A fibroblast regulatory factor, a chalcone derivative, for use in the treatment of stroma-rich tumors.

Abstract

The present invention relates to the field of oncology and provides compounds, compositions, uses and methods for treating cancer, particularly stroma-rich tumors, by selectively targeting cancer-associated fibroblasts (CAFs). CAF targeting is one strategy for enhancing the effectiveness of immunotherapy and can improve the patient response rate. Furthermore, this specificity in the target and related pathways reduces damage to normal tissues.

Description

Technical Field

[0001] The present invention relates to the field of oncology and provides compounds, compositions, uses and methods for treating cancer, particularly stroma-rich tumors, by selectively targeting cancer-associated fibroblasts (CAFs). CAF targeting is one strategy for enhancing the effectiveness of immunotherapy and can improve the patient response rate. Furthermore, this specificity in the target and related pathways reduces damage to normal tissues.

Background Art

[0002] Despite significant progress in oncology, cancer remains a major cause of death worldwide. In 2020, it accounted for nearly 10 million deaths, i.e., nearly 17% of the world's deaths. The cancers newly occurring in 2020 were breast cancer (2.26 million cases), lung cancer (2.21 million cases), and colorectal cancer (1.93 million cases). Regarding mortality, lung (1.8 million deaths), colorectal (916,000 deaths), and liver cancer (830,000 deaths) are the major cancer types. Breast cancer is also a global problem, with 685,000 deaths annually. Cancer is an abnormal growth mass composed of different cell types. Cancer cells form an ecosystem, the tumor microenvironment (TME) or stroma, i.e., other cell types and acellular matrix that can exert pre-tumor or anti-tumor effects. The TME is a dynamic and complex tissue formed by the chemical and physical interactions between the cells of the tumor and the cells of adjacent structures. It includes elements of the vascular system, immune cells [e.g., T lymphocytes and B lymphocytes, natural killer (NK) cells and macrophages], and stromal cells (including pericytes and fibroblasts), as well as the acellular extracellular matrix (ECM). The ECM functions as a scaffold for cells in the TME: it provides structural support and regulates cell motility, proliferation, and differentiation. It consists of a network of fibrous proteins, glycoproteins, proteoglycans, and polysaccharides with diverse physical and biochemical properties and is also rich in growth factors.

[0003] The tumor microenvironment (TME) is an essential and dynamic compartment that profoundly affects all stages of tumor progression and treatment response. In a healthy state, the TME can protect against and help reduce the invasiveness of tumor cells. However, the crosstalk between tumor cells and components of the TME can lead to the opposite reaction that promotes tumorigenesis, growth, drug resistance, immunosuppression, and metastasis. Therefore, recent insights in the field of oncology are that the cancer cell-centered treatment paradigm is generally not sufficient to eradicate malignancies. To improve cancer survival rates, there is an urgent unmet need for various therapeutic approaches that target the various cell types and processes that are drivers of pre-tumor events in the TME.

[0004] Among the stromal cells that occupy the tumor microenvironment, cancer-associated fibroblasts (CAFs) are the most abundant and critically involved in cancer progression. They include various fibroblast populations of different cell origins (i.e., pro-tumoral CAFs) that are activated during tumor formation. CAFs regulate the biology of tumor cells and other stromal cells via cell-cell contact, releasing a number of regulatory factors (e.g., transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), and CXC-chemokine ligand (CXCL12)), as well as synthesizing and remodeling the extracellular matrix. CAFs are drivers of pro-tumoral events such as growth, drug resistance, angiogenesis, immunosuppression, and metastasis.

[0005] The extracellular matrix (ECM) is a non-cellular component of the TME. Collagen is the most important constituent of the ECM, and its cross-linking is mainly mediated by lysyl oxidase (LOX), an amine oxidase that is frequently overexpressed in cancer. The expression of matrix metalloproteinases (MMPs) is also upregulated. These enzymes proteolytically degrade ECM components that are important in the regulation of ECM composition and enable the release of ECM-bound soluble factors (e.g., growth factors).

[0006] In addition to being a supportive structure, the ECM is also a dynamic compartment involved in promoting the malignant phenotype of cancer cells. Increased deposition of structural components (e.g., collagen, fibronectin, and proteoglycans) by CAFs and secretion of crosslinking enzymes (e.g., transglutaminase (TGM) and lysyl oxidase (LOX)) cause a denser, stiffer TME and tumor formation in a process called fibrosis. The physiological stiffness of natural tissues varies from 0.1 kPa in brain tissue to 40 kPa in bone. In cancer patients, stromal stiffness is approximately 5 kPa in invasive regions compared to 400 Pa in normal non-invasive stroma. Higher tumor stiffness correlates with the amount of fibroblasts in the stroma, as well as the stage of cancer progression, treatment resistance, immunosuppression, higher metastasis rates, and poor patient prognosis. Currently, fibroblast modulators are under investigation as potential therapeutic options for tumors with significant fibroblast presence (CAFs). An essential requirement for any CAF modulator to have practical therapeutic utility in human cancer patients is the pharmacological selectivity of CAFs with respect to fibroblasts and other relevant cell types involved in normal physiological functions. Due to this required pharmacological selectivity, the development of tumor therapies targeting CAFs faces significant obstacles. Specificity in the target and related pathways is low, so targeting CAFs without damaging normal tissues remains an unsolved problem in the field.

Prior Art Documents

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 9

Non-Patent Document 10

SUMMARY OF THE INVENTION

PROBLEM TO BE SOLVED BY THE INVENTION

[0008] An object of the present invention is to provide a compound for selectively targeting cancer-associated fibroblasts (CAFs) and thereby improving the therapeutic outcome of cancer treatment.

MEANS FOR SOLVING THE PROBLEM

[0009] Accordingly, in a first aspect, the present invention provides a compound of formula I or a salt, hydrate or solvate thereof for use in the treatment of invasive lobular carcinoma, tumors having a tumor-stroma ratio (TSR) > 50%; and / or pancreatic ductal adenocarcinoma:

CHEMICAL

CHEMICAL

Chemical formula

[0010] In certain embodiments, the compounds for use as defined herein are: R 2 and R 3 When forming a 6-membered aromatic or non-aromatic heterocyclic ring, the compound is of Formula II:

Chemical formula

[0011] In another particular embodiment, the compounds for use as defined herein, in particular compounds of Formula I or II, are:

Chemical formula

Chemical formula

[0012] In a further specific embodiment, the compounds for use as defined herein are:

Chemical formula

Chemical formula

[0013] In a further embodiment, the present invention provides the following compounds, in particular compounds of formula I or II, for use as defined herein:

Chemical formula

Chemical formula

[0014] In a further embodiment, the present invention provides the following compounds, in particular compounds of formula I or II, for use as defined herein: said R 1 , R 1 ' and R 1 " each is -H and -OCH3 Selected independently from the group consisting of; R 2 is -H; R 3 is -H; or, R 2 and R 3 together with the C atom to which they are attached form a 6-membered aromatic or non-aromatic heterocycle, R 4 is -H; Said R 5 , R 5 ' and R 5 " each is independently selected from the group consisting of -H, -OH, -F and -Cl; In the formula, said R 1 , R 1 ' and R 1 " at least one of which is -OCH 3 ; In the formula, when R 2 and R 3 do not form a heterocycle,

Chemical formula

[0015] The compounds for use as defined herein may, in certain embodiments, be represented by any one of formulas (Ia), (IIa) or (IIb):

Chemical formula

[0016] The present invention also provides a compound for use as defined herein, wherein the compound, or a salt, hydrate or solvate thereof, is shown in the following list:

Chemical formula

Chemical formula

Chemical formula

[0017] In another aspect, the present invention provides a compound shown in the following list or a salt, hydrate or solvate thereof: [Chemical formula]

[0018] The present invention also provides a pharmaceutical composition comprising a compound defined herein and a pharmaceutically acceptable excipient. In a further embodiment, the present invention provides a compound or composition defined herein for use in human or veterinary medicine; in particular for use in the treatment of cancer; more specifically for use in preventing, treating or reducing tumor-related desmoplasia in a subject having cancer; even more specifically invasive lobular breast cancer, tumors having a tumor-stroma ratio (TSR)>50%; and / or for use in the treatment of pancreatic ductal adenocarcinoma, a compound or composition is provided.

[0019] The present invention also provides a method for preventing, reducing and / or treating tumor-related fibrosis in a subject having cancer, for treating specific invasive lobular breast cancer, tumors having a tumor-stroma ratio (TSR)>50%; and / or for preventing or treating pancreatic ductal adenocarcinoma, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound or pharmaceutical composition defined herein.

[0020] In still a further aspect, the present invention provides a compound for use as an anti-invasive compound for tumor cells, the compound being represented by any one of formula (IIa) or (IIb); [Chemical formula] wherein, said R 1 , R 1 ' and R 1 " each is -H, -halo, -CF3 、 -OCF 3 and -OC 1-3 independently selected from the group consisting of alkyl; R 4 is -H, -halo, -CF 3 、 -OCF 3 and -OC 1-3 selected from the group consisting of alkyl; said R 5 、 R 5 ', and R 5 " each is -H, -OH, -CN, -C 1-3 alkyl, -OC 1-3 alkyl, -NR 6 R 7 、 -SO 2 -NH 2 、 -SO 2 -iPr, -Het 1 and -halo independently selected from the group; the -C 1-3 alkyl and -OC 1-3 alkyl each is =O, -NR 8 R 9 and -Het 1 selected from the group consisting of 1 to 3 substituents, optionally substituted; R 6 , R 7 , R 8 and R 9 is selected from the group consisting of -H and -C 1-3 alkyl; each of the -C 1-3 alkyl is selected from the group consisting of =O, -NH 2 、 and -NH-iPr 1 to 3 substituents, optionally substituted, Het 1 is a 5- or 6-membered heterocycle having 1 to 3 heteroatoms selected from S, O, and N, wherein said Het 1 each is -C 1-3 alkyl, -OC 1-3 alkyl, -C 1-3 alkyl-OH, and -OC 1-3 alkyl-OH selected from the group consisting of 1 to 3 substituents, optionally substituted; In the formula, said R1 , R 1 ', and R 1 " at least one of which is -OC 1-3 alkyl or -CF 3 .

Brief Description of the Drawings

[0021] Referring particularly to the drawings, it should be noted that the details shown are, by way of example, for the purpose of an illustrative description of different embodiments of the present invention. The description using the drawings will clarify to those skilled in the art how some forms of the present invention can actually be implemented.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Mode for Carrying Out the Invention

[0022] Next, the present invention will be further described. In the following sections, different aspects of the present invention are defined in more detail. Each aspect so defined can be combined with any other aspect, unless clearly indicated otherwise. As used in this specification and the claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, "compound" means one compound or two or more compounds. Throughout this specification and the claims, the word "comprise" and other forms of the word, such as "comprising" and "comprises", are not limiting, but are means that include, for example, other additives, components, integers, or steps, and are not intended to exclude. As used herein, "about", "approximately", "substantially", and "significantly" are understood by those skilled in the art and vary to some extent in the context in which they are used. Where there is use of terms not obvious to those skilled in the art considering the context in which they are used, "about" and "approximately" mean plus or minus 10% of a particular value or term. The above and other terms used herein are well understood by those skilled in the art. All references and teachings specifically mentioned herein are hereby incorporated by reference in their entirety into this specification.

[0023] The present invention provides CAF (cancer-associated fibroblast) modulating compounds, more specifically their use in the treatment of cancer, more specifically their use in reducing the stiffness of the tumor microenvironment (TME) (also called desmoplasia).

[0024] Fibroblasts are one of the most abundant cell types in the stroma and are often defined by a combination of their morphology, tissue location, and lack of lineage markers for epithelial cells, endothelial cells, and white blood cells.

[0025] Similar to fibroblasts in the stroma, cancer-associated fibroblasts (CAFs) are a major component of the tumor microenvironment (TME). Many of the characteristics defined for normal fibroblasts also apply to CAFs. The term "cancer-associated fibroblasts" is used as an umbrella term to describe a complex group of mesenchymal-like cells. Han et al. (Han, Liu, and Yin 2020) even consider it a general marker for "all fibroblasts found within and around the tumor tissue". After their spindle-shaped morphology, the simplest characterization is a list of negative markers. CAFs are cells that are negative for epithelial, endothelial, and leukocyte markers, as well as all mutations found within cancer cells. The latter point is important as it excludes cancer cells that have undergone epithelial-mesenchymal transition (EMT). To narrow the interpretation, lineage exclusion is typically combined with the presence of mesenchymal markers, often vimentin. Subtypes of CAFs with their respective specific markers have been shown to exist. In practice, any mesenchymal cell derived from a tumor that meets the above criteria is considered a CAF.

[0026] The compounds of the present invention are unique in that they are selective modulators of CAFs and thereby do not target or target less fibroblasts and other related cell types involved in normal physiology. Thus, the compounds of the present invention can be used to target CAFs without damaging normal tissue. The compounds provided herein have been shown to functionally inhibit CAFs at nanomolar concentrations that are highly desirable for active pharmaceutical ingredients.

[0027] Increases in tumor and ECM stiffness are well established to be associated with a negative prognosis as they promote tumor growth, progression, treatment resistance, immunosuppression, and metastasis. Stiffness is mainly regulated by the action of CAFs in the TME. Thus, stiffness modulation is an appropriate read-out of activity in screening for CAF modulators. To evaluate stiffness modulation, the stiffness of spheroids of CAF cells treated with test compounds was examined using atomic force microscope (AFM) indentation and compared to untreated cells. AFM measurement of the stiffness (elasticity) of cells and spheroids is a technique known to those skilled in the art. AFM measures the force required to generate a given deflection, and this read-out is also known as the Young's modulus. AFM is widely used in biology and cancer research: the altered physicochemical properties of living cells serve as read-outs of complex physiological processes that have changed.

[0028] The selectivity of CAFs for normal fibroblasts and other cell types was evaluated by performing the same experiments using normal fibroblasts and other cell types, where compounds were considered selective for CAFs if they only modulated CAF stiffness. The tumor stroma can limit access of therapeutic agents to the target tissue in three ways: fibrosis, high interstitial pressure, and degradation of drugs by stromal enzymes. Accumulation of a rigid ECM (fibrosis) around and throughout the tumor creates a physical barrier that reduces the diffusion of therapeutic agents to cancer cells. Similarly, the tumor stroma can limit the access and action of immune cells in two ways: by creating a physical barrier that reduces infiltration of immune cells, fibrosis, and by providing chemical and physical cues that are preferential for pre-tumor-immunosuppressive immune cell populations and induce polarization or differentiation of immune cells towards an immunosuppressive phenotype.

[0029] The present invention provides a compound as defined herein for use in treating cancer, in particular cancer having a high and / or high density stromal content, by reducing said rigid ECM and immunosuppressive cues / polarization / differentiation. In cancers where the quantitative content of fibroblasts can vary between patients, a convenient way to determine the amount of fibroblasts is to determine the amount of fibroblasts producing the components of fibroblasts. For example, cancers having a tumor-stromal ratio (TSR) of 40% or more, particularly 50% or more, particularly 60% or more, and even more particularly 70% or more are gastric cancer, colorectal cancer, cervical cancer, triple negative breast cancer, head and neck cancer, non-small cell lung cancer, liver cancer, glioblastoma and esophageal cancer. In these cancers, a higher stromal content, i.e., a higher TSR, correlates with the amount of cancer-associated fibroblasts (CAFs) and a worse prognosis, and thus identifies a population suitable for fibroblast targeted therapy. As used herein, a "tumor having a specific tumor-stromal ratio (TSR)" refers to a cancer having a high stromal content, in particular a cancer selected from the group consisting of gastric cancer, colorectal cancer, cervical cancer, triple negative breast cancer, head and neck cancer, non-small cell lung cancer, liver cancer, glioblastoma and esophageal cancer.

[0030] The amount of stroma in a patient's tumor can be easily evaluated by those skilled in the art for hematoxylin and eosin (HE) stained sections, and such evaluation is clinically applicable (Wu et al., 2016). The tumor-stroma ratio (TSR) is defined as the ratio of stroma to tumor cells and is recognized as a prognostic factor for various solid tumors. For example, a TSR score of 20% corresponds to a section in which 20% of the analyzed area consists of stroma and 80% of the area consists of tumor cells. Therefore, the analysis of TSR can be easily replicated and analyzed routinely. TSR has been shown to be a robust parameter validated by an international research group and having good inter-observer agreement. To make TSR a current guideline for clinical implementation, the UNITED study (Unified Notification for International Application of Tumor-Stroma Ratio as a Simple Diagnostic Tool) has been developed. More specifically, TSR is determined from the invasive part of the tumor where most of the stroma is present, and the area with a large amount of stroma is decisive for prognostic diagnosis. As described in detail in Kemi et al. 2018, the part of the tumor with the most stroma is identified using a low magnification (total magnification 10 - 50 times). A single field of view at a total magnification of 100x is used for analysis with tumor cells present at all four corners of the field of view. Smooth muscle, mucin, and necrosis are excluded from the analysis. The remaining area consists of tumor cells and stroma and is used as the area under analysis. The area of stroma is estimated compared to the total area under analysis and scored at 10% intervals using human evaluation or computer-assisted analysis. As described above, a score of 20% means that 20% of the area under analysis consists of stroma and 80% consists of tumor cells.

[0031] When using TSR during the diagnostic and treatment decision-making, a ratio of 50% is defined as the cut-off point. Patients are divided into stroma-poor cases (stroma ≤ 50% ratio) and stroma-rich cases (stroma > 50% ratio). The HE-stained TSR class has been shown to distinguish stroma-rich high-risk patients (TSR > 50%) in gastric cancer, colorectal cancer, cervical cancer, triple-negative breast cancer, head and neck cancer, non-small cell lung cancer, liver cancer, glioblastoma, and esophageal cancer. From the previous paragraph, in these cancers, a TSR > 50% indicates patients with fibroblast-driven tumor progression, and thus represents a target population for treatment with the compounds of the present invention.

[0032] In addition to the above list of cancers, there are other cancer types with abundant CAFs regardless of the TSR status (protumoral). These cancer types are pancreatic ductal adenocarcinoma (PDAC) and invasive lobular breast cancer (ILC). PDAC and ILC tumors show an increase in the levels of CAF markers such as platelet-derived growth factor receptor-β, α-smooth muscle actin, fibroblast activation protein-α, and fibroblast-specific protein 1 / S100A4. Therefore, patients presenting with these cancers form a target population for treatment with the compounds of the present invention.

[0033] An essential requirement for any CAF modulator to have practical therapeutic use in human cancer patients is the pharmacological selectivity of CAF with respect to fibroblasts and other related cell types involved in normal physiological functions. Examples of such related cell types are smooth muscle cells. Cancer-associated fibroblasts share most of the drug pathways and markers with normal fibroblasts and these related cell types. Normal fibroblasts serve as structural components and play an important role in wound healing. They are important in the immune response to tissue injury: fibroblasts from different anatomical sites in the body express many genes encoding immune mediators and proteins. Smooth muscle cells substantially contribute to the regulation of blood pressure and blood flow to the vascular bed, and the phasic contractions of the gastrointestinal tract.

[0034] Accordingly, in a first aspect, the present invention provides a compound of formula I or a salt, hydrate or solvate thereof for use in the treatment of invasive lobular breast cancer, tumors having a tumor-stroma ratio (TSR) > 50%; and / or pancreatic ductal adenocarcinoma;

Chemical formula

Chemical formula

Chemical formula

[0035] The term "alkyl", by itself or as part of another substituent, has the formula C x H 2x+1(wherein x is a number of 1 or more) refers to a completely saturated hydrocarbon. Generally, the alkyl group of the present invention contains 1 to 3 carbon atoms. The alkyl group may be linear or branched and may be substituted as shown herein. When a subscript is used herein after a carbon atom, the subscript refers to the number of carbon atoms that the specified group may contain. C 1-3 Alkyl includes all linear, branched or cyclic alkyl groups having 1 to 3 carbon atoms and thus includes methyl, ethyl, n-propyl, c-propyl and i-propyl.

[0036] The term "optionally substituted alkyl" or "optionally, substituted alkyl" refers to an alkyl group or an optionally substituted alkyl group that may be substituted at any available bond with one or more substituents (e.g., 1 to 3 substituents, e.g., 1, 2, or 3 substituents, or 1 to 2 substituents). Non-limiting examples of such substituents include oxo, amide, amine, aromatic and non-aromatic heterocycles, etc.

[0037] The term "halo" or "halogen" as a group or part of a group is a general term for fluoro, chloro, bromo or iodo. In certain embodiments, halo is fluoro or chloro; preferably fluoro.

[0038] The term "heterocyclyl", "heterosyl" or "heterocyclo" when used herein alone or as part of another group refers to an aromatic or non-aromatic cyclic group (e.g., a 5- to 6-membered monocyclic ring system) having 1 to 3, e.g., 1, 2 or 3 heteroatoms in a carbon atom-containing ring. The heteroatom may be selected from nitrogen, oxygen and / or sulfur atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. In certain embodiments, the heteroatom is selected from N and O atoms. The heterocyclic group may be bonded to any heteroatom or carbon atom of the ring that the valence permits. Optionally, a substituted or optionally substituted heterocyclic ring refers to a -C 1-3 alkyl, -OC 1-3 alkyl, -C 1-3 alkyl-OH and -OC 1-3 alkyl-OH, and refers to a heterocyclic ring which may have one or more substituents (e.g., 1 to 3 substituents, or, for example, 1, 2, or 3). Exemplary heterocyclic groups include piperazinyl and morpholinyl.

[0039] In the context of the present invention, when R 2 and R 3 together with the C atom to which they are attached form a 6-membered aromatic or non-aromatic heterocyclic ring, this results in the formation of a compound defined by any one of formula (IIa) or (IIb):

Chemical formula

[0040] As used herein, the term "oxo" refers to the group =O. As used herein, the term "alkoxy" or "alkyloxy" refers to a radical of the formula -OR b (wherein R b is alkyl). Preferably, the alkoxy is C 1~3 alkoxy, C 1~2 alkoxy, or methoxy. Non-limiting examples of suitable alkoxys include methoxy, ethoxy, propoxy, or isopropoxy.

[0041] The term "solvate" refers to an aggregate of a compound and one or more solvent molecules. As used herein, "hydrate" is a compound formed by hydration, i.e., the addition of water or the molecular entity of the elements of water (i.e., H and OH). The compounds of the present invention can further be provided in the form of pharmaceutically acceptable salts. As used herein, the term pharmaceutically acceptable salt or complex refers to a suitable salt or complex of an active compound according to the present invention that retains the desired biological activity of the parent compound and exhibits a limited toxic effect on normal cells. Non-limiting examples of such salts are acid addition salts formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, etc.), and salts formed with organic acids such as, inter alia, acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, and polyglutamic acid.

[0042] The compounds provided herein can be prepared using readily available starting materials, using techniques available in the art, and using reaction pathways and synthetic schemes as described below.

[0043] In certain embodiments, the present invention provides a compound of formula I or a salt, hydrate or solvate thereof for use in the treatment of invasive lobular carcinoma, tumors having a tumor-stroma ratio (TSR) > 50%; and / or pancreatic ductal adenocarcinoma;

Chemical formula

Chemical formula

Chemical formula

[0044] In certain embodiments, the compounds for use as defined herein are defined as follows: R 2 and R 3 when forming a 6-membered aromatic or non-aromatic heterocyclic ring, the compound is represented by formula II;

Chemical formula

[0045] In another specific embodiment, the compounds for use as defined herein are of formula I or II:

Chemical formula

Chemical Formula

[0046] In a further specific embodiment, for use as defined herein, in particular the compounds of formula I or II are as follows: [Chemical formula] represents a single bond or a double bond; said R 1 , R 1 ' and R 1 " each independently is selected from the group consisting of -H, -CF 3 and -OCH 3 ; R 2 is -H; R 3 is -H; or, R 2 and R 3 together with the C atom to which they are attached form a 6-membered aromatic or non-aromatic heterocycle; R 4 is -H; said R 5 , R 5 ' and R 5 " each independently is selected from the group consisting of -H, -OH, -C 1-2 alkyl, -NR 6 R 7 , -SO 2 -NH 2 , -Het 1 and -halo; each of said -C 1-2 alkyl is optionally substituted with 1 to 3 substituents selected from the group consisting of =O and -Het 1 ; R 6 and R 7 are selected from the group consisting of -H and -C 1-2 alkyl; each of said -C 1-2 alkyl is optionally substituted with 1 to 3 substituents selected from the group consisting of =O and -NH-iPr; Het 1 is selected from morpholinyl and piperazinyl; each of said Het 1 is -C 1-2 alkyl and -C 1-2Optionally substituted with 1 to 3 substituents selected from the group consisting of alkyl-OH; In the formula, the R 1 , R 1 ', and R 1 " at least one of which is -OCH 3 ; In the formula, when R 2 and R 3 do not form a heterocyclic ring,

Chemical formula

[0047] In a further embodiment, the present invention provides a compound of formula I or II, in particular, for use as defined herein:

Chemical formula

[0048] In a further embodiment, the present invention provides a compound of formula I or II for use as defined herein: Said R 1 , R 1 ' and R 1 " are each independently selected from the group consisting of -H and -OCH 3 ; R 2 is -H; R 3 is -H; or, R 2 and R 3 together with the C atom to which they are attached form a 6-membered aromatic or non-aromatic heterocyclic ring, R 4 is -H; Said R 5 , R 5 ' and R 5 " are each independently selected from the group consisting of -H, -OH, -F and -Cl; wherein at least one of said R 1 , R 1 ' and R 1 " is -OCH 3 ; wherein when R 2 and R 3 do not form a heterocyclic ring, [Chemical formula] represents a double bond.

[0049] Furthermore, it has been found that the combination of one or more methoxy groups on one aromatic ring of the molecule and one or more halogens, with or without a hydroxyl group, on another aromatic ring of the molecule is advantageous for the desired activity. Accordingly, the present invention provides a compound of formula I or II for use as defined herein: wherein, The R 1 , R 1 ', and R 1 " each is independently selected from the group consisting of -H and -OCH 3 ; R 2 is -H; R 3 is -H; or, R 2 and R 3 together with the C atom to which they are attached form a 6-membered aromatic or non-aromatic heterocyclic ring; R 4 is -H; The R 5 , R 5 ', and R 5 " each is independently selected from the group consisting of -H, -OH, -F, and -Cl; wherein at least one of the R 1 , R 1 ', and R 1 " is -OCH 3 ; R5 is -OH; wherein, when R 2 and R 3 do not form a heterocyclic ring,

Chemical formula

[0050] For use as defined herein, the compounds in certain embodiments can be represented by any one of formulas (Ia), (IIa), or (IIb), each containing R-groups and substituents as defined herein for formulas I and II.

Chemical formula

[0051] The present invention also provides a compound for use as defined herein, or a salt, hydrate, or solvate thereof, wherein the compound is included in the following list:

Chemical formula

Chem.

Chem.

Chem.

[0052] In another aspect, the present invention provides a compound, or a salt, hydrate, or solvate thereof, wherein the compound is included in the following list:

Chem.

Chem.

Chem.

Chem.

[0053] In a very specific embodiment, the present invention provides a compound, or a salt, hydrate, or solvate thereof, wherein the compound is included in the following list:

Chem.

[0054] For use of a compound of the formula provided herein for the therapeutic treatment (including prophylactic treatment) of a subject such as an animal or a human, it is usually formulated as a pharmaceutical composition according to standard pharmaceutical practices. Accordingly, the present invention also provides a pharmaceutical composition comprising a compound as defined herein and a pharmaceutically acceptable excipient.

[0055] The compounds of the present invention can be incorporated into formulations for all routes of administration, including, for example, oral, topical and parenteral (intravenous, intramuscular, ocular or including the eye), intraperitoneal, buccal, transdermal and suppository forms. Generally, it is preferred to administer the pharmaceutical composition in an orally administrable form, but for the treatment of many conditions, many other formulations can be administered via other routes including topical, parenteral, intravenous, intramuscular, transdermal, buccal, subcutaneous, suppository or other routes including the ocular or ophthalmic route. Intravenous and intramuscular formulations are preferably administered in sterile saline. Minor modifications of the compounds to render them soluble in water or other vehicles can be readily achieved, for example, by minor modifications well known to those skilled in the art (salt formulations, esterification, etc.). It is also well within the skill of the art to vary the route of administration and the method of administration of a particular compound in order to manage the pharmacokinetics of the compound for maximum beneficial effect on the patient.

[0056] The composition of the present invention can be in a form suitable for oral use (e.g., tablets, troches, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), topical use (e.g., as creams, ointments, gels, or aqueous or oily solutions or suspensions), administration by inhalation (e.g., as micronized powders or liquid aerosols), administration by insufflation (e.g., as micronized powders), or injection (e.g., as sterile aqueous or oily solutions for intravenous, subcutaneous, or intramuscular administration, or as suppositories for rectal administration). For example, a composition intended for oral use may contain, for example, one or more colorants, sweeteners, flavoring agents and / or preservatives. Pharmaceutically acceptable excipients suitable for tablet formulations include, for example, inert diluents such as lactose, sodium carbonate, calcium phosphate or calcium carbonate, granulating and disintegrating agents such as corn starch or alginic acid; binders such as starch; lubricants such as magnesium stearate, stearic acid or talc; preservatives such as ethyl p-hydroxybenzoate or propyl p-hydroxybenzoate, and antioxidants such as ascorbic acid. Tablet formulations may or may not be coated, using conventional coating agents and procedures well known in the art, to modify their disintegration and subsequent absorption of the active ingredient in the gastrointestinal tract, or to improve their stability and / or appearance.

[0057] An aqueous suspension generally contains one or more suspending agents such as sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, sodium alginate, polyvinyl pyrrolidone, tragacanth and gum acacia; a dispersant or wetting agent such as lecithin or a condensation product of an alkylene oxide and a fatty acid (for example, polyoxyethylene stearate), or a condensation product of ethylene oxide and a long-chain aliphatic alcohol, such as heptadecaethyleneoxy cetanol, or a condensation product of ethylene oxide and a partial ester derived from a fatty acid and a hexitol, such as polyoxyethylene sorbitol monooleate, or a condensation product of ethylene oxide and a partial ester derived from a fatty acid and hexitol anhydride, such as polyethylene sorbitan monooleate. The aqueous suspension may also contain one or more preservatives (for example, ethyl or propyl - p-hydroxybenzoate), antioxidants (for example, ascorbic acid), coloring agents, flavoring agents, and / or sweetening agents (for example, sucrose, saccharin or aspartame).

[0058] Additional excipients such as sweetening agents, flavoring agents and coloring agents may also be present in the pharmaceutical composition.

[0059] The pharmaceutical composition of the present invention may be in the form of an oil-in-water emulsion. The pharmaceutical composition may also be in the form of a sterile injectable aqueous solution or an oily suspension, which can be formulated according to known procedures using one or more suitable dispersants or wetting agents and suspending agents. The sterile injectable preparation may be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent.

[0060] For example, a formulation for oral administration to humans can contain, for example, from 0.5 mg to 2 g of the compound of the present invention together with a suitable and convenient amount of excipient, which can vary from about 5 to about 98% by weight of the total composition. Dosage unit forms generally contain from about 1 mg to about 500 mg of the active compound. Appropriate information regarding the route of administration and dosing schedule is shown, for example, in Chapter 25.3 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990, the content of which is incorporated herein by reference.

[0061] The magnitude of the dosage for the therapeutic or prophylactic purposes of the compounds of the formulas presented herein will, of course, vary according to well-known principles of medicine according to the nature and severity of the condition, the age and sex of the animal or patient, and the route of administration.

[0062] In a further embodiment, the present invention provides a compound or composition as defined herein; for use in human or veterinary medicine; particularly for use in the treatment of cancer; more specifically, for use in the prevention, treatment or reduction of tumor-associated fibrosis; even more specifically, infiltrating lobular carcinoma, tumors having a tumor-stroma ratio (TSR) > 50%; and / or for use in the treatment of pancreatic ductal adenocarcinoma, a compound or composition.

[0063] The present invention also provides a method for the prevention and / or treatment of cancer; more specifically for use in the prevention, treatment or reduction of tumor-associated fibrosis; even more specifically, infiltrating lobular carcinoma, tumors having a tumor-stroma ratio (TSR) > 50%; and / or for the treatment of pancreatic ductal adenocarcinoma, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound or pharmaceutical composition as defined herein.

[0064] In a further embodiment, the compounds of the invention are of particular interest for use in combination therapy for cancer, in particular in combination with a cytotoxic agent or an immunomodulatory agent, in particular an immunocyte activator such as an immune checkpoint inhibitor (e.g., a PD1 or PDL1 pathway inhibitor; in particular an anti-PD1 or anti-PDL1 antibody). The combination therapy can be administered to a patient either separately from or simultaneously with the compounds of the invention, whichever is most appropriate.

[0065] In another embodiment, it has been demonstrated herein that the compounds of Formulas IIa and IIb exhibit anti-invasive activity, and more specifically, these compounds inhibit the invasion of tumor cells into the surrounding tissue, thereby reducing metastasis (the risk of metastasis).

[0066] Accordingly, in a further aspect, the present invention provides a compound for use as an anti-invasive compound against tumor cells, the compound being represented by any one of the following Formulas IIa or IIb;

Chemical Formula

[0067] Cancer spreads from the tissue of its origin, and subsequent growth in other organs is the most life-threatening aspect of this disease. This process, called metastasis, requires cancer cells to survive and proliferate outside the tissue of their origin. The first important step in this process is the invasion of cancer cells into the tissue surrounding the tumor and the vasculature.

[0068] The compounds of the present invention can inhibit the first step, thereby preventing and / or reducing the seeding and metastasis formation of cancer cells. Accordingly, the present invention relates to the compounds of Formulas IIa and IIb described herein for use in the treatment of cancer by inhibiting the invasion of tumor cells into surrounding or adjacent tissues, cells and / or their entry into the circulatory system. Accordingly, the compounds of the present invention are particularly useful for inhibiting or preventing the spread of tumors.

[0069] In certain embodiments, the compounds of formula IIa and IIb of the invention are used as anti-invasive compounds. "Anti-invasive" refers to the ability to inhibit the invasion of tumor or cancer cells into the surrounding tissue (inside or outside the organ of origin). To determine whether a compound exhibits anti-invasive behavior, the chicken heart invasion assay as provided herein or as described by Bracke et al., 2014 can be applied. This screening method pits fragments of normal tissue against cancer cells so as not to ignore the contribution of the host tissue in the microecosystem that governs tumor behavior. The interaction between cancer cells and normal tissue is histologically evaluated and classified along a subjective scale of 5 degrees of malignancy. Degrees of malignancy III and IV are typical of invasion, while degrees of malignancy 0, I, and II correspond to the absence of invasion. A compound that inhibits the invasion of the confronting cancer cells, i.e., a compound that inhibits invasion from III / IV to 0 / I / II, is determined to be anti-invasive. Possible alternative assays generally known in the art are the type I collagen invasion assay and the Matrigel invasion assay.

Example

[0070] Materials and Methods 1 Synthetic Procedures and Physicochemical Properties Reaction Procedures for Chalcones Synthesis General Protocol: Chalcones Synthesis A solution of the appropriate acetophenone (10 mmol) and LiOH·H 2 O (10 mol%) in 10 mL of anhydrous ethanol is stirred for 10 minutes at the appropriate temperature (for reactions at 40 °C, the valve is equipped with a reflux condenser). Then, the appropriate benzaldehyde (10 mmol, 1 equivalent) is added and the system is protected from the atmosphere with a cork stopper. The progress of the reaction is monitored (LCMS). During the reaction, chalcone may precipitate. Once the maximum conversion grade is reached, the reaction mixture is quenched with 15 mL of 1% hydrochloric acid.

[0071] When chalcones precipitate, they are isolated by filtration means. To remove the residual amount of benzaldehyde, the residue is thoroughly washed with water until the filtrate becomes clear. Chalcones can be crystallized in absolute ethanol to obtain high-purity crystals.

[0072] When chalcones form a separate oily liquid at the bottom of the valve, the chalcones can be extracted from the mixture with diethyl ether. Then, the organic phase is washed with brine (2x), dried over MgSO 4 and concentrated in vacuo. Purification of the residue thus obtained can be carried out by crystallization in absolute ethanol.

[0073] 3-(4-Fluorophenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one A1 This substance was prepared according to the general protocol described above.

Chemical Structure

[0074] 3-(4-Chlorophenyl)-1-(4-methoxyphenyl)prop-2-en-1-one A2 This substance was prepared according to the above general protocol.

Chem.

[0075] 3-(4-Fluorophenyl)-1-phenylprop-2-en-1-one A3 This substance was prepared according to the above general protocol.

Chemical formula

[0076] 1-(4-Fluorophenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one A4 This substance was prepared according to the above general protocol.

Chemical formula

[0077] 3-(4-Fluorophenyl)-1-(4-methoxyphenyl)prop-2-en-1-one A5 This substance was prepared according to the above general protocol.

Chemical formula

[0078] 3-(Phenyl)-1-(4-methoxyphenyl)prop-2-en-1-one A6 This substance was prepared according to the above general protocol. [Chemical formula] 1 H NMR (CDCl 3 , 300 MHz, ppm): δ = 3.86 (s, 3H), 6.98 (d, 2H, J = 8.5 Hz), 7.33 - 7.43 (m, 3H), 7.52 (d, 1H, J = 15.8 Hz), 7.63 (dd, 2H, J = 7.4 Hz, J = 3.5 Hz), 7.81 (d, 1H, J = 15.8 Hz), 8.04 (d, 2H, J = 8.5 Hz); 13 C NMR (CDCl 3 , 75 MHz, ppm): δ = 55.3, 113.7, 121.8, 128.1, 128.7, 130.1, 130.6, 130.9, 134.9, 143.7, 163.3, 188.5. Yield: 88%, white needles (from EtOH); MW = 238.29.

[0079] 3 - Phenyl - 1 - (3,4,5 - trimethoxyphenyl)prop - 2 - en - 1 - one A7 This substance was prepared according to the above general protocol. [Chemical formula] 1 H NMR (CDCl 3 , 300 MHz, ppm): δ = 3.93 (s, 3H), 3.94 (s, 6H), 7.28 (s, 2H), 7.36 - 7.50 (m, 3H), 7.48 (d, 1H, J = 15.8 Hz), 7.58 - 7.72 (m, 2H), 7.81 (d, 1H, J = 15.8 Hz); 13 C NMR (CDCl 3, 75 MHz, ppm): δ = 56.4, 61.0, 106.1, 121.8, 128.4, 129.0, 130.6, 133.5, 134.9, 142.5, 144.9, 153.2, 189.2. Yield: 89%, yellow needles (from EtOH); MW = 298.34.

[0080] 1,3-Diphenylprop-2-en-1-one A8 This substance was prepared according to the above general protocol.

Chem.

[0081] 3-(4-Fluorophenyl)-1-(4-methoxy-3-(trifluoromethyl)phenyl)prop-2-en-1-one A14 This substance was prepared according to the above general protocol.

Chem.

[0082] Synthesis of Chalcone A9

Chem.

[0083] Scheme 1. Synthesis of Chalcone A9: Reagents and Conditions: a) 2 equiv DIPEA, 1.5 equiv MEMCl, dry CH 2 Cl 2 , THF, N 2 , 0 °C to room temperature for 24 h; b) 1 equiv 3',4',5'-trimethoxyacetophenone, 0.5 equiv LiOH·H 2 O, abs. EtOH, N 2 , room temperature, 26 h; c) 5% aq. M HCl, THF, reflux, 6 h.

[0084] The synthesis of chalcone A9 is shown in Scheme 1. A flame-dried round-bottom flask equipped with a stir bar and maintained under a nitrogen atmosphere is charged with 2-hydroxy-4-fluorobenzaldehyde 1 (4.59 mmol), dry CH 2 Cl 2 (45 mL) and dry THF (3 mL). Benzaldehyde does not dissolve in CH 2 Cl 2 but dissolves in THF. The solution is cooled to 0 °C in an ice bath, and then 2 equiv of DIPEA (9.18 mmol) and 1.5 equiv of MEMCl are added. The ice bath is removed, and the mixture is stirred at room temperature for 24 h. After completion of the reaction, the mixture is poured into aq. qat. NaHCO 3Quench by addition (3 portions of 6 mL). Extract the organics with EtOAc (2 x 6 mL), then wash the combined organic phases with 1N NaOH (20 mL) and water (20 mL). Dry the organic phase over MgSO 4 If LCMS analysis indicates residual presence of MEMCl, dissolve the product in a saturated aqueous solution mixture. Dissolve NaHCO 3 and THF in N 2 atmosphere and stir vigorously for 20 minutes, then extract with EtOAc and dry over MgSO 4 The protected benzaldehyde 2 (52% yield) is used directly in the next step.

[0085] In a round-bottom flask equipped with a stir bar, dissolve 18.18 mmol of 3',4',5'-trimethoxyacetophenone in 20 mL of abs. EtOH. After adding 0.5 eq (9.09 mmol) of LiOH·H 2 O, stir the mixture under N 2 atmosphere at room temperature for 10 minutes. After adding 1 eq of benzaldehyde 2, stir the mixture under N 2 atmosphere at room temperature for 26 hours. Quench the mixture by adding 30 mL of water to form a precipitate. Isolate the precipitate by filtration and wash with water until the filtrate is clear. Crystallization of the residue in abs. EtOH affords 3 in 93% yield as white-yellow crystals.

[0086] Next, dissolve the protected chalcone 3 (8.73 mmol) in a mixture of 5% aq. HCl (20 mL) and THF (40 mL). Then reflux the mixture for 6 hours. After completion of the deprotection step (LCMS), quench the mixture with brine (60 mL) and extract the organics with diethyl ether (2 x 60 mL). Extract the combined organic phases with aq. 1M NaOH (120 mL), then neutralize the aqueous phase with aq. HCl (pH 7). During this step, the clear solution turns into an emulsion. Extract the product from this solution with EtOAc (2 x 60 mL) and dry the organic phase over MgSO 4 Crystallization in abs. EtOH gives chalcone A9 in 55% yield.

[0087] 3-(4-Fluoro-2-hydroxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one A9 Compound A9 was synthesized according to the above protocol.

Chemical Structure

[0088] General protocol: Synthesis of hydroxychalcones Into a round-bottom flask maintained under a nitrogen atmosphere, charge 15 mL of 1,2-dimethoxyethane, 2.2 mmol of the appropriate hydroxyacetophenone, and 3.5 - 6 equivalents (0.55 g) of LiOH·H 2 O. Fill the resulting suspension. Stir the obtained suspension at room temperature for 10 minutes and add 1.5 - 3 equivalents of the appropriately substituted benzaldehyde. Equip the flask with a reflux condenser and stir the mixture at 60 °C for 48 hours. Quench the reaction by the addition of 10 mL of 10% aq. HCl;. Extract the organic layer with EtOAc (2 × 15 mL), combine the organic layers, and dry over MgSO 4 and concentrate in vacuo. Purify the obtained crude chalcone by recrystallization in anhydrous EtOH.

[0089] 3-(4-Fluorophenyl)-1-(6-hydroxy-2,3,4-trimethoxyphenyl)prop-2-en-1-one A10 Compound A10 was synthesized according to the above general protocol.

Chemical formula

[0090] 3-(4-Fluorophenyl)-1-(2-hydroxy-3,4,5-trimethoxyphenyl)prop-2-en-1-one A11 Compound A11 was synthesized according to the general protocol described above.

Chem.

[0091] 3-(4-Fluorophenyl)-1-(2-hydroxy-4-methoxyphenyl)prop-2-en-1-one A12 Compound A12 was synthesized according to the above general protocol.

Chemical Structure

[0092] 3-(4-Chlorophenyl)-1-(2-hydroxy-4-methoxyphenyl)prop-2-en-1-one A13 Compound A13 was synthesized according to the above general protocol.

Chemical Structure

[0093] Reaction procedure for the synthesis of flav(an) [flavone] General protocol: Oxidative cyclization of 2'-hydroxy chalcone to flavone To a round-bottom flask containing 5 mL of DMSO, add 0.63 mmol of the appropriate 2'-hydroxy chalcone and 4 - 11 mol% of I 2 . Heat the resulting solution to reflux temperature for 2 - 5 hours, then pour it into 10 mL of an ice-water mixture. Isolate the resulting blue residue by filtration and redissolve it in 10 mL of EtOAc. Wash the solution thus obtained with 10 mL of 10% aqueous solution. Na 2 S 2 O 3 is dried over MgSO 4 and evaporated to dryness to obtain the crude flavone as a pure solid.

[0094] 2-(4-Fluorophenyl)-5,6,7-trimethoxy-4H-chromen-4-one B1 Compound B1 was synthesized according to the general protocol for the oxidative cyclization described above. [Chemical Structure] 1 H NMR (CDCl 3 , 300 MHz, ppm): δ = 3.92(3H, s), 3.99(3H, s, 3.99(3H, s), 6.61(s, 1H), 6.80(s, 1H), 7.20(2H, dd, J = 8.8 Hz, J = 8.8 Hz), 7.88(2H, dd, J = 8.8 Hz, J = 5.2 Hz'). 13 C NMR (CDCl 3 , 75 MHz, ppm): δ = 56.4, 61.7, 62.3, 96.3, 108.3, 113.0, 116.3(d, J = 21.9 Hz), 127.9(d, J = 3.5 Hz), 128.3(d, J = 8.1 Hz), 140.6, 152.7, 154.6, 157.9, 160.3, 164.7(d, J = 252.7 Hz), 177.2. IR (ATR, cm -1 )ν =1120、1346、1420、1486、1513、1605、1653。MS (ES+): m / z (%)=331.1([m+H] + , 100), 332.1([M+H+1] + , 19). HRMS (ES+): m / z = C 18 H 15 FO 5 + H + [m+H] + calculated value of: 331.0976; measured value: 331.0974. MP (℃)= 172-173. Yield: 98%; beige powder; MW = 330.31.

[0095] 2-(4-Fluorophenyl)-6,7,8-trimethoxy-4H-chromen-4-one B2 Compound B2 was synthesized according to the general protocol for the above oxidative cyclization. [Chemical formula] 1 H NMR (CDCl 3 , 300 MHz, ppm): δ = 3.97(s, 3H), 4.05(s, 3H), 4.09(s, 3H), 6.75(s, 1H), 7.23(dd, 2H, J = 9.1 Hz, J = 9.1 Hz), 7.39(s, 1H), 7.95(dd, 2H, J = 9.1 Hz, J = 5.2 Hz). 13 C NMR (CDCl 3 , 75 MHz, ppm): δ = 56.31, 61.50, 62.08, 100.02, 106.59, 116.38(d, J = 21.9 Hz, ), 119.76, 128.15(d, J = 3.5 Hz), 128.34(d, J = 9.2 Hz, ), 142.12, 145.78, 147.54, 151.32, 161.90, 164.73(d, J = 253.8 Hz, ), 177.63. IR (ATR, cm -1 )ν = 1075, 1108, 1125, 1366, 1378, 1414, 1466(C=C), 1650(C=O). MS (ES+): m / z (%)=330.7([m+H] + , 100), 331.8([M+H+1] + , 19). Yield: 70%; off-white fibers (from EtOH); MW = 330.31.

[0096] 2-(4-Fluorophenyl)-7-methoxy-4H-chromen-4-one B3 Compound B3 was synthesized according to the general protocol for the above oxidative cyclization. The product was crystallized in EtOH to increase the purity. [Chemical formula] 1 1H NMR (CDCl 3 , 300 MHz, ppm): δ = 3.93 (s, 3H), 6.70 (s, 1H), 6.96 (d, 1H, J = 2.2 Hz), 6.99 (dd, 1H, J = 8.8 Hz, J = 2.2 Hz), 7.21 (dd, 2H, J = 8.8 Hz, J = 8.5 Hz),, 7.92 (dd, 2H, J = 8.8 Hz, J = 5.2 Hz), 8.13 (d, 1H, J = 8.8 Hz). 13 13C NMR (CDCl 3 , 75 MHz, ppm): δ = 55.9, 100.4, 107.2, 114.5, 116.2 (d, J = 23.1 Hz), 117.7, 127.0, 128.0 (d, J = 3.5 Hz), 128.3 (d, J = 9.2 Hz), 157.9, 161.9, 164.2, 164.6 (d, J = 253.8 Hz), 177.6. 19 19F NMR (CDCl 3 , 282 MHz, ppm): δ = (-)108.37? (-)108.27 (m). IR (ATR, cm -1 ) ν = 810, 827, 838, 1084, 1164, 1202, 1233, 1351, 1372, 1416, 1437, 1509, 1586, 1606, 1628, 1636. MS (ES+): m / z (%) = 271.3 ([m + H] + , 100), 272.0 ([M + H + 1] + , 15). MP (°C) = 163. Yield: 74%, off - white crystals (from EtOH); MW = 270.26.

[0097] 2-(4-Chlorophenyl)-7-methoxy-4H-chromen-4-one B4 Compound B4 was synthesized according to the general protocol for the above oxidative cyclization.

Chemical Structure

[0098] General protocol: Cyclization of 2'-hydroxy chalcone to flavanone under acidic conditions Add 0.27 mmol of the appropriate 2'-hydroxy chalcone to a round-bottom flask containing 7 mL of 3N HCl in MeOH. Heat the resulting solution to reflux temperature for 24 hours and then concentrate it in vacuo. Then add H 2 O (7 mL) and neutralize the resulting suspension with saturated aq. NaHCO 3 (7 mL). Next, perform extraction with EtOAc (15 mL). Wash the thus obtained organic layer with H 2 O (10 mL), dry over MgSO 4 and evaporate to dryness to obtain the crude flavanone as a solid. Then recrystallize in absolute EtOH to obtain the pure product.

[0099] 2-(4-Fluorophenyl)-5,6,7-trimethoxychroman-4-one B5 Compound B5 was synthesized according to the general protocol for cyclization under the above acidic conditions. [Chemical Structure] 1 1H NMR (CDCl 3, 400 MHz, ppm): δ = 2.77 (dd, 1H, J = 16.7 Hz, J = 2.8 Hz), 2.98 (dd, 1H, J = 16.7 Hz, J = 13.3 Hz), 3.82 (s, 3H), 3.88 (s, 3H), 3.94 (s, 3H), 5.38 (dd, 1H, J = 13.3 Hz, J = 2.8 Hz), 6.34 (s, 1H), 7.11 (t, 2H, J = 8.7 Hz), 7.43 (dd, 2H, J = 8.7 Hz, J = 5.3 Hz). 13 C NMR (CDCl 3 , 100 MHz, ppm): δ = 45.7, 56.3, 61.5, 61.7, 78.8, 96.4, 109.3, 115.9 (d, J = 21.9 Hz), 128.1 (d, J = 8.1 Hz), 134.7 (d, J = 2.9 Hz), 137.8, 154.4, 159.54 and 159.57 (3C). IR (ATR, cm -1 ) ν = 1083, 1103, 1264 (C - O), 1412, 1461, 1483, 1514 (C = C), 1599 (C = O). MS (ES+): m / z (%) = 333.1 ([m + H] + , 100), 334.1 ([M + 1 + H] + , 19). HRMS (ES+): m / z = C 18 H 17 FO 5 + H + [m + H] + calculated value of: 333.1133; found value: 333.1134. Yield: 44%; white powder (from EtOH); MW = 332.32.

[0100] 2-(4-Fluorophenyl)-6,7,8-trimethoxychroman-4-one B6 Compound B6 was synthesized according to the general protocol for cyclization under the above acidic conditions.

Chemical Structure

[0101] General protocol: Cyclization of 2'-hydroxy chalcone to flavanone under basic conditions Reflux condenser and CaCl 2Place 10 mL of anhydrous EtOH in a flame-dried round-bottom flask equipped with a tube. Heat the solvent to reflux, then add the appropriate 2'-hydroxy chalcone (1 mmol) and 10 equivalents of NaOAc. Maintain reflux and monitor the reaction by LC / MS. Once the maximum conversion is obtained, cool the yellow reaction mixture to room temperature and pour it into 10 mL of ice water. Extract the organic layer with EtOAc (3 × 20 mL), wash the combined organic layers with brine, dry over MgSO 4 and concentrate in vacuo. A yellow residue containing a mixture of 2'-hydroxy chalcone and flavanone is obtained. Separation is carried out using preparative TLC with an eluent mixture of petroleum ether and EtOAc (8:1). The flavanone is thus obtained as an off-white solid.

[0102] 2-(4-Fluorophenyl)-7-methoxychroman-4-one B7 Compound B7 was synthesized according to the general protocol for cyclization under the above basic conditions. [Chemical formula] 1 H NMR (CDCl 3 , 300 MHz, ppm): δ = 2.81(dd, 1H, J = 16.7 Hz, J = 2.8 Hz), 3.02(dd, 1H, J = 16.7 Hz, J = 12.9 Hz), 3.84(s, 3H), 5.46(dd, 1H, J = 12.9 Hz, J = 2.8 Hz), 6.49(d, 1H, J = 2.2 Hz), 6.63(dd, 1H, J = 8.8 Hz, J = 2.2 Hz), 7.12(dd, 2H, J = 8.5 Hz, J = 8.5 Hz), 7.46(dd, 2H, J = 8.4 Hz, J = 5.2 Hz), 7.87(d, 1H, J = 8.8 Hz). 13 C NMR (CDCl 3, 75 MHz, ppm): δ = 44.4, 55.7, 79.4, 101.0, 110.4, 114.8, 115.8(d, J = 21.9 Hz), 128.1(d, J = 8.1 Hz), 128.8, 134.8(d, J = 2.3 Hz), 162.8(d, J = 248.1 Hz), 163.4, 166.3, 190.3。 19 F NMR (CDCl 3 , 282 MHz, ppm): δ =(-)113.39 ?(-)113.30(m)。 IR (ATR, cm -1 )ν = 804, 820, 830, 841, 1112, 1160, 1200, 1220, 1232, 1256, 1442, 1514, 1572, 1602, 1671。 MS (ES+): m / z (%)=273.3([m+H] + , 100), 274.3([M+H+1] + , 29)。MP (℃)= 9。Yield: 66%, beige solid; Chromatography: Rf: 0.15(petroleum ether / EtOAc 8:1); MW =272.27。

[0103] 2-(4-Chlorophenyl)-7-methoxychroman-4-one B8 Compound B8 was synthesized according to the general protocol for cyclization under the above basic conditions.

Chemical Structure

[0104] 2 Cell culture The Michigan Cancer Foundation-7 (MCF-7) cell line is a human breast cancer cell line originally isolated from the pleural effusion of breast cancer. This cell line was further selected for invasive and metastatic activities, and the MCF-7 / 6 cell line, provided by H. Rochefort (Unite d'Endocrinologie Cellulaire et Moleculaire, Montpellier, France), was prepared. Human 13B20332 hTERT (also called 13B2 hTERT) breast CAF was derived from a tumor that was 99% estrogen receptor positive, 25% progesterone receptor positive, and HER2 negative, and was isolated as previously described (Primac et al. 2019). IF staining and flow cytometry of 13B2 hTERT CAF cells showed positive staining for α-SMA, FAP, VIM, CD44, CD105, and CD90, and negative expression of the epithelial marker CD326. Human CT5.3 colon CAF was isolated from colorectal adenocarcinoma resection specimens obtained according to the local ethics committee (Ghent University Hospital) and infected with a pBABE retroviral vector expressing the hTERT open reading frame (De Vlieghere et al. 2015). IF staining and flow cytometry of CT5.3 hTERT CAF cells showed positive staining for α-SMA, FAP, VIM, CD44, CD105, and CD90, and negative expression of the epithelial marker CD326. HCA2 hTERT skin fibroblasts were provided by C. Jones (Cardiff University, UK). 4T1-luc breast tumor cells were a kind gift from Professor Clare Isacke (Breakthrough Breast Cancer Research Centre, London, UK). This aggressive cell line resembles human metastatic triple-negative breast cancer (TNBC) and constitutively expresses firefly luciferase. Primary human atrial fibroblasts were obtained from the right atrial appendage tissue of patients in sinus rhythm undergoing open heart surgery and isolated using the tissue explant method.The epicardial layer was removed and pieces of 1 - 2 mm were cultured on plates with a polished surface using 2 ml of Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin (all from Sigma - Aldrich, Germany). 2 The resulting growing fibroblasts were considered passage zero. Primary glioblastoma cells were obtained from patients who underwent glioblastoma resection using a similar protocol. All patients gave informed consent for the use of their tissue. CAF03 glioblastoma CAF was purchased from Vitro Biopharma (USA).

[0105] MCF - 7 / 6 cells were cultured in a medium containing F12 Nut Mix (Ham) medium (catalog number 11765054, Thermo Fisher Scientific) supplemented with Dulbecco's Modified Eagle Medium (DMEM) (catalog number 41965039, Thermo Fisher Scientific) and 10% heat - inactivated fetal bovine serum (FBS) (catalog number ATCC - 30 - 2030, LGC Standards), 100 IU / ml penicillin and 100 mg / ml streptomycin (catalog number 15070063, Thermo Fisher Scientific) in a 1:1 ratio. CT5.3 hTERT, HCA2 hTERT and 4T1 - luc cells were cultured in DMEM (catalog number 41965039, Thermo Fisher Scientific) supplemented with 10% heat - inactivated fetal bovine serum (FBS) (catalog number ATCC - 30 - 2030, LGC Standards), 100 IU / ml penicillin and 100 mg / ml streptomycin (catalog number 15070063, Thermo Fisher Scientific). The 13B2 hTERT cell line was cultured in the same medium as above using the following additives: 0.05 μg / mL cholera toxin (catalog number C8052, Sigma - Aldrich), 5% human serum (catalog number P2918, Sigma - Aldrich), 0.01 μM triiodothyronine (catalog number IRM6649, Sigma - Aldrich), 0.01 μM hydrocortisone (catalog number H0888, Sigma - Aldrich), 0.01 μg / mL epidermal growth factor (EGF) (catalog number E9644, Sigma - Aldrich) and 0.05 mg / mL insulin (catalog number I6634, Sigma - Aldrich). Cells were grown and maintained as a monolayer at 37 °C in an atmosphere of 5% CO 2 (MCF - 7 / 6, 4T1 - luc) or 10% CO 2 (CT5.3 hTERT, HCA2 hTERT, 13B2 hTERT), and passaged at 80% confluence. To exclude mycoplasma contamination, all human cell lines were tested monthly using the Mycoalert Mycoplasma Detection Kit (catalog number LT07 - 318, Lonza). Primary human atrial fibroblasts were cultured in DMEM supplemented with 2 mM L - alanyl - L - glutamine (GlutaMAX, catalog number 31966021, Life Technologies), 10% fetal bovine serum (catalog number F9665, Sigma - Aldrich), and 1% penicillin / streptomycin (catalog number 15070063, Thermo Fisher Scientific). CAF03 glioblastoma CAF and primary glioblastoma cells were grown in MSC - GRO™ VitroPlus III Low Serum Complete Medium (Vitro Biopharma, US).

[0106] 3 Chicken heart infiltration test The chicken heart infiltration assay was performed as previously described (Bracke et al., 2014). Pre - cultured heart fragments from 9 - day - old chicken embryos were compared with MCF - 7 / 6 aggregates on top of semi - solid agar overnight at 37 °C, and then the pairs to be compared were incubated at 37 °C, 10% CO 2It was cultured in suspension for 8 days in the presence or absence (during duplo) of the test compound. This procedure can also be carried out using aggregates of 4T1-Luc cells s, and a paired suspension culture for comparison is carried out for 3.5 days. All compounds were evaluated at 1 μM and 100 nM; depending on the results, higher or lower concentrations were then evaluated. When fixed in Bouin-Hollande, the cultures were dehydrated and embedded in paraffin. Sections of the control cultures s were stained with hematoxylin and eosin, blinded and scored by trained personnel (Bracke et al., 2014).

[0107] In the chicken heart infiltration assay, fragments of normal tissue are compared to disregard the contribution of host tissue in the microecosystem where cancer cells dominate tumor behavior. As such, pre-cultured heart tissue fragments (PHF) dissected from 9-day-old chicken embryos can be compared to aggregates of human invasive MCF-7 / 6 breast cancer cells or other cells in the presence of a certain concentration of the test compound. After 8 days for MCF-7 / 6, or another period depending on the cell type, the interaction between cancer cells and PHF is histologically evaluated and classified along a subjective scale of 5 malignancy grades (Bracke et al., 2014). Malignancy grades III and IV are typical of infiltration, while grades 0, I, and II correspond to no infiltration. Compounds that inhibit the infiltration of the cancer cells being compared, i.e., those that inhibit infiltration from III / IV to 0 / I / II, are determined to be anti-invasive. Possible alternative assays commonly known in the art are the type I collagen infiltration assay, the wound healing assay, and the Matrigel infiltration assay.

[0108] 4 Atomic force microscopy A U-shaped 384-well ULA dish (catalog number MS-9384UZ, S-bio) was seeded with a suspension of cell-specific culture medium containing cells. The cells were allowed to form spheroids for an appropriate time under the treatment conditions before being used in the experiment. A cell culture dish (catalog number 734-2814, VWR) was filled with a collagen gel and left at 37 °C for an appropriate time to solidify. For each condition, the spheroids were pooled into one cell culture dish. The medium was removed (stored at 37 °C) to promote adhesion of the spheroids to the collagen gel at the bottom. The cell culture dish was incubated at 37 °C. After the incubation period, the stored medium was carefully added to the dish (to prevent detachment of the spheroids).

[0109] Mechanical measurements were performed using an atomic force microscope. A custom-made colloidal probe was used to indent the spheroids. Force curves were collected using appropriate set values and probe speeds (depending on the stiffness of the ellipsoid). For each ellipsoid, multiple force curves were collected. The collected curves were then processed using JPK DP software. The Young's modulus was extracted from the force curves. All experiments were performed in biological triplicates.

[0110] 5 Rigidity measurement CAFs are the dominant cell type in the TME and are involved in all stages of cancer progression. CAFs are generally larger and more metabolically active than healthy fibroblasts. They promote angiogenesis and immune evasion by producing a series of growth factors and cytokines such as transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), and CXC-chemokine ligand (CXCL12). Another important feature of CAFs is their ability to remodel the ECM, which ultimately contributes to metastasis. CAFs are the major producers of ECM components and thus widely contribute to its remodeling, including the stiffness of the ECM. To create 3D cultures that mimic the complexity and pathophysiology of in vivo tumors, spheroids were used (cfr. point 4). Spheroids are thought to better simulate cell heterogeneity, nutrient and oxygen gradients, cell-cell interactions, cell-matrix interactions, gene expression profiles, stromal interactions, treatment responses, multicellular resistance, drug penetration, and anti-apoptotic signaling.

[0111] 6 Evaluation of cell viability. The half-maximal inhibitory concentration (IC 50 ) of A1 was evaluated using the CellTiter-Glo® 2.0 Cell Viability Assay (Catalog No. G9243, Promega) and the CellTiter-Glo® 3D Cell Viability Assay (Catalog No. G9681, Promega). 13B2 hTERT cells s (2D: 2×10 5 , 3D: 8×10 4was seeded at a volume of 90 μL / well into each well of a Nunc™ MicroWell™ 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplate (catalog number 167008, Thermo Fisher Scientific) for 2D experiments or a 96-well ULA plate (catalog number MS-9096UZ, S-, bio). A1 was serially diluted up to 10-fold the final concentration in an appropriate medium. For each treatment condition, 10 μL was added to three replicate wells (final concentrations in the range of 1 - 100 μM), and the plate was incubated for 48 hours.

[0112] The CellTiter-Glo Cell Viability Assay was performed according to the manufacturer's instructions. Briefly, the plate and the CellTiter-Glo® reagent were equilibrated to room temperature (RT). The CellTiter-Glo® reagent was added to each well at a 1:1 (v / v) ratio. For 2D experiments, the plate was placed on an orbital shaker for 2 minutes, and then cell lysis was induced by stabilizing the luminescence signal for 10 minutes. For 3D experiments, the plate was placed on an orbital shaker for 5 minutes and then stabilized for 25 minutes. Subsequently, luminescence was measured using a Synergy™ HTX Multi-Mode Microplate Reader (BioTek). IC 50 values were determined using Graphpad Prism software. This was done for three biological replications.

[0113] 7 In Vivo Experiments: 4T1-Based Intraductal Model 7.1 Intraductal Inoculation of 4T1-luc Cells Experiments in mice were conducted in accordance with Good Scientific Practice - principles and approved by the Ethical Committee (EC) of the Faculty of Veterinary Medicine of Ghent University (EC 2019 - 105).

[0114] Intraductal inoculation was performed in the third mammary pair of lactating female mice. To reach the lactation period, 8 - week - old female BALB / c mice were mated with male BALB / c mice of the same age, and the pups were weaned 12 - 14 days after parturition. One hour after weaning, the lactating females were anesthetized by inhalation with a mixture of oxygen and isoflurane (2 - 3%), and subsequently, 5×10 4 individual 4T1 - luc cells suspended in 100 μl of a mixture of PBS and Matrigel® (1:10; Corning, Bedford, MA, USA) were inoculated through the nipple duct using a 32 - gauge blunt needle. All materials required for inoculation were pre - cooled to avoid Matrigel® coagulation. Analgesia was performed by intraperitoneal administration of buprenorphine (10 μg / kg, Val d'Hony Verdifarm NV - Belgium).

[0115] 7.2 Preparation and administration of the formulation The compound was dissolved in DMSO. Subsequently, a 1 mL syringe and an 18 G (pink) needle were used to aspirate the compound solution, and all the solution was expelled, including additional air, to help avoid retention of the compound in the dead volume. The solution was injected into Medigel in five parts at five different sites, a certain volume of the gel was aspirated, and the syringe dead volume was rinsed by reinjecting into the cup at the last site. Then, the drug was shaken vigorously and visually inspected for uniform dispersion of the compound. Mice received freshly prepared drug - containing cups every 48 hours, and the gel was placed in a gel - cup holder to avoid contact with the bedding material and absorbent shavings in the mouse cage. The amount of Medigel given to the mice was determined weekly and depended on the average mouse body weight and consumption. The administration level was kept constant at 100 mg / kg three times a day.

[0116] 7.3 Analysis of tumor and metastasis progression Body weight and body temperature were measured daily during the first week p.i. and then once a week for the subsequent 5 weeks (i.e., up to 6 w p.i.) using a digital weighing scale and a rectal temperature probe, respectively. As a measurement of primary tumor growth, primary tumor volume was monitored weekly using digital calipers, and 4T1-luc-derived bioluminescence in the primary tumor was monitored at 1, 3, and 6 w p.i. using an IVIS lumina III system (PerkinElmer, Zaventem, Belgium). To perform in vivo imaging, mice were injected with 200 μl of D-luciferin (2 mg / 100 μl; Gold Biotechnology, St. Louis, MO) suspended in PBS, and images were acquired under inhalant anesthesia 10 minutes later. To perform ex vivo imaging, mice were sedated with a mixture of 100 mg / kg ketamine (Ketamidor, Ecuphar nv / sa, Oostkamp, Belgium) and 10 mg / kg xylazine (Xylazini Hydrochloridum, Val d'HonyVerdifarm, Beringen, Belgium), followed by sacrifice by cervical dislocation. Images of bioluminescence signals in isolated primary tumors, spleens, and organs with metastases (including axillary lymph nodes, lungs, and livers) were rapidly acquired using an IVIS system. Primary tumors and spleens were also weighed using a digital weighing scale. Analysis of in vivo and ex vivo bioluminescence signals was performed using Living Image software 4.7.2.

[0117] 7.4 Histological and immunohistochemical staining Isolated tissues of primary tumors, axillary lymph nodes, lungs, and livers were fixed in buffered 3.5% formaldehyde for 24 hours at room temperature (RT) and embedded in paraffin wax. 5-μm sections were deparaffinized, hydrated, and stained with hematoxylin and eosin (H&E). To enable analysis, the sections were dehydrated and mounted.

[0118] For Masson trichrome staining, sections 3-5 μm thick were deparaffinized, hydrated, incubated on in Bouin's solution to improve staining quality. The next day, the slides were washed in distilled H 2 O (dH 2 O), then immersed three times in Biebrich scarlet-acid fuchsin solution, washed again in dH 2 O, and incubated for 20 minutes in phosphomolybdotungstic acid solution. After incubation in phosphomolybdic acid-phosphotungstic acid solution, the slides were not rinsed and immediately incubated in aniline blue solution for 8 minutes. After the final wash step in dH 2 O, the slides were incubated in 1% acetic acid solution for 8 minutes. The sections were then rapidly dehydrated through 94% ethyl alcohol and absolute alcohol to remove Biebrich scarlet-acid fuchsin staining and xylene, and the slides were cleared and then mounted with Tissue-Tek glass mounting medium. The staining visualizes blue collagen, dark red nuclei, and red / pink cytoplasm. Quantification of positive staining was established using color deconvolution (for Masson trichrome staining) and subsequently automatically counted in ImageJ.

[0119] For immunohistochemical staining, antigen retrieval was performed on 3-5 μm thick deparaffinized sections using a pressurized decloaking chamber NxGen (Biocare Medical, CA, USA) at 95°C for 30 minutes with citrate buffer [10 mM trisodium citrate (Santa Cruz Biotechnology, Heidelberg, Germany)], pH 6 for α-SMA, CD11b, Ly6G, and CD163, or Tris-EDTA buffer [pH 9, 10 mM Tris, 1 mM EDTA (Thermo Fisher Scientific)] for PDPN and FAP-α. The slides were cooled to RT for 30 minutes and then incubated for all blocking, rinsing (3 times for 2 minutes each at RT during each incubation step in TBS), and staining steps in a closed microscope box containing tissue paper moistened with Tris-buffered saline (TBS, Biocare Medical) on an orbital shaker at 20 rpm. To block endogenous peroxidase activity, the slides were first treated with 3% H 2 O 2 in methanol for 10 minutes. To block non-specific binding sites, the slides were then treated with serum-free protein block (Dako, Heverlee, Belgium) for 10 minutes.

[0120] Next, the slides were stained with primary antibodies diluted in antibody diluent (Dako) at RT for 1 hour, followed by incubation with secondary antibodies at RT for 30 minutes. The primary antibodies and dilutions used were anti-α-SMA (1:2000, clone EPR5368, Abcam, Cambridge, UK), anti-PDPN (1:100, clone PMab-1, Abcam), anti-FAP-α (1:100, polyclonal, Abcam), anti-CD11b (1:4000, clone EPR1344, Abcam), anti-Ly6G (1:1000, clone 1A8, Biolegend, CA, USA), anti-CD163 (1:500, clone EPR19518, Abcam). The secondary antibodies used were rat-on-mouse HRP polymer (Biocare Medical) for PDPN and Ly6G, and recycled Dako EnVision+ rabbit for α-SMA, FAP-α, CD11b, and CD163. For visualization of HRP positive staining, the slides were treated with 3,3'-diaminobenzidine (DAB)-containing buffer (Dako) at room temperature for 10 minutes. Counterstaining with hematoxylin was applied at RT for 5 minutes, followed by dehydration and mounting of the slides with Tissue-Tek glass mounting medium. Quantification of positive staining was established using color deconvolution (for DAB and hematoxylin counterstaining) followed by automated counting in ImageJ.

[0121] 7.5 Cytokine Array Analysis The Proteome Profiler Mouse XL Cytokine Array (catalog number ARY028, R&D Systems) was performed using primary tumor lysates from the syngeneic 4T1 TNBC mouse model according to the manufacturer's instructions. Analysis was performed using the ImageJ plugin "Protein Array Analyzer".

[0122] 7.6 Statistical Analysis Data are presented as mean ± standard error of the mean (SEM) on graphs. *, **, and *** represent statistically significant differences at P < 0.05, P < 0.01, and P < 0.001, respectively. Statistics were performed using Prism (GraphPad), and data were normalized using log10 normalization as needed. P values were calculated by one-sided unpaired t-test or one-way analysis of variance (ANOVA) test, followed by Newman-Keuls post hoc test for multiple comparisons.

[0123] 8 Combination therapy Compound inactivation of E-cadherin and PTEN in mouse mammary epithelium leads to the rapid formation of classical invasive lobular carcinoma (CLC). Ninety mice of the Ecad;Pten mouse model (Boelens et al. 2016) were injected intravascularly with lenticre to induce Cre conditional inactivation of E-cadherin and PTEN, leading to the formation of CLC. Animals were divided into five treatment groups: (i) Daily oral vehicle (Medigel Sucralose, ClearH 2 O) and injection vehicle, (ii) Daily A1 in oral vehicle, (iii) Oral vehicle and twice-weekly injection of anti-PD1 monoclonal antibody, (iv) A1 in oral vehicle and twice-weekly injection of anti-PD1 monoclonal antibody, and (v) Oral vehicle and daily PI3K inhibitor BEZ-235. Throughout the experiment, mouse body weights were measured every 48 hours to assess tolerance, and tumors were measured every 48 hours. Treatment was initiated when the cancer volume reached 75 millimeters and continued for 6 weeks, after which the animals were sacrificed. Tumors were weighed, and tissues (including tumors and lymph nodes) were examined using histological and other techniques to compare the tolerance and efficacy of different treatments.

[0124] Results Example 1 - Anti-invasive flavonoid The synthesis of the target flav(an)ones proceeded via the intermediate of 2'-hydroxychalcone from appropriately substituted 2'-hydroxyacetophenone and benzaldehyde. Due to the presence of the 2'-hydroxyl group, the Claisen-Schmidt condensation in the first step promoted the use of excess benzaldehyde and base in 1,2-dimethoxyethane at 60 °C.

Chem.

[0125] Scheme 2 Synthesis of 2'-hydroxychalcones A9 - A12, flavones B1 - B4 and flavanones B5 - B8. Reagents and conditions: a) 1.5 - 3 eq. of substituted benzaldehyde, 3.6 - 6 eq. of LiOH·H 2 O, 1,2-dimethoxyethane, N 2 , 60 °C, 48 h; b) 0.04 - 0.11 mol% I 2 , DMSO, reflux, 2 - 5 h, N 2 ; c) 3M HCl in MeOH, reflux, 24 h (for B5 and B6) or 10 eq. of NaOAc, abs. EtOH, reflux, 1.5 h, dry conditions (for B7 and B8).

[0126] The gentle oxidative cyclization of chalcones A9 - A12 to flavones B1 - B4 was achieved in DMSO using a catalytic amount of I 2 . This method has proven to be rapid and convenient, delivering the desired product in high yield and high purity and often not requiring further purification by crystallization.

[0127] Treatment of A9-A10 with HCl in MeOH at reflux temperature gave flavanones B5-B6 in moderate yields. The starting material was completely consumed after 24 h, but purification (extraction and crystallization in EtOH) resulted in significant product loss and moderate yields (44 - 50%) (Hsieh et al. 1998). Treatment of chalcones A11 - A12 with HCl in MeOH also led to an equilibrium with the desired flavanones B7 - B8 after 18 - 24 h (ca. 70% conversion based on LC / MS data). Thus, cyclization of A11 - A12 in the basic structural environment (NaOAc in EtOH) was also evaluated. This gave a similar reaction equilibrium (ca. 70% conversion), but the work-up was easier and thus gave moderate yields of flavanones B7 - B8.

[0128] The chick heart invasion (CHI) assay is a phenotypic assay that confronts pre-cultured heart tissue fragments (PHF, “normal tissue”) obtained from 9-day-old chick embryos with aggregates of human invasive MCF-7 / 6 breast cancer cells (Bracke et al., 2014). In the control situation, MCF-7 / 6 cells surround, invade and destroy the PHF over 8 days. Invasion in this assay is defined as the progressive occupancy and destruction of the PHF by MCF-7 / 6 cells. The three-dimensional reconstruction of this interaction is obtained by microscopic analysis of all consecutive tissue sections of the culture. The read-out is an invasion score on a 5-grade histological scale.

[0129] CHI closely recapitulates the temporal, spatial and histological invasion patterns in humans. The co-culture setup probes the effects on both tumor cells and host tissue and thus probes potential pharmacological targets in either interaction partner. The use of its histological evaluation allows discrimination of anti-invasive effects from cytotoxic or other effects.

[0130] The inventors subjected flavones B1 - B4, flavanones B5 - B8 and the intermediates 2'-hydroxy chalcones A9, A11 and A12 to a chicken heart infiltration assay using MCF-7 / 6 aggregates. All compounds were evaluated at 1 μM and 100 nM; depending on the results, higher or lower concentrations were then evaluated. Table 1 shows the lowest concentration at which an anti-invasive effect was observed (histological grade I or II - cfr. Bracke et al., 2014). This table also includes CHI data for chalcones A1, A2 and A5. Table 2 shows the anti-invasive effects of A1 and B1 in the CHI assay using 4T1-Luc aggregates.

[0131] The results show that a specific substitution pattern leads to molecules with higher activity, but all test compounds show appropriate activity. The anti-invasive activity data confirm that a combination of a (tri)methoxy decoration pattern on the left side of the molecule and a halo (e.g., fluoro, chloro) substituent on the right side is preferred. Table 1: Activity data of compounds in the CHI assay using MCF-7 / 6 aggregates.

Table 1-1

Table 1-2

[0132] Table 2: Activity data of compounds A1 and B1 at 1 μM in the CHI assay using 4T1-Luc aggregates. Structure Histological infiltration degree

Table 2

[0133] Example 2 - Regulation of the tumor microenvironment (TME) CAF selectivity Treatment with 1 μM A1 or B1 showed a significant decrease in the Young's modulus in 13B2 hTERT breast CAFs, while 1 μM of the inactive control compound A8 had no effect. Compound A14 also decreased the Young's modulus in 13B2 hTERT breast CAFs at 1 μM. A dose-response behavior was obtained using a concentration range of A1 from 30 nM to 3 μM (Figures 1A - C). Similar results were obtained for CT5.3 hTERT colon CAF spheroids, as 1 μM A1 or B1 decreased the rigidity of CT5.3 hTERT spheroids (Figure 1D). For glioblastoma CAF03 CAF spheroids, further similar results were obtained as 1 μM A1 decreased the spheroid rigidity (Figure 1E).

[0134] In the examples regarding the CAF selectivity of fibroblast modulators in the present invention, a decrease in spheroid rigidity by 1 μM A1 or 1 μM B1 was observed in CAFs (Figures 1A - E), but not in normal fibroblasts (HCA2 hTERT, Figure 1F). Treatment with A1 at 0.03 - 3 μM further did not result in a change in spheroid rigidity for epithelial cell lines such as MCF - 7 / 6 (Figure 1G), nor did it change the spheroid rigidity for primary glioblastoma cells (Figure 1H). In particular, from the perspective of avoiding the risk of cardiotoxicity, treatment with 1 μM A1 or 1 μM B1 did not change the spheroid hardness of primary human atrial fibroblasts (Figure 1I).

[0135] The novelty of this discovery is exemplified by the selectivity of the fibroblast modulators in the present invention and the behavior of the existing CAF modulators, the myosin II-inhibitor blebbistatin, the ROCK-inhibitor Y-27632 or the actin polymerization inhibitor cytochalasin D (Figure 2). A1, B1 and the existing CAF modulators were all active in CAF spheroids (13B2 hTERT, Figure 2A; CAF03, Figure 2B), but A1 and B1 had no effect on the stiffness of normal fibroblasts (HCA2 hTERT, Figure 2C), epithelial cell spheroids (MCF-7 / 6, Figure 2D), primary glioblastoma cell spheroids (Figure 2E) or primary human atrial fibroblast spheroids (Figure 2F) (Figure 2F). However, the existing CAF modulators not only reduced the stiffness of CAF spheroids, but also reduced the stiffness of normal fibroblast (HCA2 hTERT), epithelial cell (MCF-7 / 6), primary glioblastoma cell and primary human atrial fibroblast spheroids (Figures 2A-F).

[0136] Selectivity data for additional compounds of the present invention for the reduction of spheroid stiffness of CAF (13B2 hTERT) versus normal fibroblasts (HCA2 hTERT) are shown in Figure 3. The chalcones, flavones and flavanones tested are effective modulators of CAF spheroid stiffness but not of normal fibroblast stiffness. Combinations of one or more methoxy groups on one aromatic ring of the molecule and one or more halogens with or without a hydroxyl group on the other aromatic ring of the molecule may be advantageous for the desired activity.

[0137] Cell viability To examine whether the effects observed in the AFM experiments were not caused by cytotoxicity, 2D and / or 3D CellTiter-Glo® cell viability assays were performed on 13B2 hTERT, CT5.3 hTERT, HCA2 hTERT, MCF-7 / 6 and 4T1-Luc cells after treatment with A1 or B1. 48 hours after treatment, the metabolic activity of the 2D cultures was high micromolar IC 50It was inhibited in a dose-dependent manner by the value. Cells cultured as spheroids did not show a response to treatment with A1 or B1 up to 100 μM. From these results, it can be concluded that the observed activity against CAFs is not due to a decrease in the viability of these CAFs, and the observed selectivity against CAFs for normal fibroblasts or cancer cells is also not due to the higher sensitivity of CAFs to the decrease in viability by A1 or B1.

[0138] 4T1 triple-negative breast cancer model In a 4T1-based intraductal model for triple-negative breast cancer (TBNC), the antimetastatic efficacy of A1 and B1 was investigated. A1 and B1 were added to Medigel for oral intake and compared with DMSO added to Medigel as a negative control. Axillary lymph node, lung, and liver metastases were the main parameters analyzed 6 weeks (w.) after inoculation (p.i.) of the tumor cell suspension in the mammary ducts, as well as histological analysis of the microenvironment of the primary tumor and axillary lymph nodes, lungs, and livers.

[0139] Tolerance The body weights of all intraductally inoculated mice decreased during the first 3 - 4 days after inoculation (p.i.) due to a decrease in milk volume after weaning of the pups (Figure 4A), while body temperature remained stable in all mice during the first week of p.i. (Figure 4B). In weekly evaluations of body weight and body temperature over a 6-week test period, DMSO-, A1-, and B1-treated mice did not show significant fluctuations over the 6-week test period (Figures 4A - B).

[0140] Metastasis Treatment with A1 or B1 showed an overall decrease in the metastatic burden in lung, liver, and axillary lymph node metastases compared to DMSO based on ex vivo bioluminescence data (Figures 5A - C).

[0141] The H&E images of the primary tumors corroborate the aggressive character of the 4T1-based in situ model for triple-negative breast cancer (TNBC), with tumor infiltration in breast adipose tissue and the blood vascular system, areas of cell necrosis, immune infiltration showing spindle shapes characteristic of epithelial-mesenchymal transition (EMT) preceding metastasis, and the presence of tumor cells. After A1 treatment, smaller lung metastases as well as liver metastases were found, and in B1 treatment, smaller axillary lymph node metastases were found (data not shown) compared to DMSO treatment, as corroborated by bioluminescence data based on IVIS measurements.

[0142] Histology of the TME of primary tumors and metastases Based on staining for the CAF marker α-smooth muscle actin (α-SMA), the amount of CAFs in the primary tumors was significantly decreased by treatment with A1 or B1 compared to DMSO treatment (Figure 6A). Podoplanin (PDPN), an alternative marker for CAFs, confirmed the results of α-SMA and showed a significant decrease in PDPN staining in primary tumors treated with A1 and B1 (P<0.001) (Figure 6B). As a further validation of both α-SMA and PDPN staining, significantly decreased staining was observed for the CAF marker fibroblast activation protein α (FAP-α) in primary tumor tissues treated with A1 or B1 compared to DMSO (Figure 6C). This serine protease is highly expressed in reactive fibroblasts and has been shown to regulate fibroblast proliferation.

[0143] Masson's trichrome staining, which visualizes collagen fibers, showed a significant decrease in collagen deposition upon treatment with A1 or B1 compared to DMSO (Figure 6D), reconfirming the results of α-SMA, PDPN, and FAP-α of CAFs and collectively identifying a decrease in tumor stiffness upon treatment with A1 and B1 compared to DMSO.

[0144] In addition to the decrease in the number of CAFs, CD11b staining of the primary tumor tissue also showed a significant decrease in the amount of bone marrow cells during treatment with A1 or B1 compared to DMSO (Figure 6E). Ly6G staining is more specific for neutrophil precursors such as TAN and granulocyte myeloid-derived suppressor cells (G-MDSC), and this decrease was confirmed during A1 or B1 treatment compared to DMSO (Figure 6F). CD163 staining of the primary tumor tissue, which complements innate immune cell regulation, showed a significant decrease in tumor-associated macrophages (TAMs) during treatment with A1 or B1 (Figure 6G). CD163 is a marker of M2-polarized macrophages that promote tumor progression, angiogenesis, metastasis, and immunosuppression. Due to these pro-tumoral activities associated with negative patient prognosis, the M2 macrophage phenotype is called tumor-associated macrophages (TAMs). TAMs support the immunosuppressive TME through the secretion of cytokines, chemokines, and growth factors. They also induce the release of inhibitory immune checkpoint proteins in T cells.

[0145] In liver tissue, α-SMA and Masson's trichrome staining showed a significant decrease in collagen deposition at sites with CAFs and proliferating bone marrow cells during treatment with A1 or B1 compared to DMSO (Figures 7A and 7C). PDPN staining also decreased significantly during treatment with A1 or B1 compared to DMSO in liver tissue (Figure 7B). Similar decreases were seen in axillary lymph node tissue and lung tissue (Figures 7D - G) (Liu et al., 2017) (Liu et al., 2017) (Liu et al., 2017).

[0146] Cytokine profile of the primary tumor 4T1 primary tumor lysates from animals treated with DMSO or A1 were used in a cytokine array that quantifies the relative expression levels of 111 soluble mouse proteins. A total of 13 proteins were significantly affected by A1 treatment: CC-chemokine ligand (CCL)6, CD40, chitinase-3-like protein 1 (CHI3L1), chemokine (C-X-C motif) ligand (CXCL)16, fibroblast growth factor (FGF) acidic, interleukin (IL)-12p40, low density lipoprotein receptor (LDLR), MMP-3, MMP-9, myeloperoxidase (MPO), serine protease inhibitor (serpin) E1, and WNT1-inducible-signaling pathway protein 1 (WISP-1). This perturbation pattern not only is selective for CAFs but also shows an effect specific for tumor-promoting CAFs of subtypes S1 and S4 (Costa et al., 2020). CAF-S1 (CD29 Med FAP Hi FSP1 Med aSMA Hi PDGFRb Med-Hi CAV1 Low ) and CAF-S4 (CD29 Hi FAP Neg FSP1 Low -Med aSMA Hi PDGFRb Low-Med CAV1 Low ) subsets are both detected, for example, in aggressive (HER2 and TN) breast cancer subtypes.

Claims

1. A pharmaceutical product comprising a compound of formula I or a salt, hydrate, or solvate thereof, for use in the treatment of invasive lobular breast cancer, tumors with a tumor-stromal ratio (TSR) > 50%; and / or pancreatic ductal adenocarcinoma; 【Chemistry 1】 During the ceremony, 【Chemistry 1-2】 represents a single bond or a double bond; Applicable R 1 , R 1 'and R 1 Each of these is -H, -Hallo, -CF 3 , -OCF 3 and -OC 1-3 Independently selected from the group consisting of alkyl groups; R 2 is selected from the group consisting of -H, -halo, -CF 3 , -OCF 3 and -OC 1-3 alkyl; R 3 -H, -Hallo, -C 1-3 Alkyl and -CF 3 Selected from the group consisting of R 2 and R 3 These, together with the C atoms to which they are bonded, form a six-membered aromatic or non-aromatic heterocycle; R 4 -H, -Hallo, -CF 3 , -OCF 3 and -OC 1-3 Selected from the group consisting of alkyl groups; Applicable R 5 , R 5 'and R 5 Each of these is -H, -OH, -CN, -C 1-3 Alkyl, -OC 1-3 Alkyl, -NR 6 R 7 , -SO 2 -NH 2 , -SO 2 -iPr, -Het 1 and - Selected independently from the group consisting of - C 1-3 Alkyl and -OC 1-3 Each alkyl group is either =O or -NR. 8 R 9 and -Het 1 It is optionally substituted with 1 to 3 substituents selected from the group consisting of; R 6 , R 7 , R 8 and R 9 -H and -C 1-3 Selected from the group consisting of alkyl groups; the -C 1-3 Each alkyl group is either =O or -NH. 2 , and -NH-iPr are optionally substituted with 1 to 3 substituents selected from the group consisting of, Het 1 Het is a 5- or 6-membered heterocycle having 1 to 3 heteroatoms selected from S, O, and N; where Het 1 Each of these is -C 1-3 Alkyl, -OC 1-3 Alkyl, -C 1-3 Alkyl-OH and -OC 1-3 It is optionally substituted with 1 to 3 substituents selected from the group consisting of alkyl-OH groups; In the formula, the R 1 , R 1 'and R 1 "At least one of them is -OC 1-3 Alkyl or -CF 3 It is; In the formula, R 2 and R 3 If it does not form a complex ring, [Chemistry 1-3] This represents a double bond.

2. A pharmaceutical product according to claim 1, R 2 and R 3 However, if the compound forms a six-membered aromatic or non-aromatic heterocycle, the compound is represented by formula II; 【Chemistry 2】 In the formula, X is O, S, NH, or NC 1-3 Selected from the group consisting of alkyl groups.

3. A pharmaceutical product according to claim 1 or 2, During the ceremony, 【Chemistry 2-1】 represents a single bond or a double bond; Applicable R 1 , R 1 'and R 1 Each of these is -H, -CF 3 and -OC 1-2 Independently selected from the group consisting of alkyl groups; R 2 is -H; R 3 is selected from the group consisting of -H and -Me; Or, R 2 and R 3 These, together with the C atoms to which they are bonded, form a six-membered aromatic or non-aromatic heterocycle. R 4 is -H; Applicable R 5 , R 5 'and R 5 Each of these is -H, -OH, -C 1-2 Alkyl, -NR 6 R 7 , -SO 2 -NH 2 ,-Het 1 and - Selected independently from the group consisting of - C 1-2 Each of the alkyl groups is =O and -Het 1 It is optionally substituted with 1 to 3 substituents selected from the group consisting of; R 6 and R 7 -H and -C 1-2 Selected from the group consisting of alkyl groups; the -C 1-2 Each alkyl group is optionally substituted with one to three substituents selected from the group consisting of =O and -NH-iPr; Het 1 Het is a six-membered heterocycle having 1 to 3 heteroatoms selected from O and N; where Het 1 Each of these is -C 1-2 Alkyl and -C 1-2 It is optionally substituted with 1 to 3 substituents selected from the group consisting of alkyl-OH groups; In the formula, the R 1 , R 1 ', and R 1 " at least one of which is -OC 1-2 alkyl; In the formula, R 2 and R 3 If it does not form a complex ring, 【Chemistry 2-2】 This represents a double bond.

4. A pharmaceutical product according to claim 1 or 2, During the ceremony, [Chemistry 2-3] represents a single bond or a double bond; The R 1 , R 1 ', and R 1 " each is independently selected from the group consisting of -H, -CF 3 and -OCH 3 ; R 2 is -H; R 3 is -H; Or, R 2 and R 3 These, together with the C atoms to which they are bonded, form a six-membered aromatic or non-aromatic heterocycle. R 4 is -H; Applicable R 5 , R 5 'and R 5 Each of these is -H, -OH, -C 1-2 Alkyl, -NR 6 R 7 , -SO 2 -NH 2 ,-Het 1 and - Selected independently from the group consisting of - C 1-2 Each of the alkyl groups is =O and -Het 1 It is optionally substituted with 1 to 3 substituents selected from the group consisting of; R 6 and R 7 -H and -C 1-2 Selected from the group consisting of alkyl groups; the -C 1-2 Each alkyl group is optionally substituted with one to three substituents selected from the group consisting of =O and -NH-iPr; Het 1 Het is selected from morpholinil and piperazinil; where Het 1 Each of these is -C 1-2 Alkyl and -C 1-2 It is optionally substituted with 1 to 3 substituents selected from the group consisting of alkyl-OH groups; In the formula, the R 1 , R 1 'and R 1 "At least one of them is -OCH 3 It is; In the formula, R 2 and R 3 If it does not form a complex ring, 【Chemistry 2-4】 This represents a double bond.

5. A pharmaceutical product according to claim 1 or 2, During the ceremony, 【Chemistry 2-5】 represents a single bond or a double bond; Applicable R 1 , R 1 'and R 1 Each of the following is -H and -OCH 3 Independently selected from the group consisting of; R 2 is -H; R 3 is -H; Or, R 2 and R 3 However, together with the C atoms to which they are bonded, they form a 6-membered aromatic or non-aromatic heterocycle. R 4 is -H; Applicable R 5 , R 5 'and R 5 Each of these is independently selected from the group consisting of -H, -OH, and -halo; In the formula, the R 1 , R 1 'and R 1 "At least one of them is -OCH 3 It is; In the formula, R 2 and R 3 If it does not form a complex ring, 【Chemistry 2-6】 This represents a double bond.

6. A pharmaceutical product according to claim 1 or 2, During the ceremony, Applicable R 1 , R 1 'and R 1 Each of these is -H and -OCH 3 Independently selected from the group consisting of; R 2 is -H; R 3 is -H; Or, R 2 and R 3 These, together with the C atoms to which they are bonded, form a six-membered aromatic or non-aromatic heterocycle. R 4 is -H; Applicable R 5 , R 5 'and R 5 Each of these is independently selected from the group consisting of -H, -OH, -F, and -Cl; In the formula, the R 1 , R 1 'and R 1 "At least one of them is -OCH 3 It is; In the formula, R 2 and R 3 If it does not form a complex ring, 【Chemistry 2-7】 This represents a double bond.

7. The pharmaceutical product according to claim 1 or 2, wherein the compound is represented by any one of the following formulas: (Ia), (IIa), or (IIb): 【Transformation 3】

8. The pharmaceutical product according to claim 1 or 2, wherein the compound or its salt, hydrate, or solvate is selected from the list including: 【Chemistry 4-1】 【Chemistry 4-2】 【Chemistry 4-3】 【Chemistry 4-4】

9. A compound or its salt, hydrate, or solvate, selected from the following list: 【Transformation 5】

10. A pharmaceutical composition comprising the compound described in claim 9 and a pharmaceutically acceptable excipient.

11. A pharmaceutical product comprising the compound described in claim 9 or the pharmaceutical composition described in claim 10, for use in humans or in veterinary medicine.

12. A pharmaceutical product comprising the compound described in claim 9 or the pharmaceutical composition described in claim 10, for use in the treatment of cancer.

13. A pharmaceutical comprising the compound according to claim 9 or the pharmaceutical composition according to claim 10 for use in the treatment of invasive lobular breast cancer, tumors having a tumor-stromal ratio (TSR) >50%; and / or pancreatic ductal adenocarcinoma.

14. A pharmaceutical product comprising a compound represented by either formula (IIa) or (IIb) below, for use as an anti-invasive compound for tumor cells in subjects with cancer: 【Transformation 6】 During the ceremony, Applicable R 1 , R 1 'and R 1 Each of these is -H, -Hallo, -CF 3 , -OCF 3 and -OC 1-3 Independently selected from the group consisting of alkyl groups; R 4 -H, -Hallo, -CF 3 , -OCF 3 and -OC 1-3 Selected from the group consisting of alkyl groups; Applicable R 5 , R 5 'and R 5 Each of these is -H, -OH, -CN, -C 1-3 Alkyl, -OC 1-3 Alkyl, -NR 6 R 7 , -SO 2 -NH 2 , -SO 2 -iPr, -Het 1 and - Selected independently from the group consisting of - C 1-3 Alkyl and -OC 1-3 Each alkyl group is either =O or -NR. 8 R 9 and -Het 1 Substitution is made optionally with 1 to 3 substituents selected from the group consisting of; R 6 , R 7 , R 8 and R 9 -H and C 1-3 Selected from the group consisting of alkyl groups; the -C 1-3 Each alkyl group is either =O or -NH. 2 , and 1 to 3 substituents selected from the group consisting of -NH-iPr, are optionally substituted. Het 1 This is a 5- or 6-membered heterocycle having 1 to 3 heteroatoms selected from S, O, and N, where the Het 1 Each of these is -C 1-3 Alkyl, -OC 1-3 Alkyl, -C 1-3 Alkyl-OH and -OC 1-3 It is optionally substituted with 1 to 3 substituents selected from the group consisting of alkyl-OH groups; In the formula, the R 1 , R 1 'and R 1 "At least one of them is -OC 1-3 Alkyl or -CF 3 That is the case.

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