from arenes (monocyclic and polycyclic arenes) by removal of a hydrogen atom from a ring carbon atom.

[0064] "Alkyl" includes "lower alkyl" and extends to cover 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- propyl nonyl, 2-(cyclododecyl)ethyl, adamantyl, and the like.

[0065] "Lower alkyl" denotes an alkyl group having from 1 to 7 carbon atoms. Examples of lower alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec- and t-butyl, pentyl, hexyl, heptyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, 2-methylcyclopropyl, cyclopropylmethyl, and the like.

[0066] In the present invention, halogen means an element selected from the following list: fluorine (F), chlorine (CI), bromine (Br), iodine (I), astatine (At).

[0067] In the present invention, the term "heterocycle" denotes that at least one of the atoms forming the ring backbone is not carbon. Unless otherwise specified, a heterocycle can be a saturated, partially unsaturated, or fully unsaturated ring. A "saturated heterocycle" refers to a heterocycle containing only single bonds between the members of the ring. A "partially saturated heterocycle" refers to a non-aromatic heterocycle containing at least one double bond. The term "heteroaromatic ring" denotes a fully unsaturated aromatic ring in which at least one of the atoms forming the ring backbone is not carbon. Typically, a heteroaromatic ring contains no more than 4 nitrogens, no more than 1 oxygen, and no more than 1 sulfur. Unless otherwise specified, a heteroaromatic ring can be attached through any available carbon or nitrogen by replacing a hydrogen atom on said carbon or nitrogen. The term "heteroaromatic bicyclic system" denotes a ring system consisting of two fused rings, at least one of which is a heteroaromatic ring as defined above.

[0068] The term "carbocycle" denotes a ring in which the atoms forming the ring backbone are selected from carbon only. Unless otherwise specified, a carbocycle can be a saturated, partially unsaturated, or fully unsaturated ring. A fully unsaturated carbocycle is also referred to as an "aromatic ring" when it satisfies huckel's rule. A "saturated carbocycle" refers to a ring having a backbone consisting of carbon atoms connected to each other by single bonds; unless otherwise specified, hydrogen atoms occupy the remaining carbon atom valences. BRIEF DESCRIPTION OF DRAWINGS

[0069] The following drawings are provided to illustrate preferred embodiments of the present invention and should not be considered limiting of the scope of the invention.

[0070] Figure 1 The effect of benzopyranyl compounds on MCF-7 cell migration as evaluated by the wound-healing assay is shown (for each compound, the respective 1 / 2 IC 50 and IC 50Values treated for 12, 24, 48, 72 hours).

[0071] Figure 2 The effect of benzopyran-based compounds on Caki-2 cell line migration evaluated by wound-healing assay is shown (for each compound, IC 50 Values treated for 12, 24, 36, 48 hours).

[0072] Figure 3 The effect of benzopyran-based compounds on 786-O cell proliferation after 48 hours of treatment is shown.

[0073] Figure 4 Flow cytometry analysis of MCF-7 cell viability evaluated by Annexin V / PI assay is shown. Figure 4 A was treated with 0.5% DMSO (control) or with IC 50 Representative dot plots of MCF-7 cells treated for 12 and 24 hours at the IC Figure 4 B shows the quantification of the percentage of cells in each quadrant of the dot plots.

[0074] Figure 5 Flow cytometry analysis of Hs578t cell viability evaluated by Annexin V / PI assay is shown. Figure 5 A was treated with 0.5% DMSO (control) or with IC 50 Representative dot plots of Hs578t cells treated for 12 and 24 hours at the IC Figure 5 B shows the quantification of the percentage of cells in each quadrant of the dot plots.

[0075] Figure 6 Immunoblot analysis of total PARP, caspase 3 and 9, BIM and Bcl-xL after treatment of MCF-7 cells with compounds MC409, MC408, MC406 and MC421 using the respective IC 50 Immunoblot analysis of total PARP, caspase 3 and 9, BIM and Bcl-xL after treatment of MCF-7 cells with compounds MC409, MC408, MC406 and MC421 using the respective IC

[0076] Figure 7 Immunoblot analysis of total PARP, caspase 3 and 9, BIM, Bcl-xL and Bax after treatment of Hs578t cells with compounds MC409, MC408, MC406 and MC421 using the respective IC 50 Immunoblot analysis of total PARP, caspase 3 and 9, BIM, Bcl-xL and Bax after treatment of Hs578t cells with compounds MC409, MC408, MC406 and MC421 using the respective IC

[0077] Figure 8 Flow cytometry analysis showing DNA content of MCF-7 cells. Figure 8 A is treated with 0.5% DMSO (control) or IC 50 Representative histograms of cell cycle profiles of MCF-7 cells treated for 12 and 24 hours with the indicated concentrations. Figure 8 B shows quantification of cells in different phases of the cell cycle.

[0078] Figure 9 Flow cytometry analysis showing DNA content of Hs578t cells. Figure 9 A is treated with 0.5% DMSO (control) or IC 50 Representative histograms of cell cycle profiles of Hs578t cells treated for 12 and 24 hours with the indicated concentrations. Figure 9 B shows quantification of cells in different phases of the cell cycle.

[0079] Figure 10 Effect of compounds MC408 and MC421 on microtubular network of Hs578t (A, B) and MCF-7 (C, D) cells.

[0080] Figure 11 Strategy used in the determination of toxic effects in Caenorhabditis elegans (C. elegans). Worms were grown in liquid medium for 7 days using heat-inactivated bacteria (E. coli OP50) as a food source and in the presence of several concentrations of compounds MC421, MC406, MC369 and MC408, MC409, MC407. The rate of food consumption between days 3-5 was used as an indication of worm development, health and fertility, since after day 3, the offspring of the worms will also contribute to a more rapid decrease in food amount. An example is shown for hypothetical compound X. DMSO 1% and 5% were used as negative and positive controls for toxicity.

[0081] Figure 12 Compounds show lack of in vivo toxicity in Caenorhabditis elegans (C. elegans).

[0082] Figure 13 Effect of compound MC408 on C57BI / 6 mice is shown.

[0083] Figure 14 Results of a series of welfare tests performed on C57BI / 6 mice treated with compound MC408 are shown.

[0084] Figure 15 Effect of MC408 treatment on enzymatic liver function of C57BI / 6 mice is shown.

[0085] Figure 16 Effect of MC408 treatment on C57B1 / 6 mice - Experiment 2 is shown. Figure 16 A is a schematic of the experimental timeline. Figure 16 B shows the mice body weight.

[0086] Figure 17 In vivo therapeutic effect of MC408 and MC421 on breast cancer Hs578t cell line is shown. Figure 17 A shows representative photographs of the CAM assay and Figure 17 B shows the tumor growth percentage.

[0087] Figure 18 Flow cytometry analysis of 786-O cell viability assessed by Annexin V / PI assay is shown. Figure 18 A is a representative dot plot of 786-O cells treated with 0.5% DMSO (control) or at the IC 50 concentration for 24 and 48 hours. Figure 18 B shows the quantification of the percentage of cells in each quadrant of the dot plot.

[0088] Figure 19 Immunoblot analysis of KDM4C, total PARP, Hsp90, total JNK, total p53, Bid and p21 after treatment of 786-O cells (A) for 24 hours and (B) for 48 hours with compounds MC408 and MC421 using the respective IC 50 concentrations is shown. Protein levels in lysates were detected by immunoblot using 12% polyacrylamide gels.

[0089] Figure 20 Immunoblot analysis of total mTOR and phosphorylated form of mTOR, ERK, VEGFR 2 , EGFR, AKT, PTEN and phosphorylated form of AMPK, PDK1, NF-kB and pl8 and c-Myc, Hsp90 and PRAS40 proteins after a 2 hours starvation period and treatment of 786-O cells with cediranib, compounds MC350, MC415, MC412, MC408 and MC421 using a unique concentration of 2 μΜ for 6 hours is shown. Protein levels in lysates were detected by immunoblot using 10% polyacrylamide gels.

[0090] Figure 21 In vivo therapeutic effect of MC408 and MC421 on renal cancer 786-O cell line is shown. Figure 21 A shows representative photographs of the CAM assay and Figure 21 B shows the tumor growth percentage.

[0091] Figure 22 Flow cytometry analysis of DNA content in 786-O cells is shown. Figure 22 A is treated with 0.5% DMSO (control) or with the reagent's IC50. 50 Representative bar charts of cell cycle profiles of 786-O cells treated with different concentrations for 24 and 48 hours. Figure 22 B shows cell quantification at different phases of the cell cycle.

[0092] Figure 23 The results show the 1 / 2 IC value of the selected compound. 50 or IC 50 Effects of benzopyranyl compounds on A498 tolerance and cell proliferation of parental cell lines after 24 and 48 hours of treatment.

[0093] Figure 24 The results show the 1 / 2 IC value of the selected compound. 50 or IC 50 Effects of benzopyranyl compounds on Caki-2 tolerance and cell proliferation of parental cell lines after 24 and 48 hours of treatment.

[0094] Figure 25 IC50 values ​​for rapamycin and sildenafil, compounds MC350, MC413, MC408, and MC421 are shown. 50 Immunoblot analysis of total ERK and GAPDH, as well as their phosphorylated forms, HK2, LDHA, Hif2α, MCT1, PKM, PFKL, Hsp90, and c-Myc proteins was performed after 48 hours of treatment with A498 parental (P) and tolerant (R) cells. Protein levels in the lysates were detected by immunoblotting using a 10% polyacrylamide gel.

[0095] Figure 26 The in vivo effects of compounds MC408 and MC421 on angiogenesis are shown. Representative images (20× magnification) of intraocular and extraocular CAM measurements at days 13 and 17.

[0096] Figure 27 The tumor volume and body weight are shown in an orthotopic breast cancer NSG mouse xenograft model. Detailed Implementation

[0097] This invention relates to novel types of benzopyranyl anticancer compounds. The invention describes the anticancer properties of these novel types of benzopyranyl anticancer compounds. The invention also describes methods for the synthesis and isolation of these novel types of benzopyranyl anticancer compounds.

[0098] Specifically, the present invention describes a method for synthesizing novel benzopyranyl compounds by combining at least two different molecules, thereby producing compounds having a relevant biological spectrum of interest.

[0099] In one embodiment, 19 different benzopyran-imidazolium compounds were isolated. Table 1 shows the benzopyranium skeleton structures and the 19 synthesized and isolated benzopyran-imidazolium compounds.

[0100] Table 1: Structure of benzopyranyl compounds

[0101]

[0102]

[0103]

[0104] In one embodiment, as part of the evaluation of the antiproliferative activity of the separation reagent, the cell growth inhibition and IC50 of the synthesized and isolated benzopyranyl compounds were determined. 50 Structure-activity relationship studies were performed on all synthesized compounds. Antiproliferative activity against the MCF-7 breast cancer cell line was analyzed. Table 2 shows the IC50 values ​​of the compounds against the breast cancer cell line MCF-7 and the non-proliferative cell line MCF-10A. 50 Value (μM) and selectivity index (SI). The non-proliferative cell line MCF-10A was also used to determine the selectivity of the compound for tumor cells. For IC50... 50 For compounds with an IC50 value (concentration required to reduce cell viability by 50%) higher than 20 μM, the IC50 value has not been determined. 50 The selectivity index (SI) was calculated to evaluate the cytotoxicity of the test compounds.

[0105] Table 2: IC50 of the compounds against breast cancer cell line MCF-7 and non-proliferative cell line MCF-10A 50 Value (μM) and selectivity index (SI)

[0106]

[0107]

[0108] The dimerized compound provided excellent IC50 for the MCF-7 cell line. 50 The value is smaller than that of the respective monomers. Monomer compounds with a halogen atom attached to the imidazole moiety in the aromatic ring or without substituents provide a smaller IC value. 50 Value (<1 μM). Imidazolo-benzopyrans with OH or OCH3 / OCH2CH3 resulted in significantly higher IC50 values. 50values, even when halogen atoms are present in the same aromatic moiety.

[0109] promising IC 50 values and calculated high SI values. For the dimeric compounds, the toxicity on non-neoplastic cells was much lower, resulting in the generation of excellent SI values (higher than 60). The SI values are generally very promising.

[0110] In one embodiment, for comparative studies, the monomers and respective dimers were selected and further evaluated in Hs578t and MDA-MB-468 breast cell lines. Table 3 shows the IC 50 values (μΜ) and selectivity index (SI) for the selected benzopyran-based compounds on breast cancer cell lines Hs578t and MDA-MB-468. These cell lines correspond to the basal cell-like subtype of breast cancer, which is included in the known triple negative subtype, since they are negative for the molecular markers of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER 2 ) 2. The clinical presentation of these breast cancer subtypes is very aggressive and there is still no specific molecular therapy.

[0111] Table 3: IC 50 values (μΜ) and selectivity index (SI) for the selected benzopyran-based compounds on TNBC cell lines MDA-MB-468, MDA-MB-231 and Hs578t

[0112]

[0113]

[0114] The benzopyran-based compounds showed very low IC 50 values from 0.035 to 0.27 μΜ on Hs578t. The bromo-substituted benzopyran-based compound MC408 and the dimeric compound MC421 showed very low IC 50 values on Hs578t cell line and on MDA-MB 468 cell line. The compound MC408 is particularly active (IC 50 = 0.027 μΜ). In general, the compounds show even more interesting antiproliferative potential on these aggressive breast cancer subtypes and the SI values are also excellent.

[0115] In one embodiment, the anti-proliferative activity of compounds MC408, MC409, MC421 and MC406 was further determined for glioma cell lines (U87, GAMG and GL18) and acute leukemia cell lines (HL-60, KG-1 and Jurkat). This study was performed to evaluate whether the compounds exhibit the same interesting anti-cancer profile in other cancer cell models. After exposing the cells to each compound at an appropriate concentration range for 72 hours to determine the respective IC 50 values, cell viability was determined using the MTS assay. Table 4 shows the IC 50 values (μM) of compounds MC408, MC409, MC421 and MC406 against glioma and leukemia cancer cell models.

[0116] Table 4: IC 50 values (μM) of compounds MC408, MC409, MC421 and MC406 against glioma and leukemia cancer cell models.

[0117]

[0118]

[0119] The compounds showed excellent anti-proliferative ability since the IC 50 values determined for all cell lines were in the nano-molar or low micro-molar range. Compound MC421 showed very high cell growth inhibition against glioma cell lines, particularly for U87 and GAMG. Compound MC408 showed lower anti-proliferative ability when compared to the other compounds, but the IC 50 values were still in the low μM range. In the leukemia models, all compounds appeared to be more selective against the Jurkat cell line, but excellent growth inhibition was also achieved by treatment of the other leukemia cell lines with all compounds. These results indicate that these compounds are promising in several cancer cell models and can be considered as candidates for cancer therapy.

[0120] In one embodiment, the anti-proliferative activity of the compounds was further determined for renal cancer cell lines and non-tumoral kidney cell lines. Table 5 shows the IC 50 values (μM) and selectivity index (SI) of renal cancer cell lines (786-O, Caki-2, A498) and non-neoplastic cell line (HK2). Compounds M955, M1220, M1143 and M1221 did not show any biological activity in the first screening and IC 50 determination was not continued. Some compounds showed high potency and excellent selectivity index against renal cell carcinoma (RCC) cell lines.

[0121] Table 5: IC50 of renal cell carcinoma lines (786-O, Caki-2, A498) and non-neoplastic cell line (HK2) 50 Value (μM) and selectivity index (SI)

[0122]

[0123]

[0124] In one embodiment, the antiproliferative activities of compounds MC421, MC409, MC408, MC350, MC416, MC410, MC411, MC415, and MC413 against drug-resistant renal cell carcinoma lines were further determined. Table 6 shows the IC50 values ​​for rapamycin-resistant A498 and sildenafil-resistant Caki-2 cell lines and the non-proliferative cell line (HK2). 50 Value (μM) and selectivity index (SI).

[0125] Table 6: IC50 values ​​of rapamycin-resistant A498 and sildenafil-resistant Caki-2 cell lines and non-proliferative cell line (HK2) 50 Value (μM) and selectivity index (SI).

[0126]

[0127] In one implementation, the effects of the four selected compounds (MC408, MC409, MC406, and MC421) on cell migration were further evaluated. Figure 1 Using their respective ICs 50 and IC 50 Half of the values ​​were used to treat MCF-7 cells for 72 hours and a wound-healing assay was performed. For each compound, IC50 was used. 50 The concentration-dependent effect is evaluated using half the concentration. Figure 1 The effects of benzopyranyl compounds on MCF-7 cell migration, as evaluated by a wound-healing assay, were shown (for each compound, the values ​​were calculated using their respective 1 / 2 IC50 values). 50 and IC 50 Values ​​were processed at 12, 24, 48, and 72 hours. Results are expressed as mean ± SD of at least three independent experiments, compared with control (DMSO, 0.5%), *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0128] After only 12 hours of incubation, complete cell death was observed under the microscope for all compounds. At 72 hours, the medium was removed from each well before taking the pictures, as a large number of dead floating cells prevented accurate measurement of cell migration. Compound MC409 showed an early effect on cell migration; after 12 hours, for IC 50 and ½ IC 50 values, the compound was able to reduce the percentage of cell migration (relative to the 0.5% DMSO control) by about 40%. However, as observed by the slow recovery of cell migration, the cells seemed to recover from the effect of the compound. After 72 hours of treatment, using IC 50 concentrations, cell migration was about 12% less than the control. For the other 3 compounds, a lower effect was also observed after 24 or 48 hours of treatment. For compound MC408 and MC406 treatments, using IC 50 and ½ IC 50 concentrations, similar effects on cell migration were observed and, after 72 hours, cell migration was 20% less than the control. Compound MC421 showed an effect in a concentration-dependent manner. For IC 50 and ½ IC 50 concentrations, a slight effect on cell migration was noted over time. A decrease in cell migration (13% less than the control) was only observed after 72 hours of treatment.

[0129] In one embodiment, the effect of compounds MC408, MC421, MC412 and MC415 on cell migration was also evaluated in the renal cell carcinoma cell line Caki-2 at 4 different time points (24, 48, 72 and 96 hours) (Table 2). Figure 2 Sunitinib was used as a reference drug and all compounds were tested using their respective IC 50 concentration values. Figure 2 The effect of benzopyranyl compounds on the migration of the Caki-2 cell line evaluated by the wound-healing assay (for each compound, treated for 12, 24, 36, 48 hours with the respective IC 50 values) is shown. Results were normalized to the control (dashed line) and expressed as mean ± SEM. *p<0.05, **p<0.005, ***p<0.002, ****p<0.0001 compared to the control (0.5% DMSO). Significance of differences between groups was determined by Student's t-test.

[0130] In one embodiment, the effect of preferred benzopyranyl compounds on cell proliferation was evaluated. 786-O cells were treated for 48 hours with IC 50 and ½ IC 50 values. The ability to incorporate BrdU during DNA synthesis was measured and the results are shown in Table 3.Figure 3 middle. Figure 3 The effects of benzopyran compounds on the proliferation of 786-O cells after 48 hours of treatment were shown. Results are presented as mean ± SD of at least three independent experiments. *p<0.05, **p<0.01, ***p<0.0005, ****p<0.0001 compared to control (0.5% DMSO). Significance between groups was determined by Student's t-test. Generally, benzopyrans induced a significant reduction in cell proliferation in a concentration-dependent manner for 786-O cells. Exceptions were observed for compounds MC409, MC410, and MC368.

[0131] In one embodiment, the compounds show clear effects on cell growth and cell death, particularly compounds MC406, MC409, and MC408. This is evident through morphological changes (cells observed under a microscope) and the presence of round, floating cells.

[0132] In one implementation, to understand the mechanisms leading to cell death, the induction of apoptosis was studied using flow cytometry. MCF-7 and Hs578t cells were compared with the respective IC50 values ​​of the compounds. 50 Cells were cultured at different concentrations for 12 and 24 hours, and 786-O cells for 24 and 48 hours, followed by double staining with annexin V and propidium iodide (PI) to detect the externalization of phosphatidylserine. The externalization of phosphatidylserine occurs during early apoptotic events. Although annexin V binds to phosphatidylserine, PI stains only cells that have lost their membrane integrity, making it an experimental procedure that allows differentiation between live cells, early apoptotic cells, late apoptotic / necrotic cells, or necrotic cells. Compounds MC408 and MC421 demonstrated relevant anticancer effects by inducing cell death through apoptosis in three cell lines over 24 hours. Figure 9 and 10 As shown. In the Hs578t cell line, compounds MC406 and MC409 also induced cell death through apoptosis.

[0133] Figure 4 Flow cytometry analysis of MCF-7 cell viability as evaluated by annexin V / PI assay is shown. Figure 4 A shows the IC50 values ​​after treatment with 0.5% DMSO (control) or with compounds MC408, MC409, MC406, and MC421. 50 Representative punctate plots of MCF-7 cells treated with different concentrations for 12 and 24 hours. Figure 4B shows quantification of the percentage of cells in each quadrant of the dot plot. Results were obtained using cells treated with DMSO as control (100%) and are expressed as the mean ± SEM of 3 independent experiments. Annexin V / PI data were analyzed by two-way ANOVA and Bonferroni post-test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 compared to control (0.5% DMSO). Figure 5 Flow cytometry analysis showing Hs578t cell viability evaluated by Annexin V / PI assay. Figure 5 A shows representative dot plots of Hs578t cells treated with 0.5% DMSO (control) or with IC 50 Representative dot plots of Hs578t cells treated with the indicated concentrations for 12 and 24 hours. Figure 5 B shows quantification of the percentage of cells in each quadrant of the dot plot. Results were obtained using cells treated with DMSO as control (100%) and are expressed as the mean ± SEM of 3 independent experiments. Annexin V / PI data were analyzed by two-way ANOVA and Bonferroni post-test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 compared to control (0.5% DMSO). Figure 18 Flow cytometry analysis showing 786-0 cell viability evaluated by Annexin V / PI assay. Figure 18 A shows representative dot plots of 786-0 cells treated with 0.5% DMSO (control) or with IC 50 Representative dot plots of 786-0 cells treated with the indicated concentrations for 24 and 48 hours. Figure 18 B shows quantification of the percentage of cells in each quadrant of the dot plot. Results were obtained using cells treated with 0.5% DMSO as control (100%) and are expressed as the mean ± SEM of 3 independent experiments. Annexin V / PI data were analyzed by two-way ANOVA and Bonferroni post-test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 compared to control (0.5% DMSO).

[0134] In one embodiment, the induction of cell death was further investigated by immunoblot analysis of key apoptosis markers, checking the apoptosis pathway. After 24 and 48 hours of treatment of the cells with IC 50 , the induced apoptosis was evaluated as marker the poly (ADP-ribose) polymerase 1 (Parp), caspases 3 and 9 (Figure 6). Figure 6 ) after 24 and 48 hours of treatment of the cells with the compounds.

[0135] Treatment of cells with the compounds resulted in cleavage of PARP Figure 6 and 7 which is the last step of caspase activation and is considered a hallmark of apoptosis. Cleaved caspase 3 was also observed at both time points. Apoptosis was induced by all compounds as observed by the presence of these markers.

[0136] Bim protein was also used and elevated levels were noted for the test compounds, but only for the Hs578t cell line. The BH3-domain protein of Bim interacts with tubulin and, in the initial phase, tubulin is described to sequester Bim by binding to dynein light chain, thus preventing in this way the initiation of the apoptotic signaling pathway. Upon release from tubulin, Bim migrates to the mitochondria, interacts with some proteins (e.g. Bax, Bcl-2 and Bc-xL) and eventually promotes apoptosis. This indicates the release of early apoptotic signals from cells treated with the test compounds, thus affecting the levels of pro- and anti-apoptotic proteins involved in mitochondria-induced apoptosis.

[0137] 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 treated with the compounds at their respective IC 50 for 12 and 24 hours. MCF-7 and Hs578t cells were treated with the compounds at their respective IC 50 for 24 and 48 hours. Cell cycle analysis was evaluated by DNA content using flow cytometry Figure 8 , 9 and 22). Figure 8 Flow cytometry analysis of DNA content of MCF-7 cells is shown. Figure 8 A shows representative histograms of cell cycle profiles of MCF-7 cells treated with 0.5% DMSO (control) or with IC 50 for 12 and 24 hours of compounds MC408, MC409, MC406 and MC421. Figure 8 B shows quantification of cells in different phases of the cell cycle, except for compound MC409. Results are expressed as mean ± SD of 3 independent experiments. Data were analyzed by two-way ANOVA and Bonferroni post-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 compared to control (0.5% DMSO).

[0138] Figure 9Flow cytometry analysis showing DNA content of Hs578t cells. Figure 9 A shows IC50values of MC408, MC409, MC406 and MC421 on Hs578t cells treated for 12 and 24 hours. B shows quantification of cells in different phases of the cell cycle. Results are expressed as mean ± SD of 3 independent experiments. Data were analyzed by two-way ANOVA and Bonferroni post-test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 compared to control (0.5% DMSO). 50 Representative histograms of cell cycle profiles of Hs578t cells treated for 12 and 24 hours with the indicated concentrations. Figure 9 B shows quantification of cells in different phases of the cell cycle. Results are expressed as mean ± SD of 3 independent experiments. Data were analyzed by two-way ANOVA and Bonferroni post-test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 compared to control (0.5% DMSO).

[0139] Figure 22 Flow cytometry analysis showing DNA content of 786-O cells. Figure 22 A shows IC50values of MC408 and MC421 on 786-O cells treated for 24 and 48 hours. B shows quantification of cells in different phases of the cell cycle. Results are expressed as mean ± SD of 3 independent experiments. Data were analyzed by two-way ANOVA and Bonferroni post-test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 compared to control (0.5% DMSO). 50 Representative histograms of cell cycle profiles of 786-O cells treated for 24 and 48 hours with the indicated concentrations. Figure 22 B shows quantification of cells in different phases of the cell cycle. Results are expressed as mean ± SD of 3 independent experiments. Data were analyzed by two-way ANOVA and Bonferroni post-test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 compared to control (0.5% DMSO).

[0140] In one embodiment, the effect of benzopyranyl compounds on microtubule dynamics was analyzed. Figure 10 Effect of compounds MC408 and MC421 on microtubular network of Hs578t (A, B) and MCF-7 (C, D) cells. Untreated (control), paclitaxel (Hs578t 12h: 0.25 μΜ, 24h: 0.01 μΜ; MCF-7 12 / 24h: 1 μΜ) and IC 50 Cells treated for 12 (A, C) or 24h (B, D) with compounds MC408 and MC421, stained with β-tubulin and counterstained with 4,6-diamidino-2-phenylindole (DAPI). Microtubules and unassembled tubulin are shown in green. DNA stained with DAPI is shown in blue.

[0141] In one embodiment, the Caenorhabditis elegans (C. elegans) is used as a model for in vivo toxicity evaluation of the compounds. A food clearance-based assay is implemented, in which bacterial consumption over time is a proxy for worm development, health, and fertility Figure 11 Figure 11 The strategy used in the determination of the toxic effects in C. elegans is shown. Worms were grown in liquid media for 7 days using heat-inactivated bacteria (E. coli OP50) as a food source and in the presence of several concentrations of compounds MC421, MC406, MC369 and MC408, MC409, MC407. The rate of food consumption between days 3-5 was used as an indication of worm development, health, and fertility, since after day 3, the offspring of the worms would promote a faster reduction in the amount of food. An example is shown for the hypothetical compound X. DMSO 1% and 5% were used as negative and positive controls of toxicity, respectively. For this experiment, the compounds were dissolved in DMSO 1% (vehicle).

[0142] The compounds were tested at several concentrations (up to the highest soluble concentration possible - 150 mM (MC369, 75 mM)). Figure 12 Figure 12 The in vivo toxicity analysis of MC421, MC406, MC369 and MC408, MC409, MC407 in C. elegans is shown. The rate of food consumption between days 3-5 was used as an indication of worm development, health, and fertility and compared to 1% DMSO (vehicle, non-toxic). No statistical differences were observed for MC421, MC406, MC369 and MC408, MC409, MC407 (ANOVA followed by Games-Howell post-hoc test). No statistical differences in the rate of food consumption between days 3-5 were detected for any of the compounds / compound concentrations. The fact that the compounds seem to be well tolerated even at significantly higher concentrations than those used for in vitro testing can be a good predictor of toxicity in mammalian models.

[0143] In one embodiment, the safety profile of the compounds was evaluated in rodents. Figure 13 The effects of MC408 treatment on C57B1 / 6 mice - Experiment 1 are shown. Figure 13 ​​A is a schematic representation of the experimental timeline. Five-month-old female and male animals (n=3 / group / sex) were used and injected daily (i.p.) with MC408 3 mg / Kg or vehicle (saline, Tween 80 and methylcellulose) for a total of 7 injections. A series of welfare tests were performed daily. Figure 13 B shows what happened immediately after the 1st injection; for this analysis, animals were videotaped for 10 min and the immobility time and rearing counts (REARS) were counted by the experimenter. There was no difference between vehicle and MC408 mice. Figure 13 B shows the body weight of the mice monitored throughout the experiment and there was no difference between the treated groups. Figure 13 D shows the vertical exploratory activity of the mice. Activity was measured in a viewing jar and the number of rears was counted for 5 minutes. Figure 13 E shows the horizontal activity of the mice. Activity was recorded in an open field marked with squares and the number of squares was counted while the animals were allowed to freely explore for 1 minute. There was no difference in exploratory activity. Figure 13 F shows an indirect measure of anxiety in the mice. To indirectly measure anxiety, the number of fecal pellets produced by the animals during the behavioral procedure was counted and no differences were observed between groups. In Figure 13 In G and H, water and food intake, respectively, were analyzed. Every day, 0.300 g of food and 200 mL of water were placed in each cage. At the end of the experiment (day 7), the food was weighed and the water was measured and there were no differences in both parameters. Data are represented as mean ± SE.

[0144] Figure 14 The impact of MC408 treatment on C57B1 / 6 mice is shown. A series of welfare tests were performed to evaluate signs of toxicity when treated with MC408 compared to vehicle animals. All the parameters evaluated were scored as normal or abnormal, present or absent, and all animals were scored equally (MC408 and vehicle), indicating that the compound had no impact on the welfare of the mice.

[0145] In one embodiment, the impact of treatment on liver function of C57B1 / 6 mice is analyzed. Figure 15 The impact of MC408 treatment on enzymatic liver function of C57B1 / 6 mice is shown. Using standard techniques, the activity of the following enzymes was measured in the liver of the mice: Figure 15 A) female and Figure 15B) Aspartate aminotransferase (AST / TGO) and alanine aminotransferase (ALT / TGP) were measured in the serum of male animals. Blood was collected at the end of the experiment (day 7). No statistical differences were found between compound- and vehicle-treated animals within each gender. Notably, one male animal in the MC408-treated group showed higher levels of both AST and ALT compared to the other two animals in the same group. Data are expressed as mean ± SE.

[0146] In one embodiment, the effect of compound treatment on C57B1 / 6 mice is analyzed - Experiment 2. Figure 16 The effect of MC408 treatment on C57B1 / 6 mice is shown - Experiment 2. Figure 16 A is a schematic representation of the experimental timeline. Female and male animals of 3-months old (n=6 / group / gender) were used and injected daily with MC408 3 mg / Kg or vehicle (saline, Tween 80 and methylcellulose) (i.p.) for a total of 7 injections. In this experiment, the number of animals per group was increased and the experiment was designed to collect blood before and after treatment. Figure 16 B shows the body weight of the mice. Body weight was monitored throughout the experiment and no differences between groups within each gender were found. Data are expressed as mean ± SE.

[0147] In one embodiment, the in vivo therapeutic efficacy studies of the compounds are analyzed using the CAM model. Figure 17 The in vivo therapeutic efficacy of MC408 and MC421 on the breast cancer Hs578t cell line is shown. Figure 17 A shows representative pictures of the in ovo and ex ovo CAM assays (x10 magnification) (at day 13 and 17). Figure 14 B shows the percentage of tumor growth. Results are expressed as the mean percentage of tumor growth from day 13 to day 17 ± SD ****p<0.0001. Data were analyzed by one-way ANOVA test. Eggs were treated with control (DMSO 0.5%), MC408 (2xIC 50 = 0.094 μΜ) or MC421 (2xIC 50 = 0.070 μΜ).

[0148] In one embodiment, the induction of cell death is further investigated by examining the apoptosis pathway through immunoblot analysis of key apoptosis markers. After 24 and 48 hours of treatment of 786-0 cells with IC 50 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 evaluated as markers of induced apoptosis after 24 and 48 hours of treatment of 786-0 cells with ICFigure 19 ).

[0149] In one embodiment, the interaction with RTK receptors and mTOR / PI3K / AKT pathway was further investigated by evaluating mammalian targets of rapamycin (mTOR), extracellular-signal-regulated kinase (ERK), vascular endothelial growth factor receptor 2 (VEGFR 2 ), endothelial growth factor receptor (EGFR), protein kinase B (AKT), phosphatase and tensin homolog (PTEN), adenosine monophosphate-activated protein kinase (AMPK), pyruvate dehydrogenase kinase 1 (PDK1), nuclear factor kappa B (NF-kB), pl8, c-Myc, Hsp90 and 40 kDa proline-rich Akt substrate (PRAS40) proteins after treatment with Cediranib (an effective inhibitor of vascular endothelial growth factor used in the treatment of renal cell carcinoma) and with compounds MC350, MC412, MC415, MC408 and MC421 at a unique dose of 2 μΜ for 6 hours. Figure 20 ).

[0150] Treatment of 786-0 cells with several compounds resulted in a decrease of the levels of phospho-mTOR and AKT proteins. Moreover, in some cases, EGFR and VEGFR 2 both showed a decrease of levels when compared to controls and Cediranib. Some of the tested compounds affected the mTOR / PI3K / AKT pathway, interfering with different protein markers.

[0151] VEGF and EGFR are tyrosine kinase (RTK) receptors specific for the vascular endothelial (VEGF) and endothelial (EGF) growth factor family, which have an important role in tumor growth and metastasis. Their autophosphorylation stimulates downstream activation and signal transduction through a variety of other proteins associated with phosphorylated tyrosine. These downstream signaling proteins initiate some signal transduction cascades, including the Akt pathway, leading to gene expression, cell proliferation and migration, angiogenesis and vasculogenesis. Moreover, mTOR regulates cell growth, proliferation and metabolism. Its activity is controlled by two multiprotein complexes, mTORCl (sensitive to rapamycin) and mTORC2, which is considered resistant or inhibits only when provided at high doses for a period of time. Second-generation dual mTOR inhibitors have been developed and the most important advantage of those new drugs is a significant reduction of AKT phosphorylation blocked for mTORC2 and a better inhibition of mTORCl. Considering the results, the reduction of RTK, mTOR and AKT expression indicates a decrease of key regulators for all the above-mentioned functions.

[0152] In one embodiment, the in vivo therapeutic efficacy of the compounds is analyzed using the CAM model.Figure 21 The in vivo therapeutic effect of MC408 and MC421 on the renal cancer 786-0 cell line is shown. Figure 21 A shows representative photographs of in ovo and ex ovo CAM assays (x10 magnification) (at day 13 and 17). Figure 21 B shows the percentage of tumor growth. Results are expressed as the mean percentage of tumor growth from day 13 to day 17 ± SD, p<0.0001 compared to the control (statistical analysis performed using the one-way ANOVA test). The eggs were treated with control (DMSO 0.5%), MC408 (2xIC 50 = 0.128 μΜ) or MC421 (2xIC 50 = 0.154 μΜ).

[0153] In one embodiment, the effect of Cediranib, rapamycin and benzopyran-based compounds on cell proliferation was evaluated. A498 (parental and rapamycin-resistant) and Caki-2 (parental and Cediranib-resistant) cells were treated with IC 50 and ½ IC 50 values for 24 and 48 hours. The ability of BrdU incorporation during DNA synthesis was measured and Figure 23 and 24 Results are shown in Table 1. Figure 23 The effect of benzopyran-based compounds on A498 (parental and rapamycin-resistant) cell proliferation after 24 and 48 hours of treatment is shown. Figure 24 The effect of benzopyran-based compounds on Caki-2 (parental and Cediranib-resistant) cell proliferation after 24 and 48 hours of treatment is shown. Results are expressed as the mean ± SD of at least 3 independent experiments.

[0154] In general, for parental or resistant A498 cells, benzopyrans induced a significant reduction of cell proliferation in a concentration and time-dependent manner, especially compounds MC408, MC421 and MC413 Figure 23 ).

[0155] For parental and drug-resistant Caki-2 cells, Figure 24 benzopyrans MC421, MC350 and MC413 were able to reduce cell proliferation in a concentration and time-dependent manner.

[0156] In one embodiment, the interactions related to metabolic features were further investigated by evaluating total extracellular-signal-regulated kinase (ERK) and its phosphorylated form, 3-phosphoglyceraldehyde dehydrogenase (GAPDH), hexokinase 2 (HK2), lactate dehydrogenase A (LDHA), hypoxia-inducible factor 2a (Hif2a), monocarboxylate transporter 1 (MCT1), pyruvate kinase isozyme (PKM), 6-phosphofructokinase (PFKL), heat shock protein 90 (Hsp90) and c-Myc protein. IC 50 These markers were evaluated after a period of 48 hours of treatment of A498 parental (P) and resistant (R) cells. Protein levels in lysates were detected by immunoblotting using 10% polyacrylamide gels Figure 25 ).

[0157] Treatment of A498 cells with different compounds resulted in a decrease of some protein levels. In fact, rapamycin-resistant cells showed a significant decrease in the protein expression levels of GAPDH, Hif2a, c-Myc and Hsp90 compared to controls and even to parental cells. In light of the results of the present application, chromenes alter the metabolic response of cancer cells even when resistant to current available RCC therapies.

[0158] In one embodiment, the ability of the compounds to inhibit angiogenesis in vivo was analyzed using the CAM model. To evaluate the effect on angiogenesis, sterile 5-mm diameter filter discs were immersed in culture medium with a fixed concentration (2xIC 50 values) of chromenes MC408 and MC421 or 0.5% DMSO (control group) and placed on the vascular area of the CAM. Figure 26 Representative photographs of in ovo and ex ovo CAM assays are shown (x10 magnification) (at day 13 and 17).

[0159] After 4 days of treatment with the novel chromenes, the formation of new blood vessels was reduced with chromenes MC408 and MC421. In addition, the rupture of preexisting vessels was observed (black arrows in Figure 26 ), indicating that the compounds inhibit angiogenesis in the CAM model.

[0160] In one embodiment, the in vivo therapeutic efficacy of compound MC408 was analyzed using a mouse model. Figure 27This study demonstrates the in vivo therapeutic effect of TNBC cell lines MDA-MB-231 and MC408 in a mouse xenograft model of orthotopic breast cancer. MDA-MB-231 cells were injected into the mammary fat pads of NSG mice (n=6 or 7 per group). Treatment was administered for one week, 3 days post-implantation. NT = mediator; Dose 1 = 3 mg / kg; Dose 2 = 10 mg / kg; Dose 3 = 50 mg / kg. *, p<0.05; **, p<0.01; ****, p<0.0001 (compared to the NT group; two-way ANOVA, post-hoc Tukey test or unpaired t-test with Welch correction).

[0161] In a mouse model of orthotopic breast cancer xenograft, benzopyran MC408 significantly reduced tumor volume in a dose-dependent manner. Figure 27 A) and weight ( Figure 27 B). At a dose of 50 mg / kg, both tumor volume and weight were reduced by more than 40% compared to the carcass group. Importantly, throughout the experiment, the animals did not show any weight loss or other side effects with the treatment regimen tested.

[0162] In one embodiment, three human breast cancer cell lines, Hs578T, MDA-MB-468, and MCF-7, and the normal breast cell line MCF-10A, were obtained from the ATCC (American Type Culture Collection). The cancer cell lines were cultured in Dalberg's modified Eagle medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco) and 1% antibiotic solution (penicillin-streptomycin, Gibco), at 4.5 g / L glucose (DMEM, Gibco). Normal cell lines were cultured in Dalberg modified Eagle medium: a 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 (FABM2) and KG-1 (erythroleukemia-FAB M6), and a human lymphocytic leukemia (ALL) cell line, Jurkat (T-cell type), were also used. These three cell lines were obtained from the German Collection of Microorganisms and Cell Cultures (DCM). Deutsche Sammlung von Mikroorganismen und Zellkulturen). Cells were cultured in RPMI 1640 medium (Biochrom®- Merck Millipore) supplemented with 10% heat-inactivated FBS (Biochrom®- Merck Millipore) and 1% antibiotic / antimycotic mixture (Biochrom®- Merck Millipore). Three human glioblastoma (GBM) cell line models were also used, two established and commercially available cell lines (U87MG and GAMG, kindly gifted by Rui M. Reis) and one primary GBM culture (GL18). GBM cells were cultured in DMEM (Biochrom®- Merck Millipore), all supplemented with 10% FBS. Renal cell carcinoma cell lines A498, 786-0, Caki-2 and HK2 were obtained from ATCC. Cancer cell line A498 was cultured in MEM medium (Biochrom®- Merck Millipore), 786-0 in RPMI 1640 medium (Biochrom®- Merck Millipore), Caki-2 in Mc Coys medium (Biochrom®- Merck Millipore) and HK2 in RPMI 1640 medium (Biochrom®- Merck Millipore). All media were supplemented with 10% heat-inactivated FBS (Biochrom®- Merck Millipore) and 1% antibiotic / antimycotic mixture (Biochrom®- Merck Millipore). All cells were grown in a humidified incubator at 37°C and 5% C02. For all assays, DMSO (dimethyl sulfoxide, Sigma-Aldrich) controls were used.

[0163] ​​​​​​​​​In one embodiment, cell viability assays are performed. MCF-7, Hs578t and MCF-10A cells are plated in 96-well culture plates at 3000 cells per mL or for MDA-MB-468, 5000 cells per mL (100 μL / well) in triplicate. Cells are then allowed to adhere for a period of 18-20 hours in complete medium. Subsequently, cells are treated with 7 different concentrations (0.1 to 40 μM or 5 to 60 μM) of compounds in fresh medium or controls. HL-60, KG-1 and Jurkat cell lines are plated in 96-well culture plates at 50.000 cells / 100 μL per well and treated with different concentrations (0.001 to 2 μM) of the described compounds (or controls). Cells are plated in 96-well plates at an initial density of 2000 cells / well (for GAMG), 4000 cells / well (for GL18) and 6000 cells / well (for U87MG) in triplicate and allowed to adhere for 18-20 hours. Cells are treated with different concentrations of compounds (0.005 to 100 μM) or with controls. A498 and 786-O cells are plated in 96-well culture plates (100 μL / well) at 2000 cells per mL, Caki-2 cells at 3000 cells per mL and HK2 cells at 2000 cells per mL in triplicate and allowed to adhere for a period of 18-20 hours in complete medium. Then, cells are treated with 7 different concentrations (0.1 to 40 μM or 5 to 60 μM) of compounds in fresh medium or controls. After 72h of incubation, MTS (Promega) reduction assay is performed according to the manufacturer's instructions to evaluate indirectly the percentage of viable cells by metabolizing cell viability. After 1-2 hours of incubation with MTS at 37°C and 5% CO2 in a humidified atmosphere, the absorbance at 490 nm is measured. For RCC cell lines, sulforhodamine B assay is used according to the manufacturer's instructions. Data are log-transformed and the concentration of each compound that reduces the number of viable cells to 50% relative to controls (IC 50 ) is calculated using GraphPad Prism 6 software.

[0164] In one embodiment, the selectivity index (SI) values are calculated using the following mathematical formula, using the IC 50 values for MCF-7 and MCF10A cell lines for all compounds:

[0165] SI = (IC 50 normal cell line - IC 50 cancer cell line) / IC 50 cancer cell line

[0166] For SI values > 1, the cytotoxicity is higher against cancer cell lines than against non-neoplastic cell lines.

[0167] In one embodiment, cell migration is evaluated by a wound-healing assay that mimics the process of cell migration during wound-healing in vivo. The method is based on the following procedure: a scratch is prepared, mimicking a wound in a cell monolayer, images are recorded at the beginning and at regular intervals during the process of cell migration to close the wound and the images are compared to quantify the rate of cell migration. MCF-7 and Caki-2 cells were plated at a density of 9.0 x 10 5 and 3.0 x 10 5 Each 2 mL was plated in a 6-well plate and grown overnight at 37°C in a 5% C02humidified atmosphere. A 200 μL pipette tip was used to make two scratches in the confluent cell layer. Cells were gently washed once with 500 μL PBS. MCF-7 cells were treated with the respective IC 50 , ½ IC 50 MCF-7 cells were treated with the compounds or with 0.5% DMSO (control) for 72 h. At 0, 12, 24, 48 and 72 h, pictures were taken of the specific wound site (4 positions per wound). For Caki-2 cells, the respective IC 50 , including Cediranib or 0.5% DMSO (control) for 48 h and pictures were taken at 0, 12, 24, 36 and 48 h. Images were acquired using an Olympus IX51 inverted microscope equipped with an Olympus DP20 digital camera system at 100x magnification. Evaluation of the migration distance was performed using BeWound 1.7.1 and the percentage of cell migration normalized to the control was plotted using GraphPad Prism 6 software. Three independent experiments were performed for each compound. Significance of differences between groups was determined by Student's t-test.

[0168] In one embodiment, a proliferation assay is performed. 786-0 cells were plated in a 96-well plate at a density of 7000 cells / 10 μL and incubated overnight at 37°C in a 5% C02humidified atmosphere. Thereafter, cells were treated with the respective IC 50 or ½ IC 50Concentration Adherent cells were treated with compound MC350, MC408, MC412, MC415, MC409, MC410, MC413 and MC369 or with 0.5% DMSO (control) for 48h. After incubation, cells were labeled by adding 10 μl / well of BrdU labeling solution (final concentration: 40 μΜ BrdU). Then, cells were incubated for 16h to allow the incorporation of BrdU into the DNA of proliferating cells, replacing thymidine nucleosides. After labeling, the medium was removed, cells were fixed and DNA was denatured by incubation with 200 μl of FixDenat solution at room temperature. Denaturation of DNA is necessary for the binding of the antibody conjugate to the incorporated BrdU. After removal of FixDenat, and 100 μl of anti-BrdU-POD antibody was incubated for 90 min at room temperature. The anti-BrdU-POD antibody binds to the incorporated BrdU in newly synthesized cellular DNA. The antibody conjugate was removed and the wells were washed three times with washing solution. The immune complex was detected by adding substrate solution (100 μl / well) and the plate was incubated at room temperature until the color development was satisfactory for photometric detection (5-10 min). The substrate reaction was stopped by adding 25 μl of 1 M H2SO4 to each well and gently mixed. The reaction product was quantified by measuring the absorbance at 450 nm (reference wavelength: 690 nm) in a microplate reader (Tecan Infinite M200). In the absence of cells, all the above steps were performed using a blank test at each experimental time point. The results of at least 3 independent experiments (quadruplicate) were evaluated using GraphPad Prism 5 software.

[0169] In one embodiment, protein extraction and immunoblot analysis were performed. MCF-7 and Hs578t cells were grown in T25 flasks and 786-O cells were grown in 6-well plates, when cells reached 70-80% confluence, each IC 50Cells were treated with compounds or with 0.5% DMSO (control). Cells were treated for 24 hours and 48 hours. Specifically, for RTK and mTOR pathway analysis, 786-0 cells were starved for 2 hours and then treated with a single dose of 2 μΜ cediranib or compounds or with 0.5% DMSO (control). After treatment, adherent and floating cells were collected by scraping and centrifuged at 2000 rpm, 4°C for 10 min. The pellet was resuspended in PBS and centrifuged again (1200 rpm; 5 min, 4°C). The supernatant was discarded and the pellet was resuspended 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 14000 rpm, 4°C for 15 minutes; then, the supernatant was collected for protein concentration determination using the DC Protein Assay Kit (BioRad).

[0170] Briefly, 30 μg of total protein per sample was separated on 10%, 12%, or 15% polyacrylamide gels (100 V) and then transferred to nitrocellulose membranes (100 V for 30 min). Membranes were blocked with a solution of 5% milk in 1 x TBS for 60 min and then incubated overnight at 4°C with specific primary antibodies (rabbit anti-PARP antibody (Cell Signaling, #9542), 1 : 1000 5% milk; mouse anti-caspase-9 antibody (Cell Signaling, #9508), 1 : 500 5% milk; rabbit anti-caspase-3 antibody (Cell Signaling, #9665), 1 : 500 5% milk; mouse anti-Bax antibody (Santa Cruz Biotechnology, sc-7480), 1 : 500 5% milk; mouse anti-Bax antibody (Santa Cruz Biotechnology, sc-8392), 1 : 500 5% milk; rabbit anti-Bim antibody (Cell Signaling, #2933), 1 : 1000 5% BSA; rabbit anti-Bid antibody (Cell Signaling, #2002), 1 : 1000 5% BSA; rabbit anti-KDM4C antibody (Abeam, ab27532), 1 : 2000 5% BSA; rat anti-Hsp90 antibody (Calbiochem, cat # 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, #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-VEGFR 2Antibodies (Cell Signaling, #2479), 1 :500 5% BSA; Rabbit anti-phospho-VEGFR 2(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-kB p65 (Ser536) antibody (Cell Signaling, #3036), 1 :1000 5% BSA; rabbit anti-AMPK a antibody (Cell Signaling, #2532), 1 :1000 5% BSA; rabbit anti-phospho-AMPK a antibody (Thr172) (Cell Signaling, #2535), 1 :1000 5% BSA; rabbit anti-AKT antibody (Cell Signaling, #4691), 1 :1000 5% BSA; rabbit anti-phospho-AKT (Thr308) antibody (Cell Signaling, #13038), 1 :1000 5% BSA; rabbit anti-PTEN antibody (Cell Signaling, #9559), 1 :1000 5% BSA; rabbit anti-phospho-PTEN (Ser380) antibody (Cell Signaling, #9551), 1 :1000 5% BSA; rabbit anti-phospho-PDK1 (Ser241) antibody (Cell Signaling, #3438), 1 :1000 5% BSA; rabbit anti-phospho-p38 (Thr180 / Tyr182) antibody (Cell Signaling, #4511), 1 :1000 5% BSA; rabbit anti-p44 / 42 MAPK (Erkl / 2) antibody (Cell Signaling, #4695), 1 :1000 5% BSA; rabbit anti-phospho-p44 / 42 MAPK (Thr202 / Tyr204) (phospho-Erkl / 2) antibody (Cell Signaling, #4370), 1 :1000 5% BSA; rabbit anti-β-tubulin antibody (Abeam, ab6046), 1 :10000 5% BSA; mouse anti-GAPDH antibody (Santa Cruz Biotechnology, sc-32233), 1 :1000 5% BSA; mouse anti-HK2 (Abeam, ab104836), 1 :1000 5% BSA; mouse anti-LDHA antibody (Santa Cruz Biotechnology, sc-137243), 1 :1000 5% BSA;Rabbit anti-Hif2a antibody (Cell Signaling, #36169), 1 : 1000 5% BSA; Rabbit anti-MCT1 antibody (Santa Cruz Biotechnology, sc-365501), 1 : 1000 5% BSA; Rabbit anti-PKM (Abeam, ab38237), 1 : 1000 5% BSA; Rabbit anti-PFKL (Abeam, 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 minutes (twice) and another 15 minutes (once), the blots were incubated with the respective secondary antibodies for 1 hour 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 5% milk, Cell Signaling; and Rabbit-anti-rat IgG-HRP secondary antibody (Abeam, ab6734), 1 : 30000, 5% BSA). After washing with TBS / 0.1% Tween 20 for 5 minutes (twice) and another 15 minutes (once), the immunoreactive bands were detected by chemiluminescence Western Bright; TM The Sirius kit (Advansta) was used to detect the immunoreactive bands on the ChemiDoc XRS+ system (BioRad). Immunoblot quantification was performed by Quantity One 4.6.9.

[0171] In one embodiment, cell cycle distribution by flow cytometry was performed. MCF-7, Hs578t and 786-0 cells were seeded into 6-well culture plates. Cells were allowed to adhere in complete DMEM (for MCF-7 and Hs578t cells) and complete RPMI (for 786-0 cells) media for a period of 18-20 hours and treated with IC 50Test compounds at concentrations ranging from 0.1 to 10 μΜ or 0.5% DMSO (control) were treated for 12 and 24 hours. Each experiment was repeated three times. Floating and adherent cells were collected and pooled by centrifugation and fixed with cold ethanol (70%). Cells were resuspended in PBS; after centrifugation and removal of the supernatant, they were resuspended in a solution containing PBS, PI (50 μg / mL, P1304MP, Invitrogen), RNase A (20 mg / mL, 12091-021, Invitrogen) and Triton X 100. After a final incubation at 50°C for 1 hour in the dark, the PI signal was measured using a FACS LSRII flow cytometer (BD ) with a 488 nm excitation laser, recorded and FACS Diva was used as acquisition software. The percentage of cells in each phase was analyzed using FlowJo 7.6 (Tree ) software. Three independent biological replicates were performed.

[0172] In one embodiment, MCF-7, Hs578t and 786-O cells were seeded in 6-well culture plates and allowed to adhere in complete DMEM (for MCF-7 and Hs578t cells) and complete RPMI (for 786-O cells) media for 18-20 hours for the detection of apoptosis by flow cytometry analysis. Cells were treated with IC 50 Test compounds at concentrations ranging from 0.1 to 10 μΜ or 0.5% DMSO (control) were treated for 12 and 24 hours. Each experiment was repeated three times. Floating and adherent cells were collected and pooled by centrifugation. The supernatant was removed and 1 mL of binding buffer was added. To the cell pellet, 8 μL of FITC Annexin V (556419, BD Pharmingen) and 30 μL of PI (50 μg / mL, P1304MP, Invitrogen) were added. The samples were incubated for 15 min 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 ) with a 488 nm excitation laser. The Annexin V signal was collected through a 488 nm block filter, 550 nm long-pass dichroic with a 525 nm band-pass. The signal was recorded and FACS Diva was used as acquisition software. The percentage of cells in each phase was analyzed using FlowJo 7.6 (Tree ) software. Three independent biological replicates were performed.

[0173] In one embodiment, toxicity of each compound is tested using the Bristol strain N2 (CGC provided, funded by the NIH Office of Research Infrastructure Programs - P40 OD010440) based on a food clearance assay using C. elegans. The assay is performed in liquid culture on 96-well plates (Voisine et al. 2007; Teixeira-Castro et al. 2015). Each well includes the following (final volume 60 μΐ): about 20 worms at the egg stage, OP50 bacteria to a final OD of 0.6-0.8 (595 nm), and each compound at an appropriate concentration. Worms are grown for 7 days at 180 rpm / 20°C under continuous shaking (Shel lab Si series incubator) and absorbance (OD595) is measured daily on a microplate reader (Tecan Infinite M200). The effect of compounds on C. elegans physiology is monitored by the rate of consumption of the E. coli food suspension Figure 14 Age-synchronized worm eggs are obtained by "egg prep" as follows: adults are treated with an alkaline hypochlorite solution (20% bleach, 25% 1 M NaOH) for 6 min, then centrifuged and washed 2 times in M9 buffer. Then, egg pellets are resuspended in S-medium to obtain an appropriate egg concentration, then transferred to 96-well plates. OP50 bacteria are prepared by 4 freeze-thaw cycles, inactivated overnight (37°C, 180 rpm, Luria broth medium). OP50 is resuspended in supplemented S-medium (cholesterol, streptomycin, penicillin and nystatin) before use. Compounds are prepared in 100% DMSO (Sigma) and diluted up to a test concentration of 1% DMSO to prevent solvent toxicity. For each compound, several concentrations (150-10 μΜ) are tested, except for MC369 (75-10 μΜ) due to solubility issues.

[0174] For each compound, several concentrations are used and the corresponding OD595 consumption rate (slope) is calculated for days 3-5. Compounds are tested in worms grown in 96-well plates in two independent experiments (Plate Layout.xls, Supporting Information). For statistical analysis, data from both experiments are pooled together and IBM SPSS is used to apply ANOVA (Brown-Forsythe robust test for homogeneity of variances) followed by Games-Howell post-hoc test to compare different concentrations for each compound and 5% DMSO (wt / v) to the control (1% DMSO (wt / v)).

[0175] In one embodiment, C57 / B16 female and male mice (n=3 / group / sex) are injected intraperitoneally with MC408 (3 mg / Kg) or vehicle (saline, Tween 80 and methylcellulose) daily for 7 days to determine the toxicity of the compound in a mouse model. A series of tests to evaluate the welfare of the mice is performed daily. Immediately after the first drug administration, the animals are videotaped and the immediate effects of MC408 are recorded. The number of vertical movements and the time of inactivity are recorded. Water and food intake during the 7-day experiment is evaluated by adding a fixed amount of food and water in the cages of the animals on the first day and measuring the amount remaining on day 7. Body weight is evaluated in a dynamic mouse scale every day. Horizontal and vertical activity are analyzed to evaluate the intrinsic exploratory behavior of the animals; these are measured for 1 minute in an open field with marked squares and for 5 minutes in an observation jar, respectively. In addition, as a measure of anxiety, the number of fecal pellets is counted while the animals are performing the daily behavioral evaluation protocol. The experimenter applies a series of tests more related to animal welfare, including the following measurements: (i) body posture and curvature; (ii) spontaneous activity; (iii) respiratory rate; (iv) eye lid opening and reflex; (v) scratching the skin to analyze dehydration; (vi) grooming; (vii) fur standing; (viii) tremors; (ix) limbs hugging and (x) reaction to transfer. On day 7, the animals are euthanized. All animals are deeply anesthetized (ketamine hydrochloride (150 mg / kg) and medetomidine (0.3 mg / kg)) and heart perfused with a saline solution (NaCI 0.9% (wt / v)). Blood is collected from the vena cava, centrifuged at 13.000 rpm for 10 minutes and the plasma is transferred to a new tube and stored at -80°C until further processing for enzymatic liver function measurements using standard techniques. Organs (kidney, intestine, stomach, brain, liver, ovary / testis) are harvested and placed in tubes containing 4% paraformaldehyde and further processed for paraffin embedding and pathological analysis.

[0176] In one embodiment, a CAM assay is performed. Fertilized eggs of chicken are incubated at 37°C and on day 3 of development, the eggshell is windowed after piercing the air sac. The window is sealed with BTK tape and returned to the incubator. On day 9 of development, Hs578t cells (2 x 10 6 cells) or 786-O cells (3.5 x 10 6 cells) and Matrigel (10 μΐ) are placed on the CAM, allowing tumor formation and the egg is returned to the incubator. On day 14 of development, the tumors are photographed. The treatment groups receive 20 μΐ of 2x IC 50MC408 or MC421 in complete DMEM (for Hs578t cells) or complete RPMI (for 786-0 cells) medium at the concentrations and controls received 20 μΐ, of 0.5% DMSO in complete DMEM or RPMI; these were added over the tumors that were formed. After 72 hours of treatment (day 17 of development), the tumors were photographed in ovo again. The chicken embryos were sacrificed at -80°C for 10 minutes and the individual CAMs were fixed with 4% paraformaldehyde before being photographed ex ovo again.

[0177] In one embodiment, a mixture of 0.6 x 10 6 NSG (NOD Scid Gamma) female mice (3-6 months) were injected in the mammary fat pad with a mixture of MDA-MB-231 breast cancer cells and matrigel (ratio 1 : 1). Mice were randomly assigned to treatment groups (at least 6 mice / group) that started 3 days after implantation with 3 independent doses of compound MC408 (dose 1 = 3 mg / kg, dose 2 = 10 mg / kg and dose 3 = 50 mg / kg) or vehicle (PBS lx and Matrigel, ratio 1 : 1) and were administered daily for a total of 7 days. Mice were maintained under standard laboratory conditions. Animal tumor size was measured (by measuring the two largest sides and applying the formula: v = 3.14 x L1 x L1 x L2 / 6; L1 is the largest side and L2 is the other) and recorded periodically. On day 47 after implantation, animals were sacrificed and tumors were excised. All animal procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (European Directive 2010 / 63 / EU).

[0178] In one embodiment, silica gel 60 plates (Macherey-Nagel) were used to monitor the reactions of all compounds by thin layer chromatography (TLC) at 0.2 mm and with fluorescent indicators. A UV chamber with a 254 nm lamp (CN-6 Vilber Lourmat) was used for their display. Dry flash chromatography was performed using silica gel from MN Kieselgel 60 (230 ASTM) with a particle size < 0.063 mm. For reactions using temperature, a suitable magnetic stirrer was used according to the specific procedure and an electric furnace stirrer IKAMAG RCT was used at different temperatures. Solvents were evaporated in a Buchi RE 11 rotary evaporator with a vacuum degree and different bath temperatures. HNMR was performed at 400 MHz and with deuterated dimethyl sulfoxide (DMSO-d6) as solvent in a Bruker Avance III (for 1 HNMR was performed at 400 MHz and with deuterated dimethyl sulfoxide (DMSO-d6) as solvent in a Bruker Avance III (for 13NMR spectra were obtained at 100 MHz. Chemical shifts were recorded in parts per million (ppm) using the residual solvent peak as an internal standard. IR spectra were recorded in a FT-IR Bomem MB 104 using a NaCI cell. Melting points were determined in a Stuart SMP3 apparatus and are uncorrected.

[0179] Elemental analysis was performed on a LECO CHNS-932 instrument.

[0180]

[0181] 2-imino-8-methoxy-2H-chromen-3-amine (4) and chromene derivatives M1159, M955, M1220, M1221 and M1143 can be synthesized by the above method.

[0182] In one embodiment, the synthesis of 3-amino-8-methoxy-2H-chromen-2- iminium chloride (5) was performed. Concentrated HC1 (1.1 eq) was added to an orange solution of 2-imino-8-methoxy-2H-chromen-3-amine (4) (0.150 mg; 0.79 mmol) in 1 mL of CH3CN. A direct 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-chromen-2- iminium chloride (5). Orange solid; yield 91 %; mp > 300 °C; 1 H NMR (400 MHz, DMSO-d6)

[0183] 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) 56.02, 110.13, 113.52, 117.42, 122.21, 126.39, 130.57, 135.33, 146.55, 161.15; IR (NaCI cell)

[0184] 3322, 3192, 1678, 1654, 1600, 1576, 1552, 1460 cm -1 ; C 10 H 11 N2O2Cl.0.2H2O Theoretical: C, 52.16; H, 4.96; N, 12.17. Experimental: C, 52.06; H, 4.77; N, 12.40.

[0185] In one embodiment, benzopyrano[2,3-d]imidazolium was synthesized. Aldehyde (1) (1.1-1.7 equivalents) was added to a suspension of 3-amino-8-methoxy-2H-benzopyrano-2-ammonium chloride (5) (0.25-0.35 mmol) in CH3CN (1-2 mL), and the suspension was stirred at 60 °C for 24-48 hours. The solid was filtered, washed with CH3CN, and identified as a pure product. 1 When HCl contamination was observed in the 1H NMR spectrum, the solid was washed with an aqueous solution of NaHCO3 (0.05M), filtered, and washed with water, resulting in the production of a pure product.

[0186] In one embodiment, 2-(4-fluorophenyl)-5-methoxy-3,9-dihydrobenzopyrano[2,3-d]imidazole (MC409) was synthesized. 4-fluorobenzaldehyde (1) (0.0375 mg; 0.30 mmol) was added to a suspension of 3-amino-8-methoxy-2H-benzopyrano-2-ammonium chloride (5) (0.047 mg; 0.21 mmol) in CH3CN (1.6 mL), and the suspension was stirred at 60 °C for 32 hours. The solid was filtered, washed with CH3CN, and identified as a pure product. Beige solid; yield 98%; mp 191-192 °C; 1 H NMR(400MHz, DMSO-d6)3.82(s,3H),4.12(s,2H),6.94(dd,J=8.4,1.8Hz,1H),6.86(dd,J=7 .4,1.8Hz,1H),7.00(t,J=7.8Hz,1H),7.25-7.32(m,2H),7.86-7.92(m,2H),12.44(s,1H); 13 C NMR (75MHz, DMSO-d6) 23.64, 55.75, 103.64, 110.48, 115.77 (J=21.7Hz), 119.31, 121.82, 122.59, 126.29 (J=8.3Hz), 127.19 (J=3.2Hz), 138.88, 141.25, 148.19, 148.38, 161.78 (J=243.4Hz); IR (paraffin paste) 3348, 1700, 1650, 1608, 1578, 1538, 1500, 1461cm -1 C 17 H 13 Theoretical values ​​of N₂O₂F: C, 68.91; H, 4.42; N, 9.45. Experimental values: C, 68.89; H, 4.65; N, 9.56.

[0187] In one embodiment, 2-(4-bromophenyl)-5-methoxy-3,9-dihydrobenzopyrano[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-benzopyran-2- iminium chloride (5) (0.0758 mg; 0.34 mmol) in CH3CN (2 mL) and the suspension was stirred at 60 °C for 24 h. The solid was filtered, washed with CH3CN and identified as pure product. When HCl contamination was observed in the1H NMR spectrum, the solid was washed with an aqueous solution of NaHCO3(0.05 M; 2 mL), filtered and washed with water, resulting in the production of pure product. Yellow solid; yield 99%; mp 215-217 °C; 1 H NMR spectrum, the solid was washed with an aqueous solution of NaHCO3(0.05 M; 2 mL), filtered and washed with water, resulting in the production of pure product. Yellow solid; yield 99%; mp 215-217 °C; 1 H NMR (400 MHz, DMSO-d6) 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) 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 (paraffin paste)

[0188] 3435, 1717, 1639, 1603, 1576, 1536, 1463 cm -1 ; C 17 H 13 N2O2Br.1.9H2O Theor: C, 52.12; H, 4.29; N, 7.15. Found: C, 52.12; H, 3.93; N, 7.37.

[0189] In one embodiment, 2-(2-fluorophenyl)-5-methoxy-3,9-dihydrobenzopyrano[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-benzopyran-2- iminium chloride (5) (0.0571 mg; 0.25 mmol) in CH3CN (2 mL) and the suspension was stirred at 60 °C for 24 h. 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) 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 CNMR (75 MHz, DMSO-d6) 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 (paraffin paste) 3426, 1710, 1631, 1603, 1576, 1536, 1463 cm -1 ; C 17 H 13 Theoretical for N2O2F1.1 H2O: C, 64.60; H, 4.46; N, 8.87. Found: C, 64.36; H, 4.46; N, 8.91.

[0190] In one embodiment, 2-(3-fluorophenyl)-5-methoxy-3,9-dihydrobenzopyrano[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-benzopyran-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; 1H NMR (400 MHz, DMSO-d6) 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) 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 (paraffin paste) 3356, 1707, 1652, 1628, 1522, 1508, 1461 cm -1 ; C 17 H 13 N2O2F Theoretical: C, 68.91; H, 4.42; N, 9.45. Experimental: C, 68.98; H, 4.58; N, 9.66.

[0191] In one embodiment, 2-(4-chlorophenyl)-5-methoxy-3,9-dihydrobenzopyrano[2,3- d]imidazole (MC412) was synthesized. 4-chlorobenzaldehyde (0.0291 mg; 0.21 mmol) was added to a suspension of 3-amino-8-methoxy-2H-benzopyran-2-immonium chloride (5) (0.0417 mg; 0.18 mmol) in CH3CN (1 mL) and the suspension was stirred at 60 °C for 24 h. The solid was filtered, washed with CH3CN and identified as pure product. Beige solid; yield 100%; mp 212-214 °C; 1 H NMR (400 MHz, DMSO-d6) 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); 13CNMR (75MHz, DMSO-d6) 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 (paraffin paste) 3352, 1700, 1656, 1601, 1532, 1489, 1462 cm⁻¹ -1 C 17 H 13 Theoretical values ​​for N₂O₂Cl: C, 65.29; H, 4.19; N, 8.96. Experimental values: C, 65.47; H, 4.23; N, 8.74.

[0192] In one embodiment, 5-methoxy-2-phenyl-3,9-dihydrobenzopyrano[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-benzopyrano-2-ammonium chloride (5) (0.0653 mg; 0.29 mmol) in CH3CN (1 mL), and the suspension was stirred at 60 °C for 33 hours. The solid was filtered, washed with CH3CN, and identified as a pure product. 1 When HCl contamination was observed in the 1H NMR spectrum, the solid was washed with an aqueous solution of NaHCO3 (0.05M), filtered, and washed with water, resulting in a pure product. Beige solid; yield 99%; mp 162-162℃; 1 H NMR(400MHz, DMSO-d6)3.83(s,3H),4.12(s,2H),6.86(dd,J=7.6,1.6Hz,1H),6.94(dd,J=8.0,1.6Hz,1H) ,7.00(t,J=8.0Hz,1H),7.31(t,J=7.6Hz,1H),7.43(t,J=8.0Hz,2H),7.86(d,J=7.8Hz,2H),12.43(s,1H); 13 C NMR (75MHz, DMSO-d6) 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 (paraffin paste)

[0193] 3346,1702,1648,1602,1526,1496,1461cm -1 C17 H 14 Theoretical value for N2O2: C, 73.37; H, 5.07; N, 10.07. Experimental value: C, 73.58; H, 5.12; N, 10.34.

[0194] In one embodiment, 3-(5-methoxy-3,9-dihydrobenzopyrano[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-benzopyran-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 h. The solid was filtered, washed with CH3CN and identified as pure product. When a HCl contamination was observed in the H NMR spectrum, the solid was washed with an aqueous solution of NaHCO3(0.05 M), filtered and washed with water, resulting in the production of pure product. Light green solid; yield 94%; mp 162-164 °C; 1 H NMR spectrum, the solid was washed with an aqueous solution of NaHCO3(0.05 M), filtered and washed with water, resulting in the production of pure product. Light green solid; yield 94%; mp 162-164 °C; 1 H 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) 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 (in paraffin paste) 3351, 3218, 1702, 1659, 1611, 1523, 1507, 1460 cm -1 Theoretical value for C17H14N2O3: C, 69.38; H, 4.79; N, 9.52. Experimental value: C, 69.42; H, 4.88; N, 9.56.

[0195] In one embodiment, 4-(5-methoxy-3,9-dihydrobenzopyrano[2,3-d]imidazol-2- yl)phenol (MC413) was synthesized. 4-Hydroxybenzaldehyde (0.0388 mg; 0.32 mmol) was added to a suspension of 3-amino-8-methoxy-2H-benzopyran-2- iminium chloride (5) (0.0579 mg; 0.26 mmol) in CH3CN (2 mL) and the suspension was stirred at 60 °C for 48 h. The solid was filtered, washed with CH3CN and identified as pure product. When a HCl contamination was observed in the1H NMR spectrum, the solid was washed with an aqueous solution of NaHCO3(0.05 M), filtered and washed with water, resulting in the production of pure product. Yellow solid; yield 64%; mp 245-246 °C; 1 H NMR spectrum, the solid was washed with an aqueous solution of NaHCO3(0.05 M), filtered and washed with water, resulting in the production of pure product. Yellow solid; yield 64%; mp 245-246 °C; 1 H NMR (400 MHz, DMSO-d6) 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) 23.76, 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 (paraffin paste) 3338, 3246, 1706, 1645, 1612, 1548, 1503, 1463 cm -1 ; C17H14N2O3requires C, 69.38; H, 4.79; N, 9.52. Found: C, 69.39; H, 4.42; N, 9.54.

[0196] In one embodiment, 5-methoxy-2-(2-methoxyphenyl)-3,9-dihydrobenzopyrano[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-benzopyran-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 pure product. When a HCl contamination was observed in the1H NMR spectrum, the solid was washed with an aqueous solution of NaHCO3(0.05 M), filtered and washed with water, resulting in the production of pure product. Yellow solid; yield 64%; mp 245-246 °C; 1HCl contamination was observed in the H NMR spectrum, the solid was washed with an aqueous solution of NaHC03(0.05 M), filtered and washed with water, resulting in the pure product. Yellow solid; yield 85%; mp 188-190 °C; 1 H NMR (400 MHz, DMSO-d6) 3.82 (s, 3 H), 3.95 (s, 3 H), 4.10 (s, 2 H), 6.86 (dd, J = 7.6, 1.6 Hz, 1 H), 6.93 (dd, J = 8.0, 1.6 Hz, 1 H), 6.97 - 7.04 (m, 2 H), 7.13 (dd, J = 8.0, 0.8 Hz, 1 H), 7.30 (td, J = 7.7, 2.0 Hz, 1 H), 7.99 (dd, J = 8.0, 2.0 Hz, 1 H), 11.63 (s, 1 H); 13 C NMR (100 MHz, DMSO-d6)

[0197] 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 (in paraffin paste)

[0198] 3340, 1706, 1659, 1594, 1530, 1506, 1460 cm -1 ; C 18 H 16 Theoretical for N2O3: C, 70.12; H, 5.23; N, 9.09. Found: C, 70.44; H, 5.20; N, 9.16.

[0199] In one embodiment, 2,4-difluoro-6-(5-methoxy-3,9-dihydrobenzopyrano[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- benzopyran-2-chloroimine (5) (0.061 mg; 0.27 mmol) in CH3CN (2 mL) and the suspension was stirred at 60 °C for 36 h. The solid was filtered, washed with CH3CN and identified as pure product. When HCl contamination was observed in the H NMR spectrum, the solid was washed with an aqueous solution of NaHC03(0.05 M), filtered and washed with water, resulting in the pure product. Yellow solid; yield 49%; mp 260-262 °C; 1 HCl contamination was observed in the H NMR spectrum, the solid was washed with an aqueous solution of NaHC03(0.05 M), filtered and washed with water, resulting in the pure product. Yellow solid; yield 85%; mp 188-190 °C; 1H NMR (400 MHz, DMSO-d6) 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) 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 (paraffin paste) 3348, 3225, 1698, 1651, 1601, 1542, 1461 cm -1 ;C 17 H 12 Theoretical for N2O3F2O.8H2O: C, 59.23; H, 3.72; N, 8.13. Found: C, 59.13; H, 3.53; N, 8.42.

[0200] In one embodiment, 2-(4-ethoxyphenyl)-5-methoxy-3,9-dihydrobenzopyrano[2,3- d]imidazole (MC415) was synthesized. 4-Ethoxybenzaldehyde (0.0403 mg; 0.30 mmol) was added to a suspension of 3-amino-8-methoxy-2H-benzopyran-2- iminium chloride (5) (0.0614 mg; 0.27 mmol) in CH3CN (2 mL) and the suspension was stirred at 60 °C for 47 h. The solid was filtered, rinsed with CH3CN and identified as pure product. When HCl contamination was observed in the H NMR spectrum, the solid was rinsed with an aqueous solution of NaHCO3(0.05 M), filtered and rinsed with water, resulting in the production of pure product. Yellow solid; yield 96%; mp 215-217 °C; 1 H NMR (400 MHz, DMSO-d6) 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); 1H NMR (400 MHz, DMSO-d6) 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)

[0201] 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 (paraffin paste)

[0202] 3342, 1703, 1656, 1610, 1539, 1502, 1460 cm -1 ; C 19 H 18 Theoretical for N2O3: C, 70.81; H, 5.59; N, 8.70. Found: C, 70.74; H, 5.67; N, 8.77.

[0203] In one embodiment, 2-bromo-6-methoxy-3-(5-methoxy-3,9-dihydrobenzopyrano[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- benzopyran-2-immonium 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 pure product. When HCl contamination was observed in the H NMR spectrum, the solid was washed with an aqueous solution of NaHCO3(0.05 M), filtered and washed with water, resulting in the production of pure product. Beige solid; yield 71%; mp 160-161 °C; 1 H NMR (400 MHz, DMSO-d6) 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); 1H NMR (400 MHz, DMSO-d6) 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) 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 (in paraffin paste) 3338, 3212, 1703, 1647, 1535, 1497, 1461 cm -1 ; C 18 H 15 N2O4Br1.8H2O Theoretical: C, 49.61 ; H, 3.86; N, 6.43. Found: C, 49.68; H, 3.91 ; N, 6.43.

[0204] In one embodiment, 5,5'-dimethoxy-2,2'-diphenyl-1,1 ',9,9'-tetrahydro-9,9'- benzopyranophan[2,3-d]imidazole was synthesized. Aldehyde (1) (1-1.2 equiv.) was added to a solution of 2-imino-8-methoxy-2H-benzopyran-3-amine (4) in CH3CN (1-2 mL) and the solution was stirred at 80 °C for 7-24 hours. Solid product started to slowly precipitate, filtered, washed with CH3CN and identified as pure product 8.

[0205] In one embodiment, 2,2'-bis(4-fluorophenyl)-5,5'-dimethoxy-1,1 ',9,9'-tetrahydro-9,9'- benzopyranophan[2,3-d]imidazole (MC406) was synthesized. 4-Fluorobenzaldehyde (0.0629 mg; 0.51 mmol) was added to a solution of 2-imino-8-methoxy-2H-benzopyran-3-amine (4) (0.0962 mg; 0.51 mmol) in CH3CN (1 mL) and the solution was stirred at 80 °C for 7 hours. Solid product started to slowly precipitate, filtered, washed with CH3CN and identified as pure product. Beige solid; yield 18%; mp 272-274 °C; 1H NMR (400 MHz, DMSO-d6) 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); 13 C NMR (75 MHz, DMSO-d6) 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 (paraffin paste) 3348, 1700, 1650, 1608, 1538, 1500, 1461 cm -1 ; C 34 H 24 N4O4F21.2H2O Theoretical: C, 64.23; H, 4.85; N, 8.82. Found: C, 64.21; H, 4.79; N, 8.80.

[0206] In one embodiment, 2,2'-bis(4-bromophenyl)-5,5'-dimethoxy-l,l',9,9'- tetrahydro-9,9'-bipyranopheno[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-benzo pyran-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. Solid product started to slowly precipitate, filtered, washed with CH3CN and identified as pure product. Beige solid; yield 32%; mp 266-268 °C; 1 H NMR (400 MHz, DMSO-d6) 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) 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 (in paraffin paste) 3352, 1703, 1654, 1610, 1543, 1508, 1461 cm -1 ; C 34 H 24 Theoretical for N4O4Br2: C, 57.32; H, 3.40; N, 7.86. Experimental: C, 57.78; H, 3.37; N, 7.88.

[0207] In one embodiment, 2,2'-bis(2-fluorophenyl)-5,5'-dimethoxy-l,l',9,9'- tetrahydro-9,9'-bipyranopheno[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-benzopyran-3-amine (4) (0.0590 mg; 0.31 mmol) in CH3CN (2 mL) and the solution was stirred at 80 °C for 24 h. Solid product started to slowly precipitate, filtered, washed with CH3CN and identified as pure product. Beige solid; yield 15%; mp 276-278 °C; 1 H NMR (400 MHz, DMSO-d6) 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); 13 C NMR (75 MHz, DMSO-d6) 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 (in paraffin paste) 3438, 3447, 1637, 1575, 1614, 1575, 1530, 1469 cm -1 ; C 34 H 24Theoretical values for N4O4F2.0.8H2O: C, 67.51; H, 4.23; N, 9.26. Experimental values: C, 67.58; H, 4.30; N, 9.11.

[0208] Whenever used in this document, the term "comprising" is intended to mean that the mentioned features, integers, steps, components, or elements are present, but not excluding the presence of one or more other features, integers, steps, components, or elements, or groups thereof. The term "a" or "an" or "the" preceding an element are intended to be non-exclusive, meaning that a specific statement of "one," or "the only one," is not required for element to be encompassed.

[0209] Those skilled in the art will appreciate that the specific sequence in which steps are described is illustrative only and that steps can be performed in any order other than as described without departing from the application. Accordingly, except as otherwise noted, steps are presented in no particular order, indicating that when possible, steps can be performed in any convenient or desirable order.

[0210] If not specifically excluded, the use of singular forms "a," "an" and "the" in the specification and claims also include the plural unless the context clearly dictates otherwise. For example, the term "a compound" or "the compound" also includes the plural "compounds" or "the compounds." The use of the terms "one" and "the only one" are not required for an element to be encompassed unless specifically stated or otherwise clear from the context. The claims and description are considered to be satisfied if one, more than one, or all members of a group are present, used or otherwise associated with a given product or method, unless specifically stated otherwise or otherwise clear from the context. The present application includes embodiments in which exactly one member of a group is present, used in or otherwise associated with a given product or method. The present application also includes embodiments in which more than one or all members of a group are present, used in or otherwise associated with a given product or method.

[0211] Furthermore, it is to be understood that the application encompasses all alterations, combinations and permutations of one or more limitations, elements, clauses and descriptive terms as found in the respective claims or the description, including the use of any alternative or equivalent terminology. For example, where an element can be stated as including one or more of a list of alternatives, the element can include any one or more of those alternatives or any combination thereof.

[0212] Furthermore, where a composition is claimed, it is intended that the composition include the use of any and all isotopes and compounds as are present equivalent to those recited herein for any and all purposes including the preparation of the composition, unless otherwise indicated. For example, it is intended that any and all prodrugs, salts, solvates (including hydrates), coated particle forms, and the like, of the composition, unless otherwise indicated, are included within the scope of the present application.

[0213] When a range is given, the endpoints are included. Furthermore, it is understood that a value stated as a range can take any particular value within the range, unless otherwise indicated or otherwise evident to one of ordinary skill in the art, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise, and that a value stated as a mean can take any particular value within the range, unless the context clearly dictates otherwise. It is further understood that a value stated as a range can take any sub-range within the given range, with the endpoints of the sub-range being expressed to the same degree of accuracy as the lower limit of the range, unless the context clearly dictates otherwise, unless otherwise indicated or otherwise evident to one of ordinary skill in the art.

[0214] The present application should not be considered as being in any way limited to the described embodiments, and the person skilled in the art will foresee numerous possibilities of modification thereto.

[0215] The above-described embodiments are combinable.

[0216] References

[0217] (1) https: / / www.uicc.org / new-global-cancer-data-globocan-2018 (02 / 01 / 2019)

[0218] (2) http: / / gco.iarc.fr (02 / 01 / 2019)

[0219] (3) Bao, B; Mitrea, C.; Wijesinghe, P.; Marchetti, L.; Girsch, E.; Farr, R.; Boerner, J.; Mohammad, R.; Dyson, G.; Terlecky, S. and Bollig-Fischer, A. Treating triple negative breast cancer cells with erlotinib plus a select antioxidant overcomes drug resistance by targeting cancer cell heterogeneity. Scientific Reports 2017, 7, 44125.

[0220] (4) Costa, M.; Dias, T.; Brito, A. And F. Biological importance of structurally diversified chromenes. Eur. J. Med. Chem, 2016, 123, 487-507.

[0221] (5) Costa, M.; Rodrigues, A. I.; Proenca, F. Synthesis of 3-aminochromenes: the Zincke reaction revisited. Tetrahedron 2014, 70(33), 4869.

[0222] (6) Costa, M.; Proenca, F. 2-Aryl-l,9-dihydrochromeno[3,2-d]imidazoles: a facile synthesis from salicylaldehydes and arylideneaminoacetonitrile. Tetrahedron 2011, 67(10), 1799.

Claims

1. Use of a compound for the manufacture of a medicament for the treatment, therapy of breast cancer, renal cell carcinoma, leukemia or glioma, wherein, The compound is selected from the group consisting of:

2. A pharmaceutical composition comprising at least one of the compounds according to claim 1.

3. The pharmaceutical composition according to claim 2, further comprising an analgesic agent, an anti-inflammatory agent, a chemotherapeutic agent, a radiotherapeutic agent, a diuretic agent, or a mixture thereof.

4. A nanoparticle comprising a compound according to claim 1 and / or a pharmaceutical composition according to any one of claims 2 to 3.

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