Combination of cbp / p300 bromodomain inhibitors and kras inhibitors for the treatment of cancer

Abstract

The present invention particularly relates to (i) a combination of a CBP / p300 bromodomain inhibitor and a KRAS inhibitor for treating a patient with cancer, wherein the cancer exhibits carcinogenic alterations in KRAS; (ii) a kit comprising (a) a pharmaceutical formulation containing a CBP / p300 bromodomain inhibitor and (b) a pharmaceutical formulation containing a KRAS inhibitor; and (iii) a pharmaceutical formulation comprising a CBP / p300 bromodomain inhibitor and a KRAS inhibitor.

Description

Invention Field

[0001] This invention belongs to the field of cancer treatment. Therefore, this invention relates to a combination of a CBP / p300 bromodomain inhibitor and a KRAS inhibitor for treating patients with cancer exhibiting carcinogenic alterations in KRAS. This invention also relates to kits comprising pharmaceutical dosage forms containing a CBP / p300 bromodomain inhibitor and pharmaceutical dosage forms containing a KRAS inhibitor. Furthermore, this invention relates to pharmaceutical dosage forms containing both a CBP / p300 bromodomain inhibitor and a KRAS inhibitor. Background of the Invention

[0003] KRAS mutations are present in up to 25% of cancers, with oncogenic variants exhibiting varying prevalences across different cancers (see Mullard's Column (Box) 1, Nature Reviews DRUG DISCOVERY, Vol. 18, Dec. 2019: 887-891). Thus, the frequency of KRAS mutations in human cancers is 90% in the pancreas, 30-50% in the colon, 35% in the small intestine, 26% in the biliary tract, 17% in the endometrium, 19% in the lungs, 1% in the skin (melanoma), 8% in the cervix, and 5% in the urinary tract (see Li et al., Nature Reviews Cancer 18, 767-777 (2018), Table 1). Therefore, there is considerable interest in agents that block proliferation signaling induced by oncogenic KRAS variants.

[0004] Although KRAS has been considered an untreatable target for decades, at least five KRAS modulators are now available in clinical practice (see Mullard, ibid., Table 1 above). While preliminary clinical data from lead KRAS inhibitors as monotherapy for non-small cell lung cancer and colorectal cancer appear promising, combination strategies that could provide deeper and more durable responses have been considered and tested in clinical studies, such as NCT04185883 and NCT03785249. Examples of such combination therapies include combinations of AMG510 (sotorasib) or MRTX849 with pembrolizumab, combinations of KRAS inhibitors and SHP2 inhibitors, and combinations of KRAS inhibitors and CDK4 inhibitors (see Mullard, ibid., bridging paragraphs on pages 888 and 889 and full page 889).

[0005] Further combination strategies are needed to generate deeper and more durable responses, ideally those that overcome the declining efficacy of KRAS inhibitors over time when administered as monotherapy (or in combination with other anticancer therapies).

[0006] Invention Purpose and Overview

[0007] The inventors of this invention have surprisingly discovered that, when administered in combination with a KRAS inhibitor, the CBP / p300 bromodomain inhibitor, i.e., a bromodomain inhibitor that selectively binds to the bromodomain of CBP / p300, provides effective treatment for cancers exhibiting KRAS-induced oncogenic alterations, whereas, when administered alone, the CBP / p300 bromodomain inhibitor does not affect the cell proliferation of cancer cells. In other words, the inventors have surprisingly discovered that the combination of the CBP / p300 bromodomain inhibitor and the KRAS inhibitor is more effective in treating cancers exhibiting KRAS-induced oncogenic alterations than either of the two active substances alone against cancers exhibiting KRAS-induced oncogenic alterations. Therefore, as described above, the CBP / p300 bromodomain inhibitor is ineffective when administered alone (where "ineffective" specifically means no objective response as defined by the RECIST 1.1 response criteria for target or non-target lesions in the subject), while the efficacy of the KRAS inhibitor decreases over time when administered alone, possibly due to the development of resistance to the KRAS inhibitor.

[0008] In a first aspect, the present invention relates to a combination of (i) a CBP / p300 bromodomain inhibitor and (ii) a KRAS inhibitor for treating patients with cancer, wherein the cancer exhibits oncogenic alterations in KRAS. The first aspect may also be referred to as a combination of (I) a CBP / p300 bromodomain inhibitor and (ii) a KRAS inhibitor for treating patients with cancer, wherein the cancer is characterized by a KRAS mutation profile given in one or more indications of the KRAS inhibitor used in the combination (e.g., KRAS G12C), or wherein the cancer is characterized by a KRAS mutation profile targeted in a clinical trial setting by the KRAS inhibitor used in the combination (e.g., KRASG12C).

[0009] In a preferred embodiment of the first aspect, oncogenic alterations in KRAS lead to overactivation of KRAS signaling. Oncogenic alterations in KRAS may even result in constitutively active KRAS signaling (meaning that the signaling activity of GTP-bound KRAS is constitutively active).

[0010] In a further preferred embodiment of the first aspect, the carcinogenic alteration in KRAS is caused by at least one base mutation in the KRAS gene, resulting in the substitution of an amino acid selected from G12C, G12V, G12D, G13D, Q61H, Q61L, Q61R, K117N, and combinations thereof in KRAS. Preferably, the carcinogenic alteration in KRAS is caused by at least one base mutation in the KRAS gene, resulting in the substitution of an amino acid selected from G12C, G12V, and G12D in KRAS. Most preferably, the carcinogenic alteration in KRAS is caused by at least one base mutation in the KRAS gene, resulting in the substitution of G12C amino acid in KRAS.

[0011] In another embodiment of the first aspect, the cancer is selected from lung cancer, colorectal cancer, and pancreatic cancer. The lung cancer is preferably non-small cell lung cancer (NSCLC) and may be locally advanced or metastatic NSCLC, most preferably locally advanced or metastatic NSCLC with KRAS G12C-mutation (which can be alternatively described in the language used herein as treatment of a patient with NSCLC, optionally locally advanced or metastatic NSCLC, wherein the NSCLC exhibits a carcinogenic alteration G12C in KRAS).

[0012] In another embodiment of the first aspect, the CBP / p300 bromodomain inhibitor is a small molecule inhibitor. Therefore, in such an embodiment, the CBP / p300 bromodomain inhibitor is not a nucleic acid-based inhibitor, such as shRNA or RNAi targeting CBP and / or p300.

[0013] In another embodiment of the first aspect, the KRAS inhibitor is a small molecule inhibitor. Therefore, in such an embodiment, the KRAS inhibitor is not a nucleic acid-based inhibitor, such as shRNA or RNAi targeting KRAS. In another embodiment of the first aspect, the KRAS inhibitor targets KRAS G12C, i.e., targets cancers exhibiting oncogenic alterations to G12C in KRAS. Such an inhibitor can, in particular, be a covalent inhibitor that targets the cysteine ​​residue at position 12 in the G12C KRAS via covalent interactions.

[0014] CBP / p300 bromodomain inhibitors may be selected from compounds A, C, 00030, 00071, CCS1477, GNE-781, GNE-049, SGC-CBP30, CPI-637, FT-6876, 462, 424, and 515. These compounds are commercially available or publicly disclosed, as further summarized below, or their synthesis and structure are shown in the embodiments of this application. Preferably, the CBP / p300 bromodomain inhibitor is selected from compounds A, C, CCS1477, GNE-781, GNE-049, CPI-637, 462, 424, and 515.

[0015] The KRAS inhibitor may be selected from AMG510, MRTX849, JNJ-74699157 / ARS-3248, BI 1701963, BI1823911, BAY-293, GDC-6036, MRTX1133, RAS(ON) inhibitors (wherein the RAS(ON) inhibitor is preferably RMC-6291 or RMC-6236), and combinations thereof. In a more preferred embodiment, the KRAS inhibitor is AMG510 or MRTX849. Most preferably, the KRAS inhibitor is AMG510.

[0016] It should be understood that the combination of (i) and (ii) referred to herein can be an open or closed combination. Therefore, the combination of (i) and (ii) of the present invention can be used to treat patients with cancer that exhibits carcinogenic changes in the KRAS, and wherein (i) and (ii) are the only active agents (“closed” combination). However, the combination of (i) and (ii) can also be used to treat patients with cancer that exhibits carcinogenic changes in the KRAS, and wherein at least one additional active agent (iii) can be administered, for example, from the group consisting of: PD1 inhibitors, MEK inhibitors, SHP2 allosteric inhibitors, pan-ErbB tyrosine kinase inhibitors, PD-L1 inhibitors, EGFR inhibitors, chemotherapy regimens, mTOR inhibitors, CDK inhibitors, VEGF inhibitors, pembrolizumab, cetuximab, atezolizumab, bevacizumab, and any combination thereof (“open” combination).

[0017] In a preferred embodiment of the first aspect, the combination is administered to the patient during each treatment cycle.

[0018] In another embodiment of the first aspect, the CBP / p300 bromodomain inhibitor and the KRAS inhibitor are administered as separate dosage forms or contained in a single dosage form. If the CBP / p300 bromodomain inhibitor and the KRAS inhibitor are administered as separate dosage forms, the administration in each treatment cycle can be concomitant or sequential. This includes the option of administering the CBP / p300 bromodomain inhibitor first, followed by the KRAS inhibitor.

[0019] In another embodiment of the first aspect, the treatment results in a prolonged duration of therapeutic effect of the KRAS inhibitor compared to the duration of therapeutic effect when the KRAS inhibitor is administered as the sole active agent. In another embodiment, the treatment results in increased therapeutic efficacy of the KRAS inhibitor compared to the therapeutic efficacy when the KRAS inhibitor is administered as the sole active agent. In yet another embodiment, the treatment results in prevention of resistance to the KRAS inhibitor.

[0020] In another embodiment of the first aspect, the CBP / p300 bromodomain inhibitor is administered at a daily dose of about 1 mg to about 3000 mg, preferably about 10 mg to about 2000 mg, more preferably about 15 mg to about 1000 mg. The CBP / p300 bromodomain inhibitor is preferably administered at a daily dose of about 10 mg, about 15 mg, about 20 mg, about 50 mg, about 100 mg, about 250 mg, about 500 mg, about 1000 mg, about 1500 mg, about 2000 mg, about 2500 mg, or about 3000 mg. Administration can be intermittent, i.e., not daily, but the above-mentioned daily doses can be administered on the day of administration. If CCS1477, compound 462, compound 424, or compound 515 is used as the CBP / p300 bromodomain inhibitor, the corresponding compound can be administered at a daily dose of about 10 mg to about 600 mg.

[0021] In another embodiment of the first aspect, if the KRAS inhibitor is administered as the sole active agent, the KRAS inhibitor is administered at a daily dose within the typical daily dose range (particularly the daily dose of the KRAS inhibitor mentioned on the label, if any). The typical daily dose (or the stated daily dose, if any) depends on the specific EGFR inhibitor to be used. Typically, the KRAS inhibitor will be administered at a daily dose between about 10 mg and about 2000 mg. Therefore, AMG510 can be administered, for example, in combination at a daily dose between about 240 mg and about 1200 mg, between about 480 mg and about 1200 mg, or between about 600 mg and about 1200 mg, preferably between about 720 mg and about 1080 mg, more preferably between about 840 mg and about 960 mg, or about 960 mg for the purposes of this invention. MRTX849 can be administered, for example, in combination at a daily dose between about 200 mg and about 1400 mg, or between about 400 mg and about 1300 mg, preferably between about 600 mg and about 1200 mg, most preferably about 1200 mg, for the purposes of this invention.

[0022] In another embodiment of the first aspect, if the KRAS inhibitor is administered as the sole active agent, the KRAS inhibitor is administered at a daily dose lower than the typical daily dose described above. In other words, if the KRAS inhibitor is not administered as the sole active agent, but rather as part of a combination used according to the invention, the dose of the KRAS inhibitor can be lower than the dose when the KRAS inhibitor is administered as the sole active agent. This, for example, means that for the examples given above, the daily dose will be at the lower limit of a given range or even lower than those ranges.

[0023] In yet another embodiment of the first aspect, the present invention relates to a combination of (i) a CBP / p300 bromodomain inhibitor and (ii) a KRAS G12D inhibitor (preferably MRTX1133) for treating a patient with cancer in which the cancer exhibits carcinogenic alterations to G12D in KRAS. In this embodiment, it is further preferred that the cancer be selected from lung cancer, preferably NSCLC, colorectal cancer, and pancreatic cancer.

[0024] In a highly preferred embodiment of the first aspect, the present invention relates to a combination of (i) a CBP / p300 bromodomain inhibitor and (ii) a KRAS G12C inhibitor for treating a patient with cancer, wherein the cancer exhibits carcinogenic alterations in KRAS G12C. In this embodiment, it is further preferred that the cancer is selected from lung cancer, preferably NSCLC, colorectal cancer, and pancreatic cancer. Lung cancer is preferred. It is also preferred that the KRAS G12C inhibitor is selected from AMG510, MRTX849, JNJ-74699157 / ARS-3248, BI 1823911, GDC-6036, and RAS. G12C (ON) inhibitors (of which RAS) G12C The (ON) inhibitor is preferably RMC-6291 and combinations thereof; preferably, the KRAS G12C inhibitor is AMG510 or MRTX849.

[0025] In another preferred embodiment of the first aspect, the present invention relates to (i) a combination of a CBP / p300 bromine domain inhibitor and (ii) a KRAS G12C inhibitor, preferably AMG510 or MRTX849, for the treatment of a patient with locally advanced or metastatic NSCLC who has previously received at least one systemic therapy, wherein the NSCLC exhibits oncogenic alterations in KRAS G12C.

[0026] In another embodiment of the first aspect, the present invention relates to a combination of (i) compound A and (ii) a KRAS inhibitor for treating a patient with cancer, wherein the cancer exhibits carcinogenic alterations in KRAS. In this embodiment, it is preferred that the KRAS inhibitor is a KRAS G12C inhibitor, preferably AMG510 or MRTX849, and that the cancer exhibits carcinogenic alterations in KRAS G12C. In this embodiment, it is further preferred that the cancer is selected from lung cancer, preferably NSCLC, colorectal cancer, and pancreatic cancer.

[0027] In yet another embodiment of the first aspect, the present invention relates to a combination of (i) compound C and (ii) a KRAS inhibitor for treating a patient with cancer, wherein the cancer exhibits carcinogenic alterations in KRAS. In this embodiment, it is preferred that the KRAS inhibitor is a KRAS G12C inhibitor, preferably AMG510 or MRTX849, and that the cancer exhibits carcinogenic alterations in KRAS G12C. In this embodiment, it is further preferred that the cancer is selected from lung cancer, preferably NSCLC, colorectal cancer, and pancreatic cancer.

[0028] In a further embodiment of the first aspect, the present invention relates to a combination of (i) CCS1477 and (ii) a KRAS inhibitor for treating a patient with cancer, wherein the cancer exhibits carcinogenic alterations in KRAS. In this embodiment, it is preferred that the KRAS inhibitor is a KRAS G12C inhibitor, preferably AMG510 or MRTX849, and that the cancer exhibits carcinogenic alterations in KRAS G12C. In this embodiment, it is further preferred that the cancer is selected from lung cancer, preferably NSCLC, colorectal cancer, and pancreatic cancer.

[0029] In a further embodiment of the first aspect, the invention relates to a combination of (i) GNE-781 or GNE-049 and (ii) a KRAS inhibitor for treating a patient with cancer, wherein the cancer exhibits carcinogenic alterations in KRAS. Preferably, in this embodiment, the KRAS inhibitor is a KRAS G12C inhibitor, preferably AMG510 or MRTX849, and the cancer exhibits carcinogenic alterations in KRAS G12C. Further preferably, in this embodiment, the cancer is selected from lung cancer, preferably NSCLC, colorectal cancer, and pancreatic cancer.

[0030] In a further embodiment of the first aspect, the present invention relates to a combination of (i) CPI-637 and (ii) a KRAS inhibitor for treating a patient with cancer, wherein the cancer exhibits carcinogenic alterations in KRAS. Preferably, in this embodiment, the KRAS inhibitor is a KRAS G12C inhibitor, preferably AMG510 or MRTX849, and the cancer exhibits carcinogenic alterations in KRAS G12C. Further preferably, in this embodiment, the cancer is selected from lung cancer, preferably NSCLC, colorectal cancer, and pancreatic cancer.

[0031] In a further embodiment of the first aspect, the present invention relates to a combination of (i) compound 462, compound 424, or compound 515 and (ii) a KRAS inhibitor for treating a patient with cancer, wherein the cancer exhibits carcinogenic alterations in KRAS. In this embodiment, it is preferred that the KRAS inhibitor is a KRAS G12C inhibitor, preferably AMG510 or MRTX849, and that the cancer exhibits carcinogenic alterations in KRAS G12C. In this embodiment, it is further preferred that the cancer is selected from lung cancer, preferably NSCLC, colorectal cancer, and pancreatic cancer.

[0032] In a particularly preferred embodiment of the first aspect, the present invention relates to (i) a CBP / p300 bromodomain inhibitor, wherein the CBP / p300 bromodomain inhibitor is a small molecule inhibitor, and (ii) a KRAS inhibitor, wherein the KRAS inhibitor is a KRAS G12C small molecule inhibitor (preferably selected from AMG510, MRTX849, JNJ-74699157 / ARS-3248, BI1823911, GDC-6036, RAS). G12C (ON) inhibitors [preferably RMC-6291] and combinations thereof, most preferably combinations of AMG510 or MRTX849), are used to treat patients with cancer that exhibits a carcinogenic G12C alteration in KRAS. In this embodiment, it is further preferred that the cancer is selected from lung cancer, preferably NSCLC, colorectal cancer, and pancreatic cancer.

[0033] In a second aspect, the present invention relates to a kit comprising (i) a pharmaceutical formulation containing a CBP / p300 bromine domain inhibitor and (ii) a pharmaceutical formulation containing a KRAS inhibitor.

[0034] Dosage forms (i) and (ii) may be oral dosage forms, such as tablets, for example. The amount of CBP / p300 bromodomain inhibitor contained in the dosage form preferably conforms to the daily dosage described in the first aspect above. Therefore, if administered once daily, the amount of CBP / p300 bromodomain inhibitor in the dosage form may be the full daily dosage. However, if administered twice daily or once daily via two tablets, it may be less than the daily dosage, for example, half the daily dosage. The amount of KRAS inhibitor contained in the dosage form preferably conforms to the daily dosage described in the first aspect above. Therefore, if administered once daily, the amount of KRAS inhibitor in the dosage form may be the full daily dosage. However, if administered twice daily or once daily via two tablets, it may be less than the daily dosage, for example, half the daily dosage. For AMG510, this amount may in particular be 120 mg, wherein the dosage form preferably corresponds to tablets.

[0035] Each drug dosage form typically contains at least one pharmaceutically acceptable excipient, as defined in Section 3 below.

[0036] In a preferred embodiment of the second aspect, the CBP / p300 bromine domain inhibitor is selected from compound A, compound C, compound 00030, compound 00071, CCS1477, GNE-781, GNE-049, SGC-CBP30, CPI-637, FT-6876, compound 462, compound 424 and compound 515.

[0037] In another preferred embodiment of the second aspect, the KRAS inhibitor is selected from AMG510, MRTX849, JNJ-74699157 / ARS-3248, BI 1701963, BI 1823911, BAY-293, GDC-6036, MRTX1133, RAS(ON) inhibitors (preferably RMC-6291 or RMC-6236) and combinations thereof.

[0038] In another preferred embodiment of the second aspect, the kit comprises (i) a pharmaceutical formulation containing a CBP / p300 bromine domain inhibitor and (ii) a pharmaceutical formulation containing AMG510 or MRTX849.

[0039] Thirdly, the present invention relates to a pharmaceutical dosage form comprising (i) a CBP / p300 bromodomain inhibitor and (ii) a KRAS inhibitor.

[0040] The drug dosage form can be an oral dosage form, such as tablets. The amount of CBP / p300 bromodomain inhibitor contained in the dosage form preferably conforms to the daily dosage described in the first aspect above. Therefore, if administered once daily, the amount of CBP / p300 bromodomain inhibitor in the dosage form can be the full daily dosage. However, if administered twice daily or once daily via two tablets, it can be less than the daily dosage, for example, half the daily dosage. The amount of KRAS inhibitor contained in the dosage form preferably conforms to the daily dosage described in the first aspect above. Therefore, if administered once daily, the amount of KRAS inhibitor in the dosage form can be the full daily dosage. However, if administered twice daily or once daily via two tablets, it can be less than the daily dosage, for example, half the daily dosage. For AMG510, this amount can particularly be 120 mg, wherein the dosage form preferably corresponds to tablets.

[0041] This drug formulation typically contains at least one pharmaceutically acceptable excipient, as defined in Section 3 below.

[0042] In a preferred embodiment of the third aspect, the CBP / p300 bromine domain inhibitor is selected from compound A, compound C, compound 00030, compound 00071, CCS1477, GNE-781, GNE-049, SGC-CBP30, CPI-637, FT-6876, compound 462, compound 424 and compound 515.

[0043] In another preferred embodiment of the third aspect, the KRAS inhibitor is selected from AMG510, MRTX849, JNJ-74699157 / ARS-3248, BI 1701963, BI 1823911, BAY-293, GDC-6036, MRTX1133, RAS(ON) inhibitors (preferably RMC-6291 or RMC-6236) and combinations thereof.

[0044] In yet another preferred embodiment of the third aspect, the drug formulation comprises (i) a CBP / p300 bromine domain inhibitor and (ii) AMG510 or MRTX849.

[0045] In a fourth aspect, the present invention relates to a method for treating cancer in a patient in need, the method comprising administering to the patient an effective amount of (i) a CBP / p300 bromodomain inhibitor and an effective amount of (ii) a KRAS inhibitor, wherein the cancer exhibits carcinogenic alterations in KRAS.

[0046] In a fifth aspect, the present invention relates to a method for prolonging the duration of the therapeutic effect of a KRAS inhibitor in a patient in need, the method comprising administering to the patient an effective amount of (i) a CBP / p300 bromodomain inhibitor and an effective amount of (ii) a KRAS inhibitor, wherein the cancer exhibits oncogenic alterations in KRAS. In other words, in cancer treatment, the duration of the therapeutic effect of a KRAS inhibitor (when administered in combination) is prolonged compared to the duration of the therapeutic effect of a KRAS inhibitor when administered as the sole active agent.

[0047] In a sixth aspect, the present invention relates to a method for enhancing the therapeutic efficacy of a KRAS inhibitor in patients with this need, the method comprising administering to the patient an effective amount of (i) a CBP / p300 bromodomain inhibitor and an effective amount of (ii) a KRAS inhibitor, wherein the cancer exhibits carcinogenic alterations in KRAS. In other words, in cancer treatment, the therapeutic efficacy of a KRAS inhibitor (when administered in combination) is increased compared to the therapeutic efficacy of a KRAS inhibitor administered as the sole active agent.

[0048] In a seventh aspect, the present invention relates to a method for blocking the proliferation of cancer cells, the method comprising administering to cells an effective amount of (i) a CBP / p300 bromodomain inhibitor and an effective amount of (ii) a KRAS inhibitor, wherein the cancer cells exhibit carcinogenic alterations in KRAS.

[0049] In an eighth aspect, the present invention relates to a method for delaying the proliferation of cancer cells, the method comprising administering to cells an effective amount of (i) a CBP / p300 bromodomain inhibitor and an effective amount of (ii) a KRAS inhibitor, wherein the cancer cells exhibit carcinogenic alterations in KRAS.

[0050] The implementation scheme of the first aspect described above also applies to the methods of aspects four through eight. Brief description of the attached diagram

[0052] Figure 1 In determining the crystal structure of the bromine domain of human CREBBP complexed with compound 00004, the initial Fo-Fc differential electron density map (outline 4.0σ) of the model was obtained by refining the initial model before modeling the compound with REFMAC5.

[0053] Figure 2 AE: SNU-1411 confluence within 32 days, wherein cells are treated as indicated, i.e., with DMSO (control), or AMG510 alone or any different CBP / p300 bromodomain inhibitor alone, or with (i) a combination of AMG510 and (ii) any different CBP / p300 bromodomain inhibitor. See Example 6 for details.

[0054] Figure 3 AE: SNU-1411 confluence within 32 days, wherein cells are treated as indicated, i.e., with DMSO (control), or MRTX849 alone or any different CBP / p300 bromodomain inhibitor alone, or with (i) a combination of MRTX849 and (ii) any different CBP / p300 bromodomain inhibitor. See Example 7 for details.

[0055] Figure 4 AE: SW837 confluence within 49 days, wherein cells were treated as indicated, i.e., with DMSO (control), or AMG510 alone or any different CBP / p300 bromodomain inhibitor alone, or with (i) a combination of AMG510 and (ii) any different CBP / p300 bromodomain inhibitor. See Example 8 for details.

[0056] Figure 5 AB: NCI-H358 cells were proliferated for >20 days, wherein the cells were treated as indicated, i.e., with DMSO (control), or AMG510 alone, or one of two different CBP / p300 bromodomain inhibitors alone, or a combination of (i) AMG510 and (ii) one of two different CBP / p300 bromodomain inhibitors. See Example 9 for details. Invention Details

[0058] Before describing the invention in more detail, the following definitions are introduced.

[0059] 1. Definition

[0060] As used in the specification and claims, the singular forms “a” and “an” also include the corresponding plural forms, unless the context clearly specifies otherwise.

[0061] In the context of this invention, the term "about" refers to a range of precision that will be understood by those skilled in the art to still ensure the technical effectiveness of the features in question. This term typically indicates a deviation from the indicated value of ±10%, preferably ±5%.

[0062] It should be understood that the term "comprising" is not restrictive. For the purposes of this invention, the term "consisting of" is considered a preferred embodiment of the term "comprising". If a group is defined below as including at least a certain number of embodiments, this also means including a group that preferably consists only of those embodiments.

[0063] p300 (also known as EP300 and KAT3B) is a large protein with many different domains that can bind to a variety of proteins, including many DNA-binding transcription factors. The cyclic AMP response element-binding protein (CREB) (also known as CREBBP and KAT3A), which binds to the protein CBP, is a closely related protein to p300. Given their extensive sequence identity and functional similarity, these two proteins are often referred to as paralogs, and hereby referred to as "CBP / p300". CBP / p300 is a lysine acetyltransferase that has been shown to catalyze the attachment of acetyl groups to the lysine side chains of histones and other proteins. CBP / p300 has been proposed to activate transcription, with the mechanism of action appearing to involve bridging DNA-binding transcription factors to RNA polymerase mechanisms or by aiding in the assembly of the pre-transcriptional initiation complex. For this purpose, different CBP / p300 domains are thought to interact with different arrays of transcription factors assembled on promoters and enhancers for transcription of different genes (see Dyson and Wright, JBC Vol. 291, no. 13, pp. 6714-6722). Figure 2 ).

[0064] One of the many domains of CBP / p300 is the bromodomain. First identified in Drosophila in 1992, the bromodomain was described approximately 10 years later as a binding module for acetyl-lysine. In humans, there are many proteins containing bromodomains, which can be classified into eight classes based on sequence and structural similarity. It appears that all proteins containing bromodomains are involved in the regulation of transcriptional programs. Oncogene rearrangements suggest that targeting proteins containing bromodomains, especially their bromodomains, may be particularly beneficial for cancer treatment.

[0065] Several drug candidates have been developed and are currently undergoing clinical trials, targeting so-called "bromodomain and superterminal motif" proteins, commonly known as BET proteins, which constitute a group of bromodomain-containing proteins. Examples of BET-protein-targeting drugs are INCB054329 (Incyte Corporation), ABBV-075 (AbbVie), and I-BET762 (GlaxoSmithKline). Drugs also selectively target the bromodomains of CBP and p300, which are part of another group of bromodomain-containing proteins. Such inhibitors include, for example, CCS1477 (CellCentric), currently in clinical trials for the treatment of metastatic castration-resistant prostate cancer and hematologic malignancies, or FT-7051 (Forma Therapeutics Inc.), currently in investigations for the treatment of metastatic castration-resistant prostate cancer.

[0066] As used herein, the term "CBP / p300 bromine domain inhibitor" refers to a small molecule that strongly and selectively binds to the bromine domains of CBP and p300. This term is synonymous with the terms "bromine domain inhibitor that selectively binds to the bromine domains of CBP / p300" and "selective inhibitor of the bromine domains of CBP / p300." "Strong binding" in this respect means that, when binding to the bromine domains of CBP and p300, Kd is less than about 300 nM, preferably less than about 100 nM. In this respect, "selective binding" means binding to any other bromine-containing protein or BROMOscan. TM Compared to the Kd bound to the bromine domain of CBP, the Kd bound to the bromine domain of p300 by the small molecule is at most about 1 / 20, preferably at most about 1 / 30, more preferably at most about 1 / 50, and most preferably at most about 1 / 70. Preferably, when BROMOscan is performed as shown in Example 4... TM At that time, it was compared with other proteins or bromine-containing domains shown by the DiscoveRx gene symbol in the table of Example 4 of this application. For comparison, any protein or BROMOscan containing a bromine domain other than CBP and p300 was compared.TM The lowest Kd of the bromine domain is compared with the highest Kd of CBP and p300. Therefore, if, for example, the Kd of BRD4 (full-length, short iso.) is the lowest among all proteins containing bromine domains other than CBP and p300, or among bromine domains in general, and is 7100 nM, this is compared to the Kd of CBP (29 nM) (not to the Kd of p300, which is 12 nM and therefore lower than the Kd of CBP). The above examples were performed on compound A in the table of Example 4 below.

[0067] By binding selectively as described above, the interaction with the cellular interaction partner, which typically occurs through the bromine domain of CBP / p300, is inhibited; hence, the molecule is referred to as an "inhibitor." The term "inhibitory interaction" means that preferably no further interaction occurs between the bromine domain of CBP / p300 and the interaction partner (at least not to a detectable level). However, when a given interaction (set at 100%) between the bromine domain of CBP / p300 and the interaction partner is significantly reduced, for example, to about 50%, about 40%, about 30%, preferably about 20%, more preferably about 10%, or most preferably about 5% or lower, this reduced interaction is still included in the term "inhibitory interaction." For the medical use of compounds that inhibit interactions, complete inhibition of the interaction may not be necessary to achieve a sufficient therapeutic effect. Therefore, it should be understood that the term "inhibition" as used herein also refers to a reduction in the interaction sufficient to achieve the desired effect.

[0068] The term "KRAS" as used in this article refers to the protein in "Kirsten rat sarcoma". KRAS is a GTPase that is an important mediator of intracellular signaling pathways involved in tumor cell growth and survival. In normal cells, KRAS acts as a molecular switch, toggling between an inactive GDP-binding state and an active GTP-binding state. The switching between these states is facilitated by guanine nucleotide exchange factor (GEF) loading GTP and activating KRAS, and by GTP hydrolysis catalyzed by GTPase activator protein (GAP) to inactivate KRAS. GTP binding to KRAS promotes effector binding to trigger signal transduction pathways, including the RAF-MEK-ERK (MAPK) pathway. Activating mutations in KRAS in somatic cells are a hallmark of cancer and prevent GAP association, thereby stabilizing effector binding and enhancing KRAS signaling. Patients with KRAS-mutant tumors have significantly worse outcomes and prognoses.

[0069] As used herein, the term "KRAS inhibitor" refers to a molecule that acts on KRAS to inhibit downstream intracellular signaling that ultimately leads to cell proliferation. In this document, the term "inhibition" means, preferably, the cessation of downstream signaling. However, when a given downstream signal (set as 100%) is significantly reduced, for example, to about 70%, about 60%, about 50%, about 40%, about 30%, preferably about 20%, more preferably about 10%, or most preferably about 5% or lower, this reduction in downstream signaling is still covered by the term "inhibition of intracellular downstream signaling." For the medical use of compounds that inhibit downstream signaling, complete inhibition of signaling may not be necessary to achieve a sufficient therapeutic effect. Therefore, it should be understood that the term "inhibition" as used in this context also refers to a reduction in downstream signaling sufficient to achieve the desired effect. KRAS inhibitors can covalently bind to KRAS, particularly to the cysteine ​​residue at position 12 of KRAS G12C. If a KRAS inhibitor targets and / or binds to this cysteine ​​residue, the inhibitor is generally referred to as a "KRAS G12C inhibitor." Examples of such inhibitors include AMG510 (CAS-Nr.2296729-00-3), MRTX849 (CAS-Nr.2326521-71-3), JNJ-74699157 / ARS-3248, BI 1823911, GDC-6036, and RMC-6291. Recently, the first KRAS G12C modulator received FDA approval, LUMAKRAS (sotorasib, corresponding to Amgen's AMG510) tablets, for the treatment of locally advanced or metastatic non-small cell lung cancer (NSCLC) with KRAS G12C mutations. Another KRAS G12C modulator, adagrasib (corresponding to Mirati Therapeutics' MRTX849), is expected to follow soon. A "KRAS G12D inhibitor" is an inhibitor of KRAS G12D, and so on. An example of a KRAS G12D inhibitor is MRTX1133. Additionally, KRAS inhibitors can block the interaction of KRAS with other proteins, particularly the KRAS-SOS1 interaction. Examples of such KRAS-SOS1 inhibitors are BI 1701963 and BAY-293 (CAS Nr. 2244904-70-7).There are also so-called RAS(ON) inhibitors, which bind to KRAS bound to mutated GTP (e.g., G12C GTP-bound KRAS, G12V GTP-bound KRAS, G12D GTP-bound KRAS, G13D GTP-bound KRAS, Q61H GTP-bound KRAS, Q61L GTP-bound KRAS, or Q61RGTP-bound KRAS) and prevent RAF involvement by blocking the effector surface of their respective KRAS, as they form a three-component complex between the RAS(ON) inhibitor (a synthetic ligand), the KRAS, and cyclophilin A (see Revolution Medicines, e.g., WO 2021 / 091956 for more details). RMC-6291 is a RAS from Revolution Medicines. G12C (ON) inhibitors target KRAS through the above mechanism. G12C RMC-6236 is a RAS(ON) inhibitor from Revoilation Medicines that targets multiple RAS mutations, including KRAS mutations, through the mechanism described above.

[0070] As used in this article, the term "where cancer exhibits oncogenic alterations in KRAS" refers to a tumor that possesses a mutated version of KRAS, in which this mutated version is associated with cancer development. In other words, a mutated version of KRAS can be considered as associated with or causally related to cancer development, optionally including other factors as well. Mutated versions of KRAS are present in tumors due to alterations in the KRAS gene, particularly at least one base mutation in the KRAS gene that results in an amino acid substitution in KRAS. The specific alterations have been outlined above, with the most prominent being the KRAS G12C alteration. As noted above, KRAS mutations are present in up to 25% of cancers, with oncogenic variants exhibiting varying prevalences across different cancers (see Mullard, ibid., Column 1).

[0071] The term "overactivation" of KRAS as used in this article refers to the fact that KRAS is more active than wild-type KRAS, particularly in downstream activation and signal transduction, which leads to cancer cell growth.

[0072] As used herein, the term "small molecule" refers to a small organic compound having a low molecular weight. In the context of this invention, small molecules preferably have a molecular weight of less than 5000 Daltons, more preferably less than 4000 Daltons, more preferably less than 3000 Daltons, more preferably less than 2000 Daltons, or even more preferably less than 1000 Daltons. In a particularly preferred embodiment, the small molecule in the context of this invention has a molecular weight of less than 800 Daltons. In another preferred embodiment, the small molecule in the context of this invention has a molecular weight of 50 to 3000 Daltons, preferably 100 to 2000 Daltons, more preferably 100 to 1500 Daltons, and even more preferably 100 to 1000 Daltons.

[0073] As used in this article, “treatment” refers to clinical interventions aimed at curing or improving a disease, preventing disease recurrence, alleviating disease symptoms, reducing any direct or indirect pathological consequences of the disease, achieving a stable (i.e., non-deteriorating) state of the disease, preventing metastasis, slowing the rate of disease progression, and / or prolonging survival compared to expected survival without treatment.

[0074] The term “treatment cycle” as used in this article refers to a period of time after initial assessment of the patient’s condition, during which the patient’s condition is typically reassessed before the start of another treatment cycle.

[0075] Details of the CBP / p300 bromine domain inhibitors mentioned herein are as follows: The structures of compounds A, C, 00030, and 00071 are shown in the Examples section of this application. Furthermore, the synthetic routes for these compounds are shown in the Examples section of this application. CCS1477 is commercially available, for example at Aobious, with CAS number 2222941-37-7. GNE-781 is available, for example, at MCE (MedChemExpress), with CAS number 1936422-33-1. GNE-049 is available from companies such as MCE (MedChemExpress), with CAS number 1936421-41-8. SGC-CBP30 is available from companies such as MCE (MedChemExpress), with CAS number 1613695-14-9. CPI-637 is available from companies such as MCE (MedChemExpress), with CAS number 1884712-47-3. FT-6876 is available from companies such as MCE (MedChemExpress), with CAS number 2304416-91-7 (FT-6876 is also known as "CBP / p300-IN-8"). The structures of compounds 462, 424, and 515 are described below, with these structures and synthetic routes given in WO 2020 / 006483 (see in particular compound 424 on pages 33 and 34, compound 462 on pages 42 and 43, and compound 515 on pages 47 and 48):

[0076]

[0077] 2. The inventor's amazing discovery

[0078] The inventors have identified a novel compound that binds strongly to the bromine domain of CBP / p300 and demonstrated that the binding to the bromine domain of CBP / p300 is also selective, as many proteins are known to contain bromine domains.

[0079] CBP / p300 has been identified as a central node in the eukaryotic transcriptional regulatory network and interacts with over 400 transcription factors and other regulatory proteins. CBP / p300 regulates crosstalk and interference between many cellular signaling pathways and is targeted by tumor viruses to hijack cellular regulatory mechanisms (see Dyson and Wright, ibid., p. 6714, right column). CBP / p300 is a large protein containing several domains, which can be found in Dyson and Wright's... Figure 1As shown above, these domains are NRID, TAZ1, TAZ2, KIX, CRD1, BRD, CH2 (including the PHD and RING finger domains), HAT, ZZ, and NCBD domains. The size of these proteins and their diverse domains clearly demonstrate their highly diverse cellular functions; for example, CBP / p300 can interact with many different interacting partners due to its ability to perform multiple interactions. The enzymatic activity of CBP / p300 as a histone acetyltransferase is located in the HAT domain. As mentioned above, the function of this enzyme is primarily involved in transcriptional activation. CBP / p300 is also susceptible to post-translational modifications, particularly phosphorylation. Their inherent enzymatic activity, as well as the post-translational modifications, introduce another layer of complexity to the various functions and roles of CBP / p300. Goodman and Smolk, Genes & Development 2000, 14:1553-1577, summarize well in the introduction that these functions and effects can even be antagonistic, noting that a major paradox of CBP / p300 function is that these proteins appear to facilitate diametrically opposed cellular processes, and whether CBP / p300 promotes apoptosis or cell proliferation appears to be highly environment-dependent. For disease-specific, particularly cancer-specific, environmental factors will determine how CBP / p300 participates, and if they do participate at all.

[0080] Given the above, it is not surprising that CBP / p300 cannot be conferred a single function in cellular processes, as it may be affected by, for example, general "CBP / p300 inhibitors." Instead, due to its extremely high level of complexity, dissecting the various functions of CBP / p300 seems only possible when studying specific domains of CBP / p300, i.e., by analyzing the effects achieved, for example, by inhibiting the enzymatic activity of CBP / p300 in its HAT domain, or by making specific interactions with its interacting partner impossible by blocking (or "inhibiting") certain domains. Furthermore, as mentioned above, this must be considered within its respective context, such as a specific disease or cancer type.

[0081] Therefore, the inventors turned to investigate its effect under specific conditions, namely, that its inhibitors impede the interaction between the CBP / p300 bromine domain and the interacting object, and, based on a recent article by Hou et al. (Hou et al., BMC Cancer (2018) 18:641), preliminarily investigated the effect of its inhibitors in non-small cell lung cancer (NSCLC) cells. However, when the inhibitor was applied alone, the inventors failed to observe any effect on the proliferation of the tested NSCLC cell lines. Surprisingly, the inventors found that, compared with the EGFR inhibitor alone, its CBP / p300 bromine domain inhibitor prolonged the effect of the EGFR inhibitor in NSCLC cells exhibiting oncogenic alterations in EGFR. In other words, while it itself has no effect on the proliferation of NSCLC exhibiting oncogenic alterations in EGFR, the inventors' CBP / p300 bromine domain inhibitor exhibits an effect when used in conjunction with the EGFR inhibitor. Even more surprisingly, the inventors discovered that this combination concept not only works on EGFR signaling (and correspondingly with EGFR inhibitors), but also on KRAS signaling and KRAS inhibitors, which will be outlined below.

[0082] For their experiments, the inventors used colorectal cancer cell lines (SNU-1411 and SW837, both rectal adenocarcinoma cell lines carrying the KRAS G12C mutation) and the NSCLC cell line (NCI-H358, carrying the KRAS G12C mutation). Therefore, these cell lines can be considered as model systems for first-line treatment of cancer patients with mutated KRAS, particularly those with the KRAS G12C mutation. AMG510 and MRTX849 were used in combination as KRAS inhibitors and CBP / p300 bromodomain inhibitors, respectively (see examples below).

[0083] The significant proliferation inhibition observed in this combination during long-term culture in all tested cell lines is particularly noteworthy because—due to the development of resistance—proliferation inhibition does not remain complete over time when KRAS inhibitors are used alone. This was also the case with AMG510 and MRTX849 when used alone, as the data in the Experimental section below show.

[0084] Based on their results with CBP / p300 inhibitors, the inventors continued to investigate whether the observed effects could be similarly generalized to CBP / p300 inhibitors. To this end, other CBP / p300 bromodomain inhibitors were tested, namely CCS1477, FT-6876, and GNE-781. It is noteworthy that the structures of the different groups of CBP / p300 inhibitors tested by the inventors are unrelated; therefore, their common feature is only related to the effect achieved by these inhibitors, namely, selective inhibition of the CBP / p300 bromodomain. The structures of all tested CBP / p300 inhibitors are as follows:

[0085]

[0086]

[0087] It should also be mentioned that the tested KRAS inhibitors AMG510 and MRTX849 are structurally very different, but they both have the function of inhibiting KRAS G12C (by acting as covalent inhibitors).

[0088] Furthermore, the inventors not only tested single cancer cell lines carrying oncogenic mutations in KRAS, but also used three different cancer cell lines in general (SNU-1411 and SW837, two rectal adenocarcinoma cell lines, and NCI-H358, an NSCLC cell line).

[0089] 3. Pharmaceutical compositions of the compounds of the present invention

[0090] "CBP / p300 bromodomain inhibitors" and "KRAS inhibitors" are "pharmaceutical active agents" for the purposes claimed herein. As stated above, they may be present in individual dosage forms or contained in a single dosage form.

[0091] As used herein, "pharmaceutical active agent" refers to a compound that effectively modulates the response of a patient (i.e., a human or animal). The term "pharmaceuticalally acceptable excipient" as used herein refers to an excipient commonly included in a pharmaceutical composition, as is known to those skilled in the art. Such excipients are listed below. Given the definition of "pharmaceutical active agent" given above, a pharmaceutically acceptable excipient may be defined as having no pharmaceutical activity.

[0092] If a commercially available KRAS inhibitor is used in combination with a CBP / p300 bromodomain inhibitor, it is preferred to administer the inhibitor in separate dosage forms, and the KRAS inhibitor should be administered in an approved dosage form and route of administration (e.g., this applies to AMG510, which is approved in LUMAKRAS). The CBP / p300 bromodomain inhibitor may be administered in the dosage forms described below, or in the dosage forms currently undergoing clinical trials.

[0093] The dosage forms used according to the present invention can be formulated for oral, sublingual, nasal, rectal, topical, transdermal, or parenteral application. Oral application is preferred. Parenteral application is also preferred, including intravenous, intramuscular, or subcutaneous administration. The dosage forms of the present invention may also be referred to as formulations or pharmaceutical compositions.

[0094] Typically, pharmaceutical compositions according to the present invention may contain a variety of pharmaceutically acceptable excipients, selected based on the function the composition is intended to achieve. "Pharmaceutically acceptable excipients" in the sense of the present invention can be any substance used to prepare a pharmaceutical dosage form, including coating materials, film-forming materials, fillers, disintegrants, release-modifying materials, carrier materials, diluents, binders, and other adjuvants. Typical pharmaceutically acceptable excipients include substances such as sucrose, mannitol, sorbitol, starch and starch derivatives, lactose, and lubricants such as magnesium stearate, disintegrants, and buffers.

[0095] The term "carrier" refers to a pharmaceutically acceptable organic or inorganic carrier substance to which the active ingredient is bound to facilitate application. Suitable pharmaceutically acceptable carriers include, for example, water, salt solutions, alcohols, oils, preferably vegetable oils, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, surfactants, fragrance oils, monoglycerides and diglycerides of fatty acids, petroethral fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, etc. The pharmaceutical composition may be sterilized and, if desired, mixed with excipients such as lubricants, preservatives, stabilizers, humectants, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavoring agents, and / or aromatic substances, which do not react harmfully with the active compound.

[0096] If this invention considers liquid dosage forms, these dosage forms may include pharmaceutically acceptable emulsions, solutions, suspensions, and syrups containing inert diluents commonly used in the art, such as water. These dosage forms may contain, for example, microcrystalline cellulose for filling, alginate or sodium alginate as a suspending agent, methylcellulose as a thickener, and sweeteners / flavorings.

[0097] For parenteral administration, particularly suitable carriers include solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants. Parenteral drug formulations are particularly preferred and include aqueous solutions in water-soluble forms. Furthermore, suspensions can be formulated as suitable oily injectable suspensions. Suitable lipophilic solvents or carriers include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Aqueous injectable suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.

[0098] A particularly preferred dosage form is an injectable formulation of the pharmaceutical composition of the present invention. Therefore, sterile injectable aqueous or oily suspensions can be formulated, for example, using suitable dispersants, wetting agents, and / or suspending agents according to known techniques. Sterile injectable formulations can also be sterile injectable solutions or suspensions in non-toxic, parenteral-acceptable diluents or solvents. Acceptable carriers and solvents that can be used are water and isotonic sodium chloride solutions. Sterile oils are also commonly used as solvents or suspension media.

[0099] Suppositories for rectal administration of the pharmaceutical compositions of the present invention can be prepared, for example, by mixing the compound with suitable non-irritating excipients such as cocoa butter, synthetic triglycerides and polyethylene glycol, which are solid at room temperature but liquid at rectal temperature, such that they melt in the rectum and release the active agent from the suppository.

[0100] For inhalation administration, pharmaceutical compositions containing the compounds of the present invention can be conveniently delivered from pressurized packages or nebulizers in the form of an aerosol spray using a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gases). In the case of pressurized aerosols, the dosage unit can be determined by providing a valve to deliver the measured amount. Capsules and cartridges, such as gelatin, for use in inhalers or blowpipes can be formulated as powder mixtures containing the compounds and suitable powder matrices such as lactose or starch.

[0101] Oral dosage forms can be liquid or solid, including, for example, tablets, lozenges, pills, capsules, powders, effervescent formulations, sugar-coated pills, and granules. Orally administered pharmaceutical formulations can be obtained as solid excipients, optionally by grinding the resulting mixture and processing it into granules after adding suitable excipients (if desired) to obtain tablets or sugar-coated pill cores. Suitable excipients are particularly fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose formulations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrants such as croscarmellose, agar, or alginate or its salts, such as sodium alginate, can be added. Oral dosage forms can be formulated to ensure immediate or sustained release of the active agent.

[0102] 4. Further disclosure and implementation plan

[0103] The clinical antitumor effects of receptor tyrosine kinase (RTK) inhibitors and other kinase inhibitors are not durable. Resistance to these inhibitors often develops. More specifically, the clinical antitumor effects of EGFR inhibitors (EGFRi) are also not durable. Depending on the treatment and clinical situation, resistance to EGFR inhibitors typically develops within 9 to 19 months. Therefore, there is a need to develop a cancer treatment modality that can prevent cancer patients from developing resistance. Historically, most approaches to overcoming resistance have focused on the genetic drivers of recurrent tumors. To overcome established resistance, novel mutated proteins that drive tumor regeneration are targeted, either alone or in combination with drugs for the primary cancer. One mechanism of resistance to EGFRi treatment is the development of watchdog mutations in the EGFR protein—mutations that render EGFRi ineffective. The most common such watchdog mutation is the T790M mutation. Mutation-specific inhibitors, such as osimertinib, are used to overcome established resistance to first-generation EGFR inhibitors, which do not inhibit the mutated EGFR T790M. Another mechanism of resistance to EGFRi therapy is bypass signaling, which is activated by other receptor tyrosine kinases, such as through the amplification, overexpression, or activation of MET, ErbB2, HGF, ErbB3, IGF1R, AXL, NTRK1, BRAF, FGFR3, or FGFR1. Therapeutic interventions that inhibit bypass signaling have been tested in clinical trials with mixed results.

[0104] Previous publications, such as patent application WO2018022637, describe the use of CBP / p300 inhibitors as novel cancer therapies, particularly for treating cancers containing p300 mutations. WO2011085039 describes methods for treating cancer, including inhibiting the activity of CBP / p300 histone acetyltransferase (HAT), and the use of CBP / p300 HAT inhibitors in treating subjects with cancer, particularly in combination with DNA-damaging chemotherapeutic anticancer agents.

[0105] New and effective methods and compositions are needed to prevent the development of cancer drug resistance. This is addressed in particular by the implementation schemes described in Section 4 of this document.

[0106] Implementation Scheme 1: A CBP / p300 bromodomain inhibitor in a method for treating animal cancer, the method comprising administering to an animal in need a CBP / p300 bromodomain inhibitor and a receptor tyrosine kinase inhibitor selected from: EGFR, ALK, MET, HER2, ROS1, RET, NTRK1 and AXL inhibitors, or KRas (Kirsten Rat Sarcoma) or BRAF (proto-oncogene B-Raf and v-Raf murine sarcoma virus oncogene homologue B) inhibitor, wherein the cancer comprises alterations to the corresponding receptor tyrosine kinase or KRas or BRAF, and wherein the CBP / p300 bromodomain inhibitor alone does not slow cancer progression.

[0107] Implementation Scheme 2: A method for prolonging the duration of response to cancer therapy with a receptor tyrosine kinase inhibitor or a KRas or BRAF inhibitor in animals, comprising administering a CBP / p300 bromodomain inhibitor or a pharmaceutically acceptable salt thereof to an animal with cancer, wherein the duration of response to cancer therapy is prolonged upon administration of the CBP / p300 bromodomain inhibitor or a pharmaceutically acceptable salt thereof compared with the duration of response to cancer therapy without administration of the CBP / p300 bromodomain inhibitor or a pharmaceutically acceptable salt thereof, and wherein the receptor tyrosine kinase inhibitor is selected from EGFR, ALK, MET, HER2, ROS1, RET, NTRK1, and AXL inhibitors.

[0108] Implementation Scheme 3: A composition for treating cancer, the composition comprising a CBP / p300 bromodomain inhibitor or a pharmaceutically acceptable salt thereof and a synergistic combination of receptor tyrosine kinase inhibitors selected from: inhibitors of EGFR, ALK, MET, HER2, ROS1, RET, NTRK1 and AXL, or KRas or BRAF inhibitors, wherein the cancer includes alterations to the corresponding receptor tyrosine kinase or KRas or BRAF, and wherein the CBP / p300 bromodomain inhibitor alone does not slow cancer progression.

[0109] Implementation Scheme 4: A method for inhibiting cancer cell growth, comprising administering a CBP / p300 bromodomain inhibitor and a receptor tyrosine kinase inhibitor selected from: EGFR, ALK, MET, HER2, ROS1, RET, NTRK1 and AXL inhibitors, or KRas or BRAF inhibitors, wherein the cancer cells contain alterations to the corresponding receptor tyrosine kinase or KRas or BRAF, and wherein the CBP / p300 bromodomain inhibitor alone does not inhibit cancer cell growth.

[0110] Implementation Scheme 5: The CBP / p300 bromodomain inhibitor or composition for the use or method described in any of the foregoing embodiments, wherein the alteration of the receptor tyrosine kinase or KRas or BRAF is a carcinogenic alteration.

[0111] Implementation Scheme 6: A CBP / p300 bromodomain inhibitor or composition for the use or method according to any of the foregoing embodiments, wherein the receptor tyrosine kinase inhibitor is an EGFR inhibitor.

[0112] Implementation Scheme 7: A CBP / p300 bromodomain inhibitor or composition for the use or method described in Implementation Scheme 6, wherein the alteration of the receptor tyrosine kinase is a mutation in EGFR.

[0113] Implementation Scheme 8: A CBP / p300 bromodomain inhibitor or composition for the use or method described in any of the foregoing embodiments, wherein the CBP / p300 bromodomain inhibitor or a pharmaceutically acceptable salt thereof has a synergistic effect in the treatment of cancer compared to a single CBP / p300 inhibitor or a single receptor tyrosine kinase or KRas or BRAF inhibitor.

[0114] Implementation Scheme 9: A CBP / p300 bromodomain inhibitor or composition for the use or method described in any of the foregoing embodiments, wherein a combination or combination of a CBP / p300 bromodomain inhibitor or a pharmaceutically acceptable salt thereof and a receptor tyrosine kinase inhibitor or Kras or BRAF inhibitor delays or reduces the risk of cancer developing resistance to receptor tyrosine kinase inhibitors or KRas or BRAF inhibitors.

[0115] Implementation Scheme 10: A CBP / p300 bromodomain inhibitor or composition for the use or method described in any of the foregoing embodiments, wherein the CBP / p300 bromodomain inhibitor is administered in an effective amount to prevent cancer cells from developing resistance to receptor tyrosine kinase inhibitors or KRas or BRAF inhibitors.

[0116] Implementation Scheme 11: A CBP / p300 bromodomain inhibitor or composition for the use or method according to any of the foregoing implementation schemes, wherein the EGFR inhibitor is selected from: cetuximab, panitumumab, zalumab, nimotuzumab, materutuzumab, gefitinib, erlotinib, dacomitinib, lapatinib, neratinib, vandetanib, nexituzumab, osimertinib, afatinib, AP26113, EGFR inhibitor (CAS No. 879127-07-8), EGFR / ErbB2 / ErbB-4 inhibitor (CAS No. 881001-19-0), EGFR / ErbB-2 inhibitor (CAS No. 17924861-4), EGFR inhibitor II (BIBX 1382, CAS No. 196612-93-8), EGFR inhibitor III ... II (BIBX 1382, CAS No. 196612-93-8), EGFR inhibitor III (CAS No. 17924861-4), EGFR inhibitor II (BIBX 1382, CAS No. 196612-93-8), EGFR inhibitor III (CAS No. 17924861-4), EGFR / ErbB-2 / ErbB-4 inhibitor II (CAS No. 944341-54-2) or PKCβII / EGFR inhibitor (CAS No. 145915-60-2).

[0117] Implementation Scheme 12: A CBP / p300 bromine domain inhibitor or composition for the stated use or method according to any of the foregoing embodiments, wherein the CBP / p300 inhibitor is a compound of formula (I).

[0118]

[0119] in

[0120] R 1 Selected from halogens and - (optionally substituted hydrocarbon groups, which contain 1 to 20 carbon atoms and 1 to 15 heteroatoms selected from O, N and S);

[0121] R 21 Selected from hydrogen, - (optionally substituted C) 1-6 Alkyl groups, which may contain 1-3 oxygen atoms between carbon atoms, and - (optionally substituted C atoms) 3-6 cycloalkyl);

[0122] R 3 Selected from -(optionally substituted heterocyclic group), -(optionally substituted carbocyclic group), -(optionally substituted C 1-6 alkylene)-(optionally substituted heterocyclic group) and -(optionally substituted C 1-6 Alkylene group (optionally substituted carbocyclic group);

[0123] X 1 X 2 and X 3 Each is independently selected from N, CH and CRx Wherein X 1 X 2 and X 3 At least one of them is N;

[0124] R 31 Selected from – hydrogen, – C 1-6 -alkyl and -(C substituted with one or more F) 1-6 -alkyl); wherein R 3 And any R 31 Connections can be made arbitrarily; and

[0125] E does not exist or is selected from –CH2–, –CHR x –、–CR x 2–, –NH–, –NR x –、–O–、–L 1 –L 2 – and – L 2 –L 1 –, where L 1 Selected from –CH2–, –CHR x –、–CR x 2–, –NH–, –NR x –and–O–and L 2 Selected from –CH2–, –CHR x – and – CR x 2–;

[0126] R 6x For –halogen, –OH, =O, C 1-6 Alkyl, C 1-6 Haloalkyl, C substituted with one or more OH groups 1-6 Alkyl, optionally with one or more R xb Substituted monocyclic aryl group, optionally with one or more R xb Substituted monocyclic heteroaryl group, optionally with one or more R xb Substituted monocyclic cycloalkyl groups, optionally with one or more R xb Substituted monocyclic heterocyclic alkyl groups, optionally with one or more R xb The substituted monocyclic cycloalkenyl group, optionally replaced by one or more R xb Substituted monocyclic heterocyclic alkenyl groups, wherein the R xb Independently selected from -halogen, -OH, =O, C 1-4 Alkyl, C 1-2 Haloalkyl, C substituted with one or two OH groups 1-2 alkyl;

[0127] Ring A can be further divided by one or more R x Group substitution, wherein any two R groups on ring A are substituted.x The group can optionally be attached to and / or any R on ring A. x The group can optionally be with R 21 Linkage; and / or wherein ring A may be further connected by a group R x Replace, so as to be compatible with R 6x Together they form a double-ring section with the following partial structure:

[0128]

[0129] Wherein ring B is - (optionally substituted heterocyclic ring) or - (optionally substituted carbocyclic ring);

[0130] R x Each is independently selected from –halogen, –OH, –O- (optionally substituted C). 1-6 Alkyl), -NH- (optionally substituted C) 1-6 Alkyl), -N (optionally substituted C) 1-6 Alkyl)2, =O, -(optionally substituted C) 1-6 Alkyl), – (optionally substituted carbocyclic), – (optionally substituted heterocyclic), – (optionally substituted C 1-6 alkylene) – (optionally substituted carbocyclic group), - (optionally substituted C 1-6 alkylene) – (optionally substituted heterocyclic group), –O- (optionally substituted C 1-6 alkylene) – (optionally substituted carbocyclic group) and –O- (optionally substituted C 1-6 Alkylene groups – (optionally substituted heterocyclic groups), and

[0131] Among them, the optionally substituted hydrocarbon group, the optionally substituted C 3-6 Cycloalkyl, optionally substituted heterocyclic, optionally substituted heterocyclic, optionally substituted carbocyclic, optionally substituted carbocyclic and optionally substituted C 1-6 The optional substituents of the alkylene group are independently selected from - (C14 groups optionally substituted with one or more halogens). 1-6 Alkyl), -halogen, -CN, -NO2, oxo, -C(O)R*, -COOR*, -C(O)NR*R*, -NR*R*, -N(R*)-C(O)R*, -N(R*)-C(O)-OR*, -N(R*)-C(O)-NR*R*, -N(R*)-S(O)2R*, -OR*, -OC(O)R*, -OC(O)-NR*R*, -SR*, -S(O)R*, -S(O)2R*, -S(O)2-NR*R*, -N(R*)-S(O)2-NR*R*, optionally halogenated or C 1-6 Alkyl-substituted heterocyclic groups, and optionally halogenated or C 1-6Alkyl-substituted carbocyclic group; wherein each R* is independently selected from H, and optionally halogenated C. 1-6 Alkyl groups, optionally halogenated or C 1-6 Alkyl-substituted heterocyclic groups, and optionally halogenated or C 1-6 Alkyl-substituted carbocyclic group; wherein any two R* attached to the same nitrogen atom may optionally be linked, and

[0132] C can be arbitrarily substituted. 1-6 Alkyl and optionally substituted C 1-6 The optional substituents of the alkylene group are independently selected from -halogen, -CN, -NO2, oxo, -C(O)R**, -COOR**, -C(O)NR**R**, -NR**R**, -N(R**)-C(O)R**, -N(R**)-C(O)-OR**, -N(R**)-C(O)-NR**R**, -N(R**)-S(O)2R**, -OR**, -OC(O)R**, -OC(O)-NR**R**, -SR**, -S(O)R**, -S(O)2R**, -S(O)2-NR**R**, and -N(R**)-S(O)2-NR**R**, wherein R** is independently selected from H, C, and C, which are optionally substituted with halogen. 1-6 Alkyl groups, optionally halogenated or C 1-6 Alkyl-substituted heterocyclic groups, and optionally halogenated or C 1-6 Alkyl-substituted carbocyclic group; wherein any two R** attached to the same nitrogen atom may optionally be linked.

[0133] Implementation Scheme 13: A CBP / p300 bromine domain inhibitor or composition for the stated use or method according to any of the foregoing embodiments, wherein the CBP / p300 inhibitor is an arylimidazolylisoxazole of formula (A).

[0134] in

[0135] R° and R may be the same or different, each being H or C1-C6 alkyl, which is unsubstituted or substituted by OH, -OC(O)R' or OR', where R' is an unsubstituted C1-C6 alkyl;

[0136] W is either N or CH;

[0137] R 1 It is an unsubstituted or substituted group, and is selected from C-linked 4- to 6-membered heterocyclic groups; C3-C6 cycloalkyl groups; unsubstituted or C6-C... 10 aryl, 5-12 membered N-containing heteroaryl, C3-C6 cycloalkyl, OH, -OC(O)R' or OR' substituted C1-C6 alkyl, wherein R' is as defined above; and spirocyclic groups of the following formulas:

[0138]

[0139] Y is -CH2-, -CH2CH2-, or -CH2CH2CH2-;

[0140] n is 0 or 1;

[0141] R 2 It is selected from C6-C 10 A group consisting of an aryl group, a 5- to 12-membered N-heteroaryl group, a C3-C6 cycloalkyl group, and a C5-C6 cycloalkenyl group, wherein the group is unsubstituted or substituted, and wherein the C6-C6 group is substituted. 10 The aryl group can be selectively fused onto a 5- or 6-membered heterocycle;

[0142] Or a pharmaceutically acceptable salt thereof, wherein the arylimidazolyxazole preferably has the formula (Aa*):

[0143] (Aa*;CCS1477[CAS 2222941-37-7]).

[0144] Implementation Scheme 14: A CBP / p300 bromine domain inhibitor or composition for the stated use or method according to any of the foregoing embodiments, wherein the CBP / p300 inhibitor is a compound of formula (Ba).

[0145] in

[0146] R 1 -O (C1-C3 alkyl);

[0147] R 6 It is arbitrarily and independently controlled by one or more R B Substituted phenyl, wherein R B Selected from –OC 1-6 Alkyl, -OC 3-6 Cycloalkyl, -O-aryl or -O-heteroaryl, wherein each alkyl, cycloalkyl, aryl or heteroaryl group is optionally independently substituted by one or more halogens;

[0148] Or, one of the CBP / p300 inhibitors is a compound of formula (Bc).

[0149] in

[0150] R 1 For -OR 5 ;

[0151] R 5 For –C 1-6 Alkyl, -C 3-8Cycloalkyl, heterocyclic, aryl, or heteroaryl;

[0152] R 6 -OH, halogen, oxo, -NO2, -CN, -NH2, -C 1-6 Alkyl, -C 3-8 cycloalkyl, -C 4-8 Cycloalkenyl, heterocyclic, aryl, spirocycloalkyl, spiroheterocyclic, heteroaryl, -OC 3-6 Cycloalkyl, -O aryl, -O heteroaryl, -(CH2)n-OR 8 -C(O)R 8 '、-C(O)OR 8 or -C(O)NR 8 R 9 -NHC 1-6 Alkyl, -N(C) 1-6 alkyl)2、-S(O)2NH(C 1-6 Alkyl), -S(O)2N(C 1-6 Alkyl)2、-S(O)2C 1-6 Alkyl, -N(C) 1-6 Alkyl)SO2C 1-6 Alkyl, -S(O)(C 1-6 Alkyl), -S(O)N(C 1-6 alkyl)2 or -N(C 1-6 Alkyl)S(O)(C 1-6 Alkyl), wherein each alkyl, cycloalkyl, cycloalkenyl, heterocyclic, spirocycloalkyl, spiroheterocyclic, heteroaryl, or aryl group is optionally surrounded by one or more R 10 replace;

[0153] R 7 Each occurrence is independent of -H, halogen, -OH, -CN, -OC. 1-6 Alkyl, -NH2, -NH(C) 1-6 alkyl), -N(C) 1-6 Alkyl)2、-S(O)2H(C 1-6 Alkyl), -S(O)2N(C 1-6 Alkyl)2、-S(O)2(C 1-6 Alkyl group, -S(O)2OH, -C(O)C 1-6 Alkyl group, -C(O)NH2, -C(O)NH(C 1-6 Alkyl), -C(O)N(C 1-6 Alkyl)2, -C(O)OH, -C(O)OC 1-6 Alkyl, -N(C) 1-6 Alkyl)SO2C 1-6 Alkyl, -S(O)(C 1-6Alkyl), -S(O)N(C 1-6 Alkyl)2, -S(O)2NH2, -N(C 1-6 Alkyl)S(O)(C 1-6 Alkyl groups or tetrazolium;

[0154] R 10 Each occurrence is independently -C 1-6 Alkyl, -C 2-6 alkenyl, -C 2-6 alkynyl group, -C 3-8 cycloalkyl, -C 4-8 Cycloalkenyl, heterocyclic, heteroaryl, aryl, -OH, halogen, oxo, -NO2, -CN, -NH2, -OC 1-6 Alkyl, -OC 3-6 cycloalkyl, -O aryl, -O heteroaryl, -NHC 1-6 Alkyl, -N(C) 1-6 alkyl)2、-S(O)2NH(C 1-6 Alkyl), -S(O)2N(C 1-6 Alkyl)2、-S(O)2C 1-6 Alkyl, -C(O)C 1-6 Alkyl group, -C(O)NH2, -C(O)NH(-C 1-6 Alkyl), -NHC(O)C 1-6 Alkyl-C(O)N(C) 1-6 Alkyl)2、-C(O)OC 1-6 Alkyl, -N(C) 1-6 Alkyl)SO2-C 1-6 Alkyl, -S(O)(C 1-6 Alkyl), -S(O)N(C 1-6 alkyl)2 or -N(C 1-6 Alkyl)S(O)(C 1-6 Alkyl), wherein each alkyl, alkenyl, ynyl, cycloalkylcycloalkenyl, heterocyclic, heteroaryl, or aryl group is optionally surrounded by one or more -R 12 replace;

[0155] R 12 It is a halogen independently each time it appears;

[0156] m is an integer from 0 to 5;

[0157] r is an integer from 0 to 5.

[0158] Implementation Scheme 15: A CBP / p300 bromine domain inhibitor or composition for the use or method described in any of the preceding embodiments, wherein the slow progression of cancer in target or non-target lesions in animals is measured using RECIST 1.1 response criteria.

[0159] Implementation Scheme 16: A CBP / p300 bromine domain inhibitor or composition for the use or method described in any of the foregoing embodiments, wherein the cancer is non-small cell lung cancer (NSCLC).

[0160] Implementation Scheme 17: A CBP / p300 bromodomain inhibitor or composition for the use or method according to any of the foregoing embodiments, wherein the CBP / p300 bromodomain inhibitor is a compound of formula (I) of Implementation Scheme 12, the receptor tyrosine kinase inhibitor is an EGFR inhibitor, the receptor tyrosine kinase is EGFR, the cancer is NSCLC, more preferably, the NSCLC contains an EGFR T790M mutation, more preferably, wherein the receptor tyrosine kinase inhibitor is osimertinib.

[0161] Implementation Scheme 18: A CBP / p300 bromodomain inhibitor or composition for the use or method according to any of the foregoing embodiments, wherein the CBP / p300 bromodomain inhibitor is a compound of formula (A) of Implementation Scheme 13, preferably CCS1477 (CAS 2222941-37-7), the receptor tyrosine kinase inhibitor is an EGFR inhibitor, the receptor tyrosine kinase is EGFR, the cancer is NSCLC, more preferably NSCLC contains an EGFR T790M mutation, and more preferably wherein the receptor tyrosine kinase inhibitor is osimertinib.

[0162] Regarding the above-described embodiment 13, it is noted that the compound of formula (A) has been described in WO2016170324, WO2018073586 and WO2019202332, and all of the applications and their disclosures are incorporated herein by reference in their entirety, particularly with respect to the synthesis of the compound of formula (A).

[0163] In another embodiment, a method for treating animal cancer is provided, comprising administering to an animal in need a CBP / p300 bromodomain inhibitor and a receptor tyrosine kinase inhibitor, said inhibitor being selected from EGFR, ALK, MET, HER2, ROS1, RET, NTRK1, and AXL inhibitors, or KRas or BRAF inhibitors, wherein said cancer contains alterations to the corresponding receptor tyrosine kinase or KRas or BRAF, and wherein the CBP / p300 bromodomain inhibitor alone does not slow cancer progression.

[0164] In another embodiment, a method of treating cancer with a composition comprising a CBP / p300 bromodomain inhibitor or a pharmaceutically acceptable salt thereof and a synergistic combination of a receptor tyrosine kinase inhibitor or a Kras or BRAF inhibitor selected from EGFR, ALK, MET, HER2, ROS1, RET, NTRK1, and AXL inhibitors, wherein the cancer comprises alterations to the corresponding receptor tyrosine kinase or Kras or BRAF, and wherein the individual CBP / p300 bromodomain inhibitor does not slow cancer progression.

[0165] In another embodiment, a method is provided to prolong the duration of response in animals to cancer therapy with a receptor tyrosine kinase inhibitor or Kras or BRAF inhibitor, comprising administering a CBP / p300 bromodomain inhibitor or a pharmaceutically acceptable salt thereof to an animal with cancer, wherein the duration of response to cancer therapy is prolonged when the CBP / p300 inhibitor or a pharmaceutically acceptable salt thereof is not administered, and wherein the receptor tyrosine kinase inhibitor is selected from EGFR, ALK, MET, HER2, ROS1, RET, NTRK1, and AXL or a Kras or BRAF inhibitor.

[0166] In another embodiment, a method for inhibiting cancer cell growth is provided, the method comprising administering to cancer cells a CBP / p300 bromodomain inhibitor and a receptor tyrosine kinase inhibitor selected from EGFR, ALK, MET, HER2, ROS1, RET, NTRK1 and AXL inhibitors or KRas or BRAF inhibitors, wherein the cancer cells contain alterations to the corresponding receptor tyrosine kinase or KRas or BRAF, and wherein the CBP / p300 bromodomain inhibitor alone does not inhibit cancer cell growth, and wherein the CBP / p300 bromodomain inhibitor is administered in an effective amount to prevent cancer cells from developing resistance to the kinase inhibitor.

[0167] In another embodiment, a method for inducing cell death in cancer cells is provided, comprising administering a CBP / p300 bromodomain inhibitor and a receptor tyrosine kinase inhibitor or Kras or BRAF inhibitor selected from EGFR, ALK, MET, HER2, ROS1, RET, NTRK1, and AXL inhibitors to cancer cells, wherein the cancer cells contain alterations to the corresponding receptor tyrosine kinase or KRas or BRAF, and wherein the CBP / p300 bromodomain inhibitor alone does not induce cell death in cancer cells.

[0168] In one embodiment, alterations to receptor tyrosine kinases can be oncogenic alterations, wherein in this embodiment of Part 4, the term "oncogenic alteration" can refer to genetic alterations to cellular proto-oncogenes. These genetic changes / alterations may result in conferring a growth advantage to the cell. In one embodiment, genetic mechanisms such as mutation, gene amplification, gene fusion, and / or chromosomal rearrangement can activate oncogenes in human tumors.

[0169] In another embodiment, the oncogenic alteration is selected from the following EGFR gene mutations: EGFR exon 19 deletion, EGFR-L858R, EGFR-T790M, EGFR-T854A, EGFR-D761Y, EGFR-L747S, EGFR-G796S / R, EGFR-L792F / H, EGFR-L718Q, EGFR exon 20 insertion, EGFR-G719X (where X is any other amino acid), EGFR-L861X, EGFR-S768I, or EGFR amplification. In a preferred embodiment, the alteration is EGFR-T790M. In another embodiment, the cancer is NSCLC, and the alteration is a mutation involving EGFR exon 19 deletion, L858R, or T790M.

[0170] In another implementation, the carcinogenic alteration is selected from the following RET gene mutations or rearrangements: KIF5B-RET, CCDC6-RET, NCOA4-RET, TRIM33-RET, RET-V804L, RET-L730, RET-E732, RET-V738, RET-G810A, RET-Y806, RET-A807, or RET-S904F.

[0171] In another embodiment, the oncogenic alteration is a HER2 gene mutation selected from HER2 exon 20 insertion or mutation and HER2-C805S, HER2 T798M, HER2 L869R, HER2 G309E, HER2 S310F or HER2 amplification.

[0172] In another implementation, the oncogenic alteration is a ROS1 gene fusion or rearrangement selected from: CD74-ROS1, GOPC-ROS1, EZR-ROS1, CEP85L-ROS1, SLC34A2-ROS1, SDC4-ROS1, FIG-ROS1, TPM3-ROS1, LRIG3-ROS1, KDELR2-ROS1, CCDC6-ROS1, TMEM106B-ROS1, TPD52L1-ROS1, CLTC-ROS1, and LIMA1-ROS1, or mutations including ROS1 G2032R, D2033N, S1986Y / F, L2026M, and / or L1951R.

[0173] In another implementation, the carcinogenic alteration is MET gene amplification, MET gene mutation such as MET Y1230C, D1227N, D1228V, Y1248H, and MET exon 14 skipping, or gene fusion or rearrangement selected from TPR-MET, CLIP2-MET, TFG-MET fusion, KIF5B-MET fusion.

[0174] In another embodiment, the carcinogenic alteration is a KRas gene mutation selected from G12C, G12V, G12D, G13D, Q61H or L or R, K117N.

[0175] In another implementation, the carcinogenic alteration is selected from the following ALK gene mutations, gene fusions, or rearrangements: EML4-ALK, TFG-ALK, KIF5B-ALK, KLC1-ALK, STRN-ALK in NSCLC, EML4-ALK, C2orf44-ALK, EML4-ALK, TPM-ALK, VCL-ALK, TPM3-ALK, EML4-ALK, or VCL-ALK.

[0176] In another implementation, the carcinogenic alteration is a BRAF gene mutation selected from V600E or V600K.

[0177] In another implementation, the carcinogenic alteration is selected from the following NTR gene fusions or rearrangements: TPM3-NTRK1, ETV6-NTRK3, TPM3-NTRK1, TPR-NTRK1, TFG-NTRK1, PPL-NTRK1, ETV6-NTRK3, TPR-NTRK1, MPRIP-NTRK1, CD74-NTRK1, SQSTM1-NTRK1, TRIM24-NTRK2, LMNA-NTRK, ETV6-NTRK3, BCAN-NTRK1, ETV6-NTRK3, AML, GIST NFASC-NTRK1, BCAN-NTRK1, AGBL4-NTRK2, VCL-NTRK2, ETV6-NTRK3, BTBD1-NTRK3, RFWD2-NTRK1, RABGAP1L-NTRK1, TP53-NTRK1, AFAP1-NTRK2, NACC2-NTRK2, OKI-NTRK2, PAN3-NTRK2, or NTRK1 gene mutations selected from F589L, G595R, G667C / S, A608D, or NTRK3 gene mutations selected from G623R, G696A.

[0178] In another implementation, the receptor tyrosine kinase inhibitor is an EGFR inhibitor. In another embodiment, the EGFR inhibitor is selected from cetuximab, panitumumab, zalumab, nimotuzumab, materutuzumab, gefitinib, erlotinib, lapatinib, neratinib, vandetanib, nexituzumab, osimertinib, afatinib, dacomitinib, AP26113, poziotinib, EGFR inhibitor (CAS No. 879127-07-8), EGFR / ErbB2 / ErbB-4 inhibitor (CAS No. 881001-19-0), EGFR / ErbB-2 inhibitor (CAS No. 17924861-4), EGFR inhibitor II (BIBX1382, CAS No. 196612-93-8), EGFR inhibitor III (CAS No. 733009-42-2), EGFR / ErbB-2 / ErbB-4 inhibitor II (CAS No. 17924861-4), EGFR ... No. 944341-54-2) or PKCβII / EGFR inhibitor (CAS No. 145915-60-2).

[0179] In another implementation, the alteration of the receptor tyrosine kinase is a mutation in the EGFR gene.

[0180] In another embodiment, the receptor tyrosine kinase inhibitor is a RET inhibitor. In another embodiment, the RET inhibitor is selected from cabozantinib, vandetanib, lenvatinib, alectinib, apatinib, panatinib, LOXO-292, BLU-667, or RXDX-105.

[0181] In another embodiment, the receptor tyrosine kinase inhibitor is a HER2 inhibitor. In another embodiment, the HER2 inhibitor is selected from trastuzumab, hyaluronidase / trastuzumab fam-trastumzumab deruxtecan, ado-trastuzumab emtansine, lapatinib, neratinib, pertuzumab, tucatinib, poziotinib, or dacomitinib.

[0182] In another embodiment, the receptor tyrosine kinase inhibitor is a ROS1 inhibitor. In another embodiment, the ROS1 inhibitor is selected from crizotinib, ceritinib, brigatinib, loratinib, entrectinib, cabozantinib, DS-6051b, and TPX-0005.

[0183] In another embodiment, the receptor tyrosine kinase inhibitor is a MET inhibitor. In another embodiment, the MET inhibitor is selected from: crizotinib, cabozantinib, MGCD265, AMG208, octreotinib, golvatinib, glesantinib, faritinib, auvumatinib, tivatinib, cevotinib, AMG337, carmatinib and terpoxtinib, OMO-1 [JNJ38877618] or anti-MET antibodies onartuzumab and emibetuzumab [LY2875358] or anti-HGF antibodies ficlatuzumab [AV-299] and rilotumumab [AMG102].

[0184] In another embodiment, the inhibitor is a KRas inhibitor. In yet another embodiment, the KRas inhibitor is selected from AMG510, MRTX849, JNJ-74699157 / ARS-3248, BI1701963, BAY-293, or a “RAS(ON)” inhibitor.

[0185] In another embodiment, the receptor tyrosine kinase inhibitor is an ALK inhibitor. In yet another embodiment, the ALK inhibitor is selected from crizotinib, ceritinib, alectinib, lolatinib, or brigatinib.

[0186] In another embodiment, the inhibitor is a BRAF inhibitor. In yet another embodiment, the BRAF inhibitor is selected from vemurafenib, dabrafenib, encorafenib, or any nonspecific RAF inhibitor.

[0187] In another embodiment, the receptor tyrosine kinase inhibitor is an NTRK inhibitor. In yet another embodiment, the NTRK inhibitor is selected from entrectinib, larotrectinib (LOXO-101), LOCO-195, DS-6051b, cabozantinib, mesatinib, TSR-011, PLX7486, MGCD516, crizotinib, regorafenib, dovitinib, lestaurtinib, and BMS-7. 54807, Danusertib, ENMD-2076, midostaurin, PHA-848125AC, BMS-777607, altriratinib, AZD7451, MK5108, PF-03814735, SNS-314, furitinib, nintedanib, ponatinib, ONO-5390556 or TPX-0005.

[0188] In another embodiment, a combination or combination of a CBP / p300 bromodomain inhibitor or its pharmaceutically acceptable salt and a receptor tyrosine kinase inhibitor or a KRas or BRAF inhibitor has a synergistic effect in treating cancer compared to a single CBP / p300 inhibitor or a single receptor tyrosine kinase inhibitor or a KRas or BRAF inhibitor. In the embodiments described in Section 4, the term "synergistic" refers to an interaction between two or more drugs that results in a total effect greater than the sum of the individual effects of each drug. In a preferred embodiment, the synergistic effect is an increased response rate in animals to a combination of a CBP / p300 bromodomain inhibitor and a receptor tyrosine kinase inhibitor or a KRas or BRAF inhibitor. In another embodiment, the increase in response rate is measured as an increase in efficacy in cancer treatment.

[0189] In another embodiment, the anticancer effect provided by a combination or combination of a CBP / p300 bromodomain inhibitor or its pharmaceutically acceptable salt and receptor tyrosine kinase inhibitor or Kras or BRAF inhibitor is greater than the anticancer effect provided by monotherapy with the same dose of a CBP / p300 inhibitor or receptor tyrosine kinase inhibitor or Kras or BRAF inhibitor. As used in the context of the embodiments in Section 4, the term "anticancer" refers to the treatment of malignant or cancerous diseases. In another embodiment, the invention provides compositions for use or methods wherein the anticancer effect provided by a combination or combination of a CBP / p300 bromodomain inhibitor or its pharmaceutically acceptable salt and receptor tyrosine kinase inhibitor or Kras or BRAF inhibitor is at least 2, at least 3, at least 5, or at least 10 times that of monotherapy alone.

[0190] In another embodiment, a combination or combination of a CBP / p300 bromodomain inhibitor or its pharmaceutically acceptable salt and a receptor tyrosine kinase inhibitor or Kras or BRAF inhibitor delays or reduces the risk of cancer developing resistance to the receptor tyrosine kinase inhibitor or Kras or BRAF inhibitor. As used in the context of the embodiments in Section 4, the term "cancer resistance" refers to a reduction in drug efficacy; more specifically, the term may refer to the development of resistance in cancer cells. In another embodiment, cancer does not develop resistance to the receptor tyrosine kinase inhibitor or Kras or BRAF inhibitor for at least 3, 6, 9, 12, 24, 48, or 60 months. In another embodiment, the CBP / p300 bromodomain inhibitor is administered in an effective amount to prevent cancer cells from developing resistance to the receptor tyrosine kinase inhibitor or Kras or BRAF inhibitor.

[0191] In another implementation, the CBP / p300 bromodomain inhibitor inhibits the bromodomain of CBP and / or p300. p300 (also known as histone acetyltransferase p300, E1A-binding protein p300, E1A-associated protein p300) and CBP (also known as CREB-binding protein or CREBBP) are two structurally very similar transcriptional coactivators.

[0192] As used in the context of the embodiments described in Section 4, the term "CBP / p300 bromodomain inhibitor" can be considered to refer to a compound that binds to the CBP bromodomain and / or the p300 bromodomain and inhibits and / or reduces the biological activity or function of CBP and / or p300. In some embodiments, the CBP / p300 bromodomain inhibitor may bind to CBP and / or p300 primarily (e.g., alone) through contact and / or interaction with the CBP bromodomain and / or the p300 bromodomain. In some embodiments, the CBP / p300 bromodomain inhibitor may bind to CBP and / or p300 through contact and / or interaction with the CBP bromodomain and / or the p300 bromodomain, as well as additional CBP and / or p300 residues and / or domains. In some embodiments, the CBP / p300 bromodomain inhibitor may substantially or completely inhibit the biological activity of CBP and / or p300. In some embodiments, the bioactivity can be the binding of the bromodomains of CBP and / or p300 to chromatin (e.g., DNA-associated histones) and / or another acetylated protein. In some embodiments within the context of the embodiments described in Section 4, the inhibitor may have an IC50 of less than about 50 μM, less than about 1 μM, less than about 500 nM, less than about 100 nM, less than about 10 nM, or less than about 1 nM. 50 Or a binding constant. In some embodiments, the CBP / p300 bromodomain inhibitor can bind to and inhibit the CBP bromodomain. In some embodiments, the CBP / p300 bromodomain inhibitor can bind to and inhibit the p300 bromodomain. In some embodiments, the CBP / p300 bromodomain inhibitor may not inhibit the histone acetyltransferase activity of CBP / p300.

[0193] In one embodiment, the CBP / p300 bromodomain inhibitor is a compound of formula (I). In one embodiment, the CBP / p300 bromodomain inhibitor is a compound of formula (A), preferably CCS1477 (CAS 2222941-37-7). In another embodiment, the CBP / p300 bromodomain inhibitor is FT-7051. In another embodiment, the compound of formula (I), the compound of formula (A), preferably CCS1477 or FT-7051 is a daily dose of the drug, the concentration of which is selected from a list including 10 mg, 15 mg, 25 mg, 50 mg, 100 mg, 150 mg or 200 mg. In another embodiment, CCS1477 is administered 2, 3, 4, 5, 6 or 7 weeks. In another embodiment, CCS1477 is administered twice daily. In another embodiment, administration to cancer cells comprises contacting the cancer cells with a CBP / p300 inhibitor and a receptor tyrosine kinase inhibitor or a KRas or BRAF inhibitor.

[0194] In another implementation, the dosage depends on a variety of factors, including the patient's age, weight, condition, and route of administration. The daily dose can vary widely and will be adjusted according to the individual needs of each specific case. However, typically, when the compound is administered alone to adults, the dose used for each route of administration can range from 0.0001 to 50 mg / kg, most commonly from 0.001 to 10 mg / kg body weight, for example, 0.01 to 1 mg / kg. This dose can be administered, for example, 1 to 5 times daily. For intravenous administration, a suitable daily dose can be from 0.0001 to 1 mg / kg body weight, preferably from 0.0001 to 0.1 mg / kg body weight. The daily dose can be administered as a single dose or according to a fractionated dosing regimen.

[0195] In another implementation, the RECIST 1.1 response criteria can be used to measure cancer progression or the duration of response to cancer treatment in subjects / animals, whether in target or non-target lesions.

[0196] In another embodiment, the term "not slowing cancer progression" may be defined in the embodiment of Section 4 as a subject who does not achieve any RECIST 1.1 clinical response. In another embodiment, the term "not slowing cancer progression" may be defined in the embodiment of Section 4 as a subject / animal who does not achieve a partial RECIST 1.1 clinical response. In another embodiment, according to RECIST 1.1, the term "not slowing cancer progression" is measured as no objective response rate and / or no increase in progression-free survival. In another embodiment, the term "not slowing cancer progression" is measured as a reduction of less than 30% in the sum of the longest diameters of the target lesion, with the baseline sum of the longest diameters of the target lesion as a reference.

[0197] In some implementation schemes, the cancer is selected from acoustic neuroma, acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute T-cell leukemia, basal cell carcinoma, bile duct carcinoma, bladder cancer, brain cancer, breast cancer, bronchial cancer, cervical cancer, chondrosarcoma, chordoma, choriocarcinoma, chronic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, colon cancer, colorectal cancer, craniopharyngioma, cystadenocarcinoma, and diffuse... Large B-cell lymphoma, proliferative disorders, embryonal carcinoma, endometrial cancer, endothelial sarcoma, ependymoma, epithelial carcinoma, erythroleukemia, esophageal cancer, estrogen receptor-positive breast cancer, essential thrombocytosis, Ewing's sarcoma, fibrosarcoma, follicular lymphoma, germ cell testicular cancer, glioma, glioblastoma, gliosarcoma, heavy chain disease, head and neck cancer, angioblastoma, liver cancer, hepatocellular carcinoma, hormone-insensitive prostate cancer, smooth muscle cell lung cancer. Sarcoma, leukemia, liposarcoma, lung cancer, lymphangiosarcoma, lymphangiosarcoma, lymphoblastic leukemia, lymphoma, T-cell or B-cell malignant lymphoma, medullary carcinoma, medulloblastoma, melanoma, meningioma, mesothelioma, multiple myeloma, myeloid leukemia, myeloma, myxosarcoma, neuroblastoma, NUT midline carcinoma (NMC), non-small cell lung cancer (NSCLC), oligodendroglioma, oral cancer, osteosarcoma Tumors, ovarian cancer, pancreatic cancer, papillary adenocarcinoma, papillary carcinoma, pineal tumor, polycythemia vera, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, sebaceous gland carcinoma, seminoma, skin cancer, small cell lung cancer, solid tumors (carcinoma and sarcoma), small cell lung cancer, gastric cancer, squamous cell carcinoma, synovial malformation, sweat gland carcinoma, thyroid cancer, Waldenström macroglobulinemia, testicular tumors, uterine cancer, and nephroblastoma. In some embodiments, the cancer is melanoma, NSCLC, renal cancer, ovarian cancer, colon cancer, pancreatic cancer, hepatocellular carcinoma, or breast cancer. In some embodiments of any method, the cancer is lung cancer, breast cancer, pancreatic cancer, colorectal cancer, and / or melanoma. In some embodiments, the cancer is lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC). In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is colorectal cancer.

[0198] In another embodiment, the CBP / p300 bromodomain inhibitor and a receptor tyrosine kinase inhibitor or a KRas or BRAF inhibitor are administered simultaneously to animals as a single composition. In another embodiment, the CBP / p300 bromodomain inhibitor and a receptor tyrosine kinase inhibitor or a KRas or BRAF inhibitor are administered to animals separately. In another embodiment, the CBP / p300 bromodomain inhibitor and a receptor tyrosine kinase inhibitor or a KRas or BRAF inhibitor are administered simultaneously to animals. In another embodiment, the CBP / p300 bromodomain inhibitor is administered to animals before administration of the receptor tyrosine kinase inhibitor or the KRas or BRAF inhibitor. In another embodiment, the animal is a human.

[0199] In one embodiment, the term "effective amount" of a pharmaceutical agent (e.g., a pharmaceutical preparation) may refer to an effective amount that achieves the desired therapeutic or preventative effect within the necessary dosage and time period. In some embodiments, an effective amount refers to the amount of a CBP / p300 bromodomain inhibitor and a receptor tyrosine kinase inhibitor or a KRas or BRAF inhibitor that (i) treats a specific disease, condition, or disorder, (ii) attenuates, improves, or eliminates one or more symptoms of a specific disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a specific disease, condition, or disorder described herein. In some embodiments, an effective amount of a CBP / p300 bromodomain inhibitor and a receptor tyrosine kinase inhibitor or a KRas or BRAF inhibitor may reduce the number of cancer cells; may shrink tumor size; may inhibit (i.e., to some extent slow down and preferably stop) the invasion of cancer cells into peripheral organs; may inhibit (i.e., to some extent slow down and preferably stop) tumor metastasis; may inhibit tumor growth to some extent; and / or may alleviate one or more cancer-related symptoms to some extent. For cancer treatment, efficacy may be measured, for example, by assessing time to disease progression (TTP) and / or determining the response rate (RR). In some implementations, the effective amount is the amount of the CBP / p300 bromodomain inhibitor and receptor tyrosine kinase inhibitor or KRas or BRAF inhibitor entities described herein sufficient to significantly reduce the activity or number of drug-resistant or drug-resistant persistent cancer cells.

[0200] In one embodiment, the compounds of the present invention may be administered in combination with radiotherapy or another chemotherapeutic agent for the treatment of cancer to human or animal patients. In another embodiment, combination therapy may be provided in which a CBP / p300 inhibitor or RTK inhibitor or KRas or BRAF inhibitor is administered simultaneously or sequentially with radiotherapy; or simultaneously or sequentially with another or multiple chemotherapeutic agents or as a combination formulation for the treatment of cancer. The aforementioned or each other chemotherapeutic agent is typically an agent conventionally used for the type of cancer being treated. In one embodiment, the types of chemotherapeutic agents used in the combination may be, for example, androgen receptor antagonists for the treatment of prostate cancer, such as enzalutamide, and inhibitors of CYP17A1 (17α-hydroxylase / C17,20 lyase), such as abiraterone. In other embodiments, other chemotherapeutic agents in the combination therapy may include docetaxel. In one embodiment, the term "combination" in Section 4 may refer to simultaneous, separate, or sequential administration. When administered sequentially or separately, delayed administration of the second component should not result in loss of the beneficial effects of the combination.

[0201] In another embodiment, the response to CBP / p300 bromodomain inhibitors and receptor tyrosine kinase inhibitors or KRas or BRAF inhibitors is a sustained response. In one embodiment, a “sustainable response” can refer to a continued effect on reducing tumor growth after treatment is discontinued. For example, the tumor size may remain the same or smaller compared to the size at the start of the dosing phase.

[0202] In another implementation, the term "treatment" (and variations such as "treatment" or "management") may refer to a clinical intervention that attempts to alter the natural course of the treated individual or cells, and may be performed for prevention or in the course of clinicopathology. The desired effects of treatment may include one or more of the following: prevention of disease onset or recurrence; relief of symptoms; reduction of any direct or indirect pathological consequences of the disease; stabilization (i.e., non-deterioration) of the disease state; prevention of metastasis; reduction of the rate of disease progression; improvement or relief of the disease state; and prolongation of survival or improved prognosis compared to expected survival without treatment. In some implementations, CBP / p300 bromodomain inhibitors and receptor tyrosine kinases or KRas or BRAF inhibitors may be used to delay the development of or slow the progression of a disease or disorder. In one implementation, those individuals requiring treatment may include those who already have the condition or disorder and those who are predisposed to having the condition or disorder (e.g., through gene mutations or abnormal expression of genes or proteins) or those for whom the condition or disorder will be prevented.

[0203] In one implementation, the term "delay" can refer to slowing, hindering, mitigating, delaying, stabilizing, and / or postponing the development of a disease (such as cancer) or the development of drug resistance to a disease. The duration of such delay varies depending on the disease history and / or the individual receiving treatment. It will be apparent to those skilled in the art that a sufficient or significant delay can indeed include prevention, as the individual does not develop the disease. For example, the development of advanced cancer, such as metastasis, can be delayed. 5. Examples

[0204] The following examples are merely illustrative and will describe the invention in a further manner. These examples should not be construed as limiting the invention thereto.

[0205] The preparation of compounds 00003 (compound B), 00004 (compound A), 00030, 00071, and compound C are described below. Synthetic routes for intermediate compounds and / or compounds similar to the above compounds are provided if helpful.

[0206] General experimental methods

[0207] LCMS method:

[0208] Method A: Instruments: Agilent 1260Bin. Pump: G1312B; Degassing device; Autosampler, ColCom; DAD: Agilent G1315D, 220-320nm; MSD: Agilent LC / MSD G6130B ESI, pos / neg 100-800, ELSDAlltech 3300; Gas flow rate: 1.5mL / min; Gas temperature: 40℃; Column: Waters XSelect TM C18, 30 x 2.1 mm, 3.5 μm, temperature: 35 °C, flow rate: 1 mL / min, gradient: t0 = 5% A, t 1.6min =98% A,t 3min =98% A, Posttime: 1.3 min, Eluent A: 0.1% formic acid in acetonitrile, Eluent B: 0.1% formic acid in water).

[0209] Method B: Instruments: Agilent 1260Bin. Pump: G1312B; Degassing device; Autosampler, ColCom; DAD: Agilent G1315D, 220-320nm; MSD: Agilent LC / MSD G6130B ESI, pos / neg 100-800, ELSD Alltech 3300; Gas flow rate: 1.5mL / min; Gas temperature: 40℃; Column: Waters XSelect TMC18, 50 x 2.1 mm, 3.5 μm, temperature: 35℃, flow rate: 0.8 mL / min, gradient: t0 = 5% A, t 3.5min =98% A,t 6min =98% A, running time: 2 min; eluent A: 0.1% formic acid in acetonitrile, eluent B: 0.1% formic acid in water).

[0210] Method C: Instruments: Agilent 1260Bin; Pump: G1312B; Degassing device; Autosampler, ColCom; DAD: Agilent G1315C, 220-320nm; MSD: Agilent LC / MSD G6130B ESI, pos / neg 100-800; Column: Waters XSelect TM CSH C18, 30 x 2.1 mm, 3.5 μm, temperature: 25℃, flow rate: 1 mL / min, gradient: t0 = 5% A, t 1.6min =98% A,t 3min =98% A, running time: 1.3 min, eluent A: 95% acetonitrile + 5% 10mM ammonium bicarbonate aqueous solution in acetonitrile, eluent B: 10mM ammonium bicarbonate aqueous solution (pH=9.5).

[0211] Method D: Instruments: Agilent 1260Bin; Pump: G1312B; Degassing device; Autosampler, ColCom; DAD: Agilent G1315C, 220-320nm; MSD: Agilent LC / MSD G6130B ESI, pos / neg 100-800; Column: Waters XSelect TM CSH C18, 50 x 2.1 mm, 3.5 μm, temperature: 25℃, flow rate: 0.8 mL / min, gradient: t0 = 5% A, t 3.5min =98% A,t 6min =98% A, run time: 2 min, eluent A: 95% acetonitrile + 5% 10mM ammonium bicarbonate in acetonitrile aqueous solution, eluent B: 10mM ammonium bicarbonate aqueous solution (pH=9.5).

[0212] UPLC method:

[0213] Method A: Instruments: Agilent Infinity II; Bin. Pump: G7120A, Multi-purpose Sampler, VTC, DAD: Agilent G7117B, 220-320nm, PDA: 210-320nm, MSD: Agilent G6135B ESI, pos / neg 100-1000, ELSD G7102A: Evap 40℃, Neb 50℃, gasflow 1.6mL / min, Column: Waters XSelect CSHC18, 50x2.1 mm, 2.5μm, Temperature: 25℃, Flow Rate: 0.6mL / min, Gradient: t0=5% B, t 2min =98% B,t 2.7min =98% B, run time: 0.3 min, eluent A: 10 mM ammonium bicarbonate aqueous solution (pH = 9.5), eluent B: acetonitrile.

[0214] Method B: Instruments: Agilent Infinity II; Bin. Pump: G7120A, Multisampler, VTC, DAD: Agilent G7117B, 220-320nm, PDA: 210-320nm, MSD: Agilent G6135B ESI, pos / neg 100-1000, ELSD G7102A: Evap 40℃, Neb 40℃, gas flow: 1.6mL / min, column: Waters XSelect TM CSHC18, 50x2.1 mm, 2.5 μm; Temperature: 40℃; Flow rate: 0.6 mL / min; Gradient: t0 = 5% B,t 2min =98% B,t 2.7min =98% B, running time: 0.3 min, eluent A: 0.1% formic acid in water, eluent B: 0.1% formic acid in acetonitrile.

[0215] GCMS method:

[0216] Method A: Equipment: GC: Agilent 6890N G1530N and MS: MSD 5973G2577A, EI-forward; Measurement temperature: 280℃; Mass range: 50-550; Column: RXi-5MS 20m, ID 180μm, df 0.18μm; Average flow rate: 50cm / s; Injection volume: 1μl; Syringe temperature: 250℃; Split ratio: 100 / 1; Carrier gas: He; Initial temperature: 100℃; Onset time: 1.5min; Solvent delay: 1.0min; Rate: 75℃ / min; Final temperature: 250℃; Duration time: 4.3min.

[0217] Method B: Equipment: GC: Agilent 6890N G1530N, FID: Measurement temperature: 300℃ and MS: MSD5973G2577A, EI-forward, Measurement temperature: 280℃; Mass range: 50-550; Column: Restek RXi-5MS 20m, ID 180μm, df 0.18μm; Average flow rate: 50cm / s; Injection volume: 1μl; Syringe temperature: 250℃; Split ratio: 20 / 1; Carrier gas: He; Initial temperature: 60℃; Onset time: 1.5min; Solvent delay: 1.3min; Rate: 50℃ / min; Final temperature: 250℃; Duration time: 3.5min.

[0218] Method C: Equipment: GC: Agilent 6890N G1530N, FID: Measurement temperature: 300℃ and MS: MSD5973G2577A, EI-forward, Measurement temperature: 280℃; Mass range: 50-550; Column: Restek RXi-5MS 20m, ID 180μm, df 0.18μm; Average flow rate: 50cm / s; Injection volume: 1μl; Syringe temperature: 250℃; Split ratio: 20 / 1; Carrier gas: He; Initial temperature: 100℃; Onset time: 1.5min; Solvent delay: 1.3min; Rate: 75℃ / min; Final temperature: 250℃; Duration time: 4.5min.

[0219] Chiral LC:

[0220] Method A: (Instruments: Agilent 1260 Quart; Pump: G1311C; Autosampler: ColCom; DAD: Agilent G4212B, 220-320nm; Column: ...) OD-H 250x4.6 mm, temperature: 25℃, flow rate: 1 mL / min, no gradient: 90 / 10, time: 30 min, eluent A: heptane, eluent B: ethanol).

[0221] Preparative reversed-phase chromatography:

[0222] Method A: Device Type: Reveris TM Prep MPLC; Column: Phenomenex LUNA C18 (150x25 mm, 10μm); Flow rate: 40 mL / min; Column temperature: room temperature; Eluent A: 0.1% (v / v) formic acid in water, Eluent B: 0.1% (v / v) formic acid in acetonitrile; Gradient: t = 0 min 5% B, t = 1 min 5% B, t = 2 min 30% B, t = 17 min 70% B, t = 18 min 100% B, t = 23 min 100% B; Detection UV: 220 / 254 nm. Combine appropriate fractions and lyophilize.

[0223] Method B: Device Type: Reveris TM prep MPLC; column: Waters XSelect TM CSH C18 (145x25mm, 10μm); Flow rate: 40mL / min; Column temperature: room temperature; Eluent A: 10mM ammonium bicarbonate aqueous solution (pH=9.0); Eluent B: 99% acetonitrile + 1% 10mM ammonium bicarbonate aqueous solution; Gradient: t=0min 5% B, t=1min 5% B, t=2min 30% B, t=17min 70% B, t=18min 100% B, t=23min 100% B; Detection UV: 220 / 254nm. Combine appropriate fractions and lyophilize.

[0224] Chiral (Preparative) SFC

[0225] Method A: (Column: SFC instrument module: Waters Prep100q SFC system, PDA: Waters 2998, fraction collector: Waters 2767; Column: Phenomenex Lux Amylose-1 (250x20 mm, 5 μm), Column temperature: 35℃; Flow rate: 100 mL / min; ABPR: 170 bar; Eluent A: CO2, Eluent B: 20 ​​mM ammonia in methanol solution; No gradient 10% B, Time: 30 min, Detection: PDA (210-320 nm); PDA-based fraction collection).

[0226] Method B: (Column: SFC instrument module: Waters Prep100q SFC system, PDA: Waters 2998, fraction collector: Waters 2767; Column: Phenomenex Lux Celulose-1 (250x20 mm, 5μm), column temperature: 35℃; flow rate: 100 mL / min; ABPR: 170 bar; eluent A: CO2, eluent B: 20 ​​mM ammonia in methanol solution; no gradient 10% B, time: 30 min, detection: PDA (210-320 nm); fraction collection based on PDA).

[0227] Method C: (Column: SFC instrument module: Waters Prep100q SFC System, PDA: Waters2998; Column: Chiralpak IC (100x4.6 mm, 5μm), Column temperature: 35℃; Flow rate: 2.5 mL / min; ABPR: 170 bar; Eluent A: CO2, Eluent B: Methanol containing 20 mM ammonia; t = 0 min 5% B, t = 5 min 50% B, t = 6 min 50% B, Detection: PDA (210-320 nm); PDA-based fraction collection).

[0228] Method D: (Column: SFC instrument module: Waters Prep 100SFC UV / MS orientation system; Waters 2998 Photodiode Array (PDA) detector; Waters Acquity QDa MS detector; Waters 2767 sample manager; Column: Waters Torus 2-PIC 130A OBD (250x19 mm, 5μm); Column temperature: 35℃; Flow rate: 70 mL / min; ABPR: 120 bar; Eluent A: CO2; Eluent B: Methanol containing 20 mM ammonia; Linear gradient: t = 0 min 10% B, t = 4 min 50% B, t = 6 min 50% B; Detection: PDA (210-400 nm); PDATIC-based fractional collection).

[0229] Starting materials

[0230] Standard reagents and solvents were obtained and used at the highest commercial purity. The specific reagents purchased are described below.

[0231]

[0232]

[0233]

[0234] Synthesis steps of key intermediates

[0235] Intermediate 1: 1-(5-(4,6-dichloropyrimidin-2-yl)-2-methylpiperidin-1-yl) ethyl-1-one

[0236] In a 1 L steel autoclave, platinum oxide (IV) (0.5 g, 2.202 mmol) was added to a solution of methyl 6-methylnicotinate (100 g, 662 mmol) in acetic acid (250 mL), followed by stirring of the reaction mixture under a 10 bar hydrogen atmosphere at 60 °C. Rapid hydrogen consumption was observed, and the autoclave was refilled several times until hydrogen consumption ceased and reduction was complete. The mixture was cooled to room temperature and filtered through diatomaceous earth. The filtrate was concentrated to provide methyl 6-methylpiperidine-3-carboxylate acetate (143.8 g, 100%) as a diastereomer mixture, which was used as is for the next step. GCMS (Method A): tR 2.40 (80%) and 2.48 min (20%), 100%, MS (EI) 157.1 (M)+, 142.1 (M-Me)+. Sodium bicarbonate (82 g, 976 mmol) was carefully added to a solution of methyl 6-methylpiperidine-3-carboxylate acetate (53 g, 244 mmol) in a mixture of water (500 mL) and dichloromethane (500 mL) (effervescent!!), followed by the slow addition of acetic anhydride (29.9 g, 293 mmol). The reaction mixture was stirred at room temperature for 2 hours. The organic layer was separated, dried over sodium sulfate, filtered, and concentrated under vacuum to give methyl 1-acetyl-6-methylpiperidine-3-carboxylate (49 g, 100%) as a yellow oil. A solution of methyl 1-acetyl-6-methylpiperidin-3-carboxylic acid (49 g, 246 mmol) in ammonia in methanol (7 N, 500 mL, 3.5 mol) was stirred in a pressure vessel at 120 °C for 40 h. The mixture was cooled to room temperature and concentrated to give a pale yellow solid. This solid was dissolved in dichloromethane and filtered through a silica gel stopper. The filtrate was concentrated to give 1-acetyl-6-methylpiperidin-3-carboxamide, a grayish-white solid, which was used as is in the next step. A solution of 1-acetyl-6-methylpiperidin-3-carboxamide (266 mmol) obtained in the previous step in phosphorus oxychloride (500 mL, 5.37 mol) was stirred at room temperature for 16 h. The reaction mixture was evaporated under vacuum to give a heavy oil. This oil was co-evaporated twice with toluene, carefully distinguishing between cold saturated sodium carbonate (effervescent!) and ethyl acetate. The organic layer was separated from the alkaline aqueous layer, dried with sodium sulfate, filtered, and concentrated under vacuum to give a heavy oil product, which was then allowed to stand to solidify. The crude product was dissolved in dichloromethane and filtered through a silica gel stopper (eluted with a 10% methanol-dichloromethane solution). This yielded 1-acetyl-6-methylpiperidine-3-carboxynitrile (28 g, 63%) as an oily substance that solidified upon standing.GCMS (Method A): tR 3.78 (63%) and 3.89 min (378%), 100%, MS (EI) 166.1 (M)+. A solution of hydroxylamine (50% dissolved in water, 25.4 mL, 415 mmol) was added to a solution of 1-acetyl-6-methylpiperidine-3-carboximidamide (23 g, 138 mmol) in ethanol (300 mL), and the reaction mixture was stirred under reflux for 16 hours. The reaction mixture was concentrated and co-evaporated three times with ethyl acetate to dryness to give 1-acetyl-N-hydroxy-6-methylpiperidine-3-carboximidamide as a viscous solid. LCMS (Method A): tR 0.13 min, 100%, MS (ESI) 200.2 (M+H)+. Assuming a quantitative yield, the product was used as is in the next step. To a solution of 23 g (138 mmol) of 1-acetyl-N-hydroxy-6-methylpiperidine-3-formamidinium from the previous step in ethanol (500 mL), a slurry of acetic acid (23.79 mL, 416 mmol) and 50% Raney nickel in water (5 mL) was added. The reaction mixture was then stirred at 50 °C for 2 days under a hydrogen atmosphere. The mixture was filtered through diatomaceous earth, washed with some ethanol, and concentrated to give 70 g of a heavy oil. This oil was co-evaporated twice with ethyl acetate and thoroughly dried under vacuum to give 33 g (98%) of 1-acetyl-6-methylpiperidine-3-formamidinium acetate as a greenish-yellow oil for the next step. LCMS (Method A): tR 0.14 min, 90%, MS (ESI) 184.1 (M+H)+. Under a nitrogen atmosphere (60 mL), 1-acetyl-6-methylpiperidin-3-methylammonium acetate (32 g, 132 mmol) and dimethyl malonate (26.1 g, 197 mmol) were added to a solution of sodium (18.14 g, 789 mmol) in anhydrous methanol, and the reaction mixture was stirred at 50 °C for 16 h. The reaction mixture was concentrated, dissolved in water (300 mL), acidified to pH 4 with 6N hydrochloric acid, and precipitated. The precipitate was filtered off to give 1-(5-(4,6-dihydroxypyrimidin-2-yl)-2-methylpiperidin-1-yl)ethyl-1-one as a yellow solid (10.4 g, 31%), which was used as is in the next step. A suspension of 1-(5-(4,6-dihydroxypyrimidin-2-yl)-2-methylpiperidin-1-yl)ethyl-1-one (10.4 g, 41.4 mmol) in phosphorus oxychloride (200 mL, 2146 mmol) was stirred at 50 °C. The solid slowly dissolved after about 3 hours. After 5 hours, the reaction mixture was concentrated under vacuum and co-evaporated twice with toluene. The remaining oil was carefully quenched with ice, neutralized with a saturated aqueous sodium bicarbonate solution, and extracted with ethyl acetate (2 x 100 mL).The combined organic layers were dried with sodium sulfate and concentrated under vacuum to give 1-(5-(4,6-dichloropyrimidin-2-yl)-2-methylpiperidin-1-yl)ethyl-1-one (intermediate 1, 6.8 g, 57%), a yellow oily substance that solidified upon standing. LCMS (Method A): tR 1.88 min, 100%, MS (ESI) 288.1 (M+H)+.

[0237] Intermediate 2: 1-((2S,5R)-5-(4,6-dichloropyrimidin-2-yl)-2-methylpiperidin-1-yl)ethyl-1-one

[0238]

[0239] A solution of methyl 6-methylpiperidin-3-carboxylate (934 g, 2.38 mol, prepared according to intermediate 1) in ethanol (1.5 L) was added to the solution of N-acetyl-D-leucine (1 kg, 5.77 mol) in ethanol (1.5 L), and the mixture was heated to 40 °C. The resulting solution was allowed to reach room temperature over 16 hours, during which time precipitation occurred. The precipitate was filtered off, washed with diethyl ether (500 mL), and air-dried to give crude (3R,6S)-6-methylpiperidin-3-carboxylate acetyl-D-leucine salt (287 g, 34%) as a white solid. Crude (3R,6S)-6-methylpiperidin-3-carboxylate acetyl-D-leucine salt (287 g, 869 mmol) was crystallized from a hot mixture of ethanol and ethyl acetate in a 1:2 (1 L) ratio. The precipitate was filtered off, and the filter cake was ground in a mixture of diethyl ether and n-pentane in a 1:1 (500 mL) ratio. The precipitate was filtered off and air-dried to give a white solid (3R,6S)-6-methylpiperidine-3-carboxylic acid methyl ester acetyl-D-leucine salt (128 g, 44%). A saturated sodium carbonate solution (1 L) was added to a solution of (3R,6S)-6-methylpiperidine-3-carboxylic acid methyl ester acetyl-D-leucine salt (128 g, 387 mmol) in dichloromethane (1 L). The two-phase system was vigorously stirred for 10 minutes, and the layers were separated. The organic layer was dried over sodium sulfate and filtered to give a clear solution. Then, triethylamine (65 mL, 465 mmol) and acetic anhydride (44 mL, 465 mmol) were added, and the mixture was stirred at room temperature for 1 hour. The mixture was washed with saturated sodium bicarbonate solution, dried over sodium sulfate, and concentrated to give a pale yellow solid (3R,6S)-1-acetyl-6-methylpiperidine-3-carboxylic acid methyl ester (93 g). (3R,6S)-1-acetyl-6-methylpiperidine-3-carboxylic acid methyl ester (93 g, 387 mmol) dissolved in 7N ammonia water (600 mL, 4200 mmol) was added to an autoclave, and the mixture was heated to 60 °C for 3 days. The mixture was concentrated to give (3R,6S)-1-acetyl-6-methylpiperidine-3-carboxamide (102 g) as a pale yellow oil. Assuming a quantitative yield, the product was used as is in the next step. Chiral LC (Method A) tR = 12.35 min, >98% ee. Triethyloxonium tetrafluoroborate (77 g, 407 mmol) was added fractionally to a solution of (3R,6S)-1-acetyl-6-methylpiperidine-3-carboxamide (50 g, 271 mmol) in dichloromethane (500 mL), and the mixture was stirred at room temperature for 4 hours. 7N ammonia solution (200 ml, 9.15 mol) was slowly added to methanol, and the mixture was stirred at room temperature for 16 hours. The mixture was concentrated to give (3R,6S)-1-acetyl-6-methylpiperidine-3-methylamidine (50 g) as a pink solid for the next step.To a 5.4 M sodium methoxide solution in methanol (99 mL, 535 mmol), a solution of (3R,6S)-1-acetyl-6-methylpiperidin-3-methylammonium (49 g, 267 mmol) in methanol (400 mL) and dimethyl malonate (61.4 mL, 535 mmol) was added to methanol (200 mL). The mixture was heated to 50 °C and stirred for 24 hours. The mixture was acidified with concentrated hydrochloric acid (pH ~3) and concentrated to a smaller volume. The residue was filtered through silica (20% methanol-dichloromethane solution) and concentrated to give an orange oil. The crude product was purified by silica gel column chromatography (0% to 20% methanol-dichloromethane solution) to give 1-((2S,5R)-5-(4,6-dihydroxypyrimidin-2-yl)-2-methylpiperidin-1-yl)ethyl-1-one (12 g, 17%) as a colorless gel. LCMS (Method C): tR 0.17 min, 100%, MS (ESI) 252.1 (M+H)+.1-((2S,5R)-5-(4,6-dihydroxypyrimidin-2-yl)-2-methylpiperidin-1-yl)ethyl-1-one (12 g, 47.8 mmol) in phosphorus oxychloride (80 mL, 858 mmol) was stirred at 60 °C for 24 h. The reaction mixture was concentrated and co-evaporated twice with toluene to give a yellow oil. The oil was dissolved in ethyl acetate and washed with saturated sodium bicarbonate solution. The aqueous layer was extracted twice with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated to give a yellow oil. The oil was purified by silica gel column chromatography (0% to 20% tetrahydrofuran in toluene) to give 1-((2S,5R)-5-(4,6-dichloropyrimidin-2-yl)-2-methylpiperidin-1-yl)ethyl-1-one (intermediate 2, 1.5 g, 11%), as a colorless gel. LCMS (Method B): tR 3.34 min, 100%, MS (ESI) 288.0 (M+H)+; chiral UPLC (Method A): tR 2.54 min, >95% ee and de.

[0240] Intermediate 3: 1-((2S,5R)-5-(4-chloro-6-(pyrazin-2-yl)pyrimidin-2-yl)-2-methylpiperidin-1-yl) Synthesis of acetyl-1-one

[0241]

[0242] In a 1 L steel autoclave, platinum oxide (IV) (0.5 g, 2.202 mmol) was added to a solution of methyl 6-methylnicotinate (100 g, 662 mmol) in acetic acid (250 mL), and the reaction mixture was stirred at 60 °C under a 10 bar hydrogen atmosphere. Rapid hydrogen consumption was observed, and the autoclave was refilled several times until hydrogen consumption ceased. The mixture was cooled to room temperature and filtered through diatomaceous earth. The filtrate was carefully concentrated to give methyl 6-methylpiperidine-3-carboxylic acid methyl acetate (143.8 g, 100%) in the form of a diastereomer mixture for the next step. GCMS (Method A): t R 2.40 (80%) and 2.48 min (20%), 100%, MS (EI) 157.1 (M) + A mixture of diastereomers of methyl 6-methylpiperidine-3-carboxylate acetate (2.1 kg, 9924 mmol) was diluted with dichloromethane (4 L) and 4 M sodium hydroxide solution was slowly added until pH ~9. The layers were separated, and the aqueous layer was extracted twice with dichloromethane (after each extraction, the aqueous layer was alkalized with 4 M sodium hydroxide solution to pH ~9). The combined organic layers were dried over sodium sulfate and concentrated (35 °C, 450 mbar) to a smaller volume (~2 L) to give methyl 6-methylpiperidine-3-carboxylate (2.8 kg, 8905 mmol) as a yellow dichloromethane solution of about 50%. 1¹H NMR (400 MHz, CDCl₃, mixture of rotational isomers) δ 5.10 (s, .3H), 3.63 (s, 1H), 3.49–3.42 (m, 2.2H), 3.41–3.34 (m, 0.8H), 3.18–3.10 (m, 0.8H), 3.09–3.03 (m, 0.2H), 2.64–2.54 (m, 0.8H), 2.53–2.34 (m, 1.2H), 2.30–2.20 (m, 1H), 1.95–1.7 6(m, 1H), 1.53–1.36(m, 1H), 1.35–1.21(m, 1H), 1.04–0.90(m, 1H), 0.89–0.84(m, 0.8H), 0.83–0.76(m, 2.2H). A solution of methyl 6-methylpiperidin-3-carboxylate (934 g, 2.38 mol) in ethyl acetate (3 L) was added to an ethanol (1.5 L) solution of N-acetyl-D-leucine (1 kg, 5.77 mol), and the mixture was heated to 40 °C. The resulting solution was allowed to reach room temperature over 16 hours, during which time precipitation occurred. The precipitate was filtered off, washed with ether (500 mL), and air-dried to give crude (3R, 6S)-6-methylpiperidin-3-carboxylate acetyl-D-leucine salt (287 g, 34%) as a white solid. Crude (3R,6S)-6-methylpiperidin-3-carboxylic acid methyl ester acetyl-D-leucine salt (287 g, 869 mmol) was crystallized from a hot mixture of ethanol and ethyl acetate in a 1:2 (1 L) ratio. The precipitate was filtered off, and the filter cake was ground in a 1:1 (500 mL) mixture of diethyl ether and n-pentane. The precipitate was filtered off and air-dried to give (3R,6S)-6-methylpiperidin-3-carboxylic acid methyl ester acetyl-D-leucine salt (128 g, 44%) as a white solid. 1H-NMR (400MHz, DMSO-d6) δ7.80(d,J=8.2Hz,1H),5.80–5.00(s,2H),4.20–4.04(m,1H),3.63(s,3H),3.32–3.21(m,1H),2.93–2.80(m,2H),2. 73–2.65(m,1H),2.04–1.94(m,1H),1.82(s,3H),1.68–1.49(m,3H),1. 49–1.37(m,2H),1.30–1.15(m,1H),1.02(d,J=6.4Hz,3H),0.85(m,6H). A saturated sodium carbonate solution (1 L) was added to a solution of (3R,6S)-6-methylpiperidin-3-carboxylic acid methyl ester acetyl-D-leucine salt (128 g, 387 mmol) in dichloromethane (1 L). The biphase system was stirred vigorously for 10 min, and the layers were separated. The organic layer was dried over sodium sulfate and filtered to obtain a clear solution. Then, triethylamine (65 mL, 465 mmol) and acetic anhydride (44 mL, 465 mmol) were added, and the mixture was stirred at room temperature for 1 h. The mixture was washed with a saturated aqueous sodium bicarbonate solution, dried over sodium sulfate, and concentrated to give (3R,6S)-1-acetyl-6-methylpiperidin-3-carboxylic acid methyl ester (93 g) as a pale yellow solid. 1 ¹H-NMR (400MHz, CDCl₃, mixture of rotational isomers) δ 5.02–4.87 (m, 0.5H), 4.84–4.68 (m, 0.5H), 4.18–4.05 (m, 0.5H), 3.89–3.77 (m, 0.5H), 3.71 (d, J = 11.6Hz, 3H), 3.31–3.18 (m, 0.5H), 2.79–2.67 (m, 0.5H), 2.51–2.31 (m, 1H) ,2.11 (d, J = 6.7 Hz, 3H), 2.01–1.90 (m, 1H), 1.88–1.55 (m, 3H), 1.33–1.21 (m, 1.5H), 1.20–1.06 (m, 1.5H). A methanol solution of methyl (3R,6S)-1-acetyl-6-methylpiperidine-3-carboxylic acid (93 g, 387 mmol) in 7N ammonia (600 mL, 4200 mmol) was added to an autoclave and heated to 60 °C for 3 days. The mixture was concentrated to give a pale yellow oily product of (3R,6S)-1-acetyl-6-methylpiperidine-3-carboxamide (102 g). Assuming a quantitative yield, the product was used as is in the next step. 1¹H-NMR (400MHz, DMSO-d6, rotational isomer mixture) δ 7.38 (s, 1H), 6.89 (d, J = 24.7Hz, 1H), 4.76–4.59 (m, 0.5H), 4.39–4.24 (m, 0.5H), 4.16–4.01 (m, 0.5H), 3.72–3.51 (m, 0.5H), 3.14–2.99 (m, 0.5H), 2.68–2.51 (m, 0.5H), 2.30–2.12 (m, 0.5H), 2.11–1.92 (m, 3.5H), 1.78–1.38 (m, 4H), 1.23–1.11 (m, 1.5H), 1.09–0.94 (m, 1.5H); Chiral LC (Method A) t R =12.35 min, >98% ee. Triethyloxonium tetrafluoroborate (77 g, 407 mmol) was added fractionally to a solution of (3R,6S)-1-acetyl-6-methylpiperidine-3-carboxamide (50 g, 271 mmol) in dichloromethane (500 mL), and the mixture was stirred at room temperature for 4 hours. A solution of 7N ammonia in methanol (200 mL, 9.15 mol) was slowly added, and the mixture was stirred at room temperature for 16 hours. The mixture was concentrated to give (3R,6S)-1-acetyl-6-methylpiperidine-3-carboxamide (50 g) as a pink solid for the next step. To a 5.4 M sodium methoxide solution in methanol (99 mL, 535 mmol), a solution of (3R,6S)-1-acetyl-6-methylpiperidin-3-methylammonium (49 g, 267 mmol) in methanol (400 mL) and dimethyl malonate (61.4 mL, 535 mmol) was added to methanol (200 mL). The mixture was heated to 50 °C and stirred for 24 hours. The mixture was acidified with concentrated hydrochloric acid (pH ~3) and concentrated to a smaller volume. The residue was filtered through silica (20% methanol in dichloromethane solution) and concentrated to give an orange oil. The crude product was purified by silica gel column chromatography (0% to 20% methanol in dichloromethane solution) to give 1-((2S,5R)-5-(4,6-dihydroxypyrimidin-2-yl)-2-methylpiperidin-1-yl)ethyl-1-one (12 g, 17%) as a colorless gel. LCMS (Method C): t R 0.17min,100%,MS(ESI)252.1(M+H) +A solution of 1-((2S,5R)-5-(4,6-dihydroxypyrimidin-2-yl)-2-methylpiperidin-1-yl)ethyl-1-one (12 g, 47.8 mmol) in phosphorus oxychloride (80 mL, 858 mmol) was stirred at 60 °C for 24 hours. The reaction mixture was concentrated and co-evaporated twice with toluene to give a yellow oil. The oil was dissolved in ethyl acetate and washed with saturated sodium bicarbonate solution. The aqueous layer was extracted twice with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated to give a yellow oil. The oil was purified by silica gel column chromatography (0% to 20% tetrahydrofuran in toluene) to give 1-((2S,5R)-5-(4,6-dichloropyrimidin-2-yl)-2-methylpiperidin-1-yl)ethyl-1-one (1.5 g, 11%) as a colorless gel. 1 ¹H-NMR (400MHz, DMSO-d6, rotational isomer mixture) δ 7.95 (d, J = 7.3Hz, 1H), 4.85–4.72 (m, 1H), 4.69–4.62 (m, 1H), 4.23–4.13 (m, 1H), 4.07–3.98 (m, 1H), 3.97–3.88 (m, 1H), 3.00–2.89 (m, 1H), 2.81–2.67 (m, 1H), 2.09–1.72 (m, 7H), 1.71–1.58 (m, 2H), 1.25–1.14 (m, 3H), 1.12–1.05 (m, 2H); LCMS (Method B): t R 3.34 min, MS (ESI) 288.0 (M+H) + Chiral UPLC (Method: A)t R 2.54 min, >95% ee and de. Under argon, a solution of 2-tributyltinylpyrazine (607 mg, 1.65 mmol), 1-((2S,5R)-5-(4,6-dichloropyrimidin-2-yl)-2-methylpiperidin-1-yl)ethyl-1-one (500 mg, 1.74 mmol) and bis(triphenylphosphine)palladium(II) chloride (244 mg, 0.34 mmol) in 1,4-dioxane (20 mL) was heated to 100 °C and stirred for 32 hours. The mixture was diluted with dichloromethane containing 1% triethylamine and coated onto silica. Purification by silica gel column chromatography (dichloromethane containing 0% to 40% acetonitrile with 1% triethylamine) yielded 1-((2S,5R)-5-(4-chloro-6-(pyrazin-2-yl)pyrimidin-2-yl)-2-methylpiperidin-1-yl)ethyl-1-one (intermediate 3, 134 mg, 18%) as an orange gel. 1¹H-NMR (400MHz, DMSO-d6, mixture of rotational isomers) δ 9.46–9.41 (m, 1H), 8.80–8.76 (m, 1H), 8.65–8.59 (m, 1H), 8.33–8.29 (m, 1H), 7.66–7.59 (m, 1H), 4.86–4.70 (m, 0.5H), 4.27–4.17 (m, 0.5H), 4.09–3.97 (m, 0.5H),3.55–3.41(m,0.5H),3.06–2.98(m,0.5H),2.88–2.82(m,0.5H),2.10–1.90(m,6H),1.8 9–1.76(m,0.5H),1.75–1.61(m,1.5H),1.29–1.20(m,1.5H),1.17–1.10(m,1.5H); LCMS (Method C): t R 1.81 min, MS (ESI) 331.1 (M+H) + .

[0243] The synthesis process of the final product

[0244] Example 1: Synthesis of 1-((2S,5R)-2-methyl-5-(4-((5-methylpyridin-3-yl)amino)-6-(pyrazin-2-yl)pyrimidin-2-yl)piperidin-1-yl)acet-1-one (00001) and 1-((2R,5S)-2-methyl-5-(4-((5-methylpyridin-3-yl)amino)-6-(pyrazin-2-yl)pyrimidin-2-yl)piperidin-1-yl)acet-1-one (00002)

[0245]

[0246] A solution of 1M bis(trimethylsilyl)aminolithium in tetrahydrofuran (6.94 mL, 6.94 mmol) was added to a solution of 0.751 g (6.94 mmol) of 3-amino-5-methylpyridine in tetrahydrofuran (20 mL), and the mixture was stirred at room temperature for 10 minutes. Next, a solution of 1-(5-(4,6-dichloropyrimidin-2-yl)-2-methylpiperidin-1-yl)ethyl-1-one (intermediate 1.1 g, 3.47 mmol) in tetrahydrofuran (20 mL) was added, and the mixture was stirred at room temperature for 2 hours. The mixture was poured into a saturated ammonium chloride solution and extracted twice with ethyl acetate. The combined organic layers were washed once with brine, dried over sodium sulfate, and concentrated to give a yellow solid. The solid was purified by silica gel column chromatography (0% to 5% methanol in dichloromethane solution) to give 1-(5-(4-chloro-6-((5-methylpyridin-3-yl)amino)pyrimidin-2-yl)-2-methylpiperidin-1-yl)ethyl-1-one (788 mg, 60%) as a yellow foam. LCMS (Method B): tR 1.81 min, 100%, MS (ESI) 360.1 (M+H)+. Under nitrogen, 2-(tributyltinyl)pyrazine (103 mg, 0.28 mmol), 1-(5-(4-chloro-6-((5-methylpyridin-3-yl)amino)pyrimidin-2-yl)-2-methylpiperidin-1-yl)acet-1-one (50 mg, 0.14 mmol), and bis(triphenylphosphine)palladium(II) dichloride (9.75 mg, 0.01 mmol) were dissolved in N,N-dimethylformamide (3 mL). The mixture was heated to 80 °C and held for 24 hours, then cooled to room temperature. The mixture was eluted with acetonitrile through a C18 stopper, and the filtrate was purified by reversed-phase chromatography (Method B) and lyophilized to give 1-(2-methyl-5-(4-((5-methylpyridin-3-yl)amino)-6-(pyrazin-2-yl)pyrimidin-2-yl)piperidin-1-yl)acet-1-one (22 mg, 37%) as a white solid. The resulting mixture of cis-enantiomers was submitted to chiral preparation of SFC (Method A) and lyophilized to give two stereoisomers.1-((2S,5R)-2-methyl-5-(4-((5-methylpyridin-3-yl)amino)-6-(pyrazin-2-yl)pyrimidin-2-yl)piperidin-1-yl)acetyl-1-one (5 mg, 22%) LCMS (Method D): tR 3.17 min, 100%, MS (ESI) 404.1 (M+H)+; Chiral UPLC (Method A): tR 3.17 min, >95% ee and de. 1-((2R,5S)-2-methyl-5-(4-((5-methylpyridin-3-yl)amino)-6-(pyrazin-2-yl)pyrimidin-2-yl)piperidin-1-yl)acetyl-1-one (6 mg, 27%) LCMS (Method D): tR 3.17 min, 100%, MS (ESI) 404.2 (M+H)+; Chiral UPLC (Method A): tR 4.60 min, >95% ee and de.

[0247] Compounds 00003 (also referred to herein as compound B) and 00004 (also referred herein as compound A) were prepared using a method similar to that of Example 1, using suitable starting materials.

[0248]

[0249] Example 2: Synthesis of 1-((2S,5R)-5-(4-(imidazo[1,2-a]pyridin-6-ylamino)-6-(pyridin-3-yl)pyrimidin-2-yl)-2-methylpiperidin-1-yl)acet-1-one (00013)

[0250]

[0251] Under argon atmosphere, a solution of 3-(tributyltinyl)pyridine (607 mg, 1.65 mmol), 1-((2S,5R)-5-(4,6-dichloropyrimidin-2-yl)-2-methylpiperidin-1-yl)ethyl-1-one (intermediate 2,500 mg, 1.74 mmol), and bis(triphenylphosphine)palladium(II) chloride (244 mg, 0.34 mmol) in 1,4-dioxane (20 mL) was heated to 100 °C and stirred for 32 hours. The mixture was diluted with dichloromethane containing 1% triethylamine and coated onto silica. Purification by silica gel column chromatography (dichloromethane solution containing 0% to 40% acetonitrile of 1% triethylamine) yielded 1-((2S,5R)-5-(4-chloro-6-(pyridin-3-yl)pyrimidin-2-yl)-2-methylpiperidin-1-yl)ethyl-1-one (134 mg, 18%) as an orange gel. LCMS (Method C): tR 1.81 min, 100%, MS (ESI) 331.1 (M+H)+. Imidazolo[1,2-a]pyridine-6-amine (36.2 mg, 0.27 mmol) and hydrochloric acid (0.02 mL, 0.27 mmol) were added to a solution of 1-((2S,5R)-5-(4-chloro-6-(pyridin-3-yl)pyrimidin-2-yl)-2-methylpiperidin-1-yl)ethyl-1-one (30 mg, 0.09 mmol) in 2-propanol (2 mL). The mixture was stirred at 60 °C for 16 hours, poured into a saturated aqueous sodium bicarbonate solution, and extracted twice with ethyl acetate. The combined organic layers were dried over sodium sulfate and concentrated to give a yellow oil. The oil was purified by reversed-phase chromatography (Method B) and lyophilized to give 1-((2S,5R)-5-(4-(imidazo[1,2-a]pyridin-6-ylamino)-6-(pyridin-3-yl)pyrimidin-2-yl)-2-methylpiperidin-1-yl)ethane-1-one, as a blue solid. LCMS (Method B): tR 2.19 min, 100%, MS (ESI) 428.1 (M+H)+.

[0252] Compound 00030 was prepared using suitable starting materials in a manner similar to that of Example 2.

[0253]

[0254] Example 3A: Synthesis of 1-((2S,5R)-2-methyl-5-(4-((2-methylpyridin-4-yl)amino)-6-(pyridin-3-yl)pyrimidin-2-yl)piperidin-1-yl)acet-1-one (00071)

[0255]

[0256] A solution of 1M bis(trimethylsilyl)aminolithium in tetrahydrofuran (29.5 mL, 29.5 mmol) was added to a solution of 2-methylpyridin-4-amine (3.19 g, 29.5 mmol) in anhydrous tetrahydrofuran (100 mL), and the mixture was stirred for 10 minutes. Next, a solution of 1-((2S,5R)-5-(4,6-dichloropyrimidin-2-yl)-2-methylpiperidin-1-yl)ethyl-1-one (intermediate 2,850 mg, 2.95 mmol) in anhydrous tetrahydrofuran (100 mL) was added over 10 minutes, and the mixture was stirred at room temperature for 2 hours. The mixture was poured into a saturated ammonium chloride solution and extracted twice with ethyl acetate. The combined organic layers were washed once with brine, dried over sodium sulfate, and concentrated to give a brown oil. The oily substance was purified by silica gel column chromatography (80% to 100% ethyl acetate dissolved in n-heptane, then 0% to 10% methanol dissolved in dichloromethane) to give 1-((2S,5R)-5-(4-chloro-6-((2-methylpyridin-4-yl)amino)pyrimidin-2-yl)-2-methylpiperidin-1-yl)ethyl-1-one (275 mg 25%) as a yellow oil. LCMS (Method A): tR 1.49 min, 100%, MS (ESI) 360.1 (M+H)+. Under nitrogen, 1-((2S,5R)-5-(4-chloro-6-((2-methylpyridin-4-yl)amino)pyrimidin-2-yl)-2-methylpiperidin-1-yl)ethyl-1-one (275 mg, 0.76 mmol), sodium carbonate (162 mg, 1.53 mmol), pyridine-3-boronic acid (188 mg, 1.53 mmol), and PdCl2(dppf)-CH2Cl2 adduct (62.4 mg, 0.08 mmol) were dissolved in a mixture of 1,2-dimethoxyethane (6 mL) and water (2 mL). The mixture was heated to 80 °C and held for 1 hour, filtered through a C18 stopper, and concentrated to give a dark residue. The residue was purified by reversed-phase chromatography (Method B) and lyophilized to give a pale yellow solid. The product was further purified by chiral preparative SFC (Method B) and lyophilized to give 1-((2S,5R)-2-methyl-5-(4-((2-methylpyridin-4-yl)amino)-6-(pyridin-3-yl)pyrimidin-2-yl)piperidin-1-yl)acet-1-one (135 mg, 41%) as a beige solid. LCMS (Method D): tR 3.06 min, 100%, MS (ESI) 403.2 (M+H)+; Chiral SFC (Method B): tR 3.60 min, >95% ee and de.

[0257] Example 3B: Synthesis of 1-((2S,5R)-2-methyl-5-(4-((3-(1-methyl-1H-1,2,3-triazol-4-yl)phenyl)amino)-6-(pyrazin-2-yl)pyrimidin-2-yl)piperidin-1-yl)acet-1-one (Compound C)

[0258]

[0259] To a 2 mL solution of 1-((2S,5R)-5-(4-chloro-6-(pyrazin-2-yl)pyrimidin-2-yl)-2-methylpiperidin-1-yl)ethyl-1-one (intermediate 3, 120 mg, 0.36 mmol) in 2-propanol, 3-(1-methyl-1H-1,2,3-triazol-4-yl)aniline (188 mg, 1.08 mmol) and hydrochloric acid (0.08 mL, 1.08 mmol) were added. The mixture was stirred at 70 °C for 16 hours, poured into a saturated aqueous solution of sodium bicarbonate, and extracted twice with ethyl acetate. The combined organic layers were dried over sodium sulfate and concentrated to give a yellow oil. The oily substance was purified by reversed-phase chromatography (Method B) and lyophilized to give 1-((2S,5R)-2-methyl-5-(4-((3-(1-methyl1H-1,2,3-triazol-4-yl)phenyl)amino)-6-(pyrazin-2-yl)pyrimidin-2-yl)piperidin-1-yl)ethyl-1-one (compound C, 102 mg, 60%) as a white solid. 1 ¹H-NMR (400MHz, DMSO-d6, mixture of rotational isomers) δ 10.01 (d, J = 5.6 Hz, 1H), 9.56 (dd, J = 11.0, 1.1 Hz, 1H), 8.80 (d, J = 1.5 Hz, 2H), 8.54–8.42 (m, 2H), 7.72–7.54 (m, 2H), 7.53–7.39 (m, 2H), 4.86–4.76 (m, 1H), 4.27–4.16 (m, 0.5H) ,4.15–4.03(m,3.5H),3.58–3.42(m,0.5H),3.00–2.86(m,1H),2.86–2.68(m,0.5H),2.17–1.96(m,5H),1 .93–1.77(m,0.5H),1.76–1.64(m,1.5H),1.27(d,J=6.8Hz,1.5H),1.13(d,J=7.0Hz,1.5H); LCMS (Method D): t R 3.31 min, MS (ESI) 470.2 (M+H) + .

[0260] Example 4: Crystal structure of the human CREBBP bromine domain complexed with compound 00004 and BROMOscan of compounds A, C, and CCS1477 TM The result

[0261] Crystallization

[0262] Experimental setup: The construct used for crystallization contained residues 1081 to 1197. CREBBP crystals complexed with compound 00004 were obtained using a hanging drop vapor diffusion apparatus. CREBBP at a concentration of 20.3 mg / mL (10 mM Hepes, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP, pH 7.4) was pre-incubated for 1 h with 4.3 mM (3.0 molar excess) of 00004 (150 mM in DMSO). Then, 1 μL of the protein solution was mixed with 1 μL of the reservoir solution (0.1 M MgCl2, 0.1 M MES / NaOH pH 6.3, 18% (w / v) PEG 6000, and 10% (v / v) ethylene glycol) and equilibrated at 4 °C with 0.4 mL of the reservoir solution. Good diffraction crystals appeared and grew to full size within 4 days.

[0263] Data collection

[0264] Prior to installation, the crystal was cryogenically protected by adding 10% glycerol (final concentration) to the crystallization droplet. The complete crystallization of CREBBP / 00004 was collected using a Diamond Light Source (Didcot, UK, beamline i03). The dataset is integrated, analyzed, and scaled by XDS, Pointless, and Aimless within the autoPROC pipeline (Table 1).

[0265] Table 1: Data Collection Statistics

[0266]

[0267]

[0268] Structure determination and refinement

[0269] Molecular substitution was performed using the previously determined CREBBP structure as the starting model. Several rounds of alternating manual reconstruction and refinement using REFMAC5 yielded the final model (Table 2). Atom shift factors were modeled using an isotropic B factor per atom.

[0270] Table 2: Detailed Statistics

[0271]

[0272] Results: We prepared CREBBP / 00004 crystals, diffracted them to 1.6 Å resolution, and determined the three-dimensional structure of the protein-ligand complex. The clear electron density of the compound binding sites in each chain of CREBBP in the Fo-Fc omit map of the initial model reveals the binding of the entire compound. Figure 1 This allows for precise placement. Furthermore, the structure confirms the absolute stereochemistry of compound 00004 (2S,5R on the piperidine moiety).

[0273] BromoKdMAX - Measurement

[0274] BromoKdMAX was performed on DiscoverX. This assay can be used to determine whether a compound binds to the bromine domains of p300 and / or CBP at a specific Kd (e.g., 100 nM or lower).

[0275] The measurement principle is as follows: BROMOscan TM It is a novel, industry-leading platform for identifying small molecule bromine domain inhibitors. Based on the mature KINOMEscan... TM Technology, BROMOscan TM BROMOscan employs a proprietary ligand-binding site-specific competition analysis to quantitatively measure the interaction between the test compound and the bromine domain. This robust and reliable assay panel is suitable for high-throughput screening, providing quantitative ligand-binding data to facilitate the identification and optimization of potent and selective small-molecule bromine domain inhibitors. TM Measurements include trace bromine domain concentrations (<0.1 nM), thus reporting true thermodynamic inhibitor Kd values ​​(<0.1 nM to >10 μM) over a wide affinity range.

[0276] The assay was performed as follows: For the bromine domain assay, T7 phage strains exhibiting the bromine domain were grown in parallel in 24-well blocks in *E. coli* hosts derived from strain BL21. *E. coli* were grown to the logarithmic growth phase, infected with T7 phage from the cryopreservative stock solution (infection diversity = 0.4), and incubated at 32°C with shaking until lysis (90–150 min). The lysate was centrifuged (5000 x g) and filtered (0.2 μm) to remove cell debris. Streptavidin-coated magnetic beads were treated with biotinylated small molecules or acetylated peptide ligands for 30 min at room temperature to generate affinity resin for the bromine domain assay. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligands and reduce nonspecific phage binding. The binding reaction was performed by binding a bromine domain, liganding affinity beads, and the test compound (i.e., compound A, compound C, or CCS1477) in 1x binding buffer (17% SeaBlock, 0.33x PBS, 0.04% Tween 20, 0.02% BSA, 0.004% sodium azide, 7.4 mM DTT). The test compound was prepared in 100% DMSO at 1000x stock. Kd was determined using an 11-point 3-fold compound dilution series and a DMSO control point. All compounds used for Kd measurements were partitioned in 100% DMSO by acoustic transfer (non-contact partitioning). The compounds were then diluted directly into the assay to a final DMSO concentration of 0.09%. All reactions were performed in polypropylene 384-well plates. The final volume for each assay was 0.02 mL. The assay plates were incubated with shaking at room temperature for 1 hour, and the affinity beads were washed with washing buffer (1x PBS, 0.05% Tween 20). The beads were then resuspended in elution buffer (1x PBS, 0.05% Tween 20, 2 μM non-biotinylated affinity ligand) and incubated with shaking at room temperature for 30 minutes. The concentration of bromine domains in the elution buffer was measured by qPCR.

[0277] The results are as follows:

[0278]

[0279]

[0280] The relevant data are publicly available i) for SGC-CBP30, for example in the supplemental information in Wu et al., NATURE COMMUNICATIONS (2019) 10:1915 https: / / doi.org / 10.1038 / s41467-09672-2; ii) for GNE-781, for example in Romero et al., J.Med.Chem. 2017, 60, 9162-9183; and iii) for FT-6876, for example in poster #3079 of AACR Annual Meeting 2020, Virtual Conference II, June 22-24, 2020 (titled “FT-6876, a potent and selective inhibitor of CBP / p300 with antitumor activity in AR-positive breastcancer”).

[0281] Example 5

[0282] Materials and methods:

[0283] CBP bromine domain binding assay (TR-FRET):

[0284] A 10 mM DMSO solution was pre-diluted in DSMO to a 25x DMSO stock solution. It was then diluted to 4x in the assay buffer. A series of dilutions were performed in the assay buffer to maintain a stable DMSO concentration. 5 μL of the compound from the assay buffer was transferred into the assay plate (provided by the assay kit), and the TR-FRET assay (Cayman chemicals; 600850) was performed according to the manufacturer's instructions. After incubation in the dark at room temperature for 1 hour, the analysis plate was read in a Tecan M1000 plate reader using TR-FRET mode (top reading; excitation 340 nM bandwidth 20 nM; emission 620 nM bandwidth 7 nM; optimal gain determined for the first well, flashes: 5; flash frequency 100 Hz; integration time: 500 μs, lag time: 100 μs, room temperature). The TR-FRET ratio was calculated by dividing the 670 nm emission by the 620 nm emission. The EC50 was calculated based on the normalized value (DMSO = 1) and the positive control (0). The values ​​were logarithmically transformed and fitted to the dose-response curve using a nonlinear regression with a variable slope (4 parameters) to assess the EC50 values ​​(see Table 3 below).

[0285] Table 3:

[0286] EC50 specification: A* < 0.2 μM <A<1μM<B<10μM<C

[0287]

[0288] TR-FRET data indicate that compound 00003 with EC50 > 10 μM does not meet the definition of a CBP / p300 bromodomain inhibitor in this paper.

[0289] Example 6

[0290] Materials and methods

[0291] Label-free assay of cell proliferation:

[0292] 2000SNU-1411 cells [KCLB; 01411, a CRC (rectal adenocarcinoma) cell line carrying the KRAS G12C mutation] were seeded into 96-well plates (Greiner BioOne 655090) one day before drug treatment in RPMI medium containing 10% FCS and 2 mM L-glutamine. The next day, label-free imaging of the wells was performed using bright-field imaging on a CELIGO Image Cytometer to determine initial cell confluence. Cells were subsequently treated with DMSO, a single drug (AMG510) or any of the following CBP / p300 bromodomain inhibitors listed below, or a combination of drugs: (i) AMG510 and the CBP / p300 bromodomain inhibitor "Compound A", (ii) AMG510 and the CBP / p300 bromodomain inhibitor "Compound C", (iii) AMG510 and the CBP / p300 bromodomain inhibitor "CCS1477", (iv) AMG510 and the CBP / p300 bromodomain inhibitor "FT-6876", and (v) AMG510 and the CBP / p300 bromodomain inhibitor "GNE-781", at the following drug concentrations. The plates were periodically imaged over several weeks using a bright-field (CELIGO Imaging Cytometer) to track cell confluence in each well. Growth medium and treatments were replenished twice weekly. The drugs and concentrations were as follows: 300 nM AMG510 (a covalent KRAS G12C-specific inhibitor, ChemieTek#CT-AMG510), and for CBP / p300 bromine domain inhibitors: 1 μM compound A, 0.2 μM compound C, 0.2 μM CCS1477 (ChemiTek; CT-CCS1477), 1 μM FT-6876 (“CBP / P300-IN-8”, MedChemExpress; HY-136920), and 0.2 μM GNE-781 (MedChemExpress; HY-108696). In bright-field mode, the degree of fusion was determined using the built-in “fusion” analysis tool in CELIGO software.

[0293] Figure 2A through E show the assessment of SNU-1411 fusion over 32 days. CBP / p300 bromodomain inhibitors [(A) compound A, (B) compound C, (C) CCS1477, (D) FT-6876, and (E) GNE-781] did not affect the cell proliferation of KRAS G12C-mutant CRC cells in the absence of KRAS G12C inhibitors, but prevented the development of resistance to 300 nM AMG510 when used in combination with AMG510. It is important to note that the DMSO curves and time processes after 300 nM AMG510 treatment were different in the groups. Figure 2 A and D and Figure 2 B, C, and E are the same because their respective conditions (A, D, and B, C, and E) were run on the same plate (per plate: DMSO: 9 wells, CBP / p300 bromodomain inhibitor: 3 wells each, AMG510: 6 wells, and all combinations of AMG510 + CBP / p300 bromodomain inhibitor: 6 wells, mean ± SD). An example plate is shown.

[0294] Result: From Figure 2 The results from the AE studies showed that, when used alone, CBP / p300 bromodomain inhibitors had no or at most a weak effect on the confluence of SNU-1411 cells, while 300 nM AMG510 initially delayed cell proliferation by several days. In long-term cultures, SNU-1411 cells regrowed when treated with AMG510 alone, while co-treatment with AMG510 in combination with different CBP / p300 bromodomain inhibitors completely halted or significantly reduced regrowth over the 32-day study period.

[0295] Example 7

[0296] Materials and methods

[0297] Label-free assay of cell proliferation:

[0298] 2000SNU-1411 cells [KCLB; 01411, a CRC (rectal adenocarcinoma) cell line carrying the KRAS G12C mutation] were seeded into 96-well plates (Greiner BioOne 655090) one day before drug treatment in RPMI medium containing 10% FCS and 2 mM L-glutamine. The next day, label-free imaging of the wells was performed using bright-field imaging on a CELIGO Image Cytometer to determine initial cell confluence. Cells were subsequently treated with DMSO, a single drug, namely MRTX849 or any of the following CBP / p300 bromodomain inhibitors, or a combination of drugs, namely (i) MRTX849 and CBP / p300 bromodomain inhibitor "Compound A", (ii) MRTX849 and CBP / p300 bromodomain inhibitor "Compound C", (iii) MRTX849 and CBP / p300 bromodomain inhibitor "CCS1477", (iv) MRTX849 and CBP / p300 bromodomain inhibitor "FT-6876", and (v) MRTX849 and CBP / p300 bromodomain inhibitor "GNE-781", at the following drug concentrations. The plates were periodically imaged over several weeks using a bright-field (CELIGO Imaging Cytometer) to track cell confluence in each well. Growth medium and treatments were replenished twice weekly. The drugs and concentrations were as follows: 300 nM MRTX849 (a covalent KRASG12C-specific inhibitor, Selleckchem#S8884) and CBP / p300 bromine domain inhibitors: 1 μM compound A, 0.2 μM compound C, 0.2 μM CCS1477 (ChemiTek; CT-CCS1477), 1 μM FT-6876 (“CBP / P300-IN-8”, MedChemExpress; HY-136920), and 0.2 μM GNE-781 (MedChemExpress; HY-108696). In bright-field mode, the degree of blending was determined using the built-in “Blending” analysis tool in CELIGO software.

[0299] Figure 3A through E show the assessment of SNU-1411 fusion over 32 days. CBP / p300 bromodomain inhibitors [(A) compound A, (B) compound C, (C) CCS1477, (D) FT-6876, and (E) GNE-781] did not affect the cell proliferation of KRAS G12C-mutant CRC cells in the absence of KRAS G12C inhibitors, but prevented the development of resistance to 300 nM MRTX849 when used in combination with MRTX849. It is important to note that the DMSO curves and time processes of 300 nM MRTX849 treatment in the group... Figure 3 A and D and Figure 3 B, C, and E are identical because their respective conditions (A, D, and B, C, and E) were run on the same plate (per plate: DMSO: 9 wells, CBP / p300 bromodomain inhibitor: 3 wells each, MRTX849: 6 wells, and all combinations of MRTX849 + CBP / p300 bromodomain inhibitor: 6 wells, mean ± SD). An example plot is shown.

[0300] Result: From Figure 3 The results from the AE study showed that, when used alone, CBP / p300 bromodomain inhibitors had no or at most a slight effect on the confluence of SNU-1411 cells, while 300 nM MRTX849 initially delayed cell proliferation by several days. In long-term cultures, SNU-1411 cells regrowed when treated with MRTX849 alone, but co-treatment with MRTX849 in combination with different CBP / p300 bromodomain inhibitors prevented regrowth over the 32-day study period.

[0301] Example 8

[0302] Materials and methods

[0303] Label-free assay of cell proliferation:

[0304] 2000SW837 cells [ATCC; CCL-235, a CRC (rectal adenocarcinoma) cell line carrying the KRAS G12C mutation] were seeded into 96-well plates (Greiner BioOne 655090) one day before drug treatment in RPMI medium containing 10% FCS and 2 mM L-glutamine. The next day, label-free imaging of the wells was performed using bright-field imaging on a CELIGO Image Cytometer to determine initial cell confluence. Cells were subsequently treated with DMSO, a single drug (AMG510) or any of the following CBP / p300 bromodomain inhibitors listed below, or a combination of drugs: (i) AMG510 and the CBP / p300 bromodomain inhibitor "Compound A", (ii) AMG510 and the CBP / p300 bromodomain inhibitor "Compound C", (iii) AMG510 and the CBP / p300 bromodomain inhibitor "CCS1477", (iv) AMG510 and the CBP / p300 bromodomain inhibitor "FT-6876", and (v) AMG510 and the CBP / p300 bromodomain inhibitor "GNE-781", at the following drug concentrations. The plates were periodically imaged over several weeks using a bright-field (CELIGO Imaging Cytometer) to track cell confluence in each well. Growth medium and treatments were replenished twice weekly. The drugs and concentrations were as follows: 100 nM AMG510 (a covalent KRAS G12C-specific inhibitor, ChemieTek#CT-AMG510) and CBP / p300 bromine domain inhibitors: 1 μM compound A, 0.2 μM compound C, 0.2 μM CCS1477 (ChemiTek; CT-CCS1477), 1 μM FT-6876 (“CBP / P300-IN-8”, MedChemExpress; HY-136920), and 0.2 μM GNE-781 (MedChemExpress; HY-108696). In bright-field mode, the degree of blending was determined using the built-in “Blending” analysis tool in CELIGO software.

[0305] Figure 4 A through E show the assessment of SW837 fusion rate over 49 days. CBP / p300 bromodomain inhibitors [(A) compound A, (B) compound C, (C) CCS1477, (D) FT-6876, and (E) GNE-781] did not affect the cell proliferation of KRAS G12C-mutant CRC cells in the absence of KRAS G12C inhibitors, but prevented the development of resistance to 100 nM AMG510 when used in combination with AMG510. It is important to note that the DMSO curves and time processes of 100 nM AMG510 treatment were different in the groups. Figure 4 The same applies to A through E, as all conditions were run in parallel (per plate: DMSO: 18 wells, CBP / p300 bromodomain inhibitor: 6 wells each, AMG510: 12 wells, and all combinations of AMG510 + CBP / p300 bromodomain inhibitor: 12 wells, mean ± SD).

[0306] Result: From Figure 4 The results from the AE study showed that CBP / p300 bromodomain inhibitors, when used alone, had no or at most a weak effect on the confluence of SW837 cells, while 100 nM AMG510 initially inhibited cell proliferation. In long-term cultures, SW837 cells regrowed when treated with AMG510 alone, while co-treatment with AMG510 in combination with different CBP / p300 bromodomain inhibitors completely inhibited or strongly reduced regrowth over the 49-day study period.

[0307] Example 9

[0308] Materials and methods

[0309] Label-free assay of cell proliferation:

[0310] 2000 NCI-H358 cells [KCLB; 25807, a non-small cell lung cancer (NSCLC) cell line carrying a KRAS G12C mutation] were seeded into 96-well plates (Greiner BioOne 655090) one day prior to drug treatment in RPMI medium containing 10% FCS and 2 mM L-glutamine. The next day, the wells were labeled-free using bright-field imaging on a CELIGO Image Cytometer to determine the initial cell number. Cells were subsequently treated with DMSO, a single drug (AMG510) or one of the two CBP / p300 bromodomain inhibitors listed below, or a combination of drugs, namely (i) AMG510 and the CBP / p300 bromodomain inhibitor "Compound A", and (ii) AMG510 and the CBP / p300 bromodomain inhibitor "Compound C", at the following drug concentrations. Cells were imaged periodically over several weeks using bright-field mode (CELIGO Imaging Cytometer) to track cell proliferation over time in each well. Growth medium and treatments were replenished twice weekly. Drugs and concentrations were as follows: 100 nM AMG510 (ChemieTek#CT-AMG510), 1 μM compound A, 200 nM compound C, and combinations of 100 nM AMG510 + 1 μM compound A or 100 nM AMG510 + 200 nM compound C. Cell counts were determined using the Direct Cell Counting tool built into the CELIGO software in bright-field mode.

[0311] Figure 5 Figures A and B show the assessment of NCI-H385 cell numbers over time [in days]. In the absence of a KRAS G12C inhibitor, neither compound A (Figure A) nor compound C (Figure B) reduced cell proliferation in the KRAS G12C-mutant NSCLC cell line NCI-H358. However, when used in combination with a covalently specific KRAS G12C inhibitor, compounds A and C prevented the development of resistance (for...). Figure 5 A: DMSO: n=6, Compound A: n=6, AMG510: n=24, AMG510+ Compound A: n=24, Mean ± SD; For Figure 5 B: DMSO: n=6, compound C: n=6, AMG510: n=24, AMG510+ compound C: n=24, mean ± SD.

[0312] Results: Compounds A and C, when used alone, did not reduce the number of NCI-H358 cells, while 100 nAMG510 initially completely blocked cell proliferation. In long-term culture, NCI-H358 cells regrowed when treated with AMG510 alone, while combined treatment with AMG510 and either compound A (in A) or compound C (in B), which were inhibitors of the CBP / p300 bromodomain, completely prevented regrowth during the studied time period (>20 days).

Claims

1. Use of a combination of (i) a CBP / p300 bromodomain inhibitor and (ii) a KRAS inhibitor in the manufacture of a medicament for the treatment of a patient suffering from a cancer, wherein the cancer exhibits an oncogenic alteration in KRAS, wherein the oncogenic alteration is caused by at least one base mutation in the KRAS gene which results in the amino acid substitution G12C in KRAS and the KRAS inhibitor is a KRAS G12C inhibitor, wherein the CBP / p300 bromodomain inhibitor is selected from (Compound A), (Compound C), (CCS1477), (FT-6876), and (GNE-781), and the KRAS G12C inhibitor is selected from (AMG510), and (MRTX849), and wherein the cancer is selected from lung cancer, colorectal cancer and pancreatic cancer.

2. Use of the combination according to claim 1, wherein the oncogenic alteration in KRAS results in over-activation of KRAS signaling.

3. Use of the combination according to claim 1 or 2, wherein the combination is administered to the patient during each treatment cycle.

4. Use of the combination according to claim 1 or 2, wherein (i) and (ii) are administered as separate dosage forms or are comprised in a single dosage form.

5. Use of the combination according to claim 3, wherein if (i) and (ii) are administered as separate dosage forms, the administration during each treatment cycle is concomitantly or sequentially.

6. A kit comprising (i) a pharmaceutical dosage form comprising a CBP / p300 bromodomain inhibitor and (ii) a pharmaceutical dosage form comprising a KRAS inhibitor, wherein the KRAS inhibitor is a KRAS G12C inhibitor, and wherein the CBP / p300 bromodomain inhibitor is selected from (Compound A), (Compound C), (CCS1477), (FT-6876), and (GNE-781), and the KRAS G12C inhibitor is selected from (AMG510), and (MRTX849).

7. A pharmaceutical dosage form comprising (i) a CBP / p300 bromodomain inhibitor and (ii) a KRAS inhibitor, wherein the KRAS inhibitor is a KRAS G12C inhibitor, and wherein the CBP / p300 bromodomain inhibitor is selected from (Compound A), (Compound C), (CCS1477), (FT-6876), and (GNE-781), and the KRAS G12C inhibitor is selected from (AMG510) and (MRTX849).

Images