Uses of a CDK2 inhibitor in the treatment of cancer

Abstract

The invention relates to a pharmaceutical combination comprising a CDK2 inhibitor in combination with a CDK4 inhibitor, as well as methods of treating cancers using said combination. The present invention also relates to the use of CDK2 inhibitor in the treatment of a CCNE1 or CCNE2 amplified cancer.

Description

[0001] PAT059642-WO-PCT

[0002] USES OF A CDK2 INHIBITOR IN THE TREATMENT OF CANCER

[0003] FIELD OF THE DISCLOSURE

[0004] The present invention relates to a pharmaceutical combination comprising a CDK2 inhibitor in combination with a CDK4 inhibitor, as well as methods of treating cancers using said combination. The present invention also relates to the use of CDK2 inhibitor in the treatment of a CCNE1 or CCNE2 amplified cancer.

[0005] BACKGROUND

[0006] CDK2 is a serine / threonine kinase that regulates the mammalian cell cycle. After binding to cyclin- E1 , cyclin-E2, cyclin-A1 or cyclin-A2, the activated CDK2-cyclin complex phosphorylates a variety of cellular substrates (Chi et al 2020). For example, it phosphorylates and inactivates the retinoblastoma protein (pRB), thus de-represses the E2F family of transcription factors that promotes expression of cell cycle related genes (Sherr & Roberts 1999, 2004). As such, CDK2 propels the cell from the G1 phase into S phase of the cell cycle, when nuclear DNA is replicated in preparation of mitosis. CDK2 and its activity are often dysregulated in human cancers (Corsino et al 2008, Ying et al 2018, Nie et al 2019). One such dysregulation mechanism involves, but not limited to, the amplification or overexpression of cyclin-E1 , which leads to hyperactivation of CDK2 and promotes cellular proliferation (Schraml et al. 2003). Depending on lineage, dysregulation of CDK2 / E occurs in up to 40% of human cancers (cbioportal.org), including but not limited to uterine carcinosarcoma (~40%) ovarian (~20%), gastric (~12%), esophageal (~10%), endometrial (~10%), and breast cancers (~10%). Cyclin-E1 amplification and overexpression is also associated with poor prognosis across cancers (Nakayama et al 2010, Zhao et al 2019). In many of these cancers, besides radiation and chemotherapy, there is no targeted therapy available to patients. Thus, the development of a targeted agent against CDK2 would potentially benefit cancer patients.

[0007] Chi Y, Carter J.H, Swanger J., Mazin A.V., Moritz R.L, and Clurman B.E. (2020). A novel landscape of nuclear human CDk2 substrates revelaed by in situ phosphorylation. Sci. Adv 6, eaaz9899

[0008] Sherr CJ, Roberts JM. (1999). CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 13: 1501-1512.

[0009] Sherr CJ, Roberts JM. (2004). Living with or without cyclins and cyclin-dependent kinases. Genes Dev 18: 2699-2711.

[0010] Corsino PE, Davis BJ, Norgaard PH, Parker NN, Law M, Dunn W and Law BK. Mammary tumors initiated by constitutive Cdk2 activation contain an invasive basal-like component. Neoplasia 2008; 10: 1240-1252 PAT059642-WO-PCT

[0011] Ying M, Shao X, Jing H, Liu Y, Qi X, Cao J, et al. Ubiquitin-dependent degradation of CDK2 drives the therapeutic diferentiation of AML by targeting PRDX2. Blood J Am Soc Hematol. 2018;131 (24):2698-711 Nie L, Wei Y, Zhang F, Hsu Y-H, Chan L-C, Xia W, et al. CDK2-mediated site-specifc phosphorylation of EZH2 drives and maintains triplenegative breast cancer. Nat Common. 2019;10(1 ):1 — 15

[0012] Schraml P, Bucher C, Bissig H, et al. Cyclin E overexpression and amplification in human tumours. J Pathol. 2003;200(3):375-383.

[0013] Nakayama N, Nakayama K, Shamima Y, Ishikawa M, Katagiri A, lida K, et al. Gene amplificationCCNEI is related to poor survival and potential therapeutic target in ovarian cancer. Cancer 2010:NA-NA.

[0014] Guo Q, Lin X, Ye L, et al. . Comparative efficacy of Cdk4 / 6 inhibitors plus aromatase inhibitors versus fulvestrant for the first-line treatment of hormone receptor-positive advanced breast cancer: a network meta-analysis. Target Oncol 2019;14:139-48

[0015] Gao JJ, Cheng J, Bloomquist E, et al. . Cdk4 / 6 inhibitor treatment for patients with hormone receptorpositive, HER2-negative, advanced or metastatic breast cancer: a US food and drug administration pooled analysis. Lancet Oncol 2020;21 :250-60

[0016] The advent of targeted therapies for cancer has increased patient lifespan for various malignancies and helped to appreciate the complexity of tumors through the study of drug resistance mechanisms. The fact that clinical responses to targeted agents are generally incomplete and / or transient results from a multitude of factors that can be broadly put into two classes: toxicities that prevent optimal dosing of drugs and consequently limit target engagement (Brana and Siu 2012, Chapman, Solit et al. 2014), and the ability of cancers to adapt and maintain their proliferative potential against perturbations (Druker 2008, Chandarlapaty 2012, Doebele, Pilling et al. 2012, Duncan, Whittle et al. 2012, Katayama, Shaw et al. 2012, Lito, Rosen et al. 2013, Sullivan and Flaherty 2013, Solit and Rosen 2014). Combinations of drugs can address both these factors by improving overall efficacies and at the same time targeting tumor robustness and complexity to counter resistance (Robert, Karaszewska et al. 2015, Turner, Ro et al. 2015). It is not yet clear how many drugs are required, and which processes need to be targeted in combination to overcome specific types of cancer. But it is almost certain that different pathways or drivers need to be inhibited, most likely requiring two or more drugs (Bozic, Reiter et al. 2013). In spite of numerous treatment options for patients with specific types of cancer, there remains a need for effective and safe combination therapies that can be administered for the treatment of cancer.

[0017] SUMMARY

[0018] It is an object of the present invention to provide for a medicament to improve treatment of a cancer, in particular to improve treatment of cancer through inhibition of cell growth (proliferation) and / or induction of apoptosis (cell death). It is an object of the present invention to find novel PAT059642-WO-PCT combination therapies, which selectively synergize the inhibition of proliferation and / or the induction of apoptosis.

[0019] Surprisingly, it has been found that a pharmaceutical combination comprising i) a CDK2 inhibitor, and ii) a CDK4 inhibitor can both synergistically inhibit proliferation and / or induce apoptosis in cancers, as demonstrated in the Examples.

[0020] Therefore, according to a first aspect of the invention, there is hereby provided a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of: i) a compound of formula (I), or a salt and / or tautomer thereof: ii) a CDK4 inhibitor, and / or iii) an endocrine therapeutical agent.

[0021] According to a second aspect of the invention, there is hereby provided a compound of formula (I), or a salt and / or tautomer thereof: use in the treatment of cancer, wherein the treatment further comprises administration of (e.g. is in combination with): i) a CDK4 inhibitor; and / or ii) an endocrine therapeutical agent.

[0022] According to a third aspect of the invention, there is hereby provided a CDK4 inhibitor for use in the treatment of cancer, wherein the treatment further comprises administration of (e.g. is in combination with): PAT059642-WO-PCT i) a compound of formula (I), or a salt and / or tautomer thereof: ii) optionally an endocrine therapeutical agent.

[0023] According to a fourth aspect of the invention, there is hereby provided an endocrine therapeutical agent for use in the treatment of cancer, wherein the treatment further comprises administration of (e.g. is in combination with): i) a compound of formula (I), or a salt and / or tautomer thereof: ii) optionally, a CDK4 inhibitor. According to a fifth aspect of the invention, there is hereby provided a combination comprising: i) a compound of formula (I), or a salt and / or tautomer thereof: ii) a CDK4 inhibitor; and / or iii) an endocrine therapeutical agent. According to a sixth aspect of the invention, there is hereby provided a compound of formula (I), or a salt and / or tautomer thereof: PAT059642-WO-PCT use in the treatment of a CCNE1 or CCNE2 amplified tumor.

[0024] According to a seventh aspect of the invention, there is hereby provided a method of treating a CCNE1 or CCNE2 amplified tumor, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I), or a salt and / or tautomer thereof:

[0025] BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : Female nude mice bearing subcutaneous MCF7 [HR+ (ER+), HER2-] breast cancer xenograft tumors were treated for 42 days with vehicle, single agents or combinations of agents as indicated in the legend. Compound A and LEE011 (ribociclib) are both administered orally.

[0027] Figure 2: Female nude mice bearing subcutaneous MCF7 [HR+ (ER+), HER2-] breast cancer xenograft tumors were treated with vehicle, single agents or combinations of agents as indicated in the legend. Compound A and LEE011 (ribociclib) are both administered orally. After 3 days of treatment, tumors were collected at 1 hour after the last dose and analyzed for PD response using an internally developed MSD assay for pRB read-out. Data show the mean values ± SEM (n=5) for the pRB levels relative to the untreated controls at 1 , 3 and 7.5 mg / kg (BID) of Compound A, alone and in combination with LEE011 (ribociclib).

[0028] Figure 3: Female nude mice bearing subcutaneous MCF7 [HR+ (ER+), HER2-] breast cancer xenograft tumors were treated for 28 days with vehicle and combinations of agents as indicated in the legend. Compound A and LEE011 (ribociclib) are both administered orally. Fulvestrant is administered by intramuscular injection. Data show the mean tumor volume values (± SEM, n=8) for the respective treatments throughout the efficacy study. PAT059642-WO-PCT

[0029] Figure 4: Female nude mice bearing subcutaneous ST3164 / PBR [HR+ (ER+), HER2-] breast cancer patient derived xenograft (PDX) tumors were treated with vehicle and combinations of agents as indicated in the legend. Compound A and LEE011 (ribociclib) are both administered orally. Fulvestrant is administered by intramuscular injection. Data show the tumor volume change (%) compared to the day of enrollment for the respective treatments.

[0030] Figure 5: Female nude mice bearing subcutaneous ST 1337B [HR+ (ER+), HER2-] breast cancer patient derived xenograft (PDX) tumors were treated with vehicle and combinations of agents as indicated in the legend. Compound A and LEE011 (ribociclib) are both administered orally. Fulvestrant is administered by intramuscular injection. Data show the tumor volume change (%) compared to the day of enrollment for the respective treatments.

[0031] Figure 6: In vitro viability of the (ER+ HER2-) MCF7 breast cancer cell line was assessed using the CellTiterGlo assay following a 7-day treatment with Compound A and ribociclib. Growth inhibition %: 0-99 = delayed proliferation, 100= growth arrest / stasis, 101-200= reduction in cell number / cell death. (Top = Dose Matrix, Bottom = Loewe Excess Matrix.)

[0032] Figure 7: In vitro viability of the (ER+ HER2-) ST941 C ER+ breast cancer cell line was assessed using the CellTiterGlo assay following a 7-day treatment with Compound A and ribociclib. Growth inhibition %: 0-99 = delayed proliferation, 100= growth arrest / stasis, 101-200= reduction in cell number / cell death. (Top = Dose Matrix, Bottom = Loewe Excess Matrix.)

[0033] Figure 8: In vitro viability of the (ER+ HER2-) MCF7 breast cancer cell line was assessed using the CellTiterGlo assay following a 7-day treatment with Compound A and PF-07220060. Growth inhibition %: 0-99 = delayed proliferation, 100= growth arrest / stasis, 101-200= reduction in cell number / cell death. (Top = Dose Matrix, Bottom = Loewe Excess Matrix.)

[0034] Figure 9: In vitro viability of the (ER+ HER2-) MCF7 breast cancer cell line was assessed using the CellTiterGlo assay following a 7-day treatment with Compound A and the selective estrogen receptor degrader (SERD) Elacestrant. Growth inhibition %: 0-99 = delayed proliferation, 100= growth arrest / stasis, 101-200= reduction in cell number / cell death. (Top = Dose Matrix, Bottom = Loewe Excess Matrix.)

[0035] Figure 10: In vitro viability of the (ER+ HER2-) MCF7 breast cancer cell line was assessed using the CellTiterGlo assay following a 7-day treatment with Compound A and the selective estrogen receptor degrader (SERD) ARV-471 (also known as Vepdegestrant). Growth inhibition %: 0-99 = PAT059642-WO-PCT delayed proliferation, 100= growth arrest / stasis, 101-200= reduction in cell number / cell death.

[0036] (Top = Dose Matrix, Bottom = Loewe Excess Matrix.)

[0037] Figure 11 : Shows the drug concentration in plasma and level of PD modulation in tumor over 24 hours on day 3 of oral (p.o.) dosing of Compound A at 15 and 30 mg / kg (BID). Data show the mean values ± SD (n=3) for the free drug concentration in plasma (dotted lines) and mean values ± SEM (n=5) for the pRB values (solid lines) relative to the untreated controls at 30 mg / kg (BID) (top) and 15 mg / kg (BID) (bottom).

[0038] Figure 12: Shows the tumor growth and the change in body weight of immunodeficient NSG mice over 31 days. CCNE1 amplified OVCAR3 cells (10x106cells / 100 pl) were injected subcutaneously (s.c.) in the flank of immunodeficient NSG mice. After 31 days, mice were randomized into three groups and dosed (p.o.) with 15 and 30 mg / kg (BID) Compound A, formulated in 10% HPpCD (Hydroxypropyl-p-cyclodextrin) at pH 4.7 at 10 ml / kg.

[0039] DETAILED DESCRIPTION

[0040] As mentioned above, an object of the present invention is to find novel combination therapies, which selectively synergize in inhibiting proliferation and / or in inducing apoptosis.

[0041] Therefore, according to a first aspect of the invention, there is hereby provided a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of: i) a compound of formula (I), or a salt and / or tautomer thereof: ii) a CDK4 inhibitor, and / or iii) an endocrine therapeutical agent.

[0042] In an embodiment, the method comprises administering to a subject in need thereof a therapeutically effective amount of: PAT059642-WO-PCT i) a compound of formula (I), or a salt and / or tautomer thereof: ii) a CDK4 inhibitor.

[0043] In an embodiment, the method comprises administering to a subject in need thereof a therapeutically effective amount of: i) a compound of formula (I), or a salt and / or tautomer thereof: ii) an endocrine therapeutical agent.

[0044] In an embodiment, the method comprises administering to a subject in need thereof a therapeutically effective amount of: i) a compound of formula (I), or a salt and / or tautomer thereof: ii) a CDK4 inhibitor, and iii) an endocrine therapeutical agent.

[0045] According to a second aspect of the invention, there is hereby provided a compound of formula (I), or a salt and / or tautomer thereof: PAT059642-WO-PCT use in the treatment of cancer, wherein the treatment further comprises administration of (e.g. is in combination with): i) a CDK4 inhibitor; and / or ii) an endocrine therapeutical agent.

[0046] In an embodiment, the treatment further comprises administration of a CDK4 inhibitor.

[0047] In an embodiment, the treatment further comprises administration of an endocrine therapeutical agent.

[0048] In an embodiment, the treatment further comprises administration of both a CDK4 inhibitor and an endocrine therapeutical agent.

[0049] According to a third aspect of the invention, there is hereby provided a CDK4 inhibitor for use in the treatment of cancer, wherein the treatment further comprises administration of (e.g. is in combination with): i) a compound of formula (I), or a salt and / or tautomer thereof: ii) optionally an endocrine therapeutical agent.

[0050] In an embodiment, the endocrine therapeutical agent is not present. In another embodiment, the endocrine therapeutical agent is present.

[0051] According to a fourth aspect of the invention, there is hereby provided an endocrine therapeutical agent for use in the treatment of cancer, wherein the treatment further comprises administration of (e.g. is in combination with): i) a compound of formula (I), or a salt and / or tautomer thereof: PAT059642-WO-PCT ii) optionally, a CDK4 inhibitor.

[0052] In an embodiment, the CDK4 inhibitor is not present. In another embodiment, the CDK4 inhibitor is present.

[0053] According to a fifth aspect of the invention, there is hereby provided a combination comprising: i) a compound of formula (I), or a salt and / or tautomer thereof: ii) a CDK4 inhibitor; and / or iii) an endocrine therapeutical agent.

[0054] In an embodiment, the CDK4 inhibitor is present in the combination. In an embodiment, the endocrine therapeutical agent is present in the combination. In an embodiment, both the CDK4 inhibitor and the endocrine therapeutical agent are present in the combination.

[0055] In an embodiment of any one of the first to fifth aspects of the invention, the CDK4 inhibitor is: i) a mono CDK4 inhibitor; ii) a dual CDK2 / 4 inhibitor; iii) a dual CDK4 / 6 inhibitor; or iv) a triple CDK2 / 4 / 6 inhibitor. In an embodiment, the CDK4 inhibitor is a dual CDK4 / 6 inhibitor or a mono CDK4 inhibitor. In an embodiment, the CDK4 inhibitor is a dual CDK4 / 6 inhibitor. In an embodiment, the CDK4 inhibitor is a mono CDK4 inhibitor. In an embodiment, the CDK4 inhibitor is selected from PF-07220060, dalpiciclib, trilaciclib, palbociclib, abemaciclib and ribociclib. In an embodiment, the CDK4 inhibitor is ribociclib or PF-07220060. In a preferred embodiment, the CDK4 inhibitor is ribociclib. In an embodiment, the CDK4 inhibitor is PF-07220060.

[0056] In an embodiment of any one of the first to fifth aspects of the invention, the endocrine therapeutical agent is selected from at least one of the following: a) an aromatase inhibitor; b) a PAT059642-WO-PCT selective estrogen receptor degrader (SERD); c) a selective receptor modulator (SERM); and d) a gonadotropin-releasing hormone (GnRH) inhibitor. In an embodiment, the endocrine therapeutical agent is an aromatase inhibitor. In an embodiment, the aromatase inhibitor is selected from anastrozole, letrozole, exemestane, vorozole, formestane and fadrozole, e.g. is selected from anastrozole, letrozole and exemestane. In another embodiment, the endocrine therapeutical agent is a selective estrogen receptor degrader (SERD). In an embodiment, the selective estrogen receptor degrader (SERD) is selected from ARV-471 , fulvestrant and elacestrant. In another embodiment, the endocrine therapeutical agent is a selective receptor modulator (SERM). In an embodiment, the selective receptor modulator (SERM) is selected from tamoxifen, toremifene and raloxifene, e.g. wherein the SERM is tamoxifen. In yet another embodiment, the endocrine therapeutical agent is a gonadotropin-releasing hormone (GnRH) inhibitor. In an embodiment, the gonadotropin-releasing hormone (GnRH) inhibitor is selected from goserelin and leuprolide. In an embodiment, the endocrine therapeutical agent is selected from anastrozole, letrozole, exemestane, ARV-471 , Fulvestrant, elacestrant, tamoxifen, goserelin and leuprolide. In an embodiment, the endocrine therapeutical agent is selected from ARV-471 , Fulvestrant and elacestrant. In an embodiment, the endocrine therapeutical agent is Fulvestrant.

[0057] In an embodiment of any one of the first to fourth aspects of the invention, the cancer is selected from ovarian cancer, gastroesophageal cancer, uterine cancer, breast cancer (e.g. HR+ breast cancer, e.g. ER+ breast cancer, e.g. HER2- breast cancer e.g. HR+ / HER2- breast cancer, e.g. ER+ / HER2- breast cancer), prostate cancer and lung cancer. In an embodiment, the cancer is breast cancer. In an embodiment, the cancer is HR+ breast cancer. In an embodiment, the cancer is ER+ breast cancer. In an embodiment, the cancer is HER2- breast cancer. In an embodiment, the cancer is HR+ / HER2- breast cancer. In an embodiment, the cancer is ER+ / HER2- breast cancer. In an embodiment, the cancer is CCNE1 or CCNE2 amplified.

[0058] In an embodiment, the subject has already progressed on a combination of i) a CDK4 inhibitor (e.g. a dual CDK4 / 6 inhibitor, e.g. ribociclib, palbociclib or abemaciclib) and ii) an endocrine therapeutical agent (e.g. a) an aromatase inhibitor; b) a selective estrogen receptor degrader (SERD); c) a selective receptor modulator (SERM); and d) a gonadotropin-releasing hormone (GnRH) inhibitor, e.g. anastrozole, letrozole, exemestane, ARV-471 , Fulvestrant, elacestrant, tamoxifen, goserelin or leuprolide e.g. fulvestrant).

[0059] According to a sixth aspect of the invention, there is hereby provided a compound of formula (I), or a salt and / or tautomer thereof: PAT059642-WO-PCT use in the treatment of a CCNE1 or CCNE2 amplified tumor.

[0060] According to a seventh aspect of the invention, there is hereby provided a method of treating a CCNE1 or CCNE2 amplified tumor, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I), or a salt and / or tautomer thereof:

[0061] In an embodiment of the sixth or seventh aspect of the invention, the CCNE1 or CCNE2 amplified tumor is a CCNE1 amplified tumor. In an embodiment of the sixth or seventh aspectof the invention, the CCNE1 or CCNE2 (preferably CCNE1) amplified tumor is selected from ovarian cancer, cholangiocarinoma, breast cancer, bladder cancer, prostate cancer, a sarcoma, colorectal cancer, cervical cancer, endometrial cancer, gastric cancer (e.g. gastroesophageal adenocarcinoma), lung cancer, pancreatic cancer and fallopian tube carcinoma.

[0062] The invention therefore provides the following numbered embodiments:

[0063] Embodiment 1 . A method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of: i) a compound of formula (I), or a salt and / or tautomer thereof: PAT059642-WO-PCT iii) an endocrine therapeutical agent.

[0064] Embodiment 1 a. The method according to Embodiment 1 , comprising administering to a subject in need thereof a therapeutically effective amount of: i) a compound of formula (I), or a salt and / or tautomer thereof: ii) a CDK4 inhibitor.

[0065] Embodiment 1 b. The method according to Embodiment 1 , comprising administering to a subject in need thereof a therapeutically effective amount of: i) a compound of formula (I), or a salt and / or tautomer thereof: ii) an endocrine therapeutical agent.

[0066] Embodiment 1 c. The method according to Embodiment 1 , comprising administering to a subject in need thereof a therapeutically effective amount of: i) a compound of formula (I), or a salt and / or tautomer thereof: PAT059642-WO-PCT iii) an endocrine therapeutical agent.

[0067] Embodiment 2. A compound of formula (I), or a salt and / or tautomer thereof: use in the treatment of cancer, wherein the treatment further comprises administration of: i) a CDK4 inhibitor; and / or ii) an endocrine therapeutical agent.

[0068] Embodiment 2a. The compound of formula (I), or a salt and / or tautomer thereof: use according to Embodiment 2, wherein the treatment further comprises administration of a CDK4 inhibitor.

[0069] Embodiment 2b. The compound of formula (I), or a salt and / or tautomer thereof: use according to Embodiment 2, wherein the treatment further comprises administration of an endocrine therapeutical agent. PAT059642-WO-PCT

[0070] Embodiment 2c. The compound of formula (I), or a salt and / or tautomer thereof: use according to Embodiment 2, wherein the treatment further comprises administration of: i) a CDK4 inhibitor; and ii) an endocrine therapeutical agent.

[0071] Embodiment 3. A CDK4 inhibitor for use in the treatment of cancer, wherein the treatment further comprises administration of: i) a compound of formula (I), or a salt and / or tautomer thereof: ii) optionally an endocrine therapeutical agent.

[0072] Embodiment 3a. The CDK4 inhibitor for use according to Embodiment 3, wherein the treatment further comprises administration of a compound of formula (I), or a salt and / or tautomer thereof: Embodiment 3b. The CDK4 inhibitor for use according to Embodiment 3, wherein the treatment further comprises administration of a compound of formula (I), or a salt and / or tautomer thereof: PAT059642-WO-PCT endocrine therapeutical agent.

[0073] Embodiment 4. An endocrine therapeutical agent for use in the treatment of cancer, wherein the treatment further comprises administration of: i) a compound of formula (I), or a salt and / or tautomer thereof: ii) optionally, a CDK4 inhibitor.

[0074] Embodiment 4a. The endocrine therapeutical agent for use according to Embodiment 4, wherein the treatment further comprises administration of a compound of formula (I), or a salt and / or tautomer thereof:

[0075] Embodiment 4b. The endocrine therapeutical agent for use according to Embodiment 4, wherein the treatment further comprises administration of: i) a compound of formula (I), or a salt and / or tautomer thereof: PAT059642-WO-PCT

[0076] Embodiment 5. A combination comprising: i) a compound of formula (I), or a salt and / or tautomer thereof: ii) a CDK4 inhibitor; and / or iii) an endocrine therapeutical agent.

[0077] Embodiment 5a. The combination according to Embodiment 5, comprising: i) a compound of formula (I), or a salt and / or tautomer thereof: ii) a CDK4 inhibitor. Embodiment 5b. The combination according to Embodiment 5, comprising: i) a compound of formula (I), or a salt and / or tautomer thereof: ii) an endocrine therapeutical agent.

[0078] Embodiment 5c. The combination according to Embodiment 5, comprising: i) a compound of formula (I), or a salt and / or tautomer thereof: PAT059642-WO-PCT ii) a CDK4 inhibitor; and iii) an endocrine therapeutical agent.

[0079] Embodiment s. The method according to any one of Embodiments 1 , 1a and 1 c, the compound for use according to any one of Embodiments 2, 2a and 2c, the CDK4 inhibitor for use according to any one of Embodiments 3 to 3b, the endocrine therapeutical agent for use according to Embodiment 4 or Embodiment 4b, or the combination according to any one of Embodiments 5, 5a and 5c, wherein the CDK4 inhibitor is: i) a mono CDK4 inhibitor; ii) a dual CDK2 / 4 inhibitor; iii) a dual CDK4 / 6 inhibitor; or iv) a triple CDK2 / 4 / 6 inhibitor.

[0080] Embodiment 7. The method according to Embodiment 6, the compound for use according to Embodiment 6, the CDK4 inhibitor for use according to Embodiment 6, the endocrine therapeutical agent for use according to Embodiment 6, or the combination according to Embodiment 6, wherein the CDK4 inhibitor is a dual CDK4 / 6 inhibitor or a mono CDK4 inhibitor.

[0081] Embodiment 7a. The method according to Embodiment 7, the compound for use according to Embodiment 7, the CDK4 inhibitor for use according to Embodiment 7, the endocrine therapeutical agent for use according to Embodiment 7, or the combination according to Embodiment 7, wherein the CDK4 inhibitor is a dual CDK4 / 6 inhibitor.

[0082] Embodiment 7b. The method according to Embodiment 7, the compound for use according to Embodiment 7, the CDK4 inhibitor for use according to Embodiment 7, the endocrine therapeutical agent for use according to Embodiment 7, or the combination according to Embodiment 7, wherein the CDK4 inhibitor is a mono CDK4 inhibitor. PAT059642-WO-PCT

[0083] Embodiment s. The method according to any one of Embodiments 1 , 1 a, 1 c and 6, the compound for use according to any one of Embodiments 2, 2a, 2c and 6, the CDK4 inhibitor for use according to any one of Embodiments 3 to 3b and 6, the endocrine therapeutical agent for use according to any one of Embodiments 4, 4b and 6, or the combination according to any one of Embodiments 5, 5a, 5c and 6, wherein the CDK4 inhibitor is selected from PF-07220060, dalpiciclib, trilaciclib, palbociclib, abemaciclib and ribociclib.

[0084] Embodiment 9. The method according to Embodiment 8, the compound for use according to Embodiment 8, the CDK4 inhibitor for use according to Embodiment 8, the endocrine therapeutical agent for use according to Embodiment 8, or the combination according to Embodiment 8, wherein the CDK4 inhibitor is ribociclib or PF-07220060.

[0085] Embodiment 10. The method according to Embodiment 9, the compound for use according to Embodiment 9, the CDK4 inhibitor for use according to Embodiment 9, the endocrine therapeutical agent for use according to Embodiment 9, or the combination according to Embodiment 9, wherein the CDK4 inhibitor is ribociclib.

[0086] Embodiment 11 . The method according to Embodiment 9, the compound for use according to Embodiment 9, the CDK4 inhibitor for use according to Embodiment 9, the endocrine therapeutical agent for use according to Embodiment 9, or the combination according to Embodiment 9, wherein the CDK4 inhibitor is PF-07220060.

[0087] Embodiment 12. The method according to any one of Embodiments 1 to 1 c and 6 to 11 , the compound for use according to any one of Embodiments 2, 2b, 2c and 6 to 11 , the CDK4 inhibitor for use according to any one of Embodiments 3, 3b and 6 to 11 , the endocrine therapeutical agent for use according to any one of Embodiments 4 to 4b and 6 to 11 , or the combination according to any one of Embodiments 5, 5b, 5c and 6 to 11 , wherein the endocrine therapeutical agent is selected from at least one of the following: a) an aromatase inhibitor; b) a selective estrogen receptor degrader (SERD); c) a selective receptor modulator (SERM); and d) a gonadotropin-releasing hormone (GnRH) inhibitor.

[0088] Embodiment 12a. The method according to Embodiment 12, the compound for use according to Embodiment 12, the CDK4 inhibitor for use according to Embodiment 12, the endocrine PAT059642-WO-PCT therapeutical agent for use according to Embodiment 12, or the combination according to Embodiment 12, wherein the endocrine therapeutical agent is an aromatase inhibitor.

[0089] Embodiment 12b. The method according to Embodiment 12a, the compound for use according to Embodiment 12a, the CDK4 inhibitor for use according to Embodiment 12a, the endocrine therapeutical agent for use according to Embodiment 12a, or the combination according to Embodiment 12a, wherein the aromatase inhibitor is selected from anastrozole, letrozole, exemestane, vorozole, formestane and fadrozole, e.g. is selected from anastrozole, letrozole and exemestane.

[0090] Embodiment 12c. The method according to Embodiment 12, the compound for use according to Embodiment 12, the CDK4 inhibitor for use according to Embodiment 12, the endocrine therapeutical agent for use according to Embodiment 12, or the combination according to Embodiment 12, wherein the endocrine therapeutical agent is a selective estrogen receptor degrader (SERD).

[0091] Embodiment 12d. The method according to Embodiment 12c, the compound for use according to Embodiment 12c, the CDK4 inhibitor for use according to Embodiment 12c, the endocrine therapeutical agent for use according to Embodiment 12c, or the combination according to Embodiment 12c, wherein the selective estrogen receptor degrader (SERD) is selected from ARV-471 , fulvestrant and elacestrant.

[0092] Embodiment 12e. The method according to Embodiment 12, the compound for use according to Embodiment 12, the CDK4 inhibitor for use according to Embodiment 12, the endocrine therapeutical agent for use according to Embodiment 12, or the combination according to Embodiment 12, wherein the endocrine therapeutical agent is a selective receptor modulator (SERM).

[0093] Embodiment 12f. The method according to Embodiment 12e, the compound for use according to Embodiment 12e, the CDK4 inhibitor for use according to Embodiment 12e, the endocrine therapeutical agent for use according to Embodiment 12e, or the combination according to Embodiment 12e, wherein the selective receptor modulator (SERM) is selected from tamoxifen, toremifene and raloxifene, e.g. wherein the SERM is tamoxifen.

[0094] Embodiment 12g. The method according to Embodiment 12, the compound for use according to Embodiment 12, the CDK4 inhibitor for use according to Embodiment 12, the endocrine therapeutical agent for use according to Embodiment 12, or the combination according to PAT059642-WO-PCT

[0095] Embodiment 12, wherein the endocrine therapeutical agent is a gonadotropin-releasing hormone (GnRH) inhibitor.

[0096] Embodiment 12h. The method according to Embodiment 12g, the compound for use according to Embodiment 12g, the CDK4 inhibitor for use according to Embodiment 12g, the endocrine therapeutical agent for use according to Embodiment 12g, or the combination according to Embodiment 12g, wherein the gonadotropin-releasing hormone (GnRH) inhibitor is selected from goserelin and leuprolide.

[0097] Embodiment 13. The method according to any one of Embodiments 1 to 1c and 6 to 12, the compound for use according to any one of Embodiments 2, 2b, 2c and 6 to 12, the CDK4 inhibitor for use according to any one of Embodiments 3, 3b and 6 to 12, the endocrine therapeutical agent for use according to any one of Embodiments 4 to 4b and 6 to 12, or the combination according to any one of Embodiments 5, 5b, 5c and 6 to 12, wherein the endocrine therapeutical agent is selected from anastrozole, letrozole, exemestane, ARV-471 , Fulvestrant, elacestrant, tamoxifen, goserelin and leuprolide.

[0098] Embodiment 14. The method according to Embodiment 13, the compound for use according to Embodiment 13, the CDK4 inhibitor for use according to Embodiment 13, the selective estrogen receptor degrader for use according to Embodiment 13, or the combination according to Embodiment 13, wherein the endocrine therapeutical agent is selected from ARV-471 , Fulvestrant and elacestrant.

[0099] Embodiment 15. The method according to Embodiment 14, the compound for use according to Embodiment 14, the CDK4 inhibitor for use according to Embodiment 14, the selective estrogen receptor degrader for use according to Embodiment 14, or the combination according to Embodiment 14, wherein the endocrine therapeutical agent is Fulvestrant.

[0100] Embodiment 16. The method according to any one of Embodiments 1 to 1c and 6 to 15, the compound for use according to any one of Embodiments 2, 2b, 2c and 6 to 15, the CDK4 inhibitor for use according to any one of Embodiments 3, 3b and 6 to 15, the endocrine therapeutical agent for use according to any one of Embodiments 4 to 4b and 6 to 15, wherein the cancer is selected from ovarian cancer, gastroesophageal cancer, uterine cancer, breast cancer (e.g. HR+ breast cancer, e.g. ER+ breast cancer, e.g. HER2- breast cancer e.g. HR+ / HER2- breast cancer, e.g. ER+ / HER2- breast cancer), prostate cancer, lung cancer. PAT059642-WO-PCT

[0101] Embodiment 17. The method according to Embodiment 15, the compound for use according to Embodiment 15, the CDK4 inhibitor for use according to Embodiment 15 or the endocrine therapeutical agent for use according to Embodiment 15, wherein the cancer is breast cancer.

[0102] Embodiment 18. The method according to Embodiment 17, the compound for use according to Embodiment 17, the CDK4 inhibitor for use according to Embodiment 17 or the endocrine therapeutical agent for use according to Embodiment 17, wherein the cancer is HR+ breast cancer.

[0103] Embodiment 19. The method according to Embodiment 17 or Embodiment 18, the compound for use according to Embodiment 17 or Embodiment 18, the CDK4 inhibitor for use according to Embodiment 17 or Embodiment 18 or the endocrine therapeutical agent for use according to Embodiment 17 or Embodiment 18, wherein the cancer is ER+ breast cancer.

[0104] Embodiment 20. The method according to any one of Embodiments 17 to 19, the compound for use according to any one of Embodiments 17 to 19, the CDK4 inhibitor for use according to any one of Embodiments 17 to 19 or the endocrine therapeutical agent for use according to any one of Embodiments 17 to 19, wherein the cancer is HER2- breast cancer.

[0105] Embodiment 21 . The method according to any one of Embodiments 17 to 20, the compound for use according to any one of Embodiments 17 to 20, the CDK4 inhibitor for use according to any one of Embodiments 17 to 20 or the endocrine therapeutical agent for use according to any one of Embodiments 17 to 20, wherein the cancer is HR+ / HER2- breast cancer.

[0106] Embodiment 22. The method according to any one of Embodiments 17 to 21 , the compound for use according to any one of Embodiments 17 to 21 , the CDK4 inhibitor for use according to any one of Embodiments 17 to 21 or the endocrine therapeutical agent for use according to any one of Embodiments 17 to 21 , wherein the cancer is ER+ / HER2- breast cancer.

[0107] Embodiment 22a. The method according to any one of Embodiments 17 to 22, the compound for use according to any one of Embodiments 17 to 22, the CDK4 inhibitor for use according to any one of Embodiments 17 to 22 or the endocrine therapeutical agent for use according to any one of Embodiments 17 to 22, wherein the breast cancer is early-stage breast cancer (e.g. wherein the tumor is 20 mm or smaller and the cancer has not spread to more than 3 lymph nodes, or wherein tumor is 20 to 50 mm and has not spread to any lymph nodes).

[0108] Embodiment 23. The method according to any one of Embodiments 1 to 1 c, and 6 to 22, the compound for use according to any one of Embodiments 2 to 2c and 6 to 22, the CDK4 inhibitor PAT059642-WO-PCT for use according to any one of Embodiments 3 to 3b and 6 to 22 or the endocrine therapeutical agent for use according to any one of Embodiments 4 to 4b and 6 to 22, wherein the cancer is CCNE1 or CCNE2 amplified.

[0109] Embodiment 24. A compound of formula (I), or a salt and / or tautomer thereof: use in the treatment of a CCNE1 or CCNE2 amplified tumor.

[0110] Embodiment 25. A method of treating a CCNE1 or CCNE2 amplified tumor, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I), or a salt and / or tautomer thereof:

[0111] Embodiment 26. The compound for use according to Embodiment 24, or the method according to Embodiment 25, wherein the CCNE1 or CCNE2 amplified tumor is a CCNE1 amplified tumor.

[0112] Embodiment 27. The compound for use according to Embodiment 25 or Embodiment 26, or the method according to Embodiment 25 or Embodiment 26 wherein the CCNE1 or CCNE2 amplified tumor is selected from ovarian cancer, cholangiocarinoma, breast cancer, bladder cancer, prostate cancer, a sarcoma, colorectal cancer, cervical cancer, endometrial cancer, gastric cancer (e.g. gastroesophageal adenocarcinoma), lung cancer, pancreatic cancer and fallopian tube carcinoma.

[0113] Definitions PAT059642-WO-PCT

[0114] The terms "a" and "an" and "the" and similar references in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Where the plural form is used for compounds, patients, cancers and the like, this is taken to mean also a single compound, patient, or the like.

[0115] References in this specification to "the invention" are intended to reflect embodiments of the several inventions disclosed in this specification, and should not be taken as unnecessarily limiting of the claimed subject matter.

[0116] The term “pharmaceutically acceptable salts” refers to salts that retain the biological effectiveness and properties of the compound and which typically are not biologically or otherwise undesirable. The compound may be capable of forming acid addition salts by virtue of the presence of an amino group.

[0117] Unless otherwise specified, or clearly indicated by the text, reference to therapeutic agents useful in the pharmaceutical combination of the present invention includes both the free base of the compounds, and all pharmaceutically acceptable salts of the compounds.

[0118] “Combination” refers to either a fixed combination in one dosage unit form, or a combined administration where a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a combination partner (e.g. another drug as explained below, also referred to as “therapeutic agent” or “co-agent”) may be administered independently at the same time or separately within time intervals, especially where these time intervals allow that the combination partners show a cooperative, e.g. synergistic effect. The single components may be packaged in a kit or separately. One or both of the components (e.g., powders or liquids) may be reconstituted or diluted to a desired dose prior to administration. The terms “co-administration” or “combined administration” or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g. a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. The term “pharmaceutical combination” as used herein means a product that results from the mixing or combining of more than one therapeutic agent and includes both fixed and non-fixed combinations of the therapeutic agents. The term “fixed combination” means that the therapeutic agents, e.g. combination partners of the present invention, are both administered to a patient simultaneously in the form of a single entity or dosage. The term “non-fixed combination” means that the therapeutic agents, e.g. combination PAT059642-WO-PCT partners of the present invention, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g. the administration of three or more therapeutic agents.

[0119] In the combination therapies of the invention, the therapeutic agents may be manufactured and / or formulated by the same or different manufacturers. Moreover, the therapeutic agents may be brought together into a combination therapy: (i) prior to release of the combination product to physicians (e.g. in the case of a kit comprising the therapeutic agents); (ii) by the physician themselves (or under the guidance of the physician) shortly before administration; (iii) in the patient themselves, e.g. during sequential administration of the therapeutic agents.

[0120] The term "synergistic effect" as used herein refers to action of two or three therapeutic agents producing an effect, for example, slowing the progression of a proliferative disease, particularly cancer, or symptoms thereof, which is greater than the simple addition of the effects of each drug administered by themselves. A synergistic effect can be calculated, for example, using suitable methods such as the Sigmoid-Emax equation (Holford, N. H. G. and Scheiner, L. B., Clin. Pharmacokinet. 6: 429-453 (1981)), the equation of Loewe additivity (Loewe, S. and Muischnek, H., Arch. Exp. Pathol Pharmacol. 114: 313-326 (1926)) and the median effect equation (Chou, T. C. and Talalay, P., Adv. Enzyme Regul. 22: 27-55 (1984)). Each equation referred to above can be applied to experimental data to generate a corresponding graph to aid in assessing the effects of a drug combination. The corresponding graphs associated with the equations referred to above are the concentration-effect curve, isobologram curve and combination index curve, respectively.

[0121] The (pharmaceutical) combinations can further comprise at least one pharmaceutically acceptable carrier. Thus, the present invention relates to a pharmaceutical composition comprising the pharmaceutical combination of the present invention and at least one pharmaceutically acceptable carrier.

[0122] As used herein, the term “carrier’’ or "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, PAT059642-WO-PCT

[0123] 1990, pp. 1289- 1329). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0124] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0125] Generally, the term “pharmaceutical composition” is defined herein to refer to a mixture or solution containing at least one therapeutic agent to be administered to a subject, e.g., a mammal or human. The present pharmaceutical combinations can be formulated in a suitable pharmaceutical composition for enteral or parenteral administration are, for example, those in unit dosage forms, such as sugar-coated tablets, tablets, capsules or suppositories, or ampoules. If not indicated otherwise, these are prepared in a manner known per se, for example by means of various conventional mixing, comminution, direct compression, granulating, sugar-coating, dissolving, lyophilizing processes, or fabrication techniques readily apparent to those skilled in the art. It will be appreciated that the unit content of a combination partner contained in an individual dose of each dosage form need not in itself constitute an effective amount since the necessary effective amount may be reached by administration of a plurality of dosage units. The pharmaceutical composition may contain, from about 0.1 % to about 99.9%, preferably from about 1 % to about 60 %, of the therapeutic agent(s). One of ordinary skill in the art may select one or more of the aforementioned carriers with respect to the particular desired properties of the dosage form by routine experimentation and without any undue burden. The amount of each carriers used may vary within ranges conventional in the art. The following references disclose techniques and excipients used to formulate oral dosage forms. See The Handbook of Pharmaceutical Excipients, 4th edition, Rowe et al., Eds., American Pharmaceuticals Association (2003); and Remington: the Science and Practice of Pharmacy, 20th edition, Gennaro, Ed., Lippincott Williams & Wilkins (2003). These optional additional conventional carriers may be incorporated into the oral dosage form either by incorporating the one or more conventional carriers into the initial mixture before or during granulation or by combining the one or more conventional carriers with granules comprising the combination of agents or individual agents of the combination of agents in the oral dosage form. In the latter embodiment, the combined mixture may be further blended, e.g., through a V-blender, and subsequently compressed or molded into a tablet, for example a PAT059642-WO-PCT monolithic tablet, encapsulated by a capsule, or filled into a sachet. Clearly, the pharmaceutical combinations of the present invention can be used to manufacture a medicine.

[0126] The present invention relates to such pharmaceutical combinations or pharmaceutical compositions that are particularly useful as a medicine.

[0127] Specifically, the combinations or compositions of the present invention can be applied in the treatment of cancer.

[0128] The present invention also relates to use of pharmaceutical combinations or pharmaceutical compositions of the present invention for the preparation of a medicament for the treatment of a cancer, and to a method for treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a pharmaceutical combination according to the present invention, or the pharmaceutical composition according to the present invention.

[0129] The term “treatment” as used herein comprises a treatment relieving, reducing or alleviating at least one symptom in a subject, increasing progression-free survival, overall survival, extending duration of response or delaying progression of a disease. For example, treatment can be the diminishment of one or several symptoms of a disorder or complete eradication of a disorder, such as cancer. Within the meaning of the present invention, the term “treatment” also denotes to arrest, delay the onset (i.e . , the period prior to clinical manifestation of a disease) and / or reduce the risk of developing or worsening a disease in a patient, e.g., a mammal, particularly the patient is a human. The term “treatment” as used herein comprises an inhibition of the growth of a tumor incorporating a direct inhibition of a primary tumor growth and I or the systemic inhibition of metastatic cancer cells. The term “treatment” as used herein also includes reducing the risk of reoccurrence of the cancer (particularly wherein the cancer is early-stage breast cancer). The term “treatment” as used herein also includes increasing invasive disease-free survival (again, particularly wherein the cancer is early-stage breast cancer).

[0130] A "subject," "individual" or "patient" is used interchangeably herein, which refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, mice, simians, humans, farm animals, sport animals, and pets. In an embodiment, the subject is a (genetically) female human. In an embodiment, a pre-menopausal (genetically) female human. In another embodiment, a post-menopausal (genetically) female human.

[0131] As used herein, a subject is “in need of’ or “in need thereof” a treatment if such subject would benefit biologically, medically or in quality of life from such treatment. PAT059642-WO-PCT

[0132] The term “comprising” encompasses “including” as well as “consisting”; e.g., a composition comprising X may consist exclusively of X or may include additional, e.g. X and Y.

[0133] The term "a therapeutically effective amount" of a compound (e.g. chemical entity or biologic agent) of the present invention refers to an amount of the compound of the present invention that will elicit the biological or medical response of a subject, for example, reduction or inhibition of an enzyme or a protein activity, or ameliorate symptoms, alleviate conditions, slow or delay disease progression, or prevent a disease, etc. In one embodiment a therapeutically effective amount in vivo may range depending on the route of administration, between about 0.1-500 mg / kg, or between about 1-100 mg / kg.

[0134] As used herein, the term “inhibit”, "inhibition" or “inhibiting” refers to the reduction or suppression of a given condition, symptom, or disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.

[0135] As used herein, the term “CCNE1 amplification”, “CCNE1 amplified tumor” and the like refers to an abnormal increase in the copy number or expression of the CCNE1 gene. CCNE1 is a gene that codes for cyclin E1 , a protein involved in regulating the cell cycle progression. Amplification of CCNE1 can lead to elevated levels of cyclin E1 protein, which may result in uncontrolled cell division and proliferation by the tumor cell.

[0136] As used herein, the term “CCNE2 amplification”, “CCNE2 amplified tumor” and the like refers to an abnormal increase in the copy number or expression of the CCNE2 gene. CCNE2 is a gene that codes for cyclin E2, a protein involved in regulating the cell cycle progression. Amplification of CCNE2 can lead to elevated levels of cyclin E2 protein, which may result in uncontrolled cell division and proliferation by the tumor cell.

[0137] As used herein, the term “endocrine therapeutical agent” refers to agents that interrupt the stimulatory axis created by systemic pools of androgens or estrogens, inhibit hormone production or hormone binding to receptors, and ultimately block expression of genes that promote tumor growth or survival. Such agents are typically used in the treatment of hormonally responsive cancers such as breast, prostate and endometrial carcinomas.

[0138] As used herein, the term “Selective estrogen receptor degrader” or “SERD” for short, refers to agents that bind to estrogen receptors and facilitate their degradation, leading to a reduction in estrogen receptor levels. SERDs are typically devoid of estrogen agonist activity. PAT059642-WO-PCT

[0139] As used herein, the term “Selective estrogen receptor modulator” or “SERM” for short, refers to agents that bind to the ER (estrogen receptor), and exert either estrogenic or antiestrogenic effects, depending on the specific organ.

[0140] As used herein, the term “aromatase inhibitor” refers to an agent that blocks the function of aromatase, which converts androgens to estrogens. By blocking this enzymatic activity, an aromatase inhibitor thereby reduces estrogen production.

[0141] As used herein, the term “Gonadotropin releasing hormone inhibitor”, or“GnRH inhibitor” for short (which may be a GnRH agonist) refers to a substance that prevents the ovaries from making estrogen. Continuous administration of GnRH agonist downregulates GnRH receptors in the pituitary gland. This suppress the release of the gonadotropins FSH and LH from the pituitary gland and prevent follicular maturation in the ovary. Serum estrogens levels are reduced to those seen in postmenopausal women or in women after oophorectomy.

[0142] The optimal dosage of each combination partner for treatment of a cancer can be determined empirically for each individual using known methods and will depend upon a variety of factors, including, though not limited to, the degree of advancement of the disease; the age, body weight, general health, gender and diet of the individual; the time and route of administration; and other medications the individual is taking. Optimal dosages may be established using routine testing and procedures that are well known in the art. The amount of each combination partner that may be combined with the carrier materials to produce a single dosage form will vary depending upon the individual treated and the particular mode of administration. In some embodiments the unit dosage forms containing the combination of agents as described herein will contain the amounts of each agent of the combination that are typically administered when the agents are administered alone.

[0143] Frequency of dosage may vary depending on the compound used and the particular condition to be treated or prevented. In general, the use of the minimum dosage that is sufficient to provide effective therapy is preferred. Patients may generally be monitored for therapeutic effectiveness using assays suitable for the condition being treated or prevented, which will be familiar to those of ordinary skill in the art.

[0144] The term “cancer” refers to a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include but are PAT059642-WO-PCT not limited to colorectal, gastric, endometrial, prostate, adrenocortical, uterine, cervical, esophageal, breast, kidney, ovarian cancer and the like.

[0145] The terms “tumor” and “cancer” are used interchangeably herein, e.g., both terms encompass solid and liquid, e.g., diffuse or circulating, tumors. As used herein, the term “cancer” or “tumor” includes premalignant, as well as malignant cancers and tumors.

[0146] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0147] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. "such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed.

[0148] The term “Compound A” as used herein refers salt and / or tautomer thereof.

[0149] Unless stated clearly otherwise, salts and / or tautomers of all CDK4 Inhibitors as defined herein, such as ribociclib, palbociclib, abemeciclib, PF-07220060 etc. are encompassed by the invention. In an embodiment, the ribociclib is ribociclib succinate.

[0150] CDK4 Inhibitor

[0151] The term “CDK4 inhibitor” as used herein in particular in the independent claims refers to a compound which inhibits CDK4 following the method of Example B at < 5 pM, for example < 2 pM, for example < 1 pM, for example less than < 0.5 pM, for example < 0.2 pM, for example < 0.1 pM. PAT059642-WO-PCT

[0152] It should be understood that each of “mono CDK4 inhibitor”, “dual CDK4 / 6 inhibitor”, “dual CDK2 / 4 inhibitor”, and “triple CDK2 / 4 / 6 inhibitor”, as used herein as are subcategories of “CDK4 inhibitor” as used herein.

[0153] The term “mono CDK4 inhibitor” as used herein refers to a “CDK4 inhibitor” as used herein which inhibits CDK4 (in pM) at an amount of > 5 times, for example > 10 times, for example (and preferably) > 15 times, for example > 20 times greater than the inhibition of CDK2 (in pM), and which also inhibits CDK4 (in pM) at an amount of > 5 times, for example > 10 times, for example (and preferably) > 15 times, for example > 20 times greater than the inhibition of CDK6 (in pM), all following the method of Example B. In an embodiment, the term “mono CDK4 inhibitor” as used herein refers to a “CDK4 inhibitor” as used herein which inhibits CDK4 (in pM) at an amount of > 10 times greater than the inhibition of CDK2 (in pM) and which also inhibits CDK4 (in pM) at an amount of > 10 times greater than the inhibition of CDK6 (in pM), all following the method of Example B. In a particularly preferred embodiment, the term “mono CDK4 inhibitor” as used herein refers to a “CDK4 inhibitor” as used herein which inhibits CDK4 (in pM) at an amount of > 15 times greater than the inhibition of CDK2 (in pM) and which also inhibits CDK4 (in pM) at an amount of > 15 times greater than the inhibition of CDK6 (in pM), all following the method of Example B. In an embodiment, the term “mono CDK4 inhibitor” as used herein refers to a “CDK4 inhibitor” as used herein which inhibits CDK4 (in pM) at an amount of > 20 times greater than the inhibition of CDK2 (in pM) and which also inhibits CDK4 (in pM) at an amount of > 20 times greater than the inhibition of CDK6 (in pM), all following the method of Example B.

[0154] In an embodiment, the term “mono CDK4 inhibitor” as used herein refers to any “CDK4” inhibitor as used herein, which is more selective for CDK4 over CDK6 than all of ribociclib, palbociclib and abemeciclib all using the method of Example B, and which inhibits CDK4 (in pM) at an amount of > 5 times, for example > 10 times, for example (and preferably) > 15 times, for example > 20 times greater than the inhibition of CDK2 (in pM), again using the method of Example B.

[0155] The term “dual CDK4 / 6 inhibitor” as used herein refers to a “CDK4 inhibitor” as used herein which inhibits CDK4 (in pM) at an amount of > 5 times, for example > 10 times, for example (and preferably) > 15 times, for example > 20 times greater than the inhibition of CDK2 (in pM), and wherein the ratio of CDK4 to CDK6 inhibition (in pM) is from 20:1 to 1 :20, for example (and preferably) from 15:1 to 1 :15, for example from 10:1 to 1 :10, for example from 5:1 to 1 :5, all following the method of Example B. In an embodiment, the term “dual CDK4 / 6 inhibitor” as used herein refers to a “CDK4 inhibitor” as used herein which inhibits CDK4 (in pM) at an amount of > 10 times greater than the inhibition of CDK2 (in pM), wherein the ratio of CDK4 to CDK6 inhibition PAT059642-WO-PCT

[0156] (in pM) is from 10:1 to 1 :10, all following the method of Example B. In a particularly preferred embodiment, the term “dual CDK4 / 6 inhibitor” as used herein refers to a “CDK4 inhibitor” as used herein which inhibits CDK4 (in pM) at an amount of > 15 times greater than the inhibition of CDK2 (in pM), wherein the ratio of CDK4 to CDK6 inhibition (in pM) is from 15:1 to 1 :15, all following the method of Example B. In an embodiment, the term “dual CDK4 / 6 inhibitor” as used herein refers to a “CDK4 inhibitor” as used herein which inhibits CDK4 (in pM) at an amount of > 20 times greater than the inhibition of CDK2 (in pM), wherein the ratio of CDK4 to CDK6 inhibition (in pM) is from 20:1 to 1 :20, all following the method of Example B.

[0157] The term “dual CDK2 / 4 inhibitor” as used herein refers to a “CDK4 inhibitor” as used herein which inhibits CDK4 (in pM) at an amount of > 5 times, for example > 10 times, for example (and preferably) > 15 times, for example > 20 times greater than the inhibition of CDK6 (in pM), and wherein the ratio of CDK2 to CDK4 inhibition (in pM) is from 20:1 to 1 :20, for example (and preferably) from 15:1 to 1 :15, for example from 10:1 to 1 :10, for example from 5:1 to 1 :5 all following the method of Example B. In an embodiment, the term “dual CDK2 / 4 inhibitor” as used herein refers to a “CDK4 inhibitor” as used herein which inhibits CDK4 (in pM) at an amount of > 10 times greater than the inhibition of CDK6 (in pM), and wherein the ratio of CDK2 to CDK4 inhibition (in pM) is from 10:1 to 1 :10, all following the method of Example B. In a particularly preferred embodiment, the term “dual CDK2 / 4 inhibitor” as used herein refers to a “CDK4 inhibitor” as used herein which inhibits CDK4 (in pM) at an amount of > 15 times greater than the inhibition of CDK6 (in pM), and wherein the ratio of CDK2 to CDK4 inhibition (in pM) is from 15:1 to 1 :15, all following the method of Example B. In an embodiment, the term “dual CDK2 / 4 inhibitor” as used herein refers to a “CDK4 inhibitor” as used herein which inhibits CDK4 (in pM) at an amount of > 20 times greater than the inhibition of CDK6 (in pM), and wherein the ratio of CDK2 to CDK4 inhibition (in pM) is from 20:1 to 1 :20, all following the method of Example B.

[0158] The term “triple CDK2 / 4 / 6 inhibitor” as used herein refers to a “CDK4 inhibitor” as used herein wherein the ratio of CDK2 to CDK4 inhibition (in pM) is from 20:1 to 1 :20, for example (and preferably) from 15:1 to 1 :15, for example from 10:1 to 1 :10, for example from 5:1 to 1 :5, and wherein the ratio of CDK4 to CDK6 inhibition (in pM) is from 20:1 to 1 :20, for example (and preferably) from 15:1 to 1 :15, for example from 10:1 to 1 :10, for example from 5:1 to 1 :5 all following the method of Example B. In a particularly preferred embodiment, the term “triple CDK2 / 4 / 6 inhibitor” as used herein refers to a “CDK4 inhibitor” as used herein wherein the ratio of CDK2 to CDK4 inhibition (in pM) is from 15:1 to 1 :15, and wherein the ratio of CDK4 to CDK6 inhibition (in pM) is from 15:1 to 1 :15, all following the method of Example B. In an embodiment, PAT059642-WO-PCT the term “triple CDK2 / 4 / 6 inhibitor” as used herein refers to a “CDK4 inhibitor” as used herein wherein the ratio of CDK2 to CDK4 inhibition (in pM) is from 10:1 to 1 :10, and wherein the ratio of CDK4 to CDK6 inhibition (in pM) is from 10:1 to 1 :10, all following the method of Example B. In an embodiment, the term “triple CDK2 / 4 / 6 inhibitor” as used herein refers to a “CDK4 inhibitor” as used herein wherein the ratio of CDK2 to CDK4 inhibition (in pM) is from 5:1 to 1 :5, and wherein the ratio of CDK4 to CDK6 inhibition (in pM) is from 5:1 to 1 :5, all following the method of Example B.

[0159] In the above definitions, only the inhibition of CDKs 2, 4 and 6 are considered. The inhibition or lack thereof of other CDKs, such as CDK1 , CDK8, CDK9 etc. is permitted. For instance, a compound which strongly inhibits CDK4 and CDK8, and does not meaningfully inhibit CDK2 or CDK6 may be considered as a “mono CDK4 inhibitor” as used herein.

[0160] In an embodiment, the CDK4 inhibitor is a dual CDK4 / 6 inhibitor.

[0161] In an embodiment, the CDK4 inhibitor is selected from dalpiciclib, trilaciclib, palbociclib, abemaciclib, ribociclib, SPH-4336, TW-302, BPI-16350, BEBT-209, FCN-437, lerociclib, birociclib, TQB-3616, auceliciclib, BPI-1178, GLR-2007, UCT-03-008, QLS-12004, PRT-3645, XH-30002 and HS-10342.

[0162] In an embodiment, the CDK4 inhibitor is selected from dalpiciclib, trilaciclib, palbociclib, abemaciclib and ribociclib.

[0163] In a preferred embodiment, the CDK4 inhibitor is ribociclib. In an embodiment, the ribociclib is administered on the first 21 days of a 28-day treatment cycle (e.g. at 200 to 600 mg per day, e.g. at 400 to 600 mg per day, e.g. at 200, 250, 300, 350, 400, 450, 500, 550 or 600 mg per day, preferably at 400 or 600 mg per day), all expressed in terms of the free form, followed by 7 days off ribociclib treatment. In an embodiment, there are at least two (e.g. at least three, at least four etc.) consecutive 28-day treatment cycles. In an embodiment, the or pharmaceutically acceptable salt and / or tautomer thereof is administered (e.g. BID or once daily) on the same days that ribociclib is administered (i.e. on the same first 21 days of the same 28-day treatment cycle, followed by the same 7 days off treatment). In an embodiment, there are at least two (e.g. at least three, at least four etc.), consecutive 28-day treatment cycles. In PAT059642-WO-PCT alternative embodiment, the pharmaceutically acceptable salt and / or tautomer thereof is administered (e.g. BID or once daily) on every day of the 28-day treatment cycle. The pharmaceutically acceptable salt and / or tautomer thereof may be administered at a dose of from 10 mg to 2000 mg per day on each administration day, e.g. 10 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 120 mg, 125 mg, 140 mg, 150 mg, 160 mg, 175 mg, 180 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg or 2000 mg, all expressed in terms of the free form.

[0164] In an embodiment, the CDK4 inhibitor is a mono CDK4 inhibitor.

[0165] In an embodiment the CDK4 is selected from PF-07220060, BGB-43395, GRS-6209.

[0166] In an embodiment, the CDK4 inhibitor is PF-07220060.

[0167] In an embodiment, the CDK4 inhibitor is a triple CDK2 / 4 / 6 inhibitor.

[0168] In an embodiment, the CDK4 inhibitor is selected from lpY.20, SYH-2043, TY-0540 and RGT- 419B.

[0169] In an embodiment, the CDK4 inhibitor is a dual CDK2 / 4 inhibitor.

[0170] In an embodiment, the CDK4 inhibitor is IAM-C1 .

[0171] In a preferred embodiment the CDK4 inhibitor is selected from PF-07220060, dalpiciclib, trilaciclib, palbociclib, abemaciclib and ribociclib.

[0172] In a preferred embodiment the CDK4 inhibitor is selected from PF-07220060 and ribociclib.

[0173] According to a furtheraspect of the invention, there is hereby provided a method of treating cancer (preferably breast cancer, preferably HER2- breast cancer, preferably HR+ breast cancer, PAT059642-WO-PCT preferably ER+ breast cancer, preferably HR+ / HER2- breast cancer, preferably ER+ / HER2- breast cancer) comprising administering to a subject in need thereof a therapeutically effective amount of: i) a compound selected from AVZO-021 , PF-07104091 , BLU-222, INX-315, ARTS-021 , AZD- 8421 , NKT-3447, INCB-0123667, BLU-956, ETX-197, AU14-5, a compound selected from any one of compounds 1-4, 6, 12-47, 49-98 and 101-145 of W02023 / 069720 (which is incorporated by reference in its entirety), a compound selected from any one of the compounds of W02023 / 069700 and any one of the compounds of W02024 / 092039 (both of which are incorporated by reference in their entirety). ii) a CDK4 inhibitor (e.g. selected from PF-07220060, dalpiciclib, trilaciclib, palbociclib, abemaciclib and ribociclib), and optionally iii) an endocrine therapeutical agent (e.g. a selective estrogen receptor degrader, e.g. fulvestrant).

[0174] According to this aspect of the invention, in an embodiment the CDK4 inhibitor is PF-07220060, and the endocrine therapeutical agent is not present.

[0175] According to this aspect of the invention, in an embodiment the CDK4 inhibitor is PF-07220060 and the endocrine therapeutical agent (e.g. a selective estrogen receptor degrader, e.g. fulvestrant) is present.

[0176] According to this aspect of the invention, in an embodiment the CDK4 inhibitor is dalpiciclib, and the endocrine therapeutical agent is not present.

[0177] According to this aspect of the invention, in an embodiment the CDK4 inhibitor is dalpiciclib and the endocrine therapeutical agent (e.g. a selective estrogen receptor degrader, e.g. fulvestrant) is present.

[0178] According to this aspect of the invention, in an embodiment the CDK4 inhibitor is trilaciclib, and the endocrine therapeutical agent is not present.

[0179] According to this aspect of the invention, in an embodiment the CDK4 inhibitor is trilaciclib and the endocrine therapeutical agent (e.g. a selective estrogen receptor degrader, e.g. fulvestrant) is present.

[0180] According to this aspect of the invention, in an embodiment the CDK4 inhibitor is palbociclib, and the endocrine therapeutical agent is not present. PAT059642-WO-PCT

[0181] According to this aspect of the invention, in an embodiment the CDK4 inhibitor is palbociclib and the endocrine therapeutical agent (e.g. a selective estrogen receptor degrader, e.g. fulvestrant) is present.

[0182] According to this aspect of the invention, in an embodiment the CDK4 inhibitor is abemaciclib, and the endocrine therapeutical agent is not present.

[0183] According to this aspect of the invention, in an embodiment the CDK4 inhibitor is abemaciclib and the endocrine therapeutical agent (e.g. a selective estrogen receptor degrader, e.g. fulvestrant) is present.

[0184] According to this aspect of the invention, in an embodiment the CDK4 inhibitor is ribociclib, and the endocrine therapeutical agent is not present.

[0185] According to this aspect of the invention, in a preferred embodiment the CDK4 inhibitor is ribociclib and the endocrine therapeutical agent (e.g. a selective estrogen receptor degrader, e.g. fulvestrant) is present.

[0186] A yet further aspect of the invention relates to i) a compound selected from AVZO-021 , PF- 07104091 , BLU-222, INX-315, ARTS-021 , AZD-8421 , NKT-3447, INCB-0123667, BLU-956, ETX- 197, AU14-5, a compound selected from any one of compounds 1-4, 6, 12-47, 49-98 and 101- 145 of W02023 / 069720 (which is incorporated by reference in its entirety), a compound selected from any one of the compounds of W02023 / 069700 and any one of the compounds of W02024 / 092039 and any one of the compounds of W02025 / 090727 (all of which are incorporated by reference in their entirety) for use in the treatment of a CCNE1 or CCNE2 amplified tumor.

[0187] EXAMPLES

[0188] Synthesis Example A - Synthesis PAT059642-WO-PCT

[0189] The synthesis described in PCT / IB2024 / 051412, the contents of which are incorporated by reference.

[0190] Intermediate 1 Experimental details of Intermediate 1

[0191] Step-1 : Synthesis of (1R,3R)-3-((2-chloro-5-iodopyrimidin-4-yl)amino)cyclohexan-1-ol: To a stirred solution of (1 R,3R)-3-aminocyclohexan-1-ol hydrochloride (60 g, 397.3 mmol) and DIPEA (183 mL, 993.25 mmol) in isopropanol (300 ml), was added 2,4-dichloro-5-iodopyrimidine (108 g, 397.3 mmol) at room temperature and stirred for 16h . The progress of the reaction was monitored by TLC and LCMS. After completion of the reaction, the solvent was removed under reduced pressure to give the crude product. The crude compound was purified by column chromatography by using silica gel (100-200 mesh), eluting with 0-40% ethyl acetate in pet-ether to give (1 R,3R)-3-((2-chloro-5-iodopyrimidin-4- yl)amino)cyclohexan-1-ol (113 g, 81 % yield, regio-isomeric mixture; 82% and 15% isomers) as pale yellow solid. LC-MS m / z [M+H]+ = 353.98,1H NMR (400 MHz, DMSO-de) 6 = 8.28-8.40 (m, 1 H), 6.58 (d, J = 8.4 Hz, 1 H), 4.50-4.51 (m, 1 H), 4.25-4.30 (m, 1 H), 3.90-4.20 (m, 1 H), 1.71-1.80 (m, 4H), 1.20-1.70 (m, 5H). PAT059642-WO-PCT

[0192] Step-2: Synthesis of (1R,3R)-3-((2-chloro-5-((trimethylsilyl)ethynyl)pyrimidin-4- yl)amino)cyclohexan-1-ol: To a degassed solution of (1R,3R)-3-((2-chloro-5-iodopyrimidin-4- yl)amino)cyclohexan-1-ol (56.5 g, 160.5 mmol; 82% and 15% isomers), Cui (3.04 g, 16.0 mmol), triethyl amine (115 mL, 800 mmol) and Pd(PPhs)4 (1 .84 g, 1 .60 mmol) in dry THF (560 mL), was added trimethylsilyl acetylene (22.7 mL, 160.5 mmol) and heated at 55 °C for 4h. The progress of the reaction was monitored by TLC & LCMS. After completion of the reaction, the reaction mass was filtered through plug of celite, the filtrate was concentrated under reduced pressure to give the crude product. The crude compound was purified by column chromatography using silica gel (100-200 mesh), eluting with 0-20% ethyl acetate in pet-ether to give (1 R,3R)-3-((2-chloro-5-((trimethylsilyl)ethynyl)pyrimidin-4- yl)amino)cyclohexan-1-ol (35 g, 68%) as pale yellow solid. LC-MS m / z [M+H]+ = 325.66,1H NMR (400 MHz, DMSO-de) 6 = 8.16 (s, 1 H), 6.48 (d, J = 8.0 Hz, 1 H), 4.55 (d, J = 2.80 Hz, 1 H), 4.25-4.40 (m, 1 H), 3.90-4.0 (m, 1 H), 1 .61-1 .85 (m, 4H), 1 .30-1 .60 (m, 4H), 0.26 (s, 9H).

[0193] Step-3: Synthesis of (1 R,3R)-3-(2-chloro-7H-pyrrolo[2,3-d] pyrimidin-7-yl) cyclohexan-1-ol: To a stirred solution of (1 R,3R)-3-((2-chloro-5-((trimethylsilyl)ethynyl)pyrimidin-4-yl)amino)cyclohexan-1-ol (44 g, 136.2 mmol) in acetonitrile (2,200 mL), was added CS2CO3 (22.1 g, 68.1 mmol) and heated at 75 °C for 6 h. Progress of the reaction was monitored by TLC and LCMS. After completion of the reaction, the reaction mass was filtered, the filtrate was concentrated under reduced pressure to give crude product. The crude compound was purified by column chromatography using silica gel (100-200 mesh), eluting with 0-50% ethyl acetate in pet-ether to give (1 R,3R)-3-(2-chloro-7H-pyrrolo[2,3-d] pyrimidin-7-yl) cyclohexan-1-ol (29 g, 85% yield) as pale yellow solid; LC-MS m / z [M+H]+ = 252.17,1H NMR (400 MHz, DMSO-de) 6 = 8.90 (s, 1 H), 7.82 (d, J = 3.6 Hz, 1 H), 6.70 (d, J = 3.6 Hz, 1 H), 4.75 (d, J = 2.8 Hz, 1 H), 4.15 (d, J = 2.4 Hz, 1 H), 2.01-2.11 (m, 1 H), 1.40-1.99 (m, 7H).

[0194] Step-4: Synthesis of 7-((1R,3R)-3-((tert-butyldimethylsilyl) oxy)cyclohexyl)-2-chloro-7H-pyrrolo[2,3- d]pyrimidine: To a stirred solution of (1 R,3R)-3-(2-chloro-7H-pyrrolo[2,3-d] pyrimidin-7-yl) cyclohexan-1- ol (20 g, 79.68 mmol) and imidazole (10.8 g, 159.3 mmol) in DMF (200 mL), was added TBDMS-CI (18 g, 159.3 mmol) at room temperature and stirred for 16h. Progress of the reaction was monitored by TLC and LCMS. After, 16h, the reaction mass was quenched with ice-cold water (50 mL) and extracted with ethyl acetate (3 x 100 mL). The combined organic layers were washed with ice-cold water (2 x 60 mL). The organic layer was dried over Na2SO4, concentrated under reduced pressure. The crude compound was purified by column chromatography using silica gel (100-200 mesh), eluting with 0-50% ethyl acetate in pet-ether to give 7-((1 R,3R)-3-((tert-butyldimethylsilyl) oxy)cyclohexyl)-2-chloro-7H-pyrrolo[2,3- d]pyrimidine (29 g, 96% yield) as brown gummy liquid. LC-MS m / z [M+H]+=366.24,1H NMR (400 MHz, PAT059642-WO-PCT

[0195] DMSO-de) 6 = 8.88 (s, 1 H), 7.79 (d, J = 3.6 Hz, 1 H), 6.68 (d, J = 3.6 Hz, 1 H), 4.95-5.10 (m, 1 H), 4.27 (s, 1 H), 1.96-2.05 (m, 1 H), 1.80-1.95 (m, 4H), 1.64 (d, J = 8.0 Hz, 2H), 1.45-1.55 (m, 1 H), 0.90-0.98 (m, 9H), 0.01-0.50 (m, 6H),

[0196] Step-5: Synthesis of 5,5-dibromo-7-((1R,3R)-3-((tert-butyldimethylsilyl) oxy)cyclohexyl)-2-chloro- 5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one: To a stirred solution of 7-((1 R,3R)-3-((tert- butyldimethylsilyl) oxy)cyclohexyl)-2-chloro-7H-pyrrolo[2,3-d]pyrimidine (50 g, 136.9 mmol) in f-BuOH: H2O (600 mL, 4:1), was added NBS (73 g, 410.9 mmol) at room temperature and stirred for 4h. Progress of the reaction was monitored by TLC & LCMS. After completion of the reaction. The reaction mass was quenched with water (100 mL), extracted with MTBE (3 x 300 mL). The organic layer was dried over Na2SO4, concentrated under reduced pressure to get the crude 5,5-dibromo-7-((1 R,3R)-3-((tert-butyldimethylsilyl) oxy)cyclohexyl)-2-chloro-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (73 g crude) as pale yellow semisolid. The crude product was used as such for next step without further purification. LC-MS m / z [M+H]+ = 540.21

[0197] Step-6: Synthesis of 7-((1 R,3R)-3-((tert-butyldimethylsilyl)oxy)cyclohexyl)-2-chloro-5,7-dihydro-6H- pyrrolo[2,3-d]pyrimidin-6-one: To a stirred solution of 5,5-dibromo-7-((1 R,3R)-3-((tert-butyldimethylsilyl) oxy)cyclohexyl)-2-chloro-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (73g crude, 135.4 mmol) in AcOH (730 mL), was added Zn dust (44 g, 677.1 mmol) at room temperature and stirred for 1 h. Progress of the reaction was monitored by TLC & LCMS. After completion of the reaction, the reaction mass was filtered through celite pad, the filtrate was diluted with ethyl acetate (500 mL) and washed with water (100 mL), the organic layer was dried over Na2SO4, concentrated under reduced pressure to get the crude product. The crude product was purified by using column chromatography (silica gel, 100-200 mesh), eluting with 0-30% ethyl acetate: pet ether to give 7-((1 R,3R)-3-((tert-butyldimethylsilyl)oxy)cyclohexyl)-2-chloro-5,7-dihydro- 6H-pyrrolo[2,3-d]pyrimidin-6-one (33 g, 63% yield after 2 steps) as brown gummy liquid. LC-MS m / z [M+H]+ = 382.29,1H NMR (400 MHz, DMSO-de) 6 = 8.25 (s, 1 H), 4.58-4.68 (m, 1 H), 4.26 (s, 1 H), 3.63 (s, 2H), 2.30-2.40 (m, 1 H), 2.10-2.21 (m, 1 H), 1.55-1.80 (m, 5H), 1.45-1.50 (m, 1 H), 0.91 (s, 9H), 0.01-0.10 (m, 6H).

[0198] Step-7: Synthesis of 7'-((1R,3R)-3-((tert-butyldimethylsilyl)oxy)cyclohexyl)-2'- chlorospiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one: To a stirred solution of 7-((1 R,3R)- 3-((tert-butyldimethylsilyl)oxy)cyclohexyl)-2-chloro-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (11g, 28.87 mmol) and dibromoethane (7.4 mL, 86.61 mmol) in dry DMF (110 mL), was added NaH (60%, 3.4 g, 86.61 mmol) portion wise at room temperature and stirred for 4h. Progress of the reaction was monitored by TLC & LCMS. After completion of the reaction, the reaction was quenched sat.NH4CI, extracted with PAT059642-WO-PCT

[0199] MTBE (3 x 100 mL). The organic layer was dried over Na2SO4, concentrated under reduced pressure to get the crude product. The crude product was purified by column chromatography (silica gel, 100-200 mesh), eluting with 0-15% ethyl acetate in pet ether to give 7'-((1 R,3R)-3-((tert- butyldimethylsilyl)oxy)cyclohexyl)-2'-chlorospiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one (10 g, 85% yield) as dark brown gummy liquid. LC-MS m / z [M+H]+= 408.31 ,1H NMR (400 MHz, DMSO-de) 6 = 8.16 (s, 1 H), 4.65-4.75 (m, 1 H), 4.26 (s, 1 H), 3.58-3.62 (m, 1 H), 2.30-2.42 (m, 1 H), 2.15-2.25 (m, 1 H), 1.81-1.91 (m, 2H), 1.55-1.80 (m, 7H), 1.55-1.80 (m, 7H), 0.91 (s, 9H), 1.39-1.49 (m, 1 H), 0.04 (s, 6H).

[0200] Synthesis of Compound A from Intermediate 1

[0201] Intermediate 1

[0202] 3-ethoxy-4-nitro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole: To a stirred solution of 3-ethoxy-4- nitro-1 H-pyrazole (40.00 g, 254.6 mmol) and cesium carbonate (124.4 g, 381.8 mmol) in N, N- Dimethylformamide (400 mL), was added SEM-CI (90.10 mL, 509.1 mmol) at 0° C and then stirred at ambient temperature for 4 hours. The progress of the reaction was monitored by TLC and LCMS. After completion of reaction, reaction mass was quenched with ice-cold water and extracted with ethyl acetate. The organic layer was washed with ice-cold water, dried over sodium sulphate and concentrated under reduced pressure to give the crude product. The crude compound was purified by Sepa-Bean using silica gel (230-400 mesh), eluting with 0-10% ethyl acetate in pet-ether to give 3-ethoxy-4-nitro-1-((2- (trimethylsilyl)ethoxy)methyl)-1 H-pyrazole (70 g, 95% yield) as pale yellow color liquid.1H NMR (400 MHz, CDCI3) 6 = 8.13 (s, 1 H), 5.24 (s, 2H), 4.41- 4.39 (m, 2H), 3.36-3.60 (m, 2H), 1.48-1.45 (t, J = 6.8 HZ, 3H), 0.96-0.91 (m, 2H), 0.03-0.01 (s, 9H). PAT059642-WO-PCT

[0203] Synthesis of 3-ethoxy-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-amine): To a parr-shaker containing 3-ethoxy-4-nitro-1-((2-(trimethylsilyl)ethoxy)methyl)-1 H-pyrazole (70.00 g, 243.6 mmol) in methanol (250 mL), was added Pd / C (10%, 12.96 g) and stirred at ambient temperature at 80 psi hydrogen pressure for 1 hour. Progress of the reaction was monitored by TLC. After completion of reaction, reaction mass was filtered through celite pad and washed with 10% MeOH in DCM, the filtrates were concentrated under reduced pressure to give the crude product compound-3 (56 g, 89% yield) as brown liquid.1H NMR (400 MHz, CDCI3) 6 = 6.99 (s, 1 H), 5.24 (s, 2H), 5.12 (s, 2H), 4.27-4.22 (m, 2H), 3.52-3.47 (m, 2H), 2.73- 2.71 (s, 2H), 1.40-1.36 (m, 3H), 0.93-0.86 (m, 2H), 0.02-0.01 (s, 9H), LC-MS (m / z): 258.49 [M+H] +.

[0204] 7'-((1 R,3R)-3-((tert-butyldimethylsilyl) oxy) cyclohexyl)-2'-((3-ethoxy-1-((2-(trimethylsilyl) ethoxy) methyl)-1 H-pyrazol-4-yl) amino) spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d] pyrimidin]-6'(7'H)-one : To a degassed solution of 7'-((1 R,3R)-3-((tert-butyldimethylsilyl)oxy)cyclohexyl)-2'-chlorospiro[cyclopropane- 1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one (40.00 g, 98.04 mmol) and compound-3 (30.28 g, 117.6 mmol) in dry tetrahydrofuran (400 mL), was added sodium tert-butoxide (14.13 g, 147.1 mmol) and BrettphosPdG3 (4.44 g, 4.90 mmol), the reaction mass was closed under argon atmosphere and stirred at ambient temperature for 4 hours. Progress of the reaction was monitored by TLC and LCMS. After completion of the reaction, the reaction mass was filtered; the filtrate was concentrated under reduced pressure to give crude compound. The crude compound was purified by Sepa-Bean using silica gel (230-400 mesh), eluting with 20-50% ethyl acetate in pet-ether to give 7'-((1 R,3R)-3-((tert-butyldimethylsilyl) oxy) cyclohexyl)-2'-((3- ethoxy-1-((2-(trimethylsilyl) ethoxy) methyl)-1 H-pyrazol-4-yl) amino) spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d] pyrimidin]-6'(7'H)-one. (43 g, 66% yield) as brown gummy solid. 1 H NMR (400 MHz, CDCI3) 6 = 7.96 (s, 1 H), 7.63 (s, 1 H), 6.61 (s, 1 H), 5.34 (s, 2H), 4.88-4.81 (m, 1 H), 4.35-4.30 (m, 2H) ,4.26 (s, 1 H), 3.57-3.53 (m, 2H), 256 (br-s, 1 H), 2.39-2.30 (m, 1 H), 1.94-1 ,87(m, 1 H), 1.73-1.65 (m, 6H), 1.49-1.40 (m, 6H),1.27- 1.23 (m, 1 H), 0.95 (s, 9H), 0.57 (s, 6H), 0.20 (s, 9H), LC-MS (m / z): 629.78 [M+H] +

[0205] 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3-hydroxycyclohexyl)spiro[cyclopropane-1 ,5'- pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one To a stirred solution of 7'-((1 R,3R)-3-((tert-butyldimethylsilyl) oxy) cyclohexyl)-2'-((3-ethoxy-1-((2-(trimethylsilyl) ethoxy) methyl)-1 H-pyrazol-4-yl) amino) spiro[cyclopropane- 1 ,5'-pyrrolo[2,3-d] pyrimidin]-6'(7'H)-one (41.00 g, 65.19 mmol) in methanol (410 mL), was added 4M HCI in dioxane (820 mL) at 0 °C and stirred at 70 °C for 4 hours. Progress of the reaction was monitored by TLC & LCMS. After completion of reaction, reaction mass was diluted with ethyl acetate and concentrated under reduced pressure. The crude product was basified with aq. sodium bicarbonate and extracted with ethyl acetate. The organic layer was dried over sodium sulfate, concentrated under reduced pressure to get the crude compound. The crude product was triturated in ethyl acetate (10V) and stirred for 30 minutes and then filtered. The filtered solid was triturated in methanol (10V) and stirred for 1 hour, then filtered the filtered solid to give pure compound. Then compound co-distilled with acetonitrile and water to give 2'-((3- ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3-hydroxycyclohexyl)spiro[cyclopro pane-1 ,5'-pyrrolo[2,3- d]pyrimidin]-6'(7'H)-one (17.5 g, 68% yield) as pale pink solid. 1 H NMR (400 MHz, DMSO-d6) 6 = 11.61 PAT059642-WO-PCT

[0206] (s, 1 H), 8.10 (s, 1 H), 7.78 (s, 1 H), 7.63 (s, 1 H), 4.65-4.59 (m, 1 H), 4.52 (s, 1 H), 4.17-4.12 (m, 2H), 4.06 (s, 1 H), 2.49 (s, 1 H), 2.24-2.20 (m, 1 H), 1 .75-1 .46 (m, 9H), 1 .45-1 .28 (m, 4H), LC-MS (m / z):385.23 [M+H] +.

[0207] Example B: CDK Biochemical Assay

[0208] Biochemical assays measured the inhibitory effects of compounds in this disclosure on the enzymatic activity of CDK enzyme in complex with Cyclin protein partner by phosphorylation of Ser-780 (S780) on retinoblastoma protein peptide (RB1) in the presence of 1 mM adenosime-5 — triphosphate (ATP) and varying concentration of test compound in 20 mM 2-[4-(2- hydroxyethyl)paperazin-1-yl] ethanesulfonic acid (HEPES), pH 7.5, 10 mM MgCI2, 1 mM dithiothreitol (DTT), 0.01 % bovine serum albumin (BSA), 0.005% Tween 20. Total Reaction volume of 10 pL proceeded for 60 minutes at room temperature (25°C) and were quenched with 5 pL of 200 mM 2',2",2"'-(Ethane-1 ,2-diyldinitrilo)tetraacetic acid (EDTA) pH 8.0 before addition of 5 pL detection solution containing 100 nM fluorophore conjugate streptavidin allophycocyanin (SA-APC), 2 nM Europium labelled Anti-p-RB(S780)-K (Perkin Elmer, 64CUSKAY), 50 mM HEPES, pH 7.5, 400 mM potassium fluoride (KF), 0.1 % BSA, and 0.01 % Tween-20. Phosphorylation of S780 on RB1 peptide (His-MBP-RB1 [773-924] - SEQ ID NO. 9) was detected by TR-FRET after 3 hour incubation with detection solution. Percent phosphorylation activity was plotted against log concentration of compound to generate an apparent IC5o. The following CDK enzyme in complex with different cyclin proteins and protein peptide substrate were used in these assays:

[0209] CDK1 / CyclinB1 , Avi-tag, 10 pM used in the assay

[0210] CDK2 / CyclinE1 , Avi-tag, 100 pM used in the assay

[0211] CDK4 / cyclinD1 , Avi-tag, 20 pM used in the assay CDK6 / cyclinD3, Avi-tag, 10 pM used in the assay His-MBP-RB1 [773-924], Avi-tag, 200 nM used in the assay

[0212] The avi-tag is a C-terminal fusion on the CDK1 / 2 / 4 / 6 sequence in each of these two-protein complexes. The known avi-tag sequence, GLNDIFEAQKIEWHE (SEQ ID NO. 10), is a substrate for E. coli biotin ligase BirA, which covalently attaches a biotin molecule to the e-amino group of the lysine in that sequence. The avi-tag is attached with an 8 amino-acid linker sequence GGSGGGGS (SEQ ID NO. 11), resulting in the full C-terminal fusion GGSGGGGSGLNDIFEAQKIEWHE (SEQ ID NO. 12).

[0213] The sequences of the recombinant proteins used here are provided below. PAT059642-WO-PCT

[0214] Recombinant proteins were generated as N-terminal fusions of 9x His tag - E. coli Maltose Binding Protein (MBP), with or without a C-terminal Avi-tag.1CDK2 and His-MBP-RB1 [773-924]- c-avi were expressed in E. coli while the remainder were expressed in Sf21 insect cells. Genes were synthesized commercially (GeneArt and Twist Bioscience) using codon frequencies appropriate to the respective organisms and proprietary codon optimization strategies, and inserted into pET 24 (E. coli) or pDEST8 (insect) vectors already containing the 9x His-MBP tag using standard methods of PCR and Gibson Assembly.2E. coli expression was performed by auto-induction overnight at 18 or 21 C. For insect cell expression, bacmids and viruses were created,3then used to synchronously infect Sf21 insect cells with harvest at 40-48 hrs., per standard protocols.4

[0215] For CDK2 / Cyclin E and CDK1 / Cyclin B, the CDK and Cyclin proteins were expressed separately, lysed by sonication and clarified by centrifugation. The fusion proteins were each separately purified by immobilized metal ion affinity chromatography (IMAC) (5 mL HisTrap, Cytiva) and Superdex 200 26 / 60 (Cytiva) size-exclusion and complexed in vitro (1-3 hrs incubation at room temperature) prior to tag cleavage by recombinant his-tagged Tobacco Etch Virus protease or his- tagged Human rhinovirus 3C protease, as appropriate. The cleaved complex is further purified by Superdex 75 26 / 60 (Cytiva) size-exclusion followed by reverse-IMAC, in which the complex is in the column flow-through and residual His-MBP and his-tagged proteases are retained. The final complex is concentrated as desired by centrifugal concentration (Amicon Ultra 10,000 Da MWCO 15 mL concentrators, Millipore).

[0216] For E. coli expressed CDK2 and His-MBP-RB1 [773-924]-c-avi, the proteins were biotinylated in vitro after the first IMAC step.1

[0217] For CDK4 / Cyclin D1 and CDK6 / Cyclin D3, the complexes were expressed by simultaneous coinfection of insect cells by three separate viruses including one expressing untagged E. coli BirA. The expression media is supplemented with 50 uM d-biotin. The resulting protein complexes purified similarly. After lysis and clarification, the complex is purified by IMAC, Superdex 20026 / 60 size exclusion, tag cleavage by human rhinovirus 3C protease, Superdex 75 26 / 60 size exclusion and reverse IMAC. BirA is typically not retained on the first IMAC, and excess CDK or Cyclin monomer is separated on the Superdex 200 size exclusion. Cleaved His-MBP and remaining monomer are removed by the Superdex 75 size exclusion and the reverse IMAC step. Final concentration by centrifugal concentrators as described above. PAT059642-WO-PCT

[0218] For the is-MBP-RB1 [773-924]-c-avi, the protein was lysed by sonication and clarified by centrifugation. After IMAC capture and in vitro biotinylation, the fusion protein is purified by Q HP HiTrap (Cytiva) with a gradient elution after binding in low salt, followed by SP HP HiTrap (Cytiva) with a gradient elution. The material is finally purified on a Superdex 75 26 / 60 as a polishing step. Although there is a TEV protease cleavage site in this construct, it is not removed for this purpose. Final concentration by centrifugal concentrators as described above.

[0219] 9x His-MBP-3C-CDK2-c-avi (SEQ ID NO. 1)

[0220] MHHHHHHHHHMKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAAT GDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNK DLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGV DNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVL PTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELA KDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSGLEVLF QGPMENFQKVEKIGEGTYGWYKARNKLTGEWALKKIRLDTETEGVPSTAIREISLLKELNHPNI VKLLDVIHTENKLYLVFEFLHQDLKKFMDASALTGIPLPLIKSYLFQLLQGLAFCHSHRVLHRDLK PQNLLINTEGAIKLADFGLARAFGVPVRTYTHEWTLWYRAPEILLGCKYYSTAVDIWSLGCIFAE MVTRRALFPGDSEIDQLFRIFRTLGTPDEWWPGVTSMPDYKPSFPKWARQDFSKVVPPLDED GRSLLSQMLHYDPNKRISAKAALAHPFFQDVTKPVPHLRLGGSGGGGSGLNDIFEAQKIEWHE

[0221] 9x His-MBP-TEV-Cyclin E1 (SEQ ID NO. 2)

[0222] MHHHHHHHHHMKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAAT GDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNK DLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGV DNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVL PTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELA KDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSENLYFQ GGSGMPRERRERDAKERDTMKEDGGAEFSARSRKRKANVTVFLQDPDEEMAKIDRTARDQC GSQPWDNNAVCADPCSLIPTPDKEDDDRVYPNSTCKPRIIAPSRGSPLPVLSWANREEVWKIM LNKEKTYLRDQHFLEQHPLLQPKMRAILLDWLMEVCEVYKLHRETFYLAQDFFDRYMATQENV VKTLLQLIGISSLFIAAKLEEIYPPKLHQFAYVTDGACSGDEILTMELMIMKALKWRLSPLTIVSWL NVYMQVAYLNDLHEVLLPQYPQQIFIQIAELLDLCVLDVDCLEFPYGILAASALYHFSSSELMQKV SGYQWCDIENCVKWMVPFAMVIRETGSSKLKHFRGVADEDAHNIQTHRDSLDLLDKARAKKA MLSEQNRASPLPSGLLTPPQSGKKQSSGPEMA*

[0223] 9x His-MBP-TEV-CDK1-c-avi (SEQ ID NO. 3)

[0224] MHHHHHHHHHMKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAAT GDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNK DLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGV DNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVL PTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELA KDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSENLYFQ GGMEDYTKIEKIGEGTYGWYKGRHKTTGQWAMKKIRLESEEEGVPSTAIREISLLKELRHPNI PAT059642-WO-PCT

[0225] VSLQDVLMQDSRLYLIFEFLSMDLKKYLDSIPPGQYMDSSLVKSYLYQILQGIVFCHSRRVLHRD LKPQNLLIDDKGTIKLADFGLARAFGIPIRVYTHEWTLWYRSPEVLLGSARYSTPVDIWSIGTIFA ELATKKPLFHGDSEIDQLFRIFRALGTPNNEVWPEVESLQDYKNTFPKWKPGSLASHVKNLDEN GLDLLSKMLIYDPAKRISGKMALNHPYFNDLDNQIKKMGGSGGGGSGLNDIFEAQKIEWHE*

[0226] 9x His-MBP-Cyclin B1 (SEQ ID NO. 4)

[0227] MHHHHHHHHHMKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAAT GDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNK DLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGV DNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVL PTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELA KDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSENLYFQ GGMALRVTRNSKINAENKAKINMAGAKRVPTAPAATSKPGLRPRTALGDIGNKVSEQLQAKMP MKKEAKPSATGKVIDKKLPKPLEKVPMLVPVPVSEPVPEPEPEPEPEPVKEEKLSPEPILVDTAS PSPMETSGCAPAEEDLCQAFSDVILAVNDVDAEDGADPNLCSEYVKDIYAYLRQLEEEQAVRPK YLLGREVTGNMRAILIDWLVQVQMKFRLLQETMYMTVSIIDRFMQNNCVPKKMLQLVGVTAMFI ASKYEEMYPPEIGDFAFVTDNTYTKHQIRQMEMKILRALNFGLGRPLPLHFLRRASKIGEVDVE QHTLAKYLMELTMLDYDMVHFPPSQIAAGAFCLALKILDNGEWTPTLQHYLSYTEESLLPVMQH LAKNWMVNQGLTKHMTVKNKYATSKHAKISTLPQLNSALVQDLAKAVAKV*

[0228] 9x His-MBP-CDK4-c-avi (SEQ ID NO. 5)

[0229] MHHHHHHHHHMKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAAT GDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNK DLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGV DNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVL PTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELA KDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSENLYFQ GGSGMATSRYEPVAEIGVGAYGTVYKARDPHSGHFVALKSVRVPNGGGGGGGLPISTVREVAL LRRLEAFEHPNWRLMDVCATSRTDREIKVTLVFEHVDQDLRTYLDKAPPPGLPAETIKDLMRQ FLRGLDFLHANCIVHRDLKPENILVTSGGTVKLADFGLARIYSYQMALTPWVTLWYRAPEVLLQ STYATPVDMWSVGCIFAEMFRRKPLFCGNSEADQLGKIFDLIGLPPEDDWPRDVSLPRGAFPP RGPRPVQSVVPEMEESGAQLLLEMLTFNPHKRISAFRALQHSYLHKDEGNPEGGSGGGGSGL NDIFEAQKIEWHE*

[0230] 9x His-MBP-Cyclin D1 (SEQ ID NO. 6)

[0231] MHHHHHHHHHMKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAAT GDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNK DLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGV DNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVL PTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELA KDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSENLYFQ GGSGMEHQLLCCEVETIRRAYPDANLLNDRVLRAMLKAEETCAPSVSYFKCVQKEVLPSMRKI VATWMLEVCEEQKCEEEVFPLAMNYLDRFLSLEPVKKSRLQLLGATCMFVASKMKETIPLTAEK LCIYTDNSIRPEELLQMELLLVNKLKWNLAAMTPHDFIEHFLSKMPEAEENKQIIRKHAQTFVALC ATDVKFISNPPSMVAAGSWAAVQGLNLRSPNNFLSYYRLTRFLSRVIKCDPDCLRACQEQIEAL LESSLRQAQQNMDPKAAEEEEEEEEEVDLACTPTDVRDVDI* PAT059642-WO-PCT

[0232] 9x His-MBP-CDK6-c-avi (SEQ ID NO. 7)

[0233] MHHHHHHHHHMKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAAT GDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNK DLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGV DNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVL PTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELA KDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSMMGSS SSGLEVLFQGPMEKDGLCRADQQYECVAEIGEGAYGKVFKARDLKNGGRFVALKRVRVQTGE EGMPLSTIREVAVLRHLETFEHPNWRLFDVCTVSRTDRETKLTLVFEHVDQDLTTYLDKVPEPG VPTETIKDMMFQLLRGLDFLHSHRWHRDLKPQNILVTSSGQIKLADFGLARIYSFQMALTSWV TLWYRAPEVLLQSSYATPVDLWSVGCIFAEMFRRKPLFRGSSDVDQLGKILDVIGLPGEEDWPR DVALPRQAFHSKSAQPIEKFVTDIDELGKDLLLKCLTFNPAKRISAYSALSHPYFQDLERCKENLD SHLPPSQNTSELNTAGGSGGGGSGLNDIFEAQKIEWHE*

[0234] 9x His-MBP-Cyclin D3 (SEQ ID NO. 8)

[0235] MHHHHHHHHHMKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAAT GDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNK DLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGV DNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVL PTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELA KDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSLEVLQG PGSMELLCCEGTRHAPRAGPDPRLLGDQRVLQSLLRLEERYVPRASYFQCVQREIKPHMRKM LAYWM LE VC E EQ RC E E E VF P LAM N Y L D RY LSCVPT R KAQ LQ L LG AVC M L LAS KL R ETT P LT I E K LCIYTDHAVSPRQLRDWEVLVLGKLKWDLAAVIAHDFLAFILHRLSLPRDRQALVKKHAQTFLAL CATDYTFAMYPPSMIATGSIGAAVQGLGACSMSGDELTELLAGITGTEVDCLRACQEQIEAALRE SLREASQTSSSPAPKAPRGSSSQGPSQTSTPTDVTAIHL*

[0236] 9x His-MBP- RB1 [773-924]-c-avi (SEQ ID NO. 9)

[0237] MHHHHHHHHHMKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAAT GDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNK DLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGV DNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVL PTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELA KDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSENLYFQ GGSTRPPTLSPIPHIPRSPYKFPSSPLRIPGGNIYISPLKSPYKISEGLPTPTKMTPRSRILVSIGES FGTSEKFQKINQMVCNSDRVLKRSAEGSNPPKPLKKLRFDIEGSDEADGSKHLPGESKFQQKL AEMTSTRTRMQKQKMNDSMDTSNGGSGGGGSGLNDIFEAQKIEWHE*

[0238] 1 . Li, Y. & Sousa, R. Expression and purification of E. coli BirA biotin ligase for in vitro biotinylation. Protein Expr. Purif. 82, 162-7 (2012).

[0239] 2. Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343-345 (2009).

[0240] 3. Invitrogen. Bac-to-Bac® Baculovirus Expression System. An Efficient Site-Specific Transposition System to Generate Baculovirus for High-Level Expression of Recombinant PAT059642-WO-PCT

[0241] Proteins. (2010).

[0242] 4. Invitrogen life technologies. Guide to Baculovirus Expression Vector Systems (BEVS) and Insect Cell Culture Techniques. (2002). IC5o values determined in the biochemical assays are listed in the following table: PAT059642-WO-PCT

[0243] Example 1

[0244] MCF7 [HR+ (ER+), HER2-] breast cancer xenograft model is used in a pharmacology efficacy study in mice. The control group (vehicle) displays a fast tumor growth and mean tumor sizes reach approximately 1600 mm3after 24 days. While Compound A does not show single agent activity, a dose-dependent combination effect on tumor growth inhibition is observed when Compound A is combined with LEE011 (ribociclib). In combination with LEE011 (ribociclib), Compound A at 3 and 7.5 mg / kg (BID) results in a significant efficacy improvement compared to LEE011 (ribociclib) as a single agent (change in tumor volume treated over ribociclib T / C = 40% and -2%, respectively). See Figure 1. As shown in Figure 2 the depth of PD modulation correlates with efficacy.

[0245] Example 2

[0246] MCF7 [HR+ (ER+), HER2-] breast cancer xenograft model is used in a pharmacology PK / PD study in mice. Compared to the untreated controls, Compound A does not result in significant PD PAT059642-WO-PCT modulation (pRB inhibition) as a monotherapy. However, combining Compound A with LEE011 (ribociclib) results in a dose-dependent improvement in PD modulation compared to either monotherapy. See Figure 2.

[0247] MCF7 [HR+ (ER+), HER2-] breast cancer xenograft model is used in a pharmacology efficacy study in mice. The control group (vehicle) displays a fast tumor growth and mean tumor sizes reach approximately 1000 mm3after 24 days. Combining Compound A at 3 mg / kg or 7.5 mg / kg (BID) with LEE011 (ribociclib) and endocrine therapy (fulvestrant) at clinically relevant dosing levels resulted in an efficacy benefit compared to the standard of care (ribociclib + endocrine therapy) alone. See Figure 3.

[0248] Example 4

[0249] ST3164B / PBR [HR+ (ER+), HER2-] breast cancer patient derived xenograft (PDX) model is used in a pharmacology efficacy study in mice. The control group (vehicle) displays a fast tumor growth and tumor volume change is approximately 500% at 22 days after enrollment into the study. Of note, this preclinical model is not responding to the standard of care (ribociclib + endocrine therapy). Combining Compound A at 3 mg / kg (BID) with LEE011 (ribociclib) and endocrine therapy (fulvestrant) at clinically relevant dosing levels restores efficacy. See Figure 4.

[0250] Example 5

[0251] ST1337B [HR+ (ER+), HER2-] breast cancer patient derived xenograft (PDX) model is used in a pharmacology efficacy study in mice. The control group (vehicle) displays a fast tumor growth and tumor volume change is approximately 500% at 22 days after enrollment into the study. Of note, this preclinical model is not responding to the standard of care (ribociclib + endocrine therapy). Combining Compound A at 3 mg / kg (BID) with LEE011 (ribociclib) and endocrine therapy (fulvestrant) at clinically relevant dosing levels restores efficacy. See Figure 5.

[0252] Examples 6 and 7

[0253] In vitro viability of the (ER+ HER2-) MCF7 breast cancer cell line was assessed using the CellTiterGlo assay following 7-day treatment with Compound A and ribociclib. As shown in Figure 6, the Loewe’s excess plot indicates Compound A can combine with ribociclib to enhance antiproliferative activity in MCF7 breast cancer cells. The Loewe synergy score in this cell line is 12.2 (a score of greater than 4 is indicative of synergy). PAT059642-WO-PCT

[0254] As shown in Figure 7, such synergy is also observed in (ER+ HER2-) ST941 C cells which harbor an ESR1 (estrogen receptor) Y537S mutation who have developed acquired resistance to standard of care therapy (CDK4 / 6 inhibitor + SERD, e.g. ribociclib + fulvestrant). This indicates that the combination of Compound A and ribociclib may also be efficacious in a resistance setting. The Loewe synergy score in this cell line is 8.93 (a score of greater than 4 is indicative of synergy).

[0255] In vitro viability of the (ER+ HER2-) MCF7 breast cancer cell line was assessed using the CellTiterGlo assay following 7-day treatment with Compound A and PF-07220060. As shown in Figure 8, the Loewe’s excess plot indicates Compound A can combine with PF-07220060 to enhance anti-proliferative activity in MCF7 breast cancer cells. The Loewe synergy score in this cell line is 11.7 (a score of greater than 4 is indicative of synergy).

[0256] In vitro viability of the (ER+ HER2-) MCF7 breast cancer cell line was assessed using the CellTiterGlo assay following 7-day treatment with i) as shown in Figure 9, Compound A and the selective estrogen receptor degrader (SERD) Elacestrant, and ii) as shown in Figure 10, Compound A and the selective estrogen receptor degrader (SERD) ARV-471 (also known as Vepdegestrant). The Loewe synergy scores for i) and ii) were calculated as 5.74 and 5.6 respectively, indicating that the combinations are slightly synergistic (a score of greater than 4 is indicative of synergy).

[0257] CCNE1 amplified OVCAR3 cells (10x106cells / 100 pl) were injected subcutaneously (s.c.) in the flank of immunodeficient NSG mice. After 36 days, mice were randomized into three groups and dosed (p.o.) with 15 and 30 mg / kg Compound A (BID), formulated in 20% HPpCD (Hydroxypropyl- P-cyclodextrin) at pH 4.7 at 10ml / kg. On day 3, tumors and parallel plasma samples were collected at several time points after treatment and analyzed for PD and PK, respectively. Tumors were snap-frozen and analyzed for PD response using an internally developed MSD assay for pRB read-out (RD-2024-00198). As shown in Figure 11 , treatment at 15 and 30 mg / kg (BID) showed a dose-dependent pRB inhibition with the time and level of Compound A exposure above the in vitro pRB AC50 value in OVCAR3 correlating with the depth and extent of pRB inhibition. PAT059642-WO-PCT

[0258] CCNE1 amplified 0VCAR3 cells (10x106cells / 100 pl) were injected subcutaneously (s.c.) in the flank of immunodeficient NSG mice. After 31 days, mice were randomized into three groups and dosed (p.o.) with 15 and 30 mg / kg (BID) CompoundA, formulated in 10% HPpCD (Hydroxypropyl- P-cyclodextrin) at pH 4.7 at 10 ml / kg. As shown in Figure 12, a dose-dependent effect on tumor growth inhibition was observed, with Compound A at 30 mg / kg (BID) resulting in near-stasis (change in tumor volume treated over control T / C = 33%). The time over target correlates with efficacy. Treatment with Compound A was well tolerated, and all mice had body weight gain.

[0259] Example 13

[0260] In vitro viability of the following CCNE1 amplified cell lines was assessed using the CyQuant assay following 7-day treatment, with the following results:

Claims

PAT059642-WO-PCTCLAIMS1 . A method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of: i) a compound of formula (I), or a salt and / or tautomer thereof:ii) a CDK4 inhibitor, and / or iii) an endocrine therapeutical agent.

2. A compound of formula (I), or a salt and / or tautomer thereof:use in the treatment of cancer, wherein the treatment further comprises administration of: a CDK4 inhibitor; and / or an endocrine therapeutical agent.

3. A CDK4 inhibitor for use in the treatment of cancer, wherein the treatment further comprises administration of: a compound of formula (I), or a salt and / or tautomer thereof:PAT059642-WO-PCTii) optionally an endocrine therapeutical agent.

4. An endocrine therapeutical agent for use in the treatment of cancer, wherein the treatment further comprises administration of: i) a compound of formula (I), or a salt and / or tautomer thereof:optionally, a CDK4 inhibitor.A combination comprising: i) a compound of formula (I), or a salt and / or tautomer thereof:ii) a CDK4 inhibitor; and / or iii) an endocrine therapeutical agent.PAT059642-WO-PCT6. The method according to claim 1 , the compound for use according claim 2, the CDK4 inhibitor for use according to claim 3, the endocrine therapeutical agent for use according to claim 4, or the combination according to claim 5, wherein the CDK4 inhibitor is: i) a mono CDK4 inhibitor; ii) a dual CDK2 / 4 inhibitor; iii) a dual CDK4 / 6 inhibitor; or iv) a triple CDK2 / 4 / 6 inhibitor.

7. The method according to claim 6, the compound for use according to claim 6, the CDK4 inhibitor for use according to claim 6, the endocrine therapeutical agent for use according to claim 6, or the combination according to claim 6, wherein the CDK4 inhibitor is a dual CDK4 / 6 inhibitor or a mono CDK4 inhibitor.

8. The method according to claim 1 or claim 6, the compound for use according to claim 2 or claim 6, the CDK4 inhibitor for use according to claim 3 or claim 6, the endocrine therapeutical agent for use according to claim 4 or claim 6, or the combination according to claim 5 or claim 6, wherein the CDK4 inhibitor is selected from PF-07220060, dalpiciclib, trilaciclib, palbociclib, abemaciclib and ribociclib.

9. The method according to claim 8, the compound for use according to claim 8, the CDK4 inhibitor for use according to claim 8, the endocrine therapeutical agent for use according to claim 8, or the combination according to claim 8, wherein the CDK4 inhibitor is ribociclib or PF-07220060.

10. The method according to claim 9, the compound for use according to claim 9, the CDK4 inhibitor for use according to claim 9, the endocrine therapeutical agent for usePAT059642-WO-PCT according to claim 9, or the combination according to claim 9, wherein the CDK4 inhibitor is ribociclib.11 . The method according to claim 9, the compound for use according to claim 9, the CDK4 inhibitor for use according to claim 9, the endocrine therapeutical agent for use according to claim 9, or the combination according to claim 9, wherein the CDK4 inhibitor is PF-07220060.

12. The method according to any one of claims 1 and 6 to 11 , the compound for use according to any one of claims 2 and 6 to 11 , the CDK4 inhibitor for use according to any one of claims 3 and 6 to 11 , the endocrine therapeutical agent for use according to any one of claims 4 and 6 to 11 , or the combination according to any one of claims 5 to11 , wherein the endocrine therapeutical agent is selected from at least one of the following: a) an aromatase inhibitor; b) a selective estrogen receptor degrader (SERD); c) a selective receptor modulator (SERM); and d) a gonadotropin-releasing hormone (GnRH) inhibitor.

13. The method according to any one of claims 1 and 6 to 12, the compound for use according to any one of claims 2 and 6 to 12, the CDK4 inhibitor for use according to any one of claims 3 and 6 to 12, the endocrine therapeutical agent for use according to any one of claims 4 and 6 to 12, or the combination according to any one of claims 5 to12, wherein the endocrine therapeutical agent is selected from anastrozole, letrozole, exemestane, ARV-471 , Fulvestrant, elacestrant, tamoxifen, goserelin and leuprolide.PAT059642-WO-PCT14. The method according to claim 13, the compound for use according to claim 13, the CDK4 inhibitor for use according to claim 13, the selective estrogen receptor degrader for use according to claim 13, or the combination according to claim 13, wherein the endocrine therapeutical agent is selected from ARV-471 , Fulvestrant and elacestrant.

15. The method according to claim 14, the compound for use according to claim 14, the CDK4 inhibitor for use according to claim 14, the selective estrogen receptor degrader for use according to claim 14, or the combination according to claim 14, wherein the endocrine therapeutical agent is Fulvestrant.

16. The method according to any one of claims 1 and 6 to 15, the compound for use according to any one of claims 2 and 6 to 15, the CDK4 inhibitor for use according to any one of claims 3 and 6 to 15, the endocrine therapeutical agent for use according to any one of claims 4 and 6 to 15, wherein the cancer is selected from ovarian cancer, gastroesophageal cancer, uterine cancer, breast cancer (e.g. HR+ breast cancer, e.g. ER+ breast cancer, e.g. HER2- breast cancer e.g. HR+ / HER2- breast cancer, e.g. ER+ / HER2- breast cancer), prostate cancer, lung cancer.

17. The method according to claim 15, the compound for use according to claim 15, the CDK4 inhibitor for use according to claim 15 or the endocrine therapeutical agent for use according to claim 15, wherein the cancer is breast cancer.

18. The method according to claim 17, the compound for use according to claim 17, the CDK4 inhibitor for use according to claim 17 or the endocrine therapeutical agent for use according to claim 17, wherein the cancer is HR+ breast cancer.PAT059642-WO-PCT19. The method according to claim 17 or claim 18, the compound for use according to claim 17 or claim 18, the CDK4 inhibitor for use according to claim 17 or claim 18 or the endocrine therapeutical agent for use according to claim 17 or claim 18, wherein the cancer is ER+ breast cancer.

20. The method according to any one of claims 17 to 19, the compound for use according to any one of claims 17 to 19, the CDK4 inhibitor for use according to any one of claims 17 to 19 or the endocrine therapeutical agent for use according to any one of claims 17 to 19, wherein the cancer is HER2- breast cancer.21 . The method according to any one of claims 17 to 20, the compound for use according to any one of claims 17 to 20, the CDK4 inhibitor for use according to any one of claims 17 to 20 or the endocrine therapeutical agent for use according to any one of claims 17 to 20, wherein the cancer is HR+ / HER2- breast cancer.

22. The method according to any one of claims 17 to 21 , the compound for use according to any one of claims 17 to 21 , the CDK4 inhibitor for use according to any one of claims 17 to 21 or the endocrine therapeutical agent for use according to any one of claims 17 to 21 , wherein the cancer is ER+ / HER2- breast cancer.

23. The method according to any one of claims 1 and 6 to 22, the compound for use according to any one of claims 2 and 6 to 22, the CDK4 inhibitor for use according to any one of claims 3 and 6 to 22 or the endocrine therapeutical agent for use according to any one of claims 4 and 6 to 22, wherein the cancer is CCNE1 or CCNE2 amplified.

24. A compound of formula (I), or a salt and / or tautomer thereof:PAT059642-WO-PCTuse in the treatment of a CCNE1 or CCNE2 amplified tumor.

25. A method of treating a CCNE1 or CCNE2 amplified tumor, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I), or a salt and / or tautomer thereof:

26. The compound for use according to claim 24, or the method according to claim 25, wherein the CCNE1 or CCNE2 amplified tumor is a CCNE1 amplified tumor.

27. The compound for use according to claim 25 or claim 26, or the method according to claim 25 or claim 26 wherein the CCNE1 or CCNE2 amplified tumor is selected from ovarian cancer, cholangiocarinoma, breast cancer, bladder cancer, prostate cancer, a sarcoma, colorectal cancer, cervical cancer, endometrial cancer, gastric cancer (e.g. gastroesophageal adenocarcinoma), lung cancer, pancreatic cancer and fallopian tube carcinoma.

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