WO2021127456A1 - Methods of inhibiting epidermal growth factor receptor proteins - Google Patents

Methods of inhibiting epidermal growth factor receptor proteins Download PDF

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Publication number
WO2021127456A1
WO2021127456A1 PCT/US2020/066040 US2020066040W WO2021127456A1 WO 2021127456 A1 WO2021127456 A1 WO 2021127456A1 US 2020066040 W US2020066040 W US 2020066040W WO 2021127456 A1 WO2021127456 A1 WO 2021127456A1
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growth factor
epidermal growth
factor receptor
cancer
drug
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PCT/US2020/066040
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French (fr)
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Allan S. Wagman
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Rain Therapeutics Inc.
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Publication of WO2021127456A1 publication Critical patent/WO2021127456A1/en

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/545—Heterocyclic compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/70—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings condensed with carbocyclic rings or ring systems
    • C07D239/72—Quinazolines; Hydrogenated quinazolines
    • C07D239/86—Quinazolines; Hydrogenated quinazolines with hetero atoms directly attached in position 4
    • C07D239/94—Nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/10—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D498/10—Spiro-condensed systems

Definitions

  • the present disclosure relates to prodrugs of pan-HER, HER2, HER4, and/or EGFR (HER1) inhibitory agents.
  • the prodrugs can be covalently bonded to attenuating moieties such as reductive triggers (e.g., chemical moieties that can be cleaved in vivo).
  • the prodrugs are useful in methods of treatment of the human or animal body.
  • kinase inhibitors play a central role in the regulation of wide variety' of cellular processes which has led to the development of kinase inhibitors as therapeutic agents in the treatment of a wide range of disorders, including cancer. Treating cancer is challenging because it is difficult to kill cancer ceils while not affecting, or affecting to a lesser extent, normal cells. Killing or otherwise detrimentally affecting normal cells during cancer treatment can cause adverse side effects In patients.
  • EGFR TKIs such erlotimb, gefitimb and afatinib have been approved for the treatment of several cancers including non-small cell lung cancer (see e.g Felip etal Clinical Cancer Research, 2008, 14: 3867-3874; Smith et al, 2008, Rr. J.
  • cancer cells can differ from certain normal cells in their level of oxygenation and can be more hypoxic than normal cells. Hypoxia can induce adaptations in gene regulation and associated cellular functions, specifically epidermal growth factor receptor (EGFR; HER 1)
  • EGFR epidermal growth factor receptor
  • Tumor hypoxia up-regulates wild type EGFR protein and its cognate ligand TGFa via several HIE dependent mechanisms (Curr Pharm Des 2013; 19:907).
  • TKI tyrosine kinase inhibitors
  • EGFR activating mutant positive non-small cell lung cancer is often heterozygous (Soh J, etal. 2009, PLoS ONE 4(10): e7464; Bai et al, 2013, PLoS ONE 8: e54i 70), and the presence of the wild type allele is associated with limited response to TKI treatment regimens (Taniguchi et al, 2008, Cancer Sci; 99: 929-35).
  • hypoxia -activated prodrugs HAP
  • FIAP EGFR TKI a FIAP EGFR TKI
  • Hypoxia-activated prodrugs (HAP) of EGFR TKI can overcome this mechanism of intrinsic resistance.
  • the present disclosure meets these needs by providing compositions and methods related to the use of the hypoxia activated nitroimidazole prodrug of a kinase inhibitor in therapy.
  • the hypoxia-dependent metabolism provides tumor selective release of the EGFR TKI in hypoxic tumor cells.
  • the present disclosure solves the resistance problem and provides methods for determining subjects who are at risk for resistance to EGFR treatment in patients with EGFR gene polymorphisms.
  • the present disclosure satisfies this and other needs.
  • the high local concentrations provide the necessary therapeutic index to silence wild type EGFR signaling in the hypoxic tumor compartment, as summarized below.
  • the present disclosure relates to pan-HER, HER2, HER4 and EGFR (HER1) small molecule inhibitors incorporating (e.g., covalently bonded to) an attenuating moiety such as a hypoxia-activated trigger moiety' to create prodrugs of the small molecule inhibitors.
  • Compounds of the disclosure include prodrugs of known kinase inhibitors which have potent pan-HER, EGFR (FIERI), and/or HER4 activity'.
  • the inhibitors are bonded to an attenuating moiety, the inherent kinase inhibitory activity' can be greatly attenuated or reduced to a biologically insignificant level by incorporation of an attenuating moiety on an appropriate nucleophilic atom to act as a prodrug.
  • the prodrug-kinase inhibitor conjugate has little or no on-target pharmacological or biochemical activity compared with the parent compound.
  • the potent kinase inhibitor molecule can be released in cancer tumor tissue which is hypoxic and which expresses the metaloreductase 8TEAP4.
  • the prodrug-kinase inhibitor conjugate may have systemic exposure across tissues in an animal or human, it has no or little on-target activity and therefore has greatly reduced systemic pharmacological or toxic effects.
  • the active and potent kinase inhibitor with potent pan- HER, EGFR (HERl), HER2, and/or HER4 activity' can be released in the environment found typically in some solid tumors or cancers in which the cancer is driven by activated HER family receptors.
  • the active kinase inhibitor can be further released in those cancers and cancer tumors which are in hypoxic environments and winch express STEAP4.
  • 8TEAP4 donates an electron to the prodrug or trigger which can cause the prodrug to release the active kinase inhibitor payload locally in the tumor.
  • the reductive activity of STEAP4 on the prodrug or trigger can be negated, greatly reducing or completely stopping release of the active kinase inhibitor outside of the cancer. This can result in targeted tumor concentrations of the active kinase inhibitor, with very low systemic exposure.
  • the compounds of the present disclosure exert anticancer activity with attenuated systemic toxicity and/or improved tolerability, allowing higher drug doses and higher local tumor concentrations of active kinase inhibitor than previously seen by other methods.
  • the compounds described herein address an urgent and unmet medical need in mammals and humans suffering from HER receptor driven cancers. Moreover, these compounds can be administered to treat HER receptor driven cancers in which the ATP-competitive tyrosine kinase inhibitor binding site is wild type (WT).
  • Compounds of the present disclos ure can exert antineoplastic activity in the cancer or tumor tissue, but not in HER receptors in healthy tissue thus reducing toxicity, improving dosing tolerability, and allowing higher concentrations of the active kinase inhibitor in the cancer tissue, thereby inhibiting the proliferation of tumor cells that overexpress these receptors.
  • Many cancers with limited treatment options are driven by activated HER receptors with WT ATP binding sites, such as EXON20 and NRG fusions which active WT EGFR (HER1) and are highlighted in the following reviews, which are incorporated herein in their entireties: Murtuza, A. et. al. Cancer Research (2019), 79(4), 689-698; La Salvia, A. et al.
  • the present disclosure provides, EGFR inhibitors that are covalently bonded to attenuating agents, also referred to as attenuating moieties, such as reductive triggers to form prodrugs of the EGFR inhibitors.
  • the reductive triggers can comprise electrophilic heterocycles and can be cleaved under anoxic conditions to afford the active EGFR inhibitors.
  • the trigger which can be attached to the EGFR inhibitor is selected from those disclosed in WO2010/104406, which is incorporated herein by reference. Also, the prodrugs disclosed in WQ2010/NZ00174, which is incorporated herein by reference.
  • an asterisk marks a nucleophilic atom or group of atoms where an attenuating moiety, such as a reductive trigger, could be installed to form a prodrug.
  • an attenuating moiety such as a reductive trigger
  • the present disclosure also teaches one skilled m the art how to synthesize, prepare, and develop the prodrug and how to select a prodrug-kinase inhibitor conjugate which displays the necessar hypoxia-associated release of the active potent pan-HER, EGFR (FIERI), HER2 and/or HER4 kinase inhibitor.
  • * Denotes an example nucleophilic nitrogen, oxygen or sulfur where a prodrug could be attached; either as positioned or on an available tautomeric position. Examples of attenuating moieties or trigger attachments:
  • a sulfur attachment t rs 99 er wherein A, B, and C comprise the EGFR inhibitor
  • the attenuating moiety or trigger may be attached at any available nitrogen on a small molecule HER inhibitor.
  • the attenuating moiety or trigger may be attached at a nucleophilic nitrogen or a nucleophilic nitrogen which makes an ammonium salt
  • the attenuating moiety or trigger may be attached at an aromatic ring (e.g. to form a cation).
  • the attenuating moiety or trigger may be attached at a nitrogen which is part of an amide bond.
  • the attenuating moiety or trigger may be attached at a dialkyl substituted nitrogen.
  • the attenuating moiety or trigger may be attached at a monoalkyl substituted nitrogen.
  • the attenuating moiety or trigger may be attached at an aromatic heterocyclic ring such as a pyridine, pyrazole or pyrimidine.
  • the attenuating moiety or trigger may be attached at an aromatic heterocyclic ring such as a pyridine, pyrazole or pyrimidine in a position which is nucleophilic or can form a cation.
  • the attenuating moiety or trigger may be attached at any other nucleophilic atom, such as an oxygen or phenol or sulfur or thiophenol.
  • Figure 8 shows median A431 tumour growth after q3dx4 treatment with prodrugs 17, 18, and 21 (n :::: 3).
  • Figure 12 shows inhibition of erbBl (EGFR) autophosphorylation and p44/42 MAPK(Erk 1/2) phosphorylation in intact A431 cells following lhour treatment with a range of concentrations of compound 3.
  • Figure 13 shows inhibition of erbBl (EGFR) autophosphorylation and p44/42 MAPK(Erk 1/2) phosphorylation in intact A431 cells following 1 hour treatment with a range of concentrations of compound 5.
  • Figure 14 shows inhibition of erbBl (EGFR) autophosphorylation and p44/42 MAPK(Erk 1/2) phosphorylation in intact A431 cells following 1 hour treatment with a range of concentrations of compound 6.
  • Figure 15 show's inhibition of erbBl (EGFR) autophosphorylation and p44/42 MAPK(Erk 1/2) phosphorylation in intact A431 cells following lhour treatment with a range of concentrations of prodrug 15.
  • Figure 16 is a graphical plot of band desitometry analysis of relative (b-actin corrected) inhibition of erbBl (EGFR) autophosphorylation in intact A431 cells following lhour treatment with a range of concentrations of prodrug 15 and compound 3.
  • Figure 17 is a CLU-NRGl patient-derived xenograft model.
  • Figure 17A show's high expression of NRG1 m various PDX models.
  • Figure 17B show's a schematic representation for CLU-NRGl fusion.
  • Figure 18 is a plot of the in vivo antitumor activity of tarloxotinib in CLU-NRGl fusion ovarian cancer PDX (OV- 10-0050) cells.
  • Figure 19 is microscopic images of hypoxia and STEAP4 levels in CLU-NRGl PDX models.
  • Figure 19A is images of hypoxia in OV-10-0050 PDX tumors.
  • Figure 19B is STEAP4 in-situ hybridization in OV-10-0050 PDX tumors.
  • Figure 20 shows the structures for the prodrugs TH-302 (also known as evofosfamide), tirapazamme (TPZ or SR4233), and Compound C (also known as PR- 104 A or SN27858), as referred to in Example 5.
  • prodrugs TH-302 also known as evofosfamide
  • tirapazamme TPZ or SR4233
  • Compound C also known as PR- 104 A or SN27858
  • Figure 21 is a graphic showing the layout of the 96-well plate, as described in Example 5
  • Figure 22 shows the ICso values for TH-302, TPZ, and Compound C in wild-type (WT) and STEAP4-expressing cells, where the concentration of prodrug required to inhibit cell growth by 50% (ICso) is shown, as described in Example 5.
  • Figure 23 shows a schematic depicting an exemplary STEAP4-mediated activation of Compound A.
  • the compounds of the present disclosure comprise a kinase inhibitor of Formula 1 and a reductive trigger, described m WO2010/0104406 which contents are incorporated by reference in its entirety.
  • the reductive trigger can fragment when reduced to afford an active EGFR inhibitor. Fragmentation of the trigger occurs at the one-electron reduction level and is effectively suppressed by the presence of oxygen, thus providing selective activation in hypoxic environments. This suppression by oxygen may occur through reoxidation of the one- electron radical by oxygen, or by oxidation of reducing intermediates required for prodrug reduction.
  • the latter would include, for example, scavenging by oxygen of radiation-induced reducing radicals such as the aquated electron, or oxidation of reducing intermediates in the catalytic cycle of reductase enzymes.
  • radiation-induced reducing radicals such as the aquated electron
  • oxidation of reducing intermediates in the catalytic cycle of reductase enzymes may be provided by enzymes, radiation-induced radicals, or chemical reducing agents
  • the trigger is a reductive! y-aetivated aromatic nitroheterocycle or aromatic nitrocarbocycle trigger and is linked directly to a quatermsable nitrogen of a compound of Formula I such that a quaternary nitrogen is formed.
  • the EGFR inhibitor is a compound of Formula (I): wherein either:
  • Rz is (3-chlorobenzyl)oxy- and R? is chloro;
  • Rz is 2-pyridinylmethoxy and Rz is chloro:
  • Rz is chloro and Rz is bromo
  • Rz and Rz are both bromo;
  • R?. is fluoro and R3 is ethynyi;
  • Rz is chloro and R3 is ethynyi
  • R?. is bfomo and R3 is ethynyi
  • R2 is 2-pyridinylmethoxy and R3 is fluoro;
  • Ri, R? and R3 is selected from benzyloxy, 3-chlorobenzyloxy and 2- pyridinylmethoxy and when at least one of Ri, Rz and R? is not benzyloxy, 3- chlorobenzyloxy or 2-pyridinylmethoxy, each of the others is independently selected from H, halogen, and C2-C4 alkynyl, with the proviso that when one of Ri, R2 and Rs is benzyloxy or 2-pyridinylmethoxy, the other two ofRi, R2 and R3 are not H; or
  • an asterisk marks a nucleophilic atom or group of atoms where a hypoxia activated prodrug could he installed.
  • the present disclosure also teaches one skilled in the art how to synthesis, prepare, and develop the prodrug and how to select a prodrug-kinase inhibitor conjugate which displays the necessary hypoxia-associated release of the active potent pan-HER, EGFR (FIERI ), ITER2 and/or HER4 kinase inhibitor.
  • the medicament is a prodrug and the compound of Formula I: wherein either:
  • Ra is fluoro and R is ethynyl
  • Ra is chloro and Ra is ethynyl
  • Ra is bromo and Ra is ethynyl
  • (W)R2 is 2-pyndinylniethoxy and Ra is fluoro;
  • Ra is 2-pyridinylmethoxy and Ra is bromo;
  • the disclosure provides a reductive prodrug comprising a kinase inhibitor of Formula i as defined above, or a salt or solvate thereof, and a reductive trigger linked directly or indirectly to a nitrogen of the kinase inhibitor.
  • a reductive prodrug comprising a kinase inhibitor of Formula i as defined above, or a salt or solvate thereof, and a reductive trigger linked directly or indirectly to a nitrogen of the kinase inhibitor.
  • the attenuating moiety or trigger is Formula II:
  • the reductive trigger moieties of Formula 11 are suitable for use in the prodrugs of the present disclosure.
  • the reductive trigger is selected from the group consisting of Formulae Ila iia !SJ He ik! lie Hi iig
  • Rs is selected from methyl, ethyl and propyl
  • R5 is methyl
  • the reductive trigger is of Formula Ila, wherein Rs is selected from methyl, ethyl and propyl.
  • the reductive trigger is of Formula Ila, wherein Rs is methyl.
  • a-methyl halides of Formula Ila may be prepared as described previously (bromide; Stribbling et al, PCT International patent publication WO 2008/039087) (chloride; Tercel et al, J Med Chem, 2001, 44, 3511-3522).
  • Attenuating moiety or trigger can be selected from one of the following Formulae a-q: wherein:
  • * is the point of attachment to an EGFR inhibitor
  • 26 is selected from the group consisting of H, Ci-Ce alkyl, Ci-Ce alkoxy, C2-C6 alkenyl, C2-C6 alkynyi, (IT OCF3, F, Cl, Br, I, NO2, CN, COOH, COO(Cl-C6 alkyl), CONH2, CONHICi- Ce alkyl), CON(Ci-Ce alkyl)?., CO(Ci-Ce alkyl), SO2NH2, S0?NH(CI-C6 alkyl), 80?N(CI-C 6 alkyl)?, SO?(Ci-Ce alkyl), and a group of Formula ilia, as defined above, wherein * is the point of attachment to an EGFR inhibitor;
  • R?7 IS selected from the group consisting of H andCi-C6 alkyl, as defined above, wherein * is the point of attachment to a group of Formula V;
  • R?s is selected from the group consisting of H and Ci-Ce alkyl.
  • Attenuating moiety or trigger is selected from a group of one of the following Formulas Vr-Vae: y z aa ab ac ad ae
  • Ri is a group of Formula c, wherein R?6 is H and R27 is CFF
  • Ri is a group of Formula d, where R?s is selected from the group consisting of H, Ci-Ce alkyl (e.g., methyl), Ci-Ce alkoxy (e.g., OCH3), Ci-Ce alkynyl (e.g., ethynyl), CONH?, CONHMe, CF3, OCF3, Br, NO?, and CN, and R?7 is selected from the group consisting of CH3, CH?CH?C0N1:I?, and CH2CH2CN.
  • R?s is selected from the group consisting of H, Ci-Ce alkyl (e.g., methyl), Ci-Ce alkoxy (e.g., OCH3), Ci-Ce alkynyl (e.g., ethynyl), CONH?, CONHMe, CF3, OCF3, Br, NO?, and CN, and R?7 is selected from the group consisting of CH3, CH?CH?C0N1:I
  • the attenuating moiety or trigger may also be selected from groups of Formula Vd:
  • R?s is selected from H and C1-C3 alkyl
  • R27 is selected from H and Ci-Cfi alkyl.
  • R?e is H and R?? is Ci-C:? alkyl e.g., methyl.
  • R2.6 is 1-propynyl and R27 is CH3.
  • R2.6 is selected from H, Ci-Cc, alkyl (such as methyl or ethyl) and Ci-Cc, aikoxy (such as OCTI3), and R27 is CTI3.
  • R26 is selected from H, Ci-Ce alkyl (e.g., methyl or ethyl) and Ci- Ci, alkoxy (e.g., OCH3), and R27 is CH3.
  • the attenuating moiety is selected from Formulae Vd ('!) -Vd (/) :
  • R2.7 is selected from methyl, ethyl and propyl.
  • R3.7 is methyl.
  • R2 and R3 may form a ring selected from pyrrolidimum, piperidinium, piperazimum, Ni-rnethylpiperazimum and morpholinium.
  • HER1 refers to a transmembrane protein that is a receptor for members of the epidermal growth factor family.
  • the term “HER!” may also refer to FIERI, ErbB- 1 , and EGFR.
  • HER2 refers to a transmembrane protein that is a receptor for members of the epidermal growth factor family.
  • the term “HER2” ma also refer to ErbB-2, Erbb2, ERBB2, proto-oncogene Neu, and CD340.
  • HER3 refers to a transmembrane protein that is a receptor for members of the epidermal growth factor family.
  • the term “HER3” may also refer to ErbB-3 and ERBB3.
  • HER2 can activate HER3, and therefore in some embodiments inhibition of HER2 can attenuate HER3 activity.
  • HER4 refers to a transmembrane protein that is a receptor for members of the epidermal growth factor family.
  • HER4 may also refer to ErbB-4 and
  • electrophilyte refers to physiologically relevant free ions.
  • Representative such free ions include, but are not limited to sodium (Na + ), potassium ( K K calcium (Ca 2+ ), magnesium (Mg 2 ”), chloride (Cl-), phosphate P04 J ⁇ ), and bicarbonate (HCO3 ).
  • the articles “a” and “an” are used to refer to one or to more than one (f.e., to at least one) of the grammatical object of the article.
  • an element means one element or more than one element.
  • a disease or disorder is “alleviated” if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced.
  • alkyl include both straight chain and branched chain groups, and unsubstituted and substituted groups.
  • the optional substituents may include, without limitation, halogen, Ci-Ce alkoxy, CN, OH, NH2, NO2, NH(Ci- Ce alkyl), N(Ci-Ce alkyi) , CONH2, CO(Ci-G > alkyl), SO2NH2 and S() 2 (Ci-C6 alkyl).
  • aromatic mtroheterocycle means an aromatic heterocyclic moiety substituted at any ring position by one or more nitro (NO2) groups.
  • the aromatic heterocyclic moiety may be a monocyclic or bicyclic ring containing 4 to 12 atoms of which at least one atom is chosen from nitrogen, sulphur or oxygen.
  • the aromatic heterocyclic moiety may be carbon or nitrogen linked.
  • the aromatic heterocyclic moiety may additionally be substituted by one or more additional substituents at any available ring carbon or heteroatom.
  • the substituents may include, but are not limited to the groups as defined for R2.6 in Formula V.
  • aromatic nitrocarbocycle means a benzene moiety substituted at any position by one or more nitro (NO2) groups.
  • two adjacent ring carbon atoms may optionally be linked to form a fused carbocychc or heterocyclic ring.
  • the benzene moiety (and optional fused ring) may additionally be substituted by one or more additional substituents at any available carbon or heteroatom.
  • the substituents may include, but are not limited to, the groups as defined for R26 in Formula V.
  • co-administered and “co-administration” refer to administering to the subject a compound contemplated herein or salt thereof along with a compound that may also treat the disorders or diseases contemplated herein.
  • the co administered compounds are administered separately, or in any kind of combination as part of a single therapeutic approach.
  • the co-administered compound may be formulated in any kind of combinations as mixtures of solids and liquids under a variety of solid, gel, and liquid formulations, and as a solution.
  • composition refers to a mixture of at least one compound contemplated herein with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition facilitates administration of the compound to a patient or subject. Multiple techniques of administering a compound exist m the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, nasal, pulmonary and topical administration.
  • a “disease” as used herein is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.
  • a “disorder” as used herein in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favourable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.
  • the terms “effective amount,” “pharmaceutically effective amount” and “therapeutically effective amount” refer to a nontoxic but sufficient amount of a compound or agent to provide the desired biological result. That result may be reduction and/or alleviation of the signs, symptoms, or causes of a disease, 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.
  • a therapeutic benefit or improvement need not be complete ablation of any one, most or all symptoms, complications, consequences or underlying causes associated with the disorder or disease.
  • a satisfactory endpoint is achieved when there is a transient, medium or long term, incremental improvement in a subject’s condition, or a partial reduction in the occurrence, frequency, severity, progression, or duration, or inhibition or reversal, of one or more associated adverse symptoms or complications or consequences or underlying causes, worsening or progression f e.g., stabilizing one or more symptoms or complications of the condition, disorder or disease), of the disorder or disease, over a duration of time (hours, days, weeks, months, and so forth).
  • “likelihood”, “likely to”, and similar generally refers to an increase in the probability of an event.
  • “likelihood”, “likely to”, and similar, when used m reference to responsiveness to cancer therapy generally contemplates an increased probability that the individual will exhibit a reduction in the severity of cancer or the symptoms of cancer or the retardation or slowing of the cancer progression.
  • the term “likelihood”, “likely to”, and similar, when used in reference to responsiveness to cancer therapy, can also generally mean the increase of indicators that may evidence an increase in cancer treatment.
  • patient refers to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein.
  • the patient is a human.
  • the subject is a subject in need of treatment thereof.
  • 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, and is relatively non-toxic, i.e., the material may be administered to an individual 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 disclosed herein or to the patient 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 disclosed herein or to the patient 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 compounds disclosed herein, and not injurious to the patient.
  • 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; tale; 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; Ring
  • “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 compounds disclosed herein, and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions.
  • the “pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compounds disclosed herein.
  • Other additional ingredients that may be included in the pharmaceutical compositions disclosed herein 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.
  • pharmaceutically acceptable salt refers to a salt of the administered compounds prepared from pharmaceutically acceptable non-toxic acids, including inorganic acids, organic acids, solvates, hydrates, or clathrates thereof.
  • prodrug refers to a compound that, after administration, is metabolised or otherwise converted to a biologically active or more active compound (or drug) with respect to at least one property.
  • a prodrug, relative to the drug is modified chemically in a manner that renders it, relative to the drug, less active or inactive, but the chemical modification is such that the corresponding drug is generated by metabolic or other biological processes after the prodrug is administered.
  • a prodrug may have, relative to the active drug, altered metabolic stability or transport characteristics, fewer side effects or lower toxicity, or improved flavour (for example, see the reference Nogrady, 1985, Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392, incorporated herein by reference).
  • a prodrug may be synthesized using reactants other than the corresponding drug..
  • a “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs.
  • responsiveness refers to the degree of effectiveness of the treatment in lessening or decreasing the symptoms of a disease, disorder, or condition being treated.
  • increased responsiveness when used m reference to a treatment of a cell or a subject, refers to an increase in the effectiveness in lessening or decreasing the symptoms of the disease when measured using any methods known in the art.
  • the increase in the effectiveness is at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%.
  • treatment is defined as the application or administration of a therapeutic agent, i.e., a compound disclosed herein (alone or in combination with another pharmaceutical agent), to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient (e.g, for diagnosis or ex vivo applications), who has a condition contemplated herein, a symptom of a condition contemplated herein or the potential to develop a condition contemplated herein, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect a condition contemplated herein, the symptoms of a condition contemplated herein or the potential to develop a condition contemplated herein.
  • Such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenonucs.
  • the term “treatment” or “treating” refers to an action that occurs while an individual is suffering from the specified cancer, which reduces the severity of the cancer or the symptoms of the cancer, and/or retards or slows the progression of the cancer.
  • “treatment” or “treat” refers to a 5%, 10%, 25%, 50%, or 100% decrease in the rate of progress of a tumour.
  • “treatment” refers to a 5%, 10%, 25%, 50%, or 100% decrease m determined tumour burden (i.e., number of cancerous cells present in the individual, and/or the size of the tumour).
  • treatment refers to a 5%, 10%, 25%, 50%, or 100% decrease in any physical symptom(s) of a cancer. In some embodiments, “treatment” refers to a 5%, 10%, 25%, 50%, or 100% increase in the general health of the individual, as determined by any suitable means, such as cell counts, assay results, or other suitable means.
  • reference to a range of 90-100% includes 91-99%, 92-98%, 93-95%, 91-98%, 91-97%, 91-96%, 91-95%, 91-94%, 91-93%, and so forth.
  • Reference to a range of 90-100% also includes 91%, 92%, 93%, 94%, 95%, 96%, 97%, etc , as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92 1%, 922%, 92.3%, 92.4%, 92.5%, and so forth.
  • a senes of ranges are disclosed throughout this document.
  • ranges include combinations of the upper and lower ranges to provide another range. This construction applies regardless of the breadth of the range and m all contexts throughout this patent document.
  • reference to a series of ranges such as 5-10, 10-20, 20-30, 30-40, 40-50, 50-75, 75-100, 100-150, includes ranges such as 5-20, 5-30, 5-40, 5-50, 5-75, 5-100, 5-150, and 10-30, 10-40, 10-50, 10-75, 10-100, 10-150, and 20-40, 20-50, 20-75, 20-100, 20-150, and so forth. This applies regardless of the breadth of the range.
  • the disclosure provides a reductive prodrug comprising a kinase inhibitor, e.g. an EGFR inhibitor, of Formula I as defined above, or a salt or solvate thereof, and a reductive trigger linked directly or indirectly to a nitrogen of the kinase inhibitor.
  • a kinase inhibitor e.g. an EGFR inhibitor, of Formula I as defined above, or a salt or solvate thereof
  • a reductive trigger linked directly or indirectly to a nitrogen of the kinase inhibitor.
  • the reductive trigger is of Formula ⁇ : Formula II wherein * is a point of attachment to a nitrogen of said kinase inhibitor, and where in Formula II !3 ⁇ 4 is selected from Ci-Cc, alkyl and R4 is selected from H, methyl, ethyl, trifluoromethyl, -CN, - CONH2 and propyn-l-yl.
  • the reductive trigger is selected from the group consisting of Formulae Ila to ilg:
  • Rs is selected from methyl, ethyl and propyl.
  • the reductive trigger is of Formula Ila, wherein Rs is selected from methyl, ethyl and propyl.
  • the reductive trigger is of Formula Ila, wherein R5 is methyl.
  • the EGFR inhibitor bonded to an attenuating moiety comprises AC480 (aka BMS-599626) or a pharmaceutically acceptable salt thereo
  • hypoxia activated prodrug is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoe
  • hypoxia activated prodrug i wherein R 1 is an atenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug wherein R 4 and R 5 are defined above for Formula II.
  • hypoxia activated prodrug i wherein R 4 and R 5 are defined above for Formula II.
  • the EGFR inhibitor bonded to an attenuating moiety comprises AC0010MA (avitinib) or a pharmaceutically acceptable salt thereof.
  • hypoxia activated prodrug i wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • R ! is an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug wherein R 4 and R 5 are defined above for Formula II.
  • hypoxia activated prodrug the hypoxia activated prodrug
  • the EGER inhibitor bonded to an attenuating moiety comprises AEE788 or a pharmaceutically acceptable salt thereof.
  • hypoxia activated prodrug i wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug i and R 5 are defined above for Formula II.
  • the EGFR inhibitor bonded to an attenuating moiety comprises .41,6802 (SIM 6802, Simotmib) or a pharmaceutically acceptable salt thereof.
  • hypoxia activated prodrug wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug i wherein R ! is an attenuating moiety such as a reductive trigger.
  • R 4 and R 5 are defined above for Formula 11.
  • hypoxia activated prodrug the hypoxia activated prodrug
  • the EGFR inhibitor bonded to an attenuating moiety comprises Alflutinib (alflutimb mesylate) or a pharmaceutically acceptable salt thereof.
  • hypoxia activated prodrug i wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • the EGFR inhibitor bonded to an attenuating moiety comprises AiuTibng (bngatimb) or a pharmaceutically acceptable salt thereof.
  • the hypoxia activated prodrug is wherein the * indicates a point of atta chment of an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug the hypoxia activated prodrug
  • hypoxia activated prodrug the hypoxia activated prodrug
  • the EGFR inhibitor bonded to an atenuating moiety' comprises AP23464 or a pharmaceutically acceptable salt thereof.
  • the hypoxia activated prodrug wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • the hypoxia activated prodrug is wherein R 1 is an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug is wherein R 4 and R 3 are defined above for Formula II.
  • the EGFR inhibitor bonded to an attenuating moiety comprises Arava (iefiunomide) or a pharmaceutically acceptable salt thereof.
  • hypoxia activated prodrug wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug wherein R i is an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug wherein R 4 and R 5 are defined above for Formula II.
  • hypoxia activated prodrug wherein R 4 and R 5 are defined above for Formula II.
  • the EGFR inhibitor bonded to an attenuating moiety comprises
  • hypoxia activated prodrug wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug wherein R is an attenuating moiety such as a reductive trigger.
  • the EGFR inhibitor bonded to an atenuating moiety' comprises AV-412 or a pharmaceutically acceptable salt thereof.
  • hypoxia activated prodrug wherein the * indicates a point of attachment of an attenuating moiety such as a reductive tugger.
  • the hypoxia activated prodrug is wherein R f is an atenuating moiety such as a reductive trigger. and R 3 are defined above for Formula II
  • hypoxia activated prodrug [00161] In some embodiments, the hypoxia activated prodrug
  • the EGFR inhibitor bonded to an attenuating moiety comprises AZD3759 or a pharmaceutically acceptable salt thereof.
  • the hypoxia activated prodrug i wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger. bodiments, the hypoxia activated prodrug , wherein R 1 is an attenuating moiety such as a reductive trigger. [00165] In some embodiments, the hypoxia activated prodrug is or wherein R 4 and R 5 are defined above for Formula II. odiments, the hypoxia activated prodrug
  • the EGFR inhibitor bonded to an attenuating moiety comprises AZD8931 or a pharmaceutically acceptable salt thereof.
  • hypoxia activated prodrug is , wherein the
  • hypoxia activated prodrug is wherein R 1 is an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug wherein R 4 and R 5 are defined above for
  • hypoxia activated prodrug the hypoxia activated prodrug
  • the EGFR inhibitor bonded to an attenuating moiety comprises BMS-690514 or a pharmaceutically acceptable salt thereof.
  • the hypoxia activated prodrug wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • R 1 is an atenuating moiety such as a reductive trigger.
  • the hypoxia activated prodrug wherein R 4 and R 5 are defined above for
  • hypoxia activated prodrug the hypoxia activated prodrug
  • the EGFR inhibitor bonded to an attenuating moiety comprises BPI-7711 (Beta Pharma) or a pharmaceutically acceptable salt thereof.
  • hypoxia activated prodrug is BPI-7711 (Beta Pharma) or a pharmaceutically acceptable salt thereof conjugated to an attenuating moiety.
  • the EGFR inhibitor bonded to an atenuating moiety' comprises Canertinib (Cl- 1033) or a pharmaceutically acceptable salt thereof.
  • the hypoxia activated prodrug wherein R 1 is an attenuating moiety such as a reductive trigger is an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug is wherein R’ and R- are defined above for Formula ⁇ .
  • hypoxia activated prodrug the hypoxia activated prodrug
  • the EGFR inhibitor bonded to an atenuating moiety comprises Caprelsa (vandetanih) or a pharmaceutically acceptable salt thereof.
  • the hypoxia activated prodrug i wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger. diments, the hypoxia activated prodrug , wherein R 1 is an attenuating moiety such as a reductive trigger. [00187] In some embodiments, the hypoxia activated prodrug wherein R 4 and R 5 are defined above for Formula P. iments, the hypoxia activated prodrug
  • EGFR inhibitor bonded to an attenuating moiety comprises CK ⁇ 101 (RX518) or a pharmaceutically acceptable salt thereof.
  • the hypoxia activated prodrug is wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • R f is an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug wherein R 4 and R 5 are defined above for Formula II. [00193] In some embodiments, the hypoxia activated prodrug
  • the EGFR inhibitor bonded to an attenuating moiety comprises CP-724,714 or a pharmaceutically acceptable salt thereof.
  • the hypoxia activated prodrug is , wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug is attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug wherein R 4 and R 5 are defined above for Formula II.
  • hypoxia activated prodrug the hypoxia activated prodrug
  • the EGFR inhibitor bonded to an attenuating moiety comprises CUDC-101 or a pharmaceutically acceptable salt thereof.
  • the hypoxia activated prodrug is , wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • the hypoxia activated prodrug is , wherein R 1 is an attenuating moiety such as a reductive trigger.
  • the EGFR inhibitor bonded to an attenuating moiety comprises El-I.N.T. (Transition Therapeutics) or a pharmaceutically acceptable salt thereof.
  • hypoxia activated prodrug is El-I.N.T. (Transition Therapeutics) or a pharmaceutically acceptable salt thereof conjugated to an attenuating moiety.
  • the EGFR inhibitor bonded to an attenuating moiety comprises EGF816 (nazartimb) or a pharmaceutically acceptable salt thereof.
  • the hypoxia activated prodrug wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • the hypoxia activated prodrug is wherein is an attenuating moiety such as a reductive trigger. j00209j In some embodiments, the hypoxia activated prodrug is
  • the hypoxia activated prodrug is [00211]
  • the EGFR inhibitor bonded to an attenuating moiety comprises Epitmib (Hutchison China MediTech/Medi Pharma) or a pharmaceutically acceptable salt thereof.
  • the hypoxia activated prodrug is Epitimb (Hutchison China MediTech/Medi Pharma) or a pharmaceutically acceptable salt thereof conjugated to an attenuating moiety.
  • the hypoxia activated prodrug is EKB-569 (WAY172569; Pelitinib) or a pharmaceutically acceptable salt thereof.
  • the EGFR inhibitor bonded to an attenuating moiety comprises , wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug wherein R 1 is an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug wherein R 4 and R 5 are defined above for Formula II
  • hypoxia activated prodrug the hypoxia activated prodrug
  • the EGFR inhibitor bonded to an attenuating moiety comprises E8-072 (APL 103; CBT Pharmaceuticals and Zhejiang Bossan Pharmaceutical) or a pharmaceutically acceptable salt thereof.
  • hypoxia activated prodrug is ES-072 (APL 103; CBT Pharmaceuticals and Zhejiang Bossan Pharmaceutical) or a pharmaceutically acceptable salt thereof conjugated to an attenuating moiety.
  • the EGFR inhibitor bonded to an attenuating moiety comprises FV-225 or a pharmaceutically acceptable salt thereof.
  • the hypoxia activated prodrug wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug as a reductive trigger.
  • hypoxia activated prodrug wherein R 4 and R 5 are defined above for
  • the EGFR inhibitor bonded to an attenuating moiety comprises Gilotrif (afatinib, BIBW 2992) or a pharmaceutically acceptable salt thereof.
  • hypoxia activated prodrug wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • the hypoxia activated prodrug is wherein R 1 is an attenuating moiety such as a reductive trigger.
  • R 4 and R 3 are defined above for
  • hypoxia activated prodrug the hypoxia activated prodrug
  • the EGFR inhibitor bonded to an attenuating moiety comprises GNS-1480 (aka lazertmib, YH25448) or a pharmaceutically acceptable salt thereof.
  • the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • the hypoxia activated prodrug wherein R 5 is an attenuating moiety such as a reductive trigger is an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug wherein R 4 and R 1 are defined above for Formula II.
  • the EGFR inhibitor bonded to an attenuating moiety comprises HER2 Allosteric Mutant Targeted Therapy (Black Diamond) or a pharmaceutically acceptable salt thereof.
  • the hypoxia activated prodrug is HER2 Allosteric Mutant Targeted Therapy (Black Diamond) or a pharmaceutically acceptable salt thereof conjugated to an attenuating moiety .
  • the EGFR inhibitor bonded to an attenuating moiety comprises HER2GFR inhibitors or a pharmaceutically acceptable salt thereof.
  • hypoxia activated prodrug is HER2GFR inhibitors or a pharmaceutically acceptable salt thereof conjugated to an attenuating moiety.
  • the EGFR inhibitor bonded to an attenuating moiety comprises HS-10296 or a pharmaceutically acceptable salt thereof conjugated to an attenuating moiety.
  • the hypoxia activated prodrug is wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug wherein R 1 is an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug is wherein R 4 and R 5 are defined above for Formula II
  • hypoxia activated prodrug the hypoxia activated prodrug
  • the EGFR inhibitor bonded to an attenuating moiety comprises Xcotimb or a pharmaceutically acceptable salt thereof. "Y N '
  • the hypoxia activated prodrug is ill , wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug wherein R is an attenuating moiety such as a reductive trigger.
  • the hypoxia activated prodrug i the EGFR inhibitor bonded to an attenuating moiety comprises Iressa (gefitmib).
  • hypoxia activated prodrug i wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug the hypoxia activated prodrug
  • the EGFR inhibitor bonded to an attenuating moiety comprises KD020 or a pharmaceutically acceptable salt thereof.
  • the hypoxia activated prodrug is wherein the * indicates a point of atachment of an atenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug is wherein R 4 and R 5 are defined above for Formula II.
  • hypoxia activated prodrug the hypoxia activated prodrug
  • the EGFR inhibitor bonded to an attenuating moiety comprises Neriynx (neratmib, HKI-272 PB272) or a pharmaceutically acceptable salt thereof.
  • hypoxia activated prodrug i wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug wherein
  • R is an attenuating moiety such as a reductive trigger.
  • R 4 and R 5 are defined above for Formula II.
  • hypoxia activated prodrug the hypoxia activated prodrug
  • the EGFR inhibitor bonded to an attenuating moiety comprises Qhnutinib (HM-61713; BI 1482694) or a pharmaceutically acceptable salt thereof.
  • the hypoxia activated prodrug is wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug wherein R 1 is an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug i R 5 are defined above for Formula II.
  • the EGFR inhibitor bonded to an attenuating moiety comprises
  • Neratimb (aka PB357, HKI-272) or a pharmaceutically acceptable salt thereof.
  • hypoxia activated prodrug wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug wherein R 1 is an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug the hypoxia activated prodrug
  • the EGFR inhibitor bonded to an attenuating moiety comprises PF-Q6459988 or a pharmaceutically acceptable salt thereof.
  • hypoxia activated prodrug i wherein the
  • hypoxia activated prodrug is wherein R 3 is an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug defined above for Formula II In some embodiments, the hypoxia activated prodrug is
  • the EGFR inhibitor bonded to an attenuating moiety comprises Mavelertinib (PF-06747775) or a pharmaceutically acceptable salt thereof.
  • hypoxia activated prodrug i wherein the
  • * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug wherein R is an attenuating moiety such as a reductive trigger.
  • R 4 and R 5 are defined above for Formula
  • hypoxia activated prodrug the hypoxia activated prodrug
  • the EGFR inhibitor bonded to an attenuating moiety comprises Pirotimb (KBP5209; CAS# 1799730-21 -4; Sihuan Pharmaceutical Holdings Group Ltd./ XuanZhu Pharma, Hong Kong) or a pharmaceutically acceptable salt thereof.
  • the hypoxia activated prodrug is Pirotmib (KBP5209; CAS# 1799730-21-4; Sihuan Pharmaceutical Holdings Group Ltd./ XuanZhu Pharma, Hong Kong) or a pharmaceutically acceptable salt thereof conjugated to an attenuating moiety.
  • the EGFR inhibitor bonded to an attenuating moiety comprises Poziotinib or a pharmaceutically acceptable salt thereof.
  • the hypoxia activated prodrug i wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug i wherein R 1 is an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug wherein R 4 and R 5 are defined above for Formula II.
  • the EGFR inhibitor bonded to an attenuating moiety comprises Pyrotimb (SHR-1258) or a pharmaceutically acceptable salt thereof.
  • hypoxia activated prodrug wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • moiety such as a reductive trigger.
  • hypoxia activated prodrug the hypoxia activated prodrug
  • the EGFR inhibitor bonded to an attenuating moiety comprises Rociletinib (CO 1686; AVL3Q1, CNX419) or a pharmaceutically acceptable salt thereof.
  • hypoxia activated prodrug i wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug wherein R* is an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug i wherein R 4 and R 5 are defined above for Formula II.
  • the EGFR inhibitor bonded to an attenuating moiety comprises RXDX-105 (CEP 32496; AC013773, Agerafenib) or a pharmaceutically acceptable salt thereof.
  • hypoxia activated prodrug wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug wherein R is an atenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug EGFR inhibitor bonded to an attenuating moiety comprises S ⁇ 222611 (Epertimb) or a pharmaceutically acceptable salt thereof.
  • hypoxia activated prodrug is are defined above for Formula II.
  • hypoxia activated prodrug is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoe
  • the EGFR inhibitor bonded to an attenuating moiety comprises Sirotimb (Shandong Xuanzhu, acquired by Sihuan) or a pharmaceutically acceptable salt thereof.
  • the hypoxia activated prodrug is Sirotinib (Shandong Xuanzhu, acquired by Sihuan) or a pharmaceutically acceptable salt thereof conjugated to an attenuating moiety.
  • the EGFR inhibitor bonded to an attenuating moiety comprises Tagrisso (osimertinib, AZD-9291, Mereletmib) or a pharmaceutically acceptable salt thereof.
  • the hypoxia activated prodrug wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger
  • hypoxia activated prodrug wherein R 1 is an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug wherein R 4 and R 5 are defined above for Formula II.
  • hypoxia activated prodrug wherein R 4 and R 5 are defined above for Formula II.
  • the EGFR inhibitor bonded to an attenuating moiety comprises TAK-788 (AP32788; ARIAD Pharmaceuticals/Mill enmum Pharmaceuticals, Inc , USA) or a pharmaceutically acceptable salt thereof.
  • hypoxia activated prodrug (WO2015195228A1 ), wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • R 1 is an atenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug the hypoxia activated prodrug
  • the EGFR inhibitor bonded to an attenuating moiety comprises Tarceva (er!otinib, CP358774; 081-774) or a pharmaceutically acceptable salt thereof.
  • hypoxia activated prodrug i wherein the
  • * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug wherein R 1 is an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug i wherein R 4 and R 3 are defined above for Formula II.
  • hypoxia activated prodrug i wherein R 4 and R 3 are defined above for Formula II.
  • the EGFR inhibitor bonded to an attenuating moiety comprises
  • TAS0728 or a pharmaceutically acceptable salt thereof.
  • hypoxia activated prodrug is wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug wherein R 1 is an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug wherein R 4 and R: ’ are defined above for Formula II.
  • hypoxia activated prodrug the hypoxia activated prodrug
  • the EGFR inhibitor bonded to an atenuating moiety comprises TAS-121 (Taiho Pharmaceutical Co., Ltd) or a pharmaceutically acceptable salt thereof.
  • the hypoxia activated prodrug is TAS-121 (Taiho Pharmaceutical Co., Ltd) or a pharmaceutically acceptable salt thereof conjugated to an attenuating moiety.
  • the EGFR inhibitor bonded to an attenuating moiety comprises TAS6417 (WO2015025936; Taiho Pharmaceutical ) or a pharmaceutically acceptable salt thereof.
  • the hypoxia activated prodrug i wherein the
  • * indicates a point of attachment of an atenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug is wherein R 1 is an atenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug or wherein R 4 and R 5 are defined above for Formula II.
  • hypoxia activated prodrug is or
  • the EGFR inhibitor bonded to an atenuating moiety comprises Tesevatinib or a pharmaceutically acceptable salt thereof.
  • hypoxia activated prodrug wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • R 4 and R 5 are defined above for Formula II.
  • the EGFR inhibitor bonded to an attenuating moiety comprises Theiiatimb or a pharmaceutically acceptable salt thereof.
  • hypoxia activated prodrug wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • the hypoxia activated prodrug is wherein R 1 is an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug i wherein R 4 and R 5 are defined above for Formula II
  • hypoxia activated prodrug the hypoxia activated prodrug
  • the EGFR inhibitor bonded to an attenuating moiety comprises TTI-234! (Trillium Therapeutics) or a pharmaceutically acceptable salt thereof.
  • the hypoxia activated prodrug is TTI-2341 (Trillium Therapeutics) or a pharmaceutically acceptable salt thereof conjugated to an attenuating moiety.
  • the EGFR inhibitor bonded to an attenuating moiety comprises Tucatmib or a pharmaceutically acceptable salt thereof.
  • hypoxia activated prodrug i wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug or wherein R ! is an attenuating moiety such as a reductive trigger. , wherein R 4 and R 5 are defined above for Formula II. [00356] In some embodiments, the hypoxia activated prodrug
  • the EGFR inhibitor bonded to an attenuating moiety comprises Tykerb (lapatimb, GSK 572016, GW2016) or a pharmaceutically acceptable salt thereof.
  • hypoxia activated prodrug wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug wherein R 1 is an attenuating moiety such as a reductive trigger.
  • R 4 and R 5 are defined above for Formula II
  • hypoxia activated prodrug the hypoxia activated prodrug
  • the EGFR inhibitor bonded to an attenuating moiety comprises Yarlitimb (ASLAN-001; ARRY334543; ARRY543) or a pharmaceutically acceptable salt thereof.
  • hypoxia activated prodrug wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug Of In some embodiments, the hypoxia activated prodrug Of .
  • the EGFR inhibitor bonded to an attenuating moiety comprises Vizimpro (dacomitinib, PF -299804; PF-299) or a pharmaceutically acceptable salt thereof.
  • hypoxia activated prodrug wherein R 1 is an attenuating moiety such as a reductive trigger.
  • hypoxia activated prodrug i wherein R 4 and R 5 are defined above for Formula II.
  • the EGFR inhibitor bonded to an attenuating moiety comprises WZ4002 or a pharmaceutically acceptable salt thereof.
  • hypoxia activated prodrug is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoe
  • hypoxia activated prodrug is an attenuating moiety such as a reductive trigger.
  • the hypoxia activated prodrug is wherein R 4 and R 5 are defined above for Formula II.
  • hypoxia activated prodrug is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoe
  • HER1 pan-HER, HER2 and/or EGFR (HER1) driven cancer in a subject in need thereof, with a hypoxia-activated prodrug or trigger moiety.
  • the subject is mammal.
  • the subject is human.
  • the subject is rodent. In some embodiments, the subject is bovine. In some embodiments, the subject is porcine. In some embodiments, the subject is rabbit. In some embodiments, the subject is dog. In some embodiments, the subject is monkey. [00381] In one aspect, provided herein is the method of inhibiting epidermal growth factor receptor in a subject in need thereof, comprising: administering to the subject an effective amount of an epidermal growth factor receptor- mhibitmg drug, wherein the drug is covalently bonded to an attenuating moiety.
  • the attenuating moiety is a reductive trigger of the Formula (II):
  • the attenuating moiety is a reductive trigger selected from the group consisting of a compound of the Formula Ha. lib, lie, lid, lie, Ilf, or Ilg:
  • the attenuating moiety is a reductive trigger selected from the group consisting of a compound of the Formula a-ae: [00385]
  • the epidermal growth factor receptor-inhibiting drug is selected from the group consisting of:
  • a reductive trigger is an attenuating moiety such as a reductive trigger.
  • the epidermal growth factor receptor-inhibiting drug is selected from the group consisting of: no
  • the epidermal growth factor receptor-inhibiting drug is selected from the group consisting of:
  • the epidermal growth factor receptor is HEM.
  • the epidermal growth factor receptor is HER2.
  • the epidermal growth factor receptor is HER4.
  • the attenuating moiety is cleaved from the drug in vivo.
  • the attenuating moiety is cleaved from the drug under anoxic conditions.
  • the attenuating moiety is cleaved in the presence of 8T ⁇ AR4.
  • the attenuating moiet reduces or inhibits activity of the epidermal growth factor receptor-inhibiting drug outside of a tumor environment.
  • the attenuating moiety is cleaved from the drug within a tumor environment.
  • the epidermal growth factor receptor exhibits a wild-type ATP- eompetitive tyrosine kinase binding site.
  • the epidermal growth factor receptor is a mutated epidermal growth factor receptor.
  • the epidermal growth factor receptor exhibits an upregulation mutation.
  • the epidermal growth factor receptor exhibits a constitutively activating mutation. [00400] In some embodiments, the epidermal growth factor receptor exhibits a downregulation mutation.
  • a cancer in a subject m need thereof comprising: administering to the subject an effective amount of an epidermal growth factor receptor- inhibiting drug, wherein the drug is covalently bonded to an attenuating moiety.
  • the attenuating moiety is a reductive trigger of the Formula (II):
  • the attenuating moiety is a reductive trigger selected from the group consisting of a compound of the Formula Ha. lib, lie, lid, He, Ilf, or Ilg: i!a lib !ic iid i!e iif Mg
  • the attenuating moiety is a reductive trigger selected from the group consisting of a compound of the Formula a-ae:
  • the epidermal growth factor receptor-inhibiting drug is selected from the group consisting of:
  • a reductive trigger is an attenuating moiety such as a reductive trigger.
  • the epidermal growth factor receptor-inhibiting drug is selected from the group consisting of:
  • the epidermal growth factor receptor-inhibiting drug is selected from the group consisting of:
  • the drug inhibits HERE [00409] In some embodiments, the drug inhibits HER2.
  • the drug inhibits HERA
  • the attenuating moiety is cleaved m vivo.
  • the attenuating moiety is cleaved from the drug under anoxic conditions.
  • the attenuating moiety is cleaved in the presence of 8T ⁇ AR4.
  • the attenuating moiet reduces or inhibits activity of the epidermal growth factor receptor-inhibiting drug outside of a tumor environment.
  • the attenuating moiety is cleaved from the drug within a tumor environment.
  • the epidermal growth factor receptor exhibits a wild-type ATP- eompetitive tyrosine kinase binding site.
  • the epidermal growth factor receptor is a mutated epidermal growth factor receptor.
  • the epidermal growth factor receptor exhibits an upregulation mutation.
  • the epidermal growth factor receptor exhibits a constitutively activating mutation. [00420] In some embodiments, the epidermal growth factor receptor exhibits a downregulation mutation.
  • the cancer treated is selected from the group consisting of non- small cell lung cancer, esophageal cancer, pancreatic cancer, rectal cancer, squamous cell carcinoma of the head or neck, squamous cell cancer of the skin, glioblastoma, colon cancer, cervical cancer, bladder cancer, breast cancer, gastrointestinal stromal cancer (GIST), ovarian cancer, gastric cancer, endometrial cancer, uterine cancer prostate cancer, liver cancer, melanoma, brain cancer, and mesothelioma.
  • a pharmaceutical composition comprising a therapeutically effective amount of: an epidermal growth factor receptor-inhibiting drug, wherein the drug is covalently bonded to an attenuating moiety; and a pharmaceutically acceptable carrier.
  • HER! is a mutated epidermal growth factor receptor.
  • HER1 exhibits an upregulation mutation.
  • HER! exhibits a eonstitutively activating mutation.
  • HER1 exhibits a downregulation mutation.
  • HER2 is a mutated epidermal growth factor receptor.
  • HER2 exhibits an upregulation mutation.
  • HER2 exhibits a eonstitutively activating mutation.
  • HER2 exhibits a downregulation mutation.
  • HER4 is a mutated epidermal growth factor receptor.
  • HER4 exhibits an upregulation mutation.
  • HER4 exhibits a eonstitutively activating mutation.
  • HER4 exhibits a downregulation mutation.
  • the prodrug compounds of the disclosure may, for example, be prepared by reacting a kinase inhibitor comprising a nucleophilic atom, e.g , an aliphatic tertiary amine-bearing kinase inhibitor of Formula i with an appropriate nitroheterocyclic or nitrocarbocylic a-methyl electrophile such as a halide/mesylate/tosylate, in a suitable solvent and for a suitable length of time (for example jV-methyl-2-pyrrolidinone for about 15 hours), to produce a quaternary nitrogen salt comprising the nitroheterocyclic or mtrocarbocyclic reductive trigger moiety linked directly or indirectly to a nitrogen of the kinase inhibitor.
  • a kinase inhibitor comprising a nucleophilic atom, e.g , an aliphatic tertiary amine-bearing kinase inhibitor of Formula i with an appropriate nitro
  • Scheme 3 illustrates two alternate methods to the a-methyl bromide 239, from commercially available starting materials.
  • Scheme 4 illustrates a route to the a-methyl bromide 244, from commercially available starting materials.
  • Scheme 5 illustrates a route to the a-methyl bromide 250, from l,5-dimethyl-4-mtro-li/- imidazole (238) (Scheme 3).
  • Scheme 5 illustrates a route to the a-methyl bromide 261 from the commercially available oxazole (251).
  • Scheme 7 illustrates two alternate routes to the a-methyl bromide 264, from a- methyl bromide 246 (Scheme 5) and 2 ⁇ br omo ⁇ l, 5 -dimethy I -4-nitro-l //-imidazole (245) (Scheme 5), respectively.
  • Scheme 7 below also illustrates a route to the a-methyl bromide 266, from a-methyl bromide 264.
  • Scheme 8 illustrates a route to the a-methyl bromide 270, from (2-bromo-l-methyl-4- nitro-l//-imidazol-5-yl)methyl acetate (247) (Scheme 5).
  • Scheme 9 illustrates a route to quaternary nitrogen salt compounds by reacting an aliphatic tertian- amine-bearing kinase inhibitor with an appropriate nitroheterocyclic a-methyl halide/mesyiate/tosylate (Formula VIII), in a suitable solvent and for a suitable length of time (for example inA-methyl ⁇ 2 ⁇ pyrrolidmone for about 15 hours).
  • Scheme 10 illustrates the preparation of a number of prodrug compounds according to the disclosure.
  • the 4 ⁇ anilinopyndo[3,4 ⁇ i]pyrimidine effector compounds 1-9 (Scheme 2) were reacted with the a-methyl bromide 239 (Scheme 3) in A r -methyl-2-pyrrohdinone (NMP) at room temperature for approximately 15 hours, before the addition of acetonitrile, to provide the quaternary ammonium salts (12, 13, 15-21) as a fine precipitate that was collected by filtration and washed with acetonitrile, ethyl acetate and hexane.
  • NMP A r -methyl-2-pyrrohdinone
  • Schemes 12 to 14 illustrate the preparation of alkyl trigger bromides in which Rs is Ci to Ce alkyl.
  • TKs receptor tyrosine kinases
  • EGFR FIERI, erhB-I), HER2 (erbB-2), HER3 (erbB-3), and HER4 (erbB-4), and their signaling pathways regulate cell growth, survival, adhesion, migration, and differentiation through three downstream pathways: RA S/RAF/ mitogen-activated protein kinase, phosphoinositide 3- kinase/AKT, and Janus kinase/signal transducer and activator of transcription (JAK/STAT).
  • Dysregulated signaling of HER family has been associated with the development of several malignancies including NSCLC.
  • Many patients with NSCLC have somatic mutations of EGFR, first identified in 2004, which led to aberrant constitutive signaling via EGFR/HER family and their downstream protein markers.
  • the EGFR mutations including activating and resistant mutations, mostly occur in exons 18 to 21 of the EGFR gene encoding the ATP-binding pocket of the intracellular TK domain.
  • the activating EGFR mutations (actEGFRm) have been reported in ⁇ 10%-l 5% of Caucasian patients but in up to 60% of selected Asian populations with NSCLC (female, never/light smoker, and adenocarcinoma).
  • the most frequent actEGFRm in NSCLC are in-frame deletions in exon 19 (EGFRDell9, -60%) and L858R point mutation exon 21 (EGFRL858R, -30%). These oncogenic mutations interact and generate stabilization with ATP, intrinsically stimulate phosphorylation of tyrosine residues, and then result in the intracellular signal transduction activation in a ligand-independent manner.
  • the NSCLC patients with actEGFRm become apparently dependent on EGER activity to stimulate downstream signaling pathways to maintain the malignant phenotype (“oncogene addiction”). Therefore, blocking EGFR/HER family pathways with EGFR/HER TK inhibitors (TKIs) can suppress tumor cell proliferation and initiate apoptosis. (Wang, X. et. al. OncoTargets and Therapy (2016), 9, 5461- 5473.)
  • the epidermal growth factor receptor is HERE
  • the HER! is a mutated epidermal growth factor receptor. In some embodiments, the HER! is a wild-type epidermal growth factor receptor. In some embodiments, the HER! exhibits an upregulation mutation. In some embodiments, the HER! exhibits a constitutively activating mutation. In some embodiments, the HER! exhibits a downregulation mutation.
  • the epidermal growth factor receptor is HER2.
  • the HER2 is a mutated epidermal growth factor receptor. In some embodiments, the HER2 is a wild-type epidermal growth factor receptor. In some embodiments, the HER2 exhibits an upregulation mutation. In some embodiments, the HER2 exhibits a constitutively activating mutation. In some embodiments, the HER2 exhibits a downregulation mutation.
  • the epidermal growth factor receptor is HER4.
  • the HER4 is a mutated epidermal growth factor receptor. In some embodiments, the HER4 is a wild-type epidermal growth factor receptor. In some embodiments, the HER4 exhibits an upregulation mutation. In some embodiments, the FIER4 exhibits a constitutively activating mutation. In some embodiments, the HER4 exhibits a downregulation mutation.
  • the ErbB family of receptors is a subfamily of four closely related receptor tyrosine kinases: epidermal growth factor receptor or EGFR (ErbB-1; or HER! in humans), HER2/c-neu (ErbB-2), HERS (ErbB-3) and HER4 (ErbB-4).
  • EGFR is the cell-surface receptor for members of the epidermal growth factor family (EGF family) of extracellular protein ligands.
  • Mutations that lead to EGFR overexpression (upregulation) or overactivity have been associated with a number of cancers, including squamous-cell carcinoma of the lung (80% of cases), anal cancers, glioblastoma (50% of eases), and epithelial tumors of the head and neck (80-100% of cases). These somatic mutations involving EGFR lead to its constant activation, which produces uncontrolled cell division. In a non-limiting example, in glioblastoma, a more or less specific mutation of EGFR, called EGFRvIH, is often observed. Mutations, amplifications, or misregulations of EGFR or family members are implicated in about 30% of all epithelial cancers (PCT/US2Q 18/049842, incorporated herein in its entirety).
  • EGFR tyrosine kinase inhibitors include gefitinib, erlotmib, afatinib, osimertinih, and icotinib for lung cancer.
  • EGFR tyrosine kinase inhibitors include gefitinib, erlotmib, afatinib, osimertinih, and icotinib for lung cancer.
  • cetuximab, panitumumab, necitumumab, zaiutumumab, nimotuzumab and matuzumab are examples of monoclonal antibody EGFR inhibitors.
  • Gefitinib, erlotmib, afatmib, dacomitinib, osimertinib, and lapatinib are examples of small molecule EGFR kinase inhibitors.
  • Non-limiting sources of resistance are the EGFR T790M Mutation, HER2 and MET oncogenes, transformation to small cell lung cancer (SCLC), epithelial to mesenchymal transition (EMT), and fusions including those involving BRAF, NTRK1, RET, ALK, and/or ROSE Options to combat resistance are not limited, with only osimertinib being approved to treat EGFR T790M.
  • HER- driven cancers include, but are not limited to, cancers caused by a EGFR gene fusion, a EGFR kinase domain duplication, a ERBB2 gene fusion, a ERBB2 mutation, a NRG I gene fusion, a ERBB3 mutation, and/or a ERBB4 fusion.
  • HER2 HER2
  • Table L The oncogenic alterations involving a ERBB2 (HER2) proto-oncogene are illustrated in Table L and each of those alterations is contemplated herein.
  • the cancer comprises an EGFR exon 20 insertion mutation.
  • the EGFR exon 20 insertion mutation includes a mutation such as but not limited to any of the mutations described in Yasuda, et ai., 2013, Sci. Trans 1. Med. 5(216):216ral77; doi: 10.1126/scitranslmed.3007205, and Arcila, et ai, 2013, Mol. Cancer Ther. 12:220; each of which is incorporated herein in its entirety by reference.
  • Neureguiin 1 ⁇ NRG I encodes a growth factor ligand that binds to the human epidermal growth factor receptor 3 (HERS) encoded by the erb-b2 receptor tyrosine kinase 3 (ERBB3) gene and human epidermal growth factor receptor 4 (HER4) encoded by the erb-b2 receptor tyrosine kinase 4 ( ERBB4 ). Binding of NRG1 to HERS and HER4 can induce dimerization with other HER family members, including EGFR and HERS. Unlike EGER, HER2, and HER4, the HERS kinase domain is catalytically inactive but can activate EGFR or HER2 and thus can initiate oncogene signaling via its heterodimerization partner.
  • HERS human epidermal growth factor receptor 3
  • ERBB3 erb-b2 receptor tyrosine kinase 3
  • HER4 human epidermal growth factor receptor 4
  • NRG1 fusions have been identified in breast, NSCLC, cholangiocarcinoma, pancreatic cancer, and ovarian cancer (Wang, et a!., 1999, Oncogene 18:5718-21; Fernandez-Cuesta L, et ai, 2014, Cancer Discov 4:415-22; Dhanasekaran, et at, 2014, Nat Common 5:5893; Heining, et ai, 2018, Cancer Discov).
  • NRG I gene fusions have been demonstrated to be oncogenic by inducing overexpression the ligand Neureguiin 1 which induces heterodimerization of HER3 with HER2.
  • Tarloxotinib is a potent inhibitor of HER2 and thus inhibits proliferation of cancer cells that utilize HER3/HER2 signaling. (Sene fusions involving the ERBB4 gene, similar to other fusions involving receptor tyrosine kinases, induce constitutive activation of HER4 and consequently activation of MARK (Nakaoku, et ai., 2014, Clin Cancer Res 20:3087-93). In some embodiments, tarloxotinib inhibits HER4 oncogene-driven cancers. Currently there are no U.S.
  • STEAP4 has been shown to be associated with certain cancers (Gomes et al, 2012; Xue et al, 2017). STEAP4 has also been shown to be highly induced by hypoxia, i.e., low oxygen at the tissue level. Moreover, hypoxia within tumours is associated with poor prognosis of cancer patients and with treatment failure (Hunter et al., 2016), such as resistance to radiotherapy and traditional chemotherapy.
  • High levels of STEAP4 expression are associated with certain cancers.
  • the present disclosure teaches that when STEAP4 is highly expressed in cancers, STEAP4 can catalyse a reaction in HAPs leading to release of the drug payload and/or modification of the HAP to a form that is able to penetrate the cell membrane and cause cell death.
  • cancers with elevated STEAP4 expression levels respond to treatment by HAPs. Accordingly, the present disclosure teaches an effective treatment for STEAP4-associated cancers.
  • a method of treating cancer in an individual in need thereof comprising: a) providing tumour cells of the individual; b) determining the level of STEAP4 expression in the tumour cells; c) predicting the individual as being likely to be responsive to treatment by a HAP if the tumour cells exhibit an elevated level of STEAP4 expression; and d) administering a therapeutically effective amount of a HAP to the individual.
  • the present disclosure provides a method of treating cancer in an individual in need thereof, where tumour cells of the individual exhibit an elevated level of STEAP4 expression, the method comprising: a) administering a therapeutically effective amount of a HAP to the individual.
  • the present disclosure provides a method of predicting the responsiveness of an individual with cancer to treatment with a HAP, the method comprising: a) providing tumour cells of the individual; b) determining the level of STEAP4 expression in the tumour cells; and c) predicting the individual as being likely to be responsive to a treatment with the HAP if the tumour cells exhibit an elevated level of STEAP4 expression.
  • the present disclosure provides a method of predicting the responsiveness of an individual with cancer to treatment with a HAP, the method comprising: a) determining the level of STEAP4 expression in a sample from the individual, wherein the sample comprises tumour cells; and b) predicting the individual as being likely to be responsive to a treatment with the HAP if the tumour cells exhibit an elevated level of STEAP4 expression.
  • the present disclosure also provides kits for performing the disclosed methods.
  • STEAP Six-Transmembrane Epithelial Antigen of Prostate
  • the STEAP4 protein also known as STAMP2 or TIARP, is a metalloreductase that reduces iron and copper ions. 8 ⁇ AR4 has equivalent activity under either physiological or acidic pH (pH5.5 - 7.5).
  • the full length nucleotide sequence encoding the STEAP4 protein i.e., the STEAP4 gene
  • the full length amino acid sequence of the STEAP4 protein are known in the art (see, e.g., NCBI Gene ID: 79689, NCBI Ace. No. NM_024636, NCBI Ace. No. NM_ 001205315.1, NCBI Acc. No. NM 001205316.1, and UmProt Acc. No. Q687X5).
  • This isoform 1 is encoded by two variants: variant 1 and variant 2.
  • Variant 1 is the predominant nucleotide sequence encoding isoform 1 of the STEAF4 protein, and is shown in SEQ ID NO:2 (also in Figure 2).
  • Variant 2 is an alternative nucleotide sequence encoding isoform 1 of the STEAP4 protein, and is shown in SEQ ID NO: 3 (also in Figure 3).
  • Isoform 2 of the STEAP4 protein is 283 amino acids long, and is shown in SEQ ID NO:4 (also in Figure 4).
  • Variant 3 is a nucleotide sequence encoding this shorter isoforni of STEAP4 protein (isoform 2) and is shown in SEQ ID NO: 5 (also m Figure 5).
  • STEAP4 As used herein, reference to the “STEAP4”, “STEAP4 protein”, or similar, refers to isoform 1 (SEQ ID NO:l), isoform 2 (SEQ ID NO: 4). a protein encoded by variant 1 (SEQ ID NO:2), a protein encoded by variant 2 (SEQ ID NO: 3), or a protein encoded by variant 3 (SEQ ID NO: 5), and/or to any other variant thereof.
  • Variants contemplated within the scope of the present disclosure include protein variants which are substantially homologous to a native STEAP4 protein.
  • substantially homologous refers to a protein having one or more naturally or non-naturaily occurring ammo acid deletions, insertions, or substitutions (e.g., derivatives, homologs, and fragments), as compared to the amino acid sequence of a native STEAP4 protein.
  • the am o acid sequence of a STEAP4 variant may be at least about 40% identical, at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, at least about 90% identical, or at least about 95% identical to a native STEAP4 protein.
  • a “native STEAP4 protein” refers to the STEAP4 proteins which are found in nature and are not manipulated by man, and includes isoform 1 (SEQ ID NO: l), isoform 2 (SEQ ID NO:4), a protein encoded by variant 1 (SEQ ID NO:2), a protein encoded by variant 2 (SEQ ID NO:3), and a protein encoded by variant 3 (SEQ ID NO:5).
  • variant 1 SEQ ID NO: l
  • SEQ ID NO:4 isoform 1
  • variant 2 SEQ ID NO:3
  • variant 3 protein encoded by variant 3
  • Variants contemplated within the scope of the present disclosure also include proteins encoded by polynucleotide variants which have substantial sequence similarity or sequence identity to a native STEAP4 gene.
  • the polynucleotide sequence of a STEAP4 variant may have at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% sequence similarity or sequence identity with a native STEAP4 gene.
  • a “native 8 ⁇ AR4 gene” refers to genes: (a) which are found in nature and are not manipulated by man, and encode STEAP4 proteins; and (b) which encode STEAP4 proteins that are found in nature and are not manipulated by man; and includes the gene having the polynucleotide sequence encoding isoforni 1 (SEQ ID NO:l), the gene having the polynucleotide sequence encoding isoform 2 (SEQ ID NO.4 ⁇ .
  • High levels of 8TEAP4 expression are associated with certain cancers.
  • STEAP4 when STEAP4 is highly expressed in a cancer, the inventors have found that STEAP4 may catalyse conversion of an administered drug into its active form, such as a reaction leading to release of a drug payload.
  • high STEAP4 expression leads to release of the drug payload in HAPs.
  • the high STEAP4 expression is associated with hypoxic metabolism.
  • the high STEAP4 expression is associated with a hypoxic environment
  • the high STEAP4 expression is associated with a hypoxic tumour.
  • the high STEAP4 expression is not associated with low oxygen levels.
  • STEAP4 when STEAP4 is present in hypoxic tumour environments, STEAP4 catalyses one-electron reduction of cell-excluded quaternary ammonium salt HAPs, leading to their fragmentation selectively m pathophysiologicai!y hypoxic tumour tissues, releasing the active drug which can then cross the cell wall and kill the cancer cell [00479] Without wishing to be bound by theory, it is thought that STEAP4 is located on the plasma membrane. As such, in some embodiments, STEAP4 reduces the HAP extracellular] y, forming a molecule that is capable of diffusing into the cell (e.g., a reduced form of the molecule).
  • STEAP4 is on the plasma membrane and reduces the HAP extracei Marly, at which point the charged molecule undergoes fragmentation and diffuses into the cell to inhibit EGFR.
  • the charged HAP molecule undergoes fragmentation under hypoxic conditions.
  • the charged HAP molecule undergoes fragmentation in the presence of elevated levels of STEAP4.
  • the HAP is Compound A.
  • the MAP is Compound C.
  • the HAP is Compound E.
  • the HAP is selected from the group consisting of Compound A, Compound C, and Compound E.
  • HAPs While NMQ prodrugs used in the disclosed methods function by releasing an active molecule having undergone reduction in the extracellular medium, other HAPs for use in the disclosed methods do not fragment and release an active molecule, but instead are effective because the reduced form of the molecule is able to penetrate the cell membrane and then cause cell death in some embodiments, the HAPs may have a limiting or low rate of membrane penetration such that extracelluclar metabolism by STEAP4 will contribute a significant proportion of total cellular metabolism and such that intracellular reductases will contribute a less significant proportion of total cellular metabolism.
  • One such class of HAPs is the trophenyl mustards.
  • HAPs with a net neutral charge may be hydrophilic in nature, e.g., may have a low partition coefficient, which can result in a limiting rate of cell membrane penetration.
  • wdien HAPs have a limiting rate of cell membrane penetration, extracellular metabolism by STEAP4 contributes a significant proportion of total cellular metabolism, and the remainder of HAP cellular metabolism is due to intracellular reductases.
  • HAPs with a iow r pKa may be protonated at physiologically relevant pH range and thus carry a net positive charge, resulting in a low partition coefficient, which can result in a limiting rate of cell membrane penetration.
  • extracellular metabolism by STEAP4 contributes a significant proportion of total cellular metabolism, and the remainder of HAP cellular metabolism is due to intracellular reductases.
  • the present disclosure teaches methods of treating a cancer (e.g., a cancer characterized by STEAP4 activity and/or a cancer characterized by a hypoxic tumour environment) by allowing STEAP 4 to metabolize HAPs, including cell-excluded HAPs, at the cell surface.
  • a cancer e.g., a cancer characterized by STEAP4 activity and/or a cancer characterized by a hypoxic tumour environment
  • this has the effect of releasing the active drug from the prodrug, which can enable the active drug to cross the cell membrane and deliver its payload to the intracellular targets and thus kill the cancer cell.
  • this is a previously unidentified role played by the enzyme STEAP4 in hypoxic tumour environments.
  • this activity can be unique to the STEAP4 reductase enzyme, and is not shared with the other STEAP proteins or with other reductases.
  • individuals suffering from cancer can be stratified based on their 8 ⁇ AR4 expression levels, with those exhibiting elevated expression being identified as those likely to respond to HAP treatment.
  • embodiments disclosed herein are contrary to the prevailing understanding of STEAP4 that has proposed the inhibition of STEAP4 in order to treat cancer.
  • the exploitation of these elevated levels of STEAP4 can achieve better treatment outcome for patients.
  • the present application further contemplates methods of treating a subject with cancer, e.g., the cancers disclosed herein, with the HAP compositions contemplated herein, wherein the treatment is part of a maintenance therapy for subjects with recurring or refractory cancer.
  • the present application contemplates a method of treating a resistant or refractory cancer m a subject with the HAP compositions disclosed herein.
  • the treatment leads to a full response, remission, and/or complete cure in the subject with recurring or refractory cancer.
  • the treatment maintains a stable disease, leads to a partial response ( e.g ., some tumour regression), or prevents the return of tumours which have fully regressed.
  • the HAP is a compound disclosed herein.
  • a method of inhibiting tumour cell growth comprising administering to the tumour a therapeutically effective amount of a HAP, as described herein.
  • a method of inhibiting tumour cell growth comprising administering to the tumour a therapeutically effective amount of a compound disclosed herein, or a pharmaceutically acceptable salt or solvate thereof.
  • the tumour is a solid tumour.
  • the solid tumour is selected from bladder carcinoma, squamous cell carcinoma, transitional cell carcinoma, basal cell carcinoma, renal cell carcinoma, ductal cell carcinoma, and adenocarcinoma.
  • a compound which is a HAP, as described herein, for use in the treatment of cancer wherein the treatment comprises administering the compound.
  • HAP HAP, as described herein, for use in the treatment of cancer.
  • the subject is further administered at least one additional agent, or a salt or solvate thereof, that treats or prevents the cancer, as described herein.
  • At least one of a compound of any one of the compounds as disclosed herein is administered by at least one route selected from the group consisting of inhalational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal, intravesical, intrapulmonary, intraduodenal, intragastricai, intrathecal, epidural, intrapleural, intraperitoneal, intratracheal, optic, intraocular, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, in tra bronchial, inhalation, and topical in some embodiments, at least one HAP is administered orally, parenterally, rectaliy, topically, intravenously, intramuscularly, subcutaneously, or intraperitoneally.
  • At least one HAP is administered intraperitoneally. certain embodiments, at least one HAP is administered intravenously.
  • the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the subject is a human in need of treatment thereof.
  • the HAP is administered by at least one route selected from the group consisting of iniialational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal, intravesical, intrapulmonary, intraduodenal, intragastricai, intrathecal, epidural, intrapleural, intraperitoneal, intratracheal, optic, intraocular, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchiai, inhalation, and topical.
  • the HAP is administered orally, parenterally, rectaliy, topically, intravenously, intramuscularly, subcutaneously, or intraperitoneally.
  • the HAP is administered intraperitoneally. In some embodiments the HAP is administered intravenously. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the subject is a human in need of treatment thereof.
  • At least one HAP is administered intraperitoneally is administered by at least one route selected from the group consisting of iniialational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal, intravesical, intrapulmonary, intraduodenal, intragastricai, intrathecal, epidural, intrapleural, intraperitoneal, intratracheal, optic, intraocular, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchiai, inhalation, and topical.
  • kits comprising a compound of any one of the compounds as disclosed herein, an applicator and instructional material for use thereof, wherein the instructional material comprises instructions for preventing or treating a cancer.
  • kits comprising HAP, an applicator and instructional material for use thereof, wherein the instructional material comprises instructions for preventing or treating a cancer.
  • the compounds (e.g., the HAP) described herein may form salts with acids, and such salts are included in the present application in some embodiments, the salts are pharmaceutically acceptable salts.
  • salts embraces addition salts of free acids that are useful within the methods disclosed herein.
  • 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 application, such as for example utility in process of synthesis, purification or formulation of compounds useful within the methods disclosed herein.
  • Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid.
  • inorganic acids include sulphate, hydrogen sulphate, hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulphuric, and phosphoric acids (including hydrogen phosphate and dihydrogen phosphate).
  • 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, trifluorom ethanesulfonic, 2- hydroxy ethanesulfonic, p-tol uenes ulfoni c, sulfanilic, cyclohexylaminosulfonic, stearic, alginic
  • Suitable pharmaceutically acceptable base addition salts of compounds contemplated herein include, for example, 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. All of these salts may be prepared from the corresponding compound by reacting, for example, the appropriate acid or base with the compound. Additional Agents
  • the HAP compositions contemplated herein are useful m the methods of the present application when used concurrently with at least one additional compound useful for preventing and/or treating diseases and/or disorders contemplated herein.
  • the HAP compositions contemplated herein are useful in the methods of present application in combination with at least one additional agent useful for preventing and/or treating diseases and/or disorders contemplated herein.
  • These additional agents may comprise compounds of the present application or other compounds, such as commercially available compounds, known to treat, prevent, or reduce the symptoms of diseases and/or disorders contemplated herein.
  • the HAP compositions contemplated herein, or a salt or solvate thereof can be used concurrently or in combination with one or more agents known to be useful in treating or preventing cancer, such as the cancers described herein.
  • Non-limiting examples of additional anti -proliferative agents contemplated include, but are not limited to, compounds listed on the cancer chemotherapy drug regimens in the 14 to Edition of the Merck Index (2006), which is hereby incorporated by reference, such as asparaginase, bleomycin, carboplatin, carmustine, chlorambucil, cisplatin, colaspase, cyclophosphamide, cytarabme, dacarbazine, dactinomycin, daunorubicin, doxorubicin (adriamycme), epirubicin, etoposide, 5-fiuorouracii, hexamethylmelamme, hydroxyurea, ifosfamide, irmoteean, leucovorin, lomustine, mechloretharmne, 6-mercaptopurine, mesna, methotrexate, mitomycin C, mitoxantrone, prednisolone,
  • Additional anti-proliferative agents include other molecular targeted agents that modulate parallel pathways such as MEK 1/2 inhibitors, AKT inhibitors and mTOR inhibitors, monoclonal antibodies (such as Cetuximab), oxaliplatin, gemcitahine, gefinitib, taxotere, ara A, ara C, herceptm, BCNIJ, CCNIJ, DUG, and actmomycin D. Still further anti-proliferative agents include but are not limited to those compounds acknowledged to be used in the treatment of neoplastic diseases in Goodman and Gilman's The Pharmacological Basis of Therapeutics (Eleventh Edition), editor Molinoff et al, pub!.
  • a HAP composition as disclosed herein, is eo-administered with at least one additional agent.
  • the regimen of administration may affect what constitutes an effective amount.
  • the therapeutic formulations may be administered to the subject either prior to or after the onset of a disease or disorder contemplated herein. 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.
  • compositions of the present application may be carried out using known procedures, at dosages and for periods of time effective to treat a disease or disorder contemplated herein.
  • 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 patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound to treat a disease or disorder contemplated herein.
  • 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 HAP composition contemplated herein 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 w ithout undue experimentation.
  • the therapeutically effective amount or dose of a HAP composition of the present application depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of a disease or disorder contemplated herein.
  • 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.
  • physician or veterinarian could start doses of the HAP composition contemplated herein employed m 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 HAP composition of the present application 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 m 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.
  • HAP compositions contemplated herein for administration may be in the range of from about 1 pg to about 10,000 mg, about 20 pg to about 9,500 mg, about 40 pg to about 9,000 mg, about 75 pg to about 8,500 mg, about 150 pg to about 7,500 mg, about 200 pg to about 7,000 mg, about 3050 pg to about 6,000 mg, about 500 pg to about 5,000 mg, about 750 pg 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 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and any and all whole or partial increments there between.
  • the dose of a HAP composition contemplated herein is from about 1 mg and about 2,500 mg.
  • a dose of a compound contemplated herein 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 an additional agent in the HAP composition 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 thereof.
  • the HAP composition comprises a dosage of HAP of from about 0.1 mg/kg of body weight of a subject to about 200 mg/kg of body weight of a subject. In some embodiments, the HAP composition comprises a dosage of HAP of from about 0.1 mg/kg of body weight of a subject to about 100 mg/kg of body weight of a subject, from about 0.1 mg/kg of body- weight of a subject to about 50 mg/kg of body weight of a subject from about 0.1 mg/kg of body weight of a subject to about 25 mg/kg of body weight of a subject, from about 0.1 mg/kg of body- weight of a subject to about 20 mg/kg of body weight of a subject from about 0.1 mg/kg of body weight of a subject to about 15 mg/kg of body weight of a subject, from about 0.1 mg/kg of body- weight of a subject to about 10 mg/kg of body weight of a subject, from about 0.1 mg/kg of body weight of a subject to about 5 mg/
  • the HAP composition comprises a dosage of HAP of from about 1 mg/kg of body weight of a subject to about 100 mg/kg of body weight of a subject, from about 1 mg/kg of body weight of a subject to about 50 mg/kg of body weight of a subject, from about 1 mg/kg of body weight of a subject to about 25 mg/kg of body weight of a subject, from about 1 mg/kg of body weight of a subject to about 20 mg/kg of body weight of a subject, from about 1 mg/kg of body weight of a subject to about 15 mg/kg of body weight of a subject, from about 1 mg/kg of body weight of a subject to about 10 mg/kg of body weight of a subject, or from about 1 mg/kg of body weight of a subject to about 5 mg/kg of body weight of a subject.
  • the HAP composition comprises a dosage of HAP of from about 10 mg/kg of body weight of a subject to about 100 mg/kg of body weight of a subject, from about 10 mg/kg of body weight of a subject to about 50 mg/kg of body weight of a subject, from about 10 mg/kg of body weight of a subject to about 25 mg/kg of body weight of a subject, from about 10 mg/kg of body weight of a subject to about 20 mg/kg of body- weight of a subject, or from about 10 mg/kg of body weight of a subject to about 15 mg/kg of body weight of a subject.
  • the HAP composition comprises a dosage of HAP of from about 20 mg/kg of body weight of a subject to about 100 mg/kg of body weight of a subject, from about 20 mg/kg of body weight of a subject to about 50 mg/kg of body weight of a subject, or from about 20 mg/kg of body weight of a subject to about 25 mg/kg of body weight of a subject.
  • the HAP composition comprises a dosage of HAP of from about 0.1 mg/kg of body weight of a subject to about 300 mg/m 2 of body weight of a subject.
  • the HAP composition comprises a dosage of HAP of from about 0.1 mg/m 2 of body weight of a subject to about 200 mg/m 2 of body weight of a subject, from about 0.1 mg/m 2 of body weight of a subject to about 150 mg/m 2 of body weight of a subject, from about 0.1 mg/m 2 of body weight of a subject to about 100 mg/m 2 of body weight of a subject, from about 0.1 mg/m 2 of body weight of a subject to about 50 mg/m 2 of body weight of a subject, from about 0.1 mg/m 2 of body weight of a subject to about 25 mg/m 2 of body weight of a subject, from about 0.1 mg/m 2 of body weight of a subject to about 20 mg/m 2 of body weight of a subject, from about 0.1 mg/m 2 of body- weight of a subject to about 15 mg/m 2 of body weight of a subject, from about 0.1 mg/m 2 of body weight of a subject to about 10 mg/m 2 of body weight of body weight of a
  • the HAP composition comprises a dosage of HAP of from about 1 mg/m 2 of body weight of a subject to about 150 mg/m 2 of body weight of a subject, about 1 mg/m 2 of body weight of a subject to about 100 mg/m 2 of body weight of a subject, from about 1 mg/rn 2 of body weight of a subject to about 50 mg/m 2 of body weight of a subject, from about 1 mg/m 2 of body weight of a subject to about
  • the HAP composition comprises a dosage of HAS 1 of from about 10 mg/rn 2 of body eight of a subject to about 150 mg/nr of body weight of a subject, from about 10 mg/rn 2 of body weight of a subject to about 100 mg/m 2 of body weight of a subject, from about 10 mg/m 2 of body weight of a subject to about 50 mg/m 2 of body weight of a subject, from about 10 nig/m 2 of body weight of a subject to about 25 nig/m 2 of body- weight of a subject, from about 10 mg/m 2 of body weight of a subject to about 20 mg/m 2 of body weight of a subject, or from about 10 mg/kg of body weight of a subject to about 15 mg/m 2 of body weight of a subject.
  • the MAP composition comprises a dosage of HAP of from about 20 mg/m 2 of body weight of a subject to about 150 mg/m 2 of body weight of a subject, from about 20 mg/m 2 of body weight of a subject to about 100 mg/m 2 of body weight of a subject, from about 20 mg/m 2 of body weight of a subject to about 50 mg/m 2 of body weight of a subject, or from about 20 mg/m of body weight of a subject to about 25 mg/m 2 of body weight of a subject.
  • the HAP composition comprises a dosage of HAP of from about 50 mg/m 2 of body weight of a subject to about 150 mg/m 2 of body weight of a subject, from about 50 mg/m 2 of body weight of a subject to about 100 mg/m 2 of body weight of a subject, from about 50 mg/m 2 of body weight of a subject to about 80 mg/m 2 of body w r eight of a subject, or from about 50 mg/m 2 of body weight of a subject to about 75 mg/m 2 of body weight of a subject.
  • the HAP composition comprises a dosage of HAP of from about 75 mg/m 2 of body weight of a subject to about 150 mg/m 2 of body weight of a subject, or from about 75 mg/m 2 of body weight of a subject to about 100 mg/m 2 of body weight of a subject. In some embodiments, the HAP composition comprises a dosage of HAP of from about 80 mg/m 2 of body weight of a subject to about 150 mg/m 2 of body weight of a subject, or from about 80 mg/m 2 of body weight of a subject to about 100 mg/m 2 of body weight of a subject.
  • the HAP composition comprises a dosage of HAP of about 0.1 mg/m 2 , about 1 mg/m 2 , about 5 mg/m 2 , about 10 mg/m 2 , about 15 mg/m 2 , about 20 mg/m 2 , about 25 mg/m 2 , about 30 mg/m 2 , about 40 mg/m 2 , about 50 mg/m 2 , about 60 mg/m 2 , about 70 mg/m 2 , about 75 mg/m 2 , about 80 mg/m 2 , about 100 mg/m 2 , about 150 mg/m 2 , about 200 mg/m 2 , or about 300 mg/m 2 .
  • the HAP composition comprises a dosage of the prodrug of about 50 mg/m 2 of body weight of a subject. In some embodiments, the HAP composition comprises a dosage of the prodrug of from about 0.1 mg/m 2 to about 150 mg/m 2 of body weight of a subject. In some embodiments, the HAP composition comprises a dosage of the prodrug of about 150 mg/ m 2 of body weight of a subject. In some embodiments, the HAP composition comprises a dosage of the prodrug of about 75 mg/ m 2 of body weight of a subject. In some embodiments, the HAP composition comprises a dosage of the prodrug of about 80 mg/ m 2 of body weight of a subject.
  • the HAP composition comprises a dosage of the prodrug of about 120 mg/ m 2 of body weight of a subject.
  • a dosage of the potassium supplementation is from about 3 mEq/L to about 7 mEq/L. In some embodiments, a dosage of the potassium supplementation is about 6 mEq/L.
  • administration of the potassium supplementation increases the potassium concentration to from about 0.5 mEq/L to about 6.0 mEq/L, from about 0.5 mEq/L to about 5.5 mEq/L, from about 0.5 mEq/L to about 5.0 mEq/L, from about 0.5 mEq/L to about 4.5 mEq/L, from about 0.5 mEq/L to about 4.0 mEq L, from about 0.5 mEq/L to about 3.5 mEq/L, from about 0.5 mEq/L to about 3.0 mEq/L, from about 0.5 mEq/L to about 2.5 mEq/L, from about 0.5 mEq/L to about 2.0 mEq/L, from about 0.5 mEq/L to about 1.5 mEq/L, or from about 0.5 mEq/L to about 1.0 mEq/Labove the normal serum concentration.
  • the normal serum concentration of potassium is about 3.5 mEq/L
  • administration of the potassium supplementation will increase the potassium concentration from about 0.5 to about 1 0 mEq/L, such that the increased serum concentration of potassium is about to about 4.0 mEq/L to about 4 5 mEq/L
  • administration of the potassium supplementation increases the potassium concentration from about 0.5 meQ/L to about 6.0 mEq/L, from about 0.5 mEq/L. to about 5.5 mEq/L, from about 0.5 mEq/L to about 5.0 mEq/L, from about 0.5 mEq/L to about 4 5 mEq/L, from about 0.5 mEq/L to about 4 0 mEq/L, from about 0 5 mEq/L to about 3.5 mEq L, from about 0.5 mEq/L to about 3.0 mEq/L, from about 0.5 mEq/L to about 2.5 mEq/L, from about 0.5 mEq/L to about 2.0 mEq/L, from about 0.5 mEq/L to about 1.5 mEq/L, or from about 0.5 mEq/L to about 1.0 mEq/L above the serum concentration in the absence
  • the serum concentration of potassium in the absence of administration of a HAP is about 3.5 mEq/L
  • administration of the potassium supplementation will increase the potassium concentration from about 0.5 to about 1.0 mEq/L, such that the increased serum concentration of potassium is about to about 4.0 mEq/L to about 4.5 mEq/L.
  • the HAP composition contemplated herein are administered to the patient in dosages that range from one to five times per day or more.
  • the HAP composition contemplated herein are administered to the patient 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 HAP composition HAP compositions contemplated herein 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 present disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient is determined by the attending physical taking all other factors about the patient into account.
  • the amount of HAP composition 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.
  • a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.
  • the prodrug is administered once, twice, three times, four times, five times, or six times a day. In some embodiments, the prodrug is administered once, twice, three times, four times, five times, or six times a week. In some embodiments, the prodrug is administered every week, every two weeks, every three weeks, every four weeks, every five weeks, or every' six weeks
  • the administration of the HAP composition contemplated herein is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a “drug holiday”).
  • the length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days.
  • the dose reduction during a drug holiday includes from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
  • the HAP composition provided herein reduce side effects and/or toxicity effects of the administered HAP such that a drug holiday and/or dose reduction is not needed.
  • a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, is reduced, as a function of the disease or disorder, to a level at which the improved disease is retained.
  • patients require intermittent treatment on a long-term basis upon any recurrence of symptoms and/or infection.
  • the HAP composition for use in the method disclosed herein may be formulated in unit dosage form.
  • unit dosage form refers to physically discrete units suitable as unitary dosage for patients 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.
  • Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LDso (the dose lethal to 50% of the population) and the EDso (the dose therapeutically effective in 50% of the population).
  • the dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LDso and EDso.
  • the data obtained from ceil culture assays and animal studies are optionally used in formulating a range of dosage for use in human.
  • the dosage of such compounds can lie within a range of circulating concentrations that include the EDso with minimal toxicity.
  • the dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.
  • the HAP composition contemplated herein are formulated in a pharmaceutical composition using one or more pharmaceutically acceptable excipients or carriers.
  • the pharmaceutical compositions contemplated herein comprise a therapeutically effective amount of a HAP composition contemplated herein 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 can be formulated for single dosage administration.
  • the HAP is dissolved, suspended, dispersed or otherwise mixed in a selected vehicle at an effective combined concentration such that the treated condition is relieved or ameliorated.
  • Pharmaceutical carriers or vehicles suitable for administration of the HAP provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.
  • the present application is directed to a packaged pharmaceutical composition
  • a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound contemplated herein, alone or m combination with a second pharmaceutical agent; and instructions for using the compound to treat prevent, or reduce one or more symptoms of a disease or disorder contemplated in the present disclosure.
  • Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for any 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, weting 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., analgesic agents.
  • Additional dosage forms 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 also include dosage forms as described in U.S. Patent Applications Nos. 20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and 20020051820. Further dosage forms include dosage forms as described inPCT Applications Nos.
  • the formulations of the present application 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 bolus form.
  • the compounds may be formulated with a suitable polymer or hydrophobic material that provides sustained release properties to the compounds.
  • the compounds for use in the methods disclosed herein may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.
  • the compounds contemplated herein are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.
  • delayed release is used herein m 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, includes 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.
  • 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 the present disclosure and covered by the claims appended hereto.
  • 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 r educing/ oxidizing agents, with art-recognized alternatives and using no more than routine experimentation, are within the scope of the present application.
  • Example L Biological activity of EGFR compounds and release of trigger in hypoxic cancer cells; Cell Growth Inhibitory Activity
  • Example 2 Biological activity of EGFR compounds and release of trigger in hypoxic cancer cells: Cell Growth Inhibitory Activity Prodrugs of pan-erbB inhibitors
  • Prodrug compounds selected from the above-list were tested for their ability to inhibit the proliferation of three human carcinoma cell lines, selected to provide a comparison with literature precedent: A431 (epidermoid), which overexpresses erbBl (EGFR); HI 975 (non-small-cell lung), which overexpresses erbBl L858R/T790M a double mutant form of erbBl that is known to confer resistance to the approved reversible erbBl inhibitor erlotinib and SKOV3 (ovarian), which over express erbB2 (HER2).
  • A431 epidermoid
  • HI 975 non-small-cell lung
  • erbBl L858R/T790M a double mutant form of erbBl that is known to confer resistance to the approved reversible erbBl inhibitor erlotinib and SKOV3 (ovarian), which over express erbB2 (HER2).
  • the ceils were exposed to test compounds for either 24 hours under oxic conditions or for 4 hours under anoxia followed by 20 hours under oxic conditions. They were then washed tree of drug and incubated for a further 4 days, before being stained for cellular growth with suiforhodamine B The concentration of compound required to inhibit cellular growth by 50% relative to untreated control wells, termed the ICSQ value, was calculated. Results are summarised in Table 4.
  • Table 4 Inhibition of cellular proliferation A431 , H1975 and 8KOV3 cells a compound dose-response curves were determined at 10 concentrations. Ceils received a 24 hour exposure to test compounds before being washed (x3) with drug-free media. The IC50 (usnol/L) values are the concentrations required to inhibit cell growth by 50% relative to untreated controls. Values are the average of 2-8 independent determinations (%CV ⁇ 20 in all cases). 3 ⁇ 4 Experiment performed entirely under oxie conditions. c The initial 4 hours of the 24 hour drag exposure was performed under anoxic conditions. d Hypoxic Cytotoxicity Ratio - fold change in intra-experimental IC50 for cells receiving 4 hours of anoxia relative to cells that received only oxie conditions.
  • HCR hypoxic cytotoxicity ratios
  • mice Specific pathogen-free female NIH-III nude mice, derived from breeding mice supplied by Charles River Laboratories (Wilmington, MA), were housed in groups of 4-6 in a temperature- controlled room (22 ⁇ 2°C) with a 12-hour hght/dark cycle and were fed ad libitum water and a standard rodent diet (Harlan Teklad diet 2018i). All animals were uniquely identifiable by ear tag number.
  • Treatment was initiated when the tumours reached a volume of approximately 250 mm 3 , as determined by calliper measurement. All drugs were given by intraperitoneal injection at dosing volumes of 10-20 ml/kg. Mice w3 ⁇ 4re dosed at the MTD over a q3dx4, q5dx4 or q7dx4 schedule with tumour growth measured by callipers every 3-5 days over the 30-day duration of the study. Mice w r ere culled if they developed signs of toxicity or if body weight loss exceeded 20% of starting weight. All animal experiments followed protocols approved by the Animal Ethics Committee of The University of Auckland.
  • tumour bearing mice were assigned randomly to treatment groups when tumour diameter reached treatment size. Animals were rejected if xenografts show' evidence of: (i) attachment to underlying muscle (due to risk of local invasion), (ii) signs of ulceration, or (iii) indolent tumour growth. Drug administration begins on the day of assignment.
  • tumour size and body weights were measured regularly. Animals were culled if (i) the average diameter of the tumour exceeds 15 mm (survival endpoint), (ii) body weight loss exceeds 20% of pre-treatment value, (iii) there is evidence of prolonged or excessive morbidity, or (iv) tumour ulceration occurred. The experiment was terminated at day 21 (A431 tumours) or day 30 (Hi 975 tumours) after treatment initiation.
  • RTV 4 was recorded.
  • the median RTV 4 is calculated for each group and the difference in RTV 4 between control and treatment groups is described as the Tumour Growth Delay (TGD) in days.
  • TTD Tumour Growth Delay
  • Kaplan-Meier plots were constructed and median survival was calculated (TTEso). The statistical significance of any differences in overall survival time taken to reach RTV 4 between treatment groups and control was analysed by Log Rank P statistical test.
  • Weight loss nadirs time independent maxima were recorded for each treatment group. Any signs of treatment related morbidity were documented. Acceptable toxicity was defined as no mean group weight loss of over 15% during the test, no individual weight loss over 20% and no individual weight loss over 10% in any 24h period. All unscheduled deaths were recorded.
  • tumour growth delay was particularly significant for compounds 2-9 when administered at q3dx4 (P ⁇ 0.05, log- rank test) in both Hi 975 ( Figures 1-3) and A431 ( Figure 4) tumours. There was only minor body weight loss across all groups, although there was 1 death following treatment with compounds 5 and 7 in HI 975 xenograft mice (Table 5).
  • Prodrug 17 was tested at multiple dosing schedules; q3dx4, q5dx4 and q7dx4 at its q3dx4 MTD. At all 3 dosing schedules, compound 17 significantly delayed tumour growth in HI 975 tumours compared to controls ( Figure 11). There were no statistically significant differences in tumour growth between the 3 dosing schedules. Bodyweight loss was greatest after q3dx4 dosing and minimal after q7dx4 dosing (Table 6). However, 1 death w3 ⁇ 4s observed in the q7dx4 treatment group. It is not clear if tins death w3 ⁇ 4s drug-related or not.
  • Compound 17 was also administered at q3dx4 and q5dx4 at its q3dx4 MTD to A431 xenograft mice, with both dosing schedules causing similar delays in A431 tumour growth and similar losses in animal bodyweight (Table 7).
  • the in vitro and in vivo activity data illustrate the effectiveness of the compounds of the disclosure as kinase inhibitors.
  • the compounds are suitable for use in kinase-inhibitory therapy. This is the case with the reductive prodrugs and cancer therapy as tumours commonly have hypoxic regions.
  • the prodrugs are reduced under hypoxia to release the parent kinase inhibitor and produce a tumour-targeted effect.
  • Prodrugs 15, 17 and 18 exhibit effective therapeutic properties.
  • Figures 17-19 show that compounds of the disclosure can have efficacy in vivo.
  • Figure 18 shows that tarloxotinib can be effective at reducing tumors derived from a CLU-NRG1 fusion ovarian cancer in mouse xenograft models.
  • Example 5 Evaluating the cytotoxicity of three HAPs in parental (WT) neoplastic ceil lines and paired lines engineered to overexpress human STEAP4 cDNA [00567]
  • the following HAPs evaluated in this example are TH-302, Tirapazamine (TPZ), and Compound C, the structure for each prodrug is shown in Figure 20.
  • the open reading frame fORF for human STEAP4 transcript variant 1 (NlVl 024636.2) was cloned into the F279-V5 expression vector.
  • This provides transcription of a bicistronic mRN A from the human immediate-early cytomegalovirus (CMV) promoter, which encodes the open reading frames for the gene of interest and the pac (puromycin resistance) gene; the former harbouring an occult C-terminal V5-Tag inducible with TAG-On-Demand lM technology.
  • CMV immediate-early cytomegalovirus
  • Cells engineered to express STEAP4 were routinely cultured in media supplemented with 1 mM puromycin to maintain a selection pressure on the transfected cells, although puromycin was omitted from the media during experiments.
  • cells were harvested with Trypsm/EDTA and counted. The required number of cells was transferred to a 50 mL falcon tube and centrifuged to pellet cells (1000 rpm, 5 min). Excess media was removed by aspiration leaving cell pellets sitting in 50-100 m ⁇ media.
  • the cell number was calculated to ensure a seeding density of 400 cells per well of a 96-well plate, with cells resuspended in a-MEM + 10% FCS, -P/S + lOmM D-Glucose + 0.2mM 2’-deoxycytidme.
  • Cells were seeded under aerobic or anoxic conditions, the latter using a 5% H2/palladium catalyst scrubbed Bactron anaerobic chamber (Sheldon Manufacturing, Cornelius, Oregon) to achieve severe anoxia ( ⁇ 10 ppm Q?. gas phase) during prodrug exposure.
  • ICso value is the concentration of prodrug required to inhibit cell growth by 50%.
  • Example SA Evaluating the cytotoxicity of three HAPs in parental (WT) neoplastic cell lines and paired lines engineered to over express human STEAP4 cDNA
  • the open reading frame (ORF) for human STEAP4 transcript variant 1 (NM_024636.2) was cloned into the F279-V5 expression vector.
  • This provides transcription of a bicistronic mR A from the human immediate-early cytomegalovirus (CMV) promoter, which encodes the open reading frames for the gene of interest and the pac (puromycin resistance) gene; the former harbouring an occult C-terminal V5-Tag inducible with TAG-On-Demand‘ M technology.
  • CMV immediate-early cytomegalovirus
  • C33A Human cervical carcinoma (C33A) cells were transfected using Lipofectamine 3000 reagent complexed with F279-STEAP4-V 5 plasmid DNA. The transfection mix was added drop wise and after 48 hours the culture media was replaced with fresh a-MEM (with 5% FBS) medium containing 1 pM puromycin, escalating to 3 mM puromycin over time. Cells were grown to confluence m the wells before being pooled together and transferred to a cell culture flask of desired volume. Cells were maintained in a-MEM supplemented with 3 mM puromycin. Cell lines were designated (G33A % /E33A di ⁇ AR4 .
  • Table 8A Concentration ranges for prodrugs in the 96- well plate [00581] After 2 hours incubation to enable cell attachment to the plates (and equilibrate to anoxia in the ease of the anoxic samples), cells were exposed to a range of prodrug concentrations for 4 hours under aerobic or anoxic conditions. After prodrug exposure, plates were transferred to aerobic conditions. A vacuum immunowash was used to aspirate the media and wells were washed three times with 150 m ⁇ of prodrug-free aMEM 5% PCS + P/S. The plates were then incubated under aerobic conditions for 5 days for cell growth.
  • TCA trichloroacetic acid
  • ICso value is the concentration of prodrug required to inhibit cell growth by 50%.
  • Table 8B ICso values for Compound A, Compound C, Compound G (SN29176) and Compound H (8N27686) in wild-type (WT) and STEAP4-expressing C33A cells, where the concentration of prodrug required to inhibit cell growth by 50% (ICso) is shown, as described in Example 5.

Abstract

The present disclosure relates to prodrugs of pan-HER, HER2, HER4 and/or EGFR (HER1) inhibitory agents. The prodrugs can be covalently bonded to attenuating moieties such as reductive triggers (e.g., chemical moieties that can be cleaved in vivo). The prodrugs are useful in methods of treatment of the human or animal body.

Description

METHODS OF INHIBITING EPIDERMAL GROWTH FACTOR RECEPTOR
PROTEINS
Related Applications
[00011 This application claims priority to, and the benefit of, U.8. Application No. 62/950,217, filed December 19, 2019, the entire contents of which is incorporated herein by reference.
Field of the Invention
[0002] The present disclosure relates to prodrugs of pan-HER, HER2, HER4, and/or EGFR (HER1) inhibitory agents. The prodrugs can be covalently bonded to attenuating moieties such as reductive triggers (e.g., chemical moieties that can be cleaved in vivo). The prodrugs are useful in methods of treatment of the human or animal body.
Background
[00Q3] Kinases play a central role in the regulation of wide variety' of cellular processes which has led to the development of kinase inhibitors as therapeutic agents in the treatment of a wide range of disorders, including cancer. Treating cancer is challenging because it is difficult to kill cancer ceils while not affecting, or affecting to a lesser extent, normal cells. Killing or otherwise detrimentally affecting normal cells during cancer treatment can cause adverse side effects In patients. EGFR TKIs such erlotimb, gefitimb and afatinib have been approved for the treatment of several cancers including non-small cell lung cancer (see e.g Felip etal Clinical Cancer Research, 2008, 14: 3867-3874; Smith et al, 2008, Rr. J. Cancer, 98:1630-32; Hamilton et a!., 2006, Clin. Can. Res, 12: 2166-71), squamous cell cancer of the head and neck or skin (see e.g. Calvo et al Annals of Oncology 2007, J 8: 761-767); ovarian cancer (see e.g. Posada et al., Cancer, 2007, 109: 1323-1330). In addition, new drugs designed to target mutant forms of EGFR whilst sparing wild type EGFR like AZD9291, CO- 1686 and the like have been developed for the treatment of cancerous tumors (Cross etai, 2014, Can. Discov. 4; 1046).
[0004] However, cancer cells can differ from certain normal cells in their level of oxygenation and can be more hypoxic than normal cells. Hypoxia can induce adaptations in gene regulation and associated cellular functions, specifically epidermal growth factor receptor (EGFR; HER 1)
(Franovic et al 2007, PNAS; 104: 13092; Wang et al., 2009, Nature Med., 15: 319; Wang et al,
2010, Carcinogenesis, 31 :1202; Minakata et al, 2012, Cancer Sci; 1 03(11 ): 1946-1954). Tumor hypoxia up-regulates wild type EGFR protein and its cognate ligand TGFa via several HIE dependent mechanisms (Curr Pharm Des 2013; 19:907). One consequence of up-regulation of wild type EGFR protein and cognate ligand in cancer cells is induction of resistance to tyrosine kinase inhibitors (TKI) such as erlotimb, gefitmib or afatinib, which display preferential inhibitor}' activity towards specific activating EGFR mutant forms (Yun etal, 2008, Proc Natl Acad Sci; 105: 2070: Takezawa et al Cancer Discover 2012; 2: 922—33; Caniidge et al. Nat. Rev. Clin. Oncol. 2014; 11: 473- 481; Murakami et al 2014, Plos ONE 9(l):e86459). EGFR activating mutant positive non-small cell lung cancer (NSCLC) is often heterozygous (Soh J, etal. 2009, PLoS ONE 4(10): e7464; Bai et al, 2013, PLoS ONE 8: e54i 70), and the presence of the wild type allele is associated with limited response to TKI treatment regimens (Taniguchi et al, 2008, Cancer Sci; 99: 929-35). One possibility for overcoming this TKl-resistant state is to silence wild type EGFR signaling arising in hypoxic tumor regions, however conventional EGFR TKI lack the necessary therapeutic index to achieve this goal due to the dosing-limiting side effects associated with on- mechanism inhibition of wild type EGFR in the skin and gastrointestinal tract (Hynes et al, 2005, Nat Rev Can 5:341: Sharma et al, Clin. Cancer Res 2006; 12; 4392s-4395s: Sharma et al, Genes Dev. 2007 21; 3214-3231; Sharma et al, 2007 Nat Rev Can 7:169; Janjigian et al, Cancer Discovery' 2014, 4: 1 -10).
[00Q5] While progress has been made in this field, the totality of the available data indicate that significant non-responsiveness, due to EGFR genetic polymorphisms is undesirable in patients with cancer. Accordingly, new, safer and more effective methods for treating cancer that address EGFR resistance and non-responsive patients with EGFR genetic polymorphisms are needed, especially for high risk patients.
[0006] Certain agents have been made for treating cancer by targeting hypoxic cancer cells (see e.g., PCX Patent Publication Nos. WO 2010/ 104406 and WO 2011/028135, each of which is incorporated herein by reference) which disclose hypoxia -activated prodrugs (HAP) of EGFR TKI. One such example of such a FIAP EGFR TKI is ΊΉ-4000. Hypoxia-activated prodrugs (HAP) of EGFR TKI (exemplified by TH-4000) can overcome this mechanism of intrinsic resistance. [0007] There remains a need for compositions and methods for treating cancer. The present disclosure meets these needs by providing compositions and methods related to the use of the hypoxia activated nitroimidazole prodrug of a kinase inhibitor in therapy. The hypoxia-dependent metabolism provides tumor selective release of the EGFR TKI in hypoxic tumor cells. The present disclosure solves the resistance problem and provides methods for determining subjects who are at risk for resistance to EGFR treatment in patients with EGFR gene polymorphisms. The present disclosure satisfies this and other needs. The high local concentrations provide the necessary therapeutic index to silence wild type EGFR signaling in the hypoxic tumor compartment, as summarized below.
[0008| The present disclosure relates to pan-HER, HER2, HER4 and EGFR (HER1) small molecule inhibitors incorporating (e.g., covalently bonded to) an attenuating moiety such as a hypoxia-activated trigger moiety' to create prodrugs of the small molecule inhibitors. Compounds of the disclosure include prodrugs of known kinase inhibitors which have potent pan-HER, EGFR (FIERI), and/or HER4 activity'. However, because the inhibitors are bonded to an attenuating moiety, the inherent kinase inhibitory activity' can be greatly attenuated or reduced to a biologically insignificant level by incorporation of an attenuating moiety on an appropriate nucleophilic atom to act as a prodrug. The prodrug-kinase inhibitor conjugate has little or no on-target pharmacological or biochemical activity compared with the parent compound. The potent kinase inhibitor molecule can be released in cancer tumor tissue which is hypoxic and which expresses the metaloreductase 8TEAP4.
[0009] While the prodrug-kinase inhibitor conjugate may have systemic exposure across tissues in an animal or human, it has no or little on-target activity and therefore has greatly reduced systemic pharmacological or toxic effects. The active and potent kinase inhibitor with potent pan- HER, EGFR (HERl), HER2, and/or HER4 activity' can be released in the environment found typically in some solid tumors or cancers in which the cancer is driven by activated HER family receptors. The active kinase inhibitor can be further released in those cancers and cancer tumors which are in hypoxic environments and winch express STEAP4.
[0010] In the hypoxic environment, 8TEAP4 donates an electron to the prodrug or trigger which can cause the prodrug to release the active kinase inhibitor payload locally in the tumor. However, in sy stemic tissues winch are normoxic, the reductive activity of STEAP4 on the prodrug or trigger can be negated, greatly reducing or completely stopping release of the active kinase inhibitor outside of the cancer. This can result in targeted tumor concentrations of the active kinase inhibitor, with very low systemic exposure.
The compounds of the present disclosure exert anticancer activity with attenuated systemic toxicity and/or improved tolerability, allowing higher drug doses and higher local tumor concentrations of active kinase inhibitor than previously seen by other methods. The compounds described herein address an urgent and unmet medical need in mammals and humans suffering from HER receptor driven cancers. Moreover, these compounds can be administered to treat HER receptor driven cancers in which the ATP-competitive tyrosine kinase inhibitor binding site is wild type (WT). Compounds of the present disclos ure can exert antineoplastic activity in the cancer or tumor tissue, but not in HER receptors in healthy tissue thus reducing toxicity, improving dosing tolerability, and allowing higher concentrations of the active kinase inhibitor in the cancer tissue, thereby inhibiting the proliferation of tumor cells that overexpress these receptors. Many cancers with limited treatment options are driven by activated HER receptors with WT ATP binding sites, such as EXON20 and NRG fusions which active WT EGFR (HER1) and are highlighted in the following reviews, which are incorporated herein in their entireties: Murtuza, A. et. al. Cancer Research (2019), 79(4), 689-698; La Salvia, A. et al. Expert Opinion on Investigational Drugs (2019), 28(1), 29-38; Lu, X. et al. Medicinal Research Reviews (2018), 38(5), 1550-1581; Waring, M. J. et. al. Successful Drug Discovery' (2018), 3, 341-357; Tan, C.-S. et. al. Molecular Cancer (2018), 17, 29/1-29/14; Schroeder, R L. et al. Molecules (2014), 19(9), 15196-15212, 17 pp.; Wang, X. et. al. OncoTargets and Therapy (2016), 9, 5461-5473; Roskoski, R. Pharmacological Research (2019), 139, 395-411
Summary of the Invention
[0011] In some aspects, the present disclosure provides, EGFR inhibitors that are covalently bonded to attenuating agents, also referred to as attenuating moieties, such as reductive triggers to form prodrugs of the EGFR inhibitors. The reductive triggers can comprise electrophilic heterocycles and can be cleaved under anoxic conditions to afford the active EGFR inhibitors. [0012] The trigger which can be attached to the EGFR inhibitor is selected from those disclosed in WO2010/104406, which is incorporated herein by reference. Also, the prodrugs disclosed in WQ2010/NZ00174, which is incorporated herein by reference.
[0013] In the disclosed compounds (e.g, EGFR inhibitors), an asterisk marks a nucleophilic atom or group of atoms where an attenuating moiety, such as a reductive trigger, could be installed to form a prodrug. In some embodiments, the present disclosure also teaches one skilled m the art how to synthesize, prepare, and develop the prodrug and how to select a prodrug-kinase inhibitor conjugate which displays the necessar hypoxia-associated release of the active potent pan-HER, EGFR (FIERI), HER2 and/or HER4 kinase inhibitor. [0014] * Denotes an example nucleophilic nitrogen, oxygen or sulfur where a prodrug could be attached; either as positioned or on an available tautomeric position. Examples of attenuating moieties or trigger attachments:
* = N, O or S of a kinase inhibitor attached to a prodrug.
* = Kinase Inhibitor moiety attachments such as:
B B C
A-NH A— N A-V ( S
A nitrogen attachment trigges ^ trigger ^ trigger ^
A-0
An oxygen attachment Iri99er _
A-S \
A sulfur attachment trs99er wherein A, B, and C comprise the EGFR inhibitor
[0015] In some embodiments, the attenuating moiety or trigger may be attached at any available nitrogen on a small molecule HER inhibitor.
[0016] In some embodiments, the attenuating moiety or trigger may be attached at a nucleophilic nitrogen or a nucleophilic nitrogen which makes an ammonium salt
[0017] In some embodiments, the attenuating moiety or trigger may be attached at an aromatic ring (e.g. to form a cation).
[0018] In some embodiments, the attenuating moiety or trigger may be attached at a nitrogen which is part of an amide bond.
[0019] In some embodiments, the attenuating moiety or trigger may be attached at a dialkyl substituted nitrogen.
[0020] In some embodiments, the attenuating moiety or trigger may be attached at a monoalkyl substituted nitrogen.
[0021] In some embodiments, the attenuating moiety or trigger may be attached at an aromatic heterocyclic ring such as a pyridine, pyrazole or pyrimidine.
[0022] In some embodiments, the attenuating moiety or trigger may be attached at an aromatic heterocyclic ring such as a pyridine, pyrazole or pyrimidine in a position which is nucleophilic or can form a cation.
[0023] In some embodiments, the attenuating moiety or trigger may be attached at any other nucleophilic atom, such as an oxygen or phenol or sulfur or thiophenol. Brief Description of the Drawings
10024] The invention will now be described in more detail, with reference to the accompanying Figures, in which:
[0025] Figure 1 show's median HI 975 tumour growth after q3dx4 treatment with kinase inhibitors 1, 2, and 3 (n=3).
[0026] Figure 2 show's median HI 975 tumour growth after q3dx4 treatment with kinase inhibitors 4 and 5 (n=3).
[0027] Figure 3 show's median HI 975 tumour growth after q3dx4 treatment with kinase inhibitors 7 and 8 (n=3-4).
[0028] Figure 4 shows median A431 tumour growth after q3dx4 treatment with kinase inhibitors 5, 6, and 9 (n=3).
[0029] Figure 5 shows median H1975 tumour growth after q3dx4 treatment with prodrugs 12, 13, and 15 (n=3).
[0030] Figure 6 show's median H1975 tumour growth after q3dx4 treatment with prodrugs 16 and 17 (n=3).
[0031] Figure 7 show's median H1975 tumour growth after q3dx4 treatment with prodrugs 19 and 20 (n=3-4).
[0032] Figure 8 shows median A431 tumour growth after q3dx4 treatment with prodrugs 17, 18, and 21 (n::::3).
[0033] Figure 9 shows median HI 975 tumour growth after q3dx4 treatment with prodrug 15 and its cognate kinase inhibitor 3 (n==3).
[0034] Figure 10 shows median (A) and mean (B) H1975 tumour growth after q3dx4 treatment with prodrug 17 and its cognate kinase inhibitor 5 (n=3).
[0035] Figure 11 show's median H1975 tumour growth after treatment with prodrug 17 at q3dx4, q5dx4 and q7dx4 dosing schedules (n=3).
[0036] Figure 12 shows inhibition of erbBl (EGFR) autophosphorylation and p44/42 MAPK(Erk 1/2) phosphorylation in intact A431 cells following lhour treatment with a range of concentrations of compound 3. [0037] Figure 13 shows inhibition of erbBl (EGFR) autophosphorylation and p44/42 MAPK(Erk 1/2) phosphorylation in intact A431 cells following 1 hour treatment with a range of concentrations of compound 5.
[0038] Figure 14 shows inhibition of erbBl (EGFR) autophosphorylation and p44/42 MAPK(Erk 1/2) phosphorylation in intact A431 cells following 1 hour treatment with a range of concentrations of compound 6.
[0039] Figure 15 show's inhibition of erbBl (EGFR) autophosphorylation and p44/42 MAPK(Erk 1/2) phosphorylation in intact A431 cells following lhour treatment with a range of concentrations of prodrug 15.
[0040] Figure 16 is a graphical plot of band desitometry analysis of relative (b-actin corrected) inhibition of erbBl (EGFR) autophosphorylation in intact A431 cells following lhour treatment with a range of concentrations of prodrug 15 and compound 3.
[0041] Figure 17 is a CLU-NRGl patient-derived xenograft model. Figure 17A show's high expression of NRG1 m various PDX models. Figure 17B show's a schematic representation for CLU-NRGl fusion.
[0042] Figure 18 is a plot of the in vivo antitumor activity of tarloxotinib in CLU-NRGl fusion ovarian cancer PDX (OV- 10-0050) cells.
[0043] Figure 19 is microscopic images of hypoxia and STEAP4 levels in CLU-NRGl PDX models. Figure 19A is images of hypoxia in OV-10-0050 PDX tumors. Figure 19B is STEAP4 in-situ hybridization in OV-10-0050 PDX tumors.
[0044] Figure 20 shows the structures for the prodrugs TH-302 (also known as evofosfamide), tirapazamme (TPZ or SR4233), and Compound C (also known as PR- 104 A or SN27858), as referred to in Example 5.
[0045] Figure 21 is a graphic showing the layout of the 96-well plate, as described in Example 5 [0046] Figure 22 shows the ICso values for TH-302, TPZ, and Compound C in wild-type (WT) and STEAP4-expressing cells, where the concentration of prodrug required to inhibit cell growth by 50% (ICso) is shown, as described in Example 5.
[0047] Figure 23 shows a schematic depicting an exemplary STEAP4-mediated activation of Compound A. Detailed Description
[0048 | In some embodiments, the compounds of the present disclosure comprise a kinase inhibitor of Formula 1 and a reductive trigger, described m WO2010/0104406 which contents are incorporated by reference in its entirety. The reductive trigger can fragment when reduced to afford an active EGFR inhibitor. Fragmentation of the trigger occurs at the one-electron reduction level and is effectively suppressed by the presence of oxygen, thus providing selective activation in hypoxic environments. This suppression by oxygen may occur through reoxidation of the one- electron radical by oxygen, or by oxidation of reducing intermediates required for prodrug reduction. The latter would include, for example, scavenging by oxygen of radiation-induced reducing radicals such as the aquated electron, or oxidation of reducing intermediates in the catalytic cycle of reductase enzymes. The reduction equivalents required to reduce the trigger may be provided by enzymes, radiation-induced radicals, or chemical reducing agents
[0049] In some embodiments, the trigger is a reductive! y-aetivated aromatic nitroheterocycle or aromatic nitrocarbocycle trigger and is linked directly to a quatermsable nitrogen of a compound of Formula I such that a quaternary nitrogen is formed.
[005Q] In some embodiments, the EGFR inhibitor is a compound of Formula (I):
Figure imgf000009_0001
wherein either:
(T) Ri is H, and
(a) Rz is (3-chlorobenzyl)oxy- and R? is chloro;
(b) Rz and Rz, together with the carbon atoms to which they are attached, form l-(3- fluorobenzyl)- liT-pyrazole;
(c) Rz is 2-pyridinylmethoxy and Rz is chloro:
(d) Rz and Rz are both chloro;
(e) Rz is chloro and Rz is bromo;
(f) Rz and Rz are both bromo; (g) R?. is fluoro and R3 is ethynyi;
(h) Rz is chloro and R3 is ethynyi;
(1) R?. is bfomo and R3 is ethynyi;
(j) other than when R2 is in the 3-position in combination with R3 in the 4-position, R2 is bromo and R3 is fluoro;
(k) R2 is 2-pyridinylmethoxy and R3 is fluoro; or
(l) Rz is 2-pyridinylmethoxy and R3 is bromo; or
(2) at least one of Ri, R? and R3 is selected from benzyloxy, 3-chlorobenzyloxy and 2- pyridinylmethoxy and when at least one of Ri, Rz and R? is not benzyloxy, 3- chlorobenzyloxy or 2-pyridinylmethoxy, each of the others is independently selected from H, halogen, and C2-C4 alkynyl, with the proviso that when one of Ri, R2 and Rs is benzyloxy or 2-pyridinylmethoxy, the other two ofRi, R2 and R3 are not H; or
(3) two of Ri, R2 and R3, together with the carbon atoms to which they are attached, form 1- (3-fluorobenzyl)-l if-pyrazole, and the other is selected from H, halogen and C2-C4 alkynyl; or a pharmaceutically acceptable salt or solvate thereof.
[0051] In the disclosed compounds, an asterisk marks a nucleophilic atom or group of atoms where a hypoxia activated prodrug could he installed. In some embodiments, the present disclosure also teaches one skilled in the art how to synthesis, prepare, and develop the prodrug and how to select a prodrug-kinase inhibitor conjugate which displays the necessary hypoxia-associated release of the active potent pan-HER, EGFR (FIERI ), ITER2 and/or HER4 kinase inhibitor.
[0052] In some embodiments, the medicament is a prodrug and the compound of Formula I:
Figure imgf000010_0001
wherein either:
(4} Ri is H, and
(m)R · is (3-chlorobenzyl)oxy- and R3 is chloro; (n) R?. and Ra, together with the carbon atoms to which they are attached, form l-(3- fluorohenzy 1)- 1/i-pyrazole;
(o) R?. is 2-pyridinylmethoxy and Ra is chloro; ip) R2 and Ra are both chloro;
(q) R is chloro and Ra is bromo;
(r) Ra and Ra are both bromo;
(s) Ra is fluoro and R is ethynyl;
(t) Ra is chloro and Ra is ethynyl;
(u) Ra is bromo and Ra is ethynyl;
(v) other than when R is in the 3 -position in combination with Ra in the 4-position, R is bromo and R is fluoro;
(W)R2 is 2-pyndinylniethoxy and Ra is fluoro; or
(x) Ra is 2-pyridinylmethoxy and Ra is bromo; or
('5 } at least one of i, Ra and Ra is selected from benzyloxy, 3-ehiorobenzyloxy and 2- pyridmylmethoxy and when at least one of Ri, R2 and Ra is not benzyloxy, 3- chiorobenzyloxy or 2-pyridinylmethoxy, each of the others is independently selected from H, halogen, and C2-C4 alkynyl, with the proviso that when one of Ri, R2 and Ra is benzyloxy or 2-pyridinylmethoxy, the other two of Ri, R2 and Ra are not H; or (6) two of Ri, R2 and Ra, together with the carbon atoms to which they are attached, form 1- (3-fluorobenzyl)-l /-pyrazole, and the other is selected from H, halogen and C2---C4 alkynyl; or a pharmaceutically acceptable salt or solvate thereof.
[00S3] In some embodiments, the disclosure provides a reductive prodrug comprising a kinase inhibitor of Formula i as defined above, or a salt or solvate thereof, and a reductive trigger linked directly or indirectly to a nitrogen of the kinase inhibitor. j0054j In some embodiments, the attenuating moiety or trigger is Formula II:
Figure imgf000011_0001
II wherein * is a point of attachment said EGFR inhibitor, and where in Formula II Rs is selected from { :·( ··, alkyl and Rt is selected from H, methyl, ethyl, trifluoromethyl, -CN, -CONH2 and propyn-l-yl.
[0055] In some embodiments, the reductive trigger moieties of Formula 11 are suitable for use in the prodrugs of the present disclosure.
[0056] In some embodiments, the reductive trigger is selected from the group consisting of Formulae Ila
Figure imgf000012_0002
Figure imgf000012_0001
iia !!SJ He ik! lie Hi iig
[0057] In some embodiments, Rs is selected from methyl, ethyl and propyl
[0058] In some embodiments, R5 is methyl.
[0059] In some embodiments, the reductive trigger is of Formula Ila, wherein Rs is selected from methyl, ethyl and propyl.
[0060] In some embodiments, the reductive trigger is of Formula Ila, wherein Rs is methyl.
[0061] The a-methyl halides of Formula Ila may be prepared as described previously (bromide; Stribbling et al, PCT International patent publication WO 2008/039087) (chloride; Tercel et al, J Med Chem, 2001, 44, 3511-3522).
[0062] In some embodiments, attenuating moiety or trigger can be selected from one of the following Formulae a-q:
Figure imgf000012_0003
wherein:
* is the point of attachment to an EGFR inhibitor; 26 is selected from the group consisting of H, Ci-Ce alkyl, Ci-Ce alkoxy, C2-C6 alkenyl, C2-C6 alkynyi, (IT OCF3, F, Cl, Br, I, NO2, CN, COOH, COO(Cl-C6 alkyl), CONH2, CONHICi- Ce alkyl), CON(Ci-Ce alkyl)?., CO(Ci-Ce alkyl), SO2NH2, S0?NH(CI-C6 alkyl), 80?N(CI-C6 alkyl)?, SO?(Ci-Ce alkyl), and a group of Formula ilia, as defined above, wherein * is the point of attachment to an EGFR inhibitor;
R?7 IS selected from the group consisting of H andCi-C6 alkyl, as defined above, wherein * is the point of attachment to a group of Formula V; and
R?s is selected from the group consisting of H and Ci-Ce alkyl.
10063] In some embodiments, attenuating moiety or trigger is selected from a group of one of the following Formulas Vr-Vae:
Figure imgf000013_0001
y z aa ab ac ad ae
[0064] In some embodiments, Ri is a group of Formula c, wherein R?6 is H and R27 is CFF
[0065] In some embodiments, Ri is a group of Formula d, where R?s is selected from the group consisting of H, Ci-Ce alkyl (e.g., methyl), Ci-Ce alkoxy (e.g., OCH3), Ci-Ce alkynyl (e.g., ethynyl), CONH?, CONHMe, CF3, OCF3, Br, NO?, and CN, and R?7 is selected from the group consisting of CH3, CH?CH?C0N1:I?, and CH2CH2CN.
[0066] In some embodiments, the attenuating moiety or trigger may also be selected from groups of Formula Vd:
Figure imgf000013_0002
Formula Vd wherein
* is a point of attachment to the EGFR inhibitor, R?s is selected from H and C1-C3 alkyl, and R27 is selected from H and Ci-Cfi alkyl.
[0067] In some embodiments, R?e is H and R?? is Ci-C:? alkyl e.g., methyl. [0068] In some embodiments of Formula Vd, R2.6 is 1-propynyl and R27 is CH3.
[0069] In some embodiments, R2.6 is selected from H, Ci-Cc, alkyl (such as methyl or ethyl) and Ci-Cc, aikoxy (such as OCTI3), and R27 is CTI3.
[0070] In some embodiments, R26 is selected from H, Ci-Ce alkyl (e.g., methyl or ethyl) and Ci- Ci, alkoxy (e.g., OCH3), and R27 is CH3.
[0071] In some embodiments, the attenuating moiety is selected from Formulae Vd('!)-Vd(/):
Figure imgf000014_0001
Vd(1> Vd*2f Vd(3* Vd*4* Vd(5> Vd(S* Vd*7!
[0072] In some embodiments, R2.7 is selected from methyl, ethyl and propyl. In some embodiments R3.7 is methyl. R2 and R3 may form a ring selected from pyrrolidimum, piperidinium, piperazimum, Ni-rnethylpiperazimum and morpholinium.
Definitions
[0073] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, illustrative methods and materials are described.
[0074] As used herein, each of the following terms has the meaning associated with it in this section.
[0075] As used herein, the term “HER1”, refers to a transmembrane protein that is a receptor for members of the epidermal growth factor family. The term “HER!” may also refer to FIERI, ErbB- 1 , and EGFR.
[0076] .As used herein, the term “HER2”, refers to a transmembrane protein that is a receptor for members of the epidermal growth factor family. The term “HER2” ma also refer to ErbB-2, Erbb2, ERBB2, proto-oncogene Neu, and CD340.
[0077] As used herein, the term “HER3”, refers to a transmembrane protein that is a receptor for members of the epidermal growth factor family. The term “HER3” may also refer to ErbB-3 and ERBB3. Without wishing to be bound by theory', HER2 can activate HER3, and therefore in some embodiments inhibition of HER2 can attenuate HER3 activity. [0078] As used herein, the term “HER4”, refers to a transmembrane protein that is a receptor for members of the epidermal growth factor family. The term “HER4” may also refer to ErbB-4 and
ERBB4.
[0079] As used herein, the term “electrolyte”, refers to physiologically relevant free ions. Representative such free ions include, but are not limited to sodium (Na+), potassium ( K K calcium (Ca2+), magnesium (Mg2”), chloride (Cl-), phosphate P04J~), and bicarbonate (HCO3 ).
[0080] As used herein, the articles “a” and “an” are used to refer to one or to more than one (f.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
[0081] As used herein, “about,” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
[0082] A disease or disorder is “alleviated” if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced.
[0083] As used herein, the terms “alkyl”, “alkenyl”, “alkynyl” and “alkoxy” include both straight chain and branched chain groups, and unsubstituted and substituted groups. The optional substituents may include, without limitation, halogen, Ci-Ce alkoxy, CN, OH, NH2, NO2, NH(Ci- Ce alkyl), N(Ci-Ce alkyi) , CONH2, CO(Ci-G> alkyl), SO2NH2 and S()2(Ci-C6 alkyl).
[0084] As used herein, the term “aromatic mtroheterocycle” means an aromatic heterocyclic moiety substituted at any ring position by one or more nitro (NO2) groups. The aromatic heterocyclic moiety may be a monocyclic or bicyclic ring containing 4 to 12 atoms of which at least one atom is chosen from nitrogen, sulphur or oxygen. The aromatic heterocyclic moiety may be carbon or nitrogen linked. The aromatic heterocyclic moiety may additionally be substituted by one or more additional substituents at any available ring carbon or heteroatom. The substituents may include, but are not limited to the groups as defined for R2.6 in Formula V.
[0085] As used herein, the term “aromatic nitrocarbocycle” means a benzene moiety substituted at any position by one or more nitro (NO2) groups. In addition, two adjacent ring carbon atoms may optionally be linked to form a fused carbocychc or heterocyclic ring. The benzene moiety (and optional fused ring) may additionally be substituted by one or more additional substituents at any available carbon or heteroatom. The substituents may include, but are not limited to, the groups as defined for R26 in Formula V. 0086} As used herein, the terms “co-administered” and “co-administration” refer to administering to the subject a compound contemplated herein or salt thereof along with a compound that may also treat the disorders or diseases contemplated herein. In some embodiments, the co administered compounds are administered separately, or in any kind of combination as part of a single therapeutic approach. The co-administered compound may be formulated in any kind of combinations as mixtures of solids and liquids under a variety of solid, gel, and liquid formulations, and as a solution.
[0087] As used herein, the term “composition” or “pharmaceutical composition” refers to a mixture of at least one compound contemplated herein with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient or subject. Multiple techniques of administering a compound exist m the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, nasal, pulmonary and topical administration. [0088] A “disease” as used herein is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.
[0089] A “disorder” as used herein in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favourable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.
[0090] As used herein, the terms “effective amount,” “pharmaceutically effective amount” and “therapeutically effective amount” refer to a nontoxic but sufficient amount of a compound or agent to provide the desired biological result. That result may be reduction and/or alleviation of the signs, symptoms, or causes of a disease, 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. A therapeutic benefit or improvement need not be complete ablation of any one, most or all symptoms, complications, consequences or underlying causes associated with the disorder or disease. Thus, in some embodiments, a satisfactory endpoint is achieved when there is a transient, medium or long term, incremental improvement in a subject’s condition, or a partial reduction in the occurrence, frequency, severity, progression, or duration, or inhibition or reversal, of one or more associated adverse symptoms or complications or consequences or underlying causes, worsening or progression f e.g., stabilizing one or more symptoms or complications of the condition, disorder or disease), of the disorder or disease, over a duration of time (hours, days, weeks, months, and so forth).
[0091] As used herein, “likelihood”, “likely to”, and similar generally refers to an increase in the probability of an event. Thus, “likelihood”, “likely to”, and similar, when used m reference to responsiveness to cancer therapy, generally contemplates an increased probability that the individual will exhibit a reduction in the severity of cancer or the symptoms of cancer or the retardation or slowing of the cancer progression. The term “likelihood”, “likely to”, and similar, when used in reference to responsiveness to cancer therapy, can also generally mean the increase of indicators that may evidence an increase in cancer treatment.
[0092] The terms “patient,” “subject” or “individual” are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In non-limiting embodiments, the patient is a human. In some embodiments, the subject is a subject in need of treatment thereof.
[0093] 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, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
[0094] 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 disclosed herein or to the patient 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 compounds disclosed herein, and not injurious to the patient. 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; tale; 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.
[0095] 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 compounds disclosed herein, and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions.
[0096] The “pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compounds disclosed herein. Other additional ingredients that may be included in the pharmaceutical compositions disclosed herein 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.
[0097] As used herein, the language “pharmaceutically acceptable salt” refers to a salt of the administered compounds prepared from pharmaceutically acceptable non-toxic acids, including inorganic acids, organic acids, solvates, hydrates, or clathrates thereof.
[0098] The term “prevent,” “preventing” or “prevention,” as used herein, means avoiding or delaying the onset of symptoms associated with a disease or condition in a subject that has not developed such symptoms at the time the administering of an agent or compound commences. [0099] As used herein, the term “prodrug” refers to a compound that, after administration, is metabolised or otherwise converted to a biologically active or more active compound (or drug) with respect to at least one property. A prodrug, relative to the drug, is modified chemically in a manner that renders it, relative to the drug, less active or inactive, but the chemical modification is such that the corresponding drug is generated by metabolic or other biological processes after the prodrug is administered. A prodrug may have, relative to the active drug, altered metabolic stability or transport characteristics, fewer side effects or lower toxicity, or improved flavour (for example, see the reference Nogrady, 1985, Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392, incorporated herein by reference). A prodrug may be synthesized using reactants other than the corresponding drug..
[00100] A “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs.
[00101] The term “responsiveness” or “responsive,” when used in reference to a treatment, refers to the degree of effectiveness of the treatment in lessening or decreasing the symptoms of a disease, disorder, or condition being treated. For example, the term “increased responsiveness,” when used m reference to a treatment of a cell or a subject, refers to an increase in the effectiveness in lessening or decreasing the symptoms of the disease when measured using any methods known in the art. In some embodiments, the increase in the effectiveness is at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%.
[00102] As used herein, the term “treatment” or “treating” is defined as the application or administration of a therapeutic agent, i.e., a compound disclosed herein (alone or in combination with another pharmaceutical agent), to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient (e.g, for diagnosis or ex vivo applications), who has a condition contemplated herein, a symptom of a condition contemplated herein or the potential to develop a condition contemplated herein, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect a condition contemplated herein, the symptoms of a condition contemplated herein or the potential to develop a condition contemplated herein. Such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenonucs.
[00103] In some embodiments, the term “treatment” or “treating” refers to an action that occurs while an individual is suffering from the specified cancer, which reduces the severity of the cancer or the symptoms of the cancer, and/or retards or slows the progression of the cancer. For instance, m some embodiments, “treatment” or “treat” refers to a 5%, 10%, 25%, 50%, or 100% decrease in the rate of progress of a tumour. In some embodiments, “treatment” refers to a 5%, 10%, 25%, 50%, or 100% decrease m determined tumour burden (i.e., number of cancerous cells present in the individual, and/or the size of the tumour). In some embodiments, “treatment” refers to a 5%, 10%, 25%, 50%, or 100% decrease in any physical symptom(s) of a cancer. In some embodiments, “treatment” refers to a 5%, 10%, 25%, 50%, or 100% increase in the general health of the individual, as determined by any suitable means, such as cell counts, assay results, or other suitable means.
[00104] Throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description m range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the disclosure herein. Accordingly, 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. 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 etc., 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. Thus, for example, reference to a range of 90-100% includes 91-99%, 92-98%, 93-95%, 91-98%, 91-97%, 91-96%, 91-95%, 91-94%, 91-93%, and so forth. Reference to a range of 90-100% also includes 91%, 92%, 93%, 94%, 95%, 96%, 97%, etc , as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92 1%, 922%, 92.3%, 92.4%, 92.5%, and so forth. A senes of ranges are disclosed throughout this document. The use of a senes of ranges includes combinations of the upper and lower ranges to provide another range. This construction applies regardless of the breadth of the range and m all contexts throughout this patent document. Thus, for example, reference to a series of ranges such as 5-10, 10-20, 20-30, 30-40, 40-50, 50-75, 75-100, 100-150, includes ranges such as 5-20, 5-30, 5-40, 5-50, 5-75, 5-100, 5-150, and 10-30, 10-40, 10-50, 10-75, 10-100, 10-150, and 20-40, 20-50, 20-75, 20-100, 20-150, and so forth. This applies regardless of the breadth of the range.
Stmclures of Prodrugs or triggers
[00105] In some embodiments, the disclosure provides a reductive prodrug comprising a kinase inhibitor, e.g. an EGFR inhibitor, of Formula I as defined above, or a salt or solvate thereof, and a reductive trigger linked directly or indirectly to a nitrogen of the kinase inhibitor.
[00106 j In some embodiments, the reductive trigger is of Formula ΪΪ:
Figure imgf000020_0001
Formula II wherein * is a point of attachment to a nitrogen of said kinase inhibitor, and where in Formula II !¾ is selected from Ci-Cc, alkyl and R4 is selected from H, methyl, ethyl, trifluoromethyl, -CN, - CONH2 and propyn-l-yl.
[00107] In some embodiments, the reductive trigger is selected from the group consisting of Formulae Ila to ilg:
Figure imgf000021_0001
[00108] In some embodiments, Rs is selected from methyl, ethyl and propyl.
[001Q9] In some embodiments, Rs is methyl.
[00110] In some embodiments, the reductive trigger is of Formula Ila, wherein Rs is selected from methyl, ethyl and propyl.
[00111] In some embodiments, the reductive trigger is of Formula Ila, wherein R5 is methyl. [00112] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises AC480 (aka BMS-599626) or a pharmaceutically acceptable salt thereo
[00113] In some embodiments, the hypoxia activated prodrug is
Figure imgf000021_0002
, wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
[00114] In some embodiments, the hypoxia activated prodrug i
Figure imgf000022_0001
Figure imgf000022_0002
Figure imgf000022_0003
wherein R1 is an atenuating moiety such as a reductive trigger.
[00115] In some embodiments, the hypoxia activated prodrug
Figure imgf000022_0004
Figure imgf000022_0005
Figure imgf000022_0006
wherein R4 and R5 are defined above for Formula II. [00116] In some embodiments, the hypoxia activated prodrug i
Figure imgf000023_0001
Figure imgf000023_0002
[00117] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises AC0010MA (avitinib) or a pharmaceutically acceptable salt thereof.
[00118] In some embodiments, the hypoxia activated prodrug i
Figure imgf000023_0003
wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
Figure imgf000024_0001
Figure imgf000024_0002
, wherein R! is an attenuating moiety such as a reductive trigger.
[00120] In some embodiments, the hypoxia activated prodrug
Figure imgf000025_0001
Figure imgf000025_0002
Figure imgf000025_0003
, wherein R4 and R5 are defined above for Formula II.
[00121] In some embodiments, the hypoxia activated prodrug
Figure imgf000026_0001
Figure imgf000026_0002
[00122] In some embodiments, the EGER inhibitor bonded to an attenuating moiety comprises AEE788 or a pharmaceutically acceptable salt thereof.
[00123] In some embodiments, the hypoxia activated prodrug i
Figure imgf000026_0003
wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger. [00124] In some embodiments, the hypoxia activated prodrug i
Figure imgf000027_0001
Figure imgf000027_0002
wherein R1 is an attenuating moiety such as a reductive trigger.
[00125] In some embodiments, the hypoxia activated prodrug i
Figure imgf000027_0003
Figure imgf000027_0004
and R5 are defined above for Formula II.
Figure imgf000028_0001
[00127] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises .41,6802 (SIM 6802, Simotmib) or a pharmaceutically acceptable salt thereof.
[00128] In some embodiments, the hypoxia activated prodrug
Figure imgf000028_0002
, wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
[00129] In some embodiments, the hypoxia activated prodrug i
Figure imgf000028_0003
Figure imgf000028_0004
, wherein R! is an attenuating moiety such as a reductive trigger. 00130] In some embodiments, the hypoxia activated prodrug
Figure imgf000029_0001
Figure imgf000029_0002
wherein R4 and R5 are defined above for Formula 11.
[00131] In some embodiments, the hypoxia activated prodrug
Figure imgf000029_0003
Figure imgf000029_0004
[00132] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises Alflutinib (alflutimb mesylate) or a pharmaceutically acceptable salt thereof.
100133] In some embodiments, the hypoxia activated prodrug i
Figure imgf000029_0005
wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger. 00134] In some embodiments, the hypoxia activated prodrug i
Figure imgf000030_0001
Figure imgf000030_0002
wherein R1 is an attenuating moiety such as a reductive trigger.
Figure imgf000031_0001
Figure imgf000032_0001
[00137] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises AiuTibng (bngatimb) or a pharmaceutically acceptable salt thereof.
[00138] In some embodiments, the hypoxia activated prodrug is
Figure imgf000032_0002
wherein the * indicates a point of atta chment of an attenuating moiety such as a reductive trigger.
[00139] In some embodiments, the hypoxia activated prodrug
Figure imgf000033_0001
Figure imgf000033_0002
[00140] In some embodiments, the hypoxia activated prodrug
Figure imgf000034_0001
Figure imgf000034_0002
Figure imgf000035_0001
[00142] In some embodiments, the EGFR inhibitor bonded to an atenuating moiety' comprises AP23464 or a pharmaceutically acceptable salt thereof.
[00143] In some embodiments, the hypoxia activated prodrug
Figure imgf000035_0002
wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger. In some embodiments, the hypoxia activated prodrug is
Figure imgf000036_0001
Figure imgf000036_0002
wherein R1 is an attenuating moiety such as a reductive trigger.
[00145] In some embodiments, the hypoxia activated prodrug is
Figure imgf000036_0003
Figure imgf000036_0004
wherein R4 and R3 are defined above for Formula II.
Figure imgf000037_0001
[00147] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises Arava (iefiunomide) or a pharmaceutically acceptable salt thereof.
[00148] In some embodiments, the hypoxia activated prodrug
Figure imgf000037_0002
wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
[00149] In some embodiments, the hypoxia activated prodrug
Figure imgf000037_0003
wherein R i is an attenuating moiety such as a reductive trigger.
[00150] In some embodiments, the hypoxia activated prodrug
Figure imgf000037_0004
wherein R4 and R5 are defined above for Formula II. [00151] In some embodiments, the hypoxia activated prodrug
Figure imgf000038_0001
[00152] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises
ASP8273 (naquot ib) or a pharmaceutically acceptable salt thereof.
[00153] In some embodiments, the hypoxia activated prodrug
Figure imgf000038_0002
wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
00154] In some embodiments, the hypoxia activated prodrug
Figure imgf000038_0003
Figure imgf000038_0004
Figure imgf000039_0001
wherein R is an attenuating moiety such as a reductive trigger.
Figure imgf000039_0002
Formula II.
Figure imgf000040_0001
[00157] In some embodiments, the EGFR inhibitor bonded to an atenuating moiety' comprises AV-412 or a pharmaceutically acceptable salt thereof.
In some embodiments, the hypoxia activated prodrug
Figure imgf000040_0002
, wherein the * indicates a point of attachment of an attenuating moiety such as a reductive tugger. [00159] In some embodiments, the hypoxia activated prodrug is
Figure imgf000041_0001
Figure imgf000041_0002
wherein Rf is an atenuating moiety such as a reductive trigger.
Figure imgf000041_0003
and R3 are defined above for Formula II
[00161] In some embodiments, the hypoxia activated prodrug
Figure imgf000042_0001
Figure imgf000042_0002
In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises AZD3759 or a pharmaceutically acceptable salt thereof.
[001631 In some embodiments, the hypoxia activated prodrug i
Figure imgf000042_0003
, wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger. bodiments, the hypoxia activated prodrug
Figure imgf000042_0004
Figure imgf000042_0005
, wherein R1 is an attenuating moiety such as a reductive trigger. [00165] In some embodiments, the hypoxia activated prodrug is
Figure imgf000043_0001
or wherein R4 and R5 are defined above for Formula II. odiments, the hypoxia activated prodrug
Figure imgf000043_0003
Figure imgf000043_0002
[00167] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises AZD8931 or a pharmaceutically acceptable salt thereof.
Figure imgf000043_0004
[00168] In some embodiments, the hypoxia activated prodrug is , wherein the
* indicates a point of attachment of an attenuating moiety such as a reductive trigger. [00169] In some embodiments, the hypoxia activated prodrug is
Figure imgf000044_0001
Figure imgf000044_0002
wherein R1 is an attenuating moiety such as a reductive trigger.
In some embodiments, the hypoxia activated prodrug
Figure imgf000044_0003
Figure imgf000044_0004
wherein R4 and R5 are defined above for
Formula II.
In some embodiments, the hypoxia activated prodrug
Figure imgf000045_0001
Figure imgf000045_0002
[00172] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises BMS-690514 or a pharmaceutically acceptable salt thereof.
[00173] In some embodiments, the hypoxia activated prodrug
Figure imgf000045_0003
wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger. ia activated prodrug
Figure imgf000045_0004
Figure imgf000045_0005
wherein R1 is an atenuating moiety such as a reductive trigger. [00175] In some embodiments, the hypoxia activated prodrug
Figure imgf000046_0001
Figure imgf000046_0002
wherein R4 and R5 are defined above for
Formula II
[00176] In some embodiments, the hypoxia activated prodrug
Figure imgf000046_0003
Figure imgf000046_0004
[00177] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises BPI-7711 (Beta Pharma) or a pharmaceutically acceptable salt thereof.
[00178] In some embodiments, the hypoxia activated prodrug is BPI-7711 (Beta Pharma) or a pharmaceutically acceptable salt thereof conjugated to an attenuating moiety.
[00179] In some embodiments, the EGFR inhibitor bonded to an atenuating moiety' comprises Canertinib (Cl- 1033) or a pharmaceutically acceptable salt thereof. In some embodiments, the hypoxia activated prodrug
Figure imgf000046_0005
wherein the * indicates a point of attachment of an attenuating moiety such as a reductive tugger. [00181] In some embodiments, the hypoxia activated prodrug
Figure imgf000047_0001
Figure imgf000047_0002
wherein R1 is an attenuating moiety such as a reductive trigger.
[00182] In some embodiments, the hypoxia activated prodrug is
Figure imgf000047_0003
Figure imgf000047_0004
wherein R’ and R- are defined above for Formula ΪΪ.
In some embodiments, the hypoxia activated prodrug
Figure imgf000048_0001
Figure imgf000048_0002
[00184] In some embodiments, the EGFR inhibitor bonded to an atenuating moiety comprises Caprelsa (vandetanih) or a pharmaceutically acceptable salt thereof.
[00185] In some embodiments, the hypoxia activated prodrug i
Figure imgf000048_0003
wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger. diments, the hypoxia activated prodrug
Figure imgf000048_0004
Figure imgf000048_0005
, wherein R1 is an attenuating moiety such as a reductive trigger. [00187] In some embodiments, the hypoxia activated prodrug
Figure imgf000049_0001
wherein R4 and R5 are defined above for Formula P. iments, the hypoxia activated prodrug
Figure imgf000049_0003
Figure imgf000049_0002
[00189] In some embodiments, EGFR inhibitor bonded to an attenuating moiety comprises CK~ 101 (RX518) or a pharmaceutically acceptable salt thereof.
Figure imgf000050_0001
[00I9Q] In some embodiments, the hypoxia activated prodrug is wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
Figure imgf000050_0002
, wherein Rf is an attenuating moiety such as a reductive trigger.
00192] In some embodiments, the hypoxia activated prodrug
Figure imgf000050_0003
Figure imgf000050_0004
wherein R4 and R5 are defined above for Formula II. [00193] In some embodiments, the hypoxia activated prodrug
Figure imgf000051_0001
Figure imgf000051_0002
[00194] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises CP-724,714 or a pharmaceutically acceptable salt thereof.
Figure imgf000051_0003
[001951 In some embodiments, the hypoxia activated prodrug is , wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
In some embodiments, the hypoxia activated prodrug is
Figure imgf000052_0001
Figure imgf000052_0002
attenuating moiety such as a reductive trigger.
[00197] In some embodiments, the hypoxia activated prodrug
Figure imgf000052_0003
Figure imgf000052_0004
wherein R4 and R5 are defined above for Formula II.
In some embodiments, the hypoxia activated prodrug
Figure imgf000053_0001
Figure imgf000053_0002
[00199] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises CUDC-101 or a pharmaceutically acceptable salt thereof.
)] In some embodiments, the hypoxia activated prodrug is
Figure imgf000053_0003
, wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
[00201] In some embodiments, the hypoxia activated prodrug is
Figure imgf000053_0004
Figure imgf000053_0005
, wherein R1 is an attenuating moiety such as a reductive trigger. [00202] In some embodiments, the hypoxia activated prodrug IS
Figure imgf000054_0001
, or
Figure imgf000054_0002
, wherein R4 and R5 are defined above for Formula II.
[00203] In some embodiments, the hypoxia activated prodrug IS
Figure imgf000054_0004
[00204] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises El-I.N.T. (Transition Therapeutics) or a pharmaceutically acceptable salt thereof.
[00205] In some embodiments, the hypoxia activated prodrug is El-I.N.T. (Transition Therapeutics) or a pharmaceutically acceptable salt thereof conjugated to an attenuating moiety.
[00206] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises EGF816 (nazartimb) or a pharmaceutically acceptable salt thereof.
[00207] In some embodiments, the hypoxia activated prodrug
Figure imgf000054_0003
wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger. In some embodiments, the hypoxia activated prodrug is
Figure imgf000055_0001
Figure imgf000055_0002
wherein is an attenuating moiety such as a reductive trigger. j00209j In some embodiments, the hypoxia activated prodrug is
Figure imgf000055_0003
Figure imgf000055_0004
Formula 11.
[00210] In some embodiments, the hypoxia activated prodrug is
Figure imgf000055_0005
[00211] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises Epitmib (Hutchison China MediTech/Medi Pharma) or a pharmaceutically acceptable salt thereof. [00212] In some embodiments, the hypoxia activated prodrug is Epitimb (Hutchison China MediTech/Medi Pharma) or a pharmaceutically acceptable salt thereof conjugated to an attenuating moiety.
[00213] In some embodiments, the hypoxia activated prodrug is EKB-569 (WAY172569; Pelitinib) or a pharmaceutically acceptable salt thereof.
[00214] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises
Figure imgf000056_0001
, wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
[00215] In some embodiments, the hypoxia activated prodrug
Figure imgf000056_0002
Figure imgf000056_0003
wherein R1 is an attenuating moiety such as a reductive trigger.
In some embodiments, the hypoxia activated prodrug
Figure imgf000057_0001
Figure imgf000057_0002
wherein R4 and R5 are defined above for Formula II
[00217] In some embodiments, the hypoxia activated prodrug
Figure imgf000057_0003
Figure imgf000057_0004
[00218] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises E8-072 (APL 103; CBT Pharmaceuticals and Zhejiang Bossan Pharmaceutical) or a pharmaceutically acceptable salt thereof.
[00219] In some embodiments, the hypoxia activated prodrug is ES-072 (APL 103; CBT Pharmaceuticals and Zhejiang Bossan Pharmaceutical) or a pharmaceutically acceptable salt thereof conjugated to an attenuating moiety.
[00220] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises FV-225 or a pharmaceutically acceptable salt thereof. [00221] In some embodiments, the hypoxia activated prodrug
Figure imgf000058_0001
wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
[00222] In some embodiments, the hypoxia activated prodrug
Figure imgf000058_0002
Figure imgf000058_0003
as a reductive trigger.
[00223] In some embodiments, the hypoxia activated prodrug
Figure imgf000058_0004
Figure imgf000058_0005
wherein R4 and R5 are defined above for
Formula II.
Figure imgf000059_0001
[00225] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises Gilotrif (afatinib, BIBW 2992) or a pharmaceutically acceptable salt thereof.
[00226] In some embodiments, the hypoxia activated prodrug
Figure imgf000059_0002
wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
[00227] In some embodiments, the hypoxia activated prodrug is
Figure imgf000059_0003
Figure imgf000059_0004
wherein R1 is an attenuating moiety such as a reductive trigger. In some embodiments, the hypoxia activated prodrug
Figure imgf000060_0001
Figure imgf000060_0002
wherein R4 and R3 are defined above for
Formula II
In some embodiments, the hypoxia activated prodrug
Figure imgf000060_0003
Figure imgf000060_0004
[00230] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises GNS-1480 (aka lazertmib, YH25448) or a pharmaceutically acceptable salt thereof.
[00231] In some embodiments, the hypoxia activated prodrug i
Figure imgf000060_0005
, wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger. In some embodiments, the hypoxia activated prodrug
Figure imgf000061_0001
Figure imgf000061_0002
wherein R5 is an attenuating moiety such as a reductive trigger.
[00233 j In some embodiments, the hypoxia activated prodrug
Figure imgf000061_0003
Figure imgf000061_0004
wherein R4 and R1 are defined above for Formula II.
Figure imgf000061_0005
[00235] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises HER2 Allosteric Mutant Targeted Therapy (Black Diamond) or a pharmaceutically acceptable salt thereof. [00236] In some embodiments, the hypoxia activated prodrug is HER2 Allosteric Mutant Targeted Therapy (Black Diamond) or a pharmaceutically acceptable salt thereof conjugated to an attenuating moiety .
[00237] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises HER2GFR inhibitors or a pharmaceutically acceptable salt thereof.
[00238] In some embodiments, the hypoxia activated prodrug is HER2GFR inhibitors or a pharmaceutically acceptable salt thereof conjugated to an attenuating moiety.
[00239] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises HS-10296 or a pharmaceutically acceptable salt thereof conjugated to an attenuating moiety.
Figure imgf000062_0001
[00240] In some embodiments, the hypoxia activated prodrug is wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
[00241] In some embodiments, the hypoxia activated prodrug
Figure imgf000062_0002
Figure imgf000062_0003
wherein R1 is an attenuating moiety such as a reductive trigger.
[00242] In some embodiments, the hypoxia activated prodrug is
Figure imgf000062_0004
Figure imgf000063_0001
wherein R4 and R5 are defined above for Formula II
[00243] In some embodiments, the hypoxia activated prodrug
Figure imgf000063_0002
Figure imgf000063_0003
[00244] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises Xcotimb or a pharmaceutically acceptable salt thereof. "Y N'
X
Figure imgf000063_0004
[00245] In some embodiments, the hypoxia activated prodrug is ill , wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
[00246] In some embodiments, the hypoxia activated prodrug
Figure imgf000063_0005
, wherein R is an attenuating moiety such as a reductive trigger. [00247] In some embodiments, the hypoxia activated prodrug i
Figure imgf000064_0001
wherein R4 and R3 are defined above for Formula II
[00248] In some embodiments, the hypoxia activated prodrug i
Figure imgf000064_0002
[00249] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises Iressa (gefitmib).
[00250] In some embodiments, the hypoxia activated prodrug i
Figure imgf000064_0003
, wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
[00251] In some embodiments, the hypoxia activated prodrug
Figure imgf000064_0004
or
Figure imgf000064_0005
, wherein R1 is an attenuating moiety such as a reductive trigger.
[00252] In some embodiments, the hypoxia activated prodrug
Figure imgf000065_0001
Figure imgf000065_0003
[00254] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises KD020 or a pharmaceutically acceptable salt thereof.
Figure imgf000065_0002
[00255] In some embodiments, the hypoxia activated prodrug is wherein the * indicates a point of atachment of an atenuating moiety such as a reductive trigger. [00256] In some embodiments, the hypoxia activated prodrug
Figure imgf000066_0001
Figure imgf000066_0002
wherein R1 is an attenuating moiety such as a reductive trigger.
[00257] In some embodiments, the hypoxia activated prodrug is
Figure imgf000066_0003
Figure imgf000066_0004
wherein R4 and R5 are defined above for Formula II.
In some embodiments, the hypoxia activated prodrug
Figure imgf000067_0001
Figure imgf000067_0002
In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises Neriynx (neratmib, HKI-272 PB272) or a pharmaceutically acceptable salt thereof.
[00260 j In some embodiments, the hypoxia activated prodrug i
Figure imgf000067_0003
wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
In some embodiments, the hypoxia activated prodrug
Figure imgf000067_0004
Figure imgf000067_0005
wherein
R is an attenuating moiety such as a reductive trigger.
Figure imgf000068_0001
wherein R4 and R5 are defined above for Formula II.
[00263] In some embodiments, the hypoxia activated prodrug
Figure imgf000068_0002
Figure imgf000068_0003
[00264] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises Qhnutinib (HM-61713; BI 1482694) or a pharmaceutically acceptable salt thereof.
Figure imgf000069_0001
[00265] In some embodiments, the hypoxia activated prodrug is wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
In some embodiments, the hypoxia activated prodrug
Figure imgf000069_0002
Figure imgf000069_0003
wherein R1 is an attenuating moiety such as a reductive trigger.
[00267] In some embodiments, the hypoxia activated prodrug i
Figure imgf000069_0004
Figure imgf000069_0005
Figure imgf000069_0006
R5 are defined above for Formula II.
Figure imgf000070_0001
[00269] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises
Neratimb (aka PB357, HKI-272) or a pharmaceutically acceptable salt thereof.
[00270] In some embodiments, the hypoxia activated prodrug
Figure imgf000070_0002
wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
[00271] In some embodiments, the hypoxia activated prodrug
Figure imgf000071_0001
Figure imgf000071_0002
Figure imgf000071_0003
, wherein R1 is an attenuating moiety such as a reductive trigger.
[00272] In some embodiments, the hypoxia activated prodrug
Figure imgf000072_0001
Figure imgf000072_0002
.
Figure imgf000073_0001
[00274] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises PF-Q6459988 or a pharmaceutically acceptable salt thereof.
100275] In some embodiments, the hypoxia activated prodrug i
Figure imgf000073_0002
, wherein the
* indicates a point of attachment of an atenuating moiety such as a reductive trigger. [00276] In some embodiments, the hypoxia activated prodrug is
Figure imgf000074_0001
Figure imgf000074_0002
wherein R3 is an attenuating moiety such as a reductive trigger.
[00277] In some embodiments, the hypoxia activated prodrug
Figure imgf000074_0003
Figure imgf000074_0004
defined above for Formula II In some embodiments, the hypoxia activated prodrug is
Figure imgf000075_0001
Figure imgf000075_0005
[00279] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises Mavelertinib (PF-06747775) or a pharmaceutically acceptable salt thereof.
[00280] In some embodiments, the hypoxia activated prodrug i
Figure imgf000075_0002
, wherein the
* indicates a point of attachment of an attenuating moiety such as a reductive trigger.
[00281] In some embodiments, the hypoxia activated prodrug
Figure imgf000075_0003
Figure imgf000075_0004
wherein R is an attenuating moiety such as a reductive trigger. [00282] In some embodiments, the hypoxia activated prodrug
Figure imgf000076_0001
Figure imgf000076_0002
wherein R4 and R5 are defined above for Formula
II.
[00283] In some embodiments, the hypoxia activated prodrug
Figure imgf000076_0003
Figure imgf000076_0004
[00284] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises Pirotimb (KBP5209; CAS# 1799730-21 -4; Sihuan Pharmaceutical Holdings Group Ltd./ XuanZhu Pharma, Hong Kong) or a pharmaceutically acceptable salt thereof.
[00285] In some embodiments, the hypoxia activated prodrug is Pirotmib (KBP5209; CAS# 1799730-21-4; Sihuan Pharmaceutical Holdings Group Ltd./ XuanZhu Pharma, Hong Kong) or a pharmaceutically acceptable salt thereof conjugated to an attenuating moiety.
[00286] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises Poziotinib or a pharmaceutically acceptable salt thereof. [00287] In some embodiments, the hypoxia activated prodrug i
Figure imgf000077_0001
, wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
[00288] In some embodiments, the hypoxia activated prodrug i
Figure imgf000077_0002
wherein R1 is an attenuating moiety such as a reductive trigger. In some embodiments, the hypoxia activated prodrug
Figure imgf000077_0003
wherein R4 and R5 are defined above for Formula II.
[00290] In some embodiments, the hypoxia activated prodrug i
Figure imgf000077_0004
[00291] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises Pyrotimb (SHR-1258) or a pharmaceutically acceptable salt thereof.
In some embodiments, the hypoxia activated prodrug
Figure imgf000077_0005
wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
Figure imgf000078_0001
moiety such as a reductive trigger.
Figure imgf000078_0002
above for Formula II.
[00295] In some embodiments, the hypoxia activated prodrug
Figure imgf000079_0001
Figure imgf000079_0002
[00296] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises Rociletinib (CO 1686; AVL3Q1, CNX419) or a pharmaceutically acceptable salt thereof.
[00297] In some embodiments, the hypoxia activated prodrug i
Figure imgf000079_0003
wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
In some embodiments, the hypoxia activated prodrug
Figure imgf000080_0001
Figure imgf000080_0002
Figure imgf000080_0003
wherein R* is an attenuating moiety such as a reductive trigger.
[00299] In some embodiments, the hypoxia activated prodrug i
Figure imgf000081_0001
Figure imgf000081_0002
Figure imgf000081_0003
, wherein R4 and R5 are defined above for Formula II.
Figure imgf000082_0001
[00301] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises RXDX-105 (CEP 32496; AC013773, Agerafenib) or a pharmaceutically acceptable salt thereof.
[00302] In some embodiments, the hypoxia activated prodrug
Figure imgf000082_0002
wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
[00303] In some embodiments, the hypoxia activated prodrug
Figure imgf000083_0001
Figure imgf000083_0002
wherein R is an atenuating moiety such as a reductive trigger. hypoxia activated prodrug i
Figure imgf000083_0003
Figure imgf000083_0004
wherein R4 and R3 are defined above for Formula If
00305] In some embodiments, the hypoxia activated prodrug
Figure imgf000083_0007
Figure imgf000083_0005
EGFR inhibitor bonded to an attenuating moiety comprises S~ 222611 (Epertimb) or a pharmaceutically acceptable salt thereof.
[00307] In some embodiments, the hypoxia activated prodrug i
Figure imgf000083_0006
, wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger. [00308] In some embodiments, the hypoxia activated prodrug
Figure imgf000084_0001
Figure imgf000084_0002
wherein R1 is an attenuating moiety such as a reductive trigger
[003Q9] In some embodiments, the hypoxia activated prodrug is
Figure imgf000084_0003
are defined above for Formula II.
[00310] In some embodiments, the hypoxia activated prodrug is
Figure imgf000084_0004
[00311] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises Sirotimb (Shandong Xuanzhu, acquired by Sihuan) or a pharmaceutically acceptable salt thereof. [00312] In some embodiments, the hypoxia activated prodrug is Sirotinib (Shandong Xuanzhu, acquired by Sihuan) or a pharmaceutically acceptable salt thereof conjugated to an attenuating moiety.
[00313] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises Tagrisso (osimertinib, AZD-9291, Mereletmib) or a pharmaceutically acceptable salt thereof. [00314] In some embodiments, the hypoxia activated prodrug
Figure imgf000085_0001
wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger
[00315] In some embodiments, the hypoxia activated prodrug
Figure imgf000085_0002
Figure imgf000085_0003
Figure imgf000085_0004
wherein R1 is an attenuating moiety such as a reductive trigger.
[00316] In some embodiments, the hypoxia activated prodrug
Figure imgf000085_0005
Figure imgf000085_0006
Figure imgf000085_0007
wherein R4 and R5 are defined above for Formula II. [00317] In some embodiments, the hypoxia activated prodrug
Figure imgf000086_0001
Figure imgf000086_0002
[00318] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises TAK-788 (AP32788; ARIAD Pharmaceuticals/Mill enmum Pharmaceuticals, Inc , USA) or a pharmaceutically acceptable salt thereof.
[00319] In some embodiments, the hypoxia activated prodrug
Figure imgf000086_0003
(WO2015195228A1 ), wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
Figure imgf000087_0001
wherein R1 is an atenuating moiety such as a reductive trigger.
[00321] In some embodiments, the hypoxia activated prodrug
Figure imgf000087_0002
Figure imgf000087_0003
Figure imgf000088_0001
In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises Tarceva (er!otinib, CP358774; 081-774) or a pharmaceutically acceptable salt thereof.
[00324] In some embodiments, the hypoxia activated prodrug i
Figure imgf000088_0002
, wherein the
* indicates a point of attachment of an attenuating moiety such as a reductive trigger.
[00325] In some embodiments, the hypoxia activated prodrug
Figure imgf000088_0003
, wherein R1 is an attenuating moiety such as a reductive trigger.
[00326] In some embodiments, the hypoxia activated prodrug i
Figure imgf000088_0004
, wherein R4 and R3 are defined above for Formula II. [00327] In some embodiments, the hypoxia activated prodrug
Figure imgf000089_0001
[00328] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises
TAS0728 or a pharmaceutically acceptable salt thereof.
Figure imgf000089_0002
[00329] In some embodiments, the hypoxia activated prodrug is wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
In some embodiments, the hypoxia activated prodrug
Figure imgf000089_0003
Figure imgf000089_0004
wherein R1 is an attenuating moiety such as a reductive trigger.
[00331] In some embodiments, the hypoxia activated prodrug
Figure imgf000090_0001
Figure imgf000090_0002
wherein R4 and R:’ are defined above for Formula II.
[00332] In some embodiments, the hypoxia activated prodrug
Figure imgf000090_0003
Figure imgf000090_0004
[00333] In some embodiments, the EGFR inhibitor bonded to an atenuating moiety comprises TAS-121 (Taiho Pharmaceutical Co., Ltd) or a pharmaceutically acceptable salt thereof.
[00334] In some embodiments, the hypoxia activated prodrug is TAS-121 (Taiho Pharmaceutical Co., Ltd) or a pharmaceutically acceptable salt thereof conjugated to an attenuating moiety. [0033S] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises TAS6417 (WO2015025936; Taiho Pharmaceutical ) or a pharmaceutically acceptable salt thereof. [00336] In some embodiments, the hypoxia activated prodrug i
Figure imgf000091_0001
wherein the
* indicates a point of attachment of an atenuating moiety such as a reductive trigger.
[00337] In some embodiments, the hypoxia activated prodrug is
Figure imgf000091_0002
Figure imgf000091_0003
wherein R1 is an atenuating moiety such as a reductive trigger.
[00338] In some embodiments, the hypoxia activated prodrug
Figure imgf000091_0004
or
Figure imgf000091_0005
, wherein R4 and R5 are defined above for Formula II.
[00339] In some embodiments, the hypoxia activated prodrug is
Figure imgf000092_0001
or
Figure imgf000092_0002
[00340] In some embodiments, the EGFR inhibitor bonded to an atenuating moiety comprises Tesevatinib or a pharmaceutically acceptable salt thereof.
[00341] In some embodiments, the hypoxia activated prodrug
Figure imgf000092_0003
, wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
00342] In some embodiments, the hypoxia activated prodrug
Figure imgf000092_0004
or
Figure imgf000092_0005
, wherein R is an attenuating moiety such as a reductive trigger. [00343] In some embodiments, the hypoxia activated prodrug
Figure imgf000093_0001
Figure imgf000093_0002
wherein R4 and R5 are defined above for Formula II.
Figure imgf000093_0003
[00345] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises Theiiatimb or a pharmaceutically acceptable salt thereof.
[00346] In some embodiments, the hypoxia activated prodrug
Figure imgf000093_0004
wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger. [00347] In some embodiments, the hypoxia activated prodrug is
Figure imgf000094_0001
Figure imgf000094_0002
wherein R1 is an attenuating moiety such as a reductive trigger.
[00348] In some embodiments, the hypoxia activated prodrug i
Figure imgf000094_0003
Figure imgf000094_0004
wherein R4 and R5 are defined above for Formula II
[00349] In some embodiments, the hypoxia activated prodrug
Figure imgf000094_0005
Figure imgf000094_0006
[00350] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises TTI-234! (Trillium Therapeutics) or a pharmaceutically acceptable salt thereof. [00351] In some embodiments, the hypoxia activated prodrug is TTI-2341 (Trillium Therapeutics) or a pharmaceutically acceptable salt thereof conjugated to an attenuating moiety.
[00352] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises Tucatmib or a pharmaceutically acceptable salt thereof.
[00353] In some embodiments, the hypoxia activated prodrug i
Figure imgf000095_0001
, wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
[00354] In some embodiments, the hypoxia activated prodrug
Figure imgf000095_0002
or
Figure imgf000095_0003
wherein R! is an attenuating moiety such as a reductive trigger.
Figure imgf000095_0004
Figure imgf000095_0005
, wherein R4 and R5 are defined above for Formula II. [00356] In some embodiments, the hypoxia activated prodrug
Figure imgf000096_0001
Figure imgf000096_0002
[00357] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises Tykerb (lapatimb, GSK 572016, GW2016) or a pharmaceutically acceptable salt thereof.
[00358] In some embodiments, the hypoxia activated prodrug
Figure imgf000096_0003
wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
[00359] In some embodiments, the hypoxia activated prodrug
Figure imgf000096_0004
Figure imgf000096_0005
, wherein R1 is an attenuating moiety such as a reductive trigger. ents, the hypoxia activated prodrug
Figure imgf000097_0001
Figure imgf000097_0002
, wherein R4 and R5 are defined above for Formula II
[00361] In some embodiments, the hypoxia activated prodrug
Figure imgf000097_0003
Figure imgf000097_0004
Vvv
N
Figure imgf000097_0005
In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises Yarlitimb (ASLAN-001; ARRY334543; ARRY543) or a pharmaceutically acceptable salt thereof.
[00363] In some embodiments, the hypoxia activated prodrug
Figure imgf000097_0006
wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger. [00364] In some embodiments, the hypoxia activated prodrug
Figure imgf000098_0001
Of
Figure imgf000098_0003
.
Figure imgf000098_0002
[00367] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises Vizimpro (dacomitinib, PF -299804; PF-299) or a pharmaceutically acceptable salt thereof. [00368] In some embodiments, the hypoxia activated prodrug i
Figure imgf000099_0001
wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger.
[00369] In some embodiments, the hypoxia activated prodrug
Figure imgf000099_0002
Figure imgf000099_0003
wherein R1 is an attenuating moiety such as a reductive trigger.
[00370] In some embodiments, the hypoxia activated prodrug i
Figure imgf000099_0004
Figure imgf000099_0005
wherein R4 and R5 are defined above for Formula II.
[00371] In some embodiments, the hypoxia activated prodrug IS
Figure imgf000100_0003
[00372] In some embodiments, the EGFR inhibitor bonded to an attenuating moiety comprises WZ4002 or a pharmaceutically acceptable salt thereof.
[00373] In some embodiments, the hypoxia activated prodrug is
Figure imgf000100_0001
, wherein the * indicates a point of attachment of an attenuating moiety such as a reductive trigger. some embodiments, the hypoxia activated prodrug is
Figure imgf000100_0002
an attenuating moiety such as a reductive trigger. [00375] In some embodiments, the hypoxia activated prodrug is
Figure imgf000101_0001
wherein R4 and R5 are defined above for Formula II.
[00376] In some embodiments, the hypoxia activated prodrug is
Figure imgf000101_0002
[00377] In one aspect, provided herein is the method of treating or preventing a pan-HER, HER2 and/or EGFR (HER1) driven cancer in a subject in need thereof, with a hypoxia-activated prodrug or trigger moiety.
[00378] In some embodiments, the subject is mammal.
[00379] In some embodiments, the subject is human.
[00380] In some embodiments, the subject is rodent. In some embodiments, the subject is bovine. In some embodiments, the subject is porcine. In some embodiments, the subject is rabbit. In some embodiments, the subject is dog. In some embodiments, the subject is monkey. [00381] In one aspect, provided herein is the method of inhibiting epidermal growth factor receptor in a subject in need thereof, comprising: administering to the subject an effective amount of an epidermal growth factor receptor- mhibitmg drug, wherein the drug is covalently bonded to an attenuating moiety.
[00382] In some embodiments, the attenuating moiety is a reductive trigger of the Formula (II):
Figure imgf000102_0001
[00383] In some embodiments, the attenuating moiety is a reductive trigger selected from the group consisting of a compound of the Formula Ha. lib, lie, lid, lie, Ilf, or Ilg:
Figure imgf000102_0002
!ia iib Sic iid i!e ilf iig
In some embodiments, the attenuating moiety is a reductive trigger selected from the group consisting of a compound of the Formula a-ae:
Figure imgf000102_0003
[00385] In some embodiments, the epidermal growth factor receptor-inhibiting drug is selected from the group consisting of:
Figure imgf000103_0001
Figure imgf000104_0001
Figure imgf000105_0001
Figure imgf000106_0001
Figure imgf000107_0001
Figure imgf000108_0001
Figure imgf000109_0001
Figure imgf000110_0001
Figure imgf000111_0001
is an attenuating moiety such as a reductive trigger.
[00386] In some embodiments, the epidermal growth factor receptor-inhibiting drug is selected from the group consisting of: no
Figure imgf000112_0001
Figure imgf000113_0001
Figure imgf000114_0001
Figure imgf000115_0001
Figure imgf000116_0001
Figure imgf000117_0001
Figure imgf000118_0001
Figure imgf000119_0001
Figure imgf000120_0001
Figure imgf000121_0001
Figure imgf000122_0001
Figure imgf000123_0002
[00387] In some embodiments, the epidermal growth factor receptor-inhibiting drug is selected from the group consisting of:
Figure imgf000123_0001
Figure imgf000124_0001
Figure imgf000125_0001
Figure imgf000126_0001
Figure imgf000127_0001
Figure imgf000128_0001
Figure imgf000129_0001
Figure imgf000130_0001
Figure imgf000131_0001
Figure imgf000132_0001
Figure imgf000133_0001
Figure imgf000134_0001
[00388] In some embodiments, the epidermal growth factor receptor is HEM.
[00389] In some embodiments, the epidermal growth factor receptor is HER2.
[00390] In some embodiments, the epidermal growth factor receptor is HER4.
[00391] In some embodiments, the attenuating moiety is cleaved from the drug in vivo.
[00392] In some embodiments, the attenuating moiety is cleaved from the drug under anoxic conditions.
[00393] In some embodiments, the attenuating moiety is cleaved in the presence of 8TΈAR4. [00394] In some embodiments, the attenuating moiet reduces or inhibits activity of the epidermal growth factor receptor-inhibiting drug outside of a tumor environment.
[00395] In some embodiments, the attenuating moiety is cleaved from the drug within a tumor environment.
[00396] In some embodiments, the epidermal growth factor receptor exhibits a wild-type ATP- eompetitive tyrosine kinase binding site.
[00397] In some embodiments, the epidermal growth factor receptor is a mutated epidermal growth factor receptor.
[00398] In some embodiments, the epidermal growth factor receptor exhibits an upregulation mutation.
[00399] In some embodiments, the epidermal growth factor receptor exhibits a constitutively activating mutation. [00400] In some embodiments, the epidermal growth factor receptor exhibits a downregulation mutation.
[00401] In one aspect, provided herein is the method of treating a cancer in a subject m need thereof, comprising: administering to the subject an effective amount of an epidermal growth factor receptor- inhibiting drug, wherein the drug is covalently bonded to an attenuating moiety.
[00402] In some embodiments, the attenuating moiety is a reductive trigger of the Formula (II):
Figure imgf000135_0001
[00403] In some embodiments, the attenuating moiety is a reductive trigger selected from the group consisting of a compound of the Formula Ha. lib, lie, lid, He, Ilf, or Ilg:
Figure imgf000135_0002
i!a lib !ic iid i!e iif Mg
[00404] In some embodiments, the attenuating moiety is a reductive trigger selected from the group consisting of a compound of the Formula a-ae:
Figure imgf000135_0003
Figure imgf000136_0001
[00405] In some embodiments, the epidermal growth factor receptor-inhibiting drug is selected from the group consisting of:
Figure imgf000136_0002
Figure imgf000137_0001
Figure imgf000138_0001
Figure imgf000139_0001
Figure imgf000140_0001
Figure imgf000141_0001
Figure imgf000142_0001
Figure imgf000143_0001
Figure imgf000144_0001
is an attenuating moiety such as a reductive trigger.
[00406] In some embodiments, the epidermal growth factor receptor-inhibiting drug is selected from the group consisting of:
Figure imgf000145_0001
Figure imgf000146_0001
Figure imgf000147_0001
Figure imgf000148_0001
Figure imgf000149_0001
Figure imgf000150_0001
Figure imgf000151_0001
Figure imgf000152_0001
Figure imgf000153_0001
Figure imgf000154_0001
Figure imgf000155_0001
Figure imgf000156_0002
[00407] In some embodiments, the epidermal growth factor receptor-inhibiting drug is selected from the group consisting of:
Figure imgf000156_0001
Figure imgf000157_0001
Figure imgf000158_0001
Figure imgf000159_0001
Figure imgf000160_0001
Figure imgf000161_0001
Figure imgf000162_0001
Figure imgf000163_0001
Figure imgf000164_0001
Figure imgf000165_0001
Figure imgf000166_0001
Figure imgf000167_0001
[00408] In some embodiments, the drug inhibits HERE [00409] In some embodiments, the drug inhibits HER2.
[00410] In some embodiments, the drug inhibits HERA
[00411] In some embodiments, the attenuating moiety is cleaved m vivo.
[00412] In some embodiments, the attenuating moiety is cleaved from the drug under anoxic conditions.
[00413] In some embodiments, the attenuating moiety is cleaved in the presence of 8TΈAR4. [00414] In some embodiments, the attenuating moiet reduces or inhibits activity of the epidermal growth factor receptor-inhibiting drug outside of a tumor environment.
[00415] In some embodiments, the attenuating moiety is cleaved from the drug within a tumor environment.
[00416] In some embodiments, the epidermal growth factor receptor exhibits a wild-type ATP- eompetitive tyrosine kinase binding site.
[00417] In some embodiments, the epidermal growth factor receptor is a mutated epidermal growth factor receptor.
[00418] In some embodiments, the epidermal growth factor receptor exhibits an upregulation mutation.
[00419] In some embodiments, the epidermal growth factor receptor exhibits a constitutively activating mutation. [00420] In some embodiments, the epidermal growth factor receptor exhibits a downregulation mutation.
[00421] In some embodiments, the cancer treated is selected from the group consisting of non- small cell lung cancer, esophageal cancer, pancreatic cancer, rectal cancer, squamous cell carcinoma of the head or neck, squamous cell cancer of the skin, glioblastoma, colon cancer, cervical cancer, bladder cancer, breast cancer, gastrointestinal stromal cancer (GIST), ovarian cancer, gastric cancer, endometrial cancer, uterine cancer prostate cancer, liver cancer, melanoma, brain cancer, and mesothelioma.
[00422] In one aspect, provided herein is a compound selected from the group consisting of:
Figure imgf000168_0001
Figure imgf000169_0001
Figure imgf000170_0001
Figure imgf000171_0001
Figure imgf000172_0001
Figure imgf000173_0001
Figure imgf000174_0001
Figure imgf000175_0001
Figure imgf000176_0001
Figure imgf000176_0002
, trigger.
[00423] In one aspect, provided herein is a compound selected from the group consisting of:
Figure imgf000177_0001
Figure imgf000178_0001
Figure imgf000179_0001
Figure imgf000180_0001
Figure imgf000181_0001
Figure imgf000182_0001
Figure imgf000183_0001
Figure imgf000184_0001
Figure imgf000185_0001
Figure imgf000186_0001
Figure imgf000187_0001
Figure imgf000188_0001
Figure imgf000189_0001
Figure imgf000190_0001
Figure imgf000191_0001
Figure imgf000192_0001
Figure imgf000193_0001
Figure imgf000194_0001
Figure imgf000195_0001
Figure imgf000196_0001
Figure imgf000197_0001
Figure imgf000198_0001
Figure imgf000199_0001
[00425] In one aspect, provided herein is a pharmaceutical composition comprising a therapeutically effective amount of: an epidermal growth factor receptor-inhibiting drug, wherein the drug is covalently bonded to an attenuating moiety; and a pharmaceutically acceptable carrier.
[00426] In some embodiments, HER! is a mutated epidermal growth factor receptor.
[00427] In some embodiments, HER1 exhibits an upregulation mutation.
[00428] In some embodiments, HER! exhibits a eonstitutively activating mutation.
[00429] In some embodiments, HER1 exhibits a downregulation mutation.
[00430] In some embodiments, HER2 is a mutated epidermal growth factor receptor.
[00431] In some embodiments, HER2 exhibits an upregulation mutation.
[00432] In some embodiments, HER2 exhibits a eonstitutively activating mutation.
[00433] In some embodiments, HER2 exhibits a downregulation mutation.
[00434] In some embodiments, HER4 is a mutated epidermal growth factor receptor.
[00435] In some embodiments, HER4 exhibits an upregulation mutation.
[00436] In some embodiments, HER4 exhibits a eonstitutively activating mutation.
[00437] In some embodiments, HER4 exhibits a downregulation mutation.
[00438] The prodrug compounds of the disclosure may, for example, be prepared by reacting a kinase inhibitor comprising a nucleophilic atom, e.g , an aliphatic tertiary amine-bearing kinase inhibitor of Formula i with an appropriate nitroheterocyclic or nitrocarbocylic a-methyl electrophile such as a halide/mesylate/tosylate, in a suitable solvent and for a suitable length of time (for example jV-methyl-2-pyrrolidinone for about 15 hours), to produce a quaternary nitrogen salt comprising the nitroheterocyclic or mtrocarbocyclic reductive trigger moiety linked directly or indirectly to a nitrogen of the kinase inhibitor.
[00439] Scheme 3 illustrates two alternate methods to the a-methyl bromide 239, from commercially available starting materials.
Scheme 3
Figure imgf000200_0001
237 238 239
[00440] Scheme 4 illustrates a route to the a-methyl bromide 244, from commercially available starting materials.
Scheme 4
Figure imgf000200_0002
[00441] Scheme 5 illustrates a route to the a-methyl bromide 250, from l,5-dimethyl-4-mtro-li/- imidazole (238) (Scheme 3).
Scheme 5
Figure imgf000200_0003
[00442] Scheme 6 illustrates a route to the a-methyl bromide 261 from the commercially available oxazole (251).
Scheme 6
Figure imgf000201_0001
-
260 261
[00443] Scheme 7 illustrates two alternate routes to the a-methyl bromide 264, from a- methyl bromide 246 (Scheme 5) and 2~br omo~l, 5 -dimethy I -4-nitro-l //-imidazole (245) (Scheme 5), respectively. Scheme 7 below also illustrates a route to the a-methyl bromide 266, from a-methyl bromide 264.
Scheme 7
Figure imgf000201_0002
[00444] Scheme 8 illustrates a route to the a-methyl bromide 270, from (2-bromo-l-methyl-4- nitro-l//-imidazol-5-yl)methyl acetate (247) (Scheme 5).
Scheme 8
Figure imgf000202_0001
269 270
[00445] Scheme 9 illustrates a route to quaternary nitrogen salt compounds by reacting an aliphatic tertian- amine-bearing kinase inhibitor with an appropriate nitroheterocyclic a-methyl halide/mesyiate/tosylate (Formula VIII), in a suitable solvent and for a suitable length of time (for example inA-methyl~2~pyrrolidmone for about 15 hours).
Scheme 9
Figure imgf000202_0002
[00446] Scheme 10 illustrates the preparation of a number of prodrug compounds according to the disclosure. The 4~anilinopyndo[3,4~<i]pyrimidine effector compounds 1-9 (Scheme 2) were reacted with the a-methyl bromide 239 (Scheme 3) in Ar-methyl-2-pyrrohdinone (NMP) at room temperature for approximately 15 hours, before the addition of acetonitrile, to provide the quaternary ammonium salts (12, 13, 15-21) as a fine precipitate that was collected by filtration and washed with acetonitrile, ethyl acetate and hexane.
Scheme 10
Figure imgf000203_0001
[00448] Schemes 12 to 14 illustrate the preparation of alkyl trigger bromides in which Rs is Ci to Ce alkyl.
Scheme 12
Figure imgf000204_0001
Scheme 14
Figure imgf000205_0001
[00449] Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present disclosure. The following examples are, therefore, to be construed as merely illustrative and not limitative of the remainder of the disclosure in any way whatsoever.
HER and EGFR cancer biolog y
[00450] For reference, see Next-generation EGFR/HER tyrosine kinase inhibitors for the treatment of patients with non-small-cei! lung cancer harboring EGFR mutations: a review of the evidence, Wang, Xiaochun; Goldstein, David; Crowe, Philip J.; Yang, Jia-Lin, OncoTargets and Therapy (2016), 9, 5461-5473
[00451] The EGFR/HER family of receptor tyrosine kinases (TKs) has four members including
EGFR (FIERI, erhB-I), HER2 (erbB-2), HER3 (erbB-3), and HER4 (erbB-4), and their signaling pathways regulate cell growth, survival, adhesion, migration, and differentiation through three downstream pathways: RA S/RAF/ mitogen-activated protein kinase, phosphoinositide 3- kinase/AKT, and Janus kinase/signal transducer and activator of transcription (JAK/STAT).
Dysregulated signaling of HER family has been associated with the development of several malignancies including NSCLC. Many patients with NSCLC have somatic mutations of EGFR, first identified in 2004, which led to aberrant constitutive signaling via EGFR/HER family and their downstream protein markers. The EGFR mutations, including activating and resistant mutations, mostly occur in exons 18 to 21 of the EGFR gene encoding the ATP-binding pocket of the intracellular TK domain. The activating EGFR mutations (actEGFRm) have been reported in ~10%-l 5% of Caucasian patients but in up to 60% of selected Asian populations with NSCLC (female, never/light smoker, and adenocarcinoma). The most frequent actEGFRm in NSCLC are in-frame deletions in exon 19 (EGFRDell9, -60%) and L858R point mutation exon 21 (EGFRL858R, -30%). These oncogenic mutations interact and generate stabilization with ATP, intrinsically stimulate phosphorylation of tyrosine residues, and then result in the intracellular signal transduction activation in a ligand-independent manner. The NSCLC patients with actEGFRm become apparently dependent on EGER activity to stimulate downstream signaling pathways to maintain the malignant phenotype (“oncogene addiction”). Therefore, blocking EGFR/HER family pathways with EGFR/HER TK inhibitors (TKIs) can suppress tumor cell proliferation and initiate apoptosis. (Wang, X. et. al. OncoTargets and Therapy (2016), 9, 5461- 5473.)
[00452] In some embodiments, the epidermal growth factor receptor is HERE
[00453] In some embodiments, the HER! is a mutated epidermal growth factor receptor. In some embodiments, the HER! is a wild-type epidermal growth factor receptor. In some embodiments, the HER! exhibits an upregulation mutation. In some embodiments, the HER! exhibits a constitutively activating mutation. In some embodiments, the HER! exhibits a downregulation mutation.
[00454] In some embodiments, the epidermal growth factor receptor is HER2.
[00455] In some embodiments, the HER2 is a mutated epidermal growth factor receptor. In some embodiments, the HER2 is a wild-type epidermal growth factor receptor. In some embodiments, the HER2 exhibits an upregulation mutation. In some embodiments, the HER2 exhibits a constitutively activating mutation. In some embodiments, the HER2 exhibits a downregulation mutation.
[00456] In some embodiments, the epidermal growth factor receptor is HER4.
[00457] In some embodiments, the HER4 is a mutated epidermal growth factor receptor. In some embodiments, the HER4 is a wild-type epidermal growth factor receptor. In some embodiments, the HER4 exhibits an upregulation mutation. In some embodiments, the FIER4 exhibits a constitutively activating mutation. In some embodiments, the HER4 exhibits a downregulation mutation.
Exon 20 and NRG mutations in cancer [00458] The ErbB family of receptors is a subfamily of four closely related receptor tyrosine kinases: epidermal growth factor receptor or EGFR (ErbB-1; or HER! in humans), HER2/c-neu (ErbB-2), HERS (ErbB-3) and HER4 (ErbB-4). EGFR is the cell-surface receptor for members of the epidermal growth factor family (EGF family) of extracellular protein ligands. Mutations that lead to EGFR overexpression (upregulation) or overactivity have been associated with a number of cancers, including squamous-cell carcinoma of the lung (80% of cases), anal cancers, glioblastoma (50% of eases), and epithelial tumors of the head and neck (80-100% of cases). These somatic mutations involving EGFR lead to its constant activation, which produces uncontrolled cell division. In a non-limiting example, in glioblastoma, a more or less specific mutation of EGFR, called EGFRvIH, is often observed. Mutations, amplifications, or misregulations of EGFR or family members are implicated in about 30% of all epithelial cancers (PCT/US2Q 18/049842, incorporated herein in its entirety).
[00459] The identification of EGFR as an oncogene has led to the development of anticancer therapeutics directed against EGFR (called “EGFR tyrosine kinase inhibitors”), including gefitinib, erlotmib, afatinib, osimertinih, and icotinib for lung cancer. Cetuximab, panitumumab, necitumumab, zaiutumumab, nimotuzumab and matuzumab are examples of monoclonal antibody EGFR inhibitors. Gefitinib, erlotmib, afatmib, dacomitinib, osimertinib, and lapatinib (mixed EGFR and ERBB2 inhibitor) are examples of small molecule EGFR kinase inhibitors.
[00460] Many patients develop resistance to the existing EGFR inhibitors. Non-limiting sources of resistance are the EGFR T790M Mutation, HER2 and MET oncogenes, transformation to small cell lung cancer (SCLC), epithelial to mesenchymal transition (EMT), and fusions including those involving BRAF, NTRK1, RET, ALK, and/or ROSE Options to combat resistance are not limited, with only osimertinib being approved to treat EGFR T790M. While in frame deletions in exon 19 of EGFR and the L858R substitution in exon 21 of EGFR are sensitive to EGFR inhibitors (such as erlotinib, gefitinib, and afatinib), other mutations, such as in frame insertions in EGFR exon 20, demonstrate intrinsic resistance to these inhibitors. Other rare mutations in EGFR, such as G719X (exon 18) and L861Q (exon 21), have variable response to EGFR inhibitors. That said, the major reason why it is difficult to target these mutations is due to the undesirable on-target inhibition of WT EGFR m normal tissues.
[00461] There is a need in the art to identify compounds and methods that can be used to treat or prevent HER-driven drug-resistant cancers. In some embodiments, the compounds and methods can be used to treat or prevent EGFR-driven, HER2-driven, and/or NGR1 -driven drug-resistant cancers. The present application addresses and meets these needs.
[00462] HER- driven cancers include, but are not limited to, cancers caused by a EGFR gene fusion, a EGFR kinase domain duplication, a ERBB2 gene fusion, a ERBB2 mutation, a NRG I gene fusion, a ERBB3 mutation, and/or a ERBB4 fusion. The oncogenic alterations involving a ERBB2 (HER2) proto-oncogene are illustrated in Table L and each of those alterations is contemplated herein.
Table 1. Oncogenic alterations involving a ERBB2 (HER2) proto-oncogene
Figure imgf000208_0001
1. Lebeau, et ai, 2001, J Clin Oncol 19:354-63.
2. Yu, eta!., 2015, J Transl Med 13:116, 2015. 3. Bose, etal., 2013, Cancer Diseov 3:224-37.
4. Greulich, etal, 2012, Proc Natl Acad Sci U S A 109:14476-81.
5. Li, etal, 2016, J Thorac Oncol 11:414-9.
6. Cancer Genome Atlas Research N: Comprehensive molecular profiling of lung adenocarcinoma, 2014, Nature 511:543-50.
7. Kavuri, etal, 2015, Cancer Diseov 5:832-41.
8. Gordon, etal, 2013, Ann Oncol 24:1754-61.
9. Das, etal, 2014, Cancer Lett 353:167-75.
10. Ou, etal, 2017, J Thorac Oncol 12:446-457.
11. Gonzaga, etal... 2012, BMC Cancer 12:569.
12. Ross, etal, 2013, Clin Cancer Res 19:2668-76.
13. Ross, etal, 2014, Clin Cancer Res 20:68-75.
14. Chmielecki, etal, 2015, Oncologist 20:7-12.
15. Nardi, etal, 2013, Clin Cancer Res 19:480-90.
16. Williams, etal, 2010, Clin Cancer Res 16:2266-74.
17. Gao, etal, 2018, Cell Rep 23:227-238 e3
18. Zuo, et al, 2016, Clin Cancer Res 22:4859-4869.
19. Verma, et l, 2018, PLoS One 13:e0190942, 2018.
20. Tuefferd, et al, 2007, PLoS One 2:el 138.
21. Beiimunt, etal, 2015, Cancer Med 4:844-52.
22. Kloth, et al, 2016, Gut 65:1296-305.
23. Xu, et al, 2017, Clin Cancer Res 23:5123-5134.
24. Stephens, et al, 2004, Nature 431 : 525-6.
25. Morrison, etal, 2006, J Clin Oncol 24:2376-85.
26. Slomovitz, etal, 2004, J Clin Oncol 22:3126-32,
27. Chou, et al, 2013, Genome Med 5:78.
28. Shigematsu, etal, 2005, Cancer Res 65:1642-6.
29. Wang, etal, 2006, Cancer Cell 10:25-38.
[00463] In some embodiments, the cancer comprises an EGFR exon 20 insertion mutation. In some embodiments, the EGFR exon 20 insertion mutation includes a mutation such as but not limited to any of the mutations described in Yasuda, et ai., 2013, Sci. Trans 1. Med. 5(216):216ral77; doi: 10.1126/scitranslmed.3007205, and Arcila, et ai, 2013, Mol. Cancer Ther. 12:220; each of which is incorporated herein in its entirety by reference.
[00464] Neureguiin 1 {NRG I) encodes a growth factor ligand that binds to the human epidermal growth factor receptor 3 (HERS) encoded by the erb-b2 receptor tyrosine kinase 3 (ERBB3) gene and human epidermal growth factor receptor 4 (HER4) encoded by the erb-b2 receptor tyrosine kinase 4 ( ERBB4 ). Binding of NRG1 to HERS and HER4 can induce dimerization with other HER family members, including EGFR and HERS. Unlike EGER, HER2, and HER4, the HERS kinase domain is catalytically inactive but can activate EGFR or HER2 and thus can initiate oncogene signaling via its heterodimerization partner.
[00465] Gene fusions that contain sequences from NRG1 w¾re initially discovered in a breast cancer ceil line, MDA-MB-175 (Wang, el ai, 1999, Oncogene 18:5718-21). Later CD74-NRG l fusions were discovered in lung adenocarcinoma (Femandez-Cuest L, etai, 2014, Cancer Discov 4:415-22). NRG1 fusions have been identified in breast, NSCLC, cholangiocarcinoma, pancreatic cancer, and ovarian cancer (Wang, et a!., 1999, Oncogene 18:5718-21; Fernandez-Cuesta L, et ai, 2014, Cancer Discov 4:415-22; Dhanasekaran, et at, 2014, Nat Common 5:5893; Heining, et ai, 2018, Cancer Discov). NRG I gene fusions have been demonstrated to be oncogenic by inducing overexpression the ligand Neureguiin 1 which induces heterodimerization of HER3 with HER2. Similarly, NRG1 overexpression by gene amplification or other mechanisms are be predicted to activate HER3:HER2 dimers. Tarloxotinib is a potent inhibitor of HER2 and thus inhibits proliferation of cancer cells that utilize HER3/HER2 signaling. (Sene fusions involving the ERBB4 gene, similar to other fusions involving receptor tyrosine kinases, induce constitutive activation of HER4 and consequently activation of MARK (Nakaoku, et ai., 2014, Clin Cancer Res 20:3087-93). In some embodiments, tarloxotinib inhibits HER4 oncogene-driven cancers. Currently there are no U.S. FDA approved (or other regulator}' agencies) drugs for NRG l fusions, NRG1 gene amplification, NRG1 over expression, ERBB3 activation mutations, or ERBB4 fusions. [00466] The oncogenic alterations involving a NRG1, ERBB3 (HER3) and ERBB4 (HER4) proto- oncogene are illustrated in Table 2, and each of those alterations is contemplated herein.
Table 2. Oncogenic alterations involving a i Gl, ERBB3 (HER3) and ERBB4 (HER4) proto- oncogene
Figure imgf000211_0001
. Wang, el ai, 1999, Oncogene 18:5718-21. . Fernandez-Cuesta, el al., 2014, Cancer Disco v 4:415-22, . Dhanasekaran, el ai, 2014, Nat Corarnun 5:5893. . Fleming, et al ., 2018, Cancer Diseov. . Nakaoku, etal, 2014, Clin Cancer Res 20:3087-93. . Drilon, et al., 2018, Cancer Diseov 8:686-695. 7. Jaisw¾l, etal, 2013, Cancer Cell 23:603-17.
8. Yun, etal, 2018, Gastric Cancer 21 :225-236.
9. Wilson, etal., 2011, Cancer Cell 20:158-72.
10. Guo, etal., 2016, Int J Cancer 139:373-82.
11. Jung, etal, 2015, 1 Thorac Oncol 10:1107-11.
12. Xia, et al, 2017, Int I Surg Pathol 25:238-240.
13. Jones, etal, 2017, Ann Oncol 28:3092-3097.
STEAP 4
[00467] Cancer continues to be a significant unmet clinical indication. Expression of the enzyme STEAP4 has been shown to be associated with certain cancers (Gomes et al, 2012; Xue et al, 2017). STEAP4 has also been shown to be highly induced by hypoxia, i.e., low oxygen at the tissue level. Moreover, hypoxia within tumours is associated with poor prognosis of cancer patients and with treatment failure (Hunter et al., 2016), such as resistance to radiotherapy and traditional chemotherapy. Accordingly, there is a need in the art to identify cancer therapies that can be effectively used to treat or prevent cancers associated with high expression of STEAP4, and to identify patient populations with STEAP4 expressing cancers which are likely to be responsive to hypoxia-activated prodrugs (HAPs). It is therefore an object of the present disclosure to provide identification, predictive and treatment methods as provided herein based on this work, and/or at least to provide the public with a useful choice.
[00468] High levels of STEAP4 expression are associated with certain cancers. In some embodiments, the present disclosure teaches that when STEAP4 is highly expressed in cancers, STEAP4 can catalyse a reaction in HAPs leading to release of the drug payload and/or modification of the HAP to a form that is able to penetrate the cell membrane and cause cell death. Thus, in some embodiments, cancers with elevated STEAP4 expression levels respond to treatment by HAPs. Accordingly, the present disclosure teaches an effective treatment for STEAP4-associated cancers. Accordingly, in one aspect of the present disclosure there is provided a method of treating cancer in an individual in need thereof, the method comprising: a) providing tumour cells of the individual; b) determining the level of STEAP4 expression in the tumour cells; c) predicting the individual as being likely to be responsive to treatment by a HAP if the tumour cells exhibit an elevated level of STEAP4 expression; and d) administering a therapeutically effective amount of a HAP to the individual. [00469] In some embodiments, the present disclosure provides a method of treating cancer in an individual in need thereof, where tumour cells of the individual exhibit an elevated level of STEAP4 expression, the method comprising: a) administering a therapeutically effective amount of a HAP to the individual. [00470] In some embodiments, the present disclosure provides a method of predicting the responsiveness of an individual with cancer to treatment with a HAP, the method comprising: a) providing tumour cells of the individual; b) determining the level of STEAP4 expression in the tumour cells; and c) predicting the individual as being likely to be responsive to a treatment with the HAP if the tumour cells exhibit an elevated level of STEAP4 expression.
[00471] In some embodiments, the present disclosure provides a method of predicting the responsiveness of an individual with cancer to treatment with a HAP, the method comprising: a) determining the level of STEAP4 expression in a sample from the individual, wherein the sample comprises tumour cells; and b) predicting the individual as being likely to be responsive to a treatment with the HAP if the tumour cells exhibit an elevated level of STEAP4 expression.
[00472] The present disclosure also provides kits for performing the disclosed methods.
STEAP4 protein
[00473] The Six-Transmembrane Epithelial Antigen of Prostate (STEAP) protein family conta ins four members (STEAPl-4) though only STEAP2-4 have oxidoreducta.se activity.
[00474] The STEAP4 protein, also known as STAMP2 or TIARP, is a metalloreductase that reduces iron and copper ions. 8ΊΈAR4 has equivalent activity under either physiological or acidic pH (pH5.5 - 7.5).
[00475] The full length nucleotide sequence encoding the STEAP4 protein (i.e., the STEAP4 gene) and the full length amino acid sequence of the STEAP4 protein are known in the art (see, e.g., NCBI Gene ID: 79689, NCBI Ace. No. NM_024636, NCBI Ace. No. NM_ 001205315.1, NCBI Acc. No. NM 001205316.1, and UmProt Acc. No. Q687X5). At least two isoforms of the STEAP4 protein exist. Isoforni 1 is 459 amino acids in length and is shown in SEQ ID NO: 1 (also in Figure 1). This isoform 1 is encoded by two variants: variant 1 and variant 2. Variant 1 is the predominant nucleotide sequence encoding isoform 1 of the STEAF4 protein, and is shown in SEQ ID NO:2 (also in Figure 2). Variant 2 is an alternative nucleotide sequence encoding isoform 1 of the STEAP4 protein, and is shown in SEQ ID NO: 3 (also in Figure 3). Isoform 2 of the STEAP4 protein is 283 amino acids long, and is shown in SEQ ID NO:4 (also in Figure 4). Variant 3 is a nucleotide sequence encoding this shorter isoforni of STEAP4 protein (isoform 2) and is shown in SEQ ID NO: 5 (also m Figure 5).
(00476] As used herein, reference to the “STEAP4”, “STEAP4 protein”, or similar, refers to isoform 1 (SEQ ID NO:l), isoform 2 (SEQ ID NO: 4). a protein encoded by variant 1 (SEQ ID NO:2), a protein encoded by variant 2 (SEQ ID NO: 3), or a protein encoded by variant 3 (SEQ ID NO: 5), and/or to any other variant thereof. Variants contemplated within the scope of the present disclosure include protein variants which are substantially homologous to a native STEAP4 protein. The term “substantially homologous” as used herein refers to a protein having one or more naturally or non-naturaily occurring ammo acid deletions, insertions, or substitutions (e.g., derivatives, homologs, and fragments), as compared to the amino acid sequence of a native STEAP4 protein. The am o acid sequence of a STEAP4 variant may be at least about 40% identical, at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, at least about 90% identical, or at least about 95% identical to a native STEAP4 protein. As used herein, a “native STEAP4 protein” refers to the STEAP4 proteins which are found in nature and are not manipulated by man, and includes isoform 1 (SEQ ID NO: l), isoform 2 (SEQ ID NO:4), a protein encoded by variant 1 (SEQ ID NO:2), a protein encoded by variant 2 (SEQ ID NO:3), and a protein encoded by variant 3 (SEQ ID NO:5). Variants contemplated within the scope of the present disclosure also include proteins encoded by polynucleotide variants which have substantial sequence similarity or sequence identity to a native STEAP4 gene. The polynucleotide sequence of a STEAP4 variant may have at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% sequence similarity or sequence identity with a native STEAP4 gene. As used herein, a “native 8ΊΈAR4 gene” refers to genes: (a) which are found in nature and are not manipulated by man, and encode STEAP4 proteins; and (b) which encode STEAP4 proteins that are found in nature and are not manipulated by man; and includes the gene having the polynucleotide sequence encoding isoforni 1 (SEQ ID NO:l), the gene having the polynucleotide sequence encoding isoform 2 (SEQ ID NO.4}. the gene with the polynucleotide sequence of variant 1 (SEQ ID NO:2), the gene with the polynucleotide sequence of variant 2 (SEQ ID NO: 3), and the gene with the polynucleotide sequence of variant 3 (SEQ ID NO: 5).
[004771 High levels of 8TEAP4 expression are associated with certain cancers. In some embodiments, when STEAP4 is highly expressed in a cancer, the inventors have found that STEAP4 may catalyse conversion of an administered drug into its active form, such as a reaction leading to release of a drug payload. In some embodiments, high STEAP4 expression leads to release of the drug payload in HAPs. In some embodiments, the high STEAP4 expression is associated with hypoxic metabolism. In some embodiments, the high STEAP4 expression is associated with a hypoxic environment In some embodiments, the high STEAP4 expression is associated with a hypoxic tumour. In some embodiments, the high STEAP4 expression is not associated with low oxygen levels.
[00478] In some embodiments, when STEAP4 is present in hypoxic tumour environments, STEAP4 catalyses one-electron reduction of cell-excluded quaternary ammonium salt HAPs, leading to their fragmentation selectively m pathophysiologicai!y hypoxic tumour tissues, releasing the active drug which can then cross the cell wall and kill the cancer cell [00479] Without wishing to be bound by theory, it is thought that STEAP4 is located on the plasma membrane. As such, in some embodiments, STEAP4 reduces the HAP extracellular] y, forming a molecule that is capable of diffusing into the cell (e.g., a reduced form of the molecule). In some embodiments, STEAP4 is on the plasma membrane and reduces the HAP extracei Marly, at which point the charged molecule undergoes fragmentation and diffuses into the cell to inhibit EGFR. In some embodiments, the charged HAP molecule undergoes fragmentation under hypoxic conditions. In some embodiments the charged HAP molecule undergoes fragmentation in the presence of elevated levels of STEAP4. In some embodiments, the HAP is Compound A. In some embodiments, the MAP is Compound C. In some embodiments, the HAP is Compound E. In some embodiments, the HAP is selected from the group consisting of Compound A, Compound C, and Compound E.
[00480] While NMQ prodrugs used in the disclosed methods function by releasing an active molecule having undergone reduction in the extracellular medium, other HAPs for use in the disclosed methods do not fragment and release an active molecule, but instead are effective because the reduced form of the molecule is able to penetrate the cell membrane and then cause cell death in some embodiments, the HAPs may have a limiting or low rate of membrane penetration such that extracelluclar metabolism by STEAP4 will contribute a significant proportion of total cellular metabolism and such that intracellular reductases will contribute a less significant proportion of total cellular metabolism. One such class of HAPs is the trophenyl mustards. For example, in some embodiments, HAPs with a net neutral charge, e.g., nitrophenyl mustards, may be hydrophilic in nature, e.g., may have a low partition coefficient, which can result in a limiting rate of cell membrane penetration. In some embodiments wdien HAPs have a limiting rate of cell membrane penetration, extracellular metabolism by STEAP4 contributes a significant proportion of total cellular metabolism, and the remainder of HAP cellular metabolism is due to intracellular reductases.
[00481] In some embodiments, HAPs with a iowr pKa may be protonated at physiologically relevant pH range and thus carry a net positive charge, resulting in a low partition coefficient, which can result in a limiting rate of cell membrane penetration. In some embodiments when HAPs have a limiting rate of cell membrane penetration, extracellular metabolism by STEAP4 contributes a significant proportion of total cellular metabolism, and the remainder of HAP cellular metabolism is due to intracellular reductases.
[00482] In some embodiments, the present disclosure teaches methods of treating a cancer (e.g., a cancer characterized by STEAP4 activity and/or a cancer characterized by a hypoxic tumour environment) by allowing STEAP 4 to metabolize HAPs, including cell-excluded HAPs, at the cell surface. In some embodiments, this has the effect of releasing the active drug from the prodrug, which can enable the active drug to cross the cell membrane and deliver its payload to the intracellular targets and thus kill the cancer cell. Without wishing to be bound by theory, this is a previously unidentified role played by the enzyme STEAP4 in hypoxic tumour environments. In some embodiments, this activity can be unique to the STEAP4 reductase enzyme, and is not shared with the other STEAP proteins or with other reductases. In some embodiments, individuals suffering from cancer can be stratified based on their 8ΊΈAR4 expression levels, with those exhibiting elevated expression being identified as those likely to respond to HAP treatment. Without wishing to be bound by theory, embodiments disclosed herein are contrary to the prevailing understanding of STEAP4 that has proposed the inhibition of STEAP4 in order to treat cancer. In some embodiments, the exploitation of these elevated levels of STEAP4 can achieve better treatment outcome for patients. Pharmaceutical Combinations, Compositions, and Methods
[00483] Provided herein are pharmaceuticals combinations, compositions, and methods using a HAP composition as disclosed herein.
[004841 The present application further contemplates methods of treating a subject with cancer, e.g., the cancers disclosed herein, with the HAP compositions contemplated herein, wherein the treatment is part of a maintenance therapy for subjects with recurring or refractory cancer. For example, the present application contemplates a method of treating a resistant or refractory cancer m a subject with the HAP compositions disclosed herein. In some embodiments, the treatment leads to a full response, remission, and/or complete cure in the subject with recurring or refractory cancer. In some embodiments, the treatment maintains a stable disease, leads to a partial response ( e.g ., some tumour regression), or prevents the return of tumours which have fully regressed. [00485] In some embodiments, the HAP is a compound disclosed herein.
[00486] Provided herein is a method of inhibiting tumour cell growth comprising administering to the tumour a therapeutically effective amount of a HAP, as described herein. In some embodiments, provided herein is a method of inhibiting tumour cell growth comprising administering to the tumour a therapeutically effective amount of a compound disclosed herein, or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the tumour is a solid tumour. In some embodiments, the solid tumour is selected from bladder carcinoma, squamous cell carcinoma, transitional cell carcinoma, basal cell carcinoma, renal cell carcinoma, ductal cell carcinoma, and adenocarcinoma.
[00487] Provided herein is a compound which is a HAP, as described herein, for use in the treatment of cancer, wherein the treatment comprises administering the compound. In some embodiments, provided herein is a compound which is a compound disclosed herein, or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment of cancer, wherein the treatment composes administering the compound.
[00488] Pro vided herein is a HAP, as described herein, for use in the treatment of cancer.
[00489] In some embodiments, the subject is further administered at least one additional agent, or a salt or solvate thereof, that treats or prevents the cancer, as described herein.
[00490] In some embodiments, at least one of a compound of any one of the compounds as disclosed herein, is administered by at least one route selected from the group consisting of inhalational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal, intravesical, intrapulmonary, intraduodenal, intragastricai, intrathecal, epidural, intrapleural, intraperitoneal, intratracheal, optic, intraocular, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, in tra bronchial, inhalation, and topical in some embodiments, at least one HAP is administered orally, parenterally, rectaliy, topically, intravenously, intramuscularly, subcutaneously, or intraperitoneally. In some embodiments, at least one HAP is administered intraperitoneally. certain embodiments, at least one HAP is administered intravenously. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the subject is a human in need of treatment thereof.
[00491] In some embodiments, the HAP is administered by at least one route selected from the group consisting of iniialational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal, intravesical, intrapulmonary, intraduodenal, intragastricai, intrathecal, epidural, intrapleural, intraperitoneal, intratracheal, optic, intraocular, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchiai, inhalation, and topical. In some embodiments, the HAP is administered orally, parenterally, rectaliy, topically, intravenously, intramuscularly, subcutaneously, or intraperitoneally. In some embodiments the HAP is administered intraperitoneally. In some embodiments the HAP is administered intravenously. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the subject is a human in need of treatment thereof.
[00492] In some embodiments, the certain embodiments, at least one HAP is administered intraperitoneally is administered by at least one route selected from the group consisting of iniialational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal, intravesical, intrapulmonary, intraduodenal, intragastricai, intrathecal, epidural, intrapleural, intraperitoneal, intratracheal, optic, intraocular, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchiai, inhalation, and topical.
[00493] Also provided herein is a kit comprising a compound of any one of the compounds as disclosed herein, an applicator and instructional material for use thereof, wherein the instructional material comprises instructions for preventing or treating a cancer.
[00494] Further provided herein is a kit comprising HAP, an applicator and instructional material for use thereof, wherein the instructional material comprises instructions for preventing or treating a cancer. Salts
[00495] The compounds (e.g., the HAP) described herein may form salts with acids, and such salts are included in the present application in some embodiments, the salts are pharmaceutically acceptable salts. The term “salts” embraces addition salts of free acids that are useful within the methods disclosed herein. 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 application, such as for example utility in process of synthesis, purification or formulation of compounds useful within the methods disclosed herein. [00496] Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include sulphate, hydrogen sulphate, hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulphuric, and phosphoric acids (including hydrogen phosphate and dihydrogen phosphate). 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, trifluorom ethanesulfonic, 2- hydroxy ethanesulfonic, p-tol uenes ulfoni c, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, b~ hydroxybutyric, salicylic, galactanc, galacturomc acid, glycerophosphomc acids and saccharin (e.g., saceharmate, saccbarate). Saits may be comprised of a fraction of one, one or more than one molar equivalent of acid or base with respect to any compound contemplated herein.
[00497] Suitable pharmaceutically acceptable base addition salts of compounds contemplated herein include, for example, 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. All of these salts may be prepared from the corresponding compound by reacting, for example, the appropriate acid or base with the compound. Additional Agents
[00498] In some embodiments, the HAP compositions contemplated herein are useful m the methods of the present application when used concurrently with at least one additional compound useful for preventing and/or treating diseases and/or disorders contemplated herein.
[00499] In some embodiments, the HAP compositions contemplated herein are useful in the methods of present application in combination with at least one additional agent useful for preventing and/or treating diseases and/or disorders contemplated herein.
[00500] These additional agents may comprise compounds of the present application or other compounds, such as commercially available compounds, known to treat, prevent, or reduce the symptoms of diseases and/or disorders contemplated herein.
[00501] In a non-limiting example, the HAP compositions contemplated herein, or a salt or solvate thereof, can be used concurrently or in combination with one or more agents known to be useful in treating or preventing cancer, such as the cancers described herein. Non-limiting examples of additional anti -proliferative agents contemplated include, but are not limited to, compounds listed on the cancer chemotherapy drug regimens in the 14to Edition of the Merck Index (2006), which is hereby incorporated by reference, such as asparaginase, bleomycin, carboplatin, carmustine, chlorambucil, cisplatin, colaspase, cyclophosphamide, cytarabme, dacarbazine, dactinomycin, daunorubicin, doxorubicin (adriamycme), epirubicin, etoposide, 5-fiuorouracii, hexamethylmelamme, hydroxyurea, ifosfamide, irmoteean, leucovorin, lomustine, mechloretharmne, 6-mercaptopurine, mesna, methotrexate, mitomycin C, mitoxantrone, prednisolone, prednisone, procarbazine, raloxifen, streptozocin, tamoxifen, thioguamne, topotecan, vinblastine, vincristine, and vindesine. Additional anti-proliferative agents include other molecular targeted agents that modulate parallel pathways such as MEK 1/2 inhibitors, AKT inhibitors and mTOR inhibitors, monoclonal antibodies (such as Cetuximab), oxaliplatin, gemcitahine, gefinitib, taxotere, ara A, ara C, herceptm, BCNIJ, CCNIJ, DUG, and actmomycin D. Still further anti-proliferative agents include but are not limited to those compounds acknowledged to be used in the treatment of neoplastic diseases in Goodman and Gilman's The Pharmacological Basis of Therapeutics (Eleventh Edition), editor Molinoff et al, pub!. by McGraw-Hill, pages 1225-1287 (2006), which is hereby incorporated by reference, such as ammoglutethimide, L-asparaginase, azathioprine, 5-azacytidine cladribine, busulfan, diethylstilbestrol, 2',2'-difluorodeoxycytidine, docetaxel, erythrohydroxynonyladenine, ethinyl estradiol, 5-fluorodeoxyuridine, 5-fluorodeoxyuridine monophosphate, fludarabine phosphate, fluoxymesterone, flutamide, hydroxyprogesterone eaproate, idarubicin, interferon, medroxyprogesterone acetate, megestrol acetate, melphalan, mitotane, paclitaxel, pentostatin, N- phosphonoacetyl-L-aspartate (PALA), plicamycin, semustine, tempdside, testosterone propionate, thiotepa, trimethylmelamine, uridine, and vmorelbine.
[00502] In some embodiments, a HAP composition, as disclosed herein, is eo-administered with at least one additional agent.
Administration/Dosage/Formulations
[00503] The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the subject either prior to or after the onset of a disease or disorder contemplated herein. 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.
[005Q4] Administration of the compositions of the present application to a patient, for instance a mammal, e.g., a human, may be carried out using known procedures, at dosages and for periods of time effective to treat a disease or disorder contemplated herein. 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 patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound to treat a disease or disorder contemplated herein. 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 HAP composition contemplated herein 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 w ithout undue experimentation.
[00505] Actual dosage levels of the active ingredients in the pharmaceutical compositions disclosed herein 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 patient, composition, and mode of administration, without being toxic to the patient.
[00506] The therapeutically effective amount or dose of a HAP composition of the present application depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of a disease or disorder contemplated herein.
[00507] 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 could start doses of the HAP composition contemplated herein employed m 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.
[005Q8] A suitable dose of a HAP composition of the present application 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 m 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.
[00509] HAP compositions contemplated herein for administration may be in the range of from about 1 pg to about 10,000 mg, about 20 pg to about 9,500 mg, about 40 pg to about 9,000 mg, about 75 pg to about 8,500 mg, about 150 pg to about 7,500 mg, about 200 pg to about 7,000 mg, about 3050 pg to about 6,000 mg, about 500 pg to about 5,000 mg, about 750 pg 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 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and any and all whole or partial increments there between.
[00510] In some embodiments, the dose of a HAP composition contemplated herein is from about 1 mg and about 2,500 mg. in some embodiments, a dose of a compound contemplated herein 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 some embodiments, a dose of an additional agent in the HAP composition 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 thereof. [00511] In some embodiments, the HAP composition comprises a dosage of HAP of from about 0.1 mg/kg of body weight of a subject to about 200 mg/kg of body weight of a subject. In some embodiments, the HAP composition comprises a dosage of HAP of from about 0.1 mg/kg of body weight of a subject to about 100 mg/kg of body weight of a subject, from about 0.1 mg/kg of body- weight of a subject to about 50 mg/kg of body weight of a subject from about 0.1 mg/kg of body weight of a subject to about 25 mg/kg of body weight of a subject, from about 0.1 mg/kg of body- weight of a subject to about 20 mg/kg of body weight of a subject from about 0.1 mg/kg of body weight of a subject to about 15 mg/kg of body weight of a subject, from about 0.1 mg/kg of body- weight of a subject to about 10 mg/kg of body weight of a subject, from about 0.1 mg/kg of body weight of a subject to about 5 mg/kg of body weight of a subject or from about 0.1 mg/kg of body- weight of a subject to about 1 mg/kg of body weight of a subject. In some embodiments, the HAP composition comprises a dosage of HAP of from about 1 mg/kg of body weight of a subject to about 100 mg/kg of body weight of a subject, from about 1 mg/kg of body weight of a subject to about 50 mg/kg of body weight of a subject, from about 1 mg/kg of body weight of a subject to about 25 mg/kg of body weight of a subject, from about 1 mg/kg of body weight of a subject to about 20 mg/kg of body weight of a subject, from about 1 mg/kg of body weight of a subject to about 15 mg/kg of body weight of a subject, from about 1 mg/kg of body weight of a subject to about 10 mg/kg of body weight of a subject, or from about 1 mg/kg of body weight of a subject to about 5 mg/kg of body weight of a subject. In some embodiments, the HAP composition comprises a dosage of HAP of from about 10 mg/kg of body weight of a subject to about 100 mg/kg of body weight of a subject, from about 10 mg/kg of body weight of a subject to about 50 mg/kg of body weight of a subject, from about 10 mg/kg of body weight of a subject to about 25 mg/kg of body weight of a subject, from about 10 mg/kg of body weight of a subject to about 20 mg/kg of body- weight of a subject, or from about 10 mg/kg of body weight of a subject to about 15 mg/kg of body weight of a subject. In some embodiments, the HAP composition comprises a dosage of HAP of from about 20 mg/kg of body weight of a subject to about 100 mg/kg of body weight of a subject, from about 20 mg/kg of body weight of a subject to about 50 mg/kg of body weight of a subject, or from about 20 mg/kg of body weight of a subject to about 25 mg/kg of body weight of a subject. [00512] In some embodiments, the HAP composition comprises a dosage of HAP of from about 0.1 mg/kg of body weight of a subject to about 300 mg/m2 of body weight of a subject. In some embodiments, the HAP composition comprises a dosage of HAP of from about 0.1 mg/m2 of body weight of a subject to about 200 mg/m2 of body weight of a subject, from about 0.1 mg/m2 of body weight of a subject to about 150 mg/m2 of body weight of a subject, from about 0.1 mg/m2 of body weight of a subject to about 100 mg/m2 of body weight of a subject, from about 0.1 mg/m2 of body weight of a subject to about 50 mg/m2 of body weight of a subject, from about 0.1 mg/m2 of body weight of a subject to about 25 mg/m2 of body weight of a subject, from about 0.1 mg/m2 of body weight of a subject to about 20 mg/m2 of body weight of a subject, from about 0.1 mg/m2 of body- weight of a subject to about 15 mg/m2 of body weight of a subject, from about 0.1 mg/m2 of body weight of a subject to about 10 mg/m2 of body weight of a subject, from about 0.1 mg/m2 of body- weight of a subject to about 5 mg/m2 of body weight of a subject, or from about 0.1 mg/m2 of body weight of a subject to about 1 mg/m2 of body weight of a subject. In some embodiments, the HAP composition comprises a dosage of HAP of from about 1 mg/m2 of body weight of a subject to about 150 mg/m2 of body weight of a subject, about 1 mg/m2 of body weight of a subject to about 100 mg/m2 of body weight of a subject, from about 1 mg/rn2 of body weight of a subject to about 50 mg/m2 of body weight of a subject, from about 1 mg/m2 of body weight of a subject to about
25 mg/m2 of body weight of a subject, from about 1 mg/ 2 of body weight of a subject to about
20 mg/rn2 of body weight of a subject, from about 1 mg/m2 of body weight of a subject to about
15 mg/nr of body weight of a subject, from about 1 mg/m2 of body weight of a subject to about
10 mg/m2 of body weight of a subject, or from about mg/rn2 of body weight of a subject to about 5 mg/m2 of body weight of a subject. In some embodiments, the HAP composition comprises a dosage of HAS1 of from about 10 mg/rn2 of body eight of a subject to about 150 mg/nr of body weight of a subject, from about 10 mg/rn2 of body weight of a subject to about 100 mg/m2 of body weight of a subject, from about 10 mg/m2 of body weight of a subject to about 50 mg/m2 of body weight of a subject, from about 10 nig/m2 of body weight of a subject to about 25 nig/m2 of body- weight of a subject, from about 10 mg/m2 of body weight of a subject to about 20 mg/m2 of body weight of a subject, or from about 10 mg/kg of body weight of a subject to about 15 mg/m2 of body weight of a subject. In some embodiments, the MAP composition comprises a dosage of HAP of from about 20 mg/m2 of body weight of a subject to about 150 mg/m2 of body weight of a subject, from about 20 mg/m2 of body weight of a subject to about 100 mg/m2 of body weight of a subject, from about 20 mg/m2 of body weight of a subject to about 50 mg/m2 of body weight of a subject, or from about 20 mg/m of body weight of a subject to about 25 mg/m2 of body weight of a subject. In some embodiments, the HAP composition comprises a dosage of HAP of from about 50 mg/m2 of body weight of a subject to about 150 mg/m2 of body weight of a subject, from about 50 mg/m2 of body weight of a subject to about 100 mg/m2 of body weight of a subject, from about 50 mg/m2 of body weight of a subject to about 80 mg/m2 of body wreight of a subject, or from about 50 mg/m2 of body weight of a subject to about 75 mg/m2 of body weight of a subject. In some embodiments, the HAP composition comprises a dosage of HAP of from about 75 mg/m2 of body weight of a subject to about 150 mg/m2 of body weight of a subject, or from about 75 mg/m2 of body weight of a subject to about 100 mg/m2 of body weight of a subject. In some embodiments, the HAP composition comprises a dosage of HAP of from about 80 mg/m2 of body weight of a subject to about 150 mg/m2 of body weight of a subject, or from about 80 mg/m2 of body weight of a subject to about 100 mg/m2 of body weight of a subject. In some embodiments, the HAP composition comprises a dosage of HAP of about 0.1 mg/m2, about 1 mg/m2, about 5 mg/m2, about 10 mg/m2, about 15 mg/m2, about 20 mg/m2, about 25 mg/m2, about 30 mg/m2, about 40 mg/m2, about 50 mg/m2, about 60 mg/m2, about 70 mg/m2, about 75 mg/m2, about 80 mg/m2, about 100 mg/m2, about 150 mg/m2, about 200 mg/m2, or about 300 mg/m2.
[00513] In some embodiments, the HAP composition comprises a dosage of the prodrug of about 50 mg/m2 of body weight of a subject. In some embodiments, the HAP composition comprises a dosage of the prodrug of from about 0.1 mg/m2 to about 150 mg/m2 of body weight of a subject. In some embodiments, the HAP composition comprises a dosage of the prodrug of about 150 mg/ m2 of body weight of a subject. In some embodiments, the HAP composition comprises a dosage of the prodrug of about 75 mg/ m2 of body weight of a subject. In some embodiments, the HAP composition comprises a dosage of the prodrug of about 80 mg/ m2 of body weight of a subject. In some embodiments, the HAP composition comprises a dosage of the prodrug of about 120 mg/ m2 of body weight of a subject. [00514] In some embodiments, a dosage of the potassium supplementation is from about 3 mEq/L to about 7 mEq/L. In some embodiments, a dosage of the potassium supplementation is about 6 mEq/L.
[00515 [ In some embodiments, administration of the potassium supplementation increases the potassium concentration to from about 0.5 mEq/L to about 6.0 mEq/L, from about 0.5 mEq/L to about 5.5 mEq/L, from about 0.5 mEq/L to about 5.0 mEq/L, from about 0.5 mEq/L to about 4.5 mEq/L, from about 0.5 mEq/L to about 4.0 mEq L, from about 0.5 mEq/L to about 3.5 mEq/L, from about 0.5 mEq/L to about 3.0 mEq/L, from about 0.5 mEq/L to about 2.5 mEq/L, from about 0.5 mEq/L to about 2.0 mEq/L, from about 0.5 mEq/L to about 1.5 mEq/L, or from about 0.5 mEq/L to about 1.0 mEq/Labove the normal serum concentration. For example, if the normal serum concentration of potassium is about 3.5 mEq/L, administration of the potassium supplementation will increase the potassium concentration from about 0.5 to about 1 0 mEq/L, such that the increased serum concentration of potassium is about to about 4.0 mEq/L to about 4 5 mEq/L
[00516] In some embodiments, administration of the potassium supplementation increases the potassium concentration from about 0.5 meQ/L to about 6.0 mEq/L, from about 0.5 mEq/L. to about 5.5 mEq/L, from about 0.5 mEq/L to about 5.0 mEq/L, from about 0.5 mEq/L to about 4 5 mEq/L, from about 0.5 mEq/L to about 4 0 mEq/L, from about 0 5 mEq/L to about 3.5 mEq L, from about 0.5 mEq/L to about 3.0 mEq/L, from about 0.5 mEq/L to about 2.5 mEq/L, from about 0.5 mEq/L to about 2.0 mEq/L, from about 0.5 mEq/L to about 1.5 mEq/L, or from about 0.5 mEq/L to about 1.0 mEq/L above the serum concentration in the absence of administration of a HAP. For example, if the serum concentration of potassium in the absence of administration of a HAP is about 3.5 mEq/L, administration of the potassium supplementation will increase the potassium concentration from about 0.5 to about 1.0 mEq/L, such that the increased serum concentration of potassium is about to about 4.0 mEq/L to about 4.5 mEq/L.
[00517] In some embodiments, the HAP composition contemplated herein are administered to the patient in dosages that range from one to five times per day or more. In some embodiments, the HAP composition contemplated herein are administered to the patient 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 HAP composition HAP compositions contemplated herein 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 present disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient is determined by the attending physical taking all other factors about the patient into account.
[00518] It is understood that the amount of HAP composition 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. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.
[00519] In some embodiments, the prodrug is administered once, twice, three times, four times, five times, or six times a day. In some embodiments, the prodrug is administered once, twice, three times, four times, five times, or six times a week. In some embodiments, the prodrug is administered every week, every two weeks, every three weeks, every four weeks, every five weeks, or every' six weeks
[0052Q] In the case wherein the patient’s status does improve, upon the doctor’s discretion the administration of the HAP composition contemplated herein is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a “drug holiday”). The length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during a drug holiday includes from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
[00521] In some embodiments, the HAP composition provided herein reduce side effects and/or toxicity effects of the administered HAP such that a drug holiday and/or dose reduction is not needed.
[00522] Once improvement of the patient’s conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, is reduced, as a function of the disease or disorder, to a level at which the improved disease is retained. In some embodiments, patients require intermittent treatment on a long-term basis upon any recurrence of symptoms and/or infection.
[00523] The HAP composition for use in the method disclosed herein may be formulated in unit dosage form. The term “unit dosage form” refers to physically discrete units suitable as unitary dosage for patients 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.
[00524] Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LDso (the dose lethal to 50% of the population) and the EDso (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LDso and EDso. The data obtained from ceil culture assays and animal studies are optionally used in formulating a range of dosage for use in human. The dosage of such compounds can lie within a range of circulating concentrations that include the EDso with minimal toxicity. The dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.
[00525] In some embodiments, the HAP composition contemplated herein are formulated in a pharmaceutical composition using one or more pharmaceutically acceptable excipients or carriers. In some embodiments, the pharmaceutical compositions contemplated herein comprise a therapeutically effective amount of a HAP composition contemplated herein and a pharmaceutically acceptable carrier.
[00526] 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, one can include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition.
[00527] The compositions can be formulated for single dosage administration. In some embodiments, to formulate a composition, the HAP is dissolved, suspended, dispersed or otherwise mixed in a selected vehicle at an effective combined concentration such that the treated condition is relieved or ameliorated. Pharmaceutical carriers or vehicles suitable for administration of the HAP provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.
[00528] In some embodiments, the present application is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound contemplated herein, alone or m combination with a second pharmaceutical agent; and instructions for using the compound to treat prevent, or reduce one or more symptoms of a disease or disorder contemplated in the present disclosure.
[00529] Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for any 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, weting 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., analgesic agents.
Additional Administration Forms
[00530] Additional dosage forms 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 also include dosage forms as described in U.S. Patent Applications Nos. 20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and 20020051820. Further dosage forms include dosage forms as described inPCT 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 [00531] In some embodiments, the formulations of the present application 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.
[00532 j 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 bolus form.
[00533] For sustained release, the compounds may be formulated with a suitable polymer or hydrophobic material that provides sustained release properties to the compounds. As such, the compounds for use in the methods disclosed herein may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.
[00534] In some embodiments, the compounds contemplated herein are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation. [00535] The term delayed release is used herein m 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, includes a delay of from about 10 minutes up to about 12 hours
[00536] 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.
[00537] 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.
[00538] 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.
[00539] 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. [00540] 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 the present 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 r educing/ oxidizing agents, with art-recognized alternatives and using no more than routine experimentation, are within the scope of the present application.
[00541] It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present application. 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.
[00542] The following examples further illustrate aspects of the present application. However, they are in no way a limitation of the teachings or disclosure of the present appl ication as set forth herein.
Examples
[00543] The disclosure is further illustrated by the following examples, which are not to be construed as limiting this disclosure in scope or spirit to the specific procedures herein described. It is to be understood that the examples are provided to illustrate certain embodiments and that no limitation to the scope of the disclosure is intended thereby. It is to be further understood that resort may be had to various other embodiments, modifications, and equivalents thereof which may suggest themselves to those skilled in the art without departing from the spirit of the present disclosure and/or scope of the appended claims.
Example L Biological activity of EGFR compounds and release of trigger in hypoxic cancer cells; Cell Growth Inhibitory Activity
Pan-erbB kinase inhibitors
[00544] The structures below and described in WO2011/0289135 and are contemplated for use in accordance with the present disclosure.
Figure imgf000232_0001
[00545] The above kinase inhibitors of were tested for their ability to inhibit the proliferation of three human carcinoma cell lines, selected to provide a comparison with literature precedent: A431 (epidermoid), which overexpresses erbB! (EGFR); HI 975 (non-small-cell lung), which overexpresses erbBl L8S8R/T790M a double mutant form of erbBl that is known to confer resistance to the approved reversible erbBl inhibitor erlotinib and SKOV3 (ovarian), which over express erbB2 (HER2). The cells were exposed to test compounds for either 24 hours under oxic conditions or for 4 hours under anoxia followed by 20 hours under oxic conditions. They were then washed free of drug and incubated for a further 4 days, before being stained for cellular growth with suiforhodamine B. The concentration of compound required to inhibit cellular growth by 50% relative to untreated control wells, termed the IC50 value, was calculated. Results are summarised in Table 3.
Table 3. Inhibition of cellular proliferation in A431, HI 975 and SKOV3 cells
Figure imgf000233_0001
* compound dose-response curves were determined at 10 concentrations. Cells received a 24 hour exposure to test compounds before being washed (x3) with drag-free media. The IC50 (umo!/L) values are the concentrations required to inhibit cell growth by 50% relative to untreated controls. Values are the average of 2-8 independent determinations (%CV <20 in all cases). b Experiment performed entirely under oxic conditions. cThe initial 4 hours of the 24 hour drug exposure was performed under anoxic conditions a Hypoxic Cytotoxicity Ratio = fold change in intra- experimental 1C50 for cells receiving 4 hours of anoxia relative to cells that received only oxic conditions.
[00546] Irreversible erhBl, 2, 4 inhibitors 1, 2, 3, 4, 5, 6, 7, 8 and 9 more potently inhibited proliferation of aerobic .4431 cells (XC50s = 0.28 to 0.005 umol/L) than HI975 (IC50s = 1.3 to 0.15 umol/L) and SKOV3 (IC50s :::: 1.8 to 0.2 umol/L) cells and did not show any significant change in potency when the cells received 4 hours of anoxia with intraexperimental HCR ranging from 0.5 to 3.3 for all compounds across the three cell lines.
Example 2, Biological activity of EGFR compounds and release of trigger in hypoxic cancer cells: Cell Growth Inhibitory Activity Prodrugs of pan-erbB inhibitors
[00547] The structures below' and described in WO2011/0289135 and are contemplated for use in accordance with the present disclosure
Figure imgf000235_0001
Figure imgf000236_0001
Figure imgf000237_0001
Figure imgf000238_0001
[00548] Prodrug compounds selected from the above-list (compounds 12-21) were tested for their ability to inhibit the proliferation of three human carcinoma cell lines, selected to provide a comparison with literature precedent: A431 (epidermoid), which overexpresses erbBl (EGFR); HI 975 (non-small-cell lung), which overexpresses erbBl L858R/T790M a double mutant form of erbBl that is known to confer resistance to the approved reversible erbBl inhibitor erlotinib and SKOV3 (ovarian), which over express erbB2 (HER2). The ceils were exposed to test compounds for either 24 hours under oxic conditions or for 4 hours under anoxia followed by 20 hours under oxic conditions. They were then washed tree of drug and incubated for a further 4 days, before being stained for cellular growth with suiforhodamine B The concentration of compound required to inhibit cellular growth by 50% relative to untreated control wells, termed the ICSQ value, was calculated. Results are summarised in Table 4.
Table 4 Inhibition of cellular proliferation A431 , H1975 and 8KOV3 cells
Figure imgf000239_0001
a compound dose-response curves were determined at 10 concentrations. Ceils received a 24 hour exposure to test compounds before being washed (x3) with drug-free media. The IC50 (usnol/L) values are the concentrations required to inhibit cell growth by 50% relative to untreated controls. Values are the average of 2-8 independent determinations (%CV <20 in all cases). ¾ Experiment performed entirely under oxie conditions. c The initial 4 hours of the 24 hour drag exposure was performed under anoxic conditions. d Hypoxic Cytotoxicity Ratio - fold change in intra-experimental IC50 for cells receiving 4 hours of anoxia relative to cells that received only oxie conditions.
[00549] All of the prodrugs (12, 13, 14, 15, 16, 17, 18, 19, 20 and 21) of Table 4 were significantly more potent at inhibiting the growth of all three cell lines after the cells received 4 hours of anoxia. The hypoxic cytotoxicity ratios (HCR) ranged from 8 to 96 in A431 cells, 14 to 89 in HI 975 cells and 15 to 528 in SKOV3 cells, consistent with hypoxia-selective reduction of the 4-nitroimidazole reductive trigger, followed by trigger fragmentation to release an irreversible erbBl, 2, 4 inhibitor.
Example 3. la vivo efficacy of compounds of the disclosure
Methods
[00550] Specific pathogen-free female NIH-III nude mice, derived from breeding mice supplied by Charles River Laboratories (Wilmington, MA), were housed in groups of 4-6 in a temperature- controlled room (22 ± 2°C) with a 12-hour hght/dark cycle and were fed ad libitum water and a standard rodent diet (Harlan Teklad diet 2018i). All animals were uniquely identifiable by ear tag number.
[00551] Freshly harvested cell suspensions were subcutaneously inoculated (lOOpL) on the right flank with 5x106 HI 975 or A431 cells in PBS. Mean tumour diameter was averaged from the longest diameter (length) multiplied by the perpendicular measurement (width). Tumour volume (rnnd) was calculated using the formula (L x w2) x p/ό (where; L = length and w = width in mm of the carcinoma).
Growth Delay Experimental Procedure
[00552] Treatment was initiated when the tumours reached a volume of approximately 250 mm3, as determined by calliper measurement. All drugs were given by intraperitoneal injection at dosing volumes of 10-20 ml/kg. Mice w¾re dosed at the MTD over a q3dx4, q5dx4 or q7dx4 schedule with tumour growth measured by callipers every 3-5 days over the 30-day duration of the study. Mice wrere culled if they developed signs of toxicity or if body weight loss exceeded 20% of starting weight. All animal experiments followed protocols approved by the Animal Ethics Committee of The University of Auckland.
[00553] Tumour bearing mice were assigned randomly to treatment groups when tumour diameter reached treatment size. Animals were rejected if xenografts show' evidence of: (i) attachment to underlying muscle (due to risk of local invasion), (ii) signs of ulceration, or (iii) indolent tumour growth. Drug administration begins on the day of assignment.
[00554] During and after treatment, tumour size and body weights were measured regularly. Animals were culled if (i) the average diameter of the tumour exceeds 15 mm (survival endpoint), (ii) body weight loss exceeds 20% of pre-treatment value, (iii) there is evidence of prolonged or excessive morbidity, or (iv) tumour ulceration occurred. The experiment was terminated at day 21 (A431 tumours) or day 30 (Hi 975 tumours) after treatment initiation.
Efficacy Analysis
[00555] The time for individual tumours to increase in volume by 4 fold relative to treatment day-
1 (RTV4) was recorded. The median RTV4 is calculated for each group and the difference in RTV4 between control and treatment groups is described as the Tumour Growth Delay (TGD) in days. RTV4 values normalise for any bias in tumour treatment volume on day 0. Kaplan-Meier plots were constructed and median survival was calculated (TTEso). The statistical significance of any differences in overall survival time taken to reach RTV4 between treatment groups and control was analysed by Log Rank P statistical test.
Toxicity
[00556] Weight loss nadirs (time independent maxima) were recorded for each treatment group. Any signs of treatment related morbidity were documented. Acceptable toxicity was defined as no mean group weight loss of over 15% during the test, no individual weight loss over 20% and no individual weight loss over 10% in any 24h period. All unscheduled deaths were recorded.
Results
[00557] Median tumour growth curves following treatment are presented in Figures 1-11 Summary' tables of the effect of treatment on toxicity and efficacy are presented in Tables 5-7 below.
[00558] Overall, nine kinase inhibitors and nine prodrugs were administered to mice with human HI 975 or A431 tumour xenografts. The mean (± SD) tumour volume at treatment initiation was 245 mmJ ± 60 mmJ for HI 975 tumours and 268 mmJ ± 124 mnk for A431 tumours.
[00559] At tolerated dose levels, all kinase inhibitors delayed tumour growth. This tumour growth delay was particularly significant for compounds 2-9 when administered at q3dx4 (P< 0.05, log- rank test) in both Hi 975 (Figures 1-3) and A431 (Figure 4) tumours. There was only minor body weight loss across all groups, although there was 1 death following treatment with compounds 5 and 7 in HI 975 xenograft mice (Table 5).
Table 5. Summary of treatment toxicity and efficacy parameters for kinase inhibitors
Figure imgf000241_0001
Figure imgf000242_0001
“Administered in lactate buffer (pH4) by intraperitoneal injection (< 0.02ml/g); ball animal deaths considered likely to be drug related; cMean weight loss nadir (time independent maxima) relative to day 0 weight (%) for each individual; dTmnour Growth Delay calculated as % increase in time required to reach 4 times initial treatment volume (RTV4; relative to day 0 volume) relative to control growth; 'Kaplan-Meier Log Rank survival analysis of compound treated relative to buffer treated control assuming a survival endpoint of RTV4.
[00560] Similarly, the prodrugs (compounds 12, 13, 15-21) all significantly delayed tumour growth in H1975 (Figures 5-7) or A431 (Figure 8) xenograft models after treatment at a q3dx4 schedule ( <0.05, log-rank test). Again, there was only minor bodyweight loss in the mice in all treatment groups, although there was 1 death following treatment with compound 19 (Table 6).
Table 6. Summary' of treatment toxicity' and efficacy parameters for prodrugs
Figure imgf000242_0002
Figure imgf000243_0001
“Administered in lactate buffer (pH4) by intraperitoneal injection (< 0.02nd/g); hdl animal deaths considered likely to be drug related; ‘Mean weight loss nadir (time independent maxima) relative to day 0 weight (%) for each individual; dTumour Growth Delay calculated as % increase in dine required to reach 4 times initial treatment volume (RTV4; relative to day 0 volume) relative to control growth; eKaplan-Meier Log Rank survival analysis of compound treated relative to buffer treated control assuming a survival endpoint of RTV4.
[00561 j Administration of prodrugs 15 and 17, alongside their cognate kinase inhibitors, 3 and 5 respectively, revealed an extended period of growth delay in HI 975 tumours for the prodrugs compared to the kinase inhibitors (Figures 9-10).
[005621 Prodrug 17 was tested at multiple dosing schedules; q3dx4, q5dx4 and q7dx4 at its q3dx4 MTD. At all 3 dosing schedules, compound 17 significantly delayed tumour growth in HI 975 tumours compared to controls (Figure 11). There were no statistically significant differences in tumour growth between the 3 dosing schedules. Bodyweight loss was greatest after q3dx4 dosing and minimal after q7dx4 dosing (Table 6). However, 1 death w¾s observed in the q7dx4 treatment group. It is not clear if tins death w¾s drug-related or not. Compound 17 was also administered at q3dx4 and q5dx4 at its q3dx4 MTD to A431 xenograft mice, with both dosing schedules causing similar delays in A431 tumour growth and similar losses in animal bodyweight (Table 7).
Table 7 Summary of treatment toxicity and efficacy parameters for 17 at q3dx4, q5dx4 and q7dx4 schedules
Figure imgf000244_0001
Administered in lactate buffer (pH4) by intraperitoneal injection (< 0 02ml/g); ball animal deaths considered likely to be drug related; cMean weight loss nadir (time independent maxima) relative to day 0 weight (%) for each individual; clTumour Growth Delay calculated as % increase in time required to reach 4 times initial treatment volume (RTV*; relative to day 0 volume) relative to control growth; eKaplan-Meier Log Rank survival analysis of compound treated relative to buffer treated control assuming a survival endpoint of RTV4
[00563] Overall, the in vitro and in vivo activity data illustrate the effectiveness of the compounds of the disclosure as kinase inhibitors. The compounds are suitable for use in kinase-inhibitory therapy. This is the case with the reductive prodrugs and cancer therapy as tumours commonly have hypoxic regions. The prodrugs are reduced under hypoxia to release the parent kinase inhibitor and produce a tumour-targeted effect. Prodrugs 15, 17 and 18 exhibit effective therapeutic properties.
Example 4 - Efficacy of compounds in vivo
[00564] Figures 17-19 show that compounds of the disclosure can have efficacy in vivo. For example, Figure 18 shows that tarloxotinib can be effective at reducing tumors derived from a CLU-NRG1 fusion ovarian cancer in mouse xenograft models.
[00565] While the present disclosure is broadly as described above, those persons skilled in the art will appreciate that the specific description is illustrative only and that variations may be made without departing from the disclosure as defined in the following claims.
[00566] All publications referenced above are incorporated herein in their entirety .
Example 5 - Evaluating the cytotoxicity of three HAPs in parental (WT) neoplastic ceil lines and paired lines engineered to overexpress human STEAP4 cDNA [00567] The following HAPs evaluated in this example are TH-302, Tirapazamine (TPZ), and Compound C, the structure for each prodrug is shown in Figure 20.
Cloning ofSTEAP4
[00568] The open reading frame fORF) for human STEAP4 transcript variant 1 (NlVl 024636.2) was cloned into the F279-V5 expression vector. This provides transcription of a bicistronic mRN A from the human immediate-early cytomegalovirus (CMV) promoter, which encodes the open reading frames for the gene of interest and the pac (puromycin resistance) gene; the former harbouring an occult C-terminal V5-Tag inducible with TAG-On-DemandlM technology.
[00569] Human cervical carcinoma (C33A) and non-small cell lung cancer (HI 299) cells were transfected using Lipofeetamine 3000 reagent complexed with F279-STEAP4-V5 plasmid DNA. The transfection mix was added drop wise and after 48 hours the culture media was replaced with fresh a-MEM (with 5% FBS) medium containing I mM puromycin, escalating to 3 mM puromycin over time. Cells were grown to confluence in the wells before being pooled together and transferred to a cell culture flask of desired volume. Cells were maintained in a-MEM supplemented with 3 mM puromycin. Cell line pairs were designated: C33A¾1/C33ASThAP4 and H1299¾T/H1299SThAP4 In vitro cytotoxicity assays to determine the sensitivity ofWT and STEAP4 expressing cells to HAPs
[00570] Cell lines w¾re maintained in a-MEM 5% FCS in sterile tissue culture flasks prior to the experiment. Cells engineered to express STEAP4 were routinely cultured in media supplemented with 1 mM puromycin to maintain a selection pressure on the transfected cells, although puromycin was omitted from the media during experiments. On the day of the experiment, cells were harvested with Trypsm/EDTA and counted. The required number of cells was transferred to a 50 mL falcon tube and centrifuged to pellet cells (1000 rpm, 5 min). Excess media was removed by aspiration leaving cell pellets sitting in 50-100 mΐ media. The cell number was calculated to ensure a seeding density of 400 cells per well of a 96-well plate, with cells resuspended in a-MEM + 10% FCS, -P/S + lOmM D-Glucose + 0.2mM 2’-deoxycytidme. Cells were seeded under aerobic or anoxic conditions, the latter using a 5% H2/palladium catalyst scrubbed Bactron anaerobic chamber (Sheldon Manufacturing, Cornelius, Oregon) to achieve severe anoxia (<10 ppm Q?. gas phase) during prodrug exposure.
[00571] The lay out of the plate is described in Figure 21, where column 1 (the left- most column) consists of 8 media only control wells. Column 2 (second from the left) consists of 8 cells only (no prodrug) control wells. The remaining columns (3-12) consist of cells exposed to a range of prodrug concentrations. A high concentration of prodrug was added to column 12 (the right-most column) in each row, and 3-fold dilutions were performed across the plate using a multi-channel pipette. Thus, a total of four prodrugs could be evaluated on a single plate, with duplicate wells per prodrug. In this example, the three prodrugs were evaluated with the range of concentrations shown in the following table:
Table 8: Concentration ranges for prodrugs in the 96-well plate
Figure imgf000246_0001
[00572] After 2 hours incubation to enable cell attachment to the plates (and equilibrate to anoxia m the case of the anoxic samples), cells were exposed to a range of prodrug concentrations for 4 hours under aerobic or anoxic conditions. After prodrug exposure, plates were transferred to aerobic conditions. A vacuum immunowash was used to aspirate the media and wells were washed three times with 150 m! of prodrug-free aMEM 5% FCS + P/S. The plates were then incubated under aerobic conditions for 5 days for cell growth. Cells were fixed by adding 50m1 of cold 40% trichloroacetic acid (TCA) to each well by layering on top without mixing, this gave a final concentration of 10% TCA. Plates were incubated in a fridge for 1 hour before the TCA w¾s w'ashed off and the plates rinsed in running tap water. 50m1 of 0.4% SRB in 1% acetic acid was added to each well and plates were incubated for 30 minutes in the dark. Excess stain was flicked off and plates were washed four times in 2.L of tap water containing 1% acetic acid. Excess fluid was flicked from the plates and the remaining SRB stain solubilized by adding IOOmI of lOniM unbuffered Tris to each well. Plates were then left on a shaker for at least 2 hours in the dark before analysis using a plate reader (set at an optical density of 490 nM for measurement filter and 450nM for reference filter). The ICso value is the concentration of prodrug required to inhibit cell growth by 50%.
[00573] The results from the ICso assay showed that overexpression of STEAP4 increased the sensitivity of C33A and HI 299 cells to Compound C, but not to TH-302 or TPZ. Without wishing to be bound by theory, this is understood to result from the extra-cellular site of metabolism (see, e.g., Figure 23). The active (cytotoxic) metabolite of Compound C is able to cross the cell membrane and exert its biological effect (DNA cross-linking). In contrast, evofosfamide (TIT-302) releases a negatively charged Br-ίRM active metabolite that is unable to traverse the cell membrane and exert its biological effect (DNA cross-linking). Further, the active metabolite of tirapazamine (TPZ) is a short lived free-radical species that is unable to penetrate the cell.
(00574] The results are shown graphically in Figure 22. The hypoxic cytoxicity ratio (aerobic IC50 value divided by anoxic IC50 value), is indicated above the anoxic bar for each cell group. (00575] Consequently STEAP4 metabolism only results in PR- 104 A sensitivity. The prodrugs TH-302 and TPZ are consumed BUT this does not result m cell kill. Therefore STEAP4 functions as a detoxifying pathway due to futile, unproductive metabolism.
Example SA: Evaluating the cytotoxicity of three HAPs in parental (WT) neoplastic cell lines and paired lines engineered to over express human STEAP4 cDNA
(00576] The following HAPs evaluated in this example: Compound A (Tarloxotmib), Compound C (PR- 104 A), Compound G (SN29176), Compound H (SN27686), the structure for each prodrug is shown in Figure 20.
Cloning of STEAP4
(00577] The open reading frame (ORF) for human STEAP4 transcript variant 1 (NM_024636.2) was cloned into the F279-V5 expression vector. This provides transcription of a bicistronic mR A from the human immediate-early cytomegalovirus (CMV) promoter, which encodes the open reading frames for the gene of interest and the pac (puromycin resistance) gene; the former harbouring an occult C-terminal V5-Tag inducible with TAG-On-Demand‘M technology.
[00578] Human cervical carcinoma (C33A) cells were transfected using Lipofectamine 3000 reagent complexed with F279-STEAP4-V 5 plasmid DNA. The transfection mix was added drop wise and after 48 hours the culture media was replaced with fresh a-MEM (with 5% FBS) medium containing 1 pM puromycin, escalating to 3 mM puromycin over time. Cells were grown to confluence m the wells before being pooled together and transferred to a cell culture flask of desired volume. Cells were maintained in a-MEM supplemented with 3 mM puromycin. Cell lines were designated (G33A% /E33Adiί AR4.
In vitro cytotoxicity assays to determine the sensitivity of WT and STEAP4 expressing cells to HAPs [00579] Cell lines were maintained in a-MEM 5% FCS in sterile tissue culture flasks prior to the experiment. Cells engineered to express STEAP4 were routinely cultured in media supplemented with 1 mM puromycin to maintain a selection pressure on the transfected cells, although puromycin was omitted from the media during experiments. On the day of the experiment, cells were harvested with Trypsi EDTA and counted. The required number of cells w¾s transferred to a 50 ml falcon tube and centrifuged to pellet cells (1000 rpm, 5 min). Excess media was removed by aspiration leaving cell pellets sitting m 50-100 mΐ media. The cell number w¾s calculated to ensure a seeding density of 400 cells per well of a 96-well plate, with cells resuspended in a-MEM + 10% FCS, -P/S + IQmM D-Glucose + Q.2mM 2,-deoxycytidine. Cells were seeded under aerobic or anoxic conditions, the latter using a 5% H2/palladium catalyst scrubbed Bactron anaerobic chamber (Sheldon Manufacturing, Cornelius, Oregon) to achieve severe anoxia (<10 ppm O2 gas phase) during prodrug exposure.
[00580] The layout of the plate is described in Figure 21, where column 1 (the left-most column) consists of 8 media only control wells. Column 2 (second from the left) consists of 8 cells only (no prodrug) control wells. The remaining columns (3-12) consist of cells exposed to a range of prodrug concentrations. A high concentration of prodrug was added to column 12 (the right-most column) in each row, and 3-fold dilutions were performed across the plate using a multi-channel pipette. Thus, a total of four prodrugs could be evaluated on a single plate, with duplicate wells per prodrug. In this example, the three prodrugs were evaluated ith the range of concentrations shown m the following table:
Table 8A: Concentration ranges for prodrugs in the 96- well plate
Figure imgf000248_0001
[00581] After 2 hours incubation to enable cell attachment to the plates (and equilibrate to anoxia in the ease of the anoxic samples), cells were exposed to a range of prodrug concentrations for 4 hours under aerobic or anoxic conditions. After prodrug exposure, plates were transferred to aerobic conditions. A vacuum immunowash was used to aspirate the media and wells were washed three times with 150 mΐ of prodrug-free aMEM 5% PCS + P/S. The plates were then incubated under aerobic conditions for 5 days for cell growth. Cells were fixed by adding 50m1 of cold 40% trichloroacetic acid (TCA) to each well by layering on top without mixing, this gave a final concentration of 10% TCA. Plates were incubated in a fridge for 1 hour before the TCA was washed off and the plates rinsed in running tap water. 50m1 of 0.4% SRB in 1% acetic acid was added to each well and plates were incubated for 30 minutes in the dark. Excess stain was flicked off and plates were washed four times in 2L of tap water containing 1% acetic acid. Excess fluid was flicked from the plates and the remaining SRB stain solubilized by adding IOOm] of lOmM unbuffered Tris to each well. Plates were then left on a shaker for at least 2 hours in the dark before analysis using a plate reader (set at an optical density of 490 nM for measurement filter and 450nM for reference filter). The ICso value is the concentration of prodrug required to inhibit cell growth by 50%.
[00582] The results from the ICso assay showed that overexpression of STEAP4 increased the sensitivity of C33A cells to Compound A, Compound C, Compound G, and Compound H. The results are shown in Table 8B.
Table 8B: ICso values for Compound A, Compound C, Compound G (SN29176) and Compound H (8N27686) in wild-type (WT) and STEAP4-expressing C33A cells, where the concentration of prodrug required to inhibit cell growth by 50% (ICso) is shown, as described in Example 5.
Figure imgf000249_0001
Figure imgf000250_0001
Equivalents
[00583] The details of one or more embodiments of the disclosure are set forth m the accompanying description above. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. Other features, objects, and advantages of the disclosure will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents and publications cited in this specification are incorporated by reference.
[00584] The foregoing description has been presented only for the purposes of illustration and is not intended to limit the disclosure to the precise form disclosed, but by the claims appended hereto.

Claims

Claims
1. A method of inhibiting epidermal growth factor receptor in a subject in need thereof, comprising: administering to the subject an effective amount of an epidermal growth factor receptor- inhibiting drug, wherein the drug is covalently bonded to an attenuating moiety.
2. The method of claim 1, wherein the attenuating moiety is a reductive trigger of the Formula (II):
Figure imgf000251_0001
3. The method of claim 1 or 2, wherein the atenuating moiety is a reductive trigger selected from the group consisting of a compound of the Formula 11a. lib, Tic, lid, lie, Ilf, or XXg:
Figure imgf000251_0002
fii Mb Me iBd iie Of Mg
4. The method of any one of the preceding claims, wherein the attenuating moiety is a reductive trigger selected from the group consisting of a compound of the Formula a-ae:
Figure imgf000251_0003
Figure imgf000252_0001
5. The method of claim 1, wherein the epidermal growth factor receptor-inhibiting drug is selected from the group consisting of:
Figure imgf000252_0002
Figure imgf000253_0001
Figure imgf000254_0001
Figure imgf000255_0001
Figure imgf000256_0001
Figure imgf000257_0001
Figure imgf000258_0001
Figure imgf000259_0001
Figure imgf000260_0001
trigger.
6. The method of claim i . wherein the epidermal growth factor receptor-inhibiting drug is selected from the group consisting of:
Figure imgf000261_0001
Figure imgf000262_0001
Figure imgf000263_0001
Figure imgf000264_0001
Figure imgf000265_0001
Figure imgf000266_0001
Figure imgf000267_0001
Figure imgf000268_0001
Figure imgf000269_0001
Figure imgf000270_0001
Figure imgf000271_0001
Figure imgf000272_0001
7. The method of claim 1 wherein the epidermal growth factor receptor-inhibiting drug is selected from the group consisting of:
Figure imgf000272_0002
Figure imgf000273_0001
Figure imgf000274_0001
Figure imgf000275_0001
WO 2021/127456
Figure imgf000277_0001
Figure imgf000279_0001
Figure imgf000280_0001
Figure imgf000281_0001
Figure imgf000282_0001
Figure imgf000283_0001
8. The method of claim 1, wherein the epidermal growth factor receptor is HER1.
9. The method of claim 1 wherein the epidermal growth factor receptor is HER2.
10. The method of claim 1 , wherein the epidermal growth factor receptor is HER4.
11. The method of any one of the preceding claims, wherein the attenuating moiety is cleaved from the drug in vivo.
12. The method of any one of the preceding claims, wherein the attenuating moiety is cleaved from the drug under anoxic conditions.
13. The method of any one of the preceding claims, wherein the attenuating moiety is cleaved in the presence of STEAP4.
14. The method of any one of the preceding claims, wherein the attenuating moiety reduces or inhibits activity of the epidermal growth factor receptor-inhibiting drug outside of a tumor environment.
15. The method of any one of the preceding claims, wherein the attenuating moiety is cleaved from the drug within a tumor environment.
16. The method of any one of the preceding claims, wherein the epidermal growth factor receptor exhibits a whd-type ATP-competitive tyrosine kinase binding site.
17. The method of any one of the preceding claims, wherein the epidermal growth factor receptor is a mutated epidermal growth factor receptor.
18. The method of any one of the preceding claims, wherein the epidermal growth factor receptor exhibits an upregulation mutation.
19. The method of any one of the preceding claims, wherein the epidermal growth factor receptor exhibits a constitutively activating mutation.
20. The method of any one of the preceding claims, wherein the epidermal growth factor receptor exhibits a downregulation mutation.
21. A method of treating a cancer in a subject in need thereof, comprising: administering to the subject an effective amount of an epidermal growth factor receptor- inhibiting drug, wherein the drug is covalently bonded to an attenuating moiety.
22. The method of claim 21, wherein the attenuating moiety is a reductive trigger of the Formula (II):
Figure imgf000284_0001
23. The method of claim 21, wherein the attenuating moiety is a reductive trigger selected from the group consisting of a compound of the Formula Ha, 11b, 11c, lid, lie, Ilf, or Ilg:
Figure imgf000284_0002
fid Mb He lid lie Slf Hg
24. The method of any one of the preceding claims, wherein the attenuating moiety is a reductive trigger selected from the group consisting of a compound of the Formula a-ae:
Figure imgf000284_0003
Figure imgf000285_0001
25. The method of claim 21 wherein the epidermal growth factor receptor-inhibiting drug is selected from the group consisting of:
Figure imgf000286_0001
Figure imgf000287_0001
Figure imgf000288_0001
Figure imgf000289_0001
Figure imgf000290_0001
Figure imgf000291_0001
Figure imgf000292_0001
Figure imgf000293_0001
trigger.
26. The method of claim 21, wherein the epidermal growth factor receptor-inhibiting drug is selected from the group consisting of:
Figure imgf000295_0001
Figure imgf000296_0001
Figure imgf000297_0001
Figure imgf000298_0001
Figure imgf000299_0001
Figure imgf000300_0001
Figure imgf000301_0001
Figure imgf000302_0001
Figure imgf000303_0001
Figure imgf000304_0001
Figure imgf000305_0001
.
27. The method of claim 21, wherein the epidermal growth factor receptor-inhibiting drug is selected from the group consisting of:
Figure imgf000305_0002
Figure imgf000306_0001
Figure imgf000307_0001
Figure imgf000308_0001
WO 2021/127456
Figure imgf000310_0001
Figure imgf000311_0001
Figure imgf000312_0001
Figure imgf000313_0001
Figure imgf000314_0001
Figure imgf000315_0001
Figure imgf000316_0001
28. The method of claim 21, wherein the drug inhibits HER! .
29. The method of claim 21 wherein the drug inhibits HER2.
30. The method of claim 21, wherein the drug inhibits HER4.
31. The method of any one of the preceding claims, wherein the attenuating moiety is cleaved in vivo.
32. The method of any one of the preceding claims, wherein the attenuating moiety is cleaved from the drug under anoxic conditions.
33. The method of any one of the preceding claims, wherein the attenuating moiety is cleaved in the presence of STEAP4.
34. The method of any one of the preceding claims, wherein the attenuating moiety reduces or inhibits activity of the epidermal growth factor receptor-inhibiting drug outside of a tumor environment.
35. The method of any one of the preceding claims, wherein the attenuating moiety is cleaved from the drug within a tumor environment.
36. The method of any one of the preceding claims, wherein the epidermal growth factor receptor exhibits a wild-type ATP-competitive tyrosine kinase binding site.
37. The method of any one of the preceding claims, wherein the epidermal growth factor receptor is a mutated epidermal growth factor receptor.
38. The method of any one of the preceding claims, wherein the epidermal growth factor receptor exhibits an upregulation mutation.
39. The method of any one of the preceding claims, wherein the epidermal growth factor receptor exhibits a constitutively activating mutation.
40. The method of any one of the preceding claims, wherein the epidermal growth factor receptor exhibits a downregulation mutation.
41. The method of any one of the preceding claims, wherein the cancer treated is selected from the group consisting of non-small cell lung cancer, esophageal cancer, pancreatic cancer, rectal cancer, squamous cell carcinoma of the head or neck, squamous cell cancer of the skin, glioblastoma, colon cancer, cervical cancer, bladder cancer, breast cancer, gastrointestinal stromal cancer (GIST), ovarian cancer, gastric cancer, endometrial cancer, uterine cancer prostate cancer, liver cancer, melanoma, brain cancer, and mesothelioma.
42. A compound selected from the group consisting of:
Figure imgf000317_0001
Figure imgf000318_0001
Figure imgf000319_0001
Figure imgf000320_0001
Figure imgf000321_0001
Figure imgf000322_0001
Figure imgf000323_0001
Figure imgf000324_0001
Figure imgf000325_0001
trigger.
43. A compound selected from the group consisting of:
Figure imgf000327_0001
Figure imgf000328_0001
Figure imgf000329_0001
Figure imgf000330_0001
Figure imgf000331_0001
Figure imgf000332_0001
Figure imgf000333_0001
Figure imgf000334_0001
Figure imgf000335_0001
Figure imgf000336_0001
Figure imgf000337_0001
44. A compound selected from the group consisting of:
Figure imgf000337_0002
Figure imgf000338_0001
Figure imgf000339_0001
Figure imgf000340_0001
WO 2021/127456
Figure imgf000342_0001
Figure imgf000343_0001
Figure imgf000344_0001
Figure imgf000345_0001
Figure imgf000346_0001
Figure imgf000347_0001
Figure imgf000348_0001
45. A pharmaceutical composition comprising a therapeutically effective amount of: an epidermal growth factor receptor-inhibiting drug, wherein the drug is covalently bonded to an attenuating moiety; and a pharmaceutically acceptable carrier.
46. The method of claim 8, wherein HER! is a mutated epidermal growth factor receptor.
47. The method of claim 8, wherein HER! exhibits an upregulation mutation.
48. The method of claim 8, wherein HER! exhibits a constitutively activating mutation.
49. The method of claim 8, wherein HER! exhibits a downregulation mutation.
50. The method of claim 9, wherein HER2 is a mutated epidermal growth factor receptor.
51. The method of claim 9, wherein HER2 exhibits an upregulation mutation.
52. The method of claim 9, wherein HER2 exhibits a constitutively activating mutation.
53. The method of claim 9, wherein HER2 exhibits a downregulation mutation.
54. The method of claim 10, wherein HER4 is a mutated epidermal growth factor receptor.
55. The method of claim 10, wherein HER4 exhibits an upregulation mutation.
56. The method of claim 10, wherein HER4 exhibits a constitutively activating mutation.
57. The method of claim 10, wherein HER4 exhibits a downregulation mutation.
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CN113880859A (en) * 2021-11-16 2022-01-04 江西科技师范大学 2-aryl-4-arylmethylamino pyrimidine compound and application thereof
CN114957224A (en) * 2022-05-17 2022-08-30 浙大城市学院 Tumor hypoxia-targeted EGFR inhibitor and application thereof
US11548891B1 (en) * 2022-04-22 2023-01-10 Batterjee Medical College Quaternary ammonium salts of phenanthroindolizidine and phenanthroquinolizidine alkaloids as hypoxia-targeted anticancer agents

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CN113880859A (en) * 2021-11-16 2022-01-04 江西科技师范大学 2-aryl-4-arylmethylamino pyrimidine compound and application thereof
CN113880859B (en) * 2021-11-16 2022-06-21 江西科技师范大学 2-aryl-4-arylmethylamino pyrimidine compound and application thereof
US11548891B1 (en) * 2022-04-22 2023-01-10 Batterjee Medical College Quaternary ammonium salts of phenanthroindolizidine and phenanthroquinolizidine alkaloids as hypoxia-targeted anticancer agents
CN114957224A (en) * 2022-05-17 2022-08-30 浙大城市学院 Tumor hypoxia-targeted EGFR inhibitor and application thereof
CN114957224B (en) * 2022-05-17 2024-03-19 浙大城市学院 EGFR inhibitor for tumor hypoxia targeting and application thereof

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