WO2022262797A1

WO2022262797A1 - Combination of an erk inhibitor and a kras inhibitor and uses thereof

Info

Publication number: WO2022262797A1
Application number: PCT/CN2022/099091
Authority: WO
Inventors: Bing HOU; Jian Wang; Bo Shan; Xingxing Wang; Yun Liu; Peng Chen; Hui YUWEN; Bin Jiang; Jay Mei
Original assignee: Shanghai Antengene Corporation Limited; Antengene Discovery Limited
Priority date: 2021-06-18
Filing date: 2022-06-16
Publication date: 2022-12-22
Also published as: AR126166A1; TW202317130A; CN117529321A

Abstract

The present disclosure generally relates to the combination of an ERK inhibitor and a KRAS inhibitor, and use thereof.

Description

COMBINATION OF AN ERK INHIBITOR AND A KRAS INHIBITOR AND USES THEREOF

FIELD OF THE INVENTION

BACKGROUND

KRAS is one of the most commonly mutated oncogenes. Novel covalent inhibitors selective for the KRAS ^G12C mutation, such as Adagrasib (also known as MRTX849) and Sotorasib (also known as AMG510, AMG-510) , offer the unprecedented opportunity to target KRAS directly. These drugs showed certain responding rate and disease control rate in NSCLC and CRC patients carrying KRAS ^G12C mutation. However, the progression-free survival (PFS) of these patients is relatively short, perhaps due to the acquired resistant to the KRAS ^G12C inhibitors by these patients. It has been reported that feedback reactivation of wildtype RAS as a key mechanism of adaptive resistance to KRAS ^G12C inhibitors and highlighted the potential importance of vertical inhibition strategies to enhance the clinical efficacy of KRAS ^G12C inhibitors (Clin Cancer Res. 2020 Apr 1; 26 (7) : 1633-1643) .

The mitogen-activated protein kinase (MAPK) pathway, RAS-MEK1/2-ERK1/2, is a key regulator of cellular proliferation, survival, and differentiation, and alterations in this pathway are present in cancers of nearly all lineages. Reactivation of MAPK pathway causes the resistance to the KRAS ^G12C inhibitors such as Adagrasib and Sotorasib (Figure 1; Clin Cancer Res. 2020 Apr 1; 26 (7) : 1538-1540; Cancer Discov 2021, 11: 1-8) . The combination of KRAS ^G12C inhibitor and MEK inhibitor showed enhanced anti-tumor effect and pathway inhibition (Clin Cancer Res. 2020 Apr 1; 26 (7) : 1538-1540) . The combination of KRAS ^G12C inhibitor and MEK inhibitor are being evaluated in clinic trial (ClinicalTrials. gov Identifier: NCT04185883) . As the most downstream signaling node on MAPK pathway, ERK1/2, plays a key role in the signaling cascade and contributes to the cancer cell survival, proliferation, and drug resistance (Bioorg Med Chem Lett. 2015 Jan 15; 25 (2) : 192-7) . The combination of ERK inhibitor (e.g., ERK1/2 inhibitor) and KRAS inhibitor (e.g., KRAS ^G12C inhibitor) , however, has not been reported or studied.

SUMMARY OF THE INVENTION

In one aspect, the present disclosure provides a method of treating, preventing, or ameliorating a disease or disorder associated with ERK and/or KRAS in a subject in need thereof, comprising administering to the subject an effective amount of an ERK inhibitor or a pharmaceutically acceptable salt thereof, in combination with an effective amount of a KRAS inhibitor or a pharmaceutically acceptable salt thereof.

In another aspect, the present disclosure provides a method of treating, preventing, or ameliorating a disease or disorder associated with ERK and/or KRAS in a subject who is relapsed from or resistant to treatment of a KRAS inhibitor, comprising administering to the subject an effective amount of an ERK inhibitor or a pharmaceutically acceptable salt thereof, optionally in combination with an effective amount of a KRAS inhibitor or a pharmaceutically acceptable salt thereof.

In another aspect, the present disclosure provides a method of treating, preventing, or ameliorating a disease or disorder associated with ERK and/or KRAS in a subject who is relapsed from or resistant to treatment of an ERK inhibitor, comprising administering to the subject an effective amount of a KRAS inhibitor or a pharmaceutically acceptable salt thereof, optionally in combination with an effective amount of an ERK inhibitor or a pharmaceutically acceptable salt thereof.

In another aspect, the present disclosure provides a method of improving therapeutic response to a disease or disorder associated with ERK and/or KRAS in a subject previously received treatment of a KRAS inhibitor, comprising administering to the subject an effective amount of an ERK inhibitor or a pharmaceutically acceptable salt thereof, optionally in combination with an effective amount of a KRAS inhibitor or a pharmaceutically acceptable salt thereof.

In another aspect, the present disclosure provides a method of improving therapeutic response to a disease or disorder associated with ERK and/or KRAS in a subject previously received treatment of an ERK inhibitor, comprising administering to the subject an effective amount of a KRAS inhibitor or a pharmaceutically acceptable salt thereof, optionally in combination with an effective amount of an ERK inhibitor or a pharmaceutically acceptable salt thereof.

In another aspect, the present disclosure provides a method of treating, preventing, or ameliorating cancer in a subject in need thereof, comprising: (a) screening the subject to assess whether the subject carries a KRAS mutation; and (b) if the subject carries a KRAS mutation, administering to the subject an effective amount of an ERK inhibitor or a pharmaceutically acceptable salt thereof, in combination with an effective amount of a KRAS inhibitor or a pharmaceutically acceptable salt thereof.

In another aspect, the present disclosure provides a pharmaceutical composition comprising a KRAS inhibitor or a pharmaceutically acceptable salt thereof, and an ERK inhibitor or a pharmaceutically acceptable salt thereof.

In another aspect, the present disclosure provides a method of preparing the pharmaceutical composition disclosed herein, comprising mixing the KRAS inhibitor or a pharmaceutically acceptable salt thereof and the ERK inhibitor or a pharmaceutically acceptable salt thereof to form a pharmaceutical composition.

In another aspect, the present disclosure provides a kit comprising (a) a first composition comprising a KRAS inhibitor or a pharmaceutically acceptable salt thereof, and (b) a second composition comprising an ERK inhibitor or a pharmaceutically acceptable salt thereof.

BRIEF DESCFRIPTION OF THE DRAWINGS

Figure 1 illustrates KRAS signaling, mechanisms of resistance to KRAS ^G12C drugs.

Figure 2 illustrates the dose-dependent inhibition curve of NCI-H358 cell line to four tested compounds (AMG-510, MRTX849, Compound 33, and Compound 71) .

Figure 3 illustrates the synergy score values of the Compound 71+AMG-510 combo on NCI-H358 cell line.

Figure 4 illustrates the synergy score values of the Compound 71+MRTX849 combo on NCI-H358 cell line.

Figure 5 illustrates the anti-tumor activities of different treatment groups (vehicle group, Compound 33 treatment group, Compound 71 treatment group, and the Compound 71+Compound 33 Combo treatment group) in NCI-H358 subcutaneous xenograft model in BALB/c nude mice.

Figure 6A and Figure 6B show the inhibitory proliferation curves of

Compound 71, Compound 33 and AMG-510 monotherapy in different cell lines in cell viability assay.

Figure 7 shows the inhibitory proliferation curve of Compound 71 combined with AMG-510 in AMG510-R-xMIA-PaCa-2 (CP2) cell line.

DETAILED DESCRIPTION OF THE INVENTION

The following description of the disclosure is merely intended to illustrate various embodiments of the disclosure. As such, the specific modifications discussed are not to be construed as limitations on the scope of the disclosure. It will be apparent to a person skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the disclosure, and it is understood that such equivalent embodiments are to be included herein. All references cited herein, including publications, patents and patent applications are incorporated herein by reference in their entireties.

I. Definitions

Throughout the present disclosure, the articles “a, ” “an, ” and “the” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “a compound” means one compound or more than one compound.

As used herein, the term “and/or” indicates that one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

The term “amino acid” as used herein refers to an organic compound containing amine (-NH ₂) and carboxyl (-COOH) functional groups, along with a side chain specific to each amino acid. The names of amino acids are also represented as standard single letter or three-letter codes in the present disclosure, which are summarized as follows.

Names	Three-letter Code	Single-letter Code
Alanine	Ala	A
Arginine	Arg	R
Asparagine	Asn	N
Aspartic acid	Asp	D
Cysteine	Cys	C
Glutamic acid	Glu	E
Glutamine	Gln	Q
Glycine	Gly	G
Histidine	His	H
Isoleucine	Ile	I

Names	Three-letter Code	Single-letter Code
Leucine	Leu	L
Lysine	Lys	K
Methionine	Met	M
Phenylalanine	Phe	F
Proline	Pro	P
Serine	Ser	S
Threonine	Thr	T
Tryptophan	Trp	W
Tyrosine	Tyr	Y
Valine	Val	V

The terms “polypeptide” , “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally-occurring amino acid, as well as to naturally-occurring amino acid polymers and non-naturally occurring amino acid polymers.

Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 ^th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, 2 ^nd Edition, University Science Books, Sausalito, 2006; Smith and March March’s Advanced Organic Chemistry, 6 ^th Edition, John Wiley &Sons, Inc., New York, 2007; Larock, Comprehensive Organic Transformations, 3 ^rd Edition, VCH Publishers, Inc., New York, 2018; Carruthers, Some Modern Methods of Organic Synthesis, 4 ^th Edition, Cambridge University Press, Cambridge, 2004; the entire contents of each of which are incorporated herein by reference.

At various places in the present disclosure, linking substituents are described. It is specifically intended that each linking substituent includes both the forward and backward forms of the linking substituent. For example, -NR (CR’R”) -includes both -NR (CR’R”) -and - (CR’R”) NR-. Where the structure clearly requires a linking group, the Markush variables listed for that group are understood to be linking groups. For example, if the structure requires a linking group and the Markush group definition for that variable lists “alkyl” , then it is understood that the “alkyl” represents a linking alkylene group.

When a bond to a substituent is shown to cross a bond connecting two atoms in a ring, then such substituent may be bonded to any atom in the ring. When a substituent is listed without indicating the atom via which such substituent is bonded to the rest of the compound of a given formula, then such substituent may be bonded via any atom in such formula. Combinations of substituents and/or variables are permissible, but only if such combinations result in stable compounds.

When any variable (e.g., R ⁱ) occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with 0-2 R ⁱ moieties, then the group may optionally be substituted with up to two R ⁱ moieties and R ⁱ at each occurrence is selected independently from the definition of R ⁱ. Also, combinations of substituents and/or variables are permissible, but only if such combinations result in stable compounds.

As used herein, the term “C _i-j” indicates a range of the carbon atoms numbers, wherein i and j are integers and the range of the carbon atoms numbers includes the endpoints (i.e., i and j) and each integer point in between, and wherein j is greater than i. For examples, C _1-6 indicates a range of one to six carbon atoms, including one carbon atom, two carbon atoms, three carbon atoms, four carbon atoms, five carbon atoms and six carbon atoms. In some embodiments, the term “C _1-12” indicates 1 to 12, particularly 1 to 10, particularly 1 to 8, particularly 1 to 6, particularly 1 to 5, particularly 1 to 4, particularly 1 to 3 or particularly 1 to 2 carbon atoms.

As used herein the term “acyl” refers to –C (=O) -R, wherein R is a substituent such as hydrogen, alkyl, cycloalkyl, aryl or heterocyclyl, wherein the alkyl, cycloalkyl, aryl and heterocyclyl are as defined herein.

As used herein, the term “alkyl” , whether as part of another term or used independently, refers to a saturated linear or branched-chain hydrocarbon radical, which may be optionally substituted independently with one or more substituents described below. The term “C _i-j alkyl” refers to an alkyl having i to j carbon atoms. In some embodiments, alkyl groups contain 1 to 10 carbon atoms. In some embodiments, alkyl groups contain 1 to 9 carbon atoms. In some embodiments, alkyl groups contain 1 to 8 carbon atoms, 1 to 7 carbon atoms, 1 to 6 carbon atoms, 1 to 5 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms. Examples of “C _1-10 alkyl” include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl. Examples of “C _1-6 alkyl” are methyl, ethyl, propyl, isopropyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2, 3-dimethyl-2-butyl, 3, 3-dimethyl-2-butyl, and the like.

As used herein, the term “alkenyl” , whether as part of another term or used independently, refers to linear or branched-chain hydrocarbon radical having at least one carbon-carbon double bond, which may be optionally substituted independently with one or more substituents described herein, and includes radicals having “cis” and “trans” orientations, or alternatively, “E” and “Z” orientations. In some embodiments, alkenyl groups contain 2 to 12 carbon atoms. In some embodiments, alkenyl groups contain 2 to 11 carbon atoms. In some embodiments, alkenyl groups contain 2 to 11 carbon atoms, 2 to 10 carbon atoms, 2 to 9 carbon atoms, 2 to 8 carbon atoms, 2 to 7 carbon atoms, 2 to 6 carbon atoms, 2 to 5 carbon atoms, 2 to 4 carbon atoms, 2 to 3 carbon atoms, and in some embodiments, alkenyl groups contain 2 carbon atoms. Examples of alkenyl group include, but are not limited to, ethylenyl (or vinyl) , propenyl (allyl) , butenyl, pentenyl, 1-methyl-2 buten-1-yl, 5-hexenyl, and the like.

As used herein, the term “alkynyl” , whether as part of another term or used independently, refers to a linear or branched hydrocarbon radical having at least one carbon-carbon triple bond, which may be optionally substituted independently with one or more substituents described herein. In some embodiments, alkenyl groups contain 2 to 12 carbon atoms. In some embodiments, alkynyl groups contain 2 to 11 carbon atoms. In some embodiments, alkynyl groups contain 2 to 11 carbon atoms, 2 to 10 carbon atoms, 2 to 9 carbon atoms, 2 to 8 carbon atoms, 2 to 7 carbon atoms, 2 to 6 carbon atoms, 2 to 5 carbon atoms, 2 to 4 carbon atoms, 2 to 3 carbon atoms, and in some embodiments, alkynyl groups contain 2 carbon atoms. Examples of alkynyl group include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, and the like.

As used herein, the term “alkoxyl” , whether as part of another term or used independently, refers to an alkyl group, as previously defined, attached to the parent molecule through an oxygen atom. The term “C _i-j alkoxy” means that the alkyl moiety of the alkoxy group has i to j carbon atoms. In some embodiments, alkoxy groups contain 1 to 10 carbon atoms. In some embodiments, alkoxy groups contain 1 to 9 carbon atoms. In some embodiments, alkoxy groups contain 1 to 8 carbon atoms, 1 to 7 carbon atoms, 1 to 6 carbon atoms, 1 to 5 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms. Examples of “C _1-6 alkoxyl” include, but are not limited to, methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy) , t-butoxy, neopentoxy, n-hexoxy, and the like.

As used herein, the term “alkoxylalkyl” refers to a radical of the formula –R”OR’, wherein R’ and R” are independently an alkyl as defined above.

As used herein, the term “amino” refers to –NH ₂ group. Amino groups may also be substituted with one or more groups such as alkyl, aryl, carbonyl or other amino groups.

As used herein, the term “aryl” , whether as part of another term or used independently, refers to monocyclic and polycyclic ring systems having a total of 5 to 20 ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains 3 to 12 ring members. Examples of “aryl” include, but are not limited to, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bear one or more substituents. Also included within the scope of the term “aryl” , as it is used herein, is a group in which an aromatic ring is fused to one or more additional rings. In the case of polycyclic ring system, only one of the rings needs to be aromatic (e.g., 2, 3-dihydroindole) , although all of the rings may be aromatic (e.g., quinoline) . The second ring can also be fused or bridged. Examples of polycyclic aryl include, but are not limited to, benzofuranyl, indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like. Aryl groups can be substituted at one or more ring positions with substituents as described above.

As used herein, the term “carbamoyl” refers to –C (O) NH ₂.

As used herein, the term “carboxy” refers to –COOH.

As used herein, the term “cycloalkyl” , whether as part of another term or used independently, refer to a monovalent non-aromatic, saturated or partially unsaturated monocyclic and polycyclic ring system, in which all the ring atoms are carbon and which contains at least three ring forming carbon atoms. In some embodiments, the cycloalkyl may contain 3 to 12 ring forming carbon atoms, 3 to 10 ring forming carbon atoms, 3 to 9 ring forming carbon atoms, 3 to 8 ring forming carbon atoms, 3 to 7 ring forming carbon atoms, 3 to 6 ring forming carbon atoms, 3 to 5 ring forming carbon atoms, 4 to 12 ring forming carbon atoms, 4 to 10 ring forming carbon atoms, 4 to 9 ring forming carbon atoms, 4 to 8 ring forming carbon atoms, 4 to 7 ring forming carbon atoms, 4 to 6 ring forming carbon atoms, 4 to 5 ring forming carbon atoms. Cycloalkyl groups may be saturated or partially unsaturated. Cycloalkyl groups may be substituted. In some embodiments, the cycloalkyl group may be a saturated cyclic alkyl group. In some embodiments, the cycloalkyl group may be a partially unsaturated cyclic alkyl group that contains at least one double bond or triple bond in its ring system. In some embodiments, the cycloalkyl group may be monocyclic or polycyclic. Examples of monocyclic cycloalkyl group include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl and cyclododecyl. Examples of polycyclic cycloalkyl group include, but are not limited to, adamantyl, norbornyl, fluorenyl, spiro-pentadienyl, spiro [3.6] -decanyl, bicyclo [1, 1, 1] pentenyl, bicyclo [2, 2, 1] heptenyl, and the like.

As used herein, the term “cycloalkylalkyl” refers to a radical of formula –R’R”, wherein R’ is an alkyl as defined above, and R” is a cycloalkyl as defined above.

As used herein, the term “cyano” refers to –CN.

As used herein, the term “halogen” or “halo” refers to an atom selected from fluorine (or fluoro) , chlorine (or chloro) , bromine (or bromo) and iodine (or iodo) .

As used herein, the term “haloalkyl” refers to an alkyl, as defined above, that is substituted by one or more halogens, as defined above. Examples of haloalkyl include, but are not limited to, trifluoromethyl, difluoromethyl, trichloromethyl, 2, 2, 2-trifluoroethyl, 1, 2-difluoroethyl, 3-bromo-2-fluoropropyl, 1, 2-dibromoethyl, and the like.

As used herein, the term “heteroatom” refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen (including N-oxides) .

As used herein, the term “heteroaryl” , whether as part of another term or used independently, refers to an aryl group having, in addition to carbon atoms, one or more heteroatoms. The heteroaryl group can be monocyclic. Examples of monocyclic heteroaryl include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, benzofuranyl and pteridinyl. The heteroaryl group also includes polycyclic groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Examples of polycyclic heteroaryl include, but are not limited to, indolyl, isoindolyl, benzothienyl, benzofuranyl, benzo [1, 3] dioxolyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, dihydroquinolinyl, dihydroisoquinolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

As used herein, the term “heterocyclyl” refers to a saturated or partially unsaturated carbocyclyl group in which one or more ring atoms are heteroatoms independently selected from oxygen, sulfur, nitrogen, phosphorus, and the like, the remaining ring atoms being carbon, wherein one or more ring atoms may be optionally substituted independently with one or more substituents. In some embodiments, the heterocyclyl is a saturated heterocyclyl. In some embodiments, the heterocyclyl is a partially unsaturated heterocyclyl having one or more double bonds in its ring system. In some embodiments, the heterocyclyl may contains any oxidized form of carbon, nitrogen or sulfur, and any quaternized form of a basic nitrogen. “Heterocyclyl” also includes radicals wherein the heterocyclyl radicals are fused with a saturated, partially unsaturated, or fully unsaturated (i.e., aromatic) carbocyclic or heterocyclic ring. The heterocyclyl radical may be carbon linked or nitrogen linked where such is possible. In some embodiments, the heterocycle is carbon linked. In some embodiments, the heterocycle is nitrogen linked. For example, a group derived from pyrrole may be pyrrol-1-yl (nitrogen linked) or pyrrol-3-yl (carbon linked) . Further, a group derived from imidazole may be imidazol-1-yl (nitrogen linked) or imidazol-3-yl (carbon linked) .

In some embodiments, the term “3-to 12-membered heterocyclyl” refers to a 3-to 12-membered saturated or partially unsaturated monocyclic or polycyclic heterocyclic ring system having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. The fused, spiro and bridged ring systems are also included within the scope of this definition. Examples of monocyclic heterocyclyl include, but are not limited to oxetanyl, 1, 1-dioxothietanylpyrrolidyl, tetrahydrofuryl, tetrahydrothienyl, pyrrolyl, furanyl, thienyl, pyrazolyl, imidazolyl, triazolyl, oxazolyl, thiazolyl, piperidyl, piperazinyl, piperidinyl, morpholinyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, pyridonyl, pyrimidonyl, pyrazinonyl, pyrimidonyl, pyridazonyl, pyrrolidinyl, triazinonyl, and the like. Examples of fused heterocyclyl include, but are not limited to, phenyl fused ring or pyridinyl fused ring, such as quinolinyl, isoquinolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, quinoxalinyl, quinolizinyl, quinazolinyl, azaindolizinyl, pteridinyl, chromenyl, isochromenyl, indolyl, isoindolyl, indolizinyl, indazolyl, purinyl, benzofuranyl, isobenzofuranyl, benzimidazolyl, benzothienyl, benzothiazolyl, carbazolyl, phenazinyl, phenothiazinyl, phenanthridinyl, imidazo [1, 2-a] pyridinyl, [1, 2, 4] triazolo [4, 3-a] pyridinyl, [1, 2, 3] triazolo [4, 3-a] pyridinyl groups, and the like. Examples of spiro heterocyclyl include, but are not limited to, spiropyranyl, spirooxazinyl, and the like. Examples of bridged heterocyclyl include, but are not limited to, morphanyl, hexamethylenetetraminyl, 3-aza-bicyclo [3.1.0] hexane, 8-aza-bicyclo [3.2.1] octane, 1-aza-bicyclo [2.2.2] octane, 1, 4-diazabicyclo [2.2.2] octane (DABCO) , and the like.

As used herein, the term “hydroxyl” refers to –OH.

As used herein, the term “oxo” refers to =O substituent.

As used herein, the term “partially unsaturated” refers to a radical that includes at least one double or triple bond. The term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aromatic (i.e., fully unsaturated) moieties.

As used herein, the term “substituted” , whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and that the substitution results in a stable or chemically feasible compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. It will be understood by those skilled in the art that substituents can themselves be substituted, if appropriate. Unless specifically stated as “unsubstituted” , references to chemical moieties herein are understood to include substituted variants. For example, reference to an “aryl” group or moiety implicitly includes both substituted and unsubstituted variants.

II. Methods of Treatment

The present inventors unexpectedly found that an ERK inhibitor (e.g., a dual ERK1/2 inhibitor) can synergize with a KRAS inhibitor (e.g., a KRAS ^G12C inhibitor) , such that the therapeutic effects of KRAS inhibitor (s) can be improved or enhanced, and/or resistance to KRAS inhibitor (s) can be reduced, delayed or prevented. Indeed, the present inventors demonstrated that combination of an ERK inhibitor and a KRAS inhibitor (e.g., a KRAS ^G12C inhibitor) exhibited synergistic anti-tumor effect beyond what was observed with the respective monotherapies, for example, in inhibiting tumor growth.

In another aspect, the present disclosure provides a method of treating, preventing, or ameliorating cancer in a subject in need thereof, comprising: (a) screening the subject to assess whether the subject carries a KRAS mutation; and (b) if the subject carries a KRAS mutation, then administering to the subject an effective amount of an ERK inhibitor or a pharmaceutically acceptable salt thereof, in combination with an effective amount of a KRAS inhibitor or a pharmaceutically acceptable salt thereof.

1. Indications

As used herein, the term “treat” , “treating” or “treatment” , with regard to a disease, disorder or condition, refers to eliminating, reducing or ameliorating a disease, disorder or condition and/or a symptom associated therewith. For example, “treatment of cancer” includes treating, suppressing cancer, reducing its severity, reducing its risk, or inhibiting its metastasis. Although not excluded, treatment of a disease, disorder or condition does not require that the disease, disorder, condition, or symptoms associated therewith be completely eliminated. The term “treatment” as used herein may include “prophylactic treatment” , which refers to reducing the possibility of recurrence of a disease, disorder or condition, or reducing the possibility of relapse of a previously controlled disease, disorder or condition, in a subject who is not afflicted with a disease but at risk, or who is susceptible to recurrence of the disease, disorder or condition, or who is at risk or susceptible to relapse of the disease, disorder or condition. Within the meaning of the invention, “treatment” also includes prevention of relapse or prevention stages, as well as treatment of acute or chronic signs, symptoms and/or dysfunction. Treatment can target symptoms, for example, to suppress symptoms. It can function in a short period of time, for a medium period of time, or can be a long-term treatment, such as in the case of maintenance therapy.

In certain embodiments, a subject is successfully “treated” for cancer according to the methods of the present invention if the subject shows one or more of the following: a reduction in the number of or complete absence of cancer cells; a reduction in the tumor burden; inhibition of or an absence of cancer cell infiltration into peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibition of or an absence of tumor metastasis; inhibition or an absence of tumor growth; relief of one or more symptoms associated with the specific cancer; reduced morbidity and mortality; improvement in quality of life; reduction in tumorigenicity, tumorigenic frequency, or tumorigenic capacity, of a tumor; reduction in the number or frequency of cancer stem cells in a tumor; differentiation of tumorigenic cells to a non-tumorigenic state; increased progression-free survival (PFS) , disease-free survival (DFS) , or overall survival (OS) , complete response (CR) , partial response (PR) , stable disease (SD) , a decrease in progressive disease (PD) , a reduced time to progression (TTP) , or any combination thereof.

As used herein, the term “disease or disorder associated with ERK and/or KRAS” refers to a disease or disorder which is caused by, mediated by and/or accompanied by an aberrant activity or level of ERK and/or KRAS, for example, an increased activity or level of ERK and KRAS.

The term “ERK” as used herein broadly encompasses an extracellular signal-regulated kinase (ERK) protein, peptide, or polypeptide, as well as an ERK polynucleotide such as a DNA or RNA sequence encoding the ERK protein, peptide, or polypeptide, for example, the sequences of ERK GenBank Accession Nos. NM_002745.5, NM_138957.3, NM_001040056.3, NM_001109891.2, NM_002746.3, NP_001035145.1, NP_002736.3, NP_620407.1, NP_001103361.1, NP_002737.2. The term “ERK” as used herein further encompasses other ERK encoding sequences, such as other ERK isoforms, mutant ERK genes, splice variants of ERK genes, and ERK gene polymorphisms, as well as the ERK protein, peptide, or polypeptide encoded by such ERK encoding sequences. Generally, ERK includes ERK1 (also known as mitogen-activated protein kinase 3, MAPK3) and ERK2 (also known as mitogen-activated protein kinase 1, MAPK1) .

Similarly, the term “KRAS” as used herein broadly encompasses a KRAS protein, peptide, or polypeptide, as well as a KRAS polynucleotide such as a DNA or RNA sequence encoding the KRAS protein, peptide, or polypeptide, for example, the sequences of KRAS GenBank Accession Nos. NM_033360.4, NM_004985.5, NM_001369786.1, NM_001369787.1, NP_001356715.1, NP_203524.1, NP_001356716.1, and NP_004976.2. The term “KRAS” as used herein further encompasses other KRAS encoding sequences, such as other KRAS isoforms, mutant KRAS genes, splice variants of KRAS genes, and KRAS gene polymorphisms, as well as the KRAS protein, peptide, or polypeptide encoded by such KRAS encoding sequences.

As used herein, the term “a subject in need thereof” is a subject having, or being suspected of having a disease or disorder associated with ERK and/or KRAS (e.g., cancer) , or a subject having an increased risk of developing a disease or disorder associated with ERK and/or KRAS (e.g., cancer) relative to the population at large. In the case of cancer, a subject in need thereof can have a precancerous condition. A “subject” can be human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, mice, rats, cats, rabbits, sheep, dogs, cows, chickens, amphibians, and reptiles. Except when noted, the terms “patient” , “individual” or “subject” are used herein interchangeably.

As used herein, the term “effective amount” or “therapeutically effective amount” refers to an amount of the active ingredient which, when administered by the methods of the present disclosure, is sufficient to effectively deliver the active ingredient to an individual in need thereof for the treatment of a target condition. In the case of cancer or other proliferative disorders, a therapeutically effective amount of the agent can reduce undesired cell proliferation; reduce the number of cancer cells; reduce tumor size; inhibit cancer cell infiltration to surrounding organs; inhibit tumor metastasis; inhibit tumor growth to a certain extent; inhibiting the activity/level of ERK and/or KRAS in targeted cells; and/or alleviate one or more symptoms associated with cancer to a certain extent.

As used herein, the term “KRAS inhibitor” refers to an agent that is capable of down-regulating, decreasing, suppressing, inhibiting or reducing the expression of KRAS gene, or down-regulating, decreasing, suppressing, inhibiting or reducing an activity and/or level of KRAS protein, peptide, or polypeptide. Embodiments of the invention include a KRAS inhibitor that inhibits or reduces KRAS protein expression, amount of KRAS protein or level of KRAS translation, amount of KRAS transcript or level of KRAS transcription, stability of KRAS protein or KRAS transcript, half-life of KRAS protein or KRAS transcript, prevents the proper localization of an KRAS protein or transcript; reduces or inhibits the availability of KRAS polypeptide, reduces or inhibits KRAS activity; reduces or inhibits KRAS, binds KRAS protein, or inhibits or reduces the post-translational modification of KRAS, including its phosphorylation. The KRAS inhibitors of the present disclosure will be described below in details under Section II. 2 KRAS Inhibitor.

As used herein, the term “ERK inhibitor” refers to an agent that is capable of down-regulating, decreasing, suppressing, inhibiting or reducing the expression of ERK gene, or down-regulating, decreasing, suppressing, inhibiting or reducing an activity and/or level of ERK protein, peptide, or polypeptide. An ERK inhibitor may inhibit one member, several members, or all members of the family of ERK kinases. Embodiments of the invention include an ERK inhibitor that inhibits or reduces ERK protein expression, amount of ERK protein or level of ERK translation, amount of ERK transcript or level of ERK transcription, stability of ERK protein or ERK transcript, half-life of ERK protein or ERK transcript, prevents the proper localization of an ERK protein or transcript; reduces or inhibits the availability of ERK polypeptide, reduces or inhibits ERK activity; reduces or inhibits ERK, binds ERK protein, or inhibits or reduces the post-translational modification of ERK, including its phosphorylation. The ERK inhibitors of the present disclosure will be described below in details under Section II. 3 ERK Inhibitor.

As used herein, the term “pharmaceutically acceptable” indicates that the substance or composition is compatible chemically and/or toxicologically, with the other ingredients comprising a formulation, and/or the subjects being treated therewith.

As used herein, the term “pharmaceutically acceptable salt” , unless otherwise indicated, includes salts that retain the biological effectiveness of the free acids and bases of the specified compound and that are not biologically or otherwise undesirable. Contemplated pharmaceutically acceptable salt forms include, but are not limited to, mono, bis, tris, tetrakis, and so on. Pharmaceutically acceptable salts are non-toxic in the amounts and concentrations at which they are administered. The preparation of such salts can facilitate the pharmacological use by altering the physical characteristics of a compound without preventing it from exerting its physiological effect. Useful alterations in physical properties include lowering the melting point to facilitate transmucosal administration and increasing the solubility to facilitate administering higher concentrations of the drug.

Pharmaceutically acceptable salts include acid addition salts such as those containing sulfate, chloride, hydrochloride, fumarate, maleate, phosphate, sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate. Pharmaceutically acceptable salts can be obtained from acids such as hydrochloric acid, maleic acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, fumaric acid, and quinic acid.

Pharmaceutically acceptable salts also include basic addition salts such as those containing benzathine, chloroprocaine, choline, diethanolamine, ethanolamine, t-butylamine, ethylenediamine, meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium, ammonium, alkylamine, and zinc, when acidic functional groups, such as carboxylic acid or phenol are present. For example, see Remington’s Pharmaceutical Sciences, 19 ^th ed., Mack Publishing Co., Easton, PA, Vol. 2, p. 1457, 1995; “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” by Stahl and Wermuth, Wiley-VCH, Weinheim, Germany, 2002. Such salts can be prepared using the appropriate corresponding bases.

Pharmaceutically acceptable salts can be prepared by standard techniques. For example, the free-base form of a compound can be dissolved in a suitable solvent, such as an aqueous or aqueous-alcohol solution containing the appropriate acid and then isolated by evaporating the solution. Thus, if the particular compound is a base, the desired pharmaceutically acceptable salt may be prepared by any suitable method available in the art, for example, treatment of the free base with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, or with an organic acid, such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, a pyranosidyl acid, such as glucuronic acid or galacturonic acid, an alpha-hydroxy acid, such as citric acid or tartaric acid, an amino acid, such as aspartic acid or glutamic acid, an aromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid, such as p-toluenesulfonic acid or ethanesulfonic acid, or the like.

Similarly, if the particular compound is an acid, the desired pharmaceutically acceptable salt may be prepared by any suitable method, for example, treatment of the free acid with an inorganic or organic base, such as an amine (primary, secondary or tertiary) , an alkali metal hydroxide or alkaline earth metal hydroxide, or the like. Illustrative examples of suitable salts include organic salts derived from amino acids, such as L-glycine, L-lysine, and L-arginine, ammonia, primary, secondary, and tertiary amines, and cyclic amines, such as hydroxyethyl pyrrolidine, piperidine, morpholine or piperazine, and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.

Administration “in combination with” one or more further agents includes simultaneous (concurrent) and consecutive administration in any order. The combination therapy can provide “synergy” or prove “synergistic” .

The term “synergize, ” “synergy” , “synergistic” or “synergistically” as used herein refers to two or more agents providing an effect that is greater than the sum of the effects of the two or more agents when administered alone. For example, a synergistic effect of the combination of an ERK inhibitor and a KRAS inhibitor means the effect of the combination of an ERK inhibitor and a KRAS inhibitor is greater than the sum of the effects of the ERK inhibitor and the KRAS inhibitor when administered alone.

A synergistic effect can be attained when the agents are: (1) co-formulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered serially, by alternation, or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation therapy, a synergistic effect can be attained when the agents are administered or delivered sequentially, e.g., by different injections in separate syringes.

The synergistic effect of two or more agents can be analyzed by various well-known model in the art, for example, (1) the Bliss Model as described in M N Prichard and C Shipman Jr., A three-dimensional model to analyze drug-drug interactions. Antiviral Res. Oct-Nov 1990; 14 (4-5) : 181-205) ; (2) the Loewe dose-additivity model as described in Lehar J, Krueger AS, Avery W, et al., Synergistic drug combinations tend to improve therapeutically relevant selectivity. Nat Biotechnol 27 (7) : 659-66, 2009 and Rickles RJ, Tam WF, Giordano TP, 3rd, et al., Adenosine A2A and beta-2 adrenergic receptor agonists: Novel selective and synergistic multiple myeloma targets discovered through systematic combination screening. Mol Cancer Ther 11 (7) : 1432-42, 2012) ; (3) the Greco synergism model as described in Greco, W R, Park, H S, Rustum, Y M, 1990, Cancer Res. 50: 5318-5327. (4) the Chou-Talalay Method model as described in Ting-Chao Chou, Cancer Res; 70 (2) January 15, 2010.

As used herein, the term “relapsed” refers to a subject in whom the disease or disorder (e.g., cancer) has been treated and improved but in whom the disease or disorder (e.g., cancer) recurred. Unless otherwise indicated, relapsed state refers to the process of returning to or the return to illness before the previous treatment. For example, a subject who is “relapsed from treatment of a KRAS inhibitor” means the patient’s disease or disorder has been treated and improved but is recurred after the treatment of a KRAS inhibitor.

“Resistant to” the treatment of a KRAS inhibitor or an ERK inhibitor refers to the subject who is receiving the treatment of a KRAS inhibitor or an ERK inhibitor does not respond to, or poorly respond to the treatment, and thereby the disease, disorder or condition of the subject is not being treated. The term “resistant” or “resistance” as used herein refers to being refractory or non-responsive to a therapeutic agent, such as a KRAS inhibitor or an ERK inhibitor. The subject’s response to the treatment can be determined by means known in the state of the art.

The phrase “improving therapeutic response” can include, for example, delaying progression of a disease or reducing or inhibiting cancer relapse.

As used herein, “delaying progression of a disease” means to defer, hinder, slow, retard, stabilize, and/or postpone development of the disease (such as cancer) . This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed.

As used herein, “reducing or inhibiting cancer relapse” means to reduce or inhibit tumor or cancer relapse or tumor or cancer progression. As disclosed herein, cancer relapse and/or cancer progression include, without limitation, cancer metastasis.

In some embodiments, the disease or disorder is associated with an increased activity or level of ERK and/or KRAS and/or an activated MAPK pathway.

As used herein, the term “increased activity or level” of ERK and/or KRAS refers to an overall increase of 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%or greater, in the activity or level of ERK and/or KRAS, as compared to a reference activity or level of ERK and/or KRAS.

A reference activity or level of ERK and/or KRAS may be derived from one or more reference samples, wherein the reference activity or level is obtained from experiments conducted in parallel with the experiment for testing the sample of interest. Alternatively, a reference activity or level may be obtained in a database, which includes a collection of data, standard, or level from one or more reference samples or disease reference samples. In some embodiments, such collection of data, standard or level are normalized so that they can be used for comparison purpose with data from one or more samples. “Normalize” or “normalization” is a process by which a measurement raw data is converted into data that may be directly compared with other so normalized data. Normalization is used to overcome assay-specific errors caused by factors that may vary from one assay to another, for example, variation in loaded quantities, binding efficiency, detection sensitivity, and other various errors. In some embodiments, a reference activity or level of ERK and/or KRAS may be obtained from health population.

Mitogen-activated protein kinase (MAPK) pathway is a key signaling pathway that regulates a wide variety of cellular processes, including proliferation, differentiation, apoptosis and stress responses. The MAPK pathway includes three main kinases, MAPK kinase kinase, MAPK kinase and MAPK, which activate and phosphorylate downstream proteins. Many diseases can be correlated with an activated MAPK pathway.

In some embodiments, the disease or disorder is cancer. The terms “cancer” and “tumor” are used interchangeably herein and refer to a disease, disorder or condition in which cells exhibit relatively abnormal, uncontrolled, and/or autonomous growth, so that they display an abnormally elevated proliferation rate and/or aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, such cells exhibit such characteristics in part or in full due to the expression and activity of oncogenes or the defective expression and/or activity of tumor suppressor genes, such as retinoblastoma protein (Rb) . Cancer cells are often in the form of a tumor, but such cells may exist alone within an animal, or may be a non-tumorigenic cancer cell, such as a leukemia cell. As used herein, the term “cancer” includes premalignant as well as malignant cancers.

In some embodiments, the disease or disorder is a drug-resistant cancer. “Drug-resistant” as used herein refers to being refractory or non-responsive to a therapeutic agent, such as a KRAS inhibitor, an ERK inhibitor, etc. For example, the number of tumor cells is increased despite of being treated with a therapeutic agent (e.g., a KRAS inhibitor, an ERK inhibitor, etc. ) .

In some embodiments, the disease or disorder is a cancer refractory to monotherapy of a KRAS inhibitor or an ERK inhibitor.

The term “refractory” can refer to a cancer for which treatment (e.g., chemotherapy drugs, biological agents, and/or radiation therapy) has proven to be ineffective. A refractory cancer tumor may shrink, but not to the point where the treatment is determined to be effective. Typically, however, the tumor stays the same size as it was before treatment (stable disease) , or it grows (progressive disease) .

As used herein, the term “monotherapy” refers to the use of an agent individually (also referred to herein as alone) (e.g., as a single compound or agent) , e.g., without a second active ingredient to treat the same indication, e.g., cancer. For example, monotherapy of an ERK inhibitor means the use of an ERK inhibitor (e.g., Compound 71) alone, without a KRAS inhibitor to treat a disease or disorder. For clarity, monotherapy of an ERK inhibitor may encompass the use of one or more ERK inhibitors to treat a disease or disorder (e.g., cancer) , without another active ingredient targeting another target other than ERK.

In some embodiments, the disease or disorder is a metastatic cancer. As used herein, the term “metastatic cancer” refers to a cancer in which cancerous cells spread from one organ or part of a subject to another organ or part that is not adjacent to the original organ or part where the cancer initially originated.

In some embodiments, the cancer is associated with a KRAS mutation. For example, the cancer is characterized in expressing a mutated KRAS. For another example, the cancer is a KRAS-mutated cancer.

The term “KRAS-mutated cancer” is well known to the skilled person. A comprehensive overview of RAS mutations, including KRAS-mutations, in cancer was reported by Prior et al., (2012) Cancer Res; 2457 -67. KRAS-mutant cells promote oncogenesis due to being mutationally activated, in most cases, at codons 12, 13 and 61. In total forty-four separate point mutations have been characterized in RAS isoforms, with 99.2%in codons 12, 13 and 61. The protein product of the normal KRAS gene performs an essential function in normal tissue signaling, and the mutation of a KRAS gene is an essential step in the development of many cancers.

In some embodiments, the KRAS mutation comprises one or more mutations at a codon selected from the group consisting of codon 12, 13 and 61. The sequence of a wild-type KRAS is known in the art, for example, NCBI Accession No. NM_033360.4, NM_004985.5, NM_001369786.1, NM_001369787.1, NP_001356715.1, NP_203524.1, NP_001356716.1, and NP_004976.2., which are incorporated into the present disclosure by reference.

In some embodiments, the KRAS mutation comprises a mutation at codon 12. In some embodiments, the KRAS mutation comprises a mutation at codon 13. In some embodiments, the KRAS mutation comprises a mutation at codon 61.

As used herein, the term “mutation” or “mutated” with regard to an amino acid residue as used herein refers to substitution, replacement, insertion, addition, or modification of the amino acid residue. The term “substitution” or “substituted” with regard to an amino acid residue as used herein refers to the replacement of amino acid residue X (i.e., the amino acid residue before replacement, “X” ) at position p with amino acid residue Z (i.e., the amino acid residue after replacement, “Z” ) in a peptide, polypeptide or protein, and is denoted by XpZ. For example, G12C denotes that an original native glycine residue (G) at codon 12 of a wild-type protein is substituted by a cysteine residue (C) . Accordingly, KRAS ^G12C denotes that an original native glycine residue at codon 12 of a wild-type KRAS is substituted by a cysteine residue.

In some embodiments, the KRAS mutation is selected from the group consisting of G12, G13, and Q61. In some embodiments, the KRAS mutation is selected from KRAS ^G12C/D/V, KRAS ^G13C/D, or KRAS ^Q61L/H/R, which means the KRAS mutation is selected from the group consisting of KRAS G12C, KRAS G12D, KRAS G12V, KRAS G13C, KRAS G13D, KRAS Q61L, KRAS Q61H, and KRAS Q61R mutations. In some embodiments, the KRAS mutation is KRAS ^G12C/D/V, which means the KRAS mutation is selected from the group consisting of KRAS G12C, KRAS G12D, and KRAS G12V mutations. In some embodiments, the KRAS mutation comprises or is KRAS ^G12C.

In certain embodiments, the patient is diagnosed as expressing a mutated KRAS. The patient can be diagnosed by methods well known in the art, for example, by hybridization-based methods using nucleic acid probes that specifically distinguish mutant KRAS and wild-type KRAS, by nucleic acid amplification-based methods, by detection methods using antibodies that specifically distinguish between mutant KRAS and wild type KRAS, and by commercially available kit for KRAS mutation (for example,

KRAS Mutation Test (Roche Molecular Systems, Inc. ) ,

KRAS RGQ PCT Kit (Qiagen Manchester, Ltd. ) ,

CDx (Guardant Health, Inc. ) ) and the like.

It is surprisingly found in the present disclosure that the combination of an ERK inhibitor and a KRAS inhibitor provided herein is useful for the treatment of cancers having a KRAS mutation, including but not limited to cancers having the above KRAS mutation (e.g., KRAS ^G12C mutation) . In some embodiments, the ERK inhibitor is Compound 71. In some embodiments, the KRAS inhibitor is AMG-510, MRTX849 or Compound 33. In some embodiments, the ERK inhibitor is Compound 71, and the KRAS inhibitor is AMG-510. In some embodiments, the ERK inhibitor is Compound 71, and the KRAS inhibitor is MRTX849. In some embodiments, the ERK inhibitor is Compound 71, and the KRAS inhibitor is Compound 33. Without being bound to any theory, but it is believed that a relative high concentration of an ERK inhibitor (e.g., Compound 71) generally has synergistic effects in inhibiting proliferation of cancer cells when combined with a KRAS inhibitor (e.g., AMG-510, MRTX849 or Compound 33) , regardless the concentration of the KRAS inhibitor. For example, in order to achieve a synergistic effect, the concentration of the ERK inhibitor (e.g., Compound 71) used in a combination therapy may be ranged from 0.1 to 1 μM (e.g., 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM) . Without being bound to any theory, but it is also believed that the combination of an ERK inhibitor (e.g., Compound 71) and a KRAS inhibitor (e.g., AMG-510, MRTX849 or Compound 33) has anti-tumor activity and synergistic effect in treating, preventing or ameliorating a disease or disorder in a subject who is resistant to treatment of KRAS inhibitor. For example, as shown in the Examples of the present disclosure, the combination of an ERK inhibitor Compound 71 and a KRAS ^G12C inhibitor AMG-510 demonstrated anti-tumor activity and synergistic effects in inhibiting the proliferation of AMG-510 resistant cell line, AMG510-R-xMIA-PaCa-2 (CP2) .

In some embodiments, the cancer is selected from the group consisting of lung cancer, non-small-cell lung cancer (NSCLC) , small cell lung cancer (SCLC) , bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, uterine cancer, liver cancer, cholangiocarcinoma, sarcoma, hematological cancer, colorectal cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin’s Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, chronic or acute leukemia, lymphocytic lymphomas, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS) , primary CNS lymphoma, spinal axis tumors, brain stem glioma, MYH associated polyposis, or pituitary adenoma.

In some embodiments, the cancer is KRAS ^G12C-mutated non-small-cell lung cancer (NSCLC) , KRAS ^G12C-mutated colorectal cancer, or KRAS ^G12C-mutated pancreatic cancer. In some embodiments, the cancer is KRAS ^G12C-mutated non-small-cell lung cancer. In some embodiments, the cancer is KRAS ^G12C-mutated locally advanced or metastatic non-small-cell lung cancer.

In some embodiments, the subject is determined to have de novo or acquired resistance to the KRAS inhibitor or a pharmaceutically acceptable salt thereof. In some embodiments, the subject is determined to have de novo or acquired resistance to the ERK inhibitor or a pharmaceutically acceptable salt thereof.

As used herein, the term “De novo resistance” refers to resistance that exists prior to treatment with a given agent. Therefore, “de novo resistance to a KRAS inhibitor” means the subject is resistant or non-responsive to the KRAS inhibitor prior to the treatment of the KRAS inhibitor. “Acquired resistance” as used herein refers to resistance that is acquired after at least one treatment with a given agent. Prior to the at least one treatment, the disease does not possess a resistance to the agent (and, as such, the disease responds to the first treatment as would a non-resistant disorder) . For example, a subject who has acquired resistance to the KRAS inhibitor is one that initially responds to at least one treatment of the KRAS inhibitor and thereafter develops a resistance to subsequent treatments of the KRAS inhibitor.

In some embodiments, the method of the present disclosure further comprises administering to the subject an additional therapeutical agent. In some embodiments, the addition therapeutic agent is a MEK inhibitor.

As used herein, the term “MEK inhibitor” refers to an agent that is capable of down-regulating, decreasing, suppressing, inhibiting or reducing the expression of MEK gene, or down-regulating, decreasing, suppressing, inhibiting or reducing an activity and/or level of MEK protein, peptide, or polypeptide. Embodiments of the invention include a MEK inhibitor that inhibits or reduces MEK protein expression, amount of MEK protein or level of MEK translation, amount of MEK transcript or level of MEK transcription, stability of MEK protein or MEK transcript, half-life of MEK protein or MEK transcript, prevents the proper localization of an MEK protein or transcript; reduces or inhibits the availability of MEK polypeptide, reduces or inhibits MEK activity; reduces or inhibits MEK, binds MEK protein, or inhibits or reduces the post-translational modification of MEK.

In some embodiments, the MEK inhibitor is selected from the group consisting of Binimetinib, Cobimetinib, Refametinib, Selumetinib, Trametinib, mirdametinib, PD-325901, TAK-733, E6201, CI-1040, ATR-002, SHR7390, NFX-179, pimasertib, VS-6766, refametinib, HL-085, FCN-159, LNP3794, CS3006, AS703988, TQ-B3234, and GDC-0623.

2. KRAS Inhibitor

In some embodiments, the KRAS inhibitor is a chemotherapeutic agent, an antibody, or an antigen-binding fragment thereof, an RNAi molecule that targets an encoding sequence of KRAS, an antisense nucleotide that targets an encoding sequence of KRAS, or an agent that competes with KRAS protein to bind to its substrate. In some embodiments, the antibody is a monoclonal antibody or a polyclonal antibody. In some embodiments, the antibody is a humanized antibody, a chimeric antibody or a fully human antibody. In some embodiments, the RNAi molecule is a small interfering RNA (siRNA) , a small hairpin RNA (shRNA) or a microRNA (miRNA) .

In some embodiments, the KRAS inhibitor is a small molecule compound.

As used herein, the term “small molecule compound” means a low molecular weight compound that may serve as an enzyme substrate or regulator of biological processes. In general, a “small molecule compound” is a molecule that is less than about 5 kilodaltons (kD) in size. In some embodiments, the small molecule compound is less than about 4 kD, 3 kD, about 2 kD, or about 1 kD. In some embodiments, the small molecule compound is less than about 800 daltons (D) , about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, or about 100 D. In some embodiments, a small molecule compound is less than about 2000 g/mol, less than about 1500 g/mol, less than about 1000 g/mol, less than about 800 g/mol, or less than about 500 g/mol. In some embodiments, small molecule compounds are non-polymeric. In some embodiments, in accordance with the present invention, small molecule compounds are not proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, polysaccharides, glycoproteins, proteoglycans, etc.

In some embodiments, the KRAS inhibitor is a KRAS ^G12C inhibitor.

As used herein, the term “KRAS ^G12C inhibitor” or “KRAS G12C inhibitor” refers to an agent that is capable of negatively modulating or inhibiting all or a portion of the enzymatic activity of KRAS ^G12C.

In some embodiments, the KRAS inhibitor is selected from the group consisting of Sotorasib (AMG-510) , Adagrasib (MRTX849) , D-1553, JDQ443, LY3499446, RG6330, ARS-3248, JAB-21822, BPI-421286, GH35, RMC-6291, MRTX1257, ARS-853, AU-8653, GF-105, AU-10458, LY3537982, WDB178, RM-007, LC-2, RM-018, ARS-1620, RM-032, BI 1823911, APG-1842, JAB-21000, ATG-012, and YL-15293. In some embodiments, the KRAS inhibitor is Sotorasib (AMG-510) or Adagrasib (MRTX849) . In some embodiments, the KRAS inhibitor is Sotorasib (AMG-510) . In some embodiments, the KRAS inhibitor is Adagrasib (MRTX849) . In some embodiments, the KRAS inhibitor is Compound 33. In the present disclosure, the structure and chemical name of Compound 33 are shown as follows.

PCT application No. PCT/CN2021/098083, published as WO2021244603A1 on December 09, 2021, filed on June 03, 2021, the entirety of which is incorporated herein by reference, describes certain KRAS (e.g. KRAS ^G12C) inhibitors. All of the KRAS inhibitors disclosed in WO2021244603A1 can be used and incorporated in the present disclosure.

In some embodiments, the KRAS inhibitor of the present disclosure is a compound of Formula (I) or a pharmaceutically acceptable salt thereof:

wherein

Ring A is selected from the group consisting of saturated or partially unsaturated cycloalkyl, saturated or partially unsaturated heterocyclyl, and heteroaryl;

L ¹ is a bond, O, S or N (R ^a) ;

L ² is selected from the group consisting of a bond, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl;

R ¹ is selected from the group consisting of alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, saturated or partially unsaturated cycloalkyl, saturated or partially unsaturated heterocyclyl, aryl, and heteroaryl, wherein each of alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted with one or more R ^b;

R ² is selected from the group consisting of H, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, saturated or partially unsaturated cycloalkyl, saturated or partially unsaturated heterocyclyl, aryl and heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted with one or more R ^c,

R ³ is selected from the group consisting of hydrogen, oxo, halogen, cyano, hydroxyl, -NR ^dR ^e, -C (O) NR ^dR ^e, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, saturated or partially unsaturated cycloalkyl, saturated or partially unsaturated heterocyclyl, aryl and heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted with one or more R ^f; or

R ⁴ and R ⁵, R ⁴ and R ⁶, R ⁴ and R ⁷, together with the atoms to which they are each attached, form saturated or partially unsaturated cycloalkyl, or saturated or partially unsaturated heterocyclyl, wherein each of cycloalkyl and heterocyclyl is optionally substituted with cyano, halogen, hydroxy, -NR ^cR ^d, carboxy, carbamoyl, aryl or heteroaryl;

W is saturated or partially unsaturated cycloalkyl, or saturated or partially unsaturated heterocyclyl, wherein each of cycloalkyl and heterocyclyl is optionally substituted with one or more R ^g,

L ³ is a bond, alkyl or -NR ^d-;

B is an electrophilic moiety capable of forming a covalent bond with a cysteine residue at position 12 of a KRAS ^G12C mutant protein;

R ^a is independently hydrogen or alkyl;

each R ^b is independently selected from the group consisting of oxo, cyano, halogen, hydroxy, acyl, -NR ^dR ^e, carbamoyl, carboxyl, alkyl, alkenyl, alkynyl, alkoxyl, alkoxylalkyl, cycloalkylalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl;

each R ^c is independently selected from the group consisting of oxo, halogen, cyano, hydroxy, -NR ^dR ^e, -C (O) OR ^a, -C (O) N (R ^d) (R ^e) , alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, alkoxyl, saturated or partially unsaturated cycloalkyl, saturated or partially unsaturated heterocyclyl, aryl, and heteroaryl;

each of R ^d and R ^e is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl and heteroaryl is optionally substituted with cyano, halogen, hydroxy, or amino;

each R ^f is independently selected from the group consisting of oxo, halogen, cyano, hydroxy, -NR ^cR ^d, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl;

each R ^g is independently selected from the group consisting of oxo, cyano, halogen, hydroxy, -NR ^dR ^e, carbamoyl, carboxy, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, saturated or partially unsaturated cycloalkyl, and saturated or partially unsaturated heterocyclyl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, and heterocyclyl is optionally substituted with cyano, halogen, hydroxy, -NR ^dR ^e, carboxy, carbamoyl, haloalkyl, aryl or heteroaryl;

n is 0, 1, 2, 3 or 4.

In some embodiments, Ring A is saturated or partially unsaturated cycloalkyl.

In some embodiments, Ring A is saturated or partially unsaturated heterocyclyl.

In some embodiments, Ring A is heteroaryl.

In some embodiments, L ¹ is O.

In some embodiments, L ² is a bond.

In some embodiments, L ² is alkyl.

In some embodiments, L ² is methyl, ethyl or propyl.

In some embodiments, R ¹ is saturated or partially unsaturated cycloalkyl, or saturated or partially unsaturated heterocyclyl, wherein each cycloalkyl and heterocyclyl is optionally substituted with one or more R ^b. In certain embodiments, each R ^b is selected from the group consisting of oxo, cyano, halogen, hydroxy, acyl, -NR ^dR ^e, alkyl, alkoxyl, alkoxylalkyl and cycloalkylalkyl.

In some embodiments, R ¹ is saturated or partially unsaturated heterocyclyl selected from the group consisting of:

each of which is optionally substituted with one or more R ^b.

In certain embodiments, each R ^b is selected from the group consisting of oxo, halogen, acyl, -NR ^dR ^e, alkyl, alkoxyl, alkoxylalkyl, and cycloalkylalkyl. In certain embodiments, each R ^b is halogen or alkyl. In certain embodiments, each R ^b is fluoro, chloro or methyl.

In some embodiments, R ¹ is

In some embodiments, –L ¹-L ²-R ¹ is

In some embodiments, R ¹ is

In some embodiments, –L ¹-L ²-R ¹ is

In some embodiments, R ² is aryl optionally substituted with one or more R ^c. In certain embodiments, each R ^c is selected from the group consisting of halogen, cyano, hydroxyl, alkyl, alkenyl, alkoxyl, and saturated or partially unsaturated cycloalkyl.

In some embodiments, R ² is aryl selected from the group consisting of:

each of which is optionally substituted with one or more R ^c.

In certain embodiments, each R ^c is selected from the group consisting of halogen, hydroxyl, alkyl, alkenyl, alkoxyl, and saturated or partially unsaturated cycloalkyl. In certain embodiments, each R ^c is selected from the group consisting of halogen, hydroxyl, alkyl, alkenyl, alkoxyl, and saturated cycloalkyl. In certain embodiments, each R ^c is selected from the group consisting of fluoro, chloro, hydroxyl, methyl, ethyl, 2-methylpropenyl, methoxyl, and cyclopropyl.

In some embodiments, R ² is selected from the group consisting of:

In some embodiments, R ² is heteroaryl optionally substituted with one or more R ^c. In certain embodiments, each R ^c is selected from the group consisting of halogen, cyano, hydroxyl, -NR ^dR ^e, alkyl, alkenyl, alkoxyl, and saturated or partially unsaturated cycloalkyl.

In some embodiments, R ² is heteroaryl selected from the group consisting of:

each of which is optionally substituted with one or more R ^c.

In certain embodiments, each R ^c is selected from the group consisting of halogen, cyano, hydroxyl, -NR ^dR ^e, alkyl, alkenyl, alkoxyl, and saturated or partially unsaturated cycloalkyl. In certain embodiments, each R ^c is halogen or alkyl. In certain embodiments, each R ^c is selected from the group consisting of fluoro, chloro, methyl, and ethyl.

In some embodiments, R ² is selected from the group consisting of:

In some embodiments, R ³ is selected from the group consisting of oxo, alkyl and aryl, wherein alkyl and aryl is optionally substituted with one or more R ^c. In certain embodiments, R ^c is selected from the group consisting of halogen, cyano, hydroxy, -NR ^cR ^d, alkyl.

In some embodiments, R ³ is selected from the group consisting of oxo, methyl, ethyl, trifluoromethyl and phenyl.

In some embodiments, two R ³, together with the atoms to which they are each attached, form saturated or partially unsaturated cycloalkyl optionally substituted with one or more substituents selected from the group consisting of cyano, halogen, hydroxy, and -NR ^cR ^d.

In some embodiments, W is saturated or partially unsaturated heterocyclyl optionally substituted with one or more R ^g. In certain embodiments, R ^g is alkyl optionally substituted with one or more substituents selected from the group consisting of cyano, halogen, and hydroxyl.

In some embodiments, W is heterocyclyl selected from the group consisting of:

each of which is optionally substituted with one or more R ^g.

In certain embodiments, each R ^g is alkyl optionally substituted with cyano. In certain embodiments, each R ^g is methyl optionally substituted with cyano.

In some embodiments, W is selected from the group consisting of:

In some embodiments, L ³ is a bond or -NR ^d-.

In some embodiments, B is selected from the group consisting of:

In some embodiments, the KRAS inhibitor of the present disclosure is a compound having a formula selected from the group consisting of:

or a pharmaceutically acceptable salt thereof,

wherein

J ¹ is absent, CH (R ⁴) , NR ⁴, SO ₂ or P (O) CH ₃;

J ² is absent, CR ⁵, N, SO ₂ or P (O) CH ₃;

J ³ is absent, CH (R ⁶) , NR ⁶, SO ₂ or P (O) CH ₃;

J ⁴ is absent, CR ⁷, N, SO ₂ or P (O) CH ₃;

J ⁵ is absent, CH (R ⁸) , NR ⁸, SO ₂ or P (O) CH ₃;

R ⁴, R ⁵, R ⁶, R ⁷ and R ⁸ are each independently selected from the group consisting of hydrogen, oxo, halogen, cyano, hydroxyl, -NR ^dR ^e, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, saturated or partially unsaturated cycloalkyl, saturated or partially unsaturated heterocyclyl, aryl and heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted with one or more R ^f; or

R ² and any one of R ⁴, R ⁵, R ⁶, R ⁷ and R ⁸, together with the atoms to which they are each attached, form saturated or partially unsaturated cycloalkyl, or saturated or partially unsaturated heterocyclyl, wherein each of cycloalkyl and heterocyclyl is optionally substituted with cyano, halogen, hydroxy, -NR ^cR ^d, carboxy, carbamoyl, aryl or heteroaryl; or

R ³ and any one of R ⁴, R ⁵, R ⁶ and R ⁸, together with the atoms to which they are each attached, form saturated or partially unsaturated cycloalkyl, or saturated or partially unsaturated heterocyclyl, wherein each of cycloalkyl and heterocyclyl is optionally substituted with cyano, halogen, hydroxy, -NR ^cR ^d, carboxy, carbamoyl, aryl or heteroaryl; or

R ⁴ and any one of R ⁶ and R ⁸, together with the atoms to which they are each attached, form saturated or partially unsaturated cycloalkyl, or saturated or partially unsaturated heterocyclyl, wherein each of cycloalkyl and heterocyclyl is optionally substituted with cyano, halogen, hydroxy, -NR ^cR ^d, carboxy, carbamoyl, aryl or heteroaryl; or

R ⁶ and R ⁸, together with the atoms to which they are each attached, form saturated or partially unsaturated cycloalkyl, or saturated or partially unsaturated heterocyclyl, wherein each of cycloalkyl and heterocyclyl is optionally substituted with cyano, halogen, hydroxy, -NR ^cR ^d, carboxy, carbamoyl, aryl or heteroaryl.

or a pharmaceutically acceptable salt thereof.

or a pharmaceutically acceptable salt thereof, wherein m is 0, 1, 2, 3 or 4.

or a pharmaceutically acceptable salt thereof, wherein m is 0, 1, 2, 3 or 4.

or a pharmaceutically acceptable salt thereof.

In some embodiments, the KRAS inhibitor of the present disclosure is a compound having a formula of:

or a pharmaceutically acceptable salt thereof.

or a pharmaceutically acceptable salt thereof.

or a pharmaceutically acceptable salt thereof, wherein m is 0, 1, 2, 3 or 4.

or a pharmaceutically acceptable salt thereof, wherein m is 0, 1, 2, 3 or 4.

or a pharmaceutically acceptable salt thereof.

wherein

J ¹ is absent, CH (R ⁴) , NR ⁴, SO ₂ or P (O) CH ₃;

J ² is absent, CR ⁵, N, SO ₂ or P (O) CH ₃;

J ³ is absent, CH (R ⁶) , NR ⁶, SO ₂ or P (O) CH ₃;

J ⁴ is absent, CR ⁷, N, SO ₂ or P (O) CH ₃;

J ⁵ is absent, CH (R ⁸) , NR ⁸, SO ₂ or P (O) CH ₃;

In some embodiments, L ² is alkyl.

In some embodiments, R ¹ is

In some embodiments, R ³ is selected from methyl, ethyl or trifluoromethyl.

In some embodiments, the KRAS inhibitor of the present disclosure is selected from the following compounds or a pharmaceutically acceptable salt thereof:

In some embodiments, the KRAS inhibitor is

or a pharmaceutical acceptable salt thereof.

3. ERK Inhibitor

In some embodiments, the ERK inhibitor of the present disclosure can be a dual ERK1/2 inhibitor or ERK1/2 inhibitor, which means the ERK inhibitor is capable of inhibiting the expression of both ERK1 and ERK2 genes, or capable of inhibiting the activities and/or levels of both ERK1 and ERK2 proteins. Exemplary ERK inhibitors are known in the art, for example, WO2017080979A1, the entire content of which is incorporated herein by reference. Examples of ERK inhibitors are also described in detail below.

In some embodiments, the ERK inhibitor of the present disclosure is a selective ERK inhibitor, for example, a selective ERK1 inhibitor, or a selective ERK2 inhibitor.

As used herein, the term “selective ERK inhibitor” or “selectively inhibits ERK” means that a provided compound inhibits ERK in at least one assay (e.g., biochemical or cellular) . For example, the term “selective ERK1 inhibitor” or “selectively inhibits ERK1” means that a provided agent has the IC ₅₀ for ERK2 at least 5000 fold higher, at least 4000 fold higher, at least 3000 fold higher, at least 2000 fold higher, at least 1000 fold higher, at least 500 fold higher, at least 400 fold higher, at least 300 fold higher, at least 200 fold higher, at least 100 fold higher, at least 90 fold higher, at least 80 fold higher, at least 70 fold higher, at least 60 fold higher, at least 50 fold higher, at least 40 fold higher, at least 30 fold higher, at least 20 fold higher, at least 10 fold higher, than the IC ₅₀ for inhibiting ERK1.

In some embodiments, the ERK inhibitor is a chemotherapeutic agent, an antibody, or an antigen-binding fragment thereof, an RNAi molecule that targets an encoding sequence of ERK, an antisense nucleotide that targets an encoding sequence of ERK, or an agent that competes with ERK protein to bind to its substrate. In some embodiments, the antibody is a monoclonal antibody or a polyclonal antibody. In some embodiments, the antibody is a humanized antibody, a chimeric antibody or a fully human antibody. In some embodiments, the RNAi molecule is a small interfering RNA (siRNA) , a small hairpin RNA (shRNA) or a microRNA (miRNA) .

In some embodiments, the ERK inhibitor is a small molecule compound.

In some embodiments, the ERK inhibitor is a dual ERK1/2 inhibitor.

In some embodiments, the ERK inhibitor is a compound of Formula (VII) or a pharmaceutically acceptable salt thereof

wherein:

R ⁹ is hydrogen, C _1-3 alkyl or -CH ₂OMe;

R ¹⁰ is pyridinyl, optionally substituted on 1 ring carbon atom by a substituent independently selected from the group consisting of C _1-3 alkyl, difluoromethyl and trifluoromethyl; or R ¹⁰ is pyrimidinyl, optionally substituted on 1 ring carbon atom by a substituent independently selected from the group consisting of C _1-3 alkyl, difluoromethyl and trifluoromethyl; or R ¹⁰ is phenyl optionally substituted on 1 or 2 ring carbon atoms by a substituent independently selected from the group consisting of halo, difluoromethyl, trifluoromethyl, methoxy and -OCHF ₂; and

R ¹¹ is hydrogen, C _1-3 alkyl or chloro.

As used herein, the term “optionally substituted” will be understood to mean “substituted or unsubstituted” .

In some embodiments, R ⁹ is hydrogen, methyl or -CH ₂OMe. In some embodiments, R ⁹ is methyl or -CH ₂OMe. In some embodiments, R ⁹ is hydrogen. In some embodiments, R ⁹ is methyl. In some embodiments, R ⁹ is -CH ₂OMe.

In some embodiments, R ¹⁰ is pyridinyl, optionally substituted on 1 ring carbon atom by a substituent independently selected from the group consisting of methyl, difluoromethyl and trifluoromethyl; or R ¹⁰ is pyrimidinyl, optionally substituted on 1 ring carbon atom by a substituent independently selected from the group consisting of methyl, difluoromethyl and trifluoromethyl; or R ¹⁰ is phenyl optionally substituted on 1 or 2 ring carbon atoms by a substituent independently selected from the group consisting of fluoro, chloro, difluoromethyl, trifluoromethyl, methoxy and -OCHF _2.

In some embodiments, R ¹⁰ is pyridinyl, optionally substituted on 1 ring carbon atom by a substituent independently selected from the group consisting of methyl, difluoromethyl and trifluoromethyl; or R ¹⁰ is pyrimidinyl, optionally substituted on 1 ring carbon atom by trifluoromethyl; or R ¹⁰ is phenyl optionally substituted on 1 or 2 ring carbon atoms by a substituent independently selected from the group consisting of fluoro, chloro, difluoromethyl, trifluoromethyl, methoxy and -OCHF ₂.

In some embodiments, R ¹⁰ is pyridinyl, optionally substituted on 1 ring carbon atom by a substituent independently selected from the group consisting of methyl, difluoromethyl and trifluoromethyl; or R ¹⁰ is pyrimidinyl, optionally substituted on 1 ring carbon atom by trifluoromethyl; or R ¹⁰ is phenyl optionally substituted on 1 ring carbon atom by a substituent independently selected from the group consisting fluoro, chloro, difluoromethyl, trifluoromethyl, methoxy and -OCHF ₂.

In some embodiments, R ¹⁰ is pyridinyl, optionally substituted on 1 ring carbon atom by a substituent independently selected from the group consisting of methyl, difluoromethyl and trifluoromethyl; or R ¹⁰ is pyrimidinyl, optionally substituted on 1 ring carbon atom by trifluoromethyl; or

R ¹⁰ is phenyl optionally substituted on 2 ring carbon atoms by a substituent independently selected from the group consisting of fluoro, chloro, difluoromethyl, trifluoromethyl, methoxy and -OCHF _2.

In some embodiments, R ¹⁰ is pyridinyl, optionally substituted on 1 ring carbon atom by a substituent independently selected from the group consisting of methyl, difluoromethyl and trifluoromethyl; or

R ¹⁰ is pyrimidinyl, optionally substituted on 1 ring carbon atom by trifluoromethyl; or

R ¹⁰ is phenyl optionally substituted on 1 or 2 ring carbon atoms by a substituent independently selected from the group consisting of fluoro, chloro, difluoromethyl, methoxy and –OCHF _2.

In some embodiments, R ¹⁰ is pyridinyl, optionally substituted on 1 ring carbon atom by difluoromethyl; or

R ¹⁰ is phenyl optionally substituted on 1 or 2 ring carbon atoms by a substituent independently selected from the group consisting of fluoro and chloro.

In some embodiments, R ¹⁰ is pyridinyl, optionally substituted on 1 ring carbon atom by a substituent independently selected from the group consisting of methyl, difluoromethyl and trifluoromethyl.

In some embodiments, R ¹⁰ is pyridinyl, optionally substituted on 1 ring carbon atom by a methyl. In some embodiments, R ¹⁰ is pyridinyl, optionally substituted on 1 ring carbon atom by difluoromethyl. In some embodiments, R ¹⁰ is pyridinyl, optionally substituted on 1 ring carbon atom by trifluoromethyl.

In some embodiments, R ¹⁰ is pyrimidinyl, optionally substituted on 1 ring carbon atom by a substituent independently selected from the group consisting of methyl, difluoromethyl or trifluoromethyl. In some embodiments, R ¹⁰ is pyrimidinyl, optionally substituted on 1 ring carbon atom by methyl. In some embodiments, R ¹⁰ is pyrimidinyl, optionally substituted on 1 ring carbon atom by difluoromethyl. In some embodiments, R ¹⁰ is pyrimidinyl, optionally substituted on 1 ring carbon atom by trifluoromethyl.

In some embodiments, R ¹⁰ is phenyl optionally substituted on 1 or 2 ring carbon atoms by a substituent independently selected from the group consisting of fluoro, chloro, difluoromethyl, trifluoromethyl, methoxy and -OCHF _2. In some embodiments, R ¹⁰ is phenyl optionally substituted on 1 or 2 ring carbon atoms by a substituent independently selected from the group consisting of fluoro, chloro, difluoromethyl, methoxy and –OCHF _2. In some embodiments, R ¹⁰ is phenyl optionally substituted on 1 or 2 ring carbon atoms by a substituent independently selected from fluoro, chloro or methoxy _. In some embodiments, R ¹⁰ is phenyl optionally substituted on 2 ring carbon atoms by a substituent independently selected from the group consisting of fluoro, chloro and methoxy _. In some embodiments, R ¹⁰ is phenyl optionally substituted on 1 or 2 ring carbon atoms by a substituent independently selected from the group consisting fluoro, chloro and methoxy. In some embodiments, R ¹⁰ is phenyl optionally substituted on 1 or 2 ring carbon atoms by a substituent independently selected from the group consisting fluoro and chloro.

In some embodiments, R ¹⁰ is phenyl optionally substituted on 1 ring carbon atom by a substituent independently selected from the group consisting of fluoro and chloro. In some embodiments, R ¹⁰ is phenyl optionally substituted on 2 ring carbon atoms by a substituent independently selected from the group consisting of fluoro and chloro. In some embodiments, R ¹⁰ is phenyl optionally substituted on 1 or 2 ring carbon atoms by a substituent independently selected from the group consisting fluoro and methoxy. In some embodiments, R ¹⁰ is phenyl optionally substituted on 2 ring carbon atoms by a substituent independently selected from the group consisting of fluoro and methoxy.

In some embodiments, R ¹⁰ is phenyl optionally substituted on 1 or 2 ring carbon atoms by fluoro _. In some embodiments, R ¹⁰ is phenyl optionally substituted on 1 or 2 ring carbon atoms by chloro _. In some embodiments, R ¹⁰ is phenyl optionally substituted on 1 or 2 ring carbon atoms by difluoromethyl _. In some embodiments, R ¹⁰ is phenyl optionally substituted on 1 or 2 ring carbon atoms by trifluoromethyl _. In some embodiments, R ¹⁰ is phenyl optionally substituted on 1 or 2 ring carbon atoms by methoxy _. In some embodiments, R ¹⁰ is phenyl optionally substituted on 1 or 2 ring carbon atoms by -OCHF _2. In some embodiments, R ¹⁰ is phenyl optionally substituted on 2 ring carbon atoms by fluoro. In some embodiments, R ¹⁰ is phenyl optionally substituted on 1 ring carbon atom by chloro.

In some embodiments, R ¹⁰ is 6-methylpyridin-2-yl, 4- (difluoromethyl) pyridin-2-yl, 6- (difluoromethyl) pyridin-2-yl, 4- (trifluoromethyl) pyridin-2-yl, 6- (trifluoromethyl) pyridin-2-yl, 2- (trifluoromethyl) pyridin-4-yl, 2- (trifluoromethyl) pyrimidin-4-yl, 6- (trifluoromethyl) pyrimidin-4-yl, 3-chlorophenyl, 3, 4-difluorophenyl, 3, 5-difluorophenyl, 3-chloro-4-fluorophenyl, 3- (difluoromethoxy) phenyl, 3- (difluoromethyl) phenyl, 3-methoxyphenyl or 4-fluoro-3-methoxyphenyl.

In some embodiments, R ¹⁰ is 6- (difluoromethyl) pyridin-2-yl, 3-chlorophenyl, 3, 4-difluorophenyl or 3, 5-difluorophenyl. In some embodiments, R ¹⁰ is 6-methylpyridin-2-yl. In some embodiments, R ¹⁰ is 4- (difluoromethyl) pyridin-2-yl.

In some embodiments, R ¹⁰ is 6- (difluoromethyl) pyridin-2-yl. In some embodiments, R ¹⁰ is 4- (trifluoromethyl) pyridin-2-yl. In some embodiments, R ¹⁰ is 6- (trifluoromethyl) pyridin-2-yl. In some embodiments, R ¹⁰ is 2- (trifluoromethyl) pyridin-4-yl. In some embodiments, R ¹⁰ is 2- (trifluoromethyl) pyrimidin-4-yl. In some embodiments, R ¹⁰ is 6- (trifluoromethyl) pyrimidin-4-yl. In some embodiments, R ¹⁰ is 3-chlorophenyl. In some embodiments, R ¹⁰ is 3, 4-difluorophenyl. In some embodiments, R ¹⁰ is 3, 5- difluorophenyl. In some embodiments, R ¹⁰ is 3-chloro-4-fluorophenyl. In some embodiments, R ¹⁰ is 3- (difluoromethoxy) phenyl. In some embodiments, R ¹⁰ is 3- (difluoromethyl) phenyl. In some embodiments, R ¹⁰ is 3-methoxyphenyl. In some embodiments, R ¹⁰ is 4-fluoro-3-methoxyphenyl.

In some embodiments, R ¹¹ is hydrogen, methyl or chloro. In some embodiments, R ¹¹ is hydrogen or methyl. In some embodiments, R ¹¹ is hydrogen. In some embodiments, R ¹¹ is methyl. In some embodiments, R ¹¹ is chloro.

In a further aspect, there is provided a compound of Formula (VII) or a pharmaceutically acceptable salt thereof, wherein:

R ⁹ is hydrogen, methyl or -CH ₂OMe; R ¹⁰ is pyridinyl, optionally substituted on 1 ring carbon atom by a substituent independently selected from the group consisting of methyl, difluoromethyl and trifluoromethyl; or R ¹⁰ is pyrimidinyl, optionally substituted on 1 ring carbon atom by a substituent independently selected from the group consisting of methyl, difluoromethyl and trifluoromethyl; or R ¹⁰ is phenyl optionally substituted on 1 or 2 ring carbon atoms by a substituent independently selected from the group consisting of fluoro, chloro, difluoromethyl, trifluoromethyl, methoxy and -OCHF ₂; and R ¹¹ is hydrogen, methyl or chloro.

In some embodiments, the ERK inhibitor of the present disclosure is a compound of Formula (VII) or a pharmaceutically acceptable salt thereof, wherein:

R ⁹ is hydrogen, methyl or -CH ₂OMe;

R ¹⁰ is pyridinyl, optionally substituted on 1 ring carbon atom by a substituent independently selected from the group consisting of methyl, difluoromethyl and trifluoromethyl; or

R ¹⁰ is pyrimidinyl, optionally substituted on 1 ring carbon atom by a trifluoromethyl; or

R ¹⁰ is phenyl optionally substituted on 1 or 2 ring carbon atoms by a substituent independently selected from the group consisting of fluoro, chloro, difluoromethyl, trifluoromethyl, methoxy and -OCHF ₂; and

R ¹¹ is hydrogen, methyl or chloro.

R ⁹ is hydrogen, methyl or -CH ₂OMe;

R ¹⁰ is phenyl optionally substituted on 1 carbon atom by a substituent independently selected from the group consisting of fluoro, chloro, difluoromethyl, trifluoromethyl, methoxy and -OCHF ₂; and

R ¹¹ is hydrogen, methyl or chloro.

R ⁹ is hydrogen, methyl or -CH ₂OMe;

R ¹⁰ is phenyl optionally substituted on 2 ring carbon atoms by a substituent independently selected from the group consisting of fluoro, chloro, difluoromethyl, trifluoromethyl, methoxy and -OCHF ₂; and

R ¹¹ is hydrogen, methyl or chloro.

R ⁹ is hydrogen, methyl or -CH ₂OMe;

R ¹⁰ is phenyl optionally substituted on 1 or 2 ring carbon atoms by a substituent independently selected from the group consisting of fluoro, chloro, difluoromethyl, methoxy and –OCHF ₂; and

R ¹¹ is hydrogen, methyl or chloro.

R ⁹ is methyl or -CH ₂OMe;

R ¹⁰ is pyridinyl, optionally substituted on 1 ring carbon atom by difluoromethyl; or

R ¹⁰ is phenyl optionally substituted on 1 or 2 ring carbon atoms by a substituent independently selected from the group consisting of fluoro and chloro; and

R ¹¹ is hydrogen or methyl.

R ⁹ is hydrogen, methyl or -CH ₂OMe;

R ¹⁰ is pyridinyl, optionally substituted on 1 ring carbon atom by a substituent independently selected from the group consisting of methyl, difluoromethyl and trifluoromethyl; and

R ¹¹ is hydrogen, methyl or chloro.

R ⁹ is hydrogen, methyl or -CH2OMe;

R ¹⁰ is pyridinyl, optionally substituted on 1 ring carbon atom by methyl; and

R ¹¹ is hydrogen, methyl or chloro.

R ⁹ is hydrogen, methyl or -CH ₂OMe;

R ¹⁰ is pyridinyl, optionally substituted on 1 ring carbon atom by difluoromethyl; and

R ¹¹ is hydrogen, methyl or chloro.

R ⁹ is hydrogen, methyl or -CH ₂OMe;

R ¹⁰ is pyridinyl, optionally substituted on 1 ring carbon atom by trifluoromethyl; and

R ¹¹ is hydrogen, methyl or chloro.

R ⁹ is hydrogen, methyl or -CH ₂OMe;

R ¹⁰ is pyrimidinyl, optionally substituted on 1 ring carbon atom by trifluoromethyl; and

R ¹¹ is hydrogen, methyl or chloro.

R ⁹ is hydrogen, methyl or -CH ₂OMe;

R ¹¹ is hydrogen, methyl or chloro.

R ⁹ is hydrogen, methyl or -CH ₂OMe;

R ¹¹ is hydrogen, methyl or chloro.

R ⁹ is hydrogen, methyl or -CH ₂OMe;

R ¹⁰ is phenyl optionally substituted on 1 or 2 ring carbon atoms by a substituent independently selected from the group consisting of fluoro, chloro, difluoromethyl and methoxy; and

R ¹¹ is hydrogen, methyl or chloro.

R ⁹ is hydrogen, methyl or -CH ₂OMe;

R ¹⁰ is phenyl optionally substituted on 1 or 2 ring carbon atoms by a substituent independently selected from the group consisting of fluoro, chloro and methoxy; and

R ¹¹ is hydrogen, methyl or chloro.

R ⁹ is hydrogen, methyl or -CH ₂OMe;

R ¹⁰ is phenyl optionally substituted on 2 ring carbon atoms by a substituent independently selected from the group consisting of fluoro, chloro and methoxy; and

R ¹¹ is hydrogen, methyl or chloro.

R ⁹ is hydrogen, methyl or -CH ₂OMe;

R ¹¹ is hydrogen, methyl or chloro.

R ⁹ is hydrogen, methyl or -CH ₂OMe;

R ¹⁰ is phenyl optionally substituted on 1 or 2 ring carbon atoms by a substituent independently selected from the group consisting of fluoro and methoxy; and

R ¹¹ is hydrogen, methyl or chloro.

R ⁹ is hydrogen, methyl or -CH ₂OMe;

R ¹⁰ is phenyl optionally substituted on 2 ring carbon atoms by a substituent independently selected from the group consisting of fluoro and chloro; and

R ¹¹ is hydrogen, methyl or chloro.

R ⁹ is hydrogen, methyl or -CH ₂OMe;

R ¹⁰ is phenyl optionally substituted on 2 ring carbon atoms by a substituent independently selected from the group consisting of fluoro and methoxy; and

R ¹¹ is hydrogen, methyl or chloro.

R ⁹ is hydrogen, methyl or -CH ₂OMe;

R ¹⁰ is 6-methylpyridin-2-yl, 4- (difluoromethyl) pyridin-2-yl, 6- (difluoromethyl) pyridin-2-yl, 4- (trifluoromethyl) pyridin-2-yl, 6- (trifluoromethyl) pyridin-2-yl, 2- (trifluoromethyl) pyridin-4-yl, 2- (trifluoromethyl) pyrimidin-4-yl, 6- (trifluoromethyl) pyrimidin-4-yl, 3-chlorophenyl, 3, 4-difluorophenyl, 3, 5-difluorophenyl, 3-chloro-4-fluorophenyl, 3- (difluoromethoxy) phenyl, 3- (difluoromethyl) phenyl, 3-methoxyphenyl or 4-fluoro-3-methoxyphenyl; and

R ¹¹ is hydrogen, methyl or chloro.

R ⁹ is hydrogen, methyl or -CH ₂OMe;

R ¹⁰ is 6- (difluoromethyl) pyridin-2-yl, 3-chlorophenyl, 3, 4-difluorophenyl or 3, 5-difluorophenyl; and

R ¹¹ is hydrogen, methyl or chloro.

R ⁹ is methyl or -CH ₂OMe;

R ¹¹ is hydrogen or methyl.

R ⁹ is hydrogen, methyl or -CH ₂OMe;

R ¹⁰ is 6- (difluoromethyl) pyridin-2-yl; and

R ¹¹ is hydrogen, methyl or chloro.

R ⁹ is hydrogen, methyl or -CH ₂OMe;

R ¹⁰ is 3-chlorophenyl; and

R ¹¹ is hydrogen, methyl or chloro.

R ⁹ is hydrogen, methyl or -CH ₂OMe;

R ¹⁰ is 3, 4-difluorophenyl; and

R ¹¹ is hydrogen, methyl or chloro.

R ⁹ is hydrogen, methyl or -CH ₂OMe;

R ¹⁰ is 3, 5-difluorophenyl; and

R ¹¹ is hydrogen, methyl or chloro.

R ⁹ is methyl or -CH ₂OMe;

R ¹⁰ is 6- (difluoromethyl) pyridin-2-yl; and

R ¹¹ is hydrogen or methyl.

R ⁹ is methyl or -CH ₂OMe;

R ¹⁰ is 3-chlorophenyl; and

R ¹¹ is hydrogen or methyl.

R ⁹ is methyl or -CH ₂OMe;

R ¹⁰ is 3, 4-difluorophenyl; and

R ¹¹ is hydrogen or methyl.

R ⁹ is methyl or -CH ₂OMe;

R ¹⁰ is 3, 5-difluorophenyl; and

R ¹¹ is hydrogen or methyl.

In some embodiments, the ERK inhibitor of the present disclosure is a compound selected from the group consisting of:

2- (2- ( (1-Methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -7- ( (6-methylpyridin-2-yl) methyl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -7- (3-Chlorobenzyl) -6-methyl-2- (2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -7- (3-Chloro-4-fluorobenzyl) -6-methyl-2- (2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -7- (3, 4-Difluorobenzyl) -6-methyl-2- (2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

2- (5-Methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -7- ( (6- (trifluoromethyl) pyridin-2-yl) methyl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -2- (5-Chloro-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -7- (3, 4-difluorobenzyl) -6-methyl-6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -2- (5-Chloro-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -7- (3-methoxybenzyl) -6-methyl-6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -2- (5-chloro-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6-methyl-7- ( (2- (trifluoromethyl) pyrimidin-4-yl) methyl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -2- (5-Chloro-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -7- ( (6- (difluoromethyl) pyridin-2-yl) methyl) -6-methyl-6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -2- (5-Chloro-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6-methyl-7- ( (6- (trifluoromethyl) pyridin-2-yl) methyl) -6, 7-dihydroimidazo [1, 2-a] pyrazin- 8 (5H) -one;

(S) -2- (5-chloro-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6-methyl-7- ( (6-methylpyridin-2-yl) methyl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

7- (3-Chloro-4-fluorobenzyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

7- (3-Chlorobenzyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

7- (3- (Difluoromethyl) benzyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

7- ( (6- (Difluoromethyl) pyridin-2-yl) methyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(R) -7- ( (6- (Difluoromethyl) pyridin-2-yl) methyl) -6- (methoxymethyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(R) -7- (3-Chlorobenzyl) -6- (methoxymethyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(R) -7- (3, 4-Difluorobenzyl) -6- (methoxymethyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -7- (3-Chlorobenzyl) -6-methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -7- (3, 4-Difluorobenzyl) -6-methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -7- (3- (Difluoromethyl) benzyl) -6-methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -7- (3, 5-Difluorobenzyl) -6-methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -7- (3-Methoxybenzyl) -6-methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -7- (4-Fluoro-3-methoxybenzyl) -6-methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -7- ( (6- (Difluoromethyl) pyridin-2-yl) methyl) -6-methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -6-methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -7- ( (2- (trifluoromethyl) pyrimidin-4-yl) methyl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -6-Methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -7- ( (6- (trifluoromethyl) pyridin-2-yl) methyl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -7- (3- (Difluoromethoxy) benzyl) -6-methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -6-Methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -7- ( (4- (trifluoromethyl) pyridin-2-yl) methyl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -6-Methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -7- ( (2- (trifluoromethyl) pyridin-4-yl) methyl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -7- ( (4- (Difluoromethyl) pyridin-2-yl) methyl) -6-methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -6-Methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -7- ( (6- (trifluoromethyl) pyrimidin-4-yl) methyl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

7- (3, 4-Difluorobenzyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -7- (3, 4-difluorobenzyl) -6- (methoxymethyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(R) -6-Methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -7- ( (6- (trifluoromethyl) pyridin-2-yl) methyl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(R) -7- ( (6- (Difluoromethyl) pyridin-2-yl) methyl) -6-methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(R) -7- (3- (Difluoromethyl) benzyl) -6- (methoxymethyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(R) -6- (Methoxymethyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -7- ( (6- (trifluoromethyl) pyridin-2-yl) methyl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(R) -7- (3, 5-difluorobenzyl) -6- (methoxymethyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one; and

(R) -7- (3-Methoxybenzyl) -6- (methoxymethyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

or a pharmaceutically acceptable salt thereof.

In some embodiments, the ERK inhibitor of the present disclosure is selected from the group consisting of:

(S) -7- (3, 5-difluorobenzyl) -6-methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one; and

or a pharmaceutically acceptable salt thereof.

In some embodiments, the ERK inhibitor of the present disclosure is (S) -7- (3-chlorobenzyl) -6-methyl-2- (2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one; or a pharmaceutically acceptable salt thereof.

In some embodiments, the ERK inhibitor of the present disclosure is (R) -7- (3, 4-difluorobenzyl) -6- (methoxymethyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one; or a pharmaceutically acceptable salt thereof.

In some embodiments, the ERK inhibitor of the present disclosure is (S) -7- (3, 5-difluorobenzyl) -6-methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one or a pharmaceutically acceptable salt thereof.

In some embodiments, the ERK inhibitor of the present disclosure is (S) -7- ( (6- (difluoromethyl) pyridin-2-yl) methyl) -6-methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one; or a pharmaceutically acceptable salt thereof.

In some embodiments, the ERK inhibitor of the present disclosure is (R) -7- (3, 4-Difluorobenzyl) -6- (methoxymethyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one, or a pharmaceutically acceptable salt thereof.

In some embodiments, the ERK inhibitor of the present disclosure is (R) -7- (3, 4-Difluorobenzyl) -6- (methoxymethyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one Ethanesulfonic acid salt.

In some embodiments, the ERK inhibitor of the present disclosure is a crystalline form of (R) -7- (3, 4-Difluorobenzyl) -6- (methoxymethyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one Ethanesulfonic acid salt.

In some embodiments, the ERK inhibitor of the present disclosure is (R) -7- (3, 4-Difluorobenzyl) -6- (methoxymethyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one Adipic acid salt.

In some embodiments, the ERK inhibitor of the present disclosure is a crystalline form of (R) -7- (3, 4-Difluorobenzyl) -6- (methoxymethyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one Adipic acid salt.

In some embodiments, the ERK inhibitor of the present disclosure is Compound 71. In the present disclosure, the structure and chemical name of Compound 71 are shown as follows:

In some embodiments, the KRAS inhibitor of the present disclosure is selected from the group consisting of Sotorasib (AMG-510) , Adagrasib (MRTX849) ,

and a pharmaceutically acceptable salt thereof, and the ERK inhibitor of the present disclosure is (R) -7- (3, 4-Difluorobenzyl) -6- (methoxymethyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one or a pharmaceutically acceptable salt thereof.

and a pharmaceutically acceptable salt thereof, and the ERK inhibitor of the present disclosure is

.0.5 adipic acid.

4. Other Forms of Compounds

Some compounds of the present disclosure have a chiral center and it will be recognized that such compounds may be prepared, isolated and/or supplied with or without the presence, in addition, of one or more of the other 2 possible enantiomeric isomers of the compounds in any relative proportions. The preparation of enantioenriched/enantiopure compounds may be carried out by standard techniques of organic chemistry that are well known in the art, for example by synthesis from enantioenriched or enantiopure starting materials, use of an appropriate enantioenriched or enantiopure catalyst during synthesis, and/or by resolution of a racemic or partially enriched mixture of stereoisomers, for example via chiral chromatography.

The compounds of the present disclosure are described with reference to both generic formulae and specific compounds. In addition, the compounds of the present disclosure may exist in a number of different forms or derivatives, including but not limited to prodrugs, soft drugs, active metabolic derivatives (active metabolites) , and their pharmaceutically acceptable salts, all within the scope of the present disclosure.

As used herein, the term “prodrugs” refers to compounds or pharmaceutically acceptable salts thereof which, when metabolized under physiological conditions or when converted by solvolysis, yield the desired active compound. Prodrugs include, without limitation, esters, amides, carbamates, carbonates, ureides, solvates, or hydrates of the active compound. Typically, the prodrug is inactive, or less active than the active compound, but may provide one or more advantageous handling, administration, and/or metabolic properties. For example, some prodrugs are esters of the active compound; during metabolysis, the ester group is cleaved to yield the active drug. Also, some prodrugs are activated enzymatically to yield the active compound, or a compound which, upon further chemical reaction, yields the active compound. Prodrugs may proceed from prodrug form to active form in a single step or may have one or more intermediate forms which may themselves have activity or may be inactive. Preparation and use of prodrugs is discussed in T. Higuchi and V. Stella, “Pro-drugs as Novel Delivery Systems” , Vol. 14 of the A.C.S. Symposium Series, in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987; in Prodrugs: Challenges and Rewards, ed. V. Stella, R. Borchardt, M. Hageman, R. Oliyai, H. Maag, J. Tilley, Springer-Verlag New York, 2007, all of which are hereby incorporated by reference in their entirety.

As used herein, the term “soft drug” refers to compounds that exert a pharmacological effect but break down to inactive metabolites degradants so that the activity is of limited time. See, for example, “Soft drugs: Principles and methods for the design of safe drugs” , Nicholas Bodor, Medicinal Research Reviews, Vol. 4, No. 4, 449-469, 1984, which is hereby incorporated by reference in its entirety.

As used herein, the term “metabolite” , e.g., active metabolite overlaps with prodrug as described above. Thus, such metabolites are pharmacologically active compounds or compounds that further metabolize to pharmacologically active compounds that are derivatives resulting from metabolic process in the body of a subject. For example, such metabolites may result from oxidation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, and the like, of the administered compound or salt or prodrug. Of these, active metabolites are such pharmacologically active derivative compounds. For prodrugs, the prodrug compound is generally inactive or of lower activity than the metabolic product. For active metabolites, the parent compound may be either an active compound or may be an inactive prodrug.

Prodrugs and active metabolites may be identified using routine techniques know in the art. See, e.g., Bertolini et al., 1997, J Med Chem 40: 2011-2016; Shan et al., J Pharm Sci 86: 756-757; Bagshawe, 1995, DrugDev Res 34: 220-230; Wermuth, supra.

It is also to be understood that the compounds of the present disclosure and pharmaceutically acceptable salts thereof may prepared, used or supplied in amorphous form, crystalline form, or semicrystalline form and any given compound 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 disclosure encompasses any and all such solid forms of the compounds and pharmaceutically acceptable salts thereof.

As used herein, the term “solvate” or “solvated form” refers to solvent addition forms that contain either stoichiometric or non-stoichiometric amounts of solvent. Some compounds have a tendency to trap a fixed molar ratio of solvent molecules in the crystalline solid state, thus forming a solvate. If the solvent is water the solvate formed is a hydrate; and if the solvent is alcohol, the solvate formed is an alcoholate. Hydrates are formed by the combination of one or more molecules of water with one molecule of the substance in which the water retains its molecular state as H ₂O. Examples of solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

As used herein, the terms “crystal form” , “crystalline form” , “polymorphic forms” and “polymorphs” can be used interchangeably, and mean crystal structures in which a compound (or a salt or solvate thereof) can crystallize in different crystal packing arrangements, all of which have the same elemental composition. Different crystal forms usually have different X-ray diffraction patterns, infrared spectral, melting points, density hardness, crystal shape, optical and electrical properties, stability and solubility. Recrystallization solvent, rate of crystallization, storage temperature, and other factors may cause one crystal form to dominate. Crystal polymorphs of the compounds can be prepared by crystallization under different conditions.

The compounds of the present disclosure are also intended to include all isotopes of atoms in the compounds. Isotopes of an atom include atoms having the same atomic number but different mass numbers. For example, unless otherwise specified, hydrogen, carbon, nitrogen, oxygen, phosphorous, sulfur, fluorine, chlorine, bromide or iodine in the compounds of present disclosure are meant to also include their isotopes, such as but not limited to ¹H, ²H, ³H, ¹¹C, ¹²C, ¹³C, ¹⁴C, ¹⁴N, ¹⁵N, ¹⁶O, ¹⁷O, ¹⁸O, ³¹P, ³²P, ³²S, ³³S, ³⁴S, ³⁶S, ¹⁷F, ¹⁸F, ¹⁹F, ³⁵Cl, ³⁷Cl, ⁷⁹Br, ⁸¹Br, ¹²⁴I, ¹²⁷I and ¹³¹I. In some embodiments, hydrogen includes protium, deuterium and tritium. In some embodiments, carbon includes ¹²C and ¹³C.

Those of skill in the art will appreciate that compounds of the present disclosure may exist in different tautomeric forms, and all such forms are embraced within the scope of the present disclosure. The term “tautomer” or “tautomeric form” refers to structural isomers of different energies which are interconvertible via a low energy barrier. The presence and concentrations of the isomeric forms will depend on the environment the compound is found in and may be different depending upon, for example, whether the compound is a solid or is in an organic or aqueous solution. By way of examples, proton tautomers (also known as prototropic tautomers) include interconversions via migration of a proton, such as keto-enol, amide-imidic acid, lactam-lactim, imine-enamine isomerizations and annular forms where a proton can occupy two or more positions of a heterocyclic system. Valence tautomers include interconversions by reorganization of some of the bonding electrons. Tautomers can be in equilibrium or sterically locked into one form by appropriate substitution. Compounds of the present disclosure identified by name or structure as one particular tautomeric form are intended to include other tautomeric forms unless otherwise specified.

A suitable pharmaceutically acceptable salt of a compound of the present disclosure, for example, an acid addition salt. A further suitable pharmaceutically acceptable salt of a compound of the present disclosure is, for example, a salt formed within the human or animal body after administration of the compound to said human or animal body.

A suitable pharmaceutically acceptable salt of a compound of the present disclosure may also be, for example, an acid-addition salt of a compound of the present disclosure, for example an acid-addition salt with a inorganic or organic acid such as hydrochloric acid, hydrobromic acid, sulphuric acid or trifluoroacetic acid. Pharmaceutically acceptable salts of a compound of the present disclosure may also be an acid-addition salt with an acid such as one of the following: acetic acid, adipic acid, benzene sulfonic acid, benzoic acid, cinnamic acid, citric acid, D, L-lactic acid, ethane disulfonic acid, ethane sulfonic acid, fumaric acid, L-tartaric acid, maleic acid, malic acid, malonic acid, methane sulfonic acid, napadisylic acid, phosphoric acid, saccharin, succinic acid or toluene sulfonic acid (such as p-toluenesulfonic acid) . It is to be understood that a pharmaceutically acceptable salt of a compound of the present disclosure form an aspect of the present disclosure.

5. Administration

In some embodiments, the ERK inhibitor is administered before, after, simultaneously, or in an overlapping manner with the KRAS inhibitor.

Specifically, the ERK inhibitor provided herein that is administered in combination with the KRAS inhibitor provided herein may be administered simultaneously with the KRAS inhibitor provided herein, and in certain of these embodiments the ERK inhibitor and the KRAS inhibitor may be administered as part of the same pharmaceutical composition. However, the ERK inhibitor administered “in combination” with a KRAS inhibitor does not have to be administered simultaneously with or in the same composition as the agent. The ERK inhibitor administered prior to or after the KRAS inhibitor is considered to be administered “in combination” with the KRAS inhibitor as the phrase is used herein, even if the ERK inhibitor and the KRAS inhibitor are administered via different routes (for example, the ERK inhibitor is administered orally, while the KRAS inhibitor is administered by injection) . For example, the ERK inhibitor may be administered prior to the KRAS inhibitor (e.g., 5 hours, 10 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, etc. ) , either punctually or several times before the KRAS inhibitor is administered, as long as they exert effects in the subject during an overlapped timeframe. For another example, the ERK inhibitor is administered prior to the KRAS inhibitor for a certain period of time (e.g., 5 hours, 10 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 week, 3 weeks, etc. ) , and then the ERK inhibitor and the KRAS inhibitor are administered simultaneously. Where possible, the KRAS inhibitor administered in combination with the ERK inhibitor provided herein are administered according to the schedule listed in the product information sheet of the additional therapeutic agent, or according to the Physicians’ Desk Reference 2003 (Physicians’ Desk Reference, 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002) ) or protocols well known in the art.

The ERK inhibitor and the KRAS inhibitor of the present disclosure may be administered by any route known in the art, for example parenteral (e.g., subcutaneous, intraperitoneal, intravenous including intravenous infusion, intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes. In some embodiments, the ERK inhibitor and/or the KRAS inhibitor of the present disclosure is administered orally, subcutaneously, intraperitoneally, or intravenously.

III. Kits

In another aspect, the present disclosure provides a kit comprising (a) a first composition comprising a KRAS inhibitor or a pharmaceutically acceptable salt thereof, and (b) a second composition comprising an ERK inhibitor or a pharmaceutically acceptable salt thereof. In some embodiments, the first and/or the second composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the KRAS inhibitor in the kit is a KRAS ^G12C inhibitor, for example, Sotorasib (AMG-510) , Adagrasib (MRTX849) , or Compound 33. In some embodiments, the ERK inhibitor in the kit is a dual ERK1/2 inhibitor. In some embodiments, the ERK inhibitor in the kit is Compound 71.

Such kits can further include, if desired, one or more of various conventional pharmaceutical kit components, such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers etc., as will be readily apparent to a person skilled in the art. Instructions, either as inserts or as labels, indicating quantities of the components to be administered, guidelines for administration, and/or guidelines for mixing the components, can also be included in the kit.

IV. Pharmaceutical Compositions

As used herein, the term “pharmaceutical composition” refers to a formulation containing the molecules or compounds of the present disclosure in a form suitable for administration to a subject.

As used herein, the term “pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use as well as human pharmaceutical use. A “pharmaceutically acceptable excipient” as used herein includes both one and more than one such excipient. The term “pharmaceutically acceptable excipient” also encompasses “pharmaceutically acceptable carrier” and “pharmaceutically acceptable diluent” .

The particular excipient used will depend upon the means and purpose for which the compounds of the present disclosure is being applied. Solvents are generally selected based on solvents recognized by persons skilled in the art as safe to be administered to a mammal including humans. In general, safe solvents are non-toxic aqueous solvents such as water and other non-toxic solvents that are soluble or miscible in water. Suitable aqueous solvents include water, ethanol, propylene glycol, polyethylene glycols (e.g., PEG 400, PEG 300) , etc. and mixtures thereof.

In some embodiments, suitable excipients may include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol) ; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes) ; and/or non-ionic surfactants such as TWEEN ^TM, PLURONICS ^TM or polyethylene glycol (PEG) .

In some embodiments, suitable excipients may include one or more stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavoring agents and other known additives to provide an elegant presentation of the drug (i.e., a compound of the present disclosure or pharmaceutical composition thereof) or aid in the manufacturing of the pharmaceutical product (i.e., medicament) . The active pharmaceutical ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) . A “liposome” is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as the compounds disclosed herein and, optionally, a chemotherapeutic agent) to a mammal including humans. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.

The pharmaceutical compositions provided herein can be in any form that allows for the composition to be administered to a subject, including, but not limited to a human, and formulated to be compatible with an intended route of administration.

A variety of routes are contemplated for the pharmaceutical compositions provided herein, and accordingly the pharmaceutical composition provided herein may be supplied in bulk or in unit dosage form depending on the intended administration route. For example, for oral, buccal, and sublingual administration, powders, suspensions, granules, tablets, pills, capsules, gelcaps, and caplets may be acceptable as solid dosage forms, and emulsions, syrups, elixirs, suspensions, and solutions may be acceptable as liquid dosage forms. For injection administration, emulsions and suspensions may be acceptable as liquid dosage forms, and a powder suitable for reconstitution with an appropriate solution as solid dosage forms. For inhalation administration, solutions, sprays, dry powders, and aerosols may be acceptable dosage form. For topical (including buccal and sublingual) or transdermal administration, powders, sprays, ointments, pastes, creams, lotions, gels, solutions, and patches may be acceptable dosage form. For vaginal administration, pessaries, tampons, creams, gels, pastes, foams and spray may be acceptable dosage form.

The quantity of active ingredient in a unit dosage form of composition is a therapeutically effective amount and is varied according to the particular treatment involved. As used herein, the term “therapeutically effective amount” refers to an amount of a molecule, compound, or composition comprising the molecule or compound to treat, ameliorate, or prevent an identified disease or condition, or to exhibit a detectable therapeutic or inhibitory effect. The effect can be detected by any assay method known in the art. The precise effective amount for a subject will depend upon the subject’s body weight, size, and health; the nature and extent of the condition; the rate of administration; the therapeutic or combination of therapeutics selected for administration; and the discretion of the prescribing physician. Therapeutically effective amounts for a given situation can be determined by routine experimentation that is within the skill and judgment of the clinician.

In some embodiments, the pharmaceutical compositions of the present disclosure may be in a form of formulation for oral administration.

In certain embodiments, the pharmaceutical compositions of the present disclosure may be in the form of tablet formulations. Suitable pharmaceutically acceptable excipients for a tablet formulation include, for example, inert diluents such as lactose, sodium carbonate, calcium phosphate or calcium carbonate, granulating and disintegrating agents such as corn starch or algenic acid; binding agents such as starch; lubricating agents such as magnesium stearate, stearic acid or talc; preservative agents such as ethyl or propyl p-hydroxybenzoate, and anti-oxidants, such as ascorbic acid. Tablet formulations may be uncoated or coated either to modify their disintegration and the subsequent absorption of the active ingredient within the gastrointestinal tract, or to improve their stability and/or appearance, in either case using conventional coating agents and procedures well known in the art.

In certain embodiments, the pharmaceutical compositions of the present disclosure may be in a form of hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin, or olive oil.

In certain embodiments, the pharmaceutical compositions of the present disclosure may be in the form of aqueous suspensions, which generally contain the active ingredient in finely powdered form together with one or more suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as lecithin or condensation products of an alkylene oxide with fatty acids (for example polyoxethylene stearate) , or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives (such as ethyl or propyl p-hydroxybenzoate, anti-oxidants (such as ascorbic acid) , coloring agents, flavoring agents, and/or sweetening agents (such as sucrose, saccharine or aspartame) .

In certain embodiments, the pharmaceutical compositions of the present disclosure may be in the form of oily suspensions, which generally contain suspended active ingredient in a vegetable oil (such as arachis oil, olive oil, sesame oil or coconut oil) or in a mineral oil (such as liquid paraffin) . The oily suspensions may also contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set out above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

In certain embodiments, the pharmaceutical compositions of the present disclosure may be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, such as olive oil or arachis oil, or a mineral oil, such as for example liquid paraffin or a mixture of any of these. Suitable emulsifying agents may be, for example, naturally-occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soya bean, lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides (for example sorbitan monooleate) and condensation products of the said partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening, flavoring and preservative agents.

In certain embodiments, the pharmaceutical compositions provided herein may be in the form of syrups and elixirs, which may contain sweetening agents such as glycerol, propylene glycol, sorbitol, aspartame or sucrose, a demulcent, a preservative, a flavoring and/or coloring agent.

In some embodiments, the pharmaceutical compositions of the present disclosure may be in a form of formulation for injection administration.

In certain embodiments, the pharmaceutical compositions of the present disclosure may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents, which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1, 3-butanediol or prepared as a lyophilized powder. Among the acceptable vehicles and solvents that may be employed are water, Ringer’s solution and isotonic sodium chloride solution. In addition, sterile fixed oils may conventionally be employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid may likewise be used in the preparation of injectables.

In some embodiments, the pharmaceutical compositions of the present disclosure may be in a form of formulation for inhalation administration.

In certain embodiments, the pharmaceutical compositions of the present disclosure may be in the form of aqueous and nonaqueous (e.g., in a fluorocarbon propellant) aerosols containing any appropriate solvents and optionally other compounds such as, but not limited to, stabilizers, antimicrobial agents, antioxidants, pH modifiers, surfactants, bioavailability modifiers and combinations of these. The carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (Tweens, Pluronics, or polyethylene glycol) , innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols.

In some embodiments, the pharmaceutical compositions of the present disclosure may be in a form of formulation for topical or transdermal administration.

In certain embodiments, the pharmaceutical compositions provided herein may be in the form of creams, ointments, gels and aqueous or oily solutions or suspensions, which may generally be obtained by formulating an active ingredient with a conventional, topically acceptable excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

In certain embodiments, the pharmaceutical compositions provided herein may be formulated in the form of transdermal skin patches that are well known to those of ordinary skill in the art.

Besides those representative dosage forms described above, pharmaceutically acceptable excipients and carriers are generally known to those skilled in the art and are thus included in the present disclosure. Such excipients and carriers are described, for example, in “Remingtons Pharmaceutical Sciences” Mack Pub. Co., New Jersey (1991) , in “Remington: The Science and Practice of Pharmacy” , Ed. University of the Sciences in Philadelphia, 21st Edition, LWW (2005) , which are incorporated herein by reference.

In some embodiments, the pharmaceutical compositions of the present disclosure can be formulated as a single dosage form. The amount of the compounds provided herein in the single dosage form will vary depending on the subject treated and particular mode of administration.

In some embodiments, the pharmaceutical compositions of the present disclosure can be formulated so that a dosage of between 0.001-1000 mg/kg body weight/day, for example, 0.01-800 mg/kg body weight/day, 0.01-700 mg/kg body weight/day, 0.01-600 mg/kg body weight/day, 0.01-500 mg/kg body weight/day, 0.01-400 mg/kg body weight/day, 0.01-300 mg/kg body weight/day, 0.1-200 mg/kg body weight/day, 0.1-150 mg/kg body weight/day, 0.1-100 mg/kg body weight/day, 0.5-100 mg/kg body weight/day, 0.5-80 mg/kg body weight/day, 0.5-60 mg/kg body weight/day, 0.5-50 mg/kg body weight/day, 1-50 mg/kg body weight/day, 1-45 mg/kg body weight/day, 1-40 mg/kg body weight/day, 1-35 mg/kg body weight/day, 1-30 mg/kg body weight/day, 1-25 mg/kg body weight/day of the compounds provided herein, or a pharmaceutically acceptable salt thereof, can be administered. In some instances, dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect, provided that such larger doses are first divided into several small doses for administration throughout the day. For further information on routes of administration and dosage regimes, see Chapter 25.3 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board) , Pergamon Press 1990, which is specifically incorporated herein by reference.

In some embodiments, the pharmaceutical compositions of the present disclosure can be formulated as short-acting, fast-releasing, long-acting, and sustained-releasing. Accordingly, the pharmaceutical formulations of the present disclosure may also be formulated for controlled release or for slow release.

In a further aspect, there is also provided veterinary compositions comprising one or more molecules or compounds of the present disclosure or pharmaceutically acceptable salts thereof and a veterinary carrier. Veterinary carriers are materials useful for the purpose of administering the composition and may be solid, liquid or gaseous materials which are otherwise inert or acceptable in the veterinary art and are compatible with the active ingredient. These veterinary compositions may be administered parenterally, orally or by any other desired route.

The pharmaceutical compositions or veterinary compositions may be packaged in a variety of ways depending upon the method used for administering the drug. For example, an article for distribution can include a container having deposited therein the compositions in an appropriate form. Suitable containers are well known to those skilled in the art and include materials such as bottles (plastic and glass) , sachets, ampoules, plastic bags, metal cylinders, and the like. The container may also include a tamper-proof assemblage to prevent indiscreet access to the contents of the package. In addition, the container has deposited thereon a label that describes the contents of the container. The label may also include appropriate warnings. The compositions may also be packaged in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water, for injection immediately prior to use. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the kind previously described.

In another aspect, the present disclosure also provides a method of preparing the pharmaceutical composition of the present disclosure, comprising mixing the KRAS inhibitor or a pharmaceutically acceptable salt thereof and the ERK inhibitor or a pharmaceutically acceptable salt thereof to form a pharmaceutical composition.

In some embodiments, the KRAS inhibitor is a KRAS ^G12C inhibitor, and/or the ERK inhibitor is a dual ERK1/2 inhibitor.

In some embodiments, the KRAS inhibitor is selected from the group consisting of Sotorasib (AMG-510) , Adagrasib (MRTX849) ,

and a pharmaceutically acceptable salt thereof, and the ERK inhibitor is (R) -7- (3, 4- Difluorobenzyl) -6- (methoxymethyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one or a pharmaceutically acceptable salt thereof.

and a pharmaceutically acceptable salt thereof, and the ERK inhibitor is

.0.5 adipic acid.

EXAMPLES

The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. All specific compositions, materials, and methods described below, in whole or in part, fall within the scope of the present invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate specific embodiments falling within the scope of the invention. A person skilled in the art may develop equivalent compositions, materials, and methods without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures herein described while still remaining within the bounds of the present invention. It is the intention of the inventors that such variations are included within the scope of the invention.

In the following Examples, four compounds, i.e., Sotorasib (AMG-510) , Adagrasib (MRTX849) , Compound 33, and Compound 71, were used to evaluate the effect of the combination of an ERK inhibitor and a KRAS inhibitor. The information of the four compounds were listed in Table 1 below.

Table 1. Information of Tested Compounds

Example 1. Synthesis of KRAS Inhibitors AMG-510, MRTX849, Compound 33, and ERK Inhibitor Compound 71

1.1 Synthesis of KRAS Inhibitor AMG-510

AMG-510 was prepared by the method by referring to description in Compound (R) -38 of Brian A. Lanman, Jennifer R. Allen, John G. Allen, et al., Discovery of a Covalent Inhibitor of KRAS ^G12C (AMG 510) for the Treatment of Solid Tumors, J. Med. Chem. 2020, 63, 52-65.

1.2 Synthesis of KRAS Inhibitor MRTX849

MRTX849 was prepared by the method by referring to description in compound 20 of Jay B. Fel, John P. Fischer, Brian R. Baer, et al., Identification of the Clinical Development Candidate MRTX849, a Covalent KRAS ^G12C Inhibitor for the Treatment of Cancer, J. Med. Chem. 2020, 63, 13, 6679–6693.

1.3 Synthesis of KRAS Inhibitor Compound 33

Compound 33 was prepared by the following method by referring to the description in Example 32 of the specification of PCT/CN2021/098083, published as WO2021244603A1 on December 09, 2021, filed on June 03, 2021, the entirety of which is incorporated herein by reference.

Step 1: Synthesis of compound 33-3

To a solution of compound 33-1 (600 mg, 1.29 mmol, 1.0 eq. ) and compound 33-2 (274 mg, 1.55 mmol, 1.2 eq. ) in anhydrous DMF (8 mL) , was added DIEA (416 mg, 3.23 mmol, 2.5 eq. ) , followed by the addition of HATU (590 mg, 1.55 mmol, 1.2 eq.) . The mixture was stirred at 60 ℃ under argon atmosphere for 3 h. LCMS showed starting material was consumed and desired product formed. The reaction mixture was cooled to room temperature, diluted with water (30 mL) and extracted with EtOAc (30 mL × 3) . The combined organic fractions were washed with brine (30 mL) , dried over anhydrous Na ₂SO ₄ and concentrated. The residue was purified by silica column chromatography eluting with DCM/MeOH (10: 1, v/v) to obtain tert-butyl (3S, 5S) -4- (5-amino-6- ( (8-methylnaphthalen-1-yl) carbamoyl) -2- ( ( (S) -1-methylpyrrolidin-2-yl) methoxy) pyrimidin-4-yl) -3, 5-dimethylpiperazine-1-carboxylate (550 mg, 68 %yield, 33-3) .

LCMS (ESI, m/z) : [M+1] ⁺ = 624; RT = 1.429 min.

Step 2: Synthesis of compound 33-4

To a mixture of compound 33-3 (170 mg, 0.28 mmol, 1.0 eq. ) and pyridine (220 mg, 2.80 mmol, 10.0 eq. ) in ACN (4 mL) at an ice/MeOH bath under argon atmosphere was added a solution of TFAA (294 mg, 1.40 mmol, 5.0 eq. ) in ACN (1 mL) drop-wise. The mixture was stirred at about -5℃ for 30 min. LCMS showed starting material was consumed and desired product formed. The reaction mixture was cooled, quenched with aq. NaHCO ₃ (20 mL) and extracted with EA (30 mL × 3) . The combined organic fractions were dried over anhydrous Na ₂SO ₄ and concentrated to obtain crude product of tert-butyl (3S, 5S) -3, 5-dimethyl-4- (7- (8-methylnaphthalen-1-yl) -2- ( ( (S) -1-methylpyrrolidin-2-yl) methoxy) -8-oxo-6- (trifluoromethyl) -7, 8-dihydropyrimido [5, 4-d] pyrimidin-4-yl) piperazine-1-carboxylate (260 mg, 99 %yield, 33-4) , which was used directly for the next step.

LCMS (ESI, m/z) : [M+1] ⁺ = 682; RT = 1.589 min.

Step 3: Synthesis of compound 33-5

To a solution of compound 33-4 (260 mg, 0.38 mmol) in DCM (5 mL) was added TFA (1 mL) at room temperature, and the mixture was stirred at room temperature overnight. LCMS showed starting material was consumed and desired product formed. The reaction mixture was concentrated and the residue was treated with aq. NaHCO3 (sat. 20 mL) . The resulting mixture was extracted with DCM (20 mL × 3) . The combined organic fractions were dried over anhydrous Na2SO4 and concentrated to 8- ( (2S, 6S) -2, 6-dimethylpiperazin-1-yl) -3- (8-methylnaphthalen-1-yl) -6- ( ( (S) -1-methylpyrrolidin-2-yl) methoxy) -2- (trifluoromethyl) pyrimido [5, 4-d] pyrimidin-4 (3H) -one (180 mg, 81 %yield, 33-5) , which was used directly for the next step.

LCMS (ESI, m/z) : [M+1] ⁺ = 582; RT = 1.147 min.

Step 4: Synthesis of Compounds 33-a and 33-b

To a cooled (0 ℃) solution of compound 33-5 (180 mg, 0.31 mmol, 1.0 eq. ) and Et ₃N (94 mg, 0.93 mmol, 3.0 eq. ) in anhydrous DCM (4 mL) was added dropwise a solution of acryloyl chloride (41 mg, 0.46 mmol, 1.5 eq. ) in anhydrous DCM (2 mL) . After addition, the mixture was stirred at 0 ℃ for 30 min. LCMS showed starting material was consumed and desired product formed. Water (20 mL) was added and the organic layer was separated. The aqueous layer was extracted with DCM (20 mL × 3) . The combined organic fractions were dried over anhydrous Na ₂SO ₄ and concentrated. The residue was purified by prep-HPLC (ACN-H ₂O +0.1%HCOOH) and then SFC to obtain 33-a (2.4 mg, 1 %yield) and 33-b (18.5 mg, 9 %yield) .

33-a:

LCMS (ESI, m/z) : [M+1] ⁺ = 636; RT = 1.309 min;

¹H NMR (400 MHz, DMSO) δ 8.08 (d, J = 76.6 Hz, 2H) , 7.70 (d, J = 33.6 Hz, 2H) , 7.45 (d, J = 37.3 Hz, 2H) , 6.79 (s, 1H) , 6.23 (d, J = 15.5 Hz, 1H) , 5.78 (s, 1H) , 5.33 (s, 1H) , 4.31 (d, J = 65.3 Hz, 2H) , 4.03 (s, 3H) , 3.66 (d, J = 11.0 Hz, 2H) , 2.96 (s, 1H) , 2.59 (s, 1H) , 2.36 (s, 3H) , 2.23 (s, 4H) , 1.96 (s, 1H) , 1.67 (s, 3H) , 1.43 (s, 6H) .

¹⁹F NMR (400 MHz, DMSO) δ -63.57.

33-b:

LCMS (ESI, m/z) : [M+1] ⁺ = 636; RT = 1.316 min;

¹H NMR (400 MHz, DMSO) δ 8.17 (dd, J = 8.0, 1.5 Hz, 1H) , 7.97 (d, J = 8.0 Hz, 1H) , 7.72 –7.62 (m, 2H) , 7.53 –7.46 (m, 1H) , 7.39 (d, J = 7.0 Hz, 1H) , 6.79 (dd, J = 16.7, 10.4 Hz, 1H) , 6.22 (dd, J = 16.7, 2.3 Hz, 1H) , 5.77 (dd, J = 10.4, 2.2 Hz, 1H) , 5.32 (s, 1H) , 4.39 (dd, J = 10.8, 5.0 Hz, 1H) , 4.21 (dd, J = 10.8, 6.2 Hz, 1H) , 4.02 (t, J = 15.5 Hz, 3H) , 3.65 (dd, J = 14.4, 3.6 Hz, 1H) , 2.99 –2.93 (m, 1H) , 2.62 (dd, J =14.0, 5.9 Hz, 1H) , 2.36 (s, 3H) , 2.20 (s, 3H) , 2.00 –1.91 (m, 1H) , 1.72 –1.59 (m, 3H) , 1.40 (dd, J = 6.4, 4.2 Hz, 6H) .

¹⁹F NMR (400 MHz, DMSO) δ -63.53.

1.4 Synthesis of ERK Inhibitor Compound 71

Compound 71 was prepared by the method by referring to the description in Example 18 and Example 34 of the specification of WO2017080979A1.

¹H NMR (500 MHz, Methanol-d ₄, 27 ℃) δ 8.24 (1H, s) , 7.82 (1H, s) , 7.41 (1H, d) , 7.35 (1H, ddd) , 7.28 –7.20 (3H, m) , 6.31 (1H, d) , 5.18 (1H, d) , 4.52 –4.39 (3H, m) , 4.03 –4.00 (1H, m) , 3.73 (3H, s) , 3.46 –3.43 (1H, m) , 3.39 –3.35 (1H, m) , 3.23 (3H, s) , 2.53 (3H, s) , 2.30 (2H, m) , 1.63 (2H, m) .

Example 2. Biochemical Assays of Exemplary KRAS Inhibitors

2.1 Assay 1: KRAS G12C Nucleotide Exchange Assay

2.1.1 Materials and reagents

HEPES (Sigma, Cat. No. H3375-500g)

DMSO (Sigma, Cat. No. 34869-4L)

MgCl ₂ (Sigma, Cat. No. M2670-500 g)

GTP (Sigma, Cat. No. G8877)

GDP (Sigma, Cat. No. G7127)

MANT-GTP (SIGMA, 69244-1.5UMOL)

Glycerol (Sigma, Cat. No. G6279-1 L)

Tween-20 (Sigma, Cat. No. P2287-100 mL)

SOS1 Protein, aa564-1049, 6xHis tag (CYTOSKELETON, CS-GE02-XL)

EDTA, pH 8.0 (Gibco, 15575-038, 100 mL)

Pierce Coomassie (Bradford) Protein Assay Kit (Thermo Pierce, 23200)

Illustra NAP-5 Columns (GE, 17085301)

384-well plate (Corning, Product Number 3573)

KRas (1-169) G12C protein

SOS1 (594-1049) protein

SOS1 (564-1049) protein

KRAS G12C and SOS1 proteins were packed in 5 UL /tube or 20 UL /tube, and frozen in -80℃ refrigerator.

2.1.2 Experiment Method

1. Buffer preparation:

1×Loading buffer: 20 mM HEPES, pH 7.5, 50 mM NaCl, 0.5 mM MgCl2, 1 mM DTT, 5 mM EDTA

1×Equilibration buffer: 20 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM MgCl2, 1 mM DTT

1× Assay buffer: 20 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM MgCl2, 0.01%Tween-20, 1 mM DTT

2. Load mant GDP to KRAS G12C:

a. A mixed solution of 100 UL mant GDP and KRAS G12C was prepared with 1 × loading buffer: 60 um KRAS G12C, 600 um mant GTP, incubated at room temperature for 60 minutes, and the reaction was carried out in dark conditions.

b. Add 1 uL 1 m MgCl2 (final concentration 10 mm) to stop the reaction, mix the solution upside down in the centrifuge tube, centrifugate for 3-5 seconds, and incubate at room temperature for 30 minutes.

c. At the same time of incubation for 30 minutes, balance nap-5 column with 10 ml 1x equipment buffer until no liquid drops.

d. Drop the mixed solution of 100 uL mant GDP and KRAS G12C into the center of nap-5 column. After the sample completely enters nap-5 column, add 400 UL 1x equipment buffer until no liquid drops.

e. Add 500 ul1x equipment buffer for elution and collect eluant.

f. Determination of KRAS G12C mant GDP with Bradford protein quantitative Kit.

3. Nucleotide exchange experiment:

a. Transfer 50 NL DMSO /compound to 384 well plate with echo550.

b. 10 uL enzyme mix was added into 384 pore plate and incubated with DMSO /compound for 15 min.

c. Initial reaction with 10 UL Sos1 /GTP mix.

d. Immediately after reaction, read ex360 /em440 fluorescence value in kinetic mode with Nivo.

4. Data analysis:

a. Use Graphpad software to process data and draw pictures.

b. K value is obtained in Graphpad software by fitting One phase experimental deck model.

c. Z’=1-3* (SdKmax+SdKmin) / (AveKmax-AveKmin)

d. In%is calculated by the following formula:

Inh%= (Kmax-Ksample) / (Kmax-Kmin) *100

Max: KRAS-mGDP + SOS1+ GTP

Min: KRAS-mGDP + buffer

The results for exemplary compounds of Formula (I) are shown in Table 2. For the other Example compounds for which the results are not shown, all have an IC ₅₀ against KRAS G12C of no more than 60 μM. Some of these compounds have an IC ₅₀ against KRAS G12C of no more than 50 μM, some no more than 40 μM, some no more than 30 μM, some no more than 20 μM, or no more than 10 μM, or no more than 5 μM, or no more than 4 μM, or no more than 3 μM, or no more than 2 μM, or no more than 1 μM, no more than 500 nM, no more than 400 nM, no more than 300 nM, no more than 200 nM, or even no more than 100 nM.

Table 2. IC ₅₀ values of Exemplary Compounds

*Compound 31 refers to the mixture of Compound 31-a and Compound 31-b, which are two different isomers of Compound 31. This rule is applicable to the other compounds which have chiral center (s) throughout this disclosure, for example, Compound 33 refers to the mixture of Compound 33-a and Compound 33-b.

2.2 Assay 2: KRAS GDP FI assay

1. Prepare compound dilution plate.

2. Transfer Inhibitor/DMSO to assay plate by Echo.

3. Prepare 1x assay buffer.

4. Prepare KRAS G12C mix &SOS1 mix &GTP mix &detection reagent mix.

5. Add KRAS G12C mix, SOS1 mix, GTP mix.

6. Add detection reagent mix to assay plate.

7. Kinetic reading with Ex580/Em620 for 120min.

The results for exemplary compounds of Formula (I) are shown in Table 3.

Table 3. IC ₅₀ values of Exemplary Compounds

2.3 Assay 3: Tumor Cell Anti-proliferation Assay (CTG Assay)

Tested tumor cell lines (MIA-PaCa-2, NCI-H358, and A549) were seeded to the 96-well plate for overnight, then cells were treated with the test compound at 9 serially diluted concentrations in triplicate. After 3-days incubation with the test compound, the CTG assay was performed to evaluate the IC ₅₀. The 3 cell lines were tested in the same manner. Cisplatin were used as the positive control.

2.3.1 Materials and reagents

RPMI-1640 (Hyclone, Cat. No.: SH30809.01)

DMEM medium (Hyclone, Cat. No.: SH30243.01)

Ham’s F12K (Gbico, Cat. No.: 21127-022)

FBS (Cat. No. 10099-141, Gibco)

Luminescent Cell Viability Assay (Cat. No. G7572, Promega. Stored at -20℃) .

96-Well Plate, With Lid, White, Flat Bottom, TC-Treated, Polystyrene (Cat. No.: 3610,

)

0.25%Trypsin-EDTA (Cat. No. 25200072, Gibco)

2.3.2 Equipment

BMRP004; CO2 Incubator, SANYO Electric Co., Ltd (02100400059) .

Reverse microscope, Chongguang XDS-1B, Chongqing Guangdian Corp. (TAMIC0200)

Envision 2104 Multi Label Reader, PerkinElmer, USA (TAREA0011)

Vi-Cell XR, Beckman Coulter (TACEL0030)

2.3.3 Method

Day -1: Cell plating for cell lines

1. Adjust the cell concentration to the appropriated number with the medium, and for each well of a 96-well plate, add 90 μl cell suspensions (Cell concentration will be adjusted according to the data base or density optimization assay) .

2. Incubate the plates for overnight in humidified incubator at 37℃ with 5% CO ₂.

Day 0: T0 plate reading and compound treatment

3. Add 10 μL culture medium to each well of plate A for T0 reading.

4. Equilibrate the plate and its content at RT for approximately 30 min.

5. Add 50 μL

Reagent to each well for T0 reading.

6. Mix contents for 2 minutes on an orbital shaker to facilitate cell lysis.

7. Allow the plate to incubate at room temperature for 10 minutes to stabilize the luminescent signal. Note: Uneven luminescent signal within standard plates can be caused by temperature gradients, uneven seeding of cells or edge effects in multiwall plates.

8. Place a black BackSeal sticker to the bottom of each plate.

9. Record luminescence using an Envision Multi Label Reader.

10. Dilute the test compound and the positive control (Cisplatin) . Add 10 μL of 10X test compound working solutions into the corresponding wells. Incubate the test plates in the humidified incubator at 37℃ with 5%CO2.

Day 3: Plate reading for 3-day assay

11. Monitor under the microscope to make sure that the cells in control wells are healthy.

12. After three days incubation, add 50 μL

Reagent to each well.

13. Mix contents for 2 minutes on an orbital shaker to facilitate cell lysis.

14. Allow the plate to incubate at room temperature for 10 minutes to stabilize the luminescent signal.

15. Note: Uneven luminescent signal within standard plates can be caused by temperature gradients, uneven seeding of cells or edge effects in multiwall plates.

16. Place a black BackSeal sticker to the bottom of each plate.

17. Record luminescence using an Envision Multi Label Reader.

2.3.4 Data analysis

The data were displayed graphically using GraphPad Prism 5.0. In order to calculate IC ₅₀, a dose-response curve was fitted using nonlinear regression model with a sigmoidal dose response. The formula of the surviving rate is shown below, and the IC ₅₀ was automatically produced by GraphPad Prism 5.0.

The surviving rate (%) = (LumTest compound -LumMedium control) / (LumNone treated-LumMedium control) ×100%.

LumNone treated-LumMedium control is set as 100%and LumMedium control is set as 0%surviving rate. T0 value will be presented as percentage of LumNone treated.

Table 4 provides the results for exemplary compounds of Formula (I) .

Table 4. IC ₅₀ values of Exemplary Compounds

Example 3. Pharmacokinetics Study of Exemplary KRAS Inhibitors

The purpose of this study is to determine the pharmacokinetics parameters in plasma of compounds in ICR mice following intravenous or oral administration.

3.1 Test Article Preparation

The formulations were based on sponsor's recommendation and will be prepared by the Testing Facility.

Vehicles: 60%PEG400 + 10%Ethanol + 30%water (pH 7-8)

3.2 Test System

Species and Strain: ICR Mice (Male)

Source: Sino-British SIPPR Lab Animal Ltd, Shanghai

Number of Animals: Ordered: 8; Needed: 6

3.3 Study Design

*The animals were fasted prior to oral administration. Food supply to the animals dosed orally were resumed 4 hours post-dose.

3.4 Administration

The test article was be administered via a single IV or PO dosing.

3.5 Collection Intervals

IV group: Post-dose at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h and 24 h.

PO group: Post-dose at 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h and 24 h.

30～ 40 μL/sample. Samples were be placed in tubes containing heparin sodium and stored on ice until centrifuged.

3.6 Analysis Procedure

The PK blood samples were centrifuged at approximately 6800G for 6 minutes at 2-8℃ and the resulting plasma was transferred to appropriately labeled tubes within 2 hour of blood collection/centrifugation and stored frozen at approximately -70℃.

Method development and biological samples analysis for the test articles (Sodium heparin anticoagulant) were be performed by Testing Facility by means of LC-MS/MS. The analytical results were be confirmed using quality control samples for intra-assay variation. The accuracy of >66.7%of the quality control samples were be between 80 -120%of the known value (s) .

3.7 Pharmacokinetics Analysis

Standard set of parameters including Area Under the Curve (AUC (0-t) and AUC (0-∞) ) , elimination half-live (T _1/2) , maximum plasma concentration (Cmax) and time to reach maximum plasma concentration (Tmax) and other parameters were be calculated using Phoenix WinNonlin 7.0 (Pharsight, USA) by the Study Director.

Table 5 provides the results for exemplary compounds of Formula (I) .

Table 5. Pharmacokinetic Analysis Results of Exemplary Compounds

Example 4. In Vitro Study

4.1 Materials

NCI-H358 cell line, a KRAS G12C mutant non-small cell lung cancer cell line, was purchased from ATCC. The four tested compounds, i.e., Sotorasib (AMG-510) , Adagrasib (MRTX849) , Compound 33, and Compound 71, were prepared according to the methods described in Example 1.

4.2 Methods

Firstly, the in vitro 50%inhibition concentration (IC ₅₀) of all four compounds were determined in NCI-H358 cell line, using CellTiter-Glo luminescent cell viability assay. Briefly, 4×10 ³ cells/well NCI-H358 cells were seeded on a 96-well plate, incubated overnight at 37℃. AMG-510, MRTX849, Compound 33 or Compound 71 were added, respectively, at top concentration of 10 μM, 3 times dilution. The cells were incubated for 72 hrs in a CO ₂ incubator at 37℃. 50 μL CellTiter-Glo were added to each well. The contents were mixed for 5 mins on an orbital shaker to induce cell lysis. The plate was allowed to incubate at room temperature for 10 mins to stabilize luminescent signal. Then, the luminescence signal on a microplate reader was recorded. The data was displayed graphically using GraphPad Prism. In order to calculate absolute IC ₅₀, a dose-response curve was fitted using nonlinear regression model with a sigmoidal dose response. The formula for calculating surviving rate is shown below and the absolute IC ₅₀ values were calculated according to the dose-response curve generated by GraphPad Prism 5.0.

The surviving rate (%) = (LumTest article-LumMedium control) / (LumNone treated-LumMedium control) ×100%.

LumTest article: luminescence signal of tested compound

LumMedicum control: luminescence signal of medium-only control

LumNone treated: luminescence signal of vehicle control

Then, synergistic effects of the Compound 71+AMG-510 combo and Compound 71+MRTX849 combo were tested on NCI-H358 cells. The synergistic effects were calculated by the Bliss Model (Antiviral Res. Oct-Nov 1990; 14 (4-5) : 181-205) . The matrix of combo-concentration was based on the single agent IC ₅₀ shown in Figure 2. Top concentration: 4×IC ₅₀ in medium with 2-fold serial dilutions to achieve 6 dose levels.

4.3 Results

As shown in Figure 2, all tested compounds showed in vitro anti-tumor effect on NCI-H358 cells, and the IC ₅₀ value for AMG-510, MRTX849, Compound 33, and Compound 71 alone was 0.0135μM, 0.0324μM, 0.06066μM, and 0.1666μM, respectively.

The inhibitory effects (%growth inhibition) of the Compound 71+AMG-510 combo and the Compound 71+MRTX849 combo on NCI-H358 cells were shown in Table 6 and Table 7, respectively. As shown in Table 6 and Table 7, the combination of Compound 71 with a KRAS inhibitor (e.g., AMG-510, MRTX849) showed enhanced anti-tumor effects compared to mono-therapies.

The synergy score values for the Compound 71+AMG-510 combo on NCI-H358 cells were shown in Figure 3 (the values were derived from the data shown in Table 6) , and the synergy score values for the Compound 71+MRTX849 combo on NCI-H358 cells were shown in Figure 4 (the values were derived from the data shown in Table 7) . As shown in Figure 3 and Figure 4, synergism (i.e., synergy score> 0) has been observed for multiple combination doses for Compound 71+AMG-510 combo and Compound 71+MRTX849 combo; and the average synergy scores were larger than 0 for both combinations (0.810 for Compound 71+AMG-510 combo, 1.940 for Compound 71+MRTX849 combo) , suggesting a synergistic effect by combining ERK (e.g., ERK1/2) inhibition by Compound 71 and KRAS inhibition (e.g., KRAS G12C inhibition) .

Table 6. Inhibitory effects (%growth inhibition) of the Compound 71+AMG-510 combo on NCI-H358 cells.

Table 7. Inhibitory effects (%growth inhibition) of the Compound 71+MRTX849 combo on NCI-H358 cells.

Example 5. In Vivo Study

5.1 Methods

5.1.1 Cell Culture

The NCI-H358 tumor cells were cultured in RPMI-1640 medium supplemented with 10%heat inactivated fetal bovine serum, 100U/ml penicillin and 100 μg/ml streptomycin at 37℃ in an atmosphere of 5%CO2 in air. The tumor cells were routinely sub-cultured 2 to 3 times weekly. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation.

5.1.2 Tumor Inoculation, grouping and treatment

Each mouse was inoculated subcutaneously at the right flank with the NCI-H358 tumor cells (1x10 ⁷ per mouse) in 0.1 mL RPMI-1640 medium with 50%matrigel for tumor development. 54 animals were randomized using block randomization by Excel based upon their tumor volumes, which ensures that all the groups are comparable at the baseline. The tumor bearing mice were treated with vehicle control, Compound 71 (25mg/kg, PO, QD) , Compound 33 (10mg/kg, PO, QD) or the Compound 71+Compound 33 combo, respectively, for 10 days.

5.1.3 Tumor Measurements

Tumor volume was measured twice weekly in two dimensions using a caliper, and tumor growth inhibition (TGI) was evaluated compared with vehicle control group. The tumor volume was expressed in mm ³ using the formula: V = 0.5 a x b ², where a and b are the long and short diameters of the tumor, respectively.

5.2 Results

The anti-tumor activity of NCI-H358 subcutaneous xenograft model in BALB/c nude mice for each group was shown in Figure 5. As shown in Figure 5, all treatment groups showed tumor growth inhibition compared with vehicle control. The Compound 71+Compound 33 combo group showed an enhanced tumor growth inhibition of 104%, compared with monotherapy of Compound 71 (TGI: 87.43%) or Compound 33 (TGI: 70.76%) on day 10, suggesting an in vivo synergism by combining ERK (e.g., ERK1/2) inhibition by Compound 71 and KRAS (e.g., KRAS G12C) inhibition.

Example 6. Treating KRAS G12C inhibitor monotherapy resistant tumor cells and/or in vivo models by Compound 71, KRAS G12C inhibitors and Compound 71+KRAS G12C inhibitors

Compound 71, AMG-510, Compound 33 and Compound 71+AMG-510 combo were tested for the in vitro anti-proliferation effect in MIA-PaCa-2 cell line and in AMG510-R-xMIA-PaCa-2 cell line (which is a AMG510-resistant model) , by cell viability assay.

6.1 Materials

6.1.1 Cell lines and propagation

The cell lines used in this study are shown in Table 8 below.

Table 8. Cell Lines and Propagation

Note:

AA: Antibiotic-antimycotic

6.1.2 Culture medium and reagents

The culture medium and reagents used in this study are shown in Table 9 below.

Table 9. Medium and reagents

Culture medium or reagent	Manufacturer	Catalog#
DMEM/F12	GIBCO	11330-032
Dulbecco’s PBS	Thermo	SH30028.02B
FBS	Cellmax	SA211.02
Antibiotic-antimycotic	GIBCO	15240-062
0.25%Trypsin	GIBCO	25200072
DMSO	SIGMA	D2650

6.1.3 Assay plates

The assay plates used in this study are as follows.

● Assay plate: Ultra Low Cluster, 96 Well, with Lid Round Bottom, Ultra Low Attachment, Sterile, Polystyrene (Corning-7007) .

● Assay plate: Greiner

96 well plates with black wells flat bottom (with lid and micro-clear bottom) , #655090.

● Compound plate: Pinchbar Design Polypropylene, V-shape well bottoms, Sterile (Nunc-442587)

6.1.4 Cell Viability Assay Regents and Instrument

The cell viability assay regents and instruments used in this study are as follows.

● Promega CellTiter-Glo 3D Luminescent Cell Viability Assay kit (Promega #G9683) .

●

Multi-mode Plate Reader (EnVision 2104-10) .

6.2 Experimental design

The plating of cells and compounds treatments are shown below in the plate maps of Table 10 (monotherapy) and Table 11 (combination therapy) , respectively.

Table 10. Monotherapy’s plate map for cell viability assay (μM) .

Note:

Control: Solvent control, only cells, without drugs.

Blank: Blank control, only cell culture medium, without cells.

PBS: Phosphate buffered saline.

6.3 Experimental methods and procedures

6.3.1 Cell culture

The cancer cell lines were maintained in culture conditions as shown in Table 8 above at 37℃ in an atmosphere of 5%CO ₂ in air. The tumor cells were routinely subcultured. The cells growing in an exponential growth phase were harvested and counted for plating.

6.3.2 Cell plating

The cell plating was performed by the following steps:

● The cells were counted by haemocytometer with Trypan blue staining, and then the cell concentrations were adjusted to proper density (5000 cells/96-well for each cell line) .

● 135 μL of cell suspension was plated into the ULA plate according to the plate maps of Table 10 and Table 11 above. 135 μL of assay medium was added into the Blank wells.

● Immediately after plating, the ULA plate was centrifuged for 10 minutes, 1000 RPM, at room temperature. Be careful not to disturb the plate after this centrifugation step.

● The plates were incubated at 37℃, 5%CO ₂, 95%air and 100%relative humidity overnight.

6.3.3 Compound stock plate preparation

Compound stock plates (400X stock plates) were prepared as follows: serially dilute the stock solution from highest concentration down to lowest in DMSO according to the plate maps of Table 12 and Table 13 below.

Table 12. Monotherapy’s plate layout (μM) of 400X stock.

	1	2	3	4	5	6	7	8	9	10
A	4000	1333.3	444.44	148.15	49.38	16.46	5.49	1.83	0.61	Compound 71
B	4000	1333.3	444.44	148.15	49.38	16.46	5.49	1.83	0.61	Compound 33
C	4000	1333.3	444.44	148.15	49.38	16.46	5.49	1.83	0.61	AMG-510

Table 13. Combination’s plate layout (μM) of 400X stock.

	1	2	3	4	5	6	7
A	416	208	104	52	26	13	Compound 71
B	560	280	140	70	35	17.5	AMG-510

6.3.4 Compound plate (10X) preparation and compound treatment (Monotherapy)

The compound plate (10X) preparation and compound treatment for monotherapy were performed as follows.

● 10X concentrate compound plate preparation: 78 μL of assay medium was added into each well of the V-bottom plate; then 2 μL of the stock compound solution of each concentration was transferred from the stock plate (400X stock) . 2 μL of DMSO was added into the Blank and Control wells. Pipette up and down to mix well. This V-plate was designated as the 10X concentrate compound plate.

● Compound treatment: the ULA plate was removed from the incubator and 15 μL of fresh culture media containing test compound at 10x the desired final concentration was respectively added to the cell culture plate as shown in Table 10 above. 15 μL of the DMSO-medium was added into the Blank and Control wells. The final DMSO concentration is 0.25%.

● The assay plate was returned into incubator and incubated for 120 h.

6.3.5 Compound plate (20X) preparation and compound treatment (Combination therapy)

The compound plate (20X) preparation and compound treatment for combination therapy were performed as follows.

● 20X concentrate compound plate preparation: 76 μL of assay medium was added into each well of the V-bottom plate; then 4 μL of the stock compound solution of each concentration was transferred from the stock plate (400X stock) . 4 μL of DMSO was added into the Blank and Control wells. Pipette up and down to mix well. This V-plate was designated as the 20X concentrate compound plate.

● Compound treatment: the ULA plate was removed from the incubator and 7.5 μL of fresh culture media containing test compound at 20x compound working solution were respectively added to the cell culture plate as shown in Table 11 above. The desired final concentration of the compound is 10x. 15 μL of the DMSO-medium was added into the Blank and Control wells. The final DMSO concentration is 0.25%.

● The assay plate was returned into incubator and incubated for 5 days.

● Spheroids were visually monitored daily until endpoint collection.

6.3.6 CellTiter-Glo luminescent cell viability assay

An ATP endpoint viability assay (CellTiter-Glo 3D, Promega #G9683) was performed following the manufacturer’s instructions below:

● Thaw the CellTiter-Glo buffer and equilibrate to room temperature prior to use.

● Equilibrate the lyophilized CellTiter-Glo Substrate to room temperature prior to use.

● Prepare 3D celltiter-glo working fluid.

● Slow eddy oscillation causes full dissolution.

● Take out the cell culture plate and let it balance to room temperature for 30 minutes.

● Add a volume of CellTiter-Glo 3D reagent equal to the volume of cell culture medium present in each well (75 μL) . Cover the cell plate with aluminum foil to protect it from light.

● Shake the plate for five minutes.

● Mix the well contents by carefully pipetting up and down 10 times. Ensure the spheroids are fully dissociated before continuing. Transfer the lysates into black wells flat bottom plates (#655090) , and incubate for an additional 25 minutes at room temperature.

● Record luminescence on the 2104 EnVision plate reader.

6.3.7 Data analysis

Inhibition rate (IR) of the tested compounds was determined by the following formula:

IR (%) = (1– (RLU compound –RLU blank) / (RLU control –RLU blank) ) *100%.

The inhibition rates of different doses of compounds were calculated in Excel file, and then were used to plot inhibition curve and evaluate related parameters, such as Bottom, Top and IC ₅₀. The data were interpreted by GraphPad Prism.

Combination index was calculated using CompuSyn software.

6.4 Results

The inhibition parameters, including bottom (%) , top (%) and IC ₅₀, of each monotherapy were shown in Table 14 below. The inhibition proliferation curves of Compound 71, Compound 33 and AMG-510 monotherapy in different cell lines in cell viability assay were shown in Figures 6A and 6B. The inhibitory proliferation effects of Compound 71 combined with AMG-510 in AMG510-R-xMIA-PaCa-2 (CP2) tumor cell line were shown in Table 15 and Table 16 below. The inhibitory proliferation curve of Compound 71 combined with AMG-510 in AMG510-R-xMIA-PaCa-2 (CP2) cell line was shown in Figure 7.

Table 14. The inhibition parameters in cell viability assay (monotherapy)

Table 15. The inhibition parameters in cell viability assay (combination therapy)

Table 16. Combined exponential parameters of cell activity experiments

In summary, the anti-proliferation effects of Compound 71, Compound 33 and AMG-510 in MIA-PaCa-2 and AMG510-R-xMIA-PaCa-2 (CP2) pancreatic cancer lines were evaluated by cell viability assay. In the two kinds of cells, the test compounds Compound 71 and Compound 33 showed good inhibitory effect on cell proliferation and had drug concentration-dependent characteristics. In MIA-PaCa-2 human pancreatic cancer cell, the test compounds Compound 71 and Compound 33 exhibited the same anti-proliferation effect as the positive compound AMG-510; in AMG510-R-xMIA-PaCa-2 (CP2) cell line, the test compounds showed comparable inhibitory effect to the positive compound AMG-510.

The combined anti-proliferative effect of Compound 71+AMG-510 combo in AMG510-R-xMIA-PaCa-2 (CP2) pancreatic cancer cell line was evaluated by cell viability assay. The results showed that the inhibitory effects of Compound 71 and AMG-510 when applied independently were consistent with the historical data, and the combination therapy of the two had a drug concentration-dependent effect on the proliferation inhibition of AMG510-R-xMIA-PaCa-2 (CP2) cells. At the same time, the combination of the two drugs had better inhibitory effect than the single drug. Among them, Compound 71 generally has synergistic effects (CI<0.9) or additive effects (0.9<CI<1.1) when combined with different concentrations of AMG-510.

The monotherapy of the test compounds Compound 71 and AMG-510 inhibited the cell proliferation of drug-resistant human pancreatic cancer cell line AMG510-R-xMIA-PaCa-2 (CP2) . The combination therapy of Compound 71 and AMG-510 demonstrated anti-tumor activity and synergistic effects in inhibiting the proliferation of the AMG-510 resistant cell line AMG510-R-xMIA-PaCa-2 (CP2) cell line.

Compound 71, other KRAS G12C inhibitors (e.g., MRTX849) and Compound 71+KRAS G12C inhibitors combo (e.g., Compound 71+ MRTX849 combo, Compound 71+Compound 33 combo) are tested for the in vitro (2D or 3D culture) anti-tumor effect in MIA-PaCa-2 cell line and in AMG510-R-xMIA-PaCa-2 cell line. The in vitro assay is performed by steps similar to the methods described in Example 6, except for different test compounds.

Compound 71, KRAS G12C inhibitors (e.g., AMG-510, MRTX849, Compound 33) and Compound 71+KRAS G12C inhibitors combo (e.g., Compound 71+AMG-510 combo, Compound 71+ MRTX849 combo, Compound 71+Compound 33 combo) are also tested for the in vivo anti-tumor effect in MIA-PaCa-2 cell line and in AMG510-R-MIA-PaCa-2 cell line. The in vivo assay is performed by steps similar to the methods described in Example 5.1, except for different cell lines and/or test compounds.

Claims

A method of treating, preventing, or ameliorating a disease or disorder associated with ERK and/or KRAS in a subject in need thereof, comprising administering to the subject an effective amount of an ERK inhibitor or a pharmaceutically acceptable salt thereof, in combination with an effective amount of a KRAS inhibitor or a pharmaceutically acceptable salt thereof.
A method of treating, preventing, or ameliorating a disease or disorder associated with ERK and/or KRAS in a subject who is relapsed from or resistant to treatment of a KRAS inhibitor, comprising administering to the subject an effective amount of an ERK inhibitor or a pharmaceutically acceptable salt thereof, optionally in combination with an effective amount of a KRAS inhibitor or a pharmaceutically acceptable salt thereof.
A method of treating, preventing, or ameliorating a disease or disorder associated with ERK and/or KRAS in a subject who is relapsed from or resistant to treatment of an ERK inhibitor, comprising administering to the subject an effective amount of a KRAS inhibitor or a pharmaceutically acceptable salt thereof, optionally in combination with an effective amount of an ERK inhibitor or a pharmaceutically acceptable salt thereof.
A method of improving therapeutic response to a disease or disorder associated with ERK and/or KRAS in a subject previously received treatment of a KRAS inhibitor, comprising administering to the subject an effective amount of an ERK inhibitor or a pharmaceutically acceptable salt thereof, optionally in combination with an effective amount of a KRAS inhibitor or a pharmaceutically acceptable salt thereof.
A method of improving therapeutic response to a disease or disorder associated with ERK and/or KRAS in a subject previously received treatment of an ERK inhibitor, comprising administering to the subject an effective amount of a KRAS inhibitor or a pharmaceutically acceptable salt thereof, optionally in combination with an effective amount of an ERK inhibitor or a pharmaceutically acceptable salt thereof.
The method of any one of the preceding claims, wherein the disease or disorder is associated with an increased activity or level of ERK and/or KRAS and/or an activated MAPK pathway.
The method of claim 6, wherein the disease or disorder is cancer.
The method of claim 7, wherein the disease or disorder is a drug-resistant cancer.
The method of claim 8, wherein the disease or disorder is a cancer refractory to monotherapy of a KRAS inhibitor or an ERK inhibitor.
The method of claim 7, wherein the disease or disorder is a metastatic cancer.
The method of any one of claims 7 to 10, wherein the cancer is associated with a KRAS mutation (for example, a KRAS-mutated cancer) .
The method of claim 11, wherein the KRAS mutation is selected from KRAS ^G12C/D/V, KRAS ^G13C/D, or KRAS ^Q61L/H/R.
The method of claim 12, wherein the KRAS mutation is KRAS ^G12C.
The method of any one of the claims 7-13, wherein the cancer is selected from the group consisting of lung cancer, non-small-cell lung cancer (NSCLC) , small cell lung cancer (SCLC) , bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, uterine cancer, liver cancer, cholangiocarcinoma, sarcoma, hematological cancer, colorectal cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin’s Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, chronic or acute leukemia, lymphocytic lymphomas, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS) , primary CNS lymphoma, spinal axis tumors, brain stem glioma, MYH associated polyposis, or pituitary adenoma.
The method of any one of the preceding claims, wherein the subject is determined to have de novo or acquired resistance to the KRAS inhibitor or a pharmaceutically acceptable salt thereof.
The method of any one of the preceding claims, wherein the subject is determined to have de novo or acquired resistance to the ERK inhibitor or a pharmaceutically acceptable salt thereof.
The method of any one of the preceding claims, wherein the KRAS inhibitor is a chemotherapeutic agent, an antibody, or an antigen-binding fragment thereof.
The method of claim 17, wherein the KRAS inhibitor is a small molecule compound.
The method of any one of the preceding claims, wherein the KRAS inhibitor is a KRAS ^G12C inhibitor.
The method of any one of the preceding claims, wherein the KRAS inhibitor is selected from the group consisting of Sotorasib (AMG-510) , Adagrasib (MRTX849) , D-1553, JDQ443, LY3499446, RG6330, ARS-3248, JAB-21822, BPI-421286, GH35, RMC-6291, MRTX1257, ARS-853, AU-8653, GF-105, AU-10458, LY3537982, WDB178, RM-007, LC-2, RM-018, ARS-1620, RM-032, BI 1823911, APG-1842, JAB-21000, ATG-012, and YL-15293.
The method of claim 20, wherein the KRAS inhibitor is Sotorasib (AMG-510) or Adagrasib (MRTX849) .
The method of any one of claims 1-19, wherein the KRAS inhibitor is a compound of Formula (I) or a pharmaceutically acceptable salt thereof:

wherein

Ring A is selected from the group consisting of saturated or partially unsaturated cycloalkyl, saturated or partially unsaturated heterocyclyl, and heteroaryl;

L ¹ is a bond, O, S or N (R ^a) ;

L ² is selected from the group consisting of a bond, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl;

R ¹ is selected from the group consisting of alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, saturated or partially unsaturated cycloalkyl, saturated or partially unsaturated heterocyclyl, aryl, and heteroaryl, wherein each of alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted with one or more R ^b;

R ² is selected from the group consisting of H, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, saturated or partially unsaturated cycloalkyl, saturated or partially unsaturated heterocyclyl, aryl and heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted with one or more R ^c,

R ³ is selected from the group consisting of hydrogen, oxo, halogen, cyano, hydroxyl, -NR ^dR ^e, -C (O) NR ^dR ^e, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, saturated or partially unsaturated cycloalkyl, saturated or partially unsaturated heterocyclyl, aryl and heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted with one or more R ^f; or

R ⁴ and R ⁵, R ⁴ and R ⁶, R ⁴ and R ⁷, together with the atoms to which they are each attached, form saturated or partially unsaturated cycloalkyl, or saturated or partially unsaturated heterocyclyl, wherein each of cycloalkyl and heterocyclyl is optionally substituted with cyano, halogen, hydroxy, -NR ^cR ^d, carboxy, carbamoyl, aryl or heteroaryl;

W is saturated or partially unsaturated cycloalkyl, or saturated or partially unsaturated heterocyclyl, wherein each of cycloalkyl and heterocyclyl is optionally substituted with one or more R ^g,

L ³ is a bond, alkyl or -NR ^d-;

B is an electrophilic moiety capable of forming a covalent bond with a cysteine residue at position 12 of a KRAS ^G12C mutant protein;

R ^a is independently hydrogen or alkyl;

each R ^b is independently selected from the group consisting of oxo, cyano, halogen, hydroxy, acyl, -NR ^dR ^e, carbamoyl, carboxyl, alkyl, alkenyl, alkynyl, alkoxyl, alkoxylalkyl, cycloalkylalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl;

each R ^c is independently selected from the group consisting of oxo, halogen, cyano, hydroxy, -NR ^dR ^e, -C (O) OR ^a, -C (O) N (R ^d) (R ^e) , alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, alkoxyl, saturated or partially unsaturated cycloalkyl, saturated or partially unsaturated heterocyclyl, aryl, and heteroaryl;

each of R ^d and R ^e is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl and heteroaryl is optionally substituted with cyano, halogen, hydroxy, or amino;

each R ^f is independently selected from the group consisting of oxo, halogen, cyano, hydroxy, -NR ^cR ^d, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl;

each R ^g is independently selected from the group consisting of oxo, cyano, halogen, hydroxy, -NR ^dR ^e, carbamoyl, carboxy, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, saturated or partially unsaturated cycloalkyl, and saturated or partially unsaturated heterocyclyl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, and heterocyclyl is optionally substituted with cyano, halogen, hydroxy, -NR ^dR ^e, carboxy, carbamoyl, haloalkyl, aryl or heteroaryl;

n is 0, 1, 2, 3 or 4.
The method of claim 22, wherein the KRAS inhibitor has a formula selected from the group consisting of:

wherein

J ¹ is absent, CH (R ⁴) , NR ⁴, SO ₂ or P (O) CH ₃;

J ² is absent, CR ⁵, N, SO ₂ or P (O) CH ₃;

J ³ is absent, CH (R ⁶) , NR ⁶, SO ₂ or P (O) CH ₃;

J ⁴ is absent, CR ⁷, N, SO ₂ or P (O) CH ₃;

J ⁵ is absent, CH (R ⁸) , NR ⁸, SO ₂ or P (O) CH ₃;

R ⁴, R ⁵, R ⁶, R ⁷ and R ⁸ are each independently selected from the group consisting of hydrogen, oxo, halogen, cyano, hydroxyl, -NR ^dR ^e, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, saturated or partially unsaturated cycloalkyl, saturated or partially unsaturated heterocyclyl, aryl and heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted with one or more R ^f; or

R ² and any one of R ⁴, R ⁵, R ⁶, R ⁷ and R ⁸, together with the atoms to which they are each attached, form saturated or partially unsaturated cycloalkyl, or saturated or partially unsaturated heterocyclyl, wherein each of cycloalkyl and heterocyclyl is optionally substituted with cyano, halogen, hydroxy, -NR ^cR ^d, carboxy, carbamoyl, aryl or heteroaryl; or

R ³ and any one of R ⁴, R ⁵, R ⁶ and R ⁸, together with the atoms to which they are each attached, form saturated or partially unsaturated cycloalkyl, or saturated or partially unsaturated heterocyclyl, wherein each of cycloalkyl and heterocyclyl is optionally substituted with cyano, halogen, hydroxy, -NR ^cR ^d, carboxy, carbamoyl, aryl or heteroaryl; or

R ⁴ and any one of R ⁶ and R ⁸, together with the atoms to which they are each attached, form saturated or partially unsaturated cycloalkyl, or saturated or partially unsaturated heterocyclyl, wherein each of cycloalkyl and heterocyclyl is optionally substituted with cyano, halogen, hydroxy, -NR ^cR ^d, carboxy, carbamoyl, aryl or heteroaryl; or

R ⁶ and R ⁸, together with the atoms to which they are each attached, form saturated or partially unsaturated cycloalkyl, or saturated or partially unsaturated heterocyclyl, wherein each of cycloalkyl and heterocyclyl is optionally substituted with cyano, halogen, hydroxy, -NR ^cR ^d, carboxy, carbamoyl, aryl or heteroaryl.
The method of claim 22 or 23, wherein the KRAS inhibitor is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.
The method of claim 24, wherein the KRAS inhibitor is
or a pharmaceutical acceptable salt thereof.
The method of any one of the preceding claims, wherein the ERK inhibitor is a chemotherapeutic agent, an antibody, or an antigen-binding fragment thereof.
The method of claim 26, wherein the ERK inhibitor is a small molecule compound.
The method of any one of the preceding claims, wherein the ERK inhibitor is a dual ERK1/2 inhibitor.
The method of any one of the preceding claims, wherein the ERK inhibitor is a compound of Formula (VII) or a pharmaceutically acceptable salt thereof

wherein:

R ⁹ is hydrogen, C _1-3 alkyl or -CH ₂OMe;

R ¹⁰ is pyridinyl, optionally substituted on 1 ring carbon atom by a substituent independently selected from the group consisting of C _1-3 alkyl, difluoromethyl and trifluoromethyl; or R ¹⁰ is pyrimidinyl, optionally substituted on 1 ring carbon atom by a substituent independently selected from the group consisting of C _1-3 alkyl, difluoromethyl and trifluoromethyl; or R ¹⁰ is phenyl optionally substituted on 1 or 2 ring carbon atoms by a substituent independently selected from the group consisting of halo, difluoromethyl, trifluoromethyl, methoxy and -OCHF ₂; and

R ¹¹ is hydrogen, C _1-3 alkyl or chloro.
The method of claim 29, wherein:

R ⁹ is hydrogen, methyl or -CH ₂OMe;

R ¹⁰ is pyridinyl, optionally substituted on 1 ring carbon atom by a substituent independently selected from the group consisting of methyl, difluoromethyl and trifluoromethyl; or R ¹⁰ is pyrimidinyl, optionally substituted on 1 ring carbon atom by a substituent independently selected from the group consisting of methyl, difluoromethyl and trifluoromethyl; or R ¹⁰ is phenyl optionally substituted on 1 or 2 ring carbon atoms by a substituent independently selected from the group consisting of fluoro, chloro, difluoromethyl, trifluoromethyl, methoxy and -OCHF ₂; and

R ¹¹ is hydrogen, methyl or chloro.
The method of claim 29 or 30, wherein:

R ⁹ is hydrogen, methyl or -CH ₂OMe;

R ¹⁰ is pyridinyl, optionally substituted on 1 ring carbon atom by a substituent independently selected from the group consisting of methyl, difluoromethyl and trifluoromethyl; or R ¹⁰ is pyrimidinyl, optionally substituted on 1 ring carbon atom by a trifluoromethyl; or R ¹⁰ is phenyl optionally substituted on 1 or 2 ring carbon atoms by a substituent independently selected from the group consisting of fluoro, chloro, difluoromethyl and methoxy; and

R ¹¹ is hydrogen, methyl or chloro.
The method of any one of claims 29-31, wherein R ¹⁰ is 6- (difluoromethyl) pyridin-2-yl, 3-chlorophenyl, 3, 4-difluorophenyl or 3, 5-difluorophenyl.
The method of any one of claims 29-32, wherein R ¹⁰ is 3, 4-difluorophenyl.
The method of any one of claims 29-33, wherein the ERK inhibitor is a compound selected from the group consisting of:

2- (2- ( (1-Methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -7- ( (6-methylpyridin-2-yl) methyl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -7- (3-Chlorobenzyl) -6-methyl-2- (2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -7- (3-Chloro-4-fluorobenzyl) -6-methyl-2- (2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -7- (3, 4-Difluorobenzyl) -6-methyl-2- (2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

2- (5-Methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -7- ( (6- (trifluoromethyl) pyridin-2-yl) methyl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -2- (5-Chloro-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -7- (3, 4-difluorobenzyl) -6-methyl-6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -2- (5-Chloro-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -7- (3-methoxybenzyl) -6-methyl-6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -2- (5-chloro-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6-methyl-7- ( (2- (trifluoromethyl) pyrimidin-4-yl) methyl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -2- (5-Chloro-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -7- ( (6- (difluoromethyl) pyridin-2-yl) methyl) -6-methyl-6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -2- (5-Chloro-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6-methyl-7- ( (6- (trifluoromethyl) pyridin-2-yl) methyl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -2- (5-chloro-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6-methyl-7- ( (6-methylpyridin-2-yl) methyl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

7- (3-Chloro-4-fluorobenzyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

7- (3-Chlorobenzyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

7- (3- (Difluoromethyl) benzyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

7- ( (6- (Difluoromethyl) pyridin-2-yl) methyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(R) -7- ( (6- (Difluoromethyl) pyridin-2-yl) methyl) -6- (methoxymethyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(R) -7- (3-Chlorobenzyl) -6- (methoxymethyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5- yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(R) -7- (3, 4-Difluorobenzyl) -6- (methoxymethyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -7- (3-Chlorobenzyl) -6-methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -7- (3, 4-Difluorobenzyl) -6-methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -7- (3- (Difluoromethyl) benzyl) -6-methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -7- (3, 5-Difluorobenzyl) -6-methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -7- (3-Methoxybenzyl) -6-methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -7- (4-Fluoro-3-methoxybenzyl) -6-methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -7- ( (6- (Difluoromethyl) pyridin-2-yl) methyl) -6-methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -6-methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -7- ( (2- (trifluoromethyl) pyrimidin-4-yl) methyl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -6-Methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -7- ( (6- (trifluoromethyl) pyridin-2-yl) methyl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -7- (3- (Difluoromethoxy) benzyl) -6-methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -6-Methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -7- ( (4- (trifluoromethyl) pyridin-2-yl) methyl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -6-Methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -7- ( (2- (trifluoromethyl) pyridin-4-yl) methyl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -7- ( (4- (Difluoromethyl) pyridin-2-yl) methyl) -6-methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -6-Methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -7- ( (6- (trifluoromethyl) pyrimidin-4-yl) methyl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(S) -7- (3, 4-difluorobenzyl) -6- (methoxymethyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(R) -6-Methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -7- ( (6- (trifluoromethyl) pyridin-2-yl) methyl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(R) -7- ( (6- (Difluoromethyl) pyridin-2-yl) methyl) -6-methyl-2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(R) -7- (3- (Difluoromethyl) benzyl) -6- (methoxymethyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(R) -6- (Methoxymethyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -7- ( (6- (trifluoromethyl) pyridin-2-yl) methyl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

(R) -7- (3, 5-difluorobenzyl) -6- (methoxymethyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one; and

(R) -7- (3-Methoxybenzyl) -6- (methoxymethyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one;

or a pharmaceutically acceptable salt thereof.
The method of any one of claims 29-34, wherein the ERK inhibitor is (R) -7- (3, 4-Difluorobenzyl) -6- (methoxymethyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one, or a pharmaceutically acceptable salt thereof.
The method of claim 35, wherein the ERK inhibitor is (R) -7- (3, 4-Difluorobenzyl) -6- (methoxymethyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one Ethanesulfonic acid salt.
The method of claim 36, wherein the ERK inhibitor is a crystalline form of (R) -7- (3, 4-Difluorobenzyl) -6- (methoxymethyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one Ethanesulfonic acid salt.
The method of claim 35, wherein the ERK inhibitor is (R) -7- (3, 4-Difluorobenzyl) -6- (methoxymethyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one Adipic acid salt.
The method of claim 38, wherein the ERK inhibitor is a crystalline form of (R) -7- (3, 4-Difluorobenzyl) -6- (methoxymethyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one Adipic acid salt.
The method of claim 38 or 39, wherein the ERK inhibitor has the following formula:

. 0.5 adipic acid.
The method of any one of the preceding claims, wherein the KRAS inhibitor is selected from the group consisting of Sotorasib (AMG-510) , Adagrasib (MRTX849) ,
and a pharmaceutically acceptable salt thereof, and the ERK inhibitor is (R) -7- (3, 4-Difluorobenzyl) -6- (methoxymethyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one or a pharmaceutically acceptable salt thereof.
The method of any one of the preceding claims, wherein the KRAS inhibitor is selected from the group consisting of Sotorasib (AMG-510) , Adagrasib (MRTX849) ,
and a pharmaceutically acceptable salt thereof, and the ERK inhibitor is
. 0.5 adipic acid.
The method of any one of the preceding claims, wherein the ERK inhibitor is administered before, after, simultaneously, or in an overlapping manner with the KRAS inhibitor.
The method of any one of the preceding claims, wherein the ERK inhibitor and/or the KRAS inhibitor is administered orally, subcutaneously, intraperitoneally, or intravenously.
The method of any one of the preceding claims, comprising administering to the subject an additional therapeutical agent.
The method of claim 45, wherein the additional therapeutical agent is a MEK inhibitor.
The method of claim 46, wherein the MEK inhibitor is selected from the group consisting of Binimetinib, Cobimetinib, Refametinib, Selumetinib, Trametinib, mirdametinib, PD-325901, TAK-733, E6201, CI-1040, ATR-002, SHR7390, NFX-179, pimasertib, VS-6766, refametinib, HL-085, FCN-159, LNP3794, CS3006, AS703988, TQ-B3234, and GDC-0623.
A method of treating, preventing, or ameliorating cancer in a subject in need thereof, comprising: (a) screening the subject to assess whether the subject carries a KRAS mutation; and (b) if the subject carries a KRAS mutation, then administering to the subject an effective amount of an ERK inhibitor or a pharmaceutically acceptable salt thereof, in combination with an effective amount of a KRAS inhibitor or a pharmaceutically acceptable salt thereof.
The method of claim 48, wherein the KRAS mutation is selected from KRAS ^G12C/D/V, KRAS ^G13C/D, or KRAS ^Q61L/H/R.
The method of claim 49, wherein the KRAS mutation is KRAS ^G12C.
A pharmaceutical composition comprising a KRAS inhibitor or a pharmaceutically acceptable salt thereof, and an ERK inhibitor or a pharmaceutically acceptable salt thereof.
A method of preparing the pharmaceutical composition of claim 51, comprising mixing the KRAS inhibitor or a pharmaceutically acceptable salt thereof and the ERK inhibitor or a pharmaceutically acceptable salt thereof to form a pharmaceutical composition.
A kit comprising (a) a first composition comprising a KRAS inhibitor or a pharmaceutically acceptable salt thereof, and (b) a second composition comprising an ERK inhibitor or a pharmaceutically acceptable salt thereof.
The pharmaceutical composition of claim 51 or the kit of claim 53, wherein the the KRAS inhibitor is a KRAS ^G12C inhibitor, and/or the ERK inhibitor is a dual ERK1/2 inhibitor.
The pharmaceutical composition of claim 51 or the kit of claim 53, wherein the KRAS inhibitor is selected from the group consisting of Sotorasib (AMG-510) , Adagrasib (MRTX849) ,
and a pharmaceutically acceptable salt thereof, and the ERK inhibitor is (R) -7- (3, 4-Difluorobenzyl) -6- (methoxymethyl) -2- (5-methyl-2- ( (1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -6, 7-dihydroimidazo [1, 2-a] pyrazin-8 (5H) -one or a pharmaceutically acceptable salt thereof.
The pharmaceutical composition of claim 51 or the kit of claim 53, wherein the KRAS inhibitor is selected from the group consisting of Sotorasib (AMG-510) , Adagrasib (MRTX849) ,
and a pharmaceutically acceptable salt thereof, and the ERK inhibitor is
. 0.5 adipic acid.