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WO2023126951A1 - Inhibiteurs d'interactions protéine-protéine liées à l'autophagie - Google Patents

Inhibiteurs d'interactions protéine-protéine liées à l'autophagie Download PDF

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WO2023126951A1
WO2023126951A1 PCT/IL2023/050009 IL2023050009W WO2023126951A1 WO 2023126951 A1 WO2023126951 A1 WO 2023126951A1 IL 2023050009 W IL2023050009 W IL 2023050009W WO 2023126951 A1 WO2023126951 A1 WO 2023126951A1
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compound
esi
nmr
dmso
alkyl
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Adi Kimchi
Yuval Gilad
Chaim Gal NUTA
Radim NENCKA
Petra BREHOVA
Krystof SKACH
Jiri BOSERLE
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Institute Of Organic Chemistry And Biochemistry Of Czech Academy Of Sciences Iocb
Yeda Research and Development Co Ltd
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Institute Of Organic Chemistry And Biochemistry Of Czech Academy Of Sciences Iocb
Yeda Research and Development Co Ltd
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    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • A61K31/473—Quinolines; Isoquinolines ortho- or peri-condensed with carbocyclic ring systems, e.g. acridines, phenanthridines
    • A—HUMAN NECESSITIES
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • 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
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms 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
    • C07D215/38—Nitrogen atoms
    • C—CHEMISTRY; METALLURGY
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms 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
    • C07D215/48—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
    • C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
    • C07D221/04—Ortho- or peri-condensed ring systems
    • C07D221/06—Ring systems of three rings
    • C07D221/14—Aza-phenalenes, e.g. 1,8-naphthalimide
    • C—CHEMISTRY; METALLURGY
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    • 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
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    • 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
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    • C—CHEMISTRY; METALLURGY
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    • 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
    • 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
    • C07D471/12—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 three hetero rings
    • C07D471/14—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D513/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00
    • C07D513/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains two hetero rings
    • C07D513/04—Ortho-condensed systems

Definitions

  • the present application is directed to inhibitors of autophagy -related protein-protein interactions. Further, described herein is the use of these inhibitors for treating an autophagy- related disease or disorder, cancer or tumor, a cytokine release syndrome (CRS) or a cytokine storm in a subject.
  • CRS cytokine release syndrome
  • Autophagy manifests in advanced cancer and acute inflammation as two functional arms of lysosomal degradation of intracellular components and autophagy-dependent secretion of cytokines. It is well established that many types of primary and metastatic solid tumors, depend on the lysosomal-mediated recycling features of autophagy in order to manage the various stress conditions in the tumor microenvironment such as hypoxia and nutrient starvation. The dependence of various tumors on excessive levels of autophagy is termed autophagy addiction. During chemotherapy or targeted cancer therapy, autophagy is upregulated and assists the cancer cells to counteract the drug-induced toxicity caused by these treatments.
  • the secretory arm of autophagy that enables leaderless cytokines such as IL-1 ⁇ to exit the cell without entering the Endoplasmic reticulum (ER) and the Golgi pathways, promotes the cytokine storm in acute inflammation.
  • leaderless cytokines such as IL-1 ⁇
  • an autophagy inhibitor compound represented by the structure of formula (I): wherein, is a single or double bond;
  • Y is NR 4 , O or S
  • Wi- W 4 are each independently null, hydrogen,
  • R 1 is alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -alkyl-aryl, SO 2 alkyl, SO 2 aryl, or - alkyl-O-alkyl;
  • R 2 and R 3 are each independently null, OH, hydrogen, -O-alkyl, halide, amino, or R 2 and R 3 are joined together to form a 5 or 6 membered substituted or unsubstituted ring;
  • R 4 is H, alkyl or aryl
  • R 5 is hydrogen, alkyl, aryl,
  • R 6 is hydrogen, or alkyl
  • R 7 is H, -OH, O-alkyl-aryl, or -S02-alkyl; wherein the alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -alkyl-aryl, -alkyl-O-alkyl are independently substituted or unsubstituted;
  • X 1 -X 7 are each independently N or C;
  • X 8 -X 9 are each independently N, C, CH, O or S; and n is an integer between 0-5; or pharmaceutically acceptable salt thereof.
  • autophagy inhibitor compounds represented by Compound 55 or Compound 42, or pharmaceutically acceptable salt thereof: [0007] In some aspects, disclosed herein is an autophagy inhibitor compound represented by the structure of formula (VI): wherein the aryl comprises substituted or unsubstituted aryl; and
  • Q 3 -Q 9 are each independently H, halo or -OH, wherein at least one of Q 3 -Q 9 is OH; or pharmaceutically acceptable salt thereof.
  • the aryl of formula VI is a phenyl. In other embodiments, the aryl of formula VI is a naphthyl.
  • an autophagy inhibitor compound represented by the structure of formula (VII): wherein.
  • Q 1 is independently H, halo, OH, O-alkyl, alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl
  • Q 2 is H, halo, OH, O-alkyl, alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl q is an integer between 1-4;
  • Q 3 -Q 9 are each independently H , halo or -OH, wherein at least one of Q 3 -Q 9 is OH; or pharmaceutically acceptable salt thereof.
  • a compound is represented by Compound 200, 201,
  • a pharmaceutical composition comprising the autophagy inhibitor compounds provided herein, pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
  • a method of treating an autophagy-related disease or disorder in a subject comprising the step of administering to the subject a pharmaceutical composition provided herein.
  • a method of treating, suppressing or inhibiting a cancer in a subject comprising the step of administering to the subject a pharmaceutical composition provided herein.
  • the cancer comprises a solid tumor.
  • the cancer comprises a diffuse cancer.
  • ACD autophagic cell death
  • a method of decreasing or inhibiting cytokine production or secretion in a subject experiencing cytokine release syndrome or cytokine storm or vulnerable to cytokine release syndrome or cytokine storm comprising the step of administering to the subject a pharmaceutical composition provided herein.
  • the method comprises reducing production or secretion of one or more autophagy -related cytokines.
  • the one or more autophagy related cytokines are selected from IL-1 ⁇ , IL-6, IL- 18, TNF- ⁇ , IFN-y, or TGF- ⁇ , or any combination thereof.
  • FIG. 1A ATG12-ATG3 Interaction surface.
  • the crystal structure of human ATG12 blue, white and red bound to a short segment of residues 153-165 of ATG3 (cyan blue).
  • Color gradient in ATG12 refers to the electric charge of the surface residue; blue, white and red refer to positive, neutral or negative charges, respectively.
  • FIG. 1B Mutational scanning of ATG12-L1 reveals variants of Atgl2 which fail to interact with ATG3-L2 WT in the PCA platform. Bulky Trp73 and Phe87 residues were replaced with small Ala residue (W73 A and F87A). Positive Lys were replaced with negative Asp (K54D and K72D).
  • Figure 2. shows the schematic screen workflow of the Protein fragment Complementation
  • PCA Assay
  • Pre-printed plates with compounds were incubated with ATG12-L1 or with ATG12-L1 K54D lysates. Then cell lysates expressing ATG3-L2 were added to the plates, and 24h later, the luminescence was measured. After data analysis, all positive hits passed through hit validation. After exclusion of false positive hits, the remaining compounds were triaged by a medicinal chemist, re-synthesized, and subjected to dose response for determining IC50 in the PCA assay. Positive compounds were then used for cell-based assays to determine inhibitory effects of the compounds on autophagy in viable cells.
  • FIG. 3A Adapting the PCA platform for detecting small compounds which inhibit autophagy-related protein-protein interactions (PPIs). 15 pl of ATG12-L1 WT or ATG12-L1 K54D cell lysates (7 pg/pl) were mixed with 15 pl of ATG3-L2 cell lysates (7 pg/pl). Following 24h incubation, luminescence was measured.
  • PPIs autophagy-related protein-protein interactions
  • FIG. 3B Bcl-xL-Ll expressing cell lysate was mixed with ABT-737 10 ⁇ M for Ih at 4°C or with the DMSO carrier as a control. Then, Bax-L2 cell lysate was added to the mixture. Following 30 min incubation, luminescence was measured.
  • Figure 4A Cell-based GFP-LC3 puncta assay. Compounds 200 and 42 structures and their calculated IC50 in the PCA system.
  • Figure 4B Representative images of HEK293A GFP-LC3 cells tested without and with compounds. DMSO represents the basal steady state levels of autophagy. Torinl + CQ represent autophagy induction as assessed by increased number of puncta area per cell. Compounds images represent cells treated with compounds at 10 ⁇ M for 24h, followed by 2h treatment with Torinl + CQ. Scale bar 100 pm.
  • FIG. 4C Dose-response curves of Compound 200 and Compound 42 on autophagy, measured by autophagosome area/cell.
  • Cells were treated with the compounds at serial dilutions in the range of 20 - 0.31 ⁇ M for 24h, followed by 2h treatment with Torinl + CQ. After nuclei staining using Hoechst, the cells were fixated, and the samples were imaged and analyzed. An average of 2 repeats is shown.
  • Figures 5A-5E Detailed follow up of the effects of compound 200 on autophagy levels in cells.
  • Figure 5A Structure of compound 200.
  • Figure 5B Representative images of HEK 293 A GFP-LC3B cells treated without and with 10 ⁇ M compound 200 for 24 h. Basal steady state levels of autophagy are observed in DMSO treated cells. 200 nM Torinl + 20 ⁇ M CQ were used to induce autophagy in the final 2h of treatment. Scale bar, 20 pm.
  • Figure 5C Dose-response curve of compound 200 effect on autophagy.
  • Figure 6A SPR plot of compound 200 binding to recombinant ATG12-Flag. Black arrows represent association (left) and dissociation (right) points. Cycle number represents the order of injection. The 20 ⁇ M sample concentration was injected twice to ensure consistency of association levels.
  • FIG. 6B Biochemical validation of the recombinant ATG12-Flag used for the SPR assay, purified, and visualized on acrylamide gels by Coomassie blue staining (left panel), Ponceau S (middle panel) and probed with anti-ATG12 antibody (right panel).
  • Figure 7A The structure of Compound 55.
  • Compound 55 retains the inhibitory functions in the PCA lysates and on autophagy in cells while losing its inhibitory effects on CK2.
  • Figure 7C In-silico docking assay of compounds 42 and 55 binding to ATG12 surface. Compound 55 preserve the docking score of Compound 42.
  • Figure 7D Representative images of HEK293A GFP-LC3 cells tested with controls and with Compound 55. DMSO represents the basal steady state levels of autophagy. Torinl + CQ image represents activated autophagy control. Compound 55 image represents cells treated with compound 55 at 10 ⁇ M for 24h, followed by 2h treatment with Torinl + CQ. Scale bar 100 pm.
  • FIG. 7E Dose-response curve of Compound 55 effect on autophagy, measured by autophagosome area/cell.
  • Cells were treated with the Compound at serial dilutions in the range of 20 - 0.31 ⁇ M for 24h, followed by 2h treatment with Torinl + CQ. After nuclei staining using Hoechst, the cells were fixated, and the samples were imaged and analyzed. An average of 2 repeats is shown.
  • FIG. 7F Representative Western Blot (WB) of Compounds 42 and 55 effect on CK2 activity.
  • HEK293T cells were treated with Compounds 42 or 55 for 24h at 10 ⁇ M, followed by lysis and WB, blotting for pAKT S129 (Abeam, abl33458), total AKT (Cells Signaling #2938), and GAPDH (Sigma MAB374).
  • G pAKT S129 levels were quantified and compared with total AKT. Data represent average of 3 repetitions. Statistical significance was determined by one-way analysis of variance (ANOVA), *P ⁇ 0.05.
  • Figures 8A-8E Compound 200 reduces autophagy levels and the growth of autophagy- addicted pancreatic cancer cells.
  • Figure 8A Western blot showing levels of lipidated and free LC3B in PANCI and NCI-H460 cells. GAPDH was used as a loading control.
  • Figure 8B Autophagy addicted PANCI and non-autophagy addicted cancer lung NCI-H460 cell lines were treated with 5 ⁇ M compound 200, and viability was measured over the course of 4 days by CellTiter-Glo assay. CQ (10 ⁇ M) was used as a positive control for autophagy inhibition. Data represent the mean ⁇ SD of 3 repetitions, normalized to day 0, time at which drug was added.
  • FIG. 9 Compound 200 reduces the viability of autophagy-addicted melanoma cells.
  • Autophagy addicted (MM 34) and non-addicted (MM 39) melanoma cell lines were treated with the compounds at 3 concentrations with and without vemurafenib. 48h later, viability was measured using CellTiter-Glo assay. Statistical significance was determined by two-way analysis of variance (ANOVA), **P ⁇ 0.01; ***P ⁇ 0.001; ****P ⁇ 0.0001.
  • FIG. 10A Compound 200 reduces IL1 ⁇ secretion in LPS-treated RAW 264.7 mouse cells.
  • RAW 264.7 cells were treated with LPS (1 pg/ml) combined with Compound 200 (10 ⁇ M), HCQ (20 ⁇ M), or Dexamethasone (2 pg/ml) for 24h, and levels of ILip in the medium was measured using ELISA procedure. Data represent average of 4 repetitions, and was normalized to LPS-treated cells. Statistical significance was assessed using one-way analysis of variance (ANOVA), ****P ⁇ 0.0001.
  • FIG 10B Compound 55 reduces ILip secretion in LPS-treated RAW 264.7 mouse cells.
  • RAW 264.7 cells were treated with LPS (1 pg/ml) combined with Compound 55 (10 and 5 ⁇ M), HCQ (20 ⁇ M), or Dexamethasone (2 pg/ml) for 24h, and levels of ILip in the medium was measured using ELISA procedure. Data represent average of 3 repetitions, and was normalized to LPS-treated cells. Statistical significance was assessed using one-way analysis of variance (ANOVA), *P ⁇ 0.05; **P ⁇ 0.01; ****P ⁇ 0.0001.
  • FIG. 10C Compound 200 reduces ILip secretion in LPS-treated human THP-1 cells.
  • THP-1 cells were treated with 0.5 ⁇ M PMA for 3 h followed by 0.5 pg/ml LPS combined with DMSO, compound 200 (10 ⁇ M), or dexamethasone (2 pg/ml) for 4 h and ILip levels were assayed by ELISA as described in Figure 10A.
  • FIG. 11A A549 cells treated with Resveratrol (RSV) for 48h at indicated concentrations, followed by lysis and WB, blotting for LC3-I/II and VCL (vinculin) for loading control.
  • RSV Resveratrol
  • FIG 11B A549 cells treated with RSV and DMSO for 48h, followed by viability measurement using CellTiter-Glo assay. Data represent average of 4 repetitions. Statistical significance was determined by the two-tailed unpaired Student t test, ****P ⁇ 0.0001.
  • FIG 11C A549 cells transfected with either ATG7 or ATG12 siRNAs were treated with RSV (200 ⁇ M). After 48h cell viability was assessed using CellTiter-Glo assay and represented as fold-change in siRNA + RSV-treated cells compared with siRNA treated cells. Data represents four replicate experiments, and statistical significance was assessed using one-way analysis of variance (ANOVA), **P ⁇ 0.01; ***P ⁇ 0.001.
  • Figure 11D A549 cells treated with Compound 55 and with RSV (100 ⁇ M). After 48h cell viability was assessed using CellTiter-Glo assay and represented as fold-change in compound + RSV-treated cells compared to compound only treated cells.
  • Y is NR 4 , O or S
  • Wi- W 4 are each independently null, hydrogen, CO 2 R 5 , -(CH 2 ) n - CO 2 R 5 , CN, halide, -(CH 2 ) n -
  • R 1 is alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -alkyl-aryl, SO 2 alkyl, SO 2 aryl, or - alkyl-O-alkyl;
  • R 2 and R 3 are each independently null, OH, hydrogen, -O-alkyl, halide, amino, or R 2 and R 3 are joined together to form a 5 or 6 membered substituted or unsubstituted ring;
  • R 4 is H, alkyl, or aryl
  • R 6 is hydrogen, or alkyl
  • R 7 is H, -OH, O-alkyl-aryl, or -SO 2 -alkyl; wherein the alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -alkyl-aryl, -alkyl-O-alkyl are independently substituted or unsubstituted;
  • X 1 -X 7 are each independently N or C;
  • X 8 -X 9 are each independently N, C, CH, O or S; and n is an integer between 0-5.
  • Y is NR 4 , O or S
  • W 1 -W 4 are each independently null, hydrogen, CO 2 R 5 , -(CH 2 ) n - CO 2 R 5 , CN, halide, -(CH 2 ) n -
  • R 1 is alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -alkyl-aryl, SO 2 alkyl, SO 2 aryl, or - alkyl-O-alkyl;
  • R 2 and R 3 are each independently null, OH, hydrogen, -O-alkyl, halide, amino, or R 2 and R 3 are joined together to form a 5 or 6 membered substituted or unsubstituted ring;
  • R4 is H, alkyl or aryl
  • R 6 is hydrogen, or alkyl
  • R 7 is H, -OH, O-alkyl-aryl, or - SO 2 -alkyl; wherein the alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -alkyl-aryl or -alkyl-O-alkyl are independently substituted or unsubstituted;
  • X1-X 9 are each independently N or C; and n is an integer between 0-5.
  • Y is NR 4 , O or S
  • W 1 -W 4 are each independently null, hydrogen, or CO 2 R 5 , -(CH 2 ) n -CO 2 R 5 , CN, halide, -(CH 2 ) n -
  • W 5 -W 8 are each independently null, hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, or cycloheteroalkyl;
  • R 1 is alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -alkyl-aryl, SO 2 alkyl, SO 7 aryl, or - alkyl-O-alkyl;
  • R 4 is H, alkyl, or aryl
  • R 6 is hydrogen, or alkyl
  • R 7 is H, -OH, O-alkyl-aryl, or -SO 2 -alkyl; wherein the alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -alkyl-aryl, or -alkyl-O-alkyl are independently substituted or unsubstituted;
  • X 1 -X 7 are each independently N or C;
  • X 8 -X 13 are each independently N, C, CH, O or S; n is an integer between 0-5; and
  • K is 0 or 1.
  • Y is NR 4 , O or S
  • W 1 -W 4 are each independently null, hydrogen, or CO 2 R 5 , -(CH 2 ) n -CO 2 R 5 , CN, halide, -(CH 2 ) n -
  • W 6 -W 8 are each independently null, hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, or cycloheteroalkyl;
  • R 1 is alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -alkyl-aryl, SO 2 alkyl, SO 2 aryl or - alkyl-O-alkyl;
  • R4 is H, alkyl, or aryl
  • R 6 is hydrogen, or alkyl
  • R 7 is H, -OH, O-alkyl-aryl, or -SO 2 -alkyl; wherein the alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -alkyl-aryl or -alkyl-O-alkyl are independently substituted or unsubstituted;
  • X 1 -X 7 are each independently N or C;
  • X 8 -X 13 are each independently N, C, CH, O, or S; and n is an integer between 0-5.
  • W 1 -W 3 and W 4 are each independently a hydrogens or null and
  • W 2 is CO 2 R 5 , W 5 -W 8 are each independently null, hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, cycloheteroalkyl;
  • R 7 is H, -OH, O-alkyl-aryl, or -SO 2 -alkyl
  • X 1 -X 7 are each independently N or C;
  • X 8 -X 13 are each independently N, C, CH, O, or S; n is an integer between 0-5; and k is 0 or 1.
  • W 1 -W 4 are each independently null, hydrogen, or CO 2 R 5 , -(CH 2 ) n -CO 2 R 5 , CN, halide, -(CH 2 ) n -
  • W 6 -W 8 are each independently null, hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, or cycloheteroalkyl;
  • R 6 is hydrogen, or alkyl
  • R 7 is H, -OH, O-alkyl-aryl, or -SO 2 -alkyl; wherein the alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -alkyl-aryl or -alkyl-O-alkyl are independently substituted or unsubstituted;
  • X 1 -X 7 are each independently N or C;
  • X 8 -X 13 are each independently N, C, CH, O, or S; and n is an integer between 0-5.
  • the bond between X 8 -X 9 of formula I, III, IV VA or VB is a single bond. In some embodiments bond between X 8 -X 9 of formula , III, IV VA or VB is a double bond. In other embodiments, the bond between X 8 -X 13 of formula III, IV, VA or VB is a single bond. In other embodiments, the bond between X 8 -X 13 of formula III, IV, VA or VB is a double bond. In other embodiments, the bond between X 12 -X 13 of formula III, IV, VA or VB is a single bond. In other embodiments, the bond between X 12 -X 13 of formula III, IV, VA or VB is a double bond.
  • the bond between X 11 - X 12 of formula III, IV, VA or VB is a single bond. In other embodiments, the bond between X 10 -X 12 of formula III, IV VA or VB is a double bond. In other embodiments, the bond between X 10 -X 11 of formula III or VA is a single bond. In other embodiments, the bond between X 10 - X 11 of formula III or VA is a doble bond. In other embodiments, the bond between X 9 -X 11 of formula IV or VB is a single bond. In other embodiments, the bond between X 9 -X 11 of formula IV or VB is a double bond.
  • Y of formula I, II, III or IV is NR4 wherein R4 is H, alkyl, or aryl.
  • R4 is H, alkyl, or aryl.
  • Y of formula I, II, III or IV is NH.
  • Y of formula I, II, III or IV is N-alkyl.
  • Y of formula I, II, III or IV is N-aryl.
  • Y of formula I, II, III or IV is O.
  • Y of formula I, II, III or IV is S.
  • Wi- W4 of formula I, II, III, IV, VA or VB are each independently or VB is null (if X2-X7 is nitrogen (N) then W 1 -W 4 , respectively is null).
  • Wi- W4 of formula I, II, III, IV, VA or VB is hydrogen.
  • Wi- W4 of formula I, II, III, IV, VA or VB is CO 2 R 5 .
  • Wi- W4 of formula I, II, III, IV, VA or VB is -(CH 2 ) n -CO 2 R 5 .
  • Wi- W4 of formula I, II, III, IV, VA or VB is CN.
  • Wi- W4 of formula I, II, III, IV, VA or VB is
  • W 5 -W 8 of formula III, IV, VA or VB are each independently null, hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, or cycloheteroalkyl.
  • W 5 -W 8 of formula III, IV, VA or VB is null (if X 10 -X 13 is nitrogen (N) in a double bond, then W 5 -W 8 , respectively is null or if X 10 -X 13 is oxygen (O) or sulfur (S) in a single bond, then W 5 -W 8 , respectively is null).
  • W 5 -W 8 of formula III, IV, VA or VB is hydrogen.
  • W 5 -W 8 of formula III, IV, VA or VB is alkyl. In some embodiments, W 5 - W 8 of formula III, IV, VA or VB is aryl. In some embodiments, W 5 -W 8 of formula III, IV, VA or VB is heteroaryl. In some embodiments, W 5 -W 8 of formula III, IV, VA or VB is cycloalkyl. In some embodiments, W 5 -W 8 of formula III, IV, VA or VB is cycloheteroalkyl.
  • R 1 of formula I, II, III or IV is alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -alkyl-aryl or -alkyl-O-alkyl.
  • R 1 of formula I, II, III or IV is an alkyl.
  • R 1 of formula I, II, III or IV is an aryl.
  • R 1 of formula I, II, III or IV is an aryl.
  • R 1 of formula I, II, III or IV is a heteroaryl. In other embodiments, R 1 of formula I, II, III or IV is a cycloalkyl. In other embodiments, R 1 of formula I, II, III or IV is a heterocycloalkyl. In other embodiments, R 1 of formula I, II, III or IV is an -alkyl-aryl. In other embodiments, R 1 of formula I, II, III or IV is an -alkyl-O-alkyl. In other embodiments, R 1 of formula I, II, III or IV is SO 2 alkyl. In other embodiments, R 1 of formula I, II, III or IV SO 2 aryl,
  • R 2 and R 3 of formula I or II are each independently null, hydrogen, -O-alkyl, halide, amino, or R 2 and R 3 are joined together to form a 5 or 6 membered substituted or unsubstituted ring.
  • R 2 and R 3 of formula I or II are each independently null (R2 is null if X9 is nitrogen (N), R 3 is null if X 8 is nitrogen (N)).
  • R 2 and R 3 of formula I or II are each independently hydrogen.
  • R 2 and R 3 of formula I or II are each independently OH.
  • R 2 and R 3 of formula I or II are each independently -O-alkyl.
  • R 2 and R 3 of formula I or II are each independently a halide. In other embodiments, R 2 and R 3 of formula I or II are each independently an amino. In other embodiments, R 2 and R 3 of formula I or II are joined together to form a 5 or 6 membered substituted or unsubstituted ring.
  • R 5 of CO 2 R 5 or -(CH 2 ) n -CO 2 R 5 of W 1 -W 4 of formula I, II, III, IV or V is H.
  • R 5 group of CO 2 R 5 or -(CH 2 ) n -CO 2 R 5 of W 1 -W 4 of formula I, II, III, IV or V is alkyl.
  • R 5 of CO 2 R 5 or -(CH 2 ) n -CO 2 R 5 of Wi- W4 of formula I, II, III, IV or V is -(CH 2 ) n -O-CO 2 -R 7 wherein n is an integer between 0 -5.
  • R 5 group of CO 2 R 5 or -(CH 2 ) n -CO 2 R 5 of W 1 -W 4 of formula I, II, III, IV or V is wherein n is an integer between 0-5.
  • R 6 of -(CH 2 ) n -OR 6 of W 1 -W 4 of formula I, II, III or IV wherein n is an integer between 0-5, is hydrogen or alkyl.
  • W 1 -W 4 of formula I, II, III or IV wherein n is an integer between 0-5 is hydrogen.
  • R 6 of -(CH 2 ) n -OR 6 of W 1 -W 4 of formula I, II, III or IV wherein n is an integer between 0-5 is an alkyl.
  • R 7 is -OH.
  • R 7 is O-alkyl-aryl.
  • R 7 is -SO 2 -alkyl-aryl.
  • R 7 is -H.
  • X 1 -X 7 of formula I, II, III, IV, VA or VB are atoms linked by a double bond within a ring, wherein each independently are a nitrogen (N) or a carbon (C).
  • N nitrogen
  • C carbon
  • X 1 -X 7 of formula I, II, III, IV, VA or VB is a nitrogen.
  • X 1 -X 7 of formula I, II, III, IV, VA or VB is a carbon.
  • X 8 -X 13 of formula I, II, III, IV, VA or VB are atoms linked by a double bond or a single bond within a ring wherein each independently is N, C, CH, O, or S.
  • X 8 -X 13 of formula I, II, III, IV, VA or VB when any of X 8 -X 13 of formula I, II, III, IV, VA or VB is linked by a double bond, then it is not a S or O.
  • X 8 -X 13 of formula I, II, III, IV, VA or VB is linked by a double bond with the ring, then X 8 -X 13 a N, C.
  • X 8 -X 13 of formula I, II, III, IV, VA or VB is linked by a single bond with the ring, then X 8 -X 13 a N, CH, O, or S.
  • X 8 -X 13 each independently of formula I, II, III, IV, VA or VB is linked by a double bond and is a carbon.
  • X 8 -X 13 each independently of formula I, II, III, IV, VA or VB is linked by a double bond and is a nitrogen.
  • X 8 -X 13 each independently of formula I, II, III, IV, VA or VB is linked by a single bond and is a CH. In other embodiments, X 8 -X 13 each independently of formula I I, II, III, IV, VA or VB is linked by a single bond and is a nitrogen (N). In other embodiments, X 8 -X 13 each independently of formula I, II, III, IV, VA or VB is linked by a single bond and is an oxygen (O). In other embodiments, X 8 -X 13 each independently of formula I, II, III, IV, VA or VB is linked by a single bond and is an sulfur (S).
  • the compounds are autophagy inhibitors.
  • an autophagy inhibitor compound represented by the structure of formula (VI): wherein the aryl comprises substituted or unsubstituted aryl ;
  • Q 3 -Q 9 are each independently H, halo or -OH, wherein at least one of Q 3 -Q 9 is OH; or pharmaceutically acceptable salt thereof.
  • the aryl of formula VI is a phenyl. In other embodiments, the aryl of formula VI is a naphthyl.
  • an autophagy inhibitor compound represented by the structure of formula (VII):
  • Q 1 is independently H, halo, OH, O-alkyl, alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl
  • Q 2 is H, halo, OH, O-alkyl, alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl q is an integer between 1-4;
  • Q 3 -Q 9 are each independently H , halo or -OH, wherein at least one of Q 3 -Q 9 is OH; or pharmaceutically acceptable salt thereof.
  • Q 1 of formula VII is hydrogen (H). In some embodiment, Q 1 of formula VII is an alkyl. In some embodiment, Q 1 of formula VII is halo. In some embodiment, Qi of formula VII is OH In some embodiment, Q 1 of formula VII is O-alkyl. In some embodiment, Q 1 of formula VII is aryl. In some embodiment, Q 1 of formula VII is heteroaryl. In some embodiment, Q 1 of formula VII is cycloalkyl. In some embodiment, Q 1 of formula VII is heterocycloalkyl. In some embodiment, Q 2 of formula VII is a hydrogen. In some embodiments Q 2 of formula VII is an alkyl. In some embodiment, Q 2 of formula VII is halo.
  • Q 2 of formula VII is OH In some embodiment, Q 1 of formula VII is O-alkyl. In some embodiment, Q 2 of formula VII is aryl. In some embodiment, Q 2 of formula VII is heteroaryl. In some embodiment, Q 2 of formula VII is cycloalkyl. In some embodiment, Q 2 of formula VII is heterocycloalkyl.
  • Q 3 -Q 9 of Formula VI or VII are each independently H or -OH, wherein at least one of Q 3 -Q 9 is OH. In other embodiments, Q 3 -Q 9 are each independently H, wherein at least one of Q3-Q9 is a hydroxyl (OH). In other embodiments, Q 3 -Q 9 are each independently OH. In other embodiments, Q 3 -Q 9 are each independently halo. [0065] In some aspects, disclosed herein is a compound of Formula VI represented by Compound 200, 201, 202, 203 or pharmaceutically acceptable salt thereof:
  • alkyl used alone or as part of another group, refers, in one embodiment, to a “C 1 to C 12 alkyl” and denotes linear and branched, saturated or unsaturated (e.g., alkenyl, alkynyl) groups, the latter only when the number of carbon atoms in the alkyl chain is greater than or equal to two, and can contain mixed structures.
  • alkyl groups containing from 1 to 6 carbon atoms C 1 to C 6 , alkyls
  • alkyl groups containing from 1 to 4 carbon atoms C 1 to C 4 alkyls
  • saturated alkyl groups include, but are not limited to, methyl, ethyl, n- propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, amyl, tert-amyl and hexyl.
  • alkenyl groups include, but are not limited to, vinyl, allyl, butenyl and the like.
  • alkynyl groups include, but are not limited to, ethynyl, propynyl and the like.
  • the alkyl group can be unsubstituted or substituted with one or more substituents selected from the group consisting of alkyl, aryl, halogen, haloalkyl, hydroxyl, alkoxy, carbonyl, amido, alkylamido, dialkylamido, cyano, nitro, CO 2 H, amino, alkylamino, dialkylamino, carboxyl, thio, thioalkyl, C 1 -C 5 linear or branched haloalkoxy, CF 3 , CH 3 , phenyl, halophenyl, (benzyloxy )phenyl, -CH 2 CN, NH 2 , NH- alkyl, N(alkyl) 2 , -OC(O)CF 3 , -OCH 2 Ph, -NHCO-alkyl, -C(O)Ph, C(O)O-alkyl, C(O)H, -C(O)NH 2 or
  • aryl used herein alone or as part of another group refers to an aromatic ring system containing from 6-14 ring carbon atoms.
  • the aryl ring can be a monocyclic, bicyclic, tricyclic and the like.
  • Non -limiting examples of aryl groups are phenyl, naphthyl including 1- naphthyl and 2-naphthyl, and the like.
  • the aryl group can be unsubstituted or substituted through available carbon atoms with one or more groups such as halogen, alkyl, haloalkyl, hydroxyl, alkoxy, carbonyl, amido, alkylamido, dialkylamido, cyano, nitro, CO 2 H, amino, alkylamino, dialkylamino, carboxyl, thio, thioalkyl, C 1 -C 5 linear or branched haloalkoxy, CF 3 , phenyl, halophenyl, (benzyloxy)phenyl, -CH 2 CN, NH 2 , NH-alkyl, N(alkyl)2, -OC(O)CF 3 , -OCH 2 Ph, - NHCO-alkyl, -C(O)Ph, C(O)O-alkyl, C(O)H, -C(O)NH 2 or any combination thereof.
  • groups such as hal
  • heteroaryl refers herein to an aromatic ring system containing from 5-14 member ring having at least one heteroatom in the ring.
  • suitable heteroatoms include oxygen, sulfur, phospate and nitrogen.
  • heteroaryl rings include pyridinyl, pyrrolyl, oxazolyl, indolyl, isoindolyl, purinyl, furanyl, thienyl, benzofuranyl, benzothiophenyl, carbazolyl, imidazolyl, thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, quinolyl, isoquinolyl, pyridazyl, pyrimidyl, pyrazyl, etc.
  • the heteroaryl group can be unsubtituted or substituted through available carbon atoms with one or more groups such as halogen, alkyl, haloalkyl, hydroxyl, alkoxy, carbonyl, amido, alkylamido, dialkylamido, cyano, nitro, CO 2 H, amino, alkylamino, dialkylamino, carboxyl, thio, thioalkyl, C 1 -C 5 linear or branched haloalkoxy, CF 3 , phenyl, halophenyl, (benzyl oxy )phenyl, - CH 2 CN, NH 2 , NH-alkyl, N(alkyl) 2 , -OC(O)CF 3 , -OCH 2 Ph, -NHCO-alkyl, -C(O)Ph, C(O)O-alkyl, C(O)H, -C(O)NH 2 or any combination thereof.
  • groups such
  • cycloalkyl refers herein to a ring structure comprising carbon atoms as ring atoms, which may be either saturated or unsaturated, substituted, or unsubstituted, single or fused.
  • the cycloalkyl is a 3-10 membered ring.
  • the cycloalkyl is a 3-12 membered ring.
  • the cycloalkyl is a 6 membered ring.
  • the cycloalkyl is a 5-7 membered ring.
  • the cycloalkyl is a 3-8 membered ring.
  • the cycloalkyl group may be unsubstituted or substituted by a halogen, alkyl, haloalkyl, hydroxyl, alkoxy, carbonyl, amido, alkylamido, dialkylamido, cyano, nitro, CO 2 H, amino, alkylamino, dialkylamino, carboxyl, thio, thioalkyl, Ci- C5 linear or branched haloalkoxy, CF 3 , phenyl, halophenyl, (benzyloxy)phenyl, -CH 2 CN, NH 2 , NH-alkyl, N(alkyl) 2 , -OC(O)CF 3 , -OCH 2 Ph, -NHCO-alkyl, -C(O)Ph, C(O)O-alkyl, C(O)H, - C(O)NH 2 or any combination thereof.
  • the cycloalkyl ring may be fused to another saturated or unsaturated cycloalkyl or heterocyclic 3-8 membered ring. In some embodiments, the cycloalkyl ring is a saturated ring. In some embodiments, the cycloalkyl ring is an unsaturated ring.
  • Nonlimiting examples of a cycloalkyl group comprise cyclohexyl, cyclohexenyl, cyclopropyl, cyclopropenyl, cyclopentyl, cyclopentenyl, cyclobutyl, cyclobutenyl, cycloctyl, cycloctadienyl (COD), cycloctaene (COE) etc.
  • alkyl-aryl refers herein to an alkyl group as defined herein substituted by an aryl.
  • a non-limited example is a benzyl (-CH 2 -PI1).
  • alkyl-O-alkyl refers herein to an ether group.
  • Non limited examples of an - alkyl-O-alkyl includes diethyl ether, dimethyl ether, methyl-ethyl-ether.
  • alkoxy refers to -O-alkyl group wherein the alkyl group as defined above. Alkoxy refers both to linear and to branched alkoxy groups. Nonlimiting examples of alkoxy groups are methoxy, ethoxy, propoxy, iso-propoxy, tert-butoxy.
  • 5 or 6 membered substituted or unsubstituted ring refers to a cycloalkyl, heterocycloalkyl, aryl or heteroaryl ring as defined herein.
  • heterocycloalkyl used herein alone or as part of another group refers to a cycloalkyl as defined herein having at least one heteroatom such as oxygen, sulfur and/or nitrogen.
  • the disclosure includes “pharmaceutically acceptable salts” of the compounds described herein, which may be produced, by reaction of a compound described herein with an acid or base. Certain compounds, particularly those possessing acid or basic groups, can also be in the form of a salt, preferably a pharmaceutically acceptable salt.
  • pharmaceutically acceptable salt refers to those salts that retain the biological effectiveness and properties of the free bases or free acids, which are not biologically or otherwise undesirable.
  • the salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxylic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, /'/-acetylcysteine and the like.
  • Other salts are known to those of skill in the art and can readily be adapted for use in accordance with the present disclosure.
  • Suitable pharmaceutically-acceptable salts of amines of compounds described herein may be prepared from an inorganic acid or from an organic acid.
  • examples of inorganic salts of amines are bisulfates, borates, bromides, chlorides, hemisulfates, hydrobromates, hydrochlorates, 2-hydroxyethylsulfonates (hydroxyethanesulfonates), iodates, iodides, isothionates, nitrates, persulfates, phosphate, sulfates, sulfamates, sulfanilates, sulfonic acids (alkyl sulfonates, aryl sulfonates, halogen substituted alkylsulfonates, halogen substituted aryl sulfonates), sulfonates and thiocyanates.
  • examples of organic salts of amines may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which are acetates, arginines, aspartates, ascorbates, adipates, anthranilates, algenates, alkane carboxylates, substituted alkane carboxylates, alginates, benzenesulfonates, benzoates, bisulfates, butyrates, bicarbonates, bitartrates, citrates, camphorates, camphorsulfonates, cyclohexylsulfamates, cyclopentanepropionates, calcium edetates, camsylates, carbonates, clavulanates, cinnamates, dicarboxylates, digluconates, dodecyl sulfonates, dihydrochlorides, decano
  • examples of inorganic salts of carboxylic acids or hydroxyls may be selected from ammonium, alkali metals to include lithium, sodium, potassium, cesium; alkaline earth metals to include calcium, magnesium, aluminium; zinc, barium, cholines, quaternary ammoniums.
  • examples of organic salts of carboxylic acids or hydroxyl may be selected from arginine, organic amines to include aliphatic organic amines, alicyclic organic amines, aromatic organic amines, benzathines, t-butylamines, benethamines (N- benzylphenethylamine), dicyclohexylamines, dimethylamines, diethanolamines, ethanolamines, ethylenediamines, hydrabamines, imidazoles, lysines, methylamines, meglamines, N-methyl-D- glucamines, N,N’-dibenzylethylenediamines, nicotinamides, organic amines, ornithines, pyridines, picolies, piperazines, procain, tris(hydroxymethyl)methylamines, triethylamines, triethanolamines, trimethylamines, tromethamines and urea
  • the salts may be formed by conventional means, such as by reacting the free base or free acid form of the product with one or more equivalents of the appropriate acid or base in a solvent or medium in which the salt is insoluble or in a solvent such as water, which is removed in vacuo or by freeze drying or by exchanging the ions of an existing salt for another ion or suitable ion-exchange resin.
  • the present disclosure provides compounds which are useful for inhibiting autophagy. In some embodiments, the present disclosure provides compounds which are useful for inhibiting lysosomal degradation of intracellular components. In some embodiments, the present disclosure provides compounds which are useful for inhibiting autophagy-dependent secretion of cytokines. In some embodiments, the present disclosure provides compounds which are useful for treating cancer. In some embodiments, the present disclosure provides compounds which are useful for decreasing or inhibiting cytokine production or secretion in a subject experiencing cytokine release syndrome (CRS) or cytokine storm. In some embodiments, the present disclosure provides compounds which are useful for inhibiting autophagic cell death (ACD). In some embodiments, the present disclosure provides compounds which are useful for treating autophagy-related diseases or disorders.
  • CRS cytokine release syndrome
  • ACD autophagic cell death
  • the compounds described herein comprise autophagy inhibitors. In some embodiments, the compounds described herein are autophagy inhibitors. In some embodiments, the compounds are autophagy inhibitor compounds. In some embodiments, the compounds are inhibitors of autophagy-related protein-protein interactions (PPIs).
  • PPIs autophagy-related protein-protein interactions
  • autophagy -related disease or an “autophagy- related disorder” refers to a disease or disorder that results from disruption in autophagy or cellular self-digestion.
  • autophagy-related disease or disorder may present itself as a reduced or increased level of autophagy in asubject (compared to normal autophagy levels in a healthy subject).
  • autophagy -related diseases or disorders may include, for example, cancer, including metastasis of cancer, and inflammatory diseases (such as those with increased cytokine production or secretion.
  • Another aspect of the present disclosure relates to a pharmaceutical composition including a pharmaceutically acceptable carrier and a compound according to the embodiments of the present disclosure.
  • the pharmaceutical composition can contain one or more of the aboveidentified compounds.
  • the compounds and pharmaceutical compositions described herein may be administered by any suitable route, preferably in the form of a pharmaceutical composition adapted to such a route, and in a dose effective for the treatment intended.
  • the pharmaceutical composition may be administered orally, mucosally, or parentally including intravascularly, intraperitoneally, subcutaneously, intramuscularly, and intrasternally.
  • the pharmaceutical composition described herein will include a compound of the present disclosure or its pharmaceutically acceptable salt, as well as a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to any suitable adjuvants, carriers, excipients, or stabilizers, and can be in solid or liquid form such as, tablets, capsules, powders, solutions, suspensions, or emulsions.
  • the solid unit dosage forms can be of the conventional type.
  • the solid form can be a capsule and the like, such as an ordinary gelatin type containing the compounds described herein and a carrier, for example, lubricants and inert fillers such as, lactose, sucrose, or cornstarch.
  • these compounds are tabulated with conventional tablet bases such as lactose, sucrose, or cornstarch in combination with binders like acacia, cornstarch, or gelatin, disintegrating agents, such as cornstarch, potato starch, or alginic acid, and a lubricant, like stearic acid or magnesium stearate.
  • the tablets, capsules, and the like can also contain a binder such as gum tragacanth, acacia, corn starch, or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose, or saccharin.
  • a binder such as gum tragacanth, acacia, corn starch, or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose, or saccharin.
  • a liquid carrier such as a fatty oil.
  • tablets can be coated with shellac, sugar, or both.
  • a syrup can contain, in addition to active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye, and flavoring such as cherry or orange flavor.
  • these active compounds can be incorporated with excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, and the like. Such compositions and preparations should contain at least 0.1% of active compound.
  • the active compounds of the present disclosure may be orally administered, for example, with an inert diluent, or with an assimilable edible carrier, or they can be enclosed in hard or soft shell capsules, or they can be compressed into tablets, or they can be incorporated directly with the food of the diet.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form should be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
  • the compounds or pharmaceutical compositions of the present disclosure may also be administered in injectable dosages by solution or suspension of these materials in a physiologically acceptable diluent with a pharmaceutical adjuvant, carrier, or excipient.
  • a pharmaceutical adjuvant, carrier, or excipient include, but are not limited to, sterile liquids, such as water and oils, with or without the addition of a surfactant and other pharmaceutically and physiologically acceptable components.
  • Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil.
  • water, saline, aqueous dextrose and related sugar solution, and glycols, such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions.
  • These active compounds may also be administered parenterally. Solutions or suspensions of these active compounds can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solution, and glycols such as, propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the compounds described herein in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants.
  • suitable propellants for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants.
  • the materials described herein also may be administered in a non-pressurized form such as in a nebulizer or atomizer.
  • the amounts of compounds that are administered and the dosage regimen for treating a disease or condition with the compositions described herein depends on a variety of factors, including the age, weight, gender, the medical condition of the subject, the type of disease, the severity of the disease, the route and frequency of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined routinely using standard methods.
  • disclosed herein is a method of treating, suppressing, or inhibiting a cancer in a subject, comprising the step of administering to the subject a compound or composition, as described herein. In some embodiments, disclosed herein is a method of treating, suppressing, or inhibiting cancer in a subject, comprising the step of administering to the subject one or more compounds described herein.
  • the cancer comprises a solid tumor.
  • the cancer or tumor comprises a metastasis of a cancer or tumor.
  • the solid tumor comprises a sarcoma or a carcinoma, a fibrosarcoma, a myxosarcoma, a liposarcoma, a chondrosarcoma, an osteogenic sarcoma, a chordoma, an angiosarcoma, an endotheliosarcoma, a lymphangiosarcoma, a lymphangioendotheliosarcoma, a synovioma, a mesothelioma, an Ewing's tumor, a leiomyosarcoma, a rhabdomyosarcoma, a colon carcinoma, a pancreatic cancer or tumor, a breast cancer or tumor, an ovarian cancer or tumor, a prostate cancer or tumor, a squamous cell carcinoma,
  • a compound or composition as disclosed herein has a therapeutic and/or prophylactic efficacy against a cancer or a tumor, for example sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinom
  • the cancer comprises a diffuse cancer.
  • the cancer is widely spread; not localized or confined.
  • a diffuse cancer may comprise a non-solid tumor.
  • Examples of diffuse cancers include leukemias.
  • Leukemias comprise a cancer that starts in blood-forming tissue, such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the bloodstream.
  • a diffuse cancer comprises a B-cell malignancy.
  • the diffuse cancer comprises leukemia.
  • the cancer is lymphoma.
  • the lymphoma is large B-cell lymphoma.
  • the diffuse cancer or tumor comprises a hematological tumor.
  • hematological tumors are cancer types affecting blood, bone marrow, and lymph nodes. Hematological tumors may derive from either of the two major blood cell lineages: myeloid and lymphoid cell lines.
  • the myeloid cell line normally produces granulocytes, erythrocytes, thrombocytes, macrophages, and masT-cells, whereas the lymphoid cell line produces B, T, NK, and plasma cells. Lymphomas (e.g.
  • lymphocytic leukemias and myeloma are derived from the lymphoid line, while acute and chronic myelogenous leukemia (AML, CML), myelodysplastic syndromes and myeloproliferative diseases are myeloid in origin.
  • AML, CML acute and chronic myelogenous leukemia
  • myelodysplastic syndromes myeloproliferative diseases are myeloid in origin.
  • a non-solid (diffuse) cancer comprises a hematopoietic malignancy, a blood cell cancer, a leukemia, a myelodysplastic syndrome, a lymphoma, a multiple myeloma (a plasma cell myeloma), an acute lymphoblastic leukemia, an acute myelogenous leukemia, a chronic myelogenous leukemia, a Hodgkin lymphoma, a non-Hodgkin lymphoma, or plasma cell leukemia.
  • the compound or composition as disclosed herein has therapeutic and/or prophylactic efficacy against diffuse cancers, for example but not limited to leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocyte leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chain disease.
  • leukemias e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocyte leukemia, acute myelomonocytic
  • a method of treating a proliferative disorder, or suppressing or inhibiting a proliferative disorder in a subject comprising the step of administering to the subject a compound or composition, as described herein.
  • an autophagy -related disease or disorder in a subject comprising the step of administering to the subject a compound or composition, as described herein.
  • the autophagy-related disease is cancer.
  • ACD autophagic cell death
  • the ACD is caused by an infection. In some embodiments, the ACD is caused by a bacterial infection. In some embodiments, the ACD is caused by a viral infection. In some embodiments, the viral infection is an avian H5N1 Influenza virus infection. In some embodiments, avian H5N1 Influenza virus infection causes acute lung injury. In some embodiments, the ACD is caused by acute lung injury as a result of an avian H5N1 Influenza virus infection. In some embodiments, the inhibition of ACD is compared with a subject not administered the compound or composition described herein.
  • a method of decreasing or inhibiting cytokine production or secretion in a subject experiencing cytokine release syndrome or cytokine storm or vulnerable to cytokine release syndrome or cytokine storm comprising the step of administering to the subject a compound or composition, as described herein.
  • a method of decreasing or inhibiting cytokine production or secretion in a subject experiencing cytokine release syndrome or cytokine storm or vulnerable to cytokine release syndrome or cytokine storm comprising the step of administering to the subject a compound or composition, as described herein, wherein said administering decreases or inhibits cytokine production or secretion in said subject.
  • the decrease or inhibition of cytokine production or secretion is compared with a subject experiencing cytokine release syndrome or cytokine storm or vulnerable to cytokine release syndrome or cytokine storm and not administered the compound or composition described herein.
  • methods for decreasing or inhibiting cytokine production or secretion decrease or inhibit pro-inflammatory cytokine production or secretion.
  • methods for decreasing or inhibiting cytokine production or secretion decrease or inhibit production or secretion of at least one pro-inflammatory cytokine.
  • methods for decreasing or inhibiting cytokine production or secretion decrease or inhibit autophagy -related cytokine production or secretion.
  • methods for decreasing or inhibiting cytokine production or secretion decrease or inhibit production or secretion of at least one autophagy-related cytokine.
  • autophagy-related cytokine or “autophagy- dependent secretion of cytokines” may encompass cytokines which are associated with autophagy, or with autophagy-related diseases or disorders.
  • autophagy -related cytokines may include, for example IL-1 ⁇ , IL-6, IL-18, TNF- ⁇ , IFN-y, TGF- ⁇ , or CXCL8.
  • the methods described herein decrease or inhibit production or secretion of at least one pro-inflammatory cytokine. In some embodiments, the methods described herein decrease or inhibit production or secretion of at least one autophagy -related cytokine.
  • the cytokine is Interleukin- ip (IL-lp). In some embodiments of decreasing or inhibiting cytokine production or secretion, the cytokine is IL-6. In some embodiments of decreasing or inhibiting cytokine production or secretion, the cytokine is IL-18. In some embodiments of decreasing or inhibiting cytokine production or secretion, the cytokine is tumor necrosis factor-a ( TNF- ⁇ ). In some embodiments of decreasing or inhibiting cytokine production or secretion, the cytokine is interferon-y (IFN-y).
  • IFN-y interferon-y
  • the cytokine is transforming growth factor- ⁇ (TGF- ⁇ ). In some embodiments of decreasing or inhibiting cytokine production or secretion, the cytokine is CXCL8. In another embodiment, methods disclosed herein for decreasing or inhibiting cytokine production or secretion, result in reduction or inhibition of production or secretion of cytokines IL-1 ⁇ , IL-6, IL- 18, TNF- ⁇ , IFN-y, TGF- ⁇ , or CXCL8, or any combination thereof. In some embodiments, the autophagy related cytokine is selected from IL-1 ⁇ , IL-6, IL-18, TNF- ⁇ , IFN-y, TGF- ⁇ , or CXCL8, or any combination thereof.
  • the methods described herein comprise reducing production or secretion of one or more autophagy -related cytokines.
  • the one or more autophagy related cytokines are selected from IL-1 ⁇ , IL-6, IL- 18, TNF- ⁇ , FN-y, TGF- ⁇ , or CXCL8, or any combination thereof.
  • cytokine production or secretion may encompass fold decrease or inhibition of, for example, cytokine production or secretion, or a net decrease or inhibition, or percent (%) decrease or inhibition, or may encompass a rate of change of decrease or inhibition of a cytokine production or secretion.
  • a method of inhibiting lysosomal degradation of intracellular components comprising the step of administering to a subject a compound or composition, as described herein. In some embodiments, inhibiting lysosomal degradation of intracellular components is compared with a subject not administered the compound or composition described herein.
  • a method of inhibiting autophagy-dependent secretion of cytokines comprising the step of administering to a subject a compound or composition, as described herein. In some embodiments, inhibiting autophagy-dependent secretion of cytokines is compared with a subject not administered the compound or composition described herein.
  • methods of use of a compound or composition as described herein comprises reducing production or secretion of pro-inflammatory cytokines. In some embodiments, methods of use of a compound or composition as described herein, comprises reducing production or secretion of autophagy-related cytokines. In some embodiments, methods of use of a compound or composition as described herein, comprises reducing production or secretion of pro- inflammatory cytokines/chemokines, anti-inflammatory cytokines/chemokines, and autophagy- related cytokines.
  • the acute inflammatory response is caused by a bacterial infection.
  • the acute inflammatory response is caused by a viral infection.
  • the acute inflammatory response comprises cytokine release syndrome.
  • the acute inflammatory response comprises cytokine storm.
  • methods of use of a compound or composition as described herein comprise inhibiting autophagy -related protein-protein interactions (PPIs). In some embodiments, methods of use of a compound or composition as described herein, comprise inhibiting autophagy. In some embodiments, methods of use of a compound or composition as described herein, comprise inhibiting lysosomal degradation of intracellular components. In some embodiments, methods of use of a compound or composition as described herein, comprise inhibiting autophagydependent secretion of cytokines.
  • PPIs autophagy -related protein-protein interactions
  • a compound wherein the compound is represented by the structure of Formula I, Formula II, Formula III, Formula IV, Formula V, or Formula VI, for use in the treatment of a disease or disorder in a subject in need thereof.
  • a pharmaceutical composition comprising a compound wherein the compound is represented by the structure of Formula I, Formula II, Formula III, Formula IV, Formula V, or Formula VI, or any combination thereof, for use in the treatment of a disease or disorder in a subject in need thereof.
  • the disease or disorder is selected from cancer, a proliferative disorder, an autophagy-related disease or disorder, or cytokine release syndrome or cytokine storm.
  • the disease is cancer.
  • the disorder is a proliferative disorder.
  • the disease is an autophagy-related disease.
  • the disease is a cytokine release syndrome.
  • the disease is a cytokine storm.
  • the autophagy -related disease comprises autophagy-dependent tumors.
  • autophagy-dependent tumors display intrinsic or acquired addiction to autophagy.
  • the autophagy-dependent tumor displays intrinsic addiction to autophagy.
  • the autophagy-dependent tumor displays acquired addiction to autophagy.
  • tumors are intrinsically addicted to autophagy due to changes in their metabolism caused by Ras or BRAF oncogenes.
  • the intrinsically addicted autophagy-dependent tumor is a pancreatic tumor.
  • the intrinsically addicted autophagy-dependent tumor is a melanoma.
  • the intrinsically addicted autophagy-dependent tumor is a lung tumor.
  • the intrinsically addicted autophagy-dependent tumor is a colon tumor.
  • the intrinsically addicted autophagy-dependent tumor comprises a pancreatic, melanoma, lung, or colon tumor.
  • tumors acquire addiction to autophagy during chemotherapy, targeted therapy, radiotherapy, and immunotherapy, as a mechanism to cope with the stress conditions developing in these regimens.
  • the acquired addiction autophagydependent tumor comprises a solid tumor.
  • the combination of autophagy inhibition with anti-cancer drug treatment enhances drug cytotoxicity, thus reducing drug -resistant clones and preventing tumor relapse after treatment.
  • autophagy-dependent tumor may encompass tumors which rely on autophagy for tumor promotion and maintenance.
  • the autophagy-dependent tumor relies on autophagy even under basal conditions for meeting their metabolic demands.
  • tumors are addicted to autophagy either intrinsically or they acquire such addiction.
  • tumors with oncogenic mutations depend on autophagy in order to cope with oncogene-induced metabolic stress.
  • treatment encompass both therapeutic treatment and prophylactic or preventative measures including amelioration of a disease or condition, wherein the object is to prevent or lessen the targeted pathologic condition or disorder as described hereinabove.
  • treating may include directly affecting or curing, suppressing, inhibiting, preventing, reducing the severity of, delaying the onset of, reducing symptoms associated with the disease, disorder or condition, or any combination thereof.
  • “treating” refers inter alia to delaying progression, expediting remission, inducing remission, augmenting remission, speeding recovery, increasing efficacy of or decreasing resistance to alternative therapeutics, or any combination thereof.
  • “preventing” refers, inter alia, to delaying the onset of symptoms, preventing relapse to a disease, decreasing the number or frequency of relapse episodes, increasing latency between symptomatic episodes, or any combination thereof.
  • “suppressing” or “inhibiting”, refers inter alia to reducing the severity of symptoms, reducing the severity of an acute episode, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency of symptoms, ameliorating symptoms, reducing secondary symptoms, reducing secondary infections, prolonging patient survival, or any combination thereof.
  • subject may encompass a vertebrate, in some embodiments, to a mammal, and in some embodiments, a human.
  • Subject may also refer, in some embodiments, to domesticated animals such as cows, sheep, horses, cats, dogs and laboratory animals such as mice, rats, gerbils, hamsters, etc.
  • the subject is a human subject.
  • the subject is a child.
  • the child is an infant.
  • the subject is an adult.
  • Example 1 Design high-throughput screen for detection of inhibitors that target autophagy-related protein-protein interactions (PPIs)
  • PPIs protein-protein interactions
  • Goal' Design a screen for identifying small compounds that specifically inhibit autophagy by targeting a pair of autophagic proteins (ATGs) whose interaction is indispensable for the function of both degradative and secretory arms of autophagy.
  • ATGs autophagic proteins
  • PCA Protein fragment Complementation Assay
  • PCA Protein fragment Complementation Assay
  • Figure 2 shows the schematic screen workflow of the PCA.
  • the strategy design was based on a PCA platform for detecting PPI inhibitors in high throughput compound screens.
  • the strategy included mixing of cell lysates of two cells, each derived from transfection of an individual reporter gene (Figure 3A), to allow the detection of PPI inhibitors.
  • WT compounds (at 10 ⁇ M final concentrations) were loaded with ATG12-L1 or with ATG12- L1 K54D lysates and allowed to incubate with the compounds for 1 hour at 4°C. Then ATG3-L2 was added to the plates, and 24h later, the luminescence was measured. After data analysis, all positive hits passed through hit validation. After exclusion of false positive hits, the remaining compounds were triaged by a medicinal chemist, re-synthesized, and subjected to dose response for determining IC50 in the PCA assay. Positive compounds were then used for cell-based assays to determine inhibitory effects of the compounds on autophagy in viable cells.
  • the screening protocol was calibrated and optimized by pre-exposing the cell lysates expressing the ATG12 reporter to the compounds before adding the ATG3 cell lysates, and determining the optimal time length of lysate mixing incubation.
  • a proof of concept showing that the protocol is adequate for detecting small compound inhibitors was done using the well-known ABT-737 compound that successfully and specifically inhibited the luminescence.
  • Cell lysates comprising the target pair, the Bcl-xL/Bax proteins, fused to the luciferase reporters were mixed (Figure 3B).
  • the calibrated platform was then adapted to the high-throughput robotic system of the G- INCPM unit (Weizmann Institute core unit facility).
  • the autophagy cell-based assay uses HEK 293A cells stably expressing GFP-LC3, the marker for autophagic activity in cells. These cells were treated with the hit compounds in gradient concentrations for 24h, followed by induction of autophagy and quantification of autophagosomes formation under fluorescent microscopy. Altogether, two compounds were identified from phase 1 screen that were active in cells, measured by their capability to inhibit GFP-LC3 puncta formation. The structure of the two active compounds, Compound 200 and Compound 42 is shown in Figure 4A and their IC50 in the cell lysates is documented ( Figure 4 B and 4C). In the cell-based assays, these compounds showed dose-response dependent puncta inhibition with IC50 corresponding to 10.96 ⁇ M, and 3.9 ⁇ M, respectively.
  • Example 2 The biochemical and cellular functions of compound 200.
  • FIG. 5A- C The dose response curve measuring the inhibitory effects of compound 200 on GFP-LC3B positive membrane area/cell is shown in Figures 5A- C.
  • Western blot analysis provided an independent proof for the inhibitory effect of compound 200 on LC3B lipidation; incubation with compound 200 reduced the levels of lipidated LC3B (LC3B-II) relative to the unlipidated form (LC3B-I) upon induction of autophagosomes with Torinl and CQ (Figure 5D).
  • accumulation of the autophagy cargo receptor SQSTMl/p62 was enhanced by addition of compound 200 to Torinl/CQ treated cells, as expected for an inhibitor of autophagy (Figure 5E).
  • Compound 42 from the bioactive compound library, is also known as Silmitasertib (CX- 4945), a known inhibitor of the CK2 (Casein kinase II) protein (Siddiqui-Jain, A. et al. CX-4945, an orally bioavailable selective inhibitor of protein kinase CK2, inhibits prosurvival and angiogenic signaling and exhibits antitumor efficacy. Cancer Res. 70, 10288-10298, 2010).
  • the IOCB team prepared a derivative (named compound 55, Figure 7A) that lost its inhibitory effects on CK2, yet still retained its function as inhibitor of ATG12/ATG3 interaction in PC A cell lysates ( Figure 7B), and its ability to inhibit autophagy in the cell-based experiment.
  • the design of this improved derivative was conducted by comparing the available crystal structure of compound 42 docking within the CK2 catalytic site to its in-silico docking on ATG12 ( Figure 7C).
  • Goal Test the efficacy and specificity of Compound 200 in suppressing the growth of tumor cells that are addicted to autophagy.
  • Method two cell culture-based systems were used. In one system, based on the autophagy addicted tumor pancreatic cell line, the stand alone functions was tested (Yang, S. et al. Pancreatic cancers require autophagy for tumor growth. Genes Dev. 25, 717-729, 2011; Guo, J. Y. et al. Activated Ras requires autophagy to maintain oxidative metabolism and tumorigenesis. Genes Dev. 25, 460-470, 2011).
  • Example 5 Assessing compounds ability to inhibit the secretory arm of autophagy
  • Goal Test the ability of Compound 200 and Compound 55 to inhibit the secretory arm of autophagy.
  • Method RAW 264.7 cells were treated with LPS (1 pg/ml) for inducing cytokine storm, combined with Compound 200 or Compound 55, HCQ (20 ⁇ M), or Dexamethasone (2 pg/ml) for 24h, and level of IL1 ⁇ in the medium, a cytokine secreted after LPS exposure, was measured using ELISA procedure.
  • Results Compound 200 and Compound 55 inhibited IL-1 ⁇ secretion in LPS treated macrophage cultures ( Figure 10A and B) confirming their capability to inhibit the secretory arm of autophagy and possible involvement in the cytokine storm during acute inflammation. Compound 200 also reduced the secretion of IL-1 ⁇ in LPS treated THP-1 human macrophage-like cells ( Figure 10C).
  • ACD autophagic cell death
  • a model system of autophagic cell death in a lung cell line (Dasari, S. K. et al. Signalome-wide RNAi screen identifies GBA1 as a positive mediator of autophagic cell death. Cell Death Differ. 24, 1288-1302, 2017) is used to examine the compounds.
  • Goal Test the effect of Compound 55 on autophagic cell death (ACD).
  • A549 cells were treated with Resveratrol (RSV) for 48h at various concentrations of: 3.12, 6.25, 12.5, 25, 50, 100, and 200 ⁇ M, followed by lysis and WB, blotting for LC3-EII, which are markers for autophagosomes, and VCL (vinculin) for loading control ( Figure 11 A)
  • RSV Resveratrol
  • A549 cells were treated with RSV and DMSO for 48h, followed by viability measurement using CellTiter- Gio assay ( Figure 11B).
  • A549 cells transfected with either ATG7 or ATG12 siRNAs were treated with RSV (200 ⁇ M). After 48h cell viability was assessed using CellTiter-Glo assay and represented as fold-change in siRNA + RSV-treated cells compared with siRNA treated cells ( Figure 11C)
  • A549 cells were treated with compound 55 and with RSV (100 ⁇ M). After 48h cell viability was assessed using CellTiter-Glo assay and represented as fold-change in compound + RSV-treated cells compared to compound only treated cells ( Figure 11D). [0176] Results: Treatment of A549 cells with increasing concentrations of RSV cause an increase of LC3-II levels, which indicate activation of autophagy in cells ( Figure 11A). Treatment of A549 cells with RSV causes a reduction in cell viability ( Figure 11B).
  • Ethyl 5-bromo-l-methyl-1H-pyrazole-4-carboxylate (SC13b).
  • Ethyl 1-methyl-1H- pyrazole-4-carboxylate (1.4 g, 9 mmol) in dry THF (25 ml) was treated with TMPMgCl-LiCl (IM, 11.1 ml, 11 mmol) at -30 °C under argon atmosphere and the mixture was allowed to warm to 0 °C, cooled back to -50 °C and 1,2-dibromotetrachloroethane (3.9 g, 12 mmol) in dry THF (10 ml) was slowly added. The resulting mixture was left to warm to RT, quenched with sat.
  • Example 12 Preparation of Sulfonamide Compounds 200 , 201, 202 and 203.

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Abstract

L'invention concerne des inhibiteurs d'interactions protéine-protéine liées à l'autophagie et leur utilisation pour le traitement d'une maladie ou d'un trouble lié à l'autophagie, d'un cancer, d'un syndrome de libération des cytokines (SLC) ou d'un orage cytokinique chez un sujet.
PCT/IL2023/050009 2022-01-03 2023-01-03 Inhibiteurs d'interactions protéine-protéine liées à l'autophagie Ceased WO2023126951A1 (fr)

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