HK1260112B

HK1260112B - Hspc-sparing treatments for rb-positive abnormal cellular proliferation

Info

Publication number: HK1260112B
Application number: HK19119906.6A
Authority: HK
Inventors: J·C·斯特鲁姆; J·E·比希; P·J·罗伯茨; F·X·塔瓦雷斯
Original assignee: G1 治疗公司
Priority date: 2013-03-15
Filing date: 2016-09-18
Publication date: 2021-09-10

Description

HSPC-sparing therapy for Rb-positive abnormal cell proliferation

The application is a divisional application of a Chinese patent application (with the application number of 201480027269.7, the application date of 2014, 3, 14 and the invention name of 'HSPC (high speed human liver cancer) restrictive treatment aiming at Rb positive abnormal cell proliferation') of international application PCT/US2014/029429 entering the Chinese national stage.

RELATED APPLICATIONS

The present application relates to and claims the rights and benefits of U.S. provisional application No.61/798,772 filed on day 3, month 15 of 2013, U.S. provisional application No.61/861,374 filed on day 1 of 8, month 8 of 2013, U.S. provisional application No.61/911,354 filed on day 3 of 12, month 3 of 2013, and U.S. provisional application No.61/949,786 filed on day 7 of 3, month 7 of 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 support of the grant 5R44AI084284 by the National institute of Allergy and Infectious Disease.

Technical Field

The present invention relates to improved compounds and methods for treating selected RB-positive cancers and other RB-positive aberrant cell proliferation disorders while minimizing the deleterious effects on healthy cells, such as healthy Hematopoietic Stem and Progenitor Cells (HSPCs), associated with current treatment modalities. In one aspect, improved treatments for selected RB-positive cancers using particular compounds disclosed herein are disclosed. When administered to a subject, in certain embodiments, the compounds described herein act as highly selective, and in certain embodiments, transiently, acting, cyclin-dependent kinase 4/6(CDK 4/6) inhibitors.

Background

The regulation of the cell cycle is governed and controlled by specific proteins that are activated and inactivated in a precisely timed manner, mainly by phosphorylation/dephosphorylation processes. A key protein that coordinates the initiation, progression, and completion of the cell cycle program is the cyclin-dependent kinase (CDK). Cyclin-dependent kinases belong to the serine-threonine protein kinase family. They are heterodimeric complexes consisting of a catalytic kinase subunit and a regulatory cyclin subunit. CDK activity is controlled by the association of its corresponding regulatory subunits (cyclins) and CDK inhibitor proteins (Cip and Kip proteins, INK4s), by its phosphorylation status and by ubiquitin-mediated proteolytic degradation (see D.G.Johnson, C.L.Walker, Annu.Rev.Pharmacol.Toxicol 39(1999) 295-312; D.O.Morgan, Annu.Rev.cell Dev.biol.13(1997) 261-291; C.J.Sherr, Science 274(1996) 1672-1677; T.Shimamura et al, bioorg.Med.chem.Lett.16(2006) 3751-3754).

There are four CDKs that are significantly involved in cell proliferation: CDK1, which primarily regulates the transition from G2 to M phase; and CDK2, CDK4 and CDK6, which regulate the transition from G1 phase to S phase (Malumbres M, Barbacid m.cell cycle, CDKs and cancer: a chang paradigm.nat. rev. cancer 2009; 9(3): 153-. Activation of CDK4 cyclin D and CDK6 cyclin D induces phosphorylation of retinoblastoma protein (pRb) when the cell responds to mitogenic stimuli in the early to mid stages of G1. phosphorylation of pRb releases the transcription factor E2F, which enters the nucleus, activating transcription of other cyclins that promote further progression through the Cell cycle (see J.A. Diehl, Cancer biol. Ther.1(2002) 226-1065; C.J.Sherr, Cell 73(1993) 1059-1065). CDK4 and CDK6 are closely related proteins with biochemical properties that are essentially indistinguishable (see m.malumbres, m.barbacid, Trends biochem. sci.30(2005) 630-641).

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). For example, WO 03/062236 identifies a series of 2- (pyridin-2-ylamino-pyrido [2,3] pyrimidin-7-ones, including 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido- [2,3-d ] -pyrimidin-7-one (PD0332991), for use in the treatment of Rb-positive cancers that exhibit selectivity for CDK4/6, which compounds are currently being tested by Pfizer in large clinical trials as an antineoplastic agent against estrogen-positive, HER 2-negative breast cancers. Med, chem.48(2005) 2371-. WO 99/15500 filed by Glaxo Group Ltd discloses protein kinase and serine/threonine kinase inhibitors. WO 2010/020675 filed by Novartis AG describes pyrrolopyrimidine compounds as CDK inhibitors. WO 2011/101409 also 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. WO 2012/061156 filed by Tavares and assigned to G1Thapeutics describes CDK inhibitors. WO 2013/148748 filed by Francis Tavares and assigned to G1Thapeutics describes lactam kinase inhibitors.

While selective CDK4/6 inhibitors are generally designed to target CDK4/6 replication-dependent cancers, the precise fact that they inhibit CDK4/6 activity may also cause deleterious effects on CDK 4/6-dependent healthy cells, such as their growth inhibition. CDK4/6 activity is essential for bone marrow to produce healthy blood cells, as healthy Hematopoietic Stem and Progenitor Cells (HSPC) require CDK4/6 activity for proliferation (see Roberts et al Multiple leaves of cycle-Dependent Kinase 4/6Inhibitors in Cancer therapy. JNCI 2012; 104(6):476 + 487). Healthy 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). Healthy hematopoietic cells during spinal cord/erythrocyte differentiation show a progressive dependence on CDK4/6 activity for proliferation (see Johnson et al, differentiation of pathological diagnosis in micro pharmaceutical pathology induced by CDK4/6inhibition. J Clin. invest. 2010; 120(7): 2528. sup. 2536). Thus, minimally differentiated cells, such as healthy Hematopoietic Stem Cells (HSCs), multipotent progenitors (MPPs) and common spinal cord progenitors (CMPs), appear to be most dependent on CDK4/6 activity for proliferation and are therefore most adversely affected by CDK4/6 inhibitors for the treatment of CDK4/6 replication-dependent cancers or other proliferative disorders.

Accordingly, there is an ongoing need for improved compounds, methods and regimens for treating selected patients with Rb-positive cancers and abnormal cell proliferation disorders while minimizing the effects of treatment on healthy cells such as HSPCs.

Summary of The Invention

Improved compounds, methods and compositions are provided for treating selected Rb-positive abnormal cell proliferations, including Rb-positive cancers, while minimizing the deleterious effects of treatment on healthy cells, such as healthy HSPC and other CDK4/6 replication-dependent healthy cells, by administering an effective amount of a compound described herein.

In one embodiment of the invention, the compound is selected from compounds of formula I, II, III, IV or V as described herein, or a pharmaceutically acceptable composition, salt, isotopic analogue or prodrug thereof. 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 embodiment, the Rb-positive cancer may be Rb-positive adenocarcinoma. The Rb-positive cancer can be Rb-positive adenocarcinoma of the colon. The Rb-positive cancer can also be Rb-positive adenocarcinoma of the rectum.

Alternatively, the Rb-positive cancer may be Rb-positive anaplastic astrocytoma.

The Rb-positive cancer may be Rb-positive breast cancer. In one embodiment, the Rb-positive cancer is Rb-positive estrogen receptor positive, HER 2-negative advanced breast cancer. Alternatively, the Rb-positive cancer can be Rb-positive estrogen receptor negative breast cancer. The Rb-positive cancer may be Rb-positive estrogen receptor-positive breast cancer. The Rb-positive cancer may be Rb-positive advanced metastatic breast cancer. The Rb-positive cancer may be Rb-positive tubular a-type breast cancer. The Rb-positive cancer may be Rb-positive tubular B-type breast cancer. The Rb-positive cancer can be Rb-positive Her 2-negative breast cancer or Rb-positive Her 2-positive breast cancer. The Rb-positive cancer is Rb-positive male breast cancer. In one embodiment, the Rb-positive cancer is Rb-positive progesterone receptor negative breast cancer. The Rb-positive cancer may be Rb-positive progesterone receptor positive breast cancer. The Rb-positive cancer may be Rb-positive recurrent breast cancer. In one embodiment, the Rb-positive cancer is Rb-positive stage IV breast cancer. In one embodiment, the Rb-positive cancer is Rb-positive advanced HER 2-positive breast cancer.

The Rb-positive cancer may be Rb-positive bronchial cancer. The Rb-positive cancer can be Rb-positive colon cancer. The Rb-positive cancer may be Rb-positive relapsed colon cancer. The Rb-positive cancer can be Rb-positive stage IV colon cancer. In one embodiment, the Rb-positive cancer is Rb-positive colorectal cancer.

In one embodiment, the Rb-positive cancer is Rb-positive endometrial cancer.

The Rb-positive cancer may be Rb-positive extragonadal seminoma. The Rb-positive cancer may be Rb-positive stage III extragonadal seminoma. The Rb-positive cancer may be Rb-positive stage IV extragonadal seminoma.

The Rb-positive cancer may be Rb-positive germ cell cancer. The Rb-positive cancer may be an Rb-positive central nervous system germ cell tumor. The Rb-positive cancer may be Rb-positive familial testicular germ cell tumor. The Rb-positive cancer may be Rb-positive recurrent gonadal germ cell tumor. The Rb-positive cancer may be Rb-positive recurrent extragonadal non-seminoma germ cell tumors. The Rb-positive cancer may be Rb-positive extragonadal spermatogonial germ cell tumor. The Rb-positive cancer may be Rb-positive recurrent malignant testicular germ cell tumor. The Rb-positive cancer may be Rb-positive recurrent ovarian germ cell tumor. The Rb-positive cancer can be Rb-positive stage III malignant testicular germ cell tumor. The Rb-positive cancer may be Rb-positive stage III ovarian germ cell tumor. The Rb-positive cancer may be Rb-positive stage IV ovarian germ cell tumor. The Rb-positive cancer may be Rb-positive stage III extragonadal non-seminoma germ cell tumors. The Rb-positive cancer may be Rb-positive stage IV extragonadal non-seminoma germ cell tumors. In one embodiment, the Rb-positive cancer is Rb-positive germ cell cancer. In one embodiment, the Rb-positive cancer is Rb-positive cisplatin-refractory unresectable germ cell cancer.

In one embodiment, the Rb-positive cancer is Rb-positive glioblastoma.

In one embodiment, the Rb-positive cancer is Rb-positive liver cancer. The Rb-positive cancer can be Rb-positive hepatocellular carcinoma.

The Rb-positive cancer may be Rb-positive lung cancer. In one embodiment, the Rb-positive cancer is Rb-positive non-small cell lung cancer. In one embodiment, the Rb-positive cancer is Rb-positive KRAS mutant non-small cell lung cancer.

The Rb-positive cancer may be Rb-positive melanoma. In one embodiment, the Rb-positive cancer is Rb-positive relapsed melanoma. In one embodiment, the Rb-positive cancer is Rb-positive stage IV melanoma.

The Rb-positive cancer may be Rb-positive ovarian cancer. In one embodiment, the Rb-positive cancer is Rb-positive ovarian epithelial cancer.

The Rb-positive cancer can be Rb-positive pancreatic cancer.

The Rb-positive cancer can be Rb-positive prostate cancer.

In one embodiment, the Rb-positive cancer is Rb-positive rectal cancer. The Rb-positive cancer may be Rb-positive relapsed rectal cancer. The Rb-positive cancer may be Rb-positive stage IV rectal cancer.

The Rb-positive cancer can be Rb-positive sarcoma. The Rb-positive cancer may be Rb-positive gliosarcoma. The Rb-positive cancer can be Rb-positive liposarcoma. The Rb-positive cancer can be Rb-positive fibrosarcoma. The Rb-positive cancer may be Rb-positive myxosarcoma. In one embodiment, the Rb-positive cancer may be Rb-positive chondrosarcoma. The Rb-positive cancer can be Rb-positive osteosarcoma.

The Rb-positive cancer may be Rb-positive malignant fibrous histiocytoma. The Rb-positive cancer can be Rb-positive angiosarcoma. The Rb-positive cancer can be Rb-positive angiosarcoma. The Rb-positive cancer may be Rb-positive lymphangiosarcoma. The Rb-positive cancer may be Rb-positive mesothelioma. The Rb-positive cancer may be Rb-positive leiomyosarcoma. The Rb-positive cancer may be Rb-positive rhabdomyosarcoma. The Rb-positive cancer may be Rb-positive meningioma. The Rb-positive cancer may be Rb-positive schwannoma.

In one embodiment, the Rb-positive cancer is Rb-positive pheochromocytoma. The Rb-positive cancer may be Rb-positive islet cell carcinoma. The Rb-positive cancer can be Rb-positive carcinoid tumor. The Rb-positive cancer may be Rb-positive paraganglioma.

In one embodiment, the Rb-positive cancer is Rb-positive squamous cell carcinoma. The Rb-positive cancer can be Rb-positive adenocarcinoma. The Rb-positive cancer can be Rb-positive hepatocellular carcinoma. The Rb-positive cancer can be Rb-positive renal cell carcinoma. The Rb-positive cancer can be Rb-positive hepatobiliary carcinoma.

The Rb-positive cancer can be an Rb-positive refractory solid tumor.

The Rb-positive cancer may be Rb-positive neuroblastoma.

The Rb-positive cancer may be Rb-positive medulloblastoma.

In one embodiment, the Rb-positive cancer is a teratoma. The Rb-positive cancer may be Rb-positive ovarian immature teratoma. The Rb-positive cancer may be Rb-positive ovarian mature teratoma. The Rb-positive cancer may be Rb-positive ovarian specialized teratoma. The Rb-positive cancer may be Rb-positive testicular immature teratoma. The Rb-positive cancer may be Rb-positive testicular maturation teratoma. The Rb-positive cancer may be Rb-positive teratoma. The Rb-positive cancer may be Rb-positive ovarian single germ layer teratoma.

The Rb-positive cancer may be Rb-positive testicular cancer.

In one embodiment, the Rb-positive cancer is Rb-positive vaginal cancer.

In one embodiment, the Rb-positive cancer is selected from Rb-positive carcinoma, sarcoma, including (but not limited to) lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anus, stomach cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, carcinoma of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, cancer of the prostate, cancer of the bladder, cancer of the kidney or ureter, cancer of the renal cells, carcinoma of the renal pelvis, neoplasms of the Central Nervous System (CNS), primary CNS lymphoma, spinal tumor, brain stem glioma, pituitary adenoma, or a.

In one embodiment, the subject suffers from an Rb-positive abnormal cell proliferation disorder. In one embodiment, the Rb-positive aberrant cell proliferation disorder is non-cancerous.

In certain embodiments, the compounds described herein, when used to treat selected Rb-positive cell proliferation disorders such as cancer, allow healthy cells to rapidly re-enter the normal cell cycle and rapidly rejuvenate damaged tissues and progeny cells, such as blood cells. In this regard, the compounds described herein eliminate, reduce and/or minimize drug holidays and dosing delays associated with current anti-neoplastic use of CDK4/6 inhibitors when used to treat Rb-positive cancers, thereby allowing rapid recovery of damaged blood cells through replication and differentiation of progenitor and parental cells. In particular, the invention includes administering to a patient with a cancer, e.g., an Rb-positive cancer, an effective amount of a compound described herein, wherein the compound has pharmacokinetics and enzymatic half-lives that provide transiently reversible G1 arrest of CDK4/6 replication-dependent cells. The compound may be any of those described herein. Non-limiting examples of active compounds are described in table 1, or pharmaceutically acceptable compositions, salts, isotopic analogs or prodrugs thereof, as provided below.

In one embodiment, the compounds described herein may be useful in improved methods of treating cancers, such as Rb-positive cancers, where such methods have reduced or minimized effects on CDK4/6 replication-dependent healthy cells, in part because they (i) utilize compounds that exhibit pharmacokinetics and enzymatic half-lives that provide transient, transient and reversible effects of G1 arrest on CDK4/6 replication-dependent healthy cells, and (ii) allow healthy cells to rapidly re-enter the cell cycle after cessation of administration or dissipation of therapeutically effective levels in a subject. Use of the compounds described herein allows, for example, for a reduction in the replication delay of HSPCs due to CDK4/6 inhibition, and/or an acceleration in hematopoietic lineage recovery after cessation of CDK4/6 inhibitory activity, and/or a reduction in blood deficiency, as the compounds utilized act transiently and reduce the length of the non-circulation or drug holiday associated with current CDK4/6 inhibitor treatment modalities, which reduces or minimizes the promotion of tumor drug resistance. In certain embodiments, the use of the compounds described herein allows for continued treatment of a subject over a longer period of time without the need for a non-circulatory period or drug holiday.

Timely resumption of CDK4/6 replication-dependent healthy cell proliferation is essential for tissue repair, and excessively long-term cell cycle arrest of healthy cells, such as HSPC cell cycle arrest, is undesirable. Although reports indicate that the selective CDK4/6 inhibitor PD0332991 is a potent inhibitor of Rb-positive breast cancers, it has been found that such inhibitors may not be the most desirable compounds for use as chemotherapeutic agents due to the compound's excessive myelosuppressive effect. For example, PD0332991 has a relatively long-lasting intracellular effect (see Roberts et al, Multiple rounds of cycle-Dependent Kinase 4/6 inhibition in Cancer therapy. JCNI 2012; 104(6):476-487 (FIG. 2A)), prolonging the transient G1 arrest of healthy cells such as HSPC, leading to dose-limiting myelosuppression. Such long-lasting effects delay, for example, proliferation of HSPC cell lineages required to restore blood cell lines whose growth has been inhibited by treatments that may inhibit CDK4/6 activity and thus inhibit Rb phosphorylation in Rb competent cells. While desirable for its anti-neoplastic effects, the long-lasting G1 arrest provided by PD0332991 requires a prolonged non-circulatory phase to recruit red blood cells, platelets and bone marrow cells (monocytes and granulocytes) adversely affected by acute HSPC G1-arrest, thereby limiting bone marrow suppression and allowing a period of blood replication. Treatment of selected Rb-positive cancers using the compounds described herein as anti-neoplastic agents can eliminate, reduce or minimize the required length of non-circulating or drug holidays, allowing for a longer period of effective CDK4/6 inhibition of the cancer during the course of the anti-neoplastic regimen.

Thus in one embodiment, the present invention comprises administering to a subject suffering from Rb-positive cancer an effective amount of a compound described herein, including one selected from table 1, wherein (individually or in any combination thereof, each is considered to be specifically and independently described): i) a substantial portion of CDK4/6 replication-dependent healthy cells (e.g., at least 80% or greater), e.g., HSPCs, return to or near pre-treatment baseline cell cycle activity (i.e., re-enter the cell cycle) within less than 24 hours, 30 hours, or 36 hours from the last administration of a compound described herein in a human; ii) a substantial proportion of CDK4/6 replication-dependent healthy cells, e.g., HSPCs, synchronously re-enter the cell cycle within less than 24 hours, 30 hours, or 36 hours from the last administration of a compound described herein; (iii) dissipation of the inhibitory effect of the compound on CDK4/6 replication-dependent healthy cells, e.g., HSPCs, occurs in less than 24 hours, 30 hours, or 36 hours of administration of the compound; (iv) a substantial proportion of CDK4/6 replication-dependent healthy cells, e.g., HSPCs, recover to or near baseline pre-treatment cell cycle activity (i.e., re-enter the cell cycle) in less than 24 hours, 30 hours, or 36 hours of abrogation of CDK4/6 inhibitory effects of the ionomeric compounds; or (vi) a substantial portion of CDK4/6 replication-dependent healthy cells, e.g., HSPCs, return to or near pre-treatment baseline cell cycle activity (i.e., re-enter the cell cycle) in less than about 24 hours, about 30 hours, or about 36 hours from the time that the concentration level of the administered compound in the subject's blood falls to a therapeutically effective concentration.

In important embodiments of the invention, the compounds described herein may be administered in a regimen with another agent, such as a non-DNA damaging targeted antineoplastic or hematopoietic growth factor agent, to achieve a beneficial, additive, or synergistic effect against abnormal cell proliferation. It has recently been reported that untimely administration of hematopoietic growth factors may have serious side effects. For example, the use of the EPO family of growth factors has been associated with arterial hypertension, cerebral convulsions, hypertensive encephalopathy, thromboembolism, iron deficiency, influenza-like syndrome, and venous thrombosis. The G-CSF family of growth factors has been associated with enlargement and rupture of the spleen, respiratory distress syndrome, allergies and sickle cell complications. By combining the administration of the compounds described herein and the methods of the invention with the timely administration of hematopoietic growth factors, e.g., at a point in time when the diseased cells are no longer in growth arrest, the health care practitioner can reduce the amount of growth factors to minimize unwanted side effects while achieving the desired therapeutic benefit. In one embodiment, the growth factor is administered after the compound's effect on inhibition of CDK4/6 replication-dependent healthy cells, e.g., HSPCs, is discontinued. Thus, in this embodiment, use of the selective CDK4/6 inhibitors described herein in an anti-neoplastic treatment regimen allows the subject to receive a reduced amount of growth factors because the targeted hematopoietic cells will re-enter the cell cycle faster than other CDK4/6 inhibitors such as PD 0332991. In addition, the synchronous re-entry into the cell cycle following G1 arrest with the compounds described herein provides for the administration of timed hematopoietic growth factors to help reconstitute hematopoietic cell lines, thereby maximizing the ability of the growth factors 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 dissipated.

In one embodiment, the use of a compound described herein is combined with at least one other chemotherapeutic agent within a therapeutic regimen, 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). Examples of mTOR inhibitors include, but are not limited to, rapamycin (rapamycin) and its analogs, everolimus (Afinitor), temsirolimus (temsirolimus), radar (ridaforolimus), sirolimus (sirolimus), and de-folimus (deforolimus). Examples of P13 kinase inhibitors include, but are not limited to, Wortmannin (Wortmannin), desmethylviridin (demethoxyviridin), perifosine (perifosine), idealsib (idelalisib), PX-866, IPI-145, BAY 80-6946, BEZ235, RP6503, TGR 1202(RP5264), MLN1117(INK1117), Pitisib (Picilisib), Bupasib (Buparlisib), SAR 2454408 (XL147), SAR 2458409 (XL765), Palomid (Palomid)529, ZSTK474, PWT33597, 6530, 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-deoxy Geldanamycin (17AAG) and Radicicol (Radicol).

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, a compound described herein is administered to a subject less than about 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, or 4 hours or less prior to treatment with another chemotherapeutic agent to sensitize the Rb-positive cancer to the chemotherapeutic agent. In one embodiment, the compound is administered up to 4 hours prior to treatment with the other chemotherapeutic agent.

In one embodiment, the compounds described herein are administered in a manner that allows the drug to readily enter the bloodstream, e.g., via intravenous injection or sublingual, intraaortic, or other effective route of entry into the bloodstream. In one embodiment, the compounds described herein are administered in an orally available formulation. In other embodiments, the compounds described herein are administered via topical, transdermal, or other desired routes of administration.

In one embodiment, a compound described herein is administered to a subject less than about 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, or 4 hours or less prior to treatment with a hematopoietic growth factor. In one embodiment, the compound is administered up to 4 hours prior to treatment with the hematopoietic growth factor or other chemotherapeutic agent.

Compounds suitable for use in the present invention exhibit significant selectivity for inhibition of CDK4 and/or CDK6 compared to other CKDs such as CDK 2. For example, compounds suitable for use in the present invention provide dose-dependent G1 arrest in Rb-positive cancer cells in a subject, and the methods provided herein are sufficient to provide chemotherapeutic treatment and growth inhibition of Rb-positive cancer cells while not affecting CDK4/6 replication-independent cells.

In one embodiment, use of a compound described herein causes dissipation of G1 stasis such that the subject's CDK4/6 replication-dependent healthy cells return to their pre-administration baseline cell cycle activity in less than about 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 24 hours, 30 hours, 36 hours, or 40 hours.

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

In one embodiment, the compounds described herein and suitable for use in the methods may be synchronized in their termination effects, that is, CDK4/6 replication-dependent healthy cells exposed to the compounds described herein re-enter the cell cycle in a similarly timed manner after 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 the compound in the subject' S blood falls below a therapeutically effective concentration.

The rapid cell cycle reentry associated with the rapid termination effect of compounds advantageously allows a greater number of CDK4/6 replication-dependent healthy cells to begin replicating when G1 arrest dissipates compared to other CDK4/6 inhibitors such as PD 0332991. Thus, CDK4/6 replication-dependent healthy cells, such as HSPCs, can rapidly begin replication during the non-circulating phase or during the administration phase.

The use of a compound as described herein in a therapeutic regimen targeting CDK4/6 replication-dependent cancer may result in reduced anemia, reduced lymphopenia, reduced thrombocytopenia, or neutropenia as compared to what is typically expected, common, or associated with treatment with currently available antineoplastic chemotherapeutic agents. The use of a compound as described herein may result in a faster recovery from myelosuppression, 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, the use of a compound as described herein results in myelosuppression, e.g., myelosuppression, anemia, lymphopenia, leukopenia, thrombocytopenia, or granulocytopenia, e.g., neutropenia, associated with chronic use of a CDK4/6 inhibitor.

In some embodiments, the subject or subject is a mammal, including a human. The compounds can be administered to a subject by any desired route, including intravenously, sublingually, buccally, orally, intraaortic, topically, intranasally, parenterally, transdermally, systemically, intramuscularly, or via inhalation.

In summary, the invention comprises the following features:

A) optimal compounds, methods and compositions as chemotherapeutic agents that minimize the deleterious effects of CDK4/6 replication-dependent healthy cells, such as Hematopoietic Stem and Progenitor Cells (HSPCs), in a subject being treated for a selected Rb-positive cancer, comprising administering an effective amount of a compound of formula I, II, III, IV or V, including a compound selected from table 1 as described herein;

B) optimal compounds, methods and compositions as chemotherapeutic agents that minimize the deleterious effects of CDK4/6 replication-dependent healthy cells, such as Hematopoietic Stem and Progenitor Cells (HSPCs), in a subject being treated for Rb-positive cancer, comprising administering an effective amount of a selective compound described herein, wherein a substantial portion of the healthy cells recover to or near pre-treatment baseline cell cycle activity (i.e., re-entry into the cell cycle) within less than about 24 hours, 30 hours, 36 hours, or about 40 hours from the last administration of the CDK4/6 inhibitor and wherein the IC of CDK4/6 inhibitor inhibition of CDK4 ₅₀IC for CDK2 inhibition at concentrations compared to its concentration₅₀The concentration is more than about 1500 times less. In certain embodiments, the CDK4/6 replication-dependent healthy cell is a HSPC. In certain embodiments, the CDK4/6 replication-dependent healthy cell is a renal epithelial cell;

C) optimal compounds, methods and compositions as chemotherapeutic agents that minimize the deleterious effects of CDK4/6 replication-dependent healthy cells in a subject being treated for Rb-positive cancer includeAdministering an effective amount of a compound described herein, wherein a substantial portion of the CDK replication-dependent healthy cells synchronously re-enter the cell cycle less than about 24 hours, 30 hours, 36 hours, or about 40 hours after the CDK4/6 inhibitory effect of the compound dissipates, wherein the IC of the compound on CDK4 inhibition₅₀IC for CDK2 inhibition at concentrations compared to its concentration₅₀The concentration is more than about 1500 times less. In certain embodiments, the CDK4/6 replication-dependent healthy cell is a HSPC. In certain embodiments, the CDK4/6 replication-dependent healthy cell is a renal epithelial cell.

D) Optimal compounds, methods and compositions as chemotherapeutic agents that minimize the deleterious effects on CDK4/6 replication-dependent healthy cells in a subject, comprising administering to a subject having an Rb-positive abnormal cell proliferation disorder an effective amount of a selective CDK4/6 inhibitor selected from the compounds described herein. In certain embodiments, healthy cells of the subject recover to or near baseline pre-treatment cell cycle activity (i.e., re-enter the cell cycle) in less than about 24 hours, about 30 hours, about 36 hours, or about 40 hours from the time the concentration level of the compound in the subject's blood falls below a therapeutically effective concentration. In certain embodiments, the CDK4/6 replication-dependent healthy cell is a HSPC. In certain embodiments, the CDK4/6 replication-dependent healthy cell is a renal epithelial cell.

E) A compound as described herein, or a pharmaceutically acceptable composition, salt, isotopic analog or prodrug thereof, for use as a chemotherapeutic agent for treating Rb-positive aberrant cell proliferation disorders (including Rb-positive cancers);

F) a compound as described herein, and pharmaceutically acceptable compositions, salts, isotopic analogues and prodrugs thereof, for use as a chemotherapeutic regimen for the treatment of Rb-positive aberrant cell proliferation disorders (including Rb-positive cancers) which minimizes deleterious effects on CDK4/6 replication-dependent healthy cells, such as HSPCs or kidney cells;

G) a compound as described herein, and pharmaceutically acceptable compositions, salts, isotopic analogs, and prodrugs thereof, for use in combination with hematopoietic growth factors in a subject undergoing a therapeutic regimen for treating Rb-positive aberrant cell proliferation disorders, including Rb-positive cancers;

H) a compound as described herein, or a pharmaceutically acceptable composition, salt, isotopic analog or prodrug thereof, for use in combination with a second chemotherapeutic agent in a subject undergoing a therapeutic regimen for treating Rb-positive aberrant cell proliferation disorders, including Rb-positive cancers;

I) use of a compound described herein, or a pharmaceutically acceptable composition, salt, isotopic analog or prodrug thereof, for the manufacture of a medicament for use as a chemotherapeutic agent for the treatment of Rb-positive aberrant cell proliferation disorders, including Rb-positive cancers;

J) Use of a compound described herein, or a pharmaceutically acceptable composition, salt, isotopic analogue or prodrug thereof, for the manufacture of a medicament for use as a chemotherapeutic agent in the treatment of a subject suffering from an Rb-positive aberrant cell proliferation disorder (including Rb-positive cancers) in which growth is arrested or inhibited when exposed to a CDK4/6 inhibitor;

K) a method for preparing a therapeutic product containing an effective amount of a compound described herein for treating a subject having an Rb-positive abnormal cell proliferation disorder, such as cancer, and;

l) a method of manufacturing an agent selected from the compounds described herein, intended for therapeutic use as chemotherapeutic agents for the treatment of Rb-positive aberrant cell proliferation disorders responsive to CDK4/6 inhibitors, such as cancer.

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 2 is a graph comparing the time (hours) after administration of healthy mouse HSPC and healthy spinal cord progenitor cells to PD0332991 for EdU incorporation. PD0332991(150mg/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 153, a single oral dose of PD0332991 resulted in a sustained reduction in HSPC EdU incorporation (circles; LKS +) and spinal cord progenitor EdU incorporation (squares; LKS-) for over 36 hours.

Figure 3A is a graph of plasma drug concentration (ng/ml) versus time (hours) after compound T administration. Figure 3B is a graph of plasma drug concentration (ng/ml) versus Q (hours) after compound Q administration. Figure 3C is a graph of plasma drug concentration (ng/ml) versus time (hours) after compound GG administration. Figure 3D 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 by oral gavage (diamonds) or 10mg/kg by intravenous injection (squares). 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 4A 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 4B 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 4C 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. Figure 4D 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 demonstrate that the inhibitor compounds of the present invention have a transient, transient G1 arrest in different cell types. The effect on the cell cycle after elution of the compounds was determined at 24, 36, 40 and 48 hours. As described in example 155, 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 (diamonds) only) compared to cells treated with compound T (square), compound Q (triangle), compound gg (X), or compound U (X with cross).

Figure 5A is a graph of the rate of EdU incorporation of HSPCs 12 or 24 hours post-administration (compared to untreated control mice) following oral gavage of compound T, Q or GG at 150 mg/kg. Figure 5B is a graph of the percentage of EdU positive HSPC cells in mice treated with compound T at 12 or 24 hours. Mice were given 50mg/kg (triangle), 100mg/kg (square) or 150 (inverted triangle) mg/kg by oral gavage. Figure 5C is a graph of the percentage of EdU positive HSPC cells in mice treated with compound T (150mg/kg by oral gavage) at 12, 24, 36 and 48 hours. As described in example 156, compounds T and GG demonstrated a decrease in EdU incorporation at 12 hours and began to return to normal levels of cell division by 24 hours.

Figure 6 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 and the percentage of EdU positive HSPC cells was measured at 12, 24, 36 or 48 hours. As described in example 157, a single oral dose of PD0332991 causes a persistent reduction in HSPC proliferation 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 resumed.

Figure 7 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.

FIG. 8 is a graph showing tumor volume (mm) in MMTV-c-neu (Rb positive) tumor-loaded mice treated with 100mg/kg/d (squares) or 150mg/kg/d (triangles) Compound T³) Graphs comparing time (days) after compound T administration. Tumor-bearing MMTV-c-neu mice (control, n-9; compound T, 100mg/kg, n-7; compound T, 150mg/kg, n-6) were treated with compound T delivered in food or standard food (circles). Day 0 represents the first day of compound treatment. Mice were treated with compound T for 28 days (as indicated by the box around the number on the x-axis indicating the days of treatment administration). After 28 days, all mice were fed standard food. Tumor volumes (up to 56 days) were recorded weekly and graphed as mean ± standard error of mean. Trueness ofAs described in example 159, continuous treatment with compound T (100mg/kg/d or 150mg/kg/d) during the course of 28 days of therapy resulted in a significant reduction in tumor volume compared to control.

FIG. 9 is a waterfall plot of the percent change in tumor volume of MMTV-c-neu mice (Rb positive) per mouse treated with compound T at 100mg/kg (horizontal line boxes) or 150mg/kg (inclined line boxes). Tumor volume was compared to the mean tumor size of untreated animals on day 21. Tumor volumes of mice treated with compound T indicated the best response seen at or beyond day 28. Negative values indicate tumor shrinkage.

FIG. 10 is a table showing the Objective Response Rate (ORR) of MMTV-c-neu (Rb-positive) tumors in mice treated with compound T, GG or U in the MMTV-c-neu mammary gland luminal breast cancer (Rb-positive) model. All three compounds were administered orally via a drug-supplemented diet (100 mg/kg/d). The drug-added diet was administered for 28 consecutive days, followed by discontinuation. RECIST criteria are used to evaluate the objective response rate. Objective Response Rate (ORR) was based on percent change in tumor volume using the following classification of categories: CR (complete reaction) is 100% reaction; PR (partial reaction) reduction of at least 30%; SD (stable disease) ═ no change (no PR and no PD); and PD (progressive disease) increased by 20%. As described in example 160, continuous treatment with compound T, GG or U caused a significant reduction in tumor volume during the course of 28 days of therapy.

FIG. 11 is a graph showing tumor volume (mm) in MMTV-c-neu (Rb-positive) tumor-bearing mice treated with Compound T (open circles), Compound GG (diamonds), or Compound U (squares)³) The time (days) after administration of each compound was compared. Tumor-bearing MMTV-c-neu mice (control, n-9; compound T, 100mg/kg, n-7; compound GG, 100mg/kg, n-7; compound U, 100mg/kg, n-8) were treated with compounds delivered in diet or standard diet (filled circles). Day 0 represents the first day of compound treatment. Mice were treated with compound for 28 days (as indicated by the box around the number on the x-axis indicating the number of days of treatment administration). After 28 days, all mice were fed standard food. Tumor volumes were recorded weekly (up to 56 days) and graphed as mean ± mean standard errorAnd (4) poor. As described in example 160, continuous treatment with compound T, GG or U caused a significant reduction in tumor volume during the course of 28 days of therapy, with compounds T and U exhibiting 100% objective response rate, and compound GG exhibiting 85% objective response rate.

Fig. 12 is a cascade plot of the percent change in MMTV-c-neu (Rb positive) tumor volume for each mouse treated with 100mg/kg compound T (diagonal bar, n-7), 100mg/kg compound GG (black and white square bar, n-7), 100mg/kg compound U (solid bar, n-8), or no treatment (open bar, n-9). Tumor volume was compared to the mean tumor size of untreated animals on day 21. Tumor volumes of mice treated with compound T, GG or U indicated the best response seen at or beyond day 14. Negative values indicate tumor shrinkage. Negative values indicate tumor shrinkage. As described in example 160, continuous treatment with compound T, GG or U caused a significant reduction in tumor volume during the course of 28 days of therapy.

FIGS. 13-15 illustrate R of compounds of the invention²Several exemplary implementations of (a).

FIGS. 16A-16C, 17A-17D, 18A-18C, 19A-19B, and 20A-20F illustrate several exemplary embodiments of core structures of compounds of the invention.

FIG. 21 is a graph of cell proliferation (as measured by Relative Light Units (RLU)) of MCF7(Rb positive) cells (breast cancer) versus variable molar concentration (M) treated with PD0332991 (circles) or compound T (FIG. 1; squares). MCF7 cells were seeded in Costar (Tewksbury, Massachusetts) 390396 wells tissue culture treated white wall/clear bottom plates. Nine-point dose-response serial dilutions from 10uM to 1nM were performed and after six days of compound treatment, as indicated, useLuminescence cell viability assay (CTG; Promega, Madison, Wisconsin, USA), cell viability was determined according to the manufacturer's recommendations. The discs were read on a BioTek (Winooski, Vermont) Syngergy2 multi-template reader. Relative Light Units (RLU) were plotted as a result of variable molar concentrations and statistically soft using Graphpad (LaJolla, California) Prism 5The data was analyzed to determine the EC for each compound₅₀。

FIG. 22 is a graph of cell proliferation (as measured by Relative Light Units (RLU)) of MCF7(Rb positive) cells (breast cancer) versus variable molar concentration (M) treated with compound Q (FIG. 1; circles) or compound GG (FIG. 1; squares). As described in fig. 21 and example 152, use Analysis of luminescent cell viability cell proliferation was determined.

FIG. 23 is a graph of cell proliferation (as measured by Relative Light Units (RLU)) of MCF7(Rb positive) cells (breast cancer) versus variable molar concentration (M) treated with compound U (FIG. 1; circles) or compound H (FIG. 1; squares). As described in fig. 21 and example 152, useAnalysis of luminescent cell viability cell proliferation was determined.

FIG. 24 is a graph of cell proliferation (as measured by Relative Light Units (RLU)) of MCF7(Rb positive) cells (breast cancer) versus variable molar concentration (M) treated with compound MM (FIG. 1; circles) or compound OO (FIG. 1; squares). As described in fig. 21 and example 152, useAnalysis of luminescent cell viability cell proliferation was determined.

FIG. 25 is a graph of cell proliferation (as measured by Relative Light Units (RLU)) of ZR75-1(Rb positive) cells (breast cancer) versus variable molar concentration for treatment with PD0332991 or Compound T (FIG. 1; squares). As described in fig. 21 and example 152, useAnalysis of luminescent cell viability cell proliferation was determined.

FIG. 26 is a comparison of cell proliferation (as measured by Relative Light Units (RLU)) of ZR75-1(Rb positive) cells (breast cancer) with compound Q (Table 1; circles) or compound GG (FIG. 1; Squares) plot of the variable molarity of the treatments. As described in fig. 21 and example 152, useAnalysis of luminescent cell viability cell proliferation was determined.

FIG. 27 is a graph of cell proliferation (as measured by Relative Light Units (RLU)) of ZR75-1(Rb positive) cells (breast cancer) versus variable molarity treated with Compound U (Table 1; circles) or Compound H (FIG. 1; squares). As described in fig. 21 and example 152, useAnalysis of luminescent cell viability cell proliferation was determined.

FIG. 28 is a graph of cell proliferation (as measured by relative light millimeter (RLU)) of ZR75-1(Rb positive) cells (breast cancer) versus variable molarity treated with Compound MM (Table 1; circles) or Compound OO (FIG. 1; squares). As described in fig. 21 and example 152, useAnalysis of luminescent cell viability cell proliferation was determined.

Fig. 29A is a graph of the percentage of cells in G2-M phase (open circles), S phase (triangles), G0-G1 phase (squares), <2N (diamonds) versus variable concentration of compound T (nM) in tHS68 cells. CDK4/6 dependent cell line (tHS68) was treated with compound T at the indicated concentration for 24 hours. After compound T treatment, cells were harvested and analyzed for cell cycle distribution. As described in example 161, tHS68 cells exhibited complete G1 arrest with a corresponding decrease in the number of S phase cells.

Fig. 29B 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 29C 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 29D is a graph of the number of a2058 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 29E 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 161.

Fig. 29F 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) 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 161, treatment of WM2664 cells with compound T caused a loss of the S-phase peak (indicated by the arrow).

Figure 29G is a graph of the number of a2058 cells (CDK 4/6 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 161, treatment of a2058 cells with compound T did not cause S-phase peak loss (indicated by the arrow).

FIG. 30 is a Western blot showing phosphorylation levels of Rb at Ser807/811 and Ser780 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 starting 16 hours after treatment in CDK 4/6-dependent cell line (tHS68 and WM2664), but not in CDK 4/6-independent cell line (a2058), as described in example 162.

Detailed Description

Improved compounds, methods and compositions are provided as chemotherapeutic agents for treating selected RB-positive cancers that minimize or reduce the deleterious effects caused by CDK4/6 growth arrest on CDK4/6 replication-dependent healthy cells, such as hematopoietic stem and/or progenitor cells (HSPCs), in a subject, typically a human.

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 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" embraces "N-alkylamino" and "N, N-dialkylamino" in which the amino groups are independently substituted with one alkyl group 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 can 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 can have a combination of two or more of the same halo atoms or different halo groups. "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. Aryl groups may 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, for example, hydroxy, Boc, halo, haloalkyl, cyano, lower alkyl, lower aralkyl, oxo, lower alkoxy, amino, lower alkylamino, etc.

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 includes C₃-C₆And (4) a ring. Examples include cyclopentyl, cyclopropyl and cyclohexyl. Cycloalkyl groups may be optionally substituted with one or more functional groups such as halo, hydroxy, nitro, amino, cyano, haloalkyl, aryl, heteroaryl, 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.

As used herein, the term "prodrug" 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 root 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 provide the option of modulating the conditions of in vivo production of the parent drug, 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 portion 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.

The present invention relates to HSPC sparing strategies during the treatment of Rb positive proliferative disorders. Thus, as used herein, the term "HSPC" means healthy hematopoietic stem and/or progenitor cells as opposed to diseased HSPC or related blood-derived cells. HSPC includes hematopoietic stem cells, such as long-term hematopoietic stem cells (LT-HSC) and short-term hematopoietic stem cells (ST-HSC): 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).

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, 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.

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 a molar concentration that is at least about 1/500 or 1/1000 or 1/1500 or 1/1800 or 1/2000 less.

As used herein, the term "chemotherapy" or "chemotherapeutic agent" refers to treatment with cytostatic or cytotoxic agents (i.e., compounds) 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.

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 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 as a blood deficiency.

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 a CDK4/6 inhibitor compound, e.g., HSPCs re-enter the cell cycle within relatively the same focused time frame or at relatively the same rate after the action of a compound such as PD0332991 dissipates. By contrast, "non-synchronous reentry 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 reenter the cell cycle within relatively different focused time frames or at relatively different rates after the action of the compounds dissipates.

By "non-circulatory phase" 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 21 days of administration to a subject are of a chemotherapeutic agent and 7 days of non-administration are of a chemotherapeutic agent and the regimen is largely repeated, the 7 day period of non-administration is considered to be the "non-circulatory period" 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 may 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.

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 two R's in the chain in place of a carbon O or N heteroatom and 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 nitrogen atom to which they are attached may be combined to form a compound optionally independently, where valency permits, via one or more R^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 as valence 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- (alkylene)_m-NR³*R⁴- (alkylene)_m-C(O)-R⁵- (alkylene)_m-C(＝S)R⁵- (alkylene)_m-C(＝O)O R⁵- (alkylene)_m-OC(＝O)R⁵- (alkylene)_m-C(S)-OR⁵- (alkylene)_m-C(O)-NR³*R⁴- (alkylene)_m-C(S)-NR³*R⁴- (alkylene)_m-N(R³*)-C(O)-NR³*R⁴- (alkylene)_m-N(R³*)-C(S)-NR³*R⁴- (alkylene)_m-N(R³*)-C(O)-R⁵- (alkylene)_m-N(R³*)-C(S)-R⁵- (alkylene)_m-O-C(O)-NR³*R⁴- (alkylene)_m-O-C(S)-NR³*R⁴- (alkylene)_m-SO₂-NR³*R⁴- (alkylene)_m-N(R³*)-SO₂-R⁵- (alkylene)_m-N(R³*)-SO₂-NR³*R⁴- (alkylene)_m-N(R³*)-C(O)-OR⁵- (alkylene)_m-N(R³*)-C(S)-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, as 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(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 of 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, e.g., 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 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.

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 group)_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 of R are bound to the same or to adjacent atoms^xThe groups may optionally be combined to form a ring.

In some aspects, R²Is- (alkylene)_m-heterocyclyl, - (alkylene)_m-NR³R⁴- (alkylene group)_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 of the present invention, the first and second electrodes are,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 C ₁-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 ofWherein:

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. 13-15.

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. 16-20, 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 C₁-C₄Alkyl and R^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 chemosynthesis is carried out byThe compound has the 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 R ²Is 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 has 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 composition is prepared byThe compound has the 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 R ²Is composed ofWherein P is a 4 to 8 membered monocyclic or bicyclic saturated heterocyclic group.

In some aspects, the active compound is:

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 antineoplastic and antiproliferative agents

Isotopic substitution

The present invention includes the use of compounds and isotopically substituted compounds having a desired atom in an amount 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), (d²H) 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 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. At any position of the compound where a hydrogen atom may be presentThe hydrogen atom may be any isotope of hydrogen, including protium (iii)¹H) 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-tag analog "or" deuterated¹³C-labeled analog ". The term "deuterated analog" means a compound described herein wherein the H-isotope, i.e., hydrogen/protium (C)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%.

Other specific compounds that are part of the invention and that may be used in the disclosed therapeutic methods and compositions include the structures of formulas I, II, III, IV, or V set forth in table 1 below.

Rb-positive cancer and proliferative disorders

In particular, the active compounds described herein may be used to treat subjects suffering from Rb-positive cancer or other Rb-positive abnormal cell proliferation disorders. In some embodiments, the cancer or cell proliferation disorder is a CDK4/6 replication-dependent cancer or cell proliferation disorder, which refers to a cancer or cell proliferation disorder that requires CDK4/6 activity for replication or proliferation, or which may inhibit growth by the activity of a selective CDK4/6 inhibitor. Such types of cancers and conditions may be characterized (e.g., by cellular display) by the presence of a functional retinoblastoma protein. Such cancers and disorders are classified as Rb positive. Rb-positive aberrant cell proliferation disorders and variants of this term as used herein refer to disorders or diseases caused by uncontrolled or aberrant cell division characterized by the presence of functional retinoblastoma proteins, which may include cancer. In one aspect of the invention, the compounds and methods described herein can be used to treat non-cancerous Rb positive abnormal cell proliferation disorders. Examples of such conditions may include non-malignant lymphoproliferation, non-malignant breast neoplasms, psoriasis, arthritis, dermatitis, precancerous colon lesions or flesh quality, angiogenic conditions, immune-mediated and non-immune-mediated inflammatory diseases, arthritis, age-related macular degeneration, diabetes, and other non-cancerous or benign cell proliferative conditions.

Cancer targeting suitable for administration of the compounds described herein may include Rb positive: estrogen receptor positive, HER2 negative advanced breast cancer, advanced metastatic breast cancer, liposarcoma, non-small cell lung cancer, liver cancer, ovarian cancer, glioblastoma, refractory solid tumors, retinoblastoma positive breast cancer and retinoblastoma positive endometrial, vaginal and ovarian cancers as well as lung and bronchial cancers, colon adenocarcinoma, rectal adenocarcinoma, central nervous system germ cell tumor, teratoma, estrogen receptor negative breast cancer, estrogen receptor positive breast cancer, familial testicular germ cell tumor, HER2 negative breast cancer, HER2 positive breast cancer, male breast cancer, ovarian immature teratoma, ovarian mature teratoma, ovarian single and highly specialized teratomas, progesterone receptor negative breast cancer, progesterone receptor positive breast cancer, recurrent colon cancer, recurrent extragonadal germ cell tumor, recurrent extragonadal non-seminoma germ cell tumor, Recurrent extragonadal seminoma, recurrent malignant testicular germ cell tumor, recurrent melanoma, recurrent ovarian germ cell tumor, recurrent rectal cancer, stage III extragonadal non-seminoma germ cell tumor, stage III extragonadal seminoma, stage III malignant testicular germ cell tumor, stage III ovarian germ cell tumor, stage IV breast cancer, stage IV colon cancer, stage IV extragonadal non-seminoma germ cell tumor, stage IV extragonadal seminoma, stage IV melanoma, stage IV ovarian germ cell tumor, stage IV rectal cancer, immature testicular teratoma, mature testicular teratoma. In particular embodiments, the targeted cancer includes estrogen receptor positive, HER2 negative advanced breast cancer, advanced metastatic breast cancer, liposarcoma, non-small cell lung cancer, liver cancer, ovarian cancer, glioblastoma, refractory solid tumors, retinoblastoma-positive breast cancer, and retinoblastoma-positive endometrial, vaginal and ovarian cancers as well as lung and bronchial cancers, metastatic colorectal cancer, metastatic melanoma with CDK4 mutations or amplifications, or cisplatin-refractory unresectable germ cell tumors.

In one embodiment, the Rb-positive cancer is selected from Rb-positive: fibrosarcoma, myxosarcoma, chondrosarcoma, osteosarcoma, chordoma, malignant fibrous histiocytoma, angioendothelioma, angiosarcoma, lymphangiosarcoma, mesothelioma, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma; epidermoid carcinoma, malignant skin appendage tumor, adenocarcinoma, hepatocarcinoma, hepatocellular carcinoma, renal cell carcinoma, suprarenal adenoid tumor, hepatobiliary duct type liver cancer, metastatic cell carcinoma, malignant syncytium tumor, seminoma, embryonal bacteria carcinoma, and glioma; glioblastoma multiforme, neuroblastoma, medulloblastoma, malignant meningioma, malignant schwannoma, neurofibrosarcoma, parathyroid adenocarcinoma, medullary thyroid carcinoma, bronchial carcinoid tumor, pheochromocytoma, islet cell carcinoma, malignant carcinoid tumor, malignant paraganglioma, melanoma, Merkel cell neoplasms (Merkel cell neoplasms), phyllocystic sarcoma, salivary cancer, thymus cancer, bladder cancer, and Wilms tumor (Wilms tumor).

The presence or normal function of retinoblastoma (Rb) tumor suppressor protein (Rb positive) 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 FAC S (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 would be more suitable 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 may be used to determine retinoblastoma gene status. Molecular genetic tests for retinoblastoma include the tests as described in: lohmann and Gallie "Retinob lastoma. Gene Reviews" (2010) http:// www.ncbi.nlm.nih.gov/book skin f/br. fcgibook ═ gene & part ═ Retinob or Parsam et al "A complex sensitive, sensitive and environmental adaptive for the detection of kinetic ons in the RB1gene in kinetic" Journal of Genetics,88(4), 517-.

In some embodiments, the cancer to be treated is selected from estrogen receptor positive, HER2 negative advanced breast cancer, advanced metastatic breast cancer, liposarcoma, non-small cell lung cancer, liver cancer, ovarian cancer, glioblastoma, refractory solid tumors, retinoblastoma positive breast cancer, and retinoblastoma positive endometrial, vaginal and ovarian cancers, and lung and bronchial cancers.

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 replacing themselves by regulated replication in the life of an adult mammal. In addition, stem cells divide asymmetrically, producing "progeny" 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).

It has been found that certain proliferating cells, such as HSPCs, require the proliferative kinases cyclin dependent kinase 4(CDK4) and/or cyclin dependent kinase 6(CDK6) for cell replication. In contrast, the majority of 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 present invention includes methods of treating certain cancers, particularly Rb-positive cancers, while minimizing the deleterious effects of CDK4/6 replication-dependent healthy cells and particularly hematopoietic cells and/or progenitor cells (HSPCs) in a subject by administering the compounds described herein to treat a particular Rb-positive cancer.

In one embodiment, use of the compounds described herein as chemotherapeutic agents allows for accelerated blood recovery and reduced risk of blood insufficiency due to delayed HSPC replication compared to use of other CDK4/6 inhibitors such as PD 0332991. In one embodiment, the use of the compounds described herein as chemotherapeutic agents allows for the reduction or minimization of non-circulation or drug holidays during treatment compared to current treatment modalities using other CDK4/6 inhibitors such as PD 0332991. In one embodiment, the use of a compound described herein as a chemotherapeutic agent allows for elimination of the non-circulatory phase or drug holiday. In one embodiment, the use of the compounds described herein as chemotherapeutic agents allows for extended periods of administration with fewer non-circulatory or drug holidays compared to current treatment modalities using other CDK4/6 inhibitors such as PD 0332991. In one embodiment, use of the compounds described herein as chemotherapeutic agents allows for faster recovery of blood cell numbers during the non-circulatory or drug holiday compared to current modalities using other CDK4/6 inhibitors such as PD 0332991.

In certain embodiments, the compound 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 certain embodiments, the compound administered is selected from the compounds selected from table 1.

In certain aspects, compounds, methods and compositions are provided as chemotherapeutic agents that reduce or limit the deleterious effects of CDK4/6 inhibition on CDK4/6 replication-dependent healthy cells in a subject being treated for CDK4/6 inhibition against Rb-positive cancers, the methods comprising administering an effective amount of a compound described herein, wherein a substantial portion of CDK4/6 replication-dependent healthy cells return to pre-treatment baseline cell cycle activity (i.e., re-enter the cell cycle) within less than about 24, 30, 36 or 40 hours of compound administration. In certain embodiments, wherein the IC of the compound₅₀CDK4 inhibits concentration ratio IC to CDK2₅₀The inhibitory concentration is at least 1500 times less. In certain embodiments, the compound administered is selected from a compound or composition comprising formula I, formula II, formula III, formula IV, or formula V, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof. In certain embodiments, the administered compound is selected from the compounds contained in table 1, or a pharmaceutically acceptable composition, salt, isotopic analog or prodrug thereof. In one embodiment, the CDK4/6 replication-dependent cell is a hematopoietic stem and/or progenitor cell (HSPC).

In certain aspects, compounds, methods and compositions are provided for use as chemotherapeutic agents that limit the deleterious effects of CDK4/6 inhibition on CDK4/6 replication-dependent healthy cells in a subject being treated for Rb-positive cancer, the methods comprising administering an effective amount of a compound described herein, wherein a substantial portion of CDK4/6 replication-dependent healthy cells synchronously re-enter the cell cycle within less than about 24, 30, 36 or 40 hours after compound inhibition dissipates. In certain embodiments, wherein the IC of the compound₅₀CDK4 inhibits concentration ratio IC to CDK2₅₀The inhibitory concentration is at least 1500 times less. In certain embodiments, the compound administered is selected from a compound or composition comprising formula I, formula II, formula III, formula IV, or formula V, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof. In certain embodiments, the administered compound is selected from the compounds contained in table 1, or a pharmaceutically acceptable composition, salt, isotopic analog or prodrug thereof. In one embodiment, the CDK4/6 replication-dependent cell is a hematopoietic stem and/or progenitor cell (HSPC).

In certain aspects, compounds, methods, and compositions are provided for use as chemotherapeutic agents that limit the deleterious effects of CDK4/6 inhibition on CDK4/6 replication-dependent healthy cells in a subject, the methods comprising administering to a subject having an Rb-positive cancer an effective amount of a compound described herein, wherein a substantial portion of the CDK4/6 replication-dependent healthy cells synchronously re-enter the cell cycle within less than about 24, 30, 36, or 40 hours after the compound's CDK4/6 inhibition has dissipated. In one embodiment, the IC of the compound is administered ₅₀CDK4 inhibits concentration ratio IC to CDK2₅₀The inhibitory concentration was over 500 times less. In certain embodiments, a substantial portion of the CDK4/6 replication-dependent healthy cells synchronously re-enter the cell cycle less than about 24, 30, 36, or 40 hours from the time the concentration level of the compound in the subject's blood falls below a therapeutically effective concentration. In certain embodiments, the compound administered is selected from a compound or composition comprising formula I, formula II, formula III, formula IV, or formula V, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof. In certain embodiments, the administered compound is selected from the compounds contained in table 1, or a pharmaceutically acceptable composition, salt, isotopic analog or prodrug thereof. In one embodiment, the CDK4/6 replication-dependent cell is a hematopoietic stem and/or progenitor cell (HSPC). In one embodiment, the CDK4/6 replication-dependent healthy cells are renal epithelial cells.

In certain embodiments, the compound administered is selected from the group consisting of a compound or composition comprising formula I, formula II, formula III, formula IV, or formula V, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof, or a compound contained in table 1, or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof, wherein the effect of the compound is transient and transient in nature, allowing a significant portion of the CDK4/6 replication-dependent healthy cells to rapidly synchronize re-enter the cell cycle, e.g., within less than about 24, 30, 36, or 40 hours of the last administration of the ionomeric compound.

The compounds used in the described methods are highly selective potent CDK4/6 inhibitors, with minimal CDK2 inhibitory activity. In one embodiment, the compound used in the methods described herein has CDK4/CycD1IC₅₀Inhibition 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 compounds used in the methods described herein have IC for CDK4/CycD1 inhibition₅₀Concentration value of about<1.50nM、<1.25nM、<1.0nM、<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 use in the methods described herein₅₀Concentration value of about>1.0μM、>1.25μM、>1.50μM、>1.75μM、>2.0μM、>2.25μM、>2.50μM、>2.75μM、>3.0μM、>3.25μM、>3.5. mu.M or more. In one embodiment, the compound pair used in the methods described herein is CDK2/CycA IC₅₀Has an IC50 concentration value of>0.80μM、>0.85μM、>0.90μM、>0.95μM、>1.0μM、>1.25μM、>1.50μM、>1.75μM、>2.0μM、>2.25μM、>2.50μM、>2.75uM、>3.0. mu.M or more.

In certain embodiments, compounds suitable for use in the methods may provide transient and rapidly reversible G1 arrest of CDK4/6 replication-dependent healthy cells while providing growth inhibition of CDK4/6 replication-dependent cancers. By having a transient effect for a limited time, the use of such compounds as chemotherapeutic agents allows CDK4/6 replication-dependent healthy cells to re-enter the cell cycle more rapidly after treatment cessation than, for example, long-acting CDK4/6 inhibitors (e.g., PD 0332991). The faster resolution of the G1 arrest of CDK4/6 replication-dependent healthy cells makes such compounds superior to long-acting CDK4/6 inhibitors in the following cases: 1) the subject will be exposed to intensive therapy, wherein use of a long-acting CDK4/6 inhibitor will prevent CDK4/6 replication-dependent healthy cells from cycling between exposures; 2) a continuous or chronic treatment regimen, wherein long-term G1 arrest of CDK4/6 replication-dependent healthy cells is a side effect of targeted cancer growth inhibition, and the subject would benefit from CDK4/6 replication-dependent healthy cells rapidly re-entering the cell cycle after cessation of the treatment regimen, between administrations of the inhibitor in the continuous regimen, or between treatment interruptions, thereby limiting replication delay, thus reducing, limiting or ameliorating further healthy cell damage, such as bone marrow suppression, after cessation of treatment. In accordance with the present invention, chemotherapeutic regimens employing the selective compounds described herein can be achieved by a number of different dosing schedules, including cyclic/acyclic regimens and continuous treatment regimens.

In one embodiment, the compounds described herein are used in a CDK4/6 replication-dependent healthy cell cycle strategy in which subjects are exposed to conventional repeated chemotherapeutic treatment against Rb-positive cancers. Such cycling allows CDK4/6 replication-dependent cells to regenerate disrupted blood cell lineages between conventional repeat treatments and reduces the risk associated with long-term CDK4/6 inhibition. This cycling between the G1 arrested and replicated states is not possible with time-interval-limited repeat agent exposures using more long-acting CDK4/6 inhibitors (e.g., PD0332991), because the delayed G1 arrest effect of the compound inhibits CDK4/6 replication-dependent cells from significantly and meaningfully re-entering the cell cycle before the next exposure to CDK4/6 inhibitor or delays healthy cells from entering the cell cycle and recovering the damaged tissue or cells after treatment is stopped.

In one embodiment, the use of a compound described herein provides rapid reentry into the cell cycle of CDK4/6 replication-dependent healthy cells, e.g., HSPCs, such that the cells return to pre-treatment baseline cell cycle activity in less than about 40 hours, 36 hours, 30 hours, 28 hours, 24 hours, or less. In one embodiment, the compounds described herein are used to provide rapid reentry into the cell cycle of CDK4/6 replication-dependent healthy cells, e.g., HSPCs, such that the cells approach pre-treatment baseline cell cycle activity in less than about 40 hours, 36 hours, 30 hours, 28 hours, 24 hours, 18 hours, 16 hours, 14 hours, 12 hours, or less. In one embodiment, the use of a compound described herein provides rapid reentry into the cell cycle of CDK4/6 replication-dependent healthy cells, e.g., HSPCs, such that the cells return to pre-treatment baseline cell cycle activity in less than about 40 hours, 36 hours, 30 hours, 28 hours, 24 hours, 18 hours, 16 hours, 14 hours, 12 hours, or less from the last administration of the compound described herein. In one embodiment, the use of a compound described herein provides rapid reentry of CDK4/6 replication-dependent healthy cells into the cell cycle such that the cells approach pre-treatment baseline cell cycle activity in less than about 40 hours, 36 hours, 30 hours, 28 hours, 24 hours, 18 hours, 16 hours, 14 hours, 12 hours, or less from the last administration of the compound. In one embodiment, the use of a compound described herein provides rapid reentry of CDK4/6 replication-dependent healthy cells into the cell cycle such that the cells approach pre-treatment baseline cell cycle activity in less than about 40 hours, 36 hours, 30 hours, 28 hours, 24 hours, 18 hours, 16 hours, 14 hours, 12 hours, or less from the time the concentration level of the compound in the subject's blood falls below a therapeutically effective concentration. 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, the rapid re-entry into the cell cycle is synchronized.

In one embodiment, the compounds described herein are used to provide rapid reentry of CDK4/6 replication-dependent healthy cells, e.g., HSPCs, into the cell cycle such that a portion of the cells exhibit some level of cell cycle activity, or are capable of entering the cell cycle and proliferating, during a continuous treatment regimen, e.g., a treatment regimen in which the compound is administered for an extended period of time, e.g., 5 consecutive days, 7 consecutive days, 10 consecutive days, 14 consecutive days, 18 consecutive days, 21 consecutive days, 24 consecutive days, 28 consecutive days, 35 consecutive days, or more. In one embodiment, a compound suitable for use in the method is administered for a continuous period of time, e.g., 21, 28, 35 or more days, without the need for a non-circulatory period or drug holiday. In one embodiment, the use of the compounds described herein eliminates the need for a non-circulatory period, drug holiday, or reduces the concentration of antineoplastic compounds co-administered during treatment.

In accordance with the present invention, the compounds described herein can be administered as chemotherapeutic agents to subjects suffering from Rb positive proliferative disorders on any treatment schedule and at any dosage that meets the prescribed course of treatment. For example, the compound may be administered once a day, twice a day, or three times a day. The compounds may be administered on alternating days, or every three days, or every four days, or every five days, or every six days, or every other week. The compounds may be administered every other week or month.

Combination therapy

In one aspect of the invention, the compounds disclosed herein may be advantageously administered in combination with a therapeutic regimen to achieve a beneficial, additive, or synergistic effect.

In one embodiment, the compounds/methods of the present invention are used in combination with another therapy for treating Rb-positive cancer. The second therapy may be immunotherapy. As discussed in more detail below, the compound may be conjugated to an antibody, radioactive agent, or other targeting agent that directs the compound to diseased or abnormally proliferating cells. In another embodiment, the compounds are used in combination with another drug or biological agent (e.g., an antibody) to increase the efficacy of the treatment using a combination or synergistic approach. In one embodiment, the compounds may be used with T cell vaccination, which typically involves immunization with inactivated autoreactive T cells to eliminate a population of Rb-positive cancer cells as described herein. In another embodiment, the compounds are used in combination with a bispecific T cell engager (BiTE), an antibody designed to conjugate simultaneously to a specific antigen on both endogenous T cells and Rb-positive cancer cells as described herein, linking both types of cells.

In one embodiment, the other therapy is a monoclonal antibody (MAb). Some mabs stimulate an immune response that destroys cancer cells. Similar to antibodies naturally produced by B cells, these mabs "coat" the surface of cancer cells, triggering destruction thereof by the immune system. For example, bevacizumab (bevacizumab) targets Vascular Endothelial Growth Factor (VEGF), a protein secreted by tumor cells and other cells in the tumor microenvironment that promotes tumor vascular development. When conjugated to bevacizumab, VEGF fails to interact with its cellular receptors, preventing signaling that leads to new blood vessel growth. Similarly, cetuximab (cetuximab) and panitumumab (panitumumab) target Epidermal Growth Factor Receptor (EGFR), and trastuzumab (trastuzumab) targets human epidermal growth factor receptor 2 (HER-2). Mabs conjugated to cell surface growth factor receptors prevent the targeted receptor from signaling its normal growth promotion. It can also trigger apoptosis and activate the immune system to destroy tumor cells.

Another group of cancer therapeutic mabs are immunoconjugates. These mabs, sometimes referred to as antitoxins or antibody-drug conjugates, consist of an antibody attached to a cell-killing substance (e.g., a plant or bacterial toxin, a chemotherapeutic drug, or a radioactive molecule). The antibody locks to its specific antigen on the surface of the cancer cell and the killed cellular material is taken up by the cell. FDA-approved conjugated mabs that work in this manner include ado-trastuzumab maytansine (ado-trastuzumab emtansine), which targets the HER-2 molecule to deliver drug DM1, which inhibits cell proliferation, to HER-2-expressing metastatic breast cancer cells.

Immunotherapy that engineer T cells to recognize cancer cells via bispecific antibodies (bsabs) or Chimeric Antigen Receptors (CARs) is a method that is capable of separating dividing from non-dividing/slow-dividing subpopulations of cancer cells.

Bispecific antibodies that simultaneously recognize a target antigen and an activating receptor on the surface of an immune effector cell provide the opportunity to redirect immune effector cells to kill cancer cells. Another approach is to generate chimeric antigen receptors by fusion of extracellular antibodies with intracellular signaling domains. Chimeric antigen receptor engineered T cells are capable of specifically killing tumor cells in an MHC independent manner.

In some embodiments, the compound may be administered to the subject in combination with other chemotherapeutic agents. Where appropriate, the compounds described herein may be administered simultaneously with another chemotherapeutic agent to simplify the treatment regimen. In some embodiments, the compound and the other chemotherapeutic agent may be provided in a single formulation. In one embodiment, the use of the compounds described herein is combined with other agents in a therapeutic regimen. 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 (including, but not limited to, MK-2206, GSK690693, piperacillin, KRX-0401, GDC-0068, Triciribine (Triciribine), AZD5363, and magnolol (Honokiol), PF-04691502, and Miltefosine (AMP)), PD-1 inhibitors (including, but not limited to, Nivolumab (millib), valluvistin (bortezomib), MK-65514, MK-75, and nitezomib (MK) inhibitors, including, MK-406, MK-22075), and/or combinations thereof, Dolivitinib (Dovitinib), quinatinib (Quizartinib) (AC220), amutinib (Amuvatinib) (MP-470), tangtinib (Tandutinib) (MLN518), emmd-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, desmethylviridin, pirifolin, edberg, PX-866, IPI-145(Infinity), BAY 80-6946, BEZ235, RP6503, TGR 1202(RP5264), MLN1117(INK1117), Pirisib, Bupaspaler, SAR 2454408 (XL147), SAR 2458409 (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 a particular embodiment, the compounds described herein are administered in combination with letrozole and/or tamoxifen. Other chemotherapeutic agents that may be used in combination with the compounds described herein include, but are not limited to, chemotherapeutic agents that do not require cell cycle activity for anti-neoplastic effects.

In one embodiment, the CDK4/6 inhibitor described herein may be combined with a chemotherapeutic agent selected from (but not limited to) the following: imatinib mesylate (Imatinib mesylate)Dasatinib (Dasatinib)Nilotinib (Nilotinib)Bosutinib (Bosutinib)TrastuzumabPertuzumab (Pertuzumab) (Perjeta TM), Lapatinib (Lapatinib)Gefitinib (Gefitinib)Erlotinib (Erlotinib)Cetuoxi-mexAnti (Cetuximab)Panitumumab (Panitumumab)Vandetanib (Vandetanib)Weirofenib (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).

In certain aspects, the additional therapeutic agent is an anti-inflammatory agent, a chemotherapeutic agent, radiation therapy, an additional therapeutic agent, or an immunosuppressive agent.

Suitable chemotherapeutic agents include, but are not limited to, radioactive molecules, toxins (also known as cytotoxins or cytotoxic agents, which include any agent that is detrimental to cell viability), agents, and liposomes or other vesicles containing chemotherapeutic compounds. Typical anticancer drugs include: vincristine (Vincristine) Or liposomal vincristineDaunomycin (Daunorubicin) or daunomycin) Or doxorubicin) Cytarabine (Cytarabine), ara-C or) L-asparaginase (L-asparaginase)Or PEG-L-asparaginase (pegaspragase or) Etoposide (VP-16) and Teniposide (Teniposide)6-mercaptopurine (6-mercaptoprine) (6-MP or) Methotrexate (Methotrexate), Cyclophosphamide (Cyclophosphamide)Prednisone (Prednisone), Dexamethasone (Dexamethasone) (Decadron), imatinib (imatinib)) Dasatinib (dasatinib)Nilotinib (nilotinib)BosutinibAnd ponatinib (Iclusig)^TM). Examples of other suitable chemotherapeutic agents include, but are not limited to, 1-dehydrotestosterone, 5-fluorouracil (5-fluorouracil) dacarbazine (decarbazine), 6-mercaptopurine (6-mercapture), 6-thioguanine (6-thioguanine), actinomycin D (actinomycin D), adriamycin (adriamycin), aldesleukin (aldesleukin), alkylating agents, sodium allopurinol (allopurinol sodium), altretamine (altramine), amifostine (amifostine), anastrozole (anastrozole), animycin (anthramycin, mitotic), cis-dichlorodiammine (II) (DDP) (cisplatin), diamidodichloroplatinum (diamidoplanum), anthracyclines (anthracyclines), antibiotics (antibiotics), anti-neoplastics (anti-thioguanine), and anti-epididymides (anti-thioguanine) Metabolites (antimetabolite), asparaginase (asparaginase), live BCG (intravesical), betamethasone sodium phosphate (betamethasone sodium phosphate) and betamethasone acetate (betamethasone acetate), bicalutamide (bicalutamide), bleomycin sulfate (bleomycin sodium sulfate), busulfan (busulfan), calcium formyltetrahydrofolate (calcium leucovorin), calicheamicin (calicheamicin), capecitabine (capecitabine), carboplatin (carboplatin), lomustine (lomustine), carmustine (CCNU), carmustine (BSNU), Chlorambucil (chlomambucil), cisplatin, Cladribine (Cladribine), colchicine (Colchicin), actinomyces (actinomyces), cyclophosphamide (cyclophosphamide), cytarabine (dactinomycin), cytarabine (monocalcin), dactinomycin hydrochloride (dactinomycin), dactinomycin (dactinomycin), dactinomycin (e, dactinomycin, dactylosin, dactinomycin, and other, Daunorubicin citrate (daunorubicin citrate), dinierein (denileukin diftox), Dexrazoxane (Dexrazoxane), Dibromomannitol (Dibromomannitol), dihydroxyanthralin dione (dihydroanthracin dione), Docetaxel (Docetaxel), dolasetron mesylate (dolasetron mesylate), doxorubicin hydrochloride (doxorubicin HCL), cannabinol (dronabinol), escherichia coli L-asparaginase (e.coli L-aspargine), emetine (emetine), erythropoietin-a (epoetin-a), Erwinia L-asparaginase (Erwinia L-aspargine), esterified estrogen (ethisterone), estradiol (estradiol), estramustine (setastine), phosophonate (ethisterone), ethidium phosphate (etoposide), etoposide (sodium phosphate), ethisterone (ethisterone phosphate (ethisterone), ethisterone (e) and combinations thereof, Filgrastim (filgrastim), fluuridine (floxuridine), fluconazole (fluconazole), fludarabine phosphate (fludarabine phosphate), fluorouracil (fluorouracil), flutamide (flutamide), folinic acid (folinic acid), gemcitabine hydrochloride (gemcitabine HCL), glucocorticoids (glucoorticoid), goserelin hydrochloride (goserelin acetate), gramicidin D (gramicidin D) ) Gelatillon hydrochloride (granisetron HCL), hydroxyurea (hydroxyurea), idarubicin hydrochloride (idarubicin HCL), ifosfamide (ifosfamide), interferon alpha-2 b (interferon alpha-2 b), irinotecan hydrochloride (irinotecan HCL), letrozole (letrozole), leucovorin calcium (leucovorin calcium), leuprolide acetate (leuprolide acetate), levotetramisole hydrochloride (levamisole HCL), lidocaine (lidocaine), lomustine (lomustine), maytansinoid (maytansinoid), mechlorethamine hydrochloride (mechlorothiamine HCL), medroxyprogesterone acetate (medroxyprogesterone acetate), megestrol acetate (megestrol hydrochloride), melphalan (melphalan HCL), mechlorethamine (medroxyprogesterone), medroxyprogesterone hydrochloride (medroxyprogesterone), mitoxantrone (mitoxantrone), medroxypterin (mitoxantrone), medroxide (mitoxantrone), medrythrone, mitoxantrone (mitoxantrone), medrythrone hydrochloride (mitoxantrone, mitoxantrone, Octreotide acetate (octreotide acetate), ondansetron Hydrochloride (HCL), paclitaxel (paclitaxel), disodium pamidronate (sodium discodate), pentostatin (pentostatin), pilocarpine hydrochloride (pilocarpine HCL), plicamycin (plimycin), polifeprosan20with carmustine implant, porfimer sodium (porfimer sodium), procaine (procaine), procarbazine hydrochloride (procarbazine HCL), propranolol (propranolol), rituximab (rituximab), samustine (sargramostim), streptozotocin (streptazocin), tamoxifen, taxol (tenxoside), teniposide (tetrodotril), tetrodotril (oxyprotene), thioteponin (triptolide), thiotepine (oxytetracycline), thiocorazine (tetracaine), thiotepine (tetracaine), thiotepine), thiotepicine (tetracaine), thiotepine (tetracaine) and a) in (tetracalcium) and a) to form (tetracalcium) to be obtained by using a) to obtain a) and a) to obtain a, Tretinoin (tretinoin), valrubicin (valrubicin), vinblastine sulfate (vinblastine sulfate), vincristine sulfate (vincristine sulfate), and vinorelbine tartrate (vinorelbine tartrate).

Other therapeutic agents that may be administered in combination with the compounds disclosed herein may include bevacizumab (bevacizumab), sunitinib (sutinib), sorafenib (sorafenib), 2-methoxyestrol (2-methoxystradienol) or 2ME2, finasteride (finasterite), vatalanib (vatalanib), vandetanib (vandetanib), alfacalcizumab (aflibercept), valaciximab (volociximab), etalizumab (etalcizumab) (MEDI-522), selegilitide (cilentide), erlotinib (erlotinib), cetuximab (cetuximab), panitumumab (panitumumab), gefitinib (gefitinib), trastuzumab, multi-vitalizumab (vituzumab), non-glutethimide (glutethilizumab), interleukin (rituximab), aluzumab), everolimus (rituximab), everolizumab (rituximab), rituximab (rituximab), rituximab (perituzumab (rituximab), rituximab (rituximab), rituximab (rituximab), and (rituximab), Lucaizumab (lucatumumab), daclizumab (dacetuzumab), HLL1, huN901-DM1, amikamod (atiprimod), natalizumab (natalizumab), bortezomib (bortezomib), carfilzomib (carfilzomib), mozizomib (marizoib), tasomycin (tanespimamycin), saquinavir mesylate (saquinavir mesylate), ritonavir (ritonavir), nelfinavir mesylate (nelfinavir mesylate), indinavir sulfate (indinavir sulfate), belinostat (belinostat), pangolistat (panobinostat), macbetumab (mapatumumab), lesatuzumab (lexatuzumab), duronim (dulanermin), ABT-737, oblimersen (oblimersen), piceidin (plitidipsin), tammopimod (talmopimod), P276-00, enzastacin (enzastaurin), tipifarnib (tipiranib), perifosin, imatinib, dasatinib, lenalidomide (lenalidomide), thalidomide (thalidomide), simvastatin (simvastatin), and celecoxib (celecoxib).

In one aspect of the invention, the compounds described herein may be combined with at least one immunosuppressive agent. The immunosuppressant is preferably selected from calcineurin inhibitors, such as cyclosporine or ascomycin, such as cyclosporin AFK506 (tacrolimus), picrolimus (pimecrolimus)); mTOR inhibitors, e.g. rapamycin or derivatives thereof, e.g. sirolimusEverolimusTemsirolimus, zotarolimus (zotarolimus), bailinolimus-7 (biolimus-7), bailinolimus-9 (biolimus-9); rapamycin analogues (rapalogs), such as, for example, bendamustine (ridaforolimus), azathioprine (azathioprine), carmolimus 1H (campath 1H); S1P receptor modulators, such as fingolimod or an analogue thereof, an anti-IL-8 antibody, mycophenolic acid or a salt (e.g. sodium salt) thereof or a prodrug thereof (e.g. Mycophenolate Mofetil))、OKT3(ORTHOCLONE) Prednisone (Prednisone),Brequinar Sodium (Brequinar Sodium), OKT4, T10B9.A-3A, 33B3.1, 15-deoxyspergualin (15-deoxyspergualin), Trespelimus (tresperimus), Leflunomide (Leflunomide)CTLAI-Ig, anti-CD 25, anti-IL 2R, Basiliximab Daclizumab (Daclizumab)Mizobine (mizorbine), methotrexate, dexamethasone, ISAtx-247, SDZ ASM 981 (pimecrolimus),) CTLA4lg (Abatacept), bilactate (belitacept), LFA3lg, etanercept (Immunex to Enhancept)Sale), adalimumab (adalimumab)Infliximab (infliximab)anti-LFA-1 antibody, natalizumab (natalizumab)Enromab (Enlimomab), gavelizumab (gavilimob), antithymocyte immunoglobulin (antithymocyte immunoglobulin), spiraprizumab (siplizumab), alefacezumab (Alefacept efalizumab), pentasil (pentasa), mesalamine (mesalazine), asacol (asacol), codeine phosphate (codeine phosphate), acetaminophen (benorilate), biphenyl butanoic acid (fenbufen), naproxen (naprosyn), diclofenac (diclofenac), etodolac (etodolac), and indomethacin (indomethacin), aspirin (aspirin), and ibuprofen (ibuprofen).

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 some embodiments, selective compounds can be administered to a subject such that other chemotherapeutic agents can 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 in a standard chemotherapy treatment plan. The chemotherapy dose intensity represents a unit dose 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.

In one embodiment of the invention, the compounds described herein may be administered in a regimen with another agent, such as a non-DNA damaging targeted antineoplastic agent or a hematopoietic growth factor agent. It has recently been reported that untimely administration of hematopoietic growth factors may have serious side effects. For example, the use of EPO family growth factors has been associated with arterial hypertension, cerebral convulsions, hypertensive encephalopathy, thromboembolism, iron deficiency, influenza-like syndrome, and venous thrombosis. The G-CSF family of growth factors has been associated with enlargement and rupture of the spleen, respiratory distress syndrome, allergies and sickle cell complications. By combining the administration of the short term selective compounds described herein and the methods of the invention with the timely administration of hematopoietic growth factors, for example when diseased cells are no longer in growth arrest, the health care practitioner can reduce the amount of growth factors to minimize unwanted side effects while achieving the desired therapeutic benefit. In one embodiment, the growth factor is administered after cessation of the compound's effect on CDK4/6 replication-dependent healthy cells, e.g., HSPCs. Thus, in this embodiment, use of the selective compounds described herein in an anti-neoplastic treatment regimen allows the subject to receive a reduced amount of growth factors because the targeted hematopoietic cells will re-enter the cell cycle faster than other compounds such as PD 0332991. In addition, the rapid re-entry into the cell cycle following G1 arrest using the compounds described herein provides for the administration of timed hematopoietic growth factors to help reconstitute hematopoietic cell lines to maximize the ability of the growth factors to act, that is, when the growth factors will be most effective. 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 (macronuclear growth and development factor (MGDF), such as sold as Romithrasin (Romitrosmostim) and Eprobopa (Eltrompag), Interleukin (IL) -12, interleukin-3, interleukin-11 (adipogenesis inhibitory factor or Opperleukin), 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, Repoeitin, Vintor, Epofit, Erykine, Wepox, Espogen, Relipoeitin, Shanpoeitin, Zorytin, and EPIAO). 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 compound on HSPCs has dissipated. In one embodiment, the growth factor is administered at least 20 hours after administration of the compound described herein.

If desired, multiple doses of a compound described herein can be administered to a subject. Alternatively, a single dose of a compound described herein may be administered to a subject. For example, the compound may be administered such that CDK4/6 replication-dependent healthy cells arrest at G1, wherein many healthy cells re-enter the cell cycle and are able to replicate shortly after 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 compound is subsequently administered. In one embodiment, the compound 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 regimens, e.g., long-term repeat dosing regimens.

In some embodiments, CDK4/6 replication-dependent healthy cells may be arrested for a longer period, e.g., over hours, days, weeks, and/or months, by multiple time-limited separate administrations of the compounds described herein. Because CDK4/6 replication-dependent healthy cells, such as HSPC, rapidly and synchronously re-enter the cell cycle after the compound inhibitory intracellular effects dissipate, cells are able to rejuvenate cell lineages more rapidly than compound inhibitors with longer G1 arrest patterns, such as PD 0332991.

The reduction in side effects, particularly myelosuppression, provided by the compounds described herein may allow for dose escalation (e.g., more therapy may be given over a fixed period of time), which will translate into better efficacy. Thus, the disclosed methods may render chemotherapeutic regimens less toxic and more effective. Where appropriate, the small molecules may be formulated for oral, topical, intranasal, inhalation, intravenous, or any other desired form of administration.

Compounds suitable for use in the methods described herein are selective CDK4/6 inhibitors that selectively inhibit at least one of CDK4 and CDK6 or by inhibiting cell replication of Rb-positive cancers. In one embodiment, e.g., CDK4/CycD1IC₅₀IC of CDK4 by compounds described herein measured in phosphorylation assays₅₀Such as CDK2/CycE IC₅₀IC of CDK2 by the compound measured in phosphorylation assay₅₀At least 1500 or more 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 a compound as described herein can induce selective G1 arrest in CDK 4/6-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 compound induces substantially pure (i.e., "complete") G1 cell cycle arrest in CDK4/6 dependent cells (e.g., wherein treatment of the compound induces cell cycle arrest such that a majority of the cells arrest at 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 the methods of assessing the cell division phase of a cell population are known in the art (see, e.g., U.S. patent application publication No.2002/0224522) and include cytometry analysis, microscopic analysis, gradient centrifugation, panning, fluorescence techniques (including immunofluorescence), and combinations thereof the cytometry techniques include exposing the cells to a labeling agent or staining agent, for example, a DNA binding dye, such as PI, and analyzed for 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 associated with, inter alia, inhibition of kinases other than CDK4 and or CDK6 (e.g., CDK2) are reduced or substantially absent using compounds described herein because compounds described herein are poor inhibitors of CDK2 (e.g., CDK2)>1μM 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, due to the transient nature of G1 arrest, CDK4/6 replication-dependent healthy cells re-enter the cell cycle relatively more rapidly than with PD0332991, such that in one embodiment the risk of blood insufficiency is reduced during long-term treatment regimens due to HSPCs being able to replicate between chemotherapeutic treatments.

In one aspect of the invention, the compounds disclosed herein may be advantageously administered in combination with any treatment regimen requiring radiation therapy, chemotherapy, or other therapeutic agents. In other embodiments, the compounds disclosed herein may be advantageously administered in combination with a therapeutic agent that targets an autoimmune disorder.

Drug conjugates

In one embodiment, the activity of an active compound for the purposes described herein may be enhanced by conjugation to an agent that targets diseased or abnormally proliferating cells or otherwise enhances activity, delivery, pharmacokinetics, or other beneficial properties.

For example, the compound may be administered as an antibody-drug conjugate (ADC). In certain embodiments, selected compounds described herein can be conjugated or administered in combination with an antibody or antibody fragment. Fragments of antibodies may be produced by chemical or genetic mechanisms. The antibody fragment may be an antigen-binding fragment. For example, the antigen binding fragment may be selected from Fab, Fab ', (Fab')2 or Fv. The antibody fragment may be a Fab. Monovalent f (ab) fragments have one antigen binding site. The antibody may be a bivalent (Fab')2 fragment having two antigen binding regions linked by a disulfide bond. In one embodiment, the antigen fragment is (Fab'). Reduction of the F (ab ')2 fragment produces two monovalent Fab' fragments with free sulfhydryl groups suitable for conjugation to other molecules.

Selected compounds described herein can be conjugated or administered in combination with an Fv fragment. The Fv fragment is the smallest fragment made by enzymatic cleavage of antibodies of the IgG and IgM classes. The Fv fragment has an antigen binding site made up of the VH and VC regions, but it lacks the CH1 and CL regions. The VH and VL chains are bound in the Fv fragment by non-covalent interactions.

In one embodiment, the selected compound as described herein may be combined with an antibody fragment selected from an ScFv, a domain antibody, a diabody, a trifunctional antibody, a tetrafunctional antibody, a bis ScFv, a minibody, a Fab2, or a Fab3 antibody fragment. In one embodiment, the antibody fragment is a ScFv. Genetic engineering methods allow the generation of single chain variable fragments (ScFv), which are Fv-type fragments comprising VH and VL domains linked with a flexible peptide. When the linker is at least 12 residues long, the ScFv fragment is predominantly monomeric. Manipulation of the orientation of the V-domains and linker lengths produces various Fv molecule linkers that are 3-11 residues long, resulting in scFv molecules that do not fold into functional Fv domains. These molecules may be associated with a second scFv molecule to produce a bivalent diabody. In one embodiment, the antibody fragment administered in combination with the selected compound described herein is a bivalent diabody. If the linker is less than three residues in length, the scFv molecules associate to a trifunctional or tetrafunctional antibody. In one embodiment, the antibody fragment is a trifunctional antibody. In one embodiment, the antibody fragment is a tetrafunctional antibody. By further polyconjugation to two target antigens, the off-rate of the antibody fragment is reduced and the multivalent scFv has a greater functional binding affinity for its target antigen than its monovalent counterpart. In one embodiment, the antibody fragment is a minibody. Minibodies are scFv-CH3 fusion proteins that assemble into bivalent dimers. In one embodiment, the antibody fragment is a bis-scFv fragment. The bis-scFv fragment is bispecific. Miniaturized scFv fragments can be generated with two different variable domains, allowing these bis scFv molecules to bind to two different epitopes simultaneously.

In one embodiment, the selected compounds described herein are conjugated or administered in combination with a bispecific dimer (Fab2) or a trispecific dimer (Fab 3). Genetic methods are also used to generate bispecific Fab dimers (Fab2) and trispecific Fab trimers (Fab 3). These antibody fragments are capable of binding 2 (Fab2) or 3 (Fab3) different antigens simultaneously.

In one embodiment, selected compounds described herein can be conjugated or administered in combination with an rgig antibody fragment. rIgG antibody fragments refer to reduced IgG (75,000 daltons) or half of the IgG. Which is a product of selectively reducing only the disulfide bonds of the hinge region. Although several disulfide bonds are present in IgG, those in the hinge region are most accessible and readily reduced, especially with weak reducing agents such as 2-mercaptoethylamine (2-MEA). Half of the IgG is often prepared for targeting exposed hinge region sulfhydryl groups that can be targeted for conjugation, antibody immobilization, or enzyme labeling.

In other embodiments, selected active compounds described herein can be linked to a radioisotope to increase efficacy using methods well known in the art. Any radioisotope suitable for use in Rb-positive cancer cells can be incorporated into the conjugate, such as (but not limited to) ¹³¹I、¹²³I、¹⁹²Ir、³²P、⁹⁰Sr、¹⁹⁸Au、²²⁶Ra、⁹⁰Y、²⁴¹Am、²⁵²Cf、⁶⁰Co or¹³⁷Cs。

Notably, linker chemistry may be important to the efficacy and tolerability of drug conjugates. Thioether-linked T-DM1 increases serum stability relative to the disulfide linker form and appears to undergo endosomal degradation, resulting in intracellular release of cytotoxic agents, thereby improving efficacy and tolerability. See Barginear, M.F. and Budman, D.R., Trastuzumab-DM1: A review of The novel immune-conjugate for HER 2-overpressuring breaker, The Open Breast Cancer Journal,1:25-30,2009.

Examples of early and recent antibody-drug conjugates for product development that can be used in the present invention, discussing drugs, linker chemistry and target classes can be found in the following reviews: casi, G. and Neri, D., Antibody-drug conjugates, basic conjugates, examples and future perspectives, J.Control Release 161(2), 422-; chari, r.v., Targeted cancer therapy: relating specificity to cytoxic drugs, acc.chem.rev.,41(1):98-107,2008; sapra, p. and Shor, b., Monoclonal antibody-based therapeutics in cans: advances and gallens, pharmacol. ther.,138(3): 452-; schliemann, c. and Neri, d., anti-body-based targeting of the tumor vascular structure, biochim. biophysis. acta, 1776(2) 175-; sun, Y, Yu, F, and Sun, B.W., Antibody-drug conjugates as targeted cancer therapeutics, Yao Xue Xue Bao,44(9): 943-; teicher, b.a. and Chari, r.v., Antibody conjugate therapeutics, changees and potential, clin.cancer res, 17(20) 6389-; firer, M.A. and Gellerman, G.J., Targeted drug delivery for cancer therapy the other side of antibiotics, J.Hematol.Oncol.,5:70,2012; vlachakis, D. and Kossida, S., anti body Drug Conjugate biologicals, Drug delivery through the letterbox, Computt. Math. methods Med., 2013; 2013:282398, Epub 2013, 6 months and 19 days; lambert, J.M., Drug-conjugated antibodies for the treatment of cancer, Br.J.Clin.Pharmacol, 76(2): 248-; concalves, A., Tredan, O., Villanueva, C., and Dumontet, C., Antibody-drug conjugates in the interactive, from the concept to trastuzumab emtansine (T-DM1), Bull. cancer,99(12) 1183-; newland, A.M., Brentuximab vedotin, a CD-30-directed antibody-cytoxic drug conjugate, Pharmacotherapy,33(1), 93-104,2013; lopus, M., Antibody-DM1conjugates as Cancer therapeutics, Cancer Lett.,307(2) 113-; chu, Y.W. and Poison, A., Antibody-drug conjugates for the treatment of B-cell non-Hodgkin's lymphoma and Leukemia, Future Oncol.,9(3):355-368, 2013; bertholjti, I., Antibody-drug conjugate-a new for regulated cancer treatment, Chimia,65(9):746 748, 2011; vincent, K.J. and Zurini, M., Current protocols in antibody engineering, Fc engineering and pH-dependent antibody binding, biospecific antibodies and antibody drug conjugates, Biotechnol.J.,7(12): 1444-; haeuw, J.F., Caussanel, V, and Beck, A., immunoconjudges, drug-arm antibodies to light against cancer, Med.Sci.,25(12):1046-1052, 2009; and Govindan, S.V. and Goldenberg, D.M., design immunoconjugates for cancer therapy, Expert Opin biol. ther.,12(7): 873-.

Pharmaceutical compositions and dosage forms

The active compounds, or salts, isotopic analogs, or prodrugs thereof, as described herein can be administered to a subject in an effective amount using any suitable method for achieving the desired therapeutic result. The amount and timing of administration of the active compound will, of course, depend on the subject being treated, the direction of the supervising specialist, the time course of exposure, the mode of administration, the pharmacokinetic properties of the particular active compound and the judgment of the attending physician. Thus, due to variability from subject to subject, the dosages given below are guidelines and the physician can adjust the dosage of the compound to achieve the treatment the physician deems appropriate for the subject. The physician may 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 0.5mg/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, and for example, from about 0.01, 0.1, or 0.5 to about 5, 10, 20, or more microns, and optionally from about 1 to about 2 microns. The compounds as disclosed herein have demonstrated excellent pharmacokinetic and pharmacodynamic properties, e.g. when administered by oral or intravenous route.

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 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 preservatives 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 binding agents such as polyvinylpyrrolidone, sucrose, gelatin and acacia may be employed. Additionally, lubricants 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 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 which 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 lipids forming the liposomal structure, again using conventional liposome formation 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. It is desirable that the formulation can be placed in a small chamber and aerosolized. Atomization may 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. In one embodiment, the solid particles provide controlled release through the use of degradable polymers. The solid particles may be obtained by processing the solid compound or salt thereof in any suitable manner 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 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.

Also provided are pharmaceutical formulations that provide controlled release of the compounds described herein, including through the use of degradable polymers as known in the art.

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 lowers the surface tension of the formulation sufficiently 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 excessive 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 disclosed compounds. 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. The basic compounds are capable of forming a variety of different salts with a variety of inorganic and organic acids. Acid addition salts of basic compounds are prepared by contacting the free base form with a sufficient amount of the desired acid in a conventional manner to produce the salt. The free base form may be regenerated by contacting the salt form with a base and isolating the free base in a conventional manner. The free base form may differ from its corresponding salt form in certain physical properties, such as solubility in polar solvents. Pharmaceutically acceptable base addition salts may be formed with metals or amines, for example alkali metal 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. Base addition salts of acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base in a conventional manner to produce the salt. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in a conventional manner. The free acid form differs slightly from its corresponding salt form in certain physical properties, such as solubility in polar solvents.

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 for treating cancer in a subject, wherein the cancer is selected from the group consisting of breast cancer, colon cancer, ovarian cancer, non-small cell lung cancer, and Rb-positive glioblastoma, comprising administering to a subject in need thereof an effective amount of a compound of formula I, II, III, IV, or V:

or a pharmaceutically acceptable salt thereof;

wherein:

each X is independently CH or N;

each X' is independently CH or N;

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 two R's in the chain in place of a carbon O or N heteroatom and 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;

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 subject to one or more valencies permittingR is^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, where valency permits, via one or more R^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

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- (alkylene)_m-NR³*R⁴- (alkylene)_m-C(O)-R⁵- (alkylene)_m-C(＝S)R⁵- (alkylene)_m-C(＝O)O R⁵- (alkylene)_m-OC(＝O)R⁵- (alkylene)_m-C(S)-OR⁵- (alkylene)_m-C(O)-NR³*R⁴- (alkylene)_m-C(S)-NR³*R⁴- (alkylene)_m-N(R³*)-C(O)-NR³*R⁴- (alkylene)_m-N(R³*)-C(S)-NR³*R⁴- (alkylene)_m-N(R³*)-C(O)-R⁵- (alkylene)_m-N(R³*)-C(S)-R⁵- (alkylene)_m-O-C(O)-NR³*R⁴- (alkylene)_m-O-C(S)-NR³*R⁴- (alkylene)_m-SO₂-NR³*R⁴- (alkylene)_m-N(R³*)-SO₂-R⁵- (alkylene)_m-N(R³*)-SO₂-NR³*R⁴- (alkylene)_m-N(R³*)-C(O)-OR⁵- (alkylene)_m-N(R³*)-C(S)-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 a compound optionally independently, when valency permits, via one or more R^xA group-substituted heterocycle; and is

R⁶Is H or lower alkyl, - (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 of 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 substitutedC₁-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 C ₃-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 R¹³Is R^A。

Scheme 2. the method of scheme 1, wherein the compound is selected from the following formulae:

scheme 3. the method of claim 2, wherein the compound is compound Q or a pharmaceutically acceptable salt thereof.

Scheme 4. the method of claim 2, wherein the compound is compound T or a pharmaceutically acceptable salt thereof.

Scheme 5. the method of claim 2, wherein the compound is compound U or a pharmaceutically acceptable salt thereof.

Scheme 6. the method of scheme 2, wherein the compound is compound GG or a pharmaceutically acceptable salt thereof.

Scheme 7. the method of scheme 2, wherein the compound is selected from compound a to compound Z or a pharmaceutically acceptable salt thereof.

Scheme 8. the method of scheme 2, wherein the compound is selected from compounds AA to ZZ, or a pharmaceutically acceptable salt thereof.

Scheme 9. the method of scheme 2 wherein the compound is selected from compounds AAA to ZZZ or a pharmaceutically acceptable salt thereof.

Scheme 10 the method of scheme 1, wherein the cancer is breast cancer.

Scheme 11 the method of scheme 1, wherein the cancer is colon cancer.

The method of claim 1, wherein the cancer is ovarian cancer.

The method of claim 1, wherein the cancer is non-small cell lung cancer.

Technical scheme 14. the method of technical scheme 1, wherein the cancer is Rb-positive glioblastoma.

Scheme 15. the method of scheme 1, wherein the compound is conjugated to a targeting agent.

Scheme 16. the method of claim 15, wherein the targeting agent is an antibody or antibody fragment.

Scheme 17. the method of scheme 1, wherein the compound is conjugated to a radioisotope.

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

Technical scheme 19. the method of technical scheme 1, wherein the compound is administered in combination with another chemotherapeutic agent.

Scheme 20. the method of claim 19, wherein the anti-cancer activity of the chemotherapeutic agent is independent of cell proliferation.

A method for treating Rb-positive abnormal cell proliferation in a subject, the method comprising administering to a subject in need thereof an effective amount of a compound of formula I, II, III, IV, or V:

or a pharmaceutically acceptable salt thereof;

wherein:

each X is independently CH or N;

each X' is independently CH or N;

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

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, where valency permits, via one or more R^xRadical (I)Substituted heterocyclic ring, 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

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- (alkylene)_m-NR³*R⁴- (alkylene)_m-C(O)-R⁵- (alkylene)_m-C(＝S)R⁵- (alkylene)_m-C(＝O)O R⁵- (alkylene)_m-OC(＝O)R⁵- (alkylene)_m-C(S)-OR⁵- (alkylene)_m-C(O)-NR³*R⁴- (alkylene)_m-C(S)-NR³*R⁴- (alkylene)_m-N(R³*)-C(O)-NR³*R⁴- (alkylene)_m-N(R³*)-C(S)-NR³*R⁴- (alkylene)_m-N(R³*)-C(O)-R⁵- (alkylene)_m-N(R³*)-C(S)-R⁵- (alkylene)_m-O-C(O)-NR³*R⁴- (alkylene)_m-O-C(S)-NR³*R⁴- (alkylene)_m-SO₂-NR³*R⁴- (alkylene)_m-N(R³*)-SO₂-R⁵- (alkylene)_m-N(R³*)-SO₂-NR³*R⁴- (alkylene)_m-N(R³*)-C(O)-OR⁵- (alkylene)_m-N(R³*)-C(S)-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 a compound optionally independently, when valency permits, via one or more R^xA group-substituted heterocycle; 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, unsubstitutedSubstituted 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 R¹³Is R^A。

Scheme 22. the method of scheme 21, wherein the compound is selected from the following formulae:

scheme 23 the method of scheme 22, wherein the compound is compound Q or a pharmaceutically acceptable salt thereof.

Scheme 24 the method of scheme 22, wherein the compound is compound T or a pharmaceutically acceptable salt thereof.

Scheme 25 the method of scheme 22, wherein the compound is compound U or a pharmaceutically acceptable salt thereof.

Scheme 26 the method of scheme 22, wherein the compound is compound GG or a pharmaceutically acceptable salt thereof.

Technical scheme 27 the method of technical scheme 22, wherein the compound is selected from compound a to compound Z or a pharmaceutically acceptable salt thereof.

Scheme 28. the method of scheme 22, wherein the compound is selected from compounds AA to ZZ, or a pharmaceutically acceptable salt thereof.

Scheme 29. the method of scheme 22, wherein the compound is selected from compounds AAA to ZZZ or a pharmaceutically acceptable salt thereof.

Scheme 30. the method of scheme 21, wherein the compound is conjugated to a targeting agent.

Scheme 31 the method of claim 30, wherein the targeting agent is an antibody or antibody fragment.

Scheme 32. the method of scheme 21, wherein the compound is conjugated to a radioisotope.

Claim 33 the method of claim 21, wherein the subject is a human.

Use of a compound of claim 1 in the manufacture of a medicament for treating cancer in a subject, wherein the cancer is selected from the group consisting of breast cancer, colon cancer, ovarian cancer, non-small cell lung cancer, and Rb-positive glioblastoma.

Scheme 35 use of a compound according to scheme 21 in the manufacture of a medicament for treating an Rb positive abnormal cell proliferation disorder in a subject.

Preparation of active Compounds

Synthesis of

The disclosed compounds can be made by the following general scheme:

scheme 1

In scheme 1, 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 2

In scheme 2, 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 3

In scheme 3, Ref-1 is WO 2010/020675A 1.

Scheme 4

Scheme 5

Scheme 6

Scheme 7

Scheme 8

In scheme 8, 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.

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 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 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.

Scheme 9

The CDK4/6 inhibitors of the present invention may be synthesized according to general scheme 9. Specific syntheses and characterization of substituted 2-aminopyrimidines can be found, for example, in WO 2012/061156.

Compounds T, Q, GG and U were prepared as above and characterized by mass spectrometry and NMR as follows:

compound T

¹H NMR(600MHz,DMSO-d₆)δppm 1.47(br.s.,6H)1.72(br.s.,2H)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(M+H)447。

Compound 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.,1H)8.40(br.s.,1H)9.09(br.s.,1H)9.62(br.s.,1H)11.71(br.s.,1H)。LCMS ESI(M+H)433。

Compound 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。

Compound 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,2H)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。

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 N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] ethyl ] carbamic acid tert-butyl ester, 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) and triethylamine (0.757 m)L, 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]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 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 (1.0mL) were added to the acetal from the previous step (900 mg). The reaction was stirred at room temperature for 16 hours. Concentration and silica gel Using ethyl acetate/hexane (0-60%)Column chromatography to obtain foam-shaped N- [2- (2-chloro-6-formyl-pyrrolo [2,3-d ]]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]-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 at 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)δppm9.08(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. After addition of methanol (5mL), saturated NaHCO was added₃Followed 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)δppm8.26(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 (10psi) 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 2010/020675 a 1.

Example 12

Synthesis of N- [2- (Phenylmethoxycarbonylamino) -3-methyl-butyl ] carbamic acid tert-butyl ester, 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 (10.99mL, 1.1eq) followed by DBU (8.32mL, 1.2 eq). The contents were allowed to warm 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 (c, f) ((r) ())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 N- [2- (benzyloxycarbonylamino) -4-methyl-pentyl ] carbamic acid tert-butyl ester, 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 ℃ 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 (100mL), followed by 7.21g of triphenylphosphine and stirring 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 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. After drying over magnesium sulfate and concentration in vacuo, the crude product was used in the next step.¹HNMR (600MHz, chloroform-d) δ ppm 0.89(d, J ═ 6.73) Hz,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 (100mL), 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 group]Methyl radical]Carbamic acid tert-butyl ester (13.1 g).¹HNMR(600MHz,DMSO-d₆)δppm 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 mmol) 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＝2.64Hz,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)δppm 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-piperidyl) -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＝9.37Hz,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)δppm1.34-1.52(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,1H)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)δppm 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,1H)。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,1H)。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-piperidyl]Synthesized in a similar manner as used for pyridine.¹HNMR(600MHz,DMSO-d₆)δppm 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(600MHz,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)δppm 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-piperidinyl ] 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-piperidinyl) -1-piperidinyl-1-piperazines from 1-isopropyl-4- (6-nitro-3-pyridinyl) piperazine]Synthesized in a similar manner in pyridine, which is then converted to 5- (4-isopropylpiperazin-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 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 ═ 8.78 Hz) 6.45,1H)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 ═ 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 for N- [2- [ [ 2-chloro-5- (3, 3-diethoxypropan-1-)Alkynyl) 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 added ₃And 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]Pyrimidine-6-carboxylic acid (0.050g, 0.00013 mol) in DCM (1.5mL)DIC (32.7mg) and DMAP (10mg) were added thereto. 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 afford 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 a 60% dispersion of sodium hydride 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＝13.76,1.17Hz,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 blanket 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]Reaction with 5-bromo-2, 4-dichloro-pyrimidine under analogous reaction conditions as described for tert-butyl carbamate to give N- [ (2S) -2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ]-4-methyl-pentyl](iii) carbamic acid tert-butyl ester.¹HNMR (600MHz, 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,1H)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(600MHz,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 in a pressure vessel under 50psi hydrogen using 10% Pd/C gave 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]Reaction with 5-bromo-2, 4-dichloro-pyrimidine under analogous reaction conditions as described for tert-butyl carbamate to give 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 and compound 47Synthesized in a similar way.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

Compound 21 was hydrogenated under 50psi hydrogen using 10% Pd/C in a pressure vessel to give tert-butyl N- [ (2S) -2-amino-3, 3-dimethyl-butyl ] carbamate, which was 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]Reaction with 5-bromo-2, 4-dichloro-pyrimidine under analogous reaction conditions as described for tert-butyl carbamate to give N- [ (2S) -2- [ (5-bromo-2-chloro-pyrimidin-4-yl)) Amino group]-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,9H)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]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, 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]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, 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-propyl ] 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(600MHz,DMSO-d₆)δppm 1.43(s,9H)1.73(s,6H)4.06(s,2H)7.46(s,1H)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,2H)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 using 5-bromo-2, 4-dichloro-pyrimidine and intermediate K, similar reaction conditions were described.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(600MHz,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,1H)。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 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 L under similar reaction conditions.¹HNMR(600MHz,DMSO-d₆)δppm 1.34(s,9H)1.50-1.58(m,2H)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,2H)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 group]-3-methyl-butyl]Analogous reaction conditions as described for tert-butyl carbamate, treatment of N- [ (1S,2S) -2-aminocyclopentyl group 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,1H)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,1H)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(600MHz,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,1H)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 (31mg, 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. Concentration in vacuo gave the hydrochloride.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, 1.05eq) 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 (1.1eq) 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,3H)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,1H)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＝9.37Hz,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,3H)3.22-3.81(m,8H)4.01(dd,J＝13.61,4.25Hz,2H)4.59-4.72(m,1H)7.19(s,1H)7.74(s,1H)7.96-8.10(m,2H)9.08(s,1H)9.22(s,2H)。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,3H)3.07-4.14(m,10H)4.95(s,1H)7.20(s,1H)7.66(d,J＝9.66Hz,1H)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,3H)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,2H)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＝3.81Hz,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,1H)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(600MHz,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,1H)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 the deblocking step 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 the deblocking step 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,1H)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,2H)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.,2H)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,1H)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,2H)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,4H)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,5H)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,2H)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,1H)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,2H)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 use and strip described for Compound 78Synthesized and converted to the hydrochloride under similar conditions.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,6H)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,1H)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,2H)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 and conversion to the hydrochloride salt as described for compound 65. ¹HNMR(600MHz,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,2H)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.,1H)。LCMS(ESI)461(M+H)。

Example 108

Synthesis of Compound 108

Compound 108 was synthesized in a similar manner as described for compounds 64 and 65 and recovered as the hydrochloride salt. 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,2H)3.98-4.14(m,1H)7.20(s,2H)7.77(s,1H)7.97(s,2H)8.81(s,1H)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 andfollowed by conversion 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,1H)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 gas downwardsN- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino]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 beamAfter filtration, the crude reaction was subjected to column separation using hexane/ethyl acetate (0-20%) to obtain 3.9g of the desired product. The obtained 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 ℃ 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- (diethoxy) benzeneYlmethyl) 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 NaHCO₃(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 silica gel column chromatography 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]Carbamic acid tert-butyl ester (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) in AcOH (10)mL) 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]DMAP (20mg) was added to pyrimidine-6-carboxylic acid (0.1g, 0.261mmol) in DCM (4.1mL), 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,4H)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 the product (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 N- [2- [ (5-bromo-2-chloro-pyrimidin-4-yl) amino ] -2-methyl-propyl ] carbamic acid tert-butyl ester, 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 N- (2-amino-2-methyl-propyl) 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) (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-propyl ] 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 experimental conditions similar to those 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 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)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,1H)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 ] propyl ] 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. Ethyl acetate (100mL) and water (50mL) were then added to the crude reaction mixture 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, TFA was added and the contents 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 N- [ (1-aminocyclopentyl) methyl ] 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 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 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)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 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 147

Synthesis of Compound 147

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

Example 148

Synthesis of Compound 148

Compound 148 was prepared by using an assay similar to that of compound 124Under the conditions described above, chlorotricyclo amine compound 147 was treated with trans-4-aminocyclohexanol. 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 reacts 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 147 Each and having 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, CDK2/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 a 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.

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 profiling service screened compound T against 456 (395 non-mutated) kinases. A single concentration of 1000nM (IC 50 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 showed that compound T was on CDK4 compared to the other kinases testedAnd CDK 6. 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 CDK 12/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/CycD1 IC₅₀)。

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, 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

Inhibition of cell proliferation

Cell proliferation assays were performed using the following cancer cell lines: MCF7 (Breast cancer-Rb positive), ZR-75-1 (breast carcinoma-Rb positive), H69 (human Small cell Lung cancer-Rb negative) cells or A2058 (human metastatic melanoma cells-Rb negative). These cells were seeded in Costar (Tewksbury, Massachusetts) 309396 wells in tissue culture treated white wall/clear bottom plates. 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 used after four days (H69) or six days (MCF7, ZR75-1, A2058) as indicated Luminescence 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。

The results of the cytostatic analysis of two Rb-positive breast cancer cell lines (MCF7 and ZR75-1) are shown in table 4. Inhibition of EC for inhibition of MCF7 breast cancer cell proliferation₅₀Values ranged from 28nM to 257 nM. Inhibition of EC for inhibition of ZR75-1 Breast cancer cell proliferation₅₀Values ranged from 24nM to 581 nM.

Examples of representative compounds that are highly effective against MCF7 breast cancer cell proliferation are shown in fig. 21-24. The compounds tested in figures 21-24 (compounds T, Q, GG, U, H, MM, OO and PD-332991) all demonstrated significant inhibition of cell proliferation of MCF-7 cells. As can be seen in figure 21, compound T demonstrated more potent activity against MCF-7 cells than PD 0332991.

Examples of representative compounds that were highly effective against ZR75-1 (breast ductal carcinoma (Rb positive)) cell proliferation are shown in fig. 25-28. The compounds tested in figures 25-28 (compounds T, Q, GG, U, H, MM, OO, and PD-332991) all demonstrated significant inhibition of cell proliferation of ZR75-1 cells. As can be seen in figure 25, compound T demonstrated more potent activity against ZR75-1 cells than PD 0332991.

In addition to breast cancer cell lines, a number of selected compounds disclosed herein were also evaluated against a small cell lung cancer cell line (H69) and a human metastatic melanoma cell line (a2058) (two Rb deficient (Rb negative) cell lines). 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 the two Rb-positive breast cancer cell lines.

Table 4: inhibition of cancer cell proliferation

Example 153

HSPC growth inhibition study

The effect of PD0332991 on HSPC has been previously demonstrated. FIG. 2 shows EdU incorporation of mouse HSPC and spinal cord progenitors after a single dose of 150mg/kg PD0332991 by oral gavage to assess the transient CDK4/6 inhibition on bone marrow arrest, as in Roberts et al Multiple circles of cycle-Dependent Kinase 4/6Inhibitors in Cancer therapy.JCNI 2012; 104(6) 476-487 (FIG. 2A). As can be seen in figure 2, 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 154

Pharmacokinetic and pharmacodynamic properties of antineoplastic compounds

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 3.

Table 5: pharmacokinetic and pharmacodynamic properties of antineoplastic compounds

Example 155

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. Groups of samples were harvested at 24, 40 and 48 hours in triplicate.

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 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).

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 4 shows a cell elution experiment 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. 4A and 4B) or human renal proximal tubule epithelial cells (Rb positive) (fig. 4C and 4D), and the effect on the cell cycle after compound elution was determined at 24, 36, 40 and 48 hours.

As shown in figure 4 and similar to the in vivo results as shown in figure 2, PD0332991 requires cells to return to normal baseline cell division more than 48 hours after elution. This can be seen in fig. 4A and 4B, since the values obtained are correspondingly equal to the G0-G1 fraction of cell division or the value of DMSO control in S phase. In contrast, HS68 cells treated with the compounds of the invention returned to normal baseline cell division in as little as 24 hours or 40 hours, unlike PD0332991 at these same time points. The results using human renal proximal tubule epithelial cells (fig. 4C and 4D) 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 156

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

For HSPC proliferation experiments young 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), 2528-2536). Briefly, HSPC were identified by 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. 5). Six mice per group were given 150mg/kg compound T, compound Q, compound GG or vehicle only by oral gavage. 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 figure 5A 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 returned to normal starting at 24 hours. Compound Q also demonstrated a slight decrease at 12 hours and returned to baseline beginning at 24 hours, although compound Q was poorly bioavailable orally.

Other experiments were completed with compound T to test dose response and compound treatment period was 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 measured at 12, 24, 36 and 48 hours. As can be seen in fig. 5B and 5C 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 5C can be directly compared to the results for the same dose of PD0332991 shown in figure 2, 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 157

HSPC growth inhibition study comparing Compound T and PD0332991

Figure 6 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 6, a single oral dose of PD0332991 causes a persistent decrease in HSPC for more than 36 hours. In contrast, a single oral dose of compound T caused a reduction in HSPC proliferation early in 12 hours, but by 24 hours after compound T administration HSPC proliferation restarted.

Example 158

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 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. Aliquots of the reaction mixture (without cofactor) were 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 percent natural log remaining versus 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 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 function 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 7, 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. 4, 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. 4, removal of the compounds described herein from the culture medium resulted in rapid resumption of proliferation, consistent with a rapid enzyme off-rate. These differences in enzyme off-rates translate into significant differences in Pharmacodynamic (PD) effects, as shown in figures 2, 5C and 6. As shown, a single oral dose of PD0332991 arrested Hematopoietic Stem and Progenitor Cells (HSPCs) 36+ hour growth in murine bone marrow, which is greater than illustrated 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 159

Efficacy of CDK4/6 inhibitor Compound T in HER2 driven Breast tumors

HER2 expressing c-neu (mouse ortholog of human HER 2) driven by the MMTV promoter drives the breast cancer model (Rb positive) (Muller WJ, Sinn E, Pattengale PK, Wallace R, Leder P.Single-step indication of breast cancer in transgenic mouse bearing. cell 1988; 54:105-15) for the following examples. This model was chosen because of the differences in murine models (Yu Q, Geng Y, science P. specificity detection assessment by cycle D1 association. Nature 2001; 411: 1017-21; Landis MW, Pawlyk BS, Li T, science P, Hinds PW. cycle D1-dependency in muscle definition and mapping in cancer 2006; 9: 13-22; Reddy HK, science RV, rat SG, gran X, Litvin J, Reddy EP. cycle-dependency in 4expression for expression in gene-induced metabolism analysis 2005. cancer research 2005.65: 10174-8; growth Q. 20. expression in 5. cDNA, expression in 32. growth in 23. growth in III. cDNA, expression in 32. yeast strain, growth in 32. J. expression in III. cDNA, expression in 3. J. expression in 3. expression in 5. expression in B.8. expression in B.23. expression of expression in B.23. growth in B.M.23. growth in B. (ii) a Samady L, Dennis J, Budhram-Mahadeo V, Latchman DS.activation of CDK4gene expression in human Breast cancer cells by the Brn-3b POU family similarity factor 2004; 317-23 parts of (3); takano Y, Takenaka H, Kato Y, Masuda M, Mikami T, Saegusa M et al, cycle D1overexpression in innovative samples, correlation with cycle-dependent kinase 4and oestrogen receiver overexpression, and lack of correlation with atomic activity.J Cancer Res Clin Oncol 1999; 125:505-12) showed that these tumors required CDK4/6 and CCND1 to progress and maintain.

MMTV-neu mice were generated and observed post-lactation, with tumors observed at a median latency of approximately 25 weeks. Mice entered the therapy study when tumors reached a standard size (50-60mm3) that allowed easy serial assessment. Tumor-loaded mice were treated consecutively with compound T (100mg/kg/d or 150mg/kg/d) added to their diet. MMTV-c-neu (control, n-9; compound T100 mg/kg, n-7; compound T150 mg/kg, n-6) mice were examined weekly to assess tumor development by palpation. Tumor volume was calculated by the following formula: volume ═ width²X length]/2. Tumor-loaded mice were sacrificed at indicated times due to pre-determined morbidity, tumor ulceration, or tumor size exceeding 1.5cm diameter.

As shown in figures 8 and 9, continuous treatment with compound T (100mg/kg/d or 150mg/kg/d) significantly reduced tumor volume during the course of 28 days of therapy. Several tumors showed complete tumor regression and no resistance to compound T was noted during the course of 28 days of treatment. These data show that this HER2 driven mouse model 'relies on' CDK4/6 activity for proliferation and that compound T is an effective agent in CDK 4/6-dependent Rb positive tumors.

Example 160

Efficacy of CDK4/6 inhibitors (Compound T, Compound GG and Compound U) in HER 2-driven Breast tumors

Preclinical characterization of Compound T, Compound GG and Compound U indicated that they had ICs at 0.7-1.0nM and 5-6nM, respectively₅₀Inhibit CDK4 and CDK 6. In tumor cells with functional Rb proteins, these compounds effectively inhibit Rb phosphorylation, causing G1 arrest. CDK4/6 inhibitor compound T, compound GG and compound U were tested for in vivo efficacy in a genetically engineered mouse model of breast adenolumenal breast cancer. Tumors were continuously assessed weekly using caliper measurements. Once the tumor reaches 40-64mm³Therapeutic intervention is initiated. Using the formula ((width)²) X length)/2 tumor volume was calculated. All three compounds were administered orally via a drug-supplemented diet (100 mg/kg/d). The drug-added diet was administered for 28 consecutive days, followed by discontinuation. RECIST criteria are used to evaluate the objective response rate. Objective response rates were based on percent change in tumor volume, classified using the following categories: CR (complete reaction) is 100% reaction; PR (partial reaction) reduction of at least 30%; SD (stable disease) ═ no change (no PR and no PD); and PD (progressive disease) increased by 20%.

As shown in figure 10, objective responses were noted in mice treated with compound T, compound GG, and compound U. The treatment groups were well tolerated, had no clinical signs of toxicity, no weight loss, and no mortality associated with toxicity. The linear regression t-test was used to determine the statistical significance of tumor volume growth over time. All three groups were statistically significant compared to the non-treated group; compound T, p < 0.0001; compound GG, p ═ 0.0001; compound U, p < 0.0001. As shown in fig. 10, the number of animals in each objective category was determined. Compound T was found to have an objective response rate of 100% (n-7), compound GG was found to have an objective response rate of 85% (n-7), and compound U was found to have an objective response rate of 100% (n-8). In fig. 11, tumor volumes from MMTV-Neu mice treated with compound T, compound GG, and compound U are shown, where tumor volumes are measured every seven days. In fig. 12, data for the best response (14 days or later) for each individual tumor is shown. Taken together, these data demonstrate that continuous treatment with compound T (100mg/kg), compound GG (100mg/kg), or compound U (100mg/kg) during the course of 28 days of therapy caused a significant decrease in tumor volume.

Example 161

Compound T arrests cell cycle in CDK4/6 dependent cells

To test the ability of CDK4/6 inhibitors to induce complete G1 arrest, a cell-based screen consisting of two CDK4/6 dependent cell lines (tHS68 and WM 2664; Rb positive) and one CDK4/6 independent (a 2058; Rb negative) cell line was used. 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.

In fig. 29A, 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. In FIG. 29A, the results show that compound T has an EC of 80nM in CDK 4/6-dependent cell line₅₀Robust G1 cell cycle arrest was induced tHS68 cells with a corresponding reduction in S phase shown ranging from 28% at baseline to 6% at the highest concentration. Similar reductions in the S-phase population and increases in G1 arrested cells in two CDK 4/6-dependent cell lines (tHS68 (compare fig. 29B and 29E) and WM2664 (compare fig. 29C and 29F)) but not in the CDK 4/6-independent (a 2058; compare fig. 29D and 29G) cell line following treatment with compound T (300 nM). The CDK4/6 independent cell line was shown to be ineffective in the presence of inhibitors.

Example 162

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 at two sites targeted by the CDK4/6 cyclin D complex, Ser780 and Ser807/811, using species-specific antibodies. 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. 30).

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

1. Use of a selective cyclin dependent kinase 4/6 inhibitor compound in the manufacture of a medicament for the treatment of a human retinoblastoma (Rb) -protein positive cancer, comprising administering an effective amount of a selective cyclin dependent kinase 4/6 inhibitor compound selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

2. The use of claim 1, wherein treatment of the subject during a treatment cycle does not require a drug holiday.

3. The use of claim 1 or 2, wherein the compound is:

or a pharmaceutically acceptable salt thereof.

4. The use of claim 3, wherein the compound is administered in a dosage form suitable for oral delivery.

5. The use of claim 1 or 2, wherein the compound is:

or a pharmaceutically acceptable salt thereof.

6. The use of claim 5, wherein the compound is administered in a dosage form suitable for parenteral delivery.

7. The use of claim 5, wherein the compound is administered in a dosage form suitable for intravenous delivery.

8. The use of claim 5, wherein the compound is administered in a dosage form suitable for intramuscular delivery.

9. The use of claim 1 or 2, wherein the compound is administered daily.

10. The use of claim 9, wherein the compound is administered daily for 21 or more consecutive days.

11. The use of claim 9, wherein the compound is administered daily for 24 or more consecutive days.

12. The use of claim 9, wherein the compound is administered daily for 28 or more consecutive days.

13. The use of claim 9, wherein the compound is administered daily for 35 or more consecutive days.

14. The use of claim 1 or 2, wherein the compound is administered in combination with another chemotherapeutic agent.

15. The use of claim 14, wherein the anti-cancer activity of the chemotherapeutic agent is independent of cell proliferation.

16. The use of claim 14, wherein the chemotherapeutic agent is selected from tamoxifen, midazolam, letrozole, bortezomib, anastrozole, goserelin, an mTOR inhibitor, a PI3 kinase inhibitor, a dual mTOR-PI3K inhibitor, a MEK inhibitor, a RAS inhibitor, an ALK inhibitor, an HSP inhibitor, a PD-1 inhibitor, an AKT inhibitor, and a FLT3 inhibitor.

17. The use of claim 5, wherein the cancer is selected from non-small cell lung cancer, breast cancer, prostate cancer, glioblastoma, colon cancer, bladder cancer, gastric cancer, liver cancer, pancreatic cancer, esophageal cancer, thyroid cancer, and renal cancer.

18. The use of claim 17, wherein the compound is administered daily for 21 or more consecutive days.

19. The use of claim 17, wherein the compound is administered daily for 24 or more consecutive days.

20. The use of claim 17, wherein the compound is administered daily for 28 or more consecutive days.

21. The use of claim 17, wherein the compound is administered daily for 35 or more consecutive days.

22. The use of claim 17, wherein the cancer is breast cancer.

23. The use according to claim 22, wherein the breast cancer is an estrogen receptor positive breast cancer.

24. The use according to claim 22, wherein the breast cancer is estrogen receptor positive, HER-2 negative advanced breast cancer.

25. The use of claim 17, wherein the cancer is non-small cell lung cancer.

26. The use of claim 25, wherein the non-small cell lung cancer is a KRAS mutant cancer.

27. The use of claim 7, wherein the compound is administered daily for 21 or more consecutive days.

28. The use of claim 27, wherein the cancer is selected from non-small cell lung cancer, breast cancer, prostate cancer, glioblastoma, colon cancer, bladder cancer, gastric cancer, liver cancer, pancreatic cancer, esophageal cancer, thyroid cancer, skin cancer, and renal cancer.

29. The use of claim 28, wherein the cancer is breast cancer.

30. Use according to claim 29, wherein the breast cancer is an estrogen receptor positive breast cancer.

31. The use of claim 29, wherein the breast cancer is estrogen receptor positive, HER 2-negative advanced breast cancer.

32. The use of claim 28, wherein the cancer is non-small cell lung cancer.

33. The use of claim 32, wherein the non-small cell lung cancer is a KRAS mutant cancer.

34. The use of claim 3, wherein the compound is administered daily for 24 or more consecutive days.

35. The use of claim 34, wherein the cancer is selected from non-small cell lung cancer, breast cancer, prostate cancer, glioblastoma, colon cancer, bladder cancer, gastric cancer, liver cancer, pancreatic cancer, esophageal cancer, thyroid cancer, skin cancer, and renal cancer.

36. The use of claim 35, wherein the cancer is breast cancer.

37. The use according to claim 36, wherein the breast cancer is an estrogen receptor positive breast cancer.

38. The use of claim 36, wherein the breast cancer is estrogen receptor positive, HER 2-negative advanced breast cancer.

39. The use of claim 35, wherein the cancer is non-small cell lung cancer.

40. The use of claim 39, wherein the non-small cell lung cancer is a KRAS mutant cancer.

41. The use of claim 3, wherein the compound is administered daily for 28 or more consecutive days.

42. The use of claim 41, wherein the cancer is selected from non-small cell lung cancer, breast cancer, prostate cancer, glioblastoma, colon cancer, bladder cancer, gastric cancer, liver cancer, pancreatic cancer, esophageal cancer, thyroid cancer, skin cancer, and renal cancer.

43. The use of claim 42, wherein the cancer is breast cancer.

44. The use according to claim 43, wherein the breast cancer is an estrogen receptor positive breast cancer.

45. The use of claim 43, wherein the breast cancer is estrogen receptor positive, HER 2-negative advanced breast cancer.

46. The use of claim 42, wherein the cancer is non-small cell lung cancer.

47. The use of claim 46, wherein the non-small cell lung cancer is KRAS mutant non-small cell lung cancer.

48. The use of claim 3, wherein the compound is administered daily for 35 or more consecutive days.

49. The use of claim 48, wherein the cancer is selected from the group consisting of non-small cell lung cancer, breast cancer, prostate cancer, glioblastoma, colon cancer, bladder cancer, gastric cancer, liver cancer, pancreatic cancer, esophageal cancer, thyroid cancer, skin cancer, and renal cancer.

50. The use of claim 49, wherein the cancer is breast cancer.

51. The use according to claim 50, wherein the breast cancer is an estrogen receptor positive breast cancer.

52. The use of claim 50, wherein the breast cancer is estrogen receptor positive, HER 2-negative advanced breast cancer.

53. The use of claim 49, wherein the cancer is non-small cell lung cancer.

54. The use of claim 53, wherein the non-small cell lung cancer is KRAS mutant non-small cell lung cancer.