THIOCARBAMATE DERIVATIVES AS A2A INHIBITORS, PHARMACEUTICAL COMPOSITION THEREOF AND COMBINATIONS WITH ANTICANCER AGENTS

Abstract

The present invention relates to thiocarbamate derivatives of Formula (I) which are useful as A2A adenosine receptor (A2AR) inhibitors

##STR00001##

Especially, the present invention relates to a pharmaceutical composition comprising an A2A inhibitor of Formula (I) and a lipid carrier such as lauroyl macrogol-32 glycerides, D-α-tocopherol-polyethylene glycol-1000 succinate or a mixture thereof. The pharmaceutical composition of the invention is particularly useful for oral dosing in the treatment of cancers.

The present invention also relates to a combination comprising an A2A receptor inhibitor of Formula (I) and an anticancer agent. The anticancer agent is for example an immunotherapeutic agent, such as a checkpoint inhibitor. The invention further relates to a pharmaceutical composition and a kit of parts comprising such combination. Additionally, the combination of the invention is particularly useful for the treatment and/or prevention of cancers.

Claims

1. (canceled)

2. (canceled)

3. (canceled)

4. (canceled)

5. (canceled)

6. (canceled)

7. (canceled)

8. (canceled)

9. A method of treating cancer in a patient in need thereof, the method comprising administering to the patient a therapeutically acceptable effective amount of a combination comprising: (a) a compound of Formula (I): ##STR00149## or a pharmaceutically acceptable salt thereof, wherein: R.sup.1 represents 5- or 6-membered heteroaryl or 5- or 6-membered aryl, wherein heteroaryl or aryl groups are optionally substituted by one or more substituent selected from C.sub.1-C.sub.6 alkyl and halo; R.sup.2 represents 6-membered aryl or 6-membered heteroaryl, wherein heteroaryl or aryl groups are optionally substituted by one or more substituent selected from halo, alkyl, heterocyclyl, alkoxy, cycloalkyloxy, heterocyclyloxy, carbonyl, alkylcarbonyl, aminocarbonyl, hydroxycarbonyl, heterocyclylcarbonyl, alkylsulfoxide, alkylsulfonyl, aminosulfonyl, heterocyclylsulfonyl, alkylsulfonimidoyl, carbonylamino, sulfonylamino and alkylsulfonealkyl; said substituents being optionally substituted by one or more substituent selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkyne, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl alkylsulfonyl and alkylsulfonealkyl; or the heteroaryl or aryl groups are optionally substituted with two substituents that form together with the atoms to which they are attached a 5- or 6-membered aryl ring, a 5- or 6-membered heteroaryl ring, a 5- or 6-membered cycloalkyl ring or a 5- or 6-membered heterocyclyl ring; optionally substituted by one or more substituent selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkyne, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl and alkylsulfonealkyl; and (b) a checkpoint inhibitor selected from the group consisting of a PD-1 antibody and a PD-L1 antibody.

10. The method according to claim 9, wherein the compound is of Formula (Ia) ##STR00150## or a pharmaceutically acceptable salt thereof, wherein: R.sup.1 represents 5- or 6-membered heteroaryl or 5- or 6-membered aryl, wherein heteroaryl or aryl groups are optionally substituted by one or more substituent selected from C.sub.1-C.sub.6 alkyl and halo; X.sup.1 and X.sup.2 are each independently selected from C and N; R.sup.1′ is absent when X.sup.1 is N; or when X.sup.1 is C, R.sup.1′ represents H, halo, alkyl, heterocyclyl, alkoxy, cycloalkyloxy, heterocyclyloxy, carbonyl, alkylcarbonyl, aminocarbonyl, hydroxycarbonyl, heterocyclylcarbonyl, alkylsulfoxide, alkylsulfonyl, aminosulfonyl, heterocyclylsulfonyl, alkylsulfonimidoyl, carbonylamino, sulfonylamino or alkylsulfonealkyl; said substituents being optionally substituted by one or more substituent selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkyne, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl and alkylsulfonealkyl; R.sup.2 represents H, halo, alkyl, heterocyclyl, alkoxy, cycloalkyloxy, heterocyclyloxy, carbonyl, alkylcarbonyl, aminocarbonyl, hydroxycarbonyl, heterocyclylcarbonyl, alkylsulfoxide, alkylsulfonyl, aminosulfonyl, heterocyclylsulfonyl, alkylsulfonimidoyl, carbonylamino, sulfonylamino, or alkylsulfonealkyl; said substituents being optionally substituted by one or more substituent selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkyne, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl and alkylsulfonealkyl; or R.sup.1′ and R.sup.2′ form together with the atoms to which they are attached a 5- or 6-membered aryl ring, a 5- or 6-membered heteroaryl ring, a 5- or 6-membered cycloalkyl ring or a 5- or 6-membered heterocyclyl ring; optionally substituted by one or more substituent selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkyne, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl and alkylsulfonealkyl; R.sup.3′ is absent when X.sup.2 is N; or when X.sup.2 is C, R′ represents H or halo; R.sup.4′ represents H or halo; and R.sup.5′ represents H or halo.

11. (canceled)

12. (canceled)

13. (canceled)

14. (canceled)

15. (canceled)

16. (canceled)

17. The method of claim 9, wherein the cancer is selected from the group consisting of breast, carcinoid, cervical, colorectal, endometrial, glioma, head and neck, liver, lung, melanoma, ovarian, pancreatic, prostate, renal, gastric, thyroid and urothelial cancers.

18. The method of claim 9, wherein the cancer is selected from breast cancer, prostate cancer, melanoma, and solid tumor.

19. The method of claim 9, wherein the administration of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is to be administered prior to, concomitantly, or subsequent to the checkpoint inhibitor.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[1003] FIGS. 1A and 1B show the anti-tumor efficacy of A2AR inhibitor Compound 7 administered as single agent in liver Hepa 1-6 syngeneic tumor model. FIG. 1A is a graph representing the tumor volume over time after inoculation. FIG. 1B shows the distribution of tumors volume at day 22.

[1004] FIGS. 2A and 2B show the A20 tumor growth upon treatment with Compound 7 used as single agent or in combination with anti-PD-L1. FIG. 2A is a graph representing the tumor volume over time after inoculation. FIG. 2B shows the distribution of tumors volume at day 23.

[1005] FIGS. 3A-E show EMT6 tumor growth upon treatment with Compound 8a in combination with antagonist anti-CTLA4 mAb. The number of complete responders (CR) out of 10 mice per group is indicated. Tumor growth over time: Median (FIG. 3A), Vehicle (FIG. 3B) and aCTLA-4, 3 mg/kg Q3Dx2 (day 9, 12) standalone (FIG. 3C) or in combination with D) Compound 8, 0.1 mg/kg QDx25 (FIG. 3D) or Compound 8, 0.6 mg/kg QDx25 (FIG. 3E).

[1006] FIGS. 4A and 4B show that A2AR inhibitor Compound 8a in combination with anti-PD-L1 mAb modulates T cell infiltrate in the A20 tumor microenvironment. The absolute quantification of the number of CD3+ T cells (FIG. 4A) and CD8+ T cells (FIG. 4B) was measured by calculation from the average of 10 high powered fields/sections (10 HPF) from independent tumors (symbols represent individual mice; n=5 to 7 mice per/group). The median and interquartile range are shown, p values where calculated using Mann Whitney non-parametric t test.

[1007] FIGS. 5A-5E show the MCA205 tumor growth upon treatment with Compound 8b used as single agent or in combination with Oxaliplatin. FIG. 5A is a graph representing the median tumor volume over time after subcutaneous inoculation of tumor cells. FIGS. 5B-E are the individual tumor growth volumes of mice treated with Vehicle (FIG. 5B), Compound 8b at 0.6 mg/kg QDx25 (FIG. 5C), and Oxaliplatin at 10 mg/kg QDx1 (day 11) in standalone (FIG. 5D) or in combination with Compound 8b at 0.6 mg/kg QDx25 (FIG. 5E).

[1008] FIGS. 6A-G show the CT26 tumor growth upon treatment with Compound 8b at 0.6 mg/kg QDx32, anti-TIGIT mAb at 1 mg/kg Q3Dx3 (day 9, 12 and 15), Doxorubicin 6 mg/kg Q3Dx2 (day 10 and 14), and various combinations thereof. FIG. 6A is a graph representing the median tumor volume over time after subcutaneous inoculation of tumor cells. FIGS. 6B-D are the individual tumor growth volumes of mice treated with Vehicle (FIG. 6B), Doxorubicin (FIG. 6C) or anti-TIGIT mAb (FIG. 6D). FIGS. 6E-G are the individual tumor growth volumes of mice treated with a combination of Doxorubicin and anti-TIGIT (FIG. 6E), Compound 8b and Doxorubicin (FIG. 6F) and triple combination of Compound 8b, Doxorubicin and anti-TIGIT (FIG. 6G).

[1009] FIG. 7A shows the proportion of proliferating CD4.sup.+ cells as determined by CFSE dilution. FIG. 7B shows the concentration of TNFα present in the cell culture supernatants. CD39i denotes the combination of the CD39 inhibitors ARL67156 and POM-1. Dark grey bars show Compound 8a treated wells, whilst light grey bars denote concentration-matched DMSO treated wells. Results are shown as mean value from duplicate wells±standard deviation.

EXAMPLES

[1010] The present invention will be better understood with reference to the following examples.

[1011] These examples are intended to representative of specific embodiments of the invention, and are not intended as limiting the scope of the invention.

[1012] The following abbreviations are used:

[1013] BHT: butylated hydroxytoluene

[1014] BID: bis in die (i.e. twice a day)

[1015] ca.: circa

[1016] CR: complete responder

[1017] DMSO: dimethylsulfoxide

[1018] EDTA: ethylenediaminetetraacetic acid

[1019] HPLC: high-performance liquid chromatography

[1020] LC-MS: liquid chromatography-mass spectrometry

[1021] mAb: monoclonal antibody

[1022] mg: milligram

[1023] MS: mass spectrometry

[1024] PBS: phosphate buffered saline

[1025] PEG: polyethylene glycol

[1026] QD: quaque die (i.e. once a day)

[1027] Q3D: quaque 3 die (i.e. every 3 days)

[1028] rpm: revolutions per minutes

[1029] TGL: tumor growth inhibition

[1030] TILs: tumor infiltrating lymphocytes

[1031] UV: ultraviolet

[1032] μL: microliter

[1033] % v/v: percentage in volume to the total volume of the composition

[1034] % w/w: percentage in weight to the total weight of the composition

[1035] I. Compounds

[1036] The compounds of Formula (I) are prepared as described in PCT/EP2018/058301.

[1037] II. Pharmaceutical Compositions

[1038] II.1. Manufacturing of Pharmaceutical Compositions

[1039] Two composition according to the invention were prepared under capsules form, comprising the following ingredients (Table 2):

TABLE-US-00002 TABLE 2 Capsules compositions (% w/w). Components 1 2A 3 4 5 6 7 8 Compound 8a 10 10 10 Compound 8a esylate salt 10 10 10 10 Compound 8a HCl salt 10 Gelucire ® 44/14 71.9 80.9 71.9 89 90 Vitamin E TPGS 71 71 71 PEG 400 18 \ 18 PEG 3350 18 18 18 Caprylic acid \ 9 Butylated 0.1 0.1 0.1 hydroxytoluene (BHT) polyvinyl caprolactam- 1.0 1.0 1.0 polyvinyl acetate-polyethylene glycol graft copolymer Hydroxypropylmethylcellulose 1.0

[1040] Capsules 2A were prepared from a common blend using conventional mixing and capsule filling processes according to Good Manufacturing Practice. Lauroyl polyoxyl-32 glycerides is melted with a product temperature not less than 50° C. but not exceeding 80° C. Caprylic acid and then butylated hydroxytoluene (BHT) are then added to the lauroyl polyoxyl-32 glycerides and mixed together using a suitable mixer. Compound 8a is then added gradually to the lauroyl polyoxyl-32 glycerides/caprylic acid/BHT mixture being continuously mixed together using a suitable mixer to produce a visually uniform distribution of the drug substance with no observable lumps or agglomerates. Mixing is then continued for at least 30 minutes to ensure that the drug substance is homogeneously distributed as determined visually. The blend is then maintained in the molten state with continued mixing and is filled into appropriately sized gelatine capsule shells to the target capsule fill weight. Capsule filling is undertaken using conventional capsule filling methods and equipment suitable for use with molten semi-solid formulations.

[1041] A similar process was carried out for manufacturing all other capsule examples, with Vitamin E TPGS being substituted for lauroyl polyoxyl-32 glycerides. Polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer and hydroxypropylmethylcellulose are added to the molten lauroyl polyoxyl-32 glycerides or Vitamin TPGS as required.

[1042] II.2. Pharmacology Examples

[1043] II.2.i. Thermodynamic Solubility by Shake Flask—HPLC

[1044] This example aims at showing that the compounds of Formula (I) are poorly soluble in water or in aqueous buffers and thus that there is a need to provide a formulation of said compounds.

[1045] Compound 8a (2.0 mg, crystalline solid) was weighed into the lower chambers of Whatman miniuniprep vials. 450 μL of tested medium was added into each chamber. After this addition, filter pistons of miniuniprep vials were placed and compressed to the position of the liquid level to allow for contact of the medium and compound with the filter during incubation. The samples were vortexed for 2 minutes, then incubation was carried out at room temperature (ca. 22˜25° C.) for 24 hours with shaking at 880 rpm.

[1046] The miniunipreps were compressed to prepare the filtrates for injection into HPLC system. The supernatants were diluted with the medium by a factor of 50 folds to make diluents. Three UV standard solutions were injected into IPLC from low to high concentration, followed by testing of the diluents and supernatants. Testing samples were injected in duplicate.

[1047] The results are shown in Table 3:

TABLE-US-00003 TABLE 3 Solubility of Compound 8a in tested aqueous media. Thermodynamic solubility of Tested medium Compound 8a (μg/mL) water <0.6 pH 7.4 2 FaSSIF 1-10

[1048] In all tested aqueous media, the solubility is very low and the test in FaSSIF (Fasted-State Simulated Intestinal Fluid) is representative of a low intestinal solubility.

[1049] II.2.ii. Exposures in Dogs after Oral Dosing

[1050] The purpose of this assay is to determine the exposure in dogs after oral dosing with the pharmaceutical composition of the invention. Dogs are administered with pentagastrin just before administration of the capsules formulations in order to stimulate the secretion of gastric acid.

[1051] Five male Beagle dogs (>6 months of age, 7-9 kg of weight) were fed the afternoon (at 3:30 to 4:00 pm) prior to the day of oral dosing and the remaining food was removed at about 7:00 pm. Food was withheld until after the 4-hour blood collection.

[1052] Pentagastrin (Sigma, 1 mg) was dissolved in 200 μL (0.200 mL) of a solution of 10% (v/v) ammonium hydroxide (NH.sub.4OH)/90% (v/v) Phosphate Buffered Saline (PBS). 0.12 mL of this stock solution was then diluted by adding 4.88 mL of PBS solution, then the vial was vortexed. The solution was filtered (under a laminar flow hood) through a 0.22 m syringe filter into a sterile amber glass serum bottle. Animals were administered with Pentagastrin at 6 μg/kg by intramuscular injection at approximately 30 minutes (±2 min) before dosing with the capsules.

[1053] The dose capsule formulations (80 mg/dog, i.e. about 10 mg/kg of animal) were administered by placing the capsules in the far back of the dog's throat, then pushing it past the pharynx using a thumb or index finger. The capsules were moistened with water to facilitate dosing. After administering the dose, swallowing was induced, if needed, by gently stroking the dog's throat or tapping the dog under the chin. Immediately following capsule administration, water (4 mL/kg) or an aqueous HCl solution at pH 2.5 (4 mL/kg) was given to the mouth to the animals to help capsule swallowing. After administration, the animals' mouths were inspected to ensure that the dose had been swallowed.

[1054] Blood was collected at the timepoints indicated in Table 4 into a tube (Jiangsu Kangjian medical supplies co., LTD) containing Potassium (K2) EDTA*2H.sub.2O (0.85-1.15 mg) on wet ice and processed for plasma by centrifugation (3,000×g for 10 minutes at 2 to 8° C.) within one hour of collection. The plasma samples (0.2 mL) were transferred into labeled polypropylene micro-centrifuge tubes and stored frozen at −60° C. or lower until bio-analysis.

[1055] Concentrations of Compound 8a in plasma were quantified by LC-MS/MS. The concentrations measured (mean of five dogs) are indicated in Table 4, while the main pharmacokinetic parameters are indicated in Table 5.

TABLE-US-00004 TABLE 4 Concentration of Compound 8a in plasma. Timepoint Formulation 1 Formulation 2A (h) Concentration (ng/mL) Concentration (ng/mL) 0.25 94.7 94.4 0.50 431 292 1.0 242 384 2.0 161 108 4.0 9.91 10.5 8.0 3.42 3.45 12 4.68 <1

TABLE-US-00005 TABLE 5 Pharmacokinetic parameters. Formulation 1 Formulation 2A Dose (mg/dog) 80 80 (active ingredient) Additional liquid Water Aqueous HCl pH 2.5 C.sub.max (ng/mL) 481 674 T.sub.max (h) 0.9 1 AUC.sub.last (h*ng/mL) 554 1818 AUC.sub.inf (h*ng/mL) 561 1823 C.sub.max: maximum plasma concentration of the active ingredient obtained after administration; T.sub.max: time to reach C.sub.max; AUC: area under the curve, corresponding to the integral of the concentration-time curve (AUC.sub.last: AUC up to the last sample drawn; AUC.sub.inf: AUC up to infinite time)

[1056] Above results clearly evidence that the use of the pharmaceutical composition of the invention enables suitable oral bioavailability of the thiocarbamates A2A inhibitors.

[1057] III. Combinations with Anticancer Agents—In Vivo Studies

[1058] Summary of the Results:

[1059] Compound 7 at 3 mg/kg BID, significantly delayed growth of HEPA-1-6 tumors compared to vehicle control in syngeneic host.

[1060] In combination with anti-PD-L1, Compound 7 demonstrated anti-tumor activity at 0.3 mg/kg BID and, at 3 mg/kg BID, significantly delaying tumor growth versus anti-PD-L1 control in a A20 syngeneic mouse lymphoma model.

[1061] In combination with anti-CTLA-4, Compound 8a demonstrated antitumor activity at 0.1 mg/kg and starting at 0.6 mg/kg significantly delayed tumor growth in a dose dependent manner in the EMT6 syngeneic mouse breast cancer model. In addition, in cured mice, prevented growth after re-inoculation suggesting induction of a specific memory response.

[1062] Further illustrating the mechanism of action, in combination with anti-PD-L1, Compound 8a at 3 mg/kg BID significantly increased CD3+ and CD8+ cell infiltrations without an effect of FOXP3 expressing cells.

[1063] In combination with Oxaliplatin, Compound 8b demonstrated significant antitumor activity at 0.6 mg/kg by delaying tumor growth in the MCA205 syngeneic mouse fibrosarcoma cancer model.

[1064] In combination with Doxorubicin, Compound 8b demonstrated significant antitumor activity at 0.6 mg/kg by delaying tumor growth in the CT26 syngeneic mouse colon cancer model. In the same example a triple combination of Doxorubicin, anti-TIGIT mAb at 1 mg/kg and Compound 8b at 0.6 mg/kg demonstrated significantly enhanced antitumor activity compared to double combinations of Doxorubicin and Compound 8b or Doxorubicin and anti-TIGIT mAb in the CT26 syngeneic mouse colon cancer model.

[1065] III.1. Syngeneic HEPA1-6 Mouse Liver Tumor Model

[1066] This study evaluated the anti-tumor activity of Compound 7 in a mouse hepatoma model (NCR-A2A-032).

[1067] C57BL/6 female mice (8 weeks old) were inoculated subcutaneously in the right flank region with Hepa 1-6 tumor cells (on day 0). When tumor size reached about 50 mm.sup.3 (on Day 4), mice were randomly allocated into experimental groups and treatment was initiated from day 4 to 25. Mice were administered vehicle p.o. (10% DMSO, 10% Solutol HS15 in dH.sub.2O pH3) or Compound 7 at 0.3 and 3 mg/kg, p.o., BIDx 21.

[1068] At 3 mg/kg, Compound 7 demonstrated significant antitumor efficacy with a tumor growth inhibition (TGI) of 44% calculated on Day 22 (p=0.024). (FIGS. 1A and 1B).

[1069] III.2. Syngeneic A20 Experimental Lymphoma Model in Combination with Anti-PD-L1

[1070] In this study the anti-tumor efficacy of Compound 7 was assessed either as single agent (monotherapy) or in combination with antagonist anti-Programmed Death-Ligand 1 (anti-PD-L1) monoclonal antibody (1° F.9G2, BioXcell) in a model of B cell lymphoma model. (NCR-A2A-031).

[1071] BALB/c female mice (8 weeks old) were inoculated with A20 mouse B-cell tumor cells, subcutaneously in the right lower flank region (day 0). When tumors reached a size of about 50 mm.sup.3 (Day 7), mice were allocated randomly into groups and treatment was initiated. Mice were administered vehicle p.o. (10% DMSO, 10% Solutol HS15 in dH.sub.2O pH3) as control or Compound 7 at 0.3 and 3 mg/kg, p.o., BIDx 21 (Day 7 to 28) or anti-PD-L1 mAb at 5 mg/kg i.p., Q3Dx3 (day 9, 12, 15) as single agent or in combination.

[1072] Compound 7 administered p.o. at 0.3 mg/kg, BID, in combination with anti-PD-L1 mAb, demonstrated anti-tumor activity (TGI=76%, determined on day 23) although not reaching statistical significance (p=0.106) compared with anti-PD-L1 single therapy (TGI=49%).

[1073] Compound 7 administered at 3 mg/kg BID in combination with anti-PD-L1 mAb, showed significant enhancement of antitumor activity (p=0.039 for TGI and p=0.0008 for overall suppression of tumor growth) compared with single agent anti-PD-L1 mAb (TGI=82 and 49%, observed on day 23, respectively) (FIGS. 2A and 2B).

[1074] Statistical analysis also revealed that Compound 7 synergises with anti-PD-L1 mAb to significantly inhibit the growth of established A20 syngeneic tumor (p=0.017).

[1075] III.3. Syngeneic EMT6 Breast Cancer Model in Combination with Anti-CTLA-4

[1076] The anti-tumor efficacy of Compound 8a was assessed in combination with anti-cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) antagonist mAb (9H10, BioXcell) in a model of triple negative mammary cancer.

[1077] EMT6 mammary tumor cells were inoculated orthotopically into the lower right mammary fat pad of 8 week old female BALB/c mice (day 0). When tumors reached a size of about 60 mm.sup.3 (day 9), mice were randomly allocated into groups. Mice were administered with control vehicle p.o. (10% DMSO, 10% Solutol HS15 in dH.sub.2O pH3) or anti-CTLA-4 mAb at 3 mg/kg i.p., Q3Dx2 (day 9, 12) stand alone or in combination with Compound 8a at 0.1 mg/kg p.o. QDx25 or 0.6 mg/kg p.o. QDx25.

[1078] Compound 8a combined with anti-CTLA-4 significantly reduced tumor growth when administered at 0.6 mg/kg (p=0.0153, mean TGI=99% determined on day 31—complete responder (CR)=7/10) vs. anti-CTLA4 alone (CR=3, mean TGI=72%, determined on day 31) (FIGS. 3A, 3B, 3C and 3E). Although Compound 8a at 0.1 mg/kg in combination with anti-CTLA-4 mAb (as above) did not reach statistical significance (p=0.8665) vs. anti-CTLA4 mAb as single agent, it resulted in CR=5 (n=10 mice group) and mean TGI=85% (FIGS. 3A and 3D). In addition, there was also no statistically significant difference between doses. This suggests that Compound 8a already demonstrates in vivo activity starting at 0.1 mg/kg in combination with anti-CTLA4 mAb (FIGS. 1A and 1).

[1079] When complete responders (cure mice) re-challenged with EMT6 tumor cells (specific antigen) or unrelated colon CT26 cells (non-specific antigen), the mice previously treated with Compound 8a at 0.1 and 0.6 mg/kg in combination with anti-CTLA-4 mAb, were not protected against the challenge of unrelated CT26 cells (100% of tumor incidence) but fully rejected EMT6 cells (no tumor formation) in 66% (0.1 mg/kg) and 100% (0.6 mg/kg) of mice respectively.

[1080] Compound 8a therefore significantly inhibited the growth of established EMT6 syngeneic tumors in combination with anti-CTLA-4 antagonist mAb and induce long-term memory response that results in durable specific antitumor response.

[1081] III.4. Mechanism of Action of Compound 8A to Reduce Tumor Growth in A20 Lymphoma Model

[1082] Having established that Compound 8a (or its racemate mixture, Compound 7) demonstrates anti-tumor activity in several experimental tumor models at well-tolerated doses, this study evaluated the mechanism of action responsible for this effect.

[1083] Compound 8a was evaluated in B cell Lymphoma tumor model, as single agent and in combination with antagonist anti-PD-L1 mAb. A20 tumor-bearing mice (tumor size of about 70 mm.sup.3, day 12 after inoculation) were treated with control vehicle p.o. (10% DMSO, 10% Solutol HS15 in dH.sub.2O pH3) or anti-PD-L1 mAb at 1 mg/kg i.p., Q3Dx3 (day 14, 17 and 20) and Compound 8a 3 mg/kg p.o., BIDx10 as single agent or in combination with anti-PD-L1 mAb. By IHC staining, the tumor-infiltrating lymphocytes (TIL), including total CD3.sup.+ T cells, CD8.sup.+ T cells and FOXP3.sup.+ Treg cells, were characterized and compared using a semi-quantitative technique.

[1084] Compound 8a at 3 mg/kg as single agent regimen had a moderate effect on CD3.sup.+ and CD8.sup.+ TILs in the tumor environment but the increase in infiltrate did not reach significance. The combination of Compound 8a at 3 mg/kg with anti-PDL-1 mAb demonstrated a significant increase in CD3.sup.+ T cells (p=0.0068) and CD8.sup.+ T cell (p=0.0035) infiltration in tumor as compared to anti-PDL-1 mAb used as single agent (FIGS. 4A and 4B). No statistically significant change in Treg infiltrate was demonstrated with any treatment in single agent or combination.

[1085] These results strongly suggest that Compound 8a in combination with aPDL-1 mAb significantly modulates the immunosuppressive nature of the tumor microenvironment by increasing TILs infiltration.

[1086] III.5. Syngeneic MCA205 Experimental Fibrosarcoma Model in Combination with Oxaliplatin

[1087] The anti-tumor efficacy of Compound 8b was assessed in combination with Oxaliplatin, in an established murine syngeneic MCA205 fibrosarcoma tumor model.

[1088] MCA205 tumor cells were inoculated subcutaneously into the right flank of C57BL/6 mice. When tumors reached an average size of about 80 mm.sup.3, mice were randomly allocated into groups. Mice were administered vehicle p.o. (10% DMSO, 10% Solutol HS15 in dH.sub.2O pH3) as control or Compound 8b at 0.6 mg/kg QDx25 (day 11-36) or Oxaliplatin at 10 mg/kg i.p. QDx1 (day 11) as single agent or in combination.

[1089] Oxaliplatin, given as a single administration i.p. at D11, demonstrated a significant delay in tumor growth (p=0.0202) compared to the Vehicle (FIGS. 5A, 5B, and 5D).

[1090] Compound 8b, administered p.o. at 0.6 mg/kg once daily (QD) for 25 consecutive days in combination with Oxaliplatin, demonstrated significant tumor growth delay compared to Vehicle (p<0.0001) and also significant tumor growth delay when compared to Oxaliplatin monotherapy (p=0.0284) (FIGS. 5A-E).

[1091] III.6. Syngeneic CT26 Experimental Colon Cancer Model in Combination with Doxorubicin and/or Anti-TIGIT Mab

[1092] In this study the anti-tumor efficacy of Compound 8b was assessed in combination with chemotherapeutic agent, Doxorubicin and with anti-TIGIT mAb in a Colon tumor model (CT26).

[1093] BALB/c female mice (8 weeks old) were inoculated with CT26 mouse tumor cells, subcutaneously in the right lower flank region (day 0). When tumors reached a size of about 90 mm.sup.3 (Day 9), mice were allocated randomly into groups and treatment was initiated. Mice were administered vehicle p.o. (10% DMSO, 10% Solutol HS15 in dH.sub.2O pH3) as control or Doxorubicin at 6 mg/kg i.v., Q4Dx2 (day 10 and 14) or anti-TIGIT mAb, 29527 (see U.S. Ser. No. 10/329,349) at 1 mg/kg i.p. Q3Dx3 (day 9, 12 and 15), as single agent or in combinations with Compound 8b at 0.6 mg/kg, p.o., QDx 32 (Day 9 to 41).

[1094] Intraperitoneal injection of 1 mg/kg of anti-TIGIT mAb, 29527 (see U.S. Ser. No. 10/329,349) at days 9, 12 and 15 after tumor cell inoculation, significantly suppressed tumor growth compared to mice treated with vehicle (p=0.0009, FIGS. 6A and 6B)

[1095] Doxorubicin, administered twice at 6 mg/kg on day 10 and 14, had no significant effect on tumor growth (p=0.14, FIGS. 6A and 6C), while combination with anti-TIGIT mAb improved anti-tumor efficacy (p=0.008 and p=0.0002 respectively, when compared to anti-TIGIT mAb or Doxorubicin stand-alone therapy (FIGS. 6A, 6C, 6D, and 6E).

[1096] Compound 8b administered at 0.6 mg/kg QD from day 9 in combination with Doxorubicin given at 6 mg/kg on day 10 and 14 achieved significant anti-tumor effect when compared to stand alone administration of Doxorubicin (p=0.0003, FIGS. 6A, 6C, and 6F). The anti-tumor growth efficacy in mice treated with a triple combination of Compound 8b, Doxorubicin and anti-TIGIT, was significantly higher when compared to Doxorubicin+Compound 8b (p<0.0001) or when compared to anti-TIGIT+Doxorubicin (p=0.003), and resulted in 5 complete responders (CR) out of 8 mice in this triple combination treated group verses zero and one CR out of 10 mice in the Doxorubicin+Compound 8b and anti-TIGIT+Doxorubicin combinations respectively. (FIGS. 6A, 6E, 6F, and 6G).

[1097] III.7. Restoration of Human T Cells Function by a Combination with CD39 Inhibitors

[1098] Purpose. When T cells are activated, they proliferate and produce pro-inflammatory cytokines. Addition of adenosine triphosphate (ATP) to the culture provides a source of adenosine as ATP is first converted to adenosine monophosphate by CD39 and then to adenosine by CD73. Adenosine suppresses T cell proliferation and cytokine production in part by engaging the A2A receptor. The present assay thus aims to show that the inhibition of proliferation and inflammatory cytokine production by T cells in the presence of ATP may be reversed by a combination of the A2A receptor antagonist Compound 8a and the CD39 inhibitors ARL67156 and POM-1.

[1099] PBMC and CD3.sup.+ T cell isolation. Venous blood from healthy volunteers was obtained via ImmuneHealth (Centre Hospitalier Universitaire Tivoli, La Louviere, Belgium). Peripheral blood mononuclear cells (PBMCs) were collected by density gradient centrifugation, using SepMate-50 tubes (StemCell Technologies, Grenoble, France) and Lymphoprep (Axis-shield, Oslo, Norway) according to the manufacturer's instructions. PBMCs were stored in heat inactivated foetal bovine serum (hiFBS; Gibco, ThermoFisher Scientific, Merelbeke, Belgium) containing 10% DMSO in liquid nitrogen until required. PBMCs were thawed and washed into PBS (with 10% hiFBS) and labelled with 1 μM CFSE (Life Technologies) at room temperature for 5 minutes. Cells were washed into StemCell isolation buffer and CD3.sup.+ T cells were isolated by immunomagnetic negative selection, using the EasySep Human T Cell Isolation Kit (StemCell Technologies) as per manufacturer's instructions.

[1100] Human T cell activation assay. Human CD3.sup.+ T cells were washed into X-VIVO15 medium (LONZA) and distributed into a 96 round well plate at 8×10.sup.4 cells per well. Wells received either the A2A receptor antagonist compound 8a at a final concentration of 100 nM or a matched concentration of DMSO (Sigma-Aldrich). In addition, some wells received a combination of the CD39 inhibitors ARL67156 (Tocris Bioscience, 100 μM) and POM-1 (Tocris Bioscience, 10 μM), or a combination of both CD39 inhibitors and compound 8a. Cells were cultured in the presence or absence of ATP (Sigma-Aldrich) at a final concentration of 100 μM and were activated by adding anti-CD3 and anti-CD28 coated microbeads (Dynabeads human T-activator CD3/CD28; Life Technologies).

[1101] Cells were placed in a 37° C. humidified tissue culture incubator with 5% CO.sub.2 for 72 hours. After 72 hours, supernatants were sampled and stored at −20° C. Proliferation of CD4.sup.+ T cells was assessed by determining CFSE dilution by flow cytometry using a BD LSRFortessa (BD Biosciences). Supernatants were later thawed and TNFα was quantified using the AlphaLISA Human TNFα Biotin-Free Detection Kit (AL325; Perkin-Elmer, Zaventem, Belgium), according to the manufacturer's instructions.

[1102] Results. The presence of ATP significantly inhibited CD4.sup.+ T cell proliferation which was almost completely rescued by the CD39 inhibitors ARL67156 and POM-1 (FIG. 7A). The presence of compound 8a made a small further contribution to increasing proliferation of these cells. ATP also significantly inhibited the production of TNFα. Both compound 8a and the CD39 inhibitors individually could restore approximately 50% of TNFα production, with a combination resulting in complete rescue (FIG. 7B). Overall, these data illustrate the value of combining compound 8a with a CD39 inhibitor for cancer immunotherapy, aiming at full restoration of T cell function in the ATP-rich tumor microenvironment.