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WO2023141648A1 - 2-fluoroethyl procarbazine compounds - Google Patents

2-fluoroethyl procarbazine compounds Download PDF

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Publication number
WO2023141648A1
WO2023141648A1 PCT/US2023/061149 US2023061149W WO2023141648A1 WO 2023141648 A1 WO2023141648 A1 WO 2023141648A1 US 2023061149 W US2023061149 W US 2023061149W WO 2023141648 A1 WO2023141648 A1 WO 2023141648A1
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Prior art keywords
mgmt
cells
mmr
tmz
compound
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French (fr)
Inventor
Seth HERZON
Ranjit Bindra
Kingson LIN
Kyle TARANTINO
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Yale University
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Yale University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C243/00—Compounds containing chains of nitrogen atoms singly-bound to each other, e.g. hydrazines, triazanes
    • C07C243/10—Hydrazines
    • C07C243/12—Hydrazines having nitrogen atoms of hydrazine groups bound to acyclic carbon atoms
    • C07C243/16—Hydrazines having nitrogen atoms of hydrazine groups bound to acyclic carbon atoms of an unsaturated carbon skeleton
    • C07C243/18—Hydrazines having nitrogen atoms of hydrazine groups bound to acyclic carbon atoms of an unsaturated carbon skeleton containing rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/66—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D233/90—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems

Definitions

  • R 2 individually is selected from H and CH 3
  • R 3 individually is selected from H, CH 3 , CH(CH 3 ) 2 , CH 2 CF 3 , , or R 2 and R 3 may be taken together to provide – (CH 2 ) 5 -
  • R 4 is selected from - wherein R 2 is CH 3 only when R 3 is CH 3 .
  • the disclosure also provides methods of treating, ameliorating, and/or preventing cancer, particularly MGMT deficient cancers regardless of MMR status, as well as methods of treating, ameliorating, and/or preventing cancers that are both MGMT and MMR deficient, by administering to a patient in need of such treatment a therapeutically-effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.
  • FIG.1 shows RNA-sequencing data identifying cancers that have significant subpopulations displaying reduced MGMT expression. Each dot represents an individual patient sample.
  • KL-50 (4a) displays MGMT-dependent, MMR-independent cytotoxicity in multiple isogenic cell models.
  • FIG.2A IC50 values derived from short-term viability assays in LN229 MGMT+/–, MMR+/– cells treated with TMZ (1a) derivatives.
  • FIG.2B Short-term viability assay curves for TMZ (1a), CCNU (14), KL-85 (4b), and KL-50 (4a) in LN229 MGMT+/–, MMR+/– cells.
  • FIG.2C Clonogenic survival curves for TMZ (1a) in LN229 MGMT+/–, MMR+/– cells, with representative images of wells containing 1000 plated cells treated with 30 ⁇ M TMZ (1a).
  • FIG.2D Clonogenic survival curves for KL-50 (4a) in LN229 MGMT+/–, MMR+/– cells, with representative images of wells containing 1000 plated cells treated with 30 ⁇ M KL-50 (4a).
  • FIG.2E Short-term viability assay curves for TMZ (1a) in DLD1 MSH6-deficient cells pre-treated with 0.01% DMSO control (CTR) or 10 ⁇ M O 6 BG (+O 6 BG) for 1 h prior to TMZ (1a) addition to deplete MGMT.
  • FIG.2F Short-term viability assay curves for KL-50 (4a) in DLD1 MSH6-deficient cells pre-treated with 0.01% DMSO control (CTR) or 10 ⁇ M O 6 BG (+O 6 BG) for 1 h prior to KL-50 (4a) addition.
  • FIG.2G Short-term viability assay curves for TMZ (1a) in HCT116 MLH1–/– cells or HCT116 cells complemented with chromosome 3 carrying wildtype MLH1 (+Chr3) pre-treated with 0.01% DMSO control or 10 ⁇ M O 6 BG (+O 6 BG) for 1 h prior to TMZ (1a) addition.
  • FIG.2H Short-term viability assay curves for KL-50 (4a) in HCT116 MLH1–/– cells or HCT116 cells complemented with chromosome 3 carrying wildtype MLH1 (+Chr3) pre-treated with 0.01% DMSO control or 10 ⁇ M O 6 BG (+O 6 BG) for 1 h prior to KL-50 (4a) addition.
  • FIGs.2B-2H points, mean; error bars, SD; n ⁇ 3 technical replicates.
  • FIGs.3A-3F Unrepaired primary KL-50 (4a) lesions convert to DNA ICLs in the absence of MGMT.
  • FIG.3A Scatter dot plots of the %DNA in tail upon single cell alkaline gel electrophoresis performed on LN229 MGMT–/MMR+ and MGMT–/MMR– cells treated with 0.2% DMSO control, 200 ⁇ M TMZ (1a), 200 ⁇ M KL-50 (4a), or 0.1 ⁇ M MMC (MMC*) for 24 h or with 50 ⁇ M MMC (MMC**) for 2 h. After cell lysis, comet slides were irradiated with 0 or 10 Gy prior to alkaline electrophoresis. Lines, median; error bars, 95% CI; n ⁇ 160.
  • FIG.3B Representative comet images from (A).
  • FIG.3C Scatter dot plots of the %DNA in tail upon single cell alkaline gel electrophoresis performed on LN229 MGMT– /MMR– cells treated with 0.2% DMSO control, 200 ⁇ M MTZ (12a), 200 ⁇ M TMZ (1a), or 200 ⁇ M KL-50 (4a) for 2, 8, or 24 h. After cell lysis, comet slides were irradiated with 10 Gy prior to alkaline electrophoresis. Lines, median; error bars, 95% CI; n ⁇ 230. Data from samples treated with 0 Gy are shown in FIG. S4, C and D. (FIG.3D) Representative comet images from FIG.3C.
  • FIG.3E Denaturing gel electrophoresis of genomic DNA isolated from LN229 MGMT–/MMR+ cells treated with 0.2% DMSO control, 200 ⁇ M KL-50 (4a), 200 ⁇ M TMZ (1a), 200 ⁇ M KL-85 (4b), or 200 ⁇ M MTIC (1b) for 24 h or with 50 ⁇ M MMC or 200 ⁇ M CCNU (14) for 2 h.
  • FIG.3F Denaturing gel electrophoresis of linearized 100 ng pUC19 plasmid DNA treated in vitro with 100 ⁇ M Cisplatin (36 hours), 100 ⁇ M MMS (36 hours), 100 ⁇ M of KL-50 (4a) or 12b for 6–36 hours.
  • FIG.4A-4I KL-50 (4a) activates DNA damage response pathways and cycle arrest in MGMT– cells, independent of MMR, and induces sensitivity in cells deficient in ICL or HR repair.
  • FIG.4A, FIG.4B, and FIG.4C Phospho-SER139-H2AX ( ⁇ H2AX) (FIG.4A), 53BP1 (FIG.4B), and phospo-SER33-RPA2 (pRPA) (FIG.4C) foci formation quantified by % cells with ⁇ 10 foci in LN229 MGMT+/–, MMR+/– cells treated with 0.1% DMSO control, 20 ⁇ M KL-50 (4a), or 20 ⁇ M TMZ (1a) for 48 h. Columns, mean; error bars, SD; n ⁇ 5 technical replicates. Additional time course data is presented in FIG.18, 12B to 12C. (FIG.
  • FIG.4F Change in percent cells with ⁇ 1 micronuclei from baseline (DMSO control) after treatment as in FIG.4A to FIG.4C. Columns, mean; error bars, SD; n ⁇ 15 technical replicates; **** p ⁇ 0.0001; ns, not significant.
  • FIG.21A and 21B Additional validation is presented in FIG.21A and 21B.
  • FIG.4G Short-term viability assay curves for KL-50 (4a) in PD20 cells, deficient in FANCD2 (FANCD2–/–) or complemented with FANCD2 (+FANCD2).
  • FIG.4H Short-term viability assay curves for KL-50 (4a) in PEO4 (BRCA2+) and PEO1 (BRCA2–/–) cells pre-treated with 0.01% DMSO control or 10 ⁇ M O 6 BG (+O 6 BG) for 1 h prior to KL-50 (4a) addition.
  • FIG.4I Short-term viability assay curves for KL-50 (4a) in DLD1 BRCA2+/– and BRCA2–/– cells pre-treated with 0.01% DMSO control or 10 ⁇ M O 6 BG (+O 6 BG) for 1 h prior to KL-50 (4a) addition.
  • KL-50 (4a) is safe and efficacious on both MGMT–/MMR+ and MGMT–/MMR– flank tumors over a wide range of treatment regimens and conditions.
  • FIG.5B Xenograft LN229 MGMT–/MMR– flank tumors treated with 3 weekly cycles of P.O.
  • FIG.5C Mean body weight of mice during LN229 flank tumor experiments.
  • FIG.5D Kaplan-Meier analysis of LN229 MGMT–/MMR– xenograft flank tumor-bearing mice to determine survival rate based on death, removal from study if mouse body weight loss exceeded 20% of initial body weight, or if tumor volume exceeded 2000 mm 3 .
  • Both control and TMZ (1a) treated groups have a median OS of 10 weeks and KL-50 (4a) treated mice have median OS of greater than 15 weeks.
  • the study period was limited by control groups which had to be euthanized for exceeding the ethical maximum allowed tumor size, thus ending the study.
  • FIGs.6A-6C KL-50 (4a) is efficacious in an LN229 MGMT–/MMR– intracranial model and is well tolerated with limited myelosuppression at supratherapeutic doses. (FIG.
  • FIG.6B Mean body change with SEM of mice during maximum tolerated dose experiment in non-tumor bearing mice.
  • FIG.6C Complete blood counts for mice pre-treatment and 7 days post-treatment with escalations of single dose KL-50 (4a) delivered PO.
  • FIGs.7A-7F Overview of mechanistic strategy and structures of agents employed in this study.
  • FIG.7A Underlying mechanistic hypothesis.
  • TMZ (1a) is the front-line therapy for the treatment of MGMT– GBM. Under physiological conditions, TMZ (1a) converts to MTIC (1b) which decomposes to methyl diazonium (1c).
  • FIG.7C O 6 -Guanine is the most clinically-significant site of methylation by methyl diazonium (1c).
  • O 6 MeG (3) is rapidly reverted to dG (2) by MGMT (the second-order rate constant for demethylation of calf thymus DNA by MGMT is 1 ⁇ 10 9 M –1 •min –1 ), but persists in the genome of MGMT– cells, ultimately leading to MMR-dependent cytotoxicity.
  • MGMT the second-order rate constant for demethylation of calf thymus DNA by MGMT is 1 ⁇ 10 9 M –1 •min –1
  • FIG.7D It was envisioned that imidazotetrazine KL-50 (4a) could be utilized as a source of 2-fluoroethyl diazonium ion (4c).
  • FIG.7E Fluoroethylation at O 6 -guanosine would form O 6 FEtG (5), which is known to slowly rearrange (t 1/2 ⁇ 18.5 h at 37 °C) via intermediate 6 to form the dG–dC ICL 8. Based on the broad substrate scope of MGMT, it was anticipated that O 6 FEtG (5) would be readily reversed in MGMT+ cells, thereby preventing ICL formation in healthy tissue. Realization of this goal would provide the first MMR-independent agent active specifically in MGMT– glioma.
  • FIG.7F Structures of the triazenes 9–13, mitozolomide 12a, and lomustine (CCNU, 14). FIGs.8A-8H.
  • KL-50 (4a) displays MGMT-dependent, MMR-independent cytotoxicity in multiple isogenic cell models.
  • FIG.8A IC 50 values derived from short-term viability assays in LN229 MGMT+/–, MMR+/– cells treated with TMZ (1a) derivatives.
  • a MGMT TI (therapeutic index) IC50 (MGMT+/MMR+) divided by IC50 (MGMT– /MMR+).
  • b MMR RI (resistance index) IC 50 (MGMT–/MMR–) divided by IC 50 (MGMT– /MMR+).
  • FIG.8B Short-term viability assay curves for TMZ (1a), CCNU (14), KL-85 (4b), and KL-50 (4a) in LN229 MGMT+/–, MMR+/– cells.
  • FIG.8C Clonogenic survival curves for TMZ (1a) in LN229 MGMT+/–, MMR+/– cells, with representative images of wells containing 1000 plated cells treated with 30 ⁇ M TMZ (1a).
  • FIG.8D Clonogenic survival curves for KL-50 (4a) in LN229 MGMT+/–, MMR+/– cells, with representative images of wells containing 1000 plated cells treated with 30 ⁇ M KL-50 (4a).
  • FIG.8E Short-term viability assay curves for TMZ (1a) in DLD1 MSH6-deficient cells pre-treated with 0.01% DMSO control (CTR) or 10 ⁇ M O 6 BG (+O 6 BG) for 1 h prior to TMZ (1a) addition to deplete MGMT.
  • FIG.8F Short-term viability assay curves for KL-50 (4a) in DLD1 MSH6-deficient cells pre-treated with 0.01% DMSO control (CTR) or 10 ⁇ M O 6 BG (+O 6 BG) for 1 h prior to KL-50 (4a) addition.
  • FIG.8G Short-term viability assay curves for TMZ (1a) in HCT116 MLH1–/– cells or HCT116 cells complemented with chromosome 3 carrying wildtype MLH1 (+Chr3) pre-treated with 0.01% DMSO control or 10 ⁇ M O 6 BG (+O 6 BG) for 1 h prior to TMZ (1a) addition.
  • FIG.8H Short-term viability assay curves for KL-50 (4a) in HCT116 MLH1–/– cells or HCT116 cells complemented with chromosome 3 carrying wildtype MLH1 (+Chr3) pre-treated with 0.01% DMSO control or 10 ⁇ M O 6 BG (+O 6 BG) for 1 h prior to KL-50 (4a) addition.
  • FIGs.8B-8H points, mean; error bars, SD; n ⁇ 3 technical replicates.
  • FIGs.9A-9F Unrepaired primary KL-50 (4a) lesions convert to DNA ICLs in the absence of MGMT.
  • FIG.9A Scatter dot plots of the %DNA in tail upon single cell alkaline gel electrophoresis performed on LN229 MGMT–/MMR+ and MGMT–/MMR– cells treated with 0.2% DMSO control, 200 ⁇ M TMZ (1a), 200 ⁇ M KL-50 (4a), or 0.1 ⁇ M MMC (MMC*) for 24 h or with 50 ⁇ M MMC (MMC**) for 2 h. After cell lysis, comet slides were irradiated with 0 or 10 Gy prior to alkaline electrophoresis. Lines, median; error bars, 95% CI; n ⁇ 160.
  • FIG.9B Representative comet images from (FIG.9A).
  • FIG.9C Scatter dot plots of the %DNA in tail upon single cell alkaline gel electrophoresis performed on LN229 MGMT–/MMR– cells treated with 0.2% DMSO control, 200 ⁇ M MTZ (12a), 200 ⁇ M TMZ (1a), or 200 ⁇ M KL-50 (4a) for 2, 8, or 24 h. After cell lysis, comet slides were irradiated with 10 Gy prior to alkaline electrophoresis. Lines, median; error bars, 95% CI; n ⁇ 230. Data from samples treated with 0 Gy are shown in FIGs.16C and 16D. (FIG.9D) Representative comet images from (FIG.9C).
  • FIG.9E Denaturing gel electrophoresis of genomic DNA isolated from LN229 MGMT–/MMR+ cells treated with 0.2% DMSO control, 200 ⁇ M KL-50 (4a), 200 ⁇ M TMZ (1a), 200 ⁇ M KL-85 (4b), or 200 ⁇ M MTIC (1b) for 24 h or with 50 ⁇ M MMC or 200 ⁇ M CCNU (14) for 2 h.
  • FIG.9F Denaturing gel electrophoresis of linearized 100 ng pUC19 plasmid DNA treated in vitro with 100 ⁇ M Cisplatin (36 hours), 100 ⁇ M MMS (36 hours), 100 ⁇ M of KL-50 (4a) or 12b for 6–36 hours.
  • FIGs.10A-10I KL-50 (4a) activates DNA damage response pathways and cycle arrest in MGMT– cells, independent of MMR, and induces sensitivity in cells deficient in ICL or HR repair.
  • FIG.10A, FIG.10B, and FIG.10C Phospho-SER139-H2AX ( ⁇ H2AX) (FIG.10A), 53BP1 (FIG.10B), and phospo-SER33-RPA2 (pRPA) (FIG.10C) foci formation quantified by % cells with ⁇ 10 foci in LN229 MGMT+/–, MMR+/– cells treated with 0.1% DMSO control, 20 ⁇ M KL-50 (4a), or 20 ⁇ M TMZ (1a) for 48 h. Columns, mean; error bars, SD; n ⁇ 5 technical replicates. Additional time course data is presented in FIG.18, 18B to 18D.
  • FIG.10D Representative foci images of data in (FIG.10A) to (FIG.10C).
  • FIG.10F Change in percent cells with ⁇ 1 micronuclei from baseline (DMSO control) after treatment as in (FIG. 10A) to (FIG.10C).
  • FIGs.21A and 21B Short-term viability assay curves for KL-50 (4a) in PD20 cells, deficient in FANCD2 (FANCD2–/–) or complemented with FANCD2 (+FANCD2).
  • FIG.10H Short-term viability assay curves for KL-50 (4a) in PEO4 (BRCA2+) and PEO1 (BRCA2–/–) cells pre- treated with 0.01% DMSO control or 10 ⁇ M O 6 BG (+O 6 BG) for 1 h prior to KL-50 (4a) addition.
  • FIG.10I Short-term viability assay curves for KL-50 (4a) in DLD1 BRCA2+/– and BRCA2–/– cells pre-treated with 0.01% DMSO control or 10 ⁇ M O 6 BG (+O 6 BG) for 1 h prior to KL-50 (4a) addition.
  • KL-50 (4a) is safe and efficacious on both MGMT–/MMR+ and MGMT–/MMR– flank tumors over a wide range of treatment regimens and conditions.
  • FIG.11A Xenograft LN229 MGMT–/MMR+ flank tumors treated with 3 weekly cycles of P.O.
  • FIG.11C Mean body weight of mice during LN229 flank tumor experiments.
  • FIG.11D Kaplan-Meier analysis of LN229 MGMT–/MMR– xenograft flank tumor-bearing mice to determine survival rate based on death, removal from study if mouse body weight loss exceeded 20% of initial body weight, or if tumor volume exceeded 2000 mm 3 .
  • Both control and TMZ (1a) treated groups have a median OS of 10 weeks and KL-50 (4a) treated mice have median OS of greater than 15 weeks.
  • the study period was limited by control groups which had to be euthanized for exceeding the ethical maximum allowed tumor size, thus ending the study.
  • FIGs.12A-12C KL-50 (4a) is efficacious in an LN229 MGMT–/MMR– intracranial model and is well tolerated with limited myelosuppression at supratherapeutic doses. (FIG.
  • FIG.12B Mean body change with SEM of mice during maximum tolerated dose experiment in non-tumor bearing mice.
  • FIG.12C Complete blood counts for mice pre-treatment and 7 days post-treatment with escalations of single dose KL-50 (4a) delivered PO.
  • FIGs.13A-13C Literature precedent for the hydrolysis of various 2- haloethylguanosine lesions.
  • FIG.13A Kinetics of the hydrolysis of O6-(2- fluoroethylguanosine) (S1) at pH 7.4 and 37 oC l.
  • FIG.13B Kinetics of the hydrolysis of O6-(2-chloroethylguanosine) (S4) at pH 7.4 and 37 oC.
  • FIG.13C Failed hydrolysis of N7- (2-fluoroethyl)guanosine (S5) with “extensive incubation of [S5] at 37 o [C] in neutral aqueous solution”.
  • FIGs.14A-14K Additional analysis of TMZ (1a) derivatives in MGMT+/–, MMR+/– cell models.
  • FIG.14A Western blotting performed in LN229 MGMT–/MMR+ parental line, and cells complemented with wildtype MGMT (MGMT+/MMR+) and/or stable expression of MSH2 shRNA (MGMT+/MMR– and MGMT–/MMR–).
  • MSH6 expression is reduced in these lines due to destabilization in the setting of loss of its heterodimeric partner MSH2.
  • MLH1 expression is not affected by MSH2 knockdown. Vinculin serves as loading control.
  • FIG.14B, FIG.14C, FIG.14D, FIG.14E, FIG.14F, and FIG.14G Short-term viability assay curves for compounds 9, 10, 11, 12b, 13, and 12a in LN229 MGMT+/–, MMR+/– cells.
  • FIG.14H Clonogenic survival curves for lomustine (14) in LN229 MGMT+/–, MMR+/– cells.
  • FIG.14I Western blotting in HCT116 and DLD1 cells.
  • HCT116 MLH1–/– and +Chr3 lines demonstrate re-expression of MLH1 and similar levels of MGMT and other MMR proteins.
  • DLD1 BRCA2+/– and BRCA2–/– cells have known loss of MSH6 but comparable levels of MGMT and other MMR protein expression.
  • GAPDH serves as loading control.
  • FIG.14J Western blotting performed in HCT116 MLH1–/– and +Chr3 and DLD1 BRCA2+/– and BRCA2–/– cells after exposure to 0.01% DMSO or 10 ⁇ M O6BG for 24 h, demonstrating O6BG-induced MGMT depletion. Vinculin serves as loading control.
  • FIG.14K Short-term cell viability curves for KL-50 (4a) and TMZ (1a) in BJ fibroblast cells.
  • FIG.15A-15J KL-50 (4a) is effective in TMZ (1a)-resistant cells lacking other MMR proteins.
  • FIG.15A Western blotting performed in LN229 MGMT+/– cells with stable expression of shRNA targeting MSH6, MLH1, PMS2, or MSH3 to confirm depletion of the shRNA targets.
  • aMGMT TI therapeutic index
  • IC50 IC50 (MGMT+/MMR+) divided by IC50 (MGMT–/MMR+).
  • bMMR RI resistance index
  • IC50 IC50 (MGMT–/MMR–) divided by IC50 (MGMT–/MMR+).
  • FIG.15C Short-term viability assay curves for TMZ (1a) in LN229 MGMT+/–, MMR+/shMSH6 cells.
  • FIG.15D Short-term viability assay curves for KL-50 (4a) in LN229 MGMT+/–, MMR+/shMSH6 cells.
  • FIG.15E Short-term viability assay curves for TMZ (1a) in LN229 MGMT+/–, MMR+/shMLH1 cells.
  • FIG.15F Short- term viability assay curves for KL-50 (4a) in LN229 MGMT+/–, MMR+/shMLH1 cells.
  • FIG.15G Short-term viability assay curves for TMZ (1a) in LN229 MGMT+/–, MMR+/shPMS2 cells.
  • FIG.15H Short-term viability assay curves for KL-50 (4a) in LN229 MGMT+/–, MMR+/shPMS2 cells.
  • FIG.15I Short-term viability assay curves for TMZ (1a) in LN229 MGMT+/–, MMR+/shMSH3 cells.
  • FIG.15J Short-term viability assay curves for KL-50 (4a) in LN229 MGMT+/–, MMR+/shMSH3 cells.
  • FIGs.16A-16D Supplementary IR alkaline comet assay data.
  • FIG.16A Scatter dot plots of the %DNA in tail upon single cell alkaline gel electrophoresis performed on LN229 MGMT–/MMR+ cells treated with 0.1% DMSO control or 200 ⁇ M KL-85 (4b) for 24 h or with 50 ⁇ M MMC for 2 h.
  • FIG.16B Representative comet images from (FIG.16A).
  • FIG.16C Scatter dot plots of the %DNA in tail upon single cell alkaline gel electrophoresis performed on LN229 MGMT–/MMR– cells treated with 0.2% DMSO control, 200 ⁇ M MTZ (12a), 200 ⁇ M TMZ (1a), or 200 ⁇ M KL-50 (4a) for 2, 8, or 24 h. Corresponding samples treated with 10 Gy IR are shown in FIG.9C.
  • FIG.16D Representative comet images from (FIG.16C).
  • FIGs.17A-17D Representative comet images from (FIG.16C).
  • FIGs.17A-17D Representative comet images from (FIG.16C).
  • FIGs.17A-17D Representative comet images from (FIG.16C).
  • FIGs.17A-17D Representative comet images from (FIG.16C).
  • FIGs.17A-17D Representative comet images from (FIG.16C).
  • NER, BER, ROS, and altered DNA melting point do not play a major role in the mechanism of KL-50 (4a).
  • FIG.17A Short-term cell viability assays in both WT and XPA-deficient MEFs demonstrating the absence of additional sensitivity to KL- 50 (4a) in NER compromised XPA deficient cells ⁇ MGMT depletion with O6BG, in contrast to cisplatin as positive control.
  • FIG.17B EndoIV depurination assay utilizing supercoiled pUC19 plasmid DNA assessing both spontaneous and enzymatically catalyzed SSB formation resulting from depurination post-treatment, demonstrating comparable levels of depurination and SSB formation by KL-50 (4a) and TMZ (1a).
  • FIG.17C Short-term cell viability assays in LN229 MGMT+/– , MMR+/– isogenic lines pre-treated with increasing concentrations of the ROS scavenger NAC did not result in rescue of KL-50 (4a) toxicity.
  • FIG.18A Western blotting performed in LN229 MGMT+/–, MMR+/– cells following treatment with 20 ⁇ M KL-50 (4a) or TMZ (1a) for 24 or 48 h.
  • Treatment with 1 ⁇ M doxorubicin for 24 h (Doxo) served as a positive control for p-CHK1 activation.
  • FIG.18B and FIG.18C Phospho-SER139-H2AX ( ⁇ H2AX), 53BP1, and phospho-SER33- RPA2 (pRPA) foci levels over time following treatment with KL-50 (4a; 20 ⁇ M) (FIG.18B) or TMZ (1a; 20 ⁇ M) (FIG.18C) for 0, 2, 8, 24, or 48 h in LN229 MGMT+/–, MMR+/– cells. Points, mean % cells with ⁇ 10 foci; error bars, SD; n ⁇ 5 technical replicates.
  • FIG.18D Extended time course of ⁇ H2AX foci levels following treatment with KL-50 (4a; 20 ⁇ M) or TMZ (1a; 20 ⁇ M) for 0, 48, 72, or 96 h in LN229 MGMT+/–, MMR+/– cells. Points, % cells with ⁇ 10 foci, n ⁇ 250 cells per condition.
  • FIGs.19A-19B Supplementary cell cycle analysis data.
  • FIG.19A Time course analysis of cell cycle distribution measured using integrated nuclear (Hoechst) staining intensity after treatment of LN229 MGMT+/–, MMR+/– cells with KL-50 (4a; 20 ⁇ M) or TMZ (1a; 20 ⁇ M) for 2, 8, 24, or 48 h.
  • DMSO 0.1%) serves as negative control and aphidicolin (10 ⁇ M) and paclitaxel (1 ⁇ M) serve as positive controls for S-phase and G2- phase arrest, respectively.
  • Columns, mean; error bars, SD; n 3 independent analyses.
  • FIG.19B Representative histograms showing DNA content distribution from 24 h and 48 h treatment conditions as quantified in (FIG.18A).
  • FIGs.20A-20F Representative histograms showing DNA content distribution from 24 h and 48 h treatment conditions as quantified in (FIG.18A).
  • KL-50 (4a) induces DDR foci formation primarily in S and G2 cell cycle phases, and to lesser extent, in MGMT– G1 phase cells.
  • FIG.20A and FIG.20B Phospho-SER139-H2AX ( ⁇ H2AX) foci levels in LN229 MGMT+/–, MMR+/– cells in G1, S, and G2 cell cycle phases after treatment with 0.1% DMSO control, KL-50 (4a; 20 ⁇ M) or TMZ (1a; 20 ⁇ M) for 48 h. Representative foci images with nuclei labeled as G1, S, or G2 phase cells based on Hoechst staining intensity are shown on the right.
  • FIG.20A-20F points, % cells with ⁇ 10 foci; n ⁇ 500 cells per condition and cell cycle phase.
  • FIGs.21A-21G Validation of micronuclei analysis, ICL sensitivity in FANCD2–/– and BRCA2–/– cell models, and demonstration of FANCD2 ubiquitination induced by KL-50 (4a).
  • FIG.21A Representative images of micronuclei identification.
  • FIG.21B Validation of micronuclei identification using olaparib as positive control. Change in percent cells with ⁇ 1 micronuclei from baseline (DMSO control) after treatment with olaparib (10 ⁇ M) for 48 h in LN229 MGMT+/–, MMR+/– cells. Columns, mean; error bars, SD; n ⁇ 15 technical replicates; **** p ⁇ 0.0001.
  • FIG.21C Western blotting performed in PD20 cells complemented with empty vector (EV), wildtype FANCD2 (WT), or ubiquitination-mutant FANCD2 (KR), demonstrating loss of MGMT in PD20 cells and comparable expression of MMR proteins.
  • FIG.21D Short-term viability assay curves for cisplatin and mitomycin (MMC) in PD20 cells, deficient in FANCD2 (FANCD2–/–) or complemented with FANCD2 (+FANCD2), demonstrating hypersensitivity to crosslinking agents in FANCD2–/– cells.
  • FIG.21E Short-term viability assay curves for cisplatin and MMC in PEO4 (BRCA2+) and PEO1 (BRCA2–/–) cells pre- treated with 0.01% DMSO control or 10 ⁇ M O 6 BG (+O 6 BG) for 1 h prior to cisplatin or MMC addition, demonstrating hypersensitivity of PEO4 BRCA2–/– cells to crosslinking agents independent of MGMT depletion.
  • FIG.21F Short-term viability assay curves for cisplatin and MMC in DLD1 BRCA2+/– and BRCA2–/– cells pre-treated with 0.01% DMSO control or 10 ⁇ M O 6 BG (+O 6 BG) for 1 h prior to cisplatin or MMC addition, demonstrating hypersensitivity of DLD1 BRCA2–/– cells to crosslinking agents independent of MGMT depletion.
  • FIG.21G Western blot analysis of FANCD2 ubiquitination in LN229 MGMT+/–, MMR+/– cells and PD20 FANCD2-deficient cells, complemented with empty vector (FANCD2+EV), wildtype FANCD2 (PD20+FD2) or ubiquitination-mutant FANCD2 (PD20+KR).
  • the % FANCD2 ubiquitination (% FANCD2 Ub.) is quantified as the background-corrected integrated band intensity of the upper band divided by the sum of the background-corrected integrated band intensities of the upper and lower bands.
  • the fold change in % FANCD2 ubiquitination is presented for each cell line relative to DMSO-treated cells.
  • FIGs.22A-22E Spider plots tracking individual mouse tumor response to treatment.
  • FIG.22A Spider plots tracking LN229 MGMT–/MMR+ flank tumor volume of each mouse in response to treatment with P.O.10% cyclodextrin vehicle control, TMZ (1a, 5 mg/kg MWF ⁇ 3 weeks ), or KL-50 (4a, 5 mg/kg MWF ⁇ 3 weeks).
  • FIG.22B Spider plots tracking LN229 MGMT–/MMR– flank tumor volume of each mouse in response to treatment with PO 10% cyclodextrin vehicle control, TMZ (1a, 5 mg/kg MWF ⁇ 3 weeks ), or KL-50 (4a, 5 mg/kg MWF ⁇ 3 weeks).
  • FIG.22C and FIG.22D Spider plots tracking LN229 MGMT–/MMR+ and LN229 MGMT–/MMR– flank tumor volume in response to treatment with P.O 10% cyclodextrin control, P.O KL-50 (4a, 15 mg/kg MWF ⁇ 3 weeks), P.O KL-50 (4a, 25 mg/kg M–F ⁇ 1 week), or I.P. KL-50 (4a, 5 mg/kg MWF ⁇ 3 weeks).
  • FIG.22E Spider plots tracking LN229 MGMT–/MMR– intracranial tumor size as measured by relative light units (photons/sec) in response to P.O treatment with 10% cyclodextrin vehicle control, TMZ (1a, 25 mg/kg M–F ⁇ 1 week), or KL-50 (4a, 25 mg/kg M–F ⁇ 1 week).
  • TMZ 10% cyclodextrin vehicle control
  • KL-50 4a, 25 mg/kg M–F ⁇ 1 week.
  • the disclosure provides a method of treating cancer and particularly MGMT-deficient cancer and more particularly an MGMT and MMR deficient cancer in a patient in need of such treatment by administering to the patient a therapeutically-effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.
  • the present disclosure further provides pharmaceutical compositions comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
  • R 1 is selected from R 2 individually is selected from H and CH 3
  • R 3 individually is selected from H, CH 3 , CH(CH 3 ) 2 , CH 2 CF 3 , , or R 2 and R 3 may be taken together to provide – (CH 2 ) 5 -
  • Specific compounds within the scope of the disclosure include:
  • compositions comprising a compound of formula (I) or a pharmaceutically-acceptable salt thereof and a pharmaceutically-acceptable carrier: wherein: R 1 is selected from R 2 individually is selected from H and CH 3 , and R 3 individually is selected from H, CH 3 , CH(CH 3 ) 2 , CH 2 CF 3 , , or R 2 and R 3 may be taken together to provide – (CH 2 ) 5 -; and R 4 is selected from - wherein R 2 is CH 3 only when R 3 is CH 3 .
  • the compound of the composition is in certain embodiments selected from: or a pharmaceutically-acceptable salt thereof.
  • the compound of the composition is: or a pharmaceutically-acceptable salt thereof.
  • the disclosed compounds may be prepared using one of the following synthetic schemes: General scheme for amide synthesis Abbreviations: hydroxybenzotriazole (HOBt), 1-(3-Dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride hydrochloride (EDCl). Scheme for linker replacement Abbreviations: toluene (PhMe).
  • Procarbazine representative synthetic procedure General procedure for amide coupling: 4-Formylbenzoic acid (1.0 equiv.) is dissolved in DMF (0.1 M) and 1- Hydroxybenzotriazole hydrate (0.1 equiv.) is added, followed by 1-(3- Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.05 equiv.) and triethylamine (2.0 equiv.) and the reaction is stirred at room temperature for 30 minutes. Then, pyrrolidine is added (1.1 equiv.) and the reaction is stirred at room temperature until starting carboxylic acid is consumed.
  • Fluoroethylhydrazine prepared as described in SYNLETT 2004, 13, 2355–2356 Starting aldehyde (1.0 equiv.) and (2-fluoroethyl)hydrazine (1.1 equiv.) is added to anhydrous ethanol (0.1 M) and sodium cyanoborohydride (1.5 equiv.) is added at room temperature under an atmosphere of nitrogen. Once the reaction is complete, the reaction is quenched with water and extracted (4x) with ethyl acetate. Combined organics are washed with saturated sodium chloride then dried over anhydrous sodium sulfate and concentrated to dryness on a rotary evaporator.
  • the titled compound was prepared using the general procedure for amide coupling Oxidation of alcohol: Starting thiophene alcohol (1 equiv.) is dissolved in methylene chloride (0.1 M) and dess-martin periodinane (1.05 equiv.) is added and the reaction is stirred rapidly at room temperature. After the reaction is complete, the reaction is quenched with water and extracted (3x) methylene chloride. Combined organics are dried with anhydrous sodium sulfate and concentrated to dryness. Crude material is purified by silica gel column chromatography eluting with an appropriate amount of ethyl acetate and heptane. Reductive amination: The titled compound was made using the general procedure for reductive amination.
  • R 1 is selected from the group consisting of optionally substituted C 1 -C 6 alkyl and optionally substituted C1-C6 haloalkyl, wherein each optional substituent in R 1 is independently selected from the group consisting of halogen, C 1 -C 3 haloalkyl, C 1 -C 3 alkoxy, C1-C3 haloalkoxy, C1-C3 alkyl, C2-C6 alkenyl, benzyl, phenyl, and naphthyl, and C2-C12 heterocyclyl.
  • the compound is selected from the group consisting of:
  • the compounds of the disclosure may possess one or more stereocenters, and each stereocenter may exist independently in either the (R)- or (S)-configuration.
  • compounds described herein are present in optically active or racemic forms.
  • the compounds described herein encompass racemic, optically-active, regioisomeric and stereoisomeric forms, or combinations thereof that possess the therapeutically useful properties described herein. Preparation of optically active forms is achieved in any suitable manner, including by way of non-limiting example, by resolution of the racemic form with recrystallization techniques, synthesis from optically-active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase.
  • a mixture of one or more isomer is utilized as the therapeutic compound described herein.
  • compounds described herein contain one or more chiral centers. These compounds are prepared by any means, including stereoselective synthesis, enantioselective synthesis and/or separation of a mixture of enantiomers and/ or diastereomers. Resolution of compounds and isomers thereof is achieved by any means including, by way of non-limiting example, chemical processes, enzymatic processes, fractional crystallization, distillation, and chromatography.
  • the methods and formulations described herein include the use of N-oxides (if appropriate), crystalline forms (also known as polymorphs), solvates, amorphous phases, and/or pharmaceutically acceptable salts of compounds having the structure of any compound of the disclosure, as well as metabolites and active metabolites of these compounds having the same type of activity.
  • Solvates include water, ether (e.g., tetrahydrofuran, methyl tert- butyl ether) or alcohol (e.g., ethanol) solvates, acetates and the like.
  • the compounds described herein exist in solvated forms with pharmaceutically acceptable solvents such as water, and ethanol. In other embodiments, the compounds described herein exist in unsolvated form.
  • the compounds of the disclosure exist as tautomers. All tautomers are included within the scope of the compounds recited herein.
  • compounds described herein are prepared as prodrugs.
  • a "prodrug” is an agent converted into the parent drug in vivo.
  • a prodrug upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically active form of the compound.
  • a prodrug is enzymatically metabolized by one or more steps or processes to the biologically, pharmaceutically or therapeutically active form of the compound.
  • sites on, for example, the aromatic ring portion of compounds of the disclosure are susceptible to various metabolic reactions.
  • the appropriate substituent to decrease or eliminate the susceptibility of the aromatic ring to metabolic reactions is, by way of example only, a deuterium, a halogen, or an alkyl group.
  • Compounds described herein also include isotopically-labeled compounds wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
  • isotopes suitable for inclusion in the compounds described herein include and are not limited to 2 H, 3 H, 11 C, 13 C, 14 C, 36 Cl, 18 F, 123 I, 125 I, 13 N, 15 N, 15 O, 17 O, 18 O, 32 P, and 35 S.
  • isotopically-labeled compounds are useful in drug and/or substrate tissue distribution studies.
  • substitution with heavier isotopes such as deuterium affords greater metabolic stability (for example, increased in vivo half-life or reduced dosage requirements).
  • substitution with positron emitting isotopes is useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.
  • Isotopically-labeled compounds are prepared by any suitable method or by processes using an appropriate isotopically-labeled reagent in place of the non-labeled reagent otherwise employed.
  • the compounds described herein are labeled by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.
  • Salts The compositions described herein may form salts with acids or bases, and such salts are included in the present disclosure.
  • the salts are pharmaceutically acceptable salts.
  • salts embraces addition salts of free acids or free bases that are compositions of the disclosure.
  • pharmaceutically acceptable salt refers to salts that possess toxicity profiles within a range that affords utility in pharmaceutical applications. Pharmaceutically unacceptable salts may nonetheless possess properties such as high crystallinity, which have utility in the practice of the present disclosure, such as for example utility in process of synthesis, purification or formulation of compositions of the disclosure.
  • Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, and phosphoric acids.
  • organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p- toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, ⁇ -hydroxybutyric, sal
  • Suitable pharmaceutically acceptable base addition salts of compositions of the disclosure include, for example, ammonium salts and metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts.
  • Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, N,N'-dibenzylethylene- diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N- methylglucamine) and procaine.
  • Examples of pharmaceutically unacceptable base addition salts include lithium salts and cyanate salts. All of these salts may be prepared from the corresponding composition by reacting, for example, the appropriate acid or base with the composition.
  • the present compounds, compositions, and methods are useful for treatment of cancer, and particularly any cancer that is MGMT deficient, regardless of its MMR status. They are more particularly applicable to treatment of cancers that are both MGMT and MMR deficient. As shown in FIG.1, many cancers have significant subpopulations that have critically reduced MGMT expression (i.e., that are MGMT deficient). Notable cancers among these are bladder urothelial cancer, breast invasive carcinoma, colon adenocarcinoma, head and neck tumor (SCC), lung adenocarcinoma, rectum adenocarcinoma, and acute myeloid leukemia.
  • the present compounds and method are be particularly applicable to treatment of glioblastoma multiforme and brain lower grade glioma.
  • very significant subpopulations of these two cancers display critically reduced MGMT expression.
  • monofunctional alkylators such as procarbazine
  • cancers will often develop MMR deficiency and become resistant and unresponsive to further treatment.
  • This phenomenon is well-documented for many monofunctional alkylating agents ranging from temozolamide (TMZ) to methyl methanesulfonate (MMS) to procarbazine. See, for example, Clin. Cancer. Res.1998 Jan 4(1): 1-6.
  • a substantial number of cancers treated with monofunctional alkylating agents such as procarbazine develop induced hypermutation and become resistant to further treatment.
  • the present compounds, compositions, and methods provide an effective treatment for such cancers. Accordingly, in one aspect of the present disclosure provides methods for treating a patient having cancer and particularly an MGMT deficient cancer comprising administration to the patient of a therapeutically-effective amount of a compound of formula (I) or a pharmaceutically-acceptable salt thereof wherein: R 1 is selected from R 2 individually is selected from H and CH 3 , and R 3 individually is selected from H, CH 3 , CH(CH 3 ) 2 , CH 2 CF 3 , , , or R 2 and R 3 may be taken together to provide – (CH 2 ) 5 -; and R 4 is selected from - wherein R 2 is CH 3 only when R 3 is CH 3 .
  • This method is particularly applicable to treatment of cancers that are both MGMT and MMR deficient.
  • the compound used in the method is in certain embodiments selected from: , , or a pharmaceutically-acceptable salt thereof.
  • the compound used in the method is in certain embodiments : or a pharmaceutically-acceptable salt thereof.
  • DDR DNA damage response
  • DDR inhibitors include: (1) homologous recombination (HR)-defective tumors and inhibitors of poly(ADP)- ribose polymerase (PARP) and polymerase theta (Pol ⁇ ); (2) ataxia-telangiectasia mutated (ATM)-mutant tumors and ataxia telangiectasia and Rad3-related (ATR) inhibitors; and (3) mismatch repair (MMR)-deficient tumors and Werner syndrome helicase (WRN) inhibitors.
  • HR homologous recombination
  • PARP poly(ADP)- ribose polymerase
  • Poly ⁇ polymerase theta
  • ATM ataxia-telangiectasia mutated
  • ATR ataxia telangiectasia and Rad3-related
  • MMR mismatch repair
  • WRN Werner syndrome helicase
  • genotoxins could be tailored used to exploit differential sensitivities arising from specific tumor-associated DDR defects. This approach avoids the need to engage DDR proteins directly, thereby circumventing resistance mechanisms arising from mutations in the ligand binding site, while minimizing off-target effects in healthy, DDR-proficient cells.
  • a mechanistic strategy was considered wherein a single agent modifies DNA by two successive chemical steps (FIG.7A). The first chemical reaction is designed to generate a primary DNA lesion that is rapidly removed by healthy, DDR-proficient cells. The second chemical reaction is engineered to slowly transform the primary modification into a more toxic secondary lesion.
  • GBM glioblastoma multiforme
  • GBM is the most common and devastating form of brain cancer, with a five year survival rate of ⁇ 5%.
  • Approximately half of GBMs lack the DDR protein O 6 -methylguanine methyltransferase (MGMT) via promoter hypermethylation.
  • MGMT silencing occurs at an even greater frequency in grade II and III gliomas (over 70% of cases), and these tumors are also largely incurable.
  • MGMT removes O 6 - alkylguanosine adducts by transferring the alkyl adduct to an active site cysteine via an S N 2 displacement.
  • MGMT MGMT-deficient tumors
  • TTZ temozolomide
  • MTIC 3-methyl-(triazen-1-yl)-imidazole-4-carboxamide
  • N7-Methylguanosine and N3-methyladenosine are the major sites of methylation (70% and 9% respectively) but are readily resolved by the base excision repair (BER) pathway.
  • O 6 -methylguanosine (O 6 MeG, 3) adducts derived from TMZ (1a) only comprise ⁇ 5% of addition products, these lesions persist in the genome of MGMT– tumors (but are readily reversed by healthy (MGMT+) cells) (FIG.7C).
  • O 6 MeG (3) residues are thought to induce formation of DNA double-strand breaks (DSBs) and tumor cell death by an MMR-dependent mechanism.
  • MGMT status is a predictive biomarker for initial response to TMZ (1a) in GBM, with a significant overall survival (OS) benefit in the up-front setting for patients with these cancers.
  • TMZ (1a) is also frequently utilized as adjuvant therapy for grade III and high-risk grade II gliomas; however, it remains non-curative, with recurrences typically occurring over 2–10 years. In approximately 80% of patients, recurrences coincide with transformation to higher grade tumors resistant to TMZ (1a) and harboring a distinct hypermutation signature secondary to MMR deficiency, resulting in reduced survival.
  • O 6 -(2-fluoroethyl)guanosine (S1) is known to hydrolyze slowly to N1- (2-hydroxyethyl)guanosine (S3) with a half-life of 18.5 h (37 °C, pH 7.4) (FIG. S1A).
  • the G(N1)-C(N3) interstrand cross-link (ICL) 8 may form by conversion of O 6 FEtG (5) to the N1,O 6 -ethanoguanine intermediate 6 followed by ring-opening by N3 of the complementary cytosine base (7; FIG.7E).
  • MGMT reacts rapidly with alkylated DNA (a second-order rate constant of 1 ⁇ 10 9 M –1 •min –1 was measured using methylated calf thymus DNA as substrate) and can act upon a wide range of O 6 -alkylguanine substrates, it was anticipated that MGMT-proficient cells should repair the O 6 FEtG lesion (5) before it transforms into ICL 8.
  • a range of "about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range.
  • the statement “about X to Y” has the same meaning as "about X to about Y,” unless indicated otherwise.
  • the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise.
  • the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.
  • the term "about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range, and includes the exact stated value or range.
  • acyl refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom.
  • the carbonyl carbon atom is bonded to a hydrogen forming a "formyl” group or is bonded to another carbon atom, which can be part of an alkyl, aryl, aralkyl cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl group or the like.
  • An acyl group can include 0 to about 12, 0 to about 20, or 0 to about 40 additional carbon atoms bonded to the carbonyl group.
  • An acyl group can include double or triple bonds within the meaning herein.
  • An acryloyl group is an example of an acyl group.
  • An acyl group can also include heteroatoms within the meaning herein.
  • a nicotinoyl group (pyridyl-3-carbonyl) is an example of an acyl group within the meaning herein.
  • Other examples include acetyl, benzoyl, phenylacetyl, pyridylacetyl, cinnamoyl, and acryloyl groups and the like.
  • alkenyl refers to straight and branched chain and cyclic alkyl groups as defined herein, except that at least one double bond exists between two carbon atoms.
  • alkenyl groups have from 2 to 40 carbon atoms, or 2 to about 20 carbon atoms, or 2 to 12 carbon atoms or, in some embodiments, from 2 to 8 carbon atoms.
  • alkoxy refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as are defined herein.
  • linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like.
  • branched alkoxy include but are not limited to isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyloxy, and the like.
  • cyclic alkoxy include but are not limited to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like.
  • An alkoxy group can include about 1 to about 12, about 1 to about 20, or about 1 to about 40 carbon atoms bonded to the oxygen atom, and can further include double or triple bonds, and can also include heteroatoms.
  • an allyloxy group or a methoxyethoxy group is also an alkoxy group within the meaning herein, as is a methylenedioxy group in a context where two adjacent atoms of a structure are substituted therewith.
  • alkynyl refers to straight and branched chain alkyl groups, except that at least one triple bond exists between two carbon atoms.
  • alkynyl groups have from 2 to 40 carbon atoms, 2 to about 20 carbon atoms, or from 2 to 12 carbons or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to – C ⁇ CH, -C ⁇ C(CH 3 ), -C ⁇ C(CH 2 CH 3 ), -CH 2 C ⁇ CH, -CH 2 C ⁇ C(CH 3 ), and -CH 2 C ⁇ C(CH 2 CH 3 ) among others.
  • alkyl refers to straight chain and branched alkyl groups and cycloalkyl groups having from 1 to 40 carbon atoms, 1 to about 20 carbon atoms, 1 to 12 carbons or, in some embodiments, from 1 to 8 carbon atoms.
  • straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n- butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups.
  • branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups.
  • alkyl encompasses n-alkyl, isoalkyl, and anteisoalkyl groups as well as other branched chain forms of alkyl.
  • Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.
  • amine refers to primary, secondary, and tertiary amines having, e.g., the formula N(group)3 wherein each group can independently be H or non-H, such as alkyl, aryl, and the like.
  • Amines include but are not limited to R-NH2, for example, alkylamines, arylamines, alkylarylamines; R 2 NH wherein each R is independently selected, such as dialkylamines, diarylamines, aralkylamines, heterocyclylamines and the like; and R 3 N wherein each R is independently selected, such as trialkylamines, dialkylarylamines, alkyldiarylamines, triarylamines, and the like.
  • amine also includes ammonium ions as used herein.
  • amino group refers to a substituent of the form -NH2, - NHR, -NR 2 , -NR 3 + , wherein each R is independently selected, and protonated forms of each, except for -NR 3 + , which cannot be protonated. Accordingly, any compound substituted with an amino group can be viewed as an amine.
  • An “amino group” within the meaning herein can be a primary, secondary, tertiary, or quaternary amino group.
  • alkylamino includes a monoalkylamino, dialkylamino, and trialkylamino group.
  • aralkyl refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein.
  • Representative aralkyl groups include benzyl and phenylethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl.
  • Aralkenyl groups are alkenyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein.
  • aryl refers to cyclic aromatic hydrocarbon groups that do not contain heteroatoms in the ring.
  • aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl, anthracenyl, and naphthyl groups.
  • aryl groups contain about 6 to about 14 carbons in the ring portions of the groups.
  • Aryl groups can be unsubstituted or substituted, as defined herein.
  • Representative substituted aryl groups can be mono-substituted or substituted more than once, such as, but not limited to, a phenyl group substituted at any one or more of 2-, 3-, 4-, 5-, or 6-positions of the phenyl ring, or a naphthyl group substituted at any one or more of 2- to 8-positions thereof.
  • a "disease” is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated then the subject's health continues to deteriorate.
  • cycloalkyl refers to cyclic alkyl groups such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups.
  • the cycloalkyl group can have 3 to about 8-12 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 4, 5, 6, or 7.
  • Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like. Cycloalkyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined herein.
  • Representative substituted cycloalkyl groups can be mono-substituted or substituted more than once, such as, but not limited to, 2,2-, 2,3-, 2,4- 2,5- or 2,6-disubstituted cyclohexyl groups or mono-, di- or tri-substituted norbornyl or cycloheptyl groups, which can be substituted with, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.
  • cycloalkenyl alone or in combination denotes a cyclic alkenyl group.
  • a disorder in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject's state of health.
  • glioma refers to a common type of tumor originating in the brain.
  • gliomas which originate in the glial cells that surround and support neurons in the brain, including astrocytes, oligodendrocytes and ependymal cells
  • halo halogen
  • halide halide group, as used herein, by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
  • haloalkyl group includes mono-halo alkyl groups, poly- halo alkyl groups wherein all halo atoms can be the same or different, and per-halo alkyl groups, wherein all hydrogen atoms are replaced by halogen atoms, such as fluoro.
  • haloalkyl include trifluoromethyl, 1,1-dichloroethyl, 1,2-dichloroethyl, 1,3-dibromo-3,3- difluoropropyl, perfluorobutyl, and the like.
  • heteroarylkynyl refers to alkynyl groups as defined herein in which a hydrogen or carbon bond of an alkynyl group is replaced with a bond to a heteroaryl group as defined herein.
  • Representative aralkynyl groups include, but are not limited to, 2-ethynylpyridine and 2-ethynylthiophene.
  • heteroaryl refers to aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S; for instance, heteroaryl rings can have 5 to about 8-12 ring members.
  • a heteroaryl group is a variety of a heterocyclyl group that possesses an aromatic electronic structure.
  • a heteroaryl group designated as a C 2 -heteroaryl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth.
  • a C 4 -heteroaryl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms sums up to equal the total number of ring atoms.
  • Heteroaryl groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, indolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups.
  • Heteroaryl groups can be unsubstituted, or can be substituted with groups as is discussed herein. Representative substituted heteroaryl groups can be substituted one or more times with groups such as those listed herein. Additional examples of aryl and heteroaryl groups include but are not limited to phenyl, biphenyl, indenyl, naphthyl (1-naphthyl, 2-naphthyl), N-hydroxytetrazolyl, N- hydroxytriazolyl, N-hydroxyimidazolyl, anthracenyl (1-anthracenyl, 2-anthracenyl, 3- anthracenyl), thiophenyl (2-thienyl, 3-thienyl), furyl (2-furyl, 3-furyl) , indolyl, oxadiazolyl, isoxazolyl, quinazolinyl, fluorenyl, xanthenyl, isoindanyl, benzhydry
  • heterocyclylalkyl refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group as defined herein is replaced with a bond to a heterocyclyl group as defined herein.
  • Representative heterocyclyl alkyl groups include, but are not limited to, furan-2-yl methyl, furan-3-yl methyl, pyridine-3-yl methyl, tetrahydrofuran-2-yl ethyl, and indol-2-yl propyl.
  • heteroarylalkyl refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heteroaryl group as defined herein.
  • heterocyclylalkyl refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group as defined herein is replaced with a bond to a heterocyclyl group as defined herein.
  • heterocyclyl alkyl groups include, but are not limited to, furan-2-yl methyl, furan-3-yl methyl, pyridine-3-yl methyl, tetrahydrofuran-2-yl ethyl, and indol-2-yl propyl.
  • heterocyclyl refers to aromatic and non-aromatic ring compounds containing three or more ring members, of which one or more is a heteroatom such as, but not limited to, N, O, and S.
  • a heterocyclyl can be a cycloheteroalkyl, or a heteroaryl, or if polycyclic, any combination thereof.
  • heterocyclyl groups include 3 to about 20 ring members, whereas other such groups have 3 to about 15 ring members.
  • a heterocyclyl group designated as a C2-heterocyclyl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth.
  • a C 4 -heterocyclyl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth.
  • the number of carbon atoms plus the number of heteroatoms equals the total number of ring atoms.
  • a heterocyclyl ring can also include one or more double bonds.
  • a heteroaryl ring is an embodiment of a heterocyclyl group.
  • heterocyclyl group includes fused ring species including those that include fused aromatic and non-aromatic groups.
  • a dioxolanyl ring and a benzdioxolanyl ring system are both heterocyclyl groups within the meaning herein.
  • the phrase also includes polycyclic ring systems containing a heteroatom such as, but not limited to, quinuclidyl.
  • Heterocyclyl groups can be unsubstituted, or can be substituted as discussed herein.
  • Heterocyclyl groups include, but are not limited to, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, dihydrobenzofuranyl, indolyl, dihydroindolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquino
  • substituted heterocyclyl groups can be mono-substituted or substituted more than once, such as, but not limited to, piperidinyl or quinolinyl groups, which are 2-, 3-, 4-, 5-, or 6- substituted, or disubstituted with groups such as those listed herein.
  • hydrocarbon or “hydrocarbyl” as used herein refers to a molecule or functional group that includes carbon and hydrogen atoms. The term can also refer to a molecule or functional group that normally includes both carbon and hydrogen atoms but wherein all the hydrogen atoms are substituted with other functional groups.
  • Hydrocarbyl groups can be shown as (C a -C b )hydrocarbyl, wherein a and b are integers and mean having any of a to b number of carbon atoms.
  • (C 1 -C 4 )hydrocarbyl means the hydrocarbyl group can be methyl (C1), ethyl (C2), propyl (C3), or butyl (C4), and (C0- C b )hydrocarbyl means in certain embodiments there is no hydrocarbyl group.
  • the term "independently selected from” as used herein refers to referenced groups being the same, different, or a mixture thereof, unless the context clearly indicates otherwise.
  • X 1 , X 2 , and X 3 are independently selected from noble gases
  • X 1 , X 2 , and X 3 are all the same, where X 1 , X 2 , and X 3 are all different, where X 1 and X 2 are the same but X 3 is different, and other analogous permutations.
  • knockdown or "KD” as used herein refers to an experimental technique wherein the expression of one or more of an organisms genes and/or translation of the corresponding RNA is reduced.
  • a “prophylactic” or “preventive” treatment is a treatment administered to a subject who does not exhibit signs of a disease or disorder or exhibits only early signs of the disease or disorder for the purpose of decreasing the risk of developing pathology associated with the disease or disorder.
  • pharmaceutically effective amount refers to a non-toxic but sufficient amount of the composition used in the practice of the disclosure that is effective to treat, prevent, and/or ameliorate a disease or disorder in the body of a mammal.
  • the desired treatment may be prophylactic and/or therapeutic.
  • composition refers to a mixture of at least one compound useful within the disclosure with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition facilitates administration of the compound to a subject.
  • the term "pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound useful within the disclosure, and is relatively non-toxic, i.e., the material may be administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
  • the term "pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the disclosure within or to the subject such that it may perform its intended function.
  • a pharmaceutically acceptable material, composition or carrier such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the disclosure within or to the subject such that it may perform its intended function.
  • Such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the disclosure, and not injurious to the subject.
  • materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline
  • pharmaceutically acceptable carrier also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the disclosure, and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions.
  • the "pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compound useful within the disclosure.
  • Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the disclosure are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference. See also “Ansel’s Pharmaceutical Dosage Forms and Delivery Systems”, Tenth Edition (2014).
  • the language “pharmaceutically acceptable salt” refers to a salt of the administered compound prepared from pharmaceutically acceptable non-toxic acids and/or bases, including inorganic acids, inorganic bases, organic acids, inorganic bases, solvates (including hydrates) and clathrates thereof.
  • room temperature refers to a temperature of about 15 °C to about 28 °C.
  • the terms “subject” and “individual” and “patient” can be used interchangeably and may refer to a human or non-human mammal or a bird.
  • Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals.
  • the subject is human.
  • the term "substantially” as used herein refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%.
  • substantially free of can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that the composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less.
  • substantially free of can mean having a trivial amount of, such that a composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less, or about 0 wt%.
  • substituted as used herein in conjunction with a molecule or an organic group as defined herein refers to the state in which one or more hydrogen atoms contained therein are replaced by one or more non-hydrogen atoms.
  • functional group or “substituent” as used herein refers to a group that can be or is substituted onto a molecule or onto an organic group.
  • substituents or functional groups include, but are not limited to, a halogen (e.g., F, Cl, Br, and I); an oxygen atom in groups such as hydroxy groups, alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups including carboxylic acids, carboxylates, and carboxylate esters; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups; a nitrogen atom in groups such as amines, hydroxyamines, nitriles, nitro groups, N-oxides, hydrazides, azides, and enamines; and other heteroatoms in various other groups.
  • a halogen e.g., F, Cl, Br, and I
  • an oxygen atom in groups such as hydroxy groups, al
  • Non-limiting examples of substituents that can be bonded to a substituted carbon (or other) atom include F, Cl, Br, I, OR, OC(O)N(R) 2 , CN, NO, NO 2 , ONO 2 , azido, CF 3 , OCF 3 , R, O (oxo), S (thiono), C(O), S(O), methylenedioxy, ethylenedioxy, N(R) 2 , SR, SOR, SO2R, SO2N(R) 2 , SO3R, C(O)R, C(O)C(O)R, C(O)CH 2 C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R) 2 , OC(O)N(R) 2 , C(S)N(R) 2 , (CH 2 )0- 2 N(R)C(O)R, (CH 2 ) 0-2 N(R)N(R)
  • compositions and methods include the recited elements, but not excluding others.
  • Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the composition or method.
  • Consisting of shall mean excluding more than trace elements of other ingredients for claimed compositions and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure. Accordingly, it is intended that the methods and compositions can include additional steps and components (comprising) or alternatively including steps and compositions of no significance (consisting essentially of) or alternatively, intending only the stated method steps or compositions (consisting of).
  • loweralkyl means a linear or branched saturated hydrocarbon of 1 to 5 carbon atoms, including methyl, ethyl, propyl, isopropyl, butyl, 2-methylpropyl, tert- butyl, and pentyl.
  • “about” means plus or minus 10%.
  • “optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
  • the terms “individual”, “patient”, or “subject” can be an individual organism, a vertebrate, a mammal (e.g., a bovine, a canine, a feline, or an equine), or a human. In certain embodiments, the individual, patient, or subject is a human.
  • the phrases “therapeutically effective amount” and “therapeutic level” mean a compound dose or plasma concentration in a subject, respectively, that provides the specific pharmacological effect for which the compound is administered in a subject in need of such treatment, i.e., to reduce, ameliorate, or eliminate the effects or symptoms of cancer.
  • a therapeutically effective amount or therapeutic level of a drug will not always be effective in treating the conditions/diseases described herein, even though such dosage is deemed to be a therapeutically effective amount by those of skill in the art.
  • the therapeutically effective amount may vary based on the route of administration and dosage form, the age and weight of the subject, and/or the subject’s condition, including the type and stage of the cancer at the time that treatment commences, among other factors.
  • treatment or “treating” as used herein with reference to cancer refer to reducing, ameliorating or eliminating one or more symptoms or effects of the disease or condition.
  • a “therapeutic response” means an improvement in at least one measure of cancer.
  • the term “refractory” as to a particular treatment of a disease means that the disease is unresponsive to the treatment.
  • MGMT deficient or MGMT- cancers means cancers that have more than one standard deviation lower abundance of the mRNA transcript for the MGMT gene or more than one standard deviation lower abundance of the associated functional protein itself normalized to the relevant healthy control tissue. This deficiency can occur through promoter methylation, mutations in the gene, or through other methods resulting in downregulation of the gene.
  • MMR deficient or MMR- cancers means cancers that have more than one standard deviation lower abundance of the mRNA transcript for any of the MMR genes (MSH2, MSH6, MLH1, MLH3, PMS2, PMS1) or more than one standard deviation lower abundance of the respective functional protein(s) normalized to the relevant healthy control tissue.
  • MMR genes MSH2, MSH6, MLH1, MLH3, PMS2, PMS1
  • MSI-H microsatellite instability high phenotype
  • compositions suitable for use for the compounds and in the methods described herein can include a disclosed compound and a pharmaceutically acceptable carrier or diluent.
  • the composition may be formulated for intravenous, subcutaneous, intraperitoneal, intramuscular, topical, oral, buckle, nasal, pulmonary or inhalation, ocular, vaginal, or rectal administration.
  • the compounds are formulated for oral administration.
  • the pharmaceutical composition can be formulated to be an immediate-release composition, sustained-release composition, delayed-release composition, etc., using techniques known in the art.
  • Pharmacologically acceptable carriers for various dosage forms are known in the art.
  • the pharmaceutical compositions include one or more additional components, such as one or more preservatives, antioxidants, stabilizing agents and the like.
  • the disclosed pharmaceutical compositions can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
  • 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.
  • a coating such as lecithin
  • surfactants it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts 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 sterilization microfiltration.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions non- limiting methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Pharmaceutical compositions of the disclosure can be administered in combination with other therapeutics that are part of the current standard of care for cancer. In certain embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle.
  • the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding/formulating such a therapeutic compound for the treatment of a disorder in a subject.
  • the compositions of the disclosure are formulated using one or more pharmaceutically acceptable excipients or carriers.
  • the pharmaceutical compositions of the disclosure comprise a therapeutically effective amount of a compound useful within the disclosure and a pharmaceutically acceptable carrier.
  • the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity may 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 may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • compositions of the disclosure are administered to the subject in dosages that range from one to five times per day or more. In other embodiments, the compositions of the disclosure are administered to the subject in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks.
  • Compounds useful within the disclosure for administration may be in the range of from about 1 mg to about 10,000 mg, about 20 mg to about 9,500 mg, about 40 mg to about 9,000 mg, about 75 mg to about 8,500 mg, about 150 mg to about 7,500 mg, about 200 mg to about 7,000 mg, about 3050 mg to about 6,000 mg, about 500 mg to about 5,000 mg, about 750 mg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 50 mg to about 1,000 mg, about 75 mg to about 900 mg, about 100 mg to about 800 mg, about 250 mg to about 750 mg, about 300 mg to about 600 mg, about 400 mg to about 500 mg, and any and all whole or partial increments therebetween.
  • the dose of a compound useful within the disclosure is from about 1 mg and about 2,500 mg. In other embodiments, a dose of a compound useful within the disclosure used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg.
  • a dose of a second compound, as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments therebetween.
  • the present disclosure is directed to a packaged pharmaceutical composition
  • a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound useful within the disclosure, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, and/or ameliorate a disorder.
  • Granulating techniques are well known in the pharmaceutical art for modifying starting powders or other particulate materials of an active ingredient.
  • the powders are typically mixed with a binder material into larger permanent free-flowing agglomerates or granules referred to as a "granulation.”
  • a binder material for example, solvent-using "wet" granulation processes are generally characterized in that the powders are combined with a binder material and moistened with water or an organic solvent under conditions resulting in the formation of a wet granulated mass from which the solvent must then be evaporated.
  • Melt granulation generally consists in the use of materials that are solid or semi-solid at room temperature (i.e. having a relatively low softening or melting point range) to promote granulation of powdered or other materials, essentially in the absence of added water or other liquid solvents.
  • the low melting solids when heated to a temperature in the melting point range, liquefy to act as a binder or granulating medium.
  • the liquefied solid spreads itself over the surface of powdered materials with which it is contacted, and on cooling, forms a solid granulated mass in which the initial materials are bound together.
  • the resulting melt granulation may then be provided to a tablet press or be encapsulated for preparing the oral dosage form. Melt granulation improves the dissolution rate and bioavailability of an active (i.e. drug) by forming a solid dispersion or solid solution.
  • U.S. Patent No.5,169,645 discloses directly compressible wax-containing granules having improved flow properties.
  • the granules are obtained when waxes are admixed in the melt with certain flow improving additives, followed by cooling and granulation of the admixture.
  • certain flow improving additives such as sodium bicarbonate
  • the present disclosure also includes a multilayer tablet comprising a layer providing for the delayed release of one or more compounds useful within the disclosure, and a further layer providing for the immediate release of a medication for a disorder.
  • a gastric insoluble composition may be obtained in which the active ingredient is entrapped, ensuring its delayed release.
  • Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art.
  • the pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.
  • compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients that are suitable for the manufacture of tablets.
  • excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate.
  • the tablets may be uncoated or they may be coated by known techniques for elegance or to delay the release of the active ingredients.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent.
  • the compounds for use in the disclosure may be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.
  • compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations and compositions that are described herein.
  • compositions of the disclosure may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., polyvinylpyrrolidone, hydroxypropylcellulose or hydroxypropylmethylcellulose); fillers (e.g., cornstarch, lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrates (e.g., sodium starch glycollate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., polyvinylpyrrolidone, hydroxypropylcellulose or hydroxypropylmethylcellulose
  • fillers e.g., cornstarch, lactose, microcrystalline cellulose or calcium phosphate
  • lubricants e.g., magnesium stearate, talc, or silica
  • disintegrates e.g., sodium starch glycollate
  • Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions.
  • the liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxy benzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats
  • emulsifying agent e.g., lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily esters or ethyl alcohol
  • preservatives e.g., methyl or propyl p-hydroxy benzoates or sorbic acid.
  • parenteral Administration the compositions of the disclosure may be formulated for injection or in
  • Additional dosage forms of this disclosure include dosage forms as described in U.S. Patents Nos.6,340,475, 6,488,962, 6,451,808, 5,972,389, 5,582,837, and 5,007,790. Additional dosage forms of this disclosure also include dosage forms as described in U.S. Patent Applications Nos.2003/0147952, 2003/0104062, 2003/0104053, 2003/0044466, 2003/0039688, and 2002/0051820. Additional dosage forms of this disclosure also include dosage forms as described in PCT Applications Nos.
  • the formulations of the present disclosure may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.
  • sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period.
  • the period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.
  • the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds.
  • the compounds of the present disclosure may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.
  • the compounds useful within the disclosure are administered to a subject, alone or in combination with another pharmaceutical agent, using a sustained release formulation.
  • delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that may, although not necessarily, include a delay of from about 10 minutes up to about 12 hours.
  • pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.
  • immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.
  • short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.
  • rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.
  • MGMT DNA repair protein O 6 -methylguanine methyl transferase
  • MMR mismatch repair
  • the cancer is a glioma.
  • the glioma is resistant to treatment with a DNA methylation agent and/or temozolomide.
  • O 6 -methylguanine methyl transferase (MGMT)-silenced tumors are selectively killed.
  • the cancer is liver cancer.
  • the cancer is hepatocellular carcinoma.
  • the amount of compound or pharmaceutical composition administered to the subject is a therapeutically effective amount.
  • Another aspect of the disclosure provides a method in which a therapeutically- effective amount of a compound of formula (I) is administered to a patient (e.g., a human patient) suffering from a cancer and particularly an MGMT deficient cancer.
  • the present method comprises administration of a therapeutically-effective amount of a compound of formula (I) or a pharmaceutically-acceptable salt thereof to a patient suffering from an MGMT deficient, MMR deficient cancer, particularly a glioma.
  • the therapeutically effective amount of the compound is administered together with a pharmaceutically acceptable carrier.
  • Suitable pharmaceutically acceptable carriers are well-known in the art, as discussed infra.
  • a typical route of administration is oral, but other routes of administration are possible, as is well understood by those skilled in the medical arts. Administration may be by single or multiple doses.
  • the amount of compound administered and the frequency of dosing may be optimized by the physician for the particular patient.
  • the present method and compounds are useful to treat urothelial cancer, breast invasive carcinoma, colon adenocarcinoma, head and neck tumor (SCC), lung adenocarcinoma, rectum adenocarcinoma, and acute myeloid leukemia.
  • Therapeutically effective doses and dosing regimens In some embodiments, the therapeutically effective dose of the compound may be administered every day, for 21 days followed by a 7 day rest, every 7 days with a 7 day rest in between each dosage period, or for 5 continuous days followed by a 21 day rest, in each instance referring to a 28 day dosage cycle.
  • the therapeutically effective dose of compound administered to the patient should be sufficient to treat the cancer.
  • Such therapeutically effective amount may be determined by evaluating the symptomatic changes in the patient.
  • Exemplary doses can vary according to the size and health of the individual being treated, the condition being treated, and the dosage regimen adopted.
  • the effective amount of a disclosed compound per 28 day dosage cycle is about 1.5 g/m 2 ; however, in some situations the dose may be higher or lower – for example 2.0 g/m 2 or 1.0 g/m 2 .
  • the daily dose may vary depending on (inter alia) the dosage regimen adopted.
  • the daily dose would be 200 mg/m 2 .
  • the regimen is dosing for 21 days followed by a 5 day rest and the total dosage per 28 day cycle is 1.6 g/m 2 , then the daily dose would be 75 mg/m 2 .
  • the disclosed methods of treatment may also be combined with other known methods of treatment as the situation may require.
  • Therapeutically effective doses and dosing regimens of the foregoing methods may vary, as would be readily understood by those of skill in the art. Dosage regimens may be adjusted to provide the optimum desired response.
  • a single bolus dose of the compound may be administered, while in some embodiments, several divided doses may be administered over time, or the dose may be proportionally reduced or increased in subsequent dosing as indicated by the situation.
  • Applicant believes the disclosed compounds act as bifunctional alkylation agents in a two-step process.
  • the first reaction generates a primary DNA lesion (alkylation) that is rapidly removed by healthy MGMT-proficient cells.
  • the second reaction slowly transforms the primary modification (alkylation) into a more toxic lesion via a unimolecular process.
  • any of the compounds and/or compositions of the disclosure include oral, nasal, rectal, intravaginal, parenteral (e.g., IM, IV and SC), buccal, sublingual or topical.
  • the regimen of administration may affect what constitutes an effective amount.
  • several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection.
  • the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
  • Administration of the compositions of the present disclosure to a subject, such as a mammal, such as a human, may be carried out using known procedures, at dosages and for periods of time effective to treat the disorder in a subject.
  • An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the subject; the age, sex, and weight of the subject; and the ability of the therapeutic compound to treat the disease or disorder in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response.
  • an effective dose range for a therapeutic compound useful within the disclosure is from about 1 and 5,000 mg/kg of body weight/per day.
  • One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of this disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.
  • the selected dosage level depends upon a variety of factors, including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well, known in the medical arts.
  • a medical doctor e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian may start doses of the compounds useful within the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable dose of a compound of the present disclosure may be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day.
  • the dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different. For example, a dose of 1 mg per day may be administered as two 0.5 mg doses, with about a 12-hour interval between doses.
  • the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days.
  • the compounds for use in the method of the disclosure may be formulated in unit dosage form.
  • the term "unit dosage form" refers to physically discrete units suitable as unitary dosage for subjects undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier.
  • the unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.
  • reaction conditions including but not limited to reaction times, reaction size/volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g., nitrogen atmosphere, and reducing/oxidizing agents, are within the scope of the present application.
  • the pharmaceutical activity of the subject compounds may be evaluated in the following assays: Short-term cell viability assay: On day 1, Ln229 isogenic cells of varying MGMT and MMR status are seeded in 96 well format at a density of 2000 cells/well in 100 ⁇ L of DMEM media and allowed to adhere overnight. On day 2, a drug master plate is made with 100x the desired maximal concentration of test compound and serially diluted by 2 until 100x the minimal desired concentration, with one DMSO control. Then daughter plates are created with varied concentrations from 3x the minimal concentration to 3x the maximal desired concentration. Afterwards, 50 ⁇ L of daughter drug plate is added to 100 ⁇ L of the seeded cells for a final concentration of 1x in triplicate.
  • Clonogenic survival Assay Isogenic glioma (Ln 229) cells are pretreated with the test drug in culture for 48–72 hours at the specified dilutions. Cells are then transferred in media without drug to 6-well plates in triplicate at 3-fold dilutions ranging from 9,000 to 37 cells per well. After 14 days, plates are washed with PBS and stained with crystal violet. Colonies are counted by hand. Counts are normalized to plating efficiency of the corresponding treatment condition.
  • LN229 WT and LN229-MSH2- cell lines are maintained in DMEM media supplemented with 10% fetal bovine serum.
  • Three-four-week-old female athymic nude Foxnnu mice are obtained from Envigo and each mouse is inoculated subcutaneously with tumor cells (4.5-5 x 10 6 ) in 0.1 ml of PBS with Matrigel (1:1). Wild type cells are injected on the right flank and mutant cells are injected on the left flank.
  • the tumors are then grown to a mean size of approximately 50-100 mm3 and the mice are then split into groups and treated.
  • Gavage doses of 5mg/kg of test compound are prepared by diluting stocks in DMSO with 10% cyclodextrin. Compound is administered each day of dosing at a volume of 100ul/mouse. Mice are treated for 3 weeks with dosing on Mondays, Wednesdays and Fridays. Tumors are measured 3 times a week during treatment and during the washout period of 2 weeks.
  • Statistical Analysis Analysis of variance (ANOVA) is used to test for significant differences between groups. Post-hoc Bonferroni multiple comparison test analysis is used to determine significant differences among means. All statistical analysis is accomplished using Graph Pad Prism 8.2.0 software.
  • the clonogenic survival assay is a well-recognized assay with high prediction of utility of cancer treatment compounds. See, for example, Fiebig et al. – Clonogenic assay with established human tumor xenografts: correlation of in vitro to in vivo activity as a basis for anti-cancer drug discovery – European J. Cancer, 40 (2004) 802-820. Enumerated Embodiments 1.
  • a pharmaceutical composition comprising the compound of Embodiment 1 or 2 and at least one pharmaceutically acceptable carrier. 4.
  • a method of treating cancer in a subject comprising administering to the subject a compound of Embodiment 1 or 2 or the pharmaceutical composition of Embodiment 3.
  • the method of Embodiment 4, wherein the cancer is a glioma.
  • the method of Embodiment 4 or 5 wherein the glioma is resistant to treatment with a DNA methylation agent and/or temozolomide.
  • MGMT O 6 -methylguanine methyl transferase
  • Flash-column chromatography was performed as described by Still et al., employing silica gel (SiliaFlash® P60, 60 ⁇ , 40-63 ⁇ m particle size) purchased from Silicycle (Québec, Canada).
  • Analytical thin-layered chromatography (TLC) was performed using glass plates pre-coated with silica gel (250 ⁇ m, 60 ⁇ pore size) embedded with a fluorescent indicator (254 nm). TLC plates were visualized by exposure to ultraviolet (UV) light.
  • UV ultraviolet
  • Triethylamine was distilled from calcium hydride under an atmosphere of nitrogen immediately prior to use. N,N-Di-iso- propylethylamine was distilled from calcium hydride under argon immediately prior to use.
  • the diazonium S7, the imidazolyl triazene 1b, the imidazolyl triazene 4b, the imidazolyl triazene 9, the imidazolyl triazene 12b, and the imidazolyl triazene 13 were synthesized according to published procedures. Chemistry Instrumentation. Proton nuclear magnetic resonance ( 1 H NMR) were recorded at 400 or 600 megahertz (MHz) at 23 °C, unless otherwise noted.
  • Methylmethane sulfonate was purchased from Alfa-Aesir. Mitozolomide (MTZ, 12a) was purchased from Enamine. Mitomycin C (MMC), N-ethylmaleimide (NEM), N-acetyl-L-cysteine (NAC), and cisplatin were purchased from Sigma. TMZ (1a, 100 mM stock), O 6 BG (100 mM stock), MTZ (12a, 100 mM stock), MMS (500 mM stock) and NAC (100 mM stock) were dissolved in DMSO and stored at –80 oC.
  • MMC (10 mM stock), lomustine (14, 100 mM stock), doxorubicin (10 mM stock), and olaparib (18.3 mM stock) were dissolved in DMSO and stored at –20 oC.
  • NEM 400 mM stock was dissolved in EtOH and stored at –20 oC.
  • Cisplatin 5 mM stock was dissolved in H 2 O and stored at 4 oC for up to 7 days.
  • Cell Culture. LN229 MGMT– and MGMT+ cell lines were a gift from B. Kaina (Johannes Gutenberg University Mainz, Mainz, Germany) and grown in DMEM with 10% FBS (Gibco).
  • DLD1 BRCA2+/– and BRCA2–/– cell lines were grown in RPMI 1640 with 10% FBS.
  • HCT116 MLH1–/– and HCT116+Chr3 cell lines were a gift from T. Kunkel (National Institute of Environmental Health Sciences, Durham, NC) and grown in DMEM with 10% FBS, with 0.5 ⁇ g/mL G418 (Sigma) for HCT116+Chr3 cells.
  • PD20 cell lines complemented with empty vector (+EV), wildtype FANCD2 (+FD2), or K561R ubiquitination-mutant FANCD2 (+KR) were a gift from G. Kupfer and P.
  • PEO1 and PEO4 cell lines were a gift from T. Taniguchi (Fred Hutchinson Cancer Research Center, Seattle, WA) and were grown in DMEM with 10% FBS.
  • BJ fibroblasts normal human fibroblast cells
  • NER isogenic MEFs were a gift from F. Rogers (Yale University, New Haven, CT) and were grown in DMEM with 10% FBS.
  • Lentiviral particles were produced in HEK293T cells via co-transfection with lentiviral shRNA plasmid, pCMV-VSV-G envelope plasmid (Addgene, #8454) and psPAX2 packaging plasmid (Addgene, #12260), using Lipofectamine 3000 Reagent (Invitrogen, L3000001) per manufacturer’s protocol. Viral particles were harvested 48 h post-transfection and used to transduce LN229 MGMT+/– cells in the presence of 8 ⁇ g/mL polybrene. Selection of pooled cells with lentiviral expression was established with 1 ⁇ g/mL puromycin 48 h post- transduction for 3 to 4 days.
  • N-ethylmaleimide N-ethylmaleimide
  • FANCD2 ubiquitination analysis experiments Proteins were separated using NuPAGE 4–12% Bis-Tris or 3–8% Tris-Acetate Gels (Invitrogen) and transferred to Immobilon-P PVDF membrane (Millipore) for western blotting. Membranes were blocked with 5% milk in TBS-T for 1 h prior to primary antibody addition overnight at 4 °C.
  • mice anti-CHK1 Cell Signaling Technology, #2360
  • rabbit anti-CHK2 Cell Signaling Technology, #6334
  • rabbit anti- FANCD2 Cell Signaling Technology, #16323
  • HRP-conjugated anti- GAPDH ProteinTech HRP-60004
  • rabbit anti-MGMT Cell Signaling Technology, #2739
  • rabbit anti-MLH1 Cell Signaling Technology, #4256
  • mouse anti-MSH2 Cell Signaling Technology, #2850
  • Anti-mouse IgG HRP-conjugated antibody (Cell Signaling Technology, #7076) and anti- rabbit IgG HRP-conjugated antibody (Cell Signaling Technology, #93702) were added at 1/5000 in 5% milk for 1 h. Chemiluminescence detection was performed with Clarity Max Western ECL Substrate (Bio-Rad) and blots were imaged on a ChemiDoc XRS+ Molecular Imager (Bio-Rad). Where shown, bands were quantified using ImageJ software. Short-term Cell Viability Assay. Cells were seeded in 96-well plates at 1000 or 2000 cells/well and allowed to adhere at 23 oC for 60 min and then incubated overnight at 37 °C.
  • Cells were treated with indicated concentrations of compounds in triplicate for 4-6 days prior to fixation with 3.7% paraformaldehyde and nuclear staining with 1 ⁇ g/mL Hoechst 33342 dye. Cells were imaged on a Cytation 3 imaging reader (BioTek) and quantified using CellProfiler software. Clonogenic Cell Survival Assay. Cells were trypsinized, washed, counted, and diluted in a medium containing various concentrations of drug. They were then immediately seeded in six-well plates in triplicate at three-fold dilutions, ranging from 9000 to 37 cells per well. Depending on colony size, these plates were kept in the incubator for 10 to 14 days.
  • IR Alkaline Comet Assay Assay was performed utilizing the CometAssay Kit (Trevigen) according to the alkaline assay protocol, with the addition of slide irradiation post- lysis. Cells were trypsinized, washed with 1X PBS, added to melted Comet LMAgarose (Trevigen), and spread on Trevigen CometSlides at a density of 1000 cells per sample in 50 ⁇ L. Lysis solution (Trevigen) with 10% DMSO was added overnight at 4 oC.
  • Slides were removed from lysis buffer and irradiated to 0 or 10 Gy using an XRAD 320 X-Ray System (Precision X-Ray) at 320 kV, 12.5 mA, and 50.0 cm SSD, with a 2 mm Al filter and 20 cm ⁇ 20 cm collimator. Slides were then placed in alkaline buffer (200 mM NaOH, 1 mM EDTA) for 45 min, followed by electrophoresis in 850 mL alkaline buffer for 45 min at 4 oC. Slides were washed and stained with SYBR gold (Invitrogen) per Trevigen assay protocol.
  • XRAD 320 X-Ray System Precision X-Ray
  • Genomic DNA Denaturing Gel Electrophoresis Assay was adapted from. Cells were trypsinized, washed with 1X PBS, and stored at –80 oC prior to processing. Genomic DNA was extracted with the DNeasy Blood & Tissue Kit (Qiagen) per kit protocol. A 0.7% agarose gel was prepared in 100 mM NaCl-2mM EDTA (pH 8) and soaked in 40 mM NaOH–1 mM EDTA running buffer for 2 h.
  • Genomic DNA 400 ng/well was then loaded in 1X BlueJuice loading buffer (Invitrogen) and subjected to electrophoresis at 2 V/cm for 30 min, followed by 3 V/cm for 2 h.
  • the gel was neutralized in 150 mM NaCl–100 mM Tris (pH 7.4) for 30 min, twice, and then stained with 1X SYBR Gold in 150 mM NaCl–100 mM Tris (pH 7.4) for 90 min. Imaging was performed on a ChemiDoc XRS+ Molecular Imager (Bio-Rad). Plasmid Linearization Assay.
  • Linearized pUC19 DNA was used for in vitro DNA cross-linking assays. For each condition, 200 ng of linearized pUC19 DNA (15.4 ⁇ M base pairs) was incubated with the indicated concentration of drug in 20 ⁇ L. Drug stock concentrations were made in DMSO such that each reaction contained a fixed 5% DMSO concentration. Reactions were conducted in 100 mM Tris buffer (pH 7.4). Cisplatin (Sigma) and DMSO vehicle were used as positive and negative controls, respectively. Reactions were conducted between 3–96 h at 37 °C. The DNA was stored at ⁇ 80 °C until electrophoretic analysis.
  • DNA concentration was preadjusted to 10 ng/ ⁇ L.
  • Five microliters (50 ng) of the DNA solution was removed and mixed with 1.5 ⁇ L of 6 ⁇ purple gel loading dye, no SDS, and loaded onto 1% agarose Tris Borate EDTA TBE gels.
  • 5 ⁇ L (50 ng) of the DNA solution was removed and mixed with 15 ⁇ L of 0.2% denaturing buffer (0.27% sodium hydroxide, 10% glycerol, and 0.013% bromophenol blue) or 0.4% denaturing buffer (0.53% sodium hydroxide, 10% glycerol, and 0.013% bromophenol blue) in an ice bath.
  • the mixed DNA samples were denatured at 4 °C for 5 min and then immediately loaded onto a 1% agarose Tris Borate EDTA (TBE) gel. All gel electrophoresis was conducted at 90 V for 2 h (unless otherwise noted). The gel was stained with SYBR Gold (Invitrogen) for 2 h. EndoIV Depurination Assay. For each condition, 200 ng of supercoiled pUC19 DNA (15.4 ⁇ M base pairs) was incubated with the indicated concentration of drug in 20 ⁇ L for 3 hours. Drug stock concentrations were made in DMSO such that each reaction contained a fixed 5% DMSO concentration. Reactions were conducted in 100 mM Tris buffer (pH 7.4).
  • NEBuffer 3.1 New England Biolabs
  • the NEBuffer 3.1 contained 100 mM sodium chloride, 50 mM Tris-HCl, 10 mM magnesium chloride, and 100 ⁇ g/mL BSA.
  • 50 ng of processed DNA was mixed with NEBuffer 3.1, pH 7.9, in a total volume of 20 ⁇ L for 16 ⁇ 20 h (unless otherwise noted) at 37 °C.
  • the DNA was stored at ⁇ 20 °C before electrophoretic analysis.
  • High-throughput immunofluorescence foci assays were performed at the Yale Center for Molecular Discovery (YCMD). Cells were seeded at 2000 cells/well in black polystyrene flat bottom 384-well plates (Greiner Bio-One) and allowed to adhere overnight. Compound addition was performed utilizing a Labcyte Echo 550 liquid handler (Beckman Coulter), with 6 replicates per test condition and 12 replicates per control condition. Following drug incubation, cells were fixed and stained for phospho-SER139-H2AX ( ⁇ H2AX), 53BP1, or phospho-SER33-RPA2 (pRPA) as follows.
  • ⁇ H2AX protocol Cells were fixed with 4% paraformaldehyde in 1X PBS for 15 min, washed twice with 1X PBS, incubated in extraction buffer (0.5% Triton X-100 in 1X PBS) for 10 min, washed twice with 1X PBS, and incubated in blocking buffer (Blocker Casein in PBS, Thermo Scientific + 5% goat serum, Life Technologies) for 1 h. Mouse anti-phospho- histone H2A.X (Ser139) antibody (clone JBW301, Millipore, 05-636) was added 1/1000 in blocking buffer at 4 oC overnight.
  • Rabbit anti-53BP1 antibody (Novus Biologicals, NB100-904) was added 1/1000 in blocking buffer at 4 oC overnight. After washing with 1X PBS, cells were incubated with goat anti- rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 647 (Invitrogen, A- 21245) 1/500 and with 1 ⁇ g/mL Hoechst nucleic acid dye in blocking buffer for 2 h, and then washed with 1X PBS.
  • goat anti- rabbit IgG H+L
  • Alexa Fluor 647 Invitrogen, A- 21245
  • pRPA protocol Cells were washed twice with 1X PBS on ice, incubated in extraction buffer (0.5% Triton X-100 in 1X PBS) for 5 min on ice, fixed with 3% paraformaldehyde + 2% sucrose in 1X PBS for 15 min at 23 oC, incubated again in extraction buffer for 5 min on ice, and incubated in blocking buffer (2% BSA, 10% milk, 0.1% Triton X-100 in 1X PBS) for 1 h at 23 oC. Rabbit anti-phospho-RPA2 (S33) antibody (Bethyl Laboratories, A300- 246A) was added 1/1000 in blocking buffer at 4 oC overnight.
  • IF wash buffer (0.1% Triton X-100 in 1X PBS)
  • cells were incubated with goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 647 (Invitrogen, A-21245) 1/500 and with 1 ⁇ g/mL Hoechst nucleic acid dye in blocking buffer for 1 h at 37 oC.
  • Cells were washed twice with IF wash buffer and twice with 1X PBS. Imaging was performed on an InCell Analyzer 2200 Imaging System (GE Corporation) at 40X magnification. Twenty fields-of-view were captured per well. Foci analysis was performed using InCell Analyzer software (GE Corporation) as previously described.
  • Foci analysis was performed using Focinator v2 software.
  • Cell Cycle Analysis was performed using integrated Hoechst nucleic acid dye fluorescence intensity as previously described. Briefly, integrated Hoechst fluorescence intensity was log 2 transformed and histograms from DMSO-treated cells were used to identify the centers of the 2N and 4N DNA peaks. These values were used to normalize the 2N DNA peak to 1 and the 4N DNA peak to 2. Cells were then classified by normalized log 2 DNA content as G1 (0.75–1.25), S (1.25–1.75), or G2 (1.75–2.5) phase cells. The percentage of cells within each phase of the cell cycle was determined for each treatment condition.
  • the three sets of Hoechst-stained cells corresponding to the three separate DNA foci stains were treated as three independent analyses.
  • Micronuclei Analysis An automated image analysis pipeline was developed by YCMD using InCell Analyzer software to quantify micronuclei formation. Nuclei and micronuclei were segmented based on Hoechst nucleic acid dye staining channel. A perinuclear margin was applied around the nuclei to approximate the extent of the cytoplasm and identify micronuclei associated with the parent nucleus. Cells with nuclei associated with at least 1 micronucleus were considered positive.
  • Statistical analysis Statistical analysis was performed using GraphPad Prism software. Data are presented as mean or median ⁇ SD or SEM as indicated.
  • IC50 values were determined from the nonlinear regression equation, [inhibitor] vs normalized response with variable slope.
  • comparisons were made with one-way ANOVA and Sidak correction for multiple comparisons.
  • comparisons were made with Mann- Whitney test (for comparison of 2 groups) or Kruskal-Wallis test with FDR-adjusted p-values with Q set to 5% (for comparison of ⁇ 3 groups).
  • Kaplan– Meier analysis was used to evaluate survival rate based on death or removal from study when body weight loss exceeded 20% of initial body weight.
  • mice All animal use was in accordance with the guidelines of the Animal Care and Use Committee (IACUC) of Yale University and conformed to the recommendations in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, National Academy of Sciences, 1996).
  • a mouse tumor model was established by subcutaneously implanting human LN229 (MGMT–/MMR+) or LN229 (MGMT–/MMR–) cells. Cells were cultured as a monolayer in DMEM +10% FBS (Thermo Fisher) at 37 °C in a humidified atmosphere with 5% CO2 and passaged between one and three days prior to implantation and media was replaced every 2-3 days as needed to maintain cell viability.
  • Cells were not allowed to exceed 80% confluency. On the day of implantation, cells were trypsinized, washed with complete media and pelleted by centrifugation at 1200 rpm for 5 minutes. The supernatant was decanted, and cells were washed three times with sterile PBS and pelleted by centrifugation. During the final centrifugation, viability was determined using trypan blue exclusion. Cells were resuspended in sterile PBS and diluted 1:1 in Matrigel (Corning, Cat #47743-716) for a final concentration of 5 ⁇ 10 6 cells/ 100 ⁇ L.
  • mice 5 million cells were injected into the flank of female nude mice (Envigo, Hsd:Athymic Nude- Foxn1 nu , 3-4 weeks age, 15 g). Once tumors reached a minimum volume of 100 mm 3 , mice were randomized and administered either KL-50 (4a; 5 mg/kg MWF ⁇ 3 weeks), TMZ (1a; 5 mg/kg MWF ⁇ 3 weeks), or vehicle (10% cyclodextrin) by oral gavage. Caliper measurements were obtained during the dosing period and at least two weeks following treatment. Mice were euthanized if body weight loss exceeded 20% or if tumor volume increased to greater than 2000 mm 3 . Kaplan–Meier analysis was used to evaluate survival rate based on death or removal from study.
  • mice were randomized and administered either KL-50 (4a) or vehicle (10% cyclodextrin) by oral gavage or intraperitoneal injection on either M–F ⁇ 1 or MWF ⁇ 3 cycles at 5, 15, or 25 mgs/kg. Caliper measurements were obtained during the dosing period and at least two weeks following treatment. Mice were euthanized if body weight loss exceeded 20% or if tumor volume increased to greater than 2000 mm 3 .
  • the third study involved MGMT–/MMR+ and MGMT–/MSH6– (shMSH6) LN229 cells.
  • mice tumors were allowed to grow to a larger average starting volume of ⁇ 350 mm 3 before they were randomized and administered either KL-50 (4a; 25 mg/kg MWF ⁇ 3 weeks) or vehicle (10% cyclodextrin) by oral gavage. Caliper measurements were obtained during the dosing period and at least two weeks following treatment. Mice were euthanized if body weight loss exceeded 20% or if tumor volume increased to greater than 3000 mm 3 .
  • Mouse Protocol for Intracranial Study LN229 MGMT–/MMR– cells stably expressing firefly luciferase (lentivirus-plasmids from Cellomics Technology; PLV-10003), were injected intracranially using a stereotactic injector.
  • the warmed product mixture was immediately transferred to a separatory funnel.
  • the organic layer was washed sequentially with 1 N aqueous hydrochloric acid solution (100 mL, precooled to 0 °C) and saturated aqueous sodium chloride solution (100 mL, precooled to 0 °C).
  • the washed organic layer was dried over magnesium sulfate.
  • the dried solution was filtered, and the filtrate was concentrated (330 mTorr, 31 °C).
  • the unpurified isocyanate so obtained was used directly in the following step.
  • the unpurified isocyanate obtained in the preceding step (nominally 16.7 mmol, 1.75 equiv) was added dropwise via syringe to a solution of the diazonium S7 (1.31 g, 9.54 mmol, 1 equiv) in dimethyl sulfoxide (10 mL) at 23 °C.
  • the reaction vessel was covered with aluminum foil.
  • the reaction mixture was stirred for 16 h at 23 °C.
  • the product mixture was concentrated under a stream of nitrogen.
  • 1,8-Diazabicyclo(5.4.0)undec-7-ene (3.00 mL, 20.0 mmol, 2.00 equiv) was immediately added dropwise and the reaction mixture was stirred for 48 h at 23 °C under ambient atmosphere.
  • the product mixture was diluted with water (15 mL) and the resulting biphasic mixture was transferred to a separatory funnel. The layers that formed were separated and the aqueous layer was extracted with ethyl acetate (2 ⁇ 15 mL). The organic layers were combined and the combined organic layer was washed sequentially with 1 N aqueous hydrochloric acid solution (2 ⁇ 25 mL) and saturated aqueous sodium chloride solution (2 ⁇ 25 mL).
  • the washed organic layer was dried over sodium sulfate. The dried solution was then filtered and the filtrate concentrated to provide tert-butyl (2- fluoropropyl)carbamate as a clear colorless oil.
  • the unpurified product obtained in the preceding step (nominally 6 mmol, 1 equiv) was added to a mixture of dichloromethane (30 mL) and trifluoroacetic acid (10 mL) at 23°C. The reaction mixture was stirred for 12 h at 23 °C under ambient atmosphere. The product mixture was concentrated to provide 2-fluoropropylamine trifluoroacetic acid as an opaque oil with excess equivalents of trifluoroacetic acid.
  • the unpurified product obtained in this way (nominally 6 mmol) was dissolved in tetrahydrofuran (10 mL) to generate a working nominal 0.6 M solution for future reactions.
  • a solution of 2-fluoropropylamine trifluoroacetic acid in tetrahydrofuran (4.40 mL, 2.64 mmol, 1.05 equiv) and triethylamine (1.40 mL, 10 mmol, 4.00 equiv) were added sequentially dropwise via syringe to a suspension of the diazonium S7 (343 mg, 2.50 mmol, 1 equiv) in tetrahydrofuran (15 mL) at 23°C.
  • the reaction mixture was stirred for 6 h at 23 °C.
  • the precipitate that formed was collected by vacuum filtration.
  • the precipitate was washed sequentially with ethyl acetate (2 ⁇ 15 mL) and diethyl ether (2 ⁇ 15 mL).
  • the washed precipitate was dried in vacuo to afford the imidazolyl triazene 10 as a light tan powder (365 mg, 68%, based on the diazonium S7).
  • N,N-Di-iso-propyl ethylamine (834 ⁇ L, 4.55 mmol, 1.25 equiv) was added dropwise via syringe to a mixture of (3-fluoropropyl)amine hydrochloride (410 mg, 3.65 mmol, 1 equiv) and the diazonium S7 (500 mg, 3.65 mmol, 1 equiv) in tetrahydrofuran (25 mL) at 23 oC.
  • the reaction mixture was stirred for 6 h at 23 °C.
  • the precipitate that formed was collected by vacuum filtration.
  • the precipitate was washed sequentially with ethyl acetate (2 ⁇ 15 mL) and ether (2 ⁇ 15 mL). The washed precipitate was dried in vacuo to afford the imidazolyl triazene 11 as a light tan powder (251 mg, 32%).
  • Example 2 Single crystals of KL-50 (4a) suitable for X-ray analysis were obtained by vapor diffusion of dry benzene (3 mL, precipitating solvent) into a syringe filtered (Millipore Sigma, 0.22 ⁇ m, hydrophilic polyvinylidene fluoride, 33 mm, gamma sterilized, catalogue number SLGV033RS) solution of KL-50 (4a) (3.6 mg) in dry dichloromethane (3 mL, solubilizing solvent) at 23 oC. This yielded two polymorphs of KL-50 (4a) designated Polymorph I (P21/n space group, CCDC number 2122008) and Polymorph II (Cc space group, CCDC number 2122009).
  • Example 4 Imidazotetrazine 4a (aka KL-50) and the triazene 4b (aka KL-85) were synthesized as vehicles to deliver 2-fluoroethyl diazonium (4c), and a series of related agents to probe structure–activity relationships in tissue culture (FIG.7D and 7F).
  • Structure–activity studies were consistent with the mechanistic pathway shown in FIG.7E.
  • the 2,2-difluoroethyl triazene 9 and the 2-fluoropropyl triazene 10 possessed reduced potency in MGMT–/MMR– cells, in agreement with the reduced rates of displacement following introduction of an additional fluorine or alkyl substituent.
  • the 2-chloroethyl triazene 12b was modestly potent but not as selective for MGMT– cell lines which likely derives from faster, non-selective ICL formation arising from chloride displacement (vide infra).
  • the 3-fluoropropyl triazene 11 demonstrated low activity in all four cell lines, presumably due to inefficient transfer of the electrophile to DNA.
  • the ethyl triazene 13 also demonstrated low activity. This compound may undergo rapid elimination to ethylene gas following conversion to ethyl diazonium.
  • KL-50 (4a) was prepared by diazotization of 4-aminoimidazole-5-carboxamide followed by the addition of (2-fluoroethyl)isocyanate (39% overall yield, see the Supplementary Information).
  • TMZ (1a) possessed negligible activity in MGMT+ LN229 cells, irrespective of MMR status, and induced robust tumor cell killing in MGMT–, MMR+ cells that was abolished in isogenic cells lacking MMR (FIG.2C).
  • Lomustine (14) was effective in MMR– cells but was cytotoxic to MGMT+ cells.
  • KL-50 (4a) demonstrated robust antitumor activity in MGMT– cells, independent of MMR status, with minimal toxicity to MGMT+ cells at doses up to at least 200 ⁇ M (FIG.2D).
  • a similar pattern of activities was observed in several unique cell lines across different tumor types with intrinsic or induced loss of MGMT and/or MMR activity.
  • TMZ (1a) was inactive in DLD1 cells, which possess MGMT but lack functional MMR (MSH6–) with or without induced depletion of MGMT using O 6 -benzylguanine (O 6 BG; FIG.2E).
  • MMR MMR-benzylguanine
  • KL-50 (4a) was toxic to these cells, but only after O 6 BG-induced MGMT depletion (FIG. 2F).
  • TMZ (1a) was inactive in HCT116 colorectal cancer cells, which lack the MMR protein MLH1, regardless of MGMT levels (FIG.2G).
  • KL- 50 (4a) as a first-in-class molecule that overcomes MMR mutation-induced resistance while retaining selectivity for tumor cells lacking MGMT.
  • Example 7 A well-established comet assay adapted for ICL detection was used to determine if ICLs were formed in MGMT– cells treated with KL-50 (4a) (FIG.3, 3A and 3B). In this assay, cells were sequentially exposed to genotoxins and ionizing radiation, and then analyzed by single cell alkaline gel electrophoresis.
  • Attenuation of the IR-induced comet tail is indicative of ICL formation.
  • TMZ (1a, 200 ⁇ M) and KL-50 (4a, 200 ⁇ M) both induced tailing in MGMT–/MMR+ cells, while mitomycin C (MMC, 0.1 or 50 ⁇ M) did not.
  • Exposure to 50 ⁇ M MMC for 2 h completely abolished the IR-induced comet tail, whereas exposure to 0.1 ⁇ M MMC (chosen to be ⁇ 10-fold greater than the IC 50 for this drug, comparable to 200 ⁇ M KL-50 (4a) or TMZ (1a)) for 24 h caused a partial reduction in the IR-induced comet tail.
  • TMZ (1a, 200 ⁇ M) did not reduce DNA migration following IR, in agreement with its known function as a monoalkylation agent with no known crosslinking activity.
  • KL-50 (4a, 200 ⁇ M) reduced the %DNA in the tail to levels similar to those seen for 0.1 ⁇ M MMC.
  • a similar pattern of comet tail migration was observed for MMC and KL-50 (4a) in MGMT–/MMR– cells, which supports an MMR-independent crosslinking mechanism. Comparable results were observed in MGMT–/MMR+ cells treated with KL-85 (4b).
  • This assay was carried out at varying time points (2–24 h) to assess the rates of ICL formation in MGMT–/MMR– cells treated with KL-50 (4a), MTZ (12a), or TMZ (1a) (FIG. 3, 3C and 3D).
  • the chloroethyl derivative MTZ (12a) reduced DNA mobility within 2 h, consistent with the cell line selectivities above and literature reports that this agent rapidly forms ICLs by chloride displacement from other sites of alkylation.
  • TMZ (1a) did not induce a statistically significant decrease in DNA migration within 24 h.
  • TMZ (1a) and MTIC (1b) showed no evidence of ICL induction.
  • linearized pUC19 plasmid DNA treated with KL-50 (4a, 100 ⁇ M) also possessed ICLs, with delayed rates of formation relative to 12b (FIG.3F).
  • NER nucleotide excision repair
  • BER base excision repair
  • ROS reactive oxygen species
  • N7MeG lesions induced by TMZ (1a) are prone to spontaneous depurination, apurinic (AP) site formation, and single strand breaks (SSBs), which are all known BER substrates.
  • AP apurinic site formation
  • SSBs single strand breaks
  • KL-50 (4a) induced an attenuated G2 arrest in MGMT–/MMR– cells, consistent with a role of MMR in the G2-checkpoint. This effect in MGMT–/MMR– cells was absent following TMZ (1a) treatment. Both TMZ (1a) and KL-50 (4a) induced a moderate G2 arrest in MGMT+/MMR+ cells. The levels of DDR foci were quantified across the individual cell cycle phases (FIG. 20A-20E). KL-50 (4a) induced foci formation primarily in the S- and G2-phases of the cell cycle, which is consistent with replication blocking by ICLs.
  • TMZ (1a) displayed a similar pattern of foci induction in the S- and G2-phases, with smaller increases in G1-phase foci and micronuclei formation at 48 h in MGMT–/MMR+ cells. In contrast, no foci induction or micronuclei formation was observed in MGMT–/MMR– cells exposed to TMZ (1a).
  • KL-50 (4a) induces multiple successive markers of DNA damage and engagement of the DDR in MGMT– cells, independent of MMR status, whereas the effects of TMZ (1a) are similar in MGMT–/MMR+ cells but absent in MMR– cells. Coupled with the ICL kinetics data presented above, these time-course data support a slow rate of ICL induction in situ by KL-50 (4a).
  • KL-50 (4a) induces replication stress (e.g., pRPA foci formation) and DSB formation (e.g., ⁇ H2AX and 53BP1 foci, which are known to follow the formation of ICLs).
  • replication stress e.g., pRPA foci formation
  • DSB formation e.g., ⁇ H2AX and 53BP1 foci, which are known to follow the formation of ICLs.
  • BRCA2- and FANCD2-deficient cells are hypersensitive to KL-50 (4a; FIG.4, 4G to 4I).
  • BRCA2 loss enhanced the toxicity of KL-50 (4a) following MGMT depletion via O 6 BG (FIG.4, 4H and 4I).
  • FANCD2 ubiquitination was observed by KL-50 (4a) specifically in MGMT– cells, suggesting activation of the Fanconi anemia (FA) ICL repair pathway.
  • TMZ (1a) also induced FANCD2 ubiquitination but only in MGMT– /MMR+ cells.
  • the activity of KL-50 (4a) and TMZ (1a) was evaluated in vivo using murine flank tumor models derived from the isogenic LN229 MGMT– cell lines. MGMT–/MMR+ and MGMT–/MMR– flank tumors were treated with KL-50 (4a) or TMZ (1a) (5 mg/kg MWF ⁇ 3 weeks) as previously described for TMZ (1a).
  • TMZ (1a) suppressed tumor growth in the MGMT–/MMR+ tumors (FIG.5A).
  • KL-50 (4a) was statistically non-inferior to TMZ (1a), despite a 17% lower molar dosage owing to its higher molecular weight.
  • TMZ (1a) demonstrated no efficacy, while KL-50 (4a) potently suppressed tumor growth (FIG.5B).
  • KL-50 (4a) treatment resulted in no significant changes in body weight compared to TMZ (1a) or control (FIG.5C).
  • KL-50 (4a) was effective and non-toxic using different dosing regimens (5 mg/kg, 15 mg/kg, 25 mg/kg), treatment schedules (MWF ⁇ 3 weeks, M–F ⁇ 1 week), and routes of drug administration (PO, IP) in mice bearing MGMT–/MMR+ and MGMT– /MMR– flank tumors (FIG.5E).
  • KL-50 (4a; 25 mg/kg PO MWF ⁇ 3 weeks) potently suppressed the growth of large ( ⁇ 350-400 mm 3 ) MGMT–/MMR+ and MGMT–/MSH6– tumors (FIG.5F).
  • KL-50 (4a; 25 mg/kg IP M–F ⁇ 1 week) was also effective in an orthotropic, intracranial LN229 MGMT–/MMR– model, whereas TMZ (1a) only transiently suppressed tumor growth (FIG.6A).
  • a focused maximum tolerated dose study revealed KL-50 (4a) is well-tolerated.
  • mice Healthy mice were treated with escalating doses of KL-50 (4a) (0, 25, 50, 100, and 200 mg/kg ⁇ 1 dose), and monitored over time for changes in both weights and hematologic profiles. Mice in the higher dosage groups (100 or 200 mg/kg) experienced a greater than 10% weight loss after treatment administration, which regressed to baseline at the end of one week (FIG.6B). Two of three mice in the 200 mg/kg treatment group became observably ill warranting euthanasia, but no evidence of toxicity was observed in the remaining cohorts. As the main dose limiting systemic toxicity of TMZ (1a) is myelosuppression, complete blood counts for all mice were measured on day 0 before treatment and subsequently on day 7 after drug administration.
  • MGMT silencing (which occurs in ⁇ 50% of GBMs and ⁇ 70% of grade II/III gliomas) was leveraged to obtain tumor cell selectivity.
  • bifunctional agents that are specifically designed were utilized to evolve slowly to ICLs following transfer to O 6 G, thereby establishing an MMR-independent method to amplify the therapeutic impact of MGMT silencing. This strategy has led to a new class of agents for treatment of MGMT– glioma independent of MMR status.
  • MMR mutation-induced alkylator resistance has been a major barrier to treatment efficacy, likely since the introduction of TMZ (1a) into glioma treatment regimens in the early 1990s.
  • Bifunctional alkylation agents such as lomustine (14) and MTZ (12a) have been tested with the hopes of overcoming TMZ (1a) resistance over the last ⁇ 30 years, but these agents lack a therapeutic index owing to their activity in MGMT+ (normal tissue) cells.
  • Literature data supports the notion that the remarkable cell line selectivity of KL-50 (4a) derives strictly from the poor leaving group ability of fluoride.
  • Intramolecular halide displacement gives the common intermediate N1,O 6 -ethanoguanosine (S2) which undergoes ring opening attack by water to yield N1-(2-hydroxyethyl)guanosine (S3).
  • S2 N1,O 6 -ethanoguanosine
  • S3 N1-(2-hydroxyethyl)guanosine
  • attempts to hydrolyze N7-(2- fluoroethyl)guanosine (S5) to N7-(2-hydroxyethyl)guanosine (S6) in aqueous buffer (pH 7) at 37 °C were reportedly unsuccessful, likely due to an inability to form a similar cationic cyclized intermediate.
  • 2-Chloroethyl nitrosoureas e.g., lomustine, 14
  • 2-chloroethylimidazotetrazines e.g., MTZ, 12a
  • ICLs such as 8 by a pathway analogous to KL-50 (4a, see FIG.7E).
  • they can also generate ICLs via direct chloride displacement from 2-chlorethyl adducts present at other sites of DNA alkylation, which degrades the therapeutic index of these compounds.
  • MGMT silencing has been reported in 40% of colorectal cancers and 25% of non-small cell lung cancer, lymphoma, and head & neck cancers. MGMT mRNA expression is also reduced in subsets of additional cancer types, including breast carcinoma, bladder cancer, and leukemia. MMR loss, as reported by microsatellite instability, is a well-established phenomenon in multiple cancer types and leads to resistance to various standard of care agents. It therefore stands to reason that there are likely other subsets of MGMT–/MMR– tumors in both initial and recurrent settings that would be ideal targets for KL-50 (4a).
  • KL-50 (4a) will display a higher therapeutic index in tumors with MGMT deficiency and impaired ICL repair, including HR deficiency.
  • FANCD2- and BRCA2-deficient cells are hypersensitive to KL-50 (4a), particularly in the setting of MGMT depletion.
  • the therapeutic index (TI) of KL-50 (4a) in the DLD1 isogenic model was ⁇ 600-fold, vastly larger than canonical crosslinking agents such as cisplatin (42-fold) or MMC (26-fold).
  • KL-50 (4a) with DNA repair inhibitors such as checkpoint kinase inhibitors (e.g., PD-1 and PD-L1) or other immunotherapeutic agents in the setting of MMR mutations are contemplated.
  • DNA repair inhibitors such as checkpoint kinase inhibitors (e.g., PD-1 and PD-L1) or other immunotherapeutic agents in the setting of MMR mutations.
  • the findings described herein may have significant clinical implications for patients with recurrent MGMT-methylated glioma, of which up to half acquire TMZ (1a) resistance via loss of MMR.
  • TMZ (1a) derivatives KL-50 (4a) is uniquely designed to fill this therapeutic void. More broadly, incorporating the rates of DNA modification and DNA repair pathways in therapeutic design strategies may lead to the development of additional selective chemotherapies.
  • Embodiment 2 The compound of Embodiment 1, which is selected from the group consisting of:
  • Embodiment 3 The compound of any one of Embodiments 1-2, which is: , or a pharmaceutically-acceptable salt thereof.
  • Embodiment 5 The method of Embodiment 4, wherein the cancer is MGMT deficient.
  • Embodiment 6 The method of any one of Embodiments 4-5, wherein the cancer is MMR deficient.
  • Embodiment 7 The method of any one of Embodiments 4-6, wherein the cancer is liver cancer.
  • Embodiment 8 The method of any one of Embodiments 4-7, wherein the cancer is hepatocellular carcinoma.
  • Embodiment 9 The method of any one of Embodiments 4-8, wherein the compound is selected from the group consisting of:
  • Embodiment 10 The method of any one of Embodiments 4-9, wherein the compound is , or a pharmaceutically-acceptable salt thereof.
  • Embodiment 11 The method of any one of Embodiments 4-10, wherein the cancer is O 6 -methylguanine-DNA-methyltransferase (MGMT) deficient.
  • Embodiment 12 The method of any one of Embodiments 4-11, wherein the cancer is also mismatch repair (MMR) deficient.
  • Embodiment 13 The method of any one of Embodiments 4-12, wherein the cancer is O 6 -methylguanine-DNA-methyltransferase (MGMT) deficient.
  • Embodiment 14 The method of any one of Embodiments 4-13, wherein the cancer is also mismatch repair (MMR) deficient.
  • Embodiment 16 The pharmaceutical composition of Embodiment 15, wherein the compound is selected from the group consisting of:
  • Embodiment 17 The pharmaceutical composition of claim any one of Embodiments 15-16, wherein the compound is , or a pharmaceutically-acceptable salt thereof.
  • the terms and expressions employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the embodiments of the present application. Thus, it should be understood that although the present application describes specific embodiments and optional features, modification and variation of the compositions, methods, and concepts herein disclosed may be resorted to by those of ordinary skill in the art, and that such modifications and variations are considered to be within the scope of embodiments of the present application.
  • R 1 is selected from the group consisting of:
  • R 2 is H or CH 3
  • R 3 is selected from the group consisting of H, CH 3 , CH(CH 3 ) 2 , CH 2 CF 3 , R 2 and R 3 are taken together and linked by –(CH 2 ) 5 – to form a heterocyclic ring

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Abstract

Disclosed are compounds, including 2-fluoroethyl procarbazine compounds, pharmaceutical compositions comprising the compounds, and methods of treating cancers with the compounds and particularly for treating cancers that are O6-methylguanine-DNAmethyltransferase (MGMT) deficient regardless of their mismatch repair (MMR) status and more particularly for treating cancers that are both MGMT and MMR deficient.

Description

R2 individually is selected from H and CH3, and R3 individually is selected from H, CH3, CH(CH3)2, CH2CF3,
Figure imgf000003_0001
, or R2 and R3 may be taken together to provide – (CH2)5-; and R4 is selected from -
Figure imgf000003_0002
wherein R2 is CH3 only when R3 is CH3. The disclosure also provides methods of treating, ameliorating, and/or preventing cancer, particularly MGMT deficient cancers regardless of MMR status, as well as methods of treating, ameliorating, and/or preventing cancers that are both MGMT and MMR deficient, by administering to a patient in need of such treatment a therapeutically-effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof. The disclosure also provides pharmaceutical compositions comprising a compound of formula (I) or a pharmaceutically-acceptable salt thereof and a pharmaceutically-acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments of the present application. FIG.1 shows RNA-sequencing data identifying cancers that have significant subpopulations displaying reduced MGMT expression. Each dot represents an individual patient sample. FIGs.2A-2H. KL-50 (4a) displays MGMT-dependent, MMR-independent cytotoxicity in multiple isogenic cell models. (FIG.2A) IC50 values derived from short-term viability assays in LN229 MGMT+/–, MMR+/– cells treated with TMZ (1a) derivatives. aMGMT TI (therapeutic index) = IC50 (MGMT+/MMR+) divided by IC50 (MGMT– /MMR+). bMMR RI (resistance index) = IC50 (MGMT–/MMR–) divided by IC50 (MGMT– /MMR+). (FIG.2B) Short-term viability assay curves for TMZ (1a), CCNU (14), KL-85 (4b), and KL-50 (4a) in LN229 MGMT+/–, MMR+/– cells. (FIG.2C) Clonogenic survival curves for TMZ (1a) in LN229 MGMT+/–, MMR+/– cells, with representative images of wells containing 1000 plated cells treated with 30 μM TMZ (1a). (FIG.2D) Clonogenic survival curves for KL-50 (4a) in LN229 MGMT+/–, MMR+/– cells, with representative images of wells containing 1000 plated cells treated with 30 μM KL-50 (4a). (FIG.2E) Short-term viability assay curves for TMZ (1a) in DLD1 MSH6-deficient cells pre-treated with 0.01% DMSO control (CTR) or 10 μM O6BG (+O6BG) for 1 h prior to TMZ (1a) addition to deplete MGMT. (FIG.2F) Short-term viability assay curves for KL-50 (4a) in DLD1 MSH6-deficient cells pre-treated with 0.01% DMSO control (CTR) or 10 μM O6BG (+O6BG) for 1 h prior to KL-50 (4a) addition. (FIG.2G) Short-term viability assay curves for TMZ (1a) in HCT116 MLH1–/– cells or HCT116 cells complemented with chromosome 3 carrying wildtype MLH1 (+Chr3) pre-treated with 0.01% DMSO control or 10 μM O6BG (+O6BG) for 1 h prior to TMZ (1a) addition. (FIG.2H) Short-term viability assay curves for KL-50 (4a) in HCT116 MLH1–/– cells or HCT116 cells complemented with chromosome 3 carrying wildtype MLH1 (+Chr3) pre-treated with 0.01% DMSO control or 10 μM O6BG (+O6BG) for 1 h prior to KL-50 (4a) addition. For FIGs.2B-2H, points, mean; error bars, SD; n ≥ 3 technical replicates. FIGs.3A-3F. Unrepaired primary KL-50 (4a) lesions convert to DNA ICLs in the absence of MGMT. (FIG.3A) Scatter dot plots of the %DNA in tail upon single cell alkaline gel electrophoresis performed on LN229 MGMT–/MMR+ and MGMT–/MMR– cells treated with 0.2% DMSO control, 200 μM TMZ (1a), 200 μM KL-50 (4a), or 0.1 μM MMC (MMC*) for 24 h or with 50 μM MMC (MMC**) for 2 h. After cell lysis, comet slides were irradiated with 0 or 10 Gy prior to alkaline electrophoresis. Lines, median; error bars, 95% CI; n ≥ 160. (FIG.3B) Representative comet images from (A). (FIG.3C) Scatter dot plots of the %DNA in tail upon single cell alkaline gel electrophoresis performed on LN229 MGMT– /MMR– cells treated with 0.2% DMSO control, 200 μM MTZ (12a), 200 μM TMZ (1a), or 200 μM KL-50 (4a) for 2, 8, or 24 h. After cell lysis, comet slides were irradiated with 10 Gy prior to alkaline electrophoresis. Lines, median; error bars, 95% CI; n ≥ 230. Data from samples treated with 0 Gy are shown in FIG. S4, C and D. (FIG.3D) Representative comet images from FIG.3C. (FIG.3E) Denaturing gel electrophoresis of genomic DNA isolated from LN229 MGMT–/MMR+ cells treated with 0.2% DMSO control, 200 μM KL-50 (4a), 200 μM TMZ (1a), 200 μM KL-85 (4b), or 200 μM MTIC (1b) for 24 h or with 50 μM MMC or 200 μM CCNU (14) for 2 h. (FIG.3F) Denaturing gel electrophoresis of linearized 100 ng pUC19 plasmid DNA treated in vitro with 100 μM Cisplatin (36 hours), 100 μM MMS (36 hours), 100 μM of KL-50 (4a) or 12b for 6–36 hours. For FIG.3E and FIG.3F, bands representing crosslinked DNA are indicated by arrows. FIGs.4A-4I. KL-50 (4a) activates DNA damage response pathways and cycle arrest in MGMT– cells, independent of MMR, and induces sensitivity in cells deficient in ICL or HR repair. (FIG.4A, FIG.4B, and FIG.4C) Phospho-SER139-H2AX ( γH2AX) (FIG.4A), 53BP1 (FIG.4B), and phospo-SER33-RPA2 (pRPA) (FIG.4C) foci formation quantified by % cells with ≥10 foci in LN229 MGMT+/–, MMR+/– cells treated with 0.1% DMSO control, 20 μM KL-50 (4a), or 20 μM TMZ (1a) for 48 h. Columns, mean; error bars, SD; n ≥ 5 technical replicates. Additional time course data is presented in FIG.18, 12B to 12C. (FIG. 4D) Representative foci images of data in FIG.4A to FIG.4C. (FIG.4E) Percentage of cells in G1, S, and G2 cell cycle phases after treatment as in FIG.4A to FIG.4C, measured using integrated nuclear (Hoechst) staining intensity. Columns, mean; error bars, SD; n = 3 independent analyses. Additional time course data, cell cycle controls, and representative histograms are presented in FIG. S7. (FIG.4F) Change in percent cells with ≥ 1 micronuclei from baseline (DMSO control) after treatment as in FIG.4A to FIG.4C. Columns, mean; error bars, SD; n ≥ 15 technical replicates; **** p < 0.0001; ns, not significant. Additional validation is presented in FIG.21A and 21B. (FIG.4G) Short-term viability assay curves for KL-50 (4a) in PD20 cells, deficient in FANCD2 (FANCD2–/–) or complemented with FANCD2 (+FANCD2). (FIG.4H) Short-term viability assay curves for KL-50 (4a) in PEO4 (BRCA2+) and PEO1 (BRCA2–/–) cells pre-treated with 0.01% DMSO control or 10 μM O6BG (+O6BG) for 1 h prior to KL-50 (4a) addition. (FIG.4I) Short-term viability assay curves for KL-50 (4a) in DLD1 BRCA2+/– and BRCA2–/– cells pre-treated with 0.01% DMSO control or 10 μM O6BG (+O6BG) for 1 h prior to KL-50 (4a) addition. For FIG.4G, FIG.4H, and FIG.4I, points, mean; error bars, SD; n = 3 technical replicates. FIGs.5A-5F. KL-50 (4a) is safe and efficacious on both MGMT–/MMR+ and MGMT–/MMR– flank tumors over a wide range of treatment regimens and conditions. (FIG.5A) Xenograft LN229 MGMT–/MMR+ flank tumors treated with 3 weekly cycles of P.O. administration of 10% cyclodextrin control (n=7), TMZ (1a) (n=7, 5 mg/kg) or KL-50 (4a) (n=6, 5 mg/kg) on Monday, Wednesday, and Friday (individual spider plots in FIG. 22A). (FIG.5B) Xenograft LN229 MGMT–/MMR– flank tumors treated with 3 weekly cycles of P.O. administration of 10% cyclodextrin control (n=6), TMZ (1a) (n=5, 5 mg/kg) or KL-50 (4a) (n=5, 5 mg/kg) on Monday, Wednesday, and Friday (individual spider plots in FIG.22B). (FIG.5C) Mean body weight of mice during LN229 flank tumor experiments. (FIG.5D) Kaplan-Meier analysis of LN229 MGMT–/MMR– xenograft flank tumor-bearing mice to determine survival rate based on death, removal from study if mouse body weight loss exceeded 20% of initial body weight, or if tumor volume exceeded 2000 mm3. Both control and TMZ (1a) treated groups have a median OS of 10 weeks and KL-50 (4a) treated mice have median OS of greater than 15 weeks. (FIG.5E) Xenograft LN229 MGMT– /MMR+ and LN229 MGMT–/MMR– flank tumors treated with PO administration of 10% cyclodextrin control (n=7), KL-50 (4a) (n=6, 3 cycles of 15 mg/kg on Monday, Wednesday, Friday), KL-50 (4a) (n=6, 1 cycle of 25 mg/kg Monday through Friday), or intraperitoneal (I.P.) administration of KL-50 (4a) (n=7, 3 cycles of 5 mg/kg on Monday, Wednesday, Friday) revealed equal efficacy with no observable increases in toxicity as measured by mice systemic weights (individual spider plots in FIG.22C and 22D). (FIG.5F) Xenograft LN229 MGMT–/MMR+ and LN229 MGMT–/MSH6– flank tumors with a larger average starting tumor size of ~400 mm3 and ~350 mm3 respectively, treated with 3 weekly cycles of P.O administration of 10% cyclodextrin (n=4) or KL-50 (4a) (n=4, 3 cycles of 25 mg/kg on Monday, Wednesday, and Friday). The study period was limited by control groups which had to be euthanized for exceeding the ethical maximum allowed tumor size, thus ending the study. In all panels, points, mean; error bars, SEM; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, not significant. FIGs.6A-6C. KL-50 (4a) is efficacious in an LN229 MGMT–/MMR– intracranial model and is well tolerated with limited myelosuppression at supratherapeutic doses. (FIG. 6A) Mean tumor size as measured by bioluminescent imaging (BLI) as relative light units (RLU; photons/sec) with SEM of xenograft LN229 MGMT–/MMR– intracranial tumors treated with 3 weekly cycles of P.O administration with 10% cyclodextrin control (n=10), TMZ (1a) (n=11, 25 mg/kg) or KL-50 (4a) (n=11, 25 mg/kg) on Monday, Wednesday, and Friday (individual spider plots in FIG.22E). (FIG.6B) Mean body change with SEM of mice during maximum tolerated dose experiment in non-tumor bearing mice. (FIG.6C) Complete blood counts for mice pre-treatment and 7 days post-treatment with escalations of single dose KL-50 (4a) delivered PO. WBC lower limit of normal (LLN): 2.2 K/ μL; Neutrophils LLN: 0.42 K/ μL; Lymphocyte LLN: 1.7 K/ μL; RBC LLN: 3.47 M/ μL; Platelet LLN: 155 K/ μL. *, P<0.05; ****, P<0.0001. FIGs.7A-7F. Overview of mechanistic strategy and structures of agents employed in this study. (FIG.7A) Underlying mechanistic hypothesis. Systemic administration of a bifunctional agent is envisioned to form a primary lesion that is rapidly resolved by healthy (DDR+) but not DDR-deficient (DDR–) cells. The persistence of the primary lesion allows it to evolve slowly to a more toxic secondary lesion. (FIG.7B) TMZ (1a) is the front-line therapy for the treatment of MGMT– GBM. Under physiological conditions, TMZ (1a) converts to MTIC (1b) which decomposes to methyl diazonium (1c). (FIG.7C) O6-Guanine is the most clinically-significant site of methylation by methyl diazonium (1c). O6MeG (3) is rapidly reverted to dG (2) by MGMT (the second-order rate constant for demethylation of calf thymus DNA by MGMT is 1 ×109 M–1•min–1), but persists in the genome of MGMT– cells, ultimately leading to MMR-dependent cytotoxicity. (FIG.7D) It was envisioned that imidazotetrazine KL-50 (4a) could be utilized as a source of 2-fluoroethyl diazonium ion (4c). (FIG.7E) Fluoroethylation at O6-guanosine would form O6FEtG (5), which is known to slowly rearrange (t1/2 ~ 18.5 h at 37 °C) via intermediate 6 to form the dG–dC ICL 8. Based on the broad substrate scope of MGMT, it was anticipated that O6FEtG (5) would be readily reversed in MGMT+ cells, thereby preventing ICL formation in healthy tissue. Realization of this goal would provide the first MMR-independent agent active specifically in MGMT– glioma. (FIG.7F) Structures of the triazenes 9–13, mitozolomide 12a, and lomustine (CCNU, 14). FIGs.8A-8H. KL-50 (4a) displays MGMT-dependent, MMR-independent cytotoxicity in multiple isogenic cell models. (FIG.8A) IC50 values derived from short-term viability assays in LN229 MGMT+/–, MMR+/– cells treated with TMZ (1a) derivatives. aMGMT TI (therapeutic index) = IC50 (MGMT+/MMR+) divided by IC50 (MGMT– /MMR+). bMMR RI (resistance index) = IC50 (MGMT–/MMR–) divided by IC50 (MGMT– /MMR+). (FIG.8B) Short-term viability assay curves for TMZ (1a), CCNU (14), KL-85 (4b), and KL-50 (4a) in LN229 MGMT+/–, MMR+/– cells. (FIG.8C) Clonogenic survival curves for TMZ (1a) in LN229 MGMT+/–, MMR+/– cells, with representative images of wells containing 1000 plated cells treated with 30 μM TMZ (1a). (FIG.8D) Clonogenic survival curves for KL-50 (4a) in LN229 MGMT+/–, MMR+/– cells, with representative images of wells containing 1000 plated cells treated with 30 μM KL-50 (4a). (FIG.8E) Short-term viability assay curves for TMZ (1a) in DLD1 MSH6-deficient cells pre-treated with 0.01% DMSO control (CTR) or 10 μM O6BG (+O6BG) for 1 h prior to TMZ (1a) addition to deplete MGMT. (FIG.8F) Short-term viability assay curves for KL-50 (4a) in DLD1 MSH6-deficient cells pre-treated with 0.01% DMSO control (CTR) or 10 μM O6BG (+O6BG) for 1 h prior to KL-50 (4a) addition. (FIG.8G) Short-term viability assay curves for TMZ (1a) in HCT116 MLH1–/– cells or HCT116 cells complemented with chromosome 3 carrying wildtype MLH1 (+Chr3) pre-treated with 0.01% DMSO control or 10 μM O6BG (+O6BG) for 1 h prior to TMZ (1a) addition. (FIG.8H) Short-term viability assay curves for KL-50 (4a) in HCT116 MLH1–/– cells or HCT116 cells complemented with chromosome 3 carrying wildtype MLH1 (+Chr3) pre-treated with 0.01% DMSO control or 10 μM O6BG (+O6BG) for 1 h prior to KL-50 (4a) addition. For FIGs.8B-8H, points, mean; error bars, SD; n ≥ 3 technical replicates. FIGs.9A-9F. Unrepaired primary KL-50 (4a) lesions convert to DNA ICLs in the absence of MGMT. (FIG.9A) Scatter dot plots of the %DNA in tail upon single cell alkaline gel electrophoresis performed on LN229 MGMT–/MMR+ and MGMT–/MMR– cells treated with 0.2% DMSO control, 200 μM TMZ (1a), 200 μM KL-50 (4a), or 0.1 μM MMC (MMC*) for 24 h or with 50 μM MMC (MMC**) for 2 h. After cell lysis, comet slides were irradiated with 0 or 10 Gy prior to alkaline electrophoresis. Lines, median; error bars, 95% CI; n ≥ 160. (FIG.9B) Representative comet images from (FIG.9A). (FIG.9C) Scatter dot plots of the %DNA in tail upon single cell alkaline gel electrophoresis performed on LN229 MGMT–/MMR– cells treated with 0.2% DMSO control, 200 μM MTZ (12a), 200 μM TMZ (1a), or 200 μM KL-50 (4a) for 2, 8, or 24 h. After cell lysis, comet slides were irradiated with 10 Gy prior to alkaline electrophoresis. Lines, median; error bars, 95% CI; n ≥ 230. Data from samples treated with 0 Gy are shown in FIGs.16C and 16D. (FIG.9D) Representative comet images from (FIG.9C). (FIG.9E) Denaturing gel electrophoresis of genomic DNA isolated from LN229 MGMT–/MMR+ cells treated with 0.2% DMSO control, 200 μM KL-50 (4a), 200 μM TMZ (1a), 200 μM KL-85 (4b), or 200 μM MTIC (1b) for 24 h or with 50 μM MMC or 200 μM CCNU (14) for 2 h. (FIG.9F) Denaturing gel electrophoresis of linearized 100 ng pUC19 plasmid DNA treated in vitro with 100 μM Cisplatin (36 hours), 100 μM MMS (36 hours), 100 μM of KL-50 (4a) or 12b for 6–36 hours. For (FIG.9E) and (FIG.9F), bands representing crosslinked DNA are indicated by arrows. FIGs.10A-10I. KL-50 (4a) activates DNA damage response pathways and cycle arrest in MGMT– cells, independent of MMR, and induces sensitivity in cells deficient in ICL or HR repair. (FIG.10A, FIG.10B, and FIG.10C) Phospho-SER139-H2AX ( γH2AX) (FIG.10A), 53BP1 (FIG.10B), and phospo-SER33-RPA2 (pRPA) (FIG.10C) foci formation quantified by % cells with ≥10 foci in LN229 MGMT+/–, MMR+/– cells treated with 0.1% DMSO control, 20 μM KL-50 (4a), or 20 μM TMZ (1a) for 48 h. Columns, mean; error bars, SD; n ≥ 5 technical replicates. Additional time course data is presented in FIG.18, 18B to 18D. (FIG.10D) Representative foci images of data in (FIG.10A) to (FIG.10C). (FIG. 10E) Percentage of cells in G1, S, and G2 cell cycle phases after treatment as in (FIG.10A) to (FIG.10C), measured using integrated nuclear (Hoechst) staining intensity. Columns, mean; error bars, SD; n = 3 independent analyses. Additional time course data, cell cycle controls, and representative histograms are presented in FIG. S7. (FIG.10F) Change in percent cells with ≥ 1 micronuclei from baseline (DMSO control) after treatment as in (FIG. 10A) to (FIG.10C). Columns, mean; error bars, SD; n ≥ 15 technical replicates; **** p < 0.0001; ns, not significant. Additional validation is presented in FIGs.21A and 21B. (FIG. 10G) Short-term viability assay curves for KL-50 (4a) in PD20 cells, deficient in FANCD2 (FANCD2–/–) or complemented with FANCD2 (+FANCD2). (FIG.10H) Short-term viability assay curves for KL-50 (4a) in PEO4 (BRCA2+) and PEO1 (BRCA2–/–) cells pre- treated with 0.01% DMSO control or 10 μM O6BG (+O6BG) for 1 h prior to KL-50 (4a) addition. (FIG.10I) Short-term viability assay curves for KL-50 (4a) in DLD1 BRCA2+/– and BRCA2–/– cells pre-treated with 0.01% DMSO control or 10 μM O6BG (+O6BG) for 1 h prior to KL-50 (4a) addition. For (FIG.10G), (FIG.10H), and (FIG.10I), points, mean; error bars, SD; n = 3 technical replicates. FIGs.11A-11F. KL-50 (4a) is safe and efficacious on both MGMT–/MMR+ and MGMT–/MMR– flank tumors over a wide range of treatment regimens and conditions. (FIG.11A) Xenograft LN229 MGMT–/MMR+ flank tumors treated with 3 weekly cycles of P.O. administration of 10% cyclodextrin control (n=7), TMZ (1a) (n=7, 5 mg/kg) or KL-50 (4a) (n=6, 5 mg/kg) on Monday, Wednesday, and Friday (individual spider plots in FIG. 22A). (FIG.11B) Xenograft LN229 MGMT–/MMR– flank tumors treated with 3 weekly cycles of P.O. administration of 10% cyclodextrin control (n=6), TMZ (1a) (n=5, 5 mg/kg) or KL-50 (4a) (n=5, 5 mg/kg) on Monday, Wednesday, and Friday (individual spider plots in FIG.22B). (FIG.11C) Mean body weight of mice during LN229 flank tumor experiments. (FIG.11D) Kaplan-Meier analysis of LN229 MGMT–/MMR– xenograft flank tumor-bearing mice to determine survival rate based on death, removal from study if mouse body weight loss exceeded 20% of initial body weight, or if tumor volume exceeded 2000 mm3. Both control and TMZ (1a) treated groups have a median OS of 10 weeks and KL-50 (4a) treated mice have median OS of greater than 15 weeks. (FIG.11E) Xenograft LN229 MGMT– /MMR+ and LN229 MGMT–/MMR– flank tumors treated with PO administration of 10% cyclodextrin control (n=7), KL-50 (4a) (n=6, 3 cycles of 15 mg/kg on Monday, Wednesday, Friday), KL-50 (4a) (n=6, 1 cycle of 25 mg/kg Monday through Friday), or intraperitoneal (I.P.) administration of KL-50 (4a) (n=7, 3 cycles of 5 mg/kg on Monday, Wednesday, Friday) revealed equal efficacy with no observable increases in toxicity as measured by mice systemic weights (individual spider plots in FIG.22C and 22D). (FIG.11F) Xenograft LN229 MGMT–/MMR+ and LN229 MGMT–/MSH6– flank tumors with a larger average starting tumor size of ~400 mm3 and ~350 mm3 respectively, treated with 3 weekly cycles of P.O administration of 10% cyclodextrin (n=4) or KL-50 (4a) (n=4, 3 cycles of 25 mg/kg on Monday, Wednesday, and Friday). The study period was limited by control groups which had to be euthanized for exceeding the ethical maximum allowed tumor size, thus ending the study. In all panels, points, mean; error bars, SEM; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, not significant. FIGs.12A-12C. KL-50 (4a) is efficacious in an LN229 MGMT–/MMR– intracranial model and is well tolerated with limited myelosuppression at supratherapeutic doses. (FIG. 12A) Mean tumor size as measured by bioluminescent imaging (BLI) as relative light units (RLU; photons/sec) with SEM of xenograft LN229 MGMT–/MMR– intracranial tumors treated with 3 weekly cycles of P.O administration with 10% cyclodextrin control (n=10), TMZ (1a) (n=11, 25 mg/kg) or KL-50 (4a) (n=11, 25 mg/kg) on Monday, Wednesday, and Friday (individual spider plots in FIG.22E). (FIG.12B) Mean body change with SEM of mice during maximum tolerated dose experiment in non-tumor bearing mice. (FIG.12C) Complete blood counts for mice pre-treatment and 7 days post-treatment with escalations of single dose KL-50 (4a) delivered PO. WBC lower limit of normal (LLN): 2.2 K/ μL; Neutrophils LLN: 0.42 K/ μL; Lymphocyte LLN: 1.7 K/ μL; RBC LLN: 3.47 M/ μL; Platelet LLN: 155 K/ μL. *, P<0.05; ****, P<0.0001. FIGs.13A-13C. Literature precedent for the hydrolysis of various 2- haloethylguanosine lesions. (FIG.13A) Kinetics of the hydrolysis of O6-(2- fluoroethylguanosine) (S1) at pH 7.4 and 37 ºC l. (FIG.13B) Kinetics of the hydrolysis of O6-(2-chloroethylguanosine) (S4) at pH 7.4 and 37 ºC. (FIG.13C) Failed hydrolysis of N7- (2-fluoroethyl)guanosine (S5) with “extensive incubation of [S5] at 37 º [C] in neutral aqueous solution”. FIGs.14A-14K. Additional analysis of TMZ (1a) derivatives in MGMT+/–, MMR+/– cell models. (FIG.14A) Western blotting performed in LN229 MGMT–/MMR+ parental line, and cells complemented with wildtype MGMT (MGMT+/MMR+) and/or stable expression of MSH2 shRNA (MGMT+/MMR– and MGMT–/MMR–). MSH6 expression is reduced in these lines due to destabilization in the setting of loss of its heterodimeric partner MSH2. MLH1 expression is not affected by MSH2 knockdown. Vinculin serves as loading control. (FIG.14B, FIG.14C, FIG.14D, FIG.14E, FIG.14F, and FIG.14G) Short-term viability assay curves for compounds 9, 10, 11, 12b, 13, and 12a in LN229 MGMT+/–, MMR+/– cells. (FIG.14H) Clonogenic survival curves for lomustine (14) in LN229 MGMT+/–, MMR+/– cells. (FIG.14I) Western blotting in HCT116 and DLD1 cells. HCT116 MLH1–/– and +Chr3 lines demonstrate re-expression of MLH1 and similar levels of MGMT and other MMR proteins. DLD1 BRCA2+/– and BRCA2–/– cells have known loss of MSH6 but comparable levels of MGMT and other MMR protein expression. GAPDH serves as loading control. (FIG.14J) Western blotting performed in HCT116 MLH1–/– and +Chr3 and DLD1 BRCA2+/– and BRCA2–/– cells after exposure to 0.01% DMSO or 10 μM O6BG for 24 h, demonstrating O6BG-induced MGMT depletion. Vinculin serves as loading control. (FIG.14K) Short-term cell viability curves for KL-50 (4a) and TMZ (1a) in BJ fibroblast cells. For (FIG.14B), (FIG.14C), (FIG.14D), (FIG.14E), (FIG.14F), (FIG. 14G), (FIG.14H), and (FIG.14K), points, mean; error bars, SD; n = 3 technical replicates. FIGs.15A-15J. KL-50 (4a) is effective in TMZ (1a)-resistant cells lacking other MMR proteins. (FIG.15A) Western blotting performed in LN229 MGMT+/– cells with stable expression of shRNA targeting MSH6, MLH1, PMS2, or MSH3 to confirm depletion of the shRNA targets. In shMSH6 cells, there is reduced expression of MSH2, and in shMLH1 cells, there is loss of PMS2, due to destabilization in the setting of loss of their heterodimeric partners. GAPDH serves as loading control. (FIG.15B) IC50 values derived from short-term viability assays in LN229 MGMT+/– cells lines, +/–shRNA, treated with TMZ (1a) or KL-50 (4a). aMGMT TI (therapeutic index) = IC50 (MGMT+/MMR+) divided by IC50 (MGMT–/MMR+). bMMR RI (resistance index) = IC50 (MGMT–/MMR–) divided by IC50 (MGMT–/MMR+). (FIG.15C) Short-term viability assay curves for TMZ (1a) in LN229 MGMT+/–, MMR+/shMSH6 cells. (FIG.15D) Short-term viability assay curves for KL-50 (4a) in LN229 MGMT+/–, MMR+/shMSH6 cells. (FIG.15E) Short-term viability assay curves for TMZ (1a) in LN229 MGMT+/–, MMR+/shMLH1 cells. (FIG.15F) Short- term viability assay curves for KL-50 (4a) in LN229 MGMT+/–, MMR+/shMLH1 cells. (FIG.15G) Short-term viability assay curves for TMZ (1a) in LN229 MGMT+/–, MMR+/shPMS2 cells. (FIG.15H) Short-term viability assay curves for KL-50 (4a) in LN229 MGMT+/–, MMR+/shPMS2 cells. (FIG.15I) Short-term viability assay curves for TMZ (1a) in LN229 MGMT+/–, MMR+/shMSH3 cells. (FIG.15J) Short-term viability assay curves for KL-50 (4a) in LN229 MGMT+/–, MMR+/shMSH3 cells. For (FIG.15C), (FIG.15D), (FIG.15E), (FIG.15F), (FIG.15G), (FIG.15H), (FIG.15I), and (FIG.15J), points, mean; error bars, SD; n = 3 technical replicates. FIGs.16A-16D. Supplementary IR alkaline comet assay data. (FIG.16A) Scatter dot plots of the %DNA in tail upon single cell alkaline gel electrophoresis performed on LN229 MGMT–/MMR+ cells treated with 0.1% DMSO control or 200 μM KL-85 (4b) for 24 h or with 50 μM MMC for 2 h. After cell lysis, comet slides were irradiated with 0 or 10 Gy prior to alkaline electrophoresis. Lines, median; error bars, 95% CI; n ≥160. (FIG.16B) Representative comet images from (FIG.16A). (FIG.16C) Scatter dot plots of the %DNA in tail upon single cell alkaline gel electrophoresis performed on LN229 MGMT–/MMR– cells treated with 0.2% DMSO control, 200 μM MTZ (12a), 200 μM TMZ (1a), or 200 μM KL-50 (4a) for 2, 8, or 24 h. Corresponding samples treated with 10 Gy IR are shown in FIG.9C. Lines, median; error bars, 95% CI; n ≥230. (FIG.16D) Representative comet images from (FIG.16C). FIGs.17A-17D. NER, BER, ROS, and altered DNA melting point do not play a major role in the mechanism of KL-50 (4a). (FIG.17A) Short-term cell viability assays in both WT and XPA-deficient MEFs demonstrating the absence of additional sensitivity to KL- 50 (4a) in NER compromised XPA deficient cells ±MGMT depletion with O6BG, in contrast to cisplatin as positive control. (FIG.17B) EndoIV depurination assay utilizing supercoiled pUC19 plasmid DNA assessing both spontaneous and enzymatically catalyzed SSB formation resulting from depurination post-treatment, demonstrating comparable levels of depurination and SSB formation by KL-50 (4a) and TMZ (1a). (FIG.17C) Short-term cell viability assays in LN229 MGMT+/– , MMR+/– isogenic lines pre-treated with increasing concentrations of the ROS scavenger NAC did not result in rescue of KL-50 (4a) toxicity. (FIG.17D) Melting temperature experiments in linearized pUC19 plasmid DNA treated with 100 or 500 µM of MMS or KL-50 (4a) for 3 h resulted in comparable changes in measured DNA melting temperature. Columns, mean; error bars, SD; n = 2 independent analyses. For (FIG.17A) and (FIG.17C), points, mean; error bars, SD; n = 3 technical replicates. FIGs.18A-18D. KL-50 (4a) induces activation of the ATR-CHK1 and ATM-CHK2 signaling axes and delayed DNA repair foci formation in MGMT-deficient cells, independent of MMR status. (FIG.18A) Western blotting performed in LN229 MGMT+/–, MMR+/– cells following treatment with 20 μM KL-50 (4a) or TMZ (1a) for 24 or 48 h. Treatment with 1 μM doxorubicin for 24 h (Doxo) served as a positive control for p-CHK1 activation. (FIG.18B and FIG.18C) Phospho-SER139-H2AX ( γH2AX), 53BP1, and phospho-SER33- RPA2 (pRPA) foci levels over time following treatment with KL-50 (4a; 20 μM) (FIG.18B) or TMZ (1a; 20 μM) (FIG.18C) for 0, 2, 8, 24, or 48 h in LN229 MGMT+/–, MMR+/– cells. Points, mean % cells with ≥10 foci; error bars, SD; n ≥ 5 technical replicates. (FIG.18D) Extended time course of γH2AX foci levels following treatment with KL-50 (4a; 20 μM) or TMZ (1a; 20 μM) for 0, 48, 72, or 96 h in LN229 MGMT+/–, MMR+/– cells. Points, % cells with ≥10 foci, n ≥250 cells per condition. FIGs.19A-19B. Supplementary cell cycle analysis data. (FIG.19A) Time course analysis of cell cycle distribution measured using integrated nuclear (Hoechst) staining intensity after treatment of LN229 MGMT+/–, MMR+/– cells with KL-50 (4a; 20 μM) or TMZ (1a; 20 μM) for 2, 8, 24, or 48 h. DMSO (0.1%) serves as negative control and aphidicolin (10 μM) and paclitaxel (1 μM) serve as positive controls for S-phase and G2- phase arrest, respectively. Columns, mean; error bars, SD; n = 3 independent analyses. (FIG.19B) Representative histograms showing DNA content distribution from 24 h and 48 h treatment conditions as quantified in (FIG.18A). FIGs.20A-20F. KL-50 (4a) induces DDR foci formation primarily in S and G2 cell cycle phases, and to lesser extent, in MGMT– G1 phase cells. (FIG.20A and FIG.20B) Phospho-SER139-H2AX ( γH2AX) foci levels in LN229 MGMT+/–, MMR+/– cells in G1, S, and G2 cell cycle phases after treatment with 0.1% DMSO control, KL-50 (4a; 20 μM) or TMZ (1a; 20 μM) for 48 h. Representative foci images with nuclei labeled as G1, S, or G2 phase cells based on Hoechst staining intensity are shown on the right. (FIG.20C and FIG. 20D) 53BP1 foci levels and representative foci images in cells treated as in FIG.20A and FIG.20B. (FIG.20E and FIG.20F) Phospho-SER33-RPA2 (pRPA) foci levels and representative foci images in cells treated as in FIG.20A and FIG.20B. For FIG.20A-20F, points, % cells with ≥10 foci; n ≥500 cells per condition and cell cycle phase. FIGs.21A-21G. Validation of micronuclei analysis, ICL sensitivity in FANCD2–/– and BRCA2–/– cell models, and demonstration of FANCD2 ubiquitination induced by KL-50 (4a). (FIG.21A) Representative images of micronuclei identification. (FIG.21B) Validation of micronuclei identification using olaparib as positive control. Change in percent cells with ≥1 micronuclei from baseline (DMSO control) after treatment with olaparib (10 μM) for 48 h in LN229 MGMT+/–, MMR+/– cells. Columns, mean; error bars, SD; n ≥15 technical replicates; **** p <0.0001. (FIG.21C) Western blotting performed in PD20 cells complemented with empty vector (EV), wildtype FANCD2 (WT), or ubiquitination-mutant FANCD2 (KR), demonstrating loss of MGMT in PD20 cells and comparable expression of MMR proteins. Western blotting in PEO1 BRCA2–/– and PEO4 BRCA2+ cells demonstrates intact expression of MGMT and MMR proteins. (FIG.21D) Short-term viability assay curves for cisplatin and mitomycin (MMC) in PD20 cells, deficient in FANCD2 (FANCD2–/–) or complemented with FANCD2 (+FANCD2), demonstrating hypersensitivity to crosslinking agents in FANCD2–/– cells. (FIG.21E) Short-term viability assay curves for cisplatin and MMC in PEO4 (BRCA2+) and PEO1 (BRCA2–/–) cells pre- treated with 0.01% DMSO control or 10 μM O6BG (+O6BG) for 1 h prior to cisplatin or MMC addition, demonstrating hypersensitivity of PEO4 BRCA2–/– cells to crosslinking agents independent of MGMT depletion. (FIG.21F) Short-term viability assay curves for cisplatin and MMC in DLD1 BRCA2+/– and BRCA2–/– cells pre-treated with 0.01% DMSO control or 10 μM O6BG (+O6BG) for 1 h prior to cisplatin or MMC addition, demonstrating hypersensitivity of DLD1 BRCA2–/– cells to crosslinking agents independent of MGMT depletion. (FIG.21G) Western blot analysis of FANCD2 ubiquitination in LN229 MGMT+/–, MMR+/– cells and PD20 FANCD2-deficient cells, complemented with empty vector (FANCD2+EV), wildtype FANCD2 (PD20+FD2) or ubiquitination-mutant FANCD2 (PD20+KR). The % FANCD2 ubiquitination (% FANCD2 Ub.) is quantified as the background-corrected integrated band intensity of the upper band divided by the sum of the background-corrected integrated band intensities of the upper and lower bands. The fold change in % FANCD2 ubiquitination is presented for each cell line relative to DMSO-treated cells. Vinculin serves as loading control. For (FIG.21D), (FIG.21E), and (FIG.21F), points, mean; error bars, SD; n = 3 technical replicates. FIGs.22A-22E. Spider plots tracking individual mouse tumor response to treatment. (FIG.22A) Spider plots tracking LN229 MGMT–/MMR+ flank tumor volume of each mouse in response to treatment with P.O.10% cyclodextrin vehicle control, TMZ (1a, 5 mg/kg MWF × 3 weeks ), or KL-50 (4a, 5 mg/kg MWF × 3 weeks). (FIG.22B) Spider plots tracking LN229 MGMT–/MMR– flank tumor volume of each mouse in response to treatment with PO 10% cyclodextrin vehicle control, TMZ (1a, 5 mg/kg MWF × 3 weeks ), or KL-50 (4a, 5 mg/kg MWF × 3 weeks). (FIG.22C and FIG.22D) Spider plots tracking LN229 MGMT–/MMR+ and LN229 MGMT–/MMR– flank tumor volume in response to treatment with P.O 10% cyclodextrin control, P.O KL-50 (4a, 15 mg/kg MWF × 3 weeks), P.O KL-50 (4a, 25 mg/kg M–F × 1 week), or I.P. KL-50 (4a, 5 mg/kg MWF × 3 weeks). (FIG.22E) Spider plots tracking LN229 MGMT–/MMR– intracranial tumor size as measured by relative light units (photons/sec) in response to P.O treatment with 10% cyclodextrin vehicle control, TMZ (1a, 25 mg/kg M–F × 1 week), or KL-50 (4a, 25 mg/kg M–F × 1 week). DETAILED DESCRIPTION The present disclosure provides, for example, compounds, methods of treatment, and pharmaceutical compositions. In more specific embodiments, the disclosure provides a method of treating cancer and particularly MGMT-deficient cancer and more particularly an MGMT and MMR deficient cancer in a patient in need of such treatment by administering to the patient a therapeutically-effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof. In a more specific embodiments, the present disclosure further provides pharmaceutical compositions comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. These are described in more detail elsewhere herein. Compounds In accordance with the present disclosure, there are provided compounds of formula (I) and pharmaceutically-acceptable salts thereof:
Figure imgf000015_0001
wherein: R1 is selected from
Figure imgf000015_0002
Figure imgf000015_0003
R2 individually is selected from H and CH3, and R3 individually is selected from H, CH3, CH(CH3)2, CH2CF3,
Figure imgf000015_0005
, or R2 and R3 may be taken together to provide – (CH2)5-; and R4 is selected from -C(=O)CH2CH3,
Figure imgf000015_0004
wherein R2 is CH3 only when R3 is CH3. Specific compounds within the scope of the disclosure include:
Figure imgf000016_0001
or any pharmaceutically-acceptable salt thereof. A non-limiting compound of the disclosure i
Figure imgf000017_0001
pharmaceutically-acceptable salt thereof. The compounds may be part of a pharmaceutical composition. For example, one aspect of the present disclosure provides pharmaceutical compositions comprising a compound of formula (I) or a pharmaceutically-acceptable salt thereof and a pharmaceutically-acceptable carrier:
Figure imgf000017_0002
wherein: R1 is selected from
Figure imgf000017_0004
Figure imgf000017_0005
R2 individually is selected from H and CH3, and R3 individually is selected from H, CH3, CH(CH3)2, CH2CF3, , or R2 and R3 may be taken together to provide –
Figure imgf000017_0006
(CH2)5-; and R4 is selected from -
Figure imgf000017_0007
wherein R2 is CH3 only when R3 is CH3. The compound of the composition is in certain embodiments selected from:
Figure imgf000017_0003
Figure imgf000018_0001
or a pharmaceutically-acceptable salt thereof. In certain embodiments, the compound of the composition is:
Figure imgf000018_0002
or a pharmaceutically-acceptable salt thereof. The disclosed compounds may be prepared using one of the following synthetic schemes: General scheme for amide synthesis
Figure imgf000019_0001
Abbreviations: hydroxybenzotriazole (HOBt), 1-(3-Dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride hydrochloride (EDCl). Scheme for linker replacement
Figure imgf000019_0002
Abbreviations: toluene (PhMe). Procarbazine representative synthetic procedure: General procedure for amide coupling:
Figure imgf000020_0001
4-Formylbenzoic acid (1.0 equiv.) is dissolved in DMF (0.1 M) and 1- Hydroxybenzotriazole hydrate (0.1 equiv.) is added, followed by 1-(3- Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.05 equiv.) and triethylamine (2.0 equiv.) and the reaction is stirred at room temperature for 30 minutes. Then, pyrrolidine is added (1.1 equiv.) and the reaction is stirred at room temperature until starting carboxylic acid is consumed. Once complete, the reaction is diluted with ethyl acetate, washed successively with saturated aqueous sodium chloride, dried with anhydrous sodium sulfate and concentrated to dryness on rotovap. Crude residue is purified by column chromatography with silica gel using an appropriate mixture of ethyl acetate and heptane, or dichloromethane and methanol to afford the titled compound. General procedure for reductive amination:
Figure imgf000020_0002
This procedure is adapted from Bioorganic Chemistry 2019, 83, 461-467. Fluoroethylhydrazine prepared as described in SYNLETT 2004, 13, 2355–2356 Starting aldehyde (1.0 equiv.) and (2-fluoroethyl)hydrazine (1.1 equiv.) is added to anhydrous ethanol (0.1 M) and sodium cyanoborohydride (1.5 equiv.) is added at room temperature under an atmosphere of nitrogen. Once the reaction is complete, the reaction is quenched with water and extracted (4x) with ethyl acetate. Combined organics are washed with saturated sodium chloride then dried over anhydrous sodium sulfate and concentrated to dryness on a rotary evaporator. The crude material is purified by silica gel column chromatography using an appropriate ratio of ethyl acetate and heptanes or dichloromethane and methanol to afford the titled compound. Procedure for linker replacements:
Figure imgf000020_0003
The titled compound was made following the general procedure for amide replacements starting from commercially available 5-formyl-2-pyridinecarboxylic acid. Procedure for quinazolinone linker:
Figure imgf000021_0001
Synthesis of 7-formylquinazolinone: Commercially available 7-Bromo-3-methyl-4(3H)-quinazolinone (1 equiv.) is added to a round bottom flask containing THF (0.1 M) and the solution is cooled to -78 °C in a dry ice acetone bath. N-BuLi (1.05 equiv.) is added dropwise to cooled solution under an atmosphere of nitrogen. Once addition is complete the reaction is stirred at -78 °C for 30 minutes. Next, anhydrous DMF (5 equiv.) is added dropwise to the reaction. Once addition is complete, the reaction is warmed to room temperature and stirred for 1 h. After, the reaction is quenched with aqueous ammonium chloride Procedure for reductive amination:
Figure imgf000021_0002
The titled compound was made using the general procedure for reductive amination. Procedure for thiophene linker:
Figure imgf000021_0003
Starting thiophene carboxylic acid was prepared using the procedure described in WO2009144494 A1. The titled compound was prepared using the general procedure for amide coupling Oxidation of alcohol:
Figure imgf000021_0004
Starting thiophene alcohol (1 equiv.) is dissolved in methylene chloride (0.1 M) and dess-martin periodinane (1.05 equiv.) is added and the reaction is stirred rapidly at room temperature. After the reaction is complete, the reaction is quenched with water and extracted (3x) methylene chloride. Combined organics are dried with anhydrous sodium sulfate and concentrated to dryness. Crude material is purified by silica gel column chromatography eluting with an appropriate amount of ethyl acetate and heptane. Reductive amination:
Figure imgf000022_0001
The titled compound was made using the general procedure for reductive amination. Procedure for amide replacements: Thiazole
Figure imgf000022_0002
The titled compound was prepared using the general reductive amination procedure starting from commercially available 4-(2-Thiazolyl)benzaldehyde. Oxazole
Figure imgf000022_0003
Known starting oxazole-aldehyde was prepared following the procedure from Journal of Organic Chemistry 2008, 73, 7383-7386. The titled compound was prepared using the general reductive amination procedure. Additional Compounds Another aspect of the present disclosure provides a compound of formula (I-1), which is selected from the group consisting of:
Figure imgf000022_0004
wherein R1 is selected from the group consisting of optionally substituted C1-C6 alkyl and optionally substituted C1-C6 haloalkyl, wherein each optional substituent in R1 is independently selected from the group consisting of halogen, C1-C3 haloalkyl, C1-C3 alkoxy, C1-C3 haloalkoxy, C1-C3 alkyl, C2-C6 alkenyl, benzyl, phenyl, and naphthyl, and C2-C12 heterocyclyl. In certain embodiments, the compound is selected from the group consisting of:
Figure imgf000023_0001
The compounds of the disclosure may possess one or more stereocenters, and each stereocenter may exist independently in either the (R)- or (S)-configuration. In certain embodiments, compounds described herein are present in optically active or racemic forms. The compounds described herein encompass racemic, optically-active, regioisomeric and stereoisomeric forms, or combinations thereof that possess the therapeutically useful properties described herein. Preparation of optically active forms is achieved in any suitable manner, including by way of non-limiting example, by resolution of the racemic form with recrystallization techniques, synthesis from optically-active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase. In certain embodiments, a mixture of one or more isomer is utilized as the therapeutic compound described herein. In other embodiments, compounds described herein contain one or more chiral centers. These compounds are prepared by any means, including stereoselective synthesis, enantioselective synthesis and/or separation of a mixture of enantiomers and/ or diastereomers. Resolution of compounds and isomers thereof is achieved by any means including, by way of non-limiting example, chemical processes, enzymatic processes, fractional crystallization, distillation, and chromatography. The methods and formulations described herein include the use of N-oxides (if appropriate), crystalline forms (also known as polymorphs), solvates, amorphous phases, and/or pharmaceutically acceptable salts of compounds having the structure of any compound of the disclosure, as well as metabolites and active metabolites of these compounds having the same type of activity. Solvates include water, ether (e.g., tetrahydrofuran, methyl tert- butyl ether) or alcohol (e.g., ethanol) solvates, acetates and the like. In certain embodiments, the compounds described herein exist in solvated forms with pharmaceutically acceptable solvents such as water, and ethanol. In other embodiments, the compounds described herein exist in unsolvated form. In certain embodiments, the compounds of the disclosure exist as tautomers. All tautomers are included within the scope of the compounds recited herein. In certain embodiments, compounds described herein are prepared as prodrugs. A "prodrug" is an agent converted into the parent drug in vivo. In certain embodiments, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically active form of the compound. In other embodiments, a prodrug is enzymatically metabolized by one or more steps or processes to the biologically, pharmaceutically or therapeutically active form of the compound. In certain embodiments, sites on, for example, the aromatic ring portion of compounds of the disclosure are susceptible to various metabolic reactions. Incorporation of appropriate substituents on the aromatic ring structures may reduce, minimize or eliminate this metabolic pathway. In certain embodiments, the appropriate substituent to decrease or eliminate the susceptibility of the aromatic ring to metabolic reactions is, by way of example only, a deuterium, a halogen, or an alkyl group. Compounds described herein also include isotopically-labeled compounds wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes suitable for inclusion in the compounds described herein include and are not limited to 2H, 3H, 11C, 13C, 14C, 36Cl, 18F, 123I, 125I, 13N, 15N, 15O, 17O, 18O, 32P, and 35S. In certain embodiments, isotopically-labeled compounds are useful in drug and/or substrate tissue distribution studies. In other embodiments, substitution with heavier isotopes such as deuterium affords greater metabolic stability (for example, increased in vivo half-life or reduced dosage requirements). In yet other embodiments, substitution with positron emitting isotopes, such as 11C, 18F, 15O and 13N, is useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically-labeled compounds are prepared by any suitable method or by processes using an appropriate isotopically-labeled reagent in place of the non-labeled reagent otherwise employed. In certain embodiments, the compounds described herein are labeled by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels. Salts The compositions described herein may form salts with acids or bases, and such salts are included in the present disclosure. In certain embodiments, the salts are pharmaceutically acceptable salts. The term "salts" embraces addition salts of free acids or free bases that are compositions of the disclosure. The term "pharmaceutically acceptable salt" refers to salts that possess toxicity profiles within a range that affords utility in pharmaceutical applications. Pharmaceutically unacceptable salts may nonetheless possess properties such as high crystallinity, which have utility in the practice of the present disclosure, such as for example utility in process of synthesis, purification or formulation of compositions of the disclosure. Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, and phosphoric acids. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p- toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, β-hydroxybutyric, salicylic, galactaric and galacturonic acid. Suitable pharmaceutically acceptable base addition salts of compositions of the disclosure include, for example, ammonium salts and metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, N,N'-dibenzylethylene- diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N- methylglucamine) and procaine. Examples of pharmaceutically unacceptable base addition salts include lithium salts and cyanate salts. All of these salts may be prepared from the corresponding composition by reacting, for example, the appropriate acid or base with the composition. Therapeutic Applications The present compounds, compositions, and methods are useful for treatment of cancer, and particularly any cancer that is MGMT deficient, regardless of its MMR status. They are more particularly applicable to treatment of cancers that are both MGMT and MMR deficient. As shown in FIG.1, many cancers have significant subpopulations that have critically reduced MGMT expression (i.e., that are MGMT deficient). Notable cancers among these are bladder urothelial cancer, breast invasive carcinoma, colon adenocarcinoma, head and neck tumor (SCC), lung adenocarcinoma, rectum adenocarcinoma, and acute myeloid leukemia. The present compounds and method are be particularly applicable to treatment of glioblastoma multiforme and brain lower grade glioma. As can be seen in FIG. 1, very significant subpopulations of these two cancers display critically reduced MGMT expression. When treated with monofunctional alkylators such as procarbazine, cancers will often develop MMR deficiency and become resistant and unresponsive to further treatment. This phenomenon is well-documented for many monofunctional alkylating agents ranging from temozolamide (TMZ) to methyl methanesulfonate (MMS) to procarbazine. See, for example, Clin. Cancer. Res.1998 Jan 4(1): 1-6. As described in the reference, a substantial number of cancers treated with monofunctional alkylating agents such as procarbazine develop induced hypermutation and become resistant to further treatment. The present compounds, compositions, and methods provide an effective treatment for such cancers. Accordingly, in one aspect of the present disclosure provides methods for treating a patient having cancer and particularly an MGMT deficient cancer comprising administration to the patient of a therapeutically-effective amount of a compound of formula (I) or a pharmaceutically-acceptable salt thereof
Figure imgf000026_0001
wherein: R1 is selected from
Figure imgf000026_0002
Figure imgf000026_0003
R2 individually is selected from H and CH3, and R3 individually is selected from H, CH3, CH(CH3)2, CH2CF3,
Figure imgf000027_0001
, , or R2 and R3 may be taken together to provide – (CH2)5-; and R4 is selected from -
Figure imgf000027_0002
wherein R2 is CH3 only when R3 is CH3. This method is particularly applicable to treatment of cancers that are both MGMT and MMR deficient. The compound used in the method is in certain embodiments selected from:
Figure imgf000027_0003
, ,
Figure imgf000028_0001
or a pharmaceutically-acceptable salt thereof. The compound used in the method is in certain embodiments : or a pharmaceutically-acceptable salt thereof.
Figure imgf000028_0002
General Aspects Genetic instability is a hallmark of cancer and typically arises from mutations in key DNA damage repair and/or reversal proteins (collectively referred to herein as the DNA damage response, (DDR)). Intrinsic DDR defects can be exploited with DDR inhibitors via the concept of synthetic lethality, defined as a loss of viability resulting from the disruption of two genes or pathways, which, if disrupted individually, are non-lethal. Notable examples of synthetic lethal interactions between DDR inhibitors and key tumor-associated DDR defects include: (1) homologous recombination (HR)-defective tumors and inhibitors of poly(ADP)- ribose polymerase (PARP) and polymerase theta (Pol θ ); (2) ataxia-telangiectasia mutated (ATM)-mutant tumors and ataxia telangiectasia and Rad3-related (ATR) inhibitors; and (3) mismatch repair (MMR)-deficient tumors and Werner syndrome helicase (WRN) inhibitors. In each of these examples, selective tumor cell killing via the DDR protein inhibitor relies on either the induction or persistence of DNA damage or aberrant DNA structures. These findings suggested that genotoxins could be tailored used to exploit differential sensitivities arising from specific tumor-associated DDR defects. This approach avoids the need to engage DDR proteins directly, thereby circumventing resistance mechanisms arising from mutations in the ligand binding site, while minimizing off-target effects in healthy, DDR-proficient cells. To achieve this, a mechanistic strategy was considered wherein a single agent modifies DNA by two successive chemical steps (FIG.7A). The first chemical reaction is designed to generate a primary DNA lesion that is rapidly removed by healthy, DDR-proficient cells. The second chemical reaction is engineered to slowly transform the primary modification into a more toxic secondary lesion. It was anticipated that if the rate of primary lesion repair were sufficiently rapid in healthy cells, the secondary lesions would accumulate only in the DNA of DDR-deficient tumor cells. This two-step pathway would overcome resistance mechanisms that mitigate the toxicity of a primary lesion, which have been implicated in resistance to various chemotherapies including anthracyclines (impairment of nucleoside excision repair (NER), topoisomerase inhibitors (loss of nonhomologous end joining), as well as antimetabolite and platinum resistance arising from mutations in MMR. Furthermore, delivering these bespoke lesions using established chemotherapy scaffolds could facilitate rapid translation into the clinic, owing to decades of use in cancer patients. In one embodiment, the strategy described herein is exemplified in the context of glioblastoma multiforme (GBM). GBM is the most common and devastating form of brain cancer, with a five year survival rate of ~5%. Approximately half of GBMs lack the DDR protein O6-methylguanine methyltransferase (MGMT) via promoter hypermethylation. MGMT silencing occurs at an even greater frequency in grade II and III gliomas (over 70% of cases), and these tumors are also largely incurable. Mechanistically, MGMT removes O6- alkylguanosine adducts by transferring the alkyl adduct to an active site cysteine via an SN2 displacement. Patients with MGMT-deficient tumors (referred to hereafter as MGMT– tumors) are treated with temozolomide (TMZ, 1a), a prodrug that converts under physiological conditions to the potent methylating agent methyl diazonium (1c), via the intermediacy of 3-methyl-(triazen-1-yl)-imidazole-4-carboxamide (MTIC, 1b) (FIG.7B). N7-Methylguanosine and N3-methyladenosine are the major sites of methylation (70% and 9% respectively) but are readily resolved by the base excision repair (BER) pathway. In contrast, though O6-methylguanosine (O6MeG, 3) adducts derived from TMZ (1a) only comprise ~5% of addition products, these lesions persist in the genome of MGMT– tumors (but are readily reversed by healthy (MGMT+) cells) (FIG.7C). O6MeG (3) residues are thought to induce formation of DNA double-strand breaks (DSBs) and tumor cell death by an MMR-dependent mechanism. MGMT status is a predictive biomarker for initial response to TMZ (1a) in GBM, with a significant overall survival (OS) benefit in the up-front setting for patients with these cancers. However, it is now well-established that acquired clinical resistance to TMZ (1a) by MMR mutations abrogates its toxicity, leading to recurrent GBM and death in nearly all patients. TMZ (1a) is also frequently utilized as adjuvant therapy for grade III and high-risk grade II gliomas; however, it remains non-curative, with recurrences typically occurring over 2–10 years. In approximately 80% of patients, recurrences coincide with transformation to higher grade tumors resistant to TMZ (1a) and harboring a distinct hypermutation signature secondary to MMR deficiency, resulting in reduced survival. Without being bound by theory, it was believed that the strategy outlined in FIG.7A to develop agents that overcome the resistance associated with MMR loss while maintaining TMZ’s (1a) selectivity for MGMT-silenced tumors. These agents would deposit a primary lesion susceptible to SN2-mediated removal by MGMT that could undergo a further chemical transformation to a secondary lesion capable of killing MGMT-deficient tumor cells in an MMR-independent manner. To maintain the therapeutic index between MGMT– tumor cells and MGMT+ healthy cells, the primary legion must undergo MGMT-mediated repair faster than it undergoes transformation to the secondary lesion. With these considerations in mind, it was hypothesized that an agent capable of depositing a 2-fluoroethyl lesion at O6-guanine would prove ideal as O6-(2-fluoroethyl)guanosine (S1) is known to hydrolyze slowly to N1- (2-hydroxyethyl)guanosine (S3) with a half-life of 18.5 h (37 °C, pH 7.4) (FIG. S1A). Mechanistically, this occurs via N1 displacement of the pendent fluoride to provide the N1,O6-ethanoguanosine intermediate S2 which undergoes ring-opening nucleophilic attack by water to give S3. By analogy, it was reasoned that the G(N1)-C(N3) interstrand cross-link (ICL) 8 may form by conversion of O6FEtG (5) to the N1,O6-ethanoguanine intermediate 6 followed by ring-opening by N3 of the complementary cytosine base (7; FIG.7E). As MGMT reacts rapidly with alkylated DNA (a second-order rate constant of 1 ×109 M–1•min–1 was measured using methylated calf thymus DNA as substrate) and can act upon a wide range of O6-alkylguanine substrates, it was anticipated that MGMT-proficient cells should repair the O6FEtG lesion (5) before it transforms into ICL 8. In this document, the terms "a," "an," or "the" are used to include one or more than one unless the context clearly dictates otherwise. The term "or" is used to refer to a nonexclusive "or" unless otherwise indicated. The statement "at least one of A and B" or "at least one of A or B" has the same meaning as "A, B, or A and B." In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. Throughout this document, values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement "about X to Y" has the same meaning as "about X to about Y," unless indicated otherwise. Likewise, the statement "about X, Y, or about Z" has the same meaning as "about X, about Y, or about Z," unless indicated otherwise. In the methods described herein, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process. The term "about" as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range, and includes the exact stated value or range. The term "acyl" as used herein refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is bonded to a hydrogen forming a "formyl" group or is bonded to another carbon atom, which can be part of an alkyl, aryl, aralkyl cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl group or the like. An acyl group can include 0 to about 12, 0 to about 20, or 0 to about 40 additional carbon atoms bonded to the carbonyl group. An acyl group can include double or triple bonds within the meaning herein. An acryloyl group is an example of an acyl group. An acyl group can also include heteroatoms within the meaning herein. A nicotinoyl group (pyridyl-3-carbonyl) is an example of an acyl group within the meaning herein. Other examples include acetyl, benzoyl, phenylacetyl, pyridylacetyl, cinnamoyl, and acryloyl groups and the like. When the group containing the carbon atom that is bonded to the carbonyl carbon atom contains a halogen, the group is termed a "haloacyl" group. An example is a trifluoroacetyl group. The term "alkenyl" as used herein refers to straight and branched chain and cyclic alkyl groups as defined herein, except that at least one double bond exists between two carbon atoms. Thus, alkenyl groups have from 2 to 40 carbon atoms, or 2 to about 20 carbon atoms, or 2 to 12 carbon atoms or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to vinyl, -CH=C=CCH2, -CH=CH(CH3), - CH=C(CH3)2, -C(CH3)=CH2, -C(CH3)=CH(CH3), -C(CH2CH3)=CH2, cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl among others. The term "alkoxy" as used herein refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as are defined herein. Examples of linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like. Examples of branched alkoxy include but are not limited to isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyloxy, and the like. Examples of cyclic alkoxy include but are not limited to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. An alkoxy group can include about 1 to about 12, about 1 to about 20, or about 1 to about 40 carbon atoms bonded to the oxygen atom, and can further include double or triple bonds, and can also include heteroatoms. For example, an allyloxy group or a methoxyethoxy group is also an alkoxy group within the meaning herein, as is a methylenedioxy group in a context where two adjacent atoms of a structure are substituted therewith. The term "alkynyl" as used herein refers to straight and branched chain alkyl groups, except that at least one triple bond exists between two carbon atoms. Thus, alkynyl groups have from 2 to 40 carbon atoms, 2 to about 20 carbon atoms, or from 2 to 12 carbons or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to – C ≡CH, -C ≡C(CH3), -C ≡C(CH2CH3), -CH2C ≡CH, -CH2C ≡C(CH3), and -CH2C ≡C(CH2CH3) among others. The term "alkyl" as used herein refers to straight chain and branched alkyl groups and cycloalkyl groups having from 1 to 40 carbon atoms, 1 to about 20 carbon atoms, 1 to 12 carbons or, in some embodiments, from 1 to 8 carbon atoms. Examples of straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n- butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups. As used herein, the term "alkyl" encompasses n-alkyl, isoalkyl, and anteisoalkyl groups as well as other branched chain forms of alkyl. Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups. The term "amine" as used herein refers to primary, secondary, and tertiary amines having, e.g., the formula N(group)3 wherein each group can independently be H or non-H, such as alkyl, aryl, and the like. Amines include but are not limited to R-NH2, for example, alkylamines, arylamines, alkylarylamines; R2NH wherein each R is independently selected, such as dialkylamines, diarylamines, aralkylamines, heterocyclylamines and the like; and R3N wherein each R is independently selected, such as trialkylamines, dialkylarylamines, alkyldiarylamines, triarylamines, and the like. The term "amine" also includes ammonium ions as used herein. The term "amino group" as used herein refers to a substituent of the form -NH2, - NHR, -NR2, -NR3 +, wherein each R is independently selected, and protonated forms of each, except for -NR3 +, which cannot be protonated. Accordingly, any compound substituted with an amino group can be viewed as an amine. An "amino group" within the meaning herein can be a primary, secondary, tertiary, or quaternary amino group. An "alkylamino" group includes a monoalkylamino, dialkylamino, and trialkylamino group. The term "aralkyl" as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein. Representative aralkyl groups include benzyl and phenylethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl. Aralkenyl groups are alkenyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein. The term "aryl" as used herein refers to cyclic aromatic hydrocarbon groups that do not contain heteroatoms in the ring. Thus aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl, anthracenyl, and naphthyl groups. In some embodiments, aryl groups contain about 6 to about 14 carbons in the ring portions of the groups. Aryl groups can be unsubstituted or substituted, as defined herein. Representative substituted aryl groups can be mono-substituted or substituted more than once, such as, but not limited to, a phenyl group substituted at any one or more of 2-, 3-, 4-, 5-, or 6-positions of the phenyl ring, or a naphthyl group substituted at any one or more of 2- to 8-positions thereof. As used herein, a "disease" is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated then the subject's health continues to deteriorate. The term "cycloalkyl" as used herein refers to cyclic alkyl groups such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, the cycloalkyl group can have 3 to about 8-12 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 4, 5, 6, or 7. Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like. Cycloalkyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined herein. Representative substituted cycloalkyl groups can be mono-substituted or substituted more than once, such as, but not limited to, 2,2-, 2,3-, 2,4- 2,5- or 2,6-disubstituted cyclohexyl groups or mono-, di- or tri-substituted norbornyl or cycloheptyl groups, which can be substituted with, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups. The term "cycloalkenyl" alone or in combination denotes a cyclic alkenyl group. As used herein, a "disorder" in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject's state of health. The term “glioma” as used herein refers to a common type of tumor originating in the brain. About 33 percent of all brain tumors are gliomas, which originate in the glial cells that surround and support neurons in the brain, including astrocytes, oligodendrocytes and ependymal cells The terms "halo," "halogen," or "halide" group, as used herein, by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. The term "haloalkyl" group, as used herein, includes mono-halo alkyl groups, poly- halo alkyl groups wherein all halo atoms can be the same or different, and per-halo alkyl groups, wherein all hydrogen atoms are replaced by halogen atoms, such as fluoro. Examples of haloalkyl include trifluoromethyl, 1,1-dichloroethyl, 1,2-dichloroethyl, 1,3-dibromo-3,3- difluoropropyl, perfluorobutyl, and the like. The term “heteroaralkynyl” as used herein refers to alkynyl groups as defined herein in which a hydrogen or carbon bond of an alkynyl group is replaced with a bond to a heteroaryl group as defined herein. Representative aralkynyl groups include, but are not limited to, 2-ethynylpyridine and 2-ethynylthiophene. The term "heteroaryl" as used herein refers to aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S; for instance, heteroaryl rings can have 5 to about 8-12 ring members. A heteroaryl group is a variety of a heterocyclyl group that possesses an aromatic electronic structure. A heteroaryl group designated as a C2-heteroaryl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth. Likewise a C4-heteroaryl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms sums up to equal the total number of ring atoms. Heteroaryl groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, indolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Heteroaryl groups can be unsubstituted, or can be substituted with groups as is discussed herein. Representative substituted heteroaryl groups can be substituted one or more times with groups such as those listed herein. Additional examples of aryl and heteroaryl groups include but are not limited to phenyl, biphenyl, indenyl, naphthyl (1-naphthyl, 2-naphthyl), N-hydroxytetrazolyl, N- hydroxytriazolyl, N-hydroxyimidazolyl, anthracenyl (1-anthracenyl, 2-anthracenyl, 3- anthracenyl), thiophenyl (2-thienyl, 3-thienyl), furyl (2-furyl, 3-furyl) , indolyl, oxadiazolyl, isoxazolyl, quinazolinyl, fluorenyl, xanthenyl, isoindanyl, benzhydryl, acridinyl, thiazolyl, pyrrolyl (2-pyrrolyl), pyrazolyl (3-pyrazolyl), imidazolyl (1-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl), triazolyl (1,2,3-triazol-1-yl, 1,2,3-triazol-2-yl 1,2,3-triazol-4-yl, 1,2,4-triazol-3-yl), oxazolyl (2-oxazolyl, 4-oxazolyl, 5-oxazolyl), thiazolyl (2-thiazolyl, 4- thiazolyl, 5-thiazolyl), pyridyl (2-pyridyl, 3-pyridyl, 4-pyridyl), pyrimidinyl (2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl), pyrazinyl, pyridazinyl (3- pyridazinyl, 4- pyridazinyl, 5-pyridazinyl), quinolyl (2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, 6- quinolyl, 7-quinolyl, 8-quinolyl), isoquinolyl (1-isoquinolyl, 3-isoquinolyl, 4-isoquinolyl, 5- isoquinolyl, 6-isoquinolyl, 7-isoquinolyl, 8-isoquinolyl), benzo[b]furanyl (2-benzo[b]furanyl, 3-benzo[b]furanyl, 4-benzo[b]furanyl, 5-benzo[b]furanyl, 6-benzo[b]furanyl, 7- benzo[b]furanyl), 2,3-dihydro-benzo[b]furanyl (2-(2,3-dihydro-benzo[b]furanyl), 3-(2,3- dihydro-benzo[b]furanyl), 4-(2,3-dihydro-benzo[b]furanyl), 5-(2,3-dihydro-benzo[b]furanyl), 6-(2,3-dihydro-benzo[b]furanyl), 7-(2,3-dihydro-benzo[b]furanyl), benzo[b]thiophenyl (2- benzo[b]thiophenyl, 3-benzo[b]thiophenyl, 4-benzo[b]thiophenyl, 5-benzo[b]thiophenyl, 6- benzo[b]thiophenyl, 7-benzo[b]thiophenyl), 2,3-dihydro-benzo[b]thiophenyl, (2-(2,3- dihydro-benzo[b]thiophenyl), 3-(2,3-dihydro-benzo[b]thiophenyl), 4-(2,3-dihydro- benzo[b]thiophenyl), 5-(2,3-dihydro-benzo[b]thiophenyl), 6-(2,3-dihydro- benzo[b]thiophenyl), 7-(2,3-dihydro-benzo[b]thiophenyl), indolyl (1-indolyl, 2-indolyl, 3-indolyl, 4-indolyl, 5-indolyl, 6-indolyl, 7-indolyl), indazole (1-indazolyl, 3-indazolyl, 4-indazolyl, 5-indazolyl, 6-indazolyl, 7-indazolyl), benzimidazolyl (1-benzimidazolyl, 2-benzimidazolyl, 4-benzimidazolyl, 5-benzimidazolyl, 6-benzimidazolyl, 7-benzimidazolyl, 8-benzimidazolyl), benzoxazolyl (1-benzoxazolyl, 2-benzoxazolyl), benzothiazolyl (1- benzothiazolyl, 2-benzothiazolyl, 4-benzothiazolyl, 5-benzothiazolyl, 6-benzothiazolyl, 7-benzothiazolyl), carbazolyl (1-carbazolyl, 2-carbazolyl, 3-carbazolyl, 4-carbazolyl), 5H-dibenz[b,f]azepine (5H-dibenz[b,f]azepin-1-yl, 5H-dibenz[b,f]azepine-2-yl, 5H-dibenz[b,f]azepine-3-yl, 5H-dibenz[b,f]azepine-4-yl, 5H-dibenz[b,f]azepine-5-yl), 10,11-dihydro-5H-dibenz[b,f]azepine (10,11-dihydro-5H-dibenz[b,f]azepine-1-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-2-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-3-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-4-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-5-yl), and the like. The term "heterocyclylalkyl" as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group as defined herein is replaced with a bond to a heterocyclyl group as defined herein. Representative heterocyclyl alkyl groups include, but are not limited to, furan-2-yl methyl, furan-3-yl methyl, pyridine-3-yl methyl, tetrahydrofuran-2-yl ethyl, and indol-2-yl propyl. The term "heteroarylalkyl" as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heteroaryl group as defined herein. The term "heterocyclylalkyl" as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group as defined herein is replaced with a bond to a heterocyclyl group as defined herein. Representative heterocyclyl alkyl groups include, but are not limited to, furan-2-yl methyl, furan-3-yl methyl, pyridine-3-yl methyl, tetrahydrofuran-2-yl ethyl, and indol-2-yl propyl. The term "heterocyclyl" as used herein refers to aromatic and non-aromatic ring compounds containing three or more ring members, of which one or more is a heteroatom such as, but not limited to, N, O, and S. Thus, a heterocyclyl can be a cycloheteroalkyl, or a heteroaryl, or if polycyclic, any combination thereof. In some embodiments, heterocyclyl groups include 3 to about 20 ring members, whereas other such groups have 3 to about 15 ring members. A heterocyclyl group designated as a C2-heterocyclyl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth. Likewise a C4-heterocyclyl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms equals the total number of ring atoms. A heterocyclyl ring can also include one or more double bonds. A heteroaryl ring is an embodiment of a heterocyclyl group. The phrase "heterocyclyl group" includes fused ring species including those that include fused aromatic and non-aromatic groups. For example, a dioxolanyl ring and a benzdioxolanyl ring system (methylenedioxyphenyl ring system) are both heterocyclyl groups within the meaning herein. The phrase also includes polycyclic ring systems containing a heteroatom such as, but not limited to, quinuclidyl. Heterocyclyl groups can be unsubstituted, or can be substituted as discussed herein. Heterocyclyl groups include, but are not limited to, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, dihydrobenzofuranyl, indolyl, dihydroindolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Representative substituted heterocyclyl groups can be mono-substituted or substituted more than once, such as, but not limited to, piperidinyl or quinolinyl groups, which are 2-, 3-, 4-, 5-, or 6- substituted, or disubstituted with groups such as those listed herein. The term "hydrocarbon" or "hydrocarbyl" as used herein refers to a molecule or functional group that includes carbon and hydrogen atoms. The term can also refer to a molecule or functional group that normally includes both carbon and hydrogen atoms but wherein all the hydrogen atoms are substituted with other functional groups. Hydrocarbyl groups can be shown as (Ca-Cb)hydrocarbyl, wherein a and b are integers and mean having any of a to b number of carbon atoms. For example, (C1-C4)hydrocarbyl means the hydrocarbyl group can be methyl (C1), ethyl (C2), propyl (C3), or butyl (C4), and (C0- Cb)hydrocarbyl means in certain embodiments there is no hydrocarbyl group. The term "independently selected from" as used herein refers to referenced groups being the same, different, or a mixture thereof, unless the context clearly indicates otherwise. Thus, under this definition, the phrase "X1, X2, and X3 are independently selected from noble gases" would include the scenario where, for example, X1, X2, and X3 are all the same, where X1, X2, and X3 are all different, where X1 and X2 are the same but X3 is different, and other analogous permutations. The term "knockdown" or "KD" as used herein refers to an experimental technique wherein the expression of one or more of an organisms genes and/or translation of the corresponding RNA is reduced. As used herein, a "prophylactic" or "preventive" treatment is a treatment administered to a subject who does not exhibit signs of a disease or disorder or exhibits only early signs of the disease or disorder for the purpose of decreasing the risk of developing pathology associated with the disease or disorder. As used herein, the language "pharmaceutically effective amount," "therapeutically effective amount," or "effective amount" refers to a non-toxic but sufficient amount of the composition used in the practice of the disclosure that is effective to treat, prevent, and/or ameliorate a disease or disorder in the body of a mammal. The desired treatment may be prophylactic and/or therapeutic. That result may be reduction and/or alleviation of the signs, symptoms, or causes of a disease or disorder, or any other desired alteration of a biological system. An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation. As used herein, the term "pharmaceutical composition" or "composition" refers to a mixture of at least one compound useful within the disclosure with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a subject. As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound useful within the disclosure, and is relatively non-toxic, i.e., the material may be administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the disclosure within or to the subject such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the disclosure, and not injurious to the subject. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the disclosure, and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions. The "pharmaceutically acceptable carrier" may further include a pharmaceutically acceptable salt of the compound useful within the disclosure. Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the disclosure are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference. See also “Ansel’s Pharmaceutical Dosage Forms and Delivery Systems”, Tenth Edition (2014). As used herein, the language "pharmaceutically acceptable salt" refers to a salt of the administered compound prepared from pharmaceutically acceptable non-toxic acids and/or bases, including inorganic acids, inorganic bases, organic acids, inorganic bases, solvates (including hydrates) and clathrates thereof. The term "room temperature" as used herein refers to a temperature of about 15 °C to about 28 °C. As used herein, the terms "subject" and "individual" and "patient" can be used interchangeably and may refer to a human or non-human mammal or a bird. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. In certain embodiments, the subject is human. The term "substantially" as used herein refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%. The term "substantially free of" as used herein can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that the composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less. The term "substantially free of" can mean having a trivial amount of, such that a composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less, or about 0 wt%. The term "substituted" as used herein in conjunction with a molecule or an organic group as defined herein refers to the state in which one or more hydrogen atoms contained therein are replaced by one or more non-hydrogen atoms. The term "functional group" or "substituent" as used herein refers to a group that can be or is substituted onto a molecule or onto an organic group. Examples of substituents or functional groups include, but are not limited to, a halogen (e.g., F, Cl, Br, and I); an oxygen atom in groups such as hydroxy groups, alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups including carboxylic acids, carboxylates, and carboxylate esters; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups; a nitrogen atom in groups such as amines, hydroxyamines, nitriles, nitro groups, N-oxides, hydrazides, azides, and enamines; and other heteroatoms in various other groups. Non-limiting examples of substituents that can be bonded to a substituted carbon (or other) atom include F, Cl, Br, I, OR, OC(O)N(R)2, CN, NO, NO2, ONO2, azido, CF3, OCF3, R, O (oxo), S (thiono), C(O), S(O), methylenedioxy, ethylenedioxy, N(R)2, SR, SOR, SO2R, SO2N(R)2, SO3R, C(O)R, C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R)2, OC(O)N(R)2, C(S)N(R)2, (CH2)0- 2N(R)C(O)R, (CH2)0-2N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, and C(=NOR)R, wherein R can be hydrogen or a carbon-based moiety; for example, R can be hydrogen, (C1- C100) hydrocarbyl, alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl; or wherein two R groups bonded to a nitrogen atom or to adjacent nitrogen atoms can together with the nitrogen atom or atoms form a heterocyclyl. As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the composition or method. “Consisting of” shall mean excluding more than trace elements of other ingredients for claimed compositions and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure. Accordingly, it is intended that the methods and compositions can include additional steps and components (comprising) or alternatively including steps and compositions of no significance (consisting essentially of) or alternatively, intending only the stated method steps or compositions (consisting of). As used herein, “loweralkyl” means a linear or branched saturated hydrocarbon of 1 to 5 carbon atoms, including methyl, ethyl, propyl, isopropyl, butyl, 2-methylpropyl, tert- butyl, and pentyl. As used herein, “about” means plus or minus 10%. As used herein, “optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. As used herein, the terms “individual”, “patient”, or “subject” can be an individual organism, a vertebrate, a mammal (e.g., a bovine, a canine, a feline, or an equine), or a human. In certain embodiments, the individual, patient, or subject is a human. As used herein, the phrases “therapeutically effective amount” and “therapeutic level” mean a compound dose or plasma concentration in a subject, respectively, that provides the specific pharmacological effect for which the compound is administered in a subject in need of such treatment, i.e., to reduce, ameliorate, or eliminate the effects or symptoms of cancer. It is emphasized that a therapeutically effective amount or therapeutic level of a drug will not always be effective in treating the conditions/diseases described herein, even though such dosage is deemed to be a therapeutically effective amount by those of skill in the art. The therapeutically effective amount may vary based on the route of administration and dosage form, the age and weight of the subject, and/or the subject’s condition, including the type and stage of the cancer at the time that treatment commences, among other factors. The terms “treatment” or “treating” as used herein with reference to cancer refer to reducing, ameliorating or eliminating one or more symptoms or effects of the disease or condition. A “therapeutic response” means an improvement in at least one measure of cancer. As used herein, the term “refractory” as to a particular treatment of a disease means that the disease is unresponsive to the treatment. As used herein the term “MGMT deficient” (or MGMT-) cancers means cancers that have more than one standard deviation lower abundance of the mRNA transcript for the MGMT gene or more than one standard deviation lower abundance of the associated functional protein itself normalized to the relevant healthy control tissue. This deficiency can occur through promoter methylation, mutations in the gene, or through other methods resulting in downregulation of the gene. As used herein, the term “MMR deficient” (or MMR-) cancers means cancers that have more than one standard deviation lower abundance of the mRNA transcript for any of the MMR genes (MSH2, MSH6, MLH1, MLH3, PMS2, PMS1) or more than one standard deviation lower abundance of the respective functional protein(s) normalized to the relevant healthy control tissue. Alternatively, cancers that exhibit the microsatellite instability high phenotype (MSI-H) are also considered to be MMR deficient. See, for example, Li et al. – Microsatellite instability: a review of what the oncologist should know – Cancer Cell International, Article Number 16 (2020). Pharmaceutical Formulations Pharmaceutical compositions suitable for use for the compounds and in the methods described herein can include a disclosed compound and a pharmaceutically acceptable carrier or diluent. The composition may be formulated for intravenous, subcutaneous, intraperitoneal, intramuscular, topical, oral, buckle, nasal, pulmonary or inhalation, ocular, vaginal, or rectal administration. In some embodiments, the compounds are formulated for oral administration. The pharmaceutical composition can be formulated to be an immediate-release composition, sustained-release composition, delayed-release composition, etc., using techniques known in the art. Pharmacologically acceptable carriers for various dosage forms are known in the art. For example, excipients, lubricants, binders, and disintegrants for solid preparations are known; solvents, solubilizing agents, suspending agents, isotonicity agents, buffers, and soothing agents for liquid preparations are known. In some embodiments, the pharmaceutical compositions include one or more additional components, such as one or more preservatives, antioxidants, stabilizing agents and the like. Additionally, the disclosed pharmaceutical compositions can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. 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. In some embodiment, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts 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 sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that 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, non- limiting methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Pharmaceutical compositions of the disclosure can be administered in combination with other therapeutics that are part of the current standard of care for cancer. In certain embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding/formulating such a therapeutic compound for the treatment of a disorder in a subject. In certain embodiments, the compositions of the disclosure are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions of the disclosure comprise a therapeutically effective amount of a compound useful within the disclosure and a pharmaceutically acceptable carrier. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may 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 may 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, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin. In certain embodiments, the compositions of the disclosure are administered to the subject in dosages that range from one to five times per day or more. In other embodiments, the compositions of the disclosure are administered to the subject in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It is readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the disclosure varies from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any subject are determined by the attending physical taking all other factors about the subject into account. Compounds useful within the disclosure for administration may be in the range of from about 1 mg to about 10,000 mg, about 20 mg to about 9,500 mg, about 40 mg to about 9,000 mg, about 75 mg to about 8,500 mg, about 150 mg to about 7,500 mg, about 200 mg to about 7,000 mg, about 3050 mg to about 6,000 mg, about 500 mg to about 5,000 mg, about 750 mg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 50 mg to about 1,000 mg, about 75 mg to about 900 mg, about 100 mg to about 800 mg, about 250 mg to about 750 mg, about 300 mg to about 600 mg, about 400 mg to about 500 mg, and any and all whole or partial increments therebetween. In certain embodiments, the dose of a compound useful within the disclosure is from about 1 mg and about 2,500 mg. In other embodiments, a dose of a compound useful within the disclosure used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in certain embodiments, a dose of a second compound, as described herein, is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments therebetween. In certain embodiments, the present disclosure is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound useful within the disclosure, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, and/or ameliorate a disorder. Granulating techniques are well known in the pharmaceutical art for modifying starting powders or other particulate materials of an active ingredient. The powders are typically mixed with a binder material into larger permanent free-flowing agglomerates or granules referred to as a "granulation." For example, solvent-using "wet" granulation processes are generally characterized in that the powders are combined with a binder material and moistened with water or an organic solvent under conditions resulting in the formation of a wet granulated mass from which the solvent must then be evaporated. Melt granulation generally consists in the use of materials that are solid or semi-solid at room temperature (i.e. having a relatively low softening or melting point range) to promote granulation of powdered or other materials, essentially in the absence of added water or other liquid solvents. The low melting solids, when heated to a temperature in the melting point range, liquefy to act as a binder or granulating medium. The liquefied solid spreads itself over the surface of powdered materials with which it is contacted, and on cooling, forms a solid granulated mass in which the initial materials are bound together. The resulting melt granulation may then be provided to a tablet press or be encapsulated for preparing the oral dosage form. Melt granulation improves the dissolution rate and bioavailability of an active (i.e. drug) by forming a solid dispersion or solid solution. U.S. Patent No.5,169,645 discloses directly compressible wax-containing granules having improved flow properties. The granules are obtained when waxes are admixed in the melt with certain flow improving additives, followed by cooling and granulation of the admixture. In certain embodiments, only the wax itself melts in the melt combination of the wax(es) and additives(s), and in other cases both the wax(es) and the additives(s) will melt. The present disclosure also includes a multilayer tablet comprising a layer providing for the delayed release of one or more compounds useful within the disclosure, and a further layer providing for the immediate release of a medication for a disorder. Using a wax/pH- sensitive polymer mix, a gastric insoluble composition may be obtained in which the active ingredient is entrapped, ensuring its delayed release. Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents. For oral application, particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules, caplets and gelcaps. The compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients that are suitable for the manufacture of tablets. Such excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate. The tablets may be uncoated or they may be coated by known techniques for elegance or to delay the release of the active ingredients. Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent. The compounds for use in the disclosure may be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration. Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations and compositions that are described herein. Oral Administration For oral administration, the compositions of the disclosure may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., polyvinylpyrrolidone, hydroxypropylcellulose or hydroxypropylmethylcellulose); fillers (e.g., cornstarch, lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrates (e.g., sodium starch glycollate); or wetting agents (e.g., sodium lauryl sulphate). If desired, the tablets may be coated using suitable methods and coating materials such as OPADRY™ film coating systems available from Colorcon, West Point, Pa. (e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and OPADRY™ White, 32K18400). Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions. The liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxy benzoates or sorbic acid). Parenteral Administration For parenteral administration, the compositions of the disclosure may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and/or continuous infusion. Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulation agents such as suspending, stabilizing and/or dispersing agents may be used. Additional Administration Forms Additional dosage forms of this disclosure include dosage forms as described in U.S. Patents Nos.6,340,475, 6,488,962, 6,451,808, 5,972,389, 5,582,837, and 5,007,790. Additional dosage forms of this disclosure also include dosage forms as described in U.S. Patent Applications Nos.2003/0147952, 2003/0104062, 2003/0104053, 2003/0044466, 2003/0039688, and 2002/0051820. Additional dosage forms of this disclosure also include dosage forms as described in PCT Applications Nos. WO 03/35041, WO 03/35040, WO 03/35029, WO 03/35177, WO 03/35039, WO 02/96404, WO 02/32416, WO 01/97783, WO 01/56544, WO 01/32217, WO 98/55107, WO 98/11879, WO 97/47285, WO 93/18755, and WO 90/11757. Controlled Release Formulations and Drug Delivery Systems In certain embodiments, the formulations of the present disclosure may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations. The term sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period. The period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form. For sustained release, the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds. As such, the compounds of the present disclosure may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation. In certain embodiments of the disclosure, the compounds useful within the disclosure are administered to a subject, alone or in combination with another pharmaceutical agent, using a sustained release formulation. The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that may, although not necessarily, include a delay of from about 10 minutes up to about 12 hours. The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration. The term immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration. As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration. As used herein, rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration. Methods of Treatment Approximately half of glioblastoma multiforme and over two-thirds of grade II/III glioma tumors lack the DNA repair protein O6-methylguanine methyl transferase (MGMT). MGMT– tumors respond initially to the DNA methylation agent, temozolomide, but frequently acquire resistance via loss of the mismatch repair (MMR) pathway. The need exists for agents that can overcome this resistance mechanism by inducing MMR- independent cell kill selectively in MGMT-silenced tumors and methods of use thereof. To address the foregoing need, one aspect of the present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject a compound of the present disclosure or the pharmaceutical composition of the present disclosure. In certain embodiments, the cancer is a glioma. In certain embodiments, the glioma is resistant to treatment with a DNA methylation agent and/or temozolomide. In certain embodiments, O6-methylguanine methyl transferase (MGMT)-silenced tumors are selectively killed. In certain embodiments, the cancer is liver cancer. In certain embodiments, the cancer is hepatocellular carcinoma. In certain embodiments, the amount of compound or pharmaceutical composition administered to the subject is a therapeutically effective amount. Another aspect of the disclosure provides a method in which a therapeutically- effective amount of a compound of formula (I) is administered to a patient (e.g., a human patient) suffering from a cancer and particularly an MGMT deficient cancer. In another embodiment, the present method comprises administration of a therapeutically-effective amount of a compound of formula (I) or a pharmaceutically-acceptable salt thereof to a patient suffering from an MGMT deficient, MMR deficient cancer, particularly a glioma. In some embodiments, the therapeutically effective amount of the compound is administered together with a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers are well-known in the art, as discussed infra. A typical route of administration is oral, but other routes of administration are possible, as is well understood by those skilled in the medical arts. Administration may be by single or multiple doses. The amount of compound administered and the frequency of dosing may be optimized by the physician for the particular patient. In addition to gliomas such as glioblastoma multiforme and brain lower grade glioma, the present method and compounds are useful to treat urothelial cancer, breast invasive carcinoma, colon adenocarcinoma, head and neck tumor (SCC), lung adenocarcinoma, rectum adenocarcinoma, and acute myeloid leukemia. Therapeutically effective doses and dosing regimens In some embodiments, the therapeutically effective dose of the compound may be administered every day, for 21 days followed by a 7 day rest, every 7 days with a 7 day rest in between each dosage period, or for 5 continuous days followed by a 21 day rest, in each instance referring to a 28 day dosage cycle. The therapeutically effective dose of compound administered to the patient (whether administered in a single does or multiple doses) should be sufficient to treat the cancer. Such therapeutically effective amount may be determined by evaluating the symptomatic changes in the patient. Exemplary doses can vary according to the size and health of the individual being treated, the condition being treated, and the dosage regimen adopted. In some embodiments, the effective amount of a disclosed compound per 28 day dosage cycle is about 1.5 g/m2; however, in some situations the dose may be higher or lower – for example 2.0 g/m2 or 1.0 g/m2. The daily dose may vary depending on (inter alia) the dosage regimen adopted. For example, if the regimen is dosing for five days followed by a 21 day rest and the total dosage per 28 day cycle is 1.0 g/m2, then the daily dose would be 200 mg/m2. Alternatively, if the regimen is dosing for 21 days followed by a 5 day rest and the total dosage per 28 day cycle is 1.6 g/m2, then the daily dose would be 75 mg/m2. Similar results would obtain for other dosage regimens and total 28 day doses. The disclosed methods of treatment may also be combined with other known methods of treatment as the situation may require. Therapeutically effective doses and dosing regimens of the foregoing methods may vary, as would be readily understood by those of skill in the art. Dosage regimens may be adjusted to provide the optimum desired response. For example, in some embodiments, a single bolus dose of the compound may be administered, while in some embodiments, several divided doses may be administered over time, or the dose may be proportionally reduced or increased in subsequent dosing as indicated by the situation. Without wishing to be bound thereby, Applicant believes the disclosed compounds act as bifunctional alkylation agents in a two-step process. The first reaction generates a primary DNA lesion (alkylation) that is rapidly removed by healthy MGMT-proficient cells. The second reaction slowly transforms the primary modification (alkylation) into a more toxic lesion via a unimolecular process. Thus, Applicant believes the disclosed compounds first alkylate O6-guanine and thereafter evolve slowly to more toxic inter-strand cross link (ICL), thereby establishing an MMR-independent method to amplify the therapeutic impact of MGMT deficiency. Routes of administration of any of the compounds and/or compositions of the disclosure include oral, nasal, rectal, intravaginal, parenteral (e.g., IM, IV and SC), buccal, sublingual or topical. The regimen of administration may affect what constitutes an effective amount. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation. Administration of the compositions of the present disclosure to a subject, such as a mammal, such as a human, may be carried out using known procedures, at dosages and for periods of time effective to treat the disorder in a subject. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the subject; the age, sex, and weight of the subject; and the ability of the therapeutic compound to treat the disease or disorder in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound useful within the disclosure is from about 1 and 5,000 mg/kg of body weight/per day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation. Actual dosage levels of the active ingredients in the pharmaceutical compositions of this disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject. In particular, the selected dosage level depends upon a variety of factors, including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well, known in the medical arts. A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian may start doses of the compounds useful within the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In certain embodiments, a suitable dose of a compound of the present disclosure may be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day. The dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different. For example, a dose of 1 mg per day may be administered as two 0.5 mg doses, with about a 12-hour interval between doses. It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. The compounds for use in the method of the disclosure may be formulated in unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unitary dosage for subjects undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents were considered to be within the scope of this disclosure and covered by the claims appended hereto. For example, it should be understood, that modifications in reaction conditions, including but not limited to reaction times, reaction size/volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g., nitrogen atmosphere, and reducing/oxidizing agents, with art- recognized alternatives and using no more than routine experimentation, are within the scope of the present application. It is to be understood that, wherever values and ranges are provided herein, the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, all values and ranges encompassed by these values and ranges are meant to be encompassed within the scope of the present disclosure. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application. The description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range and, when appropriate, partial integers of the numerical values within ranges. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 and so forth, as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.1, 5.3, 5.5, and 6. This applies regardless of the breadth of the range. Compound Syntheses The present compounds may be synthesized according to the synthetic schemes and reference methods described above. Compound Testing The pharmaceutical activity of the subject compounds may be evaluated in the following assays: Short-term cell viability assay: On day 1, Ln229 isogenic cells of varying MGMT and MMR status are seeded in 96 well format at a density of 2000 cells/well in 100 µL of DMEM media and allowed to adhere overnight. On day 2, a drug master plate is made with 100x the desired maximal concentration of test compound and serially diluted by 2 until 100x the minimal desired concentration, with one DMSO control. Then daughter plates are created with varied concentrations from 3x the minimal concentration to 3x the maximal desired concentration. Afterwards, 50 µL of daughter drug plate is added to 100 µL of the seeded cells for a final concentration of 1x in triplicate. Cells are allowed to grow for 6 days. On day 7, the cells are fixed, and stained with Hoechst nuclear dye and imaged to determine growth inhibition. Clonogenic Survival Assay: Isogenic glioma (Ln 229) cells are pretreated with the test drug in culture for 48–72 hours at the specified dilutions. Cells are then transferred in media without drug to 6-well plates in triplicate at 3-fold dilutions ranging from 9,000 to 37 cells per well. After 14 days, plates are washed with PBS and stained with crystal violet. Colonies are counted by hand. Counts are normalized to plating efficiency of the corresponding treatment condition. Xenograft Experiments: All animal studies are approved by the Institutional Animal Use and Care Committee and performed in accordance with the Guide for the Care and Use of Laboratory Animals. LN229 WT and LN229-MSH2- cell lines are maintained in DMEM media supplemented with 10% fetal bovine serum. Three-four-week-old female athymic nude Foxnnu mice are obtained from Envigo and each mouse is inoculated subcutaneously with tumor cells (4.5-5 x 106) in 0.1 ml of PBS with Matrigel (1:1). Wild type cells are injected on the right flank and mutant cells are injected on the left flank. The tumors are then grown to a mean size of approximately 50-100 mm3 and the mice are then split into groups and treated. Gavage doses of 5mg/kg of test compound are prepared by diluting stocks in DMSO with 10% cyclodextrin. Compound is administered each day of dosing at a volume of 100ul/mouse. Mice are treated for 3 weeks with dosing on Mondays, Wednesdays and Fridays. Tumors are measured 3 times a week during treatment and during the washout period of 2 weeks. Xenograft tumors are measured by calipers and volume is calculated using the equation for ellipsoid volume: Volume = 0.523 x (length) x (width)2. Statistical Analysis: Analysis of variance (ANOVA) is used to test for significant differences between groups. Post-hoc Bonferroni multiple comparison test analysis is used to determine significant differences among means. All statistical analysis is accomplished using Graph Pad Prism 8.2.0 software. The clonogenic survival assay is a well-recognized assay with high prediction of utility of cancer treatment compounds. See, for example, Fiebig et al. – Clonogenic assay with established human tumor xenografts: correlation of in vitro to in vivo activity as a basis for anti-cancer drug discovery – European J. Cancer, 40 (2004) 802-820. Enumerated Embodiments 1. A compound of formula (I-1), which is selected from the group consisting of:
Figure imgf000055_0001
wherein R1 is selected from the group consisting of optionally substituted C1-C6 alkyl and optionally substituted C1-C6 haloalkyl, wherein each optional substituent in R1 is independently selected from the group consisting of halogen, C1-C3 haloalkyl, C1-C3 alkoxy, C1-C3 haloalkoxy, C1-C3 alkyl, C2-C6 alkenyl, benzyl, phenyl, and naphthyl, and C2-C12 heterocyclyl; or a salt, solvate, tautomer, or isotopologue thereof. 2. The compound of Embodiment 1, which is selected from the group consisting of
Figure imgf000055_0002
3. A pharmaceutical composition comprising the compound of Embodiment 1 or 2 and at least one pharmaceutically acceptable carrier. 4. A method of treating cancer in a subject, the method comprising administering to the subject a compound of Embodiment 1 or 2 or the pharmaceutical composition of Embodiment 3. 5. The method of Embodiment 4, wherein the cancer is a glioma. 6. The method of Embodiment 4 or 5, wherein the glioma is resistant to treatment with a DNA methylation agent and/or temozolomide. 7. The method of any one of Embodiments 4-6, wherein O6-methylguanine methyl transferase (MGMT)-silenced tumors are selectively killed. EXAMPLES The disclosure is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only, and the disclosure is not limited to these Examples, but rather encompasses all variations that are evident as a result of the teachings provided herein. Materials and Methods General Chemical Experimental Procedures. All reactions were performed in single-neck, flame dried round-bottom flasks fitted with rubber septa under a positive pressure of argon, unless otherwise specified. Air- and moisture-sensitive liquids were transferred via syringe or stainless-steel cannula. Organic solutions were concentrated by rotary evaporation at 31 °C, unless otherwise noted. Flash-column chromatography was performed as described by Still et al., employing silica gel (SiliaFlash® P60, 60 Å, 40-63 μm particle size) purchased from Silicycle (Québec, Canada). Analytical thin-layered chromatography (TLC) was performed using glass plates pre-coated with silica gel (250 μm, 60 Å pore size) embedded with a fluorescent indicator (254 nm). TLC plates were visualized by exposure to ultraviolet (UV) light. Chemical Materials. Commercial solvents, chemicals, and reagents were used as received with the follow exceptions. Dichloromethane, tetrahydrofuran, and toluene were purified according to the method of Pangborn et al. Triethylamine was distilled from calcium hydride under an atmosphere of nitrogen immediately prior to use. N,N-Di-iso- propylethylamine was distilled from calcium hydride under argon immediately prior to use. The diazonium S7, the imidazolyl triazene 1b, the imidazolyl triazene 4b, the imidazolyl triazene 9, the imidazolyl triazene 12b, and the imidazolyl triazene 13 were synthesized according to published procedures. Chemistry Instrumentation. Proton nuclear magnetic resonance (1H NMR) were recorded at 400 or 600 megahertz (MHz) at 23 °C, unless otherwise noted. Chemical shifts are expressed in parts per million (ppm, δ scale) downfield from tetramethylsilane and are referenced to residual proton in the NMR solvent ((CD3)SO(CHD2), δ 2.50). Data are represented as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet and/or multiple resonances, b = broad, app = apparent), coupling constant in Hertz (Hz), integration, and assignment. Proton-decoupled carbon nuclear magnetic resonance spectra (13C NMR) were recorded at 150 MHz at 23 °C, unless otherwise noted. Chemical shifts are expressed in parts per million (ppm, ^^ scale) downfield from tetramethylsilane and are referenced to the carbon resonances of the solvent (DMSO-d6, δ 39.52). 1H-1H gradient-selected correlation spectroscopy (COSY), 1H-13C heteronuclear single quantum coherence (HSQC), and 1H-13C gradient-selected heteronuclear multiple bond correlation (gHMBC) were recorded at 600 MHz at 23 °C, unless otherwise noted. Carbon- decoupled fluorine nuclear magnetic resonance spectra (19F NMR) were recorded at 396 MHz at 23 °C, unless otherwise noted. Chemical shifts are expressed in parts per million (ppm, δ scale) downfield from tetramethylsilane. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectra were obtained using a Thermo Electron Corporation Nicolet 6700 FTIR spectrometer referenced to a polystyrene standard. Data are represented as follows: frequency of absorption (cm–1), intensity of absorption (s = strong, m = medium, w = weak, br = broad). Analytical liquid chromatography-mass spectroscopy (LCMS) was performed on a Waters instrument equipped with a reverse-phase C18 column (1.7 μm particle size, 2.1 × 50 mm). Samples were eluted with a linear gradient of 5% acetonitrile–water containing 0.1% formic acid→100% acetonitrile containing 0.1% formic acid over 0.75 min, followed by 100% acetonitrile containing 0.1% formic acid for 0.75 min, at a flow rate of 800 μL/min. HRMS were obtained on a Waters UPLC/HRMS instrument equipped with a dual API/ESI high resolution mass spectrometry detector and photodiode array detector. Biological Materials. Temozolomide (TMZ, 1a), lomustine (14), O6-benzylguanine (O6BG), doxorubicin, and olaparib were purchased from Selleck Chemicals. Methylmethane sulfonate (MMS) was purchased from Alfa-Aesir. Mitozolomide (MTZ, 12a) was purchased from Enamine. Mitomycin C (MMC), N-ethylmaleimide (NEM), N-acetyl-L-cysteine (NAC), and cisplatin were purchased from Sigma. TMZ (1a, 100 mM stock), O6BG (100 mM stock), MTZ (12a, 100 mM stock), MMS (500 mM stock) and NAC (100 mM stock) were dissolved in DMSO and stored at –80 ºC. MMC (10 mM stock), lomustine (14, 100 mM stock), doxorubicin (10 mM stock), and olaparib (18.3 mM stock) were dissolved in DMSO and stored at –20 ºC. NEM (400 mM stock) was dissolved in EtOH and stored at –20 ºC. Cisplatin (5 mM stock) was dissolved in H2O and stored at 4 ºC for up to 7 days. Cell Culture. LN229 MGMT– and MGMT+ cell lines were a gift from B. Kaina (Johannes Gutenberg University Mainz, Mainz, Germany) and grown in DMEM with 10% FBS (Gibco). DLD1 BRCA2+/– and BRCA2–/– cell lines (Horizon Discovery, Cambridge, UK) were grown in RPMI 1640 with 10% FBS. HCT116 MLH1–/– and HCT116+Chr3 cell lines were a gift from T. Kunkel (National Institute of Environmental Health Sciences, Durham, NC) and grown in DMEM with 10% FBS, with 0.5 µg/mL G418 (Sigma) for HCT116+Chr3 cells. PD20 cell lines complemented with empty vector (+EV), wildtype FANCD2 (+FD2), or K561R ubiquitination-mutant FANCD2 (+KR) were a gift from G. Kupfer and P. Glazer (Yale University, New Haven, CT) and growth in DMEM with 10% FBS. PEO1 and PEO4 cell lines were a gift from T. Taniguchi (Fred Hutchinson Cancer Research Center, Seattle, WA) and were grown in DMEM with 10% FBS. BJ fibroblasts (normal human fibroblast cells) were purchased from ATCC (CRL-2522) and grown in DMEM with 10% FBS. NER isogenic MEFs were a gift from F. Rogers (Yale University, New Haven, CT) and were grown in DMEM with 10% FBS. All human cell lines were validated by short tandem repeat profiling (excluding BJ fibroblasts which were used within 6 passages of receiving from ATCC) and confirmed negative for mycoplasma by quantitative RT-PCR. MMR Protein shRNA Knockdown. pGIPZ lentiviral shRNA vectors targeting MSH2, MSH6, MLH1, PMS2, and MSH3 were purchased from Horizon Discovery (Table S2). Lentiviral particles were produced in HEK293T cells via co-transfection with lentiviral shRNA plasmid, pCMV-VSV-G envelope plasmid (Addgene, #8454) and psPAX2 packaging plasmid (Addgene, #12260), using Lipofectamine 3000 Reagent (Invitrogen, L3000001) per manufacturer’s protocol. Viral particles were harvested 48 h post-transfection and used to transduce LN229 MGMT+/– cells in the presence of 8 µg/mL polybrene. Selection of pooled cells with lentiviral expression was established with 1 µg/mL puromycin 48 h post- transduction for 3 to 4 days. Single cell cloning was performed by limiting dilution and protein knockdown was confirmed by western blotting. Table 1. pGIPZ Lentivral shRNA Vectors for MMR protein knockdown
Figure imgf000058_0001
Figure imgf000059_0001
Western Blotting. For phospho-protein analysis experiments, cells were lysed in 1X RIPA buffer (Cell Signaling Technology, #9806) supplemented with 1X Protease Inhibitor Cocktail (Roche) and 1X PhosSTOP Phosphatase Inhibitor Cocktail (Sigma). For all other western blot analyses, cells were lysed in lysis buffer (50 mM HEPES, 250 mM NaCl, 5 mM EDTA, 1% NP-40) supplemented with 1X Protease Inhibitor Cocktail (Roche). The de- ubiquitination inhibitor N-ethylmaleimide (NEM, 4 mM) was added in FANCD2 ubiquitination analysis experiments. Proteins were separated using NuPAGE 4–12% Bis-Tris or 3–8% Tris-Acetate Gels (Invitrogen) and transferred to Immobilon-P PVDF membrane (Millipore) for western blotting. Membranes were blocked with 5% milk in TBS-T for 1 h prior to primary antibody addition overnight at 4 °C. Primary antibodies were used under the following conditions: mouse anti-CHK1 (Cell Signaling Technology, #2360), 1/1000 in 5% milk; rabbit anti-CHK2 (Cell Signaling Technology, #6334), 1/1000 in 5% BSA; rabbit anti- FANCD2 (Cell Signaling Technology, #16323), 1/1000 in 5% BSA; HRP-conjugated anti- GAPDH (ProteinTech HRP-60004), 1/10,000 in 5% milk; rabbit anti-MGMT (Cell Signaling Technology, #2739), 1/1000 in 5% BSA; rabbit anti-MLH1 (Cell Signaling Technology, #4256), 1/1000 in 5% BSA; mouse anti-MSH2 (Cell Signaling Technology, #2850), 1/1000 in 5% milk; mouse anti-MSH3 (BD Biosciences, BD611390), 1/500 in 5% milk; mouse anti- MSH6 (BD Biosciences, BD610918), 1/1000 in 5% milk; rabbit anti-phospho-CHK1 (S345) (Cell Signaling Technology, #2341), 1/1000 in 5% BSA; rabbit anti-phospho-CHK2 (T68) (Cell Signaling Technology, #2661), 1/1000 in % BSA; mouse anti-PMS2 (Santa Cruz, sc- 25315), 1/100 in 5% milk; mouse anti-Vinculin (Santa Cruz, sc-25336), 1/1000 in 5% milk. Anti-mouse IgG HRP-conjugated antibody (Cell Signaling Technology, #7076) and anti- rabbit IgG HRP-conjugated antibody (Cell Signaling Technology, #93702) were added at 1/5000 in 5% milk for 1 h. Chemiluminescence detection was performed with Clarity Max Western ECL Substrate (Bio-Rad) and blots were imaged on a ChemiDoc XRS+ Molecular Imager (Bio-Rad). Where shown, bands were quantified using ImageJ software. Short-term Cell Viability Assay. Cells were seeded in 96-well plates at 1000 or 2000 cells/well and allowed to adhere at 23 ºC for 60 min and then incubated overnight at 37 °C. Cells were treated with indicated concentrations of compounds in triplicate for 4-6 days prior to fixation with 3.7% paraformaldehyde and nuclear staining with 1 µg/mL Hoechst 33342 dye. Cells were imaged on a Cytation 3 imaging reader (BioTek) and quantified using CellProfiler software. Clonogenic Cell Survival Assay. Cells were trypsinized, washed, counted, and diluted in a medium containing various concentrations of drug. They were then immediately seeded in six-well plates in triplicate at three-fold dilutions, ranging from 9000 to 37 cells per well. Depending on colony size, these plates were kept in the incubator for 10 to 14 days. After incubation, colonies were washed in phosphate-buffered saline (PBS), stained with crystal violet, counted, and quantified. IR Alkaline Comet Assay. Assay was performed utilizing the CometAssay Kit (Trevigen) according to the alkaline assay protocol, with the addition of slide irradiation post- lysis. Cells were trypsinized, washed with 1X PBS, added to melted Comet LMAgarose (Trevigen), and spread on Trevigen CometSlides at a density of 1000 cells per sample in 50 µL. Lysis solution (Trevigen) with 10% DMSO was added overnight at 4 ºC. Slides were removed from lysis buffer and irradiated to 0 or 10 Gy using an XRAD 320 X-Ray System (Precision X-Ray) at 320 kV, 12.5 mA, and 50.0 cm SSD, with a 2 mm Al filter and 20 cm × 20 cm collimator. Slides were then placed in alkaline buffer (200 mM NaOH, 1 mM EDTA) for 45 min, followed by electrophoresis in 850 mL alkaline buffer for 45 min at 4 ºC. Slides were washed and stained with SYBR gold (Invitrogen) per Trevigen assay protocol. Slides were imaged on a Cytation 3 imaging reader (BioTek), and comets were analyzed using CometScore 2.0 software (TriTek). Genomic DNA Denaturing Gel Electrophoresis. Assay was adapted from. Cells were trypsinized, washed with 1X PBS, and stored at –80 ºC prior to processing. Genomic DNA was extracted with the DNeasy Blood & Tissue Kit (Qiagen) per kit protocol. A 0.7% agarose gel was prepared in 100 mM NaCl-2mM EDTA (pH 8) and soaked in 40 mM NaOH–1 mM EDTA running buffer for 2 h. Genomic DNA (400 ng/well) was then loaded in 1X BlueJuice loading buffer (Invitrogen) and subjected to electrophoresis at 2 V/cm for 30 min, followed by 3 V/cm for 2 h. The gel was neutralized in 150 mM NaCl–100 mM Tris (pH 7.4) for 30 min, twice, and then stained with 1X SYBR Gold in 150 mM NaCl–100 mM Tris (pH 7.4) for 90 min. Imaging was performed on a ChemiDoc XRS+ Molecular Imager (Bio-Rad). Plasmid Linearization Assay. To set up the linearization reactions, 20 units of EcoRI-HF (New England Biolabs) was mixed with 20 μg 2686 bp pUC19 vector DNA in CutSmart buffer (New England Biolabs), pH 7.9, in a total volume of 1000 μL for 30 min at 37 °C. The CutSmart buffer contains 50 mM potassium acetate, 20 mM Tris acetate, 10 mM magnesium acetate, and 100 μg/mL BSA. The reacted DNA was then purified using PCR cleanup kit and quantified using the NanoDrop One (Thermo Fisher). The DNA was then stored at −20 °C before use in in vitro DNA cross-linking assays or melting temperature analysis. In Vitro DNA Cross-linking Assays. Linearized pUC19 DNA, prepared as described above, was used for in vitro DNA cross-linking assays. For each condition, 200 ng of linearized pUC19 DNA (15.4 μM base pairs) was incubated with the indicated concentration of drug in 20 μL. Drug stock concentrations were made in DMSO such that each reaction contained a fixed 5% DMSO concentration. Reactions were conducted in 100 mM Tris buffer (pH 7.4). Cisplatin (Sigma) and DMSO vehicle were used as positive and negative controls, respectively. Reactions were conducted between 3–96 h at 37 °C. The DNA was stored at −80 °C until electrophoretic analysis. For gel electrophoresis, DNA concentration was preadjusted to 10 ng/μL. Five microliters (50 ng) of the DNA solution was removed and mixed with 1.5 μL of 6× purple gel loading dye, no SDS, and loaded onto 1% agarose Tris Borate EDTA TBE gels. For denaturing gels, 5 μL (50 ng) of the DNA solution was removed and mixed with 15 μL of 0.2% denaturing buffer (0.27% sodium hydroxide, 10% glycerol, and 0.013% bromophenol blue) or 0.4% denaturing buffer (0.53% sodium hydroxide, 10% glycerol, and 0.013% bromophenol blue) in an ice bath. The mixed DNA samples were denatured at 4 °C for 5 min and then immediately loaded onto a 1% agarose Tris Borate EDTA (TBE) gel. All gel electrophoresis was conducted at 90 V for 2 h (unless otherwise noted). The gel was stained with SYBR Gold (Invitrogen) for 2 h. EndoIV Depurination Assay. For each condition, 200 ng of supercoiled pUC19 DNA (15.4 μM base pairs) was incubated with the indicated concentration of drug in 20 μL for 3 hours. Drug stock concentrations were made in DMSO such that each reaction contained a fixed 5% DMSO concentration. Reactions were conducted in 100 mM Tris buffer (pH 7.4). For each EndoIV reaction, 50 ng of processed DNA was mixed with 20 units of EndoIV in NEBuffer 3.1 (New England Biolabs), pH 7.9, in a total volume of 20 μL for 16−20 h (unless otherwise noted) at 37 °C. The NEBuffer 3.1 contained 100 mM sodium chloride, 50 mM Tris-HCl, 10 mM magnesium chloride, and 100 μg/mL BSA. For each negative control, 50 ng of processed DNA was mixed with NEBuffer 3.1, pH 7.9, in a total volume of 20 μL for 16−20 h (unless otherwise noted) at 37 °C. Following completion of the experiment, the DNA was stored at −20 °C before electrophoretic analysis. Melting Temperature Assay. Linearized pUC19 DNA (750 ng), prepared as described above, was incubated with the indicated concentration of either MMS or KL-50 (4a) adjusted in a final volume of 18 µL in 100 mM Tris buffer (pH 7.4) for 3 h. Drug stock concentrations were made in DMSO such that each reaction contained a fixed 5% DMSO concentration. Afterwards, 1 µL each of 20x SYBR Green dye (Invitrogen) and 20x ROX reference dye (Invitrogen) was added and melting temperature analysis was run on a StepOnePlus RT PCR System (Applied Biosciences) to generate melting temperature curves. Immunofluorescence Foci Assays. High-throughput immunofluorescence foci assays were performed at the Yale Center for Molecular Discovery (YCMD). Cells were seeded at 2000 cells/well in black polystyrene flat bottom 384-well plates (Greiner Bio-One) and allowed to adhere overnight. Compound addition was performed utilizing a Labcyte Echo 550 liquid handler (Beckman Coulter), with 6 replicates per test condition and 12 replicates per control condition. Following drug incubation, cells were fixed and stained for phospho-SER139-H2AX ( γH2AX), 53BP1, or phospho-SER33-RPA2 (pRPA) as follows. ^H2AX protocol: Cells were fixed with 4% paraformaldehyde in 1X PBS for 15 min, washed twice with 1X PBS, incubated in extraction buffer (0.5% Triton X-100 in 1X PBS) for 10 min, washed twice with 1X PBS, and incubated in blocking buffer (Blocker Casein in PBS, Thermo Scientific + 5% goat serum, Life Technologies) for 1 h. Mouse anti-phospho- histone H2A.X (Ser139) antibody (clone JBW301, Millipore, 05-636) was added 1/1000 in blocking buffer at 4 ºC overnight. After washing with 1X PBS, cells were incubated with goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 647 (Invitrogen, A-21236) 1/500 and with 1 µg/mL Hoechst nucleic acid dye in blocking buffer for 2 h, and then washed with 1X PBS. 53BP1 protocol: Assay was performed as previously described. Cells were fixed with 4% paraformaldehyde + 0.02% Triton X-100 in 1X PBS for 20 minutes, washed twice with 1X PBS, and incubated in blocking buffer (10% FBS, 0.5% Triton X-100 in 1X PBS) for 1 h. Rabbit anti-53BP1 antibody (Novus Biologicals, NB100-904) was added 1/1000 in blocking buffer at 4 ºC overnight. After washing with 1X PBS, cells were incubated with goat anti- rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 647 (Invitrogen, A- 21245) 1/500 and with 1 µg/mL Hoechst nucleic acid dye in blocking buffer for 2 h, and then washed with 1X PBS. pRPA protocol: Cells were washed twice with 1X PBS on ice, incubated in extraction buffer (0.5% Triton X-100 in 1X PBS) for 5 min on ice, fixed with 3% paraformaldehyde + 2% sucrose in 1X PBS for 15 min at 23 ºC, incubated again in extraction buffer for 5 min on ice, and incubated in blocking buffer (2% BSA, 10% milk, 0.1% Triton X-100 in 1X PBS) for 1 h at 23 ºC. Rabbit anti-phospho-RPA2 (S33) antibody (Bethyl Laboratories, A300- 246A) was added 1/1000 in blocking buffer at 4 ºC overnight. After washing 4 times with IF wash buffer (0.1% Triton X-100 in 1X PBS), cells were incubated with goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 647 (Invitrogen, A-21245) 1/500 and with 1 µg/mL Hoechst nucleic acid dye in blocking buffer for 1 h at 37 ºC. Cells were washed twice with IF wash buffer and twice with 1X PBS. Imaging was performed on an InCell Analyzer 2200 Imaging System (GE Corporation) at 40X magnification. Twenty fields-of-view were captured per well. Foci analysis was performed using InCell Analyzer software (GE Corporation) as previously described. Outer wells were excluded from analysis to limit variation due to edge effects. Additional small scale immunofluorescence assays used for extended time course analysis of γH2AX foci were performed in Millicell EZSLIDE 8-well chamber slides (Millipore). Cells were seeded at 10,000 cells/well and allowed to adhere overnight. Following drug treatment, cells were fixed and stained for γH2AX as described above, without the addition of Hoechst dye. Slides were mounted with Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories). Imaging was performed on a Keyence BZ-X800 fluorescence microscope at 40X magnification. Nine adjacent fields-of-view were captured per well and stitched together using a Fiji/ImageJ software plugin. Foci analysis was performed using Focinator v2 software. Cell Cycle Analysis. Cell cycle analysis was performed using integrated Hoechst nucleic acid dye fluorescence intensity as previously described. Briefly, integrated Hoechst fluorescence intensity was log2 transformed and histograms from DMSO-treated cells were used to identify the centers of the 2N and 4N DNA peaks. These values were used to normalize the 2N DNA peak to 1 and the 4N DNA peak to 2. Cells were then classified by normalized log2 DNA content as G1 (0.75–1.25), S (1.25–1.75), or G2 (1.75–2.5) phase cells. The percentage of cells within each phase of the cell cycle was determined for each treatment condition. The three sets of Hoechst-stained cells corresponding to the three separate DNA foci stains were treated as three independent analyses. Micronuclei Analysis. An automated image analysis pipeline was developed by YCMD using InCell Analyzer software to quantify micronuclei formation. Nuclei and micronuclei were segmented based on Hoechst nucleic acid dye staining channel. A perinuclear margin was applied around the nuclei to approximate the extent of the cytoplasm and identify micronuclei associated with the parent nucleus. Cells with nuclei associated with at least 1 micronucleus were considered positive. Statistical analysis. Statistical analysis was performed using GraphPad Prism software. Data are presented as mean or median ± SD or SEM as indicated. For in vitro short-term growth delay experiments, IC50 values were determined from the nonlinear regression equation, [inhibitor] vs normalized response with variable slope. For micronuclei assays, comparisons were made with one-way ANOVA and Sidak correction for multiple comparisons. For xenograft growth delay experiments, comparisons were made with Mann- Whitney test (for comparison of 2 groups) or Kruskal-Wallis test with FDR-adjusted p-values with Q set to 5% (for comparison of ≥ 3 groups). For xenograft survival analysis, Kaplan– Meier analysis was used to evaluate survival rate based on death or removal from study when body weight loss exceeded 20% of initial body weight. Mice Protocols. Animals. All animal use was in accordance with the guidelines of the Animal Care and Use Committee (IACUC) of Yale University and conformed to the recommendations in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, National Academy of Sciences, 1996). Mouse Protocols for Flank Studies. A mouse tumor model was established by subcutaneously implanting human LN229 (MGMT–/MMR+) or LN229 (MGMT–/MMR–) cells. Cells were cultured as a monolayer in DMEM +10% FBS (Thermo Fisher) at 37 °C in a humidified atmosphere with 5% CO2 and passaged between one and three days prior to implantation and media was replaced every 2-3 days as needed to maintain cell viability. Cells were not allowed to exceed 80% confluency. On the day of implantation, cells were trypsinized, washed with complete media and pelleted by centrifugation at 1200 rpm for 5 minutes. The supernatant was decanted, and cells were washed three times with sterile PBS and pelleted by centrifugation. During the final centrifugation, viability was determined using trypan blue exclusion. Cells were resuspended in sterile PBS and diluted 1:1 in Matrigel (Corning, Cat #47743-716) for a final concentration of 5×106 cells/ 100 µL. 5 million cells were injected into the flank of female nude mice (Envigo, Hsd:Athymic Nude- Foxn1nu, 3-4 weeks age, 15 g). Once tumors reached a minimum volume of 100 mm3, mice were randomized and administered either KL-50 (4a; 5 mg/kg MWF × 3 weeks), TMZ (1a; 5 mg/kg MWF × 3 weeks), or vehicle (10% cyclodextrin) by oral gavage. Caliper measurements were obtained during the dosing period and at least two weeks following treatment. Mice were euthanized if body weight loss exceeded 20% or if tumor volume increased to greater than 2000 mm3. Kaplan–Meier analysis was used to evaluate survival rate based on death or removal from study. In a second study, mice were randomized and administered either KL-50 (4a) or vehicle (10% cyclodextrin) by oral gavage or intraperitoneal injection on either M–F × 1 or MWF × 3 cycles at 5, 15, or 25 mgs/kg. Caliper measurements were obtained during the dosing period and at least two weeks following treatment. Mice were euthanized if body weight loss exceeded 20% or if tumor volume increased to greater than 2000 mm3. The third study involved MGMT–/MMR+ and MGMT–/MSH6– (shMSH6) LN229 cells. Mice tumors were allowed to grow to a larger average starting volume of ~350 mm3 before they were randomized and administered either KL-50 (4a; 25 mg/kg MWF × 3 weeks) or vehicle (10% cyclodextrin) by oral gavage. Caliper measurements were obtained during the dosing period and at least two weeks following treatment. Mice were euthanized if body weight loss exceeded 20% or if tumor volume increased to greater than 3000 mm3. Mouse Protocol for Intracranial Study. LN229 MGMT–/MMR– cells stably expressing firefly luciferase (lentivirus-plasmids from Cellomics Technology; PLV-10003), were injected intracranially using a stereotactic injector. Briefly, 1.5 million cells in 5 μl PBS were injected into the brain and the mice were imaged weekly using the IVIS Spectrum In Vivo Imaging System (PerkinElmer) according to the manufacturer’s protocol. Images were taken on a weekly basis and acquired 10 min post intraperitoneal injection with d-luciferin (150 mg/kg of animal mass). Tumors were allowed to grow to an average of 1.0 × 108 RLU before randomization and treated with 5 continuous days of P.O treatment with 10% cyclodextrin vehicle control, TMZ (1a, 25 mg/kg M–F × 1 week) or KL-50 (4a, 25 mg/kg M–F × 1 week). Quantification of BLI flux (photons/sec) was made through the identification of a region of interest (ROI) for each tumor. Example 1: Synthesis of KL-50 (4a):
Figure imgf000066_0001
A mixture of fluoroethylamine hydrochloride (3.32 g, 33.3 mmol, 1 equiv), and N,N- di-iso-propyl ethylamine (12.2 mL, 70.0 mmol, 2.10 equiv) in dichloromethane (80 mL) was added dropwise via syringe pump over 45 min to a solution of diphosgene (2.40 mL, 20.0 mmol, 0.60 equiv) in dichloromethane (80 mL) at 0 °C (CAUTION: Gas evolution!). Upon completion of the addition, the cooling bath was removed, and the reaction mixture was allowed to warm to 23 °C over 15 min. The warmed product mixture was immediately transferred to a separatory funnel. The organic layer was washed sequentially with 1 N aqueous hydrochloric acid solution (100 mL, precooled to 0 °C) and saturated aqueous sodium chloride solution (100 mL, precooled to 0 °C). The washed organic layer was dried over magnesium sulfate. The dried solution was filtered, and the filtrate was concentrated (330 mTorr, 31 °C). The unpurified isocyanate so obtained was used directly in the following step. The unpurified isocyanate obtained in the preceding step (nominally 16.7 mmol, 1.75 equiv) was added dropwise via syringe to a solution of the diazonium S7 (1.31 g, 9.54 mmol, 1 equiv) in dimethyl sulfoxide (10 mL) at 23 °C. Upon completion of the addition, the reaction vessel was covered with aluminum foil. The reaction mixture was stirred for 16 h at 23 °C. The product mixture was concentrated under a stream of nitrogen. The residue obtained was suspended in dichloromethane and purified by automated flash-column chromatography (eluting with 100% dichloromethane initially, grading to 5% methanol– dichloromethane, linear gradient) to provide KL-50 (4a) as a white crystalline powder (840 mg, 39% based on the diazonium S7). 1H NMR (400 MHz, DMSO-d6) δ 8.85 (s, 1H, H6), 7.83 (s, 1H, NH), 7.70 (s, 1H, NH), 4.82 (dt, J = 47.0, 4.9 Hz, 2H, H3b), 4.62 (dt, J = 26.0, 4.7 Hz, 2H, H3a). 13C NMR (151 MHz, DMSO-d6) δ 161.5 (C8a), 139.2 (C4), 134.2 (C9), 131.0 (C8), 128.9 (C6), 80.8 (d, J = 168.7 Hz, C3b), 49.1 (d, J = 20.8 Hz, C3a). 19F NMR (376 MHz, DMSO-d6) δ –222.66 (tt, J = 47.0, 26.1 Hz). IR (ATR-FTIR), cm–1: 3459 (w), 3119 (m), 1736 (s), 1675 (s). HRMS-ESI (m/z): [M + H]+ calcd for [C7H8FN6O2]+ 227.0688, found 227.0676. Synthesis of the imidazolyl triazene 10:
Figure imgf000067_0001
Tert-butyl (2-hydroxypropyl)carbamate (1.72 mL, 10.0 mmol, 1 equiv) was added dropwise via syringe to a mixture of PyFluor (1.77 g, 11.0 mmol, 1.10 equiv) in tetrahydrofuran (10 mL) at 23°C. 1,8-Diazabicyclo(5.4.0)undec-7-ene (3.00 mL, 20.0 mmol, 2.00 equiv) was immediately added dropwise and the reaction mixture was stirred for 48 h at 23 °C under ambient atmosphere. The product mixture was diluted with water (15 mL) and the resulting biphasic mixture was transferred to a separatory funnel. The layers that formed were separated and the aqueous layer was extracted with ethyl acetate (2 × 15 mL). The organic layers were combined and the combined organic layer was washed sequentially with 1 N aqueous hydrochloric acid solution (2 × 25 mL) and saturated aqueous sodium chloride solution (2 × 25 mL). The washed organic layer was dried over sodium sulfate. The dried solution was then filtered and the filtrate concentrated to provide tert-butyl (2- fluoropropyl)carbamate as a clear colorless oil. The unpurified product obtained in the preceding step (nominally 6 mmol, 1 equiv) was added to a mixture of dichloromethane (30 mL) and trifluoroacetic acid (10 mL) at 23°C. The reaction mixture was stirred for 12 h at 23 °C under ambient atmosphere. The product mixture was concentrated to provide 2-fluoropropylamine trifluoroacetic acid as an opaque oil with excess equivalents of trifluoroacetic acid. The unpurified product obtained in this way (nominally 6 mmol) was dissolved in tetrahydrofuran (10 mL) to generate a working nominal 0.6 M solution for future reactions. A solution of 2-fluoropropylamine trifluoroacetic acid in tetrahydrofuran (4.40 mL, 2.64 mmol, 1.05 equiv) and triethylamine (1.40 mL, 10 mmol, 4.00 equiv) were added sequentially dropwise via syringe to a suspension of the diazonium S7 (343 mg, 2.50 mmol, 1 equiv) in tetrahydrofuran (15 mL) at 23°C. The reaction mixture was stirred for 6 h at 23 °C. The precipitate that formed was collected by vacuum filtration. The precipitate was washed sequentially with ethyl acetate (2 × 15 mL) and diethyl ether (2 × 15 mL). The washed precipitate was dried in vacuo to afford the imidazolyl triazene 10 as a light tan powder (365 mg, 68%, based on the diazonium S7). 1H NMR (600 MHz, DMSO-d6) δ 12.65 (s, 1H, NH), 10.92 (s, 1H, H8), 7.54 (s, 1H, H2), 7.45 (br s, 1H, NH), 7.21 (s, 1H, NH), 4.98 (br d, J = 49 Hz, 1H, H8b), 3.87 – 3.55 (m, 2H, H8a), 1.34 (dd, J = 23.9, 6.3 Hz, 3H, H8c). 13C NMR (151 MHz, DMSO-d6) δ 161.0 (Cquat.), 149.5 – 148.9 (br s, Cquat.)a, 135.6 (C2), 115.9 (Cquat.), 87.1 (d, J = 166.6 Hz, C8b), 48.1 (d, J = 22.3 Hz, C8a), 18.8 (d, J = 21.7 Hz, C8c). 19F NMR (376 MHz, DMSO-d6) δ –174.43 (dq, J = 47.7, 23.9 Hz). IR (ATR-FTIR), cm–1: 3480 (w), 3249 (m), 3077 (m), 1638 (s), 1590 (s), 1427 (s), 1397 (s). HRMS-ESI (m/z): [M + H]+ calcd for [C7H12FN6O]+ 215.1052, found 215.1048. Note: aBroad peak tentatively attributed to a quaternary carbon from the imidazolyl triazene 10. Synthesis of imidazolyl triazene 11:
Figure imgf000068_0001
N,N-Di-iso-propyl ethylamine (834 µL, 4.55 mmol, 1.25 equiv) was added dropwise via syringe to a mixture of (3-fluoropropyl)amine hydrochloride (410 mg, 3.65 mmol, 1 equiv) and the diazonium S7 (500 mg, 3.65 mmol, 1 equiv) in tetrahydrofuran (25 mL) at 23 ºC. The reaction mixture was stirred for 6 h at 23 °C. The precipitate that formed was collected by vacuum filtration. The precipitate was washed sequentially with ethyl acetate (2 × 15 mL) and ether (2 × 15 mL). The washed precipitate was dried in vacuo to afford the imidazolyl triazene 11 as a light tan powder (251 mg, 32%). 1H NMR (400 MHz, DMSO-d6) δ 12.63 (s, 1H, NH), 10.72 (s, 1H, H8), 7.54 (s, 1H, H2), 7.46 (s, 1H, NH), 7.28 (s, 1H, NH), 4.54 (dt, J = 47.3, 5.8 Hz, 2H, H8c), 3.60 – 3.50 (m, 2H, H8b), 2.08 – 1.94 (m, 2H, H8af). 13C NMR (151 MHz, DMSO-d6) δ 161.3 (Cquat.), 155.4 (Cquat.), 149.7 (Cquat.), 135.9 (C2)a, 81.9 (d, J = 161.4 Hz, C8c), 39.6 (C8b)b, 26.7 (d, J = 19.9 Hz, C8a). 19F NMR (376 MHz, DMSO-d6) δ –219.23 (tt, J = 47.1, 26.1 Hz). IR (ATR-FTIR), cm–1: 3483 (w), 3269 (m), 3082 (m), 1640 (m), 1587 (m), 1392 (m). HRMS-ESI (m/z): [M + Na]+ calcd for [C7H11FN6NaO]+ 237.0871, found 237.0986. Notes: aSignal not observed in 1-D 13C NMR spectrum; shift obtained from weak correlation in HSQC spectrum. bSignal obscured by solvent peak in 1-D 13C NMR spectrum; shift obtained from correlation in 1H-13C HSQC spectrum. Notes: aSignal not observed in 1-D 13C NMR spectrum; shift obtained from weak correlation in 1H-13C HSQC spectrum. bSignal obscured by solvent peak in 1-D 13C NMR spectrum; shift obtained from correlation in 1H-13C HSQC spectrum. Example 2: Single crystals of KL-50 (4a) suitable for X-ray analysis were obtained by vapor diffusion of dry benzene (3 mL, precipitating solvent) into a syringe filtered (Millipore Sigma, 0.22 µm, hydrophilic polyvinylidene fluoride, 33 mm, gamma sterilized, catalogue number SLGV033RS) solution of KL-50 (4a) (3.6 mg) in dry dichloromethane (3 mL, solubilizing solvent) at 23 ºC. This yielded two polymorphs of KL-50 (4a) designated Polymorph I (P21/n space group, CCDC number 2122008) and Polymorph II (Cc space group, CCDC number 2122009). Experimental procedure for Polymorph I of KL-50 (4a): Low-temperature diffraction data (ω-scans) were collected on a Rigaku MicroMax- 007HF diffractometer coupled to a Dectris Pilatus3R detector with Mo Kα (λ = 0.71073 Å) for the structure of 007c-21083. The diffraction images were processed and scaled using Rigaku Oxford Diffraction software (CrysAlisPro; Rigaku OD: The Woodlands, TX, 2015). The structure was solved with SHELXT and was refined against F2 on all data by full-matrix least squares with SHELXL (Sheldrick, G. M. Acta Cryst.2008, A64, 112–122). All non- hydrogen atoms were refined anisotropically. Hydrogen atoms were included in the model at geometrically calculated positions and refined using a riding model. The isotropic displacement parameters of all hydrogen atoms were fixed to 1.2 times the U value of the atoms to which they are linked (1.5 times for methyl groups). Table 2. Crystal data and structure refinement for Polymorph I of KL-50 (4a):
Figure imgf000069_0001
Figure imgf000070_0001
Example 3: Experimental procedure for Polymorph II of KL-50 (4a): Low-temperature diffraction data (ω-scans) were collected on a Rigaku MicroMax- 007HF diffractometer coupled to a Saturn994+ CCD detector with Cu Kα (λ = 1.54178 Å) for the structure of 007b-21124. The diffraction images were processed and scaled using Rigaku Oxford Diffraction software (CrysAlisPro; Rigaku OD: The Woodlands, TX, 2015). The structure was solved with SHELXT and was refined against F2 on all data by full-matrix least squares with SHELXL (Sheldrick, G. M. Acta Cryst.2008, A64, 112–122). All non- hydrogen atoms were refined anisotropically. Hydrogen atoms were included in the model at geometrically calculated positions and refined using a riding model. The isotropic displacement parameters of all hydrogen atoms were fixed to 1.2 times the U value of the atoms to which they are linked (1.5 times for methyl groups). Table 3. Crystal data and structure refinement for Polymorph II of KL-50 (4a):
Figure imgf000071_0001
Figure imgf000072_0001
Example 4: Imidazotetrazine 4a (aka KL-50) and the triazene 4b (aka KL-85) were synthesized as vehicles to deliver 2-fluoroethyl diazonium (4c), and a series of related agents to probe structure–activity relationships in tissue culture (FIG.7D and 7F). The 2-fluoropropyl- and 3-fluoropropyl-triazenes (10 and 11, respectively) were prepared by diazotization of 4- aminoimdazole-5-carboxamide, followed by the addition of the respective amine. All other triazenes were prepared according to literature procedures. The cytotoxicity of compounds of formula (I) was evaluated in short-term cell viability assays against four isogenic LN229 glioblastoma cell lines engineered to be proficient or deficient in MGMT and/or MMR activity, using short hairpin RNAs (shRNAs) targeting MSH2 (referred to as MGMT+/–, MMR+/– cells hereafter; FIG. S2A). This approach allowed for rapid and rigorous interrogation of the relationship between MGMT and MMR status and compound activity. Example 5: The IC50 values of these agents are shown in Table 1 (FIG.2A) and representative dose–response curves are shown in FIG.2B. KL-85 (4b) retained potency in MGMT– /MMR– cells (IC50 = 27.5 μM), while TMZ (1a) was essentially inactive (IC50 = 837.7 μM). Structure–activity studies were consistent with the mechanistic pathway shown in FIG.7E. The 2,2-difluoroethyl triazene 9 and the 2-fluoropropyl triazene 10 possessed reduced potency in MGMT–/MMR– cells, in agreement with the reduced rates of displacement following introduction of an additional fluorine or alkyl substituent. The 2-chloroethyl triazene 12b was modestly potent but not as selective for MGMT– cell lines which likely derives from faster, non-selective ICL formation arising from chloride displacement (vide infra). The 3-fluoropropyl triazene 11 demonstrated low activity in all four cell lines, presumably due to inefficient transfer of the electrophile to DNA. The ethyl triazene 13 also demonstrated low activity. This compound may undergo rapid elimination to ethylene gas following conversion to ethyl diazonium. KL-50 (4a) was prepared by diazotization of 4-aminoimidazole-5-carboxamide followed by the addition of (2-fluoroethyl)isocyanate (39% overall yield, see the Supplementary Information). The potency of 4a mirrored that of 4b in the four cell lines examined (FIG.2B). To benchmark selectivity, evaluation of the experimental agent mitozolomide (MTZ, 12a) and the clinical nitrosourea lomustine (aka CCNU, 14) was undertaken, which have been studied with hopes of addressing TMZ (1a) resistance. However, these agents were only ~4–7-fold selective for MGMT-deficient cells, as opposed to the ~25-fold selectivity seen with KL-50 (4a). Example 6: The antitumor activity of KL-50 (4a) was validated in clonogenic survival assays (CSAs) and additional cell lines in vitro. TMZ (1a) possessed negligible activity in MGMT+ LN229 cells, irrespective of MMR status, and induced robust tumor cell killing in MGMT–, MMR+ cells that was abolished in isogenic cells lacking MMR (FIG.2C). Lomustine (14) was effective in MMR– cells but was cytotoxic to MGMT+ cells. In contrast, KL-50 (4a) demonstrated robust antitumor activity in MGMT– cells, independent of MMR status, with minimal toxicity to MGMT+ cells at doses up to at least 200 µM (FIG.2D). A similar pattern of activities was observed in several unique cell lines across different tumor types with intrinsic or induced loss of MGMT and/or MMR activity. For example, TMZ (1a) was inactive in DLD1 cells, which possess MGMT but lack functional MMR (MSH6–) with or without induced depletion of MGMT using O6-benzylguanine (O6BG; FIG.2E). In contrast, KL-50 (4a) was toxic to these cells, but only after O6BG-induced MGMT depletion (FIG. 2F). TMZ (1a) was inactive in HCT116 colorectal cancer cells, which lack the MMR protein MLH1, regardless of MGMT levels (FIG.2G). Restoration of MMR activity via complementation with chromosome 3 containing MLH1 resulted in the enhanced sensitivity to TMZ (1a), which was further potentiated by MGMT depletion (FIG.2G). In contrast, KL- 50 (4a) induced selective tumor cell killing specifically in the setting of O6BG-induced MGMT suppression, in both MLH1-deficient and MLH1-complemented cells (FIG.2H). MMR status and O6BG-induced loss of MGMT expression was confirmed by western blot analysis. The activity of KL-50 (4a) was also confirmed in MGMT– LN229 cells engineered to lack expression of other key MMR proteins including MSH6, MLH1, PMS2, and MSH3. Finally, the cytotoxicity of KL-50 (4a) and TMZ (1a) was compared in normal human fibroblast cells and observed no increase in toxicity with KL-50 (4a). These data define KL- 50 (4a) as a first-in-class molecule that overcomes MMR mutation-induced resistance while retaining selectivity for tumor cells lacking MGMT. Example 7: A well-established comet assay adapted for ICL detection was used to determine if ICLs were formed in MGMT– cells treated with KL-50 (4a) (FIG.3, 3A and 3B). In this assay, cells were sequentially exposed to genotoxins and ionizing radiation, and then analyzed by single cell alkaline gel electrophoresis. Attenuation of the IR-induced comet tail is indicative of ICL formation. In the absence of IR, TMZ (1a, 200 μM) and KL-50 (4a, 200 μM) both induced tailing in MGMT–/MMR+ cells, while mitomycin C (MMC, 0.1 or 50 μM) did not. Exposure to 50 μM MMC for 2 h completely abolished the IR-induced comet tail, whereas exposure to 0.1 μM MMC (chosen to be ~10-fold greater than the IC50 for this drug, comparable to 200 μM KL-50 (4a) or TMZ (1a)) for 24 h caused a partial reduction in the IR-induced comet tail. TMZ (1a, 200 μM) did not reduce DNA migration following IR, in agreement with its known function as a monoalkylation agent with no known crosslinking activity. In contrast, KL-50 (4a, 200 μM) reduced the %DNA in the tail to levels similar to those seen for 0.1 μM MMC. A similar pattern of comet tail migration was observed for MMC and KL-50 (4a) in MGMT–/MMR– cells, which supports an MMR-independent crosslinking mechanism. Comparable results were observed in MGMT–/MMR+ cells treated with KL-85 (4b). This assay was carried out at varying time points (2–24 h) to assess the rates of ICL formation in MGMT–/MMR– cells treated with KL-50 (4a), MTZ (12a), or TMZ (1a) (FIG. 3, 3C and 3D). The chloroethyl derivative MTZ (12a) reduced DNA mobility within 2 h, consistent with the cell line selectivities above and literature reports that this agent rapidly forms ICLs by chloride displacement from other sites of alkylation. TMZ (1a) did not induce a statistically significant decrease in DNA migration within 24 h. However, a time- dependent decrease in DNA mobility was observed in cells treated with KL-50 (4a), with the largest difference observed between 8 and 24 h, consistent with the reported half-life of O6FEtG (6; 18.5 h). In the unirradiated samples, KL-50 (4a), MTZ (12a), and TMZ (1a) all induced maximal damage at 2 h, which decreased over time, consistent with progressive DNA repair (FIG.16, 16C and 16D). Analysis of genomic DNA isolated from LN229 MGMT–/MMR+ cells treated with KL-50 (4a, 200 μM) or KL-85 (4b, 200 μM) by denaturing gel electrophoresis demonstrated the presence of crosslinked DNA (FIG.3E). TMZ (1a) and MTIC (1b) showed no evidence of ICL induction. Similarly, linearized pUC19 plasmid DNA treated with KL-50 (4a, 100 μM) also possessed ICLs, with delayed rates of formation relative to 12b (FIG.3F). Collectively, these data support a mechanism of action for KL-50 (4a) involving the slow generation of DNA ICLs in the absence of MGMT. Example 8: Alternative mechanisms of action were investigated implicating nucleotide excision repair (NER), base excision repair (BER), reactive oxygen species (ROS), and DNA duplex destabilization. Short term cell viability assays in isogenic mouse embryonic fibroblasts (MEFs) proficient or deficient in XPA, a common shared NER factor, revealed no differential sensitivity, either with or without O6BG-induced MGMT depletion. N7MeG lesions induced by TMZ (1a) are prone to spontaneous depurination, apurinic (AP) site formation, and single strand breaks (SSBs), which are all known BER substrates. To probe for potential differential induction of BER substrates by KL-50 (4a) compared to TMZ (1a), in vitro supercoiled plasmid DNA assays were performed that measure the formation of AP sites. Similar levels of spontaneous and enzyme-catalyzed SSBs were observed from AP sites with KL-50 (4a) and TMZ (1a), suggesting comparable levels of depurination. Co-treatment with increasing concentrations of the ROS scavenger N-acetylcysteine (NAC) did not rescue cell viability. Melting point analysis did not reveal any notable differences in DNA stability resulting from fluoroethylation compared to methylation. These data suggest that NER status, AP site induction, ROS, and altered DNA stability are peripheral or noncontributory to the effectiveness of KL-50 (4a). The profile of DDR activation was characterized across four isogenic cell lines after treatment with KL-50 (4a) or TMZ (1a). The prior finding that the ATR–CHK1 signaling axis is activated in response to TMZ (1a)-induced replication stress in MGMT-deficient cells prompted the analysis of the phosphorylation status of CHK1 and CHK2 in LN229 MGMT+/– and MMR+/– cells. KL-50 (4a) induced CHK1 and CHK2 phosphorylation in MGMT– cells regardless of MMR status, whereas TMZ (1a) only induced phospho-CHK1 and -CHK2 in MGMT–/MMR+ cells. Foci formation of the DDR factors phospho-SER139- H2AX ( γH2AX), p53 binding protein 1 (53BP1), and phospho-SER33-RPA2 (pRPA) were analyzed over the period of 2 to 48 h (FIG.4, 4A to 4D. KL-50 (4a) induced a maximal foci response at 48 h, specifically in MGMT– cells and irrespective of MMR status (4a). TMZ (1a) induced a comparable response in MGMT– cells, but this was abolished in the absence of functional MMR, consistent with known MMR-silencing-based resistance. A reduced level of foci formation was observed in MGMT+/MMR+ cells that was absent in MGMT+/MMR– cells, suggesting an MMR-dependent DNA damage response in these cells. However, these foci dissipate at later timepoints (72–96 h), and they are not associated with appreciable cellular toxicity (as shown earlier in FIG.2, 2C and 2D). KL-50 (4a) induced increasing G2 arrest on progression from 24 to 48 h in MGMT– /MMR+ cells, as determined by simultaneous analysis of DNA content based on nuclear (Hoechst) staining in the foci studies above (FIG.4E). KL-50 (4a) induced an attenuated G2 arrest in MGMT–/MMR– cells, consistent with a role of MMR in the G2-checkpoint. This effect in MGMT–/MMR– cells was absent following TMZ (1a) treatment. Both TMZ (1a) and KL-50 (4a) induced a moderate G2 arrest in MGMT+/MMR+ cells. The levels of DDR foci were quantified across the individual cell cycle phases (FIG. 20A-20E). KL-50 (4a) induced foci formation primarily in the S- and G2-phases of the cell cycle, which is consistent with replication blocking by ICLs. Foci increased in MGMT– G1 cells at 48 h, suggesting that a fraction of cells may progress through S-phase with unrepaired DNA damage. Consistent with this, a significant increase in micronuclei was observed at 48 h following KL-50 (4a) treatment, which was greatest in the MGMT–/MMR– cells (FIG.4F). TMZ (1a) displayed a similar pattern of foci induction in the S- and G2-phases, with smaller increases in G1-phase foci and micronuclei formation at 48 h in MGMT–/MMR+ cells. In contrast, no foci induction or micronuclei formation was observed in MGMT–/MMR– cells exposed to TMZ (1a). These findings are in agreement with the differential toxicity profiles of KL-50 (4a) and TMZ (1a): KL-50 (4a) induces multiple successive markers of DNA damage and engagement of the DDR in MGMT– cells, independent of MMR status, whereas the effects of TMZ (1a) are similar in MGMT–/MMR+ cells but absent in MMR– cells. Coupled with the ICL kinetics data presented above, these time-course data support a slow rate of ICL induction in situ by KL-50 (4a). These foci data suggest that KL-50 (4a) induces replication stress (e.g., pRPA foci formation) and DSB formation (e.g., γH2AX and 53BP1 foci, which are known to follow the formation of ICLs). Consistent with this, BRCA2- and FANCD2-deficient cells are hypersensitive to KL-50 (4a; FIG.4, 4G to 4I). In two MGMT-proficient cell models, BRCA2 loss enhanced the toxicity of KL-50 (4a) following MGMT depletion via O6BG (FIG.4, 4H and 4I). FANCD2 ubiquitination was observed by KL-50 (4a) specifically in MGMT– cells, suggesting activation of the Fanconi anemia (FA) ICL repair pathway. As previously reported, TMZ (1a) also induced FANCD2 ubiquitination but only in MGMT– /MMR+ cells. The activity of KL-50 (4a) and TMZ (1a) was evaluated in vivo using murine flank tumor models derived from the isogenic LN229 MGMT– cell lines. MGMT–/MMR+ and MGMT–/MMR– flank tumors were treated with KL-50 (4a) or TMZ (1a) (5 mg/kg MWF × 3 weeks) as previously described for TMZ (1a). TMZ (1a) suppressed tumor growth in the MGMT–/MMR+ tumors (FIG.5A). KL-50 (4a) was statistically non-inferior to TMZ (1a), despite a 17% lower molar dosage owing to its higher molecular weight. In the MGMT– /MMR– tumors, TMZ (1a) demonstrated no efficacy, while KL-50 (4a) potently suppressed tumor growth (FIG.5B). KL-50 (4a) treatment resulted in no significant changes in body weight compared to TMZ (1a) or control (FIG.5C). Representative Kaplan–Meier survival curves are shown in FIG.5D with a greater than 5-week increase in median OS for KL-50 (4a) vs TMZ (1a). KL-50 (4a) was effective and non-toxic using different dosing regimens (5 mg/kg, 15 mg/kg, 25 mg/kg), treatment schedules (MWF × 3 weeks, M–F × 1 week), and routes of drug administration (PO, IP) in mice bearing MGMT–/MMR+ and MGMT– /MMR– flank tumors (FIG.5E). KL-50 (4a; 25 mg/kg PO MWF × 3 weeks) potently suppressed the growth of large (~350-400 mm3) MGMT–/MMR+ and MGMT–/MSH6– tumors (FIG.5F). KL-50 (4a; 25 mg/kg IP M–F × 1 week) was also effective in an orthotropic, intracranial LN229 MGMT–/MMR– model, whereas TMZ (1a) only transiently suppressed tumor growth (FIG.6A). A focused maximum tolerated dose study revealed KL-50 (4a) is well-tolerated. Healthy mice were treated with escalating doses of KL-50 (4a) (0, 25, 50, 100, and 200 mg/kg × 1 dose), and monitored over time for changes in both weights and hematologic profiles. Mice in the higher dosage groups (100 or 200 mg/kg) experienced a greater than 10% weight loss after treatment administration, which regressed to baseline at the end of one week (FIG.6B). Two of three mice in the 200 mg/kg treatment group became observably ill warranting euthanasia, but no evidence of toxicity was observed in the remaining cohorts. As the main dose limiting systemic toxicity of TMZ (1a) is myelosuppression, complete blood counts for all mice were measured on day 0 before treatment and subsequently on day 7 after drug administration. Overall, neutrophils and lymphocytes experienced the most significant drops in cell count, although all blood counts were within normal physiological ranges (defined as values falling within 2 SDs of the average for healthy mice) for all cohorts (FIG. 6C). Taken together, these data demonstrate the robust in vivo efficacy, systemic tolerability, and CNS penetrance of KL-50 (4a). Example 9: Herein, the discovery of agents for the eradication of drug-resistant glioma in vitro and in vivo is described. The success of these agents arises from two factors. First, following on the seminal clinical studies of Stupp and co-workers, who established MGMT expression as a predictive biomarker for TMZ (1a) treatment, MGMT silencing (which occurs in ~50% of GBMs and ~70% of grade II/III gliomas) was leveraged to obtain tumor cell selectivity. Second, and in a departure from prior studies, bifunctional agents that are specifically designed were utilized to evolve slowly to ICLs following transfer to O6G, thereby establishing an MMR-independent method to amplify the therapeutic impact of MGMT silencing. This strategy has led to a new class of agents for treatment of MGMT– glioma independent of MMR status. MMR mutation-induced alkylator resistance has been a major barrier to treatment efficacy, likely since the introduction of TMZ (1a) into glioma treatment regimens in the early 1990s. Bifunctional alkylation agents, such as lomustine (14) and MTZ (12a), have been tested with the hopes of overcoming TMZ (1a) resistance over the last ~30 years, but these agents lack a therapeutic index owing to their activity in MGMT+ (normal tissue) cells. Literature data supports the notion that the remarkable cell line selectivity of KL-50 (4a) derives strictly from the poor leaving group ability of fluoride. While the aliphatic C–F bond is strong (~109 kcal/mol) and not normally susceptible to cleavage by bimolecular nucleophilic displacement, the appropriate positioning of hydrogen bond donors or covalently attached nucleophiles can promote substitution. The half-lives of O6-(2- fluoroethyl)guanosine (S1) and O6-(2-chloroethyl)guanosine (S4) are ~18.5 h and ~ 18 min, respectively, at 37 °C and pH 7.4. Intramolecular halide displacement gives the common intermediate N1,O6-ethanoguanosine (S2) which undergoes ring opening attack by water to yield N1-(2-hydroxyethyl)guanosine (S3). By comparison, attempts to hydrolyze N7-(2- fluoroethyl)guanosine (S5) to N7-(2-hydroxyethyl)guanosine (S6) in aqueous buffer (pH 7) at 37 °C were reportedly unsuccessful, likely due to an inability to form a similar cationic cyclized intermediate. Thus, while O6FEtG (5) lesions likely only constitute a small fraction of alkylation products derived from KL-50 (4a), without being bound by theory, it is hypothesized that the more prevalent sites of alkylation, such as N7G, do not form ICLs at a significant rate, and are readily resolved. The intramolecular displacement pathway (5 ^6) provides an essential acceleration in the formation of ICLs by KL-50 (4a) to biologically relevant timescales and enables an ample kinetic window for MGMT-mediated repair of the primary O6FEtG (5) adduct. These data also provide an explanation for the failure of related cross-linking agents to display the therapeutic index that underpins TMZ’s (1a) success. 2-Chloroethyl nitrosoureas (e.g., lomustine, 14) or 2-chloroethylimidazotetrazines (e.g., MTZ, 12a) are known to form ICLs such as 8 by a pathway analogous to KL-50 (4a, see FIG.7E). However, they can also generate ICLs via direct chloride displacement from 2-chlorethyl adducts present at other sites of DNA alkylation, which degrades the therapeutic index of these compounds. Consistent with this, the time-course analysis established a faster onset of ICLs for MTZ (12a) than KL-50 (4a), and, in turn, explains their differential MGMT selectivity (~7-fold and ~25-fold for 12a and 4a, respectively). An order-of-estimate calculation provides insight into the number of ICLs necessarily generated by KL-50 (4a) to induce toxicity. It has been reported that the mean lethal dose of ICLs in HeLa cells is 230 and TMZ (1a) has been demonstrated to yield 20,600 O6MeG (3) adducts per cell at a dose of 20 µM. Assuming a similar level of O6FEtG (5) lesions are induced by KL-50 (4a) at the IC50 (~20 μM) in MGMT–/MMR– LN229 cells, the number of adducts required to convert to ICLs to generate the mean lethal dose is ~1 in 90, or ~1.1% cross-linking efficiency. Extensive characterization of KL-50 (4a) versus TMZ (1a) activity was performed in vitro to support the hypothesis that MGMT– cells can be selectively targeted independent of MMR status. While MGMT–/MMR– cells display no signs of DNA damage or DNA repair signaling in response to TMZ (1a), we found robust, MMR-independent, activation of DNA damage checkpoint signaling, DNA repair foci formation, cell cycle arrest, and micronuclei formation following KL-50 (4a) treatment. Moreover, KL-50 (4a) retained its effectiveness in vivo in MMR-deficient flank and intracranial tumor models resistant to TMZ (1a) as well as in large MSH6-deficient tumors, a commonly lost MMR component reported in glioma patients. Beyond MGMT-silenced recurrent glioma, it is anticipated that other potential beneficial indications for selective targeting of cancer cells with KL-50 (4a). MGMT silencing has been reported in 40% of colorectal cancers and 25% of non-small cell lung cancer, lymphoma, and head & neck cancers. MGMT mRNA expression is also reduced in subsets of additional cancer types, including breast carcinoma, bladder cancer, and leukemia. MMR loss, as reported by microsatellite instability, is a well-established phenomenon in multiple cancer types and leads to resistance to various standard of care agents. It therefore stands to reason that there are likely other subsets of MGMT–/MMR– tumors in both initial and recurrent settings that would be ideal targets for KL-50 (4a). The data also suggest KL-50 (4a) will display a higher therapeutic index in tumors with MGMT deficiency and impaired ICL repair, including HR deficiency. Specifically, it was demonstrated that FANCD2- and BRCA2-deficient cells are hypersensitive to KL-50 (4a), particularly in the setting of MGMT depletion. Remarkably, the therapeutic index (TI) of KL-50 (4a) in the DLD1 isogenic model, as measured by the ratio of IC50 values in MGMT+/BRCA2+ cells compared to MGMT–/BRCA2– cells, was ~600-fold, vastly larger than canonical crosslinking agents such as cisplatin (42-fold) or MMC (26-fold). A similar amplification of the TI was seen in the PEO1/4 model with KL-50 (4a) (62-fold) vs. cisplatin (13-fold) or MMC (7-fold). HR-related gene mutations have been detected in a substantial number of tumors across multiple cancer types (17.4% in 21 cancer lineages) and methods have been developed to assess for tumor-associated HR deficiency. Thus, in the modern era of molecular precision medicine, the biomarker-guided use of KL-50 (4a) in individual cancers could result in therapeutic indices and exquisite tumor sensitivities previously only observed with synthetic lethal interactions targeting DNA repair proteins. In various embodiments, combinations of KL-50 (4a) with DNA repair inhibitors such as checkpoint kinase inhibitors (e.g., PD-1 and PD-L1) or other immunotherapeutic agents in the setting of MMR mutations are contemplated. The findings described herein may have significant clinical implications for patients with recurrent MGMT-methylated glioma, of which up to half acquire TMZ (1a) resistance via loss of MMR. As demonstrated by the analysis of related TMZ (1a) derivatives, KL-50 (4a) is uniquely designed to fill this therapeutic void. More broadly, incorporating the rates of DNA modification and DNA repair pathways in therapeutic design strategies may lead to the development of additional selective chemotherapies. Enumerated Embodiments The following exemplary embodiments are provided, the numbering of which is not to be construed as designating levels of importance: Embodiment 1: A compound of formula (I) or a pharmaceutically acceptable salt thereof,
Figure imgf000081_0001
wherein: R1 is selected from the group consisting of:
Figure imgf000081_0002
R2 is H or CH3; R3 is selected from the group consisting of H, CH3, CH(CH3)2, CH2CF3,
Figure imgf000081_0003
, and
Figure imgf000081_0004
R2 and R3 are taken together and linked by –(CH2)5– to form a heterocyclic ring; R4 is selected from the group consisting of -C(=O)CH2CH3,
Figure imgf000081_0005
, , ,
Figure imgf000081_0006
provided that R2 is CH3 only if R3 is CH3. Embodiment 2: The compound of Embodiment 1, which is selected from the group consisting of:
Figure imgf000081_0007
Figure imgf000082_0001
or a pharmaceutically-acceptable salt thereof. Embodiment 3: The compound of any one of Embodiments 1-2, which is:
Figure imgf000082_0002
, or a pharmaceutically-acceptable salt thereof. Embodiment 4: A method for treating or ameliorating cancer in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a compound of formula (I), or a pharmaceutically-acceptable salt thereof:
Figure imgf000083_0002
wherein: R1 is selected from the group consisting of
Figure imgf000083_0003
Figure imgf000083_0004
R2 is H or CH3; R3 is selected from the group consisting of H, CH3, CH(CH3)2, CH2CF3, and
Figure imgf000083_0005
Figure imgf000083_0007
, or R2 and R3 may be taken together to provide –(CH2)5-; and R4 is selected from the group consisting of -C(=O)CH2CH3,
Figure imgf000083_0006
, ,
Figure imgf000083_0001
provided that R2 is CH3 only if R3 is CH3. Embodiment 5: The method of Embodiment 4, wherein the cancer is MGMT deficient. Embodiment 6: The method of any one of Embodiments 4-5, wherein the cancer is MMR deficient. Embodiment 7: The method of any one of Embodiments 4-6, wherein the cancer is liver cancer. Embodiment 8: The method of any one of Embodiments 4-7, wherein the cancer is hepatocellular carcinoma. Embodiment 9: The method of any one of Embodiments 4-8, wherein the compound is selected from the group consisting of:
Figure imgf000084_0001
or a pharmaceutically-acceptable salt thereof. Embodiment 10: The method of any one of Embodiments 4-9, wherein the compound is
Figure imgf000085_0001
, or a pharmaceutically-acceptable salt thereof. Embodiment 11: The method of any one of Embodiments 4-10, wherein the cancer is O6-methylguanine-DNA-methyltransferase (MGMT) deficient. Embodiment 12: The method of any one of Embodiments 4-11, wherein the cancer is also mismatch repair (MMR) deficient. Embodiment 13: The method of any one of Embodiments 4-12, wherein the cancer is O6-methylguanine-DNA-methyltransferase (MGMT) deficient. Embodiment 14: The method of any one of Embodiments 4-13, wherein the cancer is also mismatch repair (MMR) deficient. Embodiment 15: A pharmaceutical composition comprising a pharmaceutically- acceptable carrier and a compound of formula (I) or a pharmaceutically acceptable salt thereof:
Figure imgf000085_0004
wherein: R1 is selected from the group consisting of
Figure imgf000085_0005
Figure imgf000085_0006
R2 is H or CH3; R3 is selected from the group consisting of H, CH3, CH(CH3)2, CH2CF3,
Figure imgf000085_0002
Figure imgf000085_0003
R2 and R3 are taken together and linked by –(CH2)5– to form a heterocyclic ring; and R4 is selected from the group consisting of C(=O)CH2CH3,
Figure imgf000086_0001
, , ,
Figure imgf000086_0002
provided that R2 is CH3 only if R3 is CH3. Embodiment 16: The pharmaceutical composition of Embodiment 15, wherein the compound is selected from the group consisting of:
Figure imgf000086_0003
Figure imgf000087_0001
or a pharmaceutically-acceptable salt thereof. Embodiment 17: The pharmaceutical composition of claim any one of Embodiments 15-16, wherein the compound is
Figure imgf000087_0002
, or a pharmaceutically-acceptable salt thereof. The terms and expressions employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the embodiments of the present application. Thus, it should be understood that although the present application describes specific embodiments and optional features, modification and variation of the compositions, methods, and concepts herein disclosed may be resorted to by those of ordinary skill in the art, and that such modifications and variations are considered to be within the scope of embodiments of the present application.
CLAIMS What is claimed is: 1. A compound of formula (I) or a pharmaceutically acceptable salt thereof,
Figure imgf000088_0001
wherein: R1 is selected from the group consisting of:
Figure imgf000088_0002
R2 is H or CH3; R3 is selected from the group consisting of H, CH3, CH(CH3)2, CH2CF3,
Figure imgf000088_0003
R2 and R3 are taken together and linked by –(CH2)5– to form a heterocyclic ring; R4 is selected from the group consisting of -C(=O)CH2CH3,
Figure imgf000088_0004
, ,
Figure imgf000088_0005
provided that R2 is CH3 only if R3 is CH3. 2. The compound of claim 1, which is selected from the group consisting of:
Figure imgf000089_0001
or a pharmaceutically-acceptable salt thereof. 3. The compound of claim 2, which is:

Claims

Figure imgf000090_0001
, or a pharmaceutically-acceptable salt thereof. 4. A method for treating or ameliorating cancer in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a compound of formula (I), or a pharmaceutically-acceptable salt thereof:
Figure imgf000090_0002
R2 is H or CH3; R3 is selected from the group consisting of H, CH3, CH(CH3)2, CH2CF3, , or R2 and R3 may be taken together to provide –(CH2)5-; and
Figure imgf000090_0005
R4 is selected from the group consisting of -C(=O)CH2CH3,
Figure imgf000090_0003
Figure imgf000090_0004
provided that R2 is CH3 only if R3 is CH3. 5. The method of claim 4, wherein the cancer is MGMT deficient.
6. The method of claim 5, wherein the cancer is MMR deficient. 7. The method of claim 4, wherein the cancer is liver cancer. 8. The method of claim 4, wherein the cancer is hepatocellular carcinoma. 9. The method of claim 4, wherein the compound is selected from the group consisting of:
Figure imgf000091_0001
Figure imgf000092_0001
or a pharmaceutically-acceptable salt thereof. 10. The method of claim 9, wherein the compound is
Figure imgf000092_0002
, or a pharmaceutically-acceptable salt thereof. 11. The method of claim 9, wherein the cancer is O6-methylguanine-DNA- methyltransferase (MGMT) deficient. 12. The method of claim 11, wherein the cancer is also mismatch repair (MMR) deficient. 13. The method of claim 10, wherein the cancer is O6-methylguanine-DNA- methyltransferase (MGMT) deficient. 14. The method of claim 10, wherein the cancer is also mismatch repair (MMR) deficient. 15. A pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically-acceptable carrier
Figure imgf000092_0003
wherein: R1 is selected from the group consisting
Figure imgf000093_0001
Figure imgf000093_0002
R2 is H or CH3; R3 is selected from the group consisting of H, CH3, CH(CH3)2, CH2CF3,
Figure imgf000093_0003
R2 and R3 are taken together and linked by –(CH2)5– to form a heterocyclic ring; and R4 is selected from the group consisting of C(=O)CH2CH3,
Figure imgf000093_0004
, ,
Figure imgf000093_0005
provided that R2 is CH3 only if R3 is CH3. 16. The pharmaceutical composition of claim 15, wherein the compound is selected from the group consisting of:
Figure imgf000093_0006
Figure imgf000094_0001
or a pharmaceutically-acceptable salt thereof. 17. The pharmaceutical composition of claim 16, wherein the compound is
Figure imgf000094_0002
, or a pharmaceutically-acceptable salt thereof.
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