WO2024158242A1 - Compound for inhibiting kras g12d mutation, and composition for preventing or treating cancer disease, comprising same as active ingredient - Google Patents

Abstract

The present invention relates to: a compound, represented by chemical formula 1, for inhibiting cell proliferation of a KRAS G12D mutation; and a pharmaceutical composition for preventing or treating cancer disease, comprising same as an active ingredient. Therefore, the present invention can exhibit significantly superior effects in targeted treatment on cancer cell lines of colon cancer, lung cancer and the like, and on KRAS G12D mutations and KRAS mutant cells thereof.

Description

Compound for inhibiting KRAS G12D mutation and composition for preventing or treating cancer disease containing the same as an active ingredient

The present invention relates to a compound for inhibiting KRAS G12D mutation and a composition for preventing or treating cancer diseases containing the same as an active ingredient. More specifically, it relates to a compound capable of inhibiting cell proliferation by targeting the KRAS G12D mutation and a composition containing the same as an active ingredient. It relates to a composition for preventing or treating cancer disease, comprising:

Cancer is a disease caused by immature, overproliferating cells that contain genetic mutations. According to Statistics Korea, the three leading causes of death in 2021 are cancer, heart disease, and pneumonia, with cancer accounting for 26% of all deaths. This value is increasing by 0.6% every year, leading to an increase in cancer-related mortality (Statistics Korea, 2021). The cancer incidence rate was 214.2 per 100,000 population in 1999 and 445.3 in 2012. The risk of developing cancer is 37.3% for people who survive to the average life expectancy (age 81), 37.5% for men (age 77), and 34.9% for women (age 84).

The main reasons for the increase in cancer-related deaths are believed to be Westernized lifestyles, aging population, smoking, drinking, and obesity. Cancer refers to a phenomenon in which abnormalities occur in the DNA of the cell proliferation control system. This DNA synthesizes cancer-related proteins due to genetic mutations and abnormal active genetic information in the DNA, so it is called a cancer gene (oncogene). Under normal conditions, oncogenes do not induce cancer development but rather regulate proliferation; These genes cause cancer when activated by genetic mutation or abnormality. Mutations in cancer genes can cause cells to lose their ability to inhibit cell growth. Uncontrolled cell proliferation causes cancer. These cells can be moved to other parts of the body through processes such as angiogenesis and metastasis.

RAS mutations occur at a high rate in colon cancer, lung cancer, and pancreatic cancer. RAS is a family of small GTPase proteins that play important roles in cell growth, differentiation, proliferation, and survival. There are three types of RAS genes: HRAS, KRAS, and NRAS. The RAS gene is the most commonly found oncogene in human cancer. KRAS is known to have the highest mutation rate compared to HRAS and NRAS in various cancer types. KRAS is also known to exhibit a unique enzymatic activity that binds GTP in its active state and converts it to GDP, thereby inactivating KRAS. The rate of turnover is generally slow but can be dramatically increased by auxiliary GTPase activating proteins (GAPs). KRAS releases GDP through a guanine nucleotide exchange factor (GEF), like SOS. GTP binding of KRAS causes several residues in the switch I region (residues 30-40) and switch II region (residues 60-70) to bind to the RAS effector protein. These switches can be regulated by GAP and GEF. After KRAS is converted from the inactive switch-off state bound to GDP to the active switch-on state bound to GTP via GEF, signal transduction required for cell differentiation, proliferation, and survival can proceed. Over time, in the presence of GAP, GTP can be converted to GDP and cycle through the ON/OFF state.

However, when KRAS is mutated, GAP binding is disrupted and remains in the GTP state, resulting in an abnormal signaling pathway that helps tumor formation and maintenance. It is known that approximately 85% of cancers with RAS mutations include KRAS mutations, including 45% of colon cancer, 86% of pancreatic cancer, and 25% of lung cancer. KRAS mutations have been reported at glycine 12 (G12), glycine 13 (G13), or glutamine 61. G12 mutations account for approximately 80% of KRAS mutations, including G12D (41%), G12C (28%), and G12C (14%). A high proportion of lung (32%), pancreatic (86%), and colorectal (41%) cancers contain mutations in codon 12 of KRAS. In the case of KRAS G12C mutation in lung cancer, glycine is mutated to cysteine. In the case of KRAS G12D mutation in pancreatic cancer, glycine is mutated to D-aspartic acid. Targeted therapy using epidermal growth factor receptor (EGFR) in patients with KRAS mutations is known to have lower treatment efficacy and lower survival rates than patients without these mutations.

The EGFR family of receptor tyrosine kinases activates RAS by promoting GDP-GTP exchange. Inhibiting EGFR prevents RAS activation, while inhibiting SOS or SHP2 reduces GDP-GTP exchange rate and thus reduces GTP binding to RAS. Because EGFR is upstream of RAS, inactivation of this receptor tyrosine kinase can inhibit RAS activity. Monoclonal antibodies against EGFR, such as cetuximab or panitumumab, can increase overall survival and progression-free survival in patients with wild-type KRAS metastatic colorectal cancer. However, this antibody is not effective against KRAS mutant tumors, especially those with codon 12 or 13 mutations. EGFR tyrosine kinase inhibitors, such as erlotinib and gefitinib, are approved for the treatment of EGFR-mutant non-small cell lung cancer, but have no single effect in KRAS-mutant non-small cell lung cancer. .

Wild-type KRAS colorectal cancer responds to cetuximab treatment. However, metastatic colorectal cancer acquires KRAS mutations that make the cancer resistant to anti-EGFR therapy. Therefore, an inhibitor that directly targets KRAS mutations is needed rather than an anticancer drug that targets EGFR. Additionally, although inhibitors of mutant KRAS G12C in lung cancer are currently being developed and evaluated, inhibitors of KRAS G12D mutations in colon and pancreatic cancer have not been extensively evaluated.

Targeted therapy using epidermal growth factor receptor (EGFR) in cancer patients with KRAS G12D mutation of the present invention selectively proliferates KRAS G12D mutant cells to solve the problem of lower treatment efficacy compared to patients without this mutation. The aim is to provide a pharmaceutical composition for preventing or treating cancer that contains an inhibitory compound and the same as an active ingredient and can be used for targeted treatment against KRAS G12D mutant cells.

According to one aspect of the present invention,

A compound for inhibiting proliferation of KRAS G12D mutant cells represented by the following formula (1) is provided.

[Formula 1]

In Formula 1,

n is the number of repeat units, which is an integer from 1 to 10,

m is the number of repeat units that is an integer from 1 to 10,

X ¹ is an oxygen atom or a sulfur atom,

R ¹ is a substituted or unsubstituted 1C to 30C alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C1 to C30 heterocycloalkyl group, a substituted or unsubstituted C6 to C30 aryl group, or a substituted or It is an unsubstituted C1 to C30 heteroaryl group,

R ² to R ⁴ are each independently a hydrogen atom, a substituted or unsubstituted 1C to 30C alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C1 to C30 heterocycloalkyl group, or a substituted or unsubstituted A C6 to C30 aryl group, or a substituted or unsubstituted C1 to C30 heteroaryl group,

R ⁵ is a substituted or unsubstituted 1C to 30C alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C1 to C30 heterocycloalkyl group, a substituted or unsubstituted C6 to C30 aryl group, or a substituted or It is an unsubstituted C1 to C30 heteroaryl group.

Preferably in Formula 1,

n is the number of repeat units that is an integer from 1 to 4,

m is the number of repeating units that is an integer from 1 to 4,

X ¹ is an oxygen atom,

R ¹ is a 1C to 10C alkyl group,

R ² to R ⁴ are each independently a hydrogen atom or a 1C to 10C alkyl group,

이고, R ⁵ is

ego,

X ² is an oxygen atom or a sulfur atom,

R ⁶ may be a hydrogen atom or a 1C to 10C alkyl group.

More preferably,

The compound represented by Formula 1 may be a compound represented by Formula 2 below.

[Formula 2]

.

According to another aspect of the present invention,

A pharmaceutical composition for preventing or treating cancer disease comprising a compound represented by the following formula (1) or a salt thereof as an active ingredient is provided.

[Formula 1]

In Formula 1,

n is the number of repeat units, which is an integer from 1 to 10,

m is the number of repeat units that is an integer from 1 to 10,

X ¹ is an oxygen atom or a sulfur atom,

R ¹ is a substituted or unsubstituted 1C to 30C alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C1 to C30 heterocycloalkyl group, a substituted or unsubstituted C6 to C30 aryl group, or a substituted or It is an unsubstituted C1 to C30 heteroaryl group,

R ² to R ⁴ are each independently a hydrogen atom, a substituted or unsubstituted 1C to 30C alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C1 to C30 heterocycloalkyl group, or a substituted or unsubstituted A C6 to C30 aryl group, or a substituted or unsubstituted C1 to C30 heteroaryl group,

R ⁵ is a substituted or unsubstituted 1C to 30C alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C1 to C30 heterocycloalkyl group, a substituted or unsubstituted C6 to C30 aryl group, or a substituted or It is an unsubstituted C1 to C30 heteroaryl group.

Preferably in Formula 1,

n is the number of repeat units that is an integer from 1 to 4,

m is the number of repeating units that is an integer from 1 to 4,

X ¹ is an oxygen atom,

R ¹ is a 1C to 10C alkyl group,

R ² to R ⁴ are each independently a hydrogen atom or a 1C to 10C alkyl group,

이고, R ⁵ is

ego,

X ² is an oxygen atom or a sulfur atom,

R ⁶ may be a hydrogen atom or a 1C to 10C alkyl group.

More preferably, the compound represented by Formula 1 may be a compound represented by Formula 2 below.

[Formula 2]

.

The pharmaceutical composition for preventing or treating cancer disease may be used to inhibit proliferation of KRAS G12D mutant cells.

The KRAS G12D mutant cells may be one or more types selected from LIM1215 KRAS G12D, SW48 KRAS G12D, NCI-H1975 KRAS G12D, NCI-H838 KRAS G12D, and LS174T KRAS G12D.

The composition for preventing or treating cancer disease may be for targeted treatment against the KRAS G12D mutant cells.

The composition for preventing or treating cancer disease may be for inhibiting the MAPK signaling pathway.

Inhibition of the MAPK signaling pathway can be performed by inhibiting C-RAF phosphorylation in the RAS-RAF-MEK-ERK pathway.

The composition for preventing or treating cancer disease may be for inhibiting the PI3K signaling pathway.

The inhibition of the PI3K signaling pathway can be performed by inhibiting AKT phosphorylation in the PI3K-AKT-mTOR pathway.

The cancer disease may be any one selected from colon cancer, colon cancer, rectal cancer, lung cancer, melanoma, thyroid cancer, uterine cancer, ovarian cancer, cervix, pancreatic cancer, stomach cancer, and liver cancer.

Since the compound of the present invention selectively inhibits cell proliferation in KRAS G12D mutant cells, it can solve the problem of low efficacy of conventional targeted therapy using epidermal growth factor receptor (EGFR), and the compound of the present invention and its salt are effective. The pharmaceutical composition containing the composition as an ingredient can be used for targeted treatment against KRAS G12D mutant cells.

Figure 1 shows the cell growth measurement results of compounds Q1a to Q11a of Experimental Example 1 on wild-type cells of LIM1215 and KRAS mutant cells.

Figure 2 shows the molecular structure and three-dimensional model structure of the Q2a compound selected in Experimental Example 1.

Figure 3 shows the results of protein thermal transfer (PTS) analysis to confirm whether the Q2a compound of Experimental Example 1 bound to KRAS.

Figure 4 shows the results of confirming whether compound Q2a has specific activity only in the KRAS G12D mutation in Experimental Example 1.

Figure 5 shows the results of comparing the cell growth rate of the wild type and KRAS G12D mutant pair of colon cancer cell line LIM1215 according to Experimental Example 2 when treated with Q2a compound.

Figure 6 shows the results of comparing the cell growth rate of the wild type and KRAS G12D mutant pair of colon cancer cell line SW48 according to Experimental Example 2 when treated with Q2a compound.

Figure 7 shows the results of comparing the cell growth rate of the wild type and KRAS G12D mutant pair of lung cancer cell line NCI-H838 according to Experimental Example 2 when treated with Q2a compound.

Figure 8 shows the results of comparing the cell growth rate of the wild type and KRAS G12D mutant pair of lung cancer cell line NCI-H1975 according to Experimental Example 2 when treated with Q2a compound.

Figure 9 shows the results of colony formation analysis for wild type (WT) LIM1215 cells and KRAS G12D mutant cells in Experimental Example 2.

Figure 10 shows the results of colony formation analysis for wild type (WT) SW48 cells and KRAS G12D mutant cells in Experimental Example 2.

Figure 11 shows the results of colony formation analysis for wild type (WT) and KRAS G12D mutant cells of NCI-H838 cells in Experimental Example 2.

Figure 12 shows the results of colony formation analysis for wild type (WT) and KRAS G12D mutant cells of NCI-H1975 cells in Experimental Example 2.

Figure 13 shows the results of colony formation analysis of LS174T KRAS G12D mutant cells in Experimental Example 2.

Figure 14 shows the results of analysis of the apoptosis effect on wild type (WT) and KRAS G12D mutant cells of LIM1215 cells in Experimental Example 3.

Figure 15 shows the results of analysis of the apoptosis effect on wild type (WT) and KRAS G12D mutant cells of SW48 cells in Experimental Example 3.

Figure 16 shows the results of analysis of the apoptosis effect on wild type (WT) and KRAS G12D mutant cells of NCI-H1975 cells in Experimental Example 3.

Figure 17 shows the results of analysis of the apoptosis effect on wild type (WT) and KRAS G12D mutant cells of NCI-H838 cells in Experimental Example 3.

Figure 18 shows the results of Western blotting of wild type (WT) LIM1215 cells and KRAS G12D mutant cells in Experimental Example 4 when treated with Q2a for 48.72 hours.

Figure 19 shows the results of Western blotting of wild type (WT) SW48 cells and KRAS G12D mutant cells in Experimental Example 4 when treated with Q2a for 48.72 hours.

Figure 20 shows the Western blot results of wild type (WT) and KRAS G12D mutant cells of NCI-H1975 cells in Experimental Example 4 when treated with Q2a for 48 and 72 hours.

Figure 21 shows the results of Western blotting of wild type (WT) and KRAS G12D mutant cells of NCI-H838 cells in Experimental Example 4 when treated with Q2a for 48.72 hours.

Figure 22 shows the results of Western blotting of LIM1215 and SW48 cells in Experimental Example 4 when wild type (WT) and KRAS G12D mutant cells were treated with Q2a for 24 hours.

Figure 23 shows the results of Western blotting of wild type (WT) and KRAS G12D mutant cells of NCI-H1975 and NCI-H838 cells in Experimental Example 4 when treated with Q2a for 24 hours.

Figure 24 is a heatmap according to microarray analysis in Experimental Example 5.

Figure 25 shows the results of Western blot analysis in Experimental Example 5.

Figure 26 shows the change in tumor size over time according to Q2a administration in mice administered LIM1215 wild-type cells and KRAS G12D mutant cells in Experimental Example 5.

Figure 27 is a graph of mouse body weight change measured while administering compound Q2a of Experimental Example 5.

Figure 28 is a photo comparing the size of the tumor after sacrificing the mouse and removing the tumor 21 days after the treatment in Experimental Example 5.

Figure 29 shows the results of measuring the weight of the tumor before and after treatment according to Experimental Example 5.

Figure 30 shows the mechanism of cancer cell inhibition according to Q2a administration in the KRAS pathway.

Since the present invention can be modified in various ways and can have various embodiments, specific embodiments will be illustrated and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all transformations, equivalents, and substitutes included in the spirit and technical scope of the present invention. In describing the present invention, if it is determined that a detailed description of related known technologies may obscure the gist of the present invention, the detailed description will be omitted.

The term “substituted” means that at least one hydrogen atom is deuterium, C1 to C30 alkyl group, C3 to C30 cycloalkyl group, C2 to C30 heterocycloalkyl group, C1 to C30 halogenated alkyl group, C6 to C30 aryl group, C1 to C30 heteroaryl group, C1 to C30 alkoxy group, C3 to C30 cycloalkoxy group, C1 to C30 heterocycloalkoxy group, C2 to C30 alkenyl group, C2 to C30 alkynyl group, C6 to C30 aryloxy group, C1 to C30 heteroaryloxy group, silyl oxide Group (-OSiH ₃ ), -OSiR ¹ H ₂ (R ¹ is a C1 to C30 alkyl group or C6 to C30 aryl group), -OSiR ¹ R ² H (R ¹ and R ² are each independently a C1 to C30 alkyl group or C6 to C30 aryl group), -OSiR ¹ R ² R ³ , (R ¹ , R ² , and R ³ are each independently a C1 to C30 alkyl group or a C6 to C30 aryl group), C1 to C30 acyl group, C2 to C30 acyl Oxy group, C2 to C30 heteroaryloxy group, C1 to C30 sulfonyl group, C1 to C30 alkylthiol group, C3 to C30 cycloalkylthiol group, C1 to C30 heterocycloalkylthiol group, C6 to C30 arylthiol group, C1 to C30 heteroarylthiol group, C1 to C30 phosphate amide group, silyl group (SiR ¹ R ² R ³ ₎ (R ¹ , R ² , and R ³ are each independently a hydrogen atom, a C1 to C30 alkyl group, or a C6 to C30 aryl group ), amine group (-NRR') (wherein R and R' are each independently a substituent selected from the group consisting of a hydrogen atom, C1 to C30 alkyl group, and C6 to C30 aryl group), carboxyl group, halogen group, It means substituted with a substituent selected from the group consisting of cyano group, nitro group, azo group, and hydroxy group.

Additionally, two adjacent substituents among the above substituents may be fused to form a saturated or unsaturated ring.

In addition, the carbon number range of the alkyl group or aryl group in the “substituted or unsubstituted C1 to C30 alkyl group” or “substituted or unsubstituted C6 to C30 aryl group” does not take into account the portion on which the substituent is substituted and is not substituted. It refers to the total number of carbon atoms constituting the alkyl portion or aryl portion when viewed as being formed. For example, a phenyl group substituted with a butyl group at the para position corresponds to an aryl group with 6 carbon atoms substituted with a butyl group with 4 carbon atoms.

As used herein, unless otherwise defined, “hetero” means that one functional group contains 1 to 4 hetero atoms selected from the group consisting of N, O, S, and P, and the remainder is carbon.

In this specification, “hydrogen” means single hydrogen, double hydrogen, or tritium hydrogen, unless otherwise defined.

In this specification, “alkyl group” means an aliphatic hydrocarbon group, unless otherwise defined.

The alkyl group may be a “saturated alkyl group” that does not contain any double or triple bonds.

The alkyl group may be an “unsaturated alkyl group” containing at least one double or triple bond.

Alkyl groups, whether saturated or unsaturated, may be branched, straight-chain, or cyclic.

The alkyl group may be a C1 to C30 alkyl group. More specifically, it may be a C1 to C20 alkyl group, a C1 to C10 alkyl group, or a C1 to C4 alkyl group.

For example, C1 to C4 alkyl groups have 1 to 4 carbon atoms in the alkyl chain, i.e., the alkyl chain is methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl and t-butyl. Indicates selection from a group consisting of

For specific examples, the alkyl group includes methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, t-butyl group, pentyl group, hexyl group, ethenyl group, propenyl group, butenyl group, cyclopropyl group, and cyclopropyl group. It means butyl group, cyclopentyl group, cyclohexyl group, etc.

“Cycloalkyl group” includes monocyclic or fused-ring polycyclic (i.e., rings splitting adjacent pairs of carbon atoms) functional groups.

“Heterocycloalkyl group” means that the cycloalkyl group contains 1 to 4 heteroatoms selected from the group consisting of N, O, S, and P, and the remainder is carbon. When the heterocycloalkyl group is a fused ring, at least one ring of the fused ring may include 1 to 4 heteroatoms.

“Aryl groups” include monocyclic or fused ring polycyclic (i.e., rings splitting adjacent pairs of carbon atoms) functional groups.

“Heteroaryl group” means that the aryl group contains 1 to 4 heteroatoms selected from the group consisting of N, O, S, and P, and the remainder is carbon. When the heteroaryl group is a fused ring, at least one ring of the fused ring may include 1 to 4 heteroatoms.

In aryl groups and heteroaryl groups, the number of ring atoms is the sum of the number of carbon atoms and the number of non-carbon atoms.

The present invention provides a compound for inhibiting proliferation of KRAS G12D mutant cells represented by the following formula (1).

[Formula 1]

In Formula 1,

n is the number of repeating units, which is an integer from 1 to 10,

m is the number of repeat units that is an integer from 1 to 10,

X ¹ is an oxygen atom or a sulfur atom,

R ¹ is a substituted or unsubstituted 1C to 30C alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C1 to C30 heterocycloalkyl group, a substituted or unsubstituted C6 to C30 aryl group, or a substituted or It is an unsubstituted C1 to C30 heteroaryl group,

R ² to R ⁴ are each independently a hydrogen atom, a substituted or unsubstituted 1C to 30C alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C1 to C30 heterocycloalkyl group, or a substituted or unsubstituted A C6 to C30 aryl group, or a substituted or unsubstituted C1 to C30 heteroaryl group,

R ⁵ is a substituted or unsubstituted 1C to 30C alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C1 to C30 heterocycloalkyl group, a substituted or unsubstituted C6 to C30 aryl group, or a substituted or It is an unsubstituted C1 to C30 heteroaryl group.

Preferably, in Formula 1,

n is the number of repeat units that is an integer from 1 to 4,

m is the number of repeating units that is an integer from 1 to 4,

X ¹ is an oxygen atom,

R ¹ is a 1C to 10C alkyl group,

R ² to R ⁴ are each independently a hydrogen atom or a 1C to 10C alkyl group,

이고, R ⁵ is

ego,

X ² is an oxygen atom or a sulfur atom,

R ⁶ may be a hydrogen atom or a 1C to 10C alkyl group.

More preferably, the compound represented by Formula 1 may be a compound represented by Formula 2 below.

[Formula 2]

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer disease, comprising a compound represented by the following formula (1) or a salt thereof as an active ingredient.

[Formula 1]

In Formula 1,

n is the number of repeating units, which is an integer from 1 to 10,

m is the number of repeat units that is an integer from 1 to 10,

X ¹ is an oxygen atom or a sulfur atom,

R ¹ is a substituted or unsubstituted 1C to 30C alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C1 to C30 heterocycloalkyl group, a substituted or unsubstituted C6 to C30 aryl group, or a substituted or It is an unsubstituted C1 to C30 heteroaryl group,

R ² to R ⁴ are each independently a hydrogen atom, a substituted or unsubstituted 1C to 30C alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C1 to C30 heterocycloalkyl group, or a substituted or unsubstituted A C6 to C30 aryl group, or a substituted or unsubstituted C1 to C30 heteroaryl group,

R ⁵ is a substituted or unsubstituted 1C to 30C alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C1 to C30 heterocycloalkyl group, a substituted or unsubstituted C6 to C30 aryl group, or a substituted or It is an unsubstituted C1 to C30 heteroaryl group.

Preferably, in Formula 1,

n is the number of repeat units that is an integer from 1 to 4,

m is the number of repeating units that is an integer from 1 to 4,

X ¹ is an oxygen atom,

R ¹ is a 1C to 10C alkyl group,

R ² to R ⁴ are each independently a hydrogen atom or a 1C to 10C alkyl group,

이고, R ⁵ is

ego,

X ² is an oxygen atom or a sulfur atom,

R ⁶ may be a hydrogen atom or a 1C to 10C alkyl group.

More preferably, the compound represented by Formula 1 may be a compound represented by Formula 2 below.

[Formula 2]

Another example provides a method for preventing and/or treating cancer disease, comprising administering a pharmaceutically effective amount of the compound represented by Formula 1 or a salt thereof to a subject in need thereof. The method may further include the step of identifying a subject in need of prevention and/or treatment of a cancer disease.

As used herein, the terms “subject,” “patient,” “individual,” and “host” and their variants are interchangeable and refer to any mammalian subject to which a compound or salt or composition thereof described herein is administered. Non-limiting examples include humans, livestock (e.g. dogs, cats, etc.), farm animals (e.g. cattle, sheep, pigs, horses, etc.), and laboratory animals (e.g. monkeys, rats, etc.) in need of diagnosis, treatment or treatment. , mice, rabbits, guinea pigs, etc.), especially humans. The methods described herein are applicable to both human prophylactic or therapeutic and veterinary applications.

As used herein, the phrase “subject in need” includes subjects such as mammalian subjects who would benefit from administration of the compositions described herein.

Another example provides the use of the compound represented by Formula 1 or a salt thereof for the treatment and/or prevention of cancer diseases.

The pharmaceutical composition for preventing or treating cancer disease may be used to inhibit proliferation of KRAS G12D mutant cells.

The KRAS G12D mutant cells may be one or more types selected from LIM1215 KRAS G12D, SW48 KRAS G12D, NCI-H1975 KRAS G12D, NCI-H838 KRAS G12D, and LS174T KRAS G12D. Here, LIM1215 KRAS G12D, SW48 KRAS G12D, and LS174T KRAS G12D are colon cancer cell lines, and NCI-H1975 KRAS G12D and NCI-H838 KRAS G12D are lung cancer cell lines.

The composition for preventing or treating cancer disease can be used for targeted treatment against the KRAS G12D mutant cells. This is because, although it has little cytostatic effect on KRAS wild-type cells, it can selectively exhibit anti-proliferation effect on KRAS G12D mutant cells.

The composition for preventing or treating cancer diseases can be used to inhibit the MAPK signaling pathway.

Inhibition of the MAPK signaling pathway can be performed by inhibiting C-RAF phosphorylation in the RAS-RAF-MEK-ERK pathway.

Additionally, the composition for preventing or treating cancer diseases can be used to inhibit the PI3K signaling pathway.

The inhibition of the PI3K signaling pathway can be performed by inhibiting AKT phosphorylation in the PI3K-AKT-mTOR pathway.

The cancer disease may be any one selected from colon cancer, colon cancer, rectal cancer, lung cancer, melanoma, thyroid cancer, uterine cancer, ovarian cancer, cervix, pancreas cancer, stomach cancer, and liver cancer, but the scope of the present invention is not limited thereto and KRAS Any carcinoma that can cause a G12D mutation is possible.

Meanwhile, in this specification, the term ‘including as an active ingredient’ means containing a sufficient amount to achieve the efficacy or activity of the compound represented by Formula 1 or a salt thereof. In one embodiment of the present invention, the compound represented by Formula 1 or its salt in the composition of the present invention is, for example, 0.001 mg/kg or more, preferably 0.1 mg/kg or more, more preferably 10 mg/kg. It may contain more than kg, more preferably more than 100 mg/kg, even more preferably more than 250 mg/kg, and most preferably more than 1 g/kg.

The quantitative lower limit and/or upper limit of the compound represented by Formula 1 or its salt can be selected within an appropriate range by a person skilled in the art.

As used herein, the term "salt", "pharmaceutical salt" or "pharmaceutically acceptable salt" refers to a compound that does not cause significant irritation to the organism to which the compound is administered and does not impair the biological activity and physical properties of the compound. It means dosage form. The pharmaceutical salts include the compounds of the present invention, inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid, sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, and p-toluenesulfonic acid, tartaric acid, formic acid, citric acid, acetic acid, and trichloroacid. It can be obtained by reacting with organic carboxylic acids such as loacetic acid, trifluoroacetic acid, capric acid, isobutanoic acid, malonic acid, succinic acid, phthalic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicylic acid, etc. In addition, the compound of the present invention can be reacted with a base to produce salts such as alkali metal salts such as ammonium salts, sodium or potassium salts, alkaline earth metal salts such as calcium or magnesium salts, dicyclohexylamine, and N-methyl-D-glue. It may be obtained by forming salts of organic bases such as carmine, tris(hydroxymethyl) methylamine, and amino acid salts such as arginine and lysine, but is not limited thereto.

The pharmaceutical composition of the present invention can be prepared using pharmaceutically suitable and physiologically acceptable auxiliaries in addition to the active ingredients, and the auxiliaries include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, and lubricants. Agents or flavoring agents can be used.

For administration, the pharmaceutical composition may be preferably formulated as a pharmaceutical composition containing one or more pharmaceutically acceptable carriers in addition to the active ingredients described above.

The pharmaceutical composition may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops, or injectable solutions. For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, etc. Additionally, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tracacance or sodium oleate, sodium stearate, magnesium stearate, sodium Includes benzoate, sodium acetate, sodium chloride, etc. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, etc.

Acceptable pharmaceutical carriers in compositions formulated as liquid solutions include those that are sterile and biocompatible, such as saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and these. One or more of the ingredients can be mixed and used, and other common additives such as antioxidants, buffers, and bacteriostatic agents can be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc., and is preferably parenteral administration. .

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity, and is usually A skilled doctor can easily determine and prescribe an effective dosage for desired treatment or prevention. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-10 g/kg.

The pharmaceutical composition of the present invention is manufactured in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person skilled in the art. Alternatively, it can be manufactured by placing it in a multi-capacity container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet, or capsule, and may additionally contain a dispersant or stabilizer.

Hereinafter, the present invention will be described with reference to specific examples.

[Example]

Ingredients Preparation

The small molecule quinazoline (Q2a) compound was obtained from KIST (Korea Institute of Science and Technology). KRAS mutant cells were purchased from Horizon Discovery. PARP, C-RAF, P-CRAF, AKT, P-AKT, ERK, P-ERK, HSP90, C-MYC, NOTCH1, and β-actin antibodies were purchased from Cell Signaling Technology. Secondary antibodies anti-rabbit Ig horseradish peroxidase (HRP) and anti-mouse Ig horseradish peroxidase (HRP) were purchased from GeneTex, and EZ-Cytox for cell proliferation measurement was purchased from Dogen Bio. ANNEXIN V-FITC apoptosis detection kit for measuring apoptosis was purchased from Koma Biotech. Protein Thermal Shift™ dye kit and Raf-1-RBD beads for pulling down active RAS and detection kit were purchased from Thermo Fisher Scientific and Cell Signaling Technology.

Below, the NMR analysis results of compound Q2a represented by Formula 2 are shown.

[Formula 2]

^1H NMR (500 MHz, MeOD) δ 8.04 (dt, J = 8.4, 1.8 Hz, 1H), 7.87 (s, 5H), 7.68 (t, J = 7.7 Hz, 1H), 7.32 (t, J = 7.8 Hz, 1H), 7.24 - 7.14 (m, 2H), 6.90 (d, J = 8.2 Hz, 1H), 6.81 (t, J = 7.4 Hz, 1H), 6.09 (d, J = 5.9 Hz, 2H), 4.77 (s, 2H), 4.72 (s, 1H), 4.70 (s, 4H), 3.78 (d, J = 1.0 Hz, 3H), 3.62 (p, J = 6.7 Hz, 2H), 3.39 - 3.33 (m , 2H), 3.16 - 3.08 (m, 2H), 2.56 (s, 1H).

¹³ C NMR (126 MHz, MeOD) δ 167.28, 163.52, 160.39, 157.57, 153.40, 139.03, 135.09, 130.50, 129.11, 128.72, 125.67, 124.84, 124.61, 123.42, 120.11, 116.47, 110.30, 54.48, 42.43, 35.59, 30.27.

Cell lines and cell culture

Wild-type cancer cells and KRAS G12D mutant cancer cell lines purchased from Horizon Discovery were used. Colon cell lines LIM1215 and SW48, and non-small cell lung cancer (NSCLC) cell lines NCI-H1975 and NCI-H838 were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin. Additionally, COLO205, LOVO, HCT8, HCT15, SW480, and SW620 were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin. All cells were cultured in a 5% CO ₂ incubator at 37°C.

Protein analysis by Western blot

Cells were seeded in a 60 mm ² cell culture dish overnight in a 5% CO ₂ incubator at 37°C. Compounds were treated at concentrations of 0 μM, 1 μM, 5 μM, and 10 μM. After obtaining the cells using trypsin-EDTA, the cells were centrifuged at 1500 rpm for 5 minutes to remove the supernatant, and washed with cold PBS for 5 minutes at 1500 rpm. After removing the supernatant, cells were lysed using 2x sample buffer (100 μl). After heating for 5 to 7 minutes using a heating block, it was quantified using a BCA protein analysis kit (Thermal Fisher Scientific). Equal amounts of protein samples were separated by SDS-PAGE and transferred to PVDF membranes using an electroblotting device (Bio-Rad). Membranes were blocked in blocking buffer (TBS-T + 5% skim milk) for 1 h and incubated with primary antibodies overnight at 4°C. The membrane was washed with TBS-T and incubated with horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature. Protein expression was visualized using the ECL kit (Amersham Biosciences).

Cell proliferation analysis

KRAS G12D mutant cancer cells and wild-type cancer cells were seeded in a 96-well plate at 5 Х 10 ³ to 1 Х 10 ⁴ cells per well and grown overnight. Afterwards, the cells were treated with quinazoline derivative Q2a compound at concentrations of 0μM, 1μM, 5μM, and 10μM. After compound treatment, WST analysis using Ez-cytox was performed to measure cell proliferation. After treatment with 10% Ez-cytox, the reaction was performed in a 5% CO ₂ incubator at 37°C for 2 hours. Optical density (OD) values were determined by measuring absorbance at 490-500 nm using a microplate reader.

Cloning analysis

Cells were seeded in 6-well plates at 1 Х 10 ³ cells per well and grown overnight. Cells were treated with Q2a compound at concentrations of 0 μM, 1 μM, 5 μM, and 10 μM, and the medium was replaced with new medium containing Q2a compound every 3 days. After 21 days of Q2a compound treatment, the medium was removed and washed with cold PBS. Cell colonies were fixed with methanol for 10 min and stained with crystal violet (0.1% in 20% methanol) for 20 min. After staining, it was carefully washed with distilled water. After at least three independent experiments, the number of surviving colonies was calculated, and the number of surviving colonies (SF) was calculated according to Equation 1 below.

[Equation 1]

SF = number of colonies treated/number of control colonies

Apoptosis analysis

Fluorescence-activated cell sorting (FACS) was performed to measure apoptosis in KRAS G12D mutant cells upon treatment with Q2a compound. KRAS G12D mutant cancer cells and wild-type cancer cells were seeded at 1 Х 10 ⁵ to 1.5 Х 10 ⁵ cells per well in a 6-well plate and grown overnight. Afterwards, the cells were treated with Q2a compound at concentrations of 0 μM, 1 μM, 5 μM, and 10 μM, and performed according to the kit protocol. Cells were obtained using trypsin EDTA, centrifuged at 1000 xg for 5 minutes to remove the supernatant, and washed with cold PBS at 1000 xg for 5 minutes. After removing the supernatant, 500 μl of 1x binding buffer was added and suspended. 1.25㎕ (5㎍/㎖) of Annexin V was added to the cells and stained for 15 minutes at room temperature in the dark. After 15 minutes, it was centrifuged at 1000 xg for 5 minutes and the supernatant was removed. After adding 500 μl of 1×binding buffer and 10 μl of PI, cell death was immediately measured using a flow cytometer (Beckman cytoflex). The percentage of viable or dead cells was analyzed by fluorescence-activated cell sorter (FACS) analysis.

Measurement of intracellular KRAS activity

The LIM1215 KRAS G12D mutant cell line was seeded in a 60 mm ² cell culture dish overnight in a 5% CO ₂ incubator at 37°C. Q2a compound was treated at concentrations of 0μM, 1μM, 5μM, and 10μM. Cellular activity of KRAS protein was measured using the active RAS pull-down and detection kit according to the manufacturer's instructions (Cell Signaling Technology). GTP-bound form of KRAS was extracted from cell lysates using GST-Raf-1 RBD beads. After washing, loading buffer was added to the sample, boiled for 5 minutes, and then SDS-PAGE and Western blotting using KRAS antibody were performed.

Colon cancer xenograft and treatment

Six-week-old female BALB/C nude mice were purchased from Nara Biotech. 3×10 ⁶ LIM1215 wild type and KRAS G12D mutant cells were administered to the subcutaneous tissue of both flanks of nude mice. Tumors were allowed to grow to an average volume of approximately 100 mm ³ before starting treatment, and mice were treated with compound Q2a by intraperitoneal administration (ip). During the treatment period, tumor volume (V) was measured with calipers once every 2 days and calculated according to the following equation 2.

[Equation 2]

Tumor volume (V) = ( L × S ² ) / 2 (L is long axis, S is short axis)

Measurement of KRAS binding of Q2a compound through thermal shift analysis

Thermal shift analysis was performed using the Protein Thermal Shift dye kit (Thermo Fisher Scientific). Colon cancer cells, LIM1215 wild type cells and KRAS G12D mutant cells, were treated with Q2a compound for 72 hours, and then protein extraction was performed using 1x lipa buffer. The extracted protein was quantified at 1㎍ through BCA analysis and reacted with the protein using Protein Thermal Shift buffer containing thermal shift dye. Afterwards, Tm (melting point) was measured using real-time PCR.

statistical analysis

All data are expressed as mean ± standard deviation (SD) from at least three independent experiments. Statistical analysis was performed using GraphPad InStat 6 software. For comparison between groups, one-way ANOVA and tukey's post-hoc test methods were used. Additionally, the t-test method was used to evaluate significance between groups. The significance of the p-value was evaluated as *p < 0.05, **p < 0.01, and ***p < 0.001.

[Experimental example]

Experimental Example 1: Screening of small molecules targeting KRAS G12D

To find compounds that react to KRAS mutant cells, a total of 11 compounds from Q1a to Q11a were treated at a concentration of 5 μM each in wild-type cells and KRAS mutant cells of the colon cancer cell line LIM1215, and cell proliferation was measured after 72 hours. The method for measuring cell growth was WST-1 analysis. After 72 hours of compound treatment, Ez-cytox reagent was added, and after 2 hours of reaction, cell growth was measured using a microplate reader, and the results are shown in Figure 1. According to this, most compounds showed an average inhibition of cell proliferation of more than 15% in KRAS mutants compared to the wild type.

Some compounds with excellent cell growth inhibition effects were selected and the degree of cell death was measured again using fluorescence-activated cell sorting (FACS). In FACS, apoptosis was measured using Annexin V and PI (Propidium Iodide) staining 72 hours after treatment with 5 μM of the compound as in WST-1. Among these, the apoptosis effect of Q2a was about 3% higher than that of other compounds, and the protein expression of PARP, an indicator of apoptosis, was also found to increase compared to other compounds. Therefore, compound Q2a was finally selected among 11 compounds and the experiment was conducted. The molecular structure and three-dimensional model structure of the Q2a compound are shown in Figure 2.

Protein thermal shift (PTS) analysis was performed to confirm that the Q2a compound bound to KRAS. 72 hours after treating colon cancer cell lines LIM1215 wild-type cells and KRAS mutant cells each with 5 μM of Q2a compound, Tm (melting point) was measured using a Protein Thermal Shift kit, and the results are shown in Figure 3. According to this, the Tm value of the wild type (WT) control group was 60.81℃, the Tm value of the wild type Q2a compound treatment group was 58.31℃, the Tm value of the KRAS G12D mutant group was 55.41℃, and the Tm value of the KRAS G12D mutant Q2a compound treatment group was 58.31℃. The value was found to increase by about 3.51℃. According to these results, it was confirmed that compound Q2a binds to KRAS.

Thereafter, in order to confirm whether the Q2a compound had specific activity only in the KRAS G12D mutant, mutant cells other than KRAS G12D were treated with the Q2a compound and cell growth was measured, and the results are shown in Figure 4. Specifically, experiments were performed on colon cancer cell lines such as wild-type COLO205, KRAS-G12D mutant LS174T and SNUC2A, KRAS-G12V mutant SW620 and SW480, and KRAS-G13D mutant LOVO and HCT15 cells. After treating each cell with 5 μM of Q2a compound, cell growth was measured using Ez-cytox 48 and 72 hours later. As a result, the wild type KRAS G12V and KRAS G13D mutants did not show any significant effect, but the KRAS G12D mutant cells LS174T and LOVO showed a cell growth inhibitory effect. In other words, it can be confirmed that the Q2a compound exhibits specific activity only in the KRAS G12D mutant.

Experimental Example 2: Analysis of growth inhibition effect of KRAS G12D mutant cells and wild-type colon cancer and lung cancer cells

For the selected Q2a compound, WST-1 analysis was performed to compare the degree of cell growth inhibition following Q2a treatment of a pair of wild-type and KRAS G12D mutant cells in the same cell line.

Q2a compound was administered at various concentrations (0 μM, 1 μM, 5 μM, 10 μM) to wild-type and KRAS G12D mutant pairs of colon cancer cell lines LIM1215 and SW48 and lung cancer cell lines NCI-H838 and NCI-H1975 for 24, 48, and 72 hours. After each treatment, the cell growth inhibition rate was measured, and the results are shown in Figures 5 to 8. After reacting with Ez-cytox for 2 hours, the cell proliferation rate was measured using a microplate reader. As a result, both colon cancer cell lines LIM1214 and SW48 showed a tendency to inhibit cell growth in the KRAS G12D mutant compared to the wild type.

According to Figure 5, LIM1215 cells showed inhibition of cell proliferation compared to the wild type in the 5μM and 10μM treatment groups when treated for 24 and 48 hours, and cell proliferation was observed at all treatment concentrations of 1μM, 5μM, and 10μM when treated for 72 hours. Inhibition appeared.

According to Figure 6, in SW48 cells, when treated for 24 hours, 48 hours, and 72 hours, cell growth was inhibited only in KRAS G12D mutant cells at concentrations of 1 μM, 5 μM, and 10 μM.

Lung cancer cell lines showed a better cell proliferation inhibition effect than colon cancer cell lines. According to Figure 7, in NCI-H838 cells, compared to wild-type cells, KRAS G12D mutant cells showed about 30% inhibition of cell proliferation in the 1 μM concentration group when treated for 48 hours, even when treated for 24 hours and 72 hours. It was confirmed that cell proliferation was inhibited in a concentration-dependent manner.

According to Figure 8, in NCI-H1975 cells, KRAS G12D mutant cells were shown to be inhibited in cell growth in a concentration-dependent manner compared to wild-type cells when treated for 24 hours, 48 hours, and 72 hours.

Meanwhile, the cell proliferation inhibitory effect of Q2a was confirmed through colony formation assay. The results of colony formation analysis for wild type (WT) LIM1215 cells and KRAS G12D mutant cells are shown in Figure 9. According to this, in LIM1215 cells, the number of colonies of KRAS G12D mutant cells was 50% lower than that of the control group.

The results of colony formation analysis for wild type (WT) SW48 cells and KRAS G12D mutant cells are shown in Figure 10. According to this, SW48 KRAS G12D mutant cells also showed a significant decrease in cell proliferation in the Q2a compound treatment group. The number of colonies of KRAS G12D mutant cells was found to be 60% lower than that of the control group, but there was no significant difference in the wild type group.

Meanwhile, in addition to colon cancer, the number of colonies was also found to be significantly reduced in lung cancer cell lines. The results of colony formation analysis for wild type (WT) and KRAS G12D mutant cells of NCI-H838 and NCI-H1975 cells are shown in Figures 11 and 12, respectively. According to this, in NCI-H838 and NCI-H1975 cells, the KRAS G12D mutant group showed a significant decrease in colonies in the Q2a compound treatment group compared to the control group. In NCI-H838 cells, the number of colonies of KRAS G12D mutant cells was found to be less than 50% compared to the control group, and in NCI-H1975 cells, the number of colonies of KRAS G12D mutant cells was found to be 70-50% less than the control group. In contrast, there was no significant difference in the wild type group.

Additionally, the results of colony formation analysis of LS174T KRAS G12D mutant cells, a colon cancer cell line, are shown in Figure 13. According to this, the number of colonies was significantly lower in the Q2a compound group compared to the control group.

Experimental Example 3: Analysis of apoptosis effect of KRAS G12D mutant cells and wild-type colon cancer and lung cancer cells

Fluorescence-activated cell sorting (FACS) was performed to confirm the apoptotic effect in KRAS G12D mutant cells when treated with Q2a compound. Wild-type and KRAS G12D mutant pairs of LIM1215, SW48, NCI-H1975, and NCI-H838 cells were treated with Q2a compound at different concentrations (0 μM, 1 μM, 5 μM, 10 μM) for 24, 48, and 72 h. Apoptosis was measured by Annexin V and PI staining. In colon cancer cell lines LIM1215 and SW48, compared to the control group, apoptosis in KRAS G12D mutant cells was at least 5% and at most 20% in the Q2a compound treatment group. However, in the wild type, there was no significant effect in the Q2a compound treatment group compared to the control group.

The results of analysis of the apoptosis effect on wild type (WT) LIM1215 cells and KRAS G12D mutant cells are shown in Figure 14. According to this, in LIM1215 KRAS G12D mutant cells, the apoptosis rate increased in a concentration-dependent manner for 24, 48, and 72 hours, and at 72 hours, 20% of apoptosis was observed in the 10μM concentration treatment group.

The results of analysis of the apoptosis effect on wild type (WT) SW48 cells and KRAS G12D mutant cells are shown in Figure 15. According to this, it was confirmed that cell death occurred in SW48 KRAS G12D mutant cells when treated for 24 hours, 48 hours, and 72 hours in the Q2a compound treatment group compared to the control group.

In lung cancer cell lines, it was confirmed that the cell death effect was better than that in colon cancer cell lines, as was the cell growth inhibition effect. In NCI-H1975 and NCI-H838 cells, KRAS G12D mutant cells showed a more significant apoptotic effect compared to the wild type, and the apoptotic effect was confirmed to be less than 10% and up to 60% in mutant cells compared to the control group. I was able to.

The results of analysis of the apoptosis effect on wild type (WT) NCI-H1975 cells and KRAS G12D mutant cells are shown in Figure 16. According to this, in NCI-H1975 KRAS G12D cells, the apoptosis effect of the Q2a compound treatment group was found to significantly increase over 24 hours, 48 hours, and 72 hours compared to the control group. In NCI-H1975 KRAS G12D cells, the apoptosis effect of the Q2a compound treatment group was significantly increased over 24, 48, and 72 hours compared to the control group, in the 5μM and 10μM treatment groups for 24 hours and the 1μM treatment group for 48 hours. An apoptosis effect of approximately 20% was observed.

The results of analysis of the apoptosis effect on wild type (WT) NCI-H838 cells and KRAS G12D mutant cells are shown in Figure 17. According to this, NCI-H838 KRAS G12D cells showed the greatest apoptosis effect among all cells used in the experiment. When treated at a concentration of 1 μM for 24 hours, an apoptosis effect of more than 60% was observed compared to the control group.

Experimental Example 4: Analysis of changes in apoptosis and KRAS downstream-related protein expression

(1) Analysis of the effect of reducing apoptosis-related protein expression in KRAS G12D mutant cells

Western blot was performed to confirm the mechanism related to cell death caused by treatment with Q2a compound. Wild type and KRAS G12D mutant pairs of LIM1215, SW48, NCI-H1975, and NCI-H838 cells were treated with Q2a compound at different concentrations (0 μM, 1 μM, 5 μM, 10 μM), and protein extraction was performed after 48 and 72 hours. After loading on 10% SDS-Page, it was transferred to a PVDF membrane, primary and secondary antibodies were attached, and it was detected with an ECL kit.

PARP poly(ADP-ribose) polymerase, a representative marker of apoptosis, is a family of 17 proteins involved in several cellular processes, including stress response, chromatin modification, DNA repair, and apoptosis. This has been confirmed to play a role in the detection and repair of DNA breaks. The presence of cleaved PARP-1 is one of the most commonly used diagnostic tools to detect apoptosis in many cell types.

Cleavage of PARP, a representative marker of apoptosis, occurred in all KRAS G12D mutant cells of LIM1215, SW48, NCI-H1975, and NCI-H838 cells. As a result of confirming apoptosis in protein expression, it was confirmed that Q2a compound induces apoptosis in KRAS G12D mutant.

Figure 18 shows the Western blot results for wild-type (WT) LIM1215 cells and KRAS G12D mutant cells treated with Q2a 48.72 for 72 hours, and Figure 19 shows Q2a 48.72 for wild-type (WT) SW48 cells and KRAS G12D mutant cells. This is the Western blot result upon time processing. According to this, PARP cleavage in LIM1215 and SW48 KRAS G12D mutant cells increased in both the 48-hour and 72-hour treatment groups compared to the wild type.

Figure 20 is a Western blot result for wild-type (WT) NCI-H1975 cells and KRAS G12D mutant cells treated with Q2a 48. 72 hours, and Figure 21 is a Western blot result for wild-type (WT) NCI-H838 cells and KRAS G12D mutant cells. Q2a 48. This is the Western blot result after 72 hours of treatment. According to this, likewise NCI-H1975. PARP cleavage also occurred in NCI-H838 KRAS G12D mutant cells and increased for both 48 and 72 hours compared to the wild type.

(2) Effect of reducing expression of KRAS downstream-related proteins in KRAS G12D mutant cells

It was confirmed whether compound Q2a also affects the inhibition of KRAS downstream activity. Downstream of KRAS are the MAPK pathway proteins C-RAF and ERK and the PI3K pathway protein AKT.

Figure 22 shows the results of Western blotting of wild type (WT) LIM1215 and SW48 cells and KRAS G12D mutant cells treated with Q2a for 24 hours. According to this, colon cancer cells LIM1215 and SW48 showed decreased phosphorylation of the MAPK pathway protein C-RAF from 48 hours, and phosphorylation of AKT, a PI3K pathway protein, was also significantly decreased compared to the control group (see Figures 18 and 19).

For lung cancer cell lines NCI-H1975 and NCI-H838, changes in KRAS downstream proteins in the KRAS G12D mutant at 48 and 72 hours did not differ from phosphorylation of AKT upon 72-hour treatment of NCI-H1975 cells (see Figures 20 and 21 ).

Figure 23 shows the results of Western blotting of wild type (WT) NCI-H1975 and NCI-H838 cells and KRAS G12D mutant cells treated with Q2a for 24 hours. According to this, unlike at 48 and 72 hours, the phosphorylation of the MAPK pathway protein C-RAF decreased at 24 hours, and the phosphorylation of the PI3K pathway protein AKT also decreased significantly compared to the control group.

In NCI-H838 cells, P-CRAF and P-AKT were significantly decreased, and P-ERK was decreased compared to the control group at 1 μM and 5 μM concentrations.

Experimental Example 5: Analysis of gene expression differences between Q2a treatment group and control group in LIM 1215 KRAS G12D cells

Gene expression differences between control and Q2a compound treatment groups were examined using LIM1215 KRAS G12D cells, which showed increased protein expression of cleaved PARP and decreased protein expression of RAS downstream-related proteins PC-RAF, P-AKT, and P-ERK in Western blot. Microarray was performed to confirm. Microarrays were performed at Macrogene.

Figure 24 is a heatmap of three control groups and three groups treated with 5 μM of Q2a compound. According to this, differences in gene expression can be confirmed. When treated with 2a, it was confirmed that the KRAS signal in KRAS mutant cells was decreased, indicating that the Q2a compound targeted KRAS.

Additionally, treatment with Q2a showed a decrease in the expression of MYC and NOTCH, which are oncogenic pathway proteins, in KRAS mutant cells.

Pull down analysis was performed based on the microarray results. RAS activity was measured using Raf-1-RBD, and expression of NOTCH1 and C-MYC proteins was confirmed by Western blot, and the results are shown in Figure 25.

According to this, KRAS-GTP binding decreased when treated with 5 μM for 48 hours and 10 μM for 72 hours, confirming that RAS activity was reduced. Similarly, C-MYC and NOTCH1, representative markers of oncogenic pathway proteins, were also found to have decreased protein expression for 24, 48, and 72 hours.

Experimental Example 6: Analysis of the inhibitory effect on mutant KRAS G12D colon cancer in a xenograft model

After confirming the inhibitory effect on cell growth and apoptosis in vitro, an animal experiment using a mouse model was performed to determine the tumor growth inhibitory effect of Q2a compound in vivo. LIM1215 wild-type cells and KRAS G12D mutant cells were injected subcutaneously into both flanks of BALB/C nude mice. After the tumor grew to a size of 100 mm ² , 100 μl of Q2a compound was injected intraperitoneally every two days at a concentration of 15 mg/kg and 30 mg/kg.

Changes in tumor size over time according to Q2a administration in mice administered LIM1215 wild-type cells and KRAS G12D mutant cells are shown in Figure 26. According to this, 100㎕ of physiological saline was injected intraperitoneally in the control group. The tumor size of the wild-type group showed an increase in both the control and Q2a compound treatment groups, but the tumor size of the KRAS G12D mutant group was found to increase over time only in the control group, and the tumor size of the group decreased in a concentration-dependent manner. It was found that

Meanwhile, the change in mouse body weight measured while administering the Q2a compound for 21 days is shown in Figure 27. According to this, there was no significant weight loss in the drug administration group compared to the control group. In other words, it can be confirmed that Q2a is not toxic.

After 21 days of treatment, the mouse was sacrificed and the tumor was removed. A photo comparing the size of the tumor is shown in Figure 28. According to this, there was no difference in tumor size between the Q2a compound treatment group and the control group in the wild type group, but there was a significant decrease in the Q2a compound treatment group in the KRAS G12D mutant group.

The results of measuring the weight of the tumor before and after treatment are shown in Figure 29. According to this, the weight of the tumor was larger in the wild-type group compared to the control group at an average of 15 mg/kg, and the weight of the tumor decreased in a concentration-dependent manner in the KRAS G12D mutant group.

A schematic diagram of the mechanism of cancer cell inhibition according to Q2a administration in the KRAS pathway is shown in Figure 30. According to this, treatment with Q2a compound reduces signaling of downstream MAPK and PI3K mechanisms along with decreased KRAS-GTP activity. As a result, cell death may occur and cell proliferation may be inhibited.

Although the embodiments of the present invention have been described above, those skilled in the art can add, change, delete or add components without departing from the spirit of the present invention as set forth in the patent claims. The present invention may be modified and changed in various ways, and this will also be included within the scope of rights of the present invention.

Since the compound of the present invention selectively inhibits cell proliferation in KRAS G12D mutant cells, it can solve the problem of low efficacy of conventional targeted therapy using epidermal growth factor receptor (EGFR), and the compound of the present invention and its salt are effective. The pharmaceutical composition containing the composition as an ingredient can be used for targeted treatment against KRAS G12D mutant cells.

Claims (14)

A compound for inhibiting proliferation of KRAS G12D mutant cells represented by the following formula (1); [Formula 1]

In Formula 1, n is the number of repeat units, which is an integer from 1 to 10, m is the number of repeat units that is an integer from 1 to 10, X ¹ is an oxygen atom or a sulfur atom, R ¹ is a substituted or unsubstituted 1C to 30C alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C1 to C30 heterocycloalkyl group, a substituted or unsubstituted C6 to C30 aryl group, or a substituted or It is an unsubstituted C1 to C30 heteroaryl group, R ² to R ⁴ are each independently a hydrogen atom, a substituted or unsubstituted 1C to 30C alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C1 to C30 heterocycloalkyl group, or a substituted or unsubstituted A C6 to C30 aryl group, or a substituted or unsubstituted C1 to C30 heteroaryl group, R ⁵ is a substituted or unsubstituted 1C to 30C alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C1 to C30 heterocycloalkyl group, a substituted or unsubstituted C6 to C30 aryl group, or a substituted or It is an unsubstituted C1 to C30 heteroaryl group. According to paragraph 1, In Formula 1, n is the number of repeat units that is an integer from 1 to 4, m is the number of repeating units that is an integer from 1 to 4, X ¹ is an oxygen atom, R ¹ is a 1C to 10C alkyl group, R ² to R ⁴ are each independently a hydrogen atom or a 1C to 10C alkyl group, R ⁵ is

ego, X ² is an oxygen atom or a sulfur atom, A compound for inhibiting proliferation of KRAS G12D mutant cells, wherein R ⁶ is a hydrogen atom or a 1C to 10C alkyl group. According to paragraph 2, The compound represented by Formula 1 is a compound for inhibiting proliferation of KRAS G12D mutant cells, characterized in that it is a compound represented by Formula 2 below; [Formula 2]

. A pharmaceutical composition for preventing or treating cancer disease comprising a compound represented by the following formula (1) or a salt thereof as an active ingredient; [Formula 1]

In Formula 1, n is the number of repeating units, which is an integer from 1 to 10, m is the number of repeating units that is an integer from 1 to 10, X ¹ is an oxygen atom or a sulfur atom, R ¹ is a substituted or unsubstituted 1C to 30C alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C1 to C30 heterocycloalkyl group, a substituted or unsubstituted C6 to C30 aryl group, or a substituted or It is an unsubstituted C1 to C30 heteroaryl group, R ² to R ⁴ are each independently a hydrogen atom, a substituted or unsubstituted 1C to 30C alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C1 to C30 heterocycloalkyl group, or a substituted or unsubstituted A C6 to C30 aryl group, or a substituted or unsubstituted C1 to C30 heteroaryl group, R ⁵ is a substituted or unsubstituted 1C to 30C alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C1 to C30 heterocycloalkyl group, a substituted or unsubstituted C6 to C30 aryl group, or a substituted or It is an unsubstituted C1 to C30 heteroaryl group. According to clause 4, In Formula 1, n is the number of repeat units that is an integer from 1 to 4, m is the number of repeating units that is an integer from 1 to 4, X ¹ is an oxygen atom, R ¹ is a 1C to 10C alkyl group, R ² to R ⁴ are each independently a hydrogen atom or a 1C to 10C alkyl group, R ⁵ is

ego, X ² is an oxygen atom or a sulfur atom, A compound for inhibiting proliferation of KRAS G12D mutant cells, wherein R ⁶ is a hydrogen atom or a 1C to 10C alkyl group. According to clause 5, A pharmaceutical composition for preventing or treating cancer disease, wherein the compound represented by Formula 1 is a compound represented by Formula 2 below; [Formula 2]

. According to paragraph 4, The pharmaceutical composition for preventing or treating cancer diseases is characterized in that it is used to inhibit proliferation of KRAS G12D mutant cells. In clause 7, The KRAS G12D mutant cells are one or more selected from LIM1215 KRAS G12D, SW48 KRAS G12D, NCI-H1975 KRAS G12D, NCI-H838 KRAS G12D, and LS174T KRAS G12D. A composition for preventing or treating cancer disease. According to clause 8, A pharmaceutical composition for preventing or treating cancer diseases, characterized in that the composition for preventing or treating cancer diseases is for targeted treatment of the KRAS G12D mutant cells. In clause 7, The composition for preventing or treating cancer diseases is a pharmaceutical composition for preventing or treating cancer diseases, wherein the composition is for inhibiting the MAPK signaling pathway. According to clause 10, A pharmaceutical composition for preventing or treating cancer diseases, wherein the inhibition of the MAPK signaling pathway is carried out by inhibiting C-RAF phosphorylation in the RAS-RAF-MEK-ERK pathway. In clause 7, A pharmaceutical composition for preventing or treating cancer diseases, characterized in that the composition for preventing or treating cancer diseases is for inhibiting the PI3K signaling pathway. According to clause 12, A pharmaceutical composition for preventing or treating cancer diseases, wherein the inhibition of the PI3K signaling pathway is carried out by inhibiting AKT phosphorylation in the PI3K-AKT-mTOR pathway. According to paragraph 4, A pharmaceutical composition for preventing or treating cancer diseases, wherein the cancer disease is any one selected from colon cancer, colon cancer, rectal cancer, lung cancer, melanoma, thyroid cancer, uterine cancer, ovarian cancer, cervix cancer, pancreatic cancer, stomach cancer, and liver cancer.

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