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WO2024263573A1 - Combination therapy methods for treating tp53-y220c mutant and tp53 wildtype leukemias - Google Patents

Combination therapy methods for treating tp53-y220c mutant and tp53 wildtype leukemias Download PDF

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WO2024263573A1
WO2024263573A1 PCT/US2024/034492 US2024034492W WO2024263573A1 WO 2024263573 A1 WO2024263573 A1 WO 2024263573A1 US 2024034492 W US2024034492 W US 2024034492W WO 2024263573 A1 WO2024263573 A1 WO 2024263573A1
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alkyl
independently
inhibitor
alkenyl
alkynyl
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Bing Z. CARTER
Michael Andreeff
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University of Texas System
University of Texas at Austin
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University of Texas System
University of Texas at Austin
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—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
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—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
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/63—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide
    • A61K31/635—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide having a heterocyclic ring, e.g. sulfadiazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia

Definitions

  • the present technology relates generally to methods for treating TP53-Y220C mutant and TP53 wildtype leukemias, such as acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS).
  • AML acute myeloid leukemia
  • MDS myelodysplastic syndrome
  • the tumor suppression network is an elaborate network that can prevent cells carrying an activated oncogene, damaged genome, or other cancer-promoting alteration from replicating.
  • a central component of the tumor suppression network is p53, one of the most potent tumor suppressors in the cell. Both the wild-type and mutant conformations of p53 are implicated in the progression of cancer.
  • TP53-Y220C is a recurrent hotspot TP53 mutation observed in solid tumors and hematological malignancies, predominantly in AML and MDS. It frequently occurs as a subclone among TP53-WT cancer cells.
  • PC14586 a p53 reactivator, exerts mainly a cytostatic effect, and thus has limited apoptotic activity in TP53-Y220C leukemia cells.
  • the present disclosure provides a method for treating a wild-type p53 or TP53-Y220C leukemia in a patient in need thereof comprising administering to the patient an effective amount of a TP53 reactivating indole derivative and an effective amount of an MDM2 inhibitor, a BCL-2 inhibitor, and/or an XPO-1 inhibitor, wherein the TP53 reactivating indole derivative has formula of formula (I): wherein: each - is independently a single bond or a double bond;
  • X 5 is CR 13 , N, or NR 13 ; wherein at least one of X 1 , X 2 , X 3 , and X 4 is a carbon atom connected to Q 1 ;
  • Y is N or O
  • the present disclosure provides a method for prolonging survival of a wild-type p53 or TP53-Y220C leukemia patient comprising administering to the patient an effective amount of a TP53 reactivating indole derivative and an effective amount of an MDM2 inhibitor, a BCL-2 inhibitor, and/or an XPO-1 inhibitor, wherein the TP53 reactivating indole derivative has formula of formula (I): wherein: each - is independently a single bond or a double bond;
  • X 5 is CR 13 , N, or NR 13 ; wherein at least one of X 1 , X 2 , X 3 , and X 4 is a carbon atom connected to Q 1 ;
  • Y is N or O
  • the present disclosure provides a method for selecting a leukemia patient for treatment with a TP53 reactivating indole derivative and an additional therapeutic agent comprising: detecting wild-type TP53 or TP53-Y220C mRNA or polypeptide expression in a biological sample obtained from a leukemia patient; and administering to the patient an effective amount of a TP53 reactivating indole derivative and an effective amount of an MDM2 inhibitor, a BCL-2 inhibitor, and/or an XPO-1 inhibitor, wherein the TP53 reactivating indole derivative has formula of formula (I): wherein: each - is independently a single bond or a double bond;
  • X 5 is CR 13 , N, or NR 13 ; wherein at least one of X 1 , X 2 , X 3 , and X 4 is a carbon atom connected to Q 1 ;
  • Y is N or O
  • Administering to the leukemia patient may include administering the effective amount of the TP53 reactivating indole derivative and the effective amount of the MDM2 inhibitor.
  • Administering to the leukemia patient may include administering the effective amount of the TP53 reactivating indole derivative and the effective amount of the BCL-2 inhibitor.
  • Administering to the leukemia patient may include administering the effective amount of the TP53 reactivating indole derivative and the effective amount of the XPO-1 inhibitor.
  • Administering to the leukemia patient may include administering the effective amount of the TP53 reactivating indole derivative, the effective amount of the MDM2 inhibitor, and the effective amount of the BCL-2 inhibitor.
  • Administering to the leukemia patient may include administering the effective amount of the TP53 reactivating indole derivative, the effective amount of the MDM2 inhibitor, and the effective amount of the XPO-1 inhibitor.
  • Administering to the leukemia patient may include administering the effective amount of the TP53 reactivating indole derivative, the effective amount of the BCL-2 inhibitor, and the effective amount of the XPO-1 inhibitor.
  • Administering to the leukemia patient may include administering the effective amount of the TP53 reactivating indole derivative, the effective amount of the MDM2 inhibitor, the effective amount of the BCL-2 inhibitor, and the effective amount of the XPO-1 inhibitor.
  • MDM2 inhibitors include, but are not limited to, RG7112, RO5045337, idasanutlin, nutlin-3a, RG7388, AMG-232, KRT-232, APG-115, BI-907828, CGM097, siremadlin, HDM201, milademetan, BI 907828, MEL23, MEL24, and DS-3032b and MDM2 degraders, such as spirooxindole MDM2 inhibitors (e.g., MI-1061) tethered to lenalidomide, nutlin derivatives tethered to lenalidomide analogues, YX-02-030 (a RG7112 derivative) and MS3227.
  • spirooxindole MDM2 inhibitors e.g., MI-1061
  • BCL-2 inhibitors include, but are not limited to venetoclax, obatoclax, subatoclax, maritoclax, gossypol, apogossypol, TW-37, UMI-77, APG2575, and BDA-366.
  • XPO-1 inhibitors include, but are not limited to KPT330 (Selinexor), XPOVIO, KPT8602 (Eltanexor), KPT8602, KPT330, KPT335, Verdinexor, and KPT185.
  • the MDM2 inhibitor, the BCL-2 inhibitor, and/or the XPO-1 inhibitor are sequentially, simultaneously, or separately administered with the TP53 reactivating indole derivative.
  • the MDM2 inhibitor, the BCL-2 inhibitor, the XPO-1 inhibitor, and/or the TP53 reactivating indole derivative is administered orally, intravenously, intramuscularly, intraperitoneally, or subcutaneously.
  • the leukemia is acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS).
  • AML acute myeloid leukemia
  • MDS myelodysplastic syndrome
  • the leukemia may be TP53-Y220C leukemia.
  • the TP53 reactivating indole derivative may have a formula of formula (IA) wherein:
  • X 5 is CR 13 , CH, or NR 13 ; wherein at least one of X 1 , X 2 , X 3 , and X 4 is a carbon atom connected to Q 1 ;
  • Y is N or O
  • the TP53 reactivating indole derivative may have a formula of formula (IB) wherein:
  • X 5 is CR 13 , CH, or NR 13 ; wherein at least one of X 1 , X 2 , X 3 , and X 4 is a carbon atom connected to Y;
  • Y is N or O
  • R 1 is -C(O)R 16 , -C(O)OR 16 , -C(O)NR 16 R 17 , -OR 16 , -R 16 OR 17 , -R 16 OR 17 R 18 , -SR 16 , -
  • each R 3 and R 4 is independently -C(O)R 19 , -C(O)OR 19 , -C(O)NR 19 R 20 , -SOR 19 , - SO2R 19 , hydrogen, alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl, wherein each of alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl is independently unsubstituted or substituted with one or two groups selected from alkyl or halogen, or R 3 and R 4 together with the Y atom
  • mRNA expression levels are detected via real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, microarray, dot or slot blots, in situ hybridization, or fluorescent in situ hybridization (FISH).
  • polypeptide expression levels are detected via Western blotting, enzyme-linked immunosorbent assays (ELISA), dot blotting, immunohistochemistry, immunofluorescence, immunoprecipitation, immunoelectrophoresis, or mass-spectrometry.
  • the patient is non- responsive to at least one prior line of cancer therapy, such as chemotherapy or immunotherapy.
  • the chemotherapy comprises one or more of trioxide, azacytidine, cerubidine, cyclophosphamide, cytarabine, daunorubicin hydrochloride, daurismo, dexamethasone, doxorubicin hydrochloride, enasidenib mesylate, gemtuzumab ozogamicin, gilteritinib fumarate, glasdegib maleate, idamycin PFS, idarubicin hydrochloride, idhifa, ivosidenib, midostaurin, mitoxantrone hydrochloride, mylotarg, olutasidenib, onureg, pemazyre, pemigatinib, prednisone, rezlidhia, Rituxan,
  • kits comprising (a) a TP53 reactivating indole derivative (e.g., PC14586), (b) at least one of an MDM2 inhibitor, a BCL-2 inhibitor, and/or an XPO-1 inhibitor, and (c) instructions for treating wild-type p53 leukemia and/or TP53- Y220C leukemia.
  • FIG. 1 PC14586 converts mutant p53 Y220C to wild-type p53 conformation
  • FIG. 1 shows a Western blot demonstrating the activity of PC14586 on mutant and wild-type p53 (upper panel) and a Western blot demonstrating the transcriptional activity of p53 (lower panel) after treatment with PC 14586.
  • FIGS. 2A-2B PC14586 primarily suppresses cell grow in TP53 Y220C, not in TP53 WT, KO, or TP53 R175H mutant AML cells.
  • FIG. 2A Dose-response viability curves of a panel of AML cell lines having TP53-WT, TP53-KO, TP53-R175H, or TP53- Y220C treated with PC 14586 for 120 hours.
  • 2B 7- Aminoactinomycin (7AAD)/annexin V (AnnV) curves of a panel of AML cell lines having TP53-WT, TP53- KO, TP53-R175H, or TP53-Y220C treated with PC14586 for 120 hours.
  • 7AAD 7- Aminoactinomycin
  • Annexin V Annexin V
  • FIGS. 3A-3H MDM2 inhibition enhances PC 14586 activity in AML cells having TP53-WT and the combination is highly synergistic in AML cells having TP53- Y220C.
  • FIG. 3A 7AAD/AnnV curves of AML cells having TP53-WT treated with an MDM2 inhibitor, PC 14586, or a combination of the MDM2 inhibitor and PC 14586 for 72 hours.
  • FIG. 3B Dose-response viability curves of AML cells having TP53-WT treated with the MDM2 inhibitor, PC 14586, or the combination of the MDM2 inhibitor and PC 14586 for 72 hours.
  • FIG. 3A 7AAD/AnnV curves of AML cells having TP53-WT treated with an MDM2 inhibitor, PC 14586, or a combination of the MDM2 inhibitor and PC 14586 for 72 hours.
  • FIG. 3B Dose-response viability curves of AML cells having TP
  • FIG. 3C 7AAD/AnnV curves of AML cells having TP53-Y220C treated with the MDM2 inhibitor, PC 14586, or the combination of the MDM2 inhibitor and PC 14586 for 72 hours.
  • FIG. 3D Dose-response viability curves of AML cells having TP53-Y220C treated with the MDM2 inhibitor, PC 14586, or the combination of the MDM2 inhibitor and PC14586 for 72 hours.
  • FIG. 3E 7AAD/AnnV curves of AML cells having TP53-KO treated with the MDM2 inhibitor, PC 14586, or the combination of the MDM2 inhibitor and PC14586 for 72 hours.
  • FIGS. 3F Dose-response viability curves of AML cells having TP53- KO treated with the MDM2 inhibitor, PC 14586, or the combination of the MDM2 inhibitor and PC 14586 for 72 hours.
  • FIG. 3G 7AAD/AnnV curves of AML cells having TP53- R175H treated with the MDM2 inhibitor, PC14586, or the combination of the MDM2 inhibitor and PC 14586 for 72 hours.
  • FIG. 3H Dose-response viability curves of AML cells having TP53-R175H treated with the MDM2 inhibitor, PC14586, or the combination of the MDM2 inhibitor and PC14586 for 72 hours. [0022] FIGS.
  • FIG. 4A-4H BCL-2 inhibition enhances PC 14586 activity in AML cells having TP53-WT and the combination is highly synergistic in AML cells having TP53- Y220C.
  • FIG. 4A 7AAD/AnnV curves of AML cells having TP53-WT treated with a BCL- 2 inhibitor, PC14586, or a combination of the BCL-2 inhibitor and PC14586 for 72 hours.
  • FIG. 4B Dose-response viability curves of AML cells having TP53-WT treated with the BCL-2 inhibitor, PC14586, or the combination of the BCL-2 inhibitor and PC14586 for 72 hours.
  • FIG. 4A-4H BCL-2 inhibition enhances PC 14586 activity in AML cells having TP53-WT and the combination is highly synergistic in AML cells having TP53- Y220C.
  • FIG. 4A 7AAD/AnnV curves of AML cells having TP53-WT treated with a BCL-
  • FIG. 4C 7AAD/AnnV curves of AML cells having TP53-Y220C treated with the BCL-2 inhibitor, PC14586, or the combination of the BCL-2 inhibitor and PC14586 for 72 hours.
  • FIG. 4D Dose-response viability curves of AML cells having TP53-Y220C treated with the BCL-2 inhibitor, PC 14586, or the combination of the BCL-2 inhibitor and PC14586 for 72 hours.
  • FIG. 4E 7AAD/AnnV curves of AML cells having TP53-KO treated with the BCL-2 inhibitor, PC 14586, or the combination of the BCL-2 inhibitor and PC14586 for 72 hours.
  • FIG. 4F Dose-response viability curves of AML cells having TP53- KO treated with the BCL-2 inhibitor, PC14586, or the combination of the BCL-2 inhibitor and PC 14586 for 72 hours.
  • FIG. 4G 7AAD/AnnV curves of AML cells having TP53- R175H treated with the BCL-2 inhibitor, PC14586, or the combination of the BCL-2 inhibitor and PC 14586 for 72 hours.
  • FIG. 4H Dose-response viability curves of AML cells having TP53-R175H treated with the BCL-2 inhibitor, PC14586, or the combination of the BCL-2 inhibitor and PC14586 for 72 hours.
  • FIGS. 5A-5H XPO-1 inhibition enhances PC14586 activity in AML cells having TP53-WT and the combination is highly synergistic in AML cells having TP53- Y220C.
  • FIG. 5A 7AAD/AnnV curves of AML cells having TP53-WT treated with an XPO-1 inhibitor, PC14586, or a combination of the XPO-1 inhibitor and PC14586 for 72 hours.
  • FIG. 5B Dose-response viability curves of AML cells having TP53-WT treated with the XPO-1 inhibitor, PC 14586, or the combination of the XPO-1 inhibitor and PC 14586 for 72 hours.
  • FIG. 5A 7AAD/AnnV curves of AML cells having TP53-WT treated with an XPO-1 inhibitor, PC14586, or a combination of the XPO-1 inhibitor and PC14586 for 72 hours.
  • FIG. 5B Dose-response viability curves of AML cells having
  • FIG. 5C 7AAD/AnnV curves of AML cells having TP53-Y220C treated with the XPO-1 inhibitor, PC14586, or the combination of the XPO-1 inhibitor and PC14586 for 72 hours.
  • FIG. 5D Dose-response viability curves of AML cells having TP53-Y220C treated with the XPO-1 inhibitor, PC 14586, or the combination of the XPO-1 inhibitor and PC14586 for 72 hours.
  • FIG. 5E 7AAD/AnnV curves of AML cells having TP53-KO treated with the XPO-1 inhibitor, PC 14586, or the combination of the XPO-1 inhibitor and PC14586 for 72 hours.
  • FIG. 5F Dose-response viability curves of AML cells having TP53- KO treated with the XPO-1 inhibitor, PC 14586, or the combination of the XPO-1 inhibitor and PC 14586 for 72 hours.
  • FIG. 5G 7AAD/AnnV curves of AML cells having TP53- R175H treated with the XPO-1 inhibitor, PC 14586, or the combination of the XPO-1 inhibitor and PC 14586 for 72 hours.
  • FIG. 5H Dose-response viability curves of AML cells having TP53-R175H treated with the XPO-1 inhibitor, PC14586, or the combination of the XPO-1 inhibitor and PC14586 for 72 hours.
  • FIGS. 6A-6D PC 14586 in combination with three other agents shows the highest synergy, followed by PC 14586 in combination with two other agents as compared to PC 14586 in combination with one agent or PC 14586 alone with respect to in vitro inhibition of AML cells having TP53-WT (WT) and in AML cells having TP53-Y220C (Y220C).
  • WT TP53-WT
  • Y220C TP53-Y220C
  • FIG. 6A 7AAD/AnnV curves of AML cells having TP53-WT treated with PC14586, a BCL-2 inhibitor (BCL201), an MDM2 inhibitor (HDM201), an XPO-1 inhibitor (KPT-330), or a combination of PC 14586 with one, two, or all three of these other agents after 72-hour incubation.
  • FIG. 6B Dose-response viability curves of AML cells having TP53-WT treated with PC14586, the BCL-2 inhibitor, the MDM2 inhibitor, the XPO-1 inhibitor, or the combination of PC 14586 with one, two, or all three of these other agents after 72-hour incubations.
  • FIG. 6B Dose-response viability curves of AML cells having TP53-WT treated with PC14586, the BCL-2 inhibitor, the MDM2 inhibitor, the XPO-1 inhibitor, or the combination of PC 14586 with one, two, or all three of these other agents after 72-hour in
  • FIG. 6C 7AAD/AnnV curves of AML cells having TP53-Y220C treated with PC14586, the BCL-2 inhibitor, the MDM2 inhibitor, the XPO-1 inhibitor, or the combination of PC 14586 with one, two, or all three of these other agents after 72-hour incubations.
  • FIG. 6D Dose-response viability curves of AML cells having TP53-Y220C treated with PC14586, the BCL-2 inhibitor, the MDM2 inhibitor, the XPO-1 inhibitor, or the combination of PC 14586 with one, two, or all three of these other agents after 72-hour incubations.
  • FIGS. 7A-7C Molml3 TP53-Y220C cells treated with PC14586-alone or in combination with XPO-1, MDM2, or BCL-2 inhibitors.
  • FIG. 7A Western blots demonstrating the protein levels of Molml3 TP53-Y220C cells treated for 24 hours with venetoclax (VEN, 5 nM or 10 nM), nutlin-3a (Nut, 2.5 pM or 5 pM), PC14586 (PC, 2 pM or 4 pM), KPT-8602 (KPT, 100 nM or 200 nM), or combinations of PC 14586 with venetoclax (VEN/PC, 5 nM VEN and 2 pM PC, 10 nM VEN and 4 pM PC), nutlin-3a (Nut/PC, 2.5 pM Nut and 2 pM PC, 5 pM Nut and 4 pM PC), or KPT-8602 (KPT/PC, 100 nM or
  • FIG. 7B Cell cycle distribution and apoptosis determined by flow cytometry of cells stained with 5-ethynyl-2’-deoxyuridine (EdU) and DNA dye.
  • Molml3 TP53-Y220C cells were treated for 72 hours with venetoclax (VEN, 10 nM), nutlin-3a (N3, 5 pM), KPT-8602 (KPT, 200 nM), PC 14586 (4 pM), or combinations of PC 14586 with venetoclax (4 pM PC 14586 and 10 nM VEN), nutlin-3a (4 pM PC14586 and 5 pM N3), or KPT (4 pM PC14586 and 200 nM KPT).
  • FIG. 1 Ventoclax
  • nutlin-3a N3, 5 pM
  • KPT-8602 KPT, 200 nM
  • PC 14586 4 pM
  • combinations of PC 14586 with venetoclax (4 pM PC 14586 and
  • FIGS. 8A-8G Cells from an AML patient peripheral blood (PB) sample with 77% TP53-Y220C under mesenchymal stroma cell (MSC) co-culture conditions (COX). Cell death and viable cells were determined by flow cytometry after the cells were stained with annexin V/7-aminoactinomycin D (Ann V/7-ADD) in the presence of counting beads.
  • FIG. 8 A CD45+, CD34+, and CD34+CD38- population after 48 hours of treatment with PC14586 at different concentrations.
  • FIG. 8B CD45+ population assessed by Ann V/7- ADD+ positive cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT- 8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC14586; or nutlin-3a, KPT-8602, and PC14586.
  • FIG. 8B CD45+ population assessed by Ann V/7- ADD+ positive cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT- 8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC14586; or nutlin-3a, KPT-8602, and PC14586.
  • FIG. 8C CD45+ viable cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC14586.
  • FIG. 8D CD34+ assessed by Ann V/7ADD+ cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC14586; venetoclax and PC14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC 14586.
  • FIG. 8D CD34+ assessed by Ann V/7ADD+ cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC14586; venetoclax and PC14586; nutlin-3
  • FIG. 8E CD34+ viable cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC14586.
  • FIG. 8E CD34+ viable cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC14586.
  • FIG. 8F CD34+CD38- population assessed by Ann V/7ADD+ cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC14586.
  • FIG. 8F CD34+CD38- population assessed by Ann V/7ADD+ cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC14586.
  • FIGS. 9A-9G Cells from an AML patient peripheral blood (PB) sample with 45.7% TP53-Y220C under MSC COX. Cell death and viable cells were determined by flow cytometry after the cells were stained with Ann V/7-ADD in the presence of counting beads.
  • FIG. 9A-9G Cells from an AML patient peripheral blood (PB) sample with 45.7% TP53-Y220C under MSC COX. Cell death and viable cells were determined by flow cytometry after the cells were stained with Ann V/7-ADD in the presence of counting beads.
  • FIG. 9A CD45+, CD34+, and CD34+CD38- population after 48 hours of treatment with PC 14586 at different concentrations.
  • FIG. 9B CD45+ population assessed by Ann V/7-ADD+ positive cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT- 8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC14586; or nutlin-3a, KPT-8602, and PC14586.
  • FIG. 9B CD45+ population assessed by Ann V/7-ADD+ positive cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT- 8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC14586; or nutlin-3a, KPT-8602, and PC14586.
  • FIG. 9B CD45+ population assessed
  • FIG. 9C CD45+ viable cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC14586.
  • FIG. 9D CD34+ assessed by Ann V/7ADD+ cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC14586; venetoclax and PC14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC 14586.
  • FIG. 9D CD34+ assessed by Ann V/7ADD+ cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC14586; venetoclax and PC14586; nutlin-3
  • FIG. 9E CD34+ viable cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC14586.
  • FIG. 9E CD34+ viable cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC14586.
  • 9G CD34+CD38- viable cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC14586; KPT-8602 and PC14586; or nutlin-3a, KPT-8602, and PC14586.
  • FIGS. 10A-10D Patient-derived xenograft cells with Y220C mutation under MSC COX. Cell death and viable cells were determined by flow cytometry after the cells were stained with Ann V/7-ADD in the presence of counting beads.
  • FIG. 10A CD45+, CD34+, and CD34+CD38- population after 96 hours of treatment with PC14586 at different concentrations.
  • FIG. 10A CD45+, CD34+, and CD34+CD38- population after 96 hours of treatment with PC14586 at different concentrations.
  • 10B CD45+ population assessed by Ann V/7-ADD+ positive cells after 96 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; nutlin-3a, KPT-8602, and PC 14586; venetoclax, KPT-8602, and PC 14586; KPT-8602, nutlin-3a, and PC14586; or venetoclax, nutlin-3a, KPT-8602, and PC14586.
  • 10C CD45+ viable cells after 96 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; nutlin-3a, KPT-8602, and PC 14586; venetoclax, KPT-8602, and PC 14586; KPT-8602, nutlin-3a, and PC14586; or venetoclax, nutlin-3a, KPT-8602, and PC14586.
  • 10D CD34+ assessed by Ann V/7ADD+ cells after 96 hours of treatment with venetoclax; nutlin-3a (N3); KPT- 8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; nutlin-3a, KPT-8602, and PC 14586; venetoclax, KPT-8602, and PC 14586; KPT- 8602, nutlin-3a, and PC14586; or venetoclax, nutlin-3a, KPT-8602, and PC14586.
  • FIG. 10E CD34+ viable cells after 96 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC14586; venetoclax and PC14586; nutlin-3a and PC14586; KPT-8602 and PC 14586; nutlin-3a, KPT-8602, and PC 14586; venetoclax, KPT-8602, and PC 14586; KPT-8602, nutlin-3a, and PC 14586; or venetoclax, nutlin-3a, KPT-8602, and PC14586.
  • FIG. 10F CD34+CD38- population assessed by Ann V/7ADD+ cells after 96 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; nutlin-3a, KPT-8602, and PC 14586; venetoclax, KPT-8602, and PC 14586; KPT-8602, nutlin-3a, and PC 14586; or venetoclax, nutlin-3a, KPT-8602, and PC 14586.
  • FIG. 10G CD34+CD38- viable cells after 96 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; nutlin-3a, KPT-8602, and PC 14586; venetoclax, KPT- 8602, and PC14586; KPT-8602, nutlin-3a, and PC14586; or venetoclax, nutlin-3a, KPT- 8602, and PC 14586.
  • FIGS. 11A-11D Normal bone marrow cells under MSC COX. Cell death and viable cells were determined by flow cytometry after the cells were stained with Ann V/7- ADD in the presence of counting beads.
  • FIG. 11 A CD45+ population assessed by Ann V/7-ADD+ positive cells after 48 hours of treatment with venetoclax; nutlin-3a; KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or venetoclax, nutlin-3a, KPT-8602, and PC14586.
  • FIG. 11 A CD45+ population assessed by Ann V/7-ADD+ positive cells after 48 hours of treatment with venetoclax; nutlin-3a; KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or venetoclax, nutlin-3a, KPT-
  • FIG. 1 IB CD45+ cell viability after 48 hours of treatment with venetoclax; nutlin-3a; KPT-8602; PC14586; venetoclax and PC14586; nutlin-3a and PC14586; KPT-8602 and PC14586; or venetoclax, nutlin-3a, KPT- 8602, and PC14586.
  • FIG. 1 IB CD45+ cell viability after 48 hours of treatment with venetoclax; nutlin-3a; KPT-8602; PC14586; venetoclax and PC14586; nutlin-3a and PC14586; KPT-8602 and PC14586; or venetoclax, nutlin-3a, KPT- 8602, and PC14586.
  • 11C CD34+ population assessed by Ann V/7-ADD+ positive cells after 48 hours of treatment with venetoclax; nutlin-3a; KPT-8602; PC14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or venetoclax, nutlin-3a, KPT-8602, and PC 14586.
  • FIG. 11C CD34+ population assessed by Ann V/7-ADD+ positive cells after 48 hours of treatment with venetoclax; nutlin-3a; KPT-8602; PC14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or venetoclax, nutlin-3a, KPT-8602, and PC 14586.
  • FIG. 12A-12F Molml3 TP53-WT and TP53-Y220C cells were treated with nutlin-3a (N3a, 5 pM), PC14586 (PC, 4 pM), or the combination for 4 hours.
  • RNA was isolated and subject to RNA sequence.
  • FIG. 12A Principal component analysis.
  • FIG. 12B Gene set enrichment analysis.
  • FIG. 12C Differentially expressed genes.
  • FIG. 12D Pathway analysis.
  • FIG. 12E Volcano plots show comparisons of increased, unchanged, and decreased gene expression in treated compared to controls.
  • FIG. 12F Western blots showing changes in several gene expression.
  • FIG. 13A-13C Patient-derived xenograft cells from an AML patient sample with TP53-Y220C (VAF 48%), TP53-P151A (VAF 47%), and NRAS (VAF 50%) mutations were injected via tail vein into NSG mice followed by treatment.
  • FIG. 13A Flow cytometry analysis of human CD45+ cells in peripheral blood (PB) after four weeks of treatment.
  • FIG. 13B Flow cytometry analysis of human CD45+ cells in peripheral blood (PB) after eight weeks of treatment.
  • FIG. 13C Kaplan-Meier estimation of mouse survival and survival data analyzed using the log-rank test.
  • FIG. 14 Western blot of p53 and BCL-2 from Molml3 TP53-WT and TP53- Y220C cells treated for 4 hours with nutlin-3a (5 pM) or PC 14586 (PC, 1.25 pM or 5 pM).
  • FIG. 15 Kaplan-Meier estimation of mouse survival and survival data analyzed using the log-rank test of mice injected with Molml3 TP53-Y220C cells, and treated daily with vehicle, PC14586 (100 mg/kg), venetoclax (50 mg/kg), or the combination.
  • FIG. 16 Western blot of p53, Lamin Bl, and tubulin from TP53-Y220C Molml3 cells treated with PC14586 (4 pM), KPT-8602 (200 nM), or both for 24 hours.
  • PC14586 4 pM
  • KPT-8602 200 nM
  • references to a certain element such as hydrogen or H is meant to include all isotopes of that element.
  • an R group is defined to include hydrogen or H, it also includes deuterium and tritium.
  • Compounds comprising radioisotopes such as tritium, C 14 , P 32 and S 35 are thus within the scope of the present technology. Procedures for inserting such labels into the compounds of the present technology will be readily apparent to those skilled in the art based on the disclosure herein.
  • the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).
  • the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including but not limited to, orally, intranasally, intrathecally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, intraocularly, intradermally, transmucosally, iontophoretically, or topically. Administration includes self-administration and the administration by another.
  • cancer or “tumor” are used interchangeably and refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells can exist alone within an animal, or can be a non-tumorigenic cancer cell. As used herein, the term “cancer cells” includes precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and non-metastatic cells.
  • cancers of virtually every tissue are known to those of skill in the art, including solid tumors such as carcinomas, sarcomas, glioblastomas, melanomas, etc., and circulating cancers such as leukemias.
  • cancer include, but are not limited to, ovarian cancer, breast cancer, colon cancer, lung cancer, prostate cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, head and neck cancer, and brain cancer.
  • cancer burden or “tumor burden” refers to the quantity of cancer cells or tumor volume in a subject.
  • Reducing cancer burden accordingly may refer to reducing the number of cancer cells, or the tumor volume in a subject.
  • cancer cell refers to a cell that exhibits cancer-like properties, e.g., uncontrollable reproduction, resistance to anti- growth signals, ability to metastasize, and loss of ability to undergo programmed cell death (e.g., apoptosis) or a cell that is derived from a cancer cell, e.g., clone of a cancer cell.
  • control is an alternative sample used in an experiment for comparison purpose.
  • a control can be "positive” or “negative.”
  • a positive control a compound or composition known to exhibit the desired therapeutic effect
  • a negative control a subject or a sample that does not receive the therapy or receives a placebo
  • the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein.
  • the amount of a composition administered to the subject will vary depending on the composition, the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
  • the compositions can also be administered in combination with one or more additional therapeutic compounds.
  • the therapeutic compositions may be administered to a subject having one or more signs or symptoms of a disease or condition described herein.
  • a "therapeutically effective amount" of a composition refers to composition levels in which the physiological effects of a disease or condition are ameliorated or eliminated.
  • a therapeutically effective amount can be given in one or more administrations.
  • expression includes one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function.
  • polypeptide As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to mean a polymer comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, z.e., peptide isosteres.
  • Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides, or oligomers, and to longer chains, generally referred to as proteins.
  • Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.
  • Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art.
  • a “sample” or “biological sample” refers to a body fluid or a tissue sample isolated from a subject.
  • a biological sample may consist of or comprise whole blood, platelets, red blood cells, white blood cells, plasma, sera, urine, feces, epidermal sample, vaginal sample, skin sample, cheek swab, sperm, amniotic fluid, cultured cells, bone marrow sample, tumor biopsies, aspirate and/or chorionic villi, cultured cells, endothelial cells, synovial fluid, lymphatic fluid, ascites fluid, interstitial or extracellular fluid and the like.
  • sample may also encompass the fluid in spaces between cells, including gingival crevicular fluid, bone marrow, cerebrospinal fluid (CSF), saliva, mucus, sputum, semen, sweat, urine, or any other bodily fluids.
  • Samples can be obtained from a subject by any means including, but not limited to, venipuncture, excretion, ejaculation, massage, biopsy, needle aspirate, lavage, scraping, surgical incision, or intervention or other means known in the art.
  • a blood sample can be whole blood or any fraction thereof, including blood cells (red blood cells, white blood cells or leukocytes, and platelets), serum and plasma.
  • the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.
  • sequential therapeutic use refers to administration of at least two active ingredients at different times. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.
  • the term “simultaneous” therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time.
  • the terms “subject”, “patient”, or “individual” can be an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the subject, patient, or individual is a human.
  • a “synergistic therapeutic effect” refers to a greater-than-additive therapeutic effect which is produced by a combination of at least two agents, and which exceeds that which would otherwise result from the individual administration of the agents. For example, lower doses of one or more agents may be used in treating a disease or disorder.
  • the term “therapeutic agent” is intended to mean a compound that, when present in an effective amount, produces a desired therapeutic effect on a subject in need thereof.
  • Treating” or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, i.e., arresting its development; (ii) relieving a disease or disorder, i.e., causing regression of the disorder; (iii) slowing progression of the disorder; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder.
  • treatment means that the symptoms associated with the disease are, e.g., alleviated, reduced, cured, or placed in a state of remission.
  • the various modes of treatment of disorders as described herein are intended to mean “substantial,” which includes total but also less than total treatment, and wherein some biologically or medically relevant result is achieved.
  • the treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.
  • references to a certain element such as hydrogen or H is meant to include all isotopes of that element.
  • an R group is defined to include hydrogen or H, it also includes deuterium and tritium.
  • Compounds comprising radioisotopes such as tritium, C 14 , P 32 and S 35 are thus within the scope of the present technology. Procedures for inserting such labels into the compounds of the present technology will be readily apparent to those skilled in the art based on the disclosure herein.
  • substituted refers to an organic group as defined below (e.g., an alkyl group) in which one or more bonds to a hydrogen atom contained therein are replaced by a bond to non-hydrogen or non-carbon atoms.
  • Substituted groups also include groups in which one or more bonds to a carbon(s) or hydrogen(s) atom are replaced by one or more bonds, including double or triple bonds, to a heteroatom.
  • a substituted group is substituted with one or more substituents, unless otherwise specified.
  • a substituted group is substituted with 1, 2, 3, 4, 5, or 6 substituents.
  • substituent groups include: halogens i.e., F, Cl, Br, and I); hydroxyls; alkoxy, alkenoxy, aryloxy, aralkyloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, and heterocyclylalkoxy groups; carbonyls (oxo); carboxylates; esters; urethanes; oximes; hydroxylamines; alkoxyamines; aralkoxyamines; thiols; sulfides; sulfoxides; sulfones; sulfonyls; pentafluorosulfanyl (i.e., SFs), sulfonamides; amines; N-oxides; hydrazines; hydrazides; hydrazones; azides; amides; ureas; amidines; guanidines; enamines; imides; isocyanates; isothio
  • Substituted ring groups such as substituted cycloalkyl, aryl, heterocyclyl and heteroaryl groups also include rings and ring systems in which a bond to a hydrogen atom is replaced with a bond to a carbon atom. Therefore, substituted cycloalkyl, aryl, heterocyclyl and heteroaryl groups may also be substituted with substituted or unsubstituted alkyl, alkenyl, and alkynyl groups as defined below.
  • Alkyl groups include straight chain and branched chain alkyl groups having from 1 to 12 carbon atoms, and typically from 1 to 10 carbons or, in some embodiments, from 1 to 8, 1 to 6, or 1 to 4 carbon atoms. Alkyl groups may be substituted or unsubstituted. Examples of straight chain alkyl groups include groups such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups.
  • branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, tert-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups.
  • Representative substituted alkyl groups may be substituted one or more times with substituents such as those listed above, and include without limitation haloalkyl (e.g., trifluoromethyl), hydroxyalkyl, thioalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, alkoxyalkyl, carboxyalkyl, and the like.
  • Cycloalkyl groups include mono-, bi- or tricyclic alkyl groups having from 3 to 12 carbon atoms in the ring(s), or, in some embodiments, 3 to 10, 3 to 8, or 3 to 4, 5, or 6 carbon atoms. Cycloalkyl groups may be substituted or unsubstituted. Exemplary monocyclic cycloalkyl groups include, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups.
  • the cycloalkyl group has 3 to 8 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 5, 3 to 6, or 3 to 7.
  • Bi- and tricyclic ring systems include both bridged cycloalkyl groups and fused rings, such as, but not limited to, bicyclo[2.1.1]hexane, adamantyl, decalinyl, and the like.
  • Substituted cycloalkyl groups may be substituted one or more times with non-hydrogen and non-carbon groups as defined above.
  • substituted cycloalkyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined above.
  • Representative substituted cycloalkyl groups may be mono-substituted or substituted more than once, such as, but not limited to, 2,2-, 2,3-, 2,4- 2,5- or 2,6-disubstituted cyclohexyl groups, which may be substituted with substituents such as those listed above.
  • Cycloalkylalkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a cycloalkyl group as defined above. Cycloalkylalkyl groups may be substituted or unsubstituted. In some embodiments, cycloalkylalkyl groups have from 4 to 16 carbon atoms, 4 to 12 carbon atoms, and typically 4 to 10 carbon atoms. Substituted cycloalkylalkyl groups may be substituted at the alkyl, the cycloalkyl or both the alkyl and cycloalkyl portions of the group. Representative substituted cycloalkylalkyl groups may be mono-substituted or substituted more than once, such as, but not limited to, mono-, di- or tri -substituted with substituents such as those listed above.
  • Alkenyl groups include straight and branched chain alkyl groups as defined above, except that at least one double bond exists between two carbon atoms. Alkenyl groups may be substituted or unsubstituted. Alkenyl groups have from 2 to 12 carbon atoms, and typically from 2 to 10 carbons or, in some embodiments, from 2 to 8, 2 to 6, or 2 to 4 carbon atoms. In some embodiments, the alkenyl group has one, two, or three carboncarbon double bonds.
  • substituted alkenyl groups may be mono-substituted or substituted more than once, such as, but not limited to, mono-, di- or tri -substituted with substituents such as those listed above.
  • Cycloalkenyl groups include cycloalkyl groups as defined above, having at least one double bond between two carbon atoms. Cycloalkenyl groups may be substituted or unsubstituted. In some embodiments the cycloalkenyl group may have one, two or three double bonds but does not include aromatic compounds. Cycloalkenyl groups have from 4 to 14 carbon atoms, or, in some embodiments, 5 to 14 carbon atoms, 5 to 10 carbon atoms, or even 5, 6, 7, or 8 carbon atoms. Examples of cycloalkenyl groups include cyclohexenyl, cyclopentenyl, cyclohexadienyl, cyclobutadienyl, and cyclopentadienyl.
  • Cycloalkenylalkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of the alkyl group is replaced with a bond to a cycloalkenyl group as defined above. Cycloalkenylalkyl groups may be substituted or unsubstituted. Substituted cycloalkenylalkyl groups may be substituted at the alkyl, the cycloalkenyl or both the alkyl and cycloalkenyl portions of the group. Representative substituted cycloalkenylalkyl groups may be substituted one or more times with substituents such as those listed above.
  • Aryl groups are cyclic aromatic hydrocarbons that do not contain heteroatoms.
  • Aryl groups herein include monocyclic, bicyclic and tricyclic ring systems.
  • Aryl groups may be substituted or unsubstituted.
  • aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, fluorenyl, phenanthrenyl, anthracenyl, indenyl, indanyl, pentalenyl, and naphthyl groups.
  • aryl groups contain 6-14 carbons, and in others from 6 to 12 or even 6-10 carbon atoms in the ring portions of the groups.
  • the aryl groups are phenyl or naphthyl.
  • aryl groups includes groups containing fused rings, such as fused aromatic-aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl, and the like).
  • Representative substituted aryl groups may be mono-substituted (e.g., tolyl) or substituted more than once.
  • monosubstituted aryl groups include, but are not limited to, 2-, 3-, 4-, 5-, or 6-substituted phenyl or naphthyl groups, which may be substituted with substituents such as those listed above.
  • Aralkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined above.
  • Aralkyl groups may be substituted or unsubstituted.
  • aralkyl groups contain 7 to 16 carbon atoms, 7 to 14 carbon atoms, or 7 to 10 carbon atoms.
  • Substituted aralkyl groups may be substituted at the alkyl, the aryl or both the alkyl and aryl portions of the group.
  • Representative aralkyl groups include but are not limited to benzyl and phenethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-indanylethyl.
  • Heterocyclyl groups include aromatic (also referred to as heteroaryl) and nonaromatic ring compounds containing 3 or more ring members, of which one or more is a heteroatom such as, but not limited to, N, O, and S. Heterocyclyl groups may be substituted or unsubstituted. In some embodiments, the heterocyclyl group contains 1, 2, 3 or 4 heteroatoms. In some embodiments, heterocyclyl groups include mono-, bi- and tricyclic rings having 3 to 16 ring members, whereas other such groups have 3 to 6, 3 to 10, 3 to 12, or 3 to 14 ring members.
  • Heterocyclyl groups encompass aromatic, partially unsaturated and saturated ring systems, such as, for example, imidazolyl, imidazolinyl and imidazolidinyl groups.
  • the phrase “heterocyclyl group” includes fused ring species including those comprising fused aromatic and non-aromatic groups, such as, for example, benzotri azolyl, 2,3-dihydrobenzo[l,4]dioxinyl, and benzo[l,3]dioxolyl.
  • the phrase also includes bridged polycyclic ring systems containing a heteroatom such as, but not limited to, quinuclidyl.
  • heterocyclyl groups that have other groups, such as alkyl, oxo or halo groups, bonded to one of the ring members, referred to as “substituted heterocyclyl groups”.
  • Heterocyclyl groups include, but are not limited to, aziridinyl, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, thiazolidinyl, tetrahydrothiophenyl, tetrahydrofuranyl, dioxolyl, furanyl, thiophenyl, pyrrolyl, pyrrolinyl, imidazolyl, imidazolinyl, pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, thiazolinyl, isothiazolyl, thiadiazolyl, o
  • substituted heterocyclyl groups may be mono-substituted or substituted more than once, such as, but not limited to, pyridyl or morpholinyl groups, which are 2-, 3-, 4-, 5-, or 6-substituted, or disubstituted with various substituents such as those listed above.
  • Heteroaryl groups are aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S. Heteroaryl groups may be substituted or unsubstituted.
  • Heteroaryl groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiophenyl, benzothiophenyl, furanyl, benzofuranyl, indolyl, azaindolyl (pyrrolopyridinyl), indazolyl, benzimidazolyl, imidazopyridinyl (azabenzimidazolyl), pyrazolopyridinyl, triazolopyridinyl, benzotri azolyl, benzoxazolyl, benzothiazolyl, benzothiadi azolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthyl, purin
  • Heteroaryl groups include fused ring compounds in which all rings are aromatic such as indolyl groups and include fused ring compounds in which only one of the rings is aromatic, such as 2,3-dihydro indolyl groups.
  • Representative substituted heteroaryl groups may be substituted one or more times with various substituents such as those listed above.
  • Heterocyclylalkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heterocyclyl group as defined above. Heterocyclylalkyl groups may be substituted or unsubstituted. Substituted heterocyclylalkyl groups may be substituted at the alkyl, the heterocyclyl or both the alkyl and heterocyclyl portions of the group.
  • heterocyclyl alkyl groups include, but are not limited to, morpholin-4-yl-ethyl, furan-2-yl-methyl, imidazol-4-yl-methyl, pyridin-3-yl-methyl, tetrahydrofuran-2-yl-ethyl, and indol-2-yl-propyl.
  • Representative substituted heterocyclylalkyl groups may be substituted one or more times with substituents such as those listed above.
  • Heteroaralkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heteroaryl group as defined above. Heteroaralkyl groups may be substituted or unsubstituted. Substituted heteroaralkyl groups may be substituted at the alkyl, the heteroaryl or both the alkyl and heteroaryl portions of the group. Representative substituted heteroaralkyl groups may be substituted one or more times with substituents such as those listed above.
  • Groups described herein having two or more points of attachment i.e., divalent, trivalent, or polyvalent
  • divalent alkyl groups are alkylene groups
  • divalent aryl groups are arylene groups
  • divalent heteroaryl groups are divalent heteroarylene groups
  • Substituted groups having a single point of attachment to the compound of the present technology are not referred to using the “ene” designation.
  • chloroethyl is not referred to herein as chloroethylene.
  • Alkoxy groups are hydroxyl groups (-OH) in which the bond to the hydrogen atom is replaced by a bond to a carbon atom of a substituted or unsubstituted alkyl group as defined above. Alkoxy groups may be substituted or unsubstituted. Examples of linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, and the like. Examples of branched alkoxy groups include but are not limited to isopropoxy, sec-butoxy, tert-butoxy, isopentoxy, isohexoxy, and the like.
  • cycloalkoxy groups include but are not limited to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like.
  • Representative substituted alkoxy groups may be substituted one or more times with substituents such as those listed above.
  • alkyloyl and alkyloyloxy can refer, respectively, to -C(O)-alkyl groups and -O-C(O)-alkyl groups.
  • aryloyl and aryloyloxy refer to -C(O)-aryl groups and -O-C(O)-aryl groups.
  • aryloxy and arylalkoxy refer to, respectively, a substituted or unsubstituted aryl group bonded to an oxygen atom and a substituted or unsubstituted aralkyl group bonded to the oxygen atom at the alkyl. Examples include but are not limited to phenoxy, naphthyloxy, and benzyloxy. Representative substituted aryloxy and arylalkoxy groups may be substituted one or more times with substituents such as those listed above.
  • carboxylate refers to a -COOH group.
  • esters refers to -COOR 70 and -C(O)O-G groups.
  • R 70 is a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heterocyclylalkyl or heterocyclyl group as defined herein.
  • G is a carboxylate protecting group.
  • Carboxylate protecting groups are well known to one of ordinary skill in the art. An extensive list of protecting groups for the carboxylate group functionality may be found in Protective Groups in Organic Synthesis, Greene, T.W.; Wuts, P. G. M., John Wiley & Sons, New York, NY, (3rd Edition, 1999) which can be added or removed using the procedures set forth therein and which is hereby incorporated by reference in its entirety and for any and all purposes as if fully set forth herein.
  • amide includes C- and N-amide groups, i.e., -C(O)NR 71 R 72 , and -NR 71 C(O)R 72 groups, respectively.
  • R 71 and R 72 are independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl or heterocyclyl group as defined herein.
  • Amido groups therefore include but are not limited to carbamoyl groups (-C(O)NH2) and formamide groups (-NHC(O)H).
  • the amide is -NR 71 C(O)-(CI-5 alkyl) and the group is termed “carbonylamino,” and in others the amide is -NHC(O)-alkyl and the group is termed "alkanoylamino.”
  • nitrile or “cyano” as used herein refers to the -CN group.
  • Urethane groups include N- and O-urethane groups, i.e., -NR 73 C(O)OR 74 and -OC(O)NR 73 R 74 groups, respectively.
  • R 73 and R 74 are independently a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl, or heterocyclyl group as defined herein.
  • R 73 may also be H.
  • amine refers to -NR 75 R 76 groups, wherein R 75 and R 76 are independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl or heterocyclyl group as defined herein.
  • the amine is alkylamino, dialkylamino, arylamino, or alkylarylamino.
  • the amine is NH2, methylamino, dimethylamino, ethylamino, diethylamino, propylamino, isopropylamino, phenylamino, or benzylamino.
  • sulfonamido includes S- and N-sulfonamide groups, i.e., -SO2NR 78 R 79 and -NR 78 SO2R 79 groups, respectively.
  • R 78 and R 79 are independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl, or heterocyclyl group as defined herein.
  • Sulfonamido groups therefore include but are not limited to sulfamoyl groups (-SO2NH2).
  • the sulfonamido is -NHSCh-alkyl and is referred to as the "alkylsulfonylamino" group.
  • thiol refers to -SH groups
  • sulfides include -SR 80 groups
  • sulfoxides include -S(O)R 81 groups
  • sulfones include -SO2R 82 groups
  • sulfonyls include -SO2OR 83
  • sulfonates include -SO3 .
  • R 80 , R 81 , R 82 , and R 83 are each independently a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.
  • the sulfide is an alkylthio group, -S-alkyl.
  • urea refers to -NR 84 -C(O)-NR 85 R 86 groups.
  • R 84 , R 85 , and R 86 groups are independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclyl, or heterocyclylalkyl group as defined herein.
  • amidine refers to -C(NR 87 )NR 88 R 89 and -NR 87 C(NR 88 )R 89 , wherein R 87 , R 88 , and R 89 are each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.
  • guanidine refers to -NR 90 C(NR 91 )NR 92 R 93 , wherein R 90 , R 91 , R 92 and R 93 are each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.
  • halogen refers to bromine, chlorine, fluorine, or iodine. In some embodiments, the halogen is fluorine. In other embodiments, the halogen is chlorine or bromine.
  • hydroxyl as used herein can refer to -OH or its ionized form, -O .
  • a “hydroxyalkyl” group is a hydroxyl -substituted alkyl group, such as HO-CH2-.
  • imide refers to -C(O)NR 98 C(O)R 99 , wherein R 98 and R 99 are each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.
  • the term “imine” refers to -CR 100 (NR 101 ) and -N(CR 100 R 101 ) groups, wherein R 100 and R 101 are each independently hydrogen or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein, with the proviso that R 100 and R 101 are not both simultaneously hydrogen.
  • nitro refers to an -NO2 group.
  • trifluoromethyl refers to -CF3.
  • trifluoromethoxy refers to -OCF3.
  • trialkyl ammonium refers to a -N(alkyl)s group.
  • a trialkylammonium group is positively charged and thus typically has an associated anion, such as halogen anion.
  • isocyano refers to -NC.
  • isothiocyano refers to -NCS.
  • pentafluorosulfanyl refers to -SF5.
  • molecular weight (also known as “relative molar mass”) is a dimensionless quantity but is converted to molar mass by multiplying by 1 gram/mole or by multiplying by 1 Da - for example, a compound with a weight-average molecular weight of 5,000 has a weight-average molar mass of 5,000 g/mol and a weight-average molar mass of 5,000 Da.
  • salts of compounds described herein are within the scope of the present technology and include acid or base addition salts which retain the desired pharmacological activity and is not biologically undesirable (e.g., the salt is not unduly toxic, allergenic, or irritating, and is bioavailable).
  • pharmaceutically acceptable salts can be formed with inorganic acids (such as hydrochloric acid, hydroboric acid, nitric acid, sulfuric acid, and phosphoric acid), organic acids (e.g., alginate, formic acid, acetic acid, benzoic acid, gluconic acid, fumaric acid, oxalic acid, tartaric acid, lactic acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, naphthalene sulfonic acid, and p-toluenesulfonic acid) or acidic amino acids (such as aspartic acid and glutamic acid).
  • inorganic acids such as hydrochloric acid, hydroboric acid, nitric acid, sulfuric acid, and phosphoric acid
  • organic acids e.g., alginate, formic acid, acetic acid, benzoic acid, gluconic acid, fumaric acid, ox
  • the compound of the present technology can form salts with metals, such as alkali and earth alkali metals (e.g., Na + , Li + , K + , Ca 2+ , Mg 2+ , Zn 2+ ), ammonia or organic amines (e.g., di cyclohexylamine, trimethylamine, tri ethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine) or basic amino acids (e.g., arginine, lysine and ornithine).
  • metals such as alkali and earth alkali metals (e.g., Na + , Li + , K + , Ca 2+ , Mg 2+ , Zn 2+ ), ammonia or organic amines (e.g., di cyclohexylamine, trimethylamine, tri ethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine) or basic amino acids
  • Tautomers refers to isomeric forms of a compound that are in equilibrium with each other. The presence and concentrations of the isomeric forms will depend on the environment the compound is found in and may be different depending upon, for example, whether the compound is a solid or is in an organic or aqueous solution. For example, in aqueous solution, quinazolinones may exhibit the following isomeric forms, which are referred to as tautomers of each other:
  • guanidines may exhibit the following isomeric forms in protic organic solution, also referred to as tautomers of each other: Because of the limits of representing compounds by structural formulas, it is to be understood that all chemical formulas of the compounds described herein represent all tautomeric forms of compounds and are within the scope of the present technology.
  • Stereoisomers of compounds include all chiral, diastereomeric, and racemic forms of a structure, unless the specific stereochemistry is expressly indicated.
  • compounds used in the present technology include enriched or resolved optical isomers at any or all asymmetric atoms as are apparent from the depictions.
  • racemic and diastereomeric mixtures, as well as the individual optical isomers can be isolated or synthesized so as to be substantially free of their enantiomeric or diastereomeric partners, and these stereoisomers are all within the scope of the present technology.
  • Leukemias are blood cancers that usually begin in the bone marrow and result in high numbers of abnormal blood cells. These blood cells are not fully developed and are called blasts or leukemia cells. Symptoms may include bleeding and bruising, bone pain, fatigue, fever, and an increased risk of infections. These symptoms occur due to a lack of normal blood cells. Diagnosis is typically made by blood tests or bone marrow biopsy.
  • ALL acute lymphoblastic leukemia
  • AML acute myeloid leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myeloid leukemia
  • TP53 Mutations in TP53 are present in approximately 10% of patients with AML and myelodysplastic syndrome (MDS), and represent a unique subtype with poor outcome.
  • MDS myelodysplastic syndrome
  • TP53 is located on chromosome 17p 13 and is essential for cell cycle control and DNA damage response.
  • TP53 mutations drive a dominant negative effect and typically occur in founding clones that expand after cytotoxic stress.
  • Patients with TP53-mutant AML have a very poor prognosis and lack durable responses to essentially all current therapies.
  • TP53- Y220C is a recurrent hotspot TP53 mutation observed in numerous solid tumors and hematological malignancies.
  • TP53 reactivating indole derivatives are known in the art and are described in US Patent No. 10,640,485, the contents of which are incorporated herein by reference in its entirety.
  • the TP53 reactivating indole derivative may have a formula of formula (I): wherein: each - is independently a single bond or a double bond;
  • X 5 is CR 13 , N, or NR 13 ; wherein at least one of X 1 , X 2 , X 3 , and X 4 is a carbon atom connected to Q 1 ;
  • Y is N or O
  • the TP53 reactivating indole derivative may have a formula of formula (IA) wherein:
  • X 5 is CR 13 , CH, or NR 13 ; wherein at least one of X 1 , X 2 , X 3 , and X 4 is a carbon atom connected to Q 1 ;
  • Y is N or O
  • the TP53 reactivating indole derivative may have a formula of formula (IB) wherein:
  • X 5 is CR 13 , CH, or NR 13 ; wherein at least one of X 1 , X 2 , X 3 , and X 4 is a carbon atom connected to Y;
  • Y is N or O
  • Non-limiting examples of TP53 reactivating indole derivatives include compounds of any of the following formulae:
  • the indole derivative is a compound of the formula
  • each is independently a single bond or a double bond
  • X 5 is CR 13 , N, or NR 13 ; wherein at least one of X 1 , X 2 , X 3 , and X 4 is a carbon atom connected to Q
  • the pattern of dashed bonds is chosen to provide an aromatic system, for example, an indole, an indolene, a pyrrolopyridine, a pyrrolopyrimidine, or a pyrrolopyrazine.
  • X 1 is CR 5 , CR 5 R 6 , or a carbon atom connected to Q 1 .
  • X 2 is CR 7 , CR 7 R 8 , or a carbon atom connected to Q 1 .
  • X 3 is CR 9 , CR 9 R 10 , or a carbon atom connected to Q 1 .
  • X 4 is CR 11 , CR n R 12 , or a carbon atom connected to Q 1 .
  • X 5 is CR 13 , N, or NR 13 .
  • X 1 is a carbon atom connected to Q 1 .
  • X 2 is a carbon atom connected to Q 1 .
  • X 3 is a carbon atom connected to Q 1 .
  • X 4 is a carbon atom connected to Q 1 .
  • X 5 is N.
  • the compound is of the formula:
  • the compound is of the formula:
  • R 1 is — C(O)R 16 , — C(O)OR 16 , — C(O)NR 16 R 17 , —OR 16 , —SR 16 , — NR 16 R 17 , — NR 16 C(O)R 16 , — OC(O)R 16 , SiR 16 R 17 R 18 , alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen.
  • m is 1, 2, 3, or 4. In some embodiments, m is 1. In some embodiments, X 3 is carbon atom connected to Q 1 , and m is 1. In some embodiments, the compound is of the formula:
  • R 1 is — C(O)R 16 , — C(O)OR 16 , — C(O)NR 16 R 17 , —OR 16 , —SR 16 , —
  • NR 16 R 17 — NR 16 C(O)R 16 , — OC(O)R 16 , SiR 16 R 17 R 18 , alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen.
  • R is alkyl, alkenyl, — C(O)R 16 , — C(O)OR 16 , or — C(O)NR 16 R 17 .
  • R 1 is a substituted alkyl.
  • R 1 can be substituted by one or more substituents selected from hydroxyl groups, sulfhydryl groups, halogens, amino groups, nitro groups, nitroso groups, cyano groups, azido groups, sulfoxide groups, sulfone groups, sulfonamide groups, carboxyl groups, carboxaldehyde groups, imine groups, alkyl groups, halo-alkyl groups, cyclic alkyl groups, alkenyl groups, halo-alkenyl groups, alkynyl groups, halo-alkynyl groups, alkoxy groups, aryl groups, aryloxy groups, aralkyl groups, arylalkoxy groups, heterocyclyl groups, acyl groups, acyloxy groups, carbamate groups, amide groups, urethane groups, and ester groups.
  • R 1 is alkyl substituted with an amine group.
  • R 1 is alkyl substituted with amine group.
  • Q 1 is alkylene, alkenylene, or alkynylene.
  • Q 1 is Ci-alkylene.
  • each R 16 and R 17 is independently alkyl, alkenyl, aryl, heteroaryl, heterocyclyl, or hydrogen.
  • Q 1 is Ci- alkylene, R 16 is aryl, and R 17 is alkyl.
  • Q 1 is Ci-alkylene, R 16 is aryl, and R 17 is hydrogen. In some embodiments, Q 1 is Ci-alkylene, R 16 is heteroaryl, and R 17 is alkyl. In some embodiments, Q 1 is Ci-alkylene, R 16 is heteroaryl, and R 17 is hydrogen. In some embodiments, Q 1 is Ci-alkylene, R 16 is substituted heteroaryl, and R 17 is hydrogen. In some embodiments, Q 1 is Ci-alkylene, R 16 is substituted alkyl, and R 17 is hydrogen. In some embodiments, R 17 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted with halogen, alkyl, or hydroxyl.
  • R 16 is hydrogen, and R 17 is aryl or heteroaryl, substituted or unsubstituted with halogen or alkyl.
  • R 16 is alkyl, and R 17 is heteroaryl substituted with halogen or alkyl.
  • R 17 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted with alkyl.
  • R 17 is aryl or heteroaryl, each of which is independently substituted with alkyl, wherein the alkyl is optionally substituted with fluorine, chlorine, bromine, iodine, or cyano.
  • R 2 is hydrogen or alkyl.
  • R 13 is alkyl, alkenyl, hydrogen, or halogen.
  • R 2 is alkyl, and R 13 is alkyl.
  • R 2 is hydrogen, and R 13 is alkyl.
  • R 2 is methyl, ethyl, propyl, iso-propyl, butyl, or tert-butyl.
  • R 13 is methyl, ethyl, propyl, iso-propyl, butyl, or tert-butyl.
  • R 2 is hydrogen, and R 13 is hydrogen.
  • R 3 is — C(O)R 19 , — C(O)OR 19 , alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen
  • R 4 is — C(O)R 19 , — C(O)OR 19 , alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen.
  • R 3 is hydrogen and R 4 is substituted alkyl.
  • R 3 is hydrogen and R 4 is alkyl substituted with aryl.
  • R 3 is alkyl and R 4 is alkyl.
  • R 3 is alkyl and R 4 is aryl.
  • R 3 is hydrogen
  • R 4 is heterocyclyl
  • the compound is of the formula:
  • R 3 and R 4 together with the nitrogen atom to which R 3 and R 4 are bound form a ring, wherein the ring is substituted or unsubstituted. In some embodiments, R 3 and R 4 together with the nitrogen atom to which R 3 and R 4 are bound form a substituted heterocycle. In some embodiments, R 3 and R 4 together with the nitrogen atom to which R 3 and R 4 are bound form a heterocycle substituted with a hydroxyl group, halogen, amino group, or alkyl group. In some embodiments, R 3 and R 4 together with the nitrogen atom to which R 3 and R 4 are bound form a heterocycle, wherein the heterocycle is substituted by a substituted or unsubstituted heterocycle.
  • R 3 and R 4 together with the nitrogen atom to which R 3 and R 4 are bound form a ring of a following formula:
  • R 16 is alkyl, alkenyl, aryl, heteroaryl, heterocyclyl, or hydrogen
  • R 17 is aryl, heteroaryl, or heterocyclyl.
  • R 17 is phenyl, indolyl, piperidinyl, imidazolyl, thiazolyl, morpholinyl, pyrrolyl, or pyridinyl.
  • the compound is of the formula:
  • the compound is of the formula:
  • the compound is of the formula:
  • each Z 1 and Z 2 is independently CR X or N; each R x is independently — C(O)R 21 , — C(O)OR 21 , — C(O)NR 21 R 22 , —OR 21 , —SR 21 , — NR 21 R 22 , — NR 21 C(O)R 22 , — OC(O)R 21 , alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, hydrogen, or halogen; each R 25 and R 26 is independently — C(O)R 21 , — C(O)OR 21 , — C(O)NR 21 R 22 , —OR 21 , —SR 21 , — SO 2 R 21 , — NR 21 R 22 , — NR 21 C(O)R 22 , — OC(O)R 21 , alkyl, alkeny
  • Z 1 is N. In some embodiments, Z 1 and Z 2 are N. In some embodiments, each R 25 and R 26 is independently a halogen. In some embodiments, R 25 is [0138] In some embodiments, R25 is SO2R 21 . In some embodiments, R 25 is SO2R 21 , wherein R 21 is alkyl. In some embodiments, R 25 is SO2R 21 , wherein R 21 is methyl.
  • Non-limiting examples of compounds of the current disclosure include the following:
  • Non-limiting examples of compounds of the current disclosure include the following:
  • Non-limiting examples of compounds of the current disclosure include the following:
  • the compound is of the formula
  • each L 1 and L 2 is independently an ester, ether, thioether, polyethyleneglycol (PEG), alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocyclyl ene, arylene, heteroarylene, or heterocycloalkylene group, any of which is substituted or unsubstituted.
  • each L 1 and L 2 is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, or heterocycloalkylene.
  • L 1 is alkylene and L 2 is an ester.
  • Compounds herein can include all stereoisomers, enantiomers, diastereomers, mixtures, racemates, atropisomers, and tautomers thereof.
  • Non-limiting examples of optional substituents include hydroxyl groups, sulfhydryl groups, halogens, amino groups, nitro groups, nitroso groups, cyano groups, azido groups, sulfoxide groups, sulfone groups, sulfonamide groups, carboxyl groups, carboxaldehyde groups, imine groups, alkyl groups, halo-alkyl groups, alkenyl groups, halo- alkenyl groups, alkynyl groups, halo-alkynyl groups, alkoxy groups, aryl groups, aryloxy groups, aralkyl groups, arylalkoxy groups, heterocyclyl groups, acyl groups, acyloxy groups, carbamate groups, amide groups, ureido groups, epoxy groups, and ester groups.
  • Non-limiting examples of alkyl and alkylene groups include straight, branched, and cyclic alkyl and alkylene groups.
  • An alkyl or alkylene group can be, for example, a Ci, C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , Cio, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, C35, C36, C37, C38, C39, C40, C41, C42, C43, C44, C45, C46, C47, C48, C49, or C50 group that is substituted or unsubstituted.
  • Non-limiting examples of straight alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl.
  • Branched alkyl groups include any straight alkyl group substituted with any number of alkyl groups.
  • Non-limiting examples of branched alkyl groups include isopropyl, isobutyl, sec-butyl, and t-butyl.
  • Non-limiting examples of substituted alkyl groups includes hydroxymethyl, chloromethyl, trifluoromethyl, aminomethyl, 1 -chloroethyl, 2-hydroxy ethyl, 1,2- difluoroethyl, and 3 -carboxypropyl.
  • Non-limiting examples of cyclic alkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptlyl, and cyclooctyl groups. Cyclic alkyl groups also include fused-, bridged-, and spiro-bicycles and higher fused-, bridged-, and spiro- systems. A cyclic alkyl group can be substituted with any number of straight, branched, or cyclic alkyl groups.
  • Non-limiting examples of cyclic alkyl groups include cyclopropyl, 2-methyl- cycloprop-l-yl, cycloprop-2-en-l-yl, cyclobutyl, 2,3-dihydroxycyclobut-l-yl, cyclobut-2- en-l-yl, cyclopentyl, cyclopent-2-en-l-yl, cyclopenta-2,4-dien-l-yl, cyclohexyl, cyclohex- 2-en-l-yl, cycloheptyl, cyclooctanyl, 2,5-dimethylcyclopent-l-yl, 3,5-dichlorocyclohex-l- yl, 4-hydroxycyclohex-l-yl, 3,3,5-trimethylcyclohex-l-yl, octahydropentalenyl, octahydro- IH-indenyl, 3a,4,5,6,7,
  • Non-limiting examples of alkenyl and alkenylene groups include straight, branched, and cyclic alkenyl groups.
  • the olefin or olefins of an alkenyl group can be, for example, E, Z, cis, trans, terminal, or exo-methylene.
  • An alkenyl or alkenylene group can be, for example, a C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , Cio, Cn, C12, C13, C i4 , C15, C i6 , C17, C i8 , C19, C20, C2I, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, C35, C36, C37, C38, C39, C40, C41, C42, C43, C44, C45, C46, C47, C48, C49, or C50 group that is substituted or unsubstituted.
  • Non-limiting examples of alkenyl and alkenylene groups include ethenyl, prop-l-en-l-yl, isopropenyl, but- 1-en -4-yl; 2-chloroethenyl, 4-hydroxybuten-l-yl, 7- hydroxy-7-methyloct-4-en-2-yl, and 7-hydroxy-7-methyloct-3,5-dien-2-yl.
  • Non-limiting examples of alkynyl or alkynylene groups include straight, branched, and cyclic alkynyl groups.
  • the triple bond of an alkylnyl or alkynylene group can be internal or terminal.
  • An alkylnyl or alkynylene group can be, for example, a C2, C3, C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, c 35 , C36, C37, C38, C39, C40, C41, C42, C43, C44, C45, C46, C47, C48, C49, or C50 group that is substituted or unsubstituted.
  • Non-limiting examples of alkynyl or alkynylene groups include ethynyl, prop-2-yn-l-yl, prop-l-yn-l-yl, and 2-methyl-hex-4-yn-l-yl; 5-hydroxy-5-methylhex-3-yn-l-yl, 6-hydroxy-6-methylhept-3- yn-2-yl, and 5-hydroxy-5-ethylhept-3-yn-l-yl.
  • a halo-alkyl group can be any alkyl group substituted with any number of halogen atoms, for example, fluorine, chlorine, bromine, and iodine atoms.
  • a halo-alkenyl group can be any alkenyl group substituted with any number of halogen atoms.
  • a halo- alkynyl group can be any alkynyl group substituted with any number of halogen atoms.
  • An alkoxy group can be, for example, an oxygen atom substituted with any alkyl, alkenyl, or alkynyl group.
  • An ether or an ether group comprises an alkoxy group.
  • alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, and isobutoxy.
  • An aryl group can be heterocyclic or non-heterocyclic.
  • An aryl group can be monocyclic or polycyclic.
  • An aryl group can be substituted with any number of substituents described herein, for example, hydrocarbyl groups, alkyl groups, alkoxy groups, and halogen atoms.
  • Non-limiting examples of aryl groups include phenyl, toluyl, naphthyl, pyrrolyl, pyridyl, imidazolyl, thiophenyl, and furyl.
  • Non-limiting examples of substituted aryl groups include 3,4-dimethylphenyl, 4-tert-butylphenyl, 4-cyclopropylphenyl, 4- diethylaminophenyl, 4-(trifluoromethyl)phenyl, 4-(difluoromethoxy)-phenyl, 4- (trifluorom ethoxy )phenyl, 3 -chlorophenyl, 4-chlorophenyl, 3, 4-di chlorophenyl, 2- fluorophenyl, 2-chlorophenyl, 2-iodophenyl, 3 -iodophenyl, 4-iodophenyl, 2-methylphenyl, 3 -fluorophenyl, 3 -methylphenyl, 3 -methoxyphenyl, 4-fluorophenyl, 4-methylphenyl, 4- methoxyphenyl, 2,3 -difluorophenyl, 3,4-difluorophenyl, 3,5-d
  • Non-limiting examples of substituted aryl groups include 2-aminophenyl, 2-(N- methylamino)phenyl, 2-(N,N-dimethylamino)phenyl, 2-(N-ethylamino)phenyl, 2-(N,N- diethylamino)phenyl, 3 -aminophenyl, 3-(N-methylamino)phenyl, 3-(N,N- dimethylamino)phenyl, 3-(N-ethylamino)phenyl, 3-(N,N-diethylamino)phenyl, 4- aminophenyl, 4-(N-methylamino)phenyl, 4-(N,N-dimethylamino)phenyl, 4-(N- ethylamino)phenyl, and 4-(N,N-diethylamino)phenyl.
  • a heterocycle can be any ring containing a ring atom that is not carbon, for example, N, O, S, P, Si, B, or any other heteroatom.
  • a heterocycle can be substituted with any number of substituents, for example, alkyl groups and halogen atoms.
  • a heterocycle can be aromatic (heteroaryl) or non-aromatic.
  • Non-limiting examples of heterocycles include pyrrole, pyrrolidine, pyridine, piperidine, succinamide, maleimide, morpholine, imidazole, thiophene, furan, tetrahydrofuran, pyran, and tetrahydropyran.
  • Non-limiting examples of heterocycles include: heterocyclic units having a single ring containing one or more heteroatoms, non-limiting examples of which include, diazirinyl, aziridinyl, azetidinyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolinyl, thiazolidinyl, isothiazolinyl, oxathiazolidinonyl, oxazolidinonyl, hydantoinyl, tetrahydrofuranyl, pyrrolidinyl, morpholinyl, piperazinyl, piperidinyl, dihydropyranyl, tetrahydropyranyl, piperidin-2-onyl, 2,3,4,5-tetrahydro-lH-azepinyl, 2,3-dihydro-lH-indole, and 1,2,3,4-tetrahydroquino
  • heteroaryl include: i) heteroaryl rings containing a single ring, non-limiting examples of which include, 1,2,3,4-tetrazolyl, [l,2,3]triazolyl, [l,2,4]triazolyl, triazinyl, thiazolyl, IH-imidazolyl, oxazolyl, isoxazolyl, isothiazolyl, furanyl, thiophenyl, pyrimidinyl, 2-phenylpyrimidinyl, pyridinyl, 3-methylpyridinyl, and 4- dimethylaminopyridinyl; and ii) heteroaryl rings containing 2 or more fused rings one of which is a heteroaryl ring, non-limiting examples of which include: 7H-purinyl, 9H-purinyl, 6-amino-9H-purinyl, 5H-pyrrolo[3,2-d]pyrimidinyl, 7H-pyr
  • the TP53 reactivating indole derivative is PC14586 (also called rezatapopt; see https://classic.clinicaltrials.gov/ct2/show/NCT04585750;
  • PC14586 is a first-in- class, oral, small molecule p53 reactivator selective for the p53 Y220C mutation.
  • PC14586 is designed to bind non-covalently to the crevice created by the Y220C mutation and restore WT p53 function.
  • the phase 1 clinical trial of PC14586 in locally advanced or metastatic solid tumors that have a TP53 Y220C mutation demonstrated only limited activity, including limited cell death.
  • PC 14586 has a structure of formula
  • Murine double minute 2 homolog also known as E3 ubiquitin-protein ligase MDM2 is a protein that in humans is encoded by the MDM2 gene. MDM2 is an important negative regulator of the p53 tumor suppressor. MDM2 protein functions both as an E3 ubiquitin ligase that recognizes the N-terminal trans-activation domain (TAD) of the p53 tumor suppressor and as an inhibitor of p53 transcriptional activation. MDM2 inhibitors target TP53 WT cells but have the potential to select/enrich TP53 mutant leukemia cells. MDM2 inhibitors activate p53, which in turn induces MDM2 and p21 levels, which can result in cell cycle arrest, but not in apoptosis.
  • TAD N-terminal trans-activation domain
  • MDM2 inhibitors bind to the MDM2 protein, thereby preventing the binding of the MDM2 protein to the transcriptional activation domain of the tumor suppressor protein p53.
  • MDM2 inhibitors can be selective or non- selective, small molecules or biomolecules.
  • MDM2 inhibitors include, but are not limited to, RG7112, RO5045337, idasanutlin, nutlin-3a, RG7388, AMG-232, KRT-232, APG-115, BI-907828, CGM097, siremadlin, HDM201, milademetan, MEL23, MEL24, and DS-3032b.
  • MDM2 inhibitors may include MDM2 degraders, which degrade MDM2 protein.
  • MDM2 inhibitors may include proteolysis targeting chimeras (PROTACs), which are heterobifunctional molecules composed of a targeting ligand tethered to an E3 ubiquitin ligase recruiting ligand to induce selective degradation of MDM2.
  • PROTACs proteolysis targeting chimeras
  • MDM2 degrader PROTACs examples include spirooxindole MDM2 inhibitors e.g., MI-1061) tethered to lenalidomide, nutlin derivatives (e.g., idasanutlin, nutlin-3a) tethered to lenalidomide analogues, YX-02-030 (a RG7112 derivative) and MS3227, as described in more detail in B. Wang et al. Development of selective small molecule MDM2 degraders based on nutlin, Eur. J. Med. Chem. 2019, 176, 476-491; C. M.
  • BCL-2 B-cell lymphoma 2 proteins are a class of proteins that are regulators of apoptosis. BCL-2 is variably highly expressed in many hematological malignancies, providing protection from cell death induced by oncogenic and external stresses.
  • BCL-2 inhibitors bind to BCL-2 protein, thereby preventing protection from apoptosis.
  • BCL-2 inhibitors can be selective or non- selective, small molecules or biomolecules.
  • Examples of BCL-2 inhibitors include, but are not limited to, venetoclax, obatoclax, subatoclax, maritoclax, gossypol, apogossypol, TW-37, UMI-77, and BDA-366.
  • XPO-1 Inhibitors include, but are not limited to, venetoclax, obatoclax, subatoclax, maritoclax, gossypol, apogossypol, TW-37, UMI-77, and BDA-366.
  • Exportin 1 also known as chromosomal region maintenance 1 (CRM1)
  • CCM1 chromosomal region maintenance 1
  • XPO-1 mediates NES-dependent protein transport. It exports several hundreds of different proteins from the nucleus.
  • XPO-1 is involved in the nuclear export of ribosomal subunits.
  • XPO-1 is affected in some cancer types and is therefore viewed as a target for development of anti-cancer drugs.
  • XPO-1 overexpression plays a role in the onset and progression of both solid tumors and hematological malignancies and is associated with a poor prognosis in patients.
  • XPO-1 inhibitors bind to the XPO-1 protein, thereby preventing or substantially reducing nuclear export, which can increase accumulation of tumor suppressor proteins, reduce oncoproteins, and increase apoptosis.
  • XPO-1 inhibitors can be selective or non- selective, small molecules or biomolecules. Examples of XPO-1 inhibitors include, but are not limited to, KPT330 (Selinexor), XPOVIO, KPT8602 (Eltanexor), KPT335, Verdinexor, and KPT 185.
  • compositions of the present technology include a TP53 reactivating indole derivative (e.g., PC 14586) and one or more additional therapeutic agents.
  • the additional therapeutic agents may include MDM2 inhibitors, BCL-2 inhibitors and/or XPO-1 inhibitors.
  • the pharmaceutical compositions include an effective amount of a TP53 reactivating indole derivative (e.g., PC14586) and an effective amount of an MDM2 inhibitor. In some embodiments, the pharmaceutical compositions include an effective amount of a TP53 reactivating indole derivative (e.g., PC14586) and an effective amount of a BCL-2 inhibitor. In some embodiments, the pharmaceutical compositions include an effective amount of a TP53 reactivating indole derivative (e.g., PC14586) and an effective amount of XPO-1 inhibitor.
  • the pharmaceutical compositions include an effective amount of a TP53 reactivating indole derivative (e.g., PC 14586) and an effective amount of an MDM2 inhibitor and a BCL-2 inhibitor. In some embodiments, the pharmaceutical compositions include an effective amount of a TP53 reactivating indole derivative (e.g., PC 14586) and an effective amount of an MDM2 inhibitor and an XPO-1 inhibitor. In some embodiments, the pharmaceutical compositions include an effective amount of a TP53 reactivating indole derivative (e.g., PC14586) and an effective amount of a BCL-2 inhibitor and an XPO-1 inhibitor.
  • the pharmaceutical compositions include an effective amount of a TP53 reactivating indole derivative (e.g., PC 14586) and an effective amount of an MDM2 inhibitor, a BCL-2 inhibitor, and an XPO-1 inhibitor.
  • the pharmaceutical compositions may include pharmaceutically acceptable excipients, diluents, or carriers that are compatible with the one or more therapeutic agent in the pharmaceutical composition and the method of administration.
  • compositions of the present technology can be manufactured by methods well known in the art such as conventional granulating, mixing, dissolving, encapsulating, lyophilizing, or emulsifying processes, among others.
  • Compositions may be produced in various forms, including granules, precipitates, or particulates, powders, including freeze dried, rotary dried or spray dried powders, amorphous powders, tablets, capsules, syrup, suppositories, injections, emulsions, elixirs, suspensions, or solutions.
  • Formulations may optionally contain solvents, diluents, and other liquid vehicles, dispersion or suspension aids, surface active agents, pH modifiers, isotonic agents, thickening or emulsifying agents, stabilizers and preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • the compositions disclosed herein are formulated for administration to a mammal, such as a human.
  • Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -butylene glycol, cyclodextrins, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art such
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3 -butanediol.
  • compositions formulated for parenteral administration may be injected by bolus injection or by timed push, or may be administered by continuous infusion.
  • the rate of compound release can be controlled.
  • biodegradable polymers include poly(orthoesters) and poly(anhydrides).
  • Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and g
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or in a certain part of the intestinal tract, optionally, in a delayed manner.
  • Examples of embedding compositions that can be used include polymeric substances and waxes.
  • the active compounds can also be in micro-encapsulated form with one or more excipients as noted above.
  • the active compound may be admixed with at least one inert diluent such as sucrose, lactose, or starch.
  • Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose.
  • the dosage forms may also comprise buffering agents.
  • opacifying agents may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or in a certain part of the intestinal tract, optionally, in a delayed manner.
  • embedding compositions include polymeric substances and waxes.
  • the present disclosure provides a method for treating a wild-type p53 or TP53-Y220C leukemia in a patient in need thereof comprising administering to the patient an effective amount of a TP53 reactivating indole derivative and an effective amount of an MDM2 inhibitor, a BCL-2 inhibitor, and/or an XPO-1 inhibitor, wherein the TP53 reactivating indole derivative has formula of formula (I): wherein: each - is independently a single bond or a double bond;
  • X 5 is CR 13 , N, or NR 13 ; wherein at least one of X 1 , X 2 , X 3 , and X 4 is a carbon atom connected to Q 1 ;
  • Y is N or O
  • the present disclosure provides a method for selecting a leukemia patient for treatment with a TP53 reactivating indole derivative and an additional therapeutic agent comprising: detecting wild-type TP53 or TP53-Y220C mRNA or polypeptide expression in a biological sample obtained from a leukemia patient; and administering to the leukemia patient an effective amount of a TP53 reactivating indole derivative and an effective amount of at least one additional therapeutic agent selected from among an MDM2 inhibitor, a BCL-2 inhibitor, and an XPO-1 inhibitor, wherein the TP53 reactivating indole derivative has formula of formula (I): wherein: each - is independently a single bond or a double bond;
  • X 5 is CR 13 , N, or NR 13 ; wherein at least one of X 1 , X 2 , X 3 , and X 4 is a carbon atom connected to Q 1 ;
  • Y is N or O
  • the biological sample may be whole blood, serum, or plasma.
  • the TP53 reactivating indole derivative is PC 14586.
  • MDM2 inhibitors include, but are not limited to, RG7112, RO5045337, idasanutlin, nutlin-3a, RG7388, AMG-232, KRT-232, APG-115, BI-907828, CGM097, siremadlin, HDM201, milademetan, MEL23, MEL24, and DS-3032b.
  • BCL-2 inhibitors include, but are not limited to venetoclax, obatoclax, subatoclax, maritoclax, gossypol, apogossypol, TW-37, UMI-77, and BDA-366.
  • XPO-1 inhibitors include, but are not limited to KPT330 (Selinexor), XPOVIO, KPT8602 (Eltanexor), KPT335, Verdinexor, and KPT 185.
  • the MDM2 inhibitor, the BCL-2 inhibitor, and/or the XPO-1 inhibitor are sequentially, simultaneously, or separately administered with the TP53 reactivating indole derivative.
  • the MDM2 inhibitor, the BCL-2 inhibitor, the XPO-1 inhibitor, and/or the TP53 reactivating indole derivative is administered orally, intravenously, intramuscularly, intraperitoneally, or subcutaneously.
  • the leukemia is acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS).
  • AML acute myeloid leukemia
  • MDS myelodysplastic syndrome
  • mRNA expression levels are detected via real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, microarray, dot or slot blots, in situ hybridization, or fluorescent in situ hybridization (FISH).
  • polypeptide expression levels are detected via Western blotting, enzyme-linked immunosorbent assays (ELISA), dot blotting, immunohistochemistry, immunofluorescence, immunoprecipitation, immunoelectrophoresis, or mass-spectrometry.
  • the method comprises administering to the patient an effective amount of a TP53 reactivating indole derivative (e.g., PC14586) and an effective amount of an MDM2 inhibitor. In certain embodiments, the method comprises administering to the patient an effective amount of a TP53 reactivating indole derivative (e.g., PC 14586) and an effective amount of a BCL-2 inhibitor. In other embodiments, the method comprises administering to the subject an effective amount of a TP53 reactivating indole derivative (e.g., PC 14586) and an effective amount of an XPO-1 inhibitor.
  • a TP53 reactivating indole derivative e.g., PC14586
  • an MDM2 inhibitor e.g., MDM2 inhibitor
  • the method comprises administering to the patient an effective amount of a TP53 reactivating indole derivative (e.g., PC 14586) and an effective amount of a BCL-2 inhibitor.
  • the method comprises administering to the
  • the MDM2 inhibitor, the BCL-2 inhibitor, the XPO-1 inhibitor and/or the TP53 reactivating indole derivative may be administered as a single composition or as separate compositions.
  • the patient is non- responsive to at least one prior line of cancer therapy, such as chemotherapy or immunotherapy.
  • the chemotherapy comprises one or more of trioxide, azacytidine, cerubidine, cyclophosphamide, cytarabine, daunorubicin hydrochloride, daurismo, dexamethasone, doxorubicin hydrochloride, enasidenib mesylate, gemtuzumab ozogamicin, gilteritinib fumarate, glasdegib maleate, idamycin PFS, idarubicin hydrochloride, idhifa, ivosidenib, midostaurin, mitoxantrone hydrochloride, mylotarg, olutasidenib, onureg, pemazyre, pemigatinib, prednisone, rezlidhia, Rituxan,
  • the time to response and/or duration of response is improved relative to that observed with TP53 reactivating indole derivative (e.g., PC 14586) monotherapy, or monotherapy with an MDM2 inhibitor, a BCL-2 inhibitor, or XPO-1 inhibitor.
  • TP53 reactivating indole derivative e.g., PC 14586
  • MDM2 inhibitor e.g., MDM2 inhibitor
  • BCL-2 inhibitor e.g., XPO-1 inhibitor
  • the TP53 reactivating indole derivative (e.g., PC 14586) and the MDM2 inhibitor are administered sequentially, simultaneously, or separately.
  • the TP53 reactivating indole derivative (e.g., PC14586) and/or the MDM2 inhibitor may be administered orally, parenterally, by inhalation spray, intranasally, buccally, or via an implanted reservoir.
  • parenteral as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intra-synovial, intrastemal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
  • compositions are administered orally, intravenously, or subcutaneously.
  • Formulations including a TP53 reactivating indole derivative (e.g., PC14586) and/or an MDM2 inhibitor disclosed herein may be designed to be short-acting, fast-releasing, or long-acting.
  • compounds can be administered in a local rather than systemic means, such as administration (e.g., by injection) at a tumor site.
  • the TP53 reactivating indole derivative (e.g., PC 14586) and the BCL-2 inhibitor are administered sequentially, simultaneously, or separately.
  • the TP53 reactivating indole derivative (e.g., PC14586) and/or the BCL-2 inhibitor may be administered orally, parenterally, by inhalation spray, intranasally, buccally, or via an implanted reservoir.
  • parenteral as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intra-synovial, intrastemal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
  • compositions are administered orally, intravenously, or subcutaneously.
  • Formulations including a TP53 reactivating indole derivative (e.g., PC14586) and/or BCL-2 inhibitor disclosed herein may be designed to be short-acting, fast-releasing, or long-acting.
  • compounds can be administered in a local rather than systemic means, such as administration (e.g., by injection) at a tumor site.
  • the TP53 reactivating indole derivative (e.g., PC 14586) and the XPO-1 inhibitor are administered sequentially, simultaneously, or separately.
  • the TP53 reactivating indole derivative (e.g., PC14586) and/or the XPO-1 inhibitor may be administered orally, parenterally, by inhalation spray, intranasally, buccally, or via an implanted reservoir.
  • parenteral as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intra-synovial, intrastemal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
  • compositions are administered orally, intravenously, or subcutaneously.
  • Formulations including a TP53 reactivating indole derivative (e.g., PC14586) and/or XPO-1 inhibitor disclosed herein may be designed to be short-acting, fast-releasing, or long-acting.
  • compounds can be administered in a local rather than systemic means, such as administration (e.g., by injection) at a tumor site.
  • a TP53 reactivating indole derivative (e.g., PC14586) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), simultaneously with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of an MDM2 inhibitor, a BCL-2 inhibitor, or a XPO-1 inhibitor to a patient with leukemia.
  • a TP53 reactivating indole derivative e.g., PC 14586
  • at least one of an MDM2 inhibitor, a BCL-2 inhibitor, or an XPO-1 inhibitor are administered to a patient, for example, a mammal, such as a human, in a sequence and within a time interval such that the inhibitor that is administered first acts together with the inhibitor that is administered second to provide greater benefit than if each inhibitor were administered alone.
  • a TP53 reactivating indole derivative e.g., PC 14586
  • at least one of an MDM2 inhibitor, a BCL-2 inhibitor, or an XPO-1 inhibitor can be administered at the same time or sequentially in any order at different points in time; however, if not administered at the same time, a TP53 reactivating indole derivative (e.g., PC14586) and at least one of an MDM2 inhibitor, a BCL-2 inhibitor, or an XPO-1 inhibitor are administered sufficiently close in time so as to provide the desired therapeutic or prophylactic effect of the combination of the at least two inhibitors.
  • a TP53 reactivating indole derivative e.g., PC14586
  • at least one of an MDM2 inhibitor, a BCL-2 inhibitor, or an XPO-1 inhibitor exert their effects at times which overlap.
  • a TP53 reactivating indole derivative e.g., PC 14586
  • at least one of an MDM2 inhibitor, a BCL-2 inhibitor, or an XPO-1 inhibitor are each administered as separate dosage forms, in any appropriate form and by any suitable route.
  • a TP53 reactivating indole derivative e.g., PC 14586
  • at least one of an MDM2 inhibitor, a BCL-2 inhibitor, or an XPO-1 inhibitor are administered simultaneously in a single dosage form.
  • the frequency with which any of these therapeutic agents can be administered can be once or more than once over a period of about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 20 days, about 28 days, about a week, about 2 weeks, about 3 weeks, about 4 weeks, about a month, about every 2 months, about every 3 months, about every 4 months, about every 5 months, about every 6 months, about every 7 months, about every 8 months, about every 9 months, about every 10 months, about every 11 months, about every year, about every 2 years, about every 3 years, about every 4 years, or about every 5 years.
  • a TP53 reactivating indole derivative e.g., PC 14586
  • an MDM2 inhibitor e.g., a BCL-2 inhibitor or an XPO-1 inhibitor
  • a TP53 reactivating indole derivative e.g., PC 14586
  • an MDM2 inhibitor e.g., a BCL-2 inhibitor or an XPO-1 inhibitor
  • a TP53 reactivating indole derivative e.g., PC14586
  • a TP53 reactivating indole derivative e.g., PC14586
  • an MDM2 inhibitor e.g., MDM2 inhibitor
  • a BCL-2 inhibitor or an XPO-1 inhibitor may be administered daily, weekly, biweekly, or monthly for a particular period of time followed by a particular period of nontreatment.
  • a TP53 reactivating indole derivative e.g., PC 14586
  • an MDM2 inhibitor, a BCL-2 inhibitor or an XPO-1 inhibitor can be administered daily for 14 days followed by seven days of non-treatment, and repeated for two more cycles of daily administration for 14 days followed by seven days of non-treatment.
  • a TP53 reactivating indole derivative e.g., PC 14586
  • an MDM2 inhibitor e.g., MDM2 inhibitor
  • a BCL-2 inhibitor e.g., BCL-2 inhibitor
  • an XPO-1 inhibitor can be administered twice daily for seven days followed by 14 days of non-treatment, which may be repeated for one or two more cycles of twice daily administration for seven days followed by 14 days of non-treatment.
  • a TP53 reactivating indole derivative e.g., PC 14586
  • the MDM2 inhibitor, the BCL-2 inhibitor or the XPO-1 inhibitor is administered daily over a period of 14 days.
  • a TP53 reactivating indole derivative e.g., PC14586
  • the MDM2 inhibitor, the BCL-2 inhibitor or the XPO-1 inhibitor is administered daily over a period of 12 days, or 11 days, or 10 days, or nine days, or eight days.
  • a TP53 reactivating indole derivative e.g., PC14586
  • the MDM2 inhibitor, the BCL-2 inhibitor or the XPO-1 inhibitor is administered daily over a period of seven days.
  • a TP53 reactivating indole derivative e.g., PC14586
  • the MDM2 inhibitor, the BCL-2 inhibitor or the XPO-1 inhibitor is administered daily over a period of six days, or five days, or four days, or three days.
  • individual doses of a TP53 reactivating indole derivative e.g., PC 14586
  • the MDM2 inhibitor, BCL-2 inhibitor, or XPO-1 inhibitor are administered within a time interval such that the two inhibitors can work together (e.g., within 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 5 days, 6 days, 1 week, or 2 weeks).
  • the treatment period during which the therapeutic agents are administered is then followed by a non-treatment period of a particular time duration, during which the therapeutic agents are not administered to the patient.
  • This non-treatment period can then be followed by a series of subsequent treatment and non-treatment periods of the same or different frequencies for the same or different lengths of time.
  • the treatment and non-treatment periods are alternated. It will be understood that the period of treatment in cycling therapy may continue until the patient has achieved a complete response or a partial response, at which point the treatment may be stopped. Alternatively, the period of treatment in cycling therapy may continue until the patient has achieved a complete response or a partial response, at which point the period of treatment may continue for a particular number of cycles. In some embodiments, the length of the period of treatment may be a particular number of cycles, regardless of patient response. In some other embodiments, the length of the period of treatment may continue until the patient relapses.
  • a TP53 reactivating indole derivative e.g., PC 14586
  • the MDM2 inhibitor, BCL-2 inhibitor, or XPO-1 inhibitor are cyclically administered to a patient.
  • Cycling therapy involves the administration of a first agent (e.g., a first prophylactic or therapeutic agent) for a period of time, followed by the administration of a second agent and/or third agent (e.g., a second and/or third prophylactic or therapeutic agent) for a period of time and repeating this sequential administration. Cycling therapy can reduce the development of resistance to one or more of the therapies, avoid or reduce the side effects of one of the therapies, and/or improve the efficacy of the treatment.
  • a first agent e.g., a first prophylactic or therapeutic agent
  • third agent e.g., a second and/or third prophylactic or therapeutic agent
  • a TP53 reactivating indole derivative e.g., PC 14586 in combination with an MDM2 inhibitor, BCL-2 inhibitor, or XPO-1 inhibitor are each administered at a dose and schedule typically used for that agent during monotherapy.
  • a TP53 reactivating indole derivative e.g., PC 14586 and one of MDM2 inhibitor, BCL-2 inhibitor, or XPO-1 inhibitor are administered concomitantly, one or both of the agents can advantageously be administered at a lower dose than typically administered when the agent is used during monotherapy, such that the dose falls below the threshold that an adverse side effect is elicited.
  • the therapeutically effective amounts or suitable dosages of the TP53 reactivating indole derivative e.g, PC 14586
  • the MDM2 inhibitor e.g., MDM2 inhibitor
  • the BCL-2 inhibitor e.g., BCL-2 inhibitor
  • the XPO-1 inhibitor in combination depends upon a number of factors, including the nature of the severity of the condition to be treated, the particular inhibitor, the route of administration and the age, weight, general health, and response of the individual patient.
  • the suitable dose level is one that achieves a therapeutic response as measured by reduction in cancer cells or other standard measures of disease progression, progression free survival, or overall survival.
  • the suitable dose level is one that achieves this therapeutic response and also minimizes any side effects associated with the administration of the therapeutic agent.
  • Suitable daily dosages of MDM2 inhibitors can generally range, in single or divided or multiple doses, from about 10% to about 120% of the maximum tolerated dose as a single agent. In certain embodiments, the suitable dosages of MDM2 inhibitors are from about 20% to about 100% of the maximum tolerated dose as a single agent. In other embodiments, suitable dosages of MDM2 inhibitors are about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,
  • I l l about 100%, about 105%, about 110%, about 115%, or about 120% of the maximum tolerated dose as a single agent.
  • Suitable daily dosages of BCL-2 inhibitors can generally range, in single or divided or multiple doses, from about 10% to about 120% of the maximum tolerated dose as a single agent.
  • the suitable dosages of BCL-2 inhibitors are from about 20% to about 100% of the maximum tolerated dose as a single agent.
  • suitable dosages of BCL-2 inhibitors are about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, or about 120% of the maximum tolerated dose as a single agent.
  • Suitable daily dosages of XPO-1 inhibitors can generally range, in single or divided or multiple doses, from about 10% to about 120% of the maximum tolerated dose as a single agent. In certain embodiments, the suitable dosages of XPO-1 inhibitors are from about 20% to about 100% of the maximum tolerated dose as a single agent. In other embodiments, suitable dosages of XPO-1 inhibitors are about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, or about 120% of the maximum tolerated dose as a single agent.
  • Suitable daily dosages of a TP53 reactivating indole derivative can generally range, in single or divided or multiple doses, from about 10% to about 120% of the maximum tolerated dose as a single agent. In certain embodiments, the suitable dosages of a TP53 reactivating indole derivative (e.g., PC 14586) are from about 20% to about 100% of the maximum tolerated dose as a single agent.
  • suitable dosages of a TP53 reactivating indole derivative are about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, or about 120% of the maximum tolerated dose as a single agent.
  • Dosage, toxicity, and therapeutic efficacy of any therapeutic agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Compounds that exhibit high therapeutic indices are advantageous. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds may be within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (z.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC50 z.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • an effective amount of a TP53 reactivating indole derivative (e.g., PC 14586), an MDM2 inhibitor, a BCL-2 inhibitor, or an XPO-1 inhibitor, sufficient for achieving a therapeutic or prophylactic effect, may range from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day.
  • the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day.
  • dosages can be 1 mg/kg body weight or 10 mg/kg body weight every day, every two days, or every three days or within the range of 1-10 mg/kg every week, every two weeks, or every three weeks.
  • a single dosage of a TP53 reactivating indole derivative ranges from 0.001-10,000 micrograms per kg body weight.
  • a TP53 reactivating indole derivative e.g., PC 14586
  • an MDM2 inhibitor e.g., a BCL-2 inhibitor
  • an XPO-1 inhibitor concentrations in a carrier range from 0.2 to 2000 micrograms per delivered milliliter.
  • An exemplary treatment regime entails administration once per day or once a week. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, or until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.
  • a therapeutically effective amount of a TP53 reactivating indole derivative may be defined as a concentration of a TP53 reactivating indole derivative (e.g., PC14586), an MDM2 inhibitor, a BCL-2 inhibitor, or an XPO-1 inhibitor at the target tissue of IO’ 12 to 10' 6 molar, e.g., approximately 10' 7 molar.
  • This concentration may be delivered by systemic doses of 0.001 to 100 mg/kg or equivalent dose by body surface area.
  • the schedule of doses would be optimized to maintain the therapeutic concentration at the target tissue, such as by single daily or weekly administration, but also including continuous administration (e.g, parenteral infusion or transdermal application).
  • treatment of a subject with a therapeutically effective amount of the therapeutic compositions described herein can include a single treatment or a series of treatments.
  • the mammal treated in accordance with the present methods can be any mammal, including, for example, farm animals, such as sheep, pigs, cows, and horses; pet animals, such as dogs and cats; laboratory animals, such as rats, mice, and rabbits.
  • the mammal is a human.
  • kits for treating leukemia comprising (a) a TP53 reactivating indole derivative (e.g, PC 14586), (b) at least one of an MDM2 inhibitor, a BCL-2 inhibitor, and XPO-1 inhibitor, and (c) instructions for treating leukemia.
  • the leukemia is AML and/or MDS. Additionally or alternatively, in some embodiments, the leukemia comprises wild-type p53 or a TP53-Y220C mutation.
  • the kit may comprise a TP53 reactivating indole derivative (e.g., PC14586) and at least one of an MDM2 inhibitor, a BCL-2 inhibitor, and XPO-1 inhibitor that has been formulated into a single pharmaceutical composition such as a tablet, or as separate pharmaceutical compositions.
  • a TP53 reactivating indole derivative e.g., PC14586
  • MDM2 inhibitor e.g., TP53 reactivating indole derivative
  • BCL-2 inhibitor e.g., XPO-1 inhibitor
  • the kit may comprise (a) a TP53 reactivating indole derivative (e.g., PC 14586), and (b) at least one of an MDM2 inhibitor, a BCL-2 inhibitor, and XPO-1 inhibitor that has been formulated as separate pharmaceutical compositions either in a single package, or in separate packages.
  • a TP53 reactivating indole derivative e.g., PC 14586
  • at least one of an MDM2 inhibitor, a BCL-2 inhibitor, and XPO-1 inhibitor that has been formulated as separate pharmaceutical compositions either in a single package, or in separate packages.
  • kits further comprise at least one chemotherapeutic agent and/or at least one immunotherapeutic agent that are useful for treating leukemia.
  • chemotherapeutic agents include arsenic trioxide, azacytidine, cerubidine, cyclophosphamide, cytarabine, daunorubicin hydrochloride, daurismo, dexamethasone, doxorubicin hydrochloride, enasidenib mesylate, gemtuzumab ozogamicin, gilteritinib fumarate, glasdegib maleate, idamycin PFS, idarubicin hydrochloride, idhifa, ivosidenib, midostaurin, mitoxantrone hydrochloride, mylotarg, olutasidenib, onureg, pemazyre, pemigatinib, prednisone, rezlidh
  • kits may further comprise pharmaceutically acceptable excipients, diluents, or carriers that are compatible with one or more kit components described herein.
  • the above-described components of the kits of the present technology are packed in suitable containers and labeled for the treatment of leukemia, including AML and/or MDS.
  • the kits may optionally include instructions customarily included in commercial packages of therapeutic products, which contain information about, for example, the indications, usage, dosage, manufacture, administration, contraindications and/or warnings concerning the use of such therapeutic products.
  • Example 1 Materials and methods
  • PC14586 was supplied by PMV Pharmaceuticals. For details, see Dumbrava
  • the BCL-2 inhibitor was venetoclax (BCL201).
  • the MDM2 inhibitor was nutlin-3a (HDM201).
  • the XPO1 inhibitors were KPT330 and KPT8602.
  • AML cells were cultured in RPML1640 medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 pg/mL streptomycin. Cells were kept at 37°C in a humidified, 5% CO2 atmosphere.
  • Example 2 PC14586 converts p53Y220C to wild-type p53 conformation and activates p53 transcriptional activity and greatly induces p21
  • FIG. 1 shows a Western blot demonstrating the activity of PC14586 on mutant and wild-type p53 (upper panel) and a Western blot demonstrating the transcriptional activity of p53 (lower panel) after treatment with PC 14586.
  • the Western blot reveals PC14586 activates p53 and p53 transcriptional activity and induces p21 expression.
  • Example 3 PC14586 primarily suppresses cell grow in TP53 Y220C, not in TP53 WT, KO, or TP53 R175H mutant AML cells
  • FIG. 2A is a graph of dose-response viability curves of the panel of AML cell lines treated with PC 14586 for 120 hours.
  • FIG. 2B is a graph of 7AAD/AnnV curves of the panel of AML cell lines treated with PC 14586 for 120 hours. Viability curves indicate the percentage of live cells in the sample and 7AAD/AnnV curves reveal the percentage of cells in apoptosis in the sample.
  • Example 4 ⁇ 1D ⁇ 12 inhibition enhances PC14586 activity in AML cells having TP53-WT and the combination is highly synergistic in AML cells having TP53-Y220C.
  • MDM2i+PC 14586 The combination of an MDM2 inhibitor and PC 14586 (MDM2i+PC 14586) was tested in vitro and compared to treatment with the MDM2 inhibitor (MDM2i) or the PC14586 alone.
  • AML cell lines having TP53-WT, TP53-KO, TP53-R175H, or TP53- Y220C were treated with the combination of MDM2 and PC14586 for 72 hours.
  • Concentrations of MDM2 were varied between 0 pM and 5.00 pM and concentrations of PC14586 were varied between 0 pM and 4.0 pM.
  • FIG. 3A is a graph of 7AAD/AnnV curves of AML cells having TP53-WT treated with an MDM2 inhibitor, PC 14586, or a combination of the MDM2 inhibitor and PC14586 for 72 hours.
  • FIG. 3B is a graph of dose-response viability curves of AML cells having TP53-WT treated with the MDM2 inhibitor, PC14586, or the combination of the MDM2 inhibitor and PC 14586 for 72 hours. Results revealed that the MDM2 inhibitor enhanced PC14586 activity in AML cells having TP53-WT, greatly increasing cell death and reducing cell growth as compared to the monotherapies.
  • FIG. 3C is a graph of 7AAD/AnnV curves of AML cells having TP53-Y220C treated with the MDM2 inhibitor, PC 14586, or the combination of the MDM2 inhibitor and PC14586 for 72 hours.
  • FIG. 3D is a graph of dose-response viability curves of AML cells having TP53-Y220C treated with the MDM2 inhibitor, PC14586, or the combination of the MDM2 inhibitor and PC 14586 for 72 hours. Results revealed that the MDM2 inhibitor enhanced PC14586 activity in AML cells having TP53-Y220C, and that the combination treatment had a synergistic affect to greatly increase cell death and reduce cell growth as compared to the monotherapies.
  • FIG. 3E is a graph of 7AAD/AnnV curves of AML cells having TP53-KO treated with the MDM2 inhibitor, PC 14586, or the combination of the MDM2 inhibitor and PC 14586 for 72 hours.
  • FIG. 3F is a graph of dose-response viability curves of AML cells having TP53-KO treated with the MDM2 inhibitor, PC 14586, or the combination of the MDM2 inhibitor and PC14586 for 72 hours.
  • FIG. 3E is a graph of 7AAD/AnnV curves of AML cells having TP53-KO treated with the MDM2 inhibitor, PC 14586, or the combination of the MDM2 inhibitor and PC14586 for 72 hours.
  • 3G is a graph of 7AAD/AnnV curves of AML cells having TP53-R175H treated with the MDM2 inhibitor, PC14586, or the combination of the MDM2 inhibitor and PC 14586 for 72 hours.
  • FIG. 3H is a graph of dose-response viability curves of AML cells having TP53-R175H treated with the MDM2 inhibitor, PC14586, or the combination of the MDM2 inhibitor and PC 14586 for 72 hours.
  • Example 5 BCL-2 inhibition enhances PC14586 activity in AML cells having TP53-WT and the combination is highly synergistic in AML cells having TP53-Y220C.
  • BCL-2i+PC 14586 The combination of an BCL-2 inhibitor and PC 14586 (BCL-2i+PC 14586) was tested in vitro and compared to treatment with the BCL-2 inhibitor (BCL-2i) or the PC14586 alone.
  • AML cell lines having TP53-WT, TP53-KO, TP53-R175H, or TP53- Y220C were treated with the combination of BCL-2 and PC14586 for 72 hours.
  • Concentrations of BCL-2 were varied between 0 nM and 20.00 nM and concentrations of PC14586 were varied between 0 pM and 4.0 pM.
  • FIG. 4A is a graph of 7AAD/AnnV curves of AML cells having TP53-WT treated with a BCL-2 inhibitor, PC 14586, or a combination of the BCL-2 inhibitor and PC14586 for 72 hours.
  • FIG. 4B is a graph of dose-response viability curves of AML cells having TP53-WT treated with the BCL-2 inhibitor, PC14586, or the combination of the BCL-2 inhibitor and PC14586 for 72 hours. Results revealed that the combination therapy had a synergistic effect on AML cells having TP53-WT to increase cell death and reduce cell growth as compared to the monotherapies.
  • FIG. 4C is a graph of 7AAD/AnnV curves of AML cells having TP53-Y220C treated with the BCL-2 inhibitor, PC 14586, or the combination of the BCL-2 inhibitor and PC14586 for 72 hours.
  • FIG. 4D is a graph of dose-response viability curves of AML cells having TP53-Y220C treated with the BCL-2 inhibitor, PC14586, or the combination of the BCL-2 inhibitor and PC14586 for 72 hours. Results revealed that the combination therapy had a synergistic effect on AML cells having TP53-Y220C to increase cell death and reduce cell growth as compared to the monotherapies.
  • FIG. 4E is a graph of 7AAD/AnnV curves of AML cells having TP53-KO treated with the BCL-2 inhibitor, PC14586, or the combination of the BCL-2 inhibitor and PC14586 for 72 hours.
  • FIG. 4F is a graph of dose-response viability curves of AML cells having TP53-KO treated with the BCL-2 inhibitor, PC14586, or the combination of the BCL-2 inhibitor and PC14586 for 72 hours.
  • FIG. 4E is a graph of 7AAD/AnnV curves of AML cells having TP53-KO treated with the BCL-2 inhibitor, PC14586, or the combination of the BCL-2 inhibitor and PC14586 for 72 hours.
  • FIG. 4G is a graph of 7AAD/AnnV curves of AML cells having TP53-R175H treated with the BCL-2 inhibitor, PC 14586, or the combination of the BCL-2 inhibitor and PC14586 for 72 hours.
  • FIG. 4H is a graph of dose-response viability curves of AML cells having TP53-R175H treated with the BCL-2 inhibitor, PC14586, or the combination of the BCL-2 inhibitor and PC14586 for 72 hours.
  • Example 6 XPO-1 inhibition enhances PC14586 activity in AML cells having TP53-WT and the combination is highly synergistic in AML cells having TP53-Y220C.
  • Concentrations of XPO-1 were varied between 0 nM and 200.00 nM and concentrations of PC14586 were varied between 0 pM and 4.0 pM.
  • FIG. 5A is a graph of 7AAD/AnnV curves of AML cells having TP53-WT treated with a XPO inhibitor, PC 14586, or a combination of the XPO inhibitor and PC14586 for 72 hours.
  • FIG. 5B is a graph of dose-response viability curves of AML cells having TP53-WT treated with the XPO inhibitor, PC 14586, or the combination of the XPO inhibitor and PC 14586 for 72 hours. Results revealed that the combination therapy had a synergistic effect on AML cells having TP53-WT to increase cell death and reduce cell growth as compared to the monotherapies.
  • FIG. 5C is a graph of 7AAD/AnnV curves of AML cells having TP53-Y220C treated with the XPO inhibitor, PC 14586, or the combination of the XPO inhibitor and PC14586 for 72 hours.
  • FIG. 5D is a graph of dose-response viability curves of AML cells having TP53-Y220C treated with the XPO inhibitor, PC 14586, or the combination of the XPO inhibitor and PC14586 for 72 hours. Results revealed that the combination therapy had a synergistic effect on AML cells having TP53-Y220C to increase cell death and reduce cell growth as compared to the monotherapies.
  • FIG. 5E is a graph of 7AAD/AnnV curves of AML cells having TP53-KO treated with the XPO inhibitor, PC14586, or the combination of the XPO inhibitor and PC14586 for 72 hours.
  • FIG. 5F is a graph of dose-response viability curves of AML cells having TP53-KO treated with the XPO inhibitor, PC 14586, or the combination of the XPO inhibitor and PC 14586 for 72 hours.
  • FIG. 5E is a graph of 7AAD/AnnV curves of AML cells having TP53-KO treated with the XPO inhibitor, PC14586, or the combination of the XPO inhibitor and PC14586 for 72 hours.
  • 5G is a graph of 7AAD/AnnV curves of AML cells having TP53-R175H treated with the XPO inhibitor, PC 14586, or the combination of the XPO inhibitor and PC14586 for 72 hours.
  • FIG. 5H is a graph of dose-response viability curves of AML cells having TP53-R175H treated with the XPO inhibitor, PC14586, or the combination of the XPO inhibitor and PC 14586 for 72 hours.
  • Example 7 Synergistic effects of combination therapies of the present technology in AML cells having TP53-WT and in AML cells having TP53-Y220C.
  • PC14586 in combination with MDM2, BCL-2, or XPO-1 inhibitor is more effective than each single agent alone
  • PC 14586 in combination with any two other agents is more effective than PC14586 in combination with one other agent
  • PC14586 in combination with MDM2, BCL-2, and XPO-1 inhibitors is the most effective in cell death induction in both TP53 wild-type and Y220C mutant AML cells.
  • FIG. 6A shows 7AAD/AnnV curves of AML cells having TP53-WT treated with PC 14586, the BCL-2 inhibitor, the MDM2 inhibitor, the XPO-1 inhibitor, or the combination of PC14586 with one, two, or all three of these inhibitors for 72 hours.
  • FIG. 6B shows dose-response viability curves of AML cells having TP53-WT treated with PC14586, the BCL-2 inhibitor, the MDM2 inhibitor, the XPO-1 inhibitor, or the combination of PC14586 with one, two, or all three of these inhibitors for 72 hours.
  • FIG. 6A shows 7AAD/AnnV curves of AML cells having TP53-WT treated with PC 14586, the BCL-2 inhibitor, the MDM2 inhibitor, the XPO-1 inhibitor, or the combination of PC14586 with one, two, or all three of these inhibitors for 72 hours.
  • FIG. 6C shows 7AAD/AnnV curves of AML cells having TP53-Y220C treated with PC 14586, the BCL-2 inhibitor, the MDM2 inhibitor, the XPO-1 inhibitor, or the combination of PC 14586 with one, two, or all three of these inhibitors for 72 hours.
  • FIG. 6D shows doseresponse viability curves of AML cells having TP53-Y220C treated with PC14586, the BCL-2 inhibitor, the MDM2 inhibitor, the XPO-1 inhibitor, or the combination of PC14586 with one, two, or all three of these inhibitors for 72 hours. Accordingly, the combination therapies and methods disclosed herein are useful for treating leukemias in a subject in need thereof.
  • Example 8 Mol ml 3 TP53-Y220C cells treated with PC14586-alone or in combination with XPO-1, MDM2, or BCL-2 inhibitors.
  • FIG. 7A shows Western blots of the protein levels in Molml3 TP53-Y220C cells treated for 24 hours with venetoclax (VEN, 5 nM or 10 nM), nutlin-3a (Nut, 2.5 pM or 5 pM), PC14586 (PC, 2 pM or 4 pM), KPT-8602 (KPT, 100 nM or 200 nM), or combinations of PC 14586 with venetoclax (VEN/PC, 5 nM VEN and 2 pM PC, 10 nM VEN and 4 pM PC), nutlin-3a (Nut/PC, 2.5 pM Nut and 2 pM PC, 5 pM Nut and 4 pM PC), or KPT-8602 (KPT/PC, 100 nM KPT and 2 pM PC, 200 nM KPT
  • FIG. 7B shows cell cycle distribution and apoptosis determined by flow cytometry of cells stained with 5-ethynyl-2’-deoxyuridine (EdU) and DNA dye.
  • Molml3 TP53-Y220C cells were treated for 72 hours with venetoclax (VEN, 10 nM), nutlin-3a (N3, 5 pM), KPT-8602 (KPT, 200 nM), PC 14586 (4 pM), or combinations of PC 14586 with venetoclax (4 pM PC14586 and 10 nMVEN), nutlin-3a (4 pM PC14586 and 5 pM N3), or KPT (4 pM PC14586 and 200 nM KPT).
  • VEN venetoclax
  • nutlin-3a N3, 5 pM
  • KPT-8602 KPT, 200 nM
  • PC 14586 4 pM
  • combinations of PC 14586 with venetoclax 4 pM PC14586 and 10 nMVEN
  • nutlin-3a (4 pM PC14586 and 5 pM N3
  • KPT 4 pM PC14586 and 200 nM KPT
  • FIG. 7C shows DNA content and PARP cleavage determined by flow cytometry of Molml3 TP53-Y220C cells treated for 72 hours with venetoclax (VEN, 10 nM), nutlin3a (N3, 5 pM), KPT-8602 (KPT, 200 nM), PC 14586 (4 pM), or combinations of PC 14586 with venetoclax (4 pM PC 14586 and 10 nM VEN), nutlin-3a (4 pM PC 14586 and 5 pM), or KPT-8602 (4 pM PC 14586 and 200 nM KPT).
  • VEN venetoclax
  • nutlin3a N3, 5 pM
  • KPT-8602 KPT, 200 nM
  • PC 14586 4 pM
  • PC 14586 4 pM
  • nutlin-3a 4 pM PC 14586 and 5 pM
  • KPT-8602 4 pM PC 14586 and 200
  • Example 9 PC14586 induced cell death in bulk AML cells and in ste /prosenitor cells in 2/3 samples in primary AML cells with Y220C mutations and the combinations were highly synergistic in AML cells and stem/prosenitor cells regardless of response to PC14586 alone
  • Cells from AML patient samples (FIGS. 8A-8G and 9A-9G), patient-derived xenograft (FIGS. 10A-10D), or normal bone marrow cells (FIGS. 11A-11D), each sample under mesenchymal stroma cell (MSC) co-culture conditions (COX) were treated for 48 hours or 96 hours with PC 14586; venetoclax; nutlin3a; KPT-8062; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC14586.
  • Cell death and cell viability were determined by flow cytometry after the cells were stained with annexin V/7-aminoactinomycin D (Ann V/7-ADD) in the presence of counting beads.
  • PC14586 as a single agent had limited activity in PDX cells derived from the patient in FIGS. 10A-10D.
  • MDM2, BCL-2, and/or XPO-1 inhibitors were synergistic in cells and stem/progenitor cells from all three AML patient samples in FIGS. 8A-8G, 9A-9G, and FIGS. 10A-10D. All treatments using primary samples were carried out under mesenchymal stroma cell (MSC) co-culture conditions (COX).
  • MSC mesenchymal stroma cell
  • FIGS. 8A-8G used 47% peripheral blood (PB) samples with 77% TP53-Y220C mutation.
  • the samples had complex cytogenetics with mutations in NF1, JAK2, SMC1A, SRSF2, TET2, and CUX1 genes.
  • Cell death and viable cells were determined by flow cytometry after the cells were stained with annexin V/7-aminoactinomycin D (Ann V/7- ADD) in the presence of counting beads.
  • FIG. 8A showed CD45+, CD34+, and CD34+CD38- populations after 48 hours of treatment with PC14586 at different concentrations.
  • Tables 1 and 2 provide ECso and IC50 data, respectively, for CD45+, CD34+, and CD34+CD38- cell populations treated with PC14586.
  • Table 1 EC50 for different cell types from PB samples as shown in FIGS. 8A-8G treated with PC 14586 for 48 hours.
  • Table 2 ICso for different cell types from PB samples as shown in FIGS. 8A-8G treated with PC 14586 for 48 hours.
  • FIG. 8B shows CD45+ population assessed by Ann V/7-ADD+ positive cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC 14586.
  • Table 3 provides combination index (CI) values from FIG. 8B FIG.
  • 8C shows CD45+ viable cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC14586; venetoclax and PC14586; nutlin-3a and PC14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC 14586.
  • Table 3 CI values for different treatment combinations for CD45+ cells.
  • FIG. 8D shows CD34+ assessed by Ann V/7ADD+ cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC14586; KPT-8602 and PC14586; or nutlin-3a, KPT-8602, and PC14586.
  • Table 4 provides combination index (CI) values from FIG. 8D.
  • FIG. 8E shows CD34+ viable cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602;
  • PC 14586 ; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC 14586.
  • Table 4 CI values for different treatment combinations for CD34+ cells.
  • FIG. 8F shows CD34+CD38- population assessed by Ann V/7ADD+ cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC14586.
  • Table 5 provides combination index (CI) values from FIG. 8F.
  • 8G shows CD34+CD38- viable cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT- 8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC14586; or nutlin-3a, KPT-8602, and PC14586.
  • Table 5 CI values for different treatment combinations for CD34+CD38- cells.
  • FIGS. 9A-9G show cell samples from an AML patient with 92% peripheral blood (PB) with 45.7% TP53-Y220C under MSC COX. The samples had complex cytogenetics with mutations in TP-53-G105fs (39.7%) and U2AF1 genes. Cell death and viable cells were determined by flow cytometry after the cells were stained with Ann V/7- ADD in the presence of counting beads.
  • FIG. 9A shows CD45+, CD34+, and CD34+CD38- population after 48 hours of treatment with PC14586 at different concentrations. PC14586 induced cell death in AML blasts and stem/progenitor cells. Tables 6 and 7 provide ECso and IC50 data, respectively, for CD45+, CD34+, and CD34+CD38- cell populations treated with PC14586.
  • Table 6 EC50 for different cell types from PB samples as shown in FIGS. 9A-9G treated with PC 14586 for 48 hours.
  • Table 7 ICso for different cell types from PB samples as shown in FIGS. 9A-9G treated with PC 14586 for 48 hours.
  • FIG. 9B shows
  • Table 8 provides combination index (CI) values from FIG. 9B.
  • 9C shows CD45+ viable cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC 14586.
  • Table 8 CI values for different treatment combinations for CD45+ cells.
  • FIG. 9D shows CD34+ assessed by Ann V/7ADD+ cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC14586; KPT-8602 and PC14586; or nutlin-3a, KPT-8602, and PC14586.
  • Table 9 provides combination index (CI) values from FIG. 9D.
  • FIG. 9E shows CD34+ viable cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602;
  • PC 14586 ; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC 14586.
  • Table 9 CI values for different treatment combinations for CD34+ cells.
  • FIG. 9F shows CD34+CD38- population assessed by Ann V/7ADD+ cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC14586.
  • Table 10 provides combination index (CI) values from FIG. 9F.
  • 9G shows CD34+CD38- viable cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC14586; venetoclax and PC14586; nutlin-3a and PC14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC 14586.
  • Table 10 CI values for different treatment combinations for CD34+CD38- cells.
  • FIGS. 10A-10D show patient-derived xenograft (PDX) spleen cells with 99.5% hCD45+ and TP53-Y220C mutation confirmed by sequencing.
  • the samples had complex cytogenetics with complex Karyotype, MECOM rearrangement, and mutations in NBAS, KRAS, TP53 Y220C, and P151A genes.
  • Cell death and viable cells were determined by flow cytometry after the cells were stained with Ann V/7-ADD in the presence of counting beads. Variants of probable somatic origin are in Table 11.
  • Table 11 Variants of probable somatic origin in PDX samples used in FIGS. 10A-10D.
  • FIG. 10A shows CD45 ⁇ , CD34 ⁇ , and CD34+CD38- populations after 96 hours of treatment with PC 14586 at different concentrations.
  • PC 14586 as a single agent induced limited cell death in AML blast and stem/progenitor cells.
  • FIG. 10B shows CD45+ population assessed by Ann V/7-ADD+ positive cells after 96 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC14586; venetoclax and PC14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; nutlin-3a, KPT-8602, and PC 14586; venetoclax, KPT-8602, and PC14586; KPT-8602, nutlin-3a, and PC14586; or venetoclax, nutlin-3a, KPT-8602, and PC 14586.
  • FIG. 10C CD45+ viable cells after 96 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC14586; venetoclax and PC14586; nutlin-3a and PC14586; KPT-8602 and PC 14586; nutlin-3a, KPT-8602, and PC 14586; venetoclax, KPT-8602, and PC 14586; KPT-8602, nutlin-3a, and PC14586; or venetoclax, nutlin-3a, KPT-8602, and PC14586.
  • Table 12 CI values for different treatment combinations for CD45+ cells.
  • FIG. 10D shows CD34+ assessed by Ann V/7ADD+ cells after 96 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; nutlin-3a, KPT-8602, and PC 14586; venetoclax, KPT-8602, and PC14586; KPT-8602, nutlin-3a, and PC14586; or venetoclax, nutlin-3a, KPT-8602, and PC 14586.
  • Table 13 provides combination index (CI) values from FIG. 10D.
  • 10E shows CD34+ viable cells after 96 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; nutlin-3a, KPT-8602, and PC 14586; venetoclax, KPT- 8602, and PC14586; KPT-8602, nutlin-3a, and PC14586; or venetoclax, nutlin-3a, KPT- 8602, and PC 14586.
  • Table 13 CI values for different treatment combinations for CD34+ cells.
  • FIG. 10F shows CD34+CD38- population assessed by Ann V/7ADD+ cells after 96 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; nutlin-3a, KPT-8602, and PC 14586; venetoclax, KPT-8602, and PC 14586; KPT-8602, nutlin-3a, and PC14586; or venetoclax, nutlin-3a, KPT-8602, and PC14586.
  • Table 14 provides combination index (CI) values from FIG. 10F.
  • 10G CD34+CD38- viable cells after 96 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; nutlin-3a, KPT-8602, and PC 14586; venetoclax, KPT-8602, and PC 14586; KPT-8602, nutlin-3a, and PC 14586; or venetoclax, nutlin-3a, KPT-8602, and PC 14586.
  • Table 14 CI values for different treatment combinations for CD34+CD38- cells.
  • FIGS. 11A-11D show normal bone marrow cells under MSC COX. Cell death and viable cells were determined by flow cytometry after the cells were stained with Ann V/7-ADD in the presence of counting beads.
  • FIG. 11A-11D show normal bone marrow cells under MSC COX. Cell death and viable cells were determined by flow cytometry after the cells were stained with Ann V/7-ADD in the presence of counting beads.
  • FIG. 11A shows CD45+ population assessed by Ann V/7-ADD+ positive cells after 48 hours of treatment with venetoclax; nutlin-3a; KPT-8602; PC14586; venetoclax and PC14586; nutlin-3a and PC14586; KPT- 8602 and PC14586; or venetoclax, nutlin-3a, KPT-8602, and PC14586.
  • FIG. 11B shows CD45+ cell viability after 48 hours of treatment with venetoclax; nutlin-3a; KPT-8602;
  • FIG. 11C shows CD34+ population assessed by Ann V/7-ADD+ positive cells after 48 hours of treatment with venetoclax; nutlin-3a; KPT-8602; PC14586; venetoclax and PC14586; nutlin-3a and PC14586; KPT- 8602 and PC14586; or venetoclax, nutlin-3a, KPT-8602, and PC14586.
  • FIG. 11C shows CD34+ population assessed by Ann V/7-ADD+ positive cells after 48 hours of treatment with venetoclax; nutlin-3a; KPT-8602; PC14586; venetoclax and PC14586; nutlin-3a and PC14586; KPT- 8602 and PC14586; or venetoclax, nutlin-3a, KPT-8602, and PC14586.
  • FIG. 11C shows CD34+ population assessed by Ann V/7-ADD+ positive cells after 48 hours of treatment with venetoclax; nutlin-3a; KPT-8602; PC14586
  • 11D shows CD34+ cell viability after 48 hours of treatment with venetoclax; nutlin-3a; KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or venetoclax, nutlin-3a, KPT-8602, and PC14586.
  • Example 10 PC14586 reactivates p53 signaling, which is further enhanced by inhibition of MDM2, induced by PC14586-activated p53
  • Molml3 7P53-WT and 7P53-Y220C cells were treated with nutlin-3a (N3a, 5 pM), PC14586 (PC, 4 pM), or the combination for 4 hours.
  • RNA was isolated and subject to RNA sequencing.
  • FIG. 12A shows a principal component analysis comparing Molml3 TP 53-WT and 7P53-Y220C cells treated with nutlin-3a (N3a, 5 pM), PC14586 (PC, 4 pM), or the combination for 4 hours.
  • FIG. 12B shows gene set enrichment analysis with 77 degrees of 2-D hierarchical clustering comparing Molml3 TP53-WT and TP53-Y22QC cells treated with nutlin-3a (N3a, 5 pM), PC 14586 (PC, 4 pM), or the combination for 4 hours.
  • Arrows in FIG. 12B indicate upregulation of p53 pathway for Molml3 TP53-WT treated with N3a or the combination of N3a and PC14586, but not PC14586 alone.
  • Arrows in FIG. 12B also indicate upregulation of p53 pathway for Molml3 TP53-Y22QC cells treated with PC14586 or the combination of N3a and PC 14586, but not N3a alone.
  • Results showed greatest upregulation for Molml3 TP53-Y220C cells treated with the combination of nutlin- 3a and PC 14586 and no change or deregulation for groups starting at Molml3 TP53-Y220C cells treated with nutlin-3a and every group to the left thereof.
  • FIG. 12D shows a pathway analysis.
  • FIG. 12E shows volcano plots comparing increased, unchanged, and decreased gene expression in treated compared to control samples.
  • FIG. 12F shows Western blots showing changes in gene expression indicated at the protein level.
  • Example 11 Combination of PC14586 and RG7388 significantly prolonged the survival in a TP53-Y220C PDX model, while PC14586 alone has no effect in the model that carries TP53-Y220C, TP53-P151A and NAS Mutations
  • PDX cells derived from an AML patient sample with TP53-Y220C (VAF 48%), TP53-P151A (VAF 47%), and NRAS (VAF 50%) mutations were injected via tail vein into NSG mice (8 week old, male) (1.6> ⁇ 10 6 /mouse).
  • mice (10/group) were treated with vehicle, PC14586 (100 mg/kg, daily), MDM2 inhibitor idasanutlin (RG7388) with 50% activity (40 mg/kg, initially 8 days on, then 2 days off/5 days on) or both PC 14586 (100 mg/kg, daily) and MDM2 inhibitor idasanutlin (RG7388) with 50% activity (40 mg/kg, initially 8 days on, then 2 days off/5 days on).
  • Disease progression and treatment responses were monitored by flow cytometry of human CD45+ cells. The Kaplan-Meier method was applied to estimate mouse survival and survival data were analyzed using the log-rank test.
  • FIG. 13A shows flow cytometry analysis of human CD45+ cells in peripheral blood (PB) after four weeks of treatment.
  • FIG. 13B shows flow cytometry analysis of human CD45+ cells in peripheral blood (PB) after eight weeks of treatment.
  • FIG. 13C Kaplan-Meier estimation of mouse survival and survival data analyzed using the log-rank test.
  • Example 12 Unlike WT p53 that binds and antagonizes antiapoptotic BCL-2, PC14586 activated p53 does not bind to BCL-2
  • Molml3 TP53-WT and TP53-Y220C cells were treated with nutlin-3a (5 pM) or PC14586 (PC, 1.25 pM or 5 pM) for 4 hours.
  • p53 was co-immunoprecipitated with antibodies selective for WT (PAbl620, Cat no. 102201, Caprico Bioscience) or mutant (PAb240, NB200-103, Novus Biosciences) conformation of p53.
  • p53 and BCL-2 were determined by Western blot after co-immunoprecipitation.
  • Example 13 Combination of PC14586 and venetoclax significantly prolonged the survival in the Moll 3 TP53-Y220C xenograft model
  • Molml3 TP53-Y220C cells 0.5> ⁇ 10 6 /mice were injected via tail vein into NSG mice (male, 6-10 weeks old). Mice (5/group) were treated daily with vehicle, PC 14586 (100 mg/kg), venetoclax (50 mg/kg), or the combination.
  • the Kaplan-Meier method was applied to estimate mouse survival and survival data were analyzed using the log-rank test.
  • FIG. 15 shows the Kaplan-Meier estimation of mouse survival and survival data analyzed using the log-rank test of mice injected with Molml3 TP53-Y220C cells, and treated daily with vehicle, PC 14586, venetoclax, or the combination.
  • Example 14 Combination of PC14586 with XPO-1 inhibitor greatly increased p53 nuclear localization and transcription activity
  • TP53-Y220C Molml3 cells were treated with PC14586 (4 pM), KPT-8602 (200 nM), or both for 24 hours.
  • p53 localization and its target proteins were determined by Western blot (MWM, molecular weight marker). For protein localization, cytosolic and nuclear proteins were fractionated. Tubulin and Lamin Bl were used as cytosolic or nuclear loading control, respectively.
  • FIG. 16 shows the Western blots of p53, Lamin Bl, and tubulin from TP53-Y220C Molml3 cells treated with PC14586 (4 pM), KPT-8602 (200 nM), or both for 24 hours.
  • a range includes each individual member.
  • a group having 1-3 cells refers to groups having 1, 2, or 3 cells.
  • a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

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Abstract

The present disclosure provides methods for treating TP53-Y220C mutant and TP53 wild-type leukemias, such as acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). In some embodiments, the methods disclosed herein comprise administering to the subject an indole derivative in combination with one or more of an MDM2 inhibitor, a BCL-2 inhibitor, or an XPO-1 inhibitor.

Description

COMBINATION THERAPY METHODS FOR TREATING TP53-Y220C MUTANT AND TP53 WILDTYPE LEUKEMIAS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The application claims the benefit of and priority to U.S. Provisional Patent Application No. 63/508,949, filed June 19, 2023, which is incorporated by reference herein in its entirety for any and all purposes.
TECHNICAL FIELD
[0002] The present technology relates generally to methods for treating TP53-Y220C mutant and TP53 wildtype leukemias, such as acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS).
BACKGROUND
[0003] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.
[0004] The tumor suppression network is an elaborate network that can prevent cells carrying an activated oncogene, damaged genome, or other cancer-promoting alteration from replicating. A central component of the tumor suppression network is p53, one of the most potent tumor suppressors in the cell. Both the wild-type and mutant conformations of p53 are implicated in the progression of cancer. TP53-Y220C is a recurrent hotspot TP53 mutation observed in solid tumors and hematological malignancies, predominantly in AML and MDS. It frequently occurs as a subclone among TP53-WT cancer cells. However, PC14586, a p53 reactivator, exerts mainly a cytostatic effect, and thus has limited apoptotic activity in TP53-Y220C leukemia cells.
[0005] Accordingly, there is an urgent need to identify clinically actionable therapeutic strategies for acute myeloid leukemia (AML) and MDS patients with TP53-Y220C mutation.
SUMMARY OF THE PRESENT TECHNOLOGY
[0006] In an aspect, the present disclosure provides a method for treating a wild-type p53 or TP53-Y220C leukemia in a patient in need thereof comprising administering to the patient an effective amount of a TP53 reactivating indole derivative and an effective amount of an MDM2 inhibitor, a BCL-2 inhibitor, and/or an XPO-1 inhibitor, wherein the TP53 reactivating indole derivative has formula of formula (I):
Figure imgf000004_0001
wherein: each - is independently a single bond or a double bond;
X1 is CR5, CR5R6, N, NR5, O, S, C=O, C=S, or a carbon atom connected to Q1;
X2 is CR7, CR7R8, N, NR7, O, S, C=O, C=S, or a carbon atom connected to Q1;
X3 is CR9, CR9R10, N, NR9, O, S, C=O, C=S, or a carbon atom connected to Q1;
X4 is CR11, CRnR12, N, NR11, O, S, C=O, C=S, or a carbon atom connected to Q1;
X5 is CR13, N, or NR13; wherein at least one of X1, X2, X3, and X4 is a carbon atom connected to Q1;
Q1 is C=O, C=S, C=CR14R15, C=NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, a bond; m is 1, 2, 3, or 4;
Y is N or O;
R1 is -C(O)R16, -C(O)OR16, -C(O)NR16R17, -OR16, -R16OR17, -R16OR17R18, -SR16, - R16SR17, -R16SR17R18, -NR16R17, -R16NR17, -R16NR17R18, -NR16C(O)R16, - OC(O)R16, C=O, C=S, -CN, -SiR16R17R18, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; each R3 and R4 is independently -C(O)R19, -C(O)OR19, -C(O)NR19R20, -SOR19, - SO2R19, alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently unsubstituted or substituted, or hydrogen; each R2, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, and R18 is independently - C(O)R21, -C(O)OR21, -C(O)NR21R22, -OR21, -SR21, -NR21R22, - NR21C(O)R22, -OC(O)R21, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently unsubstituted or substituted, or hydrogen; each R19 and R20 is C(O)R23, -C(O)OR23, -C(O)NR23R24, -OR23, -SR23, -NR23R24, - NR23C(O)R24, -OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen; each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or a pharmaceutically-acceptable salt thereof.
[0007] In an aspect, the present disclosure provides a method for prolonging survival of a wild-type p53 or TP53-Y220C leukemia patient comprising administering to the patient an effective amount of a TP53 reactivating indole derivative and an effective amount of an MDM2 inhibitor, a BCL-2 inhibitor, and/or an XPO-1 inhibitor, wherein the TP53 reactivating indole derivative has formula of formula (I):
Figure imgf000005_0001
wherein: each - is independently a single bond or a double bond;
X1 is CR5, CR5R6, N, NR5, O, S, C=O, C=S, or a carbon atom connected to Q1;
X2 is CR7, CR7R8, N, NR7, O, S, C=O, C=S, or a carbon atom connected to Q1;
X3 is CR9, CR9R10, N, NR9, O, S, C=O, C=S, or a carbon atom connected to Q1;
X4 is CR11, CRnR12, N, NR11, O, S, C=O, C=S, or a carbon atom connected to Q1; X5 is CR13, N, or NR13; wherein at least one of X1, X2, X3, and X4 is a carbon atom connected to Q1;
Q1 is C=O, C=S, C=CR14R15, C=NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, a bond; m is 1, 2, 3, or 4;
Y is N or O;
R1 is -C(O)R16, -C(O)OR16, -C(O)NR16R17, -OR16, -R16OR17, -R16OR17R18, -SR16, - R16SR17, -R16SR17R18, -NR16R17, -R16NR17, -R16NR17R18, -NR16C(O)R16, - OC(O)R16, C=O, C=S, -CN, -SiR16R17R18, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; each R3 and R4 is independently -C(O)R19, -C(O)OR19, -C(O)NR19R20, -SOR19, - SO2R19, alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently unsubstituted or substituted, or hydrogen; each R2, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, and R18 is independently - C(O)R21, -C(O)OR21, -C(O)NR21R22, -OR21, -SR21, -NR21R22, - NR21C(O)R22, -OC(O)R21, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently unsubstituted or substituted, or hydrogen; each R19 and R20 is C(O)R23, -C(O)OR23, -C(O)NR23R24, -OR23, -SR23, -NR23R24, - NR23C(O)R24, -OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen; each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or a pharmaceutically-acceptable salt thereof. [0008] In an aspect, the present disclosure provides a method for selecting a leukemia patient for treatment with a TP53 reactivating indole derivative and an additional therapeutic agent comprising: detecting wild-type TP53 or TP53-Y220C mRNA or polypeptide expression in a biological sample obtained from a leukemia patient; and administering to the patient an effective amount of a TP53 reactivating indole derivative and an effective amount of an MDM2 inhibitor, a BCL-2 inhibitor, and/or an XPO-1 inhibitor, wherein the TP53 reactivating indole derivative has formula of formula (I):
Figure imgf000007_0001
wherein: each - is independently a single bond or a double bond;
X1 is CR5, CR5R6, N, NR5, O, S, C=O, C=S, or a carbon atom connected to Q1;
X2 is CR7, CR7R8, N, NR7, O, S, C=O, C=S, or a carbon atom connected to Q1;
X3 is CR9, CR9R10, N, NR9, O, S, C=O, C=S, or a carbon atom connected to Q1;
X4 is CR11, CRnR12, N, NR11, O, S, C=O, C=S, or a carbon atom connected to Q1;
X5 is CR13, N, or NR13; wherein at least one of X1, X2, X3, and X4 is a carbon atom connected to Q1;
Q1 is C=O, C=S, C=CR14R15, C=NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, a bond; m is 1, 2, 3, or 4;
Y is N or O;
R1 is -C(O)R16, -C(O)OR16, -C(O)NR16R17, -OR16, -R16OR17, -R16OR17R18, -SR16, - R16SR17, -R16SR17R18, -NR16R17, -R16NR17, -R16NR17R18, -NR16C(O)R16, - OC(O)R16, C=O, C=S, -CN, -SiR16R17R18, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; each R3 and R4 is independently -C(O)R19, -C(O)OR19, -C(O)NR19R20, -SOR19, - SO2R19, alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently unsubstituted or substituted, or hydrogen; each R2, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, and R18 is independently - C(O)R21, -C(O)OR21, -C(O)NR21R22, -OR21, -SR21, -NR21R22, - NR21C(O)R22, -OC(O)R21, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently unsubstituted or substituted, or hydrogen; each R19 and R20 is C(O)R23, -C(O)OR23, -C(O)NR23R24, -OR23, -SR23, -NR23R24, - NR23C(O)R24, -OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen; each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or a pharmaceutically-acceptable salt thereof.
[0009] Administering to the leukemia patient may include administering the effective amount of the TP53 reactivating indole derivative and the effective amount of the MDM2 inhibitor. Administering to the leukemia patient may include administering the effective amount of the TP53 reactivating indole derivative and the effective amount of the BCL-2 inhibitor. Administering to the leukemia patient may include administering the effective amount of the TP53 reactivating indole derivative and the effective amount of the XPO-1 inhibitor. Administering to the leukemia patient may include administering the effective amount of the TP53 reactivating indole derivative, the effective amount of the MDM2 inhibitor, and the effective amount of the BCL-2 inhibitor. Administering to the leukemia patient may include administering the effective amount of the TP53 reactivating indole derivative, the effective amount of the MDM2 inhibitor, and the effective amount of the XPO-1 inhibitor. Administering to the leukemia patient may include administering the effective amount of the TP53 reactivating indole derivative, the effective amount of the BCL-2 inhibitor, and the effective amount of the XPO-1 inhibitor. Administering to the leukemia patient may include administering the effective amount of the TP53 reactivating indole derivative, the effective amount of the MDM2 inhibitor, the effective amount of the BCL-2 inhibitor, and the effective amount of the XPO-1 inhibitor.
[0010] Examples of MDM2 inhibitors include, but are not limited to, RG7112, RO5045337, idasanutlin, nutlin-3a, RG7388, AMG-232, KRT-232, APG-115, BI-907828, CGM097, siremadlin, HDM201, milademetan, BI 907828, MEL23, MEL24, and DS-3032b and MDM2 degraders, such as spirooxindole MDM2 inhibitors (e.g., MI-1061) tethered to lenalidomide, nutlin derivatives tethered to lenalidomide analogues, YX-02-030 (a RG7112 derivative) and MS3227. Examples of BCL-2 inhibitors include, but are not limited to venetoclax, obatoclax, subatoclax, maritoclax, gossypol, apogossypol, TW-37, UMI-77, APG2575, and BDA-366. Examples of XPO-1 inhibitors include, but are not limited to KPT330 (Selinexor), XPOVIO, KPT8602 (Eltanexor), KPT8602, KPT330, KPT335, Verdinexor, and KPT185.
[0011] Additionally or alternatively, in any embodiment of the methods disclosed herein, the MDM2 inhibitor, the BCL-2 inhibitor, and/or the XPO-1 inhibitor are sequentially, simultaneously, or separately administered with the TP53 reactivating indole derivative. In certain embodiments, the MDM2 inhibitor, the BCL-2 inhibitor, the XPO-1 inhibitor, and/or the TP53 reactivating indole derivative is administered orally, intravenously, intramuscularly, intraperitoneally, or subcutaneously.
[0012] In any and all embodiments of the methods disclosed herein, the leukemia is acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). The leukemia may be TP53-Y220C leukemia.
[0013] The TP53 reactivating indole derivative may have a formula of formula (IA)
Figure imgf000009_0001
wherein:
X1 is CR5, NR5, O, S, C=O, C=S, or a carbon atom connected to Q1; X2 is CR7, CH, NR7, O, S, C=O, C=S, or a carbon atom connected to Q1;
X3 is CR9, CH, NR9, O, S, C=O, C=S, or a carbon atom connected to Q1;
X4 is CR11, CH, NR11, O, S, C=O, C=S, or a carbon atom connected to Q1;
X5 is CR13, CH, or NR13; wherein at least one of X1, X2, X3, and X4 is a carbon atom connected to Q1;
Q1 is a bond; m is 1, 2, 3, or 4;
Y is N or O;
R1 is -C(O)R16, -C(O)OR16, -C(O)NR16R17, -OR16, -R16OR17, -R16OR17R18, -SR16, - R16SR17, -R16SR17R18, -NR16R17, -R16NR17, -R16NR17R18, -NR16C(O)R16, - OC(O)R16, C=O, C=S, -CN, -SiR16R17R18, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; each R3 and R4 is independently -C(O)R19, -C(O)OR19, -C(O)NR19R20, -SOR19, - SO2R19, hydrogen, alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl, wherein each of alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl is independently unsubstituted or substituted with one or two groups selected from alkyl or halogen, or R3 and R4 together with the Y atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent; each R2, R5, R7, R9, R11, R13, R16, R17, and R18 is independently - C(O)R21, - C(O)OR21, -C(O)NR21R22, -OR21, -SR21, -NR21R22, -NR21C(O)R22, -OC(O)R21, hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, wherein each alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl is independently unsubstituted or substituted with one or two groups selected from -C(O)R25, - C(O)OR25, -C(O)NR25, -CR253, -OR25, -SR25, -NR25R26, -NR25C(O)R26, -OC(O)R25, alkyl, alkenyl, or alkynyl, wherein R25 and R26 are each independently alkyl, hydrogen, or halogen; each R19 and R20 is C(O)R23, -C(O)OR23, -C(O)NR23R24, -OR23, -SR23, -NR23R24, - NR23C(O)R24, -OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen; each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or a pharmaceutically-acceptable salt thereof.
[0014] The TP53 reactivating indole derivative may have a formula of formula (IB)
Figure imgf000011_0001
wherein:
X1 is CR5, NR5, O, S, C=O, C=S, or a carbon atom connected to Y;
X2 is CR7, CH, NR7, O, S, C=O, C=S, or a carbon atom connected to Y;
X3 is CR9, CH, NR9, O, S, C=O, C=S, or a carbon atom connected to Y;
X4 is CR11, CH, NR11, O, S, C=O, C=S, or a carbon atom connected to Y;
X5 is CR13, CH, or NR13; wherein at least one of X1, X2, X3, and X4 is a carbon atom connected to Y;
Y is N or O;
R1 is -C(O)R16, -C(O)OR16, -C(O)NR16R17, -OR16, -R16OR17, -R16OR17R18, -SR16, -
R16SR17, -R16SR17R18, -NR16R17, -R16NR17, -R16NR17R18, -NR16C(O)R16, -
OC(O)R16, C=O, C=S, -CN, -SiR16R17R18, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; each R3 and R4 is independently -C(O)R19, -C(O)OR19, -C(O)NR19R20, -SOR19, - SO2R19, hydrogen, alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl, wherein each of alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl is independently unsubstituted or substituted with one or two groups selected from alkyl or halogen, or R3 and R4 together with the Y atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent; each R2, R5, R7, R9, R11, R13, R16, R17, and R18 is independently - C(O)R21, - C(O)OR21, -C(O)NR21R22, -CR253, -OR21, -SR21, -NR21R22, -NR21C(O)R22, - OC(O)R21, hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, wherein each of alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl is independently unsubstituted or substituted with one or two groups selected from -C(O)R25, -C(O)OR25, -C(O)NR25, -CR25 3, -OR25, -SR25, -NR25R26, - NR25C(O)R26, -OC(O)R25, alkyl, alkenyl, or alkynyl, wherein R25 and R26 are each independently alkyl, hydrogen, or halogen; each R19 and R20 is C(O)R23, -C(O)OR23, -C(O)NR23R24, -OR23, -SR23, -NR23R24, - NR23C(O)R24, -OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen; each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or a pharmaceutically-acceptable salt thereof. [0015] The TP53 reactivating indole derivative may have a formula
Figure imgf000013_0001
[0016] Additionally or alternatively, in any embodiment of the methods disclosed herein, mRNA expression levels are detected via real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, microarray, dot or slot blots, in situ hybridization, or fluorescent in situ hybridization (FISH). Additionally or alternatively, in certain embodiments of the methods disclosed herein, polypeptide expression levels are detected via Western blotting, enzyme-linked immunosorbent assays (ELISA), dot blotting, immunohistochemistry, immunofluorescence, immunoprecipitation, immunoelectrophoresis, or mass-spectrometry.
[0017] In any and all embodiments of the methods disclosed herein, the patient is non- responsive to at least one prior line of cancer therapy, such as chemotherapy or immunotherapy. In some embodiments, the chemotherapy comprises one or more of trioxide, azacytidine, cerubidine, cyclophosphamide, cytarabine, daunorubicin hydrochloride, daurismo, dexamethasone, doxorubicin hydrochloride, enasidenib mesylate, gemtuzumab ozogamicin, gilteritinib fumarate, glasdegib maleate, idamycin PFS, idarubicin hydrochloride, idhifa, ivosidenib, midostaurin, mitoxantrone hydrochloride, mylotarg, olutasidenib, onureg, pemazyre, pemigatinib, prednisone, rezlidhia, Rituxan, rituximab, rubidomycin, rydapt, tabloid, thioguanine, tibsovo, tisagenlecleucel, trisenox, venclexta, venetoclas, vinicristine sulfate, vyxeos, or xospata. The patient may be a child or an adult.
[0018] Also disclosed herein are kits comprising (a) a TP53 reactivating indole derivative (e.g., PC14586), (b) at least one of an MDM2 inhibitor, a BCL-2 inhibitor, and/or an XPO-1 inhibitor, and (c) instructions for treating wild-type p53 leukemia and/or TP53- Y220C leukemia. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 : PC14586 converts mutant p53 Y220C to wild-type p53 conformation
(upper panel) and activates p53 transcriptional activity and greatly induces p21 (lower panel). FIG. 1 shows a Western blot demonstrating the activity of PC14586 on mutant and wild-type p53 (upper panel) and a Western blot demonstrating the transcriptional activity of p53 (lower panel) after treatment with PC 14586.
[0020] FIGS. 2A-2B: PC14586 primarily suppresses cell grow in TP53 Y220C, not in TP53 WT, KO, or TP53 R175H mutant AML cells. FIG. 2A: Dose-response viability curves of a panel of AML cell lines having TP53-WT, TP53-KO, TP53-R175H, or TP53- Y220C treated with PC 14586 for 120 hours. FIG. 2B: 7- Aminoactinomycin (7AAD)/annexin V (AnnV) curves of a panel of AML cell lines having TP53-WT, TP53- KO, TP53-R175H, or TP53-Y220C treated with PC14586 for 120 hours.
[0021] FIGS. 3A-3H: MDM2 inhibition enhances PC 14586 activity in AML cells having TP53-WT and the combination is highly synergistic in AML cells having TP53- Y220C. FIG. 3A: 7AAD/AnnV curves of AML cells having TP53-WT treated with an MDM2 inhibitor, PC 14586, or a combination of the MDM2 inhibitor and PC 14586 for 72 hours. FIG. 3B: Dose-response viability curves of AML cells having TP53-WT treated with the MDM2 inhibitor, PC 14586, or the combination of the MDM2 inhibitor and PC 14586 for 72 hours. FIG. 3C: 7AAD/AnnV curves of AML cells having TP53-Y220C treated with the MDM2 inhibitor, PC 14586, or the combination of the MDM2 inhibitor and PC 14586 for 72 hours. FIG. 3D: Dose-response viability curves of AML cells having TP53-Y220C treated with the MDM2 inhibitor, PC 14586, or the combination of the MDM2 inhibitor and PC14586 for 72 hours. FIG. 3E: 7AAD/AnnV curves of AML cells having TP53-KO treated with the MDM2 inhibitor, PC 14586, or the combination of the MDM2 inhibitor and PC14586 for 72 hours. FIG. 3F: Dose-response viability curves of AML cells having TP53- KO treated with the MDM2 inhibitor, PC 14586, or the combination of the MDM2 inhibitor and PC 14586 for 72 hours. FIG. 3G: 7AAD/AnnV curves of AML cells having TP53- R175H treated with the MDM2 inhibitor, PC14586, or the combination of the MDM2 inhibitor and PC 14586 for 72 hours. FIG. 3H: Dose-response viability curves of AML cells having TP53-R175H treated with the MDM2 inhibitor, PC14586, or the combination of the MDM2 inhibitor and PC14586 for 72 hours. [0022] FIGS. 4A-4H: BCL-2 inhibition enhances PC 14586 activity in AML cells having TP53-WT and the combination is highly synergistic in AML cells having TP53- Y220C. FIG. 4A: 7AAD/AnnV curves of AML cells having TP53-WT treated with a BCL- 2 inhibitor, PC14586, or a combination of the BCL-2 inhibitor and PC14586 for 72 hours. FIG. 4B: Dose-response viability curves of AML cells having TP53-WT treated with the BCL-2 inhibitor, PC14586, or the combination of the BCL-2 inhibitor and PC14586 for 72 hours. FIG. 4C: 7AAD/AnnV curves of AML cells having TP53-Y220C treated with the BCL-2 inhibitor, PC14586, or the combination of the BCL-2 inhibitor and PC14586 for 72 hours. FIG. 4D: Dose-response viability curves of AML cells having TP53-Y220C treated with the BCL-2 inhibitor, PC 14586, or the combination of the BCL-2 inhibitor and PC14586 for 72 hours. FIG. 4E: 7AAD/AnnV curves of AML cells having TP53-KO treated with the BCL-2 inhibitor, PC 14586, or the combination of the BCL-2 inhibitor and PC14586 for 72 hours. FIG. 4F: Dose-response viability curves of AML cells having TP53- KO treated with the BCL-2 inhibitor, PC14586, or the combination of the BCL-2 inhibitor and PC 14586 for 72 hours. FIG. 4G: 7AAD/AnnV curves of AML cells having TP53- R175H treated with the BCL-2 inhibitor, PC14586, or the combination of the BCL-2 inhibitor and PC 14586 for 72 hours. FIG. 4H: Dose-response viability curves of AML cells having TP53-R175H treated with the BCL-2 inhibitor, PC14586, or the combination of the BCL-2 inhibitor and PC14586 for 72 hours.
[0023] FIGS. 5A-5H: XPO-1 inhibition enhances PC14586 activity in AML cells having TP53-WT and the combination is highly synergistic in AML cells having TP53- Y220C. FIG. 5A: 7AAD/AnnV curves of AML cells having TP53-WT treated with an XPO-1 inhibitor, PC14586, or a combination of the XPO-1 inhibitor and PC14586 for 72 hours. FIG. 5B: Dose-response viability curves of AML cells having TP53-WT treated with the XPO-1 inhibitor, PC 14586, or the combination of the XPO-1 inhibitor and PC 14586 for 72 hours. FIG. 5C: 7AAD/AnnV curves of AML cells having TP53-Y220C treated with the XPO-1 inhibitor, PC14586, or the combination of the XPO-1 inhibitor and PC14586 for 72 hours. FIG. 5D: Dose-response viability curves of AML cells having TP53-Y220C treated with the XPO-1 inhibitor, PC 14586, or the combination of the XPO-1 inhibitor and PC14586 for 72 hours. FIG. 5E: 7AAD/AnnV curves of AML cells having TP53-KO treated with the XPO-1 inhibitor, PC 14586, or the combination of the XPO-1 inhibitor and PC14586 for 72 hours. FIG. 5F: Dose-response viability curves of AML cells having TP53- KO treated with the XPO-1 inhibitor, PC 14586, or the combination of the XPO-1 inhibitor and PC 14586 for 72 hours. FIG. 5G: 7AAD/AnnV curves of AML cells having TP53- R175H treated with the XPO-1 inhibitor, PC 14586, or the combination of the XPO-1 inhibitor and PC 14586 for 72 hours. FIG. 5H: Dose-response viability curves of AML cells having TP53-R175H treated with the XPO-1 inhibitor, PC14586, or the combination of the XPO-1 inhibitor and PC14586 for 72 hours.
[0024] FIGS. 6A-6D: PC 14586 in combination with three other agents shows the highest synergy, followed by PC 14586 in combination with two other agents as compared to PC 14586 in combination with one agent or PC 14586 alone with respect to in vitro inhibition of AML cells having TP53-WT (WT) and in AML cells having TP53-Y220C (Y220C). FIG. 6A: 7AAD/AnnV curves of AML cells having TP53-WT treated with PC14586, a BCL-2 inhibitor (BCL201), an MDM2 inhibitor (HDM201), an XPO-1 inhibitor (KPT-330), or a combination of PC 14586 with one, two, or all three of these other agents after 72-hour incubation. FIG. 6B: Dose-response viability curves of AML cells having TP53-WT treated with PC14586, the BCL-2 inhibitor, the MDM2 inhibitor, the XPO-1 inhibitor, or the combination of PC 14586 with one, two, or all three of these other agents after 72-hour incubations. FIG. 6C: 7AAD/AnnV curves of AML cells having TP53-Y220C treated with PC14586, the BCL-2 inhibitor, the MDM2 inhibitor, the XPO-1 inhibitor, or the combination of PC 14586 with one, two, or all three of these other agents after 72-hour incubations. FIG. 6D: Dose-response viability curves of AML cells having TP53-Y220C treated with PC14586, the BCL-2 inhibitor, the MDM2 inhibitor, the XPO-1 inhibitor, or the combination of PC 14586 with one, two, or all three of these other agents after 72-hour incubations.
[0025] FIGS. 7A-7C: Molml3 TP53-Y220C cells treated with PC14586-alone or in combination with XPO-1, MDM2, or BCL-2 inhibitors. FIG. 7A: Western blots demonstrating the protein levels of Molml3 TP53-Y220C cells treated for 24 hours with venetoclax (VEN, 5 nM or 10 nM), nutlin-3a (Nut, 2.5 pM or 5 pM), PC14586 (PC, 2 pM or 4 pM), KPT-8602 (KPT, 100 nM or 200 nM), or combinations of PC 14586 with venetoclax (VEN/PC, 5 nM VEN and 2 pM PC, 10 nM VEN and 4 pM PC), nutlin-3a (Nut/PC, 2.5 pM Nut and 2 pM PC, 5 pM Nut and 4 pM PC), or KPT-8602 (KPT/PC, 100 nM KPT and 2 pM PC, 200 nM KPT and 4 pM PC). FIG. 7B: Cell cycle distribution and apoptosis determined by flow cytometry of cells stained with 5-ethynyl-2’-deoxyuridine (EdU) and DNA dye. Molml3 TP53-Y220C cells were treated for 72 hours with venetoclax (VEN, 10 nM), nutlin-3a (N3, 5 pM), KPT-8602 (KPT, 200 nM), PC 14586 (4 pM), or combinations of PC 14586 with venetoclax (4 pM PC 14586 and 10 nM VEN), nutlin-3a (4 pM PC14586 and 5 pM N3), or KPT (4 pM PC14586 and 200 nM KPT). FIG. 7C: DNA content and PARP cleavage determined by flow cytometry of Molml3 TP53- Y220C cells treated for 72 hours with venetoclax (VEN, 10 nM), nutlin-3a (N3, 5 pM), KPT-8602 (KPT, 200 nM), PC 14586 (4 pM), or combinations of PC 14586 with venetoclax (4 pM PC 14586 and 10 nM VEN), nutlin-3a (4 pM PC 14586 and 5 pM), or KPT-8602 (4 pM PC14586 and 200 nM KPT). Cells were stained with EdU, DNA dye, and antibody against cleaved PARP.
[0026] FIGS. 8A-8G: Cells from an AML patient peripheral blood (PB) sample with 77% TP53-Y220C under mesenchymal stroma cell (MSC) co-culture conditions (COX). Cell death and viable cells were determined by flow cytometry after the cells were stained with annexin V/7-aminoactinomycin D (Ann V/7-ADD) in the presence of counting beads. FIG. 8 A: CD45+, CD34+, and CD34+CD38- population after 48 hours of treatment with PC14586 at different concentrations. FIG. 8B: CD45+ population assessed by Ann V/7- ADD+ positive cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT- 8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC14586; or nutlin-3a, KPT-8602, and PC14586. FIG. 8C: CD45+ viable cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC14586. FIG. 8D: CD34+ assessed by Ann V/7ADD+ cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC14586; venetoclax and PC14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC 14586. FIG. 8E: CD34+ viable cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC14586. FIG. 8F: CD34+CD38- population assessed by Ann V/7ADD+ cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC14586. FIG. 8G: CD34+CD38- viable cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC14586; KPT-8602 and PC14586; or nutlin-3a, KPT-8602, and PC14586. [0027] FIGS. 9A-9G: Cells from an AML patient peripheral blood (PB) sample with 45.7% TP53-Y220C under MSC COX. Cell death and viable cells were determined by flow cytometry after the cells were stained with Ann V/7-ADD in the presence of counting beads. FIG. 9A: CD45+, CD34+, and CD34+CD38- population after 48 hours of treatment with PC 14586 at different concentrations. FIG. 9B: CD45+ population assessed by Ann V/7-ADD+ positive cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT- 8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC14586; or nutlin-3a, KPT-8602, and PC14586. FIG. 9C: CD45+ viable cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC14586. FIG. 9D: CD34+ assessed by Ann V/7ADD+ cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC14586; venetoclax and PC14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC 14586. FIG. 9E: CD34+ viable cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC14586. FIG. 9F: CD34+CD38- population assessed by Ann V/7ADD+ cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC14586. FIG. 9G: CD34+CD38- viable cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC14586; KPT-8602 and PC14586; or nutlin-3a, KPT-8602, and PC14586.
[0028] FIGS. 10A-10D: Patient-derived xenograft cells with Y220C mutation under MSC COX. Cell death and viable cells were determined by flow cytometry after the cells were stained with Ann V/7-ADD in the presence of counting beads. FIG. 10A: CD45+, CD34+, and CD34+CD38- population after 96 hours of treatment with PC14586 at different concentrations. FIG. 10B: CD45+ population assessed by Ann V/7-ADD+ positive cells after 96 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; nutlin-3a, KPT-8602, and PC 14586; venetoclax, KPT-8602, and PC 14586; KPT-8602, nutlin-3a, and PC14586; or venetoclax, nutlin-3a, KPT-8602, and PC14586. FIG. 10C: CD45+ viable cells after 96 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; nutlin-3a, KPT-8602, and PC 14586; venetoclax, KPT-8602, and PC 14586; KPT-8602, nutlin-3a, and PC14586; or venetoclax, nutlin-3a, KPT-8602, and PC14586. FIG. 10D: CD34+ assessed by Ann V/7ADD+ cells after 96 hours of treatment with venetoclax; nutlin-3a (N3); KPT- 8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; nutlin-3a, KPT-8602, and PC 14586; venetoclax, KPT-8602, and PC 14586; KPT- 8602, nutlin-3a, and PC14586; or venetoclax, nutlin-3a, KPT-8602, and PC14586.
[0029] FIG. 10E: CD34+ viable cells after 96 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC14586; venetoclax and PC14586; nutlin-3a and PC14586; KPT-8602 and PC 14586; nutlin-3a, KPT-8602, and PC 14586; venetoclax, KPT-8602, and PC 14586; KPT-8602, nutlin-3a, and PC 14586; or venetoclax, nutlin-3a, KPT-8602, and PC14586.
[0030] FIG. 10F: CD34+CD38- population assessed by Ann V/7ADD+ cells after 96 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; nutlin-3a, KPT-8602, and PC 14586; venetoclax, KPT-8602, and PC 14586; KPT-8602, nutlin-3a, and PC 14586; or venetoclax, nutlin-3a, KPT-8602, and PC 14586.
[0031] FIG. 10G: CD34+CD38- viable cells after 96 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; nutlin-3a, KPT-8602, and PC 14586; venetoclax, KPT- 8602, and PC14586; KPT-8602, nutlin-3a, and PC14586; or venetoclax, nutlin-3a, KPT- 8602, and PC 14586.
[0032] FIGS. 11A-11D: Normal bone marrow cells under MSC COX. Cell death and viable cells were determined by flow cytometry after the cells were stained with Ann V/7- ADD in the presence of counting beads. FIG. 11 A: CD45+ population assessed by Ann V/7-ADD+ positive cells after 48 hours of treatment with venetoclax; nutlin-3a; KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or venetoclax, nutlin-3a, KPT-8602, and PC14586. FIG. 1 IB: CD45+ cell viability after 48 hours of treatment with venetoclax; nutlin-3a; KPT-8602; PC14586; venetoclax and PC14586; nutlin-3a and PC14586; KPT-8602 and PC14586; or venetoclax, nutlin-3a, KPT- 8602, and PC14586. FIG. 11C: CD34+ population assessed by Ann V/7-ADD+ positive cells after 48 hours of treatment with venetoclax; nutlin-3a; KPT-8602; PC14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or venetoclax, nutlin-3a, KPT-8602, and PC 14586. FIG. 1 ID: CD34+ cell viability after 48 hours of treatment with venetoclax; nutlin-3a; KPT-8602; PC14586; venetoclax and PC14586; nutlin-3a and PC14586; KPT-8602 and PC14586; or venetoclax, nutlin-3a, KPT-8602, and PC14586.
[0033] FIG. 12A-12F: Molml3 TP53-WT and TP53-Y220C cells were treated with nutlin-3a (N3a, 5 pM), PC14586 (PC, 4 pM), or the combination for 4 hours. RNA was isolated and subject to RNA sequence. FIG. 12A: Principal component analysis. FIG. 12B: Gene set enrichment analysis. FIG. 12C: Differentially expressed genes. FIG. 12D: Pathway analysis. FIG. 12E: Volcano plots show comparisons of increased, unchanged, and decreased gene expression in treated compared to controls. FIG. 12F: Western blots showing changes in several gene expression.
[0034] FIG. 13A-13C: Patient-derived xenograft cells from an AML patient sample with TP53-Y220C (VAF 48%), TP53-P151A (VAF 47%), and NRAS (VAF 50%) mutations were injected via tail vein into NSG mice followed by treatment. Treatments included vehicle (negative control), PC14586 (100 mg/kg, daily), MDM2 inhibitor idasanutlin (RG7388) with 50% activity (40 mg/kg, initially 8 days on, then 2 days off/5 days on) or both PC14586 (100 mg/kg, daily), MDM2 inhibitor idasanutlin (RG7388) with 50% activity (40 mg/kg, initially 8 days on, then 2 days off/5 days on). FIG. 13A: Flow cytometry analysis of human CD45+ cells in peripheral blood (PB) after four weeks of treatment. FIG. 13B: Flow cytometry analysis of human CD45+ cells in peripheral blood (PB) after eight weeks of treatment. FIG. 13C: Kaplan-Meier estimation of mouse survival and survival data analyzed using the log-rank test.
[0035] FIG. 14: Western blot of p53 and BCL-2 from Molml3 TP53-WT and TP53- Y220C cells treated for 4 hours with nutlin-3a (5 pM) or PC 14586 (PC, 1.25 pM or 5 pM).
[0036] FIG. 15: Kaplan-Meier estimation of mouse survival and survival data analyzed using the log-rank test of mice injected with Molml3 TP53-Y220C cells, and treated daily with vehicle, PC14586 (100 mg/kg), venetoclax (50 mg/kg), or the combination.
[0037] FIG. 16: Western blot of p53, Lamin Bl, and tubulin from TP53-Y220C Molml3 cells treated with PC14586 (4 pM), KPT-8602 (200 nM), or both for 24 hours. DETAILED DESCRIPTION
[0038] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology. It is to be understood that the present disclosure is not limited to particular uses, methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
[0039] In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology, and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology, the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach,' Harlow and Lane eds. ( \ 999 Antibodies, A Laboratory Manual,' Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis,' U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization,' Anderson (1999) Nucleic Acid Hybridization,' Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir ’s Handbook of Experimental Immunology. Methods to detect and measure levels of polypeptide gene expression products (i.e., gene translation level) are well-known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also, Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)). Definitions
[0040] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.
[0041] The phrase “and/or” as used in the present disclosure will be understood to mean any one of the recited members individually or a combination of any two or more thereof - for example, “A, B, and/or C” would mean “A, B, C, A and B, A and C, B and C, or the combination of A, B, and C.”
[0042] Generally, reference to a certain element such as hydrogen or H is meant to include all isotopes of that element. For example, if an R group is defined to include hydrogen or H, it also includes deuterium and tritium. Compounds comprising radioisotopes such as tritium, C14, P32 and S35 are thus within the scope of the present technology. Procedures for inserting such labels into the compounds of the present technology will be readily apparent to those skilled in the art based on the disclosure herein.
[0043] As used herein, the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).
[0044] As used herein, the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including but not limited to, orally, intranasally, intrathecally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, intraocularly, intradermally, transmucosally, iontophoretically, or topically. Administration includes self-administration and the administration by another. [0045] As used herein, the terms “cancer” or “tumor” are used interchangeably and refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells can exist alone within an animal, or can be a non-tumorigenic cancer cell. As used herein, the term “cancer cells” includes precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and non-metastatic cells. Cancers of virtually every tissue are known to those of skill in the art, including solid tumors such as carcinomas, sarcomas, glioblastomas, melanomas, etc., and circulating cancers such as leukemias. Examples of cancer include, but are not limited to, ovarian cancer, breast cancer, colon cancer, lung cancer, prostate cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, head and neck cancer, and brain cancer. The phrase “cancer burden” or “tumor burden” refers to the quantity of cancer cells or tumor volume in a subject. Reducing cancer burden accordingly may refer to reducing the number of cancer cells, or the tumor volume in a subject. The term “cancer cell” refers to a cell that exhibits cancer-like properties, e.g., uncontrollable reproduction, resistance to anti- growth signals, ability to metastasize, and loss of ability to undergo programmed cell death (e.g., apoptosis) or a cell that is derived from a cancer cell, e.g., clone of a cancer cell.
[0046] As used herein, a "control" is an alternative sample used in an experiment for comparison purpose. A control can be "positive" or "negative." For example, where the purpose of the experiment is to determine a correlation of the efficacy of a therapeutic agent for the treatment for a particular type of disease, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or a sample that does not receive the therapy or receives a placebo) are typically employed.
[0047] As used herein, the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will vary depending on the composition, the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic compositions may be administered to a subject having one or more signs or symptoms of a disease or condition described herein. As used herein, a "therapeutically effective amount" of a composition refers to composition levels in which the physiological effects of a disease or condition are ameliorated or eliminated. A therapeutically effective amount can be given in one or more administrations.
[0048] As used herein, “expression” includes one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function.
[0049] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to mean a polymer comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, z.e., peptide isosteres. Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides, or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art.
[0050] As used herein, a “sample” or “biological sample” refers to a body fluid or a tissue sample isolated from a subject. In some cases, a biological sample may consist of or comprise whole blood, platelets, red blood cells, white blood cells, plasma, sera, urine, feces, epidermal sample, vaginal sample, skin sample, cheek swab, sperm, amniotic fluid, cultured cells, bone marrow sample, tumor biopsies, aspirate and/or chorionic villi, cultured cells, endothelial cells, synovial fluid, lymphatic fluid, ascites fluid, interstitial or extracellular fluid and the like. The term "sample" may also encompass the fluid in spaces between cells, including gingival crevicular fluid, bone marrow, cerebrospinal fluid (CSF), saliva, mucus, sputum, semen, sweat, urine, or any other bodily fluids. Samples can be obtained from a subject by any means including, but not limited to, venipuncture, excretion, ejaculation, massage, biopsy, needle aspirate, lavage, scraping, surgical incision, or intervention or other means known in the art. A blood sample can be whole blood or any fraction thereof, including blood cells (red blood cells, white blood cells or leukocytes, and platelets), serum and plasma.
[0051] As used herein, the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.
[0052] As used herein, the term “sequential” therapeutic use refers to administration of at least two active ingredients at different times. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.
[0053] As used herein, the term “simultaneous” therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time.
[0054] As used herein, the terms “subject”, “patient”, or “individual” can be an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the subject, patient, or individual is a human.
[0055] As used herein, a “synergistic therapeutic effect” refers to a greater-than-additive therapeutic effect which is produced by a combination of at least two agents, and which exceeds that which would otherwise result from the individual administration of the agents. For example, lower doses of one or more agents may be used in treating a disease or disorder.
[0056] As used herein, the term “therapeutic agent” is intended to mean a compound that, when present in an effective amount, produces a desired therapeutic effect on a subject in need thereof.
[0057] “Treating” or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, i.e., arresting its development; (ii) relieving a disease or disorder, i.e., causing regression of the disorder; (iii) slowing progression of the disorder; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. In some embodiments, treatment means that the symptoms associated with the disease are, e.g., alleviated, reduced, cured, or placed in a state of remission.
[0058] It is also to be appreciated that the various modes of treatment of disorders as described herein are intended to mean “substantial,” which includes total but also less than total treatment, and wherein some biologically or medically relevant result is achieved. The treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.
[0059] Generally, reference to a certain element such as hydrogen or H is meant to include all isotopes of that element. For example, if an R group is defined to include hydrogen or H, it also includes deuterium and tritium. Compounds comprising radioisotopes such as tritium, C14, P32 and S35 are thus within the scope of the present technology. Procedures for inserting such labels into the compounds of the present technology will be readily apparent to those skilled in the art based on the disclosure herein.
[0060] In general, “substituted” refers to an organic group as defined below (e.g., an alkyl group) in which one or more bonds to a hydrogen atom contained therein are replaced by a bond to non-hydrogen or non-carbon atoms. Substituted groups also include groups in which one or more bonds to a carbon(s) or hydrogen(s) atom are replaced by one or more bonds, including double or triple bonds, to a heteroatom. Thus, a substituted group is substituted with one or more substituents, unless otherwise specified. In some embodiments, a substituted group is substituted with 1, 2, 3, 4, 5, or 6 substituents.
Examples of substituent groups include: halogens i.e., F, Cl, Br, and I); hydroxyls; alkoxy, alkenoxy, aryloxy, aralkyloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, and heterocyclylalkoxy groups; carbonyls (oxo); carboxylates; esters; urethanes; oximes; hydroxylamines; alkoxyamines; aralkoxyamines; thiols; sulfides; sulfoxides; sulfones; sulfonyls; pentafluorosulfanyl (i.e., SFs), sulfonamides; amines; N-oxides; hydrazines; hydrazides; hydrazones; azides; amides; ureas; amidines; guanidines; enamines; imides; isocyanates; isothiocyanates; cyanates; thiocyanates; imines; nitro groups; and nitriles (i.e., CN). [0061] Substituted ring groups such as substituted cycloalkyl, aryl, heterocyclyl and heteroaryl groups also include rings and ring systems in which a bond to a hydrogen atom is replaced with a bond to a carbon atom. Therefore, substituted cycloalkyl, aryl, heterocyclyl and heteroaryl groups may also be substituted with substituted or unsubstituted alkyl, alkenyl, and alkynyl groups as defined below.
[0062] Alkyl groups include straight chain and branched chain alkyl groups having from 1 to 12 carbon atoms, and typically from 1 to 10 carbons or, in some embodiments, from 1 to 8, 1 to 6, or 1 to 4 carbon atoms. Alkyl groups may be substituted or unsubstituted. Examples of straight chain alkyl groups include groups such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, tert-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups. Representative substituted alkyl groups may be substituted one or more times with substituents such as those listed above, and include without limitation haloalkyl (e.g., trifluoromethyl), hydroxyalkyl, thioalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, alkoxyalkyl, carboxyalkyl, and the like.
[0063] Cycloalkyl groups include mono-, bi- or tricyclic alkyl groups having from 3 to 12 carbon atoms in the ring(s), or, in some embodiments, 3 to 10, 3 to 8, or 3 to 4, 5, or 6 carbon atoms. Cycloalkyl groups may be substituted or unsubstituted. Exemplary monocyclic cycloalkyl groups include, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, the cycloalkyl group has 3 to 8 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 5, 3 to 6, or 3 to 7. Bi- and tricyclic ring systems include both bridged cycloalkyl groups and fused rings, such as, but not limited to, bicyclo[2.1.1]hexane, adamantyl, decalinyl, and the like. Substituted cycloalkyl groups may be substituted one or more times with non-hydrogen and non-carbon groups as defined above. However, substituted cycloalkyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined above. Representative substituted cycloalkyl groups may be mono-substituted or substituted more than once, such as, but not limited to, 2,2-, 2,3-, 2,4- 2,5- or 2,6-disubstituted cyclohexyl groups, which may be substituted with substituents such as those listed above.
[0064] Cycloalkylalkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a cycloalkyl group as defined above. Cycloalkylalkyl groups may be substituted or unsubstituted. In some embodiments, cycloalkylalkyl groups have from 4 to 16 carbon atoms, 4 to 12 carbon atoms, and typically 4 to 10 carbon atoms. Substituted cycloalkylalkyl groups may be substituted at the alkyl, the cycloalkyl or both the alkyl and cycloalkyl portions of the group. Representative substituted cycloalkylalkyl groups may be mono-substituted or substituted more than once, such as, but not limited to, mono-, di- or tri -substituted with substituents such as those listed above.
[0065] Alkenyl groups include straight and branched chain alkyl groups as defined above, except that at least one double bond exists between two carbon atoms. Alkenyl groups may be substituted or unsubstituted. Alkenyl groups have from 2 to 12 carbon atoms, and typically from 2 to 10 carbons or, in some embodiments, from 2 to 8, 2 to 6, or 2 to 4 carbon atoms. In some embodiments, the alkenyl group has one, two, or three carboncarbon double bonds. Examples include, but are not limited to vinyl, allyl, -CH=CH(CH3), -CH=C(CH3)2, -C(CH3)=CH2, -C(CH3)=CH(CH3), -C(CH2CH3)=CH2, among others.
Representative substituted alkenyl groups may be mono-substituted or substituted more than once, such as, but not limited to, mono-, di- or tri -substituted with substituents such as those listed above.
[0066] Cycloalkenyl groups include cycloalkyl groups as defined above, having at least one double bond between two carbon atoms. Cycloalkenyl groups may be substituted or unsubstituted. In some embodiments the cycloalkenyl group may have one, two or three double bonds but does not include aromatic compounds. Cycloalkenyl groups have from 4 to 14 carbon atoms, or, in some embodiments, 5 to 14 carbon atoms, 5 to 10 carbon atoms, or even 5, 6, 7, or 8 carbon atoms. Examples of cycloalkenyl groups include cyclohexenyl, cyclopentenyl, cyclohexadienyl, cyclobutadienyl, and cyclopentadienyl.
[0067] Cycloalkenylalkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of the alkyl group is replaced with a bond to a cycloalkenyl group as defined above. Cycloalkenylalkyl groups may be substituted or unsubstituted. Substituted cycloalkenylalkyl groups may be substituted at the alkyl, the cycloalkenyl or both the alkyl and cycloalkenyl portions of the group. Representative substituted cycloalkenylalkyl groups may be substituted one or more times with substituents such as those listed above. [0068] Alkynyl groups include straight and branched chain alkyl groups as defined above, except that at least one triple bond exists between two carbon atoms. Alkynyl groups may be substituted or unsubstituted. Alkynyl groups have from 2 to 12 carbon atoms, and typically from 2 to 10 carbons or, in some embodiments, from 2 to 8, 2 to 6, or 2 to 4 carbon atoms. In some embodiments, the alkynyl group has one, two, or three carboncarbon triple bonds. Examples include, but are not limited to - C=CH, -C=CCH3, -CH2OCCH3, and -C=CCH2CH(CH2CH3)2, among others. Representative substituted alkynyl groups may be mono-substituted or substituted more than once, such as, but not limited to, mono-, di- or tri -substituted with substituents such as those listed above.
[0069] Aryl groups are cyclic aromatic hydrocarbons that do not contain heteroatoms. Aryl groups herein include monocyclic, bicyclic and tricyclic ring systems. Aryl groups may be substituted or unsubstituted. Thus, aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, fluorenyl, phenanthrenyl, anthracenyl, indenyl, indanyl, pentalenyl, and naphthyl groups. In some embodiments, aryl groups contain 6-14 carbons, and in others from 6 to 12 or even 6-10 carbon atoms in the ring portions of the groups. In some embodiments, the aryl groups are phenyl or naphthyl. The phrase “aryl groups” includes groups containing fused rings, such as fused aromatic-aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl, and the like). Representative substituted aryl groups may be mono-substituted (e.g., tolyl) or substituted more than once. For example, monosubstituted aryl groups include, but are not limited to, 2-, 3-, 4-, 5-, or 6-substituted phenyl or naphthyl groups, which may be substituted with substituents such as those listed above.
[0070] Aralkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined above. Aralkyl groups may be substituted or unsubstituted. In some embodiments, aralkyl groups contain 7 to 16 carbon atoms, 7 to 14 carbon atoms, or 7 to 10 carbon atoms. Substituted aralkyl groups may be substituted at the alkyl, the aryl or both the alkyl and aryl portions of the group. Representative aralkyl groups include but are not limited to benzyl and phenethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-indanylethyl. Representative substituted aralkyl groups may be substituted one or more times with substituents such as those listed above. [0071] Heterocyclyl groups include aromatic (also referred to as heteroaryl) and nonaromatic ring compounds containing 3 or more ring members, of which one or more is a heteroatom such as, but not limited to, N, O, and S. Heterocyclyl groups may be substituted or unsubstituted. In some embodiments, the heterocyclyl group contains 1, 2, 3 or 4 heteroatoms. In some embodiments, heterocyclyl groups include mono-, bi- and tricyclic rings having 3 to 16 ring members, whereas other such groups have 3 to 6, 3 to 10, 3 to 12, or 3 to 14 ring members. Heterocyclyl groups encompass aromatic, partially unsaturated and saturated ring systems, such as, for example, imidazolyl, imidazolinyl and imidazolidinyl groups. The phrase “heterocyclyl group” includes fused ring species including those comprising fused aromatic and non-aromatic groups, such as, for example, benzotri azolyl, 2,3-dihydrobenzo[l,4]dioxinyl, and benzo[l,3]dioxolyl. The phrase also includes bridged polycyclic ring systems containing a heteroatom such as, but not limited to, quinuclidyl. The phrase includes heterocyclyl groups that have other groups, such as alkyl, oxo or halo groups, bonded to one of the ring members, referred to as “substituted heterocyclyl groups”. Heterocyclyl groups include, but are not limited to, aziridinyl, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, thiazolidinyl, tetrahydrothiophenyl, tetrahydrofuranyl, dioxolyl, furanyl, thiophenyl, pyrrolyl, pyrrolinyl, imidazolyl, imidazolinyl, pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, thiazolinyl, isothiazolyl, thiadiazolyl, oxadiazolyl, piperidyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydropyranyl, tetrahydrothiopyranyl, oxathiane, dioxyl, dithianyl, pyranyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, dihydropyridyl, dihydrodithiinyl, dihydrodithionyl, homopiperazinyl, quinuclidyl, indolyl, indolinyl, isoindolyl,azaindolyl (pyrrolopyridyl), indazolyl, indolizinyl, benzotri azolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, benzthiazolyl, benzoxadiazolyl, benzoxazinyl, benzodithiinyl, benzoxathiinyl, benzothiazinyl, benzoxazolyl, benzothiazolyl, benzothiadi azolyl, benzo [1,3] dioxolyl, pyrazolopyridyl, imidazopyridyl (azabenzimidazolyl), tri azol opyridyl, isoxazolopyridyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, quinolizinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl, pteridinyl, thianaphthyl, dihydrobenzothiazinyl, dihydrobenzofuranyl, dihydroindolyl, dihydrobenzodioxinyl, tetrahydroindolyl, tetrahydroindazolyl, tetrahydrobenzimidazolyl, tetrahydrobenzotriazolyl, tetrahydropyrrol opy ri dy 1 , tetrahy dropy razol opy ri dy 1 , tetrahy droimi dazopy ri dy 1 , tetrahydrotriazolopyridyl, and tetrahydroquinolinyl groups. Representative substituted heterocyclyl groups may be mono-substituted or substituted more than once, such as, but not limited to, pyridyl or morpholinyl groups, which are 2-, 3-, 4-, 5-, or 6-substituted, or disubstituted with various substituents such as those listed above.
[0072] Heteroaryl groups are aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S. Heteroaryl groups may be substituted or unsubstituted. Heteroaryl groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiophenyl, benzothiophenyl, furanyl, benzofuranyl, indolyl, azaindolyl (pyrrolopyridinyl), indazolyl, benzimidazolyl, imidazopyridinyl (azabenzimidazolyl), pyrazolopyridinyl, triazolopyridinyl, benzotri azolyl, benzoxazolyl, benzothiazolyl, benzothiadi azolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Heteroaryl groups include fused ring compounds in which all rings are aromatic such as indolyl groups and include fused ring compounds in which only one of the rings is aromatic, such as 2,3-dihydro indolyl groups. Representative substituted heteroaryl groups may be substituted one or more times with various substituents such as those listed above.
[0073] Heterocyclylalkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heterocyclyl group as defined above. Heterocyclylalkyl groups may be substituted or unsubstituted. Substituted heterocyclylalkyl groups may be substituted at the alkyl, the heterocyclyl or both the alkyl and heterocyclyl portions of the group. Representative heterocyclyl alkyl groups include, but are not limited to, morpholin-4-yl-ethyl, furan-2-yl-methyl, imidazol-4-yl-methyl, pyridin-3-yl-methyl, tetrahydrofuran-2-yl-ethyl, and indol-2-yl-propyl. Representative substituted heterocyclylalkyl groups may be substituted one or more times with substituents such as those listed above.
[0074] Heteroaralkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heteroaryl group as defined above. Heteroaralkyl groups may be substituted or unsubstituted. Substituted heteroaralkyl groups may be substituted at the alkyl, the heteroaryl or both the alkyl and heteroaryl portions of the group. Representative substituted heteroaralkyl groups may be substituted one or more times with substituents such as those listed above.
[0075] Groups described herein having two or more points of attachment (i.e., divalent, trivalent, or polyvalent) within the compound of the present technology are designated by use of the suffix, “ene.” For example, divalent alkyl groups are alkylene groups, divalent aryl groups are arylene groups, divalent heteroaryl groups are divalent heteroarylene groups, and so forth. Substituted groups having a single point of attachment to the compound of the present technology are not referred to using the “ene” designation. Thus, e.g., chloroethyl is not referred to herein as chloroethylene.
[0076] Alkoxy groups are hydroxyl groups (-OH) in which the bond to the hydrogen atom is replaced by a bond to a carbon atom of a substituted or unsubstituted alkyl group as defined above. Alkoxy groups may be substituted or unsubstituted. Examples of linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, and the like. Examples of branched alkoxy groups include but are not limited to isopropoxy, sec-butoxy, tert-butoxy, isopentoxy, isohexoxy, and the like. Examples of cycloalkoxy groups include but are not limited to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. Representative substituted alkoxy groups may be substituted one or more times with substituents such as those listed above.
[0077] The terms “alkyloyl” and “alkyloyloxy” as used herein can refer, respectively, to -C(O)-alkyl groups and -O-C(O)-alkyl groups. Similarly, “aryloyl” and “aryloyloxy” refer to -C(O)-aryl groups and -O-C(O)-aryl groups.
[0078] The terms "aryloxy" and “arylalkoxy” refer to, respectively, a substituted or unsubstituted aryl group bonded to an oxygen atom and a substituted or unsubstituted aralkyl group bonded to the oxygen atom at the alkyl. Examples include but are not limited to phenoxy, naphthyloxy, and benzyloxy. Representative substituted aryloxy and arylalkoxy groups may be substituted one or more times with substituents such as those listed above.
[0079] The term “carboxylate” as used herein refers to a -COOH group.
[0080] The term “ester” as used herein refers to -COOR70 and -C(O)O-G groups. R70 is a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heterocyclylalkyl or heterocyclyl group as defined herein. G is a carboxylate protecting group. Carboxylate protecting groups are well known to one of ordinary skill in the art. An extensive list of protecting groups for the carboxylate group functionality may be found in Protective Groups in Organic Synthesis, Greene, T.W.; Wuts, P. G. M., John Wiley & Sons, New York, NY, (3rd Edition, 1999) which can be added or removed using the procedures set forth therein and which is hereby incorporated by reference in its entirety and for any and all purposes as if fully set forth herein.
[0081] The term “amide” (or “amido”) includes C- and N-amide groups, i.e., -C(O)NR71R72, and -NR71C(O)R72 groups, respectively. R71 and R72 are independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl or heterocyclyl group as defined herein. Amido groups therefore include but are not limited to carbamoyl groups (-C(O)NH2) and formamide groups (-NHC(O)H). In some embodiments, the amide is -NR71C(O)-(CI-5 alkyl) and the group is termed "carbonylamino," and in others the amide is -NHC(O)-alkyl and the group is termed "alkanoylamino."
[0082] The term “nitrile” or “cyano” as used herein refers to the -CN group.
[0083] Urethane groups include N- and O-urethane groups, i.e., -NR73C(O)OR74 and -OC(O)NR73R74 groups, respectively. R73 and R74 are independently a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl, or heterocyclyl group as defined herein. R73 may also be H.
[0084] The term “amine” (or “amino”) as used herein refers to -NR75R76 groups, wherein R75 and R76 are independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl or heterocyclyl group as defined herein. In some embodiments, the amine is alkylamino, dialkylamino, arylamino, or alkylarylamino. In other embodiments, the amine is NH2, methylamino, dimethylamino, ethylamino, diethylamino, propylamino, isopropylamino, phenylamino, or benzylamino.
[0085] The term “sulfonamido” includes S- and N-sulfonamide groups, i.e., -SO2NR78R79 and -NR78SO2R79 groups, respectively. R78 and R79 are independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl, or heterocyclyl group as defined herein. Sulfonamido groups therefore include but are not limited to sulfamoyl groups (-SO2NH2). In some embodiments herein, the sulfonamido is -NHSCh-alkyl and is referred to as the "alkylsulfonylamino" group. [0086] The term “thiol” refers to -SH groups, while “sulfides” include -SR80 groups, “sulfoxides” include -S(O)R81 groups, “sulfones” include -SO2R82 groups, “sulfonyls” include -SO2OR83, and “sulfonates” include -SO3 . R80, R81, R82, and R83 are each independently a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein. In some embodiments the sulfide is an alkylthio group, -S-alkyl.
[0087] The term “urea” refers to -NR84-C(O)-NR85R86 groups. R84, R85, and R86 groups are independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclyl, or heterocyclylalkyl group as defined herein.
[0088] The term “amidine” refers to -C(NR87)NR88R89 and -NR87C(NR88)R89, wherein R87, R88, and R89 are each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.
[0089] The term “guanidine” refers to -NR90C(NR91)NR92R93, wherein R90, R91, R92 and R93 are each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.
[0090] The term “enamine” refers to -C(R94)=C(R95)NR96R97 and -NR94C(R95)=C(R96)R97, wherein R94, R95, R96 and R97 are each independently hydrogen, a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.
[0091] The term “halogen” or “halo” as used herein refers to bromine, chlorine, fluorine, or iodine. In some embodiments, the halogen is fluorine. In other embodiments, the halogen is chlorine or bromine.
[0092] The term “hydroxyl” as used herein can refer to -OH or its ionized form, -O . A “hydroxyalkyl” group is a hydroxyl -substituted alkyl group, such as HO-CH2-.
[0093] The term “imide” refers to -C(O)NR98C(O)R99, wherein R98 and R99 are each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.
[0094] The term “imine” refers to -CR100(NR101) and -N(CR100R101) groups, wherein R100 and R101 are each independently hydrogen or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein, with the proviso that R100 and R101 are not both simultaneously hydrogen.
[0095] The term “nitro” as used herein refers to an -NO2 group.
[0096] The term “trifluoromethyl” as used herein refers to -CF3.
[0097] The term “trifluoromethoxy” as used herein refers to -OCF3.
[0098] The term “azido” refers to -N3.
[0099] The term “trialkyl ammonium” refers to a -N(alkyl)s group. A trialkylammonium group is positively charged and thus typically has an associated anion, such as halogen anion.
[0100] The term “isocyano” refers to -NC.
[0101] The term “isothiocyano” refers to -NCS.
[0102] The term “pentafluorosulfanyl” refers to -SF5.
[0103] As understood by one of ordinary skill in the art, “molecular weight” (also known as “relative molar mass”) is a dimensionless quantity but is converted to molar mass by multiplying by 1 gram/mole or by multiplying by 1 Da - for example, a compound with a weight-average molecular weight of 5,000 has a weight-average molar mass of 5,000 g/mol and a weight-average molar mass of 5,000 Da.
[0104] Pharmaceutically acceptable salts of compounds described herein are within the scope of the present technology and include acid or base addition salts which retain the desired pharmacological activity and is not biologically undesirable (e.g., the salt is not unduly toxic, allergenic, or irritating, and is bioavailable). When the compound of the present technology has a basic group, such as, for example, an amino group, pharmaceutically acceptable salts can be formed with inorganic acids (such as hydrochloric acid, hydroboric acid, nitric acid, sulfuric acid, and phosphoric acid), organic acids (e.g., alginate, formic acid, acetic acid, benzoic acid, gluconic acid, fumaric acid, oxalic acid, tartaric acid, lactic acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, naphthalene sulfonic acid, and p-toluenesulfonic acid) or acidic amino acids (such as aspartic acid and glutamic acid). When the compound of the present technology has an acidic group, such as for example, a carboxylic acid group, it can form salts with metals, such as alkali and earth alkali metals (e.g., Na+, Li+, K+, Ca2+, Mg2+, Zn2+), ammonia or organic amines (e.g., di cyclohexylamine, trimethylamine, tri ethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine) or basic amino acids (e.g., arginine, lysine and ornithine). Such salts can be prepared in situ during isolation and purification of the compounds or by separately reacting the purified compound in its free base or free acid form with a suitable acid or base, respectively, and isolating the salt thus formed.
[0105] Those of skill in the art will appreciate that compounds of the present technology may exhibit the phenomena of tautomerism, conformational isomerism, geometric isomerism, and/or stereoisomerism. As the formula drawings within the specification and claims can represent only one of the possible tautomeric, conformational isomeric, stereochemical or geometric isomeric forms, it should be understood that the present technology encompasses any tautomeric, conformational isomeric, stereochemical and/or geometric isomeric forms of the compounds having one or more of the utilities described herein, as well as mixtures of these various different forms.
[0106] Tautomers” refers to isomeric forms of a compound that are in equilibrium with each other. The presence and concentrations of the isomeric forms will depend on the environment the compound is found in and may be different depending upon, for example, whether the compound is a solid or is in an organic or aqueous solution. For example, in aqueous solution, quinazolinones may exhibit the following isomeric forms, which are referred to as tautomers of each other:
Figure imgf000036_0001
As another example, guanidines may exhibit the following isomeric forms in protic organic solution, also referred to as tautomers of each other:
Figure imgf000036_0002
Because of the limits of representing compounds by structural formulas, it is to be understood that all chemical formulas of the compounds described herein represent all tautomeric forms of compounds and are within the scope of the present technology.
[0107] Stereoisomers of compounds (also known as optical isomers) include all chiral, diastereomeric, and racemic forms of a structure, unless the specific stereochemistry is expressly indicated. Thus, compounds used in the present technology include enriched or resolved optical isomers at any or all asymmetric atoms as are apparent from the depictions. Both racemic and diastereomeric mixtures, as well as the individual optical isomers can be isolated or synthesized so as to be substantially free of their enantiomeric or diastereomeric partners, and these stereoisomers are all within the scope of the present technology.
Leukemias
[0108] Leukemias are blood cancers that usually begin in the bone marrow and result in high numbers of abnormal blood cells. These blood cells are not fully developed and are called blasts or leukemia cells. Symptoms may include bleeding and bruising, bone pain, fatigue, fever, and an increased risk of infections. These symptoms occur due to a lack of normal blood cells. Diagnosis is typically made by blood tests or bone marrow biopsy.
[0109] There are four main types of leukemia — acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML) — as well as a number of less common types. Leukemias and lymphomas both belong to a broader group of tumors that affect the blood, bone marrow, and lymphoid system, known as tumors of the hematopoietic and lymphoid tissues.
[0110] Mutations in TP53 are present in approximately 10% of patients with AML and myelodysplastic syndrome (MDS), and represent a unique subtype with poor outcome. TP53 is located on chromosome 17p 13 and is essential for cell cycle control and DNA damage response. TP53 mutations drive a dominant negative effect and typically occur in founding clones that expand after cytotoxic stress. Patients with TP53-mutant AML have a very poor prognosis and lack durable responses to essentially all current therapies. TP53- Y220C is a recurrent hotspot TP53 mutation observed in numerous solid tumors and hematological malignancies. TP53 Reactivating Indole Derivatives
[0111] TP53 reactivating indole derivatives are known in the art and are described in US Patent No. 10,640,485, the contents of which are incorporated herein by reference in its entirety.
[0112] The TP53 reactivating indole derivative may have a formula of formula (I):
Figure imgf000038_0001
wherein: each - is independently a single bond or a double bond;
X1 is CR5, CR5R6, N, NR5, O, S, C=O, C=S, or a carbon atom connected to Q1;
X2 is CR7, CR7R8, N, NR7, O, S, C=O, C=S, or a carbon atom connected to Q1;
X3 is CR9, CR9R10, N, NR9, O, S, C=O, C=S, or a carbon atom connected to Q1;
X4 is CR11, CRnR12, N, NR11, O, S, C=O, C=S, or a carbon atom connected to Q1;
X5 is CR13, N, or NR13; wherein at least one of X1, X2, X3, and X4 is a carbon atom connected to Q1;
Q1 is C=O, C=S, C=CR14R15, C=NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, a bond; m is 1, 2, 3, or 4;
Y is N or O;
R1 is -C(O)R16, -C(O)OR16, -C(O)NR16R17, -OR16, -R16OR17, -R16OR17R18, -SR16, - R16SR17, -R16SR17R18, -NR16R17, -R16NR17, -R16NR17R18, -NR16C(O)R16, - OC(O)R16, C=O, C=S, -CN, -SiR16R17R18, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; each R3 and R4 is independently -C(O)R19, -C(O)OR19, -C(O)NR19R20, -SOR19, - SO2R19, alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently unsubstituted or substituted, or hydrogen; each R2, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, and R18 is independently - C(O)R21, -C(O)OR21, -C(O)NR21R22, -OR21, -SR21, -NR21R22, - NR21C(O)R22, -OC(O)R21, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently unsubstituted or substituted, or hydrogen; each R19 and R20 is C(O)R23, -C(O)OR23, -C(O)NR23R24, -OR23, -SR23, -NR23R24, - NR23C(O)R24, -OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen; each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or a pharmaceutically-acceptable salt thereof.
[0113] The TP53 reactivating indole derivative may have a formula of formula (IA)
Figure imgf000039_0001
wherein:
X1 is CR5, NR5, O, S, C=O, C=S, or a carbon atom connected to Q1;
X2 is CR7, CH, NR7, O, S, C=O, C=S, or a carbon atom connected to Q1;
X3 is CR9, CH, NR9, O, S, C=O, C=S, or a carbon atom connected to Q1;
X4 is CR11, CH, NR11, O, S, C=O, C=S, or a carbon atom connected to Q1;
X5 is CR13, CH, or NR13; wherein at least one of X1, X2, X3, and X4 is a carbon atom connected to Q1;
Q1 is a bond; m is 1, 2, 3, or 4;
Y is N or O;
R1 is -C(O)R16, -C(O)OR16, -C(O)NR16R17, -OR16, -R16OR17, -R16OR17R18, -SR16, - R16SR17, -R16SR17R18, -NR16R17, -R16NR17, -R16NR17R18, -NR16C(O)R16, - OC(O)R16, C=O, C=S, -CN, -SiR16R17R18, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; each R3 and R4 is independently -C(O)R19, -C(O)OR19, -C(O)NR19R20, -SOR19, - SO2R19, hydrogen, alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl, wherein each of alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl is independently unsubstituted or substituted with one or two groups selected from alkyl or halogen, or R3 and R4 together with the Y atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent; each R2, R5, R7, R9, R11, R13, R16, R17, and R18 is independently - C(O)R21, - C(O)OR21, -C(O)NR21R22, -OR21, -SR21, -NR21R22, -NR21C(O)R22, -OC(O)R21, hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, wherein each alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl is independently unsubstituted or substituted with one or two groups selected from -C(O)R25, - C(O)OR25, -C(O)NR25, -CR253, -OR25, -SR25, -NR25R26, -NR25C(O)R26, -OC(O)R25, alkyl, alkenyl, or alkynyl, wherein R25 and R26 are each independently alkyl, hydrogen, or halogen; each R19 and R20 is C(O)R23, -C(O)OR23, -C(O)NR23R24, -OR23, -SR23, -NR23R24, - NR23C(O)R24, -OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen; each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or a pharmaceutically-acceptable salt thereof.
[0114] The TP53 reactivating indole derivative may have a formula of formula (IB)
Figure imgf000041_0001
wherein:
X1 is CR5, NR5, O, S, C=O, C=S, or a carbon atom connected to Y;
X2 is CR7, CH, NR7, O, S, C=O, C=S, or a carbon atom connected to Y;
X3 is CR9, CH, NR9, O, S, C=O, C=S, or a carbon atom connected to Y;
X4 is CR11, CH, NR11, O, S, C=O, C=S, or a carbon atom connected to Y;
X5 is CR13, CH, or NR13; wherein at least one of X1, X2, X3, and X4 is a carbon atom connected to Y;
Y is N or O;
R1 is -C(O)R16, -C(O)OR16, -C(O)NR16R17, -OR16, -R16OR17, -R16OR17R18, -SR16, - R16SR17, -R16SR17R18, -NR16R17, -R16NR17, -R16NR17R18, -NR16C(O)R16, - OC(O)R16, C=O, C=S, -CN, -SiR16R17R18, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; each R3 and R4 is independently -C(O)R19, -C(O)OR19, -C(O)NR19R20, -SOR19, - SO2R19, hydrogen, alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl, wherein each of alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl is independently unsubstituted or substituted with one or two groups selected from alkyl or halogen, or R3 and R4 together with the Y atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent; each R2, R5, R7, R9, R11, R13, R16, R17, and R18 is independently - C(O)R21, - C(O)OR21, -C(O)NR21R22, -CR253, -OR21, -SR21, -NR21R22, -NR21C(O)R22, - OC(O)R21, hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, wherein each of alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl is independently unsubstituted or substituted with one or two groups selected from -C(O)R25, -C(O)OR25, -C(O)NR25, -CR25 3, -OR25, -SR25, -NR25R26, - NR25C(O)R26, -OC(O)R25, alkyl, alkenyl, or alkynyl, wherein R25 and R26 are each independently alkyl, hydrogen, or halogen; each R19 and R20 is C(O)R23, -C(O)OR23, -C(O)NR23R24, -OR23, -SR23, -NR23R24, - NR23C(O)R24, -OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen; each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or a pharmaceutically-acceptable salt thereof.
[0115] Non-limiting examples of TP53 reactivating indole derivatives include compounds of any of the following formulae:
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
[0116] In some embodiments, the indole derivative is a compound of the formula
Figure imgf000045_0002
[0117] wherein each = is independently a single bond or a double bond; X1 is CR5, CR5R6, N, NR5, O, S, C=O, C=S, or a carbon atom connected to Q1; X2 is CR7, CR7R8, N, NR7, O, S, C=O, C=S, or a carbon atom connected to Q1; X3 is CR9, CR9R10, N, NR9, O, S, C=O, C=S, or a carbon atom connected to Q1; X4 is CR11, CRnR12, N, NR11, O, S, C=O, C=S, or a carbon atom connected to Q1; X5 is CR13, N, or NR13; wherein at least one of X1, X2, X3, and X4is a carbon atom connected to Q1; Q1 is C=O, C=S, C=CR14R15, C=NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond; m is 1, 2, 3, or 4; Y is N, O, or absent; R1 is — C(O)R16, — C(O)OR16, — C(O)NR16R17, —OR16, —SR16, — NR16R17, — NR16C(O)R16, — OC(O)R16, — SiR16R17R18, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; each R3 and R4is independently — C(O)R19, — C(O)OR19, — C(O)NR19R20, — SOR19, — SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent; each R2, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, and R18 is independently — C(O)R21, — C(O)OR21, — C(O)NR21R22, —OR21, —SR21, — NR21R22, — NR21C(O)R22, — OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen; each R19 and R20 is C(O)R23, — C(O)OR23, — C(O)NR23R24, —OR23, —SR23, — NR23R24, — NR23C(O)R24, — OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen; each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and each R23 and R24is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or a pharmaceutically-acceptable salt thereof.
[0118] In some embodiments, the pattern of dashed bonds is chosen to provide an aromatic system, for example, an indole, an indolene, a pyrrolopyridine, a pyrrolopyrimidine, or a pyrrolopyrazine.
[0119] In some embodiments, X1 is CR5, CR5R6, or a carbon atom connected to Q1. In some embodiments, X2 is CR7, CR7R8, or a carbon atom connected to Q1. In some embodiments, X3 is CR9, CR9R10, or a carbon atom connected to Q1. In some embodiments, X4 is CR11, CRnR12, or a carbon atom connected to Q1. In some embodiments, X5 is CR13, N, or NR13. In some embodiments, X1 is a carbon atom connected to Q1. In some embodiments, X2 is a carbon atom connected to Q1. In some embodiments, X3 is a carbon atom connected to Q1. In some embodiments, X4 is a carbon atom connected to Q1. In some embodiments, X5 is N.
[0120] In some embodiments, the compound is of the formula:
Figure imgf000047_0001
[0121] In some embodiments, the compound is of the formula:
Figure imgf000047_0002
[0122] wherein R1 is — C(O)R16, — C(O)OR16, — C(O)NR16R17, —OR16, —SR16, — NR16R17, — NR16C(O)R16, — OC(O)R16, SiR16R17R18, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen.
[0123] In some embodiments, m is 1, 2, 3, or 4. In some embodiments, m is 1. In some embodiments, X3 is carbon atom connected to Q1, and m is 1. In some embodiments, the compound is of the formula:
Figure imgf000048_0001
[0124] wherein R1 is — C(O)R16, — C(O)OR16, — C(O)NR16R17, —OR16, —SR16, —
NR16R17, — NR16C(O)R16, — OC(O)R16, SiR16R17R18, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen.
[0125] In some embodiments, R is alkyl, alkenyl, — C(O)R16, — C(O)OR16, or — C(O)NR16R17. In some embodiments, R1 is a substituted alkyl. R1 can be substituted by one or more substituents selected from hydroxyl groups, sulfhydryl groups, halogens, amino groups, nitro groups, nitroso groups, cyano groups, azido groups, sulfoxide groups, sulfone groups, sulfonamide groups, carboxyl groups, carboxaldehyde groups, imine groups, alkyl groups, halo-alkyl groups, cyclic alkyl groups, alkenyl groups, halo-alkenyl groups, alkynyl groups, halo-alkynyl groups, alkoxy groups, aryl groups, aryloxy groups, aralkyl groups, arylalkoxy groups, heterocyclyl groups, acyl groups, acyloxy groups, carbamate groups, amide groups, urethane groups, and ester groups. In some embodiments, R1 is alkyl substituted with an amine group. In some embodiments, R1 is alkyl substituted with NR16R17.
[0126] In some embodiments, Q1 is C=O, C=S, C=CR14R15, C=NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond. In some embodiments, Q1 is alkylene, alkenylene, or alkynylene. In some embodiments, Q1 is Ci-alkylene. In some embodiments, each R16 and R17 is independently alkyl, alkenyl, aryl, heteroaryl, heterocyclyl, or hydrogen. In some embodiments, Q1 is Ci- alkylene, R16 is aryl, and R17 is alkyl. In some embodiments, Q1 is Ci-alkylene, R16 is aryl, and R17 is hydrogen. In some embodiments, Q1 is Ci-alkylene, R16 is heteroaryl, and R17 is alkyl. In some embodiments, Q1 is Ci-alkylene, R16 is heteroaryl, and R17is hydrogen. In some embodiments, Q1 is Ci-alkylene, R16 is substituted heteroaryl, and R17is hydrogen. In some embodiments, Q1 is Ci-alkylene, R16 is substituted alkyl, and R17is hydrogen. In some embodiments, R17 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted with halogen, alkyl, or hydroxyl. In some embodiments, R16 is hydrogen, and R17is aryl or heteroaryl, substituted or unsubstituted with halogen or alkyl. In some embodiments, R16 is alkyl, and R17is heteroaryl substituted with halogen or alkyl. In some embodiments, R17is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted with alkyl. In some embodiments, R17 is aryl or heteroaryl, each of which is independently substituted with alkyl, wherein the alkyl is optionally substituted with fluorine, chlorine, bromine, iodine, or cyano.
[0127] In some embodiments, R2 is hydrogen or alkyl. In some embodiments, R13 is alkyl, alkenyl, hydrogen, or halogen. In some embodiments, R2 is alkyl, and R13 is alkyl. In some embodiments, R2 is hydrogen, and R13 is alkyl. In some embodiments, R2 is methyl, ethyl, propyl, iso-propyl, butyl, or tert-butyl. In some embodiments, R13 is methyl, ethyl, propyl, iso-propyl, butyl, or tert-butyl. In some embodiments, R2 is hydrogen, and R13 is hydrogen.
[0128] In some embodiments, R3 is — C(O)R19, — C(O)OR19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, and R4is — C(O)R19, — C(O)OR19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen. In some embodiments, R3 is hydrogen and R4 is substituted alkyl. In some embodiments, R3 is hydrogen and R4 is alkyl substituted with aryl. In some embodiments, R3 is alkyl and R4 is alkyl. In some embodiments, R3 is alkyl and R4 is aryl.
[0129] In some embodiments, R3 is hydrogen, and R4is heterocyclyl. In some embodiments, the compound is of the formula:
Figure imgf000050_0001
[0130] In some embodiments, R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted. In some embodiments, R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a substituted heterocycle. In some embodiments, R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a heterocycle substituted with a hydroxyl group, halogen, amino group, or alkyl group. In some embodiments, R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a heterocycle, wherein the heterocycle is substituted by a substituted or unsubstituted heterocycle.
[0131] In some embodiments, R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a ring of a following formula:
Figure imgf000051_0001
[0132] In some embodiments, R16 is alkyl, alkenyl, aryl, heteroaryl, heterocyclyl, or hydrogen, and R17is aryl, heteroaryl, or heterocyclyl. In some embodiments, R17 is phenyl, indolyl, piperidinyl, imidazolyl, thiazolyl, morpholinyl, pyrrolyl, or pyridinyl.
[0133] In some embodiments, the compound is of the formula:
Figure imgf000051_0002
[0134] In some embodiments, the compound is of the formula:
Figure imgf000052_0001
[0135] In some embodiments, the compound is of the formula:
Figure imgf000052_0002
[0136] wherein each Z1 and Z2 is independently CRX or N; each Rx is independently — C(O)R21, — C(O)OR21, — C(O)NR21R22, —OR21, —SR21, — NR21R22, — NR21C(O)R22, — OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, hydrogen, or halogen; each R25 and R26 is independently — C(O)R21, — C(O)OR21, — C(O)NR21R22, —OR21, —SR21, — SO2R21, — NR21R22, — NR21C(O)R22, — OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen.
[0137] In some embodiments, Z1 is N. In some embodiments, Z1 and Z2 are N. In some embodiments, each R25 and R26 is independently a halogen. In some embodiments, R25 is
Figure imgf000052_0003
[0138] In some embodiments, R25 is SO2R21. In some embodiments, R25 is SO2R21, wherein R21 is alkyl. In some embodiments, R25 is SO2R21, wherein R21 is methyl.
[0139] Non-limiting examples of compounds of the current disclosure include the following:
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
Figure imgf000069_0001
or a pharmaceutically-acceptable salt of any of the foregoing.
[0140] Non-limiting examples of compounds of the current disclosure include the following:
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000078_0001
Figure imgf000079_0001
Figure imgf000080_0001
pharmaceutically acceptable salt thereof.
[0141] Non-limiting examples of compounds of the current disclosure include the following:
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000087_0001
Figure imgf000088_0001
Figure imgf000089_0001
Figure imgf000090_0001
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
or a or or a pharmaceutically-acceptable salt of any of the forgoing.
[0142] In some embodiments, the compound is of the formula
Figure imgf000093_0002
[0143] wherein each Qla and Qlb is independently C=O, C=S, C=CR14' R15 , C=NR14', alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond; each Rla and Rlb is independently — C(O)R16 , — C(O)OR16 , — C(O)NR16'R7', —OR16', —SR16', — NR16'R17', — NR16'C(O)R16', — OC(O)R16', — SiR16 R17 R8 , alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; each R3aand R3bis independently alkylene, alkenylene, alkynylene, arylene, heteroarylene, or heterocyclylene, each of which is independently substituted or unsubstituted, or hydrogen; each R4a and R4b is independently absent, — C(O)R19', — C(O)OR19', — C(O)NR19'R20', — SOR19', — SO2R19', alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; each R2a, R2b, R13a, and R13b is independently — C(O)R21 , — C(O)OR21 , — C(O)NR21 R22', —OR21', —SR21', — NR21'R22', — NR21'C(O)R22', — OC(O)R21 , alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen; each R19' and R20 is independently — C(O)R23', — C(O)OR23', — C(O)NR23R24', —OR23', —SR23', — NR23'R24', — NR23 C(O)R24 , — OC(O)R23 , alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen; each R21' and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; each R23' and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; L1 is a linker moiety; and L2 is a linker moiety, or a pharmaceutically acceptable salt thereof.
[0144] In some embodiments, each L1 and L2 is independently an ester, ether, thioether, polyethyleneglycol (PEG), alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocyclyl ene, arylene, heteroarylene, or heterocycloalkylene group, any of which is substituted or unsubstituted. In some embodiments, each L1 and L2 is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, or heterocycloalkylene. In some embodiments, L1 is alkylene and L2 is an ester.
[0145] Compounds herein can include all stereoisomers, enantiomers, diastereomers, mixtures, racemates, atropisomers, and tautomers thereof.
[0146] Non-limiting examples of optional substituents include hydroxyl groups, sulfhydryl groups, halogens, amino groups, nitro groups, nitroso groups, cyano groups, azido groups, sulfoxide groups, sulfone groups, sulfonamide groups, carboxyl groups, carboxaldehyde groups, imine groups, alkyl groups, halo-alkyl groups, alkenyl groups, halo- alkenyl groups, alkynyl groups, halo-alkynyl groups, alkoxy groups, aryl groups, aryloxy groups, aralkyl groups, arylalkoxy groups, heterocyclyl groups, acyl groups, acyloxy groups, carbamate groups, amide groups, ureido groups, epoxy groups, and ester groups.
[0147] Non-limiting examples of alkyl and alkylene groups include straight, branched, and cyclic alkyl and alkylene groups. An alkyl or alkylene group can be, for example, a Ci, C2, C3, C4, C5, C6, C7, C8, C9, Cio, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, C35, C36, C37, C38, C39, C40, C41, C42, C43, C44, C45, C46, C47, C48, C49, or C50 group that is substituted or unsubstituted.
[0148] Non-limiting examples of straight alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl.
[0149] Branched alkyl groups include any straight alkyl group substituted with any number of alkyl groups. Non-limiting examples of branched alkyl groups include isopropyl, isobutyl, sec-butyl, and t-butyl.
[0150] Non-limiting examples of substituted alkyl groups includes hydroxymethyl, chloromethyl, trifluoromethyl, aminomethyl, 1 -chloroethyl, 2-hydroxy ethyl, 1,2- difluoroethyl, and 3 -carboxypropyl.
[0151] Non-limiting examples of cyclic alkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptlyl, and cyclooctyl groups. Cyclic alkyl groups also include fused-, bridged-, and spiro-bicycles and higher fused-, bridged-, and spiro- systems. A cyclic alkyl group can be substituted with any number of straight, branched, or cyclic alkyl groups. Non-limiting examples of cyclic alkyl groups include cyclopropyl, 2-methyl- cycloprop-l-yl, cycloprop-2-en-l-yl, cyclobutyl, 2,3-dihydroxycyclobut-l-yl, cyclobut-2- en-l-yl, cyclopentyl, cyclopent-2-en-l-yl, cyclopenta-2,4-dien-l-yl, cyclohexyl, cyclohex- 2-en-l-yl, cycloheptyl, cyclooctanyl, 2,5-dimethylcyclopent-l-yl, 3,5-dichlorocyclohex-l- yl, 4-hydroxycyclohex-l-yl, 3,3,5-trimethylcyclohex-l-yl, octahydropentalenyl, octahydro- IH-indenyl, 3a,4,5,6,7,7a-hexahydro-3H-inden-4-yl, decahydroazulenyl, bicyclo- [2.1.1]hexanyl, bicyclo[2.2.1]heptanyl, bicyclo[3.1.1]heptanyl, l,3-dimethyl[2.2.1]heptan- 2-yl, bicyclo[2.2.2]octanyl, and bicyclo[3.3.3]undecanyl.
[0152] Non-limiting examples of alkenyl and alkenylene groups include straight, branched, and cyclic alkenyl groups. The olefin or olefins of an alkenyl group can be, for example, E, Z, cis, trans, terminal, or exo-methylene. An alkenyl or alkenylene group can be, for example, a C2, C3, C4, C5, C6, C7, C8, C9, Cio, Cn, C12, C13, Ci4, C15, Ci6, C17, Ci8, C19, C20, C2I, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, C35, C36, C37, C38, C39, C40, C41, C42, C43, C44, C45, C46, C47, C48, C49, or C50 group that is substituted or unsubstituted. Non-limiting examples of alkenyl and alkenylene groups include ethenyl, prop-l-en-l-yl, isopropenyl, but- 1-en -4-yl; 2-chloroethenyl, 4-hydroxybuten-l-yl, 7- hydroxy-7-methyloct-4-en-2-yl, and 7-hydroxy-7-methyloct-3,5-dien-2-yl.
[0153] Non-limiting examples of alkynyl or alkynylene groups include straight, branched, and cyclic alkynyl groups. The triple bond of an alkylnyl or alkynylene group can be internal or terminal. An alkylnyl or alkynylene group can be, for example, a C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, c35, C36, C37, C38, C39, C40, C41, C42, C43, C44, C45, C46, C47, C48, C49, or C50 group that is substituted or unsubstituted. Non-limiting examples of alkynyl or alkynylene groups include ethynyl, prop-2-yn-l-yl, prop-l-yn-l-yl, and 2-methyl-hex-4-yn-l-yl; 5-hydroxy-5-methylhex-3-yn-l-yl, 6-hydroxy-6-methylhept-3- yn-2-yl, and 5-hydroxy-5-ethylhept-3-yn-l-yl.
[0154] A halo-alkyl group can be any alkyl group substituted with any number of halogen atoms, for example, fluorine, chlorine, bromine, and iodine atoms. A halo-alkenyl group can be any alkenyl group substituted with any number of halogen atoms. A halo- alkynyl group can be any alkynyl group substituted with any number of halogen atoms.
[0155] An alkoxy group can be, for example, an oxygen atom substituted with any alkyl, alkenyl, or alkynyl group. An ether or an ether group comprises an alkoxy group. Non-limiting examples of alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, and isobutoxy.
[0156] An aryl group can be heterocyclic or non-heterocyclic. An aryl group can be monocyclic or polycyclic. An aryl group can be substituted with any number of substituents described herein, for example, hydrocarbyl groups, alkyl groups, alkoxy groups, and halogen atoms. Non-limiting examples of aryl groups include phenyl, toluyl, naphthyl, pyrrolyl, pyridyl, imidazolyl, thiophenyl, and furyl. Non-limiting examples of substituted aryl groups include 3,4-dimethylphenyl, 4-tert-butylphenyl, 4-cyclopropylphenyl, 4- diethylaminophenyl, 4-(trifluoromethyl)phenyl, 4-(difluoromethoxy)-phenyl, 4- (trifluorom ethoxy )phenyl, 3 -chlorophenyl, 4-chlorophenyl, 3, 4-di chlorophenyl, 2- fluorophenyl, 2-chlorophenyl, 2-iodophenyl, 3 -iodophenyl, 4-iodophenyl, 2-methylphenyl, 3 -fluorophenyl, 3 -methylphenyl, 3 -methoxyphenyl, 4-fluorophenyl, 4-methylphenyl, 4- methoxyphenyl, 2,3 -difluorophenyl, 3,4-difluorophenyl, 3,5-difluorophenyl, 2,3- di chlorophenyl, 3,4-dichlorophenyl, 3,5-dichlorophenyl, 2-hydroxyphenyl, 3- hydroxyphenyl, 4-hydroxyphenyl, 2-methoxyphenyl, 3 -methoxyphenyl, 4-methoxyphenyl, 2,3-dimethoxyphenyl, 3,4-dimethoxyphenyl, 3,5-dimethoxyphenyl, 2,4-difluorophenyl, 2,5- difluorophenyl, 2,6-difluorophenyl, 2,3,4-trifluorophenyl, 2,3,5-trifluorophenyl, 2,3,6- trifluorophenyl, 2,4,5-trifluorophenyl, 2,4,6-trifluorophenyl, 2,4-dichlorophenyl, 2,5- di chlorophenyl, 2,6-dichlorophenyl, 3, 4-di chlorophenyl, 2,3,4-trichlorophenyl, 2,3,5- trichlorophenyl, 2,3,6-trichlorophenyl, 2,4,5-trichlorophenyl, 3,4,5-trichlorophenyl, 2,4,6- tri chlorophenyl, 2,3 -dimethylphenyl, 2,4-dimethylphenyl, 2,5-dimethylphenyl, 2,6- dimethylphenyl, 2,3,4-trimethylphenyl, 2,3,5-trimethylphenyl, 2,3,6-trimethylphenyl, 2,4,5- trimethylphenyl, 2,4,6-trimethylphenyl, 2-ethylphenyl, 3 -ethylphenyl, 4-ethylphenyl, 2,3- diethylphenyl, 2,4-diethylphenyl, 2,5-diethylphenyl, 2,6-diethylphenyl, 3, 4-di ethylphenyl, 2,3,4-triethylphenyl, 2,3,5-triethylphenyl, 2,3,6-triethylphenyl, 2,4,5-triethylphenyl, 2,4,6- triethylphenyl, 2-isopropylphenyl, 3-isopropylphenyl, and 4-isopropylphenyl.
[0157] Non-limiting examples of substituted aryl groups include 2-aminophenyl, 2-(N- methylamino)phenyl, 2-(N,N-dimethylamino)phenyl, 2-(N-ethylamino)phenyl, 2-(N,N- diethylamino)phenyl, 3 -aminophenyl, 3-(N-methylamino)phenyl, 3-(N,N- dimethylamino)phenyl, 3-(N-ethylamino)phenyl, 3-(N,N-diethylamino)phenyl, 4- aminophenyl, 4-(N-methylamino)phenyl, 4-(N,N-dimethylamino)phenyl, 4-(N- ethylamino)phenyl, and 4-(N,N-diethylamino)phenyl.
[0158] A heterocycle can be any ring containing a ring atom that is not carbon, for example, N, O, S, P, Si, B, or any other heteroatom. A heterocycle can be substituted with any number of substituents, for example, alkyl groups and halogen atoms. A heterocycle can be aromatic (heteroaryl) or non-aromatic. Non-limiting examples of heterocycles include pyrrole, pyrrolidine, pyridine, piperidine, succinamide, maleimide, morpholine, imidazole, thiophene, furan, tetrahydrofuran, pyran, and tetrahydropyran.
[0159] Non-limiting examples of heterocycles include: heterocyclic units having a single ring containing one or more heteroatoms, non-limiting examples of which include, diazirinyl, aziridinyl, azetidinyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolinyl, thiazolidinyl, isothiazolinyl, oxathiazolidinonyl, oxazolidinonyl, hydantoinyl, tetrahydrofuranyl, pyrrolidinyl, morpholinyl, piperazinyl, piperidinyl, dihydropyranyl, tetrahydropyranyl, piperidin-2-onyl, 2,3,4,5-tetrahydro-lH-azepinyl, 2,3-dihydro-lH-indole, and 1,2,3,4-tetrahydroquinoline; and ii) heterocyclic units having 2 or more rings one of which is a heterocyclic ring, non-limiting examples of which include hexahydro- 1H- pyrrolizinyl, 3a,4,5,6,7,7a-hexahydro-lH-benzo[d]imidazolyl, 3a,4,5,6,7,7a-hexahydro-H- indolyl, 1,2,3,4-tetrahydroquinolinyl, and decahydro-lH-cycloocta[b]pyrrolyl.
[0160] Non-limiting examples of heteroaryl include: i) heteroaryl rings containing a single ring, non-limiting examples of which include, 1,2,3,4-tetrazolyl, [l,2,3]triazolyl, [l,2,4]triazolyl, triazinyl, thiazolyl, IH-imidazolyl, oxazolyl, isoxazolyl, isothiazolyl, furanyl, thiophenyl, pyrimidinyl, 2-phenylpyrimidinyl, pyridinyl, 3-methylpyridinyl, and 4- dimethylaminopyridinyl; and ii) heteroaryl rings containing 2 or more fused rings one of which is a heteroaryl ring, non-limiting examples of which include: 7H-purinyl, 9H-purinyl, 6-amino-9H-purinyl, 5H-pyrrolo[3,2-d]pyrimidinyl, 7H-pyrrolo[2,3-d]pyrimidinyl, pyrido[2,3-d]pyrimidinyl, 4,5,6,7-tetrahydro-l-H-indolyl, quinoxalinyl, quinazolinyl, quinolinyl, 8-hydroxy-quinolinyl, and isoquinolinyl.
[0161] In some embodiments, the TP53 reactivating indole derivative is PC14586 (also called rezatapopt; see https://classic.clinicaltrials.gov/ct2/show/NCT04585750;
PCT/US2023/067005 filed May 15, 2023; CAS No. 2636846-41-6). PC14586 is a first-in- class, oral, small molecule p53 reactivator selective for the p53 Y220C mutation. PC14586 is designed to bind non-covalently to the crevice created by the Y220C mutation and restore WT p53 function. However, the phase 1 clinical trial of PC14586 in locally advanced or metastatic solid tumors that have a TP53 Y220C mutation (NCT04585750) demonstrated only limited activity, including limited cell death. PC 14586 has a structure of formula
Figure imgf000098_0001
MDM2 Inhibitors
[0162] Murine double minute 2 homolog (MDM2) also known as E3 ubiquitin-protein ligase MDM2 is a protein that in humans is encoded by the MDM2 gene. MDM2 is an important negative regulator of the p53 tumor suppressor. MDM2 protein functions both as an E3 ubiquitin ligase that recognizes the N-terminal trans-activation domain (TAD) of the p53 tumor suppressor and as an inhibitor of p53 transcriptional activation. MDM2 inhibitors target TP53 WT cells but have the potential to select/enrich TP53 mutant leukemia cells. MDM2 inhibitors activate p53, which in turn induces MDM2 and p21 levels, which can result in cell cycle arrest, but not in apoptosis.
[0163] MDM2 inhibitors bind to the MDM2 protein, thereby preventing the binding of the MDM2 protein to the transcriptional activation domain of the tumor suppressor protein p53. MDM2 inhibitors can be selective or non- selective, small molecules or biomolecules. Examples of MDM2 inhibitors include, but are not limited to, RG7112, RO5045337, idasanutlin, nutlin-3a, RG7388, AMG-232, KRT-232, APG-115, BI-907828, CGM097, siremadlin, HDM201, milademetan, MEL23, MEL24, and DS-3032b.
[0164] MDM2 inhibitors may include MDM2 degraders, which degrade MDM2 protein. MDM2 inhibitors may include proteolysis targeting chimeras (PROTACs), which are heterobifunctional molecules composed of a targeting ligand tethered to an E3 ubiquitin ligase recruiting ligand to induce selective degradation of MDM2. Examples of MDM2 degrader PROTACs include spirooxindole MDM2 inhibitors e.g., MI-1061) tethered to lenalidomide, nutlin derivatives (e.g., idasanutlin, nutlin-3a) tethered to lenalidomide analogues, YX-02-030 (a RG7112 derivative) and MS3227, as described in more detail in B. Wang et al. Development of selective small molecule MDM2 degraders based on nutlin, Eur. J. Med. Chem. 2019, 176, 476-491; C. M. Adams et al., Targeted MDM2 degradation reveals a new vulnerability for p53 inactivated triple negative breast cancer, Cancer Discov. 2023, 13(5), 1210-1229; and B. K. Marcellino, An MDM2 degrader for treatment of acute leukemias, Leukemia 2022, 37, 370-378.
BCL-2 Inhibitors
[0165] B-cell lymphoma 2 (BCL-2) proteins are a class of proteins that are regulators of apoptosis. BCL-2 is variably highly expressed in many hematological malignancies, providing protection from cell death induced by oncogenic and external stresses.
[0166] BCL-2 inhibitors bind to BCL-2 protein, thereby preventing protection from apoptosis. BCL-2 inhibitors can be selective or non- selective, small molecules or biomolecules. Examples of BCL-2 inhibitors include, but are not limited to, venetoclax, obatoclax, subatoclax, maritoclax, gossypol, apogossypol, TW-37, UMI-77, and BDA-366. XPO-1 Inhibitors
[0167] Exportin 1 (XPO-1), also known as chromosomal region maintenance 1 (CRM1), is a eukaryotic protein that mediates the nuclear export of various proteins and RNAs. XPO-1 mediates NES-dependent protein transport. It exports several hundreds of different proteins from the nucleus. XPO-1 is involved in the nuclear export of ribosomal subunits. XPO-1 is affected in some cancer types and is therefore viewed as a target for development of anti-cancer drugs. XPO-1 overexpression plays a role in the onset and progression of both solid tumors and hematological malignancies and is associated with a poor prognosis in patients.
[0168] XPO-1 inhibitors bind to the XPO-1 protein, thereby preventing or substantially reducing nuclear export, which can increase accumulation of tumor suppressor proteins, reduce oncoproteins, and increase apoptosis. XPO-1 inhibitors can be selective or non- selective, small molecules or biomolecules. Examples of XPO-1 inhibitors include, but are not limited to, KPT330 (Selinexor), XPOVIO, KPT8602 (Eltanexor), KPT335, Verdinexor, and KPT 185.
Formulations Including TP53 Reactivating Indole Derivatives., MDM2 Inhibitors, BCL-2 Inhibitors and/or XPO-1 Inhibitors of the Present Technology
[0169] The pharmaceutical compositions of the present technology include a TP53 reactivating indole derivative (e.g., PC 14586) and one or more additional therapeutic agents. The additional therapeutic agents may include MDM2 inhibitors, BCL-2 inhibitors and/or XPO-1 inhibitors.
[0170] In some embodiments, the pharmaceutical compositions include an effective amount of a TP53 reactivating indole derivative (e.g., PC14586) and an effective amount of an MDM2 inhibitor. In some embodiments, the pharmaceutical compositions include an effective amount of a TP53 reactivating indole derivative (e.g., PC14586) and an effective amount of a BCL-2 inhibitor. In some embodiments, the pharmaceutical compositions include an effective amount of a TP53 reactivating indole derivative (e.g., PC14586) and an effective amount of XPO-1 inhibitor. In some embodiments, the pharmaceutical compositions include an effective amount of a TP53 reactivating indole derivative (e.g., PC 14586) and an effective amount of an MDM2 inhibitor and a BCL-2 inhibitor. In some embodiments, the pharmaceutical compositions include an effective amount of a TP53 reactivating indole derivative (e.g., PC 14586) and an effective amount of an MDM2 inhibitor and an XPO-1 inhibitor. In some embodiments, the pharmaceutical compositions include an effective amount of a TP53 reactivating indole derivative (e.g., PC14586) and an effective amount of a BCL-2 inhibitor and an XPO-1 inhibitor. In some embodiments, the pharmaceutical compositions include an effective amount of a TP53 reactivating indole derivative (e.g., PC 14586) and an effective amount of an MDM2 inhibitor, a BCL-2 inhibitor, and an XPO-1 inhibitor. In any embodiments, the pharmaceutical compositions may include pharmaceutically acceptable excipients, diluents, or carriers that are compatible with the one or more therapeutic agent in the pharmaceutical composition and the method of administration.
[0171] The pharmaceutical compositions of the present technology can be manufactured by methods well known in the art such as conventional granulating, mixing, dissolving, encapsulating, lyophilizing, or emulsifying processes, among others. Compositions may be produced in various forms, including granules, precipitates, or particulates, powders, including freeze dried, rotary dried or spray dried powders, amorphous powders, tablets, capsules, syrup, suppositories, injections, emulsions, elixirs, suspensions, or solutions. Formulations may optionally contain solvents, diluents, and other liquid vehicles, dispersion or suspension aids, surface active agents, pH modifiers, isotonic agents, thickening or emulsifying agents, stabilizers and preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. In certain embodiments, the compositions disclosed herein are formulated for administration to a mammal, such as a human.
[0172] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -butylene glycol, cyclodextrins, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. [0173] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. Compositions formulated for parenteral administration may be injected by bolus injection or by timed push, or may be administered by continuous infusion.
[0174] In order to prolong the effect of a compound of the present disclosure, it is often desirable to slow the absorption of the compound from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compound then depends upon its rate of dissolution that, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered compound form is accomplished by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of compound to polymer and the nature of the particular polymer employed, the rate of compound release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.
[0175] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents such as phosphates or carbonates.
[0176] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.
[0177] The active compounds can also be in micro-encapsulated form with one or more excipients as noted above. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Methods of Treatment of the Present Technology
[0178] In one aspect, the present disclosure provides a method for treating a wild-type p53 or TP53-Y220C leukemia in a patient in need thereof comprising administering to the patient an effective amount of a TP53 reactivating indole derivative and an effective amount of an MDM2 inhibitor, a BCL-2 inhibitor, and/or an XPO-1 inhibitor, wherein the TP53 reactivating indole derivative has formula of formula (I):
Figure imgf000104_0001
wherein: each - is independently a single bond or a double bond;
X1 is CR5, CR5R6, N, NR5, O, S, C=O, C=S, or a carbon atom connected to Q1;
X2 is CR7, CR7R8, N, NR7, O, S, C=O, C=S, or a carbon atom connected to Q1;
X3 is CR9, CR9R10, N, NR9, O, S, C=O, C=S, or a carbon atom connected to Q1;
X4 is CR11, CRnR12, N, NR11, O, S, C=O, C=S, or a carbon atom connected to Q1;
X5 is CR13, N, or NR13; wherein at least one of X1, X2, X3, and X4 is a carbon atom connected to Q1;
Q1 is C=O, C=S, C=CR14R15, C=NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, a bond; m is 1, 2, 3, or 4;
Y is N or O;
R1 is -C(O)R16, -C(O)OR16, -C(O)NR16R17, -OR16, -R16OR17, -R16OR17R18, -SR16, - R16SR17, -R16SR17R18, -NR16R17, -R16NR17, -R16NR17R18, -NR16C(O)R16, - OC(O)R16, C=O, C=S, -CN, -SiR16R17R18, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; each R3 and R4 is independently -C(O)R19, -C(O)OR19, -C(O)NR19R20, -SOR19, - SO2R19, alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently unsubstituted or substituted, or hydrogen; each R2, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, and R18 is independently - C(O)R21, -C(O)OR21, -C(O)NR21R22, -OR21, -SR21, -NR21R22, - NR21C(O)R22, -OC(O)R21, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently unsubstituted or substituted, or hydrogen; each R19 and R20 is C(O)R23, -C(O)OR23, -C(O)NR23R24, -OR23, -SR23, -NR23R24, - NR23C(O)R24, -OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen; each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or a pharmaceutically-acceptable salt thereof.
[0179] In one aspect, the present disclosure provides a method for selecting a leukemia patient for treatment with a TP53 reactivating indole derivative and an additional therapeutic agent comprising: detecting wild-type TP53 or TP53-Y220C mRNA or polypeptide expression in a biological sample obtained from a leukemia patient; and administering to the leukemia patient an effective amount of a TP53 reactivating indole derivative and an effective amount of at least one additional therapeutic agent selected from among an MDM2 inhibitor, a BCL-2 inhibitor, and an XPO-1 inhibitor, wherein the TP53 reactivating indole derivative has formula of formula (I):
Figure imgf000105_0001
wherein: each - is independently a single bond or a double bond;
X1 is CR5, CR5R6, N, NR5, O, S, C=O, C=S, or a carbon atom connected to Q1;
X2 is CR7, CR7R8, N, NR7, O, S, C=O, C=S, or a carbon atom connected to Q1;
X3 is CR9, CR9R10, N, NR9, O, S, C=O, C=S, or a carbon atom connected to Q1;
X4 is CR11, CRnR12, N, NR11, O, S, C=O, C=S, or a carbon atom connected to Q1;
X5 is CR13, N, or NR13; wherein at least one of X1, X2, X3, and X4 is a carbon atom connected to Q1;
Q1 is C=O, C=S, C=CR14R15, C=NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, a bond; m is 1, 2, 3, or 4;
Y is N or O;
R1 is -C(O)R16, -C(O)OR16, -C(O)NR16R17, -OR16, -R16OR17, -R16OR17R18, -SR16, - R16SR17, -R16SR17R18, -NR16R17, -R16NR17, -R16NR17R18, -NR16C(O)R16, - OC(O)R16, C=O, C=S, -CN, -SiR16R17R18, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; each R3 and R4 is independently -C(O)R19, -C(O)OR19, -C(O)NR19R20, -SOR19, - SO2R19, alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently unsubstituted or substituted, or hydrogen; each R2, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, and R18 is independently - C(O)R21, -C(O)OR21, -C(O)NR21R22, -OR21, -SR21, -NR21R22, - NR21C(O)R22, -OC(O)R21, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently unsubstituted or substituted, or hydrogen; each R19 and R20 is C(O)R23, -C(O)OR23, -C(O)NR23R24, -OR23, -SR23, -NR23R24, - NR23C(O)R24, -OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen; each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or a pharmaceutically-acceptable salt thereof.
[0180] The biological sample may be whole blood, serum, or plasma.
[0181] Additionally or alternatively, in some embodiments of the methods disclosed herein, the TP53 reactivating indole derivative is PC 14586.
[0182] Examples of MDM2 inhibitors include, but are not limited to, RG7112, RO5045337, idasanutlin, nutlin-3a, RG7388, AMG-232, KRT-232, APG-115, BI-907828, CGM097, siremadlin, HDM201, milademetan, MEL23, MEL24, and DS-3032b. Examples of BCL-2 inhibitors include, but are not limited to venetoclax, obatoclax, subatoclax, maritoclax, gossypol, apogossypol, TW-37, UMI-77, and BDA-366. Examples of XPO-1 inhibitors include, but are not limited to KPT330 (Selinexor), XPOVIO, KPT8602 (Eltanexor), KPT335, Verdinexor, and KPT 185.
[0183] Additionally or alternatively, in some embodiments of the methods disclosed herein, the MDM2 inhibitor, the BCL-2 inhibitor, and/or the XPO-1 inhibitor are sequentially, simultaneously, or separately administered with the TP53 reactivating indole derivative. In certain embodiments, the MDM2 inhibitor, the BCL-2 inhibitor, the XPO-1 inhibitor, and/or the TP53 reactivating indole derivative is administered orally, intravenously, intramuscularly, intraperitoneally, or subcutaneously.
[0184] In any and all embodiments of the methods disclosed herein, the leukemia is acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS).
[0185] Additionally or alternatively, in some embodiments of the methods disclosed herein, mRNA expression levels are detected via real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, microarray, dot or slot blots, in situ hybridization, or fluorescent in situ hybridization (FISH). Additionally or alternatively, in certain embodiments of the methods disclosed herein, polypeptide expression levels are detected via Western blotting, enzyme-linked immunosorbent assays (ELISA), dot blotting, immunohistochemistry, immunofluorescence, immunoprecipitation, immunoelectrophoresis, or mass-spectrometry.
[0186] In some embodiments, the method comprises administering to the patient an effective amount of a TP53 reactivating indole derivative (e.g., PC14586) and an effective amount of an MDM2 inhibitor. In certain embodiments, the method comprises administering to the patient an effective amount of a TP53 reactivating indole derivative (e.g., PC 14586) and an effective amount of a BCL-2 inhibitor. In other embodiments, the method comprises administering to the subject an effective amount of a TP53 reactivating indole derivative (e.g., PC 14586) and an effective amount of an XPO-1 inhibitor. In any and all embodiments of the methods disclosed herein, the MDM2 inhibitor, the BCL-2 inhibitor, the XPO-1 inhibitor and/or the TP53 reactivating indole derivative (e.g., PC14586) may be administered as a single composition or as separate compositions.
[0187] In any and all embodiments of the methods disclosed herein, the patient is non- responsive to at least one prior line of cancer therapy, such as chemotherapy or immunotherapy. In some embodiments, the chemotherapy comprises one or more of trioxide, azacytidine, cerubidine, cyclophosphamide, cytarabine, daunorubicin hydrochloride, daurismo, dexamethasone, doxorubicin hydrochloride, enasidenib mesylate, gemtuzumab ozogamicin, gilteritinib fumarate, glasdegib maleate, idamycin PFS, idarubicin hydrochloride, idhifa, ivosidenib, midostaurin, mitoxantrone hydrochloride, mylotarg, olutasidenib, onureg, pemazyre, pemigatinib, prednisone, rezlidhia, Rituxan, rituximab, rubidomycin, rydapt, tabloid, thioguanine, tibsovo, tisagenlecleucel, trisenox, venclexta, venetoclas, vinicristine sulfate, vyxeos, or xospata. The patient may be a child or an adult. In certain embodiments, the patient is human. Additionally or alternatively, in any and all embodiments of the methods disclosed herein, the patient comprises an acquired TP53- Y220C mutation.
[0188] Additionally or alternatively, in some embodiments of the combination therapy methods disclosed herein, the time to response and/or duration of response is improved relative to that observed with TP53 reactivating indole derivative (e.g., PC 14586) monotherapy, or monotherapy with an MDM2 inhibitor, a BCL-2 inhibitor, or XPO-1 inhibitor.
[0189] Additionally or alternatively, in some embodiments of the methods disclosed herein, the TP53 reactivating indole derivative (e.g., PC 14586) and the MDM2 inhibitor are administered sequentially, simultaneously, or separately. The TP53 reactivating indole derivative (e.g., PC14586) and/or the MDM2 inhibitor may be administered orally, parenterally, by inhalation spray, intranasally, buccally, or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intra-synovial, intrastemal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. In some embodiments, the compositions are administered orally, intravenously, or subcutaneously. Formulations including a TP53 reactivating indole derivative (e.g., PC14586) and/or an MDM2 inhibitor disclosed herein may be designed to be short-acting, fast-releasing, or long-acting. In other embodiments, compounds can be administered in a local rather than systemic means, such as administration (e.g., by injection) at a tumor site.
[0190] Additionally or alternatively, in some embodiments of the methods disclosed herein, the TP53 reactivating indole derivative (e.g., PC 14586) and the BCL-2 inhibitor are administered sequentially, simultaneously, or separately. The TP53 reactivating indole derivative (e.g., PC14586) and/or the BCL-2 inhibitor may be administered orally, parenterally, by inhalation spray, intranasally, buccally, or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intra-synovial, intrastemal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. In some embodiments, the compositions are administered orally, intravenously, or subcutaneously. Formulations including a TP53 reactivating indole derivative (e.g., PC14586) and/or BCL-2 inhibitor disclosed herein may be designed to be short-acting, fast-releasing, or long-acting. In other embodiments, compounds can be administered in a local rather than systemic means, such as administration (e.g., by injection) at a tumor site.
[0191] Additionally or alternatively, in some embodiments of the methods disclosed herein, the TP53 reactivating indole derivative (e.g., PC 14586) and the XPO-1 inhibitor are administered sequentially, simultaneously, or separately. The TP53 reactivating indole derivative (e.g., PC14586) and/or the XPO-1 inhibitor may be administered orally, parenterally, by inhalation spray, intranasally, buccally, or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intra-synovial, intrastemal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. In some embodiments, the compositions are administered orally, intravenously, or subcutaneously. Formulations including a TP53 reactivating indole derivative (e.g., PC14586) and/or XPO-1 inhibitor disclosed herein may be designed to be short-acting, fast-releasing, or long-acting. In other embodiments, compounds can be administered in a local rather than systemic means, such as administration (e.g., by injection) at a tumor site.
[0192] Additionally or alternatively, in some embodiments of the methods disclosed herein, a TP53 reactivating indole derivative (e.g., PC14586) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), simultaneously with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of an MDM2 inhibitor, a BCL-2 inhibitor, or a XPO-1 inhibitor to a patient with leukemia.
[0193] In some embodiments, a TP53 reactivating indole derivative (e.g., PC 14586) and at least one of an MDM2 inhibitor, a BCL-2 inhibitor, or an XPO-1 inhibitor are administered to a patient, for example, a mammal, such as a human, in a sequence and within a time interval such that the inhibitor that is administered first acts together with the inhibitor that is administered second to provide greater benefit than if each inhibitor were administered alone. For example, a TP53 reactivating indole derivative (e.g., PC 14586) and at least one of an MDM2 inhibitor, a BCL-2 inhibitor, or an XPO-1 inhibitor can be administered at the same time or sequentially in any order at different points in time; however, if not administered at the same time, a TP53 reactivating indole derivative (e.g., PC14586) and at least one of an MDM2 inhibitor, a BCL-2 inhibitor, or an XPO-1 inhibitor are administered sufficiently close in time so as to provide the desired therapeutic or prophylactic effect of the combination of the at least two inhibitors. In one embodiment, a TP53 reactivating indole derivative (e.g., PC14586) and at least one of an MDM2 inhibitor, a BCL-2 inhibitor, or an XPO-1 inhibitor exert their effects at times which overlap. In some embodiments, a TP53 reactivating indole derivative (e.g., PC 14586) and at least one of an MDM2 inhibitor, a BCL-2 inhibitor, or an XPO-1 inhibitor are each administered as separate dosage forms, in any appropriate form and by any suitable route. In other embodiments, a TP53 reactivating indole derivative (e.g., PC 14586) and at least one of an MDM2 inhibitor, a BCL-2 inhibitor, or an XPO-1 inhibitor are administered simultaneously in a single dosage form.
[0194] It will be appreciated that the frequency with which any of these therapeutic agents can be administered can be once or more than once over a period of about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 20 days, about 28 days, about a week, about 2 weeks, about 3 weeks, about 4 weeks, about a month, about every 2 months, about every 3 months, about every 4 months, about every 5 months, about every 6 months, about every 7 months, about every 8 months, about every 9 months, about every 10 months, about every 11 months, about every year, about every 2 years, about every 3 years, about every 4 years, or about every 5 years.
[0195] For example, a TP53 reactivating indole derivative (e.g., PC 14586), an MDM2 inhibitor, a BCL-2 inhibitor or an XPO-1 inhibitor may be administered daily, weekly, biweekly, or monthly for a particular period of time. A TP53 reactivating indole derivative (e.g., PC 14586), an MDM2 inhibitor, a BCL-2 inhibitor or an XPO-1 inhibitor may be dosed daily over a 14-day time period, or twice daily over a seven-day time period. A TP53 reactivating indole derivative (e.g., PC14586), an MDM2 inhibitor, a BCL-2 inhibitor or an XPO-1 inhibitor may be administered daily for 7 days.
[0196] Alternatively, a TP53 reactivating indole derivative (e.g., PC14586), an MDM2 inhibitor, a BCL-2 inhibitor or an XPO-1 inhibitor may be administered daily, weekly, biweekly, or monthly for a particular period of time followed by a particular period of nontreatment. In some embodiments, a TP53 reactivating indole derivative (e.g., PC 14586), an MDM2 inhibitor, a BCL-2 inhibitor or an XPO-1 inhibitor can be administered daily for 14 days followed by seven days of non-treatment, and repeated for two more cycles of daily administration for 14 days followed by seven days of non-treatment. In some embodiments, a TP53 reactivating indole derivative (e.g., PC 14586), an MDM2 inhibitor, a BCL-2 inhibitor or an XPO-1 inhibitor can be administered twice daily for seven days followed by 14 days of non-treatment, which may be repeated for one or two more cycles of twice daily administration for seven days followed by 14 days of non-treatment.
[0197] In some embodiments, a TP53 reactivating indole derivative (e.g., PC 14586), the MDM2 inhibitor, the BCL-2 inhibitor or the XPO-1 inhibitor is administered daily over a period of 14 days. In another embodiment, a TP53 reactivating indole derivative (e.g., PC14586), the MDM2 inhibitor, the BCL-2 inhibitor or the XPO-1 inhibitor is administered daily over a period of 12 days, or 11 days, or 10 days, or nine days, or eight days. In another embodiment, a TP53 reactivating indole derivative (e.g., PC14586), the MDM2 inhibitor, the BCL-2 inhibitor or the XPO-1 inhibitor is administered daily over a period of seven days. In another embodiment, a TP53 reactivating indole derivative (e.g., PC14586), the MDM2 inhibitor, the BCL-2 inhibitor or the XPO-1 inhibitor is administered daily over a period of six days, or five days, or four days, or three days.
[0198] In some embodiments, individual doses of a TP53 reactivating indole derivative (e.g., PC 14586) and the MDM2 inhibitor, BCL-2 inhibitor, or XPO-1 inhibitor are administered within a time interval such that the two inhibitors can work together (e.g., within 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 5 days, 6 days, 1 week, or 2 weeks). In some embodiments, the treatment period during which the therapeutic agents are administered is then followed by a non-treatment period of a particular time duration, during which the therapeutic agents are not administered to the patient. This non-treatment period can then be followed by a series of subsequent treatment and non-treatment periods of the same or different frequencies for the same or different lengths of time. In some embodiments, the treatment and non-treatment periods are alternated. It will be understood that the period of treatment in cycling therapy may continue until the patient has achieved a complete response or a partial response, at which point the treatment may be stopped. Alternatively, the period of treatment in cycling therapy may continue until the patient has achieved a complete response or a partial response, at which point the period of treatment may continue for a particular number of cycles. In some embodiments, the length of the period of treatment may be a particular number of cycles, regardless of patient response. In some other embodiments, the length of the period of treatment may continue until the patient relapses.
[0199] In some embodiments, a TP53 reactivating indole derivative (e.g., PC 14586) and the MDM2 inhibitor, BCL-2 inhibitor, or XPO-1 inhibitor are cyclically administered to a patient. Cycling therapy involves the administration of a first agent (e.g., a first prophylactic or therapeutic agent) for a period of time, followed by the administration of a second agent and/or third agent (e.g., a second and/or third prophylactic or therapeutic agent) for a period of time and repeating this sequential administration. Cycling therapy can reduce the development of resistance to one or more of the therapies, avoid or reduce the side effects of one of the therapies, and/or improve the efficacy of the treatment.
[0200] In some embodiments, a TP53 reactivating indole derivative (e.g., PC 14586) in combination with an MDM2 inhibitor, BCL-2 inhibitor, or XPO-1 inhibitor are each administered at a dose and schedule typically used for that agent during monotherapy. In other embodiments, a TP53 reactivating indole derivative (e.g., PC 14586) and one of MDM2 inhibitor, BCL-2 inhibitor, or XPO-1 inhibitor are administered concomitantly, one or both of the agents can advantageously be administered at a lower dose than typically administered when the agent is used during monotherapy, such that the dose falls below the threshold that an adverse side effect is elicited.
[0201] The therapeutically effective amounts or suitable dosages of the TP53 reactivating indole derivative (e.g, PC 14586), the MDM2 inhibitor, the BCL-2 inhibitor and the XPO-1 inhibitor in combination depends upon a number of factors, including the nature of the severity of the condition to be treated, the particular inhibitor, the route of administration and the age, weight, general health, and response of the individual patient. In certain embodiments, the suitable dose level is one that achieves a therapeutic response as measured by reduction in cancer cells or other standard measures of disease progression, progression free survival, or overall survival. In other embodiments, the suitable dose level is one that achieves this therapeutic response and also minimizes any side effects associated with the administration of the therapeutic agent.
[0202] Suitable daily dosages of MDM2 inhibitors can generally range, in single or divided or multiple doses, from about 10% to about 120% of the maximum tolerated dose as a single agent. In certain embodiments, the suitable dosages of MDM2 inhibitors are from about 20% to about 100% of the maximum tolerated dose as a single agent. In other embodiments, suitable dosages of MDM2 inhibitors are about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,
I l l about 100%, about 105%, about 110%, about 115%, or about 120% of the maximum tolerated dose as a single agent.
[0203] Suitable daily dosages of BCL-2 inhibitors can generally range, in single or divided or multiple doses, from about 10% to about 120% of the maximum tolerated dose as a single agent. In certain embodiments, the suitable dosages of BCL-2 inhibitors are from about 20% to about 100% of the maximum tolerated dose as a single agent. In other embodiments, suitable dosages of BCL-2 inhibitors are about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, or about 120% of the maximum tolerated dose as a single agent.
[0204] Suitable daily dosages of XPO-1 inhibitors can generally range, in single or divided or multiple doses, from about 10% to about 120% of the maximum tolerated dose as a single agent. In certain embodiments, the suitable dosages of XPO-1 inhibitors are from about 20% to about 100% of the maximum tolerated dose as a single agent. In other embodiments, suitable dosages of XPO-1 inhibitors are about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, or about 120% of the maximum tolerated dose as a single agent.
[0205] Suitable daily dosages of a TP53 reactivating indole derivative (e.g., PC 14586) can generally range, in single or divided or multiple doses, from about 10% to about 120% of the maximum tolerated dose as a single agent. In certain embodiments, the suitable dosages of a TP53 reactivating indole derivative (e.g., PC 14586) are from about 20% to about 100% of the maximum tolerated dose as a single agent. In other embodiments, suitable dosages of a TP53 reactivating indole derivative (e.g., PC 14586) are about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, or about 120% of the maximum tolerated dose as a single agent. [0206] Dosage, toxicity, and therapeutic efficacy of any therapeutic agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are advantageous. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
[0207] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds may be within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (z.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to determine useful doses in humans accurately. Levels in plasma may be measured, for example, by high performance liquid chromatography.
[0208] Typically, an effective amount of a TP53 reactivating indole derivative (e.g., PC 14586), an MDM2 inhibitor, a BCL-2 inhibitor, or an XPO-1 inhibitor, sufficient for achieving a therapeutic or prophylactic effect, may range from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day. Suitably, the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day. For example dosages can be 1 mg/kg body weight or 10 mg/kg body weight every day, every two days, or every three days or within the range of 1-10 mg/kg every week, every two weeks, or every three weeks. In one embodiment, a single dosage of a TP53 reactivating indole derivative (e.g., PC14586), an MDM2 inhibitor, a BCL-2 inhibitor, or an XPO-1 inhibitor ranges from 0.001-10,000 micrograms per kg body weight. In one embodiment, a TP53 reactivating indole derivative (e.g., PC 14586), an MDM2 inhibitor, a BCL-2 inhibitor, or an XPO-1 inhibitor concentrations in a carrier range from 0.2 to 2000 micrograms per delivered milliliter. An exemplary treatment regime entails administration once per day or once a week. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, or until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.
[0209] In some embodiments, a therapeutically effective amount of a TP53 reactivating indole derivative (e.g., PC 14586), an MDM2 inhibitor, a BCL-2 inhibitor, or an XPO-1 inhibitor may be defined as a concentration of a TP53 reactivating indole derivative (e.g., PC14586), an MDM2 inhibitor, a BCL-2 inhibitor, or an XPO-1 inhibitor at the target tissue of IO’12 to 10'6 molar, e.g., approximately 10'7 molar. This concentration may be delivered by systemic doses of 0.001 to 100 mg/kg or equivalent dose by body surface area. The schedule of doses would be optimized to maintain the therapeutic concentration at the target tissue, such as by single daily or weekly administration, but also including continuous administration (e.g, parenteral infusion or transdermal application).
[0210] The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to, the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compositions described herein can include a single treatment or a series of treatments.
[0211] The mammal treated in accordance with the present methods can be any mammal, including, for example, farm animals, such as sheep, pigs, cows, and horses; pet animals, such as dogs and cats; laboratory animals, such as rats, mice, and rabbits. In some embodiments, the mammal is a human.
Kits of the Present Technology
[0212] The present disclosure provides kits for treating leukemia comprising (a) a TP53 reactivating indole derivative (e.g, PC 14586), (b) at least one of an MDM2 inhibitor, a BCL-2 inhibitor, and XPO-1 inhibitor, and (c) instructions for treating leukemia. In some embodiments, the leukemia is AML and/or MDS. Additionally or alternatively, in some embodiments, the leukemia comprises wild-type p53 or a TP53-Y220C mutation. [0213] When simultaneous administration is contemplated, the kit may comprise a TP53 reactivating indole derivative (e.g., PC14586) and at least one of an MDM2 inhibitor, a BCL-2 inhibitor, and XPO-1 inhibitor that has been formulated into a single pharmaceutical composition such as a tablet, or as separate pharmaceutical compositions. When the TP53 reactivating indole derivative (e.g., PC 14586) and one or more of the MDM2 inhibitors, BCL-2 inhibitors, and XPO-1 inhibitors disclosed herein are not administered simultaneously, the kit may comprise (a) a TP53 reactivating indole derivative (e.g., PC 14586), and (b) at least one of an MDM2 inhibitor, a BCL-2 inhibitor, and XPO-1 inhibitor that has been formulated as separate pharmaceutical compositions either in a single package, or in separate packages.
[0214] Additionally or alternatively, in some embodiments, the kits further comprise at least one chemotherapeutic agent and/or at least one immunotherapeutic agent that are useful for treating leukemia. Examples of such chemotherapeutic agents include arsenic trioxide, azacytidine, cerubidine, cyclophosphamide, cytarabine, daunorubicin hydrochloride, daurismo, dexamethasone, doxorubicin hydrochloride, enasidenib mesylate, gemtuzumab ozogamicin, gilteritinib fumarate, glasdegib maleate, idamycin PFS, idarubicin hydrochloride, idhifa, ivosidenib, midostaurin, mitoxantrone hydrochloride, mylotarg, olutasidenib, onureg, pemazyre, pemigatinib, prednisone, rezlidhia, Rituxan, rituximab, rubidomycin, rydapt, tabloid, thioguanine, tibsovo, tisagenlecleucel, trisenox, venclexta, venetoclas, vinicristine sulfate, vyxeos, and xospata. The kits may further comprise pharmaceutically acceptable excipients, diluents, or carriers that are compatible with one or more kit components described herein. Optionally, the above-described components of the kits of the present technology are packed in suitable containers and labeled for the treatment of leukemia, including AML and/or MDS. The kits may optionally include instructions customarily included in commercial packages of therapeutic products, which contain information about, for example, the indications, usage, dosage, manufacture, administration, contraindications and/or warnings concerning the use of such therapeutic products.
EXAMPLES
[0215] The present technology is further illustrated by the following Examples, which should not be construed as limiting in any way. Example 1: Materials and methods
[0216] PC14586 was supplied by PMV Pharmaceuticals. For details, see Dumbrava
EE, Johnson ML, Tolcher AW, Shapiro G, Thompson JA, El-Khoueiry AB, et al. “First-inhuman study of PC14586, a small molecule structural corrector of Y220C mutant p53, in patients with advanced solid tumors harboring a TP53 Y220C mutation.” J Clin Oncol (2022) 16 suppl:3003. doi: 10.1200/JC0.2022.40.16 suppl.3003.
[0217] The BCL-2 inhibitor was venetoclax (BCL201). The MDM2 inhibitor was nutlin-3a (HDM201). The XPO1 inhibitors were KPT330 and KPT8602.
[0218] In vitro studies were conducted with AML cells with wild-type TP53 and isogenic AML cells engineered with TP53 knockout (KO), TP53-R175H, and Y220C mutations.
[0219] Cell Culture: AML cells were cultured in RPML1640 medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 pg/mL streptomycin. Cells were kept at 37°C in a humidified, 5% CO2 atmosphere.
[0220] Cell Viability Testing: Apoptosis was assessed by flow cytometric measurement of cells stained with annexin V (AnnV). Cell membrane integrity was simultaneously assessed by 7-aminoactinomycin D (7AAD) exclusion in the annexin V-stained cells. Viable cell numbers were determined by flow cytometry using counting beads and expressed as % to the control.
Example 2: PC14586 converts p53Y220C to wild-type p53 conformation and activates p53 transcriptional activity and greatly induces p21
[0221] FIG. 1 shows a Western blot demonstrating the activity of PC14586 on mutant and wild-type p53 (upper panel) and a Western blot demonstrating the transcriptional activity of p53 (lower panel) after treatment with PC 14586. The Western blot reveals PC14586 activates p53 and p53 transcriptional activity and induces p21 expression.
Example 3: PC14586 primarily suppresses cell grow in TP53 Y220C, not in TP53 WT, KO, or TP53 R175H mutant AML cells
[0222] Treatment with PC14586 was tested on a panel of AML cell lines. The panel included AML cell lines having TP53-WT, TP53-KO, TP53-R175H, or TP53-Y220C. FIG. 2A is a graph of dose-response viability curves of the panel of AML cell lines treated with PC 14586 for 120 hours. FIG. 2B is a graph of 7AAD/AnnV curves of the panel of AML cell lines treated with PC 14586 for 120 hours. Viability curves indicate the percentage of live cells in the sample and 7AAD/AnnV curves reveal the percentage of cells in apoptosis in the sample.
[0223] Results from these tests revealed that PC 14586 primarily suppresses cell growth in AML cells having a TP53-Y220C p53 mutation. However, PC14586 did not substantially affect the percentage of cells in apoptosis in any of the samples, including AML cells having a TP53-Y220C p53 mutation, indicating that PC14586 monotreatment does not induce apoptosis in these cell lines.
Example 4: \1D\12 inhibition enhances PC14586 activity in AML cells having TP53-WT and the combination is highly synergistic in AML cells having TP53-Y220C.
[0224] The combination of an MDM2 inhibitor and PC 14586 (MDM2i+PC 14586) was tested in vitro and compared to treatment with the MDM2 inhibitor (MDM2i) or the PC14586 alone. AML cell lines having TP53-WT, TP53-KO, TP53-R175H, or TP53- Y220C were treated with the combination of MDM2 and PC14586 for 72 hours.
Concentrations of MDM2 were varied between 0 pM and 5.00 pM and concentrations of PC14586 were varied between 0 pM and 4.0 pM.
[0225] FIG. 3A is a graph of 7AAD/AnnV curves of AML cells having TP53-WT treated with an MDM2 inhibitor, PC 14586, or a combination of the MDM2 inhibitor and PC14586 for 72 hours. FIG. 3B is a graph of dose-response viability curves of AML cells having TP53-WT treated with the MDM2 inhibitor, PC14586, or the combination of the MDM2 inhibitor and PC 14586 for 72 hours. Results revealed that the MDM2 inhibitor enhanced PC14586 activity in AML cells having TP53-WT, greatly increasing cell death and reducing cell growth as compared to the monotherapies.
[0226] FIG. 3C is a graph of 7AAD/AnnV curves of AML cells having TP53-Y220C treated with the MDM2 inhibitor, PC 14586, or the combination of the MDM2 inhibitor and PC14586 for 72 hours. FIG. 3D is a graph of dose-response viability curves of AML cells having TP53-Y220C treated with the MDM2 inhibitor, PC14586, or the combination of the MDM2 inhibitor and PC 14586 for 72 hours. Results revealed that the MDM2 inhibitor enhanced PC14586 activity in AML cells having TP53-Y220C, and that the combination treatment had a synergistic affect to greatly increase cell death and reduce cell growth as compared to the monotherapies. [0227] Results revealed that neither the MDM2 inhibitor, PC 14586, nor their combination had substantial activity in TP53 KO or TP53 R175H AML cells. FIG. 3E is a graph of 7AAD/AnnV curves of AML cells having TP53-KO treated with the MDM2 inhibitor, PC 14586, or the combination of the MDM2 inhibitor and PC 14586 for 72 hours. FIG. 3F is a graph of dose-response viability curves of AML cells having TP53-KO treated with the MDM2 inhibitor, PC 14586, or the combination of the MDM2 inhibitor and PC14586 for 72 hours. FIG. 3G is a graph of 7AAD/AnnV curves of AML cells having TP53-R175H treated with the MDM2 inhibitor, PC14586, or the combination of the MDM2 inhibitor and PC 14586 for 72 hours. FIG. 3H is a graph of dose-response viability curves of AML cells having TP53-R175H treated with the MDM2 inhibitor, PC14586, or the combination of the MDM2 inhibitor and PC 14586 for 72 hours.
Example 5: BCL-2 inhibition enhances PC14586 activity in AML cells having TP53-WT and the combination is highly synergistic in AML cells having TP53-Y220C.
[0228] The combination of an BCL-2 inhibitor and PC 14586 (BCL-2i+PC 14586) was tested in vitro and compared to treatment with the BCL-2 inhibitor (BCL-2i) or the PC14586 alone. AML cell lines having TP53-WT, TP53-KO, TP53-R175H, or TP53- Y220C were treated with the combination of BCL-2 and PC14586 for 72 hours.
Concentrations of BCL-2 were varied between 0 nM and 20.00 nM and concentrations of PC14586 were varied between 0 pM and 4.0 pM.
[0229] FIG. 4A is a graph of 7AAD/AnnV curves of AML cells having TP53-WT treated with a BCL-2 inhibitor, PC 14586, or a combination of the BCL-2 inhibitor and PC14586 for 72 hours. FIG. 4B is a graph of dose-response viability curves of AML cells having TP53-WT treated with the BCL-2 inhibitor, PC14586, or the combination of the BCL-2 inhibitor and PC14586 for 72 hours. Results revealed that the combination therapy had a synergistic effect on AML cells having TP53-WT to increase cell death and reduce cell growth as compared to the monotherapies.
[0230] FIG. 4C is a graph of 7AAD/AnnV curves of AML cells having TP53-Y220C treated with the BCL-2 inhibitor, PC 14586, or the combination of the BCL-2 inhibitor and PC14586 for 72 hours. FIG. 4D is a graph of dose-response viability curves of AML cells having TP53-Y220C treated with the BCL-2 inhibitor, PC14586, or the combination of the BCL-2 inhibitor and PC14586 for 72 hours. Results revealed that the combination therapy had a synergistic effect on AML cells having TP53-Y220C to increase cell death and reduce cell growth as compared to the monotherapies.
[0231] The combination of the BCL-2 inhibitor and PC 14586 slightly increased the activity against TP53 KO and TP53 R175H AML cells. FIG. 4E is a graph of 7AAD/AnnV curves of AML cells having TP53-KO treated with the BCL-2 inhibitor, PC14586, or the combination of the BCL-2 inhibitor and PC14586 for 72 hours. FIG. 4F is a graph of dose-response viability curves of AML cells having TP53-KO treated with the BCL-2 inhibitor, PC14586, or the combination of the BCL-2 inhibitor and PC14586 for 72 hours. FIG. 4G is a graph of 7AAD/AnnV curves of AML cells having TP53-R175H treated with the BCL-2 inhibitor, PC 14586, or the combination of the BCL-2 inhibitor and PC14586 for 72 hours. FIG. 4H is a graph of dose-response viability curves of AML cells having TP53-R175H treated with the BCL-2 inhibitor, PC14586, or the combination of the BCL-2 inhibitor and PC14586 for 72 hours.
Example 6: XPO-1 inhibition enhances PC14586 activity in AML cells having TP53-WT and the combination is highly synergistic in AML cells having TP53-Y220C.
[0232] The combination of an XPO-1 inhibitor and PC 14586 (XPO-li+PC14586) was tested in vitro and compared to treatment with the XPO-1 inhibitor (XPO-1 i) or the PC14586 alone. AML cell lines having TP53-WT, TP53-KO, TP53-R175H, or TP53- Y220C were treated with the combination of XPO-1 and PC14586 for 72 hours.
Concentrations of XPO-1 were varied between 0 nM and 200.00 nM and concentrations of PC14586 were varied between 0 pM and 4.0 pM.
[0233] FIG. 5A is a graph of 7AAD/AnnV curves of AML cells having TP53-WT treated with a XPO inhibitor, PC 14586, or a combination of the XPO inhibitor and PC14586 for 72 hours. FIG. 5B is a graph of dose-response viability curves of AML cells having TP53-WT treated with the XPO inhibitor, PC 14586, or the combination of the XPO inhibitor and PC 14586 for 72 hours. Results revealed that the combination therapy had a synergistic effect on AML cells having TP53-WT to increase cell death and reduce cell growth as compared to the monotherapies.
[0234] FIG. 5C is a graph of 7AAD/AnnV curves of AML cells having TP53-Y220C treated with the XPO inhibitor, PC 14586, or the combination of the XPO inhibitor and PC14586 for 72 hours. FIG. 5D is a graph of dose-response viability curves of AML cells having TP53-Y220C treated with the XPO inhibitor, PC 14586, or the combination of the XPO inhibitor and PC14586 for 72 hours. Results revealed that the combination therapy had a synergistic effect on AML cells having TP53-Y220C to increase cell death and reduce cell growth as compared to the monotherapies.
[0235] The combination of the XPO-1 inhibitor and PC 14586 slightly increased the activity against TP53 KO and TP53 R175H AML cells. FIG. 5E is a graph of 7AAD/AnnV curves of AML cells having TP53-KO treated with the XPO inhibitor, PC14586, or the combination of the XPO inhibitor and PC14586 for 72 hours. FIG. 5F is a graph of dose-response viability curves of AML cells having TP53-KO treated with the XPO inhibitor, PC 14586, or the combination of the XPO inhibitor and PC 14586 for 72 hours. FIG. 5G is a graph of 7AAD/AnnV curves of AML cells having TP53-R175H treated with the XPO inhibitor, PC 14586, or the combination of the XPO inhibitor and PC14586 for 72 hours. FIG. 5H is a graph of dose-response viability curves of AML cells having TP53-R175H treated with the XPO inhibitor, PC14586, or the combination of the XPO inhibitor and PC 14586 for 72 hours.
Example 7. Synergistic effects of combination therapies of the present technology in AML cells having TP53-WT and in AML cells having TP53-Y220C.
[0236] While PC14586 in combination with MDM2, BCL-2, or XPO-1 inhibitor is more effective than each single agent alone, PC 14586 in combination with any two other agents is more effective than PC14586 in combination with one other agent, and PC14586 in combination with MDM2, BCL-2, and XPO-1 inhibitors is the most effective in cell death induction in both TP53 wild-type and Y220C mutant AML cells.
[0237] FIG. 6A shows 7AAD/AnnV curves of AML cells having TP53-WT treated with PC 14586, the BCL-2 inhibitor, the MDM2 inhibitor, the XPO-1 inhibitor, or the combination of PC14586 with one, two, or all three of these inhibitors for 72 hours. FIG. 6B shows dose-response viability curves of AML cells having TP53-WT treated with PC14586, the BCL-2 inhibitor, the MDM2 inhibitor, the XPO-1 inhibitor, or the combination of PC14586 with one, two, or all three of these inhibitors for 72 hours. FIG. 6C shows 7AAD/AnnV curves of AML cells having TP53-Y220C treated with PC 14586, the BCL-2 inhibitor, the MDM2 inhibitor, the XPO-1 inhibitor, or the combination of PC 14586 with one, two, or all three of these inhibitors for 72 hours. FIG. 6D shows doseresponse viability curves of AML cells having TP53-Y220C treated with PC14586, the BCL-2 inhibitor, the MDM2 inhibitor, the XPO-1 inhibitor, or the combination of PC14586 with one, two, or all three of these inhibitors for 72 hours. Accordingly, the combination therapies and methods disclosed herein are useful for treating leukemias in a subject in need thereof.
Example 8: Mol ml 3 TP53-Y220C cells treated with PC14586-alone or in combination with XPO-1, MDM2, or BCL-2 inhibitors.
[0238] PC14586-induced p53 target proteins were further upregulated when PC14586 was combined with XPO-1 or MDM2 inhibitors. FIG. 7A shows Western blots of the protein levels in Molml3 TP53-Y220C cells treated for 24 hours with venetoclax (VEN, 5 nM or 10 nM), nutlin-3a (Nut, 2.5 pM or 5 pM), PC14586 (PC, 2 pM or 4 pM), KPT-8602 (KPT, 100 nM or 200 nM), or combinations of PC 14586 with venetoclax (VEN/PC, 5 nM VEN and 2 pM PC, 10 nM VEN and 4 pM PC), nutlin-3a (Nut/PC, 2.5 pM Nut and 2 pM PC, 5 pM Nut and 4 pM PC), or KPT-8602 (KPT/PC, 100 nM KPT and 2 pM PC, 200 nM KPT and 4 pM PC).
[0239] While PC 14586 had limited activity on proliferation in TP53-Y220C AML cells, the combination with BCL-2 inhibitors largely blocked Gl-S transition and enhanced apoptosis, which were even more pronounced when PC14586 was combined with XPO-1 or MDM2 inhibitors. FIG. 7B shows cell cycle distribution and apoptosis determined by flow cytometry of cells stained with 5-ethynyl-2’-deoxyuridine (EdU) and DNA dye. Molml3 TP53-Y220C cells were treated for 72 hours with venetoclax (VEN, 10 nM), nutlin-3a (N3, 5 pM), KPT-8602 (KPT, 200 nM), PC 14586 (4 pM), or combinations of PC 14586 with venetoclax (4 pM PC14586 and 10 nMVEN), nutlin-3a (4 pM PC14586 and 5 pM N3), or KPT (4 pM PC14586 and 200 nM KPT). FIG. 7C shows DNA content and PARP cleavage determined by flow cytometry of Molml3 TP53-Y220C cells treated for 72 hours with venetoclax (VEN, 10 nM), nutlin3a (N3, 5 pM), KPT-8602 (KPT, 200 nM), PC 14586 (4 pM), or combinations of PC 14586 with venetoclax (4 pM PC 14586 and 10 nM VEN), nutlin-3a (4 pM PC 14586 and 5 pM), or KPT-8602 (4 pM PC 14586 and 200 nM KPT). Cells were stained with EdU, DNA dye, and antibody against cleaved PARP.
Example 9: PC14586 induced cell death in bulk AML cells and in ste /prosenitor cells in 2/3 samples in primary AML cells with Y220C mutations and the combinations were highly synergistic in AML cells and stem/prosenitor cells regardless of response to PC14586 alone
[0240] Cells from AML patient samples (FIGS. 8A-8G and 9A-9G), patient-derived xenograft (FIGS. 10A-10D), or normal bone marrow cells (FIGS. 11A-11D), each sample under mesenchymal stroma cell (MSC) co-culture conditions (COX) were treated for 48 hours or 96 hours with PC 14586; venetoclax; nutlin3a; KPT-8062; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC14586. Cell death and cell viability were determined by flow cytometry after the cells were stained with annexin V/7-aminoactinomycin D (Ann V/7-ADD) in the presence of counting beads.
[0241] Cells from the two patients in FIGS. 8A-8G and 9A-9G responded to PC 14586 as a single agent. PC14586 as a single agent had limited activity in PDX cells derived from the patient in FIGS. 10A-10D. The combinations of PC14586 with MDM2, BCL-2, and/or XPO-1 inhibitors were synergistic in cells and stem/progenitor cells from all three AML patient samples in FIGS. 8A-8G, 9A-9G, and FIGS. 10A-10D. All treatments using primary samples were carried out under mesenchymal stroma cell (MSC) co-culture conditions (COX).
[0242] FIGS. 8A-8G used 47% peripheral blood (PB) samples with 77% TP53-Y220C mutation. The samples had complex cytogenetics with mutations in NF1, JAK2, SMC1A, SRSF2, TET2, and CUX1 genes. Cell death and viable cells were determined by flow cytometry after the cells were stained with annexin V/7-aminoactinomycin D (Ann V/7- ADD) in the presence of counting beads. FIG. 8A showed CD45+, CD34+, and CD34+CD38- populations after 48 hours of treatment with PC14586 at different concentrations. Tables 1 and 2 provide ECso and IC50 data, respectively, for CD45+, CD34+, and CD34+CD38- cell populations treated with PC14586.
Table 1: EC50 for different cell types from PB samples as shown in FIGS. 8A-8G treated with PC 14586 for 48 hours.
Figure imgf000124_0001
Table 2: ICso for different cell types from PB samples as shown in FIGS. 8A-8G treated with PC 14586 for 48 hours.
Figure imgf000125_0001
[0243] FIG. 8B shows CD45+ population assessed by Ann V/7-ADD+ positive cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC 14586. Table 3 provides combination index (CI) values from FIG. 8B FIG. 8C shows CD45+ viable cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC14586; venetoclax and PC14586; nutlin-3a and PC14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC 14586.
Table 3: CI values for different treatment combinations for CD45+ cells.
Figure imgf000125_0002
[0244] FIG. 8D shows CD34+ assessed by Ann V/7ADD+ cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC14586; KPT-8602 and PC14586; or nutlin-3a, KPT-8602, and PC14586. Table 4 provides combination index (CI) values from FIG. 8D. FIG. 8E shows CD34+ viable cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602;
PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC 14586.
Table 4: CI values for different treatment combinations for CD34+ cells.
Figure imgf000125_0003
[0245] FIG. 8F shows CD34+CD38- population assessed by Ann V/7ADD+ cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC14586. Table 5 provides combination index (CI) values from FIG. 8F. FIG. 8G shows CD34+CD38- viable cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT- 8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC14586; or nutlin-3a, KPT-8602, and PC14586.
Table 5: CI values for different treatment combinations for CD34+CD38- cells.
Figure imgf000126_0001
[0246] FIGS. 9A-9G show cell samples from an AML patient with 92% peripheral blood (PB) with 45.7% TP53-Y220C under MSC COX. The samples had complex cytogenetics with mutations in TP-53-G105fs (39.7%) and U2AF1 genes. Cell death and viable cells were determined by flow cytometry after the cells were stained with Ann V/7- ADD in the presence of counting beads. FIG. 9A shows CD45+, CD34+, and CD34+CD38- population after 48 hours of treatment with PC14586 at different concentrations. PC14586 induced cell death in AML blasts and stem/progenitor cells. Tables 6 and 7 provide ECso and IC50 data, respectively, for CD45+, CD34+, and CD34+CD38- cell populations treated with PC14586.
Table 6: EC50 for different cell types from PB samples as shown in FIGS. 9A-9G treated with PC 14586 for 48 hours.
Figure imgf000126_0002
Table 7: ICso for different cell types from PB samples as shown in FIGS. 9A-9G treated with PC 14586 for 48 hours.
Figure imgf000126_0003
[0247] Combination of PC14586 with BCL-2, MDM2, and/or XPO-1 inhibition synergistically induced cell death in TP53 Y220C-mutant AML Blast Cells. FIG. 9B shows
CD45+ population assessed by Ann V/7-ADD+ cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC14586; KPT-8602 and PC14586; or nutlin-3a, KPT-8602, and PC14586. Table 8 provides combination index (CI) values from FIG. 9B. FIG. 9C shows CD45+ viable cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC 14586.
Table 8: CI values for different treatment combinations for CD45+ cells.
Figure imgf000127_0001
[0248] FIG. 9D shows CD34+ assessed by Ann V/7ADD+ cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC14586; KPT-8602 and PC14586; or nutlin-3a, KPT-8602, and PC14586. Table 9 provides combination index (CI) values from FIG. 9D. FIG. 9E shows CD34+ viable cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602;
PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC 14586.
Table 9: CI values for different treatment combinations for CD34+ cells.
Figure imgf000127_0002
[0249] FIG. 9F shows CD34+CD38- population assessed by Ann V/7ADD+ cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC14586. Table 10 provides combination index (CI) values from FIG. 9F. FIG. 9G shows CD34+CD38- viable cells after 48 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC14586; venetoclax and PC14586; nutlin-3a and PC14586; KPT-8602 and PC 14586; or nutlin-3a, KPT-8602, and PC 14586.
Table 10: CI values for different treatment combinations for CD34+CD38- cells.
Figure imgf000127_0003
Figure imgf000128_0001
[0250] FIGS. 10A-10D show patient-derived xenograft (PDX) spleen cells with 99.5% hCD45+ and TP53-Y220C mutation confirmed by sequencing. The samples had complex cytogenetics with complex Karyotype, MECOM rearrangement, and mutations in NBAS, KRAS, TP53 Y220C, and P151A genes. Cell death and viable cells were determined by flow cytometry after the cells were stained with Ann V/7-ADD in the presence of counting beads. Variants of probable somatic origin are in Table 11.
Table 11: Variants of probable somatic origin in PDX samples used in FIGS. 10A-10D.
Figure imgf000128_0002
[0251] FIG. 10A shows CD45±, CD34±, and CD34+CD38- populations after 96 hours of treatment with PC 14586 at different concentrations. PC 14586, as a single agent induced limited cell death in AML blast and stem/progenitor cells.
[0252] Combination of PC14586 with BCL-2, MDM2, and/or XPO-1 inhibitors synergistically induced cell death in TP53 Y220C-mutant AML blast cells. FIG. 10B shows CD45+ population assessed by Ann V/7-ADD+ positive cells after 96 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC14586; venetoclax and PC14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; nutlin-3a, KPT-8602, and PC 14586; venetoclax, KPT-8602, and PC14586; KPT-8602, nutlin-3a, and PC14586; or venetoclax, nutlin-3a, KPT-8602, and PC 14586. Table 12 provides combination index (CI) values from FIG. 10B. FIG. 10C: CD45+ viable cells after 96 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC14586; venetoclax and PC14586; nutlin-3a and PC14586; KPT-8602 and PC 14586; nutlin-3a, KPT-8602, and PC 14586; venetoclax, KPT-8602, and PC 14586; KPT-8602, nutlin-3a, and PC14586; or venetoclax, nutlin-3a, KPT-8602, and PC14586.
Table 12: CI values for different treatment combinations for CD45+ cells.
Figure imgf000129_0001
[0253] FIG. 10D shows CD34+ assessed by Ann V/7ADD+ cells after 96 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; nutlin-3a, KPT-8602, and PC 14586; venetoclax, KPT-8602, and PC14586; KPT-8602, nutlin-3a, and PC14586; or venetoclax, nutlin-3a, KPT-8602, and PC 14586. Table 13 provides combination index (CI) values from FIG. 10D. FIG. 10E shows CD34+ viable cells after 96 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; nutlin-3a, KPT-8602, and PC 14586; venetoclax, KPT- 8602, and PC14586; KPT-8602, nutlin-3a, and PC14586; or venetoclax, nutlin-3a, KPT- 8602, and PC 14586.
Table 13: CI values for different treatment combinations for CD34+ cells.
Figure imgf000129_0002
[0254] FIG. 10F shows CD34+CD38- population assessed by Ann V/7ADD+ cells after 96 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; nutlin-3a, KPT-8602, and PC 14586; venetoclax, KPT-8602, and PC 14586; KPT-8602, nutlin-3a, and PC14586; or venetoclax, nutlin-3a, KPT-8602, and PC14586. Table 14 provides combination index (CI) values from FIG. 10F. FIG. 10G: CD34+CD38- viable cells after 96 hours of treatment with venetoclax; nutlin-3a (N3); KPT-8602; PC14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; nutlin-3a, KPT-8602, and PC 14586; venetoclax, KPT-8602, and PC 14586; KPT-8602, nutlin-3a, and PC 14586; or venetoclax, nutlin-3a, KPT-8602, and PC 14586.
Table 14: CI values for different treatment combinations for CD34+CD38- cells.
Figure imgf000130_0001
[0255] PC 14586 and treatment combinations of PC 14586 with venetoclax, nutlin-3a, and/or KPT-8602 had limited toxicity in normal bone marrow cells and CD34+ stem/progenitor cells. FIGS. 11A-11D show normal bone marrow cells under MSC COX. Cell death and viable cells were determined by flow cytometry after the cells were stained with Ann V/7-ADD in the presence of counting beads. FIG. 11A shows CD45+ population assessed by Ann V/7-ADD+ positive cells after 48 hours of treatment with venetoclax; nutlin-3a; KPT-8602; PC14586; venetoclax and PC14586; nutlin-3a and PC14586; KPT- 8602 and PC14586; or venetoclax, nutlin-3a, KPT-8602, and PC14586. FIG. 11B shows CD45+ cell viability after 48 hours of treatment with venetoclax; nutlin-3a; KPT-8602;
PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or venetoclax, nutlin-3a, KPT-8602, and PC14586. FIG. 11C shows CD34+ population assessed by Ann V/7-ADD+ positive cells after 48 hours of treatment with venetoclax; nutlin-3a; KPT-8602; PC14586; venetoclax and PC14586; nutlin-3a and PC14586; KPT- 8602 and PC14586; or venetoclax, nutlin-3a, KPT-8602, and PC14586. FIG. 11D shows CD34+ cell viability after 48 hours of treatment with venetoclax; nutlin-3a; KPT-8602; PC 14586; venetoclax and PC 14586; nutlin-3a and PC 14586; KPT-8602 and PC 14586; or venetoclax, nutlin-3a, KPT-8602, and PC14586.
Example 10: PC14586 reactivates p53 signaling, which is further enhanced by inhibition of MDM2, induced by PC14586-activated p53
[0256] Molml3 7P53-WT and 7P53-Y220C cells were treated with nutlin-3a (N3a, 5 pM), PC14586 (PC, 4 pM), or the combination for 4 hours. RNA was isolated and subject to RNA sequencing.
[0257] FIG. 12A shows a principal component analysis comparing Molml3 TP 53-WT and 7P53-Y220C cells treated with nutlin-3a (N3a, 5 pM), PC14586 (PC, 4 pM), or the combination for 4 hours.
[0258] FIG. 12B shows gene set enrichment analysis with 77 degrees of 2-D hierarchical clustering comparing Molml3 TP53-WT and TP53-Y22QC cells treated with nutlin-3a (N3a, 5 pM), PC 14586 (PC, 4 pM), or the combination for 4 hours. Arrows in FIG. 12B indicate upregulation of p53 pathway for Molml3 TP53-WT treated with N3a or the combination of N3a and PC14586, but not PC14586 alone. Arrows in FIG. 12B also indicate upregulation of p53 pathway for Molml3 TP53-Y22QC cells treated with PC14586 or the combination of N3a and PC 14586, but not N3a alone.
[0259] FIG. 12C shows a heat map of differentially expressed genes. From left to right, Molml3 TP53-Y220C cells were treated with the combination of nutlin-3a and PC14586 (n=3, N3a at 5 pM, PC at 4 pM); Molml3 TP53-WT cells were treated with nutlin-3a (n=3, N3a, 5 pM); Molml3 TP53-WT cells were treated with the combination of nutlin-3a and PC14586 (n=3, N3a at 5 pM, PC at 4 pM); Molml3 TP53-Y22QC cells were treated with nutlin-3a (n=3, N3a, 5 pM); Molml3 TP53-Y22QC cells were treated with vehicle only as a control (n=3, con); Molml3 TP53-Y220C cells were treated with PC14586 (n=3, PC at 4 pM); Molml3 TP53-WT cells were treated with vehicle only as a control (n=3, con); and ); Molml3 TP53-WT cells were treated with PC14586 (n=3, PC at 4 pM). Results showed greatest upregulation for Molml3 TP53-Y220C cells treated with the combination of nutlin- 3a and PC 14586 and no change or deregulation for groups starting at Molml3 TP53-Y220C cells treated with nutlin-3a and every group to the left thereof.
[0260] FIG. 12D shows a pathway analysis. FIG. 12E shows volcano plots comparing increased, unchanged, and decreased gene expression in treated compared to control samples. FIG. 12F shows Western blots showing changes in gene expression indicated at the protein level.
Example 11: Combination of PC14586 and RG7388 significantly prolonged the survival in a TP53-Y220C PDX model, while PC14586 alone has no effect in the model that carries TP53-Y220C, TP53-P151A and NAS Mutations
[0261] PDX cells derived from an AML patient sample with TP53-Y220C (VAF 48%), TP53-P151A (VAF 47%), and NRAS (VAF 50%) mutations were injected via tail vein into NSG mice (8 week old, male) (1.6>< 106/mouse). Once human CD45 positivity in the circulation reached >1% as determined by flow cytometry, mice (10/group) were treated with vehicle, PC14586 (100 mg/kg, daily), MDM2 inhibitor idasanutlin (RG7388) with 50% activity (40 mg/kg, initially 8 days on, then 2 days off/5 days on) or both PC 14586 (100 mg/kg, daily) and MDM2 inhibitor idasanutlin (RG7388) with 50% activity (40 mg/kg, initially 8 days on, then 2 days off/5 days on). Disease progression and treatment responses were monitored by flow cytometry of human CD45+ cells. The Kaplan-Meier method was applied to estimate mouse survival and survival data were analyzed using the log-rank test.
[0262] FIG. 13A shows flow cytometry analysis of human CD45+ cells in peripheral blood (PB) after four weeks of treatment. FIG. 13B shows flow cytometry analysis of human CD45+ cells in peripheral blood (PB) after eight weeks of treatment. FIG. 13C: Kaplan-Meier estimation of mouse survival and survival data analyzed using the log-rank test.
Example 12: Unlike WT p53 that binds and antagonizes antiapoptotic BCL-2, PC14586 activated p53 does not bind to BCL-2
[0263] Molml3 TP53-WT and TP53-Y220C cells were treated with nutlin-3a (5 pM) or PC14586 (PC, 1.25 pM or 5 pM) for 4 hours. p53 was co-immunoprecipitated with antibodies selective for WT (PAbl620, Cat no. 102201, Caprico Bioscience) or mutant (PAb240, NB200-103, Novus Biosciences) conformation of p53. p53 and BCL-2 were determined by Western blot after co-immunoprecipitation. FIG. 14 shows these Western blots of p53 and BCL-2 from Molml3 TP53-WT and TP53-Y220C cells treated for 4 hours with nutlin-3a (5 pM) or PC14586 (PC, 1.25 pM or 5 pM). Without being bound by any theory, the pattern of PC14586 activated p53 not binding to BCL-2 may be related to its lack of apoptogenic activity in Molml3 TP53-Y220C cells.
Example 13: Combination of PC14586 and venetoclax significantly prolonged the survival in the Moll 3 TP53-Y220C xenograft model
[0264] Molml3 TP53-Y220C cells, 0.5>< 106/mice were injected via tail vein into NSG mice (male, 6-10 weeks old). Mice (5/group) were treated daily with vehicle, PC 14586 (100 mg/kg), venetoclax (50 mg/kg), or the combination. The Kaplan-Meier method was applied to estimate mouse survival and survival data were analyzed using the log-rank test. FIG. 15 shows the Kaplan-Meier estimation of mouse survival and survival data analyzed using the log-rank test of mice injected with Molml3 TP53-Y220C cells, and treated daily with vehicle, PC 14586, venetoclax, or the combination.
Example 14: Combination of PC14586 with XPO-1 inhibitor greatly increased p53 nuclear localization and transcription activity
[0265] TP53-Y220C Molml3 cells were treated with PC14586 (4 pM), KPT-8602 (200 nM), or both for 24 hours. p53 localization and its target proteins were determined by Western blot (MWM, molecular weight marker). For protein localization, cytosolic and nuclear proteins were fractionated. Tubulin and Lamin Bl were used as cytosolic or nuclear loading control, respectively. FIG. 16 shows the Western blots of p53, Lamin Bl, and tubulin from TP53-Y220C Molml3 cells treated with PC14586 (4 pM), KPT-8602 (200 nM), or both for 24 hours.
EQUIVALENTS
[0266] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
[0267] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.
[0268] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a nonlimiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.
[0269] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

Claims

1. A method for treating a wild-type p53 or TP53-Y220C leukemia in a patient in need thereof comprising administering to the patient an effective amount of a TP53 reactivating indole derivative and an effective amount of an MDM2 inhibitor, a BCL-2 inhibitor, and/or an XPO-1 inhibitor, wherein the TP53 reactivating indole derivative has formula of formula (I):
Figure imgf000135_0001
wherein: each - is independently a single bond or a double bond;
X1 is CR5, CR5R6, N, NR5, O, S, C=O, C=S, or a carbon atom connected to Q1;
X2 is CR7, CR7R8, N, NR7, O, S, C=O, C=S, or a carbon atom connected to Q1;
X3 is CR9, CR9R10, N, NR9, O, S, C=O, C=S, or a carbon atom connected to Q1;
X4 is CR11, CRnR12, N, NR11, O, S, C=O, C=S, or a carbon atom connected to Q1;
X5 is CR13, N, or NR13; wherein at least one of X1, X2, X3, and X4 is a carbon atom connected to Q1;
Q1 is C=O, C=S, C=CR14R15, C=NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, a bond; m is 1, 2, 3, or 4;
Y is N or O;
R1 is -C(O)R16, -C(O)OR16, -C(O)NR16R17, -OR16, -R16OR17, -R16OR17R18, -SR16, - R16SR17, -R16SR17R18, -NR16R17, -R16NR17, -R16NR17R18, -NR16C(O)R16, - OC(O)R16, C=O, C=S, -CN, -SiR16R17R18, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; each R3 and R4 is independently -C(O)R19, -C(O)OR19, -C(O)NR19R20, -SOR19, - SO2R19, alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently unsubstituted or substituted, or hydrogen; each R2, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, and R18 is independently - C(O)R21, -C(O)OR21, -C(O)NR21R22, -OR21, -SR21, -NR21R22, - NR21C(O)R22, -OC(O)R21, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently unsubstituted or substituted, or hydrogen; each R19 and R20 is C(O)R23, -C(O)OR23, -C(O)NR23R24, -OR23, -SR23, -NR23R24, - NR23C(O)R24, -OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen; each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or a pharmaceutically-acceptable salt thereof.
2. A method for prolonging survival of a wild-type p53 or TP53-Y220C leukemia patient comprising: administering to the leukemia patient an effective amount of a TP53 reactivating indole derivative and an effective amount of at least one additional therapeutic agent selected from among an MDM2 inhibitor, a BCL-2 inhibitor, and an XPO-1 inhibitor, wherein the TP53 reactivating indole derivative has formula of formula (I):
Figure imgf000136_0001
wherein: each - is independently a single bond or a double bond;
X1 is CR5, CR5R6, N, NR5, O, S, C=O, C=S, or a carbon atom connected to Q1;
X2 is CR7, CR7R8, N, NR7, O, S, C=O, C=S, or a carbon atom connected to Q1;
X3 is CR9, CR9R10, N, NR9, O, S, C=O, C=S, or a carbon atom connected to Q1;
X4 is CR11, CRnR12, N, NR11, O, S, C=O, C=S, or a carbon atom connected to Q1;
X5 is CR13, N, or NR13; wherein at least one of X1, X2, X3, and X4 is a carbon atom connected to Q1;
Q1 is C=O, C=S, C=CR14R15, C=NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, a bond; m is 1, 2, 3, or 4;
Y is N or O;
R1 is -C(O)R16, -C(O)OR16, -C(O)NR16R17, -OR16, -R16OR17, -R16OR17R18, -SR16, - R16SR17, -R16SR17R18, -NR16R17, -R16NR17, -R16NR17R18, -NR16C(O)R16, - OC(O)R16, C=O, C=S, -CN, -SiR16R17R18, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; each R3 and R4 is independently -C(O)R19, -C(O)OR19, -C(O)NR19R20, -SOR19, - SO2R19, alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently unsubstituted or substituted, or hydrogen; each R2, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, and R18 is independently - C(O)R21, -C(O)OR21, -C(O)NR21R22, -OR21, -SR21, -NR21R22, - NR21C(O)R22, -OC(O)R21, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently unsubstituted or substituted, or hydrogen; each R19 and R20 is C(O)R23, -C(O)OR23, -C(O)NR23R24, -OR23, -SR23, -NR23R24, - NR23C(O)R24, -OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen; each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or a pharmaceutically-acceptable salt thereof.
3. A method for selecting a leukemia patient for treatment with a TP53 reactivating indole derivative and an additional therapeutic agent comprising: detecting wild-type TP53 or TP53-Y220C mRNA or polypeptide expression in a biological sample obtained from a leukemia patient; and administering to the leukemia patient an effective amount of a TP53 reactivating indole derivative and an effective amount of at least one additional therapeutic agent selected from among an MDM2 inhibitor, a BCL-2 inhibitor, and an XPO-1 inhibitor, wherein the TP53 reactivating indole derivative has formula of formula (I):
Figure imgf000138_0001
wherein: each - is independently a single bond or a double bond;
X1 is CR5, CR5R6, N, NR5, O, S, C=O, C=S, or a carbon atom connected to Q1;
X2 is CR7, CR7R8, N, NR7, O, S, C=O, C=S, or a carbon atom connected to Q1;
X3 is CR9, CR9R10, N, NR9, O, S, C=O, C=S, or a carbon atom connected to Q1;
X4 is CR11, CRnR12, N, NR11, O, S, C=O, C=S, or a carbon atom connected to Q1;
X5 is CR13, N, or NR13; wherein at least one of X1, X2, X3, and X4 is a carbon atom connected to Q1; Q1 is C=0, C=S, C=CR14R15, C=NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, a bond; m is 1, 2, 3, or 4;
Y is N or O;
R1 is -C(O)R16, -C(O)OR16, -C(O)NR16R17, -OR16, -R16OR17, -R16OR17R18, -SR16, - R16SR17, -R16SR17R18, -NR16R17, -R16NR17, -R16NR17R18, -NR16C(O)R16, - OC(O)R16, C=O, C=S, -CN, -SiR16R17R18, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; each R3 and R4 is independently -C(O)R19, -C(O)OR19, -C(O)NR19R20, -SOR19, - SO2R19, alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently unsubstituted or substituted, or hydrogen; each R2, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, and R18 is independently - C(O)R21, -C(O)OR21, -C(O)NR21R22, -OR21, -SR21, -NR21R22, - NR21C(O)R22, -OC(O)R21, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently unsubstituted or substituted, or hydrogen; each R19 and R20 is C(O)R23, -C(O)OR23, -C(O)NR23R24, -OR23, -SR23, -NR23R24, - NR23C(O)R24, -OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen; each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or a pharmaceutically-acceptable salt thereof.
4. The method of any one of claims 1-3, wherein administering to the leukemia patient comprises administering the effective amount of the TP53 reactivating indole derivative and the effective amount of the MDM2 inhibitor.
5. The method of any one of claims 1-3, wherein administering to the leukemia patient comprises administering the effective amount of the TP53 reactivating indole derivative and the effective amount of the BCL-2 inhibitor.
6. The method of any one of claims 1-3, wherein administering to the leukemia patient comprises administering the effective amount of the TP53 reactivating indole derivative and the effective amount of the XPO-1 inhibitor.
7. The method of any one of claims 1-3, wherein administering to the leukemia patient comprises administering the effective amount of the TP53 reactivating indole derivative, the effective amount of the MDM2 inhibitor, and the effective amount of the BCL-2 inhibitor.
8. The method of any one of claims 1-3, wherein administering to the leukemia patient comprises administering the effective amount of the TP53 reactivating indole derivative, the effective amount of the MDM2 inhibitor, and the effective amount of the XPO-1 inhibitor.
9. The method of any one of claims 1-3, wherein administering to the leukemia patient comprises administering the effective amount of the TP53 reactivating indole derivative, the effective amount of the BCL-2 inhibitor, and the effective amount of the XPO-1 inhibitor.
10. The method of any one of claims 1-3, wherein administering to the leukemia patient comprises administering the effective amount of the TP53 reactivating indole derivative, the effective amount of the MDM2 inhibitor, the effective amount of the BCL-2 inhibitor, and the effective amount of the XPO-1 inhibitor.
11. The method of any one of claims 1-10, wherein the MDM2 inhibitor is selected from the group consisting of RG7112, RO5045337, idasanutlin, nutlin-3a, RG7388, AMG- 232, KRT-232, APG-115, BI-907828, CGM097, siremadlin, HDM201, milademetan, MEL23, MEL24, and DS-3032b.
12. The method of any one of claims 1-11, wherein the BCL-2 inhibitor is selected from the group consisting of venetoclax, obatoclax, subatoclax, maritoclax, gossypol, apogossypol, TW-37, UMI-77, and BDA-366.
13. The method of any one of claims 1-12, wherein the XPO-1 inhibitor is selected from the group consisting of KPT330, XPOVIO, KPT8602 (Eltanexor), KPT335, Verdinexor, and KPT185.
14. The method of any one of claims 1-13, wherein the MDM2 inhibitor, the BCL-2 inhibitor, and/or the XPO-1 inhibitor are sequentially, simultaneously, or separately administered with the TP53 reactivating indole derivative.
15. The method of any one of claims 1-14, wherein the MDM2 inhibitor, the BCL-2 inhibitor, the XPO-1 inhibitor, or the TP53 reactivating indole derivative is administered orally, intravenously, intramuscularly, intraperitoneally, or subcutaneously.
16. The method of any one of claims 1-15, wherein the leukemia is acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS).
17. The method of any one of claims 1-16, wherein the leukemia is TP53-Y220C leukemia.
18. The method of any one of claims 1-17, wherein the TP53 reactivating indole derivative has a formula of formula (I A)
Figure imgf000141_0001
wherein:
X1 is CR5, NR5, O, S, C=O, C=S, or a carbon atom connected to Q1;
X2 is CR7, CH, NR7, O, S, C=O, C=S, or a carbon atom connected to Q1;
X3 is CR9, CH, NR9, O, S, C=O, C=S, or a carbon atom connected to Q1;
X4 is CR11, CH, NR11, O, S, C=O, C=S, or a carbon atom connected to Q1;
X5 is CR13, CH, or NR13; wherein at least one of X1, X2, X3, and X4 is a carbon atom connected to Q1;
Q1 is a bond; m is 1, 2, 3, or 4;
Y is N or O;
R1 is -C(O)R16, -C(O)OR16, -C(O)NR16R17, -OR16, -R16OR17, -R16OR17R18, -SR16, - R16SR17, -R16SR17R18, -NR16R17, -R16NR17, -R16NR17R18, -NR16C(O)R16, - OC(O)R16, C=O, C=S, -CN, -SiR16R17R18, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; each R3 and R4 is independently -C(O)R19, -C(O)OR19, -C(O)NR19R20, -SOR19, - SO2R19, hydrogen, alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl, wherein each of alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl is independently unsubstituted or substituted with one or two groups selected from alkyl or halogen, or R3 and R4 together with the Y atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent; each R2, R5, R7, R9, R11, R13, R16, R17, and R18 is independently - C(O)R21, - C(O)OR21, -C(O)NR21R22, -OR21, -SR21, -NR21R22, -NR21C(O)R22, -OC(O)R21, hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, wherein each alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl is independently unsubstituted or substituted with one or two groups selected from -C(O)R25, - C(O)OR25, -C(O)NR25, -CR253, -OR25, -SR25, -NR25R26, -NR25C(O)R26, -OC(O)R25, alkyl, alkenyl, or alkynyl, wherein R25 and R26 are each independently alkyl, hydrogen, or halogen; each R19 and R20 is C(O)R23, -C(O)OR23, -C(O)NR23R24, -OR23, -SR23, -NR23R24, - NR23C(O)R24, -OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen; each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or a pharmaceutically-acceptable salt thereof.
19. The method of any one of claims 1-17, wherein the TP53 reactivating indole derivative has a formula of formula (IB)
Figure imgf000143_0001
wherein:
X1 is CR5, NR5, O, S, C=O, C=S, or a carbon atom connected to Y;
X2 is CR7, CH, NR7, O, S, C=O, C=S, or a carbon atom connected to Y;
X3 is CR9, CH, NR9, O, S, C=O, C=S, or a carbon atom connected to Y;
X4 is CR11, CH, NR11, O, S, C=O, C=S, or a carbon atom connected to Y;
X5 is CR13, CH, or NR13; wherein at least one of X1, X2, X3, and X4 is a carbon atom connected to Y;
Y is N or O;
R1 is -C(O)R16, -C(O)OR16, -C(O)NR16R17, -OR16, -R16OR17, -R16OR17R18, -SR16, - R16SR17, -R16SR17R18, -NR16R17, -R16NR17, -R16NR17R18, -NR16C(O)R16, - OC(O)R16, C=O, C=S, -CN, -SiR16R17R18, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; each R3 and R4 is independently -C(O)R19, -C(O)OR19, -C(O)NR19R20, -SOR19, - SO2R19, hydrogen, alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl, wherein each of alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl is independently unsubstituted or substituted with one or two groups selected from alkyl or halogen, or R3 and R4 together with the Y atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent; each R2, R5, R7, R9, R11, R13, R16, R17, and R18 is independently - C(O)R21, - C(O)OR21, -C(O)NR21R22, -CR253, -OR21, -SR21, -NR21R22, -NR21C(O)R22, - OC(O)R21, hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, wherein each of alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl is independently unsubstituted or substituted with one or two groups selected from -C(O)R25, -C(O)OR25, -C(O)NR25, -CR25 3, -OR25, -SR25, -NR25R26, - NR25C(O)R26, -OC(O)R25, alkyl, alkenyl, or alkynyl, wherein R25 and R26 are each independently alkyl, hydrogen, or halogen; each R19 and R20 is C(O)R23, -C(O)OR23, -C(O)NR23R24, -OR23, -SR23, -NR23R24, - NR23C(O)R24, -OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen; each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or a pharmaceutically-acceptable salt thereof.
20. The method of any one of claims 1-17, wherein the TP53 reactivating indole derivative has a formula
Figure imgf000144_0001
21. The method of any one of claims 3-20, wherein mRNA expression levels are detected via real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase- PCR (RT-PCR), Northern blotting, microarray, dot or slot blots, in situ hybridization, or fluorescent in situ hybridization (FISH).
22. The method of any one of claims 3-21, wherein polypeptide expression levels are detected via Western blotting, enzyme-linked immunosorbent assays (ELISA), dot blotting, immunohistochemistry, immunofluorescence, immunoprecipitation, immunoelectrophoresis, or mass-spectrometry.
23. The method of any one of claims 1-22, wherein the patient is non-responsive to at least one prior line of cancer therapy.
24. The method of claim 23, wherein the at least one prior line of cancer therapy is chemotherapy or immunotherapy.
25. The method of claim 24, wherein the chemotherapy comprises one or more of trioxide, azacytidine, cerubidine, cyclophosphamide, cytarabine, daunorubicin hydrochloride, daurismo, dexamethasone, doxorubicin hydrochloride, enasidenib mesylate, gemtuzumab ozogamicin, gilteritinib fumarate, glasdegib maleate, idamycin PFS, idarubicin hydrochloride, idhifa, ivosidenib, midostaurin, mitoxantrone hydrochloride, mylotarg, olutasidenib, onureg, pemazyre, pemigatinib, prednisone, rezlidhia, Rituxan, rituximab, rubidomycin, rydapt, tabloid, thioguanine, tibsovo, tisagenlecleucel, trisenox, venclexta, venetoclas, vinicristine sulfate, vyxeos, or xospata.
26. The method of any one of claims 1-25, wherein the patient is a child or adult.
27. The method of any one of claims 3-26, wherein the biological sample is whole blood, serum, or plasma.
PCT/US2024/034492 2023-06-19 2024-06-18 Combination therapy methods for treating tp53-y220c mutant and tp53 wildtype leukemias Pending WO2024263573A1 (en)

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US20130302248A1 (en) * 2009-03-23 2013-11-14 Eli Lilly And Company Imaging Agents for Detecting Neurological Disorders
US20220281970A1 (en) * 2018-12-20 2022-09-08 Novartis Ag Pharmaceutical Combinations
US20220315564A1 (en) * 2019-09-23 2022-10-06 Pmv Pharmaceuticals, Inc. METHODS AND COMPOUNDS FOR RESTORING MUTANT p53 FUNCTION
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