WO2015161285A1 - Tricyclic lactams for use in the protection of hematopoietic stem and progenitor cells against ionizing radiation - Google Patents

Tricyclic lactams for use in the protection of hematopoietic stem and progenitor cells against ionizing radiation Download PDF

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WO2015161285A1
WO2015161285A1 PCT/US2015/026522 US2015026522W WO2015161285A1 WO 2015161285 A1 WO2015161285 A1 WO 2015161285A1 US 2015026522 W US2015026522 W US 2015026522W WO 2015161285 A1 WO2015161285 A1 WO 2015161285A1
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alkylene
compound
groups
formula
heterocyclo
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PCT/US2015/026522
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French (fr)
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Jay Copeland Strum
John Emerson Bisi
Patrick Joseph Roberts
Ricky D. Gaston
Robert C. Gadwood
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G1 Therapeutics, Inc.
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Publication of WO2015161285A1 publication Critical patent/WO2015161285A1/en

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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
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—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
    • 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
    • C07D487/14—Ortho-condensed systems
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains three hetero rings
    • C07D471/20—Spiro-condensed systems

Definitions

  • This invention is in the area of compounds and methods for protecting healthy cells, and in particular hematopoietic stem and progenitor cells (HSPC), from the damage associated with ionizing radiation (IR) exposure using selective radioprotectants.
  • HSPC hematopoietic stem and progenitor cells
  • Ionizing radiation is an important therapeutic modality to treat a range of cancers and other proliferative disorders such as tumors. Radiation therapy uses high energy radiation to shrink tumors and kill the proliferating cells. X-rays, gamma rays, and charged particles are typical kinds of ionizing radiation used for cancer treatments. IR causes extensive DNA damage to exposed cells, including both normal cells and abnormally proliferating cells such as cancer and tumor cells.
  • Therapeutic radiation is generally applied to a defined area of the subject's body which contains abnormal proliferative tissue, in order to minimize the dose absorbed by the nearby normal tissue. It is difficult, however, to selectively administer therapeutic ionizing radiation to the abnormal tissue. Thus, normal tissue proximate to the abnormal tissue is also exposed to potentially damaging doses of ionizing radiation throughout the course of treatment. There are also some treatments that require exposure of the subject's entire body to the radiation, in a procedure called “total body irradiation" (TBI). Numerous methods have been designed to reduce normal tissue damage while still delivering effective therapeutic doses of ionizing radiation.
  • exposure to IR may occur through occupational, environmental, or disaster or terroristic events.
  • occupational doses of ionizing radiation can be received by persons whose job involves exposure to radiation, for example in the nuclear power and nuclear weapons industry.
  • Incidents such as the 1979 accident at Three Mile Island or 2011 accident at the Fukushima nuclear power plant, both of which released radioactive material into the reactor containment building and surrounding environment, illustrate the potential for harmful exposure. Intentional infliction of harmful radiation can occur during war and aggression.
  • Hematologic toxicity i.e., IR-induced bone marrow suppression
  • myelosuppression can be a limiting side-effect associated with radiation therapy treatments, resulting in a stoppage, delay, or reduction of treatment until the side-effects subside.
  • hematological toxicity is a major source of morbidity following acute exposure to high doses of radiation.
  • proliferating hematopoietic stem cells and progenitor cells (HSPCs) within the bone marrow are particularly sensitive to IR, and IR damage to these cells reduces their ability to reconstitute the hematological cell lineages.
  • TBI total body irradiation
  • IR cytotoxicity
  • S phase DNA synthesis
  • M phase mitosis
  • Gl and G2 the gaps of varying length between these periods.
  • Non-dividing cells remain in a resting or quiescence stage named GO before they re-enter into phase Gl .
  • Early Gl and late S phases are relatively radioresistant.
  • the Gl/S transition and G2/M phases are relatively radiosensitive (see Sinclair WK, Morton RA. X-ray sensitivity during cell generation cycle of cultured Chinese hamster cells. Radiat. Res.
  • Hematopoietic stem cells give rise to progenitor cells which in turn give rise to all the differentiated components of blood as shown in Figure 1 (e.g., lymphocytes, erythrocytes, platelets, granulocytes, monocytes).
  • HSPCs require the activity of CDK4/6 for proliferation (see Roberts et al. Multiple Roles of Cyclin-Dependent Kinase 4/6 Inhibitors in Cancer Therapy. JNCI 2012;104(6):476-487).
  • Hematopoietic cells display a gradient dependency on CDK4/6 activity for proliferation during myeloid/erythroid differentiation (see Johnson et al.
  • the least differentiated cells e.g., hematopoietic stem cells (HSCs), multi-potent progenitors (MPPs), and common myeloid progenitors (CMP)
  • HSCs hematopoietic stem cells
  • MPPs multi-potent progenitors
  • CMP common myeloid progenitors
  • More differentiated lineages e.g., granulocyte -monocyte progenitors (GMPs) and megakaryocyte-erythroid progenitors (MEPs)
  • GFPs granulocyte -monocyte progenitors
  • MEPs megakaryocyte-erythroid progenitors
  • CDK 4/6 inhibitors include specific pyrido[2,3- d]pyrimidines, 2-anilinopyrimidines, diaryl ureas, benzoyl-2,4-diaminothiazoles, indolo[6,7- a]pyrrolo[3,4-c]carbazoles, and oxindoles (see P.S. Sharma, R. Sharma, R. Tyagi, Curr. Cancer Drug Targets 8 (2008) 53-75).
  • WO 03/062236 identifies a series of 2-(pyridin-2- ylamino-pyrido[2,3]pyrimidin-7-ones for the treatment of Rb positive cancers that show selectivity for CDK4/6, including 6-acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-l-yl-pyridin- 2-ylammino)-8H-pyrido-[2,3-d]-pyrimidin-7-one (PD0332991), which is currently being tested by Pfizer/Onyx in clinical trials as an anti-neoplastic agent against estrogen-positive, HER2- negative breast cancer.
  • PD0332991 6-acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-l-yl-pyridin- 2-ylammino)-8H-pyrido-[2,3-d]-pyrimidin-7-one
  • VanderWel et al. describe an iodine-containing pyrido[2,3-d]pyrimidine-7-one (CKIA) as a potent and selective CDK4 inhibitor (see VanderWel et al, J. Med. Chem. 48 (2005) 2371- 2387).
  • WO 99/15500 filed by Glaxo Group Ltd discloses protein kinase and serine/threonine kinase inhibitors.
  • WO 2010/020675 filed by Novartis AG describes pyrrolopyrimidine compounds as CDK inhibitors.
  • WO 2011/101409 also filed by Novartis describes pyrrolopyrimidines with CDK 4/6 inhibitory activity.
  • WO 2005/052147 filed by Novartis and WO 2006/074985 filed by Janssen Pharma disclose additional CDK4 inhibitors.
  • U.S. Patent Publication 2011/0224221 to Sharpless et al. describes the use of certain CDK4/6 inhibitors, such as PD0332991 and 2BrIC (see Zhu, et al, J. Med. Chem., 46 (1 1) 2027- 2030 (2003); PCT/US2009/059281) to reduce or prevent the deleterious effects of ionizing radiation on HSPCs in a subject exposed to radiation. See also U.S. Patent Publication 2012/0100100. Stone, et al, Cancer Research 56, 3199-3202 (July 1, 1996) describes reversible, pie- mediated cell cycle arrest as protection from chemotherapy.
  • CDK inhibitors see also, US Pat. Nos. 8,829,012, 8,822,683, 8,598,186, 8,691,830, and 8,598,197, all assigned to Gl Therapeutics), describe CDK Inhibitors having the basic core structure:
  • WO 2013/148748 filed by Tavares and assigned to Gl Therapeutics describes Lactam Kinase inhibitors having the basic core structures:
  • Patent Publication 2014/0271466, assigned to Gl Therapeutics, describes the use of CDK4/6 inhibitors such as 2'-((5-(4-methylpiperazin-l-yl)pyridin-2-yl)amino)-7',8'-dihydro-6'Fi- spiro[cyclohexane-l,9'-pyrazino[ ,2':l ,5]pyrrolo[2,3-d]pyrimidin]-6'-one for use as an antineoplastic for the treatment of a Rb-positive proliferative disorders.
  • CDK4/6 inhibitors such as 2'-((5-(4-methylpiperazin-l-yl)pyridin-2-yl)amino)-7',8'-dihydro-6'Fi- spiro[cyclohexane-l,9'-pyrazino[ ,2':l ,5]pyrrolo[2,3-d]pyrimidin]-6'-one for use
  • CDK4/6 inhibitors such as 2'-((5-(4-methylpiperazin-l-yl)pyridin-2-yl)amino)-7',8'-dihydro-6'Fi- spiro[cyclohexane-l,9'-pyrazino[ ,2': l,5]pyrrolo[2,3-d]pyrimidin]-6'-one for use an antineoplastic for the treatment of a T- or B-cell disorder, for example a leukemia.
  • HSPCs hematopoietic stem cells and/or hematopoietic progenitor cells
  • the invention includes administering an effective amount of a compound of Formula I, II, III, IV, V, or VI, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof, to protect HSPCs in a subject during or following the subject's exposure to IR.
  • a compound can be selected from the compounds of Table 1 below, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof.
  • Compounds of the present invention can be used to protect healthy cells during ionizing radiation therapy or radiotherapy for the treatment of any malignant or non-malignant tumor or abnormal cell proliferation, for example, in a solid tumor, including a cancer of the brain, breast, cervix, larynx, lung, pancreas, prostate, skin, spine, stomach, uterus, soft tissue sarcoma, leukemia or lymphoma.
  • the invention can also be used in conjunction with radiotherapy used as a palliative treatment in the absence of a cure for local control of the tumor or symptomatic release, or as a therapeutic treatment to extend the life span of the patient, or total body irradiation performed prior to bone marrow transplant.
  • Compounds of the present invention can also be used to protect healthy cells in connection with radiotherapy for the treatment of non- malignant conditions, such as trigeminal neuralgia, thyroid eye disease, pterygium, or prevention of keloid scar growth or heterotopic ossification.
  • the present invention can also be used to protect healthy cells during ionizing radiation therapy or radiotherapy for the treatment of proliferative disorders, including but not limited to rheumatoid arthritis, lupus, scleroderma, ankylosing spondylitis, asthma, bronchitis and psoriasis. Radiation therapy is also used to treat early stage Dupuytren's disease and Ledderhose disease.
  • the present invention can further be used to protect people at imminent risk of environmental, occupational or aggression-based radiation exposure or who have recently been exposed to harmful radiation.
  • a compound described herein may provide protection of CDK-replication dependent HSPCs during or after IR exposure due in part because it (1) exhibits a transient Gl -arresting effect and (ii) displays a rapid, synchronous reentry into the cell cycle by the HSPCs following the cessation of IR exposure or mitigation of IR induced DNA damage.
  • CDK4/6-specific transient Gl -arresting compounds as radioprotectants and radiomitigants allows for an accelerated hematological recovery, reduced hematological cytotoxicity risk due to HSPC replication delay, and/or a minimization of IR induced cell death.
  • Tricyclic lactams useful in the present invention can be administered to the subject prior to exposure to IR, during exposure to IR, after exposure to IR, or a combination thereof.
  • the compounds described herein are typically administered in a manner that allows the drug facile access to the blood stream, for example via intravenous injection or sublingual, intraaortal, or other efficient blood-stream accessing route; however, oral or other desired administrative routes can be used.
  • the compound is administered to the subject less than about 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, or 4 hours, 3 hours, 2 hours, 1 hour, 1 ⁇ 2 hour or less prior to exposure to IR.
  • the compound is administered up to 4 hours prior to exposure to IR.
  • the tricyclic lactam is administered to the subject prior to exposure to IR such that the compound reaches peak serum levels before or during exposure to IR.
  • the tricyclic lactam is administered concomitantly, or closely thereto, with IR exposure.
  • the tricyclic lactam can be administered following exposure to IR in order to mitigate HSPC DNA damage associated with IR exposure.
  • the tricyclic lactam can be administered multiple times during the IR exposure to maximize inhibition, especially when the IR exposure occurs over a long period.
  • the tricyclic lactam is administered up to about 1 ⁇ 2 hour, up to about 1 hour, up to about 2 hours, up to about 4 hours, up to about 8 hours, up to about 10 hours, up to about 12 hours, up to about 14 hours, up to about 16 hours, up to about 20 hours, or up to about 24 hours or greater following IR exposure.
  • the tricyclic lactam is administered up to between about 12 hours and 20 hours following exposure to IR.
  • the tricyclic lactam is administered one or more times following exposure to IR.
  • the tricyclic lactam compounds inhibit Cyclin Dependent Kinase 4 (CDK4) and/or Cyclin Dependent Kinase 6 (CDK6).
  • the tricyclic lactams useful in the present invention may show a marked selectivity for the inhibition of CDK4 and/or CDK6 in comparison to other CDKs, for example CDK2.
  • Tricyclic lactams useful in the present invention may provide for a dose-dependent Gl-arresting effect on a subject's HSPCs sufficient to afford radioprotection to targeted HSPCs during IR exposure, while allowing for the reentry into the cell-cycle by the HSPCs after IR exposure and/or tricyclic lactam administration due to a time-limited CDK4/6 inhibitory effect.
  • tricyclic lactams useful in the present invention may provide a dose-dependent mitigating effect on HSPCs that have been exposed to IR, allowing for repair of DNA damage associated with IR exposure.
  • cell-cycle reentry following Gl arrest using a tricyclic lactam described herein may provide for the ability to time the administration of hematopoietic growth factors to assist in the reconstitution of hematopoietic cell lines to maximize the growth factor effect without forcing hematological cells into replication before DNA damage is repaired.
  • the use of the compounds described herein is combined with the use of hematopoietic growth factors including, but not limited to, granulocyte colony stimulating factor (G-CSF), granulocyte -macrophage colony stimulating factor (GM-CSF), thrombopoietin, interleukin (IL)-12, steel factor, and erythropoietin (EPO), and their derivatives.
  • G-CSF granulocyte colony stimulating factor
  • GM-CSF granulocyte -macrophage colony stimulating factor
  • thrombopoietin interleukin (IL)-12
  • steel factor erythropoietin
  • EPO erythropoietin
  • the tricyclic lactam is administered prior to administration of the hematopoietic growth factor.
  • the hematopoietic growth factor administration is timed so that the tricyclic lactam's effect on HSPCs has dissipated.
  • the use of a tricyclic lactam described herein allows for a HSPC radioprotective regimen for use during standard radio-therapeutic dosing schedules or regimens common in many anti-cancer treatments.
  • the subject is undergoing therapeutic IR for the treatment of a proliferative disorder or disease such as cancer.
  • the cancer is a CDK4/6-replication independent cancer.
  • the cancer is characterized by one or more of the group consisting of increased activity of eye Independent 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 MYC expression, increased cyclin E, and increased cyclin A.
  • CDK1 eye Independent kinase 1
  • CDK2 cyclin-dependent kinase 2
  • Rb retinoblastoma tumor suppressor protein
  • the subject is undergoing therapeutic IR for the treatment of an Rb-null or Rb-deficient cancer, including but not limited to, small cell lung cancer, triple-negative breast cancer, HPV-positive head and neck cancer, retinoblastoma, Rb-negative bladder cancer, Rb negative prostate cancer, osteosarcoma or cervical cancer.
  • an Rb-null or Rb-deficient cancer including but not limited to, small cell lung cancer, triple-negative breast cancer, HPV-positive head and neck cancer, retinoblastoma, Rb-negative bladder cancer, Rb negative prostate cancer, osteosarcoma or cervical cancer.
  • administration of the tricyclic lactam compound allows for a higher dose of ionizing radiation to be used to treat the disease than the standard dose that would be safely used in the absence of administration of the tricyclic lactam compound.
  • the subject is at risk of being exposed to IR due to an environmental, occupational or aggression-based situation, such as radiological agent exposure during warfare, a radiological terrorist attack, an industrial accident, other occupational exposure, or space travel.
  • an environmental, occupational or aggression-based situation such as radiological agent exposure during warfare, a radiological terrorist attack, an industrial accident, other occupational exposure, or space travel.
  • the subject has already been exposed to IR, for example, including but not limited to, through an environmental or occupational situation, such as radiological agent exposure during warfare, a radiological terrorist attack, an industrial accident, other occupational exposure, or space travel, and the tricyclic lactams described herein are administered for the purpose of mitigating DNA damage in HSPCs.
  • an environmental or occupational situation such as radiological agent exposure during warfare, a radiological terrorist attack, an industrial accident, other occupational exposure, or space travel
  • the tricyclic lactams described herein are administered for the purpose of mitigating DNA damage in HSPCs.
  • the protected HSPCs include hematopoietic stem cells, including long term hematopoietic stem cells (LT-HSCs) and short term hematopoietic stem cells (ST- HSCs), and hematopoietic progenitor cells, including multipotent progenitors (MPPs), common myeloid progenitors (CMPs), common lymphoid progenitors (CLPs), granulocyte-monocyte progenitors (GMPs) and megakaryocyte-erythroid progenitors (MEPs).
  • administration of the tricyclic lactam compound provides temporary, transient pharmacologic quiescence of hematopoietic stem and/or hematopoietic progenitor cells in the subject.
  • the methods described herein using a tricyclic lactam may result in reduced long-term hematologic toxicity, that is, the use of a tricyclic lactam described herein prior to, during, or after IR exposure reduces the occurrence or development of long-term hematological toxicities associated with IR exposure.
  • the reduction in long-term hematological toxicity is associated with the ability of HSPCs that are Gl -arrested during IR exposure to the cell-cycle after cessation of IR exposure and replicate, including replicating between successive or repeated IR exposures.
  • administration of a tricyclic lactam as described herein may result in reduced anemia, reduced lymphopenia, reduced thrombocytopenia, or reduced neutropenia compared to that typically expected after, common after, or associated with exposure to ionizing radiation in the absence of administration of the tricyclic lactam.
  • the tricyclic lactam is the compound of Formula I, II, III, IV, V, or VI.
  • the tricyclic lactam used in the aspects of the invention described herein is selected from the compounds of Table 1.
  • the subject or host is a mammal, including a human.
  • the present invention includes at least the following features:
  • Tricyclic lactam compounds, methods, and compositions are provided for reducing the effect of IR exposure on CDK4/6 replication dependent HSPCs in a subject who will be exposed, is being exposed, or has been exposed to IR, the method comprising administering an effective amount of a tricyclic lactam selected from the group consisting of a compound or composition comprising Formula I, Formula II, Formula III, Formula IV, Formula V, and Formula VI, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof.
  • the compound is selected from the compounds listed in Table 1 or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof.
  • the compound is selected from the compounds listed in Table 1, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof;
  • the compound is selected from the compounds listed in Table 1, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof;
  • the compound is selected from the compounds listed in Table 1, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof;
  • H Compounds of Formula I, II, III, IV, V, and VI as described herein, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof, for use in the radioprotection of HSPCs during an IR therapeutic regimen for the treatment of an Rb-null or Rb-deficient cancer.
  • the compound is selected from the compounds listed in Table 1, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof;
  • the compound is selected from the compounds listed in Table 1 , or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof;
  • the compound is selected from the compounds listed in Table 1, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof;
  • K Compounds of Formula I, II, III, IV, V, and VI as described herein, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof, for use in the forced cycling of HSPCs between Gl -arrest and replication in coordination with repeated IR exposures.
  • the compound is selected from the compounds listed in Table 1, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof;
  • the compound is selected from the compounds listed in Table 1, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof;
  • M Compounds of Formula I, II, III, IV, V, and VI as described herein, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof, for use in combination with hematopoietic growth factors in a subject that will be, is being, or has been exposed to IR.
  • the compound is selected from the compounds listed in Table 1, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof;
  • a pharmaceutical formulation comprising an effective subject-treating amount of compounds of Formula I, II, III, IV, V, and VI as described herein for the protection against ionizing radiation, or pharmaceutically acceptable compositions, salts, isotopic analog, or prodrugs thereof;
  • the compound is selected from the compounds listed in Table 1, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof
  • a method for manufacturing a medicament of Formula I, II, III, IV, V, and VI intended for therapeutic use in the radioprotection of HSPCs is selected from the compounds listed in Table 1 , or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof; and,
  • R A method for manufacturing a medicament of Formula I, II, III, IV, V, and VI intended for therapeutic use in the mitigation of DNA damage of HSPCs that have been exposed to IR.
  • the medicament is selected from the compounds listed in Table 1, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof.
  • S Compounds of Formula I, II, III, IV, V, and VI as described herein, or pharmaceutically acceptable compositions, salts, isotopic analog, or prodrugs thereof;
  • the compound is selected from the compounds listed in Table 1, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof.
  • FIG. 1 is a schematic drawing of hematopoiesis showing the hierarchical proliferation of healthy hematopoietic stem cells (HSC) and healthy hematopoietic progenitor cells with increasing differentiation upon proliferation.
  • HSC healthy hematopoietic stem cells
  • FIGS. 2-4 illustrate several exemplary embodiments of R 2 of the compounds of the invention.
  • FIGS. 5A-5C, 6A-6D, 7A-7C, 8A-8B and 9A-9F illustrate exemplary embodiments of the core structure of the compounds of the invention.
  • Tricyclic lactam compounds, methods, and compositions are provided that minimize the effect of ionizing radiation (IR) toxicity on CDK4/6 replication dependent hematopoietic stem cells and/or hematopoietic progenitor cells (together referred to as HSPCs) in subjects, typically humans, that will be, are being, or have been exposed to IR.
  • IR ionizing radiation
  • alkyl either alone or within other terms such as “haloalkyl” and
  • alkylamino embraces linear or branched radicals having one to about twelve carbon atoms.
  • “Lower alkyl” radicals have one to about six carbon atoms. Examples of such radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-bvXy ⁇ , pentyl, isoamyl, hexyl and the like.
  • alkylene embraces bridging divalent linear and branched alkyl radicals. Examples include methylene, ethylene, propylene, isopropylene and the like.
  • alkenyl embraces linear or branched radicals having at least one carbon- carbon double bond of two to about twelve carbon atoms. "Lower alkenyl” radicals having two to about six carbon atoms. Examples of alkenyl radicals include ethenyl, propenyl, allyl, propenyl, butenyl and 4-methylbutenyl.
  • alkenyl and lower alkenyl embrace radicals having "cis” and “trans” orientations, or alternatively, "E” and "Z” orientations.
  • alkynyl denotes linear or branched radicals having at least one carbon-carbon triple bond and having two to about twelve carbon atoms.
  • Alkyl, alkenyl, and alkynyl radicals may be optionally substituted with one or more functional groups such as halo, hydroxy, nitro, amino, cyano, haloalkyl, aryl, heteroaryl, heterocyclo and the like.
  • alkylamino embraces “N-alkylamino” and " ⁇ , ⁇ -dialkylamino” where amino groups are independently substituted with one alkyl radical and with two alkyl radicals, respectively.
  • “Lower alkylamino” radicals have one or two alkyl radicals of one to six carbon atoms attached to a nitrogen atom.
  • Suitable alkylamino radicals may be mono or dialkylamino such as N-methylamino, N-ethylamino, N.N-dimethylamino, ⁇ , ⁇ -diethylamino and the like.
  • halo means halogens such as fluorine, chlorine, bromine or iodine atoms.
  • haloalkyl embraces radicals wherein any one or more of the alkyl carbon atoms is substituted with one or more halo as defined above. Examples include monohaloalkyl, dihaloalkyl and polyhaloalkyl radicals including perhaloalkyl.
  • a monohaloalkyl radical for one example, may have an iodo, bromo, chloro or fluoro atom within the radical.
  • Dihalo and polyhaloalkyl radicals may have two or more of the same halo atoms or a combination of different halo radicals.
  • “Lower haloalkyl” embraces radicals having 1-6 carbon atoms.
  • haloalkyl radicals include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichloro fluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl and dichloropropyl.
  • Perfluoroalkyl means an alkyl radical having all hydrogen atoms replaced with fluoro atoms. Examples include trifluoromethyl and pentafluoroethyl.
  • aryl alone or in combination, means a carbocyclic aromatic system containing one or two rings wherein such rings may be attached together in a fused manner.
  • aryl embraces aromatic radicals such as phenyl, naphthyl, indenyl, tetrahydronaphthyl, and indanyl. More preferred aryl is phenyl.
  • Said "aryl” group may have 1 or more substituents such as lower alkyl, hydroxyl, halo, haloalkyl, nitro, cyano, alkoxy, lower alkylamino, and the like.
  • An aryl group may be optionally substituted with one or more functional groups such as halo, hydroxy, nitro, amino, cyano, haloalkyl, aryl, heteroaryl, heterocyclo and the like.
  • heterocyclyl (or “heterocyclo") embraces saturated, and partially saturated heteroatom-containing ring radicals, where the heteroatoms may be selected from nitrogen, sulfur and oxygen.
  • Heterocyclic rings comprise monocyclic 6-8 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.
  • Said "heterocyclyl” group may have 1 to 3 substituents such as hydroxyl, Boc, halo, haloalkyl, cyano, lower alkyl, lower aralkyl, oxo, lower alkoxy, amino, lower alkylamino, and the like.
  • saturated heterocyclo 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 heterocyclyl radicals include dihydrothienyl, dihydropyranyl, dihydrofuryl, dihydrothiazolyl, and the like.
  • Particular examples of partially saturated and saturated heterocyclo groups include 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-l,2,4-triazolo[3,4-a]isoquino
  • Heterocyclo groups also includes radicals where heterocyclic radicals are fused/condensed with aryl radicals: unsaturated condensed heterocyclic group containing 1 to 5 nitrogen atoms, for example, indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl, tetrazolopyridazinyl [e.g., tetrazolo [l,5-b]pyridazinyl]; unsaturated condensed heterocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms [e.g.
  • benzoxazolyl, benzoxadiazolyl unsaturated condensed heterocyclic group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms
  • benzothiazolyl, benzothiadiazolyl unsaturated condensed heterocyclic group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms
  • saturated, partially unsaturated and unsaturated condensed heterocyclic group containing 1 to 2 oxygen or sulfur atoms e.g. benzofuryl, benzothienyl, 2,3-dihydro- benzo[l,4]dioxinyl and dihydrobenzofuryl].
  • heteroaryl denotes aryl ring systems that contain one or more heteroatoms selected from the group O, N and S, wherein the ring nitrogen and sulfur atom(s) are optionally oxidized, and nitrogen atom(s) are optionally quarternized.
  • Examples include unsaturated 5 to 6 membered heteromonocyclyl group containing 1 to 4 nitrogen atoms, for example, 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-l,2,3-triazolyl]; unsaturated 5- to 6-membered heteromonocyclic group containing an oxygen atom, for example, pyranyl, 2-furyl, 3-furyl, etc.; unsaturated 5 to 6-membered heteromonocyclic group containing a sulfur atom, for example, 2- thienyl, 3-thienyl, etc.; unsaturated 5- to 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1
  • heteroarylalkyl denotes alkyl radicals substituted with a heteroaryl group. Examples include pyridylmethyl and thienylethyl.
  • sulfonyl whether used alone or linked to other terms such as alkylsulfonyl, denotes respectively divalent radicals -S0 2 -.
  • carboxy or “carboxyl”, whether used alone or with other terms, such as “carboxyalkyl”, denotes -C(0)-OH.
  • carbonyl whether used alone or with other terms, such as “aminocarbonyl”, denotes -C(O)-.
  • aminocarbonyl denotes an amide group of the Formula -C(0)-NH 2 .
  • heterocycloalkyl embrace heterocyclic-substituted alkyl radicals. Examples include piperidylmethyl and morpholinylethyl.
  • arylalkyl embraces aryl-substituted alkyl radicals. Examples include benzyl, diphenylmethyl and phenylethyl. The aryl in said aralkyl may be additionally substituted with halo, alkyl, alkoxy, halkoalkyl and haloalkoxy.
  • cycloalkyl includes saturated carbocyclic groups of 3 to 10 carbons.
  • Lower cycloalkyl groups include C 3 -C 6 rings. Examples include cyclopentyl, cyclopropyl, and cyclohexyl.
  • Cycloalkyl groups may be optionally substituted with one or more functional groups such as halo, hydroxy, nitro, amino, cyano, haloalkyl, aryl, heteroaryl, heterocyclo and the like.
  • cycloalkylalkyl embraces cycloalkyl-substituted alkyl radicals.
  • “Lower cycloalkylalkyl” radicals are cycloalkyl radicals attached to alkyl radicals having one to six carbon atoms. Examples of include cyclohexylmethyl.
  • the cycloalkyl in said radicals may be additionally substituted with halo, alkyl, alkoxy and hydroxy.
  • cycloalkenyl includes carbocyclic groups having one or more carbon-carbon double bonds including “cycloalkyldienyl” compounds. Examples include cyclopentenyl, cyclopentadienyl, cyclohexenyl and cycloheptadienyl.
  • oxo as used herein contemplates an oxygen atom attached with a double bond.
  • nitro as used herein contemplates -N0 2 .
  • cyano as used herein contemplates -CN.
  • the term "prodrug” means a compound which when administered to a host in vivo is converted into the parent drug.
  • parent drug means any of the presently described chemical compounds that are useful to treat any of the disorders described herein, or to control or improve the underlying cause or symptoms associated with any physiological or pathological disorder described herein in a host, typically a human.
  • Prodrugs can be used to achieve any desired effect, including to enhance properties of the parent drug or to improve the pharmaceutic or pharmacokinetic properties of the parent.
  • Prodrug strategies exist which provide choices in modulating the conditions for in vivo generation of the parent drug, all of which are deemed included herein.
  • Nonlimiting examples of prodrug strategies include covalent attachment of removable groups, or removable portions of groups, for example, but not limited to acylation, phosphorylation, phosphonylation, phosphoramidate derivatives, amidation, reduction, oxidation, esterification, alkylation, other carboxy derivatives, sulfoxy or sulfone derivatives, carbonylation or anhydride, among others.
  • selective CDK4/6 inhibitor and derivatives thereof means a compound that inhibits only CDK4 activity, only CDK6 activity, or both CDK4 and CDK6 activity at an IC 50 molar concentration at least about 500, or 1000, or 1500, or 1800, or 2000, or 5000, or 10,000 times less than the IC 50 molar concentration necessary to inhibit to the same degree of CDK2 activity in a standard phosphorylation assay.
  • CDK4 and/or CDK6 refers to situations where both items or conditions are present or applicable and to situations wherein only one of the items or conditions is present or applicable.
  • a CDK4 and/or CDK6 inhibitor can be a compound that inhibits both CDK4 and CDK6, a compound that inhibits only CDK4, or a compound that only inhibits CDK6.
  • hematopoietic stem and progenitor cells include, but are not limited to, long term hematopoietic stem cells (LT-HSCs), short term hematopoietic stem cells (ST- HSCs), multipotent progenitors (MPPs), common myeloid progenitors (CMPs), common lymphoid progenitors (CLPs), granulocyte-monocyte progenitors (GMPs), and megakaryocyte- erythroid progenitors (MEPs).
  • LT-HSCs long term hematopoietic stem cells
  • ST- HSCs short term hematopoietic stem cells
  • MPPs common myeloid progenitors
  • CLPs common lymphoid progenitors
  • GFPs granulocyte-monocyte progenitors
  • MEPs megakaryocyte- erythroid progenitors
  • the term "ionizing radiation” refers to radiation of sufficient energy that, when absorbed by cells and tissues, can induce formation of reactive oxygen species and DNA damage.
  • Ionizing radiation can include X-rays, gamma rays, and particle bombardment (e.g., neutron beam, electron beam, protons, mesons, and others).
  • IR is used for purposes including, but not limited to, medical testing and treatment, scientific purposes, industrial testing, manufacturing and sterilization, and weapons and weapons development, nuclear energy and can also be found as an environmental or occupational toxin or used as an assault. Radiation is generally measured in units of absorbed dose, such as the rad or gray (Gy), or in units of dose equivalence, such as rem or sievert (Sv).
  • substantially portion or “significant portion” is meant at least about 80%. In alternative embodiments, the portion may be 85%, 90% or 95% or greater.
  • inducces Gl -arrest is meant that the tricyclic lactam compound induces a quiescent state in a substantial portion of a cell population at the Gl phase of the cell cycle.
  • long-term hematological toxicity is meant hematological toxicity affecting a subject for a period lasting more than one or more weeks, months or years following exposure of IR.
  • Long-term hematological toxicity can result in bone marrow disorders that can cause the ineffective production of blood cells (i.e., myelodysplasia) and/or lymphocytes (i.e., lymphopenia, the reduction in the number of circulating lymphocytes, such as B- and T-cells).
  • Hematological toxicity can be observed, for example, as anemia, reduction in platelet count (i.e., thrombocytopenia) or reduction in white blood cell count (i.e., neutropenia).
  • myelodysplasia can result in the development of leukemia.
  • Long-term toxicity related to ionizing radiation can also damage other self-renewing cells in a subject, in addition to hematological cells. Thus, long-term toxicity can also lead to graying and frailty.
  • a tricyclic lactam compound that is "substantially free” of off-target effects can have some minor off-target effects that do not interfere with the tricyclic lactam's ability to provide protection from cytotoxic compounds in CDK4/6-dependent cells.
  • a tricyclic lactam that is "substantially free” of CDK4/6 inhibitory off-target effects can have some minor inhibitory effects on other CDKs (e.g., IC 50 s for CDK1 or CDK2 that are >0.5 ⁇ ; >1.0 ⁇ , or >5.0 ⁇ ), so long as the tricyclic lactam provides selective Gl arrest in CDK4/6-dependent cells.
  • synchronous reentry into the cell cycle is meant that HSPC cells in Gl -arrest due to the effects of a tricyclic lactam compound reenter the cell-cycle within relatively the same collective timeframe or at relatively the same rate upon dissipation of the compound's effect.
  • asynchronous reentry into the cell cycle is meant that the HSPC cells in Gl arrest reenter the cell-cycle within relatively different collective timeframes or at relatively different rates upon dissipation of the compound's effect, such as induced by PD0332991.
  • the subject treated or exposed to IR is typically a human subject, although it is to be understood the methods described herein are effective with respect to other mammals or vertebrate species.
  • the term subject can include animals such as mice, monkeys, dogs, pigs, rabbits, domesticated swine (pigs and hogs), ruminants, equine, poultry, felines, murines, bovines, canines, and the like.
  • the invention is directed to compounds or the use of such compounds of Formula I, II, III, IV, or V:
  • Z is -(CH 2 )x- wherein x is 1, 2, 3 or 4 or -0-(CH 2 ) z - wherein z is 2, 3 or 4;
  • each X is independently CH or N;
  • each X' is independently CH or N;
  • X is independently CH 2 , S or NH, arranged such that the moiety is a stable 5-membered ring;
  • R, R 8 , and R 11 are independently H, C1-C3 alkyl or haloalkyl, cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O or S; -(alkylene) m - C3-C8 cycloalkyl, -(alkylene) m - aryl, -(alkylene) m -heterocyclo, -(alkylene) m - heteroaryl, -(alkylene) m -NR 3 R 4 , -(alkylene) m -C(0)- NR 3 R 4 ; -(alkylene) m -0-R 5 , -(alkylene) m - S(0) n -R 5 , or -(alkylene) m -S(0) admir-NR 3
  • each R 1 is independently aryl, alkyl, cycloalkyl or haloalkyl, wherein each of said alkyl, cycloalkyl and haloalkyl groups optionally includes O or N heteroatoms in place of a carbon in the chain and two R x '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;
  • y is 0, 1, 2, 3 or 4;
  • 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 -0-R 5 ,
  • R 3 and R 4 at each occurrence are independently:
  • R 5 and R 5 * at each occurrence is:
  • R x at each occurrence is independently, halo, cyano, nitro, oxo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, heterocycloalkyl, -(alkylene) m -OR 5 , -(alkylene) m -0-alkylene-OR 5 , -(alkylene) m -S(0) n -R 5
  • alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, and heterocycloalkyl groups may be further independently substituted with one or more -(alkylene) m -CN, -(alkylene) m -OR 5 *, -(alkylene) m -S(0) n -R 5 *
  • n 0, 1 or 2
  • n 0, 1 or 2;
  • R 3 * and R 4 * at each occurrence are independently:
  • R 6 is H or lower alkyl, -(alkylene)m-heterocyclo, -(alkylene) m -heteroaryl, -(alkylene) m -NR 3 R 4 , -(alkylene) m -C(0)-NR 3 R 4 ; -(alkylene) m -0-R 5 , -(alkylene) m -S(0) n -R 5 , or -(alkylene) m -S(0) n -
  • NR 3 R 4 any of which may be optionally independently substituted with one or more R x groups as allowed by valence, and wherein two R x groups bound to the same or adjacent atoms may optionally combine to form a ring;
  • R 10 is (i) NHR A , wherein R A is unsubstituted or substituted Ci-Cg alkyl, cycloalkylalkyl, or -TT- RR, Ci-Cg cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O, and S; TT is an unsubstituted or substituted Ci-Cg alkyl or C 3 -C8 cycloalkyl linker; and RR is a hydroxyl, unsubstituted or substituted Ci-C 6 alkoxy, amino, unsubstituted or substituted Ci-C 6 alkylamino, unsubstituted or substituted di-Ci-C 6 alkylamino, unsubstituted or substituted C 6 -Cio aryl, unsubstituted or substituted heteroaryl comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S, unsubstituted or
  • two R 8 groups bonded to the same carbon can form an exocyclic double bond. In another embodiment, two R 8 groups bonded to the same carbon can form a carbonyl group.
  • the invention is directed to compounds or the use of such compounds of Formula VI:
  • R, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R x , Z, m, n, and y are as defined above;
  • each R 14 is independently H, C1-C3 alkyl or haloalkyl, cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O or S; -(alkylene) m - C3-C8 cycloalkyl, -(alkylene) m -aryl, - (alkylene)m-heterocyclo, -(alkylene) m - heteroaryl, -(alkylene) m -NR 3 R 4 , -(alkylene) m -C(0)- NR 3 R 4 ; -(alkylene) m -0-R 5 , -(alkylene) m - S(0) n -R 5 , or -(alkylene) m -S(0) felicit-NR 3 R 4 any of which may be optionally independently substituted with one or more R x groups as allowed by valence, and wherein two R x groups bound to the same or adjacent
  • the invention is directed to compounds or the use of such compounds of Formula I, II, III, IV, or V: wherein:
  • Z is -(CH 2 ) X - wherein x is 1, 2, 3 or 4 or -0-(CH 2 ) z - wherein z is 2, 3 or 4;
  • each X is independently CH or N;
  • each X' is independently CH or N;
  • X is independently CH 2 , S or NH, arranged such that the moiety is a stable 5-membered ring;
  • R, R 8 , and R 11 are independently H, C1-C3 alkyl (including methyl) or haloalkyl, cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O or S; -(alkylene) m - C3-C8 cycloalkyl, -(alkylene) m -aryl, -(alkylene) m -heterocyclo, -(alkylene) m - heteroaryl, -(alkylene) m - NR 3 R 4 , -(alkylene) m -C(0)-NR 3 R 4 ; -(alkylene) m -0-R 5 , -(alkylene) m - S(0) n -R 5 , or -(alkylene) m - S(0)
  • each R 1 is independently aryl, alkyl, cycloalkyl or haloalkyl, wherein each of said alkyl, cycloalkyl and haloalkyl groups optionally includes O or N heteroatoms in place of a carbon in the chain and two R x '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;
  • y is 0, 1, 2, 3 or 4;
  • 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 -0-R 5 , -(alkylene) m -S(0) n -R 5 , or - (alkylene) m -S(0) n -NR 3 R 4 any of which, other than heterocyclo, may be optionally independently substituted with one or more R x groups as allowed by valance, and wherein two R x groups bound to the same or adjacent atom may optionally combine to form a ring and wherein m is 0, 1 , or 2 and n is 0, 1 or 2;
  • heterocyclo may be optionally independently substituted with 1 to 3 R x groups as allowed by valance, and wherein two R x groups bound to the same or adjacent atom may optionally combine to form a ring;
  • R 3 and R 4 at each occurrence are independently:
  • R 5 and R 5 * at each occurrence is:
  • alkyl alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl any of which, other than heterocyclo, may be optionally independently substituted with one or more R x groups as allowed by valance;
  • R x at each occurrence is independently, halo, cyano, nitro, oxo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, heterocycloalkyl, -(alkylene) m -OR 5 , -(alkylene) m -0-alkylene-OR 5 , -(alkylene) m -S(0) n -R 5 , -(alkylene) m -NR 3 R 4 , -(alkylene) m -CN, -(alkylene) m -C(0)-R 5 , -(alkylene) m -C(S)-R 5 , -(alkylene) m -C(0)-OR 5 , -(alkylene
  • heterocycle may be further independently substituted with one to three substitutions selected from
  • n 0, 1; or 2 and
  • R 3 * and R 4 * at each occurrence are independently:
  • alkyl alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl any of which, other than heterocyclo, may be optionally independently substituted with one or more R x groups as allowed by valance; or R 3 * and R 4 * together with the nitrogen atom to which they are attached may combine to form a heterocyclo ring optionally independently substituted with one or more R x groups as allowed by valance;
  • R 6 is H, absent, or lower alkyl, -(alkylene)m-heterocyclo, -(alkylene) m -heteroaryl, -(alkylene) m - NR 3 R 4 , -(alkylene) m -C(0)-NR 3 R 4 ; -(alkylene) m -0-R 5 , -(alkyl)
  • R 10 is (i) NHR A , wherein R A is unsubstituted or substituted Ci-Cg alkyl, cycloalkylalkyl, or -TT- RR, Ci-Cg cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O, and S; TT is an unsubstituted or substituted Ci-Cg alkyl or C 3 -C8 cycloalkyl linker; and RR is a hydroxyl, unsubstituted or substituted Ci-C 6 alkoxy, amino, unsubstituted or substituted Ci-C 6 alkylamino, unsubstituted or substituted di-Ci-C 6 alkylamino, unsubstituted or substituted C 6 -Cio aryl, unsubstituted or substituted heteroaryl comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S, unsubstituted or
  • each heteroaryl is an aryl ring system that contains one or more heteroatoms selected from the group O, N and S, wherein the ring nitrogen and sulfur atom(s) are optionally oxidized, and nitrogen atom(s) are optionally quarternized;
  • each aryl is a carbocyclic aromatic system containing one or two rings, wherein such rings may be attached together in a fused manner, and wherein each aryl may have 1 or more R x substituents;
  • each heterocyclo is a saturated or partially saturated heteroatom-containing ring radical, where the heteroatoms may be selected from nitrogen, sulfur and oxygen, wherein each heterocyclo is a monocyclic 6-8 membered ring or a 5-16 membered bicyclic ring system, and wherein each heterocyclo may have 1 to 3 R x substituents; or a pharmaceutically acceptable salt, prodrug or isotopic variant, for example, partially or fully deuterated form thereof.
  • aryl means a carbocyclic aromatic system containing one or two rings wherein such rings may be attached together in a fused manner, which may have 1 or more substituents selected from lower alkyl, hydroxyl, halo, haloalkyl, nitro, cyano, alkoxy and lower alkylamino.
  • heterocyclyl or “heterocyclo” means a saturated or partially saturated heteroatom-containing ring radical, where the heteroatoms may be selected from nitrogen, sulfur and oxygen, which may have 1 to 3 substituents selected from hydroxyl, Boc, halo, haloalkyl, cyano, lower alkyl, lower aralkyl, oxo, lower alkoxy, amino and lower alkylamino, wherein the heterocyclic ring is a monocyclic 6-8 membered rings, or a 5-16 membered bicyclic ring systems which can include bridged fused and spiro- fused bicyclic ring systems, and which does not include rings containing -O-O-.-O-S- or -S-S- portion.
  • heteroaryl means an aryl ring system that contains one or more heteroatoms selected from the group O, N and S, wherein the ring nitrogen and sulfur atom(s) are optionally oxidized, and nitrogen atom(s) are optionally quarternized.
  • two R 8 groups bonded to the same carbon can form an exocyclic double bond. In another embodiment, two R 8 groups bonded to the same carbon can form a carbonyl group.
  • the invention is directed to compounds or the use of such compounds of Formula VI:
  • R, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R x , Z, m, n, and y are as defined above;
  • each R 14 is independently H, C 1 -C 3 alkyl (including methyl) or haloalkyl, cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O or S; -(alkylene) m - C 3 -C8 cycloalkyl, -(alkylene) m -aryl, -(alkylene) m -heterocyclo, -(alkylene) m - heteroaryl, -(alkylene) m - NR 3 R 4 , -(alkylene) m -C(0)-NR 3 R 4 ; -(alkylene) m -0-R 5 , -(alkylene) m - S(0) n -R 5 , or -(alkylene) m - S(0) n -NR 3 R 4 any of which, other than heterocyclo, may be optionally independently substituted with one or more R x groups as allowed by
  • each heteroaryl is an aryl ring system that contains one or more heteroatoms selected from the group O, N and S, wherein the ring nitrogen and sulfur atom(s) are optionally oxidized, and nitrogen atom(s) are optionally quarternized;
  • each aryl is a carbocyclic aromatic system containing one or two rings, wherein such rings may be attached together in a fused manner, and wherein each aryl may have 1 or more R x substituents;
  • each heterocyclo is a saturated or partially saturated heteroatom-containing ring radical, where the heteroatoms may be selected from nitrogen, sulfur and oxygen, wherein each heterocyclo is a monocyclic 6-8 membered ring or a 5-16 membered bicyclic ring system, and wherein each heterocyclo may have 1 to 3 R x substituents;
  • the compound is selected from Formula I or Formula II and R 6 is absent.
  • the compound is of Formula III:
  • R x is not further substituted.
  • 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 -0-R 5 ,
  • R 8 is hydrogen or C 1 -C 3 alkyl.
  • R is hydrogen or C 1 -C 3 alkyl.
  • R 2 is -(alkylene) m -heterocyclo, -(alkylene) m -NR 3 R 4 , -(alkylene) m -C(0)-NR 3 R 4 , -(alkylene) m -C(0)-0-alkyl or -(alkylene) m -OR 5 any of which may be optionally independently substituted with one or more R x groups as allowed by valence, and wherein two R x groups bound to the same or adjacent atom may optionally combine to form a ring.
  • R 2 is -(alkylene) m -heterocyclo, -(alkylene) m -NR 3 R 4 , -(alkylene) m -C(0)-NR 3 R 4 , -(alkylene) m -C(0)-0-alkyl or -(alkylene) m -OR 5 without further substitution.
  • n in R 2 is 1. In a further aspect, the alkylene in R 2 is methylene.
  • R 2* is a bond, alkylene, -(alkylene) m -0-(alkylene) m -, -(alkylene) m -C(0)-(alkylene) m -,
  • P is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group
  • each R xl is independently -(alkylene) m -(C(0)) m -(alkylene) m -(N(R N )) m -(alkyl) m wherein each m is independently 0 or 1 provided at least one m is 1, -(C(0))-0-alkyl, -(alkylene) m -cycloalkyl wherein m is 0 or 1, -N(R N )-cycloalkyl, -C(0)-cycloalkyl, -(alkylene) m -heterocyclyl wherein m is 0 or 1, or -N(R N )-heterocyclyl, -C(0)-heterocyclyl,- S(0) 2 -(alkylene) m wherein m is 1 or 2, wherein:
  • R N is H, Ci to C 4 alkyl or Ci to C 6 heteroalkyl
  • R xl can, together with the atoms to which they attach on P, which may be the same atom, form a ring;
  • t 0, 1 or 2.
  • each R xl is only optionally substituted by unsubstituted alkyl, halogen or hydroxy.
  • R xl is hydrogen or unsubstituted Ci-C 4 alkyl.
  • At least one R xl is -(alkylene) m -heterocyclyl wherein m is 0 or 1.
  • R 2 is wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group.
  • R 2 is
  • R 2 is wherein: R is a bond, alkylene, -(alkylene) m -0-(alkylene) m -, -(alkylene) m -C(0)-(alkylene) m -, -(alkylene) m -S(0) 2 -(alkylene) m -, or -(alkylene) m -NH-(alkylene) m - wherein each m is independently 0 or 1 ;
  • P is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group
  • PI is a 4- to 6-membered monocyclic saturated heterocyclyl group
  • each R x2 is independently hydrogen or alkyl
  • s 0, 1 or 2.
  • R 2 is
  • PI includes at least one nitrogen.
  • any alkylene in R 2 * in any previous aspect is not further substituted.
  • R 2 is selected from the structures depicted in FIGS.
  • R is
  • the compound has general Formula I and more specifically one of the general structures in FIGS. 5A-9F wherein the variables are as previously defined.
  • the compound has general Formula la:
  • R 1 , R 2 , R, R 8 , X and y are as previously defined.
  • the compound has Formula la and R is alkyl.
  • the compound has Formula la and R is H.
  • the compound has Formula la and R 2 is erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R , R xl and t are as previously defined.
  • the compound has Formula la and R 2 is erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, R xl is hydrogen or unsubstituted C 1 -C4 alkyl and R 2* is as previously defined.
  • the compound is of Formula lb:
  • R, R 2 and R 8 are as previously defined.
  • the compound is of Formula lb and R is alkyl.
  • the compound is of Formula lb and R is H.
  • the compound has Formula lb and R 2 is erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R , R xl and t are as previously defined.
  • the compound has Formula lb and R is ein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, R xl is hydrogen or C 1 -C 4 alkyl and R 2* is as previously defined.
  • the compound has Formula Ic:
  • R, R 2 and R 8 are as previously defined.
  • the compound has Formula Ic and R is alkyl.
  • the compound has Formula Ic and R is H.
  • the compound has Formula Ic and R 2 is ein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R , R xl and t are as previously defined.
  • the compound has Formula Ic and R 2 is erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, R xl is hydrogen or C 1 -C 4 alkyl and R 2* is as previously defined.
  • the compound has Formula Id:
  • R, R 2 and R 8 are as previously defined.
  • the compound has Formula Id and R is alkyl.
  • the compound has Formula Id and R is H.
  • the compound has Formula R is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R 2* , R xl and t are as previously defined.
  • the compound has Formula Id and R z is erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, R xl is hydrogen or C 1 -C4 alkyl and R 2* is as previously defined.
  • the compound has Formula Ie:
  • R, R 2 and R 8 are as previously defined.
  • the compound has Formula Ie and R is alkyl.
  • the compound has Formula Ie and R is H.
  • the compound has Formula Ie and R 2 is erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R , R xl and t are as previously defined.
  • the compound has Formula Ie and R is ein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, R xl is hydrogen or C 1 -C 4 alkyl and R 2* is as previously defined.
  • the compound has Formula If:
  • R, R 2 and R 8 are as previously defined.
  • the compound has Formula If and R is alkyl.
  • the compound has Formula If and R is H.
  • the compound has Formula If and R 2 is erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R 2* , R xl and t are as previously defined.
  • the compound has Formula If and R 2 is ein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, R xl is hydrogen or C 1 -C 4 alkyl and R 2* is as previously defined.
  • R, R 2 and R 8 are as previously defined.
  • the compound has Formula Ig and R is alkyl.
  • the compound has Formula Ig and R is H.
  • the compound has Formula Ig and R 2 is erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R 2* , R xl and t are as previously defined.
  • the compound has Formula Ig and R 2 is ein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, R xl is hydrogen or C1-C4 alkyl and R 2* is as previously defined.
  • R, R 2 and R 8 are as previously defined.
  • the compound has Formula Ih and R is alkyl.
  • the compound has Formula Ih and R is H.
  • the compound has Formula Ih and R 2 is erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R 2* , R xl and t are as previously defined.
  • the compound has Formula Ih and R is ein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, R xl is hydrogen or C 1 -C 4 alkyl and R 2* is as previously defined.
  • R, R 2 and R 8 are as previously defined.
  • the compound has Formula Ii and R is alkyl.
  • the compound has Formula Ii and R is H.
  • the compound has Formula Ii and R 2 is erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R , R xl and t are as previously defined.
  • the compound has Formula Ii and R 2 is erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, R xl is hydrogen or C 1 -C 4 alkyl and R 2* is as previously defined.
  • R, R 2 and R 8 are as previously defined.
  • the compound has Formula Ij and R is alkyl.
  • the compound has Formula Ij and R is H.
  • the compound has Formula Ij and R 2 is erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group.
  • the compound has Formula Ij and R 2 is erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, R xl is hydrogen or C 1 -C4 alkyl.
  • the compound has Formula Ij and R is H, and X is CH and N. In some embodiments the compound has the structure:
  • the compound has Formula Ik and R 2 is erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group. In some embodiments, the compound has Formula Ik and R is ein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, R xl is hydrogen or C 1 -C4 alkyl.
  • the compound has Formula II and R is erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group.
  • the compound has Formula II and R 2 is erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, R xl is hydrogen or C 1 -C4 alkyl.
  • the compound has Formula Im:
  • the compound has Formula Im and R is
  • the compound has Formula Im and R is wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, R xl is hydrogen or C 1 -C4 alkyl.
  • the compound has Formula Ila:
  • the compound has Formula Ila and R is wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group.
  • the compound has Formula Ila and R 2 is wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, R xl is hydrogen or C 1 -C4 alkyl.
  • the compound has Formula lib:
  • the compound has Formula lib and R is erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group. In some embodiments, the compound has Formula lib and R is erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, R xl is hydrogen or C1-C4 alkyl.
  • the active compound is:
  • the present invention includes compounds and the use of compounds with desired isotopic substitutions of atoms, at amounts 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 of hydrogen for example, deuterium ( 2 H) and tritium ( 3 H) may be used anywhere in described structures.
  • isotopes of carbon e.g., 13C and 14 C, may be used.
  • a preferred isotopic substitution is deuterium for hydrogen at one or more locations on the molecule to improve the performance of the drug.
  • the deuterium can be bound in a location of bond breakage during metabolism (an a-deuterium kinetic isotope effect) or next to or near the site of bond breakage (a ⁇ -deuterium kinetic isotope effect).
  • substitution with isotopes such as deuterium can afford certain therapeutic advantages resulting from greater metabolic stability, such as, for example, increased in vivo half-life or reduced dosage requirements.
  • Substitution of deuterium for hydrogen at a site of metabolic break down can reduce the rate of or eliminate the metabolism at that bond.
  • the hydrogen atom can be any isotope of hydrogen, including protium (1H), deuterium ( 2 H) and tritium ( 3 H).
  • isotopically-labeled refers to an analog that is a "deuterated analog", a " 13 C- labeled analog,” or a “deuterated/ 13 C-labeled analog.”
  • deuterated analog means a compound described herein, whereby a H-isotope, i.e., hydrogen/protium (1H), is substituted by a H-isotope, i.e., deuterium ( 2 H).
  • Deuterium substitution can be partial or complete. Partial deuterium substitution means that at least one hydrogen is substituted by at least one deuterium.
  • the isotope is 90, 95 or 99% or more enriched in an isotope at any location of interest. In some embodiments it is deuterium that is 90, 95 or 99% enriched at a desired location.
  • Tissue-specific stem cells are capable of self-renewal, meaning that they are capable of replacing themselves throughout the adult mammalian lifespan through regulated replication. Additionally, stem cells divide asymmetrically to produce "progeny" or "progenitor” cells that in turn produce various components of a given organ. For example, in the hematopoietic system, the hematopoietic stem cells give rise to progenitor cells which in turn give rise to all the differentiated components of blood (e.g., white blood cells, red blood cells, and platelets) (See FIG. 1).
  • blood e.g., white blood cells, red blood cells, and platelets
  • HSPC hematopoietic stem/progenitor cells
  • CDK4 proliferative kinases
  • CDK6 cyclin-dependent kinase 6
  • the present invention includes methods of protecting healthy cells in a subject, and in particular, hematopoietic cells and/or progenitor cells (HSPCs) from the toxic effects or mitigation of ionizing radiation by the administration of a tricyclic lactam compound.
  • the tricyclic lactam compound is a selective CDK4/6 inhibitor.
  • the use of tricyclic lactams as CDK4/6-specific Gl -arresting effect compounds as radioprotectants and radiomitigants allows for an accelerated hematological recovery and reduced hematological cytotoxicity risk due to HSPC replication delay.
  • the tricyclic lactam administered is selected from the group consisting of a compound or composition comprising Formula I, Formula II, Formula III, Formula IV, Formula V, Formula VI, or a combination thereof.
  • a compound can be selected from the compounds of Table 1 , or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof.
  • compounds, methods, and compositions are provided for reducing or limiting the effect of DNA damaging ionizing radiation on hematopoietic stem and progenitor cells in a subject undergoing treatment for a Rb-null cancer, the method comprising administering an effective amount of a tricyclic compound prior to exposure to IR.
  • a substantial portion of the hematopoietic stem and/or progenitor cells return to pre-treatment baseline cell cycle activity (i.e., reenter the cell-cycle) within less than about 48 hours of administration of the tricyclic lactam.
  • the tricyclic lactam administered is selected from the group consisting of a compound or composition comprising Formula I, Formula II, Formula III, Formula IV, Formula V, and Formula VI, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof.
  • a compound can be selected from the compounds of Table 1, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof.
  • tricyclic lactam compounds, methods, and composition are provided for reducing or limiting the effect of DNA-damaging IR on hematopoietic stem and progenitor cells in a subject that has been exposed to IR, the method comprising administering an effective amount of a tricyclic lactam following exposure to IR, wherein a substantial portion of the hematopoietic stem and/or progenitor cells reenter the cell-cycle synchronously within less than about 24, 30, 36, 40, or 48 hours following the dissipation of the compound's CDK4/6 inhibitory effect, wherein the tricyclic lactam compound has an IC 50 CDK4 inhibitory concentration that is more than 500 times less than its IC 50 inhibitory concentration for CDK2.
  • a substantial portion of the hematopoietic stem and/or progenitor cells reenter the cell-cycle synchronously within less than about 24, 30, 36, 40, or 48 hours from the point in which the tricyclic lactam's concentration level in the subject's blood drops below a therapeutic effective concentration.
  • the tricyclic lactam administered is selected from the group consisting of a compound or composition comprising Formula I, Formula II, Formula III, Formula IV, Formula V, and Formula VI or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof.
  • a compound can be selected from the compounds of Table 1 , or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof.
  • the tricyclic lactam is a CDK4/6 inhibitor selected from Formula I, II, III, IV, V, or VI, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof, wherein the protection afforded by the compound is short term and transient in nature, allowing a significant portion of the cells to synchronously renter the cell-cycle following the cessation of IR exposure.
  • Cells that are quiescent within the Gl phase of the cell cycle are more resistant to the DNA damaging effect of radiation than proliferating cells.
  • the tricyclic lactam compounds for use in the described methods are CDK4/6 inhibitors, with minimal CDK2 inhibitory activity.
  • a tricyclic lactam compound for use in the methods described herein has a CDK4/CycDl IC 50 inhibitory concentration value that is >100, >200, >300, >400, >500, >600, >700, >800, >900, >1000, >1250, >1500 times, >1800 times, >2000 times, >2200 times, >2500 times, >2700 times, >3000 times, >3200 times lower than its respective IC 50 concentration value for CDK2/CycE inhibition.
  • a tricyclic lactam for use in the methods described herein has an IC 50 concentration value for CDK4/CycDl inhibition that is about ⁇ 1.50 nM, ⁇ 1.25 nM, ⁇ 1.0 nM, ⁇ 0.90 nM, ⁇ 0.85 nM, ⁇ 0.80 nM, ⁇ 0.75 nM, ⁇ 0.70 nM, ⁇ 0.65 nM, ⁇ 0.60 nM, ⁇ 0.55 nM, or less.
  • a tricyclic lactam for use in the methods described herein has an IC 50 concentration value for CDK2/CycE inhibition that is about >1.0 ⁇ , >1.25 ⁇ , >1.50 ⁇ , >1.75 ⁇ , >2.0 ⁇ , >2.25 ⁇ , >2.50 ⁇ , >2.75 ⁇ , >3.0 ⁇ , >3.25 ⁇ , >3.5 ⁇ or greater.
  • a tricyclic lactam for use in the methods described herein has an IC 50 concentration value for CDK2/CycA IC 50 that is >0.80 ⁇ , >0.85 ⁇ , >0.90 ⁇ , >0.95 ⁇ , >.1.0 ⁇ , >1.25 ⁇ , >1.50 ⁇ , >1.75 ⁇ , >2.0 ⁇ , >2.25 ⁇ , >2.50 ⁇ , >2.75 ⁇ , >3.0 ⁇ or greater.
  • the tricyclic lactam for use in the methods described herein are selected from the group consisting of Formula I, Formula II, Formula III, Formula IV, Formula V, and Formula VI, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug, thereof.
  • a compound can be selected from the compounds of Table 1, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof.
  • radiation protection with the tricyclic lactams described herein may be achieved by a number of different dosing schedules.
  • concomitant treatment can also be effective.
  • the tricyclic lactams described herein are used in HSPC cycling strategies wherein a subject is exposed to regular, repeated IR exposures, wherein HSPCs are Gl -arrested when IR exposed and allowed to reenter the cell-cycle before the subject's next IR exposure.
  • HSPCs are Gl -arrested when IR exposed and allowed to reenter the cell-cycle before the subject's next IR exposure.
  • Such cycling allows HSPCs to regenerate damaged blood cell lineages in between regular, repeated IR exposures, for example those associated with standard IR treatments for cancer, and reduces the risk associated with long term CDK4/6 inhibition.
  • the subject is exposed to IR at least 5 times a week, at least 4 times a week, at least 3 times a week, at least 2 times a week, at least 1 time a week, at least 3 times a month, at least 2 times a month, or at least 1 time a month, wherein the subject's HSPCs are Gl arrested during treatment and allowed to cycle in between IR exposure, for example during a treatment break.
  • the subject is undergoing 5 times a week IR exposure, wherein the subject's HSPCs are Gl arrested during the IR exposure and allowed to reenter the cell-cycle during the 2 day break, for example, over the weekend.
  • the subject's HSPCs are arrested during the entirety of the IR exposure time-period for the weekly treatment, for example, during a 5 times/week IR regimen, the cells are arrested over the time period that is required to complete the IR exposure regimen for the week, and then allowed to recycle at the end of the regimen.
  • the subject's HSPCs are arrested during the entirety of the IR regimen, for example, in a 5 times a week IR regimen for 5 weeks, and rapidly reenter the cell-cycle following the completion of the IR regimen.
  • the subject has been exposed to IR, and, using a tricyclic lactam described herein, the subject's HSPCs are placed in Gl arrest following exposure in order to mitigate DNA damage.
  • the tricyclic lactam is administered at least 1 ⁇ 2 hour, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 14 hours, at least 16 hours, at least 18 hours, at least 20 hours, at least 24 hours or more post IR exposure.
  • the subject has been exposed to IR and is administered multiple tricyclic lactam doses at differing time points, for example, at 12 hours and 24 hours post IR exposure.
  • the presently disclosed subject matter provides methods for protection of mammals from the acute and chronic toxic effects of ionizing radiation by forcing hematopoietic stem and progenitor cells (HSPCs) into a quiescent state by transient (e.g., over a period of less than about 40 hours, 36 hours, 30 hours, 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, 4 hours, 3 hours, 2.5 hours, 2 hours, 1 hour, 1 ⁇ 2 hour or less prior to IR exposure) treatment with a tricyclic lactam selected from the group consisting of Formula I, Formula II, Formula III, Formula IV, Formula V, and Formula VI, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof.
  • HSPCs hematopoietic stem and progenitor cells
  • the HSPCs recover from this period of transient quiescence, and then function normally after treatment with the compound is stopped, and its intra-cellular effect dissipates. During the period of quiescence, the stem and progenitor cells are protected from the effects of ionizing radiation. The ability to protect stem/progenitor cells is desirable both in the treatment of cancer where patients are given high, repeated doses of ionizing radiation, and in environmental or occupational situations where individuals may be in danger of being exposed to large doses of radiation.
  • the HSPCs can be arrested for longer periods, for example, over a period of hours, days, and/or weeks, through multiple, time separated administrations of a tricyclic lactam described herein.
  • these compounds can be administered in a concerted regimen with a blood growth factor agent.
  • hematopoietic growth factors including, but not limited to, granulocyte colony stimulating factor (G-CSF, for example, sold as Neupogen (filgrastin), Neulasta (peg-filgrastin), or lenograstin), 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
  • G-CSF granulocyte colony stimulating factor
  • GM-CSF granulocyte colony
  • the EPO family of therapeutics has been associated with arterial hypertension, cerebral convulsions, hypertensive encephalopathy, tumor progression thromboembolism, iron deficiency, influenza like syndromes and venous thrombosis.
  • the G- CSF family of therapeutics has been associated with myelodysplasia and secondary leukemia, spleen enlargement and rupture, respiratory distress syndrome, allergic reactions and sickle cell complications.
  • the tricyclic lactam allows the patient to receive some amount of the growth factor.
  • the patient may not need as much hematopoietic growth factor because the hematopoietic cells will have been protected during the chemotherapy and not diminished to the extent without the tricyclic lactam.
  • hematopoietic cells are not forced into replicating while harboring major DNA structural damage.
  • the radio-protective methods described herein using a tricyclic lactam described herein can allow for dose intensification (e.g., more therapy can be given in a fixed period of time) in medically related IR therapies, which will translate to better efficacy. Therefore, the presently disclosed methods can result in radio-therapy regimens that are less toxic and more effective.
  • the tricyclic lactam described herein are orally available small molecules, which can be formulated for administration via a number of different routes.
  • such small molecules can be formulated for oral, topical, intranasal, inhalation, intravenous, intramuscular, or any other form of administration. Further, as opposed to biologies, stable small molecules can be more easily stockpiled and stored. Thus, the tricyclic lactam compounds can be more easily and cheaply kept on hand in emergency rooms where subjects of IR exposure can report or at sites where radiation exposure is particularly likely to occur: at nuclear power plants, on nuclear powered vessels, at military installations, near battlefields, etc.
  • the use of a tricyclic lactam as described herein can induce selective Gl arrest in CDK4/6-dependent cells (e.g., as measured in a cell-based in vitro assay).
  • the tricyclic lactam is capable of increasing the percentage of CDK4/6-dependent cells in the Gl phase, while decreasing the percentage of CDK4/6-dependent cells in the G2/M phase and S phase.
  • the tricyclic lactam induces substantially pure (i.e., "clean") Gl cell cycle arrest in the CDK4/6-dependent cells (e.g., wherein treatment with the tricyclic lactam induces cell cycle arrest such that the majority of cells are arrested in Gl as defined by standard methods (e.g.
  • Methods of assessing the cell phase of a population of cells are known in the art (see, for example, in U.S. Patent Application Publication No. 2002/0224522) and include cytometric analysis, microscopic analysis, gradient centrifugation, elutriation, fluorescence techniques including immunofluorescence, and combinations thereof.
  • Cytometric techniques include exposing the cell to a labeling agent or stain, such as DNA-binding dyes, e.g., PI, and analyzing cellular DNA content by flow cytometry.
  • Immunofluorescence techniques include detection of specific cell cycle indicators such as, for example, thymidine analogs (e.g., 5-bromo-2-deoxyuridine (BrdU) or an iododeoxyuridine), with fluorescent antibodies.
  • the use of a tricyclic lactam described herein reduces the risk of undesirable off-target effects including, but not limited to, long term toxicity, anti-oxidant effects, and estrogenic effects.
  • Anti-oxidant effects can be determined by standard assays known in the art.
  • a compound with no significant anti-oxidant effects is a compound that does not significantly scavenge free-radicals, such as oxygen radicals.
  • the anti-oxidant effects of a compound can be compared to a compound with known anti-oxidant activity, such as genistein.
  • a compound with no significant anti-oxidant activity can be one that has less than about 2, 3, 5, 10, 30, or 100 fold anti-oxidant activity relative to genistein.
  • Estrogenic activities can also be determined via known assays.
  • a non-estrogenic compound is one that does not significantly bind and activate the estrogen receptor.
  • a compound that is substantially free of estrogenic effects can be one that has less than about 2, 3, 5, 10, 20, or 100 fold estrogenic activity relative to a compound with estrogenic activity, e.g., genistein.
  • the subject has been exposed to ionizing radiation, will be exposed to ionizing radiation, or is at risk of incurring exposure to ionizing radiation as the result of radiological agent exposure during warfare, a radiological terrorist attack, an industrial accident, or space travel.
  • Subjects can further be exposed to, or be scheduled to be exposed to, ionizing radiation when undergoing therapeutic irradiation for the treatment of proliferative disorders.
  • proliferative disorders include cancerous and non-cancer proliferative diseases.
  • the compounds are effective in protecting healthy hematopoietic stem/progenitor cells during therapeutic irradiation of a broad range of tumor types, including but not limited to the following: breast, prostate, ovarian, skin, lung, colorectal, brain (i.e., glioma) and renal.
  • growth of the cancer being treated by IR should not be affected by the tricyclic lactam compound.
  • the potential sensitivity of certain tumors to 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 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 or absence of retinoblastoma (Rb) tumor suppressor protein (Rb-null), high levels of MYC expression, increased cyclin E and increased cyclin A.
  • Rb retinoblastoma
  • Rb-null 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 certain classes of Rb-positive 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 leukemias, certain classes of lymphomas, 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 cancer and certain classes of testicular cancers.
  • small cell lung cancer retinoblastoma
  • HPV positive malignancies like cervical cancer and certain head and neck cancers
  • 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, 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 of the present invention 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.
  • 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 of the present invention 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.
  • the cancer a small cell lung cancer, retinoblastoma, and triple negative (ER/PR/Her2 negative) or "basal-like" breast cancer, which almost always inactivate the retinoblastoma tumor suppressor protein (Rb), and therefore do not require CDK4/6 activity to proliferate.
  • Triple negative (basal-like) 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 which inactivates Rb making these tumors functionally Rb-null.
  • Some lung cancers are also believed to be caused by HPV.
  • the tricyclic lactams described herein can also be used in protecting healthy CDK4/6- replication dependent cells during ionizing radiation of abnormal tissues in non-cancer proliferative diseases, including but not limited to the following: psoriasis, lupus, arthritis (notably rheumatoid arthritis), hemangiomatosis in infants, multiple sclerosis, myelodegenerative disease, neurofibromatosis, ganglioneuromatosis, keloid formation, Paget's Disease of the bone, fibrocystic disease of the breast, Peyronie's and Duputren's fibrosis, restenosis, and cirrhosis.
  • psoriasis lupus
  • arthritis notably rheumatoid arthritis
  • hemangiomatosis in infants multiple sclerosis
  • myelodegenerative disease neurofibromatosis
  • ganglioneuromatosis ganglioneuromatosis
  • keloid formation Paget
  • therapeutic ionizing radiation can be administered to a subject on any schedule and in any dose consistent with the prescribed course of treatment, for example by administering a compound of Formula I, Formula II, Formula III, Formula IV, Formula V, and Formula VI, or a compound selected from Table 1, prior to or during the radiation.
  • administration of the compound is timed such that maximal Gl arrest of the HSPCs, or a significant portion thereof, occurs at the time of the IR exposure.
  • a tricyclic lactam compound described herein is administered so that a peak serum concentration for the compound is reached at or near the time of IR exposure.
  • multiple doses of the radioprotectant compound can be administered to the subject.
  • the subject can be given a single dose of the compound.
  • the course of treatment differs from subject to subject, and those of ordinary skill in the art can readily determine the appropriate dose and schedule of therapeutic radiation in a given clinical situation.
  • the term "active compound” refers to the tricyclic lactam compounds described herein or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof.
  • the active compound can be administered to the subject through any suitable approach. The amount and timing of active compound administered is dependent on the subject being treated, on the dosage of IR to which the subject is anticipated of being exposed to, on the time course of the IR exposure, on the manner of administration, on the pharmacokinetic properties of the particular active compound, and on the judgment of the prescribing physician. Thus, because of subject to subject variability, 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 subject.
  • compositions can be prepared for any desired route of administration including, but not limited to, oral, intravenous, or aerosol administration, as discussed in greater detail below.
  • a dosage from about 0.1 to about 200 mg/kg is administered, with all weights being calculated based upon the weight of the active compound, including the cases where a salt is employed.
  • a dosage can provide the amount of compound needed to provide a serum concentration of the active compound of up to between about 1 and 5, 10, 20, 30 or 40 ⁇ .
  • a dosage from about 10 mg/kg to about 50 mg/kg can be employed for oral administration.
  • a dosage from about 0.5 mg/kg to 5 mg/kg can be employed for intramuscular injection.
  • dosages can be from about 1 umol/kg to about 50 umol/kg, or, optionally, between about 22 umol/kg and about 33 umol/kg of the compound for intravenous or oral administration.
  • An oral dosage form can include any appropriate amount of active material, including for example from 5 mg to, 50, 100, 200 or 500 mg per tablet or other solid dosage form.
  • pharmaceutically active compounds as described herein can be administered orally as a solid or as a liquid, or can be administered intramuscularly, intravenously, or by inhalation as a solution, suspension, or emulsion.
  • the compounds or salts also can be administered by inhalation, intravenously, or intramuscularly as a liposomal suspension.
  • the active compound or salt can 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 present invention 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 is a 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.
  • 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 formulation 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 solutions, suspensions, tablets, pills, capsules, powders, 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 tabletting purposes.
  • Solid compositions of a similar type may be also 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 subject 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.
  • an injectable, stable, sterile formulation 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 a liquid formulation suitable for injection thereof into a subject.
  • 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.
  • compositions 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 formulation 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 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.
  • the formulation 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 formulation sufficiently to result in the formation of droplets within the desired size range when subjected to nebulization.
  • pharmaceutically acceptable salts refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with subjects (e.g., human subjects) 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 subject matter.
  • salts refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the presently disclosed subject matter. These salts can be prepared in situ 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.
  • the compounds of the presently disclosed subject matter are basic compounds, they are all 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.
  • metals used as cations include, but are not limited to, sodium, potassium, magnesium, calcium, and the like.
  • 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.
  • Tricyclic lactams of the present invention can be synthesized by any means known to those of ordinary skill in the art, including for example, according to the generalized Schemes of 1 through 11 below.
  • a method for the preparation of substituted tricyclic lactams includes efficient methods for the preparation of a tricyclic lactam ring system and subsequent displacement of an aryl sulfone with an amine.
  • diethyl succinate is employed to prepare the pyrimidine ester, 2, according to the method of A. Haidle, See, WO 2009/152027 entitled 5,7-dihydro-6H-pyrrolo[2,3- d]pyrimidin-6-one derivatives for MARK inhibition.
  • the ester intermediate 2 can be reduced by directly reacting the ester with a reducing agent such as lithium borohydride in a protic organic solvent such as ethanol to produce the corresponding primary alcohol.
  • the primary alcohol can be reacted with a reagent such as phosphorus tribromide in an organic solvent such as dimethylforamide to produce the primary bromide 3.
  • the primary bromide 3 can be condensed with the lactam 4 optionally at low temperature using a base such as lithium diisopropylamide in an organic solvent such as tetrahydrofuran to produce the lactam 5.
  • Lactam 6 can be directly reacted with an organic base such as 1,8- diazabicyclo[5.4.0]undec-7-ene in a protic solvent such as ethanol optionally at high temperature to cyclize Compound 5 to form the tricyclic lactam 7.
  • the thiol moiety can be subsequently oxidized to the sulfone 8 by directly reacting Compound 7 with an oxidizing reagent such as meta-chloroperoxybenzoic acid.
  • the sulfone, 8, can be directly reacted with an amine, 9, in the presence of a strong base such as lithium hexamethyldisilazane to form the tricyclic lactam 10.
  • the tricyclic lactam 7 is directly reacted with an oxidizing reagent such as 2,3-dichloro-5,6-dicyano-l,4-benzoquinone (DDQ) to form the alkene 11.
  • Alkene 11 can be directly reacted with an oxidizing reagent such as meta-chloroperoxybenzoic acid to form the sulfone intermediate 12.
  • the sulfone, 12, can be condensed with an amine, 13, in the presence of a strong base such as lithium hexamethyldisilazane to form the tricyclic lactam 14.
  • Scheme 3 illustrates the synthesis of a di-protected lactam useful in the preparation of tricyclic lactams.
  • Compound 15 is prepared according to the method of Arigon, J., See, US 2013/0289031 entitled Pyrimidinone derivatives, preparation thereof and pharmaceutical use thereof.
  • Compound 15 is protected with a suitable protecting group by directly reacting Compound 15 with di-tert-butyl carbonate (Boc anhydride) in the presence of an organic base such as triethylamine or diisopropylethylamine in an organic solvent such as dichloromethane to form the protected amine 16.
  • Boc anhydride di-tert-butyl carbonate
  • the protected amine 16 can be directly reacted with methyl chloroacetate in the presence of a base such as potassium carbonate in an organic solvent such as acetonitrile to form the ester 17.
  • the ester 17 can be cyclized by directly reacting the ester with an acid such as hydrochloric acid in a protic solvent such as methanol optionally at a high temperature to form the spirolactam 18.
  • the lactam 18 can be directly reacted with a protecting reagent such as chloromethyl methyl ether (MOM-C1) in the presence of an organic base such as diisopropylethylamine in an organic solvent such as dichloromethane optionally at a low or at ambient temperature to form the MOM-protected amine 19.
  • MOM-C1 chloromethyl methyl ether
  • the lactam 19 can be protected by directly reacting the lactam with a suitable protecting reagent such as chloromethyl methyl ether (MOM-C1) in the presence of a base such as sodium bis(trimethylsilyl)amide in an organic solvent such as tetrahydrofuran optionally at a low temperature.
  • a suitable protecting reagent such as chloromethyl methyl ether (MOM-C1)
  • MOM-C1 chloromethyl methyl ether
  • a base such as sodium bis(trimethylsilyl)amide
  • organic solvent such as tetrahydrofuran optionally at a low temperature.
  • Additional lactam intermediates such as Compounds 25 and 31 can be synthesized using analogous chemistry as described for the synthesis of Compound 4. The chemistry for the production of Compounds 25 and 31 is illustrated in Schemes 5 and 6.
  • Scheme 4 illustrates the coupling of a tricyclic lactam sulfone with an amine to generate compounds of Formula I, II, III, and IV.
  • Scheme 7 illustrates the preparation of the tricyclic lactam compound 33.
  • Compound 32 is prepared according to the method of Tavares, See, US 8,598,186.
  • Compound 32 is directly reacted with sulfone 8 optionally in the presence of an organic base such as lithium hexamethyldisilazane and the amine 32 to form the amine 33.
  • an organic base such as lithium hexamethyldisilazane
  • the same chemistry can be employed to produce the alkene compound 34.
  • a lactam intermediate is treated with BOC-anhydride in the presence of an organic base such as triethylamine in an organic solvent such as dichloromethane.
  • the Boc protected lactam is treated with carbon dioxide in the presence of a nickel catalyst to generate a carboxylic acid.
  • the carboxylic acid is reacted with thionyl chloride in the presence of an organic solvent such as toluene.
  • the resulting acid chloride is treated with an amine to generate an amide that can be deprotected with a strong acid such as trifluoroacetic acid to generate the final target compound.
  • the lactam can be generated by reacting the carboxylic acid with a protected amine in the presence of a strong acid and a dehydrating agent, which can be together in one moiety as a strong acid anhydride.
  • strong acid anhydrides include, but are not limited to, trifluoroacetic acid anhydride, tribromoacetic acid anhydride, trichloroacetic acid anhydride, or mixed anhydrides.
  • the dehydrating agent can be a carbodiimide based compound such as but not limited to DCC ( ⁇ , ⁇ -dicyclohexylcarbodiimide), EDC (l-ethyl-3-(3- dimethylaminopropyl)carbodiimide or DIC ( ⁇ , ⁇ -diisopropylcarbodiimide).
  • DCC ⁇ , ⁇ -dicyclohexylcarbodiimide
  • EDC l-ethyl-3-(3- dimethylaminopropyl)carbodiimide or DIC ( ⁇ , ⁇ -diisopropylcarbodiimide).
  • DIC ⁇ , ⁇ -diisopropylcarbodiimide
  • the SMe moiety bonded to the pyrimidine ring can be substituted with any leaving group that can be displaced by a primary amine, for example to create an intermediate for a final product such as Br, I, F, S0 2 Me, SOalkyl, S0 2 alkyl. See, for Example, PCT /US2013/037878 to Tavares.
  • amine intermediates and final amine compounds can be synthesized by those skilled in the art. It will be appreciated that the chemistry can employ reagents that comprise reactive functionalities that can be protected and de-protected and will be known to those skilled in the art at the time of the invention. See for example, Greene, T.W. and Wuts, P.G.M., Greene's Protective Groups in Organic Synthesis, 4 th edition, John Wiley and Sons.
  • the reaction was quenched at 0°C by the slow addition of saturated aqueous sodium sulfate (265.3 mL, 530.7 mmol) keeping the internal reaction temperature below 10°C.
  • Ethyl acetate (900 mL) was added and the reaction slowly warmed to room temperature overnight. 6M HC1 was added till the reaction mixture was slightly acidic (pH 6).
  • the reaction mixture was filtered thru a pad of Celite® and the aluminum salts were washed with ethyl acetate (1L). The filtrate was poured into a separatory funnel and washed twice with water (600 mL) and finally with brine (600 mL).
  • [4-Chloro-2-(methylthio)pyrimidin-5-yl]methanol (39.2 g, 206 mmol) was taken up in methylene chloride (520 mL) at room temperature.
  • Manganese(IV) oxide (140 g, 1.60 mol) was added in one portion and the reaction mixture stirred at room temperature overnight.
  • the reaction mixture was filtered through a pad of Celite® and washed with methylene chloride. The filtrate was concentrated under reduced pressure to afford a dark yellow semisolid.
  • the crude product was purified by reverse phase chromatography running a gradient of 1 :9 acetonitrile: water (0.1%TFA) to 100% acetonitrile (0.1%TFA).
  • Isopropylmagnesium chloride lithium chloride complex (4.43 g, 30.5 mmol, 25.4mL of a 1.2M solution) was added to tetrahydrofuran (104 mL) in a 500 mL round bottomed flask which had been flame dried and cooled under Argon. The solution was cooled to -15°C. Ethyl propiolate (3.26 mL, 32.1 mmol) was added dropwise affording a yellow solution.
  • the organic layer was washed twice with a 10% citric acid (aq) (20 mL)/ brine (60 mL) mixture.
  • the organic layer was dried over sodium sulfate, filtered and the solvent removed in vacuo to afford a yellow oil.
  • the product was purified by silica gel chromatography using a gradient from 1 :9 to 2:3 ethyl acetate :hexanes which afforded 2.48g (41% yield) of the desired product as a pale yellow oil.
  • reaction mixture was transferred to a separatory funnel with water (30 mL) and ethyl acetate (150 mL). The aqueous layer was removed and the organic layer washed with brine (50 mL). The organic layer was dried over sodium sulfate, filtered and the solvent concentrated in vacuo to afford the 2.08g (89%> yield) of the desired product as a dark yellow oil.
  • the mixture was extracted with twice with ethyl acetate (150 mL) and the combined organic layers were thrice washed with half-saturated aqueous LiCl (20 mL). The combined organic layers were dried over sodium sulfate, filtered and the solvent removed in vacuo to afford a dark yellow oil.
  • the product was purified by silica gel chromatography using a gradient from 1 :4 to 2:3 ethyl acetate:hexanes which afforded 2.39g (88% yield) of the desired product as a brown sticky solid.
  • the product was purified by reverse phase chromatography using a gradient from 100% Water (0.1% TFA) to 1 : 1 acetonitrile: water (0.1% TFA). Lyophilization of the desired fractions afforded 1 1 mg (7% yield) of the desired product as an orange powder.
  • the reaction mixture was heated to 45 °C for 30 minutes. After cooling to room temperature, the reaction mixture was filtered through a pad of Celite® washing with ethanol. The solvent was removed in vacuo affording a yellow oil.
  • the product was purified by reverse phase chromatography using a gradient from 100% water (0.1% TFA) to 3:2 acetonitrile: water (0.1% TFA). Lyophilization of the desired fractions afforded 4 mg (14% yield) of the desired product as a pale yellow powder.
  • reaction mixture was further diluted with tetrahydrofuran (160 mL) and then cooled to 0°C. Saturated aqueous sodium carbonate (100 ml) was added very slowly keeping the internal temperature below 15°C. After the addition of the carbonate solution is complete, the ice bath was left to expire and the mixture slowly warmed to room temperature overnight.
  • the reaction mixture was filtered thru a pad of Celite® washing with ethyl acetate (400 mL). The solvent was removed in vacuo to afford a wet oil which was taken up in methylene chloride (300 mL) and dried over sodium sulfate.
  • an alkyne can be treated with an organic solvent, and a base optionally at a reduced temperature and subsequently treated with an aldehyde according to methods known in the art.
  • the aldehyde in Step 1 can be treated with a base, for example, isopropylmagnesium chloride lithium chloride complex in an organic solvent, for example, tetrahydrofuran at about -15 °C and next treated with an aldehyde to generate an alkyne.
  • a desired alkynyl alcohol can be treated with a base in an organic solvent at an elevated temperature to isomerize the desired alkynyl alcohol to a desired alkene.
  • a desired alkynyl alcohol can be treated with a base, such as triethylamine in an organic solvent, for example, 1 ,4-dioxane at an elevated temperature of about 60 °C to generate an alkene.
  • a desired alkene can be treated with ammonia and a mixture of organic solvents to form a tert-butyl 2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7- carboxylate according to methods known in the art.
  • an alkene can be treated with 0.5M ammonia and a mixture of organic solvents, for example, dioxane and acetonitrile to form a tert-butyl 2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate.
  • a desired ester can be treated with a desired acid and an organic solvent to generate a desired carboxylic acid according to methods known in the art.
  • a desired ester can be treated with a desired acid, for example, trifluoroacetic acid, to generate a carboxylic acid.
  • the organic solvent is dichloromethane.
  • a desired acid can be treated with a desired amine, an organic solvent and a coupling reagent to form a desired amide according to methods known in the art.
  • a desired acid can be treated with a desired amine, an organic solvent, for example, ⁇ , ⁇ -dimethylformamide, and a coupling reagent, for example, l-[bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate, to generate an amide.
  • a silyl protected alcohol can be treated with a fluoride reagent and an organic solvent according to methods known in the art to generate a desired alcohol.
  • a silyl protected alcohol can be treated with a fluoride reagent, for example, tetrabutylammonium fluoride, and an organic solvent, for example, acetonitrile, to generate an alcohol.
  • a desired alcohol can be treated with a sulfonyl chloride to generate a desired mesylate according to methods known in the art.
  • an alcohol can be treated with a desired sulfonyl chloride, for example, methanesulfonyl chloride, to generate a mesylate.
  • an amine spontaneously reacts with said mesylate to generate a cyclic amide.
  • a desired thiol can be treated with a desired amine and an organic solvent at an elevated temperature to generate a desired amine according to methods known in the art.
  • a thiol can be treated with an amine, for example, 5-(4-methylpiperazin-l- yl)pyridin-2-amine, and an organic solvent, for example, ⁇ , ⁇ -dimethylacetamide, at an elevated temperature of about 150 °C to generate an amine.
  • the compound 5-(4-methylpiperazin-l- yl)pyridin-2-amine can be prepared as disclosed in US 8,598,186 to Tavares and Strum.
  • an alkyne can be treated with an organic solvent, and a base optionally at a reduced temperature and subsequently treated with an aldehyde according to methods known in the art.
  • the aldehyde in Step 1 can be treated with a base, for example, isopropylmagnesium chloride lithium chloride complex in an organic solvent, for example, tetrahydrofuran at about -15 °C and next treated with an aldehyde to generate an alkyne.
  • a desired alkynyl alcohol can be treated with a base in an organic solvent at an elevated temperature to isomerize the desired alkynyl alcohol to a desired alkene.
  • a desired alkynyl alcohol can be treated with a base, such as triethylamine in an organic solvent, for example, 1 ,4-dioxane at an elevated temperature of about 60 °C to generate an alkene.
  • a desired alkene can be treated with ammonia and a mixture of organic solvents to form a tert-butyl 2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7- carboxylate according to methods known in the art.
  • an alkene can be treated with 0.5M ammonia and a mixture of organic solvents, for example, dioxane and acetonitrile to form a tert-butyl 2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate.
  • an amine can be treated with a base, an organic solvent, and a cyclic sulfamidate to form an amine according to methods known in the art.
  • a desired amine can be treated with a base, for example, triethylamine, an organic solvent, for example, N,N- dimethylformamide, and a cyclic sufamidate to form an amine.
  • a protected amine can be treated with an organic acid to form an amine that can subsequently form a cyclic amide according to methods known in the art.
  • a protected amine can be treated with an organic acid, for example, trifluoroacetic acid, and subsequently react with an ester to form a cyclic amide.
  • Step 1 A round-bottomed flask inerted with a nitrogen atmosphere is charged with Compound 2, ethanol, and lithium borohydride at ambient temperature. The reaction is stirred at ambient temperature and monitored by thin layer chromatography (TLC) or high-performance liquid chromatography (HPLC). Once Compound 2 can no longer be detected, the reaction is quenched with an aqueous acid such as aqueous hydrochloric acid, diluted with ethyl acetate and the layers separated. The organic layer is dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo. The product, a primary alcohol, is purified by silica gel column chromatography eluting with a hexane - ethyl acetate gradient and used directly in the next step.
  • TLC thin layer chromatography
  • HPLC high-performance liquid chromatography
  • Step 2 A round-bottomed flask inerted with a nitrogen atmosphere is charged with the primary alcohol prepared in step 1, DMF and phosphorus tribromide. The reaction is stirred at ambient temperature and monitored by thin layer chromatography (TLC) or HPLC. Once the primary alcohol can no longer be detected, the reaction is quenched with brine and diluted with toluene. The layers are separated and the toluene layer is dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo. The bromide is purified by silica gel column chromatography eluting with a hexane - ethyl acetate gradient.
  • TLC thin layer chromatography
  • the reaction is allowed to stir at room temperature until starting material is no longer detected by thin layer chromatography or HPLC.
  • the reaction is neutralized with solid K 2 CO 3 and diluted with dichloromethane.
  • the layers are separated, the organic layer dried over anhydrous magnesium sulfate, filtered and concentrated.
  • Compound 6 is purified by silica gel column chromatography eluting with a dichloromethane - methanol gradient.
  • the tricyclic lactam 8 is combined with an amine (9, 0.9 eq) and an organic solvent such as tetrahydrofuran.
  • a strong base such as lithium hexamethyldisilazane is added and the reaction is stirred until lactam 8 is no longer detected by either thin layer chromatography or HPLC.
  • the reaction is concentrated in vacuo.
  • the product is purified by silica gel column chromatography eluting with a dichloromethane - methanol gradient.
  • a CEM Discovery microwave vessel is charged with the tricyclic lactam 8, N-methyl-2-pyrrolidone (NMP), Hunig's base, and amine 9 (0.9eq).
  • NMP N-methyl-2-pyrrolidone
  • Hunig's base Hunig's base
  • amine 9 0.9eq
  • the reaction is heated at 150 °C for 1-4 hours while being monitored by TLC. Once the tricyclic lactam 8 is no longer detected by TLC or HPLC, the reaction is concentrated in vacuo.
  • the product is purified by silica gel column chromatography eluting with a dichloromethane - methanol gradient.
  • the sulfone intermediate 12 is combined with an amine (13, 0.9 eq) in an organic solvent such as tetrahydrofuran.
  • An organic base such as lithium hexamethyldisilazane is added and the reaction is stirred until sulfone intermediate 12 can no longer be detected by thin layer chromatography or HPLC.
  • the product is purified by silica gel column chromatography eluting with a dichloromethane - methanol gradient.
  • a CEM Discovery microwave vessel is charged with the sulfone intermediate 12, N-methyl-2-pyrrolidone (NMP), Hunig's base, and amine 13 (0.9eq).
  • NMP N-methyl-2-pyrrolidone
  • Hunig's base Hunig's base
  • amine 13 0.9eq.
  • the reaction is heated at 150 °C for 1-4 hours while being monitored by TLC. Once the sulfone intermediate 12 is no longer detected by TLC or HPLC, the reaction is concentrated in vacuo.
  • Step 1 Synthesis of Compound 15 (Scheme 3)
  • Compound 15 is synthesized according to the method of Arigon, J., See, US 2013/0289031, entitled “Pyrimidinone derivatives, preparation thereof and pharmaceutical use thereof.”
  • Compound 20 is commercially available.
  • Compound 25 is synthesized according to the synthetic methodology disclosed in Example 10.
  • Example 12
  • Compound 32 5-morpholinopyrid-2-amine, is synthesized according to Tavares, F.X. and Strum, J.C., See, US 8,598,186, entitled “CDK Inhibitors”.
  • the sulfone intermediate 8 is diluted with a suitable solvent such as tetrahydrofuran and an organic base such as lithium hexamethyldisilazane is added.
  • a suitable solvent such as tetrahydrofuran
  • an organic base such as lithium hexamethyldisilazane is added.
  • the compound 32 is added and the reaction is stirred until sulfone intermediate 8 can no longer be detected by thin layer chromatography or HPLC.
  • the product is purified by silica gel column chromatography eluting with a dichloromethane - methanol gradient.
  • a CEM Discovery microwave vessel is charged with the sulfone intermediate 8, N-methyl-2-pyrrolidone (NMP), Hunig's base, and 5-morpholinopyrid-2-amine (0.9eq).
  • NMP N-methyl-2-pyrrolidone
  • Hunig's base Hunig's base
  • 5-morpholinopyrid-2-amine 0.eq.
  • the reaction is heated at 150 °C for 1-4 hours while being monitored by TLC. Once the sulfone intermediate 8 is no longer detected by TLC or HPLC, the reaction is concentrated in vacuo.
  • the product is purified by silica gel column chromatography eluting with a dichloromethane - methanol gradient.
  • Compound 32 5-morpholinopyrid-2-amine, is synthesized according to Tavares, F.X. and Strum, J.C., See, US 8,598,186, entitled “CDK Inhibitors”.
  • the sulfone intermediate 12 is combined with a suitable solvent such as tetrahydrofuran and an organic base such as lithium hexamethyldisilazane.
  • a suitable solvent such as tetrahydrofuran
  • an organic base such as lithium hexamethyldisilazane.
  • the amine 32 is added and the reaction is stirred until sulfone intermediate 12 can no longer be detected by thin layer chromatography or HPLC.
  • the product is purified by silica gel column chromatography eluting with a dichloromethane - methanol gradient.
  • a CEM Discovery microwave vessel is charged with the sulfone intermediate 12, N-methyl-2-pyrrolidone (NMP), Hunig's base, and 5-morpholinopyrid-2-amine (0.9eq).
  • NMP N-methyl-2-pyrrolidone
  • Hunig's base Hunig's base
  • 5-morpholinopyrid-2-amine 0.eq.
  • the reaction is heated at 150 °C for 1-4 hours while being monitored by TLC. Once the sulfone intermediate 12 is no longer detected by TLC or HPLC, the reaction is concentrated in vacuo.
  • the product is purified by silica gel column chromatography eluting with a dichloromethane - methanol gradient.
  • Step 1 Compound 7 is Boc protected according to the method of A. Sarkar et al. (JOC, 2011, 76, 7132-7140).
  • Step 2 Boc-protected Compound 7 is treated with 5 mol% NiCl 2 (Ph 3 )2, 0.1 eq triphenylphosphine, 3 eq Mn, 0.1 eq tetraethylammonium iodide, in DMI under C0 2 (1 arm) at 25 °C for 20 hours to convert the methyl thiol derivative into the carboxylic acid.
  • Step 3 The carboxylic acid from Step 2 is converted to the corresponding acid chloride using standard conditions.
  • Step 4 The acid chloride from Step 3 is reacted with N-methyl piperazine to generate the corresponding amide.
  • Step 5 The amide from Step 4 is deprotected using trifluoroacetic acid in methylene chloride to generate the target compound.
  • the product is purified by silica gel column chromatography eluting with a dichloromethane - methanol gradient.
  • Phosphoacceptor substrate peptide concentration used was 1.25 ⁇ for all assays (except ⁇ 10 was used for the CKD7/CycH/MATl assay and Staurosporine was used as the reference compound for all assays. Specifics of each assay are as described below: CDK2/CyclinA: Enzyme concentration: 0.2 nM; ATP concentration: 50 ⁇ ; Incubation time: 3 hr.
  • CDK2/CyclinE Enzyme concentration: 0.2 nM; ATP concentration: 100 ⁇ ; Incubation time: 3 hr.
  • CDK4/CyclinDl Enzyme concentration: 1 nM; ATP concentration: 200 ⁇ ; Incubation time: 3 hr.
  • CDK6/CyclinD3 Enzyme concentration: 10 nM; ATP concentration: 300 ⁇ ; Incubation time: 3 hr.
  • CDK5/p25 Enzyme concentration: 0.1 nM; ATP concentration: 20 ⁇ ; Incubation time:
  • CDK5/p35 Enzyme concentration: 0.07 nM; ATP concentration: 20 ⁇ ; Incubation time: 3 hr.
  • CDK7/CycH/MATl Enzyme concentration: 5 nM; ATP concentration: 50 ⁇ ; Incubation time: 3 hr.
  • CDK9/CycT Enzyme concentration: 5 nM; ATP concentration: 10 ⁇ ; Incubation time: 17 hr.
  • HS68 cells human skin fibroblast cell line (Rb-positive)
  • Rb-positive human skin fibroblast cell line
  • the fraction of cells in G0-G1 DNA cell cycle versus the fraction in S-phase DNA cell cycle is determined using Flow Jo 7.2 .2 analysis.
  • a cell based screening method consisting of two CDK4/6-dependent cell lines (tHS68 and WM2664; Rb- positive) and one CDK4/6-independent (A2058; Rb-negative) cell line. Twenty-four hours after plating, each cell line is treated with a tricyclic lactam compound in a dose dependent manner for 24 hours. At the conclusion of the experiment, cells are harvested, fixed, and stained with propidium iodide (a DNA intercalator), which fluoresces strongly red (emission maximum 637 nm) when excited by 488nm light. Samples are run on Dako Cyan flow cytometer and > 10,000 events were collected for each sample. Data are analyzed using Flow Jo 2.2 software developed by TreeStar, Inc. Example 19
  • the CDK4/6-cyclin D complex is essential for progression from Gl to the S-phase of the DNA cell cycle.
  • This complex phosphorylates the retinoblastoma tumor suppressor protein (Rb).
  • Rb retinoblastoma tumor suppressor protein
  • tricyclic lactam compounds are exposed to three cell lines, two CDK4/6 dependent (tHS68, WM2664; Rb- positive) and one CDK4/6 independent (A2058; Rb-negative). Twenty four hours after seeding, cells are treated with a tricyclic lactam compound at 300 nM final concentration for 4, 8, 16, and 24 hours. Samples are lysed and protein is assayed by western blot analysis. Rb phosphorylation is measured at two sites targeted by the CDK4/6-cyclin D complex, Ser780 and Ser807/811 using species specific antibodies.
  • RB tumor suppressor is a major negative cell cycle regulator that is inactivated in approximately 11% of all human cancers. Functional loss of RB is an obligate event in small cell lung cancer (SCLC) development. In RB competent tumors, activated CDK2/4/6 promote Gl to S phase traversal by phosphorylating and inactivating RB (and related family members). Conversely, cancers with RB deletion or inactivation do not require CDK4/6 activity for cell cycle progression.
  • Tricyclic lactam compounds are tested for their ability to block cell proliferation in a panel of SCLC cell lines with known genetic loss of RB.
  • SCLC cells are treated with DMSO or a tricyclic lactam for 24 hours.
  • the effect on proliferation is measured by EdU incorporation.
  • An RB-intact, CDK4/6-dependent cell line (WM2664 or tHS68) and a panel of RB-negative SCLC cell lines (H69, H82, H209, H345, NCI417, or SHP-77) are analyzed for growth inhibition by the various tricyclic lactams.
  • Rb-negative cancer cell lines H69 (human small cell lung cancer - Rb-negative) cells or A2058 (human metastatic melanoma cells - Rb-negative). These cells are seeded in Costar (Tewksbury, Massachusetts) 3093 96 well tissue culture treated white walled, clear bottom plates. Cells are treated with tricyclic lactam compounds at nine point dose response dilution series from 10 uM to 1 nM.
  • Cells are exposed to compounds and then cell viability is determined after either four (H69) or six (A2058) days using the CellTiter-Glo® luminescent cell viability assay (CTG; Promega, Madison, Wisconsin, United States of America) following the manufacturer's recommendations. Plates are read on a BioTek (Winooski, Vermont) Syngergy2 multi-mode plate reader. The Relative Light Units (RLU) are plotted as a result of variable molar concentration and data are analyzed using Graphpad (LaJolla, Californaia) Prism 5 statistical software to determine the EC 50 for each compound.
  • CTG CellTiter-Glo® luminescent cell viability assay
  • hematopoietic stem cell and/or hematopoietic progenitor cell (HSPC) proliferation experiments young adult female FVB/N mice are treated with a single dose of the tricyclic lactams described herein by oral gavage. Mice are then sacrificed at 0, 12, 24, 36, or 48 hours following compound administration, and bone marrow is harvested, as previously described (Johnson et al. J. Clin. Invest. (2010) 120(7), 2528-2536). Four hours before the bone marrow is harvested, mice are treated with 100 ⁇ g of EdU by intraperitoneal injection (Invitrogen). Bone marrow mononuclear cells are harvested and immunophenotyped using previously described methods and percent EdU positive cells are then determined (Johnson et al. J. Clin. Invest. (2010) 120(7), 2528-2536). In brief, HSPCs are identified by expression of lineage markers (Lin-), Seal (S+), and c-Kit (K+).
  • HS68 cells are seeded out at 40,000 cells/ well in 60mm dish on day 1 in DMEM containing 10% fetal bovine serum, 100 U/ml penicillin/ streptomycin and lx Glutamax (Invitrogen) as described (Brookes et al. EMBO J, 21(12)2936-2945 (2002) and Ruas et al. Mol Cell Biol, 27(12)4273-4282 (2007)).
  • cells are treated with a tricyclic lactam compound or DMSO vehicle alone at 300 nM final concentration of test compounds. On day 3, one set of treated cell samples are harvested in triplicate (0 Hour sample).
  • Remaining cells are washed two times in PBS-CMF and returned to culture media lacking test compound. Sets of samples are harvested in triplicate at 24, 40, and 48 hours.
  • Rb-positive Renal Proximal Tubule Epithelial Cells obtained from American Type Culture Collection (ATCC, Manassas, VA). Cells are grown in an incubator at 37°C in a humidified atmosphere of 5% C02 in Renal Epithelial Cell Basal Media (ATCC) supplemented with Renal Epithelial Cell Growth Kit (ATCC) in 37°C humidified incubator.
  • Tricyclic lactam compounds described herein can be dosed to mice at 30 mg/kg by oral gavage or 10 mg/kg by intravenous injection. Blood samples are taken at 0, 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 hours post dosing and the plasma concentrations of the tricyclic lactam compounds are determined by HPLC.
  • the metabolic stability of tricyclic lactam compounds can be determined in human, dog, rat, monkey, and mouse liver microsomes.
  • Human, mouse, and dog liver microsomes are purchased from Xenotech, and Sprague-Dawley rat liver microsomes are prepared by Absorption Systems.
  • the reaction mixture comprising 0.5 mg/mL of liver microsomes, 100 mM of potassium phosphate, pH 7.4, 5 mM of magnesium chloride, and 1 uM of test compound is prepared.
  • the test compound is added into the reaction mixture at a final concentration of 1 uM.
  • An aliquot of the reaction mixture (without cofactor) is incubated in shaking water bath at 37°C for 3 minutes.
  • the control compound, testosterone is run simultaneously with the test compound in a separate reaction.
  • the reaction is initiated by the addition of cofactor (NADPH), and the mixture is then incubated in a shaking water bath at 37°C. Aliquots (100 ⁇ ) are withdrawn at 0, 10, 20, 30, and 60 minutes for the test compound and 0, 10, 30, and 60 minutes for testosterone.
  • Test compound samples are immediately combined with 100 of ice-cold acetonitrile containing internal standard to terminate the reaction.
  • Testosterone samples are immediately combined with 800 of ice cold 50/50 acetonitrile/dH20 containing 0.1% formic acid and internal standard to terminate the reaction. The samples are assayed using a validated LC-MS/MS method.
  • Test compound samples are analyzed using the Orbitrap high resolution mass spectrometer to quantify the disappearance of parent test compound and detect the appearance of metabolites.
  • the peak area response ration (PARR) to internal standard is compared to the PARR at time 0 to determine the percent of test compound or positive control remaining at time-point.
  • mice 8-week-old female C57B1/6 mice are given a single dose of vehicle alone (20% Solutol) or a tricyclic lactam compound (150 mg/kg) by oral gavage. Ten- hours later, all mice are given a single i.p. injection of 100 meg EdU (5-ethynyl-2 ' -deoxyuridine) to label cells in S-phase of the cell cycle. All treated mice are euthanized 2 hours after EdU injection, bone marrow cells are harvested and processed for flow cytometric analysis of EdU- incorporation
  • TBI total body irradiation
  • hematologic manifestations such as granulocytopenia, anemia, thrombocytopenia and lymphopenia.
  • intestinal, cutaneous and neurologic toxicities additionally become significant contributors to morbidity and mortality, but the hematologic syndrome has been the principal complication faced by immediate survivors of a mass casualty radiologic disaster.
  • Tricyclic lactams are tested for their ability to protect cells from DNA damage and apoptosis induced by irradiation.
  • DNA damage is determined using the g-H2A.X assay and apoptosis is determined with a Caspase 3/7 assay.
  • g-H2AX assay tHS68 cells are fixed and stained using the g-H2A.X Phosphorylation Assay Kit (Flow Cytometry; Millipore, Temecula, CA) by the manufacturer's instructions.
  • g-H2AX-positive tHDF cells are then quantified using a CyAn ADP Analyzer (Beckman Coulter, Indianapolis, IN) and Flow Jo analysis software (Version 7.2.2; Tree Star, Ashland, OR).
  • tHDF cells are analyzed directly in the 96-well plates 24 hours after radiation or staurosporine treatment.
  • Caspase 3/7 activation is measured using the Caspase-Glo 3/7 Assay System (Promega, Madison, WI) by following the manufacturer's instructions.
  • 1,000 cells are plated per well in 96-well white wall clear bottom plates. Cells are incubated at 37°C in a humidified atmosphere of 5% C0 2 for 24 hours and then irradiated at 6 Gy, 8 Gy, or 10 Gy. Cells are then incubated at 37°C in a humidified atmosphere of 5% C0 2 with 100, 300, or 1,000 nM compound or dimethyl sulfoxide (Sigma- Aldrich) vehicle control for an additional 16 hours prior to analysis.
  • mice Wild-type mice, young adult (8-12 weeks of age) C57BL/6 (The Jackson Laboratory) or C3H (Harlan Sprague-Dawley) animals are used. Animals are irradiated using a 137Cs AECL GammaCell 40 Irradiator (Atomic Energy of Canada) or a XRAD320 (Precision XRay Inc.) biological irradiator. Experiments are carried out using the 137Cs source, unless otherwise noted. Mice are dosed at 150 mg/kg compound by oral gavage 12 hours post irradiation for single dose studies. Mice are dosed at 150 mg/kg of compound by oral gavage 12 hours post irradiation and 24 hours post irradiation for two dose studies. Kaplan-Meier analysis of survival over the next 30 days for both treated and control groups are determined.
  • the active compounds of the present invention can be prepared for intravenous administration using the following procedure.
  • the excipients hydroxypropyl-beta-cyclodextrin and dextrose can be added to 90% of the batch volume of USP Sterile Water for Injection or Irrigation with stirring; stir until dissolved.
  • the active compound in the hydrochloride salt form is added and stirred until it is dissolved.
  • the pH is adjusted with IN NaOH to pH 4.3 + 0.1 and IN HC1 can be used to back titrate if necessary.
  • USP sterile water for injection or irrigation can be used to bring the solution to the final batch weight.
  • the pH is next re-checked to ensure that the pH is pH 4.3 + 0.1.

Abstract

This invention is in the area of tricyclic lactam compounds and methods for protecting healthy cells, and in particular hematopoietic stem and progenitor cells (HSPC), from the damage associated with ionizing radiation (IR) exposure using selective radioprotectants.

Description

TRICYCLIC LACTAMS FOR USE IN THE PROTECTION OF HEMATOPOIETIC STEM AND PROGENITOR CELLS AGAINST IONIZING RADIATION
RELATED APPLICATIONS
This application claims the benefit of provisional U.S. Application No. 61/980,883, filed
April 17, 2014, provisional U.S. Application No. 61/980,895, filed April 17, 2014, provisional U.S. Application No. 61/980,918, filed April 17, 2014, and provisional U.S. Application No. 61/980,939, filed April 17, 2014, which are hereby incorporated by reference for all purposes. GOVERNMENT INTEREST
The U.S. Government has certain rights in this invention arising from support under Grant No. 5R44AI084284 awarded by the National Institute of Allergy and Infectious Diseases.
FIELD OF THE INVENTION
This invention is in the area of compounds and methods for protecting healthy cells, and in particular hematopoietic stem and progenitor cells (HSPC), from the damage associated with ionizing radiation (IR) exposure using selective radioprotectants.
BACKGROUND
Ionizing radiation (IR) is an important therapeutic modality to treat a range of cancers and other proliferative disorders such as tumors. Radiation therapy uses high energy radiation to shrink tumors and kill the proliferating cells. X-rays, gamma rays, and charged particles are typical kinds of ionizing radiation used for cancer treatments. IR causes extensive DNA damage to exposed cells, including both normal cells and abnormally proliferating cells such as cancer and tumor cells.
Therapeutic radiation is generally applied to a defined area of the subject's body which contains abnormal proliferative tissue, in order to minimize the dose absorbed by the nearby normal tissue. It is difficult, however, to selectively administer therapeutic ionizing radiation to the abnormal tissue. Thus, normal tissue proximate to the abnormal tissue is also exposed to potentially damaging doses of ionizing radiation throughout the course of treatment. There are also some treatments that require exposure of the subject's entire body to the radiation, in a procedure called "total body irradiation" (TBI). Numerous methods have been designed to reduce normal tissue damage while still delivering effective therapeutic doses of ionizing radiation. These techniques include brachytherapy, fractionated and hyper-fractionated dosing, complicated dosing scheduling and delivery systems, and high voltage therapy with a linear accelerator. Such techniques, however, only attempt to strike a balance between the therapeutic and undesirable effects of the radiation and full efficacy has not been achieved.
In addition, exposure to IR may occur through occupational, environmental, or disaster or terroristic events. For example, occupational doses of ionizing radiation can be received by persons whose job involves exposure to radiation, for example in the nuclear power and nuclear weapons industry. Incidents such as the 1979 accident at Three Mile Island or 2011 accident at the Fukushima nuclear power plant, both of which released radioactive material into the reactor containment building and surrounding environment, illustrate the potential for harmful exposure. Intentional infliction of harmful radiation can occur during war and aggression.
Hematologic toxicity (i.e., IR-induced bone marrow suppression), resulting in myelosuppression, can be a limiting side-effect associated with radiation therapy treatments, resulting in a stoppage, delay, or reduction of treatment until the side-effects subside. Furthermore, hematological toxicity is a major source of morbidity following acute exposure to high doses of radiation. In particular, proliferating hematopoietic stem cells and progenitor cells (HSPCs) within the bone marrow are particularly sensitive to IR, and IR damage to these cells reduces their ability to reconstitute the hematological cell lineages. For example, exposure to high levels of IR such as total body irradiation (TBI) is associated with acute and chronic myelosuppressive hematological toxicities, such as anemia, neutropenia, thrombocytopenia, and lymphcytopenia.
The cytotoxicity of IR, however, is largely cell cycle dependent. In healthy cells, cell division occurs in the context of a highly regulated concert of molecular events known as the cell cycle. The cell cycle is divided into four distinct phases: DNA synthesis (S phase), mitosis (M phase), and the gaps of varying length between these periods called Gl and G2. Non-dividing cells remain in a resting or quiescence stage named GO before they re-enter into phase Gl . Early Gl and late S phases are relatively radioresistant. Conversely, the Gl/S transition and G2/M phases are relatively radiosensitive (see Sinclair WK, Morton RA. X-ray sensitivity during cell generation cycle of cultured Chinese hamster cells. Radiat. Res. 1966; 29(3):450-474; Terasuna T, Tolmach LJ. X-ray sensitivity and DNA synthesis in synchronous populations of HeLa cells. Science, 1963; 140:490-92.). Transversing from Gl to S phase while harboring DNA damage is particularly toxic. As a result of DNA damage induced by IR, persistent proliferation in the setting of unrepaired DNA damage can be fatal to replicating cells (Little JB. Repair of sub- lethal and potentially lethal radiation damage in plateau phase cultures of human cells. Nature, 1969; 224(5221):804-806.). It has been shown that an extended period of Gl after exposure to DNA-damaging agents enhances resistance to such agents, possibly by allowing for greater DNA repair prior to Gl/S transversal (Elkind MM, Sutton H. X-ray damage and recovery in mammalian cells in culture. Nature, 1959; 184: 1293-1295; Elkind MM, Sutton H. Radiation response of mammalian cells grown in culture. 1. Repair of x-ray damage in surviving Chinese hamster cells. Radiat Res. 1960; 13: 556-593). Cell cycle arrest allows cells to properly repair these defects, thus preventing their transmission to the resulting daughter cells. If repair is unsuccessful owing to excessive DNA damage, cells may enter senescence or undergo apoptosis.
Hematopoietic stem cells give rise to progenitor cells which in turn give rise to all the differentiated components of blood as shown in Figure 1 (e.g., lymphocytes, erythrocytes, platelets, granulocytes, monocytes). HSPCs require the activity of CDK4/6 for proliferation (see Roberts et al. Multiple Roles of Cyclin-Dependent Kinase 4/6 Inhibitors in Cancer Therapy. JNCI 2012;104(6):476-487). Hematopoietic cells, however, display a gradient dependency on CDK4/6 activity for proliferation during myeloid/erythroid differentiation (see Johnson et al. Mitigation of hematological radiation toxicity in mice through pharmacological quiescence induced by CDK4/6 inhibition. J Clin. Invest. 2010; 120(7): 2528-2536). Accordingly, the least differentiated cells (e.g., hematopoietic stem cells (HSCs), multi-potent progenitors (MPPs), and common myeloid progenitors (CMP)) appear to be the most dependent on CDK4/6 activity for proliferation. More differentiated lineages (e.g., granulocyte -monocyte progenitors (GMPs) and megakaryocyte-erythroid progenitors (MEPs)) are less dependent, and even more differentiated myeloid and erythroid cells proliferate independently of CDK4/6 activity.
A number of CDK 4/6 inhibitors have been identified, including specific pyrido[2,3- d]pyrimidines, 2-anilinopyrimidines, diaryl ureas, benzoyl-2,4-diaminothiazoles, indolo[6,7- a]pyrrolo[3,4-c]carbazoles, and oxindoles (see P.S. Sharma, R. Sharma, R. Tyagi, Curr. Cancer Drug Targets 8 (2008) 53-75). For example, WO 03/062236 identifies a series of 2-(pyridin-2- ylamino-pyrido[2,3]pyrimidin-7-ones for the treatment of Rb positive cancers that show selectivity for CDK4/6, including 6-acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-l-yl-pyridin- 2-ylammino)-8H-pyrido-[2,3-d]-pyrimidin-7-one (PD0332991), which is currently being tested by Pfizer/Onyx in clinical trials as an anti-neoplastic agent against estrogen-positive, HER2- negative breast cancer. The clinical trial studies have reported rates of Grade 3/4 neutropenia and leukopenia with the use of PD0332991, resulting in 71% of patients requiring a dose interruption and 35% requiring a dose reduction; and adverse events leading to 10% of the discontinuations (see Finn, Abstract SI -6, SABCS 2012).
VanderWel et al. describe an iodine-containing pyrido[2,3-d]pyrimidine-7-one (CKIA) as a potent and selective CDK4 inhibitor (see VanderWel et al, J. Med. Chem. 48 (2005) 2371- 2387).
WO 99/15500 filed by Glaxo Group Ltd discloses protein kinase and serine/threonine kinase inhibitors.
WO 2010/020675 filed by Novartis AG describes pyrrolopyrimidine compounds as CDK inhibitors.
WO 2011/101409 also filed by Novartis describes pyrrolopyrimidines with CDK 4/6 inhibitory activity.
WO 2005/052147 filed by Novartis and WO 2006/074985 filed by Janssen Pharma disclose additional CDK4 inhibitors.
US 2007/0179118 filed by Barvian et al. teaches the use of CDK4 inhibitors to treat inflammation.
U.S. Patent Publication 2011/0224227 to Sharpless et al. describes the use of certain CDK4/6 inhibitors, such as PD0332991 and 2BrIC (see Zhu, et al, J. Med. Chem., 46 (1 1) 2027- 2030 (2003); PCT/US2009/059281) to reduce or prevent the effects of cytotoxic compounds on HSPCs in a subject undergoing chemotherapeutic treatments. See also U.S. Patent Publication 2012/0100100.
U.S. Patent Publication 2011/0224221 to Sharpless et al. describes the use of certain CDK4/6 inhibitors, such as PD0332991 and 2BrIC (see Zhu, et al, J. Med. Chem., 46 (1 1) 2027- 2030 (2003); PCT/US2009/059281) to reduce or prevent the deleterious effects of ionizing radiation on HSPCs in a subject exposed to radiation. See also U.S. Patent Publication 2012/0100100. Stone, et al, Cancer Research 56, 3199-3202 (July 1, 1996) describes reversible, pie- mediated cell cycle arrest as protection from chemotherapy.
WO 2012/061156 filed by Tavares and assigned to Gl Therapeutics describes CDK inhibitors (see also, US Pat. Nos. 8,829,012, 8,822,683, 8,598,186, 8,691,830, and 8,598,197, all assigned to Gl Therapeutics), describe CDK Inhibitors having the basic core structure:
Figure imgf000007_0001
WO 2013/148748 filed by Tavares and assigned to Gl Therapeutics describes Lactam Kinase inhibitors having the basic core structures:
Figure imgf000007_0002
U.S. Patent Publication 2014/0275066 and 2014/0275067, assigned to Gl Therapeutics, describes the use of CDK4/6 inhibitors such as 2'-((5-(4-methylpiperazin-l-yl)pyridin-2- yl)amino)-7^8'-dihydro-6Ή-spiro[cyclohexane-l,9'-pyrazino[ ,2': l ,5]pyrrolo[2,3-d]pyrimidin]- 6'-one for the protection of healthy hematopoietic stem and progenitor cells in a subject receiving a DNA-damaging chemotherapeutic agent for the treatment of a Rb-negative tumors.
U.S. Patent Publication 2014/0274896, assigned to Gl Therapeutics, describes the use of CDK4/6 inhibitors such as 2'-((5-(4-methylpiperazin-l-yl)pyridin-2-yl)amino)-7',8'-dihydro-6'H- spiro[cyclohexane-l,9'-pyrazino[ ,2':l ,5]pyrrolo[2,3-d]pyrimidin]-6'-one for the protection of healthy hematopoietic stem and progenitor cells in a subject exposed to ionizing radiation. U.S. Patent Publication 2014/0271466, assigned to Gl Therapeutics, describes the use of CDK4/6 inhibitors such as 2'-((5-(4-methylpiperazin-l-yl)pyridin-2-yl)amino)-7',8'-dihydro-6'Fi- spiro[cyclohexane-l,9'-pyrazino[ ,2':l ,5]pyrrolo[2,3-d]pyrimidin]-6'-one for use as an antineoplastic for the treatment of a Rb-positive proliferative disorders.
U.S. Patent Publication 2014/0271460, assigned to Gl Therapeutics, describes the use of
CDK4/6 inhibitors such as 2'-((5-(4-methylpiperazin-l-yl)pyridin-2-yl)amino)-7',8'-dihydro-6'Fi- spiro[cyclohexane-l,9'-pyrazino[ ,2': l,5]pyrrolo[2,3-d]pyrimidin]-6'-one for use an antineoplastic for the treatment of a T- or B-cell disorder, for example a leukemia.
Stone et al, Cancer Research 56, 3199-3202 (July 1, 1996) describes reversible, pi 6- mediated cell cycle arrest as protection from chemotherapy.
Johnson et al. have shown that pharmacological inhibition of CDK4/6 using the CDK4/6 inhibitors 6-acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-l-yl-pyridin-2-ylammino)-8H-pyrido- [2,3-d]-pyrimidin-7-one (PD0332991) and 2-bromo-12,13-dihydro-5H-indolo[2,3- a]pyrrolo[3,4]carbazole-5,6-dione (2BrIC) exhibited IR protective characteristics in CDK4/6- dependent cell lines. (Johnson et al. Mitigation of hematological radiation toxicity in mice through pharmacological quiescence induced by CDK4/6 inhibition. J Clin. Invest. 2010; 120(7): 2528-2536).
Accordingly, it is an object of the present invention to provide compounds and methods to protect healthy cells, and in particular hematopoietic stem and progenitor cells, during IR exposure.
SUMMARY OF THE INVENTION
Methods and tricyclic lactam compounds are provided to minimize the effects of ionizing radiation (IR) on hematopoietic stem cells and/or hematopoietic progenitor cells (together referred to as HSPCs) in subjects, typically humans, that will be, are being, or have been exposed to IR.
Specifically, the invention includes administering an effective amount of a compound of Formula I, II, III, IV, V, or VI, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof, to protect HSPCs in a subject during or following the subject's exposure to IR. In one non- limiting embodiment, a compound can be selected from the compounds of Table 1 below, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof.
Compounds of the present invention can be used to protect healthy cells during ionizing radiation therapy or radiotherapy for the treatment of any malignant or non-malignant tumor or abnormal cell proliferation, for example, in a solid tumor, including a cancer of the brain, breast, cervix, larynx, lung, pancreas, prostate, skin, spine, stomach, uterus, soft tissue sarcoma, leukemia or lymphoma. The invention can also be used in conjunction with radiotherapy used as a palliative treatment in the absence of a cure for local control of the tumor or symptomatic release, or as a therapeutic treatment to extend the life span of the patient, or total body irradiation performed prior to bone marrow transplant. Compounds of the present invention can also be used to protect healthy cells in connection with radiotherapy for the treatment of non- malignant conditions, such as trigeminal neuralgia, thyroid eye disease, pterygium, or prevention of keloid scar growth or heterotopic ossification.
The present invention can also be used to protect healthy cells during ionizing radiation therapy or radiotherapy for the treatment of proliferative disorders, including but not limited to rheumatoid arthritis, lupus, scleroderma, ankylosing spondylitis, asthma, bronchitis and psoriasis. Radiation therapy is also used to treat early stage Dupuytren's disease and Ledderhose disease.
The present invention can further be used to protect people at imminent risk of environmental, occupational or aggression-based radiation exposure or who have recently been exposed to harmful radiation.
A compound described herein, in a non-limiting embodiment, may provide protection of CDK-replication dependent HSPCs during or after IR exposure due in part because it (1) exhibits a transient Gl -arresting effect and (ii) displays a rapid, synchronous reentry into the cell cycle by the HSPCs following the cessation of IR exposure or mitigation of IR induced DNA damage. The use of CDK4/6-specific transient Gl -arresting compounds as radioprotectants and radiomitigants allows for an accelerated hematological recovery, reduced hematological cytotoxicity risk due to HSPC replication delay, and/or a minimization of IR induced cell death.
Tricyclic lactams useful in the present invention can be administered to the subject prior to exposure to IR, during exposure to IR, after exposure to IR, or a combination thereof. The compounds described herein are typically administered in a manner that allows the drug facile access to the blood stream, for example via intravenous injection or sublingual, intraaortal, or other efficient blood-stream accessing route; however, oral or other desired administrative routes can be used. In one embodiment, the compound is administered to the subject less than about 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, or 4 hours, 3 hours, 2 hours, 1 hour, ½ hour or less prior to exposure to IR. In one embodiment, the compound is administered up to 4 hours prior to exposure to IR. Typically, the tricyclic lactam is administered to the subject prior to exposure to IR such that the compound reaches peak serum levels before or during exposure to IR. In one embodiment, the tricyclic lactam is administered concomitantly, or closely thereto, with IR exposure. In one embodiment, the tricyclic lactam can be administered following exposure to IR in order to mitigate HSPC DNA damage associated with IR exposure. If desired, the tricyclic lactam can be administered multiple times during the IR exposure to maximize inhibition, especially when the IR exposure occurs over a long period. In one embodiment, the tricyclic lactam is administered up to about ½ hour, up to about 1 hour, up to about 2 hours, up to about 4 hours, up to about 8 hours, up to about 10 hours, up to about 12 hours, up to about 14 hours, up to about 16 hours, up to about 20 hours, or up to about 24 hours or greater following IR exposure. In a particular embodiment, the tricyclic lactam is administered up to between about 12 hours and 20 hours following exposure to IR. In one embodiment, the tricyclic lactam is administered one or more times following exposure to IR.
In one embodiment, the tricyclic lactam compounds inhibit Cyclin Dependent Kinase 4 (CDK4) and/or Cyclin Dependent Kinase 6 (CDK6). In one embodiment, the tricyclic lactams useful in the present invention may show a marked selectivity for the inhibition of CDK4 and/or CDK6 in comparison to other CDKs, for example CDK2. Tricyclic lactams useful in the present invention may provide for a dose-dependent Gl-arresting effect on a subject's HSPCs sufficient to afford radioprotection to targeted HSPCs during IR exposure, while allowing for the reentry into the cell-cycle by the HSPCs after IR exposure and/or tricyclic lactam administration due to a time-limited CDK4/6 inhibitory effect. Likewise, tricyclic lactams useful in the present invention may provide a dose-dependent mitigating effect on HSPCs that have been exposed to IR, allowing for repair of DNA damage associated with IR exposure.
In addition, in particular embodiments, cell-cycle reentry following Gl arrest using a tricyclic lactam described herein may provide for the ability to time the administration of hematopoietic growth factors to assist in the reconstitution of hematopoietic cell lines to maximize the growth factor effect without forcing hematological cells into replication before DNA damage is repaired. As such, in one embodiment, the use of the compounds described herein is combined with the use of hematopoietic growth factors including, but not limited to, granulocyte colony stimulating factor (G-CSF), granulocyte -macrophage colony stimulating factor (GM-CSF), thrombopoietin, interleukin (IL)-12, steel factor, and erythropoietin (EPO), and their derivatives. In one embodiment, the tricyclic lactam is administered prior to administration of the hematopoietic growth factor. In one embodiment, the hematopoietic growth factor administration is timed so that the tricyclic lactam's effect on HSPCs has dissipated.
In one aspect, the use of a tricyclic lactam described herein allows for a HSPC radioprotective regimen for use during standard radio-therapeutic dosing schedules or regimens common in many anti-cancer treatments. In some embodiments, the subject is undergoing therapeutic IR for the treatment of a proliferative disorder or disease such as cancer. In one embodiment, the cancer is a CDK4/6-replication independent cancer. In some embodiments, the cancer is characterized by one or more of the group consisting of increased activity of eye Independent 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 MYC expression, increased cyclin E, and increased cyclin A. In one embodiment, the subject is undergoing therapeutic IR for the treatment of an Rb-null or Rb-deficient cancer, including but not limited to, small cell lung cancer, triple-negative breast cancer, HPV-positive head and neck cancer, retinoblastoma, Rb-negative bladder cancer, Rb negative prostate cancer, osteosarcoma or cervical cancer. In some cases, administration of the tricyclic lactam compound allows for a higher dose of ionizing radiation to be used to treat the disease than the standard dose that would be safely used in the absence of administration of the tricyclic lactam compound.
In some embodiments, the subject is at risk of being exposed to IR due to an environmental, occupational or aggression-based situation, such as radiological agent exposure during warfare, a radiological terrorist attack, an industrial accident, other occupational exposure, or space travel.
In some embodiments, the subject has already been exposed to IR, for example, including but not limited to, through an environmental or occupational situation, such as radiological agent exposure during warfare, a radiological terrorist attack, an industrial accident, other occupational exposure, or space travel, and the tricyclic lactams described herein are administered for the purpose of mitigating DNA damage in HSPCs.
In some embodiments, the protected HSPCs include hematopoietic stem cells, including long term hematopoietic stem cells (LT-HSCs) and short term hematopoietic stem cells (ST- HSCs), and hematopoietic progenitor cells, including multipotent progenitors (MPPs), common myeloid progenitors (CMPs), common lymphoid progenitors (CLPs), granulocyte-monocyte progenitors (GMPs) and megakaryocyte-erythroid progenitors (MEPs). In some embodiments, administration of the tricyclic lactam compound provides temporary, transient pharmacologic quiescence of hematopoietic stem and/or hematopoietic progenitor cells in the subject.
In one aspect, the methods described herein using a tricyclic lactam may result in reduced long-term hematologic toxicity, that is, the use of a tricyclic lactam described herein prior to, during, or after IR exposure reduces the occurrence or development of long-term hematological toxicities associated with IR exposure. In some embodiments, the reduction in long-term hematological toxicity is associated with the ability of HSPCs that are Gl -arrested during IR exposure to the cell-cycle after cessation of IR exposure and replicate, including replicating between successive or repeated IR exposures.
Alternatively, administration of a tricyclic lactam as described herein may result in reduced anemia, reduced lymphopenia, reduced thrombocytopenia, or reduced neutropenia compared to that typically expected after, common after, or associated with exposure to ionizing radiation in the absence of administration of the tricyclic lactam.
In aspects of the invention, the tricyclic lactam is the compound of Formula I, II, III, IV, V, or VI. Alternatively, the tricyclic lactam used in the aspects of the invention described herein is selected from the compounds of Table 1. In some embodiments, the subject or host is a mammal, including a human.
The present invention includes at least the following features:
A. Tricyclic lactam compounds, methods, and compositions for reducing the effect of IR on CDK4/6 replication dependent healthy cells in a subject, preferably a human, exposed to IR;
B. Tricyclic lactam compounds, methods, and compositions for reducing the effect of IR on CDK4/6 replication dependent healthy cells, for example HSPCs, in a subject, preferably a human, undergoing treatment for a CDK4/6-replication independent cancer, for example a Rb-null or Rb-deficient cancer, comprising administering an effective amount of a tricyclic lactam prior to treatment with IR;
C. Tricyclic lactam compounds, methods, and compositions are provided for reducing the effect of IR exposure on CDK4/6 replication dependent HSPCs in a subject who will be exposed, is being exposed, or has been exposed to IR, the method comprising administering an effective amount of a tricyclic lactam selected from the group consisting of a compound or composition comprising Formula I, Formula II, Formula III, Formula IV, Formula V, and Formula VI, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof. In one embodiment, the compound is selected from the compounds listed in Table 1 or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof.
D. Compounds of Formula I, II, III, IV, V, and VI as described herein, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof, for use in the radioprotection of HSPCs during an IR exposure. In one embodiment, the compound is selected from the compounds listed in Table 1, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof;
E. Compounds of Formula I, II, III, IV, V, and VI as described herein, and pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof, for use in the radioprotection of HSPCs during an IR therapeutic regimen for the treatment of a proliferative disorder. In one embodiment, the compound is selected from the compounds listed in Table 1, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof;
F. Compounds of Formula I, II, III, IV, V, and VI as described herein, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof, for use in the radioprotection of HSPCs during an IR therapeutic regimen for the treatment of cancer. In one embodiment, the compound is selected from the compounds listed in Table 1, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof;
G. Compounds of Formula I, II, III, IV, V, and VI as described herein, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof, for use in the radioprotection of HSPCs during an IR therapeutic regimen for the treatment of a CDK4/6- replication independent cancer. In one embodiment, the compound is selected from the compounds listed in Table 1, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof;
H. Compounds of Formula I, II, III, IV, V, and VI as described herein, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof, for use in the radioprotection of HSPCs during an IR therapeutic regimen for the treatment of an Rb-null or Rb-deficient cancer. In one embodiment, the compound is selected from the compounds listed in Table 1, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof;
I. Compounds of Formula I, II, III, IV, V, and VI as described herein, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof, for use in the radioprotection of HSPCs during IR exposure associated with an environmental or occupational condition. In one embodiment, the compound is selected from the compounds listed in Table 1 , or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof;
J. Compounds of Formula I, II, III, IV, V, and VI as described herein, and pharmaceutically acceptable compositions, salts, isotopic analogs, and prodrugs thereof, for use in the forced cycling of HSPCs between Gl -arrest and replication in coordination with a standard IR therapeutic regimen for a proliferative disorder. In one embodiment, the compound is selected from the compounds listed in Table 1, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof;
K. Compounds of Formula I, II, III, IV, V, and VI as described herein, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof, for use in the forced cycling of HSPCs between Gl -arrest and replication in coordination with repeated IR exposures. In one embodiment, the compound is selected from the compounds listed in Table 1, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof;
L. Compounds of Formula I, II, III, IV, V, and VI as described herein, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof, for use in the mitigation of DNA damage to HSPCs following IR exposure. In one embodiment, the compound is selected from the compounds listed in Table 1, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof; M. Compounds of Formula I, II, III, IV, V, and VI as described herein, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof, for use in combination with hematopoietic growth factors in a subject that will be, is being, or has been exposed to IR. In one embodiment, the compound is selected from the compounds listed in Table 1, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof;
N. Use of Compounds of Formula I, II, III, IV, V, and VI as described herein, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof, in the manufacture of a medicament for use in the radioprotection of HSPCs. In one embodiment, the compound is selected from the compounds listed in Table 1, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof;
O. Use of Compounds of Formula I, II, III, IV, V, and VI as described herein, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof, in the manufacture of a medicament for use in the mitigation of DNA damage of HSPCs that have been exposed to IR. In one embodiment, the compound is selected from the compounds listed in Table 1, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof;
P. A pharmaceutical formulation comprising an effective subject-treating amount of compounds of Formula I, II, III, IV, V, and VI as described herein for the protection against ionizing radiation, or pharmaceutically acceptable compositions, salts, isotopic analog, or prodrugs thereof; In one embodiment, the compound is selected from the compounds listed in Table 1, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof
Q. A method for manufacturing a medicament of Formula I, II, III, IV, V, and VI intended for therapeutic use in the radioprotection of HSPCs. In one embodiment, the compound is selected from the compounds listed in Table 1 , or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof; and,
R. A method for manufacturing a medicament of Formula I, II, III, IV, V, and VI intended for therapeutic use in the mitigation of DNA damage of HSPCs that have been exposed to IR. In one embodiment, the medicament is selected from the compounds listed in Table 1, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof. S. Compounds of Formula I, II, III, IV, V, and VI as described herein, or pharmaceutically acceptable compositions, salts, isotopic analog, or prodrugs thereof; In one embodiment, the compound is selected from the compounds listed in Table 1, or pharmaceutically acceptable compositions, salts, isotopic analogs, or prodrugs thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic drawing of hematopoiesis showing the hierarchical proliferation of healthy hematopoietic stem cells (HSC) and healthy hematopoietic progenitor cells with increasing differentiation upon proliferation.
FIGS. 2-4 illustrate several exemplary embodiments of R2 of the compounds of the invention.
FIGS. 5A-5C, 6A-6D, 7A-7C, 8A-8B and 9A-9F illustrate exemplary embodiments of the core structure of the compounds of the invention. DETAILED DESCRIPTION OF THE INVENTION
Tricyclic lactam compounds, methods, and compositions are provided that minimize the effect of ionizing radiation (IR) toxicity on CDK4/6 replication dependent hematopoietic stem cells and/or hematopoietic progenitor cells (together referred to as HSPCs) in subjects, typically humans, that will be, are being, or have been exposed to IR.
Definitions
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this presently described subject matter belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety to the extent authorized by law.
Unless otherwise stated, the following terms used in this application, including the specification and claims, have the definitions given below. As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Definition of standard chemistry terms may be found in reference works, including Carey and Sundberg (2007) Advanced Organic Chemistry 5th Ed. Vols. A and B, Springer Science+Business Media LLC, New York. The practice of the present invention will employ, unless otherwise indicated, conventional methods of synthetic organic chemistry, mass spectroscopy, preparative and analytical methods of chromatography, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology. Conventional methods of organic chemistry include those included in March 's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 6th Edition, M.B. Smith and J. March, John Wiley & Sons, Inc., Hoboken, NJ, 2007.
The term "alkyl," either alone or within other terms such as "haloalkyl" and
"alkylamino," embraces linear or branched radicals having one to about twelve carbon atoms. "Lower alkyl" radicals have one to about six carbon atoms. Examples of such radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-bvXy\, pentyl, isoamyl, hexyl and the like. The term "alkylene" embraces bridging divalent linear and branched alkyl radicals. Examples include methylene, ethylene, propylene, isopropylene and the like.
The term "alkenyl" embraces linear or branched radicals having at least one carbon- carbon double bond of two to about twelve carbon atoms. "Lower alkenyl" radicals having two to about six carbon atoms. Examples of alkenyl radicals include ethenyl, propenyl, allyl, propenyl, butenyl and 4-methylbutenyl. The terms "alkenyl" and "lower alkenyl," embrace radicals having "cis" and "trans" orientations, or alternatively, "E" and "Z" orientations.
The term "alkynyl" denotes linear or branched radicals having at least one carbon-carbon triple bond and having two to about twelve carbon atoms. "Lower alkynyl" radicals having two to about six carbon atoms. Examples of such radicals include propargyl, butynyl, and the like.
Alkyl, alkenyl, and alkynyl radicals may be optionally substituted with one or more functional groups such as halo, hydroxy, nitro, amino, cyano, haloalkyl, aryl, heteroaryl, heterocyclo and the like.
The term "alkylamino" embraces "N-alkylamino" and "Ν,Ν-dialkylamino" where amino groups are independently substituted with one alkyl radical and with two alkyl radicals, respectively. "Lower alkylamino" radicals have one or two alkyl radicals of one to six carbon atoms attached to a nitrogen atom. Suitable alkylamino radicals may be mono or dialkylamino such as N-methylamino, N-ethylamino, N.N-dimethylamino, Ν,Ν-diethylamino and the like. The term "halo" means halogens such as fluorine, chlorine, bromine or iodine atoms. The term "haloalkyl" embraces radicals wherein any one or more of the alkyl carbon atoms is substituted with one or more halo as defined above. Examples include monohaloalkyl, dihaloalkyl and polyhaloalkyl radicals including perhaloalkyl. A monohaloalkyl radical, for one example, may have an iodo, bromo, chloro or fluoro atom within the radical. Dihalo and polyhaloalkyl radicals may have two or more of the same halo atoms or a combination of different halo radicals. "Lower haloalkyl" embraces radicals having 1-6 carbon atoms. Examples of haloalkyl radicals include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichloro fluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl and dichloropropyl. "Perfluoroalkyl" means an alkyl radical having all hydrogen atoms replaced with fluoro atoms. Examples include trifluoromethyl and pentafluoroethyl.
The term "aryl", alone or in combination, means a carbocyclic aromatic system containing one or two rings wherein such rings may be attached together in a fused manner. The term "aryl" embraces aromatic radicals such as phenyl, naphthyl, indenyl, tetrahydronaphthyl, and indanyl. More preferred aryl is phenyl. Said "aryl" group may have 1 or more substituents such as lower alkyl, hydroxyl, halo, haloalkyl, nitro, cyano, alkoxy, lower alkylamino, and the like. An aryl group may be optionally substituted with one or more functional groups such as halo, hydroxy, nitro, amino, cyano, haloalkyl, aryl, heteroaryl, heterocyclo and the like.
The term "heterocyclyl" (or "heterocyclo") embraces saturated, and partially saturated heteroatom-containing ring radicals, where the heteroatoms may be selected from nitrogen, sulfur and oxygen. Heterocyclic rings comprise monocyclic 6-8 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. Said "heterocyclyl" group may have 1 to 3 substituents such as hydroxyl, Boc, halo, haloalkyl, cyano, lower alkyl, lower aralkyl, oxo, lower alkoxy, amino, lower alkylamino, and the like.
Examples of saturated heterocyclo 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]. Examples of partially saturated heterocyclyl radicals include dihydrothienyl, dihydropyranyl, dihydrofuryl, dihydrothiazolyl, and the like.
Particular examples of partially saturated and saturated heterocyclo groups include 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-l,2,4-triazolo[3,4-a]isoquinolyl, 3,4-dihydro-2H- benzo[l,4]oxazinyl, benzo[l,4]dioxanyl, 2,3- dihydro-lH- '-benzo[d]isothiazol-6-yl, dihydropyranyl, dihydrofuryl and dihydrothiazolyl, and the like.
Heterocyclo groups also includes radicals where heterocyclic radicals are fused/condensed with aryl radicals: unsaturated condensed heterocyclic group containing 1 to 5 nitrogen atoms, for example, indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl, tetrazolopyridazinyl [e.g., tetrazolo [l,5-b]pyridazinyl]; unsaturated condensed heterocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms [e.g. benzoxazolyl, benzoxadiazolyl]; unsaturated condensed heterocyclic group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms [e.g., benzothiazolyl, benzothiadiazolyl]; and saturated, partially unsaturated and unsaturated condensed heterocyclic group containing 1 to 2 oxygen or sulfur atoms [e.g. benzofuryl, benzothienyl, 2,3-dihydro- benzo[l,4]dioxinyl and dihydrobenzofuryl].
The term "heteroaryl" denotes aryl ring systems that contain one or more heteroatoms selected from the group O, N and S, wherein the ring nitrogen and sulfur atom(s) are optionally oxidized, and nitrogen atom(s) are optionally quarternized. Examples include unsaturated 5 to 6 membered heteromonocyclyl group containing 1 to 4 nitrogen atoms, for example, 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-l,2,3-triazolyl]; unsaturated 5- to 6-membered heteromonocyclic group containing an oxygen atom, for example, pyranyl, 2-furyl, 3-furyl, etc.; unsaturated 5 to 6-membered heteromonocyclic group containing a sulfur atom, for example, 2- thienyl, 3-thienyl, etc.; unsaturated 5- to 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, for example, oxazolyl, isoxazolyl, oxadiazolyl [e.g., 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5- oxadiazolyl]; unsaturated 5 to 6-membered heteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms, for example, thiazolyl, thiadiazolyl [e.g., 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl].
The term "heteroarylalkyl" denotes alkyl radicals substituted with a heteroaryl group. Examples include pyridylmethyl and thienylethyl.
The term "sulfonyl", whether used alone or linked to other terms such as alkylsulfonyl, denotes respectively divalent radicals -S02-.
The terms "carboxy" or "carboxyl", whether used alone or with other terms, such as "carboxyalkyl", denotes -C(0)-OH.
The term "carbonyl", whether used alone or with other terms, such as "aminocarbonyl", denotes -C(O)-.
The term "aminocarbonyl" denotes an amide group of the Formula -C(0)-NH2.
The terms "heterocycloalkyl" embrace heterocyclic-substituted alkyl radicals. Examples include piperidylmethyl and morpholinylethyl.
The term "arylalkyl" embraces aryl-substituted alkyl radicals. Examples include benzyl, diphenylmethyl and phenylethyl. The aryl in said aralkyl may be additionally substituted with halo, alkyl, alkoxy, halkoalkyl and haloalkoxy.
The term "cycloalkyl" includes saturated carbocyclic groups of 3 to 10 carbons. Lower cycloalkyl groups include C3-C6 rings. Examples include cyclopentyl, cyclopropyl, and cyclohexyl. Cycloalkyl groups may be optionally substituted with one or more functional groups such as halo, hydroxy, nitro, amino, cyano, haloalkyl, aryl, heteroaryl, heterocyclo and the like.
The term "cycloalkylalkyl" embraces cycloalkyl-substituted alkyl radicals. "Lower cycloalkylalkyl" radicals are cycloalkyl radicals attached to alkyl radicals having one to six carbon atoms. Examples of include cyclohexylmethyl. The cycloalkyl in said radicals may be additionally substituted with halo, alkyl, alkoxy and hydroxy.
The term "cycloalkenyl" includes carbocyclic groups having one or more carbon-carbon double bonds including "cycloalkyldienyl" compounds. Examples include cyclopentenyl, cyclopentadienyl, cyclohexenyl and cycloheptadienyl.
The term "comprising" is meant to be open ended, including the indicated component but not excluding other elements.
The term "oxo" as used herein contemplates an oxygen atom attached with a double bond. The term "nitro" as used herein contemplates -N02.
The term "cyano" as used herein contemplates -CN.
As used herein, the term "prodrug" means a compound which when administered to a host in vivo is converted into the parent drug. As used herein, the term "parent drug" means any of the presently described chemical compounds that are useful to treat any of the disorders described herein, or to control or improve the underlying cause or symptoms associated with any physiological or pathological disorder described herein in a host, typically a human. Prodrugs can be used to achieve any desired effect, including to enhance properties of the parent drug or to improve the pharmaceutic or pharmacokinetic properties of the parent. Prodrug strategies exist which provide choices in modulating the conditions for in vivo generation of the parent drug, all of which are deemed included herein. Nonlimiting examples of prodrug strategies include covalent attachment of removable groups, or removable portions of groups, for example, but not limited to acylation, phosphorylation, phosphonylation, phosphoramidate derivatives, amidation, reduction, oxidation, esterification, alkylation, other carboxy derivatives, sulfoxy or sulfone derivatives, carbonylation or anhydride, among others.
Throughout the specification and claims, a given chemical formula or name shall encompass all optical and stereoisomers, as well as racemic mixtures where such isomers and mixtures exist, unless otherwise noted.
The term "selective CDK4/6 inhibitor" and derivatives thereof means a compound that inhibits only CDK4 activity, only CDK6 activity, or both CDK4 and CDK6 activity at an IC50 molar concentration at least about 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 term "and/or" when used in describing two items or conditions, e.g., CDK4 and/or CDK6, refers to situations where both items or conditions are present or applicable and to situations wherein only one of the items or conditions is present or applicable. Thus, a CDK4 and/or CDK6 inhibitor can be a compound that inhibits both CDK4 and CDK6, a compound that inhibits only CDK4, or a compound that only inhibits CDK6.
As described herein, hematopoietic stem and progenitor cells include, but are not limited to, long term hematopoietic stem cells (LT-HSCs), short term hematopoietic stem cells (ST- HSCs), multipotent progenitors (MPPs), common myeloid progenitors (CMPs), common lymphoid progenitors (CLPs), granulocyte-monocyte progenitors (GMPs), and megakaryocyte- erythroid progenitors (MEPs).
As used herein the term "ionizing radiation" refers to radiation of sufficient energy that, when absorbed by cells and tissues, can induce formation of reactive oxygen species and DNA damage. Ionizing radiation can include X-rays, gamma rays, and particle bombardment (e.g., neutron beam, electron beam, protons, mesons, and others). IR is used for purposes including, but not limited to, medical testing and treatment, scientific purposes, industrial testing, manufacturing and sterilization, and weapons and weapons development, nuclear energy and can also be found as an environmental or occupational toxin or used as an assault. Radiation is generally measured in units of absorbed dose, such as the rad or gray (Gy), or in units of dose equivalence, such as rem or sievert (Sv).
By "substantial portion" or "significant portion" is meant at least about 80%. In alternative embodiments, the portion may be 85%, 90% or 95% or greater.
By "induces Gl -arrest" is meant that the tricyclic lactam compound induces a quiescent state in a substantial portion of a cell population at the Gl phase of the cell cycle.
By "long-term hematological toxicity" is meant hematological toxicity affecting a subject for a period lasting more than one or more weeks, months or years following exposure of IR. Long-term hematological toxicity can result in bone marrow disorders that can cause the ineffective production of blood cells (i.e., myelodysplasia) and/or lymphocytes (i.e., lymphopenia, the reduction in the number of circulating lymphocytes, such as B- and T-cells). Hematological toxicity can be observed, for example, as anemia, reduction in platelet count (i.e., thrombocytopenia) or reduction in white blood cell count (i.e., neutropenia). In some cases, myelodysplasia can result in the development of leukemia. Long-term toxicity related to ionizing radiation can also damage other self-renewing cells in a subject, in addition to hematological cells. Thus, long-term toxicity can also lead to graying and frailty.
A tricyclic lactam compound that is "substantially free" of off-target effects can have some minor off-target effects that do not interfere with the tricyclic lactam's ability to provide protection from cytotoxic compounds in CDK4/6-dependent cells. For example, a tricyclic lactam that is "substantially free" of CDK4/6 inhibitory off-target effects can have some minor inhibitory effects on other CDKs (e.g., IC50s for CDK1 or CDK2 that are >0.5 μΜ; >1.0 μΜ, or >5.0 μΜ), so long as the tricyclic lactam provides selective Gl arrest in CDK4/6-dependent cells.
By "synchronous reentry into the cell cycle" is meant that HSPC cells in Gl -arrest due to the effects of a tricyclic lactam compound reenter the cell-cycle within relatively the same collective timeframe or at relatively the same rate upon dissipation of the compound's effect. Comparatively, by "asynchronous reentry into the cell cycle" is meant that the HSPC cells in Gl arrest reenter the cell-cycle within relatively different collective timeframes or at relatively different rates upon dissipation of the compound's effect, such as induced by PD0332991.
Throughout the specification and claims, a given chemical formula or name shall encompass all optical and stereoisomers, as well as racemic mixtures where such isomers and mixtures exist, unless otherwise noted.
The subject treated or exposed to IR is typically a human subject, although it is to be understood the methods described herein are effective with respect to other mammals or vertebrate species. The term subject can include animals such as mice, monkeys, dogs, pigs, rabbits, domesticated swine (pigs and hogs), ruminants, equine, poultry, felines, murines, bovines, canines, and the like.
Active Compounds
In one embodiment, the invention is directed to compounds or the use of such compounds of Formula I, II, III, IV, or V:
Figure imgf000023_0001
Figure imgf000024_0001
or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof;
wherein:
Z is -(CH2)x- wherein x is 1, 2, 3 or 4 or -0-(CH2)z- wherein z is 2, 3 or 4;
each X is independently CH or N;
each X' is independently CH or N;
X" is independently CH2, S or NH, arranged such that the moiety is a stable 5-membered ring; R, R8, and R11 are independently H, C1-C3 alkyl or haloalkyl, cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O or S; -(alkylene)m- C3-C8 cycloalkyl, -(alkylene)m- aryl, -(alkylene)m-heterocyclo, -(alkylene)m- heteroaryl, -(alkylene)m-NR3R4, -(alkylene)m-C(0)- NR3R4; -(alkylene)m-0-R5, -(alkylene)m- S(0)n-R5, or -(alkylene)m-S(0)„-NR3R4 any of which may be optionally independently substituted with one or more Rx groups as allowed by valence, and wherein two Rx groups bound to the same or adjacent atoms may optionally combine to form a ring;
each R1 is independently aryl, alkyl, cycloalkyl or haloalkyl, wherein each of said alkyl, cycloalkyl and haloalkyl groups optionally includes O or N heteroatoms in place of a carbon in the chain and two Rx'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;
y is 0, 1, 2, 3 or 4;
R2 is -(alkylene)m-heterocyclo, -(alkylene)m-heteroaryl, -(alkylene)m-NR3R4, -(alkylene)m-C(0)-NR3R4; -(alkylene)m-C(0)-0-alkyl; -(alkylene)m-0-R5,
-(alkylene)m-S(0)n-R5, or -(alkylene)m-S(0)n-NR3R4 any of which may be optionally independently substituted with one or more Rx groups as allowed by valence, and wherein two Rx groups bound to the same or adjacent atom may optionally combine to form a ring and wherein m is 0, 1 or 2 and n is 0, 1 or 2;
R3 and R4 at each occurrence are independently:
(i) hydrogen or
(ii) alkyl, cycloalkyl, heterocyclo, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl any of which may be optionally independently substituted with one or more Rx groups as allowed by valence, and wherein two Rx groups bound to the same or adjacent atom may optionally combine to form a ring; or R3 and R4 together with the nitrogen atom to which they are attached may combine to form a heterocyclo ring optionally independently substituted with one or more Rx groups as allowed by valence, and wherein two Rx groups bound to the same or adjacent atom may optionally combine to form a ring;
R5 and R5* at each occurrence is:
(i) hydrogen or
(ii) alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl any of which may be optionally independently substituted with one or more Rx groups as allowed by valence; Rx at each occurrence is independently, halo, cyano, nitro, oxo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, heterocycloalkyl, -(alkylene)m-OR5, -(alkylene)m-0-alkylene-OR5, -(alkylene)m-S(0)n-R5, -(alkylene)m-NR3R4, -(alkylene)m-CN, -(alkylene)m-C(0)-R5, -(alkylene)m-C(S)-R5, -(alkylene)m-C(0)-OR5, -(alkylene)m-0-C(0)-R5,
-(alkylene)m-C(S)-OR5, -(alkylene)m-C(0)-(alkylene)m-NR3R4, -(alkylene)m-C(S)-NR3R4, -(alkylene)m-N(R3)-C(0)-NR3R4, -(alkylene)m-N(R )-C(S)-NR3R4, -(alkylene)m-N(R3)-C(0)-R5, -(alkylene)m-N(R3)-C(S)-R5, -(alkylene)m-0-C(0)-NR3R4, -(alkylene)m-0-C(S)-NR3R4, -(alkylene)m-S02-NR R4, -(alkylene)m-N(R3)-S02-R5, -(alkylene)m-N(R3)-S02-NR3R4, -(alkylene)m-N(R3)-C(0)-OR5 ) -(alkylene)m-N(R3)-C(S)-OR5, or -(alkylene)m-N(R3)-S02-R5; wherein:
said alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, and heterocycloalkyl groups may be further independently substituted with one or more -(alkylene)m-CN, -(alkylene)m-OR5*, -(alkylene)m-S(0)n-R5*
-(alkylene)m-NR3 *R4 * , -(alkylene)m-C(0)-R5 * , -(alkylene)m-C(=S)R5 *
-(alkylene)m-C(=0)0 R5*, -(alkylene)m-OC(=0)R5*, -(alkylene)m-C(S)-OR5* -(alkylene)m-C(0)-NR3*R4*, -(alkylene)m-C(S)-NR3*R4* -(alkylene)m-N(R3^-C(0)-NR3*R4*, -(alkylene)m-N(R3*)-C(S)-NR3*R4* -(alkylene)m-N(R3*)-C(0)-R5*, -(alkylene)m-N(R3*)-C(S)-R5*
-(alkylene)m-0-C(0)-NR3:f!R4*, -(alkylene)m-0-C(S)-NR3:,!R :,! -(alkylene)m-S02-NR3*R4*, -(alkylene)m-N(R3!i:)-S02-R5:l! -(alkylene)m-N(R3 *)-S02-NR3 ^R4* , -(alkylene)m-N(R3 *)-C(0)-OR5!i:
-(alkylene)m-N(R3*)-C(S)-OR5*, or -(alkylene)m-N(R3!i:)-S02-R5*,
n is 0, 1 or 2, and
m is 0, 1 or 2;
R3* and R4* at each occurrence are independently:
(i) hydrogen or
(ii) alkyl, alkenyl, alkynyl cycloalkyl, heterocyclo, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl any of which may be optionally independently substituted with one or more Rx groups as allowed by valence; or R~"* and R4* together with the nitrogen atom to which they are attached may combine to form a heterocyclo ring optionally independently substituted with one or more Rx groups as allowed by valence; and
R6 is H or lower alkyl, -(alkylene)m-heterocyclo, -(alkylene)m-heteroaryl, -(alkylene)m-NR3R4, -(alkylene)m-C(0)-NR3R4; -(alkylene)m-0-R5, -(alkylene)m-S(0)n-R5, or -(alkylene)m-S(0)n-
NR3R4 any of which may be optionally independently substituted with one or more Rx groups as allowed by valence, and wherein two Rx groups bound to the same or adjacent atoms may optionally combine to form a ring; and
R10 is (i) NHRA, wherein RA is unsubstituted or substituted Ci-Cg alkyl, cycloalkylalkyl, or -TT- RR, Ci-Cg cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O, and S; TT is an unsubstituted or substituted Ci-Cg alkyl or C3-C8 cycloalkyl linker; and RR is a hydroxyl, unsubstituted or substituted Ci-C6 alkoxy, amino, unsubstituted or substituted Ci-C6 alkylamino, unsubstituted or substituted di-Ci-C6 alkylamino, unsubstituted or substituted C6-Cio aryl, unsubstituted or substituted heteroaryl comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S, unsubstituted or substituted C3-C10 carbocycle, or unsubstituted or substituted heterocycle comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S; or (ii) -C(0)-R12 or -C(0)0-R13, wherein R12 is NHRA or RA andR13 is RA;
when compounds comprise a double bond in the 6-membered ring fused to the pyrimidine ring, two R8 groups are present and are as defined above;
when compounds do not comprise a double bond in the 6-membered ring fused to the pyrimidine ring, four R8 groups are present and are as defined above;
or a pharmaceutically acceptable salt, prodrug or isotopic variant, for example, partially or fully deuterated form thereof.
In one embodiment, two R8 groups bonded to the same carbon can form an exocyclic double bond. In another embodiment, two R8 groups bonded to the same carbon can form a carbonyl group.
In one embodiment, the invention is directed to compounds or the use of such compounds of Formula VI:
Figure imgf000028_0001
wherein R, R1, R2, R3, R4, R5, R6, Rx, Z, m, n, and y are as defined above;
each R14 is independently H, C1-C3 alkyl or haloalkyl, cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O or S; -(alkylene)m- C3-C8 cycloalkyl, -(alkylene)m-aryl, - (alkylene)m-heterocyclo, -(alkylene)m- heteroaryl, -(alkylene)m-NR3R4, -(alkylene)m-C(0)- NR3R4; -(alkylene)m-0-R5, -(alkylene)m- S(0)n-R5, or -(alkylene)m-S(0)„-NR3R4 any of which may be optionally independently substituted with one or more Rx groups as allowed by valence, and wherein two Rx groups bound to the same or adjacent atoms may optionally combine to form a ring;
or two R14 groups bonded to the same carbon can form an exocyclic double bond;
or two R14 groups bonded to the same carbon can form a carbonyl group; and
when the compound of Formula VI has a double bond, as indicated by the (— ), in the 6- membered ring fused to the pyrimidine ring, two R14 groups are present as allowed for in
Formula VI above; or
when the compound of Formula VI does not include a double bond, as indicated by the (— ), in the 6-membered ring fused to the pyrimidine ring, four R14 groups are present as allowed for in Formula VI above;
or a pharmaceutically acceptable salt, prodrug or isotopic variant, for example, partially or fully deuterated form thereof.
In an alternative embodiment, the invention is directed to compounds or the use of such compounds of Formula I, II, III, IV, or V:
Figure imgf000029_0001
wherein:
Z is -(CH2)X- wherein x is 1, 2, 3 or 4 or -0-(CH2)z- wherein z is 2, 3 or 4;
each X is independently CH or N;
each X' is independently CH or N;
X" is independently CH2, S or NH, arranged such that the moiety is a stable 5-membered ring; R, R8, and R11 are independently H, C1-C3 alkyl (including methyl) or haloalkyl, cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O or S; -(alkylene)m- C3-C8 cycloalkyl, -(alkylene)m-aryl, -(alkylene)m-heterocyclo, -(alkylene)m- heteroaryl, -(alkylene)m- NR3R4, -(alkylene)m-C(0)-NR3R4; -(alkylene)m-0-R5, -(alkylene)m- S(0)n-R5, or -(alkylene)m- S(0)n-NR3R4 any of which, other than heterocyclo, may be optionally independently substituted with one or more Rx groups as allowed by valence, and wherein two Rx groups bound to the same or adjacent atoms may optionally combine to form a ring;
each R1 is independently aryl, alkyl, cycloalkyl or haloalkyl, wherein each of said alkyl, cycloalkyl and haloalkyl groups optionally includes O or N heteroatoms in place of a carbon in the chain and two Rx'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;
y is 0, 1, 2, 3 or 4;
R2 is -(alkylene)m-heterocyclo, -(alkylene)m-heteroaryl, -(alkylene)m-NR3R4, -(alkylene)m- C(0)-NR3R4; -(alkylene)m-C(0)-0-alkyl; -(alkylene)m-0-R5, -(alkylene)m-S(0)n-R5, or - (alkylene)m-S(0)n-NR3R4 any of which, other than heterocyclo, may be optionally independently substituted with one or more Rx groups as allowed by valance, and wherein two Rx groups bound to the same or adjacent atom may optionally combine to form a ring and wherein m is 0, 1 , or 2 and n is 0, 1 or 2;
wherein heterocyclo may be optionally independently substituted with 1 to 3 Rx groups as allowed by valance, and wherein two Rx groups bound to the same or adjacent atom may optionally combine to form a ring;
R3 and R4 at each occurrence are independently:
(i) hydrogen or
(ii) alkyl, cycloalkyl, heterocyclo, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl any of which, other than heterocyclo, may be optionally independently substituted with one or more Rx groups as allowed by valance, and wherein two Rx groups bound to the same or adjacent atom may optionally combine to form a ring; or R3 and R4 together with the nitrogen atom to which they are attached may combine to form a heterocyclo ring optionally independently substituted with one or more Rx groups as allowed by valance, and wherein two Rx groups bound to the same or adjacent atom may optionally combine to form a ring;
R5 and R5* at each occurrence is:
(i) hydrogen or
(ii) alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl any of which, other than heterocyclo, may be optionally independently substituted with one or more Rx groups as allowed by valance;
Rx at each occurrence is independently, halo, cyano, nitro, oxo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, heterocycloalkyl, -(alkylene)m-OR5, -(alkylene)m-0-alkylene-OR5, -(alkylene)m-S(0)n-R5, -(alkylene)m-NR3R4, -(alkylene)m-CN, -(alkylene)m-C(0)-R5, -(alkylene)m-C(S)-R5, -(alkylene)m-C(0)-OR5, -(alkylene)m-0-C(0)-R5,
-(alkylene)m-C(S)-OR5, -(alkylene)m-C(0)-(alkylene)m-NR3R4, -(alkylene)m-C(S)- NR3R4, -(alkylene)m-N(R3)-C(0)-NR3R4, -(alkylene)m-N(R3)-C(S)-NR3R4,
-(alkylene)m-N(R3)-C(0)-R5, -(alkylene)m-N(R3)-C(S)-R5, -(alkylene)m-0-C(0)- NR3R4, -(alkylene)m-0-C(S)-NR3R4, -(alkylene)m-S02-NR3R4, -(alkylene)m-N(R3)- S02-R5, -(alkylene)m-N(R3)-S02-NR3R4, -(alkylene)m-N(R3)-C(0)-OR5, -(alkylene)m- N(R3)-C(S)-OR5, or -(alkylene)m-N(R3)-S02-R5; wherein:
said alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, and heterocycloalkyl groups, any of which, other than heterocyclo, may be further independently substituted with one or more
-(alkylene)m-CN, -(alkylene)m-OR5*, -(alkylene)m-S(0)n-R5*,
-(alkylene)m-NR3 *R4 * , -(alkylene)m-C(0)-R5 * , -(alkylene)m-C(=S)R5 * , -(alkylene)m-C(=0)OR5 * , -(alkylene)m-OC(=0)R5 * , -(alkylene)m-C(S)-OR5 * , -(alkylene)m-C(0)-NR3 *R4 * , -(alkylene)m-C(S)-NR3 *R4 * , -(alkylene)m-N(R3^-C(0)-NR3*R4*, alkylene)m-N(R3*)-C(S)-NR3*R4* -(alkylene)m-N(R3*)-C(0)-R5*, -(alkylene)m-N(R3*)-C(S)-R5* -(alkyleneVO-C(0)-NR3*R4*, alkylene)m-0-C(S)-NR3*R4* -(alkylene)m-S02-NR3*R4*, -(alkylene)m-N(R3*)-S02-R5* -(alkylene)m-N(R3 *)-S02-NR3 *R4* , -(alkylene)m-N(R3 *)-C(0)-OR5 *
-(alkylene)m-N(R3*)-C(S)-OR5*, or -(alkylene)m-N(R3*)-S02-R5*, and wherein heterocycle may be further independently substituted with one to three substitutions selected from
-(alkylene)m-CN, -(alkylene)m-OR5 -(arkylene)m-S(0)n-R5*
-(alkylene)m -NR *R4*, -(alkylene)m -C(0)-R5*, -(alkylene)m-C(=S)R5*
-(alkylene)m- -C(=0)OR5*, -(alkylene)m- ■OC(=0)R5 * , -(alkylene)m-C(S)-OR5 *
-(alkylene)m- -C(0)-NR3*R4*, -(alkylene)m-C(S)-NR3*R4*
-(alkylene)m- -N(R3 * )-C (O)-NR3 * R4 * , alkylene)m-N(R3*)-C(S)-NR3*R4*
-(alkylene)m- -N(R3*)-C(0)-R5*, -(alkylene)m-N(R3*)-C(S)-R5*
-(alkylene)m- -0-C(0)-NR3*R4*, -(alkylene)m-0-C(S)-NR3*R4*
-(alkylene)m- -S02-NR3*R4*, -(alkylene)m-N(R *)-S02-R5*
-(alkylene)m- -N(R3*)-S02-NR3*R4*, -(alkylene)m-N(R3 *)-C(0)-OR5 *
-(alkylene)m- -N(R3*)-C(S)-OR5*, or -(alkylene)m-N(R3*)-S02-R5*; n is 0, 1 or 2, and
m is 0, 1; or 2 and
R3* and R4* at each occurrence are independently:
(i) hydrogen or
(ii) alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl any of which, other than heterocyclo, may be optionally independently substituted with one or more Rx groups as allowed by valance; or R3* and R4* together with the nitrogen atom to which they are attached may combine to form a heterocyclo ring optionally independently substituted with one or more Rx groups as allowed by valance; R6 is H, absent, or lower alkyl, -(alkylene)m-heterocyclo, -(alkylene)m-heteroaryl, -(alkylene)m- NR3R4, -(alkylene)m-C(0)-NR3R4; -(alkylene)m-0-R5, -(alkylene)m-S(0)n-R5, or -(alkylene)m-S(0)n- any of which, other than heterocyclo, may be optionally independently substituted with one or more Rx groups as allowed by valence, and wherein two Rx groups bound to the same or adjacent atoms may optionally combine to form a ring; and
R10 is (i) NHRA, wherein RA is unsubstituted or substituted Ci-Cg alkyl, cycloalkylalkyl, or -TT- RR, Ci-Cg cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O, and S; TT is an unsubstituted or substituted Ci-Cg alkyl or C3-C8 cycloalkyl linker; and RR is a hydroxyl, unsubstituted or substituted Ci-C6 alkoxy, amino, unsubstituted or substituted Ci-C6 alkylamino, unsubstituted or substituted di-Ci-C6 alkylamino, unsubstituted or substituted C6-Cio aryl, unsubstituted or substituted heteroaryl comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S, unsubstituted or substituted C3-C10 carbocycle, or unsubstituted or substituted heterocycle comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S; or (ii) -C(0)-R12 or -C(0)0-R13, wherein R12 is NHRA or RA andR13 is RA;
when the compound of Formula I, II, III, IV, or V has a double bond, as indicated by the (— ), in the 6-membered ring fused to the pyrimidine ring, two R8 groups are present as allowed for in Formula I, II, III, IV, or V above; or
when the compound of Formula I, II, III, IV, or V does not include a double bond, as indicated by the (— ), in the 6-membered ring fused to the pyrimidine ring, four R8 groups are present as allowed for in Formula I, II, III, IV, or V above;
wherein each heteroaryl is an aryl ring system that contains one or more heteroatoms selected from the group O, N and S, wherein the ring nitrogen and sulfur atom(s) are optionally oxidized, and nitrogen atom(s) are optionally quarternized;
wherein each aryl is a carbocyclic aromatic system containing one or two rings, wherein such rings may be attached together in a fused manner, and wherein each aryl may have 1 or more Rx substituents;
wherein each heterocyclo is a saturated or partially saturated heteroatom-containing ring radical, where the heteroatoms may be selected from nitrogen, sulfur and oxygen, wherein each heterocyclo is a monocyclic 6-8 membered ring or a 5-16 membered bicyclic ring system, and wherein each heterocyclo may have 1 to 3 Rx substituents; or a pharmaceutically acceptable salt, prodrug or isotopic variant, for example, partially or fully deuterated form thereof.
In an alternative embodiment, the term "aryl" means a carbocyclic aromatic system containing one or two rings wherein such rings may be attached together in a fused manner, which may have 1 or more substituents selected from lower alkyl, hydroxyl, halo, haloalkyl, nitro, cyano, alkoxy and lower alkylamino.
In an alternative embodiment, the term "heterocyclyl" or "heterocyclo" means a saturated or partially saturated heteroatom-containing ring radical, where the heteroatoms may be selected from nitrogen, sulfur and oxygen, which may have 1 to 3 substituents selected from hydroxyl, Boc, halo, haloalkyl, cyano, lower alkyl, lower aralkyl, oxo, lower alkoxy, amino and lower alkylamino, wherein the heterocyclic ring is a monocyclic 6-8 membered rings, or a 5-16 membered bicyclic ring systems which can include bridged fused and spiro- fused bicyclic ring systems, and which does not include rings containing -O-O-.-O-S- or -S-S- portion.
In an alternative embodiment, the term "heteroaryl" means an aryl ring system that contains one or more heteroatoms selected from the group O, N and S, wherein the ring nitrogen and sulfur atom(s) are optionally oxidized, and nitrogen atom(s) are optionally quarternized.
In one embodiment, two R8 groups bonded to the same carbon can form an exocyclic double bond. In another embodiment, two R8 groups bonded to the same carbon can form a carbonyl group.
In an alternative embodiment, the invention is directed to compounds or the use of such compounds of Formula VI:
Figure imgf000034_0001
VI,
wherein R, R1, R2, R3, R4, R5, R6, Rx, Z, m, n, and y are as defined above;
each R14 is independently H, C1-C3 alkyl (including methyl) or haloalkyl, cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O or S; -(alkylene)m- C3-C8 cycloalkyl, -(alkylene)m-aryl, -(alkylene)m-heterocyclo, -(alkylene)m- heteroaryl, -(alkylene)m- NR3R4, -(alkylene)m-C(0)-NR3R4; -(alkylene)m-0-R5, -(alkylene)m- S(0)n-R5, or -(alkylene)m- S(0)n-NR3R4 any of which, other than heterocyclo, may be optionally independently substituted with one or more Rx groups as allowed by valence, and wherein two Rx groups bound to the same or adjacent atoms may optionally combine to form a ring;
or two R14 groups bonded to the same carbon can form an exocyclic double bond;
or two R14 groups bonded to the same carbon can form a carbonyl group; and
when the compound of Formula VI has a double bond, as indicated by the (— ), in the 6- membered ring fused to the pyrimidine ring, two R14 groups are present as allowed for in Formula VI above; or
when the compound of Formula VI does not include a double bond, as indicated by the (— ), in the 6-membered ring fused to the pyrimidine ring, four R14 groups are present as allowed for in Formula VI above;
wherein each heteroaryl is an aryl ring system that contains one or more heteroatoms selected from the group O, N and S, wherein the ring nitrogen and sulfur atom(s) are optionally oxidized, and nitrogen atom(s) are optionally quarternized;
wherein each aryl is a carbocyclic aromatic system containing one or two rings, wherein such rings may be attached together in a fused manner, and wherein each aryl may have 1 or more Rx substituents;
wherein each heterocyclo is a saturated or partially saturated heteroatom-containing ring radical, where the heteroatoms may be selected from nitrogen, sulfur and oxygen, wherein each heterocyclo is a monocyclic 6-8 membered ring or a 5-16 membered bicyclic ring system, and wherein each heterocyclo may have 1 to 3 Rx substituents;
or a pharmaceutically acceptable salt, prodrug or isotopic variant, for example, partially or fully deuterated form thereof.
In some aspects, the compound is selected from Formula I or Formula II and R6 is absent. In some as ects, the compound is of Formula III:
Figure imgf000036_0001
and the variables are as defined for compounds of Formulae I and II and pharmaceutically acceptable salts, isotopic analogs, or prodrugs thereof.
In some aspects, Rx is not further substituted.
In some aspects, R2 is -(alkylene)m-heterocyclo, -(alkylene)m-heteroaryl, -(alkylene)m-NR3R4, -(alkylene)m-C(0)-NR3R4; -(alkylene)m-0-R5,
-(alkylene)m-S(0)n-R5, or -(alkylene)m-S(0)n-NR3R4 any of which may be optionally independently substituted with one or more Rx groups as allowed by valence, and wherein two Rx groups bound to the same or adjacent atom may optionally combine to form a ring and wherein m is 0 or 1 and n is 0, 1 or 2.
In some aspects, R8 is hydrogen or C1-C3 alkyl.
In some aspects, R is hydrogen or C1-C3 alkyl.
In some aspects, R2 is -(alkylene)m-heterocyclo, -(alkylene)m-NR3R4, -(alkylene)m-C(0)-NR3R4, -(alkylene)m-C(0)-0-alkyl or -(alkylene)m-OR5 any of which may be optionally independently substituted with one or more Rx groups as allowed by valence, and wherein two Rx groups bound to the same or adjacent atom may optionally combine to form a ring.
In some aspects, R2 is -(alkylene)m-heterocyclo, -(alkylene)m-NR3R4, -(alkylene)m-C(0)-NR3R4, -(alkylene)m-C(0)-0-alkyl or -(alkylene)m-OR5 without further substitution.
In some aspects, m in R2 is 1. In a further aspect, the alkylene in R2 is methylene.
In some aspects,
Figure imgf000036_0002
wherein: R2* is a bond, alkylene, -(alkylene)m-0-(alkylene)m-, -(alkylene)m-C(0)-(alkylene)m-,
-(alkylene)m-S(0)2-(alkylene)m-, or -(alkylene)m-NH-(alkylene)m- wherein each m is independently 0 or 1 ;
P is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group;
each Rxl is independently -(alkylene)m-(C(0))m-(alkylene)m-(N(RN))m-(alkyl)m wherein each m is independently 0 or 1 provided at least one m is 1, -(C(0))-0-alkyl, -(alkylene)m-cycloalkyl wherein m is 0 or 1, -N(RN)-cycloalkyl, -C(0)-cycloalkyl, -(alkylene)m-heterocyclyl wherein m is 0 or 1, or -N(RN)-heterocyclyl, -C(0)-heterocyclyl,- S(0)2-(alkylene)m wherein m is 1 or 2, wherein:
RN is H, Ci to C4 alkyl or Ci to C6 heteroalkyl, and
wherein two Rxl can, together with the atoms to which they attach on P, which may be the same atom, form a ring; and
t is 0, 1 or 2.
In some aspects, each Rxl is only optionally substituted by unsubstituted alkyl, halogen or hydroxy.
In some aspects, Rxl is hydrogen or unsubstituted Ci-C4 alkyl.
In some aspects, at least one Rxl is -(alkylene)m-heterocyclyl wherein m is 0 or 1.
In some aspects, R2 is
Figure imgf000037_0001
wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group.
In some aspects, R2
Figure imgf000037_0002
R2*-N j N x1
r -R
In some aspects, R2 is
In some aspects, R2 is
Figure imgf000037_0003
wherein: R is a bond, alkylene, -(alkylene)m-0-(alkylene)m-, -(alkylene)m-C(0)-(alkylene)m-, -(alkylene)m-S(0)2-(alkylene)m-, or -(alkylene)m-NH-(alkylene)m- wherein each m is independently 0 or 1 ;
P is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group;
PI is a 4- to 6-membered monocyclic saturated heterocyclyl group;
each Rx2 is independently hydrogen or alkyl; and
s is 0, 1 or 2.
In some aspects, R2 is
Figure imgf000038_0001
In some aspects, PI includes at least one nitrogen.
In some aspects, any alkylene in R2* in any previous aspect is not further substituted.
In some aspects, R2 is selected from the structures depicted in FIGS
In some aspects, R is
Figure imgf000038_0002
Figure imgf000038_0003
In some aspects, the compound has general Formula I and more specifically one of the general structures in FIGS. 5A-9F wherein the variables are as previously defined.
In some as ects, the compound has general Formula la:
Figure imgf000038_0004
wherein R1, R2, R, R8, X and y are as previously defined.
In some embodiments, the compound has Formula la and R is alkyl.
In some embodiments, the compound has Formula la and R is H.
In some embodiments, the compound has Formula la and R2 is
Figure imgf000039_0001
erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R , Rxl and t are as previously defined.
In some embodiments, the compound has Formula la and R2 is
Figure imgf000039_0002
erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, Rxl is hydrogen or unsubstituted C1-C4 alkyl and R2* is as previously defined.
In some embodiments, the compound is of Formula lb:
Figure imgf000039_0003
wherein R, R2 and R8 are as previously defined.
In some embodiments, the compound is of Formula lb and R is alkyl.
In some embodiments, the compound is of Formula lb and R is H.
In some embodiments, the compound has Formula lb and R2 is
Figure imgf000039_0004
erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R , Rxl and t are as previously defined. In some embodiments, the compound has Formula lb and R is
Figure imgf000040_0001
ein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, Rxl is hydrogen or C1-C4 alkyl and R2* is as previously defined.
In some embodiments, the compound has Formula Ic:
Figure imgf000040_0002
wherein R, R2 and R8 are as previously defined.
In some embodiments, the compound has Formula Ic and R is alkyl.
In some embodiments, the compound has Formula Ic and R is H.
In some embodiments, the compound has Formula Ic and R2 is
Figure imgf000040_0003
ein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R , Rxl and t are as previously defined.
In some embodiments, the compound has Formula Ic and R2 is
Figure imgf000040_0004
erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, Rxl is hydrogen or C1-C4 alkyl and R2* is as previously defined.
In some embodiments, the compound has Formula Id:
Figure imgf000040_0005
wherein R, R2 and R8 are as previously defined.
In some embodiments, the compound has Formula Id and R is alkyl.
In some embodiments, the compound has Formula Id and R is H.
In some embodiments, the compound has Formula R is
Figure imgf000041_0001
a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R2*, Rxl and t are as previously defined.
In some embodiments, the compound has Formula Id and Rz is
Figure imgf000041_0002
erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, Rxl is hydrogen or C1-C4 alkyl and R2* is as previously defined.
In some embodiments, the compound has Formula Ie:
Figure imgf000041_0003
wherein R, R2 and R8 are as previously defined.
In some embodiments, the compound has Formula Ie and R is alkyl.
In some embodiments, the compound has Formula Ie and R is H.
In some embodiments, the compound has Formula Ie and R2 is
Figure imgf000041_0004
erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R , Rxl and t are as previously defined. In some embodiments, the compound has Formula Ie and R is
Figure imgf000042_0001
ein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, Rxl is hydrogen or C1-C4 alkyl and R2* is as previously defined.
In some embodiments, the compound has Formula If:
Figure imgf000042_0002
wherein R, R2 and R8 are as previously defined.
In some embodiments, the compound has Formula If and R is alkyl.
In some embodiments, the compound has Formula If and R is H.
In some embodiments, the compound has Formula If and R2 is
Figure imgf000042_0003
erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R2*, Rxl and t are as previously defined.
In some embodiments, the compound has Formula If and R2 is
Figure imgf000042_0004
ein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, Rxl is hydrogen or C1-C4 alkyl and R2* is as previously defined.
In some embodiments the compound has Formula Ig:
Figure imgf000042_0005
wherein R, R2 and R8 are as previously defined.
In some embodiments, the compound has Formula Ig and R is alkyl.
In some embodiments, the compound has Formula Ig and R is H.
In some embodiments, the compound has Formula Ig and R2 is
Figure imgf000043_0001
erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R2*, Rxl and t are as previously defined.
In some embodiments, the compound has Formula Ig and R2 is
Figure imgf000043_0002
ein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, Rxl is hydrogen or C1-C4 alkyl and R2* is as previously defined.
In some embodiments the compound has Formula Ih:
Figure imgf000043_0003
wherein R, R2 and R8 are as previously defined.
In some embodiments, the compound has Formula Ih and R is alkyl.
In some embodiments, the compound has Formula Ih and R is H.
In some embodiments, the compound has Formula Ih and R2 is
Figure imgf000043_0004
erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R2*, Rxl and t are as previously defined. In some embodiments, the compound has Formula Ih and R is
Figure imgf000044_0001
ein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, Rxl is hydrogen or C1-C4 alkyl and R2* is as previously defined.
In some embodiments the compound has Formula Ii:
Figure imgf000044_0002
wherein R, R2 and R8 are as previously defined.
In some embodiments, the compound has Formula Ii and R is alkyl.
In some embodiments, the compound has Formula Ii and R is H.
In some embodiments, the compound has Formula Ii and R2 is
Figure imgf000044_0003
erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R , Rxl and t are as previously defined.
In some embodiments, the compound has Formula Ii and R2 is
Figure imgf000044_0004
erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, Rxl is hydrogen or C1-C4 alkyl and R2* is as previously defined.
In some embodiments the compound has Formula Ij :
Figure imgf000044_0005
wherein R, R2 and R8 are as previously defined. In some embodiments, the compound has Formula Ij and R is alkyl.
In some embodiments, the compound has Formula Ij and R is H.
In some embodiments, the compound has Formula Ij and R2 is
Figure imgf000045_0001
erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group.
In some embodiments, the compound has Formula Ij and R2 is
Figure imgf000045_0002
erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, Rxl is hydrogen or C1-C4 alkyl.
In some embodiments, the compound has Formula Ij and R is H, and X is CH and N. In some embodiments the compound has the structure:
Figure imgf000045_0003
In some embodiments, the compound has Formula Ik and R2 is
Figure imgf000045_0004
erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group. In some embodiments, the compound has Formula Ik and R is
Figure imgf000046_0001
ein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, Rxl is hydrogen or C1-C4 alkyl.
In some embodiments the compound has Formula II:
Figure imgf000046_0002
In some embodiments, the compound has Formula II and R is
Figure imgf000046_0003
erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group.
In some embodiments, the compound has Formula II and R2 is
Figure imgf000046_0004
erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, Rxl is hydrogen or C1-C4 alkyl.
In some embodiments, the compound has Formula Im:
Figure imgf000046_0005
In some embodiments, the compound has Formula Im and R is
Figure imgf000046_0006
is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group. In some embodiments, the compound has Formula Im and R is
Figure imgf000047_0001
wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, Rxl is hydrogen or C1-C4 alkyl.
In some embodiments, the compound has Formula Ila:
Figure imgf000047_0002
In some embodiments, the compound has Formula Ila and R is
Figure imgf000047_0003
wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group.
In some embodiments, the compound has Formula Ila and R2 is
Figure imgf000047_0004
wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, Rxl is hydrogen or C1-C4 alkyl.
In some embodiments, the compound has Formula lib:
Figure imgf000047_0005
In some embodiments, the compound has Formula lib and R is
Figure imgf000047_0006
erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group. In some embodiments, the compound has Formula lib and R is
Figure imgf000048_0001
erein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, Rxl is hydrogen or C1-C4 alkyl.
In some as ects, the active compound is:
Figure imgf000048_0002
Further specific compounds that fall within the present invention and that can be used in the disclosed methods of treatment and compositions include, but are not limited to, the structures listed in Table 1 below.
Table 1 : Structures of Tricyclic Lactams
Figure imgf000048_0003
Figure imgf000049_0001
Figure imgf000050_0001

Figure imgf000051_0001

Figure imgf000052_0001
Figure imgf000053_0001
51
Figure imgf000054_0001
52
Figure imgf000055_0001
53
Figure imgf000056_0001
54
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Isotopic Substitution
The present invention includes compounds and the use of compounds with desired isotopic substitutions of atoms, at amounts 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. By way of general example and without limitation, isotopes of hydrogen, for example, deuterium (2H) and tritium (3H) may be used anywhere in described structures. Alternatively or in addition, isotopes of carbon, e.g., 13C and 14C, may be used. A preferred isotopic substitution is deuterium for hydrogen at one or more locations on the molecule to improve the performance of the drug. The deuterium can be bound in a location of bond breakage during metabolism (an a-deuterium kinetic isotope effect) or next to or near the site of bond breakage (a β-deuterium kinetic isotope effect).
Substitution with isotopes such as deuterium can afford certain therapeutic advantages resulting from greater metabolic stability, such as, for example, increased in vivo half-life or reduced dosage requirements. Substitution of deuterium for hydrogen at a site of metabolic break down can reduce the rate of or eliminate the metabolism at that bond. At any position of the compound that a hydrogen atom may be present, the hydrogen atom can be any isotope of hydrogen, including protium (1H), deuterium (2H) and tritium (3H). Thus, reference herein to a compound encompasses all potential isotopic forms unless the context clearly dictates otherwise. The term "isotopically-labeled" analog refers to an analog that is a "deuterated analog", a "13C- labeled analog," or a "deuterated/13C-labeled analog." The term "deuterated analog" means a compound described herein, whereby a H-isotope, i.e., hydrogen/protium (1H), is substituted by a H-isotope, i.e., deuterium (2H). Deuterium substitution can be partial or complete. Partial deuterium substitution means that at least one hydrogen is substituted by at least one deuterium. In certain embodiments, the isotope is 90, 95 or 99% or more enriched in an isotope at any location of interest. In some embodiments it is deuterium that is 90, 95 or 99% enriched at a desired location.
Hematopoietic Stem Cells and Cyclin-Dependent Kinase Inhibitors
Tissue-specific stem cells are capable of self-renewal, meaning that they are capable of replacing themselves throughout the adult mammalian lifespan through regulated replication. Additionally, stem cells divide asymmetrically to produce "progeny" or "progenitor" cells that in turn produce various components of a given organ. For example, in the hematopoietic system, the hematopoietic stem cells give rise to progenitor cells which in turn give rise to all the differentiated components of blood (e.g., white blood cells, red blood cells, and platelets) (See FIG. 1).
Early hematopoietic stem/progenitor cells (HSPC) in the adult mammal require the enzymatic activity of the proliferative kinases cyclin-dependent kinase 4 (CDK4) and/or cyclin- dependent kinase 6 (CDK6) for cellular replication. In contrast, the majority of proliferating cells in adult mammals (e.g., the more differentiated blood-forming cells in the bone marrow) do not require the activity of CDK4 and/or CDK6 (i.e., CDK4/6). These differentiated cells can proliferate in the absence of CDK4/6 activity by using other proliferative kinases, such as cyclin- dependent kinase 2 (CDK2) or cyclin-dependent kinase 1 (CDK1).
The present invention includes methods of protecting healthy cells in a subject, and in particular, hematopoietic cells and/or progenitor cells (HSPCs) from the toxic effects or mitigation of ionizing radiation by the administration of a tricyclic lactam compound. In one embodiment, the tricyclic lactam compound is a selective CDK4/6 inhibitor. The use of tricyclic lactams as CDK4/6-specific Gl -arresting effect compounds as radioprotectants and radiomitigants allows for an accelerated hematological recovery and reduced hematological cytotoxicity risk due to HSPC replication delay. In certain embodiments, the tricyclic lactam administered is selected from the group consisting of a compound or composition comprising Formula I, Formula II, Formula III, Formula IV, Formula V, Formula VI, or a combination thereof. In one non-limiting embodiment, a compound can be selected from the compounds of Table 1 , or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof.
In certain aspects, compounds, methods, and compositions are provided for reducing or limiting the effect of DNA damaging ionizing radiation on hematopoietic stem and progenitor cells in a subject undergoing treatment for a Rb-null cancer, the method comprising administering an effective amount of a tricyclic compound prior to exposure to IR. In one embodiment, a substantial portion of the hematopoietic stem and/or progenitor cells return to pre-treatment baseline cell cycle activity (i.e., reenter the cell-cycle) within less than about 48 hours of administration of the tricyclic lactam. In certain embodiments, the tricyclic lactam administered is selected from the group consisting of a compound or composition comprising Formula I, Formula II, Formula III, Formula IV, Formula V, and Formula VI, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof. In one non- limiting embodiment, a compound can be selected from the compounds of Table 1, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof.
In certain aspects, tricyclic lactam compounds, methods, and composition are provided for reducing or limiting the effect of DNA-damaging IR on hematopoietic stem and progenitor cells in a subject that has been exposed to IR, the method comprising administering an effective amount of a tricyclic lactam following exposure to IR, wherein a substantial portion of the hematopoietic stem and/or progenitor cells reenter the cell-cycle synchronously within less than about 24, 30, 36, 40, or 48 hours following the dissipation of the compound's CDK4/6 inhibitory effect, wherein the tricyclic lactam compound has an IC50 CDK4 inhibitory concentration that is more than 500 times less than its IC50 inhibitory concentration for CDK2. In certain embodiments, a substantial portion of the hematopoietic stem and/or progenitor cells reenter the cell-cycle synchronously within less than about 24, 30, 36, 40, or 48 hours from the point in which the tricyclic lactam's concentration level in the subject's blood drops below a therapeutic effective concentration. In certain embodiments, the tricyclic lactam administered is selected from the group consisting of a compound or composition comprising Formula I, Formula II, Formula III, Formula IV, Formula V, and Formula VI or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof. In one non-limiting embodiment, a compound can be selected from the compounds of Table 1 , or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof.
In certain embodiments, the tricyclic lactam is a CDK4/6 inhibitor selected from Formula I, II, III, IV, V, or VI, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof, wherein the protection afforded by the compound is short term and transient in nature, allowing a significant portion of the cells to synchronously renter the cell-cycle following the cessation of IR exposure. Cells that are quiescent within the Gl phase of the cell cycle are more resistant to the DNA damaging effect of radiation than proliferating cells.
In one embodiment, the tricyclic lactam compounds for use in the described methods are CDK4/6 inhibitors, with minimal CDK2 inhibitory activity. In one embodiment, a tricyclic lactam compound for use in the methods described herein has a CDK4/CycDl IC50 inhibitory concentration value that is >100, >200, >300, >400, >500, >600, >700, >800, >900, >1000, >1250, >1500 times, >1800 times, >2000 times, >2200 times, >2500 times, >2700 times, >3000 times, >3200 times lower than its respective IC50 concentration value for CDK2/CycE inhibition. In one embodiment, a tricyclic lactam for use in the methods described herein has an IC50 concentration value for CDK4/CycDl inhibition that is about <1.50 nM, <1.25 nM, <1.0 nM, <0.90 nM, <0.85 nM, <0.80 nM, <0.75 nM, <0.70 nM, <0.65 nM, <0.60 nM, <0.55 nM, or less. In one embodiment, a tricyclic lactam for use in the methods described herein has an IC50 concentration value for CDK2/CycE inhibition that is about >1.0 μΜ, >1.25 μΜ, >1.50 μΜ, >1.75 μΜ, >2.0 μΜ , >2.25 μΜ, >2.50 μΜ, >2.75 μΜ, >3.0 μΜ, >3.25 μΜ, >3.5 μΜ or greater. In one embodiment, a tricyclic lactam for use in the methods described herein has an IC50 concentration value for CDK2/CycA IC50 that is >0.80 μΜ, >0.85 μΜ, >0.90 μΜ, >0.95 μΜ, >.1.0 μΜ, >1.25 μΜ, >1.50 μΜ, >1.75 μΜ, >2.0 μΜ, >2.25 μΜ, >2.50 μΜ, >2.75 μΜ, >3.0 μΜ or greater. In one embodiment, the tricyclic lactam for use in the methods described herein are selected from the group consisting of Formula I, Formula II, Formula III, Formula IV, Formula V, and Formula VI, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug, thereof. In one non-limiting embodiment, a compound can be selected from the compounds of Table 1, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof.
According to the presently disclosed subject matter, radiation protection with the tricyclic lactams described herein may be achieved by a number of different dosing schedules. In addition to multi-dosing schedules or single pretreatment, concomitant treatment can also be effective.
In one embodiment, the tricyclic lactams described herein are used in HSPC cycling strategies wherein a subject is exposed to regular, repeated IR exposures, wherein HSPCs are Gl -arrested when IR exposed and allowed to reenter the cell-cycle before the subject's next IR exposure. Such cycling allows HSPCs to regenerate damaged blood cell lineages in between regular, repeated IR exposures, for example those associated with standard IR treatments for cancer, and reduces the risk associated with long term CDK4/6 inhibition.
In one embodiment, the subject is exposed to IR at least 5 times a week, at least 4 times a week, at least 3 times a week, at least 2 times a week, at least 1 time a week, at least 3 times a month, at least 2 times a month, or at least 1 time a month, wherein the subject's HSPCs are Gl arrested during treatment and allowed to cycle in between IR exposure, for example during a treatment break. In one embodiment, the subject is undergoing 5 times a week IR exposure, wherein the subject's HSPCs are Gl arrested during the IR exposure and allowed to reenter the cell-cycle during the 2 day break, for example, over the weekend.
In one embodiment, using a tricyclic lactam described herein, the subject's HSPCs are arrested during the entirety of the IR exposure time-period for the weekly treatment, for example, during a 5 times/week IR regimen, the cells are arrested over the time period that is required to complete the IR exposure regimen for the week, and then allowed to recycle at the end of the regimen. In one embodiment, using a tricyclic lactam described herein, the subject's HSPCs are arrested during the entirety of the IR regimen, for example, in a 5 times a week IR regimen for 5 weeks, and rapidly reenter the cell-cycle following the completion of the IR regimen.
In one embodiment, the subject has been exposed to IR, and, using a tricyclic lactam described herein, the subject's HSPCs are placed in Gl arrest following exposure in order to mitigate DNA damage. In one embodiment, the tricyclic lactam is administered at least ½ hour, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 14 hours, at least 16 hours, at least 18 hours, at least 20 hours, at least 24 hours or more post IR exposure. In one embodiment, the subject has been exposed to IR and is administered multiple tricyclic lactam doses at differing time points, for example, at 12 hours and 24 hours post IR exposure.
In some embodiments, the presently disclosed subject matter provides methods for protection of mammals from the acute and chronic toxic effects of ionizing radiation by forcing hematopoietic stem and progenitor cells (HSPCs) into a quiescent state by transient (e.g., over a period of less than about 40 hours, 36 hours, 30 hours, 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, 4 hours, 3 hours, 2.5 hours, 2 hours, 1 hour, ½ hour or less prior to IR exposure) treatment with a tricyclic lactam selected from the group consisting of Formula I, Formula II, Formula III, Formula IV, Formula V, and Formula VI, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof. HSPCs recover from this period of transient quiescence, and then function normally after treatment with the compound is stopped, and its intra-cellular effect dissipates. During the period of quiescence, the stem and progenitor cells are protected from the effects of ionizing radiation. The ability to protect stem/progenitor cells is desirable both in the treatment of cancer where patients are given high, repeated doses of ionizing radiation, and in environmental or occupational situations where individuals may be in danger of being exposed to large doses of radiation. In some embodiments, the HSPCs can be arrested for longer periods, for example, over a period of hours, days, and/or weeks, through multiple, time separated administrations of a tricyclic lactam described herein.
In one embodiment of the invention, these compounds can be administered in a concerted regimen with a blood growth factor agent. As such, in one embodiment, the use of the compounds and methods described herein is combined with the use of hematopoietic growth factors including, but not limited to, granulocyte colony stimulating factor (G-CSF, for example, sold as Neupogen (filgrastin), Neulasta (peg-filgrastin), or lenograstin), 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 derivatives (sold as for example epoetin-a as Darbopoetin, Epocept, Nanokine, Epofit, Epogin, Eprex and Procrit; epoetin-β sold as for example NeoRecormon, Recormon and Micera), epoetin-delta (sold as for example Dynepo), epoetin omega (sold as for example Epomax), epoetin zeta (sold as for example Silapo and Reacrit) as well as for example Epocept, EPOTrust, Erypro Safe, Repoeitin, Vintor, Epofit, Erykine, Wepox, Espogen, Relipoeitin, Shanpoietin, Zyrop and EPIAO).
It has been recently reported that some of the hematopoietic growth factors can have serious side effects. For example, the EPO family of therapeutics has been associated with arterial hypertension, cerebral convulsions, hypertensive encephalopathy, tumor progression thromboembolism, iron deficiency, influenza like syndromes and venous thrombosis. The G- CSF family of therapeutics has been associated with myelodysplasia and secondary leukemia, spleen enlargement and rupture, respiratory distress syndrome, allergic reactions and sickle cell complications.
By combining the administration of the described tricyclic lactams and methods of the present invention with hematopoietic growth factors, it is possible for the health care practitioner to decrease the amount of the growth factor to minimize the unwanted adverse effects while achieving the therapeutic benefit. Thus, in this embodiment, the tricyclic lactam allows the patient to receive some amount of the growth factor. The patient may not need as much hematopoietic growth factor because the hematopoietic cells will have been protected during the chemotherapy and not diminished to the extent without the tricyclic lactam. Furthermore, by timing the administration of the growth factors, hematopoietic cells are not forced into replicating while harboring major DNA structural damage.
Several advantages can result from the radio-protective methods described herein using a tricyclic lactam described herein. The reduction in radio-toxicity afforded by the tricyclic lactam can allow for dose intensification (e.g., more therapy can be given in a fixed period of time) in medically related IR therapies, which will translate to better efficacy. Therefore, the presently disclosed methods can result in radio-therapy regimens that are less toxic and more effective. Also, in contrast to protective treatments with exogenous biological growth factors, in one embodiment, the tricyclic lactam described herein are orally available small molecules, which can be formulated for administration via a number of different routes. When appropriate, such small molecules can be formulated for oral, topical, intranasal, inhalation, intravenous, intramuscular, or any other form of administration. Further, as opposed to biologies, stable small molecules can be more easily stockpiled and stored. Thus, the tricyclic lactam compounds can be more easily and cheaply kept on hand in emergency rooms where subjects of IR exposure can report or at sites where radiation exposure is particularly likely to occur: at nuclear power plants, on nuclear powered vessels, at military installations, near battlefields, etc.
In one embodiment, the use of a tricyclic lactam as described herein can induce selective Gl arrest in CDK4/6-dependent cells (e.g., as measured in a cell-based in vitro assay). In one embodiment, the tricyclic lactam is capable of increasing the percentage of CDK4/6-dependent cells in the Gl phase, while decreasing the percentage of CDK4/6-dependent cells in the G2/M phase and S phase. In one embodiment, the tricyclic lactam induces substantially pure (i.e., "clean") Gl cell cycle arrest in the CDK4/6-dependent cells (e.g., wherein treatment with the tricyclic lactam induces cell cycle arrest such that the majority of cells are arrested in Gl as defined by standard methods (e.g. propidium iodide (PI) staining or others) with the population of cells in the G2/M and S phases combined being less than about 30%, about 25%, about 20%>, about 15%), about 10%>, about 5%, about 3% or less of the total cell population. Methods of assessing the cell phase of a population of cells are known in the art (see, for example, in U.S. Patent Application Publication No. 2002/0224522) and include cytometric analysis, microscopic analysis, gradient centrifugation, elutriation, fluorescence techniques including immunofluorescence, and combinations thereof. Cytometric techniques include exposing the cell to a labeling agent or stain, such as DNA-binding dyes, e.g., PI, and analyzing cellular DNA content by flow cytometry. Immunofluorescence techniques include detection of specific cell cycle indicators such as, for example, thymidine analogs (e.g., 5-bromo-2-deoxyuridine (BrdU) or an iododeoxyuridine), with fluorescent antibodies.
In some embodiments, the use of a tricyclic lactam described herein reduces the risk of undesirable off-target effects including, but not limited to, long term toxicity, anti-oxidant effects, and estrogenic effects. Anti-oxidant effects can be determined by standard assays known in the art. For example, a compound with no significant anti-oxidant effects is a compound that does not significantly scavenge free-radicals, such as oxygen radicals. The anti-oxidant effects of a compound can be compared to a compound with known anti-oxidant activity, such as genistein. Thus, a compound with no significant anti-oxidant activity can be one that has less than about 2, 3, 5, 10, 30, or 100 fold anti-oxidant activity relative to genistein. Estrogenic activities can also be determined via known assays. For instance, a non-estrogenic compound is one that does not significantly bind and activate the estrogen receptor. A compound that is substantially free of estrogenic effects can be one that has less than about 2, 3, 5, 10, 20, or 100 fold estrogenic activity relative to a compound with estrogenic activity, e.g., genistein.
In some embodiments, the subject has been exposed to ionizing radiation, will be exposed to ionizing radiation, or is at risk of incurring exposure to ionizing radiation as the result of radiological agent exposure during warfare, a radiological terrorist attack, an industrial accident, or space travel. Subjects can further be exposed to, or be scheduled to be exposed to, ionizing radiation when undergoing therapeutic irradiation for the treatment of proliferative disorders. Such disorders include cancerous and non-cancer proliferative diseases. The compounds are effective in protecting healthy hematopoietic stem/progenitor cells during therapeutic irradiation of a broad range of tumor types, including but not limited to the following: breast, prostate, ovarian, skin, lung, colorectal, brain (i.e., glioma) and renal. Ideally, growth of the cancer being treated by IR should not be affected by the tricyclic lactam compound. The potential sensitivity of certain tumors to 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 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 or absence of retinoblastoma (Rb) tumor suppressor protein (Rb-null), high levels of MYC expression, increased cyclin E and increased cyclin A. 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 certain classes of Rb-positive 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 leukemias, certain classes of lymphomas, 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 cancer and certain classes of testicular cancers.
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, 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 of the present invention 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".
Alternatively, 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) http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=gene&part=retinoblastoma 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 of the present invention 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.
In some embodiments, the cancer a small cell lung cancer, retinoblastoma, and triple negative (ER/PR/Her2 negative) or "basal-like" breast cancer, which almost always inactivate the retinoblastoma tumor suppressor protein (Rb), and therefore do not require CDK4/6 activity to proliferate. Triple negative (basal-like) breast cancer is also almost always genetically or functionally Rb-null. Also, certain virally induced cancers (e.g. cervical cancer and subsets of Head and Neck cancer) express a viral protein (E7) which inactivates Rb making these tumors functionally Rb-null. Some lung cancers are also believed to be caused by HPV.
The tricyclic lactams described herein can also be used in protecting healthy CDK4/6- replication dependent cells during ionizing radiation of abnormal tissues in non-cancer proliferative diseases, including but not limited to the following: psoriasis, lupus, arthritis (notably rheumatoid arthritis), hemangiomatosis in infants, multiple sclerosis, myelodegenerative disease, neurofibromatosis, ganglioneuromatosis, keloid formation, Paget's Disease of the bone, fibrocystic disease of the breast, Peyronie's and Duputren's fibrosis, restenosis, and cirrhosis.
According to the present invention, therapeutic ionizing radiation can be administered to a subject on any schedule and in any dose consistent with the prescribed course of treatment, for example by administering a compound of Formula I, Formula II, Formula III, Formula IV, Formula V, and Formula VI, or a compound selected from Table 1, prior to or during the radiation. Preferably, administration of the compound is timed such that maximal Gl arrest of the HSPCs, or a significant portion thereof, occurs at the time of the IR exposure. In certain embodiments, a tricyclic lactam compound described herein is administered so that a peak serum concentration for the compound is reached at or near the time of IR exposure. If desired, multiple doses of the radioprotectant compound can be administered to the subject. Alternatively, the subject can be given a single dose of the compound. The course of treatment differs from subject to subject, and those of ordinary skill in the art can readily determine the appropriate dose and schedule of therapeutic radiation in a given clinical situation.
Active Compounds, Salts and Formulations
As used herein, the term "active compound" refers to the tricyclic lactam compounds described herein or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof. The active compound can be administered to the subject through any suitable approach. The amount and timing of active compound administered is dependent on the subject being treated, on the dosage of IR to which the subject is anticipated of being exposed to, on the time course of the IR exposure, on the manner of administration, on the pharmacokinetic properties of the particular active compound, and on the judgment of the prescribing physician. Thus, because of subject to subject variability, 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 subject. In considering the degree of treatment desired, the physician can balance a variety of factors such as age and weight of the subject, presence of preexisting disease, as well as presence of other diseases. Pharmaceutical formulations can be prepared for any desired route of administration including, but not limited to, oral, intravenous, or aerosol administration, as discussed in greater detail below.
The therapeutically effective dosage of any of the 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. In one embodiment, a dosage from about 0.1 to about 200 mg/kg is administered, with all weights being calculated based upon the weight of the active compound, including the cases where a salt is employed. For example, a dosage can provide the amount of compound needed to provide a serum concentration of the active compound of up to between about 1 and 5, 10, 20, 30 or 40 μΜ. In some embodiments, a dosage from about 10 mg/kg to about 50 mg/kg can be employed for oral administration. Typically, a dosage from about 0.5 mg/kg to 5 mg/kg can be employed for intramuscular injection. In some embodiments, dosages can be from about 1 umol/kg to about 50 umol/kg, or, optionally, between about 22 umol/kg and about 33 umol/kg of the compound for intravenous or oral administration. An oral dosage form can include any appropriate amount of active material, including for example from 5 mg to, 50, 100, 200 or 500 mg per tablet or other solid dosage form. In accordance with the presently disclosed methods, pharmaceutically active compounds as described herein can be administered orally as a solid or as a liquid, or can be administered intramuscularly, intravenously, or by inhalation as a solution, suspension, or emulsion. In some embodiments, the compounds or salts also can be administered by inhalation, intravenously, or intramuscularly as a liposomal suspension. When administered through inhalation the active compound or salt can 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 present invention 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. If a solution is desired, water is a carrier of choice for water-soluble compounds or salts. With respect to the 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.
In addition to the active compounds or their salts, the pharmaceutical formulations can contain other additives, such as pH-adjusting additives. In particular, 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. Further, the formulations can contain antimicrobial preservatives. Useful antimicrobial preservatives include methylparaben, propylparaben, and benzyl alcohol. An antimicrobial preservative is typically employed when the formulation 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. For oral administration a pharmaceutical composition can take the form of solutions, suspensions, tablets, pills, capsules, powders, 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. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often very useful for tabletting purposes. Solid compositions of a similar type may be also employed as fillers in soft and hard- filled gelatin capsules. Materials in this connection also include lactose or milk sugar as well as high molecular weight polyethylene glycols. When aqueous suspensions and/or elixirs are desired for oral administration, the compounds of the presently disclosed subject 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.
In yet another embodiment of the subject matter described herein, there is provided an injectable, stable, sterile formulation 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 a liquid formulation suitable for injection thereof into a subject. When the compound or salt is substantially water-insoluble, 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. When the compound is an aqueous-soluble salt, using conventional liposome technology, the same can be incorporated into lipid vesicles. In such an instance, 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. When 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. In either instance, 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 formulation 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 can be obtained by processing the solid compound or a salt thereof, in any appropriate manner known in the art, such as by micronization. Optionally, the size of the solid particles or droplets can be from about 1 to about 2 microns. In this respect, 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.
When the pharmaceutical formulation suitable for administration as an aerosol is in the form of a liquid, the formulation 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 formulation sufficiently to result in the formation of droplets within the desired size range when subjected to nebulization.
The term "pharmaceutically acceptable salts" as used herein refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with subjects (e.g., human subjects) 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 subject matter.
Thus, the term "salts" refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the presently disclosed subject matter. These salts can be prepared in situ 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. In so far as the compounds of the presently disclosed subject matter are basic compounds, they are all 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. 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.
Synthesis of Tricyclic Lactams
Tricyclic lactams of the present invention can be synthesized by any means known to those of ordinary skill in the art, including for example, according to the generalized Schemes of 1 through 11 below.
Scheme 1
Figure imgf000081_0001
Figure imgf000081_0002
Figure imgf000081_0003
Scheme 2
11 12
Figure imgf000082_0002
□HMDS
14
Scheme 3
Figure imgf000082_0003
15 16 17
HCI, MeOH 1) NaHMDS
2) MOMCI
Figure imgf000082_0004
Figure imgf000082_0005
Scheme 4
Figure imgf000083_0001
IV Scheme 5
NH2
Figure imgf000084_0001
20 21 K2C03, CH3CN 22 1) NaHMDS 2) MOMCI
Figure imgf000084_0002
25
Scheme 6
Figure imgf000084_0003
Scheme 7
Figure imgf000085_0001
Ref 1 is US 8598186
Scheme 8
Figure imgf000085_0002
Ref 1 is US 8598186
A method for the preparation of substituted tricyclic lactams is provided that includes efficient methods for the preparation of a tricyclic lactam ring system and subsequent displacement of an aryl sulfone with an amine.
In Scheme 1, diethyl succinate is employed to prepare the pyrimidine ester, 2, according to the method of A. Haidle, See, WO 2009/152027 entitled 5,7-dihydro-6H-pyrrolo[2,3- d]pyrimidin-6-one derivatives for MARK inhibition. The ester intermediate 2 can be reduced by directly reacting the ester with a reducing agent such as lithium borohydride in a protic organic solvent such as ethanol to produce the corresponding primary alcohol. The primary alcohol can be reacted with a reagent such as phosphorus tribromide in an organic solvent such as dimethylforamide to produce the primary bromide 3. The primary bromide 3 can be condensed with the lactam 4 optionally at low temperature using a base such as lithium diisopropylamide in an organic solvent such as tetrahydrofuran to produce the lactam 5. Lactam 5 can be deprotected by directly reacting Compound 5 with an aqueous acid such as HC1 = pH 1 solution. Lactam 6 can be directly reacted with an organic base such as 1,8- diazabicyclo[5.4.0]undec-7-ene in a protic solvent such as ethanol optionally at high temperature to cyclize Compound 5 to form the tricyclic lactam 7. The thiol moiety can be subsequently oxidized to the sulfone 8 by directly reacting Compound 7 with an oxidizing reagent such as meta-chloroperoxybenzoic acid. The sulfone, 8, can be directly reacted with an amine, 9, in the presence of a strong base such as lithium hexamethyldisilazane to form the tricyclic lactam 10.
In Scheme 2, the tricyclic lactam 7 is directly reacted with an oxidizing reagent such as 2,3-dichloro-5,6-dicyano-l,4-benzoquinone (DDQ) to form the alkene 11. Alkene 11 can be directly reacted with an oxidizing reagent such as meta-chloroperoxybenzoic acid to form the sulfone intermediate 12. The sulfone, 12, can be condensed with an amine, 13, in the presence of a strong base such as lithium hexamethyldisilazane to form the tricyclic lactam 14.
Scheme 3 illustrates the synthesis of a di-protected lactam useful in the preparation of tricyclic lactams. Compound 15 is prepared according to the method of Arigon, J., See, US 2013/0289031 entitled Pyrimidinone derivatives, preparation thereof and pharmaceutical use thereof. Compound 15 is protected with a suitable protecting group by directly reacting Compound 15 with di-tert-butyl carbonate (Boc anhydride) in the presence of an organic base such as triethylamine or diisopropylethylamine in an organic solvent such as dichloromethane to form the protected amine 16. The protected amine 16 can be directly reacted with methyl chloroacetate in the presence of a base such as potassium carbonate in an organic solvent such as acetonitrile to form the ester 17. The ester 17 can be cyclized by directly reacting the ester with an acid such as hydrochloric acid in a protic solvent such as methanol optionally at a high temperature to form the spirolactam 18. The lactam 18 can be directly reacted with a protecting reagent such as chloromethyl methyl ether (MOM-C1) in the presence of an organic base such as diisopropylethylamine in an organic solvent such as dichloromethane optionally at a low or at ambient temperature to form the MOM-protected amine 19. The lactam 19 can be protected by directly reacting the lactam with a suitable protecting reagent such as chloromethyl methyl ether (MOM-C1) in the presence of a base such as sodium bis(trimethylsilyl)amide in an organic solvent such as tetrahydrofuran optionally at a low temperature. Additional lactam intermediates such as Compounds 25 and 31 can be synthesized using analogous chemistry as described for the synthesis of Compound 4. The chemistry for the production of Compounds 25 and 31 is illustrated in Schemes 5 and 6.
Scheme 4 illustrates the coupling of a tricyclic lactam sulfone with an amine to generate compounds of Formula I, II, III, and IV.
Scheme 7 illustrates the preparation of the tricyclic lactam compound 33. Compound 32 is prepared according to the method of Tavares, See, US 8,598,186. Compound 32 is directly reacted with sulfone 8 optionally in the presence of an organic base such as lithium hexamethyldisilazane and the amine 32 to form the amine 33. The same chemistry can be employed to produce the alkene compound 34.
In one embodiment a lactam intermediate is treated with BOC-anhydride in the presence of an organic base such as triethylamine in an organic solvent such as dichloromethane. The Boc protected lactam is treated with carbon dioxide in the presence of a nickel catalyst to generate a carboxylic acid. The carboxylic acid is reacted with thionyl chloride in the presence of an organic solvent such as toluene. The resulting acid chloride is treated with an amine to generate an amide that can be deprotected with a strong acid such as trifluoroacetic acid to generate the final target compound.
Alternatively, the lactam can be generated by reacting the carboxylic acid with a protected amine in the presence of a strong acid and a dehydrating agent, which can be together in one moiety as a strong acid anhydride. Examples of strong acid anhydrides include, but are not limited to, trifluoroacetic acid anhydride, tribromoacetic acid anhydride, trichloroacetic acid anhydride, or mixed anhydrides. The dehydrating agent can be a carbodiimide based compound such as but not limited to DCC (Ν,Ν-dicyclohexylcarbodiimide), EDC (l-ethyl-3-(3- dimethylaminopropyl)carbodiimide or DIC (Ν,Ν-diisopropylcarbodiimide). An additional step may be necessary to take off the N-protecting group and the methodologies are known to those skilled in the art
Alternatively, the SMe moiety bonded to the pyrimidine ring can be substituted with any leaving group that can be displaced by a primary amine, for example to create an intermediate for a final product such as Br, I, F, S02Me, SOalkyl, S02alkyl. See, for Example, PCT /US2013/037878 to Tavares.
Other amine intermediates and final amine compounds can be synthesized by those skilled in the art. It will be appreciated that the chemistry can employ reagents that comprise reactive functionalities that can be protected and de-protected and will be known to those skilled in the art at the time of the invention. See for example, Greene, T.W. and Wuts, P.G.M., Greene's Protective Groups in Organic Synthesis, 4th edition, John Wiley and Sons.
Scheme 9
Figure imgf000089_0001
to rt
Figure imgf000089_0002
ACN, rt
R, R = c-pentyl
R,R = H,H
R,R = c-hexyl
R,R = Me, Me
Figure imgf000089_0003
H
Figure imgf000089_0004
R,R = c-hexyl R,R = c-hexyl
R,R = Me, Me R,R = Me, Me
Figure imgf000090_0001
[4-C hlor o-2-(methylthio)pyrimidin-5-yl] methanol.
4-Chloro-2-methylsulfanyl-5-pyrimidinecarboxylate ethyl ester (62 g, 260 mmol) was dissolved in anhydrous tetrahydrofuran (500 mL) in a 3-necked 5L round bottomed flask fitted with a mechanical stirrer, addition funnel, temperature probe and nitrogen inlet. The solution was cooled to 0°C. Diisobutylaluminum hydride in tetrahydrofuran (1M solution, 800 mL) was added dropwise over a period of 2 hours. After the addition was complete, the reaction mixture was kept at 0°C for 0.5 hours. The reaction was quenched at 0°C by the slow addition of saturated aqueous sodium sulfate (265.3 mL, 530.7 mmol) keeping the internal reaction temperature below 10°C. Ethyl acetate (900 mL) was added and the reaction slowly warmed to room temperature overnight. 6M HC1 was added till the reaction mixture was slightly acidic (pH 6). The reaction mixture was filtered thru a pad of Celite® and the aluminum salts were washed with ethyl acetate (1L). The filtrate was poured into a separatory funnel and washed twice with water (600 mL) and finally with brine (600 mL). The organic layer was dried over sodium sulfate, filtered thru Celite® and the solvent concentrated in vacuo to afford 39.2g (77% crude yield) of a dark yellow oil. The material was used as is for the next step. NMR (CDCI3) δ 8.56 (s, 1H), 4.76 (s, 2H), 2.59 (s, 3H); MS (ESI+) for C6H7C1N20S m/z 191.0 (M+H)+ .
Figure imgf000090_0002
4-Chloro-2-(methylthio)pyrimidine-5-carbaldehyde.
[4-Chloro-2-(methylthio)pyrimidin-5-yl]methanol (39.2 g, 206 mmol) was taken up in methylene chloride (520 mL) at room temperature. Manganese(IV) oxide (140 g, 1.60 mol) was added in one portion and the reaction mixture stirred at room temperature overnight. The reaction mixture was filtered through a pad of Celite® and washed with methylene chloride. The filtrate was concentrated under reduced pressure to afford a dark yellow semisolid. The crude product was purified by reverse phase chromatography running a gradient of 1 :9 acetonitrile: water (0.1%TFA) to 100% acetonitrile (0.1%TFA). The desired fractions were combined and the acetonitrile was removed under reduced pressure causing precipitation of the desired product. The solids were removed by filtration and the solids washed with water and dried under vacuum at 50°C. Affords 16.6g (43% yield) of the desired product as a white solid. NMR (CDCI3) δ 10.32 (s, 1H), 8.88 (s, 1H), 2.65 (s, 3H); MS (ESI+) for C6H5C1N20S m/z 189.0 (M+H)+. General Procedure A.
Figure imgf000091_0001
Ethyl 4- [4-chloro-2-(methylthio)pyrimidin-5-yl] -4-hydroxybut-2-ynoate.
Isopropylmagnesium chloride: lithium chloride complex (4.43 g, 30.5 mmol, 25.4mL of a 1.2M solution) was added to tetrahydrofuran (104 mL) in a 500 mL round bottomed flask which had been flame dried and cooled under Argon. The solution was cooled to -15°C. Ethyl propiolate (3.26 mL, 32.1 mmol) was added dropwise affording a yellow solution. Stirring was continued at -15°C for 30 minutes and then 4-Chloro-2-(methylthio)pyrimidine-5-carbaldehyde (6.06 g, 32.1 mmol) in tetrahydrofuran (52 mL) was added rapidly. After 10 minutes, the reaction was quenched by the addition of saturated aqueous ammonium chloride (40 mL). The reaction mixture was warmed to room temperature and poured into a separatory funnel partitioning between ethyl acetate (200 mL) and water (100 mL). The organic layer removed and the aqueous layer extracted with ethyl acetate (100 mL). The combined organic layers were washed with brine (100 mL), dried over sodium sulfate, filtered and the solvent removed in vacuo to afford a dark red oil. The product was purified by silica gel chromatography using a gradient of 1 :4 to 2:3 ethyl acetate :hexanes which afforded 3.76g (37% yield) of the desired product as a light red oil. NMR (CDC13) δ 8.75 (s, 1H), 5.81 (d, 1H, J=6.0Hz), 2.72 (bs, 1H), 2.60 (s, 3H), 1.33 (t, 3H, J=7.2Hz); MS (ESI+) for C11H11CIN2O3S m/z 287.9 (M+H)+ .
Figure imgf000091_0002
tert-Butyl 4- [4-chlor o-2-(methylthio)pyrimidin-5-yl] -4-hydr oxybut-2-ynoate.
Following General Procedure A and using tert-butyl propiolate affords the desired product in 74% yield as a viscous yellow oil. NMR (CDC13) δ 8.75 (s, 1H), 5.79 (d, 1H, J=5.4Hz), 4.27 (q, 2H, J=7.2Hz), 2.97 (d, 1H, J=5.4Hz), 2.60 (s, 3H), 1.52 (s, 9H); MS (ESI+) for Ci3Hi5ClN203S m/z 314.9 (M+H)+ .
General Procedure B.
Figure imgf000092_0001
Ethyl 4- [4-chloro-2-(methylthio)pyrimidin-5-yl] -4-oxobut-2-enoate.
Ethyl 4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-hydroxybut-2-ynoate (3.40 g, 11.8 mmol) was taken up in 1,4-Dioxane (100 mL) at room temperature under argon. Triethylamine (3.3 mL, 24 mmol) was added and the mixture heated to 60°C for lh. The reaction mixture was cooled to room temperature and the solvent removed in vacuo. The resultant dark orange oil was re-evaporated twice with toluene. Affords the desired product (3: 1 E:Z double bond isomers) in 99% yield as a dark orange oil. NMR (CDC13) (major E isomer) δ 8.69 (s, 1H), 7.65 (d, 1H, J=l 8.0Hz), 6.81 (d, 1H, J=18.0Hz), 4.32 (q, 2H, J=6.0Hz), 2.64 (s, 3H), 1.36 (t, 3H, J=6.0Hz); NMR (CDC13) (minor Z isomer) δ 8.87 (s, 1H), 6.89 (d, 1H, J=12.0Hz), 6.23 (d, 1H, J=12.0Hz), 4.14 (q, 2H, J=6.0Hz), 2.63 (s, 3H), 1.23 (t, 3H, J=6.0Hz); MS (ESI+) for CnHnClN203S m/z 287.0 (M+H)+ .
Figure imgf000092_0002
tert-Butyl 4- [4-chloro-2-(methylthio)py rimidin-5-yl] -4-oxobut-2-enoate.
Isomerization of tert-butyl 4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-hydroxybut-2- ynoate using General Procedure B affords the desired product (5: 1 E:Z double bond isomers) in a 99% yield as a viscous dark yellow oil. NMR (CDC13) (major E isomer) δ 8.67 (s, 1H), 7.54 (d, 1H, J=15.6Hz), 6.72 (d, 1H, J=15.6Hz), 2.64 (s, 3H), 1.54 (s, 9H); NMR (CDC13) (minor Z isomer) δ 8.86 (s, 1H), 6.76 (d, 1H, J=12.0Hz), 6.18 (d, 1H, J=12.0Hz), 2.63 (s, 3H), 1.40 (s, 9H); MS (ESI+) for CI3HI5C1N203S m/z 314.9 (M+H)+ . General Procedure C.
Figure imgf000093_0001
Ethyl 2-(methylthio)-5-oxo-8-(l-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8- tetrahydr opyrido [2,3-d] pyrimidine-7-carboxylate.
Ethyl 4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-oxobut-2-enoate (3.40 g, 11.8 mmol) was taken up in acetonitrile (20 mL) at room temperature. 1- {[(Triisopropylsilyl)oxy]methyl}cyclopentanamine (3.86 g, 14.2 mmol) was added followed by triethylamine (3.30 mL, 23.7 mmol) . The mixture was stirred at room temperature overnight. The reaction mixture was transferred to a separatory funnel transferring with ethyl acetate (250 mL). The organic layer was washed twice with a 10% citric acid (aq) (20 mL)/ brine (60 mL) mixture. The organic layer was dried over sodium sulfate, filtered and the solvent removed in vacuo to afford a yellow oil. The product was purified by silica gel chromatography using a gradient from 1 :9 to 2:3 ethyl acetate :hexanes which afforded 2.48g (41% yield) of the desired product as a pale yellow oil. NMR (CDC13) δ 8.58 (s, 1H), 4.72 (m, 1H), 4.46 (m, 1H), 4.16 (q, 2H, J=6.9Hz), 3.52 (m, 1H), 2.96 (m, 2H), 2.54 (s, 3H), 2.31 (m, 3H), 1.81-1.52 (m, 5H), 1.24 (t, 3H, J=6.9Hz) 1.08-0.96 (m, 21H); MS (ESI+) for C26H43N304SSi m/z 522.2 (M+H)+ .
Figure imgf000093_0002
Ethyl 2-(methylthio)-5-oxo-8-(l-{[(triisopropylsilyl)oxy]methyl}cyclohexyl)-5,6,7,8- tetrahydr opyrido [2,3-d] pyrimidine-7-carboxylate.
Cyclization of ethyl 4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-oxobut-2-enoate and 1- {[(triisopropylsilyl)oxy]methyl}cyclohexanamine using General Procedure C afforded the desired product in 17% yield as a pale yellow oil. NMR (CDC13) δ 8.63 (s, 1H), 4.83 (m, 1H), 4.73 (m, 1H), 4.14 (q, 2H, J=6.0Hz), 3.86 (m, 1H), 2.97 (m, 2H), 2.55 (s, 3H), 1.97 (m, 1H), 1.72-1.48 (m, 9H), 1.23 (t, 3H, J=6.0Hz), 1.13-0.95 (m, 21H); MS (ESI+) for C27H45N304SSi m/z 536.2 (M+H)+ .
Figure imgf000094_0001
Ethyl 8-(2-{[tei-i-butyl(dimethyl)silyl]oxy}-l,l-dimethylethyl)-2-(methylthio)-5-oxo-5,6,7,8- tetrahydr opyrido [2,3-i ] pyrimidine-7-carboxylate.
Cyclization of ethyl 4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-oxobut-2-enoate and 1- {[tert-butyl(dimethyl)silyl]oxy}-2-methylpropan-2-amine using General Procedure C afforded the desired product in 52% yield as a pale yellow oil. NMR (CDC13) δ 8.62 (s, 1H), 4.94 (m, 1H), 4.18 (m, 3H), 3.63 (m, 1H), 2.93 (m, 2H), 2.57 (s, 3H), 1.71 (s, 3H), 1.55 (s, 3H), 1.23 (t, 3H, J=7.2Hz), 0.89 (s, 9H), 0.06 (s, 3H), 0.01 (s, 3H); MS (ESI+) for C2iH35N304SSi m/z 454.3 (M+H)+ .
Ethyl 2-(methylthio)-5-oxo-8-{2-[(triisopropylsilyl)oxy]ethyl}-5,6,7,8-tetrahydropyrido[2,3- d] pyrimidine-7-carboxylate.
Cyclization of ethyl 4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-oxobut-2-enoate and 2- [(triisopropylsilyl)oxy]ethanamine using General Procedure C afforded the desired product in 45% yield as a pale yellow oil. NMR (CDC13) δ 8.60 (s, 1H), 4.72 (m, 1H), 4.63 (m, 1H), 4.19 (q, 2H, J=6.0Hz), 3.97 (m, 2H), 3.22 (m, 1H), 3.01 (m, 2H), 2.54 (s, 3H), 1.28 (t, 3H, J=6.0Hz), 1.17-1.02 (m, 21H); MS (ESI+) for C22H37N304SSi m/z 468.1 (M+H)+ .
Figure imgf000095_0001
tert-Butyl 2-(methylthio)-5-oxo-5,6,7,8-tetr ahydr opyrido [2,3-d] pyrimidine-7-carboxylate.
Cyclization of tert-Butyl 4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-oxobut-2-enoate and 0.5 M ammonia/dioxane using General Procedure C afforded the desired product in 60% yield as an off-white solid. NMR (CDC13) δ 8.66 (s, 1H), 6.18 (bs, 1H), 4.34 (m, 1H), 3.05-2.80 (m, 2H), 2.56 (s, 3H), 1.51 (s, 9H); MS (ESI+) for Ci3Hi7N303S m/z 296.0 (M+H)+ .
General Procedure D.
Figure imgf000095_0002
2-(Methylthio)-5-oxo-8-(l-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8- tetrahydr opyrido [2,3-d] pyrimidine-7-carboxylic acid.
Ethyl 2-(methylthio)-5-oxo-8-( 1 - { [(triisopropylsilyl)oxy]methyl} cyclopentyl)-5 ,6,7,8- tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate (2.48 g, 4.75 mmol) was taken up in tetrahydrofuran (10 mL) and acetonitrile (10 mL) at room temperature. 1M Sodium hydroxide (10 mL, 10 mmol) was added at room temperature for 1 hour. The reaction was quenched by the addition of 10% citric acid till pH ca 6-7. The reaction mixture was transferred to a separatory funnel with water (30 mL) and ethyl acetate (150 mL). The aqueous layer was removed and the organic layer washed with brine (50 mL). The organic layer was dried over sodium sulfate, filtered and the solvent concentrated in vacuo to afford the 2.08g (89%> yield) of the desired product as a dark yellow oil. NMR (CDC13) δ 8.46 (s, 1H), 4.58 (m, 1H), 4.39 (m, 1H), 3.71 (m, 1H), 2.88 (m, 2H), 2.51 (s, 3H), 2.26 (m, 3H), 1.97-1.45 (m, 6H) 1.12-0.92 (m, 21H); MS (ESI+) for C24H39N304SSi m/z 494.2 (M+H)+.
Figure imgf000096_0001
2-(Methylthio)-5-oxo-8-(l-{[(triisopropylsilyl)oxy]methyl}cyclohexyl)-5,6,7,8- tetr ahydropyrido [2,3-d] pyrimidine-7-carboxylic acid.
Saponification of ethyl 2-(methylthio)-5-oxo-8-(l-
{[(triisopropylsilyl)oxy]methyl}cyclohexyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimi
carboxylate using General Procedure D affords the desired product in 95% yield as a dark yellow oil. NMR (CDCI3) δ 8.58 (s, 1H), 4.75 (m, 1H), 4.53 (m, 1H), 3.96 (m, 1H), 2.99 (m, 2H), 2.54 (s, 3H), 1.93-1.48 (m, 10H), 1.13-0.95 (m, 21H); MS (ESI+) for C25H41N3O4SS1 m/z 508.1 (M+H)+ .
Figure imgf000096_0002
8-(2-{[tert-Butyl(dimethyl)silyl]oxy}-l,l-dimethylethyl)-2-(methylthio)-5-oxo-5,6,7,8- tetr ahydropyrido [2,3-d] pyrimidine-7-carboxylic acid.
Saponification of ethyl 8-(2-{[fert-butyl(dimethyl)silyl]oxy}-l , l-dimethylethyl)-2- (methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-(i]pyrimidine-7-carboxylate using General Procedure D affords the desired product in 99% yield as a pale yellow foam. NMR (CDCI3) δ 8.69 (s, 1H), 4.88 (m, 1H), 4.51 (m, 1H), 3.82 (m, 1H), 3.17 (m, 1H), 2.79 (m, 1H), 2.57 (s, 3H), 1.65 (s, 3H), 1.60 (s, 3H), 0.92 (s, 9H), 0.1 1 (s, 6H); MS (ESI+) for
Figure imgf000096_0003
m/z 426.3 (M+H)+ .
Figure imgf000097_0001
2-(Methylthio)-5-oxo-8-{2-[(triisopropylsilyl)oxy]ethyl}-5,6,7,8-tetrahydropyrido[2,3- d]pyrimidine-7-carboxylic acid.
Saponification of ethyl 2-(methylthio)-5-oxo-8-{2-[(triisopropylsilyl)oxy]ethyl}-5,6,7,8- tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate using General Procedure D affords the desired product in 98% yield as an orange foam. NMR (CDC13) δ 8.56 (s, 1H), 4.71 (m, 1H), 4.54 (m, 1H), 3.99 (m, 2H), 3.32 (m, 1H), 3.01 (m, 2H), 2.53 (s, 3H), 1.16-0.98 (m, 21H); MS (ESI+) for C20H33N3O4SS1 m/z 440.2 (M+H)+ .
Figure imgf000097_0002
2-(Methylthio)-5-oxo-5,6,7,8-tetrahydr opyrido [2,3-d] pyrimidine-7-carboxylic acid.
tert-Butyl 2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate (220 mg,0.74 mmol) was taken up in trifluoroacetic acid (5 mL) at room temperature under argon. The mixture was stirred at room temperature for 45 minutes. The solvent was removed in vacuo to a pink oil which was re-evaporated first from toluene and finally methanol affording 180 mg (99% yield) of the desired product as an off-white solid. NMR (MeOH-d4) δ 8.49 (s,
1H), 4.59 (m, 1H), 3.16-2.91 (m, 2H), 2.63 (s, 3H); MS (ESI+) for C9H9N3O3S m/z 240.0
(M+H)+ .
General Procedure E.
Figure imgf000098_0001
N-(2,4-Dimethoxybenzyl)-2-(methylthio)-5-oxo-8-(l-
{ [(triisopropylsilyl)oxy] methyl} cyclopentyl)-5,6,7,8-tetrahydropyrido [2,3-d] pyrimidine-7- carboxamide.
2-(Methylthio)-5-oxo-8-(l-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8- tetrahydropyrido[2,3-d]pyrimidine-7-carboxylic acid (2.08 g, 4.21 mmol) was taken up in N,N- dimethylformamide (30 mL) at room temperature. l-(2,4-dimethoxyphenyl)methanamine (1.26 mL, 8.42 mmol) was added followed by the addition of N,N,N',N'-tetramethyl-0-(7- azabenzotriazol-l-yl)uronium hexafluorophosphate (4.80g, 12.6 mmol) and N,N- diisopropylethylamine (4.40 mL, 25.3 mmol). The reaction mixture was stirred overnight at room temperature. The product was diluted with water (50 mL) and poured into a separatory funnel. The mixture was extracted with twice with ethyl acetate (150 mL) and the combined organic layers were thrice washed with half-saturated aqueous LiCl (20 mL). The combined organic layers were dried over sodium sulfate, filtered and the solvent removed in vacuo to afford a dark yellow oil. The product was purified by silica gel chromatography using a gradient from 1 :4 to 2:3 ethyl acetate:hexanes which afforded 2.39g (88% yield) of the desired product as a brown sticky solid. NMR (CDC13) δ 8.58 (s, 1H), 7.07 (m, 1H), 6.62 (m, 1H), 6.39 (m, 2H), 4.56 (m, 1H), 4.38 (m, 2H), 4.20 (m, 1H), 3.80 (s, 3H), 3.66 (s, 3H), 3.48 (m, 1H), 3.02 (m, 2H), 2.55 (s, 3H), 2.35 (m, 2H), 2.06 (m, 1H), 1.70-1.31 (m, 5H), 1.09-0.90 (m, 21H); MS (ESI+) for C33H50N4O5SSi m/z 643.2 (M+H)+ .
Figure imgf000099_0001
N-(2,4-Dimethoxybenzyl)-2-(methylthio)-5-oxo-8-(l-
{[(triisopropylsilyl)oxy]methyl}cyclohexyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7- carboxamide.
Coupling reaction of 2-(methylthio)-5-oxo-8-(l-
{[(triisopropylsilyl)oxy]methyl}cyclohexyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimi
carboxylic acid using General Procedure E afforded the desired product in 52% yield as a yellow solid. NMR (CDC13) δ 8.60 (s, 1H), 6.87 (m, 2H), 6.37 (m, 2H), 4.71 (m, 1H), 4.49-4.14 (m, 4H), 3.79 (s, 3H), 3.70 (s, 3H), 3.18 (m, 1H), 2.86 (m, 1H), 2.65 (m, 1H), 2.54 (s, 3H), 2.20 (m, 1H), 1.98 (m, 2H), 1.61-1.48 (m, 6H), 1.08-0.92 (bs, 21H); MS (ESI+) for C34H52N405SSi m/z 651.2 (M+H)+ .
Figure imgf000099_0002
8-(2-{[tei-i-Butyl(dimethyl)silyl]oxy}-l,l-dimethylethyl)- V-(2,4-dimethoxybenzyl)-2- (methylthio)-5-oxo-5,6,7,8-tetr ahydropyrido [2,3-i ] pyrimidine-7-carboxamide.
Coupling reaction of 8-(2-{[tert-butyl(dimethyl)silyl]oxy}-l,l-dimethylethyl)-2- (methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylic acid using General Procedure E afforded the desired product in 86% yield as a viscous yellow oil. NMR (CDC13) δ 8.58 (s, 1H), 6.95 (m, 1H), 6.80 (m, 1H), 6.37 (m, 2H), 4.71 (m, 1H), 4.27 (m, 2H), 4.13 (m, 1H), 3.84 (m, 1H), 3.79 (s, 3H), 3.70 (s, 3H), 3.15 (m, 1H), 2.77 (m, 1H), 2.56 (s, 3H), 1.64 (s, 3H), 1.58 (s, 3H), 0.85 (s, 9H), 0.02 (s, 3H), -0.03 (s, 3H); MS (ESI+) for C28H42N405SSi m/z 575.4 (M+H)+ .
Figure imgf000100_0001
N-(2,4-Dimethoxybenzyl)-2-(methylthio)-5-oxo-8-{2-[(triisopropylsilyl)oxy]ethyl}-5,6,7,8- tetrahydr opyrido [2,3-d] pyrimidine-7-carboxamide.
Coupling reaction of 2-(methylthio)-5-oxo-8-{2-[(triisopropylsilyl)oxy]ethyl}-5,6,7,8- tetrahydropyrido[2,3-d]pyrimidine-7-carboxylic acid using General Procedure E afforded the desired product in 57% yield as a dark yellow solid. NMR (CDC13) δ 8.59 (s, 1H), 7.06 (m, 1H), 6.39 (m, 3H), 4.51 (m, 2H), 4.32 (m, 2H), 3.93 (m, 2H), 3.81 (s, 3H), 3.73 (s, 3H), 3.18 (m, 1H), 3.01 (m, 2H), 2.54 (s, 3H), 1.11-0.95 (m, 21H); MS (ESI+) for C29H44N405SSi m/z 589.4 (M+H)+ .
Figure imgf000100_0002
2-(Methylthio)-5-oxo-N-(l-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8- tetr ahydropyrido [2,3-d] pyrimidine-7-carboxamide.
Coupling reaction of 2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7- carboxylic acid using General Procedure E afforded the desired product in 94% yield as a dark yellow oil. NMR (CDC13) δ 8.64 (s, 1H), 6.09 (bs, 1H), 6.04 (bs, 1H), 4.25 (m, 1H), 3.68 (m, 2H), 2.86 (m, 2H), 2.54 (s, 3H), 2.07-1.54 (m, 8H), 1.33-0,96 (m, 21H); MS (ESI+) for C24H4oN403SSi m/z 493.1 (M+H)+ .
General Procedure F.
Figure imgf000101_0001
N-(2,4-Dimethoxybenzyl)-8-[l-(hydroxymethyl)cyclopentyl]-2-(methylthio)-5-oxo-5,6,7,8- tetrahydr opyrido [2,3-d] pyrimidine-7-carboxamide.
N-(2,4-Dimethoxybenzyl)-2-(methylthio)-5-oxo-8-(l -
{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7- carboxamide (2.39 g, 3.72 mmol) was taken up in tetrahydrofuran (42 mL) at room temperature. Tetra-n-butylammonium fluoride (5.6 mL, 5.6 mmol, 1M solution in THF) was added and the reaction stirred for 10 minutes at room temperature. The reaction mixture was concentrated in vacuo to an orange oil and was transferred to a separatory funnel and partitioned between ethyl acetate (200 mL) and water (50 mL). The aqueous layer was removed and the organic layer washed with water (50 mL) and brine (50 mL). The organic layer was dried over sodium sulfate, filtered and the solvent removed in vacuo to afford a dark yellow semi-solid. The product was purified by reverse phase chromatography using a gradient from 1 :9 to 3:2 acetonitrile: water (0.1% TFA). Lyophilization of the desired fractions afforded 1.8 lg (99% yield) of the desired product as a dark yellow powder. NMR (CDC13) δ 8.59 (s, 1H), 7.21 (m, 1H), 7.02 (m, 1H), 6.39 (m 2H), 4.56 (m, 1H), 4.29 (m, 2H), 3.79 (s, 3H), 3.75 (s, 3H), 3.70 (m, 2H), 3.41 (m, 1H), 3.20 (m, 1H), 2.87 (m, 1H), 2.55 (s, 3H), 2.18 (m, 1H), 1.97-1.59 (m, 7H); MS (ESI+) for C24H3oN405S m/z 487.1 (M+H)+ .
Figure imgf000102_0001
N-(2,4-Dimethoxybenzyl)-8-[l-(hydroxymethyl)cyclohexyl]-2-(methylthio)-5-oxo-5,6,7,8- tetr ahydropyrido [2,3-d] pyrimidine-7-carboxamide.
Desilylation of N-(2,4-dimethoxybenzyl)-2-(methylthio)-5-oxo-8-(l -
{[(triisopropylsilyl)oxy]methyl}cyclohexyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrim
carboxamide using General Procedure F afforded the desired product in 97% yield as a yellow powder. NMR (CDC13) δ 8.69 (s, 1H), 7.58 (m, 1H), 7.07 (m, 1H), 6.48 (m, 2H), 4.72 (m, 2H), 4.38 (m, 2H), 3.89 (s, 3H), 3.87 (s, 3H), 3.31 (m, 1H), 2.99 (m, 1H), 2.81 (m, 1H), 2.64 (s, 3H), 2.11 (m, 3H), 1.94-1.58 (m, 7H); MS (ESI+) for C25H32N405S m/z 501.1 (M+H)+ .
Figure imgf000102_0002
8-(2-Hydroxy-l,l-dimethylethyl)-N-(4-methoxybenzyl)-2-(methylthio)-5-oxo-5,6,7,8- tetr ahydropyrido [2,3-d] pyrimidine-7-carboxamide.
Desilylation of 8-(2- { [tert-butyl(dimethyl)silyl]oxy } -1,1 -dimethylethyl)-N-(2,4- dimethoxybenzyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3- ]pyrimidine-7- carboxamide using General Procedure F afforded the desired product in 98% yield as a yellow powder. NMR (CDC13) δ 8.61 (s, 1H), 7.51 (m, 1H), 7.01 (m, 1H), 6.37 (m, 2H), 4.72 (m, 1H), 4.65 (m, 1H), 4.26 (m, 2H), 3.80 (s, 3H), 3.76 (s, 3H), 3.63 (m, 1H), 3.18 (m, 1H), 2.80 (m, 1H), 2.57 (s, 3H), 1.58 (s, 3H), 1.56 (s, 3H); MS (ESI+) for C22H28N405S m/z 461.4 (M+H)+ .
Figure imgf000103_0001
N-(2,4-Dimethoxybenzyl)-8-(2-hydroxyethyl)-2-(methylthio)-5-oxo-5,6,7,8- tetr ahydropyrido [2,3-d] pyrimidine-7-carboxamide.
Desilylation of N-(2,4-dimethoxybenzyl)-2-(methylthio)-5-oxo-8- {2-
[(triisopropylsilyl)oxy]ethyl}-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide using General Procedure F afforded the desired product in 97% yield as a pale yellow powder. NMR (CDCls) δ 8.59 (s, 1H), 7.02 (m, 1H), 6.69 (m, 1H), 6.43 (m, 2H), 4.34 (m, 3H), 3.88 (m, 4H), 3.81 (s, 3H), 3.78 (s, 3H), 3.02 (m, 2H), 2.55 (s, 3H); MS (ESI+) for C2oH24N405S m/z 432.9 (M+H)+ .
Figure imgf000103_0002
N- [l-(Hydroxymethyl)cyclopentyl] -2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido [2,3- d] pyrimidine-7-carboxamide.
Desilylation of 2-(methylthio)-5-oxo-N-(l- {[(triisopropylsilyl)oxy]methyl}cyclopentyl)- 5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide using General Procedure F afforded the desired product in 31% yield as an off white powder. NMR (CDC13) δ 8.66 (s, 1H), 6.19 (bs, 1H), 5.97 (bs, 1H), 4.31 (m, 1H), 3.69 (s, 2H), 2.92 (m, 2H), 2.56 (s, 3H), 1.95-1.65 (m, 9H); MS (ESI+) for Ci5H2oN403S m/z 337.0 (M+H)+ .
General Procedure G.
Figure imgf000104_0001
8 '-(2,4-Dimethoxybenzyl)-2 '-(methylthio)-6 ',6a',8 ',9 '-tetr ahydrospir o [cyclopentane- 1,10'- yrazinoIl'^'rl^l yridoIl^-dl yrimidinel-S'^'-dione.
N-(2,4-Dimethoxybenzyl)-8-[ 1 -(hydroxymethyl)cyclopentyl]-2-(methylthio)-5-oxo-
5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide (1.81 g, 3.72 mmol) was taken up in methylene chloride (30 mL) at room temperature. Triethylamine (1.3 mL, 9.3 mmol) was added followed by the rapid addition of methanesulfonyl chloride (0.43 mL, 5.6 mmol). The reaction mixture was stirred at room temperature for 30 minutes before being heated at reflux overnight. The solvent was removed in vacuo affording a brown semi-solid. The product was purified by reverse phase chromatography using a gradient from 1 :9 to 3:2 acetonitrile: water (0.1% TFA). Lyophilization of the desired fractions gave 764 mg (44%> yield) of the desired product as a light brown powder. NMR (CDC13) δ 8.49 (s, 1H), 7.13 (m, 1H), 6.43 (m, 2H), 5.49 (m, 1H), 4.37- 4.16 (m, 4H), 3.81 (s, 3H), 3.80 (s, 3H), 3.29 (m, 1H), 2.95 (m, 1H), 2.82 (s, 3H), 2.41 (m, 1H), 1.99-1.52 (m, 7H); MS (ESI+) for C24H28N4O4S m/z 469.1 (M+H)+ .
Figure imgf000104_0002
8 '-(2,4-Dimethoxybenzyl)-2 '-(methylthio)-6 ',6a',8 ',9 '-tetr ahydrospir 0 [cyclohexane-1 , 10'- pyrazinoIl'^'rl^lpyrido^^-dlpyrimidinel-S'^'-dione.
Mesylation and cyclization of N-(2,4-dimethoxybenzyl)-8-[l-
(hydroxymethyl)cyclohexyl]-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7- carboxamide using General Procedure G afforded the desired product in 73 %> yield as a white powder. NMR (MeOH-d4) δ 8.48 (s, 1H), 7.14 (m, 1H), 6.43 (m, 1H), 6.39 (m, 1H), 5.61 (m, 1H), 4.31 (m, 1H), 4.21 (m, 2H), 3.83 (s, 3H), 3.80 (s, 3H), 3.38 (m, 1H), 3.22 (m, 1H), 2.95 (m, 1H), 2.82 (s, 3H), 2.22 (m, 1H), 1.99- 1.09 (m, 9H); MS (ESI+) for C^HSO^C S m/z 483.1 (M+H)+ .
Figure imgf000105_0001
8-(2,4-Dimethoxybenzyl)-10,10-dimethyl-2-(methylthio)-6,6a,9,10-tetrahydro-5H- pyrazino [1 ',2 ' : 1 ,6] pyrido [2,3-d] pyrimidine-5,7(8H)-dione.
Mesylation and cyclization of 8-(2-Hydroxy-l,l-dimethylethyl)-N-(4-methoxybenzyl)-2- (methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide using General Procedure G afforded the desired product in 82% yield as a yellow powder. NMR (CDCI3) δ 8.45 (s, 1H), 7.14 (m, 1H), 6.40 (m, 2H), 5.56 (m, 1H), 4.40 (m, 1H), 4.31 (m, 2H), 4.13 (m, 1H), 3.81 (s, 3H), 3.79 (s, 3H), 3.28 (m, 1H), 2.88 (m, 1H), 2.79 (s, 3H), 1.80 (s, 3H), 1.45 (s, 3H); MS (ESI+) for C22H26N4O4S m/z 443.5 (M+H)+ .
Figure imgf000105_0002
8-(2,4-Dimethoxybenzyl)-2-(methylthio)-6,6a,9, 10-tetrahydro-5H- pyrazino [1 ',2 ' : 1 ,6] pyrido [2,3-d] pyrimidine-5,7(8H)-dione.
Mesylation and cyclization of N-(2,4-dimethoxybenzyl)-8-(2-hydroxyethyl)-2- (methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide using General Procedure G afforded the desired product in 81% yield as a light brown powder. NMR (CDCI3) δ 8.52 (s, 1H), 7.11 (m, 1H), 6.44 (m, 2H), 5.31 (m, 1H), 4.68-4.29 (m, 5H), 4.04 (m, 1H), 3.81 (s, 3H), 3.80 (s, 3H), 3.20 (m, 1H), 3.01 (m, 1H), 2.82 (s, 3H); MS (ESI+) for C20H22N4O4S m/z 415.0 (M+H)+ . General Procedure H.
Figure imgf000106_0001
2,-(Methylthio)-6,,6a,,8,,9,-tetrahydrospiro[cyclopentane-l,10'- yrazinoIl'^'rl^l yridoIl^-dl yrimidinel-S'^'-dione.
8'-(2,4-Dimethoxybenzyl)-2'-(methylthio)-6',6a',8',9'-tetrahydrospiro[cyclopentane- 1,10'- pyrazino[ ,2': l,6]pyrido[2,3-d]pyrimidine]-5',7'-dione (764 mg, 1.63 mmol) was taken up in trifluoroacetic acid (10 mL) at room temperature under argon. The mixture was heated to 75 °C for 6 hours, cooled to room temperature and left to stir overnight. The solvent was removed in vacuo to afford a purple oil. The product was purified by reverse phase chromatography using a gradient from 100% water (0.1% TFA) to 1 : 1 acetonitrile: water (0.1% TFA). Lyophilization of the desired fractions afforded 117 mg (23% yield) of the desired product as a pale yellow powder. NMR (CDC13) δ 8.52 (s, 1H), 5.58 (m, 2H), 4.34 (bs 2H), 3.29 (m, 1H), 3.06 (m, 1H), 2.84 (s, 3H), 2.48 (m, 1H), 2.31 (m, 1H), 2.11-1.65 (m, 6H); MS (ESI+) for Ci5Hi8N402S m/z 319.0 (M+H)+ .
Figure imgf000106_0002
2'-(Methylthio)-6,,6a,,8,,9,-tetrahydrospiro[cyclohexane-l,10'- pyrazinoIl'^'rl^lpyridoIl^-dlpyrimidinel-S'^'-dione.
Removal of the dimethoxybenzyl group of 8'-(2,4-Dimethoxybenzyl)-2'-(methylthio)- 6',6a',8',9'-tetrahydrospiro[cyclohexane-l,10'-pyrazino[ ,2^ 1,6]pyrido[2,3-d]pyrimidine]-5',7'- dione using General Procedure H afforded the desired product in 23% yield as a white powder. NMR (MeOH-d4) δ 8.60 (s, 1H), 4.78 (m, 1H), 4.54 (m, 2H), 3.38 (m, 1H), 2.86 (s, 3H), 2.84 (m, 1H), 2.12-1.30 (m, 10H); MS (ESI+) for Ci6H2oN402S m/z 333.1 (M+H)+ .
Figure imgf000107_0001
10,10-Dimethyl-2-(methylthio)-6,6a,9,l 0-tetr ahydr o-5H-pyr azino [ 1 ',2 ' : 1 ,6] pyrido [2,3- d] pyrimidine-5,7(8H)-dione.
Removal of the dimethoxybenzyl group of 8-(2,4-dimethoxybenzyl)-10,10-dimethyl-2- (methylthio)-6,6a,9,10-tetrahydro-5H-pyrazm^
using General Procedure H afforded the desired product in 56% yield as a white powder. NMR (CDCls) δ 8.54 (s, 1H), 5.65 (m, 1H), 5.51 (bs, 1H), 4.31 (s, 2H), 3.23 (m, 1H), 3.04 (m, 1H), 2.85 (s, 3H), 1.78 (s, 3H), 1.68 (s, 3H); MS (ESI+) for Ci3Hi6N402S m/z 293.2 (M+H)+ .
Figure imgf000107_0002
2-(Methylthio)-6,6a,9,l 0-tetr ahydro-5H-pyrazino [1 ',2 ' : 1 ,6] pyrido [2,3-i ] pyrimidine- 5,7(8H)-dione.
Removal of the dimethoxybenzyl group of 8-(2,4-dimethoxybenzyl)-2-(methylthio)- 6,6a,9, 10-tetrahydro-5H-pyrazino[ ,2': l ,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dione using General Procedure H afforded the desired product in 21% yield as a brown powder. NMR (MeOH-d4) δ 8.65 (s, 1H), 4.78-4.05 (m, 5H), 3.32 (m, 2H), 3.01 (m, 1H), 2.87 (s, 3H); MS (ESI+) for CiiHi2N402 m/z 265.0 (M+H)+ .
eneral Procedure I.
Figure imgf000108_0001
2,-{[5-(4-Methylpiperazin-l-yl)pyridin-2-yl]amino}-6',6a,,8,,9'- tetrahydr ospiro [cyclopentane- 1 ,10 '-pyrazino [1 ',2 ': 1 ,6] pyrido [2,3-d] pyrimidine] -5 ',7'-dione.
2'-(Methylthio)-6',6a',8',9'-tetrahydrospiro[cyclopentane- 1 ,10'- pyrazino[ ,2': l ,6]pyrido[2,3-d]pyrimidine]-5',7'-dione (1 17 mg, 0.367 mmol) was taken up in N,N-dimethylacetamide (4.0 mL, 43 mmol) at room temperature under argon. 5-(4- methylpiperazin-l-yl)pyridin-2-amine (100 mg, 0.55 mmol) was added and the reaction mixture was heated just to 150°C and then immediately removed from the heat and cooled to room temperature. The product was purified by reverse phase chromatography using a gradient from 100% Water (0.1% TFA) to 1 : 1 acetonitrile: water (0.1% TFA). Lyophilization of the desired fractions afforded 1 1 mg (7% yield) of the desired product as an orange powder. NMR (MeOH- d4) δ 8.54 (s, 1H); 8.06 (m, 1H), 7.85 (m, 1H), 7.69 (m, 1H), 4.69 (m, 1H), 4.39 (m, 2H), 3.95 (m, 2H), 3.69 (m, 2H), 3.39-3.15 (m, 5H), 3.02 (s, 3H), 2.77 (m, 1H), 2.21 (m, 1H), 2.09-1.67 (m, 7H); MS (ESI+) for C24H3oN802 m/z 463.1 (M+H)+ .
Figure imgf000108_0002
2,-{[5-(4-Methylpiperazin-l-yl)pyridin-2-yl]amino}-6',6a,,8,,9'- tetrahydr ospiro [cyclohexane- 1 ,10 '-pyrazino [ 1 ',2 ' : 1 ,6] pyrido [2,3-d] pyrimidine] -5 ',7'-dione.
ΞΝΑΓ reaction using General Procedure I and 2'-(methylthio)-6',6a',8',9'- tetrahydrospiro [cyclohexane- 1 , 10'-pyrazino[ 1 ',2': 1 ,6]pyrido[2,3-d]pyrimidine]-5',7'-dione afforded the desired product in 38% yield as a yellow powder. NMR (MeOH-d4) δ 8.52 (s, 1H), 8.07 (m, 1H), 7.89 (bs, 1H), 7.71 (m, 1H), 4.71 (m, 1H), 4.40 (m, 2H), 3.94 (m, 2H), 3.68 (m, 2H), 3.35-3.22 (m, 5H), 3.02 (s, 3H), 2.73 (m, 1H), 2.02-1.25 (m, 10H); MS (ESI+) for C25H32N802 m/z All 2 (M+H)+ .
Figure imgf000109_0001
10,10-Dimethyl-2- { [5-(4-methylpiperazin- l-yl)pyridin-2-yl] amino}-6,6a,9,l 0-tetr ahydro- 5H-pyr azino [ 1 ',2 ' : 1 ,6] pyrido [2,3-d] pyrimidine-5,7(8H)-dione.
SNAr reaction using General Procedure I and 10,10-dimethyl-2-(methylthio)-6,6a,9,10- tetrahydro-5H-pyrazino[ ,2': l,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dione afforded the desired product in 10% yield as a yellow powder. NMR (MeOH-d4) δ 8.52 (s, 1H), 8.05 (m, 2H), 7.86 (m, 1H), 7.67 (m, 1H), 4.66 (m, 1H), 4.33 (m, 2H), 3.93 (m, 2H), 3.68 (m, 2H), 3.38-3.21 (m, 5H), 3.01 (s, 3H), 2.72 (m, 1H), 1.65 (s, 3H), 1.54 (s, 3H); MS (ESI+) for C22H28N8O2 m/z 437.4 (M+H)+ .
Figure imgf000109_0002
2-{[5-(4-methylpiperazin-l-yl)pyridin-2-yl]amino}-6,6a,9,10-tetrahydro-5H- pyrazino [1 ',2 ' : 1 ,6] pyrido [2,3- \ pyrimidine-5,7(8H)-dione.
SNAr reaction using General Procedure I and 2-(methylthio)-6,6a,9,10-tetrahydro-5H- pyrazino[l\2^ 1,6]pyrido[2,3-(i]pyrimidine-5,7(8H)-dione afforded the desired product in 15% yield as an orange powder. NMR (DMSO-d6) δ 8.41 (s, 1Η), 8.05 (m,lH), 7.93 (m, 1H), 7.82 (m, 1H), 4.59-4.34 (m, 3H), 4.03-3.84 (m, 4H), 3.49 (m, 2H), 3.30-3.09 (m, 6H), 2.80 (s, 3H), 2.80-2.67 (m, 2H); MS (ESI+) for C2oH24N802 m/z 409.1 (M+H)+ . eneral Procedure J.
Figure imgf000110_0001
2 '- { [5-(4-Methylpiper azin- l-yl)pyridin-2-yl] am
dispiro[cyclohexane-l,10'-pyrazino[1^2,:l,6]pyrido[2,3-d]pyrimidine-5,,2M-[l,3]dithian]-7'- one.
2'- {[5-(4-Methylpiperazin- 1 -yl)pyridin-2-yl]amino} -6',6a',8',9'- tetrahydrospiro[cyclohexane- 1 , 10'-pyrazino[ 1 ',2': 1 ,6]pyrido[2,3-d]pyrimidine]-5',7'-dione (90.0 mg, 0.189 mmol) and 1,3-propanedithiol (0.0379 mL, 0.378 mmol) were taken up in toluene (5 mL) at room temperature under argon. p-Toluenesulfonic acid (0.02 g, O. lmmol) was then added. The reaction vessel was fitted with a condenser and the reaction mixture heated at reflux overnight. The reaction mixture was cooled to room temperature and the solvent removed in vacuo affording a thick dark yellow oil. The product was purified by reverse phase chromatography using a gradient from 100% water (0.1% TFA) to 3 :2 acetonitrile: water (0.1% TFA). Lyophilization of the desired fractions afforded 35 mg (33% yield) of the desired product as a pale yellow powder. NMR (MeOH-d4) δ 8.52 (s, 1H), 7.90 (m, 1H), 7.84 (m, 1H), 7.52 (m, 1H), 4.64 (m, 1H), 4.53 (m, 1H), 4.16 (m, 1H), 3.60 (m, 2H), 3.41-3.26 (m, 6H), 3.01 (s, 3H), 2.91 (m, 1H), 2.75 (m, 1H), 2.61 (m, 1H), 2.21 (m, 1H), 2.11 (m, 1H), 1.95-1.72 (m, 10H), 1.61 (m, 1H), 1.33 (m 2H); MS (ESI+) for CzsHssNsOSz m/z 567.1 (M+H)+ .
Figure imgf000110_0002
10 10'-Dimethyl-2,-{[5-(4-methylpiperazin-l-yl)pyridin-2-yl]amino}-6,,6a,,9,,10'- tetrahydr ospiro [ 1 ,3-dithiane-2,5 '-pyrazino [ 1 ',2 ' : 1 ,6] pyrido [2,3-i ] pyrimidin] -7'(8 'H)-one.
Dithiane formation using General Procedure J and 10,10-dimethyl-2-{[5-(4- methylpiperazin-1 -yl)pyridin-2-yl] amino} -6,6a,9, 10-tetrahydro-5H- pyrazino[ ,2': l,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dione afforded the desired product in 43% yield as an orange powder. NMR (MeOH-d4) δ 8.53 (s, 1H), 7.88 (m, 1H), 7.82 (m, 1H), 7.47 (m, 1H), 4.47 (m, 1H), 4.41 (m, 1H), 4.16 (m, 1H), 3.92-3.15 (m, 11H), 3.00 (s, 3H), 2.90-2.81 (m, 3H), 2.21 (m, 1H), 1.87 (m, 1H), 1.60 (s, 3H), 1.48 (s, 3H); MS (ESI+) for C25H34N8OS2 m/z 527.1 (M+H)+ .
General Procedure K.
Figure imgf000111_0001
2,-{[5-(4-Methylpiperazin-l-yl)pyridin-2-yl]amino}-6',6a,,8,,9'- tetrahydr ospiro [cyclohexane- 1 ,10 '-pyrazino [ 1 ',2 ' : 1 ,6] pyrido [2,3-d] pyrimidin] -7'(5 'H)-one.
2'-{[5-(4-Methylpiperazin-l-yl)pyridin-2-yl]amino}-6',6a',8',9'-tetrahydro-7'H- dispiro [cyclohexane- 1 , 10'-pyrazino[ 1 ',2': 1 ,6]pyrido[2,3-d]pyrimidine-5',2"-[ 1 ,3]dithian]-7'-one (35 mg, 0.062 mmol) in ethanol (1ml) was added to Raney nickel (1 mL of the aqueous slurry which was washed thrice with ethanol decanting off the ethanol after each washing) in ethanol (3 mL) under argon. The reaction mixture was heated to 45 °C for 30 minutes. After cooling to room temperature, the reaction mixture was filtered through a pad of Celite® washing with ethanol. The solvent was removed in vacuo affording a yellow oil. The product was purified by reverse phase chromatography using a gradient from 100% water (0.1% TFA) to 3:2 acetonitrile: water (0.1% TFA). Lyophilization of the desired fractions afforded 4 mg (14% yield) of the desired product as a pale yellow powder. NMR (CDC13) δ 7.95 (m, 1H), 7.93 (s, 1H), 7.77 (m, 1H), 7.51 (m, 1H), 4.57 (m, 1H), 4.51 (m, 1H), 4.35 (m, 1H), 3.89 (m, 2H), 3.69 (m, 2H), 3.37 (m, 2H), 3.15 (m, 2H), 3.01 (s, 3H), 2.79 (m, 1H), 2.58 (m, 1H), 2.39 (m, 1H), 2.05-1.45 (m, 11H); MS (ESI+) for C25H34N80 m/z 463.1 (M+H)+ .
Figure imgf000112_0001
10,10-Dimethyl-2- { [5-(4-methylpiperazin- l-yl)pyridin-2-yl] amino}-6,6a,9,l 0-tetr ahydro- 5H-pyr azino [ 1 ',2 ' : 1 ,6] pyrido [2,3-d] pyrimidin-7(8H)-one.
Desulfurization using General Procedure K and 10',10'-dimethyl-2'-{[5-(4- methylpiperazin- 1 -yl)pyridin-2-yl] amino} -6',6a',9', 10'-tetrahydrospiro[l ,3-dithiane-2,5'- pyrazino[l\2^ 1,6]pyrido[2,3-(i]pyrimidin]-7'(8'H)-one afforded the desired product in 11% yield as a yellow powder. NMR (MeOH-d4) δ 7.93 (m, 2H), 7.75 (m, 1H), 7.43 (m, 1H), 4.52 (m, 1H), 4.32 (m, 2H), 3.89 (m, 2H), 3.67 (m, 2H), 3.38 (m, 2H), 3.14 (m, 2H), 3.01 (s, 3H), 2.79 (m, 1H), 2.60 (m, 1H), 2.39 (m, 1H), 2.00 (m, 1H), 1.55 (s, 3H), 1.54 (s, 3H); MS (ESI+) for C22H30N8O m/z 423.1 (M+H)+ .
General Procedure L.
Figure imgf000112_0002
(l-Aminocyclohexyl)methanol.
2M Lithium tetrahydroaluminate in tetrahydrofuran(80.0 mL, 160 mmol) was charged into a 500 mL 3 -necked round bottomed flask (oven-dried and cooled under argon) fitted with a magnetic stir bar and the solution was cooled to 0°C under argon. 1- Aminocyclohexanecarboxylic acid (7.64g, 53.3 mmol) is added portionwise over a period of 1 hour. At the end of the addition, the reaction mixture was diluted with tetrahydrofuran (60 mL), slowly warmed to room temperature, and then heated at reflux for 18 hours. The mixture was cooled to room temperature. The reaction mixture was further diluted with tetrahydrofuran (160 mL) and then cooled to 0°C. Saturated aqueous sodium carbonate (100 ml) was added very slowly keeping the internal temperature below 15°C. After the addition of the carbonate solution is complete, the ice bath was left to expire and the mixture slowly warmed to room temperature overnight. The reaction mixture was filtered thru a pad of Celite® washing with ethyl acetate (400 mL). The solvent was removed in vacuo to afford a wet oil which was taken up in methylene chloride (300 mL) and dried over sodium sulfate. Filtration and concentration of the solvent in vacuo affords 6.89g (99% yield) of the desired product as a clear colorless oil. NMR (CDCls) 3.34 (s, 2H), 1.81 (bs, 3H), 1.51-1.32 (m, 10H); MS (ESI+) for C7Hi5NO m/z 130.0 (M+H)+ .
Figure imgf000113_0001
(l-Aminocyclopentyl)methanol.
Using General Procedure L on commercially available cycloleucine affords the desired product in 99% yield as a pale yellow oil. NMR (CDC13) 3.40 (s, 2H), 1.86-1.61 (m, 9H), 1.46-1.29 (m, 2H); MS (ESI+) for C6Hi3NO m/z 116.1 (M+H)+ .
General Procedure M.
Figure imgf000113_0002
1- { [(Triisopropylsilyl)oxy] methyl} cyclohexanamine.
(l-Aminocyclohexyl)methanol (3.43 g, 26.5 mmol) was taken up in methylene chloride
(80 mL) at room temperature under argon. Triethylamine (5.6mL, 40 mmol) was added followed by the addition of triisopropylsilyl chloride (5.34 mL, 25.2 mmol). The reaction mixture was stirred at room temperature overnight during which time it became turbid. The reaction mixture was poured into a separatory funnel transferring with methylene chloride (100 mL). The organic layer was washed sequentially with water (40 mL x 2) and brine (40 mL). The organic layer was dried over sodium sulfate, filtered and the solvent concentrated in vacuo to afford 6.68g (93%> yield) of the desired product as a clear pale yellow oil. NMR (CDC13) δ 3.49 (s, 2H), 1.75-1.25 (m, 10H), 1.16-1.06 (m, 21H); MS (ESI+) for CnH27NOSi m/z 203.2 (M+H)+ .
I l l
Figure imgf000114_0001
1- { [(Triisopropylsilyl)oxy] methyl} cyclopentanamine.
Following General Procedure M and using (l-aminocyclopentyl)methanol the desired product was obtained in 85% yield as a clear dark yellow oil. NMR (CDC13) δ 3.53 (s, 2H), 1.85-1.39 (m, 8H), 1.16-1.07 (m, 21H); MS (ESI+) for Ci5H33NOSi m/z 272.2 (M+H)+ .
Figure imgf000114_0002
2- [(Triisopropylsilyl)oxy] ethanamine.
Following General Procedure M and using commercially available ethanolamine the desired product was obtained in 99% yield as a clear pale yellow oil. NMR (CDC13) δ 3.56 (t, 2H, J=6.0Hz), 2.94(t, 2H, J=6.0Hz), 1.09-0.99 (m, 21H); MS (ESI+) for CnH27NOSi m/z 217.2 (M+H)+ .
Figure imgf000114_0003
l-{[tei"i-Butyl(dimethyl)silyl]oxy}-2-methylpropan-2-amine.
Following General Procedure M and using commercially available 2-amino-2-methyl-l- propanol and using tert-butyldimethylsilyl chloride the desired product was obtained in 95% yield as a clear colorless oil. NMR (CDC13) δ 3.31 (s, 2H), 0.93 (s, 9H), 0.06 (s, 6H); MS (ESI+) for Ci0H25NOSi m/z 204.2 (M+H)+ . Scheme 10
Figure imgf000115_0001
As exemplified in Scheme 10, compounds of Formula VI can be synthesized beginning with the aldehyde illustrated above. In Step 1, an alkyne can be treated with an organic solvent, and a base optionally at a reduced temperature and subsequently treated with an aldehyde according to methods known in the art. For example, the aldehyde in Step 1 can be treated with a base, for example, isopropylmagnesium chloride lithium chloride complex in an organic solvent, for example, tetrahydrofuran at about -15 °C and next treated with an aldehyde to generate an alkyne. In Step 2, a desired alkynyl alcohol can be treated with a base in an organic solvent at an elevated temperature to isomerize the desired alkynyl alcohol to a desired alkene. For example, a desired alkynyl alcohol can be treated with a base, such as triethylamine in an organic solvent, for example, 1 ,4-dioxane at an elevated temperature of about 60 °C to generate an alkene. In Step 3, a desired alkene can be treated with ammonia and a mixture of organic solvents to form a tert-butyl 2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7- carboxylate according to methods known in the art. For example, an alkene can be treated with 0.5M ammonia and a mixture of organic solvents, for example, dioxane and acetonitrile to form a tert-butyl 2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate. In Step 4, a desired ester can be treated with a desired acid and an organic solvent to generate a desired carboxylic acid according to methods known in the art. For example, a desired ester can be treated with a desired acid, for example, trifluoroacetic acid, to generate a carboxylic acid. In one embodiment, the organic solvent is dichloromethane. In Step 5, a desired acid can be treated with a desired amine, an organic solvent and a coupling reagent to form a desired amide according to methods known in the art. For example, a desired acid can be treated with a desired amine, an organic solvent, for example, Ν,Ν-dimethylformamide, and a coupling reagent, for example, l-[bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate, to generate an amide. In Step 6, a silyl protected alcohol can be treated with a fluoride reagent and an organic solvent according to methods known in the art to generate a desired alcohol. For example, a silyl protected alcohol can be treated with a fluoride reagent, for example, tetrabutylammonium fluoride, and an organic solvent, for example, acetonitrile, to generate an alcohol. In step 7, a desired alcohol can be treated with a sulfonyl chloride to generate a desired mesylate according to methods known in the art. For example, an alcohol can be treated with a desired sulfonyl chloride, for example, methanesulfonyl chloride, to generate a mesylate. In one embodiment, an amine spontaneously reacts with said mesylate to generate a cyclic amide. In Step 8, a desired thiol can be treated with a desired amine and an organic solvent at an elevated temperature to generate a desired amine according to methods known in the art. For example, a thiol can be treated with an amine, for example, 5-(4-methylpiperazin-l- yl)pyridin-2-amine, and an organic solvent, for example, Ν,Ν-dimethylacetamide, at an elevated temperature of about 150 °C to generate an amine. The compound 5-(4-methylpiperazin-l- yl)pyridin-2-amine can be prepared as disclosed in US 8,598,186 to Tavares and Strum.
Scheme 11
Figure imgf000117_0001
As exemplified in Scheme 11, compounds of Formula VI can be synthesized beginning with the aldehyde illustrated above. In Step 1, an alkyne can be treated with an organic solvent, and a base optionally at a reduced temperature and subsequently treated with an aldehyde according to methods known in the art. For example, the aldehyde in Step 1 can be treated with a base, for example, isopropylmagnesium chloride lithium chloride complex in an organic solvent, for example, tetrahydrofuran at about -15 °C and next treated with an aldehyde to generate an alkyne. In Step 2, a desired alkynyl alcohol can be treated with a base in an organic solvent at an elevated temperature to isomerize the desired alkynyl alcohol to a desired alkene. For example, a desired alkynyl alcohol can be treated with a base, such as triethylamine in an organic solvent, for example, 1 ,4-dioxane at an elevated temperature of about 60 °C to generate an alkene. In Step 3, a desired alkene can be treated with ammonia and a mixture of organic solvents to form a tert-butyl 2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7- carboxylate according to methods known in the art. For example, an alkene can be treated with 0.5M ammonia and a mixture of organic solvents, for example, dioxane and acetonitrile to form a tert-butyl 2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate. In Step 4, an amine can be treated with a base, an organic solvent, and a cyclic sulfamidate to form an amine according to methods known in the art. For example, a desired amine can be treated with a base, for example, triethylamine, an organic solvent, for example, N,N- dimethylformamide, and a cyclic sufamidate to form an amine. In Step 5, a protected amine can be treated with an organic acid to form an amine that can subsequently form a cyclic amide according to methods known in the art. For example, a protected amine can be treated with an organic acid, for example, trifluoroacetic acid, and subsequently react with an ester to form a cyclic amide.
EXAMPLES
The patents WO 2013/148748 entitled "Lactam Kinase Inhibitors" to Tavares, F.X., WO
2013/163239 entitled "Synthesis of Lactams" to Tavares, F.X., and US 8,598,186 entitled "CDK Inhibitors" to Tavares, F.X. and Strum, J.C. are incorporated by reference herein in their entirety.
Example 1
Synthesis of Compound 2 (Scheme 1)
Compound 2 is synthesized according to the method of A. Haidle et al, See, WO 2009/152027 entitled "5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one derivatives for MARK inhibition." Example 2
Synthesis of Compound 3 (Scheme 1)
Step 1 : A round-bottomed flask inerted with a nitrogen atmosphere is charged with Compound 2, ethanol, and lithium borohydride at ambient temperature. The reaction is stirred at ambient temperature and monitored by thin layer chromatography (TLC) or high-performance liquid chromatography (HPLC). Once Compound 2 can no longer be detected, the reaction is quenched with an aqueous acid such as aqueous hydrochloric acid, diluted with ethyl acetate and the layers separated. The organic layer is dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo. The product, a primary alcohol, is purified by silica gel column chromatography eluting with a hexane - ethyl acetate gradient and used directly in the next step.
Step 2: A round-bottomed flask inerted with a nitrogen atmosphere is charged with the primary alcohol prepared in step 1, DMF and phosphorus tribromide. The reaction is stirred at ambient temperature and monitored by thin layer chromatography (TLC) or HPLC. Once the primary alcohol can no longer be detected, the reaction is quenched with brine and diluted with toluene. The layers are separated and the toluene layer is dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo. The bromide is purified by silica gel column chromatography eluting with a hexane - ethyl acetate gradient.
Example 3
Synthesis of Compound 5 (Scheme 1)
A round-bottomed flask inerted with a nitrogen atmosphere is charged with tetrahydrofuran and the lactam 4, described below. The reaction is cooled to -78 °C and lithium diisopropylamide solution (2M in THF/heptane/ethyl benzene) is added dropwise. To the resulting enolate is added Compound 3, dropwise, and the reaction is allowed to warm to room temperature overnight. The reaction is diluted with saturated brine and the layers are separated. The organic layer is dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo. The product is purified by silica gel column chromatography eluting with a dichloromethane - methanol gradient.
Example 4
Synthesis of Compound 6 (Scheme 1)
A round-bottomed flask is charged with Compound 5 and an aqueous acid, for example a pH = 1 HC1 solution. The reaction is allowed to stir at room temperature until starting material is no longer detected by thin layer chromatography or HPLC. The reaction is neutralized with solid K2CO3 and diluted with dichloromethane. The layers are separated, the organic layer dried over anhydrous magnesium sulfate, filtered and concentrated. Compound 6 is purified by silica gel column chromatography eluting with a dichloromethane - methanol gradient.
Example 5
Synthesis of Compound 7 (Scheme 1)
A round-bottomed flask inerted with a nitrogen atmosphere is charged with Compound 6, ethanol and DBU (10 eq). The reaction is monitored by thin layer chromatography or HPLC. Note: The reaction can be heated at reflux if necessary. Once Compound 6 is no longer detected, the reaction is concentrated in vacuo. The lactam 7 is purified by silica gel column chromatography eluting with a dichloromethane - methanol gradient.
Example 6
Synthesis of Compound 8 (Scheme 1)
A round-bottomed flask inerted with a nitrogen atmosphere is charged with Compound 7, meta-chloroperoxybenzoic acid, an organic solvent and stirred at ambient temperature. The reaction is monitored by thin layer chromatography or HPLC. Once Compound 7 is no longer detected, the reaction is concentrated in vacuo. Compound 8 is purified by silica gel column chromatography eluting with a dichloromethane - methanol gradient.
Example 7
Synthesis of Compound 10 (Scheme 1)
The tricyclic lactam 8 is combined with an amine (9, 0.9 eq) and an organic solvent such as tetrahydrofuran. A strong base such as lithium hexamethyldisilazane is added and the reaction is stirred until lactam 8 is no longer detected by either thin layer chromatography or HPLC. The reaction is concentrated in vacuo. The product is purified by silica gel column chromatography eluting with a dichloromethane - methanol gradient.
Alternatively, a CEM Discovery microwave vessel is charged with the tricyclic lactam 8, N-methyl-2-pyrrolidone (NMP), Hunig's base, and amine 9 (0.9eq). The reaction is heated at 150 °C for 1-4 hours while being monitored by TLC. Once the tricyclic lactam 8 is no longer detected by TLC or HPLC, the reaction is concentrated in vacuo. The product is purified by silica gel column chromatography eluting with a dichloromethane - methanol gradient. Example 8
Synthesis of Compound 11 (Scheme 2)
Compound 7 is treated with an oxidizing agent such as 2,3-dichloro-5,6-dicyano-l,4- benzoquinone (DDQ) in an organic solvent to generate the alkene intermediate 12. Example 9
Synthesis of Compound 14 (Scheme 2)
The sulfone intermediate 12 is combined with an amine (13, 0.9 eq) in an organic solvent such as tetrahydrofuran. An organic base such as lithium hexamethyldisilazane is added and the reaction is stirred until sulfone intermediate 12 can no longer be detected by thin layer chromatography or HPLC. The product is purified by silica gel column chromatography eluting with a dichloromethane - methanol gradient.
Alternatively, a CEM Discovery microwave vessel is charged with the sulfone intermediate 12, N-methyl-2-pyrrolidone (NMP), Hunig's base, and amine 13 (0.9eq). The reaction is heated at 150 °C for 1-4 hours while being monitored by TLC. Once the sulfone intermediate 12 is no longer detected by TLC or HPLC, the reaction is concentrated in vacuo.
The product is purified by silica gel column chromatography eluting with a dichloromethane - methanol gradient. Example 10
Synthesis of Compound 4
Step 1 : Synthesis of Compound 15 (Scheme 3)
Compound 15 is synthesized according to the method of Arigon, J., See, US 2013/0289031, entitled "Pyrimidinone derivatives, preparation thereof and pharmaceutical use thereof."
Step 2: Synthesis of Compound 16 (Scheme 3)
A round-bottomed flask inerted with a nitrogen atmosphere is charged with Compound 15, dichloromethane and triethylamine (1.5 eq). The reaction is cooled to 0 °C and Boc anhydride (1.5 eq) is added. The reaction is allowed to stir at room temperature until Compound 15 is no longer detected by thin layer chromatography or HPLC. The reaction is concentrated in vacuo. The product is purified by silica gel column chromatography eluting with a hexane - ethyl acetate gradient.
Step 3: Synthesis of Compound 17 (Scheme 3)
A round-bottomed flask inerted with a nitrogen atmosphere is charged with Compound 16, acetonitrile and a base such as potassium carbonate. Methyl chloroacetate is added dropwise. The reaction is allowed to stir at room temperature until Compound 16 is no longer detected by thin layer chromatography or HPLC. The reaction is concentrated in vacuo. The product is purified by silica gel column chromatography eluting with a hexane - ethyl acetate gradient.
Step 4: Synthesis of Compound 18 (Scheme 3)
Compound 17 is dissolved in a solution comprising 3M HC1 in methanol and the reaction is stirred at ambient temperature. Note: the reaction can be heated at a temperature of about 25 °C to about 60 °C to accelerate the reaction rate. Once the starting material is no longer detected by thin layer chromatography, the reaction is concentrated in vacuo. The product is purified by silica gel column chromatography using a dichloromethane - methanol gradient
Step 5: Synthesis of Compound 19 (Scheme 3)
A round-bottomed flask inerted with a nitrogen atmosphere is charged with Compound
18, dichloromethane, and diisopropylethylamine (1.2 eq). Chloromethyl methyl ether (MOM-Cl, 1.2 eq) is added dropwise. The reaction is allowed to stir at room temperature and monitored by TLC. Once the starting material is no longer detected by thin layer chromatography, the reaction is quenched with saturated brine solution. The organic layer is separated, dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo. The product is purified by silica gel column chromatography using a dichloromethane - methanol gradient.
Step 6: Synthesis of Compound 4
A round-bottomed flask inerted with a nitrogen atmosphere is charged with anhydrous tetrahydrofuran and Compound 19. The reaction is cooled to -78 °C. Sodium bis(trimethylsilyl)amide (1M in THF, 1.1 eq) is added dropwise. Chloromethyl methyl ether (MOM-Cl, 1.2 eq) is added dropwise with stirring and the reaction is allowed to warm to room temperature overnight. The reaction is quenched with saturated brine solution and the layers are separated. The organic layer is dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo. The product is purified by silica gel column chromatography using a dichloromethane - methanol gradient.
Example 11
Synthesis of Compound 25 (Scheme 5)
Compound 20 is commercially available. Compound 25 is synthesized according to the synthetic methodology disclosed in Example 10. Example 12
Synthesis of Compound 31 (Scheme 6)
Compound 31 is synthesized according to the synthetic methodology disclosed in Example 10. Example 13
Synthesis of Compound 33 (Scheme 7)
Step 1 : Synthesis of Compound 32
Compound 32, 5-morpholinopyrid-2-amine, is synthesized according to Tavares, F.X. and Strum, J.C., See, US 8,598,186, entitled "CDK Inhibitors".
Step 2: Synthesis of Compound 33
The sulfone intermediate 8 is diluted with a suitable solvent such as tetrahydrofuran and an organic base such as lithium hexamethyldisilazane is added. The compound 32 is added and the reaction is stirred until sulfone intermediate 8 can no longer be detected by thin layer chromatography or HPLC. The product is purified by silica gel column chromatography eluting with a dichloromethane - methanol gradient.
Alternatively, a CEM Discovery microwave vessel is charged with the sulfone intermediate 8, N-methyl-2-pyrrolidone (NMP), Hunig's base, and 5-morpholinopyrid-2-amine (0.9eq). The reaction is heated at 150 °C for 1-4 hours while being monitored by TLC. Once the sulfone intermediate 8 is no longer detected by TLC or HPLC, the reaction is concentrated in vacuo. The product is purified by silica gel column chromatography eluting with a dichloromethane - methanol gradient.
Example 14
Synthesis of Compound 34 (Scheme 8)
Step 1 : Synthesis of Compound 32
Compound 32, 5-morpholinopyrid-2-amine, is synthesized according to Tavares, F.X. and Strum, J.C., See, US 8,598,186, entitled "CDK Inhibitors".
Step 2: Synthesis of Compound 34
The sulfone intermediate 12 is combined with a suitable solvent such as tetrahydrofuran and an organic base such as lithium hexamethyldisilazane. The amine 32 is added and the reaction is stirred until sulfone intermediate 12 can no longer be detected by thin layer chromatography or HPLC. The product is purified by silica gel column chromatography eluting with a dichloromethane - methanol gradient.
Alternatively, a CEM Discovery microwave vessel is charged with the sulfone intermediate 12, N-methyl-2-pyrrolidone (NMP), Hunig's base, and 5-morpholinopyrid-2-amine (0.9eq). The reaction is heated at 150 °C for 1-4 hours while being monitored by TLC. Once the sulfone intermediate 12 is no longer detected by TLC or HPLC, the reaction is concentrated in vacuo. The product is purified by silica gel column chromatography eluting with a dichloromethane - methanol gradient. Example 15
Preparation of a Formula V compound
Step 1 : Compound 7 is Boc protected according to the method of A. Sarkar et al. (JOC, 2011, 76, 7132-7140).
Step 2: Boc-protected Compound 7 is treated with 5 mol% NiCl2(Ph3)2, 0.1 eq triphenylphosphine, 3 eq Mn, 0.1 eq tetraethylammonium iodide, in DMI under C02 (1 arm) at 25 °C for 20 hours to convert the methyl thiol derivative into the carboxylic acid.
Step 3 : The carboxylic acid from Step 2 is converted to the corresponding acid chloride using standard conditions.
Step 4: The acid chloride from Step 3 is reacted with N-methyl piperazine to generate the corresponding amide.
Step 5: The amide from Step 4 is deprotected using trifluoroacetic acid in methylene chloride to generate the target compound. The product is purified by silica gel column chromatography eluting with a dichloromethane - methanol gradient. Example 16
CDK4/6 Inhibition In Vitro Assay
Selected compounds disclosed herein were tested in CDK4/cyclinDl, CDK6/CycD3, CDK2/CycA, CDK2/cyclinE, CDK5/p25, CDK5/p35, CDK7/CycH/MATl, and CDK9/CycT kinase assays by Nanosyn (Santa Clara, CA) to determine their inhibitory effect on these CDKs. The assays were performed using microfluidic kinase detection technology (Caliper Assay Platform). The compounds were tested in 12-point dose-response format in singlicate at Km for ATP. Phosphoacceptor substrate peptide concentration used was 1.25 μΜ for all assays (except μΜ 10 was used for the CKD7/CycH/MATl assay and Staurosporine was used as the reference compound for all assays. Specifics of each assay are as described below: CDK2/CyclinA: Enzyme concentration: 0.2 nM; ATP concentration: 50 μΜ; Incubation time: 3 hr.
CDK2/CyclinE: Enzyme concentration: 0.2 nM; ATP concentration: 100 μΜ; Incubation time: 3 hr.
CDK4/CyclinDl : Enzyme concentration: 1 nM; ATP concentration: 200 μΜ; Incubation time: 3 hr.
CDK6/CyclinD3: Enzyme concentration: 10 nM; ATP concentration: 300 μΜ; Incubation time: 3 hr.
CDK5/p25: Enzyme concentration: 0.1 nM; ATP concentration: 20 μΜ; Incubation time:
3 hr.
CDK5/p35: Enzyme concentration: 0.07 nM; ATP concentration: 20 μΜ; Incubation time: 3 hr.
CDK7/CycH/MATl : Enzyme concentration: 5 nM; ATP concentration: 50 μΜ; Incubation time: 3 hr.
CDK9/CycT: Enzyme concentration: 5 nM; ATP concentration: 10 μΜ; Incubation time: 17 hr.
Table 2. Inhibition of CDK kinases by Tricyclic Lactam Compounds
Figure imgf000125_0001
CCCC * * * * * * * *
GGGG * * ** * * ** * *
* >100μΜ
** 10μΜ<Χ>100μΜ
*** <10μΜ Example 17
Gl Arrest (Cellular Gl and S-phase) Assay
For determination of cellular fractions in various stages of the cell cycle following various treatments, HS68 cells (human skin fibroblast cell line (Rb-positive)) are stained with propidium iodide staining solution and run on Dako Cyan Flow Cytometer. The fraction of cells in G0-G1 DNA cell cycle versus the fraction in S-phase DNA cell cycle is determined using Flow Jo 7.2 .2 analysis.
Example 18
Cell Cycle Arrest by Tricyclic Lactams in CDK4/6-Dependent Cells
To test the ability of tricyclic lactams to induce a clean Gl -arrest, a cell based screening method is used consisting of two CDK4/6-dependent cell lines (tHS68 and WM2664; Rb- positive) and one CDK4/6-independent (A2058; Rb-negative) cell line. Twenty-four hours after plating, each cell line is treated with a tricyclic lactam compound in a dose dependent manner for 24 hours. At the conclusion of the experiment, cells are harvested, fixed, and stained with propidium iodide (a DNA intercalator), which fluoresces strongly red (emission maximum 637 nm) when excited by 488nm light. Samples are run on Dako Cyan flow cytometer and > 10,000 events were collected for each sample. Data are analyzed using Flow Jo 2.2 software developed by TreeStar, Inc. Example 19
Inhibition of RB Phosphorylation
The CDK4/6-cyclin D complex is essential for progression from Gl to the S-phase of the DNA cell cycle. This complex phosphorylates the retinoblastoma tumor suppressor protein (Rb). To demonstrate the impact of CDK4/6 inhibition on Rb phosphorylation (pRb), tricyclic lactam compounds are exposed to three cell lines, two CDK4/6 dependent (tHS68, WM2664; Rb- positive) and one CDK4/6 independent (A2058; Rb-negative). Twenty four hours after seeding, cells are treated with a tricyclic lactam compound at 300 nM final concentration for 4, 8, 16, and 24 hours. Samples are lysed and protein is assayed by western blot analysis. Rb phosphorylation is measured at two sites targeted by the CDK4/6-cyclin D complex, Ser780 and Ser807/811 using species specific antibodies.
Example 20
Growth Arrest of Small Cell Lung Cancer (SCLC) Cells
The retinoblastoma (RB) tumor suppressor is a major negative cell cycle regulator that is inactivated in approximately 11% of all human cancers. Functional loss of RB is an obligate event in small cell lung cancer (SCLC) development. In RB competent tumors, activated CDK2/4/6 promote Gl to S phase traversal by phosphorylating and inactivating RB (and related family members). Conversely, cancers with RB deletion or inactivation do not require CDK4/6 activity for cell cycle progression.
Tricyclic lactam compounds are tested for their ability to block cell proliferation in a panel of SCLC cell lines with known genetic loss of RB. SCLC cells are treated with DMSO or a tricyclic lactam for 24 hours. The effect on proliferation is measured by EdU incorporation. An RB-intact, CDK4/6-dependent cell line (WM2664 or tHS68) and a panel of RB-negative SCLC cell lines (H69, H82, H209, H345, NCI417, or SHP-77) are analyzed for growth inhibition by the various tricyclic lactams.
Example 21
Growth Arrest of Rb-Negative Cancer Cells
Cellular proliferation assays are conducted using the following Rb-negative cancer cell lines: H69 (human small cell lung cancer - Rb-negative) cells or A2058 (human metastatic melanoma cells - Rb-negative). These cells are seeded in Costar (Tewksbury, Massachusetts) 3093 96 well tissue culture treated white walled, clear bottom plates. Cells are treated with tricyclic lactam compounds at nine point dose response dilution series from 10 uM to 1 nM. Cells are exposed to compounds and then cell viability is determined after either four (H69) or six (A2058) days using the CellTiter-Glo® luminescent cell viability assay (CTG; Promega, Madison, Wisconsin, United States of America) following the manufacturer's recommendations. Plates are read on a BioTek (Winooski, Vermont) Syngergy2 multi-mode plate reader. The Relative Light Units (RLU) are plotted as a result of variable molar concentration and data are analyzed using Graphpad (LaJolla, Californaia) Prism 5 statistical software to determine the EC50 for each compound.
Example 22
Bone Marrow Proliferation as Evaluated Using EdU Incorporation and Flow Cytometry Analysis
For hematopoietic stem cell and/or hematopoietic progenitor cell (HSPC) proliferation experiments, young adult female FVB/N mice are treated with a single dose of the tricyclic lactams described herein by oral gavage. Mice are then sacrificed at 0, 12, 24, 36, or 48 hours following compound administration, and bone marrow is harvested, as previously described (Johnson et al. J. Clin. Invest. (2010) 120(7), 2528-2536). Four hours before the bone marrow is harvested, mice are treated with 100 μg of EdU by intraperitoneal injection (Invitrogen). Bone marrow mononuclear cells are harvested and immunophenotyped using previously described methods and percent EdU positive cells are then determined (Johnson et al. J. Clin. Invest. (2010) 120(7), 2528-2536). In brief, HSPCs are identified by expression of lineage markers (Lin-), Seal (S+), and c-Kit (K+).
Example 23
Cellular Wash-Out Experiment
HS68 cells are seeded out at 40,000 cells/ well in 60mm dish on day 1 in DMEM containing 10% fetal bovine serum, 100 U/ml penicillin/ streptomycin and lx Glutamax (Invitrogen) as described (Brookes et al. EMBO J, 21(12)2936-2945 (2002) and Ruas et al. Mol Cell Biol, 27(12)4273-4282 (2007)). 24hrs post seeding, cells are treated with a tricyclic lactam compound or DMSO vehicle alone at 300 nM final concentration of test compounds. On day 3, one set of treated cell samples are harvested in triplicate (0 Hour sample). Remaining cells are washed two times in PBS-CMF and returned to culture media lacking test compound. Sets of samples are harvested in triplicate at 24, 40, and 48 hours. Alternatively, the same experiment is done using normal Renal Proximal Tubule Epithelial Cells (Rb-positive) obtained from American Type Culture Collection (ATCC, Manassas, VA). Cells are grown in an incubator at 37°C in a humidified atmosphere of 5% C02 in Renal Epithelial Cell Basal Media (ATCC) supplemented with Renal Epithelial Cell Growth Kit (ATCC) in 37°C humidified incubator.
Upon harvesting cells, samples are stained with propidium iodide staining solution and samples run on Dako Cyan Flow Cytometer. The fraction of cells in G0-G1 DNA cell cycle versus the fraction in S-phase DNA cell cycle is determined using Flow Jo 7.2 .2 analysis. Example 24
Pharmacokinetic and Pharmacodynamic Properties of Tricyclic Lactams
Tricyclic lactam compounds described herein can be dosed to mice at 30 mg/kg by oral gavage or 10 mg/kg by intravenous injection. Blood samples are taken at 0, 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 hours post dosing and the plasma concentrations of the tricyclic lactam compounds are determined by HPLC.
Example 25
Metabolic Stability
The metabolic stability of tricyclic lactam compounds can be determined in human, dog, rat, monkey, and mouse liver microsomes. Human, mouse, and dog liver microsomes are purchased from Xenotech, and Sprague-Dawley rat liver microsomes are prepared by Absorption Systems. The reaction mixture comprising 0.5 mg/mL of liver microsomes, 100 mM of potassium phosphate, pH 7.4, 5 mM of magnesium chloride, and 1 uM of test compound is prepared. The test compound is added into the reaction mixture at a final concentration of 1 uM. An aliquot of the reaction mixture (without cofactor) is incubated in shaking water bath at 37°C for 3 minutes. The control compound, testosterone, is run simultaneously with the test compound in a separate reaction. The reaction is initiated by the addition of cofactor (NADPH), and the mixture is then incubated in a shaking water bath at 37°C. Aliquots (100 μί) are withdrawn at 0, 10, 20, 30, and 60 minutes for the test compound and 0, 10, 30, and 60 minutes for testosterone. Test compound samples are immediately combined with 100 of ice-cold acetonitrile containing internal standard to terminate the reaction. Testosterone samples are immediately combined with 800 of ice cold 50/50 acetonitrile/dH20 containing 0.1% formic acid and internal standard to terminate the reaction. The samples are assayed using a validated LC-MS/MS method. Test compound samples are analyzed using the Orbitrap high resolution mass spectrometer to quantify the disappearance of parent test compound and detect the appearance of metabolites. The peak area response ration (PARR) to internal standard is compared to the PARR at time 0 to determine the percent of test compound or positive control remaining at time-point. Half-lives are calculated using GraphPad software, fitting to a single- phase exponential decay equation. Half-life is calculated based on tl/2 = 0.693k, where k is the elimination rate constant based on the slope plot of natural logarithm percent remaining versus incubation time.
Example 26
Inhibition of Hematopoietic Stem and/or Progenitor Cell (HSPC) Proliferation
To characterize the effect of tricyclic lactam compound treatment on proliferation of the different mouse hematopoietic cells, 8-week-old female C57B1/6 mice are given a single dose of vehicle alone (20% Solutol) or a tricyclic lactam compound (150 mg/kg) by oral gavage. Ten- hours later, all mice are given a single i.p. injection of 100 meg EdU (5-ethynyl-2'-deoxyuridine) to label cells in S-phase of the cell cycle. All treated mice are euthanized 2 hours after EdU injection, bone marrow cells are harvested and processed for flow cytometric analysis of EdU- incorporation
Example 27
Inhibition of Differentiated Hematopoietic Cell Proliferation
Using the same experimental protocol as discussed in Example 26 above, the effect of tricyclic lactam compounds on the proliferation of differentiated hematopoietic cells is investigated.
Example 28
Radiomitigation Effects of Tricyclic Lactams
The principal acute toxicities of total body irradiation (TBI) at doses less than 10 Gy are hematologic manifestations such as granulocytopenia, anemia, thrombocytopenia and lymphopenia. At higher doses of IR exposure, intestinal, cutaneous and neurologic toxicities additionally become significant contributors to morbidity and mortality, but the hematologic syndrome has been the principal complication faced by immediate survivors of a mass casualty radiologic disaster. Tricyclic lactams are tested for their ability to protect cells from DNA damage and apoptosis induced by irradiation.
DNA damage is determined using the g-H2A.X assay and apoptosis is determined with a Caspase 3/7 assay. For the g-H2AX assay, tHS68 cells are fixed and stained using the g-H2A.X Phosphorylation Assay Kit (Flow Cytometry; Millipore, Temecula, CA) by the manufacturer's instructions. g-H2AX-positive tHDF cells are then quantified using a CyAn ADP Analyzer (Beckman Coulter, Indianapolis, IN) and Flow Jo analysis software (Version 7.2.2; Tree Star, Ashland, OR). For the in vitro caspase 3/7 assay, tHDF cells are analyzed directly in the 96-well plates 24 hours after radiation or staurosporine treatment. Caspase 3/7 activation is measured using the Caspase-Glo 3/7 Assay System (Promega, Madison, WI) by following the manufacturer's instructions.
For the g-H2AX assay, 30,000 cells are plated per well in 12-well plates. For the caspase
3/7 assay, 1,000 cells are plated per well in 96-well white wall clear bottom plates. Cells are incubated at 37°C in a humidified atmosphere of 5% C02 for 24 hours and then irradiated at 6 Gy, 8 Gy, or 10 Gy. Cells are then incubated at 37°C in a humidified atmosphere of 5% C02 with 100, 300, or 1,000 nM compound or dimethyl sulfoxide (Sigma- Aldrich) vehicle control for an additional 16 hours prior to analysis.
Example 29
Radiomitigation Effects of Tricyclic Lactams in a Mouse Model
Compounds are tested for mitigation of radiation-induced death in vivo in a mouse model. Wild-type mice, young adult (8-12 weeks of age) C57BL/6 (The Jackson Laboratory) or C3H (Harlan Sprague-Dawley) animals are used. Animals are irradiated using a 137Cs AECL GammaCell 40 Irradiator (Atomic Energy of Canada) or a XRAD320 (Precision XRay Inc.) biological irradiator. Experiments are carried out using the 137Cs source, unless otherwise noted. Mice are dosed at 150 mg/kg compound by oral gavage 12 hours post irradiation for single dose studies. Mice are dosed at 150 mg/kg of compound by oral gavage 12 hours post irradiation and 24 hours post irradiation for two dose studies. Kaplan-Meier analysis of survival over the next 30 days for both treated and control groups are determined.
Example 30
Preparation of Drug Product
The active compounds of the present invention can be prepared for intravenous administration using the following procedure. The excipients hydroxypropyl-beta-cyclodextrin and dextrose can be added to 90% of the batch volume of USP Sterile Water for Injection or Irrigation with stirring; stir until dissolved. The active compound in the hydrochloride salt form is added and stirred until it is dissolved. The pH is adjusted with IN NaOH to pH 4.3 + 0.1 and IN HC1 can be used to back titrate if necessary. USP sterile water for injection or irrigation can be used to bring the solution to the final batch weight. The pH is next re-checked to ensure that the pH is pH 4.3 + 0.1. If the pH is outside of the range add IN HC1 or IN NaOH as appropriate to bring the pH to 4.3 + 0.1. The solution is next sterile filtered to fill 50 or 100 mL flint glass vials, stopper, and crimped.
This specification has been described with reference to embodiments of the invention. The invention has been described with reference to assorted embodiments, which are illustrated by the accompanying Examples. The invention can, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Given the teaching herein, one of ordinary skill in the art will be able to modify the invention for a desired purpose and such variations are considered within the scope of the invention.

Claims

We claim:
1. A method for reducing the effect of ionizing radiation exposure on cyclin-dependent kinase 4 (CDK4) replication-dependent hematopoietic stem cells and/or progenitor cells (HSPCs) in a subject exposed to ionizing radiation, the method comprising administering to the subject an effective amount of a compound of Formula I, II, III, IV, or V:
Figure imgf000133_0001
Figure imgf000134_0001
wherein:
Z is -(CH2)X- wherein x is 1, 2, 3 or 4 or -0-(CH2)z- wherein z is 2, 3 or 4;
each X is independently CH or N;
each X' is independently, CH or N;
X" is independently CH2, S or NH, arranged such that the moiety is a stable 5-membered ring; R, R8, and R11 are independently H, C1-C3 alkyl or haloalkyl, cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O or S; -(alkylene)m- C3-C8 cycloalkyl, -(alkylene)m- aryl, -(alkylene)m-heterocyclo, -(alkylene)m- heteroaryl, -(alkylene)m-NR3R4, -(alkylene)m-C(0)- NR3R4; -(alkylene)m-0-R5, -(alkylene)m- S(0)n-R5, or -(alkylene)m-S(0)n-NR3R4 any of which may be optionally independently substituted with one or more Rx groups as allowed by valance, and wherein two Rx groups bound to the same or adjacent atoms may optionally combine to form a ring;
each R1 is independently aryl, alkyl, cycloalkyl or haloalkyl, wherein each of said alkyl, cycloalkyl and haloalkyl groups optionally includes O or N heteroatoms in place of a carbon in the chain and two Rx'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;
y is 0, 1, 2, 3 or 4;
R2 is -(alkylene)m-heterocyclo, -(alkylene)m-heteroaryl, -(alkylene)m-NR3R4,
-(alkylene)m-C(0)-NR3R4; -(alkylene)m-C(0)-0-alkyl; -(alkylene)m-0-R5,
-(alkylene)m-S(0)n-R5, or -(alkylene)m-S(0)n-NR3R4 any of which may be optionally independently substituted with one or more Rx groups as allowed by valance, and wherein two Rx groups bound to the same or adjacent atom may optionally combine to form a ring and wherein m is 0, 1 or 2 and n is 0, 1 or 2;
R3 and R4 at each occurrence are independently:
(i) hydrogen or (ii) alkyl, cycloalkyl, heterocyclo, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl any of which may be optionally independently substituted with one or more Rx groups as allowed by valance, and wherein two Rx groups bound to the same or adjacent atom may optionally combine to form a ring; or R3 and R4 together with the nitrogen atom to which they are attached may combine to form a heterocyclo ring optionally independently substituted with one or more Rx groups as allowed by valance, and wherein two Rx groups bound to the same or adjacent atom may optionally combine to form a ring;
R5 and R5* at each occurrence is:
(i) hydrogen or
(ii) alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl any of which may be optionally independently substituted with one or more Rx groups as allowed by valance;
Rx at each occurrence is independently, halo, cyano, nitro, oxo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl, heteroarylalkyl,
cycloalkylalkyl, heterocycloalkyl, -(alkylene)m-OR5, -(alkylene)m-0-alkylene-OR5,
-(alkylene)m-S(0)n-R5, -(alkylene)m-NR3R4, -(alkylene)m-CN, -(alkylene)m-C(0)-R5,
-(alkylene)m-C(S)-R5, -(alkylene)m-C(0)-OR5, -(alkylene)m-0-C(0)-R5,
-(alkylene)m-C(S)-OR5, -(alkylene)m-C(0)-(alkylene)m-NR3R4, -(alkylene)m-C(S)-NR3R4, -(alkylene)m-N(R3)-C(0)-NR3R4, -(alkylene)m-N(R3)-C(S)-NR3R4,
-(alkylene)m-N(R3)-C(0)-R5, -(alkylene)m-N(R3)-C(S)-R5, -(alkylene)m-0-C(0)-NR3R4, -(alkylene)m-0-C(S)-NR3R4, -(alkylene)m-S02-NR3R4, -(alkylene)m-N(R3)-S02-R5,
-(alkylene)m-N(R3)-S02-NR3R4, -(alkylene)m-N(R3)-C(0)-OR5 )
-(alkylene)m-N(R3)-C(S)-OR5, or -(alkylene)m-N(R3)-S02-R5; wherein:
said alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclo, aryl,
heteroaryl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, and heterocycloalkyl groups may be further independently substituted with one or more
-(alkylene)m-CN, -(alkylene)m-OR5*, -(alkylene)m-S(0)n-R5*,
-(alkylene)m-NR3 *R4 * , -(alkylene)m-C(0)-R5 * , -(alkylene)m-C(=S)R5 * ,
-(alkylene)m-C(=0)0 R5*, -(alkylene)m-OC(=0)R5*, -(alkylene)m-C(S)-OR5*, -(alkylene)m-C(0)-NR3 *R4* , -(alkylene)m-C(S)-NR3 *R4* , -(alkylene)m-N(R3 *)-C(0)-NR3 *R4* , -(alkylene)m-N(R3 *)-C(S)-NR3*R4* ,
-(alkylene)m-N(R3 *)-C(0)-R5 * , -(alkylene)m-N(R3 *)-C(S)-R5 * ,
-(alkylene)m-0-C(0)-NR3*R4*, -(alkylene)m-0-C(S)-NR3*R4*,
-(alkylene)m-S02-NR3 *R4* , -(alkylene)m-N(R3 *)-S02-R5* ,
-(alkylene)m-N(R3 *)-S02-NR3 *R4* , -(alkylene)m-N(R3 *)-C(0)-OR5 * ,
-(alkylene)m-N(R3*)-C(S)-OR5*, or -(alkylene)m-N(R3*)-S02-R5*,
n is 0, 1 or 2, and
m is 0, 1 or 2;
R3* and R4* at each occurrence are independently:
(i) hydrogen or
(ii) alkyl, alkenyl, alkynyl cycloalkyl, heterocyclo, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl any of which may be optionally independently substituted with one or more Rx groups as allowed by valance; or R3* and R4* together with the nitrogen atom to which they are attached may combine to form a heterocyclo ring optionally independently substituted with one or more Rx groups as allowed by valance; and
R6 is H or lower alkyl, -(alkylene)m-heterocyclo, -(alkylene)m-heteroaryl, -(alkylene)m-NR3R4, -(alkylene)m-C(0)-NR3R4; -(alkylene)m-0-R5, -(alkylene)m-S(0)n-R5, or -(alkylene)m-S(0)n- NR3R4 any of which may be optionally independently substituted with one or more Rx groups as allowed by valance, and wherein two Rx groups bound to the same or adjacent atoms may optionally combine to form a ring; and
R10 is (i) NHRA, wherein RA is unsubstituted or substituted Ci-Cg alkyl, cycloalkylalkyl, or -TT- RR, Ci-Cg cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O, and S; TT is an unsubstituted or substituted Ci-Cg alkyl or C3-C8 cycloalkyl linker; and RRis a hydroxyl, unsubstituted or substituted Ci-C6 alkoxy, amino, unsubstituted or substituted Ci-C6 alkylamino, unsubstituted or substituted di-Ci-C6 alkylamino, unsubstituted or substituted C6-Cio aryl, unsubstituted or substituted heteroaryl comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S, unsubstituted or substituted C3-C10 carbocycle, or unsubstituted or substituted heterocycle comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S; or (ii) -C(0)-R12 or -C(0)0-R13, wherein R12 is NHRA or RA andR13 is RA; when compounds comprise a double bond in the 6-membered ring fused to the pyrimidine ring, two R8 groups are present and are as defined above;
when compounds do not comprise a double bond in the 6-membered ring fused to the pyrimidine ring, four R8 groups are present and are as defined above;
or a pharmaceutically acceptable salt thereof.
2. The method of claim 1, wherein each R8 is independently hydrogen or C1-C3 alkyl.
3. The method of claim 1, wherein the compound has the formula:
Figure imgf000137_0001
4. The method of claim 1 wherein the compound has the formula:
Figure imgf000137_0002
5. The method of claim 1 wherein the compound has the formula:
Figure imgf000137_0003
6. The method of claim 1 wherein the compound has the formula:
Figure imgf000138_0001
7. The method of claim 1, wherein the compound has the formula:
Figure imgf000138_0002
8. The method of claim 1 wherein the compound has the formula:
Figure imgf000138_0003
9. The method of claim 1 wherein the compound has the formula:
Figure imgf000138_0004
The method of claim 1 wherein the compound has the formula:
The method of claim 1 wherein the compound has the formula:
Figure imgf000139_0002
The method of claim 1 wherein the compound has the formula:
Figure imgf000139_0003
The method of claim 1, wherein the compound has the formula:
Figure imgf000139_0004
The method of claim 1, wherein the compound has the formula:
Figure imgf000139_0005
The method of claim 1 wherein the compound has the formula:
Figure imgf000140_0001
The method of claim 1, wherein the compound has the formula:
Figure imgf000140_0002
The method of claim 1, wherein the compound has the formula:
Figure imgf000140_0003
The method of claim 1, wherein the compound has the formula:
Figure imgf000140_0004
The method of claim 1 , wherein the compound is selected from the group consisting
Structure Structure
Reference
Figure imgf000141_0001
Figure imgf000142_0001
140
Figure imgf000143_0001
141
Figure imgf000144_0001
142
Figure imgf000145_0001
143
Figure imgf000146_0001
144
Figure imgf000147_0001
145
Figure imgf000148_0001
Figure imgf000149_0001
Figure imgf000150_0001
Figure imgf000151_0001
Figure imgf000152_0001
Figure imgf000153_0001
Figure imgf000154_0001
Figure imgf000155_0001
The method of claim 1, wherein the subject is a human.
The method of claim 1, wherein the subject's HSPCs return to approximately pre- treatment baseline cell cycle activity prior to the exposure to IR.
22. The method of claim 1, wherein the compound is administered to the subject prior to the exposure to IR.
23. The method of claim 1, wherein the subject is undergoing radio-therapy to treat a disease.
24. The method of claim 1, wherein the subject is being treated for a proliferative disorder.
25. The method of claim 1, wherein the subject is being treated for a CDK4/6 replication independent cancer.
26. The method of claim 1, wherein the subject is being treated for an ionizing radiation exposure associated with an environmental or occupational condition.
27. The method of claim 1, wherein administration of the compound does not affect growth of diseased cells.
28. The method of claim 1, wherein the subject is further treated with a hematopoietic growth factor upon dissipation of the CDK4/6 inhibitor's inhibitory effect.
29. The method of claim 28, wherein the hematopoietic growth factor is selected form the group consisting of granulocyte colony stimulating factor (G-CSF), granulocyte- macrophage colony stimulating factor (GM-CSF), thrombopoietin, interleukin (IL)-12, steel factor, and erythropoietin (EPO).
30. The method of claim 1, wherein the compound is administered to the subject prior to exposure to the ionizing radiation, during exposure to the ionizing radiation, after exposure to the ionizing radiation, or a combination thereof.
31. The method of claim 1, wherein the compound is administered to the subject less than about 24 hours prior to exposure to the ionizing radiation.
32. The method of claim 1, wherein the compound is administered to the subject prior to exposure to the ionizing radiation such that the compound reaches peak serum levels during exposure to the ionizing radiation.
33. The method of claim 1, wherein the compound is administered to the subject less than about 4 hours prior to exposure to the ionizing radiation.
34. The method of claim 1, wherein the compound is administered to the subject after exposure to the ionizing radiation.
35. The method of claim 1, wherein the compound is administered to the subject about 12 hours or more after exposure to the ionizing radiation.
36. Use of a compound of claim 1 for reducing the effect of ionizing radiation exposure on cyclin-dependent kinase 4 (CDK4) replication-dependent hematopoietic stem cells and/or progenitor cells (HSPCs) in a subject exposed to ionizing radiation.
37. A method for reducing the effect of ionizing radiation exposure on cyclin-dependent kinase 4 (CDK4) replication-dependent hematopoietic stem cells and/or progenitor cells (HSPCs) in a subject exposed to ionizing radiation, the method comprising administering to the subject an effective amount of a compound of Formula VI:
Figure imgf000157_0001
wherein R, R1, R2, R3, R4, R5, R6, Rx, Z, m, n, and y are as defined in claim 1 ;
each R14 is independently H, C1-C3 alkyl (including methyl) or haloalkyl, cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O or S; -(alkylene)m- C3- C8 cycloalkyl, -(alkylene)m-aryl, -(alkylene)m-heterocyclo, -(alkylene)m- heteroaryl, - (alkylene)m-NR3R4, -(alkylene)m-C(0)-NR3R4; -(alkylene)m-0-R5, -(alkylene)m- S(0)„- R5, or -(alkylene)m-S(0)n-NR3R4 any of which may be optionally independently substituted with one or more Rx groups as allowed by valence, and wherein two Rx groups bound to the same or adjacent atoms may optionally combine to form a ring; or two R14 groups bonded to the same carbon can form an exocyclic double bond;
or two R14 groups bonded to the same carbon can form a carbonyl group; and
when the compound of Formula VI has a double bond, as indicated by the (— ), in the 6- membered ring fused to the pyrimidine ring, two R14 groups are present as allowed for in Formula VI above; or
when the compound of Formula VI does not include a double bond, as indicated by the (— ), in the 6-membered ring fused to the pyrimidine ring, four R14 groups are present as allowed for in Formula VI above;
or a pharmaceutically acceptable salt thereof.
The method of claim 1, wherein the compound is selected from the group consisting of:
Figure imgf000158_0001
Figure imgf000159_0001
40. A method for reducing the effect of ionizing radiation exposure on cyclin-dependent kinase 4 (CDK4) replication-dependent hematopoietic stem cells and/or progenitor cells (HSPCs) in a subject exposed to ionizing radiation, the method comprising administering to the subject an effective amount of a compound of Formula I, II, III, IV, or V:
Figure imgf000160_0001
Figure imgf000161_0001
wherein:
Z is -(CH2)X- wherein x is 1, 2, 3 or 4 or -0-(CH2)z- wherein z is 2, 3 or 4;
each X is independently CH or N;
each X' is independently CH or N;
X" is independently CH2, S or NH, arranged such that the moiety is a stable 5-membered ring; R, R8, and R11 are independently H, C1-C3 alkyl (including methyl) or haloalkyl, cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O or S; -(alkylene)m- C3-C8 cycloalkyl, -(alkylene)m-aryl, -(alkylene)m-heterocyclo, -(alkylene)m- heteroaryl, -(alkylene)m- NR3R4, -(alkylene)m-C(0)-NR3R4; -(alkylene)m-0-R5, -(alkylene)m- S(0)n-R5, or -(alkylene)m- S(0)n-NR3R4 any of which, other than heterocyclo, may be optionally independently substituted with one or more Rx groups as allowed by valence, and wherein two Rx groups bound to the same or adjacent atoms may optionally combine to form a ring;
each R1 is independently aryl, alkyl, cycloalkyl or haloalkyl, wherein each of said alkyl, cycloalkyl and haloalkyl groups optionally includes O or N heteroatoms in place of a carbon in the chain and two Rx'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;
y is 0, 1, 2, 3 or 4;
R2 is -(alkylene)m-heterocyclo, -(alkylene)m-heteroaryl, -(alkylene)m-NR3R4, -(alkylene)m- C(0)-NR3R4; -(alkylene)m-C(0)-0-alkyl; -(alkylene)m-0-R5, -(alkylene)m-S(0)n-R5, or - (alkylene)m-S(0)n-NR3R4 any of which, other than heterocyclo, may be optionally
independently substituted with one or more Rx groups as allowed by valance, and wherein two Rx groups bound to the same or adjacent atom may optionally combine to form a ring and wherein m is 0, 1 , or 2 and n is 0, 1 or 2; wherein heterocyclo may be optionally independently substituted with 1 to 3 Rx groups as allowed by valance, and wherein two Rx groups bound to the same or adjacent atom may optionally combine to form a ring;
R3 and R4 at each occurrence are independently:
(i) hydrogen or
(ii) alkyl, cycloalkyl, heterocyclo, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl any of which, other than heterocyclo, may be optionally independently substituted with one or more Rx groups as allowed by valance, and wherein two Rx groups bound to the same or adjacent atom may optionally combine to form a ring; or R3 and R4 together with the nitrogen atom to which they are attached may combine to form a heterocyclo ring optionally independently substituted with one or more Rx groups as allowed by valance, and wherein two Rx groups bound to the same or adjacent atom may optionally combine to form a ring;
R5 and R5* at each occurrence is:
(i) hydrogen or
(ii) alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl any of which, other than
heterocyclo, may be optionally independently substituted with one or more Rx groups as allowed by valance;
Rx at each occurrence is independently, halo, cyano, nitro, oxo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, heterocycloalkyl, -(alkylene)m-OR5, -(alkylene)m-0-alkylene-OR5, -(alkylene)m-S(0)n-R5, -(alkylene)m-NR3R4, -(alkylene)m-CN, -(alkylene)m-C(0)-R5, -(alkylene)m-C(S)-R5, -(alkylene)m-C(0)-OR5, -(alkylene)m-0-C(0)-R5,
-(alkylene)m-C(S)-OR5, -(alkylene)m-C(0)-(alkylene)m-NR3R4, -(alkylene)m-C(S)- NR3R4, -(alkylene)m-N(R3)-C(0)-NR3R4, -(alkylene)m-N(R3)-C(S)-NR3R4,
-(alkylene)m-N(R3)-C(0)-R5, -(alkylene)m-N(R3)-C(S)-R5, -(alkylene)m-0-C(0)- NR3R4, -(alkylene)m-0-C(S)-NR3R4, -(alkylene)m-S02-NR3R4, -(alkylene)m-N(R3)- S02-R5, -(alkylene)m-N(R3)-S02-NR3R4, -(alkylene)m-N(R3)-C(0)-OR5, -(alkylene)m- N(R3)-C(S)-OR5, or -(alkylene)m-N(R3)-S02-R5; wherein: said alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, and heterocycloalkyl groups, any of which, other than heterocyclo, may be further independently substituted with one or more
-(alkylene)m-CN, -(alkylene)m-OR5*, -(alkylene)m-S(0)n-R5*,
-(alkylene)m-NR3 *R4 * , -(alkylene)m-C(0)-R5 * , -(alkylene)m-C(=S)R5 * , -(alkylene)m-C(=0)OR5 * , -(alkylene)m-OC(=0)R5 * , -(alkylene)m-C(S)-OR5 * , -(alkylene)m-C(0)-NR3 *R4* , -(alkylene)m-C(S)-NR3 *R4* ,
-(alkylene)m-N(R *)-C(0)-NR3 *R4* , -(alkylene)m-N(R3 *)-C(S)-NR3*R4* , -(alkylene)m-N(R3 *)-C(0)-R5 * , -(alkylene)m-N(R3 *)-C(S)-R5 * , -(alkylene)m-0-C(0)-NR35!:R4*, -(alkylene)m-0-C(S)-NR3!i:R4*, -(alkylene)m-S02-NR3!i:R4*, -(alkylene)m-N(R3>!:)-S02-R5*,
-(alkylene)m-N(R3 *)-S02-NR3 *R4* , -(alkylene)m-N(R3 *)-C(0)-OR5 * , -(alkylene)m-N(R3*)-C(S)-OR5*, or -(alkylene)m-N(R3*)-S02-R5*, and wherein heterocycle may be further independently substituted with one to three
substitutions selected from
-(alkylene)m-CN, -(alkylene)m-OR5*, -(alkylene)m-S(0)n-R5*,
-(alkylene)m-NR *R4*, -(alkylene)m-C(0)-R5*, -(alkylene)m-C(=S)R5*, -(alkylene)m-C(=0)OR5 * , -(alkylene)m-OC(=0)R5 * , -(alkylene)m-C(S)-OR5 * , -(alkylene)m-C(0)-NR3 *R4* , -(alkylene)m-C(S)-NR3 *R4* ,
-(alkylene)m-N(R3 *)-C(0)-NR3 *R4* , -(alkylene)m-N(R3 ^-C(S)-NR3*R4* , -(alkylene)m-N(R3 *)-C(0)-R5 * , -(alkylene)m-N(R3 *)-C(S)-R5 * ,
-(alkylene)m-0-C(0)-NR3*R4*, -(alkylene)m-0-C(S)-NR3*R4*, -(alkylene)m-S02-NR3 *R4* , -(alkylene)m-N(R3 *)-S02-R5* ,
-(alkylene)m-N(R3 *)-S02-NR3 *R4* , -(alkylene)m-N(R3 *)-C(0)-OR5 * , -(alkylene)m-N(R *)-C(S)-OR5 *, or -(alkylene)m-N(R3 *)-S02-R5 * ; n is 0, 1 or 2, and
m is 0, 1; or 2 and
and R4* at each occurrence are independently:
(i) hydrogen or (ii) alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl any of which, other than heterocyclo, may be optionally independently substituted with one or more Rx groups as allowed by valance; or R3* and R4* together with the nitrogen atom to which they are attached may combine to form a heterocyclo ring optionally independently substituted with one or more Rx groups as allowed by valance;
R6 is H, absent, or lower alkyl, -(alkylene)m-heterocyclo, -(alkylene)m-heteroaryl, -(alkylene)m-
NR3R4,
-(alkylene)m-C(0)-NR3R4; -(alkylene)m-0-R5, -(alkylene)m-S(0)n-R5, or -(alkylene)m-S(0)n- NR3R4 any of which, other than heterocyclo, may be optionally independently substituted with one or more Rx groups as allowed by valence, and wherein two Rx groups bound to the same or adjacent atoms may optionally combine to form a ring; and
R10 is (i) NHRA, wherein RA is unsubstituted or substituted Ci-Cg alkyl, cycloalkylalkyl, or -TT- RR, Ci-Cg cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O, and S; TT is an unsubstituted or substituted Ci-Cg alkyl or C3-C8 cycloalkyl linker; and RRis a hydroxyl, unsubstituted or substituted Ci-C6 alkoxy, amino, unsubstituted or substituted Ci-C6 alkylamino, unsubstituted or substituted di-Ci-C6 alkylamino, unsubstituted or substituted C6-Cio aryl, unsubstituted or substituted heteroaryl comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S, unsubstituted or substituted C3-C10 carbocycle, or unsubstituted or substituted heterocycle comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S; or (ii) -C(0)-R12 or -C(0)0-R13, wherein R12 is NHRA or RA andR13 is RA;
when the compound of Formula I, II, III, IV, or V has a double bond, as indicated by the (— ), in the 6-membered ring fused to the pyrimidine ring, two R8 groups are present as allowed for in Formula I, II, III, IV, or V above; or
when the compound of Formula I, II, III, IV, or V does not include a double bond, as indicated by the (— ), in the 6-membered ring fused to the pyrimidine ring, four R8 groups are present as allowed for in Formula I, II, III, IV, or V above;
wherein each heteroaryl is an aryl ring system that contains one or more heteroatoms selected from the group O, N and S, wherein the ring nitrogen and sulfur atom(s) are optionally oxidized, and nitrogen atom(s) are optionally quarternized; wherein each aryl is a carbocyclic aromatic system containing one or two rings, wherein such rings may be attached together in a fused manner, and wherein each aryl may have 1 or more Rx substituents;
wherein each heterocyclo is a saturated or partially saturated heteroatom-containing ring radical, where the heteroatoms may be selected from nitrogen, sulfur and oxygen, wherein each heterocyclo is a monocyclic 6-8 membered ring or a 5-16 membered bicyclic ring system, and wherein each heterocyclo may have 1 to 3 Rx substituents;
or a pharmaceutically acceptable salt thereof.
41. A method for reducing the effect of ionizing radiation exposure on cyclin-dependent kinase 4 (CDK4) replication-dependent hematopoietic stem cells and/or progenitor cells
(HSPCs) in a subject exposed to ionizing radiation, the method comprising administering to the subject an effective amount of a compound of Formula VI:
Figure imgf000165_0001
wherein R, R1, R2, R3, R4, R5, R6, Rx, Z, m, n, and y are as defined in claim 40;
each R14 is independently H, C1-C3 alkyl (including methyl) or haloalkyl, cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O or S; -(alkylene)m- C3-C8 cycloalkyl, -(alkylene)m-aryl, -(alkylene)m-heterocyclo, -(alkylene)m- heteroaryl, -(alkylene)m-
NR3R4, -(alkylene)m-C(0)-NR3R4; -(alkylene)m-0-R5, -(alkylene)m- S(0)n-R5, or -(alkylene)m-
S(0)n-NR3R4 any of which, other than heterocyclo, may be optionally independently substituted with one or more Rx groups as allowed by valence, and wherein two Rx groups bound to the same or adjacent atoms may optionally combine to form a ring;
or two R14 groups bonded to the same carbon can form an exocyclic double bond;
or two R14 groups bonded to the same carbon can form a carbonyl group; and
when the compound of Formula VI has a double bond, as indicated by the (— ), in the 6- membered ring fused to the pyrimidine ring, two R14 groups are present as allowed for in
Formula VI above; or when the compound of Formula VI does not include a double bond, as indicated by the (— ), in the 6-membered ring fused to the pyrimidine ring, four R14 groups are present as allowed for in Formula VI above;
wherein each heteroaryl is an aryl ring system that contains one or more heteroatoms selected from the group O, N and S, wherein the ring nitrogen and sulfur atom(s) are optionally oxidized, and nitrogen atom(s) are optionally quarternized;
wherein each aryl is a carbocyclic aromatic system containing one or two rings, wherein such rings may be attached together in a fused manner, and wherein each aryl may have 1 or more Rx substituents;
wherein each heterocyclo is a saturated or partially saturated heteroatom-containing ring radical, where the heteroatoms may be selected from nitrogen, sulfur and oxygen, wherein each heterocyclo is a monocyclic 6-8 membered ring or a 5-16 membered bicyclic ring system, and wherein each heterocyclo may have 1 to 3 Rx substituents;
or a pharmaceutically acceptable salt thereof.
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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9464092B2 (en) 2013-03-15 2016-10-11 G1 Therapeutics, Inc. Transient protection of normal cells during chemotherapy
US9717735B2 (en) 2014-04-17 2017-08-01 G1 Therapeutics, Inc. Tricyclic lactams for use in HSPC-sparing treatments for RB-positive abnormal cellular proliferation
WO2018005865A1 (en) 2016-07-01 2018-01-04 G1 Therapeutics, Inc. Synthesis of n-(heteroaryl)-pyrrolo[3,2-d]pyrimidin-2-amines
WO2019006393A1 (en) 2017-06-29 2019-01-03 G1 Therapeutics, Inc. Morphic forms of git38 and methods of manufacture thereof
US10231969B2 (en) 2014-09-12 2019-03-19 GI Therapeutics, Inc. Anti-neoplastic combinations and dosing regimens using CDK4/6 inhibitor compounds to treat RB-positive tumors
US10413547B2 (en) 2014-09-12 2019-09-17 G1 Therapeutics, Inc. Treatment of Rb-negative tumors using topoisomerase with cyclin dependent kinase 4/6 inhibitors
US10618905B2 (en) 2016-07-01 2020-04-14 G1 Therapeutics, Inc. Pyrimidine-based compounds for the treatment of cancer
US10654831B2 (en) 2016-07-01 2020-05-19 G1 Therapeutics, Inc. Antiproliferative pyrimidine-based compounds
US10709711B2 (en) 2013-03-15 2020-07-14 G1 Therapeutics, Inc. Highly active anti-neoplastic and anti-proliferative agents
US10829490B2 (en) 2016-07-01 2020-11-10 GI Therapeutics, Inc. Substituted dihydropyrazino[1 ′,2′:1,5]pyrrolo[2,3-d]pyrimidine-based antiproliferative agents
US10988479B1 (en) 2020-06-15 2021-04-27 G1 Therapeutics, Inc. Morphic forms of trilaciclib and methods of manufacture thereof
US11357779B2 (en) 2018-01-08 2022-06-14 G1 Therapeutics, Inc. G1T38 superior dosage regimes
US11395821B2 (en) 2017-01-30 2022-07-26 G1 Therapeutics, Inc. Treatment of EGFR-driven cancer with fewer side effects
US11529352B2 (en) 2016-12-05 2022-12-20 G1 Therapeutics, Inc. Preservation of immune response during chemotherapy regimens

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8691830B2 (en) 2010-10-25 2014-04-08 G1 Therapeutics, Inc. CDK inhibitors
CN107286180B (en) * 2016-04-11 2019-07-02 上海勋和医药科技有限公司 Miscellaneous generation Pyridopyrimidinone derivatives are as CDK inhibitor and its application
EP3500570B1 (en) * 2016-08-19 2022-10-05 Arena Pharmaceuticals, Inc. 5-ht2c receptor agonists and compositions and methods of use
CN110177554B (en) 2017-01-06 2023-06-02 G1治疗公司 Combination therapy for the treatment of cancer
EP3840756A4 (en) 2018-08-24 2022-04-27 G1 Therapeutics, Inc. Improved synthesis of 1,4-diazaspiro[5.5]undecan-3-one
CN113271977A (en) * 2018-11-09 2021-08-17 G1治疗公司 Therapeutic regimens for treating cancer using eribulin in combination with selective CDK4/6 inhibitors
EP3999065A4 (en) * 2019-06-21 2023-09-13 Pattern Computer, Inc. Therapeutic compositions and methods for treating cancers
EP4153597A1 (en) 2020-05-19 2023-03-29 G1 Therapeutics, Inc. Cyclin-dependent kinase inhibiting compounds for the treatment of medical disorders
CN112094241B (en) * 2020-09-19 2023-04-18 浙江凯普化工有限公司 Preparation method of 1, 4-diazaspiro [5,5] undecane-3-ketone
CN115925559A (en) * 2022-11-11 2023-04-07 上海泰坦科技股份有限公司 Preparation method of 1-amino-1-cyclopentyl methanol
CN115974703A (en) * 2022-12-21 2023-04-18 上海泰坦科技股份有限公司 Preparation method of hydroxymethyl-substituted N, N-dimethyl alicyclic amine

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013148748A1 (en) * 2012-03-29 2013-10-03 Francis Xavier Tavares Lactam kinase inhibitors
US20130289031A1 (en) * 2010-12-23 2013-10-31 Sanofi Pyrimidinone derivatives, preparation thereof and pharmaceutical use thereof
WO2013163239A1 (en) * 2012-04-26 2013-10-31 Francis Xavier Tavares Synthesis of lactams
US8598186B2 (en) * 2010-10-25 2013-12-03 G1 Therapeutics, Inc. CDK inhibitors

Family Cites Families (59)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5591855A (en) 1994-10-14 1997-01-07 Cephalon, Inc. Fused pyrrolocarbazoles
US5628984A (en) 1995-07-31 1997-05-13 University Of North Carolina At Chapel Hill Method of detecting lung disease
EP0964864B1 (en) 1997-02-05 2008-04-09 Warner-Lambert Company LLC Pyrido 2,3-d pyrimidines and 4-aminopyrimidines as inhibitors of cellular proliferation
GB9718913D0 (en) 1997-09-05 1997-11-12 Glaxo Group Ltd Substituted oxindole derivatives
US20040006074A1 (en) 1998-04-28 2004-01-08 The Government Of The United States Of America Cyclin dependent kinase (CDK)4 inhibitors and their use for treating cancer
CA2335115C (en) 1998-06-16 2009-01-27 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Fused azepinone cyclin dependent kinase inhibitors
WO2000044364A2 (en) 1999-01-29 2000-08-03 The Board Of Trustees Of The University Of Illinois P53 inhibitors and their use for the treatment of cancer, hyperthermia, hypoxia, a burn, trauma to the central nervous system, a seizure, acute inflammation, tissue ageing, preservation of organs for transplant and preparation of a host for bone marrow transplant
AU6314900A (en) 1999-07-26 2001-02-13 Banyu Pharmaceutical Co., Ltd. Biarylurea derivatives
US6387900B1 (en) 1999-08-12 2002-05-14 Pharmacia & Upjohn S.P.A. 3(5)-ureido-pyrazole derivatives process for their preparation and their use as antitumor agents
US6291504B1 (en) 1999-10-20 2001-09-18 Dupont Pharmaceuticals Company Acylsemicarbazides and their uses
WO2001044247A2 (en) 1999-12-16 2001-06-21 Eli Lilly And Company Agents and methods for the treatment of proliferative diseases
US7053070B2 (en) 2000-01-25 2006-05-30 Warner-Lambert Company Pyrido[2,3-d]pyrimidine-2,7-diamine kinase inhibitors
EE200200711A (en) 2000-06-26 2004-06-15 Pfizer Products Inc. Pyrrolo [2,3-d] pyrimidine compounds as immunosuppressive agents
ATE266031T1 (en) 2000-09-29 2004-05-15 Lilly Co Eli METHODS AND COMPOUNDS FOR TREATING PROLIFERATIVE DISEASES
WO2002044174A2 (en) 2000-12-01 2002-06-06 Bristol-Myers Squibb Pharma Company 3-(2,4-dimethylthiazol-5-yl) indeno[1,2-c]pyrazol-4-one derivatives as cdk inhibitors
JP4302986B2 (en) 2001-02-28 2009-07-29 テンプル・ユニバーシティ−オブ・ザ・コモンウェルス・システム・オブ・ハイアー・エデュケイション Protecting cells and tissues from ionizing radiation with α, β unsaturated aryl sulfones
WO2003062236A1 (en) 2002-01-22 2003-07-31 Warner-Lambert Company Llc 2-(PYRIDIN-2-YLAMINO)-PYRIDO[2,3d]PYRIMIDIN-7-ONES
AU2003221584A1 (en) 2002-05-01 2003-11-17 Chromos Molecular Systems, Inc. Methods for delivering nucleic acid molecules into cells and assessment thereof
DE602004021558D1 (en) 2003-01-17 2009-07-30 Warner Lambert Co 2-AMINOPYRIDINE SUBSTITUTED HETEROCYCLES AS INHIBITORS OF CELLULAR PROLIFERATION
CA2526578C (en) 2003-05-22 2012-01-24 Pharmacia Italia S.P.A. Pyrazolo-quinazoline derivatives, process for their preparation and their use as kinase inhibitors
EP1648889B1 (en) 2003-07-11 2008-10-29 Warner-Lambert Company LLC Isethionate salt of a selective cdk4 inhibitor
MXPA06004305A (en) 2003-10-23 2006-06-05 Hoffmann La Roche Triaza-spiropiperidine derivatives for use as glyt-1 inhibitors in the treatment of neurological and neuropsychiatric disorders.
GB0327380D0 (en) 2003-11-25 2003-12-31 Cyclacel Ltd Method
BRPI0509580A (en) 2004-03-30 2007-11-27 Pfizer Prod Inc signal transduction inhibitor combinations
WO2005100999A2 (en) 2004-04-08 2005-10-27 Cornell Research Foundation, Inc. Functional immunohistochemical cell cycle analysis as a prognostic indicator for cancer
ITMI20040874A1 (en) 2004-04-30 2004-07-30 Ist Naz Stud Cura Dei Tumori INDOLIC AND AZAINDOLIC DERIVATIVES WITH ANTI-TUMORAL ACTION
AU2006205851A1 (en) 2005-01-14 2006-07-20 Janssen Pharmaceutica N.V. 5-membered annelated heterocyclic pyrimidines as kinase inhibitors
BRPI0606319A2 (en) 2005-01-21 2009-06-16 Astex Therapeutics Ltd pharmaceutical compounds
MX2007014619A (en) 2005-05-20 2009-02-13 Vertex Pharma Pyrrolopyridines useful as inhibitors of protein kinase.
US20070049591A1 (en) 2005-08-25 2007-03-01 Kalypsys, Inc. Inhibitors of MAPK/Erk Kinase
EP1779848A1 (en) 2005-10-28 2007-05-02 Nikem Research S.R.L. V-ATPase inhibitors for the treatment of inflammatory and autoimmune diseases
CN101321761A (en) 2005-12-09 2008-12-10 霍夫曼-拉罗奇有限公司 Tricyclic amide derivatives useful for treating obesity
BRPI0620151A2 (en) 2005-12-22 2010-06-29 Wyeth Corp compound; pharmaceutical composition; method of treating or inhibiting the growth of cancerous tumor cells in a mammal in need thereof; method; and process
CN101415409B (en) 2006-04-05 2012-12-05 诺瓦提斯公司 Combinations of therapeutic agents for treating cancer
US20070270362A1 (en) 2006-05-18 2007-11-22 The University Of Washington Methods and compositions for prevention or treatment of inflammatory-related diseases and disorders
US8222256B2 (en) 2006-07-05 2012-07-17 Exelixis, Inc. Methods of using IGFIR and ABL kinase modulators
EP2109450A2 (en) 2006-12-14 2009-10-21 Panacea Pharmaceuticals, Inc. Methods of neuroprotection by cyclin-dependent kinase inhibition
EA200900799A1 (en) 2006-12-22 2009-12-30 Новартис Аг Heteroarylheteroaryl compounds as inhibitors of CDK, intended for the treatment of cancer, inflammation and the fight against viral infections
EP2162451A4 (en) 2007-06-25 2012-04-18 Neurogen Corp Piperazinyl oxoalkyl tetrahydro-beta-carbolines and related analogues
US9259399B2 (en) 2007-11-07 2016-02-16 Cornell University Targeting CDK4 and CDK6 in cancer therapy
EA201001030A1 (en) 2007-12-19 2011-02-28 Амген Инк. Condensed Compounds of Pyridine, Pyrimidine, and Triazine as Cellular Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Cycle Capsule inhibitors
ES2622559T3 (en) 2008-07-29 2017-07-06 Nerviano Medical Sciences S.R.L. Therapeutic combination comprising a cdk inhibitor and an antineoplastic agent
WO2010020675A1 (en) 2008-08-22 2010-02-25 Novartis Ag Pyrrolopyrimidine compounds as cdk inhibitors
JP2012504645A (en) 2008-10-01 2012-02-23 ザ ユニバーシティ オブ ノース カロライナ アット チャペル ヒル Pharmaceutical composition for reducing or preventing the influence of ionizing radiation on healthy cells
JP2012504646A (en) 2008-10-01 2012-02-23 ザ ユニバーシティ オブ ノース カロライナ アット チャペル ヒル Protection of the hematopoietic system against chemotherapeutic compounds using selective cyclin-dependent kinase 4/6 inhibitors
JP2012526850A (en) 2009-05-13 2012-11-01 ザ ユニバーシティ オブ ノース カロライナ アット チャペル ヒル Cyclin-dependent kinase inhibitors and uses thereof
WO2011103485A1 (en) 2010-02-18 2011-08-25 Medivation Technologies, Inc. Fused tetracyclic pyrido[4,3-b]indole and pyrido[3,4-b]indole derivatives and methods of use
UY33227A (en) 2010-02-19 2011-09-30 Novartis Ag PIRROLOPIRIMIDINE COMPOUNDS AS INHIBITORS OF THE CDK4 / 6
US8691830B2 (en) 2010-10-25 2014-04-08 G1 Therapeutics, Inc. CDK inhibitors
WO2012068381A2 (en) 2010-11-17 2012-05-24 The University Of North Carolina At Chapel Hill Protection of renal tissues from schema through inhibition of the proliferative kisses cdk4 and cdk6
ES2676180T3 (en) 2011-07-01 2018-07-17 Novartis Ag Combination therapy comprising a CDK4 / 6 inhibitor and a PI3K inhibitor for use in the treatment of cancer
US20140274896A1 (en) 2013-03-15 2014-09-18 G1 Therapeutics, Inc. Transient Protection of Hematopoietic Stem and Progenitor Cells Against Ionizing Radiation
JP6435315B2 (en) 2013-03-15 2018-12-05 ジー１、セラピューティクス、インコーポレイテッドＧ１ Ｔｈｅｒａｐｅｕｔｉｃｓ， Ｉｎｃ． Highly active antineoplastic and antiproliferative agents
CN105407889B (en) 2013-03-15 2018-06-01 G1治疗公司 For the regulated treatments of HSPC of Rb positive abnormal cell proliferations
WO2014168975A1 (en) 2013-04-08 2014-10-16 Pharmacyclics, Inc. Ibrutinib combination therapy
US20160257688A1 (en) 2013-10-24 2016-09-08 Francis Xavier Tavares Process for Synthesis of Lactams
WO2015161283A1 (en) 2014-04-17 2015-10-22 G1 Therapeutics, Inc. Tricyclic lactams for use in hspc-sparing treatments for rb-positive abnormal cellular proliferation
WO2016040848A1 (en) 2014-09-12 2016-03-17 G1 Therapeutics, Inc. Treatment of rb-negative tumors using topoisomerase inhibitors in combination with cyclin dependent kinase 4/6 inhibitors
WO2016040858A1 (en) 2014-09-12 2016-03-17 G1 Therapeutics, Inc. Combinations and dosing regimes to treat rb-positive tumors

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8598186B2 (en) * 2010-10-25 2013-12-03 G1 Therapeutics, Inc. CDK inhibitors
US20130289031A1 (en) * 2010-12-23 2013-10-31 Sanofi Pyrimidinone derivatives, preparation thereof and pharmaceutical use thereof
WO2013148748A1 (en) * 2012-03-29 2013-10-03 Francis Xavier Tavares Lactam kinase inhibitors
WO2013163239A1 (en) * 2012-04-26 2013-10-31 Francis Xavier Tavares Synthesis of lactams

Cited By (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10966984B2 (en) 2013-03-15 2021-04-06 G1 Therapeutics, Inc. Transient protection of normal cells during chemotherapy
US10434104B2 (en) 2013-03-15 2019-10-08 G1 Therapeutics, Inc. HSPC-sparing treatments for Rb-positive abnormal cellular proliferation
US10660896B2 (en) 2013-03-15 2020-05-26 GI Therapeutics, Inc. Transient protection of normal cells during chemotherapy
US11040042B2 (en) 2013-03-15 2021-06-22 G1 Therapeutics, Inc. Transient protection of normal cells during chemotherapy
US9931345B2 (en) 2013-03-15 2018-04-03 Presidents And Fellows Of Harvard College Transient protection of normal cells during chemotherapy
US10076523B2 (en) 2013-03-15 2018-09-18 G1 Therapeutics, Inc. HSPC-sparing treatments for RB-positive abnormal cellular proliferation
US10085992B2 (en) 2013-03-15 2018-10-02 G1 Therapeutics, Inc. Transient protection of normal cells during chemotherapy
US9527857B2 (en) 2013-03-15 2016-12-27 GI Therapeutics, Inc. HSPC-sparing treatments for RB-positive abnormal cellular proliferation
US9464092B2 (en) 2013-03-15 2016-10-11 G1 Therapeutics, Inc. Transient protection of normal cells during chemotherapy
US10925878B2 (en) 2013-03-15 2021-02-23 G1 Therapeutics, Inc. HSPC-sparing treatments for RB-positive abnormal cellular proliferation
US10709711B2 (en) 2013-03-15 2020-07-14 G1 Therapeutics, Inc. Highly active anti-neoplastic and anti-proliferative agents
US10376519B2 (en) 2014-04-17 2019-08-13 G1 Therapeutics, Inc. Tricyclic lactams for use in HSPC-sparing treatments for Rb-positive abnormal cellular proliferation
US9717735B2 (en) 2014-04-17 2017-08-01 G1 Therapeutics, Inc. Tricyclic lactams for use in HSPC-sparing treatments for RB-positive abnormal cellular proliferation
US11446295B2 (en) 2014-09-12 2022-09-20 G1 Therapeutics, Inc. Anti-neoplastic combinations and dosing regimens using CDK4/6 inhibitor compounds to treat Rb-positive tumors
US11090306B2 (en) 2014-09-12 2021-08-17 G1 Therapeutics, Inc. Treatment of Rb-negative tumors using topoisomerase inhibitors in combination with cyclin dependent kinase 4/6 inhibitors
US10413547B2 (en) 2014-09-12 2019-09-17 G1 Therapeutics, Inc. Treatment of Rb-negative tumors using topoisomerase with cyclin dependent kinase 4/6 inhibitors
US10231969B2 (en) 2014-09-12 2019-03-19 GI Therapeutics, Inc. Anti-neoplastic combinations and dosing regimens using CDK4/6 inhibitor compounds to treat RB-positive tumors
EP3858835A1 (en) 2016-07-01 2021-08-04 G1 Therapeutics, Inc. Pyrimidine-based antiproliferative agents
US10829490B2 (en) 2016-07-01 2020-11-10 GI Therapeutics, Inc. Substituted dihydropyrazino[1 ′,2′:1,5]pyrrolo[2,3-d]pyrimidine-based antiproliferative agents
US10654831B2 (en) 2016-07-01 2020-05-19 G1 Therapeutics, Inc. Antiproliferative pyrimidine-based compounds
WO2018005865A1 (en) 2016-07-01 2018-01-04 G1 Therapeutics, Inc. Synthesis of n-(heteroaryl)-pyrrolo[3,2-d]pyrimidin-2-amines
US10618905B2 (en) 2016-07-01 2020-04-14 G1 Therapeutics, Inc. Pyrimidine-based compounds for the treatment of cancer
US11529352B2 (en) 2016-12-05 2022-12-20 G1 Therapeutics, Inc. Preservation of immune response during chemotherapy regimens
US11395821B2 (en) 2017-01-30 2022-07-26 G1 Therapeutics, Inc. Treatment of EGFR-driven cancer with fewer side effects
WO2019006393A1 (en) 2017-06-29 2019-01-03 G1 Therapeutics, Inc. Morphic forms of git38 and methods of manufacture thereof
US11357779B2 (en) 2018-01-08 2022-06-14 G1 Therapeutics, Inc. G1T38 superior dosage regimes
US10988479B1 (en) 2020-06-15 2021-04-27 G1 Therapeutics, Inc. Morphic forms of trilaciclib and methods of manufacture thereof

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