WO2015051149A1

WO2015051149A1 - Sorafenib analogs and uses thereof

Info

Publication number: WO2015051149A1
Application number: PCT/US2014/058869
Authority: WO
Inventors: Brent R. Stockwell; Scott J. Dixon; Rachid SKOUTA
Original assignee: The Trustees Of Columbia University In The City Of New York
Priority date: 2013-10-04
Filing date: 2014-10-02
Publication date: 2015-04-09

Abstract

The present invention provides, inter alia, compounds according to formula I. Also provided are pharmaceutical compositions and kits containing such compounds. Methods for using such compounds, compositions, and kits for treating a subject having system x_c-, dysregulation for activating ferroptosis, for inhibiting system x_c- in a cell, and for monitoring treatment of a subject having system x_c- dysregulation are provided as well.

Description

SORAFENIB ANALOGS AND USES THEREOF RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Patent Application Serial No. 61/886,716, filed on October 4, 2013, which application is incorporated by reference herein in its entirety.

GOVERNMENT FUNDING

[0002] This invention was made with government support under grant nos. R01 CA097061 and 1 K99CA166517-01 awarded by the National Cancer Institute. The government has certain rights in the invention.

FIELD OF INVENTION

The present invention provides, inter alia, compounds according formula I:

provided are pharmaceutical compositions and kits containing such compounds, and methods for using such compounds, compositions, and kits. BACKGROUND OF THE INVENTION

[0004] Transporters for small molecule nutrients, including sugars, nucleotides and amino acids, are essential for cell metabolism and represent important targets for drug development (Hediger et al., 2013). System x_c ^" is a cell-surface Na⁺-independent cystine/glutamate antiporter composed of the 12-pass transmembrane transporter protein SLC7A1 1 (xCT) linked via a disulfide bridge to the single-pass transmembrane regulatory subunit SLC3A2 (4F2hc, CD98hc) (Sato et al., 1999, Conrad and Sato, 2012). System x_c ^" is essential for normal mammalian plasma redox homeostasis, pigmentation, immune system function and memory formation (Chintala et al., 2005, Sato et al., 2005, De Bundel et al., 201 1 ). When dysregulated, system x_c ^" is also implicated in tumorigenesis, cancer stem cell maintenance, drug resistance and neurological dysfunction (Okuno et al., 2003, Buckingham et al., 201 1 , Ishimoto et al., 201 1 , Yae et al., 2012). Efforts to treat gliomas and lymphomas in human patients by modulating system x_c ^" activity using an existing small molecule system x_c ^" inhibitor, sulfasalazine (SAS (Gout et al., 2001 )), met with limited success (Robe et al., 2009), likely due to the low potency and poor pharmacokinetics of this compound. Progress has been made towards the development of improved analogs based on the SAS scaffold (Shukla et al., 201 1 ), but the identification and development of more potent and drug-like small molecule system x_c ^" inhibitors based on alternative scaffolds is highly desirable.

[0005] Erastin and RSL3 are small molecules that trigger a non- apoptotic form of cell death in mammalian cells, termed ferroptosis, that is characterized by the iron-dependent accumulation of intracellular reactive oxygen species (ROS) (Dolma et al., 2003, Yagoda et al., 2007, Yang and Stockwell, 2008, Dixon et al., 2012). Both erastin and RSL3-induced ROS accumulation and cell death are prevented by iron chelators, such as deferoxamine (DFO) and ciclopirox olamine (CPX), and by lipophilic antioxidants, such as trolox and ferrostatin-1 (Fer-1 ), suggesting that lipid ROS in particular are crucial for ferroptosis (Yagoda et al., 2007, Yang and Stockwell, 2008, Wolpaw et al., 201 1 a, Dixon et al., 2012). Mechanistically, erastin prevented Na⁺-independent cystine uptake (Dixon et al., 2012), suggesting that erastin most likely inhibits system x_c ^" function. Indeed, erastin-induced cell death was strongly suppressed by beta-mercaptoethanol (β-ΜΕ) (Dixon et al., 2012), which forms mixed disulfides with extracellular cystine that are imported into cells by non-system-x_c ^" transporters (Ishii et al., 1981 ), effectively circumventing the need for system x_c ^" for cell survival. RSL3-induced death was not suppressed by β-ΜΕ and RSL3 did not inhibit cystine import, indicating that it acts on a distinct target (Dixon et al., 2012). Thus, erastin (like SAS) inhibits system x_c ^" function to trigger death.

[0006] Accordingly, there is a need, inter alia, for understanding the mechanism for inhibiting system x_c ^" and for developing effective inihibitors of system x_c ^" _. This application is directed to meeting this and other needs.

SUMMARY OF THE INVENTION

[0007] Accordingly, one embodiment of the present invention is a compound according to formula I:

wherein:

Ri, R2, R3, R4, and R6 are independently selected from the group consisting of H, halo, CF₃, OCF₃, Ci_-4alkyl, and CN;

R5 is independently selected from the group consisting of no atom, NH, O, and Ci_-4alkyl;

R₇ is selected from the group consisting of no atom, carbonyl, thiocarbonyl, and sulfonyl;

Rs is NH or no atom;

Rg is an aryl or heteroaryl optionally substituted with a moiety selected from the group consisting of H, hydroxy, Ci_-4alkyl, halo, and combinations thereof;

R10 is no atom, O, or Ci_-4alkyl;

R11 is selected from the group consisting of no atom, aryl, heterocycle, and heteroaryl optionally substituted with a halo or a Ci_-4alkyl; R-12 is selected from the group consisting of no atom, amide, CN, heteroaryl, O, Ci_-4alkoxy, and amine;

R-I 3 is selected from the group consisting of no atom, alkoxy, amine, CN, carboxy, carbocyclyl optionally substituted with d- 4alkyl, diol; and the dotted line ( ) is an optional double bond, or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof, with the proviso that formula I is not sorafenib,

SRS13-60

[0008] Another embodiment of the present invention is a pharmaceutical composition. This pharmaceutical composition comprises a pharmaceutically acceptable salt or diluent and a compound according to formula I:

wherein:

Rs is NH or no atom;

R10 is no atom, O, or Ci_-4alkyl;

R₁₁ is selected from the group consisting of no atom, aryl, heterocycle, and heteroaryl optionally substituted with a halo or a Ci_-4alkyl; R-12 is selected from the group consisting of no atom, amide, CN, heteroaryl, O, Ci_-4alkoxy, and amine;

SRS13-60

[0009] A further embodiment of the present invention is a kit. This kit comprises any compound disclosed herein together with instructions for the use of the compound.

[0010] Another embodiment of the present invention is a kit. This kit comprises any pharmaceutical composition disclosed herein together with instructions for the use of the pharmaceutical composition. [0011] An additional embodiment of the present invention is a method for treating a subject having dysregulated system x_c ^" activity. This method comprises administering to the subject an effective amount of a compound according to formula I:

wherein:

Ri , R2, R3, R4, and R6 are independently selected from the group consisting of H, halo, CF₃, OCF₃, Ci_-4alkyl, and CN;

Rs is NH or no atom;

R10 is no atom, O, or Ci_-4alkyl; Rii is selected from the group consisting of no atom, aryl, heterocycle, and heteroaryl optionally substituted with a halo or a Ci_-4alkyl;

R-I2 is selected from the group consisting of no atom, amide, CN, heteroaryl, O, Ci_-4alkoxy, and amine;

R-I 3 is selected from the group consisting of no atom, alkoxy, amine, CN, carboxy, carbocyclyl optionally substituted with d- 4alkyl, diol; and the dotted line ( ) is an optional double bond, or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.

[0012] Another embodiment of the present invention is a method for treating a subject having dysregulated system x_c ^" activity. This method comprises administering to the subject an effective amount of any pharmaceutical composition disclosed herein.

[0013] A further embodiment of the present invention is a method of activating ferroptosis in a cell. This method comprises administering to the cell an effective amount of a compound according to formula I:

wherein:

Ri , R2, R3, R₄, and R₆ are independently selected from the group consisting of H, halo, CF₃, OCF₃, Ci_-4alkyl, and CN;

R₅ is independently selected from the group consisting of no atom, NH, O, and Ci_-4alkyl;

R₈ is NH or no atom;

R₉ is an aryl or heteroaryl optionally substituted with a moiety selected from the group consisting of H, hydroxy, Ci- alkyl, halo, and combinations thereof;

R-io is no atom, O, or Ci_-4alkyl;

[0014] Another embodiment of the present invention is a method of inhibiting system x_c ^" in a cell. This method comprises administering to the cell an effective amount of a compound according to formula I:

wherein:

R5 is independently selected from the group consisting of no atom, NH, O, and Ci_-4alkyl; R₇ is selected from the group consisting of no atom, carbonyl, thiocarbonyl, and sulfonyl;

R₈ is NH or no atom;

R10 is no atom, O, or Ci_-4alkyl;

R11 is selected from the group consisting of no atom, aryl, heterocycle, and heteroaryl optionally substituted with a halo or a Ci_-4alkyl;

R12 is selected from the group consisting of no atom, amide, CN, heteroaryl, O, Ci_-4alkoxy, and amine;

[0015] An additional embodiment of the present invention is a method for monitoring treatment of a subject having system x_c ^" dysregulation. This method comprises: (a) measuring the expression level of a CHAC1 gene in a subject being treated with an amount of any compound or pharmaceutical composition disclosed herein, wherein increased expression levels of the CHAC1 gene relative to a control indicate that the cells of the subject are undergoing cystine limitation; and

(b) adjusting the amount of the compound or composition administered to the subject based on the expression levels of the CHAC1 gene measured in (a).

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0017] Figure 1 shows cell death triggered by ferroptosis inducers in multicellular tumor spheres (MCTSs). Figures 1A and 1 B show the modulatory effect (M_e) profiles of erastin- and SAS-induced death in five different cell lines in response to seven different cell death inhibitors. M_e > 0 = rescue from cell death. Figures 1 C-1 F show the relative viability of MCTSs formed over 72 hours from HT-1080 (C), Calu-1 (D), A549 (E) or HCT-1 16 (F) cells in response to erastin, RSL3 or staurosporine (STS) +/- β-ΜΕ or ferrostatin-1 (Fer-1 ). Viability was assessed by Alamar blue and represents mean+/-standard deviation (SD) from three independent biological replicate experiments. Data were analyzed by two-way ANOVA with Bonferroni tests, ^*P < 0.05, ^**P < 0.05, ^***P < 0.001 , ns = not significant. Figures 1 G-I show cell growth and glutamate release in oncogenic RAS-mutant cell lines. (G,H) Relative (G) and absolute (H) glutamate release was quantified in A549, HCT- 1 16 and Calu-1 cells. (/) The number of viable cells (grown in monolayer culture) was quantified by Vi-Cell trypan blue exclusion at baseline (t=0) and then 24 hours later in the presence of various compounds, as indicated. All values are expressed as mean+/-SD (n=3 biological replicates). Data were analyzed by two-way ANOVA (cell line x drug treatment) with Bonferroni post- hoc analysis and select interactions are highlighted ^*P < 0.05, ^**P < 0.01 , ^***P < 0.001 .

[0018] Figure 2 shows that erastin inhibits system x_c ^" function potently and specifically. Figures 2A and 2B show Na+-independent uptake of ¹⁴C- cystine (A) and ¹⁴C-L-phenylalanine (B) uptake over five minutes in HT-1080 and Calu-1 cells treated with erastin or SAS. D-Phe is included as a positive control in B. Figure 2C shows glutamate release +/- erastin in HT-1080 cells where SLC7A11 was silenced for 48 hours using two independent siRNAs. Figure 2D shows SLC7A11 mRNA levels assayed using RT-qPCR in si- SL C7/477-transfected cells. Figure 2E shows dose-response analysis of glutamate release from HT-1080 and Calu-1 cells in response to erastin and SAS. Figure 2F shows glutamate release in response to erastin, SAS, RSL3, artesunate, and PEITC, +/- beta-mercaptoethanol (β-ΜΕ). All data are from three independent biological replicates. Data are presented as mean+/-SD. Data in A and B are normalized to DMSO controls (set to 100%). Data in A, B, C and F were analyzed by ANOVA with Bonferroni post-tests, ^*P < 0.05, ^***P < 0.001 , ns = not significant. Figures 2G-I show monitoring system x_c ^~ activity by following glutamate release. Figure 2G shows an overview of the assay design. Glutamate released by the cell is detected by an enzyme-linked reaction. Erastin and SAS inhibit glutamate release from system x_c ^", but not other transporters. System x_c ^" is one of several transporters that can release glutamate, including system X_AG and others. Figure 2H shows SLC7A5 expression was silenced in HT-1080 cells for 48 hours using two independent siRNAs and then glutamate release was assayed +/- erastin. Figure 2I shows SLC7A5 mRNA levels in HT-1080 transfected as in 2H. Data in 2H and 2I represent mean +/-SD from three independent biological replicates.

[0019] Figure 3 shows inhibition of system x_c ^" by erastin is necessary for lethality. Figure 3A shows structure and lethal potency (EC50 in HT-1080 cells) of erastin and the inactive erastin analog erastin-A8. Figure 3B shows dose-dependent inhibition of glutamate release by erastin and erastin-A8 (Era-A8). Figure 3C shows total glutathione (GSH+GSSG) quantified by biochemical assay in HT-1080 cells treated with DMSO, erastin or erastin-A8 (Era-A8). Figure 3D shows total glutathione (GSH+GSSG) levels in HT-1080 cells treated with compound combinations as indicated. Figure 3E shows viability of HT-1080 cells in response to different compound combinations measured by Trypan blue exclusion. Data in Figures 3B-E are from three independent biological replicates and represent mean+/-SD. In Figures 3C-E, data were analyzed by one-way ANOVA with Bonferroni post-tests, ^*P < 0.05, ^**P < 0.01 , ^***P < 0.001 , ns = not significant. Significance is indicated relative to the DMSO control. Figures 3F-H show the role of glutathione in ferroptosis. Figure 3F shows a cartoon model of the predicted ferroptotic pathway. Erastin or SAS can inhibit the function of system x_c ^", thereby depleting the cell of cysteine and, ultimately, reduced glutathione (GSH), allowing for the accumulation of lethal lipid ROS. Lipid ROS accumulation and ferroptotic death is prevented by Fer-1 , downstream of GSH. L-buthionine sulfoximine (BS) inhibits the first enzyme in the GSH synthetic pathway. Figure 3G shows total glutathione levels measured in HT-1080 cells. Cells were treated with DMSO or BSO for 30 hours or, as an internal control, DMSO or erastin for 5 hours. Figure 3H shows cell viability measured by Trypan blue exclusion 48 or 96 hours after drug treatment. Data in Figures 3G and 3H represents mean +/- standard deviation from three independent biological replicates. Data in Figure 3G was analyzed by Student's f-test. ^***P < .001 , ^**P < .01 compared to the relevant DMSO control.

[0020] Figure 4 shows structure activity relationship (SAR) analysis of erastin. Figure 4A shows structures of 20 erastin analogs. Figure 4B shows lethal EC50 for each analog determined in HT-1080 cells in a 10-point, 2-fold dilution assay, starting at a high dose of 20 μΜ, +/- β-ΜΕ. Data represent mean and 95% confidence interval (95% CI) from three independent biological replicate experiments. Also reported are IC50 values for inhibition of glutamate release as determined in CCF-STTG1 cells. These data represent the average of two experiments. All values are in μΜ. ND: not determined.

[0021] Figure 5 shows analysis of erastin effects using RNA-Seq. Figures 5A and 5B show lists of genes upregulated (A) and downregulated (B) by erastin treatment, as detected in HT-1080 cells using RNA-Seq. The number of fragments per kilobase of exon per megabase of sequence (FPKM) were counted and are expressed as a fold-change ratio between the different conditions. E/D: Erastin/DMSO expression ratio. Ε+β-ΜΕ/D: Erastin+β- ME/DMSO ratio. ATP6V1G2^*: ATP6V1 G2-DDX39B read-through transcript. Data represent the average of two independent biological replicates for each condition. Figures 5C and 5D show mRNA expression level of CHAC1 determined by RT-qPCR in HT-1080 and Calu-1 cells in response to erastin+/- -ME treatment for 5 hr. Data are from three independent biological replicates and represented as mean+/-SD and were analyzed by one-way ANOVA with Bonferroni post-tests, ^**P < 0.01 , ^***P < 0.001 , ns = not significant. In Figure 5D significance is indicated relative to the DMSO control. Figure 5E shows CHAC1 mRNA levels in 13 different erastin- sensitive cell lines treated with erastin or STS (6 hours). Results in Figure 5E were analyzed using the Kruskal-Wallis test, ^***P < 0.001 , ns = not significant. Figures 5F-H show regulation of CHAC1 expression. Figure 5F shows Chad mRNA levels assessed by RT-qPCR in SV40-transformed MEFs treated for 6 hours with erastin or thapsigargin (a positive control for ER stress), as indicated. Figure 5G shows CHAC1 mRNA levels in HT-1080 cells treated with DMSO or erastin (10 μΜ) for 6 hours or DMSO or buthionine sulfoximine (BSO, 2.5 mM) for 30 hours +/- actinomycin D (Act D) or cycloheximide (CHX). Figure 5H shows CHAC1 mRNA levels in HT-1080 cells treated for 6 hours with compounds as indicated. Data in Figures 5F and 5G represent mean+/-SD from two independent biological replicates and data in Figure 5H represents mean+/-SD from three independent biological replicates.

[0022] Figure 6 shows the identification of sorafenib as an inhibitor of system x_c ^". Figures 6A and 6B show the modulatory profiling of (A) A549 and (B) HCT-1 16 cells in response to either buthionine sulfoximine (BSO) or erastin +/- 20 different lethal compounds. Figure 6C shows the viability of HT- 1080 cells treated for 24 hours with ferroptosis inhibitors (β-ΜΕ, Fer-1 , DFO) +/- sorafenib, erastin or STS. Figure 6D shows the quantification of the inhibition of glutamate release by sorafenib, erastin and imatinib +/- Fer-1 . Figure 6E shows the viability of HT-1080 cells treated for 24 hours with erastin, sorafenib, nilotinib, masitinib or imatinib +/- β-ΜΕ or Fer-1 . Cell viability in Figures 6C and 6E was quantified by Alamar blue. Data in Figures 6C-6E represent mean+/-SD from three independent biological replicates and were analyzed by one- and two-way ANOVA with Bonferroni post-tests, ^*P < 0.05, ^**P < 0.01 , ^***P < 0.001 , ns = not significant relative to the indicated treatments. In Figure 6D, none of the comparisons between DMSO and Fer-1 treated samples were significant (P > 0.05).

[0023] Figure 7 shows structure-activity relationship (SAR) analysis of sorafenib. Figure 7A shows the structure of Sorafenib (compound 23) and thirteen sorafenib analogs (compounds 24-36). These compounds were prepared and tested in HT-1080 cells for the induction of cell death (EC50) and the suppression of this cell death (death suppression, DS) by β-ΜΕ (18 μΜ) and ferrostatin-1 (Fer-1 , 1 μΜ) over 48 hours. DS values are expressed as a percentage relative with the level of suppression observed with sorafenib (compound 23) treatment, which was set to 100%. All values are means from three independent biological replicates. Figure 7B shows glutamate release in response to sorafenib and select analogs. Figure 7C shows the DEVDase (caspase-3/7) activity in response to sorafenib and select analogs as measured by the cleavage of a fluorescent rhodamine substrate.

[0024] Figure 8 shows a summary of adverse events reported with sorafenib and other kinase inhibitors. Analysis of adverse events across 20 physiological system categories associated with kinase inhibitor treatment. DETAILED DESCRIPTION OF THE INVENTION

[0025] One embodiment of the present invention is a compound according to formula I:

wherein:

Rs is NH or no atom;

or SRS13-60

[0026] The terms "halo" and "halogen" are used interchangeably herein and mean halogen and include chloro, fluoro, bromo, and iodo.

[0027] The term "alkyi" refers to the radical of saturated aliphatic groups, including straight-chain alkyi groups, branched-chain alkyi groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In certain embodiments, a straight chain or branched chain alkyl has 10 or fewer carbon atoms in its backbone (e.g., C1 -C10 for straight chains, C3-C10 for branched chains). Preferably, the alkyl has from 1 -4 carbon atoms. Likewise, certain cycloalkyls have from 3-8 carbon atoms in their ring structure, including 5, 6 or 7 carbons in the ring structure.

[0028] The term "C_x-y" when used in conjunction with a chemical moiety, such as, alkyl, alkenyl, or alkoxy is meant to include groups that contain from x to y carbons in the chain. For example, the term "C_x-yalkyl" refers to saturated hydrocarbon groups, including straight-chain alkyl and branched-chain alkyl groups that contain from x to y carbons in the chain.

[0029] The term "carbonyl" means a functional group composed of a carbon atom double-bonded to an oxygen atom: C=O. Carbonyls include without limitation, aldehydes, ketones, carboxylic acids, esters, and amides.

[0030] The term "thiocarbonyl" means a functional group composed of a sulfur atom double-bonded to an oxygen atom: S=O.

[0031] The term "sulfonyl" refers to the group:

[0032] The term "aryl" as used herein includes single-ring aromatic groups in which each atom of the ring is carbon. Preferably the ring is a 3- to 8-membered ring, more preferably a 6-membered ring. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

[0033] The term "heteroaryl" includes aromatic single ring structures, preferably 3- to 8-membered rings, more preferably 5- to 7-membered rings, even more preferably 5- to 6-membered rings, whose ring structures include at least one heteroatom (i.e., atoms other than C, such as, e.g., N, S, or O), preferably one to four heteroatoms, more preferably one or two heteroatoms. The term "heteroaryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, benzothiazole, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.

[0034] The term "hydroxyl" or "hydroxy," as used herein, refers to the group -OH.

[0035] The terms "heterocyclyl", "heterocycle", "heterocyclic", and the like refer to non-aromatic ring structures, preferably 3- to 8-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms, i.e., atoms other than C, such as, e.g., N, S, or O. The terms "heterocyclyl," "heterocyclic," and the like also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heterocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

[0036] The term "amide" or "amido", as used herein, refers to a group

wherein R ₄ and Ri₅ each independently represent a hydrogen or a Ci- alkyl group.

[0037] The term "alkoxy" refers to an alkyl group, preferably a lower alkyl group, having an oxygen attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, tert-butoxy and the like. Other alkoxy groups within the scope of the present invention include, for example, the following:

[0038] The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines and salts thereof, e.g., a moiety that can be represented by

wherein Ri , Ri₅, and R15 each independently represent a hydrogen or a Ci_-4alkyl group. The term "primary" amine means only one of Ri₄ and R15, or one of Ri₄, R15, and R15' is an alkyl group. Secondary amines have two alkyl groups bound to N. In tertiary amines, all three groups, Ri₄, R15, and R15 , are replaced by alkyl groups.

[0039] The terms "carbocycle", "carbocyclyl", and "carbocyclic", as used herein, refer to a non-aromatic saturated or unsaturated ring in which each atom of the ring is carbon. The ring may be monocyclic, bicyclic, tricyclic, or even of higher order. Thus, the terms "carbocycle", "carbocyclyl", and "carbocyclic", encompass fused, bridged and spirocyclic systems: Preferably a carbocycle ring contains from 3 to 14 atoms, including 3 to 8 or 5 to 7 atoms, such as for example, 6 atoms.

[0040] The terms "carboxy" and "carboxyl", as used herein, refer to a group represented by the formula -CO2R, wherein R is H or alkyl.

[0041] The term "diol" is art-recognized and refers to molecules that

contain two hydroxyl groups. Non-limiting preferred diols include

wherein B is boron. [0042] The term "N-oxide", as used herein, refers to a compound containing a N⁺-O^" functional group.

[0043] The term "crystalline form", as used herein, refers to the crystal structure of a compound of the present invention. A compound may exist in one or more crystalline forms, which may have different structural, physical, pharmacological, or chemical characteristics. Different crystalline forms may be obtained using variations in nucleation, growth kinetics, agglomeration, and breakage. Nucleation results when the phase-transition energy barrier is overcome, thereby allowing a particle to form from a supersaturated solution. Crystal growth is the enlargement of crystal particles caused by deposition of the chemical compound on an existing surface of the crystal. The relative rate of nucleation and growth determine the size distribution of the crystals that are formed. The thermodynamic driving force for both nucleation and growth is supersaturation, which is defined as the deviation from thermodynamic equilibrium. Agglomeration is the formation of larger particles through two or more particles (e.g., crystals) sticking together and forming a larger crystalline structure.

[0044] The term "hydrates", as used herein, refers to a solid or a semisolid form of a chemical compound containing water in a molecular complex. The water is generally in a stochiometric amount with respect to the chemical compound.

[0045] In one aspect of this embodiment, the compound has the structure of formula (II):

wherein:

R₂ and R3 are independently selected from the group consisting of H, halo, and CF₃;

R is H or halo;

R10 is O or Ci_-4alkyl;

A is C or N;

B is O or S;

R12 is selected from the group consisting of no atom, amide, CN, and heteroaryl; and

R-I 3 is selected from the group consisting of no atom or CN, or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof, with the proviso that formula II is not sorafenib.

[0046] In another aspect of this embodiment, the compounds has the structure of formula (III):

wherein:

R10 is O or Ci_-4alkyl;

A is C or N;

[0047] In a further aspect of this embodiment, the compound is selected from the group consisting of:

SRS 13-45 SRS 13-46

30

SRN1-25 SRN1-33

EJ1-61

EJ2-01 EJ2-02

or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.

[0048] In a further aspect of this embodiment, the compound is selected from the group consisting of:

SRS15-37 SRS13-65

and combinations thereof, or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.

[0049] In another aspect of this embodiment, the compound is selected from the group consisting of:

SRS13-45 SRS14-23

[0050] Preferably, the compound is

[0051] Another embodiment of the present invention is a pharmaceutical composition. This pharmaceutical composition comprises a pharmaceutically acceptable salt or diluent and a compound according to formula I:

wherein:

Rs is NH or no atom;

R10 is no atom, O, or Ci_-4alkyl;

SRS13-60

[0052] In this embodiment, suitable and preferred compounds are as disclosed herein.

[0053] Further embodiment of the present invention include kits. These kits comprise any compound or pharmaceutical composition disclosed herein together with instructions for the use of the compound or pharmaceutical composition. [0054] The kits of the invention may also include suitable storage containers, e.g., ampules, vials, tubes, etc., for each compound or pharmaceutical composition and other reagents, e.g., buffers, balanced salt solutions, etc., for use in administering the compound or pharmaceutical compositions to subjects. The compounds, pharmaceutical compositions and other reagents may be present in the kits in any convenient form, such as, e.g., in a solution or in a powder form. The kits may further include a packaging container, optionally having one or more partitions for housing the pharmaceutical composition and other optional reagents.

[0055] An additional embodiment of the present invention is a method for treating a subject having dysregulated system x_c ^" activity. This method comprises administering to the subject an effective amount of a compound according to formula I:

wherein:

Ri , F¾, R3, R4, and R6 are independently selected from the group consisting of H, halo, CF₃, OCF₃, Ci_-4alkyl, and CN;

R₈ is NH or no atom;

R10 is no atom, O, or Ci_-4alkyl;

[0056] Preferably, the compound of formula I is not sorafenib,

or SRS13-60

[0057] As used herein, a "s ubject" is a mammal, preferably, a human or a primate and more preferably, a human. In addition to humans and primates, categories of mammals within the scope of the present invention include, for example, farm animals, domestic animals, laboratory animals, etc. Some examples of farm animals include cows, pigs, horses, goats, etc. Some examples of domestic animals include dogs, cats, etc. Some examples of laboratory animals include rats, mice, rabbits, guinea pigs, etc.

[0058] As used herein, the terms "treat," "treating," "treatment" and grammatical variations thereof mean subjecting an individual subject to a protocol, regimen, process or remedy, in which it is desired to obtain a physiologic response or outcome in that subject, e.g., a patient. In particular, the methods and compositions of the present invention may be used to slow the development of disease symptoms or delay the onset of the disease or condition, or halt the progression of disease development. However, because every treated subject may not respond to a particular treatment protocol, regimen, process or remedy, treating does not require that the desired physiologic response or outcome be achieved in each and every subject or subjects, e.g., patient population. Accordingly, a given subject or a patient population may fail to respond or respond inadequately to treatment.

[0059] As used herein, system x_c ^" "dysregulation" means abnormal or impaired functioning of the system x_c ^", which is an amino acid antiporter that typically mediates the exchange of extracellular L-cystine and intracellular L- glutamate across the cellular plasma membrane. The system x_c ^~ dysregulation may be implicated in the following disease states or conditions: tumorigenesis, cancer stem cell maintenance, drug resistance, and neurological dysfunction.

[0060] In the present invention, an "effective amount" or a "therapeutically effective amount" of a compound or pharmaceutical composition disclosed herein is an amount of such compound or composition that is sufficient to effect beneficial or desired results as described herein when administered to a subject. Effective dosage forms, modes of administration, and dosage amounts may be determined empirically, and making such determinations is within the skill of the art. It is understood by those skilled in the art that the dosage amount will vary with the route of administration, the rate of excretion, the duration of the treatment, the identity of any other drugs being administered, the age, size, and species of mammal, e.g., human patient, and like factors well known in the arts of medicine and veterinary medicine. In general, a suitable dose of a compound or pharmaceutical composition according to the invention will be that amount of the compound or pharmaceutical composition, which is the lowest dose effective to produce the desired effect. The effective dose of a compound or pharmaceutical composition of the present invention may be administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day.

[0061] The term "tumorigenesis" refers to the formation or production of tumors or cancers.

[0062] The term cancer stem cell "maintenance" refers to the ability of cancer stem cells to continually retain those properties that are typically associated with normal stem cells, such as self-renewal and differentiation. Like normal stem cells, cancer stem cells have varying degrees of potency, including, but not limited to, pluripotency, and can give rise to numerous differentiated cell types, leading to the heterogeneity observed in many tumors.

[0063] Non-limiting examples of solid tumors and cancers include adrenocortical carcinoma, anal tumor/cancer, bladder tumor/cancer, bone tumor/cancer (such as osteosarcoma), brain tumor, breast tumor/cancer, carcinoid tumor, carcinoma, cervical tumor/cancer, colon tumor/cancer, endometrial tumor/cancer, esophageal tumor/cancer, extrahepatic bile duct tumor/cancer, Ewing family of tumors, extracranial germ cell tumor, eye tumor/cancer, gallbladder tumor/cancer, gastric tumor/cancer, germ cell tumor, gestational trophoblastic tumor, head and neck tumor/cancer, hypopharyngeal tumor/cancer, islet cell carcinoma, kidney tumor/cancer, laryngeal tumor/cancer, leukemia, lip and oral cavity tumor/cancer, liver tumor/cancer, lung tumor/cancer, lymphoma, malignant mesothelioma, Merkel cell carcinoma, mycosis fungoides, myelodysplastic syndrome, myeloproliferative disorders, nasopharyngeal tumor/cancer, neuroblastoma, oral tumor/cancer, oropharyngeal tumor/cancer, osteosarcoma, ovarian epithelial tumor/cancer, ovarian germ cell tumor, pancreatic tumor/cancer, paranasal sinus and nasal cavity tumor/cancer, parathyroid tumor/cancer, penile tumor/cancer, pituitary tumor/cancer, plasma cell neoplasm, prostate tumor/cancer, rhabdomyosarcoma, rectal tumor/cancer, renal cell tumor/cancer, transitional cell tumor/cancer of the renal pelvis and ureter, salivary gland tumor/cancer, Sezary syndrome, skin tumors (such as cutaneous t-cell lymphoma, Kaposi's sarcoma, mast cell tumor, and melanoma), small intestine tumor/cancer, soft tissue sarcoma, stomach tumor/cancer, testicular tumor/cancer, thymoma, thyroid tumor/cancer, urethral tumor/cancer, uterine tumor/cancer, vaginal tumor/cancer, vulvar tumor/cancer, and Wilms' tumor.

[0064] Preferably, the subject has a cancer selected from the group consisting of colon cancer, brain cancer, breast cancer, bone cancer, colorectal cancer, lung cancer, pancreatic cancer, bladder cancer, skin cancer, liver cancer, lymphoma, and leukemia.

[0065] The term "drug resistance" refers to a reduction in efficacy of a drug being used to treat a disease or condition. Examples of drugs for which individuals can develop drug resistance include, but are not limited to, antineoplastics, antibiotics, antimicrobials, and antivirals.

[0066] Non-limiting examples of neurological dysfunction include but are not limited to multiple sclerosis, Alzheimer's disease, Parkinson's disease, myasthenia gravis, motor neuropathy, Guillain-Barre syndrome, autoimmune neuropathy, Lambert-Eaton myasthenic syndrome, paraneoplastic neurological disease or disorder, paraneoplastic cerebellar atrophy, progressive cerebellar atrophy, Rasmussen's encephalitis, amyotrophic lateral sclerosis, Sydehann chorea, Gilles de la Tourette syndrome, autoimmune polyendocrinopathy, dysimmune neuropathy, acquired neuromyotonia, arthrogryposis multiplex, Huntington's disease, AIDS associated dementia, amyotrophic lateral sclerosis (ALS), multiple sclerosis, an inflammatory retinal disease or disorder, an inflammatory ocular disease or disorder, and optic neuritis.

[0067] Suitable and preferred compounds for use in this method are as disclosed herein.

[0068] In one aspect of this embodiment, the method further comprises administering at least one additional therapeutic agent selected from the group consisting of an antibody or fragment thereof, a cytotoxic agent, a toxin, a radionuclide, an immunomodulator, a photoactive therapeutic agent, a radiosensitizing agent, a hormone, and combinations thereof.

[0069] As used herein, an "antibody" encompasses naturally occurring immunoglobulins as well as non-naturally occurring immunoglobulins, including, for example, single chain antibodies, chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies), as well as antigen-binding fragments thereof, (e.g., Fab', F(ab')2, Fab, Fv, and rlgG). See also, e.g., Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, III.); Kuby, J., Immunology, 3rd Ed., W.H. Freeman & Co., New York (1998). The term antibody also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. The term "antibody" further includes both polyclonal and monoclonal antibodies. [0070] Examples of antibodies used as therapeutics include rituximab (Rituxan), Cetuximab (Erbitux), bevacizumab (Avastin), and Ibritumomab (Zevalin).

[0071] Cytotoxic agents may be DNA damaging agents, antimetabolites, anti-microtubule agents, antibiotic agents, etc. DNA damaging agents include alkylating agents, intercalating agents, and enzyme inhibitors of DNA replication. Non-limiting examples of DNA alkylating agents include cyclophosphamide, mechlorethamine, uramustine, melphalan, chlorambucil, ifosfamide, carmustine, lomustine, streptozocin, busulfan, temozolomide, cisplatin, carboplatin, oxaliplatin, a pharmaceutically acceptable salt thereof, a prodrug thereof, and combinations thereof. Preferably, the DNA alkylating agent is temozolomide, a prodrug thereof, or a pharmaceutically acceptable salt thereof. Non-limiting examples of intercalating agents include doxorubicin, daunorubicin, idarubicin, and mitoxantrone. Non-limiting examples of enzyme inhibitors of DNA replication include irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, and teniposide. Antimetabolites include folate antagonists such as methotrexate and premetrexed, purine antagonists such as 6-mercaptopurine, dacarbazine, and fludarabine, and pyrimidine antagonists such as 5- fluorouracil, arabinosylcytosine, capecitabine, gemcitabine, and decitabine. Anti-microtubule agents include without limitation vinca alkaloids, paclitaxel (Taxol®), docetaxel (Taxotere®), and ixabepilone (Ixempra®). Antibiotic agents include without limitation actinomycin, anthracyclines, valrubicinepirubicin, bleomycin, plicamycin, and mitomycin. Cytotoxic agents also include erastin, RSL3, and analogs of erastin or RSL3 and pharmaceutically acceptable salts of erastin and RSL3. As used herein, "analogs" means those compounds that are structurally similar. Non-limiting examples of erastin analogs are disclosed herein and are disclose in U.S. Patent No. 8,518,959 to Becklin et al.

[0072] The term "toxin" refers to an antigenic poison or venom of plant or animal origin. An example is diphtheria toxin or portions thereof.

[0073] The term "radionuclide" refers to a radioactive substance administered to the patient intravenously or orally, after which it penetrates via the patient's normal metabolism into the target organ or tissue, where it delivers local radiation for a short time. Examples of radionuclides include, but are not limited to, 1-125, At-21 1 , Lu-177, Cu-67, 1-131 , Sm-153, Re-186, P-32, Re-188, ln-1 14m, and Y-90.

[0074] The term "immunomodulator" refers to a substance that alters the immune response by augmenting or reducing the ability of the immune system to produce antibodies or sensitized cells that recognize and react with the antigen that initiated their production. Immunomodulators can be recombinant, synthetic, or natural preparations and include cytokines, corticosteroids, cytotoxic agents, thymosin, and immunoglobulins. Some immunomodulators are naturally present in the body, and certain of these are available in pharmacologic preparations. Examples of immunomodulators include, but are not limited to, granulocyte colony-stimulating factor (G-CSF), interferons, imiquimod and cellular membrane fractions from bacteria, IL-2, IL- 7, IL-12, CCL3, CCL26, CXCL7, synthetic cytosine phosphate-guanosine (CpG), and oligodeoxynucleotides. [0075] The term "photoactive therapeutic agent" refers to compounds and compositions that become active upon exposure to light. Certain examples of photoactive therapeutic agents are described in U.S. Patent Application Serial No. 201 1/0152230 A1 , "Photoactive Metal Nitrosyls For Blood Pressure Regulation And Cancer Therapy."

[0076] The term "radiosensitizing agent" refers to a compound that makes tumor cells more sensitive to radiation therapy. Examples of radiosensitizing agents include misonidazole, metronidazole, tirapazamine, and trans sodium crocetinate.

[0077] The term "hormone" refers to a substance released by cells in one part of a body that affects cells in another part of the body. Examples of hormones include, but are not limited to, prostaglandins, leukotrienes, prostacyclin, thromboxane, amylin, antimullerian hormone, adiponectin, adrenocorticotropic hormone, angiotensinogen, angiotensin, vasopressin, atriopeptin, brain natriuretic peptide, calcitonin, cholecystokinin, corticotropin- releasing hormone, encephalin, endothelin, erythropoietin, follicle-stimulating hormone, galanin, gastrin, ghrelin, glucagon, gonadotropin-releasing hormone, growth hormone-releasing hormone, human chorionic gonadotropin, human placental lactogen, growth hormone, inhibin, insulin, somatomedin, leptin, liptropin, luteinizing hormone, melanocyte stimulating hormone, motilin, orexin, oxytocin, pancreatic polypeptide, parathyroid hormone, prolactin, prolactin releasing hormone, relaxin, renin, secretin, somatostain, thrombopoietin, thyroid-stimulating hormone, testosterone, dehydroepiandrosterone, androstenedione, dihydrotestosterone, aldosterone, estradiol, estrone, estriol, Cortisol, progesterone, calcitriol, and calcidiol. [0078] Some compounds interfere with the activity of certain hormones or stop the production of certain hormones. These hormone-interfering compounds include, but are not limited to, tamoxifen (Nolvadex®), anastrozole (Arimidex®), letrozole (Femara®), and fulvestrant (Faslodex®).

[0079] Another embodiment of the present invention is a method for treating a subject having dysregulated system x_c ^" activity. This method comprises administering to the subject an effective amount of any pharmaceutical composition disclosed herein.

[0080] In this embodiment, suitable and preferred subjects and system Xc^" dysregulations are as disclosed herein.

[0081] In one aspect of this embodiment, the method further comprises administering at least one additional therapeutic agent selected from the group consisting of an antibody or fragment thereof, a cytotoxic agent, a toxin, a radionuclide, an immunomodulator, a photoactive therapeutic agent, a radiosensitizing agent, a hormone, and combinations thereof. Suitable and preferred thereapeutic agents are as disclosed herein.

[0082] A further embodiment of the present invention is a method of activating ferroptosis in a cell. This method comprises administering to the cell an effective amount of a compound according to formula I:

wherein:

Rs is NH or no atom;

R10 is no atom, O, or Ci_-4alkyl;

[0083] As used herein, "ferroptosis" means regulated cell death that is iron-dependent. See, e.g., Dixon et al., 2012. Ferroptosis is characterized by the overwhelming, iron-dependent accumulation of lethal lipid reactive oxygen species. Ferroptosis is distinct from apoptosis, necrosis, and autophagy. Assays for ferroptosis are as disclosed herein, for instance, in the Examples section.

[0084] Another embodiment of the present invention is a method of inhibiting system x_c ^" in a cell. This method comprises administering to the cell an effective amount of a compound according to formula I:

wherein:

Ri , R₂, R3, R₄, and R6 are independently selected from the group consisting of H, halo, CF₃, OCF₃, Ci_-4alkyl, and CN;

R₇ is selected from the group consisting of no atom, carbonyl, thiocarbonyl, and sulfonyl; R₈ is NH or no atom;

Rio is no atom, O, or Ci_-4alkyl;

Rii is selected from the group consisting of no atom, aryl, heterocycle, and heteroaryl optionally substituted with a halo or a Ci_-4alkyl;

[0085] An additional embodiment of the present invention is a method for monitoring treatment of a subject having system x_c ^" dysregulation. This method comprises:

(a) measuring the expression level of a CHAC1 gene in a subject being treated with an amount of any compound or pharmaceutical composition disclosed herein, wherein increased expression levels of the CHAC1 gene relative to a control indicate that the cells of the subject are undergoing cystine limitation; and

[0086] The phrase "measuring the expression level" refers to the use of those methods recognized in the art for measuring the expression level of one or multiple genes. These methods include, but are not limited to, RNA-seq (whole transcriptome shotgun sequencing), microarrays, and RT-qPCR. Preferably, the CHAC1 gene is measured using the method disclosed in Example 7.

[0087] The following examples are provided to further illustrate the methods of the present invention. These examples are illustrative only and are not intended to limit the scope of the invention in any way.

EXAMPLES

Example 1

A. Total synthesis of erastin and analogs

General information.

[0088] All reactions were carried out under a nitrogen atmosphere under anhydrous conditions unless indicated otherwise. Anhydrous methylene chloride (DCM), tetrahydrofuran (THF), Ν,Ν-dimethylformamide (DMF) was purchased from Sigma-Aldrich (St. Louis, MO). Reactions were magnetically stirred and monitored by thin layer chromatography carried out on Merck (Whitehouse Station, NJ) pre-coated 0.25 mm silica plates containing a 254 nm fluorescence indicator. Flash chromatography was preformed on a Teledyne Connbiflash companion automatic flash chromatography system (Teledyne Technologies International Corp., Thousand Oaks, CA). Preparative thin layer chromatography was performed on 1 mm plates. Spectroscopy: NMR spectra were obtained on a Bruker DPX 300 or 400 MHz spectrometer (Bruker Biosciences Corporation, Billerica, MA).

[0089] The abbreviations used herein are as follows: EDIPA = diisopropylethyl amine, EtOAc = ethyl acetate, Et₂O = diethyl ether, MeOH = methanol, EtOH = ethanol, Pd(PPh₃) tetrakis(triphenylphosphine)palladium(0), Na₂SO₄ = sodium sulfate, MgSO4 = magnesium sulfate, DMAP = 4-dimethylaminopyridine, POCI3 = phosphorous oxychloride, NaHCO3 = sodium bicarbonate, TBS-CI = tert- butylchlorodimethylsilane, NBS = N-bromosuccinimide, NaBH = sodium borohydride, NH CI = ammonium chloride, TFA = triflouroacetic acid, PBr₃ = phosphours tribromide, HBTU = O-(Benzotriazol-1 -yl)-N,N,N',N'- tetramethyluronium hexafluorophosphate.

Scheme 1. (a) TEA (1.1 eq), chloroacetyl chloride (1.1 eq), THF, 0°C to 25°C, 6 hr, ; (b) PCI₃ (1.2 eq), EDIPA (1.0 eq), then aniline (1.1 eq), dioxane, 25°C to 70°C, 6 hr; (c) piperazine (3.0 eq), THF, 25°C, 14 hr; (d) EDIPA (1.2 eq), 4-DMAP (0.5 eq), acyl chloride (1.2 eq), CH₂CI₂ 0°C to 25°C, 3 hr -or- EDIPA (1.2 eq), carboxyiic acid (1.1 eq), HBTU (1.2 eq), 30 min, then amine DMF, 12 hr; e) alkyl halide (1.5 eq), K₂C0₃ (1.2 eq), 60°C, DMF 24 hr; (f) EDIPA (1.2 eq), chloroacetyl chloride (1.2 eq), CH₂CI₂0°C to 25°C, 3 hr g) 4-chloroaniline (3.0 eq), DMF, 70°C, 12 hr.

[0090] Procedures for forming the quinzaolinone core (the synthesis of compound 2, scheme 1 from anthranillic acid derivatives) are described in Yang et al.

Scheme 2. (a) EDIPA (1.2 eq), acetyl chloride (1.2 eq), CH₂CI₂ , 0°C to 25°C, 6 hr; (b) POCI₃ (9.5 eq), DMF (3.3 eq), 0°C to 70°C, 12 hr; (c) NaBH₄ (2.0 eq), MeOH:CH₂CI₂ (1 :2), 0°C, 1 hr; (d) EDIPA (1.5 eq), TBS-CI (1.5 eq), CH ₂CI₂, 25°C, 12 hr; (e) Pd(PPh₃)₄ (10%), 2- Isopropoxyphenylboronic acid (1.5 eq) in EtOH, NaC0₃ (2M, aq) (10.0 eq), dioxane 25°C to 80°C, 24 hr; (f) TBAF (1 .1 eq), CH₂CI₂ , 0°C, 1 hr; (g) PBr₃ (3.0 eq), CH₂CI₂ , 0°C, 30 min, 25°C, 2 hr; (h) piperazine (3.0 eq), THF, 25 °C, 14 hr; (i) EDIPA, 4-DMAP, 4-chlorophenoxy acetylchloride, CH₂CI₂ 0°C to 25°C, 3 hr. Quinoline Erastin (scheme 2) - 6MEW78

[0091] N-phenylethanamide. Acetyl chloride (0.916 mL, 12.89 mmol, 1 .2 eq) was added slowly to a solution of EDIPA (2.25 mL, 12.89 mmol, 1 .2 eq) and aniline (0.98 mL, 10.74 mmol) at 0°C. The resulting mixture was stirred for an additional hour at 0°C then at 25°C for 4 hours. Upon completion, the mixture was quenched with saturated aqueous NaHCO₃ and extracted 3 times with EtOAc. The combined organic layers were dried (Na₂SO ), concentrated, and the crude material was purified by combi flash 0- > 20% EtOAc in hexanes to provide N-phenylethanamide (1 .27 g, 88% yield).

2-chloroquinoline-3-carbaldehvde.

[0092] POCIs (1 .29 mL, 14.1 mmol, 9.5 eq) was added to DMF (0.379 mL, 4.89 mmol, 3.3 eq) at 0°C. The mixture was stirred for an additional 5 minutes at 0°C before the addition of N-phenylethanamide (200 mg, 1 .48 mmol). The mixture was then heated to 75°C and stirred for 12 hours. Upon completion, the reaction was quenched very carefully with cold water and extracted 3 times with Et₂O. The combined organic layers were dried (MgSO₄), concentrated, and the crude material was purified by combiflash 0- >30% EtOAc in hexanes to provide 2-chloroquinoline-3-carbaldehyde (139 mg, 49% yield).

(2-chloroquinolin-3-yl)methanol.

[0093] NaBH₄ (0.849 g, 22.44 mmol, 2.0 eq) was added to a solution of 2-chloroquinoline-3-carbaldehyde (2.15 g, 1 1 .22 mmol) in DCM:MeOH (2:1 , 120 mL) at 0°C and stirred for 1 hour. Upon completion, the reaction was quenched with saturated aqueous NH₄CI and extracted 3 times with dichloromethane. The combined organic layers were dried (Na₂SO ), concentrated, and the crude material was purified by combi flash 0->50% EtOAc in hexanes to provide (2-chloroquinolin-3-yl)methanol (2.1 g, 97%).

3-((tert-butyldimethylsilyloxy)methyl)-2-chloroquinoline.

[0094] 3-((tert-butyldimethylsilyloxy)methyl)-2-chloroquinoline was prepared according to the general TBS protection procedure (1 .2 g, 29%).

3-((tert-butyldimethylsilyloxy)methyl)-2-(2-isopropoxyphenyl)quinoline (Suzuki coupling procedure).

[0095] Pd(PPh₃)₄ (187 mg, 0.162 mmol, 10%) was added to a solution of 3-((tert-butyldimethylsilyloxy)methyl)-2-chloroquinoline (0.5 g, 1 .62 mmol) in dioxane (10 mL) and stirred for 10 min at 25°C. To the resulting mixture, a solution of 2-isopropoxyphenylboronic acid (0.438 g, 2.43 mmol, 1 .5 eq) in EtOH (4 mL) was added and the resulting mixture was stirred at 25°C for 10 minutes before the addition of a 2 M aqueous solution of sodium carbonate (8.1 mL, 16.2 mmol, 10 eq). The resulting mixture was stirred at 25°C for an additional 5 minutes, then heated to 80°C for 24 hours. Upon completion, the mixture was diluted with water and extracted 3 times with EtOAc. The combined organic layers were dried (Na₂SO ), concentrated, and the crude material was purified by combi flash 0->20% EtOAc in hexanes to provide 3- ((tert-butyldimethylsilyloxy)methyl)-2-(2-isopropoxyphenyl)quinoline (0.57 g, 86% yield).

(2-(2-isopropoxyphenyl)quinolin-3-yl)methanol.

[0096] TBAF (1 M, 0.67 mL, 0.67 mmol, 1 .1 eq) was added to a solution of (2-(2-isopropoxyphenyl)quinolin-3-yl)methanol in THF (6 mL) at 0°C. The resulting mixture was stirred at 0°C for 1 hour. Upon completion, saturated aqueous NH CI was added and extracted 3 times with EtOAc. The combined organic layers were dried (Na₂SO₄), concentrated, and the crude material was purified by combi flash 0->40% EtOAc in hexanes to provide (2- (2-isopropoxyphenyl)quinolin-3-yl)methanol (135 mg, 75%).

3-(bromomethyl)-2-(2-isopropoxyphenyl)quinolone.

[0097] PBr₃ (0.130 ml_, 1 .38 mmol, 3 eq) was added to a solution of (2- (2-isopropoxyphenyl)quinolin-3-yl)methanol (135 mg, 0.45 mmol) in DCM (5 ml_) at 0°C. The resulting mixture was stirred at 0°C for an additional 30 minutes, then at 25°C for 2 hours. Upon completion, the reaction was quenched with saturated aqueous NaHCO₃ and extracted 3 times with EtOAc. The combined organic layers were dried (Na₂SO ), concentrated, and the crude material was purified by combi flash 0-20% EtOAc in hexanes to provide 3-(bromomethyl)-2-(2-isopropoxyphenyl)quinoline (57 mg, 35%).

2-(2-isopropoxyphenyl)-3-(piperazin-1 -ylmethvDquinoline (amine addition procedure).

[0098] To a solution of 3-(bromomethyl)-2-(2- isopropoxyphenyl)quinoline (57 mg, 0.160 mmol) in THF 2 ml_ at 0°C, piperzine (138 mg, 1 .6 mmol, 10 eq) was added and stirred for 12 hours. The reaction mixture was then concentrated and purified directly by combi flash 0- 20% MeOH in DCM to afford 2-(2-isopropoxyphenyl)-3-(piperazin-1 - ylmethyl)quinoline (51 mg, 88% yield).

6MEW78 - 2-(2-isopropoxyphenyl)-3-(piperazin-1 -ylmethvDquinoline (acyl chloride addition procedure).

[0099] To a solution of 2-(2-isopropoxyphenyl)-3-(piperazin-1 - ylmethyl)quinoline (51 mg, 0.141 mmol), EDIPA (30 uL, 0.169 mmol, 1 .2 eq), and 4-DMAP (3 mg, 0.0282 mmol, 0.2 eq) in DCM (2 mL) at 0°C, a solution of 4-chlorophenoxy acetyl chloride (32 mg, 0.155 mmol, 1 .1 eq) in DCM (1 mL) was added dropwise. The reaction slowly warmed to 25°C and was stirred for 4 hours. Upon completion, the reaction was quenched with saturated aqueous NaHCO3 and extracted 3 times with DCM. The combined organic layers were dried (Na₂SO₄), concentrated, and the crude material was purified by preparative TLC 5% MeOH in DCM (34 mg, 45% yield) ¹ H NMR (300 MHz, chloroform-d) δ 8.29 p.p.m. (s, 1 H), 8.23-8.04 (m, 1 H), 7.88 (dd, J = 8.0, 1 .5 Hz, 1 H), 7.71 (ddd, J = 8.4, 6.9, 1 .5 Hz, 1 H), 7.57 (ddd, J = 8.1 , 6.9, 1 .2 Hz, 1 H), 7.47-7.31 (m, 2H), 7.27-7.19 (m, 2H), 7.09 (td, J = 7.5, 7.4, 1 .0 Hz, 1 H), 6.99 (d, J = 8.3 Hz, 1 H), 6.91 ? 6.82 (m, 2H), 4.64 (s, 2H), 4.43 (p, J = 6.0, 6.0, 6.0, 6.0 Hz, 1 H), 3.85-3.37 (m, 6H), 2.34 (m, 4H), 1 .22 (d, J = 6.0 Hz, 3H), 1 .03 (d, J = 6.0 Hz, 3H). HRMS (m/z): [M+] calculated for C31 H32CIN3O3 530.06, found 530.22.

Scheme 3. (a) EDIPA (1.5 eq), TBS-CI (1.2 eq), DMF, 25°C, 12 hr; (b) NBS (1.0

eq), DMF 0°C, 1 hr; (c) Boc₂0 (1.8 eq), 4-DMAP (0.2 eq), CH₂CI₂, 25°C, 12 hr; (d)

Pd(PPh₃)₄ (10%), 2-lsopropoxyphenylboronic acid (1.5 eq) in EtOH, NaC0₃ (2M, aq) (10.0 eq), dioxane 25°C to 80°C, 24 hr; (e) HF-py (xs), 25°C, 6 hr; (f) DMP (1.8 eq), NaHC0₃ (10 eq), CH₂CI₂, 25°C, 3 hr; (g) piperazine (10.0 eq), ZnCI₂ (0.2 eq),

NaBH₃CN in MeOH, 40°C, 6 hr; (h) EDIPA, 4-DMAP, 4-chlorophenoxy

acetylchloride, CH₂CI₂ 0°C to 25°C, 3 hr; (i) TFA: CH₂CI₂ (1 :1 ), 25°C, 12 hr.

7MEW81 - 2-((tert-butyldimethylsilyloxy)methyl)-1 H-indole (TBS protection procedure).

[0100] TBS-CI (1 .66 g, 1 1 .04 mmol, 1 .2 eq) was added to a solution of (1 H-indol-2-yl)methanol (1 .335 g, 9.2 mmol) and EDIPA (2.4 mL, 13.8 mmol, 1 .5 eq) in DMF (30 mL) at 25°C. The mixture was stirred for an additional 5 hours at 25°C. Upon completion, the reaction contents were diluted with water and extracted 3 times with Et₂O. The combined organic layers were dried (MgSO₄), concentrated, and the crude material was purified by combi flash 0- 20% EtOAc in hexanes to provide 2-((tert-butyldimethylsilyloxy)methyl)-1 H- indole (2.04 g, 85%).

3-bromo-2-((tert-butyldimethylsilyloxy)methyl)-1 H-indole. [0101] NBS (0.34 g, 1 .91 mmol) was added to a stirred solution of 2- ((tert-butyldimethylsilyloxy)methyl)-1 H-indole (0.5 g, 1 .91 mmol) in DMF at 0°C. The mixture was stirred an additional 1 hour at 0°C. Upon completion, the reaction contents were diluted with water and extracted 3 times with Et₂O. The combined organic layers were dried (MgSO₄), concentrated, and the crude material was purified by combi flash 0->10% EtOAc in hexanes to provide 3-bromo-2-((tert-butyldimethylsilyloxy)methyl)-1 H-indole (0.514 g, 79%).

Tert-butyl 3-bromo-2-((tert-butyldimethylsilyloxy)methyl)-1 H-indole-1 - carboxylate.

[0102] Di-tert-butyl dicarbonate (2.08 g, 9.52 mmol, 1 .8 eq) and 4- DMAP (0.129 g, 1 .06 mmol, 0.2 eq) were added sequentially to a stirred solution of 3-bromo-2-((tert-butyldimethylsilyloxy)methyl)-1 H-indole (1 .8 g, 5.29 mmol) in DCM (20 ml_) at 25°C. The mixture was stirred for an additional 12 hours. Upon completion, the reaction contents were diluted with DCM, poured into water, and extracted 2 times with DCM. The crude material was pushed forward to the next step without additional purification (1 .72 g, 74% yield). Tert-butyl 2-((tert-butyldimethylsilyloxy)methyl)-3-(2-isopropoxyphenyl)-1 H- indole-1 -carboxylate

[0103] Tert-butyl 2-((tert-butyldimethylsilyloxy)methyl)-3-(2- isopropoxyphenyl)-1 H-indole-1 -carboxylate was prepared according to general Suzuki coupling procedure (0.883 g, 79%).

Tert-butyl 2-(hvdroxymethyl)-3-(2-isopropoxyphenyl)-1 H-indole-1 -carboxylate.

[0104] Hydrogen fluoride pyridine (0.66 mL, 70% as HF, 30% as py) was added to a solution of tert-butyl 2-((tert-butyldimethylsilyloxy)methyl)-3-(2- isopropoxyphenyl)-1 H-indole-1 -carboxylate (0.3 g, 1 .01 mmol) in THF:pyridine (5:1 , 10 mL) at 0°C. The resulting mixture was then brought to room temperature and stirred for an additional 6 hours. Upon completion, the reaction was quenched with saturated NH₄CI and extracted 3 times with EtOAc. The combined organic layers were dried (Na₂SO ), concentrated, and the crude material was purified by combi flash 0-30% EtOAc in hexanes to provide tert-butyl 2-(hydroxymethyl)-3-(2-isopropoxyphenyl)-1 H-indole-1 - carboxylate (0.279 g, 73%).

Tert-butyl 2-formyl-3-(2-isopropoxyphenyl)-1 H-indole-1 -carboxylate.

[0105] Dess-Martin periodinane (230 mg, 0.543 mmol, 1 .8 eq) was added to a stirred suspension of sodium bicarbonate (242 mg, 2.88 mmol, 10.0 eq) and tert-butyl 2-(hydroxymethyl)-3-(2-isopropoxyphenyl)-1 H-indole-1 - carboxylate (1 10 mg, 0.288 mmol) in DCM (3 mL). The mixture was stirred at 25°C for 2 hours, and upon completion a saturated aqueous solution of sodium sulfite (1 mL) was added, and the mixture was stirred for an additional 5 minutes. This was followed subsequently by the addition of water and extraction with DCM three times. The combined organic layers were dried (Na₂SO₄), concentrated, and the crude material was purified by combi flash 0- 40% EtOAc in hexanes to give tert-butyl 2-formyl-3-(2-isopropoxyphenyl)-1 H- indole-1 -carboxylate (quantitative yield).

Tert-butyl 3-(2-isopropoxyphenyl)-2-(piperazin-1 -ylmethyl)-1 H-indole-1 - carboxylate.

[0106] A solution of tert-butyl 2-formyl-3-(2-isopropoxyphenyl)-1 H- indole-1 -carboxylate (0.140 g, 0.369 mmol) in DCM (1 .5 mL) was added to a stirred solution of piperazine (0.318 g, 3.69 mmol, 10 eq) in MeOH (1 .5 mL) at 25°C. After an additional 15 minutes of stirring a solution of ZnC^ (10 mg, 0.0738 mmol, 0.2 eq), sodium cyanoborohydride (70 mg, 1 .1 1 mmol, 3.0 eq) in MeOH (1 .5 eq) was added. The resulting mixture was heated to 40°C and stirred for 6 hours. Upon completion, the reaction was diluted with water and extracted 3 times with EtOAc. The combined organic layers were dried (Na₂SO₄), concentrated, and the crude material was purified by combi flash 0- >20% MeOH in DCM to provide tert-butyl 3-(2-isopropoxyphenyl)-2- (piperazin-1 -ylmethyl)-1 H-indole-1 -carboxylate (73 mg, 44% yield).

Tert-butyl 2-((4-(2-(4-chlorophenoxy)ethanoyl)piperazin-1 -yl)methyl)-3-(2- isopropoxyphenyl)-1 H-indole-1 -carboxylate

[0107] Tert-butyl 2-((4-(2-(4-chlorophenoxy)ethanoyl)piperazin-1 - yl)methyl)-3-(2-isopropoxyphenyl)-1 H-indole-1 -carboxylate was prepared according to the general acyl chloride addition procedure (89 mg, 90%).

7MEW81 - 2-(4-chlorophenoxy)-1 -(4-((3-(2-isopropoxyphenyl)-1 H-indol-2- yl)methyl)piperazin-1 -yl)ethanone.

[0108] TFA (1 mL) was added to a solution of tert-butyl 2-((4-(2-(4- chlorophenoxy)ethanoyl)piperazin-1 -yl)methyl)-3-(2-isopropoxyphenyl)-1 H- indole-1 -carboxylate (85 mg, 0.138 mmol) in DCM (1 mL) and the resulting solution was stirred at 25°C for 24 hours. Upon completion, the reaction was quenched with saturated aqueous NaHCO3 and extracted 3 times with EtOAc. The combined organic layers were dried (Na₂SO₄), concentrated, and the crude material was purified by combi flash 0->5% MeOH in DCM to provide 2- (4-chlorophenoxy)-1 -(4-((3-(2-isopropoxyphenyl)-1 H-indol-2- yl)methyl)piperazin-1 -yl)ethanone (50 mg, 70% yield). ¹ H NMR (300 MHz, chloroform-d) δ 8.61 p.p.m. (s, 1 H), 7.52 (d, J = 7.9 Hz, 1 H), 7.40 (ddd, J = 7.3, 5.8, 1 .5 Hz, 2H), 7.39-7.27 (m, 1 H), 7.30-7.15 (m, 3H), 7.17-6.99 (m, 3H), 6.94-6.81 (m, 2H), 4.66 (s, 2H), 4.35 (p, J = 6.1 , 6.1 , 6.1 , 6.1 Hz, 1 H), 3.93- 3.40 (m, 7H), 2.43 (d, J = 9.7 Hz, 4H), 1 .24-1 .00 (m, 6H). HRMS (m/z): [M+] calculated for C30H32CIN3O3 518.05, found 517.21 .

2-((4-(2-chloroethanoyl)piperazin-1 -yl)methyl)-3-(2- isopropoxyphenyl)quinazolin-4(3H)-one.

[0109] To a solution of a solution of 3-(2-isopropoxyphenyl)-2- (piperazin-1 -ylmethyl)quinazolin-4(3H)-one (200 mg, 0.53 mmol) and EDIPA (82 uL, 0.635 mmol, 1 .2 eq) in DCM (5 ml_) at 0°C, a solution of chloroacetyl chloride (50 uL, 0.635 mmol, 1 .2 eq) was added and the resulting mixture was stirred at 25°C for 4 hours. Upon completion, the reaction was quenched with saturated aqueous NaHCO3 and extracted 3 times with DCM. The combined organic layers were dried (Na₂SO₄), concentrated, and the crude material was purified by combi flash 0->5% MeOH in DCM to provide 2-((4-(2- chloroethanoyl)piperazin-1 -yl)methyl)-3-(2-isopropoxyphenyl)quinazolin- 4(3H)-one (210 mg, 73% yield). 35MEW22 - 2-((4-(2-(4-chlorophenylamino)ethanoyl)piperazin-1 -yl)methyl)-3- (2-isopropoxyphenyl)quinazolin-4(3H)-one.

[0110] 4-chloroaniline (29 mg, 0.230 mmol, 5.0 eq) was added to a solution of 2-((4-(2-chloroethanoyl)piperazin-1 -yl)methyl)-3-(2- isopropoxyphenyl)quinazolin-4(3H)-one (21 mg, 0.0462 mmol) in DMF (0.5 ml_), and the resulting mixture was stirred at 60°C for 12 hours. Upon completion, the mixture was diluted with water and extracted 3 times with diethyl ether. The combined organic layers were dried (MgSO₄), concentrated, and the crude material was purified by preparative TLC 5% MeOH in DCM to provide 2-((4-(2-(4-chlorophenylamino)ethanoyl)piperazin-1 -yl)methyl)-3-(2- isopropoxyphenyl)quinazolin-4(3H)-one (8 mg, 32% yield). ¹ H NMR (400 MHz, Chloroform-d) δ 8.32 p.p.m. (dq, J = 8.0, 1 .6, 1 .6, 1 .5 Hz, 1 H), 7.87-7.71 (m, 2H), 7.64-7.39 (m, 2H), 7.31 (s, 1 H), 7.19-6.96 (m, 5H), 6.56-6.46 (m, 2H), 4.91 (d, J = 6.0 Hz, 1 H), 4.57 (dtd, J = 8.1 , 6.3, 5.8, 4.2 Hz, 1 H), 3.80 (dt, J = 3.9, 1 .8, 1 .8 Hz, 2H), 3.58 (d, J = 5.6 Hz, 2H), 3.41 -3.26 (m, 4H), 2.66- 2.43 (m, 2H), 2.28 (dt, J = 16.2, 7.1 , 7.1 Hz, 2H), 1 .26 (dt, J = 6.1 , 1 .8, 1 .8 Hz, 4H), 1 .17 (dt, J = 6.2, 1 .7, 1 .7 Hz, 3H). HRMS (m/z): [M+] calculated for C30H32CIN5O3 546.06, found 546.23.

3-(2-isopropoxyphenyl)-2-

((methyl(2(methylamino)ethyl)amino)methyl)quinazolin-4(3H)-one

[0111] 3-(2-isopropoxyphenyl)-2-

((methyl(2(methylamino)ethyl)amino)methyl)quinazolin-4(3H)-one was prepared according to the general amine addition procedure using N,N- dimethylethylene diamine in place of piperazine (27 mg, 56% yield). 35MEW26

[0112] 35MEW26 was prepared from 3-(2-isopropoxyphenyl)-2- ((methyl(2(methylamino)ethyl)amino)methyl)quinazolin-4(3H)-one (20 mg, 22% yield). ¹H NMR (400 MHz, chloroform-d) δ 8.36-8.26 p.p.m. (m, 1 H), 7.81 -7.70 (m, 2H), 7.54-7.39 (m, 2H), 7.27-7.16 (m, 3H), 7.09 (td, J = 7.9, 7.4, 4.1 Hz, 2H), 6.92-6.80 (m, 2H), 4.73 (s, 1 H), 4.61 (s, 1 H), 4.57 (ddt, J = 9.2, 6.1 , 3.1 , 3.1 Hz, 1 H), 3.49-3.30 (m, 4H), 3.01 (s, 1 H), 2.90 (s, 2H), 2.82-2.44 (m, 2H), 2.29 (d, J = 15.6 Hz, 3H), 1 .25 (d, J = 3.7 Hz, 3H), 1 .16 (dd, J = 6.3, 1 .3 Hz, 3H). HRMS (m/z): [M+] calculated for C30H33CIN4O4 549.04, found 549.22.

3-(2-isopropoxyphenyl)-2-((methylamino)methyl)quinazolin-4(3H)-one.

[0113] A 33% (wt) solution (EtOH) of methyl amine (0.15 mL) was added to a solution of 2-(chloromethyl)-3-(2-isopropoxyphenyl)quinazolin- 4(3H)-one (80 mg, 0.243 mmol) in THF (3 mL) and the resulting mixture was stirred at 25°C for 12 hours. Upon completion, the mixture was diluted with water and extracted 3 times with EtOAc. The combined organic layers were dried (Na₂SO₄), concentrated, and the crude material was purified by combi flash 0->10% MeOH in DCM to provide 3-(2-isopropoxyphenyl)-2- ((methylamino)methyl)quinazolin-4(3H)-one (43 mg, 55% yield).

35MEW27

[0114] 35MEW27 was prepared from 3-(2-isopropoxyphenyl)-2- ((methylamino)methyl)quinazolin-4(3H)-one using general acyl chloride addition procedure (71 mg, 72% yield). Mixture of atropeisomers ¹H NMR (400 MHz, chloroform-d) δ 8.31 p.p.m. (d, J = 7.9 Hz, 2H), 7.80 (dddd, J = 12.6, 8.5, 7.2, 1 .5 Hz, 2H), 7.64 (dd, J = 8.3, 1 .1 Hz, 1 H), 7.59-7.41 (m, 5H), 7.25 (dd, J = 8.2, 6.1 Hz, 3H), 7.20-7.07 (m, 6H), 7.00-6.94 (m, 2H), 6.86-6.75 (m, 2H), 4.82 (s, 2H), 4.60 (d, J = 7.6 Hz, 5H), 4.25 (s, 2H), 3.98 (d, J = 17.0 Hz, 1 H), 3.22 (s, 3H), 3.06 (s, 3H), 1 .30 (dd, J = 18.1 , 6.1 Hz, 7H), 1 .18 (dd, J = 16.3, 6.0 Hz, 6H). HRMS (m/z): [M+] calculated for C27H26CIN3O4 491 .97, found 492.13.

35MEW14

[0115] To a solution of 3-(2-isopropoxyphenyl)-2-(piperazin-1 - ylmethyl)quinazolin-4(3H)-one (30 mg, 0.079 mmol) and potassium carbonate (13 mg, 0.094 mmol, 1 .2 eq) in DMF (1 mL), 4-Chlorophenyl 2-bromoethyl ether (28 mg, 0.1 18 mmol, 1 .5 eq) was added. The resulting mixture was stirred at 60°C for 24 hours. Upon completion, the mixture was diluted with saturated aqueous NaHCO₃ and extracted 3 times with EtOAc. The combined organic layers were dried (Na₂SO ), concentrated, and the crude material was purified with preparative TLC, 10% MeOH in DCM to provide 35MEW14 (1 1 .8 mg, 28% yield). ¹ H NMR (400 MHz, chloroform-d) δ 8.32 p.p.m. (dd, J = 7.9, 1 .1 Hz, 1 H), 7.88-7.75 (m, 2H), 7.50 (dt, J = 8.1 , 4.1 , 4.1 Hz, 1 H), 7.43 (td, J = 8.1 , 7.9, 1 .7 Hz, 1 H), 7.31 (dd, J = 7.7, 1 .7 Hz, 1 H), 7.27-7.20 (m, 2H), 7.12- 7.00 (m, 2H), 6.90-6.76 (m, 2H), 4.56 (p, J = 6.0, 6.0, 6.0, 6.0 Hz, 1 H), 4.06 (t, J = 5.8, 5.8 Hz, 2H), 3.26 (d, J = 1 .4 Hz, 2H), 2.78 (t, J = 5.8, 5.8 Hz, 2H), 2.51 (s, 6H), 2.35-2.22 (m, 2H), 1 .25 (d, J = 6.0 Hz, 3H), 1 .17 (d, J = 6.0 Hz, 3H). HRMS (m/z): [M+] calculated for C30H33CIN4O3 533.06, found 533.23.

35MEW38 (amine coupling procedure).

[0116] HBTU (147 mg, 0.38 mmol, 1 .5 eq) was added to a solution of 3-chlorophenoxy acetic acid (69 mg, 0.336 mmol, 1 .3 eq) and EDIPA (67 uL, 0.38 mmol, 1 .5 eq) in DCM (3 mL) and stirred for 30 minutes at 25°C. A solution of 3-(2-isopropoxyphenyl)-2-(piperazin-1 -ylmethyl)quinazolin-4(3H)- one (100 mg, 0.258 mmol) in DCM (1 mL) was added and stirred for an additional 4 hours. Upon completion, the reaction was quenched with saturated aqueous NaHCO3 and extracted 3 times with DCM. The combined organic layers were dried (Na₂SO₄), concentrated, and the crude material was purified by combi flash 0->5% MeOH in DCM to provide 35MEW38 (120 mg, 85% yield). ¹H NMR (400 MHz, chloroform-d) δ 8.38-8.27 p.p.m. (m, 1H), 7.87-7.68 (m, 2H), 7.51 (ddd, J = 8.2, 6.6, 1.7 Hz, 1H), 7.43 (ddd, J = 8.3, 7.5, 1.7 Hz, 1H), 7.33-7.26 (m, 1H), 7.21 (t, J = 8.1, 8.1 Hz, 1H), 7.12-7.04 (m, 2H), 6.97 (ddd, J = 8.0, 1.9, 0.9 Hz, 1H), 6.92 (t, J = 2.2, 2.2 Hz, 1H), 6.83 (ddd, J = 8.5, 2.5, 0.9 Hz, 1H), 4.64 (s, 2H), 4.56 (p, J = 6.1, 6.1, 6.0, 6.0 Hz, 1H), 3.68-3.36 (m, 4H), 3.29 (s, 2H), 2.49 (dddd, J = 26.4, 11.1, 6.1, 3.2 Hz, 2H), 2.32-2.12 (m, 2H), 1.24 (d, J = 6.1 Hz, 3H), 1.16 (d, J = 6.0 Hz, 3H). HRMS (m/z): [M+] calculated for C36H35CIN4O4547.04, found 547.21.

35MEW39.

[0117] 35MEW39 was prepared using the general amine coupling procedure from 3-(2-isopropoxyphenyl)-2-(piperazin-1 -ylmethyl)quinazolin- 4(3H)-one, (132 mg, 96% yield).¹H NMR (400 MHz, chloroform-d) δ 8.36-8.25 p.p.m. (m, 1H), 7.83 ? 7.72 (m, 2H), 7.50 (ddd, J = 8.1, 6.8, 1.5 Hz, 1H), 7.47- 7.38 (m, 1H), 7.29-7.22 (m, 1H), 7.11-7.02 (m, 2H), 7.01-6.91 (m, 2H), 6.91- 6.80 (m, 2H), 4.62 (s, 2H), 4.56 (p, J = 6.1, 6.1, 6.1, 6.1 Hz, 1H), 3.58 -3.39 (m, 5H), 3.27 (s, 2H), 2.47 (dddd, J = 29.4, 10.8, 6.2, 3.5 Hz, 2H), 2.35-2.13 (m, 2H), 1.23 (d, J = 6.0 Hz, 3H), 1.15 (d, J = 6.0 Hz, 3H). HRMS (m/z): [M+] calculated for C30H31 FN4O4530.59, found 531.24. 35MEW13.

[0118] 35MEW13 was prepared using the general amine coupling procedure from 3-(2-isopropoxyphenyl)-2-(piperazin-1 -ylmethyl)quinazolin- 4(3H)-one (18 mg, 44% yield).¹H NMR (400 MHz, chloroform-d) δ 8.32 p.p.m. (ddd, J = 8.0, 1.4, 0.7 Hz, 1H), 7.84-7.74 (m, 2H), 7.52 (ddd, J = 8.2, 6.7, 1.7 Hz, 1H), 7.44 (ddd, J = 8.4, 7.5, 1.7 Hz, 1H), 7.31 (dd, J = 7.1, 1.9 Hz, 2H), 7.29-7.27 (m, 1H), 7.12-7.06 (m, 2H), 7.00 (td, J = 7.3, 7.3, 1.1 Hz, 1H), 6.97- 6.87 (m, 2H), 4.66 (s, 2H), 4.57 (p, J = 6.1, 6.1, 6.1, 6.1 Hz, 1H), 3.52 (ddt, J = 10.6, 7.6, 3.2, 3.2 Hz, 4H), 3.28 (s, 2H), 2.56-2.40 (m, 2H), 2.32-2.15 (m, 2H), 1.25 (d, J = 6.1 Hz, 3H), 1.17 (d, J = 6.0 Hz, 3H). HRMS (m/z): [M+] calculated for C30H32N4O4512.6, found 513.25.

15MEW81.

[0119] 15MEW81 was prepared using the general amine coupling procedure from 3-(2-isopropoxyphenyl)-2-(piperazin-1 -ylmethyl)quinazolin- 4(3H)-one (226 mg, 58% yield).¹ H NMR (400 MHz, chloroform-d) δ 8.36-8.30 p.p.m. (m, 1H), 7.86-7.74 (m, 2H), 7.52 (ddd, J = 8.2, 6.5, 1.8 Hz, 1H), 7.47- 7.38 (m, 1H), 7.29-7.22 (m, 3H), 7.18-7.11 (m, 2H), 7.11-6.98 (m, 2H), 4.57 (p, J = 6.0 Hz, 1H), 3.52 (dt, J = 9.7, 4.6 Hz, 2H), 3.28 (s, 4H), 2.98-2.89 (m, 2H), 2.56 (t, J = 7.7 Hz, 2H), 2.41 (t, J = 5.7 Hz, 2H), 2.25 -2.10 (m, 2H), 1.25 (d, J = 6.0 Hz, 3H), 1.17 (d, J = 6.0 Hz, 3H). HRMS (m/z): [M+] calculated for C31H33CIN4O3545.07, found 545.23.

35MEW28.

[0120] 35MEW28 was prepared using a general Suzuki coupling procedure from 21MEW26 (30 mg, 77% yield). ¹H NMR (400 MHz, chloroform-d) δ 8.38-8.32 p.p.m. (m, 1H), 7.85-7.76 (m, 2H), 7.74-7.67 (m, 2H), 7.60-7.53 (m, 4H), 7.53-7.47 (m, 2H), 7.44 (t, J = 7.6, 7.6 Hz, 2H), 7.36 (d, J = 7.1 Hz, 1H), 7.26-7.20 (m, 2H), 7.15 (d, J = 8.6 Hz, 1H), 6.87-6.78 (m, 2H), 4.68-4.62 (m, 1H), 4.60 (s, 2H), 3.63-3.23 (m, 7H), 2.49 (ddt, J = 14.5, 10.6, 4.8, 4.8 Hz, 2H), 2.23 (ddd, J = 18.4, 11.4, 5.5 Hz, 2H), 1.28 (d, J = 6.0 Hz, 3H), 1.21 (d, J = 6.1 Hz, 3H). HRMS (m/z): [M+] calculated for C36H35CIN4O4623.14, found 623.24.

35MEW29.

[0121] 35MEW29 was prepared using a general Suzuki coupling procedure from 21MEW26 (65 mg, 54%).¹H NMR (300 MHz, chloroform-d) δ 8.33 p.p.m. (ddd, J = 8.0, 1.5, 0.7 Hz, 1H), 7.85-7.72 (m, 2H), 7.68 (dd, J = 1.5, 0.9 Hz, 1H), 7.57-7.48 (m, 2H), 7.47 (t, J = 1.7, 1.7 Hz, 1H), 7.42 (d, J = 2.2 Hz, 1H), 7.27-7.20 (m, 2H), 7.08 (d, J = 8.6 Hz, 1H), 6.95-6.76 (m, 2H), 6.63 (dd, J = 1.9, 0.9 Hz, 1H), 4.61 (s, 2H), 4.56 (q, J = 6.1, 6.1, 6.1 Hz, 1H), 3.51 -3.35 (m, 4H), 3.29 (d, J = 2.2 Hz, 2H), 2.48 (dt, J = 11.0, 5.1, 5.1 Hz, 2H), 2.20 (tt, J = 10.8, 10.8, 5.1, 5.1 Hz, 2H), 1.25 (d, J = 6.0 Hz, 3H), 1.18 (d, J = 6.0 Hz, 3H). HRMS (m/z): [M+] calculated for C34H33CIN4O5 613.1, found 613.22.

14MEW31.

[0122] 14MEW31 was prepared from 5-chloro-3-(2-isopropoxyphenyl)- 2-(piperazin-1-ylmethyl)quinazolin-4(3H)-one using a general acyl chloride addition procedure (122 mg, 87% yield).¹ H NMR (300 MHz, chloroform-d) δ 7.70-7.59 p.p.m. (m, 2H), 7.50 (dd, J = 5.5, 3.6 Hz, 1H), 7.47-7.39 (m, 1H), 7.28-7.23 (m, 2H), 7.13-7.03 (m, 2H), 6.92-6.82 (m, 2H), 4.64 (s, 2H), 4.57 (p, J = 6.1, 6.1, 6.1, 6.1 Hz, 1H), 3.51 (d, J = 14.4 Hz, 4H), 3.25 (s, 2H), 2.48 (s, 2H), 2.25 (s, 2H), 1 .26 (d, J = 6.0 Hz, 3H), 1 .19 (d, J = 6.0 Hz, 3H). HRMS (m/z): [M+] calculated for C30H30CI2N4O4 581 .49, found 581 .17.

14MEW32.

[0123] 14MEW32 was prepared from 8-chloro-3-(2-isopropoxyphenyl)- 2-(piperazin-1 -ylmethyl)quinazolin-4(3H)-one using a general acyl chloride addition procedure (77 mg, 55% yield). ¹H NMR (300 MHz, chloroform-d) δ 8.23 p.p.m. (dd, J = 8.0, 1 .5 Hz, 1 H), 7.87 (dd, J = 7.8, 1 .5 Hz, 1 H), 7.52-7.38 (m, 2H), 7.28-7.21 (m, 4H), 7.13-7.01 (m, 2H), 6.93-6.81 (m, 2H), 4.66 (s, 2H), 4.64-4.47 (m, 1 H), 3.54 (s, 4H), 3.33 (d, J = 3.0 Hz, 2H), 2.64 (s, 1 H), 2.54 (s, 1 H), 2.44 (s, 1 H), 2.33 (s, 1 H), 1 .26 (d, J = 6.1 Hz, 3H), 1 .18 (d, J = 6.0 Hz, 3H). HRMS (m/z): [M+] calculated for C30H30CI2N4O4 581 .49, found 581 .17

13MEW16.

[0124] 13MEW16 was prepared from 3-(3-isopropoxyphenyl)-2- (piperazin-1 -ylmethyl)quinazolin-4(3H)-one using a general acyl chloride addition procedure (100 mg, 54%). ¹ H NMR (400 MHz, chloroform-d) δ 8.37- 8.27 p.p.m. (m, 1 H), 7.82 (ddd, J = 8.4, 6.9, 1 .5 Hz, 1 H), 7.76 (d, J = 8.4 Hz, 1 H), 7.54 (ddd, J = 8.2, 7.0, 1 .4 Hz, 1 H), 7.43 (t, J = 8.0, 8.0 Hz, 1 H), 7.28- 7.18 (m, 2H), 7.10-6.94 (m, 1 H), 6.88 (dd, J = 9.3, 2.1 Hz, 4H), 4.65 (s, 2H), 4.58 (p, J = 6.1 , 6.1 , 6.0, 6.0 Hz, 1 H), 3.53 (dt, J = 22.0, 5.0, 5.0 Hz, 4H), 3.32 (s, 2H), 2.40 (ddt, J = 27.6, 9.7, 5.0, 5.0 Hz, 4H), 1 .38 (dd, J = 6.3, 1 .5 Hz, 6H). HRMS (m/z): [M+] calculated for C30H31 CIN4O4 547.04, found 547.21 .

13MEW76.

[0125] 13MEW76 was prepared from 3-(2-isopropoxyphenyl)-2- (piperazin-1 -ylmethyl)quinazolin-4(3H)-one using a general acyl chloride addition procedure (270 mg, 62% yield).¹H NMR (300 MHz, chloroform-d) δ 8.32 p.p.m. (dd, J = 8.0, 1.4 Hz, 1H), 7.87-7.71 (m, 2H), 7.57-7.40 (m, 2H), 7.28-7.21 (m, 3H), 7.13-7.00 (m, 2H), 6.91-6.84 (m, 2H), 4.64 (s, 2H), 4.61- 4.48 (m, 1H), 3.50 (s, 4H), 3.28 (s, 2H), 2.44 (s, 2H), 2.23 (s, 2H), 1.24 (d, J = 6.0 Hz, 3H), 1.16 (d, J = 6.0 Hz, 3H). HRMS (m/z): [M+] calculated for C30H31CIN4O4547.04, found 547.21.

21MEW26.

[0126] 21MEW26 was prepared from 3-(5-bromo-2-isopropoxyphenyl)- 2-(piperazin-1-ylmethyl)quinazolin-4(3H)-one (1.87 g, 70% yield). ¹H NMR (400 MHz, chloroform-d) δ 8.36-8.26 p.p.m. (m, 1H), 7.81-7.70 (m, 2H), 7.54- 7.39 (m, 2H), 7.27-7.16 (m, 3H), 7.09 (td, J = 7.9, 7.4, 4.1 Hz, 2H), 6.92-6.80 (m, 2H), 4.73 (s, 1H), 4.61 (s, 1H), 4.57 (ddt, J = 9.2, 6.1, 3.1, 3.1 Hz, 1H), 3.49-3.30 (m, 4H), 3.01 (s, 1H), 2.90 (s, 2H), 2.82-2.44 (m, 2H), 2.29 (d, J = 15.6 Hz, 3H), 1.25 (d, J = 3.7 Hz, 3H), 1.16 (dd, J = 6.3, 1.3 Hz, 3H). HRMS (m/z): [M+] calculated for C30H33CIN4O4549.04, found 549.22

10MEW79.

[0127] 10MEW79 was prepared from 6-fluoro-3-(2-isopropoxyphenyl)- 2-(piperazin-1-ylmethyl)quinazolin-4(3H)-one using a general acyl chloride addition procedure (0.296 mg, 69% yield).¹H NMR (400 MHz, chloroform-d) δ 7.95 p.p.m. (dd, J = 8.4, 3.0 Hz, 1H), 7.76 (dd, J = 9.0, 4.8 Hz, 1H), 7.52 (td, J = 8.6, 8.5, 2.8 Hz, 1H), 7.48-7.41 (m, 1H), 7.28-7.17 (m, 3H), 7.16-7.02 (m, 2H), 6.91-6.81 (m, 2H), 4.64 (s, 2H), 4.57 (p, J = 6.1, 6.1, 6.0, 6.0 Hz, 1H), 3.61-3.39 (m, 4H), 3.27 (s, 2H), 2.46 (dq, J = 22.1, 7.0, 7.0, 4.7 Hz, 2H), 2.24 (ddt, J = 30.1, 10.7, 4.3, 4.3 Hz, 2H), 1.25 (d, J = 6.1 Hz, 3H), 1.17 (d, J = 6.0 Hz, 3H). HRMS (m/z): [M+] calculated for C30H30CIFN4O4 565.04, found 565.2.

8MEW98.

[0128] 8MEW98 was prepared from 3-(2-isopropoxyphenyl)-6,7- dimethoxy-2-(piperazin-1 -ylmethyl)quinazolin-4(3H)-one using the general acyl chloride addition procedure (54 mg, 53% yield). ¹ H NMR (300 MHz, chloroform-d) δ 7.64 p.p.m. (s, 1 H), 7.50 ? 7.39 (m, 1 H), 7.28 ? 7.16 (m, 5H), 7.13-7.02 (m, 2H), 6.92 ? 6.82 (m, 2H), 4.64 (s, 2H), 4.57 (q, J = 6.1 , 6.1 , 6.0 Hz, 1 H), 4.03 (d, J = 9.2 Hz, 5H), 3.50 (d, J = 17.6 Hz, 4H), 3.24 (d, J = 1 .8 Hz, 2H), 2.46 (s, 2H), 2.20 (s, 2H), 1 .24 (d, J = 6.1 Hz, 3H), 1 .17 (d, J = 6.0 Hz, 3H). HRMS (m/z): [M+] calculated for C32H35CIN4O6 607.1 , found 607.23.

6MEW160.

[0129] To a solution of 2-((4-(2-(4-chlorophenoxy)ethanoyl)piperazin-1 - yl)methyl)-3-(2-isopropoxyphenyl)-6-nitroquinazolin-4(3H)-one (60 mg, 0.1 mmol) in methanol (1 mL) 10% palladium on carbon (3 mg) was added. The reaction was then stirred under hydrogen gas (1 atm) for 12 hr. Upon completion the mixture was filtered over celite, concentrated, and purified by combi flash 0 to 5% MeOH in DCM to provide 6MEW160 (55 mg, 98% yield). ¹ H NMR (300 MHz, chloroform-d) δ 7.85 p.p.m. (d, J = 2.5 Hz, 1 H), 7.67 (d, J = 8.8 Hz, 1 H), 7.47-7.35 (m, 2H), 7.29-7.20 (m, 4H), 7.06 (ddt, J = 6.7, 3.6, 1 .8, 1 .8 Hz, 2H), 7.00 (s, 1 H), 6.90-6.79 (m, 2H), 6.60 (s, 1 H), 4.62 (s, 2H), 4.54 (p, J = 6.1 , 6.1 , 6.1 , 6.1 Hz, 1 H), 3.44 (tt, J = 7.3, 7.3, 3.0, 3.0 Hz, 5H), 3.24 (s, 2H), 2.49 (ddd, J = 10.3, 6.4, 3.5 Hz, 1 H), 2.40 (dt, J = 10.9, 4.8, 4.8 Hz, 1 H), 2.31 ? 2.1 1 (m, 2H), 1 .22 (d, J = 6.0 Hz, 4H), 1 .14 (d, J = 6.0 Hz, 3H). HRMS (m/z): [M+] calculated for C30H32CIN5O4 562.06, found 562.31 .

B. Total synthesis of sorafenib and analogs

Chromatography.

[0130] Merck precoated 0.25 mm silica plates containing a 254 nm fluorescence indicator were used for analytical thin-layer chromatography. [0131] Flash chromatography was performed on 230-400 mesh silica (SiliaFlash P60) from Silicycle.

Spectroscopy.

[0132] NMR spectra were obtained on a Bruker DPX 300 or 400 MHz spectrometer. CI-MS spectra were taken on a Nermag R-10-10 instrument.

Overall synthetic scheme of sorafenib (SRS13-42) and analog (CA-1 , SRS13- 65)

4a: R = H

6a: R = H; SRS13-42 4b: R = CH₃ 6b:R = CH₃; SRS13-65

Scheme 4. General synthetic route of sorafenib (SRS13-42) and analog (SRS13-65).

[0133] The starting material, picolinic acid 1 , was first converted to the 4-chloropyridine-2-carbonyl chloride hydrochloride 2 in 90% yield using thionyl chloride and a catalytic amount of dimethylformamide (DMF). Amidation of the acid chloride 2 with methylamine and dimethylamine in the presence of triethylamine as an HCI acceptor gave the amides 3a and 3b in 94% and 90% yield, respectively. In the next reaction step, the chloroamides 3a and 3b were coupled with 4-aminophenol to give the ethers 4a (also called "CA-1 ") and 4b in 70% and 69% yield, respectively. The ether side chains were introduced using potassium terf-butoxide in the presence of potassium carbonate. These conditions allowed a chemo-selective ArSN2 addition of the phenoxide; therefore, the main product was the ether, not the secondary amine. Finally, urea bond formation was performed at room temperature, in dichloromethane, between the aniline of the ethers 4a and 4b and the 4-chloro-3- (fluoromethyl)phenyl isocyanate 5. The final sorafenib products 4a and 4b were isolated in 90% and 92% yield, respectively.

Synthesis of 4-chloro-N-methylpyridine-2-carboxamide (3a and 3b, Scheme 4)

[0134] Anhydrous Λ/,/V-dimethylformamide (0.6 ml_) was slowly added to thionyl chloride (18 ml_) at a temperature range of 40-50 °C under argon. The solution was stirred in that temperature range for 10 minutes prior to portion wise addition of picolinic acid (1 , 6.00 g, 48.74 mmol) over a 5-minute period. The solution was heated to 70 °C, and vigorous SO₂ evolution was observed. A yellow solid precipitated after 17 hours. The mixture was then cooled to room temperature, diluted with toluene (50 ml_), and concentrated to 20 ml_. The mixture was filtered, washed with toluene (50 ml_), and dried under high vacuum for 4 h to afford the 4-chloropyridine-2-carbonyl chloride 2 as a white solid. [0135] The acid chloride 2 (500 mg, 2.907 mmol) was added portion wise to methylamine (2.0 M) in tetrahydrofuran (100 ml_) at 0 °C under argon. The mixture was stirred at 0 °C for 1 hour then at room temperature for 4 hours, concentrated to near dryness, and dissolved in ethyl acetate (50 ml_). The organics were washed with brine, dried over sodium sulfate, and concentrated. The crude reaction mixture was purified by column chromatography (dichloromethane/methanol) to provide the 4-chloro-/V- methylpyridine-2-carboxamide 3a (459 mg, 2.701 mmol, 94%) as a yellow, crystalline solid. ¹H NMR (DMSO-c/₆, 400 MHz): d 2.83 (s, 3H); 7.71 (dd, J = 2.2, 5.1 Hz, 1 H); 8.02 (d, J = 2.2, 1 H); 8.65 (d, J = 5.1 Hz, 1 H); 8.83 (br, 1 H); MS (APCI+, M+1 ) 171 .68.

[0136] The acid chloride 2 (500 mg, 2.87 mmol) was added portion wise to dimethylamine (2.0 M) in tetrahydrofuran (100 ml_) at 0 °C under argon. The mixture was stirred at 0 °C for 1 hour then at room temperature for 4 hours, concentrated to near dryness, and dissolved in ethyl acetate (50 ml_). The organics were washed with brine, dried over sodium sulfate, and concentrated. The crude reaction mixture was purified by column chromatography (dichloromethane/methanol) to provide the 4-chloro-/V,/V- dimethylpyridine-2-carboxamide 3b (459 mg, 2.7 mmol, 94%) as a yellow, crystalline solid. ¹H NMR (DMSO-c/₆, 400 MHz): d 2.83 (s, 6H); 7.71 (dd, J = 2.2, 5.1 Hz, 1 H); 8.02 (d, J = 2.2, 1 H); 8.65 (d, J = 5.1 Hz, 1 H); MS (APCI+, M+1 ) 184.68.

4a, CA-1

Synthesis of 4-(4-aminophenoxy)-N-methylpyndine-2-carboxamide (4a, CA-1 ) and 4-(4-aminophenoxy)-N,N-dimethylpyridine-2-carboxannide (4b) (Scheme 4)

[0137] A solution of 4-aminophenol (256 mg, 2.35 mmol) in dry N,N- dimethylformamide (40 ml_) was treated with potassium te/t-butoxide (274 mg, 2.44 mmol), and the reddish-brown mixture was stirred at room temperature for 1 hour. The contents were treated with 3a and 3b (400 mg, 2.35 mmol) and potassium carbonate (974 mg, 7.05 mmol) and then heated to 80 °C under argon for 4 h. The mixture was cooled to room temperature and poured into ethyl acetate (100 ml_) and brine (400 ml_). The combined organics were washed with brine, dried over sodium sulfate, and concentrated. The crude reaction mixture was purified by column chromatography (dichloromethane/methanol) to afford the desired 4-(4-aminophenoxy)-N- methylpyridine-2-carboxamide 4a (400 mg, 1 .65 mmol, 70%) and 4-(4- aminophenoxy)-N,N-dimethylpyridine-2-carboxamide (69%) 4b after column as a light-brown solid.

[0138] 4-(4-aminophenoxy)-N-methylpyridine-2-carboxamide (70%) 4a: ¹H NMR (DMSO-c/₆, 400 MHz): d 2.79 (d, J = 4.8 Hz, 3H); 5.17 (br, 2H); 6.60-6.90 (m 4H); 7.09 (dd, J = 2.5, 5.5 Hz, 1 H,); 7.38 (d, J = 2.5 Hz, 1 H); 8.49 (d, J = 5.5 Hz, 1 H); 8.76 (br, 1 H); MS (APCI+, M+1 ) 244.18. [0139] 4-(4-aminophenoxy)-N,N-dimethylpyridine-2-carboxamide (69%) 4b: ¹ H NMR (DMSO-c/₆, 400 MHz): d 2.79 (s, 6H); 5.17 (br, 2H); 6.60- 6.90 (m 4H); 7.09 (dd, J = 2.5, 5.5 Hz, 1 H,); 7.38 (d, J = 2.5 Hz, 1 H); 8.49 (d, J = 5.5 Hz, 1 H; MS (APCI+, M+1 ) 257.18.

Synthesis of 4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)phenoxy)-N- methylpyridine-2-carboxamide (6a, SRS13-42) and 4-(4-(3-(4-chloro-3- (trifluoromethyl) phenyl)ureido)phenoxy)-N,N-dinnethylpyridine-2-carboxannide (6b. SRS13-65) (Scheme 4)

SRS 13-42

General procedure A

[0140] To the 4-(4-aminophenoxy)-N-methylpyhdine-2-carboxamide 4a (300 mg, 1 .234 mmol) in methylene chlonde (15 mL) was added a solution of 1 -chloro-4-isocyanato-2-(trifluoromethyl)benzene 5 (273 mg, 1 .235 mmol) in methylene chloride (15 mL) at 0 °C under argon. The mixture was stirred for 17 hours and then filtered. The solids were washed with methylene chloride and dried under vacuum. The crude reaction mixture was purified by column chromatography (dichloromethane/methanol) to afford 6a (515 mg, 1 .109 mmol, 90%) as a white solid. ¹H NMR (DMSOcfe, 400 MHz): d 2.79 (d, J = 4.8 Hz, 3H); 7.19 (m, 3H); 7.41 (d, J = 2.5 Hz, 1 H); 7.67 (m, 4H); 8.13 (d, J = 2.5 Hz, 1 H); 8.48 (d, J = 5.5 Hz, 1 H); 8.78 (br, 1 H); 8.97 (s, 1 H); 9.24 (s, 1 H); MS (APCI+, M+1 ) 465.19. The NMR spectrum of the sorafenib 6a is similar to the previously reported data by Bankston. (Bankston et al., 2002).

SRS13-65

[0141] Similarly, the 4-(4-(3-(4-chloro-3-(trifluoromethyl) phenyl)ureido)phenoxy)-N,N-dimethylpyndine-2-carboxamide (62%) (6b) was prepared starting from the 4-(4-aminophenoxy)-N,N-dimethylpyridine-2- carboxamide 4b. ¹ H NMR (DMSOc/₆, 400 MHz): d 2.79 (s, 6H); 7.19 (m, 3H); 7.41 (d, J = 2.5 Hz, 1 H); 7.67 (m, 4H); 8.13 (d, J = 2.5 Hz, 1 H); 8.48 (d, J = 5.5 Hz, 1 H); 8.97 (s, 1 H); 9.24 (s, 1 H); MS (APCI+, M+1 ) 478.29.

Synthesis of Sorafenib derivatives

Entry R1 R2 R3 R4 R5 X Name

1 F H H CF₃ H 0 SRS 13-43

2 F CF₃ H H H 0 SRS 13-44

3 H CF₃ H CF₃ H 0 SRS13^5

4 CF₃ H Br H H 0 SRS13-46

5 H H H CI H 0 SRS 13-47

6 H H CI H H 0 SRS 13-48

7 CF₃ H CI H H 0 SRS 13-49

8 H H CF₃ H H 0 SRS 13-52

9 CI H H H H 0 SRS13-53

10 H CF₃ H H H 0 SRS 13-54

1 1 H H H CN H 0 SRS 13-57

12 H H H H H 0 SRS 13-59

13 CF₃ H H H H 0 SRS13-67

14 H H CI CF₃ H s SRS 13-60

Table 1 : Synthetic scheme of Sorafenib analogs.

Synthesis of 4-(4-(3-(2-fluoro-5-(tnfluoronnethyl)phenyl)ureido)phenoxy)-/\/- methylpyridine-2-carboxannide (SRS13-43, Table 1 , entry 1 )

[0142] Following the above general procedure A with the 4-(4- aminophenoxy)-/V-nnethylpyhdine-2-carboxannide (30 mg, 0.123 mmol), and 1 - fluoro-2-isocyanato-4-(trifluoromethyl)benzene (26.7 mL, 0.185 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 40:1 ) to provide the desired sorafenib analog (SRS13-43) (44.0 mg, 0.098 mmol, 80%). ¹H NMR (400 MHz, DMSO) δ 9.33 (s, 1 H), 8.94 (d, J = 2.5 Hz, 1 H), 8.79 (d, J = 4.8 Hz, 1 H), 8.68 - 8.60 (m, 1 H), 8.51 (d, J = 5.6 Hz, 1 H), 7.63 - 7.56 (m, 2H), 7.55 - 7.47 (m, 1 H), 7.40 (t, J = 6.3 Hz, 2H), 7.23 - 7.17 (m, 2H), 7.15 (dd, J = 5.6, 2.6 Hz, 1 H), 2.79 (d, J = 4.8 Hz, 3H); MS (APCI+, M+1) 448.96.

Synthesis of 4-(4-(3-(2-fluoro-3-(trifluoromethyl)phenyl)ureido)phenoxy)-/V- methylpyridine-2-carboxamide (SRS13-44, Table 1 , entry 2)

[0143] Following the above general procedure A with the 4-(4- aminophenoxy)-/V-methylpyhdine-2-carboxamide (30 mg, 0.123 mmol), and 2- fluoro-1 -isocyanato-3-(trifluoromethyl)benzene (26.7 μΙ_, 0.185 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 40:1 ) to provide the desired sorafenib analog (SRS13-44) (44.0 mg, 0.098 mmol, 80%).¹H NMR (400 MHz, DMSO, ppm) δ 9.29 (s, 1 H), 8.88 (s, 1 H), 8.78 (d, J = 4.6 Hz, 1 H), 8.49 (dd, J = 13.4, 4.3 Hz, 2H), 7.72 - 7.55 (m, 2H), 7.38 (dd, J = 12.9, 4.4 Hz, 3H), 7.30 - 7.10 (m, 3H), 5.76 (d, J = 1 .1 Hz, 1 H), 2.90 - 2.75 (m, 3H); MS (APCI+, M+1) 448.16.

SRS13-45

Synthesis of 4-(4-(3-(3,5-bis(trifluoronnethyl)phenyl)ureido)phenoxy)-/\/- methylpyridine-2-carboxannide (SRS13-45, Table 1 , entry 3)

[0144] Following the above general procedure A with the 4-(4- aminophenoxy)-/V-nnethylpyhdine-2-carboxannide (30 mg, 0.123 mmol), and 1 - isocyanato-3,5-bis(trifluoromethyl)benzene (26.7 mL, 0.185 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 40:1 ) to provide the desired sorafenib analog (SRS13-45) (52.0 mg, 0.104 mmol, 85%). ¹ H NMR (400 MHz, DMSO) δ 9.47 (s, 1 H), 9.16 (s, 1 H), 8.78 (d, J = 4.8 Hz, 1 H), 8.51 (d, J = 5.6 Hz, 1 H), 8.16 (s, 2H), 7.73 - 7.59 (m, 3H), 7.39 (d, J = 2.5 Hz, 1 H), 7.30 - 7.1 1 (m, 3H), 2.79 (d, J = 4.8 Hz, 3H); MS (APCI+, M+1) 498.36.

Synthesis of 4-(4-(3-(4-bromo-2-(trifluoromethyl)phenyl)ureido)phenoxy)-/\/- methylpyridine-2-carboxamide (SRS13-46, Table 1 , entry 4)

[0145] Following the above general procedure A with the 4-(4- aminophenoxy)-/V-methylpyhdine-2-carboxamide (30 mg, 0.123 mmol), and 4- bromo-1 -isocyanato-2-(trifluoronnethyl)benzene (28.8 mL, 0.185 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 40:1 ) to provide the desired sorafenib analog (SRS13-46) (56.0 mg, 0.1 10 mmol, 89%). ¹ H NMR (400 MHz, DMSO, ppm) δ 9.58 (s, 1 H), 8.79 (d, J = 4.8 Hz, 1 H), 8.51 (d, J = 5.6 Hz, 1 H), 8.20 (s, 1 H), 7.97 (d, J = 9.4 Hz, 1 H), 7.93 - 7.78 (m, 2H), 7.59 (d, J = 8.9 Hz, 2H), 7.39 (d, J = 2.3 Hz, 1 H), 7.28 - 7.1 1 (m, 3H), 2.79 (d, J = 4.8 Hz, 3H); MS (APCI+, M+1) 509.36.

SRS13-47

Synthesis of 4-(4-(3-(3-chlorophenyl)ureido)phenoxy)-/V-methylpyhdine-2- carboxamide (SRS13-47, Table 1 , entry 5)

[0146] Following the above general procedure A with the 4-(4- aminophenoxy)-/V-methylpyhdine-2-carboxamide (30 mg, 0.123 mmol), and 1 - chloro-3-isocyanatobenzene (28.8 mL, 0.185 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 40:1 ) to provide the desired sorafenib analog (SRS13-47) (33.7 mg, 0.1 10 mmol, 69%). ¹H NMR (400 MHz, DMSO) δ 8.93 (d, J = 1 1 .9 Hz, 2H), 8.79 (d, J = 4.8 Hz, 1 H), 8.51 (d, J = 5.6 Hz, 1 H), 7.73 (d, J = 1 .8 Hz, 1 H), 7.69 - 7.55 (m, 2H), 7.38 (d, J = 2.6 Hz, 1 H), 7.37 - 7.27 (m, 2H), 7.16 (ddd, J = 8.9, 6.3, 2.4 Hz, 3H), 7.03 (dt, J = 6.9, 2.1 Hz, 1 H), 2.79 (d, J = 4.8 Hz, 3H): MS (APCI+, M+1) 396.16.

SRS13-48

Synthesis of 4-(4-(3-(4-chlorophenyl)ureido)phenoxy)-/V-methylpyridine-2- carboxamide (SRS13-48, Table 1 . entry 6)

[0147] Following the above general procedure A with the 4-(4- aminophenoxy)-/V-nnethylpyhdine-2-carboxannide (30 mg, 0.123 mmol), and 1 - chloro-4-isocyanatobenzene (22.7 mL, 0.185 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 40:1 ) to provide the desired sorafenib analog (SRS13-48) (36.5 mg, 0.1 10 mmol, 75%). ¹ H NMR (400 MHz, DMSO) δ 8.88 (s, 2H), 8.79 (d, J = 4.8 Hz, 1 H), 8.51 (d, J = 5.6 Hz, 1 H), 7.59 (s, 1 H), 7.57 - 7.47 (m, 3H), 7.46 - 7.29 (m, 3H), 7.28 - 7.12 (m, 3H), 2.79 (d, J = 4.8 Hz, 3H); MS (APCI+, M+1) 396.16.

Synthesis of 4-(4-(3-(4-chloro-2-(trifluoromethyl)phenyl)ureido)phenoxy)-/\/- methylpyridine-2-carboxamide (SRS13-49, Table 1 , entry 7)

[0148] Following the above general procedure A with the 4-(4- aminophenoxy)-/V-methylpyhdine-2-carboxamide (30 mg, 0.123 mmol), and 4- chloro-1 -isocyanato-2-(trifluoromethyl)benzene (22.7 mL, 0.185 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 40:1 ) to provide the desired sorafenib analog (SRS13-49) (46.0 mg, 0.099 mmol, 80%). ¹ H NMR (400 MHz, DMSO) δ 9.59 (s, 1 H), 8.78 (d, J = 4.6 Hz, 1 H), 8.50 (d, J = 5.6 Hz, 1 H), 8.21 (s, 1 H), 8.02 (d, J = 8.5 Hz, 1 H), 7.72 (d, J = 9.1 Hz, 2H), 7.60 (d, J = 8.1 Hz, 2H), 7.40 (s, 1 H), 7.30 - 7.10 (m, 3H), 2.79 (d, J = 4.2 Hz, 3H); MS (APCI+, M+1) 464.16.

SRS 13-52

Synthesis of A/-methyl-4-(4-(3-(4-

(trifluoromethyl)phenyl)ureido)phenoxy)pyridine-2-carboxamide (SRS13-52, Table 1 , entry 8)

[0149] Following the above general procedure A with the 4-(4- aminophenoxy)-A/-methylpyridine-2-carboxamide (30 mg, 0.123 mmol), and 1 - isocyanato-4-(trifluoromethyl)benzene (26.4 ml_, 0.185 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 40:1 ) to provide the desired sorafenib analog (SRS13-52) (44.0 mg, 0.102 mmol, 83%). ¹ H NMR (400 MHz, DMSO) δ 9.20 (s, 1 H), 9.00 (s, 1 H), 8.79 (d, J = 4.7 Hz, 1 H), 8.51 (d, J = 5.6 Hz, 1 H), 7.65 (dt, J = 14.9, 8.8 Hz, 6H), 7.40 (d, J = 2.4 Hz, 1 H), 7.29 - 7.12 (m, 3H), 2.79 (d, J = 4.7 Hz, 3H); MS (APCI+, M+1) 430.19.

Synthesis of 4-(4-(3-(2-chlorophenyl)ureido)phenoxy)-/V-methylpyridine-2- carboxamide (SRS13-53, Table 1 . entry 9)

[0150] Following the above general procedure A with the 4-(4- aminophenoxy)-/V-nnethylpyhdine-2-carboxannide (30 mg, 0.123 mmol), and 1 - chloro-2-isocyanatobenzene (22.3 mL, 0.185 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 40:1 ) to provide the desired sorafenib analog (SRS13-53) (46.4 mg, 0.1 17 mmol, 95%). ¹ H NMR (400 MHz, DMSO) δ 9.94 (s, 1 H), 8.59 - 8.38 (m, 2H), 8.26 - 7.95 (m, 2H), 7.63 (d, J = 8.9 Hz, 2H), 7.61 - 7.42 (m, 2H), 7.42 - 7.25 (m, 1 H), 7.19 (dd, J = 5.9, 2.5 Hz, 3H), 7.16 - 6.98 (m, 1 H), 2.80 (d, J = 4.7 Hz, 3H); MS (APCI+, M+1) 396.18.

SRS 13-54

Synthesis of A/-methyl-4-(4-(3-(3-

(trifluoromethyl)phenyl)ureido)phenoxy)pyridine-2-carboxamide (SRS13-54, Table 1 . entry 10)

[0151] Following the above general procedure A with the 4-(4- aminophenoxy)-N-methylpyridine-2-carboxamide (30 mg, 0.123 mmol), and 1 - chloro-2-isocyanatobenzene (22.3 mL, 0.185 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 40:1 ) to provide the desired sorafenib analog (SRS13-54) (45.0 mg, 0.104 mmol, 85%). 1 H NMR (400 MHz, DMSO) δ 9.16 (s, 1 H), 9.01 (s, 1 H), 8.80 (d, J = 4.8 Hz, 1 H), 8.51 (d, J = 5.6 Hz, 1 H), 8.04 (s, 1 H), 7.61 (d, J = 8.9 Hz, 3H), 7.52 (t, J = 7.9 Hz, 1 H), 7.41 (d, J = 2.5 Hz, 1 H), 7.32 (d, J = 7.6 Hz, 1 H), 7.27 - 7.12 (m, 3H), 5.76 (s, 1 H), 2.80 (d, J = 4.8 Hz, 3H); MS (APCI+, M+1 ) 430.19.

SRS 13-57

Synthesis of 4-(4-(3-(3-cvanophenyl)ureido)phenoxy)-/V-methylpyndine-2- carboxamide (SRS13-57, Table 1 , entry 1 1 )

[0152] Following the above general procedure A with the 4-(4- aminophenoxy)-/V-nnethylpyhdine-2-carboxannide (30 mg, 0.123 mmol), and 3- isocyanatobenzonitrile (26.6 mg, 0.185 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 40:1 ) to provide the desired sorafenib analog (SRS13-57) (41 .0 mg, 0.106 mmol, 86%). ¹ H NMR (400 MHz, DMSO) δ 9.14 (s, 1 H), 9.07 (d, J = 32.6 Hz, 2H), 8.79 (d, J = 4.8 Hz, 1 H), 8.51 (d, J = 5.6 Hz, 1 H), 7.99 (d, J = 1 .4 Hz, 1 H), 7.70 (dd, J = 8.2, 1 .0 Hz, 1 H), 7.61 (s, 1 H), 7.60 - 7.35 (m, 4H), 7.30 - 7.12 (m, 3H), 2.79 (d, J = 4.8 Hz, 3H); MS (APCI+, M+1) 387.39.

SRS13-59

Synthesis of /V-methyl-4-(4-(3-phenylureido)phenoxy)pyhdine-2-carboxamide (SRS13-59. Table 1 , entry 12)

[0153] Following the above general procedure A with the 4-(4- aminophenoxy)-/V-methylpyhdine-2-carboxamide (30 mg, 0.123 mmol), and isocyanatobenzene (25 μΙ_, 0.185 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 40:1 ) to provide the desired sorafenib analog (SRS13-59) (39.0 mg, 0.107 mmol, 87%). ¹ H NMR (400 MHz, DMSO): d 2.79 (d, J = 4.8 Hz, 3H); 7.19 (m, 5H); 7.41 (d, J = 2.5 Hz, 1 H); 7.67 (m, 4H); 8.13 (d, J = 2.5 Hz, 1 H); 8.48 (d, J = 5.5 Hz, 1 H); 8.78 (br, 1 H); 8.97 (s, 1 H); 9.24 (s, 1 H); MS (APCI+, M+1 ) 463.09; MS (APCI+, M+1) 362.19.

Synthesis of A/-methyl-4-(4-(3-(2-

(trifluoromethyl)phenyl)ureido)phenoxy)pyridine-2-carboxamide (SRS13-67, Table 1 . entry 13)

[0154] Following the above general procedure A with the 4-(4- aminophenoxy)-/V-methylpyhdine-2-carboxamide (30 mg, 0.123 mmol), and 1 - isocyanato-2-(trifluoromethyl)benzene (18 ml_, 0.185 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 40:1 ) to provide the desired sorafenib analog (SRS13-67) (51 .0 mg, 0.1 19 mmol, 97%). ¹ H NMR (400 MHz, DMSO) δ 9.52 (s, 1 H), 8.76 (d, J = 4.6 Hz, 1 H), 8.50 (t, J = 5.1 Hz, 1 H), 8.12 (s, 1 H), 7.96 (d, J = 8.1 Hz, 1 H), 7.72 - 7.63 (m, 2H), 7.59 (t, J = 6.8 Hz, 2H), 7.42 - 7.36 (m, 1 H), 7.30 (t, J = 7.2 Hz, 1 H), 7.21 - 7.13 (m, 3H), 2.79 (t, J = 4.7 Hz, 3H); MS (APCI+, M+1) 431 .19.

SRS 13-60

Synthesis of 4-(4-(3-(4-chloro-3-(tnfluoromethyl)phenyl)thioureido)phenoxy)- /V-methylpyridine-2-carboxannide (SRS13-60, Table 1 , entry 14)

[0155] Following the above general procedure A with the 4-(4- aminophenoxy)-/V-nnethylpyhdine-2-carboxannide (30 mg, 0.123 mmol), and 1 - chloro-4-isothiocyanato-2-(trifluoromethyl)benzene (30 mL, 0.185 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 40:1 ) to provide the desired sorafenib analog (SRS13-60) (52.0 mg, 0.108 mmol, 88%). ¹ H NMR (400 MHz, DMSO) δ 10.16 (s, 2H), 8.78 (d, J = 4.8 Hz, 1 H), 8.54 (d, J = 5.6 Hz, 1 H), 8.10 (d, J = 2.4 Hz, 1 H), 7.83 (dd, J = 8.7, 2.4 Hz, 1 H), 7.69 (d, J = 8.7 Hz, 1 H), 7.59 (d, J = 8.8 Hz, 2H), 7.44 (d, J = 2.5 Hz, 1 H), 7.27 - 7.13 (m, 3H), 2.80 (d, J = 4.8 Hz, 3H); MS (APCI+, M+1) 480.79.

Entry R-| R4 R5 Name

o

H H CI CF, O SRS14-31

10 CN H CFo CFo O O SRS14-24

11 H CN CF₃ H CFo O O SRS 14-25

12 H H CFo H CF, O CH, SRS 14-32

13 H H H CI CF, CH, SRS 14-33

14 H H H CI CFo O CH, SRS 14-34

Table 2: Synthetic scheme of Sorafenib analogs.

SRS 13-97

Synthesis of 1 -(4-chloro-3-(trifluoronnethyl)phenyl)-3-(4-phenoxyphenyl)urea (SRS13-97, Table 2, entry 1 )

[0156] Following the above general procedure A with the 4- phenoxyaniline (30 mg, 0.162 mmol), and 1 -chloro-4-isocyanato-2- (trifluoromethyl)benzene (53 mg, 0.243 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 100:1 ) to provide the desired sorafenib analog (SRS13-97) (52.0 mg, 0.128 mmol, 80%). ¹ H NMR (400 MHz, DMSO) δ 9.15 (s, 2H), 8.85 (s, 2H), 8.12 (s, 2H), 7.62 (dd, J = 25.1 , 8.8 Hz, 4H), 7.50 (d, J = 7.5 Hz, 4H), 7.36 (t, J = 7.2 Hz, 4H), 7.09 (t, J = 7.3 Hz, 2H), 6.98 (t, J = 9.5 Hz, 8H); MS (APCI+, M+1) 406.49.

SRS13-98

Synthesis of 1 -(4-chloro-3-(trifluoromethyl)phenyl)-3-(4- phenoxyphenvDthiourea (SRS13-98, Table 2, entry 2)

[0157] Following the above general procedure A with the 4- phenoxyaniline (30 mg, 0.162 mmol), and 1 -chloro-4-isothiocyanato-2- (trifluoromethyl)benzene (53 mg, 0.243 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 100:1 ) to provide the desired sorafenib analog (SRS13-98) (60.0 mg, 0.142 mmol, 88%). ¹ H NMR (400 MHz, DMSO) δ 10.05 (s, 2H), 8.09 (d, J = 2.3 Hz, 1 H), 7.81 (dd, J = 8.7, 2.3 Hz, 1 H), 7.66 (d, J = 8.7 Hz, 1 H), 7.52 - 7.29 (m, 4H), 7.15 (t, J = 7.4 Hz, 1 H), 7.02 (dd, J = 8.7, 2.3 Hz, 4H); MS (APCI+, M+1) 423.1 .

SRS 14-22

Synthesis of 1 -(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(4

cvanophenoxy)phenyl)-urea (SRS14-22, Table 2, entry 3)

[0158] Following the above general procedure A with the 4-(4- aminophenoxy)benzonitrile (25 mg, 0.1 19 mmol), and 1 -chloro-4-isocyanato- 2-(trifluoromethyl)benzene (39.5 mg, 0.178 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 50:1 ) to provide the desired sorafenib analog (SRS14-22) (46.0 mg, 0.106 mmol, 90%). ¹ H NMR (400 MHz, DMSO) δ 9.13 (s, 1 H), 8.81 (s, 1 H), 8.1 1 (d, J = 2.3 Hz, 1 H), 7.63 (dt, J = 15.0, 5.6 Hz, 2H), 7.49 - 7.42 (m, 2H), 7.17 (d, J = 8.3 Hz, 2H), 6.98 - 6.84 (m, 4H); MS (APCI+, M+1) 431 .19.

SRS 14-26 Synthesis of 1 -(4-chloro-3-(trifluoronnethyl)phenyl)-3-(4-(4- cvanophenoxy)phenyl)-thiourea (SRS14-26, Table 2, entry 4)

[0159] Following the above general procedure A with the 4-(4- aminophenoxy)benzonithle (25 mg, 0.1 19 mmol), and 1 -chloro-4- isothiocyanato-2-(trifluoromethyl)benzene (42.0 mg, 0.178 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 50:1 ) to provide the desired sorafenib analog (SRS14-26) (42.0 mg, 0.094 mmol, 79%). ¹ H NMR (400 MHz, DMSO) δ 10.02 (d, J = 15.8 Hz, 2H), 8.08 (s, 1 H), 7.90 - 7.75 (m, 1 H), 7.65 (d, J = 8.7 Hz, 1 H), 7.41 (d, J = 8.8 Hz, 2H), 7.20 (d, J = 8.3 Hz, 2H), 6.94 (dd, J = 14.7, 8.6 Hz, 4H); MS (APCI+, M+1) 447.19.

SRS14-23

Synthesis of 1 -(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(3- cvanophenoxy)phenyl)-urea (SRS14-23, Table 2, entry 5)

[0160] Following the above general procedure A with the 3-(4- aminophenoxy)benzonitrile (25 mg, 0.1 19 mmol), and 1 -chloro-4-isocyanato-

2-(trifluoromethyl)benzene (39.5 mg, 0.178 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 50:1 ) to provide the desired sorafenib analog (SRS14-23) (46.0 mg, 0.106 mmol,

90%).¹ H NMR (400 MHz, DMSO) δ 9.15 (s, 1 H), 8.84 (s, 1 H), 8.1 1 (s, 1 H),

7.63 (dt, J = 14.9, 5.6 Hz, 2H), 7.47 (d, J = 8.9 Hz, 2H), 7.24 (t, J = 7.8 Hz,

1 H), 6.95 (dd, J = 25.4, 8.2 Hz, 3H), 6.87 - 6.70 (m, 2H); MS (APCI+, M+1)

431 .19.

SRS 14-27

Synthesis of 1 -(4-chloro-3-(trifluoronnethyl)phenyl)-3-(4-(3- cvanophenoxy)phenyl)-thiourea (SRS14-27, Table 2, entry 6)

[0161] Following the above general procedure A with the 3-(4- aminophenoxy)benzonithle (25 mg, 0.1 19 mmol), and 1 -chloro-4- isothiocyanato-2-(trifluoromethyl)benzene (42.0 mg, 0.178 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 50:1 ) to provide the desired sorafenib analog (SRS14-27) (42.0 mg, 0.094 mmol, 79%). ¹ H NMR (400 MHz, DMSO) δ 10.03 (s, 2H), 8.08 (s, 1 H), 7.81 (d, J = 8.6 Hz, 1 H), 7.66 (d, J = 8.7 Hz, 1 H), 7.43 (d, J = 8.7 Hz, 2H), 7.27 (t, J = 7.8 Hz, 1 H), 6.98 (dd, J = 12.8, 8.3 Hz, 3H), 6.90 - 6.73 (m, 2H); MS (APCI+, M+1) 447.19.

SRS14-30

Synthesis of 1 -(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(3- morpholinophenoxy)phenyl) urea (SRS14-30, Table 2, entry 7)

[0162] Following the above general procedure A with the 4-(3- morpholinophenoxy)aniline (37 mg, 0.137 mmol), and 1 -chloro-4-isocyanato- 2-(trifluoromethyl)benzene (45.0 mg, 0.205 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 50:1 ) to provide the desired sorafenib analog (SRS14-30) (45.0 mg, 0.094 mmol, 79%). ¹H NMR (400 MHz, DMSO) δ 9.13 (s, 1 H), 8.82 (s, 1 H), 8.1 1 (d, J = 2.3 Hz, 1 H), 7.62 (dd, J = 15.2, 5.5 Hz, 2H), 7.47 (d, J = 9.0 Hz, 2H), 7.18 (t, J = 8.2 Hz, 1 H), 6.97 (d, J = 8.9 Hz, 2H), 6.68 (dd, J = 8.3, 2.2 Hz, 1 H), 6.57 (t, J = 2.2 Hz, 1 H), 6.34 (dd, J = 7.9, 1 .9 Hz, 1 H), 3.76 - 3.66 (m, 4H), 3.13 - 3.02 (m, 4H); MS (APCI+, M+1) 491 .19.

SRS14-31

Synthesis of 1 -(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(3

morpholinophenoxy)-phenyl) thiourea (SRS14-31 , Table 2, entry 8)

[0163] Following the above general procedure A with the 4-(3- morpholinophenoxy)aniline (30 mg, 0.1 1 1 mmol), and 1 -chloro-4- isothiocyanato-2-(trifluoromethyl)benzene (39.0 mg, 0.166 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 40:1 ) to provide the desired sorafenib analog (SRS14-31) (46.0 mg, 0.090 mmol, 82%). ¹ H NMR (400 MHz, DMSO) δ 10.05 (s, 1 H), 9.99 (s, 1 H), 8.08 (d, J = 2.4 Hz, 1 H), 7.81 (dd, J = 8.7, 2.5 Hz, 1 H), 7.66 (d, J = 8.7 Hz, 1 H), 7.42 (d, J = 8.8 Hz, 2H), 7.22 (t, J = 8.2 Hz, 1 H), 7.02 - 6.95 (m, 2H), 6.74 (dd, J = 8.3, 2.2 Hz, 1 H), 6.62 (t, J = 2.2 Hz, 1 H), 6.39 (dd, J = 8.0, 2.0 Hz, 1 H), 3.76 - 3.66 (m, 4H), 3.15 - 3.05 (m, 4H); MS (APCI+, M+1) 507.19.

SRS14-29

Synthesis of 1 -(3,5-bis(trifluoromethyl)phenyl)-3-(4-(3-morpholinophenoxy)- phenvDurea (SRS14-29, Table 2, entry 9)

[0164] Following the above general procedure A with the 4-(3- morpholinophenoxy)aniline (30 mg, 0.1 1 1 mmol), and 1 -isocyanato-3,5- bis(trifluoromethyl)benzene (42.0 mg, 0.166 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 40:1 ) to provide the desired sorafenib analog (SRS14-29) (52.0 mg, 0.090 mmol, 89%). ¹ H NMR (400 MHz, DMSO) δ 9.37 (s, 1 H), 8.97 (s, 1 H), 8.14 (s, 2H), 7.62 (s, 1 H), 7.48 (d, J = 8.8 Hz, 2H), 7.19 (t, J = 8.2 Hz, 1 H), 6.98 (d, J = 8.8 Hz, 2H), 6.69 (d, J = 8.3 Hz, 1 H), 6.57 (s, 1 H), 6.35 (d, J = 8.0 Hz, 1 H), 3.77 - 3.65 (m, 4H), 3.14 - 3.02 (m, 4H); MS (APCI+, M+1) 525.29.

SRS14-24

Synthesis of 1 -(3,5-bis(trifluoromethyl)phenyl)-3-(4-(4- cvanophenoxy)phenyl)urea (SRS14-24, Table 2, entry 10)

[0165] Following the above general procedure A with the 4-(4- aminophenoxy)benzonitrile (25 mg, 0.1 19 mmol), and 1 -isocyanato-3,5- bis(trifluoromethyl)benzene (45.5 mg, 0.178 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 50:1 ) to provide the desired sorafenib analog (SRS14-24) (49.0 mg, 0.105 mmol, 88%). ¹ H NMR (400 MHz, DMSO) δ 9.37 (s, 1 H), 8.96 (s, 1 H), 8.14 (s, 2H), 7.62 (s, 1 H), 7.48 (d, J = 7.0 Hz, 2H), 7.17 (d, J = 6.9 Hz, 2H), 7.00 - 6.76 (m, 4H); MS (APCI+, M+1) 466.05.

SRS14-25

Synthesis of 1 -(3,5-bis(trifluoromethyl)phenyl)-3-(4-(3- cvanophenoxy)phenyl)urea (SRS14-25, Table 2, entry 1 1 )

[0166] Following the above general procedure A with the 3-(4- aminophenoxy)benzonitrile (25 mg, 0.1 19 mmol), and 1 -isocyanato-3,5- bis(trifluoromethyl)benzene (30.0 mg, 0.178 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 50:1 ) to provide the desired sorafenib analog (SRS14-25) (49.0 mg, 0.099 mmol, 89%). ¹ H NMR (400 MHz, DMSO) δ 9.42 (s, 1 H), 9.02 (s, 1 H), 8.15 (s, 2H), 7.64 (s, 1 H), 7.50 (d, J = 8.9 Hz, 2H), 7.25 (t, J = 7.7 Hz, 1 H), 6.99 (d, J = 8.8 Hz, 2H), 6.92 (d, J = 7.2 Hz, 1 H), 6.82 - 6.70 (m, 2H); MS (APCI+, M+1) 465.29.

SRS 14-32 Synthesis of 1 -(4-benzylphenyl)-3-(3,5-bis(tnfluoronnethyl)phenyl)urea

(SRS14-32, Table 2, entry 12)

[0167] Following the above general procedure A with the 4- benzylaniline (30 mg, 0.163 mmol), and 1 -isocyanato-3,5- bis(trifluoromethyl)benzene (63.0 mg, 0.245 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 100:1 ) to provide the desired sorafenib analog (SRS14-32) (64.0 mg, 0.146 mmol, 89%). ¹ H NMR (400 MHz, DMSO) δ 9.35 (s, 1 H), 8.91 (s, 1 H), 8.12 (s, 2H), 7.62 (s, 1 H), 7.43 - 7.1 1 (m, 9H), 3.89 (s, 2H); MS (APCI+, M+1) 438.19.

SRS14-33

Synthesis of 1 -(4-benzylphenyl)-3-(3,5-bis(thfluoromethyl)phenyl thiourea (SRS14-33. Table 2, entry 13)

[0168] Following the above general procedure A with the 4- benzylaniline (30 mg, 0.163 mmol), and 1 -chloro-4-isothiocyanato-2- (trifluoromethyl)benzene (58.0 mg, 0.245 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 100:1 ) to provide the desired sorafenib analog (SRS14-33) (60.0 mg, 0.142 mmol, 87%). 1 H NMR (400 MHz, DMSO) δ 10.06 (s, 1 H), 10.01 (s, 1 H), 8.09 (d, J = 2.5 Hz, 1 H), 7.80 (dd, J = 8.7, 2.4 Hz, 1 H), 7.65 (d, J = 8.7 Hz, 1 H), 7.41 - 7.15 (m, 9H); MS (APCI+, M+1 ) 420.29.

SRS14-34 Synthesis of 1 -(4-benzylphenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea (SRS14-34, Table 2, entry 14)

[0169] Following the above general procedure A with the 4- benzylaniline (30 mg, 0.163 mmol), and 1 -chloro-4-isocyanato-2- (trifluoromethyl)benzene (58.0 mg, 0.245 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 100:1 ) to provide the desired sorafenib analog (SRS14-34) (60.0 mg, 0.150 mmol, 92%). ¹ H NMR (400 MHz, DMSO) δ 9.1 1 (s, 1 H), 8.76 (s, 1 H), 8.1 1 (s, 1 H), 7.61 (dd, J = 22.7, 8.8 Hz, 2H), 7.39 (d, J = 8.1 Hz, 2H), 7.22 (ddd, J = 33.1 , 17.7, 7.7 Hz, 7H), 3.88 (s, 2H); MS (APCI+, M+1) 405.19.

Entry R R₂ R4 Y Name

11 Et CI CF, SRS15-37

Table 3: Synthetic scheme of Sorafenib analogs.

SRS14-04

Synthesis of 1-(3,5-bis(trifluoromethyl)phenyl)-3-(4-morpholinophenyl)urea (SRS14-04, Table 3. entry 1)

[0170] Following the above general procedure A with the 4- morpholinoaniline (50 mg, 0.280 mmol), and 1 -isocyanato-3,5- bis(trifluoromethyl)benzene (107.4 mg, 0.421 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 100:1 ) to provide the desired sorafenib analog (SRS14-04) (103.0 mg, 0.237 mmol, 85%). ¹ H NMR (400 MHz, DMSO) δ 9.29 (s, 1 H), 8.70 (s, 1 H), 8.13 (s, 2H), 7.58 (s, 1 H), 7.35 (d, J = 9.0 Hz, 2H), 6.90 (d, J = 9.0 Hz, 2H), 3.38 (s, 4H), 3.03 (d, J = 4.6 Hz, 4H); MS (APCI+, M+1) 433.28.

SRS14-05

Synthesis of 1 -(4-chloro-3-(trifluoromethyl)phenyl)-3-(4- morpholinophenvDurea (SRS14-05, Table 3, entry 2)

[0171] Following the above general procedure A with the 4- morpholinoaniline (50 mg, 0.280 mmol), and 1 -chloro-4-isocyanato-2- (trifluoromethyl)benzene (93.0 mg, 0.421 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 100:1 ) to provide the desired sorafenib analog (SRS14-05) (94.0 mg, 0.235 mmol, 84%). ¹H NMR (400 MHz, DMSO) δ 9.05 (s, 1 H), 8.57 (s, 1 H), 8.1 1 (s, 1 H), 7.60 (dd, J = 22.2, 8.8 Hz, 2H), 7.33 (d, J = 8.9 Hz, 2H), 6.89 (d, J = 8.9 Hz, 2H), 3.38 (s, 4H), 3.10 - 2.98 (m, 4H); MS (APCI+, M+1) 399.19.

SRS14-10

Synthesis of 1 -(3,5-bis(trifluoromethyl)phenyl)-3-(4-(pyridin-4- ylmethyl)phenyl)-urea (SRS14-10, Table 3, entry 3)

[0172] Following the above general procedure A with the 4-(pyridin-4- ylmethyl)aniline (30 mg, 0.163 mmol), and 1 -isocyanato-3,5- bis(trifluoromethyl)benzene (62.0 mg, 0.244 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 40:1 ) to provide the desired sorafenib analog (SRS14-10) (65.0 mg, 0.148 mmol, 90%). ¹H NMR (400 MHz, DMSO) δ 9.37 (s, 1 H), 8.94 (s, 1 H), 8.46 (d, J = 5.1 Hz, 2H), 8.13 (s, 2H), 7.60 (s, 1 H), 7.44 (d, J = 8.2 Hz, 2H), 7.21 (dd, J = 16.0, 6.7 Hz, 4H), 3.91 (s, 2H); MS (APCI+, M+1) 439.19.

SRS14-11

Synthesis of 1 -(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(pyridin-4-ylmethyl)- phenvDurea (SRS14-1 1 , Table 3, entry 4)

[0173] Following the above general procedure A with the 4-(pyridin-4- ylmethyl)aniline (30 mg, 0.163 mmol), and 1 -chloro-4-isocyanato-2- (trifluoromethyl)benzene (54.0 mg, 0.244 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 40:1 ) to provide the desired sorafenib analog (SRS14-11) (61 .0 mg, 0.150 mmol, 92%). ¹H NMR (400 MHz, DMSO) δ 9.13 (s, 1 H), 8.80 (s, 1 H), 8.50 - 8.36 (m, 2H), 8.10 (s, 1 H), 7.61 (s, 2H), 7.40 (d, J = 8.5 Hz, 2H), 7.21 (dd, J = 23.1 , 7.1 Hz, 4H), 3.92 (s, 2H); MS (APCI+, M+1) 406 .01 .

SRS14-12

Synthesis of 1 -(3,5-bis(trifluoromethyl)phenyl)-3-(6-morpholinopyridin-3- vDurea (SRS14-12, Table 3, entry 5)

[0174] Following the above general procedure A with the 6- morpholinopyridin-3-amine (25 mg, 0.139 mmol), and 1 -isocyanato-3,5- bis(trifluoromethyl)benzene (53.0 mg, 0.209 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 20:1 ) to provide the desired sorafenib analog (SRS14-12) (57.0 mg, 0.150 mmol, 95%). ¹H NMR (400 MHz, DMSO) δ 9.40 (s, 1 H), 8.74 (s, 1 H), 8.19 (d, J = 2.5 Hz, 1 H), 8.14 (s, 2H), 7.73 (dd, J = 9.0, 2.7 Hz, 1 H), 7.62 (s, 1 H), 6.84 (d, J = 9.0 Hz, 1 H), 3.77 - 3.64 (m, 4H), 3.38 (d, J = 5.0 Hz, 4H); MS (APCI+, M+1) 435.2.

SRS14-13 Synthesis of 1 -(4-chloro-3-(trifluoromethyl)phenyl)-3-(6-morpholinopyridin-3- vDurea (SRS14-13, Table 3, entry 6)

[0175] Following the above general procedure A with the 6- morpholinopyhdin-3-amine (25 mg, 0.139 mmol), and 1 -chloro-4-isocyanato- 2-(trifluoromethyl)benzene (46.0 mg, 0.209 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 20:1 ) to provide the desired sorafenib analog (SRS14-13) (50.0 mg, 0.150 mmol, 90%). ¹H NMR (400 MHz, DMSO) δ 9.16 (s, 1 H), 8.59 (s, 1 H), 8.18 (d, J = 2.6 Hz, 1 H), 8.09 (d, J = 2.4 Hz, 1 H), 7.71 (dd, J = 9.0, 2.7 Hz, 1 H), 7.62 (dd, J = 16.4, 5.6 Hz, 2H), 6.83 (d, J = 9.1 Hz, 1 H), 3.76 - 3.64 (m, 4H), 3.37 (d, J = 5.0 Hz, 4H); MS (APCI+, M+1) 435.1 1 .

SRS 14-66

Synthesis of 1 -(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(morpholinomethyl)- phenvDurea (SRS14-66, Table 3, entry 7)

[0176] Following the above general procedure A with the 4- (morpholinomethyl)aniline (25 mg, 0.130 mmol), and 1 -chloro-4-isocyanato-2- (trifluoromethyl)benzene (46.0 mg, 0.195 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 20:1 ) to provide the desired sorafenib analog (SRS14-66) (47.0 mg, 0.1 13 mmol, 88%). ¹H NMR (400 MHz, CDCI₃) δ 9.65 (s, 1 H), 9.33 (s, 1 H), 8.63 (s, 1 H), 8.21 - 8.10 (m, 2H), 7.93 (d, J = 8.3 Hz, 2H), 7.74 (d, J = 8.2 Hz, 2H), 4.09 (d, J = 3.8 Hz, 4H), 3.91 (s, 2H), 2.85 (s, 4H); MS (APCI+, M+1) 413 .29.

SRS 14-67

Synthesis of 1 -(3,5-bis(trifluoromethyl)phenyl)-3-(4-(morpholinomethyl)- phenvDurea (SRS14-67, Table 3, entry 8)

[0177] Following the above general procedure A with the 4- (morpholinomethyl)aniline (25 mg, 0.130 mmol), and 1 -isocyanato-3,5- bis(trifluoromethyl)benzene (49.7 mg, 0.195 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 20:1 ) to provide the desired sorafenib analog (SRS14-67) (51 .0 mg, 0.1 13 mmol, 87%). ¹ H NMR (400 MHz, CDCI₃) δ 9.93 (s, 1 H), 9.51 (s, 1 H), 8.66 (s, 2H), 8.17 (s, 1 H), 7.97 (d, J = 7.4 Hz, 2H), 7.76 (d, J = 8.0 Hz, 2H), 4.10 (s, 4H), 3.94 (s, 2H), 2.87 (s, 4H); MS (APCI+, M+1) 447 .29.

SRS15-35

Synthesis of 1 -(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-hvdroxyphenyl)urea (SRS15-35. Table 3. entry 9)

[0178] Following the above general procedure A with the 4- aminophenol (30 mg, 0.275 mmol), and 1 -chloro-4-isocyanato-2- (trifluoromethyl)benzene (72.9 mg, 0.330 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 20:1 ) to provide the desired sorafenib analog (SRS15-35) (72.0 mg, 0.218 mmol, 79%). ¹ H NMR (400 MHz, DMSO) δ 10.67 (s, 20H), 9.19 (s, 20H), 8.93 (s, 21 H), 8.09 (d, J = 23.7 Hz, 40H), 7.77 (d, J = 8.7 Hz, 20H), 7.51 (d, J = 8.8 Hz, 39H), 7.45 - 7.41 (m, 1 H), 7.18 (d, J = 8.7 Hz, 40H); MS (APCI+, M+1) 331 .29.

SRS15-36

Synthesis of 1 -(3,5-bis(trifluoronnethyl)phenyl)-3-(4-hvdroxyphenyl)urea (SRS15-36. Table 3. entry 10)

[0179] Following the above general procedure A with the 4- aminophenol (30 mg, 0.275 mmol), and 1 -isocyanato-3,5- bis(trifluoromethyl)benzene (84.0 mg, 0.330 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 20:1 ) to provide the desired sorafenib analog (SRS15-36) (70.0 mg, 0.197 mmol, 70%). ¹ H NMR (400 MHz, DMSO) δ 10.91 (s, 1 H), 9.47 (s, 1 H), 9.12 (s, 1 H), 8.16 (s, 4H), 7.77 (s, 1 H), 7.64 (s, 1 H), 7.55 (d, J = 8.9 Hz, 2H), 7.22 (d, J = 8.9 Hz, 2H); MS (APCI+, M+1) 366.10.

SRS15-37 Synthesis of 1 -(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-ethylphenyl)urea (SRS15-37, Table 3. entry 1 1 )

[0180] Following the above general procedure A with the 4-ethylaniline (30 mg, 0.247 mmol), and 1 -chloro-4-isocyanato-2-(trifluoromethyl)benzene (65.8 mg, 0.297 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 100:1 ) to provide the desired sorafenib analog (SRS15-37) (76.0 mg, 0.218 mmol, 90%). ¹ H NMR (400 MHz, DMSO) δ 9.12 (s, 1 H), 8.74 (s, 1 H), 8.1 1 (d, J = 2.0 Hz, 1 H), 7.61 (dd, J = 8.9, 5.5 Hz, 2H), 7.37 (d, J = 8.4 Hz, 2H), 7.13 (d, J = 8.4 Hz, 2H), 2.66 - 2.52 (m, 2H), 1 .16 (t, J = 7.6 Hz, 3H); MS (APCI+, M+1) 342.19.

SRS 15-38

Synthesis of 1 -(3,5-bis(trifluoromethyl)phenyl)-3-(4-ethylphenyl)urea (SRS15- 38, Table 3, entry 12)

[0181] Following the above general procedure A with the 4-ethylaniline (30 mg, 0.275 mmol), and 1 -isocyanato-3,5-bis(trifluoromethyl)benzene (75.8 mg, 0.297 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 20:1 ) to provide the desired sorafenib analog (SRS15-38) (81 .0 mg, 0.215 mmol, 87%). ¹ H NMR (400 MHz, DMSO) δ 9.35 (s, 1 H), 8.88 (s, 1 H), 8.13 (s, 2H), 7.63 (s, 1 H), 7.39 (d, J = 8.5 Hz, 2H), 7.14 (d, J = 8.3 Hz, 2H), 2.56 (q, J = 7.6 Hz, 2H), 1 .23 - 1 .12 (m, 3H); MS (APCI+, M+1) 376.19.

Synthesis of 1 -(3,5-bis(trifluoromethyl)phenyl)-3-(2-fluoro-4- hvdroxyphenvDurea (SRS15-39, Table 3, entry 13)

[0182] Following the above general procedure A with the 4-amino-3- fluorophenol (30 mg, 0.235 mmol), and 1 -isocyanato-3,5- bis(trifluoromethyl)benzene (72.0 mg, 0.283 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 20:1 ) to provide the desired sorafenib analog (SRS15-39) (64.0 mg, 0.167 mmol, 71 %). ¹ H NMR (400 MHz, DMSO) δ 9.71 (s, 1 H), 9.54 (s, 1 H), 8.36 (s, 1 H), 8.1 1 (s, 2H), 7.58 (dd, J = 18.6, 9.4 Hz, 2H), 6.73 - 6.50 (m, 2H); MS (APCI+, M+1) 383.19.

Synthesis of 1 -(4-chloro-3-(trifluoromethyl)phenyl)-3-(2-fluoro-4- hvdroxyphenvDurea (SRS15-40, Table 3, entry 14)

[0183] Following the above general procedure A with the 4-amino-3- fluorophenol (30 mg, 0.235 mmol), and 1 -chloro-4-isocyanato-2- (trifluoromethyl)benzene (72.0 mg, 0.283 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 20:1 ) to provide the desired sorafenib analog (SRS15-40) (62.0 mg, 0.178 mmol, 76%). ¹ H NMR (400 MHz, DMSO) δ 9.67 (s, 1 H), 9.31 (s, 1 H), 8.25 (s, 1 H), 8.10 (s, 1 H), 7.62 (d, J = 14.3 Hz, 3H), 6.61 (dd, J = 20.7, 10.6 Hz, 2H); MS (APCI+, M+1) 349.10.

Table 4: Synthetic scheme of Sorafenib analogs.

SRS 14-35

Synthesis of 4-(4-(3-cvclohexylureido)phenoxy)-/V-methylpyridine-2- carboxamide (SRS14-35, Table 4, entry 1 )

[0184] Following the above general procedure A with the 4-(4- arninophenoxy)-/V-methylpyhdine-2-carboxamide (30 mg, 0.123 mmol), and isocyanatocydohexane (19.0 μg, 0.148 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 20:1 ) to provide the desired sorafenib analog (SRS15-35) (38.0 mg, 0.103 mmol, 84%). ¹ H NMR (400 MHz, DMSO) δ 8.75 (d, J = 4.7 Hz, 1 H), 8.53 - 8.36 (m, 2H), 7.55 - 7.40 (m, 2H), 7.37 (d, J = 2.5 Hz, 1 H), 7.15 - 6.99 (m, 3H), 6.1 1 (d, J = 7.8 Hz, 1 H), 1 .81 (d, J = 12.0 Hz, 2H), 1 .67 (dd, J = 8.8, 3.9 Hz, 2H), 1 .55 (d, J = 12.1 Hz, 1 H), 1 .37 - 1 .26 (m, 2H), 1 .24 - 1 .13 (m, 3H); MS (APCI+, M+1) 369.32.

SRS13-100

Synthesis of 4-(4-(3-cvclohexylthioureido)phenoxy)-A/-methylpyridine-2- carboxamide (SRS13-100, Table 4, entry 2)

[0185] Following the above general procedure A with the 4-(4- aminophenoxy)-/V-nnethylpyhdine-2-carboxannide (30 mg, 0.123 mmol), and isothiocyanatocyclohexane (21 .0 μg, 0.148 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 20:1 ) to provide the desired sorafenib analog (SRS13-100) (39.0 mg, 0.101 mmol, 82%). ¹ H NMR (400 MHz, DMSO) δ 9.41 (s, 1 H), 8.77 (d, J = 4.3 Hz, 1 H), 8.52 (d, J = 5.4 Hz, 1 H), 7.74 (d, J = 6.9 Hz, 1 H), 7.59 (d, J = 8.6 Hz, 2H), 7.41 (d, J = 2.2 Hz, 1 H), 7.24 - 7.03 (m, 3H), 1 .93 (d, J = 9.5 Hz, 2H), 1 .77 - 1 .54 (m, 3H), 1 .35 - 1 .14 (m, 5H); MS (APCI+, M+1) 384.19.

Entry Ri R₂ Name

1 CF₃ H CF₃ SRS14-95

2 CF₃ CI H SRS15-11

Table 5: Synthetic scheme of Sorafenib analogs.

General procedure B (Amide bound formation, Table 5)

[0186] To the 3,5-bis(trifluoromethyl)benzoic acid or the 4-chloro-3- (trifluoromethyl)benzoic acid dissolved in dichloromethane, was added oxalyl chloride (1 .5 equiv.) and drops of dimethylformamide at 0 °C. The reaction mixture was stirred at room temperature for 4 hours then all solvents were removed under vaccuo. The desired 4-chloro-3-(trifluoromethyl)benzoyl chloride and 3,5-bis(trifluoromethyl)benzoyl chloride were used without further purification. To the 4-chloro-3-(trifluoromethyl)benzoyl chloride or the 3,5- bis(trifluoromethyl)benzoyl chloride in dry dichloromethane was added the 4- (4-aminophenoxy)-/V-methylpyridine-2-carboxamide (1 equiv.) and diisopropylethylamine (DIPEA) (3 equiv.). The mixture was stirred at room temperature for 17 hours then the solvent was concentrated under vaccuo. The crude reaction mixture was purified by column chromatography (dichloromethane/methanol) to provide the 4-(4-(3,5-bis-(trifluoromethyl)- phenylamidophenoxy)-/V-methylpyridine-2-carboxamide (SRS14-95) and 4-(4- (4-chloro-3-(trifluoromethyl)phenylamido)phenoxy)-/V-methylpyridin carboxamide (SRS15-11 ), respectively.

Synthesis of 4-(4-(3,5-bis(trifluoromethyl)phenylamidophenoxy)-/\/- methylpyridine-2-carboxamide (SRS14-95, Table 5, entry 1 )

[0187] Following the above general procedure B with the 4-(4- aminophenoxy)-/V-nnethylpyhdine-2-carboxannide (30 mg, 0.123 mmol), and 3,5-bis(trifluoromethyl)benzoic acid (51 .0 mg, 0.185 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 20:1 ) to provide the desired sorafenib analog (SRS14-95) (44.0 mg, 0.091 mmol, 74%). ¹ H NMR (400 MHz, CDCI₃) δ 9.89 (s, 1 H), 8.40 (dd, J = 16.7, 1 1 .9 Hz, 3H), 8.12 (d, J = 4.2 Hz, 1 H), 7.95 (s, 1 H), 7.89 - 7.68 (m, 2H), 7.45 (s, 1 H), 7.18 - 6.91 (m, 3H), 5.46 - 5.27 (m, 1 H), 2.95 (t, J = 4.5 Hz, 3H). MS (APCI+, M+1) 483.19.

Synthesis of 4-(4-(4-chloro-3-(tnfluoromethyl)phenylamido)phenoxy)-/V- methylpyridine-2-carboxamide (SRS15-1 1 , Table 5, entry 2)

[0188] Following the above general procedure B with the 4-(4- aminophenoxy)-/V-nnethylpyhdine-2-carboxannide (30 mg, 0.123 mmol), and 4- chloro-3-(trifluoromethyl)benzoic acid (44.0 mg, 0.185 mmol), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 20:1 ) to provide the desired sorafenib analog (SRS15-11 ) (41 .0 mg, 0.092 mmol, 74%). ¹ H NMR (400 MHz, DMSO) δ 10.66 (s, 1 H), 8.77 (s,

1 H), 8.52 (d, J = 5.4 Hz, 1 H), 8.41 (s, 1 H), 8.28 (d, J = 7.4 Hz, 1 H), 7.92 (t, J =

8.8 Hz, 3H), 7.42 (d, J = 1 .8 Hz, 1 H), 7.26 (d, J = 8.7 Hz, 2H), 7.20 - 7.12 (m,

1 H), 2.80 (d, J = 4.5 Hz, 3H); MS (APCI+, M+1) 449.19.

Entry Ri R₂ R₃ Name

1 CF₃ H CF₃ SRS 14-96

2 CF₃ CI H SRS14-97

Table 6: Synthetic scheme of Sorafenib analogs.

General procedure C (Sulfonamide synthesis, Table 6)

[0189] To the 3,5-bis(trifluoromethyl)benzene-1 -sulfonyl chloride or the 4-chloro-3-(trifluoromethyl)benzene-1 -sulfonyl chloride in dry dichloromethane was added the 4-(4-aminophenoxy)-/V-methylpyridine-2-carboxamide (1 equiv.) and diisopropylethylamine (DIPEA) (3 equiv.). The mixture was stirred at room temperature for 17 hours then the solvent was concentrated under vaccuo. The crude reaction mixture was purified by column chromatography (dichloromethane/methanol) to provide the 4-(4-(3,5- bis(trifluoromethyl)phenylsulfonamido)-phenoxy)-/V-methylpyridine-2- carboxamide (SRS14-96) and 4-(4-(4-chloro-3-(trifluoromethyl)- phenylsulfonamido)phenoxy)-/\/-methylpyridine-2-carboxamide (SRS14-97) respectively.

Synthesis of 4-(4-(3,5-bis(thfluoromethyl)phenylsulfonamido)-phenoxy)-/\/- methylpyridine-2-carboxamide (SRS14-96, Table 6, entry 1 )

[0190] Following the above general procedure C with the 4-(4- aminophenoxy)-/V-nnethylpyhdine-2-carboxannide (30 mg, 0.123 mmol), 3,5- bis(trifluoromethyl)benzene-1 -sulfonyl chloride (57.7 mg, 0.185 mmol) and diisopropylethylannine (DIPEA), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 20:1 ) to provide the desired sorafenib analog (SRS14-96) (51 .0 mg, 0.098 mmol, 80%). ¹H NMR (400 MHz, CDCIs) δ 8.42 (s, 3H), 8.25 (s, 2H), 8.03 (s, 1 H), 7.29 (dd, J = 6.0, 2.8 Hz, 1 H), 7.18 (dd, J = 5.8, 3.0 Hz, 2H), 7.1 1 - 7.02 (m, 3H), 3.06 (dd, J = 6.2, 2.7 Hz, 3H); MS (APCI+, M+1) 519.19.

Synthesis of 4-(4-(4-chloro-3-(tnfluoromethyl)phenylamido)phenoxy)-/V- methylpyridine-2-carboxamide (SRS14-97, Table 6, entry 2)

[0191] Following the above general procedure C with the 4-(4- aminophenoxy)-/V-nnethylpyhdine-2-carboxannide (30 mg, 0.123 mmol), 4- chloro-3-(trifluoromethyl)benzene-1 -sulfonyl chloride (51 .6 mg, 0.185 mmol) and diisopropylethylannine (DIPEA), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 20:1 ) to provide the desired sorafenib analog (SRS14-97) (50.0 mg, 0.102 mmol, 83%). ¹ H NMR (400 MHz, CDCIs) δ 8.50 (d, J = 5.5 Hz, 1 H), 8.28 (s, 2H), 8.1 1 - 8.03 (m, 3H), 7.82 (dd, J = 18.2, 5.3 Hz, 4H), 7.28 (s, 1 H), 7.16 - 7.07 (m, 5H), 3.06 (d, J = 5.0 Hz, 4H); MS (APCI+, M+1) 486.19.

Table 7: Synthetic scheme of Sorafenib analogs.

General procedure D (reductive amination reaction, Table 7) (Abdel-Magid, et al.. 1996)

[0192] The 4-(4-aminophenoxy)-/V-methylpyridine-2-carboxamide (1 equiv) and 3,5-bis(trifluoromethyl)-benzaldehyde (2 equiv) or 4-chloro-3- (trifluoromethyl)benzaldehyde (2 equiv) were dissolved in dichloroethane (DCE) and stirred at room temperature in the presence of molecular sieve (4 A), sodium triacetoxyborohydride (NaBH(OAc)s) (1 .6 equiv) and drops of acetic acid (AcOH). The reaction mixture was stirred at room temperature under a nitrogen atmosphere for 17 hours. The reaction mixture was quenched with aqueous saturated NaHCO3, and the product was extracted with EtOAc. The EtOAc extract was dried (MgSO₄), and the solvent was evaporated. The residue was purified by flash-column chromatography on silica gel to provide the desired 4-(4-(3,5- bis(trifluoromethyl)benzylamino)phenoxy)-/V-methylpyridine-2-carboxamide (SRS14-99) or the 4-(4-(4-chloro-3-(trifluoromethyl)benzylamino)phenoxy)-/V- methylpyridine-2-carboxamide (SRS14-98).

Synthesis of 4-(4-(3,5-bis(trifluoromethyl)benzylamino)phenoxy)-/V- methylpyridine-2-carboxamide (SRS14-99, Table 7, entry 1 )

[0193] Following the above general procedure D with the 4-(4- aminophenoxy)-/V-methylpyhdine-2-carboxamide (50 mg, 0.205 mmol), 3,5- bis(trifluoromethyl)benzaldehyde (59.0 μg, 0.410 mmol) and diisopropylethylannine (DIPEA), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 20:1 ) to provide the desired sorafenib analog (SRS14-99) (64.0 mg, 0.136 mmol, 66%). ¹H NMR (400 MHz, CDCIs) δ 8.60 (d, J = 3.0 Hz, 1 H), 8.40 (d, J = 5.6 Hz, 2H), 8.00 (s, 2H), 7.87 (s, 1 H), 7.34 (dd, J = 5.0, 2.7 Hz, 2H), 7.17 (dd, J = 8.2, 2.5 Hz, 2H), 6.95 - 6.90 (m, 2H), 4.50 (s, 2H), 3.03 - 3.01 (m, 3H); MS (APCI+, M+1) 469.19.

Synthesis of 4-(4-(4-chloro-3-(tnfluoromethyl)benzylamino)phenoxy)-/\/- methylpyridine-2-carboxamide (SRS14-98, Table 7, entry 2)

[0194] Following the above general procedure D with the 4-(4- aminophenoxy)-/V-nnethylpyhdine-2-carboxannide (50 mg, 0.205 mmol), 4- chloro-3-(trifluoromethyl)benzaldehyde (68.0 μg, 0.410 mmol) and diisopropylethylannine (DIPEA), the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 20:1 ) to provide the desired sorafenib analog (SRS14-98) (53.0 mg, 0.121 mmol, 59%). ¹ H NMR (400 MHz, CDCIs) δ 8.03 (s, 2H), 7.70 (d, J = 28.4 Hz, 3H), 7.52 (s, 1 H), 7.36 - 7.25 (m, 2H), 7.15 (d, J = 8.4 Hz, 1 H), 6.92 (d, J = 8.5 Hz, 2H), 6.62 (d, J = 8.5 Hz, 1 H), 4.33 (d, J = 53.7 Hz, 2H), 3.02 (d, J = 5.9 Hz, 3H); MS (APCI+, M+1) 435.19.

[0195] Sorafenib, regorafenib, imatinib and nilotenib were from SelleckChem (Houston, USA). The synthesis of sorafenib and sorafenib analogs is as set forth above. Unless otherwise indicated, all other compounds were from Sigma-Aldrich (St. Louis, USA).

SRS15-52 : Ri = H, R₂ = C0₂Et SRS15-53 : Ri = H, R₂ = C0₂H SRS15-97 : R₁ = R₂ = OCH₃ SRN1-25 : R-i = CH₂NH₂, R₂ = H SRN1-34 : R-i = R₂ = CN

EJ1-36 : R-i = H, R₂ = B(OH)₂

Scheme 5. Efficient synthesis of sorafenib analogs.

General synthesis of sorafenib analogs depicted in Scheme 5

ArSisi2 reaction (General procedure E)

[0196] To the 1 -fluoro-4-nitrobenzene (1 equiv.) in dry DMF (20 ml_) was added K2CO3 (2 equiv.) and various arylalcohol (1 .2 equiv.). The mixture was stirred for 17 hours at 70°C. The solution was poured into water and the organic layer was extracted three times with ethyl acetate. After drying with anhydrous magnesium sulfate the solvents were removed under vacuum. The residue was purified by flash column chromatography on silica gel to provide the desired nitro-ether derivatives 3 (Scheme 5). Hvdroqenolvsis (General procedure F)

[0197] The desired nitro-ether derivatives 3 (1 equiv.) were dissolved in MeOH (10 mL) and hydrogenated (H₂ gas) over 10% Pd(OH)₂ on charcoal for 17 hours at room temperature. The solution was filtered through a pad of celite and volatiles were removed under vacuum. The residue was purified by flash-column chromatography on silica gel to provide the desired amine derivatives 4.

Addition of various arylamines to various isocvanates

[0198] General procedure A.

Synthesis of ethyl 4-(4-(3-(4-chloro-3-

(trifluoromethyl)phenyl)ureido)phenoxy)benzoate (SRS15-49), Scheme 5)

[0199] Following the above general procedure A, the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 20:1 ) to provide the desired sorafenib analog (SRS15-49) (97.0 mg, 0.204 mmol, 70%). 1 H NMR (400 MHz, DMSO) δ 9.18 (s, 1 H), 8.93 (s, 1 H), 8.12 (s, 1 H), 7.95 (d, J = 8.3 Hz, 2H), 7.59 (dd, J = 34.5, 9.1 Hz, 4H), 7.13 - 6.94 (m, 3H), 4.29 (dd, J = 12.4, 5.4 Hz, 2H), 1 .31 (dd, J = 8.9, 5.0 Hz, 3H); MS (APCI+, M+1 ) 479.01 .

Synthesis of ethyl 4-(4-(3-(3,5- bis(trifluoromethyl)phenyl)ureido)phenoxy)benzoate (SRS15-52, Scheme 5)

[0200] Following the above general procedure A, the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 20:1 ) to provide the desired sorafenib analog (SRS15-52) (103.0 mg, 0.201 mmol, 69%). ¹ H NMR (400 MHz, DMSO) δ 9.23 (d, J = 135.0 Hz, 2H), 8.15 (s, 2H), 7.95 (d, J = 8.7 Hz, 2H), 7.66 - 7.47 (m, 3H), 7.09 (d, J = 8.8 Hz, 2H), 7.01 (d, J = 8.7 Hz, 2H), 4.29 (q, J = 7.0 Hz, 2H), 1 .30 (t, J = 7.1 Hz, 3H); MS (APCI+, M+1) 513.01 .

Synthesis of 4-(4-(3-(3,5-bis(trifluoromethyl)phenyl)ureido)phenoxy)benzoic acid (SRS15-53, Scheme 5)

[0201] Following the above general procedure A, the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 10:1 ) to provide the desired sorafenib analog (SRS15-53) (18.0 mg, 0.038 mmol, 65%). ¹ H NMR (400 MHz, DMSO) δ 12.59 (s, 1 H), 9.48 (s, 1 H), 9.1 1 (s, 1 H), 8.15 (s, 2H), 7.94 (d, J = 8.3 Hz, 2H), 7.68 - 7.45 (m, 3H), 7.10 (d, J = 8.3 Hz, 2H), 7.00 (d, J = 8.3 Hz, 2H); MS (APCI+, M+1) 485.1 1 .

Synthesis of 4-(4-(3-(4-chloro-3-

(trifluoromethyl)phenyl)ureido)phenoxy)benzoic acid (SRS15-54, Scheme 5) [0202] Following the above general procedure A, the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 10:1 ) to provide the desired sorafenib analog (SRS15-54) (17.0 mg, 0.038 mmol, 61 %). ¹ H NMR (400 MHz, DMSO) δ 12.59 (s, 1 H), 9.48 (s, 1 H), 9.1 1 (s, 1 H), 8.15 (s, 2H), 7.94 (d, J = 8.3 Hz, 2H), 7.68 - 7.45 (m, 3H), 7.10 (d, J = 8.3 Hz, 2H), 7.00 (d, J = 8.3 Hz, 2H); MS (APCI+, M+1 ) 451 .19.

SRS15-96

Synthesis of 1 -(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(3,4- dimethoxyphenoxy)phenyl)-urea (SRS15-96, Scheme 5)

[0203] Following the above general procedure A, the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 20:1 ) to provide the desired sorafenib analog (SRS15-96) (38.0 mg, 0.08 mmol, 50%). ¹ H NMR (400 MHz, DMSO) δ 9.16 (s, 1 H), 8.82 (s, 1 H), 8.1 1 (d, J = 2.3 Hz, 1 H), 7.63 (dt, J = 17.7, 5.5 Hz, 2H), 7.44 (d, J = 8.9 Hz, 2H), 6.93 (dd, J = 8.9, 3.0 Hz, 3H), 6.72 (d, J = 2.7 Hz, 1 H), 6.47 (dd, J = 8.7, 2.7 Hz, 1 H), 3.73 (d, J = 2.4 Hz, 6H); MS (APCI+, M+1 ) 467.21 .

Synthesis of 1 -(3,5-bis(trifluoromethyl)phenyl)-3-(4-(3,4-dimethoxyphenoxy)- phenvDurea (SRS15-97, Scheme 5)

[0204] Following the above general procedure A, the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 20:1 ) to provide the desired sorafenib analog (SRS15-97) (48.0 mg, 0.096 mmol, 59%). ¹ H NMR (400 MHz, DMSO) δ 9.37 (s, 1 H), 8.94 (s, 1 H), 8.14 (s, 2H), 7.61 (s, 1 H), 7.47 (d, J = 8.9 Hz, 3H), 6.93 (dd, J = 8.7, 5.8 Hz, 4H), 6.72 (d, J = 2.6 Hz, 1 H), 6.48 (dd, J = 8.7, 2.7 Hz, 1 H), 3.74 (s, 6H); MS (APCI+, M+1 ) 500.99.

SRN1 -16

Synthesis of 1 -(4-(3-(aminomethyl)phenoxy)phenyl)-3-(4-chloro-3-

(trifluoromethvD-phenvDurea (SRN1 -16, Scheme 5)

[0205] Following the above general procedure A, the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 40:1 ) to provide the desired sorafenib analog (SRN1 -16) (10.0 mg, 0.023 mmol, 19%). ¹H NMR (400 Mz, DMSO-d₆) δ 9.43 (1H, s), 9.22-8.98 (1H, t), 8.14-8.13 (1H, d), 8.04-7.97 (1H, d), 7.68-7.66 (1H, q), 7.63-7.60 (1H, q), 7.51-7.49 (2H, d), 7.33-7.29 (1H, t), 7.09-7.07 (1H, d), 7.00-6.99 (3H, d), 6.83- 6.80 (1 H, q), 3.74 (2H, s); MS (APCI+, M+1 ) 436.02.

SRN1-25

Synthesis of 1 -(4-(3-(aminomethyl)phenoxy)phenyl)-3-(3,5- bis(trifluoronnethyl)phenyl)urea (SRN1-25, Scheme 5)

[0206] Following the above general procedure A, the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 40:1 to 5:1) to provide the desired sorafenib analog (SRN1-25) (13.0 mg, 0.028 mmol, 25%).¹H NMR (400 Mz, DMSO-d₆) δ 9.94 (1H, s), 9.39 (1H, s), 8.16 (2H, s), 7.66 (1H, s), 7.56-7 .53 (2H, d) 7.45-7.41 (1H, t) 7.43 (1H, s), 7.21-7.19 (1H, d), 7.14 (1H, s), 7.05-7.02 (2H, d), 7.01-6.98 (1H, q); MS (APCI+, M+1) 470.12.

SRN1-33

Synthesis of 1 -(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(3,4-dicvanophenoxy)- phenvDurea (SRN1-33, Scheme 5)

[0207] Following the above general procedure A, the crude reaction mixture was purified by preparatory TLC chromatography (dichloromethane: methanol = 100:1) to provide the desired sorafenib analog (SRN1-33) (12.0 mg, 0.026 mmol, 31%). ¹H NMR(400Mz, DMSO-d₆) δ 9.32 (1 H, s), 9.12 (1 H, s), 8.13-8.12 (1H, d), 8.10 (1H, s), 7.77-7.76 (1H, d), 7.70-7.67 (1H, d), 7.65 (1H, s), 7.62 (1H, s), 7.60 (1H, s), 7.40-7.36 (1H, q), 7.18-7.16 (2H, d); MS (APCI+, M+1) 457.12.

SRN1-34

Synthesis of 1-(3,5-bis(trifluoromethyl)phenyl)-3-(4-(3,4- dicvanophenoxy)phenyl)urea (SRN1-34, Scheme 5)

[0208] Following the above general procedure A, the crude reaction mixture was purified by preparatory TLC chromatography (dichloromethane: methanol = 100:1) to provide the desired sorafenib analog (SRN1-34) (22.0 mg, 0.045 mmol, 52%).¹H NMR (400 Mz, DMSO-d₆) δ 9.60 (1H, s), 9.38 (1H, s), 8.17 (2H, s), 8.12-8.10 (1H, d), 7.78-7.77 (1H, d), 7.66 (1H, s), 7.64-7.61 (2H, d), 7.39-7.37 (1 H, q), 7.19-7.17 (2H, d); MS (APCI+, M+1) 457.12.

EJ1-36

Synthesis of 4-(4-(3-(3,5- bis(trifluoromethyl)phenyl)ureido)phenoxy)phenylboronic acid (EJ1-36, Scheme 5)

[0209] (4-(4-aminophenoxy)phenyl)boronic acid (10 mg, 0.0386 mmol ) was placed in a vial. Dichloroethane (3 ml_) was added to the vial. 1- isocyanato-3,5-bis(trifluoromethyl)-benzene (14 μΙ_, 2 eq) was then added to the solution. The solution was stirred at 70°C for 17 hours. After 17 hours, the solvent was evaporated under vaccuo. A minimal amount of dichloromethane was added to the mixture, and was purified by using a TLC preparatory plate with one run in 100:1 DCM: Methanol to provide the desired product (EJ1 -36) (4-(4-(3-(3,5-bis(trifluoromethyl)phenyl)ureido)phenoxy)phenyl)boronic acid (16.95 mg, 0.035 mmol, 91 %) as a dark yellow oil; 1 H-NMR(CDCI3, 400MHz, ppm).933 (s, 2H), 57.576 (s, 1 H), 57.041 (s, 2H), 57.866-6.821 (m, 2H), 5 6.628-6.517 (m, 3H), 5 6.027 (s, 1 H); MS (APCI+, M+1 ) 484.68.

EJ1 -61

Synthesis of 4-(4-(3-(4-chloro-3-

(trifluoromethyl)phenyl)ureido)phenoxy)phenylboronic acid (EJ1 -61 , Scheme 5}

[0210] (4-(4-aminophenoxy)phenyl)boronic acid (8 mg, 0.349 mmol ) was placed in a vial. Dichloromethane (3 ml_) was added to the vial. 1 -chloro- 4-isocyanato-2-(trifluoromethyl)benzene (1 1 .56 mg, 0.0523 mmol) was then added to the solution. The solution was stirred at room temperature for 17 hours. After 17 hours, the solution was a light yellow color. The stir-bar was removed from the vial and the mixture was rotovapped to give a dark brown oil. A minimal amount of dichloromethane was added to the mixture, and was purified by using a TLC preparatory plate with one run in 100:1 DCM:Methanol, to provide the desired product (EJ1 -61) (4-(4-(3-(4-chloro-3- (trifluoromethyl)phenyl)ureido)phenoxy)phenyl)boronic acid (12.3 mg, 0.273 mmol, 78%) as a dark yellow oil. 1 H-NMR(CDCI3, 400MHz, ppm) 57.66-7.56 (d, 1H), δ 7.61-7.54 (m, 1H), 57.42-7.40 (d, 1H), bl.21-1.25 (m, 4H), δ 6.99- 6.97 (m, 2H), 56.96 (s, 1H), 56.89-6.86 (d, 1H), 56.80 (s, 1H), 6.67-6.65 (d, 1 H), 6.52-6.49 ((d,d), 1 H); LCMS: m/z (M+) 451 ,23.

Scheme 6. efficient synthesis of sorafenib analogs.

H H

SRS15-94

Synthesis of 1 -(4-chloro-3-(trifluoronnethyl)phenyl)-3-(4-(pyridin-3- yloxy)phenyl)urea (SRS15-94, Scheme 6)

[0211] Following the above general procedure A, the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 40:1 ) to provide the desired sorafenib analog (SRS15-94) (70.0 mg, 0.172 mmol, 80%). ¹ H NMR (400 MHz, DMSO) δ 9.15 (s, 1 H), 8.88 (s, 1 H), 8.45 - 8.24 (m, 2H), 8.1 1 (s, 1 H), 7.62 (dd, J = 27.2, 8.3 Hz, 2H), 7.57 - 7.14 (m, 4H), 7.06 (d, J = 8.6 Hz, 2H); MS (APCI+, M+1) 408.12.

SRS 15-95

Synthesis of 1 -(3,5-bis(trifluoromethyl)phenyl)-3-(4-(pyridin-3- yloxy)phenyl)urea (SRS15-95, Scheme 6)

[0212] Following the above general procedure A, the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 40:1 ) to provide the desired sorafenib analog (SRS15-95) (71 .0 mg, 0.161 mmol., 75%). ¹ H NMR (400 MHz, DMSO) δ 9.15 (s, 1 H), 8.88 (s, 1 H), 8.45 - 8.1 1 (m, 3H), 7.62 (m, 2H), 7.57 - 7.14 (m, 4H), 7.06 (d, J = 8.6 Hz, 2H); MS (APCI+, M+1) 442.32.

H H

SRS 15-98

Synthesis of 1 -(4-chloro-3-(trifluoronnethyl)phenyl)-3-(4-(2- methylbenzo[c lthiazol-5-yloxy)phenyl)urea (SRS15-98, Scheme 6)

[0213] Following the above general procedure A, the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 20:1 ) to provide the desired sorafenib analog (SRS15-98) (17.0 mg, 0.036 mmol, 36%). 1 H NMR (400 MHz, DMSO) δ 9.17 (s, 1 H), 8.89 (s, 1 H), 8.1 1 (d, J = 6.7 Hz, 3H), 7.88 (d, J = 8.8 Hz, 1 H), 7.50 (d, J = 8.9 Hz, 2H), 7.17 - 7.10 (m, 2H), 7.03 (d, J = 9.0 Hz, 2H), 2.77 (s, 3H); MS (APCI+, M+1 ) 478.01 .

Synthesis of 1 -(3,5-bis(trifluoromethyl)phenyl)-3-(4-(2-methylbenzo[c 1thiazol- 5-yloxy)phenyl)urea (SRS15-99, Scheme 6)

[0214] Following the above general procedure A, the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 20:1 ) to provide the desired sorafenib analog (SRS15-99) (20.0 mg, 0.039 mmol, 40%). 1 H NMR (400 MHz, DMSO) δ 9.20 (s, 1 H), 8.91 (s, 1 H), 8.10- 7.88 (d, J = 8.8 Hz, 4H), 7.43-7.10 (m, 4H), 7.03 (m, 2H), 2.77 (s, 3H); MS (APCI+, M+1 ) 512.31 .

H H

SRS16-01

Synthesis of 1 -(4-chloro-3-(trifluoronnethyl)phenyl)-3-(4-(pyridin-4- yloxy)phenyl)-urea (SRS16-01 , Scheme 6)

[0215] Following the above general procedure A, the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 20:1 ) to provide the desired sorafenib analog (SRS16-01 ) (70.0 mg, 0.172 mmol, 78%). 1 H NMR (400 MHz, DMSO) δ 9.35 (s, 1 H), 9.19 (s, 1 H), 8.12 (s, 1 H), 7.95 (d, J = 5.6 Hz, 2H), 7.64 (dd, J = 9.1 , 6.7 Hz, 4H), 7.48 (d, J = 6.8 Hz, 2H), 6.27 - 6.16 (m, 2H); MS (APCI+, M+1 ) 408.03.

SRS16-02

Synthesis of 1 -(3,5-bis(trifluoromethyl)phenyl)-3-(4-(pyridin-4- yloxy)phenyl)urea (SRS16-02, Scheme 6)

[0216] Following the above general procedure A, the crude reaction mixture was purified by column chromatography (dichloromethane: methanol = 20:1 ) to provide the desired sorafenib analog (SRS16-02) (68.0 mg, 0.155 mmol, 95%). ¹ H NMR (400 MHz, DMSO) δ 10.30 (s, 1 H), 9.83 (s, 1 H), 8.13 (s, 2H), 7.96 (d, J = 7.0 Hz, 2H), 7.67 - 7.55 (m, 3H), 7.49 (d, J = 8.2 Hz, 2H), 6.24 (d, J = 6.8 Hz, 2H); MS (APCI+, M+1 ) 442.43.

Synthesis of 1 -(4-chloro-3-(trifluoronnethyl)phenyl)-3-(4-(quinolin-8- yloxy)phenyl)urea (EJ1 -30, Scheme 6)

[0217] 4-(quinolin-8-yloxy)aniline (50 mg, 0.212 mmol ) was placed in a vial. Dicholoromethane (3 ml_) was added to the vial. 1 -chloro-4-isocyanato-2- (t fluoromethyl)benzene (70.41 mg, 0.318 mmol) was then added to the solution. The solution was stirred at room temperature for 17 hours. After 17 hours, the solution had a light pink precipitate in it. The solvent was evaporated under vacuum. A minimal amount of dichloromethane was added to the mixture, and was purified by using a TLC preparatory plate with two runs in 100:1 DCM:Methanol, to provide the desired product (EJ1 -30) 1 -(4- chloro-3-(trifluoromethyl)phenyl)-3-(4-(quinolin-8-yloxy)phenyl)urea (57.78 mg, 0.126 mmol, 67%) as very light pink powder. 1 H-NMR(CDCI3, 400MHz, ppm) δ 8.94-8.93 (q, 1 H), δ 8.63 (s, 1 H), δ 8.35-8.32 (q, 1 H), δ 7.91 (s, 1 H), δ 7.68- 7.67 (m, 2H), δ 7.57-7.46 (m, 3H), δ 7.21 -7.19 (q, 1 H), δ 7.19-7.07 (d, 2H), δ 6.87-6.84 (d, 2H); MS (APCI+, M+1 ) 457.89.

EJ1 -33 Synthesis of 1 -(3,5-bis(trifluoronnethyl)phenyl)-3-(4-(quinolin-8- yloxy)phenyl)urea (EJ1 -33, Scheme 6)

[0218] 4-(quinolin-8-yloxy)aniline (40 mg, 0.169 mmol ) was placed in a vial. Dichloromethane (3 ml_) was added to the vial. 1 -isocyanato-3,5- bis(trifluoromethyl)benzene (58.59 uL, 0.339 mmol) was then added to the solution. The solution was stirred at 70°C for 17 hours under reflux. After 17 hours, the solution contained a light pink precipitate. The solvent was evaporated under vacuum. A minimal amount of dichloromethane was added to the mixture, and was purified by using a TLC preparatory plate with two runs in 100:1 DCM: Methanol, to provide the desired product (EJ1 -33) 1 -(3,5- bis(trifluoromethyl)phenyl)-3-(4-(quinolin-8-yloxy)phenyl)urea (51 .8 mg, 0.105 mmol, 62%) as a medium pink powder. 1 H-NMR(CDCI3, 400MHz, ppm) δ 9.1 (s, 1 H), δ 9.934-8.919 ((d,d), 1 H), δ8.346-8.325 ((d,d), 1 H), δ8.321 (s, 1 H), δ 7.660-7.638 ((d,d), 1 H), δ 7.574-7.554 (q, 1 H), δ 7.505-7.465 (t, 1 H), δ 7.419 (s, 1 H), δ7.293 (s, 1 H), 7.136-7.106 (m, 3H), δ6.876-6.854 (d, 2H);MS (APCI+, M+1 ) 492.09.

Synthesis of 1 -(3,5-bis(trifluoromethyl)phenyl)-3-(4-(5-chloroquinolin-8- yloxy)phenyl)urea (EJ1 -35, Scheme 6)

[0219] 4-((5-chloroquinolin-8-yl)oxy)aniline (30 mg, 0.1 1 1 mmol ) was placed in a vial. Dichloroethane (3 ml_) was added to the vial. 1 -isocyanato- 3,5-bis(trifluoromethyl)benzene (33 uL, 2 eq) was then added to the solution. The solution was stirred at 70°C for 17 hours. After 17 hours, the solution contained a pink precipitate. The solvent was evaporated under vacuum. A minimal amount of dichloromethane was added to the mixture, and was purified by using a TLC preparatory plate with two runs in 100:1 DCM:Methanol, to provide the desired product (EJ1 -35) 1 -(3,5- bis(trifluoromethyl)phenyl)-3-(4-((5-chloroquinolin-8-yl)oxy)phenyl)urea (45.4 mg, 0.086 mmol, 77.7%) as a pink powder; 1 H-NMR(CDCI3, 400MHz, ppm) δ 9.014-9.004 (d, 1 H), δ 8.743-8.722 (d, 1 H), δ 8.381 (s, 1 H), δ 7.712-7.681 (m, 2H), δ 7.606-7.585 (d, 1 H), δ 7.495 (s, 1 H), δ 7.182-7.123 (d, 2H), δ 7.123- 7.103 (d, 1 H), δ 6.942-6.921 (d, 2H); MS (APCI+, M+1 ) 536.20.

Synthesis of 1 -(3.5-bis(trifluoromethyl)phenyl)-3-(4-(1 .2.3.4- tetrahvdroquinolin-8-yloxy)phenyl)urea (EJ1 -38. Scheme 6)

[0220] 1 -(3,5-bis(trifluoromethyl)phenyl)-3-(4-((1 ,2,3,4- tetrahydroquinolin-8-yl)oxy)phenyl)urea (30 mg, 0.061 1 mmol) was placed in a round bottom flask. Ethyl Acetate (4 ml_) and methanol (1 ml_) was added to the flask. Palladium hydroxide on charcoal (6.1 1 mg, 10% eq) was added to the solution and a balloon filled with hydrogen gas was fitted to the flask. The solution was stirred at room temperature for 17 hours. The palladium hydroxide was filtered out using celite and the celite was washed with extra ethyl acetate and methanol. The solution mixture was a light pink color. The solvent was evaporated under vacuum to give a dark pink oil. A minimal amount of dicholoromethane was added to the mixture and the desired product was purified by a TLC preparatory plate with one run in 70:1 DCM:Methanol to provide the desired product (EJ1-38) 1 -(3,5- bis(trifluoromethyl)phenyl)-3-(4-((1 ,2,3,4-tetrahydroquinolin-8- yl)oxy)phenyl)urea (25.44 mg, 0.0514 mmol, 84%) as a dark yellow oil; 1 H- NMR(CDCI3, 400MHz, ppm) δ 9.89 (s, 1 H), δ 9.03 (s, 1 H), δ 8.05 (s, 2H), δ 7.47-7.41 (d, 3H), δ 6.98-6.91 (d, 2H), δ 6.79-6.73 (d, 1 H), δ 6.67-6.60 (d, 1 H), δ 6.57-6.50 (t, 1 H), δ 3.04-2.94 (q, 3H), δ 2.88-2.84 (t, 2H), 2.02-1 .97 (q, 2H); MS (APCI+, M+1 ) 496.00.

R Compound

Synthesis of 1 -(4-(quinolin-8-yloxy)phenyl)-3-(4-(trifluoromethoxy)phenyl)urea (EJ1 -79. Scheme 7)

[0221] 4-(quinolin-8-yloxy)aniline (50 mg, 0.21 1 mmol ) was dissolved in dichloroethane. 1 -isocyanato-4-(thfluoromethoxy)benzene (48uL, 1 .5 eq) was then added to the solution. The solution was stirred at 70 °C for 17 hours. After 17 hours, the solution had produced a light pink precipitate. The solvent was evaporated under vacuum. A minimal amount of dichloromethane was added to the mixture, and was purified by using a TLC preparatory plate with two runs in 40:1 DCM:Methanol to provide the desired product (1 EJ1-79) 1 - (4-(quinolin-8-yloxy)phenyl)-3-(4-(trifluoromethoxy)phenyl)urea (5.33 mg, 0.012 mmol, 5.8%) as yellow powder. ¹H NMR (400 MHz, CDCI₃) δ 8.97 (d, J = 3.1 Hz, 1 H), 8.29 (d, J = 7.6 Hz, 1 H), 7.65 (d, J = 8.1 Hz, 1 H), 7.57 - 7.47 (m, 2H), 7.40 (d, J = 7.8 Hz, 3H), 7.22 (dd, J = 15.0, 7.5 Hz, 3H), 7.13 (d, J = 7.6 Hz, 2H), 6.98 (d, J = 7.8 Hz, 2H); MS (APCI+, M+1 ) 441 .10.

Synthesis of 1 -(4-((5-chloroquinolin-8-yl)oxy)phenyl)-3-(4-(trifluoromethoxy)- phenvDurea (EJ1 -80, Scheme 7)

[0222] 4-((5-chloroquinolin-8-yl)oxy)aniline (50 mg, 0.185 mmol ) was placed in a vial. Dichloroethane was added to the vial. 1 -isocyanato-4- (trifluoromethoxy)benzene (42 uL, 0.28 mmol) was then added to the solution. The solution was stirred at 70 °C for 17 hours. After 17 hours, the reaction mixture produced a light pink precipitate.

[0223] The stir-bar was removed from the vial and the mixture was rotovapped. A minimal amount of dichloromethane was added to the mixture, and was purified by using a TLC preparatory plate. With one run in 40:1 DCM:MeOH, the mixture was separated. The desired product was scraped off from the TLC prep plate and was eluted from the silica gel to provide (EJ1 -80) 1 -(4-((5-chloroquinolin-8-yl)oxy)phenyl)-3-(4-(trifluoromethoxy)-phenyl)urea ( 39.87 mg, 0.084 mmol, 45%) as orange powder.

[0224] ¹ H NMR (400 MHz, DMSO) δ 9.02 (dd, J = 5.1 , 3.6 Hz, 2H), 8.95 (d, J = 45.2 Hz, 1 H), 8.61 (dd, J = 8.6, 1 .4 Hz, 1 H), 7.83 - 7.71 (m, 2H), 7.57 (d, J = 9.0 Hz, 2H), 7.48 (d, J = 8.9 Hz, 2H), 7.29 (d, J = 8.6 Hz, 2H), 7.18 (d, J = 8.4 Hz, 1 H), 7.02 (d, J = 8.9 Hz, 2H); MS (APCI+, M+1 ) 474.43.

Synthesis of 1 -(4-((1 ,2,3,4-tetrahvdroquinolin-8-yl)oxy)phenyl)-3-(4- (trifluoromethoxy)phenyl)urea (EJ1 -82, Scheme 7)

[0225] 1 -(4-((5-chloroquinolin-8-yl)oxy)phenyl)-3-(4- (trifluoromethoxy)phenyl)urea (15 mg, 0.0335 mmol) was placed in a round bottom flask. Ethyl Acetate (4 mL) and methanol (1 mL) was added to the flask. Palladium hydroxide on charcoal (31 .65 mg, 31 .6 mmol) was added to the solution and a balloon filled with hydrogen gas was fitted to the flask. The solution was stirred at room temperature for 17 hours. The palladium hydroxide was filtered out using celite and the celite was washed with extra ethyl acetate and methanol. The solution mixture was a light pink color. The solvent was evaporated to give dark brown oil. A minimal amount of dicholoromethane was added to the mixture and the desired product was purified by using a TLC preparatory plate. With one run in 40:1 DCM:Methanol the mixture was separated. The desired product was scraped off the TLC Prep plate and was eluted from the silica gel to provide (EJ1 -82) 1 -(4- ((1 ,2,3,4-tetrahydroquinolin-8-yl)oxy)phenyl)-3-(4-

(trifluoromethoxy)phenyl)urea (3.25 mg, 0.0073 mmol, 22%) as a light pink powder. ¹ H NMR (400 MHz, CDCI₃) δ 7.43 - 7.35 (m, 2H), 7.32 - 7.23 (m, 5H), 7.17 (d, J = 8.4 Hz, 2H), 7.02 - 6.95 (m, 2H), 6.83 (d, J = 7.4 Hz, 1 H), 6.73 (d, J = 26.2 Hz, 2H), 6.68 (s, 1 H), 6.57 (dd, J = 14.8, 7.1 Hz, 2H), 3.38 - 3.30 (m, 2H), 2.84 (t, J = 6.4 Hz, 2H), 1 .99 (dt, J = 1 1 .6, 6.4 Hz, 2H); MS (APCI+, M+1 ) 444.13.

^F3C L_N ^HX_N ^HJC EJ2-03 >

Synthesis of 1 -(4-(benzo[d1[1 ,31dioxol-5-yloxy)phenyl)-3-(4-(trifluoromethoxy)- phenvDurea (EJ2-03, Scheme 7)

[0226] 4-(benzo[c/][1 ,3]dioxol-5-yloxy)aniline (50 mg, 0.218 mmol ) was dissolved in dichloroethane. 1 -isocyanato-4-(trifluoromethoxy)benzene (40 uL, 0.28 mmol, 1 .2 eq) was then added to the solution. The solution was stirred at 70 °C for 17 hours. After 17 hours, the solution was a medium yellow color. The solvent was evaporated. A minimal amount of dichloromethane was added to the mixture, and was purified by using a TLC preparatory plate with one run in 5:2 Hexanes:EtOAc, to provide the desired product (EJ2-03) 1 -(41 - (4-(benzo[c/][1 ,3]dioxol-5-yloxy)phenyl)-3-(4-(trifluoromethoxy)phenyl)urea (22.1 mg, 0.051 1 mmol, 23%) as a light yellow oil.

[0227] ¹ H-NMR(CDCI3, 400MHz, ppm) δ 7.55-7.52 (t, 1 H), δ 7.48-7.45

(d, 2H), δ 7.44-7.42 (d, 4H), δ 7.38-7.36 (d, 1 H), δ 7.22-7.18 (m, 6H), δ 7.16- 7.13 (d, 1 H), δ 6.97-6.95 (m, 1 H), δ 6.05-5.99 (d, 2H); MS (APCI+, M+1 ) 433.

Synthesis of 1 -(4-(4-(adamantan-1 -yl)phenoxy)phenyl)-3-(4- (thfluoromethoxy)phenyl)urea (EJ1 -91 , Scheme 7)

[0228] 4-(4-((3r,5r,7r)-adamantan-1 -yl)phenoxy)aniline (55 mg, 0.172 mmol) was dissolved in dichloroethane. 1 -isocyanato-4- (thfluoromethoxy)benzene (40 uL, 1 .5eq) was then added to the solution. The solution was stirred at 70 °C for 17 hours.

[0229] The solvent was evaporated under vacuum. A minimal amount of dichloromethane was added to the mixture, and was purified by using a TLC preparatory plate with one run in 5:2 Hexanes:EtOac to provide the desired product (EJ1-91 ) 1 -(4-(4-(adamantan-1 -yl)phenoxy)phenyl)-3-(4- (trifluoromethoxy)phenyl)urea (23.4mg, 0.0448mmol, 26%) as pale yellow powder. ¹ H NMR (400 MHz, CDCI₃) δ 7.44 (d, J = 8.7 Hz, 7H), 7.29 (s, 2H), 7.20 (d, J = 8.8 Hz, 9H), 2.13 (s, 3H), 1 .94 (s, 6H), 1 .81 (d, J = 8.3 Hz, 6H); MS (APCI+, M+1 ) 523.

Synthesis of 1 -(4-(2-(adamantan-1 -yl)ethoxy)phenyl)-3-(4-(trifluoromethoxy)- phenvDurea (EJ2-07, Scheme 7)

[0230] 4-(2-((3r,5r,7r)-adamantan-1 -yl)ethoxy)aniline (35mg, 0.129mmol ) was dissolved in dichloroethane. 1 -isocyanato-4- (trifluoromethoxy)benzene (23uL, 1 .2 eq) was then added to the solution. The solution was stirred at 70 °C for 17 hours. The solvent was evaporated under vacuum. A minimal amount of dichloromethane was added to the mixture, and was purified by using a TLC preparatory plate with one run in 5:2 Hexanes:EtOAc to provide the desired product (EJ2-07) 1 -(4-(2-(adamantan- 1 -yl)ethoxy)phenyl)-3-(4-(trifluoromethoxy)phenyl)urea (47.6mg, 0.1 OOmmol, 78%) as dark brown oil. ¹H-NMR(CDCI3,400MHz,ppm) δ 7.54-7.42 (m, 5H), δ 7.24-7.18 (m, 4H), δ 1 .76-1 .60 (m, 13H), δ 1 .58-1 .57 (d, 3H); MS (APCI+, M+1 ) 475.

Synthesis of 1 -(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-((5-chloroquinolin-8- yl)oxy)phenyl)urea (EJ1 -69, Scheme 5)

[0231] 4-((5-chloroquinolin-8-yl)oxy)aniline (26.57 mg, 0.098mmol ) was placed in a vial. Dichloroethane was added to the vial. 1 -chloro-4- isocyanato-2-(thfluoromethyl)-benzene ( 32.7 mg, 1 .5eq) was then added to the solution. The solution was stirred at 70°C for 17 hours. After 17 hours, the solution produced a light pink precipitate. The solvent was evaporated under vacuum. A minimal amount of dichloromethane was added to the mixture, and was purified by using a TLC preparatory plate with one run in 40:1 DCM:MeOH to provide the desired product (EJ1 -69) 1 -(4-chloro-3 (trifluoromethyl)phenyl)-3-(4-((5-chloroquinolin-8-yl)oxy)phenyl)urea (14.8mg, 0.030mmol, 31 %) as light yellow powder. ¹ H-NMR(DMSO,400MHz,ppm) δ 9.02 (s, 1 H), δ 9.01 -9.00 (t, 1 H), δ 8.94 (s, 1 H), δ 8.63-8.60 (q, 1 H), δ 8.65- 8.24 (d, 1 H), δ 7.81 -7.81 (d, 2H), δ 7.68-7.65 (m, 2H), δ 7.48-7.47 (d, 4H), δ 7.19-7.14 (d, 1 H), δ 7.02-7.01 (d, 2H); MS (APCI+, M+1 ) 492.2.

Synthesis of 1 -(4-(benzo[d1H ,31dioxol-5-yloxy)phenyl)-3-(4-chloro-3- (trifluoromethvDphenvDurea (EJ2-01 , Scheme 8)

[0232] 4-(benzo[c/][1 ,3]dioxol-5-yloxy)aniline (50 mg, 0.218 mmol) was placed in a vial. Dichloroethane (3 ml_) was added to the vial. 1 -chloro-4- isocyanato-2-(trifluoronnethyl)benzene (58.03 mg, 1 .2 eq) was then added to the solution. The solution was stirred at 70 °C for 17 hours. After 17 hours, the solution was a medium yellow color. The solvent was evaporated under vacuum. A minimal amount of dichloromethane was added to the mixture, and was purified by using a TLC preparatory plate with one run in 5:2 Hexanes:EtOAc, to provide the desired product (EJ2-01 ) 1 -(4- (benzo[c/][1 ,3]dioxol-5-yloxy)phenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea (19.6mg, 0.0435 mmol, 20%) as dark yellow oil.

[0233] ¹ H-NMR(CDCI3,400MHz,ppm) δ 7.88-7.81 (d, 1 H), δ 7.84-7.83 (t, 2H), δ 7.63-7.61 (m, 2H), δ 7.59-7.51 (q, 1 H), δ 7.49-7.47 (d, 2H), δ 7.38- 7.36 (m, 1 H), δ 7.27-7.25 (m, 2H), δ 6.98-6.96 (m, 1 H), δ 6.79-6.77 (d, 1 H), δ 6.58-6.57 (d, 1 H), δ 6.51 -6.49 (q, 1 H), δ 6.05 (s, 1 H), δ 6.00 (s, 1 H); MS (APCI+, M+1 ) 452.10.

Synthesis of 1 -(4-(adamantan-1 -ylmethoxy)phenyl)-3-(4-chloro-3- (trifluoromethvDphenvDurea (EJ1 -70, Scheme 8)

[0234] 4-(((3r,5r,7r)-adamantan-1 -yl)methoxy)aniline (7 mg, 0.027 mmol) was dissolved in dichloroethane (3 ml_). 1 -chloro-4-isocyanato-2- (trifluoromethyl)benzene (9.05 mg, 0.041 mmol) was then added to the solution. The solution was stirred at 70°C for 17 hours. The solvent was evaporated under vacuum. A minimal amount of dichloromethane was added to the mixture, and was purified by using a TLC preparatory plate with one run in1 :1 Hexanes:EtOAc to provide the desired product (EJ1 -70) 1 -(4- (adamantan-1 -ylmethoxy)phenyl)-3-(4-chloro-3-(trifluoromethyl)ph (5mg, O.OI Ommol, 38%) as yellow powder. ¹H-NMR (CDCI3, 400MHz, ppm) 57.87-7.85 (m, 2H), 57.49-7.47 (d, 1 H), 57.10-7.18 (d, 2H), 56.90-6.83 (d, 2H), 56.73 (s, 1 H), 56.44 (s, 1 H), 53.52 (s, 2H), 52.21 -2.10 (m, 3H), 51 .88- 1 .65 (m, 12H); MS (APCI+, M+1 ) 479.

Synthesis of 1 -(4-(4-(adamantan-1 -yl)phenoxy)phenyl)-3-(4-chloro-3- (trifluoromethyl)phenyl)urea (EJ1 -89, Scheme 8)

[0235] 4-(4-((3r,5r,7r)-adamantan-1 -yl)phenoxy)aniline (55 mg, 0.172 mmol) was dissolved in dichloroethane (3 ml_). 1 -chloro-4-isocyanato-2- (trifluoromethyl)benzene (57.28 mg, 1 .5 eq) was then added to the solution. The solution was stirred at 70 °C for 17 hours.

[0236] The solvent was evaporated under vacuum. A minimal amount of dichloromethane was added to the mixture, and was purified by using a TLC preparatory plate with one run in 5:2 Hexanes:EtOAc to provide the desired product (EJ1-89) 1 -(4-(4-(adamantan-1 -yl)phenoxy)phenyl)-3-(4- chloro-3-(trifluoromethyl)phenyl)urea (21 .1 mg, 0.039mmol, 23%) as a pale yellow powder.¹ H-NMR(CDCI3,400MHz,ppm) 5 7.61 -7.60 (d, 1 H), 5 7.48 (s, 1 H), 57.44-7.41 (m, 1 H), 5 7.34-7.31 (m, 3H), 5 7.21 -7.19 (m, 3H), 5 6.96- 6.94 (m, 4H), 5 2.12-2 (s, 3H), 5 1 .92-1 .91 (d, 6H), 5 1 .84-1 .75 (m, 6H); MS (APCI+, M+1 ) 541 .

Synthesis of 1 -(4-(benzo[d1H ,31dioxol-5-yloxy)phenyl)-3-(3,5- bis(trifluoromethyl)phenyl)urea (EJ2-02, Scheme 8)

[0237] 4-(benzo[c/][1 ,3]dioxol-5-yloxy)aniline (50mg, 0.218mmol ) was dissolved in dichloroethane. 1 -isocyanato-3,5-bis(trifluoromethyl)benzene (45uL, 0.30 mmol, 1 .2 eq) was then added to the solution. The solution was stirred at 70 °C for 17 hours. After 17 hours, the solution was a medium yellow color. The solvent was evaporated under vacuum. A minimal amount of dichloromethane was added to the mixture, and was purified by using a TLC preparatory plate with one run in 5:2 Hexanes:EtOAc, to provide the desired product (EJ2-02) 1 -(4-(benzo[c/][1 ,3]dioxol-5-yloxy)phenyl)-3-(3,5- bis(trifluoromethyl)phenyl)urea (17.9 mg, 0.037mmol, 17%) as a light yellow oil. ¹ H-NMR(CDCI3,400MHz,ppm) δ 7.98 (m, 5H), δ 7.68 (m, 2H), δ 7.42-7.39 (d, 2H), δ 7.22-7.19 (d, 2H), δ 6.82-6.79 (m, 1 H), δ 6.73-6.52 (m, 2H), δ 6.07 (s, 2H); MS (APCI+, M+1 ) 485.

Synthesis of 1 -(4-(adamantan-1 -ylmethoxy)phenyl)-3-(3,5- bis(trifluoromethyl)phenyl)urea (EJ1 -71 , Scheme 8)

[0238] 4-(((3r,5r,7r)-adamantan-1 -yl)methoxy)aniline (7 mg, 0.027 mmol ) was was dissolved in dichloroethane (3 ml_). 1 -isocyanato-3,5- bis(trifluoromethyl)benzene (5.9 μΙ_, 1 .5 eq) was then added to the solution. The solution was stirred at 70°C for 17 hours.

[0239] The solvent was evaporated under vacuum. A minimal amount of dichloromethane was added to the mixture, and was purified by using a TLC preparatory plate. With one run in 1 :1 Hexanes:EtOAc to provide the desired product (EJ1 -71 ) 1 -(4-(adamantan-1 -ylmethoxy)phenyl)-3-(3,5- bis(trifluoromethyl)phenyl)urea (4.3 mg, 0.008 mmol, 31 %) as light yellow powder. 1 H-NMR(CDCI3,400MHz,ppm) 57.95-7.88 (d, 6H), 57.58-7.55 (d, 2H), 56.99-6.45 (m, 2H), 53.51 (s, 2H), 51 .82-1 .72 (m, 14H); MS (APCI+, M+1 ) 513.

Synthesis of 1 -(4-(4-(adamantan-1 -yl)phenoxy)phenyl)-3-(3,5- bis(trifluoromethyl)phenyl)urea (EJ1 -90, Scheme 8)

[0240] 4-(4-((3r,5r,7r)-adamantan-1 -yl)phenoxy)aniline (55 mg, 0.172 mmol) was dissolved in dichloroethane (3 ml_). 1 -isocyanato-3,5- bis(trifluoromethyl)benzene (45 μΙ_, 1 .5eq was then added to the solution. The solution was stirred at 70°C for 17 hours.

[0241] The solvent was evaporated under vacuum. A minimal amount of dichloromethane was added to the mixture, and was purified by using a TLC preparatory plate with one run in 5:2 Hexanes:EtOac to provide the desired product (EJ1-90) 1 -(4-(4-(adamantan-1 -yl)phenoxy)phenyl)-3-(3,5- bis(trifluoromethyl)phenyl)urea (14.6mg, 0.025mmol, 15%) as pale yellow powder. ¹ H-NMR(CDCI3,400MHz,ppm) δ 8.02-8.00 (d, 2H), δ 7.97 (s, 2H), δ 7.67-7.65 (d, 1 H), δ 7.45-7.43 (m, 1 H), δ 7.40-7.34 (m, 2H), δ 7.26-7.21 (m, 2H), δ 7.13-7.1 1 (m, 1 H), δ 6.98-9.96 (m, 1 H), δ 2.15-2.13 (m, 3H), δ 1 .97- 1 .93 (m, 6H), δ 1 .83-1 .81 (m, 6H); MS (APCI+, M+1 ) 575.

Synthesis of 1 -(4-(2-(adamantan-1 -yl)ethoxy)phenyl)-3-(4-(trifluoromethoxy)- phenvDurea (EJ2-06, Scheme 8)

[0242] 4-(2-((3r,5r,7r)-adamantan-1 -yl)ethoxy)aniline (35 mg, 0.129 mmol ) was dissolved in dichloroethane (3 ml_). 1 -isocyanato-3,5- bis(trifluoromethyl)benzene (27 uL, 1 .2 eq) was then added to the solution. The solution was stirred at 70 °C for 17 hours.

[0243] The solvent was evaporated under vacuum. A minimal amount of dichloromethane was added to the mixture, and was purified by using a TLC preparatory plate with one run in 5:2 Hexanes:EtOAc to provide the desired product (EJ2-06) 1 -(4-(2-(adamantan-1 -yl)ethoxy)phenyl)-3-(4- (trifluoromethoxy)phenyl)urea ( 50.0 mg, 0.095 mmol, 74%) as light yellow powder. ¹H-NMR(CDCI3, 400MHz, ppm) δ 7.924 (s, 5H), δ 7.90-7.89 (d, 2H), δ7.59 (s, 3H), δ 3.85 (s, 9H), δ 1 .78-1 .62 (m, 3H), δ 1 .57 (s, 5H); MS (APCI+, M+1 ) 527.

Cell lines and media.

[0244] BJeHLT and BJeLR cells were obtained from Robert Weinberg (Whitehead Institute, Cambridge, MA). BJeHLT cells express human telomerase (hTERT), large T antigen (LT) and small T antigen (ST); BJeLR cells express hTERT, LT, ST and an oncogenic HRAS allele (HRAS^V12). SV40-transformed MEFs were obtained from Craig Thompson (Memorial Sloan Kettering, New York, NY). 143B cells were obtained from Eric Schon (Columbia Medical School, New York, NY). HT-1080 and Calu-1 cells were obtained from American Type Culture Collection. BJeHLT and BJeLR cells were grown in DMEM High-Glucose media (Gibco/Life Technologies Corp., Carlsbad, CA) plus 20% M199 (Sigma) and 15% heat-inactivated fetal bovine serum (FBS). HT-1080 cells were grown in DMEM High-Glucose media (Gibco) supplemented with 10% FBS and 1 % non-essential amino acids (Gibco). Calu-1 and U2OS cells were grown in McCoy's 5A media (Gibco) supplemented with 10% fetal bovine serum. MEFs were grown in DMEM supplemented with 10% fetal calf serum. 143B cells were grown in DMEM High-Glucose supplemented with 10% FBS. All cell lines were grown in humidified tissue culture incubators (Thermo Scientific, West Palm Beach, FL) at 37°C with 5% CO₂. Except where indicated, all medias were supplemented with penicillin and streptomycin (Gibco).

Multicellular Tumor Sphere Assays.

[0245] Multicellular tumor spheroids (MCTSs) were grown in 96-well Corningware Ultra Low Attachment (ULA) Plates (CLS 3474). 200 μΐ of cell suspension containing 10⁴ cells/ml were added to each well of the ULA plate, after which they were incubated at 37°C/5% CO₂ for 72 hours to allow for MCTS formation. MCTSs were then treated with lethal compounds (vehicle control [DMSO], 10 μΜ Erastin, 1 μΜ RSL3, or 1 μΜ STS) +/- inhibitors (vehicle control [DMSO], 1 μΜ Ferrostatin-1 , or 25 μΜ β-mercaptoethanol) by carefully aspirating 50 μΙ_ of media from each well, and replacing with 50 μΙ_ each of media containing 4x desired treatment concentration of the lethal or inhibitor. After 72 hours of treatment, MCTS images were acquired using an EVOS fl microscope (Advanced Microscopy Group/Life Technologies Corp.) equipped with a 10x phase contrast objective. Three independent fields were acquired for each experimental condition. Viability was then measured using Alamar blue as described above and measured on a Victor3 plate reader.

Radioactive uptake assays.

[0246] 200,000 HT-1080 or Calu-1 cells/well were seeded overnight in 6-well dishes (Corning Life Sciences, Tewksbury, USA). The next day, cells were washed twice in pre-warmed Na⁺-free uptake buffer (137 mM choline chloride, 3 mM KCI, 1 mM CaCI₂, 1 mM MgCI₂, 5 mM D-glucose, 0.7 mM K₂HPO₄, 10 mM HEPES, pH 7.4), then incubated for 10 minutes at 37°C in 1 ml_ of uptake buffer, to deplete cellular amino acids. At this point, in each well the buffer was replaced with 600 μΙ_ uptake buffer containing compound and 0.12 pCi (80-1 10 mCi/mmol) of L-[3,3'-¹⁴C]-cystine or 0.2 pCi of L-[¹⁴C(U)]- phenylalanine (PerkinElmer, Waltham, USA) and incubated for 3 minutes at 37°C. Cells were then washed three times with ice-cold uptake buffer and lysed in 500 μΙ_ 0.1 M NaOH. To this lysate was added 15 mL of scintillation fluid and radioactive counts per minute were obtained using a scintillation counter. All experiments were repeated in three independent biological replicates for each condition. To control for differences in the absolute counts of radioactivity between replicates, data was first normalized to DMSO (set to 100%) within each replicate, then averaged across the three biological replicates. Medium-throughput glutamate release assay.

[0247] The release of glutamate from HT-1080 cells into the extracellular media was detected using an Amplex Red glutamate release assay kit (Molecular Probes/Life Technologies Corp.). For compound treatment experiments, 200,000 cells/well (400,000 for HCT-1 16) were seeded overnight into 6-well dishes (Corning). The next day, cells were washed twice in PBS and then incubated for an hour in Na⁺-containing, glutamine-free media (Cellgro/Corning) containing various compounds at different concentrations. For siRNA experiments, cells were transfected with siRNAs for 48 hours (see Figure 2), then washed twice in PBS and incubated for an hour in Na⁺-containing, glutamine-free media. 50 μΙ_ of medium per well was removed and transferred to a 96-well assay plate (Corning) and incubated with 50 μΙ_ of a reaction mixture containing glutamate oxidase, L- alanine, glutamate-pyruvate transaminase, horseradish peroxidase and Amplex Red reagents as per the manufacturers protocol. Glutamate release was first normalized to total cell number determined by Vi-Cell counting at the end of the experiment, then values were expressed as a percentage of no treatment (DMSO) controls. In some experiments, a glutamate standard curve was used to quantify the exact amount of glutamate release. Of note: as the media contained Na⁺, the total amount of glutamate release reflects the activity of both system x_c ^" (Na⁺-independent) and non-system x_c ^" glutamate transporters and, therefore, never reaches 100% inhibition as system x_c ^~ accounts for only a portion of the total glutamate release. High-throughput glutamate release assay.

[0248] Human astrocytoma cells (CCF-STTG1 ) were used as a source of the cystine-glutamate antiporter (x_c ^"). Cells were grown in 96-well plates; when confluent, cells were washed with Earle's Balanced Salt Solution (EBSS) to remove residual glutamate. Cells were then incubated for 2 h at 37°C with either EBSS/glucose (blanks) or EBSS/glucose containing cystine 80 μΜ (totals) ± compounds (30 nM - 100 μΜ). The known x_c ^" inhibitor, (S)-4- carboxyphenylglycine (S-4CPG), was used as positive control. Following incubation, glutamate released into media was detected using the Amplex Red system as per the manufactures instructions. siRNA reverse transfection.

[0249] HT-1080 cells were reverse transfected with siRNAs (Qiagen, Germantown, USA) using Lipofectamine RNAiMAX (LFMax, Invitrogen/Life Technologies Corp.). Briefly, 1 -10 nM (final concentration) of siRNAs were aliquoted into 250 μΙ_ Opti-MEM media (Gibco) in the bottom of each well of a 6-well dish (Corning). An additional 250 μΙ_ media + LFMax was added to each well and incubated for 15 minutes. At this point, 150,000 HT-1080 cells were added to each well in regular HT-1080 media. The plates were swirled to mix and incubated for 48 hours at 37°C in a tissue culture incubator prior to analysis.

Reverse transcription-quantitative polymerase chain reaction (RT-gPCR).

[0250] RNA was extracted using the Qiashredder and Qiagen RNeasy

Mini kits (Qiagen) according to the manufacturer's protocol. 1 -2 μg total RNA for each sample was used as input for each reverse transcription reaction, performed using the TaqMan RT kit (Applied Biosystems/Life Technologies Corp.). Primer pairs for were designed for target transcripts using Primer Express 2.0 (Applied Biosystems). Quantitative PCR reactions were performed using the Power SYBR Green PCR Master Mix (Applied Biosystems). Triplicate samples per condition were analyzed on an Applied Biosystems StepOnePlus qPCR instrument using absolute quantification settings. Differences in mRNA levels compared to ACTB internal reference control were computed between control and experimental conditions using the AACt method.

Cell viability measurements.

[0251] Cell viability was typically assessed in 384-well format by Alamar Blue (Invitrogen) fluorescence (ex/em 530/590) measured on a Victor3 platereader (PerkinElmer). In some experiments, Trypan Blue dye exclusion counting was performed using an automated cell counter (ViCell, Beckman-Coulter, Fullerton, USA). Cell viability in test conditions is reported as a percentage relative to the negative control treatment.

Glutathione level assay.

[0252] Total intracellular glutathione (GSH+GSSG) was measured using a glutathione assay kit (Cayman Chemical #703002, Ann Arbor, USA) exactly according to instructions. 200,000 HT-1080 cells per well were seeded overnight in 6-well dishes (Corning). The next day cells were treated with compounds for 5 hours, then washed once in 500 μΙ_ PBS and harvested by scraping into phosphate buffer (10 mM X, 1 mM EGTA). Cells were then lysed by sonication (7 cycles, 2 sec on, 1 sec off) and spun at 4°C for 15 minutes at 13,000 rpm to pellet membranes. Supernatants were mixed with 500 μΙ_ of a 10% solution of metaphosphohc acid (w/v), vortexed briefly, and centrifuged for 3 minutes at 4000 rpm. The supernatant was transferred to a new tube and to this was added 50 μΙ_ of triethanolamine solution (4M). This was vortexed and 50 μΙ_ per sample was aliquoted to each well of a 96-well plate. 150 μΙ_ of assay buffer containing 5,5'-dithiobis(2-nitrobenzoic acid)(DTNB, Ellman's reagent) was added to each well and the reaction was incubated for 25 minutes at room temperature with rotation, at which point absorbance was measured at 405 nM. The GSH concentration was calculated in reference to a GSH standard curve and normalized to total cell number per well, as determined from parallel plates.

RNA-Seauencinci.

[0253] RNA was isolated from compound-treated HT-1080 cells as described below for the RT-qPCR reactions. We then used poly-A pull-down to enrich mRNAs from total RNA samples (1 pg per sample, RIN>8) and prepared libraries using the lllumina TruSeq RNA prep kit (San Diego, USA). Libraries were then sequenced using an lllumina HiSeq 2000 instrument (Columbia Genome Center, Columbia University). Here, five samples were multiplexed in each lane, to yield the targeted number of single-end 100 bp reads for each sample (30 million), as a fraction of 180 million total reads for the whole lane. Short reads were mapped to the human reference genome using Tophat (Trapnell et al., 2009). The relative abundance of genes and splice isoforms was determined using Cufflinks (Trapnell et al., 2010). Differentially expressed genes and isoforms under the various experimental conditions were examined using Cuffdiff, a program included in the Cufflinks package. Modulatory effect profiling.

[0254] Modulatory effect (M_e) profiling was performed as described (Dixon et al., 2012). In Figures 1A and 1 B, the following ferroptosis inhibitors were tested (high dose of 10 point, 2-fold dilution series in brackets): cycloheximide (CHX, 50 μΜ), ferrostatin-1 (Fer-1 , 2 μΜ), trolox (300 μΜ), U0126 (15 μΜ), ciclopirox olamine (CPX, 50 μΜ) and beta-mercaptoethanol (β-ΜΕ, 20 μΜ). For the experiments, the results of which are shown in Figures 5A and 5B, the following lethal compounds were tested (high dose of 10 point, 2-fold dilution series in brackets): methotrexate (100 μΜ), bortezomib (5 μΜ), doxorubicin (50 μΜ), chlorambucil (500 μΜ), irinotecan (5 μΜ), SAHA (50 μΜ), actinomycin D (1 μg/mL), gefitinib (50 μΜ), taxol (5 μΜ), sorafenib (10 μΜ), erlotinib (5 μΜ), GX15 (10 μΜ), staurosporine (STS, 2 μΜ), phenylarsine oxide (PAO, 1 μΜ), RSL3 (5 μΜ), H₂O₂ (5 mM), ABT-263 (50 μΜ), 6-thioguanine (5 μΜ), 17-AAG (5 μΜ) and imatinib (50 μΜ).

Caspase-3/7 Activity Assay.

[0255] Cells were seeded at 1500 cells/well in a volume of 40 μΙ_ media in 384 well plates (Corning) for 24 hours prior to treatment of lethals and/or inhibitors. Following compound treatment for 24 hours, 10 μΙ_ of a 1 :100 v/v dilution of Apo-One Homogeneous Caspase 3/7 substrate solution/assay buffer (Promega, Madison, USA) was added to samples and the plate was vigorously agitated for 30 seconds. Plates were then incubated for eight hours in the dark at room temperature, allowing for caspase-3/7 cleavage of the fluorogenic consensus substrate, before measuring fluorescence at excitation/emission wavelengths of 498/521 nm using a Victor3 plate reader (Perkin Elmer).

Bioinformatics.

[0256] Gene Ontology (GO) process enrichment was computed using the web-based GOrilla tool with default settings ((Eden et al., 2009)). The erastin 36-gene RNA-Seq signature was used to query the Molecular Signatures Database (MSigDB).

Biological Data Collection and Statistical Analyses.

[0257] Except where indicated, all experiments were performed at least three times on separate days as independent biological replicates. The data shown represents the mean+/-SD of these replicates. All statistical analyses and curve fitting were performed using Prism 5.0c (GraphPad Software, La Jolla, USA).

Clinical Data Description.

[0258] The Food and Drug Administration (FDA) collects and maintains spontaneously submitted adverse event reports in the Adverse Event Reporting System (FAERS). We downloaded 2.9 million adverse events from FAERS representing reports through the fourth quarter of 201 1 . To complement FAERS we also downloaded the OFFSIDES (Tatonetti et al., 2012). In addition, we extracted the laboratory values, clinical notes, prescription orders, and diagnosis billing codes from the electronic health records (EHR) at Columbia University Medical Center/New York Presbyterian Hospital for 316 patients with at least one prescription order of sorafenib, dasatinib, eriotinib, gefetinib, imatinib, lapatinib or sunitunib. These data were employed in the analysis to identify adverse events uniquely associated with sorafenib treatment.

Example 2

System x_g ^" inhibition results in ferroptosis in monolayer and three- dimensional spheroid cultures

[0259] Erastin and SAS both inhibit system x_c ^" function and trigger ferroptosis in a similar manner in human HT-1080 fibrosarcoma cells (Dixon et al., 2012). We used a modulatory profiling strategy (Wolpaw et al., 201 1 b,

Dixon et al., 2012) to test whether this observation was generalizable to other cell types. This method allows for the simplified detection and presentation of interactions between small molecule inducers and suppressors (or enhancers) of cell death (modulatory effect, M_e < 0, sensitization; M_e = 0, no effect; M_e >

0, rescue). We observed that in five different human cancer cell lines, cell death induced by either erastin or SAS was prevented by the same canonical ferroptosis inhibitors: β-ΜΕ, CPX, Fer-1 , trolox, the MEK inhibitor U0126, or the protein synthesis inhibitor cycloheximide (CHX) (Yagoda et al., 2007,

Yang and Stockwell, 2008, Dixon et al., 2012) (Figures 1A and 1 B). Thus, the ferroptotic death phenotype, whether induced by erastin or SAS, appears to be indistinguishable in all cell lines tested, and in all cases involves inhibition of system x_c ^" function, as suggested by the consistent suppression of death by β-ΜΕ.

[0260] To date, studies of the ferroptotic phenotype have been conducted largely in cells cultured under standard (two-dimensional) conditions. We therefore tested whether the ferroptotic mechanism was generalizable to more complex cellular structures, specifically multicellular tumor spheres (MCTS), three-dimensional cellular aggregates proposed to recapitulate key aspects of the structural and metabolic heterogeneity observed in tumor fragments and micrometastases (Friedrich et al., 2009). We grew MCTSs from four suitable cancer cell lines (HT-1080, Calu-1 , A549 and HCT-1 16) for 72 hours and then investigated the effects of erastin +/- β- ME and Fer-1 on MCTS growth and viability. For comparison, we also tested RSL3 (which triggers ferroptosis without inhibiting system x_c ^") and staurosporine (STS, which triggers apoptosis, not ferroptosis) in these MCTSs. HT-1080 and Calu-1 MCTSs were killed by erastin and RSL3 (Figures 1 C and 1 D). Consistent with results obtained in monolayer cells (see above and (Dixon et al., 2012)), the effects of both erastin and RSL3 were rescued by Fer-1 , while β-ΜΕ rescued the effects of erastin, but not RSL3 (Figures 1 C and 1 D). Neither β-ΜΕ nor Fer-1 modulated the effects of STS on MCTS growth or viability (Figures 1 C-1 F), confirming that these inhibitors specifically block ferroptosis in this environment. These results demonstrate that erastin and RSL3 can activate ferroptosis in complex, three-dimensional cellular aggregates.

[0261] Erastin inhibits cystine import, while RSL3 does not (see Introduction). Results obtained with A549 and HCT-1 16 MCTSs suggest that this difference can have important consequences for the effects of these compounds on cell growth (Figures 1 E and 1 F). Erastin slowed the growth of A549 and HCT-1 16 MCTSs (Figures 1 E and 1 F), but the effects were less pronounced than in MCTSs formed from HT-1080 or Calu-1 cells. Erastin inhibited system x_c ^" activity to a similar extent in A549, HCT-1 16 and Calu-1 cells (Figure 1 G and 1 H), ruling out a differential effect on system x_c ^" activity as an explanation for the enhanced resistance of A549 and HCT-1 16 cells to erastin. We noted that the effects of erastin on A549 and HCT-1 16 MCTS growth were weakly (A549) or not at all (HCT-1 16) rescued by Fer-1 , demonstrating that in these MCTSs the inhibition of growth was not due entirely (for A549) or at all (for HCT-1 16) to the induction of ferroptosis. However, in both cases, growth was fully restored by β-ΜΕ, showing that cystine deprivation, in the absence of ferroptosis induction, is sufficient to inhibit cell growth. Thus, we hypothesized that certain cell types (e.g. HCT- 1 16, A549) may be intrinsically less prone than others (HT-1080, Calu-1 ) to accumulate death-inducing lipid ROS. Consistent with this hypothesis, A549 and HCT-1 16 cells cultured either as MCTSs or as monolayers were resistant to the effects of RSL3, which triggers lipid ROS, but is unable to block cystine import (Figure 1 1). These results confirm our prediction (Dixon et al., 2012) that, even when it is not possible to induce ferroptosis, erastin and related agents can effectively inhibit cell growth by interfering with other cystine- dependent processes in the cell.

Example 3

Erastin inhibits system x_g ^" function potently and specifically

[0262] The ability to modulate system x_c ^" activity would be clinically useful but requires more potent inhibitors of this transporter. We, therefore, compared the ability of erastin and SAS to specifically inhibit system x_c ^". Biochemically, high levels of intracellular glutamate drive its exchange for extracellular cystine in a sodium-and-ATP-independent manner (Bannai and Ishii, 1988). Consistent with our previous findings (Dixon et al., 2012), erastin treatment (5 μΜ) completely abolished system x_c ^" (SLC7A1 1 +SLC3A2)- mediated, Na⁺-independent uptake of radiolabeled [¹⁴C]-cystine in both HT- 1080 fibrosarcoma and Calu-1 lung carcinoma cancer cells, as did sulfasalazine (SAS) at 100-fold higher concentrations (500 μΜ) (Figure 2A). Conversely, erastin and SAS had no effect on system-L-(SLC7A5+SLC3A2)- mediated, Na⁺-independent [¹⁴C]-phenylalanine uptake, showing that neither erastin nor SAS are able to inhibit system L activity in these cell lines (Figure 2B). A positive control inhibitor of system L, D-phenylalanine, did suppress [¹⁴C]-phenylalanine uptake, confirming that system L activity was theoretically inhibitable under these conditions (Figure 2B). Thus, erastin (and SAS) demonstrate selectivity towards the inhibition of system x_c ^" function and are unlikely to function as broad-spectrum inhibitors of transporter activity.

[0263] To monitor system x_c ^" activity in an additional, independent format, we developed a medium-throughput assay that detects glutamate release into regular Na⁺-containing culture medium using an enzyme-coupled fluorescent assay (Figure 2G). First, we confirmed the specificity of this assay by showing that erastin treatment, as well as silencing of SLC7A11 by two independent siRNAs, both resulted in a significant, quantitatively similar inhibition of glutamate release (Figure 2C and 2D). As a control, we silenced the system L transporter subunit SLC7A5 using two independent siRNAs and found that this had no consistent effect on the basal or erastin-mediated inhibition of glutamate release (Figure 2H and 21). This result confirms the specificity of this assay and argues against the hypothesis (Dixon et al., 2012) that SLC7A5 is required for erastin to inhibit system x_c ^".

[0264] In light of recent clinical failures using SAS (Robe et al., 2009), it is desirable to identify more potent and drug-like inhibitors of system x_c ^". Using the glutamate release assay, we found that erastin was -2,500 times more potent than SAS as an inhibitor of system x_c ^" function in both HT-1080 and Calu-1 cells (HT-1080: erastin IC₅₀ = 0.20 μΜ, 95% C.I. 0.1 1 - 0.34 μΜ, SAS ICso = 450 μΜ, 95% C.I. 280-710 μΜ; Calu-1 erastin IC₅₀ = 0.14 μΜ, 95% C.I. 0.081 -0.21 μΜ, SAS IC₅₀ = 460 μΜ, 95% C.I. 350-590 μΜ) (Figure 2E). Other small molecules that trigger iron-dependent lethality, including artesunate (Hamacher-Brady et al., 201 1 ), RSL3 (Yang and Stockwell, 2008) and PEITC (Trachootham et al., 2006), did not inhibit system x_c ^" activity (Figure 2F), showing that the inhibition of system x_c ^" function by erastin and SAS is specific, and not a general feature of iron-dependent lethal compounds. Moreover, co-treatment with β-ΜΕ did not prevent erastin or SAS from inhibiting system x_c ^" activity (Figure 2F), thus ruling out the trivial possibility that β-ΜΕ prevents cell death by directly binding and inactivating erastin or SAS. Together, these results establish erastin as a potent and specific inhibitor of SLC7A1 1 -dependent system x_c ^" function.

Example 4 Inhibition of system x_g ^" causes depletion of glutathione, which is necessary for erastin-induced ferroptosis

[0265] Within cells cystine is reduced to cysteine (Bannai and Ishii, 1982), which serves as the rate-limiting precursor for the synthesis of the tripeptide glutathione (γ-L-glutamyl-L-cysteinylglycine), a key intracellular antioxidant (Lushchak, 2012). We hypothesized that inhibition of system x_c ^~- mediated cystine import (Figure 2A) would deplete cells of glutathione and that this depletion would be necessary for erastin to induce ferroptosis. Indeed, erastin (1 ) both inhibited glutamate release (i.e. system x_c ^" function) and depleted total glutathione (GSH+GSSG) from cells, while an inactive (i.e. non-lethal (Yagoda et al., 2007)) erastin analog lacking the p-chlorophenoxy moiety (erastin-A8, 2) neither inhibited glutamate release nor depleted glutathione (Figures 3A-C). Of note, complete depletion of glutathione was observed in this experiment at a time-point (5 hours) that precedes the onset of oxidative cell death at >6 hours (Dixon et al., 2012), consistent with a model whereby the early loss of glutathione allows for the subsequent iron- dependent accumulation of lethal lipid ROS (a model is presented in Figure 3F).

[0266] To test whether glutathione depletion was necessary for erastin to induce ferroptosis, we performed a compound combination testing study, measuring in parallel glutathione levels at 5 hours (when all cells are still alive) and cell viability at 24 hours (Figures 3D and 3E). In HT-1080 cells, the depletion of glutathione and loss of cell viability caused by erastin treatment were both rescued by co-treatment with β-ΜΕ, suggesting that glutathione depletion is necessary for death. To test if this was so, we examined the effect of L-buthionine sulfoximine (BSO), a specific inhibitor of γ- glutamylcysteine synthetase (γ-GCS), the rate-limiting enzyme in glutathione synthesis (Griffith and Meister, 1979). Cells treated with erastin+p-ME+BSO were depleted of glutathione at 5 hours and died by 24 hours. This death was ferroptotic, as it was prevented by the addition of the ferroptosis-specific inhibitor Fer-1 ; Fer-1 prevented ferroptosis without rescuing glutathione levels, consistent with the hypothesis (Dixon et al., 2012) that this compound acts downstream of system x_c ^" inhibition and glutathione depletion to scavenge lethal lipid ROS species (Figure 3F). In summary, glutathione depletion downstream of the inhibition of system x_c ^"-mediated cystine uptake appears to be necessary for erastin to induce ferroptosis.

[0267] Of note, compared to erastin, BSO treatment alone resulted in insignificant glutathione depletion and cell death at 5 hours and 24 hours, respectively, under these assay conditions (Figures 3D and 3E). The differential effects of erastin and BSO on glutathione levels over the short term are consistent with previous results demonstrating more rapid glutathione depletion in cells cultured in cystine-free media compared to those treated with BSO (Antunes and Cadenas, 2001 ), while the differential effects on cell viability are likely attributable to the ability of the system-x_c ^"-dependent cystine/cysteine redox cycle (which is unaffected by BSO) to preserve cell viability in the face of glutathione depletion (Banjac et al., 2008, Mandal et al., 2010). That said, consistent with the hypothesis that glutathione depletion alone is ultimately sufficient to trigger ferroptosis, we observed that BSO treatment for 30 hours did deplete cells of glutathione (Figure 3G) and caused observable Fer-1 -sensitive cell death induction in cells grown for 48 or 96 hours (Figure 3H). Thus, the inhibition of glutathione synthesis is, in addition to being necessary for the induction of ferroptosis by erastin, also sufficient to induce ferroptosis alone, albeit with slower death kinetics than the combined inhibition of system x_c ^" function and glutathione synthesis.

Example 5

Erastin structure-activity relationship (SAR) analysis and isolation of analogs with improved lethal potency

[0268] We hypothesized that it would be possible to further improve the potency of the erastin scaffold through targeted synthesis. Towards this end, we undertook a search for more potent analogs, beginning with a new erastin achiral analog (3) that lacked the methyl group at the chiral center, and that had an isoproproxy group in place of the ethoxy group of the parent erastin compound (1 ). This compound (3) was more synthetically accessible, but otherwise exhibited a similar lethal potency as 1 in HT-1080 cells. Using a novel synthetic route, we synthesized 19 analogs of 3, and tested each in HT- 1080 cells in a 10-point, 2-fold dose-response assay for lethal potency and efficacy. To assess in each case whether lethality involved inhibition of system x_c ^" function, as opposed to the induction of another form of death, experiments were performed +/- β-ΜΕ. To assess in a parallel assay the correlation between lethal potency and inhibition of system x_c ^" activity, we examined glutamate release using a high throughput, 96-well Amplex Red assay system in human CCF-STTG1 astrocytoma cells. Of note, we observed that glutamate release in CCF-STTG1 cells is overall more sensitive to erastin and analogs than HT-1080 cells (compare Figure 4B to Figure 2E), although the observed rank order of system x_c ^" inhibition in CCF-STTG1 cells and lethal potency in HT-1080 were found to be well correlated (Figures 4A and 4B), supporting the role of xCT in ferroptosis.

[0269] Erastin's quinazolinone core (Region A, Figure 4) is found in a number of biologically active compounds and is considered to be a "privileged" scaffold (Welsch et al., 2010). Modifications to this region (4-10), including substitution of the quinazolinone for quinolone (4) or indole (5), obtained using a Meth-Cohn quinoline synthesis, all resulted in moderate to severe losses of lethal potency compared to 3, suggesting that the unmodified quinazolinone scaffold is essential for the lethality of erastin. Modifications to the piperazine linker (Region B, 11 -12) were not tolerated, with 12 being completely inactive in both the HT-1080 lethality and CCF-STTG1 glutamate release assays. We speculate that rigidification of this portion of the molecule is essential for activity and that an increase in the number of rotatable bonds in this region results in a higher entropic cost of binding, decreasing the lethal potency. Single atom changes to the acetoxy spacer (Region C, 13-15) were likewise poorly tolerated, resulting in significant losses in potency that correlated with reduced inhibition of system x_c ^" activity. Strikingly, subtle modification to Region D, including replacement of the chlorine with a fluorine (16), replacement of the para-chloro substituent with a meta-chloro group (17) or elimination of it altogether (18) reduced or abrogated the lethality and system x_c ^" inhibitory activity. As suggested by the weakened potency of 16 and 17, and the inactivity of 18, we hypothesize that the chlorine atom makes a key halogen bonding interaction with a surrounding Lewis base (Wilcken et al., 2012) that is essential for binding. [0270] Finally, modifications to Region E, including the addition of a bromo group (20), a phenyl (21 ) or a furanyl substituent (22) resulted in approximately 5-fold increases in lethal potency that were mirrored by 5 to 10- fold increases in the ability of these molecules to inhibit system x_c ^" activity compared to 3; the most potent compound, 21 , inhibited glutamate release with sub-5 nM potency in the CCF-STTG1 assay. Crucially, these more potent compounds triggered this enhanced lethality in HT-1080 cells via ferroptosis, as death was fully suppressed by β-ΜΕ. These results demonstrate that it is possible to improve the potency of the erastin scaffold substantially, especially via modifications to Region E.

Example 6

The effects of erastin on cell physiology are attributable to the depletion of intracellular cystine

[0271] These results suggested that inhibition of system x_c ^" function by erastin and erastin analogs, resulting in the blockade of cystine uptake and the subsequent depletion of glutathione, was necessary for the lethal effects of erastin. To search in a global, unbiased manner for other cellular processes that might also be necessary for the lethal effects of erastin, we performed RNA sequencing (RNA-Seq) of mRNA from HT-1080 cells treated for 5 hours with DMSO, erastin (10 μΜ), β-ΜΕ (18 μΜ) or erastin + β-ΜΕ. The β-ΜΕ condition was included to enable the effects attributable to the inhibition of system x_c ^" function and consequent cystine depletion to be isolated from any effects due to perturbation of other targets, which would likely be independent of cystine levels. [0272] From two independent biological replicate experiments, we obtained an average of about 30.5 million unique mapped reads and 15,727 unique transcripts (with Fragments Per Kilobase of exon per Million reads [FPKM]>1 ) per compound treatment condition. After data processing and averaging of replicates, we identified 34 mRNAs with two-fold more counts ('up-regulated') and six mRNAs with two-fold fewer counts ('down-regulated') in erastin-treated samples versus DMSO-treated controls (Figures 5A and 5B). Erastin-induced changes in mRNA expression were reversed by co- treatment with β-ΜΕ for the 34 up-regulated genes (Mann-Whitney test, P < .0001 ) and the 6 down-regulated genes (Mann-Whitney test, P = 0.002). As β-ΜΕ co-treatment restores intracellular cysteine levels in erastin-treated cells (as indicated by the restoration of glutathione levels, Figure 4B), these results suggest that the major effects of erastin on cellular physiology are most due to depletion of intracellular cystine, downstream of the inhibition of system x_c ^~ function.

Example 7

Transcriptional changes reveal a PD marker of erastin activity

[0273] Pharmacodynamic (PD) markers can be used to determine when cells are responding to system x_c ^" inhibition, such as in response to erastin. This will be crucial for the further clinical development of such agents, to determine effective exposure of tissues in vivo. Overall, the most highly up- regulated gene observed in erastin-treated HT-1080 cells by RNA-Seq was CHAC1 (~24-fold, Figure 5A). In independent samples we confirmed using RT-qPCR that erastin induced up-regulation of CHAC1 in both HT-1080 and Calu-1 cells, as well as in erastin-sensitive mouse embryonic fibroblasts (Figure 5C, Figure 5F), validating the results of the RNA-Seq analysis. Treatment of HT-1080 cells with the transcriptional inhibitor actinomycin D (1 g/mL), or the translation inhibitor CHX, prevented erastin-induced upregulation of CHAC1 (Figure 5G), demonstrating that upregulation of CHAC1 following erastin treatment requires new transcription and translation, as opposed to protection of existing transcripts from degradation.

[0274] We next investigated the signals leading to CHAC1 upregulation. CHAC1 is an ER-stress-responsive gene upregulated downstream of the canonical ATF4-ATF3-CHOP ER stress response pathway (Gargalovic et al., 2006, Mungrue et al., 2009). Indeed, CHAC1 upregulation was observed in response to the ER stress-inducing agent thapsigargin (Figure 5F), and a number of the genes identified by RNA-seq to be up- regulated by erastin are known regulators of the ER stress response (e.g. ATF3, DDIT3 (CHOP)). In HT-1080 cells, significant CHAC1 up-regulation was observed in response to a 5 hour treatment with erastin, SAS and sorafenib (see below), but not in response to: (i) other iron-dependent, oxidative lethal agents (RSL3, artesunate), (ii) rotenone, an agent that triggers mitochondrial ROS production (Barrientos and Moraes, 1999, Dixon et al., 2012), or (iii) a 30 hour treatment with the glutathione-depleting agent BSO (Figure 5D; Figure 5G). Thus, CHAC1 upregulation displayed selectivity towards those treatments that deplete cystine, as opposed to agents that elevate ROS or deplete the cell of glutathione without affecting cystine levels. Indeed, CHAC1 upregulation in response to erastin was fully reversed by co- treatment with β-ΜΕ in both the RNA-Seq and RT-qPCR analyses (Figures 5A and 5C), but not by DFO or U0126 (Figure 5H), treatments that nonetheless prevent lethal ROS accumulation and ferroptotic cell death (Dixon et al., 2012). These data suggest that CHAC1 upregulation following erastin and SAS treatments is a specific response to the depletion of intracellular cystine or cysteine.

[0275] Finally, we tested the generality of the CHAC1 upregulation response, and observed that across a panel of 13 cancer cell lines, treatment with erastin, but not the apoptosis-inducer STS, resulted in a significant increase in CHAC1 expression (Figure 5E). These results demonstrate that erastin can trigger a number of changes in cell physiology specifically linked to cystine depletion and that CHAC1 up-regulation could be a useful transcriptional pharmacodynamic marker for exposure to erastin and other agents that deplete cells of cystine or cysteine. Example 8

Modulatory profiling identifies sorafenib as an inhibitor of system x_g-^"

[0276] High levels of system x_c ^" activity and glutathione are reported to contribute to drug resistance (Dai et al., 2007). We therefore used a modulatory profiling strategy to test whether system x_c ^" inhibition using erastin (10 μΜ), or glutathione depletion using BSO (2.5 mM), synergistically enhanced the lethality of twenty mechanistically diverse lethal compounds (see Materials and Methods, Example 1 above) in A549 and HCT-1 16 cells over 24 hours. Overall, we observed that most compound-compound modulatory effect (M_e) values clustered around zero (i.e. no supra-additive enhancement of death), indicative of mechanistically distinct effects on growth and viability (Figures 6A and 6B). However, we observed consistent supra- additive lethal effects when erastin or BSO were combined with any of three other compounds: RSL3, which like erastin induces ferroptosis, but through a non-system-Xc^" mechanism (Yang and Stockwell, 2008); phenylarsine oxide (PAO), a thiol oxidant whose lethal activity is opposed by glutathione- dependent enzymes (Lillig et al., 2004); and sorafenib (BAY 43-9006, Nexavar), a multi-kinase inhibitor, generally thought to target primarily RAF and VEGR kinases, that is clinically-approved for the treatment of renal cell carcinoma (Wilhelm et al., 2006). The similarity of the profiles observed with erastin and BSO provide further support for the hypothesis that these compounds trigger death through a common pathway. Most intriguingly, these results suggested that sorafenib, like RSL3 and PAO, could impact a glutathione-dependent process essential for cell viability. [0277] Despite the known activity of sorafenib as a multi-kinase inhibitor (Wilhelm et al., 2006), there is disagreement about whether the sorafenib cellular lethal mechanism of action involves kinase inhibition or binding to an alternative target (Wilhelm et al., 2008). Given the results of the modulatory profile experiment, we asked whether sorafenib treatment alone could trigger ferroptosis. We observed that in HT-1080 cells, sorafenib (10 μΜ, 24 hours), like the positive control erastin (10 μΜ, 24 hour), induced cell death that was significantly inhibited by co-treatment with β-ΜΕ, Fer-1 and DFO, consistent with induction of ferroptosis (Figure 6C). Death induced by the non-specific kinase inhibitor staurosporine (STS), included as a control for specificity, was not prevented by these inhibitors. The ability of β-ΜΕ to prevent sorafenib-induced cell death implied that sorafenib, like erastin and SAS (but unlike RSL3) triggers ferroptosis by inhibiting system x_c ^". Indeed, like erastin, sorafenib, but not the ABL kinase inhibitor imatinib, caused a dose-dependent inhibition of system x_c ^"-mediated glutamate release in HT- 1080 cells (Figure 6D). The ability of sorafenib and erastin to suppress system x_c ^" activity was not inhibited by co-treatment with Fer-1 , demonstrating that this effect is up-stream of Fer-1 -sensitive ROS accumulation (Figure 6D). As with erastin and SAS, we also observed a robust transcriptional up- regulation of CHAC1 in response to sorafenib treatment (Figure 5D), suggesting that like these compounds, sorafenib activates an ER stress response by depriving cells of cysteine. Thus, like erastin, sorafenib can trigger ferroptosis and does so by inhibiting system x_c ^" activity and blocking the uptake of cystine.

Example 9 Identification of sorafenib structural features required for ferroptosis

[0278] Sorafenib could conceivably inhibit system x_c ^" activity by modulating the activity of kinases that control system x_c ^" function, through modulation of a novel target (e.g. SLC7A1 1 itself, or a related regulatory protein) or through both mechanisms acting in parallel. We took two approaches to address this question. First, we examined the effects of functionally-related kinase inhibitors. The global pattern of kinase inhibition by sorafenib against 300 purified kinase domains is highly similar to that of nilotinib, masitinib and imatinib (Anastassiadis et al., 201 1 ), yet none of these latter agents trigger ferroptosis, as defined by sensitivity to ferroptosis-specific inhibitors (Figure 6E). Like sorafenib, these kinase inhibitors did trigger about 10-40% loss of cell viability that was not attenuated by ferroptosis-specific inhibitors, likely reflecting activity against kinase targets whose inhibition impairs cell viability via non-ferroptotic (e.g. apoptotic) mechanisms.

[0279] Second, we performed a structure-activity-relationship analysis of 53 sorafenib analogs with the goal of understanding which portions of the sorafenib scaffold are most relevant for the inhibition of system x_c ^". We made numerous targeted structural changes in search of analogs that displayed changes in lethal potency (EC₅o) or in the degree of death suppression (DS) by β-ΜΕ and Fer-1 at the fixed concentration of 10 μΜ (key results summarized in Figure 7A). These experiments were performed in HT-1080 cells assayed in 2-fold, 10-point dilution series assays starting at a highest concentration of 40 μΜ. We divided the scaffold into three regions of interest: the anilino aryl ring, the urea and the phenoxypyridinecarboxamide (red, blue and green, respectively, in Figure 7A). We initially focused on the anilino aryl ring. Compared to the parent sorafenib (23), analogs with only a phenyl ring (24) or lacking the CF₃ group (25) were inactive at the highest concentrations tested in our assay (EC50 > 40 μΜ), indicating that the CF₃ group is essential for the induction of ferroptosis. Analogs with CF₃ in the meta (26) or para (27) positions, or with di-metasubstituted CF₃ moieties, (28) largely retained potent lethality, while an analog with the CF₃ group in the ortho position (29) was inactive. It is likely that the binding of sorafenib to its target relevant to induction of ferroptosis is highly constrained by the position of these groups, and that the CF₃ group must fit into a specific hydrophobic pocket, much as it does when binding to BRAF (Wan et al., 2004). These results suggested the existence of specific molecular recognition events with the target of these sorafenib analogs.

[0280] We next examined the urea portion of the scaffold, which contributes substantially to sorafenib kinase inhibition via hydrogen bonding interactions (Lowinger et al., 2002, Wan et al., 2004). Substitution of the urea for a thiourea (30) was well tolerated. However, replacement of the urea with a secondary amine (31 ), sulfonamide (32) or an amide (33) eliminated or vastly reduced the lethal potency. We speculate that the key hydrogen bond donor-acceptor pairs of the urea in the target pocket are crucial for binding to the ferroptosis-relevant target. Alternatively, the urea (or thiourea) may confer a specific conformation upon the compound that is favored for activity.

[0281] Next we examined the phenoxypyridinecarboxamide. Notably, a compound lacking the pyridine (34) was inactive, but an analog with only a phenoxyphenyl (35) or, alternatively, with the inclusion of an extra methyl group on the carboxamide (36) largely retained the ability to induce ferroptosis (<2-fold loss of potency). Of note, compounds lacking the 4-pyhdyl and carboxamide moiety (35), or containing secondary amides (36), are individually each >5-fold less potent CRAF kinase inhibitors than compounds without these changes (Lowinger et al., 2002). Within this context, the only modest decreases in the ability of these compounds to induce ferroptosis provide evidence that the canonical kinase targets of sorafenib (RAF family, VEGFR) are unlikely to be relevant to the ferroptosis-inducing mechanism of action of sorafenib.

[0282] Finally, we evaluated selected sorafenib analogs for their effects on system x_c ^" function, using the glutamate release assay, and on caspase- 3/7 activity, using a fluorogenic substrate cleavage assay. Consistent with the above data, two active (e.g. lethal) sorafenib analogs (SRS13-45 (28) and SRS13-60 (30)) significantly inhibited system x_c ^" function, while two non-lethal analogs (EC₅₀ > 40 μΜ), SRS13-67 (29) and SRS14-98 (31 ), did not (Figure 7B). The ability of these analogs to induce caspase-3/7 (DEVDase) activity did not vary significantly from DMSO-treated controls, especially compared to cells treated with a positive control inducer of apoptosis, the proteasome inhibitor MG132 (Figure 7C). These results may help account for other reports of caspase-independent, sorafenib-induced death (Panka et al., 2006, Katz et al., 2009) and strongly support the hypothesis that sorafenib triggers cell death via inhibition of system x_c ^" and activation of ferroptosis.

Example 10

Association of sorafenib with a unique constellation of adverse clinical events. [0283] Sorafenib is a clinically-approved drug used to treat renal cell carcinoma and other indications. We wondered whether the effects of sorafenib on system x_c ^" uncovered here would result in a unique spectrum of clinical observations in patients treated with sorafenib. Previously, we applied a large-scale statistical analysis to the Food and Drug Administration Adverse Event Reporting System (FAERS) to systematically identify drug effects and interactions (Tatonetti et al., 2012). Here, we sought to use this approach to discover correlations between sorafenib exposure and human health unique to this drug. First, we identified those reports with the exposure to sorafenib, and a set of reports that could serve as controls; for this, we used a high dimensional propensity-score model previously validated for this use in the FAERS that has been shown to mitigate confounding bias and to improve the accuracy of statistical estimates (Tatonetti et al., 2012). Using disproportionality analysis (Bate and Evans, 2009) we identified adverse drug effects for sorafenib and for a set of comparison kinase-targeted drugs (dasatinib, erlotinib, gefitinib, imatinib, lapatinib and sunitinib) drugs for which sufficient data was available in our data-set. We then filtered out effects that could be attributed to chemotherapy and grouped the drug-effect associations by the physiological system that the adverse event affected. For example, cardiovascular-related adverse events were grouped into the cardiovascular system category. For each kinase inhibitor, we counted the number of reports in each of 20 physiological system categories that involved the above drugs. We then compared this number to the counts obtained from the selected control cohorts. Using a Fisher's exact test, we evaluated significance of associations of each kinase inhibitor to each physiological system category and then we plotted these data as a heatmap, clustering the data in an unsupervised, hierarchical manner using the computed P values (Figure 8).

[0284] Focusing on sorafenib, we observed that, compared to other clinically approved kinase inhibitors, none of which are known (e.g. imatinib, Figure 6D) or likely to inhibit system x_c ^", sorafenib has a unique adverse event profile. Most notably, sorafenib treatment was associated with a significant number of adverse events in 15/20 physiological system categories, the most observed in this analysis for any drug. Conversely, imatinib was associated with a significant number of adverse events in 0/20 categories. A unique subset of the adverse events uniquely associated with sorafenib compared to all other kinase inhibitor drugs included musculoskeletal, nervous system and pathological disorders as well as hemorrhage; this pattern was not observed for patients treated with sunitinib, which is approved for the same indication (Stein and Flaherty, 2007), making it unlikely that these events are confounded by the particular patient population under study. Rather, we hypothesize that some or all of these effects may be attributable to the off- target effect of sorafenib against system x_c ^".

[0285] Amino acid uptake is crucial for cell growth and survival, leading to efforts to target this process therapeutically. Here, we have demonstrated that erastin is a potent and specific inhibitor of system x_c ^"-mediated cystine uptake, elucidated erastin's mechanism of action, and shown that other system x_c ^" inhibitors are also able to trigger ferroptosis. Collectively, our results suggest that these compounds will be useful as probes to study the lethal and non-lethal effects of cystine depletion in a variety of physiological settings and, in the case of erastin and sorafenib, are candidate leads for the development of new drugs targeting system x_c ^~.

[0286] Several lines of evidence suggest that the depletion of intracellular cystine is necessary for the lethal effects of erastin, in both monolayer and more complex three-dimensional cellular environments. First, the lethality of active versus inactive erastin analogs correlates well with their ability to inhibit system x_c ^" function. Second, β-ΜΕ, which does not prevent the inhibition of system x_c ^" by erastin (Figure 2F), but restores intracellular cysteine levels (Ishii et al., 1981 ), rescues from erastin-induced death and restores cell growth. Third, the global effects of erastin on cellular physiology, as assayed by RNA sequencing, yield a profile consistent with amino acid depletion and the induction of an ER stress response (Harding et al., 2003). Indeed, these results indicate that the effects of erastin (detectable by RNA seq) are tied exclusively to the depletion of intracellular cystine, as we did not observe any erastin-induced changes in gene expression that were not reversed by β-ΜΕ co-treatment. Further work is required to define specific targets of erastin (as well as SAS and sorafenib) relevant to the inhibition of system x_c ^" function. The inability of erastin to inhibit System L (SLC7A5+SLC3A2)-mediated transport in HT-1080 cells appears to rule out both SLC7A5 and SLC3A2 as candidate targets, at least for this molecule. Currently, the most parsimonious explanation of our results is that erastin directly binds and inhibits SLC7A1 1 . Alternatively, our results do not rule out the possibility of erastin interacting with additional intracellular targets (e.g. VDAC2/3 (Yagoda et al., 2007)), which can then inhibit system x_c ^" function. [0287] Here we have addressed several issues relevant to the development of erastin as an anti-cancer agent. Many lethal compounds lose potency against complex three dimensional tissue structures such as MCTSs, a phenomenon termed 'multicellular resistance' (Desoize and Jardillier, 2000). The ability of system x_c ^" inhibitors like erastin to kill cells in both monolayer and three-dimensional culture is therefore an important characteristic for in vivo use. The ability of erastin, and other system x_c ^" inhibitors such as SAS and sorafenib, to access multiple growth-inhibitory mechanisms tied to the inhibition of system x_c ^"-mediated cystine import, including the inactivation of the cysine/cysteine redox cycle (Banjac et al., 2008), inhibition of glutathione synthesis and inhibition of protein synthesis may aid in overcoming resistance to any one mechanism that exists in a specific cellular context.

[0288] The most highly up-regulated gene that we observed by RNA- Seq in erastin-treated cells was CHAC1. Thus, CHAC1 up-regulation may be useful as a transcriptional biomarker for cells undergoing cystine limitation. What role CHAC1 plays in the cell or in ferroptosis remains unclear. ChaC- family proteins were recently reported to function as intracellular reduced glutathione (GSH)-degrading enzymes in yeast (Kumar et al., 2012). In light of the rapid onset of glutathione depletion observed in erastin-treated cells compared to cells treated with BSO (which does not trigger CHAC1 up- regulation) we hypothesize that CHAC1 may actively contribute to glutathione depletion in cells deprived of cyst(e)ine, although this remains to be tested. In the face of transient intracellular cysteine depletion, the ability to degrade GSH might be adaptive, as it would liberate cysteine stored in GSH. However, if such a process were prolonged in the face of continuous cysteine deprivation due to system x_c ^" blockade, this could result in the accelerated elimination of the intracellular glutathione pool and the more rapid onset of ferroptotic death. Regardless, we propose that the up-regulation of CHAC1 under these conditions could serve as a useful PD biomarker for cells exposed to agents that inhibit system x_c ^" function.

[0289] An important goal is to discover compounds capable of inhibiting system x_c ^" with greater potency than existing compounds (typified by SAS and derivatives) that also retain properties suitable for in vivo administration. One route could involve further optimization of the erastin scaffold. Indeed, we have identified several erastin analogs with improved lethal potency and ability to inhibit system x_c ^" function, suggesting that further improvements along these lines are feasible. Likewise, the development of sorafenib analogs as specific system x_c ^" inhibitors holds promise, as this scaffold is already optimized for oral delivery (Wilhelm et al., 2006). The SAR analysis of sorafenib set forth herein indicates that, at least in certain cases, the elimination of features crucial for high affinity sorafenib-RAF interaction have only modest effects on the ability of these compounds to induce ferroptosis, suggesting that it may be possible to dissociate the kinase-binding and system x_c ^"-inhibitory activity of this scaffold. Regardless, the finding that sorafenib inhibits system x_c ^" and triggers the amino acid deprivation response (as indicated by up-regulation of CHAC1) provides a mechanistic explanation for previous observations that sorafenib treatment inhibits translation (Rahmani et al., 2005), induces ER stress (Rahmani et al., 2007) and enhances sensitivity to ROS (Shiota et al., 2010). [0290] In light of these findings, combined with the emerging recognition that clear cell renal cell carcinoma (the approved indication for sorafenib) is a 'metabolic disease' involving frequent dysregulation of amino acid metabolic pathways (Hakimi et al., 2013), it may be of interest to evaluate whether the efficacy of sorafenib observed in patients is due, in part, to inhibition of system x_c ^"-mediated cystine uptake. Likewise, adverse events that are observed in a percentage of patients treated with sorafenib may involve the unwanted inhibition of system x_c ^". For example, the known role of system x_c ^" in brain cell function (De Bundel et al., 201 1 ) is consistent with the adverse events associated with the nervous system function. While not all patients treated with sorafenib experience any adverse events, we suspect that specific underlying (and unknown) sensitizing factors will render a percentage of individuals more sensitive to these events and it would be important to account for this potential toxicity in the design of future therapeutic strategies.

Example 11

[0291] The following table summarizes the results of biological activity in accordance with the assays outlined above in Example 1 . "DMSO % Gl" means growth inhibition compared to DMSO control. "B-ME % Gl" mean growth inhibition compared to β-mercaptoethanol. "Fer-1 % Gl" means growth inhibition compared to Ferrostatin-1 . "SD" refers to suppression of death. All reported values are from 10μΜ treatments.

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All documents cited in this application are hereby incorporated by reference as if recited in full herein.

[0292] Although illustrative embodiments of the present invention have been described herein, it should be understood that the invention is not limited to those described, and that various other changes or modifications may be made by one skilled in the art without departing from the scope or spirit of the invention.

Claims

WHAT IS CLAIMED IS:

1 . A compound according to formula I:

wherein:

Ri , R2, R3, R4, and R₆ are independently selected from the group consisting of H, halo, CF₃, OCF₃, Ci_-4alkyl, and CN;

R₅ is independently selected from the group consisting of no atom, NH, O, and C_1-4alkyl;

R₈ is N H or no atom;

R-io is no atom, O, or Ci_-4alkyl; Rii is selected from the group consisting of no atom, aryl, heterocycle, and heteroaryl optionally substituted with a halo or a Ci_-4alkyl;

R-I 3 is selected from the group consisting of no atom, alkoxy, amine, CN, carboxy, carbocyclyl optionally substituted with Ci_-4alkyl, diol; and the dotted line ( ) is an optional double bond, or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof, with the proviso that formula I is not sorafenib,

SRS13-60

2. A compound according to claim 1 having the structure of formula (II):

wherein:

R₂ and R₃ are independently selected from the group consisting of H, halo, and CF₃;

R₄ is H or halo;

R-io is O or Ci_-4alkyl;

A is C or N;

B is O or S;

R-I2 is selected from the group consisting of no atom, amide, CN, and heteroaryl; and

3. A compound according to claim 1 having the structure of formula (III):

wherein:

R-io is O or Ci_-4alkyl;

A is C or N;

R₁₂ is selected from the group consisting of no atom, amide, CN, and heteroaryl; and

4. A compound according to claim 1 , which is selected from the group consisting of:

SRS14-23 SRS 14-24

SRS14-26

SRS15-39 SRS15-40

1-61

EJ1-79

EJ1-80 EJ1 -69

EJ2-01 EJ2-02

EJ2-03 EJ1-70

5. A compound according to claim 1 , which is selected from the group consisting of:

SRS14-27 SRS13-97

SRS14-13 SRS15-39 and combinations thereof, or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.

6. A compound according to claim 1 , which is selected from the group consisting of:

7. A compound according to claim 1 , which is

8. A pharmaceutical composition comprising a pharmaceutically acceptable salt or diluent and a compound according to formula I:

wherein:

R-i , R2, R3, R4, and R₆ are independently selected from the group consisting of H, halo, CF₃, OCF₃, Ci_-4alkyl, and CN;

R₈ is NH or no atom;

R-I 3 is selected from the group consisting of no atom, alkoxy, amine, CN, carboxy, carbocyclyl optionally substituted with Ci_-4alkyl, diol;

and the dotted line ( ) is an optional double bond, or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof, with the proviso that formula I is not sorafenib,

SRS13-60

9. A pharmaceutical composition according to claim 8, wherein the compound has the structure of formula (II):

wherein:

R₄ is H or halo;

R-io is O or Ci_-4alkyl;

A is C or N;

B is O or S;

10. A pharmaceutical composition according to claim 8, wherein the compound has the structure of formula (III):

wherein:

R-io is O or Ci_-4alkyl;

A is C or N;

1 1 . A pharmaceutical composition according to claim 8, wherein the compound has a structure that is selected from the group consisting of:

SRS14-23 SRS 14-24

SRS14-26

SRS15-39 SRS15-40

SRS15-99 SRN1-16

1-61

EJ1-79

EJ1-80 EJ1 -69

EJ2-01 EJ2-02

EJ2-03 EJ1-70

12. A pharmaceutical composition according to claim 8, wherein compound has a structure that is selected from the group consisting of:

SRS14-27 SRS13-97

SRS14-13 SRS15-39 or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.

13. A pharmaceutical composition according to claim 8, wherein the compound has a structure that is selected from the group consisting of:

14. A pharmaceutical composition according to claim 8, wherein the compound has a structure that is

SRS 14-25 or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.

15. A kit comprising a compound according to any one of claims 1 -7 together with instructions for the use of the compound.

16. A kit comprising a pharmaceutical composition according to any one of claims 8-14 together with instructions for the use of the pharmaceutical composition.

17. A method for treating a subject having dysregulated system x_c ^" activity comprising administering to the subject an effective amount of a compound according to formula I:

wherein:

R₈ is NH or no atom;

R₉ is an aryl or heteroaryl optionally substituted with a moiety selected from the group consisting of H, hydroxy, Ci- alkyl, halo, and combinations thereof; R-io is no atom, O, or Ci_-4alkyl;

and the dotted line ( ) is an optional double bond, or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.

18. A method according to claim 17, wherein the compound has the structure of formula (II):

wherein: R₂ and R₃ are independently selected from the group consisting of H, halo, and CF₃;

R₄ is H or halo;

R-io is O or Ci_-4alkyl;

A is C or N;

B is O or S;

R-I 3 is selected from the group consisting of no atom or CN, or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.

19. A method according to claim 17, wherein the compound has the structure of formula (III):

wherein:

R₂ and R3 are independently selected from the group consisting of

H, halo, and CF₃; R-io is O or Ci_-4alkyl; A is C or N;

20. A method according to claim 17, wherein the compound is selected from the group consisting of:

SRS 13-45 SRS 13-46

SRS14-23 SRS14-24

SRS14-66 SRS14-67

239

SRS15-99 SRN1-16

EJ1-61

EJ2-01 EJ2-02

EJ2-03 ^EJ -70

21 . A method according to claim 17, wherein the compound is selected from the group consisting of:

and combinations thereof, or an crystalline form, hydrate, or pharmaceutically acceptable salt thereof.

22. A method according to claim 17, wherein the compound is selected from the group consisting of:

23. A method according to claim 17, wherein the compound is:

24. The method according to claim 17, wherein the subject is a mammal.

25. The method according to claim 24, wherein the mammal is selected from the group consisting of humans, primates, farm animals, and domestic animals.

26. The method according to claim 25, wherein the mammal is a human.

27. The method according to claim 17, wherein the system x_c ^" dysregulation is selected from the group consisting of tumorigenesis, cancer stem cell maintenance, drug resistance, and neurological dysfunction.

28. The method according to claim 27, wherein the subject has a cancer selected from the group consisting of colon cancer, brain cancer, breast cancer, bone cancer, colorectal cancer, lung cancer, pancreatic cancer, bladder cancer, skin cancer, liver cancer, lymphoma, and leukemia.

29. The method according to claim 27, wherein the subject has a neurological dysfunction selected from the group consisting of multiple sclerosis, Alzheimer's disease, Parkinson's disease, myasthenia gravis, motor neuropathy, Guillain- Barre syndrome, autoimmune neuropathy, Lambert-Eaton myasthenic syndrome, paraneoplastic neurological disease or disorder, paraneoplastic cerebellar atrophy,, progressive cerebellar atrophy, Rasmussen's encephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles de la Tourette syndrome, autoimmune polyendocrinopathy, dysimmune neuropathy, acquired neuromyotonia, arthrogryposis multiplex, Huntington's disease, AIDS associated dementia, amyotrophic lateral sclerosis (AML), multiple sclerosis, an inflammatory retinal disease or disorder, an inflammatory ocular disease or disorder, and optic neuritis.

30. The method according to claim 17 further comprising administering at least one additional therapeutic agent selected from the group consisting of an antibody or fragment thereof, a cytotoxic agent, a toxin, a radionuclide, an immunomodulator, a photoactive therapeutic agent, a radiosensitizing agent, a hormone, and combinations thereof.

31 . The method according to claim 30, wherein the additional thereapeutic agent is selected from the group consisting of erastin, RSL3, analogs of erastin or RSL3, and pharmaceutically acceptable salts thereof.

32. A method for treating a subject having dysregulated system x_c ^" activity comprising administering to the subject an effective amount of a pharmaceutical composition according to any one of claims 8-14.

33. The method according to claim 32, wherein the subject is a mammal.

34. The method according to claim 33, wherein the mammal is selected from the group consisting of humans, primates, farm animals, and domestic animals.

35. The method according to claim 33, wherein the mammal is a human.

36. The method according to claim 32, wherein the dysregulated system x_c ^~ activity is selected from the group consisting of tumorigenesis, cancer stem cell maintenance, drug resistance, and neurological dysfunction.

37. The method according to claim 36, wherein the subject has a cancer selected from the group consisting of colon cancer, brain cancer, breast cancer, bone cancer, colorectal cancer, lung cancer, pancreatic cancer, bladder cancer, skin cancer, liver cancer, lymphoma, and leukemia.

38. The method according to claim 36, wherein the subject has a neurological dysfunction selected from the group consisting of multiple sclerosis, Alzheimer's disease, Parkinson's disease, myasthenia gravis, motor neuropathy, Guillain- Barre syndrome, autoimmune neuropathy, Lambert-Eaton myasthenic syndrome, paraneoplastic neurological disease or disorder, paraneoplastic cerebellar atrophy, progressive cerebellar atrophy, Rasmussen's encephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles de la Tourette syndrome, autoimmune polyendocrinopathy, dysimmune neuropathy, acquired neuromyotonia, arthrogryposis multiplex, Huntington's disease, AIDS associated dementia, amyotrophic lateral sclerosis (AML), multiple sclerosis, an inflammatory retinal disease or disorder, an inflammatory ocular disease or disorder, and optic neuritis.

39. The method according to claim 32 further comprising administering at least one additional therapeutic agent selected from the group consisting of an antibody or fragment thereof, a cytotoxic agent, a toxin, a radionuclide, an immunomodulator, a photoactive therapeutic agent, a radiosensitizing agent, a hormone, and combinations thereof.

40. The method according to claim 39, wherein the additional thereapeutic agent is selected from the group consisting of erastin, RSL3, analogs of erastin or RSL3, and pharmaceutically acceptable salts thereof.

41 . A method of activating ferroptosis in a cell comprising administering to the cell an effective amount of a compound according to formula I:

wherein:

R₈ is NH or no atom;

R-io is no atom, O, or Ci_-4alkyl;

42. A method of inhibiting system x_c ^" in a cell comprising administering to the cell an effective amount of a compound according to formula I:

wherein:

Rs is NH or no atom;

R10 is no atom, O, or Ci_-4alkyl;

43. A method for monitoring treatment of a subject having system x_c ^~ dysregulation comprising:

(a) measuring the expression level of a CHAC1 gene in a subject being treated with an amount of a compound according to any one of claims 1 -8 or a pharmaceutical composition according to any one of claims 9-14, wherein increased expression levels of the CHAC1 gene relative to a control indicate that the cells of the subject are undergoing cystine limitation; and