Imidazo[4,5-c]quinolin-2-one Compounds and Their Use in Treating Cancer

FIELD

This specification relates to substituted imidazo[4,5-c]quinolin-2-one compounds and pharmaceutically acceptable salts thereof. These compounds and salts selectively modulate ataxia telangiectasia mutated ("ATM") kinase, and the specification therefore also relates to the use of substituted imidazo[4,5-c]quinolin- 2-one compounds and salts thereof to treat or prevent ATM mediated disease, including cancer. The specification further relates to pharmaceutical compositions comprising substituted imidazo[4,5-c]quinolin-2-one compounds and salts thereof; kits comprising such compounds and salts; methods of manufacture of such compounds and salts; and intermediates useful in such manufacture.

BACKGROUND

ATM kinase is a serine threonine kinase originally identified as the product of the gene mutated in ataxia telangiectasia. Ataxia telangiectasia is located on human chromosome 1 lq22-23 and codes for a large protein of about 350 kDa, which is characterized by the presence of a phosphatidylinositol ("PI") 3-kinase- like serine/threonine kinase domain flanked by FRAP-ATM-TRRAP and FATC domains which modulate ATM kinase activity and function. ATM kinase has been identified as a major player of the DNA damage response elicited by double strand breaks. It primarily functions in S/G2/M cell cycle transitions and at collapsed replication forks to initiate cell cycle checkpoints, chromatin modification, HR repair and pro-survival signalling cascades in order to maintain cell integrity after DNA damage (Lavin, M. F.; Rev. Mol. Cell Biol. 2008, 1 '59-769).

ATM kinase signalling can be broadly divided into two categories: a canonical pathway, which signals together with the Mrel 1-Rad50-NBS1 complex from double strand breaks and activates the DNA damage checkpoint, and several non-canonical modes of activation, which are activated by other forms of cellular stress (Cremona et ah, Oncogene 2013, 3351-3360). ATM kinase is rapidly and robustly activated in response to double strand breaks and is reportedly able to phosphorylate in excess of 800 substrates

(Matsuoka et al., Science 2007, 1160-1166), coordinating multiple stress response pathways (Kurz and Lees Miller, DNA Repair 2004, 889-900). ATM kinase is present predominantly in the nucleus of the cell in an inactive homodimeric form but autophosphorylates itself on Serl981 upon sensing a DNA double strand break (canonical pathway), leading to dissociation to a monomer with full kinase activity (Bakkenist et al., Nature 2003, 499-506). This is a critical activation event, and ATM phospho-Serl981 is therefore both a direct pharmacodynamic and patient selection biomarker for tumour pathway dependency.

ATM kinase responds to direct double strand breaks caused by common anti-cancer treatments such as ionising radiation and topoisomerase-II inhibitors (doxorubicin, etoposide) but also to topoisomerase-I inhibitors (for example irinotecan and topotecan) via single strand break to double strand break conversion during replication. ATM kinase inhibition can potentiate the activity of any these agents, and as a result ATM kinase inhibitors are expected to be of use in the treatment of cancer.

CN102372711 A reports certain imidazo[4,5-c]quinolin-2-one compounds which are mentioned to be dual inhibitors of PI 3-kinase a and mammalian target of rapam cin ("mTOR") kinase:

Certain compounds reported in CN 1023727 II A CN102399218A reports certain imidazo[4,5-c]quinolin-2-one compounds are mentioned to be PI 3-kinase a inhibitors:

1 14

Certain compounds reported in CN102399218A

While the compounds of CN102372711 A and CN102399218A are reported to possess activity against PI 3-kinase a and in some cases mTOR kinase, there remains a need to develop new compounds that are more effective against different kinase enzymes, such as ATM kinase. There further exists a need for new compounds that act against certain kinase enzymes, like ATM kinase, in a highly selective fashion (i.e. by modulating ATM more effectively than other biological targets). As demonstrated elsewhere in the specification (for example in the cell based assays described in the experimental section), the compounds of the present specification generally possess very potent ATM kinase inhibitory activity, but much less potent activity against other tyrosine kinase enzymes, such as PI 3-kinase a, mTOR kinase, ataxia telangiectasia and Rad3 -related protein ("ATR") kinase, and DNA-dependent protein kinase ("DNAPK"). As such, the compounds of the present specification not only inhibit ATM kinase, but can also be considered highly selective inhibitors of ATM kinase.

As a result of their highly selective nature, the compounds of the present specification are expected to be particularly useful in the treatment of diseases in which ATM kinase is implicated (for example, in the treatment of cancer), but where it is desirable to minimise off-target effects or toxicity that might arise due to the inhibition of other tyrosine kinase enzymes, such as class PI 3-kinase a, mTOR kinase, ATR kinase and/or DNAPK.

SUMMARY

Briefly, this specification describes, in part, a compound of Formula (I):

(I)

or a pharmaceutically acceptable salt thereof, where:

R ¹ is tetrahydropyran-3-yl or 3,3-dimethyltetrahydropyran-4-yl;

R ² is methyl or hydro;

R ³ is hydro or fluoro;

R ⁴ is hydro or fluoro; and

R ⁵ is methyl or hydro. This specification also describes, in part, a pharmaceutical composition which comprises a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

This specification also describes, in part, a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in therapy.

This specification also describes, in part, a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer.

This specification also describes, in part, the use of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of cancer.

This specification also describes, in part, a method for treating cancer in a warm blooded animal in need of such treatment, which comprises administering to said warm-blooded animal a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: X-Ray Powder Diffraction Pattern of Form A of (5)-8-(6- (Methoxymethyl)pyridin-3 -yl)-3 -methyl- 1 -(tetrahydro-2H-pyran-3 -yl)- 1 H- imidazo[4,5-c]quinolin-2(3H)-one.

Figure 2: DSC Thermogram of Form A of (5)-8-(6-(Methoxymethyl)pyridin-3-yl)- 3-methyl-l-(tetrahydro-2H-pyran-3-yl)-lH-imidazo[4,5-c]quino lin-2(3H)-one. Figure 3: X-Ray Powder Diffraction Pattern of Form A of (5)-7-Fluoro-8-(6-

(methoxymethyl)pyridin-3-yl)-3 -methyl- 1 -(tetrahydro-2H-pyran-3-yl)- 1 ,3-dihydro- 2H-imidazo[4,5-c]quinolin-2-one.

Figure 4: DSC Thermogram of Form A of (5)-7-Fluoro-8-(6- (methoxymethyl)pyridin-3-yl)-3 -methyl- 1 -(tetrahydro-2H-pyran-3-yl)- 1 ,3-dihydro- 2H-imidazo[4,5-c]quinolin-2-one. Figure 5: X-Ray Powder Diffraction Pattern of Form B of (5)-7-Fluoro-8-(6- (methoxymethyl)pyridin-3-yl)-3 -methyl- 1 -(tetrahydro-2H-pyran-3-yl)- 1 ,3-dihydro- 2H-imidazo[4,5-c]quinolin-2-one.

Figure 6: DSC Thermogram of Form B of (5)-7-Fluoro-8-(6- (methoxymethyl)pyridin-3-yl)-3 -methyl- 1 -(tetrahydro-2H-pyran-3-yl)- 1 ,3-dihydro- 2H-imidazo[4,5-c]quinolin-2-one.

ILLUSTRATIVE EMBODIMENTS

Many embodiments are detailed in this specification and will be apparent to a reader skilled in the art. The embodiments are not to be interpreted as being limiting.

In the first embodiment there is provided a compound of Formula (I):

or a pharmaceutically acceptable salt thereof, where:

R ¹ is tetrahydropyran-3-yl or 3,3-dimethyltetrahydropyran-4-yl;

R ² is methyl or hydro;

R ³ is hydro or fluoro;

R ⁴ is hydro or fluoro; and

R ⁵ is methyl or hydro.

A "hydro" group is equivalent to a hydrogen atom. Atoms with a hydro group attached to them can be regarded as unsubstituted.

The term "pharmaceutically acceptable" is used to specify that an object (for example a salt, dosage form or excipient) is suitable for use in patients. An example list of pharmaceutically acceptable salts can be found in the Handbook of

Pharmaceutical Salts: Properties, Selection and Use, P. H. Stahl and C. G. Wermuth, editors, Weinheim/zurichiWiley-VCH/VHCA, 2002. A suitable pharmaceutically acceptable salt of a compound of Formula (I) is, for example, an acid-addition salt. An acid addition salt of a compound of Formula (I) may be formed by bringing the compound into contact with a suitable inorganic or organic acid under conditions known to the skilled person. An acid addition salt may for example be formed using an inorganic acid selected from hydrochloric acid, hydrobromic acid, sulphuric acid and phosphoric acid. An acid addition salt may also be formed using an organic acid selected from trifluoro acetic acid, citric acid, maleic acid, oxalic acid, acetic acid, formic acid, benzoic acid, fumaric acid, succinic acid, tartaric acid, lactic acid, pyruvic acid, methanesulfonic acid, benzenesulfonic acid and para-toluenesulfonic acid.

Therefore, in one embodiment there is provided a compound of Formula (I) or a pharmaceutically acceptable salt thereof, where the pharmaceutically acceptable salt is a hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, trifluoro acetic acid, citric acid, maleic acid, oxalic acid, acetic acid, formic acid, benzoic acid, fumaric acid, succinic acid, tartaric acid, lactic acid, pyruvic acid, methanesulfonic acid, benzenesulfonic acid or para-toluenesulfonic acid salt. In one embodiment there is provided a compound of Formula (I) or a

pharmaceutically acceptable salt thereof, where the pharmaceutically acceptable salt is a methanesulfonic acid salt. In one embodiment there is provided a compound of Formula (I) or a pharmaceutically acceptable salt thereof, where the pharmaceutically acceptable salt is a mono-methanesulfonic acid salt, i.e. the stoichiometry of the compound of the compound of Formula (I) to methanesulfonic acid is 1 : 1.

A further embodiment provides any of the embodiments defined herein (for example the embodiment of claim 1) with the proviso that one or more specific Examples (for instance one, two or three specific Examples) selected from

Examples 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 is individually disclaimed.

Some values of variable groups in Formula (I) are as follows. Such values may be used in combination with any of the definitions, claims (for example claim 1), or embodiments defined herein to provide further embodiments,

a) R ¹ is tetrahydropyran-3-yl. b) R ¹ is (5)-tetrahydropyran-3-yl.

c) R ¹ is (R)-tetrahydropyran-3-yl.

d) R ¹ is 3,3-dimethyltetrahydropyran-4-yl.

e) R ¹ is (5)-3,3-dimethyltetrahydropyran-4-yl.

f) R ¹ is (R)-3,3-dimethyltetrahydropyran-4-yl.

g) R ² is methyl.

h) R ² is hydro.

i) R ³ is hydro.

j) R ³ is fluoro.

k) R ⁴ is hydro.

0 R ⁴ is fluoro.

m) R ⁵ is methyl.

n) R ⁵ is hydro.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, where:

R ¹ is tetrahydropyran-3-yl;

R ² is methyl or hydro;

R ³ is hydro or fluoro;

R ⁴ is hydro or fluoro; and

R ⁵ is methyl.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, where:

R ¹ is 3,3-dimethyltetrahydropyran-4-yl;

R ² is methyl or hydro;

R ³ is hydro or fluoro;

R ⁴ is hydro or fluoro; and

R ⁵ is methyl.

fin one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, where:

R ¹ is 3,3-dimethyltetrahydropyran-4-yl;

R ² is methyl;

R ³ is hydro; R ⁴ is hydro; and

R ⁵ is methyl.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

(R)-8-(6-(Methoxymethyl)pyridin-3-yl)-3-methyl- l-(tetrahydro-2H-pyran- 3-yl)-lH-imidazo[4,5-c]quinolin-2(3H)-one;

(5)-8-(6-(Methoxymethyl)pyridin-3-yl)-3-methyl- l-(tetrahydro-2H-pyran-: yl)-lH-imidazo[4,5-c]quinolin-2(3H)-one;

(5)-7-Fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(t etrahydro- 2H-pyran-3-yl)-l ,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one;

(R)-7-Fluoro-8-(2-fluoro-6-(methoxymethyl)pyridin-3-yl)-3-me thyl-l- (tetrahydro-2H-pyran-3-yl)-lH-imidazo[4,5-c]quinolin-2(3H)-o ne;

(5)-7-Fluoro-8-(2-fluoro-6-(methoxymethyl)pyridin-3-yl)-3-me thyl-l- (tetrahydro-2H-pyran-3-yl)-lH-imidazo[4,5-c]quinolin-2(3H)-o ne;

(R)-8-(2-Fluoro-6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(t etrahydro- 2H-pyran-3-yl)-lH-imidazo[4,5-c]quinolin-2(3H)-one;

(5)-8-(2-Fluoro-6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(t etrahydro- 2H-pyran-3-yl)-lH-imidazo[4,5-c]quinolin-2(3H)-one;

(R)-7-Fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(t etrahydro- 2H-pyran-3-yl)-lH-imidazo[4,5-c]quinolin-2(3H)-one;

rac-l-(3,3-Dimethyltetrahydro-2H-pyran-4-yl)-8-(6- (methoxymethyl)pyridin-3-yl)-3-methyl-l ,3-dihydro-2H-imidazo[4,5-c]quinolin-2 one;

(R)- 1 -(3 ,3-Dimethyltetrahydro-2H-pyran-4-yl)-8-(6- (methoxymethyl)pyridin-3-yl)-3-methyl-l ,3-dihydro-2H-imidazo[4,5-c]quinolin-2 one;

(S)- 1 -(3,3-Dimethyltetrahydro-2H-pyran-4-yl)-8-(6- (methoxymethyl)pyridin-3-yl)-3-methyl-l ,3-dihydro-2H-imidazo[4,5-c]quinolin-2 one; and

(5)-8-(6-(Methoxymethyl)pyridin-3-yl)- 1 -(tetrahydro-2H-pyran-3-yl)- 1 ,3- dihydro -2H-imidazo [4 , 5 -c] quino lin-2-one . In one embodiment there is provided any compound of Formula (I), or a pharmaceutically acceptable salt thereof, which may be prepared according to the experimental details in the Examples section.

In one embodiment there is provided (5)-8-(6-(methoxymethyl)pyridin-3- yl)-3-methyl-l-(tetrahydro-2H-pyran-3-yl)-lH-imidazo[4,5-c]q uinolin-2(3H)-one, or a pharmaceutically acceptable salt thereof.

In one embodiment there is provided (5)-8-(6-(methoxymethyl)pyridin-3- yl)-3-methyl-l-(tetrahydro-2H-pyran-3-yl)-lH-imidazo[4,5-c]q uinolin-2(3H)-one.

In one embodiment there is provided a pharmaceutically acceptable salt of (5)-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(tetrahydro -2H-pyran-3-yl)-lH- imidazo[4,5-c]quinolin-2(3H)-one.

In one embodiment there is provided (5)-7-fluoro-8-(6- (methoxymethyl)pyridin-3-yl)-3 -methyl- 1 -(tetrahydro-2H-pyran-3-yl)- 1 ,3-dihydro- 2H-imidazo[4,5-c]quinolin-2-one, or a pharmaceutically acceptable salt thereof.

In one embodiment there is provided (5)-7-fluoro-8-(6- (methoxymethyl)pyridin-3-yl)-3 -methyl- 1 -(tetrahydro-2H-pyran-3-yl)- 1 ,3-dihydro- 2H-imidazo[4,5-c]quinolin-2-one.

In one embodiment there is provided a pharmaceutically acceptable salt of (5)-7-fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(t etrahydro-2H-pyran- 3 -yl)- 1 , 3 -dihydro -2H-imidazo [4 , 5 -c] quino lin-2-one .

In one embodiment there is provided (5)-8-(6-(methoxymethyl)pyridin-3- yl)-l-(tetrahydro-2H-pyran-3-yl)-l,3-dihydro-2H-imidazo[4,5- c]quinolin-2-one, or a pharmaceutically acceptable salt thereof.

In one embodiment there is provided (5)-8-(6-(methoxymethyl)pyridin-3- yl)-l-(tetrahydro-2H-pyran-3-yl)-l,3-dihydro-2H-imidazo[4,5- c]quinolin-2-one.

In one embodiment there is provided a pharmaceutically acceptable salt of (5)-8-(6-(methoxymethyl)pyridin-3-yl)- 1 -(tetrahydro-2H-pyran-3-yl)- 1 ,3-dihydro- 2H-imidazo[4,5-c]quinolin-2-one.

Compounds and salts described in this specification may exist in solvated forms and unsolvated forms. For example, a solvated form may be a hydrated form, such as a hemi-hydrate, a mono-hydrate, a di-hydrate, a tri-hydrate or an alternative quantity thereof. The invention encompasses all such solvated and unsolvated forms of compounds of Formula (I), particularly to the extent that such forms possess ATM kinase inhibitory activity, as for example measured using the tests described herein.

Atoms of the compounds and salts described in this specification may exist as their isotopes. The invention encompasses all compounds of Formula (I) where an atom is replaced by one or more of its isotopes (for example a compound of Formula (I) where one or more carbon atom is an ⁿ C or ¹³ C carbon isotope, or where one or more hydrogen atoms is a ² H or ³ H isotope).

Compounds and salts described in this specification may exist as a mixture of tautomers. "Tautomers" are structural isomers that exist in equilibrium resulting from the migration of a hydrogen atom. The invention includes all tautomers of compounds of Formula (I) particularly to the extent that such tautomers possess ATM kinase inhibitory activity.

Compounds and salts described in this specification exist in optically active or racemic forms by virtue of an aymmetric carbon atom. The invention includes any optically active or racemic form of a compound of Formula (I) which possesses ATM kinase inhibitory activity, as for example measured using the tests described herein. The synthesis of optically active forms may be carried out by standard techniques of organic chemistry well known in the art, for example by synthesis using optically active materials or by resolution of a racemic form.

Therefore, in one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, which is a single optical isomer being in an enantiomeric excess (%ee) of > 95%, > 98% or > 99%. In one embodiment, the single optical isomer is present in an enantiomeric excess (%ee) of > 99%.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, which is an (5)-optical isomer being in an enantiomeric excess (%>ee) of > 95%>, > 98%> or > 99%>. In one embodiment, the (S)- optical isomer is present in an enantiomeric excess (%ee) of > 99%.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, which is an (7?)-optical isomer being in an enantiomeric excess (%>ee) of > 95%>, > 98%> or > 99%>. In one embodiment, the (R)- optical isomer is present in an enantiomeric excess (%ee) of > 99%. Compounds and salts described in this specification may be crystalline, and may exhibit one or more crystalline forms. The invention encompasses any crystalline or amorphous form of a compound of Formula (I), or mixture of such forms, which possesses ATM kinase inhibitory activity.

It is generally known that crystalline materials may be characterised using conventional techniques such as X-Ray Powder Diffraction (XRPD), Differential Scanning Calorimetry (DSC), Thermal Gravimetric Analysis (TGA), Diffuse Reflectance Infrared Fourier Transform (DRIFT) spectroscopy, Near Infrared (NIR) spectroscopy, solution and/or solid state nuclear magnetic resonance spectroscopy. The water content of crystalline materials may be determined by Karl Fischer analysis.

The crystalline forms described herein provide XRPD patterns substantially the same as the XRPD patterns shown in the Figures, and have the various 2-theta values as shown in the Tables included herein. One skilled in the art will understand that an XRPD pattern or diffractogram may be obtained which has one or more measurement errors depending on the recording conditions, such as the equipment or machine used. Similarly, it is generally known that intensities in an XRPD pattern may fluctuate depending on measurement conditions or sample preparation as a result of preferred orientation. Persons skilled in the art of XRPD will further realise that the relative intensity of peaks can also be affected by, for example, grains above 30μιη in size and non-unitary aspect ratios. The skilled person understands that the position of reflections can be affected by the precise height at which the sample sits in the diffractometer, and also the zero calibration of the diffractometer. The surface planarity of the sample may also have a small effect.

As a result of these considerations, the diffraction pattern data presented are not to be taken as absolute values (Jenkins, R & Snyder, R.L. 'Introduction to X- Ray Powder Diffractometry' John Wiley & Sons 1996; Bunn, C.W. (1948), 'Chemical Crystallography ', Clarendon Press, London; Klug, H. P. & Alexander, L. E. (1974), 'X-Ray Diffraction Procedures '). It should correspondingly be understood that the solid forms are not limited to the crystals that provide XRPD patterns that are identical to the XRPD pattern shown in the Figures, and any crystals providing XRPD patterns substantially the same as those shown in the Figures fall within the scope of the invention. A person skilled in the art of XRPD is able to judge the substantial identity of XRPD patterns. Generally, a

measurement error of a diffraction angle in an XRPD is approximately plus or minus 0.2° 2-theta, and such degree of a measurement error should be taken into account when considering the X-ray powder diffraction pattern in the Figures and when reading data contained in the Tables included herein.

The compound of Example 2 exhibits crystalline properties, and one crystalline form has been characterised.

Therefore, in one embodiment there is provided Form A of (5)-8-(6 (methoxymethyl)pyridin-3 -yl)-3 -methyl- 1 -(tetrahydro-2H-pyran-3 -yl)- 1 H- imidazo[4,5-c]quinolin-2(3H)-one.

In one embodiment there is provided a crystalline form, Form A of (5)-8-(6- (methoxymethyl)pyridin-3 -yl)-3 -methyl- 1 -(tetrahydro-2H-pyran-3 -yl)- 1 H- imidazo[4,5-c]quinolin-2(3H)-one, which has an X-ray powder diffraction pattern with at least one specific peak at about 2-theta = 7.0°.

In one embodiment there is provided a crystalline form, Form A of (5)-8-(6- (methoxymethyl)pyridin-3 -yl)-3 -methyl- 1 -(tetrahydro-2H-pyran-3 -yl)- 1 H- imidazo[4,5-c]quinolin-2(3H)-one, which has an X-ray powder diffraction pattern with at least one specific peak at about 2-theta = 9.2°.

In one embodiment there is provided a crystalline form, Form A of (5)-8-(6- (methoxymethyl)pyridin-3 -yl)-3 -methyl- 1 -(tetrahydro-2H-pyran-3 -yl)- 1 H- imidazo[4,5-c]quinolin-2(3H)-one, which has an X-ray powder diffraction pattern with at least two specific peaks at about 2-theta = 7.0 and 9.2°.

In one embodiment there is provided a crystalline form, Form A of (5)-8-(6- (methoxymethyl)pyridin-3 -yl)-3 -methyl- 1 -(tetrahydro-2H-pyran-3 -yl)- 1 H- imidazo[4,5-c]quinolin-2(3H)-one, which has an X-ray powder diffraction pattern with specific peaks at about 2-theta = 7.0, 9.2, 10.4, 13.9, 18.9, 22.3, 23.1, 23.6, 24.7, and 25.4°.

In one embodiment there is provided a crystalline form, Form A of (5)-8-(6- (methoxymethyl)pyridin-3 -yl)-3 -methyl- 1 -(tetrahydro-2H-pyran-3 -yl)- 1 H- imidazo[4,5-c]quinolin-2(3H)-one which has an X-ray powder diffraction pattern substantially the same as the X-ray powder diffraction pattern shown in Figure 1.

In one embodiment there is provided a crystalline form, Form A of (5)-8-(6- (methoxymethyl)pyridin-3 -yl)-3 -methyl- 1 -(tetrahydro-2H-pyran-3 -yl)- 1 H- imidazo[4,5-c]quinolin-2(3H)-one, which has an X-ray powder diffraction pattern with at least one specific peak at 2-theta = 7.0° plus or minus 0.2° 2-theta.

In one embodiment there is provided a crystalline form, Form A of (5)-8-(6- (methoxymethyl)pyridin-3 -yl)-3 -methyl- 1 -(tetrahydro-2H-pyran-3 -yl)- 1 H- imidazo[4,5-c]quinolin-2(3H)-one, which has an X-ray powder diffraction pattern with at least one specific peak at 2-theta = 9.2° plus or minus 0.2° 2-theta.

In one embodiment there is provided a crystalline form, Form A of (5)-8-(6- (methoxymethyl)pyridin-3 -yl)-3 -methyl- 1 -(tetrahydro-2H-pyran-3 -yl)- 1 H- imidazo[4,5-c]quinolin-2(3H)-one, which has an X-ray powder diffraction pattern with at least two specific peaks at 2-theta = 7.0 and 9.2° plus or minus 0.2° 2-theta.

In one embodiment there is provided a crystalline form, Form A of (5)-8-(6- (methoxymethyl)pyridin-3 -yl)-3 -methyl- 1 -(tetrahydro-2H-pyran-3 -yl)- 1 H- imidazo[4,5-c]quinolin-2(3H)-one, which has an X-ray powder diffraction pattern with specific peaks at 2-theta = 7.0, 9.2, 10.4, 13.9, 18.9, 22.3, 23.1, 23.6, 24.7, and 25.4° plus or minus 0.2° 2-theta.

DSC analysis of Form A of (5)-8-(6-(methoxymethyl)pyridin-3-yl)-3- methyl-l-(tetrahydro-2H-pyran-3-yl)-lH-imidazo[4,5-c]quinoli n-2(3H)-one shows a melting endotherm with an onset of 184.4°C and a peak at 186.0°C (Figure 2).

A person skilled in the art understands that the value or range of values observed in a particular compound's DSC Thermogram will show variation between batches of different purities. Therefore, whilst for one compound the range may be small, for others the range may be quite large. Generally, a measurement error of a diffraction angle in DSC thermal events is approximately plus or minus 5°C, and such degree of a measurement error should be taken into account when considering the DSC data included herein.

Therefore, in one embodiment there is provided a crystalline form, Form A of (5)-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl- 1 -(tetrahydro-2H-pyran-3-yl)- lH-imidazo[4,5-c]quinolin-2(3H)-one which has a DSC endotherm with an onset of melting at about 184.4°C and a peak at about 186.0°C.

Therefore, in one embodiment there is provided a crystalline form, Form A of (5)-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl- 1 -(tetrahydro-2H-pyran-3-yl)- lH-imidazo[4,5-c]quinolin-2(3H)-one which has a DSC endotherm with an onset of melting at 184.4°C plus or minus 5°C and a peak at 186.0°C plus or minus 5°C.

In one embodiment there is provided a crystalline form, Form A of (5)-8-(6- (methoxymethyl)pyridin-3 -yl)-3 -methyl- 1 -(tetrahydro-2H-pyran-3 -yl)- 1 H- imidazo[4,5-c]quinolin-2(3H)-one which has a DSC endotherm with an onset of melting at 184.4°C and a peak at 186.0°C.

In one embodiment there is provided a crystalline form, Form A of (5)-8-(6- (methoxymethyl)pyridin-3 -yl)-3 -methyl- 1 -(tetrahydro-2H-pyran-3 -yl)- 1 H- imidazo[4,5-c]quinolin-2(3H)-one which has a DSC thermogram substantially as shown in Figure 2.

The compound of Example 3 exhibits crystalline properties, and two crystalline forms have been characterised.

Therefore, in one embodiment there is provided Form A of (5)-7-fluoro-8- (6-(methoxymethyl)pyridin-3-yl)-3 -methyl- 1 -(tetrahydro-2H-pyran-3-yl)- 1,3- dihydro -2H-imidazo [4 , 5 -c] quino lin-2-one .

In one embodiment there is provided a crystalline form, Form A of (5)-7- fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(tetrahy dro-2H-pyran-3-yl)- l,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one, which has an X-ray powder diffraction pattern with at least one specific peak at about 2-theta = 4.6°.

In one embodiment there is provided a crystalline form, Form A of (5)-7- fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(tetrahy dro-2H-pyran-3-yl)- l,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one, which has an X-ray powder diffraction pattern with at least one specific peak at about 2-theta = 16.0°.

In one embodiment there is provided a crystalline form, Form A of (5)-7- fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(tetrahy dro-2H-pyran-3-yl)- l,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one, which has an X-ray powder diffraction pattern with at least two specific peaks at about 2-theta = 4.6 and 16.0°. In one embodiment there is provided a crystalline form, Form A of (5)-7- fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(tetrahy dro-2H-pyran-3-yl)- l,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one, which has an X-ray powder diffraction pattern with specific peaks at about 2-theta = 4.6, 8.5, 9.3, 13.8. 16.0, 18.1, 18.7, 19.9, 23.1, 24.5°.

In one embodiment there is provided a crystalline form, Form A of (5)-7- fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(tetrahy dro-2H-pyran-3-yl)- l,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one which has an X-ray powder diffraction pattern substantially the same as the X-ray powder diffraction pattern shown in Figure 3.

In one embodiment there is provided a crystalline form, Form A of (5)-7- fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(tetrahy dro-2H-pyran-3-yl)- l,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one, which has an X-ray powder diffraction pattern with at least one specific peak at 2-theta = 4.6° plus or minus 0.2° 2-theta.

In one embodiment there is provided a crystalline form, Form A of (5)-7- fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(tetrahy dro-2H-pyran-3-yl)- l,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one, which has an X-ray powder diffraction pattern with at least one specific peak at 2-theta = 16.0° plus or minus 0.2° 2-theta.

In one embodiment there is provided a crystalline form, Form A of (5)-7- fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(tetrahy dro-2H-pyran-3-yl)- l,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one, which has an X-ray powder diffraction pattern with at least two specific peaks at 2-theta = 4.6 and 16.0° plus or minus 0.2° 2-theta.

In one embodiment there is provided a crystalline form, Form A of (5)-7- fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(tetrahy dro-2H-pyran-3-yl)- l,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one, which has an X-ray powder diffraction pattern with specific peaks at 2-theta = 4.6, 8.5, 9.3, 13.8. 16.0, 18.1, 18.7, 19.9, 23.1, 24.5° plus or minus 0.2° 2-theta.

DSC analysis of Form A of (5)-7-fluoro-8-(6-(methoxymethyl)pyridin-3- yl)-3-methyl-l-(tetrahydro-2H-pyran-3-yl)-l,3-dihydro-2H-imi dazo[4,5-c]quinolin- 2-one shows a melting endotherm with an onset of 174.5°C and a peak at 177.3°C (Figure 4).

Therefore, in one embodiment there is provided a crystalline form, Form A of (5)-7-fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl- 1 -(tetrahydro-2H- pyran-3-yl)-l ,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one which has a DSC endotherm with an onset of melting at about 174.5°C and a peak at about 177.3°C.

Therefore, in one embodiment there is provided a crystalline form, Form A of (5)-7-fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl- 1 -(tetrahydro-2H- pyran-3-yl)-l ,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one which has a DSC endotherm with an onset of melting at 174.5°C plus or minus 5°C and a peak at 177.3°C plus or minus 5°C.

In one embodiment there is provided a crystalline form, Form A of (5)-7- fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(tetrahy dro-2H-pyran-3-yl)- l ,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one which has a DSC endotherm with an onset of melting at 174.5°C and a peak at 177.3°C.

In one embodiment there is provided a crystalline form, Form A of (5)-7- fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(tetrahy dro-2H-pyran-3-yl)- l ,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one which has a DSC thermogram substantially as shown in Figure 4.

In one embodiment there is provided Form B of (5)-7-fluoro-8-(6- (methoxymethyl)pyridin-3-yl)-3 -methyl- 1 -(tetrahydro-2H-pyran-3-yl)- 1 ,3-dihydro- 2H-imidazo[4,5-c]quinolin-2-one.

In one embodiment there is provided a crystalline form, Form B of (5)-7- fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(tetrahy dro-2H-pyran-3-yl)- l ,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one, which has an X-ray powder diffraction pattern with at least one specific peak at about 2-theta = 8.9°.

In one embodiment there is provided a crystalline form, Form B of (5)-7- fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(tetrahy dro-2H-pyran-3-yl)- l ,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one, which has an X-ray powder diffraction pattern with at least one specific peak at about 2-theta = 22.0°.

In one embodiment there is provided a crystalline form, Form B of (S)-T- fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(tetrahy dro-2H-pyran-3-yl)- l,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one, which has an X-ray powder diffraction pattern with at least two specific peaks at about 2-theta = 8.9 and 22.0°.

In one embodiment there is provided a crystalline form, Form B of (5)-7- fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(tetrahy dro-2H-pyran-3-yl)- l,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one, which has an X-ray powder diffraction pattern with specific peaks at about 2-theta = 8.9, 10.1, 11.0, 11.9, 13.9, 16.2, 16.5, 20.3, 22.0 and 25.9°.

In one embodiment there is provided a crystalline form, Form B of (5)-7- fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(tetrahy dro-2H-pyran-3-yl)- l,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one which has an X-ray powder diffraction pattern substantially the same as the X-ray powder diffraction pattern shown in Figure 5.

In one embodiment there is provided a crystalline form, Form B of (5)-7- fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(tetrahy dro-2H-pyran-3-yl)- l,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one, which has an X-ray powder diffraction pattern with at least one specific peak at 2-theta = 8.9° plus or minus 0.2' 2-theta.

In one embodiment there is provided a crystalline form, Form B of (5)-7- fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(tetrahy dro-2H-pyran-3-yl)- l,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one, which has an X-ray powder diffraction pattern with at least one specific peak at 2-theta = 22.0° plus or minus 0.2° 2-theta.

In one embodiment there is provided a crystalline form, Form B of (5)-7- fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(tetrahy dro-2H-pyran-3-yl)- l,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one, which has an X-ray powder diffraction pattern with at least two specific peaks at 2-theta = 8.9 and 22.0° plus or minus 0.2° 2-theta.

In one embodiment there is provided a crystalline form, Form B of (5)-7- fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(tetrahy dro-2H-pyran-3-yl)- l,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one, which has an X-ray powder diffraction pattern with specific peaks at 2-theta = 8.9, 10.1, 11.0, 11.9, 13.9, 16.2, 16.5, 20.3, 22.0 and 25.9° plus or minus 0.2° 2-theta. DSC analysis of Form B of (5)-7-fluoro-8-(6-(methoxymethyl)pyridin-3- yl)-3-methyl-l-(tetrahydro-2H-pyran-3-yl)-l,3-dihydro-2H-imi dazo[4,5-c]quinolin- 2-one shows a melting endotherm with an onset of 174.5°C and a peak at 175.5°C (Figure 6).

Therefore, in one embodiment there is provided a crystalline form, Form B of (5)-7-fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl- 1 -(tetrahydro-2H- pyran-3-yl)-l,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one which has a DSC endotherm with an onset of melting at about 174.5°C and a peak at about 175.5°C.

Therefore, in one embodiment there is provided a crystalline form, Form B of (5)-7-fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl- 1 -(tetrahydro-2H- pyran-3-yl)-l,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one which has a DSC endotherm with an onset of melting at 174.5°C plus or minus 5°C and a peak at 175.5°C plus or minus 5°C.

In one embodiment there is provided a crystalline form, Form B of (5)-7- fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(tetrahy dro-2H-pyran-3-yl)- l,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one which has a DSC endotherm with an onset of melting at 174.5°C and a peak at 175.5°C.

In one embodiment there is provided a crystalline form, Form B of (5)-7- fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(tetrahy dro-2H-pyran-3-yl)- l,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one which has a DSC thermogram substantially as shown in Figure 6.

When it is stated that an embodiment relates to a crystalline form, the degree of crystallinity may be greater than about 60%. In some embodiments the degree of crystallinity is greater than about 80%. In some embodiments the degree of crystallinity is greater than about 90%. In some embodiments the degree of crystallinity is greater than about 95%. In some embodiments the degree of crystallinity is greater than about 98%.

When it is stated that an embodiment relates to a crystalline form, the degree of crystallinity may be greater than 60%. In some embodiments the degree of crystallinity is greater than 80%. In some embodiments the degree of

crystallinity is greater than 90%. In some embodiments the degree of crystallinity is greater than 95%. In some embodiments the degree of crystallinity is greater than 98%.

Compounds of Formula (I) may for example be prepared by the reaction of a compound of Formula (II):

(Π)

Or a salt thereof, where R ¹ , R ² and R ³ are as defined in any of the embodiments herein and X is a leaving group (for example a halogen atom, or alternatively a fluorine atom) with a compound of formula (III):

(HI)

or a salt thereof, where R ⁴ and R ⁵ are as defined in any of the embodiments herein and Y is a boronic acid, boronic ester or potassium trifluoroborate group (for example boronic acid, boronic acid pinacol ester, or potassium trifluoroborate). The reaction may be performed under standard conditions well known to those skilled in the art, for example in the presence of a palladium source (for example tetrakis triphenylphosphine palladium or palladium(II) acetate), optionally a phosphine ligand (for example Xantphos or S-phos), and a suitable base (for example cesium carbonate or triethylamine).

Compounds of Formula (II) are therefore useful as intermediates in the preparation of compounds of Formula (I) and provide a further embodiment.

In one embodiment there is provided a compound of Formula (II), or a salt thereof, where:

R ¹ is tetrahydropyran-3-yl or 3,3-dimethyltetrahydropyran-4-yl;

R ² is methyl or hydro;

R ³ is hydro or fluoro; and X is a leaving group. In one embodiment X is an iodine, bromine, or chlorine atom or a triflate group. In one embodiment X is a bromine atom.

In one embodiment there is provided a compound of Formula (II), or a salt thereof, where:

R ¹ is tetrahydropyran-3-yl;

R ² is methyl or hydro;

R ³ is hydro or fluoro; and

X is a leaving group. In one embodiment X is an iodine, bromine, or chlorine atom or a triflate group. In one embodiment X is a bromine atom,

Compounds of Formula (II) are therefore useful as intermediates in the preparation of the compounds of Formula (I) and provide a further embodiment.

In one embodiment there is provided a compound of Formula (II), or a salt thereof, where:

R ¹ is 3,3-dimethyltetrahydropyran-4-yl;

R ² is methyl or hydro;

R ³ is hydro or fluoro; and

X is a leaving group. In one embodiment X is an iodine, bromine, or chlorine atom or a triflate group. In one embodiment X is a bromine atom.

Compounds of Formula (II) are therefore useful as intermediates in the preparation of the compounds of Formula (I) and provide a further embodiment.

In one embodiment there is provided a compound of Formula (II), or a salt thereof, where:

R ¹ is 3,3-dimethyltetrahydropyran-4-yl;

R ² is methyl;

R ³ is hydro; and

X is a leaving group. In one embodiment X is an iodine, bromine, or chlorine atom or a triflate group. In one embodiment X is a bromine atom.

In any of the embodiments where a compound of Formula (II) or a salt thereof is mentioned it is to be understood that such salts do not need to be pharmaceutically acceptable salts. A suitable salt of a compound of Formula (II) is, for example, an acid-addition salt. An acid addition salt of a compound of Formula (II) may be formed by bringing the compound into contact with a suitable inorganic or organic acid under conditions known to the skilled person. An acid addition salt may for example be formed using an inorganic acid selected from hydrochloric acid, hydrobromic acid, sulphuric acid and phosphoric acid. An acid addition salt may also be formed using an organic acid selected from trifluoro acetic acid, citric acid, maleic acid, oxalic acid, acetic acid, formic acid, benzoic acid, fumaric acid, succinic acid, tartaric acid, lactic acid, pyruvic acid, methanesulfonic acid, benzenesulfonic acid and para-toluenesulfonic acid.

Therefore, in one embodiment there is provided a compound of Formula (II) or a salt thereof, where the salt is a hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, trifluoro acetic acid, citric acid, maleic acid, oxalic acid, acetic acid, formic acid, benzoic acid, fumaric acid, succinic acid, tartaric acid, lactic acid, pyruvic acid, methanesulfonic acid, benzenesulfonic acid or para- toluenesulfonic acid salt.

In one embodiment there is provided a compound of Formula (II), or a salt thereof, wherein the compound is selected from:

8-Bromo-3-methyl-l-[(3R)-oxan-3-yl]imidazo[5,4-c]quinolin-2- one;

8-Bromo-7-fluoro-3-methyl-l-[(35)-oxan-3-yl]imidazo[5,4-c]qu inolin-2- one;

8-Bromo-7-fluoro-3-methyl-l-[(3R)-oxan-3-yl]imidazo[5,4-c]qu inolin-2- one;

rac-8-Bromo-3-methyl-l-(tetrahydro-2H-pyran-3-yl)-lH-imidazo [4,5- c] quino lin-2(3H)-one;

rac-8-Bromo-7-fluoro-3-methyl- 1 -(tetrahydro-2H-pyran-3-yl)- 1H- imidazo[4,5-c]quinolin-2(3H)-one;

rac-8-Bromo- 1 -(3,3-dimethyltetrahydro-2H-pyran-4-yl)-3-methyl- 1,3- dihydro -2H-imidazo [4 , 5 -c] quino lin-2-one ;

8-Bromo-3-methyl-l-[(3R)-oxan-3-yl]imidazo[5,4-c]quinolin-2- one;

8-Bromo-7-fluoro-3-methyl-l-[(35)-oxan-3-yl]imidazo[5,4-c]qu inolin-2- one;

8-Bromo-7-fluoro-3-methyl-l-[(3R)-oxan-3-yl]imidazo[5,4-c]qu inolin-2- one; rac-8-Bromo-3-methyl-l-(tetrahydro-2H-pyran-3-yl)-lH-imidazo [4,5- c]quinolin-2(3H)-one; and

rac-8-Bromo-7-fluoro-3-methyl- 1 -(tetrahydro-2H-pyran-3-yl)- 1H- imidazo[4,5-c]quinolin-2(3H)-one.

Compounds of formula (III) can be prepared by methods similar to those shown in the Examples section.

In one embodiment there is provided any one of the novel intermediates described in the experimental section.

As a result of their ATM kinase inhibitory activity, the compounds of Formula (I), and pharmaceutically acceptable salts thereof are expected to be useful in therapy, for example in the treatment of diseases or medical conditions mediated at least in part by ATM kinase, including cancer.

Where "cancer" is mentioned, this includes both non-metastatic cancer and also metastatic cancer, such that treating cancer involves treatment of both primary tumours and also tumour metastases.

"ATM kinase inhibitory activity" refers to a decrease in the activity of ATM kinase as a direct or indirect response to the presence of a compound of Formula (I), or pharmaceutically acceptable salt thereof, relative to the activity of ATM kinase in the absence of compound of Formula (I), or pharmaceutically acceptable salt thereof. Such a decrease in activity may be due to the direct interaction of the compound of Formula (I), or pharmaceutically acceptable salt thereof with ATM kinase, or due to the interaction of the compound of Formula (I), or pharmaceutically acceptable salt thereof with one or more other factors that in turn affect ATM kinase activity. For example, the compound of Formula (I), or pharmaceutically acceptable salt thereof may decrease ATM kinase by directly binding to the ATM kinase, by causing (directly or indirectly) another factor to decrease ATM kinase activity, or by (directly or indirectly) decreasing the amount of ATM kinase present in the cell or organism.

The term "therapy" is intended to have its normal meaning of dealing with a disease in order to entirely or partially relieve one, some or all of its symptoms, or to correct or compensate for the underlying pathology. The term "therapy" also includes "prophylaxis" unless there are specific indications to the contrary. The terms "therapeutic" and "therapeutically" should be interpreted in a corresponding manner.

The term "prophylaxis" is intended to have its normal meaning and includes primary prophylaxis to prevent the development of the disease and secondary prophylaxis whereby the disease has already developed and the patient is temporarily or permanently protected against exacerbation or worsening of the disease or the development of new symptoms associated with the disease.

The term "treatment" is used synonymously with "therapy". Similarly the term "treat" can be regarded as "applying therapy" where "therapy" is as defined herein.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in therapy.

In one embodiment there is provided the use of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of a disease mediated by ATM kinase.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of a disease mediated by ATM kinase, where the disease mediated by ATM kinase is cancer.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of a disease mediated by ATM kinase, where the disease mediated by ATM kinase is colorectal cancer, glioblastoma, gastric cancer, ovarian cancer, diffuse large B-cell lymphoma, chronic lymphocytic leukaemia, acute myeloid leukaemia, head and neck squamous cell carcinoma, breast cancer, hepatocellular carcinoma, small cell lung cancer or non-small cell lung cancer.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of a disease mediated by ATM kinase, where the disease mediated by ATM kinase is colorectal cancer. In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of colorectal cancer, glioblastoma, gastric cancer, ovarian cancer, diffuse large B-cell lymphoma, chronic lymphocytic leukaemia, acute myeloid leukaemia, head and neck squamous cell carcinoma, breast cancer, hepatocellular carcinoma, small cell lung cancer or non-small cell lung cancer.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of colorectal cancer.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of Huntingdon's disease.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use as a neuroprotective agent.

A "neuroprotective agent" is an agent that preserves neuronal structure and/or function.

In one embodiment there is provided the use of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a disease mediated by ATM kinase.

In one embodiment there is provided the use of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a disease mediated by ATM kinase, where the disease mediated by ATM kinase is cancer.

In one embodiment there is provided the use of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a disease mediated by ATM kinase, where the disease mediated by ATM kinase is colorectal cancer, glioblastoma, gastric cancer, ovarian cancer, diffuse large B-cell lymphoma, chronic lymphocytic leukaemia, acute myeloid leukaemia, head and neck squamous cell carcinoma, breast cancer, hepatocellular carcinoma, small cell lung cancer and non-small cell lung cancer. In one embodiment there is provided the use of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a disease mediated by ATM kinase, where the disease mediated by ATM kinase is colorectal cancer.

In one embodiment there is provided the use of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of cancer.

In one embodiment there is provided the use of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of colorectal cancer, glioblastoma, gastric cancer, ovarian cancer, diffuse large B-cell lymphoma, chronic lymphocytic leukaemia, acute myeloid leukaemia, head and neck squamous cell carcinoma, breast cancer, hepatocellular carcinoma, small cell lung cancer or non-small cell lung cancer.

In one embodiment there is provided the use of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of colorectal cancer.

In one embodiment there is provided the use of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of Huntingdon's disease.

In one embodiment there is provided the use of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use as a neuroprotective agent.

In one embodiment there is provided a method for treating a disease in which inhibition of ATM kinase is beneficial in a warm-blooded animal in need of such treatment, which comprises administering to said warm-blooded animal a therapeutically effective amount of a compound of Formula (I), or a

pharmaceutically acceptable salt thereof.

The term "therapeutically effective amount" refers to an amount of a compound of Formula (I) as described in any of the embodiments herein which is effective to provide "therapy" in a subject, or to "treat" a disease or disorder in a subject. In the case of cancer, the therapeutically effective amount may cause any of the changes observable or measurable in a subject as described in the definition of "therapy", "treatment" and "prophylaxis" above. For example, the effective amount can reduce the number of cancer or tumour cells; reduce the overall tumour size; inhibit or stop tumour cell infiltration into peripheral organs including, for example, the soft tissue and bone; inhibit and stop tumour metastasis; inhibit and stop tumour growth; relieve to some extent one or more of the symptoms associated with the cancer; reduce morbidity and mortality; improve quality of life; or a combination of such effects. An effective amount may be an amount sufficient to decrease the symptoms of a disease responsive to inhibition of ATM kinase activity. For cancer therapy, efficacy in-vivo can, for example, be measured by assessing the duration of survival, time to disease progression (TTP), the response rates (RR), duration of response, and/or quality of life. As recognized by those skilled in the art, effective amounts may vary depending on route of administration, excipient usage, and co-usage with other agents. For example, where a combination therapy is used, the amount of the compound of formula (I) or pharmaceutically acceptable salt described in this specification and the amount of the other pharmaceutically active agent(s) are, when combined, jointly effective to treat a targeted disorder in the animal patient. In this context, the combined amounts are in a "therapeutically effective amount" if they are, when combined, sufficient to decrease the symptoms of a disease responsive to inhibition of ATM activity as described above. Typically, such amounts may be determined by one skilled in the art by, for example, starting with the dosage range described in this specification for the compound of formula (I) or pharmaceutically acceptable salt thereof and an approved or otherwise published dosage range(s) of the other pharmaceutically active compound(s).

"Warm-blooded animals" include, for example, humans.

In one embodiment there is provided a method for treating a disease in which inhibition of ATM kinase is beneficial in a warm-blooded animal in need of such treatment, which comprises administering to said warm-blooded animal a therapeutically effective amount of a compound of Formula (I), or a

pharmaceutically acceptable salt thereof, and where the disease in which inhibition of ATM kinase is beneficial is cancer. In one embodiment there is provided a method for treating a disease in which inhibition of ATM kinase is beneficial in a warm-blooded animal in need of such treatment, which comprises administering to said warm-blooded animal a therapeutically effective amount of a compound of Formula (I), or a

pharmaceutically acceptable salt thereof, and where the disease in which inhibition of ATM kinase is beneficial is colorectal cancer, glioblastoma, gastric cancer, ovarian cancer, diffuse large B-cell lymphoma, chronic lymphocytic leukaemia, acute myeloid leukaemia, head and neck squamous cell carcinoma, breast cancer, hepatocellular carcinoma, small cell lung cancer or non-small cell lung cancer.

In one embodiment there is provided a method for treating a disease in which inhibition of ATM kinase is beneficial in a warm-blooded animal in need of such treatment, which comprises administering to said warm-blooded animal a therapeutically effective amount of a compound of Formula (I), or a

pharmaceutically acceptable salt thereof, and where the disease in which inhibition of ATM kinase is beneficial is colorectal cancer.

In one embodiment there is provided a method for treating a disease in which inhibition of ATM kinase is beneficial in a warm-blooded animal in need of such treatment, which comprises administering to said warm-blooded animal a therapeutically effective amount of a compound of Formula (I), or a

pharmaceutically acceptable salt thereof, and where the disease in which inhibition of ATM kinase is beneficial is Huntingdon's disease.

In one embodiment there is provided a method for treating cancer in a warm-blooded animal in need of such treatment, which comprises administering to said warm-blooded animal a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

In one embodiment there is provided a method for treating colorectal cancer, glioblastoma, gastric cancer, ovarian cancer, diffuse large B-cell lymphoma, chronic lymphocytic leukaemia, acute myeloid leukaemia, head and neck squamous cell carcinoma, breast cancer, hepatocellular carcinoma, small cell lung cancer or non-small cell lung cancer in a warm-blooded animal in need of such treatment, which comprises administering to said warm-blooded animal a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

In one embodiment there is provided a method for treating colorectal cancer in a warm-blooded animal in need of such treatment, which comprises

administering to said warm-blooded animal a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

In one embodiment there is provided a method for treating Huntingdon's disease in a warm-blooded animal in need of such treatment, which comprises administering to said warm-blooded animal a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

In one embodiment there is provided a method for effecting neuroprotection in a warm-blooded animal in need of such treatment, which comprises

administering to said warm-blooded animal a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

In one embodiment there is provided a method for treating cancer in a warm-blooded animal in need of such treatment, which comprises administering to said warm-blooded animal a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof. In one embodiment, said cancer is selected from colorectal cancer, glioblastoma, gastric cancer, ovarian cancer, diffuse large B-cell lymphoma, chronic lymphocytic leukaemia, acute myeloid leukaemia, head and neck squamous cell carcinoma, breast cancer, hepatocellular carcinoma, small cell lung cancer and non-small cell lung cancer. In one embodiment, said cancer is selected from colorectal cancer, glioblastoma, gastric cancer, ovarian cancer, diffuse large B-cell lymphoma, chronic lymphocytic leukaemia, head and neck squamous cell carcinoma and lung cancer. In one embodiment, said cancer is colorectal cancer.

In any embodiment where cancer is mentioned in a general sense, said cancer may be selected from colorectal cancer, glioblastoma, gastric cancer, ovarian cancer, diffuse large B-cell lymphoma, chronic lymphocytic leukaemia, acute myeloid leukaemia, head and neck squamous cell carcinoma, breast cancer, hepatocellular carcinoma, small cell lung cancer and non-small cell lung cancer. In any embodiment where cancer is mentioned in a general sense the following embodiments may apply:

In one embodiment the cancer is colorectal cancer.

In one embodiment the cancer is glioblastoma.

In one embodiment the cancer is gastric cancer.

In one embodiment the cancer is oesophageal cancer.

In one embodiment the cancer is ovarian cancer.

In one embodiment the cancer is endometrial cancer.

In one embodiment the cancer is cervical cancer.

In one embodiment the cancer is diffuse large B-cell lymphoma.

In one embodiment the cancer is chronic lymphocytic leukaemia.

In one embodiment the cancer is acute myeloid leukaemia.

In one embodiment the cancer is head and neck squamous cell carcinoma.

In one embodiment the cancer is breast cancer. In one embodiment the i s cancer is triple negative breast cancer.

"Triple negative breast cancer" is any breast cancer that does not test positive for the oestrogen receptor, progesterone receptor and Her2/neu. Test methods to determine a positive test with respect to each of these receptors are well known in the art.

20 In one embodiment the cancer is hepatocellular carcinoma.

In one embodiment the cancer is lung cancer. In one embodiment the lung cancer is small cell lung cancer. In one embodiment the lung cancer is non-small cell lung cancer.

In one embodiment the cancer is metastatic cancer. In one embodiment the 25 metastatic cancer comprises metastases of the central nervous system. In one

embodiment the metastases of the central nervous system comprise brain metastases. In one embodiment the metastases of the central nervous system comprise leptomeningeal metastases.

"Leptomeningeal metastases" occur when cancer spreads to the meninges, 30 the layers of tissue that cover the brain and the spinal cord. Metastases can spread to the meninges through the blood or they can travel from brain metastases, carried by the cerebrospinal fluid (CSF) that flows through the meninges. In one embodiment the cancer is non-metastatic cancer.

The anti-cancer treatment described in this specification may be useful as a sole therapy, or may involve, in addition to administration of the compound of Formula (I), conventional surgery, radiotherapy or chemotherapy; or a combination of such additional therapies. Such conventional surgery, radiotherapy or chemotherapy may be administered simultaneously, sequentially or separately to treatment with the compound of Formula (I).

Radiotherapy may include one or more of the following categories of therapy:

i. External radiation therapy using electromagnetic radiation, and

intraoperative radiation therapy using electromagnetic radiation;

ii. Internal radiation therapy or brachytherapy; including interstitial radiation therapy or intraluminal radiation therapy; or

iii. Systemic radiation therapy, including but not limited to iodine 131 and strontium 89.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer, where the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered in combination with radiotherapy. In one embodiment the

radiotherapy is selected from one or more of the categories of radiotherapy listed under points (i) - (iii) above.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of glioblastoma, lung cancer (for example small cell lung cancer or non-small cell lung cancer), breast cancer (for example triple negative breast cancer), head and neck squamous cell carcinoma, oesophageal cancer, cervical cancer or endometrial cancer, where the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered in combination with radiotherapy. In one embodiment the

radiotherapy is selected from one or more of the categories of radiotherapy listed under points (i) - (iii) above. In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of glioblastoma, where the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered in combination with radiotherapy. In one embodiment the radiotherapy is selected from one or more of the categories of radiotherapy listed under points (i) - (iii) above.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of metastatic cancer, where the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered in combination with radiotherapy. In one embodiment the radiotherapy is selected from one or more of the categories of radiotherapy listed under points (i) - (iii) above.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of metastases of the central nervous system, where the compound of Formula (I), or a

pharmaceutically acceptable salt thereof, is administered in combination with radiotherapy. In one embodiment the radiotherapy is selected from one or more of the categories of radiotherapy listed under points (i) - (iii) above.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of leptomeningeal metastases, where the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered in combination with radiotherapy. In one embodiment the radiotherapy is selected from one or more of the categories of radiotherapy listed under points (i) - (iii) above.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer, where the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered simultaneously, separately or sequentially with radiotherapy. In one embodiment the radiotherapy is selected from one or more of the categories of radiotherapy listed under points (i) - (iii) above.

In one embodiment there is provided a method of treating cancer in a warmblooded animal who is in need of such treatment, which comprises administering to said warm-blooded animal a compound of Formula (I), or a pharmaceutically acceptable salt thereof and radiotherapy, wherein the compound of Formula (I), or a pharmaceutically acceptable salt thereof, and radiotherapy are jointly effective in producing an anti-cancer effect. In one embodiment the cancer is selected from glioblastoma, lung cancer (for example small cell lung cancer or non-small cell lung cancer), breast cancer (for example triple negative breast cancer), head and neck squamous cell carcinoma, oesophageal cancer, cervical cancer and

endometrial cancer. In one embodiment the cancer is glioblastoma. In one embodiment, the cancer is metastatic cancer. In one embodiment the metastatic cancer comprises metastases of the central nervous system. In one embodiment the metastases of the central nervous system comprise brain metastases. In one embodiment the metastases of the central nervous system comprise leptomeningeal metastases. In any embodiment the radiotherapy is selected from one or more of the categories of radiotherapy listed under points (i) - (iii) above.

In one embodiment there is provided a method of treating cancer in a warmblooded animal who is in need of such treatment, which comprises administering to said warm-blooded animal a compound of Formula (I), or a pharmaceutically acceptable salt thereof and simultaneously, separately or sequentially administering radiotherapy, wherein the compound of Formula (I), or a pharmaceutically acceptable salt thereof, and radiotherapy are jointly effective in producing an anticancer effect. In one embodiment the cancer is glioblastoma. In one embodiment, the cancer is metastatic cancer. In one embodiment the metastatic cancer comprises metastases of the central nervous system. In one embodiment the metastases of the central nervous system comprise brain metastases. In one embodiment the metastases of the central nervous system comprise leptomeningeal metastases. In any embodiment the radiotherapy is selected from one or more of the categories of radiotherapy listed under points (i) - (iii) above.

Chemotherapy may include one or more of the following categories of anti- tumour substance:

i. Antineoplastic agents and combinations thereof, such as DNA alkylating agents (for example cisplatin, oxaliplatin, carboplatin, cyclophosphamide, nitrogen mustards like ifosfamide, bendamustine, melphalan, chlorambucil, busulphan, temozolamide and nitrosoureas like carmustine); antimetabolites (for example gemcitabine and antifolates such as fluoropyrimidines like 5-fluorouracil and tegafur, raltitrexed, methotrexate, cytosine arabinoside, and hydroxyurea); anti-tumour antibiotics (for example anthracyclines like adriamycin, bleomycin, doxorubicin, liposomal doxorubicin, pirarubicin, daunomycin, valrubicin, epirubicin, idarubicin, mitomycin-C, dactinomycin, amrubicin and mithramycin); antimitotic agents (for example vinca alkaloids like vincristine, vinblastine, vindesine and vinorelbine and taxoids like taxol and taxotere and polokinase inhibitors); and topoisomerase inhibitors (for example epipodophyllotoxins like etoposide and teniposide, amsacrine, irinotecan, topotecan and camptothecin); inhibitors of DNA repair mechanisms such as CHK kinase; DNA-dependent protein kinase inhibitors; inhibitors of poly (ADP-ribose) polymerase (PARP inhibitors, including olaparib); and Hsp90 inhibitors such as tanespimycin and retaspimycin, inhibitors of ATR kinase (such as AZD6738); and inhibitors of WEE 1 kinase (such as AZD1775/MK-1775);

Antiangio genie agents such as those that inhibit the effects of vascular endothelial growth factor, for example the anti- vascular endothelial cell growth factor antibody bevacizumab and for example, a VEGF receptor tyrosine kinase inhibitor such as vandetanib (ZD6474), sorafenib, vatalanib (PTK787), sunitinib (SU11248), axitinib (AG-013736), pazopanib (GW 786034) and cediranib (AZD2171); compounds such as those disclosed in International Patent Applications W097/22596, WO 97/30035, WO

97/32856 and WO 98/13354; and compounds that work by other

mechanisms (for example linomide, inhibitors of integrin ανβ3 function and angiostatin), or inhibitors of angiopoietins and their receptors (Tie-1 and Tie-2), inhibitors of PLGF, inhibitors of delta-like ligand (DLL-4);

Immunotherapy approaches, including for example ex-vivo and in-vivo approaches to increase the immunogenicity of patient tumour cells, such as transfection with cytokines such as interleukin 2, interleukin 4 or granulocyte-macrophage colony stimulating factor; approaches to decrease T-cell anergy or regulatory T-cell function; approaches that enhance T-cell responses to tumours, such as blocking antibodies to CTLA4 (for example ipilimumab and tremelimumab), B7H1, PD-1 (for example BMS-936558 or AMP-514), PD-L1 (for example MEDI4736) and agonist antibodies to CD 137; approaches using transfected immune cells such as

cytokine-transfected dendritic cells; approaches using cytokine -transfected tumour cell lines, approaches using antibodies to tumour associated antigens, and antibodies that deplete target cell types (e.g., unconjugated anti-CD20 antibodies such as Rituximab, radiolabeled anti-CD20 antibodies Bexxar and Zevalin, and anti-CD54 antibody Campath); approaches using anti-idiotypic antibodies; approaches that enhance Natural Killer cell function; and approaches that utilize antibody-toxin conjugates (e.g. anti- CD33 antibody Mylotarg); immunotoxins such as moxetumumab pasudotox; agonists of toll-like receptor 7 or toll-like receptor 9;

iv. Efficacy enhancers, such as leucovorin.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer, where the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered in combination with at least one additional anti-tumour substance. In one embodiment there is one additional anti-tumour substance. In one embodiment there are two additional anti-tumour substances. In one embodiment there are three or more additional anti-tumour substances. In any embodiment the additional anti- tumour substance is selected from one or more of the anti-tumour substances listed under points (i) - (iv) above.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer, where the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered simultaneously, separately or sequentially with at least one additional anti-tumour substance. In one embodiment there is one additional anti-tumour substance. In one embodiment there are two additional anti-tumour substances. In one embodiment there are three or more additional anti-tumour substances. In any embodiment the additional anti-tumour substance is selected from one or more of the anti-tumour substances listed under points (i) - (iv) above. In one embodiment there is provided a method of treating cancer in a warmblooded animal who is in need of such treatment, which comprises administering to said warm-blooded animal a compound of Formula (I), or a pharmaceutically acceptable salt thereof and at least one additional anti-tumour substance, wherein the amounts of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, and the additional anti-tumour substance are jointly effective in producing an anti-cancer effect. In any embodiment the additional anti-tumour substance is selected from one or more of the anti-tumour substances listed under points (i) - (iv) above.

In one embodiment there is provided a method of treating cancer in a warmblooded animal who is in need of such treatment, which comprises administering to said warm-blooded animal a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and simultaneously, separately or sequentially administering at least one additional anti-tumour substance to said warm-blooded animal, wherein the amounts of the compound of Formula (I), or pharmaceutically acceptable salt thereof, and the additional anti-tumour substance are jointly effective in producing an anti-cancer effect. In any embodiment the additional anti-tumour substance is selected from one or more of the anti-tumour substances listed under points (i) - (iv) above.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one anti-neoplastic agent for use in the treatment of cancer. In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer, where the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered in combination with at least one anti-neoplastic agent. In one embodiment the anti-neoplastic agent is selected from the list of

antineoplastic agents in point (i) above.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one anti-neoplastic agent for use in the simultaneous, separate or sequential treatment of cancer. In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer, where the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered simultaneously, separately or sequentially with at least one anti-neoplastic agent. In one embodiment the antineoplastic agent is selected from the list of antineoplastic agents in point (i) above.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer, where the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered simultaneously, separately or sequentially with at least one additional anti-tumour substance selected from cisplatin, oxaliplatin, carboplatin, valrubicin, idarubicin, doxorubicin, pirarubicin, irinotecan, topotecan, amrubicin, epirubicin, etoposide, mitomycin, bendamustine, chlorambucil, cyclophosphamide, ifosfamide, carmustine, melphalan, bleomycin, olaparib, MEDI4736, AZD1775 and AZD6738.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer, where the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered simultaneously, separately or sequentially with at least one additional anti-tumour substance selected from cisplatin, oxaliplatin, carboplatin, doxorubicin, pirarubicin, irinotecan, topotecan, amrubicin, epirubicin, etoposide, mitomycin, bendamustine, chlorambucil, cyclophosphamide, ifosfamide, carmustine, melphalan, bleomycin, olaparib, AZD1775 and AZD6738.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer, where the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered simultaneously, separately or sequentially with at least one additional anti-tumour substance selected from doxorubicin, irinotecan, topotecan, etoposide, mitomycin, bendamustine, chlorambucil, cyclophosphamide, ifosfamide, carmustine, melphalan, bleomycin and olaparib.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer, where the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered simultaneously, separately or sequentially with at least one additional anti-tumour substance selected from doxorubicin, irinotecan, topotecan, etoposide, mitomycin, bendamustine, chlorambucil, cyclophosphamide, ifosfamide, carmustine, melphalan and bleomycin.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer, where the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered simultaneously, separately or sequentially with at least one additional anti-tumour substance selected from doxorubicin, pirarubicin, amrubicin and epirubicin.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of acute myeloid leukaemia, where the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered simultaneously, separately or sequentially with at least one additional anti-tumour substance selected from doxorubicin, pirarubicin, amrubicin and epirubicin.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of breast cancer, where the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered simultaneously, separately or sequentially with at least one additional anti-tumour substance selected from doxorubicin, pirarubicin, amrubicin and epirubicin.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of triple negative breast cancer, where the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered simultaneously, separately or sequentially with at least one additional anti-tumour substance selected from doxorubicin, pirarubicin, amrubicin and epirubicin.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of hepatocellular carcinoma, where the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered simultaneously, separately or sequentially with at least one additional anti-tumour substance selected from doxorubicin, pirarubicin, amrubicin and epirubicin. In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer, where the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered simultaneously, separately or sequentially with irinotecan.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of colorectal cancer, where the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered simultaneously, separately or sequentially with irinotecan.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of colorectal cancer, where the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered simultaneously, separately or sequentially with FOLFIRI.

FOLFIRI is a dosage regime involving a combination of leucovorin, 5- fluorouracil and irinotecan.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer, where the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered simultaneously, separately or sequentially with olaparib.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of gastric cancer, where the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered simultaneously, separately or sequentially with olaparib.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer, where the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered simultaneously, separately or sequentially with topotecan.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of lung cancer, where the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered simultaneously, separately or sequentially with topotecan.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of small cell lung cancer, where the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered simultaneously, separately or sequentially with topotecan.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer, where the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered simultaneously, separately or sequentially with immunotherapy. In one embodiment the immunotherapy is one or more of the agents listed under point (iii) above.

In one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer, where the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered simultaneously, separately or sequentially with an anti-PD-Ll antibody (for example MEDI4736).

According to a further embodiment there is provided a kit comprising:

a) A compound of formula (I), or a pharmaceutically acceptable salt thereof, in a first unit dosage form;

b) A further additional anti-tumour substance in a further unit dosage form; c) Container means for containing said first and further unit dosage forms; and optionally

d) Instructions for use. In one embodiment the anti-tumour substance comprises an anti-neoplastic agent.

In any embodiment where an anti-neoplastic agent is mentioned, the antineoplastic agent is one or more of the agents listed under point (i) above.

The compounds of Formula (I), and pharmaceutically acceptable salts thereof, may be administered as pharmaceutical compositions, comprising one or more pharmaceutically acceptable excipients.

Therefore, in one embodiment there is provided a pharmaceutical composition comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

The excipient(s) selected for inclusion in a particular composition will depend on factors such as the mode of administration and the form of the composition provided. Suitable pharmaceutically acceptable excipients are well known to persons skilled in the art and are described, for example, in the Handbook of Pharmaceutical Excipients, Sixth edition, Pharmaceutical Press, edited by Rowe, Ray C; Sheskey, Paul J; Quinn, Marian. Pharmaceutically acceptable excipients may function as, for example, adjuvants, diluents, carriers, stabilisers, flavourings, colorants, fillers, binders, disintegrants, lubricants, glidants, thickening agents and coating agents. As persons skilled in the art will appreciate, certain

pharmaceutically acceptable excipients may serve more than one function and may serve alternative functions depending on how much of the excipient is present in the composition and what other excipients are present in the composition.

The pharmaceutical compositions may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular or intramuscular dosing), or as a suppository for rectal dosing. The compositions may be obtained by conventional procedures well known in the art. Compositions intended for oral use may contain additional components, for example, one or more colouring, sweetening, flavouring and/or preservative agents.

The compound of Formula (I) will normally be administered to a warm-blooded animal at a unit dose within the range 2.5-5000 mg/m ² body area of the animal, or approximately 0.05-100 mg/kg, and this normally provides a therapeutically-effective dose. A unit dose form such as a tablet or capsule will usually contain, for example 0.1-250 mg of active ingredient. The daily dose will necessarily be varied depending upon the host treated, the particular route of administration, any therapies being co -administered, and the severity of the illness being treated. Accordingly the practitioner who is treating any particular patient may determine the optimum dosage. The pharmaceutical compositions described herein comprise compounds of Formula (I), or a pharmaceutically acceptable salt thereof, and are therefore expected to be useful in therapy.

As such, in one embodiment there is provided a pharmaceutical composition for use in therapy, comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

In one embodiment there is provided a pharmaceutical composition for use in the treatment of a disease in which inhibition of ATM kinase is beneficial, comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

In one embodiment there is provided a pharmaceutical composition for use in the treatment of cancer, comprising a compound of Formula (I), or a

pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

In one embodiment there is provided a pharmaceutical composition for use in the treatment of a cancer in which inhibition of ATM kinase is beneficial, comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

In one embodiment there is provided a pharmaceutical composition for use in the treatment of colorectal cancer, glioblastoma, gastric cancer, ovarian cancer, diffuse large B-cell lymphoma, chronic lymphocytic leukaemia, acute myeloid leukaemia, head and neck squamous cell carcinoma, breast cancer, hepatocellular carcinoma, small cell lung cancer or non-small cell lung cancer, comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient. EXAMPLES

The various embodiments of the invention are illustrated by the following Examples. The invention is not to be interpreted as being limited to the Examples. During the preparation of the Examples, generally:

i. Operations were carried out at ambient temperature, i.e. in the range of about 17 to 30°C and under an atmosphere of an inert gas such as nitrogen unless otherwise stated;

ii. Evaporations were carried out by rotary evaporation or utilising Genevac equipment in vacuo and work-up procedures were carried out after removal of residual solids by filtration;

iii. Flash chromatography purifications were performed on an automated

Armen Glider Flash: Spot II Ultimate (Armen Instrument, Saint -Ave, France) or automated Presearch combiflash companions using prepacked Merck normal phase Si60 silica cartridges (granulometry: 15-40 or 40- 63μιη) obtained from Merck, Darmstad, Germany, silicycle silica cartridges or graceresolv silica cartridges;

iv. Preparative chromatography was performed on a Waters instrument

(600/2700 or 2525) fitted with a ZMD or ZQ ESCi mass spectrometers and a Waters X-Terra or a Waters X-Bridge or a Waters SunFire reverse-phase column (C-18, 5 microns silica, 19 mm or 50 mm diameter, 100 mm length, flow rate of 40 mL / minute) using decreasingly polar mixtures of water (containing 1% ammonia) and acetonitrile or decreasingly polar mixtures of water (containing 0.1% formic acid) and acetonitrile as eluents;

v. Yields, where present, are not necessarily the maximum attainable;

vi. Structures of end-products of Formula (I) were confirmed by nuclear

magnetic resonance (NMR) spectroscopy, with NMR chemical shift values measured on the delta scale. Proton magnetic resonance spectra were determined using a Bruker advance 700 (700MHz), Bruker Avance 500 (500 MHz), Bruker 400 (400 MHz) or Bruker 300 (300 MHz) instrument; 19F NMR were determined at 282 MHz or 376 MHz; 13C NMR were determined at 75 MHz or 100 MHz; measurements were taken at around 20 - 30°C unless otherwise specified; the following abbreviations have been used: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of doublets; ddd, doublet of doublet of doublet; dt, doublet of triplets; bs, broad signal;

End-products of Formula (I) were also characterised by mass spectroscopy following liquid chromatography (LCMS); LCMS was carried out using an Waters Alliance HT (2790 & 2795) fitted with a Waters ZQ ESCi or ZMD ESCi mass spectrometer and an X Bridge 5μιη C-l 8 column (2.1 x 50 mm) at a flow rate of 2.4 mL/min, using a solvent system of 95% A + 5% C to 95% B + 5% C over 4 minutes, where A = water, B = methanol, C = 1 : 1 methanol: water (containing 0.2% ammonium carbonate); or by using a Shimadzu UFLC or UHPLC coupled with DAD detector, ELSD detector and 2020 EV mass spectrometer (or equivalent) fitted with a Phenomenex Gemini-NX CI 8 3.0x50 mm, 3.0 μΜ column or equivalent (basic conditions) or a Shim pack XR - ODS 3.0 x 50 mm, 2.2 μΜ column or Waters BEH C18 2.1 x 50 mm, 1.7 μΜ column or equivalent using a solvent system of 95% D + 5% E to 95% E + 5% D over 4 minutes, where D = water (containing 0.05% TFA), E = Acetonitrile (containing 0.05% TFA) (acidic conditions) or a solvent system of 90% F + 10% G to 95% G + 5% F over 4 minutes, where F = water (containing 6.5 mM ammonium hydrogen carbonate and adjusted to pHIO by addition of ammonia), G = Acetonitrile (basic conditions);

Intermediates were not generally fully characterised and purity was assessed by thin layer chromatographic, mass spectral, HPLC and/or NMR analysis; X-ray powder diffraction spectra were determined (using a Bruker D4 Analytical Instrument) by mounting a sample of the crystalline material on a Bruker single silicon crystal (SSC) wafer mount and spreading out the sample into a thin layer with the aid of a microscope slide. The sample was spun at 30 revolutions per minute (to improve counting statistics) and irradiated with X-rays generated by a copper long-fine focus tube operated at 40kV and 40mA with a wavelength of 1.5418 angstroms. The collimated X-ray source was passed through an automatic variable divergence slit set at V20 and the reflected radiation directed through a 5.89mm antiscatter slit and a 9.55mm detector slit. The sample was exposed for 0.03 seconds per 0.00570° 2-theta increment (continuous scan mode) over the range 2 degrees to 40 degrees 2-theta in theta-theta mode. The running time was 3 minutes and 36 seconds. The instrument was equipped with a Position sensitive detector (Lynxeye). Control and data capture was by means of a Dell Optiplex 686 NT 4.0 Workstation operating with Diffrac+ software; Differential Scanning Calorimetry was performed on a TA Instruments Q1000 DSC. Typically, less than 5mg of material contained in a standard aluminium pan fitted with a lid was heated over the temperature range 25 °C to 300°C at a constant heating rate of 10°C per minute. A purge gas using nitrogen was used at a flow rate 50ml per minute

The following abbreviations have been used: h = hour(s); r.t. = room temperature (~18-25°C); cone. = concentrated; FCC = flash column chromatography using silica; DCM = dichloromethane; DIPEA = diisopropylethylamine; DMA = N,N-dimethylacetamide; DMF = N,N- dimethylformamide; DMSO = dimethylsulfoxide; Et ₂ 0 = diethyl ether; EtOAc = ethyl acetate; EtOH = ethanol; K ₂ C0 ₃ = potassium carbonate; MeOH = methanol; MeCN = acetonitrile; MTBE = Methyltertbutylether; MgS04 = anhydrous magnesium sulphate; Na ₂ S0 ₄ = anhydrous sodium sulphate; THF = tetrahydrofuran; sat. = saturated aqueous solution; and IUPAC names were generated using either ELN, a proprietary program or "Canvas" or "IBIS", AstraZeneca proprietary programs. As stated in the introduction, the compounds of the invention comprise an imidazo[4,5- c]quinolin-2-one core. However, in certain Examples the IUPAC name describes the core as an imidazo[5,4-c]quinolin-2-one or an 1H- imidazo[4,5-c]quinolin-2(3H)-one core. The imidazo[4,5-c]quinolin-2-one, lH-imidazo[4,5-c]quinolin-2(3H)-one and imidazo[5,4-c]quinolin-2-one cores are nevertheless the same, with the naming convention different because of the peripheral groups. Example 1

(R)-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(tetrahydro -2H-pyran-3- yl)-lH-imidazo[4,5-c]quinolin-2 3H)-one

Dichlorobis(di-tert-butyl(3-sulfopropyl)phosphonio)palladate (II) (0.05M in water) (8.28 mL, 0.41 mmol) was added to a degassed mixture of 2-(methoxymethyl)-5- (4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)pyridine (2.476 g, 9.94 mmol), (R)-8- bromo-3-methyl-l-(tetrahydro-2H-pyran-3-yl)-lH-imidazo[4,5-c ]quinolin-2(3H)- one (3 g, 8.28 mmol) and 2M K2CO3 solution (12.42 mL, 24.85 mmol) in 1,4- dioxane (50 mL). The mixture was heated to reflux for 5 h. The reaction mixture was diluted with water then extracted with EtOAc (3 x 100 mL). The combined organic phases were washed with water (100 mL), brine and then dried over MgSCM, filtered and concentrated in vacuo onto silica. The crude product was purified by FCC, elution gradient 0 to 10% methanolic ammonia in DCM. Pure fractions were evaporated to dryness. The solid was taken up in MeCN (25 mL), heated at 60°C for 1 h. The solid was collected by vacuum filtration to afford (R)-8- (6-(methoxymethyl)pyridin-3 -yl)-3 -methyl- 1 -(tetrahydro-2H-pyran-3 -yl)- 1 H- imidazo[4,5-c]quinolin-2(3H)-one (1.868 g, 55.8 %) as a white solid. NMR

Spectrum: ¹ H NMR (500MHz, DMSO-d6) δ 1.77 - 1.86 (2H, m), 2.16 (1H, d), 2.6 - 2.72 (1H, m), 3.34 - 3.4 (1H, m), 3.41 (3H, s), 3.49 (3H, s), 3.93 (1H, d), 4.13 (1H, dd), 4.21 (1H, t), 4.58 (2H, s), 4.93 - 5.04 (1H, m), 7.58 (1H, d), 7.99 (1H, dd), 8.17 (1H, d), 8.24 (1H, dd), 8.41 (1H, d), 8.90 (1H, s), 8.98 (1H, d). Mass Spectrum: m/z: ES+ [M+H]+ 405.

The following compounds were prepared in an analogous fashion from the appropriate boronic ester and bromo intermediates.

Examples 4 & 5 were separated from a racemic mixture by preparative chiral- HPLC (Chiralpak AS column), eluting isocratically with 80% heptane in EtOH (modified with triethylamine) as eluent, to afford Example 4 as the first eluting product and Example 5 as the second eluting product.

Examples 6 & 7 were separated by preparative HPLC (Chiralpak AD column), eluting isocratically with 70% heptane in EtOH-MeOH (modified with

triethylamine) as eluent. The material was then further purified by preparative HPLC (Chiralpak AD column), eluting isocratically with 80% heptane in EtOH- MeOH (modified with triethylamine) as eluent to afford Example 6 as the first eluting product and Example 7 as the second eluting product.

Example 2

NMR Spectrum: ¹ H NMR (500MHz, DMSO-d6) δ 1.77 - 1.88 (2H, m), 2.16 (1H, d), 2.61 - 2.73 (1H, m), 3.35 - 3.4 (1H, m), 3.41 (3H, s), 3.48 (3H, s), 3.93 (1H, d), 4.1 - 4.17 (1H, m), 4.22 (1H, t), 4.58 (2H, s), 4.93 - 5.03 (1H, m), 7.58 (1H, dd), 7.98 (1H, dd), 8.16 (1H, d), 8.24 (1H, dd), 8.41 (1H, d), 8.90 (1H, s), 8.98 (1H, dd). Mass Spectrum: m/z: ES+ [M+H]+ 405.

(5)-8-(6-(Methoxymethyl)pyridin-3-yl)-3-methyl-l-(tetrahy dro-2H-pyran-3-yl)-lH^ imidazo[4,5-c]quinolin-2(3H)-one can also be obtained as a crystalline solid by slurrying the amorphous solid in MeCN (10 mL/g). The slurry was heated at 60°C for 1 h then stirred at r.t. overnight. The crystalline solid was collected by vacuum filtration to afford (5)-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(tetrahydro - 2H-pyran-3-yl)-lH-imidazo[4,5-c]quinolin-2(3H)-one as a white crystalline solid, Crystallinity was confirmed using X-Ray Powder Diffraction (XRPD). Example 2 Form A is characterised in providing an X-ray powder diffraction pattern substantially as shown in Figure 1. Ten X-Ray powder diffraction peaks are shown in Table 1 : Table 1: Characteristic X-Ray powder diffraction peaks for Form A of Example 2, (5)-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(tetrahydro -2H-pyran-3-yl)-lH- imidazo[4,5-c]quinolin-2(3H)-one

Example 2 Form A displays the following thermal parameters: a melting endotherm with an onset of 184.4°C and a peak at 186.0°C as determined by differential scanning calorimetry (DSC) at a scanning rate of 10°C/mins (Figure 2).

Example 3

NMR Spectrum: ¹ H NMR (500MHz, DMSO-d6) δ 1.66 - 1.87 (2H, m), 2.13 (1H, d), 2.57 - 2.73 (1H, m), 3.32 - 3.41 (1H, m), 3.42 (3H, s), 3.48 (3H, s), 3.82 - 3.97 (1H, m), 4.04 - 4.15 (1H, m), 4.19 (1H, t), 4.59 (2H, s), 4.79 - 5.06 (1H, m), 7.60 (1H, dd), 7.95 (1H, d), 8.16 (1H, dt), 8.30 (1H, d), 8.86 (1H, s), 8.93 (1H, s). Mass Spectrum: m/z (ES+)[M+H]+ = 423.

This material can also be isolated as the methanesulfonic acid salt by dissolving in a small quantity of water and treating with an equivalent of methanesulfonic acid dissolved in a small quantity of water and then removing the water by

lyophilisation. NMR Spectrum: ¹ H NMR (300MHz, MeOH-d4) δ 1.83 - 1.98 (2H, m), 2.27 (1H, d), 2.71 (3H, s), 2.74 - 2.94 (1H, m), 3.46 - 3.54 (1H, m), 3.56 (3H, s), 3.61 (3H, s), 4.00 (1H, dd), 4.15 - 4.27 (1H, m), 4.38 (1H, t), 4.73 (2H, s), 5.02 - 5.19 (1H, m), 7.77 - 7.87 (1H, m), 8.02 (1H, d), 8.32 - 8.43 (1H, m), 8.59 (1H, d), 8.93 (1H, d), 9.09 (1H, s). Mass Spectrum: m/z (ES+)[M+H]+ = 423.

The free base (5)-7-fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l- (tetrahydro-2H-pyran-3-yl)-l,3-dihydro-2H-imidazo[4,5-c]quin olin-2-one can also be obtained as a crystalline solid. Example 3 Form A was produced by

recrystallising the material (20 mg) in tetrahydrofuran (2 mL) at 25 °C. Example 3 Form B was produced by slurrying the material (20 mg) in cyclohexane (2 mL) for 7 days. The solvent was removed by evaporation under ambient conditions to afford crystalline material as determined by XRPD and DSC. Example 3 Form A is characterised in providing an X-ray powder diffraction pattern substantially as shown in Figure 3. Ten X-Ray powder diffraction peaks are shown in Table 2: Table 2: Characteristic X-Ray powder diffraction peaks for Form A of Example 3, (5)-7-fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(t etrahydro-2H-pyran- 3 -yl)- 1 , 3 -dihydro -2H-imidazo [4 , 5 -c] quino lin-2-one

Example 3 Form A displays the following thermal parameters: a melting endotherm with an onset of 174.5°C and a peak at 177.3°C as determined by differential scanning calorimetry (DSC) at a scanning rate of 10°C/mins (Figure 4).

Example 3 Form B is characterised in providing an X-ray powder diffraction pattern substantially as shown in Figure 5. Ten X-Ray powder diffraction peaks are shown in Table 3 : Table 3: Characteristic X-Ray powder diffraction peaks for Form B of Example 3, (5)-7-fluoro-8-(6-(methoxymethyl)pyridin-3-yl)-3-methyl-l-(t etrahydro-2H-pyran- 3 -yl)- 1 , 3 -dihydro -2H-imidazo [4 , 5 -c] quino lin-2-one

Example 3 Form B displays the following thermal parameters: a melting endotherm with an onset of 174.5°C and a peak at 175.5°C as determined by differential scanning calorimetry (DSC) at a scanning rate of 10°C/mins (Figure 6).

Example 4

NMR Spectrum: ¹ H NMR (400MHz, DMSO-d6) δ 1.71 - 1.82 (2H, m), 2.12 (1H, d), 2.56 - 2.68 (1H, m), 3.31 - 3.41 (1H, m), 3.45 (3H, s), 3.50 (3H, s), 3.88 (1H, d), 3.99 - 4.14 (1H, m), 4.24 (1H, t), 4.56 (2H, s), 4.79 - 4.95 (1H, m), 7.59 (1H, dd), 7.97 (1H, d), 8.29 (1H, dd), 8.37 (1H, d), 8.96 (1H, s). Mass Spectrum: m/z

(ES+)[M+H]+ = 441.

Example 5

NMR Spectrum: ¹ H NMR (400MHz, DMSO-d6) δ 1.71 - 1.82 (2H, m), 2.12 (1H, d), 2.56 - 2.66 (1H, m), 3.32 - 3.42 (1H, m), 3.45 (3H, s), 3.50 (3H, s), 3.89 (1H, d), 3.99 - 4.14 (1H, m), 4.24 (1H, t), 4.56 (2H, s), 4.78 - 4.98 (1H, m), 7.58 (1H, dd), 7.97 (1H, d), 8.29 (1H, dd), 8.37 (1H, d), 8.96 (1H, s). Mass Spectrum: m/z

(ES+)[M+H]+ = 441

Example 6

NMR Spectrum: ¹ H NMR (400 MHz, DMSO-d6) δ 1.78 - 1.86 (2H, m), 2.13 - 2.19 (1H, m), 2.62 (1H, dd), 3.35 - 3.43 (1H, m), 3.44 (3H, s), 3.51 (3H, s), 3.93 (1H, d), 4.05 - 4.17 (1H, m), 4.29 (1H, t), 4.54 (2H, s), 4.89 - 4.93 (1H, m), 7.56 (1H, d), 7.90 (1H, d), 8.18 (1H, d), 8.35 (1H, dd), 8.50 (1H, s), 8.94 (1H, s). Mass

Spectrum: m/z: ES+ [M+H]+ 423

Example 7

NMR Spectrum: ¹ H NMR (400 MHz, DMSO-d6) δ 1.83 (2H, d), 2.16 (1H, d), 2.58 - 2.64 (1H, m), 3.35 - 3.41 (1H, m), 3.44 (3H, s), 3.51 (3H, s), 3.93 (1H, d), 4.10 (1H, d), 4.29 (1H, t), 4.54 (2H, s), 4.84 - 5 (1H, m), 7.56 (1H, d), 7.90 (1H, d), 8.19 (1H, d), 8.35 (1H, dd), 8.50 (1H, s), 8.94 (1H, s). Mass Spectrum: m/z: ES+

[M+H]+ 423

Example 8

NMR Spectrum: ¹ H NMR (400 MHz, MeOD-d ₄ ) δ 1.84 - 1.96 (2H, m), 2.24 (1H, d), 2.73 - 2.86 (1H, m), 3.50 - 3.58 (1H, m), 3.54 (3H, s), 3.59 (3H, s), 4.00 (1H, d), 4.19 (1H, d), 4.39 (1H, t), 4.68 (2H, s), 4.96 - 5.09 (1H, m), 7.72 (1H, d), 7.91 (1H, d), 8.20 - 8.28 (1H, m), 8.44 (1H, d), 8.87 (2H, s). Mass Spectrum: m/z (ES+), [M+H]+ = 423.

This material can also be isolated as the methanesulfonic acid salt by dissolving in a small quantity of water and treating with an equivalent of methanesulfonic acid dissolved in a small quantity of water and then removing the water by

lyophilisation. NMR Spectrum: ¹ H NMR (300 MHz, MeOD-d ₄ ) δ 1.83 - 1.97 (2H, m), 2.27 (1H, d), 2.70 (3H, s), 2.78 - 2.90 (1H, m), 3.49 - 3.59 (1H, m), 3.56 (3H, s), 3.61 (3H, s), 4.00 (1H, d), 4.15 - 4.27 (1H, m), 4.39 (1H, t), 4.73 (2H, s), 5.02 - 5.19 (1H, m), 7.82 (1H, d), 8.02 (1H, d), 8.31 - 8.42 (1H, m), 8.60 (1H, d), 8.93 (1H, s), 9.10 (1H, s). Mass Spectrum: m/z (ES+), [M+H]+ = 423. The boronic acid 2-(methoxymethyl)-5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan- 2- yl)pyridine was prepared as follows:

2-(Methoxymethyl)-5-(4,4,5,5-tetrameth l-l,3,2-dioxaborolan-2-yl)pyridine

A stirred mixture of 5-bromo-2-(methoxymethyl)pyridine (60g, 297mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(l,3,2-dioxaborolane) (94g, 371mmol), potassium acetate (87g, 891mmol) and PdCi2(dppf) (0.217g, 0.30mmol) in dioxane (1 L) under nitrogen was stirred at 100°C for 16 h. The mixture was concentrated and filtered through a Celite pad, eluted with hexane (1 L) and evaporated to give 2-(methoxymethyl)-5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan- 2-yl)pyridine (180g, 162%) as a brown oil, which was used without further purification. Mass Spectrum: m/z [M+H] ⁺ = 250. 5-Bromo-2-(methoxy methyl)pyridine

Sodium hydride (4.98 g, 207 mmol) was added portionwise to (5-bromopyridin-2- yl)methanol (30 g, 160mmol) in THF (450 mL) at 0°C over a period of 10 minutes under nitrogen. The resulting solution was stirred at 25°C for 0.5 h. Methyl iodide (12.97mL, 207mmol) was added dropwise to the solution at 0°C. The resulting mixture was stirred at 25 °C for 16 h. The reaction mixture was diluted with MeOH (20 mL). The crude product was purified by FCC, eluting with a gradient of 10% to 60% EtOAc in heptane. Pure fractions were evaporated to dryness to afford 5- bromo-2-(methoxymethyl)pyridine (30.0 g, 93%>) as a yellow liquid. Ή NMR Spectrum (300 MHz, CDC1 ₃ ): δ 3.48 (3H, s), 4.54 (2H, s), 7.34 (1H, dd), 7.83 (1H, dd), 8.61 (1H, d). The boronic acid 2-fluoro-6-(methoxymethyl)-3-(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)pyridine was prepared as follows:

2-Fluoro-6-(methoxymethyl)-3-(4,4,5,5-tetramethyl-l,3,2-d ioxaborolan-2- yl)pyridine

3-Bromo-2-fluoro-6-(methoxymethyl)pyridine (2.2 g, 10.00 mmol),

4,4,4 ^* ,4 ^, ,5,5,5 ^* ,5 ^* -octamethyl-2,2 ^* -bi(l,3,2-dioxaborolane) (2.79 g, 11.00 mmol), potassium acetate (2.94g, 30mmol) and {Ι,Γ- ¾z ^' 5(diphenylphosphino)ferrocene}palladium chloride in complex with CH2CI2 (0.243 g, 0.30 mmol) were suspended in degassed 1,4-dioxane (30 mL). The reaction was heated at 80°C for 5 h under a nitrogen atmosphere. The crude mixture was filtered over celite and washed with MeCN, then EtOAc. The filtrate was concentrated in vacuo to provide 2-fluoro-6-(methoxymethyl)-3-(4,4,5,5- tetramethyl-l,3,2-dioxaborolan-2-yl)pyridine (3.79 g, 142%) as a black solid which was used without further purification. ^! H NMR Spectrum (400 MHz, DMSO-d6): δ 1.30 (12H, s), 3.58 (3H, s), 4.45 (2H, d), 7.36 (1H, dd), 8.16 (1H, t).

3-Bromo-2-fluoro-6-(methoxymeth l)pyridine

Sodium methoxide (25% in MeOH, 2.472 mL, 10.81 mmol) was added dropwise over 15 minutes at -30°C to a stirred solution of 3-bromo-6-(bromomethyl)-2- fluoropyridine (3.06 g, 11.38 mmol) in dry DMF (20 mL) under an inert atmosphere. Additional sodium methoxide (0.15 mL) was added and the mixture was stirred until completion of the reaction. Saturated aqueous sodium bicarbonate (ca. 4mL) was added at -30°C and the mixture was stirred at r.t.. The mixture was extracted with DCM and the organic phase was concentrated in vacuo. The residue was purified by FCC using 1% to 1.5% EtOAc in hexane as an eluent to provide 3- bromo-2-fluoro-6-(methoxymethyl)pyridine (1.86g, 74%>) as a colourless solid. Ή NMR Spectrum (400 MHz, DMSO-d6): 3.37 (3H, s), 4.43 (2H, s), 7.31 (1H, d), 8.30 (lH, t).

3-Bromo-6-(bromomethyl)-2-fluoropyridine

In the dark, 3-bromo-2-fluoro-6-methylpyridine (1.5g, 7.89mmol), N- bromosuccinimide (1.62g, 9.08mmol) and benzoyl peroxide (0.191g, 0.79mmol) in carbon tetrachloride (15mL) were stirred at 80°C for 6 h in a sealed vessel. The mixture was cooled to r.t. and filtered. The filtrate was concentrated in vacuo. Purification by FCC eluting with 2% to 10% DCM in hexane afforded 3-bromo-6- (bromomethyl)-2-fluoropyridine (1.2 g, 56%) as a yellow oil, contaminated with 22% starting material. 'HNMR Spectrum (400 MHz, DMSO-d6) 4.68 (2H, s), 7.48 (1H, dd), 8.32 (1H, dd). Mass Spectrum: m/z [M-H] ^" = 268.

The bromo intermediate (5)-8-bromo-3-methyl-l-(tetrahydro-2H-pyran-3-yl)-lH- imidazo[4,5-c]quinolin-2(3H)-one was prepared as follows:

Intermediate Al: (S)-8-bromo-3-methyl-l-(tetrahydro-2H-pyran-3-yl)-lH- imidazo [4,5-c] quinolin-2(3H)-one

A solution of iodomethane (48.9 g, 344.63 mmol), tetrabutylammonium bromide (2.78 g, 8.62 mmol) and NaOH (10.34 g, 258.48 mmol) in water (200 mL) was added to a stirred mixture of (5)-8-bromo-l-(tetrahydro-2H-pyran-3-yl)-lH- imidazo[4,5-c]quinolin-2(3H)-one (30 g, 86.16 mmol) in DCM (350 mL). The resulting mixture was stirred at 25 °C for 16 h. The DCM was removed under reduced pressure. The precipitate was collected by filtration, washed with water (5 x 10 mL) and dried in the vacuum oven to afford a crude product. The crude product was diluted with EtOAc (150 mL). The resulting mixture was stirred at 50°C for 2 h. The precipitate was collected by filtration, washed with EtOAc (5 x 5 mL) and dried in the vacuum oven to afford (5)-8-bromo-3-methyl-l-(tetrahydro- 2H-pyran-3-yl)-lH-imidazo[4,5-c]quinolin-2(3H)-one (25.00 g, 80 %) as a white solid. NMR Spectrum: Ή NMR (300MHz, DMSO-d ₆ ) δ 1.82 - 1.88 (2H, m), 2.09 - 2.15 (1H, m), 2.55 -2.78 (1H, m), 3.30 - 3.47 (1H, m) 3.48 (3H, s), 3.92 (lH,d), 4.02 - 4.22 (2H, m), 4.68-4.88 (1H, m), 7.75 (1H, d), 7.99 (1H, d), 8.35 (1H, s), 8.92 (1H, s). Mass Spectrum: m/z (ES+)[M+H]+ = 362.

The following intermediates were prepared in an analogous fashion from the appropriate 3H-imidazo[4,5-c]quinolin-2-one intermediate:

Intermediate Structure Name

Intermediate 8-bromo-3-methyl- 1 -[(3R)-

Bl oxan-3-yl]imidazo[5,4-

* c]quinolin-2-one

Intermediate 8-bromo-7-fluoro-3-methyl- 1 -

CI [(35)-oxan-3-yl]imidazo[5,4-

** c]quinolin-2-one

Intermediate 8-bromo-7-fluoro-3-methyl- 1 - Dl [(3R)-oxan-3-yl]imidazo[5,4- c]quinolin-2-one

* The reaction had not proceeded to completion so additional methyl iodide, sodium hydroxide and tetrabutylammonium bromide were added and the reaction stirred for a further 16 - 18 h.

** The reaction was stirred for 72 h at ambient temperature.

*** 2M NaOH solution was used and the reaction was stirred at r.t. for 18 h.

Intermediate Bl: NMR Spectrum: ¹ H NMR (300MHz, DMSO-d ₆ ) δ 1.80-1.86 (2H, m), 2.07-2.12 (1H, m), 2.61-2.75 (1H, m), 3.32-3.46 (1H, m), 3.47 (3H, s), 3.92-3.98 (1H, m), 4.01-4.20 (2H,m), 4.72-4.83 (lH,m),7.76 (lH,dd), 8.00 (lH,d), 8.34 (lH,d), 8.92 (lH,s). Mass Spectrum: m/z (ES+)[M+H]+ = 362, 364.

Intermediate CI: NMR Spectrum: ¹ H NMR (400MHz, DMSO-d ₆ ) δ 1.88-190 (2H, m), 2.09 (1H, d), 2.70 (1H, ddd), 3.36 - 3.44 (1H, m), 3.47 (3H, s), 3.94 (1H, d), 4.07 (1H, dd), 4.15 (1H, t), 4.79 (1H, ddd), 7.97 (1H, d), 8.48 (1H, d), 8.93 (1H, s). Mass Spectrum: m/z (ES+)[M+H]+ = 380, 382.

Intermediate Dl: NMR Spectrum: ¹ H NMR (400MHz, DMSO-d6) δ 1.86 (2H, dd), 2.11 (1H, d), 2.69 (1H, ddd), 3.37 - 3.45 (1H, m), 3.48 (3H, s), 3.95 (1H, d), 4.08 (1H, dd), 4.18 (1H, t), 4.80 (1H, ddd), 7.98 (1H, d), 8.50 (1H, d), 8.94 (1H, s). Mass Spectrum: m/z (ES+)[M+H]+ = 380, 382. Intermediate El: NMR Spectrum: ¹ H NMR (400MHz, DMSO-d ₆ ) δ 1.77 - 1.92 (2H, m), 2.11 (1H, d), 2.62 - 2.77 (1H, m), 3.40 (1H, td), 3.49 (3H, s), 3.95 (1H, d), 4.08 (1H, dd), 4.18 (1H, t), 4.68 - 4.9 (1H, m), 7.77 (1H, dd), 8.01 (1H, d), 8.36 (1H, d), 8.93 (1H, s). Mass Spectrum: m/z (ES+)[M+H]+ = 362, 364.

Intermediate Fl: NMR Spectrum: ¹ H NMR (400MHz, DMSO-d6) δ 1.8 - 1.92 (2H, m), 2.11 (1H, d), 2.64 - 2.75 (1H, m), 3.39 - 3.43 (1H, m), 3.48 (3H, s), 3.95 (1H, d), 4 - 4.12 (1H, m), 4.18 (1H, t), 4.78 - 4.86 (1H, m), 7.99 (1H, d), 8.51 (1H, d), 8.95 (1H, s). Mass Spectrum: m/z (ES+)[M+H]+ = 380, 382.

The 3H-imidazo[4,5-c]quinolin-2-one intermediate (5)-8-bromo-l-(tetrahydro-2H- pyran-3-yl)-l,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one was prepared as follows:

Intermediate A2 : (S)-8-Bromo- l-(tetrahydro-2H-pyran-3-yl)- 1 ,3-dihydi imidazo [4,5-c] quinolin-2-one

Triethylamine (17.86 mL, 128.13 mmol) was added to (5)-6-bromo-4-((tetrahydro- 2H-pyran-3-yl)amino)quinoline-3-carboxylic acid (15 g, 42.71 mmol) in DMF (100 mL). The resulting suspension was stirred for 25 minutes, then cooled to 0°C. Diphenylphosphoryl azide (11.05 mL, 51.25 mmol) was added dropwise keeping the temperature below 10°C. The suspension was stirred for further 30 minutes at r.t. then at 60°C for 2 h. The reaction mixture was cooled to r.t., water (100 mL) was added and stirred for 30 minutes. The resulting precipitate was collected by filtration to afford (5)-8-bromo-l-(tetrahydro-2H-pyran-3-yl)-l,3-dihydro-2H- imidazo[4,5-c]quinolin-2-one (13.20 g, 89 %) as an off-white solid. NMR

Spectrum: ¹ H NMR (300MHz, DMSO-d6) δ 1.84-2.11 (3H, m), 2.62-2.76 (1H, 3.35-3.44 (1H, m), 3.92-4.22 (3H, m), 4.71-4.80 (lH,m), 7.76 (1H, dd), 7.98 (2H,d), 8.32 (1H, dd), 8.71 (1H, s),11.85 (1H, bs). Mass Spectrum: m/z

(ES+)[M+H]+ = 348, 350.

The following 3H-imidazo[4,5-c]quinolin-2-one intermediates were prepared in a similar fashion from the appropriate carboxylic acid intermediates:

* The reaction was stirred at 60°C for 60 - 90 mins. Intermediate B2: NMR Spectrum: ¹ H NMR (300MHz, DMSO-d ₆ ) δ 1.82-2.11 (3H, m), 2.61-2.75 (1H, m), 3.34-3.43 (1H, m), 3.91-4.21 (3H, m), 4.69-4.78 (1H, m), 7.75 (1H, dd), 7.99 (2H,d), 8.33 (1H, dd), 8.69 (1H, s), 11.70 (1H, bs). Mass Spectrum: m/z (ES+)[M+H]+ = 348, 350. Intermediate C2: NMR Spectrum: ¹ H NMR (400MHz, DMSO-d ₆ ) δ 1.77 - 1.93 (2H, m), 2.10 (1H, d), 2.68 (1H, qd), 3.34 - 3.44 (1H, m), 3.94 (1H, d), 4.08 (1H, dd), 4.18 (lH, t), 4.75 (1H, ddd), 7.94 (1H, d), 8.48 (1H, d), 8.69 (1H, s), 11.63 (1H, s). Mass Spectrum: m/z (ES+)[M+H]+ = 366, 368.

Intermediate D2: NMR Spectrum: ¹ H NMR (400MHz, DMSO-d ₆ ) δ 1.7 - 1.93 (2H, m), 2.10 (1H, d), 2.63 - 2.75 (1H, m), 3.49 - 3.61 (1H, m), 3.84 - 4.03 (1H, m), 4.08 (1H, dd), 4.19 (1H, t), 4.76 (1H, t), 7.95 (1H, d), 8.49 (1H, d), 8.70 (1H, s), 11.66 (1H, s). Mass Spectrum: m/z (ES+)[M+H]+ = 366, 368.

Intermediate E2: Mass Spectrum: m/z (ES+)[M+H]+ = 348.

The carboxylic acid intermediate (S)-6-Bromo-4-((tetrahydro-2H-pyran-3- yl)amino)quinoline-3-carboxylic acid was prepared as follows:

Intermediate A3 : (S)-6-Bromo-4-((tetrahydro-2H-pyran-3-yl)amino)quinoline- 3-carboxylic acid

A solution of ethyl (5)-6-bromo-4-((tetrahydro-2H-pyran-3-yl)amino)quinoline-3- carboxylate (27.8 g, 73.30 mmol) in THF (400 mL and water (80 mL) was treated with 2M sodium hydroxide (73.3 mL, 146.60 mmol) and stirred at 60°C for 6 h then allowed to cool to r.t.. The solvent was removed under reduced pressure and the aqueous was adjusted to pH 3 with 2 M HC1 to give a precipitate which was filtered off, washed thoroughly with water and dried to afford (5)-6-bromo-4- ((tetrahydro-2H-pyran-3-yl)amino)quinoline-3-carboxylic acid (19.05 g, 74.0 %) as a white solid. NMR Spectrum: ¹ H NMR (300MHz, DMSO-d ₆ ) δ 1.50-1.57 (1H, m), 1.61 - 1.82 (2H, m), 1.98- 2.13 (1H, m), 3.48-3.72 (3H, m), 3.89 (1H, d), 4.15 -4.26 (1H, m), 7.77 (1H, dd), 7.95 (1H, d), 8.31(1H, d), 8.90 (lH,s), 13.38 (1H, bs). Mass Spectrum: m/z (ES+)[M+H]+ = 351. The following carboxylic acid intermediates were prepared in a similar fashion from the appropriate ester precursor:

* The reaction was stirred between 60 - 70°C for 1 - 3 h.

** The reaction was performed using a mixture of THF and water as the solvent and heated at 60°C for 3 - 16 h. Intermediate B3: NMR Spectrum: ¹ H NMR (300MHz, DMSO-d ₆ ) δ 1.50-1.56 (1H, m), 1.62 - 1.83 (2H, m), 1.99- 2.12 (1H, m), 3.50-3.71 (3H, m), 3.89 (1H, d), 4.16 -4.28 (1H, m), 7.78 (1H, dd), 7.94 (1H, d), 8.30(1H, d), 8.94 (lH,s), 13.50 (1H, bs). Mass Spectrum: m/z (ES+)[M+H]+ = 351. Intermediate C3: NMR Spectrum: ¹ H NMR (300MHz, DMSO-d ₆ ) δ 1.51 (1H, m), 1.74 (2H, m), 2.04 (1H, m), 3.60 (3H, m), 3.82 (1H, d), 4.15 (1H, m), 7.73 (1H, m), 8.44 (1H, m), 8.92 (1H, s). Mass Spectrum: m/z (ES+)[M+H]+ = 369. Intermediate D3: Mass Spectrum: m/z (ES+)[M+H]+ = 369.

Intermediate E3: Mass Spectrum: m/z (ES+)[M+H]+ = 351.

The ester intermediate Ethyl (S)-6-bromo-4-((tetrahydro-2H-pyran-3- yl)amino)quinoline-3 -carboxylate was prepared as follows:

Intermediate A4: Ethyl (S)-6-bromo-4-((tetrahydro-2H-pyran-3- yl)amino)quinoline-3-carbox late

DIPEA (6.99 mL, 40.00 mmol) was added to ethyl 6-bromo-4-chloroquinoline-3- carboxylate (3.15 g, 10 mmol) and (5)-tetrahydro-2H-pyran-3-amine hydrochloride (1.376 g, 10.00 mmol) in DMA (30 mL) at 25°C under air. The resulting solution was stirred at 80°C for 16 h. The reaction mixture was diluted with water (100 mL), the precipitate was collected by filtration, washed with water (20 mL) and dissolved into 250 mL EtOAc/DCM (1 : 1). The formed mixture was dried over MgSCM, filtered and evaporated to afford crude (5)-ethyl 6-bromo-4-((tetrahydro-2H-pyran- 3 -yl)amino)quinoline-3 -carboxylate (3.16 g, 83 %) as a white solid. The product was used in the next step directly without further purification. NMR Spectrum: ^! H NMR (300MHz, DMSO-d ₆ ) δ 1.36 (3H, t), 1.70-1.74 (1H, m), 1.75-1.77 (2H, m), 2.03-2.05 (1H, m), 3.58-3.61 (3H, m), 3.80-3.85 (1H, m), 4.01-4.03 (1H, m), 4.35 (2H, q), 7.80 (1H, d), 7.89 (1H, dd), 8.58 (1H, s), 8.67 (1H, d), 8.93 (1H, s). Mass spectrum: m/z: ES+ [M+H]+ 379, 381. The following ester intermediates were prepared in an analogous fashion fi appropriate amine and either ethyl 6-bromo-4-chloro-7-fluoroquinoline-3- carboxylate or ethyl 6-bromo-4-chloroquinoline-3-carboxylate:

* The reaction was stirred at 80°C for 2 - 16 h

** The reaction was stirred at 100°C for 16 h

Intermediate B4: Mass Spectrum: m/z (ES+)[M+H]+ = 379, 381.

Intermediate C4: NMR Spectrum: ¹ H NMR (300MHz, DMSO-d6) δ 1.33 (3H, m), 1.51 (1H, m), 1.74 (2H, m), 2.04 (1H, m), 3.60 (3H, m), 3.82 (1H, d), 4.02 (1H, m), 4.35 (2H, m), 7.73 (1H, m), 8.49 (1H, m), 8.79 (1H, m), 8.88 (1H, s). Mass Spectrum: m/z (ES+)[M+H]+ = 397. Intermediate D4: NMR Spectrum: ¹ H NMR (400MHz, CDC1 ₃ ) δ 1.44 (3H,t), 1.67

- 1.77 (1H, m), 1.86 - 1.97 (2H, m), 2.17 - 2.24 (1H, m), 3.60 - 3.65 (1H, m), 3.72

- 3.78 (2H, m), 3.93 - 4.02 (1H, m), 4.12 - 4.19 (1H, m), 4.44 (2H, q) 7.72 (1H, d), 8.32 (1H, d), 9.14 (1H, s), 9.46 (1H, s). Mass Spectrum: m/z (ES+)[M+H]+ = 397.

Intermediate E4: Mass Spectrum: m/z (ES+)[M+H]+ = 379.

Intermediate A5: Ethyl 6-bromo-4-chloroquinoline-3-carboxylate

DMF (0.119 mL, 1.54mmol) was added to ethyl 6-bromo-l-[(4- methoxyphenyl)methyl]-4-oxoquinoline-3-carboxylate (160 g, 384.37 mmol) in thionyl chloride (800 mL) at ambient temperature under air. The resulting mixture was stirred at 75°C for 16 h then the solvent was removed under reduced pressure. The resulting mixture was azeotroped twice with toluene then n-hexane (500 mL) was added. The precipitate was collected by vacuum filtration, washed with n- hexane (200 mL) and dried under vacuum to ethyl 6-bromo-4-chloroquinoline-3- carboxylate (100 g, 83%) as a brown solid. NMR Spectrum: ¹ H NMR (400MHz, CDCb) δ 1.47 (3H, t), 4.51 (2H, q), 7.95 (1H, dd), 8.11 (1H, d), 8.60 (1H, d), 9.24 (1H, s). Mass Spectrum: m/z (ES+)[M+H]+ = 314, 316.

On a larger scale, ethyl 6-bromo-l-[(4-methoxyphenyl)methyl]-4-oxoquinoline-3- carboxylate (5765 g, 13.85 mol) was charged to the vessel with thionyl chloride (28.8 L). An exotherm from 20-26°C was observed. DMF (4.4 mL) was added with no observed exotherm and the batch heated to 75 °C and stirred for 17 h. HPLC showed 1.3% starting material remained with 98.0% product. The reaction was concentrated in vacuo and the residue azeotroped with toluene (25 L). The resulting solid was then slurried in heptane (18.5 L) for 2.5 h, filtered and washed with heptane (3 x 4 L). The solid was dried under vacuum at 35°C to give 4077 g of the desired material (93%> crude yield) which contained ~5% of ethyl 6-bromo-l-[(4- methoxyphenyl)methyl]-4-oxoquinoline-3-carboxylate in addition to ~4% hydrolysis product by HPLC (90% pure). The crude material (4077 g) was returned to the vessel and reprocessed with thionyl chloride (14.5 L) and DMF (2.2 mL). The mixture was heated to 75 °C for 40 h. The thionyl chloride was removed in vacuo and the residue azeotroped with toluene (10 L). The residue was slurried in heptane (18 L) for ~16 h at 20°C. The solid was collected by filtration, one portion being filtered under nitrogen and washed with heptane (3 L) to yield 2196 g of desired material (90% NMR assay, 99% by HPLC). The remainder of the batch was filtered under air and washed with heptane (3 L) to yield 1905 g of the desired material (88% NMR assay, 99% by HPLC). The yellow solids were combined for further processing (4101 g, 3653 g active, 83% yield, 99% by HPLC).

Intermediate A6: Ethyl 6-bromo-l-[(4-methoxyphenyl)methyl]-4- oxoquinoline-3-carboxylate

l,8-Diazabicyclo[5.4.0]undec-7-ene (102 mL, 679.62 mmol) was added drop-wise to ethyl 2-(5-bromo-2-fluorobenzoyl)-3-[(4-methoxyphenyl)methylamino] prop-2- enoate (296.5 g, 679.62 mmol), in acetone (1.2 L) at ambient temperature over a period of 2 minutes. The resulting solution was stirred for 16 h then the solid removed by filtration and washed with tert-butyl methyl ether to afford the desired material (180 g, 64%) as light yellow solid. NMR Spectrum: ^! H NMR (400MHz, DMSO-d6) δ 1.30 (3H ,t), 3.71 (3H, s), 4.25 (2H ,q), 5.60 ( 2H, s), 6.90-6.95 (2H, m), 7.12-7.25 (2H, m), 7.67 (1H, d), 7.80-7.90 (1H, m), 8.30 (1H, d), 8.92 (1H, s). Mass Spectrum: m/z (ES+)[M+H]+ = 418.

On a larger scale, ethyl 2-(5-bromo-2-fluorobenzoyl)-3-[(4- methoxyphenyl)methylamino]prop-2-enoate (8434 g, (7730 g assumed active), 17.71 mol) was charged to the vessel with acetone (23.2 L) at 15°C. 1,8- Diazabicyclo[5.4.0]undec-7-ene (2.8 L, 18.72 mol) was added over 25 minutes with an observed exotherm from 18-23°C over the addition. A precipitate formed after ~25 minutes and the batch continued to exotherm reaching a maximum of 37°C after 1 h. The reaction was stirred at 20°C for 16.5 h at which point HPLC indicated consumption of starting material and 96.5% product. The resulting precipitate was collected by filtration washing with tert-butyl methyl ether (4 x 3.4 L). The solid was then dried under vacuum at 40°C to give 6033 g of the desired material as a white solid (81.6% yield over 3 steps, 99.8%> purity by HPLC). Analytical data was consistent with that obtained on previous batches. Intermediate A7: Ethyl 2-(5-bromo-2-fluorobenzoyl)-3-[(4- methoxyphenyl)methylamino rop-2-enoate

(E)-Ethyl 3-(dimethylamino)acrylate (98 g, 685.00 mmol) was added portion-wise to 5-bromo-2-fluorobenzoyl chloride (163g, 685mmol) and DIPEA (120 mL, 685.00 mmol) in toluene (800 mL) at 10°C over a period of 10 minutes. The resulting solution was stirred at 70°C for 16 h then allowed to cool. (4- Methoxyphenyl)methanamine (94 g, 685 mmol) was added to the mixture over a period of 20 minutes at ambient temperature. The resulting solution was stirred for 3 h then the reaction mixture diluted with DCM (4 L), and washed with water (3 x 1L). The organic phase was dried over Na2S0 ₄ , filtered and evaporated to give the desired material (300 g, 100%) as brown oil, which was used immediately in the subsequent reaction without further purification. Mass Spectrum: m/z

(ES+)[M+H]+ = 436. On a larger scale, 5-bromo-2-fluorobenzoyl chloride (4318 g, 4205 g active, 17.71 mol) was charged to the vessel as a solution in toluene (7.5 L). DIPEA (3150 mL, 18.08 mol) was added with no observed exotherm. Ethyl-3- (dimethylamino)acrylate (2532 g, 17.71 mol) was added portionwise over 30 minutes maintaining a batch temperature <40°C. An exotherm from 21-24°C was noted over the 30 minute addition with a further slow rise to 38°C over 1 h. The reaction was stirred at 20-30°C for 16.5 h. 4-Methoxybenzylamine (2439 g, 17.78 mol) was added portionwise over 30 mins maintaining a batch temperature <40°C. An exotherm of 25-30°C was observed over the addition with cooling provided by a reduced jacket temperature of 15°C. The reaction was stirred for 4 h at 20-30°C after which HPLC indicated 93.2% of desired material. The batch was split for workup with each half of the mixture diluted with DCM (28.6 L) and washed with water (3 x 7.8 L). The organics were dried over MgSCM (-550 g) and filtered, washing with DCM (4 L). The combined organics were then concentrated to give 8444 g of the desired material as an oil (8434 g, 106% yield, 94.7% purity by HPLC). Analytical data was consistent with that obtained from previous batches.

Intermediate A8: 5-Bromo-2-fluorobenzoyl chloride

Thionyl chloride (75.0mL, 1027.36mmol) was added drop-wise to 5-bromo-2- fluorobenzoic acid (150g, 684.91mmol), in toluene (1.2 L) and DMF (12mL) at ambient temperature over a period of 1 h. The resulting mixture was stirred at 70°C for 16 h then the mixture allowed to cool and concentrated in vacuo to afford the desired material (160g, 98%>) as light yellow oil, which was used without further purification. NMR Spectrum: ¹ H NMR (400MHz, DMSO-d6) δ 7.26 - 7.31 (1H, m), 7.83 (1H, dd), 8.02 (1H, d).

On a larger scale, 3-bromo-6-fluorobenzoic acid (3888 g, 17.75 mol) was charged to the vessel at 20°C followed by toluene (29.2 L). Thionyl chloride (1950 ml, 26.88 mol) was added, followed by DMF (310 mL) with no observed exotherm. The mixture was heated to 65-75°C (solution obtained above ~45°C) with no observed exotherm and slight gas evolution. The reaction was stirred for 40 h at this temperature at which point HPLC analysis showed 87.6%> product, 3.4%> starting material. The reaction was concentrated in vacuo and azeotroped with toluene (18 L) to give 4328 g of the desired material (103% yield, 87.3% by HPLC).

Intermediate C5: Ethyl 6-bromo-4-chloro-7-fluoroquinoline-3-carboxylate

DMF (0.535 mL, 6.91 mmol) was added to ethyl 6-bromo-7-fluoro-l-[(4- methoxyphenyl)methyl]-4-oxo-quinoline-3-carboxylate (200 g, 460.56 mmol) in thionyl chloride (600 mL) at 10°C under an inert atmosphere and the resulting mixture stirred at 70°C for 3 h. The mixture was evaporated to dryness and the residue azeotroped with toluene (300 mL) to afford crude product. The crude product was purified by crystallisation from hexane to afford the desired material as a white solid (147 g, 96 %). NMR Spectrum: ¹ H NMR (400MHz, CDC1 ₃ ) δ 1.49 (3H, t), 4.51-4.56 (2H, m), 7.91 (1H, d), 8.71 (1H, d), 9.26 (1H, s). Mass Spectrum: m/z (ES+)[M+H]+ = 334.

Intermediate C6: Ethyl 6-bromo-7-fluoro-l-[(4-methoxyphenyl)methyl]-4-oxo- quinoline-3-carboxylate

l,8-Diazabicyclo[5.4.0]undec-7-ene (76 mL, 506.32 mmol) was added slowly to ethyl-2-(5-bromo-2,4-difluoro-benzoyl)-3-[(4-methoxyphenyl)m ethylamino]prop- 2-enoate (230 g, 506.32 mmol) in acetone (800 mL) at 10°C over a period of 5 minutes under an inert atmosphere and the resulting mixture stirred at ambient temperature for 16 h. The precipitate was collected by filtration, washed with Et ₂ 0 (3 x 500 mL) and dried under vacuum to afford the desired material as a white solid

(166 g, 75 %). NMR Spectrum: ¹ H NMR (400MHz, DMSO-d6) δ 1.29 (3H, t), 3.72 (3H, s), 4.22-4.27 (21H, m), 5.57 (2H, s), 6.92-6.95 (2H, m), 7.24 (2H, d), 7.79 (1H, d), 8.40 (1H, d), 8.89 (1H, s). Mass Spectrum: m/z (ES+)[M+H]+ = 434. Intermediate C7: Ethyl-2-(5-bromo-2,4-difluoro-benzoyl)-3-[(4- methoxyphenyl)methylamino]prop-2-enoate

(E)-Ethyl 3-(dimethylamino)acrylate (80 mL, 555.50 mmol) was added dropwise to a mixture of DIPEA (132 mL, 757.50 mmol) and 5-bromo-2,4-difluoro-benzoyl chloride (129 g, 505.00 mmol) in toluene (600 mL) at ambient temperature under an inert atmosphere. The resulting solution was stirred at 70°C for 17 h then allowed to cool. (4-Methoxyphenyl)methanamine (66.0 mL, 505.29 mmol) was added portionwise to the mixture and the reaction stirred for 3 h at ambient temperature. The reaction mixture was diluted with DCM (2 L), washed

sequentially with water (4 x 200 mL), saturated brine (300 mL), the organic layer dried over Na ₂ S04, filtered and evaporated to afford the desired material as a light brown solid (230 g, 100 %) which was used in the next step without further purification. NMR Spectrum: ¹ H NMR (400MHz, CDC1 ₃ ) δ 1.09 (3H, t), 3.82 (3H, s), 4.00-4.10 (2H, m), 4.55 (2H, t), 6.84-6.96 (3H, m), 7.20-7.29 (2H, m), 7.55 (1H, d), 8.18 (1H, t) Mass Spectrum: m/z (ES+)[M+H]+ = 454.

Intermediate C8: 5-Bromo-2,4-difluoro-benzoyl chloride

Thionyl chloride (55.4 mL, 759.50 mmol) was added portionwise to a mixture of DMF (7.84 mL, 101.27 mmol) and 5-bromo-2,4-difluorobenzoic acid (120 g, 506.33 mmol) in toluene (600 mL) at 15°C over a period of 5 minutes under an inert atmosphere. The resulting mixture was stirred at 70°C for 4 h then evaporated to dryness and the residue was azeotroped with toluene to afford the desired material as a brown oil (129 g, 100 %) which was used directly in the next step without purification. NMR Spectrum: ¹ H NMR (400MHz, CDCI3) δ 7.04-7.09 (1H, m), 8.34-8.42 (1H, m).

The intermediate rac-8-bromo-7-fluoro- 1 -(tetrahydro-2H-pyran-3-yl)- 1 H- imidazo[4,5-c]quinolin-2(3H)-one was prepared as follows:

Intermediate F2: rac-8-Bromo-7-fluoro-l-(tetrahydro-2H-pyran-3-yl)-lH- imidazo [4,5-c] quinolin-2(3H)-one

Trichloroisocyanuric acid (213 mg, 0.92 mmol) was added in one portion to 6- bromo-7-fluoro-4-((tetrahydro-2H-pyran-3-yl)amino)quinoline- 3-carboxamide (843 mg, 2.29 mmol) and l,8-diazabicyclo[5.4.0]undec-7-ene (0.629 mL, 4.58 mmol) in MeOH (10 mL). The resulting mixture was stirred at r.t. for 70 h. The reaction was incomplete and further trichloroisocyanuric acid (213 mg, 0.92 mmol) and 1,8- diazabicyclo[5.4.0]undec-7-ene (0.629 mL, 4.58 mmol) was added and the mixture was stirred at r.t. for a further 3 h. The resulting mixture was evaporated to dryness and the residue was purified by FCC, elution gradient 0 to 10% MeOH in DCM. Pure fractions were evaporated to dryness to afford rac-8-bromo-7-fluoro-l- (tetrahydro-2H-pyran-3-yl)-lH-imidazo[4,5-c]quinolin-2(3H)-o ne (585 mg, 69.8 %) as a cream solid. NMR Spectrum: ¹ H NMR (400MHz, DMSO-d ₆ ) δ 1.78 - 1.88 (2H, m), 2.10 (1H, d), 2.62 - 2.73 (1H, m), 3.40 (1H, td), 3.88 - 4.04 (1H, m), 4.02 - 4.12 (1H, m), 4.19 (1H, t), 4.72 - 4.80 (1H, m), 7.94 (1H, d), 8.48 (1H, d), 8.69 (1H, s), 11.62 (1H, s). Mass Spectrum: m/z (ES+)[M+H]+ = 366. Intermediate F3: rac-6-Bromo-7-fluoro-4-((tetrahydro-2H-pyran-3- yl)amino)quinoline-3-carboxamide

6-bromo-4-chloro-7-fluoroquinoline-3-carboxamide (1 g, 3.29 mmol), tetrahydro- 2H-pyran-3 -amine (0.350 g, 3.46 mmol) and DIPEA (1.435 mL, 8.24 mmol) were suspended in DMA (10 mL) and the resulting mixture was heated at 100°C overnight. The reaction mixture was diluted with water and the precipitate was collected by filtration, washed with water (10 mL) and dried under vacuum to afford rac-6-bromo-7-fluoro-4-((tetrahydro-2H-pyran-3-yl)amino)quin oline-3- carboxamide (0.843 g, 69.5 %) as a beige solid, which was used without further purification. NMR Spectrum: ¹ H NMR (400MHz, DMSO-d ₆ ) δ 1.47 - 1.55 (1H, m), 1.60 -1.72 (2H, m), 2.02 (1H, d), 3.33 - 3.48 (2H, m), 3.68 (1H, dd), 3.87 (1H, dd), 3.91 - 3.97 (1H, m), 7.56 (1H, s), 7.71 (1H, d), 8.11 (1H, s), 8.20 (1H, d), 8.62 (2H, d). Mass Spectrum: m/z (ES+)[M+H]+ = 368.

Intermediate F4: 6-Bromo-4-chloro-7-fluoro-quinoline-3-carboxamide

DMF (0.5 mL) was added to a stirred suspension of 6-bromo-7-fluoro-4-oxo-lH- quinoline-3-carboxylic acid (22.5 g, 78.66 mmol) in thionyl chloride (140 g, 1179.85 mmol) and the mixture heated to reflux for 2 h. The reaction was allowed to cool, concentrated in vacuo and the residue azeotroped twice with toluene to afford a yellow solid. This solid was added portionwise to a solution of ammonium hydroxide (147 mL, 1179.85 mmol) at 0°C. The white suspension was stirred for 15 minutes then the solid filtered, washed with water and dried under vacuum to afford the desired material (23.80 g, 100 %) as a white powder. NMR Spectrum: ^J H NMR (400MHz, DMSO-d6) δ 8.92 (1H, s), 8.59 (1H, d), 8.21 (1H, s), 8.09 (1H, d), 7.98 (1H, s). Mass Spectrum: m/z (ES+)[M+H]+ = 304.8 Intermediate F5: 6-Bromo-7-fluoro-4-oxo-lH- uinoline-3-carboxylic acid

A solution of sodium hydroxide (18.34 g, 458.44 mmol) in water (100 mL) was added to a stirred suspension of ethyl 6-bromo-7-fluoro-4-oxo-lH-quinoline-3- carboxylate (28.8 g, 91.69 mmol) in EtOH (500 mL) at ambient temperature. The reaction mixture was then stirred at 75°C for 2 h, allowed to cool and the pH adjusted to 4 using 2N hydrochloric acid. The precipitate was collected by filtration, washed with water and dried under vacuum to afford the desired material (23.30 g, 89 %) as a white powder. NMR Spectrum: ¹ H NMR (400MHz, DMSO- d6) δ 14.78 (1H, s), 13.45 (1H, s), 8.93 (1H, s), 8.46 (1H, d), 7.70 (1H, d). Mass Spectrum: m/z (ES+)[M+H]+ = 287.8

Intermediate F6: Ethyl 6-bromo-7-fluoro-4-oxo-lH-quinoline-3-carboxylate

A solution of diethyl 2-[(4-bromo-3-fluoro-anilino)methylene]propanedioate (90 g, 249.88 mmol) in diphenyl ether (600 mL, 3.79 mol) was stirred at 240°C for 2.5 h. The mixture was allowed to cool to 70°C, the solids collected by filtration and dried in a vacuum oven to afford the desired material (50g, 64%) as a white solid which was used without further purification. NMR Spectrum: ^! H NMR (500MHz, DMSO- d6, (100°C)) δ 1.26 - 1.33 (3H, m), 4.25 (2H, q), 7.52 (1H, d), 8.37 (1H, d), 8.48 (1H, s), 12.05 (1H, s). Mass Spectrum: m/z (ES+)[M+H]+ = 314 Intermediate F7: Diethyl 2-[(4-bromo-3-fluoro- anilino)methylene] propanedioate

A solution of 4-bromo-3-fluoroaniline (56.6 g, 297.87 mmol) and 1,3-diethyl 2- (ethoxymethylidene)propanedioate (72.45 g, 335.06 mmol) in EtOH (560 mL) was stirred at 80°C for 4 h. The reaction mixture was allowed to cool, the solids collected by filtration and dried in an oven to afford the desired material (90g, 84%) as an off-white solid which was used without further purification. NMR Spectrum: ¹ H NMR (400MHz, DMSO-d6) δ 1.26 (6H, q), 4.14 (2H, q), 4.22 (2H, q), 7.18 - 7.25 (1H, m), 7.57 (1H, dd), 7.64 - 7.7 (1H, m), 8.33 (1H, d), 10.62 (1H, d). Mass Spectrum: m/z (ES+)[M+H]+ = 360.

Example 9

rac-l-(3,3-Dimethyltetrahydro-2H-pyran-4-yl)-8-(6-(methox ymethyl)pyridin- 3-yl)-3-methyl- 1 ,3-dihydro-2H-imidazo 4,5-c] quinolin-2-one

Dichlorobis(di-tert-butyl(3-sulfopropyl)phosphonio)palladate (II) (0.05M in water) (31.4 ml, 1.57 mmol) was added to a degassed mixture of 2-(methoxymethyl)-5- (4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)pyridine (8.59 g, 34.50 mmol), rac-8- bromo- 1 -(3,3-dimethyltetrahydro-2H-pyran-4-yl)-3-methyl- 1 ,3-dihydro-2H- imidazo[4,5-c]quinolin-2-one (12.24 g, 31.36 mmol) and 2M K2CO3 solution (47.0 ml, 94.09 mmol) in 1,4-dioxane (188 ml) and water (47.0 ml). The reaction was heated to 80°C for 4 h. The reaction mixture was concentrated and diluted with DCM (250 mL), and washed with water (200 mL). The organic layer was dried with a phase separating cartridge and evaporated to afford crude product. The beige solid was heated in MeCN and left to cool to r.t. The solid was filtered under vacuum to afford rac-l-(3,3-dimethyltetrahydro-2H-pyran-4-yl)-8-(6- (methoxymethyl)pyridin-3-yl)-3-methyl-l,3-dihydro-2H-imidazo [4,5-c]quinolin-2- one (12 g, 88%) as a beige solid. NMR Spectrum: ¾ NMR (500 MHz, CDC1 ₃ ) δ 0.94 (3H, s), 1.35 (3H, s), 1.73 (1H, d), 3.31 - 3.41 (1H, m), 3.57 (3H, s), 3.60 (3H, s), 3.63 - 3.82 (3H, m), 4.26 (1H, dd), 4.69 (2H, s), 5.02 (1H, dd), 7.51 - 7.64 (1H, m), 7.83 (1H, dd), 8.02 (1H, dd), 8.27 (1H, d), 8.60 (1H, d), 8.74 (1H, s), 8.93 (1H, dt). Mass Spectrum: m/z (ES+)[M+H]+ = 433.

The racemic mixture described above was separated to give the individual enantiomeric components, Examples 10 and 11, as described below. The stereochemistry of these components has been assigned using Virtual Circular Dichroism (VCD).

A racemic mixture of l-(3,3-dimethyltetrahydro-2H-pyran-4-yl)-8-(6- (methoxymethyl)pyridin-3 -yl)-3 -methyl- 1 H-imidazo [4,5 -c] quino lin-2(3H)-one was separated by preparative chiral-HPLC (Chiral Technologies IC column, 20 μιη silica, 50 mm diameter, 250 mm length), eluting isocratically with 85% tert-butyl methyl ether in EtOH (modified with triethylamine) as eluent, to afford the first eluting product, Example 10, as solid (314 mg, 30%>), and the second eluting product, Example 11, as a solid (331 mg, 32 %>).

Example 10

Isomer 1: (R)-l-(3,3-dimethyltetrahydro-2H-pyran-4-yl)-8-(6- (methoxy methyl)py ridin-3-yl)-3-methyl- 1 ,3-dihydro-2H-imidazo [4,5- c]quinolin-2-one

Isomer 1 : NMR Spectrum: ¹ H NMR (500 MHz, DMSO-d6) δ 0.78 (3H, s), 1.05 (1H, t), 1.17 (3H, d), 1.64 - 1.82 (1H, m), 3.42 (4H, s), 3.50 (4H, s), 3.62 - 3.72 (1H, m), 4.06 (1H, dd), 4.59 (2H, s), 5.13 (1H, dd), 7.60 (1H, dd), 7.98 (1H, dd), 8.16 (1H, d), 8.26 (1H, dd), 8.74 (1H, d), 8.91 (1H, s), 9.00 (1H, dd). Mass

Spectrum: m/z (ES+)[M+H]+ = 433. Example 11

Isomer 2: (S)-l-(3,3-Dimethyltetrahydro-2H-pyran-4-yl)-8-(6- (methoxy methyl)py ridin-3-yl)-3-methyl- 1 ,3-dihydro-2H-imidazo [4,5- c]quinolin-2-one

Isomer 2: NMR Spectrum: ¹ H NMR (500 MHz, DMSO d-6) δ 0.78 (3H, s), 0.95 - 1.12 (1H, m), 1.16 (3H, s), 1.68 - 1.81 (1H, m), 3.42 (4H, m), 3.50 (4H, m), 3.62 - 3.7 (1H, m), 4.07 (1H, s), 4.59 (2H, s), 5.13 (1H, dd), 7.60 (1H, dd), 7.98 (1H, dd), 8.16 (1H, d), 8.26 (1H, dd), 8.74 (1H, d), 8.91 (1H, s), 9.00 (1H, dd). Mass

Spectrum: m/z (ES+)[M+H]+ = 433.

Stereochemistry was assigned using Virtual Circular Dichroism (VCD).

Experimental: The off white solid samples were dissolved in CDCh. The solutions were transferred to 0.100 mm BaF ₂ cells and VCD spectra acquired for six h each in a BioTools ChirallR instrument equipped with dual source and dual photoelastic modulator. The resolution was 4 cm ^"1 .

Computational Spectral Simulations: A Monte Carlo molecular mechanics search for low energy geometries was conducted for the S-enantiomer.

MacroModel within the Maestro graphical interface (Schrodinger Inc.) was used to generate 33 starting coordinates for conformers. All conformers within 5 kcal/mole of the lowest energy conformer were used as starting point for density functional theory (DFT) minimizations within Gaussian09. Optimized structures, harmonic vibrational frequencies/intensities, VCD rotational strengths, and free energies at STP (including zero-point energies) were determined at B3LYP/6-31G* level of theory. Four unique conformations were found with calculated populations over 10% the populations. These show identical conformations of the tetrahydropyran ring but differ in the orientation and torsion of the pyridine ring.

Fit Between Calculated and Experimental Spectra: Simulations of infrared and VCD spectra were generated using an in-house program to fit Lorentzian line shapes (12 cm ^"1 line width) to the computed spectra thereby allowing direct comparisons between simulated and experimental spectra. Bands below 1200 cm ^"1 are largely independent of conformation (and therefore reflect the absolute configuration at the centre of interest). Bands between 1400 and 1300 cm ^"1 are sensitive to the sign of the torsion of the pyridine and fused aromatic ring (a diastereomeric conformation). (The simulated spectrum of the R-enantiomer was obtained by inversion of the S-enantiomer spectrum.) There was excellent agreement between the calculated S-enantiomer and the experimental spectrum for Isomer 2 (Example 11) and also between the calculated R-enantiomer and experimental spectrum for Isomer 1 (Example 10). The fit in the region 1400 to 1300 cm ^"1 is also reasonable suggesting that the calculated conformational energies are also reasonable. rac-8-Bromo-l-(3,3-dimethyltetrahydro-2H-pyran-4-yl)-3-methy l-l,3-dihydro- 2H-imidazo [4,5-c] quinolin-2-one

l,l-Dimethoxy-N,N-dimethylmethanamine (58.8 ml, 442.79 mmol) was added to rac-8-bromo- 1 -(3,3-dimethyltetrahydro-2H-pyran-4-yl)- 1 ,3-dihydro-2H- imidazo[4,5-c]quinolin-2-one (16.66 g, 44.28 mmol) in DMF (46.4 ml) at 25°C. The resulting slurry was stirred at 80°C for 48 h. The reaction was left to cool to r.t. and the precipitate was filtered under vacuum. The solid was washed with water and dried in a vacuum oven overnight to afford rac-8-bromo-l-(3,3- dimethyltetrahydro-2H-pyran-4-yl)-3-methyl-l,3-dihydro-2H-im idazo[4,5- c]quinolin-2-one (13.24 g, 77 %). NMR Spectrum: ¹ H NMR (500 MHz, CDC1 ₃ ) 0.89 (3H, s), 1.32 (3H, s), 1.73 (1H, ddt), 3.41 (1H, d), 3.57 (3H, s), 3.58 - 3.64 (1H, m), 3.68 (1H, dd), 3.7 - 3.8 (1H, m), 4.27 (1H, ddd), 4.82 (1H, dd), 7.67 (1H, dd), 8.03 (1H, d), 8.55 (1H, d), 8.71 (1H, s). Mass Spectrum: m/z (ES+)[M+H]+ = 390. rac-8-Bromo-l-(3,3-dimethyltetrahydro-2H-pyran-4-yl)-l,3-dih ydro-2H- imidazo [4,5-c] quinolin-2-one

Triethylamine (18.51 ml, 132.81 mmol) was added to rac-6-bromo-4-((3,3- dimethyltetrahydro-2H-pyran-4-yl)amino)quinoline-3-carboxyli c acid (16.79 g, 44.27 mmol) in DMF (100 ml). The resulting suspension was stirred for 15 minutes then diphenylphosphoryl azide (11.45 ml, 53.13 mmol) was added and the suspension was stirred for another 30 minutes at r.t. then at 60°C for 2 h. The reaction was left to cool to r.t. and diluted with water. The precipitate was filtered under vacuum and dried in a vacuum oven to afford 8-bromo-l-(3,3- dimethyltetrahydro-2H-pyran-4-yl)-l,3-dihydro-2H-imidazo[4,5 -c]quinolin-2-one (18.42 g, 111 %). Material used in the subsequent step without further purification. NMR Spectrum: ¹ H NMR (500 MHz, DMSO-d6) 0.73 (3H, s), 1.13 (3H, s), 1.69 - 1.76 (1H, m), 3.33 (1H, s), 3.50 (2H, qd), 3.59 - 3.68 (1H, m), 4.04 (1H, dd), 4.84 (1H, dd), 7.72 (1H, dd), 7.94 (1H, d), 8.62 - 8.7 (2H, m), 11.60 (1H, s). Mass Spectrum: m/z (ES+)[M+H]+ = 376. rac-6-Bromo-4-((3,3-dimethyltetrahydro-2H-pyran-4-yl)amino)q uinoline-3- carboxylic acid

To rac-ethyl 6-bromo-4-((3,3-dimethyltetrahydro-2H-pyran-4-yl)amino)quino line- 3-carboxylate (19.66 g, 48.27 mmol) in THF (116 mL) was added 2M NaOH (97 mL, 193.07 mmol) and the reaction was heated to 60°C for 3 h. The reaction was cooled to r.t. and solvent removed under reduced pressure to afford a brown solution. 2M HC1 was added until pH4 and the resulting solid was filtered under vacuum and dried in a vacuum oven overnight to afford rac-6-bromo-4-((3,3- dimethyltetrahydro-2H-pyran-4-yl)amino)quinoline-3-carboxyli c acid (16.79 g, 92 %) as a pale yellow solid. Mass Spectrum: m/z (ES+)[M+H]+ = 379. rac-Ethyl 6-bromo-4-((3,3-dimethyltetrahydro-2H-pyran-4- yl)amino)quinoline-3-carboxylate

Triethylamine (26.9 mL, 193.03 mmol) was added to 3,3-dimethyltetrahydro-2H- pyran-4-amine hydrochloride (10.39 g, 62.73 mmol) and ethyl 6-bromo-4- chloroquinoline-3-carboxylate (15.18 g, 48.26 mmol) in MeCN (134 mL). The reaction mixture was heated at 90°C for 4 h. The reaction was cooled to r.t. and the precipitate was filtered under vacuum and washed with water (300 mL) to afford rac-ethyl 6-bromo-4-((3,3-dimethyltetrahydro-2H-pyran-4-yl)amino)quino line-3- carboxylate (11.0 g, 56 %) as a white solid. The filtrate was concentrated and extracted with DCM (400 mL). The organic layer was dried over a phase separator and concentrated under reduced pressure to afford a second batch of rac-ethyl 6- bromo-4-((3,3-dimethyltetrahydro-2H-pyran-4-yl)amino)quinoli ne-3-carboxylate (8.0 g, 41 %) as an orange solid. The two batches were combined and used in the subsequent step. NMR Spectrum: ¹ H NMR (500 MHz, CDC1 ₃ ) 0.85 (3H, s), 1.21 (3H, s), 1.44 (3H, t), 1.95 (2H, dd), 3.19 (1H, d), 3.48 - 3.59 (2H, m), 3.92 (1H, td), 4.04 (1H, dt), 4.42 (2H, q), 7.74 (1H, dd), 7.84 (1H, d), 8.22 (1H, d), 8.96 (1H, d), 9.12 (1H, s). Mass Spectrum: m/z (ES+)[M+H]+ = 407.2

Example 12

(S)-8-(6-(Methoxymethyl)pyridin-3-yl)-l-(tetrahydro-2H-pyran -3-yl)-l,3- dihydro-2H-imidazo [4,5-c] uinolin-2-one

Triethylamine (1.063 ml, 7.62 mmol) was added to (S)-6-(6- (methoxymethyl)pyridin-3 -yl)-4-((tetrahydro-2H-pyran-3 -yl)amino)quino line-3 - carboxylic acid (1 g, 2.54 mmol) in DMF (10 ml) under nitrogen. The resulting suspension was stirred for 15 minutes then diphenylphosphoryl azide (0.657 ml, 3.05 mmol) was added and the suspension was stirred for another 30 minutes at r.t., then at 60°C for one h. The reaction mixture was poured onto ice (-100 ml) and the solid was filtered off and dried then stirred in Et ₂ 0 for two h. The solid was filtered off and dried to afford (5)-8-(6-(methoxymethyl)pyridin-3-yl)-l-(tetrahydro-2H- pyran-3-yl)-l,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one (0.967 g, 97 %) as a white solid. NMR Spectrum: ¹ H NMR (500MHz, DMSO) δ 1.66 - 1.94 (2H, m), 2.16 (1H, d), 2.58 - 2.7 (1H, m), 3.35 - 3.4 (1H, m), 3.41 (3H, s), 3.92 (1H, d), 4.13 (1H, dd), 4.23 (1H, t), 4.58 (2H, s), 4.8 - 5.06 (1H, m), 7.49 - 7.74 (1H, m), 7.97 (1H, dd), 8.13 (1H, d), 8.23 (1H, dd), 8.40 (1H, d), 8.66 (1H, s), 8.97 (1H, d), 11.57 (1H, s). Mass Spectrum: m/z (ES+)[M+H]+ = 391.

This material can also be isolated as the methanesulfonic acid salt by dissolving in a small quantity of dichloromethane and treating with an equivalent of

methanesulfonic acid dissolved in a small quantity of dichloromethane, removing the solvent and then stirring the residue in Et ₂ 0 followed by filtration. NMR Spectrum: ¹ H NMR (500MHz, DMSO-d6) δ 1.75 - 1.93 (2H, m), 2.16 - 2.29 (1H, m), 2.36 (3H, s), 2.59 - 2.76 (1H, m), 3.31 - 3.48 (4H, m), 3.94 (1H, d), 4.13 - 4.31 (2H, m), 4.64 (2H, s), 5.05 - 5.15 (1H, m), 7.71 (1H, d), 8.22 - 8.49 (3H, m), 8.58 (1H, s), 9.06 (2H, d), 12.47 (1H, s). Mass Spectrum: m/z (ES+)[M+H]+ = 391.

(S)-6-(6-(Methoxymethyl)pyridin-3-yl)-4-((tetrahydro-2H-p yran-3- yl)amino)quinoline-3-carbox lic acid

A solution of ethyl (5)-6-(6-(methoxymethyl)pyridin-3-yl)-4-((tetrahydro-2H- pyran-3-yl)amino)quinoline-3 -carboxylate (1.64 g, 3.89 mmol) in THF (40 ml) was treated with sodium hydroxide (0.311 g, 7.78 mmol) in water (20 ml) and the solution stirred at 60°C for three h then allowed to cool. The organic solvent was removed under reduced pressure and the aqueous was adjusted to pH 1 with 2 M HC1 to give a precipitate which was filtered off, washed thoroughly with water and Et ₂ 0 then dried to afford (5)-6-(6-(methoxymethyl)pyridin-3-yl)-4-((tetrahydro-2H- pyran-3-yl)amino)quinoline-3-carboxylic acid (1.080 g, 70.5 %) as a white solid. Mass Spectrum: m/z (ES+)[M+H]+ = 394.

(S)-Ethyl 6-(6-(methoxymethyl)pyridin-3-yl)-4-((tetrahydro-2H-pyran-3- yl)amino)quinoline-3-carbox late

To a mixture of ethyl 4-chloro-6-(6-(methoxymethyl)pyridin-3-yl)quinoline-3- carboxylate (2 g, 5.61 mmol) and (5)-tetrahydro-2H-pyran-3-amine hydrochloride (0.926 g, 6.73 mmol) in DMF (10 ml) was added DIPEA (3.5 ml, 20.04 mmol) and the solution stirred at 80°C for three h then allowed to cool. The reaction mixture was stirred with water (100 ml) and the solid was filtered off, washed thoroughly with water and sucked dry. The crude product was purified by FCC, elution gradient 0 to 3% 2N methanolic ammonia in DCM and pure fractions were evaporated to dryness to afford (5)-ethyl 6-(6-(methoxymethyl)pyridin-3-yl)-4- ((tetrahydro-2H-pyran-3-yl)amino)quinoline-3-carboxylate (1.640 g, 69.4 %) as a white solid. NMR Spectrum: ¹ H NMR (500MHz, DMSO-d6) 5 1.36 (3H, t), 1.47 - 1.64 (1H, m), 1.67 - 1.94 (2H, m), 1.96 - 2.2 (1H, m), 3.40 (3H, s), 3.56 (1H, dd), 3.61 (2H, t), 3.87 (1H, dd), 4.22 (1H, dd), 4.36 (2H, q), 4.56 (2H, s), 7.54 (1H, dd), 7.95 (1H, d), 8.10 (1H, dd), 8.19 (1H, dd), 8.40 (1H, d), 8.91 (1H, s), 8.92 - 8.93 (1H, m), 8.94 (1H, dd). Mass Spectrum: m/z (ES+)[M+H]+ = 422.

Ethyl 4-chloro-6-(6-(methox methyl)pyridin-3-yl)quinoline-3-carboxylate

Tetrakis(triphenylphosphine)palladium(0) (2.204 g, 1.91 mmol) was added to ethyl 6-bromo-4-chloroquinoline-3-carboxylate (6.0 g, 19.07 mmol), 2-(methoxymethyl)- 5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)pyridine (10.30 g, 24.80 mmol) and cesium carbonate (12.43 g, 38.15 mmol) in 1,4-dioxane (100 mL) and water (20.00 mL) under nitrogen. The resulting suspension was stirred at 120°C for 3 h. The crude product was purified by FCC, elution gradient 25 to 100% EtOAc in heptane. Pure fractions were evaporated to dryness to afford ethyl 4-chloro-6-(6- (methoxymethyl)pyridin-3-yl)quinoline-3-carboxylate (3.10 g, 45.6 %) as a cream solid. NMR Spectrum: ¹ H NMR (400MHz, CDC1 ₃ ) δ 1.48 (3H, t), 3.54 (3H, s), 4.52 (2H, q), 4.68 (2H, s), 7.59 (1H, d), 8.02 - 8.09 (2H, m), 8.26 (1H, d), 8.58 (1H, d), 8.94 (1H, d), 9.22 (1H, s). Mass Spectrum: m/z (ES+)[M+H]+ = 357. l-(3,3-Dimethyltetrahydro-2H-pyran-4-yl)-8-(6-(methoxymethyl )pyridin-3-yl)- lH-imidazo [4,5-c] quinolin-2 3H)-one

Trichloroisocyanuric acid (0.567 g, 2.44 mmol) was added portionwise to a stirred suspension of 4-((3,3-dimethyltetrahydro-2H-pyran-4-yl)amino)-6-(6- (methoxymethyl)pyridin-3-yl)quinoline-3-carboxamide (2.05 g, 4.88 mmol) and l,8-diazabicyclo[5.4.0]undec-7-ene (1.458 ml, 9.75 mmol) in MeOH (2 ml) at 0°C .The resulting suspension was stirred to r.t. for 4 h. The reaction mixture was evaporated to dryness and redissolved in DCM (75 mL), and washed with water (50 mL). The organic layer was dried with a phase separating cartridge and evaporated to afford crude product. Material was progressed to the next step without further purification. Mass Spectrum: m/z (ES+)[M+H]+ = 419.

4-((3,3-Dimethyltetrahydro-2H-pyran-4-yl)amino)-6-(6- (methoxymethyl)pyridin-3-yl)quinoline-3-carboxamide

3,3-Dimethyltetrahydro-2H-pyran-4-amine (0.694 g, 5.37 mmol) was added slowly to 4-chloro-6-(6-(methoxymethyl)pyridin-3-yl)quinoline-3-carbox amide (2 g, 4.88 mmol) and triethylamine (0.408 mL, 2.93 mmol) in DMF (11 mL). The resulting slurry was stirred at 90°C for 4 h. A further 300 mg of 3,3-dimethyltetrahydro-2H- pyran-4-amine was added and stirred for a further 16 h overnight. DMF was removed under reduced pressure and the brown oil was diluted with DCM (50 mL). The organics were washed with water (50 mL) and dried over a phase separator. The solvent was removed under reduced pressure to afford brown solid. Material progressed without further purification. Mass Spectrum: m/z (ES+)[M+H]+ = 421. 4-Chloro-6-(6-(methoxymethyl)pyridin-3-yl)quinoline-3-carbox amide

A slurry of 4-chloro-6-(6-(methoxyrnethyl)pyridin-3-yl)quinoline-3-carbo nyl chloride (15.62 g, 45 mmol) in MeCN (200 mL) was added portionwise to

5 ammonium hydroxide (58.4 mL, 450.00 mmol) maintaining the internal

temperature at 0°C and stirred for 1 h. The precipitated solid was filtered, washed with water and dried to afford crude 4-chloro-6-(6-(methoxymethyl)pyridin-3- yl)quinoline-3-carboxamide (10.90 g, 73.9 %) as a beige solid. Mass Spectrum: m/z (ES+)[M+H]+ = 328.

10

4-Chloro-6-(6-(methoxymeth l)pyridin-3-yl)quinoline-3-carbonyl chloride

DMF (0.352 ml, 4.54 mmol) was added to 6-(6-(methoxymethyl)pyridin-3-yl)-4- oxo-l,4-dihydroquinoline-3-carboxylic acid (14.1 g, 45.44 mmol) and thionyl i s chloride (66.3 ml, 908.79 mmol) at r.t.. The resulting solution was stirred at 75°C for h with the gases passing through a water scrubber. The resulting mixture was evaporated to dryness and azeotroped with toluene (2x100 mL) to afford crude 4- chloro-6-(6-(methoxymethyl)pyridin-3-yl)quinoline-3-carbonyl chloride as a brown solid which was used in the next reaction without further purification.

20

6-(6-(Methoxymethyl)pyridin-3-yl)-4-oxo-l,4-dihydroquinoline -3-carboxylic acid

Sodium hydroxide (40.0 ml, 80.00 mmol) was added to a stirred suspension of 25 ethyl 6-(6-(methoxymethyl)pyridin-3-yl)-4-oxo- 1 ,4-dihydroquinoline-3-carboxylate (5.41 g, 16 mmol) in ethanol (50 mL). The resulting suspension was stirred at 100°C for 30 minutes. Water (50 mL) was added followed by HC1 (2N) to obtain pH 3. The precipitate was filtered, washed with water and dried under vacuum to afford 6-(6-(methoxymethyl)pyridin-3-yl)-4-oxo- 1 ,4-dihydroquinoline-3-carboxylic acid (4.15 g, 84 %) as a grey solid. Mass Spectrum: m/z (ES+)[M+H]+ = 311.

Ethyl 6-(6-(methoxymethyl)py ridin-3-yl)-4-oxo- 1 ,4-dihydroquinoline-3- carboxylate

Sodium tetrachloropalladate(II) (2.68 g, 9.12 mmol) and 3-(di-tert- butylphosphino)propane-l -sulfonic acid (4.89 g, 18.24 mmol) were added in one portion to ethyl 6-bromo-4-oxo-l,4-dihydroquinoline-3-carboxylate (54 g, 182.36 mmol), 2-(methoxymethyl)-5-(4,4,5,5-tetramethyl- 1 ,3,2-dioxaborolan-2-yl)pyridine (83 g, 200.60 mmol) and K2CO3 (76 g, 547.08 mmol) in thoroughly degassed dioxane (500 ml) and water (125 ml) under nitrogen. The resulting mixture was stirred at 85°C for 2 h then left to cool to r.t.. The precipitate was filtered and washed with water to give crude ethyl 6-(6-(methoxymethyl)pyridin-3-yl)-4-oxo- l,4-dihydroquinoline-3-carboxylate (38.1 g, 61.7 %) as a yellow solid. On standing the filtrate precipitated a further amount of solid material, this was filtered to give ethyl 6-(6-(methoxymethyl)pyridin-3-yl)-4-oxo- 1 ,4-dihydroquinoline-3-carboxylate (12 g, 19.45 %) as a white solid. Both samples were used without further purification. Mass Spectrum: m/z (ES+)[M+H]+ = 339.

Ethyl 6-bromo-4-oxo-lH-quinoline-3-carbox late

Diphenyl ether (870 mL) was heated to 240°C then diethyl {[(4- bromophenyl)amino]methylidene}propanedioate (75 g, 219.18 mmol) added portionwise. The mixture was stirred at 240°C for 60 minutes in a flask fitted with Dean-Stark apparatus. After cooling ,the mixture was diluted with Et ₂ 0 and the solid was collected by filtration, washed with Et ₂ 0 and dried to afford ethyl 6- bromo-4-oxo-lH-quinoline-3-carboxylate (59.9g) as a beige crystallized solid, which was used without purification or characterisation. Diethyl {[(4-bromophenyl)amino]methylidene}propanedioate

Diethyl 2-(ethoxymethylene)malonate (71.5 mL, 354 mmol) was added to 4- bromoaniline (42 g, 244 mmol) in EtOH (420 mL) and the resulting mixture stirred at 80°C overnight. After cooling to 10°C, the white solid was collected by filtration, washed with heptane and dried to afford diethyl {[(4- bromophenyl)amino]methylidene}propanedioate (75 g, 90%) as a white crystalline solid. 'HNMR Spectrum (500MHz, DMSO-d6) δ 1.25 (6H, s), 4.10 - 4.27 (4H, m), 7.38 (2H, d), 7.57 (2H, d), 8.37 (1H, br s). Mass Spectrum: m/z (ES+)[M+H] ⁺ = 342, 344.

BIOLOGICAL ASSAYS

The following assays were used to measure the effects of the compounds of the present invention: a) ATM cellular potency assay; b) PI3K cellular potency assay; c) mTOR cellular potency assay; d) ATR cellular potency assay; e): DNAPK cellular potency assay. During the description of the assays, generally:

i. The following abbreviations have been used: 4NQO = 4-Nitroquinoline N- oxide; Ab = Antibody; BSA = Bovine Serum Albumin; C0 ₂ = Carbon Dioxide; DMEM = Dulbecco's Modified Eagle Medium; DMSO =Dimethyl Sulphoxide; EDTA = Ethylenediaminetetraacetic Acid; EGTA = Ethylene Glycol Tetraacetic Acid; ELISA = Enzyme-linked Immunosorbent Assay; EMEM = Eagle's Minimal Essential Medium; FBS = Foetal Bovine Serum; h = Hour(iS); HRP = Horseradish Peroxidase; i.p. = intraperitoneal; PBS = Phosphate buffered saline; PBST = Phosphate buffered saline / Tween; TRIS = Tris(Hydroxymethyl)aminomethane; MTS reagent: [3-(4,5- dimethylthiazo l-2-yl)-5 -(3 -carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium, inner salt, and an electron coupling reagent (phenazine methosulfate) PMS; s.c. sub-cutaneously.

ii. IC50 values were calculated using a smart fitting model in Genedata. The IC50 value was the concentration of test compound that inhibited 50% of biological activity.

Assay a): ATM Cellular Potency

Rationale:

Cellular irradiation induces DNA double strand breaks and rapid intermolecular autophosphorylation of serine 1981 that causes dimer dissociation and initiates cellular ATM kinase activity. Most ATM molecules in the cell are rapidly phosphorylated on this site after doses of radiation as low as 0.5 Gy, and binding of a phosphospecific antibody is detectable after the introduction of only a few DNA double-strand breaks in the cell.

The rationale of the pATM assay is to identify inhibitors of ATM in cells. HT29 cells are incubated with test compounds for lhr prior to X-ray- irradiation, lh later the cells are fixed and stained for pATM (Serl981). The fiuorescence is read on the arrayscan imaging platform.

Method details:

HT29 cells (ECACC #85061109) were seeded into 384 well assay plates (Costar #3712) at a density of 3500 cells / well in 40μ1 EMEM medium containing 1% L glutamine and 10% FBS and allowed to adhere overnight. The following morning compounds of Formula (I) in 100%) DMSO were added to assay plates by acoustic dispensing. After lh incubation at 37°C and 5% CO2, plates (up to 6 at a time) were irradiated using the X-RAD 320 instrument (PXi) with equivalent to ~600cGy. Plates were returned to the incubator for a further lh. Then cells were fixed by adding 20μ1 of 3.7% formaldehyde in PBS solution and incubating for 20 minutes at r.t. before being washed with 50μ1 / well PBS, using a Biotek EL405 plate washer. Then 20μ1 of 0.1% Triton XI 00 in PBS was added and incubated for 20 minutes at r.t., to permeabalise cells. Then the plates were washed once with 50μ1 / well PBS, using a Biotek EL405 plate washer.

Phospho-ATM Serl981 antibody (Millipore #MAB3806) was diluted 10000 fold in PBS containing 0.05% polysorbate/Tween and 3% BSA and 20μ1 was added to each well and incubated over night at r.t. The next morning plates were washed three times with 50μ1 / well PBS, using a Biotek EL405 plate washer, and then 20μ1 of secondary Ab solution, containing 500 fold diluted Alexa Fluor® 488 Goat anti- rabbit IgG (Life Technologies, Al 1001) and 0.002mg/ml Hoeschst dye (Life technologies #H-3570), in PBS containing 0.05%> polysorbate/Tween and 3% BSA, was added. After lh incubation at r.t., the plates were washed three times with 50μ1 / well PBS, using a Biotek EL405 plate washer, and plates were sealed and kept in PBS at 4°C until read. Plates were read using an ArrayScan VTI instrument, using an XF53 filter with 10X objective. A two laser set up was used to analyse nuclear staining with Hoeschst (405nm) and secondary antibody staining of pSerl981 (488nm).

Assay b): ATR Cellular Potency Rationale:

ATR is a PI 3-kinase-related kinase which phosphorylates multiple substrates on serine or threonine residues in response to DNA damage during or replication blocks. Chkl, a downstream protein kinase of ATR, plays a key role in DNA damage checkpoint control. Activation of Chkl involves phosphorylation of Ser317 and Ser345 (the latter regarded as the preferential target for

phosphorylation/activation by ATR). This was a cell based assay to measure inhibition of ATR kinase, by measuring a decrease in phosphorylation of Chkl (Ser 345) in HT29 cells, following treatment with compound of Formula (I) and the UV mimetic 4NQO (Sigma #N8141). Method details:

HT29 cells (ECACC #85061109) were seeded into 384 well assay plates (Costar #3712) at a density of 6000 cells / well in 40μ1 EMEM medium containing 1% L glutamine and 10% FBS and allowed to adhere overnight. The following morning compound of Formula (I) in 100% DMSO were added to assay plates by acoustic dispensing. After lh incubation at 37°C and 5% CO ₂ , 40nl of 3mM 4NQO in 100%) DMSO was added to all wells by acoustic dispensing, except minimum control wells which were left untreated with 4NQO to generate a null response control. Plates were returned to the incubator for a further lh. Then cells were fixed by adding 20μ1 of 3.7% formaldehyde in PBS solution and incubating for 20 mins at r.t. Then 20μ1 of 0.1% Triton XI 00 in PBS was added and incubated for 10 minutes at r.t., to permeabalise cells. Then the plates were washed once with 50μ1 / well PBS, using a Biotek EL405 plate washer.

Phospho-Chkl Ser 345 antibody (Cell Signalling Technology #2348) was diluted 150 fold in PBS containing 0.05%> polysorbate/Tween and 15μ1 was added to each well and incubated over night at r.t. The next morning plates were washed three times with 50μ1 / well PBS, using a Biotek EL405 plate washer, and then 20μ1 of secondary Ab solution, containing 500 fold diluted Alexa Fluor 488 Goat anti- rabbit IgG (Molecular Probes #A-11008) and 0.002mg/ml Hoeschst dye (Molecular Probes #H-3570), in PBST, was added. After 2h incubation at r.t., the plates were washed three times with 50μ1 / well PBS, using a Biotek EL405 plate washer, and plates were then sealed with black plate seals until read. Plates were read using an ArrayScan VTI instrument, using an XF53 filter with 10X objective. A two laser set up was used to analyse nuclear staining with Hoeschst (405nm) and secondary antibody staining of pChkl (488nm).

Assay c): PI3K Cellular Potency

Rationale:

This assay was used to measure PI3K-a inhibition in cells. PDK1 was identified as the upstream activation loop kinase of protein kinase B (Aktl), which is essential for the activation of PKB. Activation of the lipid kinase

phosphoinositide 3 kinase (PI3K) is critical for the activation of PKB by PDKl .

Following ligand stimulation of receptor tyrosine kinases, PI3K is activated, which converts PIP2 to PIP3, which is bound by the PH domain of PDKl resulting in recruitment of PDKl to the plasma membrane where it phosphorylates AKT at Thr308 in the activation loop.

The aim of this cell-based mode of action assay is to identify compounds that inhibit PDK activity or recruitment of PDKl to membrane by inhibiting PI3K activity. Phosphorylation of phospho-Akt (T308) in BT474c cells following treatment with compounds for 2h is a direct measure of PDKl and indirect measure of PI3K activity.

Method details:

BT474 cells (human breast ductal carcinoma, ATCC HTB-20) were seeded into black 384 well plates (Costar, #3712) at a density of 5600 cells / well in DMEM containing 10% FBS and 1% glutamine and allowed to adhere overnight.

The following morning compounds in 100% DMSO were added to assay plates by acoustic dispensing. After a 2h incubation at 37°C and 5% C0 ₂ , the medium was aspirated and the cells were lysed with a buffer containing 25mM Tris, 3mM EDTA, 3mM EGTA, 50mM sodium fluoride, 2mM Sodium orthovanadate,

0.27M sucrose, lOmM β-glycerophosphate, 5mM sodium pyrophosphate, 0.5%> Triton X-100 and complete protease inhibitor cocktail tablets (Roche #04 693 116

001, used 1 tab per 50ml lysis buffer).

After 20 minutes, the cell lysates were transferred into ELISA plates (Greiner # 781077) which had been pre-coated with an anti total- AKT antibody in PBS buffer and non-specific binding was blocked with 1% BSA in PBS containing 0.05% Tween 20. Plates were incubated over night at 4°C. The next day the plates were washed with PBS buffer containing 0.05% Tween 20 and further incubated with a mouse monoclonal anti-phospho AKT T308 for 2h. Plates were washed again as above before addition of a horse anti-mouse-HRP conjugated secondary antibody. Following a 2h incubation at r.t., plates were washed and QuantaBlu substrate working solution (Thermo Scientific #15169, prepared according to provider's instructions) was added to each well. The developed fluorescent product was stopped after 60 minutes by addition of Stop solution to the wells. Plates were read using a Tecan Safire plate reader using 325nm excitation and 420nm emission wavelengths respectively. Except where specified, reagents contained in the Path Scan Phospho AKT (Thr308) sandwich ELISA kit from Cell Signalling (#7144) were used in this ELISA assay.

Assay d): mTOR Cellular Potency

Rationale:

The phospho-AKTser473 cell assay was performed in the MDA-MB-468 cell line, a PTEN null breast adenocarcinoma human cell line. As a consequence of the lack of PTEN, pAKT is constitutive ly activated which eliminates the requirement for stimulation to induce phosphorylation.

Method details:

MDA-MB-468 cells were cultured in cell media composed of DMEM (Dulbecco's modified Eagle's medium #D6546)), 10% (v/v) Foetal Calf Serum and 1%) (v/v) L-Glutamine. After harvesting, cells were dispensed into black, 384-well Costar plates (#3712, Corning) to give 1500 cells per well in a total volume of 40μ1 cell media, and were incubated overnight at 37°C, 90%> relative humidity and 5% C02 in a rotating incubator. Compounds were then tested by one of two assay protocols A or B:

Protocol A:

The cell plates were then incubated for 2 hours at 37°C before being fixed by the addition of 20μ1 3.7% formaldehyde in PBS/A (1.2% final concentration), followed by a 40 minute room temperature incubation, and then a 2x wash with 150μ1 PBS/A (phosphate buffered saline) using a BioTek ELx406 platewasher. Cells were permeabilised and blocked with 20μ1 of assay buffer (0.5%> Tween 20 in PBS/A + 1% milk powder) for lh at room temperature, and then washed lx with 50μ1 PBS/A. Primary phospho-AKT (Ser473) 736E11 rabbit monoclonal antibody (#3787, Cell Signaling Technology) was diluted 1 :500 in assay buffer, 20μ1 added per well, and plates were incubated at 4°C overnight. Cell plates were washed 3x with 200μ1 PBS/T (phosphate buffered saline containing 0.05% Tween-20), then 20μ1 1 : 1000 dilution in assay buffer of Alexa Fluor® 488 goat anti-rabbit IgG secondary antibody (#A11008, Molecular Probes, Life Technologies), with a 1 :5000 dilution of Hoechst 33342, was added per well. Following a 2 hour incubation at room temperature, plates were washed 3x with 200μ1 PBS/T, and 40μ1 PBS/A was added per well.

Stained cell plates were covered with black seals, and then read on the Acumen (TTP Labtech) plate reader. The primary channel (green fluorescence, 488nm) is used to set the intensity settings for the max/min cut off to allow for weekly variation in staining and the ΆΚΤ+: No of objects (No)' data is used for the analysis. Data was analysed and IC50's were calculated using Genedata Screener® software.

Protocol B:

The cell plates were then incubated for 2 hours at 37°C before being fixed by the addition of 20μ1 3.7% formaldehyde in PBS/A (1.2% final concentration), followed by a 30 minute room temperature incubation, and then a 2x wash with 150μ1 PBS/A using a BioTek ELx406 platewasher. Cells were permeabilised and blocked with 20μ1 of assay buffer (0.1% Triton X-100 in PBS/A + 1% BSA) for lh at room temperature, and then washed lx with 50μ1 PBS/A. Primary phospho-AKT (Ser473) D9E XP® rabbit monoclonal antibody (#4060, Cell Signaling

Technology) was diluted 1 :200 in assay buffer, 20μ1 added per well, and plates were incubated at 4°C overnight. Cell plates were washed 3x with 200μ1 PBS/T, then 20μ1 1 :750 dilution in assay buffer of Alexa Fluor® 488 goat anti-rabbit IgG secondary antibody (#A11008, Molecular Probes, Life Technologies), with a 1 :5000 dilution of Hoechst 33342, was added per well. Following a 1 hour incubation at room temperature, plates were washed 3x with 200μ1 PBS/T, and 40μ1 PBS w/o Ca, Mg and Na Bicarb (Gibco #14190-094) was added per well.

Stained cell plates were covered with black seals, and then read on the Cell Insight imaging platform (Thermo Scientific), with a lOx objective. The primary channel (Hoechst blue fluorescence 405nM, BGRFR 386 23) is used to Auto focus and to count number of events (this will provide information about cytotoxicity of the compounds tested). The secondary channel (Green 488nM, BGRFR 485 20) measures pAKT staining. Data was analysed and IC50's were calculated using Genedata Screener ^® software.

Assay e): DNAPK Cellular Potency

Compound Handling:

All compounds or DMSO (dimethyl sulphoxide) for the DNAPK cell ELISA assay were dispensed from source plates containing compounds at lOmM in 100% (v/v) DMSO or 100% DMSO, directly into assay plates using an Echo 555 Acoustic dispenser (Labcyte Inc™). lOmM compound stocks were diluted 1 : 100 using a fixed-tip 96-head Agilent VPrep liquid handler (Agilent Technologies, Santa Clara, CA) to give four intermediate dilutions (lOmM, ΙΟΟμΜ, ΙμΜ, lOnM). This intermediate plate was used by the Echo to dispense compounds and DMSO directly into the cell plates with a 12 point dose range (30, 10, 3.125, 1.25, 0.3, 0.1, 0.03125, 0.0125, 0.003, 0.001, 0.0003125, 0.00003μΜ) in order to calculate compound IC50, with a total DMSO concentration in the assay of 0.3%.

Method details:

The DNA-PK cell ELISA assay was performed in the HT29 colorectal carcinoma cell line. HT29 cells were cultured in cell media composed of MEM (Minimum Essential Medium Eagle Sigma #M2279), 10% (v/v) Foetal Calf Serum and 1%) (v/v) 200 mM L-Glutamine. After harvesting, cells were dispensed into black, 384-well Costar plates (#3712, Corning) to give 15,000 cells per well in a total volume of 40 ul cell media, and were incubated overnight at 37°C, 90%> relative humidity and 5% CO2 in a rotating incubator. Greiner 781077 all-black high-bind 384-well ELISA plates were coated with 0.5 μg/ml DNA-PK antibody (Abeam #abl832) in PBS overnight at 4°C. The following day the Greiner ELISA plates were washed 3x with PBS-T and blocked with 3%> BSA/PBS for ~2h, before a further 3x wash with PBS-T. Test compounds and reference controls were dosed directly into the cell plates using a Labcyte Echo 555 acoustic dispenser. The cell plates were then incubated for 1 hour at 37°C before receiving a radiation dose of 8 Gy (XPvAD 320, table height 65). The cells were incubated for a further 1 hour before removal of cell media. Lysis buffer (in-house preparation with addition of protease inhibitor cocktail tablets, Roche # 04 693 116 001) was dispensed at 25μ1Λνε11 and plates were incubated at 4°C for 15-20 min. Cell lysates (20μ1Λνε11) were transferred to the DNA-PK antibody-coated ELISA plates using a CyBio Felix liquid handling platform, and ELISA plates were incubated at 4°C overnight. The following day, ELISA plates were washed 3x with PBS-T and dispensed with in-house pS2056-DNA-PK antibody (O^g/ml in 3% BSA/PBS) at 20μ1Λνβ11. Plates were incubated with antibody for 1.5 hours at room temperature (RT) before 3x wash with PBS-T. Goat anti-rabbit HRP secondary antibody (1 :2000 dilution in 3% BSA/PBS; Cell Signaling #7074) was dispensed at 20 μΐ/well and plates were incubated at room temperature for 1 hour before 3x wash with PBS-T. QuantaBlu Working Substrate Solution (Thermo Scientific #15169, prepared according to manufacturer's instructions) was dispensed at 20 μΐ/well and plates were incubated at room temperature for 1 hour before a further 20 μΐ/well dispense with QuantaBlu Stop Solution provided within kit (Thermo Scientific #15169). The fluorescence intensity of individual wells was determined using a PerkinElmer EnVision plate reader. Data was analysed and IC50's were calculated using Genedata Screener® software.

Table 5 shows comparative data for certain Compounds of CN102399218A and CN102372711 A in Assays a) to e).

Table 5: Potency Data for Certain Compounds of CN102399218A and

CN102372711 A in Assays a) - e)