With regard to combination therapy the compound of the present invention may be administered separately, sequentially, simultaneously, concurrently or may be chronologically staggered with one or more standard therapeutics such as any of those mentioned above.

The present invention provides a compound of Formula (I):

[Image Omitted]

- or a pharmaceutically acceptable salt or N-oxide derivative thereof.

Preferably, there is provided the compound of formula (I), or a pharmaceutically acceptable salt or N-oxide derivative thereof, and at least one pharmaceutically acceptable excipient.

Suitable pharmaceutically acceptable excipients would be known by the person skilled in the art, for example, fats, water, physiological saline, alcohol (e.g., ethanol), glycerol, polyols, aqueous glucose solution, extending agent, disintegrating agent, binder, lubricant, wetting agent, stabilizer, emulsifier, dispersant, preservative, sweetener, colorant, seasoning agent or aromatizer, concentrating agent, diluent, buffer substance, solvent or solubilizing agent, chemical for achieving storage effect, salt for modifying osmotic pressure, coating agent or antioxidant, saccharides such as lactose or glucose; starch of corn, wheat or rice; fatty acids such as stearic acid; inorganic salts such as magnesium metasilicate aluminate or anhydrous calcium phosphate; synthetic polymers such as polyvinylpyrrolidone or polyalkylene glycol; alcohols such as stearyl alcohol or benzyl alcohol; synthetic cellulose derivatives such as methylcellulose, carboxymethylcellulose, ethylcellulose or hydroxypropylmethylcellulose; and other conventionally used additives such as gelatin, talc, plant oil and gum arabic.

Preferably, there is provided a pharmaceutical composition comprising the compound of formula (I), or a pharmaceutically acceptable salt or N-oxide derivative thereof, and at least one pharmaceutically acceptable excipient.

Preferably, there is provided a pharmaceutical composition comprising the compound of formula (I), or a pharmaceutically acceptable salt or N-oxide derivative thereof, comprising one or more further pharmaceutically active agents. Preferably, in certain embodiments, the pharmaceutical composition further comprises an anti-cancer agent, for example as a combination therapy as described herein. In such embodiments, a suitable anti-cancer agent may be any one or more of a genotoxic agent, a targeted agent and an immune-modulator.

Preferably, there Is provided the compound of Formula (I), or a pharmaceutically acceptable salt or N-oxide derivative thereof, or the pharmaceutical composition as described herein, for use in therapy.

Preferably, there is provided the compound of Formula (I), or a pharmaceutically acceptable salt or N-oxide derivative thereof, or the pharmaceutical composition as described herein, for use as a medicament.

Preferably, there is provided the compound of Formula (I), or a pharmaceutically acceptable salt or N-oxide derivative thereof, or the pharmaceutical composition as described herein, for use in treating or preventing cancer. Preferably the cancer is selected from colon and rectal (colorectal) cancer, head and neck cancer, lung cancer, oesophagus cancer, ovarian cancer and pancreas cancer. More preferably, the cancer Is colon and rectal (colorectal) cancer. Alternatively, preferably, the cancer is lung cancer, more preferably non-small cell lung cancer.

Preferably, there is provided the use of the compound of Formula (I), or a pharmaceutically acceptable salt or N-oxide derivative thereof, or the pharmaceutical composition as described herein, for the manufacture of a medicament for treating or preventing cancer. Preferably the cancer is selected from colon and rectal (colorectal) cancer, head and neck cancer, lung cancer, oesophagus cancer, ovarian cancer and pancreas cancer. More preferably, the cancer is colon and rectal (colorectal) cancer. Alternatively, preferably, the cancer is lung cancer, more preferably non-small cell lung cancer.

Preferably, there is provided a method of treating or preventing cancer in a human or animal patient comprising administering to a patient in need thereof an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt or N-oxide derivative thereof, or the pharmaceutical composition as described herein. Preferably the cancer is selected from colon and rectal (colorectal) cancer, head and neck cancer, lung cancer, oesophagus cancer, ovarian cancer and pancreas cancer. More preferably, the cancer is colon and rectal (colorectal) cancer. Alternatively, preferably, the cancer is lung cancer, more preferably non-small cell lung cancer.

Preferably, the compound of the present invention has an IC₅₀value for Wee-1 kinase of about 0.1 nM to about 1 nM. The method for determining the IC₅₀value of a compound for Wee-1 kinase is described in WO 2015/092431 (see page 350 to 351).

When introducing elements of the present disclosure or the preferred embodiments(s) thereof, the articles “a”, “an”, “the” and “said” are intended to mean that there are one or more of the elements. The terms “comprising”, “including” and “having” are intended to be Inclusive and mean that there may be additional elements other than the listed elements.

The foregoing detailed description has been provided by way of explanation and illustration, and is not intended to limit the scope of the appended claims. Many variations in the presently preferred embodiments illustrated herein will be apparent to one of ordinary skill in the art, and remain within the scope of the appended claims and their equivalents.

EXPERIMENTAL SECTION

Abbreviations

aq: aqueous; dba: dibenzylideneacetone; DCM: dichloromethane; DIPEA: diisopropylethylamine (Hunig's base); DMF: N,N-dimethylformamide; DMF: N,N-dimethylformamide dimethyl acetal; dppf: 1,1′-bis(diphenylphosphino)ferrocene; EtOAc: ethyl acetate; ESI: electrospray ionisation; h: hour; HPLC: high pressure liquid chromatography; LC: liquid chromatography; LCMS: liquid chromatography mass spectrometry; M: molar; m/z mass-to-charge ratio; mCPBA: 3-chloroperbenzoic acid; MeOH: methanol; min: minutes: MS: mass spectrometry; NMR: nuclear magnetic resonance; R_T: retention time; RB: round-bottomed; RT: room temperature; SOP: standard operating procedure; SM: starting material; TFA trifluoroacetic acid; THF: tetrahydrofuran; TLC: thin layer chromatography.

General Experimental Conditions

Solvents and Reagents

Common organic solvents that were used in reactions (e.g. THF, DMF, DCM, IPA, and methanol) were purchased anhydrous from Sigma Aldrich® in Sure/Seal® bottles and were handled appropriately under nitrogen. Water was delonised using an Elga PURELAB Option-Q. All other solvents used (i.e. for work-up procedures and purification) were generally HPLC grade and were used as supplied from various commercial sources. Unless otherwise stated, all starting materials used were purchased from commercial suppliers and used as supplied.

Flash Chromatography

Purification of compounds by flash chromatography was achieved using a Biotage isolera Four system. Unless otherwise stated, Blotage KP-Sil SNAP cartridge columns (10-340 g) were used along with the stated solvent system and an appropriate solvent gradient depending on compound polarity (determined by TLC analysis). In the case of more polar and basic compounds, Biotage KP-NH SNAP cartridge columns (11 g) were used.

NMR Spectroscopy

¹H NMR spectra were recorded at ambient temperature using a Bruker Avance (500 MHz) spectrometer or a Bruker Avance (400 MHz) spectrometer. All chemical shifts (8) are expressed in ppm. Residual solvent signals were used as an Internal standard and the characteristic solvent peaks were corrected to the reference data outlined in J. Org. Chem., 1997, 62, p 7512-7515; in other cases, NMR solvents contained tetramethylsilane, which was used as an internal standard.

High Pressure Liquid Chromatography

Liquid Chromatography Mass Spectrometry (LCMS) experiments to determine retention times (RT) and associated mass ions were performed using one of the following methods:

Method A: The system consists of an Agilent Technologies 6140 single quadrupole mass spectrometer linked to an Agilent Technologies 1290 Infinity LC system with UV diode array detector and autosampler. The spectrometer consists of a multimode ionization source (electrospray and atmospheric pressure chemical ionizations) operating in positive and negative ion mode. LCMS experiments were performed on each sample submitted using the following conditions: LC Column: Zorbax Eclipse Plus C18 RRHD 1.8 micron 50×2.1 mm maintained at 40° C. Mobile phases: A) 0.1% (v/v) formic acid in water; B) 0.1% (v/v) formic acid in acetonitrile.

Gradient Time (min)
Flow (mL/min)
% A
% B

0.00
1.0
95
5

1.80
1.0
0
100

2.20
1.0
0
100

2.21
1.0
95
5

2.50
1.0
95
5

Method B: The system consists of an Agilent Technologies 6130 quadrupole mass spectrometer linked to an Agilent Technologies 1290 Infinity LC system with UV diode array detector and autosampler. The spectrometer consists of an electrospray Ionization source operating in positive and negative ion mode. LCMS experiments were performed on each sample submitted using the following conditions: LC Column: Agilent Eclipse Plus C18 RRHD 1.8 micron 50×2.1 mm maintained at 40° C. Mobile phases: A) 0.1% (v/v) formic acid in water; B) 0.1% (v/v) formic acid in acetonitrile.

Gradient Time (min)
Flow (mL/min)
% A
% B

0.00
1.0
80
20

1.80
1.0
0
100

2.20
1.0
0
100

2.50
1.0
80
20

3.00
1.0
80
20

Intermediate 1: tert-butyl 4-(4-amino-2-methylphenyl)piperidine-1-carboxylate

[Image Omitted]

Step 1: tert-Butyl 4-(2-methyl-4-nitrophenyl)-5,6-dihydropyridine-1 (2H)-carboxylate

1-Bromo-2-methyl-4-nitrobenzene (123 g, 0.572 mol) and N-tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,8-dihydropyridine-1(2H)-carboxylate (177 g, 0.571 mmol) were dissolved in 1,4-dioxane (2.5 L). 3M Sodium carbonate solution (570 mL, 1.71 mol) was added, the mixture was degassed and placed under a nitrogen atmosphere. PdCl₂(dppf) dichloromethane adduct (9.3 g, 0.011 mol) was added and the mixture was heated at 100° C. overnight. The mixture was diluted with water and extracted with ethyl acetate (three times 1 L). The combined organic extracts were washed with brine, dried over MgSO₄, filtered and concentrated in vacuo. The residue was purified by flash chromatography (0-50% EtOAc/hex) to give 154 g (94.5%) of the title compound as yellowish crystals.

Step 2: tert-butyl 4-(4-amino-2-methylphenyl)piperidine-1-carboxylate

tert-Butyl 4-(2-methyl-4-nitrophenyl)-5,6-dihydropyridine-1(2H)-carboxylate (130 g, 0.408 mol) was dissolved in methanol (3.5 L) using a 5 L Parr reaction bottle. After the degassing the mixture, Pd/C (10%) (13 g) was added and the suspension was shaken under 55 psi hydrogen pressure overnight at RT. The mixture was filtered through a pad of Celite™ and the filtrate was concentrated under reduced pressure to provide 117 g (98.7%) of the title compound as a light beige solid.

The analytical data is in accordance with Example 37 step 4 as described in US2007/0254892A1.

EXAMPLES

The following non-limiting example further illustrates the present invention.

Example 1: 3-(2,6-dichlorophenyl)-1-methyl-7-((3-methyl-4-(1-methylpiperidin-4 ylphenyl)amino)-2,3-dihydropyrimido[4,5-d]pyrimidin-4(1H)-one

[Image Omitted]

Step 1: 4-chloro-N-(2,6-dichlorophenyl)-2-(methylthio)pyrimidine-5-carboxamide

To a solution of 2,6-dichloroaniline (41.02 g, 253 mmol) and pyridine (25.03 g, 316 mmol) In CH₂Cl₂(400 mL) a solution of 4-chloro-2-(methylthio)pyrimidine-5-carbonyl chloride (56.5 g, 253 mmol) in CH₂Cl₂(400 mL) was added dropwise at 0° C. The reaction mixture was stirred at RT for 2 days, the solvent was evaporated in vacuo and the remaining residue was washed with water twice, followed by 10% sq. HCl and water. The crude product was purified by recrystallization from EtOH to give 67.7 g (76.7%) of the title compound as a white solid. LCMS (Method B): R_T=1.53 min, m=348/350 [M+H]⁺.

Step 2: N-(2,6-dichlorophenyl)-4-(methylamino)-2-(methylthio)pyrimidine-5-carboxamide

To a solution of 4-chloro-N-(2,6-dichlorophenyl)-2-(methylthio)pyrimidine-5-carboxamide (70 g, 201 mmol) in THF (1 L) a solution of MeNH₂(40% in water, 46.74 g, 603 mmol) was added at 0° C. The resulting reaction mixture was stirred at room temperature overnight and the solvent was evaporated in vacuo. The remaining residue washed with water twice and dried to yield 68.35 g (99%) of the title compound as a white solid. LCMS (Method A): R_T=1.21 min, m/z=343/345 [M+H]⁺.

Step 3: 3-(2,6-dichlorophenyl)-1-methyl-7-(methylthio)-2,3-dihydropyrimido[4,5-d]pyrimidin-4(1H)-one

N-(2,6-Dichlorophenyl)-4-(methylamino)-2-(methylthio)pyrimidine-5-carboxamide (131 g, 382 mmol) was suspended in anhydrous acetonitrile (1.5 L), using a 5 L RB flask, and cesium carbonate (203.3 g, 1190 mmol) was added, followed by dibromomethane (132.7 g, 763 mmol). The mixture was heated to 80° C. while stirring for 7 days and the resulting mixture was filtered. The residue was washed with two portions of hot acetonitrile (200 mL) and the combined filtrates were concentrated in vacuo. The crude product was purified by crystallization from MeCN to yield 59.6 g (44%) of the title compound as a pale yellow solid. LCMS (Method A): R_T=1.26 min, m/z=355/357 [M+H]⁺.

Step 4: tert-butyl 4-(4-((6-(2,6-dichlorophenyl)-8-methyl-5-oxo-5,6,7,8-tetrahydropyrimido[4,5-d]pyrimidin-2-yl)amino)-2-methylphenyl)piperidine-1-carboxylate

To 6-(2,6-dichlorophenyl)-8-methyl-2-methylsulfanyl-7H-pyrimido[4,5-d]pyrimidin-5-one (10.0 g, 28 mmol) in DCM (440 ml) was charged a solution of 3-chloroperoxybenzoic acid (7.63 g, 31 mmol) in DCM (75 ml) at RT and the reaction was stirred for two hours. To the reaction mixture was added aq. Na₂S20 and the layers were separated using a phase separator. The organic layer was concentrated to dryness and tert-butyl 4-(4-amino-2-methylphenyl)piperidine-1-carboxylate (8.17 g, 28 mmol) and 2-propanol (625 mL) were added to the residue. The reaction was then heated at 95° C. overnight. The reaction was concentrated and the residue purified by flash chromatography (Biotage 340 g KP-Sil; 10-45% EtOAc in cyclohexane) to the title compound (11.13 g, 66%) as a white solid. LCMS (Method A): R_T1.86 min, m/z=597/599 [M+H]⁺

Step 5: 3-(2,6-dichlorophenyl)-1-methyl-7-((3-methyl-4-(piperidin-4-yl)phenyl)amino)-2,3-dihydropyrimido[4,5-d]pyrimidin-4(1H)-one hydrochloride

tert-butyl 4-[4-[[6-(2,6-dichlorophenyl)-8-methyl-5-oxo-7H-pyrimido[4,5-d]pyrimidin-2-yl]amino]-2-methyl-phenyl]piperidine-1-carboxylate (7.0 g. 11.7 mmol) was dissolved in DCM (300 mL) and 4N HCl in dioxane (35 mL, 140 mmol) was added and the mixture was stirred overnight at RT. The solid was collected by filtration through a sintered funnel and washed with DCM and dried on the sinter to afford the title compound (6.3 g, quant. yield). LCMS (Method A): R_T0.80 min, m/z=497/499 [M+H]⁺

Step 6: 3-(2,6-dichlorophenyl)-1-methyl-7-((3-methyl-4-(1-methylpiperidin-4-yl)phenyl)amino)-2,3-dihydropyrimido[4,5-d]pyrimidin-4(1H)-one

To a suspension of 6-(2,6-dichlorophenyl)-8-methyl-2-[3-methyl-4-(4-piperidyl)anilino]-7H-pyrimido[4,5-d]pyrimidin-5-one hydrochloride (6.77 g, 12.7 mmol) in acetonitrile (90 mL) was added methanol (20 mL) and 37% aqueous formaldehyde solution (12.76 mL, 63.4 mmol). The resulting solution was cooled to 0° C. and sodium triacetoxyborohydride (13.44 g, 63.4 mmol) was added portion wise. The reaction was stirred at 0° C. for 1 hr and subsequently the solvent was concentrated in vacuo. The residue was taken up in DCM and washed with 2M aq.Na₂CO₃, water and brine after which the biphasic mixture was passed through a phase separator and the resulting organic phase was concentrated to obtain a solid. Recrystallisation from ethyl acetate afforded the title compound (3.83 g, 59%) as a white solid. LCMS (Method A): R_T0.70 min, m/z=511/513 [M+H]⁺. ¹H NMR (500 MHz, DMSO-d₆): δ 9.75 (br s, 1H), 8.47 (s, 1H), 7.65 (d, 2H), 7.52-7.62 (m, 2H), 7.46-7.51 (m, 1H), 7.14 (d, 1H), 4.98 (s, 2H), 3.12 (s, 3H), 2.79-2.91 (m, 2H), 2.55-2.62 (m, 1H), 2.28 (s, 3H), 2.20 (s, 3H), 1.93-2.03 (m, 2H), 1.58-1.68 (m, 4H).

Comparative Example 1: 3-(2-chloro-6-methylphenyl)-1-methyl-7-((3-methyl-4-(1-methylpiperidin-4-yl)phenyl)amino)-2,3-dihydropyrimido[4,5-d]pyrimidin-4(1H)-one

[Image Omitted]

Step 1: 4-chloro-N-(2-chloro-6-methylphenyl)-2-(methyltho)pyrimidine-5-carboxamide

2-chloro-N-methylaniline (14 g, 62.8 mmol) was converted according to example 1 step 1 to result in the title compound (10 g; 30.5 mmol, 48%) as a white solid. LCMS (Method A): R_T=1.28 min, m/z=328/330 [M+H]⁺.

Step 2: N-(2-chloro-6-methylphenyl)-4-(methylamino)-2-(methylthio)pyrindine-5-carboxamide

4-Chloro-N-(2-chloro-6-methylphenyl)-2-(methylthio)pyrimidine-5-carboxamide (7 g, 21.33 mmol) was converted according to example 1 step 2 to result in the title compound (6 g; 18.59 mmol, 87%) as an off-white solid. LCMS (Method A): R_T=1.22 min, m/z=323/325 [M+H]⁺. The analytical data is in accordance with Example 92, step 1 as described in WO2015/092431.

Step 3: 3-(2-chloro-6-methylphenyl)-1-methyl-7-(methylthio)-2,3-dihydropyrimido[4,5-d]pyrimidin-4(1H)-one

N-(2-chloro-6-methylphenyl)-4-(methylamino)-2-(methylthio)pyrimidine-5-carboxamide (6 g, 18.59 mmol) was converted according to example 1 step 2 to result in the title compound (2.9 g; 8.66 mmol, 47%) as an off-white solid. LCMS (Method A): R_T=1.25 min, m/z=335/337 [M+H]⁺. The analytical data is in accordance with Example 92, step 2 as described in WO2015/092431.

Step 4: tert-butyl 4-(4-((6-(2-chloro-6-methylphenyl)-8-methyl-5-oxo-5,6,7,8-tetrahydropyrimido[4,5-d]pyrimidin-2-yl)amino)-2-methylphenyl)piperadine-1-carboxylate

3-(2-chloro-6-methylphenyl)-1-methyl-7-(methylthio)-2,3-dihydropyrimido[4,5-d]pyrimidin-4(1H)-one (150 mg, 0.45 mmol) was dissolved in DCM (6 mL) and mCPBA (121 mg, 0.49 mmol) was added. The reaction mixture was stirred for 30 min. at RT. The reaction mixture was diluted with DCM to 10 mL and washed with aq. thiosulfate solution. The organic layer was separated, dried (anh. MgSO₄) and the solvent was evaporated under reduced pressure. The residue was suspended in 2-propanol (6 mL) and tert-butyl 4-(4-amino-2-methylphenyl)piperidine-1-carboxylate (130 mg, 0.45 mmol) was added. The reaction mixture was stirred at 90° C. overnight. The volatiles were evaporated and the product was purified by flash chromatography (20-80% EtOAc in cyclohexane) yielding the title compound as a clear oil (206 mg, 80%). LCMS (Method B): R_T=1.80 min, m/z=577 [M+H]⁺.

Step 5: 3-(2-chloro-6-methylphenyl)-1-methyl-7-((3-methyl-4-(piperidin-4-yl)phenyl)amino)-2,3-dihydropyrimido[4,5-d]pyrimidin-4(1H)-one hydrochloride

tert-Butyl 4-[4-[[6-(2-chloro-6-methyl-phenyl)-8-methyl-5-oxo-7H-pyrimido[4,5-d]pyrimidin-2-yl]amino]-2-methyl-phenyl]piperidine-1-carboxylate (206 mg, 0.36 mmol) was dissolved in DCM (4 mL) and TFA (2 mL) was added. The reaction mixture was stirred for 30 min at RT. The volatiles were evaporated under reduced pressure and the residue was dissolved in DCM (2 mL) and loaded onto an SCX-2-cartridge (5 g; column eluted with DCM prior to use). The cartridge was washed with 20% MeOH in DCM and the desired compound was eluted with 20% 7N NH₃MeOH in DCM. The product containing fractions were evaporated under reduced pressure yielding the title compound as a white solid (138 mg, 81%). LCMS (Method A): R_T=0.80 min, m/z=477 [M+H]⁺.

Step 6: 3-(2-chloro-6-methylphenyl)-1-methyl-7-((3-methyl-4-(1-methylpiperidin-4-yl)phenyl)amino)-2,3-dihydropyrimido[4,5-d]pyrimidin-4(1H)-one

6-(2-chloro-6-methyl-phenyl)-8-methyl-2-[3-methyl-4-(4-piperidyl)anilino]-7H-pyrimido[4,5-d]pyrimidin-5-one (70 mg, 0.15 mmol) was dissolved in MeCN (3 mL)/MeOH (1 mL) mixture and 37% aqueous formaldehyde solution (0.11 mL, 1.47 mmol) was added followed by sodium triacetoxy borohydride (311 mg, 1.47 mmol). The reaction mixture was stirred at RT overnight. The volatiles were evaporated under reduced pressure and the residue was dissolved in DCM/1M NaOH (5 mL/5 mL). The organic layer was separated and the aqueous phase was extracted with DCM (2×5 mL). The organic extracts were combined, dried (anh. K₂C %₃) and evaporated under reduced pressure. The product was lyophilised (MeOH/MeCN/water) yielding the title compound as a white solid (58 mg, 80%). LCMS (Method B): R_T=0.79 min, m/z=491 [M+H]⁺. ¹H NMR (500 MHz, DMSO-d₆) δ 9.70 (s, 1H), 8.45 (s, 1H), 7.63-7.52 (m, 2H), 7.48-7.41 (m, 1H), 7.38-7.28 (m, 2H), 7.13 (d, 1H), 4.98-4.88 (m, 2H), 3.11 (s, 3H), 2.87 (d, 2H), 2.62-2.53 (m, 1H), 2.27 (s, 3H), 2.26 (s, 3H), 2.19 (s, 3H), 2.03-1.92 (m, 2H), 1.70-1.56 (m, 4H).

Comparison of the Compound of the Present Invention with Prior Art Compounds and Other Compounds:

Compound as described in Example 1 was tested for Wee-1 kinase Inhibitory effect (Wee-1 IC₅₀value), Wee-1 potency in cells (pCDC2 value), kinetic solubility, permeability in caco-2 cells (Caco-2 P_appvalue), in vivo plasma clearance (CL value) and elimination half-life (T_1/2value) in rat after intravenous administration, plasma exposure in rat after oral administration (AUC value), oral bioavailability in rat (F % value), brain plasma concentration ratio in healthy mouse, tumor:plasma concentration ratio in tumor-bearing mouse, in-vivo CYP3A4/5 time-dependent inhibition (CYP3A4/5 IC₅₀shift value) and detection of reactive metabolites in human hepatocytes. Comparative compounds were also tested using the same tests. The test methods employed to determine Wee-1 kinase inhibitory effect, Wee-1 potency in cells and kinetic solubility were the same as those taught in WO 2015/092431 (see page 350 to 358). The other test methods employed were as follows:

- i. Determination of Caco-2 permeability at pH 6.5 (pH7.4 for Comparative Example 6) was carried out at Cyprotex Ltd using standard protocols and SOPs.
- ii. Determination of pharmacokinetic parameters (CL/T_1/2/AUC/F %) in male Sprague-Dawley rats on Example 1 and Comparative Examples 6 and 165 was carried out at Axis BioServices Ltd using standard protocols and SOPs. Dosing was as follows: single oral dose 5 mg/kg; single Intravenous dose 1 mg/kg.
- iii. Determination of pharmacokinetic parameters (CL/T_1/2/AUC/F %) in male Sprague-Dawley rats on Comparative Example 18 was carried out at BioDuro using standard protocols and SOPs. Dosing was as follows: oral dosing 5 mg/kg; intravenous dosing 1 mg/kg
- iv. Determination of brain:plasma concentration ratio in the healthy mouse was carried out at Bioduro Inc. (Example 1) and at Axis Bioservices Ltd (Comparative Example 18) using standard protocols and SOPs. Dosing was as follows: repeated oral dose of 30 mg/kg, once a day for 7 days. Concentrations were measured at 4 h post dose on day 7.
- v. Determination of tumor-plasma concentration ratio in the tumor-bearing mouse was carried out at Axis Bioservices Ltd using standard protocols and SOPs. Dosing was as follows: single oral dose of 30 mg/kg.
- vi. Determination of CYP3A4/5 time-dependent inhibition (CYP3A4/5 IC₅₀shift value) was carried out at Cyprotex Ltd using standard protocols and SOPs. The in-vitro test system was: pooled human liver microsomes; a 30-min preincubation step with and without NADPH was included to observe the IC₅₀shift.
- vii. Determination of the formation of reactive metabolites was carried out at Admescope Ltd using standard protocols and SOPs. The in-vitro test system was: human hepatocytes; the duration of incubation was: 2 hours; the technique for metabolite analysis was: LC-MS/MS.

The compound described in Example 1 presents an improved chemical stability in solution (Buffer at pH 7.4 and in DMSO). It also shows an improved solubility in a fasted state simulated gastric fluid (FaSSGF).

The data is provided in Table 1a and 1b below:

TABLE 1a

Comparison of Examples 6, 18, 95, 165, 178 and 183 of WO 2015/092431, and Comparative Example 1, with Example 1 of the present invention