The specification relates to certain tetracyclic heteroaryl compounds and pharmaceutically acceptable salts thereof that inhibit G12C mutant RAS proteins and consequently exhibit anti-cancer activity. The specification also relates to use of said tetracyclic heteroaryl compounds and pharmaceutically acceptable salts thereof in methods of treatment of the human or animal body, for example in prevention or treatment of cancer. The specification also relates to processes and intermediate compounds involved in the preparation of said tetracyclic heteroaryl compounds and to pharmaceutical compositions containing them.

The KRAS, NRAS and HRAS genes encode a set of closely related small GTPase proteins KRas, NRas and HRas, collectively referred to herein as the Ras proteins or Ras, that share 82-90% overall sequence identity. The Ras proteins are critical components of signalling pathways transmitting signals from cell-surface receptors to regulate cellular proliferation, survival and differentiation. Ras functions as a molecular switch cycling between an inactive GDP-bound state and an active GTP-bound state. The GDP/GTP cycle of Ras is tightly regulated in cells by guanine nucleotide exchange factors (GEFs) such as Sos1 and Sos2, which promote the exchange of GDP for GTP, and GTPase activating proteins (GAPs) such as NF-1 and p120RasGAP which stimulate the intrinsic GTPase activity of Ras hydrolysing GTP to GDP.

The Ras proteins are 188-189 amino acids in length and have a highly conserved N-terminal G-domain containing the p-loop region, which binds nucleotide, and the switch I and switch II regions which are important for regulatory and effector protein interactions. The C-terminal region of the Ras proteins are more divergent and contain elements which regulate the association of Ras with the membrane including the conserved carboxyl terminal CAXX box motif which is necessary for post-translational prenylation modifications. On binding to GTP the switch I and switch II regions of Ras undergo a conformational change which enables its interaction and activation of effector proteins to regulate down-stream signalling pathways. The best characterised effector of Ras is the serine/threonine kinase Raf which regulates the activity of the mitogen-activate protein kinase (MAPK) pathway. The PI3K pathway is another important effector pathway down-stream of Ras with the p110 catalytic subunit of the class I phosphoinositide 3-kinases interacting with Ras. Other effectors of Ras including RaIGDS, Tiam1, PLC-ε and Rassf1 have been have also been described (Cox, et al. Nature Reviews Drug Discovery, 2014, 13:828-851).

RAS mutations are frequently found in cancer and approximately 30% of all human cancers have a mutation in KRAS, NRAS or HRAS genes. Oncogenic Ras is typically, but not exclusively, associated with mutations at glycine 12, glycine 13 or glutamine 61 of Ras. These residues are located at the active site of Ras and mutations impair intrinsic and/or GAP-catalysed GTPase activity favouring the formation of GTP bound Ras and aberrant activation of down-stream effector pathways. KRAS is the most frequently mutated RAS gene in cancer followed by NRAS and then HRAS. There are several tumour types that exhibit a high frequency of activating mutations in KRAS including pancreatic (˜90% prevalence), colorectal (˜40% prevalence) and non-small cell lung cancer (˜30% prevalence). KRAS mutations are also found in other cancer types including multiple myeloma, uterine cancer, bile duct cancer, stomach cancer, bladder cancer, diffuse large B cell lymphoma, rhabdomyosarcoma, cutaneous squamous cell carcinoma, cervical cancer, testicular germ cell cancer and others.

Glycine to cysteine mutations at residue 12 of Ras (the G12C mutation) is generated from a G.C to T.A base transversion at codon 12, a mutation commonly found in RAS genes that accounts for 14% of all KRAS, 2% of all NRAS and 2% of all HRAS mutations across cancer types. The G12C mutation is particularly enriched in KRAS mutant non-small cell lung cancer with approximately half carrying this mutation, which has been associated with the DNA adducts formed by tobacco smoke. The G12C mutation is not exclusively associated with lung cancer and is found in other RAS mutant cancer types including 8% of all KRAS mutant colorectal cancer.

To date there have been no inhibitors of G12C mutant Ras proteins which have been approved for therapeutic use. Hence there is a need for new inhibitors of G12C mutant Ras proteins that possess the required pharmaceutical properties to be suitable for clinical use. The compounds of the specification have been found to possess anti-tumour activity, being useful in inhibiting the uncontrolled cellular proliferation which arises from malignant disease. The compounds of the specification provide an anti-tumour effect by, as a minimum, acting as inhibitors of G12C mutant Ras proteins.

According to a first aspect of the specification there is provided a compound of the Formula (I):


wherein:
Ring A is selected from phenyl and bicyclic heteroaryl;
R1in each occurrence is independently selected from C1-4alkyl, halo, hydroxy, C1-4alkoxy, C1-3fluoroalkyl, C1-3fluoroalkoxy, cyano and acetylenyl;
b is 0, 1, 2 or 3;
Y is CH2or CH2CH2;
R2is cyano, halo, C1-4alkyl, C1-4alkoxy or C1-3fluoroalkyl;
R3is F, Me, Et, MeO or C1-2fluoroalkyl;
R4is H or Me;
R5is H or Me;
R6is H or CH2NMe2;
or a pharmaceutically acceptable salt thereof, provided that when Y is CH2, R2is Cl, R3is F, A is phenyl, b is 2, the groups R1are F and OH and are each ortho to the biaryl bond, and when both R4and R6are H, then R5is Me.

In a further aspect there is provided a pharmaceutical composition comprising a compound of Formula (I).

In a further aspect there is provided a method of treating cancer by administering to a subject suffering from cancer an effective amount of a compound of Formula (I).

In a further aspect there is provided a compound of Formula (I) for use as a medicament.

In a further aspect there is provided a compound of Formula (I) for use in the treatment of cancer.

In a further aspect there is provided a compound of Formula (I) for use in the manufacture of a medicament, for example a medicament for the treatment of cancer.

In a further aspect there is provided a kit comprising a pharmaceutical composition comprising a compound of Formula (I) and instructions for its use, for example for use in the treatment of cancer.

In a further aspect there is provided a method for the manufacture of a compound of Formula (I).

It has been found that the compounds of the present specification possess potent anti-tumour activity that, it is believed, derives from inhibition of the G12C mutant Ras proteins that are key mediators of proliferation and survival in certain tumour cells.

Due to their ability to bind to and inhibit the normal function of Ras G12C mutant proteins the compounds of the present specification may be of value as anti-tumour agents, in particular as selective inhibitors of the proliferation, survival, motility, dissemination and invasiveness of mammalian cancer cells leading to inhibition of tumour growth and survival and to inhibition of metastatic tumour growth. Particularly, the compounds of the present specification may be of value as anti-proliferative and anti-invasive agents in the containment and/or treatment of solid tumour disease. Particularly, the compounds of the present specification may be useful in the prevention or treatment of those tumours which are sensitive to inhibition of G12C mutant Ras and that are involved in the cell-signalling leading to the proliferation and survival of tumour cells.

It is believed that the compounds of the present specification interact with, and then covalently bind to, G12C mutant Ras through the acrylamide motif located on the upper piperazine ring of Formula (I). In binding to G12C mutant Ras, the compounds of the specification (as described herein) impair or substantially eliminate the ability of the G12C Ras proteins to access their active, pro-proliferative/pro-survival confirmation.

It has been discovered that the stereochemistry of the carbon atom of the piperazine ring that is bonded to the group Y (as marked with an asterisk in the figure below) is a key determinant of Ras G12C inhibitory activity, with the Ras G12C inhibitory activity between each enantiomer varying significantly. For the discussion of the properties of the compounds according to the specification herein the numbering shown in the figure below will be used throughout, albeit the names of the compounds as generated by the chemical naming software does not always adhere to this naming convention. The group O—Y is thus attached to C-5 of the quinazoline and may thus be referred to as a C-5 tether group. The groups R2, A and R3are attached to C-6, C-7 and C-8 of the quinazoline motif, respectively. As the group A is an aromatic group the bond between C-7 of the quinazoline and A is a biaryl bond.



In addition to the importance of the stereochemical configuration of the carbon to which Y is bound, the compounds according to the specification are atropisomeric due to the restricted rotation around the biaryl bond that links C-7 of the quinazoline ring to the aromatic ring A. It has been discovered that incorporation of a substituent R2at C-6 of the quinazoline ring and a substituent R3at C-8 of the quinazoline ring, in conjunction with substituents R1can deliver stable atropisomeric forms of the compounds of Formula (I) that can be separated and that are stable to storage. Furthermore, it has been discovered that the activity of each individual atropisomer of the compounds of the Formula (I) as inhibitors of Ras G12C mutant protein can vary to a large degree. In the examples reported herein activity differences of 10-fold or greater and sometimes 100-fold or more are observed between atropisomeric pairs of compounds of Formula (I). The activity difference between atropisomers may derive from the advantageous ability of the substituents R2and R3to hold the group A and the substituent(s) R1on A, in a conformation close to, or in, their optimal conformation for binding to G12C Ras mutant protein thus lowering the energy required for binding of the inhibitor to the target protein.

It has been established that the compounds of Formula (I), and in particular those compounds of Formula (I) with the preferred enantiomeric and atropisomeric form, can be very potent inhibitors of Ras G12C mutant protein. Where enantioselective syntheses have been performed wherein stereochemical configuration of a chiral starting material is retained through the synthesis, the preferred enantiomer has been identified to be that illustrated in the figure below. It has thus been found that optimum activity is delivered when the stereochemical configuration of the bond from the piperazine to Y differs for systems in which Y=CH2and those in which Y=CH2CH2. It has been assumed that where the syntheses are performed in a racemic manner and then the products are separated by chiral chromatography then the most active compounds have the same stereochemistry at the asymmetric piperazine carbon as presented below. Nonetheless, for the avoidance of doubt, the present disclosure encompasses all isomers and atropisomers of the compounds of Formula (I). It will be understood that the compounds having the stereochemical arrangement that gives optimal Ras G12C inhibitory activity are preferred embodiments of the specification.



It has been found that the compounds of Formula (I) having the stereochemistry shown above exhibit higher activity as inhibitors of G12C Ras mutant protein than compounds having the opposite stereochemistry or compounds lacking the group O—Y, that in compounds of Formula (I) tethers C-5 of the quinazoline ring to the piperazine ring.

In addition to the above, it has further been determined that incorporation of a substituent on the piperazine ring (i.e. R4or R5) can improve the metabolic profile of the compounds according to Formula (I). Compounds of Formula (I) in which R5is methyl have proven particularly advantageous in this respect. Incorporation of a methyl group at R5has advantageously been observed to greatly improve oral bioavailability in in vivo studies, this is believed to be due to a reduction in off-target reactivity with glutathione in rodents. For example, in the otherwise identical compounds, incorporation of a R5methyl group caused an improvement in oral bioavailability from 0 to 30% and a 30-fold reduction in clearance. This difference in oral bioavailability is seen for Example 4 (a compound in which R5is Me, see below) that has oral bioavailability in rats of 31% and the corresponding compound in which R5is H (not claimed herein, reported in WO2018/206539) that has an oral bioavailability of 0% in rat (a significant effect on clearance between the two compounds is also observed Example 4 has CI of 29 ml/min/kg cf R5desmethyl compound 962 ml/min/kg—a figure reflecting the short, 12 minute, half life of this compound in rat blood). Incorporation of a methyl group at R5has also been observed to improve cell permeability (Example 4, Caco AB of 58 Papp1E−6cm/s, the corresponding compound where R5is H that has 5.8 Papp1 E−6cm/s). In instances wherein Y=CH2CH2, incorporation of a R5methyl group likewise delivers good cell permeability, good oral bioavailability and low clearance (see e.g. Example 39 below, 72 Papp1 E−6cm/s, F(rat) 75%, Cl(rat) 22 ml/min/kg). R5methylation therefore delivers compounds with enhanced pharmacokinetic properties. In instances wherein Y=CH2CH2, the stereochemistry of the R5group has also proven to have a significant effect on the selective inhibitory effect on G12C mutant Ras.

Described herein are compounds that can bind to G12C mutant Ras. In biochemical and cell based assays the compounds of the present specification are shown to be potent G12C mutant Ras protein binders and may therefore be useful in the treatment of disorders mediated by KRas, NRas or HRas G12C mutations, in particular in the treatment of cancers expressing G12C mutated KRas, NRas or HRas proteins, such as pancreatic, colorectal, uterine, bile duct, stomach, bladder, cervical, testicular germ cell and non-small cell lung cancer and multiple myeloma, diffuse large B cell lymphoma, rhabdomyosarcoma and cutaneous squamous cell carcinoma. In binding to G12C mutant Ras protein that are critical components of signalling pathways transmitting signals from cell-surface receptors to regulate cellular proliferation, survival and differentiation, the compounds according to the specification can inhibit or abrogate cellular proliferation, survival and differentiation of cells such as tumour cells that express G12C mutant Ras protein.

Accordingly, the present specification provides a method for providing a selective inhibitory effect on G12C mutant Ras which comprises administering an effective amount of a compound of the Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore.

The present specification also relates to processes for the manufacture of compounds of Formula (I), to pharmaceutical compositions containing them, to methods of treatment comprising administering the said compounds to patients, for example humans, in need thereof, to the use of compounds of Formula (I) for the manufacture of medicaments, for example for use in the treatment of a patient suffering from a hyperproliferative disease such as cancer.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, theConcise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press;The Dictionary of Cell and Molecular Biology,3rd ed., 1999, Academic Press; and theOxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

So that the present specification may be more readily understood, certain terms are explicitly defined below. In addition, definitions are set forth as appropriate throughout the detailed description.

Units, prefixes, and symbols are denoted in their Système International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range.

The term “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of the active ingredient, and which contains no additional components which are unacceptably toxic to a subject to which the composition would be administered. Such compositions can be sterile. A pharmaceutical composition according to the present specification will comprise a compound of Formula (I) and a pharmaceutically acceptable excipient.

Terms such as “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to both (1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and (2) prophylactic or preventative measures that prevent and/or slow the development of a targeted pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented. In certain aspects, a subject is successfully “treated” for cancer according to the methods of the present disclosure if the patient shows, e.g., total, partial, or transient remission of a certain type of cancer.

The term “subject” refers to any animal (e.g., a mammal), including, but not limited to humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.

The ring A in the compounds of Formula (I) is selected from phenyl and bicyclic heteroaryl. Bicyclic heteroaryl as used herein refers to an aromatic group comprising two fused rings and containing 1, 2, 3 or 4 N atoms, or one 0 atom, or one S atom, or 1 N atom and one S atom, or 1 N atom and one 0 atom, or 2 N atoms and one S atom, or 2 N atoms and one 0 atom. Bicyclic heteroaryl groups include those groups where both fused rings are aromatic, or where one fused ring is aromatic and the other fused ring is partially or fully saturated. The said partially or fully saturated fused ring may also comprise a carbonyl group. The at least one heteroatom in the bicyclic heteroaryl group may be present in an aromatic ring or a saturated ring. The bicyclic heteroaryl groups A of the compounds of Formula (I) are [6,6] or [6,5] ring systems, examples of suitable bicyclic heteroaryl groups include indolyl, benzofuranyl, benzothienyl, benzoxazolyl, benzimidazolyl, benzotriazolyl, indazolyl, azaindolyl, azaindazolyl, pyrrolo[1,2-b]pyridazinyl and pyrrolo[2,3-b]pyridinyl, quinolinyl, isoquinolinyl, quinazolinyl, cinnolinyl, phthalazinyl, quinoxalinyl and naphthyridinyl and partially saturated derivatives thereof.

R1in each occurrence is independently selected from C1-4alkyl, halo, hydroxy, C1-4alkoxy, C1-3fluoroalkyl, C1-3fluoroalkoxy, cyano and acetylenyl. For the avoidance of doubt, C1-4alkyl refers to a straight or branched alkyl group containing from 1 to 4 carbon atoms, use of numerical subscripts throughout the specification, for example for alkoxy, fluoroalkyl and fluoroalkoxy groups is consistent with this usage.

The term halo as used herein refers to an atom selected from F, Cl, Br or I. In embodiments of the specification the halo groups in the compounds of Formula (I), and in particular for the groups R2and R1, F and Cl are preferred halo groups.

The group R2is selected from is cyano, halo, C1-4alkyl, C1-4alkoxy or C1-3fluoroalkyl. Examples of preferred R2groups include Cl, methyl and cyano, for example Cl. The group R3is selected from F, Me, Et, MeO or C1-2fluoroalkyl, for example F, Me or MeO.

In the instance where the compound of Formula (I) is such that Y is CH2, R2is Cl, R3is F, A is phenyl, b is 2, the groups R1are F and OH and are each ortho to the biaryl bond, and, furthermore, when both R4and R6are H, then the group R5is Me (and is not H). It will thus be understood that the compound 1-((8aS)-6-chloro-4-fluoro-5-(2-fluoro-6-hydroxyphenyl)-8a,9,11,12-tetrahydropyrazino[2′,1′:3,4] [1,4] oxazepino[5,6,7-de]quinazolin-10(8H)-yl)prop-2-en-1-one, as shown below, is not a compound according to Formula (I) and is not claimed herein.



For the avoidance of doubt, where multiple substituents are independently selected from a given group, the selected substituents may comprise the same substituents or different substituents from within the given group. By way of example only, where ring A is phenyl substituted with (R1)b, and where b is 2, the two R1substituents could be the same, for instance both fluoro, or could be different, for instance one fluoro and one hydroxy.

For the further avoidance of doubt, the use of “” in formulas of this specification denotes the point of attachment between different groups.

As noted above, the specification provides a compound of the Formula (I):


wherein:
Ring A is selected from phenyl and bicyclic heteroaryl;
R1in each occurrence is independently selected from C1-4alkyl, halo, hydroxy, C1-4alkoxy, C1-3fluoroalkyl, C1-3fluoroalkoxy, cyano and acetylenyl;
b is 0, 1, 2 or 3;
Y is CH2or CH2CH2;
R2is cyano, halo, C1-4alkyl, C1-4alkoxy or C1-3fluoroalkyl;
R3is F, Me, Et, MeO or C1-2fluoroalkyl;
R4is H or Me;
R5is H or Me;
R6is H or CH2NMe2;
or a pharmaceutically acceptable salt thereof, provided that when Y is CH2, R2is Cl, R3is F, A is phenyl, b is 2, the groups R1are F and OH and are each ortho to the biaryl bond, and when both R4and R6are H, then R5is Me.

In one embodiment there is provided a compound of Formula (I) as defined above.

In one embodiment there is provided a pharmaceutically acceptable salt of a compound of Formula (I).

In embodiments, the compound of Formula (I) is a compound of Formula (Ia) wherein Y is CH2.

In embodiments, the compound of Formula (I) is a compound of Formula (Ib) wherein Y is CH2CH2.

In embodiments, the compound of Formula (I), (Ia) or (Ib) is a compound of Formula (Ic) in which the group R2is selected from Cl, Me, or CN (cyano). In embodiments, the compound of Formula (Ic) is a compound of Formula (Id) in which the group R2is Cl.

In embodiments, the compound of Formula (I), (Ia), (Ib), (Ic) or (Id) is a compound of Formula (Ie) in which the group R3is selected from F, Me or MeO. In embodiments, the compound of Formula (Ie) is a compound of Formula (If) in which the group R3is F.

In embodiments, the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie) or (If) is a compound of Formula (Ig) in which the group R4is H.

In embodiments, the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If) or (Ig) is a compound of Formula (Ih) in which the group R5is H. In embodiments the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If) or (Ig) is a compound of Formula (Ii) in which the group R5is Me.

In embodiments, the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih) or (Ii) is a compound of Formula (Ij) in which the group R6is H.

In embodiments, the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii) or (Ij) is a compound of Formula (Ik) in which the group A is phenyl.

In embodiments, the compound of Formula (Ik) is a compound of Formula (Il) in which the integer b is 2 or 3 and at least one R1group is OH. In embodiments, the compound of Formula (Ik) or (Il) is a compound of Formula (Im) in which at least two substituents R1are ortho to the biaryl bond.

In embodiments, the compound of Formula (Ik) is a compound of Formula (In) in which the group A(R1)bis


optionally wherein R1is selected from Me, F, Cl and CN (cyano). In embodiments, the R1group in the compound of Formula (In) is selected from Me, Cl and CN.

In embodiments, the compound of Formula (Ik) is a compound of Formula (Io) in which the group A(R1)bis



In embodiments the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii) or (Ij) is a compound of Formula (Ip) in which the group A is bicyclic heteroaryl.

In embodiments, the compound of Formula (Ip) is a compound of Formula (Iq) in which the bicyclic heteroaryl group is selected from the group consisting of:



In embodiments, the compound of Formula (Ip) is a compound of Formula (Ir) in which the bicyclic heteroaryl group is selected from the group consisting of:



In embodiments, the compound of Formula (Ip) is a compound of Formula (Is) in which the bicyclic heteroaryl group is selected from the group consisting of:



In embodiments, the compound of Formula (Ip) is a compound of Formula (It) in which the bicyclic heteroaryl group is selected from the group consisting of:



In embodiments, the compounds of Formula (I), i.e. any of compounds of Formula (I), (Ia), (Ib) . . . to (It), is a compound of Formula (Iu) or (Iv) in which the stereochemistry is as shown below:



In embodiments, the compound of Formula (Iu) is a compound of Formula (Iui) in which Y=CH2. In embodiments, the compound of Formula (Iv) is a compound of Formula (Ivi) in which Y=CH2CH2.

In embodiments, the compounds of Formula (I), i.e. any of compounds of Formula (I), (Ia), (Ib) . . . to (Ivi), is a compound of Formula (Iw) in which R4is H and R5is Me.

In embodiments the compound of Formula (I) is selected from each enantiomeric and atropisomeric form of:7-[(8aS)-10-Acryloyl-6-chloro-4-fluoro-8,8a,9,10,11,12-hexahydropyrazino[2′,1′:3,4][1,4]oxazepino [5,6,7-de]quinazolin-5-yl]-6-methyl-2,3-dihydro-1H-isoindol-1-one;1-[(8aS,11S)-6-Chloro-4-fluoro-5-(2-fluoro-6-hydroxyphenyl)-11-methyl-8a,9,11,12-tetrahydropyrazino[2′,1′:3,4][1,4]oxazepino[5,6,7-de]quinazolin-10(8H)-yl]prop-2-en-1-one;1-[(8aS,11R)-6-Chloro-4-fluoro-5-(2-fluoro-6-hydroxyphenyl)-11-methyl-8a,9,11,12-tetrahydropyrazino[2′,1′:3,4][1,4]oxazepino[5,6,7-de]quinazolin-10(8H)-yl]prop-2-en-1-one;5-[(8aS)-10-Acryloyl-6-chloro-4-fluoro-8,8a,9,10,11,12-hexahydropyrazino[2′,1′:3,4][1,4]oxazepino[5,6,7-de]quinazolin-5-yl]-6-methylquinazolin-4(3H)-one;1-[(8aS)-6-Chloro-4-fluoro-5-(5-methyl-1H-benzimidazol-4-yl)-8a,9,11,12-tetrahydropyrazino [2′,1′:3,4][1,4]oxazepino[5,6,7-de]quinazolin-10(8H)-yl]prop-2-en-1-one;8-[(8aS)-6-Chloro-4-fluoro-10-(prop-2-enoyl)-8,8a,9,10,11,12-hexahydropyrazino[2′,1′:3,4][1,4] oxazepino[5,6,7-de]quinazolin-5-yl]isoquinolin-1(2H)-one;1-[(8aS)-6-Chloro-4-fluoro-5-(1H-indazol-3-yl)-8a,9,11,12-tetrahydropyrazino[2′,1′:3,4][1,4] oxazepino[5,6,7-de]quinazolin-10(8H)-yl]prop-2-en-1-one;1-[(8aS)-6-Chloro-4-fluoro-5-(2-hydroxy-6-methylphenyl)-8a,9,11,12-tetrahydropyrazino [2′,1′:3,4][1,4]oxazepino[5,6,7-de]quinazolin-10(8H)-yl]prop-2-en-1-one;(2E)-1-[(8aS)-6-Chloro-4-fluoro-5-(2-fluoro-6-hydroxyphenyl)-8a,9,11,12-tetrahydropyrazino [2′,1′:3,4][1,4]oxazepino[5,6,7-de]quinazolin-10(8H)-yl]-4-(dimethylamino)but-2-en-1-one;8-[(8aS)-6-Chloro-4-fluoro-10-(prop-2-enoyl)-8,8a,9,10,11,12-hexahydropyrazino[2′,1′:3,4][1,4] oxazepino[5,6,7-de]quinazolin-5-yl]-7-methylisoquinolin-1(2H)-one;1-[(8aS)-6-Chloro-4-fluoro-5-(5-methyl-1H-benzotriazol-4-yl)-8a,9,11,12-tetrahydropyrazino [2′,1′:3,4][1,4]oxazepino[5,6,7-de]quinazolin-10(8H)-yl]prop-2-en-1-one;1-((8aS)-6-chloro-4-fluoro-5-(5-fluoro-1H-benzo[d]imidazol-4-yl)-8a,9,11,12-tetrahydropyrazino [2′,1′:3,4][1,4]oxazepino[5,6,7-de]quinazolin-10(8H)-yl)prop-2-en-1-one;1-[(8aS)-6-Chloro-4-fluoro-5-(5-fluoro-1H-indazol-4-yl)-8a,9,11,12-tetrahydropyrazino[2′,1′:3,4] [1,4]oxazepino[5,6,7-de]quinazolin-10(8H)-yl]prop-2-en-1-one;1-((8aS)-6-Chloro-4-fluoro-5-(5-fluoro-1-methyl-1H-benzo[d]imidazol-4-yl)-8a,9,11,12-tetrahydropyrazino[2′,1′:3,4][1,4]oxazepino[5,6,7-de]quinazolin-10(8H)-yl)prop-2-en-1-one;1-[(8aS)-6-Chloro-4-fluoro-5-(5-fluoro-1H-benzotriazol-4-yl)-8a,9,11,12-tetrahydropyrazino[2′,1′:3,4] [1,4]oxazepino[5,6,7-de]quinazolin-10(8H)-yl]prop-2-en-1-one;8-[(8aS)-6-Chloro-4-fluoro-10-(prop-2-enoyl)-8,8a,9,10,11,12-hexahydropyrazino[2′,1′:3,4][1,4] oxazepino[5,6,7-de]quinazolin-5-yl]-7-fluoroisoquinolin-1(2H)-one;(2E)-1-[(8aS)-6-chloro-4-fluoro-5-(5-methyl-1H-indazol-4-yl)-8a,9,11,12-tetrahydropyrazino[2′,1′:3,4] [1,4]oxazepino[5,6,7-de]quinazolin-10(8H)-yl]-4-(dimethylamino)but-2-en-1-one;1-[(6aR,9S)-3-Chloro-1-fluoro-2-(2-fluoro-6-hydroxyphenyl)-9-methyl-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:5,6][1,5]oxazocino[4,3,2-de]quinazolin-8-yl]prop-2-en-1-one;1-[(6aR,9S)-3-Chloro-1-fluoro-2-(2-fluoro-6-hydroxyphenyl)-9-methyl-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:5,6][1,5]oxazocino[4,3,2-de]quinazolin-8-yl]prop-2-en-1-one;8-[3-Chloro-1-fluoro-8-(prop-2-enoyl)-6,6a,7,8,9,10-hexahydro-5H-pyrazino[1′,2′:5,6][1,5]oxazocino [4,3,2-de]quinazolin-2-yl]-7-methylisoquinolin-1(2H)-one;1-[(6aS,9R)-3-Chloro-1-fluoro-2-(2-fluoro-6-hydroxyphenyl)-9-methyl-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:5,6][1,5]oxazocino[4,3,2-de]quinazolin-8-yl]prop-2-en-1-one;1-[(6aR,9R)-3-Chloro-1-fluoro-2-(2-fluoro-6-hydroxyphenyl)-9-methyl-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:5,6][1,5]oxazocino[4,3,2-de]quinazolin-8-yl]prop-2-en-1-one;1-[(6aS,9S)-3-Chloro-1-fluoro-2-(2-fluoro-6-hydroxyphenyl)-9-methyl-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:5,6][1,5]oxazocino[4,3,2-de]quinazolin-8-yl]prop-2-en-1-one;1-[(8aS)-4-Chloro-6-fluoro-5-(2-fluoro-6-hydroxyphenyl)-8a,9,11,12-tetrahydropyrazino[2′,1′:3,4] [1,4]oxazepino[5,6,7-de]quinazolin-10(8H)-yl]prop-2-en-1-one;1-[(8aS,11R)-6-Chloro-4-fluoro-11-methyl-5-(5-methyl-1H-benzimidazol-4-yl)-8a,9,11,12-tetrahydropyrazino[2′,1′:3,4][1,4]oxazepino[5,6,7-de]quinazolin-10(8H)-yl]prop-2-en-1-one;8-[(8aS,11R)-6-Chloro-4-fluoro-11-methyl-10-(prop-2-enoyl)-8,8a,9,10,11,12-hexahydropyrazino [2′,1′:3,4][1,4]oxazepino[5,6,7-de]quinazolin-5-yl]-7-methylisoquinolin-1(2H)-one;(2E)-1-[(8aS,11R)-6-Chloro-4-fluoro-11-methyl-5-(5-methyl-1H-benzimidazol-4-yl)-8a,9,11,12-tetrahydropyrazino[2′,1′:3,4] [1,4]oxazepino[5,6,7-de]quinazolin-10(8H)-yl]-4-(dimethylamino)but-2-en-1-one; and(2E)-1-[(8aS,11R)-6-Chloro-4-fluoro-11-methyl-5-(5-methyl-1H-indazol-4-yl)-8a,9,11,12-tetrahydropyrazino[2′,1′:3,4][1,4]oxazepino[5,6,7-de]quinazolin-10(8H)-yl]-4-(dimethylamino)but-2-en-1-one;
or a pharmaceutically acceptable salt thereof.

In embodiments of the present specification there is provided a pharmaceutical composition which comprises a compound of the Formula (I) or a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable excipient, optionally further comprising one or more of the other stereoisomeric forms of the compound of Formula (I) or pharmaceutically acceptable salt thereof, wherein the compound of Formula (I) or pharmaceutically acceptable salt thereof is present within the composition with a diastereomeric excess (% d.e.) of ≥90%.

In embodiments of the present specification there is provided a pharmaceutical composition which comprises a compound of the Formula (I) or a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable excipient, optionally further comprising one or more of the other stereoisomeric forms of the compound of Formula (I) or pharmaceutically acceptable salt thereof, wherein the compound of Formula (I) or pharmaceutically acceptable salt thereof is present within the composition with an enantiomeric excess (% ee) of 90% and a diastereomeric excess (% de) of ≥90%.

The compounds of Formula (I) and pharmaceutically acceptable salts thereof may be prepared, used or supplied in amorphous form, crystalline form, or semi-crystalline form and any given compound of Formula (I) or pharmaceutically acceptable salt thereof may be capable of being formed into more than one crystalline/polymorphic form, including hydrated (e.g. hemi-hydrate, a mono-hydrate, a di-hydrate, a tri-hydrate or other stoichiometry of hydrate) and/or solvated forms. It is to be understood that the present specification encompasses any and all such solid forms of the compound of Formula (I) and pharmaceutically acceptable salts thereof.

In further embodiments of the present specification there is provided a compound of Formula (I), which is obtainable by the methods described in the ‘Examples’ section hereinafter.

The present specification is intended to include all isotopes of atoms occurring in the present compounds. Isotopes will be understood to include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include13C and14C. Isotopically labelled compounds of Formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples using appropriate isotopically labelled reagents in place of the non-labelled reagents previously employed.

A suitable pharmaceutically acceptable salt of a compound of the Formula (I) is, for example, an acid addition salt. A suitable pharmaceutically acceptable salt of a compound of the Formula (I) may be, for example, an acid-addition salt of a compound of the Formula (I), for example an acid-addition salt with an inorganic or organic acid.

A further suitable pharmaceutically acceptable salt of a compound of the Formula (I) is, for example, a salt formed within the human or animal body after administration of a compound of the Formula (I) to said human or animal body.

The compound of Formula (I) or pharmaceutically acceptable salt thereof may be prepared as a co-crystal solid form. It is to be understood that a pharmaceutically acceptable co-crystal of a compound of the Formula (I) or pharmaceutically acceptable salts thereof, form an aspect of the present specification.

For use in a pharmaceutical context it may be preferable to provide a compound of Formula (I) or a pharmaceutically acceptable salt thereof without large amounts of the other stereoisomeric forms being present.

Accordingly, in an embodiment of the present specification there is provided a composition comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof, optionally together with one or more of the other stereoisomeric forms of the compound of Formula (I) or pharmaceutically acceptable salt thereof, wherein the compound of Formula (I) or pharmaceutically acceptable salt thereof is present within the composition with a diastereomeric excess (% de) of 90%.

The compound of Formula (I), or a pharmaceutically acceptable salt thereof, will normally be administered via the oral or intravenous route, in the form of pharmaceutical preparations comprising the active ingredient or a pharmaceutically acceptable salt or solvate thereof, or a solvate of such a salt, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated and the route of administration, the compositions may be administered at varying doses, for example at a dose of from 1 mg to 1000 mg.

The pharmaceutical formulations of the compound of Formula (I) described above may conveniently be administered in unit dosage form and may be prepared by any of the methods well-known in the pharmaceutical art, for example as described in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., (1985). In one form the pharmaceutical might be in the form of a capsule, for example a two-piece hard shell capsule or a soft elastic gelatin (SEG) capsule comprising a compound of Formula (I) and, optionally, a pharmaceutically acceptable excipient. The pharmaceutical composition may contain an amount of from 1 mg to 1000 mg of the compound of Formula (I) or a pharmaceutically acceptable salt thereof.

According to a further embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore, for use as a medicament in a warm-blooded animal such as man.

According to a further embodiment, there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore for use in the production of an anti-proliferative effect in a warm-blooded animal such as man.

According to a further embodiment, there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore for use in a warm-blooded animal such as man as an anti-invasive agent in the containment and/or treatment of solid tumour disease.

According to a further embodiment, there is provided the use of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore, for the production of an anti-proliferative effect in a warm-blooded animal such as man.

According to a further embodiment, there is provided the use of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore, in the manufacture of a medicament for use in the production of an anti-proliferative effect in a warm-blooded animal such as man.

According to a further embodiment, there is provided the use of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore, in the manufacture of a medicament for use in a warm-blooded animal such as man as an anti-invasive agent in the containment and/or treatment of solid tumour disease.

According to a further embodiment, there is provided a method for producing an anti-proliferative effect in a warm-blooded animal, such as man, in need of such treatment which comprises administering to said animal an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore.

In this specification, unless otherwise stated, the phrase “effective amount” means an amount of a compound or composition which is sufficient enough to significantly and positively modify the symptoms and/or conditions to be treated (e.g., provide a positive clinical response). The effective amount of an active ingredient for use in a pharmaceutical composition will vary with the particular condition being treated, the severity of the condition, the duration of the treatment, the nature of concurrent therapy, the particular active ingredient(s) being employed, the particular pharmaceutically-acceptable excipient(s)/carrier(s) utilized, and like factors within the knowledge and expertise of the attending physician.

According to a further embodiment, there is provided a method for producing an anti-invasive effect by the containment and/or treatment of solid tumour disease in a warm-blooded animal, such as man, in need of such treatment which comprises administering to said animal an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore.

According to a further embodiment, there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore, for use in the prevention or treatment of cancer in a warm-blooded animal such as man.

According to a further embodiment, there is provided the use of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore in the manufacture of a medicament for use in the prevention or treatment of cancer in a warm-blooded animal such as man.

According to a further embodiment, there is provided a method for the prevention or treatment of cancer in a warm-blooded animal, such as man, in need of such treatment which comprises administering to said animal an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore.

According to a further embodiment, there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore for use in the prevention or treatment of solid tumour disease in a warm-blooded animal such as man.

According to a further embodiment, there is provided the use of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore, in the manufacture of a medicament for use in the prevention or treatment of solid tumour disease in a warm-blooded animal such as man.

According to a further embodiment, there is provided a method for the prevention or treatment of solid tumour disease in a warm-blooded animal, such as man, in need of such treatment which comprises administering to said animal an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore.

According to a further embodiment, there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore, for use in the prevention or treatment of tumours which are sensitive to inhibition of G12C mutant Ras.

According to a further embodiment, there is provided the use of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore, in the manufacture of a medicament for use in the prevention or treatment of those tumours which are sensitive to inhibition of G12C mutant Ras.

According to a further embodiment, there is provided a method for the prevention or treatment of those tumours which are sensitive to inhibition of G12C mutant RAS, which comprises administering to said animal an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore, to a patient in need thereof.

According to a further embodiment, there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore for use in providing an inhibitory effect on G12C mutant Ras.

According to a further embodiment, there is provided the use of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore in the manufacture of a medicament for use in providing an inhibitory effect on G12C mutant Ras.

According to a further embodiment, there is also provided a method for providing an inhibitory effect on G12C mutant RAS which comprises administering an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore, to a patient in need thereof.

According to a further embodiment, there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore, for use in providing a selective inhibitory effect on G12C mutant Ras.

According to a further embodiment, there is provided the use of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore, in the manufacture of a medicament for use in providing a selective inhibitory effect on G12C mutant Ras.

According to a further embodiment, there is also provided a method for providing a selective inhibitory effect on G12C mutant Ras which comprises administering an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore, to a patient in need thereof.

Described herein are compounds that can bind to G12C mutant Ras. In biochemical and cell based assays the compounds of the present specification are shown to be potent G12C mutant Ras protein binders and may therefore be useful in the treatment of disorders mediated by KRas, NRas or HRas G12C mutations, in particular in the treatment of cancers expressing G12C mutated KRas, NRas or HRas proteins, such as pancreatic, colorectal, uterine, bile duct, stomach, bladder, cervical, testicular germ cell and non-small cell lung cancer and multiple myeloma, diffuse large B cell lymphoma, rhabdomyosarcoma and cutaneous squamous cell carcinoma.

According to a further embodiment, there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore, for use in the treatment of disorders mediated by KRas, NRas or HRas G12C mutations.

According to a further embodiment, there is provided a method for treating disorders mediated by KRas, NRas or HRas G12C mutations, which comprises administering an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore, to a patient in need thereof.

According to a further embodiment, there is provided the use of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore, in the manufacture of a medicament for use in the treatment of disorders mediated by KRas, NRas or HRas G12C mutations.

According to a further embodiment, there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore, for use in the treatment of non-small cell lung cancer or colorectal cancer.

According to a further embodiment, there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore, for use in the treatment of non-small cell lung cancer.

According to a further embodiment, there is provided a method for treating non-small cell lung cancer or colorectal cancer, which comprises administering an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore, to a patient in need thereof.

According to a further embodiment, there is provided a method for treating non-small cell lung cancer, which comprises administering an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore.

According to a further embodiment, there is provided the use of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore, in the manufacture of a medicament for use in the treatment of breast or gynaecological cancers.

According to a further embodiment, there is provided the use of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore, in the manufacture of a medicament for use in the treatment of non-small cell lung cancer or colorectal cancer.

According to a further aspect of the specification, there is provided the use of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore, in the manufacture of a medicament for use in the treatment of non-small cell lung cancer.

The anti-cancer treatment defined herein may be applied as a sole therapy or may involve, in addition to the compounds of the specification, conventional surgery or radiotherapy or chemotherapy.

Accordingly, in one embodiment, there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and an additional anti-tumour substance for the conjoint treatment of cancer.

According to an embodiment of the specification there is provided a combination suitable for use in the treatment of cancer comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof and another anti-tumour agent.

In a further embodiment of the specification there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, in combination with another anti-tumour agent.

Although the compounds of Formula (I) are primarily of value as therapeutic agents for use in warm-blooded animals (including man), they are also useful whenever it is required to inhibit G12C mutant Ras. Thus, they are useful as pharmacological standards for use in the development of new biological tests and in the search for new pharmacological agents.

Another embodiment is based on identifying a link between the G12C KRas, HRas or NRas mutation status of a patient and potential susceptibility to treatment with a compound of Formula (I). A Ras inhibitor, such as a compound of Formula (I), may then advantageously be used to treat patients with G12C KRas, HRas or NRas mutations who may be resistant to other therapies. This therefore provides opportunities, methods and tools for selecting patients for treatment with a compound of Formula (I), particularly cancer patients. The selection is based on whether the tumour cells to be treated possess wild-type or G12C mutant KRAS, HRAS or NRAS gene. The G12C KRAS, HRAS or NRAS gene status could therefore be used as a biomarker to indicate that selecting treatment with a compound of Formula (I) may be advantageous. A patient identified as susceptible for successful treatment with a compound of Formula (I) can then be treated with such a compound. A method of treatment may thus encompass a first patient selection step and treatment of a patient in need thereof with an effective amount of a compound of Formula (I).

According to embodiments, there is provided a method for selecting a patient for treatment with a compound of Formula (I), the method comprising providing a tumour cell-containing sample from a patient; determining whether the RAS gene in the patient's tumour cell-containing sample encodes for wild-type (glycine at position 12) or mutant (cysteine at position 12) KRas, HRas or NRas protein; and selecting a patient for treatment with a compound of Formula (I) based thereon. A method of treatment may encompass such a method of patient selection.

The method may include or exclude the actual patient sample isolation step. Thus, according to one embodiment there is provided a method for selecting a patient for treatment with a compound of Formula (I), the method comprising determining whether the RAS gene in a tumour cell-containing sample previously isolated from the patient encodes for wild-type (glycine at position 12) or mutant (cysteine at position 12) KRas, HRas or NRas protein; and selecting a patient for treatment with a compound of Formula (I) based thereon.

In embodiments, the patient is selected for treatment with a compound of Formula (I) if the tumour cell DNA has a G12C mutant KRAS gene.

In embodiments, the patient is selected for treatment with a compound of Formula (I) if the tumour cell DNA has a G12C mutant HRAS gene.

In embodiments, the patient is selected for treatment with a compound of Formula (I) if the tumour cell DNA has a G12C mutant NRAS gene.

According to another embodiment, there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in treating cancers with tumour cells identified as harbouring a G12C mutant KRAS gene.

According to another embodiment, there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in treating cancers with tumour cells identified as harbouring a G12C mutant HRAS gene.

According to another aspect of the specification there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in treating cancers with tumour cells identified as harbouring a G12C mutant NRAS gene.

According to another embodiment, there is provided a method of treating cancers with tumour cells identified as harbouring a G12C mutant KRAS, HRAS or NRAS gene comprising administering an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

According to another embodiment, there is provided a pharmaceutical composition comprising a compound of Formula (I) for use in the prevention and treatment of cancer with tumour cells identified as harbouring a G12C mutant KRAS, HRAS or NRAS gene.

It will be appreciated that the following examples are provided so that the nature of the invention may be fully understood. It will also be appreciated that the following examples are not intended to limit the scope of the description in any way.

Biological Assays

The following assays were used to measure the effects of the compounds of the present specification.

KRasG12C Functional Assay

The inactive GDP loaded biotinylated KRasG12Cprotein was expressed, purified and GDP loaded in house. All enzyme and substrate solutions were prepared in assay buffer containing 20 mM HEPES (pH 7.5), 5 mM MgCl2, 150 mM NaCl, and 0.01% Tween 20. 10 nM GDP loaded biotinylated KRasG12Cand 37.5 ng/ml Streptavidin Europium Cryptate (Cisbio) were prepared in assay buffer, 50 was dispensed into each well of a 384 polystyrene, Hibase, medium binding white assay plate (Greiner, #784075) containing test and reference samples prepared in DMSO and the samples incubated for 4 hrs. In a separate mix 20 nM GST-Raf Ras binding domain (GST-Raf RBD, purified in house) and 4 μg/ml anti-GST XL665 antibody (Cisbio) was prepared in assay buffer containing 50 mM Potassium Fluoride and 0.05 mg/ml BSA and equilibrated for 4 hours before adding 0.6 μM Guanosine 5′-[γ-thio]triphosphate (GTPγS, Sigma) and 0.08 μM SOS (purified in house). 5 μl of the GST-RAF RBD mix was then dispensed into each well of the assay plate. This addition initiates the nucleotide exchange reaction and transition of inactive GDP loaded KRasG12Cto active GTPγS KRasG12C. This is detected simultaneously via the specific binding interaction between active GTPγS KRasG12Cwith GST-Raf RBD which brings the europium and XL665 into close proximity enabling an increased FRET signal to be detected on a Pherastar (BMG) plate reader equipped with the HTRF filter module. Any compound which prevents the activation of KRas via inhibiting the nucleotide exchange process, or inhibits the active KRas:Raf RBD binding interaction, will result in a reduced FRET signal. IC50values were calculated from normalised dose-response response FRET data curve fitted in Genedata screener (Basel, Switzerland).

KRasG12C Mass Spectrometry Adducting Assay

The inactive GDP loaded biotinylated KRasG12Cprotein was expressed, purified and GDP loaded in house. Enzyme solutions were prepared in assay buffer containing 20 mM HEPES (pH 7.5), 5 mM MgCl2, and 150 mM NaCl. 4 μM GDP loaded biotinylated KRasG12Cwas prepared in assay buffer and 50 μl added into each well of a 96 well polypropylene assay plate (Greiner, #651201) containing 500 nl of 1 mM test compounds (final concentration 10 μM), this was allowed to react for 4 hours before the addition of 501111% Formic acid to quench the reaction. The plate was sealed before reading on a Xevo G2 QTOF (Waters) and Acquity LC system (Waters). 10 μl of sample was injected onto a Xbridge BEH300; C4; 3.5 um; 2.1×50 mm column (Waters) running a 3 minute gradient. Blank samples were run in between each test sample.

Data was analysed in Mass Lynx software (Waters), the Total ion count (TIC) trace was used and the eluted protein peak data combined. Using the combined spectrum the data was deconvoluted using MaxEnt1 method. The peak area for apo-protein KRasG12C(APO) and KRAS+relative cmpd mass (adduct) were measured, and a percentage adduct was calculated using the following calculation:
Percent adduct=100*(area of adduct peak/(sum of APO+adduct peaks)

The data shown in Table A were generated for the Examples (the data below may be a result from a single experiment or an average of two or more experiments).

TABLE AKRasG12C functionalassay IC50KRasG12C M. S. BindingExample No.value (μM)Mean adduct %10.0199820.292530.1135440.059656.3491460.0989577.62880.0129690.2322100.06894110.20196120.03195139.698140.059941519.66160.0191170.37330180.23925190.02494200.029100211.306220.0395238.959240.097992513.852260.0511002712.037280.00592292.856300.1794316.833323.13330.229341.213358.507360.002370.180383.65390.003402.54410.674420.11195.528432.900440.062450.089460.0594711.2480.0464930500.14511.75522.14530.186

