WO2024097805A1 - Activateurs de pseudokinase de type domaine de kinase de lignée mixte (mlkl) - Google Patents

Activateurs de pseudokinase de type domaine de kinase de lignée mixte (mlkl) Download PDF

Info

Publication number
WO2024097805A1
WO2024097805A1 PCT/US2023/078420 US2023078420W WO2024097805A1 WO 2024097805 A1 WO2024097805 A1 WO 2024097805A1 US 2023078420 W US2023078420 W US 2023078420W WO 2024097805 A1 WO2024097805 A1 WO 2024097805A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
mhz
nmr
hydroxyl
mlkl
Prior art date
Application number
PCT/US2023/078420
Other languages
English (en)
Inventor
Alexei Degterev
Anantha Lakshmi Duddupudi
Gregory Cuny
Shirin SHABAHANG
Siddharth Balachandran
Seungheon Lee
Original Assignee
Trustees Of Tufts College
University Of Houston System
Institute For Cancer Research D/B/A The Research Institute Of Fox Chase Cancer Center
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Trustees Of Tufts College, University Of Houston System, Institute For Cancer Research D/B/A The Research Institute Of Fox Chase Cancer Center filed Critical Trustees Of Tufts College
Publication of WO2024097805A1 publication Critical patent/WO2024097805A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/70—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings condensed with carbocyclic rings or ring systems
    • C07D239/72—Quinazolines; Hydrogenated quinazolines
    • C07D239/78—Quinazolines; Hydrogenated quinazolines with hetero atoms directly attached in position 2
    • C07D239/84—Nitrogen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the field of the invention relates to substituted heterocycles as necroptosis activators
  • the field of the invention relates to substituted heterocycles as mixed lineage kinase domain like pseudokinase (MLKL) activating agents for the treatment of cell proliferation diseases and disorders such as cancer.
  • MLKL mixed lineage kinase domain like pseudokinase
  • substituted heterocycles which may be utilized as MLKL activating agents.
  • the disclosed heterocycles may be used in pharmaceutical compositions and methods for treating cell proliferative disorders such as cancer.
  • the disclosed substituted heterocycles may include compounds having formula of:
  • the disclosed compounds may exhibit one or more biological activities.
  • the disclosed compounds may activate necroptosis.
  • the disclosed compounds may activate MLKL.
  • the compounds activate MLKL at a concentration of less than about 100 pM, 50 ⁇ M, 10 pM, 1 pM, 0.1 pM, 0.05 pM, 0.01 pM, 0.005 pM, 0.001 pM, or less.
  • compositions comprising the disclosed compounds and a suitable pharmaceutical carrier, excipient, or diluent.
  • suitable pharmaceutical carrier excipient, or diluent.
  • the disclosed pharmaceutical compositions may comprise an effective amount of the compound for inhibiting the growth of cancer cells when administered to a subject in need thereof.
  • the methods may include administering the disclosed compounds or pharmaceutical compositions comprising the disclosed compounds to a subject in need thereof for example, to a subject having cancer.
  • the disclosed compounds or pharmaceutical compositions comprising the disclosed compounds may be administered with additional therapeutic agents, optionally in combination, in order to treat cell proliferative diseases and disorders.
  • Cell proliferative diseases and disorders treated by the disclosed methods may include, but are not limited to, cancers selected from the group consisting of multiple myeloma, leukemia, non-small cell lung cancer, colon cancer, cancer of the central nervous system, melanoma, ovarian cancer, renal cancer, prostate cancer, and breast cancer,
  • Figure 1 depicts a schematic showing of MLKL in the pathway of necroptosis.
  • Figure 2 show's that necroptosis is mediated by sequential activation of upstream sensors followed by RIPK3 and MLKL.
  • MLKL activators bypass RIPK3 activation, which is frequently lost in cancer cells.
  • Figure 3 shows that potent activators of MLKL were identified that bind the MLKL pseudokinase pocket.
  • Cell death induced by SS-1-127 is dependent on MLKL expression and independent from RIPK3.
  • Viability of transformed mouse embryonic fibroblasts with genetic deletions of RIPK3 and MLKL genes and treated with SS-1-127 are shown. Affinities of select MLKL activators against recombinant human RIPK1, RIPK3, and MLKL were determined by DiscoveRx.
  • Figure 4 show's that UH15*22 efficiently activates human MLKL.
  • Mikl mouse embryonic fibroblasts (MEFs) were stably infected with lentiviral vector encoding 3 XFL AG-human MLKL under the control of the Doxocyclin (Dox)-inducible promoter.
  • Cells were treated with 10 ng/rnl of Dox for 6 hr followed by the treatment with indicated concentrations of UH15-22 for 24 hr.
  • Cel I viability at the end of treatment was determined using CellTiter-Glo Viability assay (Promega)
  • Figure 5 shows the putative mechanism of action.
  • the pseudokinase domain is a target of the present disclosure.
  • Figure 6 demonstrates that MLKL activators induce necroptosis directly through binding MLKL and independently of RIPK3 activity. Plots show cell viability (%) versus MLKL mutant expressed.
  • Mikl"'' mouse embryonic fibroblasts (MEFs) were stably infected with lentiviral vector encoding mouse MLKL -FLAG under the control of the Doxocyclin (Dox)-inducible promoter. Cells conditionally expressing wild type and mutants of MLKL have been generated.
  • M272W mutant of MLKL is active but predicted not to bind to MLKL activators.
  • S345A/S347A mutant of MLKL lacks RIPK3 phosphorylation sites.
  • Cells were treated with 50 ng/ml of Dox for 6 hr followed by MLKL activators (left panels) or TNFa/IDN6556 (right panels). The latter combination is a canonical inducer of necroptosis through RIPK3 activation. 5 uM ALD-6-85 and SS-1-127 were used in top and bottom panels, respectively. After 24 hr, cell viability at the end of treatment was determined using CellTiter-Glo Viability assay (Promega).
  • FIG. 7 shows that E239Q mutation lowers the ICso of SS-1-127, demonstrated by a plot of percent viability versus Logio(drug, nM) of SS-1-127 and UH15-22.
  • Glu239 residue plays a key role in blocking MLKL activation.
  • E239Q mutation reduces non-covalent bonds (e.g. ionic-dipole interactions), thus resulting in partially active MLKL protein, inducing limited cell death upon expression.
  • SS-1-127 displays much improved activity when combined with E239Q mutation compared to wild type MLKL, suggesting a much better fit for SS- 1 -127 with the active conformation of the MLKL compared to UH15-22 which induces cell death to the same degree in the cells expressing wild type and E239Q mutant MLKL.
  • Mlkt'' mouse embryonic fibroblasts (MEFs) were stably infected with lentiviral vector encoding wi id type and E239Q mutant of mouse MLKL -FLAG under the control of the Doxocyclin (Dox)-inducible promoter.
  • Dox Doxocyclin
  • Figure 8 shows the results of a standard protocol NCI60 screening for SS-1-127 at 10 pM
  • Figure 9 shows the induction of cell death by various molecules in cells in the presence and absence of MLKL.
  • MLKL-/- mouse embryonic fibroblasts were stably transduced to allow’ Doxycycline-inducible re-expression of mouse MLKL.
  • the same cells were either left without Doxycycline (MLKL-/- condition, left three columns) or pre-treated with 50 ng/ml Doxycycline (MLKL+/+ condition, right three columns) for 6 hr. After that cells were treated with the indicated concentrations of drugs for 24 hr Cell viability was determined using CellTiter-Glo assay.
  • Necroptosis is a regulated inflammatory cell death pathway first described by Degterev, et al. in 2005 (Degterev A, Huang Z, Boyce M, Li Y, Jagtap P, Mizushima N, Cuny GD, Mitchison TJ, Moskowitz MA, Yuan J. Chemical inhibitor of nonapoptotic cell death with therapeutic- potential for ischemic brain injury. Nat Chem Biol 2005; 1(2): 112-9). It is initiated by the innate immune sensors tumor necrosis factor receptor 1 (TNFRI), toll like receptor 3 (TLR3), Z-DNA binding protein 1 (ZBP1 ), and driven by receptor interacting protein kinase 3 (RIPK3).
  • TNFRI tumor necrosis factor receptor 1
  • TLR3 toll like receptor 3
  • ZBP1 Z-DNA binding protein 1
  • RIPK3 receptor interacting protein kinase 3
  • RIPK3 then phosphorylates mixed lineage kinase domain like pseudokinase (MLKL) that oligomerizes, translocates to the plasma membrane, and triggers lytic cell death releasing large quantities of immune-stimulatory Danger Associated Molecular Patterns (DAMPs) (Figure 1) (Krysko O, Aaes TL, Kagan VE, D'Herde K, Bachert C, Leybaert L, Vandenabeele P, Krysko DV. Necroptotic cell death in anti-cancer therapy. Immunol Rev. 2017;280(l):207-19).
  • DAMPs Danger Associated Molecular Patterns
  • necroptosis mediators RIPK3 and MLKL in mRNA or protein form into tumor mass were directly shown to promote anticancer immunity (Snyder AG, Hubbard NW, Messmer MN, et al. Intratumoral activation of the necroptotic pathway components RIPK1 and RIPK3 potentiates antitumor immunity. Sci Immunol 2019,4, Van Hoecke L, Riederer S, Saelens X, Sutter G, Rojas JJ. Recombinant viruses delivering the necroptosis mediator MLKL induce a potent antitumor immunity in mice.
  • the compounds described herein are capable of directly activating the downstream executioner of necroptosis, MLKL, thus bypassing a requirement for RIPK3 ( Figure 2).
  • the Examples demonstrate that these molecules induce cell death in a manner that is completely dependent on MLKL and independent of RIPK3 and do so by directly binding to MLKL ( Figure 3).
  • the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising” in that these latter terms are “open” transitional terms that do not limit claims only to the recited elements succeeding these transitional terms.
  • the term “consisting of,” while encompassed by the term “comprising,” should be interpreted as a “closed” transitional term that limits claims only to the recited elements succeeding this transitional term.
  • the term “consisting essentially of,” while encompassed by the term “comprising,” should be interpreted as a “partially closed” transitional term which permits additional elements succeeding this transitional term, but only if those additional elements do not materially affect the basic and novel characteristics of the claim.
  • a “subject” may be interchangeable with “patient” or “individual” and means an animal, which may be a human or non-human animal, in need of treatment.
  • a “subject in need of treatment” may include a subject having a disease, disorder, or condition that is responsive to therapy with the presently disclosed compounds.
  • a “subject in need of treatment” may include a subject having a cell proliferative disease, disorder, or condition such as cancer (e.g., cancers such as multiple myeloma, leukemia, non-small cell lung cancer, colon cancer, cancer of the central nervous system, melanoma, ovarian cancer, renal cancer, prostate cancer, and breast cancer).
  • a “subject in need of treatment” may include a subject having a cell proliferative disease, disorder, or condition such as cancer and/or that may be treated by administering an effective amount of an agent that modulates MLKL activity.
  • the compounds described herein, and any of the compositions described herein comprising the same, can be used to treat tumors, including “cold tumors” and “hot tumors ”
  • the composition may be used to treat "cold tumors.”
  • Cold tumors are defined as a tumor that is not likely to trigger an immune response. Cold tumors tend to be surrounded by cells that are able to suppress the immune response and keep T cells from attacking the tumor cells and killing them. Cold tumors usually do not respond to immunotherapy. Inunune- excluded tumors and immune-desert tumors can be described as cold tumors. Cold tumors are also known as non -inflamed. There are several classes of cold tumors including tumors that demonstrate a lack of T cell activation or priming (e.g., melanoma), a lack of tumor antigens (e.g., prostate tumors), dense stroma (e g. pancreatic cancer). Most cancers of the breast, ovary, prostate, pancreas, and brain are considered cold tumors.
  • T cell activation or priming e.g., melanoma
  • tumor antigens e.g., prostate tumors
  • dense stroma e g
  • the composition may be used to treat, tumors that are likely to trigger an immune response or that are not considered cold tumors.
  • the presently disclosed composition may be used to sensitize tumor to an immunotherapy agent.
  • the disclosed composition may be used to sensitize a cold tumor to an immunotherapy agent.
  • the phrase “effective amount” shall mean the amount, of the compound that, provides the desired effect.
  • the effective amount may be a drug dosage that provides the specific pharmacological response for which the drug is administered in a significant number of subjects in need of such treatment.
  • An effective amount of a drug that is administered to a particular subject in a particular instance will not always be effective in treating the conditions/diseases described herein, even though such dosage is deemed to be a therapeutically effective amount by those of skill in the art.
  • modulate means decreasing or inhibiting activity and/or increasing or augmenting activity.
  • modulating MLKL activity may mean increasing or augmenting MLKL activity and/or decreasing or inhibiting MLKL activity.
  • the compounds disclosed herein may be administered to modulate MLKL activity.
  • an asterisk or a plus sign “+” may be used to designate the point of attachment for any radical group or substituent group.
  • alkyl as contemplated herein includes a straight-chain or branched alkyl radical in all of its isomeric forms, such as a straight or branched group of 1-12, 1-10, or 1-6 carbon atoms, referred to herein as C1 -C12 alkyl, C1-C10 -alkyl, and C1-C6-alkyl, respectively.
  • alkylene refers to a diradical of an alkyl group (e.g., -(CH2)n- where n is an integer such as an integer between 1 and 20)
  • n is an integer such as an integer between 1 and 20
  • An exemplary/ alkydene group is -CH2CH2-.
  • haloalkyl refers to an alkyl group that is substituted with at least one halogen.
  • haloalkyl refers to an alkyl group that is substituted with at least one halogen.
  • heteroaikyi refers to an “alkyl” group in which at least one carbon atom has been replaced with a heteroatom (e.g., an O, N, or S atom).
  • a heteroatom e.g., an O, N, or S atom
  • One type of heteroalkyl group is an “alkoxy” group.
  • alkenyl refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond, such as a straight or branched group of 2-12, 2-10, or 2-6 carbon atoms, referred to herein as C2-C12-alkenyl, C2-C10-alkenyl, and C2-C6-alkenyl, respectively.
  • alky ny 1 refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon triple bond, such as a straight or branched group of 2-12, 2-10, or 2-6 carbon atoms, referred to herein as C2-C12-alkynyl, C2-C10-alkynyl, and C2-C6-alkynyl, respectively.
  • cycloalkyl refers to a monovalent saturated cyclic, bicyclic, or bridged cyclic (e.g., adamantyl) hydrocarbon group of 3-12, 3-8, 4-8, or 4-6 carbons, referred to herein, e.g., as “C4-8-cycloalkyl,” derived from a cycloalkane.
  • cycloalkyl groups are optionally substituted at one or more ring positions with, for example, alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido, amidino, amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halo, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonato, phosphinato, sulfate, sulfide, sulfonamide, sulfonyl or thiocarbonyl.
  • the cycloalkyl group is not substituted, i.e., it is unsubstituted.
  • cycloheteroalkyl refers to a monovalent saturated cyclic, bicyclic, or bridged cyclic hydrocarbon group of 3-12, 3-8, 4-8, or 4-6 carbons in which at least one carbon of the cycloalkane is replaced with a heteroatom such as, for example, N, O, and/or S.
  • cycloalkylene refers to a cycloalkyl group that is unsaturated at one or more ring bonds.
  • partially unsaturated carbocyclyl refers to a monovalent cyclic hydrocarbon that contains at least one double bond between ring atoms where at least one ring of the carbocyclyl is not aromatic.
  • the partially unsaturated carbocyclyl may be characterized according to the number oring carbon atoms.
  • the partially unsaturated carbocyclyl may contain 5-14, 5-12, 5-8, or 5-6 ring carbon atoms, and accordingly be referred to as a 5-14, 5-12, 5-8, or 5-6 membered partially unsaturated carbocyclyl, respectively.
  • the partially unsaturated carbocyclyl may be in the form of a monocyclic carbocycle, bicyclic carbocycle, tricyclic carbocycle, bridged carbocycle, spirocyclic carbocycle, or other carbocyclic ring system.
  • exemplary partially unsaturated carbocyclyl groups include cycloalkenyl groups and bicyclic carbocyclyl groups that are partially unsaturated.
  • partially unsaturated carbocyclyl groups are optionally substituted at one or more ring positions with, for example, alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido, amidino, amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonato, phosphinato, sulfate, sulfide, sulfonamide, sulfonyl or thiocarbonyl.
  • the partially unsaturated carbocyclyl is not substituted, i.e., it is unsubstituted.
  • aryl is art-recognized and refers to a carbocyclic aromatic group. Representative aryl groups include phenyl, naphthyl, anthracenyl, and the like.
  • aryl includes polycyclic ring systems having two or more carbocyclic rings in which two or more carbons are common to two adjoining rings (the rings are “fused rings”) wherein at least one of the rings is aromatic and, e.g., the other ring(s) may be cycloalkyls, cycloalkenyls, cycloalkynyls, and/or aiyls.
  • the aromatic ring may be substituted at one or more ring positions with, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, carboxylic acid, -C(O)alkyl, -CChalkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamido, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aryl or heteroaryl moieties, -CFa, -CN, or the like.
  • halogen azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, carboxylic
  • the aromatic ring is substituted at one or more ring positions with halogen, alkyl, hydroxyl, or alkoxyl. In certain other embodiments, the aromatic ring is not substituted, i.e., it is unsubstituted. In certain embodiments, the aryl group is a 6-10 membered ring structure.
  • heterocyclyl and “heterocyclic group” are art-recognized and refer to saturated, partially unsaturated, or aromatic 3- to 10-membered ring structures, alternatively 3 -to 7-membered rings, whose ring structures include one to four heteroatoms, such as nitrogen, oxygen, and sulfur.
  • the number of ring atoms in the heterocyclyl group can be specified using 5 Cx-Cx nomenclature where x is an integer specifying the number of ring atoms
  • a C3-C7 heterocyclyl group refers to a saturated or partially unsaturated 3- to 7-membered ring structure containing one to four heteroatoms, such as nitrogen, oxygen, and sulfur.
  • the designation “C3-C7” indicates that the heterocyclic ring contains a total of from 3 to 7 ring atoms, inclusive of any heteroatoms that occupy a ring atom position.
  • amine and “amino” are art-recognized and refer to both unsubstituted and substituted amines (e.g., mono-substituted amines or di-substituted amines), wherein substituents may include, for example, alkyl, cycloalkyl, heterocyclyl, alkenyl, and aryl.
  • alkoxy or “alkoxy!” are art-recognized and refer to an alkyl group, as defined above, having an oxygen radical attached thereto.
  • Representative alkoxy groups include methoxy, ethoxy, tert-butoxy and the like.
  • O-alkylamine refers to ⁇ O ⁇ (CR 1 R 2 )m-NR 3 R 4 , wherein m is an integer between 1-6, and R 1 , R 2 , R 3 and R 4 , for example, are each independently hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, and aryl. R 3 and R 4 , together with the nitrogen they are attached to, may also be optionally joined to form a 4-8 membered cycloalkyl.
  • heterocycloalkyl refers to a monovalent saturated cyclic, bicyclic, or bridged cyclic hydrocarbon group of 3-12, 3-8, 4-8, or 4-6 carbons in which at least one carbon of the cycloalkane is replaced with a heteroatom such as, for example, N, O, and/or S(O)n, wherein n is an integer of 0-2.
  • “Four to seven membered heterocycloalkyl” refers to a heterocycloalkyl containing from four to seven atoms, including one or more heteroatoms, in the cyclic moiety of the heterocycloalkyl.
  • heterocycloalkyls examples include azetidinyl, oxetanyl, thietanyl, dihydrofuranyl, tetrahydrofuranyl, dihydrothiophenyl, tetrahydrothiophenyl, pyrrolinyl, pyrrolidinyl, imidazolinyl, imidazolidinyl, pyrazolinyl, pyrazolidinyl, thiazolinyl, isothiazolinyl, thiazolidinyl, isothiazolidinyl, dihydropyranyl, piperidinyl, morpholinyl, piperazinyl, azepinyl, oxepinyl, and diazepinyl.
  • the heterocycloalkyl described herein may be fused with a cycloalkyl, an aryl, or a heteroaryl, as described herein.
  • an “ether” is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as may be represented by one of -O-alkyl, -O-alkenyl, -O-alkynyl, and the like.
  • carbonyl refers to the radical -C(O)-.
  • oxo refers to a divalent oxygen atom -O-.
  • carboxyamide refers to the radical -C(O)NRR', where R and R' may be the same or different. R and R', for example, may be independently alkyl, aryl, arylalkyl, cycloalkyl, formyl, haloalkyl, beteroaryl, or heterocyclyl.
  • carboxy refers to the radical -COOH or its corresponding salts, e.g. -COONa, etc.
  • amide or “amido” or “amidy!” as used herein refers to a radical of the form - R 1 C(O)N(R 2 )-, -R 1 C(O)N(R 2 )R 3 -, -C(O)NR 2 R 3 , or -C(O)NH 2 , wherein R 1 , R 2 and R 3 , for example, are each independently alkoxy, alkyl, alkenyl, alkynyl, amide, amino, aiyl, arylalkyl, carbamate, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydrogen, hydroxyl, ketone, or nitro.
  • the compounds of the disclosure may contain one or more chiral centers and/or double bonds and, therefore, exist as stereoisomers, such as geometric isomers, enantiomers or diastereomers.
  • stereoisomers when used herein consist of all geometric isomers, enantiomers or diastereomers. These compounds may be designated by the symbols “R” or “5,” or “+” or depending on the configuration of substituents around the stereogenic carbon atom and or the optical rotation observed.
  • Stereoisomers include enantiomers and diastereomers.
  • compositions comprising, consisting essentially of, or consisting of an enantiopure compound, which composition may comprise, consist essential of, or consist of at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of a single enantiomer of a given compound (e.g., at least about 99% of an If enantiomer of a given compound).
  • MLKL activating compounds target a pseudokinase domain of MLKL to form the act, closed conformation ( Figure 5).
  • the disclosed compounds may be used in pharmaceutical compositions and methods for treating cell proliferation diseases and disorders, such as cancer.
  • the method for treating a subject having cancer includes administering a compound having a formula of:
  • R 6 may be independently O-alkylamine, alkyl-substituted piperazinyl, hydroxyl, alkoxy, -
  • R 0 are independently OCH2CHrN(Et)2, methyl-substituted piperazinyl, hydroxyl, methoxy, -
  • Exemplary' R 2 include CN, NO2, Cl, Br, CfoOjOH, ( t ())O-CH . CH?, hydroxyl, or H.
  • R J may be H, halo, amino, CN, alkoxy, hydroxyl, or amido.
  • Exemplary' R? include H, F, Cl, NH2, CN, OCH?, hydroxyl, or NHC(-O)CH 3 .
  • R' may be alkoxy, hydroxyl, or H.
  • Exemplary' R 5 include hydroxyl, methoxy, or H.
  • A may be N or CH
  • Z may be N of CH.
  • X-Y may be NR 4 -C( ⁇ O) or CH :::: CH and R 4 may be an alkyl or heterocycloalkyl.
  • Exemplary R 4 include CH? or piperidinyl.
  • the compound is of formula
  • the compound is of formula
  • the compound is selected from:
  • the cancer may be melanoma.
  • the methods may include administering an effective amount of a pharmaceutical composition including a compound of the above formulae for activating MLKL activity in the subject.
  • the method for treating a subject in need of a compound having MLKL activating activity including administering to the subject a compound as disclosed herein.
  • a method wherein administering the compound to the subject induces necroptosis in cancer cells within the subject.
  • the administration of the compound to the subject may induce release of danger associated molecular patterns (DAMPs)
  • DAMPs danger associated molecular patterns
  • pharmaceutical composition for the treatment of a subject in need of a compound having MLKL activating activity In one embodiment the pharmaceutical composition includes an effective amount of the compound.
  • the compound is not
  • the compound has MLKL activating activity.
  • the compound binds a MLKL pseudokinase domain.
  • compositions including of any of the aforementioned compounds or a pharmaceutically acceptable salt thereof and may include a pharmaceutically carrier, excipient, or diluent.
  • the formulae of the compounds disclosed herein should be interpreted as encompassing all possible stereoisomers, enantiomers, or epimers of the compounds unless the formulae indicates a specific stereoisomer, enantiomer, or epimer.
  • the formulae of the compounds disclosed herein should be interpreted as encompassing salts, esters, amides, or solvates thereof of the compounds Use of the Disclosed Compounds for activating MLKL Activity
  • the disclosed compounds may exhibit one or more biological activities.
  • the disclosed compounds may activate MLKL activity by altering the inactive “open’’ conformation of MLKL pseudokinase domain to an active “closed” conformation ( Figure 5).
  • human MLKL may be activated by the compounds disclosed herein.
  • MEF cells expressing human MLKL have been generated. Because mouse RIPK3 cannot phosphorylate human MLKL, human MLKL in these cells can only be activated in a manner that does not involve RIPK3 activity. Indeed, UH15-22 induces death in these cells, indicating its ability to induce MLKL-dependent death in a. manner that is independent of upstream RIPK3 step in the pathway.
  • the compounds disclosed herein induce necroptosis directly through binding MLKL.
  • the activity of ALD-6-85 was tested in the cells expressing either wild type MLKL or mutant expressing a bulky Trp residue in place of M272 gatekeeper residue. This mutation is expected to disrupt binding of the drug to MLKL.
  • ALD-6-85 induces reduced death in the cells expressing M272W MLKL compared to wild type protein. To ensure that observed reduction in killing is not due to the intrinsically lower pro-death activity of M272W mutant, cells were also stimulated with I ⁇ F 1DX. inducing necroptosis through a canonical TNFR1-RIPK1-RIPK3-MLKL pathway.
  • M272W mutation had no effect on activation of necroptosis.
  • ALD-6-85 induces death through direct binding to MLKL as it displays reduced activity against a mutant with reduced compound binding.
  • MEFs expressing S345A, S347A double mutant of mouse MLKL These residues are directly phosphorylated by RIPK3 and their mutation to the non-phopshorylatable alanines leads to the loss of killing by TNF+IDN. In contrast, it has minor effect on killing by SS-1-127, consistent with this molecule directly activating MLKL and not requiring upstream RIPK3 activity.
  • Glu239 residue plays a key role in blocking MLKL activation.
  • E239Q mutation reduces the ability of this residue to participate in non-covalent interactions (e g., ionic-dipole), thus resulting in partially active MLKL protein, inducing limited cell death upon expression.
  • SS- 1-127 displays much improved activity when combined with E239Q mutation compared to wild type MLKL, suggesting a much better fit for SS-1-127 with the active conformation of the MLKL compared to UH 15 -22 which induces cell death to the same degree in the cells expressing wild type and E239Q mutant MLKL.
  • the compounds can inhibit growth or kill tumor cells.
  • the compounds disclosed herein may be assessed using the standard protocol described by the NCI- 60 Human Tumor Cell Lines Screen, a well -documented activity screening that utilizes 60 different human tumor cell lines to identify and characterize novel compounds with growth inhibition or killing of tumor cell line (Figure 8).
  • the disclosed compounds inhibit tumor cell growth (preferably by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% at a concentration of less than about 100 p M, 50 pM, 10 pM, 1 pM, 0.1 pM, 0.05 pM, 0.01 pM, 0.005 pM, 0.001 pM, or less). Concentration ranges also are contemplated herein, for example, a. concentration range bounded by end-point concentrations selected from 0.001 pM, 0.005 pM, 0.01 pM, 0.5 pM, 0.1 pM, 1.0 pM, 10 pM, and 100 pM.
  • the disclosed compounds may be effective in inhibiting cell proliferation of cancer cells, by activating necroptosis and by activating MLKL activity
  • the disclosed compounds may be effective in inhibiting cell proliferation of one or more types of primary cancer cells as well as cancer cell lines, including: multiple myeloma cells, such as MM.
  • leukemia cells such as CCRF-CEM, HL-60(TB), MOLT-4, RPMI-8226, M0LM14, KU812 and SR
  • non-small lung cancer cells such as A549/ATCC, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H322M, NCI-H460 and NCI-H522
  • colon cancer cells such as COLO 205, HCC-2998, HCT-116, HCT- 15, HT29, KM 12 and SW-620
  • CNS CNS: SF-268, SF-295, SF-539, SNB-19, SNB-75 and 11251
  • bladder cancer cells such as D-BLC1, melanoma cancer cells, such as LOX IMVI, MALME-3M, M14, MDA-MB-435, SK-MEL-2, SK-MEL-28, SK-MEL-5, UACC-257 and UACC-62
  • Cell proliferation and inhibition thereof by the presently disclosed compounds may be assessed by cell viability methods disclosed in the art including colorimetric assays that utilize dyes such as MTT, XTT, and MTS to assess cell viability.
  • the disclosed compounds Preferably, have an ICso of less than about 10 uM, 5 pM, 1 uM, 0.5 pM, 0.01 gM, 0.005 pM, 0.001 pM or lower in the selected assay.
  • the disclosed compounds may be formulated as anti-cancer therapeutics, including anticancer therapeutics for the treatment of hematologic malignancies, breast, lung, pancreas, and prostate malignancies. In other embodiments, the disclosed compounds also may be formulated as anti-inflammation therapeutics.
  • the compounds utilized in the methods disclosed herein may be formulated as pharmaceutical compositions that include: (a) a therapeutically effective amount of one or more compounds as disclosed herein; and (b) one or more pharmaceutically acceptable carriers, excipients, or diluents.
  • the pharmaceutical composition may include the compound in a range of about 0.1 to 2000 mg (preferably about 0.5 to 500 mg, and more preferably about 1 to 100 nig).
  • the pharmaceutical composition may be administered to provide the compound at a daily dose of about 0.1 to about 1000 mg/kg body weight (preferably about 0.5 to about 500 mg/kg body weight, more preferably about 50 to about 100 mg/kg body weight).
  • the concentration of the compound at the site of action may be within a concentration range bounded by end-points selected from 0.001 pM, 0.005 pM, 0.01 uM, 0.5 pM, 0.1 pM, 1.0 pM, 10 pM, and 100 pM (e.g., 0.1 pM - 1.0 pM).
  • a subject in need thereof may include a subject having a cell proliferative disease, disorder, or condition such as cancer (e.g., cancers such as multiple myeloma, leukemia, non-small cell lung cancer, colon cancer, cancer of the central nervous system, melanoma, ovarian cancer, renal cancer, prostate cancer, and breast cancer).
  • cancer e.g., cancers such as multiple myeloma, leukemia, non-small cell lung cancer, colon cancer, cancer of the central nervous system, melanoma, ovarian cancer, renal cancer, prostate cancer, and breast cancer.
  • the subject may be administered a dose of a compound as low as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 me, 52 5 mg, 55 mg, 57.5 mg, 60 me, 62.5 me, 65 mg, 67.5 mg. 70 mg, 72.5 mg.
  • the subject may be administered a dose of a compound as high as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12,5 rag, 15 mg, 17.5 mg, 20 mg, 22 5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg, 60 mg, 62 5 mg, 65 mg, 67.5 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg, 200 mg, 500 mg, 1000 mg, or 2000 mg, once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times per week in order to treat the disease or disorder in the subject.
  • Minimal and/or maximal doses of the compounds may include doses falling within dose ranges having as end-points any of these disclosed doses (e.g., 2.5 mg - 200 mg).
  • a minimal dose level of a compound for achieving therapy in the disclosed methods of treatment may be at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, 1900. 2000, 3000, 4000. 5000, 6000, 7000, 8000, 9000. 10000, 15000. or 20000 ng/kg body weight of the subject.
  • a maximal dose level of a compound for achieving therapy in the disclosed methods of treatment may not exceed about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, or 20000 ng/kg body weight of the subject.
  • Minimal and/or maximal dose levels of the compounds for achieving therapy in the disclosed methods of treatment may include dose levels falling within ranges having as end-points any of these disclosed dose levels (e.g., 500 - 2000 ng/kg body weight of the subject).
  • the compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition in solid dosage form, although any pharmaceutically acceptable dosage form can be utilized
  • Exemplary solid dosage forms include, but are not limited to, tablets, capsules, sachets, lozenges, powders, pills, or granules
  • the solid dosage form can be, for example, a fast melt dosage form, controlled release dosage form, lyophilized dosage form, delayed release dosage form, extended-release dosage form, pulsatile release dosage form, mixed immediate release and controlled release dosage form, or a combination thereof.
  • the compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes a carrier.
  • the carrier may be selected from the group consisting of proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, and starch-gelatin paste.
  • the compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes one or more binding agents, filling agents, lubricating agents, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, and effervescent agents.
  • Filling agents may include lactose monohydrate, lactose anhydrous, and various starches;
  • binding agents are various celluloses and crosslinked polyvinylpyrrolidone, microcrystalline cellulose, such as Avicel® PH101 and Avicel® PHI 02, microcrystalline cellulose, and silicified microcrystalline cellulose (ProSolv SMCC'TM).
  • Suitable lubricants may include colloidal silicon dioxide, such as Aerosil®200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel.
  • colloidal silicon dioxide such as Aerosil®200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel.
  • sweeteners may include any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acsulfame.
  • sweeteners may include any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acsulfame.
  • flavoring agents are Magnasweet® (trademark of MAFCO), bubble gum flavor, and fruit flavors, and the like.
  • preservatives may include potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride.
  • Suitable diluents may include pharmaceutically acceptable inert fillers, such as microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and mixtures of any of the foregoing.
  • diluents include microcry stalline cellulose, such as Avicel® PHI 01 and Avicel® PHI 02; lactose such as lactose monohydrate, lactose anhydrous, and Phamiatose® DCL21; dibasic calcium phosphate such as Em compress®; mannitol; starch; sorbitol; sucrose; and glucose.
  • microcry stalline cellulose such as Avicel® PHI 01 and Avicel® PHI 02
  • lactose such as lactose monohydrate, lactose anhydrous, and Phamiatose® DCL21
  • dibasic calcium phosphate such as Em compress®
  • mannitol starch
  • sorbitol sucrose
  • glucose glucose
  • Suitable disintegrants include lightly crosslinked polyvinyl pyrrolidone, corn starch, potato starch, maize starch, and modified starches, croscarmellose sodium, cross-povidone, sodium starch glycolate, and mixtures thereof.
  • effervescent agents are effervescent couples such as an organic acid and a carbonate or bicarbonate.
  • Suitable organic acids include, for example, citric, tartaric, malic, fumaric, adipic, succinic, and alginic acids and anhydrides and acid salts.
  • Suitable carbonates and bicarbonates include, for example, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium glycine carbonate, L-lysine carbonate, and arginine carbonate.
  • sodium bicarbonate component of the effervescent couple may be present.
  • the compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition for delivery via any suitable route.
  • the pharmaceutical composition may be administered via oral, intravenous, intramuscular, subcutaneous, topical, intratumoral, and pulmonary route
  • Examples of pharmaceutical compositions for oral administration include capsules, syrups, concentrates, powders, and granules.
  • the compounds are formulated as a composition for administration orally (e.g., in a solvent such as 5% DMSO in oil such as vegetable oil).
  • the compounds utilized in the methods disclosed herein may be administered in conventional dosage forms prepared by combining the active ingredient with standard pharmaceutical carriers or diluents according to conventional procedures well known in the art. These procedures may involve mixing, granulating, and compressing or dissolving the ingredients as appropriate to the desired preparation.
  • compositions comprising the compounds may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual, ortransdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, or intradermal) route.
  • Such formulations may be prepared by any method known in the art of pharmacy', for example by bringing into association the active ingredient with the carrier(s) or excipient(s).
  • compositions adapted for oral administration may be presented as discrete units such as capsules or tablets, powders or granules; solutions or suspensions in aqueous or nonaqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions.
  • compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time.
  • the active ingredient may be delivered from the patch byiontophoresis.
  • compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, impregnated dressings, sprays, aerosols or oils and may contain appropriate conventional additives such as preservatives, solvents to assist drug penetration and emollients in ointments and creams.
  • the pharmaceutical compositions are preferably applied as a topical ointment or cream.
  • the compound When formulated in an ointment, the compound may be employed with either a paraffinic or a water- miscible ointment base.
  • the compound may be formulated in a cream with an oil- in-water cream base or a water-in-oil base.
  • Pharmaceutical compositions adapted for topical administration to the eye include eye drops where the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent.
  • compositions adapted for nasal administration where the carrier is a solid include a coarse powder having a particle size (e.g., in the range 20 to 500 microns) which is administered in the manner in which snuff is taken (/. ⁇ ?,, by rapid inhalation through the nasal passage from a container of the powder held close up to the nose)
  • Suitable formulations where the earner is a liquid, for administration as a nasal spray or as nasal drops include aqueous or oil solutions of the active ingredient.
  • compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.
  • Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine, tabletting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica, disintegrants, for example potato starch; or acceptable wetting agents such as sodium lauryl sulphate.
  • the tablets may be coated according to methods well known in normal pharmaceutical practice.
  • Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use.
  • Such liquid preparations may contain conventional additives, such as suspending agents, for example sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and, if desired, conventional flavoring or coloring agents.
  • suspending agents for example sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, or
  • the disclosed compounds or pharmaceutical compositions comprising the disclosed compounds may be administered in methods of treatment.
  • the disclosed compounds or pharmaceutical compositions comprising the disclosed compounds may be administered in methods of treating cell proliferative diseases and disorders.
  • Cell proliferative diseases and disorders treated by the disclosed methods may include, but are not limited to, cancers selected from the group consisting of leukemia (e.g., AML or CML), lung cancer (e.g., non-small cell lung cancer), cancer of the central nervous system (e.g., glioblastoma), melanoma, ovarian cancer, renal cancer, prostate cancer, head and neck, urinary tract, sarcoma, lymphoma (e.g., Hodgkin's lymphoma), and breast cancer.
  • leukemia e.g., AML or CML
  • lung cancer e.g., non-small cell lung cancer
  • cancer of the central nervous system e.g., glioblastoma
  • melanoma melanoma
  • the disclosed compounds or pharmaceutical compositions comprising the disclosed compounds may be administered with additional therapeutic agents, optionally in combination, in order to treat cell proliferative diseases and disorders.
  • additional therapeutic agents are administered with the disclosed compounds or with pharmaceutical compositions comprising the disclosed compounds, where the additional therapeutic agent is administered prior io, concurrently with, or after administering the disclosed compounds or the pharmaceutical compositions comprising the disclosed compounds
  • the disclosed pharmaceutical composition are formulated to comprise the disclosed compounds and further to comprise one or more additional therapeutic agents, for example, one or more additional therapeutic agents for treating cell proliferative diseases and disorders.
  • the disclosed compounds or pharmaceutical compositions comprising the disclosed compounds may be administered with an immunotherapy agent.
  • immunotherapy agent/ s refers to any therapeutic that is used to treat cancer in a subject by inducing and/or enhancing an immune response in that subject.
  • Immunotherapy agents may include, without limitation, checkpoint inhibitors.
  • Checkpoint inhibitors are therapeutics, such as antibodies, that block the immune checkpoint pathways in immune cells that are responsible for maintaining self-tolerance and modulating the degree of an immune response. Tumors often exploit certain immune checkpoint pathways as a major mechanism of immune resistance against T cells that are specific for tumor antigens. Many of the immune checkpoints are initiated by receptor-ligand interactions and thus may be blocked by antibodies to either the ligand or receptor or may be modulated by soluble recombinant forms of the ligands or receptors. Such immune checkpoint blockade allows tumorspecific T cells to continue to function in an otherwise immunosuppressive tumor microenvironment. Checkpoint inhibitors, however, are not effective against all cancer types. Furthermore, not every patient that is expected to respond to immune checkpoint blockade actually benefits from treatment with such agents.
  • Exemplary' checkpoint inhibitors include, without limitation, antibodies or other therapeutics targeting programmed cell death protein 1 (PDl, also known as CD279), programmed cell death 1 ligand 1 (PD-L1 , also known as CD274), PD-L2, cytotoxic T-lymphocyte antigen 4 (CTLA4, also known as CD 152), A2AR, CD27, CD28, CD40, CD80, CD86, CD122, CD137, OX40, G1TR, ICOS, TIM-3, LAG3, B7-H3, B7-H4, BTLA, IDO, KIR, or VISTA
  • Suitable anti- PD1 antibodies include, without limitation, lambrolizumab (Merck MK-3475), nivolumab (Bristol-Myers Squibb BMS-936558), AMP-224 (Merck), and pidilizumab (CureTech CT-011).
  • Suitable anti-PD-Ll antibodies include, without limitation, MDX-1105 (Medarex), MEDI4736 (Medimmune) MPDL3280A (Genentech/Roche) and BMS-936559 (Bristol-Myers Squibb).
  • Exemplary anti-CTLA4 antibodies include, without limitation, ipilimumab (Bristol-Myers Squibb) and tremelimumab (Pfizer).
  • NMR spectra were recorded at room temperature using a JEOL ECA-600 instrument (1H NMR at 600 MHz and 13C NMR at 150 MHz) with tetramethylsilane (TMS) as an internal standard. Chemical shifts (5) are given in parts per million (ppm) with reference to solvent signals [1H-NMR: CDC13 (7.26 ppm), CD3OD (3.31 ppm), DMSO-d6 (2.50 ppm), 13C-NMR: CDC13 (77.0 ppm), CD3OD (49.0 ppm), DMSO-d6 (39.5 ppm)].
  • Method I a Waters 1525 instrument equipped with Waters 2489 UV/Visible detector. Kinetex 5pm C18 100A column (250 X 4 6 mm) was used for analytical HPLC. HPLC gradient went from 2% to 98% CH 3 CN in H 2 O (both solvents contain 0.1% trifluoroacetic acid) with a total run time of 30 min and a flow rate of 1 mL/min for analytical analysis and 10 mL/min for preparative purification.
  • Method III a Waters 2545 instrument equipped with Waters 2489 UV/Visible detector. Kinetex 5 ⁇ m C18 100A column (250 X 4.6 mm) was used for analytical HPLC. HPLC gradient went from 2% to 98% MeOH in H 2 O (water containing 0.1% formic acid) with a total run time of 35 min and a flow rate of 1.2 mL/min for analytical analysis and 10 mL/min for preparative purification.
  • Method IV a Waters 2545 instrument equipped with Waters 2489 UV/Visible detector. Kinetex 5pm C18 100A column (250 X 4.6 mm) was used for analytical HPLC. HPLC gradient went from 98% to 2% IP A in H 2 O (water containing 0.1% formic acid) with a total run time of 30 min and a flow rate of 1.2 mL/min for 10 min and 0.5 mL/min for 20 min for analytical analysis and 10 mL/min for preparative purification.
  • HAMR 600 MHz, CEhOD 5 7.72 (s, 1H), 4.42 (s, 2H), 3.01 (s, 3H), 2.50 (s, 31 !) l3 C-NMR (150 MHz, CDsOD) 5 171.8, 162.3, 152.4, 113.8, 59.8, 27.7, 13.9.
  • the starting materials 1 (175 mg, 0.723 mmol), amine 2 (150 mg, 0.723 mmol) were placed in a round bottom flask and 2-propanol (3.0 mL) was added.
  • the reaction mixture was refluxed in an oil bath at 100 °C for 16 h. After completion of reaction all volatiles were removed under reduced pressure.
  • the crude compound was purified by combiflash chromatography (0-5% methanol in CH2CI2) to yield 3. Yellow solid, mp.
  • the starting materials 3 (30 mg, 0.072 mmol), boronic acid 4 (15 mg, 0.108 mmol), TBAB (23 mg, 0.072 mmol), NazCOs (2M, 0 1 mL, 0.179 mmol) were placed in a sealed tube and then ethanol (1.0 mL) and toluene (1.0 mL) was added. Then Pd(PPhfi4 (8.3 mg, 0.007 mmol) was added to the reaction mixture. The flask was flush with argon gas for 5-10 min. The reaction mixture was refluxed in an oil bath at. 100 °C for 16 h. After completion of the reaction, all volatiles were removed under reduced pressure.
  • Scheme 10 Reagents and conditions: a) Meldrum’s acid, piperidine, AcOH, Ethanol, reflux, 70 °C, 2 h, 88%, b) LiOAc, NIS, DMF/ H 2 O, MW, 110 °C, 15 min, 78%; c) m-CPBA, CH2CI2, 0 °C- rt, 1 h, 70-90%; d) NaH, DMF / THF, 0 °C- rt, 2 h, 68%; e) PdChfPPhsh, K2CO3 (3M), DMF, MW, Ih, 110 °C, 45-50%.
  • This compound can be prepared using a similar procedure as ALD-3-2-CN.
  • ALD-3-2b-CN ALD-3-2b-CN
  • This compound can be prepared using the following procedure.
  • This compound can be prepared using a similar procedure as SH3-20.
  • This compound can be prepared using a similar procedure as SH2-160.
  • This compound can be prepared using a similar procedure as SS-1-127.
  • This compound can be prepared by treating ALD-3-2-NH2CN with acetic anhydride.
  • This compound can be prepared by treating SS-1-127-CN with acetic anhydride.
  • ALD-3-2-OCH3CN SS-1-127-F
  • ALD-3-2-FCN SS-1-127-OCH3CN
  • ALD-3-2-OHCN SS- 1-127-OHCN
  • MLKL-/- mouse embryonic fibroblasts were stably transduced to allow Doxycycline-inducible re- expression of mouse MLKL.
  • the same cells were either left without Doxycycline (MLKL-/- condition) or pre-treated with 50 ng/ml Doxycycline (MLKL+/+ condition) for 6 hr. After that, cells were treated with the indicated concentrations of drugs for 24 hr. Cell viability was determined using CellTiter-Glo assay

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Sont divulgués des composés hétérocycliques substitués. Les hétérocycles substitués présentement divulgués sont utiles dans l'activation de la nécroptose par activation de MLKL et peuvent être utilisés en tant qu'agents thérapeutiques pour le traitement du cancer et de troubles prolifératifs cellulaires.
PCT/US2023/078420 2022-11-01 2023-11-01 Activateurs de pseudokinase de type domaine de kinase de lignée mixte (mlkl) WO2024097805A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202263381912P 2022-11-01 2022-11-01
US63/381,912 2022-11-01

Publications (1)

Publication Number Publication Date
WO2024097805A1 true WO2024097805A1 (fr) 2024-05-10

Family

ID=90931537

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2023/078420 WO2024097805A1 (fr) 2022-11-01 2023-11-01 Activateurs de pseudokinase de type domaine de kinase de lignée mixte (mlkl)

Country Status (1)

Country Link
WO (1) WO2024097805A1 (fr)

Similar Documents

Publication Publication Date Title
JP6903796B2 (ja) Ｆｇｆｒ４阻害剤としての二環式複素環
JP6911031B2 (ja) 免疫調節剤としての複素環化合物
RU2602071C2 (ru) Способ получения n-[5-(3,5-дифторбензил)-1н-индазол-3-ил]-4-(4-метилпиперазин-1-ил)-2-(тетрагидропиран-4-иламино)бензамида
CA2272705C (fr) Derives de pyrimidines bicycliques fusionnes
TW504508B (en) Atropisomers of 3-aryl-4(3h)-quinazolinones
JP2022058400A (ja) 免疫調節剤としての複素環化合物
US20190144439A1 (en) Heterocyclic compounds as immunomodulators
Brandt et al. Inhibitors of the RET tyrosine kinase based on a 2-(alkylsulfanyl)-4-(3-thienyl) nicotinonitrile scaffold
CN113164409A (zh) 具有雌激素受体α降解活性的新型化合物及其用途
CA2602294A1 (fr) Inhibiteurs heterobicycliques du vhc
JP2021524457A (ja) Ａ２ａ／ａ２ｂ阻害剤としての縮合ピリミジン誘導体
TWI781607B (zh) 一種免疫抑制劑、其製備方法和應用
WO2015060373A1 (fr) Dérivé de quinazolinone et d'isoquinolinone
WO2018127184A1 (fr) Inhibiteur de la kinase du lymphome anaplasique, son procédé de préparation et son utilisation
WO2021238827A1 (fr) Inhibiteur d'egfr, son procédé de préparation et son utilisation
US10501466B2 (en) WDR5 inhibitors and modulators
EP3661935A1 (fr) Pyrazolopyrimidines substituées pouvant être employées en tant qu'inhibiteurs de kinases
WO2022166741A1 (fr) Composé macrocyclique contenant un benzohétérocycle et agissant en tant qu'inhibiteur de la kinase egfr et composition pharmaceutique et utilisation de celui-ci
KR20110131190A (ko) 3 고리성 피라졸로피리미딘 유도체
CN114423762A (zh) 大环类衍生物及其制备方法和用途
WO2024097805A1 (fr) Activateurs de pseudokinase de type domaine de kinase de lignée mixte (mlkl)
RU2534804C1 (ru) ЗАМЕЩЕННЫЕ [1,2,4]ТРИАЗОЛО[4,3-a]ПИРИДИНЫ, ПРОЯВЛЯЮЩИЕ СВОЙСТВА АНТАГОНИСТОВ АДЕНОЗИНОВЫХ А2А РЕЦЕПТОРОВ, И ИХ ПРИМЕНЕНИЕ
JP2022554385A (ja) Ｗｄｒ５阻害剤及び調節剤
EP3974432A1 (fr) Dérivé hétéroaryle contenant de l'azote et composition pharmaceutique le comprenant en tant que principe actif pour prévenir ou traiter le cancer
WO2013041859A1 (fr) Agonistes du récepteur des cannabinoïdes 2