MIXED LINEAGE KINASE DOMAIN LIKE PSEUDOKINASE (MLKL) ACTIVATORS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No 63/381,912 filed on November 01, 2022, the contents of which are incorporated by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

This invention was made with government support under grant numbers AI144400 and Al 164003 awarded by the National Institutes of Health. The government has certain rights in the invention

BACKGROUND

The field of the invention relates to substituted heterocycles as necroptosis activators In particular, the field of the invention relates to substituted heterocycles as mixed lineage kinase domain like pseudokinase (MLKL) activating agents for the treatment of cell proliferation diseases and disorders such as cancer.

The non-responsiveness of ‘cold ^' tumors to immune checkpoint blockade (ICB) therapies remains a major hindrance. There are two broad strategies to increase the immunogenicity of cold tumors: 1) increase the ‘visibility’ of tumor cells to the immune system, for example by enabling neo-antigen production by tumor cells or 2) induce an immunogenic state in the tumor to break tolerance and enhance anti-tumor T cell responses. Herein are described compounds that can activate necroptosis, a potently immunogenic form of regulated cell death, for potential treatment either alone or in combination with other drugs for the treatment of cancerous tumors.

SUMMARY

Disclosed are substituted heterocycles which may be utilized as MLKL activating agents. The disclosed heterocycles may be used in pharmaceutical compositions and methods for treating cell proliferative disorders such as cancer.

The disclosed substituted heterocycles may include compounds having formula of:

wherein W is -C(=O)-alkyl, alkyl, or H; wherein R ¹ and R/’ are- independently O-alkylamine, alkyl-substituted piperazinyl, hydroxyl, alkoxy, --

NHSCh-alkyl, CN, orH or joined to form -O- (CH2)1-2.-O- or; wherein R ² is CN, NO2, halo, C( O )()M. C( ^::: O)O-alkyl, alkyl, hydroxyl, or H; wherein R ³ is H, halo, amino, CN, alkoxy, hydroxyl, or amido; wherein R? is alkoxy, hydroxyl, or H; wherein A i s N or CH, wherein Z is N of CH; wherein X-Y is Oi or CH-CH; and wherein R ⁴ is an alkyl or heterocycloalkyl; or a pharmaceutically acceptable salt thereof.

The disclosed compounds may exhibit one or more biological activities. The disclosed compounds may activate necroptosis. The disclosed compounds may activate MLKL. In some embodiments, the compounds activate MLKL at a concentration of less than about 100 pM, 50 μM, 10 pM, 1 pM, 0.1 pM, 0.05 pM, 0.01 pM, 0.005 pM, 0.001 pM, or less.

Also disclosed are pharmaceutical compositions comprising the disclosed compounds and a suitable pharmaceutical carrier, excipient, or diluent. The disclosed pharmaceutical compositions may comprise an effective amount of the compound for inhibiting the growth of cancer cells when administered to a subject in need thereof.

Also disclosed are methods for treating cell proliferation diseases and disorders such as cancer. The methods may include administering the disclosed compounds or pharmaceutical compositions comprising the disclosed compounds to a subject in need thereof for example, to a subject having cancer. The disclosed compounds or pharmaceutical compositions comprising the disclosed compounds may be administered with additional therapeutic agents, optionally in combination, in order to treat cell proliferative diseases and disorders. Cell proliferative diseases and disorders treated by the disclosed methods may include, but are not limited to, cancers selected from the group consisting of multiple myeloma, leukemia, non-small cell lung cancer, colon cancer, cancer of the central nervous system, melanoma, ovarian cancer, renal cancer, prostate cancer, and breast cancer,

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 depicts a schematic showing of MLKL in the pathway of necroptosis.

Figure 2 show's that necroptosis is mediated by sequential activation of upstream sensors followed by RIPK3 and MLKL. MLKL activators bypass RIPK3 activation, which is frequently lost in cancer cells.

Figure 3 shows that potent activators of MLKL were identified that bind the MLKL pseudokinase pocket. Cell death induced by SS-1-127 is dependent on MLKL expression and independent from RIPK3. Viability of transformed mouse embryonic fibroblasts with genetic deletions of RIPK3 and MLKL genes and treated with SS-1-127 are shown. Affinities of select MLKL activators against recombinant human RIPK1, RIPK3, and MLKL were determined by DiscoveRx.

Figure 4 show's that UH15*22 efficiently activates human MLKL. Mikl" ' mouse embryonic fibroblasts (MEFs) were stably infected with lentiviral vector encoding 3 XFL AG-human MLKL under the control of the Doxocyclin (Dox)-inducible promoter. Cells were treated with 10 ng/rnl of Dox for 6 hr followed by the treatment with indicated concentrations of UH15-22 for 24 hr. Cel I viability at the end of treatment was determined using CellTiter-Glo Viability assay (Promega)

Figure 5 shows the putative mechanism of action. The pseudokinase domain is a target of the present disclosure.

Figure 6 demonstrates that MLKL activators induce necroptosis directly through binding MLKL and independently of RIPK3 activity. Plots show cell viability (%) versus MLKL mutant expressed. Mikl"'' mouse embryonic fibroblasts (MEFs) were stably infected with lentiviral vector encoding mouse MLKL -FLAG under the control of the Doxocyclin (Dox)-inducible promoter. Cells conditionally expressing wild type and mutants of MLKL have been generated. M272W mutant of MLKL is active but predicted not to bind to MLKL activators. S345A/S347A mutant of MLKL lacks RIPK3 phosphorylation sites. Cells were treated with 50 ng/ml of Dox for 6 hr followed by MLKL activators (left panels) or TNFa/IDN6556 (right panels). The latter combination is a canonical inducer of necroptosis through RIPK3 activation. 5 uM ALD-6-85 and SS-1-127 were used in top and bottom panels, respectively. After 24 hr, cell viability at the end of treatment was determined using CellTiter-Glo Viability assay (Promega).

Figure 7 shows that E239Q mutation lowers the ICso of SS-1-127, demonstrated by a plot of percent viability versus Logio(drug, nM) of SS-1-127 and UH15-22. Glu239 residue plays a key role in blocking MLKL activation. E239Q mutation reduces non-covalent bonds (e.g. ionic-dipole interactions), thus resulting in partially active MLKL protein, inducing limited cell death upon expression. SS-1-127 displays much improved activity when combined with E239Q mutation compared to wild type MLKL, suggesting a much better fit for SS- 1 -127 with the active conformation of the MLKL compared to UH15-22 which induces cell death to the same degree in the cells expressing wild type and E239Q mutant MLKL. Mlkt'' mouse embryonic fibroblasts (MEFs) were stably infected with lentiviral vector encoding wi id type and E239Q mutant of mouse MLKL -FLAG under the control of the Doxocyclin (Dox)-inducible promoter. Cells were treated with 50 ng/ml of Dox for 6 hr followed by SS-1-127 and UH15-22. After 24 hr, cell viability at the end of treatment was determined using CellTiter-Glo Viability assay (Promega).

Figure 8 shows the results of a standard protocol NCI60 screening for SS-1-127 at 10 pM

Figure 9 shows the induction of cell death by various molecules in cells in the presence and absence of MLKL. MLKL-/- mouse embryonic fibroblasts were stably transduced to allow’ Doxycycline-inducible re-expression of mouse MLKL. For the experiments, the same cells were either left without Doxycycline (MLKL-/- condition, left three columns) or pre-treated with 50 ng/ml Doxycycline (MLKL+/+ condition, right three columns) for 6 hr. After that cells were treated with the indicated concentrations of drugs for 24 hr Cell viability was determined using CellTiter-Glo assay.

DETAILED DESCRIPTION

Necroptosis is a regulated inflammatory cell death pathway first described by Degterev, et al. in 2005 (Degterev A, Huang Z, Boyce M, Li Y, Jagtap P, Mizushima N, Cuny GD, Mitchison TJ, Moskowitz MA, Yuan J. Chemical inhibitor of nonapoptotic cell death with therapeutic- potential for ischemic brain injury. Nat Chem Biol 2005; 1(2): 112-9). It is initiated by the innate immune sensors tumor necrosis factor receptor 1 (TNFRI), toll like receptor 3 (TLR3), Z-DNA binding protein 1 (ZBP1 ), and driven by receptor interacting protein kinase 3 (RIPK3). RIPK3 then phosphorylates mixed lineage kinase domain like pseudokinase (MLKL) that oligomerizes, translocates to the plasma membrane, and triggers lytic cell death releasing large quantities of immune-stimulatory Danger Associated Molecular Patterns (DAMPs) (Figure 1) (Krysko O, Aaes TL, Kagan VE, D'Herde K, Bachert C, Leybaert L, Vandenabeele P, Krysko DV. Necroptotic cell death in anti-cancer therapy. Immunol Rev. 2017;280(l):207-19). Various methods of delivery' of necroptosis mediators RIPK3 and MLKL in mRNA or protein form into tumor mass were directly shown to promote anticancer immunity (Snyder AG, Hubbard NW, Messmer MN, et al. Intratumoral activation of the necroptotic pathway components RIPK1 and RIPK3 potentiates antitumor immunity. Sci Immunol 2019,4, Van Hoecke L, Riederer S, Saelens X, Sutter G, Rojas JJ. Recombinant viruses delivering the necroptosis mediator MLKL induce a potent antitumor immunity in mice. On coimmunology 2020;9: 1802968; Van Hoecke L, Van Lint S, Roose K, et al. Treatment with mRNA coding for the necroptosis mediator MLKL induces antitumor immunity directed against neo-epitopes. Nat Common 2018,9:3417). However, two major issues currently limit the translational potential of this immunogenic cell death modality: 1) most tumor cells inactivate necroptosis signaling, usually by silencing RIPK3 and 2) no dedicated small molecule activators of necroptosis exist The disclosed technology overcomes both these limitations.

The compounds described herein are capable of directly activating the downstream executioner of necroptosis, MLKL, thus bypassing a requirement for RIPK3 (Figure 2). The Examples demonstrate that these molecules induce cell death in a manner that is completely dependent on MLKL and independent of RIPK3 and do so by directly binding to MLKL (Figure 3).

The present invention is described herein using several definitions, as set forth below and throughout the application.

Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a compound” should be interpreted to mean “one or more compounds.”

As used herein, “about,” “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary' skill in the art and will vary’ to some extent on the context in which they are used. If there are uses of these terms which are not clear to persons of ordinary skill in the art given the context in which they are used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising” in that these latter terms are “open” transitional terms that do not limit claims only to the recited elements succeeding these transitional terms. The term “consisting of,” while encompassed by the term “comprising,” should be interpreted as a “closed” transitional term that limits claims only to the recited elements succeeding this transitional term. The term “consisting essentially of,” while encompassed by the term “comprising,” should be interpreted as a “partially closed” transitional term which permits additional elements succeeding this transitional term, but only if those additional elements do not materially affect the basic and novel characteristics of the claim.

As used herein, a “subject” may be interchangeable with “patient” or “individual” and means an animal, which may be a human or non-human animal, in need of treatment.

A “subject in need of treatment” may include a subject having a disease, disorder, or condition that is responsive to therapy with the presently disclosed compounds. For example, a “subject in need of treatment” may include a subject having a cell proliferative disease, disorder, or condition such as cancer (e.g., cancers such as multiple myeloma, leukemia, non-small cell lung cancer, colon cancer, cancer of the central nervous system, melanoma, ovarian cancer, renal cancer, prostate cancer, and breast cancer). A “subject in need of treatment” may include a subject having a cell proliferative disease, disorder, or condition such as cancer and/or that may be treated by administering an effective amount of an agent that modulates MLKL activity. The compounds described herein, and any of the compositions described herein comprising the same, can be used to treat tumors, including “cold tumors” and “hot tumors ”

In some embodiments, the composition may be used to treat "cold tumors." Cold tumors are defined as a tumor that is not likely to trigger an immune response. Cold tumors tend to be surrounded by cells that are able to suppress the immune response and keep T cells from attacking the tumor cells and killing them. Cold tumors usually do not respond to immunotherapy. Inunune- excluded tumors and immune-desert tumors can be described as cold tumors. Cold tumors are also known as non -inflamed. There are several classes of cold tumors including tumors that demonstrate a lack of T cell activation or priming (e.g., melanoma), a lack of tumor antigens (e.g., prostate tumors), dense stroma (e g. pancreatic cancer). Most cancers of the breast, ovary, prostate, pancreas, and brain are considered cold tumors.

Ln other embodiments, the composition may be used to treat, tumors that are likely to trigger an immune response or that are not considered cold tumors.

The presently disclosed composition may be used to sensitize tumor to an immunotherapy agent. Suitably the disclosed composition may be used to sensitize a cold tumor to an immunotherapy agent.

As used herein, the phrase “effective amount” shall mean the amount, of the compound that, provides the desired effect. The effective amount may be a drug dosage that provides the specific pharmacological response for which the drug is administered in a significant number of subjects in need of such treatment. An effective amount of a drug that is administered to a particular subject in a particular instance will not always be effective in treating the conditions/diseases described herein, even though such dosage is deemed to be a therapeutically effective amount by those of skill in the art.

As used herein, the term “modulate” means decreasing or inhibiting activity and/or increasing or augmenting activity. For example, modulating MLKL activity may mean increasing or augmenting MLKL activity and/or decreasing or inhibiting MLKL activity. The compounds disclosed herein may be administered to modulate MLKL activity.

Chemical Entities

New' chemical entities and uses for chemical entities are disclosed herein. The chemical entities may be described using terminology known in the art and further discussed below.

As used herein, an asterisk or a plus sign “+” may be used to designate the point of attachment for any radical group or substituent group.

The term “alkyl” as contemplated herein includes a straight-chain or branched alkyl radical in all of its isomeric forms, such as a straight or branched group of 1-12, 1-10, or 1-6 carbon atoms, referred to herein as C1 -C12 alkyl, C1-C10 -alkyl, and C1-C6-alkyl, respectively.

The term “alkylene” refers to a diradical of an alkyl group (e.g., -(CH2)n- where n is an integer such as an integer between 1 and 20) An exemplary/ alkydene group is -CH2CH2-.

The term “haloalkyl” refers to an alkyl group that is substituted with at least one halogen. For example, -CH2F. -CHF2, -CF3, -CH2CF3, -CF2CF3, and the like. The term “heteroaikyi” as used herein refers to an “alkyl” group in which at least one carbon atom has been replaced with a heteroatom (e.g., an O, N, or S atom). One type of heteroalkyl group is an “alkoxy” group.

The term “alkenyl” as used herein refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond, such as a straight or branched group of 2-12, 2-10, or 2-6 carbon atoms, referred to herein as C2-C12-alkenyl, C2-C10-alkenyl, and C2-C6-alkenyl, respectively.

The term “alky ny 1” as used herein refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon triple bond, such as a straight or branched group of 2-12, 2-10, or 2-6 carbon atoms, referred to herein as C2-C12-alkynyl, C2-C10-alkynyl, and C2-C6-alkynyl, respectively.

The term “cycloalkyl” refers to a monovalent saturated cyclic, bicyclic, or bridged cyclic (e.g., adamantyl) hydrocarbon group of 3-12, 3-8, 4-8, or 4-6 carbons, referred to herein, e.g., as “C4-8-cycloalkyl,” derived from a cycloalkane. Unless specified otherwise, cycloalkyl groups are optionally substituted at one or more ring positions with, for example, alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido, amidino, amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halo, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonato, phosphinato, sulfate, sulfide, sulfonamide, sulfonyl or thiocarbonyl. In certain embodiments, the cycloalkyl group is not substituted, i.e., it is unsubstituted.

The term “cycloheteroalkyl” refers to a monovalent saturated cyclic, bicyclic, or bridged cyclic hydrocarbon group of 3-12, 3-8, 4-8, or 4-6 carbons in which at least one carbon of the cycloalkane is replaced with a heteroatom such as, for example, N, O, and/or S.

The term “cycloalkylene” refers to a cycloalkyl group that is unsaturated at one or more ring bonds.

The term “partially unsaturated carbocyclyl” refers to a monovalent cyclic hydrocarbon that contains at least one double bond between ring atoms where at least one ring of the carbocyclyl is not aromatic. The partially unsaturated carbocyclyl may be characterized according to the number oring carbon atoms. For example, the partially unsaturated carbocyclyl may contain 5-14, 5-12, 5-8, or 5-6 ring carbon atoms, and accordingly be referred to as a 5-14, 5-12, 5-8, or 5-6 membered partially unsaturated carbocyclyl, respectively. The partially unsaturated carbocyclyl may be in the form of a monocyclic carbocycle, bicyclic carbocycle, tricyclic carbocycle, bridged carbocycle, spirocyclic carbocycle, or other carbocyclic ring system. Exemplary partially unsaturated carbocyclyl groups include cycloalkenyl groups and bicyclic carbocyclyl groups that are partially unsaturated. Unless specified otherwise, partially unsaturated carbocyclyl groups are optionally substituted at one or more ring positions with, for example, alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido, amidino, amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonato, phosphinato, sulfate, sulfide, sulfonamide, sulfonyl or thiocarbonyl. In certain embodiments, the partially unsaturated carbocyclyl is not substituted, i.e., it is unsubstituted.

The term “aryl” is art-recognized and refers to a carbocyclic aromatic group. Representative aryl groups include phenyl, naphthyl, anthracenyl, and the like The term “aryl” includes polycyclic ring systems having two or more carbocyclic rings in which two or more carbons are common to two adjoining rings (the rings are “fused rings”) wherein at least one of the rings is aromatic and, e.g., the other ring(s) may be cycloalkyls, cycloalkenyls, cycloalkynyls, and/or aiyls. Unless specified otherwise, the aromatic ring may be substituted at one or more ring positions with, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, carboxylic acid, -C(O)alkyl, -CChalkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamido, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aryl or heteroaryl moieties, -CFa, -CN, or the like. In certain embodiments, the aromatic ring is substituted at one or more ring positions with halogen, alkyl, hydroxyl, or alkoxyl. In certain other embodiments, the aromatic ring is not substituted, i.e., it is unsubstituted. In certain embodiments, the aryl group is a 6-10 membered ring structure.

The terms “heterocyclyl” and “heterocyclic group” are art-recognized and refer to saturated, partially unsaturated, or aromatic 3- to 10-membered ring structures, alternatively 3 -to 7-membered rings, whose ring structures include one to four heteroatoms, such as nitrogen, oxygen, and sulfur. The number of ring atoms in the heterocyclyl group can be specified using 5 Cx-Cx nomenclature where x is an integer specifying the number of ring atoms For example, a C3-C7 heterocyclyl group refers to a saturated or partially unsaturated 3- to 7-membered ring structure containing one to four heteroatoms, such as nitrogen, oxygen, and sulfur. The designation “C3-C7” indicates that the heterocyclic ring contains a total of from 3 to 7 ring atoms, inclusive of any heteroatoms that occupy a ring atom position.

The terms “amine"’ and “amino” are art-recognized and refer to both unsubstituted and substituted amines (e.g., mono-substituted amines or di-substituted amines), wherein substituents may include, for example, alkyl, cycloalkyl, heterocyclyl, alkenyl, and aryl.

The terras “alkoxy” or “alkoxy!” are art-recognized and refer to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxy groups include methoxy, ethoxy, tert-butoxy and the like.

The term “O-alkylamine” refers to ~O~ (CR ¹ R ² )m-NR ³ R ⁴ , wherein m is an integer between 1-6, and R ¹ , R ² , R ³ and R ⁴ , for example, are each independently hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, and aryl. R ³ and R ⁴ , together with the nitrogen they are attached to, may also be optionally joined to form a 4-8 membered cycloalkyl.

The term "heterocycloalkyl" refers to a monovalent saturated cyclic, bicyclic, or bridged cyclic hydrocarbon group of 3-12, 3-8, 4-8, or 4-6 carbons in which at least one carbon of the cycloalkane is replaced with a heteroatom such as, for example, N, O, and/or S(O)n, wherein n is an integer of 0-2. "Four to seven membered heterocycloalkyl" refers to a heterocycloalkyl containing from four to seven atoms, including one or more heteroatoms, in the cyclic moiety of the heterocycloalkyl. Examples of single-ring heterocycloalkyls include azetidinyl, oxetanyl, thietanyl, dihydrofuranyl, tetrahydrofuranyl, dihydrothiophenyl, tetrahydrothiophenyl, pyrrolinyl, pyrrolidinyl, imidazolinyl, imidazolidinyl, pyrazolinyl, pyrazolidinyl, thiazolinyl, isothiazolinyl, thiazolidinyl, isothiazolidinyl, dihydropyranyl, piperidinyl, morpholinyl, piperazinyl, azepinyl, oxepinyl, and diazepinyl. In some embodiments, the heterocycloalkyl described herein may be fused with a cycloalkyl, an aryl, or a heteroaryl, as described herein In some embodiments, the heterocycloalkyl described herein may be

An “ether” is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as may be represented by one of -O-alkyl, -O-alkenyl, -O-alkynyl, and the like.

The term “carbonyl” as used herein refers to the radical -C(O)-.

The term “oxo” refers to a divalent oxygen atom -O-. The term “carboxamide” as used herein refers to the radical -C(O)NRR', where R and R' may be the same or different. R and R', for example, may be independently alkyl, aryl, arylalkyl, cycloalkyl, formyl, haloalkyl, beteroaryl, or heterocyclyl.

The term “carboxy” as used herein refers to the radical -COOH or its corresponding salts, e.g. -COONa, etc.

The term “amide” or “amido” or “amidy!” as used herein refers to a radical of the form - R ¹ C(O)N(R ² )-, -R ¹ C(O)N(R ² )R ³ -, -C(O)NR ² R ³ , or -C(O)NH ₂ , wherein R ¹ , R ² and R ³ , for example, are each independently alkoxy, alkyl, alkenyl, alkynyl, amide, amino, aiyl, arylalkyl, carbamate, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydrogen, hydroxyl, ketone, or nitro.

The compounds of the disclosure may contain one or more chiral centers and/or double bonds and, therefore, exist as stereoisomers, such as geometric isomers, enantiomers or diastereomers. The term “stereoisomers” when used herein consist of all geometric isomers, enantiomers or diastereomers. These compounds may be designated by the symbols “R” or “5,” or “+” or depending on the configuration of substituents around the stereogenic carbon atom and or the optical rotation observed. The present invention encompasses various stereo isomers of these compounds and mixtures thereof Stereoisomers include enantiomers and diastereomers. Mixtures of enantiomers or diastereomers may be designated (±)” in nomenclature, but the skilled artisan will recognize that a structure may denote a chiral center implicitly. It is understood that graphical depictions of chemical structures, e.g , generic chemical structures, encompass all stereoisomeric forms of the specified compounds, unless indicated otherwise. Also contemplated herein are compositions comprising, consisting essentially of, or consisting of an enantiopure compound, which composition may comprise, consist essential of, or consist of at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of a single enantiomer of a given compound (e.g., at least about 99% of an If enantiomer of a given compound).

Mixed lineage kinase domain like pseudokhiase (MLKL) activators and uses thereof

Disclosed herein are compounds which may be utilized as activators of necroptosis. The disclosed compounds have been shown to activate necroptosis by activating mixed lineage kinase domain like pseudokinase (MLKL). In one embodiment, the MLKL activating compounds target a pseudokinase domain of MLKL to form the act, closed conformation (Figure 5). The disclosed compounds may be used in pharmaceutical compositions and methods for treating cell proliferation diseases and disorders, such as cancer.

Ln some embodiments, the method for treating a subject having cancer includes administering a compound having a formula of:

W may be ---C(=O)-alkyl, alkyl, orH. When W is , R ^; and

R ⁶ may be independently O-alkylamine, alkyl-substituted piperazinyl, hydroxyl, alkoxy, -

NHSCh-alkyl, CN, or H or joined to form -O- (CH O . W may be and R ⁰ are independently OCH2CHrN(Et)2, methyl-substituted piperazinyl, hydroxyl, methoxy, -

NHSO2-CH3. CN, or H or joined to form () ( Ci i • ) : -? < ) W may be C( (.?)-( I C. -CH?, or H

R ² may be CN, NO?., halo, C(=O)OH, C(=O)O-alkyl, alkyl, hydroxyl, or H. Exemplary' R ² include CN, NO2, Cl, Br, CfoOjOH, ( t ())O-CH . CH?, hydroxyl, or H.

R ^J may be H, halo, amino, CN, alkoxy, hydroxyl, or amido. Exemplary' R? include H, F, Cl, NH2, CN, OCH?, hydroxyl, or NHC(-O)CH ₃ .

R' may be alkoxy, hydroxyl, or H. Exemplary' R ⁵ include hydroxyl, methoxy, or H.

A may be N or CH

Z may be N of CH.

X-Y may be NR ⁴ -C(~O) or CH ^:::: CH and R ⁴ may be an alkyl or heterocycloalkyl.

Exemplary R ⁴ include CH? or piperidinyl.

In some embodiments, the compound is of formula

In some embodiments, the compound is of formula

In some embodiments of the disclosed compounds, the compound is selected from

or a pharmaceutically acceptable salt thereof. Also disclosed herein are methods for treating a subject with a cold tumor. In one embodiment the cancer may be melanoma.

Also disclosed herein are methods for treating diseases and disorders associated with cancer in a subject in need thereof. The methods may include administering an effective amount of a pharmaceutical composition including a compound of the above formulae for activating MLKL activity in the subject.

In some embodiments, the method for treating a subject in need of a compound having MLKL activating activity including administering to the subject a compound as disclosed herein.

Also disclosed herein, is a method wherein administering the compound to the subject induces necroptosis in cancer cells within the subject. In one embodiment, the administration of the compound to the subject may induce release of danger associated molecular patterns (DAMPs) Also disclosed herein are pharmaceutical composition for the treatment of a subject in need of a compound having MLKL activating activity. In one embodiment the pharmaceutical composition includes an effective amount of the compound.

In some embodiments, the compound is not

In some embodiments, the compound has MLKL activating activity.

In some embodiments, the compound binds a MLKL pseudokinase domain.

Also disclosed herein is a pharmaceutical composition including of any of the aforementioned compounds or a pharmaceutically acceptable salt thereof and may include a pharmaceutically carrier, excipient, or diluent.

The formulae of the compounds disclosed herein should be interpreted as encompassing all possible stereoisomers, enantiomers, or epimers of the compounds unless the formulae indicates a specific stereoisomer, enantiomer, or epimer. The formulae of the compounds disclosed herein should be interpreted as encompassing salts, esters, amides, or solvates thereof of the compounds Use of the Disclosed Compounds for activating MLKL Activity

The disclosed compounds may exhibit one or more biological activities. The disclosed compounds may activate MLKL activity by altering the inactive “open’’ conformation of MLKL pseudokinase domain to an active “closed” conformation (Figure 5).

As demonstrated in Figure 4, human MLKL may be activated by the compounds disclosed herein. MEF cells expressing human MLKL have been generated. Because mouse RIPK3 cannot phosphorylate human MLKL, human MLKL in these cells can only be activated in a manner that does not involve RIPK3 activity. Indeed, UH15-22 induces death in these cells, indicating its ability to induce MLKL-dependent death in a. manner that is independent of upstream RIPK3 step in the pathway.

As demonstrated in Figure 6, the compounds disclosed herein induce necroptosis directly through binding MLKL. The activity of ALD-6-85 was tested in the cells expressing either wild type MLKL or mutant expressing a bulky Trp residue in place of M272 gatekeeper residue. This mutation is expected to disrupt binding of the drug to MLKL. indeed, ALD-6-85 induces reduced death in the cells expressing M272W MLKL compared to wild type protein. To ensure that observed reduction in killing is not due to the intrinsically lower pro-death activity of M272W mutant, cells were also stimulated with I \F 1DX. inducing necroptosis through a canonical TNFR1-RIPK1-RIPK3-MLKL pathway. In this case, M272W mutation had no effect on activation of necroptosis. This result indicates that ALD-6-85 induces death through direct binding to MLKL as it displays reduced activity against a mutant with reduced compound binding. We also generated MEFs expressing S345A, S347A double mutant of mouse MLKL. These residues are directly phosphorylated by RIPK3 and their mutation to the non-phopshorylatable alanines leads to the loss of killing by TNF+IDN. In contrast, it has minor effect on killing by SS-1-127, consistent with this molecule directly activating MLKL and not requiring upstream RIPK3 activity.

As demonstrated in Figure 7, mutation of MLKL can lower the ICso of the compounds disclosed herein. Glu239 residue plays a key role in blocking MLKL activation. E239Q mutation reduces the ability of this residue to participate in non-covalent interactions (e g., ionic-dipole), thus resulting in partially active MLKL protein, inducing limited cell death upon expression. SS- 1-127 displays much improved activity when combined with E239Q mutation compared to wild type MLKL, suggesting a much better fit for SS-1-127 with the active conformation of the MLKL compared to UH 15 -22 which induces cell death to the same degree in the cells expressing wild type and E239Q mutant MLKL.

In some embodiments, the compounds can inhibit growth or kill tumor cells. The compounds disclosed herein may be assessed using the standard protocol described by the NCI- 60 Human Tumor Cell Lines Screen, a well -documented activity screening that utilizes 60 different human tumor cell lines to identify and characterize novel compounds with growth inhibition or killing of tumor cell line (Figure 8). In some embodiments, the disclosed compounds inhibit tumor cell growth (preferably by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% at a concentration of less than about 100 p M, 50 pM, 10 pM, 1 pM, 0.1 pM, 0.05 pM, 0.01 pM, 0.005 pM, 0.001 pM, or less). Concentration ranges also are contemplated herein, for example, a. concentration range bounded by end-point concentrations selected from 0.001 pM, 0.005 pM, 0.01 pM, 0.5 pM, 0.1 pM, 1.0 pM, 10 pM, and 100 pM.

The disclosed compounds may be effective in inhibiting cell proliferation of cancer cells, by activating necroptosis and by activating MLKL activity The disclosed compounds may be effective in inhibiting cell proliferation of one or more types of primary cancer cells as well as cancer cell lines, including: multiple myeloma cells, such as MM. IS cells; leukemia cells, such as CCRF-CEM, HL-60(TB), MOLT-4, RPMI-8226, M0LM14, KU812 and SR; non-small lung cancer cells, such as A549/ATCC, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H322M, NCI-H460 and NCI-H522; colon cancer cells, such as COLO 205, HCC-2998, HCT-116, HCT- 15, HT29, KM 12 and SW-620; CNS: SF-268, SF-295, SF-539, SNB-19, SNB-75 and 11251; bladder cancer cells, such as D-BLC1, melanoma cancer cells, such as LOX IMVI, MALME-3M, M14, MDA-MB-435, SK-MEL-2, SK-MEL-28, SK-MEL-5, UACC-257 and UACC-62; ovarian cancer cells, such as IGR-OV1, OVCAR-3, O VC ARM, OVCAR-5, OVCAR-8, NCI/ADR-RES and SK-OV-3; renal cancer cells, such as 786-0, A498, ACHN, CAKI-1 , RXF 393, SN12C, TR10 and UO-31; prostate cancer cells, such as DU-145 and PC-3; and breast cancer cells, such as MCF7, MDA-MB-231/ATCC, MDA-MB-468, HS 578T, BT-549 and T-47D.

Cell proliferation and inhibition thereof by the presently disclosed compounds may be assessed by cell viability methods disclosed in the art including colorimetric assays that utilize dyes such as MTT, XTT, and MTS to assess cell viability. Preferably, the disclosed compounds have an ICso of less than about 10 uM, 5 pM, 1 uM, 0.5 pM, 0.01 gM, 0.005 pM, 0.001 pM or lower in the selected assay.

The disclosed compounds may be formulated as anti-cancer therapeutics, including anticancer therapeutics for the treatment of hematologic malignancies, breast, lung, pancreas, and prostate malignancies. In other embodiments, the disclosed compounds also may be formulated as anti-inflammation therapeutics.

The compounds utilized in the methods disclosed herein may be formulated as pharmaceutical compositions that include: (a) a therapeutically effective amount of one or more compounds as disclosed herein; and (b) one or more pharmaceutically acceptable carriers, excipients, or diluents. The pharmaceutical composition may include the compound in a range of about 0.1 to 2000 mg (preferably about 0.5 to 500 mg, and more preferably about 1 to 100 nig). The pharmaceutical composition may be administered to provide the compound at a daily dose of about 0.1 to about 1000 mg/kg body weight (preferably about 0.5 to about 500 mg/kg body weight, more preferably about 50 to about 100 mg/kg body weight). In some embodiments, after the pharmaceutical composition is administered to a subject (e.g, after about 1, 2, 3, 4, 5, or 6 hours post-administration), the concentration of the compound at the site of action may be within a concentration range bounded by end-points selected from 0.001 pM, 0.005 pM, 0.01 uM, 0.5 pM, 0.1 pM, 1.0 pM, 10 pM, and 100 pM (e.g., 0.1 pM - 1.0 pM).

The disclosed compounds and pharmaceutical compositions comprising the disclosed compounds may be administered in methods of treating a subject in need thereof. For example, in the methods of treatment a subject in need thereof may include a subject having a cell proliferative disease, disorder, or condition such as cancer (e.g., cancers such as multiple myeloma, leukemia, non-small cell lung cancer, colon cancer, cancer of the central nervous system, melanoma, ovarian cancer, renal cancer, prostate cancer, and breast cancer).

In some embodiments of the disclosed treatment methods, the subject may be administered a dose of a compound as low as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 me, 52 5 mg, 55 mg, 57.5 mg, 60 me, 62.5 me, 65 mg, 67.5 mg. 70 mg, 72.5 mg. 75 me, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg, 200 mg, 500 mg, 1000 mg, or 2000 mg once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times per week in order to treat the disease or disorder in the subject. In some embodiments, the subject may be administered a dose of a compound as high as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12,5 rag, 15 mg, 17.5 mg, 20 mg, 22 5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg, 60 mg, 62 5 mg, 65 mg, 67.5 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg, 200 mg, 500 mg, 1000 mg, or 2000 mg, once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times per week in order to treat the disease or disorder in the subject. Minimal and/or maximal doses of the compounds may include doses falling within dose ranges having as end-points any of these disclosed doses (e.g., 2.5 mg - 200 mg).

In some embodiments, a minimal dose level of a compound for achieving therapy in the disclosed methods of treatment may be at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, 1900. 2000, 3000, 4000. 5000, 6000, 7000, 8000, 9000. 10000, 15000. or 20000 ng/kg body weight of the subject. In some embodiments, a maximal dose level of a compound for achieving therapy in the disclosed methods of treatment may not exceed about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, or 20000 ng/kg body weight of the subject. Minimal and/or maximal dose levels of the compounds for achieving therapy in the disclosed methods of treatment may include dose levels falling within ranges having as end-points any of these disclosed dose levels (e.g., 500 - 2000 ng/kg body weight of the subject).

The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition in solid dosage form, although any pharmaceutically acceptable dosage form can be utilized Exemplary solid dosage forms include, but are not limited to, tablets, capsules, sachets, lozenges, powders, pills, or granules, and the solid dosage form can be, for example, a fast melt dosage form, controlled release dosage form, lyophilized dosage form, delayed release dosage form, extended-release dosage form, pulsatile release dosage form, mixed immediate release and controlled release dosage form, or a combination thereof.

The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes a carrier. For example, the carrier may be selected from the group consisting of proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, and starch-gelatin paste.

The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes one or more binding agents, filling agents, lubricating agents, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, and effervescent agents. Filling agents may include lactose monohydrate, lactose anhydrous, and various starches; examples of binding agents are various celluloses and crosslinked polyvinylpyrrolidone, microcrystalline cellulose, such as Avicel® PH101 and Avicel® PHI 02, microcrystalline cellulose, and silicified microcrystalline cellulose (ProSolv SMCC'™). Suitable lubricants, including agents that act on the flowability of the powder to be compressed, may include colloidal silicon dioxide, such as Aerosil®200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel. Examples of sweeteners may include any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acsulfame. Examples of flavoring agents are Magnasweet® (trademark of MAFCO), bubble gum flavor, and fruit flavors, and the like. Examples of preservatives may include potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride. Suitable diluents may include pharmaceutically acceptable inert fillers, such as microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and mixtures of any of the foregoing. Examples of diluents include microcry stalline cellulose, such as Avicel® PHI 01 and Avicel® PHI 02; lactose such as lactose monohydrate, lactose anhydrous, and Phamiatose® DCL21; dibasic calcium phosphate such as Em compress®; mannitol; starch; sorbitol; sucrose; and glucose.

Suitable disintegrants include lightly crosslinked polyvinyl pyrrolidone, corn starch, potato starch, maize starch, and modified starches, croscarmellose sodium, cross-povidone, sodium starch glycolate, and mixtures thereof.

Examples of effervescent agents are effervescent couples such as an organic acid and a carbonate or bicarbonate. Suitable organic acids include, for example, citric, tartaric, malic, fumaric, adipic, succinic, and alginic acids and anhydrides and acid salts. Suitable carbonates and bicarbonates include, for example, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium glycine carbonate, L-lysine carbonate, and arginine carbonate. Alternatively, only the sodium bicarbonate component of the effervescent couple may be present.

The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition for delivery via any suitable route. For example, the pharmaceutical composition may be administered via oral, intravenous, intramuscular, subcutaneous, topical, intratumoral, and pulmonary route Examples of pharmaceutical compositions for oral administration include capsules, syrups, concentrates, powders, and granules. In some embodiments, the compounds are formulated as a composition for administration orally (e.g., in a solvent such as 5% DMSO in oil such as vegetable oil).

The compounds utilized in the methods disclosed herein may be administered in conventional dosage forms prepared by combining the active ingredient with standard pharmaceutical carriers or diluents according to conventional procedures well known in the art. These procedures may involve mixing, granulating, and compressing or dissolving the ingredients as appropriate to the desired preparation.

Pharmaceutical compositions comprising the compounds may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual, ortransdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, or intradermal) route. Such formulations may be prepared by any method known in the art of pharmacy', for example by bringing into association the active ingredient with the carrier(s) or excipient(s).

Pharmaceutical compositions adapted for oral administration may be presented as discrete units such as capsules or tablets, powders or granules; solutions or suspensions in aqueous or nonaqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions.

Pharmaceutical compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch byiontophoresis.

Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, impregnated dressings, sprays, aerosols or oils and may contain appropriate conventional additives such as preservatives, solvents to assist drug penetration and emollients in ointments and creams.

For applications to the eye or other external tissues, for example the mouth and skin, the pharmaceutical compositions are preferably applied as a topical ointment or cream. When formulated in an ointment, the compound may be employed with either a paraffinic or a water- miscible ointment base. Alternatively, the compound may be formulated in a cream with an oil- in-water cream base or a water-in-oil base Pharmaceutical compositions adapted for topical administration to the eye include eye drops where the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent.

Pharmaceutical compositions adapted for nasal administration where the carrier is a solid include a coarse powder having a particle size (e.g., in the range 20 to 500 microns) which is administered in the manner in which snuff is taken (/.<?,, by rapid inhalation through the nasal passage from a container of the powder held close up to the nose) Suitable formulations where the earner is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient.

Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented 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 injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine, tabletting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica, disintegrants, for example potato starch; or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives, such as suspending agents, for example sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and, if desired, conventional flavoring or coloring agents.

Combination Therapies and Pharmaceutical Compositions

The disclosed compounds or pharmaceutical compositions comprising the disclosed compounds may be administered in methods of treatment. For example, the disclosed compounds or pharmaceutical compositions comprising the disclosed compounds may be administered in methods of treating cell proliferative diseases and disorders. Cell proliferative diseases and disorders treated by the disclosed methods may include, but are not limited to, cancers selected from the group consisting of leukemia (e.g., AML or CML), lung cancer (e.g., non-small cell lung cancer), cancer of the central nervous system (e.g., glioblastoma), melanoma, ovarian cancer, renal cancer, prostate cancer, head and neck, urinary tract, sarcoma, lymphoma (e.g., Hodgkin's lymphoma), and breast cancer.

Optionally, the disclosed compounds or pharmaceutical compositions comprising the disclosed compounds may be administered with additional therapeutic agents, optionally in combination, in order to treat cell proliferative diseases and disorders. In some embodiments of the disclosed methods, one or more additional therapeutic agents are administered with the disclosed compounds or with pharmaceutical compositions comprising the disclosed compounds, where the additional therapeutic agent is administered prior io, concurrently with, or after administering the disclosed compounds or the pharmaceutical compositions comprising the disclosed compounds In some embodiments, the disclosed pharmaceutical composition are formulated to comprise the disclosed compounds and further to comprise one or more additional therapeutic agents, for example, one or more additional therapeutic agents for treating cell proliferative diseases and disorders.

In some embodiments, the disclosed compounds or pharmaceutical compositions comprising the disclosed compounds may be administered with an immunotherapy agent. The term “immunotherapy agent/ s)” refers to any therapeutic that is used to treat cancer in a subject by inducing and/or enhancing an immune response in that subject. Immunotherapy agents may include, without limitation, checkpoint inhibitors.

Checkpoint inhibitors are therapeutics, such as antibodies, that block the immune checkpoint pathways in immune cells that are responsible for maintaining self-tolerance and modulating the degree of an immune response. Tumors often exploit certain immune checkpoint pathways as a major mechanism of immune resistance against T cells that are specific for tumor antigens. Many of the immune checkpoints are initiated by receptor-ligand interactions and thus may be blocked by antibodies to either the ligand or receptor or may be modulated by soluble recombinant forms of the ligands or receptors. Such immune checkpoint blockade allows tumorspecific T cells to continue to function in an otherwise immunosuppressive tumor microenvironment. Checkpoint inhibitors, however, are not effective against all cancer types. Furthermore, not every patient that is expected to respond to immune checkpoint blockade actually benefits from treatment with such agents.

Exemplary' checkpoint inhibitors include, without limitation, antibodies or other therapeutics targeting programmed cell death protein 1 (PDl, also known as CD279), programmed cell death 1 ligand 1 (PD-L1 , also known as CD274), PD-L2, cytotoxic T-lymphocyte antigen 4 (CTLA4, also known as CD 152), A2AR, CD27, CD28, CD40, CD80, CD86, CD122, CD137, OX40, G1TR, ICOS, TIM-3, LAG3, B7-H3, B7-H4, BTLA, IDO, KIR, or VISTA Suitable anti- PD1 antibodies include, without limitation, lambrolizumab (Merck MK-3475), nivolumab (Bristol-Myers Squibb BMS-936558), AMP-224 (Merck), and pidilizumab (CureTech CT-011). Suitable anti-PD-Ll antibodies include, without limitation, MDX-1105 (Medarex), MEDI4736 (Medimmune) MPDL3280A (Genentech/Roche) and BMS-936559 (Bristol-Myers Squibb). Exemplary anti-CTLA4 antibodies include, without limitation, ipilimumab (Bristol-Myers Squibb) and tremelimumab (Pfizer).

EXAMPLES

The following Examples are illustrative and are not intended to limit the scope of the claimed subject matter.

Example 1:

General information

All reactions involving air-sensitive reagents were carried out with magnetic stirring and oven-dried glassware with rubber septa under argon unless otherwise stated. All commercially available chemicals and reagent grade solvents were used directly without further purification, unless otherwise specified. Reactions were monitored by thin-layer chromatography (TEC) on Baker-flex® silica gel plates (IB2-F) using UV-light (254 and 365 nm) detection or visualizing agents (e.g., iodine, ninhydrin or phosphomolybdic acid stain). Flash chromatography was conducted on a silica gel (230-400 mesh) using a Teledyne ISCO CombiFalsh® Rf. NMR spectra were recorded at room temperature using a JEOL ECA-600 instrument (1H NMR at 600 MHz and 13C NMR at 150 MHz) with tetramethylsilane (TMS) as an internal standard. Chemical shifts (5) are given in parts per million (ppm) with reference to solvent signals [1H-NMR: CDC13 (7.26 ppm), CD3OD (3.31 ppm), DMSO-d6 (2.50 ppm), 13C-NMR: CDC13 (77.0 ppm), CD3OD (49.0 ppm), DMSO-d6 (39.5 ppm)]. Signal patterns are reported as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and br (broad). Coupling constants (J) are given in Hz. High-resolution mass spectra (HRMS) were obtained using a Qstar Elite-ESI and reported as m/z (relative intensity) for the molecular ion [MJ. HPLC methods:

Method I) a Waters 1525 instrument equipped with Waters 2489 UV/Visible detector. Kinetex 5pm C18 100A column (250 X 4 6 mm) was used for analytical HPLC. HPLC gradient went from 2% to 98% CH ₃ CN in H ₂ O (both solvents contain 0.1% trifluoroacetic acid) with a total run time of 30 min and a flow rate of 1 mL/min for analytical analysis and 10 mL/min for preparative purification.

Method II) a Waters 2545 instrument equipped with Waters 2489 UV/Visible detector. Kinetex 5pm C18 100A column (250 X 4.6 mm) was used for analytical HPLC. HPLC gradient went from 2% to 98% CH ₃ CN in H ₂ O (water contain 0.1% formic acid) with a total run time of 35 min and a flow rate of 1 2 mL/min for analytical analysis and 10 mL/min for preparative purification.

Method III) a Waters 2545 instrument equipped with Waters 2489 UV/Visible detector. Kinetex 5μm C18 100A column (250 X 4.6 mm) was used for analytical HPLC. HPLC gradient went from 2% to 98% MeOH in H ₂ O (water containing 0.1% formic acid) with a total run time of 35 min and a flow rate of 1.2 mL/min for analytical analysis and 10 mL/min for preparative purification.

Method IV) a Waters 2545 instrument equipped with Waters 2489 UV/Visible detector. Kinetex 5pm C18 100A column (250 X 4.6 mm) was used for analytical HPLC. HPLC gradient went from 98% to 2% IP A in H ₂ O (water containing 0.1% formic acid) with a total run time of 30 min and a flow rate of 1.2 mL/min for 10 min and 0.5 mL/min for 20 min for analytical analysis and 10 mL/min for preparative purification.

Ethyl 4-(niethylamino)-2-(methylthio)pyrimidine-5-carboxylate (C2a)

(Method I)

To a solution of Cl (3 g, 12.9 mmol) in anhydrous THF (30 mL) at 0 °C was added aqueous methylamine (6 mL) and stirred for 30 min. The ice bath was then removed, and the reaction mixture was stirred for 16 h. The reaction was quenched with water, and the aqueous layer was extracted twice with ethyl acetate. The combined organic phases were washed with brine, dried over anhydrous NaiSCh, filtered, and concentrated. The crude product was separated by (0-10 % EtOAc in Hexane) and yielded C2a (90%) as a white solid. ^l H-NMR (600 MHz, CDCh) 8 8.61 (s, 1H), 8.18 (s, 1 H), 4.32 (q, J - 7.1 Hz, 2H), 3.08 (d, J - 5.0 Hz, 3H), 2.55 ( _s , 3H), 1.37 (t. J - 7.1 Hz, 3H). ¹³ C-NMR (150 MHz, CDCh) 6 176.0, 167.0, 160.6.0, 158.1.0, 100.9, 60.7, 27.2, 14.19, 14.15.

(4-Chloro-2-(methylthio)pyrimidin-5-yl)methanol (C2b)

To a solution of Cl (3 g, 12.9 mmol) in anhydrous THF (200 mL) was stirred at 0 °C, DIBAL (22 mL, 38.7 mmol) was added dropwise. The reaction mixture was stirred under argon at 0 °C for 4 h. The reaction was cooled to 0 °C, Dl-water (10 mL) and 15% NaOH (10 mL) was added dropwise. Another 10 mL Dl-water was added and stirred for 30 min. The mixture was then filtered, washed with DCM, and concentrated. The organic phase was washed with brine, dried over anhydrous NarSCh, filtered, and concentrated. The crude product was separated by (0-10 % EtOAc in DCM) and yielded C2b (55%) as a white solid. ¹ H-NMR (600 MHz, CDCh) 8 8.52 (s, 1H), 4.71 (s, 2H), 3.07 (s. 11 1), 2.55 (s, 3H). ¹³ C-NMR (150 MHz, CDCh) 6 172.2, 159.4, 157.0,

126.8, 59.3, 14.3.

(4-(Methylamino)-2-(methylthio)pyriinidin”5-yl)methanol (C3a)

(Method J)

A solution of LAH (2.17 g, 9.54 mmol) in anhydrous THF (15 ml) stirred a 0 °C and a solution of C2a (362 mg, 9.54 mmol) in anhydrous THF (10 mL) was added dropwise. The reaction mixture was stirred under argon at room temperature for 30 min. The reaction was cooled io 0 °C, and Dl-water (1 mL) and 15% NaOH (0.5 mL) were added dropwise and stirred for 1 h The mixture was then filtered, washed with EtOAc, and concentrated. The crude product was separated by chromatography (10-60% EtOAc in DCM) and yielded C3a (90%) as a white solid. HAMR (600 MHz, CEhOD) 5 7.72 (s, 1H), 4.42 (s, 2H), 3.01 (s, 3H), 2.50 (s, 31 !) ^l3 C-NMR (150 MHz, CDsOD) 5 171.8, 162.3, 152.4, 113.8, 59.8, 27.7, 13.9.

teH-Butyl 4-((5-(hydroxyrnethyl)-2-(methylthio)pyrimidin-4-yl)amino)pi peridme-l -carboxylate (C3b)

To a solution of C2b (16 mg, 0.086 mmol) in EtOH (2 mL) was added DIPEA (0.09 mL, 0.52 mmol) dropwise at room temperature under argon. C2c (19.3 mg, 0.096 mmol) in EtOH was added over 12 h at 80 °C and stirred for 4 d. The reaction mixture was cooled to room temperature, dried under vacuum, diluted with DCM, and washed with water. The organic phase was washed with brine, dried over anhydrous NasSCh, filtered, and concentrated under vacuum. The crude product was separated by (0-5 % MeOH in DCM) and yielded C3b (88%) as a white solid, ¹ H- NMR (600 MHz, CDCh) 3 7.54 (s, 1H), 6.06 (d, J = 7.1 Hz, 1H), 4.43 (s, 2H), 4.13 (t, J = 3.3 Hz, I H), 3.94 (s, 2H), 2.92 (s, 21 h, 2.43 (s, 3H), 1.97 (d, J - 11.0 Hz, 2H), 1.42 (s. 9H), 1 .38 (rn, 2H) ! ³ C-NMR (150 MHz, CDCh) 8 170.5, 160.2, 154.7, 151 7, 111.8, 79.7, 60.3, 47.3, 42.6, 42.0,

31.6, 28.3, 13.9.

4-(Methylamino)-2-(methylthio)pyrimidine-5-carbaldehyde (C4a)

(Method K)

To a mixture of C3a (1.7 g, 9 24 mmol) in anhydrous DCM (53 mL) was added MnCh (4.82 g, 55.44 mmol) and stirred at room temperature overnight. The reaction mixture was filtered and concentrated. The crude product was separated by chromatography (0-20% EtOAc in Hexane) and yielded C4a (83%) as a white solid. 'H-NMR (600 MHz, CDCh) 6 9.70 (s, 1H), 8.56 (br, 1H), 8 29 (s, 1 H), 3.12 (d, J == 2,4 Hz, 3H), 2,57 (s, 3H). ¹³ C-NMR (150 MHz, CDCh) 8 190 8, 177.5, 162.7, 159.4, 109.4, 27.1, 14,2. tert-Butyl 4-((5-formyi-2-(methylthio)pyrimjdin-4-yl)amino)piperidine-1 -carboxylate (C4b)

Method K; The reaction mixture was filtered and concentrated, yielding C4b (77%) as a white solid. 'H-XMR. (600 MHz, CDCls) 5 9.69 (s, 1H), 8.59 (d, J - 7.1 Hz, H i), 8.31 (s, i l l) 4.30-4.26 (m, 1H), 4.02 (s, 2H), 2.99 (s, 2H), 2.53 (s, 3H), 2.01 (d, J = 10.7 Hz, 2H), 1.54-1.49 (m, 2H). 1.46 (s, Oi l s. ^l3 C-NMR (150 MHz, CDCh) 5 190 8, 177.4, 162.9, 158.1, 154.6, 109.1, 79 8, 47.5, 42.7, 42, 31.4, 28.4, 14.2.

8-Methyl-2-(methylthio)-7-oxo-7,8-dihydropyrido[2,3-<f jpyrimidine-6-carboxylic acid (C5a) (Method L) To a solution of C4a (1 16g, 6.32 mmol) in EtOH (11 6 mL) was added isopropylidene maionate (910 mg, 6.32 mmol), piperidine (0.063 mL, 0.63 mmol) and acetic acid (0.11 mL, 1.9 mmol) and stirred for 20 min at room temperature. The reaction mixture was refluxed at 70 °C for 2 h. The reaction was cooled to room temperature, and the precipitant was filtered and washed with EtOH twice and dried under a high-pressure vacuum. Product C5a was afforded as a white solid with a 75% yield. H-XMR (600 MHz, DMSO-de) 5 9.16 (s. H l). 8.81 (s. H l).. 3.68 (s, 3H), 2.65 (s, 3H). ^!3 C-NMR (150 MHZ, DMSO-do) 5 174.9, 164.1, 162.9, 159.8, 154.1, 141 .9, 119.6, 109.1, 28.2, 14.1.

8-(l-(tert-Butoxycarbonyl)piperidin-4-yl)-2-(methy1thio)- 7-oxo-7,8-dihydropyrido[2,3- tfjpyrimidine-6-carboxylic acid (C5b)

Method L; The crude product was washed with ether multiple times. Product C5b (92%) was afforded as a white solid. ¹ H-NMR (600 MHz, CDCI3) 3 8.86 (s, 1H), 8.82 (s, 1H), 5.36-5.98 (1H), 4.42-4.29 (m, 2H), 3.05-2.85 (m, 4H), 2.64 (s, 3H), 1.68 (s, 2H), 1.50 (s, 9H). ¹³ C-NMR (150 MHz, CDCh) 8 176.9, 165.6, 163 5, 159.7, 154.3, 143 4, 1 18 1 , 109.1, 79.8, 54.2, 44.2, 43.3, 28.3, 27.7, 14.8. 6-Iodo-8-methyl-2-(niethylthio)pyrido[2,3-d]pyrimidin-7(8//) -one (C6a)

(Method M)

A mixture of C5a (26.3 m, 0.105 mmol), LiOAC (8.3 mg, 0.126 mmol), and NIS (75 4 mg, 0.335 mmol) in anhydrous DMF/DI-water (1.2/0.12 mL) was place under microwave irradiation at 1 10 °C for 15 min Dl-water was added to the reaction mixture, and the aqueous layer was extracted with EtOAc and washed with saturated aqueous solution of Na2S2O3. The organic phase was washed with brine, dried over anhydrous NazSCh, filtered, and concentrated. The crude product was separated by chromatography (5-10% EtOAc in Hexane). Product C6a (78%) was afforded as a pale-yellow solid. ’H-NMR (600 MHz, CDCh) 8 8.59 (s, 1H), 8.33 (s, 1H), 7 27 (s, 1H), 3.83 (s, 3H), 2.64 (s, 3H). ¹³ C-NMR (150 MHz, CDCh) 8 173.8, 159.8, 155.1, 154.4, 143.9, 110.68, 93.4, 29.6, 14.5.

tert-Butyl 4-(6-iodo-2-(methylthio)-7-oxopyrido[2,3-<7]pyrimidin-8(7 77)-yl)piperidine-l- carb oxy late (C6b)

Method M; The crude product was separated using chromatography (10-15% EtOAc in Hexane). Product C6b (95%) was afforded as a pale-yellow solid. ¹ H-NMR (600 MHz, CDCh) 8 8.50 (s, 1H), 8.24 (s, 1H), 5.51 (s, I H ) 4.22 (d, J - 73.8 Hz, 2H), 2,76 (d, J - 62.3 Hz, 4H), 2.52 (d, J = 21.3 Hz, 3H), 1.52 (d, J = 38.9 Hz, 2H), 1.39 (s, 9H). ^1;< C-NMR (150 MHz, CDCh) 5 172.7, 159.3, 155.4, 154.2, 154.0, 144 1 , 110.8, 79.3, 54.2, 44.0, 43.1, 28.2, 27.6, 14.5. 6-Iodo-8-methyl-2-(methylsulfonyl)pyrido[2,3-c/]pyrimidin-7( 8//)-one (C7a)

(Method N)

To a solution of C6a (100 mg, 0.3 mmol) in anhydrous DCM (3.5 mL) was added m-CPBA (155.5 mg, 0.9 mmol) at 0 “C. The reaction was slowly cooled to room temperature for 1 h. The mixture was dried under vacuum and washed with hexane and ether multiple times. Product C7a was afforded as a pale-yellow solid in 95% yield. Tl-NMR (600 MHz, CDCh) 58.97 (s, 1H), 8.55 (s, 1H), 3.92 (s, 3H), 3.42 (s, 3H). ⁿ C-NMR (150 MHz, CDCh) 6 164.5, 159.3 155.9, 155.2, 142.9, 115 8, 100.7, 39.2, 30.4.

fe/7-buty] 4-(6-lodo-2-(rnethylsulfony3)-7-oxopyrido[2,3-d]pyrimidin-8( 7//)-yl)piperidine-l- carboxylate (C7b)

Method N; The crude product was separated using chromatography (20-40% EtOAc in Hexane). Product C7b (89%) was afforded as a white solid. ’H-NMR (600 MHz, CDClr) 6 8.94 (s, 1H), 8.49 (s, 1H), 5.60 (s, 1H), 4.31 (d, J = 77.8 Hz, 2H), 3.36 (s, 3H), 2.86 (d, J = 49 1 Hz, 4H), 1.68 (s, 2H), 1.50 (d, J == ^: 18.4 Hz, 9H). ^1? C-NMR (150 MHz, CDCh) 8 163.9, 159.1, 156.3, 155.0, 154.4, 143.1, 116.2, 79.9, 55.5, 44.1 , 4.1, 39.3, 28.4, 27.9. 4-(2-(Diethylamino)ethoxy)aniline (C9a)

To a solution of C8 (1.02 g, 4.27 mmol) in MeOH (25 ml..) under Hr gas was added 30% Pd/C (305 mg) and stirred at room temperature for 16 h under Hr. The mixture was filtered through celite, washed with DCM, and dried under a vacuum. Product C9a was afforded as a brown oil with a 98% yield. 'H-NMR (600 MHz, CDCh) 6 6.75 (dd, J - 6.6, 2.2 Hz, 2H), 6.63 (dd, J = 6.6, 2.2 Hz, 2H), 3.97 (t, J = 6.5 Hz, 2H), 3.69-3.15 (br, 2H), 2.83 (t, J = 6.5 Hz, 2H), 2.62 (q, J = 7.2

Hz, 4H). ^i? C-NMR (150 MHz, CDCh) 8 152.0, 139.9, 116.3, 115.5, 66 9, 51.8, 47.7, 1 1 7. 2-((4-(2-(Diethylamino)ethoxy)phenyl)aniino)-6-iodo-8-methy! pyrido[2,3-rf]pyrimidin-7(877)- one (ClOa)

(Method ())

A mixture of C7a (30 mg, 0.082 mmol) and C9a (20.5 mg, 0.1 mmol) in anhydrous toluene (2 mL) in a sealed tube was refluxed at 110 °C for 2 days. The reaction mixture was dried under vacuum and separated by chromatography (5% MeOH in DCM and 0.1% NH4OH). Product ClOa was afforded as a yellow solid in 41% yield. ‘H-NMR (600 MHz, CDCh) 8 8.47 (s, 1H), 8. 19 (s, 1H), 7.52 (d, J == 8.3 Hz, 2H), 6.94 (d, J = 8.8 Hz, 2H), 4.07 (t, J == ^: 6.3 Hz, 2H), 3.76 (s, 3H), 2.90 (t, J = 6.3 Hz, 2H), 2.66 (q, J = 7.1 Hz, 4H), 2.06-1.72 (1H), 1.09 (t, J = 7.1 Hz, 6H). ( -?\MR (150 MHz, CDCh) 8 160.3, 157.3, 155.9, 155.6, 144.2, 131.2, 131.1 , 122.0, 114.9, 108.2, 88.1, 66.8, 51.7, 47.8, 29.6, 11.8.

2-((3-(2-(Diethylamino)ethoxy)pheny1)ainino)-6-iodo-8-met hylpyrido[2,3-d]pyrimidin-7(8/f)- one (Cl 0b)

Method O: The reaction mixture was dried under vacuum and separated by chromatography (5% MeOH in DCM and 0.1% NH-iOH). Product Cl 0b was afforded as a lightyellow solid in 26% yield. ‘H-NMR (600 MHz, CD3OD) 5 8.66 (s, 1H), 8 49 (s, 1H), 7.63 (s, 1H), 7.26-7.31 (m, 2H), 6.73 (d, J == 7.2 Hz, 1H), 4.33 (t, J - 4.9 Hz, 2H), 3.79 (s, 3H), 3.51 (s, 2H), 3 24 (m, 4H), 1 34 (t, J = 7.2 Hz, 6H). ^!3 C-NMR (150 MHz, CD3OD) 5 162.3, 160.6, 159.5, 159.3, 156.9, 146.8, 142.1, 130.7, 114.5, 109.9, 109.8, 107.5, 87.8, 63.9, 55.0, 52.4, 30.2, 9.5.

2-((4-Hydroxyphenyl)amino)-6-iodo-8-methylpyrido[2,3-i/]p yrimidin-7(8/Z)-one (ClOc)

Method O; The crude product was separated by chromatography (0-25% EtOAc in Hexane) and yielded ClOc (50%) as a yellow solid. ‘H-NMR (600 MHz, DMSO-de) 8 9.99 (s, 1H), 9.24 (s, 1H), 8.68 (s, 1 H), 8.51 (s, 1H), 7.54 (s, 21 h, 6.74 (d, J == 8.6 Hz, 21 B, 3.60 (s, 3H) i ³ C-NMR (150 MHz, DMSO-de) S 159.8, 158.9, 158.1, 155.4, 153.2, 144.9, 130.9, 121.6, 115.1,

107.4, 86 9, 29 1.

2-(Benzo[cZ][l,3]dioxol-5-ylamino)-6-iodo-8-methylpyrido[ 2,3-«7]pyrimidin-7(87f)-one (ClOd)

Method O; The crude product was separated by chromatography (5% MeOH in DCM and 0.1% NH-iOH ) and yielded ClOd (70%) as a yellow solid. ^f H~NMR (600 MHz, DMSO-ds) 5 10.14 (s, 1H), 8.72 (s, 1H), 8.55 (s, 1H), 7.50 (s, 1H), 7.18 (d, J = 6.7 Hz, 1H), 6.91-6.87 (m, 1H), 6.00 (s, 2H), 3.63 (s, 3H). ^{, 3} C-NMR (600 MHz, DMSO-de) 8 159.8, 158.7, 158.1, 155.3, 147.1, 144 8, 142.6, 139.2, 133.8, 128.1, 124.9, 108.0, 101.0, 87.6, 30.4. tert-butyl 4-(2-((4-(2-(Diethylamino)ethoxy)phenyl)amino)-6-iodo-7-oxop yrido[2,3-<7]pyriniidin- 8(7H)-yl)piperidine-l -carboxylate (ClOe)

(Method P)

To a solution of C7b (10 mg, 0.019 mmol) and C9a (4.7 mg, 0.023 mmol) in isopropyl alcohol in a sealed tube, TFA (0.003 mL, 0.037 mmol) was added at 0 °C under argon. The reaction mixture was slowly warmed to room temperature and then stirred at 95 °C for 2 d. The reaction mixture was dried under vacuum, diluted with DCM, and washed with the saturated aqueous NaHCOj. The aqueous layer was extracted with DCM. The combined organic phases were washed with brine, dried over anhydrous NazSCU, filtered, and concentrated. The crude product was separated by chromatography (5% MeOH in DCM) and yielded ClOe (83%) as a yellow solid. ⁱ H- NMR (600 MHz, COCH) 6 8.46 (s, 1 H). 8 15 (s, H I), 747 (d, J - 8.8 Hz, 2H), 6 93-6 91 (m, 2H), 5.56 (s, 1H), 4.38-4.24 (m, 2H), 4.11-4,08 (m, 2H), 2.92-2.69 (m, 10H), 1.59 (s, 2H), 1.49 (s, 9H), 1.10 (t, J == 7.1 Hz, 6H). ¹³ C-NMR (150 MHz, CDCb) 6 160.1, 158.9, 157.5, 155.9, 155.6, 154.4, 144.3, 131.1, 122.3, 114.7, 108.4, 79.6, 66.7, 54.2, 51.6, 47.7, 44.5, 43.5, 29.6, 28.4, 27.6, 11.7.

6-Iodo-2-((4-methoxyphenyl)amino)-8-methylpyrido[2,3-t/]p yrimidin-7(8/f)-one (ClOf)

Method P; The crude product was separated by chromatography (5% MeOH in DCM and 0.1 % NH4OH) and yielded ClOf (78%) as a yellow solid. ^l H-NMR (600 MHz, DMSO-de) 8 10.08 (s, 1H), 8.69 (s, 1H), 8.50 (s, 1H), 7.68 (s, 2H), 6.92 (d, J = 8 6 Hz, 2H), 3.73 (s, 3H), 3.61 (s, 3H). 1 ³ C-NMR (150 MHz, DMSO-de) 8 159.8, 158.8, 158.1, 155.3, 154.9, 144.8, 132.5, 130.9, 121 2, 115.0, 113 8, 87 2, 55.2, 29.1

4-((6-Iodo-8-methyl-7-oxo-7,8-dihydropyrido[2,3-</|pyr imidin-2-yl)amino)benzonitrile (ClOg)

Method P; Hie crude product was separated by chromatography (40-100% EtOAc in Hexane) and yielded ClOg (20%) as a yellow solid. ^X H-NMR (600 MHz, DMSO-de) 5 10.65 (s, 1H), 8.79 (s, 1 H), 8.58 (s, 1H), 7.97 (d, J = 8 6 Hz, 2H), 7.77 (d, J - 8.6 Hz, 2H), .3.65 (s, 3H). ^l3 C- NMR(150 MHz, DMSO-d&) 8 159.7, 158.2, 158.0, 155.1, 144.7, 143.9, 133.1, 119.4, 119.1, 108.8, 103.5, 89.8, 29.4.

A ^; -(4-((6-lodo-8-methyl-7-oxo-7,8-dihydropyrido[2,3-t7jp yrimidin-2- yl)amino)phenyl)methanesulfonamide (ClOh) Method P; The crude product was separated by chromatography (5% MeOH in DCM) and yielded ClOh (53%) as a yellow solid. ^! H-NMR (600 MHz, DMSO-de) 8 10.19 (s, 1H), 9.57 (s, 1 H ) 8.69 (d, J === 10.8 Hz, 1H), 8.50 (d, J - 12.9 Hz, 1H), 7.74 (s, 2H), 7.19 (d, J - 7.1 Hz, 2H), 3 61 (s, 3H), 2.95 (s, 3H). ¹³ C-NMR (150 MHz, DMSO-d ₆ ) 5 159 8, 158.6, 158.1, 155.3, 144.8, 136 1, 132.9, 121.4, 120.5, 107.8, 87.9, 39.0, 29.3.

5-(2-((4-(2-(Diethylanuno)ethoxy)phenyl)ai'nino)-8-methyl -7-oxo-7,8-dihydropyrido[2,3- (7]pyrimidin-6-yl)-2-hydroxybenzonitrile (SS-1-127) (Method Q)

To a mixture of ClOa (15 mg, 0.03 mmol), Clla (4.96 mg, 0.03 mmol), PdCb(PPhi)?. (1.07 mg, 0.0015 mmol), in anhydrous DMF (0.7 ml,) was added 3M K2CO3 (0.091 mL, 0.09 mmol) under argon. The mixture was placed in MW at 110 °C for 1 h. The reaction mixture was quenched with water and extracted with MeOH/CDM multiple times. The combined organic phases were washed with brine, dried over anhydrous Na2S()r, filtered, and concentrated. The crude product was separated by chromatography (10% MeOH in DCM and 0.1% bffiUOH). and yielded SS-1-127 (78%) as a yellow solid, mp 189-191 «C H-XMR (600 MHz, CI) ₃ OD) 8 8.66 (s, 1H), 7.84 (s, 1H), 7.84 (d, J = 1.9 Hz, 1H), 7.75-7.77 (dd, J = 1.8, 0.6 Hz, 1H), 7.67 (d, J = 8.3 Hz, 2H), 6.98 (t, J == 8.3 Hz, 3H), 4.31 (t, J = 4.8 Hz, 2H), 3.72 (d, J === 7.6 Hz, 3H), 3.53 (t, J =- 4.7 Hz, 2H), 3.27 (t, J = 7.2 Hz, 4H), 1.35 (t, J = 7.3 Hz, 6H). ⁿ C-NMR (150 MHz, DMSO-ds) 3 161 9, 160.0, 159.0, 158.6, 154.6, 153.6, 134.7, 133.5, 133.0, 132.9, 127.4, 124.5, 121.2, 117 1, 116.0, 114.6, 106.2, 98.5, 50.5, 46.9, 28.1, 10.3. HRMS (ESI) m/z: [M + Hp calculated for C27H28N6O3: 485.2296, observed 485.229. Purity >95%, > 9.78 min (HPLC Method II).

2-((4-(2-(Diethylamino)ethoxy)phenyl)amino)-6-(4-hydroxy- 3-methylphenyl)-8- met.hylpyrido[2,3-r7|pyrimidin-7(8H')-one (SS- 1-128)

Method Q; The crude product was separated by chromatography (5% MeOH in DCM and 0.1% NHiOH). and yielded SS-1-128 (55%) as a yellow solid, mp 205-207 °C. ril-NMR (600 MHz, CDCh) 6 8.53 (s, 1H), 7.56 (s, 1H), 7.54 (d, J = 8.6 Hz, 2H), 7.45 (s, 1H), 7.34 (dd, J = 8.2, 2.0 Hz, 1H), 6.93 (d, J === 8.8 Hz, 2H), 6.78 (d, J == 8.3 Hz, 1H), 4 07 (t, J == 6.2 Hz, 2H), 3.76 (s, 3H), 2.90 (t, J = 6.2 Hz, 2H), 2.67 (q, J = 7.2 Hz, 4H), 2.28 (s, 3H), 1.09 (t, J = 7.1 Hz, 6H). ^l ’C- NMR (150 MHz, CDCh) 3 163.2, 158.7, 157.9, 155 2, 155.0, 154.3, 132.0, 131 7, 131.4, 129 0, 128.2, 127.5, 124.0, 121.7, 114.9, 1 14.8, 107.1, 66.7, 51.7, 47.7, 28.5, 16 0, 11.6. HRMS (ESI) m/z: [M + H]' ^f calculated for C27H31N5O3: 474.2500; observed 474.2495. Purity >95%, = 10.18 min (HPLC Method II)

5-(2-((4-(2-(Diethylamino)ethoxy)phenyl)amino)-8-methyl-7 -oxo-7,8-dihydropyrido[2,3- </jpyrimidin-6-yl)-2-hydroxybenzoic acid (SS-1-149) Method Q; The crude product was separated by chromatography (20% MeOH in DCM and

0.1% NHiOH), and yielded SS-1-149 (40%) as a yellow solid, mp: at 180 °C started to change color. ^f H-NMR (600 MHz, DMSO-ds) 6 9.91 (s, IH), 8.79 (s, IH), 7.99 (d, J = 2.2 Hz, IH), 7.87 (s, IH), 7.71 (d, J = 7.9 Hz, 2H), 7.54 (dd, J = 8.4, 2.2 Hz, IH), 6.93 (d, J = 8.8 Hz, 2H), 6.64 (d, J - 8.4 Hz, IH), 4.01 (t, J = 6.0 Hz, 2H), 3.65 (s, 3H), 2.79 (t, J - 5.9 Hz, 2H), 2.57 (q, J = 7.1 Hz, 4H), 0 98 (t, J = 7 1 Hz, 6H). ^l3 C-NMR (150 MHz, CDnOD) 8 175.9, 170.4, 165.0, 162 7, 159.8,

156.3, 156.2, 134.5, 134.4, 134.1, 132.1 , 129.4, 127.5, 123.0, 120.1 , 1 16.8, 1 15 6, 67.1, 52.6, 48.5, 28.8, 11.4. HRMS (ESI) m/z: [M + H]* calculated for C27H29N5O5: 504.2241; observed 504.2241. Purity >95%, ?R ^::: 15.08 min (HPL.C Method II). 6-(3-Bromo-4-hydroxyphenyl)-2-((4-(2-(diethylamino)ethoxy)ph enyl)amino)-8- methylpyrido[2,3-</]pyrimidin-7(82y)-one (SS-1-152)

Method Q; The crude product was separated by chromatography (10% MeOH in DCM and 0.1% NH4OH). and yielded SS-1-152 (62%) as a yellow solid, mp 188-190 °C. 'H-NMR (600 MHz, CD3OD) 6 8.67 (s, 1H), 7.83 (d, J - 5.7 Hz, 2H), 7.68 (d, J - 8.3 Hz, 2H), 7.48 (d, J - 7.4 Hz, 1H), 6.99 (d, J = 8.8 Hz, 2H), 6.94 (d, J = 8.3 Hz, 1H), 4.27 (t, J = 4.5 Hz, 2H), 3.73 (s, 3H), 3.42 (s, 2H), 3 18 (d, J = 6.5 Hz, 4H), 1.31 (t, J - 7.1 Hz, 6H). ¹³ C-NMR (150 MHz, CD 3OD) 8

164.7. 160.5, 160.1, 156.2, 155.4, 155.4, 134.9, 134.8, 134.5, 130.3, 130.2, 127.8, 122.8, 116 8,

115.7, 110.5, 108.2, 64.9, 52.5, 49.6, 28.9, 10.0. HRMS (ESI) m/z: [M + H] ⁺ calculated for C2&H28B1-N5O3: 538.1448; observed 538 1439. Purity >95%, /<< 10.53 min (HPLC Method II).

Methyl 5-(2-((4-(2-(diethylamino)ethoxy)phenyl)amino)-8-methyl-7-ox o-7,8-dibydropyrido [2, 3-t/]pyrimidin-6-yl)-2-hydroxybenzoate (SS-l-l 54)

Method Q, The crude product was separated by chromatography (10% MeOH in DCM and 0 1% NH4OH). and yielded SS-1-154 (44%) as a yellow solid, mp 1 13-115 °C. ^l H-NMR (600 MHz, CD3OD) ) 5 8.67 (s, 1H), 8.20 (d, J = 2.2 Hz, 1H), 7.86 (s, 1H), 7.80 (dd, J = 8.6, 2.4 Hz, 1H), 7.68 (d, J - 8.8 Hz, 2H), 7.00 (q, J = 4.4 Hz, 3H), 4.25 (t, J - 5.3 Hz, 2H), 3.98 (s, 3H), 3.73 (s, 3H), 3 35 (t, J = 4.6 Hz, 2H), 3.11 (q, J = 7.1 Hz, 4H), 1.28 (t, J = 7.2 Hz, 6H). ¹³ C-NMR (150 MHz, CD ₃ OD) 5 171.7, 164.7, 162.4, 160.5, 160.1, 156.3, 155.5, 137.3, 135.0, 134.7, 131.4, 128.9, 127.9, 122.9, 118.2, 115.8, 113.3, 108.2, 64.9, 53.0, 52.5, 49.1, 28.9, 10.0. HRMS (ESI) m/z: [M + H]~ calculated for C28H31N5O3: 518.2398; observed 518.2400. Purity >95%, fe = 14.97 min (HPLC Method II).

5-(2-((4-(2-(Diethylanuno)ethoxy)phenyl)ai'nino)-8-methyl -7-oxo-7,8-dihydropyrido[2,3- 4/]pyrimidin-6-yl)-2-hydroxybenzonitrile (SS-2-35) Method Q; The crude product was separated by chromatography (5% MeOH in DCM and

0.1% NHrOH). and yielded SS-2-35 (54%) as a yellow solid, mp 163-165 °C. ^l H-NMR (600 MHz, CDaOD) 5 8.58 (s, 1H), 7.93 (dd, J - 8.8, 2.2 Hz, I H i. 7 89 (d, J - 2.2 Hz, 1 H). 7.63 (s, 1H), 7.55 (d, J - 8.6 Hz, 2H), 7.31 (s, I M), 7.03 (d, J = 8.8 Hz, 1H), 6.95 (dd, J = 6 9, 2. 1 Hz, 2H), 4.07 (t, J = 6.3 Hz, 2H), 3.98 (s, 3H), 3.76 (s, 3H), 2.89 (t, J = 6.3 Hz, 2H), 2.65 (q, J - 7.1 Hz, 4H), 1.08 (t, .1 = 7.1 Hz, 6H). ^i? C-NMR (150 MHz, CD3OD) 8 164.5, 162.2, 160.7, 160.4, 159.9, 156.4, 155.6,

136.4, 135.6, 135.1, 134.6, 130.7, 126.6, 123.0, 117.2, 115.8, 112.6, 102.1, 56.9, 52.5, 49.6, 30.8, 28.9, 10.2. HRMS (ESI) m/z: [M + TT] ⁺ calculated for C28H30N6O3: 499.2452; observed 499.2447. Purity >95%, fe = 10.30 min (HPLC Method II). 5-(2-((3-(2-(Diethylamino)ethoxy)phenyl)amino)-8-met.hyl-7-o xo-7,8-dihydropyrido[2,3- «/]pyrimidin-6-yl)-2-hydroxybenzonitrile (SS-2-37)

Method Q; The crude product was separated by chromatography (10% MeOH in DCM and 0.1% NH4OH). and yielded SS-2-37 (42%) as a yellow solid, nip 189-191 °C. ^! H-NMR (600 MHz, DMSO-ds) 5 10.17 (s, 1H), 8.82 (s, 1H), 8.07 (s, 1H), 7.95 (d, J - 2.1 Hz, 1H), 7.85 (dd, J = 8 7, 2.2 Hz, 1H), 7.66 (s, 1H), 7.33 (d, J = 8.3 Hz, 1H), 7.23 (t, J = 8.1 Hz, 1H), 7.07 (d, J = 8.8 Hz, 1 H). 6.61 (dd, J - 8.1, 1.7 Hz, 1H), 4.04 (s, 2H), 3.68 (s, 3H), 2.84 (s, 2H), 2 61 (s, -H l). 1.01 (t, J - 6.9 Hz, 6H). HRMS (ESI) m/z: [M + Hf calculated for C27H28N6O3: 485.2296; observed 485.2303. Purity >95%, fe= 9.95 min (HPLC Method II).

2-Hydroxy-5-(2-((4-methoxyphenyl)amino)-8-methyl-7-oxo-7, 8-dihydropyrido[2,3-<7]pyrimidin- 6-yl)benzonitrile (SS-3-1)

Method Q; The crude product was separated by chromatography (0-5% MeOH in DCM and 0.1% NH4OH). and yielded SS-3-1 (44%) as a yellow solid, mp > 250 °C. ‘H-NMR (600 MHz, DMSO-de) 5 11 .34 (s, 1 H), 10.02 (s, 1H), 8.75 ( s. 1H), 8.02 (s, 1H), 7.93 (s, 1H), 7.84 (d, J - 8.8 Hz, 1H), 7.71 (s, 2H), 7.06 (d, J = 8.6 Hz, 1H), 6.93 (d, J = 8.8 Hz, 2H), 3.74 (s, 3H), 3.63 (s, 3H). 1 ³ C-NMR (150 MHz, DMSO-ds) 5 161.9, 159.8, 159.0, 158 7, 154.9, 154.6, 134.9, 133.6, 133.0, 132.7, 127.6, 124.4, 121.2, 117.0, 115.9, 113.8, 98.6, 55.2, 28.1. HRMS (ESI) m/z: [M + H] ⁺ calculated for C22H17N5O3: 422.1224; observed 422.1237. Purity >95%, tp = 14.07 min (HPLC Method II).

2-Hydroxy~5-(2-((4-hydroxyphenyl)amino)-8-methyl-7-oxo-7, 8-dihydropyrido[2,3-fiOpyriniidin- 6-yl)benzonitriIe (SS-3-2) Method Q; The crude product was separated by chromatography (0-5% MeOH in DCM and 0. 1% NH iOH). and yielded SS-3-2 (44%) as a yellow solid mp: at 180 °C started to change color. ’H-NMR (600 MHz, DMSO-dc,) 5 11.34 (s, If 1), 9.92 (s, 1H), 9.23 (s, 1H), 8.73 (s, 1H), 8.01 (s, 1H), 7.93 (d, J = 1.5 Hz, 1H), 7.84 (d, J = 8.6 Hz, 1H), 7.57 (s, 2H), 7.05 (d, J = 8.8 Hz, 1 H), 6.75 (d, J - 8.6 Hz, 2H), 3.62 (s, 3H). ^{! 3} C-NMR (150 MHz, DMSO-d ₆ ) 8 161.9, 159.7, 159.0, 158.8, 154.6, 153.1, 134.9, 133.6, 133.0, 131.1, 127.6, 124.2, 121.5, 117.0, 115.9, 115.0, 106.0,

98.6, 28.0 HRMS (ESI) m/z: [M + H]" calculated for C21H15N5O3: 408.1067, observed 408 1080. Purity >95%, 12.20 min (HPLC Method II). 5-(2-((4-CyanophenyI)amino)-8-methyl-7-oxo-7,8-dihydropyrido [2,3-£7]pyrimidin-6-yI)-2- hydroxybenzonitrile (SS-3-4)

Method Q; The crude product was separated by chromatography (0-5% MeOH in DCM and 0.1% NH4OH). and yielded SS-3-4 (21%) as a yellow solid, mp > 250 °C. ^J H-NMR (600 MHz, DMSO-de) 5 10.89-1 1.84 (HI), 10.64 (s, II I), 8.88 (s, H I), 8.10 (s, M I ), 8.04 (d, J - 8.8 Hz, 211), 7.95 (d, J = 2.1 Hz, 1H), 7.86-7.84 (m, IH), 7.80 (d, J = 8.6 Hz, 2H), 7.07 (d, J = 8.8 Hz, 1H), 3.68 (s, 3H). ¹³ C-NMR (150 MHz, DMSO-de) 8 161.8, 159.9, 158.9, 158.1, 154.4, 144.1, 135.0, 133 3, 133.2, 133.1, 127.3, 126.1, 1 19 4, 1 19.1 , 117.0, 115.9, 107.4, 103.4, 98.7, 28.3. HRMS (ESI) m/z: [M + H] ⁺ calculated for C22H14N6O2: 395.1251; observed 395. 1254. Purity >95%, ?R = 14.02 min (HPLC Method II).

5-(2-(Benzo[ti][l,3]dioxol-5-ylamino)-8-methyl-7-oxo-7,8- dihydropyrido[2,3-ti]pyrimidin-6-yl)- 2-bydroxybenzonitrile (SS-3-50)

Method Q; The crude product was separated by chromatography (0-5% MeOH in DCM). and yielded SS-3-50 (52%) as a yellow solid. ‘H-NMR (600 MHz, DMSO-dr,) 8 11.31 (s, IH), 10.08 (s, IH), 8.78 (s, IH), 8.05 (s, IH), 7.94 (d, J - 2,2 Hz, IH), 7.85 (dd, J - 8.7, 2.2 Hz, IH), 7.54 (s, IH), 7.20 (d, J = 7.9 Hz, IH), 7.06 (d, J = 8.8 Hz, IH), 6.91 (d, J = 8.4 Hz, IH), 6.01 (s, 2H), 3.64 (s, 3H). ¹³ C-NMR (150 MHz, DMSO-dt.) 8 161.9, 159.7, 159.0, 154.6, 147.1, 142.5, 134.9, 134.0, 133.6, 133.0, 127.7, 124.6, 117.0, 115.9, 112.5, 108.0, 106.3, 101.8, 101.0, 98.6, 28.1 . HRMS (ESI) m/z: [M + H]“ calculated for C22H15N5O4: 414. 1197; observed 414 1205.

17.83 min (HPLC Method III). jV-(4-((6-(3-Cyano-4-hydroxyphenyl)-8-methyl-7-oxo-7,8-dihyd ropyrido[2,3-d]pyrimidin-2- yl)amino)phenyl)methanesulfonamide (SS-3-81)

Method Q; The crude product was separated by chromatography (0-5% MeOH in DCM and 0.1 % NH40H). and yielded SS-3-81 (78%) as a yellow solid. ^J H-NMR (600 MHz, DMSO-de) 10.18 (s, 1H), 9.57 (s, 1H), 8.80 (s, 1H), 8.06 (s, 1H), 7.95 (d, J = 2.2 Hz, 1H), 7.85 (dd, J = 8.8, 2.4 Hz, H i), 7.80 (d, J - 8.6 Hz, 2H), 7.21 (d, J - 9.0 Hz, 2H), 7.07 (d, .1 - 8.8 Hz, 1H), 3.66 (s,

3H), 2.95 (s, 3H). ¹³ C-NMR (150 MHz, DMSO-de) 8 161.9, 159.7, 159.0, 158.5, 154.6, 136.3, 135.0, 133.6, 133.0, 132.7, 127 6, 124.8, 121.4, 120.4, 117.0, 1 15.9, 106.4, 98.6, 38.9, 28.2. HRMS (ESI) m/z: [M + H] ⁺ calculated for C22H18N6O4S: 485.1002; observed 485.1017. h< = 16.72 min (HPL.C Method III).

tert-butyl 4-(6-(3-cyano-4-hydroxyphenyl)-2-((4-(2-(diethylamino)ethoxy )phenyl)amino)-7- oxopyrido[2,3-</|pyrimidin-8(77/)-yl)piperidine- 1 -carboxy late (ClOh)

To a mixture of ClOe (15 mg, 0.023 mmol), Cllf (5.6 mg, 0.023 mmol), PdCi2(dppf)»DCM (1.4 mg, 0.0023 mmol), and K2CO3 (0.023 mL, 0 069 mmol) in anhydrous DMF (1 mL) under argon gas at 100 °C for 30 min under microwave irradiation. The reaction was quenched with water, and the aqueous layer was extracted with DCM multiple times. The combined organic phases were washed with brine, dried over anhydrous NazSOr, filtered and concentrated The crude product was separated by chromatography (0-5% MeOH in DCM and 0.1% NH4OH) and yielded ClOh (66%) as a y ellow solid. TI-NMR (600 MHz, CD3OD) S 8.58

(s, 1H), 7.71 (s, 2H), 7.66 (dd, J - 8.7, 2.2 Hz, 1H), 7.55 (d, J - 8.8 Hz, 2H), 6.94 (d, J - 8.6 Hz, 2H), 6.91 (d, J == 8 8 Hz, 1H), 5.65 (d, J - 16.5 Hz, 1 H), 4 27 (d, J == 9.6 Hz, 2H), 4.23 (t, .1 == 4.5 Hz, 2H), 3.33 (s, 2H), 3.10 (q, J = 7.2 Hz, 4H), 2.91-2.78 (m, 4H), 1.63 (s, 2H), 1.50 (s, 9H), 1.28 (t, J - 7.1 Hz, 6H). ¹³ C-NMR (150 MHz, CD3OD) 8 164.7, 161.9, 160.5, 160.3, 156.4, 156.1, 155.3, 136 2, 135.2, 134.7, 134.6, 129.0, 128.2, 123.4, 118 0, 117.0, 1 15 8, 108 3, 100.5, 81.3,

64.0, 54.3, 52.4, 45.9, 45.1, 30.8, 28.8, 28.7, 9.4. 5-(2-((4-(2-(diethylaniino)ethoxy)phenyl)amino)-7-oxo-8-(pip eridin-4-yl)-7,8- dihydropyrido[2,3-i/|pyrimidin-6-yl)-2 -hydroxybenzonitrile (SS-3-86)

To a solution of ClOh (34.4 mg, 0.053 mmol) in anhydrous DCM at 0 °C is added TIPS (0.022 mL, 0.1 mmol) and TFA (0.45 mL) dropwise (until the solution turns clear) and stirred at room temperature for 30 min. The reaction mixture is quenched with saturated aqueous solution of NaHCOs and extracted with 20% MeOH in CHjCl multiple times. The combined organic layers were washed with brine, dried over anhydrous NhnSOr, filtered, and concentrated under vacuum. The crude product was separated by (50% MeOH in DCM and 01 % NH4OH) and yielded SS-3- 86 (60%) as a yellow solid. ^X H-NMR (600 MHz, DMSO-d6) 69.96 (br, 1H), 8.73 (s, IH), 7.93 (s, IH), 7.84 (d, J = 2.1 Hz, IH), 7.71 -7 65 (m, 3H), 6.93 (t, J - 9.0 Hz, 3H), 5.50 (s, I H), 4.0 (t, J - 5.9 Hz, 2H), 3.20 (br, 4H), 2.81-2.75 (m, 4H), 2.65 (br, 2H), 2.55 (q, J = 7.1 Hz, 4H), 1.60 (br, 2H), 1.25 (d. J 30 3 Hz, IH), 0.97 (t, J - 7.1 Hz, 6H) ⁱ³ C-NMR (150 MHz, DMSO-d6) 8 162 6, 162.3, 159.1, 158.3, 154.3, 154.2, 134.8, 133.1, 133.0, 132.6, 126.0, 125.3, 121.5, 118.1, 116.7, 1 14.4, 106.4, 98.6, 66.5, 51.4, 47.0, 46.0, 27.4, 11.8. HRMS (ESI) m/z: [M + H]+ calculated for C31H35N7O3: 554.2874; observed 554.2885. = 5.46 min (HPLC Method IV).

2-Amino-6-iodo-8-metbylpyrido[2,3-t/]pyrimidin-7(8/7)-one (Cl 2a)

To a solution of C7 (70 mg, 0.19 mmol) in isopropyl alcohol (2 mL), an excess of 30% ammonium hydroxide solution (1 .5 mL) was added at room temperature and was stirred for 16 h. After the completion of the reaction, the solvent and ammonium hydroxide solution were removed under vacuum. The crude product was separated using column chromatography (0-5% MeOH in DCM). Product C12a (73%) was afforded as a white solid. ^J H-NMR (600 MHz, CDCI3) 8 8.43 (s, IH), 8.19 (s, IH), 5.37 (s, 2H), 3.72 (s, 3H). ^]3 C-NMR (150 MHz, DMSO-do) 8 162.9, 159.8, 158.7, 155.7, 145.1, 106.7, 85.7, 28.7.

6-Iodo-8-methyl-2-(methylamino)pyrido[2,3-i7]pyrimidin-7( 877)-one (C12b)

To a solution of C7 (60 mg, 0.16 mmol) in isopropyl alcohol (1 ,5 ml), an excess of aqueous methylamine (1. 14 niL) was added at room temperature and was stirred for 15 min. After the completion of the reaction, the solvent and methylamine were removed under vacuum. The crude product was separated using column chromatography (0-5% MeOH in DCM). Product CI 2b (94%) was afforded as a white solid Rotamer is observed in both ⁱ H- and ^1J C NMR spectra . ^l H- NMR (600 MHz, CDCh) 5 8.41 (d, J = 58.4 Hz, IH), 8.16 (s, 1 H ), 5.52 (d, J = 81.6 Hz, IH), 3.73 (d, J = 58.2 Hz, 3H), 3.09 (d, J = 5.2 Hz, 3H). ⁱ³ C-NMR (150 MHz, CDCh) 3 162.1, 161.9 (rotamer), 160 2, 158.7, 158.6 (rotamer), 155.8, 155.7 (rotamer), 145.4, 107.2, 106.4 (rotamer), 85.9, 85.4 (rotamer), 28.9, 28.3 (rotamer), 28.1.

JV-(6-Iodo-8-methyl-7-oxo-7,8-dihydropyrido[2,3-</]pyr imidin-2-yl)acetamide (C12c)

C7 (30 mg, 0,1 mmol) was dissolved in excess acetic anhydrous at room temperature, then heated up to 90 °C and stirred for 16 h. After the completion of the reaction, the reaction mixture was cooled to room temperature. The precipitant was collected by vacuum filtration, washed with chloroform, and dried under vacuum. Product Cl 2c (87%) was afforded as a white solid, mp > 250 °C. ^J H-NMR (600 MHz, DMSO-ds) 5 10 88 (s, 1H), 8.88 (s, IH), 8.68 (s, 1H), 3.64 (s, 3H), 2.28 (s, 3H). ¹³ C-NMR (150 MHz, DMSO-dr.) 8 169.5, 159.7, 157.8, 156.8, 1.55.0, 144.5, 1104, 92.7, 29.3, 25.2.

5-(2-Amino-8-methyl-7-oxo-7,8-dihydropyrido[2,3-cZjpyrini idin-6-yl)-2-hydroxybenzonitrile (SS-2-57) Method Q; The crude product was separated by chromatography (0-5% MeOH in DCM and 0.1% NHiOH). and yielded SS-2-57 (45%) as a pale-yellow solid, rap >250 °C. ’H-NMR (600 MHz, DMSO-do) 5 10.41-12.00 (1H), 8.61 (s, 1H), 7.96 (d, J = 7.4 Hz, 1H), 7.91 (s, 1H), 7.81 (d, J - 8.6 Hz, 1H), 7.35 (s, 2H), 7.04 (d, J == 8.8 Hz, 1 H), 3.56 (s, 3H). '('AMR (150 MHz, DMSO- d ₆ ) 8 162.7, 162.0, 159.7, 159.6, 155.1, 134.9, 133.9, 132.9, 127.7, 123.4, 117.1, 115.9, 105.1, 98 5, 27 7. HRMS (ESI) m/z: [M + H]~ calculated for C15H11N5O2: 294.0986; observed 294 0978

Purity >95%, &<= 9.98 min (HPLC Method II). 2-Hydroxy-5-(8-methy!-2-(methy1amino)-7-oxo-7,8-di hydropyrido[2,3-</jpyrimidin-6- yl)benzonitrile (SS-2-58)

Method Q; The crude product was separated by chromatography (0-5% MeOH in DCM and 0.1 % NH4OH). and yielded SS-2-5S (46%) as a pale-yellow solid, mp >250 °C. ¹ H-NMR (600 MHz, DMSO-de) 6 11.25 (s, HI), 8.64 (d, J - 46.8 Hz, I H), 7.96 (s, 1H), 7.91 (d, J - 1.9 Hz, 1 H), 7.85 (dd, J = 4.0 Hz, 1H), 7.82 (d, J = 8.8 Hz, 1H), 7.74 (d, J = 4. 1 Hz, OH), 7.04 (d, J = 8.8 Hz, 1H), 3.57 (d, J - 42.7 Hz, 3H), 2.91 (d, J - 4.5 Hz, 3H). HRMS (ESI) m/z: [M + H] ⁺ calculated for C16H13N5O2: 308.1142, observed 308.1139. Purity >95%, fe == 11.37 min (HPLC Method II).

_J V-(6-(3-Cyano-4-hydroxyphenyl)-8-methyl-7-oxo-7,8-dihy dropyrido[2,3-J]pyrimi din-2- yl)acetamide (SS-3-15)

Method Q; The crude product was separated by chromatography (0-5% MeOH in DCM and 0. I%NH4OH). and yielded SS-3-15 (81%) as a white solid, mp > 250 °C. ¹ H-NMR (600 MHz, DMSO-d6) 6 11.39 (s, 1H), 10.84 (s, 1H), 8.92 (s, 1H), 8.13 (s, 1H), 7.96 (d, J = 2.2 Hz, 1H), 7.86 (dd, J - 8.8, 2.1 Hz, 1 H), 7.08 (d, J - 8.8 Hz, 1 H), 3.65 (s, 3 H), 2.30 (s, 3H) ¹³ C-NMR (150 MHz, DMSO- ds) 8 169.5, 161.8, 160.1, 158.7, 156.6, 154.2, 135.1, 133.3, 132.9, 128.0, 127.1, 116.9, 115.9, 109 1 , 98.7, 28.2, 25.2. HRMS (ESI) m/z: [M + H] ⁺ calculated for C17H13N5O3: 358.0911, observed 358.0922. Purity >95%, 6<= 10.23 min (HPLC Method II).

Ethyl 6-chloro-4-(methylamino)nicotinate (1)2)

Method I; The crude product was separated by chromatography (10% EtOAc in Hexane), and yielded »2 (67%) as white solid. ¹ H-NMR (600 MHz, CDCh) 8 8.64 (s, 1H), 8.10 (s, 1H), 6.52 (s, 1H), 4.32-4.36 (m, 2H), 2.90-2.92 (m, 3H), 1.37-1.40 (m, 3H). ⁾³ C-NMR (150 MHz, CDCh) 8 167.39, 156.62, 155.55, 152 65, 106.86, 104.16, 60 80, 29 00, 14.12

(6-Chloro-4-(methylamino)61 yridine-3 -yl)methanol (D3)

Method J; The crude product was separated by chromatography (5% MeOH in DCM), and yielded D3 (96%) as white solid. ^V H-NMR (600 MHz, CDsOD) 5 7.75 (s, 1H), 6.54 (s, 1H), 4.50 (s, 2H), 2.86 (s, 3H). ^1? C-NMR (150 MHz, CDJOD) 8 157.1, 152.4, 147.2, 121.0, 104.1, 60.3, 29.5.

6-Chloro-4-(methylamino)nicotinaldehyde (D4)

Method K; The crude product was separated by chromatography (5% MeOH in DCM), and yielded D4 (94%) as white solid. ^l H-NMR (600 MHz, CDCh) 6 9.84 (s, 1H), 8.56 (s, TH), 8 31 (s, 1H), 6.58 (s, H i), 2.96 id. J 4.8 Hz, 3H). ⁿ C-NMR (150 MHz, CDCh) 6 192.49, 157.82, 156.59, 155.89, 114.87, 104.31, 28.94.

Methy 1 2-( 3 -((tort-buty I di methy 1 sily 1 )oxy)phenyl)acetate (D5a)

(Method R)

To a solution of D8a (500 mg, 3 mmol) in anhydrous DCM (6 mL) at 0 °C was added DMAP (29.4 mg, 0.24 mmol) and imidazole (266.4 mg, 3.9 mmol) and stirred at 0 °C. TBSC1 (701 mg, 4.6 mmol) was added to the reaction mixture in one portion and stirred at room temperature overnight. The reaction was quenched with water and extracted with DCM. The combined organic layer was washed with brine, dried over anhydrous NaiSOt, filtered, and concentrated. The crude product rvas separated by chromatography (10% EtOAc in Hexane), and yielded D5a (97%) as a colorless oil. ^l H-NMR (600 MHz, CDCh) 5 7.17 (t, J = 7.7 Hz, 1H), 6.86 (d, J = 7.6 Hz, 1H), 6.78 (s, 1H), 6.74 (d, J === 8.3 Hz, 1 H ), 3.69 (s, 3H), 3.57 (s, 2H), 0.98 (s, 9H), 0.19 (s, 6H). ¹ ( AMR

(150 MHz, CDCh) 8 171.91, 155 7, 135.3, 129.4, 122.2, 121.1, 118.8, 52.0, 41.1, 25.7, 18 2, - 4.46.

Methyl 2-(4-((tor/-butyldimethylsilyl)oxy)phenyl)acetate (D5b)

Method R, The crude product was separated by chromatography (10% EtOAc in Hexane) and yielded D5b (92%) as a transparent oil. ^l H-NMR (600 MHz, CDCh) 6 7.13 (d, J = 8.4 Hz, 2H), 6.79 (d, J - 8.4 Hz, 2H), 3 69 (s, 311), 3.55 (s, 2H), 0.97 (s, 9H), 0.19 (s, 6H) ¹ (AMR (150 MHz, CDCh) 8 172.4, 154.7, 130.2, 126.6, 120 1, 52.0, 40.3, 25.6, 18.2, -4.46.

7-Chloro-3-(3-hydroxyphenyl)-l-methyl-l,6-naphthyridin-2( l/7)-one (D6a) (Method S)

A solution of D4 (50 mg, 0.29 mmol) and DSa (115.04 mg, 0.41 mmol) in anhydrous DMSO (2 mL) was stirred at room temperature overnight under argon gas. The reaction was quenched with water and extracted with EtOAc. The combined organic layer was washed with brine dried over anhydrous NarSOy filtered, and concentrated. The crude product was separated by chromatography (5% MeOH in DCM) and yielded D6a (93%) as a white solid. ’H-NMR (600 MHz, DMSO-ck) 6 9.54 (s, 1H), 8.78 (s, 1H), 8 16 (s, 1H), 7.65 (s, 1H), 7.24 (t, J - 7.7 Hz, 1H), 7.13 (s, 1 H), 7.09 id. J - 7.2 Hz, 1 H), 6.81 (d, J =- 8.3 Hz, H i), 3.64 (s, 3H). ¹ ( - \MR (150 MHz, DMSO-dr>) 6 160.5, 156.9, 150.4, 150.3, 145.8, 137.0, 134.1, 132.5, 129.0, 119.4, 115.8, 115.4, 108.4, 29.8

7-Chloro-3-(4-hydroxyphenyl)-l-methyl-l,6-naphthyridin-2( lH)-one (D6b)

Method S; The crude product was separated by chromatography (5% MeOH in DCM), and yielded »6b (91%) as a white solid. ‘H-NMR (600 MHz, DMSO-dA 8 9.72 (s, 1 H), 8.72 (s, 1H), 8.07 (s, 1H), 7.59 (s, 1H), 7.56 (d, J = 8.5 Hz, 2H), 6.82 (dd, J = 6.5, 2.0 Hz, 2H), 3.62 (s, 3H). ⁿ C-NMR (150 MHz, DMSO-d ₆ ) 8 160.7, 157 8, 150.0, 149.9, 145 5, 132.4, 132.3, 130.1, 126.4, 116.0, 114.9, 108.4, 29.8.

2-((4-(2-(Diethylamino)ethoxy)phenyl)amino)-6-(3-hydroxyp henyl)-8-methy1pyrjdo[2,3- if]pyrimidin-7(8//)-one (SS-2-45)

(Method T)

A mixture of D6a (14 mg, 0.049 mmol), D7 (10.2 mg, 0 049 mmol), Pd?(dba)3 (4 6 mg, 0.005 mmol), Xantphos (5.8 mg, 0.01 mmol), and CS2CO3 (32.6 mg, 0.1 mmol) in anhydrous dioxane (1 mL) was flushed and filled with argon and placed under microwave irradiation at 95 °C for 90 min. The reaction mixture was then filtered through celite, washed with DCM, and concentrated. The crude product was separated by chromatography (5% MeOH in DCM), and yielded SS-2-45 (45%) as yellow solid, mp 171-173 °C. H-NMR (600 MHz, CD3OD) 6 8.44 (s, H I). 7.85 (s, H I), 7.42 (d, J - 8.8 Hz, 2H), 7.22 (t, J === 7.8 Hz, H I ), 7 08 (d, J - 6.9 Hz, 2H), 6.97 (d, J = 9.0 Hz, 2H), 6.78 (dd, J = 8.1, 2.4 Hz, 1H), 6.56 (s, IH), 4.15 (t, J = 5.5 Hz, 2H), 3.60 (s, 3H), 3.03 (s, 2H), 2.80 (d, J - 6 7 Hz, 4H), 1 .16 (t, J - 7.1 Hz, 6H). ⁿ C-NMR (150 MHz, CD3OD) 8 163.9, 159.3, 158.3, 156.3, 151.4, 147.5, 139.4, 137.2, 135.1, 130.2, 129.2, 124.2, 121.2, 116.9, 1162, 115.8, 112.3, 90.9, 66.2, 52.6, 49 6, 29.8, 10.9. HRMS (ESI) m/z: [M + H]* calculated for C27H30N 4O3: 459.2391 ; observed 459.2401 . Method B purity >95%, /a 8.68 min.

2-((4-(2-(Diethylamino)ethoxy)phenyl)amino)-6-(4-hydroxyp henyl)-8-methylpyrido[2,3- t/]pyrimidin-7(87f)-one (SS-2-47)

Method T; The crude product was separated by chromatography (10% MeOH in DCM), and yielded SS-2-47 (61%) as yellow solid, mp 177-178 °C. ’H-NMR (600 MHz, CD ODi 5 8.40 (s, 1H), 7.78 (s, 1H), 7.48 (d, J = 8.6 Hz, 2H), 7.41 (d, J = 9.0 Hz, 2H), 6.95 (d, J = 9.0 Hz, 2H), 6.82 (d, J == 8.6 Hz, 2H), 6.53 (s, 1H), 4.14 (t, .1 - 5.5 Hz, 2H), 3 57 (s, 3H), 3.04 (I. J == 5.1 Hz, 2H), 2.81 (q, J = 7.1 Hz, 4H), 1.16 (t, J = 7.2 Hz, 6H). ^l3 C-NMR (150 MHz, CD3OD) 5 164.2, 159 1 , 158.5, 156.3, 151 0, 147.3, 135 9, 135.1 , 131.2, 129.3, 129.2, 124.1, 116.1 , 115.9, 112 5, 90.9, 66.5, 52.6, 48.7, 29.8, 11.0. HRMS (ESI) m/z: [M + H| ⁺ calculated for C27H30N4O3: 459.2391; observed 459.2391. Method B purity >95%, v 8.5 min

7-Chloro-l-methyl-2 -oxo-1, 2-dihydro-l,6-naphthyridine-3-carboxyiic acid (D9)

Method L; Product D9 was afforded as a white solid with a 93% yield. ^-NMR (600 MHz, DMSO-de) 5 8 66 (s, 1H), 7.74 (s, 1 H ), 7.51 (s, 1H), 3.54 (s, 3H). ⁿ C-NMR ( 150 MHz, DMSO- de) 6 171.6, 166.4, 160.6, 150.6, 150.5, 146.0, 134.1, 115.5, 108.5, 29.5.

7-Chl oro-3 -iodo- 1 -methyl- 1 ,6-naphthyridin-2( lH)-one (DIO)

Method M: The crude product was separated using chromatography (0-10% EtOAc in Hexane). Product DIO (80%) was afforded as a pale-yellow solid. ³ H-NMR (600 MHz, CDCh) 5 8.52 (s, 1H), 8.42 (s, IH), 7.24 (s, IH), 3.73 (s, 3H). ^!3 C-NMR (150 MHz, CDCh) 5 158.7, 152 4, 148.7, 146.5, 145.3, 116.7, 108.3, 95.6, 31.2.

5-(7-Chloro- 1 -methyl-2-oxo- 1 ,2-dihydro- 1 ,6-naphthyridin-3-yl)-2-hydroxybenzonitrile (D6c)

Method Q; The crude product was separated by chromatography (0-30% EtOAc in DCM). D6c (46%) was afforded as a light brown solid. ’'H-NMR (600 MHz, DMSO-d&) 3 11.46 (s, IH), 8.74 (s, IH), 8.24 (s, IH), 7.99 (s, IH), 7.88 (dd, J - 8.7, 2.0 Hz, IH), 7.66 (s, IH), 7.09 (d. J === 8.8 Hz, IH), 3.60 (s, 3H). ¹³ C-NMR ( 150 MHz, DMSO-d ₆ ) 6 160.5, 160.3, 150.5, 150.3, 145.8, 135.2, 133.8, 133.4, 130.2, 127.1, 116.9, 115.9, 115.8, 108.6, 98.7, 29.9.

5-(7-((4-(2-(Diethylamino)ethoxy)phenyr)amino)-l-methyl-2 -oxO“l,2-dihydro-l,6-naphthyridin-

3 -yl )-2-hy droxybenzonitri 1 e (SS-2-107)

Method T; The crude product was separated by chromatography (10% MeOH in DCM), and yielded SS-2-107 (42%) as yellow solid, mp 177-178 °C. ^l H-NMR (600 MHz, CDsOD) 5 8.43 is, IH), 7.87 (s, I H), 7.85 id. J == 2 2 Hz, IH), 7.76 (dd, J - 8.8, 2.2 Hz, I H), 7.47 id. J == 9 0 Hz, 2H), 7.00 (t, J = 8 2 Hz, 3H), 6 54 (s, IH), 4.35 (t, J = 4.8 Hz, 2H), 3.61 (t, J = 4.8 Hz, 2H), 3.58 (s, 3H), 3.37 (t, J =- 7.3 Hz, 4H), 1.39 (t, J = 7.3 Hz, 6H). ¹³ C-NMR (150 MHz, CD ₃ OD) 6 163.7, 161.1, 159.0, 155.0, 151.3, 147.4, 136.7, 136.2, 136.0, 134.6, 129.8, 126.8, 123.6, 117.8, 116.7, 116.3, 1 12.4, 100.4, 91.3, 63.6, 52.4, 49.4, 29.9, 9.1. HRMS (ESI) m/z: [M + Hf calculated for C28H29N5O3: 484.2343; observed 484.2340. Purity >95%, &= 9.05 min (HPLC Method II).

2-Chloro-A ^r -methyl-5-nitropyridin-4-amine (E2)

To a solution of El (1 g, 5.2 mmol) in anhydrous THF (15.4 mL) at 0 °C was added trimethylamine (0.73 mL, 5.2 mmol) and aqueous methylamine (3.4 mL, 6.76 mmol). The ice bath was removed, and the reaction mixture was stirred at room temperature for 16 h. After the completion of the reaction, the solvent and excess methylamine were removed under vacuum. The crude product was separated by (0-10 % EtOAc in Hexane) and yielded C2a (89%) as a yellow solid. ^f H-NMR (600 MHz, CDCh) 8 8.95 (s, IH), 8.15 (s, IH), 6.71 (s, 1H), 3.03 (d, J = 5.2 Hz, .311). ¹³ C-NMR (150 MHz, CDCh) 8 156.3, 150.3, 148.9, 129.2, 106.4, 29.5.

6-Chloro-A ^;4 -methylpyridine-3,4-diamine (E3)

To a solution of E2 (454 mg, 2 42. mmol) in acetic acid (20 mL) at 50 °C was added iron powder (540.2 mg, 9.67 mmol) portion wise. The temperature was increased to 80 °C and the reaction mixture was stirred for 1 h. The mixture was filtered through ceiite, washed with EtOAc, and dried under vacuum. The crude product was diluted with EtOAc, quenched with saturated aqueous solution of NaHCOs, and extracted with EtOAc. The organic layer was washed with water and brine, dried over anhydrous Na 2SO4, filtered, and concentrated under vacuum. The crude product was separated by (0-35 % EtOAc in DCM) and yielded E3 (95%) as an orange solid ¹ H- NMR (600 MHz, CDClr) 6 7.53 (s, 1H), 6.36 (s, 1H), 4.77 (s, 1H), 3.28 (s, 2H), 2.84-2.81 (m, 3H). ¹³ C-NMR (150 MHz, CDClr) 5 148.0, 144.2, 135.3, 128.2, 103.1, 29.5.

Methyl 2-(4-methoxyphenyl)-2-oxoacetate (E5)

A mixture of E4 (500 mg, 3.33 mmol), selenium dioxide (554.3 mg, 5 mmol), and pyridine (1.67 mL) under argon was stirred at 1 10 °C for 20 h. The mixture was filtered through ceiite, washed with DCM, and dried under vacuum. The crude product was dissolved in anhydrous DMT (6 mL) and stirred over an ice bath. K2CO3 (1.38 g, 10 mmol) and methyl iodide (0.62 mL, 10 mmol) was added to the reaction mixture at 0 °C. The ice bath was removed, and the mixture was stirred at room temperature for 2 h. The reaction was quenched with IN HC1 and extracted with EtOAc. The combined organic layers were washed with saturated aqueous solution of NaHCOr and then washed with saturated aqueous solution of Na2S2Ch. The organic layer was washed with water and brine, dried over anhydrous NaaSCh, filtered, and concentrated under vacuum. The crude product was separated by (5-10% EtOAc in Hexane) and yielded E5 (80%) as a white solid. ¹ H-

NMR (600 MHz, CDCh) 5 7.97 (d, J = 9.0 Hz, 2H), 6.94 (d, J = 9.0 Hz, 2H), 3.93 (s, 3H), 3.85 (s, 3H). ^1? C-NMR (150 MHz, CDCh) 8 184.4, 165.0, 164.3, 132.5, 125.2, 1 14.1, 55.5, 52.6.

7-Chloro-3-(4-methoxyphenyl)-l-methylpyrido[3,4-Z>]pyr azin-2(lZ/)-one (E6)

A mixture of E5 (622.24 mg, 3.2 mmol) in acetic acid (25 mL) was stirred at 80 °C E3 (756.5 mg, 4.8 mmol) in acetic acid (15 mL) was added over 12 h. The reaction mixture was cooled to room temperature and the solvent was removed under vacuum The crude product was dissolved in EtOAc and washed with Dl-water twice. The organic layer was washed with brine, dried over anhydrous Na2SOr, filtered, and concentrated under vacuum. The crude product was separated by (5-10% EtOAc in Hexane) and yielded E6 (89%) as a light-yellow solid. ^! H-NMR (600 MHz, CDC13) 8 8 85 (s, 1 H), 8.38 (d, J - 9.0 Hz, 2H ), 7 19 (s, 1 H), 6.98 (d, J === 9.0 Hz, 2H), 3 88 (s, 3H), 3.67 (s, 3H). ^l3 C-NMR (150 MHz, CDCh) 8 162.2, 154.4, 150.9, 150.6, 140.1, 131.6, 128.4, 127.5, 113.6, 107.5, 55.4, 29.2. 7-((4-(2-(Diethylamino)ethoxy)phenyl)amino)-3-(4-methoxyphen yl)-l -methylpyrido[3,4- 6]pyrazin-2(127)-one (E7, SS-2-107)

Method T; The crude product was separated by chromatography (5% MeOH in DCM ), and yielded SS-2-107 (42%) as yellow solid. ^! H-NMR (600 MHz, CDCh) 8 8.69 (s, 1H), 8.29 (d, J = 8.8 Hz, 2H), 7.27 is, I H), 6.97 (dd, J - 8.4, 5.7 Hz, 4H), 6.71 (s, 1 H ), 6.30 (s, 1H), 4.07 (t, J - 6.2 Hz, 2H), 3.87 (s, 3H), 3.52 (d, J = 10.3 Hz, 3H), 2.90 (t, J = 6.3 Hz, 2H), 2.66 (q, J = 7.1 Hz, 4H), 1.09 (t, J ^:::: 7. 1 Hz, 6H). ¹³ C-NMR (150 MHz, CDCh)) 8 161.1, 157.7, 156.6, 155.4, 151.5, 149.1, 140.8, 131.9, 130.9, 128.6, 125.2, 124.5, 115.6, 113.5, 86.9, 66.9, 55.4, 51.7, 47.9, 28.8, 11.8.

7-((4-(2-(Diethyiamino)ethoxy)phenyl)amino)-3-(4-hydroxyp henyl)-l-methylpyrido[3,4- Z>]pyrazin-2(lZ/)-one (SS-2-114)

To a solution of E7 (12.4 mg, 0.026 mmol) in anhydrous DCM (1 ml.) at 0 °C, was added BBn (0.21 mL, 0.21 mmol) dropwise under argon. The ice bath was removed, and the reaction wanned slowly to room temperature and stirred at room temperature for 16 h, the reaction mixture was cooled to 0 C, quenched with saturated aqueous solution of NaHCO?, and extracted with 20% MeOH in CHJCI multiple times. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The crude product was separated by (0-5% MeOH in DCM and 01% NH ₄ OH) and yielded SS-2-114 (56%) as a yellow solid, mp 189- 191 °C. ^l H-NMR (600 MHz, DMSO-de) 5 9.91 (s, 1H), 9.30 (s, H i), 8.56 (s, 1 H), 8.14 (d, J - 8.8 Hz, 2H), 7.53 (d, J = 9.0 Hz, 2H), 6.91 (d, J = 8.8 Hz, 2H), 6.84 (d, J = 8.8 Hz, 2H), 6.53 (s, 1H), 3 98 (t, J - 6.2 Hz, 2H), 3 50 (s, 3H), 2.76 (t, J - 6.1 Hz, 2H), 2.55 (q, J - 7.1 Hz, 4H), 0 97 (t, J - 7.1 Hz, 6H). ^l3 C-NMR (150 MHz, DMSO-dc,) 5 159.1, 156.3, 154.8, 153.8, 149.7, 147.9, 140.1, 133.9, 130.7, 126.9, 123.8, 121.1 , 114.7, 114.7, 89.9, 66.5, 51.4, 47.0, 28 5, 11.9. HRMS (ESI) m/z: [M + H] ⁺ calculated for C26H29N5O3: 460.2343; observed 460.2342. Purity >95%, fe - 8.82 min (HPLC Method II).

6-Bromo-N-(4-(2-(diethylamino)ethoxy)phenyl)quinazolin-2- amine (F13) A mixture of Fl 2 (175 mg, 0.72 mmol) and C9a (150 rag, 0 72 mmol) was dissolved in isopropyl alcohol (3mL). The reaction mixture was stirred at 100 °C for 18 h. The solvent was removed under vacuum. The crude product was purified using column chromatography (0-5% MeOH in DC M). Product F13 (56%) was afforded as a yellow solid. ^! H-NMR (100 MHz, CDCls) 8 8.95 (s, 1H), 7.83 (d, J = 2.3 Hz, 1H), 7.75 (dd, J = 9.2, 2.3 Hz, 1H), 7.66 (d, J = 9.2 Hz, 2H), 7 55 (d, J = 8.7 Hz, 1H), 7.46 (s, I H), 6.93 (d, J =- 9.2 Hz, 2H), 4. 10 (t, J === 6.2 Hz, 2H), 2.94 (t, J

= 6.2 Hz, 2H), 2.72 (q, J = 7.2 Hz, 4H), 1.11 (t, J = 7.1 Hz, 6H). ^l3 C-NMR (100 MHz, CDCh) 6 160.7, 157.1, 154.7, 150.4, 137.5, 132.4, 129.4, 127.9, 121.6, 121.2, 116.0, 1 14.9, 664, 51.6, 47 7, 1 1.4. 5-(2-((4-(2-(Diethylamino)ethoxy)phenyl)amino)quinazolin-6-y l)-2-hydroxybenzonitrile (SS-2- 108)

To a mixture of F13 (15 mg, 0.036 mmol), Clla (8.83 mg, 0.054 mmol), PdCh(PPh3)2 (4.2 mg, 0.0036 mmol), TBAB (11.6 nig, 0,036 mmol) in anhydrous toluene/EtOH (1 ml/ I mL) was added 2M NaiCOi (0.091 mL, 0.09 mmol) under argon The mixture was placed under MW irradiation at 110 °C for 1 h. The reaction mixture was quenched with water and extracted with CDM multiple times. The combined organic phases were washed with brine, dried over anhydrous NaiSOr, filtered, and concentrated. The crude product was separated by chromatography (5-10% MeOH in DCM and 0. 1% NHiOH). SS-2-108 (76%) was afforded as a yellow solid, mp 189-191 i ^J C. ^l H-NMR (600 MHz, CDiOD) 5 9.14 (s, H I). 7.99 (dd, J - 8.8, 2.1 Hz, 1 H), 7.97 (d, J - 1 .7 Hz, 1H), 7.84 (d, J = 2.2 Hz, 1H), 7.80 (dd, J = 8.8, 2.4 Hz, 1H), 7.76 (d, J = 8.8 Hz, 2H), 7.64 (d, J - 8 6 Hz, 1H), 7.02 (d, J - 8.8 Hz, 1 H), 6 97 (d, J - 9. 1 Hz, 2H), 4 25 (t J - 5.2 Hz, 2H), 3.33 (d, J = 5.2 Hz, 2H), 3.09 (q, J = 7.2 Hz, 4H), 1.28 (t, J = 7.2 Hz, 7H). ⁿ C-NMR(150 MHz, CDsOD) S 163.6, 163.0, 158.7, 155.1, 151.8, 135.6, 135.5, 134.2, 134.1, 132.3, 132,0, 127.1, 125.6, 122.4, 122.1, 118.5, 1 18.3, 115.8, 101.5, 65.0, 52.5, 10.0. HRMS (ESI) m/z: [M s- H]~ calculated for

C27H27N5O2: 454.2238, observed 454.2237. Purity >95%, r«= 9.92 min (HPLC Method II).

Scheme 9: Reagents and conditions: a) 2-Propanol, 100 °C, 16 h, 56%; b) Pd(PPh3)4, NazCOa (2M), TBAB, EtOH/Toluene (1 : 1), 120 °C, 16 h, 61%.

6-Bromo-/V-(4-(2-(diethylamino)ethoxy)phenyl)quinazoiin-2 -amine (3)

The starting materials 1 (175 mg, 0.723 mmol), amine 2 (150 mg, 0.723 mmol) were placed in a round bottom flask and 2-propanol (3.0 mL) was added. The reaction mixture was refluxed in an oil bath at 100 °C for 16 h. After completion of reaction all volatiles were removed under reduced pressure. The crude compound was purified by combiflash chromatography (0-5% methanol in CH2CI2) to yield 3. Yellow solid, mp. 103-105 °C; yield: 170 mg (56%); ¹ H NMR (400 MHz, CDClfi: 5 8.96 (s, 1H), 7.83 (d, J = 2.4 Hz, 1H), 7.77-7.74 (dd, J = 9.16 Hz, 2.4 Hz, 1H), 7.68-7.64 (m, 2H), 7.57-7.55 (m, 1H), 7.47 (s, 1H), 6.95-6.91 (m, 2H), 4.11 ft, J - 6 2 Hz, 2H), 2.95 (t, J= 62 Hz, 2H), 2.72 (q, J = 7.2 Hz, 4H), 1.12 (t, J = 7.1 Hz, 6H); ^B C NAIR (100 MHz, CDClfi: 8 160 7, 157.1, 154.7, 150.4, 137.5, 132.4, 129.4, 127.9, 121.6, 121.2, 116.0, 114.9, 66.4, 51.6, 47.7, 11.4.

4-(2-((4-(2-(Diethylamino)ethoxy)phenyl)amino)quinazolin- 6-yl)phenol (S).

The starting materials 3 (30 mg, 0.072 mmol), boronic acid 4 (15 mg, 0.108 mmol), TBAB (23 mg, 0.072 mmol), NazCOs (2M, 0 1 mL, 0.179 mmol) were placed in a sealed tube and then ethanol (1.0 mL) and toluene (1.0 mL) was added. Then Pd(PPhfi4 (8.3 mg, 0.007 mmol) was added to the reaction mixture. The flask was flush with argon gas for 5-10 min. The reaction mixture was refluxed in an oil bath at. 100 °C for 16 h. After completion of the reaction, all volatiles were removed under reduced pressure. The crude compound was purified by combiflash chromatography (0~5% methanol in CH2CI2) to yield 5 Yellow solid; mp. 199-201 °C; yield: 19 mg (61%); H \\!R (600 MHz, CD3OD): 5 9.16 (s, 1H), 8.03-8.0 (dd, J = 10.2 Hz, 3.0 Hz, 1H), 7.96 (d. ./ 2.4 Hz, 1H), 7.79-7.76 (m, 2H), 7.66-7.64 (m, I H), 7.58-7.55 (m, 2H), 7.01-6.98 (m, 2H), 6.90-6.88 (m, 2H), 4.26 (t, J= 6.0 Hz, 2H), 3.37-3.35 (m, 2H), 3.14-3.09 (q, J= 8.9 Hz, 4H), I 28 ( t ,./ 8 6 Hz, 6H); ^l3 C NMR (150 MHz, CD3OD): 5 163.5, 158.5, 155.0, 151.5, 137.7, 135.6, 134.8, 132.4, 129.1, 126.8, 125.2, 122.4, 122.2, 116.9, 116.7, 115.9, 115.7, 64.7, 52.5, 9.8. HRMS (ESI): m/z [M + H]+ cal cd for C26H28N4O2: 429 2285; found: 429.2279. Purity was determined to be 100% by analytical high-performance liquid chromatography (WATERS HPLC) using binary pump Kinetex 5 pm C18 100A column (250 x 4.6 mm). UV absorption was monitored at X = 254 nm. The injection volume was 15 pL. The gradient of acetonitrile/water (both containing 0.1% trifluoroacetic acid) was 2.98 to 90: 10 over a total run time of 30 min and a flow rate of 1 mL/min. tR ^::: 17.224.

Scheme 10: Reagents and conditions: a) Meldrum’s acid, piperidine, AcOH, Ethanol, reflux, 70 °C, 2 h, 88%, b) LiOAc, NIS, DMF/ H ₂ O, MW, 110 °C, 15 min, 78%; c) m-CPBA, CH2CI2, 0 °C- rt, 1 h, 70-90%; d) NaH, DMF / THF, 0 °C- rt, 2 h, 68%; e) PdChfPPhsh, K2CO3 (3M), DMF, MW, Ih, 110 °C, 45-50%.

8-Methyl-2-(methylthio)-7-oxo-7,8-dihydropyrido[2,4-</ ]pyrimidin-6-carboxylic acid (7)

To a solution of aldehyde 6 (350 mg, 1.912 mmol), Meldrum’s acid (275 mg, 1.91 mmol) in ethanol (4 mL) was added piperidine (16 mg, 0.187 mmol) and acetic acid (35 pL, 0.566 mmol) at room temperature. The reaction mixture was stirred for 20 min at room temperature and then the reaction mixture was refluxed at 70 °C for 2 h After completion of the reaction, the reaction mixture was filtered through sintered funnel and the filtered solid washed with ethanol. This solid was dried under vacuum to yield 7 as off-white solid; mp. 255-257 °C; yield: 423 mg (88%); Will (600 MHz, DMSO-Ds): 3 9.15 (s, I H), 8.81 (s, IH), 3.68 (s, 3H), 2.65 (s, 3H); ^B C NMR (150 MHz, DMSO-De): 8 174.96, 164.09, 162.88, 159.81, 154.00, 141.93, 119.41, 108.99, 28.19, 14.07.

6-Iodo-8-methyl-2-(methylthio)pyrido[2,3-c/Jpyrimidin-7(8 //)-one (8).

A solution of 7 (-410 mg, 1.6333 mmol), N-iodosuccinimide (1.17 g, 5 20 mmol) and LiOAc (129 mg, 1.96 mmol) in DMF / H ₂ O (20 ml, in 9: 1) was refluxed at 110 °C for 15 min under microwave. After completion of reaction, the reaction was quenched with 30 mL of water. The mixture was extracted with ethyl acetate. The combined organic layers were washed with brine solution and dried over sodium sulfate. The crude compound was purified by combiflash chromatography (0 20% ethyl acetate in hexane) to yield 8. Pale yellow solid; mp. 203-205 °C; yield: 427 mg (78%); NMR (600 MHz, CDCh): 6 8.59 (s, IH), 8.33 (s, IH), 3.83 (s, 3H), 2.64 (s. 3H); ^!3 C NMR (150 MHz, CDCh): 5 173,78, 159.79, 155.08, 154.34, 143.89, 110.67, 93.4, 29.55, 1448.

6-Iodo-8-methyl-2-(methylsulfonyl)pyrido[2,3-<7]pyrimi din-7(8H)-one (9).

To a solution of 8 (570 mg, 1.712 mmol) in dichloromethane (20 mL) was added mCPBA (1.15 g, 6.667 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 1 h. After completion of the reaction, the volatiles were removed under reduced pressure. The crude compound was washed multiple times with hexane and ether to yield 9. Yellow solid; yield: 560 mg (90%); 'H NMR (600 MHz, CDCI3): 5 8.95 (s, 1H), 8.53 (s, 1H), 3.91 (s, 3H), 3.42 (s, 3H). ¹³ C NMR (150 MHz, CDCh): 8 164.5, 159.3, 155 8, 155.2, 142.8, 115.8, 100.7, 39.2, 30.4.

2-((4-(2-(Dietbylamino)ethoxy)phenyl)amino)-6-iodo-8-meth ylpyrido[2,3-</jpyrimidin-7(8J/)- one (11).

To a solution of 10 (365 mg, 1.288 mmol) in anhydrous THF (9 mL) was added NaH (93 mg, 3.875 mmol) at 0 °C. The reaction mixture was stirred for 20 min at 0 °C. Then 9 (470 mg, 1.288 mmol dissolved in 18 mL of DMF / THF 1:1) was added drop wise to the reaction mixture at 0 °C The reaction mixture was stirred for 2 h at room temperature. After completion of the reaction, it was quenched with water (10 mL). The mixture was extracted with ethyl acetate. The combined organic layers were washed with brine solution and dried over sodium sulfate. The crude compound was purified by combiflash (0-3% methanol in CH2CI2) to yield 11 and 12. This material was used as mixture for the next reaction Yellow solid; 335 mg.

General Procedure E: preparation of the target compounds (14a-e).

A mixture of 11, 12 (0.1 mmol), the corresponding boronic acid 13 (0.1 mmol), K2CO3 (0.303 mmol, 3M) and PdC12(PPh3)2 (0.005 mmol) in DMF (1 mL) was refluxed at 110 °C for 1 h under microwave. After completion, the reaction was quenched with 5 mL of water. The mixture was extracted with ethyl acetate. The combined organic layers were washed with brine solution and dried over sodium sulfate. The crude compound was purified by combiflash chromatography (0—5% methanol in CH2CI2) to yield the products 14a-b.

6-(3-Chloro-4-hydroxyphenyl)-2-((4-(2-(diethylamino)ethox y)phenyl)amino)-8- methylpyrido[2,3-d]pyrimidin-7(8H)-one (14a).

Compound 14a was synthesized from a mixture of compounds 11 and 12 (50 mg, 0.1 mmol) according to the general procedure E. Orange solid, Yield: 22 mg (46%). 'l l NMR (600 MHz, CD3OD). 8 8.69 (s, 1H), 7.86 (s, 1H), 7 72-7.68 (m, 3H), 7.45-7.44 (dd, J ----- 8.3 Hz, 1.7 Hz, 1 H), 7.04-7.02 (m, 2H), 6.97-6.95 (m, 1H), 4.35 (t, J ------ 4.8 Hz. 2H), 3.74 (s, 3H), 3 61 (t, ,/ == 4.5 Hz, 2H), 3.39-3.32 (m, 4H), 1.38 (t, J= 7.2 Hz, 6H); ¹³ C NMR (150 MHz, CDCI.3): 5 164.8, 160.5, 160.1 , 156.3, 155.0, 154.3, 135.2, 134.9, 131.5, 130.1, 129.5. 128.1, 122.9, 121.4, 117.2, 115.9, 108.3, 63.7, 52.4, 28 9, 9.15. HRMS (ESI): m/z [M + H]+ calcd for C26H28CIN5O3: 494.1953, found: 494.1954. Purity was determined to be 95% by analytical high-performance liquid chromatography (WATERS HPLC) using binary pump Kinetex 5 pm C18 100 A column (250 x 4 6 mm). UV absorption was monitored at X ^:::: 254 nm. The injection volume was 15 pL. The gradient of aceiomtrile/water (both containing 0.1% trifluoroacetic acid) was 2.98 to 90:10 over a total run time of 30 min and a flow rate of 1 mL/rnin. tR ^:::: 19.739

2-((4-(2-(diethylamino)ethoxy)phenyl)amino)-6-(4-hydroxy- 3-nitrophenyl)-8-methylpyrido[2,3- d]pyrimidin-7(8H)-one (14b).

Compound 14b was synthesized from a mixture of compounds 11 and 12 (100 mg, 0.2 mmol) according to the general procedure E. Orange solid, Yield: 48 mg (48%). ^r H NMR (600 MHz, CDsOD): 5 8.65 (s, 1H), 8.27 (s, 1H), 7.81 (s, 1H), 7.64-7.55 (m, 3H), 6.93 (d, J= 8.9 Hz, 2H), 6.80 (d, ,/ == 8.9 Hz. H i), 4 10 (t, J ------ 5 6 Hz, 2H), 3.70 (s, 3H), 2.90 ( t . ./ 5 7 Hz, 2H), 2.68 (q, J= 7.2 Hz, 4H), 1.10 (t, J = 7.2 Hz, 6H); HRMS (ESI): m/z [M + H]+ calcd for Cd bAW: 505.2194; found: 505.2199. Purity was determined to be 96% by analytical high-performance liquid chromatography (WATERS HPLC) using binary pump Kinetex 5 pm C18 100A column (250 x 4,6 mm). UV absorption was monitored at X ^::: 254 nm. The injection volume was 15 pL,

The gradient of acetonitrile/ water (both containing 0.1% trifluoroacetic acid) was 2.98 to 90:10 over a total run time of 30 min and a flow' rate of 1 raL/min. tR ^:::: 20.744.

6-Iodo-8-methyl-2-((3-(4-mefhylpiperazin-l-yl)phenyl)amin o)pyrido[2,3-t/]pyrimidin-7(8jF/)-one (16).

Compounds 16 and 17 were synthesized from compound 15 according to the procedure used for synthesizing 11. Yellow solid; yield: 70 mg (mixture). This mixture was used for next reaction. 16 yellow solid; 'H NMR (600 MHz, CDC13): 8 8.51 is. H 1), 8.22 (s, IH), 7.38 (m, 2H) 7.08 (d, J= 7.9 Hz, IH), 6.72 (dd, J = 8.3, 2 1 Hz, 1H), 3.81 (s, 3H), 3.27 (t, J = 4.8 Hz, 4H), 2.60 (t, J ----- 4.8 Hz, 4H), 2.37 (s, 3H). FIRMS (ESI): m/z [M + H]+ calcd for CisthiNeOL 477.0894, found: 477.0906. jV-(6-iodo-8-methyl-7-oxo-7,8-dihydropyrido[2,3-J|pyrimidin- 2-yl)-N-(3-(4-methylpiperazin-l- yl)phenyl )formamide ( 17) .

Yellow solid: ¹ H NMR (600 MHz, CDCE): 8 10.10 (s, 1H), 8.63 (s, H i). 8.34 (s, IH), 7.39 ii, ./ 8.1 Hz, IH), 7.00 (dt, J ------ 8.6 Hz, 1.0 Hz, I H), 6.75 (s, IH), 6.71 id. ./ 7.9 Hz, IH), 3.64 (s, 3H), 3.2.5 (t, J= 4.8 Hz, 4H), 2.59 (t J = 4.6 Hz, 4H), 2.36 (s, 3H). ). HRMS (ESI): m/z [M + H]+ calcd for C20H21N6O2I: 505-0843; found: 505.0858.

6-(4-Hydroxyphenyl)-8-methyl-2-((3-(4-methylpiperazin-l-y l)phenyl)amino)pyrido[2,3- Jjpyrimidin-7(8//)-one (18).

Compound 18 was synthesized from a mixture of compounds 16 and 17 (25 mg) according to the general procedure E. Yellow solid; yield: 1 1 mg (47%); Td NMR (600 MHz, Methanol-Dr): δ 8.69 (s, IH), 7.80 (s, IH), 7.50 (d, <Z= 8.3 Hz, 3H), 7.27-7.21 (m, 2H), 6.83 (d, J= 8.6 Hz, 2H), 6.71 (d. J ----- 7.9 Hz, IH), 3.77 (s, 3H), 3.29 (m, 4H), 2.79 (m, 4H) 2.48 (s, 3H); ⁿ C NMR (150 MHz, CDCli): 3 163.0, 158.5, 157.9, 155.8, 154.9, 151.9, 139.5, 132.1, 130.2, 129.6, 129.1, 128.6, 115.2, 111.4, 1 1 1 0, 107.2, 107.1 , 54.9, 48.9, 46.0, 28 6. HRMS (ESI): m/z [M + H j calcd for C25H26N6O2: 443.2190; found: 443.2207. Purity was determined to be 96% by analytical high- performance liquid chromatograph}' (WATERS HPLC) using binary pump Kinetex 5 uni C18 100 A column (250 x 4 6 mm). UV absorption was monitored at λ ~ 254 nra. The injection volume was 15 μL. The gradient of acetonitrile/water (both containing 0.1% trifluoroacetic acid) was 2.98 to 90:10 over a total run time of 30 min and a flow rate of 1 mL/min. tR 17.757.

Scheme 12: Synthesis of SH2-160. (a) 2M Methylamine (in THF), TEA, THF, rt, 6 h, 87-88% (b) DBU, DMSO, rt, 16 h, 69-88%. (c) w-CPBA, DCM, 0 °C ~ rt, 84-85%. (d) SH2-137, IM LiHMDS (in THF), THF, -78 °C, 98%. (e) NIL OH, 1,4-dioxane, 70 °C, 16 h. (f) IM BBrs (in DCM), 0 °C - rt, 24 h, 66%

4-chloro-6-(m ethyl amino)-2-(methykhio)pyrimidine-5-carba!dehyde

To solution of 4,6-dichloro-2-(methylthio)pyrimidine-5-carbaJdehyde (1 g, 4.48 mmol) in THF (30 ml) was added 2M methylamine (2.68 mL, 5.37 mmol) and TEA (937 pL, 6.72 mmol) and the mixture was stirred 2 h at room temperature. After completion of the reaction, THF was concentrated, and the mixture was diluted with DCM (50 mL) and washed with HzO (50 mL). The aqueous layer was washed with DCM (2 x 25 mL), and the combined organic extracts were washed with brine, dried over anhydrous NarSO-4, filtered and concentrated The residue was purified by column chromatography on silica gel (EtOAc/hexane, 10:90 to 20:80) to afford SH2-149 (850 mg, 87%) as a light-yellow solid. ¹ H-NMR (CDCh, 600 MHz) 8 10.23 (s, 1 H), 9.21 (s, 1 H), 3.13 (d, J = 5.0 Hz, 3H), 2.56 (s, 3H); ^l3 C-NMR (CDCh, 150 MHz) 5 190.4, 176.8, 164.2, 160.6, 104.7, 27.70, 1443.

Note, the reaction was repeated with yields of 87-88%.

4-chloro-6-(4-methoxyphenyl)-8-methyl-2-(inethylthio)pyri do[2,3-d]pyrimidin-7(8H)-one

To solution of SH2-149 (30 mg, 0.137 mmol) in DMSO (3 niL) was added methyl 2-(4- methoxyphenyl)acetate (26 uL, 0.165 mmol) and DBU (25 pL, 0.165 mmol). The reaction was maintained at room temperature for overnight. After completion of the reaction, the mixture was filtered and wash with ether (5 x 5 ml.) to afford SH2-154 (42 mg, 88%) as a light-yellow solid. i lAMR (CDCh, 600 MHz) 5 7.94 (s, 1H), 7.67-7.65 (m, 2H), 6.99-6.98 (m, 2n), 3.87 (s, 3H), 3.82 (s, 3H), 2.65 (s, 3H); ¹³ C-NMR (CDCh, 150 MHz) 6 171.5, 162.2, 160.1, 158.4, 154,3, 132.7, 130.2, 129.5, 127.7, 1 13.8, 107 8, 55.3, 29.1 , 14.6.

Note, the reaction was repeated with yields of 69-88%. The combined organic layer (mother liquor) was washed with water, concentrated, and performed column to get more SH2- 154.

4-chloro-6-(4-methoxyphenyl)-8-methyl-2-(methylsulfonyl)p yrido[2,3-d]pyrimidin-7(8H)-one

To solution of SH2-154 (62 mg, 0.178 mmol ) in DCM (4 mL) at 0 °C was added OT-CPBA (92 mg, 0 534 mmol) and allowed the mixture to room temperature for 2 h. After 2 h, the mixture was concentrated and washed with hexane (3 * 3 mL) and ether (2 > 3 mL) to afford SH2-155 (58 mg, 85%) as a light-yellow solid. ‘H-NMR (CDCh, 600 .MHz) 8 8.05 (s, 1H), 7.73-7.71 (m, 2H), 7.02-7.00 (m, 2H), 3 91 (s, 3H), 3.88 (s, 31 h. 3.43 (s, 3H); ⁿ C-NMR (CDCh, 150 MHz) 5 162 3, 161.5, 161 0, 159.7, 154.8, 137.5, 130.5, 127.7, 126.6, 114.0, 1 13.8, 55 4, 39.1 , 29.9. Note, the reaction was repeated with yields of 84-85%.

4-(2-(diethylamino)ethoxy)aniline To solution of N,N-diethyl-2-(4-nitrophenoxy)ethanamine (2.5 g, 10.5 mmol) in MeOH

(30 ml) was added Pd/C (10% weight) and stirred at room temperature for overnight. .After completion of the reaction, the mixture was filtered by celite and concentrated to afford SH2-137 (2.15 g, 98%) as a dark black liquid HI-NMR (CDCh, 600 MHz) 8 6.76-6 74 (m, 2H), 6.65-6.62 (ni, 2H), 3.97 (t, J = 6.4 Hz, 2H), 3.45 (br. s, 2H), 2.84 (t, J = 6.5 Hz, 2H), 2.63 (q, J = 7.2 Hz, 4H), 1.06 (t, J - 7.1 Hz, 6H); ^l3 C-NMR (CDCh, 150 MHz) 8 152.0, 139.9, 116.3, 115.5, 67.0, 51.8,

47.7, 11.8.

4-chloro-2~((4-(2-(diethylamino)efhoxy)phenyl)amino)-6-(4 -methoxyphenyl)-8- niethylpyrido[2,3-d]pyrimidin-7(8H)-one To a solution of SH2-137 (188 mg, 0.906 mmol) in THF (20 niL) at - 78 °C was added 1

M LiHMDS in THF (906 pL, 0.906 mmol). After 15 minutes, SH2-155 (265 mg, 0.697 mmol) in THF (5 mL) was added to the mixture and stirred for 1.5 h. After completion of the reaction, the mixture was warmed up to room temperature and quenched by NH4CI (10 mL). THF was concentrated and the residue was diluted with EtOAc (50 mL) and washed with H ₂ O (50 mL). The aqueous layer was washed with EtOAc (2 x 25 mL), and the combined organic extracts were washed with brine, dried over anhydrous Na2SO4, filtered and concentrated. The residue was washed with hexane (2 x 10 mL) and ether (2 z 10 mL) to afford SH2-156 (350 mg, 98%) as a yellow solid. ^£ H-NMR (CDCh, 600 MHz) 5 7.89 (s, 1H), 7.65 (m, 2H), 7.57 (m, 2H), 6.98 (m, 2H), 6.93 (m, 2H), 4.41 (t, J = 4.6 Hz, 2H), 3.86 (s, 3H), 3.76 (s, 3H), 3.53 (t, J = 4.6 Hz, 2H), 3.30 (m, 4H), 1.40 (t, J = 7.3 Hz, 6H); ⁿ C-NMR (CDCh, 150 MHz) 8 162.6, 159.7, 159.5, 156.9, 155.9,

153.7, 132.4, 130.1, 130.1, 129.4, 128.2, 121.7, 114.9, 113.7, 63.1, 55.3, 50.4, 46.8, 29.2, 8.5. Note, the reaction was repeated with yields of 84-85%. The combined organic layer (mother liquor) was washed with water, concentrated, and performed column to get more SH2- 156.

4-amino-2-((4-(2-(diethylamino)ethoxy)phenyl)amino)-6-(4- methoxyphenyr)-8- methylpyrido[2,3-d]pyrimidin-7(8H)-one

To a solution of SH2-156 (22 mg, 0.043 mmol) in dioxane (2 mL) was added NFUOH (2 mL). The mixture was stirred at 70 °C for overnight. After completion of the reaction, the mixture was diluted with EtOAc (10 mL) and washed with HiO (10 mL). The aqueous layer was washed with EtOAc (2 x 10 mL), and the combined organic extracts were washed with brine, dried over anhydrous Na2SOr, filtered and concentrated. The residue was purified by column chromatography on silica gel (MeOH/DCM, 5:95 to 10:90) to afford SH2-157 (12 mg) as a yellow solid. The product was used for next, step without further purification. (CDCh, 600 MHz) 6 7.61-

7.57 (m, 2H), 7.53-7.50 (m, 2H), 7.44 (d, J = 8.8 Hz, H I). 6.95-6.89 (m, 511), 4.09 (t, J = 4.6 Hz, 2H), 3.84 (s, 3H), 3.73 (s, 3H), 2.92 (t, J = 4.6 Hz, 2H), 2.69 (m, 4H), 1.10 (t, J = 7.3 Hz, 6H).

4-amino-2-((4-(2-(diethylamino)ethoxy)phenyl)amino)-6-(4- hydroxyphenyf)-8- rn ethylpyrido[2,3 -d]py ri midin-7(8H)-one

To a solution of SH2-157 (30 mg, 0.06 mmol) in DCM (2 mL) was added IM BBn (1.5 mL) dropwise at 0 °C. The reaction was maintained at room temperature for overnight. After completion of the reaction, the mixture was quenched by saturated aqueous Na2CO3. The aqueous layer was washed with EtOAc (2 x 10 mL) and the combined organic extracts were washed with brine, dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by column chromatography on silica gel (MeOHZDCM, 5:95 to 10:90) to afford SH2-160 (19 mg, 66%) as a light-yellow solid. TI-NMR (DMSO, 600 MHz) S 9.57 (s, 1 H), 9.17 (s, 1 H), 8.17 (s, 1H), 7.70 (d.

J = 9.0 Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H), 7.35 (br, s, 2H), 6.87 (d, J = 9.0 Hz, 2H), 6.78 (d, J = 8.6 Hz, 2H), 3.98 (t. J = 6.2 Hz, 2H), 3.58 (s, 3H), 2.75 (t, J == 6.2 Hz, 2H), 2.54 (q, J == 7.1 Hz, 4H), 0.97 (t, J = 7.1 Hz, 6H); ^t3 C-NMR (DMSO, 150 MHz) 5 162.0, 161.5, 158.5, 156.6, 154.9, 153.4, 133 7, 129.7, 129.7, 127.8, 122.7, 120.9, 114.6, 114.2, 66.4, 51.4, 47.0, 40.0, 11.92. A sample for bioassay assessment was purified using Method A. HPLC purity via Method A: > 95%, fe = 17.5 min HRMS (ESI): rn/z [M + H]+ calculated for CridboNeCb : 475.2452, found: 475 2446.

Scheme 13. Synthesis of SH3-20. (a) 2M Methylamine (in THF), TEA, THF, rt, 6 h, 87-88%. (b) DBU, DMSO, rt, 16 h, 30-33%. (c) w-CPBA, DCM, 0 °C - rt, 93%. (d) SH2-137, IM LiHMDS (in THF), THF, -78 °C, 48-77%. (e) TFA, DCM, 0 °C - rt, 1.5 h, 80%. m et hy I 2-(4-( (4-m ethoxy b enzy I )oxy )ph eny 1 )acetate

To a solution of methyl 4-hydroxyphenylacetate (100 mg, 0.60 mmol) in DMF (2 mL) was added K2CO3 (165 mg, 1 .2 mmol) and 4-methoxy benzylchloride (97 p.L, 0.72 mmol). The mixture was stirred at room temperature for overnight. After completion of the reaction, the mixture was diluted with EtOAc (15 mL) and washed with water (20 niL). After completion of the reaction, the mixture was diluted with EtOAc (15 mL) and washed with H ₂ O (15 mL). The aqueous layer was washed with EtOAc (2 x 10 mL), and the combined organic extracts were washed with brine, dried over anhydrous NazSOu filtered and concentrated. The residue was purified by column chromatography on silica gel (EA/hexane, 15:85 to 30:70) to afford SH3-8 (140 mg, 81%) as a white solid I IAMR (CDCh, 600 MHz) 8 7 35 (d, J - 8.6 Hz, 2H), 7. 19 (d, J === 8.6 Hz, 2H), 6.94- 6.91 (m, 4H), 4.97 (s, 2H), 3.82 (s, 3H), 3.68 (s, 3H), 3.57 (s, 2H); ⁿ C-NMR (CDCh, 150 MHz) 6 172.3, 159.4, 157.9, 130.2, 129.2, 128.9, 126.2, 114.9, 113.9, 69.7, 55.3, 52.0, 40.3. Note, the reaction was repeated with yields of 81 89%.

4-chloro-6-(4”((4-methoxybenzyl)oxy)phenyl)-8-methyl-2- (methylthio)pyrido[2,3-d]pyrimidin- 7(8H)-one

To solution of SH2-149 (30 mg, 0. 137 mmol) in DMSO (2 mL) was added SH3-8 (47 mg, 0 165 mmol) and DBU (20 pL, 0.137 mmol) The reaction was maintained at room temperature for overnight. After completion of the reaction, the mixture was After completion of the reaction, the mixture was diluted with EtOAc (10 mL) and washed with HzO (10 mL). The aqueous layer was washed with EtOAc (2 x 10 mL), and the combined organic extracts were washed with brine, dried over anhydrous NazSOr, filtered and concentrated. The residue was purified by column chromatography on silica gel (EA/hexane, 15:85 to 30:70) to afford SH3-10 (17 mg, 27%) as a yellow solid. ^-NMR (CDCh, 600 MHz) 87.93 (s, 1H), 7.66 (d, J = 8.6 Hz, 2H), 7.38 (d, J = 8.4 Hz, 211). 7.05 (d, J =- 8.6 Hz, 2H), 6.93 (d, J - 8.4 Hz, 2H). 5.04 (d, J - 8.6 Hz. 21 1), 3.83 (s, 3H). 3.81 (s, 3H), 2.65 (s. 3H); ^l3 C-NMR (CDCh, 150 MHz) 6 171.5, 162.1, 159.4, 159.4, 158.4, 154.3, 132.7, 130.2, 129 5, 129.2, 128.7, 127.8, 114.7, 114.0, 107.8, 69.8, 55.3, 29.1, 14.5. Note, the reaction was repeated with yields of 27-33%.

4-chloro-6-(4-((4-methoxybenzyl)oxy)phenyl)-8-methyl-2-(m ethylsulfonyl)pyrido[2,3- d] py ri mi din- 7(8 H)-one

To solution of SH3-10 (100 mg, 022 mmol ) in DOM (4 mL) at 0 °C was added mCPBA (113 mg, 0.66 mmol) and allowed the mixture to room temperature for 2 h. After 2 h, the mixture was diluted with EtOAc (20 mL) and -washed with H O (20 mL). The aqueous layer was washed with EtOAc (2 * 20 mL), and the combined organic extracts were washed with brine, dried over anhydrous NazSOy filtered and concentrated. The residue was purified by column chromatography on silica gel (EA/hexane, 5:95 to 10:90) to afford SH3-15 (100 mg, 93%) as a light-yellow solid. H-XMR (CDCI3, 600 MHz) 5 8.05 (s, 1H), 7 72 (d, J - 8.8 Hz, 2H), 7.38 (d. J - 8.4 Hz, 2H), 7.08 (d, J = 8.6 Hz, 2H), 6.94 (d, J = 8.6 Hz, 2H), 5.07 (s, 2H), 3.91 (s, 3H), 3.83 (s, 3H), 3.42 (s, 3H); ¹³ C-NMR (CDCls, 150 MHz) 6 162.3, 161.5, 160.2, 159.7, 159.5, 154.8, 137.5, 130.5, 129.2, 128.4, 127.7, 126.7, 114.9, 114.0, 113.8, 69.9, 55.3, 39.1 , 29.9. Note, the reaction was repeated with yields of 86-93%.

4-chloro-2-((4-(2-(diethylamino)ethoxy)phenyl)amino)-6-(4 -((4-methoxybenzyl)oxy)phenyl)-8- methylpyrido[2,3-d]pyrimidin-7(8H)-one

To a solution of SH2-137 (47 mg, 0.225 mmol) in THF (2 mL) at - 78 °C was added 1 M LiHMDS in THF (246 uL, 0.246 mmol). After 15 minutes, SH3-15 (100 mg, 0.205 mmol) in THF (3 mL) was added to the mixture and stirred for 2 h. After completion of the reaction, the mixture was warmed up to room temperature and quenched by NH4CI (10 mL). THF was concentrated and the residue was diluted with EtOAc (20 mL) and washed with H ₂ O (20 mL). The aqueous layer was washed with EtOAc (2 x 15 ml..), and the combined organic extracts were washed with brine, dried over anhydrous NazSCh, filtered and concentrated. The residue was purified by column chromatography on silica gel (MeOH/DCM, 5:95 to 10:90) to afford SH3-16 (61 mg, 48%) as a yellow solid. ¹ H-NMR (CDCh, 600 MHz) 8 7.88 (s, 1H), 7.64 (d, J = 8.8 Hz, 2H), 7.52 (d, J = 9.0 Hz, 2H), 7.38 (d, J == 8.6 Hz, 2H), 7.30 (s, 1H), 7 03 (d, J = 8.8 Hz, 2H), 6.95-6.92 (m, 4H), 5.04 (s, 2H), 4.07 (t, J = 6.3 Hz, 2H), 3.82 (s, 3H), 3.76 (s, 3H), 2.90 (t, J = 6.3 Hz, 2H), 2.66 (q, J = 7.2 Hz, -H i). 1 .09 (1, 1 7 1 Hz, 6H); ¹³ C-NMR (CDCh, 150 MHz) 8 162.7, 159.4, 158.9, 157.0, 155.9, 155.5, 131.0, 130.2, 130.0, 129.2, 129.1 , 128.8, 128.4, 121.6, 1 14.8, 114.6, 114.0, 69.8, 66.8, 55.3, 51.7, 47.7, 11.7.

Note, the reaction was repeated with yields of 48-77%. 4-chloro-2-((4-(2-(diethyIamino)ethoxy)phenyl)amino)-6-(4-hy droxyphenyl)-8 methy 1 py ri do[2, 3 -d]pyrimi di n-7(8H)-one

To a solution of SH3-16 (20 mg, 0.032 mmol) in DCM (2 mL) at 0 °C was added TFA (200 11L, 0 13 mmol) dropwise. The mixture was warmed to room temperature and stirred for 2 h. After completion of the reaction, the mixture was concentrated and the residue was purified by column chromatography on silica gel (MeOH/DCM, 5:95 to 10:90) to afford SH3-20 (15 mg, 69%) as a yellow' solid. ^! H-NMR (DMSO, 600 MHz) 8 8 10.31 (s, 1 H), 9.75 (s, 1H), 7.74 (s, 1H), 7.66 (s, 2H), 7.51 (d, J = 8.4 Hz, 2H), 6.92 (d, J = 9.0 Hz, 2H), 6.82 (d, J = 8.6 Hz, 2H), 4.00 (t, J - 6. 1 Hz, 2H), 3.61 (s, 3H), 2.78 (t, J - 6.0 Hz, 2H), 2.57 (q, J - 7.1 Hz, 4H), 0.98 (t, J - 7. 1 Hz, 6H); ⁱ³ C-NMR (DMSO, 150 MHz) 8 161.8, 158.2, 157.5, 156.6, 155.5, 154.3, 132.1, 129.9, 129.2, 127 7, 126.5, 121.1, 114.9, 114.5, 66.2, 51 ,3, 47.0, 40.0, 1 1.7. A sample for bioassay assessment was purified using Method B. HPLC purity via Method B: > 94%, fe = 10.1 min. HRMS (ESI): rn/z [M + H]+ calculated for C26H28CIN5O3: 494.1953, found: 464.1955. Note, the reaction was repeated with yields of 69-80%.

SS-2-47-CN This compound can be prepared using a similar procedure as SS-2-47.

ALD-3-2-CN

This compound can be prepared using a similar procedure as ALD-3-2-CN. ALD-3-2b-CN

This compound can be prepared using the following procedure. ALD-3-2-C1CN

This compound can be prepared using a similar procedure as SH3-20.

SS-1- 127-C1 This compound can be prepared using a similar procedure as SH3-20

SS-1-127-NH2

This compound can be prepared using a similar procedure as SH2-160.

SS-1-127-CN

This compound can be prepared using a similar procedure as SS-1-127. ALD-3-2-amideCN

This compound can be prepared by treating ALD-3-2-NH2CN with acetic anhydride.

SS-l-127-amideCN

This compound can be prepared by treating SS-1-127-CN with acetic anhydride.

ALD-3-2-OCH3CN, SS-1-127-F, ALD-3-2-FCN, SS-1-127-OCH3CN, ALD-3-2-OHCN, and SS- 1-127-OHCN

These compounds can be prepared by nucleophilic aromatic substitution (SNAT) reactions outlined in the following figure

MLKL Activation

Induction of cell death by various molecules in cells in the presence and absence of MLKL. MLKL-/- mouse embryonic fibroblasts were stably transduced to allow Doxycycline-inducible re- expression of mouse MLKL. For the experiments, the same cells were either left without Doxycycline (MLKL-/- condition) or pre-treated with 50 ng/ml Doxycycline (MLKL+/+ condition) for 6 hr. After that, cells were treated with the indicated concentrations of drugs for 24 hr. Cell viability was determined using CellTiter-Glo assay

In the foregoing description, it will be readily apparent to one skilled in the art. that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and/or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

Citations to a number of patents and non-patent references may be made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.