WO2024158237A1 - Compound for inhibiting kras mutation, and composition for preventing or treating cancer, comprising same as active ingredient - Google Patents

Abstract

The present invention relates to a compound for inhibiting the expression of BRAF and CRAF in an MAPK signaling pathway represented by formula 1, and to a pharmaceutical composition for preventing or treating cancer, comprising same as an active ingredient. Accordingly, the present invention can inhibit the proliferation of cell lines of cancer, such as colon cancer and lung cancer, and their KRAS mutant cells, and can especially show a significantly high effect in targeted treatment for the KRAS mutant cells.

Description

Compound for inhibiting KRAS 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 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 KRAS mutation and a composition containing the same as an active ingredient. It relates to a composition for preventing or treating cancer diseases.

RAF is a serine/threonine kinase that plays an important role in the RAS/RAF/MEK/ERK protein kinase signaling pathway involved in cell proliferation, differentiation, survival, and angiogenesis. RAF protein exists in three isoforms: ARAF, BRAF, and CRAF. Among them, RAF1 protein is produced from the C-RAF1 gene, belongs to the proto-oncogene family, and has subtypes A-RAF1 and B-RAF1. In the signal transduction activation pathway of RAF-1, signals transmitted through the cell membrane are transmitted to RAS proteins through multiple receptor-type phosphorylation receptors in the cell membrane or non-receptor-type phosphorylation in the cytoplasm. Cell activation signals through the small GTPase Ras are transmitted to the MAPK (mitogen-activated protein kinase) signaling molecule protein.

Each member of the RAF family has similar amino acid sequences but different biochemical activities and biological functions. Abnormal mutations in vivo have been identified only in BRAF and occur at a frequency of 50-60% in malignant skin melanoma, 30-50% in thyroid cancer, 10-20% in colon cancer, and ~30% in ovarian cancer. To date, more than 45 BRAF mutations have been identified. The most frequent mutation is V600E, which converts valine 600 to glutamic acid and accounts for more than 90% of all BRAF mutations. This mutation increases the kinase activity of BRAF by 500-fold, allowing mutated BRAF to continue transmitting signals to downstream levels without upstream RAS signaling, thereby promoting cell proliferation and survival.

Conventional first-generation BRAF inhibitors are selectively active against BRAF V600E. However, in some cases rapid resistance and other cancers may develop; For example, skin squamous cell tumors and keratinocytes have been reported in patients treated with BRAF inhibitors such as bimorphemic and digraph.

To solve this problem, it is necessary to develop a second generation RAF inhibitor that can inhibit not only BRAF V600E but also CRAF.

The object of the present invention is that the first generation RAF inhibitor, which selectively inhibits BRAF V600E, causes rapid resistance and the development of other carcinomas such as skin squamous cell tumor and keratinocyte. In order to solve the problem, the aim is to provide a compound that is a second-generation RAF inhibitor that can inhibit not only BRAF but also CRAF, and a pharmaceutical composition for preventing or treating cancer disease containing it as an active ingredient.

According to one aspect of the present invention,

Compounds for inhibiting the expression of BRAF and CRAF in the MAPK signaling pathway represented by the following formula (1) are provided.

[Formula 1]

In Formula 1,

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

X ¹ is a sulfur atom or an oxygen atom,

R ¹ is 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, a substituted or unsubstituted C6 to C30 aryl group, Or a substituted or unsubstituted C1 to C30 heteroaryl group,

R ² 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,

X ¹ is a sulfur atom,

R ¹ is a hydrogen atom or a 1C to 10C alkyl group,

이고, R ² is

ego,

X ² is an oxygen atom or a sulfur atom,

R ³ 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 repeating units, which is an integer from 1 to 10,

X ¹ is a sulfur atom or an oxygen atom,

R ¹ is 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, a substituted or unsubstituted C6 to C30 aryl group, Or a substituted or unsubstituted C1 to C30 heteroaryl group,

R ² 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,

X ¹ is a sulfur atom,

R ¹ is a hydrogen atom or a 1C to 10C alkyl group,

이고, R ² is

ego,

R ³ is a hydrogen atom or a 1C to 10C alkyl group,

X ² may be an oxygen atom or a sulfur atom.

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 one or more cancer cell lines selected from LIM1215, SW48 WT, LOVO, LS174T, NCIH1975, and NCIH838 and their KRAS mutant cells.

The KRAS mutant cells may be one or more types selected from LIM1215 KRAS G12D, SW48 KRAS G12D, LOVO KRAS G13D, SW48 KRAS G12V, LS174T KRAS G12D, NCIH1975 KRAS G12D, and NCIH838 KRAS G12D.

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

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

The inhibition of the MAPK signaling pathway can be performed by simultaneously suppressing the expression of BRAF and CRAF, which are isoforms of RAF, in the RAS-RAF-MEK-ERK pathway.

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

The second-generation RAF inhibitor compound of the present invention has the function of inhibiting not only BRAF but also CRAF, so the first-generation RAF inhibitor, which exhibits a selective inhibitory effect on BRAF V600E, causes rapid resistance and skin squamous cell tumor. ) and keratinocytes, etc., not only has a proliferation inhibitory effect on cancer cell lines such as colon cancer and lung cancer, but also has a significant proliferation inhibitory effect on KRAS mutations in the cancer cell lines, making it a targeted therapeutic agent. It can be applied.

In addition, the pharmaceutical composition for preventing or treating cancer diseases of the present invention has an inhibitory ability against cancer cell lines such as LIM1215, SW48 WT, LOVO, LS174T, NCIH1975, and NCIH838 and their KRAS mutations, and has a particularly high inhibitory ability against KRAS mutations, making it a target It can be applied as a treatment.

Figures 1 and 2 show the results of measuring cell proliferation rates for LIM1215 WT and LIM1215 KRAS G12D according to treatment at different concentrations of 11 drugs according to Experimental Example 1.

Figure 3 shows the results of Western blotting in LIM1215 WT, LIM1215 KRAS G12D, and LS174T KRAS G12D cells for Q12b and Q21b according to Experimental Example 1.

Figure 4 shows the results of confirming the binding direction between CRAF of Experimental Example 1 and Q12b selected by biological screening using a molecular docking system.

Figure 5 shows the results of measuring cell proliferation rate upon Q12b treatment in KRAS WT (wild type) and KRAS G12D, G13D, and G12V cell lines of Experimental Example 2.

Figure 6 shows the results of measuring the cell proliferation rate in wild-type cells and mutant cells when treated at different Q12b concentrations.

Figures 7 and 8 show the cell viability measurement results according to crystal violet staining when treated at different Q12b concentrations in wild-type cells and mutant cells in Experimental Example 2.

Figure 9 shows the results of cell death analysis after treating LIM1215 WT and mutant cells LIM1215 KRAS G12D of Experimental Example 3 with Q12b reagent over time.

Figure 10 shows the results of cell death analysis after treating SW48 WT and mutant cells SW48 KRAS G12D of Experimental Example 3 with Q12b reagent over time.

Figure 11 shows the results of cell death analysis after treating NCIH838 WT and mutant NCIH838 KRAS G12D cells of Experimental Example 3 with Q12b reagent over time.

Figure 12 shows the results of analyzing cell death after treating NCIH1975 WT and mutant NCIH1975 KRAS G12D cells of Experimental Example 3 with Q12b reagent over time.

Figure 13 shows the results of analysis of MAPK pathway protein expression levels when LIM1215 WT and mutant cells LIM1215 KRAS G12D of Experimental Example 4 were treated at different concentrations of Q12b reagent.

Figure 14 shows the results of analysis of MAPK pathway protein expression levels when SW48 WT and mutant cells SW48 KRAS G12D of Experimental Example 4 were treated at different concentrations of Q12b reagent.

Figure 15 shows the results of analysis of MAPK pathway protein expression level when NCIH838 WT and mutant NCIH838 KRAS G12D cells of Experimental Example 4 were treated at different concentrations of Q12b reagent.

Figure 16 shows the results of analysis of MAPK pathway protein expression level when NCIH1975 WT and mutant NCIH1975 KRAS G12D cells of Experimental Example 4 were treated at different concentrations of Q12b reagent.

Figure 17 shows the hierarchical clustering analysis results of Experimental Example 5.

Figure 18 shows the distribution of genes with various numbers of zero counts in the hierarchical clustering analysis of Experimental Example 5.

Figure 19 shows the results of confirming the expression of c-MYC by Western blot in Experimental Example 5.

Figure 20 shows the results of tumor inhibition analysis of Q12b in KRAS WT (wild type) and mutant cells according to Experimental Example 6.

Figure 21 shows the change in body weight between groups in the tumor inhibition analysis of Q12b according to Experimental Example 6.

Figure 22 shows the results of measuring tumor weight according to Q12b administered concentration in the tumor inhibition analysis of Q12b according to Experimental Example 6.

Figure 23 is a schematic diagram of the mechanism of MAPK pathway inhibition by compound Q12b of the present invention.

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 or aryl moiety 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 has 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 the expression of BRAF and CRAF in the MAPK signaling pathway represented by structural formula 1 below.

[Formula 1]

In Formula 1,

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

X ¹ is a sulfur atom or an oxygen atom,

R ¹ is 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, a substituted or unsubstituted C6 to C30 aryl group, Or a substituted or unsubstituted C1 to C30 heteroaryl group,

R ² 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,

X ¹ is a sulfur atom,

R ¹ is a hydrogen atom or a 1C to 10C alkyl group,

이고, R ² is

ego,

X ² is an oxygen atom or a sulfur atom,

R ³ 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,

X ¹ is a sulfur atom or an oxygen atom,

R ¹ is 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, a substituted or unsubstituted C6 to C30 aryl group, Or a substituted or unsubstituted C1 to C30 heteroaryl group,

R ² 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,

X ¹ is a sulfur atom,

R ¹ is a hydrogen atom or a 1C to 10C alkyl group,

이고, R ² is

ego,

X ² is an oxygen atom or a sulfur atom,

R ³ 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, or a composition containing 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 one or more cancer cell lines selected from LIM1215, SW48 WT, LOVO, LS174T, NCIH1975, and NCIH838 and their KRAS mutant cells.

The LIM1215, SW48 WT, LOVO, and LS174T cell lines are colon cancer cell lines, and the NCIH1975 and NCIH838 cell lines are lung cancer cell lines.

The KRAS mutant cells may be one or more types selected from LIM1215 KRAS G12D, SW48 KRAS G12D, LOVO KRAS G13D, SW48 KRAS G12V, LS174T KRAS G12D, NCIH1975 KRAS G12D, and NCIH838 KRAS G12D.

The composition for preventing or treating cancer disease can be used for targeted treatment against the KRAS mutant cells. This is because the inhibitory effect on KRAS mutant cells is significant at low concentrations compared to KRAS wild-type 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 simultaneously suppressing the expression of BRAF and CRAF, which are isoforms of RAF, in the RAS-RAF-MEK-ERK signaling pathway.

Inhibition of the MAPK signaling pathway can be performed by inhibiting p-ERK production following phosphorylation of ERK in the RAS-RAF-MEK-ERK 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 mutations 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]

Materials and antibody preparation

Compounds Q1b to Q12b were supplied from KIST Gangneung Branch Natural Products Research Institute. PARP, BRAF, CRAF, p-CRAF, AKT, p-AKT, ERK, p-ERK, c-MYC, and p-MEK antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA), and β-actin was purchased from sigma- It was purchased from Aldrich (St. Louis, MO, USA). In addition, secondary antibodies, anti-mouse IgG horseradish peroxidase (HRP) and anti-rabbit IgG HRP, were purchased from Cell Signaling Technology. EZ-Cytoxan was purchased and used from dogend bio, and annexin V-FITC apoptosis detector kit was purchased and used from coma biotech. 4% paraformaldehyde was purchased from Tissue Pro Technology, and crystal violet solution was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Below, the chemical formulas and NMR analysis results for compound Q12b represented by Formula 2 and compound Q21b represented by Formula 3 are shown.

[Formula 2]

^1H NMR (500 MHz, MeOD) δ 7.94 (dd, J = 8.3, 1.3 Hz, 1H), 7.71 - 7.64 (m, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.30 (t, J = 7.7 Hz, 1H), 7.19 (dd, J = 5.2, 1.2 Hz, 1H), 7.04 (dd, J = 3.6, 1.1 Hz, 1H), 6.87 (dd, J = 5.1, 3.5 Hz, 1H), 6.73 (dd, J = 16.8, 10.6 Hz, 1H), 6.17 (dd, J = 16.7, 1.9 Hz, 1H), 5.71 (dd, J = 10.6, 1.9 Hz, 1H), 3.95 (s, 2H), 3.91 ( s, 2H), 3.76 (q, J = 6.0 Hz, 4H), 3.62 (dt, J = 13.2, 6.6 Hz, 5H).

¹³ C NMR (126 MHz, MeOD) δ 166.56, 166.10, 163.55, 159.69, 140.27, 134.75, 127.75, 127.35, 126.34, 126.32, 126.29, 124.92, 124.52, 122.93, 118.48, 110.04, 54.48, 37.50, 35.60, 30.27.

[Formula 3]

¹ H NMR (500 MHz, DMSO) δ 7.96 (s, 5H), 7.80 - 7.74 (m, 1H), 7.50 - 7.39 (m, 2H), 6.87 (dd, J = 16.7, 10.5 Hz, 1H), 6.16 (dd, J = 16.7, 2.3 Hz, 1H), 5.73 (dd, J = 10.4, 2.4 Hz, 1H), 3.80 (s, 3H), 3.66 (d, J = 17.0 Hz, 3H), 3.20 - 3.15 ( m, 1H).

¹³ C NMR (126 MHz, MeOD) δ 166.45, 166.09, 163.51, 158.75, 133.96, 127.56, 127.42, 125.11, 122.55, 116.21, 116.03, 110.47, 54.48, 4 8.26, 48.14, 48.09, 47.97, 47.80, 47.63, 47.46, 47.29, 47.12, 39.03, 37.48, 35.58, 31.67, 30.26, 29.36, 29.06.

cell culture

In the present invention, colon cancer cell lines LIM1215 WT, LIM1215 KRAS G12D, SW48 WT (wild type), SW48 KRAS G12D, and lung cancer cell lines NCIH1975 WT, NCIH1975 KRAS G12D, NCIH838 WT (wild type), and NCIH838 KRAS G12D were purchased from Horizon Discovery and used. LOVO KRAS G13D, SW48 KRAS G12V, and LS174T KRAS G12D were provided by the American Type Culture Collection (ATCC). The culture medium used was RPMI1640 (Guidepost, Texas, USA) and 10% fetal bovine serum (FBS, Gibco island, NY, USA), and 1% antibiotic-antifungal agent (gibbon) was used. The cells were cultured in an incubator under 5% CO ₂ and 37°C conditions, and subculture was performed when the cells reached approximately 80%.

drug screening

Grid-Based Method and 4D Tensor CNN method were used for drug screening. The grid-based method calculated docking scores based on a grid map. After identifying the top chemical conformer that binds to pocket 1 of the protein in the ESP structure, the encoded binding pocket was discovered. The 4D Tensor CNN method modeled the binding structure of Q12b and CRAF with AI and analyzed the binding energy in 4D using binding kinetics.

Cell proliferation assay

Colon cancer and lung cancer cell lines were distributed in 96-well plates. Cells were cultured in an incubator at 37°C and 5% CO ₂ for the experimental time. Thereafter, compound Q12b was treated at a predetermined concentration for 24 hours, 48 hours, and 72 hours. To analyze the proliferation rate of cells, EZ-Cytoxan (Digenic, Seoul, Korea) and serum-free medium were mixed at a ratio of 9:1 and supplied at 100㎕/well. After culturing in an incubator at 37°C and 5% CO ₂ for 2 hours, the measurement wavelength was set to 450 nm/reference wavelength to 600 nm, and the absorbance was measured. The measured values were averaged for each group, and the control group was set at 100%. The measured value was calculated by dividing by the average of the control value.

Cell viability measurements

Cells were distributed to wells in a 6-well plate according to doubling time. Then, compound Q12b was treated at a given concentration for 72 hours. Afterwards, the medium was changed to medium containing FBS every 2-3 days for a total of 2 weeks. After 2 weeks, the cells were washed once with PBS, fixed with 4% paraformaldehyde (Tissue Pro Technology) for 15 minutes, and stained with crystal violet solution (Sigma Aldrich) for 20 minutes. Afterwards, the crystal violet solution was washed with water, dissolved in acetic acid, and measured using an ELISA absorbance instrument with the wavelength set to 572 nm.

Fluorescence-activated cell sorting (FACS) analysis

When Compound Q12b was treated with wild-type (WT) cell lines and KRAS mutant cell lines in colon cancer and lung cancer, the apoptotic effect was confirmed by fluorescence activated cell sorting (FACS). Cells were distributed into 6-well plates. Afterwards, compound Q12b was treated at a predetermined concentration for 24 hours, 48 hours, and 72 hours. After drug treatment, cells were obtained with trypsin EDTA in PBS and centrifuged at 1000 rpm for 5 minutes at 4°C. The supernatant was removed and washed again with cold PBS, and all cells were suspended in 500 binding buffer diluted with D.W. Suspended cells were treated with Annexin V at a concentration of 5 μg/ml and placed in the dark at room temperature for 15 minutes. Next, after centrifugation at 1000 Measurements were analyzed using CyExpert software.

western blot

Cells were cultured in 60 mm plates/well for 24 hours. Drug compound Q12b was treated at a predetermined concentration for 24 hours, 48 hours, and 72 hours. After washing the drug-treated cells once with cold PBS, the cells were obtained using trypsin EDTA. The collected cells were centrifuged at 1500 rpm for 5 minutes, the supernatant was removed, resuspended using PBS, and centrifuged at 1500 rpm for 5 minutes. Cells were lysed using 100 μl of 2X SDS-sample buffer. The dissolved samples were boiled at 100°C for 10 minutes. BCA solution A and B reagents were diluted at a ratio of 50:1 and 98 ㎕ each was placed in a 96-well plate. 2 ㎕ of protein sample was added to each well and reacted at 60°C for 5 minutes. After 5 minutes, the amount of protein was checked. Absorbance was quantified by measuring absorbance at 572 nm using synergy htx (biotek) equipment. After the proteins were separated through SDS-PAGE, transfer was performed using a PVDF membrane. The transfer membrane was blocked by dissolving 5% skim milk in TBST for 1 hour at room temperature. For primary antibodies, 5% BSA was dissolved in TBST and then antibodies (PARP, BRAF, p-BRAF, CRAF, p-CRAF, AKT, p-AKT, ERK, p-ERK, p-MEK, β-actin) was diluted 1000:1 and reacted at 4°C for 24 hours. Secondary antibodies were reacted with 5% skim milk for 1 hour at room temperature. After antibody reaction, protein expression was measured with ECL solution using chemi doc (Amer sham image quant 800) .

RNA sequencing

Control and Q12b treated samples were prepared from SW48 KRAS G12D mutant cells and total RNA was isolated. Afterwards, DNA contamination was removed using DNase. Next, a kit was selected according to the type of RNA to be profiled in the library production step. Purified RNA was randomly fragmented for sequencing into short reads. Fragments of RNA were reverse transcribed into cDNA. Different adapters were attached to both ends of the generated cDNA fragment and ligated. After PCR amplification in an amount sufficient for sequencing, an insert size of 200 to 400 bp was secured through a size selection process. In the case of paired-end sequencing, sequencing was performed for the read length from both ends of the cDNA fragment. Sample analysis was performed by Macrogen.

in vivo experiments

As experimental animals, 5-week-old female BALB/C nude mice were purchased from Nara Biotech (Pyeongtaek, Korea). Solid feed and water were consumed freely, and the experiment was conducted after a 3-day adaptation period. The experimental animals were divided into 5 groups per group: a colon cancer induction group without drug administration, a group administered 15 mg/mL of Compound Q12b, and a group administered 30 mg/mL of Compound Q12b. To generate tumors in experimental animals, colon cancer cell lines SW48 WT and SW48 KRAS G12D mutant cells were subcutaneously injected into the experimental animals and tumors were grown for about 14 days. Physiological saline was administered intraperitoneally to the control group that did not administer the drug, and the drug was administered intraperitoneally to the compound Q12b 15 mg/ml and 30 mg/ml groups. Drug administration was conducted at 2-3 day intervals for a total of 21 days. The induced tumor size was measured with a caliper at 2-day intervals after tumor occurrence, and the tumor size was calculated using the following formula.

[Equation 1]

Tumor size = 1/2 × length × (width) ²

Mouse and tumor weights were also measured every other day and analyzed using Graph Pad Prism 9.

Statistical analysis

Statistical analysis was performed using the Graph Pad of Stat 9 software. For comparison between groups, one-way analysis of variance and Tukey's post hoc test method were used. Additionally, the t-test method was used to evaluate significance between groups. The p-value values were evaluated for significance as *p<0.05, **p<0.01, and ***p<0.001.

[Experimental example]

Experimental Example 1: Drug screening in wild-type cancer cell lines and mutant cells

Colon cancer cell lines LIM1215 WT (wild type) and LIM1215 KRAS G12D mutant cells were treated with Q12b for 72 hours with a total of 11 substances from Q12b to Q24b at concentrations of 0.5, 5, and 50 μM, and cell proliferation rates are shown in Figures 1 and 2. Subsequent WST measurements showed that most drugs reduced mutant cell lines compared to WT (wild type). In particular, Q12b and Q21b were shown to inhibit cell proliferation by approximately 80% at 0.5 and 5 μM and by approximately 70% at 50 μM in mutant cell lines.

Western blotting was performed on Q12b and Q21b, which showed good effects in WST, using colon cancer cell lines LIM1215 WT, LIM1215 KRAS G12D, and LS174T KRAS G12D, and the results are shown in Figure 3. According to this, in LIM1215 KRAS G12D mutant cells, the expression level of Q12b in c-PARP, a cell death marker, was higher than Q21b. There was no significant difference in the same mutant cell, LS174T. In the case of KRAS G12D, there was a slight decrease in the Q12b treatment group in the LIM1215 mutant compared to the control group, but there was no significant difference. In the case of LS174T, there was no difference in expression level between the control group and the drug treatment group.

It was confirmed that p-ERK decreased significantly more in the Q12b treatment group than the Q21b treatment group in the LIM1215 KRAS G12D mutant cell line. LS174T KRAS G12D also showed the same pattern as the LIM1215 KRAS G12D cell line. In the case of LIM1215 WT, there was no difference in expression level. Based on these results, Q12b (Mw 379.48) material represented by the following formula (2) was finally selected. Q12b uses quinazoline as its main backbone, and various Q compound derivatives can be synthesized by synthesizing linkers containing acrylic acid and various amines/anilines.

[Formula 2]

First, 2,4-dichloroquinazoline was synthesized through decarboxylation of benzoyleneurea, and amine/aniline was substituted under basic conditions. Afterwards, it was synthesized in five steps, including substitution of alkylamine under basic conditions, Boc deprotection, and amide coupling using HATU, resulting in an overall yield of 25-38%.

The binding direction of CRAF and Q12b selected by biological screening was confirmed using a molecular docking system, and the results are shown in Figure 4.

According to this, the binding affinity was -6.05 kcal/mol. During docking, it was confirmed that Q12b interacts with amino acid residues such as ASP486 of the CRAF protein and makes hydrophobic contact with Q12b. Since previous studies have already shown the importance of the interaction between CRAF and ASP486 residues, it was confirmed that Q12b has an intermolecular interaction between CRAF proteins.

Experimental Example 2: Analysis of cell proliferation inhibition and cell viability reduction in wild-type cancer cell lines and mutant cells

KRAS WT (wild type) and KRAS G12D, G13D, and G12V cell lines were treated with Q12b at 0.5, 5, and 10 μM for 72 hours, and the cell proliferation rate is shown in Figure 5.

According to this, cell proliferation was inhibited to about 87% at 10 μM in LOVO KRAS G13D and SW48 KRAS G12V cell lines, and slightly decreased to about 95% at 10 μM in COLO 205 KRAS WT cells. Additionally, LS174T KRAS G12D showed about 82% cell growth inhibition at 50 μM.

Paired colon cancer cell lines (LIM1215 WT and LIM1215 KRAS G12D) and lung cancer cell lines (NCIH838 WT and NCIH838 KRAS G12D) were treated with Q12b at concentrations of 1, 5, and 10 μM for 72 hours, and the cell proliferation rate was measured and the results were reported. It is shown in Figure 6.

According to this, there was almost no effect in the LIM1215 WT cell line, and a concentration-dependent decrease was confirmed in the mutant group. In the case of SW48 WT, 95% inhibition of cell proliferation was confirmed at a concentration of 10 μM, and in the mutant group, 83% proliferation inhibition was confirmed at 1 μM, 80% at 5 μM, and 79% at 10 μM. In the NCIH838 cell line, the 10 μM treatment group in mutant NCIH838 KRAS G12D showed a similar inhibition rate as SW48 KRAS G12D. The concentration-treated group of NCIH1975 WT showed a slight decrease in a concentration-dependent manner, but the mutant NCIH1975 KRAS G12D showed inhibition of proliferation at a higher rate.

Next, to confirm the degree of apoptosis of each cell line, cell survival rate was measured using crystal violet staining, and the results are shown in Figures 7 and 8.

In the case of LIM1215 KRAS G12D, the cell proliferation rate was similar in the concentration treatment groups except for the 10 μM treatment group, and a cell survival rate of about 78% was confirmed at 10 μM. SW48 WT showed a higher proliferation rate compared to the control group in the 1 and 5 μM treatment groups, and for mutant SW48 KRAS G12D, the proliferation rate was approximately 60% in the 1 μM treatment group, 58% in the 5 μM treatment group, and approximately 50% in the 10 μM treatment group. Cell survival rate was shown. The NCIH838 WT cell line was similar to the control at concentrations other than 10 μM, and the survival rate of mutant NCIH838 KRAS G12D showed a similar trend to that of SW48 KRAS G12D. NCIH1975 WT showed a cell survival rate of 79% in the 10 μM treatment group, and mutant NCIH1975 KRAS G12D showed a cell survival rate of 60% in the 10 μM treatment group.

Experimental Example 3: Analysis of each KRAS WT (wild type) cell and mutant apoptosis

The results of cell death analysis after treating LIM1215 WT and mutant LIM1215 KRAS G12D with Q12b reagent for 24 hours (a), 48 hours (b), and 72 hours (c) are shown in Figure 9.

According to this, the wild type (WT) showed little difference from the control group, but the mutant showed 3-4% cell death in the 1μM and 5μM treatment groups, and about 6% cell death in the 10μM treatment group.

In addition, the results of cell death analysis after treating SW48 WT and mutant cells SW48 KRAS G12D with Q12b reagent for 24 hours (a), 48 hours (b), and 72 hours (c) are shown in Figure 10.

According to this, in SW48 cells, when treated for 24 hours based on the 10μM treatment group, the wild type (WT) showed an apoptosis rate of 6% and the mutant showed an apoptosis rate of about 11%, and when treated for 48 hours, the wild type (WT) and the mutant showed an apoptosis rate of about 11%. Cell death rates were approximately 12% and 15%, and when treated for 72 hours, cell death rates were 16% in the wild type (WT) and 13% in the mutants.

The results of cell death analysis after treating NCIH838 WT and mutant cells NCIH838 KRAS G12D with Q12b reagent for 24 hours (a), 48 hours (b), and 72 hours (c) are shown in Figure 11.

According to this, when the NCIH838 cell line was treated with Q12b for 24 hours, the wild type (WT) showed little difference from the control group, and when treated with 10 μM in the mutant NCIH838 KRAS G12D, about 6% of cell death occurred. In addition, when treated for 48 hours, apoptosis increased to about 9% in the 10μM treated group in the mutant, and when treated for 72 hours, the apoptosis rate was almost similar at 1, 5, and 10μM.

Meanwhile, the results of cell death analysis after treating NCIH1975 WT and mutant cells NCIH1975 KRAS G12D with Q12b reagent for 24 hours (a), 48 hours (b), and 72 hours (c) are shown in Figure 12.

According to this, when NCIH1975 cells were treated for 24 hours, wild type (WT) showed approximately 3% apoptosis in the 5 and 10 μM treatment groups, and the mutant NCIH1975 KRAS G12D showed a tendency to increase concentration-dependent apoptosis. When treated for 48 hours, 10% of the cells died in the 10μM treated group in the mutant NCIH1975 KRAS G12D, and when treated for 72 hours, 20% of the cells died in the 10μM treated group, with approximately twice the number of cells compared to the 48-hour treated group. The death rate was shown.

Experimental Example 4: Analysis of MAPK pathway protein expression in wild-type cancer cell lines and mutant cells

All cell lines were treated with drugs for 24 hours each at concentrations of 1, 5, and 10 μM.

The results of analysis of MAPK pathway protein expression levels when LIM1215 WT and mutant cells LIM1215 KRAS G12D were treated at different concentrations of Q12b reagent are shown in Figure 13. According to this, in the case of LIM1215 cells, the level of BRAF protein expression in mutant cells was significantly reduced compared to the control group. Likewise, phosphorylated p-BRAF was also decreased compared to the control group. CRAF protein showed a tendency to significantly decrease compared to the mutant control group, and p-CRAF also showed the same trend. In the case of p-MEK, p-MEK protein was found to increase when the mutants were treated at different concentrations. However, in the case of p-ERK, the remaining concentration treatment groups except for 5 μM in the wild type (WT) were similar to the control group, and a concentration-dependent decrease was observed in the mutant. AKT protein was significantly decreased in mutant cells compared to the control group, and p-AKT showed a decrease in protein expression in the 10 μM treatment group.

The results of MAPK pathway protein expression level analysis when SW48 WT and mutant cells SW48 KRAS G12D were treated at different concentrations of Q12b reagent are shown in Figure 14.

According to this, the protein expression level of BRAF in the SW48 cell line was almost unchanged, but in p-BRAF mutant cells, the expression level decreased in groups treated with concentrations other than 1 μM. The expression level of p-CRAF protein was slightly decreased at 10 μM in the wild type (WT), and the expression level was decreased in all mutant cells compared to the control group. Likewise, the protein expression level of CRAF protein was decreased in mutant cells compared to the control group. p-MEK protein showed a tendency to increase protein expression in mutant cells. The protein expression level of p-ERK protein was significantly decreased in the drug-treated group compared to the control group in the mutant cells. In the case of p-AKT protein, the protein expression level tended to increase in the 5μM and 10μM drug treatment groups compared to the control group in wild type (WT), and in mutant cells, the protein expression level significantly decreased in the drug treatment group compared to the control group.

The results of analysis of MAPK pathway protein expression levels when NCIH838 WT and mutant cells NCIH838 KRAS G12D were treated at different concentrations of Q12b reagent are shown in Figure 15.

According to this, BRAF and p-BRAF proteins showed a tendency to decrease protein expression in the drug-treated group compared to the control group. In the case of p-CRAF protein, protein expression decreased in the 10 μM treatment group in wild type (WT), and protein expression decreased in all drug-treated groups in mutant cells. The expression level of p-MEK protein was found to be slightly increased in the 1μM and 5μM drug treatment groups of mutant cells. Unlike other cell lines, p-ERK protein did not show significant changes in expression level upon drug treatment. In the case of p-AKT protein, the expression level of the wild type (WT) increased in the 10 μM drug treatment group, but the expression level of the mutant group decreased in the drug treatment group.

The results of analysis of MAPK pathway protein expression levels when NCIH1975 WT and mutant cells NCIH1975 KRAS G12D were treated at different concentrations of Q12b reagent are shown in Figure 16.

According to this, the expression level of p-BRAF protein increased in the wild type (WT) drug-treated group, and decreased in the 5 and 10 μM drug-treated groups of mutant cells. In the case of BRAF protein, the expression level was found to be decreased in the 10 μM treated group of mutant cells. In the case of p-CRAF protein, the expression level decreased in the wild type (WT) 10 μM drug-treated group, and no protein expression occurred in the mutant drug-treated group. In the case of CRAF protein, the expression level was found to be decreased in both the wild type (WT) drug-treated group and mutant cells. In the case of p-MEK protein, unlike the wild type (WT) cell line, the mutant drug-treated group showed a decrease in expression compared to the control group. In the case of p-ERK protein, the expression level decreased in the wild type (WT) group treated with 10 μM drug, and the expression level decreased significantly in the 5 μM and 10 μM drug treated groups of mutant cells. In the case of AKT protein, the expression level decreased in the 10 μM drug-treated group of the mutant cell line, and p-AKT protein showed little expression in the drug-treated group of the mutant cell line.

According to the above results, BRAF and CRAF protein expression was reduced in wild type (WT) cells treated with high-concentration drug treatment, and in mutant cells, BRAF and CRAF protein expression was significantly reduced even in the 1 μM low-concentration drug treatment group. In addition, in the case of p-ERK protein, the expression level was decreased in a concentration-dependent manner in the 10 μM treatment group of wild type (WT) cells and the mutant drug treatment group, which was confirmed to inhibit cell survival. In other words, it was confirmed that the Q12b drug of the present invention affects the cell survival mechanism while suppressing RAF protein expression in the MAPK pathway.

Experimental Example 5: Gene expression analysis through RNA sequencing

RNA was extracted from three samples of the SW48 KRAS G12D cell line and three samples of the group treated with 5 μM Q12b reagent, and sequencing analysis was performed. Using the close-up value for each magnification of each sample, samples and genes with similar close-up levels are grouped through hierarchical clustering analysis (Euclidean Distance, Complete Linkage). The results are shown in Figure 17. Additionally, by increasing the count value and widening the range where the six supply voltage values were 0, a total of 46,427 were output, 27,312 were memorized, and 19,115 genes were marked as targets to begin the analysis. Here, the distribution of genes with various numbers of zero counts is shown in Figure 18.

First, as a result of double potential analysis, it was confirmed that misc RNA, which causes RAF1 amplification, increased by lithium in the Q12b treatment group compared to LEMON, which regulates the reversal of RAF1 discharge. Additionally, the level of mRNA making BRAF was decreased compared to BRAF. Additionally, in the Q12b treatment group, the discharge time of AKT, which produces mRNA, was decreased.

RNA type Transcript_Length SW48-48h-12b1_Read_Count SW48-48h-12b2_Read_Count SW48-48h-12b3_Read_Count SW48-48h-C1_Read_Count SW48-48h-C2_Read_Count SW48-48h-C3_Read_Count RAF1 misc_RNA 3304 7 54 60 268 237 173 RAF1 misc_RNA 3250 6 40 18 114 264 121 BRAF mRNA 6459 105 66 71 148 127 107 AKT2 mRNA 4956 289 291 248 614 591 378 AKT2 mRNA 4335 603 694 379 1024 1278 721 MAX mRNA 2010 854 674 393 992 968 1040 MAX mRNA 1983 959 698 815 1031 1299 973 MAZ mRNA 2593 156 124 112 272 285 228 MYCBP misc_RNA 2506 19 38 33 114 126 161

gene gene_biotype SW48-48h-12b1_Read_Count SW48-48h-12b2_Read_Count SW48-48h-12b3_Read_Count SW48-48h-C1_Read_Count SW48-48h-C2_Read_Count SW48-48h-C3_Read_Count MYCBP2 protein_coding 6880 5647 5357 7021 7864 6538 MYCBP protein_coding 540 453 491 983 957 860 MTOR protein_coding 4564 4079 3924 4095 4944 4029 RAF1 protein_coding 4778 4107 3782 4413 4633 3791

Next, as a result of gene upper limit analysis, it was confirmed that the upper limit of the Q12b treatment group remained in the remaining portion of the RAF1 lung group. It was confirmed that the mTOR repetition rate extended to PCB cell proliferation exceeded the threshold for repetition. In the past, the voltage applied against the MYC fold field became a value of about 400 or more compared to repetition, which confirmed the possibility of Q12b drug discharge in signaling to the cell envelope. Gene set enrichment analysis (GSEA) analysis based on RNA sequencing showed that compared to sequencing, the Q12b treatment group showed a significant decrease in amplification when MYC polar repeat and mTOR signaling were prolonged. Through RNA sequencing, it was confirmed that the expression of mRNA that activates RAF modification and AKT was highest in the drug treatment group. Additionally, the survival rates of MYC and mTOR, which play important roles within cells, gradually decreased, and this continued even after drug treatment. In addition, the expression of c-MYC was confirmed through Western blot, and the results are shown in Figure 19. According to this, it was confirmed that expression was reduced compared to the control group in both wild type (WT) cells and mutant cells. In other words, it indicates that Q12b can suppress MYC signaling.

Experimental Example 6: Mouse drug efficacy evaluation using WT (wild type) and KRAS mutant cells

To confirm the tumor inhibition of Q12b in KRAS WT (wild type) and mutant cells, drug evaluation was performed after tumor induction in 5 mice in each group, and the results are shown in Figure 20. At this time, the group was divided into a control group, a 15 mg/mL Q12b administration group, and a 30 mg/mL Q12b administration group. After SW48 cells were administered to mice, tumors grew for about 2 weeks, and drugs were administered at intervals of 2 to 3 days for 2 weeks after tumor formation.

According to this, the tumor size of SW48 WT continued to increase in the control group, and the tumor size did not decrease in the 15 mg/ml and 30 mg/ml treatment groups (FIG. 20 (a), (b)). In contrast, the growth of mutant SW48 KRAS G12D tumors appeared to be reduced (Figure 20(b), (c)).

Meanwhile, the change in mouse body weight between groups is shown in Figure 21. According to this, there was no significant difference in body weight between groups.

Additionally, the results of comparing tumor weights between groups are shown in Figure 22. According to this, the tumor weight increased about 4 times at 15 mg/ml and about 2 times at 30 mg/ml compared to the control group of SW48 WT. It can be seen that in the mutant SW48 KRAS G12D experimental group, the weight of the tumor was significantly reduced in the drug treatment group compared to the control group.

To summarize the results of the above experimental example, Q12b and Q21b were selected among 11 drugs targeting KRAS mutations using a biological screening (WST) method. Western blot was performed to evaluate the expression levels of the apoptosis markers PARP and p-ERK. The results showed that compared with the effect of Q21b, Q12b showed higher efficacy in increasing the expression of c-PARP and decreasing the expression of p-ERK, and Q12b was used for further analysis.

In addition to the KRAS G12D mutation, cell proliferation was inhibited by about 10 to 15% in the G12V and G13D mutants when treated at high concentrations, which means that the growth of KRAS mutant cells was reduced compared to wild type (WT) cells. In four pairs of wild-type (WT) and mutant cells, apoptosis increased over time, and most mutant cells showed a higher rate of apoptosis compared to wild-type (WT) cells.

The MAPK pathway, a RAS-RAF-MEK-ERK signaling pathway that induces cell survival, was evaluated using Western blotting. In most cell lines, wild-type (WT) cells showed decreased levels of BRAF and CRAF proteins at a high concentration of 10 μM, while mutant cells showed decreased levels of BRAF and CRAF proteins starting from a low concentration of 1 μM. Q12b was shown to inhibit RAF protein and reduce p-ERK phosphorylation, a final signaling step.

RNA sequencing results showed that the mRNA expression level of RAF was decreased in the Q12b treatment group compared to the control group. These results indicate that RAF proteins were targeted and affected cell survival mechanisms.

Additionally, by measuring the expression difference of AKT and MYC genes, it was found that Q12b affects signal transduction related to the signal transduction mechanism of cell proliferation. The MAPK pathway inhibition mechanism by which Q12b, which specifically inhibits RAF, induces survival of mutant cells was confirmed.

The MAPK signal transduction inhibition mechanism of the second-generation RAF inhibitor of the present invention is shown in Figure 23.

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 the rights of the present invention.

The second-generation RAF inhibitor compound of the present invention has the function of inhibiting not only BRAF but also CRAF, so the first-generation RAF inhibitor, which exhibits a selective inhibitory effect on BRAF V600E, causes rapid resistance and skin squamous cell tumor. ) and keratinocytes, etc., not only has a proliferation inhibitory effect on cancer cell lines such as colon cancer and lung cancer, but also has a significant proliferation inhibitory effect on KRAS mutations in the cancer cell lines, making it a targeted therapeutic agent. It can be applied.

In addition, the pharmaceutical composition for preventing or treating cancer diseases of the present invention has an inhibitory ability against cancer cell lines such as LIM1215, SW48 WT, LOVO, LS174T, NCIH1975, and NCIH838 and their KRAS mutations, and has a particularly high inhibitory ability against KRAS mutations, making it a target It can be applied as a treatment.

Claims (13)

Compounds for inhibiting the expression of BRAF and CRAF in the MAPK signaling pathway represented by Formula 1 below; [Formula 1]

In Formula 1, n is the number of repeat units, which is an integer from 1 to 10, X ¹ is a sulfur atom or an oxygen atom, R ¹ is 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, a substituted or unsubstituted C6 to C30 aryl group, Or a substituted or unsubstituted C1 to C30 heteroaryl group, R ² 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, X ¹ is a sulfur atom, R ¹ is a hydrogen atom or a 1C to 10C alkyl group, R ² is

ego, X ² is an oxygen atom or a sulfur atom, A compound for inhibiting the expression of BRAF and CRAF in the MAPK signaling pathway, wherein R ³ 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 the expression of BRAF and CRAF in the MAPK signaling pathway, 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 repeat units, which is an integer from 1 to 10, X ¹ is a sulfur atom or an oxygen atom, R ¹ is 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, a substituted or unsubstituted C6 to C30 aryl group, Or a substituted or unsubstituted C1 to C30 heteroaryl group, R ² 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 4, In Formula 1, n is the number of repeat units that is an integer from 1 to 4, X ¹ is a sulfur atom, R ¹ is a hydrogen atom or a 1C to 10C alkyl group, R ² is

ego, R ³ is a hydrogen atom or a 1C to 10C alkyl group, A pharmaceutical composition for preventing or treating cancer disease, wherein X ² is an oxygen atom or a sulfur atom. 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 for inhibiting one or more cancer cell lines selected from LIM1215, SW48 WT, LOVO, LS174T, NCIH1975 and NCIH838 and their KRAS mutant cells. A composition for preventing or treating cancer diseases. In clause 7, The KRAS mutant cells are one or more selected from LIM1215 KRAS G12D, SW48 KRAS G12D, LOVO KRAS G13D, SW48 KRAS G12V, LS174T KRAS G12D, NCIH1975 KRAS G12D, and NCIH838 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 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 disease, characterized in that the inhibition of the MAPK signaling pathway is performed by simultaneously suppressing the expression of BRAF and CRAF, which are isoforms of RAF in the RAS-RAF-MEK-ERK pathway. According to clause 10, A pharmaceutical composition for preventing or treating cancer disease, wherein the inhibition of the MAPK signaling pathway is performed by inhibiting p-ERK production following phosphorylation of ERK in the RAS-RAF-MEK-ERK signaling 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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