Therapeutic combination comprising n-{3-[4-amino-7-(1-methyl-piperidin-4-YL)-7h-pyrrolo[2,3-d]pyrimidin-5-YL]-2-fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzene sulfonamide compound and antineoplastic agents

Abstract

The present invention relates to combinations comprising N-{3-[4-Amino-7-(1-methyl-piperidin-4-yl)-7H-pyrrolo[2,3- d]pyrimidin-5-yl]-2-fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide or its pharmaceutically acceptable salt and antineoplastic agents. The invention also relates to the use of said combination in the treatment of cancer.

Description

[0001] NMS-129

[0002] THERAPEUTIC COMBINATION COMPRISING N-{3-[4-AMINO-7-(1-METHYL-PIPERIDIN-4-YL)-7H- PYRROLO[2,3-D]PYRIMIDIN-5-YL]-2-FLUORO-PHENYL}-5-CHLORO-2-FLUORO-4-M ETHOXY-BENZENE SULFONAMIDE COMPOUND AND ANTINEOPLASTIC AGENTS

[0003] Field of the invention

[0004] The present invention relates to combinations comprising N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3- d]py rimidin-5-yl]-2-fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide or its pharmaceutically acceptable salt and antineoplastic agents for treating cancer. Preferred antineoplastic agents are selected from the group consisting of antimetabolite agents, BCL2 family inhibitors, proteasome inhibitors, corticosteroids, immunomodulatory agents, microtubule binding agents, DNA damaging agents, aminoacid metabolizing agents, kinase inhibitors, PARP inhibitors and KRas inhibitors.

[0005] Background of the invention

[0006] N-(substituted-phenyl)-sulfonamide derivatives, and in particular the compound N-{3-[4-Amino-7-(1 -methyl-piperidin- 4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide of formula (I) are described in WO2017 / 220477.

[0007] Compound of formula (I) showed very good activity in inhibiting protein kinases, in particular Protein kinase R-like ER (PERK) and General Control Nondepressible 2 (GCN2) kinase and can be used for the treatment of cell proliferative disorders, such as cancer, or neurodegenerative diseases, such as Alzheimer’s disease. PERK is a sensor of Endoplasmic Reticulum (ER) stress. The endoplasmic reticulum (ER) represents the main subcellular compartment involved in folding and maturation of proteins destined for organelles and the extracellular space; several kinds of stress can alter the function of the ER, including hypoxia, alteration of protein glycosylation, depletion of luminal ER calcium, or changes in ER redox status These conditions provoke the accumulation of unfolded or misfolded proteins inside the ER which culminates in the activation of a series of adaptive mechanisms referred to as the Unfolded Protein Response (UPR) aimed to restore protein-folding homeostasis (Hetz C., Chevet E. and Harding H. P., Nat. Rev. Drug Discov. 2013, 12, 703-719).

[0008] The UPR is involved in many physiological processes, including homeostasis in specialized secretory cells, such as pancreatic (3 cells, salivary glands and plasma B cells, lipid and cholesterol metabolism, energy control, inflammation and cell differentiation (Wang M. and Kaufman R. J., Nat Rev Cancer. 2014 (9):581 -97). The large number of activities mediated by UPR reflects in the role of ER stress in the progression of diseases such as cancer, neurodegenerative disorders and diabetes.

[0009] GCN2 is involved in the adaptation to stress deriving from lack of nutrients, such as aminoacids. Both PERK and GCN2 are sensors of the Integrated Stress Response (ISR), a network of cellular pathways activated in response to a variety of stress. The ISR promotes adaptation to the stress, favoring cell survival and homeostasis; however, when stress is too severe or prolonged, it activates an apoptotic program leading to cell death. Cancer cells are often exposed to several sources of stress as lack of nutrients and oxygen, oncogenic growth, metabolic alterations, chemotherapeutic agents and ISR activation has been demonstrated in several human cancers (Tian X, Zhang S, Zhou L, Seyhan AA, Hernandez Borrero L, Zhang Y, El-Deiry WS. Targeting the Integrated Stress Response in Cancer Therapy. Front Pharmacol. 2021 Sep 24; 12:747837. doi: 10.3389 / fphar.2021 .747837. PMID: 34630117; PMCID: PMC8498116.). The modulation of ISR can alter the adaptive branch of the ISR, leading cancer cells to death.

[0010] Nevertheless, significant challenges remain, including the emergence of resistance mechanisms, variable responses across tumor types and treatment histories, and the need to improve pharmacokinetic, biodistribution, and safety profiles, particularly in combination regimens.

[0011] Therefore, there is a long existing need for development of additional safe proven combinational therapies to treat cancer.

[0012] Summary of the invention

[0013] The present invention provides, in a first aspect, a therapeutic combination comprising (a) a compound of formula (I): or a pharmaceutically acceptable salt thereof, or any hydrate, crystalline form thereof and (b) one or more antineoplastic agents selected from the group consisting of antimetabolite agents, BCL2 family inhibitors, proteasome inhibitors, corticosteroids, immunomodulatory agents, microtubule binding agents, DNA damaging agents, aminoacid metabolizing agents, kinase inhibitors, PARP inhibitors and KRas inhibitors.

[0014] The present invention also provides a combined preparation for simultaneous, separate or sequential use of the combination as described above.

[0015] In a further aspect the invention provides a combination comprising a compound of formula (I) as defined above and one or more antineoplastic agents selected from the group consisting of antimetabolite agents, BCL2 family inhibitors, proteasome inhibitors, corticosteroids, immunomodulatory agents, microtubule binding agents, DNA damaging agents, aminoacid metabolizing agents, kinase inhibitors, PARP inhibitors and KRas inhibitors, for use in the treatment of cancer. In another aspect, the invention provides a pharmaceutical composition comprising a combination according to the invention admixed with a pharmaceutically acceptable carrier, diluent or excipient.

[0016] In a further aspect, the present invention relates to the use of a therapeutic combination comprising (a) a compound of formula (I) or a pharmaceutically acceptable salt thereof, or any hydrate, crystalline form thereof and (b) one or more antineoplastic agents selected from the group consisting of antimetabolite agents, BCL2 family inhibitors, proteasome inhibitors, corticosteroids, immunomodulatory agents, microtubule binding agents, DNA damaging agents, aminoacid metabolizing agents, kinase inhibitors, PARP inhibitors and KRas inhibitors in the preparation of a medicament for the treatment of cancer, wherein said treatment comprises simultaneously, sequentially or separately administration to a subject in need thereof a compound of formula (I) and one or more antineoplastic agents.

[0017] In a still further aspect, the invention relates to a method for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of formula (I) as defined above, in combination with a therapeutically effective amount of one or more antineoplastic agents selected from the group consisting of antimetabolite agents, BCL2 family inhibitors, proteasome inhibitors, corticosteroids, immunomodulatory agents, microtubule binding agents, DNA damaging agents, aminoacid metabolizing agents, kinase inhibitors, PARP inhibitors and KRas inhibitors, wherein the compound of formula (I) and the antineoplastic agents can be administered simultaneously, sequentially or separately to a subject in need thereof.

[0018] In a still further aspect, the invention provides a gentisate salt of N-{3-[4-Amino-7-(1-methyl-piperidin-4-yl)-7H- pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide.

[0019] Brief description of the drawings

[0020] The invention is also illustrated by reference to the accompanying drawings described below.

[0021] Figure 1a shows the X-ray diffractograms (PXRD) of N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3- d]py rimidin-5-yl]-2-f luoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide free base form A.

[0022] Figure 1 b shows the Infrared (IR) analysis of N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5- yl]-2-fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide free base form A.

[0023] Figure 1c shows the Nuclear Magnetic Resonance (NMR) spectra of N-{3-[4-Amino-7-(1-methyl-piperidin-4-yl)-7H- py rrolo[2,3-d]py rimidin-5-yl]-2-fluoro-phenyl}-5-ch loro-2-f luoro-4-methoxy-benzenesulfonamide free base form A.

[0024] Figure 2a shows the X-ray diffractograms of N-{3-[4-Amino-7-( 1 -methyl-pi peridin -4-yl)-7H-pyrrolo[2 ,3-d]py rim idin-5-yl]- 2-fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide free base form B.

[0025] Figure 2b shows the IR analysis of N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro- phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide free base form B.

[0026] Figure 3a shows the X-ray diffractograms of 4 N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5- yl]-2-f luoro-pheny lj-5-ch loro-2-f luo ro-4-methoxy-benzenesulfonam ide gentisate form 1 .

[0027] Figure 3b shows the Differential Scanning Calorimetry (DSC) thermogram of N-{3-[4-Amino-7-(1 -methyl-piperidin-4- yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate form 1. The DSC showed a single endothermic event at 214.3 °C (Onset 211.6 °C) imputable to melting of the product. Figure 3c shows the thermogravimetric (TG) analysis of N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3- d]py ri midin -5-yl]-2-f I uoro-phenyl}-5-ch loro-2-fl uoro-4-methoxy-benzenesulfonamide gentisate form 1 . The TG analysis highlighted the presence of imbibition water (0.6 % w / w) lost between 40-110 °C and a decomposition event at the same temperature estimated for the melting.

[0028] Figure 3d shows the Infrared IR analysis of N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]- 2-fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate form 1 .

[0029] Figure 3e shows the NMR spectra of N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2- fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate form 1 . The1H-NMR revealed the presence of the free base and the gentisic acid in the stoichiometric ratio approx. 1 :1.

[0030] Figure 4a shows the X-ray diffractograms of N-{3-[4-Amino-7-( 1 -methyl-pi peridin -4-yl)-7H-pyrrolo[2 ,3-d]py rim idin-5-yl]- 2-fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate form 2.

[0031] Figure 4b shows the DSC thermogram of N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2- fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate form 2. The DSC profile shows an endothermic melting peak at 213.7 °C (onset 209.9 °C); Above 220 °C sample degradation occurred.

[0032] Figure 4c shows the TG analysis of N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro- phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate form 2. The analysis showed a weight loss immediately before the sample degradation of approx. 0.4% w / w. The analysis of the evolved vapors (EGA) highlighted the presence of ethyl acetate trapped into the crystalline lattice and released before the decomposition.

[0033] Figure 4d shows the Infrared IR analysis of N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]- 2-fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate form 2.

[0034] Figure 4e shows the NMR spectra of N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2- fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate form 2. From1H-NMR the stoichiometric ratio of free base to gentisic acid is determined as 1 :1 .

[0035] Figure 5a shows the X-ray diffractograms of N-{3-[4-Amino-7-( 1 -methyl-pi peridin -4-yl)-7H-pyrrolo[2 ,3-d]py rim idin-5-yl]- 2-fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate form 3.

[0036] Figure 5b shows the DSC thermogram of N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2- fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate form 3. The DSC profile shows an endothermic event at 218.2 °C (Onset 215.8 °C), imputable to melting and decomposition of the sample.

[0037] Figure 5c shows the TG analysis of N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro- phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate form 3. The analysis shows the typical profile of an anhydrous compound that decomposes above 210 °C, as confirmed by EGA where the evolution of carbon dioxide was observed at this temperature.

[0038] Figure 5d shows the Infrared IR analysis of N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]- 2-fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate form 3.

[0039] Figure 5e shows the NMR spectra of N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2- fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate form 3.1H-NMR the stoichiometric ratio of free base to gentisic acid is determined as 1 :1. Figure 6a shows the X-ray diffractograms of N-{3-[4-Amino-7-( 1 -methyl-pi peridin -4-yl)-7H-pyrrolo[2 ,3-d]py rim idin-5-yl]- 2-fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate form 4.

[0040] Figure 6b shows the DSC thermogram of N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2- fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate form 4. The DSC profile is characterized by a complex event where three different peaks partially overlapped can tentatively be observed: - 195.1 °C (onset 185.3 °C), endothermic; 201.1 °C (onset 198.1 °C), exothermic; 211.5 °C (onset 205.0 °C), endothermic.

[0041] Figure 6c shows the Infrared IR analysis of N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]- 2-fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate form 4.

[0042] Figure 6d shows the NMR spectra of 4 N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2- fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate form 4.1H-NMR the stoichiometric ratio of free base to gentisic acid is determined as 1 :1; traces of residual 2-propanol are preset (lower than 0.2% w / w).

[0043] Figure 7a shows the X-ray diffractograms of N-{3-[4-Amino-7-( 1 -methyl-pi peridin -4-yl)-7H-pyrrolo[2 ,3-d]py rim idin-5-yl]- 2-fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide digentisate form III.

[0044] Figure 7b shows the DSC thermogram of N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2- fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide digentisate form III.

[0045] Figure 8a shows the comparison of DSC thermograms of the solid forms 1 , 2, 3 and 4 of N-{3-[4-Amino-7-(1 -methyl- piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate.

[0046] Figure 9 shows the tumor volume traces after administering Compound (I), Paclitaxel, and their combination to female BALB / c nude mice bearing TOV21 G xenografts. Data points represent group mean tumor volume. Error bars represent standard error of the mean (SEM).

[0047] Figure 10 shows tumor volume trace after administering Compound (I), SN38, and their combinations to female BALB / c nude mice bearing TOV21 G xenografts. Data points represent group mean tumor volume. Error bars represent standard error of the mean (SEM).

[0048] Figure 11 shows tumor volume trace after administering Compound (I), Everolimus, and their combinations to female BALB / c nude mice bearing TOV21 G xenografts. Data points represent group mean tumor volume. Error bars represent standard error of the mean (SEM).

[0049] Detailed description of the invention

[0050] The term “synergic antineoplastic effect” as used herein is meant that by administering an effective amount of the combination of the compound of formula (I) as defined above and an antimetabolite agent, a BCL2 family inhibitor, a proteasome inhibitor, a corticosteroid, an immunomodulatory agent, a microtubule binding agent, a DNA damaging agent, an aminoacid metabolizing agent, a kinase inhibitors, a PARP inhibitors or a KRas inhibitor to mammals, including human the action of two or more agents is greater than could have been predicted based on the performance of the agents when used alone.

[0051] The term “combined preparation” as used herein defines especially a “kit of parts” in the sense that the combination partners (a) and (b) as defined above can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners (a) and (b), i.e. simultaneously or at different time points. The parts of the kit of parts can then, e.g., be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts. Very preferably, the time intervals are chosen such that the therapeutic effect of the combination of the invention on the treated disease is greater than the effect which would be obtained by use of only any one of the combination partners (a) and (b).

[0052] The ratio of the total amounts of the combination partner (a) to the combination partner (b) to be administered in the combined preparation can be varied, e.g. in order to cope with the needs of a patient subpopulation to be treated or the needs of the single patient which different needs can be due to the particular disease, age, sex, body weight, etc. of the patients.

[0053] The terms 'co-administration' or 'combined administration' or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g. a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time.

[0054] As used herein, the term “cancer” refers to all types of cancer, neoplasm or malignant tumors found in mammals.

[0055] The terms “treating” or “treatment of” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, or ameliorating abrogating, substantially inhibiting, slowing or reversing the progression of a disease, condition or disorder, substantially ameliorating or alleviating clinical or esthetical symptoms of a condition, substantially preventing the appearance of clinical or esthetical symptoms of a disease, condition, or disorder, and protecting from harmful or annoying symptoms. Treating further refers to accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting development of symptoms characteristic of the disorder(s) being treated; (c) limiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting recurrence of the disorder(s) in patients that have previously had the disorder(s); and / or (e) limiting recurrence of symptoms in patients that were previously asymptomatic for the disorder(s). The term “treating” with respect to cancer should be understood to e.g. encompass treatment resulting in a decrease in tumor size; a decrease in rate of tumor growth; stasis of tumor size; a decrease in the number of metastasis; a decrease in the number of additional metastasis; a decrease in invasiveness of the cancer; a decrease in the rate of progression of the tumor from one stage to the next; inhibition of tumor growth in a tissue of a mammal having a malignant cancer; control of establishment of metastases; inhibition of tumor metastases formation; regression of established tumors as well as decrease in the angiogenesis induced by the cancer, inhibition of growth and proliferation of cancer cells and so forth. The term “treating cancer as used herein should also be understood to encompass prophylaxis such as prevention as cancer reoccurs after previous treatment (including surgical removal) and prevention of cancer in an individual prone (genetically, due to life style, chronic inflammation and so forth) to develop cancer. As used herein, “prevention of cancer” is thus to be understood to include prevention of metastases, for example after surgical procedures or after chemotherapy.

[0056] The term "pharmaceutically acceptable carrier" refers to a non-toxic carrier, diluent, adjuvant, vehicle or excipient that does not adversely affect the pharmacological activity of the compound with which it is formulated, and which is also safe for human use. Pharmaceutically acceptable carriers that may be used in the compositions of this disclosure include, but are not limited to, ion exchangers, alumina, aluminum stearate, magnesium stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances (e.g., microcrystalline cellulose, hydroxypropyl methylcellulose and sodium carboxymethylcellulose), lactose monohydrate, sodium lauryl sulfate, polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, pregelatinized starch, and wool fat.

[0057] As used herein, a subject is “in need of a treatment” if such subject would benefit biologically, medically or in quality of life from such treatment.

[0058] As used herein, “safe and effective amount” means an amount of the compound sufficient to treat the patient’s condition but low enough to avoid serious side effects and it can nevertheless be routinely determined by the skilled artisan.

[0059] In a first aspect, the present invention provides a therapeutic combination comprising

[0060] (a) a compound of formula (I): or a pharmaceutically acceptable salt thereof, or any hydrate, crystalline form thereof, and

[0061] (b) one or more antineoplastic agents selected from the group consisting of antimetabolite agents, BCL2 family inhibitors, proteasome inhibitors, corticosteroids, immunomodulatory agents, microtubule binding agents, DNA damaging agents, aminoacid metabolizing agents, kinase inhibitors, PARP inhibitors and KRas inhibitors.

[0062] Advantageously and surprisingly, the administration of a protein kinase inhibitor, in particular PERK and GCN2 inhibitor, in particular the compound of formula (I) or a pharmaceutically acceptable salt thereof, or any hydrate, crystalline form thereof, and an antineoplastics agent as above defined provides a synergistic anti-cancer effect, inducing a strong tumor growth delay and a prolonged tumor regressions when compared to single agent.

[0063] The compound of formula (I) has the chemical name N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3- d]pyrimidin-5-yl]-2-fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide. It can be prepared as described in WO2017 / 220477, is endowed with protein kinase inhibitory activity, in particular with PERK and GCN2 inhibitory activity, and is thus useful in therapy as antitumor agent.

[0064] According to a preferred embodiment, the pharmaceutically acceptable salts of compound of formula (I) are selected from the group consisting of 2,5-dihydroxybenzoate (gentisate), hydrochloride, hydrobromide, succinate, citrate, L- tartrate, maleate, sulphate, sodium and potassium or any hydrate, any crystal form thereof.

[0065] According to a more preferred embodiment, the salt is 2,5-dihydroxybenzoate (gentisate) or any hydrate, any crystal form thereof. In one embodiment, the N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl}-5- chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate salt is in the crystalline form 1 characterized by a PXRD pattern having peaks at 13.7, 18.2, 20.1 and 24.8 degrees 2-theta +0.2 degrees 2-theta.

[0066] In an embodiment, the N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl}-5- chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate salt crystalline form 1 is characterized by a PXRD pattern having peaks at 5.5, 11.0, 12.8, 13.7, 14.2, 16.5, 18.2, 20.1 and 24.8 degrees 2-theta +0.2 degrees 2-theta.

[0067] In an embodiment, the N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl}-5- chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate salt crystalline form 1 has an X-Ray Powder diffraction pattern (PXRD) substantially as shown in Figure 3a.

[0068] In one embodiment, the N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl}-5- chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate salt is in the crystalline form 2 characterized by a PXRD pattern having peaks at 14.5, 17.0, 18.0 and 21.3 degrees 2-theta +0.2 degrees 2-theta.

[0069] In an embodiment, the N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl}-5- chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate salt crystalline form 2 is characterized by a PXRD pattern having peaks at 11.1 , 12.7, 13.2, 14.5, 17.0, 18.0, 18.8 and 21.3 degrees 2-theta ±0.2 degrees 2-theta.

[0070] In an embodiment, the N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl}-5- chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate salt crystalline form 2 has an X-Ray Powder diffraction pattern (PXRD) substantially as shown in Figure 4a.

[0071] In one embodiment, the N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl}-5- chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate salt is in the crystalline form 3 characterized by a PXRD pattern having peaks at 16.5, 18.2, 19.5 and 20.2.degrees 2-theta +0.2 degrees 2-theta.

[0072] In an embodiment, the N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl}-5- chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate salt crystalline form 3 is characterized by a PXRD pattern having peaks at 11.0, 13.7, 16.5, 18.2, 19.5, 20.2, 22.4, 23.5 degrees 2-theta +0.2 degrees 2-theta.

[0073] In an embodiment, the N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl}-5- chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate salt crystalline form 3 has an X-Ray Powder diffraction pattern (PXRD) substantially as shown in Figure 5a.

[0074] In one embodiment, the N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl}-5- chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate salt is in the crystalline form 4 characterized by a PXRD pattern having peaks at 12.7, 14.9, 17.6 and 18.8 degrees 2-theta +0.2 degrees 2-theta.

[0075] In an embodiment, the N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl}-5- chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate salt crystalline form 4 is characterized by a PXRD pattern having peaks at 11.4, 12.7, 13.2, 14.9, 17.6, 18.8, 20.8 and 25,0 degrees 2-theta +0.2 degrees 2-theta.

[0076] In an embodiment, the N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl}-5- chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate salt crystalline form 4 has an X-Ray Powder diffraction pattern (PXRD) substantially as shown in Figure 6a. The N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl}-5-chloro-2-fluoro-4- methoxy-benzenesulfonamide gentisate salt in the crystalline forms 1 , 2, 3, and 4 according to the present disclosure may have advantageous properties including at least one of: chemical or polymorphic purity, flowability, solubility, dissolution rate, bioavailability, morphology or crystal habit, stability such as chemical stability as well as thermal and mechanical stability with respect to polymorphic conversion, stability towards dehydration and / or storage stability, a lower degree of hygroscopicity, low content of residual solvents and advantageous processing and handling characteristics such as compressibility, or bulk density.

[0077] According to a preferred embodiment of the invention the antimetabolite agent is selected from the group consisting of gemcitabine, cytarabine, capecitabine, 5-fluorouracil, pemetrexed and methotrexate.

[0078] According to a more preferred embodiment of the invention, the antimetabolite agent is selected from the group consisting of gemcitabine and 5-fluorouracil (5-FU). Gemcitabine and 5-FU can be administered, e.g., in the form as they are marketed, e.g. under the trademark Gemzar, Infugem, Carac or Fluoroplex.

[0079] According to a preferred embodiment of the invention, the BCL-2 family inhibitor is venetoclax. Venetoclax can be administered, e.g., in the form as it is marketed, e.g. under the trademark Venclexta or Venclyxto.

[0080] According to a preferred embodiment of the invention, the proteasome inhibitor is bortezomib. Bortezomib can be administered, e.g., in the form as it is marketed, e.g. under the trademark Velcade.

[0081] According to a preferred embodiment of the invention, the corticosteroid is dexamethasone. Dexamethasone can be administered, e.g., in the form as it is marketed, e.g. under the trademark Decadron, Intensol, Dexasone or Solurex.

[0082] According to a preferred embodiment of the invention, the immunomodulatory agent is lenalidomide. Lenalidomide can be administered, e.g., in the form as it is marketed, e.g. under the trademark Revlimid.

[0083] According to a preferred embodiment of the invention, the microtubule binding agent is selected from the group consisting of monomethyl auristatin E (MMAE), paclitaxel, vincristine and vinblastine.

[0084] According to a more preferred embodiment of the invention, the microtubule binding agent is paclitaxel.

[0085] According to a preferred embodiment of the invention, the DNA damaging agent is selected from the group consisting of cyclophosphamide, doxorubicin, oxaliplatin, temozolomide, topoisomerase I inhibitors SN-38 (7-etil-10-idrossi- camptothecin) or irinotecan and topotecan. Cyclophosphamide, doxorubicin, oxaliplatin, temozolomide or topoisomerase I inhibitors can be administered, e.g., in the form as they are marketed, e.g. under the trademark Cytoxan, Adriamycin, Eloxatin, Temodar, Camptosar.

[0086] According to a preferred embodiment of the invention, the aminoacid metabolizing agent is asparaginase. Asparaginase can be administered, e.g., in the form as they are marketed, e.g. under the trademark Oncaspar.

[0087] According to a preferred embodiment of the invention, the kinase inhibitor agent is selected from the group consisting of afatinib, bosutinib, crizotinib, cabozatinib, entrectinib, everolimus, ibrutinib, nilotinib, osimertinib, and ruxolitinib. According to a more preferred embodiment of the invention, the kinase inhibitor agent is everolimus and osimertinib. Everolimus and osimertinib can be administered, e.g., in the form as it is marketed, e.g. under the trademark Afinitor, Tagrisso.

[0088] According to a preferred embodiment of the invention, the PARP inhibitor agent is selected from the group consisting of atamparib, AZD9574, NMS-293 (itareparib), niraparib, olaparib, veliparib, rucaparib, saruparib and talazoparib. The PARP inhibitor can be administered, e.g., in the form as they are marketed, e.g. under the trademark Zeyula, Lynparza, Rubraca, Talzenna.

[0089] According to a more preferred embodiment of the invention, the PARP inhibitor agent is atamparib, olaparib, saruparib and itareparib.

[0090] According to a preferred embodiment of the invention, the KRas inhibitor agent is adagrasib and sotorasib. Adagrasib and sotorasib can be administered, e.g., in the form as they are marketed, e.g. under the trademark Krazati and Lumakras.

[0091] In one embodiment, the present invention provides a therapeutic combination comprising

[0092] (a) a compound of formula (I): or a pharmaceutically acceptable salt thereof, or any hydrate, crystalline form thereof, and

[0093] (b) one or more antineoplastic agents selected from the group consisting of gemcitabine, cytarabine, capecitabine, 5- fluorouracil, pemetrexed, methotrexate, gemcitabine, 5-fluorouracil (5-FU), venetoclax, bortezomib, dexamethasone, lenalidomide, monomethyl auristatin E (MMAE), paclitaxel, vincristine, vinblastine, cyclophosphamide, doxorubicin, oxaliplatin, temozolomide, topoisomerase I inhibitors SN-38 (7-etil-10-idrossi-camptothecin) or irinotecan, topotecan, asparaginase, afatinib, bosutinib, crizotinib, cabozatinib, entrectinib, everolimus, ibrutinib, nilotinib, osimertinib, ruxolitinib, atamparib, AZD9574, itareparib, niraparib, olaparib, veliparib, rucaparib, saruparib, talazoparib, adagrasib and sotorasib.

[0094] In one preferred embodiment, the present invention provides a therapeutic combination comprising a) N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl}-5-chloro-2-fluoro-4- methoxy-benzenesulfonamide gentisate salt, and

[0095] (b) one or more antineoplastic agents selected from the group consisting of gemcitabine, cytarabine, capecitabine, 5- fluorouracil, pemetrexed, methotrexate, gemcitabine, 5-fluorouracil (5-FU), venetoclax, bortezomib, dexamethasone, lenalidomide, monomethyl auristatin E (MMAE), paclitaxel, vincristine, vinblastine, cyclophosphamide, doxorubicin, oxaliplatin, temozolomide, topoisomerase I inhibitors SN-38 (7-etil-10-idrossi-camptothecin) or irinotecan, topotecan, asparaginase, afatinib, bosutinib, crizotinib, cabozatinib, entrectinib, everolimus, ibrutinib, nilotinib, osimertinib, ruxolitinib, atamparib, AZD9574, itareparib, niraparib, olaparib, veliparib, rucaparib, saruparib, talazoparib, adagrasib and sotorasib.

[0096] In the present invention, each of the active ingredients of the combination is in amount effective to produce a synergic antineoplastic effect. The compound of formula (I), is administered in an amount range from 1 mg / m2to 2000mg / m2of body surface area. In a preferable embodiment is the amount is in the range from 5 mg / m2to 700 mg / m2of body surface area.

[0097] In one preferred embodiment, the present invention provides a combination comprising:

[0098] (a) a compound of formula (I) or a pharmaceutically acceptable salt thereof, or any hydrate, crystalline form thereof, and

[0099] (b) one or more antineoplastic agents selected from the group consisting of antimetabolite agents, BCL2 family inhibitors, proteasome inhibitors, corticosteroids, immunomodulatory agents, microtubule binding agents, DNA damaging agents, aminoacid metabolizing agents, kinase inhibitors, PARP inhibitors and KRas inhibitors, for simultaneous, sequential or separate use.

[0100] Administration of the compounds of the invention and their pharmaceutical compositions may be accomplished according to patient’s needs, for example, orally, nasally, parenterally and by inhalation.

[0101] The compound of formula (I) can be administered in a variety of dosage forms, e.g., orally, in the form of tablets, capsules, sugar or film coated tablets, liquid solutions or suspensions; rectally in the form of suppositories; parenterally, e.g., intramuscularly, or through intravenous and / or intrathecal and / or intraspinal injection or infusion.

[0102] In a second aspect, the present invention provides a therapeutic combination comprising (a) a compound of formula (I) or a pharmaceutically acceptable salt thereof, or any hydrate, crystalline form thereof, and

[0103] (b) one or more antineoplastic agents selected from the group consisting of antimetabolite agents, BCL2 family inhibitors, proteasome inhibitors, corticosteroids, immunomodulatory agents, microtubule binding agents, DNA damaging agents, aminoacid metabolizing agents, kinase inhibitors, PARP inhibitors and KRas inhibitors for use in the treatment of cancer.

[0104] In one embodiment, the present invention refers to the therapeutic combination as above described for use in treatment of cancer selected from the group including, but not limited to: carcinomas, hematopoietic tumors of lymphoid lineage, hematopoietic tumors of myeloid lineage, tumors of mesenchymal origin, tumors of the central and peripheral nervous system and other tumors.

[0105] In a more preferred embodiment, said carcinomas are selected from the group consisting of bladder, breast, colon, kidney, liver, lung, small cell lung cancer, esophagus, gall- bladder, ovary, pancreas, stomach, cervix, thyroid, prostate, skin and squamous cell carcinoma; said hematopoietic tumors of lymphoid lineage are selected among leukaemia, acute lymphocytic leukaemia, acute lymphoblastic leukaemia, B-cell lymphoma, T-cell-lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma and Burkett's lymphoma, multiple myeloma; said hematopoietic tumors of myeloid lineage are selected among acute and chronic myelogenous leukeamia, myelodysplastic syndrome and promyelocytic leukaemia; said tumors of mesenchymal origin are selected among fibrosarcoma and rhabdomyosarcoma; said tumors of the central and peripheral nervous system are selected among astrocytoma neuroblastoma, glioma and schwannomas; and other tumors are selected among melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, keratoacanthoma, thyroid follicular cancer and Kaposi's sarcoma. In a more preferable embodiment said tumors are selected from colon carcinoma, ovarian carcinoma, lung carcinoma, multiple myeloma, acute lymphocytic leukaemia, acute myelogenous leukaemia. non-Hodgkin's lymphoma and multiple myeloma.

[0106] In one embodiment, the present invention provides a therapeutic combination comprising (a) a compound of formula (I) or a pharmaceutically acceptable salt thereof, or any hydrate, crystalline form thereof, and

[0107] (b) one or more antineoplastic agents selected from the group consisting of DNA damaging agents for use in the treatment of ovarian carcinoma.

[0108] In a more preferred embodiment, the present invention provides a therapeutic combination comprising (a) a compound of formula (I) or a pharmaceutically acceptable salt thereof, or any hydrate, crystalline form thereof, and (b) a DNA damaging agent, wherein the DNA damaging agent is irinotecan, for use in the treatment of ovarian carcinoma.

[0109] In a even more preferred embodiment, the present invention provides a therapeutic combination comprising (a) N-{3- [4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy- benzenesulfonamide gentisate salt, and

[0110] (b) irinotecan, for use in the treatment of ovarian carcinoma. n a third aspect, the present invention provides a pharmaceutical composition comprising (a) a compound of formula (I) or a pharmaceutically acceptable salt thereof, or any hydrate, crystalline form thereof, and

[0111] (b) one or more antineoplastic agents selected from the group consisting of antimetabolite agents, BCL2 family inhibitors, proteasome inhibitors, corticosteroids, immunomodulatory agents, microtubule binding agents, DNA damaging agents, aminoacid metabolizing agents, kinase inhibitors, PARP inhibitors and KRas inhibitors.

[0112] In one embodiment, the present invention refers to a pharmaceutical composition comprising the combination of the invention as abode defined, in admixture with one or more pharmaceutically acceptable carrier or excipients, for example those described in Remington’s Pharmaceutical Sciences Handbook, XVII Ed., Mack Pub., N.Y., U.S.A..

[0113] In one preferred embodiment, the compounds of the combination of the invention as above defined may be administered in the form of two separate pharmaceutical compositions for oral administration, each containing one of the active ingredients, or in the form of a single pharmaceutical composition for oral administration, in which the active ingredients are in admixture.

[0114] In one embodiment, the pharmaceutical composition for oral administration comprising the compounds of the combination of the invention as above defined can be a solid oral dosage form such as a tablet, a gelcap, a capsule, a caplet, a granule, a lozenge and bulk powders. Such solid oral dosage form can also contain suitable pharmaceutically acceptable carriers, diluents, such as sucrose, mannitol, lactose, starches, and known excipients, including suspending agents, solubilizers, buffering agents, binders, disintegrants, preservatives, colorants, flavorants, lubricants and the like.

[0115] In a further embodiment, the pharmaceutical composition comprising the combination of the invention is a liquid oral dosage forms such as aqueous and non-aqueous solutions, emulsions, suspensions, syrups. Such liquid dosage forms can also contain suitable known inert diluents such as water and suitable known excipients such as preservatives, wetting agents, sweeteners, flavorants, as well as agents for emulsifying and / or suspending the compounds of the invention.

[0116] In a fourth aspect, the present invention provides a method for treatment of cancer administering to a subject in need thereof a combination comprising a therapeutically effective amount of (a) a compound of formula (I) or a pharmaceutically acceptable salt thereof, or any hydrate, crystalline form thereof, and a therapeutically effective amount of (b) one or more antineoplastic agents selected from the group consisting of antimetabolite agents, BCL2 family inhibitors, proteasome inhibitors, corticosteroids, immunomodulatory agents, microtubule binding agents, DNA damaging agents, aminoacid metabolizing agents, kinase inhibitors, PARP inhibitors and KRas inhibitors, wherein the compound of formula (I) and the antineoplastic agent can be administered simultaneously, sequentially or separately. The present invention also provides a method for lowering the side effects caused by antineoplastic therapy with an antineoplastic agent in mammals, including humans, in need thereof, the method comprising administering to said mammal a combined preparation comprising the compound of formula (I) as defined above and one or more antineoplastic agents selected from the group consisting of antimetabolite agents, BCL2 family inhibitors, proteasome inhibitors, corticosteroids, immunomodulatory agents, microtubule binding agents, DNA damaging agents, aminoacid metabolizing agents, kinase inhibitors, PARP inhibitors and KRas inhibitors, in amounts effective to produce a synergic antineoplastic effect.

[0117] In another aspect, the present invention relates to the use of a therapeutic combination comprising (a) a compound of formula (I) or a pharmaceutically acceptable salt thereof, or any hydrate, crystalline form thereof and (b) one or more antineoplastic agents selected from the group consisting of antimetabolite agents, BCL2 family inhibitors, proteasome inhibitors, corticosteroids, immunomodulatory agents, microtubule binding agents, DNA damaging agents, aminoacid metabolizing agents, kinase inhibitors, PARP inhibitors and KRas inhibitors in the preparation of a medicament for the treatment of cancer, wherein said treatment comprises simultaneously, sequentially or separately administration to a subject in need thereof a compound of formula (I) and one or more antineoplastic agents.

[0118] The present invention further provides a commercial package comprising, in a suitable container mean, (a) a compound of formula (I) as defined above, and (b) one or more antineoplastic agents selected from the group consisting of antimetabolite agent, BCL2 family inhibitor, proteasome inhibitor, corticosteroid, immunomodulatory agent, microtubule binding agent, DNA damaging agent, aminoacid metabolizing agent, kinase inhibitors, PARP inhibitors and KRas inhibitor, wherein the active ingredients are present in each case in free form or in the form of a pharmaceutically acceptable salt or any hydrate thereof, together with instructions for simultaneous, separate or sequential use thereof. In a package according to the invention each of partner (a) and (b) are present within a single container mean or within distinct container means.

[0119] Another embodiment of the present invention is a commercial package comprising a pharmaceutical composition or product as described above.

[0120] All preferred groups or embodiments described above for the combination of the invention may be combined among each other and apply as well mutatis mutandis.

[0121] The various aspects of the invention described in this application are illustrated by the following examples which are not meant to limit the invention in any way. The activities of the combination of the present invention are shown for instance by the following in vitro and in vivo which are intended to illustrate but not to limit the present invention.

[0122] Experimental part

[0123] General Procedure

[0124] Crystal structure of the N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl}-5- chloro-2-fluoro-4-methoxy-benzenesulfonamide as free base or its gentisate salt were characterized by powder X-Ray Diffraction (PXRD) performed using a Thermo / ARL XTRA apparatus, irradiating powder samples with a Cu-Ka source (40 kV, 40 mA, 1.6 kW - Ka1 radiation, wavelength X= 1.54060 Angstrom) between 2° and 40° 2-theta at room temperature.

[0125] The scan rate was of 1.20° / min (0.020° step with count time of 1 seconds per step).

[0126] In the X-Ray diffractograms, the angles of diffraction 2-theta are plotted on the horizontal axis (x-axis) and the line intensity on the vertical (y-axis).

[0127] It is known in the art that an X-ray powder diffraction pattern may be obtained with one or more measurement errors depending on measurement conditions (such as, for example, equipment and / or sample preparation). In particular, it is generally known that intensities in an X-ray powder diffraction pattern may vary depending on measurement conditions and sample preparation.

[0128] For example, persons skilled in the art of X-ray powder diffraction will realise that the relative intensity of peaks can be affected by, for example, grains above 30 microns in size and non-unitary aspect ratios, which may affect analysis of samples.

[0129] The skilled person will also realise that the position of reflections can be affected by the precise height at which the sample sits in the diffractometer and the zero calibration of the diffractometer.

[0130] The surface planarity of the sample may also affect the result.

[0131] Hence a person skilled in the art will appreciate that the diffraction pattern data presented herein is not to be considered as absolute (for further information see “Fundamentals of Powder Diffraction and Structural Characterization, Pecharsky and Zavalij, Kluwer Academic Publishers, 2003). Therefore, it shall be understood that the crystalline forms of the gentisate salt and free base of compound of formula (I) described in the present invention is not limited to the crystals that provide X-ray powder diffraction patterns identical to the X-ray powder diffraction patterns shown in the Figures below.

[0132] Generally, a measurement error of a diffraction angle in an X-ray powder diffractogram is about 2-theta = 0.5 deg or less (or, more suitably, about 2-theta = 0.2 deg or less) and such degree of a measurement error should be taken into account when considering the X-ray powder diffraction pattern in figures 1 -10, and when interpreting the peak positions referred to both in the text and in tables 3-12.

[0133] Therefore, where it is stated, for example, that the gentisate salt and free base of a compound of formula (I) have an X-ray powder diffraction pattern with at least one specific peak at about 2-theta = 20.1 deg (or any one of the other mentioned angles) then this can be interpreted as being 2-theta = 20.1 deg plus or minus 0.5 deg, or 2-theta = 20.1 deg plus or minus 0.2 deg. Example 1

[0134] N-{3-[4-Amino-7-(1-methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl}-5-chloro-2-fluoro-

[0135] 4-methoxy-benzenesulfonamide, compound of formula (I), as free base form A

[0136] A 1 L four-neck round-bottom flask equipped with a thermometer, condenser, and vacuum / argon inlet was charged with 5-iodo-7-(1 -methyl-4-piperidyl)pyrrolo[2,3-d]pyrimidin-4-amine (13.0 g, 36.39 mmol), 5-chloro-2-fluoro-N-[2-fluoro- 3-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)phenyl]-4-methoxy-benzenesulfonamide (21.75 g, 47.31 mmol, 1.3 eq), and cesium carbonate (35.57 g, 109.17 mmol, 3 eq). The mixture was treated with 260 mL of a 9:1 dioxane / water mixture, affording a yellow solution, and subjected to three vacuum / argon cycles. Tetrakis(triphenylphosphine)paiiadium(0) (1.3 g, 10% w / w) was then added, and two additional vacuum / argon cycles were performed. The reaction mixture was immersed in a preheated oil bath at 85 °C and stirred at this temperature (external 85 °C, internal 82-83 °C) for 5 hours. During the reaction, the solution turned yellow, and a white precipitate formed. The reaction mixture was cooled to an internal temperature of 40 °C, diluted with 250 mL of water, and acidified to pH 8-9 by dropwise addition of 2 N HCI (approximately 60 mL), with no exotherm observed. The mixture was further diluted with 250 mL of water and extracted with DCM / MeOH (8:2) (600 mL + 300 mL). The combined organic layers were dried over sodium sulfate and concentrated to dryness under reduced pressure to give a yellow / beige solid (39 g). The crude solid was suspended in MTBE / DCM (1 :1 , 500 mL) and stirred at room temperature for 4 hours. The suspension was cooled in an ice bath (0-5 °C), stirred for 1 hour, and filtered through a sintered glass funnel. The solid was washed with DCM / MTBE (1 :1 , 70 mL) and dried under vacuum at 45 °C to yield a pale-yellow solid (18.2 g). This material was resuspended in DCM (740 mL) and refluxed for 4 hours. After cooling to room temperature, the mixture was filtered through a sintered glass funnel and washed with DCM (3 x 50 mL). The solid was dried under vacuum at 45 °C to constant weight to afford the title compound as a white solid.

[0137] 1H NMR (401 MHz, DMSO-d6) 5: 1.88-1.98 (m, 2H), 2.10 (ddd, J1 = 4.1 , 12.1 , 24.3 Hz, 2H), 2.29 (br t, J1 = 11.6 Hz, 2H), 2.29 (s, 3H), 3.01 (br d, J1 = 11.6 Hz, 2H), 3.93 (s, 3H), 4.58 (tt, J1 = 4.1 12.1 Hz, 1 H), 5.65-6.3 (m, 2H), 7.11 (dt, J 1 = 7.4 Hz, J1 ,3 = 2.4 Hz, 1 H), 7.14 (t, J1 = 7.4 Hz, 1 H), 7.21 (dt, J 1 = 7.4 Hz, , J1 ,3 = 2.4 Hz, 1 H), 7.31 (s, 1 H), 7.32 (d, J1 = 11.8 Hz, 1 H), 7.70 (d, J1 = 7.4 Hz, 1 H), 8.13 (s, 1 H). Figure 1a shows the x-Ray diffractogram, Figure 1a shows the Infrared (IR) analysis of the free base form A, Figure 1c shows the NMR spectra.

[0138] Table A - X Ray Diffraction Data for the free base form A

[0139] Table B - IR data for the free base form A Example 2

[0140] N-{3-[4-Amino-7-(1-methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl}-5-chloro-2-fluoro-

[0141] 4-methoxy-benzenesulfonamide, compound of formula (I), as free base form B

[0142] N-[3-[4-amino-7-(4-methylcyclohexyl)pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl]-5-chloro-2-fluoro-4-methoxy- benzenesulfonamide in form A prepared as reported in the Example 1 (1.0 g) was suspended in methanol (10 V) and stirred at room temperature for 24 hours. The white solid was collected by filtration to furnish N-[3-[4-amino-7-(4- methylcyclohexyl)pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl]-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide in form B (1 g).

[0143] Figure 2a shows the x-Ray diffractogram, Figure 2b shows the Infrared (IR) analysis of the free base form B.

[0144] Table C - X Ray Diffraction Data for the free base form B Table D - IR data for the free base form B

[0145] Example 3

[0146] N-{3-[4-Amino-7-(1-methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl}-5-chloro-2-fluoro-

[0147] 4-methoxy-benzenesulfonamide gentisate form 1

[0148] N-[3-[4-amino-7-(4-methylcyclohexyl)pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl]-5-chloro-2-fluoro-4-methoxy- benzenesulfonamide free base form A, as prepared in example 1 (2.0 g) and 2,5-di hyd roxybenzoic acid (Gentisic acid, 1.09 g, 2.0 eq.) were weighed and transferred into an Erlenmeyer flask equipped with a magnetic stirring bar and then suspended in methanol (40 mL). The mixture was stirred at room temperature for 24 hours. The white precipitate was isolated by vacuum filtration, washed with methanol (approx. 10 mL), dried under vacuum at 40 °C and analyzed by PXRD.

[0149] 1.90 g of N-[3-[4-amino-7-(1 -methyl-4-pi peridy I) py rrolo[2 ,3-d]py rimidi n -5-y l]-2-f luoro-pheny l]-5-ch loro-2-fluo ro-4- methoxy-benzenesulfonamide gentisate form 1 was collected as white solid (Y=75%).1H NMR (401 MHz, DMSO-d6) 6: 2.03-2.15 (m, 2H), 2.22-2.38 (m, 2H), 2.67 (s, 3H), 2.93 (br t, J = 11.4 Hz, 2H), 3.33- 3.45 (m, 2H), 3.93 (s, 3H), 4.77 (br t, J = 12.0, 3.8 Hz, 1 H), 5.80-6.30 (br s, 2H), 6.56 (d, J = 8.4 Hz, 1 H), 6.71 (dd, J = 8.4 Hz, J1 ,3 = 3.0 Hz, 1 H), 7.15 (s, J = 3.0 Hz, 1 H), 7.18-7.27 (m, 3H), 7.34 (s, 1 H), 7.36 (d, J = 7.4 Hz, 1 H), 7.72 (d, J = 7.4 Hz, 1 H), 8.15 (s, 1 H), 8.40-8.90 (br s, 1 H).

[0150] 1H-NMR spectrum reveals the presence of the free base and the gentisic acid in the stoichiometric ratio approx. 1 :1. In Figures 3a-e are reported the X-ray diffractograms, the Differential Scanning Calorimetry (DSC) thermogram, the thermogravimetric (TG) analysis, the Infrared IR analysis, and the NMR spectra respectively.

[0151] Table E - X Ray Diffraction Data for the N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2- fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate form 1

[0152] Table F - IR Data for the N-{3-[4-Amino-7-(1 -methyl-pi peridi n -4-yl)-7H-py rrolo[2 ,3-d]pyri m idin-5-yl]-2-fl uoro-phenyl}- 5-chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate form 1

[0153] Example 4

[0154] N-{3-[4-Amino-7-(1-methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl}-5-chloro-2-fluoro-

[0155] 4-methoxy-benzenesulfonamide gentisate form 2

[0156] N-[3-[4-amino-7-(4-methylcyclohexyl)pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl]-5-chloro-2-fluoro-4-methoxy- benzenesulfonamide free base form B, prepared as reported in Example 2 (1.59 Kg) suspended in THF (12 V) was treated with 2,5-dihydroxybenzoic acid (gentisic acid, 2.61 Kg, 6.0 eq.). The reaction mixture was stirred at room temperature until complete dissolution of the solids. The reaction was concentrated and to the oily residue was added methanol (10 V). The obtained white solid was filtered and washed with methanol (6 V) to furnish N-[3-[4-amino-7-(1- methyl-4-piperidyl)pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl]-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide digentisate form III (2.46 kg). This solid was then treated with ethyl acetate (20 V) at reflux for 1 hour. The suspension was filtered and the solid dried under vacuum at 50 °C: 1.85 Kg of monogentisate was obtained (PXRD analysis showed form 3 of monogentisate) with a small amount of digentisate. Quantification of the amount of digentisate was made by NMR, showing a gentisic acid / free base ratio of 1 .14 / 1 . The obtained monogentisate (1.85 Kg) was suspended in THF and heated until complete dissolution (in this case dissolution was at 40 °C but, if necessary, it is possible to reach THF reflux temperature). The solution was cooled down to 20-25 °C and then filtered on GFF. The clear solution was evaporated to oil and then ethyl acetate (20 V) was added. The obtained suspension was heated to 50 °C and kept at that temperature for 1 hour before cooling down to 25 °C. The suspension was filtered, washing with ethyl acetate, and dried under vacuum at 50 °C. N-[3-[4-amino-7-(1 -methyl-4-piperidyl)pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro- phenyl]-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate form 2 (1 .60 Kg) with less than 10% of form 3 was obtained.

[0157] 1H NMR (401 MHz, DMSO-d6) 5: 8.66 (bs, 1 H), 8.15 (s, 1 H), 7.72 (d, J= 7.4 Hz, 1 H), 7.36 (d, J= 7.4 Hz, 1 H), 7.34 (s, 1 H), 7.27-7.18 (m, 3H), 7.15 (s, J= 3.0 Hz, 1 H), 6.71 (dd, J= 3.0, 8.4 Hz, 1 H), 6.56 (d, J= 8.4 Hz, 1 H), 6.06 (bs, 2H), 4.77 (br t, J = 12.0 Hz, 1 H), 3.93 (s, 3H), 3.45-3.33 (m, 2H), 2.93 (t, J= 11.4 Hz, 2H), 2.67 (s, 3H), 2.38-2.22 (m, 2H), 2.15-2.03 (m, 2H).

[0158] The stoichiometric ratio of free base to gentisic acid is determined as 1 :1 .

[0159] In Figures 4a-e are reported the X-ray diffractograms, the Differential Scanning Calorimetry (DSC) thermogram, the thermogravimetric (TG) analysis, the Infrared IR analysis, and the NMR spectra respectively.

[0160] Table G - X Ray Diffraction Data for the N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2- fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate form 2

[0161] Table H - IR Data for the N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl}-5- chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate form 2 Example 5

[0162] N-{3-[4-Amino-7-(1-methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl}-5-chloro-2-fluoro-

[0163] 4-methoxy-benzenesulfonamide gentisate form 3

[0164] N-[3-[4-amino-7-(1 -methyl-4-piperidyl)pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl]-5-chloro-2-fluoro-4-methoxy- benzenesulfonamide digentisate, prepared as reported in example 4 (1 g) was transferred into a 50-mL round bottom flask, equipped with a magnetic stirring bar and a vapor condenser. Butyl acetate (20 V) was added, and the mixture was heated to 110 °C by oil bath. The reaction was checked after 3 days and the conversion of the digentisate starting material to the desired monogentisate form 3 was appreciated. The product was isolated by vacuum filtration and dried at 40 °C and 30 mbar for 24 hours, affording N-[3-[4-amino-7-(1 -methyl-4-piperidyl)pyrrolo[2,3-d]pyrimidin-5-yl]-2- fluoro-phenyl]-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate form 3 (630 mg) that was confirmed by PXRD analysis.

[0165] 1H-NMR analysis confirms the structural integrity of the title compound as form 3 in 1 :1 stoichiometric ratio of free base : gentisic acid (see Figure 5e).

[0166] In Figures 5a-e are reported the X-ray diffractograms, the Differential Scanning Calorimetry (DSC) thermogram, the thermogravimetric (TG) analysis, the Infrared IR analysis, and the NMR spectra respectively.

[0167] Table I - X Ray Diffraction Data for the N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2- fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate form 3

[0168] Table L - IR Data for the N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl}-5- chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate form 3

[0169]

[0170] Example 6

[0171] N-{3-[4-Amino-7-(1-methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl}-5-chloro-2-fluoro-

[0172] 4-methoxy-benzenesulfonamide gentisate form 4 N-[3-[4-amino-7-(1 -methyl-4-piperidyl)pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl]-5-chloro-2-fluoro-4-methoxy- benzenesulfonamide digentisate, prepared as reported in the example 4 (100 mg) was weighed in a vial equipped with a magnetic stirring bar and 2-propanol (2 mL) was added. The suspension was stirred for 5 hours at room temperature and then a sample of the solid was analyzed by PXRD confirming the formation of the monogentisate as 2-propanol solvate. The solid was isolated by vacuum filtration and the vial rinsed with 2 mL of 2-propanol. The product was dried at 160 °C under vacuum for 30 minutes, affording N-[3-[4-amino-7-(1 -methyl-4-piperidyl)pyrrolo[2,3-d]pyrimidin-5-yl]- 2-fluoro-phenyl]-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate form 4 (60 mg) as a white poorly crystalline powder.

[0173] 1H-NMR confirms the presence of 1 equivalent of gentisic acid. Traces of residual 2-propanol are present as well (lower than 0.2% w / w).

[0174] In Figures 6a-d are reported the X-ray diffractograms, the Differential Scanning Calorimetry (DSC) thermogram, the Infrared IR analysis, and the NMR spectra respectively.

[0175] Table M - X Ray Diffraction Data for the N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2- fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate form 4 Table N - IR Data for the N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl}-5- chloro-2-fluoro-4-methoxy-benzenesulfonamide gentisate form 4

[0176] Example 7

[0177] N-{3-[4-Amino-7-(1-methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl}-5-chloro-2-fluoro-

[0178] 4-methoxy-benzenesulfonamide digentisate form III

[0179] The compound was obtained as reported in example 4 and its characterization is shown in figure 7a-b.

[0180] Table 0 - X Ray Diffraction Data for the N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]py rimidin-5-yl]-2- fluoro-phenyl}-5-chloro-2-fluoro-4-methoxy-benzenesulfonamide digentisate form III

[0181] Example 8

[0182] In vitro cytotoxic activity of the combinations: General procedure

[0183] Exponentially growing cancer cells were seeded in 96-well black plates in 200 l of appropriate medium (e.g., RPMI1640) + 10% FCS. After 24 hours, compound DMSO solutions, alone or in combination matrices, were directly administered to cells using a D300e digital dispenser (Tecan, Mannedorf, Switzerland) or a Biomek I5 robotic station (Beckman Coulter). Each cell line was seeded at a specific cell density such that the cells were still actively proliferating at the end of the experiment. Table 1 summarizes the cancer cell lines used. Generally, cells were incubated at 37 °C in a humidified 5% CO2 atmosphere 24 hours before treatment. Cell viability was determined after 72 or 144 hours of treatment by ATP determination in each test well with a luciferase-based detection system (CellTiterGlo, Promega, Madison, Wl, USA). The intensity of emitted light was measured using an EnVision reader (Perkin Elmer, Waltham, MA USA) and expressed as RLU (Relative Luminescence Units).

[0184] The effect of compound combinations (antagonism, additivity, synergism) was calculated by comparing, for each combination of doses of the matrix, the observed effect, and the expected additive effect according to the Bliss independence model. Combination Indexes were calculated according to [7], VAB = VA* VB

[0185] C.L = Observed viability (VObs) / Expected viability (VAB)

[0186] Where:

[0187] V (Viability) = % viability as compared with DMSO treated controls (V = 0%: complete cytotoxicity, no cells left alive; V = 100%: no cytotoxicity, all cells are alive);

[0188] VA = Observed viability after treatment with compound A, single agent;

[0189] VB = Observed viability after treatment with compound B, single agent;

[0190] VAB = Predicted viability after simultaneous treatment with both drug A and drug B if the combination would be additive; C.l. (Combination Index) = ratio between experimentally measured Viability (Vobs) and predicted viability (VAB);. C.l. = 1 indicates perfect additivity.

[0191] Theoretically, C.l. values just below 1 (e.g. 0.99) can be defined as synergistic and C.Ls just above 1 (e.g. 1.01) can be defined as antagonistic. However, to limit any effects due to experimental variability and to be more stringent in the definitions of antagonism and synergy, it was decided to set the following range of additivity: 0.8 C.L < 1 .2. Therefore, C.l. s 0.8 indicates synergism; 0.8 s C.l. s 1.2 indicates additivity; C.l. .2 indicates antagonism.

[0192] The compound of the formula (I) was prepared prepared as described in WO2017 / 220477.

[0193] The results show that on human tumor cells, the compound of formula (I) can be effectively combined with the compounds listed (Table 1), producing a synergic effect, since in most of the conditions C.Ls are lower than 0.8. Tablet Summary of cell lines and agents used for combination studies

[0194] Table 2. Combination indices of In vitro combinations at differ doses of Compound (I) with bortezomib in KMS-11 Multiple Myeloma cell line Table 3. Combination indices of In vitro combinations at differ doses of Compound (I) with dexamethasone in NCI-

[0195] H929 Multiple Myeloma cell line

[0196] Table 4. Combination indices of In vitro combinations at differ doses of Compound (I) with Lenalidomide in NCI-H929 Multiple Myeloma cell line

[0197] Table 5. Combination indices of In vitro combinations at differ doses of Compound (I) with Lenalidomide in AMO-1 Multiple Myeloma cell line Table 6. Combination indices of In vitro combinations at differ doses of Compound (I) with Asparaginase in CCRF- CEM ALL cancer cell line

[0198] Table 7. Combination indices of In vitro combinations at differ doses of Compound (I) with Asparaginase in MV-4-11 cancer cell line

[0199] Table 8. Combination indices of In vitro combinations at differ doses of Compound (I) with venetoclax in Kasumi AML cell line

[0200] Table 9. Combination indices of In vitro combinations at differ doses of Compound (I) with venetoclax in KG-1 AML cell line Table 10. Combination indices of In vitro combinations at differ doses of Compound (I) with venetoclax in MOLM-13

[0201] AML cell line

[0202] Table 11. Combination indices of in vitro combinations at differ doses of Compound (I) with cyclophosphamide in Su- DH-L8 DLBCL (Diffuse Large B Cell Lymphoma) cancer cell line

[0203] Table 12. Combination indices of In vitro combinations at differ doses of Compound (I) with adagrasib in SW837 colon cancer cell line

[0204] Table 13. Combination indices of In vitro combinations at differ doses of Compound (I) with everolimus in HT29 colon cancer cell line Table 14. Combination indices of In vitro combinations at differ doses of Compound (I) with everolimus in KM-12 colon cancer cell line

[0205] Table 15. Combination indices of In vitro combinations at differ doses of Compound (I) with everolimus in LoVo colon

[0206] Table 17. Combination indices of In vitro combinations at differ doses of Compound (I) with gemcitabine in COLO320 colon cancer cell line Table 18. Combination indices of In vitro combinations at differ doses of Compound (I) with gemcitabine in LoVo colon cancer cell line

[0207] Table 19. Combination indices of In vitro combinations at differ doses of Compound (I) with gemcitabine in Ls411 N colon cancer cell line

[0208] Table 20. Combination indices of In vitro combinations at differ doses of Compound (I) with oxaliplatin in LoVo colon cancer cell line

[0209] Table 21. Combination indices of In vitro combinations at differ doses of Compound (I) with paclitaxe in LoVo colon cancer cell line

[0210] Table 22. Combination indices of In vitro combinations at differ doses of Compound (I) with SN38 in LoVo colon cancer cell line

[0211] Table 23. Combination indices of In vitro combinations at differ doses of Compound (I) with SN38 in HT-29 colon cancer cell line

[0212] Table 24. Combination indices of In vitro combinations at differ doses of Compound (I) with SN38 in MCAS mucinous ovarian cancer cell line

[0213] Table 25. Combination indices of In vitro combinations at differ doses of Compound (I) with 5-fluorou racil (5-FU) in HEY ovarian cancer cell line

[0214] Table 26. Combination indices of In vitro combinations at differ doses of Compound (I) with doxorubicin in HEY ovarian cancer cell line Table 27. Combination indices of In vitro combinations at differ doses of Compound (I) with Gemcitabine in HEY ovarian cancer cell line

[0215] Table 28. Combination indices of In vitro combinations at differ doses of Compound (I) with olaparib in Ovcar8 ovarian cancer cell line

[0216] Table 29. Combination indices of In vitro combinations at differ doses of Compound (I) with Paclitaxel in HEY ovarian cancer cell line

[0217] Table 30. Combination indices of In vitro combinations at differ doses of Compound (I) with SN-38 in HEY ovarian cancer cell line

[0218] Table 31. Combination indices of In vitro combinations at differ doses of Compound (I) with Oxaliplatin in Tov21 G ovarian cancer cell line Table 32. Combination indices of In vitro combinations at differ doses of Compound (I) with SN-38 in Tov21 G ovarian cancer cell line

[0219] Table 33. Combination indices of In vitro combinations at differ doses of Compound (I) with Paclitaxel in Tov21 G ovarian cancer cell line

[0220] Table 34. Combination indices of In vitro combinations at differ doses of Compound (I) with Atamparib in A549

[0221] NSCLC cell line

[0222] Table 35. Combination indices of In vitro combinations at differ doses of Compound (I) with Atamparib in NCI-H358

[0223] NSCLC cell line Table 36. Combination indices of In vitro combinations at differ doses of Compound (I) with Osimerti nib in HCC827

[0224] NSCLC cell line

[0225] Table 37. Combination indices of In vitro combinations at differ doses of Compound (I) with osimertinib in NCI-H 1975 NSCLC cancer cell line

[0226] Table 38. Combination indices of In vitro combinations at differ doses of Compound (I) with sotorasib in NCI-H358 NSCLC cancer cell line

[0227] Table 39. Combination indices of In vitro combinations at differ doses of Compound (I) with temozolomide in NCI -

[0228] H727 NSCLC cancer cell line

[0229] Table 40. Combination indices of In vitro combinations at differ doses of Compound (I) with gemcitabine in NCI-H526 SCLC cancer cell line

[0230] Table 41. Combination indices of In vitro combinations at differ doses of Compound (I) with SN-38 in NCI-H526

[0231] SCLC cancer cell line Table 42. Combination indices of In vitro combinations at differ doses of Compound (I) with temozolomide in NCI-H82 SCLC cancer cell line

[0232] Table 43. Combination indices of In vitro combinations at differ doses of Compound (I) with topotecan in NCI-H146 SCLC cancer cell line

[0233] Table 44. Combination indices of In vitro combinations at differ doses of Compound (I) with sotorasib in Mia-paca-2 pancreatic cancer cell line

[0234] Table 45. Combination indices of In vitro combinations at differ doses of Compound (I) with temozolomide in QGP-1 pancreatic neuroendocrine cancer cell line

[0235] Table 46. Combination indices of In vitro combinations at differ doses of Compound (I) with oxaliplatin in QGP-1 pancreatic neuroendocrine cancer cell line

[0236] The results reported in tables 2-46 show that compound of formula (I) can be effectively combined with the compounds listed above, producing a synergic effect, since in most of the conditions C.I.s are lower than 0.8.

[0237] Example 9

[0238] In vivo activity of the combinations: General procedure

[0239] Tumor Inoculation and study design

[0240] Each mouse was inoculated subcutaneously at the right flank with the tumor cells (10 x 106) in 0.1 mL of PBS for tumor development. The animals were randomized and treatments started when the average tumor volume reached approximately 175 mm3.

[0241] The test articles, doses, schedules and the animal numbers in each group are shown in the below experimental design Table 47.

[0242] Compound of formula I was used as gentisate salt.

[0243] Tumor Measurements and the Endpoints

[0244] Prior to the onset of drug treatment, mice were measured for tumor size in two dimensions using a caliper, and the tumor volume (mm3) was calculated using formula V = 0.5 a x b2where a and b are the long and short diameters of the tumor in mm, respectively. Mice were randomized into different treatment groups based on the tumor volume. The major endpoint was to see if the tumor growth could be delayed or mice could be cured.

[0245] T-C is calculated with T as the median time (in days) required for the treatment group tumors to reach a predetermined size (e.g., day of first measurement ^500 mm3), and C is the median time (in days) for the control group tumors to reach the same size.

[0246] The T / C value (in percent) is an indication of antitumor effectiveness; T and C are the mean volumes of the treated and control groups, respectively. TGI was calculated for each group using the formula: TGI (i) (%) = (1 -(T-To) / (V-Vo)]*1OO and TGI (ii) (%)=((Vi- T) / Vi)*100; was the average tumor volume of a treatment group on a given day, Towas the average tumor volume of the treatment group on the day of treatment start, Vi was the average tumor volume of the vehicle control group on a given day, and Vowas the average tumor volume of the vehicle group on the day of treatment start.

[0247] Statistical Analysis

[0248] Summary statistics, including mean and the standard error of the mean (SEM), are provided for the tumor volume of each group at each time point.

[0249] Statistical analysis of difference in tumor volume among the groups was conducted on the data obtained on the last day of treatment (PG-D21) or the last day when comparison with the vehicle treated group was possible (PG-D35). All data was analyzed using Graphpad Prism 9.5.1 . p<0.05 is considered to be statistically significant.

[0250] Statistical analysis of the different median time for tumors to reach the predetermined size was performed by Log-Rank (Mantle-Cox) test. p<0.05 is considered to be statistically significant.

[0251] Table 47. Groups and Treatments Information

[0252] Notes: a. N: animal number. b. Treatments were started on the day of grouping (PG-DO) in the evening. Mice in QD groups had a dosingobservation since PG-D21 and mice in BID groups had a dosing-free observation since PG-D22. c. Compound (I) as gentisate salt was formulated in “0.5% methylcellulose 400 cP”. Paclitaxel was diluted in PBS. SN38 was formulated in “10% Tween 80 in glucose solution”. Everolimus was formulated in “2% DMSO + 98% (0.5% Carboxymethylcellulose Sodium)”. Vehicle group was dosed with “0.5% methylcellulose 400 cP”. d. The BID interval was 8 h / 16 h. e. All mice were supplemented with nutrient diet after the cell inoculation.

[0253] Tumor growth curves are shown in Figures 9-11 .

[0254] Tumor growth inhibition of Compound (I), Paclitaxel, SN38 or Everolimus and their combinations in female BALB / c nude mice bearing TOV21 G tumors was calculated based on tumor volume measured at the end of the treatment (PG- D21) and 2 weeks after treatments stopping (PG-D35). The maximum TGI, tumor growth delay and tumor regression induced by each treatment were analyzed.

[0255] Analysis at the end of treatment (PG-D21)

[0256] Tumor growth inhibition rates of the compound treated groups on PG-D21 are shown in Table 48.

[0257] Table 48. Tumor Growth Inhibition Analysis (PG-D21)

[0258] Notes: a. Data are shown as Mean ± SEM. b. Tumor Growth Inhibition (TGI) was calculated using the formula: TGI (i) (%) = (1-(T-To) / (V-Vo))*1OO, TGI (ii) (%)=((Vi-Ti) / Vi)*100, where T21 = mean tumor size at day 21 of treatment group; To = mean tumor size at day 0 of treatment group; V21 = mean tumor size at day 21 of control group; and Vo = mean tumor size at day 0 of control group. c. Antitumor activity (T / C) was determined by dividing the average tumor volume for treated group (T) by the average tumor volume for control group (C). d. All data were analyzed using Graphpad Prism. A one-way ANOVA followed by Dunnett’s multiple comparison test was performed to compare tumor volume among vehicle group and treatment groups, **** indicates p<0.0001 . e. Mouse #5-1 in G3, mouse #12-1 , #12-5 in G6 were euthanized before PG-D21. Mouse #11-3 in G6 died before PG-D21.

[0259] Analysis at 2 weeks after end of treatment (PG-D35)

[0260] Tumor growth inhibition rates of the compound treated groups on PG-D35 are shown in Table 49.

[0261] Table 49. Tumor Growth Inhibition Analysis (PG-D35)

[0262] Notes: a. Data are shown as Mean ± SEM. b. Tumor Growth Inhibition (TGI) was calculated using the formula: TGI (i) (%) = (1-(Ti-T0) / (Vi-V0))*100, TGI (ii) (%)=((Vi-Ti) / Vi)*100, where T35 = mean tumor size at day 35 of treatment group; TO = mean tumor size at day 0 of treatment group; V35 = mean tumor size at day 35 of control group; and VO = mean tumor size at day 0 of control group. c. Antitumor activity (T / C) was determined by dividing the average tumor volume for treated group (T) by the average tumor volume for control group (C). d. All data were analyzed using Graphpad Prism. A one-way ANOVA followed by Dunnett’s multiple comparison test was performed to compare tumor volume among vehicle group and treatment groups, **** indicates p<0.0001 . e. Mouse #3-1 , #4-4 in G2, mouse #5-1 , #6-2 in G3, mouse #12-1 , #12-5 in G6, mouse #15-3 in G8, mouse #17-2, #17-5 in G9 were euthanized before PG-D35. Mouse #11-3 in G6 died before PG-D35.

[0263] Tumor Growth Delay Analysis

[0264] The time of Compound (I), Paclitaxel, SN38, Everolimus and their combinations on delaying the growth of TOV21G xenografts is measured and is shown in Table 50; the statistical analysis is shown in Tables 51 -53.

[0265] Table 50. Tumor Growth Delay Analysis

[0266] Notes: a. Data are shown as the specific tumor size. b. The median time (in days) was evaluated according to the time needed for tumors to reach the specific size (500 mm3). For that, the first measurement day in which each tumor was ^500 mm3was used for the analysis. The median time for each group is reported. c. T-C is calculated with T as the median time (in days) required for the treatment group tumors to reach a predetermined size (^500 mm3), and C is the median time (in days) for the control group tumors to reach the same size. d. NR: Not reached. Tumors in G8 and G9 did not reach the median time to predetermined size by end of study (PG- D84). Table 51. Statistical Analysis of the Different Median Time for Tumors to Reach the Predetermined Size (Single Agent and Paclitaxel Combination Groups)

[0267] Note: a. Statistical analysis of the different median time for tumors to reach the predetermined size (500 mm3) was performed by Log-Rank (Mantle-Cox) test, *** indicates p<0.001 , ns indicates p>0.05.

[0268] Table 52. Statistical Analysis of the Different Median Time for Tumors to Reach the Predetermined Size (Single Agent and SN38 Combination Groups)

[0269] Note: a. Statistical analysis of the different median time for tumors to reach the predetermined size (500 mm3) was performed by Log-Rank (Mantle-Cox) test, *** indicates p<0.001 , ** indicates p< 0.01 , * indicates p<0.05. Table 53. Statistical Analysis of the Different Median Time for Tumors to Reach the Predetermined Size (Single Agent and Everolimus Combination Groups)

[0270] Note: a. Statistical analysis of the different median time for tumors to reach the predetermined size (500 mm3) was performed by Log-Rank (Mantle-Cox) test, *** indicates p<0.001.

[0271] Tumor Regression Analysis

[0272] The tumor regression of Compound (I), Paclitaxel, SN38, Everolimus and their combinations on the growth of TOV21G xenografts is reported in Table 54.

[0273] Table 54. Tumor Regression Analysis

[0274] Note: a. The mouse showing tumor regression is the mouse whose tumor size is smaller than that on the day of randomization. It also includes the tumor free mice. Numbers are reported per evaluable mice. b. The tumor free mouse is the mouse who showed complete tumor regression (tumor volume is 0 mm3). Numbers are reported per evaluable mice.

[0275] In this study, the in vivo efficacy of Compound (I), Paclitaxel, SN38, Everolimus and their combinations were investigated after treatment of BALB / c nude mice bearing TOV21 G xenografts.

[0276] Compared with vehicle group, all treatments showed significant inhibitory effect on the TOV21 G tumor growth at the end of the treatments (PG-D21) and 2 weeks after the end of treatment (PG-D35). Compound (I) single agent induced a TGI up to 88% with tumor regressions during the treatment. There was no difference between Compound (I) at 30 mg / kg QD and 15 mg / kg BID. TGI was improved in the combination groups with standard of care agents (paclitaxel, SN38 and everolimus).

[0277] To better evaluate the combination benefit and potential synergisms, an analysis of tumor growth delay and tumor regressions was performedv

[0278] The time needed for tumors to reach predetermined tumor volume (500 mm3) are shown in Table 50. Compared with vehicle group, all treatments delayed the growth of TOV21 G tumor significantly. Especially for Compound (I) dosed at 30 mg / kg QD or 15 mg / kg BID combined with SN38 dosed at 10 mg / kg Q4D, in view of the significant anti-tumor effect, all the mice did not reach the predetermined tumor size (500 mm3) during the entire study.

[0279] In the combination of Compound (I) with paclitaxel, regarding tumor growth delay, the combination was not better than paclitaxel single agent. However, the combination of Compound (I) at 30 mg / kg QD with paclitaxel induced 2 prolonged tumor regressions, with one of the mice tumor free at the end of the study (Table 54). This was not observed in any of the single agent arms.

[0280] The combination of Compound (I) with SN38 induced a strong tumor delay when compared to the single agents. Also in terms of prolonged tumor regressions, the combination was better than single agents. The combination with Compound (I) at 30 mg / kg QD resulted in 8 tumor regressions, of which 7 were complete regressions. The combination with Compound (I) at 15 mg / kg BID induced tumor regressions in 6 mice, of which 4 were tumor free mice. In contrast, in the group treated with Compound (I) single agent, there were no tumors in regression or tumor free mice at the end of the study. In the SN38 single agent group, only 2 animals had tumor regression at the end of the study, with one of them being tumor free.

[0281] Regarding the combination of Compound (I) with everolimus, the combination induced a strong tumor growth delay, in particular the combination with Compound (I) at 30 mg / kg QD. Moreover, the combination at this dose also resulted in 4 prolonged tumor regressions with one of them being a complete regression.

Claims

52Claims1. A therapeutic combination comprising (a) a compound of formula (I):or a pharmaceutically acceptable salt thereof, or any hydrate, crystalline form thereof and (b) one or more antineoplastic agents selected from the group consisting of antimetabolite agents, BCL2 family inhibitors, proteasome inhibitors, corticosteroids, immunomodulatory agents, microtubule binding agents, DNA damaging agents, aminoacid metabolizing agents, kinase inhibitors, PARP inhibitors and KRas inhibitors.

2. A combination according to claim 1 , wherein the pharmaceutically acceptable salt is 2,5-dihydroxybenzoate (gentisate), or any solvate, hydrate, crystalline form thereof.

3. A combination according to claim 2, wherein the pharmaceutically acceptable salt is 2,5-dihydroxybenzoate (gentisate) form 2, characterized by a PXRD pattern having peaks at 14.5, 17.0, 18.0 and 21.3 degrees 2-theta +0.2 degrees 2-theta.

4. A combination according to any one of claims 1 to 3, wherein the antimetabolite agent is selected from the group consisting of gemcitabine and 5-f luorouracil (5-FU).

5. A combination according to any one of claims 1 to 3, wherein the BCL-2 inhibitor is venetoclax.

6. A combination according to any one of claims 1 to 3, wherein the proteasome inhibitor is bortezomib.

7. A combination according to any one of claims 1 to 3, wherein the corticosteroid is dexamethasone.

8. A combination according to any one of claims 1 to 3, wherein the immunomodulatory agent is lenalidomide.

9. A combination according to any one of claims 1 to 3, wherein the microtubule binding agent is paclitaxel.

10. A combination according to any one of claims 1 to 3, wherein the DNA damaging agent is selected from the group consisting of cyclophosphamide, doxorubicin, oxaliplatin, temozolomide, SN-38 and irinotecan.11 . A combination according to any one of claims 1 to 3, wherein the aminoacid metabolizing agent is asparaginase.

12. A combination according to any one of claims 1 to 3, wherein the kinase inhibitor agent is selected from the group consisting of everolimus and osimertinib.

13. A combination according to any one of claims 1 to 3, wherein the PARP inhibitor agent is selected from the group consisting of olaparib and talazoparib.

14. A combination according to any one of claims 1 to 3, wherein the KRas inhibitor agent is selected from the group consisting of adagrasib and sotorasib.

15. The combination according to any one of claims 1 to 14, wherein the compound of formula (I) and the antineoplastic agents are administered independently intravenously or orally.5316. The combination according to any one of claims 1 to 14, wherein the compound of formula (I) and the antineoplastic agents are administered simultaneously, sequentially or separately.

17. A pharmaceutical composition comprising a combination according to any one of claims 1 to 14 admixed with a pharmaceutically acceptable carrier, diluent or excipient.

18. A combination according to any one of claims 1 to 14, or a pharmaceutical composition according to claim 17, for use in the treatment of cancer.

19. The combination for use according to claim 18 wherein the cancer is selected from the group consisting of: carcinomas, hematopoietic tumors of lymphoid lineage, hematopoietic tumors of myeloid lineage, tumors of mesenchymal origin, tumors of the central and peripheral nervous system and other tumors selected among melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, keratoacanthoma, thyroid follicular cancer and Kaposi's sarcoma.

20. Use of a therapeutic combination comprising (a) a compound of formula (I) or a pharmaceutically acceptable salt thereof, or any hydrate, crystalline form thereof and (b) one or more antineoplastic agents selected from the group consisting of antimetabolite agents, BCL2 family inhibitors, proteasome inhibitors, corticosteroids, immunomodulatory agents, microtubule binding agents, DNA damaging agents, aminoacid metabolizing agents, kinase inhibitors, PARP inhibitors and KRas inhibitors in the preparation of a medicament for the treatment of cancer, wherein said treatment comprises simultaneously, sequentially or separately administration to a subject in need thereof a compound of formula (I) and one or more antineoplastic agents.21 . A commercial package comprising, in a suitable container mean, (a) a compound of formula (I) as defined in claim 1 and (b) one or more antineoplastic agents selected from the group consisting of antimetabolite agents, BCL2 family inhibitors, proteasome inhibitors, corticosteroids, immunomodulatory agents, microtubule binding agents, DNA damaging agents, aminoacid metabolizing agents, kinase inhibitors, PARP inhibitors and KRas inhibitor, wherein the active ingredients are present in each case in free form or in the form of a pharmaceutically acceptable salt or any hydrate thereof, together with instructions for simultaneous, separate or sequential use thereof.

22. A gentisate salt of N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-fluoro-phenyl}-5- chloro-2-fluoro-4-methoxy-benzenesulfonamide.

23. A crystalline form 2 of the gentisate salt of N-{3-[4-Amino-7-(1 -methyl-piperidin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5- yl]-2-f luoro-phenyl}-5-chloro-2-f luoro-4-methoxy-benzenesulfonamide characterized by PXRD pattern having peaks at 14.5, 17.0,18.0 and 21.3 degrees 2-theta +0.2 degrees 2-theta.

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