WO2024073624A2 - Antiproliferative betti bases and prodrugs thereof - Google Patents

Antiproliferative betti bases and prodrugs thereof Download PDF

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WO2024073624A2
WO2024073624A2 PCT/US2023/075454 US2023075454W WO2024073624A2 WO 2024073624 A2 WO2024073624 A2 WO 2024073624A2 US 2023075454 W US2023075454 W US 2023075454W WO 2024073624 A2 WO2024073624 A2 WO 2024073624A2
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group
compound
equiv
substituted
mmol
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PCT/US2023/075454
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French (fr)
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Xinjiang Wang
Sherry R. Chemler
Rati LAMA
Samuel GALSTER
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The Research Foundation For The State University Of New York
Health Research, Inc.
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/72—Nitrogen atoms
    • C07D213/74—Amino or imino radicals substituted by hydrocarbon or substituted hydrocarbon radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/32—One oxygen, sulfur or nitrogen atom
    • C07D239/42—One nitrogen atom
    • 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
    • C07D401/14—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 three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings
    • 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
    • C07D498/04—Ortho-condensed systems

Definitions

  • Tumor suppressor p53 is a well-established drug target whose activation has been shown to induce tumor regression in several models.
  • p53 activity is inhibited mainly by MDM2 (Murine Double Minute 2) and MDM4, RING domain-containing proteins.
  • MDM4 also known as MDMX
  • MDM2 homologue which is expressed at levels higher than MDM2 due to its increased expression and protein stability in most cancer types.
  • the N-terminus of both MDM2 and MDM4 binds to p53 transactivation domain and inhibits p53-dependent transcriptional transactivation of downstream genes, while the C-terminal RING domain of MDM2 can bind to E2 ubiquitin conjugating enzymes for p53 ubiquitination and to the RING domain of MDM4 to form heterodimers.
  • Development of small molecule inhibitors of MDM2-p53 interaction has been a research focus for decades since the discovery of the first-in-class small molecule Nutlin3a and several Nultlin3-like MDM2 inhibitors are in clinical trials.
  • MDM2 and MDM4 are cancer drug targets validated in multiple models for p53-based cancer therapies.
  • the RING domains of MDM2 and non-p53-binder MDM2 splice isoforms form RING domain heterodimer polyubiquitin E3 ligases with MDM4, which regulate p53 stability in vivo and promote tumorigenesis independent of p53.
  • MDM2 RING domain in p53 regulation and cancer development, small molecule inhibitors targeting the E3 ligase activity of MDM2-MDM4 are poorly explored.
  • compositions and methods for inhibiting the growth of cells and/or inducing apoptosis and/or ferroptosis are provided.
  • the compositions and methods inhibit growth and/or induce apoptosis of cancer cells and/or ferroptosis.
  • An aspect of the present disclosure provides a prodrug of a Betti-base compound.
  • masking the phenol’s alcohol functional group may contribute to the Betti -base’s antiproliferative activity. Without intending to be bound by any particular theory, the increased antiproliferative activity is thought to be due to enhanced cell penetration.
  • the phenol moiety of Betti-base compounds is thought to be a structural weakness with respect to bioavailability, contributing to low in vivo half-life and problematic formulation and/or acute toxicity, potentially related to compound aggregation and ambient metal chelation.
  • An advantage of the present disclosure is the slower and more selective release of a Betti-base compound.
  • prodrugs of the present disclosure mask the phenol and allow for its slower and more selective release, for example, in cancer cells.
  • the composition of the present disclosure comprises prodrugs containing moi eties that can be converted to phenols in vivo.
  • the present disclosure comprises prodrugs containing moieties that can be converted to phenols selectively in cells, such as, for example, cancer cells.
  • the prodrug of the present disclosure could be applied to any drug candidate where the active form is a Betti-type base.
  • Figure 1 Structures of (A) MMRi62 and (B) MMRi67 compound series.
  • MMRi67 is a small molecule inhibitor of MDM2-MDM4 E3 ligase activity.
  • A MMRi67 inhibits ubiquitination of MDM2B (left) and MDM4 (middle) by MDM2B-MDM4 E3 complex but not NEDD4-1 (right) in in vitro E3 ligase assays. WB analysis of MDM4, MDM2B and NEDD4-1 using specific antibodies after in vitro ubiquitination reaction in the presence of solvent or indicated concentrations of MMRi67.
  • ubiquitinated MDM4 Ub-MDM4
  • ubiquitinated MDM2B Ub- MDM2B
  • ubiquitinated NEDD4-1 Ub-NEDD4-1
  • B The E3 ligase inhibitory activity of MMRi67 is not abolished by the presence of 0.01% Triton X-100. Same procedure as in A except the ubiquitination reaction was performed in the presence of 0.01% Triton X-100. Only MDM2B data were shown.
  • C The E3 ligase inhibitory activity of MMRi67 is not abolished by the presence of increasing concentrations of Zn to neutralize potential chelating capability of MMRi67.
  • FIG. 3 Assessment of the inhibitory effect of MMRi67 derivatives toward MDM2B-MDM4 E3 ligase in in vitro E3 ligase assays.
  • A Comparison of MMRi67 with hydroxyl to hydrogen substituted (67-6) and chloro to hydrogen substituted (67-1) analogs in inhibition of MDM4 and MDM2B ubiquitination in vitro. WB analysis of MDM4 or MDM2B using specific antibodies after in vitro ubiquitination reaction in the presence of solvent or indicated compound at the indicated concentrations. The smearing bands of ubiquitinated MDM4 (Ub-MDM4) and MDM2B (Ub-MDM2B) are indicated.
  • FIG. 4 Anti-proliferation activity of 67-5 is associated with downregulation of MDM2, MDM4 and FTH1 protein expression and caspase 3 activation.
  • Antiproliferation assays in NALM6 and shp53NALM6 cells The ICso in NAML6 cells was 12.8 pM for MMRi67 and 0.38 pM for 67-5.
  • the ICso in shP53NAML6 cells was 10.9 pM for MMRi67 and 0.45 pM for 67-5.
  • FIG. 1 Characterization of MMRi71 in cells.
  • A The anti-proliferation activity of MMRi71 is p53-independent with ICso of 0.23 pM in NALM6 and 0.29 pM in shp53NALM6 cells.
  • B WB analysis showing MMRi71 induces MDM4/MDM2 and FTH1 protein degradation and apoptotic effect in NALM6 and shP53NALM6 cells.
  • C The pro- apoptotic effect of MMRi71 is not affected by the presence of 0.0025% Triton X-100 in NALM-6 cells as shown by WB analysis of apoptotic PARP cleavage.
  • MMRi71 -induced MDM4 degradation is MDM2 dependent. WB analysis showing Mdm2 knockdown in Manca-mlp-Mdm2 cells abolished MMRi71 -induced MDM4 degradation.
  • E MMRi71- induced FTH1 degradation is lysosome dependent. WB showing rescue of MMRi71 -induced FTH1 degradation by 50 nM lysosome inhibitor Bafilomycin Al (Bfl) in NALM-6 cells.
  • F (G) DNA damage induction by MMRi71 and 67-7.
  • Mouse p53/Mdm2 double knockout MEFs (2KO) (F) or human 293T cells (G) were treated with indicated concentrations of MMRi71 and 67-7 for 24h at the indicated concentrations followed by WB analysis for gammaH2AX and Tubulin with a specific antibodies. Tubulin serves as protein loading control.
  • Figure 17. (A) 1 H NMR spectrum for 62-7. (B) 13 C NMR spectrum for 62-7. [0029] Figure 18. (A) X H NMR spectrum for 62-8. (B) 13 C NMR spectrum for 62-8.
  • Figure 20 (A) 1 H NMR spectrum for 62-9. (B) 13 C NMR spectrum for 62-9.
  • Figure 25 (A) ’H NMR spectrum for S-5. (B) 13 C NMR spectrum for S-5. [0037] Figure 26. (A) 1 H NMR spectrum for 67-7. (B) 13 C NMR spectrum for 67-7.
  • Figure 39 (A) 1 H NMR spectrum for SC-62-2. (B) 13 C NMR spectrum for SC-62-2. [0051] Figure 40. (A) X H NMR spectrum for SC-62-3. (B) 13 C NMR spectrum for SC-62-3.
  • Figure 48 (A) X H NMR spectrum for SC-62-12. (B) 13 C NMR spectrum for SC-62-12.
  • Figure 49 Comparison of Western Blot analysis of indicated proteins and apoptotic effects induced by SC-62-1 and prodrug SC-62-1 C.
  • Figure 50 Growth Inhibition of three melanoma cell lines by SC-62-1.
  • amounts, sizes, compositions, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error, or the like, or other factors known to those of skill in the art such that equivalent results or effects are obtained.
  • an amount, size, composition, parameter, or other quantity or characteristic, or alternative is “about” or “the like,” whether or not expressly stated to be such. It is understood that where “about,” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.
  • Ranges of values are disclosed herein.
  • the ranges set out a lower limit value and an upper limit value. Unless otherwise stated, the ranges include the lower limit value, the upper limit value, and all values between the lower limit value and the upper limit value, including, but not limited to, all values to the magnitude of the smallest value (either the lower limit value or the upper limit value) of a range. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.
  • a numerical range of “0.1% to 5%” should be interpreted to include not only the explicitly recited values of 0.1% to 5%, but also, unless otherwise stated, include individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5% to 1.1%; 0.5% to 2.4%; 0.5% to 3.2%, and 0.5% to 4.4%, and other possible sub-ranges) within the indicated range. It is also understood (as presented above) that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed.
  • Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about, it will be understood that the particular value forms a further disclosure. For example, if the value “about 10” is disclosed, then “10” is also disclosed.
  • group refers to a chemical entity that is monovalent (i.e., has one terminus that can be covalently bonded to other chemical species), divalent, or polyvalent (i.e., has two or more termini that can be covalently bonded to other chemical species).
  • group also includes radicals (e.g., monovalent and multivalent, such as, for example, divalent radicals, trivalent radicals, and the like).
  • alkyl refers to branched or unbranched, linear saturated hydrocarbon groups and/or cyclic hydrocarbon groups.
  • alkyl groups include, but are not limited to, methyl groups, ethyl groups, propyl groups, butyl groups, isopropyl groups, tert-butyl groups, cyclopropyl groups, cyclopentyl groups, cyclohexyl groups, and the like.
  • Alkyl groups are saturated groups, unless it is a cyclic group.
  • an alkyl group is a Ci to C20 alkyl group, including all integer numbers of carbons and ranges of numbers of carbons therebetween (e.g., Ci, C2, C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C9, C10, C11, C12, C13, Ci4, C15, Ci6, C17, Cis, C19, or C20).
  • the alkyl group may be unsubstituted or substituted with one or more substituents.
  • substituents include, but are not limited to, halogens (-F, -Cl, -Br, and -I), aliphatic groups (e.g., alkyl groups, alkenyl groups, alkynyl groups, and the like), halogenated aliphatic groups (e.g., trifluoromethyl group), aryl groups, halogenated aryl groups, alkoxide groups, amine groups, nitro groups, carboxylate groups, carboxylic acids, ether groups, alcohol groups, alkyne groups (e.g., acetylenyl groups and the like), and the like, and combinations thereof.
  • halogens -F, -Cl, -Br, and -I
  • aliphatic groups e.g., alkyl groups, alkenyl groups, alkynyl groups, and the like
  • halogenated aliphatic groups e.g., trifluoromethyl group
  • cycloalkyl refers to a cyclic hydrocarbon group, e.g., cyclopropyl, cyclobutyl, cyclohexyl, and cyclopentyl groups.
  • alkyl substituents may be substituted with various other functional groups. Additional non-limiting examples include aliphatic groups (e.g., alkyl groups, alkenyl groups, alkynyl groups, and the like), halogenated aliphatic groups (e.g., trifluoromethyl group), aryl groups, halogenated aryl groups, alkoxide groups, nitro groups, carboxylate groups, carboxylic acids, ether groups, alkyne groups (e.g., acetylenyl groups and the like), and the like, and combinations thereof.
  • aliphatic groups e.g., alkyl groups, alkenyl groups, alkynyl groups, and the like
  • halogenated aliphatic groups e.g., trifluoromethyl group
  • aryl groups e.g., halogenated aryl groups
  • alkoxide groups e.g., trifluoromethyl group
  • aryl groups e.g.
  • aryl refers to C5 to Ci6 aromatic or partially aromatic carbocyclic groups, including all integer numbers of carbons and ranges of numbers of carbons therebetween (e.g., C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, or Cie).
  • An aryl group may also be referred to as an aromatic group.
  • the aryl groups may comprise polyaryl groups such as, for example, fused rings, biaryl groups, or a combination thereof.
  • the aryl group may be unsubstituted or substituted with one or more substituents.
  • substituents include, but are not limited to, halogens (-F, -Cl, -Br, and -I), aliphatic groups (e.g., alkyl groups, alkenyl groups, alkynyl groups, and the like), aryl groups, alkoxides, carboxylates, carboxylic acids, ether groups, and the like, and combinations thereof.
  • aryl groups include, but are not limited to, phenyl groups, biaryl groups (e.g., biphenyl groups and the like), fused ring groups (e.g., naphthyl groups and the like), hydroxybenzyl groups, tolyl groups, xylyl groups, and the like.
  • heteroaryl refers to a monocyclic or bicyclic ring system comprising one or two aromatic rings and containing at least one nitrogen or oxygen atom in an aromatic ring. Unless otherwise indicated, a heteroaryl group can be unsubstituted or substituted with one or more, and in particular one or two, substituents.
  • Non-limiting examples of substituents include halogens (-F, -Cl, -Br, and -I), aliphatic groups (e.g., alkyl groups, alkenyl groups, alkynyl groups, and the like), halogenated aliphatic groups (e.g., trifluoromethyl group), aryl groups, halogenated aryl groups, alkoxide groups, amine groups, nitro groups, carboxylate groups, carboxylic acids, ether groups, alcohol groups, alkyne groups (e.g., acetylenyl groups and the like), and the like, and combinations thereof.
  • halogens -F, -Cl, -Br, and -I
  • aliphatic groups e.g., alkyl groups, alkenyl groups, alkynyl groups, and the like
  • halogenated aliphatic groups e.g., trifluoromethyl group
  • heteroaryl groups include, benzofuranyl, thienyl, furyl, pyridyl, oxazolyl, quinolyl, thiophenyl, isoquinolyl, indolyl, triazinyl, triazolyl, isothiazolyl, isoxazolyl, imidazolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, and thiadiazolyl groups, and substituents analogs of any of the foregoing heteroaryl groups.
  • alkoxy refers to where R a is a linear, branched or cyclic Ci-Ce alkyl group, including all integer numbers of carbons and ranges of numbers of carbons therebetween.
  • suitable alkoxy groups include methoxy, ethoxy, propoxy, /.w-propoxy, butoxy, sec-butoxy, tert-butoxy, and hexoxy groups.
  • alkyl substituents can be substituted with various other functional groups, e.g. functional groups disclosed herein.
  • amino or “amino R b group” refers to Rb where each R b is selected independently from the group consisting of hydrogen atom, substituted or unsubstituted C1-C10 alkyl, including all integer numbers of carbons and ranges of numbers of carbons therebetween, substituted or unsubstituted phenyl, substituted or unsubstituted heteroaryl, substituted carbonyl, substituted sulfonyl, haloalkyl, and substituted or unsubstituted benzyl groups.
  • benzyl or “benzyl group” refers to where R c is a substituent on the phenyl ring and n is from
  • the substituents can be the same or different.
  • the substituents on the benzyl group include substituted or unsubstituted alkyl, -NHz, phenyl, haloalkyl (e.g., -CF3), halo (e.g., -F, -Cl, -Br, -I), alkoxy (e.g., -OMe), and -OH groups.
  • halogen means fluorine, chlorine, bromine, and iodine
  • halo means fluoro, chloro, bromo, and iodo
  • phenoxy or “phenoxy group” (-OPh) refers to where each Y is independently selected from the group consisting of F, Cl, Br, and I and m can be 0, 1 or 2.
  • phenyl or “phenyl group” means where each R d is an independent substituent on the phenyl group and n is from 0 to 5.
  • the substituents at different occurrences can be the same or different.
  • the substituents on the phenyl group include substituted or unsubstituted Ci-Ce alkyl, including all integer numbers of carbons and ranges of numbers of carbons therebetween, substituted or unsubstituted amino, haloalkyl (e.g., -CF3), halo (e.g., -F, -Cl, - Br, -I), substituted or unsubstituted alkoxy (e.g., -OMe), and sulfonyl group.
  • two adjacent R groups can be connected through to form a dioxolyl group.
  • Betti bases which comprise phenolic (or hydroxyaryl) benzylic amine derivatives, have demonstrated broad anti-proliferative activity against cancer cells, including leukemia, pancreatic cancer, and melanomas.
  • Betti base refers to 2- (aminomethyl)phenol derivatives.
  • the compounds are thought to impact one or more cellular proteins that play significant roles in cancer progression, including MDM2, MDMX, p53, E3 ligase and FTH1. Structure-activity-relationship studies of these compounds indicate the phenol moiety contributes to the anti-proliferative activity.
  • Various active compounds in this family have been developed and substituted with functional groups that optimize antiproliferative activity in cells.
  • the present disclosure provides a compound with the structure:
  • R 5 and R 11 are connected such that a heterocyclic group is formed
  • the ring comprising X 1 , X 2 , X 3 , and X 4 may comprise nitrogen or -CH- at the various X positions on the ring.
  • X 1 is -CH- or nitrogen
  • X 2 is -CH- or nitrogen
  • X 3 is -CH- or nitrogen
  • X 4 is -CH- or nitrogen.
  • the ring comprising X 1 , X 2 , X 3 , and X 4 may have a structure wherein X 1 is nitrogen, X 2 is -CH-, X 3 is
  • the ring may have a structure wherein X 1 is -CH-, X 2 is -CH-, X 3 is -CH-, and X 4 is nitrogen, i.e.,
  • the compound when R 5 and R 11 are connected such that a heterocyclic group is formed, the compound may have the following structure:
  • R 3 may be a hydrogen, a substituted or unsubstituted alkyl group, a halogen group, a substituted or unsubstituted amino group, or a substituted or unsubstituted ether group.
  • R 3 groups include, but are not limited to halogens
  • R 4 may be a hydrogen, a substituted or unsubstituted alkyl group, a halogen group, a substituted or unsubstituted amino group, or a substituted or unsubstituted ether group.
  • A may be oxygen, sulfur, or a substituted or unsubstituted nitrogen.
  • R 5 may be ' , where R 1 may be a hydrogen, an acyl group, an alkyl carbonate group, an acetal group, carbonate group, carbamate group, a ketal group, or an aminal group.
  • R 6 may where R 2 may be a hydrogen, a cyano group, a substituted or unsubstituted ester group, a carboxylic acid group, a sulfonyl group (SO2R), or a substituted or unsubstituted amide group.
  • R 7 may be a hydrogen, a halogen group, a substituted or unsubstituted alkyl group, a nitro group, or an azide group; and A may be oxygen, sulfur, or a substituted or unsubstituted nitrogen and Z may be nitrogen, CH, or CR (where R is alkyl or aryl).
  • R 11 is H or an acyl group.
  • R 12 is H or a substituted alkyl group or unsubstituted alkyl group.
  • R 5 is not -OH.
  • R 5 may be -OH.
  • R 6 may be chosen
  • R 3 is chlorine
  • R 3 is hydrogen, i.e.,
  • R 5 is chosen from where M + is a cation (e.g., Li + , Na + , K + , Ca 2+ ). In embodiments R 5 is such that the structure is, for example,
  • R 6 is chosen from
  • the structure one or more of R 3 , R 7 , and R 2 is not hydrogen, chlorine, bromine, or iodine.
  • R 3 , R 7 , and R 2 is not hydrogen, chlorine, bromine, or iodine.
  • R 3 , R 7 , and R 2 is not hydrogen, chlorine, bromine, or iodine.
  • R 6 is not hydrogen, chlorine, bromine, or iodine.
  • R 3 , R 7 , and R 2 is not hydrogen, chlorine, bromine, or iodine.
  • the compound does not have the following structure::
  • R 5 is transformed to -OH in a body of an individual.
  • R 5 is a group that can be transformed to -OH under in vivo conditions (e.g., in a cell or in a biological medium). Without intending to be bound by any particular theory, it is considered that a compound where R 5 is not -OH may have an increased bioavailability relative to the compound where R 5 is -OH.
  • the compound of the present disclosure may have any of the following structures:
  • R 5 is -OH
  • R 2 , R 3 , and are R 7 are as described herein.
  • the compound of the present disclosure 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-aminoethyl
  • compositions of the disclosure may exist as salts.
  • Pharmaceutically acceptable salts of the compounds of the disclosure generally are preferred in the methods of the disclosure.
  • the term "pharmaceutically acceptable salts” refers to salts or zwitterionic forms of a compound of the present disclosure. Salts of compounds of the present disclosure can be prepared during the final isolation and purification of the compounds or separately by reacting the compound with an acid having a suitable cation.
  • the pharmaceutically acceptable salts of a compound of the present disclosure are acid addition salts formed with pharmaceutically acceptable acids.
  • acids which can be employed to form pharmaceutically acceptable salts include inorganic acids such as nitric, boric, hydrochloric, hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic, maleic, succinic, and citric.
  • Nonlimiting examples of salts of compounds of the disclosure include, the hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, 2- hydroxyethansulfonate, phosphate, hydrogen phosphate, acetate, adipate, alginate, aspartate, benzoate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerolphsphate, hemisulfate, heptanoate, hexanoate, formate, succinate, fumarate, maleate, ascorbate, isethionate, salicylate, methanesulfonate, mesitylenesulfonate, naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3- phenylproprionate, picrate, pivalate, prop
  • available amino groups present in the compounds of the disclosure can be quatemized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and steryl chlorides, bromides, and iodides; and benzyl and phenethyl bromides.
  • any reference to compounds of the present disclosure appearing herein is intended to include a compound of the present disclosure as well as pharmaceutically acceptable salts or hydrates, thereof.
  • Compounds of the present disclosure may exist in particular geometric or stereoisomeric forms.
  • the present disclosure contemplates all such compounds, including cis- and trans-i somers, R- and S-enantiomers, diastereomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the disclosure.
  • Additional asymmetric carbons may be present in a substituent, such as, for example an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this disclosure.
  • the present disclosure provides for all enantiomers, diastereomers, and isomers of the compounds, either in their enantioenriched or pure forms, or as isomeric mixtures, of the present disclosure.
  • compositions may comprise one or more compounds of the present disclosure.
  • the composition further comprises a pharmaceutically acceptable carrier.
  • the compositions may include one or more pharmaceutically acceptable carrier(s).
  • Non-limiting examples of compositions include solutions, suspensions, emulsions, solid injectable compositions that are dissolved or suspended in a solvent before use, and the like. Injections may be prepared by dissolving, suspending, or emulsifying one or more of the active ingredient(s) in a diluent.
  • diluents include distilled water (e.g., for injection), physiological saline, vegetable oil, alcohol, and the like, and combinations thereof.
  • Injections may contain, for example, stabilizers, solubilizers, suspending agents, emulsifiers, soothing agents, buffers, preservatives, and the like, and combinations thereof. Injections may be sterilized in the final formulation step or prepared by sterile procedure.
  • a pharmaceutical composition of the disclosure may also be formulated into a sterile solid preparation, for example, by freeze-drying, and may be used after sterilized or dissolved in sterile injectable water or other sterile diluent(s) immediately before use.
  • compositions include, but are not limited to, sugars, such as, for example, lactose, glucose, and sucrose; starches, such as, for example, corn starch and potato starch; cellulose, such as, for example, sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as, for example, cocoa butter and suppository waxes; oils, such as, for example, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil, and soybean oil; glycols, such as, for example, propylene glycol; polyols, such as, for example, glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as, for example, ethyl oleate and ethyl laurate; agar; buffering agents, such as, for example, g
  • compositions of the disclosure can comprise more than one pharmaceutical agent.
  • a first composition comprising a compound of the disclosure and a first pharmaceutical agent can be separately prepared from a composition which comprises the same compound of the disclosure and a second pharmaceutical agent, and such preparations can be mixed to provide a two-pronged (or more) approach to achieving the desired prophylaxis or therapy in an individual.
  • compositions of the disclosure can be prepared using mixed preparations of any of the compounds disclosed herein.
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • antioxidants examples include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), le
  • compositions of the disclosure suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present disclosure as an active ingredient.
  • a compound of the present disclosure may also be administered as a bolus, electuary or paste.
  • the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as
  • compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent.
  • the tablets, and other solid dosage forms of the pharmaceutical compositions of the present disclosure may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres.
  • compositions may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • embedding compositions which can be used include polymeric substances and waxes.
  • the active ingredient can also be in micro- encapsulated form, if appropriate, with one or more of the above-described excipients.
  • Liquid dosage forms for oral administration of a compound of the present disclosure include pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art, such as, for example, water or other solvents, so
  • the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • the composition may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • composition may be for administration to an individual in need of treatment.
  • the composition may comprise one or more additional compounds.
  • the additional compounds may be BRAF inhibitors or MEK inhibitors.
  • BRAF inhibitors include, but are not limited to, Vemurafenib (Zelboraf®), dabrafenib (TAFINLAR®), and encorafenib (BRAFTOVI®).
  • MEK inhibitors include, but are not limited to, trametinib (MEKINIST® ), binimetinib (MEKTOVI®), selumetinib (Koselugo®), and cobimetinib (COTELLIC®).
  • a method for inducing apoptosis or ferroptosis in a cell comprising administering a therapeutically effective amount of the compound or composition of the present disclosure.
  • the compounds may promote ferroptosis, a cell death associated with reactive oxygen species caused by dysregulation of cellular iron.
  • a marker of ferroptosis is degradation of the cellular iron storage protein, ferratin heavy chain 1 (FTH1).
  • the administered compound undergoes a reaction within the subject (e.g., within a cell of the subject).
  • the reaction may convert R 5 of the compound to -OH.
  • the compound and/or the converted compound may induce apoptosis of the cell.
  • the reaction occurs within a cancer cell.
  • the reaction occurs outside of a cancer cell, and the converted compound (e.g., the compound after undergoing the reaction) is subsequently in contact with the cancer cell.
  • the subject to be treated by the method of the disclosure may be human or non-human (e.g., mammal).
  • Non-human animals include ungulates such as bovines. Additional on-limiting examples of non-human mammals include pigs, mice, rats, rabbits, cats, dogs, or other agricultural mammals, pet, or service animals, and the like.
  • compositions comprising a compound of the present disclosure and a pharmaceutical agent generally depends upon the needs of the individual to whom the composition of the disclosure is to be administered. These factors include, for example, the weight, age, sex, medical history, and nature and stage of the disease for which a therapeutic or prophylactic effect is desired.
  • the compositions can be used in conjunction with any other conventional treatment modality designed to improve the disorder for which a desired therapeutic or prophylactic effect is intended, non-limiting examples of which include surgical interventions and radiation therapies.
  • the compositions can be administered once, or over a series of administrations at various intervals determined using ordinary skill in the art, and given the benefit of the present disclosure.
  • the compounds of the present disclosure can be therapeutically administered as the neat chemical, but it is preferred to administer a compound of the present disclosure as a pharmaceutical composition or formulation.
  • the present disclosure provides a pharmaceutical composition comprising a compound of the present disclosure together with a pharmaceutically acceptable diluent or carrier therefor.
  • a process of preparing a pharmaceutical composition comprising admixing a compound of the present disclosure with a pharmaceutically acceptable diluent or carrier therefor.
  • the composition may further comprise one or more additional compounds (e.g., BRAF inhibitors and/or MEK inhibitors).
  • a method of treating cancer comprising administering the compound of the present disclosure or a composition comprising the compound of the present disclosure to cancerous cells of a subject.
  • the administering of the compound may be any method whereby the compound of the present disclosure is caused to contact a cell of the subject.
  • the administering may be, for example, oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, inhaled, or buccal administration, or combinations thereof.
  • the parenteral administration comprises intravenous, intraarterial, intracranial, intradermal, subcutaneous, intraperitoneal, intramuscular, intrathecal, or intraarticular administration.
  • kits for inducing apoptosis in a cell comprising a composition comprising the compound of claim 1.
  • the steps of the method described in the various embodiments and examples disclosed herein are sufficient to carry out the methods of the present invention.
  • the method consists essentially of a combination of the steps of the methods disclosed herein.
  • the method consists of such steps.
  • R 3 is a hydrogen, a substituted or unsubstituted alkyl group, a halogen group, a substituted or unsubstituted amino group, or a substituted or unsubstituted alkoxy group;
  • R 4 is a hydrogen, a substituted or unsubstituted alkyl group, a halogen group, a substituted or unsubstituted amino group, or a substituted or
  • R 5 is x , wherein R 1 is a hydrogen, an acyl group, an alkyl carbonate group, an acetal group, carbonate group, carbamate group, a ketal group, or an aminal group; a cyano group, a substituted or unsubstituted ester group, a carboxylic acid group, a sulfonyl group (SO2R), or a substituted or unsubstituted amide group; wherein R 7 is a hydrogen, a halogen group, a substituted or unsubstituted alkyl group, a nitro group, or an azide group; and wherein A is oxygen, sulfur, or a substituted or unsubstituted nitrogen and Z is N, CH, or CR, where R is an alkyl group or an aryl group; R 11 is H or an acyl group; R 12 is H or a substituted or unsubstituted
  • Statement 5 A compound of any one of Statements 1 to 4, wherein X 1 is nitrogen, X 2 is -CH-, X 3 is -CH-, and X 4 is nitrogen.
  • Statement 6 A compound of any one of Statements 1 to 4, wherein X 1 is -CH-, X 2 is -CH-, X 3 is -CH-, and X 4 is nitrogen.
  • Statement 7 A compound of any one of Statements 1 to 6, wherein R 3 is chlorine.
  • Statement 8 A compound of any one of Statements 1 to 6, wherein R 3 is hydrogen.
  • Statement 9 A compound of any one of Statements 1 or 4 to 7, wherein R 5 is chosen from and , wherein M + is a cation chosen from Li + , Na + , + , and
  • Statement 11 A compound of any one of Statements 1 to 10, wherein R 6 is chosen from Statement 12.
  • Statement 13 A compound of Statement 1, wherein the compound does not have the following structure:
  • Statement 14 A compound of any one of Statements 1 or 3 to 13, wherein R 5 can be converted to -OH in a cell.
  • Statement 15 A compound of any one of Statements 1, 3 to 12, or 14, wherein the structure is:
  • Statement 20 A composition comprising a compound of any one of Statements 1 to 12 or 14 to 19.
  • Statement 21 A composition of Statement 20, further comprising a pharmaceutically acceptable carrier.
  • Statement 22 A method of inducing apoptosis or ferropotosis in a cell, comprising: administering a therapeutically effective amount of the compound of any one of Statements 1 to 12 or 14 to 19 to a subject.
  • Statement 23 A method of Statement 22, wherein at least some of the administered compounds undergoes a formation within the subject, and wherein the reaction converts R 5 to -OH.
  • Statement 24 A method of Statement 23, wherein the transformation occurs within a cancer cell.
  • Statement 25 A method of a subject having cancer or suspected of having cancer, comprising: administering a therapeutically effective amount of the compound of any one of Statements 1 to 12 or 14 to 19 to a subject.
  • Described herein is the synthesis and characterization of quinolinol derivatives for the identification of analogs capable of targeting the MDM2-MDM4 heterodimer E3 ligase and inducing apoptosis in cells.
  • the structure-activity-relationship (SAR) study identified structural moieties critical for the inhibitory effects toward MDM2-MDM4 E3 ligase, targeted degradation of MDM4 and FTH1 in cells and anti-proliferation activity. Lead optimization led to the development of compound MMRi71 with improved activity.
  • MMRi71 effectively kills p53-null leukemia cells, an activity conventional MDM2-p53 disrupting inhibitors lack.
  • This disclosure provides a protype structure for developing MDM4/FTH1 dual targeting inhibitors as potential cancer therapeutics.
  • NALM6 and shP53NALM6 leukemic cell lines were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 50 U/ml penicillin and 50 pg/ml streptomycin.
  • shP53NALM6 cell line was established using pLKO.l-p53 (purchased from Sigma) (Plasmid #19119) followed by puromycin selection at 1 pg/ml for 2 days then clonal expansion in puromycin-free medium.
  • MANCA, MANCA-mlp-puro and MANCA-mlp- MDM2 were gifts from Prof.
  • Jill Bargonetti (Hunter College, CUNY, NY, USA) and were generated as described previously and maintained in 10% FBS-Pen/Strep- RPMI-1640 medium. Small-molecule compounds were synthesized, purified and characterized in house and were dissolved in DMSO as 10 mM stocks for cell proliferation assays.
  • Apoptotic response to compounds was measured by western blotting using specific antibodies from Cell Signaling Technology for activated caspase 3 (Cleaved Caspase-3 (Aspl75) (5A1E) Rabbit mAb, #9664) and PARP (PARP Antibody #9542).
  • MMRi67 derivatives were associated with their effects on dual degradation of MDM4 and FTH1 compared to MMRi67.
  • the SAR results from MMRi67 derivatives identified the hydroxyl and the chloro in the quinoline ring and the carboxylic acid to be the critical structural elements that contribute to MDM2-MDM4 E3 ligase inhibitory effects of MMRi67 in vitro.
  • the ethyl ester substitution of the carboxylic acid moiety in MMRi67 generated potent analogs that exhibited strong MDM2/MDM4 and FTH1 degradation in cells.
  • MMRi71 represents a new class of dual inhibitor targeting degradation of MDM2/MDM4 and FTH1 for p53-independent apoptosis in leukemia cells.
  • MMRi6 are quinolinols identified in an enzyme-based HTS for inhibition toward the MDM2-MDM4 RING-domain mediated E3 ligase reaction.
  • MMRi62 binds to preformed MDM2-MDM4 RING heterodimers for preferential MDM4 ubiquitination and acts as a MDM4-degrader in cells with potent apoptosis inducing activity; while MMRi67 was the most potent MDM2- MDM4 E3 ligase inhibitor but lacked MDM4 degradation and pro-apoptotic activity in cells.
  • MMRi67 This analog showed weaker cell growth inhibitory effect in NALM6 and shP53NALM6 cells than MMRi62 but was more effective than MMRi67 (Tables 1, 2). However, 62-1 lost p53- independent antiproliferative activity of MMRi62. To further test the importance of quinolinol chlorine, an MMRi67 analog that lacked this functionality, 67-1 was synthesized. This compound was found to have similar weak leukemic cell growth inhibition as MMRi67. Based the structural similarity of the compounds to MMRi62, the compounds are expected to inhibit cancer cell growth to a much larger extent than non-transformed cell growth. MMRi62 is 125 times more cytotoxic to NALM6 leukemia cells than it is to normal peripheral blood mononuclear cells.
  • Methylated phenol 62-3 was 70-fold and 172-fold less active than MMRi62 in NALM6 and shP53NALM6 cells, respectively.
  • Methylated amine 62-4, cyclic aminal 62-5 and an esterase-labile ester analog, 62-6 also showed weaker antileukemic cell growth activity compared to the parent MMRi62.
  • the amide analog 62-7 lacking the amine proton showed better antiproliferative activity. The amide bond is not expected to be very metabolically labile, thus the intact 62-7 is expected to be responsible for antiproliferative activity.
  • MMRi71 is considered a dual prodrug of 67-8, as its aromatic ethyl ester is also succeptable to cellular esterases.
  • MMRi71 with IC 50 of 0.23 pM in NALM6 cells and 0.29 pM in shP53NALM6 cells showed improved p53-independent antiproliferative activity in leukemia cells and was selected as a potential candidate for further investigation.
  • MMRi67 is a small-molecule with demonstrated inhibitory activity against MDM2-MDM4 E3 ligase. Yet, MMRi67 does not promote MDM4 degradation, nor does it induce apoptosis in leukemic cells as MMRi62. . A series of MMRi67 derivatives were synthezized to identify derivatives with improved pro-apoptotic activity ( Figure 1).
  • MMRi67 is a bona fide inhibitor ofMDM2-MDM4 E3 ligase
  • MMRi67 predicts metal chelation potential. Because Zn is required for maintaining the RING domain structure, it is possible that MMRi67 chelates away Zn and collapse the RING structure leading to dissociation of RING-RING interaction between MDM2-MDM4 and loss of the E3 ligase activity. An excessive amount of Zn has the potential to neutralize the chelating activity of MMRi67 and cancel its inhibition of MDM2-MDM4 E3 ligase activity. An E3 ligase assay was performed in the presence of Zn and these results indicated the presence of Zn did not affect the effect of MMRi67 to inhibit MDM2-MDM4 E3 ligase activity.
  • MMRi62 This covalent binding potential of MMRi62 is similar to that of a macrophage migration inhibitory factor (MIF) inhibitor which shares structural similarities with MMRi62. However, the covalent binding is not involved in the inhibitory mechanism of MMRi67 toward MDM2-MDM4 E3 ligase in vitro. Taken together, the results suggest that MMRi67 bears specific inhibitory activity toward MDM2-MDM4 E3 ligase.
  • MIF macrophage migration inhibitory factor
  • MDM2-MDM4 E3 ligase [0155] To identify the pharmacophore of MMRi67 involved in inhibition of MDM2- MDM4 E3 ligase, new derivatives were evaluated in MDM2-MDM4 E3 ligase assays. Analogs 67-1 with hydrogen substituted chloro group and 67-6 with hydrogen substituted for the hydroxyl group in the quinoline ring both lost the strong E3 ligase inhibitory activity of MMRi67. However, 67-1 showed slightly weak inhibition at 10 pM concentration leading to a conclusion that the hydroxyl and chloro groups in MMRi67 are key structural groups for strong MDM2/MDM4 E3 ligase inhibition ( Figure 3 A).
  • MMRi71 with pyrimidine substitution along with ethyl ester substitution at carboxylic acid and hydroxyl groups of MMRi67 lost activity to inhibit MDM4 ubiquitination whereas retained inhibitory effect on MDM2B ubiquitination in a dose-dependent manner ( Figure 3C).
  • the hydroxyl, chloro, carboxylic acid and pyridine arms of MMRi67 are all desirable structural components that contribute to strong MDM4-MDM2B E3 ligase inhibition.
  • MMRi67 analog, MMRi71 with ethyl ester substitutions at hydroxyl and carboxylic acid group and pyrimidine ring showed strong anti-leukemic activity in cell proliferation inhibitory assay along with dose-dependent inhibition of MDM2B E3 ligase activity.
  • Structural changes in MMRi71 are expected to increase water solubility and decrease molecular self-aggregation (via hydrogen-bonding or through metals involving the phenol) while maintaining the cell permeability potential of the candidate and its optimal effect on cellular targets.
  • MMRi71 has the same potency in inhibiting growth of NALM6 and shP53NALM6 cells with ICsos of 0.2 pM ( Figure 5A), indicating a p53-independent cytotoxicity.
  • phenol ester MMRi71 is substantially more cytotoxic than phenol ester 62-6 even though their parent phenols 67-9 and MMRi62 have similar cell growth ICso’s.
  • MMRi71 treated NALM6 and shP53NALM6 cells were used in western blot analysis for changes in MDM4 and FTH1 proteins.
  • MMRi71 treatment induced effective downregulation of MDM4 and FTH1 in both NALM6 and shP53NALM6 cells, and induced apoptosis shown by PARP cleavage indicating a p53-independent mechanism ( Figure 5B).
  • Figure 5B To test whether MMRi71 -induced apoptosis was not a result of colloidal aggregation, cellular experiments in the presence of Triton X-10 were performed.
  • MDM4 MDM2-high MANCA lymphoma cells in which MDM2 was either stably knocked down by lentivirus-mediated microRNA (miRNA) or left untouched with control miRNA.
  • the elevated MDM4 expression in MDM2- knockdown cells is consistent with the report that MDM2 promotes ubiquitination and degradation of MDM4.
  • MMRi71 induces MDM2-dependent proteasomal degradation of MDM4 and lysosome-dependent degradation of FTH1, the same mechanisms of action used by MMRi62, in addition to its potential inhibitory activity toward MDM2- MDM4 E3 ligase activity.
  • the improved activity of MMRi71 over MMRi67 may be due to the increased covalent binding capability or improved permeability or both, which requires further confirmation using proper assays.
  • the neutral ester in MMRi71 is expected to imbue better plasma membrane permeability compared to the charged carboxylic acid/carb oxy late in MMRi67.
  • MMRi62 which has structural similarities to MMRi71, did show some covalent binding effect to MDM2 ( Figure 2D). Based on studies with quinolinol analog MIF inhibitor, covalent binders in this molecular class can be specific inhibitors.
  • MMRi71 may be a covalent inhibitor targeting cellular MDM2-MDM4 for degradation.
  • MMRi71 induced apoptosis at >5 pM at 24 h in NALM6 cells ( Figure 5C) and 67-7 that did not generate ROS is an inactive compound, these results conclude that ROS generation is required for the antitumor activity of MMRi71.
  • ROS- induced DNA damage itself is not the major mechanism contributing to cell killing by MMRi62-like compounds, since they have high cancer selective toxicity.
  • MMRi62 inhibits leukemic NALM6 (B-cell precursor leukemia) cell growth at 125-fold potency compared to its inhibition of normal peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • MMRi62 This 125-fold difference in MMRi62 sensitivity is not likely the result of DNA damage since DNA damage by radiation kills normal human lymphocytes and B cell lymphoma cells at comparable capability with Do of 1.95Gy for lymphocytes and of 1.38Gy for Burkitt's lymphoma cell, where Do is a radiation dose required to reduce the fraction of surviving cells to 37% of its previous value. Accordingly, like MMRi62, the mechanisms of action for MMRi71 may, in various embodiments, involve multiple drug targets that predispose cancer cells to its selective toxicity.
  • MMRi62 we did find MMRi62 to be stable in C2D5OD at 100 °C for 24 h including with added CD3CO2D and deuterated pyridine, respectively ( 1 H NMR analysis, separate experiments), so if MMRi62 and related analogs are covalent inhibitors, it is likely they selectively bind their targets and become activated to the quinone methide within the target.
  • 4,5-Dichloropyridine-3-carbaldehyde was purchased from AABlocks and used without further purification.
  • Analogs 67-2 and 67-3 were obtained as part of a compound screening library from Hit21ead Chembridge.
  • Analog 62-11 was synthesized as previously reported.
  • Analogs in the 62 and 67 series were synthesized via a 3 -component Betti reactions.
  • Ethyl 4-formylbenzoate was synthesized as previously reported.
  • MMRi62 (285 mg, 0.72 mmol, 1.0 equiv.) was dissolved in dry DMF (1 mL) under argon atmosphere.
  • N-Phenyl-bis(trifluoromethanesulfonamide) (385 mg, 1.08 mmol, 1.5 equiv.) and potassium carbonate (150 mg, 1.09 mmol, 1.5 equiv.) were added, the tube was capped and the solution was heated in an 80 °C oil bath for 2 h. The solution was then cooled to rt, diluted with EtOAc, and washed three times with H2O. The organic layer was dried over Na2SO4, and then concentrated.
  • MMRi62 50 mg, 0.13 mmol, 1.0 equiv
  • anhydrous acetonitrile (1 mL) under argon atmosphere.
  • Potassium carbonate (20 mg, 0.14 mmol, 1.1 equiv)
  • methyl iodide 9 pL, 0.14 mmol, 1.1 equiv
  • the mixture was stirred at reflux temperature for 2 h.
  • the reaction mixture was diluted with ethyl acetate and washed with water. The organic layer was dried over sodium sulfate and then concentrated.
  • MMRi62 100 mg, 0.128 mmol, 1.0 equiv
  • 2,6-lutidine 75 pL, 0.630 mmol, 5.0 equiv
  • CH2CI2 10 mL
  • TBSOTf 0.15 mL, 0.630 mmol, 5.0 equiv
  • the reaction was slowly warmed to rt and allowed to stir for an additional 2 h.
  • the reaction mixture was quenched with aqueous NaHCO 3 and extracted 3 times with CH2CI2.
  • the organic layers were combined and dried over Na2SO4.
  • intermediate S-3 (84 mg, 0.160 mmol, 1 equiv) was dissolved in dry THF (1 mL) under argon atmosphere at 0 °C.
  • a 1 M solution of TBAF in THF (0.25 mL, 0.240 mmol, 1.5 equiv) was added dropwise at 0 °C and stirred for 1 h.
  • the solution was slowly warmed to rt and stirred for 3 h.
  • the reaction mixture was concentrated and dissolved in EtOAc.
  • the solution was washed with saturated NH4Q.
  • the organic layer was dried over Na2SO4 and concentrated.
  • MMRi62 100 mg, 0.25 mmol, 1.0 equiv
  • paraformaldehyde 8.5 mg, 0.28 mmol, 1.1 equiv
  • the solution was heated to 100 °C for 24 h.
  • the solution was diluted with Et2O, and concentrated.
  • the crude mixture was purified by flash column chromatography (silica gel, 20-30% acetone: hexanes gradient) to give 62-5 as a green-white solid (30 mg, 30%).
  • MMRi62 (3 g, 7.6 mmol, 1.0 equiv) was dissolved in
  • analog 62-6 (20 mg, 0.041 mmol, 1.0 equiv) was dissolved in dry Et2O (1 mL). Hydrochloric acid (2M in ether, 0.1 mL, 0.2 mmol, 4.4 equiv) was then added, and the reaction was stirred for 2 h at rt.
  • lithium hydroxide monohydrate (55 mg, 1.3 mmol, 2.7 equiv) was dissolved in 4 mL H2O and 2 mL MeOH.
  • Ester 67-5 (194 mg, 0.49 mmol, 1.0 equiv) was dissolved in 2 mL MeOH and added to the vial.
  • the vial was capped and stirred at rt for 16 h.
  • 2M HC1 was added dropwise to the solution until a precipitate persisted. Do not add too much HC1, as the product will dissolve.
  • 67-4 (440 mg, 1.0 mmol, 1.0 equiv.) was dissolved in dry DMF (5 mL) under argon atmosphere.
  • N -Phenyl-bis(trifluoromethanesulfonamide) (537 mg, 1.51 mmol, 1.5 equiv.) and potassium carbonate (208 mg, 1.50 mmol, 1.5 equiv.) were added and the solution was 80 °C for 2 h.
  • the solution was then cooled to rt, diluted with EtOAc, and washed three times with H2O.
  • the organic layer was dried over Na2SO4, and then concentrated.
  • S-5 67-7 In a 50 mL pressure tube, S-5 (400 mg, 0.75 mmol, 1.0 equiv.), Pd(0Ac)2 (10 mg, 0.045 mmol, 0.06 equiv.), triphenylphosphine (23 mg, 0.088 mmol, 0.12 equiv.), and tri ethyl amine (0.4 mL, 0.51 mmol, 3.8 equiv.) were combined in dry DMF (5 mL) under argon atmosphere. Formic acid (70 pL, 1.86 mmol, 2.5 equiv.) was added, and the reaction was capped and stirred at 60 °C for 16 h.
  • lithium hydroxide monohydrate (30 mg, 0.71 mmol, 3.5 equiv) was dissolved in 2 mL H2O and 1 mL MeOH.
  • Ester 67-7 (86 mg, 0.21 mmol, 1.0 equiv) was dissolved in 1 mL MeOH and added to the vial.
  • the vial was capped and stirred at rt for 16 h.
  • 2M HC1 was added dropwise to the solution until a precipitate persisted.
  • SC-62-1 ( ⁇ )-7-((2-chloro-3-cyanophenyl)(pyridin-2-ylamino)methyl)quinolin-8-yl dimethylcarbamate (SC-62-1 A)
  • SC-62-1 500 mg, 1.29 mmol, 1.0 equiv
  • the flask was equipped with a stir bar and 175 mL of dry CH2CI2 was added to the flask under argon to dissolve SC-62-1.
  • 2-aminopyridine (128 mg, 1.36 mmol, 1.0 equiv) and 1-methyl- l/Z-pyrazole-5-carbaldehyde (0.13 mL, 1.36 mmol, 1.0 equiv) was dissolved in 5 mL of absolute ethanol. Once fully dissolved, 8-hydroxyquinoline (237 mg, 1.63 mmol, 1.2 equiv) was added to the solution. The mixture was stirred at reflux for 72 h. The reaction was allowed to cool to rt and then concentrated.
  • MMRI62 100 mg, 0.25 mmol, 1.0 equiv
  • triethylamine 0.1 mL, 0.72 mmol, 2.9 equiv
  • Propionoyl chloride 0.1 mL, 1.15 mmol, 4.5 equiv
  • SC-62-1F 15 mg, 39 pmol, 1.0 equiv was dissolved in 5 mL of dry CH2CI2 under argon atmosphere.
  • Potassium carbonate 14 mg, 0.1 mmol, 2.6 equiv was added and the solution was cooled to 0 °C.
  • Propionoyl chloride (5.0 pL, 57 pmol, 1.5 equiv) was then added to the solution.
  • the mixture was allowed to warm to rt and stirred for 1 h.
  • the reaction mixture was then filtered through celite and washed with CH2CI2.
  • the supernatant was then treated with 5 mg DMT -functionalized silica gel and stirred for 15 min.
  • 2-amino-4-azidopyridine 40 mg, 0.30 mmol, 1.0 equiv.
  • 2,3- dichlorobenzaldehyde 52 mg, 0.30 mmol, 1.0 equiv.
  • 8-hydroxyquinoline 43 mg, 0.30 mmol, 1.0 equiv.
  • the tube was then capped and stirred at reflux for 72 h. The reaction was allowed to cool to rt and a precipitate had formed.
  • SC-62-10 (20 mg, 0.040 mmol, 1.0 equiv) was dissolved in 1 mL of dry CH3CN under argon atmosphere.
  • K2CO3 (7 mg, 0.044 mmol, 1.1 equiv), followed by CH3I (3 pL, 0.044 mmol, 1.1 equiv) were added to the solution.
  • the mixture was stirred at reflux for 2 h.
  • the reaction mixture was diluted with EtOAc and washed with water. The organic layer was dried over Na2SO4 and then concentrated.
  • the compounds promote ferroptosis, a cell death associated with reactive oxygen species caused by dysregulation of cellular iron.
  • a marker of ferroptosis is degradation of the cellular iron storage protein, ferratin heavy chain 1 (FTH1).
  • the parent phenolic compounds e.g. SC-62-1, causes total loss of FTH1 at concentrations >2.5 pM.
  • Phenolic ester pro-drug SC-62- 1C retained the ability to induce apoptotic cleavage of PARP at comparable capacity as SC-62-1 at 10 pM in MDM4-high A375 cells, which was accompanied by MDM4 degradation.
  • SC-62-1 has strong and acute effect: causing total loss of FTH1 at concentrations >2.5 pM, while the effect of SC-62-1C on FTH1 was attenuated and exhibited good linear dose-response.
  • IC50 NALM6 of phenol SC-62-1 is ca. 0.25 pM.
  • the ICso NALM6 of phenolic methyl ether (non-hydrolyzable) 62-3 is ca. 12 pM.
  • the IC50 NALM6 of (hydrolysable) propionate ester 62-6 is ca. 1 pM.

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Abstract

Provided are compounds with the structure: Also provided are compositions of the compound. The compounds have broad anti¬ proliferative activity against cancer cells, including leukemia, pancreatic cancer, and melanomas. Also provided are methods for inhibiting the growth of cells and/or inducing apoptosis and/or ferroptosis.

Description

ANTIPROLIFERATIVE BETTI BASES AND PRODRUGS THEREOF
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This applications claims the benefit of U.S. Provisional Application Number 63/479,789, filed on January 13, 2023, and also claims the benefit of U.S. Provisional Application Number 63/377,506, filed on September 28, 2022, the disclosure of which are incorporated herein by reference.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under grant nos. CA208352 and GM078383 awarded by the National Institutes of Health. The government has certain rights in the invention.
BACKGROUND OF THE DISCLOSURE
[0003] Tumor suppressor p53 (TP53) is a well-established drug target whose activation has been shown to induce tumor regression in several models. In normal and cancer cells, p53 activity is inhibited mainly by MDM2 (Murine Double Minute 2) and MDM4, RING domain-containing proteins. MDM4 (also known as MDMX) is the only MDM2 homologue which is expressed at levels higher than MDM2 due to its increased expression and protein stability in most cancer types. The N-terminus of both MDM2 and MDM4 binds to p53 transactivation domain and inhibits p53-dependent transcriptional transactivation of downstream genes, while the C-terminal RING domain of MDM2 can bind to E2 ubiquitin conjugating enzymes for p53 ubiquitination and to the RING domain of MDM4 to form heterodimers. Development of small molecule inhibitors of MDM2-p53 interaction has been a research focus for decades since the discovery of the first-in-class small molecule Nutlin3a and several Nultlin3-like MDM2 inhibitors are in clinical trials.
These inhibitors are intended to bind to the p53-binding pocket of MDM2 that prevent MDM2 from binding to p53 thus releasing p53 from MDM2-mediated inhibition and elicit tumor suppression in a p53-dependent manner. Accordingly, p53 mutation confers intrinsic and acquired resistance to MDM2-p53 disruptor inhibitors. Further, this type of MDM2 inhibitors are challenged with resistance conferred by MDM4 overexpression in cancer cells, since MDM4 can bind and inhibit p53 transactivation domain in the absence of MDM2. [0004] MDM2 and MDM4 are cancer drug targets validated in multiple models for p53-based cancer therapies. The RING domains of MDM2 and non-p53-binder MDM2 splice isoforms form RING domain heterodimer polyubiquitin E3 ligases with MDM4, which regulate p53 stability in vivo and promote tumorigenesis independent of p53. Despite the importance of the MDM2 RING domain in p53 regulation and cancer development, small molecule inhibitors targeting the E3 ligase activity of MDM2-MDM4 are poorly explored.
SUMMARY OF THE DISCLOSURE
[0005] The present disclosure provides compositions and methods for inhibiting the growth of cells and/or inducing apoptosis and/or ferroptosis. For example, the compositions and methods inhibit growth and/or induce apoptosis of cancer cells and/or ferroptosis.
[0006] An aspect of the present disclosure provides a prodrug of a Betti-base compound. In one active series of Betti -base compounds, masking the phenol’s alcohol functional group may contribute to the Betti -base’s antiproliferative activity. Without intending to be bound by any particular theory, the increased antiproliferative activity is thought to be due to enhanced cell penetration.
[0007] Without intending to be bound by any particular theory, the phenol moiety of Betti-base compounds is thought to be a structural weakness with respect to bioavailability, contributing to low in vivo half-life and problematic formulation and/or acute toxicity, potentially related to compound aggregation and ambient metal chelation.
[0008] An advantage of the present disclosure is the slower and more selective release of a Betti-base compound. In an embodiment, prodrugs of the present disclosure mask the phenol and allow for its slower and more selective release, for example, in cancer cells.
[0009] In various embodiments, the composition of the present disclosure comprises prodrugs containing moi eties that can be converted to phenols in vivo. In embodiments, the present disclosure comprises prodrugs containing moieties that can be converted to phenols selectively in cells, such as, for example, cancer cells.
[0010] In various embodiments, the prodrug of the present disclosure could be applied to any drug candidate where the active form is a Betti-type base.
BRIEF DESCRIPTION OF THE FIGURES
[0011] For a fuller understanding of the nature and objects of the disclosure, reference should be made to the following detailed description taken in conjunction with the accompanying figures.
[0012] Figure 1. Structures of (A) MMRi62 and (B) MMRi67 compound series.
[0013] Figure 2. MMRi67 is a small molecule inhibitor of MDM2-MDM4 E3 ligase activity. (A) MMRi67 inhibits ubiquitination of MDM2B (left) and MDM4 (middle) by MDM2B-MDM4 E3 complex but not NEDD4-1 (right) in in vitro E3 ligase assays. WB analysis of MDM4, MDM2B and NEDD4-1 using specific antibodies after in vitro ubiquitination reaction in the presence of solvent or indicated concentrations of MMRi67. The smearing bands of ubiquitinated MDM4 (Ub-MDM4), ubiquitinated MDM2B (Ub- MDM2B) and ubiquitinated NEDD4-1 (Ub-NEDD4-1) are indicated. (B) The E3 ligase inhibitory activity of MMRi67 is not abolished by the presence of 0.01% Triton X-100. Same procedure as in A except the ubiquitination reaction was performed in the presence of 0.01% Triton X-100. Only MDM2B data were shown. (C) The E3 ligase inhibitory activity of MMRi67 is not abolished by the presence of increasing concentrations of Zn to neutralize potential chelating capability of MMRi67. The same procedure as in A except for the presence of indicated concentrations of Zn. Only MDM2B data were shown. (D) Preincubation of MDM2B proteins with MMRi67 does not abolish MDM2B E3 ligase activity. Same procedure as in A except MDM2B was preincubated with either 10 pM MMRi62 or MMRi67 for the indicated min before 10-times dilution and used for ubiquitination reaction and the preincubation did not affect ubiquitination of MDM2B or MDM4 by MDM2B-MDM4 E3 complex.
[0014] Figure 3. Assessment of the inhibitory effect of MMRi67 derivatives toward MDM2B-MDM4 E3 ligase in in vitro E3 ligase assays. (A) Comparison of MMRi67 with hydroxyl to hydrogen substituted (67-6) and chloro to hydrogen substituted (67-1) analogs in inhibition of MDM4 and MDM2B ubiquitination in vitro. WB analysis of MDM4 or MDM2B using specific antibodies after in vitro ubiquitination reaction in the presence of solvent or indicated compound at the indicated concentrations. The smearing bands of ubiquitinated MDM4 (Ub-MDM4) and MDM2B (Ub-MDM2B) are indicated. (B) Same analysis as in A except for comparison of MMRi67 with carboxylic acid substituted analogs 62-1 and 67-4 in inhibition of MDM4 and MDM2B ubiquitination in vitro. (C) Same analysis as in A except for comparison of MMRi67 with pyridine substituted analogs 67-8 and pyridine, hydroxyl and carboxylic acid substituted analogs 67-9 and MMRi71 in inhibition of MDM4 and MDM2B ubiquitination in vitro.
[0015] Figure 4. Anti-proliferation activity of 67-5 is associated with downregulation of MDM2, MDM4 and FTH1 protein expression and caspase 3 activation. (A) Improved antiproliferation activity of 67-4 and 67-5 compared with MMRi67. Antiproliferation assays in NALM6 and shp53NALM6 cells. The ICso in NAML6 cells was 12.8 pM for MMRi67 and 0.38 pM for 67-5. The ICso in shP53NAML6 cells was 10.9 pM for MMRi67 and 0.45 pM for 67-5. (B) Improved antiproliferation activity of 67-5 is associated with downregulation of MDM2, MDM4 and FTH1 and caspase activation and PARP cleavage. WB analysis of indicated proteins and apoptotic effect induced by 67-5 compared to MMRi67 in both NALM6 and shP53NALM6 cells.
[0016] Figure 5. Characterization of MMRi71 in cells. (A) The anti-proliferation activity of MMRi71 is p53-independent with ICso of 0.23 pM in NALM6 and 0.29 pM in shp53NALM6 cells. (B) WB analysis showing MMRi71 induces MDM4/MDM2 and FTH1 protein degradation and apoptotic effect in NALM6 and shP53NALM6 cells. (C) The pro- apoptotic effect of MMRi71 is not affected by the presence of 0.0025% Triton X-100 in NALM-6 cells as shown by WB analysis of apoptotic PARP cleavage. (D) MMRi71 -induced MDM4 degradation is MDM2 dependent. WB analysis showing Mdm2 knockdown in Manca-mlp-Mdm2 cells abolished MMRi71 -induced MDM4 degradation. (E) MMRi71- induced FTH1 degradation is lysosome dependent. WB showing rescue of MMRi71 -induced FTH1 degradation by 50 nM lysosome inhibitor Bafilomycin Al (Bfl) in NALM-6 cells. (F) (G) DNA damage induction by MMRi71 and 67-7. Mouse p53/Mdm2 double knockout MEFs (2KO) (F) or human 293T cells (G) were treated with indicated concentrations of MMRi71 and 67-7 for 24h at the indicated concentrations followed by WB analysis for gammaH2AX and Tubulin with a specific antibodies. Tubulin serves as protein loading control.
[0017] Figure 6. (A) 1 H NMR spectrum for MMRi62. (B) 13C NMR spectrum for MMRi62.
[0018] Figure 7. (A) 1 H NMR spectrum for MMRi67. (B) 13C NMR spectrum for MMRi67.
[0019] Figure 8. (A) XH NMR spectrum for 62-1. (B) 13C NMR spectrum for 62-1.
[0020] Figure 9. (A) XH NMR spectrum for S-l . (B) 13C NMR spectrum for S-l .
[0021] Figure 10. (A) 1 H NMR spectrum for 62-2. (B) 13C NMR spectrum for 62-2.
[0022] Figure 11. (A) XH NMR spectrum for 62-3. (B) 13C NMR spectrum for 62-3.
[0023] Figure 12. (A) 1 H NMR spectrum for S-2. (B) 13C NMR spectrum for S-2.
[0024] Figure 13. (A) XH NMR spectrum for S-3. (B) 13C NMR spectrum for S-3.
[0025] Figure 14. (A) 1 H NMR spectrum for 62-4. (B) 13C NMR spectrum for 62-4.
[0026] Figure 15. (A) XH NMR spectrum for 62-5. (B) 13C NMR spectrum for 62-5.
[0027] Figure 16. (A) 1 H NMR spectrum for 62-6. (B) 13C NMR spectrum for 62-6.
[0028] Figure 17. (A) 1 H NMR spectrum for 62-7. (B) 13C NMR spectrum for 62-7. [0029] Figure 18. (A) XH NMR spectrum for 62-8. (B) 13C NMR spectrum for 62-8.
[0030] Figure 19. (A) 1 H NMR spectrum for S-4. (B) 13C NMR spectrum for S-4.
[0031] Figure 20. (A) 1 H NMR spectrum for 62-9. (B) 13C NMR spectrum for 62-9.
[0032] Figure 21. (A) XH NMR spectrum for 62-10. (B) 13C NMR spectrum for 62-
10.
[0033] Figure 22. (A) 1 H NMR spectrum for 67-4. (B) 13C NMR spectrum for 67-4.
[0034] Figure 23. (A) XH NMR spectrum for 67-5. (B) 13C NMR spectrum for 67-5.
[0035] Figure 24. (A) XH NMR spectrum for 67-1. (B) 13C NMR spectrum for 67-1.
[0036] Figure 25. (A) ’H NMR spectrum for S-5. (B) 13C NMR spectrum for S-5. [0037] Figure 26. (A) 1 H NMR spectrum for 67-7. (B) 13C NMR spectrum for 67-7.
[0038] Figure 27. (A) 1 H NMR spectrum for 67-6. (B) 13C NMR spectrum for 67-6.
[0039] Figure 28. (A) 1 H NMR spectrum for 67-9. (B) 13C NMR spectrum for 67-9.
[0040] Figure 29. (A) XH NMR spectrum for 67-8. (B) 13C NMR spectrum for 67-8.
[0041] Figure 30. (A) XH NMR spectrum for MMRi71. (B) 13C NMR spectrum for
MMRi71.
[0042] Figure 31. (A) ’H NMR spectrum for SC-62-1. (B) 13C NMR spectrum for SC-62-1.
[0043] Figure 32. (A) ’H NMR spectrum for SC-62-1 A. (B) 13C NMR spectrum for SC-62-1A.
[0044] Figure 33. (A) 1 H NMR spectrum for SC-62- IB. (B) 13C NMR spectrum for SC-62-1B.
[0045] Figure 34. (A) ’H NMR spectrum for SC-62- 1C. (B) 13C NMR spectrum for SC-62-1C.
[0046] Figure 35. (A) ’H NMR spectrum for SC-62- ID. (B) 13C NMR spectrum for SC-62-1D.
[0047] Figure 36. (A) ’H NMR spectrum for SC-62- IE. (B) 13C NMR spectrum for SC-62-1E.
[0048] Figure 37. (A) ’H NMR spectrum for SC-62-1F. (B) 13C NMR spectrum for SC-62-1F.
[0049] Figure 38. (A) 1 H NMR spectrum for SC-62- 1G. (B) 13C NMR spectrum for SC-62-1G.
[0050] Figure 39. (A) 1 H NMR spectrum for SC-62-2. (B) 13C NMR spectrum for SC-62-2. [0051] Figure 40. (A) XH NMR spectrum for SC-62-3. (B) 13C NMR spectrum for SC-62-3.
[0052] Figure 41. (A) 1 H NMR spectrum for SC-62-4. (B) 13C NMR spectrum for SC-62-4.
[0053] Figure 42. (A) XH NMR spectrum for SC-62-5. (B) 13C NMR spectrum for SC-62-5.
[0054] Figure 43. (A) 1 H NMR spectrum for SC-62-6. (B) 13C NMR spectrum for SC-62-6.
[0055] Figure 44. (A) 1 H NMR spectrum for SC-62-7. (B) 13C NMR spectrum for SC-62-7.
[0056] Figure 45. (A) XH NMR spectrum for SC-62-8. (B) 13C NMR spectrum for SC-62-8.
[0057] Figure 46. (A) XH NMR spectrum for SC-62-10. (B) 13C NMR spectrum for SC-62-10.
[0058] Figure 47. (A) XH NMR spectrum for SC-62-11. (B) 13C NMR spectrum for SC-62-11.
[0059] Figure 48. (A) XH NMR spectrum for SC-62-12. (B) 13C NMR spectrum for SC-62-12.
[0060] Figure 49. Comparison of Western Blot analysis of indicated proteins and apoptotic effects induced by SC-62-1 and prodrug SC-62-1 C.
[0061] Figure 50. Growth Inhibition of three melanoma cell lines by SC-62-1.
DETAILED DESCRIPTION OF THE DISCLOSURE
[0062] Although claimed subject matter will be described in terms of certain embodiments, other embodiments, including embodiments that do not provide all of the benefits and features set forth herein, are also within the scope of this disclosure. Various structural, logical, and process step changes may be made without departing from the scope of the disclosure.
[0063] As used herein, unless otherwise indicated, “about”, “substantially”, or “the like”, when used in connection with a measurable variable (such as, for example, a parameter, an amount, a temporal duration, or the like) or a list of alternatives, is meant to encompass variations of and from the specified value including, but not limited to, those within experimental error (which can be determined by, e.g., a given data set, an art accepted standard, etc. and/or with, e.g., a given confidence interval (e.g. 90%, 95%, or more confidence interval from the mean), such as, for example, variations of +/- 10% or less, +/-5% or less, +/-1% or less, and +/-0.1% or less of and from the specified value), insofar such variations in a variable and/or variations in the alternatives are appropriate to perform in the instant disclosure. As used herein, the term “about” may mean that the amount or value in question is the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, compositions, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error, or the like, or other factors known to those of skill in the art such that equivalent results or effects are obtained. In general, an amount, size, composition, parameter, or other quantity or characteristic, or alternative is “about” or “the like,” whether or not expressly stated to be such. It is understood that where “about,” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.
[0064] Ranges of values are disclosed herein. The ranges set out a lower limit value and an upper limit value. Unless otherwise stated, the ranges include the lower limit value, the upper limit value, and all values between the lower limit value and the upper limit value, including, but not limited to, all values to the magnitude of the smallest value (either the lower limit value or the upper limit value) of a range. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “0.1% to 5%” should be interpreted to include not only the explicitly recited values of 0.1% to 5%, but also, unless otherwise stated, include individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5% to 1.1%; 0.5% to 2.4%; 0.5% to 3.2%, and 0.5% to 4.4%, and other possible sub-ranges) within the indicated range. It is also understood (as presented above) that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about, it will be understood that the particular value forms a further disclosure. For example, if the value “about 10” is disclosed, then “10” is also disclosed. [0065] As used herein, unless otherwise stated, the term “group” refers to a chemical entity that is monovalent (i.e., has one terminus that can be covalently bonded to other chemical species), divalent, or polyvalent (i.e., has two or more termini that can be covalently bonded to other chemical species). The term “group” also includes radicals (e.g., monovalent and multivalent, such as, for example, divalent radicals, trivalent radicals, and the like).
Illustrative examples of groups include:
Figure imgf000009_0001
[0066] As used herein, unless otherwise indicated, the term “alkyl” or “alkyl group” refers to branched or unbranched, linear saturated hydrocarbon groups and/or cyclic hydrocarbon groups. Examples of alkyl groups include, but are not limited to, methyl groups, ethyl groups, propyl groups, butyl groups, isopropyl groups, tert-butyl groups, cyclopropyl groups, cyclopentyl groups, cyclohexyl groups, and the like. Alkyl groups are saturated groups, unless it is a cyclic group. For example, an alkyl group is a Ci to C20 alkyl group, including all integer numbers of carbons and ranges of numbers of carbons therebetween (e.g., Ci, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, Ci4, C15, Ci6, C17, Cis, C19, or C20). The alkyl group may be unsubstituted or substituted with one or more substituents. Examples of substituents include, but are not limited to, halogens (-F, -Cl, -Br, and -I), aliphatic groups (e.g., alkyl groups, alkenyl groups, alkynyl groups, and the like), halogenated aliphatic groups (e.g., trifluoromethyl group), aryl groups, halogenated aryl groups, alkoxide groups, amine groups, nitro groups, carboxylate groups, carboxylic acids, ether groups, alcohol groups, alkyne groups (e.g., acetylenyl groups and the like), and the like, and combinations thereof.
[0067] As used herein, the term “cycloalkyl” or “cycloalkyl group” refers to a cyclic hydrocarbon group, e.g., cyclopropyl, cyclobutyl, cyclohexyl, and cyclopentyl groups. Cycloalkyl groups can be saturated or partially unsaturated ring systems optionally substituted with, for example, one to three substituents. Each substituent is independently chosen from alkyl, -NH2, oxo (=0), phenyl, haloalkyl (e.g., -CF3), halo (e.g., -F, -Cl, -Br, -I), alkoxy, and -OH groups. Additionally, alkyl substituents may be substituted with various other functional groups. Additional non-limiting examples include aliphatic groups (e.g., alkyl groups, alkenyl groups, alkynyl groups, and the like), halogenated aliphatic groups (e.g., trifluoromethyl group), aryl groups, halogenated aryl groups, alkoxide groups, nitro groups, carboxylate groups, carboxylic acids, ether groups, alkyne groups (e.g., acetylenyl groups and the like), and the like, and combinations thereof.
[0068] As used herein, unless otherwise indicated, the term “aryl” or “aryl group” refers to C5 to Ci6 aromatic or partially aromatic carbocyclic groups, including all integer numbers of carbons and ranges of numbers of carbons therebetween (e.g., C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, or Cie). An aryl group may also be referred to as an aromatic group. The aryl groups may comprise polyaryl groups such as, for example, fused rings, biaryl groups, or a combination thereof. The aryl group may be unsubstituted or substituted with one or more substituents. Examples of substituents include, but are not limited to, halogens (-F, -Cl, -Br, and -I), aliphatic groups (e.g., alkyl groups, alkenyl groups, alkynyl groups, and the like), aryl groups, alkoxides, carboxylates, carboxylic acids, ether groups, and the like, and combinations thereof. Examples of aryl groups include, but are not limited to, phenyl groups, biaryl groups (e.g., biphenyl groups and the like), fused ring groups (e.g., naphthyl groups and the like), hydroxybenzyl groups, tolyl groups, xylyl groups, and the like. [0069] As used herein, the term “heteroaryl” or “hereteroaryl” refers to a monocyclic or bicyclic ring system comprising one or two aromatic rings and containing at least one nitrogen or oxygen atom in an aromatic ring. Unless otherwise indicated, a heteroaryl group can be unsubstituted or substituted with one or more, and in particular one or two, substituents. Non-limiting examples of substituents include halogens (-F, -Cl, -Br, and -I), aliphatic groups (e.g., alkyl groups, alkenyl groups, alkynyl groups, and the like), halogenated aliphatic groups (e.g., trifluoromethyl group), aryl groups, halogenated aryl groups, alkoxide groups, amine groups, nitro groups, carboxylate groups, carboxylic acids, ether groups, alcohol groups, alkyne groups (e.g., acetylenyl groups and the like), and the like, and combinations thereof. Examples of heteroaryl groups include, benzofuranyl, thienyl, furyl, pyridyl, oxazolyl, quinolyl, thiophenyl, isoquinolyl, indolyl, triazinyl, triazolyl, isothiazolyl, isoxazolyl, imidazolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, and thiadiazolyl groups, and substituents analogs of any of the foregoing heteroaryl groups.
[0070] As used herein, unless otherwise indicated, the term “alkoxy” or “alkoxy group” refers to
Figure imgf000010_0001
where Ra is a linear, branched or cyclic Ci-Ce alkyl group, including all integer numbers of carbons and ranges of numbers of carbons therebetween. For example, suitable alkoxy groups include methoxy, ethoxy, propoxy, /.w-propoxy, butoxy, sec-butoxy, tert-butoxy, and hexoxy groups. Additionally, alkyl substituents can be substituted with various other functional groups, e.g. functional groups disclosed herein. [0071] As used herein, unless otherwise indicated, the term “amino” or “amino Rb
Figure imgf000011_0001
group” refers to Rb where each Rb is selected independently from the group consisting of hydrogen atom, substituted or unsubstituted C1-C10 alkyl, including all integer numbers of carbons and ranges of numbers of carbons therebetween, substituted or unsubstituted phenyl, substituted or unsubstituted heteroaryl, substituted carbonyl, substituted sulfonyl, haloalkyl, and substituted or unsubstituted benzyl groups.
[0072] As used herein, unless otherwise indicated, the term “benzyl” or “benzyl group” refers to
Figure imgf000011_0003
where Rc is a substituent on the phenyl ring and n is from
0 to 5. The substituents can be the same or different. For example, the substituents on the benzyl group include substituted or unsubstituted alkyl, -NHz, phenyl, haloalkyl (e.g., -CF3), halo (e.g., -F, -Cl, -Br, -I), alkoxy (e.g., -OMe), and -OH groups.
[0073] As used herein, unless otherwise indicated, halogen means fluorine, chlorine, bromine, and iodine, and halo means fluoro, chloro, bromo, and iodo.
[0074] As used herein, unless otherwise indicated, the term “phenoxy” or “phenoxy group” (-OPh) refers to
Figure imgf000011_0002
where each Y is independently selected from the group consisting of F, Cl, Br, and I and m can be 0, 1 or 2.
[0075] As used herein, unless otherwise indicated, the term “phenyl” or “phenyl group” means
Figure imgf000011_0004
where each Rd is an independent substituent on the phenyl group and n is from 0 to 5. The substituents at different occurrences can be the same or different.
For example, the substituents on the phenyl group include substituted or unsubstituted Ci-Ce alkyl, including all integer numbers of carbons and ranges of numbers of carbons therebetween, substituted or unsubstituted amino, haloalkyl (e.g., -CF3), halo (e.g., -F, -Cl, - Br, -I), substituted or unsubstituted alkoxy (e.g., -OMe), and sulfonyl group. In certain instances, two adjacent R groups can be connected through to form a dioxolyl group.
[0076] The present disclosure provides a family of small organic molecules, termed “Betti bases,” which comprise phenolic (or hydroxyaryl) benzylic amine derivatives, have demonstrated broad anti-proliferative activity against cancer cells, including leukemia, pancreatic cancer, and melanomas. As used throughout, “Betti base” refers to 2- (aminomethyl)phenol derivatives. The compounds are thought to impact one or more cellular proteins that play significant roles in cancer progression, including MDM2, MDMX, p53, E3 ligase and FTH1. Structure-activity-relationship studies of these compounds indicate the phenol moiety contributes to the anti-proliferative activity. Various active compounds in this family have been developed and substituted with functional groups that optimize antiproliferative activity in cells.
[0077] In an aspect, the present disclosure provides a compound with the structure:
Figure imgf000012_0001
In various embodiments, R5 and R11 are connected such that a heterocyclic group is formed
Figure imgf000012_0002
[0078] The ring comprising X1, X2, X3, and X4 may comprise nitrogen or -CH- at the various X positions on the ring. In embodiments, X1 is -CH- or nitrogen, X2 is -CH- or nitrogen, X3 is -CH- or nitrogen, and X4 is -CH- or nitrogen. In some embodiments, the ring comprising X1, X2, X3, and X4 may have a structure wherein X1 is nitrogen, X2 is -CH-, X3 is
-CH-, and X4 is nitrogen, i.e.,
Figure imgf000012_0003
In embodiments, the ring may have a structure wherein X1 is -CH-, X2 is -CH-, X3 is -CH-, and X4 is nitrogen, i.e.,
Figure imgf000012_0004
[0079] In various examples, when R5 and R11 are connected such that a heterocyclic group is formed, the compound may have the following structure:
Figure imgf000013_0001
[0080] In various embodiments, R3 may be a hydrogen, a substituted or unsubstituted alkyl group, a halogen group, a substituted or unsubstituted amino group, or a substituted or unsubstituted ether group. Examples of R3 groups include, but are not limited to halogens
Figure imgf000013_0002
[0081] In various embodiments, R4 may be a hydrogen, a substituted or unsubstituted alkyl group, a halogen group, a substituted or unsubstituted amino group, or a substituted or unsubstituted ether group. A may be oxygen, sulfur, or a substituted or unsubstituted nitrogen.
Y°'Ri
[0082] In various embodiments, R5 may be ' , where R1 may be a hydrogen, an acyl group, an alkyl carbonate group, an acetal group, carbonate group, carbamate group, a ketal group, or an aminal group.
[0083] In various embodiments, R6 may
Figure imgf000013_0003
Figure imgf000013_0004
where R2 may be a hydrogen, a cyano group, a substituted or unsubstituted ester group, a carboxylic acid group, a sulfonyl group (SO2R), or a substituted or unsubstituted amide group. R7 may be a hydrogen, a halogen group, a substituted or unsubstituted alkyl group, a nitro group, or an azide group; and A may be oxygen, sulfur, or a substituted or unsubstituted nitrogen and Z may be nitrogen, CH, or CR (where R is alkyl or aryl). [0084] In some embodiments, the structure is, for example,
Figure imgf000014_0001
where A is O, S, NR (R = alkyl, H, or aryl); and Z is N, CH, or CR (R = alkyl or aryl). [0085] In various embodiments, R11 is H or an acyl group. In various embodiments,
R12 is H or a substituted alkyl group or unsubstituted alkyl group.
[0086] In various embodiments, R5 is not -OH.
[0087] In various embodiments, R5 may be -OH. When R5 is -OH, R6 may be chosen
Figure imgf000014_0002
[0088] In embodiments, R3 is chlorine,
Figure imgf000015_0001
Figure imgf000015_0002
[0089] In some embodiments, R3 is hydrogen, i.e.,
Figure imgf000015_0003
[0090] In various embodiments, R5 is chosen from
Figure imgf000015_0004
Figure imgf000015_0005
where M+ is a cation (e.g., Li+, Na+, K+, Ca2+). In embodiments R5 is
Figure imgf000016_0001
such that the structure is, for example,
Figure imgf000016_0002
[0091] In various embodiments, R6 is chosen from
Figure imgf000016_0003
Figure imgf000016_0004
[0092] In some embodiments, the structure
Figure imgf000016_0005
one or more of R3, R7, and R2 is not hydrogen, chlorine, bromine, or iodine. For example, the
Figure imgf000016_0006
(67-5). Other compounds are described in Wang et al., United States Patent Number
10,624,881, which is incorporated by reference herein in its entirety.
[0093] In various embodiments, the compound does not have the following structure::
Figure imgf000017_0001
[0094] In various embodiments, R5 is transformed to -OH in a body of an individual. In some embodiments, R5 is a group that can be transformed to -OH under in vivo conditions (e.g., in a cell or in a biological medium). Without intending to be bound by any particular theory, it is considered that a compound where R5 is not -OH may have an increased bioavailability relative to the compound where R5 is -OH. [0095] In some embodiments, the compound of the present disclosure may have any of the following structures:
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000020_0001
Figure imgf000021_0001
Figure imgf000022_0001
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
[0096] Additional examples of compounds of the present disclosure where R5 is -OH include, but are not limited to:
Figure imgf000039_0002
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
, p p
Figure imgf000042_0002
Figure imgf000043_0001
, where R2, R3, and are R7 are as described herein.
[0098] In an embodiment, the compound of the present disclosure is
Figure imgf000043_0002
[0099] Compounds of the disclosure may exist as salts. Pharmaceutically acceptable salts of the compounds of the disclosure generally are preferred in the methods of the disclosure. As used herein, the term "pharmaceutically acceptable salts" refers to salts or zwitterionic forms of a compound of the present disclosure. Salts of compounds of the present disclosure can be prepared during the final isolation and purification of the compounds or separately by reacting the compound with an acid having a suitable cation. The pharmaceutically acceptable salts of a compound of the present disclosure are acid addition salts formed with pharmaceutically acceptable acids. Examples of acids which can be employed to form pharmaceutically acceptable salts include inorganic acids such as nitric, boric, hydrochloric, hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic, maleic, succinic, and citric. Nonlimiting examples of salts of compounds of the disclosure include, the hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, 2- hydroxyethansulfonate, phosphate, hydrogen phosphate, acetate, adipate, alginate, aspartate, benzoate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerolphsphate, hemisulfate, heptanoate, hexanoate, formate, succinate, fumarate, maleate, ascorbate, isethionate, salicylate, methanesulfonate, mesitylenesulfonate, naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3- phenylproprionate, picrate, pivalate, propionate, tri chloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, paratoluenesulfonate, undecanoate, lactate, citrate, tartrate, gluconate, methanesulfonate, ethanedi sulfonate, benzene sulphonate, and p-toluenesulfonate salts. In addition, available amino groups present in the compounds of the disclosure can be quatemized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and steryl chlorides, bromides, and iodides; and benzyl and phenethyl bromides. In light of the foregoing, any reference to compounds of the present disclosure appearing herein is intended to include a compound of the present disclosure as well as pharmaceutically acceptable salts or hydrates, thereof.
[0100] Compounds of the present disclosure may exist in particular geometric or stereoisomeric forms. The present disclosure contemplates all such compounds, including cis- and trans-i somers, R- and S-enantiomers, diastereomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the disclosure. Additional asymmetric carbons may be present in a substituent, such as, for example an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this disclosure. Thus, in various embodiments, the present disclosure provides for all enantiomers, diastereomers, and isomers of the compounds, either in their enantioenriched or pure forms, or as isomeric mixtures, of the present disclosure.
[0101] In another aspect of the present disclosure, a composition is provided. The composition may comprise one or more compounds of the present disclosure. In various embodiments, the composition further comprises a pharmaceutically acceptable carrier. [0102] The compositions may include one or more pharmaceutically acceptable carrier(s). Non-limiting examples of compositions include solutions, suspensions, emulsions, solid injectable compositions that are dissolved or suspended in a solvent before use, and the like. Injections may be prepared by dissolving, suspending, or emulsifying one or more of the active ingredient(s) in a diluent. Non-limiting examples of diluents include distilled water (e.g., for injection), physiological saline, vegetable oil, alcohol, and the like, and combinations thereof. Injections may contain, for example, stabilizers, solubilizers, suspending agents, emulsifiers, soothing agents, buffers, preservatives, and the like, and combinations thereof. Injections may be sterilized in the final formulation step or prepared by sterile procedure. A pharmaceutical composition of the disclosure may also be formulated into a sterile solid preparation, for example, by freeze-drying, and may be used after sterilized or dissolved in sterile injectable water or other sterile diluent(s) immediately before use. Additional examples of pharmaceutically acceptable carriers include, but are not limited to, sugars, such as, for example, lactose, glucose, and sucrose; starches, such as, for example, corn starch and potato starch; cellulose, such as, for example, sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as, for example, cocoa butter and suppository waxes; oils, such as, for example, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil, and soybean oil; glycols, such as, for example, propylene glycol; polyols, such as, for example, glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as, for example, ethyl oleate and ethyl laurate; agar; buffering agents, such as, for example, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; other non-toxic compatible substances employed in pharmaceutical formulations, and the like, and combinations thereof. Non-limiting examples of pharmaceutically acceptable carriers are found in: Remington: The Science and Practice of Pharmacy (2012) 22nd Edition, Philadelphia, PA. Lippincott Williams & Wilkins.
[0103] Compositions of the disclosure can comprise more than one pharmaceutical agent. For example, a first composition comprising a compound of the disclosure and a first pharmaceutical agent can be separately prepared from a composition which comprises the same compound of the disclosure and a second pharmaceutical agent, and such preparations can be mixed to provide a two-pronged (or more) approach to achieving the desired prophylaxis or therapy in an individual. Further, compositions of the disclosure can be prepared using mixed preparations of any of the compounds disclosed herein.
[0104] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
[0105] Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
[0106] Compositions of the disclosure suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present disclosure as an active ingredient. A compound of the present disclosure may also be administered as a bolus, electuary or paste.
[0107] In solid dosage forms of the disclosure for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
[0108] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent. [0109] The tablets, and other solid dosage forms of the pharmaceutical compositions of the present disclosure, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro- encapsulated form, if appropriate, with one or more of the above-described excipients. [0110] Liquid dosage forms for oral administration of a compound of the present disclosure include pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
[OHl] In addition to inert diluents, the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
[0112] Suspensions, in addition to a compound of the disclosure, the composition may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
[0113] The composition may be for administration to an individual in need of treatment.
[0114] The composition may comprise one or more additional compounds. The additional compounds may be BRAF inhibitors or MEK inhibitors. Examples of BRAF inhibitors include, but are not limited to, Vemurafenib (Zelboraf®), dabrafenib (TAFINLAR®), and encorafenib (BRAFTOVI®). Examples of MEK inhibitors include, but are not limited to, trametinib (MEKINIST® ), binimetinib (MEKTOVI®), selumetinib (Koselugo®), and cobimetinib (COTELLIC®).
[0115] In another aspect of the present disclosure, a method is provided for inducing apoptosis or ferroptosis in a cell, comprising administering a therapeutically effective amount of the compound or composition of the present disclosure. [0116] Without being intended to be bound by any particular theory, it is considered the compounds may promote ferroptosis, a cell death associated with reactive oxygen species caused by dysregulation of cellular iron. A marker of ferroptosis is degradation of the cellular iron storage protein, ferratin heavy chain 1 (FTH1).
[0117] In some embodiments the administered compound (e.g., the compound of the present disclosure or the composition comprising the compound of the present disclosure) undergoes a reaction within the subject (e.g., within a cell of the subject). The reaction may convert R5 of the compound to -OH. The compound and/or the converted compound may induce apoptosis of the cell. In some embodiments the reaction occurs within a cancer cell. In some embodiments the reaction occurs outside of a cancer cell, and the converted compound (e.g., the compound after undergoing the reaction) is subsequently in contact with the cancer cell.
[0118] The subject to be treated by the method of the disclosure may be human or non-human (e.g., mammal). Non-human animals include ungulates such as bovines. Additional on-limiting examples of non-human mammals include pigs, mice, rats, rabbits, cats, dogs, or other agricultural mammals, pet, or service animals, and the like.
[0119] The dose of the composition comprising a compound of the present disclosure and a pharmaceutical agent generally depends upon the needs of the individual to whom the composition of the disclosure is to be administered. These factors include, for example, the weight, age, sex, medical history, and nature and stage of the disease for which a therapeutic or prophylactic effect is desired. The compositions can be used in conjunction with any other conventional treatment modality designed to improve the disorder for which a desired therapeutic or prophylactic effect is intended, non-limiting examples of which include surgical interventions and radiation therapies. The compositions can be administered once, or over a series of administrations at various intervals determined using ordinary skill in the art, and given the benefit of the present disclosure.
[0120] The compounds of the present disclosure can be therapeutically administered as the neat chemical, but it is preferred to administer a compound of the present disclosure as a pharmaceutical composition or formulation. Thus, the present disclosure provides a pharmaceutical composition comprising a compound of the present disclosure together with a pharmaceutically acceptable diluent or carrier therefor. Also provided is a process of preparing a pharmaceutical composition comprising admixing a compound of the present disclosure with a pharmaceutically acceptable diluent or carrier therefor. The composition may further comprise one or more additional compounds (e.g., BRAF inhibitors and/or MEK inhibitors).
[0121] In another aspect of the present disclosure, a method of treating cancer is provided comprising administering the compound of the present disclosure or a composition comprising the compound of the present disclosure to cancerous cells of a subject.
[0122] In embodiments the administering of the compound may be any method whereby the compound of the present disclosure is caused to contact a cell of the subject. The administering may be, for example, oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, inhaled, or buccal administration, or combinations thereof. In embodiments the parenteral administration comprises intravenous, intraarterial, intracranial, intradermal, subcutaneous, intraperitoneal, intramuscular, intrathecal, or intraarticular administration.
[0123] In another aspect of the present disclosure, a kit for inducing apoptosis in a cell is provided, comprising a composition comprising the compound of claim 1.
[0124] The steps of the method described in the various embodiments and examples disclosed herein are sufficient to carry out the methods of the present invention. Thus, in an embodiment, the method consists essentially of a combination of the steps of the methods disclosed herein. In another embodiment, the method consists of such steps.
[0125] The following Statements provide various examples of the present disclosure. Statement 1. A compound having the following structure:
Figure imgf000049_0001
, or absent; R3 is a hydrogen, a substituted or unsubstituted alkyl group, a halogen group, a substituted or unsubstituted amino group, or a substituted or unsubstituted alkoxy group; R4 is a hydrogen, a substituted or unsubstituted alkyl group, a halogen group, a substituted or unsubstituted amino group, or a substituted or
Y°'Ri unsubstituted alkoxy group; R5 is x , wherein R1 is a hydrogen, an acyl group, an alkyl carbonate group, an acetal group, carbonate group, carbamate group, a ketal group, or an aminal group;
Figure imgf000050_0001
a cyano group, a substituted or unsubstituted ester group, a carboxylic acid group, a sulfonyl group (SO2R), or a substituted or unsubstituted amide group; wherein R7 is a hydrogen, a halogen group, a substituted or unsubstituted alkyl group, a nitro group, or an azide group; and wherein A is oxygen, sulfur, or a substituted or unsubstituted nitrogen and Z is N, CH, or CR, where R is an alkyl group or an aryl group; R11 is H or an acyl group; R12 is H or a substituted or unsubstituted alkyl group; X1 is -CH- or nitrogen; X2 is -CH- or nitrogen; X3 is -CH- or nitrogen; and X4 is -CH- or nitrogen.
Statement 2. A compound of Statmeent 1, wherein R5 is not -OH.
Statement 3. A compound of Statement 1, wherein R5 is -OH and wherein R6 is chosen from
Figure imgf000050_0002
Statement 4. A compound of any one of Statements 1 to 3, wherein
Figure imgf000050_0003
Statement 5. A compound of any one of Statements 1 to 4, wherein X1 is nitrogen, X2 is -CH-, X3 is -CH-, and X4 is nitrogen.
Statement 6. A compound of any one of Statements 1 to 4, wherein X1 is -CH-, X2 is -CH-, X3 is -CH-, and X4 is nitrogen.
Statement 7. A compound of any one of Statements 1 to 6, wherein R3 is chlorine.
Statement 8. A compound of any one of Statements 1 to 6, wherein R3 is hydrogen. Statement 9. A compound of any one of Statements 1 or 4 to 7, wherein R5 is chosen from
Figure imgf000051_0001
and , wherein M+ is a cation chosen from Li+, Na+, +, and
Statement 10. A compound of Statement 9, wherein R5 is
Figure imgf000051_0002
Statement 11. A compound of any one of Statements 1 to 10, wherein R6 is chosen from
Figure imgf000051_0003
Statement 12. A compound of Statement 1 or 4 to 11, wherein when the structure is
Figure imgf000051_0004
and at least of R3, R7, and R2 is not hydrogen, chlorine, bromine, or iodine. Statement 13. A compound of Statement 1, wherein the compound does not have the following structure:
Figure imgf000052_0001
Statement 14. A compound of any one of Statements 1 or 3 to 13, wherein R5 can be converted to -OH in a cell. Statement 15. A compound of any one of Statements 1, 3 to 12, or 14, wherein the structure is:
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
Figure imgf000069_0001
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
Statement 16. A compound of Statement 1, wherein the structure is chosen from
Figure imgf000077_0002
Statement 17. A compound of Statement 1, wherein the structure is chosen from
Figure imgf000078_0001
Statement 18. A compound of Statement 1, wherein the structure is
Figure imgf000078_0002
Statement 19. A compound of Statement 1, wherein the structure is
Figure imgf000078_0003
Statement 20. A composition comprising a compound of any one of Statements 1 to 12 or 14 to 19.
Statement 21. A composition of Statement 20, further comprising a pharmaceutically acceptable carrier.
Statement 22. A method of inducing apoptosis or ferropotosis in a cell, comprising: administering a therapeutically effective amount of the compound of any one of Statements 1 to 12 or 14 to 19 to a subject. Statement 23. A method of Statement 22, wherein at least some of the administered compounds undergoes a formation within the subject, and wherein the reaction converts R5 to -OH.
Statement 24. A method of Statement 23, wherein the transformation occurs within a cancer cell.
Statement 25. A method of a subject having cancer or suspected of having cancer, comprising: administering a therapeutically effective amount of the compound of any one of Statements 1 to 12 or 14 to 19 to a subject.
[0126] The following examples are presented to illustrate the present disclosure. They are not intended to be limiting in any matter.
EXAMPLE 1
[0127] Described herein is the synthesis and characterization of quinolinol derivatives for the identification of analogs capable of targeting the MDM2-MDM4 heterodimer E3 ligase and inducing apoptosis in cells. The structure-activity-relationship (SAR) study identified structural moieties critical for the inhibitory effects toward MDM2-MDM4 E3 ligase, targeted degradation of MDM4 and FTH1 in cells and anti-proliferation activity. Lead optimization led to the development of compound MMRi71 with improved activity. In addition to accumulating p53 proteins in wt-p53 bearing cancer cells as expected of any MDM2 inhibitors, MMRi71 effectively kills p53-null leukemia cells, an activity conventional MDM2-p53 disrupting inhibitors lack. This disclosure provides a protype structure for developing MDM4/FTH1 dual targeting inhibitors as potential cancer therapeutics.
[0128] Described is the structural activity relationship studies (SAR) leading to the identification of MMRi71 as an inhibitor of the E3 ligase activity of MDM2-MDM4, inducer of MDM4 and FTH1 dual protein degradation and p53-independent apoptosis in leukemia cells. The anti-cancer potency of MMRi67, the first lead in the development of structurally optimized prodrug MMRi71, was not readily apparent until its carboxylic acid was introduced as an ethyl ester. Conversely, inhibition of E3 ligase activity is only observed when MMRi67 is in its carboxylic acid form.
[0129] Ethyl 4-((5-chloro-8-hydroxyquinolin-7-yl)(pyrimidin ylamino)methyl)benzoate (67-9) Synthesis [0130] To a 250 mL dry round-bottomed flask equipped with a reflux condenser, 2- aminopyrimidine (4.3 g, 45.3 mmol, 1.2 equiv), ethyl 4-formylbenzoate (6.8 g, 38.2 mmol, 1.0 equiv), and 5 -chi oro-8-hydroxy quinoline (8.2 g, 45.8 mmol, 1.2 equiv) were dissolved in CH3CN (100 mL). Following the addition of formic acid (1.4 mL, 37.1 mmol 1.0 equiv), the solution was stirred at reflux for 16 h. The solution was allowed to cool to room temperature (“it”), concentrated, resuspended in acetone. The heterogenous mixture was filtered, and the precipitate was washed with cold acetone and hexanes to give 67-9 as a white solid (5.2 g, 31% yield), mp = 150-151 C; 3H NMR (300 MHz, CDCh) 5 8.80 (d, = 4.2 Hz, 1H), 8.49 (d, J= 8.6 Hz, 1H), 8.30 (d, J= 4.8 Hz, 2H), 7.99 (d, J= 7.9 Hz, 2H), 7.55 (dd, J= 19.6, 10.2 Hz, 4H), 6.79 (d, J= 8.2 Hz, 1H), 6.58 (t, J= 4.9 Hz, 1H), 6.39 (d, J= 8.3 Hz, 1H), 4.34 (q, J = 7.2 Hz, 2H), 1.36 (t, = 7.2 Hz, 3H); 13C NMR (75 MHz, CDCh) 5 166.3, 161.5, 158.2,
148.7, 148.5, 146.6, 138.7, 133.3, 129.8, 129.4, 127.0, 126.8, 125.6, 123.6, 122.5, 120.7, 111.5, 60.9, 54.8, 14.3; IR neat film: 3293, 2978, 1716, 1583, 1496 cm'1; HRMS (ESI) calculated for [C23H20CIN4O3] 390.1004, found 390.1011.
[0131] Ethyl 4-((8-(propionyloxy)quinolin-7-yl)(pyrimidin-2- ylamino)methyl)benzoate (MMRi71) Synthesis
[0132] In a 5 mL dry round-bottomed flask, analog 67-9 (5.0 g, 11.5 mmol, 1.0 equiv) was dissolved in dry CH2CI2 (100 mL) under argon atmosphere. Potassium carbonate (3.18 g, 23.0 mmol, 2.0 equiv) was added and the solution was cooled to 0 °C. Propionoyl chloride (1.0 mL, 11.5 mmol, 1.0 equiv) was then added to the solution. The mixture was allowed to warm to rt and stirred for 1 h. The reaction mixture was then filtered through Celite and washed with CH2CI2. The supernatant was then treated with 1 g DMT-functionalized silica gel and stirred for 15 min. The mixture was filtered and concentrated. The resulting crude solid was resuspended in Et2O and washed with deionized water. The organic layer was dried over Na2SO4 and then concentrated. The crude mixture was purified by flash column chromatography (silica gel, 50% ether: hexanes) to yield MMRi71 as a greenish white solid (2.53 g, 48% yield), mp = 178-179 C; ’H NMR (400 MHz, CDCh) 5 8.91 (d, J= 2.8 Hz, 1H), 8.50 (d, J= 6.8 Hz, 1H), 8.23 (d, J= 4.8 Hz, 2H), 8.00 (d, J= 8.4 Hz, 2H), 7.54 (s, 1H), 7.50 (dd, J= 8.5, 4.2 Hz, 1H), 7.43 (d, J= 8.1 Hz, 2H), 6.83 (d, J= 7.8 Hz, 1H), 6.57 (t, J= 4.8 Hz, 1H), 6.10 (d, J= 7.8 Hz, 1H), 4.36 (q, J= 7.1 Hz, 2H), 2.68 (q, J= 7.6 Hz, 2H), 1.37 (t, J= 1A Hz, 3H), 1.20 (t, J = 7.5 Hz, 3H); 13C NMR (101 MHz, CDCh) 5 172.2, 166.2, 161.3, 158.1, 151.2, 145.5, 144.6, 142.0, 134.3, 133.0, 130.0, 129.8, 129.0, 127.1, 126.7,
125.7, 122.4, 111.8, 61.0, 53.8, 27.3, 14.3, 9.0. IR neat film: 3270, 2982, 1773, 1716, 1578, 1491 cm'1; HRMS (ESI) calculated for [C26H23ClN4NaO4] 513.1300, found 513.1307. [0133] Biological Assays and Me thods
[0134] Cell culture and small-molecule compounds
[0135] NALM6 and shP53NALM6 leukemic cell lines were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 50 U/ml penicillin and 50 pg/ml streptomycin. shP53NALM6 cell line was established using pLKO.l-p53 (purchased from Sigma) (Plasmid #19119) followed by puromycin selection at 1 pg/ml for 2 days then clonal expansion in puromycin-free medium. MANCA, MANCA-mlp-puro and MANCA-mlp- MDM2 were gifts from Prof. Jill Bargonetti (Hunter College, CUNY, NY, USA) and were generated as described previously and maintained in 10% FBS-Pen/Strep- RPMI-1640 medium. Small-molecule compounds were synthesized, purified and characterized in house and were dissolved in DMSO as 10 mM stocks for cell proliferation assays.
[0136] Western blotting, in vitro ubiquitination, and apoptosis analysis
[0137] The western blotting procedure and antibodies for the target proteins were described previously. In vitro assays for ubiquitination by MDM2B-MDM4 were performed as described previously with minor modification. Briefly, reactions were carried out at 30 °C for Ih in a volume of 20 pl reaction in the presence of different concentrations of compounds or vehicle solvent DMSO, followed by WB of p53 with DO-1, MDM2 with rabbit anti- MDM2 (MDM2 (DI V2Z) Rabbit mAb #86934, Cell Signaling Technology), or MDM4 with a rabbit anti-MDM4 antibody (Proteintech, Cat no: 17914-1-AP). Apoptotic response to compounds was measured by western blotting using specific antibodies from Cell Signaling Technology for activated caspase 3 (Cleaved Caspase-3 (Aspl75) (5A1E) Rabbit mAb, #9664) and PARP (PARP Antibody #9542).
[0138] ICso measurement and analysis
[0139] The procedure was described previously. Briefly cells at 10,000/well were plated in 96-well plates at lOOpl/well and compounds of different concentrations were added to each well at lOOpl/well. After 70h treatment, 40pl of 6x resazurin stock solution was added to each well followed by 2h development of fluorescent metabolite by viable cells before reading OD600 in BioTek Synergy 2 Microplate Reader. The IC50 values were obtained by Chou-Median-Effect Equation using CompuSyn software and dose-effect curves were obtained by GraphPad using affected fractions of compound-treated wells normalized against no-drug control wells with non-liner regression model.
[0140] In summary, the anti-proliferative activity of these MMRi67 derivatives were associated with their effects on dual degradation of MDM4 and FTH1 compared to MMRi67. The SAR results from MMRi67 derivatives identified the hydroxyl and the chloro in the quinoline ring and the carboxylic acid to be the critical structural elements that contribute to MDM2-MDM4 E3 ligase inhibitory effects of MMRi67 in vitro. The ethyl ester substitution of the carboxylic acid moiety in MMRi67 generated potent analogs that exhibited strong MDM2/MDM4 and FTH1 degradation in cells. MMRi71 represents a new class of dual inhibitor targeting degradation of MDM2/MDM4 and FTH1 for p53-independent apoptosis in leukemia cells.
EXAMPLE 2
[0141] Structural requirement for the anti-proliferative activity ofMMRi62 and
MMRi67 derivatives
[0142] Primary hits MMRi6 are quinolinols identified in an enzyme-based HTS for inhibition toward the MDM2-MDM4 RING-domain mediated E3 ligase reaction. With available MMRi6 analogs from Hit2Lead library, secondary screens were performed and identified two analogs with unique properties: MMRi62 binds to preformed MDM2-MDM4 RING heterodimers for preferential MDM4 ubiquitination and acts as a MDM4-degrader in cells with potent apoptosis inducing activity; while MMRi67 was the most potent MDM2- MDM4 E3 ligase inhibitor but lacked MDM4 degradation and pro-apoptotic activity in cells. The capability of p53-independent apoptosis induction by MMRi62 promises potential application of the quinolinol derivatives as novel cancer therapies for killing p53-mutant drug-resistant cancer cells. This potential motivated SAR studies on MMRi62 and MMRi67 for lead optimization (Figure 1).
[0143] Anti-proliferation assays were performed for the compounds shown in Figure 1 using wtp53-bearing NALM6 and shP53NALM6 cells (Tables 1, 2) to determine p53- independent cell proliferation inhibitory effect. The chemical structures of MMRi62 and MMRi67 differ in a few positions. MMRi62 has two chlorines on the phenyl ring and none on the quinolinol ring while MMRi67 has a chlorine on the quinolinol ring and a carboxylic acid on the phenyl ring (Figure 1). To test the effect of the quinolinol chloro- substituent, a hydro-to-chloro-modified analog of MMRi62, namely, 62-1 was synthesized. This analog showed weaker cell growth inhibitory effect in NALM6 and shP53NALM6 cells than MMRi62 but was more effective than MMRi67 (Tables 1, 2). However, 62-1 lost p53- independent antiproliferative activity of MMRi62. To further test the importance of quinolinol chlorine, an MMRi67 analog that lacked this functionality, 67-1 was synthesized. This compound was found to have similar weak leukemic cell growth inhibition as MMRi67. Based the structural similarity of the compounds to MMRi62, the compounds are expected to inhibit cancer cell growth to a much larger extent than non-transformed cell growth. MMRi62 is 125 times more cytotoxic to NALM6 leukemia cells than it is to normal peripheral blood mononuclear cells.
[0144] The next step in design and synthesis involved modification of the carboxylic acid on the phenyl ring of MMRi67. Analog 67-2 replaces the carboxylic acid in MMRi67 with a similar electron-withdrawing nitro group and its corresponding 3-nitro analog, 67-3. Both 67-2 and 67-3 showed over 20 times better IC50 against NALM6 and shP53NALM6 cell growth than MMRi67. If the carboxylic acid in MMRi67 existed substantially in its charged carboxylate form, its cell permeability could be low, thereby reducing its ability to inhibit cancer cell growth. To test this, the ethyl ester analog 67-4 was synthesized and screened against leukemia cell proliferation. This analog proved a much more potent cell growth inhibitor with over 50-fold lower IC50 than MMRi67 of 0.22 pM and 0.21 pM in NALM6 and shP53NALM6 cells, respectively. Analog 67-5, an ethyl ester derivative that lacks the chloro functionality on the quinolinol, also showed strong antiproliferative activity.
Therefore, this data suggests that the ethyl ester substitution of acidic group solely contributes to improved cellular activity in MMRi67.
Table 1. MMRi62 compound series ICso.
Entry NALM6 ICso (pM) shP53NALM6 ICso (pM)
MMRi62 0.12 ± 0.001 0.14 ± 0.001
62-1 0.29 ± 0.01 0.46 ± 0.04
62-2 32.4 ± 2.25 35.5 ± 2.62
62-3 8.58 ± 0.83 24.2 ± 4.38
62-4 4.84 ± 0.48 4.68 ± 0.52
62-5 2.62 ± 0.36 1.51 ± 0.12
62-6 1.89 ± 0.53 1.08 ± 0.08
62-7 0.35 ± 0.01 0.26 ± 0.02
62-8 28.6 ± 2.87 28.5 ± 3.82
62-9 0.30 ± 0.02 0.36 ± 0.03
62-10 12.0 ± 1.79 27.1 ± 5.58
62-11 2.10 ± 0.22 2.66 ± 0.17
The treatment was tri replicated and repeated two times. ICso± SD were performed using non-linear regression analysis
Table 2. MMRi67 compound series ICso.
Entry _ NALM6 ICso (piM) _ shP53NALM6 ICso (piM)
MMRi67 12.80 ± 1.57 10.89 ± 1.56
67-1 8.93 ± 0.41 10.1 ± 0.51
67-2 0.40 ± 0.01 0.47 ± 0.01
67-3 0.38 ± 0.03 0.48 ± 0.02
67-4 0.53 ± 0.03 0.57 ± 0.04 67-5 0.38 ± 0.03 0.45 ± 0.01
67-6 >100 >100
67-7 18.2 ± 6.54 31.7 ± 3.52
67-8 2.13 ± 0.18 2.36 ± 0.15
67-9 0.27 ± 0.02 0.37 ± 0.003
MMRi71 0.23 ± 0.01 0.29 ± 0.02
The treatment was tri replicated and repeated two times. ICso± SD were performed using nonlinear regression analysis
[0145] The role of the phenol moiety in both MMRi62 and MMRi67 was determined. For this purpose, analogs lacking phenol, 62-2, 67-6 and 67-7 were synthesized. Without the hydroxyl group, all three analogs showed poor activity in cell proliferation assays compared to their lead compounds. Analog 67-7 completely lost its antiproliferative activity in leukemia cells with ICso >100 pM. Analog 67-6, with the ethyl ester group substitution but lacking the phenol, showed 80-150-fold weaker cell proliferation inhibitory activity compared to 67-5. Comparing 67-6 and 67-7, it appears that while the lack of phenol diminishes their cell inhibitory effect, the ethyl ester group in 67-7 may somewhat restore the compound’s antiproliferation effect in cells. Overall, these results suggest that the phenol group in the quinoline ring can contribute to potent cellular activities of both the compound series.
[0146] The importance of the respective protons on the phenol and amine were additionally tested in the 62 series. Methylated phenol 62-3 was 70-fold and 172-fold less active than MMRi62 in NALM6 and shP53NALM6 cells, respectively. Methylated amine 62-4, cyclic aminal 62-5 and an esterase-labile ester analog, 62-6 also showed weaker antileukemic cell growth activity compared to the parent MMRi62. Interestingly, the amide analog 62-7 lacking the amine proton showed better antiproliferative activity. The amide bond is not expected to be very metabolically labile, thus the intact 62-7 is expected to be responsible for antiproliferative activity. This data may support some covalent reactivity with biochemical targets (the N-acetyl-2-aminopyridine could be a better leaving group than 2- aminopyridine, vide infra). An MMRi62 analog containing the phenol but lacking quinoline heterocycle 62-8 also showed increased ICsos against cell growth.
[0147] The water solubility of these MMRi compounds estimated by calculated logP indicated that they are slightly lipophilic (MMRi62 LogP = 5.1, analog 67-4, LogP = 4.8). It was hypothesized that their water solubility, which would aid in formulation, could be enhanced by the addition of heteroatoms to the 2-aminopyridine ring. Therefore, a pyrimidine domain was substituted in MMRi62 to generate 62-9 and in MMRi67 to generate analogs 67- 8 and 67-9. The predicted LogP of pyrimidine analog 62-9 is 4.4 and for pyrimidino analog 67-9 is 4.0. Analysis of these more water-soluble analogs in antiproliferation of leukemic cells indicated that their activities were only modestly diminished, suggesting that the pyrimidine modification was tolerated. Analog 62-10, with an additional nitrogen in the 2,3- di chlorobenzene ring, substantially lost antiproliferative ability, indicating that not all changes are tolerated on this aryl ring. In addition, it should be noted that 62-11 that lacks substitution on the benzene ring is also an order of magnitude less potent than MMRi62. [0148] Although the phenol contributes to the antiproliferative activity of the MMRi compounds, it was seen as a potential source of molecular aggregation, either via hydrogen bonding network or transient metal chelation. Thus, to mitigate these potential unwanted reactivities, an esterase-labile propionate moiety was introduced to the lead pyrimidinyl analog 67-9 to generate the prodrug MMRi71 (Scheme 1). MMRi71 is considered a dual prodrug of 67-8, as its aromatic ethyl ester is also succeptable to cellular esterases. MMRi71 with IC50 of 0.23 pM in NALM6 cells and 0.29 pM in shP53NALM6 cells showed improved p53-independent antiproliferative activity in leukemia cells and was selected as a potential candidate for further investigation.
Figure imgf000085_0001
Scheme 1. Synthesis of MMRi71. A representative synthesis is shown for MMRi71.
[0149] MMRi67 is a small-molecule with demonstrated inhibitory activity against MDM2-MDM4 E3 ligase. Yet, MMRi67 does not promote MDM4 degradation, nor does it induce apoptosis in leukemic cells as MMRi62. . A series of MMRi67 derivatives were synthezized to identify derivatives with improved pro-apoptotic activity (Figure 1).
EXAMPLE 3
[0150] MMRi67 is a bona fide inhibitor ofMDM2-MDM4 E3 ligase
[0151] False positive hits in biochemical or cell-based screens with so-called PAINS (Pan Assay Interference compounds) are of concern and hinder lead optimization. MMRi62 and MMRi67 are unlikely PAINS because NEDD4-1 was used as a non-specific control E3 ligase in validation assays. This was further validated in this study by the selectivity of MMRi67 for Mdm2B versus NEDD4-1 (Figure 2 A). Moreover, MMRi62 and MMRi67 have distinct effect on MDM2-MDM4 E3 ligase activity, MDM4 degradation and apoptosis induction despite their subtle structural difference. Identification of a PAINS from a real hit requires more than simply running a filter screen. However, some chemical behaviors of PAINS can be tested including protein reactivity, colloidal aggregation, and metal chelation. To rule out colloidal aggregator activity, an E3 ligase assay was performed in the presence or absence of 0.01% non-ionic detergent Trixton-X-100, these results indicate that MMRi67- mediated inhibition of MDM2-MDM4 E3 ligase was not affected in the presence of detergent (Figure 2B).
[0152] The structure of MMRi67 predicts metal chelation potential. Because Zn is required for maintaining the RING domain structure, it is possible that MMRi67 chelates away Zn and collapse the RING structure leading to dissociation of RING-RING interaction between MDM2-MDM4 and loss of the E3 ligase activity. An excessive amount of Zn has the potential to neutralize the chelating activity of MMRi67 and cancel its inhibition of MDM2-MDM4 E3 ligase activity. An E3 ligase assay was performed in the presence of Zn and these results indicated the presence of Zn did not affect the effect of MMRi67 to inhibit MDM2-MDM4 E3 ligase activity.
[0153] Preincubation of MDM4 proteins with either MMRi62 or MMRi67 for a period of 20 to 320 minutes at 10 pM did not affect the E3 ligase activity of the MDM2- MDM4 E3 complex (data not shown). However, preincubation of MDM2B proteins with MMRi62 but not MMRi67 inhibited the MDM2-MDM4 E3 ligase activity (Figure 2D), suggesting that covalent binding contributes to some extent to MMRi62-mediated effect on MDM2B-MDM4 E3 ligase reaction (e.g. via alkylation of a quinone methide formed from 2- aminopyridine loss via solvolysis of MMRi62.). This covalent binding potential of MMRi62 is similar to that of a macrophage migration inhibitory factor (MIF) inhibitor which shares structural similarities with MMRi62. However, the covalent binding is not involved in the inhibitory mechanism of MMRi67 toward MDM2-MDM4 E3 ligase in vitro. Taken together, the results suggest that MMRi67 bears specific inhibitory activity toward MDM2-MDM4 E3 ligase.
EXAMPLE 4
[0154] Structural requirement of MM R i 67 derivatives for inhibitory activity toward
MDM2-MDM4 E3 ligase [0155] To identify the pharmacophore of MMRi67 involved in inhibition of MDM2- MDM4 E3 ligase, new derivatives were evaluated in MDM2-MDM4 E3 ligase assays. Analogs 67-1 with hydrogen substituted chloro group and 67-6 with hydrogen substituted for the hydroxyl group in the quinoline ring both lost the strong E3 ligase inhibitory activity of MMRi67. However, 67-1 showed slightly weak inhibition at 10 pM concentration leading to a conclusion that the hydroxyl and chloro groups in MMRi67 are key structural groups for strong MDM2/MDM4 E3 ligase inhibition (Figure 3 A). To check whether the carboxylic acid in MMRi67 contributes to its E3 ligase activity for MDM2 and MDM4, 62-1 with dichloro substitution and 67-4 (Figure 1) with ethyl ester substitution were evaluated with E3 ligase assays. The results showed weak inhibition of MDM2 and MDM4 ubiquitination at concentrations of 10 pM for both 62-1 and 67-4 derivatives, indicating that the E3 ligase inhibitory action of MMRi67 was lost by carboxylic acid substitution (Figure 3B).
[0156] Following the MMRi67 quinoline ring hydroxyl and chloro substitution and the carboxylic acid moiety modification, pyridine was switched to pyrimidine in E3 ligase assay. Pyridine substitution to pyrimidine in MMRi67 to generate analog 67-8 lost MDM4 E3 ligase inhibitory effect however showed partial inhibition of MDM2-B E3 ligase at high concentrations. 67-9 with pyrimidine substituted pyridine and ethyl ester substituted acidic group lost both MDM4 and MDM2-B E3 ligase effects. Similarly, MMRi71 with pyrimidine substitution along with ethyl ester substitution at carboxylic acid and hydroxyl groups of MMRi67 lost activity to inhibit MDM4 ubiquitination whereas retained inhibitory effect on MDM2B ubiquitination in a dose-dependent manner (Figure 3C). Overall, the hydroxyl, chloro, carboxylic acid and pyridine arms of MMRi67 are all desirable structural components that contribute to strong MDM4-MDM2B E3 ligase inhibition.
EXAMPLE 5
[0157] Induced MDM4 and FTH1 degradation is associated with the pro-apoptotic activity ofMMRi67 derivatives
[0158] The p53-independent pro-apoptotic activity of quinolinol compound MMRi62 was associated with induced MDM4 and FTH1 degradation in cells. To determine whether the increased antiproliferative activity of potent MMRi67 analogs is also associated with p53- independent apoptosis and MDM2/MDM4 and FTH1 protein degradation, western blot analysis for activated Caspase 3 and caspase-mediated PARP cleavage was conducted.
Consistent with its improved anti-proliferation activity, analog 67-5 with hydrogen substituted chloro and ethyl ester substituted acidic group in MMRi67 induced downregulation of MDM4/MDM2, caspase 3 activation and PARP cleavage in a concentration-dependent manner (Figures 4A, 4B). Moreover, the pro-apoptotic effect of 67- 5 was independent of p53 since the apoptotic response to 67-5 between NALM6 and shp53NALM6 cells showed no difference. Similar results were obtained with 67-4 (data not shown). These results demonstrate that improved cell killing ability of 67 derivatives use similar mechanism of action as MMRi62, i.e., MDM4 degradation is a necessary molecular event associated with apoptosis induction. Although 67-5 effectively induced downregulation of FTH1, as far as apoptosis of leukemia cells is concerned, it appeared that MDM2/MDM4 degradation, but not FTH1 degradation is associated with apoptosis induction, since MMRi67 also induced FTH1 degradation at concentrations >2.5 pM yet did not elicit any apoptotic response (Figure 4B). FTH1 degradation may play a role in predisposing cells to apoptosis since FTH1 is a ferroptosis target whose depletion increases reactive oxygen species (ROS) and induces ferroptosis in other cell types.
[0159] MMRi67 analog, MMRi71 with ethyl ester substitutions at hydroxyl and carboxylic acid group and pyrimidine ring showed strong anti-leukemic activity in cell proliferation inhibitory assay along with dose-dependent inhibition of MDM2B E3 ligase activity. Structural changes in MMRi71 are expected to increase water solubility and decrease molecular self-aggregation (via hydrogen-bonding or through metals involving the phenol) while maintaining the cell permeability potential of the candidate and its optimal effect on cellular targets. To test this, anti-proliferation assays showed that MMRi71 has the same potency in inhibiting growth of NALM6 and shP53NALM6 cells with ICsos of 0.2 pM (Figure 5A), indicating a p53-independent cytotoxicity. Interestingly, phenol ester MMRi71 is substantially more cytotoxic than phenol ester 62-6 even though their parent phenols 67-9 and MMRi62 have similar cell growth ICso’s. The greater aqueous solubility the pyrimidine lends to MMRi71 may contribute to a more rapid ester hydrolysis to its active form, phenol 67-9.
[0160] MMRi71 treated NALM6 and shP53NALM6 cells were used in western blot analysis for changes in MDM4 and FTH1 proteins. MMRi71 treatment induced effective downregulation of MDM4 and FTH1 in both NALM6 and shP53NALM6 cells, and induced apoptosis shown by PARP cleavage indicating a p53-independent mechanism (Figure 5B). To test whether MMRi71 -induced apoptosis was not a result of colloidal aggregation, cellular experiments in the presence of Triton X-10 were performed. Because the presence of 0.01% Trixton-X-100 appeared to induce apoptotic cell death in NALM6 cells and the tolerable concentrations of Triton X-100 was between 0.001-0.0025% (data not shown), cellular experiments in the presence of 0.0025% Triton X-100 showed that MMRi71 induced similar extend of apoptotic response indicated by cleaved PARP in the presence of detergent (Figure 5C), suggesting that MMRi71 -induced apoptosis is not a result of colloidal aggregation. [0161] MMRi62 induced MDM4 degradation was MDM2-dependent and MMRi62- induced FTH1 degradation was dependent on lysosomal pathway. To understand whether MMRi71 -induced MDM4 uses the same mechanisms as MMRi62, a WB analysis of MDM4 degradation by MMRi71 was performed using a pair of MDM2-high MANCA lymphoma cells in which MDM2 was either stably knocked down by lentivirus-mediated microRNA (miRNA) or left untouched with control miRNA. Results indicated that MDM4 expression levels were elevated in MDM2-knockdown MANCA-mlp-MDM2 cells, and treatment with MMRi71 did not induce MDM4 degradation. The elevated MDM4 expression in MDM2- knockdown cells is consistent with the report that MDM2 promotes ubiquitination and degradation of MDM4. Abolishment of MDM4 degradation by MMRi71 in the absence of MDM2 in MANCA-mlp-MDM2 cells suggest that MMRi71 -induced MDM4 degradation is MDM2-dependent (Figure 5D). To determine whether MMRi71 -induced FTH1 degradation is lysosome dependent, a rescue experiment was performed with lysosome inhibitor Bafilomycin Al (BAF1). The results showed that BAF1 fully rescued FTH1 in NALM6 cells (Figure 5E). These results suggest that MMRi71 induces MDM2-dependent proteasomal degradation of MDM4 and lysosome-dependent degradation of FTH1, the same mechanisms of action used by MMRi62, in addition to its potential inhibitory activity toward MDM2- MDM4 E3 ligase activity. The improved activity of MMRi71 over MMRi67 may be due to the increased covalent binding capability or improved permeability or both, which requires further confirmation using proper assays. The neutral ester in MMRi71 is expected to imbue better plasma membrane permeability compared to the charged carboxylic acid/carb oxy late in MMRi67. Additionally, MMRi62, which has structural similarities to MMRi71, did show some covalent binding effect to MDM2 (Figure 2D). Based on studies with quinolinol analog MIF inhibitor, covalent binders in this molecular class can be specific inhibitors. MMRi71 may be a covalent inhibitor targeting cellular MDM2-MDM4 for degradation.
[0162] Quinolinol compounds were reported to cause DNA damage via chelating metal ions and generating ROS. To test whether MMRi71 causes DNA damage, MMRi71 and inactive compound 67-7 that lacks metal chelating capability were used to treat p53/Mdm2-double knockout (2KO) MEFs and human 293T cell lines followed by detecting y-H2AX which is an indicator of DNA damage. Use of 2KO MEFs and 293T cells will exclude apoptosis-associated y-H2AX signal due to apoptotic DNA fragmentation, since these two cell types are resistant to apoptosis. Results showed that MMRi71 but not the inactive 67-7 increased y-H2AX slightly at 5 pM and significantly at 10 pM in both cell lines (Figure 5F and 5G). These results suggest that MMRi71 at concentrations of >5 pM has potential to induce DNA damage, possibly by MMRi71 -induced ROS. These results are consistent with our observation that MMRi62 induces ROS generation in pancreatic cells. ROS induced by MMRi62-like compounds likely involves both metal chelation and FTH1 degradation which releases ferrous iron, although metal chelation is not involved in its action on the E3 ligase activity (Figure 2C). Since MMRi71 induced apoptosis at >5 pM at 24 h in NALM6 cells (Figure 5C) and 67-7 that did not generate ROS is an inactive compound, these results conclude that ROS generation is required for the antitumor activity of MMRi71. However, without intending to be bound to any particular theory, it is considered that ROS- induced DNA damage itself is not the major mechanism contributing to cell killing by MMRi62-like compounds, since they have high cancer selective toxicity. MMRi62 inhibits leukemic NALM6 (B-cell precursor leukemia) cell growth at 125-fold potency compared to its inhibition of normal peripheral blood mononuclear cells (PBMCs). This 125-fold difference in MMRi62 sensitivity is not likely the result of DNA damage since DNA damage by radiation kills normal human lymphocytes and B cell lymphoma cells at comparable capability with Do of 1.95Gy for lymphocytes and of 1.38Gy for Burkitt's lymphoma cell, where Do is a radiation dose required to reduce the fraction of surviving cells to 37% of its previous value. Accordingly, like MMRi62, the mechanisms of action for MMRi71 may, in various embodiments, involve multiple drug targets that predispose cancer cells to its selective toxicity.
EXAMPLE 6
[0163] Discussion of potential covalent inhibition mechanism.
[0164] We have presented data that indicates that MMRi62 and perhaps some 62- and 67-based analogs (including MMRi71), might function in part as covalent inhibitors. We envision covalent inhibition could occur, as previously proposed for related phenolic benzylic amines, via a quinone methide forming reaction followed by addition of a protein’s nucleophilic functionality (e.g. amine or thiol) to the resulting electrophile. Our further efforts to probe this mechanism are underway and will be reported in due course. Thus far, a covalent protein-MMRi62 covalent adduct has not been isolated or biochemically identified. We did find MMRi62 to be stable in C2D5OD at 100 °C for 24 h including with added CD3CO2D and deuterated pyridine, respectively ( 1 H NMR analysis, separate experiments), so if MMRi62 and related analogs are covalent inhibitors, it is likely they selectively bind their targets and become activated to the quinone methide within the target.
Figure imgf000091_0001
[0165] General Information
[0166] All reagents were used out of the bottle as purchased from the supplier without further purification unless otherwise noted. 1 H NMR spectra were recorded in CDCh (using 7.26 ppm for reference of CHCh), CD2CI2 (using 5.30 ppm for reference of CH2CI2) DMSO- de (using 2.50 ppm for reference of DMSO) at 300 or 400 MHz. 13C NMR spectra were recorded in CDCh (using 77.0 ppm as internal reference), CD2CI2 (using 54.0 ppm as internal reference), or DMSO-de (using 40.0 ppm as internal reference) at 75.5 or 101 MHz. IR spectra were taken neat using a Nicolet-Impact 420 FTIR. Wave numbers in cm'1 are reported for characteristic peaks. High resolution mass spectra were obtained at SUNY Buffalo’s mass spec facility on a ThermoFinnigan MAT XL spectrometer. Melting points were obtained on an electrothermal melting point apparatus and are reported uncorrected. 2-Aminopyridine, 1,3-dichlorobenzaldehyde, 8-hydroxyquinoline, 1-napthol phenol, and 5-chloro-8- hydroxy quinoline were purchased from Acros and used without further purification. N- Phenyl-bis(trifluoromethanesulfonamide) was purchased from AK Scientific used without further purification. 4,5-Dichloropyridine-3-carbaldehyde was purchased from AABlocks and used without further purification. Analogs 67-2 and 67-3 were obtained as part of a compound screening library from Hit21ead Chembridge. Analog 62-11 was synthesized as previously reported. Analogs in the 62 and 67 series were synthesized via a 3 -component Betti reactions. Ethyl 4-formylbenzoate was synthesized as previously reported.
[0167] 7-((2, 3-Dichl orophenyl)(pyri din-2 -ylamino)methyl)quinolin-8-ol (MMRi62)
Figure imgf000092_0001
MMRi62, 21% yield
To a dry 50 mL round-bottomed flask, 2-aminopyridine (269 mg, 2.86 mmol, 1.0 equiv.), 2,3-dichlorobenzaldehyde (500 mg, 2.86 mmol, 1.0 equiv.), and 8-hydroxyquinoline (500 mg, 3.43 mmol, 1.2 equiv) were dissolved in CH3CN (30 mL). Following the addition of formic acid (86 pL, 2.29 mmol 0.80 eq), the solution was refluxed for 16 h. The solution was allowed to cool to rt, concentrated, and the crude mixture was then directly purified by flash column chromatography (silica gel, 10-20% acetone: hexanes) to give MMRi62 as a white solid (236 mg, 21%). mp = 178-179 °C; ’H NMR (300 MHz, CDCh) 5 8.76 (d, J= 4.1 Hz, 1H), 8.17 - 8.04 (m, 2H), 7.59 (d, J= 7.8 Hz, 1H), 7.47 - 7.34 (m, 4H), 7.29 (s, 1H), 7.19 (t, J= 7.9 Hz, 1H), 6.68 (d, J= 6.3 Hz, 1H), 6.61 (t, J= 6.1 Hz, 1H), 6.35 (d, J= 8.4 Hz, 1H), 5.55 (d, J= 6.4 Hz, 1H).; 13C NMR (75 MHz, CDCh) 5 157.5, 149.8, 148.2, 148.1, 141.5, 138.2, 137.8, 136.0, 133.5, 132.0, 129.4, 127.8, 127.2, 127.1, 126.9, 122.0, 121.3, 117.7, 113.7, 106.7, 53.8.; IR neat film: 3351, 3079, 1599, 1571, 1516, 1502 cm'1; HRMS (ESI) calculated for [C2iHi6ChN3O]+: 396.0665, found 396.0649.
[0168] 4-((5-Chloro-8-hydroxyquinolin-7-yl)(pyridin-2-ylamino)methyl)benzoic acid (MMRi67)
Figure imgf000092_0002
MMRi67, 11 % yield
To a dry 10 mL round-bottomed flask, 2-aminopyridine (94.11 mg, 1.0 mmol, 1.0 equiv.) and 4-carboxybenzaldehyde (150.13 mg, 1.0 mmol, 1.0 equiv.) were dissolved in EtOH (5 mL). Then 5-chloro-8-hydroxyquinoline (179.60 mg, 1.0 mmol, 1.0 equiv) was added, the flask was capped and stirred at rt for 14 d. Upon appearance of a precipitate, the stirring was stopped and the solid allowed to settle. The solid was filtered and washed with HPLC grade hexanes to give MMRi67 as an orange solid (45 mg, 11%). mp = 199-200 °C; XH NMR (400 MHz, DMSO-d6) 5 12.83 (bs, 1H), 10.40 (bs, 1H), 8.95 (s, 1H), 8.46 (d, J= 8.5 Hz, 1H), 7.89 (d, J= 6.2 Hz, 3H), 7.77 (s, 1H), 7.74 - 7.66 (m, 1H), 7.53 (d, J= 8.7 Hz, 1H), 7.48 (d, J = 6.3 Hz, 2H), 7.41 (t, J= 7.9 Hz, 1H), 6.96 (d, J= 8.4 Hz, 1H), 6.71 (d, J = 8.4 Hz, 1H), 6.51 (t, J= 6.3 Hz, 1H); 13C NMR (101 MHz, DMSO-d6) 5 167.1, 157.6, 149.4, 149.2, 148.0, 147.4, 138.7, 136.9, 132.5, 129.5, 129.3, 127.3, 126.3, 126.0, 124.9, 122.9, 118.7, 112.5, 109.0, 51.2. IR neat film: 3281, 2953, 1672, 1603, 1576, 1504 cm'1; HRMS (ESI) calculated for [C22HI7C1IN3O3]+: 406.0953, found 406.0964.
[0169] 5 -Chloro-7-((2,3-dichlorophenyl)(pyridin-2-ylamino)methyl)quinolin-8-ol
(62-1).
Figure imgf000093_0001
In a 50 mL pressure tube, 2-aminopyridine (94 mg, 1 mmol, 1.0 equiv.), 2,3- dichlorobenzaldehyde (175 mg, 1 mmol, 1.0 equiv.), and 5-chloro-8-hydroxyquinoline (180 mg, 1 mmol, 1.0 equiv) were dissolved in absolute ethanol (5 mL). The tube was capped and heated to 80 °C for 16 h. Upon cooling to rt a precipitate formed. This solid was filtered and washed with Et2O to yield pure 1-15 as a tan solid (40 mg, 9% yield), mp = 178-181 °C; ’H NMR (300 MHz, CDCh) 5 8.82 (d, J= 4.3 Hz, 1H), 8.48 (d, J= 8.5 Hz, 1H), 8.09 (d, J= 5.0 Hz, 1H), 7.60 - 7.50 (m, 2H), 7.48 (s, 1H), 7.42 (q, J= 4.0 Hz, 2H), 7.21 (t, J= 7.9 Hz, 1H), 6.70 (d, J= 6.3 Hz, 1H), 6.63 (t, J= 6.2 Hz, 1H), 6.37 (d, J= 8.4 Hz, 1H), 5.48 (d, J= 6.3 Hz, 1H); 13C NMR (75 MHz, CDCh) 5 157.2, 149.0, 148.7, 147.6, 140.8, 138.6, 138.1, 133.6, 133.3, 132.0, 129.7, 127.4, 126.8, 126.7, 125.8, 122.7, 121.8, 121.8, 120.6, 113.9, 107.1, 53.4; IR neat film: 1598, 1574, 1495 cm'1; HRMS (ESI) calculated for [C2iHi5ChN3O]+: 430.0275, found 430.0281.
[0170] 7-((2, 3 -Diehl orophenyl)(pyri din-2 -ylamino)methyl)quinolin-8-yl trifluoromethanesulfonate (S-l)
Figure imgf000094_0001
In a 10 mL reaction tube, MMRi62 (285 mg, 0.72 mmol, 1.0 equiv.) was dissolved in dry DMF (1 mL) under argon atmosphere. N-Phenyl-bis(trifluoromethanesulfonamide) (385 mg, 1.08 mmol, 1.5 equiv.) and potassium carbonate (150 mg, 1.09 mmol, 1.5 equiv.) were added, the tube was capped and the solution was heated in an 80 °C oil bath for 2 h. The solution was then cooled to rt, diluted with EtOAc, and washed three times with H2O. The organic layer was dried over Na2SO4, and then concentrated. The crude mixture was purified by flash column chromatography (silica gel, 10% acetone/hexanes gradient) to yield S-l as a white solid (152 mg, 40% yield), mp = 201-203 °C; ’H NMR (400 MHz, CDCh) 5 9.04 (d, J= 4.2 Hz, 1H), 8.19 (d, J= 8.4 Hz, 1H), 8.06 (d, J= 5.1 Hz, 1H), 7.78 (d, J = 8.6 Hz, 1H), 7.54 (dd, J= 8.4, 4.2 Hz, 1H), 7.49 - 7.32 (m, 4H), 7.19 (t, J= 7.9 Hz, 1H), 6.69 (d, J= 5.2 Hz, 1H), 6.65 (t, 1H), 6.32 (d, J= 8.4 Hz, 1H), 5.16 (d, J= 5.2 Hz, 1H); 13C NMR (101 MHz, CDCh) 5 156.9 151.3, 148.3, 144.3, 140.5, 140.0, 137.7, 135.6, 134.0, 132.6, 132.3, 130.1, 129.1, 127.8, 127.4, 126.8, 125.8, 122.8, 120.4, 117.2, 114.4, 106.8, 53.2; IR neat film: 3373, 2924, 2853, 2323, 2050, 1715, 1600, 1574 cm'1; HRMS (ESI) calculated for [C22Hi5ChF3N3O3S]+: 528.0158, found 528.0179.
[0171] 7V-((2,3-Dichlorophenyl)(quinolin-7-yl)methyl)pyridin-2-amine (62-2)
Figure imgf000094_0002
S-1 62-2
In a 10 mL pressure tube, S-l (90 mg, 0.17 mmol, 1.0 equiv), palladium acetate (4 mg, 0.017 mmol, 0.1 equiv), triphenylphosphine (9 mg, 0.034 mmol, 0.2 equiv), and triethylamine (71 pL, 0.51 mmol, 3.0 equiv) were combined in dry DMF (1 mL) under argon atmosphere. Formic acid (13 pL, 0.34 mmol, 2.0 equiv) was added, and the reaction was capped and stirred at 60 °C for 2 h. Upon cooling to rt, the reaction mixture was diluted with brine and extracted with EtOAc. The organic layer was then washed three times with brine. The organic layer was dried over Na2SO4 and then concentrated. The crude mixture was purified by flash column chromatography (silica gel, 30% EtOAc :hexanes gradient) to yield 62-2 as a white solid (60 mg, 93% yield), mp = 220-221 °C; ’H NMR (300 MHz, CDCh) 5 8.94 (d, J= 4.2 Hz, 1H), 8.37 (d, J= 8.2 Hz, 1H), 8.08 (d, J= 8.4 Hz, 2H), 7.64 - 7.55 (m, 1H), 7.50 - 7.35 (m, 4H), 7.25 - 7.18 (m, 2H), 7.01 (d, J= 6.6 Hz, 1H), 6.69 - 6.61 (m, 1H), 6.33 (d, J= 8.8 Hz, 1H), 5.00 (d, J = 6.6 Hz, 1H) ; 13C NMR (75 MHz, CDCh) 5 156.5, 151.3, 144.3, 140.5,
139.8, 138.3, 135.7, 134.0, 132.3, 130.2, 129.1, 127.9, 127.5, 126.8, 125.8, 122.8, 114.3,
107.1, 53.1; IR neat film: 3254, 3062, 3018, 2924, 2854, 2114, 1730, 1671, 1601, 1574, 1501 cm'1; HRMS (ESI) calculated for [C2iHi6ClF3N3]+: 380.0716, found 380.0714.
[0172] A-((2,3-Dichlorophenyl)(8-methoxyquinolin-7-yl)methyl)pyridin-2-amine
(62-3)
Figure imgf000095_0001
In a 10 mL pressure tube, MMRi62 (50 mg, 0.13 mmol, 1.0 equiv) was dissolved in anhydrous acetonitrile (1 mL) under argon atmosphere. Potassium carbonate (20 mg, 0.14 mmol, 1.1 equiv), followed by methyl iodide (9 pL, 0.14 mmol, 1.1 equiv) were added to the solution. The mixture was stirred at reflux temperature for 2 h. Upon cooling to rt, the reaction mixture was diluted with ethyl acetate and washed with water. The organic layer was dried over sodium sulfate and then concentrated. The crude mixture was purified by flash column chromatography (silica gel, 10%-20% acetone/hexanes gradient) to yield 62-3 as a white solid (33 mg, 66% yield), mp =122-123 °C; ’H NMR (300 MHz, CDCh) 5 8.93 (d, J= 4.6 Hz, 1H), 8.13 (d, J= 8.2 Hz, 1H), 8.07 (d, J= 4.9 Hz, 1H), 7.55 - 7.35 (m, 8H), 7.19 (t, J = 7.8 Hz, 1H), 6.77 (d, J= 6.4 Hz, 1H), 6.61 (t, J= 6.2 Hz, 1H), 6.34 (d, J= 8.4 Hz, 1H), 5.24 (d, J = 6.4 Hz, 1H), 4.05 (s, 1H); 13C NMR (75 MHz, CDCh) 5 157.4, 154.1, 149.6, 148.3, 142.8, 141.9, 137.7, 136.2, 133.6, 132.4, 132.0, 129.48, 129.45, 127.2, 127.0, 126.3, 123.2, 121.4, 113.8, 106.7, 62.5, 53.8; IR neat film: 3266, 3097, 3019, 1603, 1520, 1503 cm' l- HRMS (ESI) calculated for [C22HisChN3O]+: 410.0821, found 410.0814. [0173] 7V-((8-((tert-Butyldimethylsilyl)oxy)quinolin-7-yl)(2,3- dichlorophenyl)methyl)pyridin-2-amine (S-2)
Figure imgf000096_0001
In a 25 mL round-bottom flask, MMRi62 (100 mg, 0.128 mmol, 1.0 equiv), and 2,6-lutidine (75 pL, 0.630 mmol, 5.0 equiv) were dissolved in CH2CI2 (10 mL) under argon atmosphere at 0 °C. TBSOTf (0.15 mL, 0.630 mmol, 5.0 equiv) was added dropwise and allowed to stir for 1 h at 0 °C. The reaction was slowly warmed to rt and allowed to stir for an additional 2 h. The reaction mixture was quenched with aqueous NaHCO3 and extracted 3 times with CH2CI2. The organic layers were combined and dried over Na2SO4. The organic solution was concentrated and purified by flash column chromatography (silica gel, 10% EtOAc:hexanes gradient) to yield S-2 as a pale yellow solid (107 mg, 83% yield), mp = 74-77 °C; TH NMR (300 MHz, CDCk) 5 8.79 (dd, J= 4.1, 1.6 Hz, 1H), 8.11 - 8.01 (m, 2H), 7.54 (dd, J= 7.6, 1.3 Hz, 1H), 7.45 - 7.33 (m, 3H), 7.27 (d, J= 8.4 Hz, 1H), 7.20 (t, J= 7.9 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H), 6.72 (d, J= 4.9 Hz, 1H), 6.66 - 6.55 (m, 1H), 6.23 (d, J= 8.4 Hz, 1H), 5.21 (d, J= 4.7 Hz, 1H), 0.89 (s, 9H), 0.36 (d, J= 3.0 Hz, 6H); 13C NMR (75 MHz, CDCk) 5
157.7, 151.2, 148.0, 147.1, 141.8, 140.3, 137.7, 135.6, 133.7, 132.1, 129.4, 129.0, 127.2,
126.7, 126.4, 121.5, 119.1, 113.7, 106.6, 53.4, 26.2, 19.3, -1.8, -2.1; IR neat film: 3217, 2984, 2927, 2854, 1598, 1573, 1502 cm'1; HRMS (ESI) calculated for [C27H3oCl2N3OSi]+: 510.1524, found 510.1530.
[0174] 7V-((8-((tert-Butyldimethylsilyl)oxy)quinolin-7-yl)(2,3- dichlorophenyl)methyl)-N -methylpyridin-2-amine (S-3)
Figure imgf000096_0002
In a 5 mL round-bottom flask, intermediate S-2 (107 mg, 0.209 mmol, 1.0 equiv) was dissolved in dry DMF (2.5 mL). Once dissolved, NaH (50 mg, 2.09 mmol, 10.0 equiv) was added and stirred for 15 min at rt. After 15 min, CH3I (0.13 mL, 2.09 mmol, 10.0 equiv) was added to the solution and allowed to stir for an additional 2 h at rt. The solution was then diluted with EtOAc (10 mL), and washed 3 times with H2O (10 mL). The organic layer was then dried over Na2SO4 and concentrated. The crude mixture was then purified by flash column chromatography (silica gel, 10% EtOAc:hexanes gradient) to yield intermediate S-3 as a white solid (84 mg, 76% yield), mp = 59-61 °C; ’H NMR (300 MHz, CDCh) 5 8.79 (dd, J= 4.1, 1.6 Hz, 1H), 8.19 (dd, J= 4.9, 1.2 Hz, 1H), 8.09 (dd, J= 8.3, 1.6 Hz, 1H), 7.46 - 7.33 (m, 4H), 7.31 (s, 1H), 7.20 (s, 1H), 7.15 - 7.07 (m, 2H), 7.01 (s, 1H), 6.56 (dd, J= 7.0, 5.0 Hz, 1H), 6.46 (d, J= 8.6 Hz, 1H), 2.97 (s, 3H), 0.80 (s, 9H), 0.36 (s, 3H), 0.26 (s, 3H); 13C NMR (75 MHz, CDCh) 5 158.4, 151.4, 147.8, 146.9, 141.6, 140.3, 137.0, 135.6, 133.8, 132.8, 129.3, 129.0, 127.6, 127.4, 127.0, 126.4, 121.3, 118.8, 112.2, 106.3, 58.9, 33.0, 26.0, 19.0, -1.8, -2.0; IR neat film: 2949, 2927, 2893, 2855, 1594, 1561, 1502 cm'1; HRMS (ESI) calculated for [C2sH32ChN3OSi]+: 524.1699, found 524.1686.
[0175] 7-((2,3-Dichlorophenyl)(methyl(pyridin-2-yl)amino)methyl)quinolin-8-ol (62- 4)
Figure imgf000097_0001
S-3 62-4
In a 10 mL round-bottomed flask, intermediate S-3 (84 mg, 0.160 mmol, 1 equiv) was dissolved in dry THF (1 mL) under argon atmosphere at 0 °C. A 1 M solution of TBAF in THF (0.25 mL, 0.240 mmol, 1.5 equiv) was added dropwise at 0 °C and stirred for 1 h. The solution was slowly warmed to rt and stirred for 3 h. The reaction mixture was concentrated and dissolved in EtOAc. The solution was washed with saturated NH4Q. The organic layer was dried over Na2SO4 and concentrated. The crude mixture was purified by flash column chromatography (silica gel, 10% acetone: hexanes gradient) to yield 62-4 as a white solid (24 mg, 37% yield), mp = 169-170 °C; ’H NMR (400 MHz, CDCh) 5 8.79 (d, J= 2.7 Hz, 1H), 8.24 (d, J= 3.7 Hz, 1H), 8.16 (d, J= 8.2 Hz, 1H), 7.53 - 7.42 (m, 3H), 7.30 (t, J= 7.9 Hz, 1H), 7.22 - 7.10 (m, 3H), 6.69 - 6.53 (m, 2H), 2.99 (s, 3H); 13C NMR (101 MHz, CDCh) 5 158.5, 150.2, 148.3, 147.9, 141.0, 138.0, 137.3, 136.0, 133.7, 132.6, 129.4, 127.9, 127.5,
127.4, 127.1, 121.9, 120.7, 117.5, 112.4, 106.1, 58.3, 33.1; IR neat film: 3242, 2923, 1593, 1557, 1506 cm'1; HRMS (ESI) calculated for [C22HisChN3O]+: 410.0834, found 410.0821.
[0176] 4-(2,3-Dichlorophenyl)-3-(pyridin-2-yl)-3,4-dihydro-2Z/-[l,3]oxazino[5,6-
A]quinolone (62-5)
Figure imgf000098_0001
To a 10 mL pressure tube, MMRi62 (100 mg, 0.25 mmol, 1.0 equiv) and paraformaldehyde (8.5 mg, 0.28 mmol, 1.1 equiv) were dissolved in dioxane (1.5 mL). The solution was heated to 100 °C for 24 h. Upon cooling to rt, the solution was diluted with Et2O, and concentrated. The crude mixture was purified by flash column chromatography (silica gel, 20-30% acetone: hexanes gradient) to give 62-5 as a green-white solid (30 mg, 30%). mp = 115-117 °C; ’H NMR (400 MHz, CDCh) 5 8.90 (d, J= 2.7 Hz, 1H), 8.25 (d, J= 4.8 Hz, 1H), 8.10 (d, J= 10.0 Hz, 1H), 7.56 (t, J= 6.9 Hz, 1H), 7.46 - 7.35 (m, 4H), 7.33 (s, 2H), 7.21 (d, J= 8.4 Hz, 1H), 7.13 - 7.07 (m, 3H), 6.95 (d, J= 7.7 Hz, 1H), 6.82 - 6.74 (m, 1H), 6.14 (d, J= 12.8 Hz, 1H), 5.22 (d, J= 11.0 Hz, 1H); 13C NMR (75 MHz, CDCh) 5 157.1, 150.3, 149.7, 147.9,
142.2, 139.7, 137.9, 136.0, 134.1, 133.3, 130.3, 129.9, 128.6, 127.0, 126.6, 121.8, 119.5,
119.2, 116.4, 110.1, 73.9, 54.2; IR neat film: 2957, 1736, 1592, 1568, 1503 cm'1; HRMS (ESI) calculated for [C22Hi6ChN3O]+: 408.0682, found 408.0665.
[0177] 7-((2, 3-Dichl orophenyl)(pyri din-2 -ylamino)methyl)quinolin-8-yl propionate
(62-6) propionoyl chloride K2CO3, CH2CI2 0 °C to rt, 1 h
53%
Figure imgf000098_0003
Figure imgf000098_0002
MMRI62 62-6
In a 250 mL dry round bottomed flask, MMRi62 (3 g, 7.6 mmol, 1.0 equiv) was dissolved in
70 mL of dry CH2CI2 under argon atmosphere. Potassium carbonate (2.0 g, 14.5 mmol, 2.0 equiv) was added and the solution was cooled to 0 °C. Propionoyl chloride (0.67 mL, 7.7 mmol, 1.0 equiv) was then added to the solution. The mixture was allowed to warm to rt and stirred for 1 h. The reaction mixture was then filtered through celite and washed with CH2CI2. The supernatant was then treated with 800 mg DMT-functionalized silica gel and stirred for 15 min. The mixture was filtered and concentrated. The resulting crude solid was resuspended in ether and washed with deionized water. The organic layer was dried over Na2SO4 and then concentrated. The crude mixture was purified by flash column chromatography (silica gel, 50-100% etherhexanes gradient) to yield 62-6 as a white solid (1.8 g, 53% yield), mp = 110-111 °C; 3H NMR (300 MHz, CDCk) 5 8.92 (d, J= 4.2 Hz, 1H), 8.35 (d, J= 8.6 Hz, 1H), 8.10 (d, J= 5.2 Hz, 1H), 7.48 - 7.39 (m, 3H), 7.34 (dd, J= 16.3, 7.8 Hz, 2H), 7.24 - 7.12 (m, 2H), 7.00 (d, J= 6.5 Hz, 1H), 6.68 - 6.61 (m, 1H), 6.33 (d, J= 8.3 Hz, 1H), 5.03 (d, J= 6.5 Hz, 1H), 2.83 (q, J= 7.5 Hz, 2H), 1.35 (t, J= 7.5 Hz, 3H); 13C NMR (75 MHz, CDCk) 5 173.3, 156.7, 150.3, 148.4, 147.7, 141.7, 141.2, 137.8, 134.7, 133.8, 132.1, 132.1, 129.8, 127.8, 127.5, 127.0, 125.1, 122.1, 120.7, 114.1, 107.1, 53.7, 27.6, 9.2; IR (neat, thin film): 3384, 2925 2051, 1760, 1599, 1574, 1502, 1479, 1147, 1081, 898, 771 cm'1; HRMS (ESI) calculated for [C24H20CI2N3O2], (M+H)+: 452.0900, found 408.0928.
[0178] 7V-((2,3-Dichlorophenyl)(8-hydroxyquinolin-7-yl)methyl)-N-(pyridin-2- yl)propionamide (62-7)
Figure imgf000099_0001
62-6 62-7
In a dry 10 mL dry round bottomed flask, analog 62-6 (20 mg, 0.041 mmol, 1.0 equiv) was dissolved in dry Et2O (1 mL). Hydrochloric acid (2M in ether, 0.1 mL, 0.2 mmol, 4.4 equiv) was then added, and the reaction was stirred for 2 h at rt. The solution was then concentrated and the crude mixture was purified by flash column chromatography (silica gel, 50% ethyl aceate: hexanes) to yield 62-7 as a white solid (15 mg, 75% yield), mp = 175-176 °C; ’H NMR (300 MHz, CDCh) 5 9.89 (s, 1H), 8.73 (d, J= 4.3 Hz, 1H), 8.11 (d, J= 8.7 Hz, 1H), 7.99 (d, J= 3.4 Hz, 1H), 7.58 (d, J= 7.8 Hz, 1H), 7.50 - 7.36 (m, 4H), 7.29 (d, J= 8.6 Hz, 1H), 7.20 (s, 1H), 7.11 - 7.01 (m, 1H), 6.69 (d, J= 3.8 Hz, 1H), 6.64 - 6.57 (m, 1H), 6.44 (d, J= 8.5 Hz, 1H), 2.30 (q, J= 7.5 Hz, 2H), 1.08 (t, J= 7.5 Hz, 2H); 13C NMR (101 MHz, CDCk) 5 173.0, 155.0, 151.1, 145.7, 141.2, 139.0, 135.9, 133.7, 131.9, 131.6, 130.4, 130.2, 129.2, 128.0, 127.9, 127.7, 126.3, 124.6, 122.1, 113.3, 109.1, 53.5, 27.4, 9.2, 9.0; IR (neat, thin film): 3352, 2981, 1599, 1572 cm4; HRMS (ESI) calculated for [C24H2oC12N2N302]+: 452.0927, found 452.0932.
[0179] 2-((2,3-Dichlorophenyl)(pyridin-2-ylamino)methyl)phenol (62-8) and 4-((2,3-
Diehl orophenyl)(pyridin-2-ylamino)methyl)phenol (S-4)
Figure imgf000100_0001
To a 25 mL round-bottomed flask, 2-aminopyridine (75 mg, 0.796 mmol, 0.93 equiv) and 2,3-dichlorobenzaldehyde (150 mg, 0.857 mmol, 1.0 equiv) was dissolved in absolute ethanol (5 mL). Once fully dissolved, phenol (150 mg, 1.59 mmol, 1.9 equiv) was added to the solution. The mixture was stirred at reflux for 72 h. The reaction was allowed to cool and then concentrated. The crude mixture was purified by flash column chromatography (silica gel, 10-20% acetone: hexanes) to yield 62-8 as a white solid (70 mg, 24% yield), and S-4 as a white solid (73 mg, 25% yield).
[0180] 2-((2,3-Dichlorophenyl)(pyridin-2-ylamino)methyl)phenol (62-8): mp = 187- 188 °C; 'H NMR (300 MHz, CDCl3 5 11.30 (s, 1H), 8.08 (dd, J= 5.2, 0.9 Hz, 2H), 7.68 (d, J= 7.6 Hz, 2H), 7.54 - 7.43 (m, 2H), 7.38 (dd, J= 8.0, 0.9 Hz, 2H), 7.19 (t, J= 7.9 Hz, 2H), 7.15 - 7.06 (m, 4H), 6.87 (dd, J= 8.2, 0.7 Hz, 2H), 6.77 (t, J= 7.5 Hz, 2H), 6.72 - 6.63 (m, 2H), 6.46 (t, J= 7.9 Hz, 4H), 6.31 (d, J= 8.2 Hz, 2H); 13C NMR (75 MHz, CDCk) 5 157.0,
155.8, 146.3, 141.3, 139.0, 133.4, 131.7, 129.5, 128.9, 127.2, 126.4, 126.0, 119.7, 117.1,
113.9, 108.4, 54.8; IR neat film: 3431, 3024, 2981, 2921, 2852, 2686, 2586, 1613, 1598, 1574 cm'1; HRMS (ESI) calculated for [CisHi5C12N2O]+: 345.0558, found 345.055
[0181] 4-((2,3-Dichlorophenyl)(pyridin-2-ylamino)methyl)phenol (S-4): mp = 195- 196 °C; ’H NMR (300 MHz, CDCl) 5 8.07 (d, J= 5.2 Hz, 1H), 7.53 - 7.38 (m, 3H), 7.21 (t, J= 7.9 Hz, 1H), 7.01 (d, J= 8.4 Hz, 2H), 6.68 (dd, J= 7.2, 5.2 Hz, 1H), 6.19 (d, J= 8.3 Hz, 1H), 5.94 (d, J= 4.5 Hz, 1H), 5.32 (s, 1H); 13C NMR (75 MHz, CDCl3 5 156.4, 146.0, 141.1, 139.4, 133.7, 131.5, 130.6, 129.6, 129.2, 127.6, 126.1, 118.6, 116.0, 113.8, 107.0, 58.3; IR neat film: 3433, 3413, 2981, 2799, 2671, 2590, 1604, 1571, 1514, 1502 cm'1; HRMS (ESI) calculated for [CISHI5C12N2O]+: 345.0558, found 345.0556.
[0182] 7-((2,3-Dichlorophenyl)(pyrimidin-2-ylamino)methyl)quinolin-8-ol (62-9)
Figure imgf000101_0001
62-9
To a 50 mL pressure tube, 2-aminopyrimidine (54.6 mg, 0.57 mmol, 1.0 equiv) and 2,3- dichlorobenzaldehyde (100 mg, 0.57 mmol, 1.0 equiv) was dissolved in absolute ethanol (5 mL). Once fully dissolved, 8-hydroxyquinoline (100 mg, 0.69 mmol, 1.2 equiv) was added to the solution. The mixture was stirred at reflux for 72 h. The reaction was allowed to cool to rt and then concentrated. The crude mixture was purified by flash column chromatography (silica gel, 10%-20% acetone: hexanes gradient) to yield 62-9 as a white solid (37.5 mg, 16% yield), mp = 98-99 °C ;XH NMR (300 MHz, CDCh) 5 8.73 (d, J= 5.7 Hz, 1H), 8.25 (d, J= 4.8 Hz, 2H), 8.11 (d, J= 8.3 Hz, 1H), 7.60 (d, J= 7.8 Hz, 1H), 7.46 - 7.34 (m, 3H), 7.29 (s, 1H), 7.18 (t, J= 7.8 Hz, 1H), 7.04 (d, J= 7.7 Hz, 1H), 6.53 (t, J= 4.8 Hz, 1H), 6.35 (d, J= 7.7 Hz, 1H); 13C NMR (75 MHz, CDCh) 5 161.3, 158.1, 149.8, 148.1, 141.9, 138.2, 136.0, 133.4, 131.9, 129.2, 127.8, 127.5, 126.9, 126.6, 121.9, 121.4, 117.5, 111.3, 53.4; IR; HRMS (ESI) calculated for [C2oHi5ChN40i]+: 397.0617, found 397.0619.
[0183] 7-((4,5-Dichloropyridin-3-yl)(pyridin-2-ylamino)methyl)quinolin-8-ol (62-10)
Figure imgf000101_0002
62-10
To a 50 mL pressure tube, 2-aminopyridine (27 mg, 0.29 mmol, 1.0 equiv), 4,5- dichloronicotinaldehyde (50 mg, 0.29 mmol, 1.0 equiv.), and 8-hydroxyquinoline (50 mg, 0.34 mmol, 1.2 equiv) were dissolved in CEECN (30 mL). Following the addition of formic acid (10 pL, 0.23 mmol 0.80 equiv.), the solution was stirred at reflux for 16 h. The solution was allowed to cool to rt, concentrated, and the crude mixture was then directly purified by flash column chromatography (silica gel, 10-20% acetone: hexanes) to give 62-10 as a white solid (6 mg, 5% yield), mp = 102-103 °C; ’H NMR (400 MHz, CDCh) 5 8.83 - 8.67 (m, 2H),
8.52 (s, 1H), 8.13 (d, J= 8.3 Hz, 1H), 8.08 (d, J= 5.0 Hz, 1H), 7.55 - 7.34 (m, 3H), 7.31 (d, J = 8.5 Hz, 1H), 6.74 (d, J= 6.6 Hz, 1H), 6.61 (t, J= 7.2 Hz, 1H), 6.41 (d, J= 8.3 Hz, 1H),
5.53 (d, J = 6.5 Hz, 1H); 13C NMR (75 MHz, CDCh) 5 158.0, 149.7, 148.2, 148.1, 147.0, 143.5, 139.5, 138.0, 136.0, 127.8, 127.4, 124.9, 118.9, 118.1, 117.7, 117.6, 115.0, 113.0, 106.7, 53.2; HRMS (ESI) calcd for [C20H15CI2N4O], (M+H)+: 397.0623, found: 397.0618. [0184] Ethyl 4-((5-chloro-8-hydroxyquinolin-7-yl)(pyridin-2- ylamino)methyl)benzoate (67-4)
Figure imgf000102_0001
67-4
To a 50 mL pressure tube, 2-aminopyridine (26 mg, 0.28 mmol, 1.0 equiv) ethyl 4- formylbenzoate (50 mg, 0.28 mmol, 1.0 equiv.), and 5-chloro-8-hydroxyquinoline (60 mg, 0.34 mmol, 1.2 equiv.) were dissolved in CH3CN (3 mL). Following the addition of formic acid (10 pL, 0.28 mmol 1.0 equiv.), tube was capped and the solution was stirred at reflux for 16 h. The solution was allowed to cool to rt, concentrated, and the crude mixture was then directly purified by flash column chromatography (silica gel, 10-50% acetone: hexanes) to give 67-4 as a white solid (50 mg, 41% yield), mp = 50-51 C; XH NMR (300 MHz, CDCh) 5 8.81 (d, J = 4.3 Hz, 1H), 8.47 (d, J = 8.6 Hz, 1H), 8.10 (d, J= 6.8 Hz, 1H), 8.00 (d, J = 8.1 Hz, 2H), 7.61 (s, 1H), 7.54 (d, J= 8.3 Hz, 3H), 7.39 (t, J= 8.7 Hz, 1H), 6.65 - 6.59 (m, 1H), 6.51 (d, J= 6.5 Hz, 1H), 6.43 (d, J= 8.4 Hz, 1H), 5.59 (d, J= 6.6 Hz, 1H), 4.35 (q, J= 7.1 Hz, 2H), 1.36 (t, J= 7.1 Hz, 3H).; 13C NMR (75 MHz, CDCh) 5 166.3, 157.5, 148.8, 148.4, 148.0, 146.5, 138.8, 137.8, 133.3, 130.0, 129.7, 127.0, 126.4, 125.7, 123.8, 122.6, 121.0, 113.9, 107.4, 60.9, 54.8, 14.31. IR neat film: 3292, 2977, 1717, 1603, 1574, 1509 cm'1 HRMS (ESI) calculated for [C24H2IC1N3O3]+: 434.1278, found 434.1279.
[0185] Ethyl 4-((8-hydroxyquinolin-7-yl)(pyridin-2-ylamino)methyl)benzoate (67-5)
Figure imgf000103_0001
67-5
To a 50 mL pressure tube, 2-aminopyridine (26 mg, 0.28 mmol, 1.0 equiv) ethyl 4- formylbenzoate (50 mg, 0.29 mmol, 1.0 equiv.), and 8-hydroxyquinoline (50 mg, 0.34 mmol, 1.2 equiv.) were dissolved in CH3CN (30 mL). Following the addition of formic acid (10 pL, 0.23 mmol 0.80 equiv.), the solution was stirred at reflux for 16 h. The solution was allowed to cool to rt, concentrated, and the crude mixture was then directly purified by flash column chromatography (silica gel, 10-20% acetone: hexanes) to give 67-5 as a white solid (42 mg, 37% yield), mp = 60-63 0 C; XH NMR (400 MHz, CDCh) 5 8.76 (d, J= 2.6 Hz, 1H), 8.11 (d, J= 10.0 Hz, 1H), 8.05 (d, J= 3.5 Hz, 1H), 7.99 (d, J= 8.3 Hz, 2H), 7.57 (d, J= 8.5 Hz, 2H), 7.52 (d, J= 8.5 Hz, 1H), 7.42 (dd, J= 8.2, 4.4 Hz, 2H), 7.32 (d, J= 8.7 Hz, 1H), 6.65 - 6.58 (m, 1H), 6.57 - 6.46 (m, 2H), 6.28 (s, 1H), 4.34 (q, J= 7.2 Hz, 2H), 1.35 (t, J= 7.2 Hz, 3H); 13C NMR (101 MHz, CDCh) 5 166.3, 157.2, 149.1, 148.3, 146.7, 146.3, 138.7, 138.2, 136.1, 129.9, 129.5, 127.8, 126.9, 126.6, 122.8, 122.0, 118.4, 113.4, 107.7, 60.9, 54.9, 14.3. IR neat film: 3375, 2978, 2929, 1710, 1598, 1502 cm ; HRMS (ESI) calculated for [C24H22N3O3]+: 400.1656, found 400.1656.
[0186] 4-((8-hydroxyquinolin-7-yl)(pyridin-2-ylamino)methyl)benzoic acid (67-1)
Figure imgf000103_0002
To a 20 mL scintillation vial, lithium hydroxide monohydrate (55 mg, 1.3 mmol, 2.7 equiv) was dissolved in 4 mL H2O and 2 mL MeOH. Ester 67-5 (194 mg, 0.49 mmol, 1.0 equiv) was dissolved in 2 mL MeOH and added to the vial. The vial was capped and stirred at rt for 16 h. Then 2M HC1 was added dropwise to the solution until a precipitate persisted. Do not add too much HC1, as the product will dissolve. The precipitate was filtered, washed with 5 mL cold water, 5 mL cold Et2O, and collected to give 67-1 as a white solid (30.2 mg, 17% yield), mp = 140-142 C; ’H NMR (300 MHz, CD3OD) 5 8.77 (d, J= 5.9 Hz, 1H), 8.17 (d, J= 6.7 Hz, 1H), 7.94 (d, J= 8.2 Hz, 3H), 7.56 - 7.40 (m, 5H), 7.32 (d, J= 8.6 Hz, 1H), 6.67 (t, J= 4.3 Hz, 2H), 6.58 (t, J= 6.2 Hz, 1H); 13C NMR (75 MHz, CD3OD) 5 170.6, 159.2, 151.4, 149.8,
148.7, 147.4, 140.1, 139.6, 137.4, 131.7, 131.0, 130.8, 129.6, 128.6, 128.4, 128.0, 125.5, 123.1, 119.3, 114.3, 110.4, 55.2; IR neat film: 3315, 2925, 1652, 1608, 1574 cm'1; HRMS (ESI) calculated for [C22HI8N3O3]+: 372.1343, found 372.1343.
[0187] Ethyl 4-((5-chloro-8-(((trifluoromethyl)sulfonyl)oxy)quinolin-7-yl)(pyridin-2- ylamino)methyl)benzoate (S-5)
Figure imgf000104_0001
In a 50 mL pressure tube, 67-4 (440 mg, 1.0 mmol, 1.0 equiv.) was dissolved in dry DMF (5 mL) under argon atmosphere. N -Phenyl-bis(trifluoromethanesulfonamide) (537 mg, 1.51 mmol, 1.5 equiv.) and potassium carbonate (208 mg, 1.50 mmol, 1.5 equiv.) were added and the solution was 80 °C for 2 h. The solution was then cooled to rt, diluted with EtOAc, and washed three times with H2O. The organic layer was dried over Na2SO4, and then concentrated. The crude mixture was purified by flash column chromatography (silica gel, 30% EtOAc: hexanes) to yield S-5 as a yellow solid (451 mg, 77% yield), mp = 167-169 °C; 3H NMR (400 MHz, CDCh) 5 9.04 (d, J = 4.2 Hz, 1H), 8.55 (d, J = 7.0 Hz, 1H), 8.11 - 7.99 (m, 3H), 7.78 (s, 1H), 7.62 (dd, J= 8.5, 4.3 Hz, 1H), 7.43 (d, J= 8.2 Hz, 3H), 6.66 (dd, J= 7.2, 5.0 Hz, 1H), 6.53 (d, J= 5.1 Hz, 1H), 6.41 (d, J= 8.3 Hz, 1H), 5.25 (d, J= 5.3 Hz, 1H), 4.36 (q, J= 7.1 Hz, 2H), 1.37 (t, J = 7.1 Hz, 3H); 13C NMR (101 MHz, CDCh) 5 166.0,
156.8, 151.6, 148.3, 144.5, 137.9, 135.3, 133.0, 131.7, 130.4, 130.3, 127.4, 126.9, 125.2,
123.3, 114.7, 107.1, 61.1, 54.6, 14.3; IR neat film: 3379, 3066, 3001, 2980, 1725, 1603, 1601 cm'1; HRMS (ESI) calculated for [C25H2OC1F3N305S]+: 566.0759, found 566.0765.
[0188] Ethyl 4-((5-chloroquinolin-7-yl)(pyridin-2-ylamino)methyl)benzoate (67-7)
Figure imgf000104_0002
36%
S-5 67-7 In a 50 mL pressure tube, S-5 (400 mg, 0.75 mmol, 1.0 equiv.), Pd(0Ac)2 (10 mg, 0.045 mmol, 0.06 equiv.), triphenylphosphine (23 mg, 0.088 mmol, 0.12 equiv.), and tri ethyl amine (0.4 mL, 0.51 mmol, 3.8 equiv.) were combined in dry DMF (5 mL) under argon atmosphere. Formic acid (70 pL, 1.86 mmol, 2.5 equiv.) was added, and the reaction was capped and stirred at 60 °C for 16 h. Upon cooling to rt, the reaction mixture was diluted with brine and extracted with EtOAc. The organic layer was then washed three times with brine. The organic layer was dried over Na2SO4 and then concentrated. The crude mixture was purified by flash column chromatography (silica gel, 30% EtOAc:hexanes gradient) to yield 67-7 as a yellow solid (106 mg, 36% yield), mp = 56-57 °C; 3H NMR (300 MHz, CDCh) 5 8.91 (d, J= 4.3 Hz, 1H), 8.52 (d, J= 8.1 Hz, 1H), 8.07 (d, J= 4.4 Hz, 1H), 8.02 (d, J= 8.2 Hz, 3H), 7.60 (s, 1H), 7.51 - 7.43 (m, 3H), 7.39 (d, J= 6.8 Hz, 1H), 6.62 (d, J= 6.6 Hz, 1H), 6.39 (d, J= 8.3 Hz, 1H), 6.22 (d, J= 6.0 Hz, 1H), 5.25 (d, J= 6.1 Hz, 1H), 4.36 (q, J= 7.1 Hz, 2H), 1.37 (t, J = 7.1 Hz, 3H). 13C NMR (75 MHz, CDCh) 5 166.1, 157.1, 151.4, 148.8, 148.2, 146.3, 143.7, 137.6, 132.7, 132.0, 130.2, 130.1, 127.6, 126.6, 126.3, 125.7, 122.0, 114.1, 107.7, 61.0, 59.7, 14.3; IR neat film: 3268, 2925, 2980, 1713, 1601, 1478 cm4; HRMS (ESI) calculated for [C24H2oClN3Na02]+: 440.1136, found 440.1143.
[0189] Ethyl 4-((5-chloroquinolin-7-yl)(pyridin-2-ylamino)methyl)benzoate (67-6)
Figure imgf000105_0001
67-7 67-6
To a 20 mL scintillation vial, lithium hydroxide monohydrate (30 mg, 0.71 mmol, 3.5 equiv) was dissolved in 2 mL H2O and 1 mL MeOH. Ester 67-7 (86 mg, 0.21 mmol, 1.0 equiv) was dissolved in 1 mL MeOH and added to the vial. The vial was capped and stirred at rt for 16 h. Then 2M HC1 was added dropwise to the solution until a precipitate persisted. The precipitate was filtered, washed with 5 mL cold water, 5 mL cold ether, and collected to give 67-6 as a white solid (24.5 mg, 31% yield), mp = 136-137 C; XH NMR (400 MHz, CD3OD) 5 8.84 (d, J = 6.1 Hz, 1H), 8.59 (d, J= 8.5 Hz, 1H), 8.00 (d, J= 8.0 Hz, 2H), 7.97 - 7.87 (m, 2H), 7.70 (d, J= 1.7 Hz, 1H), 7.59 (dd, J= 8.5, 4.2 Hz, 1H), 7.47 (dd, J= 19.4, 7.4 Hz, 3H), 6.72 (d, J = 8.5 Hz, 1H), 6.59 (t, J= 6.2 Hz, 1H), 6.46 (s, 1H); 13C NMR (101 MHz, CD3OD) 5 169.8, 159.0, 152.5, 149.6, 148.1, 147.8, 146.5, 139.3, 134.6, 132.9, 131.7, 131.4, 129.2, 128.4, 127.0, 126.9, 123.6, 114.5, 111.1, 59.9; IR neat film: 3277, 2981, 1671, 1606, 1482 cm'1;
HRMS (ESI) calculated for [C22H17CIN3O2] 390.1004, found 390.1011.
[0190] Ethyl 4-((5-chloro-8-hydroxyquinolin-7-yl)(pyrimidin-2- ylamino)methyl)benzoate (67-9)
Figure imgf000106_0001
67-9
To a 250 mL dry round bottomed flask equipped with a reflux condenser, 2-aminopyrimidine (4.3 g, 45.3 mmol, 1.2 equiv), ethyl 4-formylbenzoate (6.8 g, 38.2 mmol, 1.0 equiv.), and 5- chloro-8-hydroxyquinoline (8.2 g, 45.8 mmol, 1.2 equiv.) were dissolved in CEECN (100 mL). Following the addition of formic acid (1.4 mL, 37.1 mmol 1.0 equiv.), the solution was stirred at reflux for 16 h. The solution was allowed to cool to rt, concentrated, resuspended in acetone. The heterogeneous mixture was filtered, and the precipitate was washed with cold acetone and hexanes to give 67-9 as a white solid (5.2 g, 31% yield), mp = 150-151 °C; TH NMR (300 MHz, CDCh) 5 8.80 (d, J= 4.2 Hz, 1H), 8.49 (d, J= 8.6 Hz, 1H), 8.30 (d, J= 4.8 Hz, 2H), 7.99 (d, J= 7.9 Hz, 2H), 7.55 (dd, J= 19.6, 10.2 Hz, 4H), 6.79 (d, J= 8.2 Hz, 1H), 6.58 (t, J= 4.9 Hz, 1H), 6.39 (d, J= 8.3 Hz, 1H), 4.34 (q, J= 7.2 Hz, 2H), 1.36 (t, J= 7.2 Hz, 3H); 13C NMR (75 MHz, CDCh) 5 166.3, 161.5, 158.2, 148.7, 148.5, 146.6, 138.7, 133.3, 129.8, 129.4, 127.0, 126.8, 125.6, 123.6, 122.5, 120.7, 111.5, 60.9, 54.8, 14.3; IR neat film: 3293, 2978, 1716, 1583, 1496 cm'1; HRMS (ESI) calculated for [C23H20CIN4O3] 390.1004, found 390.1011.
[0191] 4-((5-Chloro-8-hydroxyquinolin-7-yl)(pyrimidin-2-ylamino)methyl)benzoic acid (67-8)
Figure imgf000106_0002
To a 20 mL scintillation vial, lithium hydroxide monohydrate (54 mg, 1.29 mmol, 5.6 equiv.) was dissolved in 2 mL H2O and 1 mL MeOH. Ester 67-9 (100 mg, 0.21 mmol, 1.0 equiv.) was dissolved in 1 mL MeOH and added to the vial. The vial was capped and stirred at rt for 16 h. Then 2M HC1 was added dropwise to the solution until a precipitate persisted. The precipitate was filtered, washed with 5 mL cold water, 5 mL cold ether, and collected to give 67-8 as a brown solid (65 mg, 31% yield), mp = 174-176 °C; ’H NMR (300 MHz, DMS04) 5 10.47 (s, 1H), 8.95 (s, 1H), 8.46 (d, J= 8.5 Hz, 1H), 8.30 (t, J= 7.9 Hz, 3H), 8.03 - 7.79 (m, 3H), 7.79 - 7.62 (m, 1H), 7.46 (d, J= 7.9 Hz, 2H), 7.05 (d, J= 9.4 Hz, 1H), 6.63 (d, J= 4.9 Hz, 1H); 13C NMR (75 MHz, DMSO-d6 ) 5 167.4, 161.6, 158.2, 149.6, 149.1, 146.9, 138.7, 132.5, 129.4, 127.0, 126.6, 125.5, 124.9, 123.0, 111.0, 51.4; IR neat film: 3291, 1673, 1576, 1501 cm4; HRMS (ESI) calculated for [C21H16CIN4O3] 407.0905, found 407.0913.
[0192] Ethyl 4-((8-(propionyloxy)quinolin-7-yl)(pyrimidin-2- ylamino)methyl)benzoate (MMRi71)
Figure imgf000107_0001
67-9 MMRI71
In a 5 mL dry round bottomed flask, 67-9 (5.0 g, 11.5 mmol, 1.0 equiv.) was dissolved in of dry CH2CI2 (100 mL) under argon atmosphere. Potassium carbonate (3.18 g, 23.0 mmol, 2.0 equiv.) was added and the solution was cooled to 0 °C. Propionoyl chloride (1.0 mL, 11.5 mmol, 1.0 equiv.) was then added to the solution. The mixture was allowed to warm to rt and stirred for 1 h. The reaction mixture was then filtered through Celite and washed with CH2CI2. The supernatant was then treated with 1 g DMT-functionalized silica gel and stirred for 15 minutes. The mixture was filtered and concentrated. The resulting crude solid was resuspended in ether and washed with distilled water. The organic layer was dried over Na2SO4 and then concentrated. The crude mixture was purified by flash column chromatography (silica gel, 50% Et2O : hexanes) to yield MMRi71 as a greenish white solid (2.53 g, 48% yield), mp = 178-179 °C; ’H NMR (400 MHz, CDCI3) 5 8.91 (d, J= 2.8 Hz, 1H), 8.50 (d, J= 6.8 Hz, 1H), 8.23 (d, J= 4.8 Hz, 2H), 8.00 (d, J= 8.4 Hz, 2H), 7.54 (s, 1H), 7.50 (dd, J= 8.5, 4.2 Hz, 1H), 7.43 (d, J= 8.1 Hz, 2H), 6.83 (d, J= 7.8 Hz, 1H), 6.57 (t, J= 4.8 Hz, 1H), 6.10 (d, J= 7.8 Hz, 1H), 4.36 (q, J= 7.1 Hz, 2H), 2.68 (q, J= 7.6 Hz, 2H), 1.37 (t, J= 1A Hz, 3H), 1.20 (t, J = 7.5 Hz, 3H); 13C NMR (101 MHz, CDCh) 5 172.2, 166.2, 161.3, 158.1, 151.2, 145.5, 144.6, 142.0, 134.3, 133.0, 130.0, 129.8, 129.0, 127.1, 126.7, 125.7, 122.4, 111.8, 61.0, 53.8, 27.3, 14.3, 9.0. IR neat film: 3270, 2982, 1773, 1716, 1578, 1491 cm'1; HRMS (ESI) calculated for [C26H23ClN4NaO4] 513.1300, found 513.1307.
[0193] (±)-2-chl oro-3-((8-hydroxyquinolin-7-yl)(pyri din-2 -ylamino)methyl) benzonitrile (SC-62-1)
Figure imgf000108_0001
To a 250 mL round bottomed flask equipped with a magnetic stir bar, 2-chloro-3- cyanobenzaldehyde (1.0 g, 6.04 mmol, 1.0 equiv) was added along with 2-aminopyridine (568 mg, 6.04 mmol, 1.0 equiv). The solids were dissolved in 50 mL of absolute ethanol and the mixture was stirred until they fully dissolved, at which point 8-hydroxyquinoline (1.05 g, 7.25 mmol, 1.2 equiv) was added. The reaction flask was heated to 90 °C and refluxed for 24 h during which an off-white solid crashed out. The resulting solid was filtered off giving MMRi-62-1 (1.3 g, 55%) as an off-white powder. Mp = 190-192 °C; ’H NMR (300 MHz, CDCk) 5 8.77 (d, J= 3.0 Hz, 1H), 8.13 (d, J= 7.2 Hz, 1H), 8.09 (d, J= 4.1 Hz, 1H), 7.97 (d, J= 7.9 Hz, 1H), 7.59 (d, J= 6.4 Hz, 1H), 7.50 - 7.26 (m, 6H), 6.70 (d, J= 6.4 Hz, 1H), 6.66 - 6.57 (m, 1H), 6.36 (d, J= 8.4 Hz, 1H), 5.55 (d, J= 6.2 Hz, 1H); 13C NMR (75 MHz, CDCk) 5 157.2, 149.8, 148.3, 141.5, 138.2, 137.7, 136.1, 136.0, 133.0, 132.9, 127.9, 127.4, 127.0, 122.2, 120.6, 117.9, 116.2, 114.3, 114.0, 107.1, 53.6; IR (neat film): 3346, 2923, 2233, 1600, 1571, 1517, 1501 cm’1; HRMS (ESI) calculated for [C22Hi6ClN4O]+: 387.1016, found
387.1007.
[0194] (±)-7-((2-chloro-3-cyanophenyl)(pyridin-2-ylamino)methyl)quinolin-8-yl dimethylcarbamate (SC-62-1 A)
Figure imgf000108_0002
To an oven-dried 500 mL round bottomed flask, SC-62-1 (500 mg, 1.29 mmol, 1.0 equiv) was added. The flask was equipped with a stir bar and 175 mL of dry CH2CI2 was added to the flask under argon to dissolve SC-62-1. Next, K2CO3 (464 mg, 3.36 mmol, 2.6 equiv) was added along with 4-(A,A-dimethyl)pyridine (DMAP) (15.8 mg, 0.129 mmol, 0.1 equiv). Dimethyl carbamyl chloride (0.20 mL, 1.94 mmol, 1.5 equiv) was then added via syringe. After the reaction mixture was stirred at room temperature for 24 h, it was filtered through Celite. The filtrate was stirred for 15 min with 50 mg of DMT functionalized silica gel. This mixture was then filtered through Celite, then concentrated under vacuum. The resulting residue was dissolved in diethyl ether, poured into a separatory funnel and was washed with deionized water. The organic layer was then dried with Na2SO4 and concentrated under vacuum. The resulting solid was purified with flash chromatography (silica gel, 25:75-45:55 acetone : hexanes) to yield 353mg of SC-62-1A as white crystals (60% yield), mp: 120-122 °C; ’H NMR (400 MHz, CDCh) 5 8.85 (d, J= 4.3 Hz, 1H), 8.07 (d, J= 8.3 Hz, 1H), 7.93 (d, J= 5.3 Hz, 1H), 7.75 (d, J= 7.9 Hz, 1H), 7.59 (dd, J= 13.1, 8.1 Hz, 2H), 7.43 (t, J= 7.9 Hz, 1H), 7.40 - 7.25 (m, 3H), 6.67 - 6.56 (m, 2H), 2.97 (s, 3H), 2.83 (s, 3H). 13C NMR (101 MHz, CDCh) 5 156.2, 154.6, 151.2, 142.2, 135.9, 135.9, 135.7, 133.5, 133.4, 132.2, 129.2, 127.5, 125.9, 125.9, 122.0, 116.1, 114.3, 113.8, 37.1, 36.9, 36.8; IR (neat film): 3019, 2973, 2935, 2926, 2853, 2235, 1717, 1614, 1600, 1154 cm'1; HRMS (ESI) Calcd for [C25H2iN5O2Cl]+: 458.1384, found 458.1380.
[0195] (±)-((7-((2-chloro-3-cyanophenyl)(pyridin-2-ylamino)methyl)quinolin-8- yl)oxy)methyl isopropyl carbonate (SC-62- IB)
Figure imgf000109_0001
To an oven dried 100 mL round bottomed flask, SC-62-1 (500 mg, 1.29 mmol, 1.0 equiv) was added. The flask was equipped with a stir bar, and under argon 17 mL of dry DMF was added. Once the solid was dissolved, K2CO3 (357 mg, 2.59 mmol, 2 equiv) was added and the reaction mixture was stirred at room temperature for 10 min. Next, chloromethyl isopropyl carbonate (0.20 mL, 1.55 mmol, 1.2 equiv) was added while stirring under argon and the reaction mixture was heated at 80 °C for 2 h. The hot mixture was then poured into 50 mL of crushed ice and water and was stirred for 30 min.. A resulting white solid was filtered off. The white solid was then dissolved in CH2CI2 and the solution was extracted 5 times with deionized water to remove residual DMF. The organic layer was dried with Na2SO4 and concentrated under vacuum. The resulting solid was recrystallized from methanol to yield 455 mg of SC-62-1B as white crystals (59% yield).
Mp = 83-85 °C; ’H NMR (400 MHz, CDCh) 5 8.89 (s, 1H), 8.15 (d, J= 8.4 Hz, 1H), 8.09 (d, J= 5.0 Hz, 1H), 7.62-7.57 (m, 5H), 7.47 - 7.28 (m, 4H), 6.78 (d, J= 6.4 Hz, 1H), 6.63 (t, J= 6.2 Hz, 1H), 6.48 (d, J= 8.4 Hz, 1H), 6.30 (d, J= 5.6 Hz, 1H), 6.22 (d, J= 5.9 Hz, 1H), 5.32 (d, J = 15.8 Hz, 1H), 4.76 (d, J = 6.4 Hz, 1H), 1.28 - 1.16 (m, 6H); 13C NMR (101 MHz, CDCh) 5 157.2, 153.6, 150.3, 149.9, 148.3, 141.6, 141.5, 137.7, 136.2, 133.1, 133.0, 131.4, 129.6, 127.1, 126.5, 124.1, 121.8, 116.2, 114.5, 114.1, 107.4, 92.9, 53.4, 21.7, 21.6; IR neat film: 2982, 2234, 1747, 1598, 1260, 1051 cm'1; HRMS (ESI) calcd for [C27H24C1N4O4]+: 503.1486, found: 503.1483.
[0196] (±)-7-((2-chloro-3-cyanophenyl)(pyridin-2-ylamino)methyl)quinolin-8-yl propionate (SC-62- 1C)
Figure imgf000110_0001
SC-62-1 (500 mg, 1.30 mmol, 1 equiv) in a 25 mL round-bottomed flask equipped with a stir bar and under argon was dissolved in CH2CI2 (10 mL). K2CO3 (257 mg, 2.6 mmol, 2 equiv) was added and reaction mixture was then cooled to 0 °C. Propionyl chloride (0.113 mL, 1.29 mmol, 1 eq.) was added via syringe and the reaction mixture was stirred at rt for 1 h (the solution turns clear). The resulting reaction mixture was then filtered through Celite, washing with CH2CI2, and concentrated under vacuum. The crude residue was dissolved in Et2O, placed in a separatory funnel, and was washed with water. The the organic layer was dried over Na2SO4, filtered and concentrated to yield 300 mg of SC-62-1C as white crystals (52% yield). ’H NMR (400 MHz, CDCh) 5 8.83 (dd, J= 4.2, 1.7 Hz, 1H), 8.08 (dd, J= 8.4, 1.7 Hz, 1H), 8.04 (d, J= 4.6 Hz, 1H), 7.76 - 7.69 (m, 1H), 7.67 - 7.55 (m, 2H), 7.40 - 7.32 (m, 2H), 7.30 (d, J= 7.8 Hz, 1H), 7.23 (s, 1H), 6.63 (d, J= 5.3 Hz, 1H), 6.58 (t, J= 7.2 Hz, 1H), 6.40 - 6.30 (m, 1H), 5.09 (s, 1H), 2.66 - 2.42 (m, 2H), 1.05 (t, J = 7.5 Hz, 3H); 13C NMR (101 MHz, CDCh) 5 173.1, 156.8, 151.1, 147.7, 146.0, 141.4, 140.7, 137.9, 136.0, 135.9, 133.3,
133.2, 132.1, 129.1, 127.3, 126.0, 125.2, 122.1, 116.1, 114.5, 114.3, 107.9, 53.2, 30.3, 27.3, 8.9; IR (neat film): 3227, 3106, 2889, 2158, 1773, 1602, 1521 cm'1. HRMS (ESI) calcd for [C25H2OC1N402]+: 443.1275, found 443.1267.
[0197] (±)-7-((l -methyl- IT/-pyrazol-5-yl)(pyridin-2-ylamino)methyl)quinolin-8-ol
(SC-62-2)
Figure imgf000111_0001
SC-62-2 |
To a 50 mL pressure tube, 2-aminopyridine (128 mg, 1.36 mmol, 1.0 equiv) and 1-methyl- l/Z-pyrazole-5-carbaldehyde (0.13 mL, 1.36 mmol, 1.0 equiv) was dissolved in 5 mL of absolute ethanol. Once fully dissolved, 8-hydroxyquinoline (237 mg, 1.63 mmol, 1.2 equiv) was added to the solution. The mixture was stirred at reflux for 72 h. The reaction was allowed to cool to rt and then concentrated. The crude mixture was purified by flash column chromatography (silica gel, 10%-30% acetone :hexanes gradient) to yield SC-62-2 as a beige solid (124 mg, 28% yield). Mp= 188-140 °C; XH NMR (300 MHz, CDCh) 5 8.79 (d, J= 4.4 Hz, 1H), 8.15 - 8.05 (m, 2H), 7.50 - 7.35 (m, 4H), 7.32 (d, J= 8.6 Hz, 1H), 6.61 (t, J= 7.9 Hz, 2H), 6.48 (d, J= 8.3 Hz, 1H), 6.14 (d, J= 1.9 Hz, 1H), 5.54 (d, J= 6.7 Hz, 1H), 3.88 (s, 3H); 13C NMR (75 MHz, CDCh) 5 157.0, 149.4, 148.4, 147.6, 142.8, 138.4, 138.1, 138.0, 136.0, 128.0, 126.4, 122.0, 121.4, 118.4, 113.9, 107.7, 105.2, 47.1, 36.9; IR neat film: 3308, 3029, 1716, 1603, 1576, 1503 cm'1; HRMS (ESI) calculated for [Ci9Hi7N5NaO]+: 354.1325, found 354.1330.
[0198] 7-((2,3-dichlorophenyl)(pyridin-2-ylamino)methyl)-5-(2- morpholinoethyl)quinolin-8-ol (SC-62-3)
Figure imgf000112_0001
To a 50 mL pressure tube, 2-aminopyridine (12 mg, 0.13 mmol, 1.0 equiv) and 2,3- dichlorobenzaldehyde (22 mg, 0.13 mmol, 1.0 equiv) was dissolved in 5 mL of absolute ethanol. Once fully dissolved, 5-(2-morpholino)quinoline-8-ol (38 mg, 0.15 mmol, 1.2 equiv) was added to the solution. The mixture was stirred at reflux temperature for 72 h. The reaction was allowed to cool and then concentrated. The crude mixture was purified by flash column chromatography (silica gel, 3-9% CH3OH]CH2Cl gradient) to give SC-62-3 as a white solid (19 mg, 29%). Mp = 83-85 °C; ’H NMR (300 MHz, CD2CI2) 5 8.76 (d, J= 4.2 Hz, 1H), 8.34 (d, J= 8.5 Hz, 1H), 8.03 (q, J= 1.8 Hz, 1H), 7.57 (dd, J= 7.8, 1.5 Hz, 1H), 7.48 (dd, J= 8.6, 4.2 Hz, 1H), 7.43 - 7.34 (m, 2H), 7.25 (s, 1H), 7.20 (t, J= 7.9 Hz, 1H), 6.70 (d, J= 6.5 Hz, 1H), 6.62 - 6.53 (m, 1H), 6.38 (d, J= 8.4 Hz, 1H), 5.43 (d, J= 6.5 Hz, 1H), 3.61 (t, J= 4.7 Hz, 4H), 3.07 (t, 2H), 2.56 (t, J= 7.7 Hz, 2H), 2.45 (t, J= 4.7 Hz, 4H); 13C NMR (75 MHz, CD2CI2) 5 158.1, 148.8, 148.7, 148.4, 142.5, 139.1, 138.0, 133.8, 133.4, 129.8, 127.8, 127.7, 127.6, 127.5, 127.1, 122.3, 121.3, 114.2, 107.8, 67.4, 60.3, 54.3, 30.3, 29.6; IR neat film: 3334, 2961, 2859, 2815, 1602, 1574 cm'1; HRMS (ESI) calculated for [C27H27C12N4O2]+: 509.1506, found 509.1511.
[0199] 7-((2,3-dichlorophenyl)(pyridin-2-ylamino)methyl)-5-(2- hydroxyethyl)quinolin-8-ol (SC-62-4)
Figure imgf000112_0002
To a 50 mL pressure tube, 2-aminopyridine (35 mg, 0.37 mmol, 1.0 equiv.) and 2,3- dichlorobenzaldehyde (65 mg, 0.37 mmol, 1.0 equiv.) was dissolved in 5 mL of absolute
- I l l - ethanol. Once fully dissolved, 5-(2-hydroxyethyl)quinolin-8-ol (84 mg, 0.44 mmol, 1.2 equiv.) was added to the solution. The mixture was stirred at reflux temperature for 72 h. The reaction was allowed to cool to rt and then concentrated. The crude mixture was purified by flash column chromatography (silica gel, 10-50% acetone: hexanes) to give SC-62-4 as a white solid (52 mg, 32%). Mp = 98-100 °C; ’H NMR (300 MHz, CDCk) 5 8.77 (d, J= 2.8 Hz, 1H), 8.33 (d, J= 8.6 Hz, 1H), 8.04 (d, J= 4.9 Hz, 1H), 7.62 (d, J= 7.8 Hz, 1H), 7.52 - 7.35 (m, 3H), 7.33 (s, 1H), 7.20 (t, J= 7.9 Hz, 1H), 6.69 - 6.58 (m, 2H), 6.41 (d, J= 8.4 Hz, 1H), 6.10 (s, 1H), 3.86 (t, J = 5.5 Hz, 2H), 3.32 - 2.99 (m, 2H); 13C NMR (75 MHz, CDCh) 5
157.0, 148.6, 147.8, 146.7, 141.2, 138.6, 138.6, 133.5, 132.8, 131.9, 129.5, 127.7, 127.3,
126.9, 125.1, 121.8, 120.2, 113.6, 107.3, 63.1, 53.8, 34.7; IR neat film: 3325, 2936, 2867,
1601, 1503 cm'1; HRMS (ESI) calculated for [C23H2oC12N302]+: 440.0927, found 440.0932.
[0200] 2-(7-((2,3-dichlorophenyl)(pyridin-2-ylamino)methyl)-8-hydroxyquinolin-5- yl)acetic acid (SC-62-5)
Figure imgf000113_0001
To a 50 mL pressure tube, 2-aminopyridine (58 mg, 0.62 mmol, 1.0 equiv) and 2,3- dichlorobenzaldehyde (108 mg, 0.62 mmol, 1.0 equiv) was dissolved in 5 mL of absolute ethanol. Once fully dissolved, 2-(8-hydroxyquinolin-5-yl)acetic acid (142 mg, 0.77 mmol, 1.2 equiv) was added to the solution. The mixture was stirred at reflux temperature for 72 h. Upon cooling to rt a precipitate formed. The precipitate was filtered and washed with cold ethanol to yield SC-62-5 as a white solid (160 mg, 57%). Mp = 233-235 °C; ’H NMR (300 MHz, DMSO-d6) 5 12.33 (bs, 1H), 9.90 (bs, 1H), 8.86 (d, J= 4.1 Hz, 1H), 8.35 (d, J= 8.5 Hz, 1H), 7.89 (d, J= 5.1 Hz, 1H), 7.67 - 7.47 (m, 2H), 7.45 - 7.24 (m, 5H), 7.20 (s, 1H), 7.00 (d, J= 7.1 Hz, 1H), 6.66 (d, J= 8.4 Hz, 1H), 6.48 (t, J= 6.0 Hz, 1H), 3.87 (s, 2H); 13C NMR (75 MHz, DMSO-td6) 5 173.2, 158.0, 150.2, 148.4, 147.9, 144.0, 138.8, 137.1, 133.8, 132.4, 131.5, 129.4, 128.5, 128.3, 127.8, 127.2, 122.8, 122.1, 121.6, 112.8, 109.5, 51.2, 38.0; IR neat film: 3338, 3258, 1693, 1679, 1617, 1523 cm'1; HRMS (ESI) calculated for [C23Hi8C12N3O3]+: 454.0720, found 454.0718. [0201] 2-(7-((2,3-dichlorophenyl)(pyridin-2-ylamino)methyl)-8-hydroxyquinolin-5- yl)acetonitrile (SC-62-6)
Figure imgf000114_0001
SC-62-6
To a 50 mL pressure tube, 2-aminopyridine (60 mg, 0.64 mmol, 1.0 equiv.) and 2,3- dichlorobenzaldehyde (112 mg, 0.64 mmol, 1.0 equiv.) was dissolved in 5 mL of absolute ethanol. Once fully dissolved, 2-(8-hydroxyquinolin-5-yl)acetonitrile (142 mg, 0.77 mmol, 1.2 equiv) was added to the solution. The mixture was stirred at reflux temperature for 72 h. The reaction was allowed to cool and then concentrated. The crude mixture was purified by flash column chromatography (silica gel, 10-30% acetone: hexanes) to give SC-62-6 as a yellow solid (23 mg, 8%). Mp = 107-108 °C; XH NMR (300 MHz, CDCk) 5 8.83 (d, J= 4.2 Hz, 1H), 8.23 (d, J= 8.6 Hz, 1H), 8.08 (d, J= 5.0 Hz, 1H), 7.61 - 7.51 (m, 2H), 7.48 - 7.37 (m, 3H), 7.21 (td, J= 7.9, 1.3 Hz, 1H), 6.72 (d, J= 5.8 Hz, 1H), 6.62 (t, 1H), 6.38 (d, J= 8.4 Hz, 1H), 5.60 (d, J= 5.4 Hz, 1H), 3.95 (s, 2H); 13C NMR (75 MHz, CDCk) 5 157.3, 150.3, 148.4, 147.9, 141.0, 138.6, 138.0, 133.6, 131.9, 131.6, 129.6, 128.1, 127.3, 126.8, 125.7, 122.6, 121.1, 117.3, 116.0, 113.9, 107.1, 53.5, 20.8; IR (neat film): 3368, 3072, 3022, 2958, 2225, 1600, 1573, 1504 cm'1; HRMS (ESI) calculated for [C23Hi7C12N4O]+: 435.0774, found 435.0780.
[0202] 7-((2,3-dichlorophenyl)(pyridin-2-ylamino)methyl)quinolin-8-yl propionate (SC-62-7)
Figure imgf000114_0002
In a 10 mL dry round bottomed flask, MMRI62 (100 mg, 0.25 mmol, 1.0 equiv) was dissolved in 1 mL of dry DCM under argon atmosphere, triethylamine (0.1 mL, 0.72 mmol, 2.9 equiv) was added and the solution was cooled to 0 °C. Propionoyl chloride (0.1 mL, 1.15 mmol, 4.5 equiv) was then added to the solution. The mixture was allowed to warm to rt and stirred for 2 h. The reaction mixture was then diluted with DCM and washed with water. The organic layer was dried over Na2SO4 and then concentrated. The crude mixture was purified by flash column chromatography (silica gel, 50% EtOAc:hexanes gradient) to yield SC-62-7 as a white solid (17 mg, 13% yield). Mp = 68-69 °C; ’H NMR (300 MHz, CDCl) 5 8.88 (dd, J= 4.2, 1.7 Hz, 1H), 8.52 (d, J= 3.0 Hz, 1H), 8.05 (d, J= 6.6 Hz, 1H), 7.70 (s, 1H), 7.44 - 7.29 (m, 4H), 7.14 - 7.03 (m, 2H), 6.94 (d, J= 7.9 Hz, 1H), 2.94 - 2.75 (m, 2H), 2.11 (q, J = 7.4 Hz, 2H), 1.27 (t, J= 7.4 Hz, 3H), 1.06 (t, J= 7.4 Hz, 3H). 13C NMR (75 MHz, CDCh) 5 173.4, 153.5, 150.5, 148.7, 138.0, 135.8, 132.9, 129.4, 128.6, 127.3, 126.9, 124.7, 123.1, 121.7, 77.2, 28.5, 27.4, 9.3, 9.0. IR; HRMS (ESI) calculated for [C27H24ChN3O3]+: 508.1189, found 508.1181.
[0203] 2 -chloro-3-((8-hydroxyquinolin-7-yl)(pyrimidin-2- ylamino)methyl)benzonitrile (SC-62- ID)
Figure imgf000115_0001
To a 50 mL pressure tube, 2-aminopyrimidine (55 mg, 0.57 mmol, 1.0 equiv) and 2-chloro-3- formylbenzonitrile (95 mg, 0.57 mmol, 1.0 equiv) was dissolved in 5 mL of absolute ethanol. Once fully dissolved, 8-hydroxyquinoline (100 mg, 0.69 mmol, 1.2 equiv) was added to the solution. The mixture was stirred at reflux temperature for 72 h. The reaction was allowed to cool and then concentrated. The crude mixture was purified by flash column chromatography (silica gel, 20-100% acetone: hexanes, impure product elutes with 100% acetone, repurify with 50% EtOAC: hexanes) to give SC-62-1D as a white solid (20 mg, 9%). Mp = 205-206 C; XH NMR (300 MHz, Chloroform-^ 5 8.75 (dd, J= 4.2, 1.5 Hz, 1H), 8.28 (d, J= 4.8 Hz, 2H), 8.14 (d, J= 8.3 Hz, 1H), 8.02 (d, J= 7.9 Hz, 1H), 7.58 (d, J= 7.7 Hz, 1H), 7.48 - 7.40 (m, 2H), 7.34 (q, J= 8.8, 8.3 Hz, 2H), 6.98 (d, J= 7.5 Hz, 1H), 6.58 (d, J= 4.8 Hz, 1H), 6.45 (d, J= 7.6 Hz, 1H).13C NMR (75 MHz, CDCh) 5 161.09, 158.12, 149.86, 148.28, 141.66, 138.16, 136.05, 135.88, 132.73, 127.94, 127.74, 126.88, 122.17, 120.40, 117.77, 116.28, 114.26, 111.58, 53.40.; IR neat film: 3388, 3234.78, 2235, 1595, 1579, 1535 cm'1; HRMS (ESI) calculated for [C2iHi4ClN5NaO]+: 410.0779, found 410.0780.
[0204] 7-((2,3-dichlorophenyl)(pyrimidin-2-ylamino)methyl)quinolin-8-yl propionate
Figure imgf000116_0001
In a 5 mL dry round bottomed flask, 1-27 (11 mg, 28 pmol, 1.0 equiv) was dissolved in 5 mL of dry CH2CI2 under argon atmosphere. Potassium carbonate 7.8 mg, 57 pmol, 2.0 equiv) was added and the solution was cooled to 0 °C. Propionoyl chloride (4.0 pL, 46 pmol, 1.6 equiv) was then added to the solution. The mixture was allowed to warm to rt and stirred for 1 h. The reaction mixture was then filtered through celite and washed with CH2CI2. The supernatant was then treated with 5 mg DMT -functionalized silica gel and stirred for 15 minutes. The mixture was filtered and concentrated. The resulting crude solid was resuspended in ether and washed with distilled water. The organic layer was dried over Na2SO4 and then concentrated. The crude mixture was purified by flash column chromatography (silica gel, 20-50% EtOAc: hexanes) to yield 1-21 as a white solid (11.3 mg, 87% yield). Mp = 92-93 C; XH NMR (400 MHz, CDCh) 5 8.89 (dd, J= 4.2, 1.7 Hz, 1H), 8.29 (d, J= 4.8 Hz, 2H), 8.15 (d, J= 10.1 Hz, 1H), 7.79 (d, J= 8.0 Hz, 1H), 7.73 - 7.64 (m, 2H), 7.47 - 7.32 (m, 3H), 7.00 (d, J= 6.7 Hz, 1H), 6.63 (t, J= 4.8 Hz, 1H), 5.98 (d, J= 7.0 Hz, 1H), 2.73 - 2.57 (m, 2H), 1.16 (t, J= 7.5 Hz, 3H). 13C NMR (101 MHz, CDCh) 5 172.55, 160.68, 158.08, 150.91, 145.93, 141.27, 140.66, 136.11, 135.91, 133.20, 132.76, 131.58, 129.04, 127.23, 125.91, 125.36, 122.00, 116.00, 114.56, 111.94, 52.59, 27.34, 8.95; IR neat film: 3234, 2981, 2234, 1762, 1583, 1501 cm HRMS (ESI) calculated for [C24HisClN5NaO2]+: 466.1041, found 466.1035.
[0205] 2 -chloro-3-((8-hydroxyquinolin-7-yl)(pyrimidin-2- ylamino)methyl)benzonitrile (SC-62- IF)
Figure imgf000117_0001
To a 50 mL pressure tube, 2-aminopyrazine (55 mg, 0.57 mmol, 1.0 equiv) and 2-chloro-3- formylbenzonitrile (95 mg, 0.57 mmol, 1.0 equiv) was dissolved in 5 mL of absolute ethanol. Once fully dissolved, 8-hydroxyquinoline (100 mg, 0.69 mmol, 1.2 equiv) was added to the solution. The mixture was stirred at reflux temperature for 72 h. The reaction was allowed to cool and then concentrated. The crude mixture was purified by flash column chromatography (silica gel, 20-100% acetone: hexanes, impure product elutes with 100% acetone, repurify with 50% EtOAC: hexanes) to give SC-62-1F as a tan solid (27 mg, 9%). Mp = 99-100 C; TH NMR (300 MHz, CDCh) 5 8.79 (d, J= 5.8 Hz, 1H), 8.16 (d, J= 8.4 Hz, 1H), 8.01 - 7.92 (m, 3H), 7.85 (d, J= 2.7 Hz, 1H), 7.64 - 7.56 (m, 1H), 7.47 (dd, J= 8.3, 4.3 Hz, 1H), 7.44 - 7.38 (m, 2H), 7.37 - 7.31 (m, 2H), 6.83 (d, J= 6.7 Hz, 1H), 5.75 (d, J= 6.8 Hz, 1H); 13C NMR (75 MHz, CDCh) 5 153.2, 149.8, 148.4, 142.2, 142.1, 141.2, 140.9, 138.1, 137.2, 136.2, 136.2, 136.0, 133.9, 133.0, 132.7, 132.3, 128.0, 127.5, 127.0, 122.3, 120.1, 118.0, 116.1, 114.4, 53.3. ; IR (neat film): 3364, 3055, 2233, 1588, 1536, 1500 cm'1; HRMS (ESI) calculated for [C2iHi4ClN5NaO]+: 410.0779, found 410.0783.
[0206] 7-((2-chloro-3-cyanophenyl)(pyrazin-2-ylamino)methyl)quinolin-8-yl propionate (SC-62- 1G)
Figure imgf000117_0002
SC-62-1 F SC-62-1 G
In a 5 mL dry round bottomed flask, SC-62-1F (15 mg, 39 pmol, 1.0 equiv) was dissolved in 5 mL of dry CH2CI2 under argon atmosphere. Potassium carbonate 14 mg, 0.1 mmol, 2.6 equiv) was added and the solution was cooled to 0 °C. Propionoyl chloride (5.0 pL, 57 pmol, 1.5 equiv) was then added to the solution. The mixture was allowed to warm to rt and stirred for 1 h. The reaction mixture was then filtered through celite and washed with CH2CI2. The supernatant was then treated with 5 mg DMT -functionalized silica gel and stirred for 15 min. The mixture was filtered and concentrated. The resulting crude solid was resuspended in ether and washed with distilled water. The organic layer was dried over Na2SO4 and then concentrated. The crude mixture was purified by flash column chromatography (silica gel, 20%-50% EtOAc: hexanes) to yield SC-62-1G as a white solid (11.3 mg, 87% yield). Mp = 97-99 C; ’H NMR (400 MHz, CDCh) 5 8.91 (d, J= 5.9 Hz, 1H), 8.17 (dd, J= 8.4, 1.6 Hz, 1H), 7.96 (d, J= 6.2 Hz, 2H), 7.89 (d, J= 2.9 Hz, 1H), 7.81 (d, J= 7.8 Hz, 1H), 7.71 - 7.64 (m, 2H), 7.49 - 7.39 (m, 2H), 7.19 (s, 1H), 6.79 (d, J= 5.3 Hz, 1H), 5.18 (d, J= 5.5 Hz, 1H), 2.61 (dp, J= 24.5, 8.5 Hz, 2H), 1.10 (t, J= 7.5 Hz, 3H). 13C NMR (101 MHz, CDCl3 5
173.4, 153.0, 151.1, 141.9, 141.2, 140.1, 136.01, 135.97, 134.2, 133.3, 132.8, 132.7, 131.5,
129.2, 127.3, 126.1, 125.3, 122.2, 115.9, 114.7, 52.8, 27.3, 8.9; IR neat film: 3366, 2981, 2235, 1761, 1714, 1587, 1504 cm4; HRMS (ESI) calculated for [C24HisClN5NaO2]+: 466.1041, found 466.1035.
[0207] 7-((3-azido-2-chlorophenyl)(pyridin-2-ylamino)methyl)quinolin-8-ol (SC-62-
Figure imgf000118_0001
SC-62-11
To a 25 mL round-bottomed flask, 2-aminopyridine (166 mg, 1.76 mmol, 1.0 equiv.) and 3- azido-2-chlorobenzaldehyde (255 mg, 1.76 mmol, 1.0 equiv.) was dissolved in 7 mL of absolute ethanol. Once fully dissolved, 5-chloro-8-hydroxyquinoline (962 mg, 5.27 mmol, 3.0 equiv.) was added to the solution. The mixture was stirred at reflux for 72 h. The reaction was allowed to cool to rt and a precipitate had formed. The supernatant was decanted, and the solid was slurried with 20 mL of hexanes then filtered. The solid was further washed with hexanes to yield SC-62-11 as a yellow solid (200 mg, 28%). Mp = 177-179 °C; ’H NMR (300 MHz, CDCh) 5 8.78 (d, J= 2.8 Hz, 1H), 8.12 (d, J= 8.2 Hz, 1H), 8.07 (d, J= 4.3 Hz, 1H), 7.53 - 7.36 (m, 4H), 7.29 (t, J= 8.0 Hz, 2H), 7.14 (d, J= 8.0 Hz, 1H), 6.68 (d, J= 6.5 Hz, 1H), 6.65 - 6.59 (m, 1H), 6.40 (d, J= 8.2 Hz, 1H), 5.78 (s, 1H); 13C NMR (75 MHz, CDCh) 5 157.5, 149.8, 148.2, 148.1, 141.5, 138.2, 137.9, 136.0, 127.8, 127.4, 127.1, 124.8, 124.5, 122.0, 121.3, 118.5, 117.7, 113.7, 106.7, 53.2; IR neat film: 3349, 2891, 2116, 1599, 1571, 1517 cm'1; HRMS (ESI) calculated for [C2iHi5ClN6O]+: 403.1083, found 403.1069.
[0208] 7-(((4-azidopyridin-2-yl)amino)(2,3-dichlorophenyl)methyl)quinolin-8-ol
(SC-62-9)
Figure imgf000119_0001
SC-62-9
To a 50 mL pressure tube, 2-amino-4-azidopyridine (40 mg, 0.30 mmol, 1.0 equiv.) and 2,3- dichlorobenzaldehyde (52 mg, 0.30 mmol, 1.0 equiv.) were dissolved in 5 mL of absolute ethanol. Once fully dissolved, 8-hydroxyquinoline (43 mg, 0.30 mmol, 1.0 equiv.) was added to the solution. The mixture was stirred at rt until a clear yellow solution was obtained. The tube was then capped and stirred at reflux for 72 h. The reaction was allowed to cool to rt and a precipitate had formed. The supernatant was decanted, and the solid was slurried with 20 mL of hexanes then filtered. The solid was purified by flash column chromatography (silica gel, 1-3% CELOELCELCh gradient) to yield SC-62-9 as a yellow brown solid (10 mg, 8%). Mp = 139-141 °C; XH NMR (300 MHz, CDCh) 5 8.77 (d, J= 4.1 Hz, 1 H), 8.13 (d, J= 8.2 Hz, 1 H), 8.01 (d, J= 5.3 Hz, 1 H), 7.55 (d, J= 7.6 Hz, 1 H), 7.49 - 7.12 (m, 6 H), 6.67 (d, J = 6.4 Hz, 1 H), 6.33 (d, J= 5.3 Hz, 1 H), 5.95 (s, 1 H), 5.65 (d, J= 6.4 Hz, 1 H); 13C NMR (75 MHz, CDCh) 5158.9, 150.1, 149.7, 148.3, 141.2, 138.2, 136.0, 133.6, 132.0, 129.5, 127.9, 127.2, 127.1, 126.8, 122.1, 120.9, 117.8, 104.7, 96.2, 53.4; IR neat film: 3228, 3019, 2115, 1600, 1568, 1504 cm'1; HRMS (ESI) calculated for [C2iHi50iN6Cl2]+: 437.0679, found 437.0700.
[0209] 5-(2-azidoethyl)-7-(((4-azidopyridin-2-yl)amino)(2,3- dichlorophenyl)methyl)quinolin-8-ol (SC-62-10)
Figure imgf000120_0001
To a 10 mL pressure tube, 2-amino-4-azidopyridine (19 mg, 0.14 mmol, 1.0 equiv.) and 2,3- dichlorobenzaldehyde (24 mg, 0.14 mmol, 1.0 equiv.) was dissolved in 1 mL of absolute ethanol. Once fully dissolved, 5-(2-azidoethyl)quinoline-8-ol (29 mg, 0.14 mmol, 1.0 equiv.) was added to the solution. The mixture was stirred at reflux for 72 h. The reaction was allowed to cool to rt and then concentrated. The crude mixture was purified by flash column chromatography (silica gel, 10-30% acetone: hexanes) to give SC-62-10 as a brown solid (10 mg, 14%). Mp = 103-105 °C; XH NMR (300 MHz, CDCh) 5 8.80 (d, J = 4.5 Hz, 1H), 8.28 (d, J= 7.4 Hz, 1H), 8.00 (d, J= 5.6 Hz, 1H), 7.57 (d, J= 7.7 Hz, 1H), 7.50 (dd, J= 8.5, 4.1 Hz, 1H), 7.41 (d, J= 8.1 Hz, 1H), 7.32 (s, 1H), 7.21 (t, J= 7.9 Hz, 1H), 6.66 (d, J= 6.5 Hz, 1H), 6.34 (d, J= 5.8 Hz, 1H), 5.99 (s, 1H), 5.90 (s, 1H), 3.51 (t, J= 7.2 Hz, 2H), 3.32 - 3.06 (m, 2H); 13C NMR (75 MHz, CDCh) 5 158.6, 150.5, 149.1, 149.0, 148.0, 141.0, 138.6,
133.6, 132.2, 131.9, 129.6, 127.8, 127.3, 126.8, 126.5, 124.4, 122.1, 120.3, 104.8, 96.4, 53.9, 52.0, 31.2; IR neat film: 3353, 2924, 2854, 2109, 1694, 1596, 1567, 1504 cm'1; HRMS (ESI) calculated for [C23H18ClN9O]+: 506.1006, found 506.1017.
[0210] 4 -azido-7V-((5-(2-azidoethyl)-8-methoxyquinolin-7-yl)(2,3- dichlorophenyl)methyl)pyridin-2-amine (SC-62-12)
Figure imgf000120_0002
In a 10 mL pressure tube, SC-62-10 (20 mg, 0.040 mmol, 1.0 equiv) was dissolved in 1 mL of dry CH3CN under argon atmosphere. K2CO3 (7 mg, 0.044 mmol, 1.1 equiv), followed by CH3I (3 pL, 0.044 mmol, 1.1 equiv) were added to the solution. The mixture was stirred at reflux for 2 h. Upon cooling to rt, the reaction mixture was diluted with EtOAc and washed with water. The organic layer was dried over Na2SO4 and then concentrated. The crude mixture was purified by flash column chromatography (silica gel, 10%-20% acetone: hexanes gradient) to yield SC-62-12 as a pale yellow solid (9 mg, 44% yield). Mp = °C; TH NMR (300 MHz, CDCI3) 5 8.95 (d, J = 5.8 Hz, 1H), 8.29 (d, J = 7.1 Hz, 1H), 8.01 (d, J = 5.6 Hz, 1H), 7.46 (dd, J = 8.6, 4.1 Hz, 1H), 7.42 (d, J = 7.9 Hz, 2H), 7.34 (s, 1H), 7.20 (t, J = 7.9 Hz, 1H), 6.73 (d, J = 6.6 Hz, 1H), 6.34 (d, J = 5.6 Hz, 1H), 5.94 (d, J = 1.8 Hz, 1H), 5.35 (d, J = 6.7 Hz, 1H), 4.01 (s, 3H), 3.54 (t, J = 7.1 Hz, 2H), 3.23 (q, J = 6.9 Hz, 2H); 13C NMR (75 MHz, CDCk) 5 158.7, 153.4, 150.1, 149.8, 149.3, 143.2, 141.4, 133.7, 132.03, 131.95, 131.6, 129.7, 129.6, 127.9, 127.3, 127.1, 126.9, 121.4, 104.8, 96.3, 62.6, 54.0, 51.8, 31.6, 29.7; IR neat film: cm'1 HRMS (ESI) calculated for [C24Hi9ChN9O]+: 520.1162, found 520.1164.
EXAMPLE 7
[0211] The following example provides a description of uses of the compounds of the present disclosure.
[0212] Compounds used.
Figure imgf000121_0001
Figure imgf000122_0001
Figure imgf000123_0001
Figure imgf000124_0001
Figure imgf000125_0001
[0213] The compounds promote ferroptosis, a cell death associated with reactive oxygen species caused by dysregulation of cellular iron. A marker of ferroptosis is degradation of the cellular iron storage protein, ferratin heavy chain 1 (FTH1). The parent phenolic compounds, e.g. SC-62-1, causes total loss of FTH1 at concentrations >2.5 pM. [0214] Phenolic ester pro-drug SC-62- 1C retained the ability to induce apoptotic cleavage of PARP at comparable capacity as SC-62-1 at 10 pM in MDM4-high A375 cells, which was accompanied by MDM4 degradation. With regard to FTH1 degradation, SC-62-1 has strong and acute effect: causing total loss of FTH1 at concentrations >2.5 pM, while the effect of SC-62-1C on FTH1 was attenuated and exhibited good linear dose-response. A375 cells treated with 10 pM SC-62-1C had a FTH1 level higher than cells treated with 2.5 pM SC-62-1C. Therefore, SC-62-1C is predicted to have less acute toxicity and better PK profile in term of MDM4-targeting in vivo.
[0215] In preliminary in vivo experiments, it was empirically observed that prodrug SC-62- IB administration is tolerated at the injection site of mice much better than the parent phenol, SC-62-1.
[0216] Efficacy data.
Figure imgf000126_0001
[0217] Formulation: It is easier to make in vivo formulations of the prodrugs (phenol acylated) compared to the parent active compounds due to solubility (e.g. in 10% N-methyl- 2-pyrrolidone and 10% castor oil in water) and probably lack of ambient metal chelation in the case of the prodrugs. [0218] In vivo (mice): SC-62-1B was found to be non-toxic to mice at 80 mg/kg
(injected 3 days/week over 2 weeks). MMRi71 was found to be non-toxic to mice at 36 mg/kg (injected 3 days/week over 2 weeks). SC-62-1 at 50 mg/kg was modestly effective at reducing tumor spread in mice and caused modest weight loss (9% higher than control) (injected 3 days/week over two weeks). [0219] Prodrugs with hydrolysable functionalities that mask the phenol moiety (e.g. esters, carbamates) are much more active than non-hydrolyzable phenol derivatives (ethers), indicating that hydrolysable phenolic ethers convert to the active phenol more easily in cells: [0220] For reference, the ICso NALM6 of phenol MMRi-62 is ca. 0.12 mM and the
IC50 NALM6 of phenol SC-62-1 is ca. 0.25 pM. [0221] The ICso NALM6 of phenolic methyl ether (non-hydrolyzable) 62-3 is ca. 12 pM.
[0222] The IC50 NALM6 of (hydrolysable) propionate ester 62-6 is ca. 1 pM.
[0223] The IC50 NALM6 of carbamate SC-62-8 (hydrolysable) is ca. 2.6 pM. EXAMPLE 8
[0224] The following example provides a proposed synthesis of boronic carbamates.
[0225] Scheme. Boronic carbamate synthesis.
Figure imgf000127_0001
It is expected this synthesis can be adapted to make the following compounds:
Figure imgf000127_0002
[0226] Although the present disclosure has been described with respect to one or more particular embodiments and/or examples, it will be understood that other embodiments and/or examples of the present disclosure may be made without departing from the scope of the present disclosure.

Claims

CLAIMS:
1. A compound having the following structure:
Figure imgf000128_0001
R3 is a hydrogen, a substituted or unsubstituted alkyl group, a halogen group, a substituted or unsubstituted amino group, or a substituted or unsubstituted alkoxy group;
R4 is a hydrogen, a substituted or unsubstituted alkyl group, a halogen group, a substituted or unsubstituted amino group, or a substituted or unsubstituted alkoxy group; , wherein R1 is a hydrogen, an acyl group, an alkyl carbonate group, an
Figure imgf000128_0003
acetal group, carbonate group, carbamate group, a ketal group, or an aminal group;
Figure imgf000128_0002
cyano group, a substituted or unsubstituted ester group, a carboxylic acid group, a sulfonyl group (SO2R), or a substituted or unsubstituted amide group; wherein R7 is a hydrogen, a halogen group, a substituted or unsubstituted alkyl group, a nitro group, or an azide group; and wherein A is oxygen, sulfur, or a substituted or unsubstituted nitrogen and Z is N, CH, or CR, where R is an alkyl group or an aryl group;
R11 is H or an acyl group;
R12 is H or a substituted or unsubstituted alkyl group;
X1 is -CH- or nitrogen;
X2 is -CH- or nitrogen;
X3 is -CH- or nitrogen; and
X4 is -CH- or nitrogen.
2. The compound of claim 1, wherein R5 is not -OH.
Figure imgf000129_0001
3. The compound of claim 1, wherein R5 is -OH and wherein R6 is chosen from CN
Figure imgf000129_0002
4. The compound of claim 1, wherein
Figure imgf000129_0003
5. The compound of claim 1, wherein X1 is nitrogen, X2 is -CH- X3 is -CH- and X4 is nitrogen.
6. The compound of claim 1, wherein X1 is -CH- X2 is -CH- X3 is -CH-, and X4 is nitrogen.
7. The compound of claim 1, wherein R3 is chlorine.
8. The compound of claim 1, wherein R3 is hydrogen.
9. The compound of claim 1, wherein R5 is chosen from
Figure imgf000129_0004
Figure imgf000130_0001
wherein M+ is a cation chosen from Li+, Na+, K+, and Ca2+.
10. The compound of claim 9, wherein R5 is
Figure imgf000130_0002
Figure imgf000130_0003
aim 1, wherein when the structure is
Figure imgf000130_0004
and at least of R3, R7, and R2 is not hydrogen, chlorine, bromine, or iodine.
13. The compound of claim 1, wherein the compound does not have the following structure:
Figure imgf000131_0001
14. The compound of claim 1, wherein R5 can be converted to -OH in a cell.
15. The compound of claim 1, wherein the structure is:
Figure imgf000132_0001
Figure imgf000133_0001
Figure imgf000134_0001
Figure imgf000135_0001
Figure imgf000136_0001
Figure imgf000137_0001
Figure imgf000138_0001
Figure imgf000139_0001
Figure imgf000140_0001
Figure imgf000141_0001
Figure imgf000142_0001
Figure imgf000143_0001
Figure imgf000144_0001
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_0001
d of claim 1, wherein the structure is chosen from
Figure imgf000157_0001
17. The compound of claim 1, wherein the structure is chosen from
Figure imgf000157_0002
18. The compound of claim 1, wherein the structure is
Figure imgf000157_0003
19. The compound of claim 1, wherein the structure is
Figure imgf000158_0001
20. A composition comprising a compound of claim 1.
21. The composition of claim 20, further comprising a pharmaceutically acceptable carrier.
22. A method of inducing apoptosis or ferropotosis in a cell, comprising: administering a therapeutically effective amount of the compound of claim 1 to a subject.
23. The method of claim 22, wherein at least some of the administered compounds undergoes a formation within the subject, and wherein the reaction converts R5 to -OH.
24. The method of claim 23, wherein the transformation occurs within a cancer cell.
25. A method of a subject having cancer or suspected of having cancer, comprising: administering a therapeutically effective amount of the compound of claim 1 to a subject.
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