WO2020206035A1 - Traitement de troubles néoplasiques résistant aux inhibiteurs de cdk4/6 - Google Patents

Traitement de troubles néoplasiques résistant aux inhibiteurs de cdk4/6 Download PDF

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WO2020206035A1
WO2020206035A1 PCT/US2020/026263 US2020026263W WO2020206035A1 WO 2020206035 A1 WO2020206035 A1 WO 2020206035A1 US 2020026263 W US2020026263 W US 2020026263W WO 2020206035 A1 WO2020206035 A1 WO 2020206035A1
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inhibitor
cancer
cdk9
pharmaceutically acceptable
alkylene
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PCT/US2020/026263
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English (en)
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David Jung
Jay Copeland Strum
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G1 Therapeutics, Inc.
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Publication of WO2020206035A1 publication Critical patent/WO2020206035A1/fr

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    • 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
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • 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
    • A61K31/527—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim spiro-condensed
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains three hetero rings
    • C07D487/20—Spiro-condensed systems

Definitions

  • This invention is in the area of the treatment of disorders involving abnormal cellular proliferation, including but not limited to the treatment of cancers that have acquired resistance to, or are intrinsically resistant to, a selective cyclin dependent kinase 4/6 inhibitor by administering a cyclin dependent kinase 9 inhibitor (CDK9 inhibitor).
  • CDK9 inhibitor cyclin dependent kinase 9 inhibitor
  • Cell division sometimes referred to as the cell cycle, has four phases: Gi phase (synthesis of various enzymes required for DNA replication), S phase (DNA replication producing two identical sets of chromosomes), G2 (significant protein synthesis, including production of microtubules) and M phase (nuclear division, cytoplasmic division and formation of new cell membrane).
  • Gi phase synthesis of various enzymes required for DNA replication
  • S phase DNA replication producing two identical sets of chromosomes
  • G2 significant protein synthesis, including production of microtubules
  • M phase nuclear division, cytoplasmic division and formation of new cell membrane.
  • Cell division also includes a complex system of cell signaling networks that allow cells to interpret information from numerous extracellular signals, including through receptor proteins, inflammatory factors and pro-apoptotic and anti-apoptotic signals.
  • Dysfunctional signals include those from genetic mutation, infection, exposure to environmental factors including toxins, system stress, autoimmune disorders, and inflammation.
  • a range of disorders can occur when the process of cell proliferation becomes dysfunctional, including benign growths, neoplasms, tumorigenesis, cancerogenesis, autoimmune disorders, inflammatory disorders, graft-versus-host rejection, and fibrotic disorders.
  • a number of broad-spectrum anti -neoplastic agents have been developed. Cytoskeletal drugs like paclitaxel target tubulin to arrest mitotic cell division and are used to treat a variety of cancers including ovarian, breast, lung, pancreatic, and testicular tumors (See e.g., Jordan, Wilson, Nature Reviews Cancer (2004) 4: 253-265).
  • Organometallic-based drugs such as cisplatin have been used to treat lymphomas, sarcomas, germ cell tumors, and some carcinomas including bladder, small cell lung cancer, and ovarian cancer.
  • Cisplatin has the ability to bind nitrogenous bases and cause extensive DNA cross-linking that ultimately leads to apoptosis (See e.g., Siddick, Oncogene (2003) 22: 7265-7279).
  • Intercalating and alkylating agents have also been extensively used in the clinic for the treatment of various neoplasms, however, the global toxicity associated with these drugs presents a critical concern for patients requiring long-term therapy.
  • Palbociclib (PD-033299; Ibrance) is sold by Pfizer for the treatment of estrogen positive, HER2 -negative breast cancer in combination with letrozole.
  • the compound inhibits CDK4 and CDK6.
  • the structure of palbociclib is:
  • Abemaciclib (LY2835219) is a CDK 4/6 inhibitor currently in human clinical trials for the treatment of various types of cancers. It is in a phase III trial for stage IV non-small cell lung carcinoma; in combination with Fulvestrant for women with breast cancer; and with either anastrozole or letrozole for first line treatment of breast cancer.
  • the structure of abemaciclib is:
  • Ribociclib (LeeOl l; Kisqali), is a CDK 4/6 inhibitor approved for use in combination with an aromatase inhibitor to treat some metastatic breast cancers, and is in clinical trials for the treatment of certain other tumors.
  • the structure of ribociclib is:
  • Lerociclib is an oral, selective CDK4/6 inhibitor in clinical development by G1 Therapeutics for use in combination with other targeted therapies in multiple oncology indications.
  • Lerociclib is currently being evaluated in two Phase 1/2 clinical trials: a trial in combination with fulvestrant (Faslodex®) for patients with estrogen receptor-positive, HER2- negative (ER+, HER2-) breast cancer (NCT02983071) and a trial in combination with osmirtinib (Tagrisso®) in EGFRm non-small cell lung cancer.
  • Lerociclib has the structure:
  • Trilaciclib is a selective CDK4/6 inhibitor in clinical development by G1 Therapeutics for use as a first-in-class myelopreservation therapy designed to improve outcomes of patients who receive chemotherapy by preserving hematopoietic stem and progenitor cell (HSPC) and immune system function.
  • Trilaciclib is a short-acting intravenous CDK4/6 inhibitor administered prior to chemotherapy and is currently being evaluated in four randomized Phase 2 clinical trials, including in first-line SCLC trials in combination with a chemotherapy regimen of etoposide and carboplatin (NCT02499770); and in first-line SCLC trial in combination with the same chemotherapy regimen and the checkpoint inhibitor Tecentriq® (atezolizumab).
  • Trilaciclib has the structure:
  • SHR 6390 is a selective CDK4/6 inhibitor in clinical development by Jiangsu HengRui Medicine Co., Ltd. SHR6390 is currently being investigated in in combination with letrozole or anastrozole or fulvestrant in patients with HR-positive and HER2-negative advanced breast cancer. Various other pyrimidine-based agents have been developed for the treatment of hyperproliferative diseases.
  • WO 2013/148748 (U.S.S.N. 61/617,657) titled “Lactam Kinase Inhibitors”, WO 2013/163239 (U.S.S.N. 61/638,491) titled“Synthesis of Lactams” and WO 2015/061407 filed by Tavares and also assigned to G1 Therapeutics describes the synthesis of N-(heteroaryl)- pyrrolo[3,2-d]pyrimidin-2-amines and their use as lactam kinase inhibitors.
  • WO 2014/144326 filed by Strum et al. and assigned to G1 Therapeutics describes compounds and methods for protection of normal cells during chemotherapy using pyrimidine-based CDK4/6 inhibitors.
  • WO 2014/144596 filed by Strum et al. and assigned to G1 Therapeutics describes compounds and methods for protection of hematopoietic stem and progenitor cells against ionizing radiation using pyrimidine-based CDK4/6 inhibitors.
  • WO 2014/144847 filed by Strum et al. and assigned to G1 Therapeutics describes HSPC-sparing treatments of abnormal cellular proliferation using pyrimidine-based CDK4/6 inhibitors.
  • WO 2014/144740 filed by Strum et al.
  • WO 2015/161285 filed by Strum et al. and assigned to G1 Therapeutics describes tricyclic pyrimidine-based CDK inhibitors for use in radioprotection.
  • WO 2015/161287 filed by Strum et al. and assigned to G1 Therapeutics describes tricyclic pyrimidine-based CDK inhibitors for the protection of cells during chemotherapy.
  • WO 2015/161283 filed by Strum et al. and assigned to G1 Therapeutics describes tricyclic pyrimidine-based CDK inhibitors for use in HSPC-sparing treatments of RB-positive abnormal cellular proliferation.
  • WO 2015/161288 filed by Strum et al. and assigned to G1 Therapeutics describes tricyclic pyrimidine-based CDK inhibitors for use as anti-neoplastic and anti-proliferative agents.
  • WO 2016/040858 filed by Strum et al. and assigned to G1 Therapeutics describes the use of combinations of pyrimidine-based CDK4/6 inhibitors with other anti-neoplastic agents.
  • WO 2016/040848 filed by Strum et al. and assigned to G1 Therapeutics describes compounds and methods for treating certain Rb-negative cancers with CDK4/6 inhibitors and topoisomerase inhibitors.
  • WO 2018/005860, WO 2018/005533, and WO 2018/005863 filed by Strum and assigned to G1 Therapeutics describes various CDK inhibitors.
  • WO 2018/106739 filed by Sorrentino et al, and assigned to G1 Therapeutics describes the use of CDK4/6 inhibitors with specific dosage regimens.
  • WO 2018/156812 filed by Strum et al, and assigned to G1 Therapeutics describes the use of CDK4/6 inhibitors to treat EGFR-driven cancer.
  • WO 2019/199883 filed by Strum et al. and assigned to G1 Therapeutics describes compounds and methods for treating chemotherapy resistant cancer.
  • WO 2019/136451 filed by Beelen et al. and assigned to G1 Therapeutics describes dosage regimes for the administration of G1T38.
  • WO 2019/136244 filed by Strum et al. and assigned to G1 Therapeutics describes additional compounds for inhibiting CDKs.
  • the present invention provides advantageous methods to treat a patient with selective CDK4/6 inhibitor resistant cancer, including a cancer that has developed acquired resistance to a CDK4/6 inhibitor, which includes administering to the patient an effective amount of a CDK9 inhibitor, including but not limited to one described herein.
  • CDK9/Cyclin T1 is a regulator of transcription in eukaryotic cells and has been shown to be dysregulated at the level of protein and kinase activity in both hematologic and solid tumors.
  • CDK9/CyclinTl forms the active P-TEFb complex and phosphorylates Ser2 residues in the carboxy -terminal domain of RNA polymerase II (RNApol II) to initiate elongation of mRNA transcripts (see Fig.1).
  • RNApol II RNA polymerase II
  • CDK9 activity regulates transcription of a variety of short-lived transcripts that promote survival and directly suppress apoptosis in cancer cells, including MYC, CCNE, XRN2, MCL-1, and XIAP. It has been discovered that compounds capable of inhibiting CDK9 activity are capable of inducing G2 cell cycle arrest and apoptosis in a concentration and time-dependent manner in tumor cells independent of Rb-status.
  • cancers initially susceptible to selective CDK4/6 inhibitor inhibition may acquire resistance to a selective CDK4/6 inhibitor during the course of selective CDK4/6 inhibitor therapy, for example by upregulation of cyclin E and other genetic and phenotypic changes following the onset of treatment which allows G1 to S cell cycle progression through CDK2.
  • a CDK9 inhibitor provides an effective treatment for patients with cancers that have developed selective CDK4/6 inhibitor resistance over time during treatment with a selective CDK4/6 inhibitor by still allowing cell-cycle arrest in G2.
  • the methods described herein using a CDK9 inhibitor to treat a patient with a cancer initially responsive to selective CDK4/6 inhibition can extend the efficacy of cell-cycle inhibition therapy in these patients.
  • a method of treating a patient with cancer by administering a therapeutically effective amount of a CDK9 inhibitor, wherein the patient has previously received a selective CDK4/6 inhibitor, and the cancer has become selective CDK4/6 inhibitor resistant.
  • a CDK9 inhibitor following the development of selective CDK4/6 inhibitor resistance, the current methods allow continued use of a cell-cycle inhibitor to treat the cancer.
  • the CDK9 inhibitor is selected from (rel)-MC 180295, NVP-2, AZD4573, PHA-767491, LDC00067, atuveciclib (BAY 1143572), CDKi-73 (LS-007), CDK9-IN-1, CDK-IN-2, JSH-150, CDK9-IN-8, FIT- 039, LY2857785, SNS-032 (BMS-387032), or TG02, or a pharmaceutically acceptable salt thereof.
  • the CDK9 inhibitor is selected from a compound of Formula I, II, III, IV, V, or VI, or a pharmaceutically acceptable salt thereof.
  • the CDK9 inhibitor is selected from Compound 1 to Compound 43, or a pharmaceutically acceptable salt thereof. In some embodiments, the CDK9 inhibitor is Compound 1, or a pharmaceutically acceptable salt thereof. In some embodiments, the CDK9 inhibitor is selected from a CDK9 inhibitor described herein.
  • a method of treating a patient with cancer which includes:
  • the indicator of resistance is disease progression.
  • the CDK4/6 inhibitor administered is selected from, but are not limited to, palbociclib, abemaciclib, ribociclib, trilaciclib, SHR6390, or lerociclib.
  • the CDK9 inhibitor administered upon the detection of the patient’s cancer becoming resistant to the selective CDK4/6 inhibitor is selected (rel)-MC 180295, NVP-2, AZD4573, PHA-767491, LDC00067, atuveciclib (BAY 1143572), CDKi-73 (LS-007), CDK9-IN-1, CDK-IN-2, JSH-150, CDK9-IN-8, FIT-039, LY2857785, SNS-032 (BMS-387032), or TG02.
  • the CDK9 inhibitor administered upon the detection of the patient’s cancer becoming resistant to the selective CDK4/6 inhibitor is selected from a compound of Formula I, II, III, IV, V, or VI.
  • the CDK9 inhibitor administered upon the detection of the patient’s cancer becoming resistant to the selective CDK4/6 inhibitor is selected from Compounds 1 to 43. In some embodiments, the CDK9 inhibitor administered upon the detection of the patient’s cancer becoming resistant to the selective CDK4/6 inhibitor is Compound 1. In some embodiments, the CDK9 inhibitor administered upon the detection of the patient’s cancer becoming resistant to the selective CDK4/6 inhibitor is a CDK9 inhibitor described herein.
  • a method of treating a patient with cancer which includes:
  • one or more cellular signals indicating the development of selective CDK4/6 inhibitor resistance in the cancer is selected from increased activity of cyclin-dependent kinase 1 (CDK1); increased activity of cyclin-dependent kinase 2 (CDK2); loss, deficiency, or absence of retinoblastoma tumor suppressor protein (Rb)(Rb-null); high levels of pl6Ink4a expression; high levels of MYC expression; increased expression of cyclin El, cyclin E2, and/or cyclin A; CCNEl/2 amplification; E2F amplification; CDK2 amplification; amplification of CDK6; amplification of CDK4; pi 6 amplification; WEE1 overexpression; MDM2 overexpression; CDK7 overexpression; loss of FZR1; HD AC activation; activation of
  • the selective CDK4/6 inhibitor administered is selected from, but not limited to, palbociclib, abemaciclib, ribociclib, trilaciclib, SHR6390, or lerociclib.
  • the CDK9 inhibitor administered if one or more cellular signals indicate the development of selective CDK4/6 inhibitor resistance in the cancer is selected from (rel)-MC 180295, NVP-2, AZD4573, PHA-767491, LDC00067, atuveciclib (BAY 1143572), CDKi-73 (LS-007), CDK9-IN-1, CDK-IN-2, JSH-150, CDK9-IN-8, FIT- 039, LY2857785, SNS-032 (BMS-387032), or TG02.
  • the CDK9 inhibitor administered if one or more cellular signals indicate the development of selective CDK4/6 inhibitor resistance in the cancer is selected from a compound of Formula I, II, III, IV, V, or VI. In some embodiments, the CDK9 inhibitor administered if one or more cellular signals indicate the development of selective CDK4/6 inhibitor resistance in the cancer is selected from Compounds 1 to 43. In some embodiments, the CDK9 inhibitor administered if one or more cellular signals indicate the development of selective CDK4/6 inhibitor resistance in the cancer is Compound 1. In some embodiments, the CDK9 inhibitor administered if one or more cellular signals indicate the development of selective CDK4/6 inhibitor resistance in the cancer is a CDK9 inhibitor described herein.
  • a method of treating a patient with an Rb- positive cancer which includes:
  • the selective CDK4/6 inhibitor administered is selected from, but not limited to, palbociclib, abemaciclib, ribociclib, trilaciclib, SHR6390, or lerociclib.
  • the CDK9 inhibitor administered upon the detection of an increase in cyclin E levels that confers resistance upon or no longer renders the cancer susceptible to the inhibitory effects of the selective CDK4/6 inhibitor is selected from (rel)-MC 180295, NVP-2, AZD4573, PHA-767491, LDC00067, atuveciclib (BAY 1143572), CDKi-73 (LS-007), CDK9-IN-1, CDK-IN-2, JSH-150, CDK9-IN-8, FIT-039, LY2857785, SNS-032 (BMS- 387032), or TG02.
  • the CDK9 inhibitor administered upon the detection of an increase in cyclin E levels that confers resistance upon or renders the cancer susceptible to the inhibitory effects of the selective CDK4/6 inhibitor is selected from a compound of Formula I, II, III, IV, V, or VI. In some embodiments, the CDK9 inhibitor administered upon the detection of an increase in cyclin E levels that confers resistance upon or renders the cancer susceptible to the inhibitory effects of the selective CDK4/6 inhibitor is selected from Compounds 1 to 43. In some embodiments, the CDK9 inhibitor administered upon the detection of an increase in cyclin E levels that confers resistance upon or renders the cancer susceptible to the inhibitory effects of the selective CDK4/6 inhibitor is Compound 1. In some embodiments, the CDK9 inhibitor administered upon the detection of an increase in cyclin E levels that confers resistance upon or renders the cancer susceptible to the inhibitory effects of the selective CDK4/6 inhibitor is a CDK9 inhibitor described herein.
  • the subject following the development of acquired resistance in a cancer, is administered a CDK9 inhibitor for an initial time period, for example, at least 3 days, at least 5 days, at least 7 days, at least 10 days, at least 14 days, at least 21 days, at least 24 days, at least 28 days, or more, and, following the initial time period of CDK9 inhibitor administration, a CDK4/6 inhibitor is re-administered to the subject.
  • a CDK4/6 inhibitor upon re-administration of the CDK4/6 inhibitor, the CDK9 inhibitor is no longer administered.
  • the CDK9 inhibitor upon re-administration of the CDK4/6 inhibitor, is also administered.
  • CDK9 inhibitors are capable of inducing cell cycle arrest, inhibit proliferation, and induce apoptosis in cancers that are intrinsically resistant to selective CDK4/6 inhibition, for example but not limited to TNBC, which may be intrinsically resistant to currently commercially available selective CDK4/6 inhibitors.
  • Certain cancers that have an intact Rb-pathway may otherwise be intrinsically resistant to a selective CDK4/6 inhibitor due to the presence of other genetic or phenotypical abnormalities. For example, it is estimated that 40% of uterine, 20% of ovarian, 15% of bladder, 20% or prostate, and 15% of breast cancers may be intrinsically resistant to selective CDK4/6 inhibition due to the up regulation of Cyclin E, despite intact Rb.
  • CDK9 inhibitor to treat a patient with an intrinsically selective CDK4/6 inhibitor resistant cancer can drastically expand the population of cancer patients responsive to cell-cycle check point inhibitor therapy.
  • a CDK9 inhibitor is used to treat a patient with a cancer intrinsically resistant to selective CDK4/6 inhibition.
  • the CDK9 inhibitor is selected from (rel)-MC180295, NVP-2, AZD4573, PHA-767491, LDC00067, atuveciclib (BAY 1143572), CDKi-73 (LS-007), CDK9-IN-1, CDK-IN-2, JSH-150, CDK9- IN-8, FIT-039, LY2857785, SNS-032 (BMS-387032), or TG02, or a pharmaceutically acceptable salt thereof.
  • the CDK9 inhibitor is selected from a compound of Formula I, II, III, IV, V, or VI, or a pharmaceutically acceptable salt thereof. In some embodiments, the CDK9 inhibitor is selected from Compound 1 to Compound 43, or a pharmaceutically acceptable salt thereof. In some embodiments, the CDK9 inhibitor is Compound 1, or a pharmaceutically acceptable salt thereof. In some embodiments, the CDK9 inhibitor is selected from a CDK9 inhibitor described herein.
  • a method of treating a patient with cancer which includes:
  • the selective CDK4/6 inhibitor administered is selected from, but are not limited to, palbociclib, abemaciclib, ribociclib, trilaciclib, SHR6390, or lerociclib.
  • the CDK9 inhibitor administered is selected from (rel)-MC 180295, NVP-2, AZD4573, PHA-767491, LDC00067, atuveciclib (BAY 1143572), CDKi-73 (LS-007), CDK9-IN-1, CDK-IN-2, JSH-150, CDK9-IN-8, FIT-039, LY2857785, SNS-032 (BMS-387032), or TG02.
  • the CDK9 inhibitor administered is a compound selected from Formula I, II, III, IV, V, or VI.
  • the CDK9 inhibitor administered is selected from Compounds 1 to 43. In some embodiments, if the cancer’s Rb-status is negative, the CDK9 inhibitor administered is Compound 1. In some embodiments, if the cancer’s Rb-status is negative, the CDK9 inhibitor administered is a CDK9 inhibitor described herein. In one alternative aspect, provided herein is a method of treating a patient with cancer which includes:
  • one or more cellular signals indicating the intrinsic selective CDK4/6 inhibitor resistance in the cancer is selected from increased activity of cyclin-dependent kinase 1 (CDK1); increased activity of cyclin-dependent kinase 2 (CDK2); loss, deficiency, or absence of retinoblastoma tumor suppressor protein (Rb)(Rb-null); high levels of pl6Ink4a expression; high levels of MYC expression; increased expression of cyclin El, cyclin E2, and cyclin A; CCNEl/2 amplification; E2F amplification; CDK2 amplification; amplification of CDK6; amplification of CDK4; pi 6 amplification; WEE1 overexpression; MDM2 overexpression; CDK7 overexpression; loss of FZR1; HD AC activation; activation of the FG
  • CDK1 cyclin-dependent kinase 1
  • CDK2 cyclin-dependent kinase 2
  • Rb retino
  • the selective CDK4/6 inhibitor administered to the patient if one or more cellular signals are not indicative of the cancer’s susceptibility to being intrinsically resistant to a selective CDK4/6 inhibitor is selected from, but are not limited to, palbociclib, abemaciclib, ribociclib, trilaciclib, SHR6390, or lerociclib.
  • the CDK9 inhibitor administered to the patient if one or more cellular signals are indicative of the cancer’s susceptibility to being intrinsically resistant to a selective CDK4/6 inhibitor is selected from (rel)-MC180295, NVP-2, AZD4573, PHA-767491, LDC00067, atuveciclib (BAY 1143572), CDKi-73 (LS-007), CDK9-IN-1, CDK-IN-2, JSH-150, CDK9- IN-8, FIT-039, LY2857785, SNS-032 (BMS-387032), or TG02.
  • the CDK9 inhibitor administered to the patient if one or more cellular signals are indicative of the cancer’s susceptibility to being intrinsically resistant to a selective CDK4/6 inhibitor is a compound selected from Formula I, II, III, IV, V, or VI. In some embodiments, the CDK9 inhibitor administered to the patient if one or more cellular signals are indicative of the cancer’s susceptibility to being intrinsically resistant to a selective CDK4/6 inhibitor is selected from Compounds 1 to 43. In some embodiments, the CDK9 inhibitor administered to the patient if one or more cellular signals are indicative of the cancer’s susceptibility to being intrinsically resistant to a selective CDK4/6 inhibitor is Compound 1. In some embodiments the CDK9 inhibitor administered to the patient if one or more cellular signals are indicative of the cancer’s susceptibility to being intrinsically resistant to a selective CDK4/6 inhibitor is a CDK9 inhibitor described herein.
  • a method for the treatment of a disorder of abnormal cellular proliferation in a host such as a human includes administering an effective amount of a CDK9 inhibitor in combination or alternation with an additional active compound, wherein the disorder is resistant to the inhibitory effects of a selective CDK4/6 inhibitor.
  • the additional active compound is a chemotherapeutic agent.
  • the additional active compound is an immune modulator, including but not limited to a checkpoint inhibitor such as an anti- PD1, anti-PD-Ll, anti-CTLA, anti-LAG-3, anti-Tim, etc.
  • antibody small molecule, peptide, nucleotide or other inhibitor (including but not limited to ipilimumab (Yervoy), pembrolizumab (Keytruda) nivolumab (Opdivo), cemiplimab (Libtayo), atezolizumab (Tecentriq), avelumab (Bavencio), and durvalumab (Imfmzi).
  • ipilimumab Yervoy
  • pembrolizumab Keytruda
  • nivolumab Opdivo
  • cemiplimab Libtayo
  • atezolizumab Tecentriq
  • avelumab Bavencio
  • durvalumab Imfmzi
  • the additional active compound is selected from elotuzumab rituximab, lenalidomide, cytarabine, datatumumab, adalimumab, idealisib, gilteritinib, glasdegib, valaciclovir, acalabrutinib, ibrutinib, midostaurin, ruxolitinib, bortezomib, lapatinib, bendamstine, enzalutamide, azacitadine, obinutuzumab, decitabine, erdafitinib, or venetoclax.
  • the CDK9 inhibitor is selected from (rel)-MC 180295, NVP-2, AZD4573, PHA-767491, LDC00067, atuveciclib (BAY 1143572), CDKi-73 (LS-007), CDK9-IN-1, CDK-IN-2, JSH- 150, CDK9-IN-8, FIT-039, LY2857785, SNS-032 (BMS-387032), or TG02, or pharmaceutically acceptable salts thereof.
  • the CDK9 inhibitor is selected from a compound of Formula I, II, III, IV, V, or VI.
  • the CDK9 inhibitor is selected from Compounds 1 to 43.
  • the CDK9 inhibitor is Compound 1.
  • the CDK9 inhibitor is a CDK9 inhibitor described herein.
  • a CDK9 inhibitor is administered in an effective amount for the treatment of selective CDK4/6 inhibitor resistant abnormal tissue of the female reproductive system such as breast, ovarian, endometrial, or uterine cancer, in combination or alternation with an effective amount of an estrogen inhibitor including but not limited to a SERM (selective estrogen receptor modulator), a SERF) (selective estrogen receptor degrader), a complete estrogen receptor degrader, or another form of partial or complete estrogen antagonist.
  • an estrogen inhibitor including but not limited to a SERM (selective estrogen receptor modulator), a SERF) (selective estrogen receptor degrader), a complete estrogen receptor degrader, or another form of partial or complete estrogen antagonist.
  • the CDK9 inhibitor is selected from (rel)-MCl 80295, NVP- 2, AZD4573, PHA-767491, LDC00067, atuveciclib (BAY 1143572), CDKi-73 (LS-007), CDK9-IN-1, CDK-IN-2, JSH-150, CDK9-IN-8, FIT-039, LY2857785, SNS-032 (BMS- 387032), or TG02.
  • the CDK9 inhibitor is selected from a compound of Formula I, II, III, IV, V, or VI.
  • the CDK9 inhibitor is selected from Compounds 1 to 43.
  • the CDK9 inhibitor is Compound 1.
  • the CDK9 inhibitor is a CDK9 inhibitor described herein.
  • a CDK9 inhibitor is administered in an effective amount for the treatment of selective CDK4/6 inhibitor resistant of abnormal tissue of the male reproductive system such as prostate or testicular cancer, in combination or alternation with an effective amount of an androgen (such as testosterone) inhibitor including but not limited to a selective androgen receptor modulator, a selective androgen receptor degrader, a complete androgen receptor degrader, or another form of partial or complete androgen antagonist.
  • an androgen such as testosterone
  • the prostate or testicular cancer is androgen-resistant.
  • the CDK9 inhibitor is selected from (rel)-MC180295, NVP-2, AZD4573, PHA- 767491, LDC00067, atuveciclib (BAY 1143572), CDKi-73 (LS-007), CDK9-IN-1, CDK-IN- 2, JSH-150, CDK9-IN-8, FIT-039, LY2857785, SNS-032 (BMS-387032), or TG02.
  • the CDK9 inhibitor is selected from a compound of Formula I, II, III, IV, V, or VI.
  • the CDK9 inhibitor is selected from Compounds 1 to 43.
  • the CDK9 inhibitor is Compound 1.
  • the CDK9 inhibitor is a CDK9 inhibitor described herein.
  • a CDK9 inhibitor is administered in an effective amount for the treatment of a selective CDK4/6 inhibitor resistant hematological cancer in combination with a Bruton’s Tyrosine Kinase (BTK) inhibitor, for example, but not limited to ibrutinib (Imbruvica®) or acalabrutinib (Calquence®).
  • BTK Tyrosine Kinase
  • a CDK9 inhibitor is administered in an effective amount of a selective CDK4/6 inhibitor resistant cancer in combination with an EGFR inhibitor.
  • the CDK9 inhibitor is selected from (rel)-MC 180295, NVP-2, AZD4573, PHA-767491, LDC00067, atuveciclib (BAY 1143572), CDKi-73 (LS-007), CDK9-IN-1, CDK-IN-2, JSH-150, CDK9-IN-8, FIT-039, LY2857785, SNS-032 (BMS-387032), or TG02.
  • the CDK9 inhibitor is selected from a compound of Formula I, II, III, IV, V, or VI.
  • the CDK9 inhibitor is selected from Compounds 1 to 43.
  • the CDK9 inhibitor is Compound 1.
  • the CDK9 inhibitor is a CDK9 inhibitor described herein.
  • the CDK9 inhibitor administered is selected from (rel)-MC 180295, NVP-2, AZD4573, PHA-767491, LDC00067, atuvecicbb (BAY 1143572), CDKi-73 (LS-007), CDK9-IN-1, CDK-IN-2, JSH-150, CDK9- IN-8, FIT-039, LY2857785, SNS-032 (BMS-387032), or TG02, or a pharmaceutically acceptable salt thereof.
  • the CDK9 inhibitor is selected from a compound of Formula I, II, III, IV, V, or VI, or a pharmaceutically acceptable salt thereof.
  • the CDK9 inhibitor administered is selected from Compounds 1 to 43, or a pharmaceutically acceptable salt thereof.
  • the CDK9 inhibitor is a CDK9 inhibitor described herein.
  • the CDK9 inhibitor is (2'-(((lR,4R)-4-(4- (cyclopropylmethyl)piperazin-l-yl)cyclohexyl)amino)-7',8'-dihydro-6'H-spiro[cyclohexane- l,9'-pyrazino[r,2': l,5]pyrrolo[2,3-d]pyrimidin]-6'-one), which has the structure:
  • Compound 1 or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof.
  • the CDK9 inhibitor is selected from a compound of Formula I, II, III, IV, V, or VI, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof.
  • the CDK9 inhibitor administered is selected from Compounds 1 to 5 (described below in Table 1), or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof.
  • the CDK9 inhibitor administered is selected from Compounds 6 to 39 (described below in Table 2A) or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof.
  • the CDK9 inhibitor administered is selected from Compounds 40 to 43 (described below in Table 2B) or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof.
  • the CDK9 inhibitor administered is selected from a compound described herein, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof.
  • FIG. 1A is a graphical depiction of the active CDK9/Cyclin T1 (P-TEFb) function in RNA polymerase II-mediated transcription elongation.
  • FIG. IB is a graphical representation of pro-tumor signaling events that occur downstream of CDK9/Cyclin T1 activity.
  • FIG. 2 is a graphical depiction of all kinases inhibited greater than 80% by Compound 1 at a concentration of 100 nM as discussed in Example 1.
  • CDK9 is represented by the labeled circle.
  • FIG. 3 is a measure of the caspase 3/7 activity in HCC1806, BT549, Hs68, and MCF7 cell lines after 24 hours of incubation with increasing concentrations of Compound 1 as described in Example 2.
  • the x-axis is the concentration of Compound 1 measured in molarity (M) expressed as the log[M]
  • the y-axis is the luminescence measured in RLU.
  • FIG. 4B is a Western Blot analysis of HCC1806 cells treated with increasing concentrations of Compound 1 for 24 hours as described in Example 3.
  • FIG. 4C is a Western Blot analysis of BT549 cells treated with increasing concentrations of Compound 1 for 24 hours as described in Example 3.
  • FIG. 5A is a graph of the percentage of HCC1806 cells in the G0-G1 phase, the S phase, the G2 phase, and the M phase after increasing concentrations of Compound 1 as represented in Table 4 and described in Example 4.
  • the x-axis is the concentration of Compound 1 measured in nM and the y-axis is the percentage of the total cell population in each cell cycle phase.
  • FIG. 5B is a graph of the percentage of Hs68 cells in the G0-G1 phase, the S phase, the G2 phase, and the M phase after increasing concentrations of Compound 1 as represented in Table 5 and described in Example 4.
  • the x-axis is the concentration of Compound 1 measured in nM and the y-axis is the percentage of the total cell population in each cell cycle phase.
  • FIG. 5C is a representative flow gating schematic using the FxCycle DNA stain, Click-iTTM Edu to measure cellular proliferation of untreated HCC1806 cells as described in Example 4.
  • the y-axis is the Alexa Fluor 488-A Edu fluorescence.
  • the x-axis is the allophycocyanin (APC) fluorescence.
  • FIG. 5D is a representative flow gating schematic using the FxCycle DNA stain, Click- iTTM Edu to measure cellular proliferation of HCC1806 cells treated with Compound 1 as described in Example 4.
  • the y-axis is the Alexa Fluor 488-A Edu fluorescence.
  • the x-axis is the allophycocyanin (APC) fluorescence.
  • FIG. 5E is a representative flow gating schematic using Phospho-Histone H3 conjugated antibody to measure cellular proliferation of untreated HCC1806 cells as described in Example 4.
  • the y-axis is the Pacific Blue A Phospho-Histone H3 fluorescence.
  • the x-axis is the allophycocyanin (APC) fluorescence.
  • FIG. 5F is a representative flow gating schematic using Phospho-Histone H3 conjugated antibody to measure cellular proliferation of HCC1806 cells treated with Compound 1 as described in Example 4.
  • the y-axis is the Pacific Blue A Phospho-Histone H3 fluorescence.
  • the x-axis is the allophycocyanin (APC) fluorescence to measure DNA content.
  • FIG. 6 is an image of MCF7 parental cells (left) and MCF7 palbociclib-resistant cells (right) developed for four months as described in Example 5.
  • the MCF7 palbociclib-resistant cells were maintained in complete media plus palbociclib for three months at IC90 (750nM) followed by one month at 1 mM.
  • FIG. 7A is the pairwise comparison of transcript level in MCF7 palbociclib-resistant cells vs. control as described in Example 5. Genes above the dashed line were differentially expressed (6,039 genes out of 17,383), with adjusted p-value of 0.05. The x-axis is fold change expressed as log2. The y-axis is -logio(adjusted p value). The horizontal dashed line is - logio(0.05) and all points above the dashed line are statistically significant.
  • FIG. 7B is a graph of the fold change (expressed as log2) of specific genes in MCF7 palbociclib-resistant cells vs. control as described in Example 5.
  • the x-axis is fold change expressed as log 2 and the y-axis is labeled with the specific genes.
  • FIG. 7C are Western Blots of MCF7 control and MCF7 palbociclib-resistant cells that show the expression of Cyclin E and Rb (GAPDH is the control). The expression levels are also plotted on the graph below. As described in Example 5, there is an increase in the ratio of Cyclin E to Rb levels in the palbociclib-resistant cells compared to the control.
  • FIG. 8A is a graph of the percentage of MCF7 cells in the G0-G1 phase, the S phase, the G2 phase, and the M phase after increasing concentrations of Compound 1 as represented in Table 6 and described in Example 5.
  • the x-axis is the concentration of Compound 1 measured in nM and the y-axis is the percentage of the total cell population in each cell cycle phase.
  • FIG. 8B is a graph of the percentage of MCF7 palbo-R cells in the G0-G1 phase, the S phase, the G2 phase, and the M phase after increasing concentrations of Compound 1 as represented in Table 7 and described in Example 5.
  • the x-axis is the concentration of Compound 1 measured in nM and the y-axis is the percentage of the total cell population in each cell cycle phase.
  • FIG. 8C is a concentration curve comparing Compound 1 and palbociclib in the 6-Day CellTiter Glo assay in MCF7 parental cells as described in Example 5.
  • Compound 1 is inhibiting cell proliferation independent of CD4/6 (palbociclib is a selective CDK4/6 inhibitor).
  • the x-axis is concentration measured in molarity (M) expressed as the log[M]
  • the y-axis is the luminescence measured in RLU.
  • FIG. 8D is a concentration curve comparing Compound 1 and palbociclib in the 6-Day CellTiter Glo assay in MCF7 palbo-R cells as described in Example 5.
  • Compound 1 is inhibiting cell proliferation independent of CD4/6 (palbociclib is a selective CDK4/6 inhibitor).
  • the x-axis is concentration measured in molarity (M) expressed as the log[M]
  • the y-axis is the luminescence measured in RLU.
  • FIG. 9A is a Western Blot of pRPBT CTD (Ser 2) expression in MCF7 cells that have been treated with increasing concentrations of Compound 1 as described in Example 5.
  • FIG. 9B is a Western Blot of pRPBT CTD (Ser 2) expression in MCF7 palbo-R cells that have been treated with increasing concentrations of Compound 1 as described in Example 5.
  • FIG. 9C is a Western Blot of Rb and Cyclin E expression in MCF7 cells that have been treated with increasing concentrations of Compound 1 as described in Example 5.
  • FIG. 9D is a Western Blot of Rb and Cyclin E expression in MCF7 palbo-R cells that have been treated with increasing concentrations of Compound 1 as described in Example 5.
  • Compound 1 may include at least one desired isotopic substitution of an atom, at an amount above the natural abundance of the isotope, i.e., enriched.
  • Isotopes are atoms having the same atomic number but different mass numbers, i.e., the same number of protons but a different number of neutrons.
  • isotopes examples include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine and iodine such as 2 H, 3 H, n C, 13 C, 14 C, 15 N, 17 0, 18 0, 18 F 31 P, 32 P, 35 S, 36 CI, and 125 I respectively.
  • isotopically labelled compounds can be used in metabolic studies (with 14 C), reaction kinetic studies (with, for example 2 H or 3 H), detection or imaging techniques, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays, or in radioactive treatment of patients.
  • PET positron emission tomography
  • SPECT single-photon emission computed tomography
  • an 18 F labeled compound may be particularly desirable for PET or SPECT studies.
  • Isotopically labeled compounds of this invention and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and preparations described below by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.
  • isotopes of hydrogen for example, deuterium ( 2 H) and tritium ( 3 H) may be used anywhere in described structures that achieves the desired result.
  • isotopes of carbon e.g., 13 C and 14 C, may be used.
  • Isotopic substitutions for example deuterium substitutions, can be partial or complete. Partial deuterium substitution means that at least one hydrogen is substituted with deuterium.
  • the isotope is 90, 95 or 99% or more enriched in an isotope at any location of interest. In one non-limiting embodiment, deuterium is 90, 95 or 99% enriched at a desired location.
  • the substitution of one or more hydrogen atoms for a deuterium atoms can be provided in the CDK9 inhibitor described herein.
  • a CDK9 inhibitor may form a solvate with solvents (including water). Therefore, in one non-limiting embodiment, a CDK9 inhibitor includes a solvated form of the compound.
  • solvate refers to a molecular complex of a CDK9 inhibitor (including a salt thereol) with one or more solvent molecules.
  • solvents are water, ethanol, dimethyl sulfoxide, acetone and other common organic solvents.
  • hydrate refers to a molecular complex comprising a CDK9 inhibitor and water.
  • Pharmaceutically acceptable solvates in accordance with the invention include those wherein the solvent may be isotopically substituted, e.g. D2O, d6-acetone, d6-DMSO.
  • a solvate can be in a liquid or solid form.
  • CDK1 cyclin-dependent kinase 1
  • CDK2 cyclin-dependent kinase 2
  • Rb retinoblastoma tumor suppressor protein
  • the cancer may be characterized by reduced expression of the retinoblastoma tumor suppressor protein or a retinoblastoma family member protein or proteins (such as, but not limited to pi 07 and pi 30).
  • a tumor or cancer that is intrinsically resistant to selective CDK4/6 inhibitor inhibition is a tumor or cancer whose cell population, as a whole, does not experience substantial G1 cell-cycle arrest when exposed to a selective CDK4/6 inhibitor.
  • a tumor or cancer that is intrinsically resistant to CDK4/6 inhibitor inhibition is a tumor or cancer who has a cell population wherein less than 25%, 20%, 15%, 10%, or 5% of its cells experience G1 cell-cycle arrest when exposed to a selective CDK4/6 inhibitor.
  • “Acquired resistance,” as used herein, refers to a condition wherein a cancer that was or is initially sensitive to the inhibitory effects of at least one selective CDK4/6 inhibitor becomes non-responsive or less-responsive over time to the effects of that selective CDK4/6 inhibitor. Without wishing to be bound by any one theory, it is believed that acquired resistance to a CDK4/6 inhibitor occurs due to one or more additional mutations or genetic alterations in bypass signaling that develops after the onset of CDK4/6 inhibitor treatment regimen.
  • non-limiting exemplary causes of acquired resistance to a CDK4/6 inhibitor may be a result of: the development of one or more genetic aberrations associated with“intrinsic resistance.”
  • other non-limiting exemplary causes of acquired resistance to a CDK4/6 inhibitor may include an increase in cyclin E expression; CCNEl/2 amplification; E2F amplification; CDK2 amplification; amplification of CDK6; amplification of CDK4; pi 6 amplification; WEE1 overexpression; MDM2 overexpression; CDK7 overexpression; loss of FZR1; HD AC activation; activation of the FGFR pathway; activation of the PI3K/AKT/mTOR pathway; loss of ER or PR expression; higher transcriptional activity of AP-1; epithelial- mesenchymal transition; Smad 3 suppression; autophagy activation; Rbl-loss or inactivating RBI mutations; or a combination thereof
  • CDK4/6 resistant mechanisms can be found, for example, in
  • a tumor or cancer that has acquired resistance to selective CDK4/6 inhibitor inhibition is a tumor or cancer whose cell population, as a whole, no longer experiences substantial G1 cell-cycle arrest when exposed to a selective CDK4/6 inhibitor, resulting in disease progression.
  • a tumor or cancer that has acquired resistance to CDK4/6 inhibitor inhibition is a tumor or cancer who has a cell population wherein less than 50%, 40%, 30% 20%, 15%, 10%, or 5% of its cells experience G1 cell-cycle arrest when exposed to a selective CDK4/6 inhibitor, leading to disease progression.
  • CDK4/6 inhibitor used in the context of the compounds described herein includes compounds that inhibit CDK4 activity, CDK6 activity, or both CDK4 and CDK6 activity at an IC50 molar concentration at least about 300, or 400, or 500, or 1000, or 1500, or 1800, or 2000, or 5000 or 10,000 times less than the IC50 molar concentration necessary to inhibit to the same degree of CDK2 activity in a standard phosphorylation assay.
  • the patient treated is typically a human patient, although it is to be understood the methods described herein are effective with respect to other animals, such as mammals. More particularly, the term patient can include animals used in assays such as those used in preclinical testing including but not limited to mice, rats, monkeys, dogs, pigs and rabbits; as well as domesticated swine (pigs and hogs), ruminants, equine, poultry, felines, bovines, murines, canines, and the like.
  • animals used in assays such as those used in preclinical testing including but not limited to mice, rats, monkeys, dogs, pigs and rabbits; as well as domesticated swine (pigs and hogs), ruminants, equine, poultry, felines, bovines, murines, canines, and the like.
  • Alkyl is a branched or straight chain saturated aliphatic hydrocarbon group.
  • the alkyl group contains from 1 to about 12 carbon atoms, more generally from 1 to about 6 carbon atoms or from 1 to about 4 carbon atoms.
  • the alkyl contains from 1 to about 8 carbon atoms.
  • the alkyl is C1-C2, C1-C3, C1-C4, C1-C5, or C1-C6.
  • the specified ranges as used herein indicate an alkyl group having each member of the range described as an independent species.
  • C1-C6 alkyl indicates a straight or branched alkyl group having from 1, 2, 3, 4, 5, or 6 carbon atoms and is intended to mean that each of these is described as an independent species.
  • C1-C4 alkyl indicates a straight or branched alkyl group having from 1, 2, 3, or 4 carbon atoms and is intended to mean that each of these is described as an independent species.
  • alkyl examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, /-butyl, n- pentyl, isopentyl, /er/-pentyl, neopentyl, n-hexyl, 2-methylpentane, 3-methylpentane, 2,2- dimethylbutane, and 2,3-dimethylbutane.
  • the alkyl group is optionally substituted.
  • the term“Alkyl” also encompasses cycloalkyl or carbocyclic groups.
  • alkyl, -O-alkyl, haloalkyl, etc. can all be considered to include the cyclic forms of alkyl, unless unambiguously excluded by context.
  • substituted alkyl refers to an alkyl group that is substituted with the described substituents. If no substituents are explicitly described“substituted alkyl” refers to an alkyl group that is substituted with 1, 2, 3, or 4 substituents independently selected from F, Cl, Br, I, cyano, hydroxy, -O-alkyl, -SH, -Salkyl, -COOH, -COOalkyl, -COalkyl, -COH, -CONH2, -CONHalkyl, -CON(alkyl) 2 , -OC(0)alkyl, -NHC(0)alkyl, -NalkylC(0)alkyl, nitro, amino, -NHalkyl, N(alkyl) 2 , cyano, haloalkyl, aryl, heteroaryl, alkenyl, alkynyl, haloalkyl, cycloalkyl, alkyl
  • Alkenyl is a linear or branched aliphatic hydrocarbon groups having one or more carbon-carbon double bonds that may occur at a stable point along the chain.
  • alkenyl group having each member of the range described as an independent species, as described above for the alkyl moiety.
  • alkenyl radicals include, but are not limited to ethenyl, propenyl, allyl, propenyl, butenyl and 4-methylbutenyl.
  • alkenyl also embodies“cis” and“trans” alkenyl geometry, or alternatively,“E” and “Z” alkenyl geometry.
  • the alkenyl group is optionally substituted.
  • Alkenyl also encompasses cycloalkyl or carbocyclic groups possessing at least one point of unsaturation. As used herein“substituted alkenyl” can be substituted with the groups described above for alkyl.
  • Alkynyl is a branched or straight chain aliphatic hydrocarbon group having one or more carbon-carbon triple bonds that may occur at any stable point along the chain.
  • the specified ranges as used herein indicate an alkynyl group having each member of the range described as an independent species, as described above for the alkyl moiety.
  • alkynyl examples include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1- pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl and 5-hexynyl.
  • the alkynyl group is optionally substituted.
  • Alkynyl also encompasses cycloalkyl or carbocyclic groups possessing at least one point of unsaturation. As used herein“substituted alkynyl” can be substituted with the groups described above for alkyl.
  • Halo and“Halogen” is fluorine, chlorine, bromine or iodine.
  • Haloalkyl is a branched or straight-chain alkyl groups substituted with 1 or more halo atoms described above, up to the maximum allowable number of halogen atoms.
  • haloalkyl groups include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl and dichloropropyl.
  • “Perhaloalkyl” means an alkyl group having all hydrogen atoms replaced with halogen atoms. Examples include but are not limited to, trifluoromethyl and pentafluoroethyl.
  • aryl refers to a radical of a monocyclic or polycyclic (e.g ., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 p electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system ( G, aryl”).
  • an aryl group has 6 ring carbon atoms (“C6 aryl”; e.g., phenyl).
  • an aryl group has 10 ring carbon atoms (“Cio aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms (“Ci4 aryl”; e.g., anthracyl). “Aryl” also includes ring systems wherein the aryl ring, as defined above, is fused with one or more cycloalkyl or heterocycle groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system.
  • the one or more fused cycloalkyl or heterocycle groups can be 4 to 7-membered saturated or partially unsaturated cycloalkyl or heterocycle groups.
  • substituted aryl refers to an aryl group that is substituted with the described substituents.
  • substituted aryl refers to an aryl group that is substituted with 1, 2, 3, or 4 substituents independently selected from F, Cl, Br, I, cyano, hydroxy, -O-alkyl, -SH, -Salkyl, -COOH, -COOalkyl, -COalkyl, -COH, -CONIB, -CONHalkyl, -CON(alkyl) 2 , -OC(0)alkyl, -NHC(0)alkyl, -NalkylC(0)alkyl, nitro, amino, -NHalkyl, N (alky If.
  • cyano haloalkyl, aryl, heteroaryl, alkyl, alkenyl, alkynyl, haloalkyl, cycloalkyl, alkyl-aryl, alkyl-heteroaryl, alkyl- cycloalkyl, alkyl-heterocycle, heterocycle, -COOaryl, -COaryl, -CONHaryl, -CON(alkyl)(aryl), -OC(0)aryl, -NHC(0)aryl, -NalkylC(0)aryl, -COOheteroaryl, -COheteroaryl, -CONHheteroaryl, -CON(alkyl)(heteroaryl), -OC(0)heteroaryl, -NHC(0)heteroaryl, -NalkylC(0)heteroaryl, -COOheterocycle, -COheterocycle,
  • heterocyclyl and“heterocycle” include saturated, and partially saturated heteroatom-containing ring radicals, where the heteroatoms may be selected from nitrogen, sulfur, boron, silicone, and oxygen.
  • Heterocyclic rings comprise monocyclic 3-10 membered rings, as well as 5-16 membered bicyclic ring systems (which can include bridged fused and spiro-fused bicyclic ring systems). It does not include rings containing -O-O-.-O-S- or -S-S- portions.
  • saturated heterocycle groups include saturated 3- to 6-membered heteromonocyclic groups containing 1 to 4 nitrogen atoms [e.g.
  • pyrrolidinyl imidazolidinyl, piperidinyl, pyrrolinyl, piperazinyl] ; saturated 3 to 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms [e.g. morpholinyl] ; saturated 3 to 6- membered heteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms [e.g., thiazolidinyl].
  • partially saturated heterocycle radicals include but are not limited to, dihydrothienyl, dihydropyranyl, dihydrofuryl, and dihydrothiazolyl.
  • Examples of partially saturated and saturated heterocycle groups include but are not limited to, pyrrolidinyl, imidazolidinyl, piperidinyl, pyrrolinyl, pyrazolidinyl, piperazinyl, morpholinyl, tetrahydropyranyl, thiazolidinyl, dihydrothienyl, 2,3-dihydro-benzo[l,4]dioxanyl, indolinyl, isoindolinyl, dihydrobenzothienyl, dihydrobenzofuryl, isochromanyl, chromanyl, 1,2- dihydroquinolyl, 1,2, 3, 4- tetrahydro-isoquinolyl, 1 ,2,3,4-tetrahydro-quinolyl, 2, 3, 4, 4a, 9,9a- hexahydro-lH-3-aza-fluorenyl, 5,6,7- trihydro-1, 2, 4-triazolo[3,4-a]isoquino
  • substituted heterocycle refers to a heterocycle group that is substituted with the described substituents. If no substituents are explicitly described“substituted heterocycle” refers to a heterocycle group that is substituted with 1, 2, 3, or 4 substituents independently selected from oxo, F, Cl, Br, I, cyano, hydroxy, -O-alkyl, -SH, -Salkyl, -COOH, -COOalkyl, -COalkyl, -COH, -CONH2, -CONHalkyl, -CON(alkyl)2, -OC(0)alkyl, -NHC(0)alkyl, -NalkylC(0)alkyl, nitro, amino, -NHalkyl, N(alkyl)2, cyano, haloalkyl, aryl, heteroaryl, alkyl, alkenyl
  • Heterocycle also includes groups wherein the heterocyclic radical is fused/condensed with an aryl or carbocycle radical, wherein the point of attachment is the heterocycle ring.
  • partially unsaturated condensed heterocyclic group containing 1 to 5 nitrogen atoms for example, indoline, isoindoline, partially unsaturated condensed heterocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, partially unsaturated condensed heterocyclic group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms, and saturated condensed heterocyclic group containing 1 to 2 oxygen or sulfur atoms.
  • heteroaryl denotes stable aromatic ring systems that contain one or more heteroatoms selected from O, N, and S, wherein the ring nitrogen and sulfur atom(s) are optionally oxidized, and nitrogen atom(s) are optionally quartemized.
  • Examples include but are not limited to, unsaturated 5 to 6 membered heteromonocyclyl groups containing 1 to 4 nitrogen atoms, such as pyrrolyl, imidazolyl, pyrazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazolyl [e.g., 4H-l,2,4-triazolyl, IH-1 ,2,3-triazolyl, 2H- 1,2,3-triazolyl]; unsaturated 5- to 6-membered heteromonocyclic groups containing an oxygen atom, for example, pyranyl, 2-furyl, 3-furyl, etc.; unsaturated 5 to 6-membered heteromonocyclic groups containing a sulfur atom, for example, 2-thienyl, 3-thienyl, etc.; unsaturated 5- to 6-membered heteromonocyclic groups containing 1 to 2 oxygen atom
  • the “heteroaryl” group is a 8, 9, or 10 membered bi cyclic ring system.
  • 8, 9, or 10 membered bicyclic heteroaryl groups include benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, quinolinyl, isoquinolinyl, benzofuranyl, indolyl, indazolyl, and benzotriazolyl.
  • substituted heteroaryl refers to a heteroaryl group that is substituted with the described substituents.
  • substituted heteroaryl refers to a heteroaryl group that is substituted with 1, 2, 3, or 4 substituents independently selected from F, Cl, Br, I, cyano, hydroxy, -O-alkyl, -SH, -Salkyl, -COOH, -COOalkyl, -COalkyl, -COH, -CONH2, -CONHalkyl, -CON(alkyl)2, -OC(0)alkyl, -NHC(0)alkyl, -NalkylC(0)alkyl, nitro, amino, -NHalkyl, N(alkyl)2, cyano, haloalkyl, aryl, heteroaryl, alkyl, alkenyl, alkynyl, haloalkyl, cycloalkyl, alkyl-aryl, alkyl-heteroaryl, alkyl-cycloalkyl, alkyl-heterocycle
  • sulfonyl whether used alone or linked to other terms such as alkylsulfonyl, denotes respectively divalent radicals -SO2-.
  • Alkyl-heterocycle is an alkyl group as defined herein with a heterocycle substituent. Examples include but are not limited to, piperidylmethyl and morpholinylethyl.
  • Alkyl-aryl is an alkyl group as defined herein with an aryl substituent.
  • Alkyl-heteroaryl is an alkyl group as defined herein with a heteroaryl substituent.
  • alkyl-heteroaryl groups include:
  • “carbocyclyl”, “carbocyclic”, “carbocycle” or“cycloalkyl” is a saturated or partially unsaturated (i.e., not aromatic) group containing all carbon ring atoms and from 3 to 14 ring carbon atoms (“C3-14 cycloalkyl”) and zero heteroatoms in the non aromatic ring system.
  • a cycloalkyl group has 3 to 10 ring carbon atoms (“C3-10 cycloalkyl”).
  • a cycloalkyl group has 3 to 9 ring carbon atoms (“C3-9 cycloalkyl”).
  • a cycloalkyl group has 3 to 8 ring carbon atoms (“C3-8 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 7 ring carbon atoms (“C3-7 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C3-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 4 to 6 ring carbon atoms (“C4-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C5-6 cycloalkyl”).
  • a cycloalkyl group has 5 to 10 ring carbon atoms (“C5-10 cycloalkyl”).
  • C5-10 cycloalkyl ring carbon atoms
  • Exemplary C3-6 cycloalkyl groups include, without limitation, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (Cs), cyclopentenyl (Cs), cyclohexyl (G,). cyclohexenyl (G,). cyclohexadienyl (G,). and the like.
  • Exemplary C3-8 cycloalkyl groups include, without limitation, the aforementioned C3-6 cycloalkyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (Cs), cyclooctenyl (G). and the like.
  • Exemplary C3-10 cycloalkyl groups include, without limitation, the aforementioned C3-8 cycloalkyl groups as well as cyclononyl (Cs>), cyclononenyl (C9), cyclodecyl (C 10), cyclodecenyl (C10), and the like.
  • the cycloalkyl group can be saturated or can contain one or more carbon-carbon double or triple bonds.
  • “cycloalkyl” also includes ring systems wherein the cycloalkyl ring, as defined above, is fused with one heterocycle, aryl or heteroaryl ring wherein the point of attachment is on the cycloalkyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the carbocyclic ring system.
  • each instance of cycloalkyl is optionally substituted with one or more substituents.
  • the cycloalkyl group is an unsubstituted C3-14 cycloalkyl.
  • Alkyl-cycloalkyl is an alkyl group as defined herein with a cycloalkyl substituent.
  • alkyl-cycloalkyl groups include:
  • oxo as used herein contemplates an oxygen atom attached with a double bond.
  • a CDK9 inhibitor is used to treat a patient such as a human having a cancer that is intrinsically resistant to CDK/6i inhibition or has acquired resistance to a CDK4/6 inhibitor following receiving treatment with the CDK4/6 inhibitor.
  • the CDK9 inhibitor (CDK9 inhibitor) is selected from:
  • y is 0, 1, 2, 3, or 4;
  • each m is independently 0 or 1;
  • each n is independently 0, 1, or 2;
  • each Z is independently CH, CR 14 , or N;
  • Q is CH or N
  • R is hydrogen, Ci-C6alkyl, -(Co-C2alkyl)(C3-C8carbocyclyl), -(Co-C2alkyl)(C3- C8heterocyclyl),-(Co-C2alkyl)(aryl), -(Co-C2alkyl)(heteroaryl), -COOalkyl, -COOarylalkyl, or -COOH;
  • each R 1 is independently alkyl, aryl, cycloalkyl or haloalkyl, wherein each of said alkyl, cycloalkyl and haloalkyl groups optionally includes heteroatoms O, N, or S in place of a carbon in the chain and two R' s on adjacent ring atoms or on the same ring atom together with the ring atom(s) to which they are attached optionally form a 3-8-membered cycle or two R' s on adjacent ring atoms together with the ring atoms to which they are attached optionally form a 6-membered aryl ring;
  • R 2 is -(alkylene)m-heterocyclo, -(alkylene)m-heteroaryl, -(alkylene) m- NR 3 R 4 , -(alkylene)m-C(0)-NR 3 R 4 ; -(alkylene) m- C(0)-0-alkyl; -(alkylene)m-O-R 5 ,
  • R 3 and R 4 at each occurrence are independently:
  • R 5 is independently:
  • R x at each occurrence is independently selected from halo, cyano, nitro, oxo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, heterocycloalkyl, -(alkylene)m-OR 5 , -(alkylene)m-O-alkylene-OR 5 , -(alkylene)m-S(0)n-R 5 , -(alkylene) m -NR 3 R 4 , -(alkylene)m-CN, -(alkylene)m-CN, -(alkylene)m-CN,
  • R 6 is selected independently at each instance from: hydrogen, halogen, alkyl, alkenyl, alkynyl cycloalkyl, heterocyclo, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl;
  • R 7 is selected from:
  • R 7 is selected from cycloalkyl, heterocycle, and alkyl, each of which cycloalkyl, heterocycle, and alkyl groups is optionally substituted with one or more substituents selected from amino, -NHR 14 , -NR 14 R 15 , hydroxyl, OR 14 , R 6 , and R 2 ;
  • X 1 , X 2 , X 3 and X 4 are independently N or CR 8 , wherein at least one of X 1 , X 2 , X 3 , and X 4 , is CR 8 ;
  • R 8 is selected independently at each instance from: R 6 and R 2 , wherein one R 8 is R 2 ;
  • R 14 and R 15 are independently selected from: hydrogen, alkyl, alkenyl, alkynyl, -C(0)H, -C(0)alkyl, -C(S)alkyl, aryl, -SC alkyl, heteroaryl, arylalkyl, and heteroarylalkyl;
  • R 16 is selected from cycloalkyl, heterocycle, and alkyl, each of which cycloalkyl, heterocycle, and alkyl groups is optionally substituted with one or more substituents selected from amino, -NHR 14 , -NR 14 R 15 , hydroxyl, OR 14 , R 6 , and R 2 ;
  • R 19 is a heterocycle substituted with at least one substituent independently selected from amino, halogen, alkyl, -NHR 14 , -NR 14 R 15 , hydroxyl, OR 14 , R 6 , oxo, and R 2 ;
  • R 19 is a heterocycle substituted with at least one substituent independently selected from amino, halogen, alkyl, -NHR 14 , -NR 14 R 15 , hydroxyl, OR 14 , oxo, and R 2 ;
  • R 20 is selected from -C(0)alkyl, -C(0)aryl, -C(0)heteroaryl, -C(0)cycloalkyl, and -C(0)heterocycle each of which R 20 is optionally substituted with 1, 2, 3, or 4 substituents independently selected from amino, halogen, alkyl, -NHR 14 , -NR 14 R 15 , hydroxyl, OR 14 , R 6 , - C(0)R 6 , and R 2 ;
  • R 21 is selected from
  • R 22 is selected from
  • the CDK9 inhibitor is selected from:
  • the CDK9 inhibitor is selected from:
  • CDK9 inhibitor is selected from
  • the CDK9 inhibitor is selected from or a pharmaceutically acceptable salt, N-oxide, isotopic derivative, prodrug, and/or a pharmaceutically acceptable composition thereof.
  • CDK9 inhibitor is selected from
  • R 7 is selected from:
  • “alkyl” is a Ci-Cioalkyl, Ci-C9alkyl, Ci-Csalkyl, Ci-C7alkyl, Ci-Cealkyl, Ci-C 5 alkyl, Ci-C 4 alkyl, Ci-Csalkyl, or Ci-C 2 alkyl.
  • “alkyl” has one carbon.
  • “alkyl” has two carbons.
  • “alkyl” has three carbons.
  • “alkyl” has four carbons. In one embodiment“alkyl” has five carbons.
  • “alkyl” has six carbons.
  • Non-limiting examples of“alkyl” include: methyl, ethyl, propyl, butyl, pentyl, and hexyl.
  • “alkyl” examples include: isopropyl, isobutyl, isopentyl, and isohexyl.
  • alkyl examples include: sec-butyl. .sec-pentyl. and sec-hexyl.
  • alkyl examples include: tert- butyl, tert-pentyl, and tert- hexyl.
  • “alkyl” examples include: neopentyl, 3-pentyl, and active pentyl.
  • alkenyl is“substituted alkenyl”
  • alkynyl is“substituted alkynyl”
  • haloalkyl is a Ci-Ciohaloalkyl, Ci-Cshaloalkyl, Ci-Cshaloalkyl, Ci-C7haloalkyl, Ci-C6haloalkyl, Ci-Cshaloalkyl, Ci-Cdialoalkyl, Ci-C3haloalkyl, and Ci- C2haloalkyl.
  • “haloalkyl” has one carbon.
  • “haloalkyl” has one carbon and one halogen.
  • “haloalkyl” has one carbon and two halogens.
  • “haloalkyl” has one carbon and three halogens.
  • “haloalkyl” has two carbons.
  • “haloalkyl” has three carbons.
  • “haloalkyl” has four carbons.
  • “haloalkyl” has five carbons.
  • “haloalkyl” has six carbons.
  • Non-limiting examples of“haloalkyl” include: , , _ Additional non-limiting examples of“haloalkyl” include:
  • haloalkyl include:
  • haloalkyl include: C! , Cl , and Cl
  • aryl is a 6 carbon aromatic group (phenyl)
  • aryl is a 10 carbon aromatic group (napthyl)
  • “aryl” is a 6 carbon aromatic group fused to a heterocycle wherein the point of atachment is the aryl ring.
  • Non-limiting examples of“aryl” include indoline, tetrahydroquinoline, tetrahydroisoquinoline, and dihydrobenzofuran wherein the point of attachment for each group is on the aromatic ring.
  • “aryl” is a 6 carbon aromatic group fused to a cycloalkyl wherein the point of atachment is the aryl ring.
  • Non-limiting examples of“aryl” include dihydro-indene and tetrahydronaphthalene wherein the point of atachment for each group is on the aromatic ring.
  • aryl is“substituted aryl”.
  • heteroaryl is“heteroaryl”.
  • heteroaryl is a 5 membered aromatic group containing 1, 2, 3, or 4 nitrogen atoms.
  • Non-limiting examples of 5 membered“heteroaryl” groups include pyrrole, furan, thiophene, pyrazole, imidazole, triazole, tetrazole, isoxazole, oxazole, oxadiazole, oxatriazole, isothiazole, thiazole, thiadiazole, and thiatriazole.
  • 5 membered“heteroaryl” groups include:
  • heteroaryl is a 6 membered aromatic group containing 1, 2, or 3 nitrogen atoms (i.e. pyridinyl, pyridazinyl, triazinyl, pyrimidinyl, and pyrazinyl).
  • Non-limiting examples of 6 membered“heteroaryl” groups with 1 or 2 nitrogen atoms include:
  • heteroaryl is a 9 membered bicyclic aromatic group containing 1 or 2 atoms selected from nitrogen, oxygen, and sulfur.
  • heteroaryl groups that are bicyclic include indole, benzofuran, isoindole, indazole, benzimidazole, azaindole, azaindazole, purine, isobenzofuran, benzothiophene, benzoisoxazole, benzoisothiazole, benzooxazole, and benzothiazole.
  • heteroaryl is a 10 membered bicyclic aromatic group containing 1 or 2 atoms selected from nitrogen, oxygen, and sulfur.
  • heteroaryl groups that are bicyclic include quinoline, isoquinoline, quinoxaline, phthalazine, quinazoline, cinnoline, and naphthyridine.
  • heteroaryl is“substituted heteroaryl”
  • cycloalkyl is a C3-C8cycloalkyl, C3-C7cycloalkyl, C3- C6cycloalkyl, C3-C5cycloalkyl, C3-C4cycloalkyl, C4-C8cycloalkyl, Cs-Cscycloalkyl, or Ce- C8cycloalkyl.
  • “cycloalkyl” has three carbons.
  • “cycloalkyl” has four carbons.
  • “cycloalkyl” has five carbons.
  • “cycloalkyl” has six carbons.
  • “cycloalkyl” has seven carbons.
  • “cycloalkyl” has eight carbons.
  • “cycloalkyl” has nine carbons.
  • “cycloalkyl” has ten carbons.
  • Non-limiting examples of“cycloalkyl” include: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and cyclodecyl.
  • cycloalkyl include dihydro-indene and tetrahydronaphthalene wherein the point of attachment for each group is on the cycloalkyl ring.
  • cycloalkyl is a“substituted cycloalkyl”
  • heterocycle refers to a cyclic ring with one nitrogen and 3, 4, 5, 6, 7, or 8 carbon atoms.
  • heterocycle refers to a cyclic ring with one nitrogen and one oxygen and 3, 4, 5, 6, 7, or 8 carbon atoms.
  • heterocycle refers to a cyclic ring with two nitrogens and 3, 4, 5,
  • heterocycle refers to a cyclic ring with one oxygen and 3, 4, 5, 6,
  • heterocycle refers to a cyclic ring with one sulfur and 3, 4, 5, 6, 7, or 8 carbon atoms.
  • Non-limiting examples of“heterocycle” include aziridine, oxirane, thiirane, azetidine, 1,3-diazetidine, oxetane, and thietane.
  • heterocycle examples include pyrrolidine, 3-pyrroline, 2- pyrroline, pyrazolidine, and imidazolidine.
  • heterocycle examples include tetrahydrofuran, 1,3- dioxolane, tetrahydrothiophene, 1,2-oxathiolane, and 1,3-oxathiolane.
  • heterocycle examples include piperidine, piperazine, tetrahydropyran, 1,4-dioxane, thiane, 1,3-dithiane, 1 ,4-dithiane, morpholine, and thiomorpholine.
  • heterocycle examples include indoline, tetrahydroquinoline, tetrahydroisoquinoline, and dihydrobenzofuran wherein the point of attachment for each group is on the heterocyclic ring. For example, group.
  • Non-limiting examples of“heterocycle” also include:
  • Non-limiting examples of“heterocycle” also include:
  • Non-limiting examples of“heterocycle” also include:
  • heterocycle includes: In one embodiment“heterocycle” is“substituted heterocycle”.
  • the“alkyl-aryl” refers to a 1 carbon alkyl group substituted with an aryl group.
  • Non-limiting examples of“alkyl-aryl” include:
  • the“alkyl-aryl” refers to a 2 carbon alkyl group substituted with an aryl group.
  • Non-limiting examples of“alkyl-aryl” include:
  • the“alkyl-aryl” refers to a 3 carbon alkyl group substituted with an aryl group.
  • a group described herein that can be substituted with 1, 2, 3, or 4 substituents is substituted with two substituents.
  • a group described herein that can be substituted with 1, 2, 3, or 4 substituents is substituted with three substituents.
  • a group described herein that can be substituted with 1, 2, 3, or 4 substituents is substituted with four substituents.
  • CDK9 inhibitors useful in the methods described herein include, but are not limited to Compounds 1 to 5, or a pharmaceutically acceptable salt thereof.
  • Compounds 1 to 5 are provided below in Table 1.
  • the biochemical activity of Compounds 1 to 5 against CDK9 was determined using Caliper technology by Nanosyn, Inc. and is shown in Table 1.
  • CDK9 inhibitors useful in the methods described herein include, but are not limited to Compounds 6 to 39, or a pharmaceutically acceptable salt thereof.
  • Compounds 6 to 39 are provided below in Table 2A.
  • the biochemical activity of Compounds 6 to 39 against CDK9 was determined using Caliper technology by Nanosyn, Inc. and is shown in Table 2A.
  • CDK9 inhibitors useful in the methods described herein include Compounds 40 to 43 or a pharmaceutically acceptable salt thereof.
  • Compounds 40 to 43 are provided below in Table 2B. The biochemical activity of Compounds 40 to 43 against CDK9 was determined using
  • step 1 25 g of 6-1 was reacted with thionyl chloride in methanol at 60 °C to provide 26 g of 6-2.
  • step 2 20 g of 6-2 was reacted with potassium carbonate in acetonitrile at 50 °C to provide 24 g of 6-3.
  • step 3 5 g of 6-3 was reacted with benzyl bromide and potassium carbonate in acetonitrile at 70 °C to provide 4.2 g of 6-4.
  • step 4 reacted in concentrated HC1 at 85 °C to provide 2.1 g of 6-6.
  • step 6 600 mg of 6-7 was reacted with potassium acetate in an ethanol/water mixture at 75 °C to provide 500 mg of 6 8
  • step 7 500 mg of 6-8 was reacted with Raney nickel in ethanol under a hydrogen atmosphere at 55 °C to provide 130 mg of a mixture of 6-9 and 6-9’.
  • step 8 50 mg of 6-9/6-9’ was reacted with Pd/C in methanol at 55 °C to provide 20 mg of a mixture of 6-10 and 6-10’.
  • Step 9 10 mg of 6-10/6-10’ was reacted with triethylamine in ethanol in a microwave reactor at 140 °C to provide 6-11 and COMPOUND 6.
  • Step 1 100 mg of 7-1 was reacted with 4-methoxybenzylamine and N.N- diisopropylethylamine in /V./V-di methyl acetami de at 130 °C to provide 105 mg of 7-2.
  • step 2 100 mg of 7-2 was reacted with trifluoroacetic acid to provide 85 mg of 7-3.
  • Step 3 80 mg of 7-3 was reacted with the acyl chloride derivative of 7-4 and triethylamine in dichloromethane to provide 20 mg of 7-5.
  • Step 4 20 mg of 7-5 was reacted with trifluoroacetic acid in dichloromethane to provide 7.1 mg of COMPOUND 7.
  • 8-1 (1 g) was converted to 8-2 using NaBfbCN in the presence of HO Ac and DCM. The reaction was stirred overnight at room temperature. 8-2 was observed by LC-MS, but was difficult to purify due to its high polarity. Therefore, 8-2 was converted to 8-3 and purified to afford pure 8-3 (410 mg). 8-3 (410 mg) was converted to 8-2 using TFA in DCM and stirring at room temperature for 3 hours to afford 380 mg of 8-2 as a TFA salt. 8-2 TFA salt (50 mg) was converted to COMPOUND 8 using DIEA in EtOH and refluxing overnight. After purification, 5.9 mg of COMPOUND 8 was obtained and the product was confirmed via 3 ⁇ 4- NMR and HPLC.
  • 9-2 (4 g) was converted to 9-3 using B0C2O in THF and NaHCCb at room temperature overnight. After purification, 2.5 g of 9-3 was obtained.
  • 9-2 (100 mg) was converted to 9-3 using K2CO3 in NMP. The reaction was heated to 120 °C in a microwave for 0.5 hours to afford 5 mg of 9-3. 1 HNMR confirmed the structure.
  • 9-1 (100 mg) was converted to 9-4 using K2CO3 and 2-fluoropyridine in NMP and the reaction was heated to 140 °C for 12 hours. After purification, 20 mg of 9-4 was obtained.
  • S-90 (20 mg) was converted to COMPOUND 9 using TEA in EtOH. The reaction was heated to 140 °C in a microwave reactor for 30 minutes. After preparative TLC purification, 4.1 mg of COMPOUND 9 was obtained and confirmed via 1 HNMR and HPLC.
  • COMPOUND 10 is synthesized as shown below:
  • 15-1 (50 mg) was converted to 15-3 using 15-2 in the presence of isobutyl chloroformate and NMM in DMAc. The reaction was stirred at room temperature for 12 hours. After workup, 100 mg of crude 15-3 was obtained and used directly in the next step. Crude 15-3 is converted to COMPOUND 15 using TFA in DCM at room temperature for 2 hours.
  • 16-1 (100 mg) and 16-2 were converted to 16-3 using i-BuCC Cl in NMM and DMAc. The reaction was stirred at room temperature for 12 hours. After purification, 120 mg of crude 16- 3 was obtained and used directly in the next step. Crude 16-3 (30 mg) was converted to COMPOUND 16 using TFA in DCM. The reaction was stirred at room temperature for 2 hours. After purification, 8.1 mg of COMPOUND 16 was obtained.
  • 17-1 (200 mg) was converted to 17-2 in the presence of isobutyl carbonochloridate using i-BuCC Cl in NMM and DMAc. The reaction was stirred at room temperature for 1 hour. 17-3 was added and the reaction was stirred at 60 °C for 72 hours. After purification, 15 mg of 17-4 was obtained. 17-4 (15 mg) was converted to COMPOUND 17 using TFA in DCM. The reaction stirred at room temperature for 1 hour. After purification by preparative TLC and washing with DCM, 3.3 mg of COMPOUND 17 was obtained and the structure was confirmed via 'H-NMR. HPLC and LC-MS.
  • 23-1 (30 mg) was converted to COMPOUND 23 using HC1 in i-PrOH. The reaction was heated in a microwave reactor to 140 °C for 30 minutes. After purification and washing with DCM/MeOH (5/1), 6.8 mg of COMPOUND 23 was obtained and the structure was confirmed via 1 HNMR, LC-MS and HPLC.
  • 23-1 can be converted to COMPOUND 23 using Pd(OAc)2/x-phos in the presence of CS2CO3 in dioxane at 100 °C for 4 hours.
  • S-23-1 can be converted to COMPOUND 23 using TFA in i-PrOH. The reaction is refluxed for 48 hours.
  • 23-1 can be converted to COMPOUND 23 using 2 drops of concentrated. HC1 in i-PrOH. The reaction is heated in a microwave reactor to 80 °C for 20 minutes.
  • 23-1 can be converted to COMPOUND 23 by the scheme below:
  • COMPOUND 23 23-2 (10 mg) was converted to COMPOUND 23 using Pd(OAc)2/x-phos in the presence of CS2CO3 in dioxane. The reaction was heated in a microwave reactor at 100 °C for 30 minutes. Alternatively, 23-2 is converted to COMPOUND 23 using TFA in i-PrOH. The reaction is refluxed for 48 hours.
  • 24-1 (200 mg) was converted to 24-2 using 3-oxa-l,5-dichloropentane in the presence of K2CO3 and heating the reaction at 100 °C in DMF overnight. After purification, 100 mg of 24-2 was obtained. 24-2 (100 mg) was converted to 24-3 using TFA in DCM and stirring the reaction at room temperature for 1 hour. After purification, 50 mg of 24-3 was obtained. 24-3 (50 mg) is converted to COMPOUND 24 using TEA in DMAc and heating to 120 °C overnight.
  • 25-1 (1 g) was converted to 25-2 using (Bo O in the presence of NaHC03 in THF and stirring at room temperature overnight. After purification, 1.4 g of 25-2 was obtained. 25-2 (200 mg) was converted to 25-3 using NaBFECN in the presence of HO Ac in MeOH and stirring at room temperature overnight. The purification afforded 100 mg of 25-3. 25-3 (100 mg) was converted to 25-4 using TFA in DCM and stirring the reaction at room temperature overnight. After workup, 60 mg of 25-4 was obtained. 25-4 (60 mg) was converted to COMPOUND 25 using TEA in DMAc and stirring at 120 °C overnight. After purification by preparative TLC, 15.4 mg of COMPOUND 25 was obtained and the structure was confirmed via 3 ⁇ 4-NMR and HPLC.
  • 26-1 (200 mg) was converted to 26-2 using K2CO3 in EtOH at 80 °C overnight. After purification, 100 mg of 26-2 was obtained. 26-2 (100 mg) was converted to 26-3 using TFA in DCM and stirring at room temperature for 1 hour. After purification, 50 mg of 26-3 was obtained. 26-3 (50 mg) was converted to COMPOUND 26 using TEA in DMAc for 120 °C overnight. The purification afforded 6.7 mg of COMPOUND 26.
  • 31-1 (20 g) was coupled to 31-2 in the presence of K2CO3 in DMAc at 80 °C overnight to afford 31-3. After purification, 25 g of crude 31-3 was obtained. 31-3 (15 g) was converted to 31-5 in the presence of 31-4 using (PPhrirPdCh. Cul and TEA in THF at 40 °C for 4 hours. After purification, 9.3 g of crude 31-5 was obtained. 31-5 (9.3 g) was converted to 31-6 using TBAF in THF at 60 °C for 4 hours. 31-6 (5.6 g) was converted to 31-7 using HO Ac in THF/H2O at 60 °C for 6 hours. After purification, 3.5 g of 31-7 was obtained.
  • Step 1 100 mg of 35-1 was reacted with 35-3 to provide 35-3.
  • Step 2 50 mg of 35-3 was reacted with trifluoroacetic acid to provide 45 mg of 35-4.
  • Step 3 30 mg of 35-4 was reacted with the (1 -ethoxy cyclopropoxy)trimethylsilane in the presence of acetic acid and NaBfbCN in methanol overnight to provide COMPOUND 35.
  • Step 1 300 mg of 36-1 was reacted with cyclohexanone in the presence of sodium triacetoxyborohydride and acetic acid in DCM to provide 246 mg of 36-2.
  • Step 246 mg of 36-2 was hydrogenated with Palladium on carbon to provide 190 mg of 36-3.
  • Step 3 50 mg of 36-3 was reacted with the 36-4 in the presence of sodium bicarbonate at 125 C in DMAc overnight to provide COMPOUND 36.
  • Step 1 3 g of 37-1 was reacted with 37-2 in the presence of calcium carbonate in water at 100 C overnight to provide 5 5 g of 37-3 after purification.
  • Step 2 2.7 g of 37-3 was reacted with PBn in diethyl ether at 0 C to provide 1.8 g of 37-4.
  • Step 3 1.6 g of 37- 4 was reacted with 37-5 in the presence of DIEA at 100 C in DMF overnight to provide 455 mg of 37-6.
  • Step 455 mg of 37-6 was hydrogenated with palladium on carbon in methanol and ethylacetate to afford 206 mg of 37-7.
  • Step 5 50 mg of 37-7 was reacted with 37-8 at 140 C in NMP to afford 4.6 mg of COMPOUND 37.
  • Step 1 30 g of 38-1 was reacted with benzyl bromide in the presence of potassium carbonate in DMF overnight to provide 60 g of 38-2.
  • Step 2 56 g of 38-2 was reacted with (COCl)2(1.5eq) in DMSO in the presence of triethyl amine (3.2 eq) to provide 43.8 g of 38-3 after purification.
  • Step 3 1 g of 38-3 was reacted with mon-Boc protected piperazine (1.2 eq) in the presence of TsOH ( 05eq) at 120 C in Toluene to provide 780 mg of 38-4.
  • Step 4 700 mg of 38-4 was hydrogenated with palladium on carbon in isopropyl alcohol with acetic acid at 40 C to afford 500 mg of 38-5.
  • 500 mg of 38-5 was reacted with CbzCl in DCM to afford 100 mg of 38-6.
  • Step 6 100 mg of 38-6 is deprotected with trifluoroacetic acid to afford 80 mg of 38-7.
  • 80 mg of 38-7 is converted to 50 mg of 38-8 by reductive amination.
  • 50 mg of 38-8 is converted to 30 mg of 38-9 by hydrogenation with palladium on carbon.
  • 30 mg of 38-9 was reacted with 38-10 at 125 C in the presence of triethylamine in DMAc to afford 2.5 mg of COMPOUND 38.
  • Step 1 600 mg of 39-1 was reacted with 39-2, EDCI, HOBT (.5eq), and trimethylamine (2 eq) in DCM overnight to provide 500 mg of 29-3.
  • Step 2 100 mg of 39- 3 was hydrogenated with palladium on carbon (15%) in methanol for 3 hours to provide 50 mg of 39-4 after purification.
  • Step 3 50 mg of 39-4 was reacted with 39-5 at 120 °C in the presence of sodium bicarbonate (10 eq) in DMAc to afford 3.7 mg of COMPOUND 39.
  • CDK9 inhibitor useful in the methods described herein include, but are not limited to:
  • (rel)-MC 180295 is a potent and selective CDK9 inhibitor, at least 22-fold more selective for CDK9 over other CDKs, having the chemical structure:
  • NVP-2 or a pharmaceutically acceptable salt thereof.
  • NVP-2 is a selective CDK9 inhibitor having the chemical structure:
  • AZD4573 or a pharmaceutically acceptable salt thereof.
  • AZD4573 is a selective CDK9 inhibitor developed by AstraZeneca having the chemical structure:
  • PHA-767491 or a pharmaceutically acceptable salt thereof.
  • PHA-767491 is a selective CDK9 inhibitor having the chemical structure: LDC00067, or a pharmaceutically acceptable salt thereof.
  • LDC00067 is a highly specific CDK9 inhibitor having the chemical structure:
  • Atuveciclib (BAY-1143572), or a pharmaceutically acceptable salt thereof.
  • Atuveciclib is a selective CDK9 inhibitor developed by Bayer AG having the chemical structure: CDK9-IN-1, or a pharmaceutically acceptable salt thereof.
  • CDK9-IN-1 is a selective CDK9 inhibitor having the chemical structure:
  • CDK-IN-2 is a potent and specific CDK9 inhibitor having the chemical structure:
  • JSH-150 is a highly selective and potent CDK9 inhibitor having the chemical structure:
  • CDK9-IN-8 is a highly effective and selective CDK9 inhibitor having the chemical structure:
  • FIT-039 is a selective CDK9 inhibitor having the chemical structure:
  • CDKI-73 is a potent CDK9 inhibitor having the chemical structure:
  • TG02 is a pyrimidine-based multi-kinase inhibitor that inhibits CDKs 1, 2, 7 and 9 together with JAK2 and FLT3 having the chemical structure:
  • CDK4/6 inhibitors for use in the methods described herein include, but are not limited to palbociclib, abemaciclib, ribociclib, trilaciclib, SHR6390, or lerociclib.
  • the CDK4/6 inhibitor is palbociclib:
  • the CDK4/6 inhibitor is abemaciclib:
  • the CDK4/6 inhibitor is ribociclib:
  • the CDK4/6 inhibitor is lerociclib:
  • the CDK4/6 inhibitor is trilaciclib:
  • the CDK4/6 inhibitor is SHR 6390.
  • the CDK4/6 inhibitor is selected from an inhibitor described in, for example, U.S. Patent Nos. 8,822,683; 8,598,197; 8,598,186; 8,691,830; 8,829,102; 8,822,683; 9,102,682; 9,499,564; 9,481,591; and 9,260,442, filed by Tavares and Strum and assigned to G1 Therapeutics describe a class of N-(heteroaryl)-pyrrolo[3,2-d]pyrimidin-2- amine cyclin dependent kinase inhibitors including those of the formula (with variables as defined therein):
  • the CDK4/6 inhibitor is selected from an inhibitor described in, for example, U.S. Patent Nos. 9,464,092, 9,487,530, and 9,527,857 which are also assigned to G1 Therapeutics describe the use of the above pyrimidine-based agents in the treatment of cancer.
  • the CDK4/6 inhibitor is selected from an inhibitor described in, for example, WO 2013/148748 (U.S.S.N. 61/617,657) titled“Lactam Kinase Inhibitors”, WO 2013/163239 (U.S.S.N. 61/638,491) titled“Synthesis of Lactams” and WO 2015/061407 filed by Tavares and also assigned to G1 Therapeutics describes the synthesis of N-(heteroaryl)- pynOlo[3,2-d]pyrimidin-2-amines and their use as lactam kinase inhibitors.
  • the CDK4/6 inhibitor is selected from an inhibitor described in, for example, WO 2014/144326 filed by Strum et al. and assigned to G1 Therapeutics describes compounds and methods for protection of normal cells during chemotherapy using pyrimidine- based CDK4/6 inhibitors.
  • WO 2014/144596 filed by Strum et al. and assigned to G1 Therapeutics describes compounds and methods for protection of hematopoietic stem and progenitor cells against ionizing radiation using pyrimidine-based CDK4/6 inhibitors.
  • WO 2014/144847 filed by Strum et al. and assigned to G1 Therapeutics describes HSPC-sparing treatments of abnormal cellular proliferation using pyrimidine-based CDK4/6 inhibitors.
  • WO 2014/144740 filed by Strum et al. and assigned to G1 Therapeutics describes highly active anti neoplastic and anti-proliferative pyrimidine-based CDK 4/6 inhibitors.
  • WO 2015/161285 filed by Strum et al. and assigned to G1 Therapeutics describes tricyclic pyrimidine-based CDK inhibitors for use in radioprotection.
  • WO 2015/161287 filed by Strum et al. and assigned to G1 Therapeutics describes analogous tricyclic pyrimidine-based CDK inhibitors for the protection of cells during chemotherapy.
  • WO 2015/161288 filed by Strum et al. and assigned to G1 Therapeutics describes analogous tricyclic pyrimidine-based CDK inhibitors for use as anti-neoplastic and anti-proliferative agents.
  • WO 2016/040858 filed by Strum et al. and assigned to G1 Therapeutics describes the use of combinations of pyrimidine-based CDK4/6 inhibitors with other anti-neoplastic agents.
  • WO 2016/040848 filed by Strum et al. and assigned to G1 Therapeutics describes compounds and methods for treating certain Rb-negative cancers with CDK4/6 inhibitors and topoisomerase inhibitors. Methods of Treatment - Proliferative Disorders Intrinsically Resistant to a CDK4/6
  • a method of treating a proliferative disorder in a patient comprising administering an effective amount of a CDK9 inhibitor, wherein the proliferative disorder is intrinsically resistant to cell-cycle inhibition using a selective CDK4/6 inhibitor.
  • disorders include tumors, cancers, disorders related to abnormal cellular proliferation, inflammatory disorders, immune disorders, and autoimmune disorders.
  • the CDK9 inhibitor is selected from the CDK9 inhibitor is selected from (rel)-MCl 80295, NVP-2, AZD4573, PHA-767491, LDC00067, atuveciclib (BAY 1143572), CDKi-73 (LS-007), CDK9-IN-1, CDK-IN-2, JSH-150, CDK9-IN-8, FIT-039, LY2857785, SNS-032 (BMS-387032), or TG02, or a pharmaceutically acceptable salt thereof.
  • the CDK9 inhibitor can be selected from Compounds 1 to 53, or a pharmaceutically acceptable salt thereof.
  • the CDK9 inhibitor is selected from Formula I, II, III, IV, V, or VI, or a pharmaceutically acceptable salt thereof. In some embodiments, the CDK9 inhibitor is Compound 1, or a pharmaceutically acceptable salt thereof. In some embodiments, the CDK9 inhibitor is selected from Compounds 1 to 43, or a pharmaceutically acceptable salt thereof. In some embodiments, the CDK9 inhibitor is selected from a compound described herein.
  • CDK4/6 Many cancers do not depend on the activities of CDK4/6 for proliferation as they can use the proliferative kinases promiscuously (e.g., can use CDK 1/2/4/or 6) or lack the function of the retinoblastoma tumor suppressor protein (Rb), which is inactivated by the CDKs.
  • Rb retinoblastoma tumor suppressor protein
  • the potential sensitivity of certain tumors to selective CDK4/6 inhibition can be deduced based on tumor type and molecular genetics using standard techniques.
  • Cancers that are not typically affected by the inhibition of CDK4/6, that is intrinsically resistant to a selective CDK4/6 inhibitor are those that can be characterized by one or more of the group including, but not limited to, increased activity of CDK1 or CDK2, loss, deficiency, or absence of retinoblastoma tumor suppressor protein (Rb), high levels of MYC expression, increased cyclin E (e.g., El or E2) and increased cyclin A, or expression of a Rb-inactivating protein (such as HPV-encoded E7).
  • Rb retinoblastoma tumor suppressor protein
  • Such cancers can include, but are not limited to, small cell lung cancer, retinoblastoma, HPV positive malignancies like cervical cancer and certain head and neck cancers, MYC amplified tumors such as Burkitts’ Lymphoma, and triple negative breast cancer; certain classes of sarcoma, certain classes of non-small cell lung carcinoma, certain classes of melanoma, certain classes of pancreatic cancer, certain classes of leukemia, certain classes of lymphoma, certain classes of brain cancer, certain classes of colon cancer, certain classes of prostate cancer, certain classes of ovarian cancer, certain classes of uterine cancer, certain classes of thyroid and other endocrine tissue cancers, certain classes of salivary cancers, certain classes of thymic carcinomas, certain classes of kidney cancers, certain classes of bladder cancers, and certain classes of testicular cancers.
  • small cell lung cancer retinoblastoma
  • HPV positive malignancies like cervical cancer and certain head and neck cancers
  • MYC amplified tumors such as
  • Determining intrinsic resistance to selective a CDK4/6 inhibitor for example by determining the loss or absence of retinoblastoma (Rb) tumor suppressor protein (Rb-null), can be determined through any of the standard assays known to one of ordinary skill in the art.
  • Rb-status in a cancer can be determined by, for example but not limited to, Western Blot, ELISA (enzyme linked immunoadsorbent assay), IHC (immunohistochemistry), and FACS (fluorescent activated cell sorting).
  • tissue, cell line or surrogate tissue sample that is utilized e.g., for example Western Blot and ELISA may be used with any or all types of tissues, cell lines or surrogate tissues, whereas the IHC method would be more appropriate wherein the tissue utilized in the methods of described herein was a tumor biopsy.
  • FACs analysis would be most applicable to samples that were single cell suspensions such as cell lines and isolated peripheral blood mononuclear cells. See for example, US 20070212736 “Functional Immunohistochemical Cell Cycle Analysis as a Prognostic Indicator for Cancer”.
  • molecular genetic testing may be used for determination of retinoblastoma gene status.
  • Molecular genetic testing for retinoblastoma includes the following as described in Lohmann and Gallie“Retinoblastoma. Gene Reviews” (2010) or Parsam et al.“A comprehensive, sensitive and economical approach for the detection of mutations in the RBI gene in retinoblastoma” Journal of Genetics, 88(4), 517-527 (2009).
  • Increased activity of CDK1 or CDK2, high levels of MYC expression, increased cyclin E and increased cyclin A can be determined through any of the standard assays known to one of ordinary skill in the art, including but not limited to Western Blot, ELISA (enzyme linked immunoadsorbent assay), IHC (immunohistochemistry), and FACS (fluorescent activated cell sorting).
  • the selection of the assay will depend upon the tissue, cell line, or surrogate tissue sample that is utilized e.g., for example Western Blot and ELISA may be used with any or all types of tissues, cell lines, or surrogate tissues, whereas the IHC method would be more appropriate wherein the tissue utilized in the methods was a tumor biopsy.
  • FACs analysis would be most applicable to samples that were single cell suspensions such as cell lines and isolated peripheral blood mononuclear cells. Numerous methods can be utilized to measure markers believed to contribute to CDK4/6 inhibitor acquired resistance. Current methods include immunohistochemistry (IHC), immunocytochemistry, mass spectrometry. An alternative method includes the use of immunofluorescence (IF) and image analysis to determine the relative abundance of a protein of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples. The most frequently used methods for determining gene expression levels is immunohistochemistry (IHC), although western blot allows for assessment of total as well as isoform-specific expression. mRNA from the gene of interest can also be measured by reverse transcription polymerase chain reaction (RT-PCR).
  • RT-PCR reverse transcription polymerase chain reaction
  • Immunohistochemistry and immunocytochemistry (ICC) are techniques employed to localize expression and are dependent on specific epitope-antibody interactions.
  • IHC refers to the use of tissue sections
  • ICC describes the use of cultured cells or cell suspensions.
  • positive staining is visualized using a molecular label, which can be fluorescent or chromogenic. Briefly, samples are fixed to preserve cellular integrity and then subjected to incubation with blocking reagents to prevent non-specific binding of the antibodies. Samples are subsequently incubated with primary and secondary antibodies, and the signal is visualized for microscopic analysis.
  • the western blot technique uses three elements to identify specific proteins from a complex mixture of proteins extracted from cells: separation by size, transfer to a solid support, and marking target protein using a proper primary and secondary antibody to visualize.
  • the most common version of this method is immunoblotting. This technique is used to detect specific proteins in a given sample of tissue homogenate or extract. The sample of proteins is first electrophoresed by SDS-PAGE to separate the proteins based on molecular weight. The proteins are then transferred to a membrane where they are probed using antibodies specific to the target protein.
  • Genomic alterations and mRNA expression can be determined through fluorescence in situ hybridization (FISH), targeted sequencing, and microarray analysis. Commonly mutated genes, as well as differentially expressed and co-expressed genes can be identified.
  • FISH fluorescence in situ hybridization
  • Fluorescence in situ hybridization is a cytogenic technique used for the detection and localization of RNA sequences within tissues or cells. It is particularly important for defining the spatial-temporal patterns of gene expression. FISH relies on fluorescent probes that bind to complementary sequences of the RNA of interest. A series of hybridization steps are performed to achieve signal amplification of the target of interest. This amplification is then viewed using a fluorescent microscope. This technique can be used on formalin-fixed paraffin embedded (FFPE) tissue, frozen tissues, fresh tissues, cells and circulating tumor cells.
  • FFPE formalin-fixed paraffin embedded
  • RNA-Seq Targeted RNA-sequencing
  • RNA-Seq is a highly accurate method for selecting and sequencing specific transcripts of interest. It offers both quantitative and qualitative information. Targeted RNA-Seq can be achieved via either enrichment or amplicon-based approaches, both of which enable gene expression analysis in a focused set of genes of interest. Enrichment assays also provide the ability to detect both known and novel gene fusion partners in many sample types, including formalin-fixed paraffin-embedded (FFPE) tissue. RNA enrichment provides quantitative expression information as well as the detection of small variants and gene fusions.
  • FFPE formalin-fixed paraffin-embedded
  • mRNA molecules are typically collected from both an experimental sample and a reference sample.
  • the reference sample could be collected from a healthy individual
  • the experimental sample could be collected from an individual with a disease such as cancer.
  • the two mRNA samples are then converted into complementary DNA (cDNA), and each sample is labeled with a fluorescent probe of a different color.
  • the experimental cDNA sample may be labeled with a red fluorescent dye
  • the reference cDNA may be labeled with a green fluorescent dye.
  • the two samples are then mixed together and allowed to hybridize to the microarray slide. Following hybridization, the microarray is scanned to measure the expression of each gene printed on the slide.
  • the spot on the microarray appears red. In contrast, if the expression in the experimental sample is lower than in the reference sample, then the spot appears green. Finally, if there is equal expression in the two samples, then the spot appears yellow.
  • the data gathered through microarrays can be used to create gene expression profiles, which show simultaneous changes in the expression of many genes in response to a particular condition or treatment.
  • the cancer is selected from a small cell lung cancer, retinoblastoma, and triple negative (ER/PR/Her2 negative) breast cancer, which almost always have inactivated retinoblastoma tumor suppressor proteins (Rb), and therefore do not require CDK4/6 activity to proliferate.
  • Triple negative breast cancer is also almost always genetically or functionally Rb-null.
  • certain virally induced cancers e.g. cervical cancer and subsets of Head and Neck cancer
  • E7 viral protein
  • Some lung cancers are also believed to be caused by HPV.
  • the cancer to be treated through the administration of a CDK9 inhibitor is small cell lung cancer.
  • the cancer to be treated through the administration of a CDK9 inhibitor is triple negative breast cancer.
  • the cancer to be treated through the administration of a CDK9 inhibitor is retinoblastoma.
  • the cancer to be treated through the administration of a CDK9 inhibitor is an HPV positive malignancy.
  • the HPV positive malignancy is cervical cancer.
  • the HPV positive malignancy is head and neck cancers.
  • the cancer to be treated through the administration of a CDK9 inhibitor is a MYC amplified tumor.
  • the MYC amplified tumor is Burkitts’ Lymphoma.
  • the cancer to be treated through the administration of a CDK9 inhibitor is a Rb-positive tumor that has one or more genetic or phenotypic aberrations which render it intrinsically resistant to a selective CDK4/6 inhibitor.
  • the Rb-positive, intrinsically CDK4/6 inhibitor resistant cancer is CDK4/6 inhibitor treatment naive.
  • a method of treating a proliferative disorder in a patient, including a human comprising administering an effective amount of a CDK9 inhibitor, wherein the proliferative order has developed resistance to a selective CDK4/6 inhibitor.
  • the CDK9 inhibitor is selected from (rel)- MC 180295, NVP-2, AZD4573, PHA-767491, LDC00067, atuveciclib (BAY 1143572), CDKi-73 (LS-007), CDK9-IN-1, CDK-IN-2, JSH-150, CDK9-IN-8, FIT-039, LY2857785, SNS-032 (BMS-387032), or TG02, or a pharmaceutically acceptable salt thereof.
  • the CDK9 inhibitor can be selected from Compounds 1 to 43, or a pharmaceutically acceptable salt thereof.
  • the CDK9 inhibitor is selected from Formula I, II, III, IV, V, or VI, or a pharmaceutically acceptable salt thereof. In some embodiments, the CDK9 inhibitor is Compound 1, or a pharmaceutically acceptable salt thereof. In some embodiments, the CDK9 inhibitor is selected from Compounds 1 to 43, or a pharmaceutically acceptable salt thereof. In some embodiments, the CDK9 inhibitor is selected from a compound described herein.
  • Cancers initially susceptible to selective CDK4/6 inhibitor inhibition may acquire resistance to a selective CDK4/6 inhibitor by, for example but not limited to upregulation of cyclin E which allows G1 to S cell cycle progression through CDK2.
  • a CDK9 inhibitor provides an effective treatment for patients with cancers that have developed selective CDK4/6 inhibitor resistance over time during treatment with a selective CDK4/6 inhibitor.
  • cancers initially susceptible to selective CDK4/6 inhibitor inhibition generally require the activity of CDK4/6 for replication or proliferation.
  • Cancers and disorders of such type can be characterized by (e.g., that has cells that exhibit) the presence of a functional Retinoblastoma protein.
  • Such cancers and disorders are classified as being Rb-positive.
  • Rb- positive abnormal cellular proliferation disorders, and variations of this term as used herein, refer to disorders or diseases caused by uncontrolled or abnormal cellular division which are characterized by the presence of a functional Retinoblastoma protein, which can include cancers.
  • Cancers initially susceptible to selective CDK4/6 inhibitor inhibition that may acquire resistance to a selective CDK4/6 inhibitor may include Rb-positive: estrogen-receptor positive cancer, HER2 -negative advanced breast cancer, late-line metastatic breast cancer, liposarcoma, non-small cell lung cancer, liver cancer, ovarian cancer, glioblastoma, refractory solid tumors, retinoblastoma positive breast cancer as well as retinoblastoma positive endometrial, vaginal and ovarian cancers and lung and bronchial cancers, adenocarcinoma of the colon, adenocarcinoma of the rectum, central nervous system germ cell tumors, teratomas, estrogen receptor-negative breast cancer, estrogen receptor-positive breast cancer, familial testicular germ cell tumors, HER2-negative breast cancer, HER2 -positive breast cancer, male breast cancer, ovarian immature teratomas, ovarian mature teratoma, ovarian monodermal
  • the targeted cancers included estrogen-receptor positive, HER2-negative advanced breast cancer, late-line metastatic breast cancer, liposarcoma, non-small cell lung cancer, liver cancer, ovarian cancer, glioblastoma, refractory solid tumors, retinoblastoma positive breast cancer as well as retinoblastoma positive endometrial, vaginal and ovarian cancers and lung and bronchial cancers, metastatic colorectal cancer, metastatic melanoma with CDK4 mutation or amplification, or cisplatin-refractory, unresectable germ cell tumors.
  • the cancer initially susceptible to selective CDK4/6 inhibitor inhibition that may acquire resistance to a selective CDK4/6 inhibitor may include Rb-positive cancer selected from an Rb-positive carcinoma, sarcoma, including, but not limited to, lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma
  • the cancer initially susceptible to selective CDK4/6 inhibitor inhibition that may acquire resistance to a selective CDK4/6 inhibitor may include Rb-positive: fibrosarcoma, myxosarcoma, chondrosarcoma, osteosarcoma, chordoma, malignant fibrous histiocytoma, hemangiosarcoma, angiosarcoma, lymphangiosarcoma.
  • Mesothelioma Mesothelioma, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma; epidermoid carcinoma, malignant skin adnexal tumors, adenocarcinoma, hepatoma, hepatocellular carcinoma, renal cell carcinoma, hypernephroma, cholangiocarcinoma, transitional cell carcinoma, choriocarcinoma, seminoma, embryonal cell carcinoma, glioma anaplastic; glioblastoma multiforme,, neuroblastoma, medulloblastoma, malignant meningioma, malignant schwannoma, neurofibrosarcoma, parathyroid carcinoma, medullary carcinoma of thyroid, bronchial carcinoid, pheochromocytoma, Islet cell carcinoma, malignant carcinoid, malignant paraganglioma, melanoma, Merkel cell neoplasm,
  • the cancer initially susceptible to selective CDK4/6 inhibitor inhibition that may acquire resistance to a selective CDK4/6 inhibitor may include a blood disorder or a hematologic malignancy, including, but not limited to, myeloid disorder, lymphoid disorder, leukemia, lymphoma, myelodysplastic syndrome (MDS), myeloproliferative disease (MPD), mast cell disorder, and myeloma (e.g., multiple myeloma), among others.
  • a blood disorder or a hematologic malignancy including, but not limited to, myeloid disorder, lymphoid disorder, leukemia, lymphoma, myelodysplastic syndrome (MDS), myeloproliferative disease (MPD), mast cell disorder, and myeloma (e.g., multiple myeloma), among others.
  • T-cell or NK-cell lymphoma examples include T-cell or NK-cell lymphoma, for example, but not limited to: peripheral T-cell lymphoma; anaplastic large cell lymphoma, for example anaplastic lymphoma kinase (ALK) positive, ALK negative anaplastic large cell lymphoma, or primary cutaneous anaplastic large cell lymphoma; angioimmunoblastic lymphoma; cutaneous T-cell lymphoma, for example mycosis fungoides, Sezary syndrome, primary cutaneous anaplastic large cell lymphoma, primary cutaneous CD30+ T-cell lymphoproliferative disorder; primary cutaneous aggressive epidermotropic CD8+ cytotoxic T-cell lymphoma; primary cutaneous gamma-delta T-cell lymphoma; primary cutaneous small/medium CD4+ T-cell lymphoma, and lymphomatoid papulosis; Adult T-cell Leukemia/Lymphoma (AT
  • the cancer initially susceptible to selective CDK4/6 inhibitor inhibition that may acquire resistance to a selective CDK4/6 inhibitor may include a lymphoma or lymphocytic or myelocytic proliferation disorder or abnormality.
  • the compounds as described herein can be administered to a host suffering from a Hodgkin Lymphoma or a Non-Hodgkin Lymphoma.
  • the host can be suffering from a Non- Hodgkin Lymphoma such as, but not limited to: an AIDS-Related Lymphoma; Anaplastic Large-Cell Lymphoma; Angioimmunoblastic Lymphoma; Blastic NK-Cell Lymphoma; Burkitt’s Lymphoma; Burkitt-like Lymphoma (Small Non-Cleaved Cell Lymphoma); Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma; Cutaneous T-Cell Lymphoma; Diffuse Large B-Cell Lymphoma; Enteropathy-Type T-Cell Lymphoma; Follicular Lymphoma; Hepatosplenic Gamma-Delta T-Cell Lymphoma; Lymphoblastic Lymphoma; Mantle Cell Lymphoma; Marginal Zone Lymphoma; Nasal T-Cell Lymphoma; Pediatric Lymphoma; Peripheral
  • the cancer initially susceptible to selective CDK4/6 inhibitor inhibition that may acquire resistance to a selective CDK4/6 inhibitor may include a Hodgkin Lymphoma, such as, but not limited to: Nodular Sclerosis Classical Hodgkin’s Lymphoma (CHL); Mixed Cellularity CHL; Lymphocyte-depletion CHL; Lymphocyte-rich CHL; Lymphocyte Predominant Hodgkin Lymphoma; or Nodular Lymphocyte Predominant HL.
  • CHL Nodular Sclerosis Classical Hodgkin’s Lymphoma
  • Mixed Cellularity CHL Lymphocyte-depletion CHL
  • Lymphocyte-rich CHL Lymphocyte Predominant Hodgkin Lymphoma
  • Lymphocyte Predominant Hodgkin Lymphoma or Nodular Lymphocyte Predominant HL.
  • the cancer initially susceptible to selective CDK4/6 inhibitor inhibition that may acquire resistance to a selective CDK4/6 inhibitor may include a specific B-cell lymphoma or proliferative disorder such as, but not limited to: multiple myeloma; Diffuse large B cell lymphoma; Follicular lymphoma; Mucosa-Associated Lymphatic Tissue lymphoma (MALT); Small cell lymphocytic lymphoma;Mediastinal large B cell lymphoma; Nodal marginal zone B cell lymphoma (NMZL); Splenic marginal zone lymphoma (SMZL); Intravascular large B-cell lymphoma; Primary effusion lymphoma; or Lymphomatoid granulomatosis;; B-cell prolymphocytic leukemia; Hairy cell leukemia; Splenic lymphoma/leukemia, unclassifiable; Splenic diffuse red pulp small B-cell lymphoma; Hairy cell leukemia-variant; Lymph
  • the cancer initially susceptible to selective CDK4/6 inhibitor inhibition that may acquire resistance to a selective CDK4/6 inhibitor may include a leukemia, for example, but not limited to: Acute lymphoblastic leukemia (ALL); Acute myelogenous leukemia (AML); Chronic lymphocytic leukemia (CLL); Chronic myelogenous leukemia (CML); juvenile myelomonocytic leukemia (JMML); hairy cell leukemia (HCL); acute promyelocytic leukemia (a subtype of AML); large granular lymphocytic leukemia; or Adult T-cell chronic leukemia.
  • ALL Acute lymphoblastic leukemia
  • AML Acute myelogenous leukemia
  • CLL Chronic lymphocytic leukemia
  • CML Chronic myelogenous leukemia
  • JMML juvenile myelomonocytic leukemia
  • HCL hairy cell leukemia
  • acute promyelocytic leukemia
  • the cancer initially susceptible to selective CDK4/6 inhibitor inhibition may include an acute myelogenous leukemia, for example an undifferentiated AML (M0); myeloblastic leukemia (Ml; with/without minimal cell maturation); myeloblastic leukemia (M2; with cell maturation); promyelocytic leukemia (M3 or M3 variant [M3V]); myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]); monocytic leukemia (M5); erythroleukemia (M6); or megakaryoblastic leukemia (M7).
  • M0 undifferentiated AML
  • Ml myeloblastic leukemia
  • M2 myeloblastic leukemia
  • M3V promyelocytic leukemia
  • M4 or M4 variant with eosinophilia [M4E] myelomonocytic leukemia
  • M5 monoc
  • retinoblastoma retinoblastoma (Rb) tumor suppressor protein (Rb-positive), or any of the other cellular signals indicative of the development of CDK4/6 inhibitor resistance, can be determined through any of the standard assays known to one of ordinary skill in the art, including but not limited to Western Blot, ELISA (enzyme linked immunoadsorbent assay), IHC (immunohistochemistry), and FACS (fluorescent activated cell sorting).
  • tissue, cell line or surrogate tissue sample that is utilized e.g., for example Western Blot and ELISA may be used with any or all types of tissues, cell lines or surrogate tissues, whereas the IHC method would be more appropriate wherein the tissue utilized in the methods described herein I s a tumor biopsy.
  • FACs analysis would be most applicable to samples that were single cell suspensions such as cell lines and isolated peripheral blood mononuclear cells. See for example, US 20070212736“Functional Immunohistochemical Cell Cycle Analysis as a Prognostic Indicator for Cancer”.
  • molecular genetic testing may be used for determination of retinoblastoma gene status.
  • retinoblastoma Molecular genetic testing for retinoblastoma includes the following as described in Lohmann and Gallie“Retinoblastoma. Gene Reviews” (2010):“A comprehensive, sensitive and economical approach for the detection of mutations in the RBI gene in retinoblastoma” Journal of Genetics, 88(4), 517-527 (2009).
  • a method for the treatment of a disorder of abnormal cellular proliferation in a host such as a human includes administering an effective amount of CDK9 inhibitor in combination or alternation with an additional active compound.
  • the additional active compound is a chemotherapeutic agent.
  • the additional active compound is an immune checkpoint inhibitor.
  • Immune checkpoint inhibitors for use in the methods described herein include, but are not limited to PD-1 inhibitors, PD-L1 inhibitors, PD-L2 inhibitors, CTLA-4 inhibitors, LAG-3 inhibitors, TIM-3 inhibitors, and V-domain Ig suppressor of T-cell activation (VISTA) inhibitors, or combinations thereof.
  • an immune checkpoint inhibitor is administered in an effective amount in combination with a CDK9 inhibitor compound described herein to treat a cancer, including but not limited to, Hodgkin lymphoma, melanoma, non-small cell lung cancer, including NSCLC with EGFR or ALK genomic tumor aberrations, squamous cell carcinoma of the head and neck, small cell lung cancer, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma, colorectal cancer, colorectal cancer, hepatocellular carcinoma, renal cell carcinoma, small-cell lung carcinoma, bladder carcinoma, B-cell lymphoma, gastric cancer, cervical cancer, liver cancer, advanced Merkel cell carcinoma, esophageal squamous cell carcinoma, or ovarian cancer.
  • a cancer including but not limited to, Hodgkin lymphoma, melanoma, non-small cell lung cancer, including NSCLC with EGFR or ALK genomic tumor aberrations, squamous cell carcinoma of the head and neck, small cell lung cancer,
  • the immune checkpoint inhibitor is a PD-1 inhibitor that blocks the interaction of PD-1 and PD-L1 by binding to the PD-1 receptor, and in turn inhibits immune suppression.
  • the immune checkpoint inhibitor is a PD-1 immune checkpoint inhibitor selected from nivolumab (Opdivo®), pembrolizumab (Keytruda®), pidilizumab, AMP-224 (AstraZeneca and Medlmmune), PF-06801591 (Pfizer), MEDI0680 (AstraZeneca), PDR001 (Novartis), cemiplimad/REGN2810 (Libtayo® Regeneron), MGA012 (MacroGenics), BGB-A317 (BeiGene) SHR-12-1 (Jiangsu Hengrui Medicine Company and Incyte Corporation), TSR-042 (Tesaro), and the PD-L1/VISTA inhibitor CA-170 (Curis Inc.).
  • the immune checkpoint inhibitor is the PD-1 immune checkpoint inhibitor nivolumab (Opdivo®) administered in an effective amount with a compound described herein for the treatment of Hodgkin lymphoma, melanoma, non-small cell lung cancer, including NSCLC with EGFR or ALK genomic tumor aberrations, squamous cell carcinoma of the head and neck, small cell lung cancer, hepatocellular carcinoma, renal cell carcinoma, squamous cell carcinoma, urothelial carcinoma, colorectal cancer, colorectal cancer, hepatocellular carcinoma, or ovarian cancer.
  • a compound described herein for the treatment of Hodgkin lymphoma, melanoma, non-small cell lung cancer including NSCLC with EGFR or ALK genomic tumor aberrations, squamous cell carcinoma of the head and neck, small cell lung cancer, hepatocellular carcinoma, renal cell carcinoma, squamous cell carcinoma, urothelial carcinoma, colorectal cancer, colore
  • Nivolumab has been FDA approved for the use of Hodgkin lymphoma, melanoma, non-small cell lung cancer, including NSCLC with EGFR or ALK genomic tumor aberrations, squamous cell carcinoma of the head and neck, small cell lung cancer, hepatocellular carcinoma, renal cell carcinoma, squamous cell carcinoma, urothelial carcinoma, colorectal cancer, progressive classical Hodgkin lymphoma (cHL), colorectal cancer, urothelial cancer, squamous cell carcinoma of the head and neck, or ovarian cancer.
  • the immune checkpoint inhibitor is the PD-1 immune checkpoint inhibitor pembrolizumab (Keytruda®) administered in an effective amount for the treatment of melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, bladder cancer, urothelial carcinoma, renal cell carcinoma, classical Hodgkin lymphoma, gastric cancer, cervical cancer, liver cancer, primary mediastinal B-cell lymphoma, advanced Merkel cell carcinoma, esophageal squamous cell carcinoma, or urothelial cancer.
  • melanoma non-small cell lung cancer, small cell lung cancer, head and neck cancer, bladder cancer, urothelial carcinoma, renal cell carcinoma, classical Hodgkin lymphoma, gastric cancer, cervical cancer, liver cancer, primary mediastinal B-cell lymphoma, advanced Merkel cell carcinoma, esophageal squamous cell carcinoma, or urothelial cancer.
  • the immune checkpoint inhibitor is the PD-1 immune checkpoint inhibitor pidilizumab (Medivation) administered in an effective amount for refractory diffuse large B-cell lymphoma (DLBCL) or metastatic melanoma.
  • the immune checkpoint inhibitor is the PD-1 immune checkpoint inhibitor cemiplimab (Libtayo/Regeneron) administered in an effective amount for cutaneous squamous cell carcinoma.
  • the immune checkpoint inhibitor is a PD-L1 inhibitor that blocks the interaction of PD-1 and PD-L1 by binding to the PD-L1 receptor, and in turn inhibits immune suppression.
  • PD-L1 inhibitors include, but are not limited to, atezolizumab, durvalumab, KN035CA-170 (Curis Inc.), and LY3300054 (Eli Lilly).
  • the PD-L1 inhibitor is atezolizumab.
  • the PD-L1 inhibitor blocks the interaction between PD-L1 and CD80 to inhibit immune suppression.
  • the immune checkpoint inhibitor is the PD-L1 immune checkpoint inhibitor atezolizumab (Tecentriq®) administered in an effective amount for the treatment of metastatic bladder cancer, small cell lung cancer, metastatic melanoma, metastatic non-small cell lung cancer, or metastatic renal cell carcinoma.
  • the immune checkpoint inhibitor is durvalumab (Imfinzi®; AstraZeneca and Medlmmune) administered in an effective amount for the treatment of small cell lung cancer, non-small cell lung cancer, or bladder cancer.
  • the immune checkpoint inhibitor is the PD- L1 immune checkpoint inhibitor avelumab (Bavencio®; EMD Serono/Pfizer) administered in an effective amount for the treatment of Merkel cell carcinoma or urothelial carcinoma.
  • the immune checkpoint inhibitor is KN035 (Alphamab) administered in an effective amount for the treatment of PD-L1 positive solid tumors.
  • the immune checkpoint inhibitor is a CTLA-4 immune checkpoint inhibitor that binds to CTLA-4 and inhibits immune suppression.
  • CTLA- 4 inhibitors include, but are not limited to, ipilimumab, tremelimumab (AstraZeneca and Medlmmune), AGEN1884 and AGEN2041 (Agenus).
  • the CTLA-4 immune checkpoint inhibitor is ipilimumab (Yervoy®) administered in an effective amount for the treatment of metastatic melanoma, adjuvant melanoma, or non-small cell lung cancer.
  • the immune checkpoint inhibitor is a LAG-3 immune checkpoint inhibitor.
  • LAG-3 immune checkpoint inhibitors include, but are not limited to, BMS-986016 (Bristol-Myers Squibb), GSK2831781 (GlaxoSmithKline), IMP321 (Prima BioMed), LAG525 (Novartis), and the dual PD-1 and LAG-3 inhibitor MGD013 (MacroGenics).
  • the immune checkpoint inhibitor is a TIM-3 immune checkpoint inhibitor.
  • a specific TIM-3 inhibitor includes, but is not limited to, TSR-022 (Tesaro).
  • immune checkpoint inhibitors for use in the invention described herein include, but are not limited to, B7-H3/CD276 immune checkpoint inhibitors such as MGA217, indoleamine 2,3-dioxygenase (IDO) immune checkpoint inhibitors such as Indoximod and INCB024360, killer immunoglobulin-like receptors (KIRs) immune checkpoint inhibitors such as Lirilumab (BMS-986015), carcinoembryonic antigen cell adhesion molecule (CEACAM) inhibitors (e.g., CEACAM-1, -3 and/or -5).
  • B7-H3/CD276 immune checkpoint inhibitors such as MGA217
  • IDO indoleamine 2,3-dioxygenase
  • KIRs killer immunoglobulin-like receptors
  • CEACAM carcinoembryonic antigen cell adhesion molecule
  • anti-CEACAM-1 antibodies are described in WO 2010/125571, WO 2013/082366 and WO 2014/022332, e.g., a monoclonal antibody 34B1, 26H7, and 5F4; or a recombinant form thereof, as described in, e.g., US 2004/0047858, U.S. Pat. No. 7,132,255 and WO 99/052552.
  • the anti- CEACAM antibody binds to CEACAM-5 as described in, e.g., Zheng et al. PLoS One. 2010 September 2; 5(9).
  • checkpoint inhibitors can be molecules directed to B and T lymphocyte attenuator molecule (BTLA), for example as described in Zhang et al, Monoclonal antibodies to B and T lymphocyte attenuator (BTLA) have no effect on in vitro B cell proliferation and act to inhibit in vitro T cell proliferation when presented in a cis, but not trans, format relative to the activating stimulus, Clin Exp Immunol. 2011 Jan; 163(1): 77-87.
  • BTLA B and T lymphocyte attenuator molecule
  • a CDK9 inhibitor is administered in an effective amount for the treatment of abnormal tissue of the female reproductive system such as breast, ovarian, endometrial, or uterine cancer, in combination or alternation with an effective amount of an estrogen inhibitor including but not limited to a SERM (selective estrogen receptor modulator), a SERD (selective estrogen receptor degrader), a complete estrogen receptor degrader, or another form of partial or complete estrogen antagonist.
  • an estrogen inhibitor including but not limited to a SERM (selective estrogen receptor modulator), a SERD (selective estrogen receptor degrader), a complete estrogen receptor degrader, or another form of partial or complete estrogen antagonist.
  • a CDK9 inhibitor is administered in an effective amount for the treatment of abnormal tissue of the male reproductive system such as prostate or testicular cancer, in combination or alternation with an effective amount of an androgen (such as testosterone) inhibitor including but not limited to a selective androgen receptor modulator, a selective androgen receptor degrader, a complete androgen receptor degrader, or another form of partial or complete androgen antagonist.
  • an androgen (such as testosterone) inhibitor including but not limited to a selective androgen receptor modulator, a selective androgen receptor degrader, a complete androgen receptor degrader, or another form of partial or complete androgen antagonist.
  • the prostate or testicular cancer is androgen-resistant.
  • additional active compound is used to describe an agent, other than the selected compound according to the disclosure, which can be used in combination or alternation with a CDK9 inhibitor to achieve a desired result of therapy.
  • the CDK9 inhibitor and the additional active compound are administered in a manner that they are active in vivo during overlapping time periods, for example, have time-period overlapping Cmax, Tmax, AUC or other pharmacokinetic parameter.
  • a CDK9 inhibitor and the additional active compound are administered to a host in need thereof that do not have overlapping pharmacokinetic parameter, however, one has a therapeutic impact on the therapeutic efficacy of the other.
  • the additional active compound is a chemotherapeutic.
  • the additional active compound is a growth factor.
  • a CDK9 inhibitor is administered in combination with an additional active compound, wherein the additional active compound is a standard chemotherapeutic agent treatment modality.
  • the chemotherapeutic agent inhibits cell growth.
  • the cytotoxic chemotherapeutic agent administered is a DNA damaging chemotherapeutic agent.
  • the chemotherapeutic agent is a protein synthesis inhibitor, a DNA-damaging chemotherapeutic, an alkylating agent, a topoisomerase inhibitor, an RNA synthesis inhibitor, a DNA complex binder, a thiolate alkylating agent, a guanine alkylating agent, a tubulin binder, DNA polymerase inhibitor, an anticancer enzyme, RAC1 inhibitor, thymidylate synthase inhibitor, oxazophosphorine compound, integrin inhibitor such as cilengitide, camptothecin or homocamptothecin, antifolate or a folate antimetabolite.
  • the additional therapeutic agent is trastuzumab. In some embodiments, the additional therapeutic agent is lapatinib. In some embodiments, the CDK9 inhibitory compound is dosed with 2, 3, or 4 additional therapeutic agents. In some embodiments, there are t2 additional therapeutic agents. In some embodiments, the two additional therapeutic agents are lapatinib and trastuzumab.
  • the additional therapeutic agent is osimertinib.
  • the additional therapeutic agent is alectinib.
  • the additional therapeutic agent is a MEK inhibitor.
  • the additional therapeutic agent is an Androgen Receptor ligand.
  • the additional therapeutic agent is a BTK inhibitor, for example but not limited to ibrutinib (Imbruvica®) or acalabrutinib (Calquence®).
  • the additional therapeutic agents are a MEK inhibitor and a RAF inhibitor
  • the additional therapeutic agent is a RAF inhibitor.
  • the additional therapeutic agent is regorafenib.
  • the additional active compound is a cytotoxic, DNA-damaging chemotherapeutic agent.
  • Cytotoxic, DNA-damaging chemotherapeutic agents tend to be non specific and, particularly at high doses, toxic to rapidly dividing cells.
  • DNA-damaging chemotherapy or chemotherapeutic agent refers to treatment with a cytostatic or cytotoxic agent (i.e., a compound) to reduce or eliminate the growth or proliferation of undesirable cells, for example cancer cells, wherein the cytotoxic effect of the agent can be the result of one or more of nucleic acid intercalation or binding, DNA or RNA alkylation, inhibition of RNA or DNA synthesis, the inhibition of another nucleic acid-related activity (e.g., protein synthesis), or any other cytotoxic effect.
  • a cytostatic or cytotoxic agent i.e., a compound
  • DNA damaging compounds include, but are not limited to, DNA damaging compounds that can kill cells.
  • DNA damaging chemotherapeutic agents include, but are not limited to, alkylating agents, DNA intercalators, protein synthesis inhibitors, inhibitors of DNA or RNA synthesis, DNA base analogs, topoisomerase inhibitors, telomerase inhibitors, and telomeric DNA binding compounds.
  • alkylating agents include alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as a benzodizepa, carboquone, meturedepa, and uredepa; ethylenimines and methylmel amines, such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylol melamine; nitrogen mustards such as chlorambucil, chlomaphazine, cyclophosphamide, estramustine, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichine, phenesterine, prednimustine, trofosfamide, and uracil mustard; and nitroso ureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine.
  • aziridines
  • DNA- damaging chemotherapeutic agents include daunorubicin, doxorubicin, idarubicin, epirubicin, mitomycin, and streptozocin.
  • Chemotherapeutic antimetabolites include gemcitabine, mercaptopurine, thioguanine, cladribine, fludarabine phosphate, fluorouracil (5-FU), floxuridine, cytarabine, pentostatin, methotrexate, azathioprine, acyclovir, adenine b-1-D- arabinoside, amethopterin, aminopterin, 2-aminopurine, aphidicolin, 8-azaguanine, azaserine, 6-azauracil, 2'-azido-2'-deoxynucleosides, 5-bromodeoxycytidine, cytosine b-1-D- arabinoside, diazooxynorleucine, dideoxynucleosides
  • Chemotherapeutic protein synthesis inhibitors include abrin, aurintricarboxylic acid, chloramphenicol, colicin E3, cycloheximide, diphtheria toxin, edeine A, emetine, erythromycin, ethionine, fluoride, 5-fluorotryptophan, fusidic acid, guanylyl methylene diphosphonate and guanylyl imidodiphosphate, kanamycin, kasugamycin, kirromycin, and O- methyl threonine.
  • Additional protein synthesis inhibitors include modeccin, neomycin, norvaline, pactamycin, paromomycine, puromycin, ricin, shiga toxin, showdomycin, sparsomycin, spectinomycin, streptomycin, tetracycline, thiostrepton, and trimethoprim.
  • Inhibitors of DNA synthesis include alkylating agents such as dimethyl sulfate, nitrogen and sulfur mustards; intercalating agents, such as acridine dyes, actinomycins, anthracenes, benzopyrene, ethidium bromide, propidium diiodide-intertwining; and other agents, such as distamycin and netropsin.
  • alkylating agents such as dimethyl sulfate, nitrogen and sulfur mustards
  • intercalating agents such as acridine dyes, actinomycins, anthracenes, benzopyrene, ethidium bromide, propidium diiodide-intertwining
  • other agents such as distamycin and netropsin.
  • Topoisomerase inhibitors such as irinotecan, teniposide, coumermycin, nalidixic acid, novobiocin, and oxolinic acid; inhibitors of cell division, including colcemide, mitoxantrone, colchicine, vinblastine, and vincristine; and RNA synthesis inhibitors including actinomycin D, a-amanitine and other fungal amatoxins, cordycepin (3 '-deoxy adenosine), dichlororibofuranosyl benzimidazole, rifampicine, streptovaricin, and streptoly digin also can be used as the DNA damaging compound.
  • RNA synthesis inhibitors including actinomycin D, a-amanitine and other fungal amatoxins, cordycepin (3 '-deoxy adenosine), dichlororibofuranosyl benzimidazole, rifampicine, streptovaricin, and strepto
  • the chemotherapeutic agent is a DNA complex binder such as camptothecin, or etoposide; a thiolate alkylating agent such as nitrosourea, BCNU, CCNU, ACNU, or fotesmustine; a guanine alkylating agent such as temozolomide, a tubulin binder such as vinblastine, vincristine, vinorelbine, vinflunine, cryptophycin 52, halichondrins, such as halichondrin B, dolastatins, such as dolastatin 10 and dolastatin 15, hemiasterlins, such as hemiasterlin A and hemiasterlin B, colchicine, combrestatins, 2-methoxyestradiol, E7010, paclitaxel, docetaxel, epothilone, discodermolide; a DNA polymerase inhibitor such as cytarabine; an anti cancer enzyme such as asparagina
  • the topoisomerase inhibitor is a type I inhibitor. In another embodiment the topoisomerase inhibitor is a type II inhibitor.
  • DNA-damaging chemotherapeutic agents include, but are not limited to, cisplatin, hydrogen peroxide, carboplatin, procarbazine, ifosfamide, bleomycin, plicamycin, taxol, transplatinum, thiotepa, oxaliplatin, and the like, and similar acting-type agents.
  • the DNA damaging chemotherapeutic agent is selected from the group consisting of cisplatin, carboplatin, camptothecin, and etoposide.
  • chemotherapeutic agents include, but are not limited to, radioactive molecules, toxins, also referred to as cytotoxins or cytotoxic agents, which includes any agent that is detrimental to the viability of cells, agents, and liposomes or other vesicles containing chemotherapeutic compounds.
  • General anticancer pharmaceutical agents include: Vincristine (Oncovin®), liposomal vincristine (Marqibo®), Cytarabine (cytosine arabinoside, ara-C, or Cytosar®), L-asparaginase (Elspar®) or PEG-L-asparaginase (pegaspargase or Oncaspar®), Etoposide (VP-16), Teniposide (Vumon®), 6-mercaptopurine (6-MP or Purinethol®), Prednisone, and Dexamethasone (Decadron).
  • chemotherapeutic agents include but are not limited to 5-fluorouracil, dacarbazine, alkylating agents, anthramycin (AMC)), anti-mitotic agents, cis-dichlorodiamine platinum (II) (DDP) cisplatin), diamino dichloro platinum, anthracyclines, antibiotics, antimetabolites, asparaginase, BCG live (intravesical), bleomycin sulfate, calicheamicin, cytochalasin B, dactinomycin (formerly actinomycin), daunorubicin HC1, daunorubicin citrate, denileukin diftitox, dihydroxy anthracin dione, Docetaxel, doxorubicin HC1, E.
  • coli L-asparaginase Erwinia L-asparaginase, etoposide citrovorum factor, etoposide phosphate, gemcitabine HC1, idarubicin HC1, interferon a-2b, irinotecan HC1, maytansinoid, mechlorethamine HC1, melphalan HC1, mithramycin, mitomycin C, mitotane, polifeprosan 20 with carmustine implant, procarbazine HC1, streptozotocin, teniposide, thiotepa, topotecan HC1, valrubicin, vinblastine sulfate, vincristine sulfate, and vinorelbine tartrate.
  • cytotoxic chemotherapeutic agents for use with the methods described herein include: epirubicin, abraxane, taxotere, epothilone, tafluposide, vismodegib, azacytidine, doxifluridine, vindesine, and vinorelbine.
  • the chemotherapeutic agent is a DNA complex binder. In some embodiments, the chemotherapeutic agent is a tubulin binder. In some embodiments, the chemotherapeutic agent is an alkylating agent. In some embodiments, the chemotherapeutic agent is a thiolate alkylating agent.
  • Additional active compounds may include 2- methoxyestradiol or 2ME2, fmasunate, etaracizumab (MEDI-522), HLL1, huN901-DMl, atiprimod, saquinavir mesylate, ritonavir, nelfmavir mesylate, indinavir sulfate, plitidepsin, P276-00, tipifamib, lenalidomide, thalidomide, pomalidomide, simvastatin, and celecoxib.
  • Chemotherapeutic agents useful in the methods described herein include, but are not limited to, Trastuzumab (Herceptin®), Pertuzumab (PerjetaTM), Lapatinib (Tykerb®), Gefitinib (Iressa®), Erlotinib (Tarceva®), Cetuximab (Erbitux®), Panitumumab (Vectibix®), Vandetanib (Caprelsa®), Vemurafenib (Zelboraf®), Vorinostat (Zolinza®), Romidepsin (Istodax®), Bexarotene (Targretin®), Abtretinoin (Panretin®), Tretinoin (Vesanoid®), Carfilzomib (KyprobsTM), Pralatrexate (Folotyn®), Bevacizumab (Avastin®), Ziv-aflibercept (Zaltrap®), Sor
  • Additional active compounds contemplated include, but are not limited to, a calcineurin inhibitor, e.g. a cyclosporin or an ascomycin, e.g. Cyclosporin A (Neoral®), FK506 (tacrolimus), pimecrolimus, a mTOR inhibitor, e.g. rapamycin or a derivative thereof, e.g. Sirolimus (Rapamune®), Everolimus (Certican®), temsirolimus, zotarolimus, biolimus-7, biolimus-9, a rapalog, e.g.
  • Mycophenolate Mofetil (CellCept®), OKT3 (Orthoclone OKT3®), Prednisone, ATGAM®, Thymoglobulin®, Brequinar Sodium, OKT4, T10B9.A-3A, 33B3.1, 15- deoxysperguabn, tresperimus, Leflunomide Arava®, anti-CD25, anti-IL2R, Basibximab (Simulect®), Dacbzumab (Zenapax®), mizoribine, dexamethasone, ISAtx-247, SDZ ASM 981 (pimecrolimus, Elidel®), Abatacept, belatacept, LFA31g, etanercept (sold as Enbrel® by ImmuneXcite), adalimumab (Humira®), infliximab (Remicade®), an anti-LFA-1 antibody, natabzumab (Antegren®
  • the chemotherapeutic agent is an estrogen receptor ligand such as tamoxifen, raloxifene, fulvestrant, anordrin, apeledoxifene, broparestriol, chlorotrianisene, clomiphene citrate, cyclofenil, lasofoxifene, ormeloxifene, or toremifene; an androgen receptor ligand such as bicalutamide, enzalutamide, apalutamide, cyproterone acetate, chlormadinone acetate, spironolactone, canrenone, drospirenone, ketoconazole, topilutamide, abiraterone acetate, or cimetidine; an aromatase inhibitor such as letrozole, anastrozole, or exemestane; an anti-inflammatory such as prednisone; an oxidase inhibitor such as allopurinol; an anticancer antibody;
  • the CDK9 inhibitor is further combined with the use of hematopoietic growth factors including, but not limited to, granulocyte colony stimulating factor (G-CSF, for example, sold as Neupogen® (filgrastim), Neulasta® (peg-filgrastim), or lenograstim), granulocyte-macrophage colony stimulating factor (GM-CSF, for example sold as molgramostim and sargramostim (Leukine®)), M-CSF (macrophage colony stimulating factor), Thrombopoietin (megakaryocyte growth development factor (MGDF), for example sold as Romiplostim® and Eltrombopag®) interleukin (IL)-12, interleukin-3, interleukin- 11 (adipogenesis inhibiting factor or oprelvekin), SCF (stem cell factor, steel factor, kit-ligand, or KL) and erythropoietin (EPO), and their
  • Additional active compounds contemplated herein, particularly in the treatment of abnormal tissue of the female reproductive system such as breast, ovarian, endometrial, or uterine cancer include a CDK9 inhibitor described herein in combination with an estrogen inhibitor including but not limited to a SERM (selective estrogen receptor modulator), a SERD (selective estrogen receptor degrader), a complete estrogen receptor degrader, or another form of partial or complete estrogen antagonist.
  • an estrogen inhibitor including but not limited to a SERM (selective estrogen receptor modulator), a SERD (selective estrogen receptor degrader), a complete estrogen receptor degrader, or another form of partial or complete estrogen antagonist.
  • Partial anti-estrogens include raloxifene and tamoxifen retain some estrogen-like effects.
  • Complete anti-estrogens include fulvestrant.
  • Non-limiting examples of anti-estrogen compounds are provided in WO 2014/19176 assigned to Astra Zeneca, W02013/090921, WO 2014/203129, WO 2014/203132, and US2013/0178445 assigned to Olema Pharmaceuticals, W02017/100712, W02017/100715, W02018/081168, and WO2018/148576 assigned to G1 Therapeutics, and U.S. Patent Nos. 9,078,871, 8,853,423, and 8,703,810, as well as US 2015/0005286, WO 2014/205136, and WO 2014/205138.
  • anti-estrogen compounds include: SERMS such as anordrin, arzoxifene, avasene, broparestriol, clomiphene citrate, cyclofenil, droloxifene, endoxifen, idoxifene, lasofoxifene, ormeloxifene, pipendoxifene, raloxifene, tamoxifen, toremifene, and fulvestrant; aromatase inhibitors such as aminoglutethimide, testolactone, anastrozole, exemestane, fadrozole, formestane, and letrozole; and antigonadotropins such as leuprorelin, cetrorelix, allylestrenol, chloromadinone acetate, delmadinone acetate, dydrogesterone, medroxyprogesterone acetate, megestrol acetate, nomege
  • SERMS such
  • anti-estrogen compounds include: SERDS such as fulvestrant, brilanestrant (GDC0810), elacestrant (RAD1901), etacstil (GW5638), GW7604, AZD9496, GDC-0927, GDC9545 (RG6171), LSZ102, and SAR439859.
  • SERDS such as fulvestrant, brilanestrant (GDC0810), elacestrant (RAD1901), etacstil (GW5638), GW7604, AZD9496, GDC-0927, GDC9545 (RG6171), LSZ102, and SAR439859.
  • Additional active compounds for combining with a CDK9 inhibitor described herein, particularly in the treatment of abnormal tissue of the male reproductive system such as prostate or testicular cancer include, but are not limited to, an androgen (such as testosterone) inhibitor including but not limited to a selective androgen receptor modulator, a selective androgen receptor degrader, a complete androgen receptor degrader, or another form of partial or complete androgen antagonist.
  • an androgen (such as testosterone) inhibitor including but not limited to a selective androgen receptor modulator, a selective androgen receptor degrader, a complete androgen receptor degrader, or another form of partial or complete androgen antagonist.
  • the prostate or testicular cancer is androgen-resistant.
  • Non-limiting examples of anti-androgen compounds are provided in WO 2011/156518 and US Patent Nos. 8,455,534 and 8,299,112.
  • anti-androgen compounds include: chlormadinone acetate, spironolactone, canrenone, drospirenone, ketoconazole, topilutamide, abiraterone acetate, and cimetidine.
  • the additional active compound combined with a CDK9 inhibitor described herein may include a kinase inhibitor, including but not limited to a phosphoinositide 3-kinase (PI3K) inhibitor, a Bruton’s tyrosine kinase (BTK) inhibitor, or a spleen tyrosine kinase (Syk) inhibitor, or a combination thereof.
  • PI3K phosphoinositide 3-kinase
  • BTK Bruton’s tyrosine kinase
  • Syk spleen tyrosine kinase
  • the additional active compound combined with a CDK9 inhibitor described herein may include a PARP inhibitor, for example, but not limited to, olaparib, rucaparib, niraparib, talazoparib, veliparib, pamiparib, CEP-9722, E7016, and 3-aminobenzamide, or a pharmaceutically acceptable salt thereof.
  • a PARP inhibitor for example, but not limited to, olaparib, rucaparib, niraparib, talazoparib, veliparib, pamiparib, CEP-9722, E7016, and 3-aminobenzamide, or a pharmaceutically acceptable salt thereof.
  • PI3k inhibitors are well known.
  • Examples of PI3 kinase inhibitors include but are not limited to Wortmannin, demethoxyviridin, perifosine, idelalisib, pictilisib, Palomid 529, ZSTK474, PWT33597, CUDC-907, and AEZS-136, duvelisib, GS-9820, GDC-0032 (2-[4-[2- (2-Isopropyl-5-methyl-l,2,4-triazol-3-yl)-5,6-dihydroimidazo[l,2-d][l,4]benzoxazepin-9- yl]pyrazol-l-yl]-2-methylpropanamide), MLN-1117 ((2R)-l-Phenoxy-2-butanyl hydrogen (S)-methylphosphonate; or Methyl(oxo) ⁇ [(2R)-l-phenoxy-2-butanyl]oxy ⁇
  • XL-765 N-(3-(N-(3-(3,5-dimethoxyphenylamino)quinoxalin-2-yl)sulfamoyl)phenyl)-3- methoxy-4-methylbenzamide
  • GSK1059615 5-[[4-(4-Pyridinyl)-6- quinolinyl]methylene]-2,4-thiazolidenedione
  • PX886 [(3aR,6E,9S,9aR,10R,l laS)-6-
  • BTK inhibitors are well known.
  • BTK inhibitors include ibrutinib (also known as PCI-32765)(ImbruvicaTM) (l-[(3R)-3-[4-amino-3-(4-phenoxy-phenyl)pyrazolo[3,4- d] py rimidin- 1 -y ljpiperidin- 1 -y l]prop-2-en- 1 -one), acalabrutinib (Calquence), dianilinopyrimidine-based inhibitors such as AVL-101 and AVL-291/292 (N-(3-((5-fluoro-2- ((4-(2-methoxyethoxy)phenyl)amino)pyrimidin-4-yl)amino)phenyl)acrylamide) (Avila
  • Syk inhibitors are well known, and include, for example, Cerdulatinib (4- (cyclopropylamino)-2-((4-(4-(ethylsulfonyl)piperazin-l-yl)phenyl)amino)pyrimidine-5- carboxamide), entospletinib (6-(lH-indazol-6-yl)-N-(4-morpholinophenyl)imidazo[l,2- a]pyrazin-8-amine), fostamatinib ([6-( ⁇ 5-Fluoro-2-[(3,4,5-trimethoxyphenyl)amino]-4- pyrimidinyl ⁇ amino)-2,2-dimethyl-3-oxo-2,3-dihydro-4H-pyrido[3,2-b] [l,4]oxazin-4- yl]methyl dihydrogen phosphate), fostamatinib disodium salt (sodium (6-((
  • the additional active compound can also be a B-cell lymphoma 2 (Bcl-2) protein inhibitor.
  • BCL-2 inhibitors are known in the art, and include, for example, ABT-199 (4-[4- [[2-(4-Chlorophenyl)-4,4-dimethylcyclohex-l-en-l-yl]methyl]piperazin-l-yl]-N-[[3-nitro-4- [[(tetrahydro-2H-pyran-4-yl)methyl]amino]phenyl]sulfonyl]-2-[(lH- pyrrolo[2,3-b]pyridin-5- yl)oxy]benzamide), ABT-737 (4-[4-[[2-(4-chlorophenyl)phenyl]methyl]piperazin-l-yl]-N-[4- [[(2R)-4-(dimethylamino)-l-phenylsulfanylbutan-2-yl] amino]
  • ABT-263 ((R)-4-(4-((4'-chloro-4,4-dimethyl-3, 4,5,6- tetrahydro-[l, l'-biphenyl]-2-yl)methyl)piperazin-l-yl)-N-((4-((4-morpholino-l-
  • Additional active compounds for use in the methods contemplated herein include, but are not limited to, midazolam, MEK inhibitors, RAS inhibitors, ERK inhibitors, ALK inhibitors, HSP inhibitors (for example, HSP70 and HSP 90 inhibitors, or a combination thereof), RAF inhibitors, apoptotic compounds, topoisomerase inhibitors, AKT inhibitors, including but not limited to, MK-2206, GSK690693, Perifosine, (KRX-0401), GDC-0068, Triciribine, AZD5363, Honokiol, PF-04691502, and Miltefosine, or FLT-3 inhibitors, including but not limited to, P406, Dovitinib, Quizartinib (AC220), Amuvatinib (MP-470), Tandutinib (MLN518), ENMD-2076, and KW-2449, or combinations thereof.
  • HSP inhibitors for example, HSP70 and HSP 90 inhibitors,
  • Examples of MEK inhibitors include but are not limited to trametinib /GSK1120212 (N-(3- ⁇ 3-Cyclopropyl- 5-[(2-fluoro-4-iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydropyrido[4,3- d]pyrimidin-l(2H-yl ⁇ phenyl)acetamide), selumetinib (6-(4-bromo-2-chloroanilino)-7-fluoro- N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide),
  • pimasertib/AS703026/MSC1935369 ((S)-N-(2,3-dihydroxypropyl)-3-((2-fluoro-4- iodophenyl)amino)isonicotinamide), XL-518/GDC-0973 (l-( ⁇ 3,4-difluoro-2-[(2-fluoro-4- iodophenyl)amino]phenyl ⁇ carbonyl)-3-[(2S)-piperidin-2-yl]azetidin-3-ol),
  • RAS inhibitors include but are not limited to Reolysin and siG12D LODER.
  • ALK inhibitors include but are not limited to Crizotinib, AP26113, and LDK378.
  • HSP inhibitors include but are not limited to Geldanamycin or 17-N-Allylamino-17-demethoxygeldanamycin (17AAG), and Radicicol.
  • ERK inhibitors include SCH772984 (Merck/Schering-Plough), VTX-l le (Vertex), DEL-22379, Ubxertinib (BVD-523, VRT752271), GDC-0994, FR 180204, XMD8- 92, and ERK5-IN-1.
  • Raf inhibitors are well known, and include, for example, Vemurafmib (N-[3-[[5-(4- Chlorophenyl)-lH-pynOlo[2,3-b]pyridin-3-yl]carbonyl]-2,4-difluorophenyl]-l- propanesulfonamide), sorafenib tosylate (4-[4-[[4-chloro-3-
  • topoisomerase I inhibitors useful in the methods described herein include (S)- 10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-lH-pyrano[3',4':6,7]indobzino[l,2- b]quinobne-3,14(4H,12H)-dione monohydrochloride (topotecan), (S)-4-ethyl-4-hydroxy-lH- pyrano[3',4' : 6,7]indolizino[ 1 ,2-b] quinoline-3, 14-(4H, 12H)-dione (camptothecin), (1 S,9S)- 1 - Amino-9-ethyl-5-fluoro-l ,2,3,9, 12, 15-hexahydro-9-hydroxy-4-methyl-10H, 13H- benzo(de)pyrano(3',4' : 6,7)indolizino(l ,2-b)
  • the methods provided herein include methods of treating a patient with an EGFR-mutant cancer by administering a CDK9 inhibitor in combination or alternation with a EGFR-TKI as described herein.
  • EGFR-TKIs for use in the invention include, but are not limited to, erlotinib (Tarceva), gefitinib (Iressa), afatinib (Gilotrif), rociletinib (CO- 1686), osimertinib mesylate (Tagrisso), olmutinib (Olita), naquotinib (ASP8273), josartinib (EGF816), PF-06747775 (Pfizer), icotinib (BPI-2009), neratinib (HKI-272; PB272); avitinib (ACOOIO), EAI045, tarloxotinib (TH-4000; PR-610), PF-
  • EGFRi useful in the methods described herein include:
  • Erlotinib (Tarveva®), or a pharmaceutically acceptable salt thereof, which is a first- generation EGFR inhibitor and binds in a reversible fashion to the adenosine triphosphate (ATP) binding site of the EGFR receptor and has the chemical structure:
  • Gefitinib (Iressa®), or a pharmaceutically acceptable salt thereof, which is a first- generation EGFR-TKI and binds to the adenosine triphosphate (ATP)-binding site of EGFR.
  • Gefitinib has the chemical structure:
  • Afatinib (Gilotrif®), or a pharmaceutically acceptable salt thereof, which is a second- generation EGFR-TKI which irreversibly binds to and inhibits human epidermal growth factor receptors 1 and 2 (EGFR-1; HER2) and has the chemical structure:
  • Neratinib (HKI-272 or PB272), or a pharmaceutically acceptable salt thereof, which is a second-generation, orally available, 6,7-disubstituted-4-anilinoquinoline-3-carbonitrile inhibitor of EGFR having the chemical structure:
  • Dacomitinib (PF-299 and PF-00299804), or a pharmaceutically acceptable salt thereof, which is an orally bioavailable, highly selective, second-generation small-molecule inhibitor of the pan-epidermal growth factor receptor (EGFR) family of tyrosine kinases (ErbB family) with potential antineoplastic activity.
  • EGFR pan-epidermal growth factor receptor
  • ErbB family tyrosine kinases
  • Dacomitinib specifically and irreversibly binds to and inhibits human EGFR subtypes, resulting in inhibition of proliferation and induction of apoptosis in EGFR-expressing tumor cells.
  • Dacomitinib has the chemical structure:
  • Icotinib (BPI-2009; Conmana®), or a pharmaceutically acceptable salt thereof, which is a third-generation EGFR-TKI quinazoline-based inhibitor of EGFR.
  • Icotinib selectively inhibits the wild-type and several mutated forms of EGFR tyrosine kinase, and has the chemical structure:
  • Osimertinib (AZD9291; Tagrisso®), or a pharmaceutically acceptable salt thereof, which is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) for T790M mutated EGFR NSCLC and has the chemical structure:
  • Olmutinib (Olita®), or a pharmaceutically acceptable salt thereof, which is a third- generation EGFR-TKI that acts by irreversibly blocking the epidermal growth factor receptor (EGFR), and has the chemical structure:
  • Naquotinib (ASP8273), or a pharmaceutically acceptable salt thereof, which is third- generation, mutant-selective EGFR inhibitor which covalently binds to and inhibits the activity of mutant forms of EGFR, including the T790M EGFR mutant, thereby preventing EGFR- mediated signaling, and has the chemical structure:
  • Nazartinib (EGF816), or a pharmaceutically acceptable salt thereof, which is a third- generation, irreversible, mutant-selective EGFR inhibitor which covalently binds to and inhibits the activity of mutant forms of EGFR, including the T790M EGFR mutant, thereby preventing EGFR-mediated signaling.
  • Nazartinib has the chemical structure:
  • PF-06747775 or a pharmaceutically acceptable salt thereof, which is a third-generation inhibitor of the EGFR mutant form T790M.
  • PF-06747775 specifically binds to and inhibits EGFR T790M, a secondarily acquired resistance mutation, which prevents EGFR-mediated signaling and leads to cell death in EGFR T790M-expressing tumor cells.
  • PF-06747775 has the chemical structure:
  • Avitinib is a third-generation EGFR-TKI which covalently binds to and inhibits the activity of mutant forms of EGFR, including the drug-resistant T790M EGFR mutant having the chemical structure:
  • Tarloxotinib is a third-generation, irreversible EGFR-tyrosine kinase inhibitor having the chemical structure:
  • PF-06459988 or a pharmaceutically acceptable salt thereof, which is an orally available third-generation, irreversible inhibitor of EGFR which specifically binds to and inhibits mutant forms of EGFR, including the secondary acquired resistance mutation T790M, which prevents EGFR-mediated signaling and leads to cell death in EGFR-mutant-expressing tumor cells.
  • PF- 06459988 has the chemical structure:
  • Tesevatinib (XL647, EXEL-7647 and KD-019), or a pharmaceutically acceptable salt thereof, which is an orally bioavailable EGFR inhibitor having the chemical structure:
  • Transtinib or a pharmaceutically acceptable salt thereof, which is a third-generation, irreversible EGFR-TKI with activity against L858R/T790M mutant NSCLC cell lines and xenografts.
  • Transtinib has the chemical structure:
  • WZ-3146 or a pharmaceutically acceptable salt thereof, which is a third-generation, irreversible pyrimidine-based T790M EGFR-TKI having the chemical structure:
  • WZ8040 or a pharmaceutically acceptable salt thereof, which is a third-generation, irreversible T790M EGFR-mutant inhibitor having the chemical structure:
  • CNX-2006 or a pharmaceutically acceptable salt thereof, which is a third-generation mutant-selective EGFR inhibitor that selectively targets T790M substitution.
  • CNX-2006 has the chemical structure:
  • EAI045 or a pharmaceutically acceptable salt thereof, which is a fourth-generation EGFR-TKI which inhibits L858R/T790M EGFR-mutant NSCLC, as well as C797S and C797G EGFR-mutant NSCLC having the chemical structure:
  • Cruatinib or a pharmaceutically acceptable salt thereof, which is a dual ALK and EGFR inhibitor that has been shown to successfully inhibit the T790M/C797S/dell9 EGFR mutant, particularly in combination with an anti-EGFR antibody such as cetuximab or panitumumab (see Uchibori, K. et al. Nat. Commun. 2017, 8: 14768).
  • Brigatinib has the following structure:
  • Vandetanib (Caprelsa), or a pharmaceutically acceptable salt thereof, which is an inhibitor of EGFR, VEGFR, and RET-tyrosine kinase having the chemical structure:
  • Norcantharadin or a pharmaceutically acceptable salt thereof, which is an inhibitor of EGFR and c-Met having the chemical structure:
  • Canertinib or a pharmaceutically acceptable salt thereof, which is an irreversible inhibitor of EGFR, Her-2, and ErbB4 having the chemical structure:
  • Lifirafenib (BGB-283), or a pharmaceutically acceptable salt thereof, which is a potent inhibitor of EGFR and RAF having the chemical structure:
  • AZ5104 or a pharmaceutically acceptable salt thereof, which is a potent inhibitor of both wild-type and mutant (L858R/T790M, L858R, L861Q) EGFR having the chemical structure:
  • AST-1306 or a pharmaceutically acceptable salt thereof, which is an irreversible inhibitor of EGFR (including the T790M/L858R mutation) and ErbB2 having the chemical structure:
  • AEE788 (NVP-AEE788), or a pharmaceutically acceptable salt thereof, which is a potent inhibitor of EGFR and HER2/ErbB2 having the chemical structure:
  • Pelitinib or a pharmaceutically acceptable salt thereof, which is a potent irreversible inhibitor of EGFR having the chemical structure:
  • PD153035 or a pharmaceutically acceptable salt thereof, which is a potent and specific inhibitor of EGFR having the chemical structure:
  • CUDC-101 or a pharmaceutically acceptable salt thereof, which is a potent inhibitor of EGFR, HD AC, and HER2 having the chemical structure:
  • Sapitinib (AZD8931), or a pharmaceutically acceptable salt thereof, which is a reversible inhibitor of EGFR, ErbB2, and ErbB3 having the chemical structure:
  • Lapatinib (Tykerb), or a pharmaceutically acceptable salt thereof, which reversibly blocks phosphorylation of the epidermal growth factor receptor (EGFR), ErbB2, and the Erk- 1 and-2 and AKT kinases; it also inhibits cyclin D protein levels in human tumor cell lines and xenografts.
  • EGFR and ErbB2 have been implicated in the growth of various tumor types having the structure:
  • An active compound described herein, or its salt, isotopic analog, or prodrug can be administered in an effective amount to a host to treat any of the disorders described herein using any suitable approach which achieves the desired therapeutic result.
  • the amount and timing of active compound administered will, of course, be dependent on the host being treated, the instructions of the supervising medical specialist, on the time course of the exposure, on the manner of administration, on the pharmacokinetic properties of the particular active compound, and on the judgment of the prescribing physician.
  • the dosages given below are a guideline and the physician can titrate doses of the compound to achieve the treatment that the physician considers appropriate for the host.
  • the physician can balance a variety of factors such as age and weight of the host, presence of preexisting disease, as well as presence of other diseases.
  • the pharmaceutical composition may be formulated as any pharmaceutically useful form, e.g., as an aerosol, a cream, a gel, a pill, an injection or infusion solution, a capsule, a tablet, a syrup, a transdermal patch, a subcutaneous patch, a dry powder, an inhalation formulation, in a medical device, suppository, buccal, or sublingual formulation, parenteral formulation, or an ophthalmic solution.
  • Some dosage forms, such as tablets and capsules are subdivided into suitably sized unit doses containing appropriate quantities of the active components, e.g., an effective amount to achieve the desired purpose.
  • the therapeutically effective dosage of any active compound described herein will be determined by the health care practitioner depending on the condition, size and age of the patient as well as the route of delivery.
  • a dosage from about 0.1 to about 200 mg/kg has therapeutic efficacy, with all weights being calculated based upon the weight of the active compound, including the cases where a salt is employed.
  • the dosage is at about or greater than 0.1, 0.5, 1, 5, 10, 25, 50, 75, 100, 125, 150, 175, or 200 mg/kg.
  • the dosage may be the amount of compound needed to provide a serum concentration of the active compound of up to about 10 nM, 50 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 mM, 5 pM, 10 pM, 20 pM, 30 pM, or 40 pM.
  • the pharmaceutical composition is in a dosage form that contains from about 0.1 mg to about 2000 mg, from about 10 mg to about 1000 mg, from about 100 mg to about 800 mg, or from about 200 mg to about 600 mg of the active compound and optionally from about 0.1 mg to about 2000 mg, from about 10 mg to about 1000 mg, from about 100 mg to about 800 mg, or from about 200 mg to about 600 mg of an additional active agent in a unit dosage form.
  • dosage forms with at least 5, 10, 15, 20, 25, 50, 100, 200, 250, 300, 400, 500, 600, 700, or 750 mg of active compound, or its salt.
  • the pharmaceutical composition may also include a molar ratio of the active compound and an additional active agent, in a ratio that achieves the desired results.
  • compounds disclosed herein or used as described are administered once a day (QD), twice a day (BID), or three times a day (TID). In some embodiments, compounds disclosed herein or used as described are administered at least once a day for at least 21 days, at least 24 days, at least 28 days, at least 35 days, at least 45 days, at least 60 days, at least 75 days, at least 90 days, at least 120 days, at least 180 days, or longer.
  • Compounds disclosed herein or used as described herein may be administered orally, topically, parenterally, by inhalation or spray, sublingually, via implant, including ocular implant, transdermally, via buccal administration, rectally, as an ophthalmic solution, injection, including ocular injection, intravenous, intramuscular, inhalation, intra-aortal, intracranial, subdermal, intraperitoneal, subcutaneous, transnasal, sublingual, or rectal or by other means, in dosage unit formulations containing conventional pharmaceutically acceptable carriers.
  • the compound can be administered, as desired, for example, via intravitreal, intrastromal, intracameral, sub-tenon, sub-retinal, retro-bulbar, peribulbar, suprachorodial, conjunctival, subconjunctival, episcleral, periocular, transscleral, retrobulbar, posterior juxtascleral, circumcomeal, or tear duct injections, or through a mucus, mucin, or a mucosal barrier, in an immediate or controlled release fashion or via an ocular device.
  • an oral administration can be in any desired form such as a solid, gel or liquid, including a solution, suspension, or emulsion.
  • the compounds or salts are administered by inhalation, intravenously, or intramuscularly as a liposomal suspension.
  • the active compound or salt may be in the form of a plurality of solid particles or droplets having any desired particle size, and for example, from about 0.01, 0.1 or 0.5 to about 5, 10, 20 or more microns, and optionally from about 1 to about 2 microns.
  • Compounds as disclosed in the herein have demonstrated good pharmacokinetic and pharmacodynamics properties, for instance when administered by the oral or intravenous routes.
  • the pharmaceutical formulations can comprise an active compound described herein or a pharmaceutically acceptable salt thereof, in any pharmaceutically acceptable carrier.
  • water may sometimes be the carrier of choice for water-soluble compounds or salts.
  • an organic vehicle such as glycerol, propylene glycol, polyethylene glycol, or mixtures thereof, can be suitable. In the latter instance, the organic vehicle can contain a substantial amount of water.
  • the solution in either instance can then be sterilized in a suitable manner known to those in the art, and for illustration by filtration through a 0.22-micron filter. Subsequent to sterilization, the solution can be dispensed into appropriate receptacles, such as depyrogenated glass vials. The dispensing is optionally done by an aseptic method. Sterilized closures can then be placed on the vials and, if desired, the vial contents can be lyophilized.
  • Carriers include excipients and diluents and must be of sufficiently high purity and sufficiently low toxicity to render them suitable for administration to the patient being treated.
  • the carrier can be inert or it can possess pharmaceutical benefits of its own.
  • the amount of carrier employed in conjunction with the compound is sufficient to provide a practical quantity of material for administration per unit dose of the compound.
  • Classes of carriers include, but are not limited to binders, buffering agents, coloring agents, diluents, disintegrants, emulsifiers, flavorants, glidents, lubricants, preservatives, stabilizers, surfactants, tableting agents, and weting agents.
  • Some carriers may be listed in more than one class, for example vegetable oil may be used as a lubricant in some formulations and a diluent in others.
  • Exemplary pharmaceutically acceptable carriers include sugars, starches, celluloses, powdered tragacanth, malt, gelatin; talc, and vegetable oils.
  • Optional active agents may be included in a pharmaceutical composition, which do not substantially interfere with the activity of the compound of the present invention.
  • a biological buffer can be any solution which is pharmacologically acceptable and which provides the formulation with the desired pH, i.e., a pH in the physiologically acceptable range.
  • buffer solutions include saline, phosphate buffered saline, Tris buffered saline, Hank’s buffered saline, and the like.
  • the pharmaceutical compositions can be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, creams, ointments, lotions or the like, preferably in unit dosage form suitable for single administration of a precise dosage.
  • the compositions will include an effective amount of the selected drug in combination with a pharmaceutically acceptable carrier and, in addition, can include other pharmaceutical agents, adjuvants, diluents, buffers, and the like.
  • compositions of the disclosure can be administered as pharmaceutical formulations including those suitable for oral (including buccal and sub-lingual), rectal, nasal, topical, pulmonary, vaginal or parenteral (including intramuscular, intra-arterial, intrathecal, subcutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation.
  • oral including buccal and sub-lingual
  • rectal including nasal, topical, pulmonary, vaginal or parenteral (including intramuscular, intra-arterial, intrathecal, subcutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation.
  • parenteral including intramuscular, intra-arterial, intrathecal, subcutaneous and intravenous administration or in a form suitable for administration by inhalation or insufflation.
  • the preferred manner of administration is intravenous or oral using a convenient daily dosage regimen which can be adjusted according to the degree of affliction.
  • conventional nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, and the like, an active compound as described herein and optional pharmaceutical adjuvants in an excipient, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension.
  • the pharmaceutical composition to be administered can also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and the like.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and the like.
  • permeation enhancer excipients including polymers such as: polycations (chitosan and its quaternary ammonium derivatives, poly-L- arginine, aminated gelatin); polyanions (TV-carboxy methyl chitosan, poly-acrylic acid); and, thiolated polymers (carboxymethyl cellulose-cysteine, polycarbophil-cysteine, chitosan- thiobutylamidine, chitosan-thiogly colic acid, chitosan-glutathione conjugates).
  • polycations chitosan and its quaternary ammonium derivatives, poly-L- arginine, aminated gelatin
  • polyanions TV-carboxy methyl chitosan, poly-acrylic acid
  • thiolated polymers carboxymethyl cellulose-cysteine, polycarbophil-cysteine, chitosan- thiobutylamidine, chitosan
  • the composition will generally take the form of a tablet, capsule, a softgel capsule or can be an aqueous or nonaqueous solution, suspension or syrup. Tablets and capsules are preferred oral administration forms. Tablets and capsules for oral use can include one or more commonly used carriers such as lactose and com starch. Lubricating agents, such as magnesium stearate, are also typically added.
  • the compositions of the disclosure can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like.
  • suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, com sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like.
  • Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like.
  • Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.
  • the active agent can be combined with any oral, non toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like and with emulsifying and suspending agents. If desired, flavoring, coloring and/or sweetening agents can be added as well.
  • Other optional components for incorporation into an oral formulation herein include, but are not limited to, preservatives, suspending agents, thickening agents, and the like.
  • Parenteral formulations can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solubilization or suspension in liquid prior to injection, or as emulsions.
  • sterile injectable suspensions are formulated according to techniques known in the art using suitable carriers, dispersing or wetting agents and suspending agents.
  • the sterile injectable formulation can also be a sterile injectable solution or a suspension in a nontoxic parenterally acceptable diluent or solvent.
  • acceptable vehicles and solvents that can be employed are water, Ringer’s solution and isotonic sodium chloride solution.
  • sterile, fixed oils, fatty esters or polyols are conventionally employed as solvents or suspending media.
  • parenteral administration can involve the use of a slow release or sustained release system such that a constant level of dosage is maintained.
  • Parenteral administration includes intraarticular, intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, and include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • Administration via certain parenteral routes can involve introducing the formulations of the disclosure into the body of a patient through a needle or a catheter, propelled by a sterile syringe or some other mechanical device such as an continuous infusion system.
  • a formulation provided by the disclosure can be administered using a syringe, injector, pump, or any other device recognized in the art for parenteral administration.
  • the pharmaceutical formulations can contain other additives, such as pH-adjusting additives.
  • useful pH-adjusting agents include acids, such as hydrochloric acid, bases or buffers, such as sodium lactate, sodium acetate, sodium phosphate, sodium citrate, sodium borate, or sodium gluconate.
  • the formulations can contain antimicrobial preservatives.
  • Useful antimicrobial preservatives include methylparaben, propylparaben, and benzyl alcohol. An antimicrobial preservative is typically employed when the formulations is placed in a vial designed for multi dose use.
  • the pharmaceutical formulations described herein can be lyophilized using techniques well known in the art.
  • a pharmaceutical composition can take the form of a solution suspension, tablet, pill, capsule, powder, and the like.
  • Tablets containing various excipients such as sodium citrate, calcium carbonate and calcium phosphate may be employed along with various disintegrants such as starch (e.g., potato or tapioca starch) and certain complex silicates, together with binding agents such as polyvinylpyrrolidone, sucrose, gelatin and acacia.
  • binding agents such as polyvinylpyrrolidone, sucrose, gelatin and acacia.
  • lubricating agents such as magnesium stearate, sodium lauryl sulfate, and talc are often very useful for tableting purposes.
  • Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules.
  • compositions in this connection also include lactose or milk sugar as well as high molecular weight polyethylene glycols.
  • lactose or milk sugar as well as high molecular weight polyethylene glycols.
  • the compounds of the presently disclosed host matter can be combined with various sweetening agents, flavoring agents, coloring agents, emulsifying agents and/or suspending agents, as well as such diluents as water, ethanol, propylene glycol, glycerin and various like combinations thereof.
  • injectable, stable, sterile formulations comprising an active compound as described herein, or a salt thereof, in a unit dosage form in a sealed container.
  • the compound or salt is provided in the form of a lyophilizate, which is capable of being reconstituted with a suitable pharmaceutically acceptable carrier to form liquid formulation suitable for injection thereof into a host.
  • a sufficient amount of emulsifying agent which is physiologically acceptable, can be employed in sufficient quantity to emulsify the compound or salt in an aqueous carrier.
  • Particularly useful emulsifying agents include phosphatidyl cholines and lecithin.
  • Additional embodiments provided herein include liposomal formulations of the active compounds disclosed herein.
  • the technology for forming liposomal suspensions is well known in the art.
  • the compound is an aqueous-soluble salt, using conventional liposome technology, the same can be incorporated into lipid vesicles.
  • the active compound due to the water solubility of the active compound, the active compound can be substantially entrained within the hydrophilic center or core of the liposomes.
  • the lipid layer employed can be of any conventional composition and can either contain cholesterol or can be cholesterol-free.
  • the active compound of interest is water-insoluble, again employing conventional liposome formation technology, the salt can be substantially entrained within the hydrophobic lipid bilayer that forms the structure of the liposome.
  • the liposomes that are produced can be reduced in size, as through the use of standard sonication and homogenization techniques.
  • the liposomal formulations comprising the active compounds disclosed herein can be lyophilized to produce a lyophilizate, which can be reconstituted with a pharmaceutically acceptable carrier, such as water, to regenerate a liposomal suspension.
  • Pharmaceutical formulations also are provided which are suitable for administration as an aerosol by inhalation. These formulations comprise a solution or suspension of a desired compound described herein or a salt thereof, or a plurality of solid particles of the compound or salt. The desired formulations can be placed in a small chamber and nebulized.
  • Nebulization can be accomplished by compressed air or by ultrasonic energy to form a plurality of liquid droplets or solid particles comprising the compounds or salts.
  • the liquid droplets or solid particles may for example have a particle size in the range of about 0.5 to about 10 microns, and optionally from about 0.5 to about 5 microns.
  • the solid particles provide for controlled release through the use of a degradable polymer.
  • the solid particles can be obtained by processing the solid compound or a salt thereof, in any appropriate manner known in the art, such as by micronization.
  • the size of the solid particles or droplets can be from about 1 to about 2 microns.
  • commercial nebulizers are available to achieve this purpose.
  • the compounds can be administered via an aerosol suspension of respirable particles in a manner set forth in U.S. Pat. No. 5,628,984, the disclosure of which is incorporated herein by reference in its entirety.
  • compositions also are provided which provide a controlled release of a compound described herein, including through the use of a degradable polymer, as known in the art.
  • the formulations suitable for administration as an aerosol can comprise a water-soluble active compound in a carrier that comprises water.
  • a surfactant can be present, which lowers the surface tension of the formulations sufficiently to result in the formation of droplets within the desired size range when hosted to nebulization.
  • pharmaceutically acceptable salts refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with hosts (e.g., human hosts) without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the presently disclosed host matter.
  • salts refers to the relatively non-toxic, inorganic and organic acid addition salts of the presently disclosed compounds. These salts can be prepared during the final isolation and purification of the compounds or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed.
  • Basic compounds are capable of forming a wide variety of different salts with various inorganic and organic acids. Acid addition salts of the basic compounds are prepared by contacting the free base form with a sufficient amount of the desired acid to produce the salt in the conventional manner. The free base form can be regenerated by contacting the salt form with a base and isolating the free base in the conventional manner.
  • the free base forms may differ from their respective salt forms in certain physical properties such as solubility in polar solvents.
  • Pharmaceutically acceptable base addition salts may be formed with metals or amines, such as alkali and alkaline earth metal hydroxides, or of organic amines. Examples of metals used as cations, include, but are not limited to, sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines include, but are not limited to, N,N'- dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N- methylglucamine, and procaine.
  • the base addition salts of acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner.
  • the free acid form can be regenerated by contacting the salt form with an acid and isolating the free acid in a conventional manner.
  • the free acid forms may differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents.
  • Salts can be prepared from inorganic acids sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydriodic, phosphorus, and the like.
  • Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate, glucoheptonate, lactobionate, laurylsulphonate and isethionate salts, and the like.
  • Salts can also be prepared from organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. and the like.
  • organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. and the like.
  • Representative salts include acetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate, methanesulfonate, and the like.
  • Pharmaceutically acceptable salts can include cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Also contemplated are the salts of amino acids such as arginate, gluconate, galacturonate, and the like. See, for example, Berge et al, J. Pharm. Sci., 1977, 66, 1-19, which is incorporated herein by reference.
  • sterile injectable suspensions are formulated according to techniques known in the art using suitable carriers, dispersing or wetting agents and suspending agents.
  • the sterile injectable formulation can also be a sterile injectable solution or a suspension in a nontoxic parenterally acceptable diluent or solvent.
  • acceptable vehicles and solvents that can be employed are water, Ringer’s solution and isotonic sodium chloride solution.
  • sterile, fixed oils, fatty esters or polyols are conventionally employed as solvents or suspending media.
  • parenteral administration can involve the use of a slow release or sustained release system such that a constant level of dosage is maintained.
  • Preparations according to the disclosure for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions.
  • non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and com oil, gelatin, and injectable organic esters such as ethyl oleate.
  • Such dosage forms can also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They can be sterilized by, for example, filtration through a bacteria retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured using sterile water, or some other sterile injectable medium, immediately before use.
  • Sterile injectable solutions are prepared by incorporating one or more of the compounds of the disclosure in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • a parenteral composition suitable for administration by injection is prepared by stirring 1.5% by weight of active ingredient in 10% by volume propylene glycol and water. The solution is made isotonic with sodium chloride and sterilized.
  • Formulations suitable for rectal administration are typically presented as unit dose suppositories. These may be prepared by admixing the active disclosed compound with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.
  • Formulations suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil.
  • Carriers which may be used include petroleum jelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.
  • Formulations suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Formulations suitable for transdermal administration may also be delivered by iontophoresis (see, for example, Pharmaceutical Research 3 (6):318 (1986)) and typically take the form of an optionally buffered aqueous solution of the active compound.
  • microneedle patches or devices are provided for delivery of drugs across or into biological tissue, particularly the skin. The microneedle patches or devices permit drug delivery at clinically relevant rates across or into skin or other tissue barriers, with minimal or no damage, pain, or irritation to the tissue.
  • Formulations suitable for administration to the lungs can be delivered by a wide range of passive breath driven and active power driven single/-multiple dose dry powder inhalers (DPI).
  • DPI dry powder inhalers
  • the devices most commonly used for respiratory delivery include nebulizers, metered- dose inhalers, and dry powder inhalers.
  • nebulizers include jet nebulizers, ultrasonic nebulizers, and vibrating mesh nebulizers. Selection of a suitable lung delivery device depends on parameters, such as nature of the drug and its formulation, the site of action, and pathophysiology of the lung.
  • Additional non-limiting examples of drug delivery devices and methods include, for example, US20090203709 titled“Pharmaceutical Dosage Form For Oral Administration Of Tyrosine Kinase Inhibitor” (Abbott Laboratories); US20050009910 titled“Delivery of an active drug to the posterior part of the eye via subconjunctival or periocular delivery of a prodrug”, US 20130071349 titled“Biodegradable polymers for lowering intraocular pressure”, US 8,481,069 titled“Tyrosine kinase microspheres”, US 8,465,778 titled“Method of making tyrosine kinase microspheres”, US 8,409,607 titled“Sustained release intraocular implants containing tyrosine kinase inhibitors and related methods”, US 8,512,738 and US 2014/0031408 titled“Biodegradable intravitreal tyrosine kinase implants”, US 2014/0294986
  • Example 1 Compound 1 is a Potent and Selective CDK9 Inhibitor
  • Compound 1 The biochemical activity of Compound 1 was determined using Caliper technology by Nanosyn, Inc. As shown in Table 3A, Compound 1 exhibited high potency and selectivity for CDK9 regulators.
  • FIG. 1 is a graphical representation of all kinases that were inhibited greater than 80% by Compound 1 at 100 nM and Table 4 is a list of all of the kinases that were inhibited greater than 80% by Compound 1 at 100 nM along with the average % inhibition of Compound 1.
  • Example 2 Compound 1 reduces Cell Viability and induces Caspase 3/7 in TNBC
  • the activity of Compound 1 against cell viability in various normal and tumor cell lines was measured using a 6-Day CellTiter Glo analysis. Cells were plated 24 hours prior to the Compound 1 treatment (1.0 nM - 10 mM). The 6-day continuous treatment with Compound 1 reduced viability of all tested cell types (Table 5).
  • Compound 1 induced caspase 3/7 in the triple negative breast cancer cell lines HCC1806 and BT549, the ER+ breast cancer MCF7 cell line, and the primary fibroblast MCF7 cell line ( Figure 3).
  • the cells were incubated with increasing concentrations of Compound 1 and the Caspase 3/7 Glo results were measured at 24 hours.
  • Example 3 Compound 1 modulates uiRNA Protein Expression of Pro-Survival Oncogenes regulated by RNA Polymerase II in TNBC Cells
  • HCC1806 ( Figure 5A, Table 6) and Hs68 ( Figure 5B, Table 7) cell lines were treated with Compound 1 for 24 hours and the cell cycle profiles were evaluated using FlowJo (vlO.O) software. Both cell lines showed a dose-dependent decrease in S and an increase in G2. Table 6. Percentage of HCC1806 Cells in Each Phase following Compound 1 Treatment
  • Figure 5C and Figure 5D are representative flow gating schematics for FxCycle DNA stain, Click-iTTM EdU in HCC1806 cells.
  • Figure 5C is the schematic for untreated cells and Figure 5D is the schematic for cells treated with Compound 1.
  • Figure 5E and Figure 5F are representative flow gating schematics for Phospho-Histone H3 conjugated antibody in HCC1806 cells.
  • Figure 5E is the schematic for untreated cells and Figure 5F is the schematic for cells treated with Compound 1.
  • Compound 1 induces G2 Arrest in MCF7 Parental and MCF7
  • MCF7 parental cells were maintained in culture for four months in complete media (EMEM/ 10% FBS/ glutamax/insulin) as a control and MCF7 palbociclib-resistant (palbo-R) cells were maintained in complete media plus palbociclib for three months at IC90 (750nM) followed by one month at ImM ( Figure 6).
  • Whole transcriptome profiling was performed on control and palbo-R MCF7 cells by RNA-Seq. Libraries were prepared using the Illumina TruSeq Stranded mRNA assay and paired-end sequenced (2x50bp) on the Illumina HiSeq platform.
  • Figure 7A is the pairwise comparison of transcript levels in MCF7 palbo-R vs. control.
  • Figure 7B is the fold change (expressed as Log2) of the transcript levels of specific genes in MCF7 palbo-R vs. control.
  • the transcript levels of CCNE1, CCNE2, and MYC were significantly upregulated in the MCF7 palbo-R compared to the control.
  • the Western blot analysis of MCF7 parental and MCF7 palbo-R cells demonstrated an increase in the ratio of Cyclin E to Rb levels in palbo-R cells compared to the control ( Figure 7C).
  • the MCF7 and MCF7 palbo-R cell lines were treated with Compound 1 for 24 hours.
  • Cell cycle profiles following treatment were evaluated via Flow Cytometry with FxCycle DNA stain, Click-iT 1M EdU and Phospho-Histone H3 conjugated antibody.
  • Profiles of the MCF7 ceil line ( Figure 8A, Table 8) and the MCF7 palbo-R cell line ( Figure 8B, Table 9) showed a dose-dependent decrease in S and increase in G2.
  • Compound 1 also inhibits pRPBl CTD (Ser2) in both MCF7 cells ( Figure 9A) and MCF7-paJbo-R cells (Figure 9B) in addition to its downstream target Cyclin E in both MCF7 ceils ( Figure 9C) and MCF7-palbo-R cells ( Figure 9D). Compound 1 is active in MCF7-palbo- R cells.
  • Ser2 pRPBl CTD

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Abstract

La présente invention concerne des procédés de traitement de troubles impliquant une prolifération cellulaire anormale qui ont développé une résistance à un inhibiteur sélectif de CDK4/6.
PCT/US2020/026263 2019-04-01 2020-04-01 Traitement de troubles néoplasiques résistant aux inhibiteurs de cdk4/6 WO2020206035A1 (fr)

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CN115872996A (zh) * 2023-02-21 2023-03-31 山东绿叶制药有限公司 一种雌激素受体降解剂化合物及其制备方法和应用

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CN112979532A (zh) * 2021-05-20 2021-06-18 中国药科大学 邻苯二甲酰亚胺类化合物、制备方法和应用
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CN115872996A (zh) * 2023-02-21 2023-03-31 山东绿叶制药有限公司 一种雌激素受体降解剂化合物及其制备方法和应用

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