Survivin as a biomarker to predict the response to cancer treatment

Abstract

The present invention relates to a method for determining the responsiveness of a cancer patient to treatment with a compound that inhibits the KRAS protein or a mutant of the KRAS protein, or treatment with a compound that inhibits the interaction between MDM2 and p53, comprising: measuring the level of survivin in a first sample obtained from the patient before treatment with the compound; measuring the level of survivin in a second sample obtained from the patient during or after treatment with the compound; and comparing the level of survivin in the second sample with the level of survivin in the first sample, wherein if the level of survivin in the second sample is lower than the level of survivin in the first sample, the patient is determined to be responsive to treatment with the compound. The present invention further relates to the use of survivin in a method for determining the ability of a compound that inhibits the KRAS protein or a mutant of the KRAS protein, or a compound that inhibits the interaction between MDM2 and p53, or a pharmaceutical formulation containing the compound that inhibits the KRAS protein or a mutant of the KRAS protein, or a pharmaceutical formulation containing the compound that inhibits the interaction between MDM2 and p53, to treat cancer.

Description

[Technical Field]

[0001] The present invention relates to the field of targeted cancer therapy, biomarkers for evaluating the activity of specific compounds, and monitoring of treatment with said compounds using the biomarkers. In particular, the present invention relates to a method for determining the responsiveness of a cancer patient to treatment with a compound that inhibits the KRAS(K-Ras) protein or a mutant of the KRAS(K-Ras) protein, or to treatment with a compound that inhibits the interaction between MDM2 and p53, comprising: measuring the level of survivin in a first sample obtained from the patient before treatment with the compound; measuring the level of survivin in a second sample obtained from the patient during or after treatment with the compound; and comparing the level of survivin in the second sample with the level of survivin in the first sample, wherein if the level of survivin in the second sample is lower than the level of survivin in the first sample, the patient is determined to be responsive to treatment with the compound. The present invention further relates to the use of Survivin in a method for determining the ability of a pharmaceutical formulation containing a compound that inhibits the KRAS protein or a mutant of the KRAS protein, or a compound that inhibits the KRAS protein or a mutant of the KRAS protein, to treat cancer. The present invention further relates to the use of Survivin in a method for determining the ability of a pharmaceutical formulation containing a compound that inhibits the interaction between MDM2 and p53, or a compound that inhibits the interaction between MDM2 and p53, to treat cancer. [Background technology]

[0002] Cancer is the leading cause of death worldwide, and its treatment and outcomes are being dramatically revolutionized by targeted therapies. The Kirsten rat sarcoma viral oncogene homolog (KRAS and K-Ras are used synonymously in this application) is a centrally mediated and most frequently mutated oncogene. Gain-of-function mutations in it have been identified in approximately 30% of all cancer cells. KRAS mutations are closely associated with tumor onset and development and have been linked to a range of highly lethal cancers, including pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NSCLC), and colorectal cancer (CRC) (Huang et al. 2021).

[0003] KRAS is the most frequently mutated oncogene in human cancers, including pancreatic ductal adenocarcinoma (PDAC), with 95% of PDAC patients having a mutant form of KRAS. KRAS mutations induce constitutive activation of this oncogene and downstream signaling pathways, including ERK and PI3K, which promote tumorigenesis, invasiveness, metastasis, and treatment resistance (Dhirendra et al. 2017).

[0004] The KRAS gene is a member of the rat sarcoma virus oncogene family (RAS), which includes two other isoforms in humans: the Harvey mouse sarcoma virus oncogene (HRAS) and the neuroblastoma rat sarcoma virus oncogene homolog (NRAS).

[0005] The RAS gene is evolutionarily conserved in its similar structure and contains four exons distributed across the entire length of approximately 30 kb of DNA. The KRAS gene encodes two highly related protein isoforms, KRAS-4B and KRAS-4A, which consist of 188 and 189 amino acids, respectively, due to different excisions of the fourth exon (Huang et al. 2021).

[0006] RAS is a type of membrane-bound regulatory protein (G protein)-linked guanine nucleotide belonging to the family of guanosine triphosphatases (GTPases). RAS functions as a guanosine diphosphate (GDP) / triphosphate (GTP) two-component switch, which regulates important signal transduction from activated membrane receptors to intracellular molecules.

[0007] Ras family proteins, including KRAS, NRAS, and HRAS, and any of their mutants, are small GTPases present intracellularly in either a GTP-bound or GDP-bound state (McCormick et al. 2016; Nimnual et al. 2002). Ras family proteins possess weak intrinsic GTPase activity and a slow nucleotide exchange rate (Hunter et al. 2015). Binding of GTPase-activating proteins (GAPs), such as NF1, increases the GTPase activity of Ras family proteins. Binding of guanine nucleotide exchange factors (GEFs), such as SOS1 (Son of Sevenless 1), promotes the release of GDP from Ras family proteins, enabling GTP binding (Chardin et al. 1993). In the GTP-bound state, Ras family proteins are active and, in conjunction with effector proteins including C-RAF and phosphoinositide 3-kinase (PI3K), promote the RAF / mitogen or extracellular signal-regulated kinase (MEK / ERK) pathway, the PI3K / AKT / rapamycin-mammalian target (mTOR) pathway, and the RalGDS (Ral guanine nucleotide dissociation-stimulating factor) pathway (McCormick et al. 2016; Rodriguez-Viciana et al. 2005). These pathways influence a variety of cellular processes, such as proliferation, survival, metabolism, motility, angiogenesis, immunity, and growth (Young et al. 2009; Rodriguez-Viciana et al. 2005).

[0008] KRAS proteins function as precisely regulated molecular switches that control numerous signaling cascades through cycles between activated and inactivated conformations. KRAS proteins are involved in the signaling of growth factors, chemokines, and Ca 2+ Alternatively, it can be activated by receptor tyrosine kinases (RTKs). Activated KRAS proteins can activate numerous signaling pathways, including the RAF / MEK / ERK pathway, which is a canonical downstream target of KRAS signaling. Activated KRAS-GTP can recruit RAF (rapidly accelerating fibrosarcoma), a serine / threonine-specific protein kinase, from the cytoplasm to the cell membrane, induce conformational changes in RAF, and promote RAF activation by allogeneic or heterologous dimerization. The C-terminal catalytic domain of RAF binds to MEK (mitogen-activated protein kinase) 1 / 2 and activates it by phosphorylation. MEK1 / 2 phosphorylates and activates ERK (extracellularly regulated protein kinase) 1 / 2, and activated ERK phosphorylates ribosomal S6 kinase (RSK), serum response factor (SRF), E26 transformation-specific transcription factor (ETS), and ETS-like-1 protein, thereby regulating the transcription and translation of corresponding target genes and thus participating in the regulation of cell proliferation, differentiation, migration, and other life activities.

[0009] Furthermore, KRAS has been found to be involved in the phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT)-mTOR (mammalian target of rapamycin) pathway, which is thought to play an important role in cellular life activities such as cell proliferation, differentiation, apoptosis, and glucose transport, and has a significant impact on the development of tumor resistance. Activated KRAS can activate PI3K by binding to its p110 subunit. Phosphatidylinositol 4,5-bisphosphate (PIP2), catalyzed by activated PI3K, is converted to phosphatidylinositol 3,4,5-trisphosphate (PIP3). PIP3 promotes phosphoinositide-dependent kinase 1 (PDK1), phosphorylating Thr308 of AKT. mTOR complex 2 further phosphorylates the serine phosphorylation site (Ser473) of AKT, resulting in overall AKT activation. Activated AKT enters the nucleus and activates or inhibits numerous downstream pathways, regulating cell proliferation, apoptosis, and metabolic processes. On the one hand, AKT can directly activate mTOR target proteins that play crucial roles in cell proliferation, survival, metabolism, protein synthesis, and transcription. On the other hand, AKT phosphorylates and activates the Bcl-XL / Bcl-2-related cell death promoter (BAD), promoting the binding of BAD to the companion protein 14-3-3 instead of Bcl-2 / Bcl-XL, and thus inhibiting apoptosis.

[0010] Furthermore, RAL guanine nucleotide dissociation stimulator (RalGDS) is a downstream signaling protein of KRAS that functions as a GTP / GDP exchange factor that promotes GDP / GTP conversion of RAS-like protein (RAL). Downstream effector factors of RAL proteins include Rac / Cdc42 (cell division cycle 42), associated with cell migration; TANK-binding kinase 1 (TBK1), associated with viral immunity; and phospholipase D (PLD), associated with endocytosis. In addition, KRAS activates the RAC1 signaling pathway, which influences cell morphology, migration, adhesion, actin cytoskeleton formation, endocytosis, and membrane transport, by modulating TIAM1 and RAC1-specific guanine nucleotide exchange factors. Moreover, KRAS can also modulate the phosphatidylinositol signaling pathway by activating PLCε. In short, the KRAS-mediated signaling network is complex and involved in a variety of biological activities (Huang et al. 2021).

[0011] KRAS, mediated by MAP kinase (MAPK) signaling, mediates immune evasion in the tumor macroenvironment by upregulating PD-L1 expression, downregulating MHC1 expression in tumor cells, and mobilizing immunosuppressive immune cells by enhancing the secretion of various cytokines and chemokines.

[0012] Cancer-associated mutations in Ras family proteins suppress their intrinsic GTPase activity and GAP-induced GTPase activity, leading to an increase in the population of GTP-binding / active mutant Ras family proteins (McCormick et al. 2015; Hunter et al. 2015). This then leads to sustained activation of downstream effector pathways (e.g., RAF / MEK / ERK, PI3K / AKT / mTOR, RalGDS pathways) of mutant Ras family proteins. KRAS mutations (e.g., amino acids G12, G13, Q61, A146) have been found in various human cancers, including lung cancer, colorectal cancer, and pancreatic cancer (Cox et al. 2014). Furthermore, mutations in HRAS (e.g., amino acids G12, G13, Q61) and NRAS (e.g., amino acids G12, G13, Q61, A146) are found in various human cancer types, but typically at lower frequencies compared to KRAS mutations (Cox et al. 2014). In addition, changes in Ras family proteins / Ras genes (e.g., mutations, overexpression, gene amplification) have been explained as mechanisms of resistance to cancer drugs such as the EGFR antibodies cetuximab and panitumumab (Leto et al. 2014) and the EGFR tyrosine kinase inhibitor osimertinib / AZD9291 (Ortiz-Cuaran et al. 2016; Eberlein et al. 2015).

[0013] Mutations in the Ras family proteins, specifically the G12C mutation (G12C mutation, e.g., KRAS G12C, NRAS G12C, and HRAS G12C), which replace glycine with cysteine ​​at codon 12, are generated from a GC-TA transposition at codon 12. This transposition is a commonly found mutation in the Ras gene and accounts for 14% of all KRAS mutations, 2% of all NRAS mutations, and 2% of all HRAS mutations across cancer types. G12C mutations are particularly enriched in KRAS mutant non-small cell lung cancer, with approximately half of patients having this mutation, which is associated with DNA adducts formed by tobacco smoke. G12C mutations are not exclusively associated with lung cancer and are found in other RAS mutant cancer types, including 3-5% of all KRAS mutant colorectal cancers.

[0014] Inhibitors of these proteins, such as KRAS G12C, NRAS G12C, and HRAS G12C, that have the ability to covalently bind to such G12C mutant Ras family proteins are predicted to inhibit downstream cellular signaling (e.g., ERK phosphorylation) of Ras family proteins. In cancer cells associated with dependence on mutant Ras family proteins (e.g., KRAS mutant cancer cell lines), such binders / inhibitors are predicted to deliver anti-cancer efficacy (e.g., inhibition of proliferation, survival, metastasis, etc.).

[0015] Several selective drugs targeting the KRAS G12C mutant protein have moved into clinical development, and sotrasib has recently been approved for the treatment of KRAS G12C-driven lung cancer (corresponding patent applications WO2018 / 217651, WO2017 / 201161, WO2019 / 099524, WO2020 / 102730).

[0016] Significant amplification of the wild-type (WT) KRAS oncogene has been observed in one subset of tumor indications, e.g., gastric cancer, gastroesophageal junction cancer, and esophageal cancer, where it acts as a driver of alteration, making tumor models with this genotype KRAS-toxic both in vitro and in vivo (Wong et al. 2018). In contrast, unamplified KRAS WT cell lines are KRAS-independent unless they have secondary alterations in the gene that indirectly induce KRAS activation (Meyers et al. 2017). Based on these observations, drugs with KRAS WT-targeting activity offer further approaches for the treatment of KRAS-dependent cancers.

[0017] Target proteolytic chimeras (PROTACs) bind to proteins and induce their degradation by inducing their ubiquitination. PROTACs are triplicate or heterobifunctional molecules consisting of a portion that binds to the protein to be degraded, a second portion that binds to an E3 ubiquitin ligase and can artificially recruit it, and a linker connecting the two portions. Once a trimer complex is formed consisting of the target protein, PROTAC, and ligase, the tightness of the ligase to the target leads to ubiquitination of the target protein. Ubiquitination acts as a post-translational modification of proteins, among other things, leading to their recruitment to the proteasome and subsequent proteolytic degradation. Subsequently, the multi-ubiquitin chains on the target protein are recognized by the proteasome, and the target protein is degraded.

[0018] In contrast to classical small molecule drugs, PROTAC-driven degradation operates on a substoichiometric basis and therefore requires lower systemic exposure to achieve efficacy. PROTACs have been shown to exhibit greater selectivity for proteolysis than the target ligand itself due to differences in complementarity at the protein-protein interaction interface of the ternary complex they form. In addition, since degradation is not limited to the protein domain that is functionally responsible for the disease, PROTACs promise to expand the proteome in a way that could lead to the development of new drugs. In challenging cases such as multi-domain proteins, which have traditionally been considered unsuitable targets for new drug development, the most ligand-binding domain can be targeted for degradation, independently of its functionality or susceptibility to small molecule blockade.

[0019] The irreversible nature of KRAS degradation induced by the recruitment of E3 ubiquitin ligase is predicted to induce cellular effects equivalent to irreversible inhibition. Furthermore, since mutant KRAS is still predicted to depend on the cycle between GTP-binding active and GDP-binding inactive states induced by GEF / GAP, degradation of mutant KRAS, not just wt-amplified KRAS, induced by PROTACs involved in the GDP-binding state, can lead to the gradual degradation of a large fraction of the total cellular KRAS pool. Therefore, degradation of oncogenic KRAS can inhibit downstream signaling in tumors and deliver anticancer efficacy as described for KRAS inhibition. In the case of irreversible targeted degradation, the recovery of downstream signaling activity depends not only on the removal of the drug from the treated target (e.g., by efflux) but is further limited by the de novo resynthesis of the target protein by ribosomes. Irreversible inhibition is currently limited to the KRAS G12C protein, which constitutes only a portion of the complete complement of KRAS mutant tumors. In contrast, if KRAS can bind to heterobifunctional degradable molecules, induced KRAS degradation has the potential to enable irreversible inhibition of KRAS signaling for most of the remaining KRAS mutation / alteration (including amplification)-driven tumor growth. Degraders of wild-type KRAS (e.g., amplified or overexpressed) or mutated KRAS (e.g., G12C, G12D, G12V, G13D) have been used to deliver anti-cancer efficacy.

[0020] While many methods for treating patients with KRAS-targeting compounds are being pursued in this field, there are still many aspects that need to be investigated, such as providing means to measure the response of cancer patients to treatment with compounds that inhibit the KRAS protein or mutants of the KRAS protein.

[0021] The ERBB transmembrane receptor tyrosine kinase (RTK) family consists of four members: EGFR (ERBBI), HER2 (Neu, ERBB2), HER3 (ERBB3), and HER4 (ERBB4). HER2, whose ligand has not yet been identified, is a preferred dimerization partner for the other ERBB members. Once the active ligand-receptor complex is formed, the intracellular tyrosine kinase domain of EGFR, HER2, HER3, or HER4 is activated by autophosphorylation or phosphate transfer, subsequently inducing a signaling cascade, most notably involving MAP (mitogen-activated protein) kinase and / or phosphoinositide 3-kinase (PI3K) pathways.

[0022] Abnormal HER2 signaling is observed in a wide range of human malignancies. Oncogenic mutations have been described for the extracellular, membrane (near-membrane), and intracellular regions of the protein. Taken together, these mutations constitutively activate HER2, stimulating cancer initiation, tumor maintenance, and growth. Similarly, HER2 overexpression increases HER2 signaling and underlies oncochemical transformation and tumor maintenance in various indications, including breast, gastric, or lung cancer.

[0023] As a result, interference with HER2 oncogenic signaling leads to inhibition of tumor growth. Targeted therapies include antibodies targeting HER2 (trastuzumab and pertuzumab), antibody-drug conjugates targeting HER2 (trastuzumab-DM1 (T-DM1, adtrastuzumab emtansine)), and small molecules that inhibit the HER2 kinase domain.

[0024] In short, tumors driven by HER2 oncogenic mutations or HER2 wild-type overexpression (e.g., for gene amplification) may benefit from HER2-specific tyrosine kinase inhibitors (TKIs). In summary, HER2 modifications affect up to 6-7% of all human cancers (WO2021213800).

[0025] p53 inactivation is a central mechanism by which tumors evade the body's regulatory mechanisms and promote tumor growth and proliferation. In many cancer types, the TP53 gene is often mutated or deleted, which inactivates the tumor suppressor activity of the p53 protein. However, loss of p53 tumor suppressor activity can also occur through amplification of MDM2. Since MDM2 is a negative regulator of p53, it promotes p53 degradation and inhibits p53 tumor suppressor activity (Zhao et al. 2014).

[0026] Overall, approximately 5-7% of tumors exhibit MDM2 amplification. However, such amplification is more common in some tumor types than in others, with an incidence of up to 90% in some types of late soft tissue sarcoma (STS).

[0027] Therefore, blocking the MDM2-p53 interaction to reactivate wild-type p53 function is a promising cancer treatment strategy. Early compounds designed to target the MDM2-p53 interaction have been developed. These types of compounds may have a dual mechanism of action, involving direct targeting of tumor cells and exerting immunomodulatory effects. In tumor cells with wild-type TP53 status, they directly bind to MDM2, block its interaction with p53, and lead to p53 stabilization, induction of TP53 target genes, cell cycle arrest, and apoptosis. Furthermore, p53 activation promotes an anti-tumor immune response by increasing CD8+ T cell infiltration in tumors and induces anti-tumor immune memory.

[0028] The MDM2 (Mouse Double Minute) protein (or its human homolog, also known as HDM2) acts to downregulate p53 activity in an autoregulatory manner, and under normal cellular conditions (no stress), the MDM2 protein works to maintain low levels of p53 activity. MDM2 directly inhibits the transactivation function of p53, transporting p53 out of the nucleus and promoting proteasome-mediated degradation of p53.

[0029] Tumor suppressor protein p53 is a sequence-specific transcription factor that plays a central role in regulating several cellular processes, including the cell cycle and growth arrest, apoptosis, DNA repair, aging, angiogenesis, and innate immunity.

[0030] Overexpression of MDM2 or disruption of the MDM2 / p53 balance due to p53 mutation or loss leads to malignant transformation of normal cells. Currently, it is known that p53 plays a crucial role in virtually all types of human cancer, and mutations or loss of the p53 gene can be identified in more than 50% of all human cancers worldwide.

[0031] In tumors with wild-type p53, MDM2 is the major cellular inhibitor of p53 activity, and MDM2 overexpression has been found in many human tumors. Since MDM2 inhibits p53 through direct protein-protein interactions, there is a growing need to block this interaction using small molecules.

[0032] The compounds of the present invention are characterized by a potent inhibitory effect on the interaction between MDM2 and p53, and thereby by high in vitro efficacy against tumor cells, such as osteosarcoma, ALL, etc. This efficacy is mediated through the inhibition of the interaction between MDM2 and p53, which is essential for the corresponding efficacy in in vivo models and, in the future, in patients (WO2017 / 060431). [Overview of the project]

[0033] According to a first aspect, the present invention relates to a method for determining the response of a cancer patient to treatment with a compound that inhibits the KRAS protein or a mutant of the KRAS protein, - Measure the level of survivin in the first sample obtained from the patient before treatment with the compound. - Measuring the level of survivin in a second sample obtained from the patient during or after treatment with the compound. - Comparing the level of survivin in the second sample with the level of survivin in the first sample. The present invention relates to a method wherein, if the level of survivin in a second sample is reduced compared to the level of survivin in a first sample, the patient is determined to be responsive to treatment with the compound.

[0034] Furthermore, the present invention relates to a method for determining the response of cancer patients to treatment with a compound that inhibits the interaction between MDM2 and p53, - Measure the level of survivin in the first sample obtained from the patient before treatment with the compound. - Measuring the level of survivin in a second sample obtained from the patient during or after treatment with the compound. - Comparing the level of survivin in the second sample with the level of survivin in the first sample. The present invention relates to a method wherein, if the level of survivin in a second sample is reduced compared to the level of survivin in a first sample, the patient is determined to be responsive to treatment with the compound.

[0035] According to a second aspect, the present invention relates to a compound for inhibiting mutants of the KRAS protein or K-Ras protein for use in treating cancer patients, i) The KRAS inhibitor is selected from the group consisting of KRAS(G12C) inhibitor or degrader, KRAS(G12D) inhibitor or degrader, GDP-KRAS inhibitor or degrader, and HER2 inhibitor or degrader. ii) The patient has been determined to be responsive to treatment with the compound in accordance with the method described above. Regarding compounds.

[0036] Furthermore, the present invention relates to a compound for use in treating cancer patients that inhibits the interaction between MDM2 and p53, wherein the patient has been determined to be responsive to treatment with the compound in accordance with the method described above.

[0037] In a third aspect, the present invention relates to a method for treating cancer in a patient with a compound that inhibits the KRAS protein or a mutant of the KRAS protein, wherein the patient has been determined to be responsive to treatment with the compound according to any of the methods described above.

[0038] Furthermore, the present invention relates to a method for treating cancer in a patient with a compound that inhibits the interaction between MDM2 and p53, wherein the patient has been determined to be responsive to treatment with the compound according to any of the methods described above.

[0039] In accordance with a fourth aspect, the present invention relates to the use of Survivin in a method for determining the ability of a compound that inhibits the KRAS protein or a mutant of the KRAS protein, or a pharmaceutical formulation containing said compound that inhibits the KRAS protein or a mutant of the KRAS protein, to treat cancer.

[0040] Furthermore, the present invention relates to the use of Survivin in a method for determining the ability of a compound that inhibits the interaction between MDM2 and p53, or a pharmaceutical formulation containing said compound that inhibits the interaction between MDM2 and p53, to treat cancer.

[0041] According to a fifth aspect, the present invention relates to a kit of components comprising means for determining the level of survivin in a sample provided by a patient with cancer, and instructions for how to perform any of the methods described above.

[0042] The inventors unexpectedly discovered that apoptosis inhibitor (IAP) protein survivors can serve as a biomarker for selecting cancer patients who may be better treated with KRAS inhibitors, and that this biomarker can be readily evaluated and monitored by quantitative determination in blood, plasma, or serum samples provided by patients.

[0043] The use of such molecular biomarkers can avoid ineffective treatments for cancer patients who do not respond to KRAS inhibitors, thus allowing for a more rapid transition to a more effective drug category, increasing treatment efficacy, and extending survival time. Determining survivin levels as biomarkers in samples is easily provided, further reducing overall treatment costs and the workload of clinical staff. In addition, it thus avoids the need to frequently collect tumor samples to monitor treatment success or assess cancer progression.

[0044] These objectives are satisfied by the subject matter, methods, and means described in the independent claims of the present invention. Dependent claims relate to preferred embodiments.

[0045] Further details, characteristics, features and advantages of the object of the present invention are disclosed in the dependent claims, and the following descriptions of the drawings and examples illustrate preferred embodiments of the invention in an exemplary manner. However, these examples and drawings should not be understood in any way as limiting the scope of the invention. [Brief explanation of the drawing]

[0046] [Figure 1A] This figure shows survivin levels in untreated patients. Survivin is detected in exosomes released by HPAC cells, but not in exosomes from healthy subjects. [Figure 1B]This figure shows survivin levels in untreated patients. It compares plasma survivin levels in healthy controls versus cancer patients (colorectal cancer, CRC; pancreatic ductal adenocarcinoma, PDAC; and non-small cell lung cancer, NSCLC). [Figure 1C] This figure shows Survivin levels in untreated patients. It also shows Survivin plasma levels in CRC patients (placebo group) at different time points. [Figure 2] This figure shows that Survivin / BIRC5 is a biomarker for susceptibility to KRAS inhibition. In vivo (HCC461) biomarker studies show dose-dependent Survivin downregulation after 3 days of daily treatment with the KRASG12D inhibitor. [Figure 3] Figure 3A shows the ELISA assay in GP2D cells treated with KRASG12D inhibitor for 2 hours (top) and 24 hours (bottom), illustrating the downregulation of survivorin 24 hours after treatment with KRASG12D inhibitor in GP2D cells. Figure 3B shows the ELISA assay in HPAC cells treated with KRASG12D inhibitor for 2 hours (top) and 24 hours (bottom), illustrating the downregulation of survivorin 24 hours after treatment with KRASG12D inhibitor in HPAC cells. [Figure 4] This figure shows that survivin is downregulated in NCI-H358 cells treated with various KRASG12C inhibitors. ELISA assays show survivin levels in NCI-H358 cells after 72 hours of treatment with 100 nM compounds G12C-cpd 1 to G12C-cpd 11. [Figure 5] This figure shows that survivin is downregulated in the medium of NCI-H358 cells treated with various KRASG12C inhibitors. ELISA assays show survivin levels in H358 medium after 72 hours of treatment with 100 nM compounds G12C-cpd 1 to G12C-cpd 11. [Figure 6]This figure shows that survivin is downregulated in exosomes of NCI-H358 cells treated with various KRASG12C inhibitors. ELISA assays show survivin levels in exosomes of H358 cells after 72 hours of treatment with 100 nM compounds G12C-cpd 1 to G12C-cpd 11. [Figure 7] This figure shows that survivin is downregulated in SW1990 cells treated with various KRASG12D inhibitors and KRAS protac. The ELISA assay shows survivin levels in cell lysates of SW1990 cells after 72 hours of treatment with 300 nM compounds G12D-cpd 1 to G12D-cpd 9. [Figure 8] This figure shows that survivin is downregulated in the medium of SW1990 cells treated with various KRASG12D inhibitors and KRAS Protac. The ELISA assay shows the survivin levels in the supernatant of SW1990 cells after 72 hours of treatment with 300 nM compounds G12D-cpd 1 to G12D-cpd 9. [Figure 9] This figure shows that survivin is downregulated in exosomes of SW1990 cells treated with various KRASG12D inhibitors and KRAS Protac. The ELISA assay shows survivin levels in exosomes of SW1990 cells after 72 hours of treatment with 300 nM compounds G12D-cpd 1 to G12D-cpd 9. [Figure 10] This figure shows the survival levels in PC9 cells after treatment with 50 nM HER2-cpd 1 using an ELISA assay. [Figure 11] This figure shows the dose-dependent adjustment of Survivin in vivo with two different GDP-KRAS inhibitors (Cpd a and Cpd b). [Figure 12]This figure shows the dose-dependent regulation of survivin in tumors and plasma in the SW1990 CDX model after treatment with the KRASG12D inhibitor. [Figure 13] Figure 13A shows the modulation of survivorin in SNU1196 cells (KRASWT amp) treated with 11 types of GDP-KRAS inhibitors (see Table 1 for compound numbering) or DMSO for 72 hours. Cell lysates are shown. Figure 13B shows the modulation of survivorin in SNU1196 cells (KRASWT amp) treated with 11 types of GDP-KRAS inhibitors (see Table 1 for compound numbering) or DMSO for 72 hours. Exosomes are shown. [Figure 14A] This figure shows the dose-dependent modulation of Survivin in H358 tumors. [Figure 14B] Figure 14A shows the dose-dependent modulation of Survivin in the plasma of each mouse with the H358 tumor. [Figure 14C] This figure shows the modulation of Survivin in MKN1 tumors. [Figure 14D] Figure 14C shows the modulation of survivin in the plasma of each mouse with an MKN1 tumor. [Figure 15A] This figure shows the downregulation of survivin and the correlation between plasma survivin levels and tumor volume (TV) at various final days of efficacy studies in the CDX model. An exemplary GDP-KRAS inhibitor-treated KRAS-WTamp, CDX model is shown. [Figure 15B] This figure shows the downregulation of survivin and the correlation between plasma survivin levels and tumor volume (TV) at various final days of efficacy studies in the CDX model. An exemplary GDP-KRAS inhibitor-treated KRAS-WTamp, CDX model is shown. [Figure 15C]This figure shows the downregulation of survivin and the correlation between plasma survivin levels and tumor volume (TV) at various final days of efficacy studies in the CDX model. An exemplary KRAS-G12V CDX model treated with a GDP-KRAS inhibitor is shown. [Figure 15D] This figure shows the downregulation of survivin and the correlation between plasma survivin levels and tumor volume (TV) at various final days of efficacy studies in the CDX model. An exemplary KRAS-G12V CDX model treated with a GDP-KRAS inhibitor is shown. [Figure 15E] This figure shows the downregulation of survivin and the correlation between plasma survivin levels and tumor volume (TV) at various final days of efficacy studies in the CDX model. An exemplary KRAS-G12V CDX model treated with a GDP-KRAS inhibitor is shown. [Figure 15F] This figure shows the downregulation of survivin and the correlation between plasma survivin levels and tumor volume (TV) at various final days of efficacy studies in the CDX model. The figure shows the KRAS-G12D CDX model treated with the KRAS-G12D inhibitor, indicated as G12D-cpd 2 in Table 1, at the concentrations shown in the figure. [Figure 15G] This figure shows the downregulation of survivin and the correlation between plasma survivin levels and tumor volume (TV) at various final days of efficacy studies in the CDX model. The figure shows the KRAS-G12D CDX model treated with the KRAS-G12D inhibitor, indicated as G12D-cpd 2 in Table 1, at the concentrations shown in the figure. [Figure 15H] This figure shows the downregulation of survivin and the correlation between plasma survivin levels and tumor volume (TV) at various final days of efficacy studies in the CDX model. The figure shows the KRAS-G12D CDX model treated with the KRAS-G12D inhibitor, indicated as G12D-cpd 2 in Table 1, at the concentrations shown in the figure. [Figure 16A]This figure shows the downregulation of survivin levels in tumors in the HER2 mutant PC9_YMVA-5 NSCLC CDX model after treatment with the HER2 inhibitor HER2-cpd 1. [Figure 16B] This figure shows the downregulation of plasma survivin levels in the HER2 mutant PC9_YMVA-5 NSCLC CDX model after treatment with the HER2 inhibitor HER2-cpd 1. [Figure 16C] This figure shows the correlation between plasma survivin levels and tumor volume at the end of the efficacy study of the HER2 inhibitor HER2-cpd 1 in the CDX model NCI-N87. [Figure 16D] This figure shows the correlation between plasma survivin levels and tumor volume at the end of an efficacy study of the CDX model SK-GT-2 with the HER2 inhibitor HER2-cpd 1. [Figure 16E] This figure shows the correlation between plasma survivin levels and tumor volume at the end of the efficacy study of the HER2 inhibitor HER2-cpd 1 in the CDX model NCI-H2170. [Figure 17] Figure 17A shows the downregulation of survival levels (RNA) by the MDM2 inhibitor MDM2i-cpd 1 in vivo in the TP53 WT PDX model, CRC PDX model (Co10748). Figure 17B shows the downregulation of survival levels (RNA) by the MDM2 inhibitor MDM2i-cpd 1 in vivo in the TP53 WT PDX model, malignant peripheral nerve sheath tumor PDX. [Modes for carrying out the invention]

[0047] Before describing the present invention in detail, it should be understood that the present invention is not limited to specific compounds or methods, as the particular compounds or methods described may vary. It should also be understood that the terms used herein are solely for the purpose of describing specific embodiments and are not intended to be limiting. It should be noted that, as used herein and in the accompanying claims, the singular forms "a," "an," and "the" include singular and / or plural nouns unless the context clearly indicates otherwise. Where parameter ranges are indicated by numerical delimiters, it should be understood that the range includes these limiting values. Furthermore, it should be understood that ranges of numerically delimiting values ​​include the aforementioned delimiting values.

[0048] It should be further understood that the embodiments disclosed herein are not meant to be understood as separate embodiments unrelated to one another. A feature described in one embodiment is disclosed in relation to other embodiments shown herein. Where a particular feature is not disclosed in one embodiment but is disclosed in another, a person skilled in the art will understand that this does not necessarily mean that the feature is not disclosed in the other embodiment. A person skilled in the art will understand that disclosing the feature in other embodiments is an essential point of this application, but this has not been done merely for clarity and to make the specification manageable.

[0049] Furthermore, the contents of prior art documents referenced herein are incorporated by reference. This refers particularly to prior art documents disclosing standard or routine methods. In such cases, the incorporation by reference is primarily intended to provide a sufficiently practicable disclosure and to avoid redundant repetition.

[0050] According to a first aspect, the present invention relates to a method for determining the response of a cancer patient to treatment with a compound that inhibits the KRAS protein or a mutant of the KRAS protein, - Measure the level of survivin in the first sample obtained from the patient before treatment with the compound. - Measuring the level of survivin in a second sample obtained from the patient during or after treatment with the compound. - Comparing the level of survivin in the second sample with the level of survivin in the first sample. Includes, The present invention relates to a method by which a patient is determined to be responsive to treatment with the compound if the level of survivin in a second sample is lower than the level of survivin in a first sample.

[0051] Two or more samples, more than the first and second samples, may be obtained during the treatment period to continue monitoring the patient's responsiveness, or after the treatment period to monitor for potential recurrence.

[0052] The method for determining the responsiveness of cancer patients is preferably performed in vitro (ex vivo).

[0053] Furthermore, the present invention relates to a method for determining the response of cancer patients to treatment with compounds that inhibit the HER2 protein or mutants of the HER2 protein, - Measure the level of survivin in the first sample obtained from the patient before treatment with the compound. - Measuring the level of survivin in a second sample obtained from the patient during or after treatment with the compound. - Comparing the level of survivin in the second sample with the level of survivin in the first sample. Includes, The present invention relates to a method in which, if the level of survivin in a second sample is reduced compared to the level of survivin in a first sample, the patient is determined to be responsive to treatment with the compound, preferably the HER2 inhibitor being HER2-cpd 1.

[0054] As described above, two or more samples, and more than the first and second samples, may be obtained during the treatment period to continue monitoring the patient's responsiveness, or after the treatment period to monitor for potential recurrence. Furthermore, the method for determining the responsiveness of cancer patients is preferably performed in vitro (ex vivo).

[0055] The present invention relates to a method for determining the response of cancer patients to treatment with compounds that inhibit the interaction between MDM2 and p53 (also referred to herein as MDM2 inhibitors or MDM2i), - Measure the level of survivin in the first sample obtained from the patient before treatment with the compound. - Measuring the level of survivin in a second sample obtained from the patient during or after treatment with the compound. - Comparing the level of survivin in the second sample with the level of survivin in the first sample. Includes, The method further relates to a method in which, if the level of survivin in the second sample is decreased compared to the level of survivin in the first sample, the patient is determined to be responsive to treatment with the compound, preferably the compound that inhibits the interaction between MDM2 and p53 is MDM2i-cpd 1.

[0056] As described above, two or more samples, and more than the first and second samples, may be obtained during the treatment period to continue monitoring the patient's responsiveness, or after the treatment period to monitor for potential recurrence. Furthermore, the method for determining the responsiveness of cancer patients is preferably performed in vitro (ex vivo).

[0057] As used herein, the terms “inhibit” or “inhibit” refer to the reduction or prevention of the overall or partial activity and / or function of the KRAS protein or a mutant of the KRAS protein, or the HER2 protein or a mutant of the HER2 protein, respectively, by the compound. Furthermore, the terms “inhibit” or “inhibit” refer to the binding of the compound to the KRAS protein or a mutant of the KRAS protein, or to the HER2 protein or a mutant of the HER2 protein, respectively, and such binding may be direct or indirect, competitive or allosteric. Furthermore, the terms “inhibit” or “inhibit” refer, respectively, to the degradation of the KRAS protein or its mutant, or the HER2 protein or its mutant, and to the labeling of the degradation of the KRAS protein or its mutant, or the HER2 protein or its mutant, and to any other kind of neutralization of the activity and / or function, whether overall or partial, of the KRAS protein or its mutant, or the HER2 protein or its mutant. The same concept applies to the terms “inhibit” or “inhibit” when used in the context of compounds that inhibit the interaction between MDM2 and p53, as will be further explained below.

[0058] As used herein, the terms “level” or “measuring a level” of survivin refer to the level, amount, or measurement of any of the survivin RNA, mRNA, or protein. The term further refers to measuring the level or amount of survivin in a sample, where the survivin RNA, mRNA, or protein may be present intracellularly, intracellularly or on the cell membrane, extracellularly, in a fluid medium, in exosomes, and / or in a blood, plasma, or serum sample (one or more).

[0059] As used herein, the term “reduced” means a reduction or decrease of at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more, or 100%, of the level in a second (or n+1) sample compared to the level in a first (or n) sample of the biomarker survivin.

[0060] As used herein, the term “Survivin” refers to a protein having the amino acid sequence described in UniProtKB / TrEMBL accession number O15392, and any variant, isoform, splice variant, active mutant, or secondary database entry of said protein.

[0061] Survivin, encoded by the BIRC5 (Baculoviral IAP Repeat-Containing 5, also known as API4) gene, is the smallest member of the "apoptotic inhibitor (IAP)" family of proteins and acts as an inhibitor of apoptosis and a regulator of the cell cycle. Due to these functional attributes, survivin is a unique protein and exhibits a wide range of functions, including the regulation of cell death and cell proliferation.

[0062] Survivin is a small 142-amino acid protein with a multifunctional domain. Its N-terminal two-thirds contain globular baculovirus inhibitor of apoptosis repeat (BIR) domains (20-90 aa), the integrity of which depends on zinc fingers produced by C57, C60, C84, and H77, while the C-terminal one-third is an extended α-helix (98-142). While IAP family members typically contain multiple baculovirus IAP repeat (BIR) domains, the survivin protein encoded by the BIRC5 gene contains only a single BIR domain. Gene expression is high during fetal development and in most tumors, but low in adult tissues. Survivin is absent in adult cells other than activated T lymphocytes, erythroblasts, and autogenous stem cells (Wheatley and Altieri, 2019).

[0063] Survivin (BIRC5) has been shown to be involved in apoptosis inhibition and cell proliferation (LaCasse et al. 1998). Survivin is upregulated in many cancer types and is low in normal tissues (Kawasaki et al. 1998). Several publications have shown that survivin is correlated with poor prognosis (Takai et al. 2002) and resistance to chemotherapy (Zafaroni et al. 2002).

[0064] Apoptosis is the primary form of programmed cell death, which degrades cells in a controlled manner, dependent on cysteine ​​proteins called caspases. Survivin protects cells from apoptosis and autophagy, and its localization in the cytoplasm is crucial for its anti-apoptotic activity. Survivin expression has been found to reduce caspase activity; however, survivin does not bind to caspases at physiological concentrations. Instead, in a complex with XIAP-associated factor 1 (XAF1), it cooperates with XIAP and hepatitis B virus X-interacting protein (HBXIP, also known as LAMTOR5) to influence the interaction between XIAP and caspases or enhance the effects of other IAP family members.

[0065] In addition to its intracellular localization, survivins have also been found on the surface of exosomes constitutively secreted by cancer cells. The release of such survivins from tumor cells has been found to support neighboring tumor cells in evading apoptosis (Khan et al. 2011).

[0066] Due to its anti-apoptotic effect, survivin is considered a target for cancer treatment (Altieri 2003; Li et al. 2019; Wheatley and Altieri, 2019). Both mutant KRAS and survivin have been shown to contribute to carcinogenesis (Tecleab and Sebti, 2013). The combined application of a KRAS inhibitor and a beta-catenin inhibitor has been shown to synergistically arrest cancer cell proliferation, induce cell death, and downregulate survivin; however, this effect was observed only with the combined application, and the effect was not achieved with single treatment of either inhibitor (Mologni et al. 2012).

[0067] The inventors have surprisingly discovered that apoptosis inhibitor (IAP) protein survivor can be used in a method for determining the responsiveness of cancer patients to treatment with compounds that inhibit the KRAS protein or KRAS protein mutants, or the HER2 protein or HER2 protein mutants, and therefore can serve as a biomarker for selecting cancer patients who can be more beneficially treated with KRAS or HER2 inhibitors, preferably patients with KRAS-dependent cancer. Similarly, the inventors have discovered that survivor can also be used in a method for determining the responsiveness of cancer patients to treatment with compounds that inhibit the interaction between MDM2 and p53, as will be further described below. They have further discovered that this biomarker can be readily evaluated and monitored by quantitative determination in blood, plasma, or serum samples.

[0068] Furthermore, this finding was unexpected, as it has been reported that K-RAS-dependent human cancer cells strongly increase the production of exosomes enriched with survivin, and thus improve protection from apoptotic cell death for themselves, other cancer cells, and non-cancerous fibroblasts (Chang et al. 2021). Moreover, Chang et al. (2021) observed that upregulation of survivin is a means by which tumor cells make them resistant to treatment with antitumor drugs. Surprisingly, despite the aforementioned anti-apoptotic and protective effects of survivin, the inventors found that therapeutic intervention with K-RAS inhibitors was effective, and they also showed that the therapeutic efficacy could be non-invasively monitored from samples obtained from treated mice. Therefore, these experiments in mice provide evidence that evaluating survivin levels is equally suitable for determining the responsiveness of cancer patients to treatment with KRAS inhibitors or degraders, or with compounds that inhibit the interaction between MDM2 and p53.

[0069] In one or more preferred embodiments of the present invention, the first and / or second sample (one or more) is a blood, plasma, or serum sample (one or more). Preferably, all samples are blood, plasma, or serum samples.

[0070] In one or more preferred embodiments of the present invention, the step of measuring the level of survivin in a sample includes isolating exosomes from the sample and measuring the level of survivin contained in the exosomes. Means and methods for isolating exosomes and measuring the level of survivin contained in the exosomes are known in the art and are described in the experimental portion of this specification.

[0071] In one or more preferred embodiments of the present invention, the survivin level is measured using a survivin-specific assay selected from the group consisting of Western blotting, ELISA, RIA, MSD® S-PLEX technology, and FACS. Preferably, the survivin level is measured using ELISA or MSD® S-PLEX technology.

[0072] In one or more preferred embodiments of the present invention, the cancer is a KRAS-dependent cancer, preferably selected from the group consisting of pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NSCLC), and colorectal cancer (CRC).

[0073] In one or more preferred embodiments of the present invention, cancers treated with compounds that inhibit the KRAS protein or mutants of the KRAS protein, selected from the group consisting of KRAS(G12C) inhibitor or degrader, KRAS(G12D) inhibitor or degrader, and GDP-KRAS inhibitor or degrader, include brain tumors such as acoustic neuromas, astrocytomas, such as pilocytic astrocytomas, fibrous astrocytomas, and protoplasmic astrocytomas. Hypertrophic astrocytomas, anaplastic astrocytomas and glioblastomas, gliomas, cerebral lymphomas, brain metastases, pituitary tumors, such as prolactin-producing adenomas, HGH (human growth hormone)-producing tumors and ACTH-producing tumors (adrenocorticotropic hormone), craniopharyngiomas, medulloblastomas, meningiomas and oligodendrogliomas; neurotumors (neoplasms), such as tumors of the autonomic nervous system, such as neuroblastomas, sympathicomas, ganglia, paragangliomas (pheochromocytoma, chromophilia) Tumors of the carotid body, tumors of the peripheral nervous system, such as stump neuromas, neurofibromas, schwannomas (neurofibrillomas, schwann cell tumors) and malignant schwann cell tumors, and tumors of the central nervous system, such as brain and bone marrow tumors; intestinal cancers, such as rectal cancer, colon cancer, colorectal cancer, anal cancer, colorectal cancer, small intestine and duodenal tumors; eyelid tumors, such as basal cell tumors or basal cell carcinomas; pancreatic cancer or pancreatic cancer; bladder cancer or bladder cancer and other urothelial carcinomas; lung cancer (bronchial cancer), such as small cell bronchial cancer (oat cell carcinoma) and Non-small cell bronchial cancer (NSCLC), e.g., squamous cell carcinoma, adenocarcinoma and large cell bronchial cancer; breast cancer, e.g., invasive ductal carcinoma, mucinous carcinoma, lobular invasive carcinoma, tubular carcinoma, adenocystic carcinoma and papillary carcinoma, hormone receptor-positive breast cancer (estrogen receptor-positive breast cancer, progesterone receptor-positive breast cancer), Her2-positive breast cancer, triple-negative breast cancer; non-Hodgkin lymphoma (NHL), e.g., Burkitt lymphoma, low-grade non-Hodgkin lymphoma (NHL) and mycosis fungoides; uterine cancer or endometrial cancer or endometrial cancer; CUP syndrome (cancer of unknown primary origin); ovarian cancer or ovarian cancer, e.g., mucinous, endometrial or serous carcinoma; gallbladder cancer; bile duct cancer, e.g., Kratkin's tumor; testicular cancer, e.g., seminomas and nonseminomas;Lymphomas (lymphosarcomas), such as malignant lymphoma, Hodgkin's disease, non-Hodgkin lymphoma (NHL), such as chronic lymphocytic leukemia, leukemic reticuloendotheliosis, immunocytomas, plasmacytomas, multiple myeloma (MM), immunoblastic lymphoma, Burkitt lymphoma, T-zone mycosis fungoides, large cell anaplastic lymphoblastoma and lymphoblastoma; laryngeal cancers, such as tumors of the vocal cords, supraglottic, glottic and subglottic laryngeal tumors; bone cancers, such as osteochondroma, chondroma, and chondroblastoma. , chondromyxofibroma, osteoma, osteoid, osteoblastoma, eosinophilic granuloma, giant cell tumor, chondrosarcoma, osteosarcoma, Ewing's sarcoma, reticulum sarcoma, soft tissue sarcoma, liposarcoma, plasmacytoma, fibrous dysplasia, juvenile bone cysts and aneurysmal bone cysts; head and neck tumors, e.g., tumors of the lips, tongue, floor of the mouth, oral cavity, gums, palate, salivary glands, throat, nasal cavity, sinuses, larynx and middle ear; liver cancer, e.g., hepatocellular carcinoma or hepatocellular carcinoma (HCC); leukemia, e.g., acute leukemia, e.g. Lymphocytic / lymphoblastic leukemia (ALL), acute myeloid leukemia (AML); chronic leukemia, e.g., chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML); myelodysplastic syndrome (MDS); gastric cancer or gastric cancer, e.g., papillary adenocarcinoma, tubular adenocarcinoma and mucinous adenocarcinoma, signet ring cell carcinoma, adenosquamous carcinoma, small cell carcinoma and anaplastic carcinoma; melanoma, e.g., superficial spreading lentigo nodular and acral lentiginous melanoma; renal cancer, e.g., renal cell carcinoma or adrenal tumor (hypern Selected from: ephroma or Gravitz tumor; esophageal cancer or cancer of the esophagus; penile cancer; prostate cancer (e.g., castration-resistant prostate cancer); throat cancer or pharyngeal cancer, e.g., nasopharyngeal cancer, oropharyngeal cancer, and hypopharyngeal cancer; retinoblastoma, vaginal cancer, mesothelioma; squamous cell carcinoma, adenocarcinoma, carcinoma in situ, malignant melanoma, and sarcoma; thyroid cancer, e.g., papillary thyroid carcinoma, follicular thyroid carcinoma, and medullary thyroid carcinoma, as well as undifferentiated carcinoma; acanthoma, epidermoid carcinoma, and squamous cell carcinoma of the skin; thymoma, urethral cancer, cervical cancer, adenoid cystic carcinoma (AdCC), adrenocortical carcinoma, and vulvar cancer.

[0074] In another preferred embodiment, cancers treated with compounds that inhibit or degrade HER2 include: tumors / cancers of the head and neck, e.g., tumors / cancers of the nasal cavity, sinuses, nasopharynx, oral cavity (including lips, gums, alveolar ridge, retromolar triangle, floor of the mouth, tongue, hard palate, buccal mucosa), oropharynx (including base of the tongue, tonsils, tonsillar pillar, soft palate, tonsillar fossa, pharyngeal wall), middle ear, larynx (including supraglottic, glottis, subglottic, vocal cords), subpharyngeal, and salivary gland (including minor salivary glands); tumors / cancers of the lung, e.g., non-small cell lung cancer (NSCLC) (squamous cell carcinoma, spindle cell carcinoma, adenocarcinoma, large cell carcinoma, clear cell carcinoma, bronchoalveolar epithelium), small cell lung cancer (SCLC) (oat cell carcinoma, intermediate cell carcinoma, mixed oat cell carcinoma); and neoplasms of the mediastinum, e.g., neurogenic tumors (neuroembolic). tumors) (including neurofibromas, schwannomas, malignant Schwann cell tumors, neurosarcomas, ganglioblastomas, gangliomas, neuroblastomas, pheochromocytomas, and paragangliomas), germ cell tumors (including seminomas, teratomas, and nonseminomas), thymic tumors (including thymomas, thymic lipomas, thymic carcinomas, and thymic carcinoids), mesenchymal tumors (fibromas, fibrosarcomas, lipomas, liposarcomas, myxomas, mesotheliomas, leiomyomas, leiomyosarcomas, rhabdomyosarcomas, xanthogranulomas, mesenchymal tumors, hemangiomas, hemangioendotheliomas, hemangioextrinsomas, lymphangiomas, lymphangiopericytocytomas (Iymphangi (including opericytoma, lymphangiomyoma); cancers / tumors / cancers of the gastrointestinal (GI) tract, e.g., esophagus, stomach (stomach cancer), pancreas, liver and bile duct (hepatocellular carcinoma (HCC), e.g., pediatric HCC, lamellar HCC, mixed HCC, spindle cell HCC, clear cell HCC, giant cell HCC, carcinosarcoma HCC, sclerosing HCC; hepatoblastoma; cholangiocarcinoma; cholangiocarcinoma; hepatic cystadenocarcinoma; angiosarcoma, hemangioendothelioma, leiomyosarcoma, malignant Schwann cell tumor, fibrosarcoma, Kratkin's tumor); gallbladder, extrahepatic bile duct, small intestine (including duodenum, jejunum, ileum), large intestine (cecum, colon, rectum, anus); colorectal cancer, gastrointestinal stromal tumors (gastrointestinal stroma) tumors (including GIST), urogenital system (kidney, e.g., renal pelvis, renal cell carcinoma (RCC), nephroblastoma (Wilms' tumor), adrenal tumor, Gravitz tumor; ureter; bladder, e.g., urachal carcinoma, urothelial carcinoma; urethra, e.g., distal, spherical membranous, prostate;Prostate cancer (androgen-dependent, androgen-independent, castration-resistant, hormone-independent, hormone-refractory), including the penis; cancers / tumors / cancers of the testes, e.g., seminomas, non-seminomas; cancers / tumors / cancers of the gynecological system, e.g., tumors / cancers of the ovaries, fallopian tubes, peritoneum, cervix, vulva, vagina, and uterine body (including endometrium and fundus); cancers / tumors / cancers of the breast, e.g., breast cancer (invasive ductal carcinoma, gelatinous carcinoma, lobular invasive carcinoma, tubular carcinoma, adenocystic carcinoma, papillary carcinoma, medullary carcinoma, mucinous carcinoma), hormone receptor-positive breast cancer (estrogen receptor-positive breast cancer, progesterone receptor-positive breast cancer), HER2-positive breast cancer, triple-negative breast cancer, Paget's disease of the breast; cancers / tumors / cancers of the endocrine system, e.g., tumors / cancers of the endocrine glands, thyroid gland (thyroid gland). Parathyroid carcinoma / tumor (papillary, follicular, undifferentiated, medullary carcinoma), parathyroid gland (parathyroid carcinoma / tumor), adrenal cortex (adrenal cortical carcinoma / tumor), pituitary gland (including prolactin-producing tumors and craniopharyngiomas), thymus, adrenal gland, pineal gland, carotid body, islet cell tumors, paraganglia, pancreatic endocrine tumors (PET; nonfluorineunctional PET, PPoma, gastrin-producing tumors, insulin-producing tumors, VIP-producing tumors, glucagon-producing tumors, somatostatin-producing tumors, GRF-producing tumors, ACTH-producing tumors), carcinoid tumors; soft tissue sarcomas, e.g., fibrosarcoma, fibrous histiocytoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, angiosarcoma, lymphangiosarcoma, Kaposi's sarcoma, glomus tumors tumor), hemangiovascular tumor, synovial sarcoma, giant cell tumor of the tendon sheath, solitary fibrous tumor of the pleura and peritoneum, diffuse mesothelioma, malignant peripheral nerve sheath tumor (MPNST), granular cell tumor, clear cell sarcoma, melanocyte Schwann cell tumor, plexosarcoma, neuroblastoma, gangliblastoma, neuroepithelioma, extraskeletal Ewing's sarcoma, paraganglioma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, mesenchymal tumor, hydatidiform soft tissue sarcoma, epithelioid sarcoma, extrarenal rhabdoid tumor, fibroplastic small cell tumor;Bone sarcomas, e.g., myeloma, reticular sarcoma, chondrosarcoma (including central, peripheral, clear cell, and mesenchymal chondrosarcoma), osteosarcoma (including paraostemic, periosteal, highly superficial, small cell, radiation-induced osteosarcoma, and Paget's sarcoma), Ewing's tumor, malignant giant cell tumor, adamantinoma, (fibrous) histiocytoma, fibrosarcoma, chordoma, small round cell sarcoma, hemangioendothelioma, hemangioepidermoma, osteochondroma, osteoidoma, osteoblastoma, eosinophilic granuloma, chondroblastoma; mesothelioma, e.g., pleural mesothelioma, peritoneal mesothelioma; skin cancers, e.g., basal cell carcinoma, squamous cell carcinoma, Merkel cell carcinoma, Melanoma (including cutaneous, superficial spreading, malignant lentigo, acral lentiginous, nodular, and intraocular melanoma), actinic keratosis, eyelid cancer; neoplasms of the central nervous system and brain, e.g., astrocytoma (cerebral, cerebellar, diffuse, fibrillary, undifferentiated, pilocytic, protoplasmic, hypertrophic), glioblastoma, glioma, oligodendroglioma, oligodendronic astrocytoma, ependymoma, ependymocyte, choroid plexus tumor, medulloblastoma, meningioma, schwann cell tumor, hemangioblastoma, hemangioma, hemangioectocytoma, neuroma, gangliomas, neuroblastoma, retinoblastoma, schwannoma (e.g., auditory nerve), axial vertebral tumor;Lymphomas and leukemias, such as B-cell non-Hodgkin lymphoma (NHL) (including small lymphocytic lymphoma (SLL), lymphoplasmacytic lymphoma (LPL), mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large cell lymphoma (DLCL), and Burkitt lymphoma (BL)), T-cell non-Hodgkin lymphoma (anaplastic large cell lymphoma (ALCL)), and adult T-cell leukemia / lymphoma (ATLL). ), cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL)), lymphoblastic T-cell lymphoma (T-LBL), adult T-cell lymphoma, lymphoblastic B-cell lymphoma (B-LBL) , immunocytoma, chronic B-cell lymphocytic leukemia (BchlorineL), chronic T-cell lymphocytic leukemia (TchlorineL), B-cell small lymphocytic lymphoma (B-SLL), cutaneous T-cell lymphoma (CTLC), Primary Central Nervous System Lymphoma (PCNSL), Immunoblastic Lymphoma, Hodgkin's Disease (HD) (including Nodular Lymphocyte-Predominant HD (NLPHD), Nodular Sclerosis HD (NSHD), Mixed Cell Type HD (MCHD), Lymphocytogenic Classical HD, Lymphocytopenic HD (LDHD)), Large Granular Lymphocyte Leukemia (LGL), Chronic Myeloid Leukemia (CML), Acute Myeloid / Myeloid Leukemia (AML), Acute Li The following are selected from the group consisting of lymphoblastic leukemia (ALL), acute promyelocytic leukemia (APL), chronic lymphocytic leukemia (CLL), prolymphoblastic leukemia (PLL), pilocytic cell leukemia, chronic myeloid leukemia (CML), myeloma, plasmacytoma, multiple myeloma (MM), plasmacytoma, myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML); and cancer of unknown primary site (CUP).

[0075] In another preferred embodiment, cancers treated with compounds that inhibit the interaction between MDM2 and p53 include brain tumors, e.g., acoustic neuromas, astrocytomas, e.g., pilocytic astrocytomas, fibrous astrocytomas, protoplasmic astrocytomas, hypertrophic astrocytomas, anaplastic astrocytomas and glioblastomas, gliomas, cerebral lymphomas, brain metastases, pituitary tumors, e.g., prolactin-producing adenomas, HGH (human growth hormone)-producing tumors and ACTH-producing tumors (adrenocorticotropic hormone), craniopharyngiomas, medulloblastomas, meningiomas and oligodendrogliomas; and neurological tumors (neoplasms), e.g., autonomic nerve tumors. Tumors of the carotid body, such as neuroblastoma, sympathetic neuroblastoma, gangliomas, paragangliomas (pheochromocytoma, chromophilic cell tumors), and carotid body tumors; tumors of the peripheral nervous system, such as stump neuromas, neurofibromas, schwannomas (neurofibrillomas, schwann cell tumors), and malignant schwann cell tumors; and tumors of the central nervous system, such as brain and bone marrow tumors; intestinal cancers, such as rectal cancer, colon cancer, colorectal cancer, anal cancer, colorectal cancer, small intestine and duodenal tumors; eyelid tumors, such as basal cell tumors or basal cell carcinomas; pancreatic cancer or pancreatic cancer; bladder cancer or bladder cancer and other urothelial carcinomas; lung cancer (bronchial cancer), such as small cell tracheal cancer Bronchial carcinoma (oat cell carcinoma) and non-small cell bronchial carcinoma (NSCLC), e.g., squamous cell carcinoma, adenocarcinoma and large cell bronchial carcinoma; breast cancer, e.g., invasive ductal carcinoma, mucinous carcinoma, lobular invasive carcinoma, tubular carcinoma, adenocystic carcinoma and papillary carcinoma, hormone receptor-positive breast cancer (estrogen receptor-positive breast cancer, progesterone receptor-positive breast cancer), Her2-positive breast cancer, triple-negative breast cancer; non-Hodgkin lymphoma (NHL), e.g., Burkitt lymphoma, low-grade non-Hodgkin lymphoma (NHL) and mycosis fungoides; uterine cancer or endometrial cancer or endometrial cancer; CUP syndrome (primary cancer of unknown origin) n); ovarian cancer or ovarian carcinoma, e.g., mucinous, endometrial, or serous carcinoma; gallbladder cancer; bile duct cancer, e.g., Kratkin's tumor; testicular cancer, e.g., seminomas and nonseminomas; lymphoma (lymphosarcoma), e.g., malignant lymphoma, Hodgkin's disease, non-Hodgkin lymphoma (NHL), e.g., chronic lymphocytic leukemia, leukemic reticuloendotheliopathy, immunocytoma, plasmacytoma, multiple myeloma (MM), immunoblastic lymphoma, Burkitt lymphoma, T-zone mycosis fungoides, large cell anaplastic lymphoblastoma and lymphoblastoma; laryngeal cancer, e.g., tumors of the vocal cords, supraglottic, glottic and subglottic laryngeal tumors;Bone cancers, such as osteochondroma, chondroma, chondroblastoma, chondromyxofibroma, osteoma, osteoid, osteoblastoma, eosinophilic granuloma, giant cell tumor, chondrosarcoma, osteosarcoma, Ewing's sarcoma, reticulum sarcoma, soft tissue sarcoma, liposarcoma, plasmacytoma, fibrous dysplasia, juvenile bone cysts and aneurysmal bone cysts; head and neck tumors, such as tumors of the lips, tongue, floor of the mouth, oral cavity, gums, palate, salivary glands, throat, nasal cavity, sinuses, larynx and middle ear; liver cancers, such as hepatocellular carcinoma or hepatocellular carcinoma (HCC); leukemias, such as acute leukemia, such as acute lymphoblastic / lymphoblastic leukemia (ALL), acute myeloid leukemia (AML); chronic leukemias, such as chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML); myelodysplastic syndromes (MDS); gastric cancer or gastric cancer, such as breast cancer. Selected from: capillary adenocarcinoma, tubular adenocarcinoma and mucinous adenocarcinoma, signet ring cell carcinoma, adenosquamous carcinoma, small cell carcinoma and anaplastic carcinoma; melanoma, e.g., superficial spreading lentigo nodular malignant and acral lentiginous melanoma; renal cancer, e.g., renal cell carcinoma or adrenal tumor or Gravitz tumor; esophageal cancer or cancer of the esophagus; penile cancer; prostate cancer (e.g., castration-resistant prostate cancer); throat cancer or pharyngeal cancer, e.g., nasopharyngeal cancer, oropharyngeal cancer and hypopharyngeal cancer; retinoblastoma, vaginal cancer or vaginal cancer, mesothelioma; squamous cell carcinoma, adenocarcinoma, carcinoma in situ, malignant melanoma and sarcoma; thyroid cancer, e.g., papillary thyroid carcinoma, follicular thyroid carcinoma and medullary thyroid carcinoma, and anaplastic carcinoma; acanthoma, epidermoid carcinoma and squamous cell carcinoma of the skin; thymoma, urethral cancer, cervical cancer, adenoid cystic carcinoma (AdCC), adrenocortical carcinoma and vulvar cancer. Preferably, the cancer has a functional p53 and / or p53 wild-type state. Functional p53 means that p53 is capable of binding to DNA and activating the transcription of target genes. In one or more preferred embodiments of the present invention, the compound inhibiting the KRAS protein or a mutant of the KRAS protein is selected from the group consisting of KRAS(G12C) inhibitors or degraders, KRAS(G12D) inhibitors or degraders, GDP-KRAS inhibitors or degraders, and HER2 inhibitors or degraders.

[0076] In one or more preferred embodiments of the present invention, the compound that inhibits the KRAS protein or a mutant of the KRAS protein may also be a compound that inhibits or degrades the HER2 protein or a mutant of the HER2 protein, and the compound that inhibits or degrades the HER2 protein or a mutant of the HER2 protein acts as an indirect inhibitor of the KRAS protein or a mutant of the KRAS protein located downstream of the HER2 protein or a mutant of the HER2 protein in the signaling pathway.

[0077] Those skilled in the art will understand that KRAS-dependent cancers vary depending on the type of compound.

[0078] In one or more preferred embodiments of the present invention, the KRAS(G12C) inhibitor or degrader is selected from the group consisting of sotrasib (AMG510), adagrasib (MRTX849), G12C-cpd 1, G12C-cpd 2, G12C-cpd 3, G12C-cpd 4, G12C-cpd 5, G12C-cpd 6, G12C-cpd 7, G12C-cpd 8, G12C-cpd 9, G12C-cpd 10, and G12C-cpd 11.

[0079] In one or more preferred embodiments of the present invention, the KRAS(G12D) inhibitor or degrader is selected from the group consisting of MRTX1133, G12D-cpd 2, G12D-cpd 3, G12D-cpd 4, G12D-cpd 5, G12D-cpd 6, G12D-cpd 7, G12D-cpd 8, and G12D-cpd 9.

[0080] In one or more preferred embodiments of the present invention, the GDP-KRAS inhibitor or degrader is selected from the group consisting of GDP-cpd 1, GDP-cpd 2, GDP-cpd 3, GDP-cpd 4, GDP-cpd 5, GDP-cpd 6, GDP-cpd 7, GDP-cpd 8, GDP-cpd 9, GDP-cpd 10, GDP-cpd 11, GDP-cpd 12, GDP-cpd 13, and GDP-cpd 14.

[0081] In one or more preferred embodiments of the present invention, the HER2 inhibitor or degrader is the compound described in formula (F) (see below), and more preferably, the HER2 inhibitor or degrader is compound HER2-cpd 1.

[0082] In one or more preferred embodiments of the present invention, the compound that inhibits the interaction between MDM2 and p53 is MDM2i-cpd 1.

[0083] Preferably, the compound is selected from the compounds shown in Table 1. [Table 1] TIFF2026520494000003.tif236169 TIFF2026520494000004.tif232169 TIFF2026520494000005.tif229169 TIFF2026520494000006.tif218169 TIFF2026520494000007.tif245165 TIFF2026520494000008.tif205169 TIFF2026520494000009.tif205169 TIFF2026520494000010.tif228169 TIFF2026520494000011.tif208169 TIFF2026520494000012.tif202169 TIFF2026520494000013.tif175169

[0084] Anticancer drugs are sometimes evaluated based on their effects on “biomarkers” or surrogate markers that can predict the clinical benefit to the patient, such as tumor suppression. When monitoring a patient’s response to anticancer drugs, “biomarkers” can be the basis for clear demonstration that the administered compound, selected dose, and treatment schedule have the ability to efficiently inhibit the targeted cancer, and that the level achieved is sufficient for efficient inhibition of the targeted cancer. In evaluating molecularly targeted therapies, traditional clinical endpoints have proven difficult to apply, and standard clinical trial endpoints used for cytotoxic compounds have been found insufficient for evaluating molecularly targeted anticancer drugs. For their development, it is important to have usable endpoint evaluations for the efficacy of compounds in modulating the activity of molecular targets, and a relationship between target modulation and clinical response. Therefore, the usability of biomarkers that indirectly indicate the effect of treatment on a disease state is one of the important requirements for the development of minimally invasive or non-invasive techniques for evaluating the efficacy of inhibitors, and consequently for the clinical evaluation of drugs, and for monitoring the efficacy of approved drugs in treatment in the long term.

[0085] A "biomarker" can be defined as a measurable characteristic or feature that indicates a normal physiological process, a pathogenic process, or a response to exposure or therapeutic intervention. Molecular, histological, radiographic, or physiological biomarkers represent various types of biomarkers depending on the nature of the measurable characteristic (Aboy et al. 2019). The FDA and NIH further classify biomarkers into prognostic biomarkers, predictive biomarkers, responsive biomarkers, monitoring biomarkers, safety biomarkers, diagnostic biomarkers, and susceptibility / risk biomarkers (BEST Resource). According to this classification, pharmacodynamic / response biomarkers are biomarkers used to indicate that a biological response has occurred in an individual exposed to a drug or environmental agent (BEST Resource).

[0086] Pharmacodynamic / response biomarkers are biomarkers whose levels change in response to exposure to a drug or environmental agent. Changes in pharmacodynamic / response biomarkers, such as circulating small molecules or proteins, or physiological measurements, can be used to provide early evidence that a treatment may have an effect on an intended clinical endpoint, or to assess pharmacological endpoints related to safety concerns. They can also provide useful information for patient management, such as whether to continue a treatment or adjust the dose, or for drug development, such as whether a drug has had a pharmacodynamic effect that is thought to be related to clinical efficacy. Due to the continuous nature of their assessment, pharmacodynamic / response biomarkers may also fall into the category of monitoring biomarkers. Pharmacodynamic / response biomarkers are crucial in early drug development trial settings and can be used to measure the level of response to an intervention and to guide clinical dose-response studies. The primary use of pharmacodynamic / response biomarkers in clinical practice is to guide medication or the continued use of a drug or other intervention. Such biomarkers may be used to measure response levels so that individual drug doses can be varied, or to identify whether treatment needs to be added, subtracted, or replaced. In these cases, pharmacodynamic / response biomarkers may provide evidence of target involvement. In addition, these biomarkers may be used in pharmacological dose-range studies to determine which doses should be considered in trials evaluating clinical outcomes (BEST Resource).

[0087] As demonstrated in the experimental examples of this application, the inventors have found that Survivin is a suitable and reliable biomarker, preferably a pharmacodynamic / response biomarker, for evaluating the inhibitory activity of KRAS inhibitors or degraders in KRAS-dependent cancer cells and cancer types. The same applies to HER2 inhibitors or degraders in HER2 and / or KRAS-dependent cancer cells and cancer types, and the inventors have found that Survivin is a suitable and reliable biomarker for evaluating the inhibitory activity of HER2 inhibitors. Similarly, as demonstrated in Example 9.5 and Figure 17, Survivin is also downregulated upon successful treatment with MDM2i-cpd 1, and therefore Survivin can be conveniently used as a circulating biomarker in patient blood samples to monitor treatment efficacy during tumor treatment, thereby reducing the burden on patients or cumbersome imaging techniques associated with invasive tumor biopsies.

[0088] In one or more preferred embodiments of the present invention, the compound that inhibits the KRAS protein or a mutant of the KRAS protein may also be a compound that inhibits or degrades the HER2 protein or a mutant of the HER2 protein, and the compound that inhibits or degrades the HER2 protein or a mutant of the HER2 protein acts as an indirect inhibitor of the KRAS protein or a mutant of the KRAS protein located downstream of the HER2 protein or a mutant of the HER2 protein in the signaling pathway. Therefore, the term "compound that inhibits the KRAS protein or a mutant of the KRAS protein," as used herein, includes a compound that inhibits or degrades the HER2 protein or a mutant of the HER2 protein.

[0089] The present invention relates to a method for selecting cancer patients for treatment with a KRAS inhibitor or degrader, and includes at least: - A step of determining the level of survivin in a sample from a patient before administering a KRAS inhibitor or degrader to the patient, and - A step to determine a decrease in the level of Survivin in at least one second sample from a patient. Further details regarding methods including

[0090] Similarly, the present invention relates to a method for selecting cancer patients for treatment with a compound that inhibits the interaction between MDM2 and p53, and at least, - A step of determining the level of survivin in a sample from a patient before administering the compound to the patient, and - A step to determine a decrease in the level of Survivin in at least one second sample from a patient. Further details regarding methods including

[0091] The present invention relates to a method for selecting patients as described above, wherein a decrease in survivin levels is determined in several, preferably all, samples obtained from the patient after administering a KRAS inhibitor or degrader to the patient, or after administering a compound that inhibits the interaction between MDM2 and p53.

[0092] The present invention relates to a method for selecting patients as described above, further comprising the step of selecting the patients for continued treatment with a KRAS inhibitor or degrader, or a compound that inhibits the interaction between MDM2 and p53, respectively.

[0093] The present invention relates to a method for selecting cancer patients to be treated with a compound acting as a KRAS inhibitor or degrader by ex vivo determining the levels of survivin in at least one first sample and at least one second sample from the cancer patients, wherein the at least one first sample is collected before treating the cancer patient with the compound acting as a KRAS inhibitor or degrader, the at least one second sample is collected after treating the cancer patient with the compound acting as a KRAS inhibitor or degrader, and the levels of survivin in the second or one or more further samples collected from the cancer patient after treating the cancer patient with the compound acting as a KRAS inhibitor or degrader are determined to be reduced compared to the first sample or compared to the sample immediately preceding the one or more further samples.

[0094] The present invention relates to a method for selecting cancer patients to be treated with a compound that inhibits the interaction between MDM2 and p53, by ex vivo determining the levels of survivin in at least one first sample and at least one second sample from the cancer patients, wherein the at least one first sample is collected before the cancer patient is treated with the compound that inhibits the interaction between MDM2 and p53, the at least one second sample is collected after the cancer patient is treated with the compound that inhibits the interaction between MDM2 and p53, and the levels of survivin in the second or one or more further samples collected from the cancer patient after the cancer patient is treated with the compound that inhibits the interaction between MDM2 and p53 are determined to be reduced compared to the first sample or compared to the sample immediately preceding the one or more further samples.

[0095] The present invention is a method for predicting the response of cancer patients to a KRAS inhibitor or degrader, and includes at least, - The process of providing a first sample from the patient, - A step of determining the level of survivin in the first sample from the patient prior to the following steps, - The step of administering a KRAS inhibitor or degrader to a patient, and - If it is determined that the Survivin level in a second or one or more further samples provided by the patient after administration of the KRAS inhibitor or degrader is reduced compared to the first sample, or compared to the sample immediately preceding the one or more further samples, then the patient is identified as responsive to treatment with the KRAS inhibitor or degrader. Further details regarding methods including

[0096] The present invention provides a method for predicting the response of cancer patients to compounds that inhibit the interaction between MDM2 and p53, and at least, - The process of providing a first sample from the patient, - A step of determining the level of survivin in the first sample from the patient prior to the following steps, - A step of administering a compound that inhibits the interaction between MDM2 and p53 to a patient, and - If it is determined that the level of survivin in a second or one or more further samples provided by the patient after administration of the compound that inhibits the interaction between MDM2 and p53 is decreased compared to the first sample, or compared to the sample immediately preceding the one or more further samples, then the patient is identified as responsive to treatment with the compound that inhibits the interaction between MDM2 and p53. Further details regarding methods including

[0097] The present invention relates to a method for determining whether a compound that inhibits the KRAS protein or a mutant of the KRAS protein is effective in treating cancer and / or monitoring the response of cancer patients to said treatment, wherein at least, - The process of providing a first sample from the patient, - A step of measuring the level of Survivin in the first sample from the patient, - A step of administering to a patient a compound that inhibits the KRAS protein or a mutant of the KRAS protein. - The process of providing a second sample from the patient, - A step of measuring the level of survivin in the second sample, - A step of comparing the levels of survival measured in the first and second samples, and - A process in which steps 4 to 6 are repeated depending on the circumstances. The method further relates to a method that includes a decrease in the level of survivin in the second sample, indicating an effective response.

[0098] The present invention relates to a method for determining whether a compound that inhibits the interaction between MDM2 and p53 is effective in treating cancer and / or monitoring the response of cancer patients to said treatment, wherein at least, - The process of providing a first sample from the patient, - A step of measuring the level of Survivin in the first sample from the patient, - A step of administering the compound that inhibits the interaction between MDM2 and p53 to a patient, - The process of providing a second sample from the patient, - A step of measuring the level of survivin in the second sample, - A step of comparing the levels of survival measured in the first and second samples, and - A process in which steps 4 to 6 are repeated depending on the circumstances. The method further relates to a method that includes a decrease in the level of survivin in the second sample, indicating an effective response.

[0099] In any preferred embodiment of the method described above, the sample(s) is a blood, plasma, or serum sample(s).

[0100] In any preferred embodiment of the method described above, the compound that inhibits the KRAS protein or a mutant of the KRAS protein is selected from the group consisting of KRAS(G12C) inhibitor or degrader, KRAS(G12D) inhibitor or degrader, GDP-KRAS inhibitor or degrader, and HER2 inhibitor or degrader.

[0101] In a more preferred embodiment of any of the methods described above, the KRAS(G12C) inhibitor or degrader is selected from the group consisting of sotrasib (AMG510), adagrasib (MRTX849), G12C-cpd 1, G12C-cpd 2, G12C-cpd 3, G12C-cpd 4, G12C-cpd 5, G12C-cpd 6, G12C-cpd 7, G12C-cpd 8, G12C-cpd 9, G12C-cpd 10, and G12C-cpd 11.

[0102] In a more preferred embodiment of any of the methods described above, the KRAS(G12D) inhibitor or degrader is selected from the group consisting of MRTX1133, G12D-cpd 2, G12D-cpd 3, G12D-cpd 4, G12D-cpd 5, G12D-cpd 6, G12D-cpd 7, G12D-cpd 8, and G12D-cpd 9.

[0103] In a more preferred embodiment of any of the methods described above, the GDP-KRAS inhibitor or degrader is selected from the group consisting of GDP-cpd 1, GDP-cpd 2, GDP-cpd 3, GDP-cpd 4, GDP-cpd 5, GDP-cpd 6, GDP-cpd 7, GDP-cpd 8, GDP-cpd 9, GDP-cpd 10, GDP-cpd 11, GDP-cpd 12, GDP-cpd 13, and GDP-cpd 14.

[0104] In a more preferred embodiment of any of the methods described above, the HER2 inhibitor or degrader is the compound described in formula (F) (see below), and more preferably, the HER2 inhibitor or degrader is compound HER2-cpd 1.

[0105] In one or more preferred embodiments of the present invention, the compound that inhibits the KRAS protein or a mutant of the KRAS protein may also be a compound that inhibits or degrades the HER2 protein or a mutant of the HER2 protein, and the compound that inhibits or degrades the HER2 protein or a mutant of the HER2 protein acts as an indirect inhibitor of the KRAS protein or a mutant of the KRAS protein located downstream of the HER2 protein or a mutant of the HER2 protein in the signaling pathway.

[0106] In any preferred embodiment of the method described above, the compound that inhibits the interaction between MDM2 and p53 is compound MDM2i-cpd 1.

[0107] In a more preferred embodiment of any of the methods described above, the survivor is contained within an exosome.

[0108] In a more preferred embodiment of any of the methods described above, the cancer is a KRAS-dependent cancer, preferably selected from the group consisting of pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NSCLC), and colorectal cancer (CRC).

[0109] In another preferred embodiment of any of the methods described above, the cancer treated with a compound that inhibits the interaction between MDM2 and p53 is selected from any of the cancers defined herein above as preferred cancer types for treatment with such MDM2 inhibitors.

[0110] In a more preferred embodiment of any of the methods described above, determining the level of survivorbin preferably involves a survivorbin-specific assay selected from the group consisting of Western blotting, ELISA, RIA, MSD® S-PLEX technology, and FACS, more preferably the assay being ELISA or MSD® S-PLEX technology.

[0111] According to a second aspect, the present invention relates to a compound for inhibiting the KRAS protein or a mutant of the KRAS protein for use in treating cancer patients, i) The KRAS inhibitor or degrader is selected from the group consisting of KRAS(G12C) inhibitor or degrader, KRAS(G12D) inhibitor or degrader, GDP-KRAS inhibitor or degrader, and HER2 inhibitor and degrader. ii) The patient has been determined to be responsive to treatment with the compound according to any of the methods described above, Regarding compounds.

[0112] In accordance with this second aspect of the present invention, the present invention also relates to a compound for use in treating cancer patients that inhibits the interaction between MDM2 and p53, i) The compound that inhibits the interaction between MDM2 and p53 is MDM2i-cpd 1, ii) The patient has been determined to be responsive to treatment with a compound according to the method for determining the responsiveness of cancer patients to treatment with a compound that inhibits the interaction between MDM2 and p53 of the present invention, Regarding compounds.

[0113] In one or more preferred embodiments of the present invention, the compound that inhibits the KRAS protein or a mutant of the KRAS protein may also be a compound that inhibits or degrades the HER2 protein or a mutant of the HER2 protein, and the compound that inhibits or degrades the HER2 protein or a mutant of the HER2 protein acts as an indirect inhibitor of the KRAS protein or a mutant of the KRAS protein located downstream of the HER2 protein or a mutant of the HER2 protein in the signaling pathway.

[0114] The present invention further relates to a KRAS inhibitor or degrader for use in a method for treating KRAS-dependent cancer in patients exhibiting the molecular biomarker Survivin. The present invention further relates to a compound that inhibits the interaction between MDM2 and p53 for use in a method for treating cancer in patients exhibiting the molecular biomarker Survivin.

[0115] The present invention relates to a KRAS inhibitor or degrader for use in a method for treating KRAS-dependent cancer in a patient, wherein the patient has been determined to exhibit the molecular biomarker Survivin. The present invention also relates to a compound that inhibits the interaction between MDM2 and p53 for use in a method for treating cancer in a patient, wherein the patient has been determined to exhibit the molecular biomarker Survivin.

[0116] The present invention relates to a KRAS inhibitor or degrader for use in a method for treating K-ras-dependent cancer in a patient, comprising determining that the patient exhibits the molecular biomarker Survivin. The present invention also relates to a compound for use in a method for treating cancer in a patient, comprising determining that the patient exhibits the molecular biomarker Survivin.

[0117] The present invention relates to a KRAS inhibitor or degrader for use in a method for treating KRAS-dependent cancer in a patient, - The level of survivin in the first sample obtained from the aforementioned patient was measured. - The level of survivin in at least one second sample obtained from the patient during the procedure was compared with the level of survivin in the first sample, and - It was determined that the level of survivin in the second sample was decreased compared to the level of survivin in the first sample, or that the level of survivin in any further sample obtained from the patient during treatment after the second sample was decreased compared to the level of survivin in the previous sample obtained from the patient during treatment. Further relating to KRAS inhibitors or degraders, including those mentioned above.

[0118] The present invention relates to a compound for use in a method for treating cancer in a patient, which inhibits the interaction between MDM2 and p53, - The level of survivin in the first sample obtained from the aforementioned patient was measured. - The level of survivin in at least one second sample obtained from the patient during the procedure was compared with the level of survivin in the first sample, and - It was determined that the level of survivin in the second sample was decreased compared to the level of survivin in the first sample, or that the level of survivin in any further sample obtained from the patient during treatment after the second sample was decreased compared to the level of survivin in the previous sample obtained from the patient during treatment. Further relating to compounds containing these compounds.

[0119] The present invention further relates to the KRAS inhibitor or degrader for use as described above, wherein the compound is selected from the group consisting of KRAS(G12C) inhibitor or degrader, KRAS(G12D) inhibitor or degrader, GDP-KRAS inhibitor or degrader, and HER2 inhibitor and degrader. The present invention further relates to the KRAS inhibitor or degrader for use as described above, wherein the compound is MDM2i-cpd 1.

[0120] The present invention relates to a KRAS inhibitor or degrader, or a compound that inhibits the interaction between MDM2 and p53 for use as described above, wherein the survivor is contained in an exosome.

[0121] The present invention relates to a KRAS inhibitor or degrader for use as described above, or a compound that inhibits the interaction between MDM2 and p53, wherein the sample(s) is a blood, plasma, or serum sample(s).

[0122] The present invention further relates to the K-Ras inhibitor or degrader for use as described above, wherein the KRAS-dependent cancer is selected from the group consisting of pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NSCLC), and colorectal cancer (CRC). The present invention further relates to the compound for use as described above, wherein the cancer is selected from any of the cancers defined above herein as preferred cancer types for treatment with such MDM2 inhibitors.

[0123] The present invention relates to a K-Ras inhibitor or degrader, or a compound that inhibits the interaction between MDM2 and p53, for use as described above, wherein determining the level of survivin involves a survivin-specific assay selected from the group consisting of Western blotting, ELISA, RIA, MSD® S-PLEX technology, and FACS.

[0124] In its broadest sense, a "biomarker" can be defined as a measurable characteristic that is an indicator of a normal physiological process, a pathogenic process, or a response to exposure or (therapeutic) intervention. Molecular, histological, radiographic, or physiological biomarkers represent various types of biomarkers used in medicine, with blood pressure or glucose levels being direct examples of physiological and molecular biomarkers, respectively.

[0125] Various methods are used to further classify biomarkers. For example, the FDA and NIH classify them into prognostic biomarkers, predictive biomarkers, responsive biomarkers, monitoring biomarkers, safety biomarkers, diagnostic biomarkers, and susceptibility / risk biomarkers (BEST Resource 2018).

[0126] In the sense of this invention, a biomarker is used as an indicator of a biological state. It is a feature that can be objectively measured and evaluated as an indicator of a normal biological process, a pathogenic process, or a pharmacological response to a therapeutic intervention. This is consistent with the definition presented by the NIH research group in 1998.

[0127] More specifically, biomarkers indicate changes that correlate with disease risk or progression, or susceptibility to a given treatment for the disease. Once a proposed biomarker is confirmed, it can be used to diagnose disease risk, the presence of disease, or to tailor treatment for disease in an individual (selection of drug treatment or administration regimen). In evaluating potential drug therapies, biomarkers may be used as surrogates for natural endpoints such as survival or irreversible pathological conditions. If a treatment alters a biomarker, and this is directly linked to improved health, the biomarker serves as a surrogate endpoint for evaluating clinical benefit.

[0128] As used herein, the term "sample" refers to a tissue sample or a bodily fluid sample, such as a blood sample. Preferably, the sample is a blood, plasma, or serum sample.

[0129] A tissue sample is a section of an organ or tissue of the body, which typically contains several cell types and, in some cases, cytoskeletal structures that bind cells together. Tissue samples can be obtained by biopsy, including, for example, by excision, thinning, or perforation. This involves the extraction of sample cells or tissue for examination. However, the terms “providing a sample,” “a sample from a patient,” or “a sample obtained from a patient” do not include the active process of tissue or blood extraction, but rather refer to the provision of an already extracted sample, i.e., the provision of an ex vivo sample from a patient. The term “a sample obtained from a patient,” as used herein, refers to an ex vivo, i.e., post-collection sample, and is synonymous with “providing a sample from a patient” or “providing an ex vivo sample from a patient.”

[0130] Furthermore, the tissue sample may preferably be a tumor sample or a control sample from the same tissue. The blood sample may be a serum sample or a plasma sample, preferably a plasma sample. However, the blood sample may also include, for example, a whole blood count and a cell fraction for blood cell testing, such as a peripheral blood smear. In addition to blood, other bodily fluid samples include, but are not limited to, mucus, semen, saliva, sputum, bronchial lavage fluid, breast milk, bile, and urine. The sample may further be a bone marrow biopsy or aspirate or cerebrospinal fluid.

[0131] The sample may be analyzed using any method known in the art, including, but not limited to, cell chemistry such as chemical staining (dyes) that reacts with certain substances found inside or on different types of cells; flow cytometry; immunohistochemistry (IHC) such as by using antibody staining; fluorescence in situ hybridization (FISH); polymerase chain reaction (PCR); enzyme-linked immunosorbent assay (ELISA); MSD® S-PLEX technology; or Western blotting. Preferably, the analysis is performed by ELISA or MSD® S-PLEX technology.

[0132] In any of the methods described above, the level of the biomarker survivin in the second (or n+1th) sample is reduced by at least approximately 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more, or 100%, compared to the level in the first (or nth) sample.

[0133] Survivor-specific assays may be based on antibodies, particularly monoclonal antibodies, especially if they involve assays such as Western blotting, dot blotting, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), RIST (radioimmunosorbent assay), MSD® S-PLEX technology, or FACS (fluorescence-activated cell sorting). The general principles of Western blotting, dot blotting, ELISA, RIA, RIST, MSD® S-PLEX technology, and FACS are known to those skilled in the art (Coligan 2011).

[0134] Antibodies targeting survivorbin and monoclonal antibodies have been described (Fenstermaker et al. 2018; Watanuki-Miyauchi et al. 2005; Arora et al. 2012).

[0135] Survivin-specific assays are known from prior art (Naumunik et al. 2009; Derin et al. 2008).

[0136] As used herein, the term “antibody” refers to a protein comprising one or more polypeptide chains encoded by a native or recombinant immunoglobulin gene or a fragment of an immunoglobulin gene or cDNA derived therefrom. The immunoglobulin gene includes any of the light chain kappa, lambda, and heavy chain alpha, delta, epsilon, gamma, and mu constant region genes, as well as many different variable region genes.

[0137] The basic immunoglobulin (antibody) structural unit is typically a tetramer containing two identical pairs of polypeptide chains: a light chain (L, with a molecular weight of approximately 25 kDa) and a heavy chain (H, with a molecular weight of approximately 50-70 kDa). Each heavy chain has a variable heavy chain region (VH or V). H (abbreviated as) and heavy chain steady region (CH or C H The heavy chain constant region includes three domains, namely CH1, CH2, and CH3. Each light chain contains a light chain variable region (VL or V). L (Abbreviated as) and the light chain constant region (CL or CL It contains (abbreviated as ). The VH and VL regions can be further subdivided into a highly variable region called the complementarity-determining region (CDR) and a more conserved region called the framework region (FR) interspersed within it. Each VH and VL region contains three CDRs and four FRs, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus. The variable regions of the heavy and light chains form binding domains that interact with the antigen.

[0138] The CDR is most important for the binding of the antibody or its antigen-binding portion. The FR can be replaced by other sequences, as long as the three-dimensional structure required for antigen binding is maintained. Structural changes in the construct almost always result in a loss of sufficient binding to the antigen.

[0139] The term "antigen binding" in relation to antibodies (monoclonal antibodies), their antigen-binding fragments or derivatives, and antigen-binding antibody-like proteins refers to one or more parts or fragments of an antibody that retain the ability to specifically bind to an antigen in its native form. Examples of antigen-binding parts of an antibody include Fab fragments; monovalent fragments consisting of VL, VH, CL, and CH1 domains; F(ab')2 fragments; bivalent fragments containing two Fab fragments linked by disulfide crosslinks in the hinge region; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of VL and VH domains of a single arm of the antibody; and dAb fragments consisting of a VH domain and an isolated complementarity-determining region (CDR).

[0140] As used herein, the term “monoclonal antibody (mAb)” refers to an antibody composition having a homogeneous population of antibodies, i.e., a homogeneous population of total immunoglobulins or fragments or derivatives thereof. Such antibodies may be selected from the group consisting of IgG, IgD, IgE, IgA and / or IgM or fragments or derivatives thereof. Particularly preferred is the antibody IgG.

[0141] As used herein, the term antibody “fragment” refers to an antibody fragment that retains target-binding ability, such as the CDR (complementarity-determining region), hypervariable region, variable domain (Fv), IgG heavy chain (consisting of VH, CH1, hinge, CH2 and CH3 regions), IgG light chain (consisting of VL and CL regions), and / or Fab and / or F(ab)2.

[0142] As used herein, the term antibody “derivative” refers to protein constructs that are structurally different from the general concept of an antibody but still have some structural relationship to it, such as scFv, Fab and / or F(ab)2, as well as double, triple, or more multispecific antibody constructs.

[0143] When used herein, the term “KRAS-dependent cancer” means cancer, cancer cells, or cancerous tissue, which includes (i) one or more mutations in the K-ras oncogene, either alone or with one or more comutations that may affect the function of the KRAS protein and the development and growth of tumors, and / or the formation of such cancer, cancer cells, or cancerous tissue is caused by such one or more mutations in the K-ras oncogene, or (ii) amplification of the wild-type (WT) K-Ras proto-oncogene. K-ras mutations are considered to be the most common oncogene drivers in human cancers. The profile of K-ras mutations can differ significantly between different cancer types. Single-nucleotide missense mutations are dominant in K-ras mutations, with 98% found at codon 12 (G12), codon 13 (G13), or codon 61 (Q61). Mutation-induced alterations in the KRAS protein have been shown to inhibit the interaction between KRAS and GAP, as well as the hydrolysis of GTP bound to KRAS, thereby keeping KRAS constitutively active (Huang et al. 2021).

[0144] As used herein, the term “KRAS mutant” refers to the K-ras gene or the KRAS protein, respectively, including any type of mutation in the K-ras gene and at any amino acid position in the KRAS protein. In particular, a K-ras mutant is a KRAS protein containing a mutation at codon 12 (G12), codon 13 (G13), or codon 61 (Q61) in the K-ras gene. More specifically, a K-ras mutant is the KRAS(G12C) protein.

[0145] Significant amplification of the wild-type (WT) KRAS oncogene has been observed in one subset of tumor indications, such as gastric cancer, gastroesophageal junction cancer, and esophageal cancer, as described above herein.

[0146] As used herein, the term “KRAS inhibitor” or “K-Ras inhibitor” refers to any compound, drug, small molecule, peptide, polypeptide, or protein that has the ability to inhibit the activity of K-Ras, including degraders of the KRAS protein. The KRAS inhibitors according to the present invention are preferably compounds that inhibit KRAS wild-type, preferably amplified KRAS wild-type, KRAS mutated at residue 12, e.g., KRAS G12C, KRAS G12D, KRAS G12V, KRAS G12A, and KRAS G12R, preferably compounds that inhibit KRAS G12C and / or KRAS G12D, and compounds that inhibit KRAS mutated at residue 13, e.g., KRAS G13D, or KRAS mutated at residue 61, e.g., KRAS Q61H.

[0147] The inhibition may be direct or indirect, and may be competitive or allosteric.

[0148] Indirect inhibitors include, but are not limited to, compounds that inhibit K-Ras activity by interfering with upstream signaling molecules in the KRAS signaling pathway, such as receptor tyrosine kinases of the EGFR family, including EGFR (also known as ERBB1 or HER1), ERBB2 / HER2, ERBB3 / HER3, and ERBB4 / HER4. Preferably, the indirect KRAS inhibitor is a HER2 inhibitor.

[0149] In one or more preferred embodiments of the present invention, the compound that inhibits the KRAS protein or a mutant of the KRAS protein may also be a compound that inhibits or degrades the HER2 protein or a mutant of the HER2 protein, and the compound that inhibits or degrades the HER2 protein or a mutant of the HER2 protein acts as an indirect inhibitor of the KRAS protein or a mutant of the KRAS protein located downstream of the HER2 protein or a mutant of the HER2 protein in the signaling pathway.

[0150] The ERBB transmembrane receptor tyrosine kinase (RTK) family consists of four members that fulfill essential functions during development: EGFR (ERBB1), HER2 (Neu, ERBB2), HER3 (ERBB3), and HER4 (ERBB4) (Citri et al. 2006; Wang, Z. 2017). ERBB signaling is initiated by the binding of the extracellular domains of EGFR, HER3, or HER4 to their respective ligands, followed by homodimerization or heterodimerization of ERBB family members. HER2, whose ligand has not been identified, is a preferred dimerization partner for other ERBB members. Once the active ligand-receptor complex is formed, the intracellular tyrosine kinase domains of EGFR, HER2, HER3, or HER4 are activated by autophosphorylation or transphosphorylation, subsequently initiating a signaling cascade, most notably involving the K-Ras-mediated MAP (mitogen-activated protein) kinase and / or phosphoinositide 3-kinase (PI3K) pathway, as described above (Citri et al. 2006; Wang, Z. 2017).

[0151] In cancer, ERBB signaling is hyperactivated through mutations that constitutively activate RTKs by promoting dimerization or shifting equilibrium to the kinase's active conformer, and / or through RTK amplification and resulting overexpression. Both oncological mechanisms increase the net output of ERBB signaling, thereby promoting cell survival, cell growth, and proliferation.

[0152] Abnormal HER2 signaling is observed in a wide range of human malignancies. Oncogenic mutations have been described for the extracellular, membrane (near-membrane), and intracellular regions of the protein. Taken together, these mutations constitutively activate HER2, stimulating cancer initiation, tumor maintenance, and growth. Similarly, HER2 overexpression increases HER2 signaling and underlies oncochemical transformation and tumor maintenance in various indications, including breast, gastric, or lung cancer.

[0153] As a result, interference with HER2 oncogenic signaling leads to inhibition of tumor growth. Targeted therapies include antibodies targeting HER2 (e.g., trastuzumab and pertuzumab), antibody-drug conjugates targeting HER2 (trastuzumab-DM1 (T-DMl, adtrastuzumab emtansine)), and small molecules that inhibit the HER2 kinase domain (afatinib, neratinib, lapatinib).

[0154] Tumors driven by HER2 oncogenic mutations or HER2 wild-type overexpression (e.g., for gene amplification) may benefit from HER2-specific tyrosine kinase inhibitors (TKIs). In summary, HER2 modifications affect up to 6–7% of all human cancers, and EGFR wild-type-preserving TKIs (tyrosine kinase inhibitors) may emerge as an effective treatment option.

[0155] HER2 exon 20 mutations constitute a subset of HER2 gain-of-function mutations, which result in enhanced kinase activity. This enhanced HER2 kinase activity feeds into downstream signaling cascades, stimulating neoplastic transformation by promoting the growth, proliferation, and survival of mutated cells. Selective inhibitors of HER2 exon 20 have been developed and, compared to prior art compounds, exhibit improved wild-type EGFR-preserving efficacy profiles in addition to higher selectivity compared to wild-type EGFR. Furthermore, some compounds of this type show improved pharmacokinetic and pharmacological profiles, such as superior metabolic stability (WO2021 / 213800).

[0156] KRAS inhibitors include, but are not limited to, compounds known in the art, such as sotrasib (AMG510) and adaglasib (MRTX849), and compounds detailed herein.

[0157] The KRAS inhibitor is preferably of formula (A) [ka] {wherein, R 1a , R 1b , R 2a , R 2b , Z, R 3 ~R 5 , A, p, U, V, W, L, and E have the following meanings: [A0] R 1a and R 1b are both independently selected from the group consisting of hydrogen, C 1~4 alkyl, C 1~4 haloalkyl, C 1~4 alkoxy, C 1~4 haloalkoxy, halogen, -NH2, -NH(C 1~4 alkyl), -N(C 1~4 alkyl)2, C 3~5 cycloalkyl, and 3- to 5-membered heterocyclyl, R 2a and R 2b are both independently selected from the group consisting of hydrogen, C 1~4 alkyl, C 1~4 haloalkyl, C 1~4 alkoxy, C 1~4 haloalkoxy, halogen, -NH2, -NH(C 1~4 alkyl), -N(C 1~4 alkyl)2, C 3~5 cycloalkyl, and 3- to 5-membered heterocyclyl, and / or, optionally, one of R 1a or R 1b and one of R 2a or R 2b forms a cyclopropane ring together with the carbon atom to which they are attached; [B0] Z is -(CR 6a R 6b ) n -, each R 6a and R 6b is independently hydrogen, C 1~4 alkyl, C 1~4 haloalkyl, C 1~4 alkoxy, C 1~4Haloalkoxy, halogen, -NH2, -NH(C) 1~4 Alkyl), -N(C 1~4 Alkyl)2, C 3~5 Selected from the group consisting of cycloalkyl and 3-5 membered heterocyclines, n is selected from the group consisting of 0, 1, and 2; [C0] R 3 is hydrogen, C 1~6 Alkyl, C 1~6 Haloalkyl, C 1~6 Alkoxy, C 1~6 Haloalkoxy, cyano-C 1~6 Alkyl, halogen, -OH, -NH2, -NH(C 1~4 Alkyl), -N(C 1~4 Alkyl)2,-CN,C 3~5 Selected from the group consisting of cycloalkyl and 3- to 5-membered heterocyclines; [D0] Ring A is a ring selected from the group consisting of pyrrole, furan, thiophene, imidazole, pyrazole, oxazole, isoxazole, thiazole, isothiazole, and triazole; [E0] Each R 4 If present, independently, C 1~6 Alkyl, C 1~6 Haloalkyl, C 1~6 Alkoxy, C 1~6 Haloalkoxy, cyano-C 1~6 Alkyl, halogen, -OH, -NH2, -NH(C 1~4 Alkyl), -N(C 1~4 Alkyl)2,-CN,C 3~5 Selected from the group consisting of cycloalkyl and 3-5 membered heterocyclines, p is selected from the group consisting of 0, 1, 2, and 3; [F0] U is nitrogen (=N-) and R A The carbon that is replaced by (=C(R A Selected from the group consisting of )-), V is nitrogen (=N-) and R B The carbon that is replaced by (=C(RB )-) selected from the group consisting of W is nitrogen (=N-) and R C carbon (=C(R C )-) selected from the group consisting of R A , R B and R C are each independently hydrogen, C 1~6 haloalkyl, C 3~5 cycloalkyl optionally substituted by C 2~6 alkynyl, C 1~6 alkoxy, C 1~6 haloalkoxy, halogen, -CN, -OH, -NH2, -NH(C 1~4 alkyl), -N(C 1~4 alkyl)2, -C(=O)NH2, -C(=O)NH(C 1~4 alkyl), -C(=O)N(C 1~4 alkyl)2, -S-C 1~6 alkyl, -S(=O)2-C 1~6 alkyl, C 3~5 cycloalkyl, 3-5 membered heterocyclyl, and C 1~6 alkoxy, -CN, -OH, -NH2, -NH(C 1~4 alkyl), -N(C 1~4 alkyl)2, -C(=O)NH2, -C(=O)NH(C 1~4 alkyl) and -C(=O)N(C 1~4 alkyl)2 selected from the group consisting of substituents optionally substituted by 1~6 alkyl selected from the group consisting of; [G0] R 5 is selected from the group consisting of R a1 and R b1 and is selected from the group consisting of R a1 is C 1~6 alkyl, C 1~6 haloalkyl, C 2~6 alkenyl, C 2~6 alkynyl, C 3~10 cycloalkyl, C 4~10 cycloalkenyl, 3-11 membered heterocyclyl, C 6~10Selected from the group consisting of aryls and 5-10 membered heteroaryls, C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenil, C 2~6 Alkinyl, C 3~10 Cycloalkyl, C 4~10 Cycloalkenyl, 3-11 member heterocyclyl, C 6~10 All aryls and 5-10 member heteroaryls are one or more identical or different R b1 and / or R c1 Depending on the circumstances, it may be replaced by Each R b1 These are, independently, -OR c1 , -NR c1 R c1 , halogen, -CN, -C(=O)R c1 , -C(=O)OR c1 -C(=O)NR c1 R c1 -S(=O)2R c1 -S(=O)2NR c1 R c1 , -NHC(=O)R c1 , -N(C 1~4 Alkyl)C(=O)R c1 , -NHS(=O)2R c1 , -N(C 1~4 Alkyl)S(=O)2R c1 , -NHC(=O)OR c1 , -N(C 1~4 Alkyl)C(=O)OR c1 And selected from the group consisting of divalent substituents = O, Each R c1 These are, independently, hydrogen and C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenil, C 2~6 Alkinyl, C 3~10 Cycloalkyl, C 4~10 Cycloalkenyl, 3-11 member heterocyclyl, C 6~10 Selected from the group consisting of aryls and 5-10 membered heteroaryls, C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenil, C2~6 Alkinyl, C 3~10 Cycloalkyl, C 4~10 Cycloalkenyl, 3-11 member heterocyclyl, C 6~10 All aryls and 5-10 member heteroaryls are one or more identical or different R d1 and / or R e1 Depending on the circumstances, it may be replaced by Each R d1 These are, independently, -OR e1 , -NR e1 R e1 , halogen, -CN, -C(=O)R e1 , -C(=O)OR e1 -C(=O)NR e1 R e1 -S(=O)2R e1 -S(=O)2NR e1 R e1 , -NHC(=O)R e1 , -N(C 1~4 Alkyl)C(=O)R e1 , -NHS(=O)2R c1 , -N(C 1~4 Alkyl)S(=O)2R c1 , -NHC(=O)OR e1 , -N(C 1~4 Alkyl)C(=O)OR e1 And selected from the group consisting of divalent substituents = O, Each R e1 These are, independently, hydrogen and C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenil, C 2~6 Alkinyl, C 3~10 Cycloalkyl, C 4~10 Cycloalkenyl, 3-11 member heterocyclyl, C 6~10 Selected from the group consisting of aryls and 5-10 membered heteroaryls, C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenil, C 2~6 Alkinyl, C 3~10 Cycloalkyl, C 4~10 Cycloalkenyl, 3-11 member heterocyclyl, C 6~10All aryl and 5-10 membered heteroaryls are C 1~6 Alkyl, C 1~6 Haloalkyl, C 3~10 Cycloalkyl, one or more identical or different C 1~4 3-11 member heterocyclines, optionally substituted with alkyl groups, C 6~10 Aryl, 5-10 member heteroaryl, -OH, C 1~6 Alkoxy, C 1~4 Alkoxy-C 1~4 Alkyl, hydroxy-C 1~4 Alkyl, halogen, -CN, -NH2, -C(=O)C 1~4 Alkyl, -NH(C 1~4 Alkyl), -N(C 1~4 Optionally substituted by one or more identical or different substituents selected from the group consisting of alkyl)2 and divalent substituents = O; [H0] L is -L 1 -L 2 -L 3 - and L 1 It is connected to E, L 1 is a bond, -NH-, -N(C 1~4 Alkyl)-, -O-, -C(=O)-, -NH-C(=O)-, -N(C 1~4 Alkyl)-C(=O)-, -C(=O)-NH-, -C(=O)-N(C 1~4 Alkyl)-, -C(=O)-, C 1~6 Alkilen, C 3~7 Selected from the group consisting of cycloalkylene, phenylene, 4-12 member heterocyclylene, and 5-10 member heteroarylene, L 2 C 1~6 Alkilen, C 3~7 Selected from the group consisting of cycloalkylene, phenylene, 4-12 member heterocyclylene, and 5-10 member heteroarylene, L 3 is a bond, -NH-, -N(C 1~4 Alkyl)-, -O-, -C(=O)-, -NH-C(=O)-, -N(C 1~4Alkyl)-C(=O)-, -C(=O)-NH-, -C(=O)-N(C 1~4 Alkyl)-, -C(=O)-, C 1~6 Alkilen, C 3~7 Selected from the group consisting of cycloalkylene, phenylene, 4-12 member heterocyclylene, and 5-10 member heteroarylene, Here, L 1 , L 2 and L 3 Each C 1~6 Alkilen, C 3~7 Cycloalkylenes, phenylenes, 4-12 member heterocyclylenes, and 5-10 member heteroarylenes are, optionally and independently, C 2~6 Alkinyl, C 1~6 Haloalkyl, C 3~7 Cycloalkyl, phenyl, 5-6 member heteroaryl, halogen, -OH, -CN, C 1~6 Alkoxy, -NH2, -NH(C 1~4 Alkyl), -N(C 1~4 Alkyl)2, -C(=O)OH, -C(=O)-OC 1~6 Alkyl, -C(=O)NH2, -C(=O)NH(C 1~4 Alkyl), -C(=O)N(C 1~4 Alkyl)2, divalent substituents = O, and halogens, -OH, -CN, C 1~4 Alkoxy, -NH2, -NH(C 1~4 Alkyl), -N(C 1~4 Alkyl)2, -C(=O)OH, -C(=O)-OC 1~6 Alkyl, -C(=O)NH2, -C(=O)NH(C 1~4 Alkyl) and -C(=O)N(C 1~4 C is optionally substituted by one or more identical or different substituents selected from the group consisting of alkyl)2. 1~6 Substituted by one or more identical or different substituents selected from the group consisting of alkyl groups; [I0] E is [ka] And, [ka] This represents a double bond or a triple bond. Q 1 The bonds are -CH2-, -CH(OH)-, -C(=O)-, and -C(=O)N(R). G1 )-, -C(=O)O-, -S(=O)2-, -S(=O)2N(R G1 )- and -C(=NR H1 Selected from the group consisting of )-, Each R G1 These are, independently, hydrogen and C 1~6 Alkyl, C 1~6 Haloalkyl, hydroxy-C 1~6 Alkyl, H2N-C 1~6 Alkyl, cyano-C 1~6 Alkyl, (C 1~4 Alkyl)HN-C 1~6 Alkyl, (C 1~4 Alkyl)2N-C 1~6 Alkyl, C 1~6 Alkoxy-C 1~6 Alkyl, C 3~7 Selected from the group consisting of cycloalkyl and 3-11 membered heterocyclyl, Each R H1 These are independently hydrogen, -OH, and C. 1~6 Alkoxy, -CN, and C 1~6 Selected from the group consisting of alkyl groups, [ka] When represents a double bond, R D is hydrogen, C 3~7 Cycloalkyl, phenyl, halogen, -CN, C 1~6 Alkoxy, -C(=O)OC 1~6 Alkyl, -NHC(=O)-C 1~6 Alkyl, as well as phenyl, 3-11 member heterocyclyl, C 1~6 Alkyl, halogen, -OH, -NH2, -NH(C) 1~6Alkyl), -N(C 1~6 Alkyl)2, -C(=O)OH, -C(=O)OC 1~6 Alkyl, -C(=O)NH(C 1~6 Alkyl), -NHC(=O)-C 1~6 Alkyl, -OC(=O)-C 1~6 Alkyl and phenyl-C 1~6 C is optionally substituted by one or more identical or different substituents selected from the group consisting of alkoxys. 1~6 Selected from the group consisting of alkyl groups, R E and R F Each of them operates independently, R a2 and R b2 Selected from the group consisting of, R a2 is hydrogen, C 1~6 Alkyl, C 1~6 Haloalkyl, C 3~10 Cycloalkyl, 3-11 member heterocyclyl, C 6~10 Selected from the group consisting of aryls and 5-10 membered heteroaryls, C 1~6 Alkyl, C 1~6 Haloalkyl, C 3~10 Cycloalkyl, 3-11 member heterocyclyl, C 6~10 All aryls and 5-10 member heteroaryls are one or more identical or different R b2 and / or R c2 Depending on the circumstances, it may be replaced by Each R b2 These are, independently, -OR c2 , -NR c2 R c2 , halogen, -CN, -C(=O)R c2 , -C(=O)OR c2 -C(=O)NR c2 R c2 -S(=O)2R c2 -S(=O)2NR c2 R c2 , -NHC(=O)R c2 , -N(C 1~4 Alkyl)C(=O)R c2 , -NHC(=O)OR c2 , -N(C1~4 Alkyl)C(=O)OR c2 And selected from the group consisting of divalent substituents = O, Each R c2 These are, independently, hydrogen and C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenil, C 2~6 Alkinyl, C 3~10 Cycloalkyl, C 4~10 Cycloalkenyl, 3-11 member heterocyclyl, C 6~10 Selected from the group consisting of aryls and 5-10 membered heteroaryls, C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenil, C 2~6 Alkinyl, C 3~10 Cycloalkyl, C 4~10 Cycloalkenyl, 3-11 member heterocyclyl, C 6~10 All aryl and 5-10 membered heteroaryls are C 1~6 Alkyl, C 1~6 Alkoxy, halogen, -OH, -C(=O)OH, -C(=O)OC 1~6 Alkyl, -C(=O)C 1~6 Alkyl, -C(=O)NH2, -C(=O)NH(C 1~6 Alkyl), -C(=O)N(C 1~6 It may be substituted by one or more identical or different substituents selected from the group consisting of alkyl)2 and divalent substituents = O, or R D and R E These, together with the carbon atoms to which they are bonded, form a 4-7 membered unsaturated alicyclic ring or a 4-7 membered unsaturated heterocyclic ring, and this 4-7 membered unsaturated alicyclic ring or 4-7 membered unsaturated heterocyclic ring may, in some cases, R F In addition, C 1~6 Alkyl, C 1~6 Haloalkyl, -OH, C 1~6 Alkoxy, C 1~4 Alkoxy-C 1~4 Alkyl, -NH2, -CN, -NH(C 1~4 Alkyl), -N(C 1~4Alkyl)2, halogen, -C(=O)OC 1~6 It is substituted with one or more identical or different substituents selected from the group consisting of alkyl and divalent substituents = O, or Q 1 -C(=O)N(R G1 )- If -C(=O)N(R G1 )- of R G1 and R F Together, they form a linker selected from the group consisting of -C(=O)-, -CH2-, -CH2-C(=O)-, -C(=O)-CH2-, and -C2H4-. [ka] When this represents a triple bond, R D and R E Both are nonexistent, R F is R a2 And, R a2 is hydrogen, C 1~6 Alkyl, C 1~6 Haloalkyl, C 3~10 Cycloalkyl, 3-11 member heterocyclyl, C 6~10 Selected from the group consisting of aryls and 5-10 membered heteroaryls, C 1~6 Alkyl, C 1~6 Haloalkyl, C 3~10 Cycloalkyl, 3-11 member heterocyclyl, C 6~10 All aryls and 5-10 member heteroaryls are one or more identical or different R b2 and / or R c2 Depending on the circumstances, it may be replaced by Each R b2 These are, independently, -OR c2 , -NR c2 R c2 , halogen, -CN, -C(=O)R c2 , -C(=O)OR c2 -C(=O)NR c2 R c2 -S(=O)2Rc2 -S(=O)2NR c2 R c2 , -NHC(=O)R c2 , -N(C 1~4 Alkyl)C(=O)R c2 , -NHC(=O)OR c2 , -N(C 1~4 Alkyl)C(=O)OR c2 And selected from the group consisting of divalent substituents = O, Each R c2 These are, independently, hydrogen and C 1~6 Alkyl, C 1~6 Haloalkyl, C 3~10 Cycloalkyl, 3-11 member heterocyclyl, C 6~10 Selected from the group consisting of aryls and 5-10 membered heteroaryls, or E is [ka] And, Q 2 The bonds are -CH2-, -CH(OH)-, -C(=O)-, and -C(=O)N(R). G2 )-, -C(=O)O-, -S(=O)2-, -S(=O)2N(R G2 )- and -C(=NR H2 Selected from the group consisting of )-, Each R G2 These are, independently, hydrogen and C 1~6 Alkyl, C 1~6 Haloalkyl, hydroxy-C 1~6 Alkyl, H2N-C 1~6 Alkyl, cyano-C 1~6 Alkyl, (C 1~4 Alkyl)HN-C 1~6 Alkyl, (C 1~4 Alkyl)2N-C 1~6 Alkyl, C 1~6 Alkoxy-C 1~6 Alkyl, C 3~7 Selected from the group consisting of cycloalkyl and 3-11 membered heterocyclyl, Each R H2 These are independently hydrogen, -OH, and C.1~6 Alkoxy, -CN, and C 1~6 Selected from the group consisting of alkyl groups, R I It is selected from the group consisting of hydrogen and halogens, R J is hydrogen, or R I and R J Together with the carbon atoms to which they are bonded, they form a cyclopropane or oxirane ring. R K is hydrogen, C 1~6 Selected from the group consisting of alkyl, -CN, and halogen, R L is hydrogen, C 1~6 Alkyl, -CN, halogen, and -C(=O)-C 1~6 Selected from the group consisting of alkyl groups, or E is [ka] And, Q 3 -C(=O)-, -C(=O)N(R G3 )-, -C(=O)O-, -S(=O)2-, -S(=O)2N(R G3 )- and -C(=NR H3 Selected from the group consisting of )-, Each R G3 These are, independently, hydrogen and C 1~6 Alkyl, C 1~6 Haloalkyl, hydroxy-C 1~6 Alkyl, H2N-C 1~6 Alkyl, cyano-C 1~6 Alkyl, (C 1~4 Alkyl)HN-C 1~6 Alkyl, (C 1~4 Alkyl)2N-C 1~6 Alkyl, C 1~6 Alkoxy-C 1~6 Alkyl, C 3~7 Selected from the group consisting of cycloalkyl and 3-11 membered heterocyclyl, Each R H3These are independently hydrogen, -OH, and C. 1~6 Alkoxy, -CN, and C 1~6 Selected from the group consisting of alkyl groups, R M These are halogens, -CN and -OC(=O)-C 1~6 Selected from the group consisting of alkyl groups, or E is [ka] And, Q 4 is a bond, -C(=O)-, -C(=O)O-, -C(=O)NH-, -C(=O)N(C 1~4 Selected from the group consisting of alkyl)-, -S(=O)2-, and -S(=O)2NH-, Ring B is selected from the group consisting of phenyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, and 5-membered heteroaryl compounds. q is selected from the group consisting of 1, 2, 3, and 4. Each R N Independently, C 1~4 Alkyl, C 1~4 Haloalkyl, vinyl, ethynyl, halogen, -CN, nitro and C 1~4 Selected from the group consisting of alkoxys. It comprises cyclic 2-amino-3-cyanothiophene and its derivatives, or salts thereof.

[0158] Furthermore, the KRAS inhibitor is preferably of formula (B) [ka] {In the formula, R 1a and R 1b Both are independent of hydrogen and C 1~4 Alkyl, C 1~4 Haloalkyl, C 1~4 Alkoxy, C 1~4 Haloalkoxy, halogen, -NH2, -NH(C) 1~4 Alkyl), -N(C1~4 Alkyl)2, C 3~5 Selected from the group consisting of cycloalkyl and 3-5 membered heterocyclines, R 2a and R 2b Both are independent of hydrogen and C 1~4 Alkyl, C 1~4 Haloalkyl, C 1~4 Alkoxy, C 1~4 Haloalkoxy, halogen, -NH2, -NH(C) 1~4 Alkyl), -N(C 1~4 Alkyl)2, C 3~5 Selected from the group consisting of cycloalkyl and 3-5 membered heterocyclines, And / or depending on the circumstances, R 1a Or R 1b one of the and R 2a Or R 2b One of them, together with the carbon atoms to which they are bonded, forms a cyclopropane ring. Z is -(CR 6a R 6b ) n -and, Each R 6a and R 6b These are, independently, hydrogen and C 1~4 Alkyl, C 1~4 Haloalkyl, C 1~4 Alkoxy, C 1~4 Haloalkoxy, halogen, -NH2, -NH(C) 1~4 Alkyl), -N(C 1~4 Alkyl)2, C 3~5 Selected from the group consisting of cycloalkyls and 3- to 5-membered heterocyclines, Or R 6a and R 6b Together with the carbon atoms to which they are bonded, they form a cyclopropane ring. n is selected from the group consisting of 0, 1, and 2. -L- is a bond, or -O-, -S- and -N(R 13 )- Selected from, R 13 is hydrogen or C 1~6 It is alkyl, R 3is replaced by E, -L- is -O-, -S- and -N(R 13 )- If selected from, R 3 C 1~6 Alkyl, C 1~6 Selected from the group consisting of alkoxys, 5-10 membered heteroaryls, and 3-11 membered heterocyclines, C 1~6 Alkyl, 5-10 member heteroaryl, C 1~6 Alkoxy and 3- to 11-membered heterocyclines are all optionally and independently halogenated with C 1~6 Alkyl, -OH, -NH2, -NH(C 1~4 Alkyl), -N(C 1~4 Alkyl)2, C 3~5 Substituted with one or more identical or different substituents selected from the group consisting of cycloalkyl and 3- to 11-membered heterocyclines, If -L- is a bond, then R 3 C 1~6 Alkyl, C 1~6 Haloalkyl, C 3~10 Cycloalkyl, 3-11 member heterocyclyl, C 6~10 Selected from the group consisting of aryls and 5-10 membered heteroaryls, C 1~6 Alkyl, C 1~6 Haloalkyl, C 3~10 Cycloalkyl, 3-11 member heterocyclyl, C 6~10 All aryls and 5- to 10-membered heteroaryls may and independently be one or more identical or different R 7 and / or R 8 Replaced by, Each R 7 These are, independently, halogen, -CN, -OH, and C 1~6 Alkoxy, -NR 8 R 8 -C(=O)R 8 , -C(=O)OR 8 -C(=O)NR 8 R 8 , -NHC(=O)OR 8 And selected from the group consisting of divalent substituents = O, Each R 8These are, independently, hydrogen and C 1~6 Alkyl, C 3~10 Selected from the group consisting of cycloalkyl, 3-11 member heterocyclyl, phenyl, and 5-10 member heteroaryl, C 1~6 Alkyl, C 3~10 Cycloalkyls, 3-11 membered heterocyclyls, phenyls, and 5-10 membered heteroaryls all have one or more identical or different R 9 and / or R 10 Depending on the circumstances, it may be replaced by Each R 9 These are, independently, -OR 10 And, Each R 10 These are, independently, hydrogen and C 1~6 Selected from the group consisting of alkyl, 3-11 membered heterocyclyl, and 5-10 membered heteroaryl, W is nitrogen (-N=) or -CH=, V is nitrogen (-N=) or -CH=, U is nitrogen (-N=) or -C(R 11 )= and R 11 It consists of hydrogen, halogens and C 1~4 Selected from alkoxy, Ring A is a ring selected from the group consisting of pyrrole, furan, thiophene, imidazole, pyrazole, isoxazole, isothiazole, and triazole. Each R 4 If present, independently, C 1~6 Alkyl, C 1~6 Haloalkyl, C 1~6 Alkoxy, C 1~6 Haloalkoxy, cyano-C 1~6 Alkyl, halogen, -OH, -NH2, -NH(C 1~4 Alkyl), -N(C 1~4 Alkyl)2,-CN,C 3~5 Selected from the group consisting of cycloalkyl and 3-5 membered heterocyclines, p is selected from the group consisting of 0, 1, 2, and 3. R 5 This is one or more identical or different C1~6 Alkyl, C 1~6 A 3-11 member heterocycline that may be substituted with an alkoxy or a 5-6 member heterocycline, C 1~6 Alkyl is optionally substituted with cyclopropyl, Or R 5 -OC is substituted by 3-11 member heterocyclyl groups. 1~6 Alkyl, 3-11 member heterocyclyl, one or more identical or different R 12 Depending on the circumstances, it may be replaced by Each R 12 C 1~6 Alkyl, C 1~6 Selected from the group consisting of alkoxys, halogens, and 3- to 11-membered heterocyclines, E is [ka] And, [ka] This represents a double bond or a triple bond. Q 1 The bonds are -CH2-, -CH(OH)-, -C(=O)-, and -C(=O)N(R). G1 )-, -C(=O)O-, -S(=O)2-, -S(=O)2N(R G1 )- and -C(=NR H1 Selected from the group consisting of )-, Each R G1 These are, independently, hydrogen and C 1~6 Alkyl, C 1~6 Haloalkyl, hydroxy-C 1~6 Alkyl, H2N-C 1~6 Alkyl, cyano-C 1~6 Alkyl, (C 1~4 Alkyl)HN-C 1~6 Alkyl, (C 1~4 Alkyl)2N-C 1~6 Alkyl, C 1~6 Alkoxy-C 1~6 Alkyl, C 3~7Selected from the group consisting of cycloalkyl and 3-11 membered heterocyclyl, Each R H1 These are independently hydrogen, -OH, and C. 1~6 Alkoxy, -CN, and C 1~6 Selected from the group consisting of alkyl groups, [ka] When represents a double bond, R D is hydrogen, C 3~7 Cycloalkyl, phenyl, halogen, -CN, C 1~6 Alkoxy, -C(=O)OC 1~6 Alkyl, -NHC(=O)-C 1~6 Alkyl, as well as phenyl, 3-11 member heterocyclyl, C 1~6 Alkyl, halogen, -OH, -NH2, -NH(C) 1~6 Alkyl), -N(C 1~6 Alkyl)2, -C(=O)OH, -C(=O)OC 1~6 Alkyl, -C(=O)NH(C 1~6 Alkyl), -NHC(=O)-C 1~6 Alkyl, -OC(=O)-C 1~6 Alkyl and phenyl-C 1~6 C is optionally substituted by one or more identical or different substituents selected from the group consisting of alkoxys. 1~6 Selected from the group consisting of alkyl groups, R E and R F Each of them operates independently, R a2 and R b2 Selected from the group consisting of, R a2 is hydrogen, C 1~6 Alkyl, C 1~6 Haloalkyl, C 3~10 Cycloalkyl, 3-11 member heterocyclyl, C 6~10 Selected from the group consisting of aryls and 5-10 membered heteroaryls, C 1~6 Alkyl, C 1~6 Haloalkyl, C 3~10Cycloalkyl, 3-11 member heterocyclyl, C 6~10 All aryls and 5-10 member heteroaryls are one or more identical or different R b2 and / or R c2 Depending on the circumstances, it may be replaced by Each R b2 These are, independently, -OR c2 , -NR c2 R c2 , halogen, -CN, -C(=O)R c2 , -C(=O)OR c2 -C(=O)NR c2 R c2 -S(=O)2R c2 -S(=O)2NR c2 R c2 , -NHC(=O)R c2 , -N(C 1~4 Alkyl)C(=O)R c2 , -NHC(=O)OR c2 , -N(C 1~4 Alkyl)C(=O)OR c2 And selected from the group consisting of divalent substituents = O, Each R c2 These are, independently, hydrogen and C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenil, C 2~6 Alkinyl, C 3~10 Cycloalkyl, C 4~10 Cycloalkenyl, 3-11 member heterocyclyl, C 6~10 Selected from the group consisting of aryls and 5-10 membered heteroaryls, C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenil, C 2~6 Alkinyl, C 3~10 Cycloalkyl, C 4~10 Cycloalkenyl, 3-11 member heterocyclyl, C 6~10 All aryl and 5-10 membered heteroaryls are C 1~6 Alkyl, C 1~6 Alkoxy, halogen, -OH, -C(=O)OH, -C(=O)OC 1~6 Alkyl, -C(=O)C 1~6Alkyl, -C(=O)NH2, -C(=O)NH(C 1~6 Alkyl), -C(=O)N(C 1~6 It may be substituted by one or more identical or different substituents selected from the group consisting of alkyl)2 and divalent substituents = O, or R D and R E These, together with the carbon atoms to which they are bonded, form a 4-7 membered unsaturated alicyclic ring or a 4-7 membered unsaturated heterocyclic ring, and this 4-7 membered unsaturated alicyclic ring or 4-7 membered unsaturated heterocyclic ring may, in some cases, R F In addition, C 1~6 Alkyl, C 1~6 Haloalkyl, -OH, C 1~6 Alkoxy, C 1~4 Alkoxy-C 1~4 Alkyl, -NH2, -CN, -NH(C 1~4 Alkyl), -N(C 1~4 Alkyl)2, halogen, -C(=O)OC 1~6 It is substituted with one or more identical or different substituents selected from the group consisting of alkyl and divalent substituents = O, or Q 1 -C(=O)N(R G1 )- If -C(=O)N(R G1 )- of R G1 and R F Together, they form a linker selected from the group consisting of -C(=O)-, -CH2-, -CH2-C(=O)-, -C(=O)-CH2-, and -C2H4-. [ka] When this represents a triple bond, R D and R E Both are nonexistent, R F is R a2 And, R a2 is hydrogen, C 1~6 Alkyl, C 1~6Haloalkyl, C 3~10 Cycloalkyl, 3-11 member heterocyclyl, C 6~10 Selected from the group consisting of aryls and 5-10 membered heteroaryls, C 1~6 Alkyl, C 1~6 Haloalkyl, C 3~10 Cycloalkyl, 3-11 member heterocyclyl, C 6~10 All aryls and 5-10 member heteroaryls are one or more identical or different R b2 and / or R c2 Depending on the circumstances, it may be replaced by Each R b2 These are, independently, -OR c2 , -NR c2 R c2 , halogen, -CN, -C(=O)R c2 , -C(=O)OR c2 -C(=O)NR c2 R c2 -S(=O)2R c2 -S(=O)2NR c2 R c2 , -NHC(=O)R c2 , -N(C 1~4 Alkyl)C(=O)R c2 , -NHC(=O)OR c2 , -N(C 1~4 Alkyl)C(=O)OR c2 And selected from the group consisting of divalent substituents = O, Each R c2 These are, independently, hydrogen and C 1~6 Alkyl, C 1~6 Haloalkyl, C 3~10 Cycloalkyl, 3-11 member heterocyclyl, C 6~10 Selected from the group consisting of aryls and 5-10 membered heteroaryls, or E is [ka] And, Q 2 The bonds are -CH2-, -CH(OH)-, -C(=O)-, and -C(=O)N(R). G2)-, -C(=O)O-, -S(=O)2-, -S(=O)2N(R G2 )- and -C(=NR H2 Selected from the group consisting of )-, Each R G2 These are, independently, hydrogen and C 1~6 Alkyl, C 1~6 Haloalkyl, hydroxy-C 1~6 Alkyl, H2N-C 1~6 Alkyl, cyano-C 1~6 Alkyl, (C 1~4 Alkyl)HN-C 1~6 Alkyl, (C 1~4 Alkyl)2N-C 1~6 Alkyl, C 1~6 Alkoxy-C 1~6 Alkyl, C 3~7 Selected from the group consisting of cycloalkyl and 3-11 membered heterocyclyl, Each R H2 These are independently hydrogen, -OH, and C. 1~6 Alkoxy, -CN, and C 1~6 Selected from the group consisting of alkyl groups, R I It is selected from the group consisting of hydrogen and halogens, R J is hydrogen, or R I and R J Together with the carbon atoms to which they are bonded, they form a cyclopropane or oxirane ring. R K is hydrogen, C 1~6 Selected from the group consisting of alkyl, -CN, and halogen, R L is hydrogen, C 1~6 Alkyl, -CN, halogen, and -C(=O)-C 1~6 Selected from the group consisting of alkyl groups, or E is [ka] And, Q 3-C(=O)-, -C(=O)N(R G3 )-, -C(=O)O-, -S(=O)2-, -S(=O)2N(R G3 )- and -C(=NR H3 Selected from the group consisting of )-, Each R G3 These are, independently, hydrogen and C 1~6 Alkyl, C 1~6 Haloalkyl, hydroxy-C 1~6 Alkyl, H2N-C 1~6 Alkyl, cyano-C 1~6 Alkyl, (C 1~4 Alkyl)HN-C 1~6 Alkyl, (C 1~4 Alkyl)2N-C 1~6 Alkyl, C 1~6 Alkoxy-C 1~6 Alkyl, C 3~7 Selected from the group consisting of cycloalkyl and 3-11 membered heterocyclyl, Each R H3 These are independently hydrogen, -OH, and C. 1~6 Alkoxy, -CN, and C 1~6 Selected from the group consisting of alkyl groups, R M These are halogens, -CN and -OC(=O)-C 1~6 Selected from the group consisting of alkyl groups, or E is [ka] And, Q 4 is a bond, -C(=O)-, -C(=O)O-, -C(=O)NH-, -C(=O)N(C 1~4 Selected from the group consisting of alkyl)-, -S(=O)2-, and -S(=O)2NH-, Ring B is selected from the group consisting of phenyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, and 5-membered heteroaryl compounds. q is selected from the group consisting of 1, 2, 3, and 4. Each R N Independently, C 1~4Alkyl, C 1~4 Haloalkyl, vinyl, ethynyl, halogen, -CN, nitro and C 1~4 Selected from the group consisting of alkoxys. It contains compounds of or salts thereof.

[0159] Furthermore, the KRAS inhibitor is preferably of formula (C) [ka] {In the formula, R 1a and R 1b Both are independent of hydrogen and C 1~4 Alkyl, C 1~4 Haloalkyl, C 1~4 Alkoxy, C 1~4 Haloalkoxy, halogen, -NH2, -NH(C) 1~4 Alkyl), -N(C 1~4 Alkyl)2, C 3~5 Selected from the group consisting of cycloalkyl and 3-5 membered heterocyclines, R 2a and R 2b Both are independent of hydrogen and C 1~4 Alkyl, C 1~4 Haloalkyl, C 1~4 Alkoxy, C 1~4 Haloalkoxy, halogen, -NH2, -NH(C) 1~4 Alkyl), -N(C 1~4 Alkyl)2, C 3~5 Selected from the group consisting of cycloalkyl and 3-5 membered heterocyclines, And / or depending on the circumstances, R 1a Or R 1b one of the and R 2a Or R 2b One of them, together with the carbon atoms to which they are bonded, forms a cyclopropane ring. Z is -(CR 6a R 6b ) n -and, Each R 6a and R 6b These are, independently, hydrogen and C1~4 Alkyl, C 1~4 Haloalkyl, C 1~4 Alkoxy, C 1~4 Haloalkoxy, halogen, -NH2, -NH(C) 1~4 Alkyl), -N(C 1~4 Alkyl)2, C 3~5 Selected from the group consisting of cycloalkyls and 3- to 5-membered heterocyclines, Or R 6a and R 6b Together with the carbon atoms to which they are bonded, they form a cyclopropane ring. n is selected from the group consisting of 0, 1, and 2. R 3 is halogen, C 1~6 Alkyl, C 1~6 Haloalkyl, -N3, C 3~10 Cycloalkyl, 3-11 member heterocyclyl, C 6~10 Selected from the group consisting of aryls and 5-10 membered heteroaryls, C 1~6 Alkyl, C 1~6 Haloalkyl, C 3~10 Cycloalkyl, 3-11 member heterocyclyl, C 6~10 All aryls and 5- to 10-membered heteroaryls may and independently be one or more identical or different R 7 and / or R 8 Replaced by, Each R 7 These are, independently, halogen, -CN, and -OR. 8 , -NR 8 R 8 -C(=O)R 8 , -C(=O)OR 8 -C(=O)NR 8 R 8 , -NHC(=O)OR 8 And selected from the group consisting of divalent substituents = O, Each R 8 These are, independently, hydrogen and C 1~6 Alkyl, C 3~10 Cycloalkyl, 3-11 member heterocyclyl, C 6~10 Selected from the group consisting of aryls and 5-10 membered heteroaryls, C 1~6Alkyl, C 3~10 Cycloalkyl, 3-11 member heterocyclyl, C 6~10 All aryls and 5-10 member heteroaryls are one or more identical or different R 9 and / or R 10 Depending on the circumstances, it may be replaced by Each R 9 These are, independently, -OR 10 , -NR 10 R 10 and -C(O)NR 10 R 10 Selected from the group consisting of, Each R 10 These are, independently, hydrogen and C 1~6 Alkyl, C 3~10 Selected from the group consisting of cycloalkyl, 3-11 membered heterocyclyl, and 5-10 membered heteroaryl, C 1~6 Alkyl is C 1~6 Alkoxy, C 3~10 Cycloalkyl, and C 1~6 Optionally substituted with substituents selected from the group consisting of 3- to 11-membered heterocyclines that are optionally substituted with alkyl groups, W is nitrogen (-N=) or -CH=, V is nitrogen (-N=) or -CH=, U is nitrogen (-N=) or -C(R 11 )= and R 11 It consists of hydrogen, halogens and C 1~4 Selected from alkoxy, Ring A is a ring selected from the group consisting of pyrrole, furan, thiophene, imidazole, pyrazole, oxazole, isoxazole, thiazole, isothiazole, and triazole. Each R 4 If present, independently, C 1~6 Alkyl, C 1~6 Haloalkyl, C 1~6 Alkoxy, C 1~6 Haloalkoxy, cyano-C 1~6 Alkyl, halogen, -OH, -NH2, -NH(C 1~4 Alkyl), -N(C1~4 Alkyl)2,-CN,C 3~5 Selected from the group consisting of cycloalkyl and 3-5 membered heterocyclines, p is selected from the group consisting of 0, 1, 2, and 3. R 5 This is one or more identical or different C 1~6 Alkyl, C 1~6 A 3-11 member heterocycline that may be substituted with an alkoxy or a 5-6 member heterocycline, C 1~6 Alkyl is optionally substituted with cyclopropyl, Or R 5 -OC is substituted by 3-11 member heterocyclyl groups. 1~6 Alkyl, 3-11 member heterocyclyl, one or more identical or different R 12 Depending on the circumstances, it may be replaced by Each R 12 C 1~6 Alkyl, C 1~6 Selected from the group consisting of alkoxys, halogens, and 3- to 11-membered heterocyclines. It contains compounds of or salts thereof.

[0160] Furthermore, the KRAS inhibitor is preferably of formula (D) [ka] {In the formula, R 1a and R 1b Both are independent of hydrogen and C 1~4 Alkyl, C 1~4 Haloalkyl, C 1~4 Alkoxy, C 1~4 Haloalkoxy, halogen, -NH2, -NH(C) 1~4 Alkyl), -N(C 1~4 Alkyl)2, C 3~5 Selected from the group consisting of cycloalkyl and 3- to 5-membered heterocycloalkyl groups, R 2a and R 2b Both are independent of hydrogen and C 1~4Alkyl, C 1~4 Haloalkyl, C 1~4 Alkoxy, C 1~4 Haloalkoxy, halogen, -NH2, -NH(C) 1~4 Alkyl), -N(C 1~4 Alkyl)2, C 3~5 Selected from the group consisting of cycloalkyl and 3- to 5-membered heterocycloalkyl groups, And / or depending on the circumstances, R 1a Or R 1b one of the and R 2a Or R 2b One of them, together with the carbon atoms to which they are bonded, forms a cyclopropane ring. Z is -(CR 3a R 3b ) n -and, Each R 3a and R 3b These are, independently, hydrogen and C 1~4 Alkyl, C 1~4 Haloalkyl, C 1~4 Alkoxy, C 1~4 Haloalkoxy, halogen, -NH2, -NH(C) 1~4 Alkyl), -N(C 1~4 Alkyl)2, C 3~5 Selected from the group consisting of cycloalkyl and 3- to 5-membered heterocycloalkyl groups, Or R 3a and R 3b Together with the carbon atoms to which they are bonded, they form a cyclopropane ring. n is selected from the group consisting of 0, 1, and 2. R 4 is hydrogen, C 1~6 Alkyl, C 1~6 Haloalkyl, C 1~6 Alkoxy, C 1~6 Haloalkoxy, cyano-C 1~6 Alkyl, halogen, -OH, -NH2, -NH(C 1~4 Alkyl), -N(C 1~4 Alkyl)2,-CN,C 3~5 Selected from the group consisting of cycloalkyl and 3- to 5-membered heterocycloalkyl groups, Ring A is a 5-membered heteroarylene, Each R 5 If present, independently, C 1~6 Alkyl, C 1~6 Haloalkyl, C 1~6 Alkoxy, C 1~6 Haloalkoxy, cyano-C 1~6 Alkyl, halogen, -OH, -NH2, -NH(C 1~4 Alkyl), -N(C 1~4 Alkyl)2,-CN,C 3~5 Selected from the group consisting of cycloalkyl and 3- to 5-membered heterocycloalkyl groups, m is selected from the group consisting of 0, 1, 2, and 3. W is nitrogen (-N=) or -CH=, V is nitrogen (-N=) or -CH=, U is nitrogen (-N=) or -C(R 11 )= and R 11 It consists of hydrogen, halogens and C 1~4 Selected from alkoxy, Ring B contains one or more identical or different C 1~6 Alkyl, C 1~6 A 3-11 member heterocycloalkylene that is optionally substituted with an alkoxy or a 5-6 member heterocycloalkyl, C 1~6 Alkyl groups are optionally substituted with cyclopropyl groups. L is bond, C 1~8 Alkilen, C 2~8 Alkenylene, C 2~8 Alkynylene and C 1~8 Selected from the group consisting of alkoxylenes, X is either -(CH2)- or -O-, Y is a 5-membered heteroarylene or -C(O)(NR 12 )- and the 5-membered heteroarylene contains at least one nitrogen atom, and the -C(O)(NR 12 )- is linked to X via a C atom, R 9 is C 1~4 It is alkyl, R 10 is selected from the group consisting of hydrogen, C 1~6 alkyl, C 1~6 alkoxy, -C(O)R 12 and -C(O)OR 12 wherein the C 1~6 alkyl is optionally substituted by -OH or -OP(O)(OH)2, each R 12 is independently hydrogen or C 1~4 alkyl, q is selected from the group consisting of 0, 1 and 2, each R 6 is, when present, independently at each occurrence, halogen or C 1~3 alkyl, R 7 is selected from the group consisting of halogen, C 1~3 alkyl, -CN and 5-membered heteroaryl, the 5-membered heteroaryl containing at least one nitrogen atom, and is optionally substituted by R 8 ; R 8 is C 1~3 alkyl or C 1~3 hydroxyalkyl}, and includes a compound thereof, or a salt thereof.

[0161] Also, the KRAS inhibitor preferably has the formula (E)

Chemical formula

[0162] The HER2 inhibitor is preferably of formula (F) [ka] {In the formula, R 1 It is selected from the group consisting of hydrogen, -CH3, -CCH, -OCH3, and halogens. R 2 is hydrogen or halogen, R 3 This is expressed in equations (i.1), (i.2), (i.3), and (i.4) [Table 2] Selected from the group consisting of, R 4 R 4.a and R 4.b [ka] Selected from the group consisting of, Here, Q represents a 4-6 membered heterocycline containing one N atom, and one carbon atom of the ring is optionally substituted by a methyl atom. Z represents a 4-6 membered heterocycline containing one N atom, and one carbon atom of the ring is optionally substituted by a methyl group. R 5 is -H or -CH3, R 1 and R 2 At least one of them is not hydrogen. This includes [1,3]diazino[5,4-d]pyrimidine and its derivatives.

[0163] Statistical methods for predicting clinical outcomes are well known in the art and can be readily adapted to the methods of the present invention.

[0164] As used herein, the terms “monitoring a treatment” or “monitoring” mean monitoring the degree of response, monitoring the duration of response, monitoring the rate of response, monitoring the rate of stabilization, monitoring the duration of stabilization, monitoring the time to disease progression, monitoring progression-free survival, or monitoring overall survival.

[0165] In a third aspect, the present invention relates to a method for treating cancer in a patient with a compound that inhibits the KRAS protein or a mutant of the KRAS protein, wherein the patient has been determined to be responsive to treatment with the compound according to any of the methods described above. Similarly, the present invention relates to a method for treating cancer in a patient with a compound that inhibits the interaction between MDM2 and p53, wherein the patient has been determined to be responsive to treatment with the compound according to any of the methods described above.

[0166] The present invention relates to a method for treating KRAS-dependent cancer in a patient with a KRAS inhibitor or degrader, - To determine that the patient exhibits the molecular biomarker Survivin, and - Administer the patient a pharmacokinetically effective amount of KRAS inhibitor or degrader. Further details regarding methods including

[0167] The present invention relates to a method for treating cancer in a patient using a compound that inhibits the interaction between MDM2 and p53, - To determine that the patient exhibits the molecular biomarker Survivin, and - Administer to the patient a pharmacokinetically effective amount of a compound that inhibits the interaction between MDM2 and p53. Further details regarding methods including

[0168] The present invention relates to a method for treating KRAS-dependent cancer in patients with a KRAS inhibitor or degrader, - The level of survivin in the first sample obtained from the aforementioned patient was measured. - The level of survivin in at least one second sample obtained from the patient during the procedure was compared with the level of survivin in the first sample, and - It was determined that the level of survivin in the second sample obtained from the patient during treatment was lower than the level of survivin in the first sample, or that the level of survivin in any further sample obtained from the patient during treatment after the second sample was lower than the level of survivin in the previous sample obtained from the patient during treatment, and - Continue administering a pharmacokinetically effective dose of KRAS inhibitor or degrader to the patient. Further details regarding methods including

[0169] The present invention relates to a method for treating cancer in patients using a compound that inhibits the interaction between MDM2 and p53, - The level of survivin in the first sample obtained from the aforementioned patient was measured. - The level of survivin in at least one second sample obtained from the patient during the procedure was compared with the level of survivin in the first sample, and - It was determined that the level of survivin in the second sample obtained from the patient during treatment was lower than the level of survivin in the first sample, or that the level of survivin in any further sample obtained from the patient during treatment after the second sample was lower than the level of survivin in the previous sample obtained from the patient during treatment, and - Continue administering a pharmacokinetically effective dose of a compound that inhibits the interaction between MDM2 and p53 to the patient. Further details regarding methods including

[0170] The aforementioned cancer patients may be those selected according to either the method for determining the cancer patient's responsiveness to a KRAS inhibitor or a compound that inhibits the interaction between MDM2 and p53, as described above.

[0171] As used herein, the term "monotherapy" refers to treatment with the compounds of the present invention without additional anti-cancer treatment such as radiotherapy or chemotherapy.

[0172] As used herein, the term “combination therapy” as defined herein can be achieved by means of simultaneous, sequential, or separate administration of the individual components of the treatment. The combination treatment as defined herein may be applied as a single treatment, or may include surgery, radiotherapy, or additional chemotherapy or targeted agents in addition to the combination treatment of the present invention. Surgery may include the step of partial or complete tumor resection before, during, or after administration of the combination treatment described herein.

[0173] The treatment of KRAS-dependent cancer in a patient may be a combination therapy of the KRAS inhibitor and an additional anticancer drug, such as an immune checkpoint inhibitor. Furthermore, the treatment of cancer responsive to compounds that inhibit the interaction between MDM2 and p53 may be a combination therapy of the compound that inhibits the interaction between MDM2 and p53 and an additional anticancer drug, such as an immune checkpoint inhibitor.

[0174] An "immune checkpoint" is a receptor on the cell membrane of a T lymphocyte that modulates the immune response of the aforementioned cells. There are anti-inflammatory (suppressive) and pro-inflammatory (activating) immune checkpoints expressed on the cell membrane of T cells, which interact with their respective ligands, which are either soluble or cell-binding ligands. Tumor cells often activate the anti-inflammatory immune checkpoint pathway via their respective ligands that suppress the anti-tumor immune response, thus evading immune surveillance and allowing tumor growth to progress. "Immune checkpoint inhibitors (ICIs)" are drugs, particularly monoclonal antibodies, that can block this tumor suppression by binding to anti-inflammatory immune checkpoints or their ligands, reactivating the immune system and thus re-establishing its ability to fight the tumor. ICIs have been shown to be clinically effective in various tumor types (Dyck and Mills, 2017).

[0175] In other words, immune checkpoint inhibitors contain an antagonist of an immunosuppressive receptor, such as PD-1, which inhibits PD-1 or PD-L1 in the PD-1 / PD-L1 pathway. Examples of PD-1 or PD-L1 inhibitors include, but are not limited to, human or humanized antibodies that block human PD-1, such as pembrolizumab or pidilizumab, or human or humanized antibodies that block PD-L1, such as avelumab, durvalumab, and atezolizumab, as well as fully human antibodies, such as PD-1 blocker nivolumab.

[0176] The term "therapeutic dose" is used to refer to the amount of an activator that reduces or improves one or more symptoms of the disorder being treated. In another embodiment, the therapeutic dose refers to the target serum concentration of an activator that has been shown to be effective, for example, in slowing disease progression. Effectiveness may be measured by conventional means depending on the condition being treated.

[0177] When used herein, terms such as “treatment” and “therapy” include means of treatment, prevention, or suppression of a disease or disorder that produce any clinically desired or beneficial effect, and the effect includes, but is not limited to, the alleviation or reduction, regression, slowing or cessation of the progression of one or more symptoms of the disease or disorder. For example, the term “treatment” includes the administration of a drug before or after the onset of symptoms of a disease or disorder, and thereby preventing or eliminating one or more signs of the disease or disorder. Another example is the administration of a drug after the onset of clinical symptoms of a disease, to combat the symptoms of the disease.

[0178] Furthermore, the administration of drugs after initiation and after the onset of clinical symptoms includes, as used herein, “treatment” or “therapy” if the administration affects clinical parameters of the disease or disorder, such as the degree of tissue damage or the amount or extent of metastasis, regardless of whether the treatment results in improvement of the disease. Moreover, the treatment, either alone or in combination with another therapeutic agent, should be considered an effective treatment of the underlying disorder, regardless of whether all symptoms of the disorder are relieved, as long as it alleviates or improves at least one symptom of the disorder being treated compared to the symptoms in the absence of the use of each drug.

[0179] In accordance with a fourth aspect, the present invention relates to the use of Survivin in a method for determining the ability of a compound that inhibits the KRAS protein or a mutant of the KRAS protein, or a pharmaceutical formulation containing said compound that inhibits the KRAS protein or a mutant of the KRAS protein, to treat cancer.

[0180] The present invention further relates to the use described above, wherein the level of survivin is determined in a sample obtained from the patient before treatment and in at least one sample obtained from the patient after treatment with the compound that inhibits the KRAS protein or a mutant of the KRAS protein, and the decrease in the level of survivin after treatment with the compound that inhibits the KRAS protein or a mutant of the KRAS protein indicates the ability of the compound to treat the cancer.

[0181] Furthermore, in accordance with this aspect of the present invention, the present invention also relates to the use of Survivin in a method for determining the ability of a compound that inhibits the interaction between MDM2 and p53, or a pharmaceutical formulation containing said compound that inhibits the interaction between MDM2 and p53, to treat cancer. Preferably, in accordance with the said use, the level of Survivin is determined in a sample obtained from the patient before treatment and in at least one sample obtained from the patient after treatment with said compound that inhibits the interaction between MDM2 and p53, and a decrease in the level of Survivin after treatment with said compound that inhibits the interaction between MDM2 and p53 indicates the ability of said cancer-treating compound.

[0182] The present invention relates to the use of a molecular biomarker for selecting patients with KRAS-dependent cancer in relation to treatment with a KRAS inhibitor or degrader, or a pharmaceutical formulation containing the said KRAS inhibitor or degrader, wherein the molecular biomarker is Survivin. The present invention also relates to the use of a molecular biomarker for selecting patients with cancer in relation to treatment with a compound that inhibits the interaction between MDM2 and p53, or a pharmaceutical formulation containing the said compound that inhibits the interaction between MDM2 and p53, wherein the molecular biomarker is Survivin.

[0183] The present invention relates to the use of a molecular biomarker in a method for determining or confirming the ability of a KRAS inhibitor or degrader, or a pharmaceutical formulation containing said KRAS inhibitor or degrader, to inhibit KRAS-dependent cancer, wherein the molecular biomarker is Survivin. The present invention also relates to the use of a molecular biomarker in a method for determining or confirming the ability of a compound that inhibits the interaction between MDM2 and p53, or a pharmaceutical formulation containing said compound that inhibits the interaction between MDM2 and p53, to inhibit cancer, wherein the molecular biomarker is Survivin.

[0184] The present invention relates to the use of the molecular biomarker described above, wherein the level of the molecular biomarker survivin is determined in a sample provided by the patient before treatment and in at least one sample provided by the patient after treatment with the KRAS inhibitor or degrader, and it is identified that the level of survivin decreased after treatment with the KRAS inhibitor or degrader. The present invention also relates to the use of the molecular biomarker described above, wherein the level of the molecular biomarker survivin is determined in a sample provided by the patient before treatment and in at least one sample provided by the patient after treatment with the compound that inhibits the interaction between MDM2 and p53, and it is identified that the level of survivin decreased after treatment with the compound that inhibits the interaction between MDM2 and p53.

[0185] The present invention further relates to the use of the molecular biomarkers described above, in vitro.

[0186] In any preferred embodiment of the use described above, the sample(s) is a blood, plasma, or serum sample(s).

[0187] In any more preferred embodiment of the use described above, the compound that inhibits the KRAS protein or a mutant of the KRAS protein is selected from the group consisting of KRAS(G12C) inhibitor or degrader, KRAS(G12D) inhibitor or degrader, GDP-KRAS inhibitor or degrader, and HER2 inhibitor and degrader. In yet another preferred embodiment of any of the use described above, the compound that inhibits the interaction between MDM2 and p53 is MDM2i-cpd 1.

[0188] Those skilled in the art will understand that KRAS-dependent cancers vary depending on the type of compound.

[0189] In any more preferred embodiment of the use described above, the KRAS(G12C) inhibitor or degrader is selected from the group consisting of sotrasib (AMG510), adagrasib (MRTX849), G12C-cpd 1, G12C-cpd 2, G12C-cpd 3, G12C-cpd 4, G12C-cpd 5, G12C-cpd 6, G12C-cpd 7, G12C-cpd 8, G12C-cpd 9, G12C-cpd 10, and G12C-cpd 11.

[0190] In any more preferred embodiment of the use described above, the KRAS(G12D) inhibitor or degrader is selected from the group consisting of MRTX1133, G12D-cpd 2, G12D-cpd 3, G12D-cpd 4, G12D-cpd 5, G12D-cpd 6, G12D-cpd 7, G12D-cpd 8, and G12D-cpd 9.

[0191] In any more preferred embodiment of the use described above, the GDP-KRAS inhibitor or degrader is selected from the group consisting of GDP-cpd 1, GDP-cpd 2, GDP-cpd 3, GDP-cpd 4, GDP-cpd 5, GDP-cpd 6, GDP-cpd 7, GDP-cpd 8, GDP-cpd 9, GDP-cpd 10, GDP-cpd 11, GDP-cpd 12, and GDP-cpd 13.

[0192] In any more preferred embodiment of the use described above, the HER2 inhibitor or degrader is the compound described in formula (F) (see above), and more preferably, the HER2 inhibitor or degrader is compound HER2-cpd 1.

[0193] In one or more preferred embodiments of the present invention, the compound that inhibits the KRAS protein or a mutant of the KRAS protein may also be a compound that inhibits or degrades the HER2 protein or a mutant of the HER2 protein, and the compound that inhibits or degrades the HER2 protein or a mutant of the HER2 protein acts as an indirect inhibitor of the KRAS protein or a mutant of the KRAS protein located downstream of the HER2 protein or a mutant of the HER2 protein in the signaling pathway.

[0194] In any more preferred embodiment of the use described above, the survivor is contained within an exosome.

[0195] In any more preferred embodiment of the use described above, the KRAS-dependent cancer is selected from the group consisting of pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NSCLC), and colorectal cancer (CRC). In yet another preferred embodiment of any of the use described above, the cancer treated with a compound that inhibits the interaction between MDM2 and p53 is selected from any of the cancers defined herein above as preferred cancer types for treatment with such MDM2 inhibitors.

[0196] In any more preferred embodiment of the use described above, determining the level of survivorbin involves a survivorbin-specific assay selected from the group consisting of Western blotting, ELISA, RIA, MSD® S-PLEX technology, and FACS.

[0197] In any more preferred embodiment of the use described above, the treatment is a combination therapy of the KRAS inhibitor or degrader, or the compound that inhibits the interaction between MDM2 and p53, and an additional anticancer drug and / or standard treatment.

[0198] In accordance with a fifth aspect, the present invention relates to a kit of components including means for determining the level of survivin in a sample provided by a patient suffering from cancer, for example, KRAS-dependent cancer, and instructions for use on how to carry out any of the methods described above.

[0199] The present invention further relates to a kit of components, which includes means for determining the level of survivin in a sample provided by a patient suffering from cancer, for example, KRAS-dependent cancer, for performing any of the methods described above. [Examples]

[0200] Although the present invention is illustrated and described in detail in the drawings and the above description, such illustrations and descriptions should be considered illustrative or typical, not limiting, and the present invention is not limited to the embodiments disclosed. Other modifications to the disclosed embodiments in the claimed implementation of the present invention can be understood and implemented by those skilled in the art from a review of the drawings, this disclosure and the accompanying claims. In the claims, the phrase “comprising” does not exclude other elements or processes, and the indefinite article “a” or “an” does not exclude the plural. The mere fact that certain measurements are enumerated in different dependent claims does not imply that combinations of these measurements cannot be used for benefit. No reference numeral in the claims should be construed as limiting the scope.

[0201] All amino acid sequences disclosed herein are shown from the N-terminus to the C-terminus, and all nucleic acid sequences disclosed herein are shown from the 5'-terminus to the 3'-terminus.

[0202] Materials and methods (Example 1) compound The KRAS inhibitors and HER2 inhibitors are shown in Table 1 above, respectively.

[0203] Compound synthesis (Example 2) Synthesis of the compound described in formula (A) The synthesis of the compound shown in formula (A) is described in WO2021 / 245051.

[0204] (Example 3) Synthesis of the compound shown in formula (B) [Table 3] TIFF2026520494000037.tif249169 TIFF2026520494000038.tif149169 Examples The characteristics and advantages of the present invention will become apparent from the following detailed examples, which illustrate the principles of the present invention by the methods of the examples without limiting the scope of the present invention.

[0205] Preparation of the compound of the present invention Unless otherwise indicated, all reactions are carried out using commercially available equipment and methods commonly used in chemical laboratories. Starting materials sensitive to air and / or moisture are stored under a protective gas, and their corresponding reactions and operations are carried out under a protective gas (nitrogen or argon).

[0206] If a compound is represented by both a structural formula and its nomenclature, and a contradiction arises, the structural formula shall prevail.

[0207] The microwave reaction is carried out in a sealed container (preferably 2, 5, or 20 mL) with stirring, preferably in an initiator / reactor manufactured by Biotage, or in an Explorer manufactured by CEM, or in a Synthos 3000 or Monowave 3000 manufactured by Anton Paar.

[0208] chromatography Thin-layer chromatography is performed on a ready-made silica gel 60 TLC glass plate manufactured by Merck (with fluorescence index F-254).

[0209] Preparative high-pressure chromatography (RP HPLC) of the compounds of the examples of the present invention is performed in an Agilent or Gilson system with columns manufactured by Waters (name: SunFire® Prep C18, OBD® 10 μm, 50 × 150 mm, or SunFire® Prep C18 OBD® 5 μm, 30 × 50 mm, or XBridge® Prep C18, OBD® 10 μm, 50 × 150 mm, or XBridge® Prep C18, OBD® 5 μm, 30 × 150 mm, or XBridge® Prep C18, OBD® 5 μm, 30 × 50 mm) and columns manufactured by YMC (name: Actus-Triart Prep C18, 5 μm, 30 × 50 mm).

[0210] Compounds are eluted using various H2O / acetonitrile gradients. For the Agilent system, a 5% acid modifier (20 mL of HCOOH to 1 L of H2O / acetonitrile (1 / 1)) is added to water (acidic conditions). For the Gilson system, 0.1% HCOOH is added to water.

[0211] For chromatography under basic conditions using the Agilent system, an H2O / acetonitrile gradient is used, while the water is made alkaline by adding a 5% basic modifier (50g of NH4HCO3 + 50mL of NH3 (25% of H2O) diluted to 1L with H2O). For the Gilson system, the water is made alkaline as follows: 5mL of NH4HCO3 solution (158g in 1L of H2O) and 2mL of NH3 (28% of H2O) are diluted to 1L with H2O.

[0212] Supercritical fluid chromatography (SFC) of the intermediates and compounds of the examples of the present invention is performed in a JASCO SFC system with the following columns: Chiralcel OJ (250×20mm, 5μm), Chiralpak AD (250×20mm, 5μm), Chiralpak AS (250×20mm, 5μm), Chiralpak IC (250×20mm, 5μm), Chiralpak IA (250×20mm, 5μm), Chiralcel OJ (250×20mm, 5μm), Chiralcel OD (250×20mm, 5μm), and Phenomenex Lux C2 (250×20mm, 5μm).

[0213] The analysis of intermediates and final compounds by controlled HPLC (reaction control) is performed using columns manufactured by Waters (name: XBridge® C18, 2.5 μm, 2.1 × 20 mm, or XBridge® C18, 2.5 μm, 2.1 × 30 mm, or Aquity UPLC BEH C18, 1.7 μm, 2.1 × 50 mm), YMC (name: Triart C18, 3.0 μm, 2.0 × 30 mm), and Phenomenex (name: Luna C18, 5.0 μm, 2.0 × 30 mm). The analytical equipment is equipped with a mass detector in each case.

[0214] HPLC-Mass Spectrometry / UV Spectroscopy Retention time / MS-ESI for characterizing the compounds of the examples of the present invention + This is generated using an HPLC-MS (high-performance liquid chromatography with a mass detector). The compound eluted at the injection peak is determined by the retention time t Ret. The value = 0.00 is given.

[0215] SFC method (for preparative separation) Preparative SFC will be performed on the Waters Thar SFC 80 system. Column: Chiralpak AD-H (21×250mm), 5μm Flow rate: 25g / min Mobile phase: 75% CO2 + 25% MeOH (0.5% isopropylamine) ABPR: 120 bar Temperature: 35℃ UV: 220nm Stack time: 8 minutes

[0216] HPLC method (for analysis) Method A The samples were analyzed using an Agilent 1200 series LC system connected to an Agilent 6140 mass spectrometer. Purity was determined by UV detection with a bandwidth of 170 nm within the range of 230–400 nm. The LC parameters were as follows: Waters Xbridge C18 column, 3.5 μm particle size, 2.1 × 30 mm; Flow rate 1mL / min; Column temperature: 60°C; Injection 5μL injection; Solvent A: 20 mM NH4HCO3 / NH3 pH 9 B: MS grade acetonitrile; Gradient 0.0~1.5 min 10%~95%B 1.5~2.0 minutes 95%B 2.0~2.1 minutes 95%~10%B

[0217] Method B HPLC Agilent 1100 / 1200 Series MS Agilent LC / MSD SL Column: Waters X-Bridge BEH C18, 2.5 μm, 2.1 × 30 mm XP Solvent A: 20 mM NH4HCO3 / 28 mM NH3 in H2O; B: Acetonitrile (HPLC grade) Detection MS: Positive and negative modes Mass range 100~750m / z Flow rate 1.40mL / min Column temperature 45℃ Gradient: 0.00~1.00 min: 15%B → 95%B 1.00~1.30 minutes: 95%B

[0218] Method F HPLC Agilent 1100 / 1200 System MS 1200 Series LC / MSD (API-ES+ / -3000 / 3500V, Quadruple, G6140A) MSD signal setting: Scan positive / negative 150-750 Column YMC; Part number TA12S03-0302WT; Triart C18, 3μm, 12nm; 30×2.0mm column Eluent A: H2O + 0.11% formic acid B: MeCN + 0.1% Formic Acid (HPLC Grade) Detection signals: UV 254nm, 230nm, 214nm (bandwidth 10, reference off) Spectrum range: 190-400 nm; Slit: 4 nm Peak duration: over 0.0031 minutes (response time: 0.063 seconds, 80Hz) Injection Standard injection 0.5μL Flow rate 1.4mL / min Column temperature 45℃ Gradient 0.0~1.0 min 15%→95%B 1.0~1.1 minutes 95%B Stop time: 1.23 minutes

[0219] The compounds and intermediates of the present invention are prepared by the synthetic methods described herein, in which substituents of the general formula have the meanings set forth herein. These methods are intended as examples of the present invention and do not limit the subject matter or the scope of compounds claimed for these examples. Where the preparation of the starting compounds is not described, they are either commercially available, or their synthesis is described in the prior art, or they can be prepared similarly to known prior art compounds or methods described herein, i.e., the synthesis of these compounds is within the scope of the organic chemist's skill. Substances described in the literature can be prepared according to the published synthetic methods. Where the following chemical structures are shown without the exact arrangement of stereocenters, e.g., asymmetrically substituted carbon atoms, both arrangements should be considered included and disclosed in such representation. The representation of the stereocenters of a racemic mixture is always considered to include and disclose both enantiomers (if no other defined stereocenters are present) or all other potential diastereomers and enantiomers (if additional, defined or undefined stereocenters are present).

[0220] Experimental Procedure for the Synthesis of A-2a [ka] To a suspension of 5-chloropentanenitrile (22.9 g, 194.8 mmol, 1.0 equivalent) in anhydrous EtOH (136 mL), acetyl chloride (111.3 mL, 1.558 mol, 8.0 equivalents) is added dropwise at 0°C. The reaction mixture is allowed to rise to ambient temperature and stirred for 12 hours. The mixture is concentrated under reduced pressure, washed with Et2O, and the crude product A-2a is used directly as the HCl salt in the next step without further purification.

[0221] Experimental Procedure for the Synthesis of A-3a [ka] Unpurified A-2a (HCl salt) (28 g, 139.9 mmol, 1.0 equivalent) and ethylene glycol (7.382 g, 118.94 mmol, 0.9 equivalents) are dissolved in DCM (300 mL) and stirred at ambient temperature for 6 days. The resulting suspension is concentrated under reduced pressure, diluted with Et2O (200 mL), and filtered. The filtrate is concentrated under reduced pressure, placed in DCM (200 mL), and treated with 2N KOH solution (150 mL). The mixture is stirred overnight at ambient temperature while maintaining the intact phase. The phases are separated, the aqueous phase is extracted twice with DCM, the mixed organic phase is dried over MgSO4, filtered, and concentrated under reduced pressure. Unpurified orthoester A-3a is used in the next step without further purification.

[0222] Experimental Procedure for the Synthesis of A-4a [ka] Unpurified A-3a (22.3 g, 106.9 mmol, 1.0 equivalent), 1-cyclohexenyloxytrimethylsilane (16.42 mL, 82.3 mmol, 0.8 equivalent), and zinc chloride (10.195 g, 74.8 mmol, 0.7 equivalent) are dissolved in DCM (120 mL) and stirred at ambient temperature for 5 hours. The reaction mixture is treated by adding saturated sodium bicarbonate solution. The organic phase is separated, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product is purified by NP chromatography (gradient elution: 0%~50% siRNA in cHexane) to obtain the desired compound A-4a.

[0223] Experimental Procedure for the Synthesis of A-5a [ka] Dissolve A-4a (14.9 g, 57.14 mmol, 1.0 equivalent) and sodium iodide (25.954 g, 171.4 mmol, 3.0 equivalents) in acetone (120 mL) and stir under reflux for 16 hours. Concentrate the reaction mixture under reduced pressure, dilute with DCM, and wash with saturated sodium thiosulfate solution. Separate the organic phase, dry it over MgSO4, filter it, and concentrate it under reduced pressure. Use the crude product A-5a in the next step without further purification.

[0224] Experimental Procedure for the Synthesis of A-6b [ka] Dissolve A-5a (30 g, 85.0 mmol, 1.0 equivalent) in THF. Treat the mixture with potassium tert.-butoxide (28.67 g, 256.0 mmol, 3.0 equivalents) at 0°C and stir overnight at ambient temperature. Quench the reaction mixture by adding water (2 mL) and dilute by adding Et2O and saturated sodium bicarbonate solution. Separate the organic phase, dry it over MgSO4, filter it, and concentrate it under reduced pressure. The crude product was purified by NP chromatography (gradient elution: 0%~50% siRNA in cHexane) to obtain (racemic) compound A-6a (the reaction sequence A-1a → A-6a is based on Marko et al., THL 2003, 44, 3333~3336 and Maulide et al., Eur.J.Org.Chem. 2004, 19:3962~3967).

[0225] Subsequently, the desired enantiomer A-6b can be obtained after chiral separation by SFC (e.g., using a CHIRACEL OX-3 column and acetonitrile as a cosolvent).

[0226] Experimental procedure for the synthesis of E-4c (Method C) [ka] To a stirred solution of E-1c (10.20 g, 57.22 mmol) in DCM (60.0 mL), piperazine-1-carboxylic acid tert-butyl ester (11.22 g, 57.22 mmol, 1.0 equivalent) is added. Then, DIPEA (20.71 g, 160.21 mmol, 2.8 equivalents) is added, and the reaction mixture is stirred at 60°C for 1 hour. After complete conversion, the mixture is dissolved in ethyl acetate and washed with water (3×). The organic phase is dried, filtered, and concentrated under reduced pressure. The crude product is purified by column chromatography (DCM / MeOH) to obtain E-4c.

[0227] The following (additional) intermediate E-4 (Table 4) can be obtained in a similar manner using different amine PG-LH and intermediate E-1 according to methods A-E. Crude product E-4 may be purified by chromatography as needed. [Table 4]

[0228] Experimental Techniques for the Synthesis of E-8d [ka] To a solution of E-1a (600 mg, 3.21 mmol, 93% purity, 1.0 equivalent) in anhydrous DMSO (6 mL), cesium fluoride (1.218 g, 8.02 mmol, 2.5 equivalents) is added, and the resulting mixture is stirred at ambient temperature for 1 hour until complete conversion of the starting materials is observed. The resulting suspension is filtered, and the filtered solid is washed with anhydrous DMSO (2 mL). The filtrate (8 mL) is added to (S)-1-((S)-1-methylpyrrolidine-2-yl)ethane-1-ol (453 mg, 3.51 mmol, 1.1 equivalents), and DIPEA (1.085 mL, 6.38 mmol, 2 equivalents) is added. The mixture is stirred at ambient temperature for 1 hour. After complete conversion of the starting materials is observed, a solution of tert-butylpiperazine-1-carboxylate (674 mg, 3.51 mmol, 97% purity, 1.1 equivalents) and DIPEA (1.085 mL, 6.38 mmol, 2 equivalents) in anhydrous DMSO (3 mL) is added to the mixture. The mixture is stirred at ambient temperature for 30 minutes. After complete conversion is observed, the reaction is diluted with acetonitrile and water, filtered, and purified by basic reverse-phase chromatography (gradient elution: 30% to 98% acetonitrile in water) to obtain the desired product E-8d.

[0229] The following intermediates E-8 (Table 5) can be obtained in a similar manner without the isolation of the corresponding intermediates E-5 and E-7. The crude product E-8 is purified by chromatography as needed. [Table 5]

[0230] Experimental procedure for the synthesis of E-8p (Method J) [ka] A mixture of E-4r (200 mg, 0.59 mmol, 1.0 equivalent) and (S)-1-((S)-1-methylpyrrolidine-2-yl)ethane-1-ol (91.8 mg, 0.71 mmol, 1.2 equivalents) in acetonitrile (1.5 mL) is mixed with trimethylamine (149.8 mg, 1.48 mmol, 2.5 equivalents). The mixture is stirred at 40°C for 2 hours. The mixture is stirred at 80°C for 16 hours. The solvent is removed under reduced pressure, and the crude product is purified by normal-phase chromatography (gradient elution: DCM + 0%~90% MeOH in ammonia) to obtain the desired product E-8p.

[0231] Intermediate E-8, indicated as "J" (Table 6), is available in a similar form. The crude product E-8 is purified by chromatography as needed. [Table 6]

[0232] Experimental technique for synthesizing E-8ch (Method M) [ka] E-6h (100.0 mg, 0.31 mmol, 1.0 equivalent) and (S)-1,3-dimethylpiperazine (42.5 mg, 0.37 mmol, 1.2 equivalents) are dissolved in DMSO (1 mL) at ambient temperature, and DIPEA (115.0 μL, 0.62 mmol, 2.0 equivalents) is added. The mixture is stirred for 1 hour. The mixture is diluted with acetonitrile and water, and purified by acid reverse-phase chromatography to obtain E-8ch.

[0233] Intermediate E-8, indicated as "M" (Table 7), is available in a similar form. The crude product E-8 is purified by chromatography as needed. [Table 7]

[0234] Additional nitrile building blocks E-8 not expressly disclosed herein are disclosed in WO2021 / 245051 and WO2021 / 245055 (including synthesis), both of which are incorporated herein by reference, with respect to such building blocks E-8, their synthesis, and their synthetic use. These building blocks may also be used in the synthesis of additional compounds of formula (I) of the present invention not specifically disclosed herein.

[0235] Scheme 3a: [ka] Experimental Procedure for the Synthesis of E-12a [ka] To a solution of E-8aq (1.776 g, 4.26 mmol, 1 equivalent) in MeOH (35 mL), add a solution of sodium hydroxide in water (16 mL, 4 M, 63.96 mmol, 15.0 equivalents), and stir the resulting mixture at 65°C for 1.5 hours. Reduce the reaction volume under reduced pressure to remove most of the MeOH, and carefully neutralize the remaining aqueous solution with an aqueous solution of HCl (8 M). Dilute the mixture with acetonitrile and purify it by acid reverse-phase chromatography (gradient elution: 10% to 85% acetonitrile in water) to obtain the desired product E-12a.

[0236] Experimental Procedure for the Synthesis of E-12e [ka] To a solution of E-8c (2.2 g, 4.97 mmol, 1 equivalent) in MeOH, a solution of sodium hydroxide in water (6.2 mL, 4 M, 40 mmol, 5.0 equivalents) is added, and the resulting mixture is stirred at 65°C for 4 hours. The reaction mixture is concentrated under reduced pressure, suspended in MeOH, filtered, and purified by acid reverse-phase chromatography (gradient elution: 10% to 85% acetonitrile in water). The fractions containing the product are mixed, concentrated under reduced pressure, and freeze-dried to obtain the desired product E-12e.

[0237] The following intermediates E-12 / E-12* (Table 8) can be obtained in a similar manner starting from different intermediates E-8 / E-8*. The crude product E-8 / E-8* is purified by chromatography as needed. [Table 8]

[0238] Scheme 4a: [ka] Experimental Procedure for the Synthesis of B-1a [ka] Dissolve CDI (18.781 g, 112.352 mmol, 2.0 equivalents) in anhydrous THF and heat to 50°C. In a second flask, stir E-12d (13.021 g, 28.088 mmol, 0.5 equivalents) and activated molsieve in anhydrous THF at ambient temperature for 10 minutes, then add to the CDI solution. Stir the reaction mixture at 50°C for 15 minutes. In a third flask, dissolve A-6b (15 g, 56.176 mmol, 1.0 equivalent) in 1 M LiHMDS solution in THF (117.969 mL, 117.969 mmol, 2.1 equivalents), stir at ambient temperature for 10 minutes, then add to the activated ester. Stir the reaction mixture at 50°C overnight. After cooling to ambient temperature, the reaction mixture is concentrated under reduced pressure, diluted with DCM, and washed with saturated sodium bicarbonate solution. The aqueous phase is extracted with EtOAx (3 × 100 mL). The mixed organic phase is dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product is purified by NP chromatography (using a MeOH / DCM 0-10% gradient under basic conditions). The fractions containing the product are mixed and freeze-dried to obtain B-1a.

[0239] The following intermediates B-1 / B-1* (Table 9) can be obtained in a similar manner starting from different intermediates E-12 / E-12*. ​​The crude product B-1 / B-1* is purified by chromatography as needed. [Table 9] TIFF2026520494000059.tif58169

[0240] Scheme 5a: [ka] Experimental Procedure for the Synthesis of B-6a and B-7a [ka] To a solution of B-1a (12.7 g, 19 mmol, 1.0 equivalent) in EtOH / water, hydroxylamine hydrochloride (50% of the water content, 3.131 g, 47 mmol, 2.5 equivalents) is added, and the reaction mixture is heated to 50°C for 2 hours. The reaction mixture is concentrated under reduced pressure, dissolved in MeOH (40 mL), and treated with concentrated hydrochloric acid (40 mL). The reaction mixture is stirred at 60°C for 1 hour, concentrated under reduced pressure, dissolved in ethyl acetate, and neutralized by careful addition of a saturated solution of sodium carbonate. The aqueous phase is extracted with ethyl acetate (three times), the mixed organic phase is dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product is purified by RP chromatography (using an ACN / water 30-80% gradient under basic conditions). The fractions containing the product are mixed and freeze-dried to obtain B-6a and other isoxazole isomers B-7a.

[0241] The following intermediates B-6 / B-6* and B-7 / B-7* (Table 10) can be obtained in a similar manner starting from a different intermediate B-1 / B-1*. The crude product is purified by chromatography as needed. [Table 10] TIFF2026520494000063.tif153169

[0242] Scheme 6a: [ka] Experimental Procedure for the Synthesis of C-3a [ka] To a solution of B-6a (1.2 g, 2.3 mmol, 1.0 equivalent) in EtOH (10 mL), malononitrile (95% purity, 798.2 mg, 11 mmol, 5.0 equivalents), beta-alanine (95% purity, 646 mg, 6.9 mmol, 3.0 equivalents), and activated molecular sieve (Roth, 200 mg) are added at ambient temperature under nitrogen gas. The reaction mixture is heated to 80°C for 3 hours. During the complete condensation reaction, which is monitored by HPLC-MS, sulfur (220.9 mg, 6.9 mmol, 3.0 equivalents) is added, and the reaction mixture is stirred at 80°C for 15 minutes. The reaction mixture is cooled to ambient temperature, dissolved with water and RINKAN, and filtered. The layers are separated. 4N NaOH solution (10 mL) is added to the aqueous phase, and the mixture is extracted three times with RINKAN. The mixed organic phase is dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product is purified by silica gel chromatography using a gradient under basic conditions. The fractions containing the product are mixed and freeze-dried to obtain C-3a.

[0243] The following intermediates C-3 / C-3* and C-4 / C-4* (Table 11) are available in a similar manner, starting from different intermediates B-6 / B-6* and B-7 / B-7*. The crude product is purified by chromatography as needed. [Table 11] TIFF2026520494000067.tif107169

[0244] Synthesis of compound (I) described in the present invention Scheme 7: [ka] Experimental Techniques for the Synthesis of Compound Ia-1 [ka] To a solution of 2-fluoroacrylic acid (136 mg, 1.51 mmol, 2.6 equivalents) and HATU (552 mg, 1.452 mmol, 2.5 equivalents) in DMF (0.6 mL), TEA (353 mg, 3.484 mmol, 6.0 equivalents) is added, and the reaction mixture is stirred at ambient temperature for 2 minutes. To the reaction mixture, a solution of C-3a (350 mg, 581 μmol, 1.0 equivalent) dissolved in DMF (3 mL) is added, and the mixture is stirred at ambient temperature for 15 minutes. After the reaction is complete, the mixture is diluted with acetonitrile and water, filtered, and purified by basic reverse-phase chromatography (gradient elution: 30% to 98% acetonitrile in water) to obtain the desired compound Ia-1.

[0245] Experimental Techniques for the Synthesis of Compound Ia-2 [ka] To a solution of sodium carbonate (154 mg, 1.45 mmol, 2.5 equivalents) and C-3a (350 mg, 581 μmol, 1.0 equivalent) in acetone / water (8:1), a freshly prepared solution of acryloyl chloride in acetone (81.3 mg, 871 μmol, 1.5 equivalents) is added. After the reaction is complete, the mixture is diluted with acetonitrile and water, filtered, and purified by basic reverse-phase chromatography (gradient elution: 30% to 98% acetonitrile in water) to obtain the desired compound Ia-2. [Table 12] TIFF2026520494000072.tif118169

[0246] (Example 4) Synthesis of the compound shown in formula (C) [Table 13] TIFF2026520494000074.tif245169 Examples The characteristics and advantages of the present invention will become apparent from the following detailed examples, which illustrate the principles of the present invention by the methods of the examples without limiting the scope of the present invention.

[0247] Preparation of the compound of the present invention Unless otherwise indicated, all reactions are carried out using commercially available equipment and methods commonly used in chemical laboratories. Starting materials sensitive to air and / or moisture are stored under a protective gas, and their corresponding reactions and operations are carried out under a protective gas (nitrogen or argon).

[0248] If a compound is represented by both a structural formula and its nomenclature, and a contradiction arises, the structural formula shall prevail.

[0249] The microwave reaction is carried out in a sealed container (preferably 2, 5, or 20 mL) with stirring, preferably in an initiator / reactor manufactured by Biotage, or in an Explorer manufactured by CEM, or in a Synthos 3000 or Monowave 3000 manufactured by Anton Paar.

[0250] chromatography Thin-layer chromatography is performed on a ready-made silica gel 60 TLC glass plate manufactured by Merck (with fluorescence index F-254).

[0251] Preparative high-pressure chromatography (RP HPLC) of the compounds of the examples of the present invention is performed in an Agilent or Gilson system with columns manufactured by Waters (name: SunFire® Prep C18, OBD® 10 μm, 50 × 150 mm, or SunFire® Prep C18 OBD® 5 μm, 30 × 50 mm, or XBridge® Prep C18, OBD® 10 μm, 50 × 150 mm, or XBridge® Prep C18, OBD® 5 μm, 30 × 150 mm, or XBridge® Prep C18, OBD® 5 μm, 30 × 50 mm) and columns manufactured by YMC (name: Actus-Triart Prep C18, 5 μm, 30 × 50 mm).

[0252] Compounds are eluted using various H2O / ACN gradients. For the Agilent system, a 5% acid modifier (20 mL of HCOOH to 1 L of H2O / ACN (1 / 1)) is added to water (acidic conditions). For the Gilson system, 0.1% HCOOH is added to water.

[0253] For chromatography under basic conditions using the Agilent system, an H2O / ACN gradient is used, while the water is made alkaline by adding a 5% basic modifier (50g of NH4HCO3 + 50mL of NH3 (25% of H2O) mixed with 1L of H2O). For the Gilson system, the water is made alkaline as follows: 5mL of NH4HCO3 solution (158g in 1L of H2O) and 2mL of NH3 (28% of H2O) are mixed with 1L of H2O.

[0254] Supercritical fluid chromatography (SFC) of the intermediates and compounds of the examples of the present invention is performed in a JASCO SFC system with the following columns: Chiralcel OJ (250×20mm, 5μm), Chiralpak AD (250×20mm, 5μm), Chiralpak AS (250×20mm, 5μm), Chiralpak IC (250×20mm, 5μm), Chiralpak IA (250×20mm, 5μm), Chiralcel OJ (250×20mm, 5μm), Chiralcel OD (250×20mm, 5μm), and Phenomenex Lux C2 (250×20mm, 5μm).

[0255] The analysis of intermediates and final compounds by controlled HPLC (reaction control) is performed using columns manufactured by Waters (name: XBridge® C18, 2.5 μm, 2.1 × 20 mm, or XBridge® C18, 2.5 μm, 2.1 × 30 mm, or Aquity UPLC BEH C18, 1.7 μm, 2.1 × 50 mm), YMC (name: Triart C18, 3.0 μm, 2.0 × 30 mm), and Phenomenex (name: Luna C18, 5.0 μm, 2.0 × 30 mm). The analytical equipment is equipped with a mass detector in each case.

[0256] HPLC-Mass Spectrometry / UV Spectroscopy Retention time / MS-ESI for characterizing the compounds of the examples of the present invention + This is generated using an HPLC-MS (high-performance liquid chromatography with a mass detector). The compound eluted at the injection peak is determined by the retention time t Ret. The value = 0.00 is given.

[0257] Method A HPLC Agilent 1100 System MS 1200 Series LC / MSD (API-ES+ / -3000V, Quadruple, G6140) MSD signal settings: Scan positive / negative, 120-900 MHz Detection signal 315nm (bandwidth 170nm, reference off) Spectral range: 230-400 nm Peak width less than 0.01 minutes Column: Waters, Xbridge C18, 2.5 μm, 2.1 × 20 mm column Column temperature: 60°C Solvent A: 20 mM NH4HCO3 / NH3 aqueous solution pH 9 B: ACN HPLC Grade Flow rate 1.00mL / min Gradient 0.00~1.50 min 10%~95%B 1.50~2.00 minutes 95%B 2.00~2.10 minutes 95%~10%B

[0258] Method B HPLC Agilent 1260 System MS 1200 Series LC / MSD (MM-ES + APCI + / - 3000V, Quadruple, G6130) Detection UV: 254nm (bandwidth 8, reference off) UV: 230nm (bandwidth 8, reference off) UV spectral range: 190-400nm; step size: 4nm MS: Positive and negative modes Mass range 100~800 m / z Column Waters; Part number 186003389; XBridge BEH C18, 2.5 μm, 30 × 2.1 mm Column temperature 45℃ Solvent A: 5 mM NH4HCO3 / 19 mM NH3 in H2O; B: ACN (HPLC grade) Flow rate 1.40mL / min Gradient 0.00~1.00 min: 5%B~100%B 1.00~1.37 minutes: 100%B 1.37~1.40 minutes: 100%B~5%B

[0259] Method C Agilent HPLC 1260 Series MS Agilent LC / MSD Quadrupole Detection MS: Positive and negative modes Mass range 100~750m / z Column: Waters X-Bridge BEH C18, 2.5 μm, 2.1 × 30 mm XP Column temperature 45℃ Solvent A: 20 mM NH4HCO3 / 30 mM NH3 in H2O; B: ACN (HPLC grade) Flow rate 1.40mL / min Gradient 0.00~1.00 min: 15%B~95%B 1.00~1.30 minutes: 95%B

[0260] Method E HPLC Agilent 1100 / 1200 System MS 1200 Series LC / MSD (MM-ES + APCI + / - 3000V, Quadruple, G6130B) MSD signal setting: Scan positive / negative 150-750 Detection signals: UV 254nm, 230nm, 214nm (bandwidth 8, reference off) Spectrum range: 190-400 nm; Slit: 4 nm Peak duration: over 0.0031 minutes (response time: 0.063 seconds, 80Hz) Column (Waters, part number 186003389, XBridge BEH C18, 2.5 μm, 2.1 × 30 mm) Column temperature 45℃ Solvent A: 5 mM NH4HCO3 / 18 mM NH3 in H2O (pH=9.2) B: ACN (HPLC grade) Flow rate 1.4mL / min Gradient 0.0-1.0 min 15%-95%B 1.0~1.1 minutes 95%B Stop time: 1.3 minutes

[0261] Method SFC-1 Manufacturing Waters UPC 2 -MS Software Empower3 MS QDa Column: CHIRALCEL OX-3 (4.6*150MM) 3μm A - Solvent CO2 B-solvent ACN Total flow rate 3g / min % 15 relative to the cosolvent ABPR 1500psi Column temperature: 30°C PDA range: 200nm~400nm Resolution 1.2nm MS Parameters - QDa MS scan range: 100Da to 1000Da Ion intake pore voltage Positive scan 20V Negative scan 15V Negative scan 15V

[0262] The compounds and intermediates of the present invention are prepared by the synthetic methods described herein, in which substituents of the general formula have the meanings set forth herein. These methods are intended as examples of the present invention and do not limit the subject matter or the scope of compounds claimed for these examples. Where the preparation of the starting compounds is not described, they are either commercially available, or their synthesis is described in the prior art, or they can be prepared similarly to known prior art compounds or methods described herein, i.e., the synthesis of these compounds is within the scope of the organic chemist's skill. Substances described in the literature can be prepared according to the published synthetic methods. Where the following chemical structures are shown without the exact arrangement of stereocenters, e.g., asymmetrically substituted carbon atoms, both arrangements should be considered included and disclosed in such representation. The representation of the stereocenters of a racemic mixture is always considered to include and disclose both enantiomers (if no other defined stereocenters are present) or all other potential diastereomers and enantiomers (if additional, defined or undefined stereocenters are present).

[0263] Synthesis of spiroketone intermediate A Experimental Procedure for the Synthesis of A-2a [ka] To a suspension of 5-chloropentanenitrile (22.9 g, 195 mmol, 1.00 equivalent) in EtOH (136 mL), acetyl chloride (111 mL, 1.56 mol, 8.00 equivalent) is added dropwise at 0°C. The reaction mixture is warmed to ambient temperature and stirred for 12 hours. The mixture is concentrated under reduced pressure and washed with Et2O. Crude product A-2a is used directly as the HCl salt in the next step without further purification (HPLC method: A;t ret =1.03 minutes;[M+H] + =164).

[0264] Experimental Procedure for the Synthesis of A-3a [ka] Dissolve unpurified A-2a (HCl salt) (28.0 g, 140 mmol, 1.00 equivalent) and ethylene glycol (7.38 g, 119 mmol, 0.90 equivalent) in DCM (300 mL) and stir at ambient temperature for 6 days. Concentrate the resulting suspension under reduced pressure, dilute with Et2O (200 mL), and filter. Concentrate the filtrate under reduced pressure, transfer to DCM (200 mL), and treat with KOH solution (2 M in water, 150 mL). Stir the mixture overnight at ambient temperature while keeping the phase intact. Separate the phases, extract the aqueous phase with DCM (2 ×), dry the mixed organic phase over magnesium sulfate, filter, and concentrate under reduced pressure. Use the unpurified orthoester A-3a in the next step without further purification (HPLC method: A;t ret =1.37 minutes;[M+H] + =163).

[0265] Experimental Procedure for the Synthesis of A-4a [ka] Unpurified A-3a (22.3 g, 107 mmol, 1.00 equivalent), 1-cyclohexenyloxytrimethylsilane (16.4 mL, 82.3 mmol, 0.80 equivalent), and zinc chloride (10.2 g, 74.8 mmol, 0.70 equivalent) are dissolved in DCM (120 mL) and stirred at ambient temperature for 5 hours. The reaction mixture is treated by adding saturated sodium bicarbonate solution. The organic phase is separated, dried on magnesium sulfate, filtered, and concentrated under reduced pressure. The crude product is purified by NP-chromatography to obtain the desired compound A-4a (HPLC method: A;t ret =1.25 minutes;[M+H] + =283).

[0266] Experimental Procedure for the Synthesis of A-5a [ka] Dissolve A-4a (14.9 g, 57.1 mmol, 1.00 equivalent) and sodium iodide (25.9 g, 171 mmol, 3.00 equivalent) in acetone (120 mL) and stir under reflux for 16 hours. Concentrate the reaction mixture under reduced pressure, dilute with DCM, and wash with saturated sodium thiosulfate solution. Separate the organic phase, dry on magnesium sulfate, filter, and concentrate under reduced pressure. Use the crude product A-5a in the next step without further purification.

[0267] Experimental Procedure for the Synthesis of A-6b [ka] Dissolve A-5a (30.0 g, 85.0 mmol, 1.00 equivalent) in THF. Treat the mixture with potassium tert.-butoxide (28.7 g, 256 mmol, 3.0 equivalent) at 0°C and stir overnight at ambient temperature. Quench the reaction mixture by adding water (2 mL) and dilute by adding Et2O and saturated sodium bicarbonate solution. Separate the organic phase, dry on magnesium sulfate, filter, and concentrate under reduced pressure. Purify the crude product by NP-chromatography to obtain (racemic) compound A-6a (HPLC method: A;t ret =1.17 minutes;[M+H] + =225).

[0268] The reaction sequence A-1a□A-6a is based on Marko et al., THL 2003, 44, 3333-3336 and Maulide et al., Eur.J.Org.Chem. 2004, 19:3962-3967.

[0269] Subsequently, enantiomer A-6b can be obtained after chiral separation by SFC (Lux Cellulose-4 column (250 × 30 mm, 5 μm), column temperature 30°C, 90% CO2, using 10% ACN as a cosolvent), and enantiomer A-6b elutes as a second peak after the other enantiomers (HPLC method: A;t ret =1.17 minutes;[M+H] + =225 / SFC method:SFC-1;t ret =2.99 minutes).

[0270] Alternative procedure for the synthesis of A-6b Process 1 [ka] Add toluene (234 L) to a dry, clean reactor under nitrogen (Note: a total of 2.5V of toluene for this reaction). Add water (1.56 kg, 85.5 mol, maintaining H2O:Pd=160:1), then rinse the feed pipe under nitrogen with 1,1,3,3-tetramethylguanidine (175.5 kg, 1527.6 mol, 2.0 equivalents) and then with toluene (13 L). Add A-7a (130.0 kg, 763.8 mol) under nitrogen, then rinse with toluene (13 L). Add allyl acetate (98.8 kg, 992.9 mol, 1.3 equivalents) under nitrogen, then rinse with toluene (13 L). Cool the mixture to 10°C for 0.5 hours with stirring. Degas the batch by injecting the solution with nitrogen for approximately 30 minutes. Add (S,S)-DACH-Ph Trost ligand (0.429 kg, 0.619 mol, 0.081 mol%) (Note: Maintain Pd:ligand = 1:1.15) in degassed toluene (13 L), then rinse with degassed toluene (13 L). Add allyl palladium(II) chloride dimer (97.5 g, 267 mol, 0.035 mol%) in degassed toluene (13 L), then rinse with degassed toluene (13 L). Maintain the batch at 10-15°C for at least 8 hours. After confirming the completion of the reaction by HPLC, add a solution of N-acetyl-L-cysteine ​​(3.9 kg, 22.9 mol, 0.03 equivalents) in water (260 L) below 25°C. Warm the resulting solution to 20-25°C and maintain at 20-25°C for at least 1 hour. After separating the phases and discarding the lower aqueous layer, 260 L of 10% NH4Cl aqueous solution is added. The mixture is stirred for 10 minutes, and then the lower aqueous layer is drained. The organic phase is further washed with water (130 L). The organic layer is filtered through a very short pad of Celite, and the reactor and Celite bed are rinsed with toluene (65 L). The filtrate is placed in a clean reactor, and then the toluene is removed under vacuum at 40-50°C. The crude product can be used directly in the next step, or the product can be drained into a container with a minimum amount of toluene (65 L) and stored at 20-23°C. 150 kg of A-8a is usually obtained as a pale yellow oil in 96% yield with an enantiomer ratio of 90:10 or higher. 1H NMR (500 MHz, CDCl3): δ 5.75 (ddt, J = 14.8, 9.4, 7.5 Hz, 1H), 5.06 - 5.00 (m, 2H), 4.19 (q, J = 7.1 Hz, 2H), 2.61 (dd, J = 13.9, 7.1 Hz, 1H), 2.51 - 2.43 (m, 3H), 2.33 (dd, J = 13.9, 7.9 Hz, 1H), 2.03 - 1.98 (m, 1H), 1.78 - 1.60 (m, 3H), 1.50 - 1.42 (m, 1H), 1.25 (t, J = 7.1 Hz, 3H). 13C NMR (125 MHz, CDCl3): δ 207.7, 171.6, 133.5, 118.4, 61.3, 61.0, 41.3, 39.4, 35.9, 27.7, 22.6, 14.3. ESI-MS: m / z 211 [M+H] + .

[0271] Project 2

Chem.

[0272] Process 3 [ka] Add 9-BBN (688.5 kg, 401 mol, 1.2 equivalents) to a dry, clean reactor under nitrogen. Cool the solution to 0-5°C to obtain a slurry. Add A-9a (85.0 kg, 334.2 mol) from step 2 at 0-5°C and rinse with THF (40 L). Heat the mixture to 20-23°C for 1 hour and maintain at 20-23°C for at least 1 hour. Then cool the mixture to -45--40°C and add methyl chloroacetate (69.6 kg, 1.3 equivalents) all at once, followed by LiHMDS (909.5 kg, 1102.9 mol) dropwise, maintaining the temperature below -35°C during this time. Then heat the batch to 20-23°C for 1 hour and maintain at 20-23°C for at least 18 hours. Remove the solvent of approximately 12-13V by distillation under vacuum while heating (35°C). Add EtOH (255 kg), followed by a solution of NaOH (13.4 kg) in H2O (212.5 L). Heat the mixture under reflux (at 66-70°C) for at least 14 hours.

[0273] Subsequently, approximately 5-6V of solvent is removed by distillation under reflux, the batch is cooled to 20-25°C, then filtered through a short Celite pad to remove insoluble substances, and rinsed with heptane (160L). Approximately 5-6V of solvent (or most of the residual THF and ethanol) is distilled under vacuum at 40-50°C. The batch is cooled to 20-25°C. Then, water (255L) is added, and the crude product is extracted twice with heptane (2364.8kg). The mixed heptane layer is washed once with water (85L). After solvent removal by distillation under vacuum at 40-50°C, the crude product (52.7kg, 87.5% by mass) is obtained as yellow oil in a 52.6% assay yield. Crude product A-6b is used directly in the next step. 1H NMR (500 MHz, CDCl3): δ 4.01-3.82 (m, 4H), 2.50-2.44 (m, 1H), 2.382.34 (m, 1H), 2.28-2.22 (m, 1H), 2.11-2.05 (m, 1H), 2.01-1.95 (m, 1H), 1.92-1.86 (m, 1H), 1.81-1.58 (m, 6H), 1.54-1.43 (m, 3H), 1.27-1.18 (m, 1H). ESI-MS: m / z 225 [M+H] + .

[0274] Synthesis of alcohol-, pyrazole-, and tosylate-intermediate B Experimental Techniques for the Synthesis of B-2a [ka] Dissolve B-1a (4.92 g, 19.1 mmol, 1.00 equivalent), N,N'-carbonyldiimidazole (5.14 g, 28.6 mmol, 1.50 equivalent), and molecular sieve (3A, 500 mg) in DCM (29.5 mL) and stir at ambient temperature for 40 minutes. After complete activation, add N,O-dimethylhydroxylamine hydrochloride (2.79 g, 28.6 mmol, 1.50 equivalent) and stir the reaction again at ambient temperature for 2 hours. After complete conversion, add water (100 mL) and DCM (150 mL) to separate the phases, and extract the aqueous phase with DCM (2 ×). Wash the mixed organic phase with brine and concentrate under reduced pressure. Purify the residue by NP chromatography to obtain product B-2a.

[0275] The following intermediate B-2 (Table 14) is available in a similar form. The crude product is purified by chromatography as needed. [Table 14]

[0276] Experimental Procedure for the Synthesis of B-3a [ka] Dissolve B-2a (4.88 g, 16.9 mmol, 1.00 equivalent) in THF (15 mL) under an argon atmosphere and cool to -10°C. Add bromo(methyl)magnesium (3.4 M in MeTHF, 6.46 mL, 22.0 mmol, 1.3 equivalents) and stir at -10°C for 1 hour. After complete conversion, cool the reaction mixture to -20°C and quench by adding brine. Extract the resulting mixture by DCM (3×). Concentrate the mixed organic phase under reduced pressure to obtain B-3a.

[0277] The following intermediate B-3 (Table 15) is available in a similar form. The crude product should be purified by chromatography as needed. [Table 15]

[0278] Experimental Procedure for the Synthesis of B-4a [ka] (R)-methyloxazaborolidine (0.99 g, 3.3 mmol, 0.20 equivalents) is dissolved in THF (2 mL) under an argon atmosphere and cooled to -5°C. Borane-dimethyl sulfide complex (1.0 M, 22 mL, 22.0 mmol, 1.3 equivalents) is added. The mixture is stirred at ambient temperature for 30 minutes. The mixture is cooled to -5°C and B-3a (4.1 g, 17 mmol, 1 equivalent) is slowly added dropwise. The reaction is stirred at ambient temperature for 1 hour. After the complete conversion of the starting materials, the reaction is cooled to -10°C and quenched by the addition of MeOH. The mixture is concentrated under reduced pressure. The residue is dissolved in water (150 mL) and formic acid (0.5 mL) and extracted by DCM (3×). The mixed organic phase is concentrated under reduced pressure and purified by NP chromatography to obtain product B-4a.

[0279] The following intermediate B-4 (Table 16) is available in a similar form. The crude product is purified by chromatography as needed. [Table 16]

[0280] Experimental Procedure for the Synthesis of B-5a [ka] Dissolve B-4a (306 mg, 12.5 mmol, 1.00 equivalent) in THF (30.6 mL) under an argon atmosphere. Slowly add lithium aluminum hydride (1 M in THF, 24.9 mL, 25.0 mmol, 2.00 equivalent). Stir the reaction at 60°C for 1 hour. After complete conversion, cool the reaction to ambient temperature, add Rochelle salt solution and KOH, and stir for 1 hour. Extract the resulting suspension by DCM (3×), and concentrate the mixed organic phase under reduced pressure to obtain B-5a.

[0281] The following intermediate B-5 (Table 17) is available in a similar form. The crude product should be purified by chromatography as needed. [Table 17]

[0282] Synthesis of alcohol-, pyrazole-, and tosylate-intermediate B Experimental Procedure for the Synthesis of B-11a [ka] Dissolve 1H-pyrazole-3-carboxylic acid (500 mg, 4.46 mmol, 1.00 equivalent) in ACN (4.5 mL). Add pyrrolidine (745 μL, 8.92 mmol, 2.00 equivalent), DIPEA (1.50 mL, 8.92 mmol, 2.00 equivalent), and 1-propanephosphonic anhydride (2.00 mL, 6.69 mmol, 1.50 equivalent), and stir the reaction mixture at ambient temperature for 1 hour until complete conversion is achieved. Dilute the reaction mixture with saturated NaHCO3, extract by DCM, dry the organic phase, filter, and remove the solvent under vacuum. Purify the crude product by NP chromatography to obtain B-11a (HPLC method: C, t ret =0.14 minutes;[M+H] + =166).

[0283] Experimental Procedure for the Synthesis of B-15a [ka] Mix 3-ethinyloxetan-3-ol (120 mg, 1.16 mmol, 1.00 equivalent) and (trimethylsilyl)diazomethane (2 M solution in hexane, 2.00 mL, 4.00 mmol, 3.46 equivalents) and stir in a closed vial at 50°C for 3 hours until complete conversion is achieved. Cool the reaction mixture to ambient temperature, dilute with MeOH, and remove the solvent under vacuum to obtain unpurified B-15a (HPLC method: C, t ret =0.08min;[M+H] + (=141). The crude product is used in the next step without purification.

[0284] Synthesis of esters and acid E Experimental procedure for the synthesis of intermediate E-9a: [ka] Dissolve 4,6-dichloropyrimidine-2-carboxylic acid E-8a (900 mg, 4.66 mmol, 1.00 equivalent) in DMSO (2 mL), and add DIPEA (1.5 mL, 8.8 mmol, 2.0 equivalent) and (S)-tert-butyl 3-methyl-1,4-diazepane-1-carboxylate (1.04 g, 4.896 mmol, 95% purity, 1.05 equivalent) dropwise. Then, stir the reaction mixture at 40°C for 18 hours. Dilute the mixture with ACN and purify by RP chromatography to obtain the desired product E-9a (HPLC method: A, t ret =0.82 minutes;[M+H] + =371).

[0285] Experimental procedure for the synthesis of intermediate E-11a: [ka] Dissolve E-10a (3.00 g, 14.5 mmol, 1.00 equivalent) in DCM (30 mL), and add DIPEA (5.34 mL, 29 mmol, 2.0 equivalent) and B-5b (3.20 g, 21.8 mmol, 1.5 equivalent). Then, stir the reaction mixture at ambient temperature for 18 hours. After complete conversion, concentrate the mixture, add water, extract the mixture with dimethyl phosphate, wash the organic phase with brine, dry, filter, and concentrate. Purify the crude product by NP chromatography to obtain E-11a.

[0286] The following intermediate E-11 (Table 18) is available in a similar form. Crude product E-11 is purified by chromatography as needed. [Table 18]

[0287] Experimental procedure for the synthesis of E-11e: [ka] Dissolve B-5a (100 mg, 0.48 mmol, 1.00 equivalent) in THF (500 μL), add LiHMDS (591 μL, 0.59 mmol, 1.10 equivalent), and stir for 5 minutes. Meanwhile, dissolve methyl 4,6-dichloropyrimidine-2-carboxylate (170 mg, 0.81 mmol, 1.5 equivalent) in THF (500 μL). Add the B-5a solution to the methyl 4,6-dichloropyrimidine-2-carboxylate solution dropwise over 5 minutes. Stir the reaction for 25 minutes. After complete conversion of the starting materials is observed, filter the reaction and purify by RP chromatography to obtain E-11e (HPLC method: A, t ret =1.08 minutes;[M+H] + =330).

[0288] Synthesis of diketone F If multiple HPLC retention times are reported, it means that different tautomers exist.

[0289] Experimental Techniques for the Synthesis of the F-4a [ka] Dissolve A-6b (1.4g, 5.31 mmol, 1.1 equivalents) and magnesium bromide diethyl etherate (2.5g, 9.66 mmol, 2.0 equivalents) in DCM (10.0 mL). Add F-3a (1.0g, 4.83 mmol, 1.0 equivalent) dissolved in DCM (10 mL) dropwise. Add DIPEA (2.1 mL, 12.08 mmol, 2.5 equivalents) and stir the reaction mixture at ambient temperature for 7 hours. Quench the reaction with 1 M HCl and dilute with DCM and water. Separate the organic phase, evaporate, and purify the resulting residue by RP chromatography to obtain F-4a (HPLC method: C, t ret =0.633 minutes;[M+H] + =399).

[0290] Experimental Techniques for the Synthesis of F-5a [ka] 4,6-Dichloropyrimidine-2-carboxylate methyl ester (2.00 g, 9.67 mmol, 1.00 equivalent) is dissolved in anhydrous ACN (5 mL) under a nitrogen atmosphere. Magnesium bromide diethyl etherate (2.99 g, 11.6 mmol, 1.20 equivalent), a solution of A-6b in ACN (5 mL) (2.38 g, 10.6 mmol, 1.10 equivalent), and DIPEA (2.67 mL, 14.5 mmol, 1.50 equivalent) are added, and the reaction mixture is stirred at 50°C for 20 hours. After complete conversion, the reaction mixture is carefully quenched with HCl (1 M), diluted with water, extracted by DCM, the organic phase is dried, filtered, and concentrated to obtain unpurified F-5a. The unpurified compound is purified by normal-phase chromatography (HPLC method: H, t ret =2.50 minutes; [M+H]=399 / 401).

[0291] Experimental Techniques for the Synthesis of the F-8a [ka] Dissolve F-4a (1.27 g, 2.77 mmol, 1.0 equivalent) in dioxane (10 mL), add aqueous cesium carbonate solution (2 M, 3.46 mL, 6.93 mmol, 2.5 equivalents), and stir at 80°C for 15 minutes. Then, add pyridine-4-boronic acid (357 mg, 2.91 mmol, 1.1 equivalents) and Pd(dppf)Cl2CH2Cl2 (238 mg, 0.28 mmol, 0.1 equivalents) to the reaction mixture and stir at 90°C for 30 minutes until complete conversion of the starting materials is observed. Filter the reaction mixture, dilute with water, and extract three times by DCM. Evaporate the organic phase, dissolve the residue in DMF, and purify by RP chromatography to obtain the desired product F-8a (HPLC method: C, t ret =0.80 / 86 minutes; [M+H]=440).

[0292] Experimental Techniques for the Synthesis of F-9a [ka] Dissolve F-5a (10.0 g, 19.4 mmol, 1.00 equivalent) in DMSO (10 mL), add (1S)-1-[(2S)-1-methylpyrroloridine-2-yl]ethanol (2.76 g, 21.4 mmol, 1.10 equivalent) and DIPEA (6.78 mL, 38.8 mmol, 2.0 equivalent), and stir the solution overnight at ambient temperature. Dilute the reaction mixture with DCM and water. Separate the organic phase, evaporate, and purify the resulting residue by RP chromatography to obtain F-9a (HPLC method: A, t ret =1.58 / 1.66 minutes; [M+H]=492).

[0293] Experimental Techniques for Synthesizing the F-11a [ka] Under an argon atmosphere, dissolve E-9a (1.05 g, 2.83 mmol, 1.00 equivalent) and 1-(1H-imidazole-1-carbonyl)-1H-imidazole (918 mg, 5.66 mmol, 2.00 equivalent) in THF (5 mL) and stir at ambient temperature for 1 hour. After complete activation of the acid, add a solution of A-6b (1.34 mg, 5.98 mmol, 2.00 equivalent) and LiHMDS (1.0 M in THF, 5.95 mL, 5.95 mmol, 2.10 equivalent) to the reaction mixture and wash with THF (5 mL). Stir the resulting mixture overnight at 60°C. After complete conversion, dilute the reaction mixture with an aqueous saturated NaHCO3 solution and extract three times by DCM. Mix the organic phases, dry, filter, and concentrate under reduced pressure. The crude product is dissolved in ACN and water, filtered, and purified by basic RP chromatography to obtain the desired product F-11a (HPLC method: C, t ret =0.888 / 0.936 / 0.978 min; [M+H]=557).

[0294] Experimental Techniques for Synthesizing the F-12a [ka] Dissolve E-11e (1.80 g, 0.01 mol, 1 equivalent) in THF (18 mL), add 3 Å of activated molecular sieve (200 mg per 1 mL of solvent), and stir at 50°C for 20 minutes under an argon atmosphere. Then, add magnesium bromide ethyl etherate (2.11 g, 0.01 mol, 1.5 equivalents) and stir further at 50°C for 30 minutes. Meanwhile, prepare a second solution using A-6b (1.47 g, 0.01 mol, 1.5 equivalents), and pre-dry this using 3 Å of activated molecular sieve in THF (8 mL) at 50°C for 20 minutes. Then, add LiHMDS (1 M in THF, 13.7 mL, 0.01 mol, 2.5 equivalents) and stir for 15 minutes. Then, add the second solution to the first solution and stir at 50°C for 1 hour. After complete conversion, the reaction mixture is carefully quenched with water and THF is removed under reduced pressure. The pH of the residue is adjusted to 7-8 using 1N HCl, extracted with 5% MeOH(2×) in DCM, the mixed organic layer is washed with a salt solution, dried over Na2SO4, filtered, and concentrated to obtain unpurified F-12a. The unpurified compound is purified by NP chromatography.

[0295] The following intermediate F-12 (Table 19) is available in a similar form. The crude product is purified by chromatography as needed. [Table 19] TIFF2026520494000104.tif71169

[0296] Synthesis of isoxazole-intermediate G Experimental Procedure for the Synthesis of G-9a and G-10a [ka] Dissolve F-11a (1.10 g, 1.91 mmol, 1.0 equivalent) in 1,4-dioxane (3 mL), and add hydroxylamine (50% of the volume in water, 140 μL, 2.29 mmol, 1.2 equivalents). Stir the reaction mixture overnight at ambient temperature. After complete conversion, dilute the reaction mixture with a saturated aqueous NaHCO3 solution and extract three times by DCM. Mix the organic phases, dry, filter, and concentrate under reduced pressure to obtain the crude product.

[0297] An unpurified mixture of G-9a and G-10a (1.0 g, 1.68 mmol, 1.0 equivalent) is dissolved in 1,4-dioxane (6 mL), and HCl (4 M in water, 2.11 mL, 8.44 mmol, 5.0 equivalents) is added. The reaction mixture is stirred at ambient temperature for 3 hours. After complete conversion, the reaction is diluted with saturated aqueous NaHCO3 solution and extracted three times by DCM. The organic phases are mixed, dried, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product is dissolved in ACN and water, filtered, and purified by basic RP chromatography to obtain the desired products G-11a and G-12a.

[0298] The following intermediates G-11 and G-12 (Table 20) are available in similar forms. The crude product should be purified by chromatography as needed. [Table 20]

[0299] Experimental Procedure for the Synthesis of G-35a [ka] G-12b (140 mg, 0.31 mmol, 1.0 equivalent) is dissolved in dioxane (2 mL). [1-(oxetan-3-yl)-1H-pyrazole-3-yl]boronic acid (B-9c) (66.3 mg, 0.38 mmol, 1.19 equivalents), XPHOS PD G3 (29.9 mg, 0.03 mmol, 0.1 equivalent), and cesium carbonate (400 μl, 0.80 mmol, 2.54 equivalents) are added. The reaction is stirred at 80°C for 10 minutes under an argon atmosphere. After complete conversion is observed, the reaction is extracted with DCM / water. The mixed organic phase is concentrated under reduced pressure, dissolved in ACN / water, and purified by RP chromatography to obtain the desired product G-35a.

[0300] Experimental Procedure for the Synthesis of A-10a [ka] Add 9-BBN (387 mL, 193.5 mmol, 1.2 equivalents, 0.5 M in THF) to a dry, clean reactor under nitrogen. Cool the solution to 0-5°C to obtain a slurry. Add A-9a (41.0 g, 161.2 mmol) at 0-5°C and rinse with THF (20.5 mL). Heat the mixture to 20-23°C for 1 hour and maintain at 20-23°C for at least 1 hour. After cooling the mixture to -45--40°C, add methyl chloroacetate all at once, followed by LiHMDS (355 mL, 532.0 mmol, 3.3 equivalents) dropwise, maintaining the temperature below -35°C during this time. Then, heat the batch to 20-23°C for 1 hour and maintain at 20-23°C for at least 18 hours. Cool the batch to 5-10°C, and add AcOH (30.4 mL, 3.3 equivalents) below 20°C, followed by water (41 mL) below 20°C. Add AcOH (30.4 mL, 3.3 equivalents) below 20°C until the pH reaches approximately 6-7. Remove approximately 15-16 V of THF under vacuum at below 35°C. Add MTBE (246 mL) and water (205 mL). Separate the phases and discard the lower aqueous layer. Cool the mixture to 0-5°C, and add a solution of sodium percarbonate (37.2 g, 322.4 mmol, 2.0 equivalents) in water (320 mL) below 20°C. After 1 hour at 20-23°C, add 20% sodium sulfite solution (31 mL). After 15 minutes at 20-23°C, separate and discard the lower aqueous layer. The organic layer is washed with 5% by mass ammonium chloride solution (123 mL) and water (328 mL). The organic layer is treated with 5% activated carbon for 30 minutes, and then filtered. After removing approximately 4-5V of solvent under vacuum at a temperature below 35°C, the crude product A-10a (80% yield, HPLC method: C, t) is obtained. ret =0.84 minutes;[M+H] + (=283) is obtained as an orange-brown oil.

[0301] Experimental Procedure for the Synthesis of G-70a [ka] Add A-10a (45.5g, 161.2 mmol), ethanol (91.0 mL), NaOAc (39.7g, 483.6 mmol, 3.0 equivalents), water (45.5 mL), and NH2OH·HCl (33.6g, 483.6 mmol, 3.0 equivalents) to the reactor. Heat the mixture at 73-78°C for at least 16 hours. Cool the batch to 20-23°C and add water (227.6 mL) over 0.5 hours. Then add MTBE (136.5 mL) over 0.5 hours, followed by heptane (113.8 mL) over 1 hour. After 0.5 hours at 20-23°C, collect the solid by filtration. Wash the solid sequentially with MTBE (45 mL) and water (91.0 mL). The solid was dried under vacuum to obtain product G-70a as an off-white solid (18.27 g, 93.2 mass%) in 40% yield. 1H NMR (500 MHz, DMSO-d6): δ 11.48 (br s, 1H), 4.04 (q, J = 6.0 Hz, 1H), 3.89-3.80 (m, 2H), 3.60 (q, J = 6.8 Hz, 1H), 2.10-1.95 (m, 2H), 1.92-1.76 (m, 4H), 1.69-1.39 (m, 8H). ESI-MS: m / z 266 [M+H] + .

[0302] Experimental Procedure for the Synthesis of G-71a [ka] In a clean reactor, add G-70a (100.0 g, 376.9 mmol, 1.0 equivalent) and K3PO4 (240.0 g, 1130.8 mmol, 3.0 equivalents) in water (499.0 g, 500.0 mL) and toluene (432.5 g, 500.0 mL). Stir until the two-phase mixture is thoroughly mixed. After cooling the mixture to 0-5°C, add Tf2O (186.0 g, 110.9 mL, 659.6 mmol, 1.750 equivalents) using a syringe pump over 2 hours at below 5°C. After phase separation, the organic layer is filtered through a Celite bed with Na2SO4. After rinsing with toluene (50 mL), the crude product G-71a (149.8 g, 100% yield) is used directly in the next step. 1H NMR (500 MHz, CDCl3): δ 3.95-3.91(m, 3H), 3.77-3.74 (m, 1H), 2.51-2.44 (m, 2H), 2.16-1.80 (m, 4H), 1.77-1.48 (m, 8H). ESI-MS: m / z 398 [M+H] + .

[0303] Experimental Procedure for the Synthesis of G-72a [ka] Add G-71a (750g, 1.89mol, 1 equivalent), Pd(OAc)2 (8.48g, 37.7mmol, 0.02 equivalents), rac-BINAP (23.5g, 37.7mmol, 0.02 equivalents), 2-MeTHF (3L), EtOH (870g, 18.9mol, 10 equivalents), and DIPEA (293g, 2.26mol, 1.2 equivalents) to a dry, clean autoclave reactor. Purge the reactor twice with nitrogen (100psi), then purge twice with CO (100psi). Pressurize the reactor to 200psi CO and heat at 55-60°C for at least 12 hours. Transfer the mixture to the reactor, rinse the autoclave reactor with 2-MeTHF (0.75L), and place it in the reactor. The mixture is washed with water (3.75 L). After filtration through a short Celite pad, the solvent is removed by vacuum distillation to obtain crude product G-72a (531.9 g, 87.7% yield), which is used in the next step without purification. 1H NMR (400 MHz, CDCl3): δ 4.38 (q, J = 7.1 Hz, 2H), 3.95-3.85 (m, 3H), 3.76-3.73 (m, 1H), 2.85 (dt, J = 17.5, 5.5 Hz, 1H), 2.64 (ddd, J = ESI-MS: m / z 322 [M+H] + .

[0304] Experimental Procedure for the Synthesis of G-73a [ka] In a dry, clean reactor, add G-72a (482.0 g, 1.5 mol, 1 equivalent) and EtOH (3V), and remove residual 2-MeTHF from the previous carbonylation step by vacuum distillation at approximately 3V. Add EtOH (1.45 L) and NH4OH (1.93 L). Maintain the mixture at 20-25°C for at least 15 hours. Add water (1.69 L) over 30 minutes. After 30 minutes at 20-25°C, collect the solid and wash with 1:2 EtOH / water (0.96 L) and water (0.48 L). Slurry the solid in 1:1 MTBE / hexane (0.96 L) for 1 hour. The solid was collected by filtration and dried overnight under vacuum at 40-45°C to obtain product G-73a (332.4 g, 75.8% yield, water content less than 0.5% based on Karl Fischer titration) as a yellowish-brown solid. 1H NMR (500 MHz, DMSO-d6): δ 8.05 (s, 1H), 7.78 (s, 1H), 3.94-3.72 (m, 4H), 2.78 (dt, J = 17.1, 5.0 Hz, 1H), 2.54-2.48 (m, 1H), 2.20-2.14 (m, 1H), 1.93-1.78 (m, 3H), 1.70-1.42 (m, 8H). ESI-MS: m / z 293 [M+H] + .

[0305] Experimental Procedure for the Synthesis of G-74a [ka] In a dry, clean reactor, G-73a (383 g, 86.7% by mass, 1.137 mol, 1 equivalent), MeCN (1.15 L), and pyridine (216 g, 0.19 L, 2.4 equivalents) are added. After cooling the mixture to 0-5°C, trifluoroacetic anhydride (287 g, 1.36 mol, 1.2 equivalents) is added at a temperature below 5°C. After 5 minutes at 0-5°C, water (1.54 L) is added at a temperature below 15°C. The product is extracted with MTBE (1.92 L) and washed with 5% sodium bicarbonate solution (1.15 L). The organic layer is filtered through a silica gel pad (380 g) and rinsed with MTBE (0.58 L). After removing the solvent by distillation under vacuum, product G-74a (421.8 g, 97.8% yield) is obtained as an orange-brown oil. 1H NMR (500 MHz, CDCl3): δ 3.98-3.85 (m, 3H), 3.80-3.75 (m, 1H), 2.72 (dt, J = 17.0, 5.2 Hz, 1H), 2.60 (ddd, J = 17.0, 9.5, 5.8 Hz, 1H), 2.20-2.12 (m, 1H), 2.07-1.94 (m, 3H), 1.82-1.48 (m, 8H). ESI-MS: m / z 275 [M+H] + .

[0306] Experimental Procedure for the Synthesis of G-76a [ka] In a dry flask, add unpurified G-74a (265 g, 72.3% by mass, 698.4 mmol) and catalyst NaOMe (8.0 mL, 25% of MeOH, 34.9 mmol) in MeOH (1590 mL). Stir the mixture at ambient temperature for 1 hour to achieve more than 99% conversion. After adding solid NH4Cl (52.0 g, 977.8 mmol, 1.4 equivalents), stir the resulting mixture at ambient temperature to achieve more than 95% conversion (add more NH4Cl if not). After adding dimethyl malonate (168 g, 1047.7 mmol, 1.5 equivalents) at ambient temperature, add NaOMe (377 g, 25% of MeOH, 2.5 equivalents). Heat the resulting mixture for 4 hours until refluxed to achieve more than 95% conversion. After cooling the mixture to 23°C, water (795 mL) is added, followed by 6N HCl (349 mL) slowly at a temperature below 20°C until the pH reaches approximately 3. MTBE (530 mL) is added to the slurry. After 1 hour at ambient temperature, the solid is collected by filtration and washed with 3V water (796 mL) and MTBE (530 mL) to obtain product G-76a (178 g) as an off-white solid with a 71% unpurified yield. The crude product is used directly in the next step. 1H NMR (500 MHz, CDCl3): δ 5.82 (s, 1H), 3.96-3.74 (m, 4H), 2.74-2.70 (m, 1H), 2.62-2.59 (m, 1H), 2.22-2.10 (m, 1H), 2.12-1.90 (m, 3H), 1.80-1.48 (m, 8H). ESI-MS: m / z 360 [M+H] + .

[0307] Experimental Procedure for the Synthesis of G-77a [ka] In a dry flask, add G-76a (80.0 g, 253.7 mmol), DMAP (4.0 g), tetramethylammonium chloride (4.0 g), and POCl3 (400 mL). Heat the mixture at 80°C for 1.5 hours to achieve a conversion of over 99%. Remove POCl3 under vacuum to obtain a viscous, pale yellow slurry. Add MTBE (160 mL). Then, cool the mixture to 5°C. Slowly add water (800 mL). Stir the resulting white slurry at 23°C for 1 hour. Collect the solid by filtration and then wash sequentially with water (480 mL) and MTBE (160 mL). After drying overnight at 60°C under vacuum, isolate 84.3 g of product G-77a as a white solid with a purity of over 99% and a yield of approximately 93%. 1H NMR (600 MHz, DMSO-d6): δ 8.05(s, 1H),2.96-2.91 (m, 1H), 2.76-2.69 (m, 2H), 2.53-2.48 (m, 2H), 2.37-2.34 (m, 1H), 1.97-1.96 (m, 2H), 1.88-1.82 (m, 4H), 1.70-1.61 (m,1H), 1.52-1.41(m, 1H). 13C NMR (125 MHz, DMSO-d6): δ 209.8, 164.3,161.4, 157.3, 155.7, 120.8, 120.2, 50.3, 38.1, 37.5, 31.0, 26.6, 20.7,19.9, 18.0. ESI-MS: m / z 353 [M+H] + .

[0308] Experimental Procedure for the Synthesis of G-78a [ka] Add LiHMDS (1M in THF) (406.4 kg, 456.1 mol, 1.1 equivalents) to a dry, clean reactor. Cool the solution to 0-5°C, add unpurified A-6b (93.0 kg, 414.6 mol) at below 5°C, and rinse with THF (46.5 kg) to aid in the transfer. After 30 minutes at 0-5°C, add diethyl oxalate (72.5 kg, 497.5 mol, 1.2 equivalents) at below 5°C. Heat the mixture to 20-25°C for 1 hour, then maintain the mixture at 20-25°C for at least 3 hours. After cooling the batch to 10-15°C, the cooled HCl solution [prepared by adding acetyl chloride (73.6 kg, 932.9 mol, 2.25 equivalents) to EtOH (293.9 kg) at 0-5°C] is added to the batch at a temperature below 25°C until a yellow slurry with a final pH of approximately 6-7 is reached. Solid NH2OH·HCl (28.8 kg, 414.4 mol, 1.05 equivalents) is added all at once, and the resulting mixture is heated under reflux at 66-70°C for 6-10 hours. Subsequently, the 5V solvent is removed by distillation under reflux at 66-70°C. EtOH (73.5 kg) is used to remove any remaining THF. Water (372.0 kg) and EtOH (293.9 kg) are added. After 3-6 hours at 70-75°C, the mixture is cooled to 30-35°C. 0.5-1% G-78a crystals are added as a seed. After 2-4 hours at 30-35°C, heptane (63.2 kg) is added over 1 hour or more. After 60 minutes at 20-25°C, water (279.0 kg) is added over 4-6 hours. After 1 hour at 20-25°C, the solid is collected and washed twice with a 1:2 EtOH / water mixture (51.2 kg EtOH and 130.2 kg water), followed by heptane (63.2 kg). The solid is dried under vacuum in a nitrogen stream to obtain product G-78a (93.0 kg) in 65% yield. 1H NMR (500 MHz, CDCl3): δ 4.42 (q, J = 7.1 Hz, 2H), 2.73 (dt, J = 16.8, 5.1 Hz, 1H), 2.64 (dt, J = 14.3, 6.0 Hz, 1H), 2.60-2.51 (m, 2H), 2.43-2.30 (m, 2H), 2.09-1.96 (m, 3H), 1.91-1.81 (m, 3H), 1.76-1.67 (m, 1H), 1.65-1.58 (m, 1H), 1.40 (t, J = 7.1 Hz, 3H). ESI-MS: m / z 278 [M+H] + .

[0309] Experimental Procedure for the Synthesis of G-79a [ka] In a dry, clean reactor, G-78a (72.0 kg, 259.6 mol), EtOH (56.9 kg), and NH4OH (aqueous solution) (280.8 kg) were added. The mixture was maintained at 20-25°C for at least 16 hours. Water (144.0 kg) was added over 30 minutes, and the slurry was maintained at 20-25°C for 30 minutes. The solid was collected by filtration and washed with a 1:3 EtOH / water mixture (28.5 kg EtOH and 108 kg water), followed by heptane (97.9 kg). After drying under vacuum at 23°C for 1 hour, the solid was dried overnight under vacuum at 50-55°C to obtain product G-79a (61.4 kg, 87.2% yield, enantiomer ratio ≥95:5 (254 nm), water content ≤0.5% based on Karl Fischer titration).

[0310] Add unrefined G-79a (60.0 kg, 1.0 equivalent), 1,4-dioxane (240.0 kg), and activated carbon (3.0 kg, 5% by mass) to a dry, clean reactor. Stir the mixture at 55-65°C for 2-4 hours. After filtration at high temperature (55-65°C), wash the filtrate with 1,4-dioxane (33.0 kg). Transfer the filtrate to a clean reactor. Adjust the temperature to 45-55°C and stir at 45-55°C for 1-2 hours. Add water (240.0 kg) over 2 hours. Adjust the temperature to 45-55°C and stir at 45-55°C for 1-2 hours. Cool the mixture to 35-45°C and stir at 35-45°C for 2-4 hours. Add water (87.0 kg) over 4 hours. The mixture is cooled to 15-25°C and stirred at 15-25°C for 12-14 hours. The solid is collected by centrifugation, washed with water (120.0 kg), and dried overnight under vacuum at 50-55°C to obtain product G-79a (44.8 kg, 71% yield) as a pale yellow to off-white solid. Unwanted isomers should be less than 0.5%. 1H NMR (500 MHz, DMSO-d6): δ 7.99 (s, 1H), 7.71 (s, 1H), 2.80-2.69 (m, 1H), 2.60-2.53 (m, 1H), 2.50-2.42 (m, 1H), 2.40-2.28 (m, 2H), 2.26-2.18 (m, 1H), 2.05-1.70 (m, 7H), 1.48-1.39 (m, 1H). ESI-MS: m / z 249 [M+H] + .

[0311] Experimental Procedure for the Synthesis of G-80a [ka] In a dry, clean reactor, G-79a (40.0 kg, 161.1 mol), MeCN (96.0 kg), and pyridine (30.8 kg, 386.6 mol, 2.4 equivalents) are added. After cooling the mixture to 0-5°C, TFAA (40.8 kg, 193.3 mol, 1.2 equivalents) is slowly added at a temperature below 5°C. After 5 minutes at 0-5°C, water (120.0 kg) is added over 30 minutes at 0-5°C, and 0.5% G-80a crystals are added as a seed. After 15 minutes at 0-5°C, water (120.0 kg) is added over 30 minutes. After 30 minutes at 0-5°C, the solid is collected by filtration and washed with a 1:3 MeCN / water mixture (15.6 kg acetonitrile and 60.0 kg water), followed by water (80.0 kg). The solid is dried under vacuum to obtain a crude product (33.0 kg, 93.6% yield) as a yellowish-brown solid.

[0312] Unpurified G-80a (32.5 kg, 1.0 equivalent) and MTBE (48.1 kg) were added to a dry, clean reactor, and the slurry was stirred at 20-25°C for 30 minutes. Heptane (132.6 kg) was added over 1 hour. After 30 minutes at 20-25°C, the solid was collected and dried under vacuum to obtain product G-80a (26.6 kg, 82.0% yield) as a white solid with an enantiomer ratio of over 99:1 (254 nm) and a purity of over 98% (220 nm). 1H NMR (500 MHz, DMSO-d6): δ 2.83-2.73 (m, 1H), 2.60-2.40 (m, 3H), 2.34-2.20 (m, 2H), 2.06-1.75 (m, 7H), 1.53-1.43 (m, 1H). ESI-MS: m / z 231 [M+H] + .

[0313] Experimental Procedure for the Synthesis of G-82a [ka] To a stirred solution of G-80a (25.0 g, 108.6 mmol, 1.0 equivalent) in MeOH (150 mL), NaOMe (30% of MeOH, 4.89 g, 27.1 mmol, 0.25 equivalents) is added, and the resulting mixture is stirred at ambient temperature for 2 hours. Then, NH4Cl (6.39 g, 119.4 mmol, 1.1 equivalents) is added, and the mixture is stirred at ambient temperature for 16 hours. After complete conversion to the desired amidine, the mixture is filtered through a Celite bed and concentrated. The residue is dissolved in DMF (125 mL), and 1,8-diazabicyclo[5.4.0]undec-7-ene (32.3 g, 212.3 mmol, 2.1 equivalents) and diethyl malonate (13.4 g, 101.1 mmol, 1.0 equivalent) are added at 0°C. The resulting mixture is stirred at 90°C for 16 hours. After complete conversion, ice-cold water is added, the mixture is acidified with 1N HCl, and the precipitate is collected by filtration. The precipitate is dried under reduced pressure and unpurified G-82a (HPLC method: H, t ret =1.51 min; [M+H]=316) is obtained and used in the next step without purification.

[0314] Experimental Procedure for the Synthesis of G-83a [ka] Mix G-82a (10.0 g, 30.1 mmol, 1.0 equivalent) and POCl3 (48.0 g, 310.0 mmol, 10.3 equivalents) at 0°C and stir for 5 minutes. Add DIPEA (8.2 g, 63.2 mmol, 2.1 equivalents) and stir the resulting mixture at 80°C for 3 hours. After complete conversion, slowly add ice-cold water (1 L) to the mixture at 0°C, then allow the mixture to reach ambient temperature and stir for 1 hour. Collect the precipitate by filtration, wash with water and hexane, and dry under vacuum to obtain G-83a (HPLC method: H, t ret =2.22 mins; [M+H]=352 / 354). The crude product is used in the next step without purification.

[0315] Experimental Procedure for the Synthesis of G-84a [ka] Dissolve B-5d (694 mg, 4.09 mmol, 1.2 equivalents) in anhydrous THF (13 mL) and cool to 0°C. Add LiHMDS (1.0 M in THF, 5.11 mL, 5.11 mmol, 1.5 equivalents) dropwise at 0°C and stir the mixture for a further 15 minutes. Dissolve G-77a (1.20 g, 3.41 mmol, 1.0 equivalent) in anhydrous THF (13 mL) and add dropwise at 0°C. Stir the mixture at 65°C for 1.5 hours. After complete conversion, dilute the mixture with aqueous saturated NaHCO3 solution and extract three times by DCM. Mix the organic phases, filter, and concentrate under reduced pressure to obtain G-84a. Use the crude product in the next step without purification.

[0316] The following intermediate G-84 (Table 21) is available in a similar form. The crude product should be purified by chromatography as needed. [Table 21]

[0317] Experimental Procedure for the Synthesis of G-15a [ka] Dissolve F-8a (356 mg, 0.804 mmol, 1.0 equivalent) in dioxane (2 mL) and add hydroxylamine solution (50% of water, 98.6 μL, 1.61 mmol, 2.0 equivalents). Stir the resulting solution at 40°C until complete conversion is observed. Evaporate the solvent and purify the resulting residue by RP chromatography to obtain G-13a (G-14a is observed as a byproduct and separated by chromatography). Dissolve G-13a (136.0 mg, 0.29 mmol, 1.0 equivalent) in DCM (2 mL) and add DIPEA (114.38 μL, 0.65 mmol, 2.2 equivalents) and methanesulfonyl chloride (34.2 μL, 0.45 mmol, 1.5 equivalents). Stir the resulting solution at ambient temperature until complete conversion is observed. The reaction mixture is concentrated under reduced pressure and extracted with DCM (3×) and water. The organic solvent is evaporated, and the resulting residue is purified by RP chromatography to obtain G-15a.

[0318] The following intermediate G-15 (Table 22) is available in a similar form. The crude product should be purified by chromatography as needed. [Table 22] TIFF2026520494000125.tif50169

[0319] Experimental Procedure for the Synthesis of G-29a [ka] (1S)-1-[(2S)-1-methylpyrrolidine-2-yl]ethane-1-ol (122 μL mg, 0.865 mmol, 3.0 equivalents) and potassium tert.-butoxide (97.0 mg, 0.865 mmol, 3.0 equivalents) are dissolved in THF (2 mL) and stirred at 50°C for 30 minutes. G-15b (165 mg, 0.28 mmol, 1 equivalent) is added and the solution is stirred at 85°C for 3 hours. The solvent is evaporated and the resulting residue is purified by HPLC chromatography to obtain G-29a. (HPLC method: C, t) ret =1.12 minutes; [M+H]=665).

[0320] Experimental Procedure for the Synthesis of G-30a [ka] Dissolve G-29a (183 mg, 275 μmol, 1.0 equivalent) and HCl (8 M, 172 μL, 1.38 mmol, 5.0 equivalents) in MeOH (2.0 mL) and stir at 60°C until complete conversion is achieved. Concentrate the reaction mixture under reduced pressure and extract with siRNA / NaHCO3. Concentrate the mixed organic phase under reduced pressure to obtain G-30a (HPLC method: A, t ret =1.41 minutes; [M+H]=521).

[0321] Experimental Procedure for the Synthesis of G-34a [ka] G-12b (100 mg, 0.22 mmol, 1.0 equivalent), (S)-5-methyl-4,7-diazaspiro[2.5]octane 2HCl (141 mg, 0.67 mmol, 3.0 equivalents), and DIPEA (230 μL, 0.67 mmol, 6.0 equivalents) are dissolved in DMSO (1 mL). The reaction is stirred at 90°C for 18 hours. After the reaction is complete, the solvent is removed under reduced pressure, and the residue is purified by basic RP chromatography to obtain the desired product G-34a.

[0322] The following intermediate G-34 (Table 23) is available in a similar form. The crude product should be purified by chromatography as needed.

[0323] The diastereomer mixture G-34c can be separated by chiral HPLC (Chiralpack IE, 250 × 20 mm, 5 μm; solvent: ethanol / heptane 60:40 + 0.1% diethylamine) to obtain G-34c1 (first to elute as peak 1) and G-34c2 (later to elute as peak 2). [Table 23] TIFF2026520494000130.tif87169

[0324] Experimental Procedure for the Synthesis of G-45a [ka] Dissolve G-11a (217 mg, 0.505 mmol, 1.0 equivalent) in DMSO (2 mL), and add DIPEA (172 μL, 1.01 mmol, 2.0 equivalents) and N-methylpiperazine (75.8 mg, 0.757 mmol, 1.5 equivalents). Stir the reaction mixture at 90°C until complete conversion is observed. Dilute the mixture with aqueous saturated NaHCO3 solution and extract three times by DCM. Mix the organic phases, filter, and concentrate under reduced pressure. Dissolve the resulting residue in ACN and purify by basic RP chromatography to obtain the desired product G-45a.

[0325] The following intermediate G-45 (Table 24) is available in a similar form. The crude product should be purified by chromatography as needed.

[0326] The diastereomer mixture G-45I was separated by chiral HPLC (column: Chiralpack IE, 250 × 20 mm, 5 μm; solvent: ethanol / heptane 1:1 + 0.1% diethylamine) to obtain G-45I1 (first to elute as peak 1) and G-45I2 (later to elute as peak 2). [Table 24]

[0327] Experimental Procedure for the Synthesis of G-46a [ka] Dissolve 4-(1H-pyrazole-3-yl)pyridine (73.4 mg, 0.51 mmol, 1.50 equivalents) in DMF (1 mL), add NaH (51.7 mg, 1.35 mmol, 4.0 equivalents), and stir at ambient temperature for 20 minutes. Add G-11b (150 mg, 0.34 mmol, 1.0 equivalent) and stir the reaction at 40°C for 1 hour. After complete conversion, extract the reaction with siRNA / water. Concentrate the organic phase under reduced pressure and purify by RP chromatography to obtain the desired product G-46a.

[0328] The following intermediate G-46 (Table 25) is available in a similar form. The crude product should be purified by chromatography as needed. [Table 25]

[0329] Experimental Procedure for the Synthesis of G-48a [ka] G-11d (150 mg, 0.32 mmol, 1.0 equivalent) is dissolved in dioxane (1.5 mL). 1-Methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1h-pyrazole (82.7 mg, 0.39 mmol, 1.2 equivalents), XPHOS PD G3 (26.0 mg, 0.03 mmol, 0.09 equivalents), and cesium carbonate (0.4 mL, 0.80 mmol, 2.46 equivalents) are added. The reaction is stirred at 80°C for 2 hours. After complete conversion is observed, the reaction is extracted with DCM / water. The mixed organic phase is concentrated under reduced pressure, dissolved in ACN / water, and purified by RP chromatography to obtain the desired product G-48a.

[0330] The following intermediate G-48 (Table 26) is available in a similar form. The crude product should be purified by chromatography as needed. [Table 26]

[0331] Experimental Procedure for the Synthesis of G-51a [ka] Dissolve G-11b (150 mg, 0.34 mmol, 1.0 equivalent) in dioxane (18 mL), and add 2-oxazolidone (59.9 mg, 0.67 mmol, 2.0 equivalents), Pd(dppf)Cl2 (24.7 mg, 0.03 mmol, 0.1 equivalent), and NaOtBu (2.0 M in THF, 185 μL, 0.37 mmol, 1.1 equivalent). Stir the reaction at 60°C for 3 days. After complete conversion is observed, filter the reaction and concentrate under reduced pressure. Extract the residue with DCM / water. Concentrate the mixed organic phase under reduced pressure and purify by RP chromatography to obtain G-51a (HPLC method: C, t ret =0.78 min; [M+H]=496).

[0332] Experimental Procedure for the Synthesis of G-63a [ka] Dissolve G-45a (124 mg, 0.251 mmol, 1.0 equivalent) in DCM (1 mL) under argon and cool to 0°C. Add formaldehyde (22.5 μL, 0.301 mmol, 1.2 equivalents), followed by sodium triacetoxyborohydride (224 mg, 1.01 mmol, 4.0 equivalents). Stir the solution at 0°C for 30 minutes. After complete consumption of the starting material, quench the reaction by adding water. Extract the aqueous phase by DCM. Dry the mixed organic phase, filter it, and concentrate under reduced pressure. Purify the residue by RP chromatography to obtain the desired product G-63a.

[0333] The following intermediate G-63 (Table 27) is available in a similar manner. Deuterated intermediate G-63 is obtained similarly, except that sodium triacetoxyborohydride is replaced with sodium triacetoxydeuteride. The crude product is purified by chromatography as needed. [Table 27]

[0334] Experimental Procedure for the Synthesis of G-86a [ka] Dissolve G-12b (2.00 g, 4.23 mmol, 1 equivalent), ethyl 1H-pyrazole-5-carboxylate (936 mg, 6.34 mmol, 1.5 equivalents), and cesium carbonate (4.59 g, 8.46 mmol, 2 equivalents) in THF (20 mL). Stir the reaction at 70°C for 2 hours. After complete conversion, add DCM and wash the solution with water. Concentrate the organic phase under reduced pressure and purify by RP chromatography to obtain G-86a.

[0335] The following intermediate G-86 (Table 28) is available in a similar form. The crude product should be purified by chromatography as needed. [Table 28]

[0336] Experimental Procedure for the Synthesis of G-88a [ka] Dissolve G-11b (4.00 g, 8.99 mmol, 1 equivalent), 2-(1H-pyrazole-3-yl) acetate hydrochloride (1.73 g, 10.34 mmol, 1.15 equivalents), and cesium carbonate (8.79 g, 26.97 mmol, 3 equivalents) in DMSO (20 mL). Stir the reaction at 90°C for 1.5 hours. After complete conversion to the desired intermediate, cool the reaction mixture to ambient temperature, add isopropylamine (1.55 mL, 17.98 mmol, 2.0 equivalents), 1-methylimidazole (1.43 mL, 17.98 mmol, 2.0 equivalents), and chloro-N,N,N',N'-tetramethylformamidinium hexafluorophosphate (5.15 g, 17.98 mmol, 2.0 equivalents), and stir the mixture at ambient temperature for 15 minutes. After complete conversion, DCM is added, and the solution is washed with water and brine. The organic phase is concentrated under reduced pressure and purified by RP chromatography to obtain G-88a.

[0337] The following intermediate G-88 (Table 29) is available in a similar form. The crude product should be purified by chromatography as needed. [Table 29]

[0338] Synthesis of aminocyanothiophene I and II Experimental Techniques for the Synthesis of I-2 [ka] G-30a (80.0 mg, 154 μmol, 1.00 equivalent), malononitrile (64.2 mg, 953 μmol, 6.20 equivalent), sulfur (23.1 mg, 791 μmol, 4.70 equivalent), β-alanine (60.9 mg, 684 μmol, 4.50 equivalent), and magnesium sulfate (23.5 mg, 195 μmol, 1.30 equivalent) were suspended in EtOH (2.0 mL) and stirred at 80°C for 18 hours. The reaction mixture was diluted with HCl, filtered, and washed with aqueous saturated NaHCO3. The organic phase was separated, and the remaining aqueous phase was extracted with HCl (2×). The mixed organic phase was dried on magnesium sulfate and evaporated, and the resulting residue was purified by RP chromatography to obtain I-2.

[0339] The following final compound I (Table 30) is available in a similar form. The crude product should be purified by chromatography as needed. [Table 30]

[0340] Experimental Procedure for the Synthesis of I-37 [ka] G-34h (91.0 mg, 0.160 mmol, 1.00 equivalent), ammonium acetate (26.3 mg, 0.320 mmol, 2.00 equivalent), and sulfur (10.3 mg, 0.320 mmol, 2.00 equivalent) are suspended in EtOH (1.0 mL) and stirred at 60°C for 15 minutes. Malon nitrile (22.3 mg, 0.320 mmol, 2.00 equivalent) is added. The reaction is stirred at 80°C for 5 hours. After complete conversion, the mixture is diluted with DMSO, filtered, and purified by RP chromatography to obtain I-37.

[0341] The following final compound I (Table 31) is available in a similar form. The crude product is purified by chromatography as needed. [Table 31] TIFF2026520494000148.tif181169

[0342] Experimental Procedure for the Synthesis of I-45 [ka] Dissolve I-38 (225 mg, 0.31 mmol, 1.0 equivalent) in DCM / TFA (1:1, 2.0 mL) and stir the reaction at ambient temperature for 3 hours. After complete conversion, concentrate the reaction mixture under vacuum and purify by RP chromatography to obtain I-45 (HPLC method: A, t ret =1.47 minutes; [M+H]=619).

[0343] Experimental Procedure for the Synthesis of I-51 [ka] To a suspension of I-46 (2.73 g, 4.14 mmol, 1.0 equivalent) in ethanol (47 mL), potassium hydroxide (1.91 g, 29.0 mmol, 7.0 equivalents) dissolved in water (53 mL) is added, and the mixture is stirred at ambient temperature for 2 hours. After complete conversion, the mixture is acidified to pH 6, the ethanol is removed under reduced pressure, and the resulting precipitate is collected by repeated centrifugation and washing with water, and dried under reduced pressure to obtain the desired product I-51. The crude product is used without further purification.

[0344] The following final compound I (Table 32) is available in a similar form. The crude product is purified by chromatography as needed. [Table 32]

[0345] Experimental Procedure for the Synthesis of I-53 [ka] To a solution of I-51 (90.1 mg, 0.14 mmol, 1.0 equivalent) in DMSO (0.7 mL), (R)-tetrahydrofuran-3-amine hydrochloride (21.9 mg, 0.17 mmol, 1.2 equivalents), 1-methylimidazole (45.6 μL, 0.57 mmol, 4.0 equivalents), and chloro-N,N,N',N'-tetramethylformamidinium hexafluorophosphate (57.3 mg, 0.20 mmol, 1.4 equivalents) were added, and the mixture was stirred at ambient temperature for 1 hour. After complete conversion, the mixture was diluted with ACN, and the product was isolated by RP chromatography to obtain the desired product I-53.

[0346] The following final compound I (Table 33) is available in a similar form. The crude product is purified by chromatography as needed. [Table 33] TIFF2026520494000154.tif55169

[0347] Experimental Techniques for the Synthesis of II-1 [ka] Dissolve G-11c (1.20 g, 2.59 mmol, 1.00 equivalent), ammonium acetate (319 mg, 4.15 mmol, 1.60 equivalent), and sulfur (133 mg, 4.15 mmol, 1.60 equivalent) in EtOH (12 mL) and stir at 60°C for 15 minutes. Slowly add malon nitrile (3.77 mL, 4.28 mmol, 1.65 equivalent) as a solution in EtOH dropwise (8 mL / hour). Stir the reaction at 80°C for 5 hours. After complete conversion, concentrate the reaction and purify by NP chromatography. Concentrate the product fraction and extract with DCM and saturated NaHCO3. Concentrate the organic phase under reduced pressure to obtain II-1.

[0348] The following final compound II (Table 34) is available in a similar form. The crude product should be purified by chromatography as needed. [Table 34] TIFF2026520494000157.tif146169

[0349] Experimental Techniques for the Synthesis of II-17 [ka] II-1 (0.10 g, 0.18 mmol, 1.0 equivalent) is suspended in DMSO (0.50 ml). DIPEA (0.11 mL, 0.57 mmol, 3.1 equivalents) and (R)-5-methyl-4,7-diazaspiro[2.5]octanedihydrochloride (42 mg, 0.20 mmol, 1.1 equivalents) are added, and the reaction mixture is stirred at 80°C for 2 hours. After complete conversion, the reaction mixture is purified by RP chromatography.

[0350] The following final compound II (Table 35) is available in a similar form. The crude product should be purified by chromatography as needed. [Table 35]

[0351] Experimental Techniques for the Synthesis of II-19 [ka] II-3 (80 mg, 0.15 mmol, 1.0 equivalent) and B-11a (50.3 mg, 0.31 mmol, 2.0 equivalents) are dissolved in THF (1.0 mL), cesium carbonate (123 mg, 0.38 mmol, 2.5 equivalents) is added, and the mixture is stirred at 65°C for 3 hours. After complete conversion, saturated NaHCO3 solution is added, and the product is extracted by DCM. The organic phase is dried, filtered, and concentrated under reduced pressure. The crude product is purified by RP chromatography to obtain the desired final product II-19.

[0352] The following final compound II (Table 36) is available in a similar form. The crude product should be purified by chromatography as needed. [Table 36]

[0353] Experimental Procedure for the Synthesis of II-24 [ka] To a solution of II-23 (100 mg, 0.159 mmol, 1.0 equivalent) in 1-propanol (1 mL), sodium hydroxide (4 M in water, 99.4 μL, 0.40 mmol, 2.5 equivalents) is added, and the mixture is stirred at ambient temperature for 30 minutes. After complete conversion, saturated NaHCO3 is added, the mixture is washed with DCM, and then the aqueous phase is acidified with HCl and extracted with DCM. The organic phase is dried, filtered, concentrated, and the crude product is purified by RP chromatography to obtain the desired product II-24.

[0354] The following final compound II (Table 37) is available in a similar form. The crude product should be purified by chromatography as needed. [Table 37]

[0355] Experimental Procedure for the Synthesis of II-25 [ka] To a solution of II-24 (40 mg, 0.067 mmol, 1.0 equivalent) in DMF (0.4 mL), oxetane-3-amine hydrochloride (15 mg, 0.133 mmol, 2.0 equivalents), DIPEA (22.3 μL, 0.166 mmol, 2.5 equivalents), and 1-propanephosphonic anhydride (29.7 μL, 1.00 mmol, 1.5 equivalents) were added, and the mixture was stirred at ambient temperature for 3 hours. After complete conversion, saturated NaHCO3 was added, and the mixture was extracted by DCM. The organic phase was dried, filtered, concentrated, and the crude product was purified by RP chromatography to obtain the desired product II-25.

[0356] The following final compound II (Table 38) is available in a similar form. The crude product should be purified by chromatography as needed. [Table 38] TIFF2026520494000166.tif98169

[0357] Experimental Procedure for the Synthesis of II-30 [ka] To a solution of G-63a (94.0 mg, 0.18 mmol, 1.0 equivalent) and molecular sieve (3 Å) in anhydrous EtOH (2 mL) under an argon atmosphere, malononitrile (64.4 mg, 0.97 mmol, 5.0 equivalents), sulfur (23.8 mg, 0.74 mmol, 4.0 equivalents), and β-alanine (69.5 mg, 0.78 mmol, 4.0 equivalents) are added. The reaction mixture is stirred overnight at 80°C. After complete conversion, the mixture is cooled to ambient temperature, filtered, and extracted with DCM and aqueous saturated NaHCO3. The organic phases are mixed and concentrated under reduced pressure. The residue is dissolved in ACN and water and purified by basic RP chromatography to obtain the desired product II-30.

[0358] The following final compound II (Table 39) is available in a similar form. The crude product is purified by chromatography as needed. In the case of II-87, Boc deprotection is observed during the reaction using G-48r as the starting material. [Table 39]

[0359] Experimental Procedure for the Synthesis of II-143 [ka] G-51a (90 mg, 0.18 mmol, 1.0 equivalent), ammonium acetate (22.4 mg, 0.29 mmol, 1.6 equivalents), and sulfur (9.32 mg, 0.29 mmol, 1.6 equivalents) are dissolved in EtOH (1.20 mL) and stirred at 60°C for 15 minutes. Malon nitrile (0.26 mL, 0.3 mmol, 1.65 equivalents) is slowly added dropwise as a solution in EtOH. The reaction is stirred at 80°C for 5 hours. After complete conversion, DCM is added and extracted three times with water. The mixed organic phase is concentrated under reduced pressure, dissolved in DMF / ACN / water, and purified by RP chromatography to obtain II-143.

[0360] The following final compound II (Table 40) is available in a similar form. The crude product should be purified by chromatography as needed. [Table 40] TIFF2026520494000171.tif126169

[0361] Experimental Procedure for the Synthesis of II-160 [ka] To a solution of II-146 (210 mg, 0.29 mmol, 1.0 equivalent) in dioxane (3 mL), HCl (4 M in dioxane, 0.29 mL, 1.17 mmol, 4.0 equivalents) is added, and the reaction mixture is stirred at ambient temperature for 18 hours. The reaction mixture is heated to 50°C and stirred for 6 hours. The solvent is removed, and the residue is purified by RP chromatography to obtain II-160.

[0362] The following final compound II (Table 41) is available in a similar form. The crude product is purified by chromatography as needed. [Table 41]

[0363] (Example 5) Synthesis of the compound shown in formula (D) [Table 42] TIFF2026520494000175.tif250169 TIFF2026520494000176.tif201169 Chemical example Unless otherwise indicated, all reactions are carried out using commercially available equipment and methods commonly used in chemical laboratories. Starting materials sensitive to air and / or moisture are stored under a protective gas, and their corresponding reactions and operations are carried out under a protective gas (nitrogen or argon).

[0364] If a compound is represented by both a structural formula and its nomenclature, and a contradiction arises, the structural formula shall prevail.

[0365] chromatography Thin-layer chromatography is performed on a ready-made silica gel 60 TLC glass plate manufactured by Merck (with fluorescence index F-254).

[0366] Preparative high-pressure chromatography (RP HPLC) of the compounds in the examples of the present invention is performed in an Agilent or Gilson system with columns manufactured by Waters (name: SunFire® Prep C18, OBD® 10 μm, 50 × 150 mm, or SunFire® Prep C18 OBD® 5 μm, 30 × 50 mm, or XBridge® Prep C18, OBD® 10 μm, 50 × 150 mm, or XBridge® Prep C18, OBD® 5 μm, 30 × 150 mm, or XBridge® Prep C18, OBD® 5 μm, 30 × 50 mm) and columns manufactured by YMC (name: Actus-Triart Prep C18, 5 μm, 30 × 50 mm) and Chiralpak IE (5 μm, 250 × 20 mm).

[0367] Compounds are eluted using various H2O / acetonitrile gradients. For the Agilent system, a 5% acid modifier (20 mL of HCOOH to 1 L of H2O / acetonitrile (1 / 1)) is added to water (acidic conditions). For the Gilson system, 0.1% HCOOH is added to water.

[0368] For chromatography under basic conditions using the Agilent system, an H2O / acetonitrile gradient was used, while the water was made alkaline by adding a 5% basic modifier (50 mL of NH4HCO3 + NH3 (25% of H2O) mixed with 1 L of H2O). For the Gilson system, the water was made alkaline as follows: 5 mL of NH4HCO3 solution (158 g in 1 L of H2O) and 2 mL of NH3 (28% of H2O) were mixed with 1 L of H2O. The Gilson system was also used under isocratic conditions (60% EtOH / 40% EtOH + 0.1% DEA).

[0369] Supercritical fluid chromatography (SFC) of the intermediates and compounds of the examples of the present invention was performed using an Agilent 1260 SFC system with the following columns: Chiralcel OJ (250×20mm, 5μm), Chiralpak AD-H (21×250mm), 5μm, Chiralpak AD (250×20mm, 5μm), Chiralpak AS (250×20mm, 5μm), Chiralpak IC (250×20mm, 5μm), Chiralpak IA (250×20mm, 5μm), Chiralcel OJ (250×20mm, 5μm), Chiralcel OD (250×20mm, 5μm), Chiralcel OX-3 (150×4.6mm, 3μm), and Phenomenex Lux C2 (250×20mm, 5μm), JASCO. SFC system, or Sepiatec SFC system, or Waters Thar SFC system or Waters UPC 2 -This is done in the MS SFC system.

[0370] Analytical SFC / UV spectroscopy SFC method: SFC-1 SFC: Agilent 1260 (Binary Pump) SFC Column: Chiralpak AD-H (250×4.6mm), 5μm Flow rate: 2ml / min Mobile phase: A:CO2+B:MeOH ABPR: 120 bar Temperature: 37.5℃ UV: 220nm Gradient 80%A + 20%B (Isocratic) Stop time: 10 minutes

[0371] The analysis of intermediates and final compounds by controlled HPLC (reaction control) is performed using columns manufactured by Waters (name: XBridge® C18, 2.5 μm, 2.1 × 20 mm, or XBridge® C18, 2.5 μm, 2.1 × 30 mm, or Aquity UPLC BEH C18, 1.7 μm, 2.1 × 50 mm), YMC (name: Triart C18, 3.0 μm, 2.0 × 30 mm), and Phenomenex (name: Luna C18, 5.0 μm, 2.0 × 30 mm). The analytical equipment is equipped with a mass detector in each case.

[0372] HPLC-Mass Spectrometry / UV Spectroscopy Retention time / MS-ESI for characterizing the compounds of the examples of the present invention + This is generated using an HPLC-MS (high-performance liquid chromatography with a mass detector). The compound eluted at the injection peak is determined by the retention time t Ret. The value = 0.00 is given.

[0373] Method A HPLC Agilent 1100 System MS 1200 Series LC / MSD (API-ES+ / -3000V, Quadruple, G6140) MSD signal settings: Scan positive / negative, 120-1500 m / z Detection signal 315nm (bandwidth 170nm, reference off) Spectral range: 230-400 nm Peak width less than 0.01 minutes Column: Waters, Xbridge C18, 2.5 μm, 2.1 × 20 mm column Column temperature 60℃ Solvent A: 20 mM NH4HCO3 / NH3 aqueous solution pH 9 B: ACN HPLC Grade Flow rate 1.00mL / min Gradient 0.00~1.50 min 10%~95%B 1.50~2.00 minutes 95%B 2.00~2.10 minutes 95%~10%B

[0374] Method E UPLC-MS Waters Acquity-UPLC-SQ Detector-2 MSD signal settings: Scan positive and negative values ​​100-1500. Source voltage: Capillary voltage (kV) -3.50, Ion incorporation pore (V): 50 Source temperature: Desolvation temperature (°C): 350 Source airflow: Desolvation (L / Hr): 750, Ion uptake pores (L / Hr): 50 Detection signal diode array Spectral range: 200-400 nm; Resolution: 1.2 nm Sampling rate: 10 points / second Column AQUITY UPLC BEH C18 1.7μm, 2.1×50mm Column temperature: 35°C Solvent A: 0.07% formic acid in ACN B: 0.07% formic acid in water Flow rate 0.6mL / min Gradient 0.0-0.30 min 97%B 0.30~2.20 minutes 97%~2%B 2.20~3.30 minutes 2%B 3.30~4.50 minutes 2%~97%B 4.50~4.51 minutes 97%B

[0375] Method H HPLC Agilent 1100 / 1200 System MS 1200 Series LC / MSD (MM-ES + APCI + / - 3000V, Quadruple, G6130B) MSD signal setting: Scan positive 700-1350 Column (Waters, part number 186003389, XBridge BEH C18, 2.5 μm, 2.1 × 30 mm) Eluent A: 5mM NH4HCO3 / 18mM NH3(pH=9.2) B: Acetonitrile (HPLC grade) Detection signals: UV 254nm, 230nm, 214nm (bandwidth 8, reference off) Spectrum range: 190-400 nm; Slit: 4 nm Peak duration: over 0.0031 minutes (response time: 0.063 seconds, 80Hz) Injection 0.5μL standard injection Flow rate 1.4mL / min Column temperature 45℃ Gradient 0.0~1.0 min 15% a 95% B 1.0~1.1 minutes 95%B Stop time: 1.3 minutes

[0376] HPLC / UV spectroscopy Method I HPLC Agilent 1100 / 1200 System Chiralpak column; part number 85394; IE, 5 μm; 150 × 2.1 mm Eluent A: n-heptane B: EtOH + 0.1% DEA Detection signal: UV315nm (bandwidth 170, reference off) Spectrum range: 190-400 nm; Slit: 4 nm Peak duration: over 0.0031 minutes (response time: 0.063 seconds, 80Hz) Injection 0.5μL standard injection Flow rate 1.2mL / min Column temperature 45℃ Isocratic 70%B Stop time: 5 minutes

[0377] The compounds of the present invention and their intermediates may be obtained using synthetic methods known to those skilled in the art and described in the literature of organic synthesis. Preferably, the compounds are obtained in a manner similar to the preparation methods fully described below herein, where substituents of the general formula have the meanings set forth herein. These methods are intended as examples of the present invention and do not limit the scope of the subject matter and the compounds claimed for these examples. In some cases, the order in which the reaction steps are carried out may vary. Variants of reaction methods known to those skilled in the art but not described in detail herein may also be used.

[0378] If the preparation of the starting compounds is not described, they are either commercially available, their synthesis is described in the prior art, or they can be prepared in a manner similar to known prior art compounds or methods described herein; that is, the synthesis of these compounds is within the scope of the organic chemist's skill. Substances described in the literature can be prepared according to published synthetic methods. Any functional group of the starting material or intermediate can be protected using conventional protecting groups. These protecting groups can be cleaved again at a suitable step in the reaction sequence using methods well known to those skilled in the art. Where the following chemical structures are shown without the precise arrangement of stereocenters, e.g., asymmetrically substituted carbon atoms, both arrangements should be considered included and disclosed in such representation. The representation of the stereocenters of a racemic mixture is always considered to include and disclose both enantiomers (if no other defined stereocenters are present) or all other potential diastereomers and enantiomers (if additional, defined or undefined stereocenters are present).

[0379] Scheme 1: [ka] Scheme 2: [ka] Experimental Procedure for the Synthesis of K-1a [ka] To a solution of ethyl 1-methyl-2-oxocyclohexane-1-carboxylate (108.00 g, 586.2 mmol) in toluene (1.03 L), malononitrile (58.04 g, 879.3 mmol, 1.5 equivalents), followed by ammonium acetate (9.04 g, 117.2 mmol, 0.2 equivalents) and acetic acid (13.41 mL, 234.5 mmol, 0.4 equivalents) were added at ambient temperature. The mixture was stirred at 110°C for 16 hours. After complete conversion, the mixture was diluted with SiO2, washed with water and brine, dried over sodium sulfate, and concentrated under reduced pressure to obtain crude product K-1a. This unpurified material was used in the next step without further purification (see also Naumann et al., Pharmazie 51 (1996), 4). [Table 43]

[0380] Experimental Techniques for the Synthesis of K-2a [ka] To a solution of K-1a (250.0 g, 1.1 mol) in DMF (3.0 L), sulfur (68.9 g, 2.2 mol, 2.0 equivalents) and L-proline (24.8 g, 0.22 mol, 0.2 equivalents) are added, and the resulting mixture is stirred at 80°C for 12 hours. After complete conversion, the mixture is partitioned into RINKAN and water, and the organic layer is collected. The aqueous layer is further extracted with RINKAN, the mixed organic layer is washed with water and brine, dried over sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product is purified by column chromatography to obtain K-2a. [Table 44]

[0381] Experimental Procedure for the Synthesis of K-3a [ka] Dissolve K-2a (78.0 mg, 0.3 mmol, 1.0 equivalent) in EtOH (1.5 mL) and add potassium hydroxide (4 M in water, 0.37 mL, 1.5 mmol, 5.0 equivalents). Stir the mixture at 78°C for 16 hours. After complete conversion, add water and siRNA to the reaction mixture, set the pH of the aqueous phase to pH 4 using KHSO4 solution (10% in water), and extract the product with siRNA. Dry the mixed organic layer, filter, and concentrate. Purify the crude product by acid reverse-phase chromatography (gradient elution: 20%-90% acetonitrile in water) to obtain K-3a.

[0382] Enantiomers can be separated by preparative SFC chromatography. For example, K-3a to K-3b and its enantiomers. (Analytical SFC method SFC-1:t) ret = 4.9 min (K-3b), 7.9 min (other enantiomers). [Table 45]

[0383] Experimental Techniques for the Synthesis of K-9a [ka] To a solution of (S)-tert-butyl-3-methyl-1,4-diazepane-1-carboxylate (846.0 mg, 214.30 mmol, 1.0 equivalent) and 2-chloropyrimidine-4-carbonitride (528.9 mg, 139.54 mmol, 1.0 equivalent) in DMSO (4 ml, 4.5 V), TEA (1.1 ml, 101.19 mmol, 2.0 equivalent) is added at ambient temperature. The reaction mixture is stirred at 80 °C for 1 hour. After complete conversion, the reaction mixture is cooled to ambient temperature, and water and siRNA are added. The phases are separated. The organic layer is washed with water, dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product, which is purified by chromatography to obtain K-9a. [Table 46]

[0384] Experimental Procedure for the Synthesis of K-10a [ka] To a solution of K-9a (33.85 g, 106.65 mmol, 1.0 equivalent) in EtOH (270 ml), add 50% hydroxylamine solution in water (13.05 ml, 213.30 mmol, 2.0 equivalents) at ambient temperature. Stir the reaction mixture at 60°C for 1 hour. After complete conversion, concentrate the reaction mixture under reduced pressure to obtain K-10a, which is used in the next step without further purification. [Table 47]

[0385] Experimental Procedure for the Synthesis of K-11a [ka] To a stirred solution of K-3b (2.53 g, 10.70 mmol, 1.0 equivalent) in DMSO (10 ml), TEA (2.17 g, 21.40 mmol, 2.0 equivalent) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 4.27 g, 11.24 mmol, 1.10 equivalent) are added at ambient temperature. The mixture is stirred at ambient temperature for 15 minutes. K-10a (3.75 g, 10.70 mmol, 1.0 equivalent) is added at ambient temperature and stirred overnight. After complete conversion, the reaction mixture is diluted with water and siRNA. The phases are separated. The organic layer is washed with water, dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product.

[0386] The crude product K-11 is purified by chromatography as needed. [Table 48]

[0387] Experimental Procedure for the Synthesis of K-12a [ka] To a stirred solution of K-11a (2.00 g, 3.51 mmol, 1.0 equivalent) in THF (40 mL), DBU (1.98 mL, 14.04 mmol, 4.0 equivalents) is added at ambient temperature. The reaction mixture is stirred overnight at 70 °C. After complete conversion, the reaction mixture is concentrated under reduced pressure to obtain the crude product. The crude product is purified by column chromatography to obtain K-12a.

[0388] The crude product K-12 is purified by chromatography as needed. [Table 49]

[0389] Experimental Procedure for the Synthesis of K-13a [ka] To a stirred solution of K-12a (20.00 g, 34.52 mmol, 1.0 equivalent) in MeOH (350 mL), concentrated HCl (32.88 mL, 345.21 mmol, 10.0 equivalent) is added at ambient temperature. The reaction mixture is stirred at 50 °C for 2 hours. After complete conversion, the reaction mixture is concentrated under reduced pressure and diluted with water. The aqueous phase is extracted by DCM. The mixed organic layer is dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain K-13a, which is used in the next step without further purification.

[0390] The crude product K-13 is purified by chromatography as needed. [Table 50]

[0391] Experimental Procedure for the Synthesis of E-1a [ka] To a stirred solution of methyl 2-(3-hydroxy-1,2-oxazole-5-yl)-3-methylbutanoate (15.00 g, 0.08 mol, 1.0 equivalent) in DMF (75.0 mL), potassium carbonate (31.17 g, 0.23 mol, 3.0 equivalent) was added at 0°C. 1,3-dibromopropane (15.20 g, 0.08 mol, 1.0 equivalent) was added dropwise, and the reaction mixture was stirred at 0°C for 9 hours. After complete conversion, the reaction mixture was quenched with water and extracted with RINKAN. The organic layer was washed with ice water, dried on sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The resulting unpurified compound was purified by chromatography to obtain E-1a.

[0392] The following intermediate E-1 (Table 43) is available in a similar form. Purify the crude product E-1 by chromatography as needed. [Table 51]

[0393] Experimental Techniques for the Synthesis of E-2a [ka] To a stirred solution of K-13a (4.50 g, 9.99 mmol, 1.0 equivalent) and E-1a (3.72 g, 11.03 mmol, 1.1 equivalent) in acetonitrile (45.0 mL), potassium carbonate (2.76 g, 19.98 mmol, 2.0 equivalent) is added, and the mixture is stirred under argon at 60°C for 22 hours. After complete conversion, the reaction mixture is cooled to ambient temperature, filtered, and the solid is washed with acetonitrile. The mixed solution is concentrated under reduced pressure and purified by chromatography to obtain E-2a.

[0394] The following intermediate E-2 (Table 52) can be obtained in a similar manner starting from different intermediates K-13 and E-1 or alternative bromides. The crude product E-2 is purified by chromatography as needed. [Table 52]

[0395] Experimental Techniques for the Synthesis of E-3a [ka] To a stirred solution of E-2a (4.26 g, 6.18 mmol, 1.0 equivalent) in methanol (21.0 mL), sodium hydroxide solution (2 M in water, 6.18 mL, 12.35 mmol, 2.0 equivalents) is added, and the reaction mixture is stirred at 45°C for 1 hour. After complete conversion, the reaction mixture is concentrated under reduced pressure. The crude product is purified by chromatography to obtain E-3a.

[0396] The following intermediate E-3 (Table 53) can be obtained in a similar manner starting from a different intermediate E-2. The crude product E-3 is purified by chromatography as needed. [Table 53]

[0397] Experimental Techniques for the Synthesis of I-1 [ka] To a stirred solution of E-3a (219 mg, 0.32 mmol, 1.0 equivalent), (2S,4R)-4-hydroxy-N-{[4-(4-methyl-1,3-thiazole-5-yl)phenyl]methyl}pyrrolidine-2-carboxamide (113 mg, 0.36 mmol, 1.1 equivalent), and HATU (184 mg, 0.48 mmol, 1.3 equivalent) in DMF (1.0 mL), DIPEA (0.16 mL, 0.97 mmol, 3.0 equivalent) is added, and the reaction mixture is stirred at ambient temperature for 30 minutes. After complete conversion, the reaction mixture is quenched with water, diluted with acetonitrile, and purified by chromatography.

[0398] Compound I (Table 54) below is available in a similar manner, starting from different intermediates E-3 and A-4. [Table 54] TIFF2026520494000203.tif54169

[0399] Chiral separation of compound I by chiral column chromatography: When compound I is obtained as a mixture of diastereomers, they can be separated into a single stereoisomer by chiral chromatography, as shown for example for I-3 separated into I-26 and I-27 (Table 55). [Table 55] TIFF2026520494000205.tif102169

[0400] (Example 6) Synthesis of the compound shown in formula (E) [Table 56] TIFF2026520494000207.tif249169 TIFF2026520494000208.tif218169 Preparation of the compound of the present invention Unless otherwise indicated, all reactions are carried out using commercially available equipment and methods commonly used in chemical laboratories. Starting materials sensitive to air and / or moisture are stored under a protective gas, and their corresponding reactions and operations are carried out under a protective gas (nitrogen or argon).

[0401] If a compound is represented by both a structural formula and its nomenclature, and a contradiction arises, the structural formula shall prevail.

[0402] The microwave reaction is carried out in a sealed container (preferably 2, 5, or 20 mL) with stirring, preferably in an initiator / reactor manufactured by Biotage, or in an Explorer manufactured by CEM, or in a Synthos 3000 or Monowave 3000 manufactured by Anton Paar.

[0403] chromatography Thin-layer chromatography is performed on a ready-made silica gel 60 TLC glass plate manufactured by Merck (with fluorescence index F-254).

[0404] Preparative high-pressure chromatography (RP HPLC) of the compounds of the examples of the present invention is performed in an Agilent or Gilson system with columns manufactured by Waters (name: SunFire® Prep C18, OBD® 10 μm, 50 × 150 mm, or SunFire® Prep C18 OBD® 5 μm, 30 × 50 mm, or XBridge® Prep C18, OBD® 10 μm, 50 × 150 mm, or XBridge® Prep C18, OBD® 5 μm, 30 × 150 mm, or XBridge® Prep C18, OBD® 5 μm, 30 × 50 mm) and columns manufactured by YMC (name: Actus-Triart Prep C18, 5 μm, 30 × 50 mm).

[0405] Compounds are eluted using various H2O / acetonitrile gradients. For the Agilent system, a 5% acid modifier (20 mL of HCOOH to 1 L of H2O / acetonitrile (1 / 1)) is added to water (acidic conditions). For the Gilson system, 0.1% HCOOH is added to water.

[0406] For chromatography under basic conditions using the Agilent system, an H2O / acetonitrile gradient is used, while the water is made alkaline by adding a 5% basic modifier (50g of NH4HCO3 + 50mL of NH3 (25% of H2O) diluted to 1L with H2O). For the Gilson system, the water is made alkaline as follows: 5mL of NH4HCO3 solution (158g in 1L of H2O) and 2mL of NH3 (28% of H2O) are diluted to 1L with H2O.

[0407] Supercritical fluid chromatography (SFC) of the intermediates and compounds of the examples of the present invention is performed in a JASCO SFC system with the following columns: Chiralcel OJ (250×20mm, 5μm), Chiralpak AD (250×20mm, 5μm), Chiralpak AS (250×20mm, 5μm), Chiralpak IC (250×20mm, 5μm), Chiralpak IA (250×20mm, 5μm), Chiralcel OJ (250×20mm, 5μm), Chiralcel OD (250×20mm, 5μm), and Phenomenex Lux C2 (250×20mm, 5μm).

[0408] The analysis of intermediates and final compounds by controlled HPLC (reaction control) is performed using columns manufactured by Waters (name: XBridge® C18, 2.5 μm, 2.1 × 20 mm, or XBridge® C18, 2.5 μm, 2.1 × 30 mm, or Aquity UPLC BEH C18, 1.7 μm, 2.1 × 50 mm), YMC (name: Triart C18, 3.0 μm, 2.0 × 30 mm), and Phenomenex (name: Luna C18, 5.0 μm, 2.0 × 30 mm). The analytical equipment is equipped with a mass detector in each case.

[0409] HPLC-Mass Spectrometry / UV Spectroscopy Retention time / MS-ESI for characterizing the compounds of the examples of the present invention + This is generated using an HPLC-MS (high-performance liquid chromatography with a mass detector). The compound eluted at the injection peak is determined by the retention time t Ret. The value = 0.00 is given.

[0410] Method A HPLC Agilent 1100 System MS 1200 Series LC / MSD (API-ES+ / -3000V, Quadruple, G6140) MSD signal settings: Scan positive / negative, 120-900 m / z Detection signal 315nm (bandwidth 170nm, reference off) Spectral range: 230-400 nm Peak width less than 0.01 minutes Column: Waters, Xbridge C18, 2.5 μm, 2.1 × 20 mm column Column temperature 60℃ Solvent A: 20 mM NH4HCO3 / NH3 pH 9 in H2O B: ACN HPLC Grade Flow rate 1.00mL / min Gradient 0.00~1.50 min 10%~95%B 1.50~2.00 minutes 95%B 2.00~2.10 minutes 95%~10%B

[0411] Method C Agilent HPLC 1260 Series MS Agilent LC / MSD Quadrupole Detection MS: Positive and negative modes Mass range 100~750m / z Column: Waters X-Bridge BEH C18, 2.5 μm, 2.1 × 30 mm XP Column temperature 45℃ Solvent A: 20 mM NH4HCO3 / 30 mM NH3 in H2O; B: ACN (HPLC grade) Flow rate 1.40mL / min Gradient 0.00~1.00 min: 15%B~95%B 1.00~1.30 minutes: 95%B

[0412] Method H UPLC-MS Waters Acquity-Binary Solvent Manager-UPLC-SQ Detector-2 MSD signal settings: Scan positive and negative values ​​100-1500. Source voltage: Capillary voltage (kV) -3.50, Ion incorporation pore (V): 50 Source temperature: Desolvation temperature (°C): 350 Source airflow: Desolvation (L / Hr): 750, Ion uptake pores (L / Hr): 50 Detection signal diode array Spectral range: 200-400 nm; Resolution: 1.2 nm Sampling rate: 10 points / second Column AQUITY UPLC BEH C18 1.7μm, 2.1×50mm Column temperature: 35°C Solvent A: 0.07% formic acid in ACN B: 0.07% formic acid in water Flow rate 0.6mL / min Gradient 0.0-0.40 min 97%B 0.40~2.50 minutes 97%~2%B 2.50~3.40 minutes 2%B 3.40~3.50 minutes 2%~97%B 3.50~4.0 minutes 97%B

[0413] GCMS Method U GC Agilent Technologies-7890B GC system, with 7693 autosampler and 5977A MSD Injection temperature 230℃ Column flow rate: 2.0 mL / min Solvent delay: 1.5 minutes Split ratio: 10:01 Column oven temperature program: 100°C / 1 min, 20°C / 310°C / 5 min Total runtime: 16 minutes Interface temperature 150℃ Ion source temperature: 230℃ gaseous He Column and column dimensions: ZB-5MS (30m x 0.32mm; 1μm) MSD scan range 50-900

[0414] Method V GC Agilent Technologies-7890B GC system, with 7693 autosampler and 5977A MSD Injection temperature 230℃ Column flow rate: 2.0 mL / min Solvent delay: 1.5 minutes Split ratio: 10:01 Column oven temperature programs: 40°C / 2 min, 15°C / min / 200°C / 1 min, 25°C / min / 310°C / 0 min. Total runtime: 18 minutes Interface temperature 150℃ Ion source temperature: 230℃ gaseous He Column and column dimensions: ZB-5MS (30m x 0.32mm; 1μm) MSD scan range 50-900

[0415] Method W GC Agilent Technologies-7890B GC system, with 7693 autosampler and 5977A MSD Injection temperature 230℃ Column flow rate: 2.0 mL / min Solvent delay: 1.5 minutes Split ratio: 10:01 Column oven temperature program: 60°C / 3 min, 20°C / min / 310°C / 2 min Total runtime: 18 minutes Interface temperature 150℃ Ion source temperature: 230℃ gaseous He Column and column dimensions: ZB-5MS (30m x 0.32mm; 1μm)

[0416] Method SFC-1 Manufacturing Waters UPC 2 -MS Software Empower3 MS QDa Column: CHIRALCEL OX-3 (4.6*150MM) 3μm A - Solvent CO2 B-solvent ACN Total flow rate 3g / min % 15 relative to the cosolvent ABPR 1500psi Column temperature: 30°C PDA range: 200nm~400nm Resolution 1.2nm MS Parameters - QDa MS scan range: 100Da to 1000Da Ion intake pore voltage Positive scan 20V Negative scan 15V

[0417] The compounds and intermediates of the present invention are prepared by the synthetic methods described herein, in which substituents of the general formula have the meanings set forth herein. These methods are intended as examples of the present invention and do not limit the scope of the subject matter and the compounds claimed for these examples. Where the preparation of the starting compounds is not described, they are either commercially available, or their synthesis is described in the prior art, or they can be prepared similarly to known prior art compounds or methods described herein; that is, synthesizing these compounds is within the scope of the organic chemist's skill. Substances described in the literature can be prepared according to the published synthetic methods. Where the following chemical structures are shown without the exact arrangement of stereocenters, e.g., asymmetrically substituted carbon atoms, both arrangements should be considered included and disclosed in such representation. The representation of the stereocenters of a racemic mixture is always considered to include and disclose both enantiomers (if no other defined stereocenters (one or more) are present) or all other potential diastereomers and enantiomers (if additional, defined or undefined stereocenters are present).

[0418] Synthesis of spiroketone intermediate A Experimental Procedure for the Synthesis of A-2a [ka] To a suspension of 5-chloropentanenitrile (22.9 g, 195 mmol, 1.00 equivalent) in EtOH (136 mL), acetyl chloride (111 mL, 1.56 mol, 8.00 equivalent) is added dropwise at 0°C. The reaction mixture is warmed to ambient temperature and stirred for 12 hours. The mixture is concentrated under reduced pressure and washed with Et2O. The crude product A-2a is used directly in the next step as an HCl salt without further purification (HPLC method: A;t ret =1.03 minutes;[M+H] + =164).

[0419] Experimental Procedure for the Synthesis of A-3a [ka] Dissolve unpurified A-2a (HCl salt) (28.0 g, 140 mmol, 1.00 equivalent) and ethylene glycol (7.38 g, 119 mmol, 0.90 equivalent) in DCM (300 mL) and stir at ambient temperature for 6 days. Concentrate the resulting suspension under reduced pressure, dilute with Et2O (200 mL), and filter. Concentrate the filtrate under reduced pressure, transfer to DCM (200 mL), and treat with KOH solution (2 M in water, 150 mL). Stir the mixture overnight at ambient temperature while maintaining the phase intact. Separate the phases, extract the aqueous phase with DCM (2 ×), dry the mixed organic phase over magnesium sulfate, filter, and concentrate under reduced pressure. Use the unpurified orthoester A-3a in the next step without further purification (HPLC method: A;t ret =1.37 minutes;[M+H] + =163).

[0420] Experimental Procedure for the Synthesis of A-4a [ka] Unpurified A-3a (22.3 g, 107 mmol, 1.00 equivalent), 1-cyclohexenyloxytrimethylsilane (16.4 mL, 82.3 mmol, 0.80 equivalent), and zinc chloride (10.2 g, 74.8 mmol, 0.70 equivalent) are dissolved in DCM (120 mL) and stirred at ambient temperature for 5 hours. The reaction mixture is treated by adding saturated sodium bicarbonate solution. The organic phase is separated, dried on magnesium sulfate, filtered, and concentrated under reduced pressure. The crude product is purified by NP chromatography to obtain the desired compound A-4a (HPLC method: A;t ret = 1.25 minutes; [M+Na] + =283).

[0421] Experimental Procedure for the Synthesis of A-8a [ka] Dissolve A-4a (14.9 g, 57.1 mmol, 1.0 equivalent) and sodium iodide (26.0 g, 171 mmol, 3.0 equivalents) in acetone (120 mL) and stir under reflux for 16 hours. Concentrate the reaction mixture under reduced pressure, dilute with DCM, and wash with saturated sodium thiosulfate solution. Separate the organic phase, dry it over MgSO4, filter it, and concentrate it under reduced pressure. Use the crude product A-5a in the next step without further purification.

[0422] Dissolve A-5a (30 g, 85.0 mmol, 1.0 equivalent) in THF. Treat the mixture with potassium tert.-butoxide (28.7 g, 256 mmol, 3.0 equivalents) at 0°C and stir overnight at ambient temperature. Quench the reaction mixture by adding water (2 mL) and dilute by adding Et2O and saturated sodium bicarbonate solution. Separate the organic phase, dry it over MgSO4, filter it, and concentrate it under reduced pressure. Purify the crude product by NP chromatography to obtain compound A-6a (racemic mixture) (reaction sequences A-1a to A-6a are based on Marko et al., THL 2003, 44, 3333-3336 and Maulide et al., Eur. J. Org. Chem. 2004, 19:3962-3967).

[0423] Subsequently, enantiomer A-6b was subjected to chiral separation by SFC using the following conditions (column: Lux; Cellulose-4 (250mm × 30mm × 5μm), 90% CO2, 10% ACN, flow rate: 90g / min, temperature: 30℃), followed by enantiomer A-6b (SFC method: SFC-1; t ret Peak 2 (=2.99 mins) can be obtained as peak 2 after enantiomer elution.

[0424] Synthesis of diketone F If multiple HPLC retention times are reported, it means that different tautomers exist.

[0425] Experimental Techniques for the Synthesis of the F-1a [ka] 4,6-Dichloropyrimidine-2-carboxylate methyl ester E-4a (2.00 g, 9.67 mmol, 1.00 equivalent) is dissolved in anhydrous ACN (5 mL) under a nitrogen atmosphere. Magnesium bromide diethyl etherate (2.99 g, 11.6 mmol, 1.20 equivalent), a solution of A-6b (2.38 g, 10.6 mmol, 1.10 equivalent) in ACN (5 mL), and DIPEA (2.67 mL, 14.5 mmol, 1.50 equivalent) are added, and the reaction mixture is stirred at 50°C for 20 hours. After complete conversion, the reaction mixture is carefully quenched with 1 M HCl, diluted with water, extracted by DCM, the organic phase is dried, filtered, and concentrated to obtain unpurified F-1a. The unpurified compound is purified by NP chromatography. (HPLC method: H, t) ret =2.50 minutes; [M+H]=399 / 401).

[0426] Experimental Techniques for Synthesizing the F-2a [ka] Dissolve F-1a (10.0 g, 19.4 mmol, 1.00 equivalent) in DMSO (10 mL), add (1S)-1-[(2S)-1-methylpyrroloridine-2-yl]ethanol (2.76 g, 21.4 mmol, 1.10 equivalent) and DIPEA (6.78 mL, 38.8 mmol, 2.0 equivalent), and stir the solution overnight at ambient temperature. Dilute the reaction mixture with DCM and water. Separate the organic phase, evaporate, and purify the resulting residue by RP chromatography to obtain F-2a. (HPLC method: A, t) ret =1.58 / 1.66 minutes; [M+H]=492).

[0427] Synthesis of isoxazole intermediate G Experimental procedures for the synthesis of intermediates G3 and G4 [ka] Dissolve F-3a (1.10 g, 1.91 mmol, 1.0 equivalent) in 1,4-dioxane (3 mL) and add 50% aqueous hydroxylamine (140 μL, 2.29 mmol, 1.2 equivalents). Stir the reaction mixture overnight at ambient temperature. After complete conversion of the starting materials, dilute the reaction with aqueous saturated NaHCO3 solution and extract three times by DCM. Mix the organic phases, dry, filter, and concentrate under reduced pressure to obtain the crude product. Dissolve an unpurified mixture of G-1a and G-2a (1.00 g, 1.68 mmol, 1.0 equivalent) in 1,4-dioxane (6 mL) and add 4 M aqueous HCl (2.11 mL, 8.44 mmol, 5.0 equivalents). Stir the reaction mixture at ambient temperature for 3 hours. After complete conversion of the starting materials is observed, dilute the reaction with aqueous saturated NaHCO3 solution and extract three times by DCM. The organic phases are mixed, dried, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product is dissolved in ACN and water, filtered, and purified by basic RP chromatography to obtain the desired product G-3a and the corresponding isoxazole positional isomer G-4a.

[0428] The following intermediates G-3 and G-4 (Table 57) can be obtained from the preferred intermediate F in a similar manner. The crude product is purified by chromatography as needed. [Table 57]

[0429] Experimental procedure for the synthesis of G-9 and G-10 (Method IV) [ka] G-4b (150 mg, 0.3 mmol, 1.0 equivalent), 2-hydroxythiazole (39.4 mg, 0.39 mmol, 1.30 equivalents), and t-BuONa (2M in THF, 210 μL, 0.42 mmol, 1.4 equivalents) are dissolved in THF (1.5 mL) and stirred at 80°C for 18 hours. After complete conversion, the reaction mixture is extracted three times with DCM / H2O. The mixed organic phase is concentrated under reduced pressure and purified by RP chromatography to obtain the desired product G-10a.

[0430] The following intermediates G-9 and G-10 (Table 58) can be obtained from G-3b and G-4b in a similar manner. The crude product is purified by chromatography as needed. [Table 58]

[0431] Experimental Techniques for the Synthesis of G-9 [ka] G-9h (297 mg, 476 μmol, 1.0 equivalent) is dissolved in DCM (0.91 mL) and trifluoroacetic acid (0.99 mL, 4.76 mmol, 10.0 equivalents). The reaction is stirred at ambient temperature for 4 hours. After complete conversion, the dissolved material is removed under reduced pressure. The residue is dissolved in DCM and extracted with aqueous saturated Na2CO3. The mixed organic phase is dried, filtered, and concentrated under reduced pressure. The residue is purified by RP chromatography to obtain G-9s.

[0432] The following intermediate G-9 (Table 59) can be obtained from G-9h and G-9i in a similar manner. The crude product is purified by chromatography as needed. [Table 59]

[0433] Synthesis of aminocyanothiophenes H, I, and II Experimental Techniques for Converting from G-9 to II [ka] To a solution of G-9a (75.0 mg, 0.149 mmol, 1.0 equivalent) and molecular sieve (3 Å) in anhydrous methanol (4 mL) under an argon atmosphere, malononitrile (20.7 mg, 0.297 mmol, 2.0 equivalents), sulfur (7.15 mg, 0.223 mmol, 1.5 equivalents), and β-alanine (16.7 mg, 0.178 mmol, 1.2 equivalents) are added. The reaction mixture is stirred overnight at 80°C. After complete conversion, the mixture is cooled to ambient temperature, filtered, and extracted with DCM and aqueous saturated NaHCO3. The organic phases are mixed and concentrated under reduced pressure. The residue is dissolved in acetonitrile and water and purified by basic RP chromatography to obtain the desired product II-1.

[0434] Compound II (Table 60) below can be obtained from the corresponding ketone G-9 in a similar manner. The crude product is purified by chromatography as needed. [Table 60] TIFF2026520494000223.tif51169

[0435] (Example 7) Synthesis of the compound shown in formula (F) The synthesis of the compound shown in formula (F) is described in WO2021 / 213800.

[0436] (Example 8) In vitro and in vivo assessment of survival levels - Materials and methods Cell Titer Glow (CTG) assay The Cell Titer Glow cell viability assay (Promega) was performed in two different cell lines using 384-well plates (VIEWPLATE-384TC, Perkin Elmer, catalog no. 60007480) (Table 1). Eleven different KRASG12C and nine KRASG12D inhibitors were used. Both cell lines were grown according to the ATCC standard protocol, as shown in Table 61. [Table 61]

[0437] NCI-H358 and SW1990 cell lines were seeded in 384-well plates in 40 μL of total medium at a density of 500 cells / well, in three sets each. The following day, the cells were treated for 120 hours with KRASG12D and KRASG12C inhibitors, starting at a concentration of 3 μM and then diluted 1:3. On day 5, CTG reagent was added to each well, and luminescence was measured at 490 nm using an Enspire spectrophotometer.

[0438] IC50 values ​​were measured using the built-in BI statistics program MegaLab. Based on the results, compounds with high IC50 values ​​(IC50 greater than 100 nM for G12C inhibitors and IC50 greater than 300 nM for G12D inhibitors) were excluded. The remaining compounds were then tested using the Human Survivin ELISA kit (Abcam, ab183361).

[0439] Human Survivin ELISA Assay Cells were grown in T175 flasks to 90% confluence. H358 cells were treated with 100 nM G12C inhibitor, and SW1990 cells were treated with 300 nM G12D inhibitor. Exosome-depleted FBS was added to the culture medium (Gibco, A2720801). 72 hours after treatment, the cell culture supernatant was collected, and cells were harvested according to the Human Survivin ELISA kit protocol.

[0440] Exosome Extraction Exosomes were isolated from cell culture medium using the exoEasy Maxi kit (QIAGEN, 76064) as described in the manufacturer's instructions. Briefly, the cell culture supernatant was first filtered through a 0.8 μm filter (Sartorius Minisart NML, catalog number 16592). Then, a 1:1 volume of the filtered cell culture supernatant was mixed with a 1:1 volume of buffer XBP. The mixture was then added to an exoEasy spin column and centrifuged. The column was subsequently washed, and the exosomes were eluted into 400 μL of buffer XE.

[0441] RNA extraction from tumors Frozen tumors from in vivo studies were dissected with a scalpel into approximately 5 x 5 mm sections. The tumors were then transferred to 2 ml Eppendorf tubes containing one steel bead. 1 ml of Trizole (Quiazol 79306, 200 ml Qiagen) was added, and each sample was homogenized with tissueLyser II (Qiagen). The tubes were centrifuged, the supernatant was transferred to a new tube, and gDNA Eliminator solution and bromo-3-chloropropane were added to each sample. The samples were centrifuged again, and subsequent RNA isolation was performed using the RNeasy Mini-Kit column system ((250) 74106 Qiagen) according to the manufacturer's instructions. Briefly, after centrifugation, the aqueous phase was mixed with 70% ethanol in a 1:1 ratio and added to the supplied column. The column was centrifuged and washed several times as suggested. RNA was eluted into 40 μl of nuclease-free H2O. RNA content was measured using the QIAxpert Slide-40(25) 990700-RNeasy method, and RIN values ​​were established for Tape Station (Agilent). Subsequently, the isolated RNA was further processed for sequencing.

[0442] RNA extraction from exosomes Exosomal RNA was isolated from plasma using the exoRNeasy serum / plasma Maxi kit (catalog number 77064) according to the manufacturer's instructions. Up to 4 ml of plasma per mouse group was pooled, filtered, and conjugated with XBP buffer in a 1:1 ratio. The mixture was then added to a feed column, centrifuged, and washed. QIAzol was then added to the membrane, the tube was centrifuged, and the collected lysate was transferred to a new tube. Chloroform was added to the mixture, the tube was centrifuged, and the upper aqueous phase was transferred to a new collection tube. Then, twice the volume of 100% ethanol was added to the tube and pipetted onto an RNeasy MinElute spin column. After centrifugation, the column was washed, and the RNA was eluted in 14 μl of nuclease-free H2O. Similar to the tumors, the RNA was measured using QIAxpert Slide and TapeStation, followed by sequencing.

[0443] MSD® 96-well S-PLEX Survivin Assay for the Analysis of Plasma Samples Plasma samples from mouse in vivo efficacy and biomarker studies, or human plasma samples, were centrifuged at 10,000 rcf for 5 minutes and then tested in the MSD® 96-well S-PLEX assay for human survivorbin detection at a 2-fold dilution according to the manufacturer's instructions. Briefly, the assay was assembled by (1) washing the plate and incubating it with a coating solution containing a specific survivorbin capture antibody at ambient temperature with shaking for 1 hour. After the washing step, blocking solution, followed by the standard substance and sample solution, was added. The assay was incubated at ambient temperature with shaking for 16-18 hours. The next day, the plate was washed and incubated with (2) a TURBO-BOOST solution containing a specific survivorbin capture antibody on a shaker at ambient temperature for 1 hour. After another washing step, (3) S-PLEX Enhancement solution was added and shaken at ambient temperature for 30 minutes. The plate was then washed again and incubated with (4) S-PLEX detection solution at 27°C with shaking for 1 hour. Finally, the plate is read using an MSD instrument after the (5.) washing process and the addition of MSD GOLD reading buffer B. (6.) Data analysis is performed using MSD Discovery software and visualized in GraphPad Prism.

[0444] MSD® 96-well S-PLEX Survivin Assay for In Vitro Experimental Analysis SNU1196 cells were grown in T175 flasks to 90% confluence. SNU1196 cells were treated with 11 different GDPi inhibitors (500 nM). Exosome-depleted FBS was added to the culture medium (Gibco, A2720801). 72 hours after treatment, the cell culture supernatant was collected, and cells were recovered using the cell lysis buffer provided in the Abcam Human Survivin ELISA Kit protocol. Exosomes were isolated from the supernatant according to the exoEasy Maxi Kit (QIAGEN, 76064), and the protein concentrations of isolated exosomes and cell lysates were determined by the Quick Start® Bradford protein assay (Bio-Rad, 500-0202). Survivin levels were then analyzed using the MSD® 96-well S-PLEX assay as described above.

[0445] Human plasma sample Human plasma samples from various indications were analyzed to assess differences in median baseline survival levels and biological variance between subjects. Plasma samples were also obtained from healthy subjects. Plasma samples from CRC patients (n=21) were purchased from the vendor (BioIVT) or from viable samples from previous clinical studies conducted under the biomarker research proposal (2023-brp-0004). PDAC plasma samples (n=4) were purchased from the vendor (BioIVT). NSCLC serum samples (n=31) were leftover samples from completed clinical studies (1280.16).

[0446] (Example 9) In vitro and in vivo survival level evaluation - results (Example 9.1) Survival as a biomarker Survivin expression was analyzed in tumorigenic and healthy cells. As shown in Figure 1A, survivin was detected in exosomes released by cells from the pancreatic adenocarcinoma epithelial cell line HPAC, but not in exosomes from healthy subjects. Plasma survivin levels were further measured in healthy controls (n=30) and cancer patients (CRC (n=21), PDAC (n=4), and NSCLC (n=31)) using the MSD S-Plex ELISA assay. The results show that the cancer population exhibits approximately 2.5 times higher baseline (median baseline) survivin levels compared to healthy subjects (Figure 1B). Thus, survivin is upregulated in cancer patients compared to healthy controls. In addition, CRC patients in the placebo group of the nintedanib trial (vivo-preserved samples from 1199.52) showed stable or increasing survivin levels, thereby suggesting a possible correlation between survivin and progressive disease (Figure 1C).

[0447] These findings are consistent with earlier published data (Chang et al. 2021), which showed survivin expression in tumors and, more importantly, high survivin levels associated with treatment resistance.

[0448] To further analyze the expression patterns of survivin in response to treatment with various anti-KRAS drugs, an in vivo NSCLC CDX model (a cell line-derived xenograft model using the non-small cell lung cancer HCC461 cell line) was used. Survivin was found to be significantly downregulated in a dose-dependent manner (Figure 2). Therefore, the in vivo biomarker study demonstrated dose-dependent survivin downregulation after 3 days of daily treatment with the KRASG12D inhibitor compound G12D-cpd 2 (see Table 1).

[0449] Survivin levels encoded by BIRC5 were further examined by ELISA in GP2D and HPAC (G12D mutant) cells treated with increasing doses of KRASG12D inhibitor for 2 hours (upper panel) and 24 hours (lower panel) (Figure 3). Survivin expression decreased with respect to IC50 dose at 24 hours (8 nM for GP2D and 46 nM for HPAC), but not at 2 hours, suggesting that survivin may be a delayed biomarker of the KRAS response.

[0450] The inventors further demonstrated that after treatment with 11 different KRASG12C inhibitors at 100 nM (Figures 4-6) or 9 different KRASG12D inhibitors at 300 nM (Figures 7-9) (Figures 7-9), survival was downregulated in cell lysates (Figures 4, 7), culture medium (Figures 5, 8), and exosomes (Figures 6, 9) of NCI-H358 cells (Figures 4-6) and SW1990 cells (Figures 7-9), respectively (Figures 4-9).

[0451] Furthermore, survivin was found to be downregulated in the human adenocarcinoma cell line PC9 YMVA-5 after treatment with a HER2 inhibitor (Figure 10). Similar data were obtained using two different GDP-KRAS inhibitors, compounds Cpd a and Cpd b (Figure 11). Further experiments demonstrated survivin modulation in SNU1196 cells (KRAS-WT amp) treated with various GDP-KRAS inhibitors. SNU1196 cells with KRAS-WT amplification were treated with 11 different GDP-KRAS inhibitors (500 nM) for 72 hours. Survivin levels in cell lysates and exosomes were measured using the S-Plex survivin assay. As shown in Figures 13A and B, strong survivin downregulation was observed in cell lysates and exosomes with all the compounds tested, thereby confirming inhibition of ...

Claims

1. A method for determining the response of cancer patients to treatment with compounds that inhibit the KRAS protein or mutants of the KRAS protein, - Measure the level of survivin in the first sample obtained from the patient before treatment with the compound. - To measure the level of survivin in a second sample obtained from the patient during or after treatment with the compound. - Compare the level of survivin in the second sample with the level of survivin in the first sample. A method comprising determining that a patient is responsive to treatment with the compound if the level of survivin in the second sample is reduced compared to the level of survivin in the first sample.

2. A method for determining the response of cancer patients to treatment with a compound that inhibits the interaction between MDM2 and p53, - Measure the level of survivin in the first sample obtained from the patient before treatment with the compound. - Measure the level of survivin in a second sample obtained from the patient during or after treatment with the compound. - Compare the level of survivin in the second sample with the level of survivin in the first sample. A method comprising determining that a patient is responsive to treatment with the compound if the level of survivin in the second sample is reduced compared to the level of survivin in the first sample.

3. The method according to claim 1 or 2, wherein the first and / or second sample is a blood, plasma, or serum sample.

4. The method according to any one of claims 1 to 3, wherein the step of measuring the level of survivin in a sample includes isolating exosomes from the sample and measuring the level of survivin contained in the exosomes.

5. The method according to any one of claims 1 to 4, wherein the survivin level is measured using a survivin-specific assay selected from the group consisting of Western blotting, ELISA, RIA, FACS, and MSD® S-PLEX technology.

6. The method according to any one of claims 1 to 5, wherein the cancer is a KRAS-dependent cancer, preferably selected from the group consisting of pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NSCLC), and colorectal cancer (CRC).

7. The method according to any one of claims 1 and 3 to 6, wherein the compound that inhibits the KRAS protein or a mutant of the KRAS protein is selected from the group consisting of KRAS(G12C) inhibitor or degrader, KRAS(G12D) inhibitor or degrader, GDP-KRAS inhibitor or degrader, and HER2 inhibitor or degrader.

8. The method according to any one of claims 1 and 3 to 7, wherein the KRAS (G12C) inhibitor or degrader is selected from the group consisting of sotrasib (AMG510), adagrasib (MRTX849), G12C-cpd 1, G12C-cpd 2, G12C-cpd 3, G12C-cpd 4, G12C-cpd 5, G12C-cpd 6, G12C-cpd 7, G12C-cpd 8, G12C-cpd 9, G12C-cpd 10, and G12C-cpd 11.

9. The method according to any one of claims 1 and 3 to 7, wherein the KRAS (G12D) inhibitor or degrader is selected from the group consisting of MRTX1133, G12D-cpd 2, G12D-cpd 3, G12D-cpd 4, G12D-cpd 5, G12D-cpd 6, G12D-cpd 7, G12D-cpd 8 and G12D-cpd 9.

10. The method according to any one of claims 1 and 3 to 7, wherein the GDP-KRAS inhibitor or degrader is selected from the group consisting of GDP-cpd 1, GDP-cpd 2, GDP-cpd 3, GDP-cpd 4, GDP-cpd 5, GDP-cpd 6, GDP-cpd 7, GDP-cpd 8, GDP-cpd 9, GDP-cpd 10, GDP-cpd 11, GDP-cpd 12, GDP-cpd 13 and GDP-cpd 14.

11. The method according to any one of claims 1 and 3 to 7, wherein the HER2 inhibitor is a compound described in formula F, preferably the compound is HER2-cpd 1.

12. The method according to any one of claims 2 to 6, wherein the compound that inhibits the interaction between MDM2 and p53 is MDM2i-cpd 1.

13. Compounds that inhibit the KRAS protein or mutants of the KRAS protein for use in treating cancer patients, i) The KRAS inhibitor is selected from the group consisting of KRAS(G12C) inhibitor or degrader, KRAS(G12D) inhibitor or degrader, GDP-KRAS inhibitor or degrader, and HER2 inhibitor or degrader. ii) A compound in which a patient has been determined to be responsive to treatment with the compound according to the method described in any one of claims 1 to 11.

14. A compound that inhibits the interaction between MDM2 and p53 for use in treating cancer patients, i) The compound that inhibits the interaction between MDM2 and p53 is MDM2i-cpd 1, ii) A compound in which a patient has been determined to be responsive to treatment with the compound according to the method described in claim 12.

15. A method for determining whether a compound that inhibits the KRAS protein or a mutant of the KRAS protein, or a compound that inhibits the interaction between MDM2 and p53, is effective in treating cancer and / or monitoring the response of cancer patients to said treatment, wherein at least, - The process of providing the first sample from the patient, - A step of measuring the level of survivin in the first sample from the patient, - A step of administering the compound that inhibits the KRAS protein or a mutant of the KRAS protein to a patient, and then - The process of obtaining a second sample from the patient, - A step of measuring the level of survivin in the second sample, - A step of comparing the levels of survival measured in the first and second samples, and - A process in which steps 4 to 6 are repeated depending on the circumstances. A method comprising a second sample in which a decrease in the level of survivin indicates an effective response.

16. Use of Survivin in a method for determining the ability of a compound that inhibits the KRAS protein or a mutant of the KRAS protein, or a pharmaceutical formulation containing said compound that inhibits the KRAS protein or a mutant of the KRAS protein, to treat cancer.

17. The use according to claim 16, wherein the survivin level is determined in at least one sample obtained from the patient before and after treatment with the compound that inhibits the KRAS protein or a mutant of the KRAS protein, and the decrease in the survivin level after treatment with the compound that inhibits the KRAS protein or a mutant of the KRAS protein indicates the ability of the compound to treat the cancer.

18. Use of Survivin in a method for determining the ability of a compound that inhibits the interaction between MDM2 and p53, or a pharmaceutical formulation containing said compound that inhibits the interaction between MDM2 and p53, to treat cancer.

19. The use according to claim 18, wherein the survivin level is determined in at least one sample obtained from the patient before and after treatment with the compound that inhibits the interaction between MDM2 and p53, and the decrease in the survivin level after treatment with the compound that inhibits the interaction between MDM2 and p53 indicates the ability of the compound to treat the cancer.

20. A kit of components comprising means for determining the level of survivin in a sample provided by a patient suffering from cancer, preferably KRAS-dependent cancer, and instructions for use on how to carry out the method described in any one of claims 1 to 12 and 15.

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