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

Abstract

The present invention discloses novel compounds showing anti-cancer activity for treating subjects with chronic diseases, the compounds comprising a structure according to Formula 1 or any derivative thereof or a stereoisomer or tautomer thereof, a pharmaceutically acceptable salt thereof or a combination thereof, wherein X is H or an alkyl group; Y is a carbonyl group connected to a substituted / unsubstituted homocyclic / heterocyclic ring through at least one double bond or a carboxyl group connected to an alkyl group; Z is a carbonyl group / carboxyl group connected to a substituted / unsubstituted homocyclic / heterocyclic ring through at least one double bond or an alkene or an o-alkyl-substituted carbamate or a carbonyl group connected to a branched alkyl group, and W is H or an amide group or a carbonyl group connected to a branched alkyl group.

Description

Technical Field

[0001] The present invention relates to pharmaceutical molecules. More specifically, the present invention relates to novel compounds that exhibit anti-cancer activity for use in treating subjects suffering from chronic conditions. Background Art

[0002] Cancer is a major health problem and a leading cause of death. Cancer prognosis is closely related to the stage of the disease. In the most advanced stages, when metastasis occurs, the prognosis is often worse and treatment is most likely to fail. Genomic instability and mutation are pointed out as the main mechanisms for acquiring these hallmark features. One of the most commonly mutated oncogenes in cancer is KRAS. The KRAS gene encodes a small GTPase that cycles between GDP and GTP-bound states due to stimulation of certain 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% to 20% of non-small cell lung cancers (NSCLC), are most common in adenocarcinomas (30%-50%), are rare in small cell lung cancers, and are more common in smokers.

[0003] KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) is an oncogenic driver with mutations in 30% of non-small cell lung cancer (NSCLC). However, even though it has been identified as an oncogene for 30 years, there is no effective clinical drug. KRAS is one of the most frequently mutated oncogenes in cancer, an effective initiator of tumorigenesis, a strong inducer of malignant tumors, and a predictive biomarker for response to therapy. In addition to maintaining this distinction, unsuccessful attempts to target this protein have also led to RAS being characterized as 'non-drugable'. The RAS signaling network is built at the membrane of the cell, bridging extracellular cues to cellular events such as cell growth, proliferation, differentiation, and survival [Cox et al., 2015], and plays a decisive role in the transition of healthy cells to cancer [Welsch ME et al., 2017]. All these discoveries have prompted the scientific community to use RAS or its downstream effectors as therapeutic targets, hoping to impair tumor growth and survival [Affolter et al., 2012]. However, to date, effective KRAS-directed signaling inhibition has not been successfully achieved. Mutations that render KRAS constitutively active will lead to uncontrolled cell growth and cancer. However, despite aggressive efforts in recent years, there are no drugs on the market that directly target KRAS and inhibit its abnormal function [Westcott et al., 2015].

[0004] However, preclinical studies indicate that “K-Ras addiction” is reduced in mesenchymal cancer cells, meaning that direct inhibition of KRAS may not be effective for all patients [Yin et al., 2019]. Alternatively, inhibitors targeting downstream kinases of KRAS, such as BRAF and MEK, have shown promising activity in metastatic melanoma, but are largely ineffective in combination with chemotherapy against KRAS mutations in cancer [Janne et al., 2017]. In addition to intrinsic resistance, for example due to differentiation of mesenchymal cancer cells [Peng et al., 2019], the efficacy of MEK inhibitors is limited by the development of acquired resistance [Haigis et al., 2017]. Another factor that contributes to the difficulty of treating cancer cells is the heterogeneity of different KRAS mutations, which are defined by the corresponding amino acid substitutions. These alter the protein structure and GTPase activity of KRAS and significantly affect tumor biology and response to chemotherapy [Ambrogio et al., 2018]. Therefore, an unmet need remains to develop more effective targeted therapeutic strategies for patients with KRAS-mutated lung cancer.

[0005] References

[0006] 1. Overmeyer JH, Maltese WA. Death pathways triggered by activated Ras in cancer cells. Front Biosci (Landmark ed.). 2011;16:1693–713.

[0007] 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: 878–889e829.

[0008] 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.

[0009] 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.

[0010] 5. ADCox, CJDer, MR Philips Targeting RAS membrane association: back to the future for anti-RAS drug discovery? Clin Cancer Res, 21 (2015), pp. 1819-1827

[0011] 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

[0012] 7. Friday BB, Adjei AA. KRAS as a target for cancer therapy. Biochim Biophysi Acta. 2005; 1756: 127-144.

[0013] 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-Mutanta advanced non-small cell lung cancer: the SELECT-1 randomized clinical trial. JAMA, 317(18) (2017), pp. 1844-1853.

[0014] 9. DHPeng, STKundu, JJFradette, L.Diao, P.Tong, LAByers, et al. ZEB1 suppression sensitizeskras mutant cancers to mek inhibition by an IL17RD-dependent mechanism. Sci Transl Med, 11(483)(2019)

[0015] 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)857-68e15

[0016] 11. KM Haigis KRAS alleles: the devil is in the detail. Trends Cancer, 3(10) (2017), pp. 686-697

[0017] 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

[0018] Purpose of the invention

[0019] The main objective of the present invention is to develop novel compounds that exhibit anti-cancer activity for use in treating subjects suffering from chronic conditions.

[0020] Another object of the present invention is to synthesize said novel compounds showing anti-cancer activity for use in treating subjects suffering from chronic conditions.

[0021] Yet another object of the present invention is to synthesize anti-cancer active phenotype compounds that selectively target cancer stem cells and are effective in KRAS protein degradation.

[0022] Another object of the present invention is to utilize novel compounds exhibiting anticancer activity synthesized for use in treating subjects suffering from chronic conditions.

[0023] It is a further object of the present invention to provide pharmaceutical compositions comprising an effective amount of one or more of the compounds described herein.

[0024] Yet another object of the present invention is to provide a method of modulating, inhibiting or degrading KRAS in a cell by administering to the cell an effective amount of one or more of the compounds or pharmaceutical compositions described herein, or any combination thereof.

[0025] Yet another object provided herein is a method of treating a disease, disorder or condition mediated by KRAS in a subject in need thereof, the method comprising administering to the subject an effective amount of one or more of the compounds or pharmaceutical compositions described herein.

[0026] Yet another object provided herein is an article of manufacture (e.g., a kit) comprising (i) an effective amount of one or more of the compounds or pharmaceutical compositions described herein, and (ii) instructions for use in treating a disease, disorder, or condition mediated by KRAS. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The foregoing and following information and other features of the present disclosure will become more fully apparent through the following description and the appended claims in conjunction with the accompanying drawings. It should be understood that these drawings depict only several embodiments according to the present disclosure and therefore should not be considered as limiting the scope of the present disclosure, which will be described with additional features and details through the use of the accompanying drawings.

[0028] Figure 1 The spectrum of Compound I is depicted.

[0029] a. Mass spectrometry

[0030] b.IR spectrum

[0031] c. 1 H NMR spectroscopy

[0032] d. 13 C NMR spectroscopy

[0033] Figure 2The spectrum of compound II is depicted.

[0034] a. Mass spectrometry

[0035] b.IR spectrum

[0036] c. 1 H NMR spectroscopy

[0037] d. 13 C NMR spectroscopy

[0038] Figure 3 The spectrum of compound III is depicted.

[0039] a. Mass spectrometry

[0040] b.IR spectrum

[0041] c. 1 H NMR spectroscopy

[0042] d. 13 C NMR spectroscopy

[0043] Figure 4 The spectrum of compound IV is depicted.

[0044] a. Mass spectrometry

[0045] b.IR spectrum

[0046] c. 1 H NMR spectroscopy

[0047] d. 13 C NMR spectroscopy

[0048] Figure 5 The spectrum of Compound V is depicted.

[0049] a. Mass spectrometry

[0050] b.IR spectrum

[0051] c. 1 H NMR spectroscopy

[0052] d. 13 C NMR spectroscopy

[0053] Figure 6 Compound I is depicted to induce apoptosis in KRAS-dependent cancer cell lines.

[0054] Figure 6 (A) - Morphological changes after 24 h exposure to Compound I captured by fluorescence microscopy (0 nm, 50 nm, 100 nm and 250 nm), with magnification ×100, scale bar: 20 μm.

[0055] Figure 6 (B) - Cells were treated with 250 nm of Compound I for 24 hours and apoptosis levels were quantitatively measured by flow cytometry after staining the cells with Annexin V / Propidium Iodide (PI), and triplicate data were plotted as bar graphs.

[0056] Figure 6 (C) - Representative Western blot data of different apoptosis-related proteins (PARP, Bcl-2 and Bax) confirming the induction of apoptosis after 24 hours of treatment (0 nm, 50 nm, 100 nm and 250 nm). Densitometric measurements of the ratio of Bax / Bcl2 to cleaved PARP are shown as bar graphs.

[0057] All data represent at least three independent experiments and are presented as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001 compared with the control group.

[0058] Figure 7 Compound I is depicted to inhibit cancer cells expressing KRAS mutations.

[0059] Figure 7 A- Proliferation curves of KRAS expressing cancer cells after treatment with increasing concentrations of compound, Compound I, monitored by cell viability assay.

[0060] Figure 7 B- Relative growth of KRAS mutant and wild-type cancer cells after treatment with IC50 concentration of compound, Compound I.

[0061] Data are shown as mean ± SD; significance relative to SKLU-1 was estimated by one-way ANOVA: * = P < 0.02, ** = P < 0.005, *** = P < 0.0001.

[0062] Figure 8 Structural modeling of Compound 1 is depicted.

[0063] Fig. 9 Depicted is the blocking of GTP-KRAS formation by a compound, Compound I, in KRAS mutant cells.

[0064] Fig.10 Compound I is depicted to inhibit tumor growth in a xenograft model, and Western blot analysis of KRAS mutant protein in an in vivo animal model is also depicted.

[0065] Fig.11Compound I targeting p53 is depicted (mRNA levels of p53, Puma and Noxa in cells at 6 hours after Compound I treatment. mRNA levels were quantified by qRT-PCR. Data were normalized to GAPDH expression and plotted relative to cells treated with DMSO as control).

[0066] Fig.12 Depicted is the finding that compound 1 significantly inhibited cancer cell viability in all mutant cells tested in a dose-dependent manner.

[0067] Fig.13 Depicted is the discovery that compound 1 selectively inhibits the formation of GTP-KRAS (relative to the total amount of KRAS) in KRAS G12C, G12V, G12D, and G13D mutant cells.

[0068] Fig.14 Compound 1 blocks downstream effectors of RAS signaling in KRAS mutant cells.

[0069] Fig.15 Depicted is the discovery that compound 1 degrades RAS and inhibits stemness marker expression.

[0070] Fig.16 Depicted is a comparison of RAS reactivation in KRAS G12C mutant cell lines following treatment with compound 1 and two known KRAS G12C inhibitors.

[0071] Fig.17 Depicted is a comparison of cell viability in KRAS G12C mutant cell lines following treatment with Compound 1 and two known KRAS G12C inhibitors.

[0072] Fig.18 Regression of all mutant KRAS-driven cancers in the PDTX animal model is depicted. Treatment with Compound 1 showed a 95% reduction in tumor growth within 28 days, with no evidence of recurrence for 6-8 months.

[0073] Fig.19 Regression of all mutant KRAS-driven cancers in the PDTX animal model is depicted. Treatment with Compound 1 showed a 95% reduction in tumor growth within 28 days, with no evidence of recurrence for 6-8 months.

[0074] Fig. 20 Depicted are the results of in vivo toxicity studies of Compound 1. Organ weights, body weight, liver weight, hematological parameters, serum electrolytes LFT, KFT, lipid parameters were found to be within normal ranges compared to the control group. Histopathology of the collected normal tissues (heart, liver, spleen, skeletal muscle, kidney and intestine) after treatment with a specific dose of Compound 1 showed no evidence of normal tissue toxicity. Summary of the invention

[0075] The present invention discloses novel compounds that exhibit anticancer activity for use in treating subjects with chronic conditions, the compounds comprising a structure according to Formula 1:

[0076]

[0077] or any derivative thereof or stereoisomer or tautomer thereof, a pharmaceutically acceptable salt thereof or a combination thereof, wherein

[0078] X is H or alkyl;

[0079] Y is a carbonyl group connected to a substituted / unsubstituted homocyclic / heterocyclic ring through at least one double bond or a carboxyl group connected to an alkyl group;

[0080] Z is a carbonyl / carboxyl group connected to a substituted / unsubstituted homocyclic / heterocyclic ring through at least one double bond or an alkene or o-alkyl substituted carbamate or a carbonyl group connected to a branched alkyl group; and

[0081] W is H or an amide group or a carbonyl group connected to a branched alkyl group. DETAILED DESCRIPTION

[0082] definition:

[0083] "Alkyl" or "alkyl group" refers to a fully saturated straight or branched hydrocarbon chain having one to fifteen carbon atoms and attached to the rest of the molecule by a single bond. Unless otherwise specifically stated in the specification, an alkyl group may be optionally substituted.

[0084] "Olefin" or "olefin group" refers to a straight or branched hydrocarbon chain having two to fifteen carbon atoms and having one or more carbon-carbon double bonds. Each olefin group is connected to the rest of the molecule by a single bond. Unless otherwise specifically stated in the specification, the olefin chain may be optionally substituted.

[0085] "Homocyclic ring" refers to a cyclic compound having at least three atoms of only one element (usually carbon) in the ring. Unless specifically stated otherwise in the specification, a homocyclic ring may be optionally substituted.

[0086] "Heterocyclic ring" refers to at least three-membered cyclic compounds having at least two different elements, usually carbon, and nitrogen or sulfur or oxygen in the ring. Unless specifically stated otherwise in the specification, heterocyclic rings may be optionally substituted.

[0087] "Heteroalkyl" refers to a fully saturated straight or branched hydrocarbon chain having one to fifteen carbon atoms and connected to the rest of the molecule by a single bond, wherein at least one carbon is substituted by a heteroatom including nitrogen. Unless otherwise specifically stated in the specification, heteroalkyl may be optionally substituted.

[0088] "Carbonyl" means

[0089] Carboxyl refers to

[0090] "O-alkyl substituted carbamate" refers to a fully saturated straight or branched hydrocarbon chain having a carbamate group substituted with from one to fifteen carbon atoms at the o position.

[0091] A "carbamate" group refers to -HNCOO-.

[0092] As used herein, the term "substituted" refers to any of the above groups in which at least one hydrogen atom is replaced by a bond to a non-hydrogen atom. (may be referred to as "point of bond attachment" hereinafter) represents a bond that is a point of attachment between two chemical entities, one of which is depicted as being attached to the point of bond attachment and the other of which is not depicted as being attached to the point of bond attachment.

[0093] Compounds provided herein that have the same molecular formula but differ in the type or order of connection of their atoms or in the spatial arrangement of their atoms are referred to as "isomers". Isomers with different spatial arrangements of their atoms are referred to as "stereoisomers". The compounds disclosed herein may also exist in different tautomeric forms. The term "tautomer" means that structural isomers are interconvertible. For example, in one variation, the compounds provided herein may exhibit keto-enol tautomerism. In another variation, the compounds provided herein may exhibit imine-enamine tautomerism.

[0094] "Pharmaceutically acceptable salts" include pharmaceutically acceptable base addition salts and acid addition salts, as appropriate. As used herein, the term "pharmaceutically acceptable" infers that the salt is not biologically or otherwise undesirable; for example, the material can be added to a pharmaceutical composition and administered to a subject without causing significant adverse effects.

[0095] The term "therapeutically effective" as applied to dosage or amount refers to an amount of a compound or pharmaceutical formulation sufficient to produce the desired clinical benefit after administration to a patient in need thereof. It should be understood that an effective amount can be one or more doses, for example, a single dose or multiple doses may be required to achieve the desired therapeutic endpoint.

[0096] As used herein, the terms "treating" or "treatment" refer 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 extent of a disease, disorder, or condition; (3) slowing or stopping the development 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) relieving a disease (e.g., improving the disease state, enhancing the effect of another drug, delaying or stopping the progression of a disease, improving quality of life, and / or prolonging survival), e.g., by causing regression of one or more clinical symptoms.

[0097] In the following detailed description, reference is made to the accompanying drawings, which form a part of the detailed description. In the accompanying drawings, similar symbols generally identify similar components unless the context indicates 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 is readily understood that the various aspects of the present disclosure, as generally described herein and shown in the accompanying drawings, may be arranged, replaced, combined, separated, and designed in a variety of different configurations, all of which are expressly contemplated herein.

[0098] The present invention discloses novel compounds that exhibit anti-cancer activity for use in treating subjects suffering from chronic conditions.

[0099] In one of the preferred embodiments, the present invention discloses novel compounds exhibiting anticancer activity for use in treating subjects suffering from chronic conditions, the compounds comprising a structure according to Formula 1:

[0100]

[0101] or any derivative thereof or stereoisomer or tautomer thereof, a pharmaceutically acceptable salt thereof, or a combination thereof,

[0102] in

[0103] X is H or alkyl;

[0104] Y is a carbonyl group connected to a substituted / unsubstituted homocyclic / heterocyclic ring through at least one double bond or a carboxyl group connected to an alkyl group;

[0105] Z is a carbonyl / carboxyl group connected to a substituted / unsubstituted homocyclic / heterocyclic ring through at least one double bond or an alkene or o-alkyl substituted carbamate or a carbonyl group connected to a branched alkyl group; and

[0106] W is H or an amide group or a carbonyl group connected to a branched alkyl group.

[0107] According to the present invention, in the compound of formula 1, X is selected from the group consisting of H or C1-C5 alkyl.

[0108] According to the present invention, in the compound of formula 1, Y is selected from the group consisting of:

[0109] i. a carbonyl group connected to cyclopent-3-ene;

[0110] ii. a carboxyl group connected to a C1-C5 alkyl group;

[0111] iii. a carbonyl group linked to 5-carbamoyl, 4,5-dihydro-1H-pyrrole;

[0112] iv. A carbonyl group linked to a 4,5-dihydro-1H-pyrrole-2-carbamoyl group.

[0113] According to the present invention, in the compound of formula 1, Z is selected from the group consisting of:

[0114] i. o-C1-C5 alkyl carbamate;

[0115] ii. a carbonyl group linked to 5-carbamoyl, 2,3, dihydro-1H-pyrrole;

[0116] iii. a carboxyl group connected to pyridine;

[0117] iv. C1-C5 olefins;

[0118] v. A carbonyl group connected to a C1-C6 branched alkyl group.

[0119] According to the present invention, in the compound of formula 1, W is selected from the group consisting of H or a carbonyl group connected to a C1-C5 branched alkyl group.

[0120] According to the present invention, in the compound of formula 1,

[0121] X is H;

[0122] Y is a carbonyl group connected to cyclopent-3-ene;

[0123] Z is o-methyl carbamate;

[0124] W is an amide group.

[0125] According to the present invention, in the compound of formula 1,

[0126] X is H;

[0127] Y is

[0128] Z is

[0129] W is

[0130] According to the present invention, in the compound of formula 1,

[0131] X is butyl;

[0132] Y is a carboxyl group connected to an ethyl group;

[0133] Z is a carbonyl group connected to 5-carbamoyl, 2,3, dihydro-1H-pyrrole;

[0134] W is H.

[0135] According to the present invention, in the compound of formula 1,

[0136] X is

[0137] Y is

[0138] Z is

[0139] W is H.

[0140] According to the present invention, in the compound of formula 1,

[0141] X is H;

[0142] Y is a carbonyl group linked to 5-carbamoyl, 4,5-dihydro-1H-pyrrole;

[0143] Z is a carboxyl group connected to pyridine;

[0144] W is H.

[0145] According to the present invention, in the compound of formula 1,

[0146] X is H;

[0147] Y is

[0148] Z is

[0149] W is H.

[0150] According to the present invention, in the compound of formula 1,

[0151] X is H;

[0152] Y is a carbonyl group connected to a 4,5-dihydro-1H-pyrrole-2-carbamoyl group;

[0153] Z is ethylene;

[0154] W is a carbonyl group connected to a secondary propyl group.

[0155] According to the present invention, in the compound of formula 1,

[0156] X is H;

[0157] Y is

[0158] Z is

[0159] W is

[0160] According to the present invention, in the compound of formula 1,

[0161] X is H;

[0162] Y is a carbonyl group connected to a 4,5-dihydro-1H-pyrrole-2-carbamoyl group;

[0163] Z is a carbonyl group connected to a tert-butyl group;

[0164] W is H.

[0165] According to the present invention, in the compound of formula 1,

[0166] X is H;

[0167] Y is

[0168] Z is

[0169] W is H.

[0170] In another preferred embodiment, the present invention discloses an effective amount of a pharmaceutical composition, which comprises one of the compounds described above or its stereoisomers or tautomers and a pharmaceutically acceptable carrier thereof.

[0171] In another preferred embodiment, the present invention discloses an article of manufacture comprising: (i) an effective amount of any one of the compounds described above or its stereoisomers or tautomers or the pharmaceutical composition described above; and (ii) instructions for use in treating a disease, disorder or condition mediated by KRAS.

[0172] According to the present invention, in the preparation, KRAS comprises a G12C mutant, a G12D mutant, a G13D mutant or a G12V mutant or any combination thereof.

[0173] According to the present invention, in the article of manufacture, the disease, disorder or condition is cancer.

[0174] According to the present invention, in the preparation, the cancer is non-small cell lung cancer, colorectal cancer, triple-negative breast cancer or pancreatic cancer or any combination thereof.

[0175] In yet another embodiment, the present invention discloses a kit comprising: (i) an effective amount of any one of the compounds described above or its stereoisomers or tautomers or the pharmaceutical composition described above; and (ii) instructions for treating a disease, disorder or condition mediated by KRAS.

[0176] According to the present invention, in the kit, KRAS comprises a G12C mutant, a G12D mutant, a G13D mutant or a G12V mutant or any combination thereof.

[0177] According to the present invention, in the kit, the disease, disorder or condition is cancer.

[0178] 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.

[0179] 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 exposing the cell to (i) an effective amount of a compound described above or a stereoisomer or tautomer thereof, or a pharmaceutical composition described above.

[0180] According to the present invention, in the method described above, KRAS comprises a G12C mutant, a G12D mutant, a G13D mutant or a G12V mutant or any combination thereof.

[0181] 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, the method comprising administering to the subject: (i) an effective amount of the compound described above or its stereoisomer or tautomer or the pharmaceutical composition described above.

[0182] According to the present invention, in the method discussed above, KRAS comprises a G12C mutant, a G12D mutant, a G13D mutant or a G12V mutant or any combination thereof.

[0183] According to the present invention, in the methods discussed above, the disease, disorder or condition is cancer.

[0184] According to the present invention, in the method discussed above, the cancer is non-small cell lung cancer, triple-negative breast cancer, colorectal cancer or pancreatic cancer or any combination thereof.

[0185] The chemical structures of compounds of Formula 1 can be prepared by conventional chemistry based on the exemplary structures provided herein.

[0186] In one embodiment, exemplary compound II can be prepared by Scheme I provided below:

[0187]

[0188] Scheme I

[0189] The synthesized compound of formula 1 was then subjected to molecular characterization to determine the structure of the compound.

[0190] Molecular Characterization:

[0191] By melting point, thin layer chromatography, HPLC, IR, 13 C. Mass spectrometry and NMR analysis verified the purity of the synthesized product. Figure 1-5 The structures of the synthesized compounds of formula 1 are illustrated as compounds I to V, as shown in the following Table 1.

[0192] Table 1:

[0193]

[0194]

[0195] In some embodiments, the compound of Formula 1 and compound IV can encompass cis and trans isomers. In some embodiments, the compound of Formula 1 and compound IV can be a mixture of cis and trans isomers. In some embodiments, the compound of Formula 1 and compound IV can be cis isomers. In some embodiments, the compound of Formula 1 and compound V can be trans isomers.

[0196] In some embodiments, compounds of Formula 1 and Compound IV may encompass R or S stereoisomers and mixtures of stereoisomers. In some embodiments, compounds of Formula (I) may encompass both racemic isomers and enantiomeric isomers.

[0197] The compounds of the invention may be used to perform or provide any of the biological functions described herein.

[0198] Pharmaceutical composition

[0199] The present disclosure also includes a pharmaceutical composition 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 a pharmaceutically acceptable salt thereof. In other embodiments, the pharmaceutical composition comprises a therapeutically effective amount of one or more compounds selected from Table 1 or a pharmaceutically acceptable salt thereof. In some embodiments, the aforementioned pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0200] In various aspects, the amount of the compound of Formula 1 (including the compounds in 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 herein is administered at about 200 mg / kg to about 2000 mg / kg—for example, at about 200 mg / kg, about 500 mg / kg, about 1000 mg / kg, or about 2000 mg / kg. In some embodiments, the compound herein is administered at about 100 mg / kg to about 600 mg / kg—for example, at about 100 mg / kg, about 200 mg / kg, about 300 mg / kg, about 450 mg / kg, or about 600 mg / kg.

[0201] The concentration of the disclosed compounds in the pharmaceutically acceptable mixture will vary depending on several factors, including the dose of the compound to be administered, the pharmacokinetic properties of the compound employed, and the route of administration. The medicament can be administered in a single dose or repeated doses. The dosage regimen utilizing the compounds of the present invention is selected based on a variety of factors, including the type, species, age, weight, sex, and medical condition of the patient; the severity of the condition to be treated; the route of administration; the patient's renal and liver function; and the specific compound or salt thereof employed. Treatment can be administered once a day or more frequently depending on a number of factors, including the patient's overall health, as well as the formulation and route of administration of the selected compound.

[0202] The compounds or pharmaceutical compositions of the present disclosure may be manufactured and / or administered in single or multiple unit dosage forms.

[0203] The compounds or pharmaceutical compositions of the present disclosure may be manufactured and / or administered in single or multiple unit dosage forms.

[0204] The compounds or pharmaceutical compositions of the present disclosure can be administered by various methods, including, for example, oral, rectal, buccal, intranasal and transdermal routes. In certain embodiments, the pharmaceutical compositions can be administered intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, orally, topically or as an inhalant.

[0205] The compounds and pharmaceutical compositions of the present disclosure can be administered to an individual in any form of generally accepted oral compositions (eg, tablets, coated tablets, soft shell or gel capsules in soft shells, emulsions, or suspensions).

[0206] Treatment:

[0207] Also provided herein is a method of treating a chronic condition in a person in need thereof, the method comprising administering to the person a compound of Formula 1, (I), (II), (III), (IV) or (V), including, for example, Compounds 1-5 or pharmaceutically acceptable salts thereof. In some embodiments, the compounds of the present disclosure (compounds of Formula 1 and compounds in Table 1) are administered to patients suffering from chronic conditions.

[0208] In some embodiments, provided herein is a method of regulating, inhibiting or degrading KRAS in a cell or any combination thereof, the method comprising exposing the cell to: (i) an effective amount of a compound of Formula 1, (I), (II), (III), (IV) or (V), including, for example, Compound 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), including, for example, Compound 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, provided herein is a method of regulating KRAS in a cell. In some embodiments, provided herein is a method of inhibiting KRAS in a cell. In still other embodiments, provided herein is a method of degrading KRAS in a cell. In some embodiments, provided herein is a method of regulating and degrading KRAS in a cell. In some embodiments, provided herein is a method of inhibiting and degrading KRAS in a cell. In some of the foregoing embodiments, KRAS comprises a G12C mutant, a G12D mutant, a G13D mutant, or a G12V mutant, or any combination thereof. In some embodiments, KRAS comprises a G12C mutant.

[0209] In some embodiments, provided herein is a method of treating a disease, disorder or condition mediated by KRAS in a subject in need thereof, the method comprising administering to the subject: (i) an effective amount of a compound of Formula 1, (I), (II), (III), (IV) or (V), including, for example, Compound 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), including, for example, Compound 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.

[0210] In some embodiments, the treatment methods described herein result in a change (%) in the sum of the diameters of the target lesions of the subject. In some embodiments, the change is measured at day 1, day 14, day 21, day 28, or 6 months after treatment, or any combination of the foregoing (up to 6 cycles). In some embodiments, the change is measured at day 21 of treatment (up to 6 cycles).

[0211] In some embodiments, the treatment methods described herein result in a decrease in the number of circulating tumor cells in a subject. In one variation, the treatment methods result in a decrease in the number of circulating tumor cells in a subject with breast cancer, pancreatic cancer, or non-small cell lung cancer up to 6 months after treatment.

[0212] In some embodiments, the treatment methods described herein result in a change in the level of one or more apoptosis markers of the subject. In some embodiments, the treatment methods described herein result in a change in the level of one or more inflammatory markers of the subject. In some embodiments, the treatment methods described herein result in a change in the level of one or more pathway markers of the subject (as measured by immunohistochemistry (IHC)). In some embodiments, the treatment methods described herein result in a change in the immune response of the subject. For example, in some embodiments, the treatment methods described herein result in a change in the level of T cells, cytotoxic T lymphocytes (CTL) responses, cytokines (IFN, IL-4, IL-10) or activation markers or any combination thereof producing interferon gamma (IFN). In some of the aforementioned variations, changes can be measured at the 1st day, the 14th day, the 21st day, the 28th day or 6 months or any combination of the aforementioned (up to 6 cycles) after treatment.

[0213] In some of the foregoing embodiments, the disease, disorder or condition is mediated by a KRAS G12C mutant, a KRAS G12D mutant, a KRAS G13D mutant, or a KRAS G12V mutant, or any combination thereof. In some embodiments, the disease, disorder or condition is mediated by a KRAS G12C mutant. In some of the foregoing embodiments, the disease, disorder or condition is cancer.

[0214] In the context of 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 lymphoblastic, acute lymphoblastic 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 brain astrocytoma, cervical cancer, bile duct cancer, chondrosarcoma, chronic lymphoblastic or chronic lymphoblastic leukemia, chronic myeloid or chronic myeloid leukemia, chronic myeloproliferative disorder, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, sarcoma), extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid, gastrointestinal stromal tumor (GIST), gestational trophoblastic tumor, brain stem glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular melanoma, islet cell carcinoma, Kaposi sarcoma, laryngeal cancer, leukemia, lip and oral cancer, liposarcoma, lymphoma, male breast cancer, malignant mesothelioma, medulloblastoma, melanoma, Merkel cell skin cancer carcinoma, mesothelioma, metastatic squamous neck cancer, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma / plasma cell neoplasms, mycosis fungoides, myelodysplasia / myeloproliferative disorders, nasal and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-melanoma skin cancer, non-small cell lung cancer, oligodendroglioma, oral cancer 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 cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germ cell tumor, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary adenoma, plasmacytoma, pleuropulmonary blastoma, primary carcinoma, primary central nervous system lymphoma, primary liver cancer, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Sezary syndromesyndrome), small cell lung cancer, small intestinal cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, supratentorial primitive neuroectodermal tumors, testicular cancer, laryngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, uterine sarcoma, vaginal cancer, optic pathway and hypothalamic gliomas, perineal cancer, Waldenstrom macroglobulinemia, Wilms tumor, Parkinson's disease and Parkinsonian disorder, 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, Alzheimer's disease, other cognitive disorders, depression, onychomycosis (fungal infection of the nail), gingivitis and periodontitis (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. Formula 1, (I), (II), (III), (IV) or (V) compounds, including any one or more of compounds 1-5, or a pharmaceutically acceptable salt thereof, may also prove useful in treating SARS and COVID-19 and treating KRAS oncogene mutations in different cancers (such as those described in the above list).

[0215] 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.

[0216] In certain embodiments, the methods, compounds, and compositions described herein are administered in combination with one or more of the following: other antibody molecules; chemotherapy; other anti-cancer therapies (e.g., targeted anti-cancer therapies, gene therapy, viral therapy, RNA therapy, bone marrow transplantation, nanotherapy, or oncolytic drugs); cytotoxic agents; immune-based therapies (e.g., cytokines or cell-based immunotherapy); surgery (e.g., lumpectomy or mastectomy) or radiosurgery; or a combination of any of the foregoing.

[0217] Alternatively, or in combination with the foregoing combinations, the methods and compositions described herein can be administered in combination with one or more of: a vaccine, e.g., a therapeutic cancer vaccine; or other forms of cellular immunotherapy.

[0218] In some embodiments, provided herein are compounds or pharmaceutical compositions as described elsewhere herein for use in any of the methods described elsewhere herein. In other embodiments, provided herein are compounds or pharmaceutical compositions as 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 are compounds or pharmaceutical compositions as described elsewhere herein for use in any of the methods described herein.

[0219] The present invention aims to invent powerful therapeutic small molecules that can target mutant KRAS at the active site. Small molecule compound I that binds to the GTP / GDP binding pocket of KRAS is identified herein. For this purpose, the strong correlation between the measurements of a larger group of tumor cell lines expressing KRAS mutations was studied, and the efficacy of RAS activated to inhibit tumor cell growth was analyzed. As demonstrated by colony formation and apoptosis assays, the growth inhibitory effect of compound I is persistent and irreversible. Compound I has good binding affinity to KRAS in vitro, and exhibits selective cytotoxicity in oncogenic KRAS-expressing cell lines, and has no or minimal toxic effects on normal cell lines. Further mechanistic studies have shown that compound I can block the formation of a complex of guanosine triphosphate (GTP) and KRAS in vitro. In addition, compound I inhibits KRAS downstream signaling pathways RAF / MEK / ERK and RAF / PI3K / AKT. As measured by cell cycle analysis of DNA content and phosphorylated histone H3B immunofluorescence, compound I induces mitotic arrest. Further analysis revealed that compound I interferes with the localization of the mitotic inducing protein PLK1 to the nucleus and reduces the nuclear localization of its substrate Cdc25C, which is a downstream target of RAS-RAF signaling involved in both mitotic entry and exit checkpoints. Compound I also inhibits PD-L1 expression and activates anti-tumor immunity, which may contribute to its anti-tumor activity and suggest potential benefits in combination with immunotherapy. According to these studies, a new type of RAS inhibitor was identified that effectively and selectively inhibits RAS-driven tumor growth by disrupting downstream signaling, leading to cell cycle arrest and apoptosis. Therefore, compound I can be regarded as a potential KRAS inhibitor for the treatment of cancer cells carrying the KRAS oncogene.

[0220] Products

[0221] Also provided herein are articles, wherein the articles include a compound of Formula 1, (I), (II), (III), (IV) or (V), or any variation or embodiment thereof, as described elsewhere herein, including, for example, Compounds 1-5 or stereoisomers or tautomers thereof, or a pharmaceutically acceptable salt of any of the foregoing, in a suitable container. Also provided herein are articles, which include a pharmaceutical composition in a suitable container, the pharmaceutical composition including a compound of Formula 1, (I), (II), (III), (IV) or (V), or any variation or embodiment thereof, as described elsewhere herein, including, for example, Compounds 1-5 or stereoisomers or tautomers thereof, or a pharmaceutically acceptable salt of any of the foregoing. The container can be a vial, a can, an ampoule, a preloaded syringe, or an intravenous bag.

[0222] The present disclosure further provides a kit for performing the methods of the present invention. The kit may include a compound as described herein or a pharmaceutically acceptable salt thereof and suitable packaging. The kit may include one or more containers comprising any compound described herein. In one aspect, the kit comprises a compound of the present disclosure or a pharmaceutically acceptable salt thereof, and a label and / or instructions for using the compound in treating a disease or condition described herein. The kit may include a unit dosage form of the compound.

[0223] Provided herein are articles (such as kits) comprising: (i) an effective amount of a compound of Formula 1, (I), (II), (III), (IV) or (V), including, for example, Compound 1-5 or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any of the foregoing; and (ii) instructions for treating a disease, disorder or condition mediated by KRAS. Also provided herein are articles (such as kits) comprising: (i) a pharmaceutical composition comprising an effective amount of a compound of Formula 1, (I), (II), (III), (IV) or (V), including, for example, Compound 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 treating a disease, disorder or condition mediated by KRAS.

[0224] Examples

[0225] Biological Example 1

[0226] Compound 1 induces apoptosis in KRAS-dependent cancer cell lines ( Figure 6 )

[0227] Compound 1 was subjected to a cancer induction model and it was determined whether the observed growth inhibition was due to apoptosis or necrosis. Figure 6As shown in a, after treatment with compound 1, the cell morphology of the mutant KRAS cell line changed in a concentration-dependent manner, and a certain proportion of cells detached from the culture dish and aggregated, which are predictive apoptotic features. Next, standard Annexin 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 when compared to untreated cells ( Figure 6 C). In addition, to examine compound-induced apoptosis in KRAS mutant cells, the expression levels of several well-characterized apoptotic proteins were analyzed by Western blot. The results showed that increased expression of the pro-apoptotic protein Bax and decreased expression of the anti-apoptotic Bcl-2 were observed in Compound 1-treated cells. In addition, Compound 1 treatment achieved induction of PARP cleavage ( Figure 6 B), which is also consistent with apoptosis. Densitometry quantification of Bax / Bcl2 protein expression and the ratio of PARP cleavage relative to GAPHD was performed in Figure 6 Shown as a bar graph in B. Taken together, these results reveal that compound 1 induces apoptosis in KRAS mutant cell lines.

[0228] Biological Example 2

[0229] Compound 1 inhibits cancer cells expressing KRAS mutants ( Figure 7 and 12 )

[0230] To further investigate if compound 1 could also induce cytotoxic effects against other cancer cells expressing KRAS mutations, a panel of cancer cell lines containing KRAS G12D, G12V, and G12C point mutations were selected and used together with a KRAS wild-type (WT) control. Figure 7 As shown, after 24 hours of treatment with compound 1, compound 1 significantly inhibited cancer cell and stem cell viability in all mutant cells in a dose-dependent manner, and the IC50 value was found to be millimolar. Similarly, minimal toxicity was observed in normal cell lines. In summary, the data from cell line studies indicate that compound 1 more effectively inhibits signaling through KRAS, which is consistent with its tight binding to activated KRAS and its effect on KRAS-Raf interaction.

[0231] The cytotoxicity of compound 1 was initially evaluated in a panel of 30+ cell lines with 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. IC50 values ​​for compound 1 were found to be in the micromolar to nanomolar concentration range (see Fig.12 ).

[0232] Biological Example 3

[0233] Structural modeling of compound 1 in the GTP / GDP binding pocket of KRAS protein. Figure 8 )

[0234] Compound 1 binds to both WT and oncogenic KRAS mutants with high affinity. Figure 8 The chemical structure and predicted complex of compound 1 with KRAS are shown, indicating that the ligand may form multiple favorable interactions with residues in the p1 pocket.

[0235] Biological Example 4

[0236] Compound 1 blocks GTP-KRAS formation in KRAS mutant cells ( Fig. 9 and 13 )

[0237] In fact, mutant KRAS disturbs the balance between GEF and GAP, resulting in a locked active GTP-bound KRAS state and abnormal stimulation of its downstream signaling. Therefore, KRAS inhibitors should reduce the formation of GTP-KRAS to disrupt mutant KRAS function. In order to understand whether compound 1 can inhibit the activation of KRAS, a RAS activation assay was performed to examine the formation of GTP-bound KRAS in k-RAS mutant cells after treatment with a series of concentrations of compound 1 at 24 hours. Compared with the total amount of KRAS, compound 1 treatment inhibited the formation of GTP-KRAS in KRAS mutant cells, indicating that this small molecule can partially rescue this imbalance caused by mutant KRAS.

[0238] 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, and no effect was observed in cells with wild-type (WT) KRAS (see Fig.13 ).

[0239] Biological Example 5

[0240] Compound 1 inhibits the activation of KRAS downstream signaling pathways ( Fig. 9 and 14 )

[0241] Active KRAS stimulates downstream signaling pathways, particularly for the RAF / MEK / ERK and RAF / PI3K / AKT pathways, and then induces cell proliferation. Therefore, to study the effect of compound 1, the phosphorylation levels of CRAF, AKT, and ERK in cell lines were detected to monitor the impact of KRAS signaling after 48 hours of 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 ( Fig. 9 and 14 ), indicating that the molecule could block oncogenic KRAS function by inhibiting its downstream signaling pathways.

[0242] Biological Example 6

[0243] Compound 1 inhibits tumor growth in xenograft models ( Fig.10 )

[0244] Next, the in vivo efficacy of compound 1 in the context of mutant KRAS was evaluated. Cells were injected into nude mice and tumors were allowed to grow to approximately 60 mm. 3 size and were treated daily with Compound 1 for 14-21 days. Fig.10 Compound 1 was shown to inhibit tumor growth from day 9, and showed significant inhibition from day 10 to day 14 when compared to vehicle-treated controls. On the day of collection, the net tumor mass was determined, and the average tumor weight of the compound 1-treated group was 89% lower than the average tumor weight of the control group. After treatment with compound 1, mice (n=6) did not show significant weight loss or obvious toxicity. The effects of compound 1 treatment on KRAS-mediated RAF, MEK / ERK, and PI3K / AKT cascades in protein extracts from tumors treated with vehicle and compound 1 were further examined. Consistent with the in vitro data, significant inhibition of CRAF, ERK, and AKT phosphorylation was observed. Immunohistochemical analysis also showed that treatment with compound 1 reduced the levels of ERK and AKT phosphorylation, indicating that compound 1-induced growth inhibition was associated with KRAS-mediated signaling inhibition. In addition, immunohistochemical analysis of tumors from mice treated with compound 1 showed a significant reduction in cell proliferation as indicated by Ki-67 staining, and a significant increase in apoptotic cells as indicated by cleaved caspase-3 staining. Altogether, these data demonstrate that compound 1 is effective in inhibiting KRAS-driven lung tumor growth.

[0245] Biological Example 7

[0246] Compound 1 targets p53 ( Fig.11 )

[0247] The tumor suppressor gene TP53 (better known as p53) controls several cellular stresses, including DNA damage, hypoxia, and oncogene activation. Functionally, the p53 protein acts as a transcription factor and regulates gene expression by binding to specific DNA sequences. Typically, p53 is involved in controlling genes involved in apoptosis, aging, cell cycle arrest, and plays a role in necrosis, autophagy, metabolism, accumulation of reactive oxygen species (ROS), and stem cell maintenance. Mutations and loss of p53 are common in a variety of cancers, including lung cancer, head and neck cancer, bladder cancer, breast cancer, and prostate cancer.

[0248] It has been shown that in androgen-dependent and independent cancer cells, compound 1 induces apoptosis by raising the expression of p53, Bax, Bak, PUMA, Noxa and Bim and lowering Bcl-2 and Bcl-XL, but it has no effect on normal fibroblasts and epithelial cells. In addition, compound 1 induces p53 translocation to mitochondria, and Smac is released to cytoplasm, which strongly indicates its cancer chemoprevention properties. By changing p53 and its downstream proteins (p21, cyclin B1, CDK1, Cdc25C) and some apoptosis-related proteins (Bcl-2, Bax, Bid, Bad, Apaf1, AIF and Cyt c), compound 1 also lowers heat shock proteins (HSP) and histone deacetylase 6 (HDAC6) induced by oxidative stress, which further leads to apoptosis. Compound 1 shows the inhibitory activity of in vitro and in vivo preclinical models of p53 mutation or overexpression.

[0249] Biological Example 8

[0250] Compound 1 targets EGFR and HER2

[0251] The activity of compound 1 on various mutant EGFR, HER2 and HER4 was examined. The effects of compound 1 on different genetic characteristics and related molecular mechanisms were examined using MTT assay, flow cytometry and western blotting. A nude mouse xenograft model with cells was used to evaluate the in vivo antitumor activity of compound 1. The results showed that compound 1 effectively inhibited the enzymatic activity of EGFR family members, including drug-sensitive EGFR mutations as well as EGFR C797S mutations and wild-type (WT) HER2. Compound 1 blocked EGFR phosphorylation, thereby downregulating downstream PI3K / AKT and MAPK / ERK signaling and inducing G0 / G1 arrest in different cells. Compound 1 inhibited tumor growth in a mouse xenograft model. In summary, the results of this study indicate that compound 1 has the potential to become an oral antitumor drug for treatment and is worthy of further development.

[0252] Due to impaired homologous recombination (HR) repair, BRCA 1 and BRCA 2 mutant tumors accumulate DNA damage and genomic rearrangements that favor tumor progression. In an effort to identify drugs that specifically target BRCA 2-deficient cells, it was determined that compound 1 was specifically toxic to BRCA 1 / 2-deficient cells (cisplatin-resistant cells), suggesting that compound 1 has potential clinical use in diseases that have become resistant to these drugs.

[0253] Table 2: IC50 of compound 1 on various breast cells and corresponding EGFR and HER2 expression levels as measured by Western blot

[0254] Cell lines EGFR HER2 IC 50 (μM) MDA-MB-468 ++++ - 4.21±0.30 BT-549 ++ - 3.24±0.08 MDA-MB-231 + - 4.89±0.37 HCC1937 + - 7.52±1.26 T-47D + + 5.19±0.28 BT-474 - ++++ 4.45±0.10 MDA-MB-453 - ++ 3.65±0.44 ZR-75-1 - + 1.69±0.14 MCF-7 - - 2.98±0.60 MCF-10A +++ + 105.56±3.14

[0255] ++++ means that the expression of EGFR or HER2 is very strong; +++ means that the expression of EGFR or HER2 is relatively strong; ++ means that the expression of EGFR or HER2 is moderate; + means that the expression of EGFR or HER2 is relatively low; - means that there is no expression of EGFR or HER2.

[0256] Biological Example 9

[0257] Compound 1 and KRAS GTP Interaction

[0258] Based on these findings, the novel small molecule compound 1 competitively binds to the GTP binding pocket of K-Ras with high affinity in the switch I region and inhibits the binding of KRAS to its downstream effectors, such as RAF / PI3K involved in cancer cell progression. Based on its ability to compete with GTP bound to KRas, it blocks K-Ras signaling and inhibits cell proliferation in several KRAS tumor-derived human cell lines, and also shows a 95% reduction in tumor growth inhibition in an in vivo animal model within 28 days compared to standard drugs, and no evidence of recurrence for 6-8 months.

[0259] Table 3: Interaction of compound 1 with KRAS GTP

[0260]

[0261]

[0262] Biological Example 10

[0263] Effects of compound 1 on wild-type downstream signaling

[0264] In wild-type KRAS, compound 1 had no effect on GTP binding affinity, and the molecule did not alter the expression of KRAS downstream signaling proteins such as MEK, RAF, and PI3K, effector proteins were intact with highly regulated GEF and GAP levels, as observed by protein and gene expression analysis. Significant increases in expression of immune cells were observed in compound 1 treatment.

[0265] Table 4: Effects of Compound 1 - Downstream Effects - Wild Type

[0266] protein Comparison Compound 1 pCRAF 1.52±0.025 1.42±0.25 pERK 1.42±0.069 1.46±0.23 Pk 1.89±0.63 1.72±0.14 pMEK 1.96±0.22 1.85±0.32 P1K 1.85±0.32 1.62±0.63

[0267] Biological Example 11

[0268] Figure 3 :Effect of compound 1 on RAS degradation

[0269] Interestingly, compound 1 degraded RAS through a cross-talk mechanism with the Wnt / β-catenin pathway through its negative regulators such as APC, Axin, and glycogen synthase kinase 3β (GSK3) via GSK3β phosphorylation. Fig.15 and Table 5)

[0270] Table 5: Effect of Compound 1 on RAS degradation

[0271] protein Comparison Compound 1 GSk3β 0.69±0.02 1.89±0.23 APC 0.63±0.03 0.96±0.44 Axin 0.25±0.05 1.96±0.02 β-catenin 1.32±0.09 0.66±0.09 RAS 1.96±0.06 0.25±0.03

[0272] Biological Example 12

[0273] Role of miR-30c and miR-21 in RAS degradation and cancer stem cells.

[0274] Mutated KRAS induces significant upregulation of miR-30c and miR-21. MiR-30c and miR-21 are significantly upregulated by both KRAS isoforms and induce drug resistance and enhance cell migration / invasion by inhibiting key tumor suppressor genes such as NF1, RASA1, BID, and RASSF8. The effects of compound 1 on the expression of miR 30c and miR-21 in mutant forms of KRAS and wild type were observed. Microarray analysis showed that the expression of these miRs was significantly reduced in the treatment of compound 1, and the expression of tumor suppressor genes was significantly increased in mutant forms at a dose-dependent level, and no significant expression was observed in the wild type. Based on these observations, molecular targeting of miRNAs in KRAS mutant models, which serve as attractive therapeutic tools in cancer medicine, because these miRNAs can be multiple gene silencing, and thus shut down different pathways at the same time, which is impossible in protein-based drugs and covalent inhibitors of other different KRAS mutant forms, and ultimately compound 1 has no toxicity or off-target effects compared to other standard drugs.

[0275] Table 6: Effect of Compound 1 on miRNA upregulation

[0276] protein Comparison Compound 1 miR30c 0.96±0.025 1.89±0.25 miR21 0.72±0.02 1.58±0.32 NF1 1.56±0.06 1.21±0.033 RASA1 1.69±0.36 0.32±0.031 BID 1.56±0.36 0.32±0.012

[0277] Biological Example 13

[0278] Oral bioavailability data / blood-brain barrier cross-talk data of compound 1

[0279] Table 7: Pharmacokinetic data of compound 1

[0280]

[0281] Table 8: Cross-blood-brain barrier data of compound 1

[0282]

[0283] Biological Example 14

[0284] Comparison of Compound 1 with known KRAS inhibitors

[0285] The reactivation of RAS signaling was compared for the administration of compound 1 and two known KRAS inhibitors (AMG510 and MRTX849). In all KRAS G12C mutant cell lines, compound 1, AMG510 and MRTX849 inhibited MAPK pathway signaling, a key downstream effector pathway of KRAS, as measured by inhibition of phospho-MEK, phospho-ERK and phospho-AKT at 4 hours. By 24-48 hours, RAS-MAPK pathway signaling began to rebound in cells treated with AMG510 and MRTX849, leading to pathway reactivation and incomplete inhibition of pMEK, pERK, pAKT and MYC at 72 hours. The rapid and consistent reactivation of signaling observed after KRAS G12C inhibition suggests that adaptive feedback may have the potential to limit the efficacy of these inhibitors. In contrast, the administration of compound 1 resulted in a significant reduction in the phosphorylated form, and no rebound activity was observed by 72 hours (see Fig.16 ). Densitometric measurements of phospho-ERK, MEK, AKT and MYC were normalized to GAPDH. Results represent the average across 8 cell lines.

[0286] The cytotoxic effects of compound 1, AMG510, and MRTX849 were also compared against an NCI panel of 10 KRAS G12C mutant cell lines. Compound 1 induced apoptosis in a dose-dependent manner and showed a significant reduction in the number of cancer cells compared to AMG510 and MRTX849 (see Fig.17 ).

[0287] Biological Example 15

[0288] Compound 1: In vivo tumor model

[0289] Compound 1 was found to cause regression of all mutant ARAS-driven cancers in immunocompetent mice. Compound 1 inhibited tumor growth starting from 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, and no evidence of recurrence for 6-8 months. See Fig.18 and Fig.19 .

[0290] Biological Example 16

[0291] Compound 1: Toxicity studies

[0292] Histopathology of normal tissues collected (brain, heart, lung, liver, spleen, kidney and intestine) revealed no evidence of normal tissue toxicity after treatment with the specified dose of Compound 1. Tests on blood cells (WBC, RBC and PLT) for bone marrow, RFT for kidney and ALT / AST for liver function provided results within normal range. See Fig. 20 .

[0293] Biological Example 17

[0294] Compound 1: Clinical trial protocol

[0295] This study is an open-label, two-part, first-in-human (FIH) dose-finding study designed to determine the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and proof-of-concept (POC) of compound 1 in patients with advanced or metastatic solid tumors. This study consists of two components:

[0296] Part 1: Dose escalation in patients with advanced or metastatic solid tumors, including Compound 1 dose levels. This study is planned to escalate the dose starting from 50 mg, followed by 100 mg, 200 mg, 300 mg, 450 mg and 600 mg as tentatively designated escalating dose groups. Part 1 will recruit approximately 11 to 24 patients in total, covering 5 dose levels.

[0297] The primary objectives were to determine the safety and tolerability of compound 1 and to define an appropriate dose for further evaluation in Part 2.

[0298] The study started with an accelerated titration dose-escalation schedule, enrolling one evaluable patient in each cohort for the first 2 dose levels; pending a safety signal, the classic 3+3 design followed.

[0299] Part 2: Dose expansion, in which at least 3 parallel groups of patients with advanced non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), and pancreatic cancer (PANC) are 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.

[0300] Approximately 18 (15 evaluable) patients will be enrolled in each of the 3 parallel patient groups in Part 2 (NSCLC, TNBC, PANC).

[0301] Compound 1 is administered by continuous dosing once a day, with dose escalation to the maximum tolerated dose (MTD) until progression or cessation. Each cycle of this study lasts 28 days. This study can be extended to up to 6 cycles to confirm safety and efficacy.

[0302] It can be clearly inferred from the above biological examples that compound 1, as a new class of KRAS, selectively targets the GTP / GDP binding pocket. The binding of compounds 1 to 5 to KRAS may promote the accumulation of GTP-KRAS by preventing cleavage from GTP to GDP. Superactivation of mutant KRAS induced by compounds 1 to 5 promotes apoptotic cell death in mutant KRAS cancer cells. Combining the results with detailed structural analysis, it is possible to describe key ligand-receptor interactions associated with activity. Therefore, it is shown that the screening technology is very successful in producing KRAS binders that have an effect on signaling in cells. It is known that compound compound 1 is the first known nanomolar binder of KRAS, which disrupts the interaction with CRaf, reduces p-ERK levels and proliferates cells. Therefore, the compound, namely compound 1, is a promising hit for the development of a new non-covalent KRAS inhibitor. The development of this new anticancer drug provides a potentially effective strategy for treating cancers with KRAS mutations and / or cancers driven by mutant KRAS. Furthermore, compound 1 inhibited KRAS GTPase and activated degradation of GSK3β through ubiquitin-mediated pathways via miR-30c and miR-21 regulation leading to programmed cell death.

[0303] According to the above, it should be understood that the various embodiments of the present disclosure have been described herein for illustrative purposes, and various modifications can be made without departing from the scope and spirit of the present disclosure. Therefore, the various embodiments disclosed herein are not intended to be limiting, and the true scope and spirit are indicated by the appended claims.

Claims

1. Compound of formula 1: or a pharmaceutically acceptable salt thereof or a combination thereof, in: X is an unsubstituted C1-C5 alkyl group; Y is a carboxyl group connected to an unsubstituted C1-C5 alkyl group; Z is a carbonyl group linked to 5-carbamoyl, 2,3, dihydro-1H-pyrrole; and W is H.

2. A pharmaceutical composition comprising an effective amount of the compound according to claim 1 and a pharmaceutically acceptable carrier thereof.

3. A product comprising: (i) an effective amount of the compound according to claim 1 or the pharmaceutical composition according to claim 2; and (ii) instructions for use in treating a disease, disorder or condition mediated by KRAS.

4. A kit comprising: (i) an effective amount of the compound according to claim 1 or the pharmaceutical composition according to claim 2; and (ii) instructions for use in treating a disease, disorder or condition mediated by KRAS.

5. Use of the compound according to 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.

6. The compound according to claim 1, wherein the compound is 7. The kit according to claim 4, wherein the compound is 8. The use according to claim 5, wherein the compound is

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