EA050570B1 - Combination of a CBP/P300 bromodomain inhibitor and a KRAS inhibitor for the treatment of cancer - Google Patents

Abstract

The present invention relates, inter alia, to: (i) a combination of a CBP/p300 bromodomain inhibitor and a KRAS inhibitor for use in treating a patient suffering from cancer, wherein the cancer is characterized by an oncogenic alteration in KRAS; (ii) a kit comprising: (a) a pharmaceutical dosage form comprising a CBP/p300 bromodomain inhibitor, and (b) a pharmaceutical dosage form comprising a KRAS inhibitor; and (iii) a pharmaceutical dosage form comprising a CBP/p300 bromodomain inhibitor and a KRAS inhibitor.

Description

Field of technology to which the present invention relates

The present invention relates to the field of cancer treatment. In particular, the present invention relates to a combination of a CBP/p300 bromodomain inhibitor and a KRAS inhibitor for use in the treatment of a patient suffering from cancer, wherein the cancer is characterized by an oncogenic alteration in KRAS. The present invention also relates to a kit comprising a pharmaceutical dosage form comprising a CBP/p300 bromodomain inhibitor and a pharmaceutical dosage form comprising a KRAS inhibitor. Furthermore, the present invention relates to a pharmaceutical dosage form comprising a CBP/p300 bromodomain inhibitor and a KRAS inhibitor.

Prior art of the present invention

KRAS mutations occur in 25% of cancers, with oncogenic variants having varying prevalence across cancer types (see Box 1 in Mullard, Nature Reviews DRUG DISCOVERY, Vol. 18, December 2019: 887–891). For example, KRAS mutations in human cancers occur at a frequency of 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 lung, 1% in the skin (melanoma), 8% in the cervix, and 5% in the urinary tract (see Table 1 in Li et al., Nature Reviews Cancer 18, 767–777 (2018)). Accordingly, there is considerable interest in agents that block proliferative signaling induced by oncogenic KRAS variants.

KRAS has been considered an untreatable target for decades, but at least five KRAS-modulating agents are now in clinical use (see Table 1 in the Mullard publication cited above). While preliminary clinical data on the use of promising KRAS inhibitors as monotherapies in non-small cell lung cancer and colorectal cancer show promise, combination strategies that may provide deeper and more durable responses are already being explored and tested in clinical trials such as NCT04185883 and NCT03785249. Examples of such combination therapies include the combination of AMG510 (sotorasib) or MRTX849 with pembrolizumab, the combination of a KRAS inhibitor and an SHP2 inhibitor, and a KRAS inhibitor and a CDK4 inhibitor (see paragraph from p. 888 to p. 889 of the Mullard publication cited above).

Thus, there is a need for further combination strategies that also result in deeper and more durable responses, and ideally a combination strategy that overcomes the decline in effect of the KRAS inhibitor over time when administered as a single agent (or when administered in combination with other anticancer agents).

Objectives and summary of the present invention

The present invention is directed to the use of a combination of: (i) a CBP/p300 bromodomain inhibitor and (ii) a KRAS inhibitor for the treatment of a patient suffering from cancer, wherein the cancer is characterized by an oncogenic alteration in KRAS, wherein the oncogenic alteration is caused by at least one base mutation in the KRAS gene resulting in a G12C amino acid substitution in KRAS, and the KRAS inhibitor is a KRAS G12C inhibitor.

In another aspect, the present invention relates to a kit for treating a patient suffering from cancer, 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.

The present inventors have unexpectedly discovered that a CBP/p300 bromodomain inhibitor, i.e., a bromodomain inhibitor that selectively binds to the CBP/p300 bromodomain, provides effective treatment of cancer with an oncogenic alteration in KRAS when administered in combination with a KRAS inhibitor, while the CBP/p300 bromodomain inhibitor does not affect the cellular proliferation of cancer cells when administered alone. In other words, the present inventors have unexpectedly discovered that the combination of a CBP/p300 bromodomain inhibitor and a KRAS inhibitor is more effective in the treatment of cancer characterized by an oncogenic alteration in KRAS than the effect of either of these two active agents alone on cancer characterized by an oncogenic alteration in KRAS. Thus, as noted above, the CBP/p300 bromodomain inhibitor has no effect when administered alone (where lack of effect specifically means the absence of objective responses, as defined by the RECIST 1.1 response criteria, in target lesions or non-target lesions in a subject), and the effect of the KRAS inhibitor, when administered alone, diminishes over time, likely due to the development of resistance to the KRAS inhibitor.

In a first aspect, the present invention is directed to a combination of: (i) a CBP/p300 bromodomain inhibitor and (ii) a KRAS inhibitor for use in the treatment of a patient suffering from cancer, wherein the cancer is characterized by an oncogenic alteration in KRAS. The first aspect may also be a combination of (i) a CBP/p300 bromodomain inhibitor and (ii) a KRAS inhibitor for use in the treatment of a patient suffering from cancer, wherein the cancer is characterized by a KRAS mutational profile, in one or more indications for use of a KRAS inhibitor (such as KRAS G12C) specified on the label.

- 1 050570 ke/package insert of the combination, or where the cancer is characterized by a KRAS mutational profile that is targeted in a clinical trial setting by a KRAS inhibitor (such as KRAS G12C) used in the combination.

In a preferred embodiment of the first aspect, the oncogenic alteration in KRAS results in overactivation of the KRAS signaling pathway. The oncogenic alteration in KRAS may even result in a constitutively active KRAS signaling pathway (meaning that KRAS signaling activity, namely KRAS binding to GTP, is constitutively active).

In another preferred embodiment of the first aspect, the oncogenic alteration in KRAS is caused by at least one base mutation in the KRAS gene resulting in an amino acid substitution in KRAS selected from the group consisting of G12C, G12V, G12D, G13D, Q61H, Q61L, Q61R, K117N, and a combination thereof. It may be preferable that the oncogenic alteration is caused by at least one base mutation in the KRAS gene resulting in an amino acid substitution in KRAS selected from the group consisting of G12C, G12V, and G12D. Most preferably, the oncogenic alteration in KRAS is caused by at least one mutation in the KRAS gene resulting in an amino acid substitution G12C in KRAS.

In another embodiment of the first aspect, the cancer is selected from the group consisting of 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 a G12C mutation in KRAS (as used herein, this is equivalent to the alternative formulation as treating a patient suffering from NSCLC, optionally locally advanced or metastatic NSCLC, wherein the NSCLC is characterized by an oncogenic G12C change in KRAS).

In another embodiment of the first aspect of the present invention, the CBP/p300 bromodomain inhibitor is a small molecule inhibitor. Thus, in this embodiment, the CBP/p300 bromodomain inhibitor is not a nucleic acid-based inhibitor, such as, for example, shRNA or RNAi, which targets CBP and/or p300.

In another embodiment of the first aspect of the present invention, the KRAS inhibitor is a small molecule inhibitor. Thus, in such an embodiment of the present invention, the KRAS inhibitor is not a nucleic acid-based inhibitor, such as, for example, shRNA or RNAi targeting KRAS. In a further embodiment of the first aspect of the present invention, the KRAS inhibitor targets KRAS G12C, i.e., is aimed at the treatment of a cancer characterized by an oncogenic G12C alteration in KRAS. Such an inhibitor may, in particular, be a covalent inhibitor that targets the cysteine at position 12 present in KRAS G12C through covalent interactions.

The CBPφ300 bromodomain inhibitor may be selected from the group consisting of compound A, compound C, compound 00030, compound 00071, compound CCS1477, compound GNE-781, compound GNE-049, compound SGC-CBP30, compound CPI-637, compound FT-6876, compound 462, compound 424, and compound 515. These compounds are either commercially available or publicly disclosed as described below, or their synthesis and structures are shown in the examples of the present application. Preferably, the CBPφ300 bromodomain inhibitor is selected from the group consisting of compound A, compound C, compound CCS1477, compound GNE-781, compound GNE-049, compound CPI-637, compound 462, compound 424, and compound 515.

The KRAS inhibitor may be selected from the group consisting of AMG510, MRTX849, JNJ-74699157/ARS-3248, BI 1701963, BI 1823911, BAY-293, GDC-6036, MRTX1133, a RAS(ON) inhibitor (where the RAS(ON) inhibitor is preferably RMC-6291 or RMC-6236), and combinations thereof. In a more preferred embodiment of the present invention, the KRAS inhibitor is AMG510 or MRTX849. Most preferably, the KRAS inhibitor is AMG510.

It should be understood that the combination (i) and (ii) mentioned herein may be an open or closed combination. Thus, the combination (i) and (ii) of the present invention can be used to treat a patient suffering from cancer, wherein the cancer is characterized by an oncogenic alteration in KRAS, and wherein (i) and (ii) are the only active agents (closed combination). However, alternatively, the combination of (i) and (ii) may be used to treat a patient suffering from a cancer, wherein the cancer is characterized by an oncogenic alteration in KRAS, and wherein at least one additional active agent (iii) may be administered, for example, an active agent (iii) selected from the group consisting of a PD1 inhibitor, a MEK inhibitor, an allosteric SHP2 inhibitor, a Pan-ErbB tyrosine kinase inhibitor, a PD-L1 inhibitor, an EGFR inhibitor, a chemotherapeutic drug, an mTOR inhibitor, a CDK inhibitor, a VEGF inhibitor, pembrolizumab, cetuximab, atezolizumab, bevacizumab, and combinations of any of the above (open-label combination).

In a preferred embodiment of the first aspect of the present invention, the combination is administered to the patient during each treatment cycle.

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In another embodiment of the first aspect of the present invention, the CBP/p300 bromodomain inhibitor and the KRAS inhibitor are administered as separate dosage forms or are included in a single dosage form. If the CBP/p300 bromodomain inhibitor and the KRAS inhibitor are administered as separate dosage forms, administration during each treatment cycle may be simultaneous or sequential. This includes an option in which the CBP/p300 bromodomain inhibitor is administered first, followed by the KRAS inhibitor.

In another embodiment of the first aspect of the present invention, the treatment results in an increased duration of therapeutic effect of the KRAS inhibitor compared to the duration of therapeutic effect of the KRAS inhibitor when administered as the sole active agent. In another embodiment of the present invention, the treatment results in an increased therapeutic efficacy of the KRAS inhibitor compared to the therapeutic efficacy of the KRAS inhibitor when administered as the sole active agent. In another embodiment of the present invention, the treatment results in the prevention of resistance to the KRAS inhibitor.

In another embodiment of the first aspect of the present invention, the CBP/p300 bromodomain inhibitor is administered at a daily dose of from about 1 mg to about 3000 mg, preferably from about 10 mg to about 2000 mg, more preferably from about 15 mg to about 1000 mg. It may be preferable to administer the CBP/p300 bromodomain inhibitor 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 may be intermittent, i.e. not every day, but the above-mentioned daily amount may be administered on the day of administration. If compound CCS1477, compound 462, compound 424, or compound 515 is used as the CBP/p300 bromodomain inhibitor, the corresponding compound may be administered at a daily dose of from about 10 mg to about 600 mg.

In another embodiment of the first aspect of the present invention, the KRAS inhibitor is administered at a daily dose that is within the typical daily dose range (in particular, the daily dose indicated for the KRAS inhibitor on the label/package insert, if indicated) when the KRAS inhibitor is administered as the sole active agent. The typical daily dose (or the indicated daily dose, if indicated) depends on the particular KRAS inhibitor to be used. Typically, the KRAS inhibitor is administered at a daily dose of from about 10 mg to about 2000 mg. For example, AMG510 can be administered in a combination for use according to the present invention at a daily dose of from about 240 mg to about 1200 mg, from about 480 mg to about 1200 mg, or from about 600 mg to about 1200 mg, preferably from about 240 mg to about 1200 mg, preferably from about 720 mg to about 1080 mg, and more preferably from about 840 mg to about 960 mg, or at a dose of about 960 mg. For example, MRTX849 can be administered in a combination for use according to the present invention at a daily dose of from about 200 mg to about 1400 mg or from about 400 mg to about 1300 mg, preferably from about 600 mg to about 1200 mg, and most preferably at a dose of about 1200 mg.

In another embodiment of the first aspect of the present invention, the KRAS inhibitor is administered at a daily dose that is lower than the typical daily dose specified above when the KRAS inhibitor is administered as the sole active agent. In other words, if the KRAS inhibitor is administered not as the sole active agent, but in a combination for use in accordance with the present invention, the KRAS inhibitor may be administered in an amount lower than that used when the KRAS inhibitor is administered as the sole active agent. For example, for the above examples, this means that the daily amount will be at the lower limits of the specified ranges or even below the limits of these ranges.

In another embodiment of the first aspect of the present invention, the invention is directed to a combination of (i) a CBPf300 bromodomain inhibitor and (ii) a KRAS G12D inhibitor (preferably MRTX1133) for use in the treatment of a patient suffering from cancer, wherein the cancer is characterized by an oncogenic G12D alteration in KRAS. Furthermore, in this embodiment of the present invention, it may be preferable that the cancer is selected from the group consisting of lung cancer, preferably NSCLC, colorectal cancer, and pancreatic cancer.

In a most preferred embodiment of the first aspect of the present invention, the invention is directed to a combination of: (i) a CBPf300 bromodomain inhibitor and (ii) a KRAS G12C inhibitor for use in the treatment of a patient suffering from cancer, wherein the cancer is characterized by an oncogenic G12C alteration in KRAS. Furthermore, in this embodiment of the present invention, it may be preferred that the cancer is selected from the group consisting of lung cancer, preferably NSCLC, colorectal cancer, and pancreatic cancer. Lung cancer may be preferred. It may also be preferred that the KRAS G12C inhibitor is selected from the group consisting of

- 3 050570 consisting of the compounds AMG510, MRTX849, JNJ-74699157/ARS-3248, BI 1823911, GDC-6036, a RAS G12C (ON) inhibitor (where the RAS G12C (ON) inhibitor is preferably RMC-6291) and combinations thereof; preferably, the KRAS G12C inhibitor is AMG510 or MRTX849.

In another preferred embodiment of the first aspect of the present invention, the invention is directed to a combination of: (i) a bromodomain inhibitor of CBPf300 and (ii) a KRAS G12C inhibitor, preferably AMG510 or MRTX849, for use in the treatment of a patient suffering from locally advanced or metastatic NSCLC who has received at least one prior systemic therapy, wherein the NSCLC is characterized by an oncogenic G12C alteration in KRAS.

In another embodiment of the first aspect of the present invention, the invention is directed to a combination of: (i) compound A and (ii) a KRAS inhibitor for use in the treatment of a patient suffering from cancer, wherein the cancer is characterized by an oncogenic alteration in KRAS. In this embodiment of the present invention, it may be preferred that the KRAS inhibitor is a KRAS G12C inhibitor, preferably AMG510 or MRTX849, and that the cancer is characterized by an oncogenic alteration in G12C in KRAS. Furthermore, in this embodiment of the present invention, it may be preferred that the cancer is selected from the group consisting of lung cancer, preferably NSCLC, colorectal cancer, and pancreatic cancer.

In another embodiment of the first aspect of the present invention, the invention is directed to a combination of: (i) compound C and (ii) a KRAS inhibitor for use in the treatment of a patient suffering from cancer, wherein the cancer is characterized by an oncogenic alteration in KRAS. In this embodiment of the present invention, it may be preferred that the KRAS inhibitor is a KRAS G12C inhibitor, preferably AMG510 or MRTX849, and that the cancer is characterized by an oncogenic alteration G12C in KRAS. Furthermore, in this embodiment of the present invention, it may be preferred that the cancer is selected from the group consisting of lung cancer, preferably NSCLC, colorectal cancer, and pancreatic cancer.

In another embodiment of the first aspect of the present invention, the invention is directed to a combination of: (i) the compound CCS1477 and (ii) a KRAS inhibitor for use in the treatment of a patient suffering from cancer, wherein the cancer is characterized by an oncogenic alteration in KRAS. In this embodiment of the present invention, it may be preferred that the KRAS inhibitor is a KRAS G12C inhibitor, preferably AMG510 or MRTX849, and that the cancer is characterized by an oncogenic alteration G12C in KRAS. Furthermore, in this embodiment of the present invention, it may be preferred that the cancer is selected from the group consisting of lung cancer, preferably NSCLC, colorectal cancer, and pancreatic cancer.

In another embodiment of the first aspect of the present invention, the invention is directed to a combination of (i) a GNE-781 or GNE-049 compound and (ii) a KRAS inhibitor for use in the treatment of a patient suffering from cancer, wherein the cancer is characterized by an oncogenic alteration in KRAS. In this embodiment of the present invention, it may be preferred that the KRAS inhibitor is a KRAS G12C inhibitor, preferably AMG510 or MRTX849, and that the cancer is characterized by an oncogenic alteration in G12C in KRAS. Furthermore, in this embodiment of the present invention, it may be preferred that the cancer is selected from the group consisting of lung cancer, preferably NSCLC, colorectal cancer, and pancreatic cancer.

In another embodiment of the first aspect of the present invention, the invention is directed to a combination of (i) the compound CPI-637 and (ii) a KRAS inhibitor for use in the treatment of a patient suffering from cancer, wherein the cancer is characterized by an oncogenic alteration in KRAS. In this embodiment of the present invention, it may be preferred that the KRAS inhibitor is a KRAS G12C inhibitor, preferably AMG510 or MRTX849, and that the cancer is characterized by an oncogenic alteration in G12C in KRAS. Furthermore, in this embodiment of the present invention, it may be preferred that the cancer is selected from the group consisting of lung cancer, preferably NSCLC, colorectal cancer, and pancreatic cancer.

In another embodiment of the first aspect of the present invention, the invention is directed to a combination of (i) compound 462, compound 424, or compound 515 and (ii) a KRAS inhibitor for use in the treatment of a patient suffering from cancer, wherein the cancer is characterized by an oncogenic alteration in KRAS. In this embodiment of the present invention, it may be preferred that the KRAS inhibitor is a KRAS G12C inhibitor, preferably AMG510 or MRTX849, and that the cancer is characterized by an oncogenic alteration G12C in KRAS. Furthermore, in this embodiment of the present invention, it may be preferred that the cancer is selected from the group consisting of lung cancer, preferably NSCLC, colorectal cancer, and pancreatic cancer.

In a particularly preferred embodiment of the first aspect of the present invention, the invention is directed to a combination of: (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 small molecule inhibitor of KRAS G12C (preferably selected

- 4 050570 from the group consisting of AMG510, MRTX849, JNJ-74699157/ARS-3248, BI 1823911, GDC-6036, a RAS G12C inhibitor (ON) [preferably RMC-6291] and combinations thereof, most preferably AMG510 or MRTX849), for use in the treatment of a patient suffering from cancer, wherein the cancer is characterized by an oncogenic G12C alteration in KRAS. Furthermore, in this embodiment, it may be preferred that the cancer is selected from the group consisting of lung cancer, preferably NSCLC, colorectal cancer and pancreatic cancer.

In a second aspect of the present invention, the invention relates to a kit comprising: (i) a pharmaceutical dosage form comprising a CBPf300 bromodomain inhibitor, and (ii) a pharmaceutical dosage form comprising a KRAS inhibitor.

The pharmaceutical dosage forms (i) and (ii) may be oral dosage forms, such as, for example, tablets. The amount of the CBPf300 bromodomain inhibitor contained in the dosage form preferably corresponds to the daily amount to be administered, as indicated above with respect to the first aspect of the present invention. Thus, the amount of the CBPf300 bromodomain inhibitor in the dosage form may constitute the full daily dose when administered once a day. However, it may also be less than the daily amount, for example, half the daily amount if administered twice a day or administered once a day in the form of two tablets. The amount of the KRAS inhibitor contained in the dosage form preferably corresponds to the daily amount to be administered, as indicated above with respect to the first aspect of the present invention. Thus, the amount of the KRAS inhibitor in the dosage form may constitute the full daily dose when administered once a day. However, it may also be less than the daily dose, for example, half the daily dose if administered twice daily or once daily as two tablets. For AMG510, the dose may be 120 mg, and the dosage form is preferably a tablet.

Each pharmaceutical dosage form typically contains at least one pharmaceutically acceptable excipient, as defined in Section 3 of this description below.

In a preferred embodiment of the second aspect of the present invention, the CBPf300 bromodomain inhibitor is selected from the group consisting of compound A, compound C, compound 00030, compound 00071, compounds CCS1477, GNE-781, GNE-049, SGC-CBP30, CPI-637, FT-6876, compound 462, compound 424 and compound 515.

In another preferred embodiment of the second aspect of the present invention, the KRAS inhibitor is selected from the group consisting of compounds AMG510, MRTX849, JNJ-74699157/ARS3248, BI 1701963, BI 1823911, BAY-293, GDC-6036, MRTX1133, a RAS(ON) inhibitor (preferably compounds RMC-6291 or RMC-6236) and combinations thereof.

In another preferred embodiment of the second aspect of the present invention, the kit comprises: (i) a pharmaceutical dosage form comprising a CBP/p300 bromodomain inhibitor, and (ii) a pharmaceutical dosage form comprising an AMG510 or MRTX849 compound.

In a third aspect of the present invention, the invention relates to a pharmaceutical dosage form comprising: (i) a CBPf300 bromodomain inhibitor and (ii) a KRAS inhibitor.

The pharmaceutical dosage form may be an oral dosage form, such as, for example, a tablet. The amount of the CBPf300 bromodomain inhibitor contained in the dosage form preferably corresponds to the daily amount to be administered, as set forth above in relation to the first aspect of the present invention. Thus, the amount of the CBPf300 bromodomain inhibitor in the dosage form may constitute the full daily dose when administered once daily. However, it may also be less than the daily amount, for example, half the daily amount when administered twice daily or when administered once daily in the form of two tablets. The amount of the KRAS inhibitor contained in the dosage form preferably corresponds to the daily amount to be administered, as set forth above in relation to the first aspect of the present invention. Thus, the amount of the KRAS inhibitor in the dosage form may constitute the full daily dose when administered once daily. However, it may also be less than the daily dose, for example, half the daily dose if administered twice daily or once daily as two tablets. For AMG510, the dose may be 120 mg, and the dosage form is preferably a tablet.

The pharmaceutical dosage form typically contains at least one pharmaceutically acceptable excipient as defined in Section 3 of this specification below.

In a preferred embodiment of the third aspect of the present invention, the CBPf300 bromodomain inhibitor is selected from the group consisting of compound A, compound C, compound 00030, compound 00071, compounds CCS1477, GNE-781, GNE-049, SGC-CBP30, CPI-637, FT-6876, compound 462, compound 424 and compound 515.

In another preferred embodiment of the third aspect of the present invention,

- 5 050570 KRAS inhibitor is selected from the group consisting of compounds AMG510, MRTX849, JNJ-74699157/ARS3248, BI 1701963, BI 1823911, BAY-293, GDC-6036, MRTX1133, RAS(ON) inhibitor (preferably compounds RMC-6291 or RMC-6236) and combinations thereof.

In yet another preferred embodiment of the third aspect of the present invention, the pharmaceutical dosage form comprises: (i) the bromodomain inhibitor CBPf300 and (ii) the compounds AMG510 or MRTX849.

In a fourth aspect of the present invention, the invention is directed to a method of treating cancer in a patient in need thereof, wherein said method comprises administering to the patient an effective amount of: (i) a CBPf300 bromodomain inhibitor and an effective amount of (ii) a KRAS inhibitor, wherein the cancer is characterized by an oncogenic alteration in KRAS.

In a fifth aspect of the present invention, the invention is directed to a method for increasing the duration of the therapeutic effect of a KRAS inhibitor in a patient in need thereof, said method comprising administering to the patient an effective amount of: (i) a CBPf300 bromodomain inhibitor and (ii) an effective amount of a KRAS inhibitor, wherein the cancer is characterized by an oncogenic alteration in KRAS. In other words, the duration of the therapeutic effect of the KRAS inhibitor (when administered in combination) is increased compared to the duration of the therapeutic effect of the KRAS inhibitor when administered as a single active agent in the treatment of cancer.

In a sixth aspect of the present invention, the invention is directed to a method for enhancing the therapeutic efficacy of a KRAS inhibitor in a patient in need thereof, said method comprising administering to the patient an effective amount of: (i) a CBPf300 bromodomain inhibitor and (ii) an effective amount of a KRAS inhibitor, wherein the cancer is characterized by an oncogenic alteration in KRAS. In other words, the therapeutic efficacy of the KRAS inhibitor (when administered in combination) is increased compared to the therapeutic efficacy of the KRAS inhibitor when administered as a single active agent in the treatment of cancer.

In a seventh aspect of the present invention, the invention is directed to a method for blocking the proliferation of cancer cells, comprising administering to a cell an effective amount of: (i) a CBPf300 bromodomain inhibitor and an effective amount of (ii) a KRAS inhibitor, wherein the cancer cell is characterized by an oncogenic change in KRAS.

In an eighth aspect of the present invention, the invention is directed to a method for slowing the proliferation of cancer cells, wherein said method comprises administering to a cell an effective amount of: (i) a CBPf300 bromodomain inhibitor and an effective amount of (ii) a KRAS inhibitor, wherein the cancer cell is characterized by an oncogenic alteration in KRAS.

The embodiments described above for the first aspect of the invention are equally applicable to the methods according to the fourth to eighth aspects of the invention.

Brief description of the figures

Fig. 1 - This figure shows a map of the initial Fo-Fc electron density difference model (contour at 4.0σ) obtained by refining the initial model prior to REFMAC5 modeling for the crystal structure of the human CREBBP bromodomain in complex with compound 00004.

Fig. 2A-E - This figure shows the results of fusion of SNU-1411 cells for 32 days, where the cells were treated as indicated in the graphs, namely, with either DMSO (control), or AMG510 alone, or any of the various CBPf300 bromodomain inhibitors used alone, or a combination of (i) AMG510 and (ii) any of the various CBPf300 bromodomain inhibitors. Details are provided in Example 6.

Fig. 3A-E - This figure shows the results of fusion of SNU-1411 cells for 32 days, where the cells were treated as indicated in the graphs, namely, with either DMSO (control), or MRTX849 alone, or any of the various CBPφ300 bromodomain inhibitors used alone, or a combination of (i) MRTX849 and (ii) any of the various CBPφ300 bromodomain inhibitors. Details are provided in Example 7.

Fig. 4A-E - This figure shows the results of SW837 cell fusion for 49 days, wherein the cells were treated as indicated in the graphs, namely, with either DMSO (control), or AMG510 alone, or with any of the various CBPf300 bromodomain inhibitors used alone, or with a combination of: (i) AMG510 and (ii) any of the various CBPf300 bromodomain inhibitors. Details are provided in Example 8.

Fig. 5A, B - This figure shows the results of proliferation of NCI-H358 cells for >20 days, where the cells were treated as indicated in the graphs, namely, with either DMSO (control), or AMG510 alone, or one of two different CBPf300 bromodomain inhibitors, or a combination of (i) AMG510 and (ii) one of two different CBPf300 bromodomain inhibitors. Details are provided in Example 9.

Detailed disclosure of the present invention

Before describing the present invention in detail, definitions of terms are provided below.

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1. Definitions.

As used in the description and claims, terms presented in the singular also include the corresponding plural forms unless the context clearly dictates otherwise.

The term "approximately," as used herein, denotes an accuracy range understood by those skilled in the art to ensure the technical effect and result associated with a specific feature. This term typically indicates a deviation from the stated specific numerical value of ±10%, and preferably ±5%.

It should be understood that the term "comprising" is not limiting. For the purposes of the present invention, the term "consisting of" is considered a preferred variant of the term "comprising." If a group of features is defined to include at least a certain number of embodiments, this also encompasses the group, which preferably consists only of these embodiments.

The p300 protein (also known as EP300 and KAT3B) is a large protein with multiple distinct domains that binds to a variety of proteins, including many DNA-binding transcription factors. Cyclic AMP-responsive element-binding protein (CREB) (also known as CREBBP and KAT3A) is a protein that is very closely related to p300, and the two proteins are commonly referred to as paralogs due to their high sequence identity and functional similarity, and are here referred to as CBP/p300. The CBP/p300 proteins are lysine acetyltransferases that are known to catalyze the addition of an acetyl group to the lysine side chain of histones and other proteins. CBP/p300 has been proposed to activate transcription, with the mechanism of action apparently involving the binding of DNA-binding transcription factors to RNA polymerase or by facilitating the assembly of the transcription preinitiation complex. For this purpose, the various domains of CBP/p300 are thought to interact with sets of different transcription factors assembled at promoters and enhancers for the transcription of different genes (see Dyson and Wright, JBC Vo. 291, no. 13, pp. 6714-6722, Fig. 2).

One of the many domains in CBP/p300 is the bromodomain. The bromodomain itself was first identified in Drosophila in 1992, and approximately 10 years later, it was described as an acetyl-lysine-binding module. Humans possess numerous bromodomain-containing proteins, which can be divided into eight groups based on sequence and structural similarity. All bromodomain-containing proteins appear to be involved in the regulation of transcriptional programs. Based on evidence of oncogenic rearrangements, targeting bromodomain-containing proteins, and more specifically their bromodomains, is expected to be useful, particularly in cancer treatment.

Based on this understanding, several drug candidates currently in clinical trials have been developed that target the so-called bromodomain and extraterminal motif proteins, commonly referred to as BET proteins, which comprise a group of proteins containing bromodomains. Examples of BET-targeting drugs include INCB054329 (Incyte Corporation), ABBV-075 (AbbVie), and 1-BET762 (GlaxoSmithKline). There are also drugs that selectively target the bromodomains of CBP and p300, which are part of a separate group of bromodomain-containing proteins. Such inhibitors include, for example, CCS1477 (CellCentric), which is currently in clinical trials for the treatment of metastatic castration-resistant prostate cancer and hematological malignancies, or FT-7051 (Forma Therapeutics Inc.), which is currently in trials for the treatment of metastatic castration-resistant prostate cancer.

The term "CBP/p300 bromodomain inhibitor" as used herein means a small molecule compound that binds strongly and selectively to the CBP bromodomain and to the p300 bromodomain. This term is synonymous with the terms "a bromodomain inhibitor that selectively binds to the CBP/p300 bromodomain" and "a bromodomain inhibitor that selectively inhibits CBP/p300." Strong binding in this regard means that the Kd value for the compound, when binding to the CBP bromodomain and the p300 bromodomain, is less than approximately 300 nM, and preferably less than approximately 100 nM. Selective binding in this regard means that the small molecule binds to the CBP bromodomain and the p300 bromodomain with a Kd that is at least about 20-fold lower, preferably at least about 30-fold lower, more preferably at least about 50-fold lower, and most preferably at least about 70-fold lower than the Kd value for binding any other bromodomain-containing protein or for binding the bromodomain from BROMOscan™, preferably compared to additional bromodomain-containing proteins or bromodomains identified in the number of gene elements according to DiscoveRx, in the table in Example 4 provided herein, and when performing the BROMOscan™ assay as set forth in Example 4.

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For comparison purposes, the lowest Kd value for any bromodomain-containing protein or bromodomain from BROMOscan™, except for CBP and p300, is compared with the highest Kd value for CBP and p300. Thus, for example, if the Kd value for BRD4 (full-length, short, iso) is the lowest Kd value for all bromodomain-containing proteins or bromodomains, except for CBP and p300, and it is 7100 nM, then this value is compared with the Kd for CBP, which is 29 nM (not with the Kd value for p300, which is 12 nM and thus lower than the Kd for CBP). The above example is for compound A, presented in the table of Example 4 below.

As indicated above, interactions with interaction partners in the cell that normally occur due to strong and selective binding via the CBPφ300 bromodomain are inhibited, and therefore the compound of the invention is referred to herein as an inhibitor. The term "inhibiting interactions" means that preferably no interaction at all (at least at a detectable level) occurs between the CBPφ300 bromodomain and the interaction partner. However, when this interaction between the CBPφ300 bromodomain and the interaction partner (taken as 100%) is significantly reduced, for example, to a level of about 50%, about 40%, about 30%, preferably about 20%, more preferably about 10%, or most preferably about 5% or less, such a reduced interaction is also encompassed by the term "inhibitory interactions." From the point of view of the medical use of a compound that inhibits said interaction, complete inhibition of the interaction may not be required to achieve the necessary and sufficient therapeutic effect. It should therefore be understood that the term inhibition as used herein also refers to a reduction in the interaction that is sufficient to achieve the desired effect and result.

The term KRAS, as used in this document, refers to the Kirsten rat sarcoma viral oncogene homolog protein. KRAS is a GTPase that is an important mediator of intracellular signaling pathways involved in tumor cell growth and survival. In normal cells, KRAS functions as a molecular switch, alternating between an inactive state bound to GDP and an active state bound to GTP. The transition between these states is facilitated by guanine nucleotide exchange factors (GEFs), which load GTP and activate KRAS, and by GTP hydrolysis, catalyzed by GTPase-activating proteins (GAPs), which inactivates KRAS. GTP binding to KRAS facilitates the association of effectors, triggering signaling pathways, including the RAF-MEK-ERK (MAPK) signaling pathway. Somatically activating mutations in KRAS are a hallmark of cancer and prevent GAP association, thereby stabilizing effector binding and enhancing KRAS signaling. Patients with tumors harboring mutant KRAS have significantly worse outcomes and prognosis.

The term "KRAS inhibitor" as used herein refers to molecules capable of acting on KRAS in such a way that the downstream intracellular signaling that ultimately leads to cell proliferation is inhibited. The term "inhibited" in this context means that downstream signaling preferably no longer occurs. However, when this downstream signaling (taken as 100%) is significantly reduced, for example to a level of about 70%, about 60%, about 50%, about 40%, about 30%, preferably about 20%, more preferably 10%, or most preferably about 5% or less, such a reduction in downstream signaling is still encompassed by the term "inhibition of downstream intracellular signaling." From the standpoint of medical use of a compound of the invention that inhibits downstream signaling, complete inhibition of signaling may not be required to achieve the necessary and sufficient therapeutic effect. Thus, it should be understood that the term inhibition, as used herein and in this context, also refers to a reduction in the level of downstream signaling sufficient to achieve the desired effect. A KRAS inhibitor can covalently bind to KRAS, in particular to the cysteine at position 12 in KRAS G12C. If a KRAS inhibitor targets and/or binds to this cysteine, the inhibitor is typically referred to as a KRAS G12C inhibitor, and examples of such inhibitors include AMG510 (CAS No. 2296729-00-3), MRTX849 (CAS No. 2326521-71-3), JNJ-74699157/ARS-3248, BI 1823911, GDC-6036, and RMC-6291. Recently, the first KRAS G12C-modulating agent, LUMAKRAS (sotorasib, equivalent to Amgen's AMG510), received FDA approval for the treatment of locally advanced or metastatic non-small cell lung cancer (NSCLC) with a KRAS G12C mutation. Another KRAS G12C-modulating agent, adagrasib (equivalent to Mirati Therapeutics' MRTX849), is expected to be available soon. A KRAS G12D inhibitor is an inhibitor specific for KRAS G12D, etc. An example of a KRAS G12D inhibitor is MRTX1133. Alternatively, a KRAS inhibitor can block the interaction of KRAS with other proteins, in particular, block the KRAS-SOS1 interaction. Such inhibitors that block the KRAS-SOS1 interaction are, for example, BI 1701963 and BAY-293 (CAS number 2244904-70-7). The so-called

- 8 050570 RAS(ON) inhibitors that bind to mutated GTP-bound KRAS (e.g. GTP-bound KRAS G12C or GTP-bound KRAS G12V or GTP-bound KRAS G12D or GTP-bound KRAS G13D or GTP-bound KRAS Q61H or GTP-bound KRAS Q61L or GTP-bound KRAS Q61R) and that prevent the engagement of RAF by blocking the effector surface of the corresponding KRAS, as they form a ternary complex between the RAS(ON) inhibitor (synthetic ligand), KRAS and cyclophilin A (for further information see publications from Revolution Medicines, e.g. WO 2021/091956). RMC-6291 is a RAS G12C (ON) inhibitor from Revolution Medicines that targets KRAS G12C via the mechanism described above. RMC-6236 is a RAS(ON) inhibitor from Revolution Medicines that targets multiple RAS mutations, including KRAS mutations, via the mechanism described above.

The term "a cancer characterized by an oncogenic alteration in KRAS," as used herein, means that the tumor harbors a mutant variant of KRAS, and this mutant variant of KRAS is implicated in cancer development. In other words, a mutant variant of KRAS can be considered a factor associated with cancer development or as a cause of its development, not necessarily among other factors. A mutant variant of KRAS is present in a tumor due to alterations in the KRAS gene, where such an alteration specifically represents a mutation of at least one base in the KRAS gene, resulting in an amino acid substitution in the KRAS protein. The specific alterations involved are described above, with the KRAS G12C alteration being, in particular, a significant and relevant alteration. As noted above, KRAS mutations are present in 25% of cancer cases, with oncogenic variants having varying prevalence across different cancer types (see Box 1 of the Mullard publication cited above).

The term KRAS overactivation used here means that KRAS is more active than the wild type, and in particular, it is more active in subsequent activation and signaling that leads to cancer cell growth.

The term "low molecular weight compound" as used herein refers to a small organic compound having a low molecular weight. In the context of the present invention, the low molecular weight compound preferably has a molecular weight of less than 5000 Da, more preferably less than 4000 Da, more preferably less than 3000 Da, more preferably less than 2000 Da, or even more preferably less than 1000 Da. In the context of the present invention, the low molecular weight compound in a particularly preferred embodiment has a molecular weight of less than 800 Da. In another preferred embodiment of the present invention, the low molecular weight compound in the context of the present invention has a molecular weight of from 50 to 3000 Da, preferably from 100 to 2000 Da, more preferably from 100 to 1500 Da, and even more preferably from 100 to 1000 Da.

As used herein, the term treatment refers to a clinical intervention with the goal of curing a disease or alleviating the severity of a disease, preventing recurrence of a disease, alleviating the symptoms of a disease, reducing any direct or indirect pathological consequences of a disease, achieving stabilization (i.e., without worsening) of the disease, preventing metastasis, reducing the rate of disease progression, and/or prolonging survival compared to the expected survival without treatment.

As used herein, the term treatment cycle means that the drug is administered for a specified period of time after the initial assessment of the patient's condition, with the patient's condition then typically re-assessed before starting the next treatment cycle.

Detailed information on the CBP/p300 bromodomain inhibitors mentioned herein is provided in the Examples section of this application. In particular, the structures of compound A, compound C, compound 00030, and compound 00071 are shown in this section. In addition, the synthetic routes for these compounds are shown in the Examples section of this application. Compound CCS1477 is commercially available, for example, from Aobious, and it has the CAS number 2222941-37-7. Compound GNE-781 is commercially available, for example, from MCE (MedChemExpress), and it has the CAS number 1936422-33-1. Compound GNE-049 is commercially available, for example, from MCE (MedChemExpress), and it has the CAS number 1936421-41-8. The compound SGC-CBP30 is commercially available, for example, from MCE (MedChemExpress), and it has the CAS number 161369514-9. The compound CPI-637 is commercially available, for example, from MCE (MedChemExpress), and it has the CAS number 1884712-47-3. The compound FT-6876 is commercially available, for example, from MCE (MedChemExpress), and it has the CAS number 2304416-91-7 (FT-6876 is also referred to as CBP/p300-IN8). The structures of compounds 462, 424, and 515 are shown below; These structures and synthetic routes are disclosed in International Application Publication No. WO 2020/006483 (see, in particular, pages 33 and 34 for compound 424; pages 42 and 43 for compound 462; and pages 47 and 48 for compound 515):

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2. Discoveries of the authors of the present invention.

The present inventors have identified novel compounds that bind strongly to the CBP/p300 bromodomain and have shown that binding to the CBP/p300 bromodomain is also selective, as it is well known that there are many proteins that contain bromodomains.

CBP/p300 have been identified as central nodes in eukaryotic transcriptional regulatory networks, interacting with more than 400 transcription factors and other regulatory proteins. CBP/p300 regulates crosstalk and interference between numerous cellular signaling pathways and is a target of tumor viruses for cellular regulatory hijacking (see Dyson and Wright, above, p. 6714, right column). CBP/p300 are large proteins that contain multiple domains, as seen in Figure 1 of the Dyson and Wright publication (see above). These domains are the NRID, TAZ1, TAZ2, KIX, CRD1, BRD, CH2 (with the PHD domain and the RING finger domain), HAT, ZZ, and NCBD domains. The size of these proteins and the presence of various domains make it clear that their cellular functions are highly diverse, for example, due to interactions with many different interaction partners due to the diversity of interactions that the CBP/p300 protein is capable of. The enzymatic activity of CBP/p300 as a histone acetyltransferase is localized to the HAT domain. As noted above, this enzymatic function is primarily associated with transcriptional activation. CBP/p300 is also subject to post-translational modifications, particularly phosphorylation. The intrinsic enzymatic activity of CBP/p300, as well as that of proteins with post-translational modifications, adds another layer of complexity to the diverse functions and effects mediated by CBP/p300 proteins. That these functions and effects can be counteracted is demonstrated in general terms in the introductory section to the relevant chapter in Goodman and Smolk, Genes & Development 2000, 14:1553–1577, where it is noted that one of the major paradoxes of CBP/p300 function is that these proteins appear to contribute to diametrically opposed cellular processes, and that their functions and effects appear to be highly dependent on the context of their action, specifically whether CBP/p300 promotes apoptosis or cell proliferation. With regard to the impact on diseases, and cancer in particular, this means that the context of a particular disease and a particular cancer type will be critical to how, and whether, CBP/p300 proteins are involved in a cellular process.

Given the above, it is not surprising that the CBP/p300 protein cannot be assigned to a single function in a cellular process that could be targeted, for example, by a general CBP/p300 inhibitor. Rather, due to the vast array of actions of this protein, analysis of the various functions of the CBP/p300 protein is only possible by examining specific domains of CBP/p300—that is, by analyzing the effects achieved, for example, by inhibiting the enzymatic activity of CBP/p300 in its HAT domains or by preventing certain interactions with interaction partners by blocking (or inhibiting) certain domains. Furthermore, this must be considered in the relevant context, for example, for a specific disease or cancer type, as noted above.

Therefore, we began to study the effects of their action in specific conditions where the inhibitors prevent their interaction with their interaction partners through the CBP/p300 bromodomain and initially investigated the effect of our proposed inhibitors on non-small cell lung cancer (NSCLC) cells, following the data presented in the publication by Hou et al. (Hou et al., BMC Cancer (2018) 18:641). However, we did not observe an effect on the proliferation of the tested NSCLC cell lines when using the inhibitor alone. However, we unexpectedly found that our proposed CBP/p300 bromodomain inhibitors prolong the effect of the EGFR inhibitor in NSCLC cells characterized by an oncogenic alteration in EGFR, compared to the administration of the EGFR inhibitor alone. In other words, although the CBP/p300 bromodomain inhibitors used by the present inventors alone had no effect on the proliferation of NSCLC characterized by the presence of an oncogenic alteration in EGFR, these CBP/p300 bromodomain inhibitors achieved the desired effect when used in combination with an EGFR inhibitor. Furthermore, unexpectedly, this combination proposed by the inventors was found to be effective not only against EGFR signaling and, accordingly, with EGFR inhibitors, but also against KRAS signaling and, accordingly, with KRAS inhibitors, as described below.

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The present inventors used colorectal cancer cell lines (SNU-1411 and SW837, which are rectal adenocarcinoma cell lines carrying the KRAS G12C mutation), as well as an NSCLC cell line (NCI-H358 cells carrying the KRAS G12C mutation) in their experiments. These cell lines can therefore be considered as a model system for the first-line treatment of cancer patients whose cancer cells carry a KRAS mutation, in particular the KRAS G12C mutation. AMG510 and MRTX849 were used as KRAS inhibitors, respectively, in combination with CBP/p300 bromodomain inhibitors (see the Examples below).

The observed significant inhibition of proliferation during prolonged incubation of the combination in all cell lines tested is particularly noteworthy, as the development of resistance means that proliferation inhibition will not remain complete over time when using a single KRAS inhibitor. As the data presented below in the experimental section demonstrate, this applies to both AMG510 and MRTX849 when used separately.

Given the results for the CBP/p300 inhibitors used by the present inventors, the study was continued to determine whether the observed effect could be generalized to all CBP/p300 inhibitors as such. For this purpose, additional CBP/p300 bromodomain inhibitors were tested, namely CCS1477, FT-6876, and GNE-781. It should be noted that the structures of the various CBP/p300 inhibitors tested by the present inventors are unrelated, so their common feature relates solely to the effect exerted by these inhibitors, namely, selective inhibition of the CBP/p300 bromodomain. The structures of the tested CBP/p300 inhibitors are presented below.

It should also be noted that the tested KRAS inhibitors AMG510 and MRTX849 are quite different in their structure, but all share a common inhibitory function against KRAS G12C (acting as covalent inhibitors).

In addition, the present inventors tested not only one cancer cell line carrying an oncogenic mutation in KRAS, but also three other different cancer cell lines (colorectal adenocarcinoma cell lines SNU-1411 and SW837, and NSCLC cell line NCI-H358).

3. Pharmaceutical composition of the compounds according to the invention.

The CBP/p300 bromodomain inhibitor and the KRAS inhibitor are pharmaceutically active agents for the claimed use. As noted above, they may be presented in separate dosage forms or contained in a single dosage form.

As used herein, the term "pharmaceutically active agents" means compounds capable of modulating a response in a patient, i.e., a human or animal, in vivo. As used herein, the term "pharmaceutically acceptable excipient" refers to excipients known to those skilled in the art as commonly included in pharmaceutical compositions. Examples of such excipients are listed below. Given the definition of "pharmaceutically active agents" provided above, a pharmaceutically acceptable excipient may be defined as a pharmaceutically inactive agent.

If a commercially available KRAS inhibitor is used in combination with a CBP/p300 bromodomain inhibitor, it is preferred that they be administered as separate dosage forms and that the KRAS inhibitor be administered in a dosage form and by an officially approved/approved route of administration (this applies, for example, to AMG510 (as approved for LUMAKRAS)). The CBP/p300 bromodomain inhibitor can be administered in the dosage form described below or in the dosage form in which it is currently undergoing clinical trials.

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The dosage form for use according to the present invention may be prepared for oral, buccal, nasal, rectal, topical, transdermal, or parenteral administration. Oral administration may be preferred. Parenteral administration may also be preferred, and includes intravenous, intramuscular, or subcutaneous administration. The dosage form according to the invention may also be referred to herein as a dosage form or pharmaceutical composition.

In general, the pharmaceutical composition of the invention may contain various pharmaceutically acceptable excipients, which are selected depending on the functionality they are intended to provide for the composition. In the context of the present invention, a pharmaceutically acceptable excipient may be any substance used to prepare pharmaceutical dosage forms, including coating materials, film-forming materials, fillers, disintegrating agents, release-modifying materials, carrier materials, diluents, binders, and other auxiliary substances. Typical pharmaceutically acceptable excipients include substances such as sucrose, mannitol, sorbitol, starch and starch derivatives, lactose, as well as lubricating agents such as magnesium stearate, disintegrants/disintegrants, and buffering agents.

The term "carrier" denotes pharmaceutically acceptable organic or inorganic carrier substances with which the active ingredient is combined to facilitate application/administration. Suitable pharmaceutically acceptable carriers include, for example, water, salt solutions, alcohols, oils, preferably vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, surfactants, perfume oil, monoglycerides and diglycerides of fatty acids, esters of fatty acids in petroleum ether, hydroxymethylcellulose, polyvinylpyrrolidone, etc. Pharmaceutical compositions can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, osmotic pressure regulating salts, buffers, colorants, flavoring and/or aromatic substances, etc., which do not have a harmful effect on the body and do not interact with the active compound.

If the present invention envisages the use of liquid dosage forms, such 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 to impart bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners/flavoring agents.

For parenteral administration, particularly suitable carriers include solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants. Pharmaceutical compositions for parenteral administration are particularly preferred and include water-soluble forms in the form of an aqueous solution. Suspensions can also be prepared in the form of suitable oily injection 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 injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran.

Particularly preferred dosage forms are injectable preparations of the pharmaceutical composition according to the invention. Thus, sterile aqueous or oily injection suspensions can, for example, be prepared according to methods known in the art using suitable dispersing agents, wetting agents, and/or suspending agents. The sterile injectable preparation can also be a sterile injection solution or suspension in a non-toxic diluent or solvent acceptable for parenteral administration. Acceptable carriers and solvents that can be used within the framework of the present invention include water and isotonic sodium chloride solution. Sterile oils are also commonly used as a solvent or suspending medium.

For rectal administration of the pharmaceutical composition of the invention, suppositories can be prepared, for example, by mixing the compound with a suitable non-irritating excipient, such as cocoa butter, synthetic triglycerides and polyethylene glycols, which are solid at room temperature but liquid at the rectal temperature, so that they will melt in the rectum and release the active agent from said suppositories.

For inhalation administration, a pharmaceutical composition containing a compound of the invention is conveniently delivered as an aerosol spray from pressurized closures or from nebulizers using a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or another suitable gas. In the case of an aerosol obtained from a pressurized closure, the dose can be metered by using a valve to deliver a predetermined amount. For administration of the composition using an inhaler or insufflator,

- 12 050570 capsules and cartridges are made, for example, from gelatin, using a powder mix of the active compound and a suitable powder base such as lactose or starch.

Dosage forms for oral administration may be liquid or solid and may include, for example, tablets, lozenges, pills, capsules, powders, effervescent formulations, dragees, and granules. Pharmaceutical preparations for oral administration can be prepared using a solid excipient, optionally grinding the resulting mixture and processing the mixture of granules, after adding suitable auxiliary substances, if desired, to obtain tablet or dragee cores. In this case, suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or its salt, such as sodium alginate, may be added. Oral dosage forms may be formulated to provide immediate or sustained release of the active agent.

4. Further disclosure of the invention and further embodiments of the present invention.

The clinical antitumor effect of receptor tyrosine kinase inhibitors (RTK) and other kinase inhibitors is short-lived. Resistance to these inhibitors commonly develops. More specifically, the clinical antitumor effect of EGFR inhibitors (EGFRi) is short-lived. Resistance to EGFR inhibitors typically develops within 9-19 months, depending on the therapeutic agent used and the clinical setting. Therefore, developing a cancer treatment that prevents drug resistance in cancer patients is desirable. Historically, most approaches to combating drug resistance have focused on the genetic factors underlying recurrent tumors. In an attempt to overcome established drug resistance, a newly mutated protein that triggers tumor regrowth will serve as a therapeutic target for a drug, used alone or in combination with a primary anticancer drug. One mechanism of resistance to EGFRi treatment is the development of a gatekeeper mutation in the EGFR protein, i.e., a mutation that renders EGFRi ineffective. Most commonly, this gatekeeper mutation is the T790M mutation. Mutation-specific inhibitors, such as osimertinib, are used to overcome drug resistance to first-generation EGFR inhibitors, which do not inhibit the T790M mutant EGFR. Another mechanism of resistance to EGFRi treatment is bypass signaling, which is activated through other receptor tyrosine kinases, such as through amplification, overexpression, or activation of MET, ErbB2, HGF, ErbB3, IGF1R, AXL, NTRK1, BRAF, FGFR3, or FGFR1. Therapeutic interventions to suppress bypass signaling have been tested clinically, but with mixed results.

Prior art disclosures, such as WO 2018/022637, describe the use of CBP/p300 inhibitors as novel cancer treatments, particularly for the treatment of cancers with mutations in p300. WO 2011/085039 describes methods for treating cancer that include inhibiting the activity of the histone acetyltransferase CBP/c300 (HAT) and the use of CBP/c300 HAT inhibitors to treat a subject suffering from cancer, particularly in combination with DNA-damaging chemotherapeutic anticancer agents.

Thus, there is a need for new, effective methods and compositions for preventing the development of drug resistance in cancer. This is addressed, among other things, by the embodiments of the present invention presented in Section 4 of this description.

An embodiment of the present invention 1. A CBP/c300 bromodomain inhibitor for use in a method of treating cancer in an animal, wherein the method comprises administering to an animal in need thereof a CBP/c300 bromodomain inhibitor and a receptor tyrosine kinase inhibitor selected from the group consisting of EGFR, ALK, MET, HER2, ROS1, RET, NTRK1 and AXL inhibitors, or a KRAS (Kirsten rat sarcoma viral oncogene homolog) or BRAF (B-Raf proto-oncogene and v-Raf murine sarcoma viral oncogene B homolog), wherein the cancer involves an alteration in the corresponding receptor tyrosine kinase, or in KRAS, or in BRAF, and wherein the CBP/c300 bromodomain inhibitor does not by itself slow down the progression of the cancer.

Embodiment 2 of the present invention. A CBP/c300 bromodomain inhibitor for use in a method for increasing the duration of response to cancer treatment in an animal with a receptor tyrosine kinase inhibitor or a KRAS or BRAF inhibitor, wherein the method comprises administering to an animal with cancer a CBP/c300 bromodomain inhibitor or a pharmaceutically acceptable salt thereof, and wherein the duration of response to anti-cancer therapy is increased upon administration of the CBP/c300 bromodomain inhibitor or a pharmaceutically acceptable salt thereof, compared to the duration of response to anti-cancer therapy in the absence of administration of the CBP/c300 bromodomain inhibitor or a pharmaceutically acceptable salt thereof, and wherein the receptor tyrosine kinase inhibitor is selected from the group consisting of EGFR, ALK, MET, HER2, ROS1, RET, NTRK1 and AXL inhibitors.

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Embodiment 3 of the present invention. A composition for treating cancer comprising a synergistic combination of a CBPp300 bromodomain inhibitor or a pharmaceutically acceptable salt thereof and a receptor tyrosine kinase inhibitor selected from the group consisting of EGFR, ALK, MET, HER2, ROS1, RET, NTRK1 and AXL inhibitors, or a KRAS or BRAF inhibitor, wherein the cancer involves an alteration in the corresponding receptor tyrosine kinase, or in KRAS or BRAF, and wherein the CBP p300 bromodomain inhibitor alone does not slow down the progression of the cancer.

Embodiment 4 of the present invention. A method for inhibiting the growth of a cancer cell, comprising administering a CBPf300 bromodomain inhibitor and a receptor tyrosine kinase inhibitor selected from the group consisting of EGFR, ALK, MET, HER2, ROS1, RET, NTRK1 and AXL inhibitors, or a KRAS or BRAF inhibitor, and wherein the cancer cell has alterations in the corresponding receptor tyrosine kinase or KRAS or BRAF, and wherein the CBPf300 bromodomain inhibitor itself does not inhibit the growth of the cancer cell.

Embodiment 5 of the present invention. A CBPf300 bromodomain inhibitor or composition for use or method according to any previous embodiment, wherein the alteration in the receptor tyrosine kinase, or in KRAS or BRAF is an oncogenic alteration.

Embodiment 6 of the present invention. A CBPf300 bromodomain inhibitor or a composition for use or a method according to any previous embodiment, wherein the receptor tyrosine kinase inhibitor is an EGFR inhibitor.

Embodiment 7 of the present invention. A CBPf300 bromodomain inhibitor or composition for use or method according to embodiment 6, wherein the alteration in the receptor tyrosine kinase is a mutation in EGFR.

Embodiment 8 of the present invention. A CBPf300 bromodomain inhibitor or a composition for use or a method according to any previous embodiment, wherein the composition or combination of a CBPf300 bromodomain inhibitor or a pharmaceutically acceptable salt thereof and a receptor tyrosine kinase inhibitor or a KRAS or BRAF inhibitor is synergistic in the treatment of cancer, compared to a CBPf300 or receptor tyrosine kinase inhibitor or a KRAS or BRAF inhibitor alone.

Embodiment 9 of the present invention. A CBPf300 bromodomain inhibitor or a composition for use or a method according to any previous embodiment, wherein the composition or combination of a CBPf300 bromodomain inhibitor or a pharmaceutically acceptable salt thereof and a receptor tyrosine kinase inhibitor or a KRAS or BRAF inhibitor delays or reduces the risk of cancer resistance to a receptor tyrosine kinase inhibitor or to a KRAS or BRAF inhibitor.

Embodiment 10 of the present invention. A CBPf300 bromodomain inhibitor or a composition for use or a method according to any previous embodiment, wherein the CBPf300 bromodomain inhibitor is administered in an amount effective to prevent resistance of a cancer cell to a receptor tyrosine kinase inhibitor or to a KRAS or BRAF inhibitor.

Embodiment 11 of the present invention. The CBPf300 bromodomain inhibitor or composition for use or method according to any preceding embodiment, wherein the EGFR inhibitor is selected from the group consisting of cetuximab, panitumumab, zalutumumab, nimotuzumab, matuzumab, gefitinib, erlotinib, dacomitinib, lapatinib, neratinib, vandetanib, necitumumab, 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 II inhibitor (BIBX 1382, CAS No. 196612-93-8), an EGFR III inhibitor (CAS No. 733009-42-2), an EGFR/ErbB-2/ErbB-4 II inhibitor (CAS No. 944341-54-2), or a PKCeII/EGFR inhibitor (CAS No. 145915-60-2).

Embodiment 12 of the present invention. A CBP/p300 bromodomain inhibitor or a composition for use or a method according to any preceding embodiment, wherein the CBP/p300 inhibitor is a compound of formula (I)

in which R ¹ is selected from halogen and an optionally substituted hydrocarbon group that contains from 1 to 20 carbon atoms, and optionally from 1 to 15 heteroatoms selected from O, N and S;

R ²¹ is selected from hydrogen, optionally substituted C _1-6 αalkyl, which may contain from one to three oxygen atoms between carbon atoms, and optionally substituted C _3-6 cycloαalkyl;

- 14 050570

R ³ is selected from optionally substituted heterocyclyl, optionally substituted carbocyclyl, optionally substituted C _1-6 alkylene, optionally substituted heterocyclyl, and optionally substituted C _1-6 alkylene-optionally substituted carbocyclyl;

each of X ¹ , X ² and X ³ is independently selected from N, CH and CR ^x , wherein at least one of said X ¹ , X ² and X ³ is N;

R ³¹ is selected from hydrogen, C _1-6 alkyl, and C _1-6 alkyl substituted with one or more F; wherein R ³ and any R ³¹ may be optionally joined; and

E is either absent or selected from _CH2 , CHR ^x , CR ^x2 , NH, NR ^x , O, L1- ^L2 and L2- ^L1 , where ^L1 is selected from CH2, CHR ^x , CR ^x2 , NH, NR ^x and O, and ^L2 is selected from CH2, CHR ^x and CR ^x2 _;

R ^6x is halogen, OH, =O, C1-6alkyl, C1-6haloalkyl, C1.6okuh substituted with one or more OH, monocyclic aryl optionally substituted with one or more R ^xb , monocyclic heteroaryl optionally substituted with one or more R ^xb , monocyclic cycloalkyl optionally substituted with one or more R ^xb , monocyclic heterocycloalkyl optionally substituted with one or more R ^xb , monocyclic cycloalkenyl optionally substituted with one or more R ^xb , monocyclic heterocycloalkenyl optionally substituted with one or more R ^xb , wherein said R ^xb is independently selected from halogen, OH, =O, C14alkyl, C _1-2haloalkyl , C _1-2alkyl substituted with one or two OH;

wherein ring A may be further substituted with one or more R ^x groups, wherein any two R ^x groups in ring A may be optionally connected, and/or any R ^x group in ring A may be optionally connected to R ²¹ ; and/or wherein ring A may be further substituted with one R ^x group to form, together with R ^6x , a bicyclic moiety having the following structure:

in which ring B is an optionally substituted heterocycle or an optionally substituted carbocycle;

each R ^x is independently selected from halogen, OH, O-optionally substituted C _1-6 alkyl, NH(optionally substituted C _1-6 alkyl), ((optionally substituted C _1-6 alkyl) ₂ , =O, optionally substituted C _1-6 alkyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, (optionally substituted C _1-6 alkylene)-(optionally substituted carbocyclyl), (optionally substituted C _1-6 alkylene)-(optionally substituted heterocyclyl), O-(optionally substituted C _1-6 alkylene)-(optionally substituted carbocyclyl), and O-(optionally substituted C _1-6 alkylene)-(optionally substituted heterocyclyl), and where the optional substituent of the optionally substituted hydrocarbon group is optionally substituted C _3-6 cycloalkyl, optionally substituted heterocyclyl, optionally substituted heterocycle, optionally substituted carbocyclyl, optionally substituted carbocycle and optionally substituted C _1-6 alkylene are independently selected from C _1-6 alkyl which is optionally substituted with one or more halogen, halogen, CN, NO ₂ , 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) ₂ R*, OR*, OC(O)R*, OC(O)-NR*R*, SR*, S(O)R*, S(O) ₂ R*, S(O) ₂ -NR*R*, N(R*)-S(O) ₂ -NR*R*, heterocyclyl optionally substituted with halogen or C _1-6 alkyl, and carbocyclyl optionally substituted with halogen or C _1-6 alkyl; wherein each R* is independently selected from H, C _1-6 alkyl which is optionally substituted with halogen, heterocyclyl which is optionally substituted with halogen or C _1-6 alkyl, and carbocyclyl which is optionally substituted with halogen or C _1-6 alkyl; wherein any two R* bonded to the same nitrogen atom may be optionally joined, and wherein the optional substituent of the optionally substituted C _1-6 alkyl and the optionally substituted C _1-6 alkylene is independently selected from halogen, CN, NO ₂ , 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) ₂ R**, 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**; where R** is independently selected from H, C _1-6 alkyl which is optionally substituted with halogen, heterocyclyl which is optionally substituted with halogen or C _1-6 alkyl, and carbocyclyl which is optionally substituted with halogen or C _1-6 alkyl, where any two R** bonded to the same nitrogen atom may be optionally joined.

Embodiment 13 of the present invention. A CBP/p300 bromodomain inhibitor or a composition for use or a method according to any previous embodiment, wherein the CBP/p300 inhibitor is an arylimidazolylisoxazole of formula (A)

- 15 050570 ft*

(A), in which each R° and R, the same or different, is H or C1- _C6 alkyl, unsubstituted or substituted by OH, OC(O)R' or OR', where R' is unsubstituted C1- _C6 alkyl;

W represents N or CH;

R ¹ is a group which is unsubstituted or substituted and which is selected from C-linked 4-6-membered heterocyclyl; C ₃ -C ₆ -cycloalkyl; C 1 -C ₆ -alkyl, unsubstituted or substituted C ₆ -C ₁₀ -aryl, 5-12-membered N-containing heteroaryl, C ₃ -C ₆ -cycloalkyl, OH, OC(O)R' or OR', where R' is a group as defined above; and a spiro group of the following formula:

in which Y is _CH2 , _CH2CH2 or _{CH2CH2CH2 ;}

n is 0 or 1;

R ² is a group selected from C ₆ -C ₁₀ -aryl, 5-12-membered N-containing heteroaryl, C ₃ -C ₆ -cycloalkyl and C ₅ -C ₆ -cycloalkenyl, wherein this group is unsubstituted or substituted, and wherein C ₆ -C ₁₀ -aryl is optionally fused with a 5- or 6-membered heterocyclic ring;

or a pharmaceutically acceptable salt thereof, wherein said arylimidazolylisoxazole preferably has the formula (Aa*):

OMe ό ^F (Aa*; CCS1477 [CAS No. 2222941-37-7]).

Embodiment 14 of the present invention. A CBP/p300 bromodomain inhibitor or a composition for use or a method according to any preceding embodiment, wherein the CBP/p300 inhibitor is a compound of formula (Ba)

(Ba), in which R ¹ is O(C1-C ₃ -alkyl);

R ⁶ is phenyl optionally substituted independently with one or more R ^B , wherein R ^B is selected from OC1.6 _alkyl , _{OC3.6 cycloalkyl} , O-aryl or O-heteroaryl, wherein each alkyl, cycloalkyl, aryl or heteroaryl is optionally independently substituted with one or more halogens;

or where the CBP/p300 inhibitor is a compound of formula (Bc)

- 16 050570

(Вс), in which R ¹ is OR ⁵ ;

R ⁵ is C1-6alkyl, C _{3-8cycloalkyl} , heterocyclyl, aryl or heteroaryl;

R6 is OH, halogen, oxo, NO2, CN, NH2, C _1-6 alkyl, C _3-8 cycloalkyl, C _4-8 cycloalkenyl, heterocyclyl, aryl, spirocycloalkyl, spiroheterocyclyl, heteroaryl, OC _3-6 cycloalkyl, O-aryl, O-heteroaryl, (CH2)N-OR ⁸ , C(O)R ⁸ ', C(O)OR ⁸ or C(O)NR8R ⁹ , NHC _1- ^rn, ^C _1-6 alkyl) ₂ , S(O) ₂ NH(C _1-6 alkyl), S(O) ₂ N(C _1-6 alkyl) ₂ , S(O) ₂ C _1-6 alkyl, ^C _1-6 alkyl^O ₂ C _1-6 alkyl, S(O)(C _1-6 alkyl), S(O)N(C _1-6 alkyl) ₂ or ^C _1-6 alkyl^(O)(C _1-6 alkyl), wherein each alkyl, cycloalkyl, cycloalkenyl, heterocyclyl, spirocycloalkyl, spiroheterocyclyl, heteroaryl or aryl is optionally substituted with one or more R ¹⁰ ;

R ⁷ in each occurrence is independently H, halogen, OH, CN, OC _1-6 alkyl, NH2, NH(C _1-6 alkyl), (C _1-6 alkyl) ₂ , S(O) ₂ H(C _1-6 alkyl), S(O) ₂ N(C _1-6 alkyl) ₂ , S(O) ₂ (C _1-6 alkyl), S(O) ₂ OH, C(O)C _1-6 alkyl, C(O)NH ₂ , C(OH(C _1-6 alkyl), C(OH(C _1-6 alkyl) ₂ , C(O)OH, C(O)OC _1-6 alkyl, MC _1-6 alkyl^O ₂ C _1-6 alkyl, S(O)(C _1-6 alkyl), S(O)N(C _1-6 alkyl) ₂ , S(O)2NH2, ^C _1-6 alkyl^(O)(C _1-6 alkyl) or tetrazole;

R ¹⁰ in each occurrence is independently C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _3-8 cycloalkyl, C _4-8 cycloalkenyl, heterocyclyl, heteroaryl, aryl, OH, halogen, oxo, NO2, CN, NH2, OC _1-6 alkyl, OC _3-6 cycloalkyl, O-aryl, O-heteroaryl, NHC _1-6 alkyl, (C _1-6 alkyl) ₂ , S(O) ₂ NH(C _1-6 alkyl), S(O) 2 N(C 1-6 alkyl) 2, S(O) ₂ C 1-6 alkyl, C(O) C 1-6 alkyl, C(O) NH(C 1-6 alkyl), NHC(O) C 1-6 alkyl, C(O) C _1-6 alkyl _, C(O) _OC1-6 alkyl, ^ _C1-6 alkyl^ _O2 - _C1-6 alkyl, S(O)( _C1-6 alkyl), S(O)N( _C1-6 alkyl) ₂ or ^ _C1-6 alkyl^(O)( _C1-6 alkyl), wherein each alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl or aryl is optionally substituted with one or more ^R12 ;

R ¹² in each case independently represents a halogen;

m is an integer between 0 and 5;

r is an integer between 0 and 5.

Embodiment 15 of the present invention. A CBP/p300 bromodomain inhibitor or composition for use or method according to the previous embodiment, wherein slow cancer progression is measured using RECIST 1.1 response criteria for target lesions or non-target lesions in an animal.

Embodiment 16 of the present invention. A CBP/p300 bromodomain inhibitor or composition for use or method according to any preceding embodiment, wherein the cancer is non-small cell lung cancer (NSCLC).

Embodiment 17 of the present invention. A CBP/p300 bromodomain inhibitor or a composition for use or a method according to the previous embodiment, wherein the CBP/p300 bromodomain inhibitor is a compound of formula (I) according to embodiment 12, the receptor tyrosine kinase inhibitor is an EGFR inhibitor, the receptor tyrosine kinase is EGFR, the cancer is NSCLC, and more preferably, when the NSCLC comprises the T790M mutation in EGFR, and more preferably, when the receptor tyrosine kinase inhibitor is osimertinib.

Embodiment 18 of the present invention. A CBP/p300 bromodomain inhibitor or a composition for use or a method according to the previous embodiment, wherein the CBP/p300 bromodomain inhibitor is a compound of formula (A) according to embodiment 13, preferably compound CCS1477 (CAS No. 222941-37-7), the receptor tyrosine kinase inhibitor is an EGFR inhibitor, the receptor tyrosine kinase is EGFR, the cancer is NSCLC, and more preferably, wherein the NSCLC comprises the T790M mutation in EGFR, and more preferably, wherein the receptor tyrosine kinase inhibitor is osimertinib.

With respect to the above embodiment 13, it should be noted that compounds of formula (A) have been described in applications WO 2016/170324, WO 2018/073586 and WO 2019/202332; the contents of all these applications and their descriptions in their entirety, and in particular the descriptions with respect to the synthesis of a compound of formula (A), are incorporated herein by reference.

In another embodiment of the present invention, there is provided a method of treating cancer in an animal comprising administering to the animal in need thereof a bromodomain inhibitor.

- 17 050570

CBP/p300 and a receptor tyrosine kinase inhibitor selected from the group consisting of EGFR, ALK, MET, HER2, ROS1, RET, NTRK1 and AXL inhibitors, or a KRAS or BRAF inhibitor, where the cancer involves an alteration of the corresponding receptor tyrosine kinase or KRAS or BRAF, and where the CBP/p300 bromodomain inhibitor does not by itself slow down the progression of the cancer.

In another embodiment of the present invention, there is provided a method of treating cancer using a composition comprising a synergistic combination of a CBPf300 bromodomain inhibitor or a pharmaceutically acceptable salt thereof and a receptor tyrosine kinase inhibitor selected from the group consisting of EGFR, ALK, MET, HER2, ROS1, RET, NTRK1 and AXL inhibitors, or a KRAS or BRAF inhibitor, wherein the cancer includes an alteration in the corresponding receptor tyrosine kinase, KRAS or BRAF, and wherein the CBPf300 bromodomain inhibitor does not by itself slow down the progression of the cancer.

In another embodiment of the present invention, there is provided a method for increasing the duration of response to cancer therapy in an animal with a receptor tyrosine kinase inhibitor or a KRAS or BRAF inhibitor, comprising administering to an animal with cancer a CBP/p300 bromodomain inhibitor or a pharmaceutically acceptable salt thereof, wherein the duration of response to anti-cancer therapy upon administration of the CBP/p300 inhibitor or a pharmaceutically acceptable salt thereof is prolonged compared to the duration of response to anti-cancer therapy in the absence of administration of the CBP/p300 inhibitor or a pharmaceutically acceptable salt thereof, and wherein the receptor tyrosine kinase inhibitor is selected from the group consisting of EGFR, ALK, MET, HER2, ROSS1, RET, NTRK1 and AXL inhibitors, or it is a KRAS or BRAF inhibitor.

In another embodiment of the present invention, there is provided a method for inhibiting the growth of a cancer cell, which comprises administering to a cancer cell a CBP/p300 bromodomain inhibitor and a receptor tyrosine kinase inhibitor selected from the group consisting of EGFR, ALK, MET, HER2, ROS1, RET, NTRK1 and AXL inhibitors, or a KRAS or BRAF inhibitor, wherein the cancer cell has changes in the corresponding receptor tyrosine kinase, or in KRAS or BRAF, and wherein the CBP/p300 bromodomain inhibitor itself does not inhibit the growth of the cancer cell, and wherein the CBP/p300 bromodomain inhibitor is administered in an amount effective to prevent resistance of the cancer cell to the kinase inhibitor.

In another embodiment of the present invention, there is provided a method for inducing cancer cell death, comprising administering to a cancer cell a CBP/p300 bromodomain inhibitor and a receptor tyrosine kinase inhibitor selected from the group consisting of EGFR, ALK, MET, HER2, ROS1, RET, NTRK1 and AXL inhibitors, or a KRAS or BRAF inhibitor, wherein the cancer cell has alterations in the corresponding receptor tyrosine kinase, or in KRAS or BRAF, and wherein the CBP/p300 bromodomain inhibitor itself does not cause cancer cell death.

In one embodiment of the present invention, the alteration in the receptor tyrosine kinase may be an oncogenic alteration, wherein the term "oncogenic alteration" in this embodiment, as specified in Section 4, may refer to genetic alterations of cellular proto-oncogenes. These genetic alterations/mutations may result in a growth advantage for the cell. In one embodiment of the present invention, genetic mechanisms of mutation, gene amplification, gene fusion, and/or chromosomal rearrangements may activate oncogenes in human neoplasms.

In another embodiment of the present invention, the oncogenic alteration is an EGFR gene mutation selected from the group consisting of an EGFR exon 19 deletion, EGFR mutations L858R, T790M, T854A, D761Y, L747S, G796S/R, L792F/H, L718Q, an EGFR exon 20 insertion, an EGFR mutation G719X (where X is any other amino acid), L861X, S768I, or EGFR amplification. In a preferred embodiment of the invention, the alteration is an EGFR T790M mutation. In another embodiment of the present invention, the cancer is NSCLC, and the alteration is a mutation comprising an EGFR exon 19 deletion, an EGFR L858R, or a T790M mutation.

In another embodiment of the present invention, the oncogenic alteration is a mutation or rearrangement of the RET gene selected from the group consisting of KIF5B-RET, CCDC6RET, NCOA4-RET, TRIM33-RET, RET-V804L, RET-L730, RET-E732, RET-V738, RET-G810A, RET-Y806, RET-A807 or RET-S904F.

In another embodiment, the oncogenic change is a HER2 gene mutation selected from the group consisting of an insertion or mutation of exon 20 in HER2 and a mutation of HER2-C805S, HER2 T798M, HER2 L869R, HER2 G309E, HER2 S310F, or HER2 amplification.

In another embodiment of the present invention, the oncogenic alteration is a fusion or rearrangement of the ROS1 gene selected from the group consisting of CD74-ROS1, GOPCROS1, 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 a mutation in ROS1 comprising G2032R, D2033N, S1986Y/F, L2026M and/or L1951R.

In another embodiment of the present invention, the oncogenic alteration is an amplification of the MET gene, a mutation of the MET gene such as Y123OC, D1227N, D1228V, Y1248H in MET,

- 18 050570 as well as skipping of exon 14 in MET, or gene fusions or rearrangements selected from the group comprising TPR-MET, CLIP2-MET, TFG-MET fusion, KIF5B-MET fusion.

In another embodiment of the present invention, the oncogenic alteration is a KRAS gene mutation selected from the group consisting of G12C, G12V, G12D, G13D, Q61H or L or R, K117N.

In another embodiment of the present invention, the oncogenic alteration is an ALK gene mutation, gene fusion or rearrangement selected from the group consisting of EML4ALK, TFG-ALK, KIF5B-ALK, KLC1-ALK, STRN-ALK in NSCLC, EML4-ALK, C2orf44-ALK, EML4ALK, TPM-ALK, VCL-ALK, TPM3-ALK, EML4-ALK or VCL-ALK.

In another embodiment of the present invention, the oncogenic alteration is a BRAF gene mutation selected from the group consisting of V600E or V600K.

In another embodiment of the present invention, the oncogenic alteration is a fusion or rearrangement of the NTKR gene selected from the group consisting of TPM3-NTRK1, ETV6NTRK3, TPM3-NTRK1, TPR-NTRK1, TFG-NTRK1, PPL-NTRK1, ETV6-NTRK3, TPR-NTRK1, MPRIPNTRK1, CD74-NTRK1, SQSTM1-NTRK1, TRIM24-NTRK2, LMNA-NTRK, ETV6-NTRK3, BCAN-NTRK1, ETV6-NTRK3, AML, GTST, NFASC-NTRK1, BCAN-NTRK1, AGBL4-NTRK2, VCL-NTRK2, ETV6NTRK3, BTBD1-NTRK3, RFWD2-NTRK1, RABGAP1L-NTRK1, TP53-NTRK1, AFAP1-NTRK2, NACC2NTRK2, OKI-NTRK2, PAN3-NTRK2, or a mutation of the NTKR1 gene selected from the group consisting of F589L, G595R, G667C/S, A608D, or a mutation of the NTRK3 gene selected from the group consisting of G623R, G696A.

In another embodiment of the present invention, the receptor tyrosine kinase inhibitor is an EGFR inhibitor. In another embodiment, the EGFR inhibitor is selected from the group consisting of cetuximab, panitumumab, zalutumumab, nimotuzumab, matuzumab, gefitinib, erlotinib, lapatinib, neratinib, vandetanib, necitumumab, 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 II inhibitor (BIBX 1382, CAS No. 196612-93-8), EGFR III inhibitor (CAS No. 733009-42-2 in CAS), EGFR/ErbB2/ErbB-4 II inhibitor number 944341-54-2 in CAS) or PKCeII/EGFR inhibitor (number 145915-60-2 in CAS).

In another embodiment of the present invention, the alteration in the receptor tyrosine kinase is a mutation in the EGFR gene.

In another embodiment of the present invention, the receptor tyrosine kinase inhibitor is a RET inhibitor. In another embodiment of the present invention, the RET inhibitor is selected from the group consisting of cabozantinib, vandetanib, lenvatinib, alectinib, apatinib, ponatinib, LOXO-292, BLU-667, or RXDX-105.

In another embodiment of the present invention, the receptor tyrosine kinase inhibitor is a HER2 inhibitor. In another embodiment of the present invention, the HER2 inhibitor is selected from the group consisting of trastuzumab, hyaluronidase/trastuzumab fam-trastuzumab deruxtecan, ado-trastuzumab emtansine, lapatinib, neratinib, pertuzumab, tucatinib, poziotinib, or dacomitinib.

In another embodiment of the present invention, the receptor tyrosine kinase inhibitor is a ROS1 inhibitor. In another embodiment of the present invention, the ROS1 inhibitor is selected from the group consisting of crizotinib, ceritinib, brigatinib, lorlatinib, etrectinib, cabozantinib, DS-6051b, TPX-0005.

In another embodiment of the present invention, the receptor tyrosine kinase inhibitor is a MET inhibitor. In another embodiment of the present invention, the MET inhibitor is selected from the group consisting of crizotinib, cabozantinib, MGCD265, AMG208, altiratinib, golvatinib, glesantinib, foretinib, avumatinib, tivatinib, savolitinib, AMG337, capmatinib and tepotinib, OMO-1 [JNJ38877618] or MET antibodies such as onartuzumab and mibetuzumab [LY2875358], or HGF antibodies such as ficlatuzumab [AV-299] and rilotumumab [AMG102].

In another embodiment of the present invention, the inhibitor is a KRAS inhibitor. In another embodiment of the present invention, the KRAS inhibitor is selected from the group consisting of AMG510, MRTX849, JNJ-74699157/ARS-3248, BI1701963, BAY-293, or RAS(ON) inhibitors.

In another embodiment of the present invention, the receptor tyrosine kinase inhibitor is an ALK inhibitor. In another embodiment of the present invention, the ALK inhibitor is selected from the group consisting of crizotinib, ceritinib, alectinib, loratinib, or brigatinib.

In another embodiment of the present invention, the inhibitor is a BRAF inhibitor. In another embodiment of the present invention, the BRAF inhibitor is selected from the group consisting of vemurafenib, dabrafenib, encorafenib, or any non-specific RAF inhibitor.

In another embodiment of the present invention, the receptor tyrosine kinase inhibitor is an NTRK inhibitor. In another embodiment of the present invention, the NTRK inhibitor is selected from the group consisting of entrectinib, larotrectinib (LOXO-101), LOCO-195, DS

- 19 050570

6051b, cabozantinib, merestinib, TSR-011, PLX7486, MGCD516, crizotinib, regorafenib, dovitinib, lestaurtinib, BMS-754807, danusertib, ENMD-2076, midostaurin, PHA-848125 AC, BMS-777607, altriratinib, AZD7451, MK5108, PF-03814735, SNS-314, foretinib, nintedanib, ponatinib, ONO5390556, or TPX-0005.

In another embodiment of the present invention, a composition or combination of a CBP/p300 bromodomain inhibitor or a pharmaceutically acceptable salt thereof and a receptor tyrosine kinase inhibitor, or a KRAS or BRAF inhibitor, exhibits synergism in the treatment of cancer compared to a CBP/p300 inhibitor used alone, a receptor tyrosine kinase inhibitor, or a KRAS or BRAF inhibitor used alone. As used in the context of the description of the embodiments of the present invention presented in Section 4, the term synergistic refers to an interaction between two or more drugs that results in the total effect of the drugs exceeding the sum of the individual effects of each drug. In a preferred embodiment of the present invention, the synergistic effect is an increase in the level of response of an animal to a combination of a CBP/p300 bromodomain inhibitor and a receptor tyrosine kinase inhibitor, or a KRAS or BRAF inhibitor. In another embodiment of the present invention, an increase in the response rate is measured as an increase in the effectiveness of the cancer treatment.

In another embodiment of the present invention, the anti-cancer effect provided by a composition or combination of a CBP/p300 bromodomain inhibitor or a pharmaceutically acceptable salt thereof and a receptor tyrosine kinase, or a KRAS or BRAF inhibitor, exceeds the anti-cancer effect provided by monotherapy at the same dose of a CBP/p300 inhibitor or a receptor tyrosine kinase, or a KRAS or BRAF inhibitor. As used in the context of the embodiments of the present invention presented in Section 4, the term "anti-cancer" refers to the treatment of a malignant or cancerous disease. In another embodiment of the present invention, there is provided a composition for use or a method, wherein the anti-cancer effect provided by the composition or combination of a CBP/p300 bromodomain inhibitor or a pharmaceutically acceptable salt thereof and a receptor tyrosine kinase inhibitor, or a KRAS or BRAF inhibitor, is at least 2 times greater, at least 3 times greater, at least 5 times greater, or at least 10 times greater than monotherapy.

In another embodiment of the present invention, a composition or combination of a CBP/p300 bromodomain inhibitor or a pharmaceutically acceptable salt thereof and a receptor tyrosine kinase inhibitor, or a KRAS or BRAF inhibitor, delays or reduces the risk of cancer resistance to a receptor tyrosine kinase inhibitor or a KRAS or BRAF inhibitor. As used in the context of the embodiments of the present invention presented in Section 4, the term "cancer resistance" refers to a decrease in the effectiveness of a drug; and more specifically, this term can refer to the development of drug resistance in cancer cells. In another embodiment, the cancer does not become resistant to a receptor tyrosine kinase inhibitor or a KRAS or BRAF inhibitor for at least 3 months, 6 months, 9 months, 12 months, 24 months, 48 months, or 60 months. In another embodiment of the present invention, the CBP/p30() bromodomain inhibitor is administered in an amount effective to prevent resistance of the cancer cell to the receptor tyrosine kinase inhibitor or to the KRAS or BRAF inhibitor.

In another embodiment of the present invention, a CBP/p30() bromodomain inhibitor inhibits the bromodomain of CBP and/or p300 (also called histone acetyltransferase p300, E1A-binding protein p300, E1A-associated protein p300) and CBP (also known as CREB-binding protein or CREBBP), which are two structurally very similar proteins that coactivate transcription.

As used herein in the context of the embodiments of the present invention presented in Section 4 of the description of the present invention, 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 of the present invention, a CBP/p300 bromodomain inhibitor may bind to CBP and/or p300 preferentially (e.g., exclusively) through contacts and/or interactions with the CBP bromodomain and/or the p300 bromodomain. In some embodiments, a CBP/p300 bromodomain inhibitor may bind to CBP and/or p300 through contacts and/or interactions with the CBP bromodomain and/or p300 bromodomain, as well as with additional residues and/or domains of CBP and/or p300. In some embodiments, a CBP/p300 bromodomain inhibitor may substantially or completely inhibit the biological activity of CBP and/or p300. In some embodiments, the biological activity may be binding of the CBP and/or p300 bromodomain to chromatin (e.g., histones associated with DNA) and/or another acetylated protein. In some embodiments, in the context of the embodiments described in Section 4, the inhibitor may have an IC50 value or binding constant of less than about 50 μM, less than

- 20 050570 about 1 μM, less than about 500 nM, less than about 100 nM, less than about 10 nM, or less than about 1 nM. In some embodiments of the present invention, the CBPf300 bromodomain inhibitor may bind to and inhibit the CBP bromodomain. In some embodiments of the present invention, the CBPf300 bromodomain inhibitor may bind to and inhibit the p300 bromodomain. In some embodiments of the present invention, the CBPf300 bromodomain inhibitor may not inhibit the histone acetyltransferase activity of CBPf300.

In one embodiment of the present invention, the CBPφ300 bromodomain inhibitor is a compound of formula (I). In one embodiment of the present invention, the CBPφ300 bromodomain inhibitor is a compound of formula (A), preferably compound CCS1477 (CAS number 2222941-37-7). In another embodiment, the CBP/p300 bromodomain inhibitor is FT-7051. In another embodiment of the present invention, the compound of formula (I), the compound of formula (A), preferably compound CCS1477 or compound FT-7051, is present in a daily dose of the drug in an amount selected from the group consisting of 10 mg, 15 mg, 25 mg, 50 mg, 100 mg, 150 mg or 200 mg. In another embodiment of the present invention, compound CCS1477 is administered 2, 3, 4, 5, 6 or 7 days per week. In another embodiment of the present invention, the CCS1477 compound is administered twice daily. In another embodiment of the present invention, administration to a cancer cell comprises contacting the cancer cell with a CBPΦ300 inhibitor and a receptor tyrosine kinase inhibitor, or a KRAS or BRAF inhibitor.

In another embodiment of the present invention, the dosage depends on a variety of factors, including the age, body weight and condition of the patient, as well as the route of administration. Daily dosages can vary widely and will be adjusted to the individual needs in each specific case. However, typically, the dose adopted for each route of administration when the compound is administered alone to an adult human can be in the range of 0.0001 to 50 mg/kg body weight, most often in the range of 0.001 to 10 mg/kg body weight, for example, from 0.01 to 1 mg/kg body weight. Such a dose can be administered, for example, from 1 to 5 times a day. For intravenous injection, 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 divided dose schedule.

In another embodiment of the present invention, cancer progression or duration of response to cancer therapy can be measured using RECIST 1.1 response criteria to target or non-target lesions in a subject/animal.

In another embodiment of the present invention, the term does not slow down the progression of cancer described in the embodiments of the invention presented in Section 4 may be defined as a response in subjects who do not have any clinical response according to the RECIST 1.1 criteria. In another embodiment of the present invention, the term does not slow down the progression of cancer described in the embodiments of the invention presented in Section 4 may be defined as a response in subjects/animals who do not have a partial clinical response according to the RECIST 1.1 criteria. In another embodiment of the present invention, the term does not slow down the progression of cancer is measured as the absence of an objective response rate and/or the absence of an increase in progression-free survival according to the RECTST 1.1 response criteria. In another embodiment of the present invention, the term does not slow down the progression of cancer is measured as a decrease of less than 30% in the sum of the longest diameters of the target lesions, taking the initial sum of the longest diameters of the target lesions as a reference.

In some embodiments of the present invention, the cancer is selected from auditory neuroma, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute T-cell leukemia, basal cell carcinoma, bile duct cancer, bladder cancer, brain cancer, breast cancer, bronchogenic carcinoma, cervical cancer, chondrosarcoma, chordoma, choriocarcinoma, chronic leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, craniopharyngioma, cystadenocarcinoma, diffuse large B-cell lymphoma, dysproliferative changes, embryonal cancer, endometrial cancer, endotheliosarcoma, ependymoma, epithelial carcinoma, erythroleukemia, esophageal cancer, estrogen receptor-positive breast cancer, essential thrombocythemia, Ewing's tumor, fibrotic coma, follicular lymphoma, testicular germ cell cancer, glioma, glioblastoma, gliosarcoma, heavy chain disease, head and neck cancer, hemangioblastoma, hepatoma, hepatocellular carcinoma, hormone-insensitive prostate cancer, leiomyosarcoma, leukemia, liposarcoma, lung cancer, lymphagioendotheliosarcoma, lymphangiosarcoma, lymphoblastic leukemia, lymphoma, lymphoid malignancies of T- or B-cell origin, medullary carcinoma, medulloblastoma, melanoma, meningioma, mesothelioma, multiple myeloma, myelogenous leukemia, myeloma, myxosarcoma, neuroblastoma, midline NUT cancer (NMC), non-small cell lung cancer (NSCLC), oligomerol

- 21 050570 godendrogliomas, oral cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillary adenocarcinoma, papillary carcinoma, pinealoma, polycythemia vera, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcomas, sebaceous gland cancer, seminoma, skin cancer, small cell lung cancer, solid tumors (carcinomas and sarcomas), small cell lung cancer, gastric cancer, squamous cell carcinoma, synovioma, sweat gland cancer, thyroid cancer, Waldenstrom's macroglobulinemia, testicular tumors, uterine cancer and Wilms' tumor. In some embodiments of the present invention, the cancer is melanoma, NSCLC, renal cancer, ovarian cancer, colon cancer, pancreatic cancer, hepatocellular carcinoma, or breast cancer. In some embodiments of any of the methods of the present invention, the cancer is lung cancer, breast cancer, pancreatic cancer, colorectal cancer, and/or melanoma. In some embodiments of the present invention, the cancer is lung cancer. In some embodiments of the present invention, the lung cancer is non-small cell lung cancer (NSCLC). In some embodiments of the present invention, the cancer is breast cancer. In some embodiments of the present invention, the cancer is melanoma. In some embodiments of the present invention, the cancer is colorectal cancer.

In another embodiment of the present invention, the CBPf300 bromodomain inhibitor and the receptor tyrosine kinase inhibitor, or the KRAS or BRAF inhibitor, are administered to the animal simultaneously as a single composition. In another embodiment of the present invention, the CBPf300 bromodomain inhibitor and the receptor tyrosine kinase inhibitor, or the KRAS or BRAF inhibitor, are administered to the animal separately. In another embodiment of the present invention, the CBPf300 bromodomain inhibitor and the receptor tyrosine kinase inhibitor, or the KRAS or BRAF inhibitor, are administered to the animal simultaneously. In another embodiment of the present invention, the CBPf300 bromodomain inhibitor is administered to the animal before the administration of the receptor tyrosine kinase inhibitor, or the KRAS or BRAF inhibitor. In another embodiment of the present invention, the animal is a human.

In one embodiment of the present invention, the term "effective amount" of an agent, such as a pharmaceutical composition, may refer to an amount effective, at doses and for times necessary, to achieve the desired therapeutic or prophylactic result. In some embodiments of the present invention, an effective amount refers to an amount of a CBP/p300 bromodomain inhibitor and a receptor tyrosine kinase inhibitor, or a KRAS or BRAF inhibitor, that: (i) treats a particular disease, condition, or disorder, (ii) reduces, improves, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder, as described herein. In some embodiments of the present invention, 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 reduce the size of a tumor; may inhibit (i.e., slow to some extent and preferably stop) the infiltration of cancer cells into peripheral organs; may inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; may inhibit tumor growth to some extent; and/or may alleviate to some extent one or more symptoms associated with cancer. For cancer therapy, efficacy can be measured, for example, by assessing time to disease progression (TTP) and/or by determining the response rate (RR). In some embodiments of the present invention, the effective amount is an amount of the CBPφ300 bromodomain inhibitor and the receptor tyrosine kinase inhibitor, or the KRAS or BRAF inhibitor described herein, sufficient to significantly reduce the activity or number of drug-resistant or persistent drug-resistant cancer cells.

In one embodiment of the present invention, a compound of the invention may be administered to a human or animal patient in combination with radiation therapy or another chemotherapeutic agent for the treatment of cancer. In another embodiment of the present invention, there is provided a combination therapy in which a CBP/P300 inhibitor, or an RTK inhibitor, or a KRAS or BRAF inhibitor is administered simultaneously or sequentially with radiation therapy; or administered simultaneously or sequentially, or as a combination preparation with another chemotherapeutic agent or agents for the treatment of cancer. The chemotherapeutic agent is typically an agent commonly used to treat the relevant type of cancer. Classes of chemotherapeutic agents for combination may, in one embodiment of the present invention, include, for example, androgen receptor antagonists used to treat prostate cancer, such as enzalutamide, and CYP17A1 (17a-hydroxylase/C17,20 lyase) inhibitors, such as abiraterone. In other embodiments of the present invention, other chemotherapeutic agents in the combination therapy may include docetaxel.

- 22 050570

In one embodiment of the present invention, the term "combination," as used in the embodiments of the invention presented in Section 4, may refer to simultaneous, separate, or sequential administration. If administration is sequential or separate, the delay in administration of the second component should not be such that the beneficial effect of the combination is lost.

In another embodiment of the present invention, the response to a CBP/p300 bromodomain inhibitor and a receptor tyrosine kinase inhibitor, or a KRAS or BRAF inhibitor, is a durable response. In one embodiment, a durable response may refer to a sustained effect, expressed as a reduction in tumor growth, after cessation of treatment. For example, the tumor size may remain the same or smaller than its size at the beginning of the infusion phase.

In another embodiment of the present invention, the term "treatment" (and variants such as "treat" and other derivatives) may refer to a clinical intervention in an attempt to alter the natural course of a pathology in an individual or in a cell being treated, and the treatment may be performed either prophylactically or during the course of a clinical pathology. Desirable effects of treatment may include one or more of the following: preventing the onset or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, stabilizing (i.e., not worsening) the condition of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, increasing survival compared to the expected survival when not receiving treatment, and remission or improving prognosis. In some embodiments of the present invention, an inhibitor of the CBP/p300 bromodomain and receptor tyrosine kinase, or an inhibitor of KRAS or BRAF, may be used to delay the development of a disease or disorder, or to slow the progression of a disease or disorder. In one embodiment of the present invention, the persons in need of treatment may include persons who already have a pathological condition or disorder, as well as persons who are prone to developing such a pathological condition or disorder (e.g., as a result of a genetic mutation or aberrant expression of a gene or protein), or persons in whom it is necessary to prevent such a condition or disorder.

In one embodiment of the present invention, the term "delay" may refer to postponing, hindering, slowing, arresting, stabilizing, and/or delaying the progression of a disease (such as cancer) or resistance to the disease. This delay may vary in duration, depending on the disease history and/or the individual being treated. Those skilled in the art will appreciate that a sufficient or significant delay may, in essence, involve prevention, as the individual does not develop the disease. For example, advanced cancer, such as the development of metastases, may be delayed.

5. Examples.

The following examples are illustrative and further describe the present invention. These examples should not be construed as limiting the present invention.

The preparation of compound 00003 (compound B), compound 00004 (compound A), 00030, 00071, and compound C is described below. A description of the synthesis route of an intermediate compound and/or a compound close to the above compounds would also be helpful.

General experimental methods.

Liquid chromatography-mass spectrometry (LC-MS) methods.

Method A. Setup: Agilent 1260. Binary gradient pump: G1312B, degasser; autosampler: ColCom, diode array detector (DAD): Agilent G1315D, 220-320 nm, mass selective detector (MSD): Agilent LC/MSD G6130B ESI, posit./neg. 100-800, evaporative light scattering detector (ELSD) Alltech 3300, gas flow 1.5 mL/min, gas temperature: 40°C; Column: Waters XSelect™ C18, 30 x 2.1 mm, 3.5 µm, temperature: 35°C, flow: 1 mL/min, gradient: t ₀ = 5% A, ti.e _min = 98% A, t _{3 min} = 98 % A. Column recovery time: 1.3 min. Eluent A: 0.1% formic acid in acetonitrile, eluent B: 0.1% formic acid in water.

Method B. Setup: Agilent 1260. Binary gradient pump: G1312B, degasser; autosampler: ColCom, DAD: Agilent G1315D, 220-320 nm, MSD: Agilent LC/MSD G6130B ESI, posit./neg. 100-800, ELSD Alltech 3300, gas flow 1.5 mL/min, gas temperature: 40°C; Column: Waters XSelect™ C18, 50 x 2.1 mm, 3.5 μm, temperature: 35°C, flow: 0.8 mL/min, gradient: t ₀ = 5 % A, _{t 3.5 min} = 98 % A, t _{6 min} = 98 % A. Column recovery time: 2 min. Eluent A: 0.1% formic acid in acetonitrile, eluent B: 0.1% formic acid in water.

Method C. Setup: Agilent 1260. Binary gradient pump: G1312B, degasser; autosampler: ColCom, DAD: Agilent G1315C, 220-320 nm, MSD: Agilent LC/MSD G6130B ESI, posit./neg. 100-800; Column: Waters XSelect™ CSH C18, 30 x 2.1 mm, 3.5 μm, temperature: 25°C, flow: 1 mL/min, gradient: t ₀ = 5% A, t1, _{6 min} = 98% A, t _{3 min} = 98% A, column recovery time:

- 23 050570

1.3 min. Eluent A: 95% acetonitrile + 5% 10 mM ammonium bicarbonate in water in acetonitrile, eluent B: 10 mM ammonium bicarbonate in water (pH 9.5).

Method D. Setup: Agilent 1260. Binary gradient pump: G1312B, degasser; autosampler: ColCom, DAD: Agilent G1315C, 220-320 nm, MSD: Agilent LC/MSD G6130B ESI, posit./neg. 100-800; Column: Waters XSelect™ CSH C18, 50x2.1 mm, 3.5 μm, temperature: 25°C, flow: 0.8 mL/min, gradient: t0 = ₅ % A, _{t3.5 min} = 98% A, t6 _min = 98% A, column recovery time: 2 min. Eluent A: 95% acetonitrile + 5% 10 mM ammonium bicarbonate in water in acetonitrile. Eluent B: 10 mM ammonium bicarbonate in water (pH 9.5).

Ultra-Performance Liquid Chromatography (UPLC) Methods. Method A: Setup: Agilent Infinity II, Binary Gradient Pump: G7120A, Multisampler, Vacuum Temperature Chamber (VTC), DAD: Agilent G7117B, 220-320 nm, Photodiode Array Detector (PDA): 210-320 nm, MSD: Agilent G6135B ESI, Positive/Negative. 100-1000, ELSD G7102A: inlet 40°C, nebulizer 50°C, gas flow 1.6 mL/min, column: Waters XSelect CSH C18, 50 x 2.1 mm, 2.5 μm, temperature: 25°C, flow: 0.6 mL/min, gradient: t0 = 5% B, t2 _min = 98 % B, _t2 , _{7 min} = 98% B, column recovery time: 0.3 min. Eluent A: 10 mM ammonium bicarbonate in water (pH 9.5), eluent B: acetonitrile.

Method B. Setup: Agilent Infinity II. Binary gradient pump: G7120A, multisampler, VTC, DAD: Agilent G7117B, 220-320 nm, PDA: 210-320 nm, MSD: Agilent G6135B ESI, posit./neg. 100-1000, ELSD G7102A: inlet 40°C, nebulizer 40°C, gas flow 1.6 mL/min, column: Waters XSelect™ CSH C18, 50 x 2.1 mm, 2.5 μm, temperature: 40°C, flow: 0.6 mL/min, gradient: t0 = 5 % B, t2 _min = 98 % B, _{t2.7 min} = 98 % B, column recovery time: 0.3 min. Eluent A: 0.1% formic acid in water, eluent B: 0.1% formic acid in acetonitrile.

Gas chromatography-mass spectrometry (GC-MS) methods. Method A: setup: GC: Agilent 6890N G1530N; MS setup: MSD 5973 G2577A, EI-positive, hold temperature: 280°C, mass range: 50-550. Column: RXi-5MS 20 m, internal diameter 180 μm, pore size (df) 0.18 μm; average velocity: 50 cm/s; injection volume: 1 μl; injector temperature: 250°C; separation factor: 100/1; carrier gas: He; initial temperature: 100°C; initial time: 1.5 min; solvent hold-up: 1.0 min; flow rate 75°C/min; final temperature 250°C; column recovery time 4.3 min.

Method B. Setup: GC: Agilent 6890N G1530N. Flame ionization detector (FID): detection temp.: 300°C and MS: MSD 5973 G2577A, EI-pos., hold temperature: 280°C, mass range: 50-550. Column: Restek RXi-5MS 20 m, internal diameter 180 μm, df 0.18 μm; average speed: 50 cm/s; injection volume: 1 μL; injector temperature: 250°C; separation factor: 20/1; carrier gas: He; initial temperature: 60°C; initial time: 1.5 min; solvent hold-up: 1.3 min; flow rate 50°C/min; final temperature 250°C; column recovery time 3.5 min.

Method C. Setup: GC: Agilent 6890N G1530N, FID: detection temp: 300°C and MS: MSD 5973 G2577A, EI-positive, hold temperature: 280°C. Mass range: 50-550; Column: Restek RXi-5MS 20 m, internal diameter 180 μm, df 0.18 μm; average speed: 50 cm/s; injection volume: 1 μL; injector temperature: 250°C; separation factor: 20/1; carrier gas: He; initial temperature: 100°C; initial time: 1.5 min; solvent hold-up: 1.3 min; rate 75°C/min; final temperature 250°C; column recovery time 4.5 min.

Chiral liquid chromatography (LC).

Method A. Setup: Agilent 1260 Quart. Pump: G1311C, Autosampler, ColCom, DAD: Agilent G4212B, 220-320 nm, Column: Chiralcel® OD-H 250 x 4.6 mm, Temperature: 25°C, Flow Rate: 1 mL/min, Isocracy: 90/10, Time: 30 min. Eluent A: Heptane, Eluent B: Ethanol.

Preparative reversed-phase chromatography.

Method A. Setup: Reveleris™ Prep MPLC; Column: Phenomenex LUNA C18 (150 x 25 mm, 10 μm); 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; UV detection: 220/254 nm. The appropriate fractions were pooled and lyophilized.

Method B. Setup: Reveleris™ Prep MPLC; Column: Waters XSelect™ CSH C18 (145 x 25 mm, 10 µm); Flow Rate: 40 mL/min; Column Temperature: RT. Eluent A: 10 mM ammonium bicarbonate in water, pH 9.0; eluent B: 99% acetonitrile + 1% 10 mM ammonium bicarbonate in water; 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; UV Detection: 220/254 nm. The appropriate fractions were pooled and lyophilized.

Chiral (preparative) ultra-performance fluid chromatography (UPFC).

Method A. Column: SFC instrument modules: Waters Prep100q SFC System, PDA: Waters 2998, fraction collector: Waters 2767; column: Phenomenex Lux Amylose-1 (250 x 20 mm, 5 μm), column temperature: 35°C; flow: 100 mL/min, automated back pressure regulator (ABPR): 170 bar. Eluent A: CO2, eluent B: 20 mM ammonia in methanol, isocratic 10% B, time: 30 min, detection: PDA (210-320 nm), fraction collection by PDA signal.

- 24 050570

Method B. Column: SFC instrument modules: Waters PreplOOq SFC System, PDA: Waters 2998, fraction collector: Waters 2767; column: Phenomenex Lux Celulose-1 (250 x 20 mm, 5 μm), column temperature: 35°C; flow: 100 mL/min, ABPR: 170 bar. Eluent A: CO2, eluent B: 20 mM ammonia in methanol, isocratic 10% B, time: 30 min, detection: PDA (210-320 nm), fraction collection by PDA signal.

Method C. Column: SFC instrument modules: Waters Prep100q SFC System, PDA: Waters 2998; column: Chiralpak IC (100 x 4.6 mm, 5 μm), column temperature: 35°C; flow: 2.5 mL/min; ABPR: 170 bar. Eluent A: CO2, eluent B: methanol with 20 mM ammonia, t=0 min 5% B, t=5 min 50% B, t=6 min 50% B, detection: PDA (210-320 nm); fraction collection by PDA signal.

Method D. Column: SFC instrument modules: Waters Prep 100 SFC UV/MS directed system; Waters 2998 photodiode array (PDA) detector; Waters Acquity QDa MS detector. Waters 2767 Sample Management System; column: Waters Torus 2-PIC 130A OBD (250 x 19 mm, 5 μm); column temperature: 35°C; flow rate: 70 mL/min; ABPR: 120 bar. Eluent A: CO2, eluent B: 20 mM ammonia in methanol; linear gradient: t=0 min 10% B, t=4 min 50% B, t=6 min 50% B; detection: PDA (210-400 nm); fraction collection based on the PDA signal for tentatively identified compounds (TIC).

Source materials.

Commercially available standard reagents and solvents were of the highest commercial purity and were used as such; these reagents are described below.

Connection name Supplier CAS number Tetrakis(triphenylphosphine)palladium(0) Sigma-Aldrich 14221-01-3 1,1- bis(diphenylphosphino)ferrocenepalladium(II) dichloride Sigma-Aldrich 72287-26-4 2-dicyclohexylphosphino-2',4',6'triisopropylbiphenyl Sigma-Aldrich 564483-18-7 Bis(triphenylphosphine)palladium dichloride (TT) Fluorochem 13965-03-2

- 25 050570

2-tributylstanylpyrazine Combi-Blocks 205371-27-3 N-acetyl-O-leucine Accela Chembio 19764-30-8 Methyl 6-methylpiperidine-3-carboxylate Combi-Blocks 908245-03-4 3-bromo-5-fluoroaniline Combi-Blocks 134168-97-1 1-methyl-4-(tributylstannyl)-1H-imidazole Synthonix 446285-73-0 3-fluoro-5-iodaniline Combi-Blocks 660-49-1 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)1H-pyrazole Combi-Blocks 269410-08-4 3-bromoaniline Combi-Blocks 591-19-5 1,3,5-trimethyl-4-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)-1 H-pyrazole Combi-Blocks 844891-04-9 3-fluoro-5-nitrobenzoic acid Combi-Blocks 14027-75-9 Acetohydrazide Combi-Blocks 1068-57-1 N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride Fluorochem 25952-53-8 1-hydroxy-7-azabenzotriazole Enamine 39968-33-7 (Methoxycarbonylsulfamoyl)triethylammonium hydroxide (Burgess reagent) Combi-Blocks 29684-56-8 3-nitrophenylacetylene Combi-Blocks 3034-94-4 Sodium L-ascorbic acid Sigma-Aldrich 134-03-2 2-azidopropane, 2.5 M in DMF Enamine 691-57-6 Azidoxetane, 0.5 M in MTBE Enamine 81764-67-2 Azidotrimethylsilane Acros 4648-54-8 1-fluoro-3-iodo-5-nitrobenzene Combi-Blocks 3819-88-3 1-bromo-3-chloro-5-nitrobenzene Combi-Blocks 219817-43-3 2-iodo-1-methyl-4-nitrobenzene Fluorochem 7745-92-8 3-bromo-5-nitrotoluene Combi-Blocks 52488-28-5 4-bromo-1-methyl-1,2,3-triazole Combi-Blocks 13273-53-5 3-nitrobenzaldehyde Acros 99-61-6 3-nitrophenylacetylene Combi-Blocks 3034-94-4 Chloro(pentamethylcyclopentadienyl)bis(triphenylphosphine)ruthenium(II) ST REM chemicals 92361-49-4 Tetrabutylammonium fluoride 1.0 M solution in THF Fluorochem 429-41-4 3-ethynyl-4-fluoroaniline Synthonix 77123-60-5 Tert-butyl 3-cyanopiperidine-1-carboxylate Combi-Blocks 91419-53-3 Raney Nickel®, 50% suspension in water Acros Organics 7440-02-0 Tris(dibenzylideneacetone) dipalladium (0) Sigma-Aldrich 51364-51-3 Exphos Sigma-Aldrich 564483-18-7 2-(tributylstannyl)-pyrimidine Sigma-Aldrich 153435-63-3 10% palladium on activated carbon ACROS 7440-05-3

- 26 050570

Synthetic procedures for basic intermediates.

Intermediate 1: 1-(5-(4,6-dichloropyrimidin-2-yl)-2-methylpiperidin-1-yl)ethan-1-one

0.,.. .0..

0.,. ,0,

NHj

Acai

Ml I C Ac

DCM, water, RT, hours 'Λϋ-i -,ul 40· hours

-CL.·

INCHL·

Hi 16 hours

LIOH . 5-c hours

HN

H %H

A-OH

I-j 1i -1t.

t to*I -j:j from day •NOAc

It. Ill I.

I. »CMe di methyl mal on ate

HO... ..N

I’ >' I.

CL. .4 .’J. ,.0

Ι.'ζΠΗ RUS hours hours _: .h

EL·.

RT - room temperature

To a solution of methyl 6-methylnicotinate (100 g, 662 mmol) in acetic acid (250 mL) in a 1 L steel autoclave was added 0.5 g, 2.202 mmol of platinum oxide (C1), and the reaction mixture was stirred under a hydrogen atmosphere at 10 bar and 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 celite. The filtrate was concentrated to give methyl 6-methylpiperidine-3-carboxylate acetate as a mixture of diastereoisomers (143.8 g, 100%), which was used as such in the next step. GC-MS (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 (effervescence!) to a solution of methyl 6-methylpiperidine-3-carboxylate acetate (53 g, 244 mmol) in a mixture of water (500 mL) and dichloromethane (DCM; 500 mL) followed by slow addition of acetic anhydride (29.9 g, 293 mmol). The reaction mixture was stirred at room temperature for 2 h. The organic layer was separated, dried over sodium sulfate, filtered, and concentrated in vacuo to give methyl 1-acetyl-6-methylpiperidine-3-carboxylate (49 g, 100%) as a yellow oil. A solution of methyl 1-acetyl-6-methylpiperidine-3-carboxylate (49 g, 246 mmol) in ammonia in methanol (7 N, 500 mL, 3.5 mol) was stirred in an autoclave at 120 °C for 40 h. The mixture was cooled to room temperature and concentrated to give a light yellow solid. This solid was dissolved in dichloromethane and filtered through a pad of silica. The filtrate was concentrated to give 1-acetyl-6-methylpiperidine-3-carboxamide as an off-white solid, which was used as such in the next step. A solution of 1-acetyl-6-methylpiperidine-3-carboxamide (266 mmol) from the previous step in phosphorus oxychloride (500 mL, 5.37 mol) was stirred at room temperature for 16 h. The reaction mixture was evaporated in vacuo to yield a thick oil. This oil was coevaporated twice with toluene and carefully partitioned between cold saturated sodium carbonate (boiling!) and ethyl acetate. The organic layer was separated from the main aqueous layer, dried over sodium sulfate, filtered, and concentrated in vacuo to yield the product as a thick oil that solidified on standing. The crude product was dissolved in dichloromethane and filtered through a pad of silica (eluting with 10% methanol in dichloromethane). 1-Acetyl-6-methylpiperidine-3-carbonitrile (28 g, 63%) was obtained as an oil that solidified on standing. GC-MS (Method A): tR 3.78 (63%) and 3.89 min (378%), 100%, MS (EI) 166.1 (M) ⁺ . To a solution of 1-acetyl-6-methylpiperidine-3-carbonitrile (23 g, 138 mmol) in ethanol (300 mL) was added a solution of hydroxylamine (50% in water, 25.4 mL, 415 mmol), and the reaction mixture was stirred at reflux for 16 h. The reaction mixture was concentrated and co-evaporated three times with ethyl acetate to dryness to afford 1-acetyl-N-hydroxy-6-methylpiperidine-3-carboximidamide as a sticky solid. LC-MS (Method A): tR 0.13 min, 100%, MS (ESI) 200.2 (M+H) ⁺ . Assuming quantitative yield, the product was used as such in the next step. To a solution of 1-acetyl-N-hydroxy-6-methylpiperidine-3-carboximidamide (23 g, 138 mmol) from the previous step in ethanol (500 mL) and acetic acid (23.79 mL, 416 mmol) was added a 50% suspension of Raney®-Nickel in water (5 mL), and the reaction mixture was stirred under hydrogen atmosphere for 2 days at 50°C. The mixture was filtered through Celite, washed with a small amount of ethanol, and concentrated to give 70 g of a viscous oil. This was co-evaporated twice with ethyl acetate and dried vigorously in vacuo to give

- 27 050570 1-Acetyl-6-methylpiperidine-3-carboximidamide acetate (33 g, 98%) as a greenish-yellow oil, which was used as such in the next step. LC-MS (Method A): t _R 0.14 min, 90%, MS (ESI) 184.1 (M+H)+. To a solution of sodium (18.14 g, 789 mmol) in dry methanol under nitrogen atmosphere (60 mL) were added 1-acetyl-6-methylpiperidine-3-carboximidamide acetate (32 g, 132 mmol) and dimethyl malonate (26.1 g, 197 mmol), 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 using 6 N hydrochloric acid, and allowed to precipitate. The precipitate was filtered off to give 1-(5-(4,6-dihydroxypyrimidin-2-yl)-2-methylpiperidin-1-yl)ethan-1-one as a yellow solid (10.4 g, 31%), which was used as such in the next step. A suspension of 1-(5-(4,6-dihydroxypyrimidin-2-yl)-2-methylpiperidin-1-yl)ethan-1-one (10.4 g, 41.4 mmol) in phosphorus oxychloride (200 mL, 2146 mmol) was stirred at 50 °C. The solids dissolved slowly after approximately 3 h. After 5 h, the reaction mixture was concentrated in vacuo and coevaporated twice with toluene. The residual oil was carefully quenched with ice, neutralized with saturated aqueous sodium bicarbonate, and extracted with ethyl acetate (2 x 100 mL). The combined organic layers were dried over sodium sulfate and concentrated in vacuo to give 1-(5-(4,6-dichloropyrimidin-2-yl)-2-methylpiperidin-1-yl)ethan-1-one (intermediate 1, 6.8 g, 57%) as a yellow oil that solidified on standing. LC-MS (Method A): t _R 1.88 min, 100%, MS (ESI) 288.1 (M+H) ⁺ .

Intermediate 2: 1-((2S,5R)-5-(4,6-dichloropyrimidin-2-yl)-2-methylpiperidin-1 yl)ethan-1-one

RT - room temperature

To a solution of X-acetyl-D-leucine (1 kg, 5.77 mol) in ethanol (1.5 L) was added a solution of methyl 6-methylpiperidine-3-carboxylate (934 g, 2.38 mol, obtained in intermediate 1) in ethyl acetate (3 L), and the mixture was heated to 40°C. The resulting solution was allowed to reach room temperature over 16 h, during which time precipitation occurred. The precipitate was filtered, washed with diethyl ether (500 mL), and air-dried to yield crude methyl (3R,6S)-6-methylpiperidine-3-carboxylateacetyl-D-leucine (287 g, 34%) as a white solid. Crude methyl (3R,6S)-6-methylpiperidine-3-carboxylate acetyl-D-leucinate (287 g, 869 mmol) was crystallized from a hot mixture of ethanol and ethyl acetate 1:2 (1 L). The precipitate was filtered off, and the residue was triturated in a mixture of diethyl ether and n-pentane 1:1 (500 mL). The precipitate was filtered off and air-dried, yielding methyl (3R,6S)-6-methylpiperidine-3-carboxylate acetyl-D-leucinate (128 g, 44%) as a white solid. To a solution of methyl (3R,6S)-6-methylpiperidine-3-carboxylate acetyl D-leucinate (128 g, 387 mmol) in dichloromethane (DCM; 1 L) was added saturated sodium carbonate solution (1 L). The two-phase system was vigorously stirred for 10 min and the layers were separated. The organic layer was dried over sodium sulfate and filtered to give a clear solution. Triethylamine (65 mL, 465 mmol) and acetic anhydride (44 mL, 465 mmol) were then added, and the mixture was stirred at room temperature for 1 h. The mixture was washed with saturated sodium bicarbonate solution, dried over sodium sulfate, and concentrated to give methyl (3R,6S)-1-acetyl-6-methylpiperidine-3-carboxylate (93 g) as a light yellow solid. An autoclave was charged with methyl (3R,6S)-1-acetyl-6-methylpiperidine-3-carboxylate (93 g, 387 mmol) in 7N ammonia in methanol (600 mL, 4200 mmol) and 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 quantitative yield, the product was used as such in the next step. Chiral LC (Method A) t _R = 12.35 min, ee >98%. To a solution of (3R,6S)-1-acetyl-6-methylpiperidine-3-carboxamide (50 g, 271 mmol) in dichloromethane (500 mL) was added triethyloxonium tetrafluoroborate (77 g, 407 mmol) portionwise, and the mixture was stirred at room temperature for 4 h. 7 N ammonia in methanol (200 mL, 9.15 mol) was slowly added, and the mixture was stirred at room temperature for 16 h. The mixture was concentrated to give (3R,6S)-1-acetyl-6-methylpiperidine-3-carboximidamide (50 g) as a pink solid, which was used as such in the next step. To a solution of 5.4 M sodium methoxide in methanol (99 ml, 535 mmol) in methanol (200 ml) was added (3R,6S)-1-acetyl-6

- 28 050570 methylpiperidine-3-carboximidamide (49 g, 267 mmol) in methanol (400 mL) and dimethyl malonate (61.4 mL, 535 mmol). The mixture was heated to 50°C and stirred for 24 h. The mixture was acidified (pH ~3) with concentrated hydrochloric acid and concentrated to a smaller volume. The residue was filtered through silica (20% methanol in dichloromethane) and concentrated to give an orange oil. The crude product was purified by silica gel column chromatography (0% to 20% methanol in dichloromethane) to give 1-((2S,5R)-5-(4,6-dihydroxypyrimidin-2-yl)-2-methylpiperidin-1-yl)ethan-1-one (12 g, 17%) as a colorless gum. LC-MS (Method C): t _R 0.17 min, 100%, MS (ESI) 252.1 (M+H)+. A solution of 1-((2S,5R)-5-(4,6-dihydroxypyrimidin-2-yl)-2-methylpiperidin-1-yl)ethan-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 coevaporated 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 saturated brine, dried over sodium sulfate, and concentrated to give a yellow oil. The oil was purified by column chromatography on silica gel (0% to 20% tetrahydrofuran in toluene) to give 1-((2S,5R)-5-(4,6-dichloropyrimidin-2-yl)-2-methylpiperidin-1-yl)ethan-1-one (intermediate 2, 1.5 g, 11%) as a colorless gum. LC-MS (method B): t _R 3.34 min, 100%, MS (ESI) 288.0 (M+H) ⁺ ; Chiral UHPLC (method A) t _R 2.54 min, ee and de >95%.

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

JL. 4*.'.1 .—nes-s '.'i--'·- stad “ L ·'

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I · ^G 1

Fuel and lubricants. FT.

hour

BUT U ⁵ g

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RT - room temperature

Platinum(IV) oxide (0.5 g, 2.202 mmol) was added to a solution of methyl 6-methylnicotinate (100 g, 662 mmol) in acetic acid (250 mL) in a 1 L steel autoclave, and the reaction mixture was stirred under a hydrogen atmosphere at 10 bar and 60 °C. 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 celite. The filtrate was carefully concentrated to give methyl 6-methylpiperidine-3-carboxylate acetate as a mixture of diastereoisomers (143.8 g, 100%), which was used as such in the next step. GC-MS (Method A): t _R 2.40 (80%) and 2.48 min (20%), 100%, MS (EI) 157.1 (M) ⁺ . Methyl 6-methylpiperidine-3-carboxylate acetate as a mixture of diastereoisomers (2.1 kg, 9924 mmol) was diluted with dichloromethane (DCM; 4 L) and 4 M sodium hydroxide solution was slowly added to pH ~9. The layers were separated and the aqueous layer was extracted twice with dichloromethane (after each extraction, the aqueous layer was basified 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 ~50% yellow solution in dichloromethane. 1H NMR (400 MHz, CDC13, rotamer mixture) 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), 1.95-1.76 (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). To a solution of N-acetyl-D-leucine (1 kg, 5.77 mol) in ethanol (1.5 L) was added a solution of methyl 6-methylpiperidine-3-carboxylate (934 g, 2.38 mol) in ethyl acetate (3 L), and the mixture was heated to 40°C. The resulting solution was allowed to reach room temperature over 16 h, during which time precipitation occurred. The precipitate was filtered, washed with diethyl ether (500 mL), and air-dried to yield crude methyl (3R,6S)-6-methylpiperidine-3-carboxylate acetyl-D-leucine (287 g, 34%) as a white solid. Crude methyl (3R,6S)-6-methylpiperidine-3-carboxylateacetyl-D-leucinate (287 g, 869 mmol) was crystallized from a hot mixture of ethanol and ethyl acetate 1:2 (1 L). The precipitate was filtered off, and the residue was triturated in a mixture of diethyl ether and n-pentane 1:1 (500 mL). The precipitate was filtered off and air-dried to give methyl (3R,6S)-6-methylpiperidine-3-carboxylateacetyl-D-leucinate.

- 29 050570 leucinate (128 g, 44%) as a white solid. 'H-NMR (400 MHz, DMSO-dg) δ 7.80 (d, J = 8.2 Hz, 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), 3H), 1.49-1.37 (m, 2H), 1.30-1.15 (m, 1H), 1.02 (d, J = 6.4 Hz, 3H), 0.85 (m, 6H). To a solution of methyl (3R,6S)-6-methylpiperidine-3-carboxylate acetyl D-leucinate (128 g, 387 mmol) in dichloromethane (1 L) was added saturated sodium carbonate solution (1 L). The two-phase system was vigorously stirred for 10 min and the layers were separated. The organic layer was dried over sodium sulfate and filtered to give a clear solution. Triethylamine (65 mL, 465 mmol) and acetic anhydride (44 mL, 465 mmol) were then added, and the mixture was stirred at room temperature for 1 h. The mixture was washed with saturated aqueous sodium bicarbonate solution, dried over sodium sulfate, and concentrated to give methyl (3R,6S)-1-acetyl-6-methylpiperidine-3-carboxylate (93 g) as a light yellow solid. ¹ H NMR (400 MHz, CDCl ₃ , rotamer mixture) 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= 11.6 Hz, 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). An autoclave was charged with methyl (3R,6S)-1-acetyl-6-methylpiperidine-3-carboxylate (93 g, 387 mmol) in 7 N ammonia in methanol (600 mL, 4200 mmol) and 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 quantitative yield, the product was used as such in the next step. A-NMR (400 MHz, DMSO-dfe rotamer mixture) δ 7.38 (s, 1H), 6.89 (d, J=24.7 Hz, 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, ee >98%. Triethyloxonium tetrafluoroborate (77 g, 407 mmol) was added portionwise 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 h.

A 7N solution of ammonia in methanol (200 mL, 9.15 mol) was slowly added, and the mixture was stirred at room temperature for 16 h. The mixture was concentrated to give (3R,6S)-1-acetyl-6-methylpiperidine-3-carboximidamide (50 g) as a pink solid, which was used as such in the next step. To a solution of 5.4 M sodium methoxide in methanol (99 mL, 535 mmol) in methanol (200 mL) was added (3R,6S)-1-acetyl-6-methylpiperidine-3-carboximidamide (49 g, 267 mmol) in methanol (400 mL) and dimethyl malonate (61.4 mL, 535 mmol). The mixture was heated to 50 °C and stirred for 24 h. The mixture was acidified (pH ~3) with concentrated hydrochloric acid and concentrated to a low volume. The residue was filtered through silica (20% methanol in dichloromethane) and concentrated to give an orange oil. The crude product was purified by silica gel column chromatography (0% to 20% methanol in dichloromethane) to give 1-((2S,5R)-5-(4,6-dihydroxypyrimidin-2-yl)-2-methylpiperidin-1-yl)ethan-1-one (12 g, 17%) as a colorless gum. LC-MS (Method C): tR 0.17 min, 100%, MS (ESI) 252.1 (M+H)+. A solution of 1-((2S,5R)-5-(4,6-dihydroxypyrimidin-2-yl)-2-methylpiperidin-1-yl)ethan-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 coevaporated 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 saturated brine, dried over sodium sulfate, and concentrated to give a yellow oil. The oil was purified by column chromatography on silica gel (0% to 20% tetrahydrofuran in toluene) to give 1-((2S,5R)-5-(4,6-dichloropyrimidin-2-yl)-2-methylpiperidin-1-yl)ethan-1-one (1.5 g, 11%) as a colorless gum. ¹ H NMR (400 MHz, DMSO-6, rotamer mixture) δ 7.95 (d, J = 7.3 Hz, 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); LC-MS (Method B): tR 3.34 min, MS (ESI) 288.0 (M+H)+; Chiral UHPLC (Method: A) tR 2.54 min, ee and de >95%. A mixture of 2-tributylstannylpyrazine (607 mg, 1.65 mmol), 1-((2S,5R)-5-(4,6-dichloropyrimidin-2-yl)-2-methylpiperidin-1-yl)ethan-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 under argon and stirred for 32 h. The mixture was diluted with dichloromethane containing 1% triethylamine and loaded onto silica gel. The mixture was purified by silica gel column chromatography (0% to 40% acetonitrile in dichloromethane containing 1% triethylamine) to give 1-((2S,5R)-5-(4-chloro-6-(pyrazin-2-yl)pyrimidin-2-yl)-2-methylpiperidin-1-yl)ethan-1-one (intermediate 3, 134 mg, 18%) as an orange gum. ¹ H-NMR (400 MHz, DMSO^6, mixture of rotamers) 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, 0.5N), 4.27-4.17 (m, 0.5N), 4.09-3.97 (m, 0.5N), 3.55-3.41 (m, 0.5N), 3.06-2.98 (m, 0.5N), 2.88-2.82 (m, 0.5N), 2.10-1.90 (m, 6H), 1.89-1.76 (m, 0.5H), 1.75-1.61 (m, 1.5H), 1.29-1.20 (m, 1.5H), 1.17-1.10 (m, 1.5H); LC-MS (method C): tR 1.81 min, MS (ESI) 331.1 (M+H)+.

- 30 050570

Synthesis procedures for final products.

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)ethan-1-one (compound 00001) and 1-((2R,5S)-2-methyl-5-(4-((5-methylpyridin-3-yl)amino)-6-(pyrazin-2-yl)pyrimidin-2-yl)piperidin-1-yl)ethan-1-one (compound 00002)

RT - room temperature

To a solution of 3-amino-5-methylpyridine (0.751 g, 6.94 mmol) in tetrahydrofuran (TNF; 20 ml) was added 1 M lithium bis(trimethylsilyl)amide in tetrahydrofuran (6.94 ml, 6.94 mmol), and the mixture was stirred at room temperature for 10 min. Then 1-(5-(4,6-dichloropyrimidin-2-yl)2-methylpiperidin-1-yl)ethan-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 h. The mixture was poured into 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) to give 1-(5-(4-chloro-6-((5-methylpyridin-3yl)amino)pyrimidin-2-yl)-2-methylpiperidin-1-yl)ethan-1-one (788 mg, 60%) as a yellow foam. LCMS (Method B): t _R 1.81 min, 100%, MS (ESI) 360.1 (M+H)+. A mixture of 2-(tributylstannyl)pyrazine (103 mg, 0.28 mmol), 1-(5-(4-chloro-6-((5-methylpyridin-3-yl)amino)pyrimidin-2-yl)-2-methylpiperidin-1yl)ethan-1-one (50 mg, 0.14 mmol) and bis-(triphenylphosphine)palladium(II) dichloride (9.75 mg, 0.01 mmol) under nitrogen atmosphere was dissolved in N,N-dimethylformamide (3 mL). The mixture was heated to 80°C for 24 h and cooled to room temperature. The mixture was eluted through a C18 plug with acetonitrile, the filtrate was purified by reverse-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)ethan-1-one (22 mg, 37%) as a white solid. The resulting mixture of cis-enantiomers was subjected to chiral preparative superperformance fluid chromatography (SPC, Method A) and lyophilized to afford both stereoisomers. 1-((2S,5R)-2-methyl-5-(4-((5-methylpyrudin-3-yl)amino)-6-(pyrazin-2-yl)pyrimudin2-yl)piperidin-1-yl)ethan-1-one (5 mg, 22%): LC-MS (Method D): t _R 3.17 min, 100%, MS (ESI) 404.1 (M+H) ⁺ ; chiral UHPLC (Method A): t R 3.17 min, ee and de >95%. 1-((2R,5S)-2-methyl-5-(4-((5methylpyridin-3-yl)amino)-6-(pyrazin-2-yl)pyrimidin-2-yl)piperidin-1-yl)ethan-1-one (6 mg, 27%): LC-MS (Method D): tR 3.17 min, 100%, MS (ESI) 404.2 (M+H) ⁺ ; chiral UHPLC (Method A): tR 4.60 min, ee and de >95%.

Compound 00003 (which is also referred to herein as Compound B) and compound 00004 (which is also referred to herein as Compound A) were prepared using similar procedures as in Example 1, using the appropriate starting materials.

- 31 050570

Example 2: synthesis of 1-((2S,5R)-5-(4-(uMuga3o[1,2-a]nupuguH-6-uTaMUHo)-6-(nupuguH-3-uT)nupuMuguH2-yl)-2-methylpiperidin-1-yl)ethan-1-one (compound 00013)

A mixture of 3-(tributylstannyl)pyridine (607 mg, 1.65 mmol), 1-((2S,5R)-5-(4,6-dichloropyrimidin-2-yl)-2-methylpiperidin-1-yl)ethan-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) under argon was heated to 100°C and stirred for 32 h. The mixture was diluted with dichloromethane containing 1% triethylamine and supported on silica. The mixture was purified by column chromatography on silica gel (0% to 40% acetonitrile in dichloromethane containing 1% triethylamine) to give 1-((2S,5R)-5-(4-chloro-6-(pyridin-3-yl)pyrimidin-2-yl)-2-methylpiperidin-1-yl)ethan-1-one (134 mg, 18%) as an orange gum. LC-MS (Method C): t _R 1.81 min, 100%, MS (ESI) 331.1 (M+H)+. To a solution of 1-((2S,5R)-5-(4-chloro-6-(pyridin-3-yl)pyrimidin-2-yl)-2-methylpiperidin-1-yl)ethan-1-one (30 mg, 0.09 mmol) in 2-propanol (2 mL) were added imidazo[1,2-a]pyridin-6-amine (36.2 mg, 0.27 mmol) and hydrochloric acid (0.02 mL, 0.27 mmol). The mixture was stirred at 60°C for 16 h, poured into 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[1,2a]pyridin-6-ylamino)-6-(pyridin-3-yl)pyrimidin-2-yl)-2-methylpiperidin-1-yl)ethan-1-one as a bluish solid. LC-MS (Method B): t _R 2.19 min, 100%, MS (ESI) 428.1 (M+H) ⁺ .

Compound 00030 was prepared using similar procedures as in Example 2, using the appropriate starting materials.

O' ^Ν Αγ ^Ν γ'

A,ν o

N

Connection 00030

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)ethan-1-one (compound 00071)

RT - room temperature

To a solution of 2-methylpyridin-4-amine (3.19 g, 29.5 mmol) in dry tetrahydrofuran (TNP; 100 ml) was added 1 M lithium bis(trimethylsilyl)amide in tetrahydrofuran (29.5 ml, 29.5 mmol), and the mixture was stirred for 10 min. Then 1-((2S,5R)-5-(4,6-dichloropyrimidin-2-yl)-2methylpiperidin-1-yl)ethan-1-one (intermediate 2, 850 mg, 2.95 mmol) in dry tetrahydrofuran (100 ml) was added over 10 min, and the mixture was stirred at room temperature for 2 h. The mixture was poured into 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 oil was purified by silica gel column chromatography (80% to 100% ethyl acetate in n-heptane, then 0% to 10% methanol in dichloromethane) to give 1-((2S,5R)-5-(4-chloro-6-((2-methylpyridin-4-yl)amino)pyrimidin-2-yl)-2methylpiperidin-1-yl)ethan-1-one (275 mg, 25%) as a yellow oil. LC-MS (Method A): t _R 1.49 min, 100%, MS (ESI) 360.1 (M+H) ⁺ . A mixture of 1-((2S,5R)-5-(4-chloro-6-((2-methylpyridin-4-yl)amino)pyrimidin-2yl)-2-methylpiperidin-1-yl)ethan-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) under nitrogen atmosphere was dissolved in a mixture of 1,2-dimethoxyethane (DME, 6 mL) and water (2 mL). The mixture was heated to

- 32 050570

80°C for 1 h, filtered through a C18 filter and concentrated to give a dark residue. The residue was purified by reverse phase chromatography (Method B) and lyophilized to give a light 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)ethan-1-one (135 mg, 41%) as a beige solid. LC-MS (Method D): t _R 3.06 min, 100%, MS (ESI) 403.2 (M+H)+; chiral SFC (method B): t _R 3.60 min, ee and de >95%.

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)ethan-1-one (Compound C)

N-N

To a solution of 1-((2S,5R)-5-(4-chloro-6-(pyrazin-2-yl)pyrimidin-2-yl)-2-methylpiperidin-1-yl)ethan-1-one (intermediate 3, 120 mg, 0.36 mmol) in 2-propanol (2 ml), 3-(1-methyl-1H1,2,3-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 h, poured into 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 reverse phase chromatography (Method B) and lyophilized to give 1-((2S,5R)-2-methyl-5-(4-((3-(1-methyl-1H-1,2,3-triazol-4yl)phenyl)amino)-6-(pyrazin-2-yl)pyrimidin-2-yl)piperidin-1-yl)ethan-1-one (compound C, 102 mg, 60%) as a white solid. ^1H -NMR (400 MHz, DMSO^ ₆ , rotamer mixture) 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=6.8 Hz, 1.5H), 1.13 (d, J=7.0 Hz, 1.5H); LC-MS (method D): tR 3.31 min, MS (ESI) 470.2 (M+H) ⁺ .

Example 4. Crystal structure of the human CREBBP bromodomain in complex with compound 00004, and BROMOscan™ assay results for compound A, compound C, and compound CCS1477.

Crystallization.

Experimental setup: The construct used for crystallization contained residues 1081 to 1197. Crystals of CREBBP complexed with compound 00004 were obtained using a hanging drop vapor diffusion setup. 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 preincubated with 4.3 mM (3.0-fold molar excess) of compound 00004 (150 mM in DMSO) for 1 h. Then 1 μl of the protein solution was mixed with 1 μl of reservoir solution (0.1 M _MgCl2 , 0.1 M EMS/NaOH, pH 6.3, 18% (w/v) PEG 6000, and 10% (v/v) ethylene glycol) and equilibrated at 4°C over 0.4 ml of reservoir solution. Well-diffracting crystals grew to full size within 4 days.

Receiving data.

Crystals were cryoprotected by adding 10% glycerol (final concentration) to the crystallization drop before mounting the specimen for analysis. A complete data set for the 1.6 Å crystal CREBBP/00004 was acquired using the Diamond Light Source (Didcot, UK) with an i03 beamline; the data were summarized, analyzed, and scaled using the XDS, Pointless, and Aimless procedures of the autoPROC pipeline (see Table 1).

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Table 1

Statistics for the obtained data

Space group P21 Unit cell parameters [A] a=70.4, b=58.6, c=73.2 α-90.0, β=115.4, γ~90.0 Resolution [A] 66.14-1.60(1.63-1.60) Number of unique reflected signals 68872(2664) 1/o(l) 14.9 (2.2) Completeness [%] 97.2 (75.5) Multiplicity 3.3 (2.1) Rh3M. 0.050 (0.460)

Definition and refinement of the structure.

Molecular replacement was performed using the previously determined CREBBP structure as the initial model. Several rounds of alternating manual rearrangement and refinement using REFMAC5 yielded the final model (Table 2). Atomic displacement factors were modeled using one isotropic B-factor per atom.

Table 2

Statistics with clarifications

Resolution 35.00-1.60 (1.64-1.60)

Yawork. 0.151 (0.305)

P-free 0.190 (0.351)

Completeness [%] 97.2 (77.6)

Results. Crystals of CREBBP/00004 were obtained and diffracted at 1.6 Å resolution, and the three-dimensional structure of the protein-ligand complex was determined. The net electron density in the Fo-Fc gap map of the initial model at the binding site of the compound in each CREBBP chain revealed the binding of the entire compound (Fig. 7) and its precise placement. Furthermore, the experimentally determined structure also confirms the absolute stereochemistry of compound 00004 (2S,5R at the piperidine moiety).

BromoKdMAX analysis.

The BromoKdMAX assay was performed in the format provided by DiscoveRX. This assay can be used to determine whether compounds bind to the p300 bromodomain and/or the CBP bromodomain with a given Kd value (e.g., 100 nM or less).

The assay principle is as follows: BROMOscan™ is a new, industry-leading platform for the identification of small molecule bromodomain inhibitors. Based on the proven KINOMEscan™ technology, BROMOscan™ uses a proprietary site-directed ligand binding competition assay to quantitatively measure interactions between test compounds and bromodomains. This robust and reliable assay panel is suitable for high-throughput screening and provides quantitative ligand binding data to facilitate the identification and optimization of potent and selective small molecule bromodomain inhibitors. BROMOscan™ assays incorporate trace concentrations of bromodomain (<0.1 nM) and, thus, provide true thermodynamic inhibitor Kd values over a wide affinity range (from <0.1 nM to >10 µM).

The assay was performed as follows. For bromodomain analysis, T7 phage strains displaying bromodomains were grown in parallel in 24-well blocks in BL21-derived E. coli host cells. E. coli cells were grown to log phase and then infected with T7 phage from frozen stock (MOI 0.4) and incubated with shaking at 32°C until lysis (90-150 min). Lysates were centrifuged (5000 g) and filtered (0.2 μm) to remove cellular debris. Streptavidin-coated magnetic beads were treated for 30 min at room temperature with biotinylated low molecular weight or acetylated peptide ligands to generate affinity resins for bromodomain analysis. Ligand 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 ligand and reduce non-specific phage binding. Binding reactions were performed by combining bromodomains, ligand-affinity beads, and test compounds (i.e., compound A, compound C, or compound CCS1477) in 1x binding buffer (17% SeaBlock, 0.33x PBS, 0.04% Tween 20, 0.02% BSA, 0.004% sodium azide,

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7.4 mM DTT). Test compounds were prepared as 1000x stock solutions in 100% DMSO. Kd values were determined using an 11-point series of 3-fold compound dilutions with a single DMSO stop point. All compounds for Kd measurements were dispensed using the acoustic transfer method (contactless dispensing) in 100% DMSO. Compounds were then diluted directly into the assays to give a final DMSO concentration of 0.09%. All reactions were performed in polypropylene 384-well plates. Each had a final volume of 0.02 mL. The assay plates were incubated at room temperature with shaking for 1 h, and the affinity beads were washed with wash 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 at room temperature with shaking for 30 minutes. The bromodomain concentration in the eluates was measured by qPCR. The following results were obtained.

Compound A Connection C Connection CCS1477 DiscoveRx designations of genes encoding the corresponding proteins Kd [nM] Kd [nM] Kd [nM] ATAD2A >10000 >10000 >10000 ATAD2B >10000 >10000 >10000 BAZ2A >10000 >10000 >10000 BAZ2B >10000 >10000 >10000 BRD1 >10000 >10000 >10000 BRD2(1) >10000 3700 230 BRD2(1,2) 7600 4500 610 BRD2(2) >10000 >10000 2100

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BRD3(1) >10000 3700 320 BRD3(1,2) >10000 7300 1400 BRD3(2) >10000 >10000 3900 BRD4(1) >10000 2500 250 BRD4(1,2) >10000 >10000 6900 BRD4(2) >10000 >10000 5200 BRD4 (full-length short protein isoform) 7100 1200 440 In RD 7 >10000 8000 5100 BRD8(1) >10000 8400 >10000 BRD8(2) >10000 >10000 >10000 BRD9 >10000 6300 790 BRDT(l) >10000 3600 390 BRDT(1,2) >10000 8500 2400 BRDT(2) >10000 >10000 8900 BRPF1 >10000 7800 1400 BRPF3 >10000 >10000 >10000 CECR2 >10000 9800 >10000 CREBBP 29 3.2 0.47 EP300 12 2.1 0.26 FALZ >10000 >10000 5500 GCN5L2 >10000 >10000 >10000 PBRM1(2) >10000 >10000 >10000 PBRM1(5) >10000 >10000 3100 PCAF >10000 9400 >10000 SMARCA2 >10000 >10000 >10000 SMARCA4 >10000 >10000 >10000 TAF1(2) >10000 230 7900 TAF1L(2) >10000 1600 >10000 TRIM24 (Bromo.) >10000 7900 680 TRIM24 (PHD, Bromo.) >10000 >10000 1900 rub. TRTM33 (PHD, Bromo.) >10000 >10000 >10000 WDR9(2) >10000 4100 >10000

The relevant data are publicly available: i) for SGC-CBP30, e.g., in the supplementary information provided in Wu et al., NATURE COMMUNICATIONS (2019) 10:1915 https://doi.org/10.1038/s41467-09672-2; ii) for GNE-781, e.g., in Romero et al., J. Med. Chem. 2017, 60, 9162–9183; and iii) for FT-6876, for example, in poster #3079 entitled FT-6876, a potent and selective inhibitor of CBPf300 with antitumor activity in AR-positive breast cancer at the 2020 American Association for Cancer Research Annual Meeting (AACR Annual Meeting 2020, Virtual Meeting II, June 22-24, 2020).

Example 5.

Materials and methods.

CBP bromodomain binding assay (TR-FRET).

Solutions of compounds in DMSO with a concentration of 10 mM were pre-diluted 25-fold in DMSO to obtain DMSO stock solutions. They were then diluted 4-fold with assay buffer. A series of dilutions in assay buffer was performed while maintaining a stable DMSO concentration. Five microliters of compound in assay buffer and assay reagents from the TR-FRET kit (Cayman; 600850) were added to the assay plate (from the assay kit) according to the manufacturer's instructions. After 1 hour of incubation at room temperature in the dark, the assay plates were read in a Tecan plate reader.

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M1000 using the mode defined in TR-FRET (reading from top; excitation 340 nM, bandwidth 20 nM; emission 620 nM, bandwidth 7 nM; optimal gain determined for the first well, number of flashes: 5, flash rate 100 Hz, integration time 500 μs, delay time 100 μs, room temperature). According to the TRFRET assay manual, the EF50 was calculated by dividing the emission at 670 nm by the emission at 620 nm. EC50 values were calculated from normalized values (DMSO = 1) and the positive control (0). The values of the parameters were transformed to logarithmic values, and nonlinear regression with variable slope (4 parameters) was used to fit the values into the dose-response curve to estimate EC50 values (see Table 3 below).

Table 3

EC50 legend: A* < 0.2 µM

From the TR-FRET assay data, it appears that compound 00003, which has an EC50 value of >10 μM, does not meet the definition of a CBPf300 bromodomain inhibitor as defined within the framework of the present invention.

Example 6.

Materials and methods.

Label-free determination of cell proliferation.

Two thousand SNU-1411 [KCLB;01411, a colorectal adenocarcinoma cell line harboring the KRAS G12C mutation] cells were seeded in 96-well plates (Greiner BioOne 655090) in RPMI medium containing 10% FCS and 2 mM L-glutamine one day before treatment with drug compound. The following day, wells were imaged using label-free brightfield imaging on a CELIGO Imaging cytometer to determine initial cell confluence. Cells were treated with either DMSO, individual compounds, or combinations of compounds and then imaged regularly for several weeks using brightfield imaging (CELIGO Imaging cytometer) to monitor cell confluence in each well over time. The cells were then treated with either DMSO or a single drug, namely either AMG510 or any of the CBPφ300 bromodomain inhibitors listed below, or a combination of drugs, namely: (i) AMG510 and the CBPφ300 bromodomain inhibitor Compound A, (ii) AMG510 and the CBPφ300 bromodomain inhibitor Compound C, (iii) AMG510 and the CBP/p300 bromodomain inhibitor Compound CCS1477, (iv) AMG510 and the CBPφ300 bromodomain inhibitor Compound FT6876, and (v) AMG510 and the CBPφ300 bromodomain inhibitor Compound GNE-781, with the drug concentrations listed below. Plates were imaged regularly for several weeks using brightfield mode (CELIGO Imaging cytometer), monitoring cell confluence in each well over time. Growth and treatment medium were replenished twice weekly. The compounds used and their concentrations were as follows: for AMG510 (covalent specific inhibitor of KRAS G12C, ChemieTek #CT-AMG510): 300 nM; For CBP/p300 bromodomain inhibitors: 1 μM compound A, 0.2 μM compound C, 0.2 μM CCS1477 (ChemiTek; CT-AMG510), 1 μM compound FT-6876 (CBP/P300-IN-8, MedChemExpress; HY-136920), and 0.2 μM compound GNE781 (MedChemExpress; HY-108696). Cell fusion was determined using the CELIGO built-in brightfield fusion analysis software.

Figures 2A-E show the results of the fusion assay in SNU-1411 cells for 32 days. The bromodomain inhibitors CBPφ300 [(A) compound A, (B) compound C, (C) compound CCS1477, (D) compound FT-6876, and (E) compound GNE-781] do not affect the proliferation of KRAS G12C-mutated CRC cells in the absence of the KRAS G12C inhibitor, but prevent the development of drug resistance when 300 nM AMG510 is combined with the bromodomain inhibitor CBPφ300. Note that the DMSO curves and the time courses obtained with 300 nM AMG510 treatment are identical in panels of Figures 2A and D, as well as in Figure 2B, C, and E, as all conditions (for A, D, and for B, C, and E) were the same for all plates (DMSO: 9 wells for each plate; bromodomain inhibitor CBPf300: 3 wells for each plate; AMG510: 6 wells for each plate; and all combinations of AMG510 + bromodomain inhibitor CBPf300: 6 wells for each plate; mean ± standard deviation (SD) is shown). One example plate is shown.

Results: Figures 2A-E show that CBPf300 bromodomain inhibitors have no effect, or at best, a weak effect, on SNU-1411 cell fusion when used alone, whereas

- 37 050570, how application of 300 nM AMG510 initially delayed cell proliferation for several days. In long-term cultures, SNU-1411 cells regrew when treated with AMG510 alone, whereas co-treatment with AMG510 in combination with various CBPf300 bromodomain inhibitors completely prevented or significantly reduced cell regrowth over the studied period of 32 days.

Example 7.

Materials and methods

Label-free determination of cell proliferation.

Two thousand SNU-1411 [KCLB;01411, a colorectal adenocarcinoma cell line harboring the KRAS G12C mutation] cells were seeded in 96-well plates (Greiner BioOne 655090) in RPMI medium containing 10% FCS and 2 mM L-glutamine one day before treatment with the drug compound. The following day, wells were imaged without labeling using brightfield imaging on a CELIGO Image cytometer to determine initial cell confluence. The cells were then treated with DMSO, or with a single drug, namely either MRTX849 or any of the CBPφ300 bromodomain inhibitors listed below, or with a combination of drugs, namely: (i) MRTX849 and the CBPφ300 bromodomain inhibitor Compound A, (ii) MRTX849 and the CBPφ300 bromodomain inhibitor Compound C, (iii) MRTX849 and the CBPφ300 bromodomain inhibitor Compound CCS1477, (iv) MRTX849 and the CBPφ300 bromodomain inhibitor Compound FT-6876, and (v) MRTX849 and the CBPφ300 bromodomain inhibitor Compound GNE-781, at the drug concentrations listed below. Plates were imaged regularly for several weeks using brightfield mode (CELIGO Imaging cytometer), monitoring cell confluence in each well over time. Growth and treatment medium were replenished twice weekly. The compounds used and their concentrations were as follows: for MRTX849 (covalent specific inhibitor of KRAS G12C, Selleckchem #S8884): 300 nM; For the CBPf300 bromodomain inhibitors: 1 μM compound A, 0.2 μM compound C, 0.2 μM compound CCS1477 (ChemiTek; CT-CCS1477), 1 μM compound FT-6876 (CBP/P300-IN-8, MedChemExpress; HY-136920), and 0.2 μM compound GNE-781 (MedChemExpress; HY-108696). Cell fusion was determined using the CELIGO built-in brightfield fusion analysis software.

Figures 3A-E show the results of the fusion assay in SNU-1411 cells for 32 days. The CBP/p300 bromodomain inhibitors [(A) compound A, (B) compound C, (C) compound CCS1477, (D) compound FT-6876, and (E) compound GNE-781] do not affect the proliferation of KRAS G12C-mutated CRC cells in the absence of the KRAS G12C inhibitor, but prevent the development of drug resistance when 300 nM MRTX849 is combined with the bromodomain inhibitor CBPp300. Note that the DMSO curves and the time courses obtained with 300 nM MRTX849 treatment are identical in panels of Figures 3A and D, as well as in Figure 3B, C, and E, as all conditions (for A and D, and for B, C, and E) were the same for all plates (DMSO: 9 wells for each plate; bromodomain inhibitor CBPf300: 3 wells for each plate, AMG510: 6 wells for each plate; and all combinations of MRTX849 + bromodomain inhibitor CBP/c300: 6 wells for each plate; mean ± standard deviation (SD) is shown). One plate is shown as an example.

Results. Figures 3A–E show that CBP/p300 bromodomain inhibitors have little or no effect on SNU-1411 cell fusion when used alone, whereas treatment with 300 nM MTRX894 initially delayed cell proliferation for several days. In long-term cultures, SNU-1411 cells regrew when treated with MRTX849 alone, whereas co-treatment with MRTX849 and various CBP/p300 bromodomain inhibitors prevented regrowth over the 32-day time period studied.

Example 8.

Materials and methods

Label-free determination of cell proliferation.

Two thousand SW837 [ATCC; CCL-235, a colorectal adenocarcinoma cell line harboring the KRAS G12C mutation] cells were seeded in 96-well plates (Greiner BioOne 655090) in RPMI medium containing 10% FCS and 2 mM L-glutamine. One day before drug treatment, wells were imaged label-free using bright-field imaging on a CELIGO Image cytometer to determine initial cell confluence. The cells were then treated with DMSO, or with a single drug, namely either AMG510 or any of the CBP/p300 bromodomain inhibitors listed below, or a combination of drugs, namely: (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, with the drug concentrations listed below. The plates were imaged regularly for several weeks using bright field mode (CELIGO Imaging cytometer),

- 38 050570 monitoring cell fusion in each well over time. Growth and treatment medium were replenished twice a week. The compounds used and their concentrations were as follows: for AMG510 (covalent specific inhibitor of KRAS G12C, ChemieTek #CT-AMG510): 100 nM; for the bromodomain inhibitors CBPφ300: 1 μM compound A, 0.2 μM compound C, 0.2 μM compound CCS1477 (ChemieTek; CT-AMG510), 1 μM compound FT-6876 (CBP/P300-IN-8, MedChemExpress; HY-136920) and 0.2 μM compound GNE-781 (MedChemExpress; HY-108696). Cell fusion was determined using the CELIGO built-in brightfield fusion analysis software.

Figures 4A-E show the results of SW837 cell fusion assay for 49 days. The CBP/p300 bromodomain inhibitors [(A) compound A, (B) compound C, (C) compound CCS 1477, (D) compound FT-6876, and (E) compound GNE-781] do not affect the proliferation of KRAS G12C-mutated CRC cells in the absence of the KRAS G12C inhibitor, but prevent the development of drug resistance when 100 nM AMG510 is combined with the CBP/p300 bromodomain inhibitor.

Note that the DMSO curves and time courses obtained with 100 nM AMG510 treatment are identical in panels of Fig. 4A-E because all conditions were the same and the experiments were performed in parallel (DMSO: 18 wells for each plate, CBP/p300 bromodomain inhibitor: 6 wells for each plate, AMG510: 12 wells for each plate; and all AMG510 + CBPp300 bromodomain inhibitor combinations: 12 wells for each plate; mean ± standard deviation (SD) is shown).

Results. Figures 4A–E show that CBPf300 bromodomain inhibitors have little or no effect on SW837 cell fusion when used alone, whereas treatment with 100 nM AMG510 initially delayed cell proliferation for several days. In long-term cultures, SW837 cells regrowth when treated with AMG510 alone, whereas co-treatment with AMG510 and various CBPf300 bromodomain inhibitors completely prevented or greatly reduced regrowth over the 49-day study period.

Example 9.

Materials and methods.

Label-free determination of cell proliferation.

Two thousand NCI-H358 [KCLB; 25807; non-small cell lung cancer (NSCLC) cell line with the KRAS G12C mutation] cells were seeded into 96-well plates (Greiner BioOne 655090) in RPMI medium containing 10% FCS and 2 mM L-glutamine one day before drug treatment. The following day, wells were imaged label-free using bright-field imaging on a CELIGO Image cytometer to determine initial cell confluence. Cells were then treated with DMSO, or with a single drug, namely either AMG510 or any of the CBPφ300 bromodomain inhibitors listed below, or with a combination of drugs, namely (i) AMG510 and the CBPφ300 bromodomain inhibitor Compound A, and (ii) AMG510 and the CBPφ300 bromodomain inhibitor Compound C, at the drug concentrations listed below. Plates were imaged regularly over several weeks using brightfield mode (CELIGO Imaging Cytometer), monitoring cell confluence in each well over time. Growth and treatment medium were replenished twice weekly. Compounds used and their concentrations were as follows: for AMG510 (ChemieTek #CT-AMG510): 100 nM; for CBP/p300 bromodomain inhibitors: 1 μM compound A, 200 nM compound C, and for the combination: 100 nM AMG510 + 1 μM compound A or 100 nM AMG510 + 200 nM compound C. Cell numbers were determined using the CELIGO built-in brightfield direct cell counting analysis software.

Figures 5A and B show the results of NCI-H385 cell counts over time [days]. Neither compound A (panel A) nor compound C (panel B) reduced the proliferation of NCI-H358 cells, a KRAS G12C-mutated NSCLC cell line, in the absence of a KRAS G12C inhibitor. However, compounds A and C prevented the development of drug resistance when used in combination with the covalent specific inhibitor of KRAS G12C (for Fig. 5A: DMSO: n=6, compound A: n=6, AMG510: n=24, AMG510 + compound A: n=24, mean ± standard deviation (SD) is shown; for Fig. 5B: DMSO: n=6, compound C: n=6, AMG510: n=24, AMG510 + compound C: n=24, mean ± standard deviation (SD) is shown).

Results. Compound A and compound C were shown not to reduce NCIH358 cell numbers when used alone, whereas 100 nM AMG510 initially completely blocked cell proliferation. In long-term cultures, NCI-H358 cells regrew when treated with AMG510 alone, whereas co-treatment with AMG510 in combination with CBP/P300 bromodomain inhibitors, such as compound A (Fig. 5A) or compound C (Fig. 5B), respectively, completely prevented cell regrowth over the studied time period (>20 days).

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Claims (11)

CLAUSES OF THE INVENTION 1. The use of a combination of: (i) a CBPf300 bromodomain inhibitor and (ii) a KRAS inhibitor for the treatment of a patient suffering from cancer, wherein the cancer is characterized by an oncogenic alteration in KRAS, wherein the oncogenic alteration is caused by at least one base mutation in the KRAS gene resulting in a G12C amino acid substitution in KRAS, and the KRAS inhibitor is a KRAS G12C inhibitor. 2. The use according to claim 1, wherein the oncogenic change in KRAS results in overactivation of the KRAS signaling pathway. 3. The use according to claim 1 or 2, wherein the CBPf300 bromodomain inhibitor is selected from the group consisting of compound 00030, compound 00071, compound CCS1477, compound GNE-781, compound CPI-637, compound FT-6876, compound GNE-049 compounds, SGC-CBP30 compounds, 4. The use according to any of the preceding claims, wherein the KRAS inhibitor is selected from the group consisting of the compound AMG510, the compound MRTX849, and combinations thereof. 5. The use according to any of the preceding claims, wherein the cancer is selected from the group consisting of - 40 050570 lung cancer, colorectal cancer and pancreatic cancer. 6. The use according to any of the preceding claims, wherein the combination is administered to the patient during each treatment cycle. 7. The use according to any of the preceding claims, wherein (i) and (ii) are administered as separate dosage forms or are included in a single dosage form. 8. Use according to claim 6, wherein administration during each treatment cycle is carried out simultaneously or sequentially if (i) and (ii) are administered as separate dosage forms. 9. A kit for treating a patient suffering from cancer, 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. 10. The kit according to claim 9, wherein the CBP/p300 bromodomain inhibitor is selected from the group consisting of CCS1477 connections, GNE-781 connections, GNE-049 connections, SGC-CBP30 connections, CPI-637 connections, FT-6876 connections, 462 connections, 424 connections connections 515 . 11. The kit according to claim 9 or 10, wherein the KRAS inhibitor is selected from the group consisting of the compound AMG510, the compound MRTX849, and combinations thereof. -