Novel small molecule KRAS inhibitors: Undruggable targets for cancer therapeutics

Abstract

The present invention provides compounds of structural formula I: TIFF2026500301000012.tif40159 or a pharmaceutically acceptable salt thereof; where W is selected from the group consisting of substituted saturated / unsaturated branched / straight chain aliphatic amines, substituted aromatic amines, unsubstituted saturated / unsaturated branched / straight chain aliphatic amines, unsubstituted aromatic amines; X is selected from the group consisting of H, a substituted / unsubstituted saturated / unsaturated branched / straight-chain aliphatic alkyl group having at least one carbon atom, a substituted / unsubstituted saturated / unsaturated branched / straight-chain aliphatic alkoxy group having at least one carbon atom, a substituted / unsubstituted aryl group, a substituted / unsubstituted aryloxy group; Y is selected from the group consisting of H, substituted / unsubstituted saturated / unsaturated branched / straight chain aliphatic alkyl groups bonded to a carbonyl group, substituted / unsubstituted aryl groups bonded to a carbonyl group; Z is selected from groups including H and OH.

Description

[Technical Field]

[0001] The present invention relates to novel molecules that exhibit anticancer activity. More specifically, the present invention relates to novel small molecules that can inhibit downstream Ras signaling and cell proliferation in cancer cells expressing wild-type and K-Ras G12C, G12D, and G12S mutants, and N-Ras Q61K mutants. [Background technology]

[0002] RAS genes encode small GTPases essential for mammalian cell growth, differentiation, and survival. RAS gene mutations are associated with approximately 30% of all human cancers. However, based on measurements reported 30 years ago that show that the affinity of Ras proteins for GTP is in the 10-20 picomolar range, the scientific and medical communities have come to recognize Ras proteins as undruggable targets.

[0003] Ras proteins are encoded by three ubiquitously expressed genes (H-RAS, K-RAS, and N-RAS) (Parker and Mattos, 2018). They couple cell surface receptors to intracellular effector pathways and are master regulators of cell proliferation and differentiation (Simanshu et al., 2017; Stephen et al., 2014). Binding of GTP and GDP cycles Ras proteins between on and off signaling conformations, respectively. Under physiological conditions, the transition between these two states has been shown to be regulated by guanine nucleotide exchange factors (GEFs), which promote Ras protein activation by stimulating the release of bound GDP and the binding of GTP, and by GTPase-activating proteins (GAPs), which promote Ras-mediated GTP hydrolysis (Simanshu et al., 2017; Milburn et al., 1990; Shieh, 2019; Hunter et al., 2015). Ras proteins are a subset of the Ras GTPase superfamily, which consists of over 150 human members that show high sequence homology in their GTP-binding sites and include Rac-1, Rho-A, and cdc42 (Wennerberg et al., 2005; Cox and Der, 2010). Approximately 30% of all human cancers are associated with mutations in Ras proteins (Prior et al., 2012; Hobbs et al., 2016; Pylayeva-Gupta et al., 2011). K-Ras is the most mutated Ras isoform. K-Ras mutations are frequently detected in pancreatic, colorectal, and lung tumors (Prior et al., 2012; Hobbs et al., 2016; Cox et al., 2014). The most frequent K-Ras mutations are at residues Gly12, Gly13, and Gln61 (Hobbs et al., 2016; Li et al., 2018). N-Ras is mutated in approximately 20% of all melanoma patients (Jenkins and Sullivan, 2016). H-Ras mutations are relatively rare (Prior et al., 2012). There remains a significant unmet medical need for patients with diseases associated with mutated RAS genes, particularly cancer patients.Numerous small molecules that specifically target the K-Ras G12C mutant via a covalent binding mechanism have been identified, two of which are currently in clinical development (McCormick, 2020; Grapsa and Syrigos, 2020). However, no small molecules have been reported that could potentially target other mutant or wild-type Ras proteins. This is likely primarily due to the generally accepted paradigm that small molecules cannot be developed that can compete with guanine nucleotides for binding to the Ras GTP / GDP binding site (a concept based on studies in the 1990s reporting that the GTP binding affinity for Ras proteins is in the 10–20 pM range (John et al., 1990; John et al., 1993)). Therefore, we sought to develop small molecules that could inhibit Ras downstream signaling and cell proliferation in cancer cells expressing wild-type and K-Ras G12C, G12D, and G12S mutants, as well as the N-Ras Q61K mutant.

[0004] Goal of the Invention The primary focus of the present invention is on novel small molecules that exhibit anti-cancer activity.

[0005] Another goal of this invention is to synthesize novel small molecules that exhibit anti-cancer activity.

[0006] Another goal of the present invention is to synthesize novel small molecules that can inhibit Ras downstream signaling and cell proliferation in human pancreatic cancer cells and non-small cell lung cancer cells expressing wild-type and K-Ras G12C, G12D, and G12S mutants, and N-Ras Q61K mutants.

[0007] A further goal of the present invention is to use the small molecules developed above for subjects suffering from cancer. [Brief explanation of the drawings]

[0008] [Figure 1] Cell proliferation assay (MTT) is shown. [Figure 2]Figure 1 shows the effect of Compound I on apoptosis induction (immunoblot assay). [Figure 3] Figure 1 shows the effect of Compound I on the induction of cell cycle arrest. [Figure 4] 1 shows the effect of Compound I on the increase of cellular cyclin inhibitors. [Figure 5] 1 shows the effect of Compound I on cyclin checkpoints in cells. [Figure 6] 1 shows the effect of Compound I on the mTor pathway. [Figure 7] 1 shows the effect of Compound I on KRAS mutant cell lines. [Figure 8] 1 shows the effect of Compound I on signal transduction downstream of Ras. [Figure 9] 1 shows the effect of Compound I on the inhibition of the Ras-GTP complex. [Figure 10] 1 shows the 1H NMR spectrum of Compound I of the present invention. [Figure 11] 1 shows the 13C NMR spectrum of Compound I of the present invention. [Figure 12] 1 shows the 2D COSY spectrum of Compound I of the present invention. [Figure 13] 1 shows the IR spectrum of Compound I of the present invention. Summary of the Invention

[0009] The present invention provides compounds of structural formula I: [ka] or a pharmaceutically acceptable salt thereof; W is selected from the group comprising substituted saturated / unsaturated branched / straight chain aliphatic amines, substituted aromatic amines, unsubstituted saturated / unsaturated branched / straight chain aliphatic amines, unsubstituted aromatic amines; X is selected from the group consisting of H, a substituted / unsubstituted saturated / unsaturated branched / straight chain aliphatic alkyl group having at least one carbon atom, a substituted / unsubstituted saturated / unsaturated branched / straight chain aliphatic alkoxy group having at least one carbon atom, a substituted / unsubstituted aryl group, a substituted / unsubstituted aryloxy group; Y is selected from the group consisting of H, substituted / unsubstituted saturated / unsaturated branched / straight chain aliphatic alkyl groups bonded to a carbonyl group, substituted / unsubstituted aryl groups bonded to a carbonyl group; Z is selected from groups including H and OH. DETAILED DESCRIPTION OF THE INVENTION

[0010] definition "Alkyl" means saturated carbon chains which may be linear or branched, or combinations thereof, unless the carbon chain is defined otherwise. Other groups having the prefix "alk," such as alkoxy and alkanoyl, may also be linear or branched, or combinations thereof, unless the carbon chain is defined otherwise. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec- and tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, and the like. In one embodiment of the invention, alkyl is methyl.

[0011] "Aryl" means a monocyclic, bicyclic, or tricyclic carbocyclic aromatic ring or ring system containing 5 to 14 carbon atoms, wherein at least one of the rings is aromatic. Examples of aryl include phenyl and naphthyl. In one embodiment of the invention, aryl is phenyl.

[0012] "Alkoxy" has the chemical formula -OR a where R ais alkyl, alkenyl, or alkynyl as defined above containing 1 to 12 carbon atoms. Unless stated otherwise in the specification, an alkoxy group may be optionally substituted.

[0013] "Aryloxy" refers to a group of formula -OAr, where Ar is a monocyclic, bicyclic, or tricyclic carbocyclic aromatic ring or ring system containing 5 to 14 carbon atoms, where at least one of the rings is aromatic. Examples of aryl include phenyl and naphthyl. In one embodiment of the invention, aryl is phenyl.

[0014] In selecting compounds of the present invention, one skilled in the art will understand that the various substituents are selected in accordance with well-known principles of connectivity and stability of chemical structures.

[0015] The term "substituted" is considered to include various degrees of substitution by a particular substituent. Where multiple substituent moieties are disclosed or claimed, the substituted compound may be independently substituted, singly or multiply, with one or more of the disclosed or claimed substituent moieties. "Independently substituted" means that the (two or more) substituents may be the same or different.

[0016] The expression "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, salts and / or dosage forms that are safe and suitable for administration to humans or animals using sound medical judgment and in accordance with all applicable government regulations.

[0017] References herein to the compounds of the invention also include their pharmaceutically acceptable salts, and, when they are used as free compounds or precursors to their pharmaceutically acceptable salts, or in other synthetic operations, will be understood to also include salts that are not pharmaceutically acceptable.

[0018] The compounds of the present invention may be administered in the form of pharmaceutically acceptable salts. The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases and acids, including inorganic or organic bases and inorganic or organic acids. The salts of basic compounds encompassed by the term "pharmaceutically acceptable salts" refer to non-toxic salts of the compounds of the present invention, which are generally prepared by reacting the free base with a suitable organic or inorganic acid. Representative salts of the basic compounds of the present invention include acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycolylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothioate, lactate, lactobionate, lauryl hydroxybenzoate, ... Examples of suitable pharmaceutically acceptable salts include, but are not limited to, phosphate, malate, maleate, mandelate, mesylate, methyl bromide, methyl nitrate, methyl sulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, sulfate, hypoacetite, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, and valerate. Furthermore, when the compound of the present invention has an acidic moiety, suitable pharmaceutically acceptable salts include, but are not limited to, salts derived from inorganic bases, including aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, zinc, etc. Particularly preferred are ammonium, calcium, magnesium, potassium, and sodium salts.Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, salts of cyclic amines, and salts of basic ion exchange resins (arginine, betaine, caffeine, choline, N,N-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like).

[0019] Additionally, for carboxylic acid (—COOH) or alcohol groups present in the compounds of the invention, pharmaceutically acceptable esters of carboxylic acid derivatives, such as methyl, ethyl, or pivaloyloxymethyl, or acyl derivatives of alcohols, such as O-acetyl, O-pivaloyl, O-benzoyl, and O-aminoacyl, can be used. Included among these are esters and acyl groups known in the art for adjusting the solubility or hydrolysis characteristics for use as sustained-release or prodrug formulations.

[0020] The term "therapeutically effective" as applied to a dose or amount refers to an amount of a compound or pharmaceutical preparation that is sufficient to produce a desired clinical effect following administration to a patient in need thereof.

[0021] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. The illustrative embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments may be used, 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 outlined herein and shown in the drawings, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein.

[0022] Generally, the present invention discloses novel therapeutic compounds that exhibit anti-cancer activity.

[0023] In a preferred embodiment, the present invention provides a compound of structural formula I: [ka] or a pharmaceutically acceptable salt thereof; W is selected from the group comprising substituted saturated / unsaturated branched / straight chain aliphatic amines, substituted aromatic amines, unsubstituted saturated / unsaturated branched / straight chain aliphatic amines, unsubstituted aromatic amines; X is selected from the group consisting of H, a substituted / unsubstituted saturated / unsaturated branched / straight chain aliphatic alkyl group having at least one carbon atom, a substituted / unsubstituted saturated / unsaturated branched / straight chain aliphatic alkoxy group having at least one carbon atom, a substituted / unsubstituted aryl group, a substituted / unsubstituted aryloxy group; Y is selected from the group consisting of H, substituted / unsubstituted saturated / unsaturated branched / straight chain aliphatic alkyl groups bonded to a carbonyl group, substituted / unsubstituted aryl groups bonded to a carbonyl group; Z is selected from groups including H and OH.

[0024] According to the present invention, in the compound of Formula 1, W is a substituted aromatic amine or a pharmaceutically acceptable salt thereof.

[0025] According to the present invention, in the compound of formula 1, W is an N-linked p-hydroxyaniline.

[0026] According to the present invention, in the compound of Formula 1, X is an unsubstituted saturated linear aliphatic alkoxy group having at least one carbon atom or a pharmaceutically acceptable salt thereof.

[0027] According to the present invention, in the compound of formula 1, X is a methoxy group.

[0028] According to the present invention, in the compound of Formula 1, Y is an unsubstituted saturated straight-chain aliphatic alkyl group attached to a carbonyl group, or a pharmaceutically acceptable salt thereof.

[0029] According to the present invention, in the compound of formula 1, Y is an acetyl group.

[0030] According to the present invention, in the compound of Formula 1, Z is a hydroxyl group or a pharmaceutically acceptable salt thereof.

[0031] According to the present invention, in the compound of formula 1, Z is a hydroxyl group.

[0032] In another preferred embodiment, the present invention provides compound I: [ka] Disclosed is a compound comprising the structure:

[0033] In yet another preferred embodiment, the present invention discloses a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0034] In a further preferred embodiment, the present invention discloses a pharmaceutical composition comprising a therapeutically effective amount of Compound I and a pharmaceutically acceptable carrier.

[0035] Any chemical structure of Compound I can be prepared by conventional chemistry based on the exemplary structures provided herein.

[0036] In one embodiment, exemplary compounds of Formula 1 can be prepared by the process provided below.

[0037] [1.1] Stage 1: Preparation of ABCD001 Manufacturing process overview: (5-(2-amino-6-oxo-1H-purin-9(6H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl triphosphate is reacted with BOC-anhydride in the presence of base, THF and water to give ABCD001.

[0038] Synthesis Route [ka]

[0039] [1.2] Stage 2: Preparation of ABCD002

[0040] Manufacturing process overview ABCD001 is reacted with 2-(4-acetyl-2-methoxyphenyl)-2-oxoethyl 4-methylbenzenesulfonate in the presence of tetrabutylammonium bromide and acetonitrile to give ABCD002.

[0041] Synthesis Route [ka]

[0042] [1.3] Stage 3: Preparation of ABCD003 Manufacturing process overview ABCD002 is reacted with hydrochloric acid in dioxane to give ABCD003.

[0043] Synthesis Route [ka]

[0044] [1.4] Stage 4: Preparation of ABCD004 / compound I (target molecule) Manufacturing process overview ABCD003 is reacted with 4-bromophenol in the presence of a copper catalyst, potassium carbonate and methanol to give ABCD004 / compound I.

[0045] Synthesis Route [ka]

[0046] The synthesized compounds are then subjected to molecular characterization to confirm the structure of the compounds.

[0047] Molecular characterization The purity of the synthesized compound I was confirmed by melting point, thin layer chromatography, HPLC, IR, and NMR analyses. Figures 10 to 13 confirm that the structure of the synthesized compound I is as follows. [ka]

[0048] In some embodiments, Compound I may include both cis and trans isomers. In some embodiments, Compound I may be a mixture of cis and trans isomers. In some embodiments, Compound I may be a cis isomer. In some embodiments, Compound I may be a trans isomer.

[0049] In some embodiments, Compound I can include either the R or S stereoisomer, as well as mixtures of stereoisomers. In some embodiments, Compound I can include both racemic and optical isomers.

[0050] Compounds I of the present invention can be used to exert or provide any of the biological functions described herein.

[0051] Pharmaceutical Composition The present disclosure also includes pharmaceutical compositions comprising a therapeutically effective amount of Compound I disclosed herein. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of Compound I or a pharmaceutically acceptable salt thereof.

[0052] In various aspects, the amount of Compound I or a pharmaceutically acceptable salt thereof can be administered in an amount of from about 0.001 mg / kg to about 100 mg / kg of body weight (e.g., from about 0.01 mg / kg to about 10 mg / kg of body weight, or from about 0.1 mg / kg to about 5 mg / kg of body weight).

[0053] The concentration of the compound of the present disclosure in the pharmaceutically acceptable mixture varies depending on several factors, including the dosage of the compound to be administered, the pharmacokinetic properties of the compound used, and the route of administration.The drug can be administered in a single dose or multiple doses.The dosage regimen utilizing the compound of the present invention is selected according to various factors, including the type, species, age, weight, sex, and condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the specific compound or its salt used.Treatment can be administered once or more times a day, depending on many factors, including the overall health of the patient, and the formulation and route of administration of the compound selected.

[0054] The compounds or pharmaceutical compositions of the disclosure may be prepared and / or administered in single or multiple unit dosage forms.

[0055] In some embodiments, Compound I of the present disclosure is administered to patients with cancer and its associated complications.

[0056] In certain embodiments, the compounds and compositions described herein are administered in combination with one or more anti-cancer drugs. Compound I of the present invention may be used in combination with other drugs that are also useful in the treatment or amelioration of diseases or conditions for which the compounds of the present invention are useful. Such other drugs may be administered simultaneously with the compounds of the present invention or sequentially, by a route and in an amount commonly used therefor.

[0057] When compound I of the present invention is used simultaneously with one or more other drugs, a pharmaceutical composition in unit dosage form containing such other drugs and the compound of the present invention is preferred.However, combination therapy also includes treatment in which compound I of the present invention and one or more other drugs are administered on different overlapping schedules.It is also understood that when used in combination with one or more other active ingredients, the compound of the present invention and other active ingredients may be used in lower doses than when each is used alone.Therefore, the pharmaceutical composition of the present invention includes pharmaceutical compositions containing one or more other active ingredients in addition to the compound of the present invention.

[0058] Examples of other active ingredients that may be administered individually or in combination with Compound I described herein in the same pharmaceutical composition include, but are not limited to, altretamine, bendamustine, busulfan, carboplatin, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, mechlorethamine, melphalan, oxaliplatin, temozolomide, thiotepa, and trabectedin.

[0059] The present invention also provides a method for the treatment of cancer, the method comprising administering to a patient a therapeutically effective amount of compound I of the present invention and an amount of one or more active ingredients, whereby together they provide effective relief.

[0060] In a further aspect of the present invention, there is provided a pharmaceutical composition comprising Compound I of the present invention together with at least one pharmaceutically acceptable carrier or excipient.

[0061] Thus, according to a further aspect of the present invention, there is provided the use of compound I of the present invention for the manufacture of a therapeutic medicament for the treatment of cancer. In a further or alternative aspect of the present invention, there is therefore provided a product comprising compound I of the present invention and one or more active ingredients as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer.

[0062] It will be appreciated that for the treatment of cancer, the compounds of the invention may be used in conjunction with another pharmaceutical agent effective to treat that disorder.

[0063] The present invention also provides a method for the treatment of cancer, the method comprising administering to a patient in need of such treatment an amount of a compound I of the present invention and an amount of another pharmaceutical agent effective to treat that disorder, whereby together they provide effective relief.

[0064] Biological Examples Example 1: Cell viability for human adenocarcinoma cell lines Multiple cell lines, including human breast cancer (MCF7), colon adenocarcinoma (HT-29), liver adenocarcinoma (HEP G2), and lung adenocarcinoma (A549), were treated with various compounds I for 48 hours, and the percentage of cell viability was determined by MTT assay. The mean total viable cell counts were accurately calculated to determine the antiproliferative effect. Compound I inhibited the proliferation of all cell lines in a dose-dependent manner (Figure 1). Dose-dependent growth inhibition was observed at concentrations ranging from 250 to 0.97 nmol / L, with each cell line exhibiting different sensitivities. The drugs were also tested on monkey normal renal epithelial cells. As expected, normal monkey renal epithelial cells were less sensitive to induced cytotoxicity.

[0065] Example 2: The effect of Compound I is to trigger the apoptotic pathway To confirm that compound I induces cell apoptosis, a DNA fragmentation assay was performed. Exposure of cells to compound I for 48 hours resulted in DNA fragmentation, as indicated by a typical DNA ladder pattern in agarose gels, at all concentrations tested, whereas the control showed no DNA fragmentation. Quantitative analysis revealed a significant increase in apoptosis in cells treated with compound I, and higher doses of the drug were found to exhibit pronounced cytotoxicity and antiproliferative effects. Furthermore, the expression levels of proteins related to apoptosis and cell cycle progression were examined by Western blot. Cells were treated with compound I at different concentrations, and total protein was isolated. β-actin was used as an internal control. Supplementation with compound I downregulated the expression of Bcl-2, a potent suppressor of apoptosis, and induced the expression of proapoptotic proteins Bax, caspase-9, and caspase-3 in a dose-dependent manner (Figure 2). The effects of the drug on the cleavage of procaspase-9 and procaspase-3 were examined. Western blot analysis revealed that procaspase-9 and procaspase-3 cleavage was induced in a dose-dependent manner (Figure 2). Caspase-3 is a key regulator of the mitochondria-dependent independent apoptotic pathway, and activation of caspase-3 leads to the cleavage of multiple substrates, including PARP. Compound I then induced the cleavage of PARP into 116 kDa and 84 kDa fragments, as determined by Western blot analysis.

[0066] Example 3: Effect of Compound I on cell cycle arrest Flow cytometry analysis was performed to determine whether compound I-induced apoptosis was associated with cell cycle arrest. Cell cycle distribution in KRAS mutant cell lines was quantified after treatment with different concentrations of compound I. Flow cytometry analysis revealed an increased proportion of G2 / M arrested cells compared with control cell lines. The number of cells in G2 / M phase increased (76.89%) in a dose-dependent manner in cells treated with compound I (Figure 3). To further evaluate the effect of compound I on cell progression through G2 / M phase, its effect was examined at the protein levels of p53, p21, p27 (Figure 4), cyclin B1, and cdc2 genes (all of which regulate transition through the G2 checkpoint). As shown in Figure 5, the expression levels of cyclin B, cdc2, and CDK2 were significantly decreased in a dose-dependent manner after compound I treatment. Higher expression levels of p21, p27, and p53 were recorded in G2 cells of KRAS mutant cell lines treated with compound I. Although an increased population of cells in the G2 / M phase was found, 23.98% of the cells showed G1 / S phase arrest (Figure 3). Therefore, we attempted to determine whether compound I treatment could also affect the expression levels of G1 / S phase cell cycle regulatory proteins. As shown in Figure 4, the expression levels of CDK4, CDK6, cyclin D1, cyclin D3, and pRb were significantly reduced after treatment with compound I in a dose-dependent manner. Control cell lines treated with compound I were better protected against alterations in cell cycle control and apoptosis compared to KRAS mutant cell lines treated with compound I.

[0067] Example 4: Effect of Compound I on the mTOR pathway To better understand the molecular basis of compound I-induced cell cycle arrest and apoptosis, we examined the expression of Akt and mTOR at various concentrations of compound I. Akt / mTOR is an important cell survival pathway, and activated Akt inhibits apoptosis and leads to cell survival. As shown in Figure 6, the activation of both AKT and mTOR was inhibited by compound I in a concentration-dependent manner, while the overall levels of Akt and mTOR remained constant. Compound I was found to inhibit the phosphorylation of Akt and mTOR at serine 473 and serine 2448, respectively. ERK phosphorylation was also inhibited by compound I treatment at both threonine 202 and tyrosine 204. We further examined whether the effect of compound I involved the ERK signaling pathway and found reduced ERK activation. These results suggest that in addition to downregulating anti-apoptotic gene products, compound I also mediates its effects by downregulating the Akt / mTOR cell signaling pathway.

[0068] Example 5: Antiproliferative effects induced by selected compounds The inhibitory effect of compound I on cell proliferation in six cell lines was evaluated: three cell lines in signal transduction studies (PANC-1, MIA PaCa-2, and NCIH1975), and the non-small cell lung cancer lines A549 (K-Ras G12S mutant) and NCI-H1299 (N-Ras Q61K mutant). Figure 7 and IC50 measurements were obtained in these studies. Compound I inhibited cell proliferation in a dose-dependent manner in all tested cell lines, with similar IC50 values ​​ranging from approximately 0.45 μM to 1.20 μM. Consistent with the concept that these compounds are pan-Ras superfamily inhibitors, it is reasonable to assume that they may inhibit downstream pathways of multiple members of the Ras superfamily.

[0069] Example 6: Inhibition of Ras downstream pathways The inhibitory effects of compound I on Ras-related signaling pathways were tested in three different human cell lines: the pancreatic cell line PANC-1 (K-Ras G12D mutant), MIA PaCa-2 (K-Ras G12C mutant), and the non-small cell lung cancer cell line NCI-H1975 (KRas WT). Figure 8 shows the inhibitory effects of compound I on the phosphorylation and activation of MEK, Erk1 / 2, and Akt in these three cell lines. As shown in Figure 2, compound I dose-dependently inhibited the phosphorylation and activation of MEK, Erk1 / 2, and Akt in PANC-1, MIA PaCa-2, and NCI-H1975 cell lines with similar IC50 values ​​(approximately 1–3 μM). These effects are expected from a Ras inhibitor that acts as an upstream blocker of the two pathways and support the notion that compound I induces an inactive conformation of Ras through a consistent binding mode, regardless of the different mutations. In contrast, the IC50 of Compound I for phosphorylation and activation of MEK and Erk1 / 2 was approximately 2 μM in PANC-1 and MIA PaCa-2 and 3 μM in NCI-H1975; the IC50 for Akt was approximately 5 μM in PANC-1 and MIA PaCa-2 and 5 μM in NCI-H1975 (Figure 8).

[0070] Example 7: Inhibition of Ras-GTP complex As a follow-up to the downstream assays, the ability of compound I to inhibit Ras-GTP complex formation was tested in three cell lines (PANC-1, MIA PaCa-2, and NCIH1975) using a Ras pull-down assay. As shown in Figure 4, the compound inhibits Ras-GTP complex formation with IC50 values ​​in the range of 2–3 μM. These values ​​correlate well with the IC50 values ​​obtained for the inhibition of MEK, Erk1 / 2, and Akt phosphorylation shown in Figures 2 and 3. The detected inhibition of Ras-GTP complex formation strongly suggests that both compounds bind directly to the GTP-binding site of the Ras protein and that, upon binding, the tested compounds induce an inactive conformation of Ras due to similar effects on the GDP-bound state.

[0071] From the foregoing description, it will be understood that the various embodiments of the present disclosure have been described for purposes of illustration and that various modifications may be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to limit the true scope of the present disclosure, which is set forth in the appended claims.

Claims

1. Structural formula I: 【Chemistry 1】 or a pharmaceutically acceptable salt thereof; wherein W is selected from the group consisting of substituted saturated / unsaturated branched / straight chain aliphatic amines, substituted aromatic amines, unsubstituted saturated / unsaturated branched / straight chain aliphatic amines, unsubstituted aromatic amines; X is selected from the group consisting of H, a substituted / unsubstituted saturated / unsaturated branched / straight chain aliphatic alkyl group having at least one carbon atom, a substituted / unsubstituted saturated / unsaturated branched / straight chain aliphatic alkoxy group having at least one carbon atom, a substituted / unsubstituted aryl group, a substituted / unsubstituted aryloxy group; Y is selected from the group consisting of H, a substituted / unsubstituted saturated / unsaturated branched / straight chain aliphatic alkyl group bonded to a carbonyl group, a substituted / unsubstituted aryl group bonded to a carbonyl group; Z is selected from the group including H, OH; A compound or a pharmaceutically acceptable salt thereof.

2. W is a substituted aromatic amine or a pharmaceutically acceptable salt thereof; 10. The compound claimed in claim 1.

3. W is an N-linked p-hydroxyaniline; 10. The compound claimed in claim 1.

4. X is an unsubstituted saturated straight-chain aliphatic alkoxy group having at least one carbon atom or a pharmaceutically acceptable salt thereof; 10. The compound claimed in claim 1.

5. X is a methoxy group; 10. The compound claimed in claim 1.

6. Y is an unsubstituted saturated straight-chain aliphatic alkyl group attached to a carbonyl group, or a pharmaceutically acceptable salt thereof; 10. The compound claimed in claim 1.

7. Y is an acetyl group; 10. The compound claimed in claim 1.

8. Z is a hydroxyl group or a pharmaceutically acceptable salt thereof; 10. The compound claimed in claim 1.

9. Z is a hydroxyl group; 10. The compound claimed in claim 1.

10. Compound I: 【Chemistry 2】 It consists of the structure of 10. The compound claimed in claim 1.

11. 10. A pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

12. 11. A pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 10 and a pharmaceutically acceptable carrier.

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