Use of a MPS1 inhibitor for the treatment of hepatocellular carcinoma

Abstract

The invention provides a low molecular weight ATP-competitive MPS1 inhibitor for use in the treatment of hepatocellular carcinoma (HCC).

Description

[0001] USE OF A MPS1 INHIBITOR FOR THE TREATMENT OF HEPATOCELLULAR CARCINOMA

[0002] Field of the invention

[0003] The present invention relates to the treatment of hepatocellular carcinoma (HCC) patients through the administration of a low molecular weight ATP-competitive MPS1 (Monopolar Spindle 1) Kinase inhibitor.

[0004] Background of the invention

[0005] Liver cancer is the sixth most commonly diagnosed cancer and the third leading cause of cancer-related mortality (Sung H 2021 CA Cancer J Clin, 71 (3): 209-249). The number of new cases and deaths from liver cancer is predicted to rise by more than 55% by 2040 (Rumgay H 2022 J Hepatol, 77(6): 1598-1606). Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer, accounting for up to 75- 85% of all cases (Sung H 2021 CA Cancer J Clin, 71 (3): 209-249). Over 90% of HCC cases occur in the setting of chronic liver disease. The major risk factors for HCC include chronic alcohol consumption, diabetes or obesity-related non-alcoholic steatohepatitis (NASH), and infections by HBV or HCV (Llovet JM 2021 Nat Rev Dis Primers, 7(1 ):6).

[0006] Numerous mutations have been identified in human HCC samples thus far, with telomerase reverse transcriptase (TERT) (in 50%-60% of patients), TP53 (28%-36%), CTNNB1 (17%-37%) AXIN1 (4%-14%), ARID1A (16.8%), and ARID2 (5.6%) being the most frequent ones, affecting telomere maintenance, cellcycle control, the Wnt / p-catenin pathway, and epigenetic machinery (Gallage S 2021 Med, 2(5):505-552).

[0007] A large proportion of patients with HCC are diagnosed at advanced stages of disease and, therefore, are only amenable to systemic therapies. Unfortunately, HCC does not respond to classical cytotoxic chemotherapy. Over a decade, the multikinase inhibitor (MKI) sorafenib was the only approved systemic therapy for HCC. Unfortunately, this treatment only modestly increases survival and is accompanied by toxicities in most patients what leads to treatment discontinuation (Gallage S 2021 Med, 2(5):505-552). Recently, the therapeutic landscape of HCC has been expanded and, today, the United States Food and Drug Administration (US-FDA) and European Medical Agency (EMA) approved systemic therapies include the combination of atezolizumab-bevacizumab, sorafenib, and lenvatinib in the first line setting, while cabozantinib, regorafenib, ramicirumab (in patients with alfa-fetoprotein [AFP]>400 ng / mL) are approved in second line settings. (Telia SH 2022 Biomedicines, 10(6):1304). Despite remarkable advances with these new therapeutic strategies that inhibit angiogenesis and oncogenic kinases, or activate the immune system, only a small subset of patients can obtain durable clinical benefit and, therefore, new therapeutic approaches with novel antitumor mechanisms and the potential to offer improved treatment effects in patients with advanced HCC are urgently needed (from Yang C 2022 Nat Rev Gastroenterol Hepatol, 20(4): 203-222). Unfortunately, the identification of novel and efficient therapies for HCC remains a huge clinical challenge. One of the major obstacles being the high heterogeneity at the genetic, epigenetic, histopathological, and immunological levels. In fact, tumors can differ not only between patients but also between distinct lesions found within the same liver or even within different regions of the same tumor. (Gallage S 2021 Med, 2(5):505-552).

[0008] Monopolar spindle 1 (MPS1 / TTK) is a serine / threonine kinase that regulates the spindle assembly checkpoint and its inhibition leads to cancer death through excess mitotic instability (Dominguez-Brauer C 2015 Mol Cell, 60(4): 524-536). MPS1 is found overexpressed in several cancers and high MPS1 overexpression is associated with a poor prognosis (Liu X et al 2015 Oncotarget, 6(33): 34309-34320).

[0009] Summary of the invention

[0010] The invention provides a low molecular weight compound able to inhibit the spindle assembly checkpoint (SAC) through inhibition of MPS1 kinase mediated signalling pathways and efficacious in inhibiting HCC proliferation.

[0011] The compound of the present invention showing the desired activity is a pyrazoloquinazoline designed to target the ATP pocket of protein kinases. The compound has revealed to be a potent ATP-competitive inhibitor of MPS1 kinase.

[0012] In view of its biological activity, the compound of the invention offers a new path for the development of a treatment for the patient population suffering from hepatocellular carcinoma.

[0013] Brief description of the drawings

[0014] Figure 1 illustrates in vivo antitumor efficacy of compound (I) on the Hepa 1-6 syngeneic mouse model of HCC. Mean tumor volume (+ / - SEM) over time of vehicle treated animals and compound (I) treated animals at 13 mg / kg or 20 mg / kg every four days (Q4D).

[0015] Figure 2 illustrates in vivo antitumor efficacy of compound (I) on the Hepa 1-6 syngeneic mouse model of HCC. Individual tumor volume over time, during treatment and dosing-free observation period, of mice treated with vehicle and mice treated with compound (I) at 13 mg / kg or 20 mg / kg Q4D for 10 doses.

[0016] Figure 3 Compound (I) plasma concentration at steady state at Cycle 1 , Day 15 (C1 D15).

[0017] Detailed description of the invention

[0018] In a first aspect, the present invention relates to a compound of formula (I)

[0019] or a pharmaceutically acceptable salt thereof for use in a method for treating a hepatocellular carcinoma (HCC).

[0020] The compound of formula (I) has the chemical name N-(2,6-diethylphenyl)-8-({4-[4-(dimethylamino)piperidin- 1 -yl]-2-methoxyphenyl}amino)-1 -methyl-4,5-dihydro-1 H-pyrazolo[4,3-h]quinazoline-3-carboxamide. It can be prepared as described in W02019002454, it is endowed with protein kinase inhibitory activity and it is thus useful in therapy as antitumor agent. In particular, the preferred preparation of the compound (I) is that described in example 2, Step A of the above mentioned International Patent Application.

[0021] Pharmaceutically acceptable salts of the compound (I) include the acid addition salts with inorganic or organic acids, e.g., nitric, hydrochloric, hydrobromic, sulphuric, perchloric, phosphoric, acetic, trifluoroacetic, propionic, glycolic, lactic, oxalic, malonic, malic, maleic, tartaric, citric, benzoic, cinnamic, mandelic, methanesulphonic, benzensulphonic, isethionic and salicylic acid and the like.

[0022] In a preferred embodiment the salts are selected from phosphate, methansulphonate (tosylate) and benzensulphonate (besylate). In a more preferred embodiment, the salt is phosphate.

[0023] Another object of the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in a method of treating cancer, wherein the cancer is preferably a malignant hepatocellular carcinoma.

[0024] Within the scope of the claimed invention is the use of all the possible isomers and their admixtures and of both the metabolites and the pharmaceutically acceptable bio-precursors (otherwise referred to as pro-drugs) of the compound (I). Prodrugs are any covalently bonded compounds, which release the active parent drug, according to formula (I), in vivo.

[0025] A therapeutically effective amount of the compound according to formula (I) may be administered to a subject upon determination of the subject as having a disease or unwanted condition that would benefit by treatment with said compound. Medical or clinical personnel may make the determination as part of a diagnosis of a disease or condition in a subject. The compound may also be used in the prevention of such conditions, which may be viewed as reducing the probability of a subject having one or more of the conditions.

[0026] As used herein, a “therapeutically effective amount” of a compound refers to an amount sufficient to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art based upon the achievement of a desired effect. An effective amount will depend on factors including, but not limited to, the size of a subject and / or the degree to which the disease or unwanted condition from which a subject suffers has progressed. The effective amount will also depend on whether the compound is administered to the subject in a single dosage or periodically over time.

[0027] The compound (I) of the present invention is intended for the treatment of subjects. As used herein, the term "subject" encompasses mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish and the like.

[0028] The term "treating" as used herein includes achieving a therapeutic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. For example, in a cancer subject, therapeutic benefit includes eradication or amelioration of the underlying cancer. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding the fact that the subject may still be afflicted with the underlying disorder.

[0029] Another object of the present invention relates to a method for the treatment of malignant hepatocellular carcinoma in a subject in need thereof comprising administering to said subject a therapeutically effective amount of a compound of formula (I). In a particular embodiment, the subject is a mammal.

[0030] Another object of the present invention relates to a pharmaceutical composition comprising a compound of formula (I), for treating a malignant hepatocellular carcinoma in a subject in need thereof. In a particular embodiment, the subject is a mammal. In another particular embodiment, the pharmaceutical composition is admixed with a pharmaceutically acceptable carrier, diluent or excipient.

[0031] Another object of the present invention relates to a pharmaceutical composition comprising a compound of formula (I), for treating a malignant hepatocellular carcinoma in a subject in need thereof. In a particular embodiment, the subject is a mammal.

[0032] The pharmaceutical compositions containing the compound of the invention are usually prepared following conventional methods and are administered in a suitable pharmaceutical form.

[0033] For example, the solid oral forms may contain, together with the active compound, diluents, e.g., lactose, dextrose, saccharose, sucrose, cellulose, corn starch or potato starch; lubricants, e.g., silica, talc, stearic acid, magnesium or calcium stearate, and / or polyethylene glycols; binding agents, e.g., starches, arabic gum, gelatine, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disintegrating agents, e.g., starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations. These pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film-coating processes.

[0034] The liquid dispersions for oral administration may be, e.g., syrups, emulsions or suspensions.

[0035] As an example, the syrups may contain, as carrier, saccharose or saccharose with glycerine and / or mannitol and sorbitol.

[0036] The suspensions and the emulsions may contain, as examples of carriers, natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose or polyvinyl alcohol.

[0037] In therapeutic use, the compound of formula (I) is administered to a subject at dosage levels of from about 20 mg / m2 / week to about 400 mg / m2 / week of body surface. A dosage level of from about 20 mg / m2 / week to 200 mg / m2 / week constitutes a particularly suitable range. In a preferable embodiment dosage level is from 20 mg / m2 / week-to 150 mg / m2 / week. A preferred schedule of treatment consists of a dose lower than 135 mg / m2 day once a week for three weeks followed by one week rest in a four-week cycle. Cycles can be repeated as long as requested to control the diseases.

[0038] Lower or higher doses than those disclosed herein may be used, as required, accordingly to dose optimization and benefit / risk continuous assessments. Such dosages, however, may be altered depending on a number of variables, not limited to the activity of the compound used, the condition to be treated, the mode of administration, the regimen of treatment, the requirements of the individual subject, the severity of the condition being treated and the judgment of the practitioner. The foregoing ranges are merely suggestive, as the number of variables in regard to an individual treatment regime is large and considerable excursions from these recommended values are not uncommon.

[0039] Administration of the compound disclosed herein may be affected by any method that enables delivery of the compounds of the site of action. These methods include oral routes, intraduodenal routes parental injections (including intravenous, subcutaneous, intramuscular, intravascular or infusion) topical and rectal administration.

[0040] The term “comprising” as used herein is synonymous with “including,” or “containing,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0041] All numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth herein are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of any claims in any application claiming priority to the present application, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches. The above description discloses several methods and materials of the present invention. This invention is susceptible to modifications in the methods and materials, as well as alterations in the fabrication methods and equipment. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein. Consequently, it is not intended that this invention be limited to the specific embodiments disclosed herein, but that it covers all modifications and alternatives coming within the true scope and spirit of the invention.

[0042] All references cited herein, including but not limited to published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and are hereby made a part of this specification. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

[0043] With the aim to better illustrate the present invention, without posing any limitation to it, the following examples are now given.

[0044] Experimental part

[0045] Example 1. Antiproliferative activity of compound of formula (I) at 240 hours treatment on a panel of HCC cell lines

[0046] This example evaluated the antiproliferative activity of compound of formula (I) at 240 hours (10 days) treatment on a panel of six HCC cell lines (HuH-7, Hep3B, SNU-475, SNU-423, PLC-PRF-5 and SNU-449). Cytotoxicity assay

[0047] Exponentially growing cells were seeded at 300 cells / well in 96-well cell culture microplates and incubated at 37° C in a humidified 5% CO2 atmosphere 48 hours before treatment. Compound’s solutions were directly administered to the cells using the D300e digital dispenser (Tecan) to obtain scalar final concentrations ranging from 2 nM to 10 p M (duplicates wells for each point), with 200 p L final volume per well. The final DMSO concentration in test wells was adjusted to 0.1% (v / v); control wells were mock-treated and contained the same final concentration of DMSO. Cells were exposed to the compounds for 240 hours.

[0048] Cell viability was determined at the end point by ATP determination in each test well with a luciferase-based detection system (CellTiterGlo, Promega, Madison, Wl, USA), following manufacturer’s instructions (Hook B, Bratz M and Schagat T. Gain More Informative Data by Multiplexing a Fluorescent Real-Time Cytotoxicity Assay with Luminescent, Fluorescent or Colorimetric Viability Assays. Promega Corporation Web site. http: / / ita.promega.com / resources / pubhub / multiplexing-a-fluorescent-real-time-cytotoxicity-assay-with- luminescent-fluorescent-or-colorimetric / Updated June 2013). The intensity of emitted light was measured using an EnVision 2102 reader (PerkinElmer, Walthman, MA USA) and expressed as RLU. Independent experiments were performed for each cell line (N=3 or 4, as indicated). Analysis of antiproliferative data

[0049] Cell growth inhibitory activity was evaluated comparing treated versus control data using the Assay Explorer Software suite (Symix Technologies Inc.). IC50S values were calculated by sigmoidal fitting interpolation using the following quadriparametric Hill equation:

[0050] Top - Bottom

[0051] Activity = Bottom + -

[0052] 1 + 10(Log IC50 - Cone) HillSlope

[0053] The Top, Bottom and Hill slope parameters were left free to assume any value, but only curves whose Hill slope had values between 0.5 and 4 were arbitrarily considered acceptable and used to calculate the average IC50S. Curves too steep (HillSlope 4) or too slight (HillSlope s 0.5) were not included. Percent average bottom of dose-response curves are shown. N represents the number of independent experiments.

[0054] The results showed that after continuous treatment for 240 hours, compound of formula (I) has a marked in vitro antiproliferative activity in six HCC cell lines with an average IC50 of 12 nM, ranging from 7 nM to 15 nM (Table 1).

[0055] Table 1

[0056] Example 2. In vivo antitumor activity of compound (I)

[0057] This example evaluated the in vivo antitumor activity of compound (I) on the Hepa 1 -6 syngeneic mouse model of HCC (Figure 1).

[0058] Hepa 1-6 murine HCC cancer cells were cultured in DMEM (Dulbecco's Modified Eagle Medium), supplemented with 10% heat inactivated fetal bovine serum at 37°C in an atmosphere of 5% CO2 in air. The cells in exponential growth phase were harvested and quantitated by cell counter before inoculation in C57BL / 6 mice. Each mouse was inoculated subcutaneously in the right flank with 5x106cells Hepa 1-6 cells in 0.1 ml of PBS for tumor development.

[0059] Eight days after cell injection, animals having a tumor volume of about 95 mm3were randomized and allocated to 3 experimental groups (n=10 mice / group) for intravenous treatment with vehicle (1% Tween 80 in glucose solution), Compound (I) at 13 mg / kg or 20 mg / kg every 4 days (Q4D) for 10 doses (Days 0, 4, 8, 12, 16, 20, 24, 28, 32 and 36).

[0060] During the study, animals were checked daily for morbidity and mortality. Effect of treatment on tumor growth and body weight was evaluated three times per week. Mortality and observed clinical signs were recorded for individual animals. Tumors were measured in two dimensions using a caliper, and the tumor volume was expressed in mm3using the formula: V=(L x W2) / 2, where V is tumor volume, L is tumor length (the longest tumor dimension) and W is tumor width (the longest tumor dimension perpendicular to L). Dosing as well as tumor and body weight measurements were conducted in a Laminar Flow Cabinet. The tumor volumes and body weights were measured by using StudyDirector™ software (version 3.1.399.19). Animals were followed until ethical endpoint was reached (individual mice were euthanized if they lost over 20% of body weight relative to the weight on the first day of treatment or if tumor volume exceeded 3000 mm3).

[0061] On day 33, tumors in vehicle treated mice had a large tumor burden with a mean tumor volume of 1760.3 (+ / - 334.6), whereas mice treated with compound of formula (I) at 13 mg / kg had a mean tumor volume of 480.2 (+ / - 108.4) and mice treated with compound formula (I) at 20 mg / kg had a mean tumor volume of 401 .6 (+ / - 92.1). This indicates that treatment with compound of formula (I) at 13 mg / kg and 20 mg / kg induced a strong antitumor activity in the Hepa 1 -6 syngeneic mouse model of HCC with a tumor growth inhibition (TGI) at Day 33 of 72.7% and 77.2%, respectively. Treatments were well tolerated.

[0062] The TGI could not be calculated beyond Day 33 because some vehicle treated animals had to be euthanized due to that the ethical endpoint was reached (tumor volume >3000 mm3) and the MTV for this group was not representative of the group anymore. All mice in the vehicle treated group reached the ethical endpoint by Day 49 (Figure 2a).

[0063] In mice treated with compound of formula (I), tumor growth delay in individual mice was still appreciated during the dosing-free observation period. The tumor of one mouse that had been treated with compound of formula (I) at 13 mg / kg remained stabilized up to Day 82. Treatment at 20 mg / kg resulted with one tumor free mouse at Day 82 and one mouse with tumor regression, when compared to the tumor volume on the first day of treatment (Figure 2b-c).

[0064] The good in vivo antitumor activity of compound of formula (I) in the Hepa 1-6 mouse model of HCC and the good tolerability clearly shows the advantage to use the compound of formula (I) in the treatment of HCC.

[0065] Preliminary clinical data

[0066] A dose escalation study of N-(2,6-diethylphenyl)-8-({4-[4-(dimethylamino)piperidin-1-yl]-2- methoxyphenyl}amino)-1 -methyl-4,5-dihydro- 1 H-pyrazolo[4,3-h]quinazoline-3-carboxamide (compound (I)) was conducted in human patient with advanced solid tumors, showing good result in patient with hepatocellular carcinoma.

[0067] In particular, two HCC patients were treated in a non-randomized, non-comparative, dose-escalation Clinical study of compound (I) as monotherapy in adult patients with advanced / metastatic solid tumors. Both HCC patients had three prior lines of anti-tumoral systemic therapy, one was treated at 20 mg / m2 / week and another at 135 mg / m2 / week, and both achieved stable disease lasting 6 months.

[0068] Phase l / ll trial are ongoing with compound (I) administered as single agent in patients with unresectable HCC previously treated with systemic therapy. The Phase I part of the trial ended with the declaration of an Recommended Phase 2 Dose (RP2D) of 100 mg / m2 / week, and recommendation from an independent Data Safety Monitoring Board (DSMB) to continue the trial, moving to the Phase II part (the open label, single arm, expansion).

[0069] Phase l / ll part of the trial is ongoing dosing the compound (I) through IV infusion once a week for three consecutive weeks (D1 , D8, D15), followed by one week rest, starting from 100 mg / m2 / week.

[0070] A total of 19 patients were enrolled as of end of April 2023, 13 of them dosing the compound (I) at 100 mg / m2 / week and 6 of them at 135 mg / m2 / week.

[0071] Validated LC / MS-MS methods were developed to determine the plasma concentrations of compound (I) and its metabolite during the clinical studies. In plasma, the lower and upper limits of quantitation were 1 and 500 ng / mL for both analytes. In the range of doses tested in the patients, (4 to 135 mg / m2 / week, for 3 weeks in a 4-week cycle) compound (I) exposure increased proportionally to the dose.

[0072] The pharmacokinetic profiles of compound (I) in patients with unresectable hepatocellular shows a polyexponential decay with a rapid decline of plasma concentrations after the end of infusion. D15 Mean Cend nf (Day 15 plasma Concentration media at the end of infusion) at the dose of 135 mg / m2 was 1.59 p M (CV% 83) with corresponding AUCweekly of 39.1 p M-h (CV% 38) (Figure 3). The compound shows long half-life (i.e., around 100 hours), low clearance (mean D1 and D15: 10.1 and 8.51 L / h at 100 mg / m2; mean D1 and D15: 10.2 and 9.27 L at 135 mg / m2) and a high volume of distribution (mean D1 and D15: 1230 L and 1180 L at 100 mg / m2, respectively; mean D1 and D15: 1210 and 1370 L at 135 mg / m2).

[0073] The safety profile for 19 HCC patients treated with compound (I) as single agent has been evaluated.

[0074] Table 2 reports data collected at data cut-off (end of April 2023) referring to Drug Related Treatment Emergent Adverse Events (TEAEs). The most frequent drug related adverse events were hematological ones (neutropenia, anemia and platelet count decreased) and the most relevant one was neutropenia (in dose escalation, G3 in 2 / 6 patients (pts) at dose of 100 mg / m2 / week, G3 in 2 / 6 pts at 135 mg / m2 / week, and G4 in 2 / 6 pts at 135 mg / m2 / week, and in the phase 2 part, out of 7 patients treated at R no G3-4 ).The effect on neutrophils mainly appeared after the third drug infusion of cycle 1 and remained consistent over time, with no evidence of a cumulative effect. Neutropenia usually resolved spontaneously in about 7-10 days. An overview is reported in Table 2 below.

[0075] Table 2

[0076] Results

[0077] Of the 19 patients treated so far, 12 were in the dose escalation part, and 7 in the single agent expansion arm. In the phase 1 part of the study, 12 patients were evenly enrolled within the two dose levels: 100 and 135 mg / m2 / wk. Out of the 12 treated patients, 11 were evaluable for efficacy; two patients (0002, at 100 mg / m2 / w, and 0004, at 135 mg / m2 / whad confirmed investigator-assessed PRs as by RECIST1.1 with a median DoR lasting 2.6 and 9.3 months, respectively. It is notable that pt 002 missed an early dose and that either of these patients had prior therapies potentially sensitizing tumors to chemotherapy or potentially carrying over with long half-life, such as immunotherapy.

[0078] Patient 0002 was a black, 29 year-old man with active and treated hepatitis B infection (genotype A treated with tenofovir) with primary diagnosis as localized disease in 12 / 2019, who underwent surgery in 12 / 19 and then three subsequent lines of anti-tumoral systemic therapy, namely lenvatinib, sorafenib and regorafenib in palliative settings. At entrance he had pulmonary and lymphonodal metastasis with primary liver recurrence and large tumor burden. He had SD at Cycle 2 assessment that improved to PR at Cycle 4, confirmed then at Cycle 5. Duration of response was 2.6 months when he progressed for increase in target lesions from nadir at cycle 6.

[0079] Patient 0004 was a white 66 year-old male with treated hepatitis C infection with primary diagnosis in 2017 treated with loco-regional and radiation therapy and, afterwards, three subsequent lines of anti-tumoral systemic treatment (sorafenib, capecitabine, cabozantinib). He started treatment presenting with 2 target liver lesions and one non-target lymphonodal lesion and large tumor burden. He had -52% reduction in target lesions on Cycle 2 that was maintained until Cycle 13 when he was withdrawn due to progressive disease for appearance of a new liver lesion. DoR was 9.3 months. Two further patients i.e: 0003, at 100 mg / m2 / week, still on treatment at data cut-off, and 0007, at 135 mg / m2 / week, had SD lasting 25.3 (ongoing) and 10.9 months, respectively. Patient 0003 is a 71 years-old male with a treated dysmetabolic liver disease and one line of prior systemic anti-tumoral therapy with sorafenib. He presented, at entrance, a single target primary liver recurrence. Patient 0007 is a 64 years-old male treated with lenvatinib as only line of prior systemic anti-tumoral therapy. He had a locally advanced multinodal liver disease. In the phase 2 part of the study, 4 patients had at least one radiological assessment on treatment, with 3 out of 4 showing stable disease.

[0080] The good in vivo antitumor activity of compound (I) in the Hepa 1 -6 mouse model of HCC, supported by the positive anti-proliferative data on HCC cell-lines and the good preclinical tolerability clearly shows the advantage to use the compound (I) in the treatment of HCC. Preliminary clinical data on HCC patients treated with single agent compound (I) showed suitable safety features and very promising preliminary level of efficacy. Prognosis in pretreated HCC patients worsen with increase of prior lines of therapy and, indeed, patients that have undergone two lines of prior systemic therapies will have very dismal one with median progression free survival (PFS) around 2 months and an Overall survival lower than 12 months.

Claims

Claims1. A compound of formula (I)N-(2,6-diethylphenyl)-8-({4-[4-(dimethylamino)piperidin-1 -yl]-2-methoxyphenyl}amino)-1 -methyl-4,5-dihydro- 1 H-pyrazolo[4,3-h]quinazoline-3-carboxamide or a pharmaceutically acceptable salt thereof for use in a method for treating cancer, wherein the cancer is a malignant hepatocellular carcinoma.

2. The compound for use according to claim 1 , wherein the pharmaceutically acceptable salt is selected from the group consisting of phosphate, tosylate and besylate.

3. Use of a compound of formula (I)N-(2,6-diethylphenyl)-8-({4-[4-(dimethylamino)piperidin-1 -yl]-2-methoxyphenyl}amino)-1 -methyl-4,5-dihydro- 1 H-pyrazolo[4,3-h]quinazoline-3-carboxamide or a pharmaceutically acceptable salt thereof in themanufacture of a medicament for treating a malignant hepatocellular carcinoma.

4. A Method for the treatment of malignant hepatocellular carcinoma in a subject in need thereof comprising administering to said subject a therapeutically effective amount of a compound of formula (I)N-(2,6-diethylphenyl)-8-({4-[4-(dimethylamino)piperidin-1 -yl]-2-methoxyphenyl}amino)-1 -methyl-4,5-dihydro-1 H-pyrazolo[4,3-h]quinazoline-3-carboxamide or a pharmaceutically acceptable salt thereof.

5. A pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof as defined in claims 1 or 2, for treating a malignant hepatocellular carcinoma.

6. A pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof as defined in claims 1 or 2 admixed with a pharmaceutically acceptable carrier, diluent or excipient, for use in the treatment of malignant hepatocellular carcinoma.

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