Tricyclic Lactams for Use in the Protection of Hematopoietic Stem and Progenitor Cells Against Ionizing Radiation

- G1 THERAPEUTICS, INC.

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.

Skip to: Description  ·  Claims  · Patent History  ·  Patent History
Description
RELATED APPLICATIONS

This application claims the benefit of provisional U.S. Application No. 61/980,883, filed Apr. 17, 2014, provisional U.S. Application No. 61/980,895, filed Apr. 17, 2014, provisional U.S. Application No. 61/980,918, filed Apr. 17, 2014, and provisional U.S. Application No. 61/980,939, filed Apr. 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 G1 and G2. Non-dividing cells remain in a resting or quiescence stage named G0 before they re-enter into phase G1. Early G1 and late S phases are relatively radioresistant. Conversely, the G1/S transition and G2/M phases are relatively radiosensitive (see Sinclair W K, Morton R A. X-ray sensitivity during cell generation cycle of cultured Chinese hamster cells. Radiat. Res. 1966; 29(3):450-474; Terasuna T, Tolmach L J. X-ray sensitivity and DNA synthesis in synchronous populations of HeLa cells. Science, 1963; 140:490-92.). Transversing from G1 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 J B. 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 G1 after exposure to DNA-damaging agents enhances resistance to such agents, possibly by allowing for greater DNA repair prior to G1/S transversal (Elkind M M, Sutton H. X-ray damage and recovery in mammalian cells in culture. Nature, 1959; 184: 1293-1295; Elkind M M, 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 FIG. 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-1-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 S1-6, 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 (11) 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 (11) 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 (Jul. 1, 1996) describes reversible, p16-mediated cell cycle arrest as protection from chemotherapy.

WO 2012/061156 filed by Tavares and assigned to G1 Therapeutics describes CDK inhibitors (see also, U.S. Pat. Nos. 8,829,012, 8,822,683, 8,598,186, 8,691,830, and 8,598,197, all assigned to G1 Therapeutics), describe CDK Inhibitors having the basic core structure:

WO 2013/148748 filed by Tavares and assigned to G1 Therapeutics describes Lactam Kinase inhibitors having the basic core structures:

U.S. Patent Publication 2014/0275066 and 2014/0275067, assigned to G1 Therapeutics, describes the use of CDK4/6 inhibitors such as 2′-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-7′,8′-dihydro-6′H-spiro[cyclohexane-1,9′-pyrazino[1′,2′:1,5]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 G1 Therapeutics, describes the use of CDK4/6 inhibitors such as 2′-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-7′,8′-dihydro-6′H-spiro[cyclohexane-1,9′-pyrazino[1′,2′:1,5]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 G1 Therapeutics, describes the use of CDK4/6 inhibitors such as 2′-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-7′,8′-dihydro-6′H-spiro[cyclohexane-1,9′-pyrazino[1′,2′:1,5]pyrrolo[2,3-d]pyrimidin]-6′-one for use as an anti-neoplastic for the treatment of a Rb-positive proliferative disorders.

U.S. Patent Publication 2014/0271460, assigned to G1 Therapeutics, describes the use of CDK4/6 inhibitors such as 2′-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-7′,8′-dihydro-6′H-spiro[cyclohexane-1,9′-pyrazino[1′,2′:1,5]pyrrolo[2,3-d]pyrimidin]-6′-one for use an anti-neoplastic for the treatment of a T- or B-cell disorder, for example a leukemia.

Stone et al., Cancer Research 56, 3199-3202 (Jul. 1, 1996) describes reversible, p16-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-1-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 G1-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 G1-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 G1-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 G1 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 radio-protective 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 cyclin-dependent kinase 1 (CDK1), increased activity of cyclin-dependent kinase 2 (CDK2), loss, deficiency, or absence of retinoblastoma tumor suppressor protein (Rb)(Rb-null), high levels of 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 G1-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 G1-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 G1-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, N.J., 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-butyl, 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 “N,N-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, N,N-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, dichlorofluoromethyl, 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[1,4]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-1H-3-aza-fluorenyl, 5,6,7-trihydro-1,2,4-triazolo[3,4-a]isoquinolyl, 3,4-dihydro-2H-benzo[1,4]oxazinyl, benzo[1,4]dioxanyl, 2,3-dihydro-1H-1λ′-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[1,5-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[1,4]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-1,2,4-triazolyl, IH-1,2,3-triazolyl, 2H-1,2,3-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 —SO2—.

The terms “carboxy” or “carboxyl”, whether used alone or with other terms, such as “carboxyalkyl”, denotes —C(O)—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(O)—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 —NO2.

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 G1-arrest” is meant that the tricyclic lactam compound induces a quiescent state in a substantial portion of a cell population at the G1 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 μM; >1.0 μM, or >5.0 μM), so long as the tricyclic lactam provides selective G1 arrest in CDK4/6-dependent cells.

By “synchronous reentry into the cell cycle” is meant that HSPC cells in G1-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 G1 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:

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 —O—(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(O)—NR3R4; -(alkylene)m-O—R5, -(alkylene)m-S(O)n—R5, or -(alkylene)m-S(O)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;
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 R1'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(O)—NR3R4; -(alkylene)m-C(O)—O-alkyl; -(alkylene)m-O—R5, -(alkylene)m-S(O)n—R5, or -(alkylene)m-S(O)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-O-alkylene-OR5, -(alkylene)m-S(O)n—R5, -(alkylene)m-NR3R4, -(alkylene)m-CN, -(alkylene)m-C(O)—R5, -(alkylene)m-C(S)—R5, -(alkylene)m-C(O)—OR5, -(alkylene)m-O—C(O)—R5, -(alkylene)m-C(S)—OR5, -(alkylene)m-C(O)-(alkylene)m-NR3R4, -(alkylene)m-C(S)—NR3R4, -(alkylene)m-N(R3)—C(O)—NR3R4, -(alkylene)m-N(R3)—C(S)—NR3R4, -(alkylene)m-N(R3)—C(O)—R5, -(alkylene)m-N(R3)—C(S)—R5, -(alkylene)m-O—C(O)—NR3R4, -(alkylene)m-O—C(S)—NR3R4, -(alkylene)m-SO2—NR3R4, -(alkylene)m-N(R3)—SO2—R5, -(alkylene)m-N(R3)—SO2—NR3R4, -(alkylene)m-N(R3)—C(O)—OR5) -(alkylene)m-N(R3)—C(S)—OR5, or -(alkylene)m-N(R3)—SO2—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(O)n—R5*, -(alkylene)m-NR3*R4*, -(alkylene)m-C(O)—R5*, -(alkylene)m-C(═S)R5*, -(alkylene)m-C(═O)OR5*, -(alkylene)m-OC(═O)R5*, -(alkylene)m-C(S)—OR5*, -(alkylene)m-C(O)—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(O)—R5*, -(alkylene)m-N(R3*)—C(S)—R5*, -(alkylene)m-O—C(O)—NR3*R4*, -(alkylene)m-O—C(S)—NR3*R4*, -(alkylene)m-SO2—NR3*R4*, -(alkylene)m-N(R3*)—SO2—R5*, -(alkylene)m-N(R3*)—SO2—NR3*R4*, -(alkylene)m-N(R3*)—C(O)—OR5*, -(alkylene)m-N(R3*)—C(S)—OR5*, or -(alkylene)m-N(R3*)—SO2—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 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
      R6 is H or lower alkyl, -(alkylene)m-heterocyclo, -(alkylene)m-heteroaryl, -(alkylene)m-NR3R4, -(alkylene)m-C(O)—NR3R4; -(alkylene)m-O—R5, -(alkylene)m-S(O)n—R5, or -(alkylene)m-S(O)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 1 (i) NHRA, wherein RA is unsubstituted or substituted C1-C8 alkyl, cycloalkylalkyl, or -TT-RR, C1-C8 cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O, and S; TT is an unsubstituted or substituted C1-C8 alkyl or C3-C8 cycloalkyl linker; and RR is a hydroxyl, unsubstituted or substituted C1-C6 alkoxy, amino, unsubstituted or substituted C1-C6 alkylamino, unsubstituted or substituted di-C1-C6 alkylamino, unsubstituted or substituted C6-C10 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(O)—R12 or —C(O)O—R13, wherein R12 is NHRA or RA and R13 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:

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(O)—NR3R4; -(alkylene)m-O—R5, -(alkylene)m-S(O)n—R5, or -(alkylene)m-S(O)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, 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:

or a pharmaceutically acceptable salt thereof;
wherein:
Z is —(CH2)x— wherein x is 1, 2, 3 or 4 or —O—(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(O)—NR3R4; -(alkylene)m-O—R5, -(alkylene)m-S(O)n—R5, or -(alkylene)m-S(O)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 R1'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(O)—NR3R4; -(alkylene)m-C(O)—O-alkyl; -(alkylene)m-O—R5, -(alkylene)m-S(O)n—R5, or -(alkylene)m-S(O)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-O-alkylene-OR5, -(alkylene)m-S(O)n—R5, -(alkylene)m-NR3R4, -(alkylene)m-CN, -(alkylene)m-C(O)—R5, -(alkylene)m-C(S)—R5, -(alkylene)m-C(O)—OR5, -(alkylene)m-O—C(O)—R5, -(alkylene)m-C(S)—OR5, -(alkylene)m-C(O)-(alkylene)m-NR3R4, -(alkylene)m-C(S)—NR3R4, -(alkylene)m-N(R3)—C(O)—NR3R4, -(alkylene)m-N(R3)—C(S)—NR3R4, -(alkylene)m-N(R3)—C(O)—R5, -(alkylene)m-N(R3)—C(S)—R5, -(alkylene)m-O—C(O)—NR3R4, -(alkylene)m-O—C(S)—NR3R4, -(alkylene)m-SO2—NR3R4, -(alkylene)m-N(R3)—SO2—R5, -(alkylene)m-N(R3)—SO2—NR3R4, -(alkylene)m-N(R3)—C(O)—OR5, -(alkylene)m-N(R3)—C(S)—OR5, or -(alkylene)m-N(R3)—SO2—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(O)n—R5*, -(alkylene)m-NR3*R4*, -(alkylene)m-C(O)—R5*, -(alkylene)m-C(═S)R5*, -(alkylene)m-C(═O)OR5*, -(alkylene)m-OC(═O)R5*, -(alkylene)m-C(S)—OR5*, -(alkylene)m-C(O)—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(O)—R5*, -(alkylene)m-N(R3*)—C(S)—R5*, -(alkylene)m-O—C(O)—NR3*R4*, -(alkylene)m-O—C(S)—NR3*R4*, -(alkylene)m-SO2—NR3*R4* , -(alkylene)m-N(R3*)—SO2—R5*, -(alkylene)m-N(R3*)—SO2—NR3*R4*, -(alkylene)m-N(R3*)—C(O)—OR5*, -(alkylene)m-N(R3*)—C(S)—OR5*, or -(alkylene)m-N(R3*)—SO2—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(O)n—R5*, -(alkylene)m-NR3*R4*, -(alkylene)m-C(O)—R5*, -(alkylene)m-C(═S)R5*, -(alkylene)m-C(═O)OR5*, -(alkylene)m-OC(═O)R5*, -(alkylene)m-C(S)—OR5*, -(alkylene)m-C(O)—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(O)—R5*, -(alkylene)m-N(R3*)—C(S)—R5*, -(alkylene)m-O—C(O)—NR3*R4*, -(alkylene)m-O—C(S)—NR3*R4*, -(alkylene)m-SO2—NR3*R4*, -(alkylene)m-N(R3*)—SO2—R5*, -(alkylene)m-N(R3*)—SO2—NR3*R4*, -(alkylene)m-N(R3*)—C(O)—OR5*, -(alkylene)m-N(R3*)—C(S)—OR5*, or -(alkylene)m-N(R3*)—SO2—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(O)—NR3R4; -(alkylene)m-O—R5, -(alkylene)m-S(O)n—R5, or -(alkylene)m-S(O)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 1 (i) NHRA, wherein RA is unsubstituted or substituted C1-C8 alkyl, cycloalkylalkyl, or -TT-RR, C1-C8 cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O, and S; TT is an unsubstituted or substituted C1-C8 alkyl or C3-C8 cycloalkyl linker; and RR is a hydroxyl, unsubstituted or substituted C1-C6 alkoxy, amino, unsubstituted or substituted C1-C6 alkylamino, unsubstituted or substituted di-C1-C6 alkylamino, unsubstituted or substituted C6-C10 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(O)—R12 or —C(O)O—R13, wherein R12 is NHRA or RA and R13 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:

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(O)—NR3R4; -(alkylene)m-O—R5, -(alkylene)m-S(O)n—R5, or -(alkylene)m-S(O)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 aspects, the compound is of Formula III:

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(O)—NR3R4; -(alkylene)m-O—R5, -(alkylene)m-S(O)n—R5, or -(alkylene)m-S(O)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(O)—NR3R4, -(alkylene)m-C(O)—O-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(O)—NR3R4, -(alkylene)m-C(O)—O-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, R2 is

wherein:
R2* is a bond, alkylene, -(alkylene)m-O-(alkylene)m-, -(alkylene)m-C(O)-(alkylene)m-, -(alkylene)m-S(O)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 Rx1 is independently -(alkylene)m-(C(O))m-(alkylene)m-(N(RN))m-(alkyl)m wherein each m is independently 0 or 1 provided at least one m is 1, —(C(O))—O-alkyl, -(alkylene)m-cycloalkyl wherein m is 0 or 1, —N(RN)-cycloalkyl, —C(O)-cycloalkyl, -(alkylene)m-heterocyclyl wherein m is 0 or 1, or —N(RN)-heterocyclyl, —C(O)-heterocyclyl, —S(O)2-(alkylene)m wherein m is 1 or 2, wherein:

    • RN is H, C1 to C4 alkyl or C1 to C6 heteroalkyl, and
    • wherein two Rx1 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 Rx1 is only optionally substituted by unsubstituted alkyl, halogen or hydroxy.

In some aspects, Rx1 is hydrogen or unsubstituted C1-C4 alkyl.

In some aspects, at least one Rx1 is -(alkylene)m-heterocyclyl wherein m is 0 or 1.

In some aspects, R2 is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group.

In some aspects, R2 is

In some aspects, R2 is

In some aspects, R2 is

wherein:
R2* is a bond, alkylene, -(alkylene)m-O-(alkylene)m-, -(alkylene)m-C(O)-(alkylene)m-, -(alkylene)m-S(O)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;
P1 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

In some aspects, P1 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. 2-4.

In some aspects, R2 is

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 aspects, the compound has general Formula Ia:

wherein R1, R2, R, R8, X and y are as previously defined.

In some embodiments, the compound has Formula Ia and R is alkyl.

In some embodiments, the compound has Formula Ia and R is H.

In some embodiments, the compound has Formula Ia and R2 is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R2*, Rx1 and t are as previously defined.

In some embodiments, the compound has Formula Ia and R2 is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, Rx1 is hydrogen or unsubstituted C1-C4 alkyl and R2* is as previously defined.

In some embodiments, the compound is of Formula Ib:

wherein R, R2 and R8 are as previously defined.

In some embodiments, the compound is of Formula Ib and R is alkyl.

In some embodiments, the compound is of Formula Ib and R is H.

In some embodiments, the compound has Formula Ib and R2 is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R2*, Rx1 and t are as previously defined.

In some embodiments, the compound has Formula Ib and R2 is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, Rx1 is hydrogen or C1-C4 alkyl and R2* is as previously defined.

In some embodiments, the compound has Formula Ic:

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

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R2*, Rx1 and t are as previously defined.

In some embodiments, the compound has Formula Ic and R2 is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, Rx1 is hydrogen or C1-C4 alkyl and R2* is as previously defined.

In some embodiments, the compound has Formula Id:

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 Id and R2 is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R2*, Rx1 and t are as previously defined.

In some embodiments, the compound has Formula Id and R2 is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, Rx1 is hydrogen or C1-C4 alkyl and R2* is as previously defined.

In some embodiments, the compound has Formula Ie:

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

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R2*, Rx1 and t are as previously defined.

In some embodiments, the compound has Formula Ie and R2 is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, Rx1 is hydrogen or C1-C4 alkyl and R2* is as previously defined.

In some embodiments, the compound has Formula If:

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

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R2*, Rx1 and t are as previously defined.

In some embodiments, the compound has Formula If and R2 is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, Rx1 is hydrogen or C1-C4 alkyl and R2* is as previously defined.

In some embodiments, the compound has Formula Ig:

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

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R2*, Rx1 and t are as previously defined.

In some embodiments, the compound has Formula Ig and R2 is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, Rx1 is hydrogen or C1-C4 alkyl and R2* is as previously defined.

In some embodiments, the compound has Formula Ih:

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

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R2*, Rx1 and t are as previously defined.

In some embodiments, the compound has Formula Ih and R2 is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, Rx1 is hydrogen or C1-C4 alkyl and R2* is as previously defined.

In some embodiments, the compound has Formula Ii:

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

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group and R2*, Rx1 and t are as previously defined.

In some embodiments, the compound has Formula Ii and R2 is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, Rx1 is hydrogen or C1-C4 alkyl and R2* is as previously defined.

In some embodiments, the compound has Formula Ij:

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

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group.

In some embodiments, the compound has Formula Ij and R2 is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, Rx1 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:

In some embodiments, the compound has the structure Ik:

In some embodiments, the compound has Formula Ik and R2 is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group.

In some embodiments, the compound has Formula Ik and R2 is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, Rx1 is hydrogen or C1-C4 alkyl.

In some embodiments, the compound has Formula Il:

In some embodiments, the compound has Formula Il and R2 is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group.

In some embodiments, the compound has Formula Il and R2 is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, Rx1 is hydrogen or C1-C4 alkyl.

In some embodiments, the compound has Formula Im:

In some embodiments, the compound has Formula Im and R2 is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group.

In some embodiments, the compound has Formula Im and R2 is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, Rx1 is hydrogen or C1-C4 alkyl.

In some embodiments, the compound has Formula IIa:

In some embodiments, the compound has Formula IIa and R2 is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group.

In some embodiments, the compound has Formula IIa and R2 is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, Rx1 is hydrogen or C1-C4 alkyl.

In some embodiments, the compound has Formula IIb:

In some embodiments, the compound has Formula IIb and R2 is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group.

In some embodiments, the compound has Formula IIb and R2 is

wherein P* is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group, Rx1 is hydrogen or C1-C4 alkyl.

In some aspects, the active compound is:

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 Structure Reference Structure A B C D E F G H I J K L M N O P Q R S T U V W X Y Z AA BB CC DD EE FF GG HH II JJ KK LL MM NN OO PP QQ RR SS TT UU VV WW XX YY ZZ AAA BBB CCC DDD EEE FFF GGG HHH III JJJ KKK LLL MMM NNN OOO PPP QQQ RRR SSS TTT UUU VVV WWW XXX YYY ZZZ AAAA BBBB CCCC DDDD EEEE FFFF GGGG HHHH

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 α-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 G1-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 G1 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/CycD1 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/CycD1 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 μM, >1.25 μM, >1.50 μM, >1.75 μM, >2.0 μM, >2.25 μM, >2.50 μM, >2.75 μM, >3.0 μM, >3.25 μM, >3.5 μM 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 μM, >0.85 μM, >0.90 μM, >0.95 μM, >0.1.0 μM, >1.25 μM, >1.50 μM, >1.75 μM, >2.0 μM, >2.25 μM, >2.50 μM, >2.75 μM, >3.0 μM 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 G1-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 G1 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 G1 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 G1 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-α 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 biologics, 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 G1 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 G1 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”) G1 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 G1 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 RB 1 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 G1 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 μM. 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.

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 HCl=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-1,4-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-Cl) 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-Cl) 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, U.S. Pat. No. 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 (N,N-dicyclohexylcarbodiimide), EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or DIC (N,N-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, SO2Me, SOalkyl, SO2alkyl. 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.

[4-Chloro-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 5 L 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 HCl 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 (1 L). 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 39.2 g (77% crude yield) of a dark yellow oil. The material was used as is for the next step. NMR (CDCl3) δ 8.56 (s, 1H), 4.76 (s, 2H), 2.59 (s, 3H); MS (ESI+) for C6H7ClN2OS m/z 191.0 (M+H)+.

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 16.6 g (43% yield) of the desired product as a white solid. NMR (CDCl3) δ 10.32 (s, 1H), 8.88 (s, 1H), 2.65 (s, 3H); MS (ESI+) for C6H5ClN2OS m/z 189.0 (M+H)+.

General Procedure A.

Ethyl 4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-hydroxybut-2-ynoate

Isopropylmagnesium chloride:lithium chloride complex (4.43 g, 30.5 mmol, 25.4 mL 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 3.76 g (37% yield) of the desired product as a light red oil. NMR (CDCl3) δ 8.75 (s, 1H), 5.81 (d, 1H, J=6.0 Hz), 2.72 (bs, 1H), 2.60 (s, 3H), 1.33 (t, 3H, J=7.2 Hz); MS (ESI+) for C11H11ClN2O3S m/z 287.9 (M+H)+.

tert-Butyl 4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-hydroxybut-2-ynoate

Following General Procedure A and using tert-butyl propiolate affords the desired product in 74% yield as a viscous yellow oil. NMR (CDCl3) δ 8.75 (s, 1H), 5.79 (d, 1H, J=5.4 Hz), 4.27 (q, 2H, J=7.2 Hz), 2.97 (d, 1H, J=5.4 Hz), 2.60 (s, 3H), 1.52 (s, 9H); MS (ESI+) for C13H15ClN2O3S m/z 314.9 (M+H)+.

General Procedure B.

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 1 h. 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 (CDCl3) (major E isomer) δ 8.69 (s, 1H), 7.65 (d, 1H, J=18.0 Hz), 6.81 (d, 1H, J=18.0 Hz), 4.32 (q, 2H, J=6.0 Hz), 2.64 (s, 3H), 1.36 (t, 3H, J=6.0 Hz); NMR (CDCl3) (minor Z isomer) δ 8.87 (s, 1H), 6.89 (d, 1H, J=12.0 Hz), 6.23 (d, 1H, J=12.0 Hz), 4.14 (q, 2H, J=6.0 Hz), 2.63 (s, 3H), 1.23 (t, 3H, J=6.0 Hz); MS (ESI+) for C11H11ClN2O3S m/z 287.0 (M+H)+.

tert-Butyl 4-[4-chloro-2-(methylthio)pyrimidin-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 (CDCl3) (major E isomer) δ 8.67 (s, 1H), 7.54 (d, 1H, J=15.6 Hz), 6.72 (d, 1H, J=15.6 Hz), 2.64 (s, 3H), 1.54 (s, 9H); NMR (CDCl3) (minor Z isomer) δ 8.86 (s, 1H), 6.76 (d, 1H, J=12.0 Hz), 6.18 (d, 1H, J=12.0 Hz), 2.63 (s, 3H), 1.40 (s, 9H); MS (ESI+) for C13H15ClN2O3S m/z 314.9 (M+H)+.

General Procedure C.

Ethyl 2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8-tetrahydropyrido[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 2.48 g (41% yield) of the desired product as a pale yellow oil. NMR (CDCl3) δ 8.58 (s, 1H), 4.72 (m, 1H), 4.46 (m, 1H), 4.16 (q, 2H, J=6.9 Hz), 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=6.9 Hz) 1.08-0.96 (m, 21H); MS (ESI+) for C26H43N3O4SSi m/z 522.2 (M+H)+.

Ethyl 2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclohexyl)-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 1-{[(triisopropylsilyl)oxy]methyl}cyclohexanamine using General Procedure C afforded the desired product in 17% yield as a pale yellow oil. NMR (CDCl3) δ 8.63 (s, 1H), 4.83 (m, 1H), 4.73 (m, 1H), 4.14 (q, 2H, J=6.0 Hz), 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=6.0 Hz), 1.13-0.95 (m, 21H); MS (ESI+) for C27H45N3O4SSi m/z 536.2 (M+H)+.

Ethyl 8-(2-{[tert-butyl(dimethyl)silyl]oxy}-1,1-dimethylethyl)-2-(methylthio)-5-oxo-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 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 (CDCl3) 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=7.2 Hz), 0.89 (s, 9H), 0.06 (s, 3H), 0.01 (s, 3H); MS (ESI+) for C21H35N3O4SSi 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 (CDCl3) δ 8.60 (s, 1H), 4.72 (m, 1H), 4.63 (m, 1H), 4.19 (q, 2H, J=6.0 Hz), 3.97 (m, 2H), 3.22 (m, 1H), 3.01 (m, 2H), 2.54 (s, 3H), 1.28 (t, 3H, J=6.0 Hz), 1.17-1.02 (m, 21H); MS (ESI+) for C22H37N3O4SSi m/z 468.1 (M+H)+.

tert-Butyl 2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[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 (CDCl3) 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 C13H17N3O3S m/z 296.0 (M+H)+.

General Procedure D.

2-(Methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8-tetrahydropyrido[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 2.08 g (89% yield) of the desired product as a dark yellow oil. NMR (CDCl3) 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 C24H39N3O4SSi m/z 494.2 (M+H)+.

2-(Methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclohexyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylic acid

Saponification of ethyl 2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclohexyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate using General Procedure D affords the desired product in 95% yield as a dark yellow oil. NMR (CDCl3) 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 C25H41N3O4SSi m/z 508.1 (M+H)+.

8-(2-{[tert-Butyl(dimethyl)silyl]oxy}-1,1-dimethylethyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylic acid

Saponification of ethyl 8-(2-{[tert-butyl(dimethyl)silyl]oxy}-1,1-dimethylethyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate using General Procedure D affords the desired product in 99% yield as a pale yellow foam. NMR (CDCl3) 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), 0.11 (s, 6H); MS (ESI+) for C19H31N3O4SSi m/z 426.3 (M+H)+.

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 (CDCl3) 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 C20H33N3O4SSi m/z 440.2 (M+H)+.

2-(Methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[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.

N-(2,4-Dimethoxybenzyl)-2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

2-(Methylthio)-5-oxo-8-(1-{[(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. 1-(2,4-dimethoxyphenyl)methanamine (1.26 mL, 8.42 mmol) was added followed by the addition of N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (4.80 g, 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 2.39 g (88% yield) of the desired product as a brown sticky solid. NMR (CDCl3) 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)+.

N-(2,4-Dimethoxybenzyl)-2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclohexyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

Coupling reaction of 2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclohexyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylic acid using General Procedure E afforded the desired product in 52% yield as a yellow solid. NMR (CDCl3) 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 C34H52N4O5SSi m/z 657.2 (M+H)+.

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-d]pyrimidine-7-carboxamide

Coupling reaction of 8-(2-{[tert-butyl(dimethyl)silyl]oxy}-1,1-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 (CDCl3) 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 C28H42N4O5SSi m/z 575.4 (M+H)+.

N-(2,4-Dimethoxybenzyl)-2-(methylthio)-5-oxo-8-{2-[(triisopropylsilyl)oxy]ethyl}-5,6,7,8-tetrahydropyrido[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 (CDCl3) 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 C29H44N4O5SSi m/z 589.4 (M+H)+.

2-(Methylthio)-5-oxo-N-(1-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8-tetrahydropyrido[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 (CDCl3) 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 C24H40N4O3SSi m/z 493.1 (M+H)+.

General Procedure F.

N-(2,4-Dimethoxybenzyl)-8-[1-(hydroxymethyl)cyclopentyl]-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

N-(2,4-Dimethoxybenzyl)-2-(methylthio)-5-oxo-8-(1-{[(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 1.81 g (99% yield) of the desired product as a dark yellow powder. NMR (CDCl3) 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 C24H30N4O5S m/z 487.1 (M+H)+.

N-(2,4-Dimethoxybenzyl)-8-[1-(hydroxymethyl)cyclohexyl]-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

Desilylation of N-(2,4-dimethoxybenzyl)-2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclohexyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide using General Procedure F afforded the desired product in 97% yield as a yellow powder. NMR (CDCl3) 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 C25H32N4O5S m/z 501.1 (M+H)+.

8-(2-Hydroxy-1,1-dimethylethyl)-N-(4-methoxybenzyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[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-d]pyrimidine-7-carboxamide using General Procedure F afforded the desired product in 98% yield as a yellow powder. NMR (CDCl3) 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 C22H28N4O5S m/z 461.4 (M+H)+.

N-(2,4-Dimethoxybenzyl)-8-(2-hydroxyethyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[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 (CDCl3) 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 C20H24N4O5S m/z 432.9 (M+H)+.

N-[1-(Hydroxymethyl)cyclopentyl]-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

Desilylation of 2-(methylthio)-5-oxo-N-(1-{[(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 (CDCl3) 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 C15H20N4O3S m/z 337.0 (M+H)+.

General Procedure G.

8′-(2,4-Dimethoxybenzyl)-2′-(methylthio)-6′,6a′,8′,9′-tetrahydrospiro[cyclopentane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-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 (CDCl3) 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)+.

8′-(2,4-Dimethoxybenzyl)-2′-(methylthio)-6′,6a′,8′,9′-tetrahydrospiro[cyclohexane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione

Mesylation and cyclization of N-(2,4-dimethoxybenzyl)-8-[1-(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 C25H30N4O4S m/z 483.1 (M+H)+.

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-1,1-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 (CDCl3) 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)+.

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 (CDCl3) 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.

2′-(Methylthio)-6′,6a′,8′,9′-tetrahydrospiro[cyclopentane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione

8′-(2,4-Dimethoxybenzyl)-2′-(methylthio)-6′,6a′,8′,9′-tetrahydrospiro[cyclopentane-1,10′-pyrazino[1′,2′:1,6]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 (CDCl3) 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 C15H18N4O2S m/z 319.0 (M+H)+.

2′-(Methylthio)-6′,6a′,8′,9′-tetrahydrospiro[cyclohexane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione

Removal of the dimethoxybenzyl group of 8′-(2,4-Dimethoxybenzyl)-2′-(methylthio)-6′,6a′,8′,9′-tetrahydrospiro[cyclohexane-1,10′-pyrazino[1′,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 C16H20N4O2S m/z 333.1 (M+H)+.

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

Removal of the dimethoxybenzyl group of 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 using General Procedure H afforded the desired product in 56% yield as a white powder. NMR (CDCl3) 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 C13H16N4O2S m/z 293.2 (M+H)+.

2-(Methylthio)-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dione

Removal of the dimethoxybenzyl group of 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 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 C11H12N4O2 m/z 265.0 (M+H)+.

General Procedure I.

2′-{[5-(4-Methylpiperazin-1-yl)pyridin-2-yl]amino}-6′,6a′,8′,9′-tetrahydrospiro[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[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione (117 mg, 0.367 mmol) was taken up in N,N-dimethylacetamide (4.0 mL, 43 mmol) at room temperature under argon. 5-(4-methylpiperazin-1-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 11 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 C24H30N8O2 m/z 463.1 (M+H)+.

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

SNAr 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 C25H32N8O2 m/z 477.2 (M+H)+.

10,10-Dimethyl-2-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6,6a,9,10-tetrahydro-5H-pyrazino[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[1′,2′:1,6]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)+.

2-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dione

SNAr reaction using General Procedure I and 2-(methylthio)-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dione afforded the desired product in 15% yield as an orange powder. NMR (DMSO-d6) δ 8.41 (s, 1H), 8.05 (m, 1H), 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 C20H24N8O2 m/z 409.1 (M+H)+.

General Procedure J.

2′-{[5-(4-Methylpiperazin-1-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

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, 0.1 mmol) 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 C28H38N8OS2 m/z 567.1 (M+H)+.

10′,10′-Dimethyl-2′-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6′,6a′,9′,10′-tetrahydrospiro[1,3-dithiane-2,5′-pyrazino[1′,2′:1,6]pyrido[2,3-d]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[1′,2′:1,6]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.

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]pyrimidin]-7′(5′H)-one

2′-{[5-(4-Methylpiperazin-1-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 (1 ml) 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 (CDCl3) 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 C25H34N8O m/z 463.1 (M+H)+.

10,10-Dimethyl-2-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6,6a,9,10-tetrahydro-5H-pyrazino[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[1,3-dithiane-2,5′-pyrazino[1′,2′:1,6]pyrido[2,3-d]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.

(1-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 (7.64 g, 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 6.89 g (99% yield) of the desired product as a clear colorless oil. NMR (CDCl3) 3.34 (s, 2H), 1.81 (bs, 3H), 1.51-1.32 (m, 10H); MS (ESI+) for C7H15NO m/z 130.0 (M+H)+.

(1-Aminocyclopentyl)methanol

Using General Procedure L on commercially available cycloleucine affords the desired product in 99% yield as a pale yellow oil. NMR (CDCl3) 3.40 (s, 2H), 1.86-1.61 (m, 9H), 1.46-1.29 (m, 2H); MS (ESI+) for C6H13NO m/z 116.1 (M+H)+.

General Procedure M.

1-{[(Triisopropylsilyl)oxy]methyl}cyclohexanamine

(1-Aminocyclohexyl)methanol (3.43 g, 26.5 mmol) was taken up in methylene chloride (80 mL) at room temperature under argon. Triethylamine (5.6 mL, 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×2) and brine (40 mL). The organic layer was dried over sodium sulfate, filtered and the solvent concentrated in vacuo to afford 6.68 g (93% yield) of the desired product as a clear pale yellow oil. NMR (CDCl3) δ 3.49 (s, 2H), 1.75-1.25 (m, 10H), 1.16-1.06 (m, 21H); MS (ESI+) for C11H27NOSi m/z 203.2 (M+H)+.

1-{[(Triisopropylsilyl)oxy]methyl}cyclopentanamine

Following General Procedure M and using (1-aminocyclopentyl)methanol the desired product was obtained in 85% yield as a clear dark yellow oil. NMR (CDCl3) δ 3.53 (s, 2H), 1.85-1.39 (m, 8H), 1.16-1.07 (m, 21H); MS (ESI+) for C15H33NOSi m/z 272.2 (M+H)+.

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 (CDCl3) δ 3.56 (t, 2H, J=6.0 Hz), 2.94 (t, 2H, J=6.0 Hz), 1.09-0.99 (m, 21H); MS (ESI+) for C11H27NOSi m/z 217.2 (M+H)+.

1-{[tert-Butyl(dimethyl)silyl]oxy}-2-methylpropan-2-amine

Following General Procedure M and using commercially available 2-amino-2-methyl-1-propanol and using tert-butyldimethylsilyl chloride the desired product was obtained in 95% yield as a clear colorless oil. NMR (CDCl3) δ 3.31 (s, 2H), 0.93 (s, 9H), 0.06 (s, 6H); MS (ESI+) for C10H25NOSi m/z 204.2 (M+H)+.

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, N,N-dimethylformamide, and a coupling reagent, for example, 1-[bis(dimethylamino)methylene]-1H-1,2,3-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-1-yl)pyridin-2-amine, and an organic solvent, for example, N,N-dimethylacetamide, at an elevated temperature of about 150° C. to generate an amine. The compound 5-(4-methylpiperazin-1-yl)pyridin-2-amine can be prepared as disclosed in U.S. Pat. No. 8,598,186 to Tavares and Strum.

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 U.S. Pat. No. 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 HCl 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 (0.9 eq). 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-1,4-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 (0.9 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.

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 HCl 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, U.S. Pat. No. 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 (0.9 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.

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, U.S. Pat. No. 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 (0.9 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.

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 CO2 (1 atm) 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/cyclinD1, CDK6/CycD3, CDK2/CycA, CDK2/cyclinE, CDK5/p25, CDK5/p35, CDK7/CycH/MAT1, and CDK9/CycT kinase assays by Nanosyn (Santa Clara, Calif.) 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 μM for all assays (except μM 10 was used for the CKD7/CycH/MAT1 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 μM; Incubation time: 3 hr.

CDK2/CyclinE: Enzyme concentration: 0.2 nM; ATP concentration: 100 μM; Incubation time: 3 hr.

CDK4/CyclinD1: Enzyme concentration: 1 nM; ATP concentration: 200 μM; Incubation time: 3 hr.

CDK6/CyclinD3: Enzyme concentration: 10 nM; ATP concentration: 300 μM; Incubation time: 3 hr.

CDK5/p25: Enzyme concentration: 0.1 nM; ATP concentration: 20 μM; Incubation time: 3 hr.

CDK5/p35: Enzyme concentration: 0.07 nM; ATP concentration: 20 μM; Incubation time: 3 hr.

CDK7/CycH/MAT1: Enzyme concentration: 5 nM; ATP concentration: 50 μM; Incubation time: 3 hr.

CDK9/CycT: Enzyme concentration: 5 nM; ATP concentration: 10 μM; Incubation time: 17 hr.

TABLE 2 Inhibition of CDK kinases by Tricyclic Lactam Compounds Cdk7/ Compound Cdk2/ Cdk2/ Cdk4/ Cdk5/ Cdk5/ Cdk6/ CycH/ Cdk9/ No. CycA CycE CycD1 p25 p35 CycD3 MAT1 Cyc T ZZZ * * * * * * * YYY * * *** * * ** * BBBB ** * ** * * ** * * AAAA * * * * * * * * CCCC * * * * * * * * GGGG * * ** * * ** * * * >100 μM ** 10 μM < X > 100 μM *** <10 μM

Example 17 G1 Arrest (Cellular G1 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 FlowJo 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 G1-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 488 nm light. Samples are run on Dako Cyan flow cytometer and >10,000 events were collected for each sample. Data are analyzed using FlowJo 2.2 software developed by TreeStar, Inc.

Example 19 Inhibition of RB Phosphorylation

The CDK4/6-cyclin D complex is essential for progression from G1 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 G1 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, Mass.) 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, Wis., United States of America) following the manufacturer's recommendations. Plates are read on a BioTek (Winooski, Vt.) 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, Calif.) 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−), Sca1 (S+), and c-Kit (K+).

Example 23 Cellular Wash-Out Experiment

HS68 cells are seeded out at 40,000 cells/well in 60 mm dish on day 1 in DMEM containing 10% fetal bovine serum, 100 U/ml penicillin/streptomycin and 1× 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)). 24 hrs 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% CO2 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 FlowJo 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 μL) 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 μL of ice-cold acetonitrile containing internal standard to terminate the reaction. Testosterone samples are immediately combined with 800 μL of ice cold 50/50 acetonitrile/dH2O 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 t½=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 C57Bl/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 mcg 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, Calif.) by the manufacturer's instructions. g-H2AX-positive tHDF cells are then quantified using a CyAn ADP Analyzer (Beckman Coulter, Indianapolis, Ind.) and FlowJo analysis software (Version 7.2.2; Tree Star, Ashland, Oreg.). 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, Wis.) 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% CO2 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% CO2 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 1N NaOH to pH 4.3+0.1 and 1N HCl 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 1N HCl or 1N 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

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: wherein: or a pharmaceutically acceptable salt thereof.

Z is —(CH2)x— wherein x is 1, 2, 3 or 4 or —O—(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(O)—NR3R4; -(alkylene)m-O—R5, -(alkylene)m-S(O)n—R5, or -(alkylene)m-S(O)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 R1'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(O)—NR3R4; -(alkylene)m-C(O)—O-alkyl; -(alkylene)m-O—R5, -(alkylene)m-S(O)n—R5, or -(alkylene)m-S(O)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-O-alkylene-OR5, -(alkylene)m-S(O)n—R5, -(alkylene)m-NR3R4, -(alkylene)m-CN, -(alkylene)m-C(O)—R5, -(alkylene)m-C(S)—R5, -(alkylene)m-C(O)—OR5, -(alkylene)m-O—C(O)—R5, -(alkylene)m-C(S)—OR5, -(alkylene)m-C(O)-(alkylene)m-NR3R4, -(alkylene)m-C(S)—NR3R4, -(alkylene)m-N(R3)—C(O)—NR3R4, -(alkylene)m-N(R3)—C(S)—NR3R4, -(alkylene)m-N(R3)—C(O)—R5, -(alkylene)m-N(R3)—C(S)—R5, -(alkylene)m-O—C(O)—NR3R4, -(alkylene)m-O—C(S)—NR3R4, -(alkylene)m-SO2—NR3R4, -(alkylene)m-N(R3)—SO2—R5, -(alkylene)m-N(R3)—SO2—NR3R4, -(alkylene)m-N(R3)—C(O)—OR5) -(alkylene)m-N(R3)—C(S)—OR5, or -(alkylene)m-N(R3)—SO2—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(O)n—R5*, -(alkylene)m-NR3*R4*, -(alkylene)m-C(O)—R5*, -(alkylene)m-C(═S)R5*, -(alkylene)m-C(═O)OR5*, -(alkylene)m-OC(═O)R5*, -(alkylene)m-C(S)—OR5*, -(alkylene)m-C(O)—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(O)—R5*, -(alkylene)m-N(R3*)—C(S)—R5*, -(alkylene)m-O—C(O)—NR3*R4*, -(alkylene)m-O—C(S)—NR3*R4*, -(alkylene)m-SO2—NR3*R4*, -(alkylene)m-N(R3*)—SO2—R5*, -(alkylene)m-N(R3*)—SO2—NR3*R4*, -(alkylene)m-N(R3*)—C(O)—OR5*, -(alkylene)m-N(R3*)—C(S)—OR5*, or -(alkylene)m-N(R3*)—SO2—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(O)—NR3R4; -(alkylene)m-O—R5, -(alkylene)m-S(O)n—R5, or -(alkylene)m-S(O)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 C1-C8 alkyl, cycloalkylalkyl, or -TT-RR, C1-C8 cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O, and S; TT is an unsubstituted or substituted C1-C8 alkyl or C3-C8 cycloalkyl linker; and RR is a hydroxyl, unsubstituted or substituted C1-C6 alkoxy, amino, unsubstituted or substituted C1-C6 alkylamino, unsubstituted or substituted di-C1-C6 alkylamino, unsubstituted or substituted C6-C10 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(O)—R12 or —C(O)O—R13, wherein R12 is NHRA or RA and R13 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;

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:

4. The method of claim 1, wherein the compound has the formula:

5. The method of claim 1, wherein the compound has the formula:

6. The method of claim 1, wherein the compound has the formula:

7. The method of claim 1, wherein the compound has the formula:

8. The method of claim 1, wherein the compound has the formula:

9. The method of claim 1, wherein the compound has the formula:

10. The method of claim 1, wherein the compound has the formula:

11. The method of claim 1, wherein the compound has the formula:

12. The method of claim 1, wherein the compound has the formula:

13. The method of claim 1, wherein the compound has the formula:

14. The method of claim 1, wherein the compound has the formula:

15. The method of claim 1, wherein the compound has the formula:

16. The method of claim 1, wherein the compound has the formula:

17. The method of claim 1, wherein the compound has the formula:

18. The method of claim 1, wherein the compound has the formula:

19. The method of claim 1, wherein the compound is selected from the group consisting of: Structure Reference Structure A B C D E F G H I J K L M N O P Q R S T U V W X Y Z AA BB CC DD EE FF GG HH II JJ KK LL MM NN OO PP QQ RR SS TT UU VV WW XX YY ZZ AAA BBB CCC DDD EEE FFF GGG HHH III JJJ KKK LLL MMM NNN OOO PPP QQQ RRR SSS TTT UUU VVV WWW XXX

20. The method of claim 1, wherein the subject is a human.

21. 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. 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: wherein R, R1, R2, R3, R4, R5, R6, Rx, Z, m, n, and y are as defined in claim 1; or a pharmaceutically acceptable salt thereof.

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(O)—NR3R4; -(alkylene)m-O—R5, -(alkylene)m-S(O)n—R5, or -(alkylene)m-S(O)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;

37. The method of claim 1, wherein the compound is selected from the group consisting of:

38. The method of claim 36, wherein the compound is selected from the group consisting of:

39. 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: wherein:

Z is —(CH2)x— wherein x is 1, 2, 3 or 4 or —O—(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(O)—NR3R4; -(alkylene)m-O—R5, -(alkylene)m-S(O)n—R5, or -(alkylene)m-S(O)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 R1'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(O)—NR3R4; -(alkylene)m-C(O)—O-alkyl; -(alkylene)m-O—R5, -(alkylene)m-S(O)n—R5, or -(alkylene)m-S(O)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-O-alkylene-OR5, -(alkylene)m-S(O)n—R5, -(alkylene)m-NR3R4, -(alkylene)m-CN, -(alkylene)m-C(O)—R5, -(alkylene)m-C(S)—R5, -(alkylene)m-C(O)—OR5, -(alkylene)m-O—C(O)—R5, -(alkylene)m-C(S)—OR5, -(alkylene)m-C(O)-(alkylene)m-NR3R4, -(alkylene)m-C(S)—NR3R4, -(alkylene)m-N(R3)—C(O)—NR3R4, -(alkylene)m-N(R3)—C(S)—NR3R4, -(alkylene)m-N(R3)—C(O)—R5, -(alkylene)m-N(R3)—C(S)—R5, -(alkylene)m-O—C(O)—NR3R4, -(alkylene)m-O—C(S)—NR3R4, -(alkylene)m-SO2—NR3R4, -(alkylene)m-N(R3)—SO2—R5, -(alkylene)m-N(R3)—SO2—NR3R4, -(alkylene)m-N(R3)—C(O)—OR5, -(alkylene)m-N(R3)—C(S)—OR5, or -(alkylene)m-N(R3)—SO2—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(O)n—R5*, -(alkylene)m-NR3*R4*, -(alkylene)m-C(O)—R5*, -(alkylene)m-C(═S)R5*, -(alkylene)m-C(═O)OR5*, -(alkylene)m-OC(═O)R5*, -(alkylene)m-C(S)—OR5*, -(alkylene)m-C(O)—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(O)—R5*, -(alkylene)m-N(R3*)—C(S)—R5*, -(alkylene)m-O—C(O)—NR3*R4*, -(alkylene)m-O—C(S)—NR3*R4*, -(alkylene)m-SO2—NR3*R4*, -(alkylene)m-N(R3*)—SO2—R5*, -(alkylene)m-N(R3*)—SO2—NR3*R4*, -(alkylene)m-N(R3*)—C(O)—OR5*, -(alkylene)m-N(R3*)—C(S)—OR5*, or -(alkylene)m-N(R3*)—SO2—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(O)n—R5*, -(alkylene)m-NR3*R4*, -(alkylene)m-C(O)—R5*, -(alkylene)m-C(═S)R5*, -(alkylene)m-C(═O)OR5*, -(alkylene)m-OC(═O)R5*, -(alkylene)m-C(S)—OR5*, -(alkylene)m-C(O)—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(O)—R5*, -(alkylene)m-N(R3*)—C(S)—R5*, -(alkylene)m-O—C(O)—NR3*R4*, -(alkylene)m-O—C(S)—NR3*R4*, -(alkylene)m-SO2—NR3*R4*, -(alkylene)m-N(R3*)—SO2—R5*, -(alkylene)m-N(R3*)—SO2—NR3*R4*, -(alkylene)m-N(R3*)—C(O)—OR5*, -(alkylene)m-N(R3*)—C(S)—OR5*, or -(alkylene)m-N(R3*)—SO2—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(O)—NR3R4; -(alkylene)m-O—R5, -(alkylene)m-S(O)n—R5, or -(alkylene)m-S(O)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 1 (i) NHRA, wherein RA is unsubstituted or substituted C1-C8 alkyl, cycloalkylalkyl, or -TT-RR, C1-C8 cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O, and S; TT is an unsubstituted or substituted C1-C8 alkyl or C3-C8 cycloalkyl linker; and RR is a hydroxyl, unsubstituted or substituted C1-C6 alkoxy, amino, unsubstituted or substituted C1-C6 alkylamino, unsubstituted or substituted di-C1-C6 alkylamino, unsubstituted or substituted C6-C10 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(O)—R12 or —C(O)O—R13, wherein R12 is NHRA or RA and R13 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.

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 VI: wherein R, R1, R2, R3, R4, R5, R6, Rx, Z, m, n, and y are as defined in claim 39;

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(O)—NR3R4; -(alkylene)m-O—R5, -(alkylene)m-S(O)n—R5, or -(alkylene)m-S(O)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.
Patent History
Publication number: 20150297606
Type: Application
Filed: Apr 17, 2015
Publication Date: Oct 22, 2015
Applicant: G1 THERAPEUTICS, INC. (Research Triangle Park, NC)
Inventors: Jay Copeland Strum (Hillsborough, NC), John Emerson Bisi (Apex, NC), Patrick Joseph Roberts (Durham, NC), Ricky D. Gaston (Kalamazoo, MI), Robert C. Gadwood (Portage, MI)
Application Number: 14/690,206
Classifications
International Classification: A61K 31/5377 (20060101); A61K 31/527 (20060101); A61K 45/06 (20060101);