Patent Description:
AZD5305 (<NUM>-[<NUM>-[(<NUM>-ethyl-<NUM>-oxo-<NUM>-<NUM>,<NUM>-naphthyridin-<NUM>-yl)methyl]piperazin-<NUM>-yl]-N-methylpyridine-<NUM>-carboxamide) is a small molecule drug that acts by selectively inhibiting and trapping PARP1 at the sites of DNA single cell breaks (SSB). This both prevents the DNA repair and, during DNA replication, leads to the generation of the more deleterious DNA double strand breaks (DSB), when the DNA replication machinery collides with the PARP1-DNA non-covalent complexes. In contexts where the accurate DNA repair pathways are effective, such as in cells with a proficient homologous recombination repair (HRR), DSBs are accurately repaired. In contrast, in cells with deficiencies in the repair pathways, such as those with deleterious mutation in the BRCA genes, AZD5305 treatments lead to selective accumulation of genome instability which ultimately selectively kill cancer cells, while sparing normal cells.

Platinum chemotherapeutic agents are drugs used to treat cancer, frequently in the first line treatment setting. Examples of platinum chemotherapeutic agents include cisplatin, oxaliplatin, and carboplatin.

<NPL>) teaches the identification of amentoflavone as a potent highly selective PARP-<NUM> inhibitor and its potentiation on carboplatin in human non-small cell lung cancer.

The present invention is directed to AZD5305, or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer in a subject, wherein said treatment comprises the separate, sequential or simultaneous administration of i) said AZD5305, or a pharmaceutically acceptable salt thereof, and ii) a platinum chemotherapeutic agent, or a pharmaceutically acceptable salt thereof, to said subject.

The invention is further directed to a pharmaceutical product comprising i) AZD5305 or a pharmaceutically acceptable salt thereof, and ii) a platinum chemotherapeutic agent or a pharmaceutically acceptable salt thereof.

In some embodiments, the cancer is ovary, breast, pancreas, prostate, hematological, gastrointestinal such as gastric and colorectal, or lung cancer.

In some embodiments the cancer is homologous recombination deficient (HRD) cancer. For example, whether the cancer is HRD positive can be determined by Myriad Genetics myChoice® HRD or myChoice® HRD Plus assay.

In certain embodiments, the cancer cells comprise HRD gene mutation selected from BRCA1, BRCA2, ATM, BRIP1, BARD1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, and RAD54L gene mutation. In certain embodiments, the cancer cells comprise a BRCA1, a BRCA2, and/or an ATM gene mutation. In certain embodiments, the cancer cells comprise a BRCA1 and/or a BRCA2 gene mutation. For example, in certain embodiments, the cancer cells comprise a tBRCA gene mutation.

In certain embodiments, the cancer comprises homologous recombination deficiency (HRD)-positive status defined by a deleterious or suspected deleterious BRCA mutation and/or genomic instability.

In certain embodiments, the cancer is ovarian cancer or breast cancer. In certain embodiments, the cancer is ovarian cancer. In certain embodiments, the cancer is advanced epithelial ovarian cancer. In certain embodiments, the cancer is high-grade serous ovarian cancer. In certain embodiments, the cancer is high-grade endometrioid ovarian cancer. In certain embodiments, the cancer is epithelial ovarian cancer comprising a gBRCA1 or a gBRCA2 mutation. In certain embodiments, the cancer is fallopian tube cancer. In certain embodiments, the cancer is primary peritoneal cancer.

In certain embodiments, the cancer is ovarian (such as advanced epithelial ovarian), fallopian tube, or primary peritoneal cancer.

In certain embodiments, the cancer is ovarian (such as advanced epithelial ovarian), fallopian tube, or primary peritoneal cancer, the cancer comprising homologous recombination deficiency (HRD)-positive status defined by a deleterious or suspected deleterious BRCA mutation and/or genomic instability.

In certain embodiments, the cancer is breast cancer. In certain embodiments the cancer is triple negative breast cancer.

In some embodiments, the cancer is platinum-resistant.

In some embodiments, disclosed is a kit comprising a first pharmaceutical composition comprising AZD5305 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier; and a second pharmaceutical composition comprising a platinum chemotherapeutic agent or a pharmaceutically acceptable salt thereof and instructions for use.

The references to methods of treatment in the detailed description of the invention in this description are to be interpreted as references to the compounds, pharmaceutical compositions and medicaments of the present invention for use in a method for treatment of the human (or animal) body by therapy.

In some embodiments, disclosed is a method of treating cancer by a combination therapy of AZD5305 and a platinum chemotherapeutic agent. In some embodiments, the method comprises administering to a subject in need thereof a first amount of AZD5305 or a pharmaceutically acceptable salt thereof and a second amount of a platinum chemotherapeutic agent or a pharmaceutically acceptable salt thereof, wherein the first amount and the second amount together comprises a therapeutically effective amount. In some embodiments, the platinum chemotherapeutic agent includes any one of carboplatin, cisplatin and oxaliplatin. In some embodiments, the platinum chemotherapeutic agent includes carboplatin. In an embodiment, the platinum chemotherapeutic agent or a pharmaceutically acceptable salt thereof is administered first, and AZD5305 or a pharmaceutically acceptable salt thereof is administered second. In an embodiment, AZD5305 or a pharmaceutically acceptable salt is administered first, and the platinum chemotherapeutic agent or a pharmaceutically acceptable salt is administered second.

The term "AZD5305" refers to a compound with the chemical name of <NUM>-[<NUM>-[(<NUM>-ethyl-<NUM>-oxo-<NUM>-<NUM>,<NUM>-naphthyridin-<NUM>-yl)methyl]piperazin-<NUM>-yl]-N-methyl-pyridine-<NUM>-carboxamide and structure shown below:
<CHM>.

Preparation of AZD5305 is disclosed herein (see Example <NUM>). In some embodiments, a free base of AZD5305, is administered to a subject. In some embodiments, a pharmaceutically acceptable salt of AZD5305 is administered to a subject. In some embodiments, a crystalline AZD5305 is administered to a subject. In some embodiments, crystalline Form A AZD5305 is administered to a subject.

The term "platinum-containing chemotherapeutic agent" includes drugs that contain the metal platinum, such as cisplatin, carboplatin, and oxaliplatin.

In some embodiments, AZD5305 or a pharmaceutically acceptable salt thereof and a platinum chemotherapeutic agent or a pharmaceutically acceptable salt thereof are administered separately, sequentially or simultaneously. In an embodiment, the platinum chemotherapeutic agent or a pharmaceutically acceptable salt thereof is administered first, and AZD5305 or a pharmaceutically acceptable salt thereof is administered second. In an embodiment, AZD5305 or a pharmaceutically acceptable salt is administered first, and the platinum chemotherapeutic agent or a pharmaceutically acceptable salt is administered second.

In some embodiments, AZD5305 or a pharmaceutically acceptable salt thereof and a platinum chemotherapeutic agent or a pharmaceutically acceptable salt thereof are administered separately, sequentially or simultaneously in a treatment cycle.

A "cycle", "treatment cycle" or "dosing schedule", as used herein, refers to a period of combination treatment that is repeated on a regular schedule. For example, the treatment can be given for one week, two weeks, or three weeks wherein AZD5305 and a platinum chemotherapeutic agent are administered in a coordinated fashion. In some embodiments, a treatment cycle is about <NUM> week to about <NUM> months. In some embodiments, a treatment cycle is about <NUM> days to about <NUM> month. In some embodiments, a treatment cycle is about <NUM> week to about <NUM> weeks. In some embodiments, a treatment cycle is about <NUM> week, about <NUM> days, about <NUM> weeks, about <NUM> weeks, about <NUM> weeks, about <NUM> months, or about <NUM> months.

In some embodiments, AZD5305 or a pharmaceutically acceptable salt thereof and a platinum chemotherapeutic agent or a pharmaceutically acceptable salt thereof are administered to the human subject in one or more treatment cycles, e.g., a treatment course. A "treatment course" comprises multiple treatment cycles, which can be repeated on a regular schedule, or adjusted as a tapered schedule as the patient's disease progression is monitored. For example, a patient's treatment cycles can have longer periods of treatment and/or shorter periods of rest at the beginning of a treatment course (e.g., when the patient is first diagnosed), and as the cancer enters remission, the rest period lengthens, thereby increasing the length of one treatment cycle. The period of time for treatment and rest in a treatment cycle, the number of treatment cycles, and the length of time for the treatment course can be determined and adjusted throughout the treatment course by the skilled artisan based on the patient's disease progression, treatment tolerance, and prognosis. In some embodiments, the method comprises <NUM> to <NUM> treatment cycles. In some embodiments, the method comprises <NUM> to <NUM> treatment cycles.

In some embodiments, AZD5305 or a pharmaceutically acceptable salt thereof is administered orally. In some embodiments, AZD5305 or a pharmaceutically acceptable salt thereof is in capsule dosage form. In some embodiments, AZD5305 or a pharmaceutically acceptable salt thereof is in tablet dosage form.

The language "treat," "treating" and "treatment" includes the reduction or inhibition of enzyme or protein activity related to, PARP or cancer in a subject, amelioration of one or more symptoms of cancer in a subject, or the slowing or delaying of progression of cancer in a subject. The language "treat," "treating" and "treatment" also includes the reduction or inhibition of the growth of a tumor or proliferation of cancerous cells in a subject.

The language "inhibit," "inhibition" or "inhibiting" includes a decrease in the baseline activity of a biological activity or process.

The term "cancer" includes, but is not limited to a disease caused by an uncontrolled division of abnormal cells in a part of the body. In some embodiments, the cancer includes cancers that are susceptible to treatment with PARP inhibitors (e.g., AZD5305). In some embodiments, the cancer is ovarian cancer, breast cancer, pancreatic cancer, and prostate cancer. In some embodiments the cancer is hematological, gastrointestinal such as gastric and colorectal, or lung cancer. In some embodiments, the cancer is relapsed or refractory cancer. In some embodiments, the cancer is platinum-resistant cancer.

The language "pharmaceutical composition" includes compositions comprising an active ingredient and a pharmaceutically acceptable excipient, carrier or diluent, wherein the active ingredient is AZD5305 or a pharmaceutically acceptable salt thereof, or a platinum chemotherapeutic agent or a pharmaceutically acceptable salt thereof.

In this specification, unless otherwise stated, the term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions, salts, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The language "pharmaceutically acceptable excipient, carrier or diluent" includes compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, as ascertained by one of skill in the art. In some embodiments, the pharmaceutical compositions are in solid dosage forms, such as capsules, tablets, granules, powders, sachets, etc. In some embodiments, the pharmaceutical compositions are in the form of a sterile injectable solution in one or more aqueous or non-aqueous non-toxic parenterally-acceptable buffer systems, diluents, solubilizing agents, co-solvents, or carriers. A sterile injectable preparation may also be a sterile injectable aqueous or oily suspension or suspension in a non-aqueous diluent, carrier or co-solvent, which may be formulated according to known procedures using one or more of the appropriate dispersing or wetting agents and suspending agents. The pharmaceutical compositions could be a solution for iv bolus/infusion injection or a lyophilized system (either alone or with excipients) for reconstitution with a buffer system with or without other excipients. The lyophilized freeze-dried material may be prepared from non-aqueous solvents or aqueous solvents. The dosage form could also be a concentrate for further dilution for subsequent infusion.

The term "subject" includes warm-blooded mammals, for example, primates, dogs, cats, rabbits, rats, and mice. In some embodiments, the subject is a primate, for example, a human. In some embodiments, the subject is suffering from cancer, such as ovarian cancer, breast cancer, pancreatic cancer, and prostate cancer. In some embodiments the subject is suffering from cancer, such as hematological cancer, gastrointestinal such as gastric and colorectal cancer, or lung cancer. In some embodiments, the subject is suffering from ovarian cancer or breast cancer. In some embodiments, the subject is suffering from relapsed or refractory ovarian cancer. In some embodiments, the subject is suffering from relapsed or refractory breast cancer. In some embodiments, the subject is suffering from cancer and is treatment naïve (e.g., has never received treatment for cancer). In some embodiments, the subject is suffering from cancer and is platinum-resistant. Platinum-resistant disease is defined by progression within <NUM> months following the last administered platinum-based regimen. Platinumrefractory disease is defined by lack of at least a partial response while on platinum-containing regimens. A platinum-based regimen includes drugs that contain the metal platinum, such as cisplatin and carboplatin.

The language "therapeutically effective amount" includes that amount of AZD5305 and that amount of platinum chemotherapeutic agent which together will elicit a biological or medical response in a subject, for example, the reduction or inhibition of enzyme or protein activity related to PARP, or cancer; amelioration of symptoms of cancer; or the slowing or delaying of progression of cancer. In some embodiments, the language "therapeutically effective amount" includes the amount of AZD5305 and a platinum chemotherapeutic agent together that is effective to at least partially alleviate, inhibit, and/or ameliorate cancer or inhibit PARP, and/or reduce or inhibit the growth of a tumor or proliferation of cancerous cells in a subject. In some embodiments, the language "therapeutically effective amount" includes the amount of AZD5305 and a platinum chemotherapeutic agent together that is effective to at least partially reduce or inhibit the growth of a tumor or proliferation of cancerous cells in a subject.

In some embodiments, disclosed is a kit comprising: a first pharmaceutical composition comprising AZD5305 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier; and a second pharmaceutical composition comprising carboplatin or a pharmaceutically acceptable salt; and instructions for using the first and second pharmaceutical compositions in combination. In some embodiments, the first pharmaceutical composition comprises a first amount of AZD5305 or a pharmaceutically acceptable salt thereof, and the second pharmaceutical composition comprises a second amount of a platinum chemotherapeutic agent or a pharmaceutically acceptable salt thereof; wherein the first amount and the second amount together comprise a therapeutically effective amount.

The combination therapy disclosed herein will now be further explained by reference to the following non-limiting examples.

<NUM>H NMR spectra were obtained using a Bruker <NUM>, <NUM> or <NUM> spectrometer at <NUM> unless otherwise noted; chemical shifts are expressed in parts per million (ppm, δ units) and are referenced to the residual mono-<NUM>H isotopologue of the solvent (CHCl<NUM>: <NUM> ppm; CHDCl<NUM>: <NUM> ppm; CD<NUM>S(=O)CD<NUM>H: <NUM> ppm). Coupling constants are given in units of hertz (Hz). Splitting patterns describe apparent multiplicities and are designated as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and br s (broad singlet). LC-MS was carried out using a Waters UPLC fitted with a Waters SQD mass spectrometer or Shimadzu LC-20AD LC-20XR LC-30AD with a Shimadzu <NUM> mass spectrometer. Reported molecular ions correspond to [M+H]+ unless otherwise noted; for molecules with multiple isotopic patterns (Br, Cl, etc.) the reported value is the one obtained for the lowest isotope mass unless otherwise specified.

Flash chromatography was performed using straight phase flash chromatography on a SP1™ Purification system from Biotage™, CombiFlash®Rf from ISCO or on Gilson system from Thermo Fisher using normal phase silica FLASH+™ (<NUM>, <NUM> or <NUM>) or SNAP™ KP-Sil Cartridges (<NUM>, <NUM>, <NUM> or <NUM>), Flash Column silica-CS columns from Agela, with C18-flash columns or standard flash chromatography. In general, all solvents used were commercially available and of analytical grade. Anhydrous solvents were routinely used for reactions. Phase Separators used in the examples are ISOLUTE® Phase Separator columns. The intermediates and examples named below were named using ACD/Name <NUM> from Advanced Chemistry Development, Inc. (ACD/Labs). The starting materials were obtained from commercial sources or made via literature routes.

XRPD analysis was performed using a Bruker D8 diffractometer, which is commercially available from Bruker AXS Inc™ (Madison, Wisconsin). The XRPD spectra were obtained by mounting a sample (approximately <NUM>) of the material for analysis on a single silicon crystal wafer mount (e.g., a Bruker silicon zero background X-ray diffraction sample holder) and spreading out the sample into a thin layer with the aid of a microscope slide. The sample was spun at <NUM> revolutions per minute (to improve counting statistics) and irradiated with X-rays generated by a copper long-fine focus tube operated at <NUM> kV and <NUM> mA with a wavelength of <NUM> angstroms (i.e., about <NUM> angstroms). The sample was exposed for <NUM> second per <NUM> degree <NUM>-theta increment (continuous scan mode) over the range <NUM> degrees to <NUM> degrees <NUM>-theta in theta-theta mode. The running time was ~<NUM> for D8.

XRPD 2θ values may vary with a reasonable range, e.g., in the range ± <NUM>° and that XRPD intensities may vary when measured for essentially the same crystalline form for a variety of reasons including, for example, preferred orientation. Principles of XRPD are described in publications, such as, for example,<NPL>;<NPL>; and <NPL>.

The following abbreviations are used: aq = aqueous; CH2Cl2 = dichloromethane; DCM = dichloromethane; DDQ = <NUM>,<NUM>-Dichloro-<NUM>,<NUM>-dicyano-<NUM>,<NUM>-benzoquinone; DIPEA = N,N-diisopropylethylamine; DMSO = dimethylsulfoxide; DMSO-d<NUM> = deuterated dimethylsulfoxide; ESI = electrospray ionization; MeCN or CH3CN = acetonitrile; NMR = nuclear magnetic resonance; Pd/C = Palladium on carbonTFA = trifluoroacetic acid; THF = tetrahydrofuran; TLC = thin layer chromatography; HCl = Hydrochloric acid; HBr= Hydrobromic acid; Cs2CO3 = Cesium carbonate; MgSO4 = Magnesium sulfate; NaHCO3 = Sodium bicarbonate; SOCl2 = Thionyl chloride; NH<NUM>Cl = ammonium chloride; Na<NUM>SO<NUM> = sodium sulfate; H2 = hydrogen gas.

A mixture of ethyl <NUM>-methyl-<NUM>-nitro-pyridine-<NUM>-carboxylate (intermediate <NUM>, <NUM>, <NUM> mmol) and selenium dioxide (<NUM>, <NUM> mmol) in <NUM>,<NUM>-dioxane (<NUM>) was stirred at <NUM> for <NUM>. The reaction mixture was cooled to room temperature, filtered through a pad of celite and the celite was washed with ethyl acetate. The combined filtrate was concentrated, and the resulting residue was purified by flash silica chromatography, elution gradient <NUM> to <NUM>% ethyl acetate in hexanes. Product fractions were concentrated under reduced pressure to afford ethyl <NUM>-formyl-<NUM>-nitro-pyridine-<NUM>-carboxylate (Intermediate <NUM>, <NUM>, <NUM> %) as a brown oil. <NUM> NMR (<NUM>, CHLOROFORM-d) <NUM> (<NUM>, t), <NUM> (<NUM>, q), <NUM> (<NUM>, d), <NUM> (<NUM>, d), <NUM> (<NUM>, s); m/z (ES+) [M]+ = <NUM>.

To a stirred solution of sodium hydride (<NUM>, <NUM> mmol) (<NUM>% in mineral oil) in anhydrous THF (<NUM>) was added ethyl <NUM>-(diethoxyphosphoryl)butanoate (<NUM>, <NUM> mmol) dropwise with an addition funnel at <NUM> to give a grey colored mixture. The resulting mixture was stirred at <NUM> for <NUM> and warmed to room temperature over <NUM> minutes and stirred at <NUM> for <NUM> minutes. The reaction mixture was cooled to -<NUM> and to this cooled reaction mixture was then slowly added solution of ethyl <NUM>-formyl-<NUM>-nitro-pyridine-<NUM>-carboxylate (Intermediate <NUM>, <NUM>, <NUM> mmol) in <NUM> THF. The mixture was quenched with sat. NH<NUM>Cl solution, extracted with ethyl acetate. The combined the organic layers were dried over sodium Na<NUM>SO<NUM>, filtered and concentrated to give crude product. the resulting residue was purified by flash silica chromatography, elution gradient <NUM> to <NUM>% ethyl acetate in hexanes. Product fractions were concentrated under reduced pressure to afford ethyl <NUM>-[(E)-<NUM>-ethoxycarbonylbut-<NUM>-enyl]-<NUM>-nitropyridine-<NUM>-carboxylate (Intermediate <NUM>, <NUM>, <NUM> %) as a yellow oil (<NUM>:<NUM> and mixture of E/Z isomer). <NUM> NMR (<NUM>, CHLOROFORM-d) <NUM> (<NUM>, t), <NUM> (<NUM>, t), <NUM> (<NUM>, t), <NUM> (<NUM>, t), <NUM> (<NUM>, q), <NUM> (<NUM>, qd), <NUM> (<NUM>, q), <NUM> - <NUM> (<NUM>, m), <NUM> (<NUM>, q), <NUM> - <NUM> (<NUM>, m), <NUM> (<NUM>, s), <NUM> (<NUM>, s), <NUM> (<NUM>, dd), <NUM> (<NUM>, d), <NUM> (<NUM>, d); m/z (ES+) [M]+ = <NUM>.

A mixture of ethyl <NUM>-[(E)-<NUM>-ethoxycarbonylbut-<NUM>-enyl]-<NUM>-nitro-pyridine-<NUM>-carboxylate (<NUM>:<NUM> mixture of E/Z isomers) (Intermediate <NUM>, <NUM>, <NUM> mmol), Pd/C (<NUM>, <NUM> mmol) (<NUM>%) in ethanol (<NUM>) was degassed, filled up with H2 (balloon), and the reaction was stirred at room temperature for overnight under H2 atmosphere. The mixture was filtered through a celite bed and the celite bed washed with ethanol. After concentration, <NUM> HCl in dioxanes (<NUM>) was added to the resulting residue and the mixture was stirred at room temperature for <NUM>. The mixture was diluted with ether and the solid was filtered off, washed with diethyl ether and dried under vacuum to afford ethyl <NUM>-ethyl-<NUM>-oxo-<NUM>,<NUM>-dihydro-<NUM>-<NUM>,<NUM>-naphthyridine-<NUM>-carboxylate (Intermediate <NUM>, <NUM>, <NUM> %) as a white solid. <NUM> NMR (<NUM>, DMSO-d6) <NUM> (<NUM>, t), <NUM> (<NUM>, t), <NUM> - <NUM> (<NUM>, m), <NUM> - <NUM> (<NUM>, m), <NUM> - <NUM> (<NUM>, m), <NUM> (<NUM>, dd), <NUM> (<NUM>, dd), <NUM> (<NUM>, t), <NUM> (<NUM>, d), <NUM> (<NUM>, d), <NUM> (<NUM>, s); m/z (ES+) [M+H]+ = <NUM>.

Ethyl <NUM>-ethyl-<NUM>-oxo-<NUM>,<NUM>-dihydro-<NUM>-<NUM>,<NUM>-naphthyridine-<NUM>-carboxylate (Intermediate <NUM>, <NUM>, <NUM> mmol) was dissolve into <NUM>,<NUM>-dioxane (<NUM>), DDQ (<NUM>, <NUM> mmol) was added and the mixture was stirred at reflux for <NUM>. Solvent was removed under reduced pressure, sat. NaHCO<NUM> solution was added and the residue stirred at room temperature for <NUM> hr. The solid was filtered off, washed with water followed by <NUM> of diethyl ether. The resulting solid was dried under vacuum afford ethyl <NUM>-ethyl-<NUM>-oxo-<NUM>-<NUM>,<NUM>-naphthyridine-<NUM>-carboxylate (Intermediate <NUM>, <NUM>, <NUM> %) as a light brown solid.

<NUM> NMR (<NUM>, DMSO-d6) <NUM> - <NUM> (<NUM>, m), <NUM> (<NUM>, t), <NUM> (<NUM>, q), <NUM> (<NUM>, q), <NUM> (<NUM>, s), <NUM> (<NUM>, s), <NUM> (<NUM>, s), <NUM> (<NUM>, s); m/z (ES+) [M+H]+ = <NUM>.

Lithium aluminum hydride, <NUM> in THF (<NUM>, <NUM> mmol) was added dropwise to ethyl <NUM>-ethyl-<NUM>-oxo-<NUM>-<NUM>,<NUM>-naphthyridine-<NUM>-carboxylate (Intermediate <NUM>, <NUM>, <NUM> mmol) in tetrahydrofuran (<NUM>) at <NUM> over a period of <NUM> minutes under nitrogen. The resulting mixture was stirred at <NUM> for <NUM> hours. The reaction mixture was quenched by dropwise addition of <NUM> aq HCl (<NUM>). The reaction mixture was concentrated and the solid was diluted with water (~ <NUM>) and <NUM> of <NUM> HCl solution gave a yellow suspension. The solid was collected by filtration, washed with water, diethyl ether and dried to yield the crude product as a yellow solid (contaminated by some inorganic salt). This solid was suspended in a mixture of methanol and DCM (<NUM>:<NUM>) (<NUM>) and heated to reflux. The solid was filtered off. This solid was resuspended in methanol/DCM mixture and repeated this procedure <NUM> times to get most of the product out from this mixture. The combined filtrate was then concentrated until about <NUM> and the solid was collected by filtration, washed with ether, dried under vacuum to yield <NUM>-ethyl-<NUM>-(hydroxymethyl)-<NUM>-<NUM>,<NUM>-naphthyridin-<NUM>-one (Intermediate <NUM>, <NUM>, <NUM> %) as yellow solid. <NUM> NMR (<NUM>, DMSO-d6) <NUM> (<NUM>, t), <NUM> - <NUM> (<NUM>, m), <NUM> (<NUM>, d), <NUM> (<NUM>, t), <NUM> (<NUM>, s), <NUM> (<NUM>, s), <NUM> (<NUM>, s), <NUM> (<NUM>, br s); m/z (ES+) [M+H]+ = <NUM>.

Thionyl chloride (<NUM>, <NUM> mmol) was added dropwise to a suspension of <NUM>-ethyl-<NUM>-(hydroxymethyl)-<NUM>,<NUM>-naphthyridin-<NUM>(<NUM>)-one (Intermediate <NUM>, <NUM>, <NUM> mmol) and N,N-dimethylformamide (<NUM>, <NUM> mmol) in CH2CI2 (<NUM>) at <NUM> and the resulting solution was stirred at room temperature for <NUM> hours. The mixture was concentrated to dryness to give crude <NUM>-(chloromethyl)-<NUM>-ethyl-<NUM>-<NUM>,<NUM>-naphthyridin-<NUM>-one.

DIPEA (<NUM>, <NUM> mmol) was added to a stirred solution of <NUM>-(chloromethyl)-<NUM>-ethyl-<NUM>-<NUM>,<NUM>-naphthyridin-<NUM>-one (crude from above), potassium iodide (<NUM>, <NUM> mmol) and N-methyl-<NUM>-piperazin-<NUM>-yl-pyridine-<NUM>-carboxamide, 2HCl (Intermediate <NUM>, <NUM>, <NUM> mmol) in acetonitrile (<NUM>) at <NUM>. The resulting solution was stirred at <NUM> for <NUM> hours. Solvent was removed under vacuum. Crude material was diluted with water, basified with aq. NaHCO3 solution and extracted with ethyl acetate. Organic layer was dried over sodium sulphate and concentrated to give crude product. The resulting residue was purified by flash silica chromatography, elution gradient <NUM> to <NUM>% MeOH in DCM. Product fractions were concentrated under reduced pressure to afford <NUM>-[<NUM>-[(<NUM>-ethyl-<NUM>-oxo-<NUM>-<NUM>,<NUM>-naphthyridin-<NUM>-yl)methyl]piperazin-<NUM>-yl]-N-methyl-pyridine-<NUM>-carboxamide (Example <NUM>, <NUM>, <NUM> %) as an off white partially crystalline solid. <NUM> NMR (<NUM>, DMSO-d6) <NUM> (<NUM>, t), <NUM> - <NUM> (<NUM>, m), <NUM> (<NUM>, d), <NUM> - <NUM> (<NUM>, m), <NUM> (<NUM>, s), <NUM> (<NUM>, dd), <NUM> (<NUM>, s), <NUM> (<NUM>, s), <NUM> (<NUM>, d), <NUM> (<NUM>, d), <NUM> - <NUM> (<NUM>, m), <NUM> (<NUM>, d), <NUM> (<NUM>, s); m/z (ES+) [M]+ = <NUM>.

In Example <NUM>, <NUM>-[<NUM>-[(<NUM>-ethyl-<NUM>-oxo-<NUM>-<NUM>,<NUM>-naphthyridin-<NUM>-yl)methyl]piperazin-<NUM>-yl]-N-methylpyridine-<NUM>-carboxamide was obtained as a partially crystalline solid by evaporating a methanol/dichloromethane solution under reduced pressure. The crystalline material soobtained was characterised as crystalline Form A.

In the case of poor crystallinity, crystalline Form A was obtainable by suspending <NUM> of the crude sample in <NUM> of water, methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, ethyl acetate or other solvent for <NUM> day at the ambient temperature or <NUM>.

Form A was analysed by XRPD and the results are tabulated below (Table <NUM>) and shown in <FIG>.

Form A is characterized in providing at least one of the following <NUM>θ values measured using CuKα radiation: <NUM>, <NUM>, and <NUM>°.

This in vivo study was carried out to investigate the anti-tumour effects of AZD5305 in combination with carboplatin (a platinum chemotherapeutic agent) in a BRCA wild type tumour model, HBCx-<NUM>, in vivo. AZD5305 monotherapy and carboplatin monotherapy arms were included in the study design as controls.

HBCx-<NUM> is a patient-derived tumour explant (PDX) model established in XenTech without prior in vitro culture. Tumour fragments were transplanted subcutaneously (SC) onto donor mice. When tumour volume reached <NUM> to <NUM><NUM>, donor mice were sacrificed, and tumours were aseptically excised and dissected. After removing necrotic areas, tumours were cut into fragments measuring approximately <NUM><NUM> and transferred in culture medium before grafting SC into the recipient (experimental) Nude female mice. Tumours were measured (length x width) two times weekly by bilateral Vernier calliper measurements and tumour volume was calculated using elliptical formula (π/<NUM>×width×width×length). Animal bodyweight and tumour condition were monitored throughout the study. Mice were randomised into treatment groups when mean tumour volume reached approximately <NUM><NUM>. Animals were treated from the day after the randomisation. Control animals were treated orally (PO) once daily (QD) with vehicle (deionised water acidified with HCl to pH <NUM>-<NUM>). AZD5305 was dosed PO QD at <NUM>/kg. Carboplatin was dosed intraperitoneally (IP) once weekly (QW) at <NUM>/kg. In the combination group, mice were dosed with carboplatin first, followed by AZD5305 administration within <NUM> minutes.

Tumour growth inhibition from start of treatment was assessed by comparison of the mean change in tumour volume of the control and treated groups and represented as percent tumour growth inhibition (TGI, when TV ≥starting TV) or regression (reg, when TV <starting TV). Mean percent body weight change from start of treatment was also calculated for all the groups. Statistical significance was evaluated using a one-tailed t test. Statistical significance is indicated as follows: * p ≤<NUM>, ** p ≤<NUM>, *** p ≤<NUM>.

Tolerance studies using AZD5305 at <NUM>/kg PO QD in combination with carboplatin at <NUM>/kg IP QWwere run in SCID female mice prior to commencement of the efficacy study and showed no adverse effects to animal condition or significant changes to bodyweight.

AZD5305 was formulated for oral dosing in water/HCl pH3. <NUM>-<NUM> vehicle to be administered at <NUM>/<NUM> dose volume. Solid was diluted by adding appropriate volume of 1N HCl to about <NUM>:<NUM> drug/HCl molar ratio and <NUM>% of final volume of sterile deionized water with stirring/vortex/sonication until a solution was obtained. The required volume of 1N HCl was calculated according to the following equation: <MAT>.

The pH of the solution was adjusted to <NUM> to <NUM> with 1N HCl or 1N NaOH. The final volume was made up using sterile water for injections or deionized water and the final pH was measured and recorded. Dosing preparations were formulated once per week and protected from light. Carboplatin (Teva®) was formulated fresh on each day of dosing by diluting the stock solution in <NUM>% NaCl to <NUM>/ml for <NUM>/kg dose (<NUM>/<NUM> dose volume).

HBCx-<NUM> is a TNBC PDX model available in XenTech, France. It has been reported as BRCA wt model, with BRCA1 methylation [<NUM>]. HBCx-<NUM> was also shown to have a moderate sensitivity to olaparib monotherapy [<NUM>].

AZD5305 at <NUM>, <NUM>, and <NUM>/kg QD and carboplatin at <NUM>/kg QW were tested in the efficacy study as monotherapies and in combination. The treatment period lasted for <NUM> days. All treatments were well tolerated throughout the duration of the study and no significant body weight loss was observed (<FIG> &<NUM>). AZD5305 monotherapy showed dose-dependent anti-tumour efficacy, delivering <NUM>% TGI and <NUM>% TGI was observed in carboplatin monotherapy group. AZD5305 and carboplatin dosed together demonstrated clear combination benefit by delivering <NUM>% tumour regression (<FIG> and Table <NUM>). Moreover, all the animals in the combination group (<NUM>/<NUM>) reached a complete response (CR; defined as TV<<NUM><NUM>) (<FIG>).

In conclusion, AZD5305 in combination with carboplatin demonstrated improved anti-tumour effect compared to each monotherapy in HBCx-<NUM> PDX model in vivo. Combination of AZD5305 and carboplatin led to complete responses in <NUM>% of animals and was well tolerated in mice.

This study was carried out to investigate the anti-tumour effects of AZD5305 in combination with carboplatin in a BRCA1 mutant tumour model, SUM149PT, in vivo. AZD5305 monotherapy and carboplatin monotherapy arms were included in the study design as controls.

SUM149PT cells (<NUM> × <NUM><NUM>) were implanted with <NUM>% Matrigel into mammary fat pad (MFP) of female SCID mice (weight greater than <NUM>). Tumours were measured (length x width) two times weekly by bilateral Vernier calliper measurements and tumour volume was calculated using elliptical formula (π/<NUM>×width×width×length). Animal bodyweight and tumour condition were monitored throughout the studies. Mice were randomised into treatment groups when mean tumour volume reached approximately <NUM><NUM>. Animals were treated from the day after the randomisation. Control animals were treated orally (PO) once daily (QD) with vehicle (deionised water acidified with HCl to pH <NUM>-<NUM>) and intraperitoneally (IP) once weekly (QW) with PBS. AZD5305 was dosed PO QD at <NUM>, <NUM> or <NUM>/kg. Carboplatin was dosed IP once weekly (QW) at <NUM>/kg. In the combination group, mice were dosed with carboplatin first, followed by AZD5305 administration within <NUM> minutes.

Tumour growth inhibition from start of treatment was assessed by comparison of the mean change in tumour volume of the control and treated groups and represented as percent tumour growth inhibition (TGI, when TV ≥starting TV) or regression (reg, when TV <starting TV). Mean percent body weight change from start of treatment was also calculated for all the groups. Statistical significance was evaluated using a one-tailed t test. Statistical significance is indicated as follows: * p ≤<NUM>, ** p --<<NUM>, *** p --<<NUM>.

The pH of the solution was adjusted to <NUM>-<NUM> with 1N HCl or 1N NaOH. The final volume was made up using sterile water for injections or deionized water and the final pH was measured and recorded. Dosing preparations were formulated once per week and protected from light. Carboplatin (Sigma Aldrich) was formulated fresh on each day of dosing by diluting the stock solution in <NUM>% NaCl to <NUM>/ml for <NUM>/kg dose (<NUM>/<NUM> dose volume).

AZD5305 at <NUM>, <NUM> and <NUM>/kg QD and carboplatin at <NUM>/kg QW were tested in the efficacy study in SUM149PT TNBC BRCA1m xenograft model as monotherapies and in combination. All treatments were well tolerated throughout the duration of the study (<NUM> days) and no significant body weight loss was observed (<FIG>). AZD5305 monotherapy showed dose-dependent anti-tumour efficacy, delivering <NUM>% TGI and <NUM>% TGI at <NUM>/kg QD and <NUM>/kg QD, respectively. AZD5305 dosed at <NUM>/kg QD did not inhibit tumour growth. Carboplatin monotherapy resulted in <NUM>% TGI. Combination of <NUM>/kg AZD5305 and carboplatin demonstrated a combination benefit and delivered <NUM>% TGI. This anti-tumour effect was maintained even when dose level of AZD5305 in the combination with carboplatin was reduced to <NUM>/kg or <NUM>/kg (<NUM>% TGI and <NUM>% TGI, respectively) (<FIG> and Table <NUM>).

In conclusion, AZD5305 in combination with carboplatin demonstrated improved anti-tumour effect compared to monotherapy groups in SUM149PT xenograft model in vivo. Decreasing dose level of AZD5305 by <NUM>-fold (from <NUM>/kg to <NUM>/kg) in combination with carboplatin did not affect the tumour growth inhibition effect.

This study was carried out to investigate the anti-tumour effects of a dose range of AZD5305 in combination with carboplatin in a BRCA1/<NUM> wild type tumour model, HBCx-<NUM>, in vivo. AZD5305 monotherapy and carboplatin monotherapy arms were included in the study design as controls.

HBCx-<NUM> is a patient-derived tumour explant (PDX) model established in XenTech without prior in vitro culture. Tumour fragments were transplanted subcutaneously (SC) onto donor mice. When tumour volume reached <NUM> to <NUM> mm3, donor mice were sacrificed, and tumours were aseptically excised and dissected. After removing necrotic areas, tumours were cut into fragments measuring approximately <NUM> mm3 and transferred in culture medium before grafting SC into the recipient (experimental) Nude female mice. Tumours were measured (length x width) two times weekly by bilateral Vernier calliper measurements and tumour volume was calculated using elliptical formula (π/<NUM>×width×width×length). Animal bodyweight and tumour condition were monitored throughout the study. Mice were randomised into treatment groups when mean tumour volume reached approximately <NUM> mm3. Animals were treated from the day after the randomisation. Control animals were treated orally (PO) once daily (QD) with vehicle (deionised water acidified with HCl to pH <NUM>-<NUM>). AZD5305 was dosed PO QD at <NUM>, <NUM>, or <NUM>/kg. Carboplatin was dosed IP once weekly (QW) at <NUM>/kg. In the combination group, mice were dosed with carboplatin first, followed by AZD5305 administration within <NUM> minutes.

Tolerance studies using AZD5305 at <NUM>/kg PO QD in combination with carboplatin at <NUM>/kg IP QW were run in SCID female mice prior to commencement of the efficacy study and showed no adverse effects to animal condition or significant changes to bodyweight.

Samples for plasma PK were obtained by collecting <NUM>-<NUM>µL of whole blood by intra-cardiac puncture under xylazine-ketamine anaesthesia. Blood was transferred into BD Microtainer Hep-Li with separator gel tubes and centrifuged at <NUM> RPM for <NUM>-<NUM> minutes at <NUM>. Separated plasma was collected and stored at -<NUM>. To determine compound levels in plasma samples, each plasma sample (<NUM>µl) was prepared using an appropriate dilution factor and compared against an <NUM>-point standard calibration curve (<NUM>-<NUM>) prepared in DMSO and spiked into blank plasma. Acetonitrile (<NUM>µl) was added with the internal standard, followed by centrifugation at <NUM> RPM for <NUM> minutes. Supernatant (<NUM>µl) was then diluted in <NUM>µl water and analysed via UPLC-MS/MS.

The pH of the solution was adjusted to <NUM>-<NUM> with 1N HCl or 1N NaOH. The final volume was made up using sterile water for injections or deionized water and the final pH was measured and recorded. Dosing preparations were formulated once per week and protected from light. Carboplatin (Sandoz) was formulated fresh on each day of dosing by diluting the stock solution in <NUM>% NaCl to <NUM>/ml for <NUM>/kg dose (<NUM>/<NUM> dose volume).

HBCx-<NUM> is a TNBC PDX model available in XenTech, France. It has been reported as BRCA1/<NUM> wt model with BRCA1 methylation, as well as "BRCAness features" [<NUM>]. HBCx-<NUM> was also shown to have a moderate sensitivity to olaparib monotherapy [<NUM>].

AZD5305 at <NUM>, <NUM>, and <NUM>/kg QD and carboplatin at <NUM>/kg QW were tested in the efficacy study as monotherapies and in combination. All treatments were well tolerated throughout the duration of the study (<NUM> days) and no significant body weight loss was observed. AZD5305 monotherapy showed dose-dependent anti-tumour efficacy, delivering <NUM>%, <NUM>%, and <NUM>% TGI at <NUM>, <NUM>, and <NUM>/kg QD, respectively. Carboplatin monotherapy resulted in <NUM>% TGI. Treatment with <NUM>/kg AZD5305 and carboplatin demonstrated a combination benefit and delivered <NUM>% regression. This anti-tumour effect was maintained even when dose level of AZD5305 in the combination with carboplatin was reduced to <NUM>/kg (<NUM>% regression). The effect was slightly reduced when carboplatin was combined with <NUM>/kg of AZD5305 (<NUM>% TGI). However, even this group deliver a combination benefit comparing to either monotherapy arm.

In this experiment the treatment was continued for <NUM> days and then the duration of response was monitored off treatment. The graph in <FIG> depicts that in the most efficacious groups (carboplatin combined with AZD5305 at <NUM> or <NUM>/kg) tumours started to regrow around day <NUM> (<NUM> weeks after cessation of treatment), and the rate of the regrowth was similar between the two groups. Additionally, in carboplatin + <NUM>/kg of AZD5305 group tumours started to regrow about <NUM> weeks after treatment withdrawal.

In conclusion, AZD5305 in combination with carboplatin demonstrated improved anti-tumour effect compared to monotherapy groups in HBCx-<NUM> PDX model in vivo. Decreasing dose level of AZD5305 by <NUM>-fold (from <NUM>/kg to <NUM>/kg) in combination with carboplatin did not affect the tumour regression effect.

Claim 1:
AZD5305, or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer in a subject, wherein said treatment comprises the separate, sequential or simultaneous administration of i) said AZD5305, or a pharmaceutically acceptable salt thereof, and ii) a platinum chemotherapeutic agent, or a pharmaceutically acceptable salt thereof, to said subject.