WO2012162025A1 - Procédés de sélection de patients cancéreux pour traitement par des composés de n,n'-diarylurée et de n,n'-diarlythiourée - Google Patents

Procédés de sélection de patients cancéreux pour traitement par des composés de n,n'-diarylurée et de n,n'-diarlythiourée Download PDF

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WO2012162025A1
WO2012162025A1 PCT/US2012/037913 US2012037913W WO2012162025A1 WO 2012162025 A1 WO2012162025 A1 WO 2012162025A1 US 2012037913 W US2012037913 W US 2012037913W WO 2012162025 A1 WO2012162025 A1 WO 2012162025A1
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compound
hri
cancer
compounds
cells
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PCT/US2012/037913
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Bertal Huseyin Aktas
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President And Fellows Of Harvard College
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/17—Amides, e.g. hydroxamic acids having the group >N—C(O)—N< or >N—C(S)—N<, e.g. urea, thiourea, carmustine

Definitions

  • the present invention relates to methods of identifying cancer patients for treatment with diarylurea and diarylthiourea compounds.
  • 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.
  • eIF2 eukaryotic initiation factor 2
  • GTP eukaryotic initiation factor 2
  • Met-tRNAj Met-tRNAj
  • 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.
  • eIF4E eukaryotic initiation factor 4E
  • 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.
  • 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.
  • heme regulated inhibitor kinase an eIF2 - kinase
  • HRI heme regulated inhibitor kinase
  • eIF2 - kinase couples hemoglobin synthesis to heme availability and influences the severity of hemolytic anemia such as ⁇ -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 ⁇ -cells, causing permanent neonatal diabetes in the human Wolcott-Rallison syndrome.
  • 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.
  • 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.
  • translation initiation inhibitors such as clotrimazole (CLT) inhibit translation initiation by sustained depletion of intracellular Ca 2+ stores.
  • EPA polyunsaturated fatty acid eicosapentaenoic acid
  • EPA is a ligand of peroxisome proliferator-activated receptor gamma (PPARy), a fatty acid-activated transcription factor.
  • PPARy peroxisome proliferator-activated receptor gamma
  • 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).
  • Embodiments of the present disclosure are directed to methods of identifying cancer patients for treatment with an arylurea compound or an aryl thiourea compound.
  • the term arylyurea compound includes a diarylurea compound and vice versa.
  • the term arylthiourea compound includes a diarylthiourea compound and vice versa.
  • 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.
  • 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.
  • the compounds described herein will have a greater therapeutic efficacy in cells with higher levels of HRl.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the activity of HRI may be assayed in cancer cells from an individual and used as a standard.
  • 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.
  • 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.
  • a method of identifying a candidate patient for administration of a diarylurea or a diarylthiourea compound as a treatment for cancer 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.
  • 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.
  • the compounds are of substituted diarylureas, more particularly, substituted N.N'-diarylurea compounds.
  • the compounds are substituted thioureas, more particularly, substituted ⁇ , ⁇ '- diarylthiourea compounds.
  • substituted ⁇ , ⁇ '- diarylurea and/or substituted ⁇ , ⁇ '-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.
  • the substituted N.N'-diarylurea and/or substituted ⁇ , ⁇ '- diarylthiourea compounds cause phosphorylation of eIF2 .
  • the substituted ⁇ , ⁇ '-diarylurea and/or substituted N,N'-diarylthiourea compounds activate HRI thereby causing phosphorylation of eIF2a.
  • substituted ⁇ , ⁇ '-diarylurea and/or substituted N,N'-diarylthiourea compounds are effective to inhibit translation initiation.
  • substituted ⁇ , ⁇ '-diarylurea and/or substituted ⁇ , ⁇ '-diarylthiourea compounds activate downstream effectors of eIF2 phosphorylation, reduce the expression of oncogenic proteins and potently inhibit proliferation of human cancer cell lines.
  • the compounds are non-toxic.
  • 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.
  • 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.
  • a mammal e.g., a human or a non-human (e.g., a non-human primate).
  • the proliferative disorder is cancer.
  • kits are provided for the treatment of (1) proliferative disorders, such as cancer.
  • the kits comprise an ⁇ , ⁇ '- diarylurea and/or ⁇ , ⁇ '-diarylthiourea or a substituted ⁇ , ⁇ '-diarylurea and/or a substituted ⁇ , ⁇ '-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.
  • a kit includes reagents and instructions for determining HRI levels and/or HRI activity in cancer cells, such as from an individual.
  • 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 ⁇ 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
  • F/R ratio of TG or TU treated wells was normalized to the F/R ratio of vehicle treated wells in the same plate
  • 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.
  • 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 ⁇ , ⁇ '- 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.
  • FIG. 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.
  • Supplementary Figure 4 is a full gel image of Figure 2a.
  • FIGS 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 ⁇ TM- 1 and tTA plasmids. Cells were treated with compound BTdCPU or with DMSO and the normalized F/R ratio was determined by DLR.
  • 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.
  • 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.
  • Supplementary Figure 6 is a panel showing the quantification of the western blot of Figure 3c.
  • Supplementary Figure 7 is a full gel image of Figure 3c.
  • 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 ⁇ ), or 4EGI-1 (500 ⁇ ) for 2h and at 4°C, followed by digestion with subtilisin at room temperature.
  • CRL-2813 cells were loaded with 100 ⁇ 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).
  • FIG. 4 ⁇ -4 ⁇ 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.
  • 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.
  • 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).
  • 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.
  • FIG. 5 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 ⁇ -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 ⁇ -actin levels) in the cancer cell lines.
  • IC50 concentration of the three active N,N'-diarylureas that inhibit proliferation of these cells by 50%
  • mice Five female nude mice each were treated with 200 mg/kg/d, 100 mg/kg/d and 350 mg/kg/d BTdCPU in 15 ⁇ DMSO or 15 ⁇ 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 ⁇ DMSO or DMSO alone.
  • 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.
  • cancer cells from a patient are assayed to determine the expression level of HRI or the activity of HRI.
  • cancer cells from a patient are assayed for HRI activity.
  • the patient is identified as a candidate for treatment with diarylurea compounds, diarylthiourea compounds, ⁇ , ⁇ '-diarylurea compounds, N,N'-diarylthiourea compounds, substituted ⁇ , ⁇ '-diarylurea compounds, substituted N.N'-diarylthiourca compounds or compounds of Formula I and/or Formula II and/or Formula III and/or Formula IV.
  • the individual may be identified as a suitable candidate for treatment with diarylurea compounds, diarylthiourea compounds, ⁇ , ⁇ '-diarylurea compounds, ⁇ , ⁇ '-diarylthiourea compounds, substituted ⁇ , ⁇ '-diarylurea compounds, substituted ⁇ , ⁇ '-diarylthiourea compounds or compounds of Formula I and/or Formula II and/or Formula III and/or Formula IV.
  • the compounds are administered to an individual in a manner to activate HRI thereby causing phosphorylation of eIF2a and inhibition of translation initiation.
  • compositions described herein 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.
  • compounds of the invention include diarylurea compounds, diarylthiourea compounds, ⁇ , ⁇ '-diarylurea compounds, ⁇ , ⁇ '- diarylthiourea compounds, substituted ⁇ , ⁇ '-diarylurea compounds, substituted ⁇ , ⁇ '- diarylthiourea compounds.
  • Certain exemplary embodiments are represented by the generic formula set forth below.
  • 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;.
  • 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, hal
  • Ci_s- alkoxy C2-6-alkenyl, Ci_s- alkoxy, C 2 -6-alkenyloxy, Ci-6-alkoxycarbonyl, C 1-6 -alkylcarbonyl, Ci -6 - alkylcarbonyloxy, ⁇ , ⁇ -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, heteroarylcarbonyl,
  • 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.
  • CN C ⁇ CH, N-methylacetamido, l-[l,2,3]triazolyl, 4-[l,2,3]triazolyl, 5- [ 1.2,3.4]tctrazolyl.
  • 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.
  • 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-alky
  • 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-
  • 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.
  • Ci_alkenyl Ci -6 - alkoxy, C2-6-alkenyloxy, Ci_6-alkoxycarbonyl, Ci_6-alkylcarbonyl, C 1-6 - alkylcarbonyloxy, ⁇ , ⁇ -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, heteroarylcarbonyl,
  • 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-
  • 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.
  • Ci_6- alkoxy C2-6-alkenyloxy, Ci-6-alkoxycarbonyl, Ci-6-alkylcarbonyl, Ci_6- alkylcarbonyloxy, ⁇ , ⁇ -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, heteroarylcarbonyl, heteroarylsulfon
  • 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-alkylaminocarbon
  • 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.
  • 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 ,.
  • 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.
  • 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.
  • 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.
  • R i is O, S, NH or NR19.
  • R13 is preferably S,
  • R, 5 is NH. S or NHNC ' I I.
  • 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 ,
  • 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 , -
  • R i 9 is CH 3 , C(CH 3 ) 3 , C
  • R n and R 12 are absent and a covalent linkage is present between the nitrogenes that is / ⁇ or
  • 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.
  • a covalent linkage is present between Ri and Ri5 that is I .
  • 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
  • a covalent linkage is present between R6 and R 7 that is N .
  • a c ovalent linkage is present between R9 and Rio that is or
  • a covalent linkage is present between Rio and Ri 2 that is ⁇ 1 .
  • t linkage is present between R14 and R15 that is
  • R is S or O.
  • 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.
  • 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.
  • 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.
  • 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)).
  • 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).
  • 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.
  • blastic tumors i.e., a typically malignant tumor which resembles an immature or embryonic tissue
  • 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.
  • 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,
  • 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.,
  • 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 my
  • 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.
  • 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.
  • kits for treating one or more cellular proliferative disorders such as cancer are provided.
  • the kit may comprise one or more diarylurea compounds, diarylthiourea compounds, ⁇ , ⁇ '- diarylurea compounds, ⁇ , ⁇ '-diarylthiourea compounds, substituted N,N'-diarylurea compounds, substituted ⁇ , ⁇ '-diarylthiourea compounds or compounds of Formulae I and II as described herein.
  • the kit may comprise a pharmaceutically acceptable carrier.
  • the kit may also include instructions for treating one or more cellular proliferative disorders.
  • the kit may also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent.
  • 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.
  • compositions suitable for administration typically comprise the compounds disclosed here and a pharmaceutically acceptable carrier.
  • 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.
  • 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.
  • 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.
  • 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.
  • suitable carriers include physiological saline, bacteriostatic water, CREMPHOR ELTM (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • compositions of the invention comprise one or more diarylurea compounds, diarylthiourea compounds, ⁇ , ⁇ '- diarylurea compounds and/or ⁇ , ⁇ '-diarylthiourea compounds covalently linked to a peptide (i.e., a polypeptide comprising two or more amino acids).
  • 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.
  • 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), ⁇ , ⁇ -bis (2-oxo-3-oxazolidinyl)amido phosphoryl chloride (BOP- Cl), bromo-tris-pyrrolidinophosphonium hexafluorophosphate (PyBrop), diphen
  • HBTU O-benzotriazolyl- ⁇ , ⁇ , ⁇ ', ⁇ '-tetramethyluronium salts
  • TATU O-azabenzotriazolyl- ⁇ , ⁇ , ⁇ ', ⁇ '- tetramethyluronuim salts
  • DEPCN diethylphosphoryl cyanide
  • HOTDO 2,5- diphenyl-2,3-dihydro-3-oxo-4-hydroxythiophene dioxide
  • CDI 1, 1 , 1 '-carbonyldi imidazole
  • 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.
  • DMAP N,N-dimethyl-4- aminopyridine
  • HOBt N-hydroxy-benzotriazole
  • HOOBt N-hydroxybenzotriazine
  • HOSu N-hydroxysuccinimide
  • 2-hydroxypyridine 2-hydroxypyridine and the like.
  • 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 ⁇ , ⁇ '-diarylurea and/or ⁇ , ⁇ '- 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).
  • an individual afflicted with a disease or disorder is intended to include both human and non-human mammals.
  • 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.
  • the present invention provides for both prophylactic and therapeutic methods of treating a subject for one or more cellular proliferative disorders, such as cancer.
  • 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 ⁇ , ⁇ '-diarylurea and/or ⁇ , ⁇ '-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.
  • a therapeutic method of the invention involves contacting a subject with a ⁇ , ⁇ '- diarylurea and/or ⁇ , ⁇ '-diarylthiourea compound that therapeutically treats one or more cellular proliferative disorders.
  • 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.
  • a subject is administered a therapeutically and/or prophylactically effective amount that is effective to deplete intracellular calcium stores.
  • a therapeutically and/or prophylactically effective amount of agent 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.
  • agent i.e., an effective dosage
  • 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.
  • 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
  • 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.
  • 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).
  • tTA tetracycline-regulated transactivator
  • Stable KLN cells expressing tTA were generated, which were then transfected with pBISA-DL (ATF”4) to establish stable KLN-tTA/pBISA-DL (ATF”4) cell lines.
  • ATF pBISA-DL
  • thapsigargin TG
  • TU tunicamycin
  • 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 (20 ⁇ ) on F-luc/R-luc ratio score in the ternary complex assay.
  • 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
  • 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).
  • 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.
  • eI F2B the eIF2 guanine nucleotide exchange factor.
  • the three active ⁇ , ⁇ -diarylureas caused phosphorylation of eIF2 , whereas, the inactive NN -diary lurea, NCPdCPU, had a negligible effect (Fig. 2a, Supplementary Fig. 4).
  • 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.
  • ⁇ , ⁇ '-diarylurea compounds specifically activate heme regulated inhibitor (HRI)
  • PKR PKR-like endoplasmic reticulum kinase
  • GCN2 general control derepressible kinase 2
  • HRI heme regulated inhibitor
  • 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 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.
  • addition of aqueous buffers reduced BTdCPU specific signals below the detection limit of NMR, likely due to gradual compound aggregation on NMR time-scale.
  • 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.
  • 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).
  • 4EGI- 1 protects recombinant eIF4E from subtilisin digestion confirming the specificity of DARTS assay.
  • 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 .
  • cytoplasmic stress-inducing agents such as arsenate or H 2 0 2 1S ' 31 .
  • 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.
  • 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.
  • 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.
  • 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.
  • Activity ⁇ , ⁇ '- ⁇ iarylu reas correlates with HRI expression.
  • 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 ⁇ -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).
  • AyV'-diarylureas inhibit tumor growth without toxicity.
  • 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.
  • 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 ⁇ , four hour plasma concentration of 0.4 ⁇ and twenty four hour plasma concentration of 0.3 ⁇ of BTdCPU, the mice were expected to attain a steady state plasma concentration of -0.4-2 ⁇ .
  • the anti-cancer efficacy of BTdCPU was tested against xenografted breast tumors.
  • mice carrying human breast tumors xenografts were treated with 175 mg/kg/d BTdCPU in 1 5 ⁇ DM SO or 15 ⁇ 1 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.
  • BTdCPU had no effect on macroscopic and microscopic appearance of any organs (Supplementary Fig. 12).
  • 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).
  • Cell growth assay Cell growth was measured by the SRB assay as described elsewhere 1 .
  • 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.
  • TRE tetracycline regulated transactivator response element
  • CMV cytomegalovirus
  • 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.
  • CMV human cytomegalovirus
  • This plasmid 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) .
  • actin (Santa Cruz Biotechnology, CA) as described elsewhere .
  • 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 ⁇ /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 ⁇ /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.
  • Luminescence counting was conducted on a Microbeta Trilux using a 1 second read time. Renilla luciferase reporter activity was measured following addition of 30 ⁇ 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.
  • mice [ 129] In vivo toxicity and efficacy testing. Five female nude mice each were treated with 200 mg/kg, 100 mg/kg BTdCPU in 15 ⁇ DMSO or 15 ⁇ 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- ⁇ 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.
  • mice in the treatment group received 175 mg/kg compound BTdCPU in 15 ⁇ 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.
  • Plasma concentration-time profiles were determined by treating mice with a 175 mg/kg of compound BTdCPU by IP injection in 15 ⁇ , 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.
  • 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.
  • 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.
  • Reagents and conditions (i) ⁇ ,. DEAD, THF, 0 °C; (ii) SnCl 2 , EtOH, 90 °C; (iii) phenylisocyanates, dioxane, 55 °C.
  • 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.
  • 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.
  • Compound 25 4- ⁇ 2-[5-( ⁇ [3,5-bis(trifluoromethyl)phenyl]carbamoyl ⁇ amino)-2- inethylphenoxy
  • 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.
  • Boyce, M., et al. A pharmacoproteomic approach implicates eukaryotic elongation factor 2 kinase in ER stress-induced cell death. Cell Death Differ 15, 589-599 (2008). Boyce, M., et al. A selective inhibitor of eIF2alpha dephosphorylation protects cells from ER stress. Science 307, 935-939 (2005).

Abstract

L'invention concerne un procédé d'identification de patients candidats au traitement d'un cancer par un composé d'arylurée ou un composé d'arylthiourée par la détermination du niveau d'expression de HRI ou de l'activité de HRI dans des cellules cancéreuses provenant de l'individu. Des modes de réalisation de la présente invention concernent des procédés d'identification de patients cancéreux pour le traitement par un composé d'arylurée ou un composé d'arylthiourée. Selon un aspect, l'expression composé d'arylurée comprend un composé de diarylurée et réciproquement. Selon un aspect supplémentaire, l'expression composé d'arylthiourée comprend un composé de diarylthiourée et réciproquement.
PCT/US2012/037913 2011-05-20 2012-05-15 Procédés de sélection de patients cancéreux pour traitement par des composés de n,n'-diarylurée et de n,n'-diarlythiourée WO2012162025A1 (fr)

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WO2018010856A1 (fr) * 2016-07-13 2018-01-18 Universitat De Barcelona Activateurs de hri utiles pour le traitement de maladies cardiométaboliques
CN109053497A (zh) * 2018-07-17 2018-12-21 香港浸会大学深圳研究院 一种羟基化三氯卡班的制备方法与应用
CN109734677A (zh) * 2019-03-07 2019-05-10 四川大学 抑制组蛋白赖氨酸甲基转移酶nsd2的小分子化合物及其应用
CN112218851A (zh) * 2017-11-02 2021-01-12 卡里科生命科学有限责任公司 整合应激通路的调节剂
CN112759564A (zh) * 2019-11-05 2021-05-07 中国医学科学院药物研究所 二芳基脲类化合物及其制法和药物用途
CN113355086A (zh) * 2021-06-03 2021-09-07 临沂大学 一种用于onoo-检测的比率型时间分辨荧光探针及其制备方法、应用
US11179379B2 (en) 2017-01-30 2021-11-23 The Children's Hospital Of Philadelphia Compositions and methods for hemoglobin production

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Publication number Priority date Publication date Assignee Title
WO2018010856A1 (fr) * 2016-07-13 2018-01-18 Universitat De Barcelona Activateurs de hri utiles pour le traitement de maladies cardiométaboliques
US11179379B2 (en) 2017-01-30 2021-11-23 The Children's Hospital Of Philadelphia Compositions and methods for hemoglobin production
CN112218851A (zh) * 2017-11-02 2021-01-12 卡里科生命科学有限责任公司 整合应激通路的调节剂
CN109053497A (zh) * 2018-07-17 2018-12-21 香港浸会大学深圳研究院 一种羟基化三氯卡班的制备方法与应用
CN109053497B (zh) * 2018-07-17 2021-06-01 香港浸会大学深圳研究院 一种羟基化三氯卡班的制备方法与应用
CN109734677A (zh) * 2019-03-07 2019-05-10 四川大学 抑制组蛋白赖氨酸甲基转移酶nsd2的小分子化合物及其应用
CN112759564A (zh) * 2019-11-05 2021-05-07 中国医学科学院药物研究所 二芳基脲类化合物及其制法和药物用途
CN112759564B (zh) * 2019-11-05 2023-09-26 中国医学科学院药物研究所 二芳基脲类化合物及其制法和药物用途
CN113355086A (zh) * 2021-06-03 2021-09-07 临沂大学 一种用于onoo-检测的比率型时间分辨荧光探针及其制备方法、应用
CN113355086B (zh) * 2021-06-03 2023-01-24 临沂大学 一种用于onoo-检测的比率型时间分辨荧光探针及其制备方法、应用

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