HK1258477B - Transient protection of normal cells during chemotherapy - Google Patents

Description

Transient protection of normal cells during chemotherapy

The application is a divisional application of a Chinese patent application (with the application number of 201480027281.8, the application date of 2014 being 3 months and 14 days, and the invention name being 'instant protection of normal cells during chemotherapy') of an international application PCT/US2014/028685 entering the Chinese national stage.

RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application No. 61/798,772 filed on day 3, 15 in 2013, U.S. provisional application No.61/861,374 filed on day 8, 1 in 2013, U.S. provisional application No.61/911,354 filed on day 12, 3 in 2013, and U.S. provisional application No.61/949,786 filed on day 3, 7 in 2014. Each of these applications is incorporated by reference herein in its entirety for all purposes.

Benefits of government

The U.S. government has rights in this invention in view of the support of grant number 5R44AI084284 issued by the National institute of Allergy and Infectious Disease.

Technical Field

The present invention relates to improved compounds, compositions and methods that transiently protect healthy cells and particularly Hematopoietic Stem and Progenitor Cells (HSPCs) and kidney cells from damage associated with DNA damaging chemotherapeutic agents. In one aspect, improved protection of healthy cells using the disclosed compounds that act as highly selective and transiently acting cyclin-dependent kinase 4/6(CDK 4/6) inhibitors when administered to a subject undergoing a DNA-disrupting chemotherapeutic regimen for treating a proliferative disorder is disclosed.

Background

Chemotherapy refers to the use of cytotoxic (typically DNA damaging) drugs to treat a range of proliferative disorders, including cancer, tumors, psoriasis, arthritis, lupus and multiple sclerosis, among others. Chemotherapeutic compounds tend to be non-specific and, especially at high doses, toxic to normal rapidly dividing cells. This often causes a variety of side effects in patients undergoing chemotherapy.

Myelosuppression (a severe reduction in blood cell production in the bone marrow) is one such side effect. It is characterized by myelosuppression (anemia, neutropenia, agranulocytosis and thrombocytopenia) and lymphopenia. Neutropenia is characterized by a selective decrease in the number of circulating neutrophils and an increased sensitivity to bacterial infections. In the united states, anemia (reduction in the number of red blood cells or erythrocytes, the amount of hemoglobin, or the volume of packed red blood cells (characterized by a measurement of hematocrit)) affects approximately 67% of cancer patients undergoing chemotherapy. See BioWorld Today, page 4, 23/7/2002. Thrombocytopenia is a decrease in platelet number and an increase in sensitivity to bleeding. Lymphopenia is a common side effect of chemotherapy and is characterized by a reduced number of circulating lymphocytes (also known as T and B cells). Patients with lymphopenia are susceptible to various types of infection.

Myelosuppression continues to represent the major dose-limiting toxicity of cancer chemotherapy, causing considerable morbidity, and the potential need to reduce the chemotherapeutic dose intensity, which may compromise disease control and survival. Considerable evidence from prospective and retrospective randomized clinical trials clearly suggests that Chemotherapy-induced myelosuppression impairs long-term disease control and survival (Lyman, g.h., Chemotherapy-induced dose intervention and quality cancer care (oncology (williston park),2006.20(14 suppl 9): pages 16-25)). Furthermore, the treatment regimens recommended in the National Comprehensive Cancer Network (National Comprehensive Cancer Network guidelines), for example, for lung, breast and colorectal Cancer, are increasingly associated with significant myelosuppression, while treatment for early stage as well as advanced or metastatic disease is increasingly recommended (Smith, R.E., Trends in surgery for myelosuppression therapy for the treatment of the disease of solid tumors. J Natl computer Cancer Net, 2006.4(7): pages 649-58). This trend to make treatment intensity greater in cancer patients requires improved measures to minimize the risk of myelosuppression and complications while optimizing relative dose intensity.

In addition to myelosuppression, chemotherapeutic agents may adversely affect other healthy cells, such as renal epithelial cells, possibly leading to acute renal injury due to tubular epithelial death. Acute kidney injury can cause long-term kidney disease, multiple organ failure, sepsis and death.

One mechanism to minimize myelosuppression, nephrotoxicity, and other chemotherapy cytotoxicity is to reduce the planned dose intensity of chemotherapy. However, dose reduction or cycle delay reduction is effective and ultimately compromises long-term disease control and survival.

Small molecules have been used to reduce some of the side effects of certain chemotherapeutic compounds. For example, leucovorin (leukovicin) has been used to reduce the effects of methotrexate (methotrexate) on bone marrow cells and gastrointestinal mucosal cells. Amifostine (amifosine) has been used to reduce the incidence of neutropenia-associated fever and mucositis in patients receiving alkylated or platinum-containing chemotherapeutic agents. In addition, dexrazoxane has been used to provide cardiac protection from the destruction of anthracycline anticancer compounds. Unfortunately, it is noted that many chemoprotectants such as delazolamide and amifostine may reduce the efficacy of the concomitant chemotherapy.

Other chemoprotective therapies, particularly chemotherapy-associated anemia and neutropenia, include the use of growth factors. Hematopoietic growth factors are commercially available as recombinant proteins. These proteins include granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) and derivatives thereof for the treatment of neutropenia, and Erythropoietin (EPO) and derivatives thereof for the treatment of anemia. However, these recombinant proteins are expensive. In addition, EPO has significant toxicity in cancer patients, causing increased thrombosis, relapse, and death in several large randomized trials. G-CSF and GM-CSF may increase the risk of late stages (> 2 years post-treatment) of secondary myeloid disorders such as leukemia and myelodysplasia. Thus, its use is limited and no longer readily available to all patients in need thereof. Furthermore, while growth factors may accelerate the recovery of some blood cell lineages, there are no therapies that treat platelet, macrophage, T cell or B cell inhibition.

Roberts et al, 2012 reported that Pfizer compound PD-0332991 induces transient cell cycle arrest in a CDK4/6 Dependent subset of healthy cells such as HSPC (see Roberts et al Multiple circles of cycle-Dependent Kinase 4/6 Inhibitors in Cancer therapy. JNCI 2012; 104(6): 476-487). This compound is currently being tested by Pfizer in clinical trials as an anti-neoplastic agent against estrogen positive, HER2 negative breast cancers.

Hematopoietic stem cells produce progenitor cells, which in turn produce all the differentiated components of the blood, as shown in fig. 1 (e.g., lymphocytes, erythrocytes, platelets, granulocytes, monocytes). HSPC requires the activity of CDK4/6 for proliferation (see Roberts et al Multiple circles of cycle-Dependent Kinase 4/6Inhibitors in Cancer therapy. JNCI 2012; 104(6): 476-487). In healthy kidneys, renal epithelial cells occasionally enter the cell cycle (about 1% epithelial cells). However, epithelial proliferation increases steadily following renal injury (see hummphreys, b.d. et al, Intrinsic epithelial cells repair the kidney after in j oury, Cell Stem Cell 2,284-91 (2008)). Importantly, after renal injury, the surviving renal epithelial cells replicate to Repair the damage to the renal tubular epithelium (see Humphreys, B.D. et al, Repair of injected proximal tissue patients not injected specific progenerators, Proc Natl Acad Sci U S A108,9226-31 (2011)). See also WO 2010132725 filed by Sharpless et al.

A number of CDK4/6 inhibitors have been identified, including specific pyrido [2,3-d ] pyrimidines, 2-anilinopyrimidines, diaryl ureas, benzoyl-2, 4-diaminothiazole, indolo [6,7-a ] pyrrolo [3,4-c ] carbazole, and oxindoles (see p.s.sharma, r.sharma, r.tyagi, curr. Cancer Drug Targets 8(2008) 53-75). WO 03/062236 identifies a series of 2- (pyrid-2-ylamino-pyrido [2,3] pyrimidin-7-ones for the treatment of Rb-positive cancers that exhibit selectivity for CDK4/6, including 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido- [2,3-d ] -pyrimidin-7-one (PD0332991) clinical trial studies have reported the rate of neutropenia and leukopenia with a scale of 3/4 under PD0332991, resulting in 71% of patients requiring discontinuation of dosing and 35% requiring dose reduction, and adverse events that result in 10% discontinuation (see Finn, abstract S1-6, SABCS 2012).

VanderWel et al describe iodine-containing pyrido [2,3-d ] pyrimidin-7-ones (CKIA) as potent and selective inhibitors of CDK4 (see VanderWel et al, J.Med.chem.48 (2005) 2371-2387).

WO 99/15500 filed by Glaxo Group Co., 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 proposed by Novartis describes pyrrolopyrimidines with CDK4/6 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 barvias et al teaches the use of CDK4 inhibitors to treat inflammation.

WO 2012/061156 filed by Tavares and assigned to G1Therapeutics describes CDK inhibitors. WO 2013/148748 filed by Tavares and assigned to G1Therapeutics describes lactam kinase inhibitors.

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-one 2030 (2003); PCT/US2009/059281) to reduce or prevent the effects of cytotoxic compounds on HSPC in subjects undergoing chemotherapy. See also U.S. patent publication 2012/0100100.

Stone et al, Cancer Research 56,3199-3202(1996, 7/1) describe reversible p 16-mediated cell cycle arrest, thereby avoiding chemotherapy disruption.

Accordingly, it is an object of the present invention to provide novel compounds, compositions and methods for treating patients during chemotherapy.

Summary of The Invention

In one embodiment, improved compounds, methods and compositions are provided to minimize the effect of chemotherapeutic agent toxicity on CDK4/6 replication-dependent healthy cells, such as hematopoietic stem cells and hematopoietic progenitor cells (collectively HSPCs) and/or renal epithelial cells, in a subject (typically a human) that will be exposed to, is being exposed to, or has been exposed to a chemotherapeutic agent (typically a DNA damaging agent).

Specifically, the present invention includes administering an effective amount of a selected compound of formula I, II, III, IV or V as described herein, a pharmaceutically acceptable composition, salt, isotopic analogue or prodrug thereof, which provides optimal transient G1 arrest in a subject of healthy cells, e.g., HSPC and/or renal epithelial cells, during or after exposure of the subject to a chemotherapeutic agent, e.g., a DNA damaging chemotherapeutic agent:

in one non-limiting example, the compound may be selected from the compounds of table 1 below, or a pharmaceutically acceptable composition, salt, isotopic analog or prodrug thereof. In one non-limiting example, the compound may be selected from the group consisting of compound T, Q, GG, U, or AAAA described below, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof.

The compounds provide improved protection of CDK replication-dependent healthy cells during chemotherapeutic treatment, in part because they (i) exhibit transient, transient G1 arrest, and (ii) exhibit rapid, synchronized cell reentry into the cell cycle after cessation of chemotherapeutic disruption. The use of these CDK 4/6-specific transient, transient G1 arrest compounds as chemoprotectants allows, for example, accelerated restoration of cell lineages, reduced risk of cytotoxicity, and/or minimization of chemotherapeutic-induced cell death due to delayed replication.

Although there are reports demonstrating chemical protection using known CDK4/6 inhibitors such as 2BrIC and PD0332991, it has been found that these inhibitors may not be the most desirable compounds for use in a pharmacological rest (PQ) strategy. For example, the use of 2BrIC in vivo is limited by its limited bioavailability, and although PD0332991 shows relative selectivity to CDK4/6, the compounds have a relatively long-lasting intracellular effect (see Roberts et al Multiple circles of cycle-Dependent Kinase 4/6Inhibitors in Cancer therapy, JCNI 2012; 104(6):476-487 (FIG. 2A)), prolonging the transient of G1 arrest beyond that which is necessary to adequately protect against chemotherapeutic treatment injury. Such long-lasting effects delay proliferation of the HSPC cell lineage required to restore, for example, blood cell lines that are adversely affected by chemotherapeutic agents or that circulate during the natural life cycle. In subjects with chemotherapy treatment regimens that require rapid reentry of HSPCs into the cell cycle, thereby restoring red blood cells, platelets, and bone marrow cells (monocytes and granulocytes) adversely affected by chemotherapy or acute HSPC G1 arrest to limit myelosuppressive or hematologic effects, the long-lasting G1 arrest provided by PD0332991 may limit its use as a potential chemoprotectant. Furthermore, the use of PD0332991 as a chemoprotectant in subjects exposed to chemotherapeutic agents at regular and repeated intervals, e.g., on a schedule repeated every few days, is limited because it may limit the ability of HSPCs of these subjects to rapidly re-enter the cell cycle before they need to be restated before the next chemotherapeutic cycle in the subject. With respect to other diseased tissues, such as renal cells, timely resumption of proliferation is critical for tissue repair, such as renal tubular epithelium repair, due to nephrotoxic agents, and thus, excessively long-term PQ is undesirable.

Thus, in an alternative embodiment, the invention comprises administering to a subject in need thereof an effective amount of a compound described herein, such compound exhibiting one or any combination of the following factors (alone or in any combination thereof, each considered to be specifically and independently described) that provides an improved therapeutic effect: i) wherein a substantial portion (e.g., at least 80% or more) of the CDK4/6 replication-dependent healthy cells recover to or near pre-treatment baseline cell cycle activity (i.e., re-enter cell cycle) within less than 24 hours, 30 hours, or 36 hours from the last administration of the active compound or, for example, using the protocol described in the examples below in a human; ii) wherein a substantial proportion of healthy cells synchronously re-enter the cell cycle within less than 24 hours, 30 hours or 36 hours from the last administration of the active compound; (iii) wherein the elimination of CDK4/6 inhibitory effect of the active compound occurs in less than 24 hours, 30 hours, or 36 hours from the administration of the inhibitor; (iv) wherein the IC50 of the active compound inhibited CDK4 and/or CDK6 is more than 1500-fold less than its IC50 concentration inhibited CDK 2; (v) wherein a substantial proportion of healthy cells recover to or near baseline cell cycle activity (i.e. re-enter the cell cycle) prior to treatment in less than 24 hours, 30 hours or 36 hours from the abrogation of CDK4/6 inhibitory effect of the active compound; (vi) wherein the pre-treatment baseline cell cycle activity (i.e., re-entry into the cell cycle) is within less than about 24 hours, about 30 hours, or about 36 hours of the time at which the concentration level of the CDK4/6 inhibitor in the blood of the subject falls to a therapeutically effective concentration; or (vii) wherein a substantial proportion of healthy cells re-enter the cell cycle synchronously within less than 24 hours, 30 hours or 36 hours of the last administration of the ionotherapeutic agent.

The compounds described herein can be administered to a subject prior to treatment with a chemotherapeutic agent, during treatment with a chemotherapeutic agent, after exposure to a chemotherapeutic agent, or a combination thereof. The compounds described herein are typically administered in a manner that allows the drug to readily enter the bloodstream, e.g., via intravenous injection or sublingual, intraaortic, or other effective entry into the bloodstream; however, oral, topical, transdermal, intranasal, intramuscular, or by inhalation may be used, e.g., by solution, suspension, or emulsion or other desired route of administration. 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, 2.5 hours, 2 hours, 1 hour, 1/2 hours, or less prior to treatment with the chemotherapeutic agent. Typically, the active compounds described herein are administered to a subject prior to treatment with a chemotherapeutic agent such that the compounds reach peak serum levels prior to or during treatment with the chemotherapeutic agent. In one embodiment, the active compound is administered concomitantly with or following exposure to the chemotherapeutic agent. If desired, the active compound may be administered multiple times during the course of treatment with the chemotherapeutic agent to maximize inhibition, especially when the chemotherapeutic agent is administered for a prolonged period of time or has a long half-life. If it is desired to mitigate healthy cell damage associated with exposure to a chemotherapeutic agent, the active compounds described herein may be administered after exposure to the chemotherapeutic agent. In certain embodiments, the active compound is administered up to about 1/2 hours, 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, or up to about 20 hours or more after chemotherapeutic agent exposure. In a specific embodiment, the active compound is administered between up to about 12 hours and 20 hours after exposure to the chemotherapeutic agent.

The CDK4/6 inhibitors described herein exhibit significant selectivity for inhibiting CDK4 and/or CDK6 compared to other CKDs such as CDK 2. For example, the CDK4/6 inhibitors described herein provide dose-dependent G1 arrest of CDK4/6 replication-dependent healthy cells, e.g., HSPC or renal epithelial cells, in a subject, and the methods provided herein are sufficient to provide chemoprotection of targeted CDK4/6 replication-dependent healthy cells during chemotherapeutic exposure, e.g., during a time period in which a DNA-destroying chemotherapeutic is capable of performing DNA-destroying action on CDK4/6 replication-dependent healthy cells in a subject, while allowing these cells to be synchronized and rapidly re-enter the cell cycle shortly after the chemotherapeutic dissipates, as compared to, e.g., PD0332991, due to the time-limiting CDK4/6 inhibition provided by the compounds described herein. Likewise, CDK4/6 inhibitors useful in the present invention provide dose-dependent attenuation of healthy cells that have been exposed to toxic levels of chemotherapeutic agents, such as occasional overdoses of CDK4/6 replication-dependent, allowing repair of DNA damage associated with chemotherapeutic agent exposure and simultaneous rapid re-entry into the cell cycle after CDK4/6 inhibition dissipates, as compared to, for example, PD 0332991. In one embodiment, use of a CDK4/6 inhibitor described herein causes G1 arrest in CDK4/6 replication-dependent healthy cells of a subject, dissipates after administration of the CDK4/6 inhibitor such that the healthy cells of the subject return to or near their pre-administration baseline cell cycle activity in less than about 24 hours, 30 hours, 36 hours, or 40 hours of administration. In one embodiment, G1 stasis dissipates such that the subject's CDK4/6 replication-dependent healthy cells return to their pre-administration baseline cell cycle activity in less than about 24 hours, 30 hours, 36 hours, or 40 hours.

In one embodiment, use of the CDK4/6 inhibitor described herein causes dissipation of the G1 stasis such that the CDK4/6 dependent healthy cells of the subject return to or near their pre-administration baseline cell cycle activity in less than about 24 hours, 30 hours, 36 hours, or 40 hours of the effect of the chemotherapeutic agent. In one embodiment, dissipation of G1 stasis allows the subject's CDk4/6 replication-dependent cells to return to their pre-administration baseline cell cycle activity in less than about 24 hours, 30 hours, 36 hours, or 40 hours of administration of the chemotherapeutic agent, or about 48 hours of administration. In one embodiment, the CDK4/6 replication-dependent healthy cell is HSPC. In one embodiment, the CDK4/6 replication-dependent healthy cells are renal epithelial cells.

In one embodiment, use of the CDK4/6 inhibitor described herein causes dissipation of G1 stasis such that the subject's CDK4/6 replication-dependent healthy cells recover to or near their pre-administration baseline cell cycle activity within less than about 24 hours, 30 hours, 36 hours, 40 hours, or less than about 48 hours from the time the concentration level of the CDK4/6 inhibitor in the subject's blood falls to a therapeutically effective concentration.

In one embodiment, the CDK4/6 inhibitor described herein is used to protect renal epithelial cells during exposure to a chemotherapeutic agent, e.g., a DNA damaging chemotherapeutic agent, wherein the renal epithelial cells are transiently prevented from entering S phase in response to chemotherapeutic agent-induced disruption of the renal tubular epithelium, the time at which the concentration level of the CDK4/6 inhibitor in the blood of the subject falls to a therapeutically effective concentration, the cessation of the effect of the ionizing chemotherapeutic agent, or the administration of the CDK4/6 administration does not exceed about 24 hours, about 30 hours, about 36 hours, about 40 hours, or about 48 hours.

The effects of the discontinuation of CDK4/6 inhibitors suitable for use in the methods described may be synchronized, that is, CDK4/6 replication-dependent healthy cells exposed to the CDK4/6 inhibitor described herein re-enter the cell cycle in a similarly timed manner as the G1 arrest dissipates. The replication-dependent healthy cells of CDK4/6 that re-enter the cell cycle do so such that the normal ratio of G1 and S cells is rapidly and effectively reconstituted less than about 24 hours, 30 hours, 36 hours, 40 hours, or about 48 hours from the time the concentration level of CDK4/6 inhibitor in the subject' S blood falls to a therapeutically effective concentration.

This advantageously allows a larger number of healthy cells to begin to replicate when G1 arrest dissipates, as compared to an asynchronous CDK4/6 inhibitor such as PD 0332991.

In addition, the synchronous re-entry into the cell cycle following G1 arrest with the CDK4/6 inhibitor described herein provides for the administration of timed hematopoietic growth factors to help reconstitute hematopoietic cell lines, thereby maximizing the ability of the growth factor to act. Thus, in one embodiment, the use of the compounds or methods 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, graying factor, and Erythropoietin (EPO) or derivatives thereof. In one embodiment, the CDK4/6 inhibitor is administered prior to the administration of the hematopoietic growth factors. In one embodiment, the hematopoietic growth factor administration is timed such that the effect of the CDK4/6 inhibitor on HSPCs has been abolished.

In one aspect, the use of the CDK4/6 inhibitor described herein allows chemoprotective regimens to be used during standard chemotherapeutic dosing schedules or regimens common in many anti-cancer treatments. For example, a CDK4/6 inhibitor may be administered such that CDK4/6 replication-dependent healthy cells arrest at G1 during chemotherapeutic exposure, wherein many healthy cells reenter the cell cycle and are able to replicate shortly after chemotherapeutic exposure, e.g., within less than about 24, 30, 40, or 48 hours, due to the rapid dissipation of the compound's G1 arresting effect, and continue to replicate until the CDK4/6 inhibitor is administered prior to the next chemotherapeutic treatment. In one embodiment, the CDK4/6 inhibitor is administered to allow CDK4/6 replication-dependent healthy cells to cycle between G1 arrest and re-entry into the cell cycle, thereby accommodating repeat dosing of chemotherapeutic treatment regimens, including for example (but not limited to) treatment regimens in which a chemotherapeutic is administered as follows: every 21 days, on days 1-3; every 28 days, on days 1-3; every 3 weeks on day 1; every 28 days, on days 1, 8 and 15; every 28 days, on days 1 and 8; every 21 days, on days 1 and 8; every 21 days, from day 1 to day 5; 1 day a week for 6-8 weeks; on days 1, 22 and 43; weekly, day 1 and day 2; days 1-4 and 22-25; day 1-4; day 22-day 25; and days 43-46; and similar types of protocols in which CDK4/6 replication-dependent cells arrest at G1 during chemotherapeutic exposure and a significant portion of the cells re-enter the cell cycle between chemotherapeutic exposures. In one embodiment, the CDK4/6 inhibitor may be administered such that the subject's CDK4/6 replication-dependent cells arrest at G1 during daily chemotherapeutic exposure, e.g., a continuous multi-day chemotherapeutic regimen, but a significant portion of the CDK4/6 replication-dependent cells reenter the cell cycle and replicate between daily treatments. In one embodiment, the CDK4/6 inhibitor may be administered such that the subject's CDK4/6 replication-dependent cells arrest at G1 during daily chemotherapeutic exposure, e.g., a continuous multi-day regimen, but a significant portion of healthy cells re-enter the cell cycle and replicate during the interim period prior to the next chemotherapeutic exposure. In one embodiment, the CDK4/6 inhibitor is administered such that G1 arrest of CDK4/6 replication-dependent cells of the subject is provided during a daily chemotherapeutic treatment regimen, e.g., a continuous multi-day treatment regimen, and the arrested cells are able to re-enter the cell cycle shortly after the end of the multi-day regimen. In one embodiment, the cancer is small cell lung cancer and the CDK4/6 inhibitor is administered on days 1, 2, and 3 during a 21-day treatment cycle, wherein the DNA damaging agent administered is selected from carboplatin (carboplatin), cisplatin (cissplatin), and etoposide (etoposide), or a combination thereof.

A subject treated according to the invention may be subjected to therapeutic chemotherapy for the treatment of a proliferative disorder or disease, such as cancer. The cancer may be characterized by one or a combination of: increased activity of cyclin dependent kinase 1(CDK 1); increased activity of cyclin dependent kinase 2(CDK 2); loss, insufficiency or absence of retinoblastoma tumor suppressor protein (Rb) (Rb null); high level MYC performance; increase in cyclin E1, E2; and an increase in cyclin a. The cancer may be characterized by reduced expression of retinoblastoma tumor suppressor protein or retinoblastoma family member proteins, such as (but not limited to) p107 and p 130. In one embodiment, the subject is undergoing chemotherapeutic treatment to treat Rb-null or Rb-deficient cancers 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 one embodiment, the cancer is CDK 4/6-independent cancer. Administration of the inhibitor compound may allow for higher doses of chemotherapeutic agents to be used to treat diseases than would be safe for use in a standard dose in the absence of CDK4/6 inhibitor compound administration.

A subject or subject, including a human, may be treated with a chemotherapeutic agent for a non-malignant proliferative disorder or other abnormal cellular proliferation (e.g., tumor, multiple sclerosis, lupus or arthritis).

Protected HSPCs include hematopoietic stem cells, such as long term hematopoietic stem cells (LT-HSCs) and short term hematopoietic stem cells (ST-HSCs): and hematopoietic progenitors including multipotent progenitors (MPP), common spinal cord progenitors (CMP), Common Lymphoid Progenitors (CLP), granulocyte-monocyte progenitors (GMP), and megakaryocyte-erythrocyte progenitors (MEP). Administration of the inhibitor compound provides short-term transient pharmacological quiescence of hematopoietic stem cells and/or hematopoietic progenitor cells in the subject.

Administration of a CDK4/6 inhibitor as described herein may result in reduced anemia, reduced lymphopenia, reduced thrombocytopenia, or neutropenia as compared to that typically expected, common, or associated with treatment with chemotherapeutic agents in the absence of CDK4/6 inhibitor administration. The use of a CDK4/6 inhibitor as described herein allows for faster recovery from bone marrow depression, e.g., myelosuppression, anemia, lymphopenia, thrombocytopenia, or neutropenia, associated with chronic use of a CDK4/6 inhibitor after cessation of CDK4/6 inhibitor use. In some embodiments, use of a CDK4/6 inhibitor as described herein results in reduced or limited bone marrow suppression, e.g., bone marrow suppression, anemia, lymphopenia, thrombocytopenia, or neutropenia, associated with chronic use of a CDK4/6 inhibitor.

In an alternative aspect, the CDK4/6 inhibitors described herein may be used in combination with chemotherapeutic agents for the treatment of Rb-negative cancers or other Rb-negative aberrant proliferation for their anti-cancer, anti-tumor or anti-proliferative effects. In one embodiment, the CDK4/6 inhibitor described herein provides an additive or synergistic effect with the anti-cancer or anti-proliferative activity of a chemotherapeutic agent. The chemotherapeutic agents that may be combined with the CDK4/6 inhibitors described herein are any chemotherapeutic agent effective or suitable for treating RB null cancers or abnormal cell proliferation. In a particular embodiment, the use of a compound described herein is combined within a therapeutic regimen with at least one other chemotherapeutic agent, and may be a compound that does not rely on proliferation or progression through the cell cycle to achieve anti-proliferative activity. Such agents may include, but are not limited to, tamoxifen (tamoxifen), midazolam (midazolam), letrozole (letrozole), bortezomib (bortezomib), anastrozole (anastrozole), goserelin (goserelin), mTOR inhibitors, PI3 kinase inhibitors, dual mTOR-PI3K inhibitors, MEK inhibitors, RAS inhibitors, ALK inhibitors, HSP inhibitors (e.g., HSP70 and HSP 90 inhibitors or combinations thereof), BCL-2 inhibitors, apoptosis inducing compounds, AKT inhibitors, PD-1 inhibitors, or FLT-3 inhibitors, or combinations thereof. Examples of mTOR inhibitors include, but are not limited to, rapamycin (rapamycin) and its analogs, everolimus (Afinitor), temsirolimus (temsirolimus), radar limus (ridaforolimus), sirolimus (sirolimus), and de-folimus (deforolimus). Examples of P13 kinase inhibitors include, but are not limited to, Wortmannin (Wortmannin), desmethylcollagenase (demethoxyviridin), piperacillin (perifosine), idealsib (idelalisib), PX-866, IPI-145(Infinity), BAY 80-6946, BEZ235, RP6503, TGR 1202(RP5264), MLN1117(INK1117), Piticib (Picilisib), Buparlisib (Buparlisib), SAR 2454408 (XL147), SAR 2458409 (XL765), Palomid (Palomid)529, ZSTK474, PWT33597, RP6530, CUDC-907, and AEZS 136. Examples of MEK inhibitors include, but are not limited to, trametinib (Tametinib), semetinib (Selumetinib), MEK162, GDC-0973 (XL518), and PD 0325901. Examples of RAS inhibitors include, but are not limited to, relisin (Reolysin) and siG12D LODER. Examples of ALK inhibitors include, but are not limited to, Crizotinib (Crizotinib), AP26113, and LDK 378. HSP inhibitors include, but are not limited to, Geldanamycin (Geldadamycin) or 17-N-allylamino-17-deoxygeldanamycin (17AAG) and Radicicol (Radicol). The CDK4/6 inhibitor in combination with a chemotherapeutic agent is selected from a compound or composition comprising formula I, formula II, formula III, formula IV, or formula V described above, or a pharmaceutically acceptable composition, salt, isotopic analog or prodrug thereof. In one embodiment, the compound may be selected from the compounds in table 1 below, or a pharmaceutically acceptable composition, salt, isotopic analog or prodrug thereof. In one embodiment, the compound is selected from the group consisting of compound T, Q, GG, U, and AAAA, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof.

In certain embodiments, a compound described herein is administered to a subject prior to treatment with another chemotherapeutic agent, during treatment with another chemotherapeutic agent, after administration of another chemotherapeutic agent, or a combination thereof. In one embodiment, the CDK4/6 inhibitor is selected from the compounds described in table 1. In one embodiment, the compound is selected from the group consisting of compounds T, Q, GG, U, and AAAA.

In some embodiments, the subject or subject is a mammal, including a human.

In summary, the invention comprises the following features:

A. compounds of formulae I, II, III, IV and V as described herein, and pharmaceutically acceptable compositions, salts, isotopic analogues or prodrugs thereof, for use in the chemoprotection of CDK4/6 replication-dependent healthy cells, e.g., HSPCs and/or renal epithelial cells, during exposure to chemotherapeutic agents. In one embodiment, the compound is selected from the compounds described in table 1, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof. In one embodiment, the compound is selected from the group consisting of compound T, Q, GG, U, or AAAA, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof;

B. Compounds of formulae I, II, III, IV and V as described herein, and pharmaceutically acceptable compositions, salts, isotopic analogues and prodrugs thereof, for use in the chemoprotection of CDK4/6 replication-dependent healthy cells, e.g., HSPCs and/or renal epithelial cells, during a chemotherapeutic regimen for treating a proliferative disorder. In one embodiment, the compound is selected from the compounds described in table 1, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof. In one embodiment, the compound is selected from compound T, Q, GG, U, or AAAA, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof;

C. compounds of formulae I, II, III, IV and V as described herein, and pharmaceutically acceptable compositions, salts, isotopic analogues and prodrugs thereof, for use in the chemoprotection of CDK4/6 replication-dependent healthy cells, such as HSPCs and/or renal epithelial cells, during a chemotherapeutic regimen for treating a cancer. In one embodiment, the compound is selected from the compounds described in table 1, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof. In one embodiment, the compound is selected from compound T, Q, GG, U, or AAAA, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof;

D. Compounds of formulae I, II, III, IV, and V as described herein, and pharmaceutically acceptable compositions, salts, isotopic analogs, and prodrugs thereof, are used in combination with hematopoietic growth factors in subjects who will be exposed to, are being exposed to, or have been exposed to chemotherapeutic agents. In one embodiment, the compound is selected from the compounds described in table 1, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof. In one embodiment, the compound is selected from the group consisting of compound T, Q, GG, U, and AAAA, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof;

E. use of a compound of formulae I, II, III, IV and V as described herein, and pharmaceutically acceptable compositions, salts, isotopic analogues and prodrugs thereof, for the manufacture of a medicament for the chemoprotection of CDK4/6 replication-dependent healthy cells, e.g., HSPCs and/or renal epithelial cells. In one embodiment, the compound is selected from the compounds described in table 1, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof. In one embodiment, the compound is selected from the group consisting of compound T, Q, GG, U, or AAAA, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof;

F. Use of a compound of formulae I, II, III, IV and V as described herein, and pharmaceutically acceptable compositions, salts, isotopic analogues and prodrugs thereof, for the manufacture of a medicament for reducing DNA damage in CDK4/6 replication-dependent healthy cells, e.g., HSPCs and/or renal epithelial cells, that have been exposed to chemotherapeutic exposure. In one embodiment, the compound is selected from the compounds described in table 1, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof. In one embodiment, the compound is selected from compound T, Q, GG, U, or AAAA, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof;

G. a pharmaceutical formulation comprising a therapeutically effective amount of a compound of formulae I, II, III, IV and V as described herein, or pharmaceutically acceptable compositions, salts and prodrugs thereof, for the chemoprotection of healthy cells. In one embodiment, the compound is selected from the compounds described in table 1, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof. In one embodiment, the compound is selected from compound T, Q, GG, U, or AAAA, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof;

H. A process for the preparation of a therapeutic product containing an effective amount of a compound of formulae I, II, III, IV and V as described herein. In one embodiment, the compound is selected from the compounds described in table 1, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof. In one embodiment, the compound is selected from compound T, Q, GG, U, or AAAA, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof;

I. a method for the manufacture of an agent of formulae I, II, III, IV and V intended for therapeutic use in the chemoprotection of CDK4/6 replication-dependent healthy cells, such as HSPCs and/or renal epithelial cells. In one embodiment, the agent is selected from the compounds described in table 1, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof. In one embodiment, the agent is selected from the group consisting of compound T, Q, GG, U, or AAAA, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof;

J. a method for the manufacture of an agent of formulae I, II, III, IV and V intended for therapeutic use in reducing DNA damage of CDK4/6 replication-dependent healthy cells, such as HSPCs and/or renal epithelial cells, that have been exposed to a chemotherapeutic agent. In one embodiment, the agent is selected from the compounds described in table 1, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof. In one embodiment, the agent is selected from the group consisting of compound T, Q, GG, U, or AAAA, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof;

K. A method of inhibiting the development of an Rb negative cancer or proliferative condition by administering a compound of formula I, II, III, IV or V, or a pharmaceutically acceptable composition, salt, isotope analog, or prodrug thereof; in combination with a chemotherapeutic agent, provides an additive or synergistic effect with the chemotherapeutic agent. In one embodiment, the compound is selected from the compounds described in table 1, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof. In one embodiment, the compound is selected from the group consisting of compound T, Q, GG, U, and AAAA, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof. In one embodiment, the CDK4/6 inhibitor is combined with a chemotherapeutic agent selected from: a MEK inhibitor, a PI3 kinase delta inhibitor, a BCL-2 inhibitor, an AKT inhibitor, an apoptosis inducing compound, an AKT inhibitor, a PD-1 inhibitor, a FLT-3 inhibitor, an HSP90 inhibitor, or an mTOR inhibitor, or a combination thereof.

Brief Description of Drawings

Fig. 1 is a schematic diagram showing the layered proliferation and hematopoiesis with increased differentiation after proliferation of healthy Hematopoietic Stem Cells (HSCs) and healthy hematopoietic progenitor cells.

Figure 2A is a graph of the percentage of cells in G2-M phase (open circles), S phase (triangles), G0-G1 phase (squares), <2N (diamonds) versus the variable concentration of compound T in tHS68 cells (nM). Cdk4/6 dependent cell line (tHS68) was treated with compound T at indicated concentrations for 24 hours. After compound T treatment, cells were harvested and analyzed for cell cycle distribution. As described in example 152, tHS68 cells exhibited complete G1 arrest with a corresponding decrease in the number of cells in S phase. Figure 2B is a graph of the number of tHS68 cells (CDK4/6 dependent cell line) versus the DNA content of the cells (as measured by propidium iodide). Cells were treated with DMSO for 24 hours, harvested and analyzed for cell cycle distribution. Figure 2C is a plot of the number of WM2664 cells (CDK4/6 dependent cell line) versus the DNA content of the cells (as measured by propidium iodide). Cells were treated with DMSO for 24 hours, harvested and analyzed for cell cycle distribution. Figure 2D is a graph of the number of a2058 cells (CDK 4/6-independent cell line) versus the DNA content of the cells (as measured by propidium iodide). Cells were treated with DMSO for 24 hours, harvested and analyzed for cell cycle distribution. Figure 2E is a graph of the number of tHS68 cells (CDK4/6 dependent cell line) versus the DNA content of the cells (as measured by propidium iodide) after treatment with compound T. Cells were treated with compound T (300nM) for 24 hours, harvested and analyzed for cell cycle distribution. Treatment of tHS68 cells with compound T caused a loss of the S phase peak (indicated by the arrow) as described in example 152. Figure 2F is a graph of WM2664 cell (CDK4/6 dependent cell line) number versus cell DNA content (as measured by propidium iodide) after treatment with compound T. Cells were treated with compound T (300nM) for 24 hours, harvested and analyzed for cell cycle distribution. As described in example 152, treatment of WM2664 cells with compound T caused a loss of the S-phase peak (indicated by the arrow). Figure 2G is a graph of the number of a2058 cells (CDK4/6 independent cell line) versus the DNA content of the cells (as measured by propidium iodide) after treatment with compound T. Cells were treated with compound T (300nM) for 24 hours, harvested and analyzed for cell cycle distribution. As described in example 152, treatment of a2058 cells with compound T did not cause loss of the S-phase peak (indicated by the arrow).

FIG. 3 is a Western blot showing phosphorylation levels of Ser807/811 and Ser780Rb following treatment with Compound T. Cdk4/6 dependent (tHS68 or WM2664) and Cdk4/6 independent cell lines (A2058) were treated with compound T (300nM) for the indicated times (0, 4, 8, 16 and 24 hours). MAPK levels are shown as a control for protein levels. After treatment, cells were harvested and analyzed for Rb phosphorylation by western blot analysis. Compound T treatment caused a decrease in Rb phosphorylation after treatment in Cdk 4/6-dependent cell lines (tHS68 and WM2664), but not in Cdk 4/6-independent cell line (a2058), as described in example 153.

FIG. 4A is a graph of the percentage of S phase cells after treatment with DMSO (dark bars) or PD0332991 (light bars) in an Rb-positive cell line (WM2664) or an Rb-negative small cell lung cancer cell line (H345, H69, H209, SHP-77, NCI417 or H82). Cells were treated with PD0332991(300nM) or DMSO control for 24 hours. Cell proliferation was measured by EdU incorporation and flow cytometry. Data represent 100,000 cellular events per cell treatment. As described in example 154, RB null SCLC cell lines were resistant to Cdk4/6 inhibition, as no change in the percentage of S-phase cells was seen upon treatment with PD 0332991.

FIG. 4B is a graph of the percentage of S phase cells after treatment with DMSO (dark bars) or compound GG (lighter bars) in the Rb-positive cell line (tHS68) or the Rb-negative small cell lung cancer cell line (H345, H69, SHP-77, or H82). Cells were treated with compound GG (300nM or 1000nM) or DMSO control for 24 hours. Cell proliferation was measured by EdU incorporation and flow cytometry. Data represent 100,000 cellular events per cell treatment. As described in example 154, RB null SCLC cell lines were resistant to Cdk4/6 inhibition as no change in the percentage of S-phase cells was seen upon treatment with compound GG.

FIG. 4C is a graph of the percentage of S phase cells after treatment with DMSO (dark bars) or compound T (lighter bars) in the Rb positive cell line (tHS68) or in the Rb negative small cell lung cancer cell line (H345, H209, or SHP-77). Cells were treated with compound T (300nM or 1000nM) or DMSO control for 24 hours. Cell proliferation was measured by EdU incorporation and flow cytometry. Data represent 100,000 cellular events per cell treatment. As described in example 154, RB null SCLC cell lines were resistant to Cdk4/6 inhibition, as no change in the percentage of S-phase cells was seen upon treatment with compound T.

Figure 5 is a graph of time (hours) after EdU incorporation versus PD0332991 administration of healthy mouse HSPC and healthy spinal cord progenitors. PD0332991(150 mg/kg) was administered by oral gavage to assess the transient effects of transient CDK4/6 inhibition on bone marrow arrest, as in Roberts et al Multiple circles of cycles-Dependent Kinase 4/6Inhibitors in Cancer therapy.jcni 2012; 104(6), 476-487 (FIG. 2A). As described in example 156, a single oral dose of PD0332991 resulted in a sustained reduction in HSPC EdU incorporation (round; LKS +) and spinal cord progenitor EdU incorporation (square; LKS-) for over 36 hours.

Figure 6A is a graph of the rate of EdU incorporation into HSPCs (compared to untreated control mice) 12 or 24 hours after administration following oral gavage of compound T, Q or GG at 150 mg/kg. Figure 6B is a graph of the percentage of EdU positive HSPC cells at 12 or 24 hours in mice treated with compound T. Mice were given 50mg/kg (triangle), 100mg/kg (square) or 150mg/kg (inverted triangle) by oral gavage. Figure 6C is a graph of the percentage of EdU positive HSPC cells at 12, 24, 36 and 48 hours in mice treated with compound T (150mg/kg by oral gavage). As described in example 157, compounds T and GG demonstrated a decrease in EdU incorporation at 12 hours and, by 24 hours, began to return to normal levels of cell division.

Figure 7 is a graph of the percentage of EdU positive HSPC cells in mice treated with PD0332991 (triangles) or compound T (inverted triangles) versus time (hours) after compound administration. Both compounds were administered by oral gavage at 150mg/kg and the percentage of EdU positive HSPC cells was measured at 12, 24, 36 or 48 hours. As described in example 158, a single oral dose of PD0332991 causes a sustained reduction in HSPC proliferation for more than 36 hours. In contrast, a single oral dose of compound T caused an initial decrease in HSPC proliferation by 12 hours, but by 24 hours after compound T administration HSPC proliferation resumed.

Figure 8A is a graph of the percentage of cells in the G0-G1 phase of the cell cycle versus time (hours) after compound elution in human fibroblasts (Rb positive) cells. Figure 8B is a graph of the percentage of cells in the S phase of the cell cycle versus time (hours) after compound elution in human fibroblasts (Rb positive) cells. Figure 8C is a graph of the percentage of cells in the G0-G1 phase of the cell cycle versus time (hours) after compound elution in human renal proximal tubule epithelial (Rb positive) cells. Fig. 8D is a graph of the percentage of cells in the S phase of the cell cycle versus time (hours) after compound elution in human renal proximal tubule epithelial (Rb positive) cells. These cell elution experiments demonstrated that the inhibitor compounds of the present invention have a transient G1 arrest in different cell types. The effect on the cell cycle after elution of the compound was determined at 24, 36, 40 and 48 hours. As described in example 159, the results show that cells treated with PD0332991 (circle) take significantly longer to reach the baseline level of cell division (see cells treated with DMSO (diamond) only) compared to cells treated with compound T (square), compound Q (triangle), compound gg (X), or compound U (X with cross).

Figure 9A is a graph of plasma drug concentration (ng/ml) versus time (hours) after compound T administration. Figure 9B is a graph of plasma drug concentration (ng/ml) versus time (hours) after compound Q administration. Figure 9C is a graph of plasma drug concentration (ng/ml) versus time (hours) after compound GG administration. Figure 9D is a graph of plasma drug concentration (ng/ml) versus time (hours) after compound U administration. The compounds were administered to mice at 30mg/kg (diamonds) by oral gavage or 10mg/kg (squares) by intravenous injection. Blood samples were taken at 0, 0.25, 0.5, 1.0, 2.0, 4.0 and 8.0 hours post-dose and plasma concentrations were determined by HPLC.

Figure 10 provides the half-lives (in minutes) of compound T and PD0332991 in human and animal (monkey, dog, rat and mouse) liver microsomes. As described in example 158, PD0332991 has a half-life of more than 60 minutes in each tested species. Compound T had a shorter half-life than PD0332991 in each species tested.

Figure 11A is a graph of cell viability of cells treated with 5uM etoposide versus indicated amounts of compound T. Viable cells were assayed 24 hours after treatment. Compound T was shown to protect tHS68 cells from chemotherapeutic-induced cell death as described in example 162. Figure 11B is a graph of cell viability of cells treated with 100 μ M carboplatin versus indicated amounts of compound T. Viable cells were assayed 24 hours after treatment. Compound T protected tHS68 cells from chemotherapeutic-induced cell death as described in example 162.

Figure 12A is a graph of activity versus variable concentration of compound T (nM) versus H2AX in HS68 cells treated with compound T (100nM, 300nM, or 1000nM) and chemotherapy (etoposide, doxorubicin (doxorubicin), carboplatin, paclitaxel (paclitaxel), or camptothecin (camptothecin)). Cdk 4/6-dependent HS68 cells were treated with indicated doses of compound T and chemotherapy. H2AX focus formation was measured to assess chemotherapy-induced DNA damage. As described in example 162, cells treated with compound T and various chemotherapeutic compounds were protected from chemotherapy-induced DNA damage.

Figure 12B is a graph of the activity of HS68 cells treated with compound T (100nM, 300nM, or 1000nM) and chemotherapy (etoposide, doxorubicin, carboplatin, paclitaxel, or camptothecin) versus the variable concentration of compound T (nM) versus caspase 3/7 activity. Cdk 4/6-dependent HS68 cells were treated with indicated doses of compound T and chemotherapy. Caspase 3/7 activity was measured to assess chemotherapy-induced apoptosis. As described in example 162, cells treated with compound T and various chemotherapeutic compounds were protected from chemotherapy-induced activation of caspase 3/7.

Figure 13 is a series of contour plots showing proliferation (as measured by EdU incorporation after 12 hours) versus cellular DNA content (as measured by DAPI staining). Representative contour plots show proliferation in WBM (whole bone marrow; top) and HSPC (hematopoietic stem and progenitor cells; LSK; bottom) as measured by EdU incorporation 12 hours after no treatment, EdU treatment alone or EdU plus compound T treatment. Compound T reduces the proliferation of whole bone marrow and hematopoietic stem and/or progenitor cells as described in example 163.

FIG. 14A is a graph of the percentage of EdU positive cells in Whole Bone Marrow (WBM) and various hematopoietic stem and progenitor cells (Lin-, LSK, HSC, MPP, or CD28+ LSK cell lineages) treated or untreated with compound T (open bars) (solid bars). Treatment with compound T inhibited proliferation of WBM and all HSPC lineages tested as described in example 163. P <0.05, P < 0.01.

Fig. 14B is a graph of the percentage of EdU positive cells in Whole Bone Marrow (WBM) and various lineage restricted progenitor cells (MP, GMP, MEP, CMP, or CLP cell lineages) treated or untreated (solid bars) with compound T (open bars). Treatment with compound T inhibited proliferation of the tested WBMs and all lineage restricted progenitor cells as described in example 163. P <0.05, P < 0.01.

Fig. 15A is a graph of the percentage of EdU positive cells in a population of T cells (total, CD4+, CD8+, DP, DN1, DN2, DN3, or DN4) treated with compound T (open bars) or untreated (solid bars). Treatment with compound T inhibited proliferation of CD4+, CD8+, DP, DN1, DN2, DN3 or DN4T cell populations as described in example 164. P <0.05, P < 0.01.

FIG. 15B is a graph of the percentage of EdU positive cells in a B cell population (B220+, B220+ sIgM +, Pre-Pro-B sIgM-, Pro-B, Pre-B) treated or untreated (solid bars) with Compound T (open bars). Treatment with compound T inhibited proliferation of various B cell populations (B220+, B220+ smim +, Pre-Pro-B smim-, Pro-B, and Pre-B) as described in example 164. P <0.05, P < 0.01.

Fig. 15C is a graph of the percentage of EdU positive cells in a population of bone marrow cells (Mac1+ Gr1+, Ter119+, or CD41+) treated or untreated with compound T (open bars) (solid bars). Treatment with compound T inhibited proliferation of Mac1+ Gr1+, Ter119+, or CD41+ bone marrow cell populations as described in example 164. P <0.05, P < 0.01.

Figure 16 shows the pharmacodynamic evaluation of compound GG in bone marrow. To evaluate the effect of transient CDK4/6 inhibition of compound GG in bone marrow on carboplatin-induced cytotoxicity, FVB/n mice (n ═ 3 per group) were treated with vehicle control, intraperitoneal injection of 90mg/kg carboplatin, or oral gavage of 150 mg/kg compound GG plus intraperitoneal injection of 90mg/kg carboplatin. 24 hours post-treatment, bone marrow was harvested and the percentage of circulating bone marrow progenitors was measured by EdU incorporation as explained earlier.

Figure 17A is a graph of complete blood counts versus time (days) after administration of 5-fluorouracil (5FU) (triangles), 5FU plus compound T (squares), or untreated control (circles). FVB wild type mice were treated with compound T (150mg/kg) or vehicle control by oral gavage thirty minutes prior to administration of 150mg/kg of 5-fluorouracil (5FU) by intraperitoneal injection. Complete blood counts were measured every two days, starting on day six. As described in example 166, whole blood cells recovered more rapidly from chemotherapy (5FU) when pretreated with compound T. Figure 17B is a graph of neutrophil count versus time (days) after administration of 5-fluorouracil (5FU) (triangles), 5FU plus compound T (squares), or untreated control (circles). The experiment was performed as described in fig. 17A. As described in example 166, neutrophils recover more rapidly from chemotherapy (5FU) when pretreated with compound T. Figure 17C is a graph of lymphocyte counts versus time (days) after administration of 5-fluorouracil (5FU) (triangles), 5FU plus compound T (squares), or untreated control (circles). The experiment was performed as described in fig. 17A. As described in example 166, lymphocytes recovered more rapidly from chemotherapy (5FU) when pretreated with compound T. Figure 17D is a graph of platelet cell count versus time (days) after administration of 5-fluorouracil (5FU) (triangles), 5FU plus compound T (squares), or untreated control (circles). The experiment was performed as described in fig. 17A. As described in example 166, platelet cells recovered more rapidly from chemotherapy (5FU) when pretreated with compound T. Figure 17E is a graph of red blood cell count versus time (days) after administration of 5-fluorouracil (5FU) (triangles), 5FU plus compound T (squares), or untreated control (circles). The experiment was performed as described in fig. 17A. As described in example 166, erythrocytes recover more rapidly from chemotherapy (5FU) when pretreated with compound T. Figure 17F is a graph of hematocrit (%) versus time (days) after administration of 5-fluorouracil (5FU) (triangles), 5FU plus compound T (squares), or untreated control (circles). The experiment was performed as described in fig. 17A. As described in example 166, the hematocrit percentage recovered more rapidly from chemotherapy (5FU) when pretreated with compound T.

Figure 18A is a graph of complete blood cell counts 14 days after administration of 5-fluorouracil (5FU), 5FU plus compound T, or untreated controls. FVB wild type mice were treated with compound T (150mg/kg) or vehicle control by oral gavage thirty minutes prior to administration of 150mg/kg of 5-fluorouracil (5FU) by intraperitoneal injection. Complete blood cell counts were measured on day 14. Boxes represent 5% -95% distribution, whisker lines represent minima and maxima, and the median represents median. Steward's t test was performed to calculate the double-sided P values. As described in example 166, whole blood cells recovered more rapidly from chemotherapy (5FU) when pretreated with compound T. Figure 18B is a graph of neutrophil counts 14 days after administration of 5-fluorouracil (5FU), 5FU plus compound T, or untreated controls. The experiment was performed as described in fig. 18A. As described in example 166, neutrophils recover more rapidly from chemotherapy (5FU) when pretreated with compound T. Figure 18C is a graph of lymphocyte counts 14 days after administration of 5-fluorouracil (5FU), 5FU plus compound T, or untreated controls. The experiment was performed as described in fig. 18A. As described in example 166, lymphocytes recovered more rapidly from chemotherapy (5FU) when pretreated with compound T. Figure 18D is a graph of red blood cell counts 14 days after administration of 5-fluorouracil (5FU), 5FU plus compound T, or untreated controls. The experiment was performed as described in fig. 18A. As described in example 166, erythrocytes recover more rapidly from chemotherapy (5FU) when pretreated with compound T. Figure 18E is a graph of platelet cell counts 14 days after administration of 5-fluorouracil (5FU), 5FU plus compound T, or untreated controls. The experiment was performed as described in fig. 18A. As described in example 166, platelet cells recovered more rapidly from chemotherapy (5FU) when pretreated with compound T.

FIG. 19A is a graph of whole blood cells (1000 cells/microliter) in untreated mice (circles), 5-fluorouracil (5FU) plus compound T treated mice (squares), or 5-FU treated mice (triangles) on day 10 of cycle 3 (day 52). FVB wild type mice were treated with compound T (150mg/kg) or vehicle control by oral gavage thirty minutes prior to administration of 150mg/kg of 5-fluorouracil (5FU) by intraperitoneal injection. On day 1 of the 21-day cycle, mice received 3 cycles of compound T or vehicle control +5 FU. Complete blood cell counts were measured on day 10 after the second dose (day 52 after the first dose (day 10 of cycle 3)). As described in example 167, whole blood cells showed improved recovery from chemotherapy (5FU) when treated with several cycles of compound T. Figure 19B is a graph of neutrophils (1000 cells/microliter) in untreated mice (circles), 5-fluorouracil (5FU) plus compound T treated mice (squares), or 5-FU treated mice (triangles) on day 10 of cycle 3 (day 52). The experiment was performed as described in fig. 19A. As described in example 167, neutrophils display improved recovery from chemotherapy (5FU) when treated with several cycles of compound T. Figure 19C is a graph of lymphocytes (1000 cells/microliter) in untreated mice (circles), 5-fluorouracil (5FU) plus compound T treated mice (squares), or 5-FU treated mice (triangles) on day 10 of cycle 3 (day 52). The experiment was performed as described in fig. 19A. As described in example 167, lymphocytes showed improved recovery from chemotherapy (5FU) when treated with several cycles of compound T. Figure 19D is a graph of red blood cells (1000 cells/microliter) in untreated mice (circles), 5-fluorouracil (5FU) plus compound T treated mice (squares), or 5-FU treated mice (triangles) on day 10 of cycle 3 (day 52). The experiment was performed as described in fig. 19A. As described in example 167, red blood cells showed improved recovery from chemotherapy (5FU) when treated with several cycles of compound T. Figure 19E is a graph of platelet cells (1000 cells/microliter) in untreated mice (circles), 5-fluorouracil (5FU) plus compound T treated mice (squares), or 5-FU treated mice (triangles) on day 10 of cycle 3 (day 52). The experiment was performed as described in fig. 19A. As described in example 167, platelet cell display was improved from recovery of chemotherapy (5FU) when treated with several cycles of compound T.

Figure 20 is a graph of the percentage of cells in G2-M phase (x), S phase (triangle), G0-G1 phase (square), or <2N (diamond) versus the variable concentration (nM) of compound T in human renal proximal tubule cells. Cells were treated with indicated concentrations of compound T for 24 hours. After compound T treatment, cells were harvested and analyzed for cell cycle distribution. As described in example 168, human renal proximal tubule cells exhibited complete G1 arrest with a corresponding decrease in the number of S phase cells.

FIG. 21 is a graph of the percentage of cells in G2-M phase (x), S phase (triangle), G0-G1 phase (square), or <2N (diamond) versus the variable concentration (nM) of Compound T in human renal proximal tubule cells treated with DMSO, etoposide, or cisplatin. Cells were treated with indicated concentrations of compound T in combination with DMSO, etoposide or cisplatin for 24 hours. After compound T treatment, cells were harvested and analyzed for cell cycle distribution. As described in example 169, treatment of human renal proximal tubular cells with compound T protected these cells from chemotherapy-induced destruction of etoposide and cisplatin.

FIG. 22 is a graph of relative γ -H2AX activity versus variable concentration (nM) of compound T in human renal proximal tubule cells treated with compound T and chemotherapy (cisplatin). Cells were treated with indicated doses of compound T (10nM, 30nM, 100nM, 300nM or 1000nM) and chemotherapy (25uM cisplatin). Focus formation was measured for γ -H2AX to assess chemotherapy-induced DNA damage. As described in example 170, cells treated with compound T were protected from chemotherapy (cisplatin) -induced DNA damage.

FIG. 23 is a graph of caspase 3/7 activation (as measured by relative light units) in renal tubular epithelial cells treated with indicated concentrations of compound T and DMSO or cisplatin (25uM, 50uM, or 100 uM). Normal renal proximal tubular epithelial cells were obtained from the American Type Culture Collection (ATCC, Manassas, Va.). Cells were grown in an incubator at 37 ℃ in a 5% CO2 humid atmosphere at 37 ℃ in a humid incubator supplemented with a renal epithelial cell growth kit (ATCC) in a renal epithelial cell basal medium (ATCC). Cells were treated with DMSO or 30nM, 100nM, 300nM or 1uM compound T in the absence or presence of 25uM, 50uM or 100uM cisplatin. After 24 hours, Caspase 3/7 activation was measured using the Caspase-Glo 3/7 assay system (Promega, Madison, Wis.) according to the manufacturer's instructions. Compound T demonstrated a dose-dependent reduction in caspase 3/7 activation in these cells as described in example 170.

FIGS. 24-26 illustrate R of compounds of the invention²Several exemplary embodiments of (a).

FIGS. 27A-27C, 28A-D, 29A-29C, 30A-30B, and 31A-31F illustrate several exemplary embodiments of the core structures of the compounds of the present invention.

Detailed Description

Improved compounds, methods and compositions are provided to minimize the effect of chemotherapeutic toxicity on CDK4/6 replication-dependent healthy cells such as hematopoietic stem cells and hematopoietic progenitor cells (collectively HSPCs) and/or renal epithelial cells in a subject, typically a human, who will be exposed to, is being exposed to, or has been exposed to a chemotherapeutic agent, typically a DNA damaging agent.

Definition of

Unless otherwise indicated, the following terms used in this application, including the specification and claims, have the definitions set forth below. As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Definitions of standardized chemical terms can be found in the references, including Carey and Sundberg (2007) Advanced Organic Chemistry, Vol.5A 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 spectrometry, chromatographic preparation and analysis, protein chemistry, biochemistry, recombinant DNA technology and pharmacology. Conventional methods of organic chemistry include the methods included in: march's Advanced Organic Chemistry: Reactions, mechanics, and Structure, 6 th edition, M.B. Smith and J.March, John Wiley & Sons, Inc., Hoboken, NJ, 2007.

The term "alkyl", alone or within other terms such as "haloalkyl" and "alkylamino", encompasses straight or branched chain groups having one to about twelve carbon atoms. "lower alkyl" has one to about six carbon atoms. Examples of such groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, and the like. The term "alkylene" encompasses bridging divalent straight and branched chain alkyl groups. Examples include methylene, ethylene, propylene, isopropylene, and the like.

The term "alkenyl" encompasses straight or branched chain groups having at least one carbon-carbon double bond and two to about twelve carbon atoms. "lower alkenyl" has two to about six carbon atoms. Examples of alkenyl groups include ethenyl, propenyl, allyl, propenyl, butenyl and 4-methylbutenyl. The terms "alkenyl" and "lower alkenyl" encompass groups having "cis" and "trans" orientations or "E" and "Z" orientations.

The term "alkynyl" denotes a straight or branched chain group having at least one carbon-carbon triple bond and having from two to about twelve carbon atoms. "lower alkynyl" groups have two to about six carbon atoms. Examples of such groups include propargyl, butynyl, and the like.

Alkyl, alkenyl, and alkynyl groups can be optionally substituted with one or more functional groups such as halo, hydroxy, nitro, amino, cyano, haloalkyl, aryl, heteroaryl, heterocyclyl, and the like.

The term "alkylamino" encompasses "N-alkylamino" and "N, N-dialkylamino" wherein the amino groups are independently substituted with one alkyl and two alkyl groups, respectively. "lower alkylamino" has one or two alkyl groups having one to six carbon atoms attached to the nitrogen atom. Suitable alkylamino groups may be monoalkylamino or dialkylamino groups such as N-methylamino, N-ethylamino, N-dimethylamino, N-diethylamino, and the like.

The term "halo" means a halogen, such as a fluorine, chlorine, bromine or iodine atom.

The term "haloalkyl" encompasses groups wherein any one or more of the alkyl carbon atoms is substituted with one or more halo groups as defined above. Examples include monohaloalkyl, dihaloalkyl, and polyhaloalkyl, including perhaloalkyl. For example, a monohaloalkyl group can have an iodo group, a bromo group, a chloro group, or a fluoro group atom within the group. Dihaloalkyl and polyhaloalkyl groups may have two or more of the same halogen atom or a combination of different halogens. "lower haloalkyl" encompasses groups having 1-6 carbon atoms. Examples of haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl and dichloropropyl. "perfluoroalkyl" means an alkyl group in which all hydrogen atoms have been replaced with fluorine 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 fashion. The term "aryl" encompasses aromatic groups such as phenyl, naphthyl, indenyl, tetrahydronaphthyl, and indanyl. More preferred aryl is phenyl. The "aryl group" may have 1 or more substituents such as lower alkyl, hydroxy, halo, haloalkyl, nitro, cyano, alkoxy, lower alkylamino, and the like. The aryl group can be optionally substituted with one or more functional groups such as halo, hydroxy, nitro, amino, cyano, haloalkyl, aryl, heteroaryl, heterocyclyl, and the like.

The term "heterocyclyl" (or "heterocycle") encompasses saturated and partially saturated heteroatom-containing cyclic groups in which the heteroatoms may be selected from nitrogen, sulfur, and oxygen. The heterocyclic ring comprises a monocyclic 6-8 membered ring and a 5-16 membered bicyclic ring system (which may include bridged fused and spiro fused bicyclic ring systems). It does not include rings containing-O-, -O-S-or-S-moieties. The "heterocyclic group" may have 1 to 3 substituents, such as hydroxy, Boc, halo, haloalkyl, cyano, lower alkyl, lower aralkyl, oxo, lower alkoxy, amino, lower alkylamino, and the like.

Examples of the saturated heterocyclic group include a saturated 3 to 6-membered heteromonocyclic group 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 heterocyclic groups include dihydrothienyl, dihydropyranyl, dihydrofuranyl, dihydrothiazolyl, and the like.

Specific examples of partially saturated and saturated heterocyclic groups include pyrrolidinyl, imidazolidinyl, piperidinyl, pyrrolinyl, pyrazolidinyl, piperazinyl, morpholinyl, tetrahydropyranyl, thiazolidinyl, dihydrothienyl, 2, 3-dihydro-benzo [1,4] dioxanyl, indolinyl, isoindolinyl, dihydrobenzothienyl, dihydrobenzofuranyl, isochromanyl, chromanyl, 1, 2-dihydroquinolinyl, 1,2,3, 4-tetrahydro-isoquinolinyl, 1,2,3, 4-tetrahydro-quinolinyl, 2,3,4,4a,9,9 a-hexahydro-1H-3-aza-fluorenyl, 5,6, 7-trihydro-l, 2, 4-triazolo [3,4-a ] isoquinolinyl, 3, 4-dihydro-2H-benzo [1,4] oxazinyl, benzo [1,4] dioxanyl, 2, 3-dihydro-1H-l λ' -benzo [ d ] isothiazol-6-yl, dihydropyranyl, dihydrofuranyl, and dihydrothiazolyl, and the like.

Heterocyclic groups also include groups in which the heterocyclic group is fused/condensed with an aryl group: condensed heterocyclic groups containing 1 to 5 nitrogen atoms, such as indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl, tetrahydropyridazinyl [ 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 groups containing 1 to 2 oxygen or sulfur atoms [ e.g., benzofuranyl, benzothienyl, 2, 3-dihydrobenzo [1,4] dioxanyl and dihydrobenzofuranyl ].

The term "heteroaryl" denotes an aryl ring system containing one or more heteroatoms selected from the group of O, N and S, wherein the ring nitrogen and sulfur atoms are optionally oxidized and the nitrogen atoms are optionally quaternized. Examples include unsaturated 5-to 6-membered heteromonocyclic groups containing 1 to 4 nitrogen atoms, such as pyrrolyl, imidazolyl, pyrazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl [ e.g., 4H-1,2, 4-triazolyl, 1H-1,2, 3-triazolyl, 2H-1,2, 3-triazolyl ]; unsaturated 5-to 6-membered heteromonocyclic group containing an oxygen atom such as pyranyl, 2-furyl, 3-furyl and the like; unsaturated 5-to 6-membered heteromonocyclic group containing a sulfur atom, such as 2-thienyl, 3-thienyl, etc.; unsaturated 5-to 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, such as 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 an alkyl group substituted with a heteroaryl group. Examples include pyridylmethyl and thienylethyl.

The term "sulfonyl", whether used alone or in conjunction with other terms, such as alkylsulfonyl, shall denote the divalent radical-SO₂-。

The term "carboxy (or carboxyl)" whether used alone or in combination with other terms, such as "carboxyalkyl", denotes-C (O) -OH.

The term "carbonyl", whether used alone or in combination with other terms, such as "aminocarbonyl", denotes-C (O) -.

The term "aminocarbonyl" denotes a compound of formula-C (O) -NH₂Amide group of (2).

The term "heterocycloalkyl" encompasses alkyl groups substituted with a heterocyclyl group. Examples include piperidinylmethyl and morpholinylethyl.

The term "arylalkyl" encompasses alkyl groups substituted with aryl groups. Examples include benzyl, benzhydryl and phenethyl. The aryl group of the arylalkyl group can be additionally substituted with halo, alkyl, alkoxy, haloalkyl, and haloalkoxy groups.

The term "cycloalkyl" includes saturated carbocyclic groups having from 3 to 10 carbons. Lower cycloalkyl radicals including C₃-C₆And (4) a ring. Examples include cyclopentyl, cyclopropyl and cyclohexyl. The cycloalkyl group can be optionally substituted with one or more functional groups such as halo, hydroxy, nitro, amino, cyano, haloalkyl, aryl, heteroaryl, heterocyclyl, and the like.

The term "cycloalkylalkyl" encompasses alkyl substituted with cycloalkyl. "lower cycloalkylalkyl" is cycloalkyl attached to an alkyl group having one to six carbon atoms. Examples include cyclohexylmethyl. The cycloalkyl groups in the groups may additionally be 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" means open, including the indicated components but not excluding other elements.

As used herein, the term "oxo" encompasses an oxygen atom attached with a double bond.

As used herein, the term "nitro" encompasses-NO₂。

As used herein, the term "cyano" encompasses-CN.

The term "prodrug" as used herein means a compound that is converted to the parent drug when administered to a subject in vivo. As used herein, the term "parent drug" means any compound described herein that is suitable for use in treating any of the conditions described herein, or controlling or ameliorating the underlying cause or symptoms associated with any of the physiological or pathological conditions described herein, in a subject, typically a human. Prodrugs can be used to achieve any desired effect, including enhancing the properties of the parent drug or improving the drug or pharmacokinetic properties of the parent drug. Prodrug strategies exist that offer the option of modulating the conditions under which the parent drug is produced in vivo, all of which are considered to be included herein. Non-limiting examples of prodrug strategies include the following covalent attachments: a removable group or removable moiety of a group such as, but not limited to, acylation, phosphorylation, phosphonation, phosphoramidate derivatives, amidation, reduction, oxidation, esterification, alkylation, other carboxyl derivatives, sulfoxy or sulfone derivatives, carbonylation, or anhydride.

In the specification and claims, unless otherwise indicated, a given chemical formula or name shall encompass all optical and stereoisomers as well as racemic mixtures in which such isomers and mixtures exist.

In some embodiments, the CDK4/6 replication-dependent healthy cells are hematopoietic stem cell progenitors. 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), pluripotent progenitor cells (MPPs), common spinal cord progenitor Cells (CMPs), common lymphoid progenitor Cells (CLPs), granulocyte-monocyte progenitor cells (GMPs), and megakaryocyte-erythrocyte progenitor cells (MEPs). In some embodiments, the CDK4/6 replication-dependent healthy cells may be cells in non-hematopoietic tissues such as (but not limited to) liver, kidney, pancreas, brain, lung, adrenal gland, intestine, gut, stomach, skin, auditory system, bone, bladder, ovary, uterus, testis, gall bladder, thyroid, heart, pancreatic islets, blood vessels, and the like. In some embodiments, the CDK4/6 replication-dependent healthy cells are renal cells, and in particular renal epithelial cells, such as renal proximal tubule epithelial cells. In some embodiments, the CDK4/6 replication-dependent healthy cells are hematopoietic stem cell progenitors. In some embodiments, the CDK4/6 replication-dependent healthy cells may be cells in non-hematopoietic tissues such as (but not limited to) liver, kidney, pancreatic gland, brain, lung, adrenal gland, intestine, gut tube, stomach, skin, auditory system, bone, bladder, ovary, uterus, testis, gall bladder, thyroid, heart, pancreatic islets, blood vessels, and the like.

The term "selective CDK4/6 inhibitor" as used in the context of the compounds described herein is included as IC in standard phosphorylation assays₅₀Molar concentration ratio IC required to inhibit CDK2 activity to the same extent₅₀Compounds that inhibit CDK4 activity, CDK6 activity or CDK4 and CDK6 activity at molar concentrations that are at least 1/500 or 1/1000 or 1/1500 or 1/1800, 1/2000, 1/5000 or 1/10,000.

By "inducing G1 arrest" is meant that the inhibitor compound induces a substantial portion of the cell population to be quiescent in the G1 phase of the cell cycle.

By "hematopoietic insufficiency" is meant an insufficient blood cell lineage count or the production of blood cells (i.e., myelodysplasia) and/or lymphocytes (i.e., lymphopenia, a reduction in the number of circulating lymphocytes such as B cells and T cells). Bone marrow suppression in the form of blood deficiencies, such as anemia, a decrease in platelet count (i.e., thrombocytopenia), or a decrease in white blood cell count (i.e., leukopenia) or granulocytopenia (e.g., neutropenia) can be observed.

By "synchronized re-entry into the cell cycle" is meant CDK4/6 replication-dependent healthy cells that are in G1 arrest due to the action of CDK4/6 inhibitor compounds, e.g., HSPCs re-enter the cell cycle within a relatively same focused time frame or at a relatively same rate after the action of the compounds dissipates. By contrast, "non-synchronous re-entry into the cell cycle" means CDK4/6 replication-dependent healthy cells that are in G1 arrest due to the action of CDK4/6 inhibitor compounds, e.g., HSPCs re-enter the cell cycle within a relatively different time frame of concentration or at a relatively different rate after the action of compounds such as PD0332991 dissipates.

By "cessation of circulation" or "drug holiday" is meant a period of time during which the subject is not administered or exposed to a chemotherapeutic agent. For example, in a treatment regimen in which a subject chemotherapeutic agent is administered for 21 days and no chemotherapeutic agent is administered for 7 days and the regimen is largely repeated, the 7-day period of non-administration is considered to be a "stop cycle" or "drug holiday". Off-target and drug holidays can also refer to discontinuation of a treatment regimen in which the subject is not administered a chemotherapeutic agent for a period of time due to adverse side effects, such as myelosuppression.

The subject treated is typically a human subject, although it is understood that the methods described herein are effective for other animal species, such as mammals and vertebrates. More specifically, the term subject can include animals used in assays, such as animals used in preclinical testing, including (but not limited to) mice, rats, monkeys, dogs, pigs, and rabbits; and domestic swine (pigs and hogs), ruminants, horses, poultry, felines, bovines, murines, canines, and the like.

By "substantial portion" or "substantial portion" is meant at least 80%. In alternative embodiments, the fraction may be at least 85%, 90% or 95% or greater.

In some embodiments, the term "CDK 4/6 replication-independent cancer" refers to a cancer that does not significantly require CDK4/6 activity for replication. Such types of cancer are often, but not always, characterized by (e.g., cell-displayed) increased levels of CDK2 activity or decreased expression of retinoblastoma tumor suppressor proteins or retinoblastoma family member proteins (such as, but not limited to, p107 and p 130). Increased levels of CDK2 activity or reduced expression or absence of retinoblastoma tumor suppressor protein or retinoblastoma family member protein may be increased or decreased, e.g., as compared to normal cells. In some embodiments, an increased level of CDK2 activity may be associated with (e.g., may be caused by or observed with) MYC proto-oncogene amplification or overexpression. In some embodiments, the increased level of CDK2 activity may be associated with overexpression of cyclin E1, cyclin E2, or cyclin a.

As used herein, the term "chemotherapy" or "chemotherapeutic agent" refers to treatment with a cell growth inhibitory agent or cytotoxic agent (i.e., a compound) to reduce or eliminate the growth or proliferation of undesirable cells, such as cancer cells. Thus, as used herein, "chemotherapy" or "chemotherapeutic agent" refers to a cytotoxic or cytostatic agent used to treat proliferative disorders such as cancer. The cytotoxic effect of the agent can be, but need not be, the result of one or more of nucleic acid insertion or binding, DNA or RNA alkylation, RNA or DNA synthesis inhibition, another nucleic acid-related activity (e.g., protein synthesis), or inhibition of any other cytotoxic effect.

Thus, a "cytotoxic agent" can be any compound or any combination of compounds that is also described as an "antineoplastic" agent or a "chemotherapeutic agent. Such compounds include, but are not limited to, DNA damaging compounds and other chemicals that can kill cells. "DNA-disrupting chemotherapeutic agents" include, but are not limited to, alkylating agents, DNA intercalating agents, protein synthesis inhibitors, inhibitors of DNA or RNA synthesis, DNA base analogs, topoisomerase inhibitors, and telomerase inhibitors or telomere DNA binding compounds. For example, alkylating agents include alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines (aziridines), such as benzotepa (benzodizepa), carbopol quinone (carboquone), metodepa (uretepa), and urethanimine (uretepa); ethyleneimine and methyl melamine such as altretamine (altretamine), triethylenemelamine (triethyleneamine), triethylenephosphoramide (triethylenephosphoramide), triethylenethiophosphoramide (triethylenethiophosphoramide), and trimethylolmelamine (trimethylenemelamine); nitrogen mustards such as chlorambucil (chlorambucil), chlorambucil (chloranaphazine), cyclophosphamide (cyclophosphamide), estramustine (estramustine), ifosfamide (iphosphamide), mechlorethamine (mechlorethamine), mechlorethamine oxide hydrochloride (mechlorethamine oxide hydrochloride), melphalan (melphalan), norvasicine (novacine), benzene mustard (pherenesterol), prednimustine (prednimustine), chloroacetoamide (trofosfamide), and uracil mustard (uracil mustard); and nitrosoureas (nitrourea ureas), such as carmustine (carmustine), chlorouracil (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine), and ramustine (ranimustine).

Antibiotics used for the treatment of cancer include actinomycin D (dactinomycin), daunorubicin (daunorubicin), doxorubicin (doxorubicin), idamycin (idarubicin), bleomycin sulfate (bleomycin sulfate), mitomycin (mitomycin), mithramycin (plicamycin) and streptozotocin (streptozocin). Chemotherapeutic antimetabolites include mercaptopurine (mercanopirine), thioguanine (thioguanine), cladribine (cladribine), fludarabine phosphate (fludarabine phosphate), fluorouracil (fluorouracil, 5-FU)), floxuridine (floxuridine), cytarabine (cytarabine), pentostatin (pentostatin), methotrexate (methotrexate), and azathioprine (azathioprine), acyclovir (acyclovir), adenine beta-1-D-arabinoside (adenin beta-1-D-arabinoside), methotrexate (amethopterin), aminopterin (aminopterin), 2-aminopurine (2-aminopurine), aphidicolin (apidioxin), 8-azaguanine (8-azaguananine), azaserine (6-azauracil), 2-azauracil (2' -azathioprine), 2-azathioprine (azathioprine), azadeoxyriboside (azathioprine), and pharmaceutically acceptable salts thereof, 5-bromodeoxycytidine, cytosine beta-1-D-arabinoside, diazoxynorleucine (dioxaxynaleucine), dideoxynucleoside (dideoxynucleoside), 5-fluorodeoxycytidine (5-fluorodeoxycytidine), 5-fluorodeoxyuridine (5-fluorodeoxyuridine), and hydroxyurea (hydroxyurea).

Chemotherapeutic protein synthesis inhibitors include abrin (abrin), aurintricarboxylic acid (aurintricarboxylic acid), chloraminophen (chloremphenicol), colicin E3 (colicin E3), cycloheximide (cycloheximide), diphtheria toxin (diphenoxy toxin), ivermectin A (edene A), emetine (emetine), erythromycin (erythromycin), ethionine (ethionine), fluoride (fluoride), 5-fluorotryptophan (5-fluorotryptophane), fusidic acid (fusidic acid), guanylmethylenediphosphate (guanylmethylenediphosphate), and guanyliminodiphosphate (guanyliminodiphosphate), kanamycin (kanamymycin), kasugamycin (calicheamicin), xanthomycin (yellow-methyl-threonine), and methylthreonine (O). Other protein synthesis inhibitors include modeccin, neomycin, norvaline (norvaline), pactamycin (pactamycin), paromomycin (paromomycin), puromycin (puromycin), ricin (ricin), shiga toxin (shiga toxin), oxytetracycline (showdomacin), sparsomycin (sparsomycin), spectinomycin (spectinomycin), streptomycin (streptamycin), tetracycline (tetracycline), thiostrepton (thiostrepton), and trimethoprim (trimethoprim). DNA synthesis inhibitors include alkylating agents such as dimethyl sulfate, mitomycin c (mitomycin c), nitrogen mustard and sulfur mustard; intercalating agents, such as acridine dye (acridine dye), actinomycin (actinomycins), doxorubicin (adriamycin), anthracene (anthracenes), benzopyrene (benzopyrene), ethidium bromide (ethidium bromide), promethidium iodide (propidium iodide); and other agents, such as distamycin (distamycin) and fusin (netropsin). Topoisomerase inhibitors such as coumaromycin (coumermycin), nalidixic acid (nalidixic acid), novobiocin (novobiocin), and oxolinic acid (oxolinic acid); cell division inhibitors including colchicine (colcemide), colchicine (colchicine), vinblastine (vinblastine) and vincristine (vincristine); and inhibitors of RNA synthesis including actinomycin D, alpha-amanitine and other fungal properdins, cordycepin (3' -deoxyadenosine), dichlororibofuranone acylbenzimidazoles, rifampin (rifapencine), streptovaricin (streptovaricin), and liberidin (streptoldigain) may also be used as DNA damaging compounds.

Current chemotherapeutic agents whose toxic effects may be mitigated by the selective CDK4/6 inhibitors disclosed herein include, but are not limited to, doxorubicin (adriamycin), 5-fluorouracil (5FU), 6-mercaptopurine, gemcitabine (gemcitabine), melphalan, chlorambucil, mitomycin, irinotecan (irinotecan), mitoxantrone (mitoxantrone), etoposide, camptothecin (camptothecin), actinomycin-D (actinomycin-D), mitomycin, cisplatin, hydrogen peroxide (hydroxaoxide), carboplatin, procarbazine (procarbazine), methylene chloride, cyclophosphamide, ifosfamide, melphalan, chlorambucil, busulfan, nitrosourea (nitrourea), actinomycin D (dactinomycin), daunomycin, doxorubicin, bleomycin (taxol), taxol (taxol), and taxol (doxorubicin), Trans-platinum (transplatinum), vinblastine (vinblasttine), vinblastine (vinblastine), carmustine (vinblasttin), carmustine, cytarabine, mechlorethamine, chlorambucil, streptozotocin (streptacin), lomustine (lomustine), temozolomide (temozolomide), thiotepa (thiotepa), altretamine, oxaliplatin (oxaliplatin), camptothecin (camptothecin), methotrexate and the like and similar action type agents. In one embodiment, the DNA-disrupting chemotherapeutic is selected from cisplatin, carboplatin, camptothecin, doxorubicin, and etoposide.

In certain alternative embodiments, the CDK4/6 inhibitors described herein are used in combination with chemotherapeutic agents for the treatment of CDK4/6 replication-independent, e.g., Rb-negative cancers or proliferative disorders, for anti-cancer or anti-proliferative effects. The CDK4/6 inhibitors described herein may provide additive or synergistic effects with chemotherapeutic agents with greater anticancer effects than are seen with chemotherapeutic agents alone. In one embodiment, the CDK4/6 inhibitor described herein may be combined with one or more chemotherapeutic compounds described above. In one embodiment, the CDK4/6 inhibitor described herein may be combined with a chemotherapeutic agent selected from (but not limited to) the following: tamoxifen, midazolam, letrozole, bortezomib, anastrozole, goserelin, mTOR inhibitors, PI3 kinase inhibitors, dual mTOR-PI3K inhibitors, MEK inhibitors, RAS inhibitors, ALK inhibitors, HSP inhibitors (e.g., HSP70 and HSP 90 inhibitors or combinations thereof), BCL-2 inhibitors, apoptosis inducing compounds, AKT inhibitors (including but not limited to MK-2206, GSK690693, perifosin, KRX-0401, GDC-0068, Triciribine (tricirile), AZD5363, and magnolol (Honokiol), PF-04691502, and Miltefosine (tefosine)), PD-1 inhibitors (including but not limited to Nivolumab (Nivolumab), CT-011, MK-3475, BMS 6558, and BMS-93514), or FLT-3 inhibitors (including but not limited to P, dolitinib (dovidib), Quinazatinib (Quizartinib) (AC220), Amuvatinib (MP-470), Tangdultinib (Tandatinib) (MLN518), ENMD-2076 and KW-2449), or combinations thereof. Examples of mTOR inhibitors include, but are not limited to, rapamycin and its analogs, everolimus (Afinitor), temsirolimus, bendamustine, sirolimus, and de-folimus. Examples of P13 kinase inhibitors include, but are not limited to, wortmannin, desmethylcollagenase, piriforin, edberg, PX-866, IPI-145(Infinity), BAY 80-6946, BEZ235, RP6503, TGR 1202(RP5264), MLN1117(INK1117), Pirisib, Bupaspaler, SAR 2454408 (XL147), SAR 24409 (XL765), Palomid 529, ZSTK474, PWT33597, RP6530, CUDC-907, and AEZS-136. Examples of MEK inhibitors include, but are not limited to, trametinib, semetinib, MEK162, GDC-0973 (XL518), and PD 0325901. Examples of RAS inhibitors include, but are not limited to, relisin and siG12D LODER. Examples of ALK inhibitors include, but are not limited to, crizotinib, AP26113, and LDK 378. HSP inhibitors include, but are not limited to, geldanamycin or 17-N-allylamino-17-deoxygeldanamycin (17AAG) and radicicol. In one embodiment, the CDK4/6 inhibitor in combination with a chemotherapeutic agent is selected from a compound or composition comprising formula I, formula II, formula III, formula IV, or formula V described above, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof. In one embodiment, the compound is selected from the compounds in table 1 below, or a pharmaceutically acceptable composition, salt, isotopic analog or prodrug thereof. In one embodiment, the compound is selected from the group consisting of compound T, Q, GG, U, and AAAA, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof.

In one embodiment, the CDK4/6 inhibitor described herein may be combined with a chemotherapeutic agent selected from (but not limited to): imatinib mesylate (Imatinib mesylate)Dasatinib (Dasatinib)Nilotinib (Nilotinib)Bosutinib (Bosutinib)Trastuzumab (Trastuzumab)Pertuzumab (Pertuzumab) (Perjeta TM), Lapatinib (Lapatinib)Gefitinib (Gefitinib)Erlotinib (Erlotinib)Cetuximab (Cetuximab)Panitumumab (Panitumumab)Vandetanib (Vandetanib)Weiluofini (Vemurafenib)Vorinostat (Vorinostat)Romidepsin (Romidepsin)Bexarotene (Bexarotee)Aliretin A acid (Alitretinoin)Retinoic acid (Tretinoin)Carfilzomib (Kyprolis TM), Pralatrexate (Pralatrexate)Bevacizumab (Bevacizumab)(Ziv-aflibercept)Sorafenib (Sorafenib)Sunitinib (Sunitinib)Pazopanib (Pazopanib)Ruighfenib (Regorafenib)And Cabozantinib (Cabozantinib) (CometriqTM).

By "long-term hematologic toxicity" is meant hematologic toxicity that affects a subject for a period of more than one week or more, one or more months, or one or more years following administration of a chemotherapeutic agent. Long-term blood toxicity can cause bone marrow disorders that can cause inefficient production of blood cells (i.e., myelodysplasia) and/or lymphocytes (i.e., lymphopenia, a reduction in the number of circulating lymphocytes such as B cells and T cells). Hematological toxicities such as, for example, anemia, a decrease in platelet count (i.e., thrombocytopenia), or a decrease in white blood cell count (i.e., neutropenia) can be observed. In some cases, myelodysplasia can cause the development of leukemia. The long-term toxicity associated with chemotherapeutic agents may also destroy other self-renewing cells in the subject in addition to blood cells. Thus, prolonged toxicity may also cause pale complexion and weakness.

Active compound

In one embodiment, the invention is directed to the use of a compound of formula I, II, III, IV or V:

wherein:

z is- (CH)₂)_x-, where x is 1, 2, 3 or 4, or-O- (CH)₂)_z-, wherein z is 2, 3 or 4;

each X is independently CH or N;

each X' is independently CH or N;

x' is independently CH₂S or NH, configured such that the moiety is a stable 5-membered ring;

R、R⁸and R¹¹Independently H, C₁-C₃Alkyl or haloalkyl, cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O or S; - (alkylene) m-C₃-C₈Cycloalkyl, - (alkylene)_mAryl, - (alkylene)_m-heterocyclyl, - (alkylene)_m-heteroaryl, - (alkylene)_m-NR³R⁴- (alkylene group)_m-C(O)-NR³R⁴(ii) a - (alkylene)_m-O-R⁵- (alkylene group)_m-S(O)_n-R⁵Or- (alkylene)_m-S(O)n-NR³R⁴Any of which may be optionally independently substituted, where valency permits, with one or more R groups, and wherein two R groups are bound to the same or adjacent atoms^xThe groups may optionally be combined to form a ring;

each R¹Independently is aryl, alkyl, cycloalkyl or haloalkyl, wherein each of said alkyl, cycloalkyl and haloalkyl optionally includes an O or N heteroatom in the chain in place of carbon and two Rs on adjacent ring atoms or on the same ring atom ¹Optionally forming a 3-8 membered ring together with the ring atoms to which they are attached;

y is 0, 1, 2, 3 or 4;

R²is- (alkylene)_m-heterocyclyl, - (alkylene)_m-heteroaryl, - (alkylene)_m-NR³R⁴- (alkylene group)_m-C(O)-NR³R⁴(ii) a - (alkylene)_m-C (O) -O-alkyl; - (alkylene)_m-O-R⁵- (alkylene group)_m-S(O)_n-R⁵Or- (alkylene)_m-S(O)_n-NR³R⁴Any of which may optionally independently pass through one or more R, where valence permits^xSubstituted by radicals, and in which two of R are bound to the same or to adjacent atoms^xThe groups may optionally combine to form a ring and wherein m is 0 or 1 and n is 0, 1 or 2;

R³and R⁴Independently for each occurrence:

(i) hydrogen or

(ii) Alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl, any of which may optionally be independently interrupted, as valence permits, by one or more R^xSubstituted by radicals, and in which two of R are bound to the same or to adjacent atoms^xThe groups may optionally be combined to form a ring; or R³And R⁴Together with the connection thereofCan be combined to form a ring optionally independently via one or more R, as valence permits^xA heterocycle substituted by a group, and wherein two of R are bound to the same or adjacent atom ^xThe groups may optionally be combined to form a ring;

R⁵and R⁵Each occurrence of

(i) Hydrogen or

(ii) Alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl, any of which may optionally be independently interrupted by one or more R where valency permits^xSubstituted by groups;

R^xindependently for each occurrence is halo, cyano, nitro, oxo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, heterocycloalkyl, - (alkylene)_m-OR⁵- (alkylene group)_m-O-alkylene-OR⁵- (alkylene group)_m-S(O)_n-R⁵- (alkylene group)_m-NR³R⁴- (alkylene group)_m-CN, - (alkylene)_m-C(O)-R⁵- (alkylene group)_m-C(S)-R⁵- (alkylene group)_m-C(O)-OR⁵- (alkylene group)_m-O-C(O)-R⁵- (alkylene group)_m-C(S)-OR⁵- (alkylene group)_m-C (O) - (alkylene)_m-NR³R⁴- (alkylene group)_m-C(S)-NR³R⁴- (alkylene group)_m-N(R³)-C(O)-NR³R⁴- (alkylene group)_m-N(R³)-C(S)-NR³R⁴- (alkylene group)_m-N(R³)-C(O)-R⁵- (alkylene group)_m-N(R³)-C(S)-R⁵- (alkylene group)_m-O-C(O)-NR³R⁴- (alkylene group)_m-O-C(S)-NR³R⁴- (alkylene group)_m-SO₂-NR³R⁴- (alkylene group))_m-N(R³)-SO₂-R⁵- (alkylene group)_m-N(R³)-SO₂-NR³R⁴- (alkylene group)_m-N(R³)-C(O)-OR⁵ ₎- (alkylene group)_m-N(R³)-C(S)-OR⁵Or- (alkylene)_m-N(R³)-SO₂-R⁵(ii) a Wherein:

the alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, and heterocycloalkyl may be further independently substituted with one or more of the following:

- (alkylene)_m-CN, - (alkylene)_m-OR⁵- (alkylene)_m-S(O)_n-R⁵[ alkyl ] - (alkylene)_m-NR³*R⁴- (alkylene)_m-C(O)-R⁵- (alkylene)_m-C(＝S)R⁵[ alkyl ] - (alkylene)_m-C(＝O)O R⁵- (alkylene)_m-OC(＝O)R⁵- (alkylene)_m-C(S)-OR⁵'x' (alkylene)_m-C(O)-NR³*R⁴- (alkylene)_m-C(S)-NR³*R⁴[ alkyl ] - (alkylene)_m-N(R³*)-C(O)-NR³*R⁴- (alkylene)_m-N(R³*)-C(S)-NR³*R⁴[ alkyl ] - (alkylene)_m-N(R³*)-C(O)-R⁵- (alkylene)_m-N(R³*)-C(S)-R⁵[ alkyl ] - (alkylene)_m-O-C(O)-NR³*R⁴- (alkylene)_m-O-C(S)-NR³*R⁴[ alkyl ] - (alkylene)_m-SO₂-NR³*R⁴- (alkylene)_m-N(R³*)-SO₂-R⁵[ alkyl ] - (alkylene)_m-N(R³*)-SO₂-NR³*R⁴- (alkylene)_m-N(R³*)-C(O)-OR⁵[ alkyl ] - (alkylene)_m-N(R³*)-C(S)-OR⁵Or- (alkylene)_m-N(R³*)-SO₂-R⁵*，

n is 0, 1 or 2, and

m is 0 or 1;

R³a and R⁴Independently at each occurrence:

(i) hydrogen or

(ii) Alkyl, alkenyl, alkynyl cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl, any of which may optionally be independently interrupted by one or more R as valence permits^xSubstituted by groups; or R³A and R⁴Together with the nitrogen atom to which they are attached may be combined to form, optionally independently, one or more R, where valence permits^xA group-substituted heterocycle; and is

R⁶Is H or lower alkyl, - (alkylene) m-heterocyclyl, - (alkylene)_m-heteroaryl, - (alkylene)_m-NR³R⁴- (alkylene group)_m-C(0)-NR³R⁴(ii) a - (alkylene)_m-0-R⁵- (alkylene group)_m-S(0)_n-R⁵Or- (alkylene) _m-S(0)_n-NR³R⁴Any of which may optionally independently pass through one or more R, where valence permits^xSubstituted by radicals, and in which two R are bound to the same or to adjacent atoms^xThe groups may optionally be combined to form a ring; and is

R¹⁰Is (i) NHR^AWherein R is^AIs unsubstituted or substituted C₁-C₈Alkyl, cycloalkylalkyl or-TT-RR, C₁-C₈Cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O and S; TT is unsubstituted or substituted C₁-C₈Alkyl or C₃-C₈A cycloalkyl linking group; and RR is hydroxy, unsubstituted or substituted C₁-C₆Alkoxy, amino, unsubstituted or substituted C₁-C₆Alkylamino, unsubstituted or substituted di-C₁-C₆Alkylamino, unsubstituted or substituted C₆-C₁₀Aryl, unsubstituted or substituted heteroaryl comprising one or two 5-or 6-membered rings and 1 to 4 heteroatoms selected from N, O and S, unsubstituted or substituted C₃-C₁₀A carbocycle or an unsubstituted or substituted heterocycle comprising one or two 5-or 6-membered rings and 1-4 heteroatoms selected from N, O and S; or (ii) -C (O) -R¹²or-C (O) O-R¹³Wherein R is¹²Is NHR^AOr R^AAnd R is¹³Is R^A；

Or a pharmaceutically acceptable salt, prodrug or isotopic variant thereof, for example a partially or fully deuterated form.

In some aspects, the compound has formula I or formula II and R⁶Is absent.

In some aspects, the compound has formula III:

and the variables are as defined for the compounds of formulae I and II and pharmaceutically acceptable salts thereof.

In some aspects, R^xWithout further substitution.

In some aspects, R²Is- (alkylene)_m-heterocyclyl, - (alkylene)_m-heteroaryl, - (alkylene)_m-NR³R⁴- (alkylene group)_m-C(O)-NR³R⁴(ii) a - (alkylene)_m-O-R⁵- (alkylene group)_m-S(O)_n-R⁵Or- (alkylene)_m-S(O)_n-NR³R⁴Any of which may optionally independently pass through one or more R, where valence permits^xSubstituted by radicals, and in which two R are bound to the same or to adjacent atoms^xThe groups may optionally combine to form a ring and wherein m is 0 or 1 and n is 0, 1 or 2.

In some aspects, R⁸Is hydrogen or C₁-C₃An alkyl group.

In some aspects, R is hydrogen or C₁-C₃An alkyl group.

In some aspects, R²Is- (alkylene)_m-heterocyclyl, - (alkylene)_m-NR³R⁴- (Alkylene)_m-C(O)-NR³R⁴- (alkylene group)_m-C (O) -O-alkyl or- (alkylene)_m-OR⁵Any of which may optionally independently pass through one or more R, where valence permits^xSubstituted by radicals, and in which two R radicals are bound to the same or to adjacent atoms^xThe groups may optionally combine to form a ring.

In some aspects, R²Is- (alkylene)_m-heterocyclyl, - (alkylene)_m-NR³R⁴- (Alkylene)_m-C(O)-NR³R⁴- (alkylene group)_m-C (O) -O-alkyl or- (alkylene)_m-OR⁵No further substitution.

In some aspects, R²M in (1). In another aspect, R²The alkylene group in (1) is a methylene group.

In some aspects, R²Is composed ofWherein:

R^2*is a bond, alkylene, - (alkylene)_m-O- (alkylene)_m-, - (alkylene)_m-C (O) - (alkylene)_m-, - (alkylene)_m-S(O)₂- (alkylene)_m-and- (alkylene)_m-NH- (alkylene)_m-, wherein each m is independently 0 or 1;

p is a 4-to 8-membered monocyclic or bicyclic saturated heterocyclyl;

each R^x1Independently is- (alkylene)_m-(C(O))_m- (alkylene)_m-(N(R^N))_m- (alkyl)_mWherein each m is independently 0 or 1, provided that at least one m is 1; - (C (O)) O-alkyl; - (alkylene)_m-cycloalkyl, wherein m is 0 or 1;-N(R^N) -a cycloalkyl group; -c (o) -cycloalkyl; - (alkylene)_m-heterocyclyl, wherein m is 0 or 1; or-N (R)^N) -a heterocyclic group; -c (o) -heterocyclyl; -S (O)₂- (alkylene)_mWherein m is 1 or 2, wherein:

R^Nis H, C₁To C₄Alkyl or C₁To C₆A heteroalkyl group, and

wherein two R are^x1May form a ring together with the atom on P to which it is attached which may be the same atom; and is

t is 0, 1 or 2.

In some aspects, each R is ^x1Only optionally substituted with unsubstituted alkyl, halogen or hydroxy.

In some aspects, R^x1Is hydrogen or unsubstituted C₁-C₄An alkyl group.

In some aspects, at least one R^x1Is- (alkylene)_m-heterocyclyl, wherein m is 0 or 1.

In some aspects, R²Is composed ofWherein P is a 4 to 8 membered monocyclic or bicyclic saturated heterocyclic group.

In some aspects, R²Is composed of

In some aspects, R²Is composed of

In some aspects, R²Is composed ofWherein:

R^2*is a bond, alkylene, - (alkylene)_m-O- (alkylene)_m-, - (alkylene)_m-C (O) - (alkylene)_m-, - (alkylene)_m-S(O)₂- (alkylene)_m-and- (alkylene)_m-NH- (alkylene)_m-, wherein each m is independently 0 or 1;

p is a 4-to 8-membered monocyclic or bicyclic saturated heterocyclyl;

p1 is a 4 to 6 membered monocyclic saturated heterocyclyl;

each R^x2Independently hydrogen or alkyl; and is

s is 0, 1 or 2.

In some aspects, R²Is composed of

In some aspects, P1 includes at least one nitrogen.

In some aspects, R in any of the previous aspects²Any alkylene in the group is not further substituted.

In some aspects, R²Selected from the structures depicted in fig. 24-26.

In some aspects, R²Is composed of

In some aspects, the compounds have the general formula I and, more specifically, one of the general structures in fig. 27-31, wherein the variables are as previously defined.

In some aspects, the compound has general formula Ia:

wherein R is¹、R²R 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 R²Is composed ofWherein P is a 4-to 8-membered monocyclic or bicyclic saturated heterocyclic group and R^2*、R^x1And t is as previously defined.

In some embodiments, the compound has formula Ia and R²Is composed ofWherein P is a 4-to 8-membered monocyclic or bicyclic saturated heterocyclic group, R^x1Is hydrogen or unsubstituted C₁-C₄Alkyl and R^2*As previously defined.

In some embodiments, the compound has formula Ib:

wherein R is²And R is as previously defined.

In some embodiments, the compound has formula Ib and R is alkyl.

In some embodiments, the compound has formula Ib and R is H.

In some embodiments, the compound has formula Ib and R²Is composed ofWherein P is a 4-to 8-membered monocyclic or bicyclic saturated heterocyclic group and R^2*、R^x1And t is as previously defined.

In some embodiments, the compound has formula Ib and R²Is composed ofWherein P is a 4-to 8-membered monocyclic or bicyclic saturated heterocyclic group, R^x1Is hydrogen or C ₁-C₄Alkyl radicalAnd R is^2*As previously defined.

In some embodiments, the compound has formula Ic:

wherein R is²And R is 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 R²Is composed ofWherein P is a 4-to 8-membered monocyclic or bicyclic saturated heterocyclic group and R^2*、R^x1And t is as previously defined.

In some embodiments, the compound has formula Ic and R²Is composed ofWherein P is a 4-to 8-membered monocyclic or bicyclic saturated heterocyclic group, R^x1Is hydrogen or C₁-C₄Alkyl and R^2*As previously defined.

In some embodiments, the compound has formula Id:

wherein R is²And R is 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 R²Is composed ofWherein P is a 4-to 8-membered monocyclic or bicyclic saturated heterocyclic group and R^2*、R^x1And t is as previously defined.

In some embodiments, the compound has formula Id and R²Is composed ofWherein P is a 4-to 8-membered monocyclic or bicyclic saturated heterocyclic group, R ^x1Is hydrogen or C₁-C₄Alkyl and R^2*As previously defined.

In some embodiments, the compound has formula Ie:

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 R²Is composed ofWherein P is a 4-to 8-membered monocyclic or bicyclic saturated heterocyclic group and R^2*、R^x1And t is as previously defined.

In some embodiments, the compound has formula Ie and R²Is composed ofWherein P is a 4-to 8-membered monocyclic or bicyclic saturated heterocyclic group, R^x1Is hydrogen or C₁-C₄Alkyl and R^2*As previously defined.

In some embodiments, the compound has formula If:

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 R²Is composed ofWherein P is a 4-to 8-membered monocyclic or bicyclic saturated heterocyclic group and R^2*、R^x1And t is as previously defined.

In some embodiments, the compound has formula If and R²Is composed ofWherein P is a 4-to 8-membered monocyclic or bicyclic saturated heterocyclic group, R^x1Is hydrogen or C₁-C₄Alkyl and R^2*As previously defined.

In some embodiments, the compound has the formula Ig:

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 R²Is composed ofWherein P is a 4-to 8-membered monocyclic or bicyclic saturated heterocyclic group and R^2*、R^x1And t is as previously defined.

In some embodiments, the compound has formula Ig and R²Is composed ofWherein P is a 4-to 8-membered monocyclic or bicyclic saturated heterocyclic group, R^x1Is hydrogen or C₁-C₄Alkyl and R^2*As previously defined.

In some embodiments, the compound has formula Ih:

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 R²Is composed ofWherein P is a 4-to 8-membered monocyclic or bicyclic saturated heterocyclic group and R^2*、R^x1And t is as previously defined.

In some embodiments, the compound has formula Ih and R²Is composed ofWherein P is a 4-to 8-membered monocyclic or bicyclic saturated heterocyclic group, R^x1Is hydrogen or C₁-C₄Alkyl and R^2*As previously defined.

In some embodiments, the compound has formula Ii:

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 R²Is composed ofWherein P is a 4-to 8-membered monocyclic or bicyclic saturated heterocyclic group and R^2*、R^x1And t is as previously defined.

In some embodiments, the compound has formula Ii and R²Is composed ofWherein P is a 4-to 8-membered monocyclic or bicyclic saturated heterocyclic group, R^x1Is hydrogen or C₁-C₄Alkyl and R^2*As previously defined.

In some embodiments, the compound has formula Ij:

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 R²Is composed ofWherein P is a 4 to 8 membered monocyclic or bicyclic saturated heterocyclic group.

In some embodiments, the compound has formula Ij and R²Is composed ofWherein P is a 4-to 8-membered monocyclic or bicyclic saturated heterocyclic group, R^x1Is hydrogen or C₁-C₄An alkyl group.

In some embodiments, the compound has formula Ij and R is H, and both X are N.

In some embodiments, the compound has the following structure:

In some embodiments, the compound has formula Ik and R²Is composed ofWherein P is a 4 to 8 membered monocyclic or bicyclic saturated heterocyclic group.

In some embodiments, the compound has formula Ik and R²Is composed ofWherein P is a 4-to 8-membered monocyclic or bicyclic saturated heterocyclic group, R^x1Is hydrogen or C₁-C₄An alkyl group.

In some embodiments, the compound has formula ii:

in some embodiments, the compound has formula Il and R²Is composed ofWherein P is a 4 to 8 membered monocyclic or bicyclic saturated heterocyclic group.

In some embodiments, the compound has formula Il and R²Is composed ofWherein P is a 4-to 8-membered monocyclic or bicyclic saturated heterocyclic group, R^x1Is hydrogen or C₁-C₄An alkyl group.

In some embodiments, the compound has formula Im:

in some embodiments, the compound hasHaving the formula Im and R²Is composed ofWherein P is a 4 to 8 membered monocyclic or bicyclic saturated heterocyclic group.

In some embodiments, the compound has formula Im and R²Is composed ofWherein P is a 4-to 8-membered monocyclic or bicyclic saturated heterocyclic group, R^x1Is hydrogen or C₁-C₄An alkyl group.

In some embodiments, the compound has formula IIa:

in some embodiments, the compound has formula IIa and R²Is composed ofWherein P is a 4 to 8 membered monocyclic or bicyclic saturated heterocyclic group.

In some embodiments, the compound has formula IIa and R²Is composed ofWherein P is a 4-to 8-membered monocyclic or bicyclic saturated heterocyclic group, R^x1Is hydrogen or C₁-C₄An alkyl group.

In some embodiments, the compound has formula IIb:

in some embodiments, the compound has formula Im and R²Is composed ofWherein P is a 4-to 8-membered unitA cyclic or bicyclic saturated heterocyclic group.

In some embodiments, the compound has formula Im and R²Is composed ofWherein P is a 4-to 8-membered monocyclic or bicyclic saturated heterocyclic group, R^x1Is hydrogen or C₁-C₄An alkyl group.

In some aspects, the active compound is:

in certain embodiments, the compound is selected from:

wherein R is C (H) X, NX, C (H) Y or C (X)₂，

Wherein X is a linear, branched or cyclic C₁To C₅Alkyl groups including methyl, ethyl, propyl, cyclopropyl, isopropyl, butyl, sec-butyl, tert-butyl, isobutyl, cyclobutyl, pentyl, isopentyl, neopentyl, tert-pentyl, sec-pentyl and cyclopentyl; and is

Y is NR₁R₂Wherein R is₁And R₂Independently X, or wherein R₁And R₂Alkyl groups which together form a bridge comprising one or two heteroatoms (N, O or S);

and wherein two X groups may together form an alkyl bridge or bridge comprising one or two heteroatoms (N, S or O) to form a spiro compound.

The IUPAC name of formula T is 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; formula Q is 2'- ((5- (piperazin-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; formula GG is 2'- ((5- (4-isopropylpiperazin-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; and formula U is 2'- ((5- (4-morpholinopiperidin-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.

Other specific compounds within the present invention and that may be used in the disclosed therapeutic methods and compositions include the structures listed in table 1 below.

Table 1: structure of CDK4/6 inhibitor

Isotopic substitution

The present invention includes the use of compounds and isotopically substituted compounds having a desired amount of atoms in excess of the natural abundance (i.e., enrichment) of the isotope. Isotopes are atoms having the same atomic number but different mass numbers, i.e. the same number of protons but different numbers of neutrons. By way of general example and not limitation, isotopes of hydrogen may be used anywhere in the structure, such as deuterium (g), (b), (c), (d) and (d) in a) an ²H) And tritium (f)³H) In that respect Alternatively or additionally, isotopes of carbon may be used, for example¹³C and¹⁴C. one preferred isotopic substitution is the substitution of deuterium for hydrogen at one or more positions on the molecule to improve the performance of the drug. Deuterium can be bound in the position of bond cleavage during metabolism (alpha-deuterium kinetic isotope effect) or close or near to the bond cleavage site (beta-deuterium kinetic isotope effect).

Substitution with heavy isotopes such as deuterium can afford certain therapeutic advantages resulting from greater metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements. Deuterium substitution of hydrogen at the site of metabolic breakdown may reduce the rate or elimination of this bond metabolism. In any position of the compound where a hydrogen atom may be present, the hydrogen atom may be any isotope of hydrogen, including protium (l¹H) Deuterium (1)²H) And tritium (f)³H) In that respect Thus, unless the context clearly dictates otherwise, reference herein to a compound encompasses all potential isotopic forms.

The term "isotopically labeled" analog is meant to be "deuterated analog",¹³c-marker analog "or" deuteration¹³C-labeled analog ". The term "deuterated analog" means a compound described herein wherein the H-isotope, i.e., hydrogen/protium ((I)) ¹H) By the H isotope (i.e. deuterium: (²H) ) is substituted. Deuterium substitution may be partial or complete. Partial deuterium substitution means that at least one hydrogen is substituted with at least one deuterium. In certain embodiments, the isotope is enriched in isotopes at any relevant position by 90%, 95%, or 99% or more. In some embodiments, it is deuterium enriched at a predetermined position by 90%, 95% or 99%.

CDK replication-dependent cell and cyclin dependent kinase inhibitors

Tissue-specific stem cells and other subsets of intrinsically proliferating cells are capable of self-renewal, meaning that they are capable of replacement themselves by regulated replication in the life of an adult mammal. In addition, stem cells divide asymmetrically, producing "daughter" or "progenitor" cells, which in turn produce various components of a given organ. For example, in the hematopoietic system, hematopoietic stem cells give rise to progenitor cells, which in turn give rise to all the differentiated components of the blood (e.g., white blood cells, red blood cells, and platelets). See fig. 1.

Certain proliferating cells, such as HSPCs, require the enzymatic activity of the proliferative kinases cyclin-dependent kinase 4(CDK4) and/or cyclin-dependent kinase 6(CDK6) for cell replication. In contrast, most proliferating cells in adult mammals (e.g., the more differentiated blood-forming cells in bone marrow) do not require the activity of CDK4 and/or CDK6 (i.e., CDK 4/6). These differentiated cells can be propagated in the absence of CDK4/6 activity by using other proliferative kinases such as cyclin dependent kinase 2(CDK2) or cyclin dependent kinase 1(CDK 1).

The CDK4/6 inhibitor administered is selected from a compound or composition comprising formula I, formula II, formula III, formula IV or formula V, or a combination thereof. In one embodiment, the compound is selected from the compounds described in table 1.

In certain embodiments, the CDK4/6 inhibitor is a CDK4/6 inhibitor of formula I, II, III, IV or V or a pharmaceutically acceptable composition, salt, isotopic analog or prodrug thereof, wherein the protection afforded by the compound is transient and transient in nature, allowing a significant portion of the cells to rapidly synchronize re-entry into the cell cycle after cessation of the action of the chemotherapeutic agent, e.g., within less than about 24, 30, 36, or 40 hours. In one embodiment, the compound is selected from the compounds described in table 1, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof. In one embodiment, the compound is selected from the group consisting of compound T, Q, GG, U, and AAAA, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof. Cells that are quiescent within the cell cycle G1 are more resistant to the damaging effects of chemotherapeutic agents than are proliferating cells. The CDK4/6 inhibitory compounds used in the described methods are highly selective potent CDK4/6 inhibitors, with minimal CDK2 inhibitory activity. In one embodiment, the CDK4/CycD1IC of the CDK4/6 compound for use in the methods described herein ₅₀Inhibition concentration value is compared with the IC corresponding to CDK2/CycE inhibition₅₀Low concentration value>1500 times of,>1800 times of,>2000 times of,>2200 times of,>2500 times of,>2700 times of,>3000 times of,>3200 times or more. In one embodiment, the IC of CDK4/6 inhibitor on CDK4/CycD1 inhibition for methods described herein₅₀Concentration value of about<1.50nM、<1.25nM、<1.0 nM、<0.90nM、<0.85nM、<0.80nM、<0.75nM、<0.70nM、<0.65nM、 <0.60nM、<0.55nM or less. In one embodiment, the IC of CDK4/6 inhibitor on CDK2/CycE inhibition for methods described herein₅₀Concentration value of about>1.0 μM、>1.25μM、>1.50μM、>1.75μM、>2.0μM、>2.25μM、>2.50 μM、>2.75μM、>3.0μM、>3.25μM、>3.5. mu.M or more. In one embodiment, the CDK4/6 inhibitor used in the methods described herein is directed to CDK2/CycA IC₅₀IC of₅₀Concentration value>0.80μM、>0.85μM、>0.90μM、>0.95μM、>.1.0 μM、>1.25μM、>1.50μM、>1.75μM、>2.0μM、>2.25μM、>2.50 μM、>2.75uM、>3.0. mu.M or more. In one embodiment, the CDK4/6 inhibitor for use in the methods described herein is selected from the group consisting of formula I, formula II, formula III, formula IV, or formula V, or a pharmaceutically acceptable composition, salt, or prodrug thereof. In one embodiment, the compound is selected from the compounds described in table 1, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof. In one embodiment, the compound is selected from the group consisting of compound T, Q, GG, U, and AAAA, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof.

In one embodiment, the CDK4/6 inhibitor described herein is used in a CDK4/6 replication-dependent healthy cell cycle in which a subject is exposed to conventional repeated chemotherapeutic treatment, wherein healthy cells arrest at G1 when exposed to the chemotherapeutic and re-enter the cell cycle before the subject is treated with the next chemotherapeutic. Such cycling allows CDK4/6 to replicate dependent cells to regenerate disrupted blood cell lineages between conventional repeat treatments, such as those associated with standard chemotherapeutic treatments for cancer, and reduces the risk associated with long-term CDK4/6 inhibition. This cycle between the G1 arrested state and the replicated state is not possible with time-interval-limited repeated chemotherapeutic exposures using longer-acting CDK4/6 inhibitors (e.g., PD0332991) because the delayed G1 arrest effect of the compound prohibits significant and meaningful re-entry into the cell cycle before the next chemotherapeutic exposure or delays healthy cells from entering the cell cycle and recovering the damaged tissue or cells after treatment is stopped.

Proliferative disorders treated with chemotherapy include cancerous and non-cancerous diseases. In a typical embodiment, the proliferative disorder is a CDK4/6 replication independent disorder. The compounds are effective in protecting healthy CDK4/6 replication-dependent cells, such as HSPCs, during chemotherapeutic treatment of a wide range of tumor types, including (but not limited to): breast, prostate, ovary, skin, lung, colorectal, brain (i.e., glioma) and kidney. Preferably, selective CDK4/6 inhibitors should not impair the efficacy of chemotherapeutic agents or arrest G1, arresting cancer cells. Many cancers do not rely on the activity of CDK4/6 to proliferate as it may confoundly use proliferative kinases (e.g., CDK 1/2/4/or 6 may be used) or lack the function of retinoblastoma tumor suppressor protein (Rb) inactivated by CDK. The potential sensitivity of certain tumors to CDK4/6 inhibition can be inferred based on tumor type and molecular genetics using standard techniques. Cancers that are typically not affected by CDK4/6 inhibition are cancers that may be characterized by one or more of the group that includes (but is not limited to): increased CDK1 or CDK2 activity, loss, deficiency or absence of retinoblastoma tumor suppressor protein (Rb), high MYC expression levels, increased cyclin E (e.g., E1 or E2) and increased cyclin a or expression of Rb inactivating protein (e.g., HPV-encoded E7). Such cancers may include, but are not limited to, small cell lung cancer, retinoblastoma, HPV positive malignancies (such as cervical cancer and certain head and neck cancers), MYC-amplified tumors (such as Burkitts' Lymphoma), and triple negative breast cancer; certain classes of sarcoma, certain classes of non-small cell lung cancer, certain classes of melanoma, certain classes of pancreatic cancer, certain classes of leukemia, certain classes of lymphoma, certain classes of brain cancer, certain classes of colon cancer, certain classes of prostate cancer, certain classes of ovarian cancer, certain classes of uterine cancer, certain classes of thyroid cancer and other cancers of endocrine tissues, certain classes of salivary cancer, certain classes of thymic carcinoma, certain classes of renal cancer, certain classes of bladder cancer, and certain classes of testicular cancer.

Loss or absence of retinoblastoma (Rb) tumor suppressor protein (Rb empty) can be determined by any standard assay known to those skilled in the art, including, but not limited to, western blot, ELISA (enzyme linked immunosorbent assay), IHC (immunohistochemistry), and FACS (fluorescence activated cell sorting). The choice of analysis will depend on the tissue, cell line or surrogate tissue sample utilized, e.g., western blotting and ELISA can be used for any or all types of tissue, cell line or surrogate tissue, whereas IHC methods will be more applicable where the tissue utilized in the methods of the invention is a tumor biopsy. FACs analysis will be mostly applicable to samples in single cell suspensions, such as cell lines and isolated peripheral blood mononuclear cells. See, for example, US 20070212736, "Functional immunological Cell Cycle Analysis as a protective Indicator for Cancer".

Alternatively, molecular genetic tests can be used to determine retinoblastoma gene status. Molecular genetic tests for retinoblastoma include the tests as described in: lohmann and Gallie "Reinforcement. Gene Reviews" (2010) http:// www.ncbi.nlm.nih.gov/book shelf/br. fcgibook ═ gene & part ═ regeneration: "A comprehensive, sensitive and environmental adaptive for the detection of events in the RB1gene in regeneration" Journal of Genetics,88 (2009), 517-.

Increased activity of CDK1 or CDK2, high MYC expression levels, increased cyclin E and increased cyclin a may be determined by any standard assay known to those skilled in the art, including, but not limited to, western blotting, ELISA (enzyme-linked immunosorbent assay), IHC (immunohistochemistry), and FACS (fluorescence activated cell sorting). The choice of assay will depend on the tissue, cell line or surrogate tissue sample utilized, e.g., western blotting and ELISA can be used for any or all types of tissue, cell line or surrogate tissue, whereas IHC methods would be more applicable where the tissue utilized in the methods of the invention is a tumor biopsy. FACs analysis will be mostly applicable to samples in single cell suspensions, such as cell lines and isolated peripheral blood mononuclear cells.

In some embodiments, the cancer is selected from small cell lung cancer, retinoblastoma, and triple negative (ER/PR/Her2 negative) or "basal-like" breast cancer, which nearly always inactivates retinoblastoma tumor suppressor protein (Rb), and thus does not require CDK4/6 activity to proliferate. Triple negative (baseline-like) breast cancer has also almost always been genetically or functionally Rb empty. In addition, certain virus-induced cancers (e.g., cervical cancer and a subset of head and neck cancers) express viral proteins that inactivate Rb (E7), making these tumors functionally Rb-blank. Some lung cancers are also thought to be caused by HPV. In a specific embodiment, the cancer is small cell lung cancer and the patient is treated with a DNA damaging agent selected from etoposide, carboplatin, and cisplatin, or a combination thereof.

The selected CDK4/6 inhibitors described herein may also be used to protect healthy CDK4/6 replication-dependent cells during chemotherapeutic treatment of abnormal tissue in non-cancer proliferative disorders including (but not limited to): psoriasis, lupus, arthritis (particularly rheumatoid arthritis), angiomatosis in infants, multiple sclerosis, myelodegenerative diseases, neurofibromatosis, gangliomases, keloid formation, Paget's Disease of the skeleton, fibrocystic diseases of the breast, Peyronie's and dupurten's fibrosis, restenosis and cirrhosis of the liver. In addition, selective CDK4/6 inhibitors may be used to improve the effect of chemotherapeutic agents if accidental irradiation or overdose (e.g., overdose of methotrexate) occurs.

According to the invention, the active compound can be administered to the subject over any chemotherapeutic treatment period and at any dose that meets the prescribed treatment period. The selective CDK4/6 inhibitor compound is administered before, during or after administration of the chemotherapeutic agent. In one embodiment, the CDK4/6 inhibitor described herein may be administered to a subject during a time period ranging from 24 hours prior to chemotherapeutic treatment up to 24 hours post-exposure. However, this period of time may be extended to a period of time that is earlier than 24 hours prior to exposure to the agent (e.g., based on the time it takes for the chemotherapeutic agent to achieve a suitable plasma concentration and/or the plasma half-life of the compound). Furthermore, the time period may be extended to be longer than 24 hours after exposure to the chemotherapeutic agent, as long as the subsequent administration of the CDK4/6 inhibitor produces at least some protection. Such post-exposure treatments may be particularly useful in the event of accidental radiation or overdose.

In some embodiments, the selective CDK4/6 inhibitor may be administered to a subject at a time period prior to administration of the chemotherapeutic agent such that the plasma levels of the selective CDK4/6 inhibitor peak at the time of administration of the chemotherapeutic agent. Where appropriate, selective CDK4/6 inhibitors may be administered concurrently with chemotherapeutic agents to simplify the treatment regimen. In some embodiments, the chemoprotectant and chemotherapeutic agent may be provided in a single formulation.

In some embodiments, the selective CDK4/6 inhibitor may be administered to a subject such that the chemotherapeutic agent may be administered at higher doses (increasing chemotherapeutic agent dose intensity) or more frequently (increasing chemotherapeutic agent dose density). Dose intensive chemotherapy is a chemotherapy treatment plan in which the drug is administered less time between treatments than is possible with standard chemotherapy treatment plans. The chemotherapy dose intensity means a unit dose amount of chemotherapy administered per unit time. The dose intensity can be increased or decreased by varying the dose administered, the time interval between administrations, or both. Myelosuppression continues to represent the major dose-limiting toxicity of cancer chemotherapy, causing considerable morbidity and mortality, as well as frequently reducing the chemotherapeutic dose intensity, which can compromise disease control and survival. The compounds and uses thereof as described herein represent a means to increase the dose density and/or dose intensity of chemotherapy while mitigating adverse events such as, but not limited to, myelosuppression.

If desired, multiple doses of the selected CDK4/6 inhibitor compound may be administered to the subject. Alternatively, the subject may be administered a single dose of the selected CDK4/6 inhibitor. For example, a CDK4/6 inhibitor may be administered such that CDK4/6 replication-dependent healthy cells arrest at G1 during chemotherapeutic exposure, wherein many healthy cells reenter the cell cycle and are able to replicate shortly after chemotherapeutic exposure, e.g., within about 24-48 hours or less, due to the rapid dissipation of the compound's G1 arresting effect, and continue to replicate until the CDK4/6 inhibitor is administered prior to the next chemotherapeutic treatment. In one embodiment, the CDK4/6 inhibitor is administered to allow CDK4/6 replication-dependent healthy cells to cycle between G1 arrest and re-entry into the cell cycle, thereby accommodating repeat dosing chemotherapeutic treatment regimens, including for example (but not limited to) treatment regimens in which a chemotherapeutic is administered as follows: every 21 days, on days 1-3; every 28 days, on days 1-3; every 3 weeks on day 1; every 28 days, on days 1, 8 and 15; every 28 days, on days 1 and 8; every 21 days, on days 1 and 8; every 21 days, from day 1 to day 5; 1 day a week, 6-8 weeks in the course of the week; on days 1, 22 and 43; weekly, day 1 and day 2; days 1-4 and 22-25; day 1-4; day 22-day 25; and days 43-46; and similar types of protocols in which CDK4/6 replication-dependent cells arrest at G1 during chemotherapeutic exposure and a significant portion of the cells re-enter the cell cycle between chemotherapeutic exposures.

In one embodiment, the CDK4/6 inhibitor described herein is used to provide chemoprotection to CDK4/6 replication-dependent healthy cells of a subject during a CDK4/6 replication-independent small cell lung cancer treatment regimen. In one embodiment, the CDK4/6 inhibitor is administered to provide chemoprotection in a small cell lung cancer treatment regimen such as (but not limited to) the following: cisplatin 60mg/m2IV on day 1 and etoposide 120mg/m2IV on days 1-3 every 21 days, and circulating for 4 times; cisplatin 80mg/m2IV on day 1 and etoposide 100mg/m2IV on days 1-3 every 28 days, circulating for 4 times; cisplatin 60-80mg/m2IV on day 1 and etoposide 80-120mg/m2IV on day 1-3 every 21-28 days (4 cycles maximum); carboplatin AUC 5-6IV on day 1 plus etoposide 80-100mg/m2IV on days 1-3 every 28 days (4 cycles maximum);

cisplatin 60-80mg/m2IV on day 1 and etoposide 80-120mg/m2IV on day 1-3 every 21-28 days; carboplatin AUC 5-6IV on day 1 plus etoposide 80-100mg/m2IV on days 1-3 every 28 days (6 cycles maximum); cisplatin 60mg/m2IV on day 1 plus irinotecan 60mg/m2IV on days 1, 8 and 15 every 28 days (6 cycles maximum); cisplatin 30mg/m2IV on days 1 and 8 or 80mg/m2IV on day 1 plus irinotecan 65mg/m2IV on days 1 and 8 every 21 days (6 cycles maximum); carboplatin AUC 5IV on day 1 plus irinotecan 50mg/m2IV on days 1, 8 and 15 every 28 days (6 cycles maximum); carboplatin AUC 4-5IV on day 1 plus irinotecan 150-200mg/m2IV on day 1 every 21 days (6 cycles maximum); cyclophosphamide 800-1000mg/m2IV on day 1 plus doxorubicin 40-50mg/m2IV on day 1 plus vincristine 1-1.4mg/m2IV on day 1 every 21-28 days (6 cycles maximum); etoposide 50mg/m2PO daily every 4 weeks for 3 weeks; topotecan 2.3mg/m2PO on days 1-5 every 21 days; topotecan 1.5mg/m2IV on days 1-5 every 21 days; carboplatin AUC 5IV on day 1 plus irinotecan 50mg/m2IV on days 1, 8, and 15 every 28 days; carboplatin AUC 4-5IV on day 1 plus irinotecan 150-200mg/m2IV on day 1 every 21 days; cisplatin 30mg/m2IV on days 1, 8 and 15 plus irinotecan 60mg/m2IV on days 1, 8 and 15 every 28 days; cisplatin 60mg/m2IV on day 1 plus irinotecan 60mg/m2IV on days 1, 8 and 15 every 28 days; cisplatin 30mg/m2IV on days 1 and 8 or 80mg/m2IV on day 1 plus irinotecan 65mg/m2IV on days 1 and 8 every 21 days; paclitaxel 80mg/m2IV weekly every 8 weeks for 6 weeks; paclitaxel 175mg/m2IV on day 1 every 3 weeks; etoposide 50mg/m2PO daily every 4 weeks for 3 weeks; topotecan 2.3mg/m2PO on days 1-5 every 21 days; topotecan 1.5mg/m2IV on days 1-5 every 21 days; carboplatin AUC 5IV every 28 days at day 1 plus irinotecan 50mg/m2IV at days 1, 8, and 15; carboplatin AUC 4-5IV on day 1 plus irinotecan 150-200mg/m2IV on day 1 every 21 days; cisplatin 30mg/m2IV on days 1, 8 and 15 plus irinotecan 60mg/m2IV on days 1, 8 and 15 every 28 days; cisplatin 60mg/m2IV on day 1 plus irinotecan 60mg/m2IV on days 1, 8 and 15 every 28 days; cisplatin 30mg/m2IV on days 1 and 8 or 80mg/m2IV on day 1 plus irinotecan 65mg/m2IV on days 1 and 8 every 21 days; paclitaxel 80mg/m2IV weekly every 8 weeks for 6 weeks; and paclitaxel 175mg/m2IV on day 1 every 3 weeks.

In one embodiment, the CDK4/6 inhibitor described herein is administered to a subject having small cell lung cancer on days 1, 2, and 3 of a treatment regimen wherein the DNA damaging agent is selected from the group consisting of: carboplatin, etoposide, and cisplatin, or a combination thereof, were administered every 21 days on days 1, 2, and 3.

In one embodiment, the CDK4/6 inhibitor described herein is used to provide chemoprotection to CDK4/6 replication-dependent healthy cells of a subject during a CDK4/6 replication-independent head and neck cancer treatment regimen. In one embodiment, the CDK4/6 inhibitor is administered to provide chemoprotection in a CDK4/6 replication-independent head and neck cancer treatment regimen such as (but not limited to) the following: cisplatin 100mg/m2IV or 40-50mg/m2IV per week for 6-7 weeks on days 1, 22 and 43; cetuximab 400mg/m2IV loading dose 1 week before starting radiotherapy, followed by 250mg/m2 weekly (pre-surgery for dexamethasone, diphenhydramine and ranitidine); cisplatin 20mg/m2IV for 7 weeks on day 2 per week plus paclitaxel 30mg/m2IV for 7 weeks on day 1 per week; cis-platinum 20mg/m 2/day IV on days 1-4 and 22-25 plus 5-FU 1000mg/m 2/day on days 1-4 and 22-25 by continuous intravenous infusion; given on the day of radiation, 5-FU 800 mg/m2 plus hydroxyurea 1g PO q12h (11 doses per cycle) by continuous intravenous infusion on days 1-5; chemotherapy and radiation were given every other week for a total of 13 weeks; carboplatin 70mg/m 2/day IV on days 1-4, 22-25, and 43-46 plus 5-FU 600mg/m 2/day by continuous intravenous infusion on days 1-4, 22-25, and 43-46; carboplatin AUC 1.5IV weekly on day 1 plus paclitaxel 45mg/m2IV weekly on day 1; cisplatin 100mg/m2IV or 40-50mg/m2IV per week for 6-7 weeks on days 1, 22 and 43; during radiotherapy docetaxel 75mg/m2IV on day 1 plus cisplatin 100mg/m2IV on day 1 every 3 weeks for 3 cycles of 5-FU 100mg/m 2/day by continuous intravenous infusion over 1-4 days, followed by 3-8 weeks followed by carboplatin AUC 1.5IV weekly for 7 weeks; docetaxel 75mg/m2IV on day 1 plus cisplatin 75mg/m2IV on day 1 plus 5-FU 750mg/m 2/day by continuous intravenous infusion for 4 cycles on day 1-4 every 3 weeks; cisplatin 100mg/m2IV on day 1 every 3 weeks, 6 cycles, plus 5-FU 1000mg/m 2/day on day 1-4 by continuous intravenous infusion every 3 weeks, 6 cycles, a cetuximab 400mg/m2IV loading dose on day 1, then 250mg/m2IV weekly until disease progression (dexamethasone, diphenhydramine, and ranitidine pre-operative medication); carboplatin AUC 5IV every 3 weeks on day 1, 6 cycles, plus 6 cycles by continuous intravenous infusion on days 1-4 of 5-FU 1000mg/m 2/day every 3 weeks, plus a loading dose of cetuximab 400mg/m2IV on day 1, followed by 250mg/m2IV weekly until disease progression (dexamethasone, diphenhydramine, and ranitidine pre-operative dosing); every 3 weeks cisplatin 75mg/m2IV on day 1 plus docetaxel 75mg/m2IV on day 1; cisplatin 75mg/m2IV on day 1 plus paclitaxel 175mg/m2IV on day 1 every 3 weeks; carboplatin AUC 6IV on day 1 plus docetaxel 65mg/m2IV on day 1 every 3 weeks; carboplatin AUC 6IV on day 1 plus paclitaxel 200mg/m2IV on day 1 every 3 weeks; cisplatin 75-100mg/m2IV at day 1 plus cetuximab 400mg/m2IV loading dose at day 1 every 3-4 weeks, followed by 250mg/m2IV weekly (dexamethasone, diphenhydramine, and ranitidine pre-operative); cisplatin 100mg/m2IV on day 1 every 3 weeks plus 5-FU 1000mg/m 2/day by continuous intravenous infusion on days 1-4; methotrexate 40mg/m2IV weekly (3 weeks equals 1 cycle); paclitaxel 200mg/m2IV every 3 weeks; docetaxel 75mg/m2IV every 3 weeks; cetuximab 400mg/m2IV loading dose on day 1, followed by 250mg/m2IV weekly until disease progression (dexamethasone, diphenhydramine, and ranitidine pre-treatment); cisplatin 100mg/m2IV on day 1 every 3 weeks for 6 cycles plus 5-FU 1000mg/m 2/day by continuous intravenous infusion on day 1-4 every 3 weeks for 6 cycles plus cetuximab 400mg/m2IV loading dose on day 1, followed by 250mg/m2IV per week (pre-operative administration of dessertmethasone, diphenhydramine and ranitidine); carboplatin AUC 5IV every 3 weeks on day 1, 6 cycles, plus 6 cycles by continuous intravenous infusion every 3 weeks on days 1-4 of 5-FU 1000mg/m 2/day, 6 cycles, plus a 400mg/m2IV loading dose of cetuximab on day 1, followed by 250mg/m2IV weekly (pre-operative administration of dexamethasone, diphenhydramine, and ranitidine); every 3 weeks cisplatin 75mg/m2IV on day 1 plus docetaxel 75mg/m2IV on day 1; cisplatin 75mg/m2IV on day 1 plus paclitaxel 175mg/m2IV on day 1 every 3 weeks; carboplatin AUC 6IV on day 1 plus docetaxel 65mg/m2IV on day 1 every 3 weeks; carboplatin AUC 6IV on day 1 plus paclitaxel 200mg/m2IV on day 1 every 3 weeks; cisplatin 75-100mg/m2IV at day 1 plus cetuximab 400mg/m2IV loading dose at day 1 every 3-4 weeks, followed by 250mg/m2IV per week (dexamethasone, diphenhydramine, and ranitidine pre-operative); cisplatin 100mg/m2IV on day 1 every 3 weeks plus 5-FU 1000mg/m 2/day by continuous intravenous infusion on days 1-4; methotrexate 40mg/m2IV weekly (3 weeks equals 1 cycle); paclitaxel 200mg/m2IV every 3 weeks; docetaxel 75mg/m2IV every 3 weeks; cetuximab 400mg/m2IV loading dose on day 1, followed by 250mg/m2IV weekly until disease progression (dexamethasone, diphenhydramine, and ranitidine pre-surgery); cisplatin 100mg/m2IV at days 1, 22 and 43 under radiation, followed by cisplatin 80mg/m2IV every 4 weeks on day 1 plus 5-FU 1000mg/m2 by continuous intravenous infusion on days 1-4, 3 cycles; every 3 weeks cisplatin 75mg/m2IV on day 1 plus docetaxel 75mg/m2IV on day 1; cisplatin 75mg/m2IV on day 1 plus paclitaxel 175mg/m2IV on day 1 every 3 weeks; carboplatin AUC 6IV on day 1 plus docetaxel 65mg/m2IV on day 1 every 3 weeks; carboplatin AUC 6IV on day 1 plus paclitaxel 200mg/m2IV on day 1 every 3 weeks; cisplatin 100mg/m2IV on day 1 every 3 weeks plus 5-FU 1000mg/m 2/day by continuous intravenous infusion on days 1-4; every 4 weeks cisplatin 50-70mg/m2IV added gemcitabine 1000mg/m2IV on day 1, and day 15; gemcitabine 1000mg/m2IV on days 1, 8 and 15 every 4 weeks or gemcitabine 1250mg/m2IV on days 1 and 8 every 3 weeks; methotrexate 40mg/m2IV weekly (3 weeks equals 1 cycle); paclitaxel 200mg/m2IV every 3 weeks; docetaxel 75mg/m2IV every 3 weeks; every 3 weeks cisplatin 75mg/m2IV on day 1 plus docetaxel 75mg/m2IV on day 1; cisplatin 75mg/m2IV on day 1 plus paclitaxel 175mg/m2IV on day 1 every 3 weeks; carboplatin AUC 6IV on day 1 plus docetaxel 65mg/m2IV on day 1 every 3 weeks; carboplatin AUC 6IV on day 1 plus paclitaxel 200mg/m2IV on day 1 every 3 weeks; cisplatin 100mg/m2IV on day 1 every 3 weeks plus 5-FU 1000mg/m 2/day by continuous intravenous infusion on days 1-4; every 4 weeks cisplatin 50-70mg/m2IV added gemcitabine 1000mg/m2IV on day 1, and day 15; gemcitabine, 1000mg/m2IV on days 1, 8 and 15 every 4 weeks or 1250mg/m2IV on days 1 and 8 every 3 weeks; methotrexate 40mg/m2IV weekly (3 weeks equals 1 cycle); paclitaxel 200mg/m2IV every 3 weeks; and docetaxel 75mg/m2IV every 3 weeks.

In one embodiment, the CDK4/6 inhibitor described herein is used to provide chemoprotection to CDK4/6 replication-dependent healthy cells of a subject during a CDK4/6 replication-independent triple negative breast cancer treatment regimen. In one embodiment, the CDK4/6 inhibitor is administered to provide chemoprotection in a CDK4/6 replication-independent triple negative breast cancer treatment regimen such as (but not limited to) the following: four cycles of two-week dosing of doxorubicin (adriamycin) and cyclophosphamide (cyclophosphamide), followed by four cycles of two-week dosing of paclitaxel (taxol); doxorubicin/paclitaxel/cyclophosphamide every three weeks for a total of four cycles; doxorubicin/paclitaxel/cyclophosphamide every two weeks for a total of four cycles; doxorubicin/cyclophosphamide every three weeks followed by paclitaxel (taxol), four cycles each; and doxorubicin/cyclophosphamide followed by paclitaxel (taxol) every two weeks, four cycles each.

Triple Negative Breast Cancer (TNBC) is defined as a lack of estrogen receptor, progestin receptor and HER2/neu staining. TNBC is less sensitive to some of the most effective therapies available for breast cancer treatment, including HER 2-directed therapies, such as trastuzumab, and endocrine therapies, such as tamoxifen or aromatase inhibitors. Combination cytotoxic chemotherapy administered in a dose-intensive or circadian time course remains the standard therapy for early stage TNBC. Platinum agents have recently emerged as relevant drugs for the treatment of TNBC, with carboplatin added to paclitaxel and doxorubicin plus cyclophosphamide chemotherapy in a neoadjuvant setting. Inhibitors of poly (ADP-ribose) polymerase (PARP) have emerged as promising therapeutic agents for TNBC. PARP is a family of enzymes involved in many cellular processes, including DNA repair.

By way of non-limiting illustration, the subject is exposed to the chemotherapeutic agent 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 CDK4/6 replication-dependent healthy cells stagnate at G1 during the treatment and are allowed to circulate in between chemotherapeutic agent exposures, e.g., during an interruption of the treatment. In one embodiment, the subject is treated with the chemotherapeutic agent 5 times a week, wherein the subject's CDK4/6 replication-dependent healthy cells arrest at G1 during chemotherapeutic agent exposure and are allowed to re-enter the cell cycle during a 2 day interruption, e.g., over the weekend.

In one embodiment, the CDK4/6 inhibitor described herein is used to arrest CDK4/6 replication-dependent healthy cells of a subject during the entire period of exposure to a chemotherapeutic agent, e.g., during a continuous multi-day regimen, the cells are arrested for more than the period of time required to complete the continuous multi-day course, then allowed to recycle at the end of the continuous multi-day course. In one embodiment, using the CDK4/6 inhibitor described herein, a subject's CDK4/6 replication dependent healthy cells are arrested throughout a chemotherapeutic regimen, e.g., during daily chemotherapeutic exposure for three weeks, and rapidly re-enter the cell cycle after completion of the therapeutic regimen.

In one embodiment, the subject has been exposed to a chemotherapeutic agent and the CDK4/6 inhibitor described herein is used to subject CDK4/6 replication-dependent healthy cells to G1 arrest following exposure to reduce, for example, DNA damage. In one embodiment, the CDK4/6 inhibitor is administered at least 1/2 hours, 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, or more following chemotherapeutic agent exposure.

In some embodiments, the present invention provides methods of protecting a mammal (particularly a human) from the acute and chronic toxic effects of chemotherapeutic agents by transiently (e.g., in less than about 40, 36, 30, 24 hours or less) treating with an inhibitor of CDK4/6 selected from formula I, formula II, formula III, formula IV, or formula V, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof, forcing CDK4/6 to replicate-dependent healthy cells, such as Hematopoietic Stem and Progenitor Cells (HSPCs) and/or renal epithelial cells, into a quiescent state. In one embodiment, the compound is selected from the compounds described in table 1, or a pharmaceutically acceptable composition, salt, isotope analog, or prodrug thereof. In one embodiment, the compound is selected from the group consisting of compound T, Q, GG, U, and AAAA, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof. CDK4/6 replication-dependent cells typically recover from this transient quiescent period and then function after inhibitor treatment ceases and their intracellular effects dissipate. During quiescence, CDK4/6 replication-dependent cells were protected from the effects of chemotherapeutic agents.

In some embodiments, CDK4/6 replication-dependent healthy cells may be arrested for a longer period, e.g., over hours, days, and/or weeks, by multiple separate administrations of the CDK4/6 inhibitor described herein. Because CDK4/6 replication-dependent healthy cells, such as HSPC, rapidly and synchronously re-enter the cell cycle after intracellular action of CDK4/6 inhibitors dissipates, cells are able to restore cell lineages more rapidly than CDK4/6 inhibitors, such as PD0332991, which have a longer G1 arrest pattern.

The reduction in chemotoxicity provided by selective CDK4/6 inhibitors may allow for dose escalation in medically relevant chemotherapy (e.g., more therapy may be administered within a fixed time period), which would translate into better efficacy. Thus, the methods disclosed herein may render chemotherapeutic regimens less toxic and more effective. Furthermore, the selective CDK4/6 inhibitors described herein are small orally administrable molecules that can be formulated for administration via a number of different routes, as compared to protective treatment with exogenous biological growth factors. Where appropriate, the small molecules may be formulated for oral, topical, intranasal, inhalation, intravenous, or any other desired form of administration.

A CDK4/6 inhibitor suitable for use in the methods described herein is a selective CDK4/6 inhibitor compound that selectively inhibits at least one of CDK4 and CDK6, or the primary mode of action is inhibition by CDK4 and/or CDK 6. In one embodiment, e.g., CDK4/CycD1IC₅₀IC of Selective CDK4/6 inhibitor on CDK4 measured in phosphorylation assay₅₀Such as CDK2/CycE IC₅₀IC of CDK2 by the compound measured in phosphorylation assay₅₀At least 1500, 2000, 5000, or even 10,000 times lower. In one embodiment, the CDK4/6 inhibitor is at least about 10-fold or much more effective than PD0332991 (i.e., IC in CDK4/CycD1 phosphorylation assays)₅₀At least 10 times lower or more).

Use of selected CDK4/6 inhibitors as described herein may induce selective G1 arrest in CDK4/6 dependent cells (e.g., as measured in a cell-based in vitro assay). In one embodiment, the CDK4/6 inhibitor is capable of increasing the percentage of G1 phase CDK4/6 dependent cells while decreasing the percentage of G2/M phase and S phase CDK4/6 dependent cells. In one embodiment, the selective CDK4/6 inhibitor induces substantially pure (i.e., "complete") G1 cell cycle arrest in CDK 4/6-dependent cells (e.g., wherein treatment with the selective CDK4/6 inhibitor induces cell cycle arrest such that a majority of the cells arrest in G1, as defined by standard methods (e.g., Propidium Iodide (PI) staining or otherwise), wherein the population of combined G2/M and S phase cells is less than about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 3% or less of the total cell population E.g., a DNA binding dye such as PI, and analyzing cellular DNA content by flow cytometry. Immunofluorescence techniques involve the detection of specific cell cycle indicators, such as thymidine analogs (e.g., 5-bromo-2-deoxyuridine (BrdU) or iododeoxyuridine), with fluorescent antibodies.

In some embodiments, off-target effects particularly associated with inhibition of kinases other than CDK4 and or CDK6 (e.g., CDK2) are reduced or substantially absent using the selective CDK4/6 inhibitors described herein because the selective CDK4/6 inhibitors described herein are poor inhibitors of CDK2 (e.g., CDK 2/6 inhibitors are poor inhibitors of CDK2)>1uM IC₅₀). Furthermore, due to the high selectivity of CDK4/6, cell cycle arrest of CDK 4/6-independent cells should not be induced using the compounds described herein. In addition, because of the transient nature of G1 arrest, CDK4/6 replication-dependent cells re-enter the cell cycle relatively more rapidly than with PD0332991, such that in one embodiment HSPC can be chemotherapeutically treated during a long-term treatment regimenReplication between therapy treatments reduces the risk of development of hematologic toxicity.

In some embodiments, the use of a selective CDK4/6 inhibitor described herein reduces the risk of adverse off-target effects, including (but not limited to) long-term toxicity, antioxidant effects, and estrogenic effects. The antioxidant effect can be determined by standard assays known in the art. For example, a compound without significant antioxidant effect is a compound that does not significantly remove free radicals, such as oxygen free radicals. The antioxidant effect of the compounds can be compared to compounds with known antioxidant activity, such as genistein. Thus, a compound without significant antioxidant activity may be a compound having less than about 2, 3, 5, 10, 30, or 100 times antioxidant activity relative to genistein. Estrogenic activity can also be determined via known assays. For example, a non-estrogenic compound is a compound that does not significantly bind to and activate the estrogen receptor. Thus, a compound that is substantially free of estrogenic effects may be a compound having less than about 2, 3, 5, 10, 20, or 100 times estrogenic activity relative to a compound having estrogenic activity, such as genistein.

Synthesis of selected CDK4/6 inhibitors

The CDK4/6 inhibitors of the present invention may be synthesized by any means known to those skilled in the art, including, for example, according to the general schemes of 1 to 9 below. Specific syntheses can be found in WO2012/061156 (corresponding to 5- (4-isopropylpiperazin-1-yl) pyridin-2-amine and 5- (4-morpholino-1-piperidinyl) pyridin-2-amine). Formula I and formula II can be synthesized according to scheme 1 using the corresponding substituted 2-aminopyrimidines or as described in WO 2012/061156.

Scheme 1

Scheme 2

In scheme 2, Ref-1 is WO 2010/020675A 1; ref-2 is White, J.D. et al J.org.chem.1995,60,3600; and Ref-3 is Presser, A. and Hufner, A. Monatsheftete fur Chemie 2004,135,1015.

Scheme 3

In scheme 3, Ref-1 is WO 2010/020675A 1; ref-4 is WO 2005/040166A 1; and Ref-5 is Schoenauer, K and Zbiral, E.tetrahedron Letters 1983,24, 573.

Scheme 4

In scheme 4, Ref-1 is WO 2010/020675A 1.

Scheme 5

Scheme 6

Scheme 7

Scheme 8

Scheme 9

In scheme 9, Ref-1 is WO 2010/020675A 1; ref-2 is WO 2005/040166A 1; and Ref-3 is Schoenauer, K and Zbiral, E.tetrahedron Letters 1983,24, 573.

Scheme 10

In one embodiment, the lactam intermediate is treated with the 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 produce 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 produce an amide that can be deprotected with a strong acid, such as trifluoroacetic acid, to produce the final target inhibitor compound.

Alternatively, the lactam may be produced by reacting a carboxylic acid with a protected amine in the presence of a strong acid and a dehydrating agent, which may together be in one moiety as a strong acid anhydride. Examples of strong acid anhydrides include, but are not limited to, trifluoroacetic anhydride, tribromoacetic anhydride, trichloroacetic anhydride, or mixed anhydrides. The dehydrating agent may 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).

Alternatively, the halogen moiety bonded to the pyrimidine ring may be substituted with any leaving group that can be displaced by a primary amine, e.g., an intermediate that produces a final product, e.g., Br, I, F, SMe, SO₂Me, SO alkyl, SO₂An alkyl group. See, e.g., PCT/US2013/037878 to Tavares.

Other amine intermediates and final amine compounds can be synthesized by those skilled in the art. It is to be understood that in the context of the present invention, the chemistry may employ reagents that contain reactive functional groups that can be protected and deprotected and that are known to those skilled in the art. See, e.g., Greene, T.W. and Wuts, P.G.M., Greene's Protective Groups in Organic Synthesis, 4 th edition, John Wiley and Sons.

Formulas T, Q, GG and U prepared above were characterized by mass spectrometry and NMR as shown below:

formula T

7.25(s,1H)7.63(br.s.,2H)7.94(br.s.,1H)8.10(br.s.,1H)8.39 (br.s.,1H)9.08(br.s.,1H)11.59(br.s.,1H)。LCMS ESI(M+H)447。

Formula Q

1H NMR(600MHz,DMSO-d₆)δppm 0.82(d,J＝7.32Hz,2H) 1.08-1.37(m,3H)1.38-1.64(m,2H)1.71(br.s.,1H)1.91(br.s.,1H) 2.80(br.s.,1H)3.12(s,1H)3.41(br.s.,4H)3.65(br.s.,4H)4.09(br. s.,1H)7.26(s,1H)7.52-7.74(m,2H)7.94(br.s.,1H)8.13(br.s.,1 H)8.40(br.s.,1H)9.09(br.s.,1H)9.62(br.s.,1H)11.71(br.s.,1H)。 LCMS ESI(M+)433

Formula GG

1H NMR(600MHz,DMSO-d₆)δppm 0.85(br.s.,1H)1.17-1.39 (m,7H)1.42-1.58(m,2H)1.67-1.84(m,3H)1.88-2.02(m,1H) 2.76-2.93(m,1H)3.07-3.22(m,1H)3.29-3.39(m,1H)3.41-3.61 (m,4H)3.62-3.76(m,4H)3.78-3.88(m,1H)4.12(br.s.,1H)7.28(s, 1H)7.60-7.76(m,2H)7.98(s,1H)8.13(br.s.,1H)8.41(s,1H)9.10 (br.s.,1H)11.21(br.s.,1H)11.54(s,1H)。LCMS ESI(M+H)475

Formula U

1H NMR(600MHz,DMSO-d₆)δppm 0.84(t,J＝7.61Hz,2H)1.13 -1.39(m,4H)1.46(d,J＝14.05Hz,2H)1.64-1.99(m,6H)2.21(br.s., 1H)2.66-2.89(m,2H)3.06(br.s.,1H)3.24-3.36(m,1H)3.37- 3.50(m,2H)3.56-3.72(m,2H)3.77-4.00(m,4H)4.02-4.19(m,2 H)7.25(s,1H)7.50-7.75(m,2H)7.89(d,J＝2.93Hz,1H)8.14(d, J＝7.32Hz,1H)8.38(br.s.,1H)9.06(s,1H)11.53(br.s.,1H)。LCMS ESI(M+H)517

Active compounds, salts and formulations

As used herein, the term "active compound" refers to a selective CDK 4/6 inhibitor compound described herein, or a pharmaceutically acceptable salt or isotopic analog thereof. The active compound may be administered to the subject by any suitable method. The amount and timing of the active compound administered may, of course, depend on the subject being treated, the dosage of the chemotherapy to which the subject is expected to be exposed, the time course of exposure to the chemotherapeutic agent, the mode of administration, the pharmacokinetic properties of the particular active compound, and the judgment of the attending physician. Thus, as affected by subject variability, the dosages given below are guidelines and a physician can adjust the dosage of the compound to achieve a treatment that the physician deems appropriate for the subject. The physician can balance factors such as the age and weight of the subject, the presence of pre-existing disease, and the presence of other diseases, when considering the degree of treatment desired. The pharmaceutical formulations can be prepared for any desired route of administration, including, but not limited to, oral, intravenous, or aerosol administration, as discussed in more detail below.

A therapeutically effective dose of any of the active compounds described herein will be determined by the health care practitioner, depending on the condition, size and age of the patient and the route of delivery. In one non-limiting embodiment, a dosage of about 0.1 to about 200mg/kg has therapeutic efficacy, wherein all weights are based on the weight of the active compound, including where a salt is employed. In some embodiments, a dose may be that amount of compound necessary to provide a serum concentration of active compound of up to between about 1 and 5, 10, 20, 30, or 40 μ M. In some embodiments, a dose of about 10mg/kg to about 50mg/kg may be used for oral administration. Typically, a dose of about 0.5 mg/kg to 5mg/kg may be used for intramuscular injection. In some embodiments, the dose may be from about 1 to about 50 μmol/kg, or optionally, between about 22 and about 33 μmol/kg of the compound for intravenous or oral administration. Oral dosage forms may include any suitable amount of the active substance, including 5mg to 50, 100, 200 or 500mg per tablet or other solid dosage form.

In accordance with the presently disclosed methods, the pharmaceutically active compounds as described herein can be administered orally in solid form or in liquid form, or can be administered intramuscularly, intravenously or by inhalation in the form of a solution, suspension or emulsion. In some embodiments, the compound or salt may also be administered by inhalation, intravenously or intramuscularly in the form of a liposomal suspension. When administered by inhalation, the active compound or salt may be in the form of a plurality of solid particles or droplets having any desired particle size, 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. The compounds as disclosed herein have demonstrated excellent pharmacokinetic and pharmacodynamic properties, e.g. when administered by oral or intravenous route.

In one embodiment of the invention, these improved CDK4/6 inhibitors may be administered in a scheduled regimen with a blood growth factor agent. Thus, 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) the following: granulocyte colony stimulating factor (G-CSF, such as sold as Neupogen (filgrastin), Neulasta (pegylated non-filgrastin) or lenograstin (lenograstin), granulocyte-macrophage colony stimulating factor (GM-CSF, such as sold as Moraxestin (molgramostim) and Sargramostim (Leukine), M-CSF (macrophage colony stimulating factor), thrombopoietin (megakaryocyte growth and development factor (MGDF), such as sold as Romithrastsin (Romipross) and Epimepa (Eltrombogopag), Interleukin (IL) -12, interleukin-3, interleukin-11 (adipogenesis inhibitor or Opproleukin), SCF (stem cell factor, brusin, erythropoietin and kits (EPO) and derivatives thereof, such as Erythrox-alpha-erythropoietin, Leukolin, and EPO (erythropoietin), Epocept, nanogine, eposit, Epogin, Eprex and Procrit; erythropoietin-beta is sold, for example, as NeoRecormon, Recormon, and Micera), erythropoietin-delta (sold, for example, as Dynepo), erythropoietin-omega (sold, for example, as Epomax), erythropoietin-zeta (sold, for example, as Silapo and Reacrit), and also, for example, Epocept, EPOTTrust, Erypro Safe, Repoetin, Vintor, Epofit, Erykine, Wepox, Espogen, Relipoeitin, Shanpoetin, Zyro, and EPIAO).

The pharmaceutical formulation may comprise an active compound described herein, or a pharmaceutically acceptable salt thereof, in any pharmaceutically acceptable carrier. If a solution is desired, water may be the carrier of choice for the water-soluble compound or salt. As regards the water-soluble compounds or salts, organic vehicles such as glycerol, propylene glycol, polyethylene glycol or mixtures thereof may be suitable. In the latter case, the organic vehicle may contain a substantial amount of water. The solution in either case can then be sterilized in a suitable manner known to those skilled in the art, and for purposes of illustration, sterilized by filtration through a 0.22 micro-porous filter. After sterilization, the solution may be dispensed into a suitable container, such as a depyrogenated glass vial. Dispensing is optionally performed by aseptic methods. A sterile cover plate can then be placed over the vial and the vial contents can be lyophilized if necessary.

In addition to the active compound or its salts, the pharmaceutical preparations may contain further additives, for example pH-adjusting additives. Specifically, suitable 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. In addition, the formulation may contain an antimicrobial preservative. Suitable antimicrobial and preservative agents include methylparaben, propylparaben and benzyl alcohol. Antimicrobial preservatives are typically employed when the formulations are placed in vials intended for multi-dose use. The pharmaceutical formulations described herein can be lyophilized using techniques well known in the art.

For oral administration, the pharmaceutical compositions may 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, various disintegrants such as starch (e.g. potato or tapioca starch) and certain complex silicates, and binders such as polyvinylpyrrolidone, sucrose, gelatin and acacia may be employed. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate, and talc are often well suited for tableting purposes. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules. Substances in this respect also include lactose or milk sugar and 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 diluents such as water, ethanol, propylene glycol, glycerin and the like, and various similar combinations thereof.

In yet another embodiment of the subject matter described herein, there is provided an injectable stable sterile formulation comprising an active compound or salt thereof as described herein in a unit dosage form in a sealed container. The compound or salt is provided in the form of a lyophilizate that can be 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 a physiologically acceptable emulsifier sufficient to emulsify the compound or salt in an aqueous carrier can be employed. Particularly suitable emulsifiers include phosphatidyl choline and lecithin.

Other embodiments provided herein include liposomal formulations of the active compounds disclosed herein. Techniques for forming liposomal suspensions are well known in the art. When the compound is a water-soluble salt, it can be incorporated into lipid vesicles using conventional liposome technology. In such cases, the active compound may be substantially entrapped within the hydrophilic center or core of the liposome due to the water solubility of the active compound. The lipid layer used may be of any conventional composition and may or may not contain cholesterol. When the relevant active compound is insoluble in water, the salt may be substantially entrapped within the hydrophobic bilayer lipid forming the liposomal structure, again using conventional liposome forming techniques. In either case, the size of the liposomes produced can be reduced, such as by using standard sonication and homogenization techniques. 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 the liposomal suspension.

Also provided are pharmaceutical formulations suitable for administration by inhalation in aerosol form. These formulations comprise a solution or suspension of the desired compound or salt thereof or a plurality of solid particles of the compound or salt described herein. The desired formulation can be placed in a small chamber and atomized. Atomization can be achieved by compressed air or by ultrasonic energy to form a plurality of droplets or solid particles comprising the compound or salt. The droplets or solid particles may, for example, have a particle size in the range of about 0.5 to about 10 microns, and optionally about 0.5 to about 5 microns. The solid particles may be obtained by processing the solid compound or salt thereof by any suitable means known in the art, for example by micronisation. Optionally, the solid particles or droplets may be from about 1 to about 2 microns in size. In this regard, commercial sprayers may be used to achieve this. The compounds may be administered via an aerosol suspension of respirable particles in the manner set forth in U.S. patent No.5,628,984, the disclosure of which is incorporated herein by reference in its entirety.

When a pharmaceutical formulation suitable for administration as an aerosol is in liquid form, the formulation may comprise a water-soluble active compound in a carrier comprising water. A surfactant may be present which reduces the surface tension of the formulation sufficient to form droplets in the desired size range when subjected to atomization.

As used herein, the term "pharmaceutically acceptable salt" refers to a salt that is, within the scope of sound medical judgment, suitable for use in contact with a subject (e.g., a human subject), without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for its intended use, as well as the zwitterionic forms of the compounds of the presently disclosed subject matter, where possible.

Thus, the term "salt" refers to the relatively non-toxic inorganic and organic acid addition salts of the compounds of the presently disclosed subject matter. These salts may be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Pharmaceutically acceptable base addition salts may be formed with metals or amines, for example alkali and alkaline earth metal hydroxides or 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' -benzhydrylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, and procaine.

The salts can be formed from inorganic acid sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides (e.g., hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, and the like). Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthoate, mesylate, glucoheptanoate, lactobionate, lauryl sulfate, isethionate and the like. Salts may also be prepared from organic acids such as aliphatic monocarboxylic and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedicarboxylic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Representative salts include acetate, propionate, octanoate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, tosylate, phenylacetate, citrate, lactate, maleate, tartrate, methanesulfonate, and the like. Pharmaceutically acceptable salts can include cations based on alkali and alkaline earth metals, such as sodium, lithium, 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 salts of amino acids such as arginate, gluconate, galacturonate, and the like. See, e.g., Berge et al, j.pharm.sci.,1977,66, 1-19, incorporated herein by reference.

For purposes of example only, the present invention includes, but is not limited to, the following:

technical solution 1. a method of reducing the effect of chemotherapy on healthy cells in a subject being treated for a cyclin dependent kinase 4/6 (CDK4/6) replication independent cancer or abnormal cell proliferation, wherein the healthy cells are hematopoietic stem cells, hematopoietic progenitor cells, or renal epithelial cells, the method comprising administering to the subject an effective amount of a compound selected from formula I, II, III, IV, or V:

wherein:

z is- (CH)₂)_x-, where x is 1, 2, 3 or 4, or-O- (CH)₂)_z-, wherein z is 2, 3 or 4;

each X is independently CH or N;

each X' is independently CH or N;

x' is independently CH₂S or NH, configured such that the moiety is a stable 5-membered ring;

R、R⁸and R¹¹Independently H, C₁-C₃Alkyl or haloalkyl, cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O or S; - (alkylene) m-C₃-C₈Cycloalkyl, - (alkylene)_mAryl, - (alkylene)_m-heterocyclyl, - (alkylene)_m-heteroaryl, - (alkylene)_m-NR³R⁴- (alkylene group)_m-C(0)-NR³R⁴(ii) a - (alkylene)_m-0-R⁵- (alkylene group)_m-S(0)_n-R⁵Or- (alkylene)_m-S(0)n-NR³R⁴Any of which may be optionally independently substituted, where valency permits, with one or more R groups, and wherein two R groups bound to the same or adjacent atoms ^xThe groups may optionally be combined to form a ring;

each R¹Independently is aryl, alkyl, cycloalkyl or haloalkyl, wherein each of said alkyl, cycloalkyl and haloalkyl optionally includes an O or N heteroatom in the chain in place of carbon and two Rs on adjacent ring atoms or on the same ring atom¹Optionally forming a 3-8 membered ring together with the ring atoms to which they are attached;

y is 0, 1, 2, 3 or 4;

R²is- (alkylene)_m-heterocyclyl, - (alkylene)_m-heteroaryl, - (alkylene)_m-NR³R⁴- (alkylene group)_m-C(O)-NR³R⁴(ii) a - (alkylene)_m-C (O) -O-alkyl; - (alkylene)_m-O-R⁵- (alkylene group)_m-S(O)_n-R⁵Or- (alkylene)_m-S(O)_n-NR³R⁴Any of which may optionally independently pass through one or more R, where valence permits^xSubstituted by radicals, and in which two of R are bound to the same or to adjacent atoms^xThe groups may optionally combine to form a ring and wherein m is 0 or 1 and n is 0, 1 or 2;

R³and R⁴Independently for each occurrence:

(i) hydrogen or

(ii) Alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl, any of which may optionally be independently interrupted, as valence permits, by one or more R^xSubstituted by radicals, and in which two of R are bound to the same or to adjacent atoms ^xThe groups may optionally be combined to form a ring; or R³And R⁴Together with the nitrogen atom to which they are attached may be combined to form a compound optionally independently via one or more R, as valence permits^xA heterocycle substituted by a group, and wherein two of the groups are bound to the sameOr R of adjacent atoms^xThe groups may optionally be combined to form a ring;

R⁵and R⁵Each occurrence of

(i) Hydrogen or

(ii) Alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl, any of which may optionally be independently interrupted by one or more R where valency permits^xSubstituted by groups;

R^xindependently for each occurrence is halo, cyano, nitro, oxo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, heterocycloalkyl, - (alkylene)_m-OR⁵- (alkylene group)_m-O-alkylene-OR⁵- (alkylene group)_m-S(O)_n-R⁵- (alkylene group)_m-NR³R⁴- (alkylene group)_m-CN, - (alkylene)_m-C(O)-R⁵- (alkylene group)_m-C(S)-R⁵- (alkylene group)_m-C(O)-OR⁵- (alkylene group)_m-O-C(O)-R⁵- (alkylene group)_m-C(S)-OR⁵- (alkylene group)_m-C (O) - (alkylene)_m-NR³R⁴- (alkylene group)_m-C(S)-NR³R⁴- (alkylene group) _m-N(R³)-C(O)-NR³R⁴- (alkylene group)_m-N(R³)-C(S)-NR³R⁴- (alkylene group)_m-N(R³)-C(O)-R⁵- (alkylene group)_m-N(R³)-C(S)-R⁵- (alkylene group)_m-O-C(O)-NR³R⁴- (alkylene group)_m-O-C(S)-NR³R⁴- (alkylene group)_m-SO₂-NR³R⁴- (alkylene group)_m-N(R³)-SO₂-R⁵- (alkylene group)_m-N(R³)-SO₂-NR³R⁴- (alkylene group)_m-N(R³)-C(O)-OR⁵ ₎- (alkylene group)_m-N(R³)-C(S)-OR⁵Or- (alkylene)_m-N(R³)-SO₂-R⁵(ii) a Wherein:

the alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, and heterocycloalkyl may be further independently substituted with one or more of the following:

- (alkylene)_m-CN, - (alkylene)_m-OR⁵- (alkylene)_m-S(O)_n-R⁵[ alkyl ] - (alkylene)_m-NR³*R⁴- (alkylene)_m-C(O)-R⁵- (alkylene)_m-C(＝S)R⁵[ alkyl ] - (alkylene)_m-C(＝O)OR⁵- (alkylene)_m-OC(＝O)R⁵- (alkylene)_m-C(S)-OR⁵'x' (alkylene)_m-C(O)-NR³*R⁴- (alkylene)_m-C(S)-NR³*R⁴[ alkyl ] - (alkylene)_m-N(R³*)-C(O)-NR³*R⁴- (alkylene)_m-N(R³*)-C(S)-NR³*R⁴[ alkyl ] - (alkylene)_m-N(R³*)-C(O)-R⁵- (alkylene)_m-N(R³*)-C(S)-R⁵[ alkyl ] - (alkylene)_m-O-C(O)-NR³*R⁴- (alkylene)_m-O-C(S)-NR³*R⁴[ alkyl ] - (alkylene)_m-SO₂-NR³*R⁴- (alkylene)_m-N(R³*)-SO₂-R⁵[ alkyl ] - (alkylene)_m-N(R³*)-SO₂-NR³*R⁴- (alkylene)_m-N(R³*)-C(O)-OR⁵[ alkyl ] - (alkylene)_m-N(R³*)-C(S)-OR⁵Or- (alkylene)_m-N(R³*)-SO₂-R⁵*，

n is 0, 1 or 2, and

m is 0 or 1;

R³a and R⁴Independently at each occurrence:

(i) hydrogen or

(ii) Alkyl, alkenyl, alkynyl cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl, any of which may optionally be independently interrupted by one or more R as valence permits ^xSubstituted by groups; or R³A and R⁴Together with the nitrogen atom to which they are attached may be combined to form, optionally independently, one or more R, where valence permits^xA group-substituted heterocycle; and is

R⁶Is H or lower alkyl, - (alkylene) m-heterocyclyl, - (alkylene)_m-heteroaryl, - (alkylene)_m-NR³R⁴- (alkylene group)_m-C(0)-NR³R⁴(ii) a - (alkylene)_m-0-R⁵- (alkylene group)_m-S(0)_n-R⁵Or- (alkylene)_m-S(0)_n-NR³R⁴Any of which may optionally independently pass through one or more R, where valence permits^xSubstituted by radicals, and in which two R are bound to the same or to adjacent atoms^xThe groups may optionally be combined to form a ring; and is

R¹⁰Is (i) NHR^AWherein R is^AIs unsubstituted or substituted C₁-C₈Alkyl, cycloalkylalkyl or-TT-RR, C₁-C₈Cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O and S; TT is unsubstituted or substituted C₁-C₈Alkyl or C₃-C₈A cycloalkyl linking group; and RR is hydroxy, unsubstituted or substituted C₁-C₆Alkoxy, amino, unsubstituted or substituted C₁-C₆Alkylamino, unsubstituted or substituted di-C₁-C₆Alkylamino, unsubstituted or substituted C₆-C₁₀Aryl, unsubstituted containing one or two 5-or 6-membered rings and 1 to 4 heteroatoms selected from N, O and S Substituted or substituted heteroaryl, unsubstituted or substituted C₃-C₁₀A carbocycle or an unsubstituted or substituted heterocycle comprising one or two 5-or 6-membered rings and 1-4 heteroatoms selected from N, O and S; or (ii) -C (O) -R¹²or-C (O) O-R¹³Wherein R is¹²Is NHR^AOr R^AAnd R is¹³Is R^A。

Scheme 2. the process of scheme 1, wherein R⁸Is hydrogen or C₁-C₃An alkyl group.

Scheme 3. the method of scheme 1, wherein the compound has a formula selected from the structures shown in figure 27.

Scheme 4. the method of scheme 1, wherein the compound has a formula selected from the structures shown in figure 28.

Scheme 5. the method of scheme 1, wherein the compound has a formula selected from the structures shown in figure 29.

Scheme 6. the method of scheme 1, wherein the compound has a formula selected from the structures shown in figure 30.

Scheme 7. the method of scheme 1, wherein the compound has a formula selected from the structures shown in figure 31.

Scheme 8. the method of scheme 1, wherein the compound has the formula:

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

Scheme 10. the method of scheme 1, wherein the compound has the formula:

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

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

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

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

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

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

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

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

wherein R is C (H) X, NX, C (H) Y or C (X)₂；

Wherein X is hydrogen, straight, branched or cyclic C₁To C₅Alkyl groups including methyl, ethyl, propyl, cyclopropyl, isopropyl, butyl, sec-butyl, tert-butyl, isobutyl, cyclobutyl, pentyl, isopentyl, neopentyl, tert-pentyl, sec-pentyl and cyclopentyl; and is

Y is NR₁R₂Wherein R is₁And R₂Independently X, or wherein R₁And R₂Alkyl groups which together form a bridge comprising one or two heteroatoms (N, O or S); and is

Wherein two X groups may together form an alkyl bridge or bridge comprising one or two heteroatoms (N, S or O) to form a spiro compound; or

A pharmaceutically acceptable salt thereof.

The method of claim 1, wherein the compound is selected from the following formulae:

scheme 20. the method of scheme 19, wherein the compound is:

the method of claim 19, wherein the compound is:

scheme 22. the method of scheme 19, wherein the compound is:

scheme 23. the method of scheme 19, wherein the compound is:

technical scheme 24. the method of technical scheme 1 wherein one X is N and one X is C.

Technical scheme 25 the method of any one of technical schemes 1 to 24, wherein the subject is a human.

The method of any one of claims 1-24, wherein the compound is administered to the subject 24 hours or less prior to exposure to the cytotoxic compound.

The method of any one of claims 1 to 26, wherein the subject has cancer.

The method of any one of claims 1 to 26, wherein the subject has abnormal cell proliferation.

Technical scheme 29 the method of any one of technical schemes 1 to 28, wherein the cancer or abnormal cell proliferation is characterized by the loss or absence of retinoblastoma tumor suppressor protein (RB).

Scheme 30 the method of any of claims 1 to 26, wherein the cancer is small cell lung cancer, retinoblastoma, triple negative breast cancer, Human Papilloma Virus (HPV) positive head and neck cancer, or HPV positive cervical cancer.

Scheme 31 the method of any of claims 1 to 30, wherein the chemotherapy is selected from alkylating agents, DNA intercalating agents, protein synthesis inhibitors, inhibitors of DNA or RNA synthesis, DNA base analogues, topoisomerase inhibitors, telomerase inhibitors or telomere DNA binding compounds.

The method of any of claims 1-30, wherein the cancer is small cell lung cancer and the chemotherapy is selected from etoposide, cisplatin, and carboplatin, or a combination thereof.

Technical scheme 33 the method of any one of technical schemes 1 to 32, wherein at least 80% or more of the HSPCs re-enter the cell cycle less than 36 hours from the last administration of the compound.

The method of any one of claims 1 to 33, wherein the healthy cells are hematopoietic stem cells or hematopoietic progenitor cells.

Scheme 35 the method of any of claims 1 to 33, wherein the healthy cells are renal epithelial cells.

A method of treating an Rb-negative cancer or abnormal cell proliferation in a subject with a combination therapy comprising administering to the subject an effective amount of chemotherapy in combination with another chemotherapeutic compound in combination with a cyclin-dependent kinase 4/6(CDK4/6) compound selected from formula I, II, III, IV or V, or a pharmaceutically acceptable salt thereof:

wherein:

z is- (CH)₂)_x-, where x is 1, 2, 3 or 4, or-O- (CH)₂)_z-, wherein z is 2, 3 or 4;

each X is independently CH or N;

each X' is independently CH or N;

x' is independently CH₂S or NH, configured such that the moiety is a stable 5-membered ring;

R、R⁸and R¹¹Independently H, C₁-C₃Alkyl or haloalkyl, cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O or S; - (alkylene) m-C ₃-C₈Cycloalkyl, - (alkylene)_mAryl, - (alkylene)_m-heterocyclyl, - (alkylene)_m-heteroaryl, - (alkylene)_m-NR³R⁴- (alkylene group)_m-C(0)-NR³R⁴(ii) a - (alkylene)_m-0-R⁵- (alkylene group)_m-S(0)_n-R⁵Or- (alkylene)_m-S(0)n-NR³R⁴Any of which may be optionally independently substituted, where valency permits, with one or more R groups, and wherein two R groups bound to the same or adjacent atoms^xThe groups may optionally be combined to form a ring;

each R¹Independently is aryl, alkyl, cycloalkyl or haloalkyl, wherein each of said alkyl, cycloalkyl and haloalkyl optionally includes an O or N heteroatom in the chain in place of carbon and two Rs on adjacent ring atoms or on the same ring atom¹Optionally forming a 3-8 membered ring together with the ring atoms to which they are attached;

y is 0, 1, 2, 3 or 4;

R²is- (alkylene)_m-heterocyclyl, - (alkylene)_m-heteroaryl, - (alkylene)_m-NR³R⁴- (alkylene group)_m-C(O)-NR³R⁴(ii) a - (alkylene)_m-C (O) -O-alkyl; - (alkylene)_m-O-R⁵- (alkylene group)_m-S(O)_n-R⁵Or- (alkylene)_m-S(O)_n-NR³R⁴Any of which may optionally independently pass through one or more R, where valence permits^xSubstituted by radicals, and in which two of R are bound to the same or to adjacent atoms^xThe groups may optionally combine to form a ring and wherein m is 0 or 1 and n is 0, 1 or 2;

R³And R⁴Independently for each occurrence:

(i) hydrogen or

(ii) Alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl, any of which may optionally be independently interrupted, as valence permits, by one or more R^xSubstituted by radicals, and in which two of R are bound to the same or to adjacent atoms^xThe groups may optionally be combined to form a ring; or R³And R⁴Together with the nitrogen atom to which they are attached may be combined to form a compound optionally independently via one or more R, as valence permits^xA heterocycle substituted by a group, and wherein two of R are bound to the same or adjacent atom^xThe groups may optionally be combined to form a ring;

R⁵and R⁵Each occurrence of

(i) Hydrogen or

(ii) Alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl, any of which may optionally be independently interrupted by one or more R where valency permits^xSubstituted by groups;

R^xindependently for each occurrence is halo, cyano, nitro, oxo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, heterocycloalkyl, - (alkylene) _m-OR⁵- (alkylene group)_m-O-alkylene-OR⁵- (alkylene group)_m-S(O)_n-R⁵- (alkylene group)_m-NR³R⁴- (alkylene group)_m-CN, - (alkylene)_m-C(O)-R⁵- (alkylene group)_m-C(S)-R⁵- (alkylene group)_m-C(O)-OR⁵- (alkylene group)_m-O-C(O)-R⁵- (alkylene group)_m-C(S)-OR⁵- (alkylene group)_m-C (O) - (alkylene)_m-NR³R⁴- (alkylene group)_m-C(S)-NR³R⁴- (alkylene group)_m-N(R³)-C(O)-NR³R⁴- (alkylene group)_m-N(R³)-C(S)-NR³R⁴- (alkylene group)_m-N(R³)-C(O)-R⁵- (alkylene group)_m-N(R³)-C(S)-R⁵- (alkylene group)_m-O-C(O)-NR³R⁴- (alkylene group)_m-O-C(S)-NR³R⁴- (alkylene group)_m-SO₂-NR³R⁴- (alkylene group)_m-N(R³)-SO₂-R⁵- (alkylene group)_m-N(R³)-SO₂-NR³R⁴- (alkylene group)_m-N(R³)-C(O)-OR⁵ ₎- (alkylene)_m-N(R³)-C(S)-OR⁵Or- (alkylene)_m-N(R³)-SO₂-R⁵(ii) a Wherein:

the alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, cycloalkylalkyl, and heterocycloalkyl may be further independently substituted with one or more of the following:

- (alkylene)_m-CN, - (alkylene)_m-OR⁵- (alkylene)_m-S(O)_n-R⁵[ alkyl ] - (alkylene)_m-NR³*R⁴- (alkylene)_m-C(O)-R⁵- (alkylene)_m-C(＝S)R⁵[ alkyl ] - (alkylene)_m-C(＝O)OR⁵- (alkylene)_m-OC(＝O)R⁵- (alkylene)_m-C(S)-OR⁵'x' (alkylene)_m-C(O)-NR³*R⁴- (alkylene)_m-C(S)-NR³*R⁴[ alkyl ] - (alkylene)_m-N(R³*)-C(O)-NR³*R⁴- (alkylene)_m-N(R³*)-C(S)-NR³*R⁴[ alkyl ] - (alkylene)_m-N(R³*)-C(O)-R⁵- (alkylene)_m-N(R³*)-C(S)-R⁵[ alkyl ] - (alkylene)_m-O-C(O)-NR³*R⁴- (alkylene)_m-O-C(S)-NR³*R⁴[ alkyl ] - (alkylene)_m-SO₂-NR³*R⁴- (alkylene)_m-N(R³*)-SO₂-R⁵[ alkyl ] - (alkylene)_m-N(R³*)-SO₂-NR³*R⁴- (alkylene)_m-N(R³*)-C(O)-OR⁵[ alkyl ] - (alkylene) _m-N(R³*)-C(S)-OR⁵Or- (alkylene)_m-N(R³*)-SO₂-R⁵*，

n is 0, 1 or 2, and

m is 0 or 1;

R³a and R⁴Independently at each occurrence:

(i) hydrogen or

(ii) Alkyl, alkenyl, alkynyl cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl, any of which may optionally be independently interrupted by one or more R as valence permits^xSubstituted by groups; or R³A and R⁴Together with the nitrogen atom to which they are attached may be combined to form, optionally independently, one or more R, where valence permits^xA group-substituted heterocycle; and is

R⁶Is H or lower alkyl, - (alkylene) m-heterocyclyl, - (alkylene)_m-heteroaryl, - (alkylene)_m-NR³R⁴- (alkylene group)_m-C(0)-NR³R⁴(ii) a - (alkylene)_m-0-R⁵- (alkylene group)_m-S(0)_n-R⁵Or- (alkylene)_m-S(0)_n-NR³R⁴Any one of themOptionally independently via one or more R, where valency permits^xSubstituted by radicals, and in which two R are bound to the same or to adjacent atoms^xThe groups may optionally be combined to form a ring; and is

R¹⁰Is (i) NHR^AWherein R is^AIs unsubstituted or substituted C₁-C₈Alkyl, cycloalkylalkyl or-TT-RR, C₁-C₈Cycloalkyl or cycloalkyl containing one or more heteroatoms selected from N, O and S; TT is unsubstituted or substituted C ₁-C₈Alkyl or C₃-C₈A cycloalkyl linking group; and RR is hydroxy, unsubstituted or substituted C₁-C₆Alkoxy, amino, unsubstituted or substituted C₁-C₆Alkylamino, unsubstituted or substituted di-C₁-C₆Alkylamino, unsubstituted or substituted C₆-C₁₀Aryl, unsubstituted or substituted heteroaryl comprising one or two 5-or 6-membered rings and 1 to 4 heteroatoms selected from N, O and S, unsubstituted or substituted C₃-C₁₀A carbocycle or an unsubstituted or substituted heterocycle comprising one or two 5-or 6-membered rings and 1-4 heteroatoms selected from N, O and S; or (ii) -C (O) -R¹²or-C (O) O-R¹³Wherein R is¹²Is NHR^AOr R^AAnd R is¹³Is R^A。

Claim 37. the method of claim 36, wherein the CDK4/6 inhibitor is:

wherein R is C (H) X, NX, C (H) Y or C (X)₂；

Wherein X is hydrogen, straight, branched or cyclic C₁To C₅Alkyl groups including methyl, ethyl, propyl, cyclopropyl, isopropyl, butyl, sec-butyl, tert-butyl, isobutyl, cyclobutyl, pentyl, isopentyl, neopentyl, tert-pentyl, sec-pentyl and cyclopentyl; and is

Y is NR₁R₂Wherein R is₁And R₂Independently X, or wherein R₁And R₂Alkyl groups which together form a bridge comprising one or two heteroatoms (N, O or S); and is

Wherein two X groups may together form an alkyl bridge or a bridge comprising one or two heteroatoms (N, S or O) to form a spiro compound; or

A pharmaceutically acceptable salt or prodrug thereof.

The method of claim 36, wherein the compound is selected from the following formulae:

the method of claim 36, wherein the compound is:

scheme 40. the method of scheme 36, wherein the compound is:

scheme 41. the method of scheme 36, wherein the compound is:

scheme 42. the method of scheme 36, wherein the compound is:

technical scheme 43 the method of technical scheme 36, wherein the chemotherapeutic compound is selected from the group consisting of mTOR inhibitors, PI3 kinase inhibitors, dual mTOR-PI3K inhibitors, MEK inhibitors, RAS inhibitors, ALK inhibitors, AKT inhibitors, HSP inhibitors, BCL-2 inhibitors, apoptosis inducing compounds, PD-1 inhibitors, and FLT-3 inhibitors, or combinations thereof.

Technical scheme 44. a compound having the formula:

or a pharmaceutically acceptable salt thereof.

Technical scheme 45. a pharmaceutical composition comprising a compound according to technical scheme 1, or a pharmaceutically acceptable salt thereof, in a suitable dosage form to achieve chemoprotection of healthy cells during chemotherapy.

Technical scheme 46. use of any of the compounds described herein for reducing the effect of chemotherapy in a subject being treated for a cyclin dependent kinase 4/6(CDK4/6) replication independent cancer or abnormal cell proliferation, wherein the healthy cells are hematopoietic stem cells, hematopoietic progenitor cells, or renal epithelial cells.

Technical scheme 47. use of any of the compounds described herein in combination with a chemotherapeutic agent for treating Rb negative cancer or abnormal cell proliferation in a subject.

Scheme 48. the use according to scheme 46, wherein the compound is described in scheme 19.

Scheme 49. the use according to scheme 47, wherein the compound is described in scheme 19.

Examples

Intermediates B, E, K, L, 1A, 1F and 1CA were synthesized according to Tavares, f.x. and Strum, j.c. US 8,598,186 titled CDK Inhibitors.

Patents WO 2013/148748 to Tavares, f.x. entitled Lactam Kinase Inhibitors, Tavares, WO 2013/163239 to f.x. entitled Synthesis of Lactams and Tavares, f.x. and US 8,598,186 to Strum, j.c. entitled CDK Inhibitors are incorporated herein by reference in their entirety.

Example 1

Synthesis of tert-butyl N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] ethyl ] carbamate, Compound 1

To a solution of 5-bromo-2, 4-dichloropyrimidine (3.2g, 0.0135mol) in ethanol (80mL) was added henig's base (3.0mL), followed by a solution of N- (tert-butoxycarbonyl) -1, 2-diaminoethane (2.5g, 0.0156 mol) in ethanol (20 mL). The contents were stirred overnight for 20 hours. The solvent was evaporated under vacuum. Ethyl acetate (200mL) and water (100mL) were added and the layers were separated. The organic layer was dried over magnesium sulfate and then concentrated in vacuo. Silica gel column chromatography using hexane/ethyl acetate (0-60%) to give N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino group]Ethyl radical](iii) carbamic acid tert-butyl ester.¹HNMR(d6-DMSO)δppm 8.21(s,1H),7.62(brs,1H),7.27(brs,1H),3.39(m,2H),3.12(m,2H), 1.34(s,9H)。LCMS(ESI)351(M+H)。

Example 2

Synthesis of tert-butyl N- [2- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] amino ] ethyl ] carbamate, Compound 2

To a compound containing N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino group]Ethyl radical]To tert-butyl carbamate (1.265g, 3.6mmol) in THF (10mL) were added acetal (0.778mL, 5.43mmol), Pd (dppf) CH₂Cl₂(148mg) andtriethylamine (0.757mL, 5.43 mmol). The contents were degassed and then purged with nitrogen. CuI (29mg) was then added thereto. The reaction mixture was heated at reflux for 48 hours. After cooling, the contents are passed through CELITE ^TMFiltered and concentrated. The residue obtained was subjected to column chromatography using hexane/ethyl acetate (0-30%) to give N- [2- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl]Amino group]Ethyl radical](iii) carbamic acid tert-butyl ester.¹HNMR(d6-DMSO)δppm 8.18(s,1H),7.63(brs,1H),7.40(brs,1H), 5.55(s,1H),3.70(m,2H),3.60(m,2H),3.42(m,2H),3.15(m,2H), 1.19-1.16(m,15H)。LCMS(ESI)399(M+H)。

Example 3

Synthesis of tert-butyl N- [2- [ 2-chloro-6- (diethoxymethyl) pyrrolo [2,3-d ] pyrimidin-7-yl ] ethyl ] carbamate, Compound 3

To a solution of the coupling product (2.1g, 0.00526 mol) in THF (30mL) was added TBAF solid (7.0 g). The contents were heated to and maintained at 65 ℃ for 2 hours. Concentration followed by column chromatography using ethyl acetate/hexane (0-50%) gave N- [2- [ 2-chloro-6- (diethoxymethyl) pyrrolo [2,3-d ] as a light brown liquid]Pyrimidin-7-yl]Ethyl radical]Tert-butyl carbamate (1.1 g).¹HNMR(d6-DMSO)δppm 8.88(s,1H),6.95(brs, 1H),6.69(s,1H),5.79(s,1H),4.29(m,2H),3.59(m,4H),3.34(m,1H), 3.18(m,1H),1.19(m,9H),1.17(m,6H)。LCMS(ESI)399(M+H)。

Example 4

Synthesis of tert-butyl N- [2- (2-chloro-6-formyl-pyrrolo [2,3-d ] pyrimidin-7-yl) ethyl ] carbamate, Compound 4

AcOH (8.0mL) and water (1.0 mL) were added to the acetal from the previous step (900 mg). The reaction was stirred at room temperature for 16 hours. Concentration and use of ethyl acetateHexane (0-60%) and subjected to silica gel column chromatography to give N- [2- (2-chloro-6-formyl-pyrrolo [2,3-d ] in the form of foam ]Pyrimidin-7-yl) ethyl]Carbamic acid tert-butyl ester (0.510 g).¹HNMR (d6-DMSO)δppm 9.98(s,1H),9.18(s,1H),7.66(s,1H),6.80(brs,1H), 4.52(m,2H),4.36(m,2H),1.14(s,9H)。LCMS(ESI)325(M+H)。

Example 5

Synthesis of 7- [2- (tert-Butoxycarbonylamino) ethyl ] -2-chloro-pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid, Compound 5

To DMF (in 4 mL) containing the aldehyde from the previous step (0.940g) was added oxone (1.95g, 1.1 eq). The contents were stirred at room temperature for 7 hours. Silica gel column chromatography using hexane/ethyl acetate (0-100%) to give 7- [2- (tert-butoxycarbonylamino) ethyl ] ethyl]-2-chloro-pyrrolo [2,3-d]Pyrimidine-6-carboxylic acid (0.545 g).¹HNMR(d6-DMSO)δppm 9.11 (s,1H),7.39(s,1H),4.38(m,2H),4.15(m,2H),1.48(m,9H)。LCMS (ESI)341(M+H)。

Example 6

Synthesis of methyl 7- [2- (tert-butoxycarbonylamino) ethyl ] -2-chloro-pyrrolo [2,3-d ] pyrimidine-6-carboxylate, Compound 6

To the 2-chloro-7-propyl-pyrrolo [2,3-d ] from the previous step]To a solution of pyrimidine-6-carboxylic acid (0.545g, 0.00156 mol) in toluene (3.5mL) and MeOH (1mL) was added TMS-diazomethane (1.2 mL). After stirring at room temperature overnight, excess TMS-diazomethane was quenched with acetic acid (3mL) and the reaction was concentrated in vacuo. The residue was purified by silica gel column chromatography using hexane/ethyl acetate (0-70%) to give 7- [2- (tert-butoxycarbonylamino) ethyl ] as an off-white solid]-2-chloro-pyrrolo [2,3-d]Pyrimidine-6-carboxylic acid methyl ester (0.52 g). ¹HNMR(d6-DMSO)δppm 9.10(s,1H),7.45(s,1H),6.81(brs,1H)4.60 (m,2H),3.91(s,3H),3.29(m,2H),1.18(m,9H)LCMS(ESI)355(M+ H)。

Example 7

Synthesis of chlorotricyclo amide, Compound 7

To the solution containing 7- [2- (tert-butoxycarbonylamino) ethyl group from the previous step]-2-chloro-pyrrolo [2,3-d]To methyl pyrimidine-6-carboxylate (0.50g, 0.0014 mol) in dichloromethane (2.0mL) was added TFA (0.830 mL). The contents were stirred at room temperature for 1 hour. Concentration in vacuo afforded the crude amino ester, which was suspended in toluene (5mL) and henniger base (0.5 mL). The contents were heated under reflux for 2 hours. Concentration followed by silica gel column chromatography using hexane/ethyl acetate (0-50%) gave the desired chlorotricycloamide (0.260 g).¹HNMR(d6-DMSO)δppm 9.08(s,1H),8.48(brs,1H),7.21(s,1H)4.33(m,2H),3.64(m,2H)。 LCMS(ESI)223(M+H)。

Example 8

Synthesis of chloro-N-methyltricyclic acid amide, Compound 8

To a solution of chlorotricyclic lactam compound 7(185mg, 0.00083 mol) in DMF (2.0mL) was added sodium hydride (55% suspension in oil, 52 mg). After stirring for 15 min, iodomethane (62 μ L, 1.2 eq). The contents were stirred at room temperature for 30 minutes. Methanol (5mL) was added followed by saturated NaHCO₃Followed by the addition of ethyl acetate. The organic layer was separated, dried over magnesium sulfate and concentrated in vacuo to give the N-methylated amide in quantitative yield.¹HNMR(d6-DMSO)δppm 9.05(s,1H),7.17(s,1H)4.38(m,2H),3.80 (m,2H),3.05(s,3H)。LCMS(ESI)237(M+H)。

Example 9

Synthesis of 1-methyl-4- (6-nitro-3-pyridyl) piperazine, Compound 9

To 5-bromo-2-nitropyridine (4.93g, 24.3 mmol) in DMF (20mL) was added N-methylpiperazine (2.96g, 1.1eq) followed by DIPEA (4.65mL, 26.7 mmol). The contents were heated at 90 ℃ for 24 hours. After addition of ethyl acetate (200mL), water (100mL) was added and the layers were separated. Drying followed by concentration gave the crude product, which was purified by silica gel column chromatography using (0-10%) DCM/methanol.¹HNMR(d6-DMSO)δppm 8.26(s,1H),8.15(1H,d,J＝9.3Hz),7.49(1H,d,J＝9.4Hz),3.50(m, 4H),2.49(m,4H),2.22(s,3H)。

Example 10

Synthesis of 5- (4-methylpiperazin-1-yl) pyridin-2-amine, Compound 10

To ethyl acetate (100mL) containing 1-methyl-4- (6-nitro-3-pyridyl) piperazine (3.4g) and ethanol (100mL) was added 10% Pd/C (400mg) and the reaction was then stirred under hydrogen (10 psi) overnight. Warp CELITE^TMAfter filtration, the solvent was evaporated and the crude product was purified by silica gel column chromatography using DCM/7N ammonia in MeOH (0-5%) to give 5- (4-methylpiperazin-1-yl) pyridin-2-amine (2.2 g).¹HNMR(d6-DMSO)δppm 7.56(1H, d,J＝3Hz),7.13(1H,m),6.36(1H,d,J＝8.8Hz),5.33(brs,2H),2.88 (m,4H),2.47(m,4H),2.16(s,3H)。

Example 11

Synthesis of tert-butyl 4- (6-amino-3-pyridyl) piperazine-1-carboxylate, Compound 11

This compound is prepared as described in WO 2010/020675a 1.

Example 12

Synthesis of tert-butyl N- [2- (benzyloxycarbonylamino) -3-methyl-butyl ] carbamate, Compound 12

To a solution containing N- [1- (hydroxymethyl) -2-methyl-propyl group cooled to 0 DEG C ]To benzyl carbamate (11.0g, 0.0464 mol) in dioxane (100mL) was added diphenyl azidophosphate (10.99 mL, 1.1eq) followed by DBU (8.32mL, 1.2 eq). The contents were warmed to room temperature and stirred for 16 hours. After addition of ethyl acetate (300mL) and water (100mL), the organic layer was separated and saturated NaHCO₃(100mL) washing. The organic layer was then dried (magnesium sulfate) and concentrated in vacuo. To DMSO (100mL) containing this intermediate was added sodium azide (7.54g) and then the contents were heated to 90 ℃ for 2 hours. After addition of ethyl acetate and water, the layers were separated. The organic layer was dried over magnesium sulfate and then concentrated in vacuo to give an oil, which was purified by silica gel column chromatography using hexane/ethyl acetate (0-70%) to give 6.9g of N- [1- (azidomethyl) -2-methyl-propyl ] as a colorless oil]Benzoic acid methyl ester.

To N- [1- (azidomethyl) -2-methyl-propyl ] carbamic acid benzyl ester (6.9g, 0.0263 mol) in THF (100mL) was added triphenylphosphine (7.59g, 1.1 eq). The contents were stirred for 20 hours. After water (10mL) was added and stirred for another 6 hours, ethyl acetate was added and the layers were separated. After drying over magnesium sulfate and concentration in vacuo, the crude product was purified by silica gel column chromatography using DCM/MeOH (0-10%) to give benzyl N- [1- (aminomethyl) -2-methyl-propyl ] carbamate as a yellow oil.

To a mixture containing N- [1- (aminomethyl) -2-methyl-propyl]To benzyl carbamate (4.65g, 0.019 mol) in THF (70mL) was added 2N NaOH (20mL) followed by di-tert-butyl dicarbonate (5.15g, 1.2 eq). After stirring for 16 hours, ethyl acetate was added and the layers were separated. After drying over magnesium sulfate and concentration in vacuo, the crude product was purified using hexane/ethyl acetate (0-40%) over a silica gel column to give intermediate A, N- [2- (benzyloxycarbonylamino) -3-methyl-butyl]Carbamic acid tert-butyl ester (6.1 g).¹HNMR (600MHz, chloroform-d) δ ppm 0.89(d, J ═ 6.73Hz,3H)0.92(d, J ═ 6.73Hz,3H)1.38(s,9H)1.70-1.81(m, 1H) 3.18(d, J ═ 5.56Hz,2H)3.47-3.60(m,1H)4.76(s,1H)4.89(d, J ═ 7.90Hz,1H)5.07(s,2H)7.25-7.36(m, 5H). LCMS (ESI)337(M + H).

Example 13

Synthesis of tert-butyl N- [2- (benzyloxycarbonylamino) -4-methyl-pentyl ] carbamate, Compound 13

To a solution of benzyl N- [1- (hydroxymethyl) -3-methyl-butyl ] carbamate (6.3g, 0.025 mole) in DCM (100mL) at 0 ℃ was added diisopropylethylamine (5.25mL, 1.2eq) followed by methanesulfonyl chloride (2.13mL, 1.1 eq). After stirring for 3 hours, water (100mL) was added and the organic layer was separated. After drying over magnesium sulfate and concentration in vacuo, the crude [2- (benzyloxycarbonylamino) -4-methyl-pentyl ] methanesulfonate was obtained and used directly in the next step.

To DMF (50mL) containing crude [2- (benzyloxycarbonylamino) -4-methyl-pentyl ] methanesulfonate from the above reaction was added 2.43g of sodium azide. The reaction mixture was then heated to 85 ℃ and held for 3 hours. After cooling, ethyl acetate (300mL) and water were added. The organic layer was separated, dried over magnesium sulfate and then concentrated in vacuo to give crude benzyl N- [1- (azidomethyl) -3-methyl-butyl ] carbamate. To this crude intermediate was added THF (100 mL), followed by 7.21g of triphenylphosphine and stirring under nitrogen for 16 h. After water (10 mL) was added and stirred for another 6 hours, ethyl acetate was added and the layers were separated. After drying over magnesium sulfate and concentration in vacuo, the crude product was column-separated using DCM/MeOH (0-10%) to give methyl N- [1- (aminomethyl) -3-methyl-butyl ] carbamate (4.5 g).

To a mixture containing N- [1- (aminomethyl) -3-methyl-butyl]To benzyl carbamate (4.5g, 0.018 mole) in THF (60mL) was added 2N NaOH (18mL), followed by di-tert-butyl dicarbonate (4.19g, 1.07 eq). After stirring for 16 hours, ethyl acetate was added and the layers were separated. By sulfuric acidAfter drying of the magnesium and concentration in vacuo, the crude product was used in the next step.¹HNMR (600MHz, chloroform-d) δ ppm 0.89(d, J ═ 6.73Hz,6H)1.25-1.34(m,1H)1.39(s,9H) 1.57-1.71(m,2H)3.04-3.26(m,2H)3.68-3.80(m,1H)4.72-4.89 (m,2H)5.06(s,2H)7.25-7.38(m, 5H). LCMS (ESI)351(M + H).

Example 14

Synthesis of tert-butyl N- [ (2R) -2- (benzyloxycarbonylamino) -3-methyl-butyl ] carbamate, Compound 14

Compound 14 was synthesized from benzyl N- [ (1R) -1- (hydroxymethyl) -2-methyl-propyl ] carbamate using analogous synthetic procedures as described for compound 13. Analytical data (NMR and mass spectrum) are consistent with compound 12.

Example 15

Synthesis of tert-butyl N- [ (2S) -2- (benzyloxycarbonylamino) -3-methyl-butyl ] carbamate, Compound 15

Compound 15 was synthesized from benzyl N- [ (1S) -1- (hydroxymethyl) -2-methyl-propyl ] carbamate using analogous synthetic procedures as described for compound 13. Analytical data (NMR and mass spectrum) are consistent with compound 12.

Example 16

Synthesis of tert-butyl N- [ (1S) -1- (aminomethyl) -2-methyl-propyl ] carbamate, Compound 16

To a solution of N- [ (1S) -1- (hydroxymethyl) -2-methyl-propyl ] carbamate tert-butyl carbamate (6.3g, 0.025 mole) in THF (100mL) was added diisopropylethylamine (5.25mL, 1.2eq) followed by methanesulfonyl chloride (2.13mL, 1.1eq) at 0 ℃. After stirring for 3 hours, water (100mL) was added and the organic layer was separated. After drying over magnesium sulfate and concentration in vacuo, the crude [ (2S) -2- (tert-butoxycarbonylamino) -3-methyl-butyl ] methanesulfonate was used directly in the next step.

To DMSO (50mL) containing crude [ (2S) -2- (tert-butoxycarbonylamino) -3-methyl-butyl ] methanesulfonate from the above reaction was added sodium azide (2.43 g). The reaction mixture was then heated to 85 ℃ for 3 hours. After cooling, ethyl acetate (300mL) and water were added. The organic layer was separated, dried over magnesium sulfate and then concentrated in vacuo to give crude benzyl N- [1- (azidomethyl) -3-methyl-butyl ] carbamate. To this crude intermediate was added THF (100 mL), followed by triphenylphosphine (7.21g) and the reaction was stirred under nitrogen for 16 h. After water (10mL) was added and stirred for another 6 hours, ethyl acetate was added and the layers were separated. After drying over magnesium sulfate and concentration in vacuo, the crude product was purified by silica gel column chromatography using DCM/MeOH (0-10%) to give benzyl N- [1- (aminomethyl) -3-methyl-butyl ] carbamate (4.5 g). LCMS (ESI)203(M + H).

Example 17

Synthesis of tert-butyl N- [ (1R) -1- (aminomethyl) -2-methyl-propyl ] carbamate, Compound 17

Compound 17 was synthesized from tert-butyl N- [ (1R) -1- (hydroxymethyl) -2-methyl-propyl ] carbamate using a similar synthetic sequence as described for compound 16. Analytical data (NMR and mass spectra) are consistent with compound 16.

Example 18

Synthesis of tert-butyl N- [ (2S) -2- (benzyloxycarbonylamino) -4-methyl-pentyl ] carbamate, Compound 18

Compound 18 was synthesized from benzyl N- [ (1S) -1- (hydroxymethyl) -3-methyl-butyl ] carbamate using a similar synthetic sequence as described for compound 13. The analytical data (NMR and mass spectrum) are consistent with compound 13.

Example 19

Synthesis of tert-butyl N- [ (2S) -2- (benzyloxycarbonylamino) -2-phenyl-ethyl ] carbamate, Compound 19

Compound 19 is prepared from N- [ (1S) -2-hydroxy-1-phenyl-ethyl]Benzyl carbamate, synthesized using a similar synthetic sequence as described for compound 13.¹HNMR(600MHz, DMSO-d₆)δppm 1.20-1.33(m,9H)3.11(t,J＝6.29Hz,2H)4.59- 4.68(m,1H)4.88-5.01(m,2H)6.81(t,J＝5.42Hz,1H)7.14-7.35(m, 10H)7.69(d,J＝8.49Hz,1H)。LCMS(ESI)371(M+H)。

Example 20

Synthesis of tert-butyl N- [ (2S) -2- (benzyloxycarbonylamino) -3-methyl-pentyl ] carbamate, Compound 20

Compound 20 is prepared from N- [ (1S) -1- (hydroxymethyl) -2-methyl-butyl]Benzyl carbamate, synthesized using a similar synthetic sequence as described for compound 13.¹HNMR (600MHz, chloroform-d) δ ppm 0.85-0.92(m,6H)1.05-1.15(m,1H)1.35-1.41(m, 9H) 1.45-1.56(m,2H)3.14-3.24(m,2H)3.54-3.64(m,1H)4.78(s, 1H)4.96(d, J ═ 7.91Hz,1H)5.06(s,2H)7.27-7.37(m, 5H). LCMS (ESI)351(M + H).

Example 21

Synthesis of tert-butyl N- [ (2S) -2- (benzyloxycarbonylamino) -3, 3-dimethyl-butyl ] carbamate, Compound 21

Compound 21 was synthesized from benzyl N- [ (1S) -1- (hydroxymethyl) -2, 2-dimethyl-propyl ] carbamate using a similar synthetic sequence as described for compound 13. LCMS (ESI) 351.

Example 22

Synthesis of tert-butyl N- [ [1- (benzyloxycarbonylamino) cyclohexyl ] methyl ] carbamate, Compound 22

To N- [1- (aminomethyl) cyclohexyl]To a solution of benzyl carbamate (10.0g, 0.0381 mol) in THF (150mL) was added di-tert-butyl dicarbonate (9.15g, 1.1eq) and the contents were stirred at room temperature for 16 hours. Ethyl acetate and water were then added. The organic layer was separated, dried over magnesium sulfate and then concentrated in vacuo to give N- [ [1- (benzyloxycarbonylamino) cyclohexyl ] amino]Methyl radical]Carbamic acid tert-butyl ester (13.1 g).¹HNMR(600MHz,DMSO-d₆)δ ppm 0.92-1.54(m,17H)1.76-2.06(m,2H)3.09(d,J＝6.15Hz,2H) 4.92(s,2H)6.63(d,J＝17.27Hz,1H)7.16-7.49(m,6H)。LCMS(ESI) 363(M+H)。

Example 23

Synthesis of tert-butyl N- [ [1- (benzyloxycarbonylamino) cyclopentyl ] methyl ] carbamate, Compound 23

Tert-butyl N- [ [1- (benzyloxycarbonylamino) cyclopentyl ] methyl ] carbamate was synthesized in a similar manner to tert-butyl N- [ [1- (benzyloxycarbonylamino) cyclohexyl ] methyl ] carbamate. LCMS (ESI)349(M + H).

Example 24

Synthesis of 2-nitro-5- [4- (1-piperidinyl) -1-piperidinyl ] pyridine, Compound 24

To 5-bromo-2-nitropyridine (1.2g, 5.9mmol) in DMSO (4mL) was added 1- (4-piperidinyl) piperidine (1.0g, 5.9mmol) and triethylamine (0.99mL, 7.1 mmol). The contents were heated to 120 ℃ in a CEM Discovery microwave system for 3 hours. The crude reaction was then purified by silica gel column chromatography using DCM/MeOH (0-20%) to give 2-nitro-5- [4- (1-piperidinyl) -1-piperidinyl) as an oil ]Pyridine (457 mg).¹HNMR (600MHz,DMSO-d₆)δppm 1.26-1.36(m,2H)1.43(m,6H)1.76(m, 2H)2.37(m,5H)2.94(t,J＝12.74Hz,2H)4.06(d,J＝13.47Hz,2H) 7.41(dd,J＝9.37,2.64Hz,1H)8.08(d,J＝9.37Hz,1H)8.20(d,J＝2.64 Hz,1H)。

Example 25

Synthesis of 5- [4- (1-piperidinyl) -1-piperidinyl ] pyridin-2-amine, Compound 25

5- [4- (1-piperidinyl) -1-piperidinyl]Pyridin-2-amine was prepared in a similar manner to that used in the synthesis of 5- (4-methylpiperazin-1-yl) pyridin-2-amine.¹HNMR(600MHz,DMSO-d₆)δ ppm 1.13-1.37(m,6H)1.40-1.63(m,6H)1.71(m,2H),2.24(m,1H) 2.43(m,2H)3.33(d,J＝12.30Hz,2H)5.31(s,2H)6.33(d,J＝8.78Hz, 1H)7.10(dd,J＝8.78,2.93Hz,1H)7.55(d,J＝2.64Hz,1H)。LCMS (ESI)261(M+H)。

Example 26

Synthesis of 4- [1- (6-nitro-3-pyridyl) -4-piperidyl ] morpholine, Compound 26

4- [1- (6-Nitro-3-pyridyl) -4-piperidyl]Morpholine can be used for synthesizing 2-nitro-5- [4- (1-piperadinyl) -1-piperidyl]Synthesized in a similar manner as used for pyridine.¹HNMR(600MHz, DMSO-d₆)δppm 1.41(m,2H)1.82(m,2H)2.42(m,5H)2.98(t, J＝12.44Hz,2H)3.52(s,4H)4.04(d,J＝12.88Hz,2H)7.42(d,J＝9.37 Hz,1H)8.08(d,J＝9.08Hz,1H)8.21(s,1H)。

Example 27

Synthesis of 5- (4-morpholino-1-piperidinyl) pyridin-2-amine, Compound 27

5- (4-Morpholino-1-piperidinyl) pyridin-2-amine is prepared in a similar manner to that used in the synthesis of 5- (4-methylpiperazin-1-yl) pyridin-2-amine.¹HNMR(600MHz,DMSO-d₆)δppm 1.34-1.52(m,2H)1.78(m,2H)2.14(m,1H)2.43(m,4H)3.32(d, J＝12.30Hz,4H)3.47-3.59(m,4H)5.32(s,2H)6.34(d,J＝8.78Hz,1 H)7.11(dd,J＝8.93,2.78Hz,1H)7.47-7.62(m,1H)。LCMS(ESI)263 (M+H)。

Example 28

Synthesis of 4- [1- (6-nitro-3-pyridyl) -4-piperidyl ] thiomorpholine, Compound 28

4- [1- (6-Nitro-3-pyridyl) -4-piperidyl]Thiomorpholine for synthesizing 2-nitro-5- [4- (1-piperidyl) -1-piperidyl]Synthesized in a similar manner as used for pyridine.¹HNMR(600MHz, DMSO-d₆)δppm 1.40-1.52(m,2H)1.71(m,2H)2.49-2.55(m,4H) 2.56-2.63(m,1H)2.68-2.75(m,4H)2.88-2.98(m,2H)4.09(d, J＝13.18Hz,2H)7.42(dd,J＝9.22,3.07Hz,1H)8.08(d,J＝9.37Hz,1H) 8.20(d,J＝3.22Hz,1H)。

Example 29

Synthesis of 5- (4-Thiomolinyl-1-piperidinyl) pyridin-2-amine, Compound 29

5- (4-Thiomolinyl-1-piperidinyl) pyridin-2-amine is prepared in a similar manner to that used in the synthesis of 5- (4-methylpiperazin-1-yl) pyridin-2-amine. ¹HNMR(600MHz,DMSO-d₆)δ ppm 1.47-1.59(m,2H)1.65(m,2H)2.22-2.38(m,1H)2.50-2.59 (m,6H)2.68-2.82(m,4H)3.33(d,J＝12.00Hz,2H)5.31(s,2H)6.33 (d,J＝9.08Hz,1H)7.10(dd,J＝8.78,2.93Hz,1H)7.55(d,J＝2.64Hz,1 H)。LCMS(ESI)279(M+H)。

Example 30

Synthesis of 2-nitro-5- (1-piperidinyl) pyridine, Compound 30

Synthesis of 2-nitro-5- (1-piperidinyl) pyridine and use thereof]Synthesized in a similar manner in pyridine.¹HNMR(600MHz,DMSO-d₆)δppm 1.56 (m,6H)3.49(d,J＝4.39Hz,4H)7.30-7.47(m,1H)8.02-8.12(m,1H) 8.15-8.26(m,1H)。

Example 31

Synthesis of 5- (1-piperidinyl) pyridin-2-amine, Compound 31

5- (1-piperidinyl) pyridin-2-amine is prepared in a similar manner to that used in the synthesis of 5- (4-methylpiperazin-1-yl) pyridin-2-amine.¹HNMR(600MHz,DMSO-d₆)δppm 1.39-1.46 (m,2H)1.51-1.62(m,4H)2.75-2.92(m,4H)5.30(s,2H)6.34(d, J＝8.78Hz,1H)7.09(dd,J＝8.78,2.93Hz,1H)7.54(d,J＝2.93Hz,1 H)。LCMS(ESI)178(M+H)。

Example 32

Synthesis of 4- (6-nitro-3-pyridyl) thiomorpholine, Compound 32

4- (6-nitro-3-pyridyl) thiomorpholine for the synthesis of 2-nitro-5- [4- (1-piperidyl) -1-piperazinyl]Synthesized in a similar manner as used for pyridine.¹HNMR(600MHz,DMSO-d₆)δ ppm 2.56-2.69(m,4H)3.79-3.92(m,4H)7.43(dd,J＝9.22,3.07Hz, 1H)8.10(d,J＝9.37Hz,1H)8.20(d,J＝2.93Hz,1H)。

Example 33

Synthesis of 5-Thiomorpholinopyridin-2-amine, Compound 33

5-Thiomolinylpyridin-2-amine was prepared in a similar manner to that used in the synthesis of 5- (4-methylpiperazin-1-yl) pyridin-2-amine.¹HNMR(600MHz,DMSO-d₆)δppm 2.59-2.73 (m,4H)3.04-3.20(m,4H)5.41(s,2H)6.35(d,J＝8.78Hz,1H)7.10 (dd,J＝8.78,2.93Hz,1H)7.57(d,J＝2.64Hz,1H)。LCMS(ESI)196(M +H)。

Example 34

Synthesis of tert-butyl (4R) -5- (6-nitro-3-pyridyl) -2, 5-diazabicyclo [2.2.1] heptane-2-carboxylate, Compound 34

(4R) -5- (6-Nitro-3-pyridinyl) -2, 5-diazabicyclo [2.2.1]Heptane-2-carboxylic acid tert-butyl ester for the synthesis of 2-nitro-5- [4- (1-piperidinyl) -1-piperidinyl]Synthesized in a similar manner as used for pyridine.¹HNMR(600MHz,DMSO-d₆)δppm 1.33(d,J＝32.21Hz,11H) 1.91(m,2H)3.15(d,J＝10.25Hz,1H)3.58(m,1H)4.46(m,1H)4.83 (s,1H)7.16(s,1H)7.94(s,1H)8.05-8.16(m,1H)。

Example 35

Synthesis of tert-butyl (4R) -5- (6-amino-3-pyridyl) -2, 5-diazabicyclo [2.2.1] heptane-2-carboxylate, Compound 35

(4R) -5- (6-amino-3-pyridinyl) -2, 5-diazabicyclo [2.2.1]Tert-butyl heptane-2-carboxylate was prepared in a similar manner to that used in the synthesis of 5- (4-methylpiperazin-1-yl) pyridin-2-amine.¹HNMR(600MHz,DMSO-d₆)δppm 1.31(d,J＝31.91Hz,11H)1.83 (m,2H)2.71-2.82(m,1H)3.44(m,1H)4.30(d,2H)5.08(s,2H)6.35 (d,J＝8.78Hz,1H)6.77-6.91(m,1H)7.33(s,1H)。LCMS(ESI)291 (M+H)。

Example 36

Synthesis of N, N-dimethyl-1- (6-nitro-3-pyridyl) piperidin-4-amine, Compound 36

Synthesis of 2-nitro-5- [4- (1-piperidinyl) -1-piperidinyl-4-amine from N, N-dimethyl-1- (6-nitro-3-pyridinyl) piperidin-4-amine]Synthesized in a similar manner as used for pyridine.¹HNMR(600 MHz,DMSO-d₆)δppm 1.30-1.45(m,2H)1.79(m,2H)2.14(s,6H) 2.33(m,1H)2.92-3.04(m,2H)4.03(d,J＝13.76Hz,2H)7.42(dd, J＝9.22,3.07Hz,1H)8.04-8.11(m,1H)8.21(d,J＝2.93Hz,1H)。

Example 37

Synthesis of 5- [4- (dimethylamino) -1-piperidinyl ] pyridin-2-amine, Compound 37

5- [4- (dimethylamino) -1-piperidinyl group]Pyridin-2-amine was prepared in a similar manner to that used in the synthesis of 5- (4-methylpiperazin-1-yl) pyridin-2-amine.¹HNMR(600MHz,DMSO-d₆)δ ppm 1.35-1.50(m,2H)1.69-1.81(m,2H)2.00-2.10(m,1H)2.11- 2.22(s,6H)3.17-3.36(m,4H)5.19-5.38(s,2H)6.34(d,J＝8.78Hz, 1H)7.10(dd,J＝8.78,2.93Hz,1H)7.55(d,J＝2.63Hz,1H)。LCMS (ESI)221(M+H)。

Example 38

Synthesis of 4- (6-nitro-3-pyridinyl) morpholine, Compound 38

4- (6-Nitro-3-pyridyl) morpholine was synthesized in a similar manner to that used in the synthesis of 2-nitro-5- [4- (1-piperidinyl) -1-piperidiny l ] pyridine.

Example 39

Synthesis of 5-morpholinopyridin-2-amine, Compound 39

5-Morpholinopyridin-2-amine was prepared in a similar manner to that used in the synthesis of 5- (4-methylpiperazin-1-yl) pyridin-2-amine.¹HNMR (600MHz, chloroform-d) δ ppm 2.91-3.00(m, 4H)3.76-3.84(m,4H)4.19(br.s.,2H)6.45(d, J ═ 8.78Hz,1H)7.12 (dd, J ═ 8.78,2.93Hz,1H)7.72(d, J ═ 2.93Hz, 1H).

Example 40

Synthesis of 5- (4-isobutylpiperazin-1-yl) pyridin-2-amine, Compound 40

Synthesis of 1-isobutyl-4- (6-nitro-3-pyridyl) piperazine and 2-nitro-5- [4- (1-piperidyl) -1-piperidyl]Synthesized in a similar manner in pyridine, which is then converted to 5- (4-isobutylpiperazin-1-yl) pyridin-2-amine in a similar manner to that used in the synthesis of 5- (4-methylpiperazin-1-yl) pyridin-2-amine.¹HNMR (600MHz, chloroform-d) δ ppm 0.88(d, J ═ 6.73Hz,6H) 1.71-1.84 (m,1H)2.10(d, J ═ 7.32Hz,2H)2.46-2.58(m,4H)2.97-3.07 (m,4H)4.12(s,2H)6.45(d, J ═ 8.78Hz,1H)7.14(dd, J ═ 8.78,2.93Hz, 1H)7.75(d, J ═ 2.93Hz, 1H). LCMS (ESI)235(M + H).

EXAMPLE 41

Synthesis of 5- (4-isopropylpiperazin-1-yl) pyridin-2-amine, Compound 41

Synthesis of 2-nitro-5- [4- (1-piperidyl) -1-piperidyl from 1-isopropyl-4- (6-nitro-3-pyridyl) piperazine]Synthesized in a similar manner in pyridine, followed byConversion to 5- (4-isopropylpiperazin-1-yl) pyridin-2-amine is carried out in a similar manner to that used in the synthesis of 5- (4-methylpiperazin-1-yl) pyridin-2-amine.¹HNMR (600MHz, chloroform-d) δ ppm 1.06(d, J ═ 6.44Hz,6H) 2.59-2.75 (m,5H)2.97-3.10(m,4H)4.13(s,2H)6.45(d, J ═ 8.78Hz,1H) 7.15(dd, J ═ 9.08,2.93Hz,1H)7.76(d, J ═ 2.93Hz, 1H). LCMS (ESI) 221(M + H).

Example 42

Synthesis of 5- [ (2R,6S) -2, 6-dimethylmorpholin-4-yl ] pyridin-2-amine, Compound 42

(2S,6R) -2, 6-dimethyl-4- (6-nitro-3-pyridyl) morpholine for the synthesis of 2-nitro-5- [4- (1-piperidyl) -1-piperidyl]Synthesized in a similar manner to that used for pyridine, which is then converted to 5- [ (2R,6S) -2, 6-dimethylmorpholin-4-yl) in a similar manner to that used for the synthesis of 5- (4-methylpiperazin-1-yl) pyridin-2-amine]Pyridin-2-amine.¹HNMR (600MHz, chloroform-d) δ ppm 1.20(d, J ═ 6.44Hz,6H)2.27-2.39(m,2H)3.11-3.21(m, 2H) 3.70-3.84(m,2H)4.15(s,2H)6.45(d, J ═ 8.78Hz,1H)7.12(dd, J ═ 8.78,2.93Hz,1H)7.72(d, J ═ 2.63Hz, 1H). LCMS (ESI)208(M + H).

Example 43

Synthesis of 5- [ (3R,5S) -3, 5-dimethylpiperazin-1-yl ] pyridin-2-amine, Compound 43

(3S,5R) -3, 5-dimethyl-1- (6-nitro-3-pyridyl) piperazine for the synthesis of 2-nitro-5- [4- (1-piperidyl) -1-piperidyl]Synthesis in a similar manner as in pyridine, followed by its conversion to 5- [ (3R,5S) -3, 5-dimethylpiperazin-1-yl) in a similar manner as used in the synthesis of 5- (4-methylpiperazin-1-yl) pyridin-2-amine]Pyridin-2-amine.¹HNMR (600MHz, chloroform-d) δ ppm 1.09(d, J ═ 6.44Hz,6H)2.20(t, J ═ 10.83Hz,2H)2.95-3.08 (m,2H)3.23(dd, J ═ 11.71,2.05Hz,2H)4.13(s,2H)6.45(d, J ═ 8.78Hz,1H) 7.14(dd, J ═ 8.78,2.93Hz,1H)7.73(d, J ═ 6.44Hz,6H) ＝2.63Hz,1H)。LCMS (ESI)207(M+H)。

Example 44

Synthesis of Compound 44

N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] -3-methyl-butyl ] carbamic acid tert-butyl ester

A solution of intermediate A in ethanol (100mL) was hydrogenated under 30psi of hydrogen in a pressure gauge for 7 hours using 10% Pd/C (0.7 g). The reaction mixture is passed over CELITE^TMAfter filtration, the organic layer was concentrated in vacuo to give tert-butyl N- (2-amino-3-methyl-butyl) carbamate (3.8 g).

To a solution of 5-bromo-2, 4-dichloro-pyrimidine (7.11g, 0.0312 mol) in ethanol (100mL) was added diisopropylethylamine (5.45mL, 1.0eq) and tert-butyl N- (2-amino-3-methyl-butyl) carbamate (6.31g, 0.0312 mol). The reaction mixture was stirred at room temperature for 20 hours. After concentration in vacuo, ethyl acetate and water were added. The organic layer was separated, dried over magnesium sulfate and then concentrated in vacuo. The crude product was purified by silica gel column chromatography using hexane/ethyl acetate (0-30%) to give N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino group]-3-methyl-butyl](iii) carbamic acid tert-butyl ester.¹HNMR(600MHz,DMSO-d₆)δppm 0.77- 0.85(d,J＝6.5Hz,3H)0.87(d,J＝6.73Hz,3H)1.31-1.39(m,9H)1.82 -1.93(m,1H)2.94(d,J＝5.56Hz,1H)3.08-3.22(m,2H)3.98(d, J＝8.20Hz,1H)6.96(d,J＝8.78Hz,1H)8.21(s,1H)。LCMS(ESI)393 (M+H)。

N- [2- [ 2-chloro-6- (diethoxymethyl) pyrrolo [2,3-d ] pyrimidin-7-yl ] -3-methyl-butyl ] carbamic acid tert-butyl ester

N- [2- [ 2-chloro-6- (diethoxymethyl) pyrrolo [2,3-d]Pyrimidin-7-yl ]-3-methyl-butyl]By reacting tert-butyl carbamate with N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino]-3-methyl-butyl]The tert-butyl carbamate is subjected to a treatment such as that directed against N- [2- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl]Amino group]Ethyl radical]Sonogoshira conditions as described for tert-butyl carbamate, followed by synthesis of N- [2- [ 2-chloro-6- (diethoxymethyl) pyrrolo [2,3-d]Pyrimidin-7-yl]Ethyl radical]Synthesis by treatment with TBAF as described in tert-butyl carbamate.¹HNMR(600MHz, DMSO-d₆)δppm 1.11(d,J＝6.44Hz,3H)1.18(t,J＝7.03Hz,6H)1.21- 1.26(m,12H)2.88(br.s.,1H)3.43-3.78(m,6H)3.97-4.08(m,1H) 5.61(s,1H)6.65(s,1H)6.71-6.78(m,1H)8.87(s,1H)。LCMS(ESI) 441(M+H)。

7- [1- [ (tert-Butoxycarbonylamino) methyl ] -2-methyl-propyl ] -2-chloro-pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid

To N- [2- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl group]Amino group]Ethyl radical]TBAF was added to a solution of tert-butyl carbamate in THF and the contents were heated at reflux for 3 hours. Ethyl acetate and water were then added and the organic layer was separated, dried over magnesium sulfate and then concentrated in vacuo. To this crude reaction was added acetic acid/water (9:1) and the contents were stirred at room temperature for 12 hours. After concentration in vacuo, saturated NaHCO was added₃And ethyl acetate. The organic layer was separated, dried and then concentrated in vacuo. The crude reaction product thus obtained was dissolved in DMF, followed by addition of oxone and stirring of the contents for 3 hours. After addition of ethyl acetate, the reaction mixture is passed over CELITE ^TMFiltered and concentrated in vacuo. The crude product was subjected to silica gel column chromatography using hexane/ethyl acetate (0-100%) to give 7- [1- [ (tert-butoxycarbonylamino) methyl ] ethyl ester]-2-methyl-propyl]-2-chloro-pyrrolo [2,3-d]Pyrimidine-6-carboxylic acid.¹HNMR(600MHz, DMSO-d₆)δppm 0.85(d,J＝7.03Hz,3H)0.97(d,J＝6.73Hz,3H)1.52 (s,9H)1.99-2.23(m,1H)3.98(dd,J＝14.05,3.51Hz,1H)4.47-4.71 (m,2H)7.47(s,1H)9.17(s,1H)。LCMS(ESI)383(M+H)。

Compound 44

To a solution containing 7- [1- [ (tert-butoxycarbonylamino) methyl group]-2-methyl-propyl]-2-chloro-pyrrolo [2,3-d]DIC (32.7mg) and DMAP (10mg) were added to pyrimidine-6-carboxylic acid (0.050g, 0.00013 mol) in DCM (1.5 mL). The contents were stirred for 2 hours. Trifluoroacetic acid (0.4mL) was then added and stirring was continued for an additional 30 minutes. Addition of saturated NaHCO₃After neutralizing the excess acid, ethyl acetate was added and the organic layer was separated, dried over magnesium sulfate and then concentrated in vacuo. The crude product was purified by silica gel column chromatography using hexane/ethyl acetate (0-100%) to give the product.¹HNMR(600MHz,DMSO-d₆)δppm 0.72(d,J＝6.73Hz,3H)0.97(d, J＝6.73Hz,3H)2.09-2.22(m,1H)3.57(dd,J＝13.18,4.98Hz,1H) 3.72(dd,J＝13.61,4.25Hz,1H)4.53(dd,J＝8.05,3.95Hz,1H)7.20(s, 1H)8.34(d,J＝4.98Hz,1H)9.08(s,1H)。LCMS(ESI)265(M+H)。

Example 45

Synthesis of Compound 45

Hydrogenation of compound 14 with 10% Pd/C provided intermediate N- [ (2R) -2-amino-3-methyl-butyl ] carbamic acid tert-butyl ester, which was then treated with 5-bromo-2, 4-dichloro-pyrimidine using similar reaction conditions as described for compound 44 to provide compound 45. The analytical data are consistent with those reported for the racemate (intermediate 1A).

Example 46

Synthesis of Compound 46

Hydrogenation of compound 15 with 10% Pd/C provided intermediate N- [ (2S) -2-amino-3-methyl-butyl ] carbamic acid tert-butyl ester, which was then treated with 5-bromo-2, 4-dichloro-pyrimidine using similar reaction conditions as described for compound 44 to provide compound 46. Analytical data (NMR and LCMS) are consistent with that reported for racemate 44.

Example 47

Synthesis of Compound 47

To a solution of compound 44(80mg, 0.00030 mol) in DMF (3mL) was added sodium hydride in 60% dispersion in oil (40 mg). After stirring for 15 min, iodomethane (37 μ L, 2eq) was added. The contents were stirred at room temperature for 30 minutes. Then saturated NaHCO was added₃Followed by ethyl acetate. The organic layer was dried over magnesium sulfate and then concentrated in vacuo to give the product.¹HNMR(600MHz,DMSO-d₆)δppm 0.74(d,J＝6.73Hz,3H)0.91(d, J＝6.73Hz,3H)2.04-2.20(m,1H)3.04(s,3H)3.69(dd,J＝13.76,1.17 Hz,1H)3.96(dd,J＝13.76,4.68Hz,1H)4.58(dd,J＝7.32,3.51Hz,1H) 7.16(s,1H)9.05(s,1H)。LCMS(ESI)279(M+H)。

Example 48

Synthesis of Compound 48

N- [ (2S) -2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] -4-methyl-pentyl ] carbamic acid tert-butyl ester

Hydrogenation of Compound 18 with 10% Pd/C in ethanol under a 50psi hydrogen cap in a manometer afforded N- [ (2S) -2-amino-4-methyl-pentyl](iv) carbamic acid tert-butyl ester, which is subsequently used with a compound directed against N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino]-3-methyl-butyl]Carbamic acid tert-butyl ester similar reaction conditions described for the reaction with 5-bromo-2, 4-dichloro-pyrimidine gave N- [ (2S) -2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ]-4-methyl-pentyl](iii) carbamic acid tert-butyl ester.¹HNMR (600 MHz, chloroform-d) δ ppm 0.91(d, J ═ 6.44Hz,3H)0.94(d, J ═ 6.44Hz,3H) 1.32-1.51(m,11H)1.55-1.67(m,1H)3.28(t, J ═ 5.86Hz,2H) 4.21-4.42 (m,1H)4.84(s,1H)5.84(d, J ═ 7.32Hz,1H)8.07(s, 1H). LCMS (ESI)407(M + H).

To N- [ (2S) -2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino under nitrogen]-4-methyl-pentyl]To a solution of tert-butyl carbamate (5.0g, 12.3 mmol) in toluene (36mL) and triethylamine (7.2mL) was added 3, 3-diethoxyprop-1-yne (2.8mL, 19.7 mmol), Pd₂(dba)₃(1.1g, 1.23 mmol) and triphenylarsine (3.8g, 12.3 mmol). The contents were heated to 70 ℃ for 24 hours. After cooling to room temperature, the reaction mixture is passed over CELITE^TMFiltered and then concentrated in vacuo. The crude product was purified by silica gel column chromatography using hexane/ethyl acetate (0-30%) to give (2S) -N2- [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl]-4-methyl-pentane-1, 2-diamine. LCMS (ESI)455(M + H).

7- [ (1S) -1- [ (tert-butoxycarbonylamino) methyl group]-3-methyl-butyl]-2-chloro-pyrrolo [2,3-d]Pyrimidine-6-carboxylic acid derivatives and their use as inhibitors of 7- [1- [ (tert-butoxycarbonylamino) methyl ] carbonyl]-2-methyl-propyl ]-2-chloro-pyrrolo [2,3-d]Pyrimidine-6-carboxylic acid was synthesized in a similar synthetic sequence.¹HNMR(600MHz,DMSO-d₆)δppm 0.88(d,J＝6.44Hz,3H)0.97(d, J＝6.44Hz,3H)1.47(s,9H)1.49-1.54(m,1H)1.56(t,J＝7.17Hz,2H) 3.98(dd,J＝13.91,3.07Hz,1H)3.76(dd,J＝13.31,4.13Hz,1H)4.38(d, J＝14.05Hz,1H)4.90(t,J＝7.17Hz,1H)7.41(s,1H)9.11(s,1H)。LCMS(M+H)397。

Compound 48 was synthesized using a similar synthetic sequence as described for compound 44.¹HNMR(600MHz,DMSO-d₆)δppm 0.82(d,J＝6.73Hz,3H)0.97(d, J＝6.44Hz,3H)1.34-1.46(m,1H)1.48-1.65(m,2H)3.40(dd, J＝13.32,5.42Hz,1H)3.76(dd,J＝13.47,4.10Hz,1H)4.76-4.92(m,1 H)7.17(s,1H)8.34(d,J＝5.27Hz,1H)9.04(s,1H)。LCMS(ESI)279 (M+H)。

Example 49

Synthesis of Compound 49

Compound 49 was synthesized in a similar manner as described for compound 47.¹HNMR(600 MHz,DMSO-d₆)δppm 0.82(d,J＝6.44Hz,3H)0.97(d,J＝6.44Hz,3H) 1.37-1.68(m,3H)3.04(s,3H)3.56(d,J＝13.47Hz,1H)4.00(dd, J＝13.32,4.25Hz,1H)4.82-4.94(m,1H)7.16(s,1H)9.03(s,1H)。 LCMS(ESI)293(M+H)。

Example 50

Synthesis of Compound 50

N- [ (2S) -2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] -3-methyl-pentyl ] carbamic acid tert-butyl ester

Hydrogenation of Compound 20 using 10% Pd/C under 50psi of hydrogen in a pressure vessel to give N- [ (2S) -2-amino-3-methyl-pentyl]Tert-butyl carbamate, its use and its use against N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino]-3-methyl-butyl]Similar reaction conditions as described for tert-butyl carbamate with 5-bromo-2, 4-dichloro-pyrimidine gave N- [ (2S) -2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino]-3-methyl-pentyl](iii) carbamic acid tert-butyl ester.¹HNMR (600MHz, chloroform-d) δ ppm 0.88-0.95(m,6H)1.11-1.20(m,1H)1.34(s,9H) 1.44-1.54 (m,1H)1.64-1.72(m,1H)3.17-3.27(m,1H)3.33-3.43(m, 1H) 4.11-4.21(m,1H)4.81(s,1H)5.92(d, J ═ 8.20Hz,1H)8.05(s, 1H). LCMS (ESI) 407.

N- [ (2S) -2- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] amino ] -3-methyl-pentyl ] carbamic acid tert-butyl ester

N- [ (2S) -2- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl]Amino group]-3-methyl-pentyl]Synthesis of (2S) -N2- [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl]-4-methyl-pentane-1, 2-diamine using similar experimental conditions.¹HNMR(600MHz,DMSO-d₆)δppm 0.76-0.89(m,6H)1.03(q, J＝7.22Hz,3H)1.10-1.17(m,3H)1.25-1.42(m,11H)1.59-1.73(m, 1H)3.35-3.47(m,4H)3.51-3.73(m,2H)3.99-4.11(m,1H)5.52- 5.56(m,1H)6.76-7.03(m,2H)8.12-8.23(m,1H)。LCMS(ESI)455 (M+H)。

7- [ (1S) -1- [ (tert-butoxycarbonylamino) methyl ] -2-methyl-butyl ] -2-chloro-pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid

7- [ (1S) -1- [ (tert-butoxycarbonylamino) methyl group]-2-methyl-butyl]-2-chloro-pyrrolo [2,3-d]Pyrimidine-6-carboxylic acid derivatives and their use as inhibitors of 7- [1- [ (tert-butoxycarbonylamino) methyl ] carbonyl]-2-methyl-propyl]-2-chloro-pyrrolo [2,3-d]Pyrimidine-6-carboxylic acid was synthesized in a similar synthetic sequence.¹HNMR(600MHz,DMSO-d₆)δppm 0.80(t,J＝7.47Hz,3H)0.86(d, J＝7.03Hz,3H)1.06-1.30(m,2H)1.48(s,9H)1.79-1.96(m,1H) 3.95(dd,J＝14.05,3.22Hz,1H)4.52(d,J＝14.35Hz,1H)4.61-4.73(m, 1H)7.43(s,1H)9.13(s,1H)。LCMS(ESI)397(M+H)。

Compound 50 was synthesized using a similar synthetic sequence as described for compound 44.¹HNMR(600MHz,DMSO-d₆)δppm 0.74(t,J＝7.32Hz,3H)0.89(d, J＝6.73Hz,3H)1.00-1.12(m,2H)1.82-1.94(m,1H)3.55(dd, J＝13.91,4.83Hz,1H)3.70(dd,J＝13.61,4.25Hz,1H)4.57(dd,J＝7.91, 4.10Hz,1H)7.17(s,1H)8.31(d,J＝5.27Hz,1H)9.05(s,1H)。LCMS (ESI)279(M+H)。

Example 51

Synthesis of Compound 51

Compound 51 was synthesized in a similar manner to compound 47.¹HNMR(600MHz, DMSO-d₆)δppm 0.77(t,J＝7.47Hz,3H)0.84(d,J＝6.73Hz,3H)1.07- 1.16(m,2H)1.82-1.95(m,1H)3.03(s,3H)3.68(d,J＝13.76Hz,1H) 3.96(dd,J＝13.76,4.39Hz,1H)4.59-4.70(m,1H)7.16(s,1H)9.04(s, 1H)。LCMS(ESI)293(M+H)。

Example 52

Synthesis of Compound 52

N- [ (2S) -2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] -3, 3-dimethyl-butyl ] carbamic acid tert-butyl ester

Hydrogenation of compound 21 using 10% Pd/C under 50psi hydrogen in a pressure vessel to give N- [ (2S) -2-amino-3, 3-dimethyl-butyl ] carbamic acid tert-butyl ester, which is then reacted with 5-bromo-2, 4-dichloro-pyrimidine using similar reaction conditions as described for tert-butyl N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] -3-methyl-butyl ] carbamate to give tert-butyl N- [ (2S) -2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] -3, 3-dimethyl-butyl ] carbamate. LCMS (ESI) 407(M + H).

N- [ (2S) -2- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] amino ] -3, 3-dimethyl-butyl ] carbamic acid tert-butyl ester

N- [ (2S) -2- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] amino ] -3, 3-dimethyl-butyl ] carbamic acid tert-butyl ester was synthesized using similar experimental conditions to those used in the synthesis of (2S) -N2- [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] -4-methyl-pentan-1, 2-diamine. LCMS (ESI)455(M + H).

7- [ (1S) -1- [ (tert-butoxycarbonylamino) methyl ] -2, 2-dimethyl-propyl ] -2-chloro-pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid

7- [ (1S) -1- [ (tert-butoxycarbonylamino) methyl ] -2, 2-dimethyl-propyl ] -2-chloro-pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid was synthesized using a synthetic sequence analogous to that described for 7- [1- [ (tert-butoxycarbonylamino) methyl ] -2-methyl-propyl ] -2-chloro-pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid. LCMS (ESI)397(M + H).

Intermediate 1F was synthesized using a similar synthetic sequence as described for intermediate 1A. LCMS (ESI)279(M + H).

Example 53

Synthesis of Compound 53

Compound 53 was synthesized in a similar manner as described for intermediate 1 CA. LCMS (ESI)293(M + H).

Example 54

Synthesis of Compound 54

N- [ (2S) -2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] -2-phenyl-ethyl ] carbamic acid tert-butyl ester

Hydrogenation of Compound 21 in a pressure vessel under 50psi hydrogen using 10% Pd/C gave N- [ (2S) -2-amino-2-phenyl-ethyl](iv) carbamic acid tert-butyl ester, which is subsequently used with a compound directed against N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino]-3-methyl-butyl]Similar reaction conditions as described for tert-butyl carbamate with 5-bromo-2, 4-dichloro-pyrimidine gave N- [ (2S) -2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino]-2-phenyl-ethyl](iii) carbamic acid tert-butyl ester.¹HNMR(600MHz, DMSO-d₆)δppm 1.32(s,9H)3.29-3.50(m,2H)5.12-5.24(m,1H) 7.10(t,J＝5.27Hz,1H)7.21(t,J＝6.88Hz,1H)7.26-7.34(m,4H) 7.89(d,J＝7.32Hz,1H)8.24(s,1H)。LCMS(ESI)427(M+H)。

N- [ (2S) -2- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] amino ] -2-phenyl-ethyl ] carbamic acid tert-butyl ester

N- [ (2S) -2- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl]Amino group]-2-phenyl-ethyl]Synthesis of (2S) -N2- [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl]-4-methyl-pentane-1, 2-diamine using similar experimental conditions.¹HNMR(600MHz,DMSO-d₆)δppm 1.14(t,J＝7.03Hz,6H)1.32(s,9 H)3.39(s,2H)3.52-3.61(m,2H)3.64-3.73(m,2H)5.17-5.26(m, 1H)5.57(s,1H)7.07-7.14(m,1H)7.20-7.25(m,1H)7.26-7.33(m, 4H)7.90(d,J＝7.61Hz,1H)8.19(s,1H)。LCMS(ESI)475(M+H)。

7- [ (1S) -2- (tert-Butoxycarbonylamino) -1-phenyl-ethyl ] -2-chloro-pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid

7- [ (1S) -2- (tert-butoxycarbonylamino) -1-phenyl-ethyl ] -2-chloro-pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid was synthesized using a synthetic sequence analogous to that described for 7- [1- [ (tert-butoxycarbonylamino) methyl ] -2-methyl-propyl ] -2-chloro-pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid. LCMS (ESI)417 (M + H).

Compound 54

Compound 54 was synthesized using a similar synthetic sequence as described for compound 44.¹HNMR(600MHz,DMSO-d₆)δppm 3.58-3.69(m,1H)4.13(dd, J＝13.47,4.39Hz,1H)6.07(d,J＝3.81Hz,1H)6.85(d,J＝7.32Hz,2H) 7.19-7.31(m,3H)7.34(s,1H)8.27(d,J＝5.27Hz,1H)9.13(s,1H)。 LCMS(ESI)299(M+H)。

Example 55

Synthesis of Compound 55

N- [ (1S) -1- [ [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] methyl ] -2-methyl-propyl ] carbamic acid tert-butyl ester

N- [ (1S) -1- [ [ (5-bromo-2-chloro-pyrimidin-4-yl) amino]Methyl radical]-2-methyl-propyl]Use of tert-butyl carbamate against N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino]-3-methyl-butyl]Tert-butyl carbamate using 5-bromo-2, 4-dichloro-pyrimidine and intermediate E, analogous reaction conditions to those described for tert-butyl carbamate.¹HNMR (600MHz, chloroform-d) δ ppm 0.95-1.02(m,6H) 1.35-1.45(m,9H)1.75-1.90(m,1H)3.35-3.48(m,1H)3.52-3.61 (m,1H)3.64-3.76(m,1H)4.56(d, J ═ 8.49Hz,1H)6.47(s,1H)8.07 (s, 1H). LCMS (ESI)393(M + H).

N- [ (1S) -1- [ [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] amino ] methyl ] -2-methyl-propyl ] carbamic acid tert-butyl ester

N- [ (1S) -1- [ [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl]Amino group]Methyl radical]-2-methyl-propyl]Preparation of tert-butyl carbamateSynthesis of (2S) -N2- [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl]Synthesis of 4-methyl-pentane-1, 2-diamine under similar experimental conditions. ¹HNMR (600MHz, chloroform-d) δ ppm 0.90-1.00(m,6H) 1.18-1.25 (m,6H)1.34-1.36(m,9H)1.69-1.90(m,1H)3.34-3.82(m, 6H) 4.53-4.77(m,1H)5.45-5.55(m,1H)6.37(dd, J ═ 15.37,6.59Hz, 1H) 6.56(s,1H)8.05(s, 1H). LCMS (ESI)441(M + H).

7- [ (2S) -2- (tert-Butoxycarbonylamino) -3-methyl-butyl ] -2-chloro-pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid

7- [ (2S) -2- (tert-Butoxycarbonylamino) -3-methyl-butyl]-2-chloro-pyrrolo [2,3-d]Use of pyrimidine-6-carboxylic acid with a compound directed against 7- [1- [ (tert-butoxycarbonylamino) methyl group]-2-methyl-propyl]-2-chloro-pyrrolo [2,3-d]Pyrimidine-6-carboxylic acid was synthesized in a similar synthetic sequence.¹HNMR (600MHz, chloroform-d) δ ppm 0.90(d, J ═ 6.73Hz,3H)0.96(d, J ═ 7.03Hz,3H) 1.55-1.66(m,10H)4.14(dd, J ═ 13.61,3.95Hz,1H)4.52-4.63(m, 1H) 4.84(dd, J ═ 13.61,1.32Hz,1H)7.37(s,1H)8.95(s, 1H). LCMS (ESI)383(M + H).

Compound 55

Compound 55 was synthesized using a similar synthetic sequence as described for compound 44. LCMS (ESI)265(M + H).

Example 56

Synthesis of Compound 56

Compound 56 was synthesized using 5-bromo-2, 4-dichloro-pyrimidine and compound 17 as starting materials and according to a synthetic sequence analogous to compound 55. The analytical data are consistent with the enantiomers described for them (compound 55). ¹HNMR(600MHz,DMSO-d₆)δppm 0.88 (d,J＝6.44Hz,6H)1.73-1.86(m,1H)3.67-3.76(m,2H)4.11-4.21 (m,1H)7.13-7.19(m,1H)8.56(s,1H)9.05(s,1H)。LCMS(ESI)265 (M+H)。

Example 57

Synthesis of Compound 57

N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] -2-methyl-propyl ] carbamic acid tert-butyl ester

N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] -2-methyl-propyl ] carbamic acid tert-butyl ester was synthesized using 5-bromo-2, 4-dichloro-pyrimidine and N- (2-amino-2-methyl-propyl) carbamic acid tert-butyl ester using similar reaction conditions as described for N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] -3-methyl-butyl ] carbamic acid tert-butyl ester. LCMS (ESI)379(M + H).

N- [2- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] amino ] -2-methyl-prop-yl ] carbamic acid tert-butyl ester

N- [2- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl]Amino group]-2-methyl-propyl]Synthesis of (2S) -N2- [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl]-4-methyl-pentane-1, 2-diamine using similar experimental conditions.¹HNMR(600MHz,DMSO-d₆)δppm 1.11-1.22(m,6H)1.31-1.45(m, 15H)3.10-3.24(m,2H)3.51-3.76(m,4H)5.60(s,1H)6.94(s,1H) 7.33(t,J＝6.44Hz,1H)8.18(s,1H)。LCMS(ESI)427(M+H)。

7- [2- (tert-Butoxycarbonylamino) -1, 1-dimethyl-ethyl ] -2-chloro-pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid

7- [2- (tert-Butoxycarbonylamino) -1, 1-dimethyl-ethyl]-2-chloro-pyrrolo [2,3-d]Pyrimidine-6-carboxylic acid derivatives and their use as inhibitors of 7- [1- [ (tert-butoxycarbonylamino) methyl ] carbonyl]-2-methyl-propyl]-2-chloro-pyrrolo [2,3-d ]Pyrimidine-6-carboxylic acid was synthesized in a similar synthetic sequence.¹HNMR(600 MHz,DMSO-d₆)δppm 1.43(s,9H)1.73(s,6H)4.06(s,2H)7.46(s,1 H)9.23(s,1H)。LCMS(ESI)369(M+H)。

Compound 57

Compound 57 was synthesized using a similar synthetic sequence as described for compound 44.¹HNMR(600MHz,DMSO-d₆)δppm 1.73(s,6H)3.50(d,J＝2.93Hz,2 H)7.25(s,1H)8.46-8.55(m,1H)9.07(s,1H)。LCMS(ESI)251(M+ H)。

Example 58

Synthesis of Compound 58

N- [ [1- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] cyclohexyl ] methyl ] carbamic acid tert-butyl ester

N- [ [1- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino group]Cyclohexyl radical]Methyl radical]Use of tert-butyl carbamate with a compound directed against N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino group]-3-methyl-butyl]Tert-butyl carbamate similar reaction conditions were used for the synthesis using 5-bromo-2, 4-dichloro-pyrimidine and intermediate K.¹HNMR(600MHz,DMSO-d₆)δppm 1.18-1.54(m,17H)2.23(d, J＝14.35Hz,2H)3.36(d,J＝6.44Hz,2H)5.82(s,1H)6.93(s,1H)8.22 (s,1H)。LCMS(ESI)419(M+H)。

N- [ [1- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] amino ] cyclohexyl ] methyl ] carbamic acid tert-butyl ester

N- [ [1- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl]Amino group]Cyclohexyl radical]Methyl radical]Synthesis of (2S) -N2- [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl]-4-methyl-pentane-1, 2-diamine using similar experimental conditions.¹HNMR(600MHz,DMSO-d₆)δppm 1.08-1.16(m,6H)1.17-1.54(m, 17H)2.13(br.s.,2H)3.36(d,J＝6.73Hz,2H)3.50-3.69(m,4H)5.72 (s,1H)6.94(s,1H)5.72(br.s.,1H)8.17(s,1H)。LCMS(ESI)467(M +H)。

7- [1- [ (tert-Butoxycarbonylamino) methyl ] cyclohexyl ] -2-chloro-pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid

7- [1- [ (tert-Butoxycarbonylamino) methyl group]Cyclohexyl radical]-2-chloro-pyrrolo [2,3-d]Pyrimidine-6-carboxylic acid, the use thereof with a compound directed against 7- [1- [ (tert-butoxycarbonylamino) methyl group ]-2-methyl-propyl]-2-chloro-pyrrolo [2,3-d]Pyrimidine-6-carboxylic acid was synthesized in a similar synthetic sequence.¹HNMR(600 MHz,DMSO-d₆)δppm 1.37-1.54(m,13H)1.75(br.s.,4H)2.74(br. s.,2H)3.78-3.84(m,2H)7.44-7.51(m,1H)8.23(s,1H)9.11(s,1 H)。LCMS(ESI)409(M+H)。

Compound 58

Compound 58 was synthesized using a similar synthetic sequence as described for compound 44.¹HNMR(600MHz,DMSO-d₆)δppm 1.28(br.s.,2H)1.42(br.s.,2H) 1.70(br.s.,4H)1.85-1.95(m,2H)2.69(m,2H)7.16-7.25(m,1H) 8.41(br.s.,1H)9.04(s,1H)。LCMS 291(M+H)。

Example 59

Synthesis of Compound 59

N- [ [1- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] cyclopentyl ] methyl ] carbamic acid tert-butyl ester

N- [ [1- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino group]Cyclopentyl group]Methyl radical]Use of tert-butyl carbamate with a compound directed against N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino group]-3-methyl-butyl]Tert-butyl carbamate using 5-bromo-2, 4-dichloro-pyrimidine and intermediate L.¹HNMR(600MHz,DMSO-d₆)δppm 1.34(s,9H)1.50-1.58(m,2 H)1.63-1.78(m,4H)1.96-2.06(m,2H)3.25(d,J＝6.15Hz,2H)6.71 (s,1H)7.18(t,J＝6.29Hz,1H)8.20(s,1H)。LCMS(ESI)405(M+H)。

N- [ [1- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] amino ] cyclopentyl ] methyl ] carbamic acid tert-butyl ester

N- [ [1- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] amino ] cyclopentyl ] methyl ] carbamic acid tert-butyl ester was synthesized using experimental conditions similar to those used in the synthesis of (2S) -N2- [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] -4-methyl-pentan-1, 2-diamine. LCMS (ESI)453(M + H).

7- [1- [ (tert-Butoxycarbonylamino) methyl ] cyclopentyl ] -2-chloro-pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid

7- [1- [ (tert-Butoxycarbonylamino) methyl group ]Cyclopentyl group]-2-chloro-pyrrolo [2,3-d]Use of pyrimidine-6-carboxylic acid with a compound directed against said 7- [1- [ (tert-butoxycarbonylamino) methyl group]-2-methyl-propyl]-2-chloro-pyrrolo [2,3-d]Pyrimidine-6-carboxylic acid was synthesized in a similar synthetic sequence.¹HNMR(600MHz, DMSO-d₆)δppm 1.47(s,9H)1.74(br.s.,2H)1.88(br.s.,2H)2.04(br. s.,2H)2.41-2.45(m,2H)4.06(s,2H)7.45(s,1H)9.11(s,1H)。 LCMS(ESI)395(M+H)。

Compound 59

Compound 59 was synthesized using a similar synthetic sequence as described for compound 44.¹HNMR(600MHz,DMSO-d₆)δppm 1.72(br.s.,2H)1.86-1.93(m,2 H)1.99(d,J＝3.81Hz,2H)2.40(br.s.,2H)3.48(d,J＝2.34Hz,2H) 7.22(s,1H)8.53(br.s.,1H)9.05(s,1H)。LCMS(ESI)277(M+H)。

Example 60

Synthesis of Compound 60

N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] -4-methyl-pentyl ] carbamic acid tert-butyl ester

N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] -4-methyl-pentyl ] carbamic acid tert-butyl ester was synthesized using 5-bromo-2, 4-dichloro-pyrimidine and intermediate B using similar reaction conditions as described for N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] -3-methyl-butyl ] carbamic acid tert-butyl ester. The analytical data are in agreement with those described for the L-enantiomer.

N- [2- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] amino ] -4-methyl-pentyl ] carbamic acid tert-butyl ester

N- [2- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl]Amino group]-4-methyl-pentyl]Synthesis of N- [2- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] carbamic acid tert-butyl ester]Amino group]Ethyl radical]Synthesis of tert-butyl carbamate using similar experimental conditions. ¹HNMR (600MHz, chloroform-d) δ ppm 1.21-1.31(m,12H)1.38-1.46(m, 11H)1.70(m,1H)3.24(m,2H)3.65-3.82(m,4H)4.86(br s.,1H), 5.65(s,1H)5.85(br s.,1H)6.94(s,1H)8.21(s, 1H). LCMS (ESI)455 (M + H).

7- [1- [ (tert-Butoxycarbonylamino) methyl ] -3-methyl-butyl ] -2-chloro-pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid

7- [1- [ (tert-butoxycarbonylamino) methyl ] -3-methyl-butyl ] -2-chloro-pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid was synthesized using a synthetic sequence analogous to that described for 7- [1- [ (tert-butoxycarbonylamino) methyl ] -2-methyl-propyl ] -2-chloro-pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid. The analytical data are in agreement with those described for the L-isomer.

Compound 60

Compound 60 was synthesized using a similar synthetic sequence as described for compound 44. The analytical data are in agreement with those described for the L-isomer.

Example 61

Synthesis of Compound 61

To a solution of compound 60(100mg, 0.00024 mol) in DMF (3.0mL) was added sodium hydride (60% dispersion in oil) (27.6mg, 3 eq). After stirring for 15 minutes, iodomethane (30, 2eq) was added. The contents were stirred at room temperature for 30 minutes. Addition of saturated NaHCO₃After that, ethyl acetate was added. The organic layer was separated, then dried over magnesium sulfate and concentrated in vacuo to give the product. Analytical data were similar to the compound 49.

Example 62

Synthesis of Compound 62

N- [ (1S,2S) -2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] cyclopentyl ] carbamic acid tert-butyl ester

N- [ (1S,2S) -2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino]Cyclopentyl group]By using tert-butyl carbamate with a compound directed against N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino]-3-methyl-butyl]Similar reaction conditions as described for tert-butyl carbamate, N- [ (1S,2S) -2-aminocyclopentyl group was treated with 5-bromo-2, 4-dichloro-pyrimidine]And (3) synthesizing tert-butyl carbamate.¹HNMR(600MHz, DMSO-d₆)δppm 1.27(s,9H)1.42-1.54(m,2H)1.56-1.65(m,2H) 1.80-1.88(m,1H)1.96-2.01(m,1H)3.88-3.96(m,1H)4.03-4.09 (m,1H)6.91(d,J＝8.20Hz,1H)7.41(d,J＝7.32Hz,1H)8.18(s,1H)。 LCMS(ESI)391(M+H)。

N- [ (1S,2S) -2- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] amino ] cyclopentyl ] carbamic acid tert-butyl ester

N- [ (1S,2S) -2- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl]Amino group]Cyclopentyl group]Synthesis of (2S) -N2- [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl]-4-methyl-pentane-1, 2-diamine using similar experimental conditions.¹HNMR(600MHz,DMSO-d₆)δppm 1.13(t,6H)1.28(s,9H)1.42- 1.52(m,2H)1.58-1.65(m,2H)1.81-1.90(m,1H)1.99-2.08(m,1 H)3.49-3.60(m,2H)3.63-3.71(m,2H)3.84-3.93(m,1H)3.96- 4.04(m,1H)5.53(s,1H)6.96(d,J＝7.90Hz,1H)7.34(d,J＝7.03Hz,1 H)8.14(s,1H)。LCMS(ESI)439(M+H)。

7- [ (1S,2S) -2- (tert-Butoxycarbonylamino) cyclopentyl ] -2-chloro-pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid

7- [ (1S,2S) -2- (tert-butoxycarbonylamino) cyclopentyl group]-2-chloro-pyrrolo [2,3-d]Pyrimidine-6-carboxylic acid derivatives and their use as inhibitors of 7- [1- [ (tert-butoxycarbonylamino) methyl ] carbonyl]-2-methyl-propyl]-2-chloro-pyrrolo [2,3-d ]Pyrimidine-6-carboxylic acid was synthesized in a similar synthetic sequence.¹HNMR(600 MHz,DMSO-d₆)δppm 1.41-1.52(m,9H)1.55-1.68(m,1H)1.88- 2.00(m,2H)2.05-2.15(m,1H)2.26-2.35(m,1H)2.71-2.89(m,1 H)4.01-4.16(m,1H)4.28-4.45(m,1H)7.41(s,1H)9.11(s,1H)。 LCMS(ESI)381(M+H)。

Compound 62

Compound 62 was synthesized using a similar synthetic sequence as described for compound 44.¹HNMR(600MHz,DMSO-d₆)δppm 1.48-1.60(m,1H)1.88-1.98(m, 3H)1.99-2.08(m,1H)2.66-2.75(m,1H)3.63-3.74(m,1H)3.99- 4.12(m,1H)7.21(s,1H)8.89(s,1H)9.04(s,1H)。LCMS(ESI)263 (M+H)。

Example 63

Synthesis of Compound 63

To a chlorotricyclic lactam (0.050g, 0.225 mmol) in dioxane (2.0mL) under nitrogen was added 5- (4-methylpiperazin-1-yl) pyridin-2-amine (0.052g, 1.2eq, 0.270 mmol) followed by Pd₂(dba)₃(18.5mg), BINAP (25mg) and sodium tert-butoxide (31 mg, 0.324 mmol). The contents of the flask were degassed for 10 minutes and then heated to 100 ℃ for 12 hours. The crude reaction was loaded onto a silica gel column and eluted with DCM/MeOH (0-15%) to give the desired product (26 mg). To this compound dissolved in DCM/MeOH (10%) was added a 3N HCl solution in isopropanol (2eq) and the reaction was stirred overnight. The hydrochloride salt was obtained by vacuum concentration.¹HNMR(d6-DMSO)δppm 11.13(brs,1H),9.07(s,1H), 8.42(s,1H),8.03(br m 1H),7.99(s,1H),7.67(brm,1H),7.18(s,1H), 4.33(m,2H),3.79(m,2H),3.64(m,2H),3.50(m,2H),3.16(m,4H), 2.79(s,3H)。LCMS(ESI)379(M+H)。

Example 64

Synthesis of Compound 64

To a chlorotricyclic lactam (0.075g, 0.338 mmol) in dioxane (3.5mL) under nitrogen was added tert-butyl 4- (6-amino-3-pyridinyl) piperazine-1-carboxylate (0.098g, 1.05 eq) followed by Pd₂(dba)₃(27mg), BINAP (36mg) and sodium tert-butoxide (45 mg). The contents were heated at reflux for 11 hours. The crude reaction was loaded onto a silica gel column and eluted with DCM/MeOH (0-10%) to give the desired product (32 mg). ¹HNMR(d6-DMSO) δppm 9.48(s,1H),8.84(s,1H),8.29(s,1H),8.18(s,1H),7.99(s,1H), 7.42(m,1H),6.98(s,1H),4.23(m,2H),3.59(m,2H),3.45(m,4H), 3.50(m,2H),3.05(m,4H)。LCMS(ESI)465(M+H)。

Example 65

Synthesis of Compound 65

To a solution of compound 64(23mg) in 10% DCM/MeOH was added 10mL of 3M HCl in isopropanol. The contents were stirred for 16 hours. The reaction mixture was concentrated to give the hydrochloride salt.¹HNMR(d6-DMSO)δppm 9.01(s,1H),7.94(m,1H),7.86(m, 1H),7.23(s,1H),4.30(m,2H),3.64(m,2H),3.36(m,4H),3.25(m, 4H)。LCMS(ESI)465(M+H)。

Example 66

Synthesis of Compound 66

To a chloro-N-methyltricyclic amide (0.080g, 0.338 mmol) containing dioxane (3.5mL) was added under nitrogen 4- (6-amino-3-pyridinyl) piperazine-1-carboxylic acid tert-butyl ester (0.102g (1.1 eq) followed by Pd₂(dba)₃(27mg), BINAP (36mg) and sodium tert-butoxide (45 mg). The contents were heated at reflux for 11 hours. The crude product was purified using silica gel column chromatography using the eluent dichloromethane/methanol (0-5%) to give the desired product (44 mg).¹HNMR (d6-DMSO)δppm 9.49(s,1H),8.85(s,1H),8.32(m,1H),8.02(s,1H), 7.44(m,1H),7.00(s,1H),4.33(m,2H),3.80(m,2H),3.48(m,4H), 3.07(m,4H),3.05(s,3H),1.42(s,9H)。LCMS(ESI)479(M+H)。

Example 67

Synthesis of Compound 67

To compound 66(32mg) was added a solution of 3N HCL (10mL) in isopropanol and the contents were stirred at room temperature overnight for 16 hours. And concentrating to obtain the hydrochloride.¹HNMR (d6-DMSO)δppm 9.13(m,2H),8.11(m,1H),8.10(s,1H),7.62(m,1H), 7.21(s,1H),4.43(m,2H),3.85(m,2H),3.41(m,4H),3.28(m,4H), 3.08(s,3H)。LCMS(ESI)379(M+H)。

Example 68

Synthesis of Compound 68

Compound 68 was synthesized using similar experimental conditions as described for compound 64.¹HNMR(600MHz,DMSO-d₆)δppm 0.79(d,J＝7.03Hz,3H)1.01(d, J＝6.73Hz,3H)1.35-1.48(m,9H)2.16(dd,J＝14.64,6.73Hz,1H) 3.00-3.14(m,4H)3.40-3.51(m,4H)3.51-3.60(m,1H)3.63-3.74 (m,1H)4.44(dd,J＝7.90,3.81Hz,1H)6.99(s,1H)7.46(dd,J＝8.93, 2.78Hz,1H)7.94-8.09(m,2H)8.31(dd,J＝9.08,1.46Hz,1H)8.85(s, 1H)9.46(s,1H)。LCMS(ESI)507(M+H)。

Example 69

Synthesis of Compound 69

Compound 69 was synthesized using similar experimental conditions as described for compound 63 and recovered as the hydrochloride salt.¹HNMR(600MHz,DMSO-d₆)δppm 0.77-0.86(m,3 H)0.96(d,J＝7.03Hz,3H)2.10-2.24(m,1H)3.07(s,3H)3.37-3.79 (m,8H)4.00(dd,J＝13.61,4.54Hz,2H)4.63-4.73(m,1H)7.20(s,1 H)7.58-7.71(m,1H)7.99(d,J＝2.34Hz,1H)8.12(d,J＝9.37Hz,1H) 9.11(s,1H)9.41(br.s.,2H)11.76(br.s.,1H)。LCMS(ESI)421(M+ H)。

Example 70

Synthesis of Compound 70

Compound 70 was synthesized using similar experimental conditions as described for compounds 64 and 65 and recovered as the hydrochloride salt. The characterization data (NMR and LCMS) are consistent with those reported for compound 71.

Example 71

Synthesis of Compound 71

Compound 71 was synthesized using similar experimental conditions as described for compounds 64 and 65 and recovered as the hydrochloride salt.¹HNMR(600MHz,DMSO-d₆)δppm 0.79(d, J＝6.73Hz,3H)1.01(d,J＝6.73Hz,3H)2.18(dd,J＝14.49,7.17Hz,1H) 3.18-3.84(m,10H)4.53-4.71(m,1H)7.24(s,1H)7.65(d,J＝9.37 Hz,1H)8.01(d,J＝2.64Hz,1H)8.14(d,J＝1.46Hz,1H)8.35(d, J＝5.27Hz,1H)9.14(s,1H)9.46(s,2H)11.80(s,1H)LCMS(ESI) 407(M+H)。

Example 72

Synthesis of Compound 72 (Compound UUU)

Compound 72 was synthesized using similar experimental conditions as described for compounds 64 and 65 and recovered as the hydrochloride salt.¹HNMR(600MHz,DMSO-d₆)δppm 0.77(d, J＝7.03Hz,3H)0.99(d,J＝6.73Hz,3H)2.10-2.24(m,1H)3.18-3.81 (m,10H)4.54-4.69(m,1H)7.22(s,1H)7.63(d,J＝9.08Hz,1H)7.99 (d,J＝2.63Hz,1H)8.11(s,1H)8.33(d,J＝5.27Hz,1H)9.12(s,1H) 9.43(s,2H)11.77(s,1H)。LCMS(ESI)407(M+H)。

Example 73

Synthesis of Compound 73

Compound 73 was synthesized using similar experimental conditions as described for compounds 64 and 65 and was recovered as the hydrochloride salt.¹HNMR(600MHz,DMSO-d₆)δppm 0.84(d, J＝6.73Hz,3H)0.98(d,J＝6.73Hz,3H)2.12-2.26(m,1H)3.09(s,3 H)3.22-3.81(m,8H)4.01(dd,J＝13.61,4.25Hz,2H)4.59-4.72(m,1 H)7.19(s,1H)7.74(s,1H)7.96-8.10(m,2H)9.08(s,1H)9.22(s,2 H)。LCMS(ESI)421(M+H)。

Example 74

Synthesis of Compound 74

Compound 74 was synthesized using similar experimental conditions as described for compound 63 and recovered as the hydrochloride salt.¹HNMR(600MHz,DMSO-d₆)δppm 0.85(d,J＝4.98Hz, 3H)0.95(d,J＝4.98Hz,3H)1.42-1.70(m,3H)2.77(d,J＝2.93Hz,3 H)3.07-4.14(m,10H)4.95(s,1H)7.20(s,1H)7.66(d,J＝9.66Hz,1 H)7.94(s,1H)8.08-8.16(m,1H)8.33(d,J＝4.68Hz,1H)9.09(s,1H) 11.38(s,1H)11.71(s,1H)。LCMS(ESI)435(M+H)。

Example 75

Synthesis of Compound 75

Compound 75 was synthesized using similar experimental conditions as described for compounds 64 and 65 and recovered as the hydrochloride salt.¹HNMR(600MHz,DMSO-d₆)δppm 0.87(d, J＝6.15Hz,3H)0.94(d,J＝6.15Hz,3H)1.57(d,J＝84.61Hz,3H)3.05 (s,3H)3.13-3.55(m,8H)3.69(d,J＝78.17Hz,2H)4.90(s,1H)7.15 (s,1H)7.63-7.85(m,1H)7.93(s,1H)8.26(s,1H)9.03(s,1H)9.20 (s,2H)。LCMS(ESI)421(M+H)。

Example 76

Synthesis of Compound 76

Compound 76 was synthesized using similar experimental conditions as described for compound 63 and recovered as the hydrochloride salt.¹HNMR(600MHz,DMSO-d₆)δppm 0.85(d,J＝6.44Hz, 3H)0.95(d,J＝6.44Hz,3H)1.43-1.70(m,3H)2.78(d,J＝2.93Hz,3 H)3.05(s,3H)3.24-3.84(m,8H)4.01(d,J＝9.66Hz,2H)4.89-5.01 (m,1H)7.15(s,1H)7.77(s,1H)7.91-8.05(m,2H)9.03(s,1H) 10.96-11.55(m,2H)。LCMS(ESI)449(M+H)。

Example 77

Synthesis of Compound 77

Compound 77 was synthesized using similar experimental conditions as described for compounds 64 and 65 and recovered as the hydrochloride salt.¹HNMR(600MHz,DMSO-d₆)δppm 0.83-0.88 (d,J＝6.15Hz,3H)0.95(d,J＝6.15Hz,3H)1.40-1.71(m,3H)3.28- 3.83(m,8H)4.00(d,J＝3.22Hz,2H)4.91-5.08(m,1H)7.17(s,1H) 7.68(d,J＝9.66Hz,1H)7.93(s,1H)8.07(s,1H)9.06(s,1H)9.40(s,2 H)11.59(s,1H)。LCMS(ESI)435(M+H)。

Example 78

Synthesis of Compound 78

To compound 500.060 g (0.205 mmol) was added 5- (4-methylpiperazin-1-yl) pyridin-2-amine (35.42mg, 0.9eq) followed by 1, 4-dioxane (3 mL). Degassing with nitrogen, and adding Pd₂dba₃(12mg), BINAP (16mg) and sodium tert-butoxide (24 mg). The contents were then heated in a CEM Discovery microwave at 90 ℃ for 3 hours. The reaction was then loaded onto a silica gel column and purified by eluting with DCM/MeOH (0-15%).¹HNMR(600MHz,DMSO-d₆)δppm 0.75(t,J＝7.47Hz,3H)0.91(d, J＝6.73Hz,3H)1.04-1.20(m,2H)1.80-1.98(m,1H)2.77(d,J＝3.81 Hz,3H)2.94-3.90(m,10H)4.54-4.68(m,1H)7.06-7.23(m,2H) 7.56-7.75(m,1H)7.90-8.12(m,2H)8.29(s,1H)9.07(s,1H)10.98 -11.74(m,2H)。LCMS(ESI)435(M+H)。

Example 79

Synthesis of Compound 79

Compound 79 was synthesized and converted to the hydrochloride salt in a similar manner as described for compound 78, followed by the deblocking step described for compound 65.¹HNMR(600MHz, DMSO-d₆)δppm 0.75(t,J＝7.32Hz,3H)0.90(d,J＝6.73Hz,3H)1.07- 1.15(m,2H)1.85-1.94(m,1H)3.17-3.75(m,10H)4.58-4.67(m,1 H)7.17(s,1H)7.71(s,1H)7.96(s,1H)7.98-8.05(m,1H)8.28(d, J＝4.10Hz,1H)9.06(s,1H)9.39(s,2H)。LCMS(ESI)421(M+H)。

Example 80

Synthesis of Compound 80

Compound 80 was synthesized in a similar manner as described for compound 78.¹HNMR(600 MHz,DMSO-d₆)δppm 0.78(t,J＝7.32Hz,3H)0.86(d,J＝6.73Hz,3H) 1.13-1.21(m,2H)1.84-1.96(m,1H)2.77(d,J＝4.39Hz,3H)3.04(s, 3H)3.11-3.84(m,8H)3.98(dd,J＝13.61,4.25Hz,2H)4.66-4.74(m, 1H)7.17(s,1H)7.64(s,1H)7.96(d,J＝2.34Hz,1H)8.03-8.13(m,1 H)9.08(s,1H)11.26(s,1H)11.66(s,1H)。LCMS(ESI)449(M+H)。

Example 81

Synthesis of Compound 81

The compound was synthesized and converted to the hydrochloride salt in a similar manner as described for compound 78, followed by a deblocking step as described for compound 65.¹HNMR(600MHz, DMSO-d₆)δppm 0.78(t,J＝7.32Hz,3H)0.85(d,J＝6.73Hz,3H)1.10- 1.27(m,2H)1.82-1.99(m,1H)3.04(s,3H)3.28-3.77(m,8H)3.97 (dd,J＝13.91,4.54Hz,2H)4.62-4.75(m,1H)7.07-7.24(m,1H)7.62 -7.75(m,1H)7.94(d,J＝2.34Hz,1H)7.97-8.08(m,1H)9.05(s,1H) 9.29(s,2H)。LCMS(ESI)435(M+H)。

Example 82

Synthesis of Compound 82

The compound was synthesized and converted to the hydrochloride salt in a similar manner as described for compound 78, followed by a deblocking step as described for compound 65. ¹HNMR(600MHz, DMSO-d₆)δppm 0.96(s,9H)3.15-3.87(m,10H)4.42-4.53(m,1H) 6.99(s,1H)7.24(s,1H)8.06(s,1H)8.11-8.21(m,1H)8.79-8.98 (m,2H)9.25(s,2H)9.88(s,1H)。LCMS(ESI)421(M+H)。

Example 83

Synthesis of Compound 83

Compound 83 was synthesized and converted to the hydrochloride salt in a similar manner as described for compound 78, followed by the deblocking step described for compound 65.¹HNMR(600MHz, DMSO-d₆)δppm 0.95(s,9H)2.79(d,J＝4.10Hz,3H)3.06-3.86(m, 10H)4.56-4.67(m,1H)7.17(s,1H)7.70(s,1H)7.96(d,J＝2.63Hz, 1H)7.99-8.08(m,1H)8.26(s,1H)9.06(s,1H)10.80(s,1H)。LCMS (ESI)435(M+H)。

Example 84

Synthesis of Compound 84

Compound 84 was synthesized and converted to the hydrochloride salt in a similar manner as described for compound 78.¹HNMR(600MHz,DMSO-d₆)δppm 2.75-2.81(m,3H)3.12- 3.16(m,2H)3.46-3.54(m,4H)3.60-3.69(m,2H)3.72-3.79(m,1 H)4.07-4.18(m,2H)6.06-6.09(m,1H)6.90(d,J＝7.61Hz,2H)7.20 -7.31(m,3H)7.33(s,1H)7.49-7.55(m,1H)7.62-7.70(m,1H) 7.92(d,J＝2.93Hz,1H)8.22(s,1H)9.14(s,1H)。LCMS(ESI)455(M +H)。

Example 85

Synthesis of Compound 85

Compound 85 was synthesized and converted to the hydrochloride salt in a similar manner as described for compound 78, followed by the deblocking step described for compound 65.¹HNMR(600MHz, DMSO-d₆)δppm 3.21(s,4H)3.35-3.67(m,5H)4.07-4.20(m,2H) 6.13(s,1H)6.90(d,J＝7.32Hz,2H)7.22-7.31(m,3H)7.36(s,1H) 7.48(d,J＝9.37Hz,1H)7.93(d,J＝2.34Hz,1H)8.04-8.11(m,1H) 8.25(d,J＝4.98Hz,1H)9.17(s,1H)11.77(br,s.,1H)。LCMS(ESI)441 (M+H)。

Example 86

Synthesis of Compound 86

Compound 86 was synthesized and converted to the hydrochloride salt in a similar manner as described for compound 78, followed by the deblocking step described for compound 65.¹HNMR(600MHz, DMSO-d₆)δppm 0.90(d,J＝6.15Hz,6H)1.72-1.89(m,1H)3.15- 3.92(m,9H)4.10-4.46(m,2H)7.18(s,1H)7.59(d,J＝8.78Hz,1H) 8.00(s,1H)8.13(d,J＝9.37Hz,1H)8.55(s,1H)9.09(s,1H)9.67(s,2 H)11.91(s,1H)。LCMS(ESI)407(ESI)。

Example 87

Synthesis of Compound 87

Compound 87 was synthesized and converted to the hydrochloride salt in a similar manner to compound 86. The characterization data (NMR and LCMS) are similar to that obtained for enantiomer for compound 86.

Example 88

Synthesis of Compound 88

Compound 88 was synthesized and converted to the hydrochloride salt in a similar manner as described for compound 78, followed by the deblocking step described for compound 65.¹HNMR(600MHz, DMSO-d₆)δppm 1.78(s,6H)3.40-3.53(m,6H)3.64-3.73(m,4H) 7.27(s,1H)7.66(d,J＝9.37Hz,1H)7.98(d,J＝2.34Hz,1H)8.12(br.s., 1H)8.47(br.s.,1H)9.11(s,1H)9.45(br.s.,2H)11.62(br.s.,1H)。 LCMS(ESI)393(M+H)。

Example 89

Synthesis of Compound 89 (also known as Compound T)

Compound 89 was synthesized and converted to the hydrochloride salt in a similar manner as described for compound 78. ¹HNMR(600MHz,DMSO-d₆)δppm 1.47(br.s.,6H)1.72(br.s.,2 H)1.92(br.s.,2H)2.77(br.s.,3H)3.18(br.s.,2H)3.46(br.s.,2H) 3.63(br.s.,2H)3.66(d,J＝6.15Hz,2H)3.80(br.s.,2H)7.25(s,1H) 7.63(br.s.,2H)7.94(br.s.,1H)8.10(br.s.,1H)8.39(br.s.,1H)9.08 (br.s.,1H)11.59(br.s.,1H)。LCMS(ESI)447(M+H)。

Example 90

Synthesis of Compound 90 (also known as Compound Q)

Compound 90 was synthesized and converted to the hydrochloride salt in a similar manner as described for compound 78, followed by the deblocking step described for compound 65.¹HNMR(600MHz, DMSO-d₆)δppm 1.27-1.64(m,6H)1.71(br.s.,2H)1.91(br.s.,2H) 2.80(br.s.,1H)3.17-3.24(m,2H)3.41(br.s.,4H)3.65(br.s.,4H) 7.26(br.s.,1H)7.63(br.s.,1H)7.94(br.s.,1H)8.13(br.s.,1H)8.40 (br.s.,1H)9.09(br.s.,1H)9.62(br.s.,1H)11.71(br.s.,1H)。LCMS (ESI)433(M+H)。

Example 91

Synthesis of Compound 91 (also known as Compound ZZ)

Compound 91 was synthesized and converted to the hydrochloride salt using conditions similar to those described for compound 78.¹HNMR(600MHz,DMSO-d₆)δppm 1.64-1.75(m,2H) 1.83-1.92(m,2H)1.96-2.06(m,2H)2.49-2.58(m,2H)2.79(d, J＝3.81Hz,3H)3.06-3.18(m,4H)3.59-3.69(m,2H)3.73-3.83(m, 2H)4.04-4.12(m,2H)7.17(br.s.,1H)7.60-7.70(m,2H)7.70- 7.92(m,2H)7.96(br.s.,1H)8.41(br.s.,1H)8.98(br.s.,1H)10.77 (br.s.,1H)。LCMS(ESI)433(M+H)。

Example 92

Synthesis of Compound 92

Compound 92 was synthesized and converted to the hydrochloride salt in a similar manner as described for compound 78, followed by a deblocking step as described for compound 65.¹HNMR(600MHz, DMSO-d₆)δppm 1.64-1.75(m,2H)1.84-1.92(m,2H)1.96-2.05(m, 2H)2.48-2.56(m,2H)3.22(br.s.,4H)3.42-3.48(m,4H)3.60- 3.69(m,2H)4.05-4.13(m,1H)7.18(s,1H)7.65(d,J＝13.47Hz,1H) 7.70-7.77(m,1H)7.94(d,J＝1.76Hz,1H)8.42(br.s.,1H)9.00(s,1 H)9.15(br.s.,2H)。LCMS(ESI)419(M+H)。

Example 93

Synthesis of Compound 93

Compound 93 was synthesized and converted to the hydrochloride salt in a similar manner as described for compound 78, followed by a deblocking step as described for compound 65.¹HNMR(600MHz, DMSO-d₆)δppm 1.76(br.s.,2H)1.89(br.s.,2H)2.03(br.s.,2H)2.47 -2.58(m,2H)3.04(s,3H)3.22(br.s.,4H)3.39(br.s.,4H)3.66(s,2 H)7.21(s,1H)7.67(d,J＝9.37Hz,1H)7.93(br.s.,1H)7.98-8.09(m, 1H)9.04(s,1H)9.34(br.s.,2H)11.31(br.s.,1H)。LCMS(ESI)433 (M+H)。

Example 94

Synthesis of Compound 94

Compound 94 was synthesized and converted to the hydrochloride salt using similar conditions as described for compound 78.¹HNMR(600MHz,DMSO-d₆)δppm 1.66-1.77(m,2H)1.84- 1.94(m,2H)1.96-2.08(m,2H)2.48-2.57(m,2H)3.36-3.52(m,4 H)3.60-3.80(m,6H)7.21(s,1H)7.53-7.74(m,2H)7.86(s,1H) 8.02(s,1H)8.45(s,1H)9.03(s,1H)11.19(br.s.,1H)。LCMS(ESI) 420(M+H)。

Example 95

Synthesis of Compound 95

Compound 95 was synthesized and converted to the hydrochloride salt using similar conditions as described for compound 78.¹HNMR(600MHz,DMSO-d₆)δppm 1.65-1.79(m,2H)1.85- 1.95(m,2H)1.97-2.08(m,2H)2.47-2.54(m,2H)3.40-3.58(m,5 H)3.65(dd,J＝21.67,5.56Hz,1H)3.69-3.78(m,4H)7.24(s,1H) 7.97-8.17(m,2H)8.48(s,1H)9.08(s,1H)11.81(s,1H)。LCMS(ESI) 421(M+H)。

Example 96

Synthesis of Compound 96

Compound 96 was synthesized and converted to the hydrochloride salt using similar conditions as described for compound 78. ¹HNMR(600MHz,DMSO-d₆)δppm 1.55-1.74(m,2H)1.80- 1.98(m,4H)2.48-2.60(m,2H)3.40-3.50(m,4H)3.57-3.72(m,2 H)3.90-4.20(m,4H)7.08(s,1H)7.37-7.57(m,2H)7.70(m,2H) 8.32(s,1H)8.88(s,1H)9.98(s,1H)。LCMS(ESI)419(M+H)。

Example 97

Synthesis of Compound 97 (also known as Compound III)

Compound 97 was synthesized and converted to the hydrochloride salt using similar conditions as described for compound 78.¹HNMR(600MHz,DMSO-d₆)δppm 1.30(d,J＝5.27Hz,6H) 1.65-1.78(m,2H)1.83-1.95(m,2H)1.97-2.10(m,2H)2.45-2.55 (m,2H)3.25-3.36(m,1H)3.39-3.48(m,4H)3.60-3.70(m,4H) 3.75-4.15(m,2H)7.24(s,1H)7.54-7.75(m,2H)7.95(s,1H)8.10 (s,1H)8.49(s,1H)9.07(s,1H)11.25(s,1H)11.48(s,1H)。LCMS (ESI)461(M+H)。

Example 98

Synthesis of Compound 98

Compound 98 was synthesized and converted to the hydrochloride salt using similar conditions as described for compound 78.¹HNMR(600MHz,DMSO-d₆)δppm 0.99(d,J＝6.15Hz,6H) 1.65-1.78(m,2H)1.90(m,2H)1.97-2.08(m,2H)2.08-2.17(m,1 H)2.45-2.55(m,2H)2.88-3.02(m,2H)3.33-3.48(m,4H)3.50- 3.90(m,6H)7.24(s,1H)7.67(s,2H)7.94(s,1H)8.12(s,1H)8.49(s, 1H)9.07(s,1H)10.77(s,1H)11.51(s,1H)。LCMS(ESI)475(M+H)。

Example 99

Synthesis of Compound 99

Compound 99 was synthesized and converted to the hydrochloride salt using conditions similar to those described for compound 78.¹HNMR(600MHz,DMSO-d₆)δppm 1.13(d,J＝5.86Hz,6H) 1.66-1.77(m,2H)1.84-1.94(m,2H)1.97-2.09(m,2H)2.40-2.53 (m,2H)3.37-3.49(m,2H)3.50-3.59(m,2H)3.59-3.73(m,4H) 7.23(s,1H)7.64(m,3H)7.85(s,1H)8.11(s,1H)8.47(s,1H)9.05(s, 1H).11.35(br s.,1H)。LCMS(ESI)448(M+H)。

Example 100

Synthesis of Compound 100

Compound 100 was synthesized and converted to the hydrochloride salt using similar conditions as described for compound 78.¹HNMR(600MHz,DMSO-d₆)δppm 1.50-1.57(m,2H)1.62 -1.68(m,3H)1.68-1.75(m,2H)1.84-1.92(m,2H)1.97-2.08(m,2 H)2.48-2.53(m,2H)3.14-3.23(m,4H)3.43-3.47(m,2H)3.58- 3.70(m,2H)7.22(s,1H)7.58-7.70(m,2H)7.85-8.00(m,1H)8.16 (d,1H)8.46(s,1H)9.04(s,1H)11.37(br s.,1H)。LCMS(ESI)418(M +H)。

Example 101

Synthesis of Compound 101 (also known as Compound WW)

Compound 101 was synthesized and converted to the hydrochloride salt using conditions similar to those described for compound 78.¹HNMR(600MHz,DMSO-d₆)δppm 1.72(s,2H)1.90(s, 4H)2.03(s,2H)2.21(s,2H)2.48-2.54(m,2H)2.73(s,2H)3.03(s, 2H)3.25-3.35(m,1H)3.38-3.48(m,4H)3.65-3.99(m,5H)7.23(s, 1H)7.63(d,J＝9.66Hz,1H)7.90(s,1H)8.13(s,1H)8.47(s,1H) 9.06(s,1H)10.50(br s.,1H)。LCMS(ESI)503(M+H)。

Example 102

Synthesis of Compound 102 (also known as Compound HHH)

Compound 102 was synthesized and converted to the hydrochloride salt using conditions similar to those described for compound 78.¹HNMR(600MHz,DMSO-d₆)δppm 1.63-1.85(m,6H) 1.87-1.92(m,2H)1.99-2.06(m,2H)2.15-2.23(m,2H)2.47-2.53 (m,1H)2.69-2.79(m,2H)2.81-2.91(m,2H)2.98-3.08(m,2H) 3.32-3.48(m,4H)3.57-3.72(m,4H)3.77-3.85(m,2H)7.22(s,1H) 7.60-7.68(m,2H)7.90(s,1H)8.07(s,1H)8.46(s,1H)9.04(s,1H). 11.41(br s.,1H)。LCMS(ESI)501(M+H)。

Example 103

Synthesis of Compound 103

Compound 103 was synthesized and converted to the hydrochloride salt using conditions similar to those described for compound 78.¹HNMR(600MHz,DMSO-d₆)δppm 1.64-1.76(m,2H) 1.87-1.93(m,2H)2.00-2.07(m,2H)2.48-2.53(m,2H)2.67-2.72 (m,4H)3.44-3.47(m,2H)3.50-3.55(m,4H)7.24(s,1H)7.61(d, J＝9.37Hz,2H)7.86(d,J＝2.63Hz,1H)8.09(d,J＝12.88Hz,1H)8.48 (s,1H)9.06(s,1H)11.41(br s.,1H)。LCMS(ESI)436(M+H)。

Example 104

Synthesis of Compound 104

Compound 104 was synthesized and converted to the hydrochloride salt using conditions similar to those described for compound 78. ¹HNMR(600MHz,DMSO-d₆)δppm 1.29(d,J＝6.73Hz,6 H)1.66-1.79(m,2H)1.84-1.95(m,2H)1.98-2.09(m,2H)2.46- 2.55(m,2H)3.29-3.39(m,2H)3.58-3.70(m,4H)3.77-3.86(m,4H) 7.24(s,1H)7.66(d,J＝9.37Hz,1H)7.96(d,J＝2.93Hz,1H)8.08(s,1 H)8.48(s,1H)9.06(s,1H)9.28(s,1H)9.67(s,1H)11.36(s,1H)。 LCMS(ESI)447(M+H)。

Example 105

Synthesis of Compound 105

Compound 105 was synthesized and converted to the hydrochloride salt using conditions similar to those described for compound 78.¹HNMR(600MHz,DMSO-d₆)δppm 1.73(s,2H)1.76- 1.85(m,2H)1.85-1.94(m,2H)1.98-2.07(m,2H)2.19-2.26(m,2 H)2.48-2.52(m,1H)2.70-2.81(m,4H)3.13-3.20(m,1H)3.30- 3.48(m,3H)3.58-3.71(m,4H)3.78-3.84(m,4H)7.24(s,1H)7.62 (d,J＝9.37Hz,2H)7.89(d,J＝1.17Hz,1H)8.09-8.18(m,1H)8.48(s, 1H)9.06(s,1H)11.46(br s.,1H)。LCMS(ESI)519(M+H)。

Example 106

Synthesis of Compound 106

Compound 106 was synthesized using conditions similar to those described for compound 78, followed by a deblocking step as described for compound 65 and conversion to the hydrochloride salt.¹HNMR(600 MHz,DMSO-d₆)δppm 1.65-1.75(m,2H)1.85-1.93(m,2H)1.93- 1.99(m,1H)2.00-2.06(m,2H)2.08-2.14(m,1H)2.47-2.55(m,2 H)3.07-3.25(m,2H)3.25-3.69(m,5H)4.46(s,1H)4.67(s,1H) 7.22(s,1H)7.58-7.69(m,2H)8.46(s,1H)9.02(s,1H)9.34(s,1H) 9.65(s,1H)。LCMS(ESI)431(M+H)。

Example 107

Synthesis of Compound 107 (also known as Compound YY)

Compound 107 was synthesized and converted to the hydrochloride salt using conditions similar to those described for compound 78.¹HNMR(600MHz,DMSO-d₆)δppm 1.65-1.82(m,3H) 1.89(br.s.,2H)1.98-2.08(m,2H)2.13(br.s.,2H)2.47-2.55(m,2H) 2.68(d,J＝4.98Hz,6H)2.71-2.80(m,2H)3.29-3.71(m,10H)7.16- 7.26(m,1H)7.67(d,J＝9.66Hz,2H)7.91(d,J＝2.05Hz,1H)8.14(br. s.,1H)8.48(br.s.,1H)9.05(s,1H)11.14(br.s.,1H)11.43(br.s.,1 H)。LCMS(ESI)461(M+H)。

Example 108

Synthesis of Compound 108

Compound 108 was synthesized and recovered as the hydrochloride salt in a similar manner as described for compounds 64 and 65. Analytical data are consistent with that described for enantiomeric compound 75.

Example 109

Synthesis of Compound 109

Compound 109 was synthesized and recovered as the hydrochloride salt in a similar manner as described for compounds 64 and 65. Analytical data are consistent with that described for enantiomeric compound 75.

Example 110

Synthesis of Compound 110

Compound 110 was synthesized and then converted to its hydrochloride salt in a similar manner as described for compound 78.¹HNMR(600MHz,DMSO-d₆)δppm 1.50-1.65(m,1H) 1.92-2.02(m,3H)2.06-2.15(m,1H)2.78(d,J＝3.81Hz,4H)3.10- 3.20(m,4H)3.47-3.51(m,2H)3.64-3.71(m,1H)3.76-3.83(m,2 H)3.98-4.14(m,1H)7.20(s,2H)7.77(s,1H)7.97(s,2H)8.81(s,1 H)9.03(s,1H)10.97(br s.,1H)。LCMS(ESI)419(M+H)。

Example 111

Synthesis of Compound 111

Compound 111 was synthesized in a similar manner as described for compound 78 and then converted to its hydrochloride salt. ¹HNMR(600MHz,DMSO-d₆)δppm 1.54-1.59(m,1H) 1.92-2.01(m,3H)2.06-2.15(m,1H)2.76-2.84(m,1H)3.17-3.24 (m,6H)3.64-3.71(m,2H)4.02-4.11(m,2H)7.22(s,2H)7.64(s,1 H)7.97(s,2H)8.75(s,1H)8.97(s,1H)9.21(s,1H)。LCMS(ESI)405 (M+H)。

Example 112

Synthesis of Compound 112

Compound 112 was synthesized using similar experimental conditions as described for compound 64.

Example 113

Synthesis of tert-butyl N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] ethyl ] carbamate, Compound 113

To a solution of 5-bromo-2, 4-dichloropyrimidine (12.80g, 0.054 moles) in ethanol (250mL) was added henniger's base (12.0mL), followed by a solution of N- (tert-butoxycarbonyl) -1, 2-diaminoethane (10g, 0.0624 moles) in ethanol (80 mL). The contents were stirred overnight for 20 hours. The solvent was evaporated under vacuum. Ethyl acetate (800mL) and water (300mL) were added and the layers were separated. The organic layer was dried over magnesium sulfate and then concentrated in vacuo. Silica gel column chromatography using hexane/ethyl acetate (0-60%) afforded N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] ethyl ] carbamic acid tert-butyl ester. LCMS (ESI)351(M + H).

Example 114

Synthesis of tert-butyl N- [2- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] amino ] ethyl ] carbamate, Compound 114

Under nitrogen to a mixture containing N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino group]Ethyl radical]Tert-butyl carbamate (5g, 14.23 mmol) in toluene (42mL) and triethylamine (8.33mL) were added triphenylarsine (4.39g), 3-diethoxyprop-1-yne (3.24mL) and Pddba (1.27 g). The contents were heated at 70 ℃ for 24 hours. Warp beam After filtration, the crude reaction was subjected to column separation using hexane/ethyl acetate (0-20%) to obtain 3.9g of the desired product. The resulting residue was subjected to column chromatography using hexane/ethyl acetate (0-30%) to give N- [2- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] amine]Amino group]Ethyl radical](iii) carbamic acid tert-butyl ester. LCMS (ESI)399(M + H).

Example 115

Synthesis of tert-butyl N- [2- [ 2-chloro-6- (diethoxymethyl) pyrrolo [2,3-d ] pyrimidin-7-yl ] ethyl ] carbamate, Compound 115

To a solution of compound 114(3.9g, 0.00976 mol) in THF (60mL) was added TBAF (68.3mL, 7 eq). The contents were heated to 45 deg.CKept at the temperature for 2 hours. Concentration followed by column chromatography using ethyl acetate/hexane (0-50%) gave N- [2- [ 2-chloro-6- (diethoxymethyl) pyrrolo [2,3-d ] as a light brown liquid]Pyrimidin-7-yl]Ethyl radical]Carbamic acid tert-butyl ester (1.1 g).¹HNMR(d6-DMSO)δppm 8.88(s,1H),6.95(brs,1H), 6.69(s,1H),5.79(s,1H),4.29(m,2H),3.59(m,4H),3.34(m,1H),3.18 (m,1H),1.19(m,9H),1.17(m,6H)。LCMS(ESI)399(M+H)。

Example 116

Synthesis of tert-butyl N- [2- [ 2-chloro-6- (diethoxymethyl) -5-iodo-pyrrolo [2,3-d ] pyrimidin-7-yl ] ethyl ] carbamate, Compound 116

To a solution containing N- [2- [ 2-chloro-6- (diethoxymethyl) pyrrolo [2,3-d]Pyrimidin-7-yl]Ethyl radical]To acetonitrile (2mL) of tert-butyl carbamate (0.1g, 0.00025mol) were added 1, 3-diiodo-5, 5-dimethylhydantoin (95mg, 1eq) and solid NaHCO ₃(63mg, 3 eq). The reaction was stirred at room temperature for 16 hours. The reaction was filtered and concentrated in vacuo. The product was purified by column chromatography on silica gel using hexane/ethyl acetate (0-50%) to give N- [2- [ 2-chloro-6- (diethoxymethyl) -5-iodo-pyrrolo [2,3-d ] as a pale yellow solid]Pyrimidin-7-yl]Ethyl radical]Tert-butyl carbamate (0.03 g). LCMS (ESI)525(M + H).

Example 117

Synthesis of tert-butyl N- [2- [ 2-chloro-6- (diethoxymethyl) -5- (o-tolyl) pyrrolo [2,3-d ] pyrimidin-7-yl ] ethyl ] carbamate, Compound 117

To N- [2- [ 2-chloro-6- (diethoxymethyl) -5-iodo-pyrrolo [2,3-d ] pyrimidin-7-yl ] ethyl ] carbamic acid tert-butyl ester (0.1g,0.19 mmol) in dioxane (3mL) was added water (0.3mL) containing 2-methylphenylboronic acid (28mg), tetrakis (triphenylphosphine) palladium (25mg), and potassium phosphate (250 mg). The reaction was heated in a CEM Discovery microwave at 90 ℃ for 3 hours. The crude reaction was loaded on silica gel and subjected to column separation using hexane/ethyl acetate (0-30%) to give tert-butyl N- [2- [ 2-chloro-6- (diethoxymethyl) -5- (o-tolyl) pyrrolo [2,3-d ] pyrimidin-7-yl ] ethyl ] carbamate (0.06 g). LCMS (ESI)489(M + H).

Example 118

Synthesis of 7- [2- (tert-Butoxycarbonylamino) ethyl ] -2-chloro-5- (o-tolyl) pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid, Compound 118

To a solution containing N- [2- [ 2-chloro-6- (diethoxymethyl) -5- (o-tolyl) pyrrolo [2,3-d]Pyrimidin-7-yl]Ethyl radical]Tert-butyl carbamate (0.85g, 1.74 mmol) to AcOH (10mL) was added water (1.5 mL). The reaction was stirred at room temperature for 16 hours. The crude reaction was then concentrated in vacuo. After addition of ethyl acetate (50mL), the organic layer was washed with saturated NaHCO₃And (6) washing. The organic layer was dried over magnesium sulfate and then concentrated in vacuo to give the crude intermediate, N- [2- [ 2-chloro-6-formyl-5- (o-tolyl) pyrrolo [2,3-d]Pyrimidin-7-yl]Ethyl radical](iii) carbamic acid tert-butyl ester. To DMF (5mL) containing this crude intermediate was added oxone (1.3 g). After stirring for 2.5 hours, water (20mL) and ethyl acetate (100mL) were added. The organic layer was separated, dried and then concentrated in vacuo to give the crude product, which was subjected to silica gel column separation using hexane/ethyl acetate (0-50%) to give 7- [2- (tert-butoxycarbonylamino) ethyl ] ethyl]-2-chloro-5- (o-tolyl) pyrrolo [2,3-d]Pyrimidine-6-carboxylic acid (0.112 g). LCMS (ESI)431(M + H).

Example 119

Synthesis of Compound 119

To a solution containing 7- [2- (tert-butoxycarbonylamino) ethyl group ]-2-chloro-5- (o-tolyl) pyrrolo [2,3-d]Pyrimidine-6-carboxylic acid (0.1g, 0.261 mm)ol) in DCM (4.1mL) was added DMAP (20 mg) followed by N, N' -diisopropylcarbodiimide (0.081mL, 2 eq). After stirring for 3 hours, TFA (0.723mL) was added. Stirring was then continued for a further 30 minutes. The reaction mixture was saturated NaHCO₃And (4) neutralizing. DCM (20mL) was then added and the organic layer was separated, dried over magnesium sulfate and then concentrated in vacuo to give the crude product which was subjected to column separation using hexane/ethyl acetate (0-100%) to give chlorotricycloamide compound 119(0.65 g). LCMS (ESI)313 (M + H).

Example 120

Synthesis of Compound 120

To a solution of the chlorotricyclic amide (0.040g, 0.128 mmol) (compound 119) in dioxane (2.5mL) under nitrogen was added Pd₂(dba)₃(12mg), sodium tert-butoxide (16mg), BINAP (16mg) and 4-morpholinoaniline (22.7mg, 1 eq). The reaction mixture was heated in a CEM Discovery microwave at 90 ℃ for 3.0 hours. The crude reaction was loaded onto a silica gel column and the contents were eluted with DCM/MeOH (0-6%) to give the product (10 mg). LCMS (ESI) 455(M + H).¹HNMR(600MHz,DMSO-d₆)δppm 2.14(s,3H)3.23- 3.50(m,2H)3.57-3.73(m,2H),3.81-3.92(m,8H),7.11-7.31(m,4 H)7.31-7.48(m,1H)7.58-7.73(m,1H)7.77-7.95(m,2H)8.05- 8.21(m,1H)8.44(s,1H)9.85-10.01(m,1H)。

Example 121

Synthesis of Compound 121

To N-methyl-2-pyrrolidone (NMP) (1.5mL) containing a trichloro-amide (0.024g) (compound 119) was added trans-4-aminocyclohexanol (0.0768mmol, 26.54mg, 3eq) and henniger's base (0.4 mL). The reaction was heated in a CEM Discovery microwave vessel at 150 ℃ for 1.2 hours. The crude reaction was loaded onto a silica gel column and the contents were eluted with DCM/MeOH (0-10%) to give product Substance (21 mg). LCMS (ESI)392(M + H).¹HNMR (600MHz,DMSO-d₆)δppm 1.23(d,J＝8.78Hz,4H)1.84(br.s.,4H) 2.11(s,3H)3.34-3.43(m,1H)3.55(br.s.,2H)3.72(br.s.,1H)4.13 (br.s.,2H)4.50(br.s.,1H)7.03(br.s.,1H)7.12-7.28(m,4H)7.96 (br.s.,1H)8.18(br.s.,1H)。

Example 122

Synthesis of 7- [2- (tert-Butoxycarbonylamino) ethyl ] -2-chloro-pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid, Compound 122

7- [2- (tert-Butoxycarbonylamino) ethyl ] -2-chloro-pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid was synthesized using a similar experimental procedure as described for the synthesis of 7- [2- (tert-butoxycarbonylamino) ethyl ] -2-chloro-5- (o-tolyl) pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid. LCMS (ESI)341(M + H).

Example 123

Synthesis of Compound 123

The chlorotricyclic amide compound 123 was synthesized using an experimental procedure similar to that described for the synthesis of chlorotricyclic amide (compound 119). LCMS (ESI)223(M + H).

Example 124

Synthesis of Compound 124

To a solution of chlorotricyclic amide, compound 123(0.035g, 0.00157 mol) in NMP (1.5mL) was added henniger's base (0.3mL) followed by trans-4-aminocyclohexanol (54.2 mg). The reaction mixture was heated at 150 ℃ for 1.5 hours. The crude reaction was loaded onto a silica gel column and the column was eluted with DCM/MeOH (0-10%) to give the product (5 mg). LCMS (ESI) 302(M + H).

Example 125

Synthesis of tert-butyl N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] -2-methyl-propyl ] carbamate, Compound 125

N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] -2-methyl-propyl ] carbamic acid tert-butyl ester was synthesized by treating 5-bromo-2, 4-dichloropyrimidine with tert-butyl N- (2-amino-2-methyl-propyl) carbamate using experimental conditions similar to those described for the synthesis of N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] ethyl ] carbamic acid tert-butyl ester. LCMS (ESI) (M + H) 379.

Example 126

Synthesis of N- [2- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] amino ] -2-methyl-propyl ] carbamic acid tert-butyl ester, Compound 126

N- [2- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] amino ] -2-methyl-prop-yl ] carbamic acid tert-butyl ester Using similar experimental conditions as described for the synthesis of N- [2- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] amino ] ethyl ] carbamic acid tert-butyl ester, synthesized by treating tert-butyl N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] -2-methyl-propyl ] carbamate with 3, 3-diethoxyprop-1-yne in the presence of a catalyst such as Pddba.

LCMS(ESI)(M+H)427。

Example 127

Synthesis of tert-butyl N- [2- [ 2-chloro-6- (diethoxymethyl) pyrrolo [2,3-d ] pyrimidin-7-yl ] -2-methyl-propyl ] carbamate, Compound 127

Tert-butyl N- [2- [ 2-chloro-6- (diethoxymethyl) pyrrolo [2,3-d ] pyrimidin-7-yl ] -2-methyl-propyl ] carbamate was synthesized by treating tert-butyl N- [2- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] amino ] -2-methyl-propyl ] carbamate with TBAF using similar experimental conditions as described for the synthesis of tert-butyl N- [2- [ 2-chloro-6- (diethoxymethyl) pyrrolo [2,3-d ] pyrimidin-7-yl ] ethyl ] carbamate. LCMS (ESI) (M + H) 427.

Example 128

Synthesis of 7- [2- (tert-Butoxycarbonylamino) -1, 1-dimethyl-ethyl ] -2-chloro-pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid, Compound 128

7- [2- (tert-Butoxycarbonylamino) -1, 1-dimethyl-ethyl ] -2-chloro-pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid was synthesized using an experimental procedure similar to that described for the synthesis of 7- [2- (tert-butoxycarbonylamino) ethyl ] -2-chloro-5- (o-tolyl) pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid. LCMS (ESI) 369(M + H).

Example 129

Synthesis of Compound 129

The chlorotricyclic amide compound 129 is synthesized using a procedure similar to that described for the synthesis of chlorotricyclic amide compound 119. LCMS (ESI)251(M + H).

Example 130

Synthesis of Compound 130

Compound 130 was synthesized by treating chlorotricyclic amine compound 129 with trans-4-aminocyclohexanol using similar experimental conditions as compound 124. LCMS (ESI)330(M + H).¹HNMR(600MHz,DMSO-d₆)δppm 1.07-1.34(m,4H)1.47- 2.05(m,10H)3.09(m,1H)3.51(d,J＝2.91Hz,2H)3.57(m,1H)4.50 (br.s.,1H)6.89(s,1H)6.94-7.05(m,1H)8.04(br.s.,1H)8.60(s,1 H)9.00(br.s.,1H)。

Example 131

Synthesis of benzyl N- [1- [ [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] methyl ] propyl ] carbamate, Compound 131

Benzyl N- [1- [ [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] methyl ] propyl ] carbamate was synthesized by treating 5-bromo-2, 4-dichloropyrimidinyl with benzyl N- [1- (aminomethyl) propyl ] carbamate using experimental conditions similar to those described for the synthesis of tert-butyl N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] ethyl ] carbamate. LCMS (ESI) (M + H) 413.

Example 132

Synthesis of benzyl N- [1- [ [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] amino ] methyl ] propyl ] carbamate, Compound 132

Benzyl N- [1- [ [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] amino ] methyl ] prop-yl ] carbamate by using similar experimental conditions as described for the synthesis of tert-butyl N- [2- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] amino ] ethyl ] carbamate, prepared by treating benzyl N- [1- [ [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] methyl ] propyl ] -carbamate with 3, 3-diethoxyprop-1-yne in the presence of a catalyst such as Pddba. LCMS (ESI) (M + H) 461.

Example 133

Synthesis of benzyl N- [1- [ [ 2-chloro-6- (diethoxymethyl) pyrrolo [2,3-d ] pyrimidin-7-yl ] methyl ] propyl ] carbamate, Compound 133

Benzyl N- [1- [ [ 2-chloro-6- (diethoxymethyl) pyrrolo [2,3-d ] pyrimidin-7-yl ] methyl ] propyl ] carbamate was synthesized by treating benzyl N- [1- [ [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] amino ] methyl ] propyl ] carbamate with TBAF using experimental conditions similar to those described for the synthesis of tert-butyl N- [2- [ 2-chloro-6- (diethoxymethyl) pyrrolo [2,3d ] pyrimidin-7-yl ] ethyl ] carbamate. LCMS (ESI) (M + H) 461.

Example 134

Synthesis of 7- [2- (benzyloxycarbonylamino) butyl ] -2-chloro-pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid, Compound 134

7- [2- (Benzoxycarbonylamino) butyl ] -2-chloro-pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid was synthesized using an analogous experimental procedure as described for the synthesis of 7- [2- (tert-butoxycarbonylamino) ethyl ] -2-chloro-5- (o-tolyl) pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid. LCMS (ESI)403(M + H).

Example 135

Synthesis of Compound 135

To 7- [2- (benzyloxycarbonylamino) butyl]-2-chloro-pyrrolo [2,3-d]HBr was added to a solution of pyrimidine-6-carboxylic acid in dichloromethane and the reaction was stirred at 45 ℃ for 3 hours. After concentration, 2N NaOH was added to basify (pH 8.0) the reaction followed by addition of THF (20 mL). Followed by the addition of Boc₂O (1.2eq) and the reaction was stirred for 16 hours. To the crude reaction mixture was then added ethyl acetate (100mL) and water (50mL) and the organic phase was separated, dried (magnesium sulfate) and then concentrated in vacuo. To the crude product was added dichloromethane (30mL) followed by DIC and DMAP. After stirring for 2 hours, the mixture was stirred,TFA was added and the contents were stirred for one hour. The solvent was evaporated in vacuo and saturated NaHCO₃Basifying the residue. Ethyl acetate was then added and the organic layer was separated, dried (magnesium sulfate) and then concentrated in vacuo. Column chromatography with hexane/ethyl acetate (0-100%) gave the desired chlorotricyclic core, compound 135. LCMS (ESI)251 (M + H).

Example 136

Synthesis of Compound 136

Compound 136 was synthesized by treating chlorotricyclic amine compound 135 with trans-4-aminocyclohexanol using similar experimental conditions as compound 124. LCMS (ESI)330(M + H).¹HNMR(600MHz,DMSO-d₆)δppm 0.80-0.95(m,3H)1.35- 1.92(m,10H)3.66(br.m.,3H)4.17(br.s.,2H)4.47(br.s.,1H)6.85 (s,1H)6.96(br.s.,1H)8.15(br.s.,1H)8.62(br.s.,1H)。

Example 137

Synthesis of tert-butyl N- [1- [ [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] methyl ] cyclopentyl ] carbamate, Compound 137

N- [1- [ [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] methyl ] cyclopentyl ] carbamic acid tert-butyl ester was synthesized by treating 5-bromo-2, 4-dichloropyrimidine with N- [1- (aminomethyl) cyclopentyl ] carbamic acid tert-butyl ester using experimental conditions similar to those described for the synthesis of N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] ethyl ] carbamic acid tert-butyl ester. LCMS (ESI)405(M + H).

Example 138

Synthesis of tert-butyl N- [1- [ [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] amino ] methyl ] cyclopentyl ] carbamate, Compound 138

Tert-butyl N- [1- [ [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] amino ] methyl ] cyclopentyl ] carbamate by using similar experimental conditions as described for the synthesis of tert-butyl N- [2- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] amino ] ethyl ] carbamate, synthesized by treating tert-butyl N- [1- [ [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] methyl ] cyclopentyl ] carbamate with 3, 3-diethoxyprop-1-yne in the presence of a catalyst such as Pddba. LCMS (ESI)453(M + H).

Example 139

Synthesis of tert-butyl N- [1- [ [ 2-chloro-6- (diethoxymethyl) pyrrolo [2,3-d ] pyrimidin-7-yl ] methyl ] cyclopentyl ] carbamate, Compound 139

Tert-butyl N- [1- [ [ 2-chloro-6- (diethoxymethyl) pyrrolo [2,3-d ] pyrimidin-7-yl ] methyl ] cyclopentyl ] carbamate was synthesized by treating tert-butyl N- [2- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] amino ] -2-methyl-propyl ] carbamate with TBAF using experimental conditions similar to those described for the synthesis of tert-butyl N- [2- [ 2-chloro-6- (diethoxymethyl) pyrrolo [2,3d ] pyrimidin-7-yl ] ethyl ] carbamate. LCMS (ESI)453(M + H).

Example 140

Synthesis of 7- [ [1- (tert-butoxycarbonylamino) cyclopentyl ] methyl ] -2-chloro-pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid, Compound 140

7- [ [1- (tert-butoxycarbonylamino) cyclopentyl ] methyl ] -2-chloro-pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid was synthesized using a similar experimental procedure as described for the synthesis of 7- [2- (tert-butoxycarbonylamino) ethyl ] -2-chloro-5- (o-tolyl) pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid. LCMS (ESI)395 (M + H).

Example 141

Synthesis of Compound 141

The chlorotricyclic core compound 141 was synthesized using an experimental procedure similar to that described for the synthesis of chlorotricyclic amide compound 119. LCMS (ESI)277(M + H).

Example 142

Synthesis of Compound 142

Compound 142 was synthesized by treating chlorotricyclic amine compound 141 with trans-4-aminocyclohexanol using similar experimental conditions as compound 124. LCMS (ESI)356(M + H).¹HNMR(600MHz,DMSO-d₆)δppm 1.08-1.32(m,8H)1.60- 2.09(m,8H)3.03-3.17(m,1H)3.35(s,2H)3.54-3.62(m,1H)4.51 (d,J＝4.39Hz,1H)6.88(s,1H)6.96(br.s.,1H)8.07(br.s.,1H)8.58 (s,1H)。

Example 143

Synthesis of tert-butyl N- [ [1- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] cyclopentyl ] methyl ] carbamate, Compound 143

N- [ [1- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] cyclopentyl ] methyl ] carbamic acid tert-butyl ester was synthesized by treating 5-bromo-2, 4-dichloropyrimidine with tert-butyl N- [ (1-aminocyclopentyl) methyl ] carbamate using experimental conditions similar to those described for the synthesis of N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] ethyl ] carbamic acid tert-butyl ester. LCMS (ESI)405(M + H).

Example 144

Synthesis of N- [2- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] amino ] -2-methyl-propyl ] carbamic acid tert-butyl ester, Compound 144

N- [ [1- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] amino ] cyclopentyl ] methyl ] carbamic acid tert-butyl ester by using experimental conditions similar to those described for the synthesis of N- [2- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] amino ] ethyl ] carbamic acid tert-butyl ester, synthesized by treating tert-butyl N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] -2-methyl-propyl ] carbamate with 3, 3-diethoxyprop-1-yne in the presence of a catalyst such as Pddba.

LCMS(ESI)453(M+H)。

Example 145

Synthesis of tert-butyl N- [ [1- [ 2-chloro-6- (diethoxymethyl) pyrrolo [2,3-d ] pyrimidin-7-yl ] cyclopentyl ] methyl ] carbamate, Compound 145

Tert-butyl N- [ [1- [ 2-chloro-6- (diethoxymethyl) pyrrolo [2,3-d ] pyrimidin-7-yl ] cyclopentyl ] methyl ] carbamate was synthesized by treating tert-butyl N- [2- [ [ 2-chloro-5- (3, 3-diethoxyprop-1-ynyl) pyrimidin-4-yl ] amino ] -2-methyl-propyl ] carbamate with TBAF using experimental conditions similar to those described for the synthesis of tert-butyl N- [2- [ 2-chloro-6- (diethoxymethyl) pyrrolo [2,3d ] pyrimidin-7-yl ] ethyl ] carbamate. LCMS (ESI)4534(M + H).

Example 146

Synthesis of 7- [2- (tert-Butoxycarbonylamino) -1, 1-dimethyl-ethyl ] -2-chloro-pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid, Compound 146

7- [2- (tert-Butoxycarbonylamino) -1, 1-dimethyl-ethyl ] -2-chloro-pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid was synthesized using an experimental procedure similar to that described for the synthesis of 7- [2- (tert-butoxycarbonylamino) ethyl ] -2-chloro-5- (o-methylphenyl) pyrrolo [2,3-d ] pyrimidine-6-carboxylic acid. LCMS (ESI) 395(M + H).

Example 147

Synthesis of Compound 147

The chlorotricycloamide compound 147 was synthesized using a similar experimental procedure as described for the chlorotricycloamide compound 119. LCMS (ESI)277(M + H).

Example 148

Synthesis of Compound 148

Compound 148 was synthesized by treating chlorotricyclo amine compound 147 with trans-4-aminocyclohexanol using similar experimental conditions as compound 124. LCMS (ESI)356(M + H).¹HNMR(600MHz,DMSO-d₆)δppm 1.06-1.35(m,8H)1.45- 1.95(m,8H)3.10(m,1H)3.58(br.s.,2H)3.95(br.s.,1H)4.49(br.s., 1H)6.84(s,1H)6.85-6.93(m,1H)8.29(s,1H)8.61(br.s.,1H)。

Example 149

Synthesis of Compound 149

Step 1: compound 59 was Boc protected according to the procedure of a. sarkar et al (JOC,2011,76, 7132-.

Step 2: boc protected Compound 59 with 5 mol% NiCl₂(Ph₃)₂0.1eq triphenylphosphine, 3eq Mn, 0.1eq tetraethylammonium iodide in DMI in CO₂(1atm) at 25 ℃ for 20 hours to convert the aryl halide derivative to formic acid.

And step 3: the formic acid from step 2 is converted to the corresponding acid chloride using standard conditions.

And 4, step 4: the acid chloride from step 3 is reacted with N-methylpiperazine to give the corresponding amide.

And 5: deprotection of the amide from step 4 using methylene chloride containing trifluoroacetic acid yields the target compound. Compound 149 was purified by silica gel column chromatography eluting with a dichloromethane-methanol gradient to afford compound 149.

Compounds 119 to 149 each and with various Rs⁸、R¹And Z may be reacted with sodium hydride and an alkyl halide or other halide to insert the desired R substitution prior to reaction with the amine, such as described above for synthetic compound 120, to yield the desired product of formula I, II, III, IV or V.

Example 150

CDK4/6 inhibition in vitro assay

Selected compounds disclosed herein were tested in CDK 4/cyclin D1, CDK6/CycD3CDK2/CycA and CDK 2/cyclin E kinase assays by Nanosyn (Santa Clara, CA) to determine their inhibitory effect on these CDKs. The analysis was performed using the microfluidic kinase detection technique (Caliper Assay Platform). Compounds were tested individually for ATP in a 12-point dose-response format at Km. For all assays, the concentration of phosphorylated receptor substrate peptide used was 1 μ M, and for all assays staurosporine was used as reference compound. The details of each assay are as follows:

CDK 2/cyclin a: enzyme concentration: 0.2 nM; ATP concentration: 50 mu M; incubation time: for 3 hours.

CDK 2/cyclin E: enzyme concentration: 0.28 nM; ATP concentration: 100 mu M; incubation time: for 1 hour.

CDK 4/cyclin D1: enzyme concentration: 1 nM; ATP concentration: 200 mu M; incubation time: for 10 hours.

CDK 6/cyclin D3: enzyme concentration: 1 nM; ATP concentration: 300 mu M; incubation time: for 3 hours.

Inhibition IC of CDK4/CycD1, CDK2/CycE, CDK2/CycA by the compounds in Table 1₅₀The values and the fold selectivities are presented in table 2.

Table 2: selective inhibition of CDK4

To further characterize its kinase activity, Discovexs KINOMEscan was used^TMThe spectral analysis service screened compound T against 456 (395 non-mutated) kinases. A single concentration of 1000 nM (of IC50 for Cdk 4) was used>1000 fold) screening compounds. The results from this screen demonstrate high potency against Cdk4 and high selectivity relative to Cdk 2. In addition, kinase profiling analysis showed that compound T was relatively selective for Cdk4 and Cdk6 compared to the other kinases tested. Specifically, when using an inhibition threshold of 65%, 90% or 99%, compound T inhibited 92 (23.3%), 31 (7.8%) or 6 (1.5%) of 395 non-mutated kinases, respectively.

In addition to CDK4 kinase activity, several compounds were tested for CDK6 kinase activity. The results of the CDK6/CycD3 kinase assay and CDK 4/cyclin D1, CDK2/CycA and CDK 2/cyclin E kinase assays are shown in table 3 for PD0332991 (reference) and compounds T, Q, GG and U. IC for CDK 4/cyclin D110 nM and for CDK12/cyclin E10 uM₅₀In good agreement with previous published reports on PD0332991 (Molecular Cancer Therapeutics (2004) 3(11) 1427-. Compounds T, Q, GG and U were more potent (IC) than the reference compound (PD0332991) ₅₀Lower) and demonstrates a higher fold selectivity relative to the reference compound (CDK2/CycE IC)₅₀Divided by CDK4/CycD1IC₅₀)。

Table 3: inhibition of CDK kinase by Compounds T, Q, GG and U

Example 151

G1 arrest (cell G1 and S phase) analysis

To determine the cell fraction at various stages of the cell cycle after various treatments, HS68 cells (human skin fibroblast cell line (Rb positive)) were stained with propidium iodide staining solution and run on a Dako Cyan flow cytometer. The fraction of cells in the G0-G1DNA cell cycle versus the fraction in the S phase DNA cell cycle was determined using the FlowJo 7.2.2 assay.

The compounds listed in table 1 were tested for their ability to arrest HS68 cells in the G1 phase of the cell cycle. From the results of the cell G1 arrest assay, the inhibition of EC required for G1 arrest of HS68 cells₅₀Values ranged from 22nM to 1500nM (see Table 4 entitled "cell G1 arresting EC)₅₀"column).

Example 152

Cdk4/6 dependent cell compound T arrests cell cycle

To test the ability of CDK4/6 inhibitors to induce complete G1 arrest, a cell-based screening method was used consisting of two CDK 4/6-dependent cell lines (tHS68 and WM 2664; Rb positive) and one CDK 4/6-independent (a 2058; Rb negative) cell line. Twenty-four hours after plating, each cell line was treated with compound T in a dose-dependent manner for 24 hours. At the end of the experiment, cells were harvested, fixed and stained with propidium iodide (DNA intercalator) and emitted intense red fluorescence (emission maximum 637nm) when excited by 488nm light. Samples were run on a Dako Cyan flow cytometer and >10,000 events were collected for each sample. Data were analyzed using FlowJo 2.2 software developed by TreeStar, inc.

Figure 2B is a graph of the number of tHS68 cells (CDK4/6 dependent cell line) versus the DNA content of the cells (as measured by propidium iodide). Cells were treated with DMSO for 24 hours, harvested and analyzed for cell cycle distribution. FIG. 2C is a plot of the number of WM2664 cells (CDK4/6 dependent cell line) versus the DNA content of the cells (as measured by propidium iodide). Cells were treated with DMSO for 24 hours, harvested and analyzed for cell cycle distribution. Figure 2D is a graph of the number of a2058 cells (CDK 4/6-independent cell line) versus the DNA content of the cells (as measured by propidium iodide). Cells were treated with DMSO for 24 hours, harvested and analyzed for cell cycle distribution. Figure 2E is a graph of the number of tHS68 cells (CDK4/6 dependent cell line) versus the DNA content of the cells (as measured by propidium iodide) after treatment with compound T. Cells were treated with compound T (300nM) for 24 hours, harvested and analyzed for cell cycle distribution. Treatment of tHS68 cells with compound T caused a loss of the S phase peak (indicated by the arrow) as described in example 152. Figure 2F is a graph of the number of WM2664 cells (CDK4/6 dependent cell line) versus the DNA content of the cells (as measured by propidium iodide) after treatment with compound T. Cells were treated with compound T (300nM) for 24 hours, harvested and analyzed for cell cycle distribution. As described in example 152, treatment of WM2664 cells with compound T caused a loss of the S-phase peak (indicated by the arrow). Figure 2G is a graph of the number of a2058 cells (CDK4/6 independent cell line) versus the DNA content of the cells (as measured by propidium iodide) after treatment with compound T. Cells were treated with compound T (300nM) for 24 hours, harvested and analyzed for cell cycle distribution. As described in example 152, treatment of a2058 cells with compound T did not cause loss of the S-phase peak (indicated by the arrow).

Example 153

Compound T inhibits phosphorylation of RB

The Cdk 4/6-cyclin D complex is essential for the progression of the DNA cell cycle from G1 to S phase. This complex phosphorylates retinoblastoma tumor suppressor protein (Rb). To demonstrate the effect of Cdk4/6 inhibition on Rb phosphorylation (pRb), compound T was exposed to three cell lines, two Cdk4/6 dependent (tHS68, WM 2664; Rb positive) and one Cdk4/6 independent (a 2058; Rb negative). Twenty-four hours after inoculation, cells were treated with compound T at 300nM final concentration for 4, 8, 16 and 24 hours. The samples were solubilized and analyzed by western blot analysis. Rb phosphorylation was measured using species-specific antibodies at two sites Ser780 and Ser807/811 targeted by the Cdk4/6 cyclin D complex. The results demonstrate that compound T blocks Rb phosphorylation in Rb-dependent cell lines by 16 hours post-exposure while having no effect on Rb-independent cells (fig. 3).

Example 154

Small Cell Lung Carcinoma (SCLC) cells resistant to CDK4/6 inhibitors

Retinoblastoma (RB) tumor suppressor is the major negative cell cycle regulator that is inactivated in approximately 11% of all human cancers. Functional loss of RB is an obligatory event in the development of Small Cell Lung Cancer (SCLC). In RB-type tumors, activated Cdk2/4/6 promotes progression of G1 to S phase by phosphorylating and inactivating RB (and related family members). In contrast, Cdk4/6 activity is not required for cell cycle progression in RB deleted or inactivated cancers. Since RB inactivation is an obligate event for SCLC development, this tumor type is highly resistant to CDK4/6 inhibitors, and co-administration of CDK4/6 inhibitors with DNA-destroying chemotherapeutic agents such as those used in SCLC should not antagonize the efficacy of such agents.

Several compounds (PD0332991, compound GG and compound T) were tested for their ability to block cell proliferation in a panel of SCLC cell lines with known genetic loss of RB. SCLC cells were treated with DMSO or indicated CDK4/6 inhibitor for 24 hours. The effect of Cdk4/6 inhibition on proliferation was measured by EdU incorporation. Growth inhibition of RB intact Cdk4/6 dependent cell lines (WM2664 or tHS68) and a panel of RB negative SCLC cell lines (H69, H82, H209, H345, NCI417 or SHP-77) by various CDK4/6 inhibitors were analyzed.

As shown in FIG. 4, Rb negative SCLC cells were resistant to Cdk4/6 inhibition. In fig. 4A, PD0332991 inhibited the growth of Rb-positive cell lines (WM2664), but did not affect the growth of Rb-negative small cell lung cancer cell lines (H345, H69, H209, SHP-77, NCI417 and H82). In FIG. 4B, compound GG inhibited the growth of Rb-positive cell lines (tHS68), but not Rb-negative cell lines (H345, H69, SHP-77, and H82). In FIG. 4C, compound T inhibited the growth of the Rb-positive cell line (tHS68), but did not affect the growth of the Rb-negative cell lines (H69, SHP-77, and H209). This analysis demonstrated that RB-null SCLC cell lines were resistant to Cdk4/6 inhibition, since no change in the percentage of S-phase cells was seen after treatment with any Cdk4/6 inhibitor tested, including compound T and compound GG, whereas RB-sufficient cell lines in each experiment had high sensitivity to Cdk4/6 inhibition, with almost no cells remaining in S-phase 24 hours after treatment.

Example 155

Rb negative cancer cells are resistant to CDK4/6 inhibitors

Cell proliferation assays were performed using the following Rb negative cancer cell lines: h69 (human small cell lung cancer-Rb negative) or a2058 (human metastatic melanoma cells-Rb negative). These cells were seeded in tissue culture treated white wall/clear trays in Costar (Tewksbury, Massachusetts) 309396 wells. Cells were treated with the compound of table 1 in nine-point dose response serial dilutions of 10uM to 1 nM. Cells were exposed to the compounds and then four days (H69) or six days (A2058) later, as indicated, CellTiter-Electroluminescent cell viability assay (CTG; Promega, Madison, Wisconsin, United States of America), cell viability was determined according to the manufacturer's recommendations. The discs were read on a BioTek (Winooski, Vermont) Syngergy2 multimode disc reader. Relative Light Units (RLU) were plotted as a result of variable molar concentrations, and the data was analyzed using Graphpad (LaJolla, california) Prism 5 statistical software to determine the EC of each compound₅₀。

Evaluation of the disclosure herein against small cell lung cancer cell line (H69) and human metastatic melanoma cell line (a2058) (two Rb deficient (Rb negative) cell lines) Of (a) a selected compound of (b). The results of these cytostatic assays are shown in table 4. Inhibition of EC for H69 small cell lung carcinoma cell inhibition₅₀Values range from 2040nM to>3000 nM. Inhibition of EC for inhibition of proliferation of A2058 malignant melanoma cells₅₀The value ranges from 1313nM to>3000 nM. The tested compounds were found not to be significantly effective in inhibiting proliferation of small cell lung cancer or melanoma cells, compared to the significant inhibition seen on Rb positive cell lines.

Table 4: resistance of Rb negative cancer cells to CDK4/6 inhibitors

Example 156

HSPC growth inhibition study

The effect of PD0332991 on HSPC has been previously demonstrated. FIG. 5 shows EdU incorporation of mouse HSPC and spinal cord progenitor cells following a single dose of 150mg/kg PD0332991 by oral cavity tube feeding to assess the transient CDK4/6 inhibition on the temporal effects of bone marrow arrest, e.g., Multiple circles of cycle-Dependent Kinase 4/6Inhibitors in Cancer therapy.JCNI 2012 Roberts et al; 104(6) 476, 487. As can be seen in figure 5, a single oral dose of PD0332991 caused a sustained reduction in HSPC (LKS +) and spinal cord progenitor cells (LKS-) for more than 36 hours. Until 48 hours after oral administration, HSPCs and spinal cord progenitors do not restore baseline cell division.

Example 157

Bone marrow proliferation as assessed using EdU incorporation and flow cytometry analysis

For HSPC proliferation experiments, young adult female FVB/N mice were treated by oral gavage with a single dose of compound T, compound Q, compound GG or PD0332991 as indicated. Mice were then sacrificed at the indicated times (0, 12, 24, 36 or 48 hours post compound administration) and bone marrow harvested as previously described (n ═ 3 mice per time point) (Johnson et al j. clin. invest. (2010)120(7), 2528-. Four hours prior to bone marrow harvest, mice were treated with 100 μ g EdU by intraperitoneal injection (Invitrogen). Using the methods described previously, bone marrow mononuclear cells were harvested and immunophenotypic analysis was performed, and then the percent EdU-positive cells determined (Johnson et al j. clin. invest. (2010)120(7), 2528-. Briefly, HSPC were identified by the expression of lineage markers (Lin-), Sca1(S +) and c-Kit (K +).

Analysis in mice determined that compound T, compound Q and compound GG demonstrated dose-dependent, transient and reversible G1 arrest of bone marrow stem cells (HSPCs) (fig. 6). Six mice per group were dosed by oral gavage at 150mg/kg compound T, compound Q, compound GG or vehicle only. Four hours prior to sacrifice and bone marrow harvest, mice were treated with 100 μ g EdU by intraperitoneal injection. Three mice per group were sacrificed at 12 hours and the remaining three animals per group were sacrificed at 24 hours. The results are shown in fig. 6A as the ratio of EdU positive cells for treated animals compared to controls at 12 or 24 hour time points. Compounds T and GG demonstrated a decrease in EdU incorporation at 12 hours, which began to return to normal at 24 hours. Compound Q also demonstrated a slight decrease at 12 hours and began to return to baseline at 24 hours, although compound Q was poorly bioavailable orally.

Longer. Compound T was administered at 50, 100 or 150mg/kg by oral gavage and EdU incorporation in bone marrow was determined at 12 and 24 hours as described above. Alternatively, 150mg/kg of compound T was administered by oral gavage and EdU incorporation in bone marrow was determined at 12, 24, 36 and 48 hours. As can be seen in fig. 6B and 6C and similar to the cell elution experiments, bone marrow cells and in particular HSPCs were restored to normal cell division in 24 hours after oral gavage with multiple doses as determined by EdU incorporation. The 150mg/kg oral dose of compound T in figure 6C can be directly compared to the results for the same dose of PD0332991 shown in figure 5, where cells still did not divide at 24 and 36 hours (as determined by low EdU incorporation), returning to normal values only at 48 hours.

Example 158

HSPC growth inhibition study comparing Compound T and PD0332991

Figure 7 is a graph of the percentage of EdU positive HSPC cells of mice treated with PD0332991 (triangles) or compound T (inverted triangles) compared to the time (hours) after compound administration. Both compounds were administered at 150mg/kg by oral gavage. One hour prior to bone marrow harvest, EdU was injected intraperitoneally to label circulating cells. Bone marrow was harvested 12, 24, 36 and 48 hours after compound treatment and at each time point the percentage of EdU positive HSPC cells was determined.

As seen in figure 7, a single oral dose of PD0332991 caused a persistent decrease in HSPC for more than 36 hours. In contrast, a single oral dose of compound T caused a decrease in HSPC proliferation early in 12 hours, but by 24 hours of compound T administration HSPC proliferation began anew.

Example 159

Cell elution experiment

HS68 cells were crystallized on day 1 at 40,000 cells/well in 60mm dishes in DMEM containing 10% fetal bovine serum, 100U/ml penicillin (penicillin)/streptomycin (streptomycin), and 1 XGlutamax (Invitrogen) as described (Brooks et al EMBO J,21(12) 2936-. 24 hours after inoculation, cells were treated with compound T, compound Q, compound GG, compound U, PD0332991, or DMSO vehicle alone at a final concentration of 300nM of test compound. On day 3, a set of treated cell samples (0 hour samples) were harvested in triplicate. The remaining cells were washed twice in PBS-CMF and returned to the medium lacking the test compound. Samples were harvested in triplicate at 24, 40 and 48 hours into each group.

Alternatively, the same experiment was performed using normal renal proximal tubule epithelial cells (Rb positive) obtained from american type culture collection (ATCC, Manassas, VA). Cells were grown in a kidney epithelial cell basal medium (ATCC) supplemented with a kidney epithelial cell growth kit (ATCC) in a humidified incubator at 37 ℃ in a 5% CO2 humidified atmosphere at 37 ℃.

After harvesting the cells, the samples were stained with propidium iodide staining solution and the samples were run on a Dako Cyan flow cytometer. The fraction of cells in the G0-G1DNA cell cycle versus the fraction in the S phase DNA cell cycle was determined using the FlowJo 7.2.2 assay.

Figure 8 shows cell elution experiments demonstrating that the inhibitor compounds of the invention have a transient, transient G1 arrest in different cell types. Compounds T, Q, GG and U were compared to PD0332991 in human fibroblasts (Rb positive) (fig. 8A and 8B) or human renal proximal tubule epithelial cells (Rb positive) (fig. 8C and 8D), and the effect on the cell cycle after compound elution was determined at 24, 36, 40 and 48 hours.

As shown in figure 8 and similar to the in vivo results as shown in figure 5, PD0332991 requires cells to return to normal baseline cell division more than 48 hours after elution. This is seen in fig. 8A and 8B, since the values obtained are correspondingly equal to the G0-G1 fraction of cell division or the value of DMSO control for S phase. In contrast, HS68 cells treated with the compounds of the invention returned to normal baseline cell division within as little as 24 hours or 40 hours, at these same time points, unlike PD 0332991. The results using human renal proximal tubule epithelial cells (fig. 8C and 8D) also show that cells treated with PD0332991 took significantly longer to return to baseline levels compared to cells treated with compound T, Q, GG or U.

Example 160

Pharmacokinetic and pharmacodynamic Properties of CDK4/6 inhibitors

The compounds of the present invention demonstrate superior pharmacokinetic and pharmacodynamic properties. Compounds T, Q, GG and U were administered at 30mg/kg by oral gavage or 10mg/kg by intravenous injection. Blood samples were taken at 0, 0.25, 0.5, 1.0, 2.0, 4.0 and 8.0 hours post-dose and plasma concentrations of compound T, Q, GG or U were determined by HPLC. As shown in table 5, compounds T, GG and U demonstrated excellent oral pharmacokinetic and pharmacodynamic properties. This includes a very high oral bioavailability (F (%)) of 52% to 80% and a plasma half-life of 3 to 5 hours after oral administration. Compounds T, Q, GG and U demonstrated excellent oral pharmacokinetic and pharmacodynamic properties when delivered by intravenous administration. Representative IV and oral PK profiles for all four compounds are shown in figure 9.

Table 5: pharmacokinetic and pharmacodynamic Properties of CDK4/6 inhibitors

Mouse PK	Compound T	Compound Q	Compound GG	Compound U
					CL(mL/min/kg)	35	44	82	52
Vss(L/kg)	2.7	5.2	7.5	3.4
					t1/2(h)p.o.	5	0.8	3.5	3
AUC 0-inf(uM*h)i.v.	1.3	0.95	1.1	0.76
					AUC(uM*h)p.o.	2.9	0.15	1.9	3.3
Cmax(uM)p.o.	2.5	0.16	1.9	4.2
					Tmax(h)p.o.	1	0.5	1	0.5
F(％)	80	2	52	67

Example 161

Metabolic stability

The metabolic stability of compound T compared to PD0332991 was determined in human, canine, rat, monkey and mouse liver microsomes. Human, mouse and canine liver microsomes were purchased from Xenotech and schorge-dory rat (Sprague-Dawley rat) liver microsomes were prepared by means of the Absorption Systems. A reaction mixture containing 0.5mg/mL liver microsomes, 100mM potassium phosphate pH 7.4, 5mM magnesium chloride and 1uM test compound was prepared. Test compound was added to the reaction mixture at a final concentration of 1 uM. An aliquot of the reaction mixture (without cofactor) was incubated in a shaking water bath at 37 ℃ for 3 minutes. The control compound testosterone was operated in a separate reaction simultaneously with the test compound. The reaction was initiated by addition of cofactor (NADPH) and the mixture was then incubated at 37 ℃ in a shaking water bath. Aliquots (100 μ L) were taken at 0, 10, 20, 30 and 60 minutes for the test compounds, and at 0, 10, 30 and 60 minutes for testosterone (100 μ L). A sample of the test compound was immediately combined with 100 μ L of ice-cold acetonitrile containing an internal standard to terminate the reaction. The testosterone samples were immediately combined with 800 μ L of ice cold 50/50 acetonitrile/dH 2O containing 0.1% formic acid and an internal standard to stop the reaction. Samples were analyzed using an efficient LC-MS/MS method. Test compound samples were analyzed using an Orbitrap high resolution mass spectrometer to quantify the disappearance of the parent test compound and detect the appearance of the metabolite. Peak Area Response Ratio (PARR) to internal standard was compared to PARR at 0 to determine the percentage of test compound or positive control retained at the time point. Half-lives were calculated using GraphPad software fitted to a monophasic exponential decay equation.

Half-life was calculated based on t1/2 ═ 0.693k, where k is the rate constant of elimination based on the slope of the plot of the residual natural log percentage versus the comparative incubation time. When the calculated half-life is longer than the duration of the experiment, the half-life is expressed as > maximum incubation time. The calculated half-lives are also listed in parentheses. If the calculated half-life > duration of experiment 2x, then no half-life is reported. Timely resumption of cell proliferation is necessary for tissue repair, and thus excessive long-term arrest in healthy cells such as HSPC is undesirable. CDK4/6 inhibitors that determine their duration of stasis are characterized by their Pharmacokinetics (PK) and enzyme half-life. Once triggered, G1 stasis in vivo will be maintained as long as circulating compounds remain at inhibitory levels and as long as the compounds occupy the enzyme. PD032991, for example, has an overall long PK half-life and a rather slow enzyme off-rate. In humans, PD0332991 shows a PK half-life of 27 hours (see Schwartz, GK et al (2011) BJC,104: 1862-. In humans, a single administration of PD0332991 produces cell cycle arrest with HSPC for approximately one week. This reflects 6 days of clearance of the compound (5 half-lives x 27 hours half-life) and an additional 1.5 to 2 days of inhibition of the function of the enzyme CDK 4/6. This calculation indicates that normal bone marrow functional recovery takes a total of 7+ days during which the production of new blood is reduced. These observations can account for the severe granulocytopenia seen clinically under PD 0332991.

Other experiments were performed with compound T and PD0332991 to compare metabolic stability (half-life) in human, canine, rat, monkey and mouse liver microsomes. As shown in figure 10, when compounds were analyzed for stability across species in liver microsomes, the measurable half-life of compound T in each species was shorter than that reported for PD 0332991. Furthermore, as previously described and shown in fig. 8, it appears that PD0332991 also has a prolonged enzyme half-life, as evidenced by significant cell cycle arrest production in human cells lasting more than forty hours, even after compound removal from the cell culture medium (i.e. in vitro elution experiments). As further shown in fig. 8, removal of the compounds described herein from the culture medium resulted in rapid resumption of proliferation, consistent with a rapid enzymatic dissociation rate. These differences in enzyme off-rates translate into significant differences in Pharmacodynamic (PD) effects, as shown in figures 5, 6C and 7. As shown, a single oral dose of PD0332991 arrested Hematopoietic Stem and Progenitor Cells (HSPCs) 36+ hour growth in murine bone marrow, which was beyond what was demonstrated by the 6 hour PK half-life of PD0332991 in mice. In contrast, compound T has a much shorter effect, allowing rapid re-entry into the cell cycle, providing good in vivo control of HSPC proliferation.

Example 162

Compound T prevents chemotherapy-induced cell death, DNA damage and caspase activation

To demonstrate that pharmacological quiescence induced by compound T treatment provides resistance to chemotherapeutic agents with different mechanisms of action, in vitro models were developed using terminally pelleted human diploid fibroblasts (tHDF; human foreskin fibroblast line immortalized with human telomerase). Proliferation of these cells is highly Cdk4/6 Dependent as evidenced by their complete G1 arrest after treatment with CDK4/6 Inhibitors (see Roberts PJ et al, Multiple Roles of cycle-Dependent Kinase 4/6Inhibitors in Cancer therapy. J Natl Cancer Inst 2012; 3 months 21; 104(6): 476-87). Cell viability was determined by Cell TiterGlo analysis, as recommended by the manufacturer. For the γ -H2AX and caspase 3/7 assays, cells were plated and allowed to attach for 24 hours. Cells were then treated with compound T (indicated concentration) or vehicle control for 16 hours, at which time indicated chemotherapy was added to the pre-treated cells. For γ -H2AX, cells were harvested for analysis at 8 hours after chemotherapy exposure. For the γ -H2AX assay, cells were fixed, permeabilized and stained with anti- γ H2AX according to the γ -H2AX flow kit (Millipore) and quantified by flow cytometry. Data were analyzed using FlowJo 2.2 software developed by TreeStar, inc. For the in vitro caspase 3/7 assay, cells were harvested 24 hours after chemotherapy treatment. Use of Caspase- 3/7 analytical System (Promega), according to the manufacturer's recommendations, measured the caspase 3/7 activation.

As shown in figure 11, compound T provided selective protection against carboplatin and etoposide-induced cell death. Treatment of tHS68 human fibroblasts with increasing concentrations of compound T in the presence of etoposide (5. mu.M; FIG. 11A) or carboplatin (100. mu.M; FIG. 11B) selectively induced dose-dependent Cell survival as determined by Cell TiterGlo.

Treatment with compound T prior to treatment with several DNA damaging agents (e.g., carboplatin, doxorubicin, etoposide, camptothecin) or antimitotic agents (paclitaxel) reduced DNA damage as measured by γ -H2AX formation (fig. 12A). In addition, treatment of tHDF cells with compound T prior to carboplatin, doxorubicin, etoposide, camptothecin, and paclitaxel exposure resulted in a robust decrease in caspase 3/7 activation in a dose-dependent manner (fig. 12B). These data show that transient G1 cell cycle arrest induced by Cdk4/6 inhibition reduces the toxicity of many commonly used cytotoxic chemotherapeutic agents associated with myelosuppression of Cdk 4/6-sensitive cells.

Example 163

Compound T inhibits the proliferation of hematopoietic stem and/or progenitor cells (HSPC)

To characterize the effect of compound T treatment on the proliferation of hematopoietic cells in different mice, a single dose of either vehicle alone (20% Solutol) or compound T (150mg/kg) was administered by oral gavage to 8 week old female C57Bl/6 mice. Ten hours later, all mice were given a single intraperitoneal injection of 100mcg EdU (5-ethynyl-2' -deoxyuridine) to label cells in the S phase of the cell cycle. At 2 hours post EdU injection, all treated mice were sacrificed, bone marrow cells were harvested and processed for flow cytometry analysis of EdU incorporation (fig. 13).

In FIG. 13, representative contour plots show proliferation in WBM (whole bone marrow; top) and HSPC (hematopoietic stem and progenitor cells; LSK; bottom) as measured by EdU incorporation of untreated, EdU only treated or EdU plus compound T treated cells. Compound T was found to reduce the proliferation of whole bone marrow and hematopoietic stem and progenitor cells.

Compound T-treated mice showed significantly less EdU positivity in all hematopoietic lineages analyzed compared to vehicle-treated mice (EdU)⁺) A cell. EdU⁺The reduction in cell frequency is likely due to a reduction in S phase entry, consistent with the fact that compound T effectively inhibits Cdk4/6 activity. Overall, compound T treatment caused a decrease in EdU + cell frequency of about 70% in unfractionated whole bone marrow cells (see fig. 13 and 14). Among Hematopoietic Stem and Progenitor Cells (HSPCs), compound T treatment resulted in hematopoietic stem cells (HSC, 74% inhibition, the most primitive in the entire hematopoietic lineage hierarchy Cells) as well as pluripotent progenitor cells (MPP, 90% suppression, immediately downstream progeny of HSC) were effectively arrested in the cell cycle (fig. 14A).

As shown in figure 14B, downstream of the lineage differentiation hierarchy, proliferation of lineage restricted spinal cord cells (CMP, GMP and MEP) and lymphoid progenitor Cells (CLP) was also significantly inhibited by compound T, demonstrating EdU⁺The cell frequency was reduced by 76-92%.

Example 164

Compound T inhibits proliferation of differentiated hematopoietic cells

The effect of compound T on the proliferation of differentiated hematopoietic cells was investigated using the same experimental protocol as discussed in example X above and shown in fig. 13 and 14. The resulting effect of compound T in differentiating hematopoietic cells is more variable than seen in HSPCs. Although T and B cell progenitors are highly sensitive to Compound T (EdU, respectively)⁺Reduction of cell frequency>99% and>80%) but proliferation of differentiated myeloid erythrocytes is more resistant to compound T, Mac1 of which⁺G1⁺Bone marrow cells display EdU⁺Cell frequency decreased by 46%, and Ter119⁺Erythrocyte-like cell display EdU⁺The cell frequency was reduced by 58% (fig. 15). Taken together, these data indicate that while all hematopoietic cells are sensitive to compound T-induced cell cycle arrest, the degree of inhibition varies among different cell lineages, with bone marrow cells displaying less effect of compound T on cell proliferation than seen in other cell lineages.

Example 165

Compound GG protects myeloid progenitor cells

To evaluate the effect of transient CDK4/6 inhibition of compound GG in bone marrow on carboplatin-induced cytotoxicity, FVB/n mice (n-3 per group) were treated with vehicle control, intraperitoneal injection of 90mg/kg carboplatin, or oral gavage of 150mg/kg compound GG plus intraperitoneal injection of 90mg/kg carboplatin. 24 hours post-treatment, bone marrow was harvested and the percentage of circulating bone marrow progenitors was measured by EdU incorporation as explained earlier. As shown in figure 16, administration of compound GG concurrently with carboplatin administration resulted in significant protection of bone marrow progenitor cells. EdU incorporation in control animals was normalized to 100% and compared to the EdU incorporation from bone marrow of carboplatin-treated animals or carboplatin and compound GG-treated animals.

Example 166

Compound T decreases 5 FU-induced myelosuppression

To determine the ability of compound T to modulate chemotherapy-induced myelosuppression, a well-characterized single dose 5-fluorouracil (5FU) regimen known to be highly myelosuppressive in mice was utilized. FVB/n female mice were administered a single oral dose of 150mg/kg vehicle or compound T, 30 minutes later with a single intraperitoneal dose of 150mg/kg 5 FU. Complete blood counts were measured every two days, starting on day six.

Co-administration of compound T positively affected the recovery of all hematopoietic lineages from 5-FU induced myelosuppression. Figure 17 demonstrates the recovery time course of different blood cell types treated with compound T or vehicle control prior to 5FU administration. It was determined that compound T provided more rapid recovery in each of the hematopoietic cell lineages tested (whole blood cells, neutrophils, lymphocytes, platelets, and erythrocytes) than cells treated with 5FU alone. These data show that compound T treatment may reduce 5 FU-induced DNA damage in HSPCs, leading to accelerated recovery of blood counts after chemotherapy.

Figure 18 shows data from day 14 of the myelosuppression study described above and shown in figure 17. Whole blood cell counts were analyzed on day 14. Fig. 18 shows the results for white blood cells (fig. 18A), neutrophils (fig. 18B), lymphocytes (fig. 18C), red blood cells (fig. 18D), and platelets (fig. 18E). In all cases, compound T caused significant protection of each cell type on day 14 when administered with 5FU, compared to myelosuppression of 5FU treatment alone.

Example 167

Compound T reduces 5 FU-induced myelosuppression by repeated cycles of 5FU treatment

To determine the ability of compound T to modulate chemotherapy-induced myelosuppression, a well-characterized 5-fluorouracil (5FU) regimen known to be highly myelosuppressive in mice was utilized. Female C57Bl/6 mice of 8 weeks of age were administered a single oral dose of 150mg/kg vehicle (20% Solutol) or compound T, 150mg/kg intraperitoneal dose of 5FU after 30 minutes. This protocol was repeated every 21 days, with 3 cycles. On day 10 of cycles 1-3, blood samples were taken for hematological analysis.

Co-administration of compound T reduced myelosuppression on day 10 of the third cycle (figure 19) as well as other cycles (data not shown). These data demonstrate that compound T treatment may reduce 5 FU-induced DNA damage in HSPCs, leading to improved hematopoietic counts, according to the single dose study described above.

Example 168

DNA cell cycle analysis in human renal proximal tubule cells

To test the ability of CDK4/6 inhibitors to induce complete G1 arrest in nonhematopoietic cells, G1 arrest was examined in human renal proximal tubule cells. Cells were treated with compound T in a dose-dependent manner for 24 hours. At the end of the experiment, cells were harvested, fixed and stained with propidium iodide (DNA intercalator) and emitted intense red fluorescence (emission maximum 637nm) when excited by 488nm light. Samples were run on a Dako Cyan flow cytometer. Data were analyzed using FlowJo 2.2 software developed by TreeStar, inc. The analysis was performed in triplicate and error bars were undetectable. As seen in fig. 20, the results show that compound T induces robust G1 cell cycle arrest in human renal proximal tubule cells, as almost all cells were found in G0-G1 phase after treatment with increasing amounts of compound T.

Example 169

Compound T protects renal proximal tubule epithelial cells from chemotherapy-induced DNA damage

Etoposide and cisplatin were used to analyze the ability of CDK4/6 inhibitors to protect human renal proximal tubule cells from chemotherapy-induced DNA damage. Cells were treated with compound T in a dose-dependent manner (10nM, 30nM, 100nM, 300nM or 1000 nM). At the end of the experiment, cells were harvested, fixed and stained with propidium iodide (DNA intercalator) and emitted intense red fluorescence (emission maximum 637nm) when excited by 488nm light. Samples were run on a Dako Cyan flow cytometer. Data were analyzed using FlowJo 2.2 software developed by TreeStar, inc. As seen in figure 21, the results demonstrate that compound T protects renal proximal tubular epithelial cells from chemotherapy-induced DNA damage, as increasing doses of compound T in combination with etoposide or cisplatin caused a decrease in the percentage of cells in the S phase, and correspondingly, an increase in the percentage of cells in the G0-G1 phases.

Example 170

Compound T prevents chemotherapy-induced cell death, DNA damage and caspase activation in human renal proximal tubule cells

To demonstrate that CDK4/6 inhibitor treatment-induced pharmacological quiescence provides resistance to chemotherapeutic agents in non-hematopoietic cells, compound T was assayed for its protective effect on human renal proximal tubule cells. Normal renal proximal tubular epithelial cells were obtained from the American Type Culture Collection (ATCC, Manassas, Va.). Cells were grown in an incubator at 37 ℃ in a 5% CO2 humid atmosphere at 37 ℃ in a humid incubator supplemented with a renal epithelial cell growth kit (ATCC) in a renal epithelial cell basal medium (ATCC). Cells were treated with DMSO or 10nM, 30nM, 100nM, 300nM or 1uM compound T in the absence or presence of 25uM cisplatin. For the γ -H2AX assay, cells were fixed, permeabilized and stained with anti- γ H2AX according to the γ -H2AX flow kit (Millipore) and quantified by flow cytometry. Data were analyzed using FlowJo 2.2 software developed by TreeStar, inc. Caspase 3/7 activation was measured using the Caspase-Glo 3/7 assay system (Promega, Madison, WI) by following the manufacturer's instructions.

Treatment of renal proximal tubule cells with compound T in combination with cisplatin attenuated DNA damage as measured by γ -H2AX formation (fig. 22). As seen in figure 22, DNA damage caused by cisplatin decreased in a dependent manner following compound T treatment.

The ability of compound T to protect renal proximal tubular epithelial cells from cisplatin-induced apoptosis (caspase 3/7 activation) was also investigated. As shown in figure 23, compound T demonstrated a dose-dependent reduction in caspase 3/7 activation in these cells. This reduction in caspase 3/7 activity was seen at all three levels of cisplatin tested (25uM, 50uM, or 100 uM). These data show that transient G1 cell cycle arrest induced by Cdk4/6 inhibition can protect renal proximal tubule cells from chemotherapy-induced DNA damage.

Example 171

Preparation of pharmaceutical products

The active compounds of the present invention can be prepared for intravenous administration using the following procedure. The excipients hydroxypropyl- β -cyclodextrin and dextrose may be added to 90% batch volume USP sterile injection or irrigation water with agitation; stirring until dissolved. The active compound in the form of the hydrochloride salt is added and stirred until it is dissolved. The pH is adjusted to pH 4.3+0.1 with 1N NaOH, and 1N HCl can be used for back titration if necessary. USP sterile injection water or irrigation water can be used to bring the solution to the final batch weight. The pH was then checked again to ensure that the pH was pH 4.3+ 0.1. If the pH is outside the range, 1N HCl or 1N NaOH is optionally added to bring the pH to 4.3+ 0.1. The solution was then sterile filtered to fill 50 or 100 mL flint glass vials, capped and crimped.

The present specification has been described with reference to embodiments of the invention. The invention has been described with reference to classification embodiments illustrated in the accompanying examples. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Given the teachings herein, one skilled in the art will be able to modify the invention to achieve the desired objectives and such modifications are considered to be within the scope of the invention.

Claims (22)

1. Cyclin dependent kinase 4/6(CDK4/6) inhibitor compounds of the formula

Or a pharmaceutically acceptable salt thereof, for use in the manufacture of a medicament for reducing the effect of chemotherapy on healthy cells in a human being treated for cyclin dependent kinase 4/6(CDK4/6) replication independent small cell lung cancer, wherein the healthy cells are hematopoietic stem cells or hematopoietic progenitor cells, the chemotherapy comprising administering an effective amount of a chemotherapeutic selected from carboplatin, cisplatin, etoposide, or a combination thereof, wherein the CDK4/6 inhibitor is administered to the human about 24 hours or less prior to administration of the chemotherapeutic.

2. The use of claim 1, wherein the chemotherapeutic agent is carboplatin and etoposide.

3. The use of claim 2, wherein the CDK4/6 inhibitor is administered to the human about 4 hours or less prior to administration of carboplatin and etoposide.

4. The use of claim 1, wherein the chemotherapeutic agent is cisplatin and etoposide.

5. The use of claim 4, wherein the CDK4/6 inhibitor is administered to the human about 4 hours or less prior to the administration of cisplatin and etoposide.

6. CDK4/6 inhibitor compounds of the formula

Or a pharmaceutically acceptable salt thereof, and at least one chemotherapeutic agent in the manufacture of a medicament for the treatment of cancer in a human, wherein the inhibitor of CDK4/6 reduces the effect of the chemotherapeutic agent on CDK 4/6-dependent healthy cells and the inhibitor of CDK4/6 is administered to the human about 24 hours or less prior to the administration of the chemotherapeutic agent.

7. The use of claim 6, wherein the CDK4/6 inhibitor is administered to the human about 4 hours or less prior to the administration of the chemotherapeutic.

8. The use of claim 6 or 7, wherein the at least one chemotherapeutic agent is selected from alkylating agents, DNA intercalating agents, protein synthesis inhibitors, inhibitors of DNA or RNA synthesis, DNA base analogs, topoisomerase inhibitors, telomerase inhibitors, or telomere DNA binding compounds.

9. The use of claim 6 or 7, wherein the chemotherapeutic agent is selected from carboplatin, etoposide, cisplatin, gemcitabine, topotecan, paclitaxel, docetaxel, doxorubicin, cyclophosphamide, vincristine, vinblastine, irinotecan, camptothecin, fluorouracil, or a combination thereof.

10. The use of claim 6 or 7, wherein the cancer is selected from lung cancer, breast cancer, cervical cancer, colorectal cancer, HPV-positive head and neck cancer, prostate cancer, retinoblastoma, osteosarcoma, ovarian cancer, and Rb-negative bladder cancer.

11. The use of claim 10, wherein the cancer is lung cancer.

12. The use of claim 11, wherein the lung cancer is small cell lung cancer.

13. The use of claim 10, wherein the cancer is breast cancer.

14. The use of claim 13, wherein the breast cancer is triple negative breast cancer.

15. The cancer of claim 10, wherein the cancer is colorectal cancer.

16. The use of claim 10, wherein the cancer is cervical cancer.

17. The use of claim 10, wherein the cancer is an HPV-positive head and neck cancer.

18. The use of claim 10, wherein the cancer is prostate cancer.

19. The use of claim 10, wherein the cancer is retinoblastoma.

20. The use of claim 10, wherein the cancer is osteosarcoma.

21. The use of claim 10, wherein the cancer is ovarian cancer.

22. The use of claim 10, wherein the cancer is Rb-negative bladder cancer.