Chalcone derivatives, modulators of fibroblasts for treatment of interstitial-rich tumors

Abstract

The present invention relates to the field of oncology and provides compounds, compositions, uses and methods for treating cancer, in particular mesenchymal-rich tumors, by selectively targeting cancer-associated fibroblasts (CAFs). CAF targeting is a strategy to enhance the efficacy of immunotherapy and may increase patient response. Furthermore, damage to normal tissue is reduced by this specificity of targets and related pathways.

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 a strategy to enhance the efficacy of immunotherapy and can improve patient response rates. In addition, through this specificity of targets and related pathways, damage to normal tissues is reduced. Background Art

[0002] Despite major advances in oncology, cancer remains the leading cause of death worldwide. In 2020, it caused nearly 10 million deaths, accounting for nearly 17% of global mortality. The most common cancers in terms of new cases in 2020 were breast cancer (2.26 million cases), lung cancer (2.21 million cases) and colorectal cancer (1.93 million cases). In terms of mortality, lung cancer (1.8 million deaths), colorectal cancer (916,000 deaths) and liver cancer (830,000 deaths) were the main cancer types. Breast cancer also remains a worldwide problem, with 685,000 deaths per year.

[0003] Cancer is an abnormal growth composed of different cell types. Cancer cells form an ecosystem with their tumor microenvironment (TME) or matrix: other cell types and acellular matrix that can play a pro-tumor or anti-tumor role. TME is a dynamic and complex organization of cellular and non-cellular components shaped by chemical and physical interactions between the cells of the tumor and the adjacent structures. It includes elements of the vascular system, immune cells (such as T and B lymphocytes, natural killer (NK) cells and macrophages) and stromal cells (including pericytes and fibroblasts) and acellular extracellular matrix (ECM). ECM plays the role of cell scaffold in TME: it provides structural support and regulates cell movement, proliferation and differentiation. It consists of a network of fibrins, glycoproteins, proteoglycans and polysaccharides with diverse physical and biochemical properties, and is also rich in growth factors.

[0004] The TME is an important and dynamic compartment that has a profound impact on all steps of tumor progression and therapeutic response. In a healthy state, the TME can help protect and reduce the invasiveness of tumor cells. However, crosstalk between tumor cells and components of the TME can lead to opposing responses that promote tumor development, growth, drug resistance, immunosuppression, and metastasis. Therefore, the latest insight in the field of oncology is that the cancer cell-centric treatment paradigm is often insufficient to eradicate malignancies. To improve cancer survival, there is an urgent unmet need for a range of therapeutic approaches that target various cell types and processes that are drivers of proto-tumor events in the TME.

[0005] Among the stromal cells that occupy the tumor microenvironment, cancer-associated fibroblasts (CAFs) are the most abundant and are closely associated with cancer progression. They contain various fibroblast populations of different cell origins that are activated during tumorigenesis (i.e., protumor CAFs). CAFs regulate the biology of tumor cells and other stromal cells through cell-cell contacts, the release of multiple regulatory factors (e.g., transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), and CXC-chemokine ligand (CXCL12)), and the synthesis and remodeling of the extracellular matrix. CAFs are drivers of protumor events such as growth, drug resistance, angiogenesis, immunosuppression, and metastasis.

[0006] The ECM is a non-cellular component of the TME. Collagen is the most important component 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, which is important in the regulation of ECM composition and can release ECM-bound soluble factors, such as growth factors.

[0007] In addition to being a supporting structure, ECM is also responsible for the dynamic compartmentalization part that promotes the malignant phenotype of cancer cells. The deposition of structural components (such as collagen, fibronectin and proteoglycan) increased by CAF and the secretion of cross-linking enzymes (such as transglutaminase (TGM) and lysyl oxidase (LOX)) lead to the formation of denser and harder TME and tumors in the process referred to as connective tissue formation. The physiological hardness of native tissue varies from 0.1kPa in brain tissue to 40kPa in bone. In cancer patients, the interstitial hardness in the invasion area is about 5kPa, and 400Pa in normal non-invasive interstitium. The higher hardness of tumor and the amount of fibroblasts in interstitium and related to the late stage of cancer, treatment resistance, immunosuppression, higher metastasis rate and poor patient prognosis. At present, fibroblast regulators are being studied as suitable treatment options for tumors with important fibroblasts (CAF).

[0008] An essential requirement for any CAF modulator to have practical therapeutic use in human cancer patients is pharmacological selectivity for CAFs relative to fibroblasts and other related cell types involved in normal physiology. Due to this desired pharmacological selectivity, the development of oncology therapeutics targeting CAFs faces significant obstacles. Targeting CAFs without damaging normal tissues remains an unanswered challenge in the field due to poor specificity of the target and related pathways.

[0009] It is an object of the present invention to provide compounds for selectively targeting CAFs and thereby improving the therapeutic outcome of cancer treatment. Summary of the invention

[0010] Therefore, in a first aspect, the present invention provides a compound of formula I or a salt, hydrate or solvate thereof for use in treating invasive lobular breast cancer, tumors with a tumor-stroma ratio (TSR)>50%; and / or pancreatic ductal adenocarcinoma,

[0011]

[0012] in

[0013] represents a single bond or a double bond;

[0014] Each of said R1, R1' and R1" is independently selected from the group consisting of: -H, -halo, -CF3, -OCF3 and -OC 1-3 alkyl;

[0015] R2 is selected from the group consisting of: -H, -halo, -CF3, -OCF3, and -OC 1-3 alkyl;

[0016] R3 is selected from the group consisting of: -H, -halo, -C 1-3 Alkyl and -CF3;

[0017] Or R2 and R3 together with the C atoms to which they are attached form a 6-membered aromatic or non-aromatic heterocyclic ring;

[0018] R4 is selected from the group consisting of: -H, -halo, -CF3, -OCF3, and -OC 1-3 alkyl;

[0019] Each of said R5, R5' and R5" is independently selected from the group consisting of: -H, -OH, -CN, -C 1-3 Alkyl, -OC 1-3 Alkyl, -NR6R7, -SO2-NH2, -SO2-iPr, -Het1 and -halogen; the -C 1-3 Alkyl and -OC 1-3 Each of the alkyl groups is optionally substituted with 1 to 3 substituents selected from the group consisting of =O, -NR8R9 and -Het1;

[0020] R6, R7, R8 and R9 are selected from the group consisting of: -H and -C 1-3 Alkyl; the -C 1-3 Each of the alkyl groups is optionally substituted with 1 to 3 substituents selected from the group consisting of =O, -NH2 and -NH-iPr,

[0021] Het1 is a 5-membered or 6-membered heterocyclic ring having 1 to 3 heteroatoms selected from S, O and N; wherein each of said Het1 is optionally replaced by 1 to 3 heteroatoms selected from -C 1-3 Alkyl, and -OC 1-3 Alkyl, -C 1-3 Alkyl -OH and -OC 1-3 Substitution by a substituent of the group consisting of alkyl-OH;

[0022] wherein at least one of R1, R1′ and R1″ is -OC 1-3 Alkyl or -CF3;

[0023] When R2 and R3 do not form a heterocyclic ring, Represents a double bond.

[0024] In a particular embodiment, the compounds for use as defined herein are those compounds wherein:

[0025] When R2 and R3 form a 6-membered aromatic or non-aromatic heterocyclic ring, the compound is represented by formula (II):

[0026]

[0027] Wherein X is selected from O, S, NH or NC 1-3 Alkyl group.

[0028] In another specific embodiment, the compounds for use as defined herein, in particular compounds of formula I or II, are those compounds wherein:

[0029] represents a single bond or a double bond;

[0030] Each of said R1, R1' and R1" is independently selected from the group consisting of: -H, -CF3 and -OC 1-2 alkyl;

[0031] R2 is -H;

[0032] R3 is selected from the group consisting of: -H and -Me;

[0033] Or R2 and R3 together with the C atoms to which they are attached form a 6-membered aromatic or non-aromatic heterocyclic ring, and R4 is -H;

[0034] Each of said R5, R5' and R5" is independently selected from the group consisting of: -H, -OH, -C 1-2 Alkyl, -NR6R7, -SO2-NH2, -Het1 and -halo; the -C 1-2Each of the alkyl groups is optionally substituted with 1 to 3 substituents selected from the group consisting of =0 and -Het1;

[0035] R6 and R7 are selected from the group consisting of: -H and -C 1-2 Alkyl; the -C 1-2 Each of the alkyl groups is optionally substituted with 1 to 3 substituents selected from the group consisting of =O and -NH-iPr,

[0036] Het1 is a 6-membered heterocyclic ring having 1 to 3 heteroatoms selected from O and N; wherein each of said Het1 is optionally replaced by 1 to 3 heteroatoms selected from -C 1-2 Alkyl and -C 1-2 Substitution by a substituent of the group consisting of alkyl-OH;

[0037] wherein at least one of R1, R1′ and R1″ is -OC 1-2 alkyl;

[0038] When R2 and R3 do not form a heterocyclic ring, Represents a double bond.

[0039] In yet another specific embodiment, the compounds for use as defined herein are those compounds, wherein:

[0040] represents a single bond or a double bond;

[0041] Each of said R1, R1' and R1" is independently selected from the group consisting of: -H, -CF3 and -OCH3;

[0042] R2 is -H;

[0043] R3 is -H;

[0044] Or R2 and R3 together with the C atoms to which they are attached form a 6-membered aromatic or non-aromatic heterocyclic ring, and R4 is -H;

[0045] Each of said R5, R5' and R5" is independently selected from the group consisting of: -H, -OH, -C 1-2 Alkyl, -NR6R7, -SO2-NH2, -Het1 and -halo; the -C 1-2 Each of the alkyl groups is optionally substituted with 1 to 3 substituents selected from the group consisting of =0 and -Het1;

[0046] R6 and R7 are selected from the group consisting of: -H and -C 1-2 Alkyl; the -C 1-2 Each of the alkyl groups is optionally substituted with 1 to 3 substituents selected from the group consisting of =O and -NH-iPr,

[0047] Het1 is selected from morpholinyl and piperazinyl; wherein each of said Het1 is optionally substituted by 1 to 3 selected from -C 1-2 Alkyl and -C 1-2 Substitution by a substituent of the group consisting of alkyl-OH;

[0048] wherein at least one of R1, R1' and R1" is -OCH3;

[0049] When R2 and R3 do not form a heterocyclic ring, Represents a double bond.

[0050] In another embodiment, the invention provides a compound as defined herein for use, in particular a compound of formula I or II, wherein:

[0051] represents a single bond or a double bond;

[0052] Each of said R1, R1' and R1" is independently selected from the group consisting of: -H and -OCH3;

[0053] R2 is -H;

[0054] R3 is -H;

[0055] Or R2 and R3 together with the C atoms to which they are attached form a 6-membered aromatic or non-aromatic heterocyclic ring, and R4 is -H;

[0056] Each of said R5, R5' and R5" is independently selected from the group consisting of: -H, -OH and -halo;

[0057] wherein at least one of R1, R1' and R1" is -OCH3;

[0058] When R2 and R3 do not form a heterocyclic ring, Represents a double bond.

[0059] In a further embodiment, the invention provides a compound as defined herein for use, in particular a compound of formula I or II, wherein:

[0060] Each of said R1, R1' and R1" is independently selected from the group consisting of: -H and -OCH3;

[0061] R2 is -H;

[0062] R3 is -H;

[0063] Or R2 and R3 together with the C atoms to which they are attached form a 6-membered aromatic or non-aromatic heterocyclic ring, and R4 is -H;

[0064] Each of said R5, R5' and R5" is independently selected from the group consisting of: -H, -OH, -F and -Cl;

[0065] wherein at least one of R1, R1' and R1" is -OCH3;

[0066] When R2 and R3 do not form a heterocyclic ring, Represents a double bond.

[0067] The compound for use as defined herein may be represented by any one of formula (Ia), (IIa) or (IIb) in a specific embodiment

[0068]

[0069]

[0070] The present invention also provides a compound for use as defined herein, wherein the compound or a salt, hydrate or solvate thereof is selected from the list comprising:

[0071]

[0072]

[0073]

[0074]

[0075] In another aspect, the present invention provides a compound or a salt, hydrate or solvate thereof selected from the list comprising:

[0076]

[0077] The present invention also provides a pharmaceutical composition comprising a compound as defined herein and a pharmaceutically acceptable excipient.

[0078] In another embodiment, the invention provides compounds and compositions as defined herein; for use in human or veterinary medicine; in particular for use in the treatment of cancer; more particularly for use in preventing, treating or reducing tumor-associated desmoplasia in a subject suffering from cancer, even more particularly for use in the treatment of invasive lobular breast cancer, tumors with a tumor-stroma ratio (TSR)>50%; and / or pancreatic ductal adenocarcinoma.

[0079] The present invention also provides a method for preventing, reducing and / or treating tumor-associated desmoplasia in a subject suffering from cancer, in particular preventing or treating invasive lobular breast cancer, tumors with a tumor-stroma ratio (TSR)>50%; and / or pancreatic ductal adenocarcinoma, the method comprising administering to a patient in need thereof a therapeutically effective amount of a compound or pharmaceutical composition as defined herein.

[0080] In another aspect, the present invention provides a compound for use as an anti-invasive compound for tumor cells; the compound is represented by any one of formula (IIa) or (IIb)

[0081]

[0082]

[0083] in

[0084] Each of said R1, R1' and R1" is independently selected from the group consisting of: -H, -halo, -CF3, -OCF3 and -OC 1-3 alkyl;

[0085] R4 is selected from the group consisting of: -H, -halo, -CF3, -OCF3 and -OC 1-3 alkyl;

[0086] Each of said R5, R5' and R5" is independently selected from the group consisting of: -H, -OH, -CN, -C 1-3 Alkyl, -OC 1-3 Alkyl, -NR6R7, -SO2-NH2, -SO2-iPr, -Het1 and -halogen; the -C 1-3 Alkyl and -OC 1-3 Each of the alkyl groups is optionally substituted with 1 to 3 substituents selected from the group consisting of =O, -NR8R9 and -Het1;

[0087] R6, R7, R8 and R9 are selected from the group consisting of: -H and -C 1-3 Alkyl; the -C 1-3 Each of the alkyl groups is optionally substituted with 1 to 3 substituents selected from the group consisting of =O, -NH2 and -NH-iPr,

[0088] Het1 is a 5- or 6-membered heterocyclic ring having 1 to 3 heteroatoms selected from O and N; wherein each of said Het1 is optionally replaced by 1 to 3 heteroatoms selected from -C 1-3 Alkyl, -OC 1-3 Alkyl, -C 1-3 Alkyl -OH and -OC 1-3Substitution by a substituent of the group consisting of alkyl-OH;

[0089] wherein at least one of R1, R1′ and R1″ is -OC 1-3 Alkyl or -CF3. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] With specific reference to the drawings, it should be noted that the details shown are by way of example and for the purpose of illustrative discussion of various embodiments of the invention. The description taken in conjunction with the drawings makes apparent to those skilled in the art how several forms of the invention may be implemented in practice.

[0091] Figure 1 .Stiffness measurements of spheroids treated with A1, A8, A14, or B1 for 48 h during spheroid formation. AC: 13B2hTERT breast CAFs, D: CT5.3 hTERT colon CAFs, E: CAF03 glioblastoma CAFs, F: HCA2 hTERT dermal fibroblasts, G: MCF-7 / 6 breast cancer cells, H: primary glioblastoma (GBM) cells, I: primary human atrial fibroblasts. Data are presented as mean ± SD of three technical replicates. *P < 0.05, **P < 0.01.

[0092] Figure 2 .Stiffness measurement of spheroids treated with 1 μM A1, 1 μM B1, 10 μM blebbistatin, 10 μM cytochalasin D, or 10 μM Y-27632 for 48 h during spheroid formation. A: 13B2 hTERT breast CAFs, B: CAF03 glioblastoma CAFs, C: HCA2 hTERT dermal fibroblasts, D: MCF-7 / 6 breast cancer cells, E: primary glioblastoma cells, F: primary human atrial fibroblasts. Data are presented as mean ± SD of three technical replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0093] Figure 3 Additional selectivity data for compounds of the invention: Comparison of stiffness measurements of spheroids of 13B2 hTERT breast CAFs and HCA2 hTERT dermal fibroblasts treated for 48 h with 1 μM A1, 1 μM A9, 1 μM A10, 1 μM A3, 1 μM A7, 1 μM B3, 1 μM B7 or 10 μM Y-27632 during spheroid formation. Data are presented as mean ± SD of three technical replicates.

[0094] Figure 4 : 4T1 animal model: Tolerance of A1 or B1 treatment compared with DMSO control. A: body weight; B: body temperature.

[0095] Figure 5 .4T1 animal model: A1 or B1 treatment reduced metastatic burden compared with DMSO. A: Axillary lymph node ex vivo bioluminescence; B: Lung ex vivo bioluminescence; C: Liver ex vivo bioluminescence.

[0096] Figure 6 .4T1 animal model: Immunohistological staining of primary tumor material when treated with A1 or B1 compared with control animals. A: α-SMA; B: PDPN; C: FAP-α; D: Masson's trichrome staining (collagen deposition); E: CD11b; F: Ly6G; G: CD163. *P<0.05, **P<0.01, ***P<0.001.

[0097] Figure 7 .4T1 animal model: Immunohistochemical staining of target organ metastasis when treated with A1 or B1 compared with control animals. A: α-SMA, liver; B: PDPN, liver; C: Masson trichrome staining (collagen deposition), liver; D: α-SMA, axillary lymph nodes; E: PDPN, axillary lymph nodes; F: Masson trichrome staining, axillary lymph nodes; G: Masson trichrome staining, lung. *P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0098] The present invention will now be further described. In the following paragraphs, different aspects of the present invention are defined in more detail. Unless explicitly indicated to the contrary, each aspect so defined can be combined with any one or more aspects. Unless the context clearly specifies otherwise, the singular forms "one", "a kind" and "the" as used in the specification and claims include plural indicators. For example, "a compound" means one compound or more than one compound. Throughout the specification and claims herein, the word "includes / comprising" and other forms of the word, such as "comprising / comprises" are meant to include but are not limited to, and are not intended to exclude, for example, other additives, components, integers or steps. As used herein, "about", "approximately", "substantially" and "significantly / obviously" will be understood by those of ordinary skill in the art and will vary to a certain extent in the context in which they are used. If there is a person of ordinary skill in the art who is not clear about the use of the term in the context given its use, "about" and "approximately" will mean plus or minus 10% of a specific value or term. The above and other terms used herein are well understood by those skilled in the art. All references cited herein and the teachings specifically mentioned are incorporated herein by reference in their entirety.

[0099] The present invention provides CAF (cancer associated fibroblast) modulating compounds, and more particularly provides their use in treating cancer, more particularly provides their use in reducing the stiffness (also known as desmoplasia) of the tumor microenvironment (TME).

[0100] Fibroblasts represent one of the most abundant cell types in the stroma and are generally defined by their combination of morphology, tissue location, and lack of lineage markers of epithelial cells, endothelial cells, and leukocytes.

[0101] Like fibroblasts in the stroma, cancer-associated fibroblasts (CAFs) are a major component in the tumor microenvironment (TME). Many of the features defined for normal fibroblasts also apply to CAFs. The expression "cancer-associated fibroblasts" is used as an umbrella term to describe a complex population of mesenchymal-like cells. In Han et al. (Han, Liu, and Yin 2020), it is even considered a universal label for "all fibroblasts found in and around tumor tissues." In addition to their elongated 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 in cancer cells. The latter point is important because it excludes cancer cells that have undergone epithelial to mesenchymal transition (EMT). To make the interpretation narrower, lineage exclusion is typically combined with the presence of mesenchymal markers (usually vimentin). It is already clear that there are subtypes of CAFs, each with its specific markers. In practice, any mesenchymal cell derived from a tumor that meets the above criteria is considered a CAF.

[0102] The uniqueness of the compounds of the present invention is that they are selective modulators of CAFs, thereby not targeting or less targeting fibroblasts and other related cell types involved in normal physiology. Therefore, the compounds of the present invention can be used to target CAFs without damaging normal tissues. It has been shown that the compounds provided herein functionally inhibit CAFs at nanomolar concentrations, which is highly desirable for active pharmaceutical ingredients.

[0103] It is well known that increased tumor and ECM hardness are associated with negative prognosis because it promotes tumor growth, progression, treatment resistance, immunosuppression and metastasis. Hardness is mainly regulated by the effect of CAF in TME. Therefore, stiffness regulation is the fitting readout (fitting read-out) of activity in CAF regulator screening. In order to evaluate stiffness regulation, the hardness of the spheroid of CAF cells treated with test compounds is explored using atomic force microscopy (AFM) indentation, and compared with untreated cells. The AFM measurement of the hardness (elasticity) of cells and spheroids is a technology well known to those skilled in the art. AFM measures the force required for the generation of a given deflection, and the reading is also referred to as Young's modulus. AFM is widely used in biology and cancer research: the physicochemical properties of the changes in living cells serve as the readings of the complex physiological processes of change.

[0104] The selectivity of CAFs over normal fibroblasts and other cell types was assessed by performing the same experiments with normal fibroblasts and other cell types, where compounds were considered selective for CAFs if they modulated only CAF stiffness.

[0105] The tumor stroma can limit the access of therapeutic agents to their target tissues in three ways: fibrosis, high interstitial pressure, and degradation of drugs by interstitial enzymes. The accumulation of rigid ECM (fibrosis) around and throughout tumors creates a physical barrier that reduces the diffusion of therapeutic agents into cancer cells. Similarly, the tumor stroma can limit the access and action of immune cells in two ways: fibrosis, which creates a physical barrier that reduces immune cell infiltration, and chemical and physical cues that favor protumor immunosuppressive immune cell populations and induce immune cell polarization or differentiation into an immunosuppressive phenotype.

[0106] The present invention provides compounds as defined herein for treating cancer by reducing the rigid ECM and immunosuppressive clues / polarization / differentiation, particularly in cancers with high and / or dense stroma content. In cancers where the quantitative content of fibroblasts may vary between patients, a convenient method for determining the amount of fibroblasts producing desmoplastic stroma components is to determine stroma content. Cancers with high stroma content, such as tumor stroma ratios (TSR) of 40% or more, particularly 50% or more, more particularly 60% or more, and even more particularly 70% or more cancers 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, higher stroma content, i.e., higher TSR, is associated with the amount of cancer-associated fibroblasts (CAF) and poor prognosis, and therefore a suitable population for fibroblast targeted therapy is determined. As used herein, "a tumor with a particular tumor-stroma ratio (TSR)" refers to a cancer with a high stromal content, particularly 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.

[0107] The amount of stroma in a patient's tumor can be easily assessed by a person skilled in the art on a hematoxylin-eosin (HE)-stained section, and its assessment is clinically applied (Wu et al., 2016). The tumor stroma ratio (TSR) is defined as the ratio of stroma to tumor cells and has been considered 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 is composed of stroma and 80% of the area is composed of tumor cells. Therefore, the analysis of TSR can be easily repeated and routinely analyzed. TSR was validated by an international research group and was shown to be a robust parameter with good interobserver consistency. The UNITED study (Uniform Noting for International Application of the Tumor-stromaratio as Easy Diagnostic tool) is being developed to make TSR consistent with current clinical implementation guidelines. In more detail, TSR is determined from the invasive part of the tumor where the most stroma is present, because areas with a large amount of stroma are decisive for prognosis. As described in detail by Kemi et al. 2018, low magnification (total magnification 10-50x) was used to identify tumor portions with a large portion of stroma. A single field of view with a total magnification of 100x in which tumor cells were present at all four corners of the field of view was used for analysis. Smooth muscle, mucin, and necrosis were excluded from the analysis. The remaining area consisted of tumor cells and stroma and was used as the analysis area. The area of ​​the stroma compared to the entire analysis area was estimated and scored in 10% intervals using manual assessment or computer-assisted analysis. As previously described, a score of 20% means that 20% of the analysis area consists of stroma and 80% consists of tumor cells.

[0108] When TSR is used during diagnosis and treatment decision-making, a ratio of 50% is defined as the cutoff point. Patients are divided into cases with poor stroma (the proportion of stroma ≤50%) and cases with rich stroma (the proportion of stroma>50%). HE-stained TSR classification has been shown to distinguish high-risk patients with rich stroma (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. As can be seen from the previous paragraphs, in these cancers, a TSR of>50% indicates patients with fibroblast-driven tumor progression, and therefore indicates a target population for treatment with the compounds of the present invention.

[0109] In addition to the above cancer list, there are other cancer types in which (proto-tumor) CAFs are present in large quantities, regardless of TSR status. These cancer types are pancreatic ductal adenocarcinoma (PDAC) and invasive lobular breast cancer (ILC). PDAC and ILC tumors show elevated 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 with these cancers also form a target population for treatment with the compounds of the present invention.

[0110] The basic requirement for any CAF regulator with practical therapeutic use in human cancer patients is the pharmacological selectivity of CAF relative to fibroblasts and other related cell types involved in normal physiology. The example of such a related cell type is smooth muscle cells. Cancer-associated fibroblasts share most druggable pathways and markers with normal fibroblasts and these related cell types. Normal fibroblasts play an important role as structural components and in wound healing. They are crucial in the immune response to tissue damage: fibroblasts from different anatomical parts of the body express many genes encoding immune mediators and proteins. Smooth muscle cells mainly help regulate blood pressure and blood flow to vascular beds, and help the phasic contraction of the digestive tract.

[0111] Therefore, in a first aspect, the present invention provides a compound of formula I or a salt, hydrate or solvate thereof for use in treating invasive lobular breast cancer, tumors with a tumor-stroma ratio (TSR)>50%; and / or pancreatic ductal adenocarcinoma,

[0112]

[0113] in

[0114] represents a single bond or a double bond;

[0115] Each of said R1, R1' and R1" is independently selected from the group consisting of: -H, -halo, -CF3, -OCF3 and -OC 1-3 alkyl;

[0116] R2 is selected from the group consisting of: -H, -halo, -CF3, -OCF3 and -OC 1-3 alkyl;

[0117] R3 is selected from the group consisting of: -H, -halo, -C 1-3 Alkyl and -CF3;

[0118] or R2 and R3 together with the C atoms to which they are attached form a 6-membered aromatic or non-aromatic heterocyclic ring;

[0119] R4 is selected from the group consisting of: -H, -halo, -CF3, -OCF3 and -OC 1-3 alkyl;

[0120] Each of said R5, R5' and R5" is independently selected from the group consisting of: -H, -OH, -CN, -C 1-3 Alkyl, -OC 1-3 Alkyl, -NR6R7, -SO2-NH2, -SO2-iPr, -Het1 and -halogen; the -C 1-3 Alkyl and -OC 1-3 Each of the alkyl groups is optionally substituted with 1 to 3 substituents selected from the group consisting of =O, -NR8R9 and -Het1;

[0121] R6, R7, R8 and R9 are selected from the group consisting of: -H and -C 1-3 Alkyl; the -C 1-3 Each of the alkyl groups is optionally substituted with 1 to 3 substituents selected from the group consisting of =O, -NH2 and -NH-iPr,

[0122] Het1 is a 5-membered or 6-membered heterocyclic ring having 1 to 3 heteroatoms selected from S, O and N; wherein each of said Het1 is optionally replaced by 1 to 3 heteroatoms selected from -C 1-3 Alkyl, -OC 1-3 Alkyl, -C 1-3 Alkyl -OH and -OC 1-3 Substitution by a substituent of the group consisting of alkyl-OH;

[0123] wherein at least one of R1, R1′ and R1″ is -OC 1-3 Alkyl or -CF3;

[0124] When R2 and R3 do not form a heterocyclic ring, Represents a double bond.

[0125] The term "alkyl" by itself or as part of another substituent refers to a radical of the formula C x H 2x+1 A fully saturated hydrocarbon of , wherein x is a number greater than or equal to 1. Typically, the alkyl groups of the present invention contain 1 to 3 carbon atoms. Alkyl groups may be straight or branched and may be substituted as indicated herein. When a subscript is used after a carbon atom herein, the subscript refers to the number of carbon atoms that the group may contain. 1-3 Alkyl includes all straight-chain, branched or cyclic alkyl groups having 1 to 3 carbon atoms and thus includes methyl, ethyl, n-propyl, cyclopropyl and isopropyl.

[0126] The term "optionally substituted alkyl" refers to an alkyl group optionally substituted with one or more substituents (e.g., 1 to 3 substituents, e.g., 1, 2 or 3 substituents or 1 to 2 substituents) at any available point of attachment. Non-limiting examples of such substituents include oxo, amides, amines, aromatic and non-aromatic heterocycles, and the like.

[0127] The term "halo" or "halogen" as a group or part of a group is a general term for fluoro, chloro, bromo or iodo. In a specific embodiment, halo is fluoro or chloro; preferably fluoro.

[0128] The terms "heterocyclyl", "heterocycle" or "heterocyclic" as used herein alone or as part of another group refer to aromatic or non-aromatic cyclic groups (e.g., 5-6 membered monocyclic ring systems) having 1 to 3, such as 1, 2 or 3 heteroatoms in a ring containing carbon atoms. The heteroatoms may be selected from nitrogen atoms, oxygen atoms and / or sulfur atoms, wherein the nitrogen and sulfur heteroatoms may be optionally oxidized and the nitrogen heteroatoms may be optionally quaternized. In a specific embodiment, the heteroatoms are selected from N and O atoms. Where valence permits, the heterocyclic group may be attached to any heteroatom or carbon atom of the ring. An optionally substituted heterocycle refers to a heterocycle that optionally has one or more substituents (e.g., 1 to 3 substituents, or, for example, 1, 2 or 3), such as selected from -C 1-3 Alkyl, -OC 1-3 Alkyl, -C 1-3 Alkyl -OH and -OC 1-3 Alkyl-OH. Exemplary heterocyclic groups include piperazinyl and morpholinyl.

[0129] When, in the context of the present invention, R2 and R3 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 compounds defined by either of the formulae (IIa) or (IIb):

[0130]

[0131]

[0066] The term "oxo" as used herein refers to the group =0.

[0132] As used herein, the term "alkoxy" or "alkyloxy" refers to a radical having the formula -OR b The residues, R b Preferably, the alkoxy group is C 1-3 Alkoxy, C 1-2 Alkoxy or methoxy. Non-limiting examples of suitable alkoxy groups include methoxy, ethoxy, propoxy or isopropoxy.

[0133] 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., adding water or water elements (i.e., H and OH) to a molecular entity. The compound of the present invention can also be provided in the form of a pharmaceutically acceptable salt. 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, which retains the desired biological activity of the parent compound and exhibits limited toxicological effects on normal cells. The 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 acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, and polyglutamic acid.

[0134] The compounds provided herein can be prepared using the reaction schemes and synthetic schemes described below, employing techniques available in the art, using readily available starting materials.

[0135] In a specific embodiment, the present invention provides a compound of formula I or a salt, hydrate or solvate thereof for use in treating invasive lobular breast cancer, tumors with a tumor-stroma ratio (TSR)>50%; and / or pancreatic ductal adenocarcinoma,

[0136]

[0137] One or more of the following applies:

[0138] represents a single bond or a double bond;

[0139] Each of said R1, R1' and R1" is independently selected from the group consisting of: -H, -halo, -CF3, -OCF3 and -OC 1-3 Alkyl; especially -H, -CF3 and -OC 1-2 Alkyl; more particularly -H and -OCH3;

[0140] R2 is selected from the group consisting of: -H, -halo, -CF3, -OCF3 and -OC 1-3 Alkyl; especially -H;

[0141] R3 is selected from the group consisting of: -H, -halo, -C 1-3 Alkyl and -CF3; especially -H and -Me;

[0142] Or R2 and R3 together with the C atoms to which they are attached form a 6-membered aromatic or non-aromatic heterocyclic ring;

[0143] R4 is selected from the group consisting of: -H, -halo, -CF3, -OCF3 and -OC 1-3 Alkyl; especially -H,

[0144] Each of said R5, R5' and R5" is independently selected from the group consisting of: -H, -OH-CN, -C 1-3 Alkyl, -OC 1-3 Alkyl, -NR6R7, -SO2-NH2, -SO2-iPr, -Het1 and -halogen; the -C 1-3 Alkyl and -OC 1-3 Each of the alkyl groups is optionally substituted with 1 to 3 substituents selected from the group consisting of =O, -NR8R9 and -Het1; in particular, each of said R5, R5' and R5" is independently selected from the group consisting of: -H, -OH, -C 1-2 Alkyl, -NR6R7, -SO2-NH2, -Het1 and -halo; the -C 1-2 Each of the alkyl groups is optionally substituted with 1 to 3 substituents selected from the group consisting of =O and -Het1; more particularly each of said R5, R5' and R5" is independently selected from the group consisting of: -H, -OH and -halo, such as -Cl and -F;

[0145] R6, R7, R8 and R9 are selected from the group consisting of: -H and -C 1-3 Alkyl; the -C 1-3 Each of the alkyl groups is optionally substituted with 1 to 3 substituents selected from the group consisting of =O, -NH2 and -NH-iPr, in particular R6 and R7 are selected from the group consisting of: -H and -C 1-2 Alkyl; the -C 1-2 Each of the alkyl groups is optionally substituted with 1 to 3 substituents selected from the group consisting of =O and -NH-iPr,

[0146] Het1 is a 5-membered or 6-membered heterocyclic ring having 1 to 3 heteroatoms selected from S, O and N; wherein each of said Het1 is optionally replaced by 1 to 3 heteroatoms selected from -C 1-3 Alkyl, and -OC 1-3 Alkyl, -C 1-3 Alkyl -OH and -OC 1-3 substituted with a substituent of the group consisting of alkyl-OH; in particular a 6-membered heterocycle having 1 to 3 heteroatoms selected from O and N; wherein each of said Het1 is optionally substituted with 1 to 3 substituents selected from -C 1-2 Alkyl and -C 1-2 substituted by a substituent consisting of: -C 1-2 Alkyl and -C1-2 Alkyl-OH substituted morpholinyl and piperazinyl,

[0147] wherein at least one of R1, R1′ and R1″ is -OC 1-3 Alkyl or -CF3; especially -OC 1-2 Alkyl; more particularly -OCH3,

[0148] When R2 and R3 do not form a heterocyclic ring, Represents a double bond.

[0149] In a particular embodiment, the compounds for use as defined herein are those compounds wherein:

[0150] When R2 and R3 form a 6-membered aromatic or non-aromatic heterocyclic ring, the compound is represented by formula (II):

[0151]

[0152] Wherein X is selected from O, S, NH or NC 1-3 Alkyl group.

[0153] In another specific embodiment, the compounds for use as defined herein, in particular of formula I or II, are those compounds wherein:

[0154] represents a single bond or a double bond;

[0155] Each of said R1, R1' and R1" is independently selected from the group consisting of: -H, -CF3 and -OC 1-2 alkyl;

[0156] R2 is -H;

[0157] R3 is selected from the group consisting of: -H and -Me;

[0158] Or R2 and R3 together with the C atoms to which they are attached form a 6-membered aromatic or non-aromatic heterocyclic ring, and R4 is -H;

[0159] Each of said R5, R5' and R5" is independently selected from the group consisting of: -H, -OH, -C 1-2 Alkyl, -NR6R7, -SO2-NH2, -Het1 and -halo; the -C 1-2 Each of the alkyl groups is optionally substituted with 1 to 3 substituents selected from the group consisting of =0 and -Het1;

[0160] R6 and R7 are selected from the group consisting of: -H and -C 1-2 Alkyl; the -C 1-2Each of the alkyl groups is optionally substituted with 1 to 3 substituents selected from the group consisting of =O and -NH-iPr,

[0161] Het1 is a 6-membered heterocyclic ring having 1 to 3 heteroatoms selected from O and N; wherein each of said Het1 is optionally replaced by 1 to 3 heteroatoms selected from -C 1-2 Alkyl and -C 1-2 Substitution by a substituent of the group consisting of alkyl-OH;

[0162] wherein at least one of R1, R1′ and R1″ is -OC 1-2 alkyl;

[0163] When R2 and R3 do not form a heterocyclic ring, Represents a double bond.

[0164] In yet another specific embodiment, the compounds for use as defined herein, in particular of formula I or II, are those compounds wherein:

[0165] represents a single bond or a double bond;

[0166] Each of said R1, R1' and R1" is independently selected from the group consisting of: -H, -CF3 and -OCH3;

[0167] R2 is -H;

[0168] R3 is -H;

[0169] Or R2 and R3 together with the C atoms to which they are attached form a 6-membered aromatic or non-aromatic heterocyclic ring, and R4 is -H;

[0170] Each of said R5, R5' and R5" is independently selected from the group consisting of: -H, -OH, -C 1-2 Alkyl, -NR6R7, -SO2-NH2, -Het1 and -halo; the -C 1-2 Each of the alkyl groups is optionally substituted with 1 to 3 substituents selected from the group consisting of =0 and -Het1;

[0171] R6 and R7 are selected from the group consisting of: -H and -C 1-2 Alkyl; the -C 1-2 Each of the alkyl groups is optionally substituted with 1 to 3 substituents selected from the group consisting of =O and -NH-iPr,

[0172] Het1 is selected from morpholinyl and piperazinyl; wherein each of said Het1 is optionally substituted by 1 to 3 selected from -C 1-2 Alkyl and -C 1-2Substitution by a substituent of the group consisting of alkyl-OH;

[0173] wherein at least one of R1, R1' and R1" is -OCH3;

[0174] When R2 and R3 do not form a heterocyclic ring, Represents a double bond.

[0175] In another embodiment, the invention provides a compound as defined herein for use, in particular of formula I or II, wherein:

[0176] represents a single bond or a double bond;

[0177] Each of said R1, R1' and R1" is independently selected from the group consisting of -H, - and -OCH3;

[0178] R2 is -H;

[0179] R3 is -H;

[0180] or R2 and R3 together with the C atoms to which they are attached form a 6-membered aromatic or non-aromatic heterocyclic ring,

[0181] R4 is -H;

[0182] Each of said R5, R5' and R5" is independently selected from the group consisting of: -H, -OH and -halo;

[0183] wherein at least one of R1, R1' and R1" is -OCH3;

[0184] When R2 and R3 do not form a heterocyclic ring, Represents a double bond.

[0185] In a further embodiment, the invention provides compounds as defined herein for use, in particular of formula I or II, wherein:

[0186] Each of said R1, R1' and R1" is independently selected from the group consisting of: -H, - and -OCH3;

[0187] R2 is -H;

[0188] R3 is -H;

[0189] Or R2 and R3 together with the C atoms to which they are attached form a 6-membered aromatic or non-aromatic heterocyclic ring, and R4 is -H;

[0190] Each of said R5, R5' and R5" is independently selected from the group consisting of: -H, -OH, -F and -Cl;

[0191] wherein at least one of R1, R1' and R1" is -OCH3;

[0192] When R2 and R3 do not form a heterocyclic ring, Represents a double bond.

[0193] Furthermore, it has been found that a combination of one or more methoxy groups on one aromatic ring of the molecule and one or more halogens on another aromatic ring of the molecule, with or without hydroxyl groups, is advantageous for the desired activity.

[0194] Accordingly, the present invention provides a compound of formula I or II for use as defined herein, wherein:

[0195] Each of said R1, R1' and R1" is independently selected from the group consisting of: -H, - and -OCH3;

[0196] R2 is -H;

[0197] R3 is -H;

[0198] or R2 and R3 together with the C atoms to which they are attached form a 6-membered aromatic or non-aromatic heterocyclic ring,

[0199] R4 is -H;

[0200] Each of said R5, R5' and R5" is independently selected from the group consisting of: -H, -OH, -F and -Cl;

[0201] wherein at least one of R1, R1′ and R1″ is —OCH3; and wherein R5 is —OH.

[0202] When R2 and R3 do not form a heterocyclic ring, Represents a double bond.

[0203] The compound for use as defined herein may in a particular embodiment be represented by any one of formula (Ia), (IIa) or (IIb), including the R groups and substituents as defined herein for formula I and II, respectively.

[0204]

[0205] The present invention also provides a compound as defined herein for use or a salt, hydrate or solvate thereof, wherein the compound is selected from the list comprising:

[0206]

[0207]

[0208]

[0209]

[0210] In another aspect, the present invention provides a compound or a salt, hydrate or solvate thereof, wherein the compound is selected from the list comprising:

[0211]

[0212]

[0213]

[0214]

[0215] In a very specific embodiment, the present invention provides a compound or a salt, hydrate or solvate thereof, wherein the compound is selected from the list comprising:

[0216]

[0217] In order to use the compound of formula as provided herein for therapeutic treatment (including prophylactic treatment) of subjects such as animals or humans, it is usually formulated as a pharmaceutical composition according to standard pharmaceutical practice. Therefore, the present invention also provides a pharmaceutical composition comprising a compound as defined herein and a pharmaceutically acceptable excipient.

[0218] The compounds of the present invention can be introduced into preparations for all routes of administration, including, for example, oral, topical and parenteral, including intravenous, intramuscular, ocular or ophthalmic, 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 preparations can be administered via topical, parenteral, intravenous, intramuscular, transdermal, buccal, subcutaneous, suppository or other routes, including ocular or ophthalmic routes. Intravenous and intramuscular preparations are preferably administered in sterile saline. The compounds of the present invention are subjected to small modifications to make them more soluble in water or other vehicles, for example, small modifications (salt preparations, esterification, etc.) well known to those skilled in the art can be easily achieved. It is also well within the technical scope of those skilled in the art to modify the route of administration and dosage regimen of a particular compound to control the pharmacokinetics of the compounds of the present invention to obtain the maximum beneficial effect on the patient.

[0219] The compositions of the invention may be in a form suitable for oral use (e.g., as tablets, lozenges, 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 finely divided powders or liquid aerosols), administration by insufflation (e.g., as finely divided powders), or for parenteral administration (e.g., as sterile aqueous or oily solutions for intravenous, subcutaneous, or intramuscular administration, or as suppositories for rectal administration). For example, compositions intended for oral use may contain, for example, one or more coloring agents, sweeteners, flavoring agents, and / or preservatives. Suitable pharmaceutically acceptable excipients for tablet formulations include, for example, inert diluents such as lactose, sodium carbonate, calcium phosphate or calcium carbonate, granulating agents and disintegrants such as corn starch or alginic acid; binders such as starch; lubricants such as magnesium stearate, stearic acid or talc; preservatives such as ethyl or propyl parabens, and antioxidants such as ascorbic acid. Tablet formulations may be uncoated or coated to alter their disintegration and subsequent absorption of the active ingredient in the gastrointestinal tract, or to improve their stability and / or appearance, in either case, using conventional coating agents and procedures well known in the art.

[0220] Aqueous suspensions usually contain the compound in fine powder form together with one or more suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum arabic; dispersants or wetting agents, such as lecithin or condensation products of olefin oxides with fatty acids (e.g. polyoxyethylene stearate), or condensation products of ethylene oxide with long-chain aliphatic alcohols, such as heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol, such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, such as polyethylene sorbitan monooleate. Aqueous suspensions may also contain one or more preservatives (e.g. ethyl or propyl parahydroxybenzoate), antioxidants (e.g. ascorbic acid), colorants, flavorings and / or sweeteners (e.g. sucrose, saccharin or aspartame).

[0221] Additional excipients such as sweetening, flavoring and coloring agents may also be present in the pharmaceutical compositions.

[0222] The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions.

[0223] The pharmaceutical composition can also be in the form of a sterile injectable aqueous or oily suspension, which can be prepared according to known procedures using one or more suitable dispersants or wetting agents and suspending agents. The sterile injectable formulation can also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent.

[0224] For example, it is contemplated that a formulation for oral administration to humans may contain, for example, 0.5 mg to 2 g of a compound of the invention and an appropriate and convenient amount of excipient, which may be about 5 to about 98 weight percent of the total composition. Dosage unit forms will typically contain about 1 mg to about 500 mg of active compound. Suitable information about routes of administration and dosage regimens is, for example, provided in Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of the Editorial Board), Pergamon Press 1990, Vol. 5, Chapter 25.3, incorporated herein by reference.

[0225] According to well-known medical principles, the size of the dose used for therapeutic or prophylactic purposes of the compounds of the formulae presented herein will naturally vary depending on the nature and severity of the condition, the age and sex of the animal or patient and the route of administration.

[0226] In another embodiment, the invention provides a compound or composition as defined herein; for use in human or veterinary medicine; in particular for use in the treatment of cancer; more particularly for use in the prevention, treatment or reduction of tumor-associated desmoplasia, even more particularly for use in the treatment of invasive lobular breast cancer, tumors with a tumor-stroma ratio (TSR)>50%; and / or pancreatic ductal adenocarcinoma.

[0227] The present invention also provides a method for preventing and / or treating cancer; more particularly for preventing, treating or reducing tumor-associated connective tissue formation, and even more particularly for treating invasive lobular breast cancer, tumors with a tumor-stroma ratio (TSR)>50%; and / or pancreatic ductal adenocarcinoma; the method comprises administering to a subject in need thereof a therapeutically effective amount of a compound or pharmaceutical composition as defined herein.

[0228] In another embodiment, the compounds of the present invention are particularly interesting in combination therapies for cancer, particularly with cytotoxic agents or immunomodulators, particularly with immune cell activators such as immune checkpoint inhibitors (e.g., PD1 or PDL1 pathway inhibitors; particularly anti-PD1 or anti-PDL1 antibodies). The combination therapy can be administered to the patient separately or simultaneously with the compounds of the present invention, whichever is most appropriate.

[0229] In another embodiment, compounds of Formulae IIa and IIb have been demonstrated herein to exert anti-invasive activity, more specifically, these compounds inhibit tumor cells from invading surrounding tissues, thereby reducing metastasis.

[0230] Therefore, in yet another aspect, the present invention provides a compound for use as an anti-invasive compound for tumor cells; the compound is represented by any one of formula (IIa) or (IIb)

[0231]

[0232] in

[0233] Each of said R1, R1' and R1" is independently selected from the group consisting of: -H, -halo, -CF3, -OCF3 and -OC 1-3 alkyl;

[0234] R4 is selected from the group consisting of: -H, -halo, -CF3, -OCF3 and -OC 1-3 alkyl;

[0235] Each of said R5, R5' and R5" is independently selected from the group consisting of: -H, -OH, -CN, -C 1-3 Alkyl, -OC 1-3 Alkyl, -NR6R7, -SO2-NH2, -SO2-iPr, -Het1 and -halogen; the -C 1-3 Alkyl and -OC 1-3 Each of the alkyl groups is optionally substituted with 1 to 3 substituents selected from the group consisting of =O, -NR8R9 and -Het1;

[0236] R6, R7, R8 and R9 are selected from the group consisting of: -H and -C 1-3 Alkyl; the -C 1-3 Each of the alkyl groups is optionally substituted with 1 to 3 substituents selected from the group consisting of =O, -NH2 and -NH-iPr,

[0237] Het1 is a 5-membered or 6-membered heterocyclic ring having 1 to 3 heteroatoms selected from S, O and N; wherein each of said Het1 is optionally replaced by 1 to 3 heteroatoms selected from -C 1-3 Alkyl, -OC 1-3 Alkyl, -C 1-3 Alkyl -OH and -OC 1-3 Substitution by a substituent of the group consisting of alkyl-OH;

[0238] The spread of cancer from its tissue of origin and its subsequent growth in other organs is the most life-threatening aspect of the disease. This process, called metastasis, requires cancer cells to survive and proliferate outside their tissue of origin. The first critical step in this process is the invasion of cancer cells into the tissue and vasculature surrounding the tumor.

[0239] The compounds of the present invention are capable of inhibiting the first step, thereby preventing and / or reducing cancer cell proliferation and metastasis formation. Therefore, the present invention relates to compounds of formula IIa and IIb as described herein, which are used to treat cancer by inhibiting tumor cells from invading surrounding or adjacent tissues, cells and / or their entry into the circulatory system. Therefore, the compounds of the present invention are particularly useful for inhibiting or preventing tumor proliferation.

[0240] In a specific aspect, the compounds of formula IIa and IIb of the present invention are used as anti-invasive compounds. "Anti-invasion" refers to the ability to inhibit tumors or cancer cells from invading surrounding tissues (inside or outside the organ of origin). In order to determine whether a compound exhibits anti-invasive behavior, a chicken heart invasion assay as provided herein or as described in Bracke et al., 2014 can be applied. This screening method antagonizes cancer cells with fragments of normal tissue so as not to ignore the contribution of host tissue in the microecological system that dominates tumor behavior. The interaction between cancer cells and normal tissue is evaluated histologically and classified on a 5-level subjective scale. Grades III and IV are typical invasions, while grades 0, I, and II correspond to the absence of invasion. Compounds that inhibit invasion against cancer cell invasion (i.e., III / IV to 0 / I / II) are determined to be anti-invasive. Possible alternative assays commonly known in the art are type I collagen invasion assays and matrigel invasion assays.

[0241] Example

[0242] Materials and methods

[0243] 1. Synthesis process and physicochemical properties

[0244] Reaction process for synthesizing chalcone

[0245] General Scheme: Synthesis of Chalcone

[0246] A solution of the appropriate acetophenone (10 mmol) and LiOH.H2O (10 mol%) in 10 mL of anhydrous ethanol was stirred at an appropriate temperature for 10 min (for reactions at 40°C, the spherical flask was equipped with a reflux condenser). Then, the appropriate benzaldehyde (10 mmol, 1 eq.) was added and the system was isolated from the atmosphere with a cork. The progress of the reaction was monitored (LCMS). During the reaction, chalcone may precipitate. When the maximum degree of conversion was reached, the reaction mixture was quenched with 15 mL of 1% hydrochloric acid.

[0247] If the chalcone has precipitated, it is separated by means of filtration. To remove residual amounts of benzaldehyde, the residue is washed thoroughly with water until the filtrate becomes clear. The chalcone can be crystallized in absolute ethanol to obtain high-purity crystals.

[0248] If the chalcone forms a separate oily liquid at the bottom of the spherical bottle, it can be extracted from the mixture with ether. The organic phase is subsequently washed with saline (2x) and dried over MgSO4, and the solution is then concentrated in a vacuum. The purification of the residue thus obtained can be carried out by crystallization in absolute ethanol.

[0249] 3-(4-Fluorophenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one A1

[0250] This material was prepared according to the general protocol described above.

[0251]

[0252] 1 H NMR (CDCl3, 300MHz, ppm): δ = 3.94 (s, 3H), 3.95 (s, 6H), 7.11 (dd, 1H, J = 8.5Hz, J = 8.5Hz), 7. 28(s,2H),7.42(d,1H,J=15.4Hz),7.64(dd,2H,J=8.5Hz,J=5.2Hz),7.78(d,1H,J=15.4Hz); 13 C NMR (CDCl3, 75MHz, ppm): δ = 56.3, 61.0, 106.0, 116.1 (d, J = 21.9Hz), 121.3, 130.4 (d, J = 8.1Hz), 131.2 (d, J = 3.5Hz), 133.4, 142.51, 143.4, 153.2, 164.0 (d, J = 251.5Hz), 188.9; 19 F NMR (CDCl3, 282MHz, ppm): δδ = (-) 109.57-(-) 109.35 (m); IR (ATR, cm -1 ): v=1124,1157,1227,1338,1504,1580,1599,1611, 1660 (C=O); MS(ES+): m / z (%): 317.1 ([M+H] + , 100), 318.1 (18), 339.1 (8), 655.2 (8); MP (℃): 124-125. Yield: 69%, off-white needles (from EtOH); MW = 316.32.

[0253] 3-(4-Chlorophenyl)-1-(4-methoxyphenyl)prop-2-en-1-one A2

[0254] This material was prepared according to the general protocol described above.

[0255]

[0256] 1 H NMR (CDCl3, 300MHz, ppm): δ = 3.83 (s, 3H), 6.89 (d, 2H, J = 8.7Hz), 7.30 (d, 2H, J = 8.7Hz), 7. 41(d,1H,J=16.9Hz), 7.49(d,2H,J=8.1Hz), 7.69(d,1H,J=16.9Hz), 7.99(d,2H,J=8.7Hz); 13 C NMR (CDCl3, 75MHz, ppm): δ = 55.6, 113.9, 122.3, 129.2, 129.4, 130.8, 133.6, 136.0, 142.4, 163.5, 188.4. MP (°C): 127-130°C. Yield: 85%, white needles (from EtOH); MW = 272.73.

[0257] 3-(4-Fluorophenyl)-1-phenylprop-2-en-1-one A3

[0258] This material was prepared according to the general protocol described above.

[0259]

[0260] 1 H NMR (CDCl3, 300MHz, ppm): δ = 7.08-7.12 (m, 2H), 7.43-7.52 (m, 3H), 7.56-7.66 (m, 3H), 7.76 (d, 1H, J = 15.8Hz), 7.99-8.01 (m, 2H); 13 CNMR (CDCl3, 75MHz, ppm): δ = 116.0 (d, J = 21.7Hz), 121.7, 128.3, 128.5, 130.2 (d, J = 8.1Hz), 131.0 (d, J = 3.6Hz), 132.7, 138.0, 143.4, 164.0 (d, J = 251.9Hz), 190.2. MP (℃): 86-88℃. Yield: 92%, yellow needles (from EtOH); MW = 226.25.

[0261] 1-(4-Fluorophenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one A4

[0262] This material was prepared according to the general protocol described above.

[0263]

[0264] 1H NMR (CDCl3, 300MHz, ppm): δ = 3.80 (s, 3H), 3.92 (s, 6H), 7.21 (s, 2H), 7.32 (dd, 2H, J = 8.9Hz, J=8.9Hz),7.75(d,1H,J=15.7Hz),7.85(d,1H,J=15.7Hz),8.23(dd,2H,J=8.9Hz,J=5.6Hz); 13 C NMR (CDCl3, 75MHz, ppm): δ = 56.7, 60.8, 107.3, 116.5 (d, J = 21.9Hz), 121.8, 131.5, 132.3 (d, J = 9.2Hz), 135.9 (J = 2.7Hz), 141.7, 145.7, 154.7, 166.5 (d, J = 252.3Hz), 188.5. MP (°C): 102-104. Yield: 85%, white needles (from EtOH); MW = 316.32.

[0265] 3-(4-Fluorophenyl)-1-(4-methoxyphenyl)prop-2-en-1-one A5

[0266] This material was prepared according to the general protocol described above.

[0267]

[0268] 1 H NMR (CDCl3, 300MHz, ppm): δ = 3.96 (s, 3H), 6.97 (d, 2H, J = 8.5Hz), 7.11 (dd, 2H, J = 8.5Hz, J = 8.5Hz), 7. 49 (d, 1H, J = 15.7Hz), 7.64 (dd, 2H, J = 8.5Hz, J = 5.2Hz), 7.72 (d, 1H, J = 15.7Hz), 8.03 (d, 2H, J = 8.5Hz); 13 C NMR (CDCl3, 75MHz, ppm): =56.0, 114.5, 116.3 (d, J = 21.9Hz), 122.4, 130.9, 131.4, 131.5 (d, J = 8.5Hz), 132.0 (d, J = 3.5Hz), 142.3, 163.7, 163.8 (d, J = 248.7Hz), 187.8. MP (℃): 126-127℃. Yield: 91%, white needles (from EtOH); MW = 256.28.

[0269] 3-(Phenyl)-1-(4-methoxyphenyl)prop-2-en-1-one A6

[0270] This material was prepared according to the general protocol described above.

[0271]

[0272] 1 H NMR (CDCl3, 300MHz, ppm): δ = 3.86 (s, 3H), 6.98 (d, 2H, J = 8.5Hz), 7.33-7.43 (m, 3H), 7.52 (d, 1 H, J=15.8Hz), 7.63 (dd, 2H, J=7.4Hz, J=3.5Hz), 7.81 (d, 1H, J=15.8Hz), 8.04 (d, 2H, J=8.5Hz); 13 C NMR (CDCl3, 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.

[0273] 3-Phenyl-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one A7

[0274] This material was prepared according to the general protocol described above.

[0275]

[0276] 1 H NMR (CDCl3, 300MHz, 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=15.8Hz),7.58-7.72(m,2H),7.81(d,1H,J=15.8Hz); 13 C NMR (CDCl3, 75 MHz, ppm): v = 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.

[0277] 1,3-Diphenylprop-2-en-1-one A8

[0278] This material was prepared according to the general protocol described above.

[0279]

[0280] 1H NMR (CDCl3, 300MHz, ppm): δ = 7.39-7.46 (m, 3H), 7.46-7.62 (m, 4H), 7.62-7.69 (m, 2H), 7.76-7.89 (m, 1H), 8.00-8.05 (m, 2H); 13 C NMR (CDCl3, 75 MHz, ppm): δ = 122.3, 128.7, 128.8, 129.1, 130.8, 133.0, 135.1, 138.4, 145.0, 190.7. Yield: 92%, white needles (from EtOH); MW = 224.30.

[0281] 3-(4-Fluorophenyl)-1-(4-methoxy-3-(trifluoromethyl)phenyl)prop-2-en-1-one A14

[0282] This material was prepared according to the general protocol described above.

[0283]

[0284] 1 H NMR (CDCl3, 300 MHz, ppm): δ = 4.01 (s, 3H), 7.11-7.15 (m, 3H), 7.45 (d, 1H), 7.66 (dd, 2H), 7.81 (d, 1H), 8.18-8.33 (m, 2H). Yield: 53%, white needles (from EtOH); MW = 324.27.

[0285] Synthesis of Chalcone A9

[0286]

[0287] Scheme 1. Synthesis of chalcone A9. Reactants and conditions: a) 2 eq DIPEA, 1.5 eq MEMCl, anhydrous CH2Cl2, THF, N2, 0°C to room temperature, 24 h; b) 1 eq 3',4',5'-trimethoxyacetophenone, 0.5 eq LiOH.H2O, anhydrous EtOH, N2, room temperature, 26 h; c) 5% M aqueous HCl, THF, reflux, 6 h.

[0288] The synthesis of chalcone A9 is described in Scheme 1. 2-hydroxy-4-fluorobenzaldehyde 1 (4.59mmol), anhydrous CH2Cl2 (45mL) and anhydrous THF (3mL) were loaded into a flame-dried round-bottom flask equipped with a stirring bar and maintained under a nitrogen atmosphere. Benzaldehyde is insoluble in CH2Cl2, but is soluble in THF. The solution is cooled to 0°C in an ice bath, followed by addition of 2 equivalents of DIPEA (9.18mmol) and 1.5 equivalents of MEMCl. The ice bath is removed and the mixture is stirred at room temperature for 24h. After the reaction is complete, the mixture is quenched by adding NaHCO3 (3 parts, 6mL) saturated aqueous solution. Organic matter is extracted with EtOAc (2×6mL), and then the combined organic phase is washed with 1N NaOH (20mL) and water (20mL). The organic phase is dried over MgSO4. If LCMS analysis indicated the residual presence of MEMCl, the product was dissolved in a mixture of NaHCO 3 saturated THF and stirred vigorously under N 2 atmosphere for 20 min and extracted with EtOAc and dried over MgSO 4. The protected benzaldehyde 2 (yield 52%) was used as such in the next step.

[0289] In a round-bottom flask equipped with a stirring bar, 18.18mmol 3',4',5'-trimethoxyacetophenone was dissolved in 20mL anhydrous EtOH. After adding 0.5 equivalents (9.09mmol) of LiOH.H2O, the mixture was stirred at room temperature for 10min under N2 atmosphere. After adding 1 equivalent of benzaldehyde 2, the mixture was stirred at room temperature for 26h under N2 atmosphere. The mixture was quenched by adding 30mL of water, resulting in the formation of a precipitate. The precipitate was separated by filtration and washed with water until the filtrate was clear. The residue was crystallized in anhydrous EtOH to produce 3 in the form of white-yellow crystals with a yield of 93%.

[0290] Next, the protected chalcone 3 (8.73mmol) was dissolved in a mixture of 5% HCl aqueous solution (20mL) and THF (40mL), and the mixture was refluxed for 6h. After completing the deprotection step (LCMS), the mixture was quenched with salt water (60mL), and the organic matter was extracted with ether (2×60mL). The organic phase merged was extracted with 1M NaOH aqueous solution (120mL), and the aqueous phase was neutralized (pH 7) with the HCl aqueous solution subsequently. During this step, the clear solution became a milky fluid. Extracted product from this fluid with EtOAc (2×60mL), and the organic phase was dried with MgSO4. Crystallization in anhydrous EtOH generated chalcone A9 with a productive rate of 55%.

[0291] 3-(4-Fluoro-2-hydroxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one A9

[0292] Compound A9 was synthesized according to the scheme described above.

[0293]

[0294] 1 H NMR (CDCl3, 300MHz, ppm): δ = 3.95 (s, 6H), 3.95 (s, 3H), 6.67 (ddd, 1H, J = 8.5Hz, J = 8.5Hz, J = 2.2Hz), 6.76 (dd, 1H, J = 9.9H z,J=2.2Hz),7.30(s,2H),7.58(dd,1H,J=8.5Hz,J=6.6Hz),7.60(d,1H,J=15.4Hz),8.21(d,1H,J=15.4Hz),8.35(s,1H); 13 CNMR (CDCl3, 75MHz, ppm): δ = 56.4, 61.1, 104.2 (d, J = 24.2Hz), 106.4, 108.0 (d, J = 23.1Hz), 118.7 (d, J = 2.3Hz) ,121.2,130.7(d,J=10.4Hz),133.4,141.2,142.6,153.1,158.2(d,J=11.5Hz),165.0(d,J=252.7Hz),191.7; 19 F NMR (CDCl3, 282MHz, ppm): δ = (-) 107.36-(-) 107.28 (m); IR (ATR, cm -1 )ν=717,801,853,976,1093,1123,1161,1269,1289,1314,1342,1414,1495,1505, 1568,1643(C=C),1656(C=O),3268(OH).MS(ES+):m / z(%)=315.3(5),333.3([M+H] + ,100),334.0([M+H+1] + ,17),396.3(5),687.3(14).MP(℃)=160.Yield: 55%.Brown crystals;MW=332.33.

[0295] General Scheme: Synthesis of Hydroxychalcone

[0296] A round-bottom flask maintained under a nitrogen atmosphere was charged with 15 mL of 1,2-dimethoxyethane, 2.2 mmol of the appropriate hydroxyacetophenone, and 3.5-6 equivalents (0.55 g) of LiOH.H2O. The resulting suspension was stirred at room temperature for 10 min, and then 1.5-3 equivalents of the appropriately substituted benzaldehyde were added. The flask was equipped with a reflux condenser, and the mixture was stirred at 60°C for 48 h. The reaction was quenched by the addition of 10 mL of 10% aqueous HCl. Hydrochloric acid. The organics were extracted with EtOAc (2×15 mL); the organic layers were combined, dried over MgSO4, and concentrated in vacuo. The resulting crude chalcone was purified by recrystallization from anhydrous EtOH.

[0297] 3-(4-Fluorophenyl)-1-(6-hydroxy-2,3,4-trimethoxyphenyl)prop-2-en-1-one A10

[0298] Compound A10 was synthesized according to the general scheme described above.

[0299]

[0300] 1 H NMR (CDCl3, 400MHz, ppm): δ = 3.82 (3H, s), 3.88 (3H, s), 3.91 (3H, s), 6.28 (1H, s), 7.08 (2H, dd, J = 8.5Hz, J =8.5Hz), 7.60 (2H, dd, J = 8.5Hz, J = 5.5Hz), 7.76 (1H, d, J = 15.7Hz), 7.86 (1H, d, J = 15.7Hz), 13.63 (1H, s). 13 C NMR (CDCl3, 100MHz, ppm): δ = 56.2, 61.3, 61.9, 96.7, 108.7, 116.2 (d, J = 21.9Hz), 126.3 (d, J = 2.2Hz), 130.3 (d, J=8.3Hz),131.7(d,J=3.2Hz),135.4,141.9,155.0,160.3,162.8(3C),164.0(d,J=251.3Hz),192.8.IR(ATR,cm -1 )ν=1493,1507,1576,1601,1610,1629.MS(ES+):m / z(%)=333.1([M+H] + ,100),334.1([M+H+1] + ,20),355.0([M+Na] + ,5).MP(℃)=103.2.Yield: 76%; bright orange crystals (from EtOH); MW=332.32.

[0301] 3-(4-Fluorophenyl)-1-(2-hydroxy-3,4,5-trimethoxyphenyl)prop-2-en-1-one A11

[0302] Compound A11 was synthesized according to the general scheme described above.

[0303]

[0304] 1 H NMR (CDCl3, 300MHz, ppm): δ = 3.90 (3H, s), 3.95 (s, 3H), 4.07 (s, 3H), 7.10 (s, 1H), 7.14 (dd, 2H, J = 8 ,7Hz,J=8,5Hz),7.44(d,1H,J=15.4Hz),7.66(dd,2H,J=8.7Hz,J=5.5Hz),7.88(d,1H,J=15.4Hz). 13 CNMR (CDCl3, 75MHz, ppm): δ = 57.2, 61.2, 61.5, 107.0, 114.6, 116.4 (d, J = 21.9Hz), 120.1, 130.7 (d, J = 8 .1Hz),131.1(d,J=2.3Hz),141.7,144.0,145.2,150.3,154.8,164.4(d,J=252.7Hz),192.4.IR(ATR,cm -1 )ν=1010,1039,1096,1194,1270,1277,1359,1407,1485,1446,1505,1563,1634.MS(ES+):m / z(%)=333.1([M+H] + ,100),333,7([M+H+1] + ,20),354,7([M+Na] + ,9).MP(℃)=67-68.Yield: 63%; orange-red crystals (from EtOH); MW=332.32.

[0305] 3-(4-Fluorophenyl)-1-(2-hydroxy-4-methoxyphenyl)prop-2-en-1-one A12

[0306] Compound A12 was synthesized according to the general scheme described above.

[0307]

[0308] 1H NMR (CDCl3, 300MHz, ppm): δ = 3.86 (s, 3H), 6.48 (d, 1H, J = 2.2Hz), 6.49 (dd, 1H, J = 9.4Hz, J = 2.2Hz), 7.12 (dd, 2H, J = 8.5 Hz, J=8.5Hz), 7.50 (d, 1H, J=15.7Hz), 7.65 (dd, 2H, J=8.5Hz, J=5.2Hz), 7.82 (d, 1H, J=9.4Hz), 7.85 (d, 1H, J=15.7Hz). 13 C NMR (CDCl3, 75MHz, ppm): δ = 55.6, 101.1, 107.9, 114.1, 116.2 (d, J = 21.9Hz), 120.1, 130.5 ( d,J=9.2Hz),131.1(d,J=2.3Hz),131.3,143.1,164.2(d,J=252.7Hz),166.3,166.8,191.6. 19 F NMR (CDCl3, 282MHz, ppm): δ = (-) 109.70-(-) 109.00 (m).IR (ATR, cm -1 )ν=802,837,958,1018,1137,1166,1204,1218,1365,1510,1568,1600,1622,1633.MS(ES+):m / z(%)=273.3([M+H] + ,100),274.3([M+H+1] + ,16),308.3(25).MP(℃)=135.Yield: 71%, light yellow crystals (from EtOH); MW=272.27.

[0309] 3-(4-Chlorophenyl)-1-(2-hydroxy-4-methoxyphenyl)prop-2-en-1-one A13

[0310] Compound A13 was synthesized according to the general scheme described above.

[0311]

[0312] 1H NMR (CDCl3, 300MHz, ppm): δ = 3.86 (s, 3H), 4.77 (br.s, 1H), 6.48 (d, 1H, J = 2.2Hz), 6.49 (dd, 1H, J = 8.8Hz, J = 2.2Hz), 7. 40(d,2H,J=8.8Hz),7.54(d,1H,J=15.4Hz),7.58(d,1H,J=8.8Hz),7.81(d,1H,J=8.8Hz),7.82(d,1H,J=15.4Hz).13C NMR (CDCl3, 75MHz, ppm): δ = 55.7, 101.2, 108.0, 114.1, 120.9, 129.4, 129.8, 131.3, 133.4, 136.7, 143.0, 166.5, 166.9, 191.6. IR (ATR, cm -1 )ν=790,824,958,1085,1122,1202,1218,1273,1359,1562,1577,1618,1636,3364.MS(ES+):m / z(%)=289.1([M+H] + ,100),290.0([M+1+H] + ,19),291.0([M+2+H] + ,37),292.0([M+3+H] + ,7).HRMS(ES+):m / z=C 16 H 13 ClO3+H + Calculated value [M+H] + :289.0626; Found: 289.0624.MP(℃)=131.Yield: 69%, bright yellow fiber (from EtOH); MW=288.73.

[0313] Reaction process for the synthesis of flavonoids

[0314] General scheme: Oxidative cyclization of 2'-hydroxychalcone to flavonoids

[0315] To the round-bottom flask containing 5mL of DMSO, add 0.63mmol of suitable 2'-hydroxychalcone and 4-11mol% of I2. The resulting solution is heated to reflux temperature for 2-5h, then poured onto 10mL of ice-water mixture. The gained blue residue is separated by filtration and redissolved in 10mL of EtOAc. The solution thus obtained is washed with 10mL of 10% Na2S2O3 aqueous solution, dried and evaporated to dryness through MgSO, to obtain the crude flavonoids in pure solid form.

[0316] 2-(4-Fluorophenyl)-5,6,7-trimethoxy-4H-chromen-4-one B1

[0317] Compound Bl was synthesized according to the general scheme for oxidative cyclization described above.

[0318]

[0319] 1 H NMR (CDCl3, 300MHz, 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 = 8.8Hz , J=8.8Hz),7.88(2H,dd,J=8.8Hz, J=5.2Hz ’ ). 13 C NMR (CDCl3, 75MHz, ppm): δ = 56.4, 61.7, 62.3, 96.3, 108.3, 113.0, 116.3 (d, J = 21.9Hz), 127.9 (d, J = 3. 5Hz),128.3(d,J=8.1Hz),140.6,152.7,154.6,157.9,160.3,164.7(d,J=252.7Hz),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 FO5+H + Calculated value [M+H] + :331.0976; Measured value: 331.0974.MP(℃)=172-173.Yield: 98%; Beige powder; MW=330.31.

[0320] 2-(4-Fluorophenyl)-6,7,8-trimethoxy-4H-chromen-4-one B2

[0321] Compound B2 was synthesized according to the general scheme for oxidative cyclization described above.

[0322]

[0323] 1H NMR (CDCl3, 300MHz, ppm): δ = 3.97 (s, 3H), 4.05 (s, 3H), 4.09 (s, 3H), 6.75 (s, 1H) ,7.23(dd,2H,J=9.1Hz,J=9.1Hz),7.39(s,1H),7.95(dd,2H,J=9.1Hz,J=5.2Hz). 13 CNMR (CDCl3, 75MHz, ppm): δ = 56.31, 61.50, 62.08, 100.02, 106.59, 116.38 (d, J = 21.9Hz,), 119.76, 128.15 (d, J = 3. 5Hz),128.34(d,J=9.2Hz,),142.12,145.78,147.54,151.32,161.90,164.73(d,J=253.8Hz,),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.

[0324] 2-(4-Fluorophenyl)-7-methoxy-4H-chromen-4-one B3

[0325] Compound B3 was synthesized according to the general scheme for oxidative cyclization described above. The product was crystallized in EtOH to improve purity.

[0326]

[0327] 1 H NMR (CDCl3, 300MHz, ppm): δ = 3.93 (s, 3H), 6.70 (s, 1H), 6.96 (d, 1H, J = 2.2Hz), 6.99 (dd, 1H, J = 8.8Hz, J=2.2Hz), 7.21(dd,2H,J=8.8Hz,J=8.5Hz),,7.92(dd,2H,J=8.8Hz,J=5.2Hz), 8.13(d,1H,J=8.8Hz). 13C NMR (CDCl3, 75MHz, ppm): δ = 55.9, 100.4, 107.2, 114.5, 116.2 (d, J = 23.1Hz), 117.7, 127.0, 128.0(d,J=3.5Hz),128.3(d,J=9.2Hz),157.9,161.9,164.2,164.6(d,J=253.8Hz),177.6. 19 F NMR (CDCl3, 282MHz, 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(℃)=163.Yield: 74%, off-white crystals (from EtOH); MW=270.26.

[0328] 2-(4-Chlorophenyl)-7-methoxy-4H-chromen-4-one B4

[0329] Compound B4 was synthesized according to the general scheme for oxidative cyclization described above.

[0330]

[0331] 1 H NMR (CDCl3, 300MHz, ppm): δ = 3.94 (s, 3H), 6.73 (s, 1H), 6.96 (d, 1H, J = 2.2Hz), 7.00 (dd, 1H, J = 8.8Hz, J = 2. 2Hz), 7.50 (d, 2H, J = 8.8Hz), 7.85 (d, 2H, J = 8.8Hz), 8.13 (d, 1H, J = 8.8Hz). MS (ES+): m / z (%) = 271.3 ([M+H] + ,100),272.0([M+H+1] + ,15). Yield: 97%, beige powder; MW = 286.71.

[0332] General scheme: Cyclization of 2'-hydroxychalcone to flavanone under acidic conditions

[0333] To a round-bottom flask containing 7 mL of 3N HCl in MeOH was added 0.27 mmol of the appropriate 2'-hydroxychalcone. The resulting solution was heated to reflux temperature for 24 h and then concentrated in vacuo. H2O (7 mL) was then added and the resulting suspension was neutralized with a saturated aqueous solution of NaHCO3 (7 mL). Next, EtOAc (15 mL) was extracted. The organic layer thus obtained was washed with H2O (10 mL), dried over MgSO4 and evaporated to dryness to obtain a solid, crude flavanone. Recrystallization was subsequently performed in anhydrous EtOH to obtain the pure product.

[0334] 2-(4-Fluorophenyl)-5,6,7-trimethoxychroman-4-one B5

[0335] Compound B5 was synthesized according to the general scheme for cyclization under acidic conditions described above.

[0336]

[0337] 1 H NMR (CDCl3, 400MHz, ppm): δ = 2.77 (dd, 1H, J = 16.7Hz, J = 2.8Hz), 2,98 (dd, 1H, J = 16.7Hz, J = 13.3Hz), 3,82 (s, 3H), 3,88 (s, 3H), 3,94 (s, 3H), 5, 38 (dd, 1H, J = 13.3Hz, J = 2.8Hz), 6, 34 (s, 1H), 7, 11 (t, 2H, J = 8.7Hz), 7, 43 (dd, 2H, J = 8.7Hz, J = 5.3Hz). 13 C NMR (CDCl3, 100MHz, ppm): δ = 45.7, 56.3, 61.5, 61.7, 78.8, 96.4, 109.3, 115.9 ( d,J=21.9Hz),128.1(d,J=8.1Hz),134.7(d,J=2.9Hz),137.8,154.4,159.54and 159.57(3C),162.9(d,J=247.6Hz),189.1.IR(ATR,cm -1 )ν=1083,1103,1264(CO),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 FO5+H + Calculated value [M+H] +:333.1133; Found: 333.1134. Yield: 44%; White powder (from EtOH); MW = 332.32.

[0338] 2-(4-Fluorophenyl)-6,7,8-trimethoxychroman-4-one B6

[0339] Compound B6 was synthesized according to the general scheme for cyclization under acidic conditions described above.

[0340]

[0341] 1 H NMR (CDCl3, 300MHz, ppm): δ = 2.87 (dd, 1H, J = 16.9Hz, J = 3.3Hz), 3.03 (dd, 1H, J = 16.9Hz, J = 12.8Hz), 3.87 (s, 3H), 3.90 (s, 3H), 4.01(s,3H),5.46(dd,1H,J=12.8Hz,J=3.3Hz),7.12(t,2H,J=8.7Hz,J=8.7Hz),7.17(s,1H),7,47(dd,2H,J=8.7Hz,J=5.2Hz). 13 C NMR (CDCl3, 75MHz, ppm): δ = 44.6, 56.3, 61.5, 61.6, 79.6, 102.5, 115.9 (d, J = 21.9Hz), 116.0, 128.1 (d, J =9.2Hz),134.8(d,J=3.5Hz),142.2,148.2,149.7,151.1(3C),162.9(d,J=246.9Hz),190.9.IR(ATR,cm -1 )ν=1068,1111,1157,1219,1283,1354,1418,1455,1470(C=C),1681(C=O).MS(ES+):m / z(%)=332.8([M+H] + ,100),333.7([M+1+H] + ,20),354.7([M+Na] + ,20),395.7(5). Yield: 50%; pale yellow solid (from EtOH); MW = 332.32.

[0342] General scheme: Cyclization of 2'-hydroxychalcone to flavanone under alkaline conditions

[0343] 10mL of anhydrous EtOH was charged into a flame-dried round-bottom flask equipped with a reflux condenser and a CaCl2-tube. The solvent was heated to reflux, and then the appropriate 2'-hydroxychalcone (1mmol) and 10 equivalents of NaOAc were added. Reflux was maintained and the reaction was monitored by LC / MS. When the maximum conversion rate was obtained, the yellow reaction mixture was cooled to room temperature and poured into 10mL of ice water. The organic matter was extracted with EtOAc (3×20mL), and the combined organic layer was dried and washed with brine, dried over MgSO4 and concentrated in vacuo. A yellow residue containing a mixture of 2'-hydroxychalcone and flavanone was obtained. Separation was performed using a preparative TLC using a mixture of petroleum ether and EtOAc (8:1) eluents. Flavanones were thus obtained in the form of beige solids.

[0344] 2-(4-Fluorophenyl)-7-methoxychroman-4-one B7

[0345] Compound B7 was synthesized according to the general scheme for cyclization under basic conditions described above.

[0346]

[0347] 1 H NMR (CDCl3, 300MHz, ppm): δ = 2.81 (dd, 1H, J = 16.7Hz, J = 2.8Hz), 3.02 (dd, 1H, J = 16.7Hz, J = 12.9Hz), 3.84 (s, 3H), 5.46 (dd, 1H, J = 12.9Hz, J = 2.8Hz) , 6.49 (d, 1H, J = 2.2Hz), 6.63 (dd, 1H, J = 8.8Hz, J = 2.2Hz), 7.12 (dd, 2H, J = 8.5Hz, J = 8.5Hz), 7.46 (dd, 2H, J = 8.4Hz, J = 5.2Hz), 7.87 (d, 1H, J = 8.8Hz). 13 C NMR (CDCl3, 75MHz, ppm): δ = 44.4, 55.7, 79.4, 101.0, 110.4, 114.8, 115.8 (d, J = 21.9Hz), 1 28.1(d,J=8.1Hz),128.8,134.8(d,J=2.3Hz),162.8(d,J=248.1Hz),163.4,166.3,190.3. 19 FNMR(CDCl3,282MHz,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(℃)=93.Yield: 66%, beige solid; Chromatography: Rf: 0.15 (petroleum ether / EtOAc 8:1); MW=272.27.

[0348] 2-(4-Chlorophenyl)-7-methoxychroman-4-one B8

[0349] Compound B8 was synthesized according to the general scheme for cyclization under basic conditions described above.

[0350]

[0351] 1 H NMR (CDCl3, 300MHz, ppm): δ = 2.82 (dd, 1H, J = 16.7Hz, J = 3.0Hz), 2.99 (dd, 1H, J = 16.7Hz, J = 13.1Hz), 3.85 (s ,3H),5.45(dd,1H,J=13.1Hz,J=3.0Hz),6.50(d,1H,J=2.2Hz),6.63(dd,1H,J=8.8Hz,J=2.2Hz),7.41(s,4H ’ ),7.87(d,1H,J=8.8Hz).IR(ATR,cm -1 )ν=811,825,832,1015,1062,1116,1226,1446,1574,1615,1674.MS(ES+):m / z(%)=289.0([M+H] + ,100),290.0([M+H+1] + ,16),291.1([M+H+2] + ,36),292.0([M+H+3] + ,7). Yield: 52%, beige solid; chromatography: Rf: 0.18 (petroleum ether / EtOAc 8:1); MW = 288.73.

[0352] 2 Cell culture

[0353] The Michigan Cancer Foundation-7 (MCF-7) cell line is a human breast cancer cell line originally isolated from pleural effusion of breast adenocarcinoma. The cell line was further selected for its invasiveness and metastatic activity, and the MCF-7 / 6 cell line donated by H.Rochefort (Unité d'Endocrinologie Cellulaire et Moléculaire, Montpellier, France) was obtained. Human 13B20332 hTERT (also known as 13B2 hTERT) breast CAFs were derived from 99% estrogen receptor-positive, 25% progesterone receptor-positive, and HER2-negative tumors and were isolated as previously described (Primac et al., 2019). IF staining and flow cytometry of 13B2 hTERTCAF cells showed positive staining of α-SMA, FAP, VIM, CD44, CD105, and CD90 and negative expression of epithelial marker CD326. Human CT5.3 colon CAFs were isolated from colorectal adenocarcinoma resection specimens obtained in accordance with the local ethics committee (Ghent University Hospital) and infected with the pBABE retroviral vector expressing the hTERT open reading frame (De Vlieghere et al., 2015). IF staining and flow cytometry of CT5.3hTERT CAF cells showed positive staining for α-SMA, FAP, VIM, CD44, CD105 and CD90 and negative expression of the epithelial marker CD326. HCA2hTERT dermal fibroblasts were donated by C. Jones (Cardiff University, UK). 4T1-luc breast tumor cells were kindly donated by 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 right atrial appendage tissue of patients with sinus rhythm undergoing open heart surgery and isolated using the tissue explant method. The epicardial layer was removed and cultured in plates with scraped surfaces for 1-2 mm in 2 mL Dulbecco's modified Eagle's medium (all Sigma-Aldrich, Germany) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. 2 The resulting outgrowth fibroblasts were considered passage 0. Primary glioblastomas were obtained from patients undergoing glioblastoma resection using a similar protocol. All patients gave informed consent for the use of the tissue. CAF03 glioblastoma CAFs were purchased from Vitro Biopharma (US).

[0354] MCF-7 / 6 cells were cultured in a medium containing Dulbecco's modified Eagle's medium (DMEM) (Catalog No. 41965039, Thermo Fisher Scientific) and F12 Nut mix (Ham) medium (Catalog No. 11765054, Thermo Fisher Scientific) in a 1:1 ratio, supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Catalog No. ATCC-30-2030, LGC Standards), 100 IU mL-1 penicillin, and 100 mg mL-1 streptomycin (Catalog No. 15070063, Thermo Fisher Scientific). CT5.3hTERT, HCA2 hTERT and 4T1-luc cells were cultured in DMEM (Cat. No. 41965039, Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Cat. No. ATCC-30-2030, LGC Standards), 100 IU mL-1 penicillin and 100 mg mL-1 streptomycin (Cat. No. 15070063, Thermo Fisher Scientific). The 13B2 hTERT cell line was cultured in the same medium as described above with the addition of the following additives: 0.05 μg / mL cholera toxin (Catalog No. C8052, Sigma-Aldrich), 5% human serum (Catalog No. P2918, Sigma-Aldrich), 0.01 μM triiodothyronine (Catalog No. IRM6649, Sigma-Aldrich), 0.01 μM hydrocortisone (Catalog No. H0888, Sigma-Aldrich), 0.01 μg / mL epidermal growth factor (EGF) (Catalog No. E9644, Sigma-Aldrich), and 0.05 mg / mL insulin (Catalog No. I6634, Sigma-Aldrich). Cells were expanded and maintained as monolayers at 37°C in an atmosphere of 5% CO2 (MCF-7 / 6, 4T1-luc) or 10% CO2 (CT5.3hTERT, HCA2 hTERT, 13B2 hTERT) in air and passaged at 80% confluence. All human cell lines were tested monthly using the Mycoalert Mycoplasma Detection Kit (Cat. No. LT07-318, Lonza) to exclude mycoplasma contamination.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 CAFs and primary glioblastoma cells were cultured in MSC-GRO. TM The cells were grown in VitroPlus III reduced serum complete medium (Vitro Biopharma, USA).

[0355] 3 Chicken heart invasion assay

[0356] Chicken heart invasion assay was performed as previously described (Bracke et al., 2014). Pre-cultured heart fragments of 9-day-old chicken embryos were confronted with MCF-7 / 6 aggregates on top of semi-solid agar at 37°C overnight, after which the confrontation pairs were cultured in suspension at 37°C, 10% CO2 in the presence or absence of test compounds (in duplo) for 8 days. This procedure can also be performed with aggregates of 4T1-Luc cells, for which suspension cultures of the confrontation pairs were cultured for 3.5 days. All compounds were evaluated at 1 μM and 100 nM; higher or lower concentrations were subsequently evaluated depending on the results. After fixation in Bouin-Hollande, the cultures were dehydrated and embedded in paraffin. Sections of the confrontation cultures were stained with hematoxylin-eosin, blinded and scored by trained personnel (Bracke et al., 2014).

[0357] In the chicken heart invasion assay, cancer cells are confronted with fragments of normal tissue so as not to ignore the contribution of host tissue in the microecological system that dominates tumor behavior. Therefore, in the presence of a certain concentration of test compounds, pre-cultured heart tissue fragments (PHF) dissected from 9-day-old chicken embryos can be confronted with aggregates of human invasive MCF-7 / 6 breast cancer cells or other cells. After 8 days of MCF-7 / 6, or after another time period depending on the cell type, the interaction between cancer cells and PHF is evaluated histologically and classified according to a 5-level subjective scale (Bracke et al., 2014). Grades III and IV are typical invasions, while grades 0, I and II correspond to the absence of invasion. Compounds that inhibit the invasion of cancer cells (i.e., III / IV to 0 / I / II) are determined to be anti-invasive. Possible alternative assays commonly known in the art are type I collagen invasion assays, wound healing assays, and matrigel invasion assays.

[0358] 4Atomic force microscopy

[0359] U-shaped 384-well ULA plates (catalog number MS-9384UZ, S-bio) were inoculated with a cell-specific culture medium suspension with cells. Before being used in the experiment, the cells were allowed to form spheroids under the treatment conditions for an appropriate time. Cell culture dishes (catalog number 734-2814, VWR) were filled with collagen gel and placed at 37°C for an appropriate time to allow solidification. For each condition, spheroids were pooled in a cell culture dish. The culture medium (stored at 37°C) was removed to promote the attachment of the bottom of each spheroid to the collagen gel. The cell culture dish was incubated at 37°C. After the incubation period, the stored culture medium was carefully added to the culture dish (to prevent spheroid separation).

[0360] Mechanical measurements were performed using an atomic force microscope. To indent the spheroids, an in-house manufactured colloidal probe was used. Force curves were collected using appropriate set points and probe speeds (depending on the hardness of the spheroid). Multiple force curves were collected on each spheroid. The collected curves were then processed using JPK DP software. Young's modulus was extracted from the force curves. All experiments were performed in biological triplicate.

[0361] 5. Hardness measurement

[0362] CAFs are the main 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 range 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 main producers of ECM components and therefore contribute extensively to its remodeling, including the stiffness of the ECM.

[0363] To generate 3D cultures that mimic the complexity and pathophysiology of in vivo tumors, spheroids are used (see point 4). Spheroids are thought to better mimic cellular heterogeneity, nutrient and oxygen gradients, cell-cell interactions, cell-matrix interactions, gene expression profiles, stromal interactions, therapeutic responses, multicellular resistance, drug penetration, and anti-apoptotic signaling.

[0364] 6. Evaluation of cell viability.

[0365] use 2.0 Cell viability assay (Catalog No. G9243, Promega) and The half maximal inhibitory concentration (IC) of A1 was evaluated by 3D cell viability assay (Catalog No. G9681, Promega). 5013B2 hTERT cells (2D: 2 x 10 5 , 3D: 8x 10 4 ) were plated at a volume of 90 μL / well on Nunc TM MicroWell TM 96-well, Nunclon Delta-Treated flat-bottom microplates (Catalog No. 167008, ThermoFisher Scientific, for 2D experiments) or 96-well ULA plates (Catalog No. MS-9096UZ, S-bio) were added to each well. A1 was serially diluted to 10 times the final concentration in appropriate culture medium. For each treatment condition, 10 μL was added to 3 replicate wells (final concentration range of 1-100 μM) and the plate was incubated for 48 h.

[0366] The CellTiter-Glo cell viability assay was performed according to the manufacturer's instructions. Briefly, the plates and Equilibrate the reagent to room temperature (RT). Reagent was added to each well at a 1:1 (v / v) ratio. For 2D experiments, cell lysis was induced by placing the plate on an orbital shaker for 2 min and then stabilizing the luminescent signal for 10 min. For 3D experiments, the plate was placed on an orbital shaker for 5 min and then stabilized for 25 min. Then, Synergy TM Luminescence was measured using an HTX multi-mode microplate reader (BioTek). IC was determined using Graphpad Prism software. 50 This was repeated for 3 biological replicates.

[0367] 7 In vivo experiments: 4T1-based intraductal model

[0368] 7.1 Intraductal inoculation of 4T1-luc cells

[0369] Experiments in mice were performed according to the principles of Good Scientific Practice and approved by the Ethics Committee (EC) of the Faculty of Veterinary Medicine, University of Ghent (EC 2019-105).

[0370] Intraductal inoculation was performed in the third mammary gland of lactating female mice. For lactation, 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, lactating female animals were anesthetized by inhalation using a mixture of oxygen and isoflurane (2-3%), and then inoculated through the teat duct using a 32-gauge blunt needle with a suspension of PBS and 5 × 10 in 100 μl of a mixture of 4 4T1-luc cells. Pre-cool all materials needed for inoculation to avoid Buprenorphine (10 μg / kg, Val d'Hony Verdifarm NV - Belgium) was administered by intraperitoneal injection to provide analgesia.

[0371] 7.2 Treatment Preparation and Administration

[0372] Compound is dissolved in DMSO.Use 1mL syringe and 18G (pink) needle to draw compound solution subsequently, comprise extra air to help discharge all solution and avoid compound to be trapped in dead volume.Solution is divided into five parts and is injected in Medigel at five different positions, draws a certain volume of gel and is re-injected in cup to flush the dead volume of syringe at last position.Then medicine is shaken violently and visually inspects the uniform dispersion of compound.Mice receive the cup with medicine of fresh preparation every 48h, and gel is placed in gel cup holder to avoid contact with bedding and absorbent wood shavings in mouse cage.Measure weekly and depend on the amount of Medigel given to mice of average mouse body weight and consumption.Dosage level remains constant at 100mg / kg, three times a day.

[0373] 7.3 Analysis of tumor and metastasis progression

[0374] Use digital weighing scale and rectal temperature probe respectively to measure body weight and body temperature every day during the first week post-inoculation (pi), and once a week during the subsequent 5 weeks (i.e., after inoculation for up to 6 weeks). As the measure of primary tumor growth, digital caliper is used to monitor primary tumor volume weekly, and IVIS lumina III system (PerkinElmer, Zaventem, Belgium) is used to monitor the bioluminescence derived from 4T1-luc in primary tumor after 1,3 and 6 weeks inoculation. In order to carry out in vivo imaging, 200 μ l of D-fluorescein (2mg / 100 μ l suspended in PBS is injected to mice; Gold Biotechnology, St.Louis, MO), and images are collected under inhalation anesthesia after 10 minutes. For 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 (XylaziniHydrochloridum, Val d'HonyVerdifarm, Beringen, Belgium) and subsequently killed by cervical dislocation. Images of bioluminescent signals in isolated primary tumors, spleens, and organs with metastases (including axillary lymph nodes, lungs, and liver) were quickly acquired using the IVIS system. Primary tumors and spleens were also weighed using a digital weighing scale. Analysis of in vivo and ex vivo bioluminescent signals was performed using living image software 4.7.2.

[0375] 7.4 Histological and immunohistological staining

[0376] Isolated tissues of primary tumors, axillary lymph nodes, lungs, and livers were fixed in buffered 3.5% formaldehyde at room temperature for 24 h and embedded in paraffin. 5 μm sections were dewaxed, hydrated, and stained with hematoxylin and eosin (H&E). To allow analysis, sections were dehydrated and mounted.

[0377] For Masson's trichrome staining, 3-5 μm thick sections were dewaxed, hydrated, and incubated ON in Bouin's solution to improve the quality of staining. On the next day, slides were washed in distilled H2O (dH2O), then immersed in Biebrich scarlet-acid fuchsin solution 3 times, washed again in dH2O, and incubated in phosphomolybdic acid-phosphotungstic acid solution for 20 min. After incubation in phosphomolybdic acid-phosphotungstic acid solution, slides were not rinsed but immediately incubated in aniline blue solution for 8 min. After a final washing step with dH2O, slides were incubated in 1% acetic acid solution for 8 min. The sections were then rapidly dehydrated through 94% ethanol, absolute ethanol to remove Biebrich scarlet-acid fuchsin stain, and xylene to clear the slides before mounting with Tissue-Tek glass mounting medium. The staining showed collagen as blue, nuclei as dark red, and cytoplasm as red / pink. Quantification of positive staining was established using color deconvolution (for Masson's trichrome staining) followed by automated counting in ImageJ.

[0378] For immunohistochemical staining, 3-5 μm thick dewaxed sections were subjected to antigen retrieval with citrate buffer at pH 6 (10 mM trisodium citrate (Santa Cruz Biotechnology, Heidelberg, Germany) for α-SMA, CD11b, Ly6G and CD163) or with Tris-EDTA buffer [pH 9, 10 mM Tris, 1 mM EDTA (Thermo Fisher Scientific) for PDPN and FAP-α) at ​​95°C using a pressurized Decloaking Chamber NxGen (Biocare Medical, CA, USA) for 30 min. The slides were cooled to room temperature for 30 min and subsequently incubated with a tissue moistened with tris-buffered saline (TBS, Biocare Medical) on an orbital shaker at 20 rpm in a closed microscope box for all blocking, rinsing (3 times, 2 min each at room temperature between each incubation step with TBS) and staining steps. To block endogenous peroxidase activity, slides were first treated with 3% H2O2 in methanol for 10 min. To block nonspecific binding sites, slides were subsequently treated with serum-free protein blocking reagent (Dako, Heverlee, Belgium) for 10 min.

[0379] The slides were then stained with primary antibodies diluted in antibody diluent (Antibody Diluent, Dako) at room temperature for 1 h, and then incubated with secondary antibodies at room temperature for 30 min. The primary antibodies and diluents 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 antibody, 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 to mouse HRP-polymer (Biocare Medical) for PDPN and Ly6G, and ready-to-use Dako EnVision+Rabbit for α-SMA, FAP-α, CD11b, and CD163. For visualization of HRP-positive staining, slides were treated with a buffer containing 3,3'-diaminobenzidine (DAB) (Dako) for 10 min at room temperature. Counterstaining with hematoxylin was performed for 5 min at room temperature, followed by dehydration and mounting of slides with Tissue-Tek glass mounting medium. Quantification of positive staining was established using color deconvolution (for DAB and hematoxylin counterstaining) followed by automatic counting in ImageJ.

[0380] 7.5 Cytokine Array Analysis

[0381] Proteome Profiler Mouse XL Cytokine Array (Cat. No. ARY 028, R&D Systems) was performed with 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".

[0382] 7.6 Statistical analysis

[0383] Data are presented in the graphs as mean + / - standard error of the mean (SEM). *, **, and *** indicate statistically significant differences at the levels of values ​​P < 0.05, P < 0.01, and P < 0.001, respectively. Statistics were performed using Prism (Graphpad), and data were normalized using log10 normalization when necessary. P values ​​were calculated by one-tailed unpaired t-test or one-way analysis of variance (ANOVA) test, followed by multiple comparisons by Newman-Keuls post hoc test.

[0384] 8Combination therapy

[0385] Combined inactivation of E-cadherin and PTEN in the mouse mammary epithelium leads to the rapid formation of classical invasive lobular carcinoma (CLC). 90 Ecad mice;PTEN mouse model (Boelens et al., 2016) were injected intraductally 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, ClearH2O) and injection of 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 oral PI3K inhibitor BEZ-235. Throughout the experiment, mice were weighed every 48 h to evaluate tolerance, and tumors were measured every 48 h. When tumor volume reached 75 mm 3 Treatment was started at 4 pm and continued for six weeks, after which the animals were sacrificed. Tumors were weighed, and tissues (including tumors and lymph nodes) were examined using histology and other techniques to compare the tolerability and efficacy of the different treatments.

[0386] result

[0387] Example 1 - Anti-Invasion Flavonoids

[0388] The synthesis of the target flavones was carried out from appropriately substituted 2'-hydroxyacetophenones and benzaldehydes via 2'-hydroxychalcone intermediates. Due to the presence of the 2'-hydroxy group, the Claisen-Schmidt condensation in the first step was promoted by using excess benzaldehyde and a base in 1,2-dimethoxyethane at 60 °C.

[0389]

[0390] Scheme 2. Synthesis of 2'-hydroxychalcone A9-A12, flavonoids B1-B4 and flavanones B5-B8. Reactants and conditions: a) 1.5-3 eq. substituted benzaldehyde, 3.6-6 eq. LiOH.H2O, 1,2-dimethoxyethane, N2, 60°C, 48h; b) 0.04-0.11 mol%.I2, DMSO, reflux, 2-5h, N2; c) 3M HCl in MeOH, reflux, 24h (for B5 and B6) or 10 eq. NaOAc, anhydrous EtOH, reflux, 1.5h, dry conditions (for B7 and B8).

[0391] A mild oxidative cyclization of chalcone A9-A12 to flavonoids B1-B4 was accomplished in DMSO using a catalytic amount of I2. This method proved to be rapid and convenient and produced the desired products in high yields and purity, often without the need for additional purification by crystallization.

[0392] Treatment of A9-A10 with HCl in MeOH at reflux temperature provided flavanones B5-B6 in moderate yields. The starting material was completely consumed after 24 h, but purification (extraction and crystallization in EtOH) resulted in considerable product losses and moderate yields (44-50%) (Hsieh et al., 1998). Treatment of chalcone A11-A12 with HCl in MeOH also resulted in equilibrium with the desired flavanones B7-B8 after 18-24 h (about 70% conversion based on LC / MS data). Therefore, we also evaluated the cyclization of A11-A12 in an alkaline environment (NaOAc in EtOH). Although this resulted in a similar reaction equilibrium (~70% conversion), the workup proved to be easier, providing flavanones B7-B8 in moderate yields.

[0393] The chicken heart invasion (CHI) assay is a phenotypic test that pits pre-cultured heart tissue fragments (PHFs, "normal tissue") obtained from 9-day-old chicken embryos against aggregates of human invasive MCF-7 / 6 breast cancer cells (Bracke et al., 2014). In control conditions, MCF-7 / 6 cells surround, invade and destroy PHFs over an 8-day period. In this assay, invasion is defined as the gradual occupation and destruction of PHFs by MCF-7 / 6 cells. A three-dimensional reconstruction of this interaction is obtained by microscopic analysis of all serial histological sections of the culture. The readout is the invasion score according to a 5-level histological scale.

[0394] CHI closely reproduces temporal, spatial and histological invasion patterns in humans. The co-culture device probes effects on both tumor cells and host tissues and thus investigates potential pharmacological targets in either interaction partner. Its use in histological evaluation allows discerning anti-invasive effects from cytotoxic or other effects.

[0395] We have submitted flavonoids B1-B4, flavanones B5-B8 and intermediate 2'-hydroxychalcone A9, A11 and A12 to a chicken heart invasion 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 subsequently evaluated. Table 1 shows the lowest concentrations at which an anti-invasive effect was observed (histological grade I or II - see Bracke et al., 2014). The table also includes CHI data for chalcone classes A1, A2 and A5. Table 2 shows the anti-invasive effects of A1 and B1 in the CHI assay using 4T1-Luc aggregates.

[0396] The results showed that all compounds tested displayed suitable activity, although specific substitution patterns resulted in molecules with higher activity.

[0397] The anti-invasive activity data demonstrate that the combination of a (tri)methoxy decoration pattern on the left side of the molecule with a halo (eg fluorine, chlorine) substituent on the right side can be advantageous.

[0398] Table 1. Compound activity data in the CHI assay using MCF-7 / 6 aggregates

[0399]

[0400]

[0401] Table 2. Activity data of compounds A1 and B1 at 1 μM in the CHI assay using 4T1-Luc aggregates.

[0402]

[0403] Example 2 - Modulation of the Tumor Microenvironment (TME)

[0404] CAF Selectivity

[0405] 13B2 hTERT breast CAFs showed a significant decrease in Young's modulus after treatment with 1 μM of A1 or B1, while 1 μM of the inactive control compound A8 had no effect. Compound A14 also reduced the Young's modulus of 13B2 hTERT breast CAFs at 1 μM. Using a concentration range of 30 nM-3 μM of A1, a dose-response behavior was obtained ( Figure 1 AC). Similar results were obtained for CT5.3 hTERT colon CAF spheroids, as 1 μM of A1 or B1 reduced the stiffness of CT5.3 hTERT spheroids ( Figure 1 D). Further similar results were obtained for glioblastoma CAF03 CAF spheroids, as 1 μM A1 reduced spheroid stiffness ( Figure 1 E).

[0406] In the example of CAF selectivity of the fibroblast regulator in the present invention, a decrease in spheroid stiffness due to 1 μM A1 or 1 μM B1 was observed in CAFs ( Figure 1 AE), but not in normal fibroblasts (HCA2 hTERT, Figure 1 Furthermore, treatment with 0.03-3 μM A1 did not result in changes in spheroid stiffness in epithelial cell lines such as MCF-7 / 6 ( Figure 1G) nor did it change the spheroid stiffness of primary glioblastoma cells ( Figure 1 H). Of note, and in light of the risk of avoiding cardiotoxicity, treatment with 1 μM A1 or 1 μM B1 did not alter spheroid stiffness of primary human atrial fibroblasts ( Figure 1 I).

[0407] By comparing the selectivity of the fibroblast regulators of the present invention with the behavior of existing CAF regulators myosin II inhibitor blebbistatin, ROCK inhibitor Y-27632 or actin polymerization inhibitor cytochalasin D ( Figure 2 ), illustrating the novelty of this discovery. While A1, B1 and the existing CAF regulators were all active in CAF spheroids (13B2 hTERT, Figure 2 A;CAF03, Figure 2 B), Effects of A1 and B1 on normal fibroblast spheroids (HCA2 hTERT, Figure 2 C), epithelial cell spheroids (MCF-7 / 6, Figure 2 D) Primary glioblastoma cell spheroids ( Figure 2 E) or primary human atrial fibroblast spheroids ( Figure 2 However, the existing CAF regulators not only reduced the hardness of CAF spheroids, but also reduced the hardness of spheroids of normal fibroblasts (HCA2hTERT), epithelial cells (MCF-7 / 6), primary glioblastoma cells, and primary human atrial fibroblasts ( Figure 2 AF).

[0408] Data showing that additional compounds of the invention selectively reduce CAF (13B2 hTERT) versus normal fibroblast (HCA2 hTERT) spheroid stiffness are shown in Figure 3 The tested chalcones, flavonoids and flavanones were effective modulators of CAF sphere stiffness, but not of normal fibroblast stiffness. The combination of one or more methoxy groups on one aromatic ring of the molecule and one or more halogens on another aromatic ring of the molecule, with or without hydroxyl groups, may be advantageous for the desired activity.

[0409] Cell viability

[0410] To investigate whether the effects observed in the AFM experiments were not due to cytotoxicity, 13B2 hTERT, CT5.3 hTERT, HCA2 hTERT, MCF-7 / 6, and 4T1-Luc cells were subjected to 2D and / or 3D FACS following treatment with A1 or B1. Cell viability assay. After 48 h of treatment, metabolic activity of 2D cultures was inhibited in a dose-dependent manner with a high micromolar IC 50 Cells cultured as spheroids did not respond 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 reduction in the viability of these CAFs, nor is the observed selectivity of CAFs over normal fibroblasts or cancer cells due to a higher sensitivity of CAFs to reduced viability by A1 or B1.

[0411] 4T1 triple-negative breast cancer model

[0412] The anti-metastatic efficacy of A1 and B1 was studied in a 4T1-based triple-negative breast cancer (TBNC) intraductal model. A1 and B1 were added to Medigel for oral ingestion and compared with DMSO added to Medigel as a negative control. Axillary lymph node, lung and liver metastasis were the main parameters analyzed 6 weeks (w) after inoculation (pi) of tumor cell suspensions in the mammary ducts and histological analysis of the microenvironment of the primary tumor and axillary lymph nodes, lungs and liver.

[0413] Tolerance

[0414] Due to decreased milk production after weaning of pups, the body weight of all intraductally inoculated mice decreased during the first 3-4 days post-inoculation (pi). Figure 4 A), while body temperature remained stable in all mice during the first week pi ( Figure 4 B). Following weekly assessment of body weight and temperature throughout the 6-week study period, mice treated with DMSO, A1, and B1 showed no significant changes throughout the 6-week study period ( Figure 4 AB).

[0415] Transfer

[0416] Based on ex vivo bioluminescence data, treatment with A1 or B1 showed an overall reduction in metastatic burden in the lungs, liver, and axillary lymph nodes compared with DMSO ( Figure 5 AC).

[0417] H&E images of primary tumors confirmed the invasive characteristics of the 4T1-based intraductal model of triple-negative breast cancer (TNBC), with tumor invasion into mammary adipose tissue and vasculature, areas of cellular necrosis, the presence of immune infiltration, and tumor cells showing a spindle shape characteristic of pre-metastatic epithelial to mesenchymal transition (EMT). Smaller lung and liver metastases were found after A1 treatment, and smaller axillary lymph node metastases were found after B1 treatment compared to DMSO treatment (data not shown), confirming the bioluminescence data measured based on IVIS.

[0418] TME Histology of Primary Tumors and Metastases

[0419] Based on staining for the CAF marker α-smooth muscle actin (α-SMA), the amount of CAFs in primary tumors treated with A1 or B1 was significantly reduced compared with DMSO treatment ( Figure 6 A).

[0420] Podoplanin (PDPN) as a surrogate marker for CAFs confirmed the α-SMA results, showing a significant decrease in PDPN staining in primary tumors treated with A1 and B1 (P < 0.001) ( Figure 6 B). As an additional validation of both α-SMA and PDPN staining, a significant decrease in staining for the CAF marker fibroblast activation protein α (FAP-α) was observed in primary tumor tissues treated with A1 or B1 compared to DMSO ( Figure 6 C) This serine protease is highly expressed in reactive fibroblasts and has been shown to regulate fibroblast growth.

[0421] Masson's trichrome staining, which visualizes collagen fibers, showed that collagen deposition was significantly reduced after A1 or B1 treatment compared with DMSO ( Figure 6 D), reconfirmed the α-SMA, PDPN, and FAP-α results for CAFs and collectively identified reduced tumor stiffness after treatment with A1 and B1 compared to DMSO.

[0422] In addition to the reduction in the number of CAFs, CD11b staining of primary tumor tissues also showed a significant decrease in the amount of myeloid cells after treatment with A1 or B1 compared with DMSO ( Figure 6 E). Ly6G staining was more specific for TANs and neutrophil precursors such as granulocyte myeloid-derived suppressor cells (G-MDSCs) compared with DMSO, confirming this reduction after A1 or B1 treatment ( Figure 6 F). Complementing innate immune cell regulation, CD163 staining of primary tumor tissues showed a significant reduction in tumor-associated macrophages (TAMs) after treatment with A1 or B1 ( Figure 6 G). CD163 is a marker for M2 polarized macrophages, which promote tumor progression, angiogenesis, metastasis, and immunosuppression. Due to these pro-tumor activities associated with negative patient prognosis, the M2 macrophage phenotype is called tumor-associated macrophages (TAMs). TAMs support the immunosuppressive TME by secreting cytokines, chemokines, and growth factors. They also trigger the release of inhibitory immune checkpoint proteins in T cells.

[0423] In liver tissue, α-SMA and Masson's trichrome staining showed that CAFs and collagen deposition in the sites of myeloid cell proliferation were significantly reduced after A1 or B1 treatment compared with DMSO ( Figure 7 A and 7C). PDPN staining was also significantly reduced after A1 or B1 treatment compared with DMSO in liver tissue ( Figure 7 B). Similar reductions were seen in axillary lymph node tissue and in lung tissue ( Figure 7 DG)(Liu et al., 2017)(Liu et al., 2017)(Liu et al., 2017).

[0424] Cytokine profile of primary tumors

[0425] 4T1 primary tumor lysates from animals treated with DMSO or A1 were used in a cytokine array to quantify 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 (CXC 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 protein 1 (WISP-1). This perturbation pattern showed not only selectivity for CAFs, but also specific effects on protumor CAFs of the S1 and S4 subtypes (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 ) subgroups.

[0426] References

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[0428] Kemi,N.,Eskuri,M.,Herva,A.et al.Tumour-stroma ratio and prognosis in gastric adenocarcinoma.Br J Cancer 119,435-439(2018).

[0429] Boelens, Mirjam C., Micha Nethe, Sjoerd Klarenbeek, Julian R. de Ruiter, Eva Schut, Nicola Bonzanni, Amber L. Zeeman, et al. 2016. “PTEN Loss in E-Cadherin-Deficient Mouse Mammary Epithelial Cells Rescues Apoptosis and Results in Development of Classical Invasive Lobular Carcinoma.” Cell Reports 16(8):2087-2101. https: / / doi.org / 10.1016 / j.celrep.2016.07.059.

[0430] Bracke,Marc E.,Virinder S.Parmar,Anthony L.DePass,ChristianV.Stevens,Barbara W.Vanhoecke,and Marc M.Mareel.2014.Chick Heart InvasionAssay.Edited by Miriam Dwek,Udo Schumacher,and Susan A.Brooks.Methods inMolecular Biology,Vol.1070.New York:Springer Science+Business Media.https: / / doi.org / 10.1007 / 978-1-4614-8244-4_7.

[0431] Costa,A.,Kieffer,Y.,Scholer-Dahirel,A.,Pelon,F.,Bourachot,B.,Cardon,M.,Sirven,P.,Magagna,I.,Fuhrmann,L.,Bernard,C.,Bonneau,C.,Kondratova,M.,Kuperstein,I.,Zinovyev,A.,Givel,A.M.,Parrini,M.C.,Soumelis,V.,Vincent-Salomon,A.,&Mechta-Grigoriou,F.(2018)."Fibroblast Heterogeneity andImmunosuppressive Environment in Human Breast Cancer."Cancer cell,33(3),463-479.e10. https: / / doi.org / 10.1016 / j.ccell.2018.01.011

[0432] Han,Chencheng,Tongyan Liu,and Rong Yin.2020.“Biomarkers for Cancer-Associated Fibroblasts.”Biomarker Research 8(1):64.https: / / doi.org / 10.1186 / s40364-020-00245-w.

[0433] Hsieh,HK,TH Lee,JP Wang,JJ Wang,and CN Lin.1998.“Synthesis andAnti-Inflammatory Effect of Chalcones and RelatedCompounds.”PharmaceuticalResearch 15(1):39-46.https: / / doi.org / 10.1023 / a:1974.1044.

[0434] Liu,Qiang,Hongfei Zhang,Xiaoli Jiang,Caiyun Qian,Zhuoqi Liu,and DayaLuo.2017.“Factors Involved in Cancer Metastasis:A BetterUnderstanding to'Seedand Soil'Hypothesis.”Molecular Cancer 16(1):176.https: / / doi.org / 10.1186 / s12943-017-0742-4.

[0435] Primac I,Maquoi E,Blacher S,Heljasvaara R,Van Deun J,SmelandHY,CanaleA,Louis T,Stuhr L,Sounni NE,Cataldo D,Pihlajaniemi T,Pequeux C,De Wever O,Gullberg D,Noel A."Stromal integrin alpha11GFR regulates and promotes breakthrough signaling. cancerprogression."J Clin Invest 2019:129:4609-28.https: / / doi.org / 10.1172 / JCI125890.

[0436] Vlieghere,Elly De,Félix Gremonprez,Laurine Verset,Lore Christopher J Jones,Bram De Craene,Geert Berx,et al.2015.“Tumor-EnvironmentBiomimetics Delay Peritoneal Metastasis Formation byDeceiving and RedirectingDisseminated Cancer Cells.”Biomaterials 54:148-57.https: / / doi.org / 10.1016 / j.biomaterials.2015.03.012.

Claims

1. 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-stroma ratio (TSR)>50%; and / or pancreatic ductal adenocarcinoma; in represents a single bond or a double bond; Each of said R1, R1' and R1" is independently selected from the group consisting of: -H, -halo, -CF3, -OCF3 and -OC 1-3 alkyl; R2 is selected from the group consisting of: -H, -halo, -CF3, -OCF3 and -OC 1-3 alkyl; R3 is selected from the group consisting of: -H, -halo, -C 1-3 Alkyl and -CF3; Or R2 and R3 together with the C atoms to which they are attached form a 6-membered aromatic or non-aromatic heterocyclic ring; R4 is selected from the group consisting of: -H, -halo, -CF3, -OCF3 and -OC 1-3 alkyl; Each of said R5, R5' and R5" is independently selected from the group consisting of: -H, -OH, -CN, -C 1-3 Alkyl, -OC 1-3 Alkyl, -NR6R7, -SO2-NH2, -SO2-iPr, -Het1 and -halogen; the -C 1-3 Alkyl and -OC 1-3 Each of the alkyl groups is optionally substituted with 1 to 3 substituents selected from the group consisting of =O, -NR8R9 and -Het1; R6, R7, R8 and R9 are selected from the group consisting of: -H and -C 1-3 Alkyl; the -C 1-3 Each of the alkyl groups is optionally substituted with 1 to 3 substituents selected from the group consisting of =O, -NH2 and -NH-iPr, Het1 is a 5-membered or 6-membered heterocyclic ring having 1 to 3 heteroatoms selected from S, O and N; wherein each of said Het1 is optionally replaced by 1 to 3 heteroatoms selected from -C 1-3 Alkyl, -OC 1-3 Alkyl, -C 1-3 Alkyl -OH and -OC 1-3 Substitution by a substituent of the group consisting of alkyl-OH; wherein at least one of R1, R1′ and R1″ is -OC 1-3 Alkyl or -CF3; When R2 and R3 do not form a heterocyclic ring, Represents a double bond.

2. A compound for use as defined in claim 1, wherein When R2 and R3 form a 6-membered aromatic or non-aromatic heterocyclic ring, the compound is represented by formula (II): Wherein X is selected from O, S, NH or NC 1-3 Alkyl group.

3. A compound for use as defined in any one of claims 1 or 2, wherein represents a single bond or a double bond; Each of said R1, R1' and R1" is independently selected from the group consisting of: -H, -CF3 and -OC 1-2 alkyl; R2 is -H; R3 is selected from the group consisting of: -H and -Me; or R2 and R3 together with the C atoms to which they are attached form a 6-membered aromatic or non-aromatic heterocyclic ring, R4 is -H; Each of said R5, R5' and R5" is independently selected from the group consisting of: -H, -OH, -C 1-2 Alkyl, -NR6R7, -SO2-NH2, -Het1 and -halo; the -C 1-2 Each of the alkyl groups is optionally substituted with 1 to 3 substituents selected from the group consisting of =0 and -Het1; R6 and R7 are selected from the group consisting of: -H and -C 1-2 Alkyl; the -C 1-2 Each of the alkyl groups is optionally substituted with 1 to 3 substituents selected from the group consisting of =0 and -NH-iPr, Het1 is a 6-membered heterocyclic ring having 1 to 3 heteroatoms selected from O and N; wherein each of said Het1 is optionally replaced by 1 to 3 heteroatoms selected from -C 1-2 Alkyl and -C 1-2 Substitution by a substituent of the group consisting of alkyl-OH; wherein at least one of R1, R1′ and R1″ is -OC 1-2 alkyl; When R2 and R3 do not form a heterocyclic ring, Represents a double bond.

4. A compound for use as defined in any one of claims 1 to 3, wherein represents a single bond or a double bond; Each of said R1, R1' and R1" is independently selected from the group consisting of: -H, -CF3 and -OCH3; R2 is -H; R3 is -H; or R2 and R3 together with the C atoms to which they are attached form a 6-membered aromatic or non-aromatic heterocyclic ring, R4 is -H; Each of said R5, R5' and R5" is independently selected from the group consisting of: -H, -OH, -C 1-2 Alkyl, -NR6R7, -SO2-NH2, -Het1 and -halo; the -C 1-2 Each of the alkyl groups is optionally substituted with 1 to 3 substituents selected from the group consisting of =0 and -Het1; R6 and R7 are selected from the group consisting of: -H and -C 1-2 Alkyl; the -C 1-2 Each of the alkyl groups is optionally substituted with 1 to 3 substituents selected from the group consisting of =0 and -NH-iPr, Het1 is selected from morpholinyl and piperazinyl; wherein each of said Het1 is optionally substituted by 1 to 3 selected from -C 1-2 Alkyl and -C 1-2 Substitution by a substituent of the group consisting of alkyl-OH; wherein at least one of R1, R1' and R1" is -OCH3; When R2 and R3 do not form a heterocyclic ring, Represents a double bond.

5. A compound for use as defined in any one of claims 1 or 4, wherein represents a single bond or a double bond; Each of said R1, R1' and R1" is independently selected from the group consisting of: -H and -OCH3; R2 is -H; R3 is -H; or R2 and R3 together with the C atoms to which they are attached form a 6-membered aromatic or non-aromatic heterocyclic ring, R4 is -H; Each of said R5, R5' and R5" is independently selected from the group consisting of: -H, -OH and -halo; wherein at least one of R1, R1' and R1" is -OCH3; When R2 and R3 do not form a heterocyclic ring, Represents a double bond.

6. A compound for use as defined in any one of claims 1 to 5, wherein Each of said R1, R1' and R1" is independently selected from the group consisting of: -H, - and -OCH3; R2 is -H; R3 is -H; or R2 and R3 together with the C atoms to which they are attached form a 6-membered aromatic or non-aromatic heterocyclic ring, R4 is -H; Each of said R5, R5' and R5" is independently selected from the group consisting of: -H, -OH, -F and -Cl; wherein at least one of R1, R1' and R1" is -OCH3; When R2 and R3 do not form a heterocyclic ring, Represents a double bond.

7. A compound for use as defined in any one of claims 1 to 5; wherein the compound is represented by any one of formula (Ia), (IIa) or (IIb) 8. A compound for use as defined in any one of claims 1 to 5, wherein the compound or a salt, hydrate or solvate thereof is selected from the list comprising:

9. A compound or a salt, hydrate or solvate thereof selected from the list comprising:

10. A pharmaceutical composition comprising a compound as defined in claim 9 and a pharmaceutically acceptable excipient.

11. A compound as defined in claim 9 or a composition as defined in claim 10; for use in human or veterinary medicine.

12. A compound as defined in claim 9 or a composition as defined in claim 10 for use in the treatment of cancer.

13. A compound as defined in claim 9 or a composition as defined in claim 10; for use in the treatment of invasive lobular breast cancer, tumors with a tumor-stroma ratio (TSR) > 50%; and / or pancreatic ductal adenocarcinoma.

14. A compound for use as an anti-infiltration compound for tumor cells in a subject suffering from cancer; the compound is represented by any one of formula (IIa) or (IIb) in Each of said R1, R1' and R1" is independently selected from the group consisting of: -H, -halo, -CF3, -OCF3 and -OC 1-3 alkyl; R4 is selected from the group consisting of: -H, -halo, -CF3, -OCF3 and -OC 1-3 alkyl; Each of said R5, R5' and R5" is independently selected from the group consisting of: -H, -OH, -CN, -C 1-3 Alkyl, -OC 1-3 Alkyl, -NR6R7, -SO2-NH2, -SO2-iPr, -Het1 and -halogen; the -C 1-3 Alkyl and -OC 1-3 Each of the alkyl groups is optionally substituted with 1 to 3 substituents selected from the group consisting of =O, -NR8R9 and -Het1; R6, R7, R8 and R9 are selected from the group consisting of: -H and -C 1-3 Alkyl; the -C 1-3 Each of the alkyl groups is optionally substituted with 1 to 3 substituents selected from the group consisting of =O, -NH2 and -NH-iPr, Het1 is a 5-membered or 6-membered heterocyclic ring having 1 to 3 heteroatoms selected from S, O and N; wherein each of said Het1 is optionally replaced by 1 to 3 heteroatoms selected from -C 1-3 Alkyl, -OC 1-3 Alkyl, -C 1-3 Alkyl -OH and -OC 1-3 Substitution by a substituent of the group consisting of alkyl-OH; wherein at least one of R1, R1′ and R1″ is -OC 1-3 Alkyl or -CF3.

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