Survivin as biomarker for predicting responsiveness to cancer treatment

Abstract

The present invention relates to a method of determining the responsiveness of a cancer patient to treatment with a compound that inhibits a KRAS protein or a KRAS protein mutant or treatment with a compound that inhibits the interaction between MDM2 and p53, the method comprising measuring the survivin level in a first sample obtained from the patient prior to 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 to the level of survivin in the first sample, and determining the level of survivin in the second sample when compared to the level of survivin in the first sample. When the survivin level in the second sample decreases, it is determined that the patient is responsive to treatment with the compound. The invention further relates to the use of survivin in compounds for determining the inhibition of KRAS protein or KRAS protein mutants, or in compounds for inhibiting the interaction between MDM2 and p53, or a pharmaceutical formulation comprising said compound that inhibits a KRAS protein or a KRAS protein mutant or said compound that inhibits an interaction between MDM2 and p53 in a method of treating cancer.

Description

Technical Field

[0001] This invention relates to the field of targeted cancer therapy, to biomarkers for evaluating the activity of specific compounds, and to monitoring treatment with said compounds using biomarkers. In particular, this 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. The method includes measuring the level of a surviving protein in a first sample obtained from the patient prior to treatment with said compound, measuring the level of a surviving protein in a second sample obtained from the patient during or after treatment with said compound, comparing the level of a surviving protein in the second sample to the level of a surviving protein in the first sample, wherein when the level of a surviving protein in the second sample is lower than the level of a surviving protein in the first sample, it is determined that the patient has responded to treatment with said compound. This invention further relates to the use of surviving proteins in methods for determining the ability of compounds that inhibit the KRAS protein or KRAS protein mutants, or pharmaceutical formulations containing said compounds that inhibit the KRAS protein or KRAS protein mutants, to treat cancer. The present invention further relates to the use of the survival protein 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. Background Technology

[0002] Cancer is a leading cause of death worldwide, and its treatment and outcomes have been dramatically transformed by targeted therapies. K Irsten rat sarcoma ( K irsten ra t s KRAS (K-Ras) viral oncogene homologs (the terms “KRAS” and “K-Ras” are used synonymously in this application) play a central role in signal transduction and are among the most frequently mutated oncogenes. Gain-of-function mutations of KRAS have been identified in approximately 30% of all cancer cells. KRAS mutations are strongly associated with tumorigenesis and development and are 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 patients having a mutated form of RAS. KRAS mutations induce constitutive activation of this oncogene and downstream signaling, including ERK and PI3K, which promote tumorigenesis, invasiveness, metastasis, and therapy resistance (Dhirendra et al. 2017).

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

[0005] The RAS gene is evolutionarily conserved, has a similar structure, and consists of four exons distributed across approximately 30 kb of DNA. Due to different splicing of the fourth exon, the KRAS gene encodes two highly related protein variants (KRAS-4B and KRAS-4A), which consist of 188 and 189 amino acids, respectively (Huang et al. 2021).

[0006] RAS is a membrane-bound regulatory protein (G protein) that binds to guanine nucleotides and belongs to the guanosine triphosphatase (GTP) family. RAS functions as a guanosine diphosphate (GDP) / triphosphate (GTP) binary switch, controlling important signal transduction from activated membrane receptors to intracellular molecules.

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

[0008] KRAS proteins function as finely regulated molecular switches, controlling multiple signal transduction cascades by cycling between activated and inactivated conformations. KRAS proteins can be activated by growth factors, chemokines, and calcium... 2+KRAS proteins can be activated by receptor tyrosine kinases (RTKs). Activated KRAS proteins can activate multiple signaling pathways, including the RAF / MEK / ERK pathway, which is a typical downstream target of KRAS signaling. Activated KRAS-GTP can recruit rapidly accelerating fibrosarcoma (RAF) (serine / threonine-specific protein kinases) from the cytoplasm to the plasma membrane, induce conformational changes in RAF, and promote RAF activation through homodimerization or heterodimerization. The C-terminal catalytic domain of RAF binds to mitogen-activated protein kinases (MEK1 / 2) and activates MEK1 / 2 through phosphorylation. MEK1 / 2 phosphorylates and activates extracellular regulatory protein kinases (ERK1 / 2), and activated ERK phosphorylates ribosomal S6 kinase (RSK), serum response factor (SRF), E26 transformation-specific transcription factor (ETS), and ETS-like-1 protein to regulate the transcription and translation of corresponding target genes, thereby participating in the regulation of cell proliferation, differentiation, migration, and other vital activities.

[0009] KRAS was also found to participate in the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT)-mammalian target of rapamycin (mTOR) pathway, which is believed 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. Activated PI3K catalyzes the conversion of phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3). PIP3 promotes the phosphorylation of AKT by phosphatidylinositol-dependent kinase 1 (PDK1) at Thr308. mTOR complex 2 further phosphorylates the serine phosphorylation site of AKT (Ser473), leading to complete activation of AKT. Activated AKT enters the nucleus, activating or inhibiting many downstream pathways and regulating cell proliferation, apoptosis, and metabolic processes. On the one hand, AKT can directly activate mTOR targets, which play an important role in cell proliferation, survival, metabolism, protein synthesis, and transcription. On the other hand, AKT phosphorylates and activates the Bcl-XL / Bcl-2-related death promoter (BAD), promoting the binding of BAD to chaperone protein 14-3-3 instead of Bcl-2 / Bcl-XL, thereby inhibiting apoptosis.

[0010] Furthermore, RAL (guanine nucleotide dissociation stimulator) (RalGDS) is a downstream signaling protein of KRAS, functioning as a GTP / GDP exchanger to promote the GDP / GTP conversion of RAS-like protein (RAL). Downstream effectors of RAL proteins include Rac / cell cycle 42 (Cdc42), associated with cell migration; TANK-binding kinase 1 (TBK1), associated with viral immunity; and phospholipase D (PLD), associated with endocytosis. KRAS also regulates TIAM1 and RAC1-specific guanine nucleotide exchangers to activate the RAC1 signaling pathway, which influences cell shape, migration, adhesion, actin cytoskeleton formation, endocytosis, and membrane transport. Additionally, KRAS can regulate the phosphatidylinositol signaling pathway by activating PLCε. In short, the KRAS-mediated signaling network is complex and involved in a wide range of life activities (Huang et al. 2021).

[0011] KRAS mediates immune escape in the tumor microenvironment by upregulating PD-L1 expression, downregulating MHC1 expression, and enhancing the secretion of various cytokines and chemokines in tumor cells through MAP kinase (MAPK) signaling, thereby recruiting immunosuppressive immune cells.

[0012] Cancer-associated mutations in Ras family proteins suppress their intrinsic and GAP-induced GTPase activity, leading to an increased population of GTP-binding / active mutant Ras family proteins (McCormick et al. 2015; Hunter et al. 2015). This, in turn, results in the sustained activation of downstream effector pathways of mutant Ras family proteins (e.g., RAF / MEK / ERK, PI3K / AKT / mTOR, RalGDS pathway). KRAS mutations (e.g., amino acids G12, G13, Q61, A146) have been found in a variety of human cancers, including lung, colorectal, and pancreatic cancers (Cox et al. 2014). Mutations in HRAS (e.g., amino acids G12, G13, Q61) and NRAS (e.g., amino acids G12, G13, Q61, A146) have also been found in a variety of human cancer types, but are typically less frequent than KRAS mutations (Cox et al. 2014). Alterations in Ras family proteins / Ras genes (e.g., mutations, overexpression, gene amplification) have also been described as mechanisms of resistance to cancer drugs such as EGFR antibodies cetuximab and panitumumab (Leto et al. 2014) and EGFR tyrosine kinase inhibitors osimertinib / AZD9291 (Ortiz-Cuaran et al. 2016; Eberlein et al. 2015).

[0013] Mutations at residue 12 of Ras family proteins, specifically glycine-to-cysteine ​​transitions (i.e., G12C mutations, such as KRASG12C, NRAS G12C, and HRAS G12C), are caused by a GC-to-TA base transversion at codon 12. These mutations are common in RAS genes, accounting for 14% of all KRAS mutations, 2% of all NRAS mutations, and 2% of all HRAS mutations across cancer types. G12C mutations are particularly prevalent in KRAS-mutant non-small cell lung cancer, affecting approximately half of all cases, and are associated with DNA adducts formed from tobacco smoke. G12C mutations are not only associated with lung cancer but have also been found in other RAS-mutant cancer types, for example, accounting for 3-5% of all KRAS-mutant colorectal cancers.

[0014] Inhibitors of G12C mutant Ras family proteins, such as covalent binders of KRAS G12C, NRAS G12C, and HRAS G12C, capable of covalently binding to G12C mutant Ras family proteins, are expected to inhibit downstream signaling of Ras family proteins in cells (e.g., ERK phosphorylation). In cancer cells associated with dependence on mutant Ras family proteins (e.g., KRAS mutant cancer cell lines), such binders / inhibitors are expected to produce anticancer effects (e.g., inhibition of proliferation, survival, metastasis, etc.).

[0015] Several selective drugs targeting the KRAS G12C mutant protein have entered clinical development; sotorasib has recently been approved for the treatment of KRAS G12C-driven lung cancer (corresponding patent applications WO 2018 / 217651, WO 2017 / 201161, WO 2019 / 099524, WO 2020 / 102730).

[0016] Significant amplification of the wild-type (WT) KRAS proto-oncogene has been observed in subgroups of tumor indications such as gastric cancer, gastroesophageal junction cancer, and esophageal cancer. This (WT) KRAS proto-oncogene acts as a driver of alterations and causes KRAS addiction in tumor models carrying this genotype both in vitro and in vivo (Wong et al. 2018). In contrast, non-amplified KRASWT cell lines are KRAS-independent unless they carry secondary alterations in their genes that indirectly induce KRAS activation (Meyers et al. 2017). Based on these observations, agents with KRAS WT targeting activity offer further avenues for treating KRAS-dependent cancers.

[0017] Proteolytic targeting chimeras (PROTACs) bind to proteins, thereby inducing protein degradation through ubiquitination. A PROTAC is a three-part or heterobifunctional molecule consisting of: a part that binds to the protein to be degraded, a second part that binds to and can be artificially recruited by an E3 ubiquitin ligase, and a linker connecting the two parts. Whenever a trimeric complex of the target protein, PROTAC, and ligase is formed, the close proximity of the ligase to the target leads to ubiquitination of the target protein. Ubiquitination acts as a post-translational modification of proteins, which in particular induces their recruitment to the proteasome, resulting in proteolytic degradation. The polyubiquitin chains on the target protein are then recognized by the proteasome, and the target protein is degraded.

[0018] Compared to classic small molecule drugs, PROTACs drive degradation function with substoichiometric properties, thus requiring lower systemic exposure to achieve efficacy. Due to the complementary differences in the protein-protein interaction interfaces of the formed ternary complexes, PROTACs have demonstrated a higher degree of selectivity for protein degradation than the target ligands themselves. Furthermore, PROTACs hold promise for expanding the druggable proteome, as degradation is not limited to functionally disease-causing protein domains. In the case of challenging multi-domain proteins traditionally considered undrugifiable targets, the most ligandable domains can be targeted for degradation, regardless of their functionality or vulnerability to small molecule blockade.

[0019] The inherently irreversible induced degradation of KRAS by recruiting E3 ubiquitin ligases is expected to induce cellular effects comparable to irreversible inhibition. Furthermore, since mutant KRAS is still expected to cycle between GEF / GAP-induced GTP-binding activity and GDP-binding inactivity, degradation of not only wt-amplified KRAS but also mutant KRAS induced by PROTAC involved in the GDP-binding state may lead to the gradual degradation of a large portion of the entire cellular KRAS pool. Therefore, degradation of oncogenic KRAS can inhibit downstream signaling in tumors, thereby producing the anticancer efficacy described for KRAS inhibition. Following irreversible targeted degradation, the recovery of downstream signaling activity depends not only on drug elimination from the treated subject (e.g., by clearance) but also further on 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 all KRAS mutant tumors. In contrast, the induced degradation of KRAS has the potential to achieve irreversible inhibition of KRAS signaling against most remaining KRAS mutations / alterations (including amplifications) that drive tumor growth, provided that such mutations / alterations can be bound by heterobifunctional degradative molecules. Degradative agents of wild-type (e.g., amplified or overexpressed) or mutant KRAS (e.g., G12C, G12D, G12V, G13D) have been used to produce anticancer efficacy.

[0020] Although many approaches have been explored in this field to treat patients with compounds that target KRAS, many aspects remain to be investigated, such as providing means to measure the responsiveness of cancer patients to treatment with compounds that inhibit the KRAS protein or KRAS protein mutants.

[0021] The ERBB transmembrane receptor tyrosine kinase (RTK) family consists of four members: EGFR (ERBB1), HER2 (Neu, ERBB2), HER3 (ERBB3), and HER4 (ERBB4). HER2 has not yet been identified as the preferred dimerizing partner for the other ERBB members. Once an active ligand-receptor complex is formed, the intracellular tyrosine kinase domains of EGFR, HER2, HER3, or HER4 are activated via autophosphorylation or transphosphorylation, subsequently triggering a signal transduction cascade, most notably involved in the mitogen-activated protein (MAP) kinase and / or phosphatidylinositol 3-kinase (PI3K) pathways.

[0022] Aberrant HER2 signaling has been observed in a variety of human malignancies. Oncogenic mutations in the extracellular, (near) membrane, and intracellular regions of the protein are described. In general, these mutations endow HER2 with constitutive activity, promoting cancer development, tumor maintenance, and growth. Similarly, HER2 overexpression increases HER2 signaling and serves as a basis for tumorigenic transformation and tumor maintenance in various indications, including breast, gastric, and lung cancer.

[0023] Therefore, interfering with HER2 oncogenic signaling can lead to the suppression of tumor growth. Targeted therapies include antibodies against HER2 (including trastuzumab and pertuzumab), antibody-drug conjugates against HER2 (trastuzumab-DM1 (T-DM1, ado emtansine trastuzumab)), and small molecules that inhibit the HER2 kinase domain.

[0024] In summary, tumors driven by HER2 oncogenic mutations or HER2 wild-type overexpression (e.g., due to gene amplification) may benefit from HER2-specific tyrosine kinase inhibitors (TKIs). Overall, HER2 alterations affect up to 6%–7% of all human cancers (WO 2021213800).

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

[0026] Overall, approximately 5%–7% of tumors exhibit MDM2 amplification. However, this type of amplification is more common in some tumor types than others, occurring in up to 90% of some types of advanced soft tissue sarcoma (STS).

[0027] Therefore, blocking the MDM2-p53 interaction to reactivate wild-type p53 function is a promising cancer treatment strategy. Initial compounds designed to target the MDM2-p53 interaction have been developed. These compounds can have a dual mechanism of action: directly targeting tumor cells and exerting immunomodulatory effects; they bind directly to MDM2 and block the interaction between MDM2 and p53, leading to p53 stabilization, TP53 target gene induction, cell cycle arrest, and apoptosis in tumor cells with wild-type TP53. Activation of p53 also promotes anti-tumor immune responses by increasing CD8+ T cell infiltration in tumors and induces anti-tumor immune memory.

[0028] Mouse double microsome 2 (MDM2) protein (or its human homology, also known as HDM2) downregulates p53 activity in an autoregulatory manner, and under normal cellular conditions (in the absence of stress), MDM2 protein is used to maintain p53 activity at low levels. MDM2 directly inhibits the transactivation function of p53, exporting p53 to the extranuclear space, and promotes proteasome-mediated p53 degradation.

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

[0030] An imbalance in the MDM2 / p53 ratio, caused by MDM2 overexpression or p53 mutation or deletion, leads to the malignant transformation of normal cells. p53 is known to play a crucial role in almost all types of human cancer, and mutations or deletions of the p53 gene can be identified in over 50% of all human cancers worldwide.

[0031] In tumors carrying wild-type p53, MDM2 is a major cellular inhibitor of p53 activity, and overexpression of MDM2 has been found in many human tumors. Since MDM2 inhibits p53 through direct protein-protein interactions, there has been recent exploration of using small molecules to block these interactions.

[0032] The compounds involved in this invention are characterized by a strong inhibitory effect on the interaction between MDM2 and p53, and consequently high in vitro efficacy against tumor cells (e.g., osteosarcoma), ALL, etc., said in vitro efficacy being mediated by the inhibition of the interaction between MDM2 and p53, and being a prerequisite for the corresponding efficacy in in vivo models and future patients (WO 2017 / 060431). Summary of the Invention

[0033] According to a first aspect, the present invention relates to a method for determining the responsiveness of a cancer patient to treatment with a compound that inhibits KRAS protein or a KRAS protein mutant, the method comprising:

[0034] - Prior to treatment with the compound, the level of viable proteins in a first sample obtained from the patient was measured.

[0035] -Measure the level of viable proteins in a second sample obtained from the patient during or after treatment with the compound.

[0036] - Compare the level of surviving proteins in the second sample with the level of surviving proteins in the first sample.

[0037] When the level of the surviving protein in the second sample is lower than that in the first sample, it is determined that the patient has responded to treatment with the compound.

[0038] Furthermore, the present invention relates to a method for determining the responsiveness of a cancer patient to treatment with a compound that inhibits the interaction between MDM2 and p53, the method comprising:

[0039] - Prior to treatment with the compound, the level of viable proteins in a first sample obtained from the patient was measured.

[0040] -Measure the level of viable proteins in a second sample obtained from the patient during or after treatment with the compound.

[0041] - Compare the level of surviving proteins in the second sample with the level of surviving proteins in the first sample.

[0042] When the level of the surviving protein in the second sample is lower than that in the first sample, it is determined that the patient has responded to treatment with the compound.

[0043] According to a second aspect, the present invention relates to a compound for use in treating cancer patients by inhibiting a mutant of KRAS protein or K-Ras protein.

[0044] i) The KRAS inhibitor is selected from KRAS (G12C) inhibitors or degraders, KRAS (G12D) inhibitors or degraders, GDP-KRAS inhibitors or degraders, and HER2 inhibitors or degraders; and

[0045] ii) Where the patient has been determined to be responsive to treatment with the compound in accordance with the method described above.

[0046] Furthermore, the present invention relates to a compound for use in treating cancer patients that inhibits the interaction between MDM2 and p53, wherein the patients have been determined to respond to treatment with the compound according to the methods described above.

[0047] According to a third aspect, the present invention relates to a method of treating a patient’s cancer with a compound that inhibits KRAS protein or a KRAS protein mutant, wherein the patient has been determined to be responsive to treatment with the compound according to any of the methods described above.

[0048] Furthermore, the present invention relates to a method for treating a patient’s cancer 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.

[0049] According to a fourth aspect, the present invention relates to the use of a survival protein in a method for determining the ability of a compound or a pharmaceutical formulation comprising a compound that inhibits KRAS protein or a KRAS protein mutant to treat cancer.

[0050] Furthermore, the present invention relates to the use of the survival protein 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.

[0051] According to a fifth aspect, the present invention relates to a multi-component kit comprising means for determining the level of viable proteins in a sample provided from a patient with cancer, and instructions on how to perform any of the methods described above.

[0052] The inventors unexpectedly discovered that the inhibitor of apoptosis (IAP) protein survival protein can serve as a biomarker for selecting cancer patients who can be more advantageously treated with KRAS inhibitors, and that this biomarker can be easily assessed and monitored by quantitative determination in blood, plasma, or serum samples provided from said patients.

[0053] By using this molecular biomarker, ineffective treatment can be avoided in cancer patients who do not respond to KRAS inhibitors, allowing for a faster switch to more effective drug classes, increasing treatment efficacy, and prolonging survival. Determining the levels of survival proteins as biomarkers in readily available samples further reduces overall treatment costs and clinical workload. Furthermore, this approach avoids the need for frequent tumor sample collection to monitor treatment success and assess cancer progression.

[0054] The subject matter, method, and apparatus according to the independent claims of the invention satisfy these objectives. The dependent claims relate to preferred embodiments.

[0055] Further details, features, characteristics, and advantages of the present invention are disclosed in the dependent claims and in the following description of the corresponding drawings and embodiments, which illustrate preferred embodiments of the invention by way of example. However, these embodiments and drawings should not be construed as limiting the scope of the invention. Attached Figure Description

[0056] Figure 1. Survival protein levels in untreated patients; (A) Survival protein was detected in exosomes released from HPAC cells but not in exosomes from healthy volunteers; (B) Comparison of plasma survival proteins in healthy controls and cancer patients (colorectal cancer, CRC; pancreatic ductal adenocarcinoma, PDAC; and non-small cell lung cancer, NSCLC); (C) Plasma survival protein levels in CRC patients (placebo group) at different time points.

[0057] Figure 2 Survival protein / BIRC5 is a biomarker of sensitivity to KRAS inhibition. An in vivo biomarker study (HCC461) showed that survival protein was dose-dependently downregulated after 3 days of daily treatment with a KRASG12D inhibitor.

[0058] Figure 3 ELISA assays of (A) GP2D cells and (B) HPAC cells treated with KRASG12D inhibitor for 2 hours (top) and 24 hours (bottom), showing downregulation of survival proteins in GP2D cells (left) and HPAC cells (right) 24 hours after treatment with KRASG12D inhibitor.

[0059] Figure 4 Survival proteins were downregulated in NCI-H358 cells treated with different KRASG12C inhibitors. ELISA assays showed the levels of survival proteins in NCI-H358 cells after treatment with 100 nM compounds G12C-cpd#1 to G12C-cpd#11 for 72 hours.

[0060] Figure 5 Survival proteins were downregulated in the culture medium of NCI-H358 cells treated with different KRASG12C inhibitors. ELISA assays showed the levels of survival proteins in the culture medium of H358 cells after treatment with 100 nM compounds G12C-cpd#1 to G12C-cpd#11 for 72 hours.

[0061] Figure 6 Survival proteins were downregulated in exosomes of NCI-H358 cells treated with different KRASG12C inhibitors. ELISA assays showed the levels of survival proteins in exosomes of H358 cells after treatment with 100 nM compounds G12C-cpd#1 to G12C-cpd#11 for 72 hours.

[0062] Figure 7Survival proteins were downregulated in SW1990 cells treated with different KRASG12D inhibitors and KRAS Protac. ELISA assays showed the levels of survival proteins in cell lysates of SW1990 cells after treatment with 300 nM compounds G12D-cpd#1 to G12D-cpd#9 for 72 hours.

[0063] Figure 8 Survival proteins were downregulated in the culture medium of SW1990 cells treated with different KRASG12D inhibitors and KRAS Protac. ELISA assays showed the levels of survival proteins in the supernatant of SW1990 cells after treatment with 300 nM compounds G12D-cpd#1 to G12D-cpd#9 for 72 hours.

[0064] Figure 9 Survival proteins were downregulated in exosomes of SW1990 cells treated with different KRASG12D inhibitors and KRAS Protac. ELISA assays showed the levels of survival proteins in exosomes of SW1990 cells after treatment with 300 nM compounds G12D-cpd#1 to G12D-cpd#9 for 72 hours.

[0065] Figure 10 ELISA assays were used to determine the levels of survival proteins in PC9 cells after treatment with 50 nM HER2-cpd#1.

[0066] Figure 11 Dose-dependent regulation of in vivo surviving proteins after treatment with two different GDP-KRAS inhibitors (Cpd#a, Cpd#b).

[0067] Figure 12 Dose-dependent regulation of survival proteins in tumors and plasma in the SW1990 CDX model after treatment with a KRASG12D inhibitor.

[0068] Figure 13 Regulation of survival proteins in SNU1196 cells (KRASWT amp) treated with 11 GDP-KRAS inhibitors (compound numbers see Table 1) or DMSO for 72 hours. (A) Cell lysates; (B) Exosomes.

[0069] Figure 14 Dose-dependent regulation of survival proteins in H358 tumors (A) and corresponding mouse plasma (B), and regulation of survival proteins in MKN1 tumors (C) and corresponding mouse plasma (D).

[0070] Figure 15Downregulation of survival proteins at the end of various efficacy studies in CDX models and the correlation between plasma survival protein levels and tumor volume (TV). (A, B) = KRAS-WTamp, CDX models treated with exemplary GDP-KRAS inhibitors; (CE) = KRAS-G12V CDX models treated with exemplary GDP-KRAS inhibitors; and (FH) = KRAS-G12D CDX models treated with the KRAS-G12D inhibitor shown as G12D-cpd#2 in Table 1 (concentrations shown in the figure).

[0071] Figure 16 Downregulation of survival protein levels in tumors (A) and plasma (B) in a HER2 mutant PC9_YMVA-5 NSCLC CDX model after treatment with the HER2 inhibitor HER2-cpd#1; and the correlation between plasma survival protein levels and tumor volume at the end of efficacy studies of the HER2 inhibitor HER2-cpd#1 in CDX models (C) = NCI-N87, (D) = SK-GT-2, and (E) = NCI-H2170.

[0072] Figure 17 In the TP53 WT PDX model, the MDM2 inhibitor MDM2i-cpd#1 downregulated the level of survival protein (RNA) in vivo. (A) = CRC PDX model (Co10748); (B) = Malignant peripheral nerve sheath tumor PDX. Detailed Implementation

[0073] Before describing the invention in detail, it should be understood that the invention is not limited to the specific compounds or methods described, as such compounds or methods can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It must be noted that, as used in the specification and appended claims, the singular forms “a / an” and “the” include singular and / or plural indicators unless the context clearly specifies otherwise. Furthermore, it should be understood that where a parameter range is given by numerical value, the range is considered to include those limit values. It should also be understood that a value range defined by numerical value should be understood to include the defined value.

[0074] It should be further understood that the embodiments disclosed herein are not intended to be construed as separate, unrelated embodiments. Features discussed with respect to one embodiment are also intended to be disclosed in conjunction with other embodiments shown herein. If, in a case, a particular feature is not disclosed in one embodiment but is disclosed in another, those skilled in the art will understand that this does not necessarily mean that the feature was not intended to be disclosed in the other embodiment. Those skilled in the art will understand that disclosing the feature for other embodiments is the essence of this application, but this has not been done merely for clarity and to keep the specification at a manageable size.

[0075] Furthermore, the content of prior art documents cited herein is incorporated by reference. This is especially true for prior art documents that disclose standards or conventional methods. In such cases, the main purpose of incorporating references is to provide sufficient authorized disclosure and to avoid lengthy repetition.

[0076] According to a first aspect, the present invention relates to a method for determining the responsiveness of a cancer patient to treatment with a compound that inhibits KRAS protein or a KRAS protein mutant, the method comprising:

[0077] - Prior to treatment with the compound, the level of viable proteins in a first sample obtained from the patient was measured.

[0078] -Measure the level of viable proteins in a second sample obtained from the patient during or after treatment with the compound.

[0079] - Compare the level of surviving proteins in the second sample with the level of surviving proteins in the first sample.

[0080] When the level of the surviving protein in the second sample is lower than that in the first sample, it is determined that the patient has responded to treatment with the compound.

[0081] More than one sample, as well as more than the first and second samples, can be obtained during the treatment period to continue monitoring the patient's responsiveness, or the samples can be obtained after the treatment is completed to monitor for potential recurrence.

[0082] The method for determining the reactivity of cancer patients is preferably performed in vitro (ex vivo).

[0083] The present invention also relates to a method for determining the responsiveness of a cancer patient to treatment with a compound that inhibits the HER2 protein or a mutant of the HER2 protein, the method comprising:

[0084] - Prior to treatment with the compound, the level of viable proteins in a first sample obtained from the patient was measured.

[0085] -Measure the level of viable proteins in a second sample obtained from the patient during or after treatment with the compound.

[0086] - Compare the level of surviving proteins in the second sample with the level of surviving proteins in the first sample.

[0087] When the level of the surviving protein in the second sample is lower than that in the first sample, the patient is determined to be responsive to treatment with the compound, preferably wherein the HER2 inhibitor is HER2-cpd#1.

[0088] As described above, more than one sample and more than the first and second samples can be obtained during the treatment period to continue monitoring the patient's responsiveness, or the samples can be obtained after treatment to monitor for potential recurrence. Furthermore, the method for determining the responsiveness of cancer patients is preferably performed in vitro (ex vivo).

[0089] The present invention further relates to a method for determining the responsiveness of a cancer patient to treatment with a compound that inhibits the interaction between MDM2 and p53 (also referred to herein as an MDM2 inhibitor or MDM2i), the method comprising:

[0090] - Prior to treatment with the compound, the level of viable proteins in a first sample obtained from the patient was measured.

[0091] -Measure the level of viable proteins in a second sample obtained from the patient during or after treatment with the compound.

[0092] - Compare the level of surviving proteins in the second sample with the level of surviving proteins in the first sample.

[0093] When the level of the surviving protein in the second sample is lower than that in the first sample, it is determined that the patient is responsive to treatment with the compound, preferably wherein the compound that inhibits the interaction between MDM2 and p53 is MDM2i-cpd#1.

[0094] As described above, more than one sample and more than the first and second samples can be obtained during the treatment period to continue monitoring the patient's responsiveness, or the samples can be obtained after treatment to monitor for potential recurrence. Furthermore, the method for determining the responsiveness of cancer patients is preferably performed in vitro (ex vivo).

[0095] As used herein, the term "inhibit" or "inhibiting" means that the compound, individually or in part, reduces or prevents the 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. The term "inhibit" or "inhibiting" also refers 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, wherein the binding can be direct or indirect, competitive or allosteric. The term “inhibit” or “inhibiting” also refers to the degradation of the KRAS protein or a mutant of the KRAS protein, or the HER2 protein or a mutant of the HER2 protein, and to the degradation of the KRAS protein or a mutant of the KRAS protein, or the HER2 protein or a mutant of the HER2 protein, and to any other type of neutralization of all or part of the 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. The same considerations apply to the term “inhibit” or “inhibiting” when used in the context of compounds that inhibit the interaction between MDM2 and p53, as further described below.

[0096] As used herein, the term “level” or “measurement level” for a surviving protein refers to the level, amount, or measure of any one of the surviving protein’s RNA, mRNA, or protein. The term further refers to measuring the level or amount of a surviving protein in a sample, wherein the surviving protein’s RNA, mRNA, or protein may be intracellular, in or on the cell membrane, extracellular, in a fluid culture medium, in exosomes, and / or in one or more blood, plasma, or serum samples.

[0097] As used herein, the term “reduction” means that the level of the biomarker surviving protein in the second (or n+1) sample is reduced or decreased by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the level of the biomarker surviving protein in the first (or n) sample.

[0098] As used herein, the term “survival protein” refers to a protein having an amino acid sequence according to UniProtKB / TrEMBL entry O15392, and any variants, allotypes, splice variants, active mutants, or secondary database entries of said protein.

[0099] Survival protein, encoded by the BIRC5 gene (containing the baculovirus IAP repeat sequence protein 5, also known as API4), is the smallest member of the "inhibitor of apoptosis" (IAP) protein family and is both an inhibitor of apoptosis and a regulator of the cell cycle. These functional properties make survival protein a unique protein, exhibiting diverse functions, including regulating cell death and cell proliferation.

[0100] Survivin is a small protein of 142 amino acids with a multifunctional domain. Its N-terminal two-thirds contain a baculovirus apoptosis inhibitor repeat (BIR) domain (aa 20-90), the integrity of which depends on zinc fingers generated by C57, C60, C84, and H77; the C-terminal third is an extended α-helix (98-142). Members of the IAP family typically contain multiple baculovirus IAP repeat (BIR) domains, but the survivin 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 except for activated T lymphocytes, erythroblasts, and self-renewing stem cells (Wheatley and Altieri, 2019).

[0101] Survival protein (BIRC5) has been shown to be involved in the inhibition of apoptosis and cell proliferation (LaCasse et al., 1998). Survival protein is upregulated in many cancer types and expressed at low levels in normal tissues (Kawasaki et al., 1998). Some publications have shown that survival protein is associated with poor prognosis (Takai et al., 2002) and chemotherapy resistance (Zafaroni et al., 2002).

[0102] Apoptosis is the primary form of programmed cell death and depends on cysteine ​​proteases (called caspases) to degrade cells in a controlled manner. Survival proteins protect cells from apoptosis and autophagy; the intracytoplasmic localization of survival proteins is crucial for their anti-apoptotic activity. Survival protein expression has been found to reduce caspase activity; however, at physiological concentrations, survival proteins do not bind to caspase but rather synergistically with XIAP and hepatitis B virus X interacting protein (HBXIP, also known as LAMTOR5) in a complex containing XIAP-associated factor 1 (XAF1) to influence the interaction between XIAP and caspase or enhance the effects of other IAP family members.

[0103] In addition to their intracellular localization, survival proteins have also been found on the surface of exosomes constitutively secreted by cancer cells. It has been found that the release of survival proteins from tumor cells in this way supports neighboring tumor cells in escaping apoptosis (Khan et al. 2011).

[0104] Survival proteins are considered targets for cancer therapy due to their anti-apoptotic effects (Altieri 2003; Li et al. 2019; Wheatley and Altieri, 2019). Both mutant KRAS and survival proteins have been shown to contribute to carcinogenesis (Tecleab and Sebti, 2013). Combination therapy with KRAS inhibitors and β-catenin inhibitors has shown synergistic effects in cancer cell growth arrest, cell death, and survival protein downregulation; however, this effect was only observed with combination therapy and not with monotherapy of either inhibitor (Mologni et al. 2012).

[0105] The inventors have surprisingly discovered that the cell apoptosis inhibitor (IAP) protein survival protein can be used in methods for determining the responsiveness of cancer patients to treatment with compounds that inhibit KRAS protein or KRAS protein mutants, or inhibit HER2 protein or HER2 protein mutants, and thus serve as a biomarker for selecting cancer patients, preferably those with KRAS-dependent cancers, who can be more advantageously treated with KRAS or HER2 inhibitors. Similarly, the inventors have found that the survival protein can also be used in methods for determining the responsiveness of cancer patients to treatment with compounds that inhibit the interaction between MDM2 and p53, as further described below. They have further found that this biomarker can be readily assessed and monitored by quantitative determination in blood, plasma, or serum samples.

[0106] This finding was also unexpected, as K-RAS-dependent human cancer cells have reportedly significantly increased the production of exosomes rich in survival proteins, thereby enhancing protection against apoptotic cell death from themselves and other cancer cells, as well as non-cancerous fibroblasts (Chang et al., 2021). Furthermore, Chang et al. (2021) observed that upregulation of survival proteins is a means by which tumor cells develop resistance to antitumor drug treatment. Surprisingly, despite the aforementioned anti-apoptotic and protective effects of survival proteins, the inventors of this invention found that therapeutic interventions with K-RAS inhibitors were effective, and they also demonstrated that the therapeutic efficacy could be monitored non-invasively from samples obtained from treated mice. Therefore, the evidence provided by these experiments in mice suggests that the assessment of survival protein levels is equally applicable to determining the responsiveness of cancer patients to treatment with KRAS inhibitors or degraders, or to treatment with compounds that inhibit the interaction between MDM2 and p53.

[0107] In one or more preferred embodiments of the present invention, one or more of the first sample and / or the second sample are one or more blood, plasma, or serum samples. Preferably, all samples are blood, plasma, or serum samples.

[0108] In one or more preferred embodiments of the invention, the step of measuring the level of viable proteins in a sample includes isolating exosomes from the sample and measuring the level of viable proteins contained in the exosomes. The means and methods for isolating exosomes and measuring the level of viable proteins contained in the exosomes are known in the art and have been described in the experimental section herein.

[0109] In one or more preferred embodiments of the invention, the level of viable proteins is measured using a viable protein-specific assay selected from Western blotting, ELISA, RIA, MSD® S-PLEX technology, and FACS. Preferably, the level of viable proteins is measured using ELISA or MSD® S-PLEX technology.

[0110] In one or more preferred embodiments of the invention, the cancer is a KRAS-dependent cancer, preferably selected from pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NSCLC), and colorectal cancer (CRC).

[0111] In one or more preferred embodiments of the present invention, the cancer to be treated with a compound that inhibits KRAS protein or a KRAS protein mutant (selected from KRAS (G12C) inhibitors or degraders, KRAS (G12D) inhibitors or degraders, and GDP-KRAS inhibitors or degraders) is selected from: brain tumors, such as acoustic neuroma, astrocytomas (e.g., pilocytic astrocytoma, fibrous astrocytoma, protoplasmic astrocytoma, gemistocytary astrocytoma, anaplastic astrocytoma, and glioblastoma), gliomas, brain lymphomas, brain metastases, pituitary tumors (e.g., prolactinomas, HGH-producing tumors, and ACTH-producing tumors), craniopharyngiomas, medulloblastomas, etc. Meningiomas, basal cell tumors, and oligodendrogliomas; neuronal tumors (vesicles), such as tumors of the autonomic nervous system (e.g., sympathoblastoma, ganglioneuroma, paraganglioma (pheochromocytoma, chromaffinoma), and carotid bulb tumors), tumors of the peripheral nervous system (e.g., stump neuroma, neurofibroma, schwannoma (schwannoma, Schwannoma), and malignant Schwannoma), and tumors of the central nervous system (e.g., brain tumors and myeloma); colorectal cancers, such as rectal cancer, colon cancer, colorectal cancer, anal cancer, large intestine cancer, small intestine and duodenum tumors; eyelid tumors, such as basal cell tumors or basal cell carcinomas; pancreatic cancer. Cancer or carcinoma of the pancreas; bladder cancer (or carcinoma of the bladder) and other urothelial carcinomas; lung cancer (bronchial cancer), such as small cell bronchial carcinoma (oat cell carcinoma) and non-small cell bronchial carcinoma (NSCLC) (such as plate epithelial carcinomas, adenocarcinomas, and large cell bronchial carcinomas); breast cancer, such as mammary carcinoma (such as invasive ductal carcinoma, colloid carcinoma, invasive lobular carcinoma, tubular carcinoma, adenoid cystic 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), such as Burkitt's lymphoma, low-grade non-Hodgkin lymphoma (NHL), and mycosis fungoides; uterine cancer or endometrial cancer or uterine corpus cancer; CUP syndrome (cancer of unknown primary origin);Ovarian cancer (or ovarian carcinoma), such as mucinous carcinoma, endometrial carcinoma, or serous carcinoma; gallbladder cancer; bile duct cancer, such as kratzkinoma; testicular cancer, such as seminoma and non-seminomatous carcinoma; lymphoma (lymphosarcoma), such as malignant lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), such as chronic lymphocytic leukemia, leukemic reticuloendotheliosis, immunocytoma, plasmacytoma, multiple myeloma (MM), immunoblastic carcinoma, Burkitt lymphoma, T-zone mycosis fungoides, large cell anaplastic lymphoblastoma, and lymphoblastoma; laryngeal cancer, such as vocal cord cancer. Tumors, supraglottic, glottic, and subglottic laryngeal tumors; bone cancers, such as osteochondroma, chondroma, chondrocyte-mediated chondromycinous fibroma, osteoma, osteoid osteoma, osteoblastoma, eosinophilic granuloma, giant cell tumor, chondrosarcoma, osteosarcoma, Ewing's sarcoma, reticulum cell sarcoma, soft tissue sarcoma, liposarcoma, plasmacytoma, fibrous dysplasia, juvenile bone cysts, and aneurysmal bone cysts; tumors of the head and neck, such as tumors of the lips, tongue, floor of mouth, oral cavity, gingiva, palate, salivary glands, larynx, nasal cavity, paranasal sinuses, larynx, and middle ear; liver cancers, such as hepatocellular carcinoma (liver cancer). Cell carcinoma or hepatocellular carcinoma (HCC); leukemia, such as acute leukemia, such as acute lymphoblastic / lymphoblastic leukemia (ALL), acute myeloid leukemia (AML); chronic leukemia, such as chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML); myelodysplastic syndrome (MDS); stomach cancer (or gastric carcinoma), such as papillary, tubular and mucinous adenocarcinoma, signet ring cell carcinoma, adenosquamous carcinoma, small cell carcinoma and undifferentiated carcinoma; melanoma, such as superficial spreading, nodular, malignant lentigines and acral lentigines melanoma; kidney cancer, such as renal cell carcinoma or adrenoidoma or Grawitz's tumor; esophageal cancer (or carcinoma of the esophagus); penile cancer; prostate cancer (e.g. castration-resistant prostate cancer); throat cancer (or carcinomas of the esophagus). The pharynx, such as nasopharyngeal carcinoma, oropharyngeal carcinoma, and laryngeal carcinoma; retinoblastoma, vaginal carcinoma, mesothelioma; lamellar carcinoma, adenocarcinoma, carcinoma in situ, malignant melanoma, and sarcoma; thyroid cancer, such as papillary, follicular, and medullary thyroid carcinoma, as well as anaplastic carcinoma;Squamous cell carcinoma, epidermoid carcinoma, and lamellar carcinoma of the skin; thymoma, urethral cancer, cervical cancer, adenoid cystic carcinoma (AdCC), adrenocortical carcinoma, and vulvar cancer.

[0112] In another preferred embodiment, the cancer to be treated with the compound that inhibits or degrades HER2 is selected from: cancers / tumors / carcinomas of the head and neck: for example, tumors / carcinomas / carcinomas of the nasal cavity, paranasal sinuses, nasopharynx, oral cavity (including lips, gingiva, alveolar ridge, retromolar triangle, floor of mouth, tongue, hard palate, buccal mucosa), oropharynx (including floor of tongue, tonsils, tonsillar columns, soft palate, tonsillar fossa, pharyngeal wall), middle ear, larynx (including superior larynx, glottis, subglottis, vocal cords), hypopharynx, and salivary glands (including minor salivary glands); cancers / tumors / carcinomas of the lungs: for example, non-small cell lung cancer (NSCLC). (Squamous cell carcinoma, spindle cell carcinoma, adenocarcinoma, large cell carcinoma, clear cell carcinoma, bronchioloalveolar carcinoma), small cell lung cancer (SCLC) (oat cell carcinoma, intermediate cell carcinoma, mixed oat cell carcinoma); mediastinal vegetations: for example, neurogenic tumors (including neurofibroma, schwannoma, malignant schwannoma, neurosarcoma, ganglioneuroma, ganglioneuroma, neuroblastoma, pheochromocytoma, paraganglioma), germ cell tumors (including seminoma, teratoma, non-seminomatous tumor), thymic tumors (including thymoma, thymic lipoma, thymic carcinoma, etc.). Thymic carcinoid tumors, mesenchymal tumors (including fibroma, fibrosarcoma, lipoma, liposarcoma, myxoma, mesothelioma, leiomyoma, leiomyosarcoma, rhabdomyosarcoma, xanthogranuloma, mesenchymal tumor, hemangioma, hemangioendothelioma, hemangiopericytoma, lymphangioma, lymphangiopericytoma, lymphangiomyoma); cancers / tumors / carcinomas of the gastrointestinal (GI) tract: e.g., esophagus, stomach (gastric cancer), pancreas, liver and biliary tract (including hepatocellular carcinoma (HCC), e.g., childhood HCC, fibrolamellar HCC, mixed HCC, spindle cell HCC, clear cell HCC, giant cell HCC, etc.). Hepatocellular carcinoma (HCC), carcinosarcoma HCC, sclerosing HCC; hepatoblastoma; cholangiocarcinoma; cholangiocarcinoma; hepatic cystadenocarcinoma; angiosarcoma, hemangioendothelioma, leiomyosarcoma, malignant schwannoma, fibrosarcoma, kratzkinoma), gallbladder, extrahepatic bile ducts, small intestine (including duodenum, jejunum, ileum), large intestine (including cecum, colon, rectum, anus); colorectal cancer, gastrointestinal stromal tumor (GIST)), genitourinary system (including kidney, such as renal pelvis, renal cell carcinoma (RCC), nephroblastoma) (Wilms) Tumors: adrenoid tumors, glaucoma; ureter; bladder, such as urachal carcinoma, urothelial carcinoma; urethra, such as distal, bulbar, prostatic tumors; prostatic (androgen-dependent, androgen-independent, castration-resistant, hormone-independent, hormone-resistant) tumors / cancers of the penis; testicular cancers: such as seminoma, non-seminomatous tumors; gynecological cancers / tumors / cancers: such as tumors / cancers of the ovary, fallopian tubes, peritoneum, cervix, vulva, vagina, and uterine body (including endometrium and basal layer);Breast cancers / tumors / cancers: e.g., breast cancer (invasive ductal, colloidal, lobular, tubular, cystic, papillary, medullary, mucinous), 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; cancers / tumors / cancers of the endocrine system: e.g., the following tumors / cancers / cancers: endocrine glands, thyroid gland (thyroid cancer / tumor; papillary, follicular, anaplastic, medullary), parathyroid gland (parathyroid cancer / tumor), adrenal cortex (adrenal cortical cancer / tumor), pituitary gland (including prolactinoma, cranial... Pharynx duct tumor, thymus, adrenal gland, pineal gland, carotid body, islet cell tumor, paraganglionic tumor, pancreatic endocrine tumor (PET; non-functional PET, pancreatic polypeptide tumor, gastrinoma, insulinoma, vasodilator intestinal peptide tumor, glucagonoma, somatostatinoma, growth hormone-releasing factor tumor, adrenocorticotropic hormone tumor), carcinoid tumor; soft tissue sarcomas: for example, fibrosarcoma, fibrous histiocytoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, angiosarcoma, lymphangiosarcoma, Kaposi's sarcoma, glomus tumor, hemangiopericytoma, synovial sarcoma, giant cell tumor of tendon sheath, solitary fibrous tumor of pleura and peritoneum, diffuse interstitial tumors Skin tumors, malignant peripheral nerve sheath tumors (MPNST), granular cell tumors, clear cell sarcomas, melanocytic schwannomas, plexosarcomas, neuroblastomas, ganglioneuromas, neuroepithelial tumors, extraosseous Ewing's sarcoma, paragangliomas, extraosseous chondrosarcomas, extraosseous osteosarcomas, mesenchymal tumors, alveolar soft tissue sarcomas, epithelioid sarcomas, extrarenal rhabdomyosarcomas, proliferative small cell tumors; osteosarcomas: for example, myeloma, reticulum cell sarcoma, chondrosarcoma (including central, peripheral, clear cell, and mesenchymal chondrosarcomas), osteosarcomas (including paraosseous, periosteal, and highly malignant ones). Cancers of the skin include: surface, small cell, radiation-induced osteosarcoma, Paget's sarcoma, Ewing's tumor, malignant giant cell tumor, ameloma, (fibro) histiocytoma, fibrosarcoma, chordoma, small round cell sarcoma, hemangioendothelioma, hemangiopericytoma, osteochondroma, osteoid osteoma, 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, malignant lentigines, acral lentigines, nodular, intraocular melanoma), actinic keratosis, eyelid cancer;Central nervous system and brain vegetations: e.g., astrocytomas (cerebral, cerebellar, diffuse, fibrotic, anaplastic, pilocytic, protoplasmic, obese), glioblastoma, glioma, oligodendroglioma, oligoastrocytoma, ependymoma, ependymoblastoma, choroid plexus tumors, medulloblastoma, meningioma, schwannoma, hemangioblastoma, hemangioma, hemangiopericytoma, neuroma, gangliocytoma, neuroblastoma, retinoblastoma, schwannoma (e.g., auditory), spinal cord axis tumors; lymphomas and leukemias: e.g., B-cell non-Hodgkin lymphoma (NH). L (including small lymphocytic lymphoma (SLL), lymphoplasmacytic lymphoma (LPL), mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large cell lymphoma (DLCL), Burkitt lymphoma (BL)), T-cell non-Hodgkin lymphoma (including anaplastic large cell lymphoma (ALCL), 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), immunocytomas, chronic B-cell lymphoblastic leukemia (B-CLL), chronic T-cell lymphoblastic leukemia (T-CLL), B-cell small lymphoblastic lymphoma (B-SLL), cutaneous T-cell lymphoma (CTLC), primary central nervous system lymphoma (PCNSL), immunoblastoma, Hodgkin's disease (HD) (including nodular lymphocyte-dominant HD (NLPHD), tuberous sclerosis HD (NSHD), mixed cellularity HD (MCHD), lymphocyte-rich classic HD, lymphocyte-depleted HD (LDHD)), large granular lymphocytes Leukemia of the 1,000-1,000-1,000 years (LGL), chronic myeloid leukemia (CML), acute myeloid / myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), acute promyelocytic leukemia (APL), chronic lymphocytic / lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia, chronic myeloid / myeloid leukemia (CML), myeloma, plasmacytoma, multiple myeloma (MM), plasmacytoma, myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML); cancer of unknown primary origin (CUP).

[0113] In another preferred embodiment, the cancer to be treated with the compound intended to inhibit the interaction between MDM2 and p53 is selected from: brain tumors, such as acoustic neuroma, astrocytomas (e.g., pilocytic astrocytoma, fibrous astrocytoma, protoplasmic astrocytoma, gemistocytary astrocytoma, anaplastic astrocytoma, and glioblastoma), gliomas, brain lymphomas, brain metastases, pituitary tumors (e.g., prolactinomas, HGH-producing tumors, and ACTH-producing tumors), craniopharyngiomas, medulloblastomas, meningiomas, and oligodendrogliomas; and neuronal tumors (vesicles), such as tumors of the autonomic nervous system (e.g., sympathetic neuroblastomas, ganglioneuromas, paragangliomas (pheochromocytomas)). a. Chromaffinoma and carotid bulb tumors); tumors of the peripheral nervous system (such as stump neuroma, neurofibroma, schwannoma (schwannoma, Schwannoma, and malignant Schwannoma) and tumors of the central nervous system (such as brain tumors and myeloma); colorectal cancers, such as rectal cancer, colon cancer, colorectal cancer, anal cancer, large intestine cancer, small intestine and duodenum tumors; eyelid tumors, such as basal cell tumors or basal cell carcinoma; pancreatic cancer (pancreatic cancer or carcinoma) of the pancreas; bladder cancer (or carcinoma of the bladder) and other urothelial carcinomas; lung cancer (bronchial cancer), such as small cell bronchial carcinoma (oat cell carcinoma) and non-small cell bronchial carcinoma (NSCLC) (such as plate epithelial carcinomas, adenocarcinomas, and large cell bronchial carcinomas); breast cancer, such as mammary carcinoma (such as invasive ductal carcinoma, colloid carcinoma, invasive lobular carcinoma, tubular carcinoma, adenoid cystic 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), such as Burkitt's lymphoma. Lymphoma, low-grade non-Hodgkin's lymphoma (NHL) and mycosis fungoides; uterine cancer or endometrial cancer or uterine corpus cancer; CUP syndrome (cancer of unknown primary origin); ovarian cancer (ovarian cancer or ovarian carcinoma), such as mucinous carcinoma, endometrial carcinoma or serous carcinoma; gallbladder cancer; bile duct cancer, such as kratzkinoma; testicular cancer, such as seminoma and non-seminomatous carcinoma;Lymphoma (lymphosarcoma), such as malignant lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), such as chronic lymphocytic leukemia, leukemic reticuloendotheliosis, immunocytoma, plasmacytoma, multiple myeloma (MM), immunoblastic carcinoma, Burkitt lymphoma, T-zone mycosis fungoides, large cell anaplastic lymphoblastoma, and lymphoblastoma; laryngeal cancer, such as tumors of the vocal cords, supraglottic, glottic, and subglottic laryngeal tumors; bone cancer, such as osteochondroma, chondroma, and osteomalacia. Chondrocyte tumor, chondromycinous fibroma, osteoma, osteoid osteoma, osteoblastoma, eosinophilic granuloma, giant cell tumor, chondrosarcoma, osteosarcoma, Ewing's sarcoma, reticulum cell sarcoma, soft tissue sarcoma, liposarcoma, plasmacytoma, fibrous dysplasia, juvenile bone cysts, and aneurysmal bone cysts; tumors of the head and neck, such as those of the lips, tongue, floor of mouth, oral cavity, gingiva, palate, salivary glands, larynx, nasal cavity, paranasal sinuses, larynx, and middle ear; liver cancer, such as hepatocellular carcinoma. Cell carcinoma or hepatocellular carcinoma (HCC); leukemia, such as acute leukemia, such as acute lymphoblastic / lymphoblastic leukemia (ALL), acute myeloid leukemia (AML); chronic leukemia, such as chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML); myelodysplastic syndrome (MDS); stomach cancer (or gastric carcinoma), such as papillary, tubular and mucinous adenocarcinoma, signet ring cell carcinoma, adenosquamous carcinoma, small cell carcinoma and undifferentiated carcinoma; melanoma, such as superficial spreading, nodular, malignant lentigines and acral lentigines melanoma; kidney cancer, such as renal cell carcinoma or adrenal adenoma or Grawitz's tumor; esophageal cancer (or carcinoma of theoesophagus); penile cancer; prostate cancer (e.g. castration-resistant prostate cancer); throat cancer (or carcinomas of the... Pharynx, such as nasopharyngeal carcinoma, oropharyngeal carcinoma, and laryngopharyngeal carcinoma; retinoblastoma, vaginal cancer (vaginal cancer or vaginal carcinoma), mesothelioma; lamellar epithelial carcinoma, adenocarcinoma, carcinoma in situ, malignant melanoma, and sarcoma; thyroid cancer, such as papillary, follicular, and medullary thyroid carcinoma, as well as anaplastic carcinoma; and squamous cell carcinoma, epidermoid carcinoma, and lamellar epithelial carcinoma of the skin.Thymoma, urethral cancer, cervical cancer, adenoid cystic carcinoma (AdCC), adrenocortical carcinoma, and vulvar cancer. More preferably, the tumor has functional p53 and / or p53 wild-type status. Functional p53 means that p53 can bind to DNA and activate the transcription of target genes.

[0114] In one or more preferred embodiments of the present invention, the compound that inhibits the KRAS protein or a KRAS protein mutant is selected from KRAS (G12C) inhibitors or degraders, KRAS (G12D) inhibitors or degraders, GDP-KRAS inhibitors or degraders, and HER2 inhibitors or degraders.

[0115] In one or more preferred embodiments of the present invention, the compound that inhibits the KRAS protein or a KRAS protein mutant may be a compound that inhibits or degrades the HER2 protein or a mutant of the HER2 protein, wherein 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 KRAS protein mutant in the signal transduction pathway, and is located downstream of the HER2 protein or a mutant of the HER2 protein.

[0116] Those skilled in the art will understand that KRAS-dependent cancers will vary depending on the type of compound.

[0117] In one or more preferred embodiments of the present invention, the KRAS (G12C) inhibitor or degrader is selected from: sotorasidib (AMG510), adagraxidib (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.

[0118] In one or more preferred embodiments of the present invention, the KRAS (G12D) inhibitor or degrader is selected from 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.

[0119] In one or more preferred embodiments of the present invention, the GDP-KRAS inhibitor or degrader is selected from 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.

[0120] In one or more preferred embodiments of the invention, the HER2 inhibitor or degrader is a compound according to formula (F) (see below), more preferably the HER2 inhibitor or degrader is compound HER2-cpd#1.

[0121] In one or more preferred embodiments of the present invention, the compound that inhibits the interaction between MDM2 and p53 is MDM2i-cpd#1.

[0122] Preferably, the compound is selected from the compounds shown in Table 1.

[0123] Table 1

[0124]

[0125] Anticancer drugs are sometimes judged based on their effect on “biomarkers” or surrogate markers that can predict clinical benefits to patients, such as tumor regression. When monitoring patient responses to anticancer drugs, “biomarkers” can be the basis for definitively demonstrating that the administered compound and the chosen dose and treatment regimen can and will reach levels sufficient to effectively inhibit the targeted cancer. In the evaluation of molecularly targeted therapies, traditional clinical endpoints have proven difficult to apply; standard clinical trial endpoints for cytotoxic compounds have been found insufficient for evaluating molecularly targeted anticancer agents. For their development, it is important to have endpoints that can be used to assess the efficacy of compounds in modulating the activity of their molecular targets and the relationship between target modulation and clinical response. Therefore, the availability of biomarkers that indirectly indicate the effect of treatment on disease status is one of the key prerequisites for developing minimal or non-invasive techniques for assessing inhibitor efficacy, and thus for both clinical evaluation of drugs and long-term monitoring of the efficacy of approved drugs in therapy.

[0126] A “biomarker” can be defined as a measurable characteristic or property that is an indicator of normal physiological processes, pathogenic processes, or responses to exposure or therapeutic interventions. Molecular, histological, radiological, or physiological biomarkers represent different types of biomarkers based on the nature of the measurable characteristic (Aboy et al. 2019). The FDA and NIH further classify biomarkers into prognostic, predictive, responsive, surveillance, safety, diagnostic, and susceptibility / risk biomarkers (BEST resources). According to this classification, pharmacodynamic / responsive biomarkers are used to indicate a biological response that has occurred in an individual who has been exposed to a pharmaceutical product or environmental agent (BEST resource).

[0127] Pharmacodynamic / response biomarkers are biomarkers whose levels change in response to exposure to pharmaceutical products or environmental agents. Changes in pharmacodynamic / response biomarkers (such as circulating small molecules or proteins) or physiological measurements provide early evidence that treatment may affect a target clinical endpoint or can be used to assess pharmacological endpoints related to safety concerns. They can also provide useful information for patient management, such as whether to continue treatment or adjust the dosage, or for pharmaceutical product development, such as whether a drug has a pharmacodynamic effect considered relevant to clinical efficacy. Due to the serial nature of their assessment, pharmacodynamic / response biomarkers can also fall into the category of surveillance biomarkers. Pharmacodynamic / response biomarkers are crucial in the setup of early-stage drug development trials and can be used to measure response levels to interventions and guide clinical dose-response studies. The primary utility of pharmacodynamic / response biomarkers in clinical practice is to guide the administration or continuation of drugs or other interventions. Such biomarkers can be used to measure response levels, allowing for changes in individual drug dosages, or to identify whether to add, subtract, or replace therapy. In these cases, pharmacodynamic / response biomarkers can provide evidence of target involvement. In addition, these biomarkers can be used in pharmacological dose range studies to determine which doses should be considered in trials evaluating clinical outcomes (BEST resource).

[0128] As shown in the experimental embodiments of this application, the inventors have found that survival proteins are suitable and reliable biomarkers, preferably pharmacodynamic / response biomarkers, for evaluating the inhibitory activity of KRAS inhibitors or degraders against KRAS-dependent cancer cells and cancer types. This also applies to HER2 inhibitors or degraders against HER2 and / or KRAS-dependent cancer cells and cancer types; the inventors have found that survival proteins are suitable and reliable biomarkers for evaluating the inhibitory activity of HER2 inhibitors. Similarly, as in Examples 9.5 and... Figure 17 As shown, the survival protein was also downregulated after successful treatment with MDM2i-cpd#1, and therefore, the survival protein can be conveniently used as a circulating biomarker in patient blood samples to monitor treatment efficiency during tumor treatment, thereby reducing the burden on patients associated with invasive tumor biopsies or cumbersome imaging methods.

[0129] In one or more preferred embodiments of the invention, the compound that inhibits the KRAS protein or a KRAS protein mutant may be a compound that inhibits or degrades the HER2 protein or a mutant of the HER2 protein, wherein 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 KRAS protein mutant in the signal transduction pathway, and is located downstream of the HER2 protein or a mutant of the HER2 protein. Therefore, as used herein, the term "compound that inhibits the KRAS protein or a KRAS protein mutant" encompasses compounds that inhibit or degrade the HER2 protein or a mutant of the HER2 protein.

[0130] The present invention further relates to a method for selecting patients with cancer to be treated with KRAS inhibitors or degrading agents, said method comprising at least the following steps:

[0131] - Before administering KRAS inhibitors or degraders to patients, determine the levels of viable proteins in samples from patients, and

[0132] - Identify a decrease in the level of viable proteins in at least one second sample from the patient.

[0133] Similarly, the present invention further relates to a method for selecting a patient with cancer to treat with a compound that inhibits the interaction between MDM2 and p53, the method comprising at least the following steps:

[0134] - Before administering the compound to the patient, determine the level of viable proteins in samples from the patient, and

[0135] - Identify a decrease in the level of viable proteins in at least one second sample from the patient.

[0136] The present invention further relates to a method for selecting patients as described above, wherein, after administering a KRAS inhibitor or a degrading agent to the patient, or after administering a compound that inhibits the interaction between MDM2 and p53, the level of the surviving protein is determined to be reduced in several samples obtained from the patient, preferably in all samples.

[0137] The present invention further relates to a method for selecting a patient as described above, the method further comprising the step of selecting the patient to continue treatment with a KRAS inhibitor or a degrading agent or with a compound that inhibits the interaction between MDM2 and p53, respectively.

[0138] The present invention further relates to a method for selecting cancer patients to be treated with a compound that acts as a KRAS inhibitor or degrader, the method being performed by in vitro determination of the levels of viable proteins in at least a first sample and at least a second sample from the cancer patient, wherein the at least first sample is collected prior to treatment of the cancer patient with the compound that acts as a KRAS inhibitor or degrader, and the at least second sample is collected after treatment of the cancer patient with the compound that acts as a KRAS inhibitor or degrader, wherein the levels of viable proteins in the second or one or more additional samples collected from the cancer patient have been determined to be reduced after treatment of the cancer patient with the compound that acts as a KRAS inhibitor or degrader, compared with the first sample or with the penultimate sample of one or more additional samples.

[0139] The present invention further relates to a method for selecting cancer patients to be treated with a compound that inhibits the interaction between MDM2 and p53, the method being performed by in vitro determination of the levels of surviving proteins in at least a first sample and at least a second sample from the cancer patient, wherein the at least first sample was collected prior to treatment of the cancer patient with the compound that inhibits the interaction between MDM2 and p53, and the at least second sample was collected after treatment of the cancer patient with the compound that inhibits the interaction between MDM2 and p53, wherein, compared with the first sample or with the penultimate sample of one or more other samples, it has been determined that the levels of surviving proteins in the second or one or more other samples collected from the cancer patient are reduced after treatment with the compound that inhibits the interaction between MDM2 and p53.

[0140] The present invention further relates to a method for predicting the responsiveness of cancer patients to KRAS inhibitors or degraders, the method comprising at least the following steps:

[0141] - Provide a first sample from the patient.

[0142] - Determine the level of viable proteins in the first sample from the patient, then

[0143] - Administer KRAS inhibitors or degrading agents to the patients, and

[0144] - When it has been determined that the level of viable protein in a second or one or more additional samples provided by the patient is reduced after administration of the KRAS inhibitor or degrader, compared with a first sample or with the penultimate sample in one or more other samples, the patient is identified as having responded to treatment with the KRAS inhibitor or degrader.

[0145] The present invention further relates to a method for predicting the responsiveness of cancer patients to compounds that inhibit the interaction between MDM2 and p53, the method comprising at least the following steps:

[0146] - Provide a first sample from the patient.

[0147] - Determine the level of viable proteins in the first sample from the patient, then

[0148] - Administering a compound to the patient that inhibits the interaction between MDM2 and p53, and

[0149] - When it has been determined that, after administration of the compound that inhibits the interaction between MDM2 and p53, the level of the viable protein in the second or one or more additional samples provided by the patient is reduced, the patient is identified as having responded to treatment with the compound that inhibits the interaction between MDM2 and p53.

[0150] The present invention further relates to a method for determining whether a compound that inhibits KRAS protein or KRAS protein mutants is effective in cancer treatment and / or in monitoring the response of cancer patients to said treatment, said method comprising at least the following steps:

[0151] - Provide a first sample from the patient.

[0152] - Measure the level of viable proteins in the first sample from the patient.

[0153] - Administer the compound that inhibits KRAS protein or KRAS protein mutants to the patient.

[0154] - Provide a second sample from the patient.

[0155] - Measure the level of viable proteins in the second sample.

[0156] - Compare the levels of viable proteins measured in the first and second samples, and

[0157] -Optionally repeat steps four through six.

[0158] The decreased level of surviving proteins in the second sample indicates an effective response.

[0159] The present invention further relates to a method for determining whether a compound that inhibits the interaction between MDM2 and p53 is effective in cancer treatment and / or in monitoring the response of cancer patients to said treatment, the method comprising at least the following steps:

[0160] - Provide a first sample from the patient.

[0161] - Measure the level of viable proteins in the first sample from the patient.

[0162] - Administer the compound that inhibits the interaction between MDM2 and p53 to the patient.

[0163] - Provide a second sample from the patient.

[0164] - Measure the level of viable proteins in the second sample.

[0165] - Compare the levels of viable proteins measured in the first and second samples, and

[0166] -Optionally repeat steps four through six.

[0167] The decreased level of surviving proteins in the second sample indicates an effective response.

[0168] In a preferred embodiment of any of the methods described above, one or more of the samples are one or more blood, plasma, or serum samples.

[0169] In a preferred embodiment of any of the methods described above, the compound that inhibits the KRAS protein or a KRAS protein mutant is selected from KRAS (G12C) inhibitors or degraders, KRAS (G12D) inhibitors or degraders, GDP-KRAS inhibitors or degraders, and HER2 inhibitors or degraders.

[0170] In a further preferred embodiment of any of the methods described above, the KRAS (G12C) inhibitor or degrader is selected from: sotorasidib (AMG510), adagrasip (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.

[0171] In a further preferred embodiment of any of the methods described above, the KRAS (G12D) inhibitor or degrader is selected from 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.

[0172] In a further preferred embodiment of any of the methods described above, the GDP-KRAS inhibitor or degrader is selected from 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.

[0173] In a further preferred embodiment of any of the methods described above, the HER2 inhibitor or degrader is a compound according to formula (F) (see below), and more preferably the HER2 inhibitor or degrader is compound HER2-cpd#1.

[0174] In one or more preferred embodiments of the present invention, the compound that inhibits the KRAS protein or a KRAS protein mutant may be a compound that inhibits or degrades the HER2 protein or a mutant of the HER2 protein, wherein 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 KRAS protein mutant in the signal transduction pathway, and is located downstream of the HER2 protein or a mutant of the HER2 protein.

[0175] In a further preferred embodiment of any of the methods described above, the compound that inhibits the interaction between MDM2 and p53 is compound MDM2i-cpd#1.

[0176] In a further preferred embodiment of any of the methods described above, the survival protein is contained in exosomes.

[0177] In a further preferred embodiment of any of the methods described above, the cancer is a KRAS-dependent cancer, preferably selected from pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NSCLC), and colorectal cancer (CRC).

[0178] 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 cancer as defined above as a preferred cancer type to be treated with such an MDM2 inhibitor.

[0179] In a further preferred embodiment of any of the methods described above, determining the level of surviving proteins includes a surviving protein-specific assay, preferably selected from Western blotting, ELISA, RIA, MSD® S-PLEX technology and FACS, more preferably, wherein said assay is ELISA or MSD® S-PLEX technology.

[0180] According to a second aspect, the present invention relates to a compound for use in treating cancer patients that inhibits KRAS protein or KRAS protein mutants.

[0181] i) The KRAS inhibitor or degrader is selected from KRAS (G12C) inhibitors or degraders, KRAS (G12D) inhibitors or degraders, GDP-KRAS inhibitors or degraders, and HER2 inhibitors and degraders; and

[0182] ii) Where it has been determined that the patient has responded to treatment with the compound according to any of the methods described above.

[0183] According to this second aspect of the invention, the invention also relates to a compound for use in treating cancer patients that inhibits the interaction between MDM2 and p53.

[0184] i) The compound that inhibits the interaction between MDM2 and p53 is MDM2i-cpd#1; and

[0185] ii) Wherein, in accordance with the method for determining the responsiveness of a cancer patient to treatment with the compound of the present invention that inhibits the interaction between MDM2 and p53, it has been determined that the patient is responsive to treatment with said compound.

[0186] In one or more preferred embodiments of the present invention, the compound that inhibits the KRAS protein or a KRAS protein mutant may be a compound that inhibits or degrades the HER2 protein or a mutant of the HER2 protein, wherein 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 KRAS protein mutant in the signal transduction pathway, and is located downstream of the HER2 protein or a mutant of the HER2 protein.

[0187] The present invention further relates to KRAS inhibitors or degraders used in methods for treating KRAS-dependent cancers in patients exhibiting the molecular biomarker survival protein. The present invention further relates to compounds that inhibit the interaction between MDM2 and p53, used in methods for treating cancers in patients exhibiting the molecular biomarker survival protein.

[0188] The present invention further relates to KRAS inhibitors or degraders used in methods for treating patients with KRAS-dependent cancer, wherein the patients have been identified as exhibiting the molecular biomarker survival protein. The present invention further relates to compounds that inhibit the interaction between MDM2 and p53, used in methods for treating cancer in patients, wherein the patients have been identified as exhibiting the molecular biomarker survival protein.

[0189] The present invention further relates to KRAS inhibitors or degraders used in a method for treating a patient with K-ras-dependent cancer, the method comprising determining that the patient exhibits a molecular biomarker, the survival protein. The present invention further relates to compounds that inhibit the interaction between MDM2 and p53, used in a method for treating a patient with cancer, the method comprising determining that the patient exhibits a molecular biomarker, the survival protein.

[0190] The present invention further relates to a KRAS inhibitor or degrader used in a method for treating a patient with KRAS-dependent cancer, the method comprising...

[0191] - The level of viable proteins has been measured in the first sample obtained from the patient.

[0192] - The levels of surviving proteins in at least a second sample obtained from the patient during treatment have been compared with the levels of surviving proteins in the first sample, and

[0193] - It has been determined that the level of surviving proteins in the second sample is lower than the level of surviving proteins in the first sample, or that the level of surviving proteins in any other sample obtained from the patient after the second sample during treatment is lower than the level of surviving proteins in the penultimate sample obtained from the patient during treatment.

[0194] The present invention further relates to compounds that inhibit the interaction between MDM2 and p53, used in a method for treating cancer in patients, the method comprising...

[0195] - The level of viable proteins has been measured in the first sample obtained from the patient.

[0196] - The levels of surviving proteins in at least a second sample obtained from the patient during treatment have been compared with the levels of surviving proteins in the first sample, and

[0197] - It has been determined that the level of surviving proteins in the second sample is lower than the level of surviving proteins in the first sample, or that the level of surviving proteins in any other sample obtained from the patient after the second sample during treatment is lower than the level of surviving proteins in the penultimate sample obtained from the patient during treatment.

[0198] The present invention further relates to the KRAS inhibitors or degraders for the uses described above, wherein the compound is selected from KRAS (G12C) inhibitors or degraders, KRAS (G12D) inhibitors or degraders, GDP-KRAS inhibitors or degraders, and HER2 inhibitors and degraders. The present invention further relates to the KRAS inhibitors or degraders for the uses described above, wherein the compound is MDM2i-cpd#1.

[0199] The present invention further relates to the KRAS inhibitors or degraders, or compounds that inhibit the interaction between MDM2 and p53, for use as described above, wherein the surviving protein is contained in exosomes.

[0200] The present invention further relates to the KRAS inhibitors or degraders, or compounds that inhibit the interaction between MDM2 and p53, for use as described above, wherein one or more of the samples are one or more blood, plasma or serum samples.

[0201] The present invention further relates to the K-Ras inhibitors or degraders for use as described above, wherein the KRAS-dependent cancers are selected from pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NSCLC), and colorectal cancer (CRC). The present invention further relates to the compounds for use as described above that inhibit the interaction between MDM2 and p53, wherein the cancers are selected from any cancers defined above as preferred cancer types treated with such MDM2 inhibitors.

[0202] The present invention further relates to the K-Ras inhibitors or degraders, or compounds that inhibit the interaction between MDM2 and p53, for use as described above, wherein determining the level of surviving proteins includes surviving protein-specific assays, preferably selected from Western blotting, ELISA, RIA, MSD® S-PLEX technology and FACS.

[0203] In its broadest sense, a "biomarker" can be defined as a measurable characteristic that is an indicator of normal physiological processes, pathogenic processes, or responses to exposure or (therapeutic) interventions. Molecular, histological, radiological, or physiological biomarkers represent different types of biomarkers and are already in use in medicine: blood pressure or glucose levels are direct examples of physiological and molecular biomarkers, respectively.

[0204] Various methods are being used to further classify biomarkers. For example, the FDA and NIH classify them into prognostic, predictive, reactive, surveillance, safety, diagnostic, and susceptibility / risk biomarkers (BEST Resources 2018).

[0205] Within the meaning of this invention, biomarkers are used as indicators of biological state. They are objectively measured and evaluated characteristics that serve as indicators of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic interventions. This is consistent with the definition given by the NIH research group in 1998.

[0206] More specifically, biomarkers indicate changes associated with the risk or progression of disease or with susceptibility to a given treatment. Once a proposed biomarker has been validated, it can be used to diagnose disease risk, the presence of disease in an individual, or to tailor treatment (selection of drug therapy or administration regimen) for the disease in an individual. In evaluating potential drug therapies, biomarkers can be used as surrogate endpoints for natural endpoints such as survival or irreversible disease. If a treatment alters a biomarker directly associated with improved health, the biomarker is used as a surrogate endpoint for evaluating clinical benefit.

[0207] As used herein, the term "sample" refers to a tissue sample or a body fluid sample, such as a blood sample. Preferably, the sample is a blood, plasma, or serum sample.

[0208] A tissue sample is a slice of an organ or tissue from the body, typically comprising several cell types and optionally having a cytoskeletal structure that holds the cells together. Tissue samples can be obtained through biopsy (e.g., by cutting, slicing, or punching). This involves extracting sample cells or tissue for examination. However, the terms “providing a sample,” “sample from a patient,” or “sample obtained from a patient” do not include the active step of extracting tissue or blood, but rather refer to providing a sample that has already been extracted, i.e., providing an ex vivo sample from the patient. As used herein, the term “sample obtained from a patient” 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.”

[0209] In addition, tissue samples can be tumor samples or corresponding control samples, preferably from the same tissue. Blood samples can be serum or plasma samples, and preferably plasma samples. However, blood samples may also include cellular fractions, for example, for complete blood counts and blood cell examinations, such as peripheral blood smears. Other body fluid samples besides blood include, but are not limited to, mucus, semen, saliva, sputum, bronchoalveolar lavage fluid, breast milk, bile, and urine. Samples may further be bone marrow biopsies or aspirates or cerebrospinal fluid.

[0210] Samples can be analyzed using any method known in the art, including, but not limited to, cytochemistry, such as chemical staining agents (dyes) that react with certain substances found in different types of cells, flow cytometry, and 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, analysis is performed by ELISA or MSD® S-PLEX counting.

[0211] In any of the methods described above, the level of the biomarker survival protein in the second (or n+1) sample is reduced by 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%, compared to the level of the biomarker survival protein in the first (or n) sample.

[0212] Survival protein-specific assays, particularly those involving Western blotting, dot blotting, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), RIST (radioimmunoassay), MSD® S-PLEX technology, and FACS (fluorescence-activated cell sorting), can be antibody-based, especially monoclonal antibodies. 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).

[0213] Antibodies and monoclonal antibodies targeting survival proteins have been described (Fenstermaker et al. 2018; Watanuki-Miyauchi et al. 2005; Arora et al. 2012).

[0214] Survival protein-specific assays are known in the prior art (Naumunik et al. 2009; Derin et al. 2008).

[0215] As used herein, the term "antibody" refers to a protein consisting of one or more polypeptide chains encoded by a natural or recombinant immunoglobulin gene or a fragment of an immunoglobulin gene or cDNA derived therefrom. The immunoglobulin gene includes any of the light chain κ, λ and heavy chain α, δ, ε, γ and μ constant region genes, as well as many different variable region genes.

[0216] Basic immunoglobulin (antibody) structural units are typically tetramers composed of two pairs of identical polypeptide chains (light chain (L, with a molecular weight of approximately 25 kDa) and heavy chain (H, with a molecular weight of approximately 50-70 kDa)). Each heavy chain consists of a heavy chain variable region (abbreviated as VH or V). H ) and heavy chain constant region (abbreviated as CH or C) H The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated as VL or V). L ) and light chain constant region (abbreviated as CL or C) L The VH and VL regions can be further subdivided into hypervariable regions, also known as complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL region consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains form binding domains that interact with the antigen.

[0217] The CDR (Containing Derivative) is crucial for the binding of the antibody or its antigen-binding portion. The FR (Containing Receptor) can be replaced by other sequences, provided that the three-dimensional structure required for antigen binding is preserved. Structural changes to the construct most often result in the loss of adequate antigen binding.

[0218] The term (monoclonal) antibody, its antigen-binding fragment or derivative, and the term "antigen binding" of antigen-binding antibody-like proteins refer to one or more portions or fragments of an antibody that retain the ability to specifically bind to an antigen in its native form. Examples of antigen-binding portions of an antibody include the Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1 domains, the F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by disulfide bonds in a hinge region, the Fd fragment consisting of VH and CH1 domains, the Fv fragment consisting of the VL and VH domains of an antibody single arm, and the dAb fragment consisting of a VH domain and a separated complementarity-determining region (CDR).

[0219] As used herein, the term "monoclonal antibody (mAb)" should refer to an antibody composition having a homogeneous antibody population, i.e., a homogeneous population consisting of intact immunoglobulins or fragments or derivatives thereof. Such antibodies may be selected from IgG, IgD, IgE, IgA, and / or IgM or fragments or derivatives thereof. Particularly preferred is IgG.

[0220] As used herein, the term "fragment" of an antibody should refer to a fragment of an antibody that retains the ability to bind to a target, such as a CDR (complementarity-determining region), a hypervariable region, a variable domain (Fv), an IgG heavy chain (composed of VH, CH1, hinge, CH2 and CH3 regions), an IgG light chain (composed of VL and CL regions), and / or Fab and / or F(ab)2.

[0221] As used herein, the term antibody “derivative” should refer to a protein construct that is structurally different from the common antibody concept but still has some structural relationship with the common antibody concept, such as scFv, Fab and / or F(ab)2, as well as bispecific, trispecific or higher specific antibody constructs.

[0222] As used herein, the term “KRAS-dependent cancer” refers to cancer, cancer cells, or cancerous tissue comprising (i) one or more mutations in the K-ras oncogene, said mutation alone or together with one or more co-mutations that can affect the function of the KRAS protein and the occurrence and development of tumors, and / or said formation of the cancer, cancer cells, or cancerous tissue is caused by 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 the most common oncogene drivers in human cancers. The K-ras mutation spectrum can vary significantly across different cancer types. K-ras mutations are predominantly single-base missense mutations, with 98% found at codon 12 (G12), codon 13 (G13), or codon 61 (Q61). Mutations in the KRAS protein have been shown to impede the interaction of KRAS with GAP and the hydrolysis of GTP bound to KRAS, leaving KRAS in a constitutively active state (Huang et al. 2021).

[0223] As used herein, the term "KRAS mutant" refers to a K-ras gene or KRAS protein containing any kind of mutation, either in the K-ras gene or at any amino acid position in the KRAS protein. Specifically, 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.

[0224] Significant amplification of the wild-type (WT) KRAS proto-oncogene has been observed in subgroups of cancer indications (such as gastric cancer, gastroesophageal junction cancer, and esophageal cancer), as described above.

[0225] As used herein, the term "KRAS inhibitor" or "K-Ras inhibitor" refers to any compound, agent, small molecule, peptide, polypeptide, or protein capable of inhibiting K-Ras activity, including degraders of KRAS proteins. The KRAS inhibitor according to the invention is preferably a compound that inhibits wild-type (preferably amplified) KRAS. KRAS mutants at residue 12, such as KRAS G12C, KRAS G12D, KRAS G12V, KRAS G12A, and KRAS G12R, are preferably inhibited by KRAS G12C and / or KRAS G12D, as well as compounds that inhibit KRAS mutants at residue 13, such as KRAS G13D, or KRAS mutants at residue 61, such as KRASQ61H.

[0226] The inhibition can be direct or indirect, competitive or allosteric.

[0227] Indirect inhibitors include, but are not limited to, compounds that inhibit K-Ras activity by interfering with signaling molecules upstream of the KRAS signaling pathway, such as, for example, 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.

[0228] In one or more preferred embodiments of the present invention, the compound that inhibits the KRAS protein or a KRAS protein mutant may be a compound that inhibits or degrades the HER2 protein or a mutant of the HER2 protein, wherein 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 KRAS protein mutant in the signal transduction pathway, and is located downstream of the HER2 protein or a mutant of the HER2 protein.

[0229] The ERBB transmembrane receptor tyrosine kinase (RTK) family consists of four members: EGFR (ERBB1), HER2 (Neu, ERBB2), HER3 (ERBB3), and HER4 (ERBB4), which perform essential functions during development (Citri et al. 2006; Wang, Z. 2017). ERBB signaling is initiated upon the binding of the extracellular domain of EGFR, HER3, or HER4 to their respective ligands, followed by homodimerization or heterodimerization of ERBB family members. HER2, whose ligand has not yet been identified, is the preferred dimerizing partner for other ERBB members. Once an active ligand-receptor complex is formed, the intracellular tyrosine kinase domains of EGFR, HER2, HER3, or HER4 are activated via autophosphorylation or transphosphorylation, subsequently triggering a signal transduction cascade, most notably involved in the mitogen-activated protein (MAP) kinase and / or phosphatidylinositol 3-kinase (PI3K) pathway (Citri et al. 2006; Wang, Z. 2017), which involves K-Ras, as described above.

[0230] In cancer, ERBB signaling is overactivated by mutations that promote dimerization or shift the balance towards the active conformational isomer of the kinase and / or confer constitutive activity to the RTK through amplification and subsequent RTK overexpression. Both oncogenic mechanisms increase the net output of ERBB signaling and thus promote cell survival, cell growth, and proliferation.

[0231] Aberrant HER2 signaling has been observed in a variety of human malignancies. Oncogenic mutations in the extracellular, (near) membrane, and intracellular regions of the protein are described. In general, these mutations endow HER2 with constitutive activity, promoting cancer development, tumor maintenance, and growth. Similarly, HER2 overexpression increases HER2 signaling and serves as a basis for tumorigenic transformation and tumor maintenance in various indications, including breast, gastric, and lung cancer.

[0232] Therefore, interfering with HER2 oncogenic signaling can lead to the suppression of tumor growth. Targeted therapies include antibodies against HER2 (e.g., trastuzumab and pertuzumab), antibody-drug conjugates against HER2 (trastuzumab-DM1 (T-DM1, ado-emtansine-trastuzumab)), and small molecules that inhibit the HER2 kinase domain (afatinib, neratinib, lapatinib).

[0233] Tumors driven by HER2 oncogenic mutations or HER2 wild-type overexpression (e.g., due to gene amplification) may benefit from HER2-specific tyrosine kinase inhibitors (TKIs). In general, HER2 alterations affect up to 6-7% of all human cancers, and EGFR-sparing TKIs may become an effective treatment option.

[0234] HER2 exon 20 mutations constitute a subset of HER2 gain-of-function mutations that lead to enhanced kinase activity. This enhanced HER2 kinase activity enters into a downstream signaling cascade that stimulates tumorigenic transformation by promoting the growth, proliferation, and survival of mutant cells. Selective inhibitors of HER2 exon 20 have been developed and, compared to prior art compounds, have shown improved wild-type EGFR-retaining efficacy characteristics in addition to high selectivity relative to wild-type EGFR. Furthermore, some compounds of this type have shown improved pharmacokinetic and pharmacological characteristics, such as good metabolic stability (WO 2021 / 213800).

[0235] KRAS inhibitors include, but are not limited to, compounds known in the art, such as sotorasibu (AMG510) and adagraxibu (MRTX849), as well as compounds detailed herein.

[0236] KRAS inhibitors preferably comprise cyclic 2-amino-3-cyanothiophene of formula (A) and its derivatives:

[0237]

[0238] Where R 1a R 1b R 2a R 2b Z, R 3 To R 5 A, p, U, V, W, L, and E have the following meanings:

[0239] [A0]

[0240] R 1a and R 1b All are independently selected from hydrogen and C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, halogens, -NH2, -NH(C) 1-4 alkyl), -N(C) 1-4 Alkyl)2, C 3-5 cycloalkyl and 3-5 membered heterocyclic groups;

[0241] R 2a and R 2b All are independently selected from hydrogen and C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, halogens, -NH2, -NH(C) 1-4 alkyl), -N(C) 1-4 Alkyl)2, C 3-5 cycloalkyl and 3-5 membered heterocyclic groups;

[0242] And / or, optionally, R 1a or R 1b One of them and R 2a or R 2b One of them, together with the carbon atoms to which they are attached, forms a cyclopropane ring;

[0243] [B0]

[0244] Z is -(CR) 6a R 6b ) n -;

[0245] Each R 6a and R 6b Independently selected from hydrogen and C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, halogens, -NH2, -NH(C) 1-4 alkyl), -N(C) 1-4 Alkyl)2, C 3-5 cycloalkyl and 3-5 membered heterocyclic groups;

[0246] n is selected from 0, 1, and 2;

[0247] [C0]

[0248] R 3 Selected from hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy groups, cyano-C 1-6 Alkyl, halogen, -OH, -NH2, -NH(C) 1-4 alkyl), -N(C) 1-4 Alkyl)2, -CN, C 3-5 cycloalkyl and 3-5 membered heterocyclic groups;

[0249] [D0]

[0250] Ring A is selected from the following rings: pyrrole, furan, thiophene, imidazole, pyrazole, oxazole, isoxazole, thiazole, isothiazole, and triazole;

[0251] [E0]

[0252] Each R 4 If it exists, then it is independently selected from C. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy groups, cyano-C 1-6 Alkyl, halogen, -OH, -NH2, -NH(C) 1-4 alkyl), -N(C) 1-4 Alkyl)2, -CN, C 3-5 cycloalkyl and 3-5 membered heterocyclic groups;

[0253] p is selected from 0, 1, 2, and 3;

[0254] [F0]

[0255] U is selected from nitrogen (=N-) and R. A Substituted carbon (=C(R) A )-);

[0256] V is selected from nitrogen (=N-) and R. B Substituted carbon (=C(R) B )-);

[0257] W is selected from nitrogen (=N-) and R. C Substituted carbon (=C(R) C )-);

[0258] R A R B and R C Each is independently selected from hydrogen and C. 1-6 Halogenated alkyl, optionally C 3-5 Cycloalkyl-substituted C 2-6 alkynyl group, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy groups, halogens, -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, -SC 1-6 Alkyl group, -S(=O)2-C 1-6 Alkyl, C 3-5 Cycloalkyl, 3-5 membered heterocyclic groups, and C groups optionally substituted with substituents selected from the following 1-6Alkyl: 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;

[0259] [G0]

[0260] R 5 Selected from R a1 and R b1 ;

[0261] R a1 Selected from C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 cycloalkyl, C 4-10 Cycloalkenyl, 3-11 membered heterocyclic groups, C 6-10 Aryl and 5-10 membered heteroaryl, of which C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 cycloalkyl, C 4-10 Cycloalkenyl, 3-11 membered heterocyclic groups, C 6-10 Aryl and 5-10 heteroaryl groups are optionally separated by one or more identical or different R groups. b1 and / or R c1 replace;

[0262] Each R b1 Independently selected from -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)ORc1 -N(C 1-4 Alkyl)C(=O)OR c1 and divalent substituent = O;

[0263] Each R c1 Independently selected from hydrogen and C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 cycloalkyl, C 4-10 Cycloalkenyl, 3-11 membered heterocyclic groups, C 6-10 Aryl and 5-10 membered heteroaryl, of which C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 cycloalkyl, C 4-10 Cycloalkenyl, 3-11 membered heterocyclic groups, C 6-10 Aryl and 5-10 heteroaryl groups are optionally separated by one or more identical or different R groups. d1 and / or R e1 replace;

[0264] Each R d1 Independently selected from -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 divalent substituent = O;

[0265] Each R e1 Independently selected from hydrogen and C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6alkynyl group, C 3-10 cycloalkyl, C 4-10 Cycloalkenyl, 3-11 membered heterocyclic groups, C 6-10 Aryl and 5-10 membered heteroaryl, of which C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 cycloalkyl, C 4-10 Cycloalkenyl, 3-11 membered heterocyclic groups, C 6-10 The aryl and 5-10 heteroaryl groups are optionally substituted by one or more identical or different substituents selected from the following: C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-10 Cycloalkyl groups, 3-11 membered heterocyclic groups—which may optionally be substituted by one or more of the same or different of the following groups: C 1-4 Alkyl, C 6-10 Aryl, 5-10 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 Alkyl group (2) and divalent substituent = O;

[0266] [H0]

[0267] L is -L 1 -L 2 -L 3 -, where L 1 Connect to E;

[0268] L 1 Selected from bonds, -NH-, -N(C 1-4 Alkyl group, -O group, -C(=O) group, -NH-C(=O) group, -N(C) group 1-4 Alkyl)-C(=O)-, -C(=O)-NH-, -C(=O)-N(C 1-4 Alkyl)-, -C(=O)-, C 1-6 Alkylene, C 3-7 Cycloalkylene, phenylene, 4-12-membered heterocyclic cycloalkylene and 5-10-membered heteroarylene;

[0269] L 2 Selected from C 1-6 Alkylene, C 3-7 Cycloalkylene, phenylene, 4-12-membered heterocyclic cycloalkylene and 5-10-membered heteroarylene;

[0270] L 3 Selected from bonds, -NH-, -N(C 1-4 Alkyl group, -O group, -C(=O) group, -NH-C(=O) group, -N(C) group 1-4 Alkyl)-C(=O)-, -C(=O)-NH-, -C(=O)-N(C 1-4 Alkyl)-, -C(=O)-, C 1-6 Alkylene, C 3-7 Cycloalkylene, phenylene, 4-12-membered heterocyclic cycloalkylene and 5-10-membered heteroarylene;

[0271] Where L 1 L 2 and L 3 Each C in 1-6 Alkylene, C 3-7 Cycloalkylene, phenylene, 4-12-membered heterocyclic and 5-10-membered heteroarylene groups are optionally and independently substituted by one or more identical or different substituents selected from the following: C 2-6 Alkyne group, C 1-6 Haloalkyl, C 3-7 Cycloalkyl, phenyl, 5-6 heteroaryl, halogen, -OH, -CN, C 1-6 Alkoxy, -NH2, -NH(C) 1-4 alkyl), -N(C) 1-4 Alkyl group 2, -C(=O)OH, -C(=O)-OC 1-6 Alkyl group, -C(=O)NH2, -C(=O)NH(C 1-4 Alkyl), -C(=O)N(C 1-4 Alkyl group 2, divalent substituent =O, and C group optionally substituted with one or more identical or different substituents selected from the following. 1-6 Alkyl groups: halogens, -OH, -CN, C 1-4 Alkoxy, -NH2, -NH(C) 1-4 alkyl), -N(C) 1-4 Alkyl group 2, -C(=O)OH, -C(=O)-OC 1-6 Alkyl group, -C(=O)NH2, -C(=O)NH(C 1-4 Alkyl) and -C(=O)N(C 1-4 Alkyl)2;

[0272] [I0]

[0273] E is

[0274]

[0275] Indicates a double or triple bond;

[0276] Q 1 Selected from bonds, -CH2-, -CH(OH)-, -C(=O)-, -C(=O)N(R) G1 )-, -C(=O)O-, -S(=O)2-, -S(=O)2N(R G1 - and -C(=NR) H1 )-;

[0277] Each R G1 Independently selected from hydrogen and C 1-6 Alkyl, C 1-6 Halogenated alkyl, 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 cycloalkyl groups and 3-11 membered heterocyclic groups;

[0278] Each R H1 Independently selected from hydrogen, -OH, C 1-6 Alkoxy, -CN and C 1-6 alkyl;

[0279] if To represent a double bond, then

[0280] R D Selected from hydrogen, C 3-7 Cycloalkyl, phenyl, halogen, -CN, C 1-6 Alkyl group, -C(=O)OC 1-6 Alkyl group, -NHC(=O)-C 1-6 Alkyl groups and C groups optionally substituted with one or more identical or different substituents selected from the following 1-6 Alkyl groups: phenyl, 3-11 membered heterocyclic groups, C 1-6 Alkoxy, halogen, -OH, -NH2, -NH(C) 1-6 alkyl), -N(C) 1-6 Alkyl group 2, -C(=O)OH, -C(=O)OC 1-6 Alkyl group, -C(=O)NH(C) 1-6 Alkyl), -NHC(=O)-C 1-6 Alkyl, -OC(=O)-C 1-6 Alkyl and phenyl-C 1-6 Alkoxy;

[0281] R E and R F Each independently selected from R a2 and R b2 ;

[0282] R a2 Selected from hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-10 Cycloalkyl, 3-11 membered heterocyclic groups, C 6-10 Aryl and 5-10 membered heteroaryl, of which C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-10 Cycloalkyl, 3-11 membered heterocyclic groups, C 6-10 Aryl and 5-10 heteroaryl groups are optionally separated by one or more identical or different R groups. b2 and / or R c2 replace;

[0283] Each R b2 Independently selected from -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 divalent substituent = O;

[0284] Each R c2 Independently selected from hydrogen and C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 cycloalkyl, C 4-10 Cycloalkenyl, 3-11 membered heterocyclic groups, C 6-10 Aryl and 5-10 membered heteroaryl, of which C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6alkynyl group, C 3-10 cycloalkyl, C 4-10 Cycloalkenyl, 3-11 membered heterocyclic groups, C 6-10 The aryl and 5-10 heteroaryl groups are optionally substituted by one or more identical or different substituents selected from the following: C 1-6 Alkyl, C 1-6 Alkyl groups, halogens, -OH, -C(=O)OH, -C(=O)OC 1-6 Alkyl group, -C(=O)C 1-6 Alkyl, -C(=O)-NH2, -C(=O)-NH-(C 1-6 Alkyl), -C(=O)-N(C 1-6 Alkyl group (2) and divalent substituent = O;

[0285] or

[0286] R D and R E Together with the carbon atoms attached to them, they form 4-7 membered unsaturated alicyclic rings or 4-7 membered unsaturated heterocyclic rings, wherein the 4-7 membered unsaturated alicyclic rings or 4-7 membered unsaturated heterocyclic rings and R F Optionally substituted by one or more identical or different substituents selected from the following: C 1-6 Alkyl, C 1-6 Halogenated alkyl, -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 group 2, halogen, -C(=O)OC 1-6 Alkyl group and divalent substituent = O;

[0287] or

[0288] If Q 1 It is -C(=O)N(R) G1 )-, then -C(=O)N(R G1 )- of R G1 and R F Together they form a connector selected from the following: -C(=O)-, -CH2-, -CH2-C(=O)-, -C(=O)-CH2- and -C2H4-;

[0289] if To represent a triple bond, then

[0290] R D and R E None of them exist;

[0291] R F It is Ra2 ;

[0292] R a2 Selected from hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-10 Cycloalkyl, 3-11 membered heterocyclic groups, C 6-10 Aryl and 5-10 membered heteroaryl, of which C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-10 Cycloalkyl, 3-11 membered heterocyclic groups, C 6-10 Aryl and 5-10 heteroaryl groups are optionally separated by one or more identical or different R groups. b2 and / or R c2 replace;

[0293] Each R b2 Independently selected from -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 divalent substituent = O;

[0294] Each R c2 Independently selected from hydrogen and C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-10 Cycloalkyl, 3-11 membered heterocyclic groups, C 6-10 Aryl and 5-10 heteroaryl groups;

[0295] or

[0296] E is

[0297]

[0298] Q 2 Selected from bonds, -CH2-, -CH(OH)-, -C(=O)-, -C(=O)N(R) G2)-, -C(=O)O-, -S(=O)2-, -S(=O)2N(R G2 - and -C(=NR) H2 )-;

[0299] Each R G2 Independently selected from hydrogen and C 1-6 Alkyl, C 1-6 Halogenated alkyl, 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 cycloalkyl groups and 3-11 membered heterocyclic groups;

[0300] Each R H2 Independently selected from hydrogen, -OH, C 1-6 Alkoxy, -CN and C 1-6 alkyl;

[0301] R I Selected from hydrogen and halogens;

[0302] R J It is hydrogen; or

[0303] R I and R J Together with the carbon atoms attached to them, they form cyclopropane or ethylene oxide rings;

[0304] R K Selected from hydrogen, C 1-6 Alkyl groups, -CN groups, and halogens;

[0305] R L Selected from hydrogen, C 1-6 Alkyl groups, -CN, halogens, and -C(=O)-C 1-6 alkyl;

[0306] or

[0307] E is

[0308]

[0309] Q 3 Selected from -C(=O)- and -C(=O)N(R) G3 )-, -C(=O)O-, -S(=O)2-, -S(=O)2N(R G3 - and -C(=NR)H3 )-;

[0310] Each R G3 Independently selected from hydrogen and C 1-6 Alkyl, C 1-6 Halogenated alkyl, 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 cycloalkyl groups and 3-11 membered heterocyclic groups;

[0311] Each R H3 Independently selected from hydrogen, -OH, C 1-6 Alkoxy, -CN and C 1-6 alkyl;

[0312] R M Selected from halogens, -CN and -OC(=O)-C 1-6 alkyl;

[0313] or

[0314] E is

[0315]

[0316] Q 4 Selected from bonds, -C(=O)-, -C(=O)O-, -C(=O)NH-, -C(=O)N(C 1-4 Alkyl group, -S(=O)2- and -S(=O)2NH-;

[0317] Ring B is selected from phenyl, pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, and 5-membered heteroaryl;

[0318] q is selected from 1, 2, 3, and 4;

[0319] Each R N Selected independently from C 1-4 Alkyl, C 1-4 Halogenated alkyl, vinyl, ethynyl, halogen, -CN, nitro and C 1-4 Alkoxy;

[0320] Or its salt.

[0321] KRAS inhibitors preferably also include compounds of formula (B):

[0322]

[0323] in

[0324] R 1a and R 1b All are independently selected from hydrogen and C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, halogens, -NH2, -NH(C) 1-4 alkyl), -N(C) 1-4 Alkyl)2, C 3-5 cycloalkyl and 3-5 membered heterocyclic groups;

[0325] R 2a and R 2b All are independently selected from hydrogen and C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, halogens, -NH2, -NH(C) 1-4 alkyl), -N(C) 1-4 Alkyl)2, C 3-5 cycloalkyl and 3-5 membered heterocyclic groups;

[0326] And / or, optionally, R 1a or R 1b One of them and R 2a or R 2b One of them, together with the carbon atoms to which they are attached, forms a cyclopropane ring;

[0327] Z is -(CR) 6a R 6b ) n -;

[0328] Each R 6a and R 6b Independently selected from hydrogen and C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, halogens, -NH2, -NH(C) 1-4 alkyl), -N(C) 1-4 Alkyl)2, C 3-5 cycloalkyl and 3-5 membered heterocyclic groups;

[0329] or R 6a and R 6b Together with the carbon atoms to which they are attached, they form a cyclopropane ring;

[0330] n is selected from 0, 1, and 2;

[0331] -L- is a key or a selection from -O-, -S-, and -N(R). 13 )-, where R 13 Is it hydrogen or C? 1-6 alkyl;

[0332] R 3 Replaced by E and

[0333] When -L- is selected from -O-, -S- and -N(R) 13 When )-, then R 3 Selected from C 1-6 Alkyl, C 1-6 Alkoxy, 5-10 membered heteroaryl and 3-11 membered heterocyclic, wherein C 1-6 Alkyl, 5-10 membered heteroaryl, C 1-6 The alkoxy group and the 3-11 membered heterocyclic group are optionally and independently substituted by one or more identical or different substituents selected from the following: halogen, C 1-6 Alkyl, -OH, -NH2, -NH(C) 1-4 alkyl), -N(C) 1-4 Alkyl)2, C 3-5 cycloalkyl groups and 3-11 membered heterocyclic groups;

[0334] When -L- is a key, then R 3 Selected from halogens, C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-10 Cycloalkyl, 3-11 membered heterocyclic groups, C 6-10 Aryl and 5-10 membered heteroaryl, of which C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-10 Cycloalkyl, 3-11 membered heterocyclic groups, C 6-10 Aryl and 5-10 heteroaryl groups are optionally and independently bound by one or more identical or different R groups. 7 and / or R 8 replace

[0335] Each R 7 Independently selected from halogens, -CN, -OH, 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 divalent substituent = O;

[0336] Each R8 Independently selected from hydrogen and C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-11 membered heterocyclic, phenyl and 5-10 membered heteroaryl, wherein C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-11 membered heterocyclic, phenyl, and 5-10 membered heteroaryl groups are optionally separated by one or more identical or different R groups. 9 and / or R 10 replace;

[0337] Each R 9 Independently is -OR 10 ;

[0338] Each R 10 Independently selected from hydrogen and C 1-6 Alkyl, 3-11 membered heterocyclic and 5-10 membered heteroaryl;

[0339] W represents nitrogen (-N=) or -CH=;

[0340] V represents nitrogen (-N=) or -CH=;

[0341] U is nitrogen (-N=) or -C(R) 11 =

[0342] R 11 Selected from hydrogen, halogens and C 1-4 Alkoxy;

[0343] Ring A is selected from the following rings: pyrrole, furan, thiophene, imidazole, pyrazole, isoxazole, isothiazole and triazole;

[0344] Each R 4 If it exists, then it is independently selected from C. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy groups, cyano-C 1-6 Alkyl, halogen, -OH, -NH2, -NH(C) 1-4 alkyl), -N(C) 1-4 Alkyl)2, -CN, C 3-5 cycloalkyl and 3-5 membered heterocyclic groups;

[0345] p is selected from 0, 1, 2, and 3;

[0346] R 5 It is optionally construed by one or more identical or different Cs 1-6 Alkyl, C 1-6 3-11 membered heterocyclic groups substituted with alkoxy or 5-6 membered heterocyclic groups, wherein C 1-6 The alkyl group is optionally replaced by a cyclopropyl group;

[0347] or R 5 It is -OC replaced by 3-11 membered heterocyclic groups. 1-6 Alkyl groups, wherein 3-11 membered heterocyclic groups are optionally surrounded by one or more identical or different R groups. 12 replace;

[0348] Each R 12 Selected from C 1-6 Alkyl, C 1-6 Alkoxy groups, halogens, and 3-11 membered heterocyclic groups;

[0349] E is

[0350]

[0351] Indicates a double or triple bond;

[0352] Q 1 Selected from bonds, -CH2-, -CH(OH)-, -C(=O)-, -C(=O)N(R) G1 )-, -C(=O)O-, -S(=O)2-, -S(=O)2N(R G1 - and -C(=NR) H1 )-;

[0353] Each R G1 Independently selected from hydrogen and C 1-6 Alkyl, C 1-6 Halogenated alkyl, 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 cycloalkyl groups and 3-11 membered heterocyclic groups;

[0354] Each R H1 Independently selected from hydrogen, -OH, C 1-6 Alkoxy, -CN and C 1-6 alkyl;

[0355] if To represent a double bond, then

[0356] R D Selected from hydrogen, C 3-7 Cycloalkyl, phenyl, halogen, -CN, C 1-6 Alkyl group, -C(=O)OC1-6 Alkyl group, -NHC(=O)-C 1-6 Alkyl groups and C groups optionally substituted with one or more identical or different substituents selected from the following 1-6 Alkyl groups: phenyl, 3-11 membered heterocyclic groups, C 1-6 Alkoxy, halogen, -OH, -NH2, -NH(C) 1-6 alkyl), -N(C) 1-6 Alkyl group 2, -C(=O)OH, -C(=O)OC 1-6 Alkyl group, -C(=O)NH(C) 1-6 Alkyl), -NHC(=O)-C 1-6 Alkyl, -OC(=O)-C 1-6 Alkyl and phenyl-C 1-6 Alkoxy;

[0357] R E and R F Each independently selected from R a2 and R b2 ;

[0358] R a2 Selected from hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-10 Cycloalkyl, 3-11 membered heterocyclic groups, C 6-10 Aryl and 5-10 membered heteroaryl, of which C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-10 Cycloalkyl, 3-11 membered heterocyclic groups, C 6-10 Aryl and 5-10 heteroaryl groups are optionally separated by one or more identical or different R groups. b2 and / or R c2 replace;

[0359] Each R b2 Independently selected from -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 divalent substituent = O;

[0360] Each R c2 Independently selected from hydrogen and C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 cycloalkyl, C 4-10 Cycloalkenyl, 3-11 membered heterocyclic groups, C 6-10 Aryl and 5-10 membered heteroaryl, of which C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 cycloalkyl, C 4-10 Cycloalkenyl, 3-11 membered heterocyclic groups, C 6-10 The aryl and 5-10 heteroaryl groups are optionally substituted by one or more identical or different substituents selected from the following: C 1-6 Alkyl, C 1-6 Alkyl groups, halogens, -OH, -C(=O)OH, -C(=O)OC 1-6 Alkyl group, -C(=O)C 1-6 Alkyl, -C(=O)-NH2, -C(=O)-NH-(C 1-6 Alkyl), -C(=O)-N(C 1-6 Alkyl group (2) and divalent substituent = O;

[0361] or

[0362] R D and R E Together with the carbon atoms attached to them, they form 4-7 membered unsaturated alicyclic rings or 4-7 membered unsaturated heterocyclic rings, wherein the 4-7 membered unsaturated alicyclic rings or 4-7 membered unsaturated heterocyclic rings and R F Optionally substituted by one or more identical or different substituents selected from the following: C 1-6 Alkyl, C 1-6 Halogenated alkyl, -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 group 2, halogen, -C(=O)OC 1-6 Alkyl group and divalent substituent = O;

[0363] or

[0364] If Q1 It is -C(=O)N(R) G1 )-, then -C(=O)N(R G1 )- of R G1 and R F Together they form a connector selected from the following: -C(=O)-, -CH2-, -CH2-C(=O)-, -C(=O)-CH2- and -C2H4-;

[0365] if To represent a triple bond, then

[0366] R D and R E None of them exist;

[0367] R F It is R a2 ;

[0368] R a2 Selected from hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-10 Cycloalkyl, 3-11 membered heterocyclic groups, C 6-10 Aryl and 5-10 membered heteroaryl, of which C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-10 Cycloalkyl, 3-11 membered heterocyclic groups, C 6-10 Aryl and 5-10 heteroaryl groups are optionally separated by one or more identical or different R groups. b2 and / or R c2 replace;

[0369] Each R b2 Independently selected from -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 divalent substituent = O;

[0370] Each Rc2 Independently selected from hydrogen and C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-10 Cycloalkyl, 3-11 membered heterocyclic groups, C 6-10 Aryl and 5-10 heteroaryl groups;

[0371] or

[0372] E is

[0373]

[0374] Q 2 Selected from bonds, -CH2-, -CH(OH)-, -C(=O)-, -C(=O)N(R) G2 )-, -C(=O)O-, -S(=O)2-, -S(=O)2N(R G2 - and -C(=NR) H2 )-;

[0375] Each R G2 Independently selected from hydrogen and C 1-6 Alkyl, C 1-6 Halogenated alkyl, 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 cycloalkyl groups and 3-11 membered heterocyclic groups;

[0376] Each R H2 Independently selected from hydrogen, -OH, C 1-6 Alkoxy, -CN and C 1-6 alkyl;

[0377] R I Selected from hydrogen and halogens;

[0378] R J It is hydrogen; or

[0379] R I and R J Together with the carbon atoms attached to them, they form cyclopropane or ethylene oxide rings;

[0380] R K Selected from hydrogen, C 1-6 Alkyl groups, -CN groups, and halogens;

[0381] RL Selected from hydrogen, C 1-6 Alkyl groups, -CN, halogens, and -C(=O)-C 1-6 alkyl;

[0382] or

[0383] E is

[0384]

[0385] Q 3 Selected from -C(=O)- and -C(=O)N(R) G3 )-, -C(=O)O-, -S(=O)2-, -S(=O)2N(R G3 - and -C(=NR) H3 )-;

[0386] Each R G3 Independently selected from hydrogen and C 1-6 Alkyl, C 1-6 Halogenated alkyl, 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 cycloalkyl groups and 3-11 membered heterocyclic groups;

[0387] Each R H3 Independently selected from hydrogen, -OH, C 1-6 Alkoxy, -CN and C 1-6 alkyl;

[0388] R M Selected from halogens, -CN and -OC(=O)-C 1-6 alkyl;

[0389] or

[0390] E is

[0391]

[0392] Q 4 Selected from bonds, -C(=O)-, -C(=O)O-, -C(=O)NH-, -C(=O)N(C 1-4 Alkyl group, -S(=O)2- and -S(=O)2NH-;

[0393] Ring B is selected from phenyl, pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, and 5-membered heteroaryl;

[0394] q is selected from 1, 2, 3, and 4;

[0395] Each R N Selected independently from C 1-4 Alkyl, C 1-4 Halogenated alkyl, vinyl, ethynyl, halogen, -CN, nitro and C 1-4 Alkoxy;

[0396] Or its salt.

[0397] KRAS inhibitors preferably also include compounds of formula (C):

[0398]

[0399] in

[0400] R 1a and R 1b All are independently selected from hydrogen and C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, halogens, -NH2, -NH(C) 1-4 alkyl), -N(C) 1-4 Alkyl)2, C 3-5 cycloalkyl and 3-5 membered heterocyclic groups;

[0401] R 2a and R 2b All are independently selected from hydrogen and C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, halogens, -NH2, -NH(C) 1-4 alkyl), -N(C) 1-4 Alkyl)2, C 3-5 cycloalkyl and 3-5 membered heterocyclic groups;

[0402] And / or, optionally, R 1a or R 1b One of them and R 2a or R 2b One of them, together with the carbon atoms to which they are attached, forms a cyclopropane ring;

[0403] Z is -(CR) 6a R 6b ) n -;

[0404] Each R 6a and R6b Independently selected from hydrogen and C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, halogens, -NH2, -NH(C) 1-4 alkyl), -N(C) 1-4 Alkyl)2, C 3-5 cycloalkyl and 3-5 membered heterocyclic groups;

[0405] or R 6a and R 6b Together with the carbon atoms to which they are attached, they form a cyclopropane ring;

[0406] n is selected from 0, 1, and 2;

[0407] R 3 Selected from halogens, C 1-6 Alkyl, C 1-6 Haloalkyl, -N3, C 3-10 Cycloalkyl, 3-11 membered heterocyclic groups, C 6-10 Aryl and 5-10 membered heteroaryl, of which C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-10 Cycloalkyl, 3-11 membered heterocyclic groups, C 6-10 Aryl and 5-10 heteroaryl groups are optionally and independently bound by one or more identical or different R groups. 7 and / or R 8 replace;

[0408] Each R 7 Independently selected from halogen, -CN, -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 divalent substituent = O;

[0409] Each R 8 Independently selected from hydrogen and C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-11 membered heterocyclic groups, C 6-10 Aryl and 5-10 membered heteroaryl, of which C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-11 membered heterocyclic groups, C 6-10 Aryl and 5-10 heteroaryl groups are optionally separated by one or more identical or different R groups. 9 and / or R10 replace;

[0410] Each R 9 Independently selected from -OR 10 -NR 10 R 10 and -C(O)NR 10 R 10 ;

[0411] Each R 10 Independently selected from hydrogen and C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-11 membered heterocyclic and 5-10 membered heteroaryl, wherein C 1-6 The alkyl group is optionally substituted with a substituent selected from the following: optionally with C 1-6 Alkyl-substituted C 1-6 Alkoxy, C 3-10 cycloalkyl groups and 3-11 membered heterocyclic groups;

[0412] W represents nitrogen (-N=) or -CH=;

[0413] V represents nitrogen (-N=) or -CH=;

[0414] U is nitrogen (-N=) or -C(R) 11 =

[0415] R 11 Selected from hydrogen, halogens and C 1-4 Alkoxy;

[0416] Ring A is selected from the following rings: pyrrole, furan, thiophene, imidazole, pyrazole, oxazole, isoxazole, thiazole, isothiazole, and triazole;

[0417] Each R 4 If it exists, then it is independently selected from C. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy groups, cyano-C 1-6 Alkyl, halogen, -OH, -NH2, -NH(C) 1-4 alkyl), -N(C) 1-4 Alkyl)2, -CN, C 3-5 cycloalkyl and 3-5 membered heterocyclic groups;

[0418] p is selected from 0, 1, 2, and 3;

[0419] R 5 It is optionally construed by one or more identical or different Cs 1-6 Alkyl, C 1-6 3-11 membered heterocyclic groups substituted with alkoxy or 5-6 membered heterocyclic groups, wherein C1-6 The alkyl group is optionally replaced by a cyclopropyl group;

[0420] or R 5 It is -OC replaced by 3-11 membered heterocyclic groups. 1-6 Alkyl groups, wherein 3-11 membered heterocyclic groups are optionally surrounded by one or more identical or different R groups. 12 replace,

[0421] Each R 12 Selected from C 1-6 Alkyl, C 1-6 Alkoxy groups, halogens, and 3-11 membered heterocyclic groups;

[0422] Or its salt.

[0423] KRAS inhibitors preferably also include compounds of formula (D):

[0424]

[0425] in

[0426] R 1a and R 1b All are independently selected from hydrogen and C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, halogens, -NH2, -NH(C) 1-4 alkyl), -N(C) 1-4 Alkyl)2, C 3-5 Cycloalkyl and 3-5 membered heterocyclic alkyl groups;

[0427] R 2a and R 2b All are independently selected from hydrogen and C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, halogens, -NH2, -NH(C) 1-4 alkyl), -N(C) 1-4 Alkyl)2, C 3-5 Cycloalkyl and 3-5 membered heterocyclic alkyl groups;

[0428] And / or, optionally, R 1a or R 1b One of them and R 2a or R 2b One of them, together with the carbon atoms to which they are attached, forms a cyclopropane ring;

[0429] Z is -(CR) 3a R 3b ) n-;

[0430] Each R 3a and R 3b Independently selected from hydrogen and C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, halogens, -NH2, -NH(C) 1-4 alkyl), -N(C) 1-4 Alkyl)2, C 3-5 Cycloalkyl and 3-5 membered heterocyclic alkyl groups;

[0431] or R 3a and R 3b Together with the carbon atoms to which they are attached, they form a cyclopropane ring;

[0432] n is selected from 0, 1, and 2;

[0433] R 4 Selected from hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy groups, cyano-C 1-6 Alkyl, halogen, -OH, -NH2, -NH(C) 1-4 alkyl), -N(C) 1-4 Alkyl)2, -CN, C 3-5 Cycloalkyl and 3-5 membered heterocyclic alkyl groups;

[0434] Cyclone A is a 5-membered heteroarylene;

[0435] Each R 5 If it exists, then it is independently selected from C. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy groups, cyano-C 1-6 Alkyl, halogen, -OH, -NH2, -NH(C) 1-4 alkyl), -N(C) 1-4 Alkyl)2, -CN, C 3-5 Cycloalkyl and 3-5 membered heterocyclic alkyl groups;

[0436] m is selected from 0, 1, 2, and 3;

[0437] W represents nitrogen (-N=) or -CH=;

[0438] V represents nitrogen (-N=) or -CH=;

[0439] U is nitrogen (-N=) or -C(R) 11 =

[0440] R 11 Selected from hydrogen, halogens and C 1-4 Alkoxy;

[0441] Ring B is optionally bounded by one or more identical or different Cs. 1-6 Alkyl, C 1-6 3-11-membered heterocyclic alkyl groups substituted with alkoxy or 5-6-membered heterocyclic alkyl groups, wherein C 1-6 The alkyl group is optionally replaced by a cyclopropyl group;

[0442] L is selected from key, C 1-8 Alkylene, C 2-8 imidene group, C 2-8 etyne and C 1-8 Alkyloxy;

[0443] X is -(CH2)- or -O-;

[0444] Y is a 5-membered heteroaryl group or -C(O)(NR) 12 )-, wherein the 5-membered heteroarylene contains at least one nitrogen atom and wherein -C(O)(NR 12 - Linked to X via a C atom;

[0445] R 9 It is C 1-4 alkyl;

[0446] R 10 Selected from hydrogen, C 1-6 Alkyl, C 1-6 Alkoxy, -C(O)R 12 and -C(O)OR 12 Wherein C 1-6 Alkyl groups may optionally be substituted with -OH or -OP(O)(OH)2;

[0447] Each R 12 Independently hydrogen or C 1-4 alkyl;

[0448] q is selected from 0, 1, and 2;

[0449] Each R 6 If present, it is either halogen or carbon each time it appears. 1-3 alkyl;

[0450] R 7 Selected from halogens, C 1-3 Alkyl, -CN, and 5-membered heteroaryl, wherein the 5-membered heteroaryl comprises at least one nitrogen atom and is optionally R 8 replace;

[0451] R 8It is C 1-3 Alkyl or C 1-3 Hydroxyalkyl;

[0452] Or its salt.

[0453] KRAS inhibitors preferably also include compounds of formula (E):

[0454]

[0455] in

[0456] R 1a and R 1b Both are independently selected from hydrogen and C. 1-4 Alkyl, C 1-4 Halogenated alkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, halogens, -NH2, -NH(C) 1-4 alkyl), -N(C) 1-4 Alkyl)2, C 3-5 cycloalkyl and 3-5 membered heterocyclic groups;

[0457] R 2a and R 2b All are independently selected from hydrogen and C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, halogens, -NH2, -NH(C) 1-4 alkyl), -N(C) 1-4 Alkyl)2, C 3-5 cycloalkyl and 3-5 membered heterocyclic groups;

[0458] And / or, optionally, R 1a or R 1b One of them and R 2a or R 2b One of them, together with the carbon atoms to which they are attached, forms a cyclopropane ring;

[0459] Z is -(CR) 6a R 6b ) n -;

[0460] Each R 6a and R 6b Independently selected from hydrogen and C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy groups, halogens, -NH2, -NH(C) 1-4 alkyl), -N(C) 1-4 Alkyl)2, C3-5 cycloalkyl and 3-5 membered heterocyclic groups;

[0461] or R 6a and R 6b Together with the carbon atoms to which they are attached, they form a cyclopropane ring;

[0462] n is selected from 0, 1, and 2;

[0463] L is selected from -O-, -S-, and -N(R) 7 )-, where R 7 Is it hydrogen or C? 1-6 alkyl;

[0464] R 3 Selected from C 1-6 Alkyl, C 1-6 Alkoxy, 5-10 membered heteroaryl and 3-11 membered heterocyclic, wherein C 1-6 Alkyl, 5-10 membered heteroaryl, C 1-6 The alkoxy group and the 3-11 membered heterocyclic group are optionally and independently substituted by one or more of the same or different of the following groups: halogen, C 1-6 Alkyl, C 1-6 Alkoxy, -OH, -NH2, -NH(C) 1-4 alkyl), -N(C) 1-4 Alkyl)2、-C(O)OC 1-6 Alkyl, C 3-5 cycloalkyl or optionally -N(C) 1-4 alkyl)2-substituted 3-11 membered heterocyclic groups;

[0465] W represents nitrogen (-N=) or -CH=;

[0466] V represents nitrogen (-N=) or -CH=;

[0467] U is nitrogen (-N=) or -C(R) 11 =

[0468] R 11 Selected from hydrogen, halogens and C 1-4 Alkoxy;

[0469] Ring A is selected from the following rings: pyrrole, furan, thiophene, imidazole, pyrazole, oxazole, isoxazole, thiazole, isothiazole, and triazole;

[0470] Each R 4 If it exists, then it is independently selected from C. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy groups, cyano-C 1-6Alkyl, halogen, -OH, -NH2, -NH(C) 1-4 alkyl), -N(C) 1-4 Alkyl)2, -CN, C 3-5 cycloalkyl and 3-5 membered heterocyclic groups;

[0471] p is selected from 0, 1, 2, and 3;

[0472] R 5 It is optionally construed by one or more identical or different Cs 1-6 Alkyl, C 1-6 3-11 membered heterocyclic groups substituted with alkoxy or 5-6 membered heterocyclic groups, wherein C 1-6 The alkyl group is optionally replaced by a cyclopropyl group;

[0473] or R 5 It is -OC replaced by 3-11 membered heterocyclic groups. 1-6 Alkyl groups, wherein 3-11 membered heterocyclic groups are optionally surrounded by one or more identical or different R groups. 12 replace,

[0474] Each R 12 Selected from C 1-6 Alkyl, C 1-6 Alkoxy groups, halogens, and 3-11 membered heterocyclic groups;

[0475] Or its salt.

[0476] HER2 inhibitors preferably comprise [1,3]diazino[5,4-d]pyrimidine and derivatives of formula (F):

[0477]

[0478] in

[0479] R 1 Selected from hydrogen, -CH3, -CCH, -OCH3, and halogens;

[0480] R 2 It is hydrogen or halogen;

[0481] R 3 Selected from formulas (i.1), (i.2), (i.3) and (i.4);

[0482] Table 2

[0483]

[0484] R 4 Selected from R 4.a and R 4.b

[0485] R 4.a

[0486] or

[0487] R 4.b

[0488] in

[0489] Q represents a 4-6 membered heterocyclic group containing one nitrogen atom, wherein one carbon atom of the ring is optionally replaced by a methyl group;

[0490] Z represents a 4-6 membered heterocyclic group containing one nitrogen atom, wherein one carbon atom of the ring is optionally replaced by a methyl group;

[0491] R 5 It is -H or -CH3;

[0492] And R 1 and R 2 At least one of them is not hydrogen.

[0493] Statistical methods for predicting clinical outcomes are known in the art and can be readily applied to the methods according to the present invention.

[0494] As used in this article, the terms “monitoring therapy” or “monitoring” refer to monitoring the degree of response, monitoring the duration of response, monitoring the response rate, monitoring the stability rate, monitoring the duration of stability, monitoring the time to disease progression, monitoring progression-free survival, or monitoring overall survival.

[0495] According to a third aspect, the present invention relates to a method of treating a patient's cancer with a compound that inhibits the KRAS protein or a KRAS protein mutant, wherein the patient has been determined to respond to treatment with the compound according to any of the methods described above. Similarly, the present invention relates to a method of treating a patient's cancer with a compound that inhibits the interaction between MDM2 and p53, wherein the patient has been determined to respond to treatment with the compound according to any of the methods described above.

[0496] The present invention further relates to a method for treating a patient with KRAS-dependent cancer using a KRAS inhibitor or a degrading agent, the method comprising:

[0497] - Determined that the patient exhibited the molecular biomarker survival protein, and

[0498] - Administer a pharmaceutically effective amount of a KRAS inhibitor or degrader to the patient.

[0499] The present invention further relates to a method for treating a patient's cancer with a compound that inhibits the interaction between MDM2 and p53, the method comprising:

[0500] - Determined that the patient exhibited the molecular biomarker survival protein, and

[0501] - Administer to patients a pharmaceutically effective amount of a compound that inhibits the interaction between MDM2 and p53.

[0502] The present invention further relates to a method for treating a patient with KRAS-dependent cancer using a KRAS inhibitor or a degrading agent, the method comprising:

[0503] - The level of viable proteins has been measured in the first sample obtained from the patient.

[0504] - The levels of surviving proteins in at least a second sample obtained from the patient during treatment have been compared with the levels of surviving proteins in the first sample, and

[0505] - It has been determined that the level of surviving proteins in the second sample obtained from the patient during treatment is lower than the level of surviving proteins in the first sample, or that the level of surviving proteins in any other sample obtained from the patient after the second sample during treatment is lower than the level of surviving proteins in the penultimate sample obtained from the patient during treatment.

[0506] Continue administering pharmaceutically effective doses of KRAS inhibitors or degraders to the patient.

[0507] The present invention further relates to a method for treating a patient's cancer with a compound that inhibits the interaction between MDM2 and p53, the method comprising:

[0508] - The level of viable proteins has been measured in the first sample obtained from the patient.

[0509] - The levels of surviving proteins in at least a second sample obtained from the patient during treatment have been compared with the levels of surviving proteins in the first sample, and

[0510] - It has been determined that the level of surviving proteins in the second sample obtained from the patient during treatment is lower than the level of surviving proteins in the first sample, or that the level of surviving proteins in any other sample obtained from the patient after the second sample during treatment is lower than the level of surviving proteins in the penultimate sample obtained from the patient during treatment.

[0511] - Continue administering to the patient a pharmaceutically effective amount of a compound that inhibits the interaction between MDM2 and p53.

[0512] The cancer patient may be selected according to any method described above for determining the responsiveness of a cancer patient to a KRAS inhibitor or to a compound that inhibits the interaction between MDM2 and p53.

[0513] As used herein, the term "monotherapy" refers to treatment with the compounds of this invention in the absence of other anticancer therapies, such as radiotherapy or chemotherapy.

[0514] As used herein, the term "combination therapy" as defined herein can be achieved by administering the individual components of the treatment simultaneously, sequentially, or separately. Combination therapy as defined herein can be used as a single therapy, or may involve surgery, radiation therapy, or additional chemotherapy agents or targeted agents in addition to the combination therapy of the present invention. Surgery may include the step of partial or complete tumor resection before, during, or after the administration of the combination therapy as described herein.

[0515] The treatment of KRAS-dependent cancers in patients may be a combination therapy using the KRAS inhibitor and another anticancer agent (e.g., an immune checkpoint inhibitor). Furthermore, the treatment of cancers that respond to compounds that inhibit the interaction between MDM2 and p53 may also be a combination therapy with the compound that inhibits the interaction between MDM2 and p53 and another anticancer agent (e.g., an immune checkpoint inhibitor).

[0516] "Immune checkpoints" are receptors on the cell membrane of T lymphocytes that regulate the immune reactivity of these cells. There are anti-inflammatory (suppressive) and pro-inflammatory (activating) immune checkpoints, expressed on the T cell membrane and interacting with corresponding ligands (soluble or cell-bound ligands). Tumor cells typically activate the anti-inflammatory immune checkpoint pathway via corresponding ligands that suppress the anti-tumor immune response, thus evading immune surveillance and promoting tumor growth. "Immune checkpoint inhibitors (ICIs)" are agents that bind to anti-inflammatory immune checkpoints or their ligands, particularly monoclonal antibodies, and can disrupt this tumor-suppressive strategy by reactivating the immune system, thus restoring its ability to fight tumors. ICIs have shown clinical efficacy against a variety of tumor types (Dyck and Mills, 2017).

[0517] In other words, immune checkpoint inhibitors include antagonists of immunosuppressive receptors (such as PD-1), which in this case inhibit 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 pildizumab), or antibodies that block PD-L1 (such as avelumab, durvalumab, and atezolizumab), as well as fully human antibodies (such as PD-1 blocker nivolumab).

[0518] The term "therapeutic effective dose" refers to the amount of an active agent that relieves or improves one or more symptoms of the treated disorder. On the other hand, a therapeutic effective dose refers to the target serum concentration of an active agent that has shown effectiveness, for example, in slowing disease progression. Efficacy can be measured in a conventional manner depending on the condition being treated.

[0519] As used herein, the terms “treatment” and “therapeutic approach” mean therapeutic, preventative, or inhibitory measures of a disease or disorder that result in any clinically necessary or beneficial effect, including but not limited to improving or alleviating one or more symptoms, eliminating, slowing, or stopping the progression of a disease or disorder. Thus, for example, the term “treatment” includes administering an agent before or after the onset of symptoms of a disease or disorder to prevent or remove one or more signs of said disease or disorder. As another example, the term includes administering an agent after the clinical manifestation of a disease to combat its symptoms.

[0520] Furthermore, in cases where the treatment affects clinical parameters of the disease or disorder (e.g., the extent of tissue damage or the amount or degree of metastasis), regardless of whether the treatment results in improvement of the disease, administration of the agent after the onset of the disease and after clinical symptoms have developed constitutes "treatment" or "therapy" as used herein. Additionally, if a therapeutic agent, alone or in combination with another therapeutic agent, reduces or improves at least one symptom of the treated disorder compared to the symptoms without the respective agent, the result should be considered an effective treatment for the underlying disorder, regardless of whether all symptoms of the disorder are relieved.

[0521] According to a fourth aspect, the present invention relates to the use of a survival protein in a method for determining the ability of a compound or a pharmaceutical formulation comprising a compound that inhibits KRAS protein or a KRAS protein mutant to treat cancer.

[0522] The present invention further relates to the use as described above, wherein the level of a viable protein in the following samples is determined: a sample obtained from a patient prior to treatment and at least one sample obtained from a patient after treatment with the compound that inhibits KRAS protein or a KRAS protein mutant, and wherein a decrease in the level of a viable protein after treatment with the compound that inhibits KRAS protein or a KRAS protein mutant indicates that the compound has the ability to treat the cancer.

[0523] Further according to this aspect of the invention, the invention also relates to the use of the survival protein 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, according to said use, the level of the survival protein is determined in at least one sample obtained from a patient before treatment and after treatment with said compound that inhibits the interaction between MDM2 and p53, wherein a decrease in the level of the survival protein after treatment with said compound that inhibits the interaction between MDM2 and p53 indicates the ability of the compound to treat said cancer.

[0524] The present invention further relates to the use of molecular biomarkers for selecting patients with KRAS-dependent cancers for treatment with KRAS inhibitors or degraders or pharmaceutical formulations containing said KRAS inhibitors or degraders, wherein the molecular biomarkers are survival proteins. The present invention further relates to the use of molecular biomarkers for selecting patients with cancers for treatment with compounds that inhibit the interaction between MDM2 and p53 or pharmaceutical formulations containing said compounds that inhibit the interaction between MDM2 and p53, wherein the molecular biomarkers are survival proteins.

[0525] The present invention further relates to the use of molecular biomarkers in methods for determining or confirming the ability of KRAS inhibitors or degraders, or pharmaceutical formulations containing said KRAS inhibitors or degraders, to inhibit KRAS-dependent cancers, wherein the molecular biomarkers are survival proteins. The present invention further relates to the use of molecular biomarkers in methods for determining or confirming the ability of compounds that inhibit the interaction between MDM2 and p53, or pharmaceutical formulations containing said compounds that inhibit the interaction between MDM2 and p53, to inhibit cancer, wherein the molecular biomarkers are survival proteins.

[0526] The present invention further relates to the use of the molecular biomarkers described above, wherein the levels of the surviving proteins of the following molecular biomarkers are determined: samples provided by the patient before treatment and at least one sample provided by the patient after treatment with the KRAS inhibitor or degrading agent, and wherein the levels of the surviving proteins are identified as reduced after treatment with the KRAS inhibitor or degrading agent. The present invention further relates to the use of the molecular biomarkers described above, wherein the levels of the surviving proteins of the following molecular biomarkers are determined: samples provided by the patient before treatment and at least one sample provided by the patient after treatment with the compound that inhibits the interaction between MDM2 and p53, and wherein the levels of the surviving proteins are identified as reduced after treatment with the compound that inhibits the interaction between MDM2 and p53.

[0527] The present invention further relates to the use of the molecular biomarkers described above, wherein the use is an in vitro use.

[0528] In a preferred embodiment of any of the uses described above, one or more of the samples are one or more blood, plasma, or serum samples.

[0529] In a further preferred embodiment of any of the uses described above, the compound that inhibits the KRAS protein or a KRAS protein mutant is selected from KRAS (G12C) inhibitors or degraders, KRAS (G12D) inhibitors or degraders, GDP-KRAS inhibitors or degraders, and HER2 inhibitors and degraders. In yet another further preferred embodiment of any of the uses described above, the compound that inhibits the interaction between MDM2 and p53 is MDM2i-cpd#1.

[0530] Those skilled in the art will understand that KRAS-dependent cancers will vary depending on the type of compound.

[0531] In a further preferred embodiment of any of the uses described above, the KRAS (G12C) inhibitor or degrader is selected from: sotorasidib (AMG510), adagraxib (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.

[0532] In a further preferred embodiment of any of the uses described above, the KRAS (G12D) inhibitor or degrader is selected from 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.

[0533] In a further preferred embodiment of any of the uses described above, the GDP-KRAS inhibitor or degrader is selected from 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.

[0534] In a further preferred embodiment of any of the uses described above, the HER2 inhibitor or degrader is a compound according to formula (F) (see above), and more preferably the HER2 inhibitor or degrader is compound HER2-cpd#1.

[0535] In one or more preferred embodiments of the present invention, the compound that inhibits the KRAS protein or a KRAS protein mutant may be a compound that inhibits or degrades the HER2 protein or a mutant of the HER2 protein, wherein 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 KRAS protein mutant in the signal transduction pathway, and is located downstream of the HER2 protein or a mutant of the HER2 protein.

[0536] In a further preferred embodiment for any of the uses described above, the survival protein is contained in exosomes.

[0537] In a further preferred embodiment of any of the uses described above, the KRAS-dependent cancer is selected from pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NSCLC), and colorectal cancer (CRC). In yet another further preferred embodiment of any of the uses described above, the cancer treated with a compound that inhibits the interaction between MDM2 and p53 is selected from any cancer defined above as a preferred cancer type treated with such an MDM2 inhibitor.

[0538] In a further preferred embodiment of any of the uses described above, determining the level of surviving proteins includes a surviving protein-specific assay selected from Western blotting, ELISA, RIA, MSD® S-PLEX technology, and FACS.

[0539] In a further preferred embodiment of any of the uses 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 with additional anticancer therapeutic agents and / or standard care.

[0540] According to a fifth aspect, the present invention relates to a multi-component kit comprising means for determining the level of viable proteins in a sample provided from a patient with cancer (e.g., KRAS-dependent cancer), and instructions on how to perform the method as described above.

[0541] The present invention further relates to a multi-component kit comprising means for determining the level of viable proteins in a sample provided from a patient with cancer (e.g., KRAS-dependent cancer), for performing any of the methods described above.

[0542] Example

[0543] Although the invention has been detailed and described in the accompanying drawings and the foregoing description, such description and description are to be considered illustrative or exemplary rather than restrictive; the invention is not limited to the disclosed embodiments. Other variations of the disclosed embodiments can be understood and implemented by those skilled in the art in practicing the claimed invention, by studying the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a or an" does not exclude multiple. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be beneficial. Any reference numerals in the claims should not be construed as limiting the scope.

[0544] All amino acid sequences disclosed herein are shown from the N-terminus to the C-terminus; all nucleic acid sequences disclosed herein are shown from the 5' end to the 3' end.

[0545] Materials and methods

[0546] Example 1: Compound

[0547] The KRAS inhibitory compounds and HER2 inhibitory compounds are shown in Table 1 above.

[0548] Compound Synthesis

[0549] Example 2 Synthesis of compounds according to formula (A)

[0550] The synthesis of the compound according to formula (A) has been described in WO 2021 / 245051.

[0551] Example 3 Synthesis of compounds according to formula (B)

[0552] List of abbreviations ( Table 3 )

[0553]

[0554] Example

[0555] The features and advantages of the invention will become apparent from the following detailed embodiments, which illustrate the principles of the invention by way of example and do not limit its scope:

[0556] Preparation of compounds according to the present invention

[0557] Unless otherwise stated, all reactions were carried out in commercially available apparatus using methods typically employed in chemical laboratories. Starting materials sensitive to air and / or moisture were stored under a protective gas, and the corresponding reactions and operations were performed under a protective gas (nitrogen or argon).

[0558] If a compound can be represented by both its structural formula and its nomenclature, then in the event of a conflict, the structural formula shall prevail.

[0559] The microwave reaction is carried out in a starter / reactor manufactured by Biotage, or in an Explorer manufactured by CEM, or in a Synthos 3000 or Monowave 3000 manufactured by Anton Paar, in a sealed container (preferably 2, 5 or 20 mL), preferably under stirring.

[0560] Chromatography

[0561] Thin-layer chromatography was performed on off-the-shelf silica 60 TLC plates on glass (with fluorescent indicator F-254) manufactured by Merck.

[0562] Preparative high-performance liquid chromatography (RP HPLC) of the compounds according to embodiments of the present invention was performed on an Agilent or Gilson system using columns manufactured by Waters (names: SunFire™ Preparative C18, OBD™ 10 µm, 50 x 150 mm or SunFire™ Preparative C18 OBD™ 5 µm, 30 x 50 mm or XBridge™ Preparative C18, OBD™ 10 µm, 50 x 150 mm or XBridge™ Preparative C18, OBD™ 5 µm, 30 x 150 mm or XBridge™ Preparative C18, OBD™ 5 µm, 30 x 50 mm) and columns manufactured by YMC (names: Actus-Triart Prep C18, 5 µm, 30 x 50 mm).

[0563] Different H2O / acetonitrile gradients were used to elute the compounds. For the Agilent system, 5% acid modifier (20 mL HCOOH to 1 L H2O / acetonitrile (1 / 1)) was added to the water (acidic conditions). For the Gilson system, 0.1% HCOOH was added to the water.

[0564] For chromatography under alkaline conditions, the Agilent system also uses an H2O / acetonitrile gradient, while making the water alkaline by adding 5% alkaline modifier (50 g NH4HCO3 + 50 mL NH3 (25%, in H2O) to 1 L (with H2O)). For the Gilson system, the water is made alkaline as follows: 5 mL of NH4HCO3 solution (158 g, in 1 L H2O) and 2 mL of NH3 (28%, in H2O) are added to 1 L.

[0565] Supercritical fluid chromatography (SFC) of the intermediates and compounds of the present invention was performed on a JASCO SFC system using the following columns: Chiralcel OJ (250 x 20 mm, 5 µm), Chiralpak AD (250 x 20 mm, 5 µm), Chiralpak AS (250 x 20 mm, 5 µm), Chiralpak IC (250 x 20 mm, 5 µm), Chiralpak IA (250 x 20 mm, 5 µm), Chiralcel OJ (250 x 20 mm, 5 µm), Chiralcel OD (250 x 20 mm, 5 µm), and Phenomenex Lux C2 (250 x 20 mm, 5 µm).

[0566] Use a column manufactured by Waters (name: XBridge) TM C18, 2.5 µm, 2.1 x 20 mm; or XBridge TM Analytical HPLC (reaction control) of intermediates and final compounds was performed using columns manufactured by YMC (name: Triart C18, 3.0 µm, 2.0 x 30 mm) and Phenomenex (name: Luna C18, 5.0 µm, 2.0 x 30 mm). In each case, the analytical apparatus was also equipped with a mass spectrometer detector.

[0567] HPLC-mass spectrometry / UV-spectroscopy

[0568] Retention time / MS-ESI for characterizing compounds according to embodiments of the present invention + It was generated using an HPLC-MS apparatus (high-performance liquid chromatography with a mass spectrometer detector). The retention time t of the compound eluting at the injection peak was given. Ret. =0.00.

[0569] SFC method (preparative)

[0570] Preparative SFC was performed in the Waters Thales SFC 80 system.

[0571] Column: Chiralpak AD-H (21 x 250 mm), 5 µm

[0572] Flow rate: 25 g / min

[0573] Mobile phase: 75% CO2 + 25% MeOH (0.5% isopropylamine)

[0574] ABPR: 120 bar

[0575] Temperature: 35ºC

[0576] UV: 220 nm

[0577] Stacking time: 8 min

[0578] HPLC method (analytical)

[0579] Method A

[0580] The samples were analyzed on an Agilent 1200 series LC system coupled with an Agilent 6140 mass spectrometer. Purity was determined by UV detection with a 170 nm bandwidth in the 230–400 nm range. LC parameters are as follows:

[0581] Waters Xbridge C18 column, 3.5 µm particle size, 2.1 x 30 mm;

[0582] Flow rate 1 mL / min;

[0583] Column temperature 60ºC;

[0584] Inject 5 µL.

[0585] Solvent A: 20 mM NH4HCO3 / NH3 pH 9

[0586] B: MS grade acetonitrile;

[0587] Gradient 0.0-1.5 min 10%-95% B

[0588] 1.5-2.0 min 95% B

[0589] 2.0-2.1 min 95%-10% B

[0590]

[0591]

[0592] The compounds and intermediates according to the invention are prepared by the synthetic methods described below, wherein the substituents of the general formula have the meanings given above. These methods are intended to illustrate the invention and not to limit its subject matter or the scope of the compounds claimed for these embodiments. Where the preparation of starting compounds is not described, they are commercially available or their synthesis is described in the prior art, or they can be prepared similarly to the known prior art compounds or methods described herein, i.e., the synthesis of these compounds is within the skill of an organic chemist. The substances described in the literature can be prepared according to the published synthetic methods. If the chemical structures depicted below do not have a precise configuration of the stereocenter, such as an asymmetrically substituted carbon atom, then both configurations should be considered to be included and disclosed in such illustrations. Stereocenter illustrations in racemic form should always be considered to include and disclose two enantiomers (if no other defined stereocenter exists) or all other potential diastereomers and enantiomers (if an additionally defined or undefined stereocenter exists).

[0593] Experimental procedure for the synthesis of A-2a

[0594]

[0595] Acetyl chloride (111.3 mL, 1.558 mol, 8.0 equivalent) was added dropwise to a suspension of 5-chloropentanones (22.9 g, 194.8 mmol, 1.0 equivalent) in dry EtOH (136 mL) at 0ºC. The reaction mixture was allowed to reach room temperature and stirred for 12 h. The mixture was concentrated under reduced pressure and washed with Et2O, and the crude product A-2a was used directly as an HCl salt for the next step without further purification.

[0596] Experimental procedure for the synthesis of A-3a

[0597]

[0598] Crude A-2a (HCl salt) (28 g, 139.9 mmol, 1.0 equivalent) and ethylene glycol (7.382 g, 118.94 mmol, 0.9 equivalent) were dissolved in DCM (300 mL) and stirred at room temperature for 6 days. The resulting suspension was concentrated under reduced pressure, diluted with Et2O (200 mL), and filtered. The filtrate was concentrated under reduced pressure, absorbed into DCM (200 mL), and treated with 2 N KOH solution (150 mL). The mixture was stirred overnight at room temperature to maintain phase integrity. The phases were separated, the aqueous phase was extracted twice with DCM, and the combined organic phases were dried over MgSO4, filtered, and concentrated under reduced pressure. Crude orthoester A-3a was used in the next step without further purification.

[0599] Experimental procedure for the synthesis of A-4a

[0600]

[0601] Crude 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) were dissolved in DCM (120 mL) and stirred at room temperature for 5 h. The reaction mixture was treated with the addition of a saturated sodium bicarbonate solution. The organic phase was separated, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by NP chromatography (gradient elution: 0% to 50% EtOAc in cHexane) to give the desired compound A-4a.

[0602] Experimental procedures for the synthesis of A-5a

[0603]

[0604] A-4a (14.9 g, 57.14 mmol, 1.0 equivalent) and sodium iodide (25.954 g, 171.4 mmol, 3.0 equivalent) were dissolved in acetone (120 mL) and stirred under reflux for 16 h. The reaction mixture was concentrated under reduced pressure, diluted with DCM, and washed with a saturated sodium thiosulfate solution. The organic phase was separated, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product A-5a was used in the next step without further purification.

[0605] Experimental procedures for the synthesis of A-6b

[0606]

[0607] A-5a (30 g, 85.0 mmol, 1.0 equivalent) was dissolved in THF. The mixture was treated with potassium tert-butoxide (28.67 g, 256.0 mmol, 3.0 equivalent) at 0ºC and stirred overnight at room temperature. The reaction mixture was quenched by adding water (2 mL) and diluted by adding Et2O and saturated sodium bicarbonate solution. The organic phase was separated, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by NP chromatography (gradient elution: 0% to 50% EtOAc in cHexane) to give (racemic) compound A-6a (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).

[0608] The desired enantiomer A-6b can then be obtained by chiral separation via SFC (e.g., using a CHIRACEL OX-3 column and acetonitrile as a co-solvent).

[0609] Experimental procedure for synthesizing E-4c (Method C)

[0610]

[0611] tert-butyl piperazine-1-carboxylate (11.22 g, 57.22 mmol, 1.0 equivalent) was added to a stirred solution of E-1c (10.20 g, 57.22 mmol) in DCM (60.0 mL). DIPEA (20.71 g, 160.21 mmol, 2.8 equivalent) was then added, and the reaction mixture was stirred at 60ºC for 1 h. After complete conversion, the mixture was dissolved in EtOAc and washed with water (3 x). The organic phase was dried, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (DCM / MeOH) to give E-4c.

[0612] According to methods A through E, the following (additional) intermediate E-4 (Table 4) can be obtained in a similar manner using different amines PG-LH and intermediate E-1. If necessary, the crude product E-4 can be purified by chromatography.

[0613] Table 4

[0614]

[0615] Experimental procedures for synthesizing E-8d

[0616]

[0617] Cesium fluoride (1.218 g, 8.02 mmol, 2.5 equivalents) was added to a solution of E-1a (600 mg, 3.21 mmol, 93% purity, 1.0 equivalent) in anhydrous DMSO (6 mL), and the resulting mixture was stirred at room temperature for 1 h until complete conversion of the starting material was observed. The resulting suspension was filtered, and the filtered solids were washed with anhydrous DMSO (2 mL). The filtrate (8 mL) was added to (S)-1-((S)-1-methylpyrrolid-2-yl)ethanol-1-ol (453 mg, 3.51 mmol, 1.1 equivalents), and DIPEA (1.085 mL, 6.38 mmol, 2 equivalents) was added. The mixture was stirred at room temperature for 1 h. After complete conversion of the starting material was observed, a solution of piperazine-1-carboxylic acid tert-butyl ester (674 mg, 3.51 mmol, 97% purity, 1.1 equivalents) in anhydrous DMSO (3 mL) and DIPEA (1.085 mL, 6.38 mmol, 2 equivalents) was added to the mixture. The mixture was stirred at room temperature for 30 min. After complete conversion was observed, the reaction mixture was diluted with acetonitrile and water, filtered, and purified by alkaline reversed-phase chromatography (gradient elution: 30% to 98% acetonitrile in water) to give the desired product E-8d.

[0618] The following intermediate E-8 (Table 5) can be obtained in a similar manner without separating the corresponding intermediates E-5 and E-7 separately. If necessary, the crude product E-8 can be purified by chromatography.

[0619] Table 5

[0620]

[0621] Experimental procedure for synthesizing E-8p (Method J)

[0622]

[0623] Trimethylamine (149.8 mg, 1.48 mmol, 2.5 equivalents) was added to a mixture of E-4r (200 mg, 0.59 mmol, 1.0 equivalents) and (S)-1-((S)-1-methylpyrrolidone-2-yl)ethanol-1-ol (91.8 mg, 0.71 mmol, 1.2 equivalents) in acetonitrile (1.5 mL). The mixture was stirred at 40ºC for 2 h. The mixture was then stirred at 80ºC for 16 h. The solvent was removed under reduced pressure, and the crude product was purified by normal-phase chromatography (gradient elution: 0% to 90% MeOH (in DCM + ammonia)) to give the desired product E-8p.

[0624] Intermediate E-8, labeled “J” (Table 6), can be obtained in a similar manner. If necessary, crude product E-8 can be purified by chromatography.

[0625] Table 6

[0626]

[0627] Experimental procedure for synthesizing E-8ch (Method M)

[0628]

[0629] E-6h (100.0 mg, 0.31 mmol, 1.0 equivalent) and (S)-1,3-dimethylpiperazine (42.5 mg, 0.37 mmol, 1.2 equivalent) were dissolved in DMSO (1 mL) at room temperature, and DIPEA (115.0 µL, 0.62 mmol, 2.0 equivalent) was added, and the mixture was stirred for 1 h. The mixture was diluted with acetonitrile and water, and purified by acid reversed-phase chromatography to give E-8ch.

[0630] Intermediate E-8, labeled “M” (Table 7), can be obtained in a similar manner. If necessary, crude product E-8 can be purified by chromatography.

[0631] Table 7

[0632]

[0633] Additional nitrile structural units E-8, not explicitly disclosed herein, are disclosed in WO 2021 / 245051 and WO 2021 / 245055 (including synthesis), the disclosures of which, by reference, relating to such structural units E-8, their synthesis, and their synthetic uses, are incorporated herein by reference. These structural units may also be used to synthesize other compounds of formula (I) according to the present invention, not explicitly disclosed herein.

[0634] Option 3a:

[0635]

[0636] Experimental procedures for the synthesis of E-12a

[0637]

[0638] A solution of sodium hydroxide in water (16 mL, 4 M, 63.96 mmol, 1 equivalence) was added to a solution of E-8aq (1.776 g, 4.26 mmol, 1 equivalence) in MeOH (35 mL), and the resulting mixture was stirred at 65ºC for 1.5 h. The reaction volume was reduced under reduced pressure to remove most of the MeOH, and the remaining aqueous solution was carefully neutralized with an aqueous solution of HCl (8 M). The mixture was diluted with acetonitrile and purified by acidic reversed-phase chromatography (gradient elution: 10% to 85% acetonitrile in water) to give the desired product E-12a.

[0639] Experimental procedures for the synthesis of E-12e

[0640]

[0641] A solution of sodium hydroxide in water (6.2 mL, 4 M, 40 mmol, 5.0 equivalents) was added to a solution of E-8c (2.2 g, 4.97 mmol, 1 equivalent) in MeOH, and the resulting mixture was stirred at 65ºC for 4 h. The reaction mixture was concentrated under reduced pressure, suspended in MeOH, filtered, and purified by acidic reversed-phase chromatography (gradient elution: 10% to 85% acetonitrile in water). The products containing fractions were combined, concentrated under reduced pressure, and lyophilized to give the desired product E-12e.

[0642] The following intermediates E-12 / E-12* (Table 8) can be obtained in a similar manner from different intermediates E-8 / E-8*. If necessary, the crude product E-8 / E-8* can be purified by chromatography.

[0643] Table 8

[0644]

[0645] Option 4a:

[0646]

[0647] Experimental procedures for the synthesis of B-1a

[0648]

[0649] CDI (18.781 g, 112.352 mmol, 2.0 equivalents) was dissolved in dry THF and heated to 50ºC. In a second flask, E-12d (13.021 g, 28.088 mmol, 0.5 equivalents) and the activated molecular sieve were stirred in dry THF at room temperature for 10 min, and then added to the CDI solution. The reaction mixture was stirred at 50ºC for 15 min. In a third flask, A-6b (15 g, 56.176 mmol, 1.0 equivalents) was dissolved in 1 M LiHMDS solution (117.969 mL, 117.969 mmol, 2.1 equivalents) in THF and stirred at room temperature for 10 min, and then added to the active ester. The reaction mixture was stirred at 50ºC overnight. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure, diluted with DCM, and washed with saturated sodium bicarbonate solution. The aqueous phase was extracted with EtOAx (3 x 100 mL). The combined organic phases were dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by NP chromatography (using a MeOH / DCM 0-10% gradient under alkaline conditions). The fractions were combined and lyophilized to give B-1a.

[0650] The following intermediates B-1 / B-1* (Table 9) can be obtained in a similar manner from different intermediates E-12 / E-12*. ​​If necessary, the crude product B-1 / B-1* can be purified by chromatography.

[0651] Table 9

[0652]

[0653] Option 5a:

[0654]

[0655] Experimental procedures for the synthesis of B-6a and B-7a

[0656]

[0657] Hydroxylamine hydrochloride (50% concentration in water, 3.131 g, 47 mmol, 2.5 equivalents) was added to a solution of B-1a (12.7 g, 19 mmol, 1.0 equivalent) in EtOH / water, and the reaction mixture was heated to 50ºC for 2 h. The reaction mixture was concentrated under reduced pressure, dissolved in MeOH (40 mL), and treated with concentrated HCl (40 mL). The reaction mixture was stirred at 60ºC for 1 h, concentrated under reduced pressure, dissolved in EtOAc, and neutralized by careful addition of a saturated sodium carbonate solution. The aqueous phase was extracted with EtOAc (three times), the combined organic phases were dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by RP chromatography (using an ACN / water gradient of 30%–80% under alkaline conditions). The products containing fractions were combined and lyophilized to give B-6a and the other isoxazole isomer B-7a.

[0658] The following intermediates B-6 / B-6* and B-7 / B-7* (Table 10) can be obtained in a similar manner from different intermediates B-1 / B-1*. If necessary, the crude product can be purified by chromatography.

[0659] Table 10

[0660]

[0661] Option 6a:

[0662]

[0663] Experimental procedure for synthesizing C-3a

[0664]

[0665] Under nitrogen atmosphere at room temperature, malononitrile (95% purity, 798.2 mg, 11 mmol, 5.0 equivalent), β-alanine (95% purity, 646 mg, 6.9 mmol, 3.0 equivalent), and activated molecular sieve (from Roth, 200 mg) were added to a solution of B-6a (1.2 g, 2.3 mmol, 1.0 equivalent) in EtOH (10 mL). The reaction mixture was heated to 80ºC for 3 h. After the condensation reaction was complete as monitored by HPLC-MS, sulfur (220.9 mg, 6.9 mmol, 3.0 equivalent) was added, and the reaction mixture was stirred at 80ºC for 15 min. The reaction mixture was cooled to room temperature, dissolved in water and EtOAc, and filtered. The layers were separated. 4 N NaOH solution (10 mL) was added to the aqueous phase, and the mixture was extracted 3 times with EtOAc. The combined organic phases were dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by chromatography using silica gel (gradient chromatography under alkaline conditions). The fractions were combined and freeze-dried to obtain C-3a.

[0666] The following intermediates C-3 / C-3* and C-4 / C-4* (Table 11) can be obtained in a similar manner from different intermediates B-6 / B-6* and B-7 / B-7*. If necessary, the crude product can be purified by chromatography.

[0667] Table 11

[0668]

[0669] Synthesis of compound (I) according to the present invention:

[0670] Option 7:

[0671]

[0672] Experimental procedure for the synthesis of compound Ia-1

[0673]

[0674] TEA (353 mg, 3.484 mmol, 2.6 equivalents) was added 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), and the reaction mixture was stirred at room temperature for 2 min. A solution of C-3a dissolved in DMF (350 mg, 581 µmol, 1.0 equivalents) was added to the reaction mixture, and the mixture was stirred at room temperature for 15 min. After the reaction was complete, the mixture was diluted with acetonitrile and water, filtered, and purified by basic reversed-phase chromatography (gradient elution: 30% to 98% acetonitrile in water) to give the desired compound Ia-1.

[0675] Experimental procedure for the synthesis of compound Ia-2

[0676]

[0677] A freshly prepared solution of acryloyl chloride in acetone (81.3 mg, 871 µmol, 1.5 equivalents) was added to a solution of sodium carbonate (154 mg, 1.45 mmol, 2.5 equivalents) in acetone / water (8:1) and C-3a (350 mg, 581 µmol, 1.0 equivalents). After the reaction was complete, the mixture was diluted with acetonitrile and water, filtered, and purified by alkaline reversed-phase chromatography (gradient elution: 30% to 98% acetonitrile in water) to give the desired compound Ia-2.

[0678] Table 12

[0679]

[0680]

[0681] Example 4 Synthesis of compounds according to formula (C)

[0682] List of abbreviations (Table 13)

[0683]

[0684]

[0685] Example

[0686] The features and advantages of the invention will become apparent from the following detailed embodiments, which illustrate the principles of the invention by way of example and do not limit its scope:

[0687] Preparation of compounds according to the present invention

[0688] Unless otherwise stated, all reactions were carried out in commercially available apparatus using methods typically employed in chemical laboratories. Starting materials sensitive to air and / or moisture were stored under a protective gas, and the corresponding reactions and operations were performed under a protective gas (nitrogen or argon).

[0689] If a compound can be represented by both its structural formula and its nomenclature, then in the event of a conflict, the structural formula shall prevail.

[0690] The microwave reaction is carried out in a starter / reactor manufactured by Biotage, or in an Explorer manufactured by CEM, or in a Synthos 3000 or Monowave 3000 manufactured by Anton Paar, in a sealed container (preferably 2, 5 or 20 mL), preferably under stirring.

[0691] Chromatography

[0692] Thin-layer chromatography was performed on off-the-shelf silica 60 TLC plates on glass (with fluorescent indicator F-254) manufactured by Merck.

[0693] Preparative high-performance liquid chromatography (RP HPLC) of the compounds according to embodiments of the present invention was performed on an Agilent or Gilson system using columns manufactured by Waters (names: SunFire™ Preparative C18, OBD™ 10 µm, 50 x 150 mm or SunFire™ Preparative C18 OBD™ 5 µm, 30 x 50 mm or XBridge™ Preparative C18, OBD™ 10 µm, 50 x 150 mm or XBridge™ Preparative C18, OBD™ 5 µm, 30 x 150 mm or XBridge™ Preparative C18, OBD™ 5 µm, 30 x 50 mm) and columns manufactured by YMC (names: Actus-Triart Prep C18, 5 µm, 30 x 50 mm).

[0694] Different H2O / ACN gradients were used to elute the compounds. For the Agilent system, 5% acid modifier (20 mL HCOOH to 1 L H2O / ACN (1 / 1)) was added to the water (acidic conditions). For the Gilson system, 0.1% HCOOH was added to the water.

[0695] For chromatography under alkaline conditions, the Agilent system also uses an H2O / ACN gradient, while making the water alkaline by adding 5% alkaline modifier (50 g NH4HCO3 + 50 mL NH3 (25% in H2O) to 1 L (with H2O)). For the Gilson system, the water is made alkaline as follows: 5 mL NH4HCO3 solution (158 g, in 1 L H2O) and 2 mL NH3 (28%, in H2O) are added to make 1 L.

[0696] Supercritical fluid chromatography (SFC) of the intermediates and compounds of the present invention was performed on a JASCO SFC system using the following columns: Chiralcel OJ (250 x 20 mm, 5 µm), Chiralpak AD (250 x 20 mm, 5 µm), Chiralpak AS (250 x 20 mm, 5 µm), Chiralpak IC (250 x 20 mm, 5 µm), Chiralpak IA (250 x 20 mm, 5 µm), Chiralcel OJ (250 x 20 mm, 5 µm), Chiralcel OD (250 x 20 mm, 5 µm), and Phenomenex Lux C2 (250 x 20 mm, 5 µm).

[0697] Use a column manufactured by Waters (name: XBridge) TM C18, 2.5 µm, 2.1 x 20 mm; or XBridge TM Analytical HPLC (reaction control) of intermediates and final compounds was performed using columns manufactured by YMC (name: Triart C18, 3.0 µm, 2.0 x 30 mm) and Phenomenex (name: Luna C18, 5.0 µm, 2.0 x 30 mm). In each case, the analytical apparatus was also equipped with a mass spectrometer detector.

[0698] HPLC-mass spectrometry / UV-spectroscopy

[0699] Retention time / MS-ESI for characterizing compounds according to embodiments of the present invention + It was generated using an HPLC-MS apparatus (high-performance liquid chromatography with a mass spectrometer detector). The retention time t of the compound eluting at the injection peak was given. Ret. =0.00.

[0700] Method A

[0701] HPLC Agilent 1100 system

[0702] MS1200 Series LC / MSD (API-ES+ / -3000V, Quadrupole, G6140)

[0703] MSD signal settings: Scan positive / negative ions 120-900 m / z

[0704] Detected signal 315 nm (bandwidth 170 nm, reference off)

[0705] Spectral range 230-400 nm

[0706] Peak width < 0.01 min

[0707] Column Waters, Xbridge C18, 2.5 µm, 2.1x20 mm column

[0708] Column temperature 60ºC

[0709] Solvent A: 20 mM aqueous solution of NH4HCO3 / NH3, pH 9

[0710] B: ACN HPLC grade

[0711] Flow rate 1.00 mL / min

[0712] Gradient 0.00-1.50 min 10% to 95% B

[0713] 1.50-2.00 min 95% B

[0714] 2.00-2.10 min 95% to 10% B

[0715] Method B

[0716] HPLC Agilent 1260 system

[0717] MS1200 series LC / MSD (MM-ES+APCI + / - 3000 V, Quad Pole, G6130)

[0718] UV detection: 254 nm (bandwidth 8, reference off)

[0719] UV: 230 nm (bandwidth 8, reference off)

[0720] UV spectral range: 190-400 nm; Step: 4 nm

[0721] MS: Positive Mode and Negative Mode

[0722] Mass range 100-800 m / z

[0723] Waters column; catalog number 186003389; XBridge BEH C18; 2.5 µm; 30 x 2.1 mm

[0724] Column temperature 45ºC

[0725] Solvent A: 5 mM NH4HCO3 / 19 mM NH3 in H2O; Solvent B: ACN (HPLC grade)

[0726] Flow rate 1.40 mL / min

[0727] Gradient 0.00-1.00 min: 5% B to 100% B

[0728] 1.00-1.37 min: 100% B

[0729] 1.37-1.40 min: 100% B to 5% B

[0730] Method C

[0731] HPLC Agilent 1260 series

[0732] MSAgilent LC / MSD quadrupole

[0733] MS detection: positive and negative modes

[0734] Mass range 100-750 m / z

[0735] Column Waters X-Bridge BEH C18, 2.5 µm, 2.1 x 30 mm XP

[0736] Column temperature 45ºC

[0737] Solvent A: 20 mM NH4HCO3 / 30 mM NH3 in H2O; Solvent B: ACN (HPLC grade)

[0738] Flow rate 1.40 mL / min

[0739] Gradient 0.00-1.00 min: 15% B to 95% B

[0740] 1.00-1.30 min: 95% B

[0741] Method E

[0742] HPLC Agilent 1100 / 1200 system

[0743] MS1200 Series LC / MSD (MM-ES + APCI + / - 3000 V, Quadrupole, G6130B)

[0744] MSD signal setting: scan positive / negative ions 150-750

[0745] Detection signals: UV 254 nm, 230 nm, 214 nm (bandwidth 8, reference off)

[0746] Spectral range: 190-400 nm; slit width: 4 nm

[0747] Peak width > 0.0031 min (0.063 s response time, 80 Hz)

[0748] Waters column, catalog number 186003389, XBridge BEH C18, 2.5 µm, 2.1 x 30 mm)

[0749] Column temperature 45ºC

[0750] Solvent A: 5 mM NH4HCO3 / 18 mM NH3 in H2O (pH = 9.2)

[0751] B: ACN (HPLC grade)

[0752] Flow rate 1.4 mL / min

[0753] Gradient 0.0-1.0 min 15% to 95% B

[0754] 1.0-1.1 min95% B

[0755] Stop time: 1.3 min

[0756] Method SFC-1

[0757] Preparation of Waters UPC 2 -MS

[0758] Empower3 software

[0759] MSQDa

[0760] Column Chiralcell OX-3 (4.6*150 MM) 3 µm

[0761] A- Solvent CO2

[0762] B-solvent ACN

[0763] Total flow rate 3 g / min

[0764] 15% of cosolvent

[0765] ABPR 1500psi

[0766] Column temperature 30ºC

[0767] PDA range 200 nm to 400 nm

[0768] 1.2 nm resolution

[0769] MS parameters -

[0770] QDa MS scan range: 100 Da to 1000 Da

[0771] Conical hole voltage

[0772] Positive ion scan 20 V

[0773] Negative ion scanning 15 V

[0774] Negative ion scanning 15 V

[0775] The compounds and intermediates according to the invention are prepared by the synthetic methods described below, wherein the substituents of the general formula have the meanings given above. These methods are intended to illustrate the invention and not to limit its subject matter or the scope of the compounds claimed for these embodiments. Where the preparation of starting compounds is not described, they are commercially available or their synthesis is described in the prior art, or they can be prepared similarly to the known prior art compounds or methods described herein, i.e., the synthesis of these compounds is within the skill of an organic chemist. The substances described in the literature can be prepared according to the published synthetic methods. If the chemical structures depicted below do not have a precise configuration of the stereocenter, such as an asymmetrically substituted carbon atom, then both configurations should be considered to be included and disclosed in such illustrations. Stereocenter illustrations in racemic form should always be considered to include and disclose two enantiomers (if no other defined stereocenter exists) or all other potential diastereomers and enantiomers (if an additionally defined or undefined stereocenter exists).

[0776] Synthesis of spirone intermediate A

[0777] Experimental procedure for the synthesis of A-2a

[0778]

[0779] Acetyl chloride (111 mL, 1.56 mol, 8.00 equivalent) was added dropwise to a suspension of 5-chloropentanones (22.9 g, 195 mmol, 1.00 equivalent) in EtOH (136 mL) at 0ºC. The reaction mixture was allowed to be warmed to room temperature and stirred for 12 h. The mixture was concentrated under reduced pressure and washed with Et2O, and the crude product A-2a was used directly as an HCl salt for the next step without further purification (HPLC method: A; t ret = 1.03 min; [M+H] + = 164).

[0780] Experimental procedure for the synthesis of A-3a

[0781]

[0782] Crude A-2a (HCl salt) (28.0 g, 140 mmol, 1.00 equivalent) and ethylene glycol (7.38 g, 119 mmol, 0.90 equivalent) were dissolved in DCM (300 mL) and stirred at room temperature for 6 days. The resulting suspension was concentrated under reduced pressure, diluted with Et2O (200 mL), and filtered. The filtrate was concentrated under reduced pressure, absorbed in DCM (200 mL), and treated with KOH solution (2 M in water, 150 mL). The mixture was stirred overnight at room temperature to maintain phase integrity. The phases were separated, the aqueous phase was extracted with DCM (2 x), and the combined organic phases were dried over magnesium sulfate, filtered, and concentrated under reduced pressure. Crude orthoester A-3a was used in the next step without further purification (HPLC method: A;t). ret = 1.37 min; [M+H] + = 163).

[0783] Experimental procedure for the synthesis of A-4a

[0784]

[0785] Crude 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) were dissolved in DCM (120 mL) and stirred at room temperature for 5 h. The reaction mixture was treated with the addition of saturated sodium bicarbonate solution. The organic phase was separated, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by NP chromatography to give the desired compound A-4a (HPLC method: A;t). ret = 1.25 min; [M+H]+ = 283).

[0786] Experimental procedures for the synthesis of A-5a

[0787]

[0788] A-4a (14.9 g, 57.1 mmol, 1.00 equivalent) and sodium iodide (25.9 g, 171 mmol, 3.00 equivalent) were dissolved in acetone (120 mL) and stirred under reflux for 16 h. The reaction mixture was concentrated under reduced pressure, diluted with DCM, and washed with a saturated sodium thiosulfate solution. The organic phase was separated, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The crude product A-5a was used in the next step without further purification.

[0789] Experimental procedures for the synthesis of A-6b

[0790]

[0791] A-5a (30.0 g, 85.0 mmol, 1.00 equivalent) was dissolved in THF. The mixture was treated with potassium tert-butoxide (28.7 g, 256 mmol, 3.0 equivalent) at 0ºC and stirred overnight at room temperature. The reaction mixture was quenched by adding water (2 mL) and diluted by adding Et2O and saturated sodium bicarbonate solution. The organic phase was separated, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by NP chromatography to give (racemic) compound A-6a (HPLC method: A;t ret = 1.17 min; [M+H] + = 225).

[0792] 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.

[0793] The enantiomer A-6b can then be obtained by chiral separation via SFC (using a Lux cellulose-4 column (250x30 mm, 5 µm), column temperature 30ºC, 90% CO2, 10% ACN as a cosolvent). The enantiomer A-6b (HPLC method: A; t) is further obtained after this chiral separation. ret = 1.17 min; [M+H] + = 225 / SFC method: SFC-1; t ret= 2.99 min) was eluted as the second peak after another enantiomer.

[0794] Alternative procedures for synthesizing A-6b

[0795] Step 1

[0796]

[0797] Toluene (234 L) was charged into a dry and clean reactor under nitrogen atmosphere (Note: a total of 2.5 V of toluene is used for this reaction). Water (1.56 kg, 85.5 mol, maintaining H₂O : Pd = 160 : 1) was added, followed by 1,1,3,3-tetramethylguanidine (175.5 kg, 1527.6 mol, 2.0 equivalent) under nitrogen atmosphere and flushing the feed line with toluene (13 L). A-7a (130.0 kg, 763.8 mol) was added under nitrogen atmosphere and flushed with toluene (13 L). Allyl acetate (98.8 kg, 992.9 mol, 1.3 equivalent) was added under nitrogen atmosphere and flushed with toluene (13 L). The mixture was cooled to 10ºC over 0.5 h with stirring. The batch was degassed by bubbling the solution under nitrogen atmosphere for approximately 30 min. (S,S)-DACH-Ph Trost ligand (0.429 kg, 0.619 mol, 0.081 mol%) was added to degassed toluene (13 L) (Note: maintain Pd: ligand = 1: 1.15), followed by washing with degassed toluene (13 L). Allyl palladium(II) chloride dimer (97.5 g, 267 mol, 0.035 mol%) was added to degassed toluene (13 L), followed by washing with degassed toluene (13 L). The batch was kept at 10ºC–15ºC for at least 8 h. After the reaction was confirmed to be complete by HPLC, a solution of N-acetyl-L-cysteine ​​(3.9 kg, 22.9 mol, 0.03 equivalents) was added to water (260 L) at below 25ºC. The resulting solution was heated to 20ºC–25ºC and maintained at 20ºC–25ºC for at least 1 h. After phase separation, the bottom aqueous layer was discarded, and 10 wt% NH4Cl aqueous solution (260 L) was added. The mixture was stirred for 10 min, and the bottom aqueous layer was drained. The organic phase was further washed with water (130 L). The organic layer was filtered through a very short diatomaceous earth mat, and the reactor and diatomaceous earth bed were rinsed with toluene (65 L). The filtrate was packed into a clean reactor, and the toluene was then distilled off under vacuum at 40ºC–50ºC. The crude product was used directly for the next step or discharged into a container with the aid of a minimal amount of toluene (65 L) and stored at 20ºC–23ºC. Typically, 150 kg of A-8a, a pale yellow oil, was obtained in 96% yield and with an enantiomeric ratio of ≥ 90:10.

[0798] 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] + 。

[0799] Step 2

[0800]

[0801] Ethylene glycol (600 L) was added to a reactor containing A-8a (150 kg, 713.4 mol) from step 1 (less than 1 V if toluene was used) to obtain a yellow biphase mixture. After cooling the mixture to 10°C–15°C, TMSCl (193.5 kg, 1783.5 mol, 2.5 equivalents) was added at a rate maintaining an internal temperature between 20°C and 30°C for at least 15 min (to obtain an orange biphase mixture). Thorough agitation was required to achieve mixing. After maintaining the batch at 20°C–25°C for 2 h, agitation was stopped and the batch was maintained at 20°C–25°C for at least 15 min. The batch was cooled to 0–5°C. NaOH (96 kg, 1854.8 mol, 2.6 equivalents) in water (600 L) was added at a rate maintaining an internal temperature below 20°C (to obtain a pale yellow, turbid biphase mixture). Add toluene (300 L) and then stir the batch for 10 min. After phase separation, drain the bottom aqueous layer (note: some precipitate may form in the intermediate phase), and wash the organic layer twice with water (300 L). Filter the organic phase through a short diatomaceous earth mat to remove insoluble solids / intermediate phase. Load the organic solution into a clean and dry reactor and then distill off the solvent at 40ºC–50ºC to minimize the stirable volume. With the aid of a minimum amount of toluene, discharge the crude product A-9a (189 kg, 95.2 wt%, 100% yield) into a vessel.

[0802] 1H NMR (500 MHz, CDCl3): δ 5.65 (ddt, J = 14.7, 8.1, 6.6 Hz, 1H), 5.07-4.98 (m, 2H), 4.20-4.10 (m, 2H), 3.97-3.88 (m, 4H), 2.81 (dd, J = 13.9,6.6 Hz, 1H), 2.35 (dd, J = 13.9, 8.1 Hz, 1H), 2.04-1.98 (m, 1H), 1.75-1.35(m, 7H), 1.26 (t, J = 7.1 Hz, 3H). 13C NMR (125 MHz, CDCl3): δ 173.7, 134.3,117.6, 110.9, 65.0, 64.7, 60.5, 54.6, 36.2, 32.3, 30.3, 23.3, 20.9, 14.4. ESI-MS: m / z 255 [M+H] + .

[0803] Step 3

[0804]

[0805] 9-BBN (688.5 kg, 401 mol, 1.2 equivalents) was added to a dry and clean reactor under nitrogen atmosphere. The solution was cooled to 0–5°C to obtain a slurry. A-9a (85.0 kg, 334.2 mol) from step 2 was added at 0–5°C and washed with THF (40 L). The mixture was warmed to 20–23°C over 1 h and maintained at 20–23°C for at least 1 h. After cooling the mixture to -45°C to -40°C, methyl chloroacetate (69.6 kg, 1.3 equivalents) was added in a single batch, followed by dropwise addition of LiHMDS (909.5 kg, 1102.9 mol) while maintaining the temperature below -35°C. The batch was then warmed to 20–23°C over 1 h and maintained at 20–23°C for at least 18 h. The solvent, approximately 12–13 V, was removed by distillation under vacuum (35°C) with simultaneous heating. EtOH (255 kg) was added, followed by a solution of NaOH (13.4 kg) in H₂O (212.5 L). The mixture was heated under reflux (at 66–70°C) for at least 14 h.

[0806] The batch was then cooled to 20°C–25°C by removing approximately 5–6 V of solvent through distillation under reflux, and then filtered through a short diatomaceous earth mat to remove insoluble material, followed by washing with heptane (160 L). The solvent (or most of the residual THF and ethanol) was distilled off under vacuum at 40°C–50°C. The batch was cooled to 20°C–25°C. Water (255 L) was then added, and the crude product was extracted twice with heptane (2364.8 kg). The combined heptane layers were washed once with water (85 L). A crude product (52.7 kg, 87.5 wt%) in a yellow oily state was obtained in an analytical yield of 52.6% after solvent removal by distillation under vacuum at 40°C–50°C. Crude product A-6b was used directly in the next step.

[0807] 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] + .

[0808] Synthesis of intermediate B of alcohol, pyrazole and toluenesulfonate

[0809] Experimental procedures for the synthesis of B-2a

[0810]

[0811] 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) were dissolved in DCM (29.5 mL) and stirred at room temperature for 40 min. After complete activation, N,O-dimethylhydroxylamine hydrochloride (2.79 g, 28.6 mmol, 1.50 equivalent) was added, and the reaction was stirred at room temperature for another 2 h. After complete conversion, water (100 mL) and DCM (150 mL) were added, and the phases were separated. The aqueous phase was extracted with DCM (2 x). The combined organic phases were washed with brine and concentrated under reduced pressure. The residue was purified by NP chromatography to give product B-2a.

[0812] The following intermediate B-2 (Table 14) can be obtained in a similar manner. If necessary, the crude product can be purified by chromatography.

[0813] Table 14

[0814]

[0815] Experimental procedures for the synthesis of B-3a

[0816]

[0817] B-2a (4.88 g, 16.9 mmol, 1.00 equivalent) was dissolved in THF (15 mL) under an argon atmosphere and cooled to -10ºC. Magnesium bromo(methyl)magnesium (3.4 M in MeTHF, 6.46 mL, 22.0 mmol, 1.3 equivalent) was added and the mixture was stirred at -10ºC for 1 h. After complete conversion, the reaction mixture was cooled to -20ºC and quenched by adding brine. The resulting mixture was extracted with DCM (3 x). The combined organic phases were concentrated under reduced pressure to give B-3a.

[0818] The following intermediate B-3 (Table 15) can be obtained in a similar manner. If necessary, the crude product can be purified by chromatography.

[0819] Table 15

[0820]

[0821] Experimental procedures for the synthesis of B-4a

[0822]

[0823] (R)-methyloxazolylborane (0.99 g, 3.3 mmol, 0.20 equivalent) was dissolved in THF (2 mL) under an argon atmosphere and cooled to -5ºC. Borane-dimethyl sulfide complex (1.0 M, 22 mL, 22.0 mmol, 1.3 equivalent) was added. The mixture was stirred at room temperature for 30 min. The mixture was cooled to -5ºC, and B-3a (4.1 g, 17 mmol, 1 equivalent) was slowly added dropwise. The reaction was stirred at room temperature for 1 h. After complete conversion of the starting material, the reaction was cooled to -10ºC and quenched by the addition of MeOH. The mixture was concentrated under reduced pressure. The residue was dissolved in water (150 mL) and formic acid (0.5 mL) and extracted with DCM (3 x). The combined organic phases were concentrated under reduced pressure and purified by NP chromatography to give product B-4a.

[0824] The following intermediate B-4 (Table 16) can be obtained in a similar manner. If necessary, the crude product can be purified by chromatography.

[0825] Table 16

[0826]

[0827] Experimental procedures for the synthesis of B-5a

[0828]

[0829] B-4a (306 mg, 12.5 mmol, 1.00 equivalent) was dissolved in THF (30.6 mL) under an argon atmosphere. Lithium aluminum hydride (1 M in THF, 24.9 mL, 25.0 mmol, 2.00 equivalent) was added slowly. The reaction was stirred at 60ºC for 1 h. After complete conversion, the reaction was cooled to room temperature, Rochelle salt solution and KOH were added, and the mixture was stirred for 1 h. The existing suspension was extracted with DCM (3 x), and the combined organic phases were concentrated under reduced pressure to give B-5a.

[0830] The following intermediate B-5 (Table 17) can be obtained in a similar manner. If necessary, the crude product can be purified by chromatography.

[0831] Table 17

[0832]

[0833] Synthesis of intermediate B of alcohol, pyrazole and toluenesulfonate

[0834] Experimental procedures for the synthesis of B-11a

[0835]

[0836] 1H-pyrazole-3-carboxylic acid (500 mg, 4.46 mmol, 1.00 equivalent) was dissolved in ACN (4.5 mL). Pyrrolidine (745 µL, 8.92 mmol, 2.00 equivalent), DIPEA (1.50 mL, 8.92 mmol, 2.00 equivalent), and 1-propylphosphoric anhydride (2.00 mL, 6.69 mmol, 1.50 equivalent) were added, and the reaction mixture was stirred at room temperature for 1 h until complete conversion. The reaction mixture was diluted with saturated NaHCO3 and extracted with DCM. The organic phase was dried, filtered, and the solvent was removed under vacuum. The crude product was purified by NP chromatography to give B-11a (HPLC method: C, t). ret = 0.14 min; [M+H] + =166).

[0837] Experimental procedures for the synthesis of B-15a

[0838]

[0839] 3-ethynyloxetane-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 equivalent) were combined and stirred in a sealed vial at 50ºC for 3 h until complete conversion. The reaction mixture was cooled to room temperature, diluted with MeOH, and the solvent was removed under vacuum to give crude B-15a (HPLC method: C, t). ret = 0.08 min; [M+H] + = 141). The crude product is used in the next step without purification.

[0840] Synthesis of esters and acid E

[0841] Experimental procedure for synthesizing intermediate E-9a:

[0842]

[0843] 4,6-Dichloropyrimidin-2-carboxylic acid E-8a (900 mg, 4.66 mmol, 1.00 equivalent) was dissolved in DMSO (2 mL) and DIPEA (1.5 mL, 8.8 mmol, 2.0 equivalent), and (S)-tert-butyl-3-methyl-1,4-diazacycloheptane-1-carboxylate (1.04 g, 4.896 mmol, 95% purity, 1.05 equivalent) was added dropwise. The reaction mixture was then stirred at 40ºC for 18 h. The mixture was diluted with ACN and purified by RP chromatography to give the desired product E-9a (HPLC method: A, t ret = 0.82 min; [M+H] + = 371).

[0844] Experimental procedure for synthesizing intermediate E-11a:

[0845]

[0846] E-10a (3.00 g, 14.5 mmol, 1.00 equivalent) was dissolved in DCM (30 mL) and DIPEA (5.34 mL, 29 mmol, 2.0 equivalent), and B-5b (3.20 g, 21.8 mmol, 1.5 equivalent) was added. The reaction mixture was then stirred at room temperature for 18 h. After complete conversion, the mixture was concentrated, water was added, and the mixture was extracted with EtOAc. The organic phase was washed with brine, dried, filtered, and concentrated. The crude product was purified by NP chromatography to give E-11a.

[0847] The following intermediate E-11 (Table 18) can be obtained in a similar manner. If necessary, the crude product E-11 can be purified by chromatography.

[0848] Table 18

[0849]

[0850] Experimental procedure for synthesizing E-11e:

[0851]

[0852] B-5a (100 mg, 0.48 mmol, 1.00 equivalent) was dissolved in THF (500 µL), and LiHMDS (591 µL, 0.59 mmol, 1.10 equivalent) was added and stirred for 5 min. Simultaneously, methyl 4,6-dichloropyrimidine-2-carboxylate (170 mg, 0.81 mmol, 1.5 equivalent) was dissolved in THF (500 µL). The B-5a solution was added dropwise to the methyl 4,6-dichloropyrimidine-2-carboxylate solution over 5 min. The reaction was stirred for 25 min. After complete conversion of the starting material was observed, the reaction was filtered and purified by RP chromatography to obtain E-11e (HPLC method: A, t). ret = 1.08 min; [M+H] + = 330).

[0853] Synthesis of diketone F

[0854] When multiple HPLC retention times are reported, it means that different tautomers exist.

[0855] Experimental procedures for synthesizing F-4a

[0856]

[0857] A-6b (1.4 g, 5.31 mmol, 1.1 equivalents) and magnesium bromide in diethyl ether (2.5 g, 9.66 mmol, 2.0 equivalents) were dissolved in DCM (10.0 mL). F-3a (1.0 g, 4.83 mmol, 1.0 equivalents) dissolved in DCM (10 mL) was added dropwise. DIPEA (2.1 mL, 12.08 mmol, 2.5 equivalents) was added, and the reaction mixture was stirred at room temperature for 7 h. The reaction was quenched with 1 M HCl and diluted with DCM and water. The organic phase was separated, evaporated, and the resulting residue was purified by RP chromatography to give F-4a (HPLC method: C, t). ret = 0.633 min; [M+H] += 399).

[0858] Experimental procedures for synthesizing the F-5a

[0859]

[0860] Methyl 4,6-dichloropyrimidine-2-carboxylate (2.00 g, 9.67 mmol, 1.00 equivalent) was dissolved in dry ACN (5 mL) under a nitrogen atmosphere. A solution of magnesium diethyl ether bromide (2.99 g, 11.6 mmol, 1.20 equivalent), 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) were added, and the reaction mixture was stirred at 50ºC for 20 h. After complete conversion, the reaction mixture was carefully quenched with HCl (1 M), diluted with water, extracted with DCM, and the organic phase was dried, filtered, and concentrated to give crude F-5a. The crude compound was purified by normal-phase chromatography (HPLC method: H, t ret = 2.50min; [M+H] = 399 / 401).

[0861] Experimental procedures for synthesizing the F-8a

[0862]

[0863] F-4a (1.27 g, 2.77 mmol, 1.0 equivalent) was dissolved in dioxane (10 mL), and cesium carbonate aqueous solution (2 M, 3.46 mL, 6.93 mmol, 2.5 equivalent) was added and stirred at 80ºC for 15 min. Then, pyridine-4-boronic acid (357 mg, 2.91 mmol, 1.1 equivalent) and Pd(dppf)Cl2CH2Cl2 (238 mg, 0.28 mmol, 0.1 equivalent) were added to the reaction mixture and stirred at 90ºC for 30 min until complete conversion of the starting material was observed. The reaction mixture was filtered, diluted with water, and extracted three times with DCM. The organic phase was evaporated, and the residue was dissolved in DMF and purified by RP chromatography to give the desired product F-8a (HPLC method: C, t). ret = 0.80 / 86 min; [M+H] = 440).

[0864] Experimental procedures for synthesizing the F-9a

[0865]

[0866] F-5a (10.0 g, 19.4 mmol, 1.00 equivalent) was dissolved in DMSO (10 mL), and (1S)-1-[(2S)-1-methylpyrrolidone-2-yl]ethanol (2.76 g, 21.4 mmol, 1.10 equivalent) and DIPEA (6.78 mL, 38.8 mmol, 2.0 equivalent) were added. The solution was stirred overnight at room temperature. The reaction mixture was diluted with DCM and water. The organic phase was separated, evaporated, and the resulting residue was purified by RP chromatography to give F-9a. (HPLC method: A, t) ret = 1.58 / 1.66min; [M+H] = 492).

[0867] Experimental procedures for synthesizing the F-11a

[0868]

[0869] E-9a (1.05 g, 2.83 mmol, 1.00 equivalent) and 1-(1H-imidazol-1-carbonyl)-1H-imidazolium (918 mg, 5.66 mmol, 2.00 equivalent) were dissolved in THF (5 mL) under an argon atmosphere and stirred at room temperature for 1 h. After complete acid activation, 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) was added to the reaction mixture and washed with THF (5 mL). The resulting mixture was stirred overnight at 60ºC. After complete conversion, the reaction mixture was diluted with a saturated aqueous solution of NaHCO3 and extracted three times with DCM. The organic phases were combined, dried, filtered, and concentrated under reduced pressure. The crude product was dissolved in ACN and water, filtered, and purified by alkaline RP chromatography to obtain the desired product F-11a (HPLC method: C, t). ret = 0.888 / 0.936 / 0.978 min; [M+H] = 557).

[0870] Experimental procedures for synthesizing the F-12a

[0871]

[0872] E-11e (1.80 g, 0.01 mol, 1 equivalent) was dissolved in THF (18 mL), and activated molecular sieve 3 Å (200 mg / 1 mL solvent) was added and stirred at 50ºC under an argon atmosphere for 20 min. Then, diethyl ether magnesium bromide (2.11 g, 0.01 mol, 1.5 equivalent) was added and further stirred at 50ºC for 30 min. Simultaneously, a second solution was prepared using A-6b (1.47 g, 0.01 mol, 1.5 equivalent), which was also dried in THF (8 mL) at 50ºC for 20 min using activated molecular sieve 3 Å. Then, LiHMDS (1 M in THF, 13.7 mL, 0.01 mol, 2.5 equivalent) was added and stirred for 15 min. The second solution was then added to the first solution and stirred at 50ºC for 1 h. After complete conversion, the reaction mixture was carefully quenched with water, and THF was removed under reduced pressure. The pH of the residue was adjusted to 7-8 using 1 N HCl and extracted with 5% MeOH in DCM (2x). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated to give crude F-12a. The crude compound was purified by NP chromatography.

[0873] The following intermediate F-12 (Table 19) can be obtained in a similar manner. If necessary, the crude product can be purified by chromatography.

[0874] Table 19

[0875]

[0876] Synthesis of isoxazole intermediate G

[0877] Experimental procedures for the synthesis of G-9a and G-10a

[0878]

[0879] F-11a (1.10 g, 1.91 mmol, 1.0 equivalent) was dissolved in 1,4-dioxane (3 mL) and hydroxylamine (50% in water, 140 µL, 2.29 mmol, 1.2 equivalent) was added. The reaction mixture was stirred overnight at room temperature. After complete conversion, the reaction mixture was diluted with saturated aqueous NaHCO3 solution and extracted three times with DCM. The organic phases were combined, dried, filtered, and concentrated under reduced pressure to give the crude product.

[0880] A crude mixture of G-9a and G-10a (1.0 g, 1.68 mmol, 1.0 equivalent) was dissolved in 1,4-dioxane (6 mL), and HCl (4 M in water, 2.11 mL, 8.44 mmol, 5.0 equivalent) was added. The reaction mixture was stirred at room temperature for 3 h. After complete conversion, the reaction mixture was diluted with saturated NaHCO3 aqueous solution and extracted three times with DCM. The organic phases were combined, dried, filtered, and concentrated under reduced pressure to give the crude product. The crude product was dissolved in ACN and water, filtered, and purified by alkaline RP chromatography to give the desired products G-11a and G-12a.

[0881] The intermediates G-11 and G-12 (Table 20) can be obtained in a similar manner. If necessary, the crude product can be purified by chromatography.

[0882] Table 20

[0883]

[0884] Experimental procedures for the synthesis of G-35a

[0885]

[0886] G-12b (140 mg, 0.31 mmol, 1.0 equivalent) was dissolved in dioxane (2 mL). [1-(oxetane-3-yl)-1Hpyrazol-3-yl]boronic acid (B-9c) (66.3 mg, 0.38 mmol, 1.19 equivalent), XPHOS PD G3 (29.9 mg, 0.03 mmol, 0.1 equivalent), and cesium carbonate (400 µl, 0.80 mmol, 2.54 equivalent) were added. The reaction was stirred at 80ºC under an argon atmosphere for 10 min. After complete conversion was observed, the reaction was extracted with DCM / water. The combined organic phases were concentrated under reduced pressure, dissolved in ACN / water, and purified by RP chromatography to give the desired product G-35a.

[0887] Experimental procedures for the synthesis of A-10a

[0888]

[0889] 9-BBN (387 mL, 193.5 mmol, 1.2 equivalents, 0.5 M in THF) was charged into a dry and clean reactor under nitrogen atmosphere. The solution was cooled to 0–5°C to obtain a slurry. A-9a (41.0 g, 161.2 mmol) was added at 0–5°C and washed with THF (20.5 mL). The mixture was warmed to 20–23°C over 1 h and maintained at 20–23°C for at least 1 h. After cooling the mixture to -45°C to -40°C, methyl chloroacetate was added in a single batch, followed by dropwise addition of LiHMDS (355 mL, 532.0 mmol, 3.3 equivalents) while maintaining the temperature below -35°C. The batch was then warmed to 20–23°C over 1 h and maintained at 20–23°C for at least 18 h. The batch was cooled to 5°C–10°C, and AcOH (30.4 mL, 3.3 equivalents) was added below 20°C, followed by water (41 mL) below 20°C. AcOH (30.4 mL, 3.3 equivalents) was added below 20°C to bring the pH to approximately 6–7. Approximately 15–16 V of THF was removed under vacuum at below 35°C. MTBE (246 mL) and water (205 mL) were added. After phase separation, the bottom aqueous layer was discarded, and the mixture was cooled to 0–5°C. A solution of sodium percarbonate (37.2 g, 322.4 mmol, 2.0 equivalents) in water (320 mL) was added below 20°C. After 1 h at 20°C–23°C, a 20 wt% sodium sulfite solution (31 mL) was added. After 15 min at 20°C–23°C, the bottom aqueous layer was separated and discarded. The organic layer was washed with 5 wt% ammonium chloride solution (123 mL) and water (328 mL). The organic layer was treated with 5% activated carbon for 30 min and then filtered. After removing the solvent by approximately 4-5 V under vacuum at below 35ºC, crude product A-10a (80% yield, HPLC method: C, t) was obtained as an orange-brown oil. ret = 0.84 min; [M+H] + = 283).

[0890] Experimental procedures for the synthesis of G-70a

[0891]

[0892] A reactor was charged with A-10a (45.5 g, 161.2 mmol), ethanol (91.0 mL), NaOAc (39.7 g, 483.6 mmol, 3.0 equivalent), water (45.5 mL), and NH₂OH·HCl (33.6 g, 483.6 mmol, 3.0 equivalent). The mixture was heated at 73ºC–78ºC for at least 16 h. After cooling the batch to 20ºC–23ºC, water (227.6 mL) was added over 0.5 h. Then, MTBE (136.5 mL) was added over 0.5 h, followed by heptane (113.8 mL) over 1 h. After 0.5 h at 20ºC–23ºC, the solids were collected by filtration. The solids were washed successively with MTBE (45 mL) and water (91.0 mL). The solid was dried under vacuum to obtain product G-70a (18.27 g, 93.2 wt%), a grayish-white solid in a yield of 40%.

[0893] 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] + .

[0894] Experimental procedures for the synthesis of G-71a

[0895]

[0896] G-70a (100.0 g, 376.9 mmol, 1.0 equivalent) and K3PO4 (240.0 g, 1130.8 mmol, 3.0 equivalent) in water (499.0 g, 500.0 mL) and toluene (432.5 g, 500.0 mL) were charged into a clean reactor. The two-phase mixture was stirred to ensure thorough mixing. After cooling the mixture to 0–5°C, Tf2O (186.0 g, 110.9 mL, 659.6 mmol, 1.750 equivalent) was added via syringe pump after 2 h at below 5°C. After phase separation, the organic layer was filtered through a diatomaceous earth bed containing Na2SO4. After washing with toluene (50 mL), the crude product G-71a (149.8 g, 100% yield) was used directly for the next step.

[0897] 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] + .

[0898] Experimental procedures for the synthesis of G-72a

[0899]

[0900] A dry and clean autoclaved reactor was charged with G-71a (750 g, 1.89 mol, 1 equivalent), Pd(OAc)2 (8.48 g, 37.7 mmol, 0.02 equivalent), racemic-BINAP (23.5 g, 37.7 mmol, 0.02 equivalent), 2-MeTHF (3 L), EtOH (870 g, 18.9 mol, 10 equivalent), and DIPEA (293 g, 2.26 mol, 1.2 equivalent). The reactor was purged twice with nitrogen (100 psi) and twice with CO (100 psi). The reactor was pressurized to 200 psi CO and heated at 55ºC–60ºC for at least 12 h. The mixture was transferred to the reactor, and the autoclaved reactor was flushed with 2-MeTHF (0.75 L) to flush the mixture into the reactor. The mixture was washed with water (3.75 L). After filtration through a short diatomaceous earth pad, the solvent was removed by vacuum distillation to give crude product G-72a (531.9 g, 87.7% yield), which was used in the next step without purification.

[0901] 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 = 17.5,9.6, 6.0 Hz, 1H), 2.22-2.14 (m, 1H), 2.04-1.88 (m, 3H), 1.78-1.45 (m, 8H), 1.37 (t, J = 7.1 Hz, 3H). ESI-MS: m / z 322 [M+H] + .

[0902] Experimental procedures for the synthesis of G-73a

[0903]

[0904] G-72a (482.0 g, 1.5 mol, 1 equivalent) and EtOH (3 V) were charged into a dry and clean reactor, and vacuum distilled for about 3 V to remove residual 2-MeTHF from the previous carbonylation step. EtOH (1.45 L) and NH4OH (1.93 L) were added. The mixture was kept at 20ºC–25ºC for at least 15 h. Water (1.69 L) was added after 30 min. After 30 min at 20ºC–25ºC, the solid was collected and washed with a 1:2 EtOH / water mixture (0.96 L) and water (0.48 L). The solid was slurried in a 1:1 MTBE / hexane mixture (0.96 L) for 1 h. The solid was collected by filtration and dried overnight under vacuum at 40ºC–45ºC to give product G-73a (332.4 g, 75.8% yield, based on Karl Fischer titration, water content ≤ 0.5%) as a brown solid.

[0905] 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] + .

[0906] Experimental procedures for the synthesis of G-74a

[0907]

[0908] G-73a (383 g, 86.7 wt%, 1.137 mol, 1 equivalent), MeCN (1.15 L), and pyridine (216 g, 0.19 L, 2.4 equivalent) were charged into a dry and clean reactor. The mixture was cooled to 0–5°C, and trifluoroacetic anhydride (287 g, 1.36 mol, 1.2 equivalent) was added below 5°C. After 5 min at 0–5°C, water (1.54 L) was added below 15°C. The product was extracted with MTBE (1.92 L) and washed with 5% sodium bicarbonate solution (1.15 L). The organic layer was filtered through a silica gel pad (380 g) and washed with MTBE (0.58 L). After removing the solvent by distillation under vacuum, product G-74a (421.8 g, 97.8% yield) was obtained as an orange-brown oil.

[0909] 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] + .

[0910] Experimental procedures for the synthesis of G-76a

[0911]

[0912] Crude G-74a (265 g, 72.3 wt%, 698.4 mmol) was packed in MeOH (1590 mL) and catalytic NaOMe (8.0 mL, 25% in MeOH, 34.9 mmol) in a dry flask. The mixture was stirred at room temperature for 1 h to achieve >99% conversion. After adding solid NH4Cl (52.0 g, 977.8 mmol, 1.4 equivalents), the resulting mixture was stirred at room temperature to achieve >95% conversion (NH4Cl was added if not achieved). After adding dimethyl malonate (168 g, 1047.7 mmol, 1.5 equivalents) at room temperature, NaOMe (377 g, 25% in MeOH, 2.5 equivalents) was added. The resulting mixture was heated to reflux for 4 h to achieve >95% conversion. After cooling the mixture to 23ºC, water (795 mL) was added, followed by slow addition of 6 N HCl (349 mL) at below 20ºC to achieve a pH of approximately 3. MTBE (530 mL) was added to the slurry. After 1 h at room temperature, the solids were collected by filtration and washed with 3 N water (796 mL) and MTBE (530 mL) to obtain product G-76a (178 g) as a grayish-white solid with a crude yield of 71%. The crude product was used directly in the next step.

[0913] 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] + .

[0914] Experimental procedures for the synthesis of G-77a

[0915]

[0916] A dry flask was packed with G-76a (80.0 g, 253.7 mmol), DMAP (4.0 g), tetramethylammonium chloride (4.0 g), and POCl3 (400 mL). The mixture was heated at 80ºC for 1.5 h to achieve >99% conversion. POCl3 was removed under vacuum to give a viscous, pale yellow slurry. MTBE (160 mL) was added. The mixture was then cooled to 5ºC. Water (800 mL) was added slowly. The resulting white slurry was stirred at 23ºC for 1 h. The solids were collected by filtration and then washed successively with water (480 mL) and MTBE (160 mL). After drying under vacuum at 60ºC overnight, 84.3 g of product G-77a as a white solid was separated with >99% purity and approximately 93% yield.

[0917] 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): δ209.8, 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] + .

[0918] Experimental procedures for the synthesis of G-78a

[0919]

[0920] LiHMDS (1 M in THF) (406.4 kg, 456.1 mol, 1.1 equivalents) was loaded into a dry and clean reactor. The solution was cooled to 0–5°C, and crude A-6b (93.0 kg, 414.6 mol) was added below 5°C and washed with THF (46.5 kg) to aid transfer. After 30 min at 0–5°C, diethyl oxalate (72.5 kg, 497.5 mol, 1.2 equivalents) was added below 5°C. The mixture was warmed to 20–25°C over 1 h, and then maintained at 20–25°C for at least 3 h. After cooling the batch to 10ºC–15ºC, a cooled HCl solution [prepared by adding acetyl chloride (73.6 kg, 932.9 mol, 2.25 equivalents) to EtOH (293.9 kg) at 0–5ºC] was added to the batch at below 25ºC to achieve a final pH of approximately 6–7 for the yellow slurry. Solid NH₂OH·HCl (28.8 kg, 414.4 mol, 1.05 equivalents) was added in a single addition, and the resulting mixture was heated to reflux at 66ºC–70ºC for 6–10 h. Solvent was then removed by distillation at reflux at 66ºC–70ºC. EtOH (73.5 kg) was used to remove residual THF. Water (372.0 kg) and EtOH (293.9 kg) were added. After 3–6 h at 70ºC–75ºC, the mixture was cooled to 30ºC–35ºC. 0.5%–1% of G-78a crystals were inoculated. After incubation at 30°C–35°C for 2–4 h, heptane (63.2 kg) was added for at least 1 h. After incubation at 20°C–25°C for 60 min, water (279.0 kg) was added for 4–6 h. After incubation at 20°C–25°C for 1 h, the solid was collected and washed with a 1:2 EtOH / water mixture (51.2 kg EtOH and 130.2 kg water) and then washed twice with heptane (63.2 kg). The solid was dried under vacuum under a nitrogen stream to give product G-78a (93.0 kg) in a yield of 65%.

[0921] 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] + .

[0922] Experimental procedures for the synthesis of G-79a

[0923]

[0924] A dry and clean reactor was charged with G-78a (72.0 kg, 259.6 mol), EtOH (56.9 kg), and NH4OH (aqueous solution) (280.8 kg). The mixture was kept at 20ºC–25ºC for at least 16 h. After adding water (144.0 kg) for 30 min, the slurry was kept at 20ºC–25ºC for 30 min. The solids were collected by filtration, washed with a 1:3 EtOH / water mixture (28.5 kg EtOH and 108 kg water), and then washed with heptane (97.9 kg). After drying, the solid was dried under vacuum at 23ºC for 1 h, and then dried under vacuum at 50ºC-55ºC overnight to obtain product G-79a (61.4 kg, 87.2% yield, enantiomeric ratio ≥ 95:5 (254 nm), water content ≤ 0.5% based on Karl Fischer titration).

[0925] A dry and clean reactor was loaded with crude G-79a (60.0 kg, 1.0 equivalent), 1,4-dioxane (240.0 kg), and activated carbon (3.0 kg, 5 wt%). The mixture was stirred at 55ºC–65ºC for 2–4 h. After filtration at high temperature (55ºC–65ºC), the filter cake was washed with 1,4-dioxane (33.0 kg). The filtrate was transferred to a clean reactor. The temperature was adjusted to 45ºC–55ºC and stirred at 45ºC–55ºC for 1–2 h. Water (240.0 kg) was added after 2 h. The temperature was adjusted to 45ºC–55ºC and stirred at 45ºC–55ºC for 1–2 h. The mixture was cooled to 35ºC–45ºC and stirred at 35ºC–45ºC for 2–4 h. Water (87.0 kg) was added after 4 h. The mixture was cooled to 15ºC–25ºC and stirred at 15ºC–25ºC for 12–14 h. The solids were collected by centrifugation, washed with water (120.0 kg), and dried overnight under vacuum at 50ºC–55ºC to give product G-79a (44.8 kg, 71% yield) as a pale yellow to grayish-white solid. Undesirable isomers should be less than 0.5%.

[0926] 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] + .

[0927] Experimental procedures for the synthesis of G-80a

[0928]

[0929] A dry and clean reactor was charged with G-79a (40.0 kg, 161.1 mol), MeCN (96.0 kg), and pyridine (30.8 kg, 386.6 mol, 2.4 equivalents). The mixture was cooled to 0–5°C, and TFAA (40.8 kg, 193.3 mol, 1.2 equivalents) was slowly added below 5°C. After 5 min at 0–5°C, water (120.0 kg) was added after 30 min at 0–5°C, and 0.5% G-80a crystals were inoculated. After 15 min at 0–5°C, water (120.0 kg) was added after 30 min at 0–5°C. After 30 min at 0–5°C, the solids were collected by filtration, washed with a 1:3 MeCN / water mixture (15.6 acetonitrile and 60.0 kg water), and then washed with water (80.0 kg). The solid was dried under vacuum to obtain a crude product (33.0 kg, 93.6% yield) that was a brownish-brown solid.

[0930] Crude G-80a (32.5 kg, 1.0 equivalent) and MTBE (48.1 kg) were charged into a dry and clean reactor, and the slurry was stirred at 20ºC–25ºC for 30 min. Heptane (132.6 kg) was added after 1 h. After 30 min at 20ºC–25ºC, the solid was collected and dried under vacuum to give product G-80a (26.6 kg, 82.0% yield) as a white solid with an enantiomer ratio > 99:1 (254 nm) and a purity > 98% (220 nm).

[0931] 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] + .

[0932] Experimental procedures for the synthesis of G-82a

[0933]

[0934] To a stirred solution of G-80a (25.0 g, 108.6 mmol, 1.0 equivalent) in MeOH (150 mL), NaOMe (30% in MeOH, 4.89 g, 27.1 mmol, 0.25 equivalent) was added, and the resulting mixture was stirred at room temperature for 2 h. Then, NH4Cl (6.39 g, 119.4 mmol, 1.1 equivalent) was added, and the mixture was stirred at room temperature for 16 h. After complete conversion to the desired amidine, the mixture was filtered through a diatomaceous earth bed and concentrated. The residue was dissolved in DMF (125 mL), and 1,8-diazabicyclo[5.4.0]undec-7-ene (32.3 g, 212.3 mmol, 2.1 equivalent) and diethyl malonate (13.4 g, 101.1 mmol, 1.0 equivalent) were added at 0°C, and the resulting mixture was stirred at 90°C for 16 h. After complete conversion, ice water was added, the mixture was acidified with 1 N HCl, and the precipitate was collected by filtration. The precipitate was dried under reduced pressure to give crude G-82a (HPLC method: H, t). ret = 1.51 min; [M+H] = 316), which was then used in the next step without purification.

[0935] Experimental procedures for the synthesis of G-83a

[0936]

[0937] G-82a (10.0 g, 30.1 mmol, 1.0 equivalent) and POCl3 (48.0 g, 310.0 mmol, 10.3 equivalent) were combined at 0ºC and stirred for 5 min. DIPEA (8.2 g, 63.2 mmol, 2.1 equivalent) was added and the resulting mixture was stirred at 80ºC for 3 h. After complete conversion, ice water (1 L) was slowly added to the mixture at 0ºC, and the mixture was then allowed to reach room temperature and stirred for 1 h. The precipitate was collected by filtration, washed with water and hexane, and dried under vacuum to obtain G-83a (HPLC method: H, t). ret = 2.22 min; [M+H] = 352 / 354). The crude product was used in the next step without purification.

[0938] Experimental procedures for the synthesis of G-84a

[0939]

[0940] B-5d (694 mg, 4.09 mmol, 1.2 equivalents) was dissolved in dry THF (13 mL) and cooled to 0ºC. LiHMDS (1.0 M in THF, 5.11 mL, 5.11 mmol, 1.5 equivalents) was added dropwise at 0ºC, and the mixture was stirred for another 15 min. G-77a (1.20 g, 3.41 mmol, 1.0 equivalents) was dissolved in dry THF (13 mL) and added dropwise at 0ºC. The mixture was stirred at 65ºC for 1.5 h. After complete conversion, the mixture was diluted with saturated NaHCO3 aqueous solution and extracted three times with DCM. The organic phases were combined, filtered, and concentrated under reduced pressure to give G-84a. The crude product was used for the next step without purification.

[0941] The intermediate G-84 (Table 21) can be obtained in a similar manner. If necessary, the crude product can be purified by chromatography.

[0942] Table 21

[0943]

[0944] Experimental procedures for the synthesis of G-15a

[0945]

[0946] F-8a (356 mg, 0.804 mmol, 1.0 equivalent) was dissolved in dioxane (2 mL), and hydroxylamine solution (50% in water, 98.6 µL, 1.61 mmol, 2.0 equivalent) was added. The resulting solution was stirred at 40ºC until complete conversion was observed. The solvent was evaporated, and the resulting residue was purified by RP chromatography to give G-13a (G-14a was observed as a byproduct and separated by chromatography). G-13a (136.0 mg, 0.29 mmol, 1.0 equivalent) was dissolved in DCM (2 mL) and DIPEA (114.38 µL, 0.65 mmol, 2.2 equivalent), and methanesulfonyl chloride (34.2 µL, 0.45 mmol, 1.5 equivalent) was added. The resulting solution was stirred at room temperature until complete conversion was observed. The reaction mixture was concentrated under reduced pressure and extracted with DCM (3x) and water. The organic solvent was evaporated, and the resulting residue was purified by RP chromatography to obtain G-15a.

[0947] The following intermediate G-15 (Table 22) can be obtained in a similar manner. If necessary, the crude product can be purified by chromatography.

[0948] Table 22

[0949]

[0950] Experimental procedures for the synthesis of G-29a

[0951]

[0952] (1S)-1-[(2S)-1-methylpyrrolidone-2-yl]ethanol-1-ol (122 µL mg, 0.865 mmol, 3.0 equivalent) and potassium tert-butoxide (97.0 mg, 0.865 mmol, 3.0 equivalent) were dissolved in THF (2 mL) and stirred at 50ºC for 30 min. G-15b (165 mg, 0.28 mmol, 1 equivalent) was added and the solution was stirred at 85ºC for 3 h. The solvent was evaporated and the resulting residue was purified by RP chromatography to obtain G-29a (HPLC method: C, t ret = 1.12 min; [M+H] = 665).

[0953] Experimental procedures for the synthesis of G-30a

[0954]

[0955] G-29a (183 mg, 275 µmol, 1.0 equivalent) and HCl (8 M, 172 µL, 1.38 mmol, 5.0 equivalent) were dissolved in MeOH (2.0 mL) and stirred at 60ºC until complete conversion. The reaction mixture was concentrated under reduced pressure and extracted with EtOAc / NaHCO3. The combined organic phases were concentrated under reduced pressure to give G-30a (HPLC method: A, t ret = 1.41 min; [M+H] = 521).

[0956] Experimental procedures for the synthesis of G-34a

[0957]

[0958] 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 equivalent), and DIPEA (230 µL, 0.67 mmol, 6.0 equivalent) were dissolved in DMSO (1 mL). The reaction was stirred at 90ºC for 18 h. After the reaction was complete, the solvent was removed under reduced pressure and the residue was purified by basic RP chromatography to give the desired product G-34a.

[0959] The intermediate G-34 (Table 23) can be obtained in a similar manner. If necessary, the crude product can be purified by chromatography.

[0960] The diastereomer mixture G-34c can be separated by chiral HPLC (Chiralpack IE, 250 X 20 mm, 5 µ; solvent: ethanol / heptane 60 : 40 + 0.1% diethylamine) to obtain G-34c1 (eluted first as peak 1) and G-34c2 (eluted subsequently as peak 2).

[0961] Table 23

[0962]

[0963] Experimental procedures for the synthesis of G-45a

[0964]

[0965] G-11a (217 mg, 0.505 mmol, 1.0 equivalent) was dissolved in DMSO (2 mL) and DIPEA (172 µL, 1.01 mmol, 2.0 equivalent), and N-methylpiperazine (75.8 mg, 0.757 mmol, 1.5 equivalent) was added. The reaction mixture was stirred at 90ºC until complete conversion was observed. The mixture was diluted with saturated aqueous NaHCO3 solution and extracted three times with DCM. The organic phases were combined, filtered, and concentrated under reduced pressure. The resulting residue was dissolved in ACN and purified by basic RP chromatography to give the desired product G-45a.

[0966] The following intermediate G-45 (Table 24) can be obtained in a similar manner. If necessary, the crude product can be purified by chromatography.

[0967] The diastereomer mixture G-45l was separated by chiral HPLC (column: Chiralpack IE, 250 X 20 mm, 5 µm; solvent: ethanol / heptane 1:1 + 0.1% diethylamine) to obtain G-45I1 (eluted first as peak 1) and G-45I2 (eluted subsequently as peak 2).

[0968] Table 24

[0969]

[0970] Experimental procedures for the synthesis of G-46a

[0971]

[0972] 4-(1H-pyrazol-3-yl)pyridine (73.4 mg, 0.51 mmol, 1.50 equivalent) was dissolved in DMF (1 mL), and NaH (51.7 mg, 1.35 mmol, 4.0 equivalent) was added and the mixture was stirred at room temperature for 20 min. G-11b (150 mg, 0.34 mmol, 1.0 equivalent) was added, and the reaction was stirred at 40ºC for 1 h. After complete conversion, the reaction was extracted with EtOAc / water. The organic phase was concentrated under reduced pressure and purified by RP chromatography to give the desired product G-46a.

[0973] The intermediate G-46 (Table 25) can be obtained in a similar manner. If necessary, the crude product can be purified by chromatography.

[0974] Table 25

[0975]

[0976] Experimental procedures for the synthesis of G-48a

[0977]

[0978] G-11d (150 mg, 0.32 mmol, 1.0 equivalent) was dissolved in dioxane (1.5 mL). 1-Methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaneborane-2-yl)-1h-pyrazole (82.7 mg, 0.39 mmol, 1.2 equivalent), XPHOS PD G3 (26.0 mg, 0.03 mmol, 0.09 equivalent), and cesium carbonate (0.4 mL, 0.80 mmol, 2.46 equivalent) were added. The reaction was stirred at 80ºC for 2 h. After complete conversion was observed, the reaction was extracted with DCM / water. The combined organic phases were concentrated under reduced pressure, dissolved in ACN / water, and purified by RP chromatography to give the desired product G-48a.

[0979] The following intermediate G-48 (Table 26) can be obtained in a similar manner. If necessary, the crude product can be purified by chromatography.

[0980] Table 26

[0981]

[0982] Experimental procedures for the synthesis of G-51a

[0983]

[0984] G-11b (150 mg, 0.34 mmol, 1.0 equivalence) was dissolved in dioxane (18 mL), and 2-oxazolidinone (59.9 mg, 0.67 mmol, 2.0 equivalence), Pd(dppf)Cl2 (24.7 mg, 0.03 mmol, 0.1 equivalence), and NaOtBu (2.0 M in THF, 185 µL, 0.37 mmol, 1.1 equivalence) were added. The reaction was stirred at 60ºC for 3 days. After complete conversion was observed, the reaction was filtered and concentrated under reduced pressure. The residue was extracted with DCM / water. The combined organic phases were concentrated under reduced pressure and purified by RP chromatography to give G-51a (HPLC method: C, t). ret = 0.78 min; [M+H] = 496).

[0985] Experimental procedures for the synthesis of G-63a

[0986]

[0987] G-45a (124 mg, 0.251 mmol, 1.0 equivalent) was dissolved in DCM (1 mL) under argon atmosphere and cooled to 0ºC. Formaldehyde (22.5 µL, 0.301 mmol, 1.2 equivalent) was added, followed by sodium triacetoxyborohydride (224 mg, 1.01 mmol, 4.0 equivalent). The solution was stirred at 0ºC for 30 min. After the starting material was completely consumed, the reaction was quenched by adding water. The aqueous phase was extracted with DCM. The combined organic phases were dried, filtered, and concentrated under reduced pressure. The residue was purified by RP chromatography to give the desired product G-63a.

[0988] The following intermediate G-63 (Table 27) can be obtained in a similar manner. Deuterated intermediate G-63 is obtained similarly, but sodium triacetoxyborohydride is replaced with sodium triacetoxyborodeuteride. If necessary, the crude product is purified by chromatography.

[0989] Table 27

[0990]

[0991] Experimental procedures for the synthesis of G-86a

[0992]

[0993] G-12b (2.00 g, 4.23 mmol, 1 equivalent), ethyl 1H-pyrazole-5-carboxylate (936 mg, 6.34 mmol, 1.5 equivalent), and cesium carbonate (4.59 g, 8.46 mmol, 2 equivalent) were dissolved in THF (20 mL). The reaction was stirred at 70ºC for 2 h. After complete conversion, DCM was added, and the solution was washed with water. The organic phase was concentrated under reduced pressure and purified by RP chromatography to give G-86a.

[0994] The intermediate G-86 (Table 28) can be obtained in a similar manner. If necessary, the crude product can be purified by chromatography.

[0995] Table 28

[0996]

[0997] Experimental procedures for the synthesis of G-88a

[0998]

[0999] G-11b (4.00 g, 8.99 mmol, 1 equivalent), 2-(1H-pyrazol-3-yl)acetic acid hydrochloride (1.73 g, 10.34 mmol, 1.15 equivalent), and cesium carbonate (8.79 g, 26.97 mmol, 3 equivalent) were dissolved in DMSO (20 mL). The reaction mixture was stirred at 90ºC for 1.5 h. After complete conversion to the desired intermediate, the reaction mixture was cooled to room temperature, and isopropylamine (1.55 mL, 17.98 mmol, 2.0 equivalent), 1-methylimidazole (1.43 mL, 17.98 mmol, 2.0 equivalent), and chloro-N,N,N',N'-tetramethylformamidinium hexafluorophosphate (5.15 g, 17.98 mmol, 2.0 equivalent) were added. The mixture was stirred at room temperature for 15 min. After complete conversion, DCM was added, and the solution was washed with water and brine. The organic phase was concentrated under reduced pressure and purified by RP chromatography to obtain G-88a.

[1000] The intermediate G-88 (Table 29) can be obtained in a similar manner. If necessary, the crude product can be purified by chromatography.

[1001] Table 29

[1002]

[1003] Synthesis of aminocyanothiophene I and II

[1004] Experimental procedures for synthesizing I-2

[1005]

[1006] 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 80ºC for 18 h. The reaction mixture was diluted with EtOAc, filtered, and washed with a saturated aqueous solution of NaHCO3. The organic phase was separated, and the remaining aqueous phase was extracted with EtOAc (2 x). The combined organic phases were dried over magnesium sulfate, evaporated, and the resulting residue was purified by RP chromatography to give I-2.

[1007] The final compound I (Table 30) can be obtained in a similar manner. If necessary, the crude product can be purified by chromatography.

[1008] Table 30

[1009]

[1010] Experimental procedures for synthesizing I-37

[1011]

[1012] 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) were suspended in EtOH (1.0 mL) and stirred at 60ºC for 15 min. Malononitrile (22.3 mg, 0.320 mmol, 2.00 equivalent) was added. The reaction was stirred at 80ºC for 5 h. After complete conversion, the mixture was diluted with DMSO, filtered, and purified by RP chromatography to obtain I-37.

[1013] The final compound I (Table 31) can be obtained in a similar manner. If necessary, the crude product can be purified by chromatography.

[1014] Table 31

[1015]

[1016] Experimental procedures for the synthesis of I-45

[1017]

[1018] I-38 (225 mg, 0.31 mmol, 1.0 equivalent) was dissolved in DCM / TFA (1:1, 2.0 mL) and the reaction was stirred at room temperature for 3 h. After complete conversion, the reaction mixture was concentrated under vacuum and purified by RP chromatography to obtain I-45 (HPLC method: A, t ret = 1.47 min; [M+H] = 619).

[1019] Experimental procedures for synthesizing I-51

[1020]

[1021] Potassium hydroxide (1.91 g, 29.0 mmol, 7.0 equivalent) dissolved in water (53 mL) was added to a suspension of I-46 (2.73 g, 4.14 mmol, 1.0 equivalent) in ethanol (47 mL), and the mixture was stirred at room temperature for 2 h. After complete conversion, the mixture was acidified to pH 6, the ethanol was removed under reduced pressure, and the resulting precipitate was collected by repeated centrifugation and washing with water, and dried under reduced pressure to give the desired product I-51. The crude product was used without further purification.

[1022] The final compound I (Table 32) can be obtained in a similar manner. If necessary, the crude product can be purified by chromatography.

[1023] Table 32

[1024]

[1025] Experimental procedures for synthesizing I-53

[1026]

[1027] 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 equivalent), 1-methylimidazole (45.6 µL, 0.57 mmol, 4.0 equivalent), and chloro-N,N,N',N'-tetramethylformamidinium-hexafluorophosphate (57.3 mg, 0.20 mmol, 1.4 equivalent) were added, and the mixture was stirred at room temperature for 1 h. After complete conversion, the mixture was diluted with ACN, and the product was separated by RP chromatography to give the desired product I-53.

[1028] The final compound I (Table 33) can be obtained in a similar manner. If necessary, the crude product can be purified by chromatography.

[1029] Table 33

[1030]

[1031] Experimental procedures for the synthesis of II-1

[1032]

[1033] 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) were dissolved in EtOH (12 mL) and stirred at 60ºC for 15 min. Malononitrile was slowly added dropwise (8 mL / h) as a solution in EtOH (3.77 mL, 4.28 mmol, 1.65 equivalent). The reaction was stirred at 80ºC for 5 h. After complete conversion, the reaction was concentrated and purified by NP chromatography. The product fraction was concentrated and extracted with DCM and saturated NaHCO3. The organic phase was concentrated under reduced pressure to give II-1.

[1034] The following final compound II (Table 34) can be obtained in a similar manner. If necessary, the crude product can be purified by chromatography.

[1035] Table 34

[1036]

[1037] Experimental procedures for the synthesis of II-17

[1038]

[1039] II-1 (0.10 g, 0.18 mmol, 1.0 equivalent) was suspended in DMSO (0.50 mL). DIPEA (0.11 mL, 0.57 mmol, 3.1 equivalent) and (R)-5-methyl-4,7-diazaspiro[2.5]octane dihydrochloride (42 mg, 0.20 mmol, 1.1 equivalent) were added, and the reaction mixture was stirred at 80ºC for 2 h. After complete conversion, the reaction mixture was purified by RP chromatography.

[1040] The following final compound II (Table 35) can be obtained in a similar manner. If necessary, the crude product can be purified by chromatography.

[1041] Table 35

[1042]

[1043] Experimental procedures for the synthesis of II-19

[1044]

[1045] II-3 (80 mg, 0.15 mmol, 1.0 equivalent) and B-11a (50.3 mg, 0.31 mmol, 2.0 equivalent) were dissolved in THF (1.0 mL), cesium carbonate (123 mg, 0.38 mmol, 2.5 equivalent) was added, and the mixture was stirred at 65ºC for 3 h. After complete conversion, a saturated NaHCO3 solution was added, and the product was extracted with DCM. The organic phase was dried, filtered, and concentrated under reduced pressure. The crude product was purified by RP chromatography to obtain the desired final product II-19.

[1046] The following final compound II (Table 36) can be obtained in a similar manner. If necessary, the crude product can be purified by chromatography.

[1047] Table 36

[1048]

[1049] Experimental procedures for the synthesis of II-24

[1050]

[1051] Sodium hydroxide (4 M in water, 99.4 µL, 0.40 mmol, 2.5 equivalents) was added to a solution of II-23 (100 mg, 0.159 mmol, 1.0 equivalent) in 1-propanol (1 mL), and the mixture was stirred at room temperature for 30 min. After complete conversion, saturated NaHCO3 was added, the mixture was washed with DCM, and the aqueous phase was acidified with HCl and extracted with DCM. The organic phase was dried, filtered, and concentrated, and the crude product was purified by RP chromatography to give the desired product II-24.

[1052] The following final compound II (Table 37) can be obtained in a similar manner. If necessary, the crude product can be purified by chromatography.

[1053] Table 37

[1054]

[1055] Experimental procedures for the synthesis of II-25

[1056]

[1057] 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 equivalent), DIPEA (22.3 µL, 0.166 mmol, 2.5 equivalent), and 1-propylphosphoric anhydride (29.7 µL, 1.00 mmol, 1.5 equivalent) were added, and the mixture was stirred at room temperature for 3 h. After complete conversion, saturated NaHCO3 was added, and the mixture was extracted with DCM. The organic phase was dried, filtered, and concentrated, and the crude product was purified by RP chromatography to give the desired product II-25.

[1058] The following final compound II (Table 38) can be obtained in a similar manner. If necessary, the crude product can be purified by chromatography.

[1059] Table 38

[1060]

[1061] Experimental procedures for the synthesis of II-30

[1062]

[1063] Under an argon atmosphere, malononitrile (64.4 mg, 0.97 mmol, 5.0 equivalent), sulfur (23.8 mg, 0.74 mmol, 4.0 equivalent), and β-alanine (69.5 mg, 0.78 mmol, 4.0 equivalent) were added 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). The reaction mixture was stirred overnight at 80ºC. After complete conversion, the mixture was cooled to room temperature, filtered, and extracted with DCM and a saturated aqueous solution of NaHCO3. The organic phases were combined and concentrated under reduced pressure. The residue was dissolved in ACN and water and purified by alkaline RP chromatography to give the desired product II-30.

[1064] The final compound II (Table 39) can be obtained in a similar manner. The crude product is purified by chromatography if necessary. In the case of II-87, Boc deprotection was observed during the reaction using G-48r as the starting material.

[1065] Table 39

[1066]

[1067] Experimental procedures for the synthesis of II-143

[1068]

[1069] G-51a (90 mg, 0.18 mmol, 1.0 equivalent), ammonium acetate (22.4 mg, 0.29 mmol, 1.6 equivalent), and sulfur (9.32 mg, 0.29 mmol, 1.6 equivalent) were dissolved in EtOH (1.20 mL) and stirred at 60ºC for 15 min. Malononitrile, a solution in EtOH (0.26 mL, 0.3 mmol, 1.65 equivalent), was slowly added dropwise. The reaction was stirred at 80ºC for 5 h. After complete conversion, DCM was added and the mixture was extracted three times with water. The combined organic phases were concentrated under reduced pressure, dissolved in DMF / ACN / water, and purified by RP chromatography to give II-143.

[1070] The final compound II (Table 40) can be obtained in a similar manner. If necessary, the crude product can be purified by chromatography.

[1071] Table 40

[1072]

[1073] Experimental procedures for the synthesis of II-160

[1074]

[1075] To a solution of II-146 (210 mg, 0.29 mmol, 1.0 equivalence) in dioxane (3 mL), HCl (4 M in dioxane, 0.29 mL, 1.17 mmol, 4.0 equivalence) was added, and the reaction mixture was stirred at room temperature for 18 h. The reaction mixture was then heated to 50ºC and stirred for 6 h. The solvent was removed, and the residue was purified by RP chromatography to give II-160.

[1076] The following final compound II (Table 41) can be obtained in a similar manner. If necessary, the crude product can be purified by chromatography.

[1077] Table 41

[1078]

[1079] Example 5 Synthesis of compounds according to formula (D)

[1080] List of abbreviations ( Table 42 )

[1081]

[1082] Chemical Examples

[1083] Unless otherwise stated, all reactions were carried out in commercially available apparatus using methods typically employed in chemical laboratories. Starting materials sensitive to air and / or moisture were stored under a protective gas, and the corresponding reactions and operations were performed under a protective gas (nitrogen or argon).

[1084] If a compound can be represented by both its structural formula and its nomenclature, then in the event of a conflict, the structural formula shall prevail.

[1085] Chromatography

[1086] Thin-layer chromatography was performed on off-the-shelf silica 60 TLC plates on glass (with fluorescent indicator F-254) manufactured by Merck.

[1087] Preparative high-pressure chromatography (RP HPLC) of the example compounds according to the invention was performed on an Agilent or Gilson system using columns manufactured by Waters (name: SunFire™ preparative C18, OBD™ 10 µm, 50 x 150 mm; or SunFire™ preparative C18 OBD™ 5 µm, 30 x 50 mm; or XBridge™ preparative C18, OBD™ 10 µm, 50 x 150 mm; or XBridge™ preparative C18, OBD™ 5 µm, 30 x 150 mm; or XBridge™ preparative C18, OBD™ 5 µm, 30 x 50 mm) and columns manufactured by YMC (name: Actus-Triart preparative C18, 5 µm, 30 x 50 mm) and Chiralpak IE (5 µm, 250 x 20 mm).

[1088] Different H2O / acetonitrile gradients were used to elute the compounds. For the Agilent system, 5% acid modifier (20 mL HCOOH to 1 L H2O / acetonitrile (1 / 1)) was added to the water (acidic conditions). For the Gilson system, 0.1% HCOOH was added to the water.

[1089] For chromatography under alkaline conditions, the Agilent system also uses an H2O / acetonitrile gradient, while making the water alkaline by adding 5% alkaline modifier (50 g NH4HCO3 + 50 mL NH3 (25%, in H2O) to 1 L (with H2O)). For the Gilson system, the water is made alkaline as follows: 5 mL NH4HCO3 solution (158 g, in 1 L H2O) and 2 mL NH3 (28%, in H2O) are added to 1 L. The Gilson system is also used under isocratic conditions (60% EtOH / 40% EtOH + 0.1% DEA).

[1090] In Agilent 1260 SFC system, JASCO SFC system, Sepiatec SFC system, Waters Thar SFC system, or Waters UPC system with the following columns 2Supercritical fluid chromatography (SFC) of the intermediates and example compounds according to the present invention performed on an MS SFC system: Chiralcel OJ (250 x 20 mm, 5 µm), Chiralpak AD-H (21 x 250 mm, 5 µm), Chiralpak AD (250 x 20 mm, 5 µm), Chiralpak AS (250 x 20 mm, 5 µm), Chiralpak IC (250 x 20 mm, 5 µm), Chiralpak IA (250 x 20 mm, 5 µm), Chiralcel OJ (250 x 20 mm, 5 µm), Chiralcel OD (250 x 20 mm, 5 µm), Chiralcel OX-3 (150 x 4.6 mm, 3 µm), Phenomenex Lux C2 (250 x 20 mm, 5 µm).

[1091] Analytical SFC / UV-spectroscopy methods

[1092] SFC Method: SFC-1

[1093] SFC: Agilent 1260 (binary pump) SFC

[1094] Column: Chiralpak AD-H (250 x 4.6 mm), 5 µm

[1095] Flow rate: 2 ml / min

[1096] Mobile phase: A: CO2 + B: MeOH

[1097] ABPR: 120 bar

[1098] Temperature: 37.5ºC

[1099] UV: 220 nm

[1100] Gradient 80% A + 20% B (isotropic)

[1101] Stop time 10 min

[1102] Use a column manufactured by Waters (name: XBridge) TM C18, 2.5 µm, 2.1 x 20 mm; or XBridge TMAnalytical HPLC (reaction control) of intermediates and final compounds was performed using columns manufactured by YMC (name: Triart C18, 3.0 µm, 2.0 x 30 mm) and Phenomenex (name: Luna C18, 5.0 µm, 2.0 x 30 mm). In each case, the analytical apparatus was also equipped with a mass spectrometer detector.

[1103] HPLC-mass spectrometry / UV-spectroscopy

[1104] Retention time / MS-ESI for characterizing compounds according to embodiments of the present invention + It was generated using an HPLC-MS apparatus (high-performance liquid chromatography with a mass spectrometer detector). The retention time t of the compound eluting at the injection peak was given. Ret. =0.00.

[1105] Method A

[1106] HPLC Agilent 1100 system

[1107] MS 1200 Series LC / MSD (API-ES+ / -3000V, Quadrupole, G6140)

[1108] MSD signal settings: scan positive / negative ions 120-1500 m / z

[1109] Detected signal 315 nm (bandwidth 170 nm, reference off)

[1110] Spectral range 230-400 nm

[1111] Peak width < 0.01 min

[1112] Column Waters, Xbridge C18, 2.5 µm, 2.1x20 mm column

[1113] Column temperature 60ºC

[1114] Solvent A: 20 mM aqueous solution of NH4HCO3 / NH3, pH 9

[1115] B: ACN HPLC grade

[1116] Flow rate 1.00 mL / min

[1117] Gradient 0.00-1.50 min 10% to 95% B

[1118] 1.50-2.00 min 95% B

[1119] 2.00-2.10 min 95% to 10% B

[1120] Method E

[1121] UPLC-MS Waters Acquity-UPLC-SQ Detector-2

[1122] MSD signal settings: scan positive and negative ions 100-1500.

[1123] Source voltage: Capillary voltage (kV) -3.50, Taper voltage (V): 50

[1124] Source temperature: Desolvation temperature (ºC): 350

[1125] Source gas flow rate: Desolvation (L / Hr): 750, Conical orifice (L / Hr): 50

[1126] Detection signal diode array

[1127] Spectral range: 200-400 nm; Resolution: 1.2 nm

[1128] Sampling rate 10 points / second

[1129] Column AQUITY UPLC BEH C18 1.7 µm, 2.1 X 50 mm

[1130] Column temperature 35ºC

[1131] Solvent A: 0.07% formic acid in ACN

[1132] B: 0.07% formic acid in water

[1133] Flow rate 0.6 mL / min

[1134] Gradient 0.0-0.30 min 97% B

[1135] 0.30-2.20 min 97% to 2% B

[1136] 2.20-3.30 min 2% B

[1137] 3.30-4.50 min 2% to 97% B

[1138] 4.50-4.51 min 97% B

[1139] Method H

[1140] HPLC Agilent 1100 / 1200 system

[1141] MS 1200 Series LC / MSD (MM-ES + APCI + / - 3000 V, quadrupole, G6130B)

[1142] MSD signal setting: Scan positive ions 700-1350

[1143] Waters column, catalog number 186003389, XBridge BEH C18, 2.5 µm, 2.1 x 30 mm)

[1144] Eluent A: 5 mM NH4HCO3 / 18 mM NH3 (pH = 9.2)

[1145] B: Acetonitrile (HPLC grade)

[1146] Detection signals: UV 254 nm, 230 nm, 214 nm (bandwidth 8, reference off)

[1147] Spectral range: 190-400 nm; slit width: 4 nm

[1148] Peak width > 0.0031 min (0.063 s response time, 80 Hz)

[1149] Inject 0.5 µL standard injection

[1150] Flow rate 1.4 mL / min

[1151] Column temperature 45ºC

[1152] Gradient 0.0-1.0 min 15% à 95% B

[1153] 1.0-1.1 min 95% B

[1154] Stop time: 1.3 min

[1155] HPLC / UV-spectroscopy

[1156] Method I

[1157] HPLC Agilent 1100 / 1200 system

[1158] Chiralpak column; catalog number 85394; IE, 5 µm; 150 x 2.1 mm

[1159] Eluent A: n-Heptane

[1160] B: EtOH + 0.1% DEA

[1161] Detection signal UV 315 nm (bandwidth 170, reference off)

[1162] Spectral range: 190-400 nm; slit width: 4 nm

[1163] Peak width > 0.0031 min (0.063 s response time, 80 Hz)

[1164] Inject 0.5 µL standard injection

[1165] Flow rate 1.2 mL / min

[1166] Column temperature 45ºC

[1167] 70% B

[1168] Stop time: 5 min

[1169] The compounds and intermediates according to the invention can be obtained using synthetic methods known to those skilled in the art and described in the organic synthesis literature. Preferably, the compounds are obtained in a manner similar to the preparation methods explained more fully below (wherein the substituents of the general formula have the meanings given above). These methods are intended to illustrate the invention and not to limit its subject matter or the scope of the compounds claimed for these embodiments. In some cases, the order of the reaction steps may vary. Variations of reaction methods known to those skilled in the art but not described in detail herein may also be used.

[1170] Where the preparation of the starting compounds is not described, they are commercially available, their synthesis is described in the prior art, or they can be prepared similarly to the known prior art compounds or methods described herein, i.e., the synthesis of these compounds is within the skill of an organic chemist. The substances described in the literature can be prepared according to the published synthetic methods. Any functional group in the starting material or intermediate can be protected using conventional protecting groups. These protecting groups can be further cleaved at appropriate stages in the reaction sequence using methods familiar to those skilled in the art. If the chemical structures depicted below do not have a precise configuration of the stereocenter, such as an asymmetrically substituted carbon atom, both configurations should be considered included and disclosed in such illustrations. Stereocenter illustrations in racemic form should always be considered to include and disclose two enantiomers (if no other defined stereocenter exists) or all other potential diastereomers and enantiomers (if an additional defined or undefined stereocenter exists).

[1171] Option 1:

[1172]

[1173] Option 2:

[1174]

[1175] Experimental procedure for synthesizing K-1a

[1176]

[1177] At room temperature, malononitrile (58.04 g, 879.3 mmol, 1.5 equivalents) was added to a solution of ethyl 1-methyl-2-oxocyclohexane-1-carboxylate (108.00 g, 586.2 mmol) in toluene (1.03 L), followed by the addition of ammonium acetate (9.04 g, 117.2 mmol, 0.2 equivalents) and acetic acid (13.41 mL, 234.5 mmol, 0.4 equivalents). The mixture was stirred at 110ºC for 16 h. After complete conversion, the mixture was diluted with EtOAc and washed with water and brine, dried over sodium sulfate, and concentrated under reduced pressure to give crude product K-1a. This crude material was used for the next step without further purification (see also Naumann et al., Pharmazie 51 (1996), 4).

[1178] Table 43

[1179]

[1180] Experimental procedure for synthesizing K-2a

[1181]

[1182] Sulfur (68.9 g, 2.2 mol, 2.0 equivalent) and L-proline (24.8 g, 0.22 mol, 0.2 equivalent) were added to a solution of K-1a (250.0 g, 1.1 mol) in DMF (3.0 L), and the resulting mixture was stirred at 80ºC for 12 h. After complete conversion, the mixture was partitioned between EtOAc and water, and the organic layer was collected. The aqueous layer was further extracted with EtOAc, and the combined organic layers were washed with water and brine, dried over sodium sulfate, and concentrated under reduced pressure to give the crude product. The crude product was purified by column chromatography to give K-2a.

[1183] Table 44

[1184]

[1185] Experimental procedure for synthesizing K-3a

[1186]

[1187] K-2a (78.0 mg, 0.3 mmol, 1.0 equivalent) was dissolved in EtOH (1.5 mL), and potassium hydroxide (4 M, in water, 0.37 mL, 1.5 mmol, 5.0 equivalent) was added. The mixture was stirred at 78ºC for 16 h. After complete conversion, water and EtOAc were added to the reaction mixture, the pH of the aqueous phase was adjusted to pH 4 using KHSO4 solution (10%, in water), and the product was extracted using EtOAc. The combined organic layers were dried, filtered, and concentrated. The crude product was purified by acid reversed-phase chromatography (gradient elution: 20% to 90% acetonitrile in water) to give K-3a.

[1188] Enantiomers can be separated using preparative SFC chromatography. For example, K-3a to K-3b and their enantiomers. (Analytical SFC method SFC-1: for K-3b, t...) ret = 4.9 min, compared to 7.9 min for other enantiomers).

[1189] Table 45

[1190]

[1191] Experimental procedures for synthesizing K-9a

[1192]

[1193] TEA (1.1 ml, 101.19 mmol, 2.0 equivalent) was added to a solution of (S)-tert-butyl-3-methyl-1,4-diazacycloheptane-1-carboxylic acid (846.0 mg, 214.30 mmol, 1.0 equivalent) and 2-chloropyrimidin-4-carboxynitrile (528.9 mg, 139.54 mmol, 1.0 equivalent) in DMSO (4 ml, 4,5V). The reaction mixture was stirred at 80ºC for 1 h. After complete conversion, the reaction mixture was cooled to room temperature and water and EtOAc were added. The phases were separated. The organic layer was washed with water, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product, which was purified by chromatography to obtain K-9a.

[1194] Table 46

[1195]

[1196] Experimental procedures for the synthesis of K-10a

[1197]

[1198] A 50% hydroxylamine solution (13.05 ml, 213.30 mmol, 2.0 equivalent) in water was added to a solution of K-9a (33.85 g, 106.65 mmol, 1.0 equivalent) in EtOH (270 ml) at room temperature. The reaction mixture was stirred at 60ºC for 1 h. After complete conversion, the reaction mixture was concentrated under reduced pressure to give K-10a, which was used in the next step without further purification.

[1199] Table 47

[1200]

[1201] Experimental procedures for the synthesis of K-11a

[1202]

[1203] TEA (2.17 g, 21.40 mmol, 2.0 equivalent) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylureonium hexafluorophosphate (HATU, 4.27 g, 11.24 mmol, 1.10 equivalent) were added to a stirred solution of K-3b (2.53 g, 10.70 mmol, 1.0 equivalent) in DMSO (10 ml) at room temperature. The mixture was stirred at room temperature for 15 min. K-10a (3.75 g, 10.70 mmol, 1.0 equivalent) was added at room temperature and stirred overnight. After complete conversion, the reaction mixture was diluted with water and EtOAc. The phases were separated. The organic layer was washed with water, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product.

[1204] If necessary, the crude product K-11 can be purified by chromatography.

[1205] Table 48

[1206]

[1207] Experimental procedures for the synthesis of K-12a

[1208]

[1209] DBU (1.98 mL, 14.04 mmol, 4.0 equivalent) was added to a stirred solution of K-11a (2.00 g, 3.51 mmol, 1.0 equivalent) in 40 mL of THF at room temperature. The reaction mixture was stirred overnight at 70ºC. After complete conversion, the reaction mixture was concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography to give K-12a.

[1210] If necessary, the crude product K-12 can be purified by chromatography.

[1211] Table 49

[1212]

[1213] Experimental procedures for the synthesis of K-13a

[1214]

[1215] Concentrated HCl (32.88 mL, 345.21 mmol, 1.0 equivalent) was added to a stirred solution of K-12a (20.00 g, 34.52 mmol, 1.0 equivalent) in MeOH (350 mL) at room temperature. The reaction mixture was stirred at 50ºC for 2 h. After complete conversion, the reaction mixture was concentrated under reduced pressure and diluted with water. The aqueous phase was extracted with DCM. The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give K-13a, which was used in the next step without further purification.

[1216] If necessary, the crude product K-13 can be purified by chromatography.

[1217] Table 50

[1218]

[1219] Experimental procedures for the synthesis of E-1a

[1220]

[1221] Potassium carbonate (31.17 g, 0.23 mol, 3.0 equivalent) was added to a stirred solution of methyl 2-(3-hydroxy-1,2-oxazol-5-yl)-3-methylbutyrate (15.00 g, 0.08 mol, 1.0 equivalent) in DMF (75.0 mL) at 0ºC. 1,3-Dibromopropane (15.20 g, 0.08 mol, 1.0 equivalent) was added dropwise, and the reaction mixture was stirred at 0ºC for 9 hours. After complete conversion, the reaction mixture was quenched with water and extracted with EtOAc. The organic layer was washed with ice water, dried over sodium sulfate, and concentrated under reduced pressure to give the crude product. The obtained crude compound was purified by chromatography to give E-1a.

[1222] The following intermediate E-1 (Table 43) can be obtained in a similar manner. If necessary, the crude product E-1 can be purified by chromatography.

[1223] Table 51

[1224]

[1225] Experimental procedure for the synthesis of E-2a

[1226]

[1227] Potassium carbonate (2.76 g, 19.98 mmol, 2.0 equivalent) was added 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), and the mixture was stirred at 60ºC under argon for 22 h. After complete conversion, the reaction mixture was allowed to cool to room temperature, filtered, and the solids were washed with acetonitrile. The combined solutions were concentrated under reduced pressure and purified by chromatography to give E-2a.

[1228] The following intermediate E-2 (Table 52) can be obtained in a similar manner from different intermediates K-13 and E-1 or alternative bromides. If necessary, the crude product E-2 can be purified by chromatography.

[1229] Table 52

[1230]

[1231] Experimental procedures for the synthesis of E-3a

[1232]

[1233] A sodium hydroxide solution (2 M, in water, 6.18 mL, 12.35 mmol, 2.0 equivalent) was added to a stirred solution of E-2a (4.26 g, 6.18 mmol, 1.0 equivalent) in methanol (21.0 mL), and the reaction mixture was stirred at 45ºC for 1 h. After complete conversion, the reaction mixture was concentrated under reduced pressure. The crude product was purified by chromatography to give E-3a.

[1234] The following intermediate E-3 (Table 53) can be obtained in a similar manner from different intermediates E-2. If necessary, the crude product E-3 can be purified by chromatography.

[1235] Table 53

[1236]

[1237] Experimental procedures for synthesizing I-1

[1238]

[1239] DIPEA (0.16 mL, 0.97 mmol, 3.0 equivalent) was added 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-thiazolyl-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), and the reaction mixture was stirred at room temperature for 30 min. After complete conversion, the reaction mixture was quenched with water, diluted with acetonitrile, and purified by chromatography.

[1240] The following compound I (Table 54) can be obtained in a similar manner from different intermediates E-3 and A-4.

[1241] Table 54

[1242]

[1243] Chiral separation of compound I by chiral column chromatography:

[1244] If a mixture of compounds I as diastereomers is obtained, they can be separated into individual stereoisomers by chiral chromatography, for example, as shown below: I-3 is separated into I-26 and I-27 (Table 55).

[1245] Table 55

[1246]

[1247] Example 6 Synthesis of compounds according to formula (E)

[1248] List of abbreviations ( Table 56 )

[1249]

[1250]

[1251] Preparation of compounds according to the present invention

[1252] Unless otherwise stated, all reactions were carried out in commercially available apparatus using methods typically employed in chemical laboratories. Starting materials sensitive to air and / or moisture were stored under a protective gas, and the corresponding reactions and operations were performed under a protective gas (nitrogen or argon).

[1253] If a compound can be represented by both its structural formula and its nomenclature, then in the event of a conflict, the structural formula shall prevail.

[1254] The microwave reaction is carried out in a starter / reactor manufactured by Biotage, or in an Explorer manufactured by CEM, or in a Synthos 3000 or Monowave 3000 manufactured by Anton Paar, in a sealed container (preferably 2, 5 or 20 mL), preferably under stirring.

[1255] Chromatography

[1256] Thin-layer chromatography was performed on off-the-shelf silica 60 TLC plates on glass (with fluorescent indicator F-254) manufactured by Merck.

[1257] Preparative high-performance liquid chromatography (RP HPLC) of the compounds according to embodiments of the present invention was performed on an Agilent or Gilson system using columns manufactured by Waters (names: SunFire™ Preparative C18, OBD™ 10 µm, 50 x 150 mm or SunFire™ Preparative C18 OBD™ 5 µm, 30 x 50 mm or XBridge™ Preparative C18, OBD™ 10 µm, 50 x 150 mm or XBridge™ Preparative C18, OBD™ 5 µm, 30 x 150 mm or XBridge™ Preparative C18, OBD™ 5 µm, 30 x 50 mm) and columns manufactured by YMC (names: Actus-Triart Prep C18, 5 µm, 30 x 50 mm).

[1258] Different H2O / acetonitrile gradients were used to elute the compounds. For the Agilent system, 5% acid modifier (20 mL HCOOH to 1 L H2O / acetonitrile (1 / 1)) was added to the water (acidic conditions). For the Gilson system, 0.1% HCOOH was added to the water.

[1259] For chromatography under alkaline conditions, the Agilent system also uses an H2O / acetonitrile gradient, while making the water alkaline by adding 5% alkaline modifier (50 g NH4HCO3 + 50 mL NH3 (25%, in H2O) to 1 L (with H2O)). For the Gilson system, the water is made alkaline as follows: 5 mL of NH4HCO3 solution (158 g, in 1 L H2O) and 2 mL of NH3 (28%, in H2O) are added to 1 L.

[1260] Supercritical fluid chromatography (SFC) of the intermediates and compounds of the present invention was performed on a JASCO SFC system using the following columns: Chiralcel OJ (250 x 20 mm, 5 µm), Chiralpak AD (250 x 20 mm, 5 µm), Chiralpak AS (250 x 20 mm, 5 µm), Chiralpak IC (250 x 20 mm, 5 µm), Chiralpak IA (250 x 20 mm, 5 µm), Chiralcel OJ (250 x 20 mm, 5 µm), Chiralcel OD (250 x 20 mm, 5 µm), and Phenomenex Lux C2 (250 x 20 mm, 5 µm).

[1261] Use a column manufactured by Waters (name: XBridge) TM C18, 2.5 µm, 2.1 x 20 mm; or XBridge TM Analytical HPLC (reaction control) of intermediates and final compounds was performed using columns manufactured by YMC (name: Triart C18, 3.0 µm, 2.0 x 30 mm) and Phenomenex (name: Luna C18, 5.0 µm, 2.0 x 30 mm). In each case, the analytical apparatus was also equipped with a mass spectrometer detector.

[1262] HPLC-mass spectrometry / UV-spectroscopy

[1263] Retention time / MS-ESI for characterizing compounds according to embodiments of the present invention + It was generated using an HPLC-MS apparatus (high-performance liquid chromatography with a mass spectrometer detector). The retention time t of the compound eluting at the injection peak was given. Ret. =0.00.

[1264] Method A

[1265] HPLC Agilent 1100 system

[1266] MS 1200 Series LC / MSD (API-ES+ / -3000V, Quadrupole, G6140)

[1267] MSD signal settings: Scan positive / negative ions 120-900 m / z

[1268] Detected signal 315 nm (bandwidth 170 nm, reference off)

[1269] Spectral range 230-400 nm

[1270] Peak width < 0.01 min

[1271] Column Waters, Xbridge C18, 2.5 µm, 2.1x20 mm column

[1272] Column temperature 60ºC

[1273] Solvent A: 20 mM NH4HCO3 / NH3 in H2O, pH 9

[1274] B: ACN HPLC grade

[1275] Flow rate 1.00 mL / min

[1276] Gradient 0.00-1.50 min 10% to 95% B

[1277] 1.50-2.00 min 95% B

[1278] 2.00-2.10 min 95% to 10% B

[1279] Method C

[1280] HPLC Agilent 1260 series

[1281] MS Agilent LC / MSD quadrupole

[1282] MS detection: positive and negative modes

[1283] Mass range 100-750 m / z

[1284] Column Waters X-Bridge BEH C18, 2.5 µm, 2.1 x 30 mm XP

[1285] Column temperature 45ºC

[1286] Solvent A: 20 mM NH4HCO3 / 30 mM NH3 in H2O; Solvent B: ACN (HPLC grade)

[1287] Flow rate 1.40 mL / min

[1288] Gradient 0.00-1.00 min: 15% B to 95% B

[1289] 1.00-1.30 min: 95% B

[1290] Method H

[1291] UPLC-MS Waters Acquity-Binary Solvent Manager-UPLC-SQ Detector-2

[1292] MSD signal settings: scan positive and negative ions 100-1500.

[1293] Source voltage: Capillary voltage (kV) -3.50, Taper voltage (V): 50

[1294] Source temperature: Desolvation temperature (ºC): 350

[1295] Source gas flow rate: Desolvation (L / Hr): 750, Conical orifice (L / Hr): 50

[1296] Detection signal diode array

[1297] Spectral range: 200-400 nm; Resolution: 1.2 nm

[1298] Sampling rate 10 points / second

[1299] Column AQUITY UPLC BEH C18 1.7 µm, 2.1 X 50 mm

[1300] Column temperature 35ºC

[1301] Solvent A: 0.07% formic acid in ACN

[1302] B: 0.07% formic acid in water

[1303] Flow rate 0.6 mL / min

[1304] Gradient 0.0-0.40 min 97% B

[1305] 0.40-2.50 min 97% to 2% B

[1306] 2.50-3.40 min 2% B

[1307] 3.40-3.50 min 2% to 97% B

[1308] 3.50-4.0 min 97% B

[1309] GCMS

[1310] Method U

[1311] The Agilent Technologies-7890B GC system features the 7693 Auto Sampler. and 5977A MSD

[1312] Injection temperature 230ºC

[1313] Column flow rate: 2.0 mL / min

[1314] Solvent delay 1.5 min

[1315] The split ratio is 10:01.

[1316] Column oven temperature program: 100ºC / 1 min, 20ºC / min / 310°C / 5 min

[1317] Total running time: 16 min

[1318] Interface temperature 150ºC

[1319] Ion source temperature 230ºC

[1320] He gas

[1321] Column and column dimensions ZB-5MS (30 m x 0.32 mm; 1 µm)

[1322] MSD scan range 50-900

[1323] Method V

[1324] The Agilent Technologies-7890B GC system features 7693

[1325] Auto Sampler and 5977A MSD

[1326] Injection temperature 230ºC

[1327] Column flow rate: 2.0 mL / min

[1328] Solvent delay 1.5 min

[1329] The split ratio is 10:01.

[1330] The column oven temperature program is 40ºC / 2 min, 15ºC / min / 200° / 1 min, and 25ºC / min / 310° / 0 min.

[1331] Total running time: 18 min

[1332] Interface temperature 150ºC

[1333] Ion source temperature 230ºC

[1334] He gas

[1335] Column and column dimensions ZB-5MS (30 m x 0.32 mm; 1 µm)

[1336] MSD scan range 50-900

[1337] Method W

[1338] The Agilent Technologies-7890B GC system features the 7693 Auto Sampler. and 5977A MSD

[1339] Injection temperature 230ºC

[1340] Column flow rate: 2.0 mL / min

[1341] Solvent delay 1.5 min

[1342] The split ratio is 10:01.

[1343] Column oven temperature program: 60ºC / 3 min, 20ºC / min / 310°C / 2 min

[1344] Total running time: 18 min

[1345] Interface temperature 150ºC

[1346] Ion source temperature 230ºC

[1347] He gas

[1348] Column and column dimensions ZB-5MS (30 m x 0.32 mm; 1 µm)

[1349] Method SFC-1

[1350] Preparation of Waters UPC 2 -MS

[1351] Empower3 software

[1352] MS QDa

[1353] Column Chiralcell OX-3 (4.6*150 MM) 3 µm

[1354] A- Solvent CO2

[1355] B-solvent ACN

[1356] Total flow rate 3 g / min

[1357] 15% of cosolvent

[1358] ABPR 1500psi

[1359] Column temperature 30ºC

[1360] PDA range 200 nm to 400 nm

[1361] 1.2 nm resolution

[1362] MS parameters -

[1363] QDa MS scan range: 100 Da to 1000 Da

[1364] Conical hole voltage

[1365] Positive ion scan 20 V

[1366] Negative ion scanning 15 V

[1367] The compounds and intermediates according to the invention are prepared by the synthetic methods described below, wherein the substituents of the general formula have the meanings given above. These methods are intended to illustrate the invention and not to limit its subject matter or the scope of the compounds claimed for these embodiments. Where the preparation of starting compounds is not described, they are commercially available or their synthesis is described in the prior art, or they can be prepared similarly to the known prior art compounds or methods described herein, i.e., the synthesis of these compounds is within the skill of an organic chemist. The substances described in the literature can be prepared according to the published synthetic methods. If the chemical structures depicted below do not have a precise configuration of the stereocenter, such as an asymmetrically substituted carbon atom, then both configurations should be considered to be included and disclosed in such illustrations. Stereocenter illustrations in racemic form should always be considered to include and disclose two enantiomers (if no other defined stereocenters or one or more stereocenters are present) or all other potential diastereomers and enantiomers (if other defined or undefined stereocenters are present).

[1368] Synthesis of spirone intermediate A

[1369] Experimental procedure for the synthesis of A-2a

[1370]

[1371] Acetyl chloride (111 mL, 1.56 mol, 8.00 equivalent) was added dropwise to a suspension of 5-chloropentanones (22.9 g, 195 mmol, 1.00 equivalent) in EtOH (136 mL) at 0ºC. The reaction mixture was allowed to be warmed to room temperature and stirred for 12 h. The mixture was concentrated under reduced pressure and washed with Et2O, and the crude product A-2a was used directly as an HCl salt for the next step without further purification (HPLC method: A; t ret = 1.03 min; [M+H] + = 164).

[1372] Experimental procedure for the synthesis of A-3a

[1373]

[1374] Crude A-2a (HCl salt) (28.0 g, 140 mmol, 1.00 equivalent) and ethylene glycol (7.38 g, 119 mmol, 0.90 equivalent) were dissolved in DCM (300 mL) and stirred at room temperature for 6 days. The resulting suspension was concentrated under reduced pressure, diluted with Et2O (200 mL), and filtered. The filtrate was concentrated under reduced pressure, absorbed in DCM (200 mL), and treated with KOH solution (2 M in water, 150 mL). The mixture was stirred overnight at room temperature to maintain phase integrity. The phases were separated, the aqueous phase was extracted with DCM (2 x), and the combined organic phases were dried over magnesium sulfate, filtered, and concentrated under reduced pressure. Crude orthoester A-3a was used in the next step without further purification (HPLC method: A;t). ret = 1.37 min; [M+H] + = 163).

[1375] Experimental procedure for the synthesis of A-4a

[1376]

[1377] Crude 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) were dissolved in DCM (120 mL) and stirred at room temperature for 5 h. The reaction mixture was treated with the addition of saturated sodium bicarbonate solution. The organic phase was separated, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by NP chromatography to give the desired compound A-4a (HPLC method: A;t). ret = 1.25 min; [M+Na] + = 283).

[1378] Experimental procedures for the synthesis of A-8a

[1379]

[1380] A-4a (14.9 g, 57.1 mmol, 1.0 equivalent) and sodium iodide (26.0 g, 171 mmol, 3.0 equivalent) were dissolved in acetone (120 mL) and stirred under reflux for 16 h. The reaction mixture was concentrated under reduced pressure, diluted with DCM, and washed with saturated sodium thiosulfate solution. The organic phase was separated, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product A-5a was used in the next step without further purification.

[1381] A-5a (30 g, 85.0 mmol, 1.0 equivalent) was dissolved in THF. The mixture was treated with potassium tert-butoxide (28.7 g, 256 mmol, 3.0 equivalent) at 0ºC and stirred overnight at room temperature. The reaction mixture was quenched by adding water (2 mL) and diluted by adding Et2O and saturated sodium bicarbonate solution. The organic phase was separated, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by NP chromatography to give (racemic) compound A-6a (reaction sequence A-1a à A-6a based on Marko et al., THL 2003, 44, 3333-3336 and Maulide et al., Eur. J. Org. Chem. 2004, 19:3962-3967).

[1382] The enantiomer A-6b can then be obtained after chiral separation via SFC under the following conditions: column: Lux; cellulose-4 (250 mm x 30 mm x 5 µm), 90% CO2, 10% ACN, flow rate: 90 g / min, temperature: 30ºC, enantiomer A-6b (SFC method: SFC-1, t ret = 2.99 min) was eluted as peak 2 after enantiomerization.

[1383] Synthesis of diketone F

[1384] When multiple HPLC retention times are reported, it means that different tautomers exist.

[1385] Experimental procedures for synthesizing F-1a

[1386]

[1387] Methyl 4,6-dichloropyrimidin-2-carboxylate E-4a (2.00 g, 9.67 mmol, 1.00 equivalent) was dissolved in dry ACN (5 mL) under a nitrogen atmosphere. A solution of magnesium diethyl ether bromide (2.99 g, 11.6 mmol, 1.20 equivalent), 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) were added, and the reaction mixture was stirred at 50ºC for 20 h. After complete conversion, the reaction mixture was carefully quenched with 1 M HCl, diluted with water, extracted with DCM, the organic phase was dried, filtered, and concentrated to give crude F-1a. The crude compound was purified by NP chromatography. (HPLC method: H, t) ret = 2.50 min; [M+H] = 399 / 401).

[1388] Experimental procedures for the synthesis of F-2a

[1389]

[1390] F-1a (10.0 g, 19.4 mmol, 1.00 equivalent) was dissolved in DMSO (10 mL), and (1S)-1-[(2S)-1-methylpyrrolidone-2-yl]ethanol (2.76 g, 21.4 mmol, 1.10 equivalent) and DIPEA (6.78 mL, 38.8 mmol, 2.0 equivalent) were added. The solution was stirred overnight at room temperature. The reaction mixture was diluted with DCM and water. The organic phase was separated, evaporated, and the resulting residue was purified by RP chromatography to give F-2a. (HPLC method: A, t) ret = 1.58 / 1.66min; [M+H] = 492).

[1391] Synthesis of isoxazole intermediate G

[1392] Experimental procedures for the synthesis of intermediates G3 and G4

[1393]

[1394] F-3a (1.10 g, 1.91 mmol, 1.0 equivalent) was dissolved in 1,4-dioxane (3 mL) and 50% hydroxylamine aqueous solution (140 µL, 2.29 mmol, 1.2 equivalent) was added. The reaction mixture was stirred overnight at room temperature. After complete conversion of the starting material, the reaction was diluted with saturated NaHCO3 aqueous solution and extracted three times with DCM. The organic phases were combined, dried, filtered, and concentrated under reduced pressure to give the crude product. A crude mixture of G-1a and G-2a (1.00 g, 1.68 mmol, 1.0 equivalent) was dissolved in 1,4-dioxane (6 mL) and 4 M HCl aqueous solution (2.11 mL, 8.44 mmol, 5.0 equivalent) was added. The reaction mixture was stirred at room temperature for 3 h. After complete conversion of the starting material was observed, the reaction was diluted with saturated NaHCO3 aqueous solution and extracted three times with DCM. The organic phases were combined, dried, filtered, and concentrated under reduced pressure to give the crude product. The crude product was dissolved in ACN and water, filtered, and purified by alkaline RP chromatography to obtain the desired product G-3a and the corresponding isoxazole reticulomer G-4a.

[1395] The following intermediates G-3 and G-4 (Table 57) can be obtained from suitable intermediate F in a similar manner. If necessary, the crude product can be purified by chromatography.

[1396] Table 57

[1397]

[1398] Experimental procedures for the synthesis of G-9 and G-10 (Method IV)

[1399]

[1400] G-4b (150 mg, 0.3 mmol, 1.0 equivalent), 2-hydroxythiazole (39.4 mg, 0.39 mmol, 1.30 equivalent), and t-BuONa (2 M in THF, 210 µL, 0.42 mmol, 1.4 equivalent) were dissolved in THF (1.5 mL) and stirred at 80ºC for 18 h. After complete conversion, the reaction mixture was extracted three times with DCM / H2O. The combined organic phases were concentrated under reduced pressure and purified by RP chromatography to give the desired product G-10a.

[1401] The following intermediates G-9 and G-10 (Table 58) can be obtained from suitable intermediates G-3b and G-4b in a similar manner. If necessary, the crude product can be purified by chromatography.

[1402] Table 58

[1403]

[1404] Experimental procedures for synthesizing G-9

[1405]

[1406] G-9h (297 mg, 476 µmol, 1.0 equivalent) was dissolved in DCM (0.91 mL) and trifluoroacetic acid (0.99 mL, 4.76 mmol, 10.0 equivalent). The reaction was stirred at room temperature for 4 h. After complete conversion, the dissolution was removed under reduced pressure. The residue was dissolved in DCM and extracted with saturated aqueous Na₂CO₃ solution. The combined organic phases were dried, filtered, and concentrated under reduced pressure. The residue was purified by RP chromatography to give G-9s.

[1407] The following intermediate G-9 (Table 59) can be obtained from G-9h and G-9i in a similar manner. If necessary, the crude product can be purified by chromatography.

[1408] Table 59

[1409]

[1410] Synthesis of aminocyanothiophene H, I and II

[1411] Experimental procedure for the conversion from G-9 to II

[1412]

[1413] Under an argon atmosphere, malononitrile (20.7 mg, 0.297 mmol, 2.0 equivalent), sulfur (7.15 mg, 0.223 mmol, 1.5 equivalent), and β-alanine (16.7 mg, 0.178 mmol, 1.2 equivalent) were added 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). The reaction mixture was stirred overnight at 80ºC. After complete conversion, the mixture was cooled to room temperature, filtered, and extracted with DCM and a saturated aqueous solution of NaHCO3. The organic phases were combined and concentrated under reduced pressure. The residue was dissolved in acetonitrile and water and purified by alkaline RP chromatography to give the desired product II-1.

[1414] The following compounds II (Table 60) can be obtained from the corresponding ketone G-9 in a similar manner. If necessary, the crude product can be purified by chromatography.

[1415] Table 60

[1416]

[1417] Example 7 Synthesis of compounds according to formula (F)

[1418] The synthesis of compounds according to formula (F) has been described in WO 2021 / 213800.

[1419] Example 8 Assessment of viable protein levels in vitro and in vivo - Materials and Methods

[1420] Cell Titer Glow (CTG) Measurement

[1421] Cell Titer Glow cell viability assays (Promega) were performed on two different cell lines using 384-well plates (VIEWPLATE-384 TC, Perkin Elmer, catalog number 60007480) (Table 1). Eleven different KRASG12C and nine KRASG12D inhibitors were used. Both cell lines were cultured according to the ATCC standard protocol, as shown in Table 61.

[1422] Table 61. Cell Culture Conditions

[1423]

[1424] NCI-H358 and SW1990 cell lines were seeded triplicate at a density of 500 cells / well in 40 µL of total culture medium in 384-well plates. The following day, cells were treated with KRASG12D and KRASG12C inhibitors for 120 hours, starting at 3 µM and subsequently diluted 1:3. On day 5, CTG reagent was added to each well, and luminescence was measured at 490 nm using an Enspire spectrophotometer.

[1425] IC50 values ​​were measured using the internal BI statistical program MegaLab. Based on the results, compounds with high IC50 values ​​(over 100 nM for G12C inhibitors and over 300 nM for G12D inhibitors) were excluded. The remaining compounds were then tested using a human survival protein ELISA kit (Abcam, ab183361).

[1426] Human survival protein ELISA assay

[1427] Cells were grown to 90% confluence in T175 flasks. H358 cells were treated with 100 nM of G12C inhibitor and SW1990 cells with 300 nM of 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 viability protein ELISA kit protocol.

[1428] Exosome extraction

[1429] Exosomes were isolated from cell culture medium using the exoEasy Maxi kit (QIAGEN, 76064) as described in the manufacturer's instructions. In brief, the cell culture supernatant was first filtered through a 0.8 µm filter (Sartorius Minisart NML, catalog number 16592). Then, 1 volume of the filtered cell culture supernatant was mixed with 1 volume of buffer XBP. The mixture was then added to an exoEasy centrifuge column and centrifuged. The column was subsequently washed, and exosomes were eluted in 400 µL of buffer XE.

[1430] RNA extraction from tumors

[1431] Frozen tumors from in vivo studies were surgically cut to approximately 5x5 mm size. The tumors were then transferred to 2 ml Eppendorf tubes containing one steel ball. 1 mL of Trizol (Quiazol #79306 200 ml Qiagen) was added, and each sample was homogenized with tissueLyser II (Qiagen). The tubes were centrifuged, and the supernatant was transferred to new tubes. gDNA elimination solution and bromo-3-chloropropane were added to each sample. The samples were centrifuged again, and RNA was subsequently isolated using the RNeasy MiniKit column system ((250) #74106 Qiagen) following the manufacturer's instructions. In short, after centrifugation, the aqueous phase was mixed with 70% ethanol at a 1:1 ratio and added to the provided column. The column was centrifuged and washed several times as recommended. RNA was eluted in 40 µl of nuclease-free H2O. RNA content was measured using QIAxpert Slide - 40(25) #990700-RNeasy modus, and RIN values ​​were established on Tape Station (Agilent). The isolated RNA was then further processed for sequencing.

[1432] RNA extraction from exosomes

[1433] 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 from each group of mice was pooled, filtered, and conjugated with XBP buffer at a 1:1 ratio. The mixture was then added to the provided 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. Two volumes of 100% ethanol were then added to the tube and pipetteed onto an RNeasy MinElute centrifuge column. After centrifugation, the column was washed, and RNA was eluted in 14 μl of nuclease-free H2O. RNA was measured using QIAxpert slides and TapeStation, as with tumors, and subsequently sequenced.

[1434] MSD® 96-well S-PLEX Survival Protein Assay for Plasma Sample Analysis

[1435] Plasma samples from mouse efficacy and biomarker studies or human plasma samples were centrifuged at 10,000 rcf for 5 minutes and then tested at a 2-fold dilution in the MSD® 96-well S-PLEX assay for detecting human survival proteins according to the manufacturer's instructions. In short, (1.) the assay was assembled by washing the plate and incubating with a coating solution containing specific survival protein capture antibodies at room temperature with shaking for 1 hour. After the washing step, blocking solution was added, followed by calibrators and sample solutions. The assay was incubated with shaking at room temperature for 16–18 hours. The next day, the plate was washed and incubated with (2.) TURBO-BOOST solution containing specific survival protein capture antibodies on a shaker at room temperature for 1 hour. After another washing step, (3.) S-PLEX enhancement solution was added with shaking at room temperature for 30 minutes. The plate was then washed again and incubated with (4.) S-PLEX detection solution at 27ºC with shaking for 1 hour. Finally, (5.) the plate was read on the MSD instrument after the washing step and the addition of MSDGOLD read buffer B. (6.) Use MSD Discovery software for data analysis and visualize the data in GraphPad Prism.

[1436] MSD® 96-well S-PLEX Survival Protein Assay for In Vitro Laboratory Analysis

[1437] SNU1196 cells were grown to 90% confluence in T175 flasks. SNU1196 cells were treated with 11 different GDPi inhibitors (500 nM). Exosome-depleted FBS was added to the culture medium (Gibco, A2720801). Cell culture supernatant was collected 72 h post-treatment, and cells were harvested using the cell lysis buffer provided in the Abcam Human Survival Protein ELISA Kit protocol. Exosomes were isolated from the supernatant using the exoEasy Maxi kit (QIAGEN, 76064), and protein concentrations of isolated exosomes and cell lysates were determined by the Quick Start™ Bradford protein assay (Bio-Rad, 500-0202). Survival protein levels were then analyzed using the MSD® 96-well S-PLEX assay as described above.

[1438] Human plasma samples

[1439] Human plasma samples from various indications were analyzed to assess differences in median baseline survival protein levels and inter-subject biological variability. Plasma samples from healthy volunteers were also obtained. Plasma samples from CRC patients (n = 21) were purchased from a commercial supplier (BioIVT) or from biobank samples from previous clinical studies conducted under a biomarker research proposal (2023-brp-0004). PDAC plasma samples (n = 4) were purchased from a commercial supplier (BioIVT). NSCLC serum samples (n = 31) were residual samples from completed clinical studies (1280.16).

[1440] Example 9 Assessment of viable protein levels in vitro and in vivo - result

[1441] Example 9.1 Survival proteins as biomarkers

[1442] The expression of survival proteins in tumorigenic cells and healthy cells was analyzed, such as... Figure 1A As shown, survival proteins were detected in exosomes released from the pancreatic adenocarcinoma epithelial cell line HPAC, but not in exosomes from healthy volunteers. Additionally, plasma survival protein levels were 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. Results showed that the cancer population exhibited approximately 2.5-fold (median) higher baseline survival protein levels compared to healthy volunteers. Figure 1B Therefore, survival proteins were upregulated in cancer patients compared to healthy controls. Furthermore, CRC patients in the placebo group of the nintedanib trial (from a biobank sample of 1199.52) showed stable or elevated survival protein levels, suggesting a possible correlation between survival proteins and disease progression. Figure 1C ).

[1443] These findings are consistent with earlier published data (Chang et al. 2021), which showed the expression of survival proteins in tumors, and more importantly, high levels of survival proteins were associated with therapy resistance.

[1444] To further analyze the expression patterns of survival proteins after treatment with various anti-KRAS drugs, an in vivo NSCLCCDX model (a xenograft model derived from the non-small cell lung cancer HCC461 cell line) was used. It was found that survival proteins were significantly downregulated in a dose-dependent manner. Figure 2Therefore, in vivo biomarker studies showed that treatment with the KRASG12D inhibitor compound G12D-cpd#2 (see Table 1) for 3 days daily resulted in a dose-dependent downregulation of the surviving protein.

[1445] The levels of BIRC5-encoded survival protein were further tested by ELISA in GP2D and HPAC (G12D mutant) cells treated with escalating doses of KRASG12D inhibitor for 2 hours (top inset) and 24 hours (bottom inset). Figure 3 Survival protein expression decreased at 24 hours rather than 2 hours at IC50 doses (8 nM for GP2D and 46 nM for HPAC), suggesting that survival protein may be a late biomarker of the KRAS response.

[1446] The inventors further demonstrated that, using 100 nM ( Figures 4-6 ) or 300 nM ( Figures 7-9 11 different KRASG12C ( Figures 4-6 ) or 9 different KRASG12D inhibitors ( Figures 7-9 After treatment, NCI-H358 cells ( Figures 4-6 ) and SW1990 cells ( Figures 7-9 Cell lysates ( Figure 4 , Figure 7 ), culture medium ( Figure 5 , Figure 8 ) and exosomes ( Figure 6 , Figure 9 The survival proteins in ) were downregulated respectively ( Figures 4-9 ).

[1447] A downregulation of survival proteins was also observed after treating PC9 YMVA-5 cell line adenocarcinomas 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 experi...

Claims

1. A method for determining the responsiveness of a cancer patient to treatment with a compound that inhibits KRAS protein or a KRAS protein mutant, the method comprising: - Prior to treatment with the compound, the level of viable proteins in a first sample obtained from the patient was measured. -Measure the level of viable proteins in a second sample obtained from the patient during or after treatment with the compound. - Compare the level of surviving proteins in the second sample with the level of surviving proteins in the first sample. When the level of the surviving protein in the second sample is lower than that in the first sample, it is determined that the patient has responded to treatment with the compound.

2. A method for determining the responsiveness of a cancer patient to treatment with a compound that inhibits the interaction between MDM2 and p53, the method comprising: - Prior to treatment with the compound, the level of viable proteins in a first sample obtained from the patient was measured. -Measure the level of viable proteins in a second sample obtained from the patient during or after treatment with the compound. - Compare the level of surviving proteins in the second sample with the level of surviving proteins in the first sample. When the level of the surviving protein in the second sample is lower than that in the first sample, it is determined that the patient has responded to treatment with the compound.

3. The method according to claim 1 or 2, wherein one or more of the first sample and / or the second sample are one or more blood, plasma or serum samples.

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

5. The method according to any one of claims 1-4, wherein a protein blotting, ELISA, RIA, FACS, and MSD method is used. ® S-PLEX technology is used to measure the level of viable proteins through viable protein-specific assays.

6. The method according to any one of claims 1-5, wherein the cancer is a KRAS-dependent cancer, preferably selected from 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 3-6, wherein the compound that inhibits the KRAS protein or a KRAS protein mutant is selected from KRAS (G12C) inhibitors or degraders, KRAS (G12D) inhibitors or degraders, GDP-KRAS inhibitors or degraders, and HER2 inhibitors or degraders.

8. The method according to any one of claims 1 and 3-7, wherein the KRAS (G12C) inhibitor or degrader is selected from: sotorasidib (AMG510), adagraxib (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 3-7, wherein the KRAS (G12D) inhibitor or degrader is selected from 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 3-7, wherein the GDP-KRAS inhibitor or degrader is selected from 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 3-7, wherein the HER2 inhibitor is a compound according to formula F, preferably wherein the compound is HER2-cpd#1.

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

13. A compound for use in treating cancer patients that inhibits KRAS protein or KRAS protein mutants. i) The KRAS inhibitor is selected from KRAS (G12C) inhibitors or degraders, KRAS (G12D) inhibitors or degraders, GDP-KRAS inhibitors or degraders, and HER2 inhibitors or degraders; and ii) wherein the patient has been determined to be responsive to treatment with the compound according to the method according to any one of claims 1 to 11.

14. 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; and ii) wherein the patient has been determined to be responsive to treatment with the compound according to the method according to claim 12.

15. A method for determining whether a compound that inhibits KRAS protein or a KRAS protein mutant, or a compound that inhibits the interaction between MDM2 and p53, is effective in said treatment of cancer and / or in monitoring the response of a cancer patient to said treatment, said method comprising at least the following steps: - Provide a first sample from the patient. - Measure the level of viable proteins in the first sample from the patient. - Administer the compound that inhibits KRAS protein or KRAS protein mutants to the patient, then - Provide a second sample from the patient. - Measure the level of viable proteins in the second sample. - Compare the levels of viable proteins measured in the first and second samples, and -Optionally repeat steps four through six. The decreased level of surviving proteins in the second sample indicates an effective response.

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

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

18. Use of the survival protein 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 level of the survival protein in the following samples is determined: a sample obtained from the patient prior to the treatment and at least one sample obtained from the patient after treatment with the compound that inhibits the interaction between MDM2 and p53, and wherein a decrease in the level of the survival protein after treatment with the compound that inhibits the interaction between MDM2 and p53 indicates that the compound has the ability to treat the cancer.

20. A multi-component kit comprising means for determining the level of viable proteins in a sample provided from a patient with cancer, preferably KRAS-dependent cancer, and instructions on how to perform the method according to any one of claims 1-12 and 15.

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