METHODS OF SELECTING CANCER PATIENTS FOR TREATMENT WITH Ν,Ν'- DIARYLUREA COMPOUNDS AND N,N ' - D I A R V LTH I OUREA COMPOUNDS

RELATED APPLICATION DATA

[01] This application claims priority to U.S. Provisional Patent Application No.

61/488,250, filed on May 20, 2011 and is hereby incorporated herein by reference in its entirety for all purposes.

STATEMENT OF GOVERNMENT INTERESTS

[02] This invention was made with government support under #R21AG032546 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD

[03] The present invention relates to methods of identifying cancer patients for treatment with diarylurea and diarylthiourea compounds.

BACKGROUND

[04] Translation, the mRNA-directed synthesis of proteins, occurs in three distinct steps: initiation, elongation and termination. Translation initiation is a complex process in which the two ribosomal subunits and methionyl /RNA (Met-ZRNAj) assemble on a properly aligned mRNA to commence chain elongation at the AUG initiation codon. The established scanning mechanism for initiation involves the formation of a ternary complex among eukaryotic initiation factor 2 (eIF2), GTP and Met-tRNAj. The ternary complex recruits the 40S ribosomal subunit to form the 43S pre-initiation complex. This complex recruits mRNA in cooperation with other initiation factors such as eukaryotic initiation factor 4E (eIF4E), which recognizes the 7-methyl- guanidine cap (m- ⁷ GTP cap) in an mRNA molecule and forms the 48S pre-initiation complex. Cap recognition facilitates the 43 S complex entry at the 5' end of a capped mRNA. Subsequently, this complex migrates linearly until it reaches the first AUG codon, where a 60S ribosomal subunit joins the complex, and the first peptide bond is formed (Pain (1996) Eur. J. Biochem., 236:747-771). After each initiation, the GTP in the ternary complex is converted to GDP. The eIF2 GDP binary complex must be converted to eIF2 GTP by the guanidine exchange factor, eIF2B for a new round of translation initiation to occur. Inhibition of this exchange reaction by phosphorylation of eIF2a reduces the abundance of the ternary complex and inhibits translation initiation. Forced expression of non-phosphorylatable eIF2a or Met-tRNAj causes transformation of normal cells (Marshall (2008) Cell 133 :78; Bems (2008) Cell 133:29). In contrast, pharmacologic agents that restrict the amount of eIF2 GTP-Met- tRNAj ternary complex inhibit proliferation of cancer cells in vitro and tumors in vivo (Aktas (1998) Proc. Natl. Acad. Sci. U.S.A. 95:8280), Palakurthi (2000) Cancer Res. 60:2919, Palakurthi (2001) Cancer Res. 61 : 6213). These findings indicate that more potent and specific agents that reduce amount of ternary complex are potent anticancer agents.

[05] The ternary complex plays critical roles in normal physiology and participates in the pathogenesis of several human disorders. For example, forced expression of eIF2a- S51A, a non-phosphorylatable eIF2 mutant or of Met-tRNAj transforms normal cells. Consistently, overexpression of eIF2 and inactivating mutations eIF2a kinases that cause unrestricted translation has been reported in various cancers. The ternary complex also plays an important role in the development and/or progression of other human disorders. For example, heme regulated inhibitor kinase (HRI), an eIF2 - kinase, couples hemoglobin synthesis to heme availability and influences the severity of hemolytic anemia such as β-thalassemia by regulating the abundance of the ternary complex. The eIF2cc-kinases Protein Kinase R (PKR), General Control Nonderepressible (GCN) 2, and PKR-like Kinase (PERK) are activated to shut down protein synthesis in response to viral infections, amino acid starvation or ER-stress, respectively. Inactivating mutations of PERK allow uncontrolled insulin synthesis, induces ER-stress and apoptosis of pancreatic β-cells, causing permanent neonatal diabetes in the human Wolcott-Rallison syndrome.

[06] Several features of the mRNA structure influence the efficiency of its translation.

These include the m- ⁷ GTP cap, the primary sequence surrounding the AUG codon and the length and secondary structure of the 5' untranslated region (5' UTR). Indeed, a moderately long, unstructured 5' UTR with a low G and C base content seems to be optimal to ensure high translational efficiency. Surprisingly, sequence analysis of a large number of vertebrate cDNAs has shown that although most transcripts have features that ensure translational fidelity, many do not appear to be designed for efficient translation (Kozak (1991) J. Cell. Biol, 1 15 :887-903). Many vertebrate mR As contain 5' UTRs that are hundreds of nucleotides long with a remarkably high GC content, indicating that they are highly structured because G and C bases tend to form highly stable bonds. Because highly structured and stable 5' UTRs are the major barrier to translation, mRNAs with stable secondary structure in their 5' IJTR are translated inefficiently and their translation is highly dependent on the activity of translation initiation factors. mRNAs with complex, highly structured 5' UTRs include a disproportionately high number of proto-oncogenes such as the Gl cyclins, transcription and growth factors, cytokines and other critical regulatory proteins. In contrast, mRNAs that encode globins, albumins, histones and other housekeeping proteins rarely have highly structured, GC-rich 5' UTRs (Kozak (1994) Biochimie, 76; 815-21 ; Kozak (1999) Gene, 234: 187-208). The fact that genes encoding for regulatory but not for housekeeping proteins frequently produce transcripts with highly structured 5' UTRs indicates that extensive control of the expression of regulatory genes occurs at the level of translation. In other words, low efficiency of translation is a control mechanism which modulates the yield of proteins such as cyclins, mos, c-myc, VEGF, TNF, among others, that could be harmful if overproduced.

Translation initiation is a critical step in the regulation of cell growth, such as cellular homeostasis, proliferation, differentiation and malignant transformation, because the expression of most oncogenes and cell growth regulatory proteins is translationally regulated. Consistently, increasing the abundance of the eIF2 GTP Met-tRNAj translation initiation complex transforms normal cells and contributes to cancer initiation and the severity of some anemia. One approach to inhibiting translation initiation has recently been identified using small molecule known as translation initiation inhibitors. Translation initiation inhibitors such as clotrimazole (CLT) inhibit translation initiation by sustained depletion of intracellular Ca ²⁺ stores. Depletion of intracellular Ca ²⁺ stores activates "interferon-inducible" "double- stranded RNA activated" protein kinase (PKR) which phosphorylates and thereby inhibits the a subunit of eIF2. Since the activity of eIF2 is required for translation initiation, its inhibition by compounds such as CLT reduces the overall rate of protein synthesis. Because most cell regulatory proteins are encoded for by mRNAs containing highly structured 5' UTRs, they are poorly translated and their translation depends heavily on translation initiation factors such as eIF2 and eIF4. Therefore, inhibition of translation initiation preferentially affects the synthesis and expression of growth regulatory proteins such as Gl cyclins. Sequential synthesis and expression of Gl cyclins (Dl , E and A) is necessary to drive the cell cycle beyond the restriction point in late G l . Thus, the decreased synthesis and expression of Gl cyclins resulting from CLT-induced inhibition of translation initiation causes cell cycle arrest in Gl and inhibits cancer cell and tumor growth (Aktas et al. (1998) Proc. Natl. Acad. Sci. USA, 95 :8280-8285, incorporated herein by reference in its entirety for all purposes).

[09] Like CLT, the n-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) depletes internal calcium stores, and exhibits anti-carcinogenic activity. Unlike CLT, however, EPA is a ligand of peroxisome proliferator-activated receptor gamma (PPARy), a fatty acid-activated transcription factor. Although EPA and other ligands of PPARy, such as troglitazone and ciglitazone, inhibit cell proliferation, they do so in a PPARy- independent manner (Palakurthi et al. (2000) Cancer Research, 60:2919; and Palakurthi et al. (2001) Cancer Research, 61 :6213, incorporated herein by reference in their entirety for all purposes).

SUMMARY

[10] Embodiments of the present disclosure are directed to methods of identifying cancer patients for treatment with an arylurea compound or an aryl thiourea compound. According to one aspect, the term arylyurea compound includes a diarylurea compound and vice versa. According to an additional aspect, the term arylthiourea compound includes a diarylthiourea compound and vice versa. According to this aspect, patients are identified as being candidates for treatment with a diarylurea compound or a diarylthiourea compound if the cancer cells of the patient express HRI. According to this aspect of the present disclosure, diarylurea or diarylthiourea compounds are more effective at treating cancer cells that have a higher level of HRI expression compared with cancer cells which do not express HRI or which express low levels of HRI. Accordingly, a method is provided for identifying a level, such as a substantial or significant level, of HRI expression in cancer cells from an individual and then treating the individual with a diary lurea or diarylthiourea compound. According to one aspect, the level of HRl expression exceeds a threshold level so as to indicate that treatment with a diarylurea or diarylthiourea compound would be advantageous. According to an additional aspect, the diarylurea or diarylthiourea compound inhibits translation initiation of the cancer cell or other cell characterized by abnormal proliferation.

[11] According to one aspect of the present disclosure, the compounds described herein will have a greater therapeutic efficacy in cells with higher levels of HRl. In this manner, HRl is a marker for individuals susceptible for treatment of cancer with the compounds described herein. Levels of HRl within the scope of the present disclosure sufficient to select a patient for treatment include a level within the detection limit of the device or method being used to detect the level of HRL According to one aspect, the level of HRl in a particular cancer cell or cancer cells is determined. The amount of a compound or compounds as described herein sufficient to kill a significant number of cancer cells, such as 25%, 30%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, or 99%, in an in vitro assay is determined. The patient is then administered an amount of the compound or compounds sufficient to establish a plasma concentration equal to the amount of compound used in the in vitro assay. According to another aspect, an individual is selected for treatment based upon the level of expression of HRl in a sample of cancer cells from the individual. Suitable individuals will have cancer cells expressing HRl in an amount between the limit of detection for a given method and above.

[12] An exemplary threshold HRl expression level for identifying individuals for treatment with the compounds described herein includes the level of HRl expression in PC-3 human prostate cancer cells per unit of total protein. According to one aspect, individuals having cancer cells that express HRl at levels about equal to or about greater than PC-3 human prostate cancer cells per unit of total protein are selected for treatment with one or more of the compounds described herein. HRl levels in cancer cells may be determined by methods readily available to those of ordinary skill in the art such as Western blot, in-cell western, dot blot, Elisa, sandwich Elisa, mass spectroscopy, immune-electrophoresis or any other method known for the detection of protein level. Additional methods include those for detecting HRl mRNA by real time PGR. in-situ PGR, RT-PCR, Northern blot, RNAse protection assay or any other methods known to those skilled in the art. Using well known methods, the level of HRI expression in PC-3 human prostate cancer cells per unit of total protein is determined and may be used as a standard. PC-3 human prostate cancer cells are available from the ATCC. Using well known methods, the level of HRI expression in cancer cells from an individual per unit of total protein is determined and then compared with the level of HRI expression in PC-3 human prostate cancer cells per unit of total protein. A patient is selected for treatment with the compounds described herein if the level of HRI expression in cancer cells from the individual per unit of total protein is about equal to or about greater than the level of HRI expression in PC- 3 human prostate cancer cells per unit of total protein.

[13] In an alternative embodiment, individuals may be identified for treatment with the compounds identified herein by determining the activity of HRI to phosphorylate eIF2a in cancer cells from an individual. According to one aspect, individuals having cancer cells that exhibit an HRI activity to phosphorylate eIF2a about equal to or about greater than the HRI activity of PC-3 human prostate cancer cells to phosphorylate eIF2a are selected for treatment with one or more of the compounds described herein. HRI activity in cancer cells to phosphorylate eIF2a may be determined by methods readily available to those of ordinary skill in the art such as by using an in-gel kinase assay, in vitro-kinase assay, enzyme-linked kinase assay, indirect reporter gene assay or any other method known to those of skill in the art useful for determining HRI activity. Using well known methods, the activity of HRI may be assayed in cancer cells from an individual and used as a standard. Using well known methods, the activity of HRI may be assayed in cancer cells from an individual that have been contacted with one or more compounds described herein and then compared with the activity of HRI from cancer cells that have not been contacted with one or more compounds described herein. A patient is selected for treatment with the compound or compounds described herein if the compound or compounds increase the activity of HRI in the cancer cells from the individual. According to one aspect, the compounds described herein activate HRI thereby causing phosphorylation of eIF2a and inhibition of translation initiation. Accordingly, if the compound or compounds increase activation of HRI in cancer cells relative to HRI activity in cancer cells which have not been contacted with a compound or compounds described herein or relative to HRI activity in PC -3 human prostate cancer cells, then the individual is selected for treatment with a compound or compounds described herein.

[14] According to a certain aspect, a method of identifying a candidate patient for administration of a diarylurea or a diarylthiourea compound as a treatment for cancer is provided including the steps of obtaining cancer cells from the individual, and determining a substantial or significant level of expression of HRI in the cancer cells from the individual or determining an increased activity of HRI in cancer cells contacted with a compound or compounds described herein. Determining the expression level of HRI in any given cancer cell can be accomplished using methods known to those of ordinary skill in the art and described herein. Should the cancer cells express a significant or substantial level of HRI, or an increased activity of HRI, the patient is a candidate patient for treatment with a diarylurea compound or diarylthiourea compound. Treatment with a diarylurea compound or diarylthiourea compound is more effective with cancer cells that express higher levels of HRI compared to lower levels of H RI . According to certain additional aspects, the method includes the step of administering a diarylurea compound or diarylthiourea compound to the individual. The compound or compounds may be administered by inhalation, transdermally, orally, rectally, transmucosally, intestinally, parenterally, intramuscularly, subcutaneously or intravenously. The compound or compounds may be administered in an amount effective to activate H RI and phosphorylate eIF2a.

[15] In at least certain examples, the compounds are of substituted diarylureas, more particularly, substituted N.N'-diarylurea compounds. In other examples, the compounds are substituted thioureas, more particularly, substituted Ν,Ν'- diarylthiourea compounds. In certain exemplary embodiments, substituted Ν,Ν'- diarylurea and/or substituted Ν,Ν'-diarylthiourea compounds include compounds comprising Formula I, Formula II, Formula III, Formula IV and/or compounds set forth in Tables 1-6, Figures 1-12 and the Appendix.

[16] In certain examples, the substituted N.N'-diarylurea and/or substituted Ν,Ν'- diarylthiourea compounds cause phosphorylation of eIF2 . In accordance with other examples, the substituted Ν,Ν'-diarylurea and/or substituted N,N'-diarylthiourea compounds activate HRI thereby causing phosphorylation of eIF2a. In other examples, substituted Ν,Ν'-diarylurea and/or substituted N,N'-diarylthiourea compounds are effective to inhibit translation initiation. In other examples, substituted Ν,Ν'-diarylurea and/or substituted Ν,Ν'-diarylthiourea compounds activate downstream effectors of eIF2 phosphorylation, reduce the expression of oncogenic proteins and potently inhibit proliferation of human cancer cell lines. According to one aspect, the compounds are non-toxic. According to one aspect of the present disclosure, N,N'-diarylureas or NN'-diarylthioureas interact with I IRI leading to phosphorylation of eIF2 and promote advantageous downstream effects in the treatment of cancer.

In accordance with a method aspect, a method of treating a proliferative disorder by providing and/or administering a compound of Formula I and/or Formula II and/or Formula III and/or Formula IV to a mammal, e.g., a human or a non-human (e.g., a non-human primate), is provided. In one example, the proliferative disorder is cancer.

In accordance with an additional aspect, kits are provided for the treatment of (1) proliferative disorders, such as cancer. In one aspect, the kits comprise an Ν,Ν'- diarylurea and/or Ν,Ν'-diarylthiourea or a substituted Ν,Ν'-diarylurea and/or a substituted Ν,Ν'-diarylthiourea compound or a compound of Formula I and/or Formula II and/or Formula III and/or Formula IV, a pharmaceutically acceptable carrier, and optionally, instructions for use. The pharmaceutical composition can be administered to a human subject or a non-human subject depending on the disorder to be treated. In another aspect, a kit includes reagents and instructions for determining HRI levels and/or HRI activity in cancer cells, such as from an individual.

It will be recognized by the person of ordinary skill in the art that the compounds, compositions, methods and kits disclosed herein provide significant advantages over prior technology. Compounds, compositions, methods and kits can be designed or selected to relieve and/or alleviate symptoms in a patient suffering from one or more disorders. These and other aspects and examples are described below.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. [211 Figures 1.4- IE a) F-luc and R-luc ORFs were cloned into pBISA plasmid to transcribe two reporter mRNAs. The 5'UTR of the mouse ATF-4 mRNA including first two codons of bona-fide ORF was cloned in frame with respect to the start codon of F-luc ORF (pBISA-DL ^(ATF"4) ). The mRNA products of pBISA-DL ^(ATF"4) plasmid are shown, b) The structure of three active (a) and one inactive (i) N,N'-diarylureas. c) KLN-tTA/pBISA-DL ^(ATF"4) cells were incubated with the indicated concentrations of each N,N'-diarylurea and the normalized F/R ratio was determined by DLR assay, d) KLN-tTA/pBISA-DL ^(ATF"4) cells were incubated with the indicated concentrations of each N,N -diary lurea and expression of endogenous CHOP protein was determined by Western blot analysis, e) KLN-tTA/pBISA-DL ^(ATF"4) cells were incubated with 5 or 20 μΜ of each N,N ^J -diarylurea and expression of endogenous CHOP mRNA was determined by real-time PCR. 3 replicates in each experimental and control group and each experiment was independently performed 3 times.

[22] Supplementary Figure 1 a) pBISA-DL ^{l VI F~41} construct was stably transt ^' ected into KLN-tTA cells and responsiveness of these cells to DMSO (vehicle), TG, or TU was evaluated by DLR assay. F/R ratio of TG or TU treated wells was normalized to the F/R ratio of vehicle treated wells in the same plate, b) The second uORF in the ATF-4 5 'UTR was fused in frame to the AUG start codon of firefly luciferase, KLN-tTA cells were transfected with this plasmid, treated with DMSO TG (100 nM) or TU (1 μg/ml) and the normalized F/R ratio was determined by DLR assay, c) The stable KLN-tTA/pBISA-DL ^(ATF"4) cell line in B was plated into a 384- we 11 plate, half the plate was treated with TG and other half with the vehicle (DMSO). The normalized F/R ratio was determined by DLR assay and was spread along the X-axis for clarity. Shown is the triplicate analysis of each experimental and control group, repeated on three different days.

[23] Supplementary Figure 2 is a full gel image of Figure I d.

[24] Supplementary Figure 3 a-c) I luman PC-3 prostate (a), CRL-2351 breast (b), and

CRL-2813melanoma (c) cancer cell lines were co-transfected with pBISA-DL ^{l VI F~4} ' and ptTA plasmids. One day after transfection the cells were treated with the indicated concentrations of N,N'-diarylureas. The normalized F/R ratio was determined by DLR assay 8 hour after treatment, d) PC-3. CR-L2351, and CRL-2813 human cancer cell lines were treated with the indicated concentrations of Ν,Ν'- diarylureas for 6 hours and expression of endogenous CHOP mRNA was determined by real-time PGR. Shown is triplicate analysis of each experimental and control group, repeated on three different days.

[25] Figure 2A-2C a) KLN-tTA/pBISA-DL ^(ATF"4) (left) or PC-3 cell (right) lines were incubated with selected N,N '-diarylureas, levels of phosphorylated (p-eIF2oc) and total eIF2ot (eIF2oc) were determined by Western blot analysis with pS51-eIF2a specific rabbit monoclonal antibodies or with total eIF2oc specific mouse monoclonal antibodies; respectively, b) The PC-3 cells in which endogenous eIF2a is replaced by recombinant WT or non-phosphorylatable eIF2cx-S5 1 A mutant were co-transfected with tTA and pBISA-DL ^{l Vn 41} dual luciferase expression vector and treated with the indicated concentrations of N,N '-diarylureas. The normalized F R ratio was determined by DLR assay and standard error of mean are shown, c) Genetically engineered PC-3 cells in (b) were treated with N,N '-diarylureas (10 μΜ) and the expression of CHOP mRNA was determined by real-time PGR. The experiment was conducted in triplicates and each experiment was independently performed three times.

[26] Supplementary Figure 4 is a full gel image of Figure 2a.

[27] Supplementary Figure 5 a) Mouse KLN cells (left panel) were incubated with 20 μΜ and human PC3 cancer cell (right panel) lines were incubated with 5 μΜ of each N,N'-diarylureas. Expression of cyclin Dl, p27 ^Klpl , and β-actin was determined by Western blot analysis, b) KLN and PC3 cells were treated as in a and expression of cyclin Dl and p27 ^Kipl mRNA was determined by real-time PGR. Levels of these mRNAs were normalized to 18S mRNA. 3 replicates in each experimental and control group and each experiment was independently performed 3 times.

[28] Figures 3A-3E a) KLN-tTA/pBISA-DL ^(ATF"4) cells were transfected with mock siRNA or siRNA targeting PKR, PERK, GCN2, or HRI individually or simultaneously in all combinations (only PKR, PERK, and GCN2 combination is shown). CRL-2813 cells were transfected in the same manner except that the transfection mixture also contained the pBISA-DL^™- ¹ and tTA plasmids. Cells were treated with compound BTdCPU or with DMSO and the normalized F/R ratio was determined by DLR. b) KLN-tTA pBISA-DL ^(ATF"4) or CRL-2813 cells were transfected with siRNAs targeting each of the eIF2a kinases and treated with compound BTdCPU or with DMSO. Expression of CHOP mRNA was determined by real-time PCR. c) CRL-2813 cells were transfected with mock, PERK or HRI siRNA, treated with tunicamycin, compound BTdCPU or vehicle, and the levels of phosphorylated (p-eIF2a) and total eIF2a (eIF2a) were determined by Western blot, d) KLN-tTA/pBISA-DL ^(ATF"4) cells were transfected with mock or HRI-targeting siRNA, treated with four N,N'-diarylurea compounds or vehicle and the normalized F/R ratio was determined by DLR. e) CRL-2813 cells were transfected with mock or H RI targeting siRNA, treated with four N,N'-diarylurea compounds or vehicle and the normalized F/R ratio was determined by DLR.

[29] Supplementary Figure 6 is a panel showing the quantification of the western blot of Figure 3c.

[30] Supplementary Figure 7 is a full gel image of Figure 3c.

[31] Supplementary Figure 9 (a) Purified recombinant HRI was incubated with DMSO and with the indicated concentrations of BTdCPU, and (b) purified recombinant eIF4E was incubated with DMSO, BTdCPU (500 μΜ), or 4EGI-1 (500 μΜ) for 2h and at 4°C, followed by digestion with subtilisin at room temperature. Samples were separated by SDS-PAGE and stained with coomassie brilliant blue, (c) CRL-2813 cells were loaded with 100 μΜ DCHF-DA (2', V-Dichlorodihydrofluorescin diacetate) and treated with DMSO, Sodium Arsenite, and the indicated concentrations of BTdCPU for the indicated times. The fluorescence was read with an ENVISION 2100 plate reader at 480 nm (ex)/530 nm (em). Note that all concentrations of BTdCPU yield a straight line and are superimposed with vehicle control, (d) and (e) Heme supplemented rabbit reticulocyte (d) or in-house prepared human melanoma cancer cell lysates (e) were incubated with the indicated concentration of BTdCPU for 30 minutes at 37°C and phosphorylation of eIF2a was determined by Western blot analysis. Right panels shows quantification of data from three different gels run independently on three different days.

[32] Figures 4Λ-4Β a) The PC-3 human prostate cancer cells in which endogenous eIF2a is replaced by recombinant WT or the non-phosphorylatable eIF2a-S51A mutant were treated with the indicated concentrations of NN'-diarylureas and cell proliferation was measured by SRB assay. Panel a shows the growth inhibition curve for one active (BTCtFPU) and one inactive (NCPdCPU) NN ^J -diarylurea. Calculated IC ₅₀ for all four compounds in these genetically engineered cell lines are shown in Supplementary Fig. 10a. b) CRL-2813 human melanoma cancer cells were transfected with HRI or mock siRNA, treated with the indicated concentrations of N,N'-diarylureas and cell proliferation was measured by SRB assay. The panel b shows the growth inhibition curve for one active (BTCtFPU) and one inactive (NCPdCPU) N,N'-diarylurea, calculated IC50 for all four compounds in cells transfected with HRI or mock siRNA is shown in Supplementary Fig. 10b. The experiment was conducted in triplicates and each experiment was independently performed three times.

Supplementary Figure 8 (a) MCF-7 cells were co-transfected with tTA and pBISA- DL ^(ATW ' dual luciferase expression vector and scrambled or HRI siRNA, and treated with the indicated concentrations of N,N'-diarylureas 48 hours after siRNA transfection. The normalized F/R ratio was determined by DLR assay after overnight incubation with the compounds. Shown are mean values with the standard error of the means, (b) MCF-7 cells were transfected with scrambled or HRI siRNA, 48 hours later the cells were treated with the indicated concentrations of N,N'-diarylureas. After 6 hours of incubation with each drug, expression of endogenous CHOP mRNA was determined by real-time PCR. Experiment was independently performed three times in triplicates.

Supplementary Figure 10 Panel a shows the calculated IC5 ₀ for all four compounds in genetically engineered PC-3 cell lines (see also Figure 4a). Panel b shows the calculated IC5 ₀ for all four compounds in CRL-2813 cells transfected with HRI or mock siRNA (se also Figure 4b). (c) MCF-7 cells were transfected with scrambled or HRI siRNA. After 24 hours, cells were treated with the indicated concentrations of NN'-diarylureas for 3 days and cell proliferation was measured by SRB assay. The calculated IC5 ₀ S for all four compounds are shown in the right panel.

Figures 5.4-5 D a) Lysates were prepared from KLN mouse squamous cell carcinoma, HTB-26, 1 1TB- 128. and CRL-2351 human breast, PC-3 human prostate, and CRL-2813 human melanoma cancer cell lines were separated by SDS-PAGE and probed with antibodies specific to HRI or β-actin and quantified. The concentration of the three active N,N'-diarylureas that inhibit proliferation of these cells by 50% (IC50) were plotted against the levels of HR1 (normalized for β-actin levels) in the cancer cell lines. Each experiment was independently performed three times, b) Five female nude mice each were treated with 200 mg/kg/d, 100 mg/kg/d and 350 mg/kg/d BTdCPU in 15 μΐ DMSO or 15 μΐ DMSO daily for seven days, weighed every other day for total of 15 days and then necropsy was performed. The average body weight of each group is plotted against the time, c) Female nude mice xenografted with MCF-7 human breast cancer cells were randomly distributes to two groups and treated with 175 mg/kg/d BTdCPU in 15 μΐ DMSO or DMSO alone. Mice were observed daily, and tumor dimensions were measured weekly, d) Lysates prepared from three excised tumors in the treatment and control groups, separated by the SDS-PAGK and blotted with antibodies specific to phosphorylated (P-eIF2 ) or total eIF2ot (eIF2cc) and ratio of phosphorylated eIF2a to total eIF2oc was quantified (see Supplementary Fig. 11).

Supplemantary Figure 11 (a) is a full gel image of Figure 5d and (b) is a graph of the data of Figure 5d.

Supplementary Figure 12 are tissue sections of female nude mice treated with 1 75 mg/kg, BTdCPU for three weeks which were necropsied at the end of a 21 days treatment, and major organs were analyzed by hematoxcylin/eosine staining.

Supplementary Figure 13 are graphs of blood collection data from each female nude mice treated with 175 mg/kg, BTdCPU for twenty one days and analyzed at the hematology core facility. WBC: white blood cells, RBC: red blood cells, HGB: hemoglobin, HCT: hematocrit, PLT: platelets, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, HDW: hemoglobin distribution width.

It will be recognized that the results and examples in the figures are only illustrative and other examples and illustrations will be readily recognized by the person of ordinary skill in the art, given the benefit of this disclosure.

DETAILED DESCRIPTION

In accordance with certain examples, methods are provided of identifying cancer patients for treatment with diarylurea compounds, diarylthiourea compounds, Ν,Ν'- diarylurea compounds, Ν,Ν'-diarylthiourea compounds, substituted N,N'-diarylurea compounds, substituted Ν,Ν'-diarylthiourea compounds or compounds of Formula I and/or Formula II and/or Formula III and/or Formula IV. According to one aspect of the present disclosure, cancer cells from a patient are assayed to determine the expression level of HRI or the activity of HRI. According to another aspect, cancer cells from a patient are assayed for HRI activity. Based on the expression level of HRI or the activity of HRI responsive to a compound or compounds described herein, the patient is identified as a candidate for treatment with diarylurea compounds, diarylthiourea compounds, Ν,Ν'-diarylurea compounds, N,N'-diarylthiourea compounds, substituted Ν,Ν'-diarylurea compounds, substituted N.N'-diarylthiourca compounds or compounds of Formula I and/or Formula II and/or Formula III and/or Formula IV.

Certain examples are described below with reference to various chemical formulae. The chemical formulae referred to herein can exhibit the phenomena of tautomerism, conformational isomerism, stereo isomerism or geometric isomerism. As the formulae drawings within this specification can represent only one of the possible tautomeric, conformational isomeric, enantiomeric or geometric isomeric forms, it should be understood that the invention encompasses any tautomeric, conformational isomeric, enantiomeric or geometric isomeric forms which exhibit biological or pharmacological activity as described herein.

Once the HRI expression level and/or HRI activity of cancer cells from an individual is determined, such as by methods described herein, the individual may be identified as a suitable candidate for treatment with diarylurea compounds, diarylthiourea compounds, Ν,Ν'-diarylurea compounds, Ν,Ν'-diarylthiourea compounds, substituted Ν,Ν'-diarylurea compounds, substituted Ν,Ν'-diarylthiourea compounds or compounds of Formula I and/or Formula II and/or Formula III and/or Formula IV. According to one aspect, the compounds are administered to an individual in a manner to activate HRI thereby causing phosphorylation of eIF2a and inhibition of translation initiation.

The compounds and compositions described herein certain exemplary embodiments are provided below are effective to activate HRI thereby causing phosphorylation of eIF2 and inhibition of translation initiation at least to the extent necessary for effective treatment of cancer cells. While in certain examples translation may be substantially inhibited such that little or no activity results, in other examples the inhibition is at least sufficient to relieve and or alleviate the symptoms from cancer.

In accordance with certain embodiments, compounds of the invention include diarylurea compounds, diarylthiourea compounds, Ν,Ν'-diarylurea compounds, Ν,Ν'- diarylthiourea compounds, substituted Ν,Ν'-diarylurea compounds, substituted Ν,Ν'- diarylthiourea compounds. Certain exemplary embodiments are represented by the generic formula set forth below.

Formula I I I

[45] In certain exemplary embodiments with respect to Formula I, 11 or 111. Ri is I I. CI, CH ₃ , OCH ₃ , N0 ₂ , Oi l, F, CF ₃ , OCF ₃ , Br. CH S, Acl IN. (CH ₃ ) ₂ N, CO- NH-NI I;. SO2 H2, C(CH ₃ ) ₃ , COOCH ₂ CH ₃ , COCH ₃ , 0(CH ₂ ) ₂ CH ₃ , CHO, C0 ₂ H, OCONH2, CN, C≡CH, N-methylacetamido, l-[l,2,3]triazolyl, 4-[l,2,3]triazolyl, 5- [l,2,3,4]tetrazolyl, guanidine, Ci-6-alkyl, Ci_6-alkyl amino substituted with: hydroxyl, Ci-6-alkoxy, amino, mono- and di-(Ci_6-alkyl)amino, carboxy, C _1-6 - alkylcarbonylamino, Ci-6-alkylaminocarbonyl, aminosulfonyl, mono- and di-(Ci _-6 - alkyl)aminosulfonyl, carbamido, mono- and di-(Ci_6-alkyl)aminocarbonylamino, halogen(s), aryl, arylheterocycle, heterocycle, and heteroaryl. C2-6-alkenyl, Ci_s- alkoxy, C ₂ -6-alkenyloxy, Ci-6-alkoxycarbonyl, C _1-6 -alkylcarbonyl, Ci _-6 - alkylcarbonyloxy, Ν,Ν-dimethylamino, N,N-di(Ci_6-alkyl)amino, mono- and di-(Ci _-6 - alkyl)aminocarbonyl, Ci.6-alkylcarbonylamino, Ci-6-alkylsulfonylamino, C _1-6 - alkylthio, Ci-6-alkylsulphinyl, aryl, aryloxy, arylcarbonyl, arylamino, arylsulfonylamino, hetrocyclyl, hetrocyclyloxy, heterocyclylamino, heterocyclylcarbonyl, heteroaryl, heteroaryloxy, heteroarylamino, heteroarylcarbonyl, heteroarylsulfonylamino, 0-(CH ₂ ) ₂ -4-morpholino, 0-(CH ₂ ) ₂ -4-(piperazin-l-yl), O- (CH ₂ )2 _-4 -(4-methylpiperazin-l-yl), 0-(CH ₂ ) ₂ -4-mono- and di-(Ci_6-alkyl)amino, O- (CH ₂ )2-4-lH-[l,2,3]triazol-l-yl), 0-(CH ₂ ) ₂ -4-4(lH-[l,2,3]triazol-l-yl), 0-(CH ₂ ) ₂ - ₄ -(4- -alkyl)-lH-[l,2,3]triazol-l-yl), 0-(CH ₂ )2-4-4-(l-(Ci- ₆ -alkyl)-lH-[l,2,3]triazol-l-

R ₂ is H, CI. CH ;, OCH ₃ , N0 ₂ , OH, F, CF ₃ , OCF ₃ , Br. CH ₃ S, AcHN, (CH ₃ ) ₂ N, CO- NI I-NI I2. SO2NH2. C(CH ₃ ) ₃ , COOCI I2CI I ;. COCH . 0(CH ₂ ) ₂ CH ₃ , CI 10. C0 ₂ H, OCONH2. CN, C≡CH, N-methylacetamido, l-[l,2,3]triazolyl, 4-[l,2,3]triazolyl, 5- [ 1.2,3.4]tctrazolyl. guanidine, Ci_6-alkyl, Ci_6-alkyl amino substituted with: hydroxyl, Ci-6-alkoxy, amino, mono- and di-(Ci_6-alkyl)amino, carboxy, Ci _-6 - alkylcarbonylamino, Ci-6-alkylaminocarbonyl, aminosulfonyl, mono- and di-(C] _6- alkyl)aminosulfonyl, carbamido, mono- and di-(Ci_6-alkyl)aminocarbonylamino, halogen(s), aryl, arylheterocycle, heterocycle, and heteroaryl. C _2- 6-alkenyl, C _1-6 - alkoxy, C2-6-alkenyloxy, Ci_6-alkoxycarbonyl, C _1-6 -alkylcarbonyl, Ci _-6 - alkylcarbonyloxy, N . K-d i me thy 1 am i no. N,N-di(Ci_6-alkyl)amino, mono- and di-(C _1-6 - alkyl)aminocarbonyl, Ci.6-alkylcarbonylamino, Ci.6-alkylsulfonylamino, Ci _-6 - alkylthio, Ci-6-alkylsulphinyl, aryl, aryloxy, arylcarbonyl, arylamino, arylsulfonylamino, hetrocyclyl, hetrocyclyloxy, heterocyclylamino, heterocyclylcarbonyl, heteroaryl, heteroaryloxy, heteroarylamino, heteroarylcarbonyl, heteroarylsulfonylamino, 0-( C H ; );. ₄ -morphol i no, 0-(CH ₂ )2-4-(piperazin-l-yl), O- (CH ₂ )2-4-(4-methylpiperazin-l-yl), 0-(CH ₂ )2-4-mono- and di-(Ci- ₆ -alkyl)amino, O- (CH ₂ )_-4- 1 //-[ 1.2.3]tria/.ol- 1 -yl), 0-(CI I ) _2-4 -4( 1 //-[ 1.2.3 |triazol- 1 -yl), 0-(CH ₂ ) ₂ -4-(4- (C , . -alkyl )- 1 //-[ 1.2,3 |triazol- 1 -yl). 0-(CI I ₂ )2-4-4-( 1 -(Ci. -alkyl)-l //-[ 1.2.3 |triazol- 1 -

R; is I I. CI, CH ₃ , OCH ₃ , N0 ₂ , OH, F, CF ₃ , OCF ₃ , Br, CH ₃ S, AcI IN, (CH ₃ ) ₂ N, CO- NH-NH ₂ , SO2NH2, C(CH ₃ ) ₃ , COOCH ₂ CH ₃ , COCH ₃ , 0(CH ₂ ) ₂ CH ₃ , CMC), C0 ₂ H, OCONH ₂ , CN, C≡CH, N-methylacetamido, l-[l,2,3]triazolyl, 4-[l ,2,3]triazolyl, 5- [l,2,3,4]tetrazolyl, guanidine, Ci-6-alkyl, Ci.6-all yl amino substituted with: hydroxyl, Ci-6-alkoxy, amino, mono- and di-(Ci_6-alkyl)amino, carboxy, C] _6- alkylcarbonylamino, Ci-6-alkylaminocarbonyl, aminosulfonyl, mono- and di-(Ci,6- alkyl)aminosulfonyl, carbamido, mono- and di-(Ci.6-alkyl)aminocarbonylamino, halogen(s), aryl, arylheterocycle, heterocycle, and heteroaryl. C _2- 6-alkenyl, C^- alkoxy, C ₂ _6-alkenyloxy, Ci.6-alkoxycarbonyl, C i_ ₆ -alkylcarbonyl, C1.6- alkylcarbonyloxy, Ν,Ν-dimethylamino, N,N-di(Ci_6-alkyl)amino, mono- and di-(Ci_6- alkyl)aminocarbonyl, Ci_6-alkylcarbonylamino, Ci.6-alkylsulfonylamino, Ci _-6 - alkylthio, Ci-6-alkylsulphinyl, aryl, aryloxy, arylcarbonyl, arylamino, arylsulfonylamino, hetrocyclyl, hetrocyclyloxy, heterocyclylamino, heterocyclylcarbonyl, heteroaryl, heteroaryloxy, heteroarylamino, heteroarylcarbonyl, heteroarylsulfonylamino, 0-(CH ) . ₄ -morpholino, 0-(CH ₂ ) ₂ _4-(piperazin-l-yl), O- (CH ₂ ) ₂ _4-(4-methylpiperazin-l-yl), 0-(CH ₂ ) ₂ _ ₄ -mono- and di-(C] _6-alkyl)amino, O- (CH ₂ ) _2- 4- ,2,3]triazol- 1 -yl), 0-(CH ₂ ) ₂ . ₄ -4( lH-[ 1 ,2,3]triazol- 1 -yl), 0-(CH ₂ ) ₂ - ₄ -(4- -alkyl)-lH-[l,2,3]triazol-l-yl), 0-(CH ₂ ) _2-4 -4-(l-(C, _-6 -alkyl)-lH-[l,2,3]triazol-l-

R ₄ is H. CI, CI I ₃ , OCI I , N0 ₂ , OH, F, CF ₃ , OCF ₃ , Br, CH ₃ S, AcHN, (CH ₃ ) ₂ N, CO- NH-NH ₂ , SO2NH2, C(CH ₃ ) ₃ , COOCH2CH3, COCH3. 0(CH ₂ ) ₂ CH ₃ , CHO, C0 ₂ H, OCONH2, CN, C≡CH, N-methylacetamido. l-[l,2,3]triazolyl, 4-| 1.2.3 |triazolyl. 5- [l,2,3,4]tetrazolyl, guanidine, Ci-6-alkyl, Ci_6-alkyl amino substituted with: hydroxy!, C] _6-alkoxy, amino, mono- and d i -( C 1.,,-a 1 ky 1 jam i n 0, carboxy, Ci_6- alkylcarbonylamino, Ci_6-alkylaminocarbonyl, aminosulfonyl, mono- and di-(Ci_6- alkyl)aminosulfonyl, carbamido, mono- and di-(Ci-6-alkyl)aminocarbonylamino, halogen(s), aryl, arylheterocycle, heterocycle, and heteroaryl. C2-6-alkenyl, Ci _-6 - alkoxy, C2-6-alkenyloxy, Ci_6-alkoxycarbonyl, Ci_6-alkylcarbonyl, C _1-6 - alkylcarbonyloxy, Ν,Ν-dimethylamino, N,N-di(Ci-6-alkyl)amino, mono- and di-(Ci _-6 - alkyl)aminocarbonyl, Ci_6-alkylcarbonylamino, Ci-6-alkylsulfonylamino, C _1-6 - alkylthio, Ci_6-alkylsulphinyl, aryl, aryloxy, arylcarbonyl, arylamino, arylsulfonylamino, hetrocyclyl, hetrocyclyloxy, heterocyclylamino, heterocyclylcarbonyl, heteroaryl, heteroaryloxy, heteroarylamino, heteroarylcarbonyl, heteroarylsulfonylamino, 0-(CI I:)2. ₄ -morpholino, 0-(CH ₂ )2-4-(piperazin-l -yl), O- (CH ₂ )2 _-4 -(4-methylpiperazin-l -yl), 0-(CH ₂ )2-4-mono- and di-(Ci_6-alkyl)amino, O- (CH ₂ ) ₂ -4- 1.2.3]tria/ol- 1 -yl), 0-(CH ₂ ) ₂ -4-4( lH-[ 1 ,2,3]triazol- 1 -yl), 0-(CH ₂ ) ₂ - ₄ -(4- -alkyl)-lH-[l ,2,3]triazol- l-yl), 0-(CH ₂ )2-4-4-(l-(C ₁ . ₆ -alkyl)-lH-[l,2,3]triazol- l-

R ₅ is H. CI, CH ₃ , OCH ₃ , N0 ₂ , OH, F. CF ₃ , OCF ₃ , Br. CH ₃ S, AcHN, (CH ₃ ) ₂ N, CO-

NH-NI I2, SO2NH2, C(CH ₃ ) ₃ , COOCH ₂ CH ₃ , COCH ₃ , 0(CH ₂ ) ₂ CH ₃ , CMC ⁾ . C0 ₂ H,

OCONH2, CN, C≡CH, N-methylacetamido, l-[l,2,3]triazolyl, 4-[ 1.2,3]tria/.olyl. 5-

[l ,2,3,4]tetrazolyl, guanidine, Ci-6-alkyl, Ci.6-alkyl amino substituted with: hydroxyl,

Ci_6-alkoxy, amino, mono- and di-(Ci-6-alkyl)amino, carboxy, Q.6- alkylcarbonylamino, Ci-6-alkylaminocarbonyl, aminosulfonyl, mono- and di-(C ₁ _6- alkyl)aminosulfonyl, carbamido, mono- and di-(Ci-6-alkyl)aminocarbonylamino, halogen(s), aryl, arylheterocycle, heterocycle, and heteroaryl. C _2- 6-alkenyl, Ci_6- alkoxy, C2-6-alkenyloxy, Ci-6-alkoxycarbonyl, Ci-6-alkylcarbonyl, Ci_6- alkylcarbonyloxy, Ν,Ν-dimethylamino, N,N-di(Ci_6-alkyl)amino, mono- and di-(Ci-6- alkyl)aminocarbonyl, Ci-6-alkylcarbonylamino, Ci-6-alkylsulfonylamino, C] .6- alkylthio, Ci-6-alkylsulphinyl, aryl, aryloxy, arylcarbonyl, arylamino, arylsulfonylamino, hetrocyclyl, hetrocyclyloxy, heterocyclylamino, heterocyclylcarbonyl, heteroaryl, heteroaryloxy, heteroarylamino, heteroarylcarbonyl, heteroarylsulfonylamino, 0-(CH2) ₂ -4-morpholino, 0-(CH ₂ )2-4-(piperazin-l-yl), O-

(CH ₂ )2-4-(4-methylpiperazin-l-yl), 0-(CH ₂ )2-4-mono- and di-(Ci. ₍ ,-alkyl)amino. O-

(CH ₂ ) ₂ -4- lH-[ 1 ,2,3]triazol- 1 -yl), 0-(CH ₂ ) ₂ - ₄ -4( lH-[ 1 ,2,3]triazol- 1 -yl), 0-(CH ₂ ) ₂ -4-(4- -(C ₁ . ₆ -alkyl)-lH-[l,2,3]triazol-l - R ₆ is H, CI, CH ₃ , OCH ₃ , N0 ₂ , OH, F, CF ₃ , OCF ₃ , Br, CH ,S, Ac I IN. (CH ₃ ) ₂ N, CO- NH-NH ₂ , SO2NH2, C(CH ₃ ) ₃ , COOCH ₂ CH ₃ , COCH ₃ , 0(CH ₂ ) ₂ CH ₃ , CHO, C0 ₂ H, OCONH ₂ , CN, C≡CH, N-methylacetamido, l-[l,2,3]triazolyl, 4-[l,2,3]triazolyl, 5- [l,2,3,4]tetrazolyl, guanidine, Ci_6-alkyl, Ci-6-alkyl amino substituted with: hydroxyl, Ci-6-alkoxy, amino, mono- and di-(Ci.6-alkyl)amino, carboxy, Ci-6- alkylcarbonylamino, Ci-6-alkylaminocarbonyl, aminosulfonyl, mono- and di-(Ci_6- alkyl)aminosulfonyl, carbamido, mono- and di-(Ci-6-all<yl)aminocarbonylamino, halogen(s), aryl, arylheterocycle, heterocycle, and heteroaryl. C _2- 6-alkenyl, C _1-6 - alkoxy, C ₂ _6-alkenyloxy, Ci_6-alkoxycarbonyl, Ci.6-alkylcarbonyl, Ci_6- alkylcarbonyloxy, Ν,Ν-dimethylamino, N,N-di(Ci_6-alkyl)amino, mono- and di-(C _1-6 - alkyl)aminocarbonyl, Ci_6-alkylcarbonylamino, Ci_6-alkylsulfonylamino, Ci _-6 - alkylthio, Ci-6-alkylsulphinyl, aryl, aryloxy, arylcarbonyl, arylamino, arylsulfonylamino, hetrocyclyl, hetrocyclyloxy, heterocyclylamino, heterocyclylcarbonyl, heteroaryl, heteroaryloxy, heteroarylamino, heteroarylcarbonyl, heteroarylsulfonylamino, 0-(CH ₂ )2-4-morpholino, 0-(CH ₂ )2-4-(piperazin- l-yl), O- (CH2)2-4-(4-methylpiperazin- l-yl), 0-(CH ₂ )2-4-mono- and di-(Ci-6-alkyl)amino, O- (CH ₂ ) ₂ -4- 1 H-[ 1 ,2,3 ]triazol- 1 -yl), 0-(CH ₂ ) ₂ - ₄ -4( lH-[ 1 ,2,3]triazol- 1 -yl), 0-(CH ₂ ) ₂ -4-(4- -alkyl)-lH-[l,2,3]triazol-l-yl), 0-(CH ₂ )2-4-4-(l-(Ci. ₆ -alkyl)-lH-[l,2,3]triazol-l -

R ₇ is I I, CI, CH ₃ , OCH ₃ , N0 ₂ , OH, F, CF ₃ , OCF ₃ , Br, CH ₃ S, AcHN, (CH ₃ ) ₂ N, CO- NH-NH ₂ , SO2NH2, C(CH ₃ ) ₃ , COOCH ₂ CH ₃ , COCH ₃ , 0(CH ₂ ) ₂ CH ₃ , CHO, C0 ₂ H, OCONH2, CN, OCH, N-methylacetamido, l-[l,2,3]triazolyl, 4-[l,2,3]triazolyl, 5- [l ,2,3,4]tetrazolyl, guanidine, Ci_6-alkyl, Ci-6-alkyl amino substituted with: hydroxyl, Ci-6-alkoxy, amino, mono- and di-(Ci_6-alkyl)arnino, carboxy, C _1-6 - alkylcarbonylamino, Ci-6-alkylaminocarbonyl, aminosulfonyl, mono- and di-(Ci_6- alkyl)aminosulfonyl, carbamido, mono- and di-(Ci_6-alkyl)aminocarbonylamino, halogen(s), aryl, arylheterocycle, heterocycle, and heteroaryl. C _2- 6-alkenyl, C _1-6 - alkoxy, C ₂ -6-alkenyloxy, C _1-6 -alkoxycarbonyl, Ci_6-alkylcarbonyl, Ci_ ₆ - alkylcarbonyloxy, Ν,Ν-dimethylamino, N,N-di(Ci_6-alkyl)amino, mono- and di-(Ci_6- alkyl)aminocarbonyl, Ci^-alkylcarbonylamino, Ci-6-alkylsulfonylamino, C _1-6 - alkylthio, C _1-6 -alkylsulphinyl, aryl, aryloxy, arylcarbonyl, arylamino, arylsulfonylamino, hetrocyclyl, hetrocyclyloxy, heterocyclylamino, heterocyclylcarbonyl, heteroaryl, heteroaryloxy, heteroarylamino, heteroarylcarbonyl, heteroarylsulfonylamino, 0-(CH ₂ )2-4-morpholino, 0-(CH2) ₂ -4-(piperazin-l -yl), O- (CH ₂ ) ₂ _4-(4-methylpiperazin-l-yl), 0-(CH2) ₂ -4-mono- and di-(Ci.6-alkyl)amino, O- (CH ₂ ) _2- 4-lH-[l,2,3]triazol-l-yl), 0-(CH ₂ ) ₂ - ₄ -4(lH-[l,2,3]triazol-l-yl), 0-(CH ₂ ) _2-4 -(4- (Ci-6-alkyl)-lH-[l,2,3]triazol-l-yl), 0-(CH ₂ ) _2- 4-4-(l -(C _1-6 -alkyl)-lH-[l ,2,3]triazol-l -

R ₈ is H. CI, CH ₃ , OCH ₃ , N0 ₂ , OH, F. CF ₃ , OCF ₃ , Br. CH ₃ S, AcHN, (CH ₃ ) ₂ N, CO- NH-NH ₂ , S0 ₂ NH ₂ , C(CH ₃ ) ₃ , COOCH ₂ CH ₃ , COCH ₃ , 0(CH ₂ ) ₂ CH ₃ , CI 10, C0 ₂ H, OCONH ₂ , CN, C≡CH, N-methylacetamido, l -[l,2,3]triazolyl, 4-[l ,2,3]triazolyl, 5- [l ,2,3,4]tetrazolyl, guanidine, Ci-6-alkyl, Ci-6-alkyl amino substituted with: hydroxyl, Ci-6-alkoxy, amino, mono- and di-(Ci_6-alkyl)amino, carboxy, Ci _-6 - alkylcarbonylamino, Ci.6-alkylaminocarbonyl, aminosulfonyl, mono- and di-(Ci _-6 - alkyl)aminosulfonyl, carbamido, mono- and di-(Ci-6-alkyl)aminocarbonylamino, halogen(s), aryl, arylheterocycle, heterocycle, and heteroaryl. C2-6-alkenyl, C _1-6 - alkoxy, C2-6-alkenyloxy, Ci_6-alkoxycarbonyl, Ci_6-alkylcarbonyl, C _1-6 - alkylcarbonyloxy, Ν,Ν-dimethylamino, N,N-di(Ci.6-alkyl)amino, mono- and di-(Ci_6- alkyl)aminocarbonyl, Ci_6-alkylcarbonylamino, C]_6-alkylsulfonylamino, Ci _-6 - alkylthio, Ci_5-alkylsulphinyl, aryl, aryloxy, arylcarbonyl, arylamino, arylsulfonylamino, hetrocyclyl, hetrocyclyloxy, heterocyclylamino, heterocyclylcarbonyl, heteroaryl, heteroaryloxy, heteroarylamino, heteroarylcarbonyl, heteroarylsulfonylamino, ()-( C H 2 J^-morpho lino. ()-( C H 2 ) -H p i pe raz i n - 1 -y 1 ) , O- (CI l2 )2- _t -(4-methylpiperazin-l -yl), ()-(CH2 (2-4-1110110- and di-(Ci.„-alky (amino, O- (CH ₂ ) ₂ -4- lH-[ 1 ,2,3]triazol- 1 -yl), 0-(CH ₂ ) ₂ -4-4( lH-[ 1 ,2,3]triazol- 1 -yl), 0-(CH ₂ ) ₂ - ₄ -(4- -alkyl)-lH-[l,2,3]triazol-l-yl), 0-(CH ₂ )2-4-4-(l-(C _1-6 -alkyl)-lH-[l,2,3]triazol-l-

[54] Rg is I I, CI. CH ₃ , OCH ₃ , N0 ₂ , OH. F, CF ₃ , OCF ₃ , Br. CH ₃ S, AcHN, (CH ₃ ) ₂ N, CO- NH-NH ₂ , S0 ₂ NH ₂ , C(CH ₃ ) ₃ , COOCH ₂ CH ₃ , COCI 1 ,. 0(CH ₂ ) ₂ CH ₃ , CHO, C0 ₂ H, OCONH ₂ , CN, C≡CH, N-methylacetamido, l-[l,2,3]triazolyl, 4-[l,2,3]triazolyl, 5- [l ,2,3,4]tetrazolyl, guanidine, Ci-6-alkyl, Ci-6-alkyl amino substituted with: hydroxyl,

Ci_6-alkoxy, amino, mono- and di-(Ci.6-alkyl)amino, carboxy, Cl-6- alkylcarbonylamino, Ci-6-alkylaminocarbonyl, aminosulfonyl, mono- and di-(Ci_6- alkyl)aminosulfonyl, carbamido, mono- and di-(Ci_6-alkyl)aminocarbonylamino, halogen(s), aryl, arylheterocycle, heterocycle, and heteroaryl. C _2- 6-alkenyl, C _1-6 - alkoxy, C _2- 6-alkenyloxy, C _1-6 -alkoxycarbonyl, Ci-6-alkylcarbonyl, Ci_ ₆ - alkylcarbonyloxy, Ν,Ν-dimethylamino, N,N-di(C ₁ _6-alkyl)amino, mono- and di-(Ci-6- alkyl)aminocarbonyl, Ci- ₆ -alkylcarbonylamino, Ci_6-alkylsulfonylamino, Ci _-6 - alkylthio, Ci- ₆ -alkylsulphinyl, aryl, aryloxy, arylcarbonyl, arylamino, arylsulfonylamino, hetrocyclyl, hetrocyclyloxy, heterocyclylamino, heterocyclylcarbonyl, heteroaryl, heteroaryloxy, heteroarylamino, heteroarylcarbonyl, heteroarylsulfonylamino, 0-(CH ₂ ) ₂ . -morpholino, 0-(CH ₂ ) ₂ _ -(piperazin- 1 -yl), O- (CH ₂ ) ₂ _4-(4-methylpiperazin-l-yl), 0-(CH ₂ ) ₂ . -mono- and di-(Ci- ₆ -alkyl)amino, O- (CH ₂ ) ₂ .4-lH-[l,2,3]triazol-l-yl), 0-(CH ₂ ) ₂ _ ₄ -4(lH-[l,2,3]triazol-l-yl), 0-(CH ₂ ) ₂ . ₄ -(4- (C,_ ₆ -alkyl)-lH-[l,2,3]triazol-l-yl), 0-(CH ₂ ) ₂ . ₄ -4-(l-(Ci. ₆ -alkyl)-lH-[l,2,3]triazol-l-

Rio is I I, CI. Cl k OCH ₃ , NO _: . OH. F, CF ₃ , OCF ₃ , Br. Cl S. AcHN, (CH ₃ ) ₂ N, CO- NH-NH2, S0 ₂ NH ₂ , C(CH ₃ ) ₃ , COOCI bCI h. COCH ₃ , 0(CH ₂ ) ₂ CH ₃ , CI 10, C0 ₂ H, OCONH2, CN, C≡CH, N-methylacetamido, l-[l,2,3]triazolyl, 4-[l ,2,3]triazolyl, 5- [l,2,3,4]tetrazolyl, guanidine, Ci.6-alkyl, Cu,-alkyl amino substituted with: hydroxyl, Cj_6-alkoxy, amino, mono- and d i-( C 1. ₆ -a 1 ky 1 )am ino. carboxy, Ci^- alkylcarbonylamino, Ci-6-alkylaminocarbonyl, aminosulfonyl, mono- and di-(C _1- e- alkyl)aminosulfonyl, carbamido, mono- and di-(Ci_6-alkyl)aminocarbonylamino, halogen(s), aryl, arylheterocycle, heterocycle, and heteroaryl. C2-6-alkenyl, Ci _-6 - alkoxy, C2-6-alkenyloxy, Ci_6-alkoxycarbonyl, Ci.6-alkylcarbonyl, Ci _-6 - alkylcarbonyloxy, Ν,Ν-dimethylamino, N,N-di(Ci.6-alkyl)amino, mono- and di-(Ci _-6 - alkyl)aminocarbonyl, Ci.6-alkylcarbonylamino, Ci.6-alkylsulfonylamino, Ci _-6 - alkylthio, Ci-6-alkylsulphinyl, aryl, aryloxy, arylcarbonyl, arylamino, arylsulfonylamino, hetrocyclyl, hetrocyclyloxy, heterocyclylamino, heterocyclylcarbonyl, heteroaryl, heteroaryloxy, heteroarylamino, heteroarylcarbonyl, heteroarylsulfonylamino, 0-(CH2)2-4-morpholino, 0-(CI l2):-4-(pipcrazin- 1 -yl). O- (CH ₂ )2-4-(4-methylpiperazin-l -yl), 0-(CH ₂ )2-4-mono- and di-(Ci_6-alkyl)amino, O- (CH ₂ )2-4- lH-[l,2,3]triazol-l-yl), 0-(CH ₂ ) ₂ -4-4(lH-[l ,2,3]triazol- l -yl), 0-(CH ₂ ) ₂ -4-(4- -alkyl)-lH-[l,2,3]triazol-l -yl), 0-(CH ₂ )2-4-4-(l-(Ci_ ₆ -alkyl)-lH-[l ,2,3]triazol-l-

R i ; is O, S, NH or NR19. For compounds of Formulae II and III, R13 is preferably S,

160] R, 5 is NH. S or NHNC ^' I I.

[61] Ri6 is C.

R, 7 is H. CH ₃ , -[(CH ₂ ) ₂ -0],. ₃ H, -[(CH ₂ ) ₂ -0],. ₃ CH ₃ , -(CH ₂ ) ₂ -NH ₂ , -(CH ₂ ) ₂ -NHCH ₃ ,

-KCH ₂ ) ₂ 03 _{1 -2} — (CH [63] R, 8 is H, CH ₃ , -[(CH ₂ ) ₂ -0] ₁ . ₃ H, -[(CH ₂ ) ₂ -0] _1-3 CH3, -(CH ₂ ) ₂ -NH ₂ , -(CH ₂ ) ₂ -NHCH ₃ , -

-KCH ₂ ) ₂ 0] ₁ . ₂ — (CH ₂ ) ₂ -N^^ —

164] R i 9 is CH ₃ , C(CH ₃ ) ₃ , C|.,,-alkyl, Ci _-6 -alkyl substituted with: hydroxyl, C _U6 -alkoxy, amino, mono- and carboxy, Ci.6-alkylcarbonylamino, Ci _-6 - alkylaminocarbonyl, aminosulfonyl, mono- and di-(Ci_6-alkyl)aminosulfonyl, carbamido, mono- and di-(Ci_6-alkyl)aminocarbonylamino, halogen(s), aryl, arylheterocycle, heterocycle, and heteroaryl. C ₂ -6-alkenyl, -(CH ₂ ) _2- 4-morpholino, - (CH ₂ ) ₂ - ₄ -(piperazin-l -yl), -(CH ₂ ) _2-4 -(4-methylpiperazin- l -yl), -(CH ₂ ) _2-4 -mono- and di- (C _1-6 -alkyl)amino, -(CH ₂ ) _2-4 -lH-[l,2,3]triazol-l-yl, -(CH ₂ ) ₂ _ ₄ -lH-[l,2,3]triazol-4-yl, - (CH ₂ ) ₂ .4-(4-(C i-6-alkyl)- 1 H-[ 1 ,2,3 ]triazol- 1 -yl), or -(CH ₂ ) _2-4 -( 1 -(Ci _-6 -alkyl)- 1H- [l ,2,3]triazol-4-yl).

[65] According to one particular embodiment of Formula I, II or III, R n and R ₁₂ are absent and a covalent linkage is present between the nitrogenes that is / ^ or

According to another particular embodiment Rn and Rjg are absent

N— O

and replaced by either \— / or \— / covalently linked to the nitrogen atom.

In certain exemplary embodiments, compounds within the scope of Formula I, II or III are those where, optionally, at least one atom is covalently linked between two R groups. In certain exemplary embodiments, a covalent linkage is present between Ri and Ri5 that is I . In certain exemplary embodiments, a covalent linkage is present between R ₂ and R ₃ that is . [67] lent linkage is present between R ₇ and Rs that is

In certain exemplary embodiments, a covalent

\

linkage is present between Ri and R ₂ that is ^" " . In certain exemplary

\ ,0 embodiments, a covalent linkage is present between R6 and R ₇ that is N . In and R9 that is "N / her diments, a c ovalent linkage is present between R9 and Rio that is or In other embodiments, a covalent linkage is present between Rio and Ri ₂ that is ^^1 . In t linkage is present between R14 and R15 that is

ge is present

between R15 With respect to the substituents identified above, it is to be understood that the substituents are to be covalently linked to an atom or atoms and so one of skill in the art would understand that the terminal lines in the moieties for the various R groups in this application may indicate linkage points to an atom and not the presence of an atom itself.

[68] In accordance with certain embodiments, compounds of the invention are represented by the generic formula set forth below.

Formula IV

[69] In certain exemplary embodiments with respect to Formula IV above, [70] R , is S or O.

[73] Specific compounds within the scope of the present invention include the following/those set forth in Table 1 and derivatives thereof, below.

[74] Table 1. Compounds according to certain exemplary embodiments. Compound Number Structure

1482

ΊΓΥΥΎ -

1522

XT YTT T





 1799

1800

ΙΎΪΎΥ

T

[75] An example of derivatives included within the scope of the present disclosure includes derivatives of BAY 43-9066 in Table 1 above as described in PCT/US2009/053595 hereby incorporated by reference herein in its entirety. In at least certain examples, the compounds disclosed here can be used in the treatment of cellular proliferative disorders, such as cancer and non-cancerous cellular proliferative disorders. Treatment of cellular proliferative disorders is intended to include, but is not limited to, inhibition of proliferation including rapid proliferation. As used herein, the term "cellular proliferative disorder" includes, but is not limited to, disorders characterized by undesirable or inappropriate proliferation of one or more subset(s) of cells in a multicellular organism. The term "cancer" refers to various types of malignant neoplasms, most of which can invade surrounding tissues, and may metastasize to different sites (see, for example, PDR Medical Dictionary 1 st edition (1995)). The terms "neoplasm" and "tumor" refer to an abnormal tissue that grows by cellular proliferation more rapidly than normal and continues to grow after the stimuli that initiated proliferation is removed. Id. Such abnormal tissue shows partial or complete lack of structural organization and functional coordination with the normal tissue which may be either benign (i.e., benign tumor) or malignant (i.e., malignant tumor).

Examples of general categories of cancer include, but are not limited to, carcinomas (i.e., malignant tumors derived from epithelial cells such as, for example, common forms of breast, prostate, lung, kidney, and colon cancer), sarcomas (i.e., malignant tumors derived from connective tissue or mesenchymal cells), lymphomas (i.e., malignancies derived from hematopoietic cells), leukemias (i.e., malignancies derived from hematopoietic cells), germ cell tumors (i.e., tumors derived from totipotent cells. In adults most often found in the testicle or ovary; in fetuses, babies and young children, most often found on the body midline, particularly at the tip of the tailbone), blastic tumors (i.e., a typically malignant tumor which resembles an immature or embryonic tissue) and the like. One of skill in the art will understand that this list is exemplary only and is not exhaustive, as one of skill in the art will readily be able to identify additional cancers based on the disclosure herein.

Examples of specific neoplasms intended to be encompassed by the present invention include, but are not limited to, acute lymphoblastic leukemia; myeloid leukemia, acute myeloid leukemia, childhood; adrenocortical carcinoma; AIDS-related cancers; AIDS-related lymphoma; anal cancer; appendix cancer; astrocytoma (e.g., cerebellar, cerebral); atypical teratoid/rhabdoid tumor; basal cell carcinoma; bile duct cancer, extrahepatic; bladder cancer; bone cancer, osteosarcoma and malignant fibrous histiocytoma; brain tumor (e.g., brain stem glioma, central nervous system atypical teratoid/rhabdoid tumors, central nervous system embryonal tumors, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma, medulloepithelioma, pineal parenchymal tumors of intermediate differentiation, supratentorial primitive neuroectodermal tumors and/or pineoblastoma, visual pathway and/or hypothalamic glioma, brain and spinal cord tumors); breast cancer; bronchial tumors; Burkitt lymphoma; carcinoid tumor (e.g., gastrointestinal); carcinoma of unknown primary; central nervous system (e.g., atypical teratoid/rhabdoid tumor, embryonal tumors

(e.g., lymphoma, primary); cerebellar astrocytoma; cerebral astrocytoma/malignant glioma; cervical cancer; chordoma; chronic lymphocytic leukemia; chronic myelogenous leukemia; chronic myeloproliferative disorders; colon cancer; colorectal cancer; craniopharyngioma; cutaneous T-cell lymphoma; embryonal tumors, central nervous system; endometrial cancer; ependymoblastoma; ependymoma; esophageal cancer; Ewing family of tumors; extracranial germ cell tumor; extragonadal germ cell tumor; extrahepatic bile duct cancer; eye cancer (e.g., intraocular melanoma, retinoblastoma); gallbladder cancer; gastric cancer; gastrointestinal tumor (e.g., carcinoid tumor, stromal tumor (gist), stromal cell tumor); germ cell tumor (e.g., extracranial, extragonadal, ovarian); gestational trophoblastic tumor; glioma (e.g., brain stem, cerebral astrocytoma); hairy cell leukemia; head and neck cancer; hepatocellular cancer; Hodgkin lymphoma; hypopharyngeal cancer; hypothalamic and visual pathway glioma; intraocular melanoma; islet cell tumors; Kaposi sarcoma; kidney cancer; large cell tumors; laryngeal cancer (e.g., acute lymphoblastic, acute myeloid); leukemia (e.g., acute myeloid, chronic lymphocytic, chronic myelogenous, hairy cell); lip and/or oral cavity cancer; liver cancer; lung cancer (e.g., non-small cell, small cell); lymphoma (e.g., AIDS-related, Burkitt, cutaneous Tcell, Hodgkin, non-Hodgkin, primary central nervous system); macroglobulinemia, Waldenstrom; malignant fibrous histiocytoma of bone and/or osteosarcoma; medulloblastoma; medulloepithelioma; melanoma; merkel cell carcinoma; mesothelioma; metastatic squamous neck cancer; mouth cancer; multiple endocrine neoplasia syndrome; multiple myeloma/plasma cell neoplasm; mycosis fungoides; myelodysplasia syndromes; myelodysplastic/myeloproliferative diseases; myelogenous leukemia

(e.g., chronic, acute, multiple); myeloproliferative disorders, chronic; nasal cavity and/or paranasal sinus cancer; nasopharyngeal cancer; neuroblastoma; non-Hodgkin lymphoma; non-small cell lung cancer; oral cancer; oral cavity cancer, oropharyngeal cancer; osteosarcoma and/or malignant fibrous histiocytoma of bone; ovarian cancer (e.g., ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor); pancreatic cancer (e.g., islet cell tumors); papillomatosis; paranasal sinus and/or nasal cavity cancer; parathyroid cancer; penile cancer; pharyngeal cancer; pheochromocytoma; pineal parenchymal tumors of intermediate differentiation; pineoblastoma and supratentorial primitive neuroectodermal tumors; pituitary tumor; plasma cell neoplasm/multiple myeloma; pleuropulmonary blastoma; primary central nervous system lymphoma; prostate cancer; rectal cancer; renal cell cancer; renal, pelvis and/or ureter, transitional cell cancer; respiratory tract carcinoma involving the nut gene on chromosome 15; retinoblastoma; rhabdomyosarcoma; salivary gland cancer; sarcoma (e.g., Ewing family of tumors, Kaposi, soft tissue, uterine); Sezary syndrome; skin cancer (e.g., non-melanoma, melanoma, merkel cell); small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma; squamous neck cancer with occult primary, metastatic; stomach cancer; supratentorial primitive neuroectodermal tumors; T-cell lymphoma, cutaneous; testicular cancer; throat cancer; thymoma and/or thymic carcinoma; thyroid cancer; transitional cell cancer of the renal, pelvis and/or ureter; trophoblastic tumor; unknown primary site carcinoma; urethral cancer; uterine cancer, endometrial; uterine sarcoma; vaginal cancer; visual pathway and/or hypothalamic glioma; vulvar cancer; clear cell carcinoma, Waldenstrom macroglobulinemia;

Wilms tumor and the like. For a review, see the National Cancer Institute's Worldwide Website (cancer.gov/cancertopics/alphalist). One of skill in the art will understand that this list is exemplary only and is not exhaustive, as one of skill in the art will readily be able to identify additional cancers and/or neoplasms based on the disclosure herein.

Examples of noncancerous cellular proliferative disorders includes fibroadenoma, adenoma, intraductal papilloma, nipple adenoma, adenosis, fibrocystic disease or changes of breast, plasma cell proliferative disorder (PCPD), restenosis, atherosclerosis, rheumatoid arthritis, myofibromatosis, fibrous hamartoma, granular lymphocyte proliferative disorders, benign hyperplasia of prostate, heavy chain diseases (HCDs), lymphoproliferative disorders, psoriasis, idiopathic pulmonary fibrosis, sclroderma, cirrhosis of the liver, IgA nephropathy, mesangial proliferative glomerulonephritis, membranoproliferative glomerulonephritis, hemangiomas, vascular and non-vascular intraocular proliferative disorders and the like. One of skill in the art will understand that this list is exemplary only and is not exhaustive, as one of skill in the art will readily be able to identify additional noncancerous cellular proliferative disorders based on the disclosure herein.

[80] The language "treatment of cellular proliferative disorders" is intended to include, but is not limited to, the prevention of the growth of neoplasms in a subject or a reduction in the growth of pre-existing neoplasms in a subject, as well as the prevention or reduction of increased or uncontrollable cell growth. The inhibition also can be the inhibition of the metastasis of a neoplasm from one site to another.

[81] In accordance with certain other examples, kits for treating one or more cellular proliferative disorders such as cancer are provided. In one example, the kit may comprise one or more diarylurea compounds, diarylthiourea compounds, Ν,Ν'- diarylurea compounds, Ν,Ν'-diarylthiourea compounds, substituted N,N'-diarylurea compounds, substituted Ν,Ν'-diarylthiourea compounds or compounds of Formulae I and II as described herein. In another example, the kit may comprise a pharmaceutically acceptable carrier. In an additional example, the kit may also include instructions for treating one or more cellular proliferative disorders. In some examples, the kit may also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent. In other examples, the kit may also contain a control sample or a series of control samples which can be assayed and compared to the test sample contained. Other suitable components for including in the kit will be selected by the person of ordinary skill in the art, given the benefit of this disclosure.

[82] In accordance with certain examples, compounds of the present invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the compounds disclosed here and a pharmaceutically acceptable carrier. As used herein the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[83] In accordance with certain examples, a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Such pharmaceutical compositions may be administered by inhalation, transdermally, orally, rectally, transmucosally, intestinally, parenterally, intramuscularly, subcutaneously, intravenously or other suitable methods that will be readily selected by the person of ordinary skill in the art, given the benefit of this disclosure. For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.

[84] In accordance with other examples, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMPHOR EL™ (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

In accordance with other examples, sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation can be vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. [86] In at least certain examples, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,81 1, incorporated herein by reference in its entirety for all purposes.

[87] In accordance with certain examples, pharmaceutical compositions of the invention comprise one or more diarylurea compounds, diarylthiourea compounds, Ν,Ν'- diarylurea compounds and/or Ν,Ν'-diarylthiourea compounds covalently linked to a peptide (i.e., a polypeptide comprising two or more amino acids). Peptides may be assembled sequentially from individual amino acids or by linking suitable small peptide fragments. In sequential assembly, the peptide chain is extended stepwise, starting at the C-terminus, by one amino acid per step. In fragment coupling, fragments of different lengths can be linked together, and the fragments can also be obtained by sequential assembly from amino acids or by fragment coupling of still shorter peptides.

[88] In both sequential assembly and fragment coupling it is necessary to link the units (e.g., amino acids, peptides, compounds and the like) by forming an amide linkage, which can be accomplished via a variety of enzymatic and chemical methods. The methods described herein for formation of peptidic amide linkages are also suitable for the formation of non-peptidic amide linkages.

[89] Chemical methods for forming the amide linkage are described in detail in standard references on peptide chemistry, including Miiller, Methoden der organischen Chemie Vol. XV/2, 1-364, Thieme Verlag, Stuttgart, (1974); Stewart and Young, Solid Phase Peptide Synthesis, 31-34 and 71-82, Pierce Chemical Company, Rockford, 111. (1984); Bodanszky et al., Peptide Synthesis, 85-128, John Wiley & Sons, New York, (1976); Practice of Peptide Synthesis, M. Bodansky, A. Bodansky, Springer- Verlag, 1994 and other standard works in peptide chemistry. Methods include the azide method, the symmetric and mixed anhydride method, the use of in situ generated or preformed active esters, the use of urethane protected N-carboxy anhydrides of amino acids and the formation of the amide linkage using coupling reagents, such as dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1 -ethoxycarbonyl- 2-ethoxy-l,2-dihydroquinoline (EEDQ), pivaloyl chloride, l -ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDCI), n-propane-phosphonic anhydride (PPA), Ν,Ν-bis (2-oxo-3-oxazolidinyl)amido phosphoryl chloride (BOP- Cl), bromo-tris-pyrrolidinophosphonium hexafluorophosphate (PyBrop), diphenylphosphoryl azide (DPP A), Castro's reagent ( BOP. PyBop), O-benzotriazolyl- Ν,Ν,Ν',Ν'-tetramethyluronium salts (HBTU), O-azabenzotriazolyl-Ν,Ν,Ν',Ν'- tetramethyluronuim salts (TATU), diethylphosphoryl cyanide (DEPCN), 2,5- diphenyl-2,3-dihydro-3-oxo-4-hydroxythiophene dioxide (Steglich's reagent; HOTDO), 1 , 1 '-carbonyldi imidazole (CDI) and the like. The coupling reagents can be employed alone or in combination with additives such as N,N-dimethyl-4- aminopyridine (DMAP), N-hydroxy-benzotriazole (HOBt), N-hydroxybenzotriazine (HOOBt), N-hydroxysuccinimide (HOSu), 2-hydroxypyridine and the like.

In accordance with other examples, methods of modulating translation initiation for therapeutic purposes are disclosed. In one example, a method involves contacting a cell with an agent that activates HRI thereby causing phosphorylation of cIF2 and inhibits translation initiation. An agent that inhibits translation initiation can be any one of the compounds described herein, such as a Ν,Ν'-diarylurea and/or Ν,Ν'- diarylthiourea compounds. Methods of modulating translation initiation can be performed in vitro (e.g., by culturing a cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). Certain examples disclosed herein are directed to methods of treating an individual afflicted with a disease or disorder characterized by aberrant translation initiation. Examples of such disorders are described herein. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that inhibits translation initiation. As used herein, an individual afflicted with a disease or disorder is intended to include both human and non-human mammals. Examples of non-human mammals include, but are not limited to, non-human primates, horses, cows, goats, sheep, dogs, cats, mice, rats, hamsters, guinea pigs and the like.

[91] The present invention provides for both prophylactic and therapeutic methods of treating a subject for one or more cellular proliferative disorders, such as cancer. In one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with one or more proliferative disorders by administering, to the subject one or more Ν,Ν'-diarylurea and/or Ν,Ν'-diarylthiourea compounds described herein to modulate one or more proliferative disorders. Administration of a prophylactic agent can occur prior to the manifestation of symptoms, such that a disease or disorder is prevented or, alternatively, delayed in its progression.

[92] Another aspect of the invention pertains to therapeutic methods of treating one or more cellular proliferative disorders. Accordingly, in an exemplary embodiment, a therapeutic method of the invention involves contacting a subject with a Ν,Ν'- diarylurea and/or Ν,Ν'-diarylthiourea compound that therapeutically treats one or more cellular proliferative disorders.

[93] One embodiment of the present invention involves a method of treating a translation initiation-associated disease or disorder which includes the step of administering a therapeutically and/or prophylactically effective amount of an agent which activates I I RI thereby causing phosphorylation of eIF2 and inhibits translation initiation to a subject. In another embodiment, a subject is administered a therapeutically and/or prophylactically effective amount that is effective to deplete intracellular calcium stores. As defined herein, a therapeutically and/or prophylactically effective amount of agent (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, from about 0.01 to 25 mg/kg body weight, from about 0.1 to 20 mg/kg body weight, from about 1 to 10 mg/kg, from about 2 to 9 mg/kg, from about 3 to 8 mg/kg, from about 4 to 7 mg/kg, or from about 5 to 6 mg/kg body weight. The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Treatment of a subject with a therapeutically and/or prophylactically effective amount of an inhibitor can include a single treatment or can include a series of treatments. It will also be appreciated that the effective dosage of in used for treatment may increase or decrease over the course of a particular treatment.

[94] It is to be understood that the embodiments of the present invention which have been described are merely illustrative of some of the applications of the principles of the present invention. Numerous modifications may be made by those skilled in the art based upon the teachings presented herein without departing from the true spirit and scope of the invention. The contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference in their entirety for all purposes.

[95] The following examples are set forth as being representative of the present invention.

These examples are not to be construed as limiting the scope of the invention as these and other equivalent embodiments will be apparent in view of the present disclosure, figures, and accompanying claims.

EXAMPLE I

Ν,Ν'-Diarylurea Translation Initiation Inhibitors

Design and Development of a Ternary Complex Assay

[96] A ternary complex assay was developed by constructing a bi-directional plasmid in which a common tetracycline-regulated transactivator (tTA) ²⁷ dependent promoter/enhancer complex drives the transcription of the firefly luciferase (F-luc) ORF fused to the 5'UTR of ATF-4 on one side and of a renilla luciferase (R-luc) ORF fused to a 90-nucleotide 5'UTR on the other side (pBISA-DL ^(ATF"4) , Fig. la). Stable KLN cells expressing tTA (KLN-tTA) were generated, which were then transfected with pBISA-DL ^(ATF"4) to establish stable KLN-tTA/pBISA-DL ^(ATF"4) cell lines. For assay validation, thapsigargin (TG) or tunicamycin (TU), two ER-stress inducing agents that cause phosphorylation of eIF2a ⁸ ' ²⁴ were used. Treatment with either TG or TU increased the ratio of F-luc to R-luc activity that resulted from the increased expression of F-luc and the reduced expression of R-luc (Supplementary Results, Supplementary Fig. la). The activity of TG or TU in the ternary complex assay is due to the presence and organization of multiple uORFs in the 5'UTR of ATF-4 because elimination of uORF-2 by insertion of a single nucleotide that puts it in-frame with the bona-fide ORF completely reversed the increase in the normalized F-luc R- luc ratio induced by TG or TU (Supplementary Fig. lb). Furthermore, this activity is not secondary to inhibition of cell growth because other anti-proliferative agents such as ctoposide had no activity in the ternary complex assay. See Supplementary Table 1 showing the effect of anti-cancer agents (20μΜ) on F-luc/R-luc ratio score in the ternary complex assay.

Assay validation studies with TG and DMSO in a 384-well format demonstrated that the assay has a high signal to background ratio (-100 for both luciferases), and an excellent Z factor of 0.58 as calculated from the scattered plots (Supplementary Fig. l c).

Screening

Screening was conducted in 384 well white opaque plates (Nalge Nunc), 100 μΐ volume RPMI + 10% fetal bovine serum. Cells were plated at the sub-confluent density of 10,000 cells/well, and allowed to attach for a period of 16-18 hours at 37 °C, 5% C0 ₂ . Compounds were added as 1 μΐ of a 1 niM DMSO stock solution for a final screening concentration of 10 μΜ, using low-volume tips for transfer (Molecular Bioproducts). Cells were then incubated in the presence of compound for an additional sixteen hours, again at 37°C, 5% C0 ₂ . Following incubation 70 μΐ of the culture medium was removed from each well to allow for reagent addition and plates were allowed to equilibrate to room temperature for thirty minutes. Firefly luciferase reporter activity was then read by the addition of thirty microliters of Dual Glo Luciferase reagent (Promega), followed by one hour incubation at room temperature to allow for adequate signal buildup. Luminescence counting was conducted on a Microbeta Trilux using a 1 second read time. Renilla luciferase reporter activity was measured following addition of 30 μΐ Stop and Glo Luciferase reagent (Promega) and incubation identical to the one carried out for the firefly luciferase one.

[99] Compound scores were interpreted as firefly luciferase activity divided by renilla luciferase activity, normalized to the plate's DM SO control. Using a preliminary screen of the NCI Diversity set as a guide (1990 compounds), a hit threshold of three times the DM SO control readout was chosen to achieve a target hit rate of 1%; wells with this threshold value typically fell three standard deviations from the plate mean. All data analysis was conducted using the BioAssay I I I S software package (CambridgeSoft).

[100] With this format, signal-to-noise and signal-to-background typically ran at 100 and 10 respectively, with thapsigargin (TG) an agent known to induce eIF2cc phosphorylation at 100 nM.

[101] 102,709 compounds in the NCI Open Chemical Repository were then screened using this H I S assay. Of these, approximately 1200 compounds were identified as hits in the primary screen (1.2% hit rate). Initial hits were confirmed by repeating the same dual luciferase assay in 96 well plates. Briefly, 20,000 cells/well were plated in triplicate for each concentration (10, 5 and 2.5 μΜ) of the compounds. The compounds that increased firefly/renilla luciferase ratio at least three-fold above the same ratio in the DM SO treated wells were considered confirmed hits. The final number of confirmed hits was 606. Lead Scaffolds

[102] Analysis of the screening data revealed a high prevalence of N,N -diarylureas, a known privileged scaffold, among the hits. To further assess this scaffold, a diversity library of 180 NN -diarylureas was assembled and tested in the ternary complex assay. Based on their activity as well as on structural features, one inactive ccompound was selected, l-(2-chloro-5-nitrophenyl)-3-(3,4-dichlorophenyl)urea (1, NCPdCPU), and three active compounds were selected, l-(benzo[d][l,2,3]thiadiazol- 6-yl)-3-(3,4-dichlorophenyl)urea (2, BTdCPU), l-(benzo[d][l,2,3]thiadiazol-6-yl)-3- (4-chloro-3-(trifluoromethyl)phenyl)urea (3, BTCtFPU), 1 -(benzo[c][ 1,2,5 Joxadiazol- 5-yl)-3-(4-chlorophenyl)urea (4, BOCPU), NN-diarylureas for further evaluation (Fig. lb). Dose-dependent activities of these NJV-diarylureas in the ternary complex assay are shown in Figure lc.

[103] To validate these compounds as bona fide inhibitors of the ternary complex formation, the effect of the N.N -diarylureas on the expression of CHOP mRNA was measured by real time PGR and CHOP protein by Western blot of KLN-tTA/pBISA- DL ^{(ATF" )} cells. Results of these secondary assays showed that NN-diarylureas active in the ternary complex assay also induce expression of both CHOP protein, and mRNA (Fig. Id, Fig. le, Supplementary Fig. 2) without any effect on the expression of the housekeeping protein β-actin. These N,N'-diarylureas displayed similar activities in the ternary complex and secondary assays in CRL-2351 breast, PC-3 prostate, and CRL-2813 melanoma human cancer cell lines that were co-transfected with the tTA and the pBISA-DL ^{IA IT"4)} dual luciferase expression vector (Supplementary Fig. 3a-d).

N'N'-Diarylurea Compounds Induce Phosphorylation of eIF2

[104] The availability of the ternary complex can be reduced by phosphorylation of eIF2 , by reduced expression of Met-tRNA; or by eIF2 phosphorylation-independent reduction in the activity of eI F2B, the eIF2 guanine nucleotide exchange factor. To explore these possibilities, the effect of NN -diarylureas on phosphorylation of eIF2 was determined by Western blot analysis of K I .N-tTA/pB I S A-DL ^{(ATr 4)} and PC-3 human prostate cancer cells. The three active Ν,Ν -diarylureas caused phosphorylation of eIF2 , whereas, the inactive NN -diary lurea, NCPdCPU, had a negligible effect (Fig. 2a, Supplementary Fig. 4). To determine if phosphorylation of eIF2a is necessary for the activity of NN -diary lureas in the ternary complex assay, the transgenic PC-3 human prostate cancer cell lines in which endogenous eIF2a is replaced by either a non-phosphorylatable eIF2oc mutant (eIF2a-S51A) or a recombinant wild type eIF2a (eIF2a-WT) were used. These cells were genetically engineered by transducing PC-3 cells with lentiviral expression vectors that co- express an shRNA that specifically targets only the endogenous eIF2a and HA-tagged recombinant eIF2a-S51A or eIF2a-WT. These transgenic cells were co-transfected with tTA and pBISA-DL ^lATI and treated with four NN'-diarylureas or vehicle. Replacement of endogenous eIF2oc by the non-phosphorylatable eIF2oc-S51A mutant, but not with eIF2oc-WT, significantly reduced the activity of NJV-diarylureas in the ternary complex assay (Fig. 2b). Similarly, expression of the el F2ot-S5 I A mutant but not eIF2a-WT compromised the induction of CHOP mRNA expression by these agents (Fig. 2c). These findings demonstrate that phosphorylation of eIF2a mediates the activity of the NN-diarylureas in the ternary complex assay. Phosphorylation of eIF2oc and inhibition of translation initiation selectively reduces the expression of many oncogenic proteins such as cyclin Dl with less prominent effect on that of housekeeping proteins ^10,28 . Consistently, active NJV-diarylureas reduced the expression of cyclin Dl with minimal effect on expression of proteins such p27 ^Kipl or β-actin (Supplementary Fig. 5). These findings indicate that NJV-diarylureas preferentially reduce the expression of oncogenic proteins.

Ν,Ν'-diarylurea compounds specifically activate heme regulated inhibitor (HRI)

Four distinct kinases are shown to specifically phosphorylate eIF2a in response to the metabolic state of the cells or external stimuli. These are PKR, PKR-like endoplasmic reticulum kinase (PERK), general control derepressible kinase 2 (GCN2), and heme regulated inhibitor (HRI). To elucidate the mechanism by which NN -diary lureas induce eIF2a phosphorylation, the expression of each one of the four eIF2oc kinases individually or in all combinations was knocked down by transfecting mouse KLN-tTA/pBISA-DL ^{(ATF" )} and human CRL-2813 melanoma cells with siRNAs targeting PKR, GCN2, PERK, HRI or combinations thereof. The knocked down efficiency was 70-80% for all four kinases. See Supplementary Table 3 which shows efficiency of siRNAs in knocking down the expression of e! F2cx kinase mRNAs in KLN-tT/pBISA-DL ^{(ATF_ )} and CRL-2813 cancer cells .

6] Co-transfected cells were treated with vehicle or BTdCPU, an active NN'-diarylurea, and determined the normalized F-luc/R-luc ratio. Reducing the expression of HRI significantly interfered with the activity of BTdCPU in the ternary complex assay. In contrast, knocking down PKR, PERK, or GCN2 expression either individually or in double or triple combination had no effect on the activity of BTdCPU (Fig. 3a). Consistently, silencing HRI but not the other eIF2a kinases significantly reduced the increased expression of CHOP mRNA in cells treated with BTdCPU (Fig. 3b). The effect of knocking down PERK or HRI expression on the induction of eIF2a phosphorylation by tunicamycin or BTdCPU was compared. Knocking down HRI expression significantly reduced BTdCPU induced eIF2a phosphorylation without any apparent effect on the tunicamycin induced eIF2a phosphorylation. In contrast, knocking down PERK expression significantly reduced tunicamycin induced eIF2a phosphorylation without any apparent effect on BTdCPU induced eIF2a phosphorylation (Fig. 3c and Supplementary Fig. 6, Supplementary Fig. 7). Furthermore, studies in KLN-tTA/pBISA-DL ^{(ATF" )} and CRL-2813 cell lines with all four NN'-diarylureas showed that knocking-down expression of HRI, but not other eIF2a kinases significantly reduced the effect of all three active NN'-diarylureas on the ternary complex abundance (Fig. 3d and Fig. 3e, respectively). Knocking down HRI expression in MCF-7 human breast cancer cells fully abrogated the effect of all three active N,N'-diarylureas on the ternary complex abundance and reduced the induction of CHOP mRNA (Supplementary Fig. 8a and 8b), consistent with the very high HRI knockdown efficiency in MCF-7 cells. Taken together, these data demonstrate that activation of HRI specifically mediates NN'-diarylurea-induced phosphorylation of el F2a, reduces the abundance of the ternary complex and its downstream effects. [107] Λ ^? ,Λ ^? '-dia rylu reas interact directly with HRI. To determine directly if active NN'- diarylureas directly interact with HRI, recombinant HRI was expressed and interactions of BTdCPU with HRI was investigated by proton NMR and by drug affinity responsive target stability (DARTS) assay. The proton NMR relies on the fact that BTdCPU has a unique NMR signature that would be lost upon binding to HRI because the ligand/receptor interaction causes broadening of compound specific proton signals. However, addition of aqueous buffers reduced BTdCPU specific signals below the detection limit of NMR, likely due to gradual compound aggregation on NMR time-scale. As an alternative approach, the DARTS assay was carried out in which binding of a small molecule to a protein target imparts on to the protein resistance to certain bacterial proteases such as thermolysin and subtilisin ²⁹ . HRI was digested with subtilisin in the presence of increasing concentrations of BTdCPU or vehicle. To demonstrate the specificity of the DARTS assay, recombinant eIF4E was incubated with 4EGI- 1 , a small molecule that interacts with eIF4E ³⁰ or BTdCPU. BTdCPU renders recombinant HRI but not eIF4E resistant to proteolysis (Supplementary Figure 9a and 9b). In contrast 4EGI- 1 protects recombinant eIF4E from subtilisin digestion confirming the specificity of DARTS assay. These data indicate that BTdCPU directly interacts with HRI.

[ 108] 7V,7V'-diarylureas do not cause oxidative stress. HRI can be activated in intact cells but not in cell lysates by cytoplasmic stress-inducing agents such as arsenate or H ₂ 0 ₂ ^1S ' ³¹ . To determine directly if NN'-diarylureas activate HRI by causing oxidative stress, CRL-2813 cells were incubated with various doses of BTdCPU using sodium arsenite and H ₂ 0 ₂ as positive controls. As shown in Supplementary Figure 9c, BTdCPU does not cause oxidative stress, ruling out the possibility that oxidative stress mediates activation of HRI by active NN'-diarylureas.

[109] jV,/V'-diarylureas induce eIF2oc phosphorylation in cell-free lysates. Cytoplasmic stress-inducing agents activate HRI thereby cause eIF2 phosphorylation in intact cells but not in cell-free extracts ³¹ indicating that activation of HRI is secondary to the perturbations in cellular homeostasis. To determine if the NN-diarylureas activate HRI directly or as a consequence of their effects on cellular stress, lysates of CRL- 2813 human melanoma cancer cells or rabbit reticulocytes were treated with BTdCPU and determined phosphorylation of eIF2 by Western blot analysis. BTdCPU caused phosphorylation of eIF2a in cell-free extracts in a dose dependent manner (Supplementary Figure 9d and Supplementary Figure 9e), ruling out the possibility that NN-diarylureas activate 1 1 R I by causing cellular stress. Taken together with the demonstration that BTdCPU interacts directly with HRI but does not cause oxidative stress, these data demonstrate that direct interaction of N,N'- diarylureas with HRI (or HRI containing molecular complexes) is responsible for their activity.

[ 1 10] Specificity of 7V,7V'-diarylureas. Cell proliferation was selected as a biological response parameter to demonstrate target specificity of the N,N ^J -diarylureas. The effects of the NN'-diarylureas were tested on the proliferation of KLN mouse squamous cell carcinoma, CRL-2351 human breast, CRL-2813 melanoma, A549 lung and PC-3 prostate cancer cell lines. The N,N' -diary lureas active in the ternary complex assay were also potent inhibitors of cancer cell proliferation (Table 1 below).

Table 1. Effect of N.N'-diar lureas on roliferation of human cancer cells.

Concentration of compound that inhibit cell proliferation by 50%.

[Ill] To determine if the NN-diarylureas inhibit cell proliferation by reducing the abundance of the ternary complex, their effect on the proliferation of the transgenic PC-3 human prostate cancer cell lines expressing either the non-phosphorylatable eIF2a-S51A mutant or the eIF2a-WT was compared. The results of these studies (Fig. 4a and Supplementary Fig. 10a) demonstrate that PC-3 cancer cells expressing the non-phosphorylatable eIF2a-S51A mutant were resistant, while those expressing cl F2a-WT were sensitive to the inhibition of cell proliferation by the ¾JV- diarylureas that induce eIF2a phosphorylation. Reducing the expression of HRI, the eIF2a kinase that mediates the phosphorylatation of eIF2a by the NN-diarylureas, also significantly reduced the inhibition of cell proliferation by these agents in CRL- 2813 human melanoma (Fig. 4b and Supplementary Fig. 10b) and MCF-7 human breast cancer cells (Supplementary Fig. 10c).

[ 1 12] Activity Ν,Ν'-ύ iarylu reas correlates with HRI expression. According to aspects of the present disclosure, cancer cells expressing high levels of HRI can be more susceptible to inhibition of cell proliferation than those expressing low levels of HRI. The level of HRI expression in a panel of breast, melanoma and prostate cancer cell lines was determined by Western blot analysis using β-actin levels as internal standard. The potency of the N,N'-diarylureas in abrogating proliferation of these cells was determined by SRB assay. Results showed that the sensitivity of the various cancer cell lines to the anti-proliferative effects of the NN-diarylureas correlates well with the expression of HRI. KLN cells, which express undetectable levels of HRI are least sensitive to inhibition of cell proliferation by the N,N -diary lure as whereas CRL- 2813 or CRL-2351 cells that express high level of HRI are most sensitive (Fig. 5a).

[ 1 13] AyV'-diarylureas inhibit tumor growth without toxicity. To further demonstrate that the N,N'-diarylureas can be utilized for studying the biology of the HRI and/or the ternary complex in-vivo, inhibition of tumor growth was used as an in vivo paradigm. The in vivo safety of N,N '-diary lureas was investigated. Briefly, mice were treated with various doses of BTdCPU or vehicle for seven consecutive days, the weight of animals was measured and the mice were observed for frank signs of toxicity. Treatment with 100, 200 or 350 mg/kg/d of BTdCPU had no discernable adverse effect on weight gain and mice did not display any outward signs of toxicity even at the highest dose (Fig. 5b). To determine the plasma drug levels, mice were treated with a single 175mg/kg dose of BTdCPU and the plasma drug concentrations were measured by liquid chromatography mass-spectroscopy (LC-MS). Based on the one hour plasma concentration of 1.4 μΜ, four hour plasma concentration of 0.4 μΜ and twenty four hour plasma concentration of 0.3 μΜ of BTdCPU, the mice were expected to attain a steady state plasma concentration of -0.4-2 μΜ. The anti-cancer efficacy of BTdCPU was tested against xenografted breast tumors. Briefly, mice carrying human breast tumors xenografts (-150 mm ³ ) were treated with 175 mg/kg/d BTdCPU in 1 5 μΐ DM SO or 15μ1 DMSO alone; both by i.p. injection. Mice were observed daily, and weighed twice weekly, and tumor dimensions were measured weekly. Administration of 175 mg/kg/d of BTdCPU caused a total tumor stasis starting one week after the first injection (Fig. 5c). This complete tumor stasis persisted for the remainder of the 3-week study. Western blot analysis of tumors treated for three weeks demonstrated that treatment with compound BTdCPU significantly elevated phosphory lation of eIF2a (Fig. 5d. Supplementary Fig. 11), suggesting that in vivo and in vitro anti-tumor effects of the N,N'-diarylureas are mediated by the same mechanism.

[114] To determine if long term (21 days) administration of BTdCPU causes any macro- or micro-toxicity, blood from tumor-bearing mice was collected on day 21st of treatment, the animals were sacrificed and submitted for necropsy. Blood was processed in the Hematology core facility and full necropsy and histopathology was carried out at the core Rodent Pathology Laboratory. This analysis demonstrated that BTdCPU had no effect on macroscopic and microscopic appearance of any organs (Supplementary Fig. 12). The results showed further that the administration of BTdCPU did not have any negative effect on red and white blood cells, platelet and reticulocyte counts, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin or any other blood parameters measured (Supplementary Fig. 13).

EXAMPLE 11

Materials and Methods

[115] Cell growth assay: Cell growth was measured by the SRB assay as described elsewhere ¹ .

[ 1 16] Plasmids: The pBISA plasmid contains tetracycline regulated transactivator response element (TRE), flanked on both sides by minimal human cytomegalovirus (CMV) minimal promoters, allowing bi-directional transcription and two multiple cloning sites (MCS) ²⁷ . Firefly and renilla luciferases were subcloned into MCS-I and MCS-II, respectively (Fig. 1). Generation of this expression plasmid, called pBISA-DL ^(ATF"4) , is described as follows. The bi-directional mammalian expression vector pBI (Clontech, CA) was modified to expand the multiple cloning sites MCSs. This vector contains seven copies of the tetracyline regulated transactivator response element (TRE), which together act as core promoter/enhancer. The TRE is flanked on both sides by human cytomegalovirus (CMV) minimal promoters allowing bi-directional transcription of the ORF inserted into two multiple cloning sites (MCS). Firefly and re n ilia luciferases were subcloned into MCS-I and MCS-II, respectively. This plasmid, designated pBISA-DL, transcribes two mR As that contain the 90 nucleotide plasmid derived 5 'UTR (same sequence in both mRNAs), and the ORF encoding either firefly or renilla luciferase followed by a polyadenylation sequence. This plasmid was further modified by inserting the 5 'UTR of ATF-4 into MCS-I in front of the firefly luciferase mRNA. Transcription from this direction generates an mRNA that contains the firefly luciferase ORF preceded by a 5 'UTR composed of 90 nucleotides derived from the plasmid and 267 nucleotides derived from the 5 'UTR of ATF-4 mRNA. The first two codons of the ATF-4 ORF are in frame with the firefly luciferase ORF in this mRNA. Transcription from the other direction generates an mRNA that contains the renilla luciferase ORF preceded only by the 90-nucleotide plasmid-derived sequence in the 5 'UTR (Figure 1). This expression plasmid is called pBISA-DL ^(ATF"4) .

[117] Stable and transient transfection. Cells were seeded at a density of 2x10 ⁵ in 60-mm (stable transfection) or 10 ⁴ cells per well in 96-well plates (transient transfection) and transfected using the Qiagen transfectamine transfection kit. For selection of stable cell lines, transfected cells were transferred to 100-mm plates and selected with appropriate antibiotics.

[118] Western blotting. Cell extracts were separated by SDS-PAGE and probed with anti- phosphoserine-51-eIF2a (pS51 -eIF2a, Epitomics Inc, CA), anti-total eIF2a-specific antibodies (eIF2a, Biosource International, Hopkinton, MA), anti-CHOP, or anti-β-

42

actin (Santa Cruz Biotechnology, CA) as described elsewhere .

[119] Real time PGR. Total RNA was extracted with TaqMan Gene Expression Cells-to-

Ct™ Kit (Applied Biosystems, Branchburg, NJ) and DNAse I treated according to manufacturer's recommendations. 1-Step Real-time PC R was performed on a Bio-Rad iCycler IQ5 system by using B-R 1 -Step SYBR Green qRT-PCR Kit (Quanta

Biosciences, Gaithersburg, MD) according to manufacturer's specifications. The thermal cycler conditions and the primers utilized are detailed as follows. For real time PGR, total RNA was extracted with TaqMan Gene Expression Cells-to-CtTM

Kit (Applied Biosystems, Branchburg, NJ) according to the manufacturer's protocol.

Contaminating DNA was removed by DNase I treatment. 1 -Step Real-time PGR was performed on a Bio-Rad iCycler IQ5 system by using B-R 1-Step SYBR Green qRT- PCR Kit (Quanta Biosciences, Gaithersburg, MD) according to the manufacturer's specifications. The thermal cycler conditions were as follows: 10 minutes at 50°C, hold for 5 minutes at 95°C, followed by 2-step PCR for 45 cycles of 95°C for 15 seconds followed by 60°C for 30 seconds. All PCRs were performed in triplicate in at least 2 independent PCR runs. Mean values of these repeated measurements were used for calculation. To calibrate the results, all the transcripts quantities were normalized to 18S rRNA (was 18S ribosomal RNA-like mRNA in mouse). The following primers were used in real-time PCR reactions:

[120] Human CHOP

5 ' AGAACCAGGAAACGGAAACAGA 3 ' (SEQ ID NO: l)

5' TCTCCT rCATGCGCTGCTTT 3' (SEQ ID NO:2) [121 ] Mouse CHOP

5 ' CATACACCACCACACCTGAAAG 3' (SEQ ID NO:3)

5' CCGTT rCCTAGTTCTTCCTTGC 3 ' (SEQ ID NO:4) [ 122] Human Cyclin D l

5' CGGAGGAGAACAAACAGA 3' (SEQ ID NO:5)

5 ' TGAGGCGGTAGTAGGACA 3 ' (SEQ ID NO:6) 11231 Mouse Cyclin D l

5 ' TACCGCACAACGCACTTTCTT 3' (SEQ ID NO: 7)

5 ' CGCAGGCTTGACTCCAGAAG 3' (SEQ ID NO: 8) [124] Human/Mouse 18s rRNA

5 ' CGGCGACGACCCATTCGAAC 3 ' (SEQ ID NO:9)

5 ' GAATCGAACCCTGATTCCCCGTC 3 ' (SEQ ID NO: 10) [1251 Primers for Human and Mouse p27Kip l , PKR. PERK, GCN2, HRI were purchased from Qiagen (QuantiTect Primer Assay), Human p27Kip l (QT00998445), Human PKR (QT01883280), Human PERK (QT00066003), Human GCN2 (QT01036350), Human HRI (QT01018920), Mouse p27Kipl (QTO 1058708), Mouse PKR (QT00162715), Mouse PERK (QT01565753), Mouse GCN2 (QT00138677), and Mouse HRI (QT01039171). All PCRs were performed in triplicate in at least two independent PCR runs. Mean values of these repeated measurements were used for calculation. To calibrate the results, all the transcript quantities were normalized to 18S rRNA (18S ribosomal RNA-like mRNA in mouse).

[126] RNAi transfection. The siRNA pools against Human PKR, PERK, GCN2 and HRI and Mouse PKR, PERK, GCN2 and HRI were obtained from Dharmacon. Cells were plated in 96-well plates (l x lO ⁴ cells/well) together with 25nM of siRNA Smartpool and 0.2 μΐ/well Lipofectamine RNAiMax (Invitrogen) incubated for 24 hours, then treated with compounds, and harvested at 6, 16, and 72 h after treatment for Real-time PCR, luciferase, and viability assays. The siRNA pools and transfections reagents are further described as follows. The siRNA pools are ON -T A R G ETp 1 u s SMARTpool designed siRNA pools against Human PKR (L-003527-00), Human PERK (L- 044901-00), Human GCN2 ( 1.-005314-00), Human HRI (L-005007-00), Mouse PKR (L-040807-00), Mouse PERK (L-044901 -00), Mouse GCN2 (L-044353-00), and Mouse HRI (L-045523-00) from Thermo Fisher Scientific, Dharmacon Products. Cells obtained from ATCC were cultured under recommended media conditions and plated in 96-well plates (l x lO ⁴ cells/well) in serum-containing media without antibiotics and with 25nM of siRNA Smartpool and 0.2 μΐ/well Lipofectamine RNAiMax (Invitrogen). Cells were grown at 37°C for 24 hours, then treated with compounds, and harvested at 6, 24, and 72 h after treatment for Real-time PCR, luciferase, and viability assays.

[127] High throughput screening and dual luciferase assay. Liquid handling was conducted on a Biomek FX (Beckman Coulter). Luminescence measurements were conducted on a Microbeta Trilux (Perkin Elmer). Screening was conducted in 384- well white opaque plates (Nalge Nunc), 100 μΐ RPMI + 10% fetal bovine serum. The details of screening procedure and dual luciferase assay are described as follows. Cells were plated at the subconfluent density of 10 ⁴ cells/well, and allowed to attach for a period of 16-18 hours at 37°C, 5% CO2. Compounds were added in Ι μΐ of a I mM DM SO stock solution for a final screening concentration of 10 μΜ, using low- volume tips for transfer (Molecular Bioproducts). Cells were then incubated in the presence of compound for an additional sixteen hours, again at 37°C, 5% C( Following incubation 70 μΐ of the culture medium was removed from each well to allow for reagent addition and plates were allowed to equilibrate to room temperature for thirty minutes. Firefly luciferase (F-luc) reporter activity was then read by the addition of 30 μΐ of Dual Glo Luciferase reagent (Promega Inc., Madison, WI), followed by one hour incubation at room temperature to allow for adequate signal buildup. Luminescence counting was conducted on a Microbeta Trilux using a 1 second read time. Renilla luciferase reporter activity was measured following addition of 30 μΐ Stop and Glo Luciferase reagent (Promega) and incubation identical to the one carried out for the firefly luciferase. The F-luc/R-luc (F/R) ratio in each well of a plate was normalized to the F/R ratio of vehicle treated wells of that plate.

[ 128] DARTS assay. Twelve μg recombinant I I RI or 5 μg recombinant eIF4e was incubated with DMSO, BTdCPU (5, 50, and 500 μΜ) or 4EGI1 (500 μΜ) for 2 h at 4 °C, followed by digestion with subtilisin at room temperature. 1 :800 (wt:wt) subtilisin:! I RI or 1 :500 (wt:wt) subtilisin :eIF4E for 1 h. The reactions were stopped by adding 12 μΐ SDS loading buffer and boiling for 5 min. Samples were loaded onto a 12% acrylamide SDS-PAGE gel, followed by staining with Coomassie brilliant blue to visualize the banding pattern.

[ 129] In vivo toxicity and efficacy testing. Five female nude mice each were treated with 200 mg/kg, 100 mg/kg BTdCPU in 15 μΐ DMSO or 15 μΐ DMSO daily for seven days. Mice were observed daily for signs of toxicity and weighed every other day for total of 15 days and then necropsy was performed. The average body of each group is plotted against the time. Female nude mice were implanted with a slow release estradiol 17-β pellet in the sub-scapular region. The MCF-7 human breast cancer cells were transplanted to the mammary fat pad of the 4 ^th inguinal gland of these mice. Tumor bearing mice were randomly distributed to the vehicle or treatment group, mice in the treatment group received 175 mg/kg compound BTdCPU in 15 μΐ DMSO and those in the vehicle group received the same amount of DMSO alone. Mice were observed daily, and weighed twice weekly, and tumor dimensions were measured weekly.

[ 130] Pharmacokinetics studies. Plasma concentration-time profiles were determined by treating mice with a 175 mg/kg of compound BTdCPU by IP injection in 15 μΙ, of DM SO. Blood samples were obtained from sacrificed mice at 1, 4, and 24 hours postinjection. Plasma was prepared by spinning the fresh blood containing 1000 unit/ml heparin. Analytical methods based upon high performance liquid chromatography coupled with electrospray ionization mass spectrometry were developed and validated for the determination of compounds BTdCPU in mouse plasma.

EXAMPLE III

Method of Selecting Patients for Treatment with Compounds

[131] The following protocol is used to identify or select patients for treatment with compounds described herein. 1) Obtain cancer sample (by biopsy or surgery in solid tumors or by drawing blood for blood-born disorders such as acute or chronic myologenic leukemia). Samples may be naive or treated with Ν,Ν'-diarylurea in vivo or in vitro as needed. 2) Lyse the samples and determine protein concentration. 3) Aliquot 0.1, 0.3, 1, 3, and 10 micrograms of tumor protein or PC-3 human prostate cancer cell protein to micro-centrifuge tubes with 50 microliter of 50% slurry of protein A- and/or protein G-agarose beads. 4) Rotate in cold room for one hour, centifuge at 5 thousand G, remove supernatant to fresh tubes. 5) Add between about 1 to about 10 microgram anti-HRI antibodies to between about 20 to about 50 microliter of 50% slurry of protein A- and/or protein G-agarose beads to each tube with supernatant. 6) Incubate for between about 1 to about 24 hours at between about 4°C to about 37°C by rotation. 7) Centrifuge, wash with kinase buffer 3X or as needed. 8) Remove supernatant and suspend beads in 20 microliter kinase buffer. 9) In a separate tube, mix between about 1 to about 10 micrograms of recombinant eIF2a, 10 microCi alpha-32PATP, between about 1 to about 100 micromole nonradioactive ATP, between about 1 to about 100 micromole dithiotrathiol (DTT) per each tube and bring total volume to 10 microliter per tube in step 3 plus 20 microliter. 10) Dispense 10 microliter into each tube plus 10 microliter into a tube that has only 50% slurry of protein A- and/or protein G-agarose beads (negative control). 1 1) Incubate at 37°C for between about 15 to about 60 min. 12) Run all samples on a 10% SDS-PAGE gel, dry gel, expose to phosohoroimager or photographic film for 1- 72 hours. 1 1) Develop as needed, reduce negative signal from all others. 12) Calculate the signal intensity per microgram of starting cancer or PC-3 cell protein and determine the ratio of signal intensity of cancer sample to signal intensity of PC-3 sample. If the ratio is about 0.5 or greater, the patient is selected for treatment with one or more of the compounds described herein. Exemplary, if the ratio is about 1.5 or greater, the patient is selected for treatment with one or more of the compounds described herein.

EXAMPLE IV

Method of Selecting Patients for Treatment with Compounds

The following protocol is used to identify or select patients for treatment with compounds described herein. 1) Obtain cancer sample (by biopsy or surgery in solid tumors or by drawing blood for blood-born disorders such as acute or chronic myologenic leukemia). 2) Lyse the samples and determine protein concentration. 3) Bind 0.1, 0.3, 1, 3, and 10 microgram of tumor protein or PC-3 human prostate cancer cell protein to ELISA plate. 4) Block plates for non-specific binding using any one of the available or known blocking agents such as non-fat dry milk and wash 3X or as needed. 5) Add antibodies in 1 : 100, 1 :300, 1 : 1000, 1 :3000, 1 : 10000, or 1 :30000 dilutions and incubate for between about 1 to about 24 hours at between about 4°C to about 37°C. 6) Wash plates 3X or as needed. 7) Add secondary antibodies conjugated to horseradish peroxidase, alkaline phosphatase, fluorescent dyes or any other tag known to one of skill in the art. If secondary antibodies are biotin labeled then add enzyme-linked or fluorescent tagged streptavidin. 8) Wash 3x or as needed, add enzyme substrate as needed and read the signal as needed. 9) Calculate the signal intensity per microgram of protein. Determine the ratio of the signal intensity of the cancer sample to the signal intensity of the PC-3 sample. If the ratio is about 0.5 or greater, the patient is selected for treatment with one or more of the compounds described herein. Exemplary, if the ratio is about 1.5 or greater, the patient is selected for treatment with one or more of the compounds described herein. EXAMPLE V

Synthesis

[133] Preparation of l -( 1.2,3-Bcnzothiadiazol-6-yl-3-( 3.4-dichlorophenyl)-iirea

[134] Step 1. ( 2-Λι ino-5 -nitrophcny 1 ) disulphide 2: 25 g (139 mmol) 6-nitrobenzothiazole 1 was suspended in abs. ethanol (500 mL). Hydrazine hydrate (50 mL, 1 mol) was added and the mixture was refluxed for 2 h, converting from a yellow mixture to a dark red solution. The reaction was cooled to 30 °C and 30% hydrogen peroxide (45 mL) was added in small portions, maintaining the temperature with an ice water bath. On completion, the red color disappeared and a yellow precipitate formed. The suspension was stirred for 1 h, the precipitate was collected, washed with water and diethyl ether, and dried to give 18.3 g of product 2. Yield: 78%

[135] Step 2. 6-Nitro- 1,2, 3 -benzothiadiazole 3 : 18.3 g (54 mmol) (2-amino-5-nitrophenyl) disulphide 2 was dissolved in concentrated sulfuric acid (150 mL), chilled to 0 °C, and 8.6 g (136 mmol) sodium nitrite was added in small portions, while the temperature was kept under 10 °C. The reaction was allowed to warm to room temperature and stirred for 18 h. The reaction mixture was poured into a mixture of 1 kg ice and water (100 mL), stirred for 1 h, and the pale brown precipitate was collected, then washed with water and diethyl ether. The crude product was taken up in dichloromethane (1 x 400 mL), filtered, and the filtrate was treated with a small amount of charcoal, filtered, and evaporated to give 12.3 g of product 3. Yield: 63%

[ 136] Step 3. 6-Amino-l,2,3-thiadiazole 4: 12.3 g (67.9 mmol) 6-nitro- 1 ,2,3- benzothiadiazole 3 was added to a solution of 65 g tin (II) chloride in concentrated hydrochloric acid (100 mL) at 55 °C, using a water bath to maintain that temperature. The reaction mixture was heated to 70 °C for 10 min, then cooled to 4 °C and let stand for 18 h. The crystalline precipitate was collected and washed with ice cold water. The solid was dissolved in water (200 mL) and 10 N sodium hydroxide (17 mL) was added followed by ethyl acetate (100 mL) and the mixture was stirred for 15 min. The layers were separated; the aqueous layer was extracted with, then ethyl acetate (1 x 50 mL, 1 x 30 mL). The organic layers were combined, washed with saturated brine (1 x 20 mL), water (1 x 20 mL) and evaporated. The crude product was taken up in diethyl ether (200 mL), the pale green precipitate was filtered off and extracted with diethyl ether (1 x 100 mL). The combined filtrates were treated with charcoal, filtered, and evaporated to give 5.91 g of product 4. Yield: 58%

[137] Step 4. l-(l,2,3-Benzothiadiazol-6-yl-3-(3,4-dichlorophenyl)-urea : 4.88 g (32.3 mmol) 6-Amino-l,2,3-thiadiazole 4, and 6.68 g (35.7 mmol) 3,4-dichlorophenyl isocyanate 5 were dissolved in tetrahydrofuran (125 mL), and stirred at room temperature for 2.5 h; a white precipitate formed. Methanol (3 mL) was added and the mixture was stirred for 15 min, cooled to 0 °C in an ice-water bath, stirred for 30 min, and the precipitate was collected and washed with ice cold tetrahydrofuran to give 4.69 g of product. Yield: 43% .Mp.: 267 - 269 °C (decomp.) ,LCMS: 100%; NMR: 98%.

[138] The general synthetic approaches to produce N.N'-diarylurea compounds of the present invention are set forth below.

[139] The first series of molecules in this example, most of which are symmetrical Ν,Ν'- diarylureas substituted by heteroatoms or groups of heteroatoms, was prepared by using appropriate commercially available aryl isocyanates and aryl amines according to Scheme 1.

[140] Scheme 1 :

1 (R-, = 3-CI R ₂ = 4-CI)

2 (R ₁ = 3-CF ₃ R ₂ = 4-CI)

3 (Ri = 3-CF3 R ₂ = 5-CF3)

Reagents and conditions: (i) anilines, 1,4-dioxane, 55 °C.

[141] The synthesis of compounds 1-3 was carried out in one step in 1,4-dioxane at 55 °C overnight. The same simple procedure using 5-aminocresol or 3-methoxy-4- methylaniline as new starting aryl amines was followed for the elaboration of the unsymmetrical N.N ^' -diarylureas 4-9 according to Scheme 2. [142] Scheme 2:

4 (R _† = 3-CI R ₂ = 4-CI R ₃ = H)

5 (R ₁ = 3-CF ₃ R ₂ = 4-CI R ₃ = H)

6 (R ₁ = 3-CF3 R ₂ = 5-CF3 R ₃ = H)

7 (R-i = 3-CI R ₂ = 4-CI R ₃ = CH ₃ )

8 (R-i = 3-CF3 R ₂ = 4-CI R ₃ = CH ₃ )

9 (R ₁ = 3-CF3 R ₂ = 5-CF3 R ₃ = CH ₃ )

Reagents and conditions: (i) anilines, 1,4-dioxane, 55 °C.

[143] Analogs 11-13 and 15-17 were prepared in a slightly different manner. The synthesis began by the elaboration of two different substituted anilines starting from 2-methyl- 5-nitrophenol. Compound 10, which was the precursor of N.N ^' -diarylureas 11-13, was obtained via a classic Mitsunobu coupling reaction in presence of N,N- dimethylethanolamine according to Scheme 3.

[144] Scheme 3:

11 (R = 3-CI R ₂ = 4-CI)

12 (R-i = 3-CF3 R ₂ = 4-CI)

13 (R-i = 3-CF3 R ₂ = 5-CF3)

Reagents and conditions: (i) Ν,Ν-dimethylethanolamine, PPh ₃ , DEAD, THF, 0 °C; (ii) SnC ^' b- EtOH, 90 °C; (iii) phenylisocyanates, dioxane, 55 °C. [145] In the same way, compound 14, which was the precursor of N.N'-diarylureas 15-17 was obtained starting from 4-(2-hydroxymethyl)morpholine according to Scheme 4.

1146] Scheme 4:

14

15 (Ri = 3-CI R ₂ = 4-CI)

16 (Ri = 3-CF ₃ R ₂ = 4-CI)

17 (Ri = 3-CF3 R ₂ = 5-CF3)

Reagents and conditions: (i) ΡΡΙι ,. DEAD, THF, 0 °C; (ii) SnCl ₂ , EtOH, 90 °C; (iii) phenylisocyanates, dioxane, 55 °C.

[147] After reduction of the nitro group in amine by the use of tin chloride in ethanol at 90 °C, the substituted anilines were directly coupled to the same various isocyanates in 1 ,4-dioxane at 55 °C overnight to produce 11-13 and 15-17.

[148] In order to couple piperazine with 2-methyl-5-nitrophenol via a Mitsunobu reaction (compound 19 ). the secondary amine was first protected by a benzyloxycarbonyl group. The protection was carried out with benzylclhoro formate and a solution of NaOH 4N to afford 18 according to Scheme 5.

[149] Scheme 5 :

23 (R-, = 3-CI R ₂ = 4-CI)

24 (R-, = 3-CF ₃ R ₂ = 4-CI)

25 (Ri = 3-CF3 R ₂ = 5-C F3)

Reagents and conditions: (i) benzylchloro formate, NaOH 4N, CH ₃ CN H ₂ 0; (ii) 2- methyl-5-nitrophenol, PPh ₃ , DEAD, THF, 0 °C; (iii) SnCl ₂ , EtOH, 90 °C; (iv) phenylisocyanates, dioxane, 55 °C; (v) H ₂ , Pd-C, MeOH, 1 atm; (vi) HCl 4N, dioxane.

[ 150] After the coupling reaction using triphenylphosphine and DEAD in THF, the nitro group was, as previously described, reduced in amine and coupled to the same various isocyanates to produce protected intermediates 20-22. Finally, after a hydrogenolysis carried out at atmospheric pressure under hydrogen and in presence of palladium on carbon, a precipitation in a solution of HCl 4N in 1 ,4-dioxane allowed the isolation of N.K ^' -diar\ lureas 23-25 as salts.

[151] The last series of molecules in this example, in which heteroatoms were included in the aromatic ring, was prepared starting from several substituted pyridine and pyrimidine and using the same general procedure in 1,4-dioxane at 55°C overnight according to Scheme 6. 11521 Scheme 6:

26 (R-, 3-CI R ₂ = 4-CI)

27 (R-, 3-CF3 R ₂ = 4-CI)

28 (R-i 3-CF3 R ₂ = 5-CF3)

Reagents and conditions: (i) 2-amino-3-hydroxypyridine, dioxane 55 °C.

[153] While compounds 26 and 27 appeared to be easily isolable by crystallization or purification by preparative HPLC, compound 28, which was derivated from 2-amino- 4-hydroxy-6-methylpyrimidine, appeared to be not soluble in any solvent. Because it could not be purified, this compound was removed from the structure-activity relationship (SAR) study.

General Procedure A for the Synthesis of Compounds 1-9 l,3-bis(3,4-dichlorophenyI)urea (Compound 1):

[154] As a non-limiting example, 3,4-dichlorophenylisocyanate (188 mg, 1 mmol) and 3,4- dichloroaniline (178 mg, 1.1 mmol) were dissolved in 10 mL of anhydrous dioxane. The reaction mixture was warmed to 55 °C, stirred under nitrogen over night and then cooled to room temperature. The solvent was removed under vacuum and the crude was purified twice by crystallization in ethyl acetate/hexane to afford 1 ( 262 mg, 75%) as a white powder. l,3-bis[4-chloro-3-(trifluoromethyI)phenyl]urea (Compound 2):

[155] As another non-limiting example, 4-chloro-3(trifluoromethyl) phenylisocyanate (222 mg, 1 mmol) and 4-chloro-3-(trifluoromethyl)aniline (215 mg, 1.1 mmol) were used following the general procedure A to isolate 2 (250 mg, 60 %) as a white powder. l,3-bis[3,5-bis(trifluoromethy!)phenyl]urea (Compound 3): [156] As another non-limiting example, 3,5-bis(trifluoromethyl)phenylisocyanate (600 mg, 2.353 mmol) and 3,5-bis(trifluoromethyl)aniline (647 mg, 2.824 mmol) were used following the general procedure A to isolate 3 (1 140 mg, 78%) as a white powder. ^l H NMR (500 MHz, CD ₃ OD, δ): 8.12 (s, I I I. CI I _arom ), 7.59 (s, 1H, CI I _arom ). ¹³ C NMR (400 MHz, CD ₃ OD, δ): 152.89, 141.32, 132.55, 131.80, 124.9, 122.2, 1 18.4, 1 15.24.

3-(3,4-dichloropheiiyl)-l -(3-liydroxy-4-niethylpheiiyl)urea (Compound 4):

[157] As another non-limiting example, 3,4dichlorophenylisocyanate (202 mg, 1.073 mmol) and 5-aminocresol (120 mg, 0.976 mmol) were used following the general procedure A to isolate 4 (244 mg, 81%) as a white powder. Ή NMR (500 MHz, DMSO _d6 , δ): 9.24 (s, 1H, OH), 8.82 (s, 1H, NH), 8.57 (s, 1H, NH), 7.85 (s, 1H, CH _arom ), 7.47 (d, J = 1 1 Hz, 1H, CH _arom ), 7.28 (d, ./ = 1 1 Hz, 1H, CH _arom .), 7.04 (s, 1H, CH _arom .), 6.90 (d, J = 10 Hz, 1H, CH _arom ), 6.69 (d, J = 10 Hz, 1H, CH _arom ), 2.02 (s, 3H, CH ₃ ). ¹³ C NMR (400 MHz, DMSO _d6 , δ): 156.09, 152.84, 140.77, 138.45, 131.68, 131.19, 13 1.06, 123.54, 1 19.78, 1 18.84, 1 18.35, 109.71 , 105.96, 16.09.

3-[4-chloro-3-(trifluoromethyl)phenyl]-l-(3-hydroxy-4-met hylphenyl)urea

(Compound 5):

[158] As another non-limiting example, 4-chloro-3-(trifluoromethyl)phenylisocyanate (198 mg, 0.894 mmol) and 5-aminocresol (100 mg, 0.813 mmol) were used following the general procedure A to isolate 5 (102 mg, 46%) as a white powder. Ή NMR (500 MHz, DMSO _d6 , δ): 9.26 (s, 1H, OH), 9.03 (s, 1H, NH), 8.64 (s, 1H, NH), 8.12 (s, 1H, CH _arom ), 7.58 (m, 2H, CH _arom ), 7.10 (s, 1H, CH _arom ), 6.92 (d, J = 8 Hz, 1H, CH a _rom ), 6.69 (d, J = 8 Hz, 1H, CH _arom ), 2.04 (s, 3H, CH ₃ ). ¹³ C NMR (400 MHz, DMSO _d6 , δ): 156.09, 125.93, 140.17, 138.38, 132.62, 131.05, 123.53, 122.72, 1 18.40, 109.79, 106.04, 16.07.

3-[3,5-bis(trifluoromethyl)phenyl]-l-(3-hydroxy-4-methylp henyl)urea (Compound 6):

[159] As another non-limiting example, 3,5-bis(trifluoromethyl) phcnylisocyanate (200 mg, 0.784 mmol) and 5-aminocresol (106 mg, 0.862 mmol) were used following the general procedure A to synthesize 9. At the end of the reaction, the crude was purified by flash chromatography (15 to 30% of ethyl acetate in cyclohexane). After concentration of the pure fractions, the white solid was crystallized in hexane to afford 6 (260 mg, 52.5 %) as a white powder. Ή NMR (500 MHz, DMSO _d6 , δ): 9.27 (s, 1H, OH). 9.25 (s, 1 H, Ni l). 8.79 (s, 1H, NH), 8.1 1 (s, 2H, CH _arom> ), 7.61 (s, 1H, CH _arom ), 7.12 (s, 1H, CH _arom ), 6.94 (d, J = 8 Hz, 1H, CH _arom ), 6.72 (d, J = 8 Hz, I H, CI I _arom ), 2.05 (s, 31 1. CH ₃ ). ¹³ C NMR (500 MHz, DMSO _d6 , δ): 1 56.1 1. 152.97. 142.26, 138.20, 13 1.48, 131.23, 130.97, 127.27, 125.10, 122.93, 1 18.76, 114.85, 110.05, 106.30, 16.09.

Compound 7:

[160] As another non-limiting example, 3 ,4-dich loropheny 1 isocyanate (151 mg, 0.803 mmol) and 3-methoxy-4-methylaniline (100 mg, 0.730 mmol) were used following the general procedure A. to isolate 7 (204 mg, 86%) as a white powder. Ή NMR (500 MHz, DMSO _d6 , δ): 8.91 (s, 1H, NH), 8.70 (s, 1H, NH), 7.88 (s, 1H, CH _arom ), 7.50 (d, J = 9 Hz, 1H, CH _arom ), 7.30 (d, J = 9 Hz, 1H, CH _arom ), 7.19 (s, 1H, CH _arom ), 7.01 (d, J = 8 Hz, 1H, CH _arom ), 6.82 (d, J = 8 Hz, 1H, CH _arom ), 3.76 (s, 3H, OCH ₃ ), 2.07 (s, 31 1. CH ₃ ). ¹³ C NMR (400 MHz, DMSO _d6 , δ): 157.99, 152.97, 140.71, 139.02, 131.70, 131.22, 130.87, 123.67, 119.91, 1 19.84, 119.00, 110.72, 102.14, 55.72, 16.15.

Compound 8:

[161] As another non-limiting example, 4-chloro-3-(trifluoromethyl)phenyl isocyanate (151 mg, 0.682 mmol) and 3-methoxy-4-methylaniline (85 mg, 0.620 mmol) were used following the general procedure A to isolate 8 (102 mg, 46%) as a white powder. 1H NMR (500 MHz, DMSO _d6 , δ): 9.07 (s, I I I, NH), 8.74 (s, I H, NH). 8.07 (s, I I I. CH arom.), 7.60 (m, 2H, CH arom.), 7.17 (s, IH, CH arom.), 7.00 (d, J = 10 Hz, IH, CH arom.), 6.82 (d, J = 10 Hz, IH, CH arom.), 3.74 (s, 3H, OCH3), 2.06 (s, 3H, CH3). 13C NMR (400 MHz, DMSO _d6 , δ): 157.99, 153.05, 140.10, 138.95, 132.64, 130.87, 123.70, 1 19.95, 1 17.39, 1 10.85, 102.22, 55.70, 16.14.

Compound 9:

[162] As another non-limiting example, 3,5-bis(trifluoromethyl)phenylisocyanate (143 mg, 0.562 mmol) and 3-methoxy-4-methylaniline (70 mg, 0.511 mmol) were used following the general procedure A. to isolate 9 (92 mg, 46%) as a white powder. Ή NMR (500 MHz, DMSO _d6 , δ): 9.32 (s, 1H, NH), 8.90 (s, I I I, NH), 8.10 (m, 21 1. CI I arom.), 7.61 (s, 1H, CH _arom ), 7.18 (s, 1H, CH _arom ), 7.00 (d, J = 10 Hz, 1H, CH _arom .), 6.85 (d, J = 10 Hz, I I I, CH _arom .), 3.74 (s, 3H, OCH ₃ ), 2.06 (s, 31 1. CH ₃ ). ¹³ C NMR (400 MHz, DMSO _d6 , δ): 157.98, 153.05, 142.59, 138.74, 131.50, 131.18, 130.86, 128.06, 125.35, 122.64, 120.16, 1 18.59, 1 14.92, 1 1 1.08, 102.42, 55.73, 16.15.

General procedure B (for the synthesis of compounds 10, 14 and 19)

Compound 10:

[163] As another non-limiting example, 2-methyl-5-nitrophenol (2.00 g, 13.07 mmol), N,N- dimethylethanolamine (1.31 mL, 13.07 mmol) and triphenylphosphine (4.46 g, 16.99 mmol) were placed in a 100 mL round-bottomed flask under nitrogen. 40 mL of anhydrous TI IF were added via syringe at 0°C. After stirring the reaction mixture at this temperature for 10 minutes, 7.32 mL of a solution of diethylazodicarboxylate 40% in toluene (2.93 g, 16.99 mmol) were added via syringe. The reaction was warmed to room temperature and stirred under nitrogen for two hours. The solvents were removed under vacuum. Triphenylphosphine oxide formed during the reaction was precipitated in a mixture of ethyl acetate/hexane and filtrated. The crude was then purified by flash chromatography (0 to 2% of MeOH in DCM) to afford 10 (2.1 1 g, 70%) as a yellow oil.

General procedure C (for the synthesis of compounds 11-13, 14-16 and 20-22)

3-(3,4-dichlorophenyl)-l-{3-[2-(dimethylamino)ethoxy]-4-m ethylphenyl}urea (Compound 11 ):

[164] As another non-limiting example, Compound 10 (262 mg, 1.169 mmol) was dissolved in 10 mL of EtOH. SnCLTLO (1316 mg, 5.848 mmol) was added and the temperature was increased to 90 °C. The reaction mixture was stirred for 1.5 hours, cooled to room temperature and poured into iced water. The solution was made alkaline with solid NaOH and then extracted with DCM (3 x 30 mL). Organic extracts were combined, washed with water (60 mL) and brine (60 mL), dried over sodium sulfate, concentrated and finally dried under high vacuum over night to afford the substituted aniline (202 mg, 1.041 mmol) as a light-yellow oil. This compound was then dissolved in 10 mL of anhydrous dioxane and 3,4-dichlorophenylisocyanate (254 mg, 1.353 mmol) was added. The reaction mixture was warmed to 55 °C, stirred under nitrogen overnight and then cooled to room temperature. The crude was purified by flash chromatography (0 to 2% of MeOH in DCM). After concentration of the pure fractions, the obtained white solid was crystallized in hexane to afford 11 (260 mg, 58 %) as a white powder. Ή NMR (300 MHz, DMSO _d6 , δ): 8.92 (s, IH, NH), 8.69 (s, IH, NH), 7.85 (s, IH, CH _arom ), 7.45 (d, ./ = 8.7 Hz, IH, CH _arom ), 7.27 (d, ./ = 8.7 Hz, IH, CH arom.), 7.15 (m, IH, CH„.), 6.98 (d, ./ = 7.8 Hz, I I I, CH _arom ), 6.80 (d, J = 7.8 Hz, IH, CH _arom ), 3.98 (t, J = 5.7 Hz, 2H, OCH ₂ ), 2.63 (t, J = 5.7 Hz, 2H, CH ₂ N), 2.2 1 (s, 61 1. N(CH ₃ ) ₂ ), 2.04 (s, 31 1. CH ₃ ). ¹³ C NMR (400 MHz, DMSO _d6 , δ): 157.22, 152.97, 140.67, 138.88, 131.68, 131.19, 130.90, 123.66, 120.1 1, 1 19.89, 1 18.99, 1 10.88, 103.09, 66.72, 58.30, 46.30, 16.1 1.

3-[4-chloro-3-(trifluoromethyl)phenyl]-l-{3-[2-(dimethyla mino)ethoxy]-4- methylpheiiyl} urea (Compound 12):

[165] As another non-limiting example, Compound 10 (155 mg, 0.692 mmol) and 4-chloro- 3-(trifluoromethyl) phenyl isocyanate (151 mg, 0.682 mmol) were used following the general procedure B to synthesize 12. At the end of the reaction, the mixture was precipitated in a solution of HCl 4N in dioxane. After filtration, the white solid was dissolved in acetic acid and purified by preparative HPLC (10 to 40% of acetonitrile in water with 0.1% of acetic acid) to afford 12 (161 mg, 52 %) as a white powder. ^J H NMR (500 MHz, DMSO _d6 , δ): 9.26 (s, IH, NH), 8.86 (s, IH, NH), 8.09 (s, I I I, CH a _rom ), 7.62 (m, 2H, CH _arom ), 7.18 (s, IH, CH _arom ), 7.15 (d, J = 8 Hz, IH, CH _arom ), 6.85 (d, J = 8 Hz, IH, CH _arom ), 4.02 (t, J = 5.5 Hz, 2H, OCH ₂ ), 2.71 (t, J = 5.5 Hz, 2H, CH ₂ N), 2.27 (s, 6H, N(CH ₃ ) ₂ ), 2.08 (s, 3H, CH ₃ ).

[166] 3-[3,5-bis(trifluoromethyl)phenyl]-l-{3-[2-(dimethylamino)et hoxy]-4- methylpheiiyl}urea (Compound 13):

[167] As another non-limiting example, Compound 10 (248 mg. 1.107 mmol) and 3,5- bis(trifluoromethyl) phenylisocyanate (326 mg, 1.280 mmol) were used following the general procedure B to synthesize 13. At the end of the reaction, the crude was purified by flash chromatography (2 to 8% of MeOH in DCM). After concentration of the pure fractions, the white solid was crystallized in hexane to afford 13 (260 mg, 52.5 %) as a white powder. Ή NMR (500 MHz, DMSO _d6 , δ): 9.37 (s, IH, NH), 8.90 (s, 1H, NH), 8.12 (s, 2H, CH _arom. ), 7.62 (m, 1H, CH _arom. ), 7.19 (s, 1H, CH _arom. ), 7.03 (d, J = 8 Hz, 1H, CH _arom ), 6.88 (d, J = 8 Hz, 1H, CH _arom ), 4.03 (t, J = 5.5 Hz, 2H, OCH ₂ ), 2.68 (t, J - 5.5 Hz, 2H, CH ₂ N), 2.25 (s, 6H, N(CH ₃ ) ₂ ), 2.09 (s, 3H. CH ₃ ). ¹³ C NMR (400 MHz, DMSO _d6 , δ): 157.24, 153.05, 142.58, 138.61, 131.51 , 131.19, 130.90, 127.78, 125.34, 122.62, 120.46, 1 18.53, 1 14.89, 1 1 1.26, 103.40, 66.77, 58.30, 46.28, 15.95.

Compound 14:

[168] As another non-limiting example, 2-methyl-5-nitrophenol (2.00 g, 13.07 mmol), 4(2- hydroxyethyl)morpholine (1.71 mg, 13.07 mmol), triphenylphosphine (4.46 g, 16.99 mmol) and 7.32 mL of a solution of diethylazodicarboxylate 40% in toluene (2.93 g, 16.99 mmol) were used following the general procedure B to synthesize 14. After treatments, the crude was purified by flash chromatography (0 to 3% of MeOH in DCM) to afford 14 (0.85 g, 23%) as a brown oil.

3-(3,4-diclilorophenyl)-l-{4-metliyl-3-|2-(morp oliii-4-yl)ethoxy] phenyl} urea (Compound 15):

[169] As another non-limiting example, Compound 14 (278 mg, 1 .045 mmol) and 3,4- dichlorophenylisocyanate (285 mg, 1.1 18 mmol) were used following the general procedure C to synthesize 14. At the end of the reaction, the crude was purified by flash chromatography in normal phase (10 to 0%> of cyclohexane in ethyl acetate). After concentration of the pure fractions, the obtained white solid was crystallized in hexane to afford 15 (217 mg, 49 %) as a white powder. Ή NMR (500 MHz, DMSO _d6 , δ): 8.94 (s, 1H, NH), 8.70 (s, 1H, NH), 7.89 (s, I I I. CH _arom .), 7.51 (d, J = 8.5 Hz, 1H, CH _arom ), 7.32 (d, J = 8.5 Hz, 1H, CH _arom ), 7.20 (s, 1H, CH _arom .), 7.02, (d, J = 8 Hz, 1H, CH _arom ), 6.81 (d, J = 8 Hz, 1H, CH _arom ), 4.05 (t, J = 5.5 Hz, 2H, OCH ₂ ), 3.58 (m, 4H, CH ₂ OCH ₂ ), 2.73 (t, ./ = 5.5 Hz, 21 1. CH ₂ N), 2.50 (m, 41 1. N(CH ₂ ) ₂ ), 2.08 (s, 31 1, CH ₃ ). ¹³ C NMR (400 MHz, DMSO _d6 , δ): 1 57.19. 152.96. 140.66, 138.88, 131.68, 131.19, 130.91, 123.67, 120.15, 1 19.89, 1 18.98, 110.95, 103.22, 66.87, 66.46, 57.62, 54.62, 15.98.

3-|4-chloro-3-(trifluoromethyl)phenyl]-l-{4-methyl-3-|2-( morpholin-4- yl)ethoxy]phenyl}urea (Compound 16): [ 170] As another non-limiting example. Compound 14 (267 mg, 1.004 mmol) and 4-chloro- 3-(trifluoromethyl) phenylisocyanate (241 mg, 1.091 mmol) were used following the general procedure C to synthesize 16. At the end of the reaction, the crude was purified by flash chromatography (10 to 0% of cyclohexane in ethyl acetate). After concentration of the pure fractions, the obtained white solid was crystallized in hexane to afford 16 (263 mg, 58 %) as a white powder. Ή NMR (500 MHz, DMSO _d e, δ): 9.1 1 (s, 1H, NH), 8.74 (s, 1H, NH), 8.09 (s, 1H, CH _arom ), 7.61 (m, 2H, CH _arom ), 7.20 (s, 1H, CH _arom ), 7.02 (d. J = 8 Hz, 1H, CH _arom ), 6.83 (d, J = 8 Hz, 1 1 1, CH _arom ), 4.05 (t, ./ = 5.5 Hz, 2H, OCH ₂ ), 3.58 (m, 4H, CH ₂ OCH ₂ ), 2.73 (t. J = 5.5 Hz, 2H, CH ₂ N), 2.50 (m, 4H, N(CH ₂ ) ₂ ), 2.08 (s, 31 1. CH ,). ¹³ C NMR (400 MHz, DMSO _d6 , δ): 157.23, 153.07, 140.1 1, 138.85, 1321.65, 130.92, 123.71, 120.27, 117.35, 1 10.12, 103.40, 66.77, 66.37, 57.65, 54.28, 15.98.

3-[3,5-bis(trifluoromethyl)plienyl]-l-{4-methyl-3-[2-(mor pholin-4- yl)ethoxy| phenyl} urea (Compound 17):

[171 1 As another non-limiting example, Compound 14 (273 mg, 1.026 mmol) and 3.5- bis(trifluoromethyl) phenylisocyanate (285 mg, 1.1 18 mmol) were used following the general procedure C to synthesize 17. At the end of the reaction, the crude was purified by flash chromatography in normal phase (20 to 10% of cyclohexane in ethyl acetate). After concentration of the pure fractions, the obtained white solid was crystallized in hexane to afford 17 (235 mg, 48 %) as a white powder. Ή NMR (500 MHz, DMSO _d e, δ): 10.1 1 (s, 1H, NH), 9.34 (s, 1H, NH), 8.1 1 (s, 2H, CH _arom ), 7.60 (s, 1H, CH _arom ), 7.22, (s, 1H, CH _arom ), 7.02 (d, J= 7.5 Hz, 1H, CH _arom ), 6.85 (d, J = 7.5 Hz, 1H, CH _arom ), 4.05 (m, 2H, OCH ₂ ), 3.58 (m, 4H, CH ₂ OCH ₂ ), 2.73 (m, 2H, CH ₂ N), 2.51 (m, 4H, N(CH ₂ ) ₂ ), 2.08 (s, 3H, CH ₃ ).

Compound 18:

[172] As another non-limiting example, piperazine (3.00 g, 23.04 mmol) was dissolved in 15 mL of water in a three-neck round-bottomed flask. A solution of benzylchloroformate (3.95 mL, 27.65 mmol) in 15 mL of acetonitrile was added drop wise via isobar cylindrical funnel. In order to maintain the pH around 9, a solution of NaOH 4N was added drop wise via a second isobar cylindrical funnel. The reaction mixture was stirred over night at room temperature and then extracted with DCM (2 x 75 mL). The aqueous phase containing the final compound was acidified with HC1 3N and extracted with DCM (3 x 75 mL). Organic extracts were combined, washed with brine (150 mL), dried over sodium sulfate and concentrated under vacuum. The crude was purified by flash chromatography (0 to 2% of MeOH in DCM) to afford 18 (5.41g, 90%) as a colorless oil.

Compound 19:

[173] As another non-limiting example, 2-methyl-5-nitrophenol (1.70 g, 1 1.1 1 mmol), compound 18 (2.94 mg, 1 1.1 mmol), triphenylphosphine (3.79 g, 14.44 mmol) and 6.27 mL of a solution of diethylazodicarboxylate 40% in toluene (2.51 g, 14.44 mmol) were used following the general procedure B to synthesize 19. After treatments, the crude was then purified by flash chromatography (0 to 3% of MeOH in DCM) to afford 19 (3.82 g, 86%) as a yellow oil.

Compound 20:

[174] As another non-limiting example, Compound 9 (1.172 g, 2.937 mmol) and 3.4- dichlorophenylisocyanate (0.545 g, 2.778 mmol) were used following the general procedure C to synthesize 20. After treatments, the crude was purified by flash chromatography (40 to 0% of cyclohexane in ethyl acetate) to afford 20 (0.984 g, 60 %>) as a white powder.

Compound 21 :

[175] As another non-limiting example, Compound 19 (1.042 g, 2.609 mmol) and 4-chloro- 3-(trifluoromethyl) phenylisocyanate (0.545 g, 2.459 mmol) were used following the general procedure C to synthesize 21. After treatments, the crude was purified by flash chromatography (40 to 0% of cyclohexane in ethyl acetate) to afford 21 (1.045 g, 68 %>) as a white powder.

Compound 22:

[176] As another non-limiting example, Compound 19 (992 mg, 2.486 mmol) and 3,5- bis(trifluoromethyl) phenylisocyanate (600 mg, 2.352 mmol) were used following the general procedure C to synthesize 22. After treatments, the crude was purified by flash chromatography (5 to 0% of cyclohexane in ethyl acetate) to afford 22 (945 mg, 71 %) as a white powder.

General procedure D (for the synthesis of compounds 23-25)

4-[2-(5-{|(3,4-dichlorophcnyl)carbamoyl]amino}-2- mcthylpheiioxy)ethyl|pipcrazinc-l ,4-diiuni dichloride (Compound 23):

[177] As another non-limiting example, Compound 20 (870 mg, 1.561 mmol) was dissolved in 20 mL of MeOH and Pd-C (10% by weight, 93 mg) was carefully added. The reaction mixture was stirred under a flux of hydrogen for 2 hours at atmospheric pressure and room temperature (the reaction was monitored by LCMS) and then filtered through a pad of celite. The filtrate was concentrated and purified by HPLC (10 to 45% of acetonitrile in water with 0.1% of acetic acid). After concentration of the pure fractions, the compound was precipitated in a solution of HCl 4N in dioxane to afford 23 (379 mg, 49%) as a white powder. Ή NMR (500 MHz, DMSO _d6 , δ): 12.03 (s, 1H, NH), 9.77 (s, 1H, NH), 9.57 (m, 2H, NH), 9.36 (s, 1H, NH), 7.89 (s, 1H, CH _arom ), 7.50 (d, J = 8.5 Hz, 1H, CH _arom ), 7.32 (m, 2H, CH _arom ), 7.05 (d, J = 8 Hz, 1H, CH _arom ), 6.83 (d, J = 8 Hz, 1H, CH _arom ), 4.33 (m, 2H, OCH ₂ ), 3.74-3.39 (m, 10H, CH ₂ N), 2.13 (s, 3H, CH ₃ ).

4-{2-[5-({[4-chloro-3-(trifluoromcthyl)phcnyl]carbanioyl} amino)-2- etfaylphenoxy] ethyl} piperazinc-1 ,4-diiu in dichloride (Compound 24):

[178] As another non-limiting example, Compound 21 (930 mg, 1.573 mmol) was treated following the general procedure I) and purified by HPLC (15 to 45% of acetonitrile in water with 0.1 % of acetic acid). After concentration of the pure fractions, the compound was precipitated in a solution of HCl 4N in dioxane to afford 24 (600 mg, 72%) as a white powder. Ίΐ NMR (500 MHz, DMSO _d6 , δ): 12.00 (s, 1H, NH), 9.94 (s, 1H, NH), 9.61 (m, 2H, NH), 9.40 (s, 1H, NH), 8.1 1 (s, 1H, CH _arom ), 7.61 (m, 2H, CH _arom ), 7.29 (s, 1H, CH _arom ), 7.06 (d, J = 8 Hz, 1H, CH _arom ), 6.85 (d, J = 8 Hz, 1H, CH _arom ), 4.37 (m, 2H, OCH ₂ ), 3.85-3.46 (m, 10H, CH ₂ N), 2.13 (s, 3H, CH ₃ ).

4-{2-[5-({[3,5-bis(trifluoromethyl)phenyl]carbamoyl}amino )-2- inethylphenoxy|ethyl}piperazine-l ,4-diium dichloride (Compound 25): [179] As another non-limiting example, Compound 22 (840 mg, 1.345 mmol) was treated following the general procedure D and purified by HPLC (15 to 45% of acetonitrile in water with 0.1% of acetic acid). After concentration of the pure fractions, the compound was precipitated in a solution of HC1 4N in dioxane to afford 25 (318 mg, 42%) as a white powder. Ή NMR (500 MHz, DMSO _d6 , δ): 12.13 (s, 1H, NH), 10.45 (s, I I I, NH), 9.71 (m, 2H, NH), 9.58 (s, H I, Ni l), 8. 10 (s, 21 1, CH _arom ), 7.61 (m, 121 1. CH _arom ), 7.29 (s, I I I, CH _arom ), 7.07 (d. ../ = 8 Hz, 1H, CH _arom .), 6.87 (d, J = 8 Hz, 1H, CH _arom .), 4.37 (m, 21 1. OCI I ₂ ), 3.77-3.40 (m, 101 1, CH ₂ N), 2.14 (s, 31 1, CH ₃ ).

General procedure E (for the synthesis of compounds 26-28)

Compound 26:

[180] As another non-limiting example, 3,4-dichlorophenylisocyanate (250 mg, 1.330 mmol) and 2-amino-3-hydroxypyridine (146 mg, 1.330 mmol) were dissolved in 10 mL of anhydrous dioxane. The reaction mixture was warmed to 55°C, stirred under nitrogen over night and then cooled to room temperature. The crude was purified twice by crystallization in EtOH to afford 26 (178 mg, 45 %) as a white powder.

Compound 27:

[181] As another non-limiting example, 3,4-dichlorophenylisocyanate (250 mg, 1.330 mmol) and 4-amino-6-methoxypyrimidine (166 mg, 1.330 mmol) were used following the general procedure E to synthesize 27. At the end of the reaction, the crude was dissolved in acetic acid and purified by HPLC (70 to 100 % of acetonitrile in water) to afford 27 (224 mg, 54 %) as a white powder.

Compound 28:

[182] As another non-limiting example, 3,4-dichlorophenylisocyanate (250 mg, 1.330 mmol) and 2-amino-4-hydroxy-6-methylpyrimidine (166 mg, 1.330 mmol) were used following the general procedure E to synthesize 28. Unfortunately, at the end of the reaction the crude wasn't purified as it wasn't soluble in any solvent tested. [ 183] Tabic 2. ATF-4 assays in CRL-2351 cell line.

U ^" ΎΥΎΎΤ Ϊ 7.5 20 8 28 YT T II J 5 40 40

12 _RL ^ 7 10 7

a Maximal activity corresponding to the ratio of firefly over renilla luciferase expression. * Concentration (in μΜ) corresponding to the maximal activity. ⁰ Concentration (in μΜ) corresponding to an activity threshold of 5.

[ 184] Table 3. Determination of the IC ₅ o (μΜ) of the compounds by SRB assay.

Compound ^ICs °

235 1 2813 KLN

1 2.6 0.8 >20

2 2.8 1 .7 ND

3 0.6 0.1 3.9

4 9 1 .8 >20

5 9.3 1 .6 20

6 3.2 1 13.5

1 1 1 >20 >20

8 2.5 J 17.2 2 2.2 0.7 20

11 5.8 5.3 13.5

12 2.5 2.8 12.3

13 2.9 ND 12.4

15 2.4 ND 20

16 2.7 0.9 >20

JLZ 2.3 0.8 18.6

23 4.5 5.1 >20

24 2 3.5 >20

25 5.2 3.1 ND

26 2 1.8 >20

27 1.2 0.8 12.5

28 0.7 0.7 15

Table 4. In vitro Structure Activity Relationship of N.N'-Diary Ithioureas.

Structure Code Ternary Ternary Growth

Complex* Complex* Inhibition

IC ₅₀ **

10 μηι 3 μιη

(μπι)

1806 3.5 2.5 0.9

[186] NA: Not active; *Fold over basal; ** Concentration of compound that inhibits proliferation of CRL-2351 human breast cancer cells by 50%.

[187] Table 5. Effect of anti-cancer agents (20 μΜ) on F-luc/R-luc ratio in the ternary complex assay.

Agent Mechanism of Action Normalized F-luc/R- luc Ratio (+/- SEM)

Camptothecin Topoisomerase inhibitor 1.3 ^{+ "} 0.3

Colchicine Inhibitor of tubulin polymerization 1.2 ^+/" 0.3

Threo- 1 -phenyl Glucolipid synthase inhibitor 1.5 ^{+ "} 0.4

M itomycin C Alkylating agent, DNA synthesis inhibitor 0.9 ^{+ ~} 0.3

11-89 PK-A inhibitor 1.5 ^+/" 0.6

5-fluorouracil Thymidilate synthase inhibitor l ^+/" 0.2

Epigallocatechin Laminin Receptor 1 activation 1.0 ^+/" 0.1

3-isobutyl-l- Phoshodiesterase inhibitor l .l ^+/" 0.2

methylxanthine Dilthiazem Ca++ channel blocker 1.4 ⁺ ' ^" 0.4

Amiloride Na+ channel blocker l . l ^{+ "} 0.3

Okadaic acid Protein Phoshatase 1 inhibitor l ^{+ "} 0.3

Somatostatin Inhibitor of growth hormone secretion 0.9 ^+/" 0.2

Glycyl-l-histidyl acetate Not known i ^+/ -o. i

Etoposite Topoisomerase I inhibitor l ^{+ "} 0.2

CLT Ca ⁺⁺ store depletion 6 ^+/ - l . l

[188] Other embodiments will be evident to those of skill in the art. It should be understood that the foregoing description is provided for clarity only and is merely exemplary. The spirit and scope of the present invention are not limited to the above examples, but are encompassed by the following claims. All publications and patent applications cited above are incorporated by reference herein in their entirety for all purposes to the same extent as if each individual publication or patent application was specifically indicated to be so incorporated by reference.

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