Compounds identified as CDK4 inhibitors for use as medications

Abstract

The invention relates to compounds that have been identified as CDK4 (cyclin-dependent kinase enzyme) inhibitors for the first time, for use as a medication, in particular, for the treatment of cancers e.g. those with overexpression of the enzyme CDK4, and to pharmaceutical compositions comprising same. Said compounds target the interaction interface between CDK4 and its activating cyclin, whereas those drugs already approved for the treatment of cancers with overexpression of the enzyme CDK4 target the ATP-binding site.

Description

[0001] COMPOUNDS IDENTIFIED AS CDK4 INHIBITORS FOR USE AS

[0002] MEDICINES

[0003] TECHNICAL SECTOR

[0004] This document addresses compounds that have been identified for the first time as inhibitors of CDK4, a cyclin-dependent kinase enzyme, for use as a medicinal product and pharmaceutical compositions comprising the same.

[0005] BACKGROUND OF THE INVENTION

[0006] The cyclin-dependent kinase enzyme CDK4 is important in cell cycle transitions, making it a therapeutic target. The FDA and EMA have approved the drugs palbociclib, ribociclib, and abemaciclib, which target the enzyme's ATP-binding site. However, this ATP-binding site is highly conserved across different kinases within the kinase superfamily, and therefore this therapeutic strategy may offer limited specificity to CDK4.

[0007] This report identifies a family of compounds, as described in detail below, that bind to CDK4 in distinct ways, specifically to the CDK4-cyclin binding interface, namely cyclin D1 (also referred to as Cyc D1), illustrated in Figure 1. Specifically, it has been identified that compounds in this family bind to CDK4 with greater affinity than cyclin D1 and therefore block the binding of Cyc D1 to CDK4, preventing CDK4 activation by this cyclin and thus acting as selective CDK4 inhibitors. Furthermore, this family of compounds offers the advantage of greater selectivity for CDK4 than other types of inhibitors, such as those targeting the ATP binding site.

[0008] GENERAL DESCRIPTION

[0009] This specification is addressed to any compound with a structure according to formula (I) for use as a medicament: where

[0010] - A is an atom independently selected from O and S; preferably A is O or S; and most preferably A is O;

[0011] R 1 and R 2 They are selected independently from among H, -CONH-R a , aryl and alkyl, or R 1 and R 2 together they form a ring;

[0012] R 3 is selected from -CONH-R b , aryl and alkyl;

[0013] R a and R b being independently selected from aryl and alkyl; and the aryl and alkyl groups and the ring being optionally substituted, preferably substituted by a group selected from aryl, alkyl, halogen, ether and amine, more preferably selected from aryl, alkyl and halogen, and still more preferably selected from aryl and alkyl; and the alkyl groups being linear or branched.

[0014] Furthermore, this document addresses formulations comprising such compounds and in particular a pharmaceutical composition comprising a compound with a structure as defined above and detailed below and at least one pharmaceutically acceptable excipient.

[0015] This report also addresses methods of administration of the compound and the pharmaceutical composition.

[0016] BRIEF DESCRIPTION OF THE FIGURES

[0017] To complement the description being made and in order to help a better understanding of the characteristics of the invention, a set of figures is included as an integral part of said description, in which, for illustrative and non-limiting purposes, the following has been represented:

[0018] Figure 1.- Schematic of the CDK4 enzyme with the target of the inhibitors of the present memory interacting with its natural substrate Cyc D1 and with one of said inhibitors, resulting, respectively, in an enzyme-substrate complex that activates the enzyme (active enzyme) and in an enzyme-inhibitor complex that inactivates the enzyme (inactive enzyme).

[0019] Figure 2.— Structure of CDK4 (Protein Data Bank code 6P8E) showing the protein surface with a color intensity code indicating the hydrophobicity of the surface amino acids. The cube superimposed on the protein indicates the interaction site with Cyc D1, showing a highly hydrophobic CDK4 / Cyc D1 interface where the compounds described in this document also bind to CDK4.

[0020] Figure 3.- Effect on cell proliferation of HGUE-GB-39 cells treated with different concentrations of compounds (la), (Ib) and (le) and the CDK4 inhibitors abemaciclib and palbociclib, showing the percentages of viable cells after treatment with the different compounds.

[0021] Figure 4A.- Percentage of dead cells obtained from the HGUE-GB-39 cell line treated with compound (la) and the CDK4 inhibitor abemaciclib.

[0022] Figure 4B.- Effect of the CDK4 inhibitors abemaciclib and palbociclib compared with that of compounds (la), (Ib) and (le), all at a concentration of 10 pM, on the enzymatic phosphorylation activity of the recombinant CDK4-Cyc D1 complex, on a synthetic peptide obtained from the retinoblastoma protein (natural substrate of CDK4).

[0023] Figure 5.- Intracellular localization of CDK4 and Cyc D1 proteins by immunofluorescence for different types of tumor cells: HGUE-GB-42, HGUE-GB-39, RWP-1 and SW-480.

[0024] Figure 6.- Colocalization analysis of the CDK4 and Cyc D1 proteins using Pearson correlation for different cell types: HGUE-GB-42, HGUE-GB-39, RWP-1 and SW-480 corresponding to the photographs in Figure 5.

[0025] Figure 7A.—Interaction of CDK4 and Cyc D1 proteins with compound (la). Proximity ligation fluorescence assay (PLA), where fluorescent spots indicate the binding of CDK4 and Cyc D1 proteins. The images show a decrease in the number of spots due to disruption of the binding caused by compound (la) compared to the control situation where only the vehicle (DMSO) was added. This was observed for different cell types: HGUE-GB-42, HGUE-GB-39, RWP-1, and SW-480.

[0026] Figure 7B.—Interaction of CDK4 and Cyc D1 proteins with compounds (Ib and le). PLA assay, where the images show a decrease in the number of spots resulting from disruption of the binding caused by compounds (Ib and le) compared to the control situation where only the vehicle (DMSO) was added. This has been observed for different types of glioblastoma (GBM) cells: HGUE-GB-39, HGUE-GB-42.

[0027] Figure 8.- Quantification of PLA fluorescence spots per cell in the absence (control) and in the presence of potential inhibitors. Compound (la) has been tested on different cell types (HGUE-GB-42, HGUE-GB-39, RWP-1 and SW-480) corresponding to different tumor types (GBM, pancreas and colon) while compounds (le) and (Ib) only for GBM cells.

[0028] DETAILED DESCRIPTION

[0029] Compounds with a structure according to formula (I) have been identified as binding to the CDK4-Cyc D1 interface and can therefore be used as CDK4 inhibitors, making them potentially useful as drugs. While not linked to any specific theory, compounds with a structure according to formula (I) possess a combination of aromatic rings, hydrophobic groups, and hydrogen bond acceptors that would favor their binding to the CDK4-Cyc D1 interface, given the high hydrophobicity of this interface, as illustrated in Figure 2. Therefore, this dissertation focuses on compounds with a structure according to formula (I) for use as drugs.

[0030] Formula (I) where

[0031] - A is an atom independently selected from O and S; preferably A is O or S; and most preferably A is O;

[0032] R 1 and R2 They are selected independently from among H, -CONH-R a , aryl and alkyl, or R 1 and R 2 together they form a ring;

[0033] R 3 is selected from -CONH-R b , aryl and alkyl;

[0034] R a and R b being independently selected from aryl and alkyl; and the aryl and alkyl groups and the ring being optionally substituted, preferably substituted by a group selected from aryl, alkyl, halogen, ether and amine, more preferably selected from aryl, alkyl and halogen, and still more preferably selected from aryl and alkyl; and the alkyl groups being linear or branched.

[0035] This report also addresses hydrates, or solvates of said compound.

[0036] As can be seen from the structure of formula (I), the family of defined compounds has hydrophobic groups, notably the aromatic rings shown and also the aryl and alkyl groups of the R substituent groups 1 , R 2 , R 3 , R a and R bFurthermore, the structure also includes hydrogen bond acceptor groups, such as the A atoms and the -CONH- groups, which may also be present and can act as hydrogen bond donor groups. While not tied to any specific theory, the combination of hydrophobic groups and hydrogen bond acceptor and donor groups may favor the binding of this family of compounds to the CDK4-Cyc D1 interface. This is because, although the CDK4 surface at the CDK4-Cyc D1 interface has been identified as highly hydrophobic, it may also contain hydrogen bond donor and acceptor groups, such as those present in the functional groups of amino acids located on the CDK4 surface. The corresponding groups of the identified family of compounds can interact with these groups, thus promoting their binding.

[0037] In particular, in formula (I) A is an atom independently selected from O and S; preferably A is O or S; and most preferably A is O. The oxygen (O) and sulfur (S) atoms can act as hydrogen bond acceptor groups.

[0038] In formula (I) R 1 and R 2 They are selected independently from among H, -CONH-R a , aryl and alkyl, or R 1 and R 2 together they form a ring; R 3 is selected from -CONH-R b , aryl and alkyl; R a and R b being selected independently from aryl and alkyl. As previously stated, although the -CONH- groups can act as both hydrogen bond acceptors and donors.

[0039] In formula (I) the aryl and alkyl groups and the ring (if present) formed by R 1 and R 2) may be optionally substituted. In certain embodiments of the invention, they may not be substituted. In other embodiments, some may be substituted and others not. If one or more of the aryl and alkyl groups and the ring (formed by R 1 and R 2 ) are substituted, may preferably be substituted by a group selected from aryl, alkyl, halogen, ether and amine, more preferably selected from aryl, alkyl, and halogen, and even more preferably selected from aryl and alkyl.

[0040] By way of example: aryl groups can be selected from phenyl, phthalimidyl, biphenyl, naphthalenyl, and benzyl; alkyl groups can be linear or branched and, in particular, can be selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl; a halogen group can be selected from fluorine, chlorine, bromine, and iodine; an ether group can include substituents selected from -OR and -R and R are independent; an alkyl group, preferably an alkyl group as defined above; and an amine group may include substituents selected from -NH2, -R¡-NH2, -NH-R¡, -R¡-NH-R¡, -N(R¡)-R¡¡ and -R¡-N(R¡¡)-R¡¡, wherein R¡, R¡ and R¡¡ are independently an alkyl group, preferably an alkyl group as defined above.

[0041] These aryl or alkyl groups within formula (I) can be the aryl or alkyl groups of R 1 , R 2 , R3 , R a and R b Or, if one or more of these groups are substituted by an aryl or alkyl group, they can be the groups of that substitution. For example, if R 1 It is a -CONH-R group a and R a It is an aryl group, such as a phthalimidyl group. The phthalimidyl group can be substituted by an aryl group, such as a phenyl or naphthalenyl group, and this phenyl or naphthalenyl group can be found, for example, at the N position of the phthalimide group. More specifically, R a It can be an N-phenyl-phthalimide group or an N-naphthalenyl-phthalimide group, preferably an N-phenyl-phthalimide group. More specifically, R a It can be a 5-(N-phenyl-phthalimide) or a 5-(N-naphthalenyl-phthalimide), that is, an N-phenyl-phthalimide or N-naphthalenyl-phthalimide group attached at its 5 position, as illustrated by the following structures (Ha) and (llb) respectively:

[0042] Structure (lia) Structure (llb)

[0043] For example, if R 2 It is a -CONH-R group a and R a is an aryl group, such as a phenyl group; the phenyl group can be substituted by another aryl group, such as a second phenyl group, and this phenyl group can be located, for example, at position 2 of the first phenyl group. More specifically, R a It can be a biphenyl and even more specifically it can be a 2-(biphenyl), that is, a biphenyl group attached at its 2 position, as illustrated by the following structure (He):

[0044] Structure (lie) As an example, if R 3 It is a -CONH-R group b , R b It can be a substituted aryl group as described above for R a , and R 3 It could be, for example, an N-phenyl-phthalimido group or a biphenyl group, as also described above for R 1 and R 2 .

[0045] In some embodiments, in formula (I) the R groups 1 and R 2 Together they can form a ring. For example, the ring formed can be an aromatic ring, preferably an aromatic ring selected from a 6-membered or 5-membered aromatic ring. By way of illustration, the ring formed by R 1 and R 2 can form an aromatic complex with the aromatic ring to which they are attached. For example, a compound with a structure of formula (I) where R 1 and R 2 Together they form a 6-member ring that can have the following structure (lid):

[0046] Structure (lid)

[0047] In some realizations in formula (I) R 1 and / or R 2 it can be -CONH-R a In some particular implementations, preferably R aIt may be aryl. More preferably, the aryl group may be selected from phenyl, biphenyl, phthalimidyl, naphthalenyl, or benzyl. Optionally, the aryl group may be substituted with a group preferably selected from phenyl, benzyl, naphthalenyl, or ethyl. Even more preferably, the aryl group may be selected from biphenyl, naphthalenyl, N-phenyl-phthalimide, or N-naphthalenyl-phthalimide.

[0048] In some particular realizations, one of R 1 and R 2 It is H and the other one is from R 1 and R 2 es -CONH-R a .

[0049] In more specific realizations, preferably: when R 1 is H then R 2 es -CONH-R a and R a is selected from biphenyl and naphthalene; and preferably is biphenyl; or when R 2 is H then R 1 es -CONH-R a and R aIt is selected from N-phenyl-phthalimide and N-naphthalenyl-phthalimide; and preferably it is N-phenyl-phthalimide.

[0050] In some realizations, in formula (I) R 3 is CONH-R b In some particular implementations, preferably R b is aryl. More preferably, the aryl group can be selected from phenyl, biphenyl, phthalimidyl, naphthalenyl, and benzyl. Optionally, the aryl group can be substituted with a group preferably selected from phenyl, benzyl, naphthalenyl, and ethyl. Even more preferably, the aryl group can be selected from biphenyl, naphthalenyl, N-phenyl-phthalimide, and N-naphthalenyl-phthalimide; and still more preferably, it can be selected from biphenyl and N-phenyl-phthalimide.

[0051] In other realizations, in formula (I) R 3is aryl. In some particular embodiments, the aryl group may preferably be selected from phenyl, biphenyl, phthalimidyl, naphthalenyl, or benzyl. Optionally, the aryl group may be substituted with a group preferably selected from phenyl, benzyl, naphthalenyl, or ethyl. Even more preferably, the aryl group may be selected from biphenyl, naphthalenyl, N-phenyl-phthalimide, or N-naphthalenyl-phthalimide; and still more preferably, the aryl group is N-naphthalenyl-phthalimide.

[0052] Compounds with a structure according to formula (I) may preferably have a molecular weight of 175 to 800 Da, particularly from 200 to 775 Da. Compounds with molecular weights in these ranges have been observed to bind to CDK4 and show inhibitory activity both in vitro and in cellular systems, and therefore would have the ability to penetrate plasma membranes.

[0053] Alternatively or additionally, compounds with a structure according to formula (I) may preferably have a logP of 1.0 to 10.0, particularly from 1.5 to 9.0. In particular embodiments, the logP may be from 5.0 to 9.0, particularly from 6.0 to 8.5. Surprisingly, it has been observed that compounds with a logP within this latter range exhibit activity in cellular systems despite being outside the logP range of less than 5.0, considered in the art to be the most suitable logP for pharmaceutical uses, for example, according to Lipinsky's rule of five.

[0054] The logP can be known, for example, from the compound supplier, or it can be determined by methods known to an expert in the field. For example, the logP of a compound can be determined using specialized software, such as Datawarrior 5.0 (Sander et al., J. Chem. Inf. Model 2015, 55, 460-473, [DOI: 10.1021 / c¡500588j]). As an example, compound (la) has a logP of 6.44, compound (Ib) of 8.10, and compound (le) of 7.37, determined with Datawarrior 5.0.

[0055] The compound with structure according to formula (I) may preferably be selected from a compound with structure according to formula (Ia), formula (Ib), and formula (Ie), as detailed in the following table. The table also includes the SMILES notation and the English IUPAC name of each compound:

[0056] The compound according to formula (I) may preferably be the compound of formula (la). Surprisingly, compound (la), despite having a molecular weight of 752 Da, and therefore being above 500 Da, this being the maximum molecular weight according to Lipinsky's rule of five, has been observed to show inhibitory activity of CDK4 in cellular systems, which would indicate that it has penetrability of cell membranes.

[0057] These compounds are commercially available. They can be obtained, for example, from the supplier Molport (accessible online at: https: / / www.molport.com / , with the access codes: Molport-001-025-593 for compound (la); Molport-002-134-975 for compound (Ib); and Molport-001-024-020 for compound (le)). Other compounds with structures according to formula (I) are also commercially available and / or can be prepared by known methods.

[0058] Compounds with a structure according to formula (I), including the particular and preferred embodiments detailed above, have been identified as potentially useful as a medicament. Specifically, compounds with a structure according to formula (I), particularly including the particular and preferred embodiments detailed above, advantageously exhibit activity against cancer cells, particularly against human GBM cell lines (e.g., HGUE-GB-42 and HGUE-GB-39), human pancreatic adenocarcinoma cell lines (e.g., RWP-1), and colorectal cancer cell lines (e.g., SW-480). While not linked to a specific theory, their anticancer activity can be attributed to the interaction of these molecules with CDK4, particularly at the Cyc D1 binding site, where they act as CDK4 inhibitors.Given its affinity for binding to CDK4 and its effect as CDK4 inhibitors, it opens the door to the treatment of diseases associated with CDK4 overexpression such as cancer, particularly GBM, pancreatic cancer, colorectal cancer and other types of cancer such as breast cancer, mantle cell lymphoma, Kaposi sarcoma, prostate cancer, lung cancer, and gynecological cancer.

[0059] Therefore, in particular embodiments, the drug may be for the treatment of a disease. More preferably, a compound having a structure according to formula (I), as described above, may be used as a drug for the treatment of cancer. In particular, the drug may be for the treatment of, for example, GBM, breast cancer, pancreatic cancer, colon cancer, mantle cell lymphoma, Kaposi's sarcoma, prostate cancer, lung cancer, and / or gynecological cancer; in particular for the treatment of GBM, breast cancer, pancreatic cancer, and / or colon cancer; and more particularly for the treatment of GBM.

[0060] This specification also addresses the use of compounds with a structure according to formula (I) in a treatment method. Such a method may include, among other things, administering a compound with a structure according to formula (I) to a subject, as described above. In particular, the subject may be human or non-human. For example, the subject may be a mammal, preferably a human, although use in animals is not excluded. The subject may have cancer.

[0061] In particular embodiments, a compound with a structure according to formula (I) may be used as a medicament or treatment. This may comprise administering the compound, or a pharmaceutical composition comprising the compound and a pharmaceutically acceptable excipient, to a subject suffering from cancer, in particular glioblastoma (GBM), breast cancer, pancreatic cancer, colon cancer, mantle cell lymphoma, Kaposi's sarcoma, prostate cancer, lung cancer, and / or gynecological cancer, more particularly GBM, breast cancer, pancreatic cancer, and / or colon cancer, and still more particularly GBM.

[0062] This specification also relates to a pharmaceutical composition comprising a compound of structure according to formula (I), as described above, and at least one pharmaceutically acceptable excipient.

[0063] Pharmaceutically acceptable excipients include fillers, absorbents, wetting agents, binders, disintegrants, lubricants, vehicles (e.g., solvents such as water or solvent mixtures), solubilizers, surfactants, pH regulators, isotonic agents, preservatives, antioxidants, and the like.

[0064] In some embodiments of this disclosure, the pharmaceutical composition includes, but is not limited to, a formulation suitable for oral and parenteral administration (e.g., an injectable formulation). In some embodiments, the pharmaceutical composition is a formulation suitable for oral administration. In some embodiments, the pharmaceutical composition is a solid formulation, and in particular a solid formulation suitable for oral administration. In some embodiments, the pharmaceutical composition is a formulation suitable for parenteral administration. In some embodiments, the pharmaceutical composition is a liquid formulation, and in particular a liquid formulation suitable for parenteral administration, for example, an injectable formulation.

[0065] The compound may be the sole therapeutic agent or active ingredient of the pharmaceutical composition or may be combined with other therapeutic agents, within the same pharmaceutical composition or used in combination with other separately formulated therapeutic agents.

[0066] In some embodiments, the pharmaceutical composition may further comprise an additional therapeutic agent. The additional therapeutic agent may be a known therapeutic agent in the treatment of cancer, preferably a therapeutic agent for GBM, breast cancer, pancreatic cancer, and / or colon cancer. By way of example, the additional therapeutic agent may be selected from inhibitors of, e.g., CDK4, CDK5, and / or CDK6; preferably a compound selected from: palbociclib, ribociclib, abemaciclib, gemcitabine, and carmustine (BCN1).

[0067] In some embodiments, the pharmaceutical composition may be formulated for the controlled release of the compound and / or additional therapeutic agent.

[0068] The pharmaceutical composition described in this document can be manufactured using methods well known in the art, such as conventional mixing, dissolution, granulation, tablet making, leviga, emulsification, and lyophilization.

[0069] A solid oral composition can be prepared by conventional mixing, filling, or tableting. For example, it can be obtained by the following method: mixing the active compounds with solid excipients, optionally milling the resulting mixture, adding additional suitable excipients if desired, and processing the mixture into granules to obtain the core portions of tablets or coated tablets. Suitable excipients include, but are not limited to: binders, diluents, disintegrants, lubricants, glidants, sweeteners, flavoring agents, and the like.

[0070] A liquid parenteral composition can be prepared by dissolution, suspension, and mixing. For example, it can be obtained by the following method: mixing the compound of structure according to formula (I) with a suitable vehicle (e.g., water or a suitable solvent mixture), mixing and adding additional suitable excipients if desired, and processing the solution and / or suspension to obtain an injectable formulation. Suitable excipients include, but are not limited to: solubilizers, surfactants, pH regulators, isotonic agents, preservatives, and antioxidants.

[0071] The compound or pharmaceutical composition may be administered via multiple routes of administration, including, but not limited to, oral, parenteral, intravenous, intra-arterial, intramuscular, transdermal, intraperitoneal, sublingual, rectal, transoral, intranasal, inhalation, vaginal, intraocular, topical, subcutaneous, intraadipose, intra-articular, intraperitoneal, and intrathecal. In specific embodiments, the compound or pharmaceutical composition may be administered orally, intraperitoneally, and / or intravenously.

[0072] The amount of compound administered can be determined according to the severity of the disease, the response to the disease, any treatment-related toxicity, and the patient's age and health status.

[0073] In this text, the words "comprises," "includes," and their variants (such as "comprising," "including," etc.) should not be interpreted in an exclusive way; that is, they do not exclude the possibility that what is described includes other elements, steps, etc. In this text, "a" refers to at least one, that is, one or more.

[0074] Furthermore, the invention is not limited to the specific embodiments described but also encompasses, for example, those that can be carried out by the average person skilled in the art (for example, regarding the choice of materials, dimensions, components, configuration, etc.), within the scope of the claims.

[0075] This disclosure is further illustrated by, but not limited to, the following examples.

[0076] EXAMPLES

[0077] In vitro experiments.

[0078] The activity of the three compounds with structures according to the formula (la), (Ib) and (le), referred to respectively as compounds (la), (Ib) and (le), was determined in vitro by different experiments with the CDK4 and Cyc D1 proteins and cancer cell lines as described below.

[0079] Compounds (la), (Ib) and (le) were obtained from the supplier MolPort (accessible through its online portal: https: / / www.molport.com / , with the access codes: Molport-001-025-593 for compound (la); Molport-002-134-975 for compound (Ib); and Molport-001-024-020 for compound (le).

[0080] For the assays, the compounds were dissolved in DMSO and diluted with water to the assay concentration for cell treatment. The maximum concentration of the solvent vehicle (DMSO) of each tested compound at the assay concentration was used as a control.

[0081] Human GBM cell lines HGUE-GB-42 and HGUE-GB-39 were used. Isolation of the human GBM cell lines HGUE-GB-42 and HGUE-GB-39 was performed from surgical washings, as described by Ventero et al. (PLOS ONE 2019, 14(4), e0215714; [DOI: 10.1371 / journal.pone.0212581]). The cells were then cultured in Dulbecco's modified Eagle medium: nutrient mixture F-12 (DMEM F-12) (Biowest, MO, USA). The Municipal Institute of Medical Research (IMIM, Barcelona, ​​Spain) donated the human pancreatic adenocarcinoma (RWP-1) and colorectal cancer (SW-480) cell lines. The RWP-1 and SW-480 cell lines were cultured in Dulbecco's modified Eagle medium: high glucose (DEMEM-HG) (Biowest, MO, USA). Both media were supplemented with 10% (v / v) heat-inactivated fetal bovine serum (FBS) (Capricorn Scientific, Ebsdorfergrund, Germany) and 1% (v / v) of a penicillin / streptomycin mixture (Biowest, MO, USA).). The cells were incubated at 37 °C in a humidified atmosphere with 5% CO2, as described by Fuentes-Baile et al. (Biomolecules 2020, 10(2), 222; [DOI: 10.3390 / biom 10020222] and Int. J. Mol. Sel. 2021 , 22(3), 1477; [DOI : doi.org / 10.3390 / ijms22031477]).

[0082] Study of cell proliferation:

[0083] The antiproliferative capacity of the different compounds (1a), (1b), and (1e) was analyzed using the MTT colorimetric assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) based on the reduction reaction catalyzed by the mitochondrial enzyme succinate dehydrogenase. Upon addition of the yellow MTT compound, succinate dehydrogenase in viable cells catalyzes the opening of the tetrazolium ring, generating formazan salts ((E,Z)-5-(4,5-dimethylthiazol-2-yl)-1,3-diphenylformazan), which are insoluble, impermeable to the plasma membrane, and purple in color. Thus, the number of viable cells can be quantified, as it is proportional to the amount of formazan generated.

[0084] Initially, HGUE-GB-39 cells were seeded in 96-well plates at a density of 3,000–4,000 cells per well, depending on the size and growth rate of the cell line. After 24 hours, the corresponding treatments were added in sextuplicate with increasing concentrations of each of the compounds (la), (Ib), and (le), ranging from 0.1–25 pM, and the cells were in culture for 72 hours. After the treatment period, MTT reagent (Sigma-Aldrich®, St. Louis, MO, USA) was added at a final concentration of 0.25 mg / mL for 3 hours. Throughout the assay, the cells were incubated at 37°C with 5% CO2. Finally, the contents of the wells were removed and 200 ml of dimethyl sulfoxide (DMSO, Sigma Aldrich®, St. Louis, MO, USA) were added and the plates were kept for 30 minutes, at room temperature and under intense agitation to dissolve the formazan crystals.Finally, the absorbance of the plates was measured at 570 nm in a Gen5™ plate reader (BioTeK®, Winooski, VT, USA), as described by Grasso et al. (BMC Cancer 2015, 15, 240; [DOI: 10.1186 / s 12885-015-1183-3]).

[0085] As a control, the cells were treated with the maximum concentration of the solvent vehicle (DMSO) of each tested compound, following the same process described for conducting the study.

[0086] The effects on proliferation were compared with those obtained at the same concentration range with the CDK4 inhibitor drugs abemaciclib and palbociclib.

[0087] Figure 3 shows the results obtained, representing the percentages of viable cells after treatment with the different compounds, in a concentration range of 0.1 to 25 pM, obtained in MTT assays, and referring to 100% viable cells as those cells only treated with vehicle (DMSO), added at 1% by volume.

[0088] In addition, different concentrations in the range of 0.1-50 pM were used to obtain the 50% inhibitory concentration, IC50, of compounds (la), (Ib) and (le) by evaluating the viability of HGUE-GB-39 cell line in the presence of these compounds, and were compared with the viability of the cells in the presence of palbociclib, obtaining the following IC50 values: 5.9 pM for compound (la), 25.2 pM for compound (Ib), 17.7 pM for compound (le), and 7.5 pM for palbociclib.

[0089] Cell cycle analysis:

[0090] To determine the percentage of cells in each phase of the cell cycle, titration with propidium iodide was used. Propidium iodide is a fluorescent compound impermeable to the plasma membrane that intercalates into the genetic material of cells. It has an excitation peak at 535 nm (blue-green) and its maximum emission occurs at 617 nm (red).

[0091] The cell cycle is divided into four phases: G1, S, G2, and M. During the G1 phase, cells grow, synthesize proteins and RNA, and the number of certain cytoplasmic structures and organelles increases. In the S phase, DNA replication occurs. In the G2 phase, cells continue to grow and produce the proteins necessary for cell division. The M phase corresponds to mitosis, or cell division. The G1 phase is the longest, so in their normal state, most cells are in this phase. Once the RNA is removed by ribonuclease (RNase), it is possible to differentiate between the phases of the cell cycle based on their DNA content, since cells in the G2 or M phase have twice the amount of DNA as those in the G1 phase.Distillation of cells with propidium iodide and subsequent analysis by flow cytometry allows differentiation between the various phases of the cell cycle based on fluorescence intensity. This type of assay makes it possible to detect dead cells, whether due to apoptosis or necrosis, since their DNA is fragmented, resulting in a lower fluorescence intensity than that observed in live cells; this is known as the SubG1 phase. Therefore, by analyzing the cell cycle, we can determine not only if the treatment has an effect on the cells, but also whether this effect is cytotoxic (cell death) or cytostatic (cell cycle arrest in one of the phases), as described by Al-Malky et al. Cancer Cell Int. 2019. 19:191., [DOI: 10.1186 / s 12935-019- 0912-0].

[0092] To carry out this type of study, between 250,000-350,000 cells per well were seeded in 6-well plates and, after 24 hours of incubation, the corresponding treatment was added, a concentration of compound (la) of 10 pM and a concentration of abemaciclib of 10 and 25 pM, which was maintained for a minimum time of 24 hours, which is approximately the duration of the cell cycle in mammals.

[0093] After the treatment period, the cells were trypsinized, centrifuged at 400 x g for 5 minutes, resuspended in 1 mL of cold 75% ethanol, and incubated at -20°C for a minimum of 1 hour to fix them. The ethanol was then removed by another 5-minute centrifugation at 400 x g, and the cells were resuspended in 500 pL of a PBS solution containing 0.5% (v / v) Triton X-100 (Sigma Aldrich®, St. Louis, MO, USA) to permeabilize the plasma membrane, 25 pg / mL RNase A (Sigma Aldrich®, St. Louis, MO, USA) to remove RNA, and 25 pg / mL propidium iodide (Promocell®, Heidelberg, Germany). Finally, after 30 minutes of incubation in darkness, cell cycle analysis was performed using the BD FACSCanto™ II flow cytometer (BD Biosciences, San Jose, CA, USA), as described by Castro-Galache et al. (Int. J. Cancer 2003, 104(5), 579-586; [DOI: 10.1002 / ¡jc.10998]).

[0094] To evaluate the induction of cell death in the HGUE-GB-39 and HGUE-GB-42 cell lines by compound (la) and compare it with that of the inhibitor abemaciclib, the effect of abemaciclib at 10 pM in each cell line was used as the 100% reference value. Figure 4A shows the results obtained, representing the percentage of dead cells. As can be seen, at the same concentration of 10 pM, compound (la) showed a greater number of dead cells than abemaciclib for both cell lines. Furthermore, for the HGUE-GB-39 cell line, compound (la) showed a greater number of dead cells than abemaciclib at a higher concentration (25 pM).

[0095] Therefore, the compound (la) would have superior activity against GBM cell lines, indicating potential efficacy for the treatment of GBM in living organisms.

[0096] Phosphorylation assay:

[0097] The effect of the CDK4 inhibitors abemaciclib and palbociclib, and of compounds (la), (Ib), and (le), at a concentration of 10 pM, on the phosphorylation activity of the recombinant CDK4-Cyc D1 complex was evaluated using a synthetic peptide derived from retinoblastoma protein (Rb) (the natural substrate of CDK4) via the Promega ADP-Glo ​​kit (Madison, W1, USA), following the manufacturer's instructions. The results are shown in Figure 4B. 100% represents the maximum enzymatic activity observed with the recombinant complex in the presence of the highest dose of the vehicle (1% v / v DMSO) used to solubilize the compounds. Three experiments were averaged, and the mean ± SD (standard deviation) is shown.

[0098] The CDK4 / Cyc D1 enzyme was briefly diluted to 0.1 mg / ml in a buffer solution containing 20 mM Ths (pH 7.5), 0.02% Triton X-100, 0.1 mg / ml BSA, 2 mM DTT, 0.5 mM Na3VO4, and 10% glycerol. Another buffer solution (100 ml) was prepared with 2 ml of 1 M Ths, 20 ml of Triton X-100, 10 mg BSA, 30.84 mg DTT, 9.2 mg Na3VO4, 10 ml glycerol, and 88 ml H2O. CDK4 / Cyc D1 0.35 mg / ml was prepared, and 71.43 µl of the prepared buffer solution was added to dilute the CDK4 / Cyc D1 to 0.1 mg / ml. The mixture was then aliquoted into five 20 µl Eppendorf tubes. Water was added to prepare a 10 pg / ml aliquot. ATP was then added, and the plate was incubated for 1 h at room temperature. Five µl of ADP-Glo ​​reagent was added to stop the reaction and deplete any unconsumed ATP, followed by 10 µl of kinase detection reagent to convert ADP to ATP and introduce luciferase and luciferin for ATP detection. The plate was incubated at room temperature according to the manufacturer's instructions. Finally, luminescence was measured.

[0099] Compounds (la), (Ib), and (le) were found to inhibit the kinase activity of the CDK4 / Cyc D1 enzyme by percentages ranging from 60% (compound le), 95% (compound Ib), and 90% (compound la) when added at a concentration of 10 pM. These values ​​are very similar to the effects exerted by palbociclib or abemaciclib, as shown in Figure 4B.

[0100] Immunofluorescence:

[0101] To verify whether both proteins (CDK4 and Cyc D1) were expressed and colocalized in the same cellular compartments in the different cell lines (Fig. 1), immunofluorescence assays were performed. The cell lines evaluated were: HGUE-GB-42 and HGUE-GB-39 from GBM, RWP-1 from pancreatic cancer, and SW-480 from colorectal cancer.

[0102] Thirty-five thousand cells from the HGUE-GB-42, HGUE-GB-39, RWP-1, and SW-480 cell lines were seeded in twenty-four well plates on 12 mm coverslips. After 24 h, they were fixed with 4% paraformaldehyde (PFA) and blocked with FBS / PBS (1x) (50 pL / mL). The cells were then incubated with anti-CDK4 antibodies (1:100, mouse; NeoMarkers, California, USA) and anti-CyD1 antibodies (1:100, rabbit; Cell Signaling, Massachusetts, USA).

[0103] After washing off the first antibody, the cells were incubated with Alexa Fluor 568 (1:500) labeled anti-mouse and Alexa Fluor 488 (1:500) labeled anti-rabbit secondary antibodies (Invitrogen, Barcelona, ​​Spain). DAPI reagent was used to stain the nucleus. Coverslips were mounted on Prolong Gold Antifade Reagent (Invitrogen, Barcelona, ​​Spain) and analyzed using an LSM900 confocal microscope with an Airyscan 2 microscope (Carl Zeiss, Oberkochen, Germany) at 63x magnification.

[0104] The results of the protein localization are shown in Figure 5 for the different cell types evaluated HGUE-GB-42, HGUE-GB-39, RWP-1 and SW-480.

[0105] CDK4 was found to be highly expressed in the nucleus of GBM cells (HGUE-GB-42 and HGUE-GB-39) as shown by colocalization with DAPI, and also in the cytoplasm. However, it is predominantly expressed in the cytoplasm of pancreatic tumor cells (RWP-1) and colon adenocarcinoma cells (COAD) (SW-480).

[0106] Similarly, Cyc D1 was expressed in both the cytoplasm and nucleus of GBM cells, but also in the rest of the tumor cell lines.

[0107] Colocalization of these two proteins was measured using the Pearson correlation coefficient (PRV), as illustrated in Figure 6, which confirmed the fact that in GBM, CDK4 and Cyc D1 have a higher level of co-expression (with a PRV of 0.451 for HGUE-GB-39 and 0.495 for HGUE-GB-42), than in pancreatic cancer (with a PRV of 0.373 for RWP-1) and colorectal cancer (with a PRV of 0.285 for SW-480).

[0108] Proximity ligation assay (PLA):

[0109] To determine the ability of compounds (la), (Ib), and (le) to block the interaction between CDK4 and Cy-D1, the interaction between these two proteins was studied in different cell lines. Thirty-five thousand cells from the HGUE-GB-42, HGUE-GB-39, RWP-1, and SW-480 cell lines were seeded in twenty-four well plates on coverslips. The cells were treated with compounds (la), (Ib), and (le) (10 pM) for 6 hours, then washed in PBS (1x), fixed with 4% PFA, washed again, permeabilized in PBS (1x) with 0.2% Triton X-100, and blocked with blocking solution for 1 h at 37 °C before immunostaining with DuoLink using PLA technology (Merck, Madrid, Spain), following the manufacturer's protocol. Primary antibodies used were anti-CDK4 (1:100, mouse; NeoMarkers, California, USA) and anti-CyD1 (1:100, rabbit; Cell Signaling, Massachusetts, USA).The slides were processed for in situ PLA using sequentially DuoLink In Situ PLA Probe Anti-Mouse MINUS, DuoLink In Situ PLA Probe Anti-Rabbit PLUS and DuoLink In Situ Red detection reagents (Merck, Madrid, Spain).

[0110] In these experiments, the fluorescent spots (red) correspond to positive signals for PLA, indicating that the two proteins (CDK4 and Cyc D1) are bound, forming a protein complex; conversely, the blue fluorescent spots correspond to nuclei (DAPI staining). This technique reveals protein binding that occurs at distances of less than 16 Å.

[0111] Both negative and positive control experiments were performed, the former omitting one of the primary antibodies.

[0112] Image acquisition was performed using an LSM900 confocal microscope with Airyscan 2 (Carl Zeiss, Oberkochen, Germany) with a magnification of x 63. The number of red dots was counted using Fiji software (Image J2).

[0113] Results are presented as the mean ± standard deviation (SD) of three independent experiments (n = 5 images). The Shapiro-Wilk test or Student's t-test was used to assess the normality of the data and to analyze the association between variables. Differences were considered statistically significant with a p-value <0.05. Statistical analysis was performed using GraphPad Prism v7.0a software (GraphPad Software Inc., San Diego, CA, USA).

[0114] All cell lines were treated with a 10 pM concentration of compound (la) for 6 hours and with the control. Examples of the images obtained with compound (la) are shown in Figure 7A. The fluorescent dots (red) corresponding to PLA signals (indicated in the figure as “PLA: CDK4 / Cyc D1”) indicate that CDK4 interacted efficiently with Cyc D1, mostly within the nucleus, in all tumor cell lines under the control, as expected from the immunofluorescence experiments (Figure 5-6). The HGUE-GB-42 and HGUE-GB-39 cell lines were also treated with a 10 pM concentration of compounds (Ib) and (le) for 6 hours and with the control. The results obtained with compounds (Ib) and (le) are shown in Figure 7B.

[0115] Figure 8 shows the quantification, in the form of a bar graph, of the number of fluorescent spots per cell, treated with the control (DMSO) or with compounds (la), (Ib), and (le). Fiji software was used to count the number of red spots. Data represent the mean ± SEM. One-way ANOVA was performed, and Tukey's post hoc test was used. *p < 0.05, **p < 0.01, ***p < 0.001; ****p < 0.0001.

[0116] Treatment with compound (la) confirmed that the interaction between CDK4 and CyD1 was displaced by it in all tested cell lines. Compound (la) was even more effective in the HGUE-GB-42 cell line than in the other GBM cell line (HGUE-GB-39), which was the line where the nuclear interactions between CDK4 and CyD1 were least affected. In the other two cancer types, the reduction of the interaction between the two proteins was also significantly affected by the compound.

[0117] (la), which is an indicator of the effectiveness of this type of compound not only in a therapy against GBM, but can also affect other types of cancer (Figure 7-8).

[0118] It is observed that the number of spots per cell decreases in the presence of the compound

[0119] (lb) and (le). While compound (le) showed a similar decrease in the number of points per cell for both types of cell lines, compound (Ib) showed a more marked decrease for HGUE-GB-39 cells, but perhaps more surprising is that it even showed a more significant decrease for these cells than compound (le).

[0120] Computer simulations:

[0121] Other properties of the three compounds with structures according to the formula (la), (Ib) and (le), referred to respectively as compounds (la), (Ib) and (le), were determined in silico by different simulations.

[0122] ADMET Properties:

[0123] The in silico absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties were calculated using the AdmetSAR web application, as described by Cheng et al. (J. Chem. Inf. Model. 2012, 52, 11, 3099-3105; [DOI: 10.1021 / c¡300367a]), and the DataWarrior software (v. 5.0), as described by Sander et al. (J. Chem. Inf. Model. 2015, 55, 2, 460-473; [DOI: 10.1021 / c¡500588j]). The parameters evaluated were: total surface area, topological polar surface area, calculated LogS, molecular weight, calculated logP, hydrogen bond acceptors, violations of Lipinski's rule of five, hydrogen bond donors, drug similarity, drug score, calculated Caco-2 permeability, calculated acute toxicity in rats, calculated toxicity in Tetrahynema pyriformis, and calculated toxicity in fish (data not shown).

[0124] Compounds (la), (Ib), and (le) exhibited ADMET properties that would not always be considered optimal. For example, they violate one or more of Lipinsky's five rules. Compound (la) has a molecular weight (752 Da) greater than 500 Da, a calculated logP greater than 5 (la = 6.44, lb = 7.37, and lc = 8.10), and le has 13 hydrogen bond acceptors (when Lipinsky's rule states no more than 10). The number of donors is less than 3 for all three compounds. However, as demonstrated, compounds (IA), (Ib), and (le) show inhibitory activity when used to treat cell lines, even better than other known CDK4 inhibitors.

[0125] Molecular coupling:

[0126] Molecular docking simulations of compounds (la), (Ib) and (le) were carried out using YASARA structure v22.5.22 software, running the AutoDock 4 algorithm with AMBER99 as the force field, as described by Encinar et al. (Drug Des Devel Ther. 2015, 9, 5877-5895; [DOI: 10.2147 / DDDT.S93449]), Galiano et al. (Drug Des Devel Ther. 2016, 10, 3163-3181 ; [DOI: 10.2147 / DDDT.S117369]), and Ruiz-Torres et al. (Mar. Drugs 2018, 16, 385; [DOI: 10.3390 / md16100385]). A total of 100 flexible coupling runs were established and grouped (7 Å) around the interaction surface between CDK4 and Cyc D1, i.e., two compounds coupled to CDK4 belonged to different groupings if the mean square deviation of the ligand from its atomic positions was greater than 7 Å, as described by Cuyás et al. (Food and Chemical Toxicology 2019, 132, 110645; [DOI: 10.1016 / j.fct.2019.110645]).

[0127] The YASARA software calculated the variation of Gibbs free energy (AG, Kcal / mol), where more positive energy values ​​indicate a stronger binding to CDK4 as described by Rubio-Camacho et al. (Biomolecules 2020, 10, 1015; [DOI:

[0128] 10.3390 / biom10071015]). To calculate this parameter, Autodock Vina uses a force field scoring function that considers the strength of electrostatic interactions, hydrogen bonds between all atoms of the two bonding partners in the complex, van der Waals intermolecular forces, and solvation and entropy contributions, as described by Morris et al. (J. Comp. Chem. 2009, 30, 16, 2785-2791 ; [DOI: 10.1002 / jcc.21256]).

[0129] For the human CDK4 enzyme (UniProt code: P11802 ■ CDK4_HUMAN), the 6P8E structure (https: / / www.rcsb.org / structure / 6P8E) deposited in the Protein Data Bank database was used. In this structure, CDK4 is co-crystallized with Cyc D1 and the regulatory protein P27. The grid dimensions were 23 x 23 x 23 Å. The profanation state was set at pH 7.4 for the CDK4 side chains using YASARA structure v22.5.22 software, as described by Krieger et al. (Bioinformatics 2014, 30, 20 2981-2982 [DOI: 10.1093 / bioinformatics / btu426]). After each docking simulation, the YASARA software generated a file containing the molecular coordinates of different poses of each library compound docked to the binding site on the CDK4 protein, as well as the Gibbs free energy variation (AG, kcal / mol) for each pose.Compounds (la), (Ib) and (le) showed an AG > 9.5 kcal / mol, which is indicative of a good coupling of the compounds to CDK4, particularly in their interface zone with Cyc D1, and of their potential inhibitory activity, as has also been demonstrated in in vitro experiments.

[0130] Molecular dynamics:

[0131] Molecular dynamics simulations were carried out using YASARA structure v22.5.22 software, with AMBER14 as the force field, as described by Encinar et al. (Viruses 2020,10, 12(5), 525; [DOI: 10.3390 / v12050525]), Marroquí et al. (Chemosphere 2021 , 265, 129051 ; [DOI: 10.1016 / j.chemosphere.2020.129051]), Verdura et al. (Aging 2020, 12(1), 8-34; [DOI: 10.18632 / aging.102646]), and Fernández-Ginés et al. (Redox Biology 2022, 55,102396; [DOI: 10.1016 / j.redox.2022.102396]). The simulation cell was allowed to include 20 Å around the CDK4 protein, which was filled with water at a density of 0.997 g / ml. Initial energy minimization was performed under relaxed constraints using steepest descent minimization. Simulations were carried out in water at constant pressure (1 atm) and temperature (25 °C). To mimic a physiological environment, counterions were added to neutralize the system (Na+). +or OI' in place of water to obtain a final NaCl concentration of 0.9% and a pH maintained at 7.4). Hydrogen atoms were added to the protein structure at the appropriate ionizable groups according to the calculated pKa and the simulation pH (i.e., a hydrogen atom was added if the calculated pKa was higher than the pH). The pKa was calculated for each residue according to the Ewald method, as described by Krieger et al. (J. Mol. Graph. Model. 2006, 25 (4), 481-486; [DOI: 10.1016 / j.jmgm.2006.02.009]). Data were recorded every 100 ps. Molecular mechanics Poisson-Boltzmann surface (MM / PBSA) calculations were used to determine the binding free energy of candidate compounds against CDK4 inhibitors using the YASARA macro md_analyzebindenergy.mcr, as described by Encinar et al. (Viruses 2020,10, 12(5), 525; [DOI: 10.3390 / v12050525]), Marroquí et al. (Chemosphere 2021 , 265, 129051 ; [DOI: 10.1016 / j.chemosphere.2020.129051]), Verdura et al. (Aging 2020, 12(1), 8-34; [DOI: 10.18632 / aging.102646]), and Fernández-Ginés et al. (Redox Biology 2022, 55,102396; [DOI: 10.1016 / j.redox.2022.102396]). When a compound remains bound to the CDK4-Cyc D1 interface surface for the entire duration of the MD simulation (100 ns), the measurement of its trajectory should not exceed an RMSD value of 10 Å. Compounds (la), (Ib), and (le) showed an RMSD value lower than 10 Å (i.e., their binding to CDK4 was stable for 100 ns). Furthermore, the solvation bond energy value MM|PBSA was taken into account in this process, as described by Gnheden et al. (Expert Opinion on Drug Discovery 2015, 10(5), 449-461; [DOI: 10.1517 / 17460441.2015.1032936]) and Wang et al. (Comp. Chem. 2016, 37(27), 2436-2446; [DOI: 10.1002 / jcc.24467]). Compounds (la), (Ib), and (le) showed MM|PBSA values ​​greater than 20 kcal / mol for the last 50 ns of the MD simulation.

Claims

CLAIMS 1. A compound with a structure according to formula (I) for use as a medicament: Formula (I) where - A is an atom independently selected from O and S; preferably A is O or S; and most preferably A is O; R 1 and R 2 They are selected independently from among H, -CONH-R a , aryl and alkyl, or R 1 and R 2 together they form a ring; R 3 is selected from -CONH-R b , aryl and alkyl; R a and R b being independently selected from aryl and alkyl; and the aryl and alkyl groups and the ring being optionally substituted by a group selected from aryl, alkyl, and halogen; and the alkyl groups being linear or branched.

2. The compound of claim 1, wherein the aryl groups are selected from phenyl, phthalimidyl, biphenyl, naphthalenyl, benzyl; the alkyl groups are selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl; and / or the halogen is selected from fluorine, chlorine, bromine, and iodine.

3. The compound of claim 1 or 2, wherein R 1 and R 2 form a ring, preferably an aromatic ring, more preferably an aromatic ring selected from a 6-membered or 5-membered aromatic ring.

4. The compound according to any of claim 1 or 2 wherein R 1 and / or R 2 is - CONH-R a and preferably R ais aryl; more preferably the aryl is selected from phenyl, biphenyl, phthalimidyl, naphthalenyl, benzyl, optionally substituted with a group preferably selected from phenyl, benzyl, naphthalenyl, ethyl; and still more preferably the aryl is selected from biphenyl, naphthalenyl, N-phenyl-phthalimide and N-naphthalenyl-phthalimide.

5. The compound of claim 4 wherein one of R 1 and R 2 It is H and the other one is from R 1 and R 2 es -CONH-R a , preferably where: when R 1 is H then R 2 es -CONH-R a and R a is selected from biphenyl and naphthalene; and preferably is biphenyl; or when R 2 is H then R 1 es -CONH-R a and R a It is selected from N-phenyl-phthalimide and N-naphthalenyl-phthalimide; and preferably it is N-phenyl-phthalimide.

6. The compound of any of claims 1 to 5 wherein R 3 is CONH-R b and preferably R b is aryl; more preferably the aryl is selected from phenyl, biphenyl, phthalimidyl, naphthalenyl, benzyl, optionally substituted with a group preferably selected from phenyl, benzyl, naphthalenyl, and ethyl; still more preferably the aryl is selected from biphenyl, naphthalenyl, N-phenyl-phthalimide and N-naphthalenyl-phthalimide; and still more preferably it is selected from biphenyl and N-phenyl-phthalimide.

7. The compound of any of claims 1 to 5 wherein R 3is aryl; preferably the aryl is selected from phenyl, biphenyl, phthalimidyl, naphthalenyl, benzyl, optionally substituted with a group preferably selected from phenyl, benzyl, naphthalenyl, and ethyl; more preferably the aryl is selected from biphenyl, naphthalenyl, N-phenyl-phthalimide and N-naphthalenyl-phthalimide; and still more preferably the aryl is N-naphthalenyl-phthalimide.

8. The compound of claim 1 wherein formula (I) is according to formula (a):

9. The compound of claim 1 wherein formula (I) is according to formula (Ib): Formula (Ib) 10. The compound of claim 1 wherein formula (I) is according to formula (le):

11. The compound of any one of claims 1 to 10 wherein the medicament is for the treatment of a disease, preferably for the treatment of cancer, and more preferably for the treatment of a cancer that overexpresses CDK4 kinase and Cyc D1, in particular GBM, breast cancer, pancreatic cancer, colon cancer, mantle cell lymphoma, Kaposi sarcoma, prostate cancer, lung cancer, and / or gynecological cancer; more particularly for the treatment of GBM, breast cancer, pancreatic cancer, and / or colon cancer; and still more particularly for the treatment of GBM.

12. The compound of any one of claims 1 to 11 wherein the use as a medicament or treatment comprises administering the compound or a pharmaceutical composition comprising the compound and a pharmaceutically acceptable excipient, to a subject, the subject being human or non-human and in particular a human, suffering from a disease, particularly wherein the subject suffers from cancer, more particularly suffering from GBM, breast cancer, pancreatic cancer, and / or colon cancer, and still more particularly suffering from GBM.

13. A pharmaceutical composition comprising the compound of any one of claims 1 to 10 and a pharmaceutically acceptable excipient.

14. The pharmaceutical composition of claim 13 wherein the formulation comprises the compound as the sole therapeutic agent or comprises an additional therapeutic agent, in particular selected from CDK4, CDK5 and / or CDK6 inhibitors, preferably a compound selected from palbociclib, ribociclib, abemaciclib, gemcitabine and carmustine (BCNU).

15. The pharmaceutical composition of claim 13 or 14 that is formulated for the controlled release of the compound and / or additional therapeutic agent.

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