Novel small molecules for targeted degradation of untargetable KRAs in cancer therapy

Abstract

The present invention discloses a compound comprising the structure represented by Formula 1, wherein X is H or an alkyl group, Y is a carbonyl group bonded to a substituted / unsubstituted mono / heterocyclic ring having at least one double bond or a carboxyl group bonded to an alkyl group, Z is a carbonyl / carboxyl group bonded to a substituted / unsubstituted mono / heterocyclic ring having at least one double bond or a carbonyl group bonded to an alkene or an o-alkyl substituted carbamate or a branched alkyl group, and W is H or an amide group, or a carbonyl group bonded to a branched alkyl group, or any derivative thereof, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt thereof, or a combination thereof, which exhibits novel anticancer activity for treating subjects with chronic disorders. JPEG2023529354000044.jpg4333

Description

[Technical Field]

[0001] The present invention relates to pharmaceutical molecules, more particularly to compounds that exhibit novel anti-cancer activity for treating subjects with chronic disorders. [Background technology]

[0002] Cancer is a major health concern and a leading cause of death. Cancer prognosis is closely related to the stage of the disease. In the most advanced stages, where metastasis occurs, prognosis generally worsens and treatment is more likely to fail. Genomic instability and mutations have been identified as the primary mechanisms underlying the acquisition of these hallmarks. One of the most frequently mutated oncogenes in cancer is KRAS. The KRAS gene encodes a small GTPase that cycles between GDP- and GTP-bound states as a result of stimulation of specific cell surface receptors, such as EGFR. The most common KRAS mutations include G12C, G12D, G12R, G12S, G12V, G13D, and Q61H [Meng et al., 2013]. The most common mutation sites are codon 12 (80% of tumors), codon 13, and codon 61 [Friday et al., 2015]. Activating KRAS mutations are most common in pancreatic cancer (72%–90%), colorectal cancer (28%–57%), and lung cancer (15%–50%). In lung cancer, KRAS mutations are detected in 15%–20% of non-small cell lung cancers (NSCLCs), are most common in adenocarcinomas (30%–50%), are rare in small cell lung cancer, and are more common in smokers.

[0003] KRAS (v-Ki-ras2, Kirsten rat sarcoma viral oncogene homolog) is an oncogenic driver mutated in 30% of non-small cell lung cancers (NSCLCs). However, despite being identified as an oncogene for 30 years, no effective clinical drugs exist. KRAS is a potent initiator of tumorigenesis, a potent inducer of malignancy, and a predictive biomarker of response to therapy. It is one of the most frequently mutated oncogenes in cancer. This characteristic, combined with the failure of attempts to target this protein, has led to RAS being characterized as "undruggable." The RAS signaling network, organized at the cell membrane, bridges extracellular cues to cellular events such as cell growth, proliferation, differentiation, and survival [Cox et al., 2015] and plays a crucial role in the transition of healthy cells to cancer [Welsch ME et al., 2017]. All these findings have led the scientific community to exploit RAS or its downstream effectors as therapeutic targets in the hopes of impairing tumor growth and survival [Affolter et al., 2012]. However, effective inhibition of KRAS signaling has not been successful to date. Mutations that constitutively activate KRAS would lead to uncontrolled cell growth and cancer. However, despite vigorous efforts in recent years, no drugs that directly target KRAS and inhibit its abnormal function are commercially available [Westcott et al., 2015].

[0004] However, preclinical studies suggest that "K-Ras addiction" is reduced in mesenchymal cancer cells, indicating that direct KRAS inhibition may not be effective in all patients [Yin et al., 2019]. Alternatively, inhibitors targeting KRAS downstream kinases, such as BRAF and MEK, have shown promising activity in metastatic melanoma, but in combination with chemotherapy, KRAS mutations in cancers have been largely ineffective [Janne et al., 2017]. Apart from intrinsic resistance due to mesenchymal cancer cell differentiation, for example [Peng et al., 2019], the effectiveness of MEK inhibitors is limited by the development of acquired resistance [Haigis et al., 2017]. Another factor contributing to the difficulty of treating cancer cells is the heterogeneity of different KRAS mutations, defined by their respective amino acid substitutions. These alter the protein structure and GTPase activity of KRAS, substantially affecting tumor biology and response to chemotherapy [Ambrogio et al., 2018]. Therefore, there remains an unmet need to develop more effective targeted therapeutic strategies for patients with KRAS-mutated lung cancer. References 1.Overmeyer JH, Maltese WA.Death pathways triggered by activated Ras in cancer cells.Front Biosci(Landmark Ed).2011;16:1693-713. 2.Welsch ME, Kaplan A, Chambers JM, Stokes ME, Bos PH, Zask A, Zhang Y, Sanchez-Martin M, Badgley MA, Huang CS, et al.Multivalent small-molecule pan-RAS inhibitors.Cell.2017;168:878-889 e829 3.Affolter A,Drigotas M,Fruth K,Schmidtmann I,Brochhausen C,Mann WJ,Brieger J.Increased radioresistance via G12S K-Ras by compensatory upregulation of MAPK and PI3K pathways in epithelial cancer.Head Neck.2012. 4.Meng D,Yuan M,Li X,Chen L,Yang J,Zhao X,Ma W,Xin J.Prognostic value of K-RAS mutations in patients with non-small cell lung cancer:a systematic review with meta-analysis.Lung Cancer.2013;81:1-10. 5.A.D.Cox,C.J.Der,M.R.Philips Targeting RAS membrane association:back to the future for anti-RAS drug discovery? Clin Cancer Res,21(2015),pp.1819-1827 6.P.M.Westcott,K.D.Halliwill,M.D.To,M.Rashid,A.G.Rust,T.M.Keane,et al.The mutational landscapes of genetic and chemical models of KRAS-driven lung cancer Nature,517(2015),pp.489-492 7.Friday BB,Adjei AA.KRAS as a target for cancer therapy.BiochimBiophysi Acta.2005;1756:127-144. 8.P.A.Janne,M.M.van den Heuvel,F.Barlesi,M.Cobo,J.Mazieres,L.Crino,et al.Selumetinib plus docetaxel compared with docetaxel alone and progression-free survival in patients with KRAS-Mutant advanced non-small cell lung cancer:the SELECT-1 randomized clinical trial JAMA,317(18)(2017),pp.1844-1853 9.D.H.Peng,S.T.Kundu,J.J.Fradette,L.Diao,P.Tong,L.A.Byers,et al.ZEB1 suppression sensitizes kras mutant cancers to mek inhibition by an IL17RD-dependent mechanism Sci Transl Med,11(483)(2019) 10.C.Ambrogio,J.Kohler,Z.W.Zhou,H.Wang,R.Paranal,J.Li,et al.KRAS dimerization impacts mek inhibitor sensitivity and oncogenic activity of mutant kras Cell,172(4)(2018) 857-68 e15 11.K.M.Haigis KRAS alleles:the devil is in the detail Trends Cancer,3(10)(2017),pp.686-697 12. N. Yin, Y. Liu, A. Khoor, X. Wang, E. A. Thompson, M. Leitges, et al. Protein kinase ciota and wnt / beta-catenin signaling: alternative pathways to kras / trp53-driven lung adenocarcinoma Cancer Cell, 36(2)(2019)156 - 67.e7 [Prior Art Documents] [Non-Patent Documents]

[0005] [Non-Patent Document 1] Overmeyer JH, Maltese WA. Death pathways triggered by activated Ras in cancer cells. Front Biosci (Landmark Ed). 2011;16:1693 - 713. [Non-Patent Document 2] Welsch ME, Kaplan A, Chambers JM, Stokes ME, Bos PH, Zask A, Zhang Y, Sanchez-Martin M, Badgley MA, Huang CS, et al. Multivalent small-molecule pan-RAS inhibitors. Cell . 2017;168:878 - 889 e829 [Non-Patent Document 3] Affolter A, Drigotas M, Fruth K, Schmidtmann I, Brochhausen C, Mann WJ, Brieger J. Increased radioresistance via G12S K-Ras by compensatory upregulation of MAPK and PI3K pathways in epithelial cancer. Head Neck. 2012. [Non-Patent Document 4] Meng D, Yuan M, Li X, Chen L, Yang J, Zhao X, Ma W, Xin J. Prognostic value of K-RAS mutations in patients with non-small cell lung cancer: a systematic review with meta-analysis. Lung Cancer. 2013;81:1-10.

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

[0006] Objectives of the present invention: The primary objective of the present invention is to develop compounds that exhibit novel anti-cancer activity for treating subjects with chronic disorders.

[0007] The primary objective of the present invention is to synthesize compounds that exhibit novel anti-cancer activity for treating subjects with chronic disorders.

[0008] Yet another object of the present invention is to synthesize compounds that selectively target cancer stem cells and exhibit efficient anti-cancer activity in KRAS proteolysis.

[0009] A further object of the present invention is to utilize synthesized novel compounds exhibiting anti-cancer activity for treating subjects with chronic disorders.

[0010] It is a further object of the present invention to provide pharmaceutical compositions containing an effective amount of one or more of the compounds described herein.

[0011] It is yet another object of the present invention to provide a method for modulating, inhibiting, or degrading KRAS in a cell, or any combination thereof, by administering to the cell an effective amount of one or more compounds or pharmaceutical compositions described herein.

[0012] Yet another object provided herein is a method of treating a disease, disorder, or condition mediated by KRAS in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds or pharmaceutical compositions described herein.

[0013] In yet another object provided herein, an article of manufacture (e.g., a kit) comprises (i) an effective amount of one or more compounds or pharmaceutical compositions described herein, and (ii) instructions for use in treating a disease, disorder, or condition mediated by KRAS.

[0014] The foregoing and following information, as well as other features of the present disclosure, will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings, in which: The present disclosure will be described with additional specificity and detail through the use of the accompanying drawings, with the understanding that these drawings depict only some embodiments in accordance with the present disclosure and therefore are not to be considered limiting of its scope. [Brief explanation of the drawings]

[0015] [Figure 1] Figure 1 shows the spectra of Compound I: a. Mass spectrum b. IR spectrum c. 1H NMR spectrum d. 13C NMR spectrum [Figure 2] Figure 2 shows the spectra of Compound II: a. Mass spectrum b. IR spectrum c. 1H NMR spectrum d. 13C NMR spectrum [Figure 3] Figure 3 shows the spectra of compound III: a. Mass spectrum b. IR spectrum c. 1H NMR spectrum d. 13C NMR spectrum [Figure 4] Figure 4 shows the spectra of compound IV: a. Mass spectrum b. IR spectrum c. 1H NMR spectrum d. 13C NMR spectrum [Figure 5]Figure 5 shows the spectra of compound V: a. Mass spectrum b. IR spectrum c. 1H NMR spectrum d. 13C NMR spectrum [Figure 6] Figure 6 shows that Compound I induces apoptosis in KRAS-dependent cancer cell lines. Figure 6(A)—Morphological changes after 24 hours of exposure to Compound I (0, 50, 100, and 250 nm) were captured by epifluorescence microscopy at 100x magnification (scale bar: 20 μm). Figure 6(B)—Cells were treated with 250 nm Compound I for 24 hours, and apoptosis levels were quantitatively measured by flow cytometry after staining the cells with Annexin V / propidium iodide (PI). The triplicate data are plotted as a bar graph. Figure 6(C)—Representative Western blot data for different apoptosis-related proteins (PARP, Bcl-2, and Bax) confirmed the induction of cell apoptosis after 24 hours of treatment (0, 50, 100, and 250 nm). Densitometry of the ratio of Bax / Bcl-2 to cleaved PARP is shown as a bar graph. All data are representative of at least three independent experiments and presented as mean ± SD, *P<0.05, **P<0.01, ***P<0.001 compared with control. [Figure 7] Figure 7 shows Compound I inhibiting KRAS mutant expressing cancer cells. Figure 7A - Growth profile of KRAS-expressing cancer cells upon treatment with increasing concentrations of compound Compound I, monitored by cell viability assay. Figure 7B - Relative growth of KRAS mutant and wild-type cancer cells after treatment with IC50 concentration of compound Compound I. Data are presented as mean ± SD, significance was estimated by one-way ANOVA for sata vs. SKLU-1: *=P<0.02, **=P<0.005, ***=P<0.0001. [Figure 8] FIG. 8 shows the structural modeling of Compound I. [Figure 9] FIG. 9 shows that compound Compound I blocks GTP-KRAS formation in KRAS mutant cells. [Figure 10]FIG. 10 shows Compound I suppressing tumor growth in a xenograft model and Western blotting analysis of KRAS mutant protein in an in vivo animal model. [Figure 11] FIG. 11 shows Compound I targeting p53 (p53, Puma, and Noxa mRNA levels in cells 6 hours after Compound I treatment. mRNA levels were quantified by qRT-PCR. Data were normalized to GAPDH expression and plotted against cells treated with DMSO as a control). [Figure 12-1] FIG. 12 shows that Compound 1 was found to significantly inhibit cancer cell viability in all tested mutant cells in a dose-dependent manner. [Figure 12-2] Same as above. [Figure 13-1] FIG. 13 shows that Compound 1 was found to selectively inhibit the formation of GTP-KRAS (relative to total KRAS) in KRAS G12C, G12V, G12D, and G13D mutant cells. [Figure 13-2] Same as above. [Figure 14] FIG. 14 shows that Compound 1 blocked downstream effectors of RAS signaling in KRAS mutant cells. [Figure 15] FIG. 15 shows that Compound 1 was found to degrade RAS and inhibit the expression of stemness markers. [Figure 16] FIG. 16 shows a comparison of RAS reactivation in KRAS G12C mutant cell lines after treatment with Compound 1 and two known KRAS G12C inhibitors. [Figure 17-1] FIG. 17 shows a comparison of cell viability in KRAS G12C mutant cell lines after treatment with Compound 1 and two known KRAS G12C inhibitors. [Figure 17-2] Same as above. [Figure 18]Figure 18 shows regression of all mutant KRAS-driven cancers in a PDTX animal model. Treatment with compound 1 showed a 95% reduction in tumor growth within 28 days, with no evidence of recurrence for 6-8 months. [Figure 19] Figure 19 shows regression of all mutant KRAS-driven cancers in a PDTX animal model. Treatment with compound 1 showed a 95% reduction in tumor growth within 28 days, with no evidence of recurrence for 6-8 months. [Figure 20-1] Figure 20 shows the results of in vivo toxicity testing of Compound 1. Organ weights, body weight, liver weight, hematological parameters, serum electrolytes, LFT, KFT, and lipid parameters were found to be within normal ranges compared to the control group. Histopathology of harvested normal tissues (heart, liver, spleen, skeletal muscle, kidney, and intestine) showed no evidence of normal tissue toxicity after treatment with specific doses of Compound 1. [Figure 20-2] Same as above. [Figure 20-3] Same as above. [Figure 20-4] Same as above. [Figure 20-5] Same as above.

[0016] The present invention provides compounds of Formula 1: [ka] During the ceremony, X is H or an alkyl group; Y is a carbonyl group bonded to a substituted or unsubstituted mono- or heterocyclic ring having a carboxyl group bonded to at least one double bond or alkyl group; Z is a carbonyl / carboxyl group attached to a substituted / unsubstituted mono / heterocyclic ring having at least one double bond or a carbonyl group attached to an alkene or an o-alkyl substituted carbamate or branched alkyl group; W is H or an amide group or a carbonyl group attached to a branched alkyl group; or any derivative thereof, or a stereoisomer or tautomer thereof, a pharmaceutically acceptable salt thereof, or a combination thereof. DETAILED DESCRIPTION OF THE INVENTION

[0017] Definition: "Alkyl" or "alkyl group" refers to a fully saturated, straight or branched hydrocarbon chain having from one to fifteen carbon atoms and attached to the rest of the molecule by a single bond. Unless stated otherwise in the specification, an alkyl group can be optionally substituted.

[0018] An "alkene" or "alkene group" refers to a straight or branched hydrocarbon chain having two to fifteen carbon atoms and one or more carbon-carbon double bonds. Each alkene group is attached to the rest of the molecule by a single bond. Unless otherwise stated herein, the alkene chain may be optionally substituted.

[0019] A "homocycle" refers to a cyclic compound having at least three atoms of only one element, usually carbon, in the ring. Unless stated otherwise in the specification, a homocycle may be optionally substituted.

[0020] "Heterocycle" refers to a ring compound of at least three members having at least two different elements in the ring, usually carbon and either nitrogen or suphur or oxygen. Unless stated otherwise in the specification, heterocycles can be optionally substituted.

[0021] "Heteroalkyl group" refers to a fully saturated, straight or branched hydrocarbon chain having from one to fifteen carbon atoms, with at least one carbon replaced by a heteroatom, including a nitrogen, and attached to the remainder of the molecule by a single bond. Unless stated otherwise in the specification, a heteroalkyl group can be optionally substituted.

[0022] The carbonyl group is [ka] Refers to...

[0023] The carboxyl group is [ka] Refers to...

[0024] "o-Alkyl substituted carbamate" refers to a fully saturated, straight or branched hydrocarbon chain having from one to fifteen carbon atoms substituted at the o-position with a carbamate group.

[0025] A "carbamate" group refers to -HNCOO-.

[0026] As used herein, the term "substituted" means any of the above groups wherein at least one hydrogen atom is replaced by a bond to a non-hydrogen atom. [ka] (hereinafter may be referred to as "bond point") refers to a point of attachment that is a bond between two chemical entities, one of which is shown as being attached to the bond point and the other of which is not shown as being attached to the bond point.

[0027] Compounds provided herein that have the same molecular formula but differ in the type or sequence of bonds of their atoms or the arrangement of their atoms in space are referred to as "isomers." Isomers that differ in the arrangement of their atoms in space are referred to as "stereoisomers." Compounds of the present disclosure may also exist in different tautomeric forms. The term "tautomer" refers to interconvertible structural isomers. For example, in one variation, compounds provided herein may exhibit keto-enol tautomerism. In another variation, compounds provided herein may exhibit imine-enamine tautomerism.

[0028] "Pharmaceutically acceptable salts" includes pharmaceutically acceptable base addition salts and acid addition salts, where appropriate; as used herein, the term "pharmaceutically acceptable" implies that the salt is not biologically or otherwise undesirable, e.g., the material may be added to a pharmaceutical composition and administered to a subject without causing significant undesirable effects.

[0029] The term "therapeutically effective" as applied to a dose or amount refers to that amount of a compound or pharmaceutical preparation sufficient to result in a desired clinical benefit following administration to a patient in need thereof. It should be understood that an effective amount may be one or more doses, e.g., a single dose or multiple doses may be required to achieve a desired therapeutic endpoint.

[0030] As used herein, the term "treating" or "treatment" refers to a method or procedure for obtaining a beneficial or desired result, e.g., a clinical result. Beneficial or desired results may include (1) alleviating one or more symptoms caused by or associated with a disease, disorder, or condition, (2) reducing the severity of a disease, disorder, or condition, (3) slowing or halting the onset or progression of one or more symptoms caused by or associated with a disease, disorder, or condition (e.g., stabilizing a disease, disorder, or condition), and (4) alleviating a disease, e.g., by causing regression of one or more clinical symptoms (e.g., improving a disease state, enhancing the effect of another drug, slowing or halting the progression of a disease, increasing quality of life, and / or prolonging survival).

[0031] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like symbols typically identify like elements unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that aspects of the present disclosure, as generally described herein and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein.

[0032] The present invention discloses compounds that exhibit novel anti-cancer activity for treating subjects with chronic disorders.

[0033] In one preferred embodiment, the present invention provides a compound of Formula 1: [ka] During the ceremony, X is H or an alkyl group; Y is a carbonyl group bonded to a substituted or unsubstituted mono- or heterocyclic ring having a carboxyl group bonded to at least one double bond or alkyl group; Z is a carbonyl / carboxyl group attached to a substituted / unsubstituted mono / heterocyclic ring having at least one double bond or a carbonyl group attached to an alkene or an o-alkyl substituted carbamate or branched alkyl group; W is H or an amide group or a carbonyl group attached to a branched alkyl group; or any derivative thereof, or a stereoisomer or tautomer thereof, a pharmaceutically acceptable salt thereof, or a combination thereof.

[0034] According to the present invention, in the compound of formula 1, X is selected from a group comprising H or a C1-C5 alkyl group.

[0035] According to the present invention, in the compound of formula 1, Y is i. a carbonyl group attached to cyclopent-3-ene; ii. a carboxyl group bonded to a C1-C5 alkyl group; iii. Carbonyl group attached to 5-carbamoyl, 4,5-dihydro-1H-pyrrole iv. A carbonyl group bonded to 4,5-dihydro-1H-pyrrole-2-carbamoyl.

[0036] According to the present invention, in the compound of formula 1, Z is i-C1-C5 alkyl carbamates, ii.5 Carbamoyl, a carbonyl group attached to 2,3, dihydro 1H-pyrrole; iii. a carboxyl group attached to a pyridine; iv.C 1~ C5 alkene, v. A carbonyl group bonded to a C1-C6 branched alkyl group.

[0037] According to the present invention, in the compound of formula 1, W is selected from an amide group or a group containing a carbonyl group bonded to H or a C1-C5 branched alkyl group.

[0038] According to the present invention, in the compound of formula 1 X is H Y is a carbonyl group attached to cyclopent-3-ene Z is o-methyl carbamate W is an amide group.

[0039] According to the present invention, in the compound of formula 1 X is H Y is [ka] and Z is [ka] and W is [ka] is.

[0040] According to the present invention, in the compound of formula 1 X is a butyl group Y is a carboxyl group attached to an ethyl group Z is a carbonyl group attached to a 5-carbamoyl, 2,3-dihydro-1H-pyrrole W is H.

[0041] According to the present invention, in the compound of formula 1 X is [ka] and Y is [ka] and Z is [ka] and W is H.

[0042] According to the present invention, in the compound of formula 1 X is H Y is a carbonyl group attached to a 5-carbamoyl, 4,5-dihydro-1H-pyrrole Z is a carboxyl group attached to pyridine W is H.

[0043] According to the present invention, in the compound of formula 1 X is H Y is [ka] and Z is [ka] and W is H.

[0044] According to the present invention, in the compound of formula 1 X is H Y is a carbonyl group attached to 4,5-dihydro-1H-pyrrole-2-carbamoyl Z is an ethylene group W is a carbonyl group attached to a sec-propyl group.

[0045] According to the present invention, in the compound of formula 1 X is H Y is [ka] and Z is [ka] and W is [ka] is.

[0046] According to the present invention, in the compound of formula 1 X is H Y is a carbonyl group attached to 4,5-dihydro-1H-pyrrole-2-carbamoyl Z is a carbonyl group attached to a tertiary butyl group W is H.

[0047] According to the present invention, in the compound of formula 1 X is H Y is [ka] and Z is [ka] and W is H.

[0048] In a further preferred embodiment, the present invention discloses a pharmaceutical composition comprising an effective amount of one of the above-mentioned compounds, or a stereoisomer or tautomer thereof, and a pharmaceutically acceptable carrier thereof.

[0049] In another preferred embodiment, the present invention discloses an article of manufacture comprising (i) an effective amount of any of the above-described compounds, or stereoisomers or tautomers thereof, or the above-described pharmaceutical compositions, and (ii) instructions for use in treating a disease, disorder, or condition mediated by KRAS.

[0050] According to the present invention, KRAS comprises a G12C mutation, a G12D mutation, a G13D mutation, or a G12V mutation, or any combination thereof, in the product.

[0051] According to the present invention, in the product, the disease, disorder, or condition is cancer.

[0052] According to the present invention, in the product, the cancer is non-small cell lung cancer, colorectal cancer, triple-negative breast cancer, or pancreatic cancer, or any combination thereof.

[0053] In yet another preferred embodiment, the present invention discloses a kit comprising (i) an effective amount of any of the above-described compounds, or stereoisomers or tautomers thereof, or the above-described pharmaceutical compositions, and (ii) instructions for use in treating a disease, disorder, or condition mediated by KRAS.

[0054] According to the present invention, the KRAS comprises a G12C mutant, a G12D mutant, a G13D mutant, or a G12V mutant, or any combination thereof, in the kit.

[0055] According to the present invention, in the kit, the disease, disorder, or condition is cancer.

[0056] According to the present invention, in the kit, the cancer is non-small cell lung cancer, colorectal cancer, triple-negative breast cancer, or pancreatic cancer, or any combination thereof.

[0057] In another preferred embodiment, the present invention discloses a method for regulating, inhibiting, or degrading KRAS in a cell, or any combination of the foregoing, comprising (i) exposing the cell to an effective amount of the compound described above, or a stereoisomer or tautomer thereof, or the pharmaceutical composition described above.

[0058] According to the present invention, KRAS comprises a G12C mutation, a G12D mutation, a G13D mutation, or a G12V mutation, or any combination thereof, in the above methods.

[0059] In another preferred embodiment, the present invention discloses a method for treating a disease, disorder, or condition mediated by KRAS in a subject in need thereof, comprising administering to the subject (i) an effective amount of the compound described above, or a stereoisomer or tautomer thereof, or the pharmaceutical composition described above.

[0060] According to the present invention, KRAS comprises a G12C mutation, a G12D mutation, a G13D mutation, or a G12V mutation, or any combination thereof, in the above-mentioned methods.

[0061] According to the present invention, in the above-mentioned method, the disease, disorder or condition is cancer.

[0062] According to the present invention, in the above-mentioned method, the cancer is non-small cell lung cancer, triple-negative breast cancer, colorectal cancer, or pancreatic cancer, or any combination thereof.

[0063] The chemical structures of compounds of Formula 1 can be prepared by routine chemistry based on the exemplary structures provided herein.

[0064] In one embodiment, exemplary compound II can be prepared according to Scheme I provided below. [ka]

[0065] The synthesized compound of formula 1 was then subjected to molecular characterization to confirm the structure of the compound.

[0066] Molecular characterization: Melting point, thin layer chromatography, HPLC, IR, 13 C, Purity of the synthesized products confirmed by mass spectrometry and NMR analysis. From Figures 1-5, the structures of the synthesized compounds of Formula 1 are revealed as compounds I-V as shown in Table 1 below. [Table 1-1] [Table 1-2]

[0067] In some embodiments, the compound of Formula 1 and compounds I-V may include both cis and trans isomers. In some embodiments, the compound of Formula 1 and compounds I-V may be a mixture of cis and trans isomers. In some embodiments, the compound of Formula 1 and compounds I-V may be a cis isomer. In some embodiments, the compound of Formula 1 and compounds I-V may be a trans isomer.

[0068] In some embodiments, the compounds of Formula 1 and compounds I-V may include either R or S stereoisomers and mixtures of stereoisomers. In some embodiments, the compounds of Formula (I) may include both racemic and enantiomeric isomers.

[0069] The compounds of the present invention can be used to perform or provide any of the biological functions described herein.

[0070] Pharmaceutical Composition The present disclosure also includes pharmaceutical compositions comprising a therapeutically effective amount of one or more compounds disclosed herein. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of one or more compounds of Formula 1, or pharmaceutically acceptable salts thereof. In other embodiments, the pharmaceutical composition comprises a therapeutically effective amount of one or more compounds selected from Table 1, or pharmaceutically acceptable salts thereof. In some embodiments, the aforementioned pharmaceutical compositions further comprise one or more pharmaceutically acceptable excipients.

[0071] In various embodiments, the compound of Formula 1 (including the compounds of Table 1) or a pharmaceutically acceptable salt thereof can be administered at about 0.001 mg / kg to about 100 mg / kg body weight (e.g., about 0.01 mg / kg to about 10 mg / kg or about 0.1 mg / kg to about 5 mg / kg). In some embodiments, the compound of the present invention is administered at about 200 mg / kg to about 2000 mg / kg, e.g., about 200 mg / kg, about 500 mg / kg, about 1000 mg / kg, or about 2000 mg / kg. In some embodiments, the compound of the present invention is administered at about 100 mg / kg to about 600 mg / kg, e.g., about 100 mg / kg, about 200 mg / kg, about 300 mg / kg, about 450 mg / kg, or about 600 mg / kg.

[0072] The concentration of the disclosed compounds in a pharmaceutically acceptable mixture will vary depending on several factors, including the dosage of the compound administered, the pharmacokinetic characteristics of the compound used, and the route of administration.The drug can be administered in a single dose or multiple doses.The administration regimen utilizing the compounds of the present invention is selected according to various factors, including the type, species, age, weight, sex, and medical condition of the patient, the severity of the condition being treated, the route of administration, the patient's renal and hepatic function, and the specific compound or its salt used.Treatment can be administered once a day or more frequently, depending on many factors, including the patient's overall health, and the formulation and route of administration of the selected compound.

[0073] The compounds or pharmaceutical compositions of the disclosure may be prepared and / or administered in single or multiple unit dosage forms.

[0074] The compounds or pharmaceutical compositions of the disclosure may be prepared and / or administered in single or multiple unit dosage forms.

[0075] The compounds or pharmaceutical compositions of the present disclosure may be administered by a variety of methods, including, for example, oral, rectal, buccal, intranasal, and transdermal routes. In certain embodiments, the pharmaceutical compositions may be administered intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, orally, topically, or as an inhalant.

[0076] The compounds and pharmaceutical compositions of the present disclosure may be administered to an individual in any form of commonly accepted oral composition (e.g., tablets, coated tablets, gel capsules in hard or soft shell, emulsions or suspensions).

[0077] Treatment method: Also provided herein are methods for treating a chronic disorder in a human in need thereof, comprising administering to the human a compound of Formula 1, (I), (II), (III), (IV), or (V), including, for example, compounds 1-5, or a pharmaceutically acceptable salt thereof. In some embodiments, a compound of the present disclosure (a compound of Formula 1 and Table 1) is administered to a patient with a chronic condition.

[0078] In some embodiments, provided herein are methods of modulating, inhibiting, or degrading KRAS in a cell, or any combination thereof, comprising exposing the cell to (i) an effective amount of a compound of Formula 1, (I), (II), (III), (IV), or (V), e.g., compounds 1-5, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any of the foregoing, or (ii) a pharmaceutical composition comprising an effective amount of a compound of Formula 1, (I), (II), (III), (IV), or (V), e.g., compounds 1-5, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any of the foregoing, and one or more pharmaceutically acceptable excipients. In some of the foregoing embodiments, methods of modulating KRAS in a cell are provided. In some embodiments, methods of inhibiting KRAS in a cell are provided. In still other embodiments, methods of degrading KRAS in a cell are provided. In some embodiments, methods of modulating and degrading KRAS in a cell are provided. In some embodiments, the present application provides a method for inhibiting and degrading KRAS in cells.In some embodiments, KRAS comprises G12C mutation, G12D mutation, G13D mutation, or G12V mutation, or any combination thereof.In some embodiments, KRAS comprises G12C mutation.

[0079] In some embodiments, provided herein are methods of treating a disease, disorder, or condition mediated by KRAS in a subject in need thereof, comprising administering to the subject (i) an effective amount of a compound of Formula 1, (I), (II), (III), (IV), or (V), e.g., including compounds 1-5, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any of the foregoing, or (ii) a pharmaceutical composition comprising an effective amount of a compound of Formula 1, (I), (II), (III), (IV), or (V), e.g., including compounds 1-5, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any of the foregoing, and one or more pharmaceutically acceptable excipients.

[0080] In some embodiments, the treatment methods described herein result in a change (%) in the total diameter of target lesions in a subject.In some embodiments, the change is measured on the 1st day, 14th day, 21st day, 28th day, or 6th month after treatment, or any combination of the above (up to 6 cycles).In some embodiments, the change is measured on the 21st day of treatment (up to 6 cycles).

[0081] In some embodiments, the methods of treatment described herein result in a reduction in the number of circulating tumor cells in a subject. In one variation, the methods of treatment result in a reduction in the number of circulating tumor cells in a subject with breast cancer, pancreatic cancer, or non-small cell lung cancer up to six months after treatment.

[0082] In some embodiments, the treatment methods described herein result in a change in the level of one or more markers of apoptosis in a subject. In some embodiments, the treatment methods described herein result in a change in the level of one or more markers of inflammation in a subject. In some embodiments, the treatment methods described herein result in a change in the level of one or more pathway markers in a subject (as determined by immunohistochemistry (IHC)). In some embodiments, the treatment methods described herein result in a change in the immune response in a subject. For example, in some embodiments, the treatment methods described herein result in a change in the level of interferon gamma (IFN)-producing T cells, cytotoxic T lymphocyte (CTL) responses, cytokines (IFN, IL-4, IL-10), or activation markers, or any combination thereof. In some of the foregoing changes, the change may be measured at 1 day, 14 days, 21 days, 28 days, or 6 months after treatment, or any combination of the foregoing (up to 6 cycles).

[0083] In some embodiments, the disease, disorder, or symptom is mediated by KRAS G12C mutation, KRAS G12D mutation, KRAS G13D mutation, or KRAS G12V mutation, or any combination thereof.In some embodiments, the disease, disorder, or symptom is mediated by KRAS G12C mutation.In some embodiments, the disease, disorder, or symptom is cancer.

[0084] In connection with some embodiments of the present invention, the term "chronic disorder" or the term "disease, disorder, or condition" refers to, but is not limited to, acute lymphocytic, acute lymphocytic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, appendix cancer, basal cell carcinoma, bladder cancer, brain cancer, brain stem glioma, breast cancer, bronchial adenoma / carcinoid, Burkitt's lymphoma, carcinoid tumor, cerebellar or cerebral astrocytoma, cervical cancer, bile duct carcinoma, chondrosarcoma, chronic lymphocytic or chronic lymphocytic leukemia, chronic myelogenous or chronic myelogenous leukemia, chronic myeloproliferative disorder, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma , Esophageal cancer, Ewing's sarcoma, Extracranial germ cell tumors, Extragonadal germ cell tumors, Extrahepatic bile duct cancer, Gallbladder cancer, Gastric (stomach) cancer, Gastrointestinal carcinoid tumors, Gastrointestinal stromal tumors (GIST), Gestational trophoblastic tumors, Brain stem glioma, Hairy cell leukemia, Head and neck cancer, Heart cancer, Hepatocellular (liver) cancer, Hodgkin's lymphoma, Hypopharyngeal cancer, Hypothalamic and optic glioma, Intraocular melanoma, Pancreatic islet cell carcinoma, Kaposi's sarcoma, Laryngeal cancer, Leukemia, Lip and oral cancer, Liposarcoma, Lymphoma, Male breast cancer, Malignant mesothelioma, Medulloblastoma, Melanoma, Merkel cell skin cancer, Mesothelioma, Metastatic squamous cell carcinoma of the neck, Oral Cavity cancer, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic / myeloproliferative disorders, nasal and paranasal cavity cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-melanoma skin cancer, non-small cell lung cancer, oligodendroglioma, oral cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma, ovarian cancer, ovarian germ cell tumor, ovarian epithelial cancer (surface epithelial-stromal tumor), ovarian low malignant potential tumor, pancreatic cancer, paranasal sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma and supratentorial genitourinary Primary neuroectodermal tumors, pituitary adenomas, plasma cell tumors, pleuropulmonary blastomas, primary carcinomas, primary central nervous system lymphomas, primary liver cancer, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Sézary syndrome, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, supratentorial primitive neuroectodermal tumors, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, vulvar cancer, Waldenstrom's macroglobulinemia,Wilms' tumor, Parkinson's disease and parkinsonian disorders, Huntington's disease, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, Shy-Drager syndrome, progressive supranuclear palsy, Lewy body disease, spinal cord ischemia, spinal cord injury, ischemic stroke, cerebral infarction, spinal cord injury, and cancer-related brain and spinal cord injury, multi-infarct dementia, senile dementia, other cognitive disorders, depression, onychomycosis (fungal infection of the nails, gingivitis and periodontal disease (gum disease)), obesity, and diabetes. In some embodiments, the disease, disorder, or condition is non-small cell lung cancer, triple-negative breast cancer, or pancreatic cancer, or any combination thereof. Compounds of Formula 1, (I), (II), (III), (IV), or (V), including one or more of Compounds 1-5, or pharmaceutically acceptable salts thereof, may also prove useful in the treatment of SARS and COVID-19, as well as the treatment of KRAS oncogene mutations in the different cancers listed above.

[0085] In some embodiments, the chronic condition is cancer. In some embodiments, the cancer is colon cancer, prostate cancer, breast cancer, or leukemia. In some embodiments, the cancer is stage 4 cancer. In some embodiments, the colon cancer, prostate cancer, breast cancer, or leukemia is stage 4. In some embodiments, the chronic condition is a KRAS oncogene mutation in a different cancer.

[0086] In certain embodiments, the methods, compounds, and compositions described herein are administered in combination with one or more other antibody molecules, chemotherapy, other anti-cancer therapies (e.g., targeted anti-cancer therapies, gene therapy, viral therapy, RNA therapy, bone marrow transplant, nanotherapy, or oncolytic drugs), cytotoxic drugs, immune-based therapies (e.g., cytokines or cell-based immunotherapy), surgery (e.g., lumpectomy or mastectomy) or radiation therapy, or a combination of any of the foregoing.

[0087] Alternatively, or in combination with the aforementioned combinations, the methods and compositions described herein may be administered in combination with one or more vaccines, such as, for example, a therapeutic cancer vaccine, or other forms of cellular immunotherapy.

[0088] In some embodiments, provided herein is the use of a compound or pharmaceutical composition described elsewhere herein in any of the methods described elsewhere herein. In other embodiments, provided herein is a compound or pharmaceutical composition described elsewhere herein for use in the manufacture of a medicament for use in any of the methods described elsewhere herein. In other embodiments, provided herein is a compound or pharmaceutical composition described elsewhere herein for use in any of the methods described herein.

[0089] The present invention aims to invent potent therapeutic small molecules capable of targeting mutant KRAS at its active site. Here, we identified Compound I, a small molecule that binds to the GTP / GDP-binding pocket of KRAS. To this end, we investigated the strong correlation of measurements across a larger panel of tumor cell lines expressing KRAS mutations to analyze its potency in inhibiting tumor cell growth-activated RAS. The growth inhibitory effect of Compound I was sustained and irreversible, as demonstrated by colony formation and apoptosis assays. Compound I possessed good binding affinity to KRAS in vitro and exhibited selective cytotoxicity in oncogenic KRAS-expressing cell lines, with no or minimal toxic effects on normal cell lines. Further mechanistic studies demonstrated that Compound I could block the formation of a complex between guanosine triphosphate (GTP) and KRAS in vitro. Furthermore, Compound I inhibited the KRAS downstream signaling pathways RAF / MEK / ERK and RAF / PI3K / AKT. Compound I induced mitotic arrest as measured by cell cycle analysis of DNA content and phosphohistone H3B immunofluorescence. Further analysis revealed that compound I interfered with the localization of mitogenic proteins, PLK1 to kinetochores, and reduced the nuclear localization of its substrate Cdc25C, downstream targets of RAS-RAF signaling involved in both mitotic entry and exit checkpoints. Compound I also suppressed PD-L1 expression and activated antitumor immunity, which may contribute to its antitumor activity and suggest potential benefits of combining it with immunotherapy. These studies identified a novel class of RAS inhibitors that potently and selectively inhibit RAS-driven tumor growth by disrupting downstream signaling, resulting in cell cycle arrest and apoptosis. Therefore, compound I may be considered a potential KRAS inhibitor for the treatment of cancer cells carrying the KRAS oncogene.

[0090] product Articles of manufacture are also provided herein and comprise a compound of Formula 1, (I), (II), (III), (IV), or (V) as described elsewhere herein, or any variation or embodiment thereof, including, for example, compounds 1-5, or stereoisomers or tautomers thereof, or pharmaceutically acceptable salts of any of the foregoing, in a suitable container. Articles of manufacture are also provided herein and comprise a pharmaceutical composition comprising a compound of Formula 1, (I), (II), (III), (IV), or (V) as described elsewhere herein, or any variation or embodiment thereof, including, for example, compounds 1-5, or stereoisomers or tautomers thereof, or pharmaceutically acceptable salts of any of the foregoing, in a suitable container. The container may be a vial, bottle, ampoule, pre-loaded syringe, or intravenous bag.

[0091] The present disclosure further provides a kit for carrying out the method of the present invention. The kit may include a compound described herein or a pharmaceutically acceptable salt thereof and suitable packaging. The kit may include one or more containers containing any compound described herein. In one embodiment, the kit includes a compound of the present disclosure or a pharmaceutically acceptable salt thereof, and a label and / or instructions for using the compound in the treatment of a disease or disorder described herein. The kit may also include a unit dosage form of the compound.

[0092] Provided herein are articles of manufacture (e.g., kits) that include (i) an effective amount of a compound of Formula 1, (I), (II), (III), (IV), or (V), including, for example, compounds 1-5, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any of the foregoing, and (ii) instructions for use in treating a disease, disorder, or condition mediated by KRAS. Provided herein are articles of manufacture (e.g., kits) that include (i) an effective amount of a compound of Formula 1, (I), (II), (III), (IV), or (V), including, for example, compounds 1-5, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any of the foregoing, and one or more pharmaceutically acceptable excipients, and (ii) instructions for use in treating a disease, disorder, or condition mediated by KRAS. [Example]

[0093] Biological Example 1 Compound 1 induces apoptosis in KRAS-dependent cancer cell lines (Figure 6). We subjected a cancer induction model to compound 1 to determine whether the observed growth inhibition was due to apoptosis or necrosis. As shown in Figure 6A, after treatment with compound 1, the cell morphology of mutant KRAS cell lines changed in a concentration-dependent manner, and the percentage of cells that detached from the culture dish and collected was significantly higher, a predictive feature of apoptosis. Next, standard Annex in V-FITC / PI staining was applied, followed by flow cytometry analysis to quantitatively measure the percentage of apoptotic and necrotic cells. The results showed that compound 1 significantly induced apoptosis in mutant cancer cells compared to untreated cells (Figure 6C). To further investigate compound-induced apoptosis in KRAS mutant cells, we analyzed the expression levels of several well-characterized apoptotic proteins by Western blotting. Results showed that increased expression of the pro-apoptotic protein Bax and decreased expression of the anti-apoptotic Bcl-2 were observed with compound 1 in treated cells. Furthermore, compound 1 treatment led to the induction of PARP cleavage, consistent with apoptosis (Figure 6B). Densitometric quantification of the ratio of Bax / Bcl2 protein expression and PARP cleavage to GAPHD is shown as a bar graph in Figure 6B. Collectively, these results demonstrated that compound 1 induces apoptosis in KRAS mutant cell lines.

[0094] Biological Example 2 Compound 1 inhibits KRAS mutant-expressing cancer cells (Figures 7 and 12). To further investigate whether compound 1 can also induce cytotoxic effects in other cancer cells expressing KRAS mutations, a panel of cancer cell lines harboring KRAS G12D, G12V, and G12C point mutations, respectively, was selected and used alongside a KRAS wild-type (WT) control. As shown in Figure 7, 24 hours after treatment with compound 1, compound 1 significantly inhibited cancer cell and stem cell viability in all mutant cells in a dose-dependent manner, with IC50 values ​​found to be nanomolar. Similarly, minimal toxicity was observed in normal cell lines. Collectively, the data from the cell line studies suggested that compound 1 more efficiently inhibited signaling through KRAS, consistent with its tight binding to activated KRAS and its effect on the KRAS-Raf interaction.

[0095] The cytotoxicity of Compound 1 was initially evaluated in a panel of 30+ cell lines harboring wild-type (WT) KRAS or known KRAS mutations. Compound 1 was found to significantly inhibit cancer cell viability in all mutant cells in a dose-dependent manner. The IC50 value of Compound 1 was found to be in the micromolar to nanomolar range (see Figure 12).

[0096] Biological Example 3 Structural modeling of compound 1 in the GTP / GDP binding pocket of the KRAS protein (Figure 8). Compound 1 binds to wild-type and oncogenic KRAS mutants with high affinity. Figure 8 shows the chemical structure and predicted complex of compound 1 with KRAS, suggesting that the ligand potentially forms multiple favorable interactions with residues in the p1 pocket.

[0097] Biological Example 4 Compound Compound 1 blocked GTP-KRAS formation in KRAS mutant cells (Figures 9 and 13) In fact, mutant KRAS disrupts the balance between GEFs and GAPs, resulting in the fixation of active GTP-bound KRAS and the abnormal stimulation of downstream signaling. Therefore, KRAS inhibitors should reduce the formation of GTP-KRAS to disrupt mutant KRAS function. To determine whether Compound 1 can inhibit KRAS activation, a RAS activation assay was performed to examine the formation of GTP-bound KRAS in KRAS mutant cells after 24 hours of treatment with a range of concentrations of Compound 1. Compound 1 treatment inhibited the formation of GTP-KRAS in KRAS mutant cells compared with the total amount of KRAS, suggesting that this small molecule can partially restore the imbalance caused by mutant KRAS.

[0098] Compound 1 was found to inhibit the formation of GTP-KRAS (relative to the total amount of KRAS) in KRAS G12C, G12V, G12D, and G13D mutant cells. Compound 1 was found to selectively target mutant forms of KRAS, with no effect observed in cells with wild-type (WT) KRAS (see Figure 13).

[0099] Biological Example 5 Compound 1 inhibited the activation of the KRAS downstream signaling pathway (Figures 9 and 14). Active KRAS stimulates downstream signaling pathways, particularly the RAF / MEK / ERK and RAF / PI3K / AKT pathways, which then induces cell proliferation. Therefore, to investigate the effects of compound 1, we examined the phosphorylation levels of CRAF, AKT, and ERK in cell lines and monitored the impact of KRAS signaling upon 48-hour treatment with this compound. As expected, the molecule reduced the phosphorylation levels of CRAF and AKT in a time-dependent manner in KRAS-mutant cell lines (Figures 9 and 14), indicating that the molecule can block oncogenic KRAS function by inhibiting its downstream signaling pathways.

[0100] Biological Example 6 Compound 1 inhibits tumor growth in xenograft models (Figure 10) The in vivo efficacy of Compound 1 in the context of mutant KRAS was next evaluated. Nude mice were injected with cells and tumors grew to approximately 60 mm 3 The tumors were grown to a size of 100 μg / ml and treated daily with Compound 1 for 14 to 21 days. Figure 10 shows that Compound 1 inhibited tumor growth compared to vehicle-treated controls, starting on day 9 and continuing to show significant inhibition from days 10 to 14. On the day of harvest, net tumor mass was determined, and the mean tumor weight in the Compound 1-treated group was 89% lower than that in the control group. Mice (n = 6) did not exhibit significant weight loss or overt toxicity after treatment with Compound 1. We further examined the effects of Compound 1 treatment on KRAS-mediated RAF, MEK / ERK, and PI3K / AKT cascades in protein extracts derived from vehicle- and Compound 1-treated tumors. Consistent with the in vitro data, significant inhibition of phosphorylation of CRAF, ERK, and AKT was observed. Immunohistochemical analysis also demonstrated that treatment with Compound 1 reduced the levels of ERK and AKT phosphorylation, indicating that growth inhibition induced by Compound 1 is associated with the suppression of KRAS-mediated signaling. Furthermore, immunohistochemical analysis of tumors from mice treated with compound 1 showed a significant decrease in cell proliferation, as indicated by Ki-67 staining, and a significant increase in apoptotic cells, as indicated by cleaved caspase-3 staining. Collectively, these data demonstrated that compound 1 is effective in suppressing KRAS-driven lung tumor growth.

[0101] Biological Example 7 Compound 1 targets p53 (Figure 11) The tumor suppressor gene TP53 (better known as p53) regulates several cellular stresses, including DNA damage, hypoxia, and oncogene activation. Functionally, the p53 protein acts as a transcription factor, controlling gene expression by binding to specific DNA sequences. Classically, p53 has been implicated in the regulation of genes involved in apoptosis, senescence, and cell cycle arrest, and plays a role in necrosis, autophagy, metabolism, reactive oxygen species (ROS) accumulation, and stem cell maintenance. Mutations and deletions of p53 are common in a variety of cancers, including those of the lung, head and neck, bladder, breast, and prostate.

[0102] Compound 1 has been shown to induce apoptosis by upregulating the expression of p53, Bax, Bak, PUMA, Noxa, and Bim, and downregulating Bcl-2 and Bcl-XL in androgen-dependent and -independent cancer cells, but it had no effect on normal fibroblasts or epithelial cells. Furthermore, compound 1 induced p53 translocation to mitochondria and Smac release into the cytoplasm, strongly suggesting its cancer chemopreventive properties. By modifying p53 and its downstream proteins (p21, cyclin B1, CDK1, Cdc25C) and several apoptosis-related proteins (Bcl-2, Bax, Bid, Bad, Apaf1, AIF, and Cyt c), compound 1 also downregulated oxidative stress-induced heat shock proteins (HSPs) and histone deacetylase 6 (HDAC6), which further led to apoptosis. Compound 1 demonstrated inhibitory activity in p53 mutant or overexpressed in vitro and in vivo preclinical models.

[0103] Biological Example 8 Compound 1 targets EGFR and HER2 The activity of compound 1 against various mutant EGFRs, HER2, and HER4 was investigated. The effects of compound 1 on different genetic features and associated molecular mechanisms were investigated using MTT assays, flow cytometry, and Western blotting. The in vivo antitumor activity of compound 1 was evaluated using nude mouse xenograft models bearing cells. The results showed that compound 1 effectively inhibited the enzymatic activity of EGFR family members, including drug-sensitive EGFR mutations, EGFR C797S mutations, and wild-type (WT) HER2. Compound 1 blocked EGFR phosphorylation, thereby downregulating the downstream PI3K / AKT and MAPK / ERK signaling pathways and inducing G0 / G1 arrest in different cell lines. Compound 1 inhibited tumor growth in mouse xenograft models. In summary, these findings suggest that compound 1 has potential as an oral antitumor agent for treatment and is worthy of further development.

[0104] Due to defects in homologous recombination (HR) repair, BRCA1- and BRCA2-mutated tumors accumulate DNA damage and genomic rearrangements that promote tumor progression. To identify drugs that specifically target BRCA2-deficient cells, compound 1 was demonstrated to be particularly toxic to cisplatin-resistant BRCA1 / 2-deficient cells, suggesting its potential clinical use in diseases that have become resistant to these drugs. [Table 2]

[0105] ++++ means very strong expression of EGFR or HER2. +++ means strong expression of EGFR or HER2. ++ means moderate expression of EGFR or HER2. + means low expression of EGFR or HER2. + means no expression of EGFR or HER2.

[0106] Biological Example 9 Interaction of compound 1 with KRAS GTP Based on these findings, a novel small molecule compound, 1, competitively bound to the K-Ras pocket in the switch I region with high affinity, inhibiting KRAS binding to downstream effectors such as RAF / PI3K, which are involved in cancer cell progression. Based on its ability to compete with GTP binding to KRas, it blocked K-Ras signaling and inhibited cell proliferation in several KRAS tumor-derived human cell lines, and demonstrated a 95% reduction in tumor growth inhibition in in vivo animal models within 28 days, with no evidence of recurrence for 6-8 months, compared to standard drugs. [Table 3]

[0107] Biological Example 10 Compound 1 downstream signaling – effects of wild type Compound 1 has no effect on wild-type KRAS GTP binding affinity, and the molecule does not alter the expression of downstream KRAS signaling proteins, such as MEK, RAF, and PI3K. Effector proteins, such as GEFs and GAPs, are intact at highly regulated levels, as observed by protein and gene expression analysis. A significant increase in immune cell expression was observed with Compound 1 treatment. [Table 4]

[0108] Biological Example 11 Figure 3: Effect of compound 1 on RAS degradation Interestingly, Compound 1 degrades RAS through negative regulators such as APC, axin, and glycogen synthase kinase 3β (GSK3B) via phosphorylation of GSK3β through a crosstalk mechanism with the Wnt / β-catenin pathway (Figure 15 and Table 5). [Table 5]

[0109] Biological Example 12 The role of miR-30c and miR-21 in RAS degradation and cancer stem cells. Mutant KRAS induced significant upregulation of miR-30c and miR-21. miR-30c and miR-21 are significantly upregulated by both KRAS isoforms, induce drug resistance, and enhance cell migration / invasion through the inhibition of key tumor suppressor genes, such as NF1, RASA1, BID, and RASSF8. We observed the effect of Compound 1 on the expression of miR-30c and miR-21 in mutant and wild-type KRAS. Microarray analysis showed that the expression of these miRs was significantly reduced with Compound 1 treatment, significantly increasing the expression of tumor suppressor genes at a dose-dependent level in mutant KRAS, while no significant expression was observed in wild-type KRAS. Based on these observations, our molecule targets miRNAs in KRAS mutant models, serving as attractive therapeutic tools in cancer medicine because these miRNAs can silence multiple genes and therefore block different pathways simultaneously, which is not possible with protein-based drugs and other covalent inhibitors of different KRAS mutant types. Finally, compound 1 has no toxicity or off-target effects compared to other standard drugs. [Table 6]

[0110] Biological Example 13 Oral bioavailability data / Crossing blood-brain barrier data for compound 1 [Table 7] [Table 8]

[0111] Biological Example 14 Comparison of Compound 1 with known KRAS inhibitors We compared the reactivation of RAS signaling following administration of compound 1 and two known KRAS inhibitors (AMG510 and MRTX849). In all KRAS G12C mutant cell lines, compound 1, AMG510, and MRTX849 suppressed MAPK pathway signaling, a key downstream effector pathway of KRAS, as measured by inhibition of phosphor-MEK, phosphor-ERK, and phosphor-AKT at 4 hours. At 24–48 hours, RAS-MAPK pathway signaling began to rebound in cells treated with AMG510 and MRTX849, resulting in pathway reactivation and incomplete suppression of pMEK, pERK, pAKT, and MYC by 72 hours. The rapid and consistent reactivation of signaling observed after KRAS G12C inhibition suggests that adaptive feedback may limit the effectiveness of these inhibitors. In contrast, administration of compound 1 resulted in a significant decrease in the phosphorylated forms, with no rebound activity observed by 72 hours (see Figure 16). Densitometry of phosphor-ERK, MEK, AKT, and MYC is normalized to GAPDH. Results represent the average across eight cell lines.

[0112] The cytotoxic effects of Compound 1, AMG510, and MRTX849 on an NCI panel of 10 KRAS G12C mutant cell lines were also compared. Compound 1 induced apoptosis in a dose-dependent manner and showed a significant reduction in cancer cell numbers compared to AMG510 and MRTX849 (see Figure 17).

[0113] Biological Example 15 Compound 1: In vivo tumor model Compound 1 was found to cause regression of all mutant KRAS-driven cancers in immune-competent mice. Compound 1 suppressed tumor growth beginning on day 9 and showed significant inhibition from days 10-14 in all mutant KRAS-expressing animal models. Compound 1 was also found to inhibit downstream signaling. Treatment with Compound 1 showed a 95% reduction in tumor growth within 28 days, with no evidence of recurrence for 6-8 months. See Figures 18 and 19.

[0114] Biological Example 16 Compound 1: Toxicity testing Histopathology of harvested normal tissues (brain, heart, lung, liver, spleen, kidney, and intestine) showed no evidence of normal tissue toxicity after treatment with the specified doses of Compound 1. Tests of blood cells (WBC, RBC, and PLT) for bone marrow, RFT for kidney, and ALT / AST for liver function all had results within the normal range. See Figure 20.

[0115] Biological Example 17 Compound 1: Clinical Trial Protocol This study is an open-label, two-part, first-in-human (FIH) dose-ranging study designed to determine the safety, tolerability, pharmacokinetics (PK), pharmacology (PD), and proof-of-concept (POC) of Compound 1 in patients with advanced or metastatic solid tumors. The study consists of two parts: Part 1: Dose escalation in patients with advanced or metastatic solid tumors, including dose levels of Compound 1. The study is planned to begin dose escalation at 50 mg, followed by tentatively designated escalation dose groups of 100 mg, 200 mg, 300 mg, 450 mg, and 600 mg. Part 1 will enroll approximately 11-24 patients in total, covering five dose levels.

[0116] The primary objectives are to determine the safety and tolerability of Compound 1 and to define an appropriate dose for further evaluation in Part 2.

[0117] The trial will begin with an accelerated titration dose escalation scheme enrolling one evaluable patient per cohort for the first two dose levels, followed by a classic 3+3 design pending safety signals.

[0118] Part 2: Dose expansion, in which at least three parallel arms of patients with advanced non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), and pancreatic cancer (PANC) will be treated with the recommended phase 2 dose (RP2D) of compound 1 to further characterize the safety, tolerability, PK, PD, and antitumor activity of compound 1.

[0119] Part 2 will enroll approximately 18 patients (15 evaluable) in each of three parallel groups of patients (NSCLC, TNBC, PANC).

[0120] Compound 1 will be administered once daily with continuous dose escalation to the maximum tolerated dose (MTD) until progression or discontinuation. Each cycle of the study will last 28 days. The study may be extended for up to six cycles to confirm safety and efficacy.

[0121] From the above biological examples, compound 1 is clearly inferred as a novel class of KRAS inhibitors that selectively targets the GTP / GDP-binding pocket. Binding of compounds 1-5 to KRAS promotes the accumulation of GTP-KRAS, likely by preventing the cleavage of GTP to GDP. Compounds 1-5-induced overactivation of mutant KRAS promotes apoptotic cell death in mutant KRAS cancer cells. Combining the results with detailed structural analysis allows us to delineate key ligand-receptor interactions that correlate with activity. Therefore, our screening technique demonstrates its remarkable success in generating KRAS binders that affect signal transduction in cells. To our knowledge, compound 1 is the first known nanomolar binder of KRAS that disrupts its interaction with CRaf, resulting in decreased p-ERK levels and cell proliferation. Therefore, compound 1 is a promising candidate for the development of novel noncovalent KRAS inhibitors. The development of this new class of anticancer drug offers a potentially effective strategy for the treatment of cancers with KRAS mutations and / or mutant KRAS-driven cancers. Furthermore, compound 1 inhibits KRAS GTP and activates its degradation via GSK3ab by a ubiquitin-mediated pathway via miR30c and miR21 regulation, thereby leading to programmed cell death.

[0122] From the foregoing, it will be understood that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications thereof can be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope and spirit being indicated by the following claims. The present invention provides, for example, the following items. (Item 1) Formula 1: [ka] During the ceremony, X is H or an alkyl group; Y is a carbonyl group bonded to a substituted or unsubstituted mono- or heterocyclic ring having a carboxyl group bonded to at least one double bond or alkyl group; Z is a carbonyl / carboxyl group attached to a substituted / unsubstituted mono / heterocyclic ring having at least one double bond or a carbonyl group attached to an alkene or an o-alkyl substituted carbamate or branched alkyl group; W is H or an amide group or a carbonyl group attached to a branched alkyl group; or any derivative thereof, or a stereoisomer or tautomer thereof, a pharmaceutically acceptable salt thereof, or a combination thereof. (Item 2) In the formula, X is H or C 1 ~C 5 alkyl groups, The compound according to item 1, selected from the group comprising: (Item 3) In the formula, Y is i. a carbonyl group attached to cyclopent-3-ene; I C 1 ~C 5 a carboxyl group bonded to an alkyl group; ii. Carbonyl group attached to 5-carbamoyl, 4,5-dihydro-1H-pyrrole iii. A compound according to item 1, wherein the compound is selected from the group comprising a carbonyl group bonded to a 4,5 dihydro-1H-pyrrole-2 carbamoyl. (Item 4) In the formula, Z is io-C 1 ~C 5 alkyl carbamates, i. 5-carbamoyl, a carbonyl group attached to 2,3,dihydro-1H-pyrrole; ii. a carboxyl group attached to a pyridine; iii.C 1~ C 5 Alkenes, iv.C 1 ~C 6 a carbonyl group bonded to a branched alkyl group. (Item 5) In the formula, W is Amide group or H or C 1 ~C 5 Item 1. The compound according to item 1, wherein the carbonyl group is selected from the group comprising a carbonyl group attached to a branched alkyl group. (Item 6) During the ceremony, X is H Y is a carbonyl group attached to cyclopent-3-ene Z is o-methyl carbamate 6. The compound according to items 1 to 5, wherein W is an amide group. (Item 7) During the ceremony, X is H Y is

change

change

change

change

change

change

change

change

change

change

change

change

change

Claims

1. Formula 1: 【Chemistry 20】 or a pharmaceutically acceptable salt thereof, or a combination thereof, During the ceremony, X is C 1 -C 5 alkyl; Y is a carboxyl group bonded to a C 1 -C 5 alkyl; Z is a carbonyl group attached to 5-carbamoyl, 2,3, dihydro-1H-pyrrole; W is H, or a pharmaceutically acceptable salt thereof, or a combination thereof.

2. The compound of claim 1, wherein X is a butyl group.

3. The compound of claim 1, wherein Y is a carboxyl group bonded to an ethyl group.

4. A pharmaceutical composition comprising an effective amount of a compound defined in claim 1 and a pharmaceutically acceptable carrier thereof.

5. A product comprising: (i) an effective amount of a compound as defined in claim 1 or a pharmaceutical composition as defined in claim 4; and (ii) instructions for use in the treatment of cancer mediated by KRAS.

6. A kit comprising: (i) an effective amount of a compound as defined in claim 1 or a pharmaceutical composition as defined in claim 4, and (ii) instructions for use in the treatment of cancer mediated by KRAS.

7. Use of a compound of claim 1 in the manufacture of a medicament for treating non-small cell lung cancer, triple-negative breast cancer, pancreatic cancer, or a combination thereof.

8. The compound 【Transformation 34】 2. The compound of claim 1, wherein:

9. The compound 【Chemistry 35】 The kit according to claim 6, wherein 10. The compound of claim 1, 【Transformation 36】 The use according to claim 7, wherein

Images