7-alkoxyquinazoline-based egfr inhibitors, processes for their preparation and medical uses

Abstract

This invention, entitled "EGFR Inhibitor Based on 7-Alkoxyquinazoline, its Preparation Method, and Pharmaceutical Use," belongs to the field of pharmaceutical technology. The technical problem to be solved is to provide an EGFR inhibitor based on 7-alkoxyquinazoline that can efficiently, broadly, and safely inhibit EGFR activity. The key technical solution is a 7-alkoxyquinazoline-based EGFR inhibitor that, by employing an α-chlorofluoroacetamide warhead and introducing an ethoxy group at the 7-position, obtains a novel EGFR inhibitor with excellent comprehensive performance in terms of efficacy spectrum, resistance overcoming potential, and safety, capable of safely, potently, and persistently inhibiting both wild-type and mutant EGFR.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to EGFR inhibitors, specifically to EGFR inhibitors based on 7-alkoxyquinazoline, their preparation methods, and pharmaceutical uses. Background Technology

[0002] For understanding the technical content of this invention: Epidermal growth factor receptor (EGFR), an important receptor tyrosine kinase, plays a central role in regulating cell proliferation, differentiation, survival, and migration. Aberrant activation of EGFR, especially persistent activation caused by gene mutations, is closely related to the occurrence and development of various epithelial-derived malignancies, such as non-small cell lung cancer. Therefore, EGFR has become one of the key targets for anti-tumor drug development.

[0003] In the development of EGFR inhibitors, covalent irreversible inhibitors represent an important technological direction. These inhibitors achieve potent and sustained inhibition of kinase activity by forming a covalent bond with the Cys797 residue at the edge of the ATP-binding site of the EGFR kinase domain. Second-generation EGFR covalent inhibitors, represented by afatinib, have been used clinically; however, their core reactive group—the Michael receptor—has issues such as high reactivity and a significant potential off-target risk, which may lead to side effects based on wild-type EGFR inhibition, such as diarrhea and rashes.

[0004] Furthermore, with the widespread use of third-generation EGFR inhibitors (targeting the T790M mutation), such as osimertinib, new acquired resistance has emerged in clinical practice. Mutations at the EGFR C797S site are one of the most significant mechanisms leading to the failure of these third-generation drugs. This mutation renders third-generation drugs that rely on C797 for covalent binding ineffective, highlighting the urgent need to develop next-generation inhibitors or new strategies to overcome this resistance.

[0005] Relevant patent documents retrieved: The publishing agency is the International Bureau of the World Intellectual Property Organization (WIPO), publication number WO2018084321A1, publication date May 11, 2018. This document discloses novel compounds for EGFR inhibition and tumor therapy, aiming to develop a covalent drug with low side effect risk and high safety by searching for a reactive group that can replace the Michael receptor and has milder reactivity, and using this group to design drug molecules. This invention relates to, for example, compounds derived from... The compound referred to is a pharmacologically acceptable salt thereof. This literature uses the α-chlorofluoroacetamide group as an alternative covalent reactive group, which has milder reactivity and higher selectivity, aiming to reduce off-target effects and associated toxicity.

[0006] Relevant non-patent literature retrieved: The journal *Chongqing Medical Journal*, with the article titled "Correlation between EGFR C797S Mutation and Osimertinib Resistance," volume 2021, 50(09), and publication date 2021, discloses that the C797S mutation of the epidermal growth factor receptor (EGFR) is confirmed to be one of the most common mechanisms of osimertinib resistance, manifesting as simple C797S mutation, C797S / T790M cis mutation, and C797S / T790M trans mutation. Currently, strategies for overcoming osimertinib resistance associated with EGFR C797S mutations exist, and good efficacy has been achieved through continuous exploration. For simple C797S mutations, a first-generation EGFR tyrosine kinase inhibitor (EGFR-TKI) can be used for treatment. For C797S / T790M cis mutations, a regimen of brigatinib combined with cetuximab can be used for treatment. For C797S / T790M trans mutations, a combination of first-generation and third-generation EGFR inhibitors can be used for treatment.

[0007] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: (1) The inhibitory efficacy and activity spectrum need to be improved: For example, some compounds provided in patent document WO2018084321A1 have insufficient inhibitory efficacy against wild type and common activating mutants (such as L858R / Del19, T790M), resulting in limited anti-tumor efficacy, narrow therapeutic window, and difficulty in achieving an ideal balance between efficacy and safety.

[0008] (2) Inability to overcome key clinical resistance mutations: Existing technologies lack effective activity against acquired resistance mutations (especially the C797S mutation) that occur after treatment with third-generation EGFR inhibitors. When faced with triple mutants such as EGFR T790M / C797S, existing drugs are largely ineffective and cannot meet the urgent need to address clinical resistance, resulting in a significant treatment gap. For example, while the non-patent literature "Correlation between EGFR C797S mutation and osimertinib resistance" provides strategies to overcome osimertinib resistance related to the EGFR C797S mutation, these are all combination therapies, lacking a single-agent targeted compound or treatment method that can effectively overcome this resistance mutation.

[0009] (3) The overall performance is insufficient to meet the needs of next-generation drugs: While pursuing highly selective covalent warheads, existing technologies have failed to synergistically address the multiple challenges of efficacy, broad-spectrum resistance to drug resistance mutations, and ideal pharmacokinetic / safety. Their overall performance has shortcomings and cannot become a next-generation inhibitor that is significantly superior in terms of efficacy spectrum, drug resistance overcoming potential, and safety. Summary of the Invention

[0010] The inventors discovered that an EGFR inhibitor (hereinafter referred to as compound 8) disclosed in patent document publication number WO2018084321A1 has the following chemical formula: Although existing technologies, exemplified by Compound 8, have shown potential in terms of safety through the introduction of mild covalent warheads, their overall inhibitory efficacy and therapeutic window still require further optimization, and they are insufficient to effectively address the pressing clinical problem of drug resistance. Specifically, the following clear defects and shortcomings exist: (1) The inhibitory efficacy urgently needs to be improved, and there is a clear technical bottleneck: The biological activity data of compound 8 are mediocre. In the proliferation inhibition experiments against various EGFR-driven lung cancer cell lines, its half-maximal inhibitory concentration (IC50) was low. 50 The efficacy value is not ideal. This indicates that its binding affinity to the target and the intensity of functional inhibition have inherent limitations. The insufficient efficacy directly leads to the need for higher dosages in clinical applications, which not only increases the metabolic burden on patients but may also amplify the potential off-target risks, thereby offsetting the safety advantages envisioned based on the mild warhead, ultimately resulting in a narrow "therapeutic window" and limited practical value.

[0011] (2) Lack of effective activity against drug-resistant mutations (such as C797S), failing to meet the most pressing clinical needs: More importantly, existing technologies, such as compound 8 mentioned above, were not designed to overcome the resistance problem of third-generation EGFR inhibitors represented by the C797S mutation. These traditional quinazoline backbone covalent inhibitors are generally ineffective or have extremely weak activity against EGFR C797S mutants. This means that existing technologies have a fundamental functional deficiency in the face of this major clinical challenge and cannot serve as an ideal starting point for subsequent development.

[0012] (3) Insufficient molecular structure optimization and design gaps at key sites: Taking compound 8 in the prior art as an example, the simplest methoxy group (-OCH3) is retained at the 7-position substituent of the quinazoline core. This choice is too conservative and rudimentary in the optimization of medicinal chemical structures. The methoxy group has inherent deficiencies in terms of spatial volume, hydrophobicity, and conformational flexibility, making it unable to interact optimally with the "pocket" composed of hydrophobic amino acid residues adjacent to the EGFR kinase ATP binding cavity. This suboptimal space filling and hydrophobic contact directly limits the stability and efficiency of the drug molecule's binding mode with the target protein, which is a key structural bottleneck restricting the breakthrough of its biological efficacy.

[0013] (4) Mediocre overall performance: In the prior art (WO2018084321A1), the activity of compound 8 is far lower than that of other preferred compounds in this patent (such as NS-062), and its efficacy against common mutants is insufficient, making it even more difficult to solve the C797S drug resistance problem.

[0014] The purpose of this invention is to provide: EGFR inhibitors based on 7-alkoxyquinazoline, their preparation methods and pharmaceutical applications, and related technologies, aim to solve technical problems in existing technologies such as insufficient inhibitory activity against a wide range of EGFR types, limited anti-tumor efficacy, inability to overcome key clinical drug resistance mutations, and poor overall performance, or combinations thereof.

[0015] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0016] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0017] Definitions of the standard terminology can be found in the references “Bio-targeted Therapy for Lung Cancer (2nd Edition), People’s Medical Publishing House, authors: Wu Caicun, Wu Yilong, et al., 2016-06”, “Modern Molecular Biology (5th Edition), Higher Education Press, authors: Zhu Yuxian, Li Yi, Zheng Xiaofeng, and Guo Hongwei, 2019-06-19”, and “Genetic Engineering, Higher Education Press, 2013-08-01”.

[0018] Unless otherwise stated, conventional methods within the scope of the art, such as filtration, washing, neutralization, extraction, etc., shall be used.

[0019] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0020] The term "drug resistance" used in this article refers to the tolerance of tumor cells and other cells to the effects of drugs. Once drug resistance develops, the effectiveness of the drug decreases significantly. Drug resistance can be divided into acquired resistance and natural resistance based on its cause.

[0021] The term “potential spectrum” as used in this article refers to the range and intensity of biological activity exhibited by a drug or compound in different targets, subtypes, mutants, or disease models.

[0022] The term "stereoisomer" as used in this article refers to isomers of compounds with the same molecular formula, in which atoms or groups of atoms are connected in the same order but arranged in different spatial arrangements.

[0023] The term "solvent" as used in this article refers to a compound formed by the combination of solvent molecules and solute during the dissolution of a substance, the formation of which alters the original state of the solute.

[0024] The term "prodrug" used in this article refers to a compound that, after chemical modification, is inactive or has low activity in vitro but releases its active drug effect in vivo through enzymatic or non-enzymatic conversion.

[0025] In a first aspect, the present invention provides: an EGFR inhibitor based on 7-alkoxyquinazoline, said EGFR inhibitor being selected from at least one of compound 42, a pharmaceutically acceptable salt of said compound 42, a stereoisomer of said compound 42, a solvate of said compound 42, or a prodrug of said compound 42; Compound 42 is a quinazoline derivative in which the 4-position is 4-fluoro-3-chlorophenylamino, the 7-position is ethoxy, and the 6-position is N-chlorofluoroacetylamino.

[0026] For example, compound 42 is 4-(4-fluoro-3-chlorophenylamino)-7-ethoxy-6-(N-chlorofluoroacetyl)aminoquinazoline. The structural formula of compound 42 is shown in formula (I): (I).

[0027] Existing technologies (such as compound 8) commonly and conventionally use a methoxy group at the 7-position. This invention is the first to discover and demonstrate that introducing an ethoxy group with a slightly larger spatial volume and hydrophobicity at this critical site can produce a synergistic effect by optimizing the interaction with the hydrophobic region within the EGFR kinase ATP binding cavity, resulting in a significant and universal enhancement of inhibitory activity.

[0028] Secondly, the present invention provides a method for preparing the above-mentioned EGFR inhibitor, comprising the following steps: S1. Reaction of 4-chloro-6-nitro-7-fluoroquinazoline with 4-fluoro-3-chloroaniline yields 4-(4-fluoro-3-chlorophenylamino)-7-fluoro-6-nitroquinazoline intermediate; S2. The 4-(4-fluoro-3-chlorophenylamino)-7-fluoro-6-nitroquinazoline intermediate is reacted with an ethoxy donor to obtain a quinazoline derivative with 4-fluoro-3-chlorophenylamino at the 4-position and ethoxy at the 7-position of the quinazoline core. S3. The quinazoline derivative, reducing agent, and acidic medium are mixed and subjected to a nitro reduction reaction to obtain a 4-(4-fluoro-3-chlorophenylamino)-7-ethoxy-6-aminoquinazoline intermediate; S4. A tetrahydrofuran solution of chloroacetyl chloride is added dropwise to the 4-(4-fluoro-3-chlorophenylamino)-7-ethoxy-6-aminoquinazoline intermediate to react and obtain the EGFR inhibitor.

[0029] Further, in step S1, the molar ratio of the 4-chloro-6-nitro-7-fluoroquinazoline to the 4-fluoro-3-chloroaniline is 1:1.0-1.2. This molar ratio can be, for example, 1:1.0, 1:1.05, 1:1.1, 1:1.15, 1:1.2, or any value within the range of any two of the above values; the preferred molar ratio is 1:1.1.

[0030] Furthermore, in step S1, the reaction further includes a reaction solvent and a reaction auxiliary agent. The reaction solvent is selected from at least one of isopropanol, tetrahydrofuran, and dioxane, and the reaction auxiliary agent is selected from at least one of triethylamine, pyridine, and N,N-diisopropylethylamine.

[0031] Further, in step S1, the reaction temperature is 50℃-70℃, and the reaction time is 5 h-7 h. The reaction temperature can, for example, be 50℃, 60℃, 70℃, or any value within the range of any two of the above values; preferably, the reaction temperature is 60℃. The reaction time can, for example, be 5 h, 6 h, 7 h, or any value within the range of any two of the above values; preferably, the reaction time is 6 h.

[0032] Further, in step S2, the molar ratio of the 4-(4-fluoro-3-chlorophenylamino)-7-fluoro-6-nitroquinazoline intermediate to the ethoxy group in the ethoxy donor is 1:1.1-1.3. This molar ratio can, exemplarily, be 1:1.1, 1:1.15, 1:1.2, 1:1.25, 1:1.3, or any value within the range of any two of the above values; preferably, the molar ratio is 1:1.2.

[0033] Furthermore, in step S2, the ethoxy donor includes sodium ethoxide.

[0034] Further, in step S2, the reaction temperature is 80℃ - 120℃, and the reaction time is 6 h - 10 h. The reaction temperature can, for example, be 80℃, 90℃, 100℃, 110℃, 120℃, or any value within the range of any two of the above values; preferably, the reaction temperature is 100℃. The reaction time can, for example, be 6 h, 7 h, 8 h, 9 h, 10 h, or any value within the range of any two of the above values; preferably, the reaction time is 8 h.

[0035] Furthermore, the reaction system in step S2 may also contain a solvent. The solvent may, exemplarily, be selected from at least one of ethanol, dioxane, and tetrahydrofuran.

[0036] Furthermore, in step S3, the reducing agent is selected from at least one of reduced iron powder and zinc powder.

[0037] Furthermore, in step S3, the acidic medium is selected from at least one of ammonium chloride and acetic acid.

[0038] Further, in step S3, the temperature of the nitro reduction reaction is 80℃-100℃, and the time of the nitro reduction reaction is 4 h-8 h. The temperature of the nitro reduction reaction can, for example, be 80℃, 90℃, 100℃, or any value within the range of any two of the above values; preferably, the temperature of the nitro reduction reaction is 90℃. The time of the nitro reduction reaction can, for example, be 4 h, 5 h, 6 h, 7 h, 8 h, or any value within the range of any two of the above values; preferably, the time of the nitro reduction reaction is 6 h.

[0039] Further, in step S4, the molar ratio of the 4-(4-fluoro-3-chlorophenylamino)-7-ethoxy-6-aminoquinazoline intermediate to the chloroacetyl chloride is 1:1.1-1.3. This molar ratio can, exemplarily, be 1:1.1, 1:1.15, 1:1.2, 1:1.25, 1:1.3, or any value within the range of any two of the above values; preferably, the molar ratio is 1:1.2.

[0040] Further, in step S4, the reaction temperature is 20℃ - 30℃, and the reaction time is 3 h - 5 h. The reaction temperature can, for example, be 20℃, 22℃, 25℃, 28℃, 30℃, or any value within the range of any two of the above values; preferably, the reaction temperature is 25℃. The reaction time can, for example, be 3 h, 4 h, 5 h, or any value within the range of any two of the above values; preferably, the reaction time is 4 h.

[0041] The key step in the preparation method provided by this invention lies in the nucleophilic substitution of the 7-halogen precursor (4-(4-fluoro-3-chlorophenylamino)-7-fluoro-6-nitroquinazoline intermediate) with an ethoxy donor in step S2. Furthermore, the solvent system in step S2 is optimized. Targeted and adaptive modifications and optimizations are made to both the nucleophile and the solvent system. These specific process conditions are crucial for the successful preparation of the core compound of this invention.

[0042] Thirdly, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the above-described EGFR inhibitor.

[0043] Specifically, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0044] Furthermore, the carriers include, but are not limited to, nanoparticles, liposomes, polymers, micelles, cyclodextrins, exosomes, etc.

[0045] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0046] Furthermore, the pharmaceutically acceptable excipients are selected from one or more of the following: excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, wetting agents, emulsifiers, preservatives, antioxidants, buffers, solubilizers, thickeners, stabilizers, and sweeteners.

[0047] Furthermore, the dosage form of the pharmaceutical composition is a tablet, capsule, injection, powder for injection, or inhalation.

[0048] Fourthly, the present invention provides the use of the above-mentioned EGFR inhibitor in the preparation of medicaments for the prevention or treatment of EGFR-mediated diseases.

[0049] Furthermore, the disease includes tumors.

[0050] Furthermore, the tumor is selected from at least one of lung cancer, breast cancer, colorectal cancer, kidney cancer, prostate cancer, and pancreatic cancer.

[0051] Furthermore, the tumor is selected from at least one of lung cancer, breast cancer, and colorectal cancer.

[0052] Abnormal activation of EGFR, especially persistent activation of EGFR due to gene mutations, is closely related to the occurrence and development of various epithelial-derived malignancies (Research Progress on the Role of EGFR in Tumor Development and Treatment, Clinical Medical Progress, 2024, 14(12), 796-803), such as in renal cell carcinoma (Research Progress on EGFR in Renal Cell Carcinoma, Journal of Clinical Urology, 2012, 27(11):873-876), lung cancer (Research Progress on EGFR-related Inhibitors for Lung Cancer Driver Genes, Inner Mongolia Medical Journal, 2018, 50(08):908-911), prostate cancer (Research on Novel EGFR Tyrosine Kinase Inhibitors for the Treatment of Prostate Cancer, Kunming Medical University, 2019), and pancreatic cancer (Research on EGFR-ERK in Pancreatic Cancer). EGFR is overexpressed in various cancers, including breast cancer ("The Role of EGFR in Triple-Negative Breast Cancer and Research Progress in Targeted Therapy", Oncology, 2023, 43(10):829-838) and colorectal cancer ("Research Progress on Anti-EGFR Therapy Resistance Mechanism in Metastatic Colorectal Cancer", Chinese Journal of General Surgery, 2024, 33(06):996-1011). EGFR inhibitors can be used in the preparation of drugs to prevent or treat the above-mentioned EGFR-mediated diseases.

[0053] Specifically, the EGFR in the EGFR-mediated disease is selected from at least one of EGFR wild-type and EGFR mutant.

[0054] For example, the EGFR mutant is selected from at least one of the following: EGFR L858R mutant, EGFR Exon19 Del mutant, EGFR T790M mutant, EGFR C797S mutant, EGFR T790M / C797S complex mutant, and EGFR Del19 / T790M / C797S complex mutant.

[0055] Example 1 of this invention at least supports the protection scope of "EGFR inhibitors".

[0056] "EGFR inhibitor" is defined as the compound 42 described above and / or in Example 1. Therefore, those skilled in the art can reasonably presume that "EGFR inhibitor," its subordinate concepts, its substantially equivalent technical means, and technical means that can replace it within the scope of conventional and common knowledge based on the existing level of technology should all fall within the protection scope of "EGFR inhibitor."

[0057] Example 1 of this invention at least supports the protection scope of "method for preparing EGFR inhibitor".

[0058] The term "preparation method of EGFR inhibitor" is summarized by the foregoing explanation and / or the corresponding "specific preparation method" in Example 1. Therefore, those skilled in the art can reasonably presume that the "preparation method of EGFR inhibitor," its subordinate concepts, its substantially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing level of technology should all fall within the protection scope of the "preparation method of EGFR inhibitor."

[0059] Examples 1-5 of this invention at least support the scope of protection of "pharmaceutical composition".

[0060] "Pharmaceutical composition" is derived from the foregoing explanation and / or the corresponding pharmaceutical generalizations in Examples 1-5. Therefore, those skilled in the art can reasonably presume that "pharmaceutical composition," its subordinate concepts, its substantially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing level of technology should all fall within the protection scope of "pharmaceutical composition."

[0061] Examples 1-5 of this invention at least support the scope of protection for "the use of EGFR inhibitors in the preparation of medicaments for the prevention or treatment of EGFR-mediated diseases".

[0062] The term "use of EGFR inhibitors in the preparation of medicaments for the prevention or treatment of EGFR-mediated diseases" is summarized from the foregoing explanation and / or the corresponding CCK-8 assay, plate colony formation assay, flow cytometry detection of apoptosis, scratch assay, and in vivo safety assay in Examples 1-5. Therefore, those skilled in the art can reasonably presume that "use of EGFR inhibitors in the preparation of medicaments for the prevention or treatment of EGFR-mediated diseases," its subordinate concepts, its substantially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing level of technology should all fall within the protection scope of "use of EGFR inhibitors in the preparation of medicaments for the prevention or treatment of EGFR-mediated diseases."

[0063] The present invention has at least the following beneficial effects: Compared with existing technologies, this invention provides an EGFR inhibitor based on 7-alkoxyquinazoline, its preparation method, and its pharmaceutical applications, which have better technical effects, specifically reflected in the following aspects: (1) Significantly Enhanced Universality and Efficacy: In various mutant lung cancer cell lines, including EGFR wild-type (A549), EGFR-sensitive mutant PC-9 cells (Exon19 Del), and EGFR double mutant NCI-H1975 cells (L858R / T790M), the EGFR inhibitor of this invention showed significantly better proliferation inhibition than compound 8, demonstrating that its enhanced efficacy is broad-spectrum and fundamental. This is because the present invention introduces an ethoxy group, moderately increasing the hydrophobicity and steric extension of the substituent, enabling it to interact more fully and stably with the hydrophobic region surrounding the EGFR kinase ATP-binding domain. This optimized binding mode universally enhances the binding affinity of the compound to different EGFR types (wild-type and mutant), thereby leading to a fundamental improvement in basic inhibitory efficacy.

[0064] (2) Superior Functional Inhibition Effect: In adherent cell models, plate colony formation assays and cell scratch assays confirmed that the EGFR inhibitor of this invention significantly outperformed compound 8 in inhibiting the long-term proliferation and migration of tumor cells. Furthermore, in a suspension-cultured BaF3 EGFR-Del19-T790M-C797S triple mutant cell model, the EGFR inhibitor of this invention also exhibited significantly superior proliferation inhibition compared to compound 8. More importantly, flow cytometry apoptosis analysis showed that treatment with compound 42 induced a higher proportion of apoptosis in this drug-resistant cell model, demonstrating its stronger pro-apoptotic ability and potential to overcome highly resistant mutations. The EGFR inhibitor of this invention has superior potential in blocking the key pathological process of tumor invasion and metastasis. This is because the EGFR inhibitor of this invention can more effectively block EGFR and its downstream signaling pathways (such as MAPK and PI3K / Akt), which regulate cell proliferation, survival, and migration, thus leading to a more thorough inhibition of its malignant phenotype, reflecting the synergistic biological effects brought about by ethoxylation modification.

[0065] (3) Significant potential for inhibition and overcoming resistance mutations to third-generation EGFR inhibitors: In experiments targeting the most challenging third-generation EGFR inhibitor resistance model—BaF3 EGFR-Del19-T790M-C797S triple mutant cells—the EGFR inhibitor of this invention exhibited unexpectedly superior activity. CCK-8 experimental results showed that the EGFR inhibitor of this invention significantly inhibited the proliferation of this resistant cell line better than compound 8. Further functional analysis (flow cytometry apoptosis detection) revealed that the EGFR inhibitor of this invention could effectively induce apoptosis in this highly resistant cell line, and its pro-apoptotic ability was significantly stronger than that of the almost ineffective compound 8. This directly proves that the compound of this invention has a strong potential to overcome acquired resistance to third-generation EGFR inhibitors mediated by the C797S mutation, while the prior art compound 8 is weak in this regard. This effect is the most unexpected major breakthrough of this invention. The C797S mutation renders all existing covalent inhibitors that rely on this site for covalent binding ineffective. This invention, through precise modification of the 7-ethoxy group, may fundamentally optimize the interaction mode between the compound and the EGFR kinase domain. This allows it to maintain sufficient target occupancy and inhibitory efficacy, and even activate apoptosis pathways, even when covalent binding with the C797 residue is blocked (or weakened). This is not the result of conventional homologue substitution of a known structure (compound 8), but rather a unique solution to one of the most challenging clinical problems presented by the specific structural modification of this invention.

[0066] (4) Excellent safety: In a 14-day repeated-dose in vivo safety evaluation in healthy mice, the EGFR inhibitor of this invention did not cause weight loss, abnormal liver and kidney biochemical indicators, or histopathological changes at the effective dose, demonstrating a good safety profile comparable to compound 8. This achieved synergistic optimization of "enhanced efficacy without increased toxicity" and expanded the therapeutic window. This proves that the 7-ethoxy modification, while achieving broad-spectrum and highly effective inhibition (including overcoming drug resistance), did not simultaneously increase systemic toxicity in vivo. This optimization is highly inclined to enhance its targeting, expanding the therapeutic boundary while substantially widening the therapeutic window, providing a better safety margin and stronger confidence for the clinical development of complex drug-resistant tumors.

[0067] In summary, this invention, through precise and crucial structural modification of the 7-position substituent of the quinazoline core from methoxy to ethoxy, has successfully developed a new generation of EGFR inhibitors that comprehensively outperform the closest existing technologies in four core dimensions: inhibitory efficacy (covering wild-type to highly resistant mutants), functional activity (anti-proliferation, anti-migration, and pro-apoptosis), overcoming key clinical resistance, and biosafety. Particularly noteworthy is that this subtle change unexpectedly endowed the compound with the potential to overcome the key resistance mutation (C797S) of third-generation EGFR inhibitors—a significant breakthrough that was completely unforeseen by those skilled in the art based on existing technology. Attached Figure Description

[0068] Figure 1 For CCK-8 experimental results (A549 and NCI-H1975 cell lines), there was no significant difference in ns; ***P≤0.001; ****P≤0.0001.

[0069] Figure 2 For CCK-8 experiments (PC9, EGFR Del19-T790M-C797S / BaF3 cell lines), there was no significant difference in ns; **P≤ 0.01; ***P≤ 0.001; ****P≤ 0.0001.

[0070] Figure 3 The results for the plate colony formation experiment (A549 cell line) showed no significant difference in ns; *P≤ 0.05; ****P≤0.0001.

[0071] Figure 4 The results of the plate colony formation experiment (PC9 cell line) showed no significant difference in ns; ***P≤ 0.001.

[0072] Figure 5 The results of the plate colony formation experiment (NCI-H1975 cell line) showed no significant difference in ns; ***P≤0.001; ****P≤0.0001.

[0073] Figure 6 The results of apoptosis were detected by flow cytometry (EGFR Del19 - T790M - C797S / BaF3 cell line - control group and compound 42 group).

[0074] Figure 7 Apoptosis results were detected by flow cytometry (EGFR Del19 - T790M - C797S / BaF3 cell line - compound 8 group).

[0075] Figure 8The bar chart shows the apoptosis rate of EGFR Del19-T790M-C797S / BaF3 cells. There was no significant difference in ns; **P≤ 0.01.

[0076] Figure 9 For scratch assay results (A549 cell migration inhibition), there was no significant difference in ns; ***P≤ 0.001; ****P≤ 0.0001.

[0077] Figure 10 The results of the scratch assay (NCI-H1975 cell migration inhibition) showed no significant difference in ns; *P≤ 0.05.

[0078] Figure 11 The results of the scratch assay (PC 9 cell migration inhibition) showed no significant difference in ns; ****P≤ 0.001.

[0079] Figure 12 This is the curve showing the change in mouse body weight.

[0080] Figure 13 The results are the biochemical indicators of liver and kidney function in mice.

[0081] Figure 14 HE-stained sections of mouse liver and kidney tissue. Detailed Implementation

[0082] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0083] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0084] Data analysis and statistical analysis were performed using professional data processing software. One-way ANOVA was used for significance analysis, and P ≤ 0.05 was considered to indicate a significant difference.

[0085] Example 1: EGFR inhibitor based on 7-alkoxyquinazoline and its preparation method An EGFR inhibitor based on 7-alkoxyquinazoline: 4-(4-fluoro-3-chlorophenylamino)-7-ethoxy-6-(N-chlorofluoroacetyl)aminoquinazoline, named compound 42, has the structural formula shown in formula (I): (I).

[0086] The basic reaction process for the preparation of compound 42 is as follows:

[0087] The specific preparation method of compound 42 includes the following steps: (1) Preparation of 4-(4-fluoro-3-chlorophenylamino)-7-fluoro-6-nitroquinazoline: 2.3 g of 4-chloro-6-nitro-7-fluoroquinazoline and 100 mL of isopropanol were added to a 250 mL round-bottom flask. After stirring evenly, 5 mL of triethylamine and 1.6 g of 4-fluoro-3-chloroaniline were added. The mixture was reacted at 60 °C for 6 hours. After the reaction was completed, the mixture was cooled and filtered to obtain a filter cake. The filter cake was washed with a small amount of methanol aqueous solution to obtain 3.0 g of the product 4-(4-fluoro-3-chlorophenylamino)-7-fluoro-6-nitroquinazoline. MS (m / z): 337.0 (M+1)+.

[0088] (2) Preparation of 4-(4-fluoro-3-chlorophenylamino)-7-ethoxy-6-nitroquinazoline: 3.3 g of 4-(4-fluoro-3-chlorophenylamino)-7-fluoro-6-nitroquinazoline and 100 mL of ethanol were added to a 250 mL round-bottom flask. After stirring evenly, 1 g of sodium ethoxide was added. The reaction was carried out at 100 °C for 8 hours. After the reaction was completed, the reaction solution was poured into cold water and neutralized with hydrochloric acid aqueous solution. The filter cake was obtained by suction filtration and washed with a small amount of ethanol aqueous solution to obtain 3 g of product 4-(4-fluoro-3-chlorophenylamino)-7-ethoxy-6-nitroquinazoline. MS (m / z): 363.1 (M+1)+.

[0089] (3) Preparation of 4-(4-fluoro-3-chlorophenylamino)-7-ethoxy-6-aminoquinazoline: 3.6 g of 4-(4-fluoro-3-chlorophenylamino)-7-ethoxy-6-nitroquinazoline, 150 mL of ethanol, 10 mL of water, 3 g of reduced iron powder and 2 g of ammonium chloride were added to a 250 mL round-bottom flask. After stirring evenly, the mixture was slowly heated and reacted at 90 °C for 6 hours. After the reaction was completed, the excess iron powder was removed by hot filtration, the solvent was evaporated under reduced pressure, 100 mL of distilled water was added, and the mixture was extracted with dichloromethane, dried with anhydrous sodium sulfate, and the solvent was recovered under reduced pressure to dryness to obtain 2.8 g of the product 4-(4-fluoro-3-chlorophenylamino)-7-ethoxy-6-aminoquinazoline. MS (m / z): 333.1 (M+1)+.

[0090] (4) Preparation of 4-(4-fluoro-3-chlorophenylamino)-7-ethoxy-6-(N-chlorofluoroacetyl)aminoquinazoline: Add 3.3 g of 4-(4-fluoro-3-chlorophenylamino)-7-ethoxy-6-aminoquinazoline, 120 mL of tetrahydrofuran, and 5 mL of triethylamine to a 250 mL round-bottom flask. Place the flask in an ice bath, stir well, and then slowly add 30 mL of a THF solution containing 1.7 g of chlorofluoroacetyl chloride dropwise using a funnel. The addition is completed in 30 minutes. Continue the reaction at room temperature for 4 hours. After the reaction is complete, pour the reaction solution into cold water and adjust the pH with sodium hydroxide solution until a large amount of solid precipitates. Filter to obtain the crude product, and further purify it by column chromatography to obtain the product 4-(4-fluoro-3-chlorophenylamino)-7-ethoxy-6-(N-chlorofluoroacetyl)aminoquinazoline 3 g. gram, MS(m / z): 427.1 (M+1)+.

[0091] Compound 8 was prepared by referring to the basic reaction process and specific preparation method of compound 42. The structural formula of compound 8 is shown in formula (II): (II).

[0092] The compounds described in this invention include, but are not limited to, at least one of compound 42, its pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug.

[0093] Example 1: CCK-8 Experiment Lung cancer cells in logarithmic growth phase, including A549 (Shanghai Jinyuan Biotechnology Co., Ltd., JY135), PC-9 (also known as PC9, Shanghai Jinyuan Biotechnology Co., Ltd., JY123), NCI-H1975 (also known as 1975, Shanghai Jinyuan Biotechnology Co., Ltd., JY102), and EGFR Del19-T790M-C797S / BaF3 (Nanjing Kebai Biotechnology Co., Ltd., CBP73173), were washed three times with sterile 1×PBS. An appropriate amount of 0.25% trypsin was added to digest the cells. When the intercellular spaces widened and the cells became rounded and shrunken, an equal volume of complete cell culture medium containing 10% fetal bovine serum was added to terminate the digestion. The cells were centrifuged at 300g for 5 minutes, and then seeded evenly in each well of a 96-well plate at a count of 5000 cells / well. After cell adhesion, the culture medium was discarded. Add 200 μL of the prepared compound 8 and compound 42 solutions of the corresponding concentrations to each well (the drug concentrations used for A549, PC-9, and NCI-H1975 cells were 0, 250 nM, and 500 nM, respectively; the drug concentrations used for EGFR Del19-T790M-C797S / BaF3 cells were 0, 500 nM, and 1000 nM, respectively). After treating the cells with each drug for 72 hours, remove the 96-well plates of each group of cells from the cell culture incubator, add CCK8 working solution to each well, incubate for 1 h, and measure and count the cell absorbance at 450 nm using a full-spectrum microplate reader.

[0094] The results showed that compound 42 exhibited significantly better inhibitory activity than the control (compound 8) in all four cell models (as shown in Figures 1-2).

[0095] Example 2: Plate colony formation experiment Three human lung cancer cell lines (A549, PC-9, and NCI-H1975) in logarithmic growth phase were digested with trypsin and prepared into single-cell suspensions. The cell suspensions were seeded at a density of 500 cells / well in 6-well plates, with 2 mL of cell suspension added to each well. The plates were gently shaken to ensure even distribution of cells at the bottom, and then incubated at 37°C in a 5% CO2 incubator until cell attachment. After cell attachment, the culture medium was discarded. 2 mL of the corresponding concentrations of drug solutions (0, 250 nM, and 500 nM solutions of compound 8 and compound 42, respectively) were added to the appropriate wells. After 72 h of drug treatment, the medium was replaced with normal culture medium. The medium was then changed every 5 days until visible cell colonies formed. After cell clones have formed, discard the culture supernatant in the 6-well plates. Gently wash the plates three times with PBS. Add 500 μL of 4% paraformaldehyde to each well and fix at room temperature for 15 minutes. After fixation, add 500 μL of crystal violet staining solution to each well and stain at room temperature for 15 minutes to ensure thorough staining of the cell clones. After staining, wash the plates repeatedly with PBS until the background is clean. Invert the plates to dry and then photograph the results.

[0096] The results showed that, compared with the control group drug (compound 8), compound 42 of the present invention significantly reduced the number of cell clones with increasing drug concentration, indicating that compound 42 had a better inhibitory effect on cell proliferation than compound 8 (as shown in Figure 3). Figure 5 (As shown).

[0097] Example 3: Flow cytometry detection of apoptosis Log-phase EGFR Del19-T790M-C797S / BaF3 cells were seeded in 6-well plates and cultured overnight. The supernatant was discarded, and the cells were washed once with PBS. The cells were then divided into a control group (complete culture medium without the drug), a compound 8 group, and a compound 42 group. 2 mL of the prepared drug solution (1000 nM) was added to each well. After co-incubation with the drug for 24 h, the cell suspension was aspirated into centrifuge tubes, centrifuged at 1000 g for 5 minutes, the supernatant was discarded, and the cells were collected. The cells were gently resuspended in 195 μL of Annexin V-FITC binding solution. Then, 2.5 μL of Annexin V-FITC and 5 μL of propidium iodide staining solution were added and gently mixed. The cells were incubated at room temperature in the dark for 10-20 minutes, then placed on ice and protected from light with aluminum foil. The apoptosis rate of each group was then measured and statistically analyzed using flow cytometry.

[0098] The results showed that in EGFR Del19-T790M-C797S / BaF3 cells, compound 42 induced apoptosis significantly more strongly than compound 8 in the control group. This indicates that compound 42 has a higher pro-apoptotic capacity (as shown in Figures 6-8).

[0099] Example 4: Scratch Test Collect cells in the logarithmic growth phase (A549, PC-9, NCI-H1975), wash three times with sterile 1×PBS, add an appropriate amount of 0.25% trypsin for digestion, and when the intercellular spaces widen and the cells become rounded and wrinkled, add an equal volume of complete cell culture medium containing 10% fetal bovine serum to terminate the digestion; collect the cells in sterile centrifuge tubes, centrifuge at 300 g for 5 minutes to collect and count the cells; after counting, divide the cells into 5×10⁻⁶ cells per cell line. 6 Cells were evenly seeded into 6-well plates. When the cell confluence reached 80%, the cells were streaked vertically into the 6-well plates using a 200 μL pipette tip. The cells were washed 2-3 times with sterile 1×PBS to remove detached cells and cell debris. The cells were then cultured in serum-free medium containing compound 8 (250 nM and 500 nM) and compound 42 (250 nM and 500 nM), respectively, and the culture time was recorded as 0 h. Subsequently, the cells were photographed and the data were saved at 12 h for statistical analysis.

[0100] The experimental results showed that, compared with the control drug (compound 8), compound 42 significantly inhibited cell migration in three types of human lung cancer cells and the effect was statistically significant (as shown in Figures 9-11).

[0101] Example 5: In vivo safety experiment Male C57 mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd., strain name: C57BL / 6JGpt, strain number: N000013) aged 6-8 weeks and weighing approximately 22 grams were used in four groups of five each: a normal control group (no treatment), a solvent control group (containing 3% DMSO, 2% propylene glycol, 2% ethanol, and 2.5% polyoxyethylene 40 hydrogenated castor oil), compound 8, and compound 42. Each mouse was administered 100 μL of the corresponding drug intraperitoneally daily at a concentration of 10 mg / kg / day (based on mouse body weight) for 14 consecutive days, during which mouse body weight was monitored. On day 14 after drug administration, blood samples were collected from mice to detect liver function (ALT and AST) and kidney function (BUN and CRE) biochemical indicators (the kits were all purchased from Nanjing Jiancheng Bioengineering Institute Co., Ltd., with catalog numbers: ALT: C009-2-1, AST: C010-2-1, BUN: C013-2-1, CRE: C011-2-1). In addition, liver and kidney tissues were subjected to histopathological HE staining.

[0102] The results showed that, compared with the normal control and solvent control groups, the experimental groups treated with compounds 42 and 8 had no significant effect on mouse body weight; in fact, the mice's body weight showed an increasing trend, and their condition remained good during this period. Biochemical tests showed no significant changes in any of the treated groups compared with the normal control and solvent control groups. Pathological HE staining showed no obvious abnormalities in liver and kidney tissues. These results suggest that compound 42 has good in vivo safety (e.g., ...). Figure 12 - As shown in Figure 14).

[0103] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. EGFR inhibitors based on 7-alkoxyquinazolines characterised in that, The EGFR inhibitor is selected from at least one of compound 42, a pharmaceutically acceptable salt of compound 42, a stereoisomer of compound 42, a solvate of compound 42, or a prodrug of compound 42; Compound 42 is a quinazoline derivative in which the 4-position is 4-fluoro-3-chlorophenylamino, the 7-position is ethoxy, and the 6-position is N-chlorofluoroacetylamino.

2. The EGFR inhibitor according to claim 1, wherein Compound 42 is 4-(4-fluoro-3-chlorophenylamino)-7-ethoxy-6-(N-chlorofluoroacetyl)aminoquinazoline.

3. A method for preparing the EGFR inhibitor according to any one of claims 1-2, characterized in that, Includes the following steps: S1. Reaction of 4-chloro-6-nitro-7-fluoroquinazoline with 4-fluoro-3-chloroaniline yields 4-(4-fluoro-3-chlorophenylamino)-7-fluoro-6-nitroquinazoline intermediate; S2. The 4-(4-fluoro-3-chlorophenylamino)-7-fluoro-6-nitroquinazoline intermediate is reacted with an ethoxy donor to obtain a quinazoline derivative with 4-fluoro-3-chlorophenylamino at the 4-position and ethoxy at the 7-position of the quinazoline core. S3. The quinazoline derivative, reducing agent, and acidic medium are mixed and subjected to a nitro reduction reaction to obtain a 4-(4-fluoro-3-chlorophenylamino)-7-ethoxy-6-aminoquinazoline intermediate; S4. A tetrahydrofuran solution of chloroacetyl chloride is added dropwise to the 4-(4-fluoro-3-chlorophenylamino)-7-ethoxy-6-aminoquinazoline intermediate to react and obtain the EGFR inhibitor.

4. The preparation method according to claim 3, characterized in that, In step S1, the molar ratio of the 4-chloro-6-nitro-7-fluoroquinazoline to the 4-fluoro-3-chloroaniline is 1:1.0-1.2; and / or In step S1, the reaction further includes a reaction solvent and a reaction auxiliary agent. The reaction solvent is selected from at least one of isopropanol, tetrahydrofuran, and dioxane, and the reaction auxiliary agent is selected from at least one of triethylamine, pyridine, and N-N-diisopropylethylamine; and / or In step S1, the reaction temperature is 50℃ - 70℃, and the reaction time is 5 h - 7 h.

5. The preparation method according to claim 3, characterized in that, In step S2, the molar ratio of the 4-(4-fluoro-3-chlorophenylamino)-7-fluoro-6-nitroquinazoline intermediate to the ethoxy group in the ethoxy donor is 1:1.1-1.3; and / or In step S2, the ethoxy donor includes sodium ethoxide; and / or In step S2, the reaction temperature is 80℃ - 120℃, and the reaction time is 6 h - 10 h.

6. The preparation method according to claim 3, characterized in that, In step S3, the reducing agent is selected from at least one of reduced iron powder and zinc powder; and / or In step S3, the acidic medium is selected from at least one of ammonium chloride and acetic acid; and / or In step S3, the temperature of the nitro reduction reaction is 80℃ - 100℃, and the time of the nitro reduction reaction is 4 h - 8 h.

7. The preparation method according to claim 3, characterized in that, In step S4, the molar ratio of the 4-(4-fluoro-3-chlorophenylamino)-7-ethoxy-6-aminoquinazoline intermediate to the chloroacetyl chloride is 1:1.1-1.3; and / or In step S4, the reaction temperature is 20℃ - 30℃, and the reaction time is 3 h - 5 h.

8. A pharmaceutical composition, characterized in that, The EGFR inhibitor comprising a therapeutically effective amount as described in any one of claims 1-2.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to any one of claims 8-9, characterized in that, The dosage form of the pharmaceutical composition is tablets, capsules, injections, powder for injection, or inhalation.

11. Use of the EGFR inhibitor of any one of claims 1-2 in the preparation of a medicament for the prevention or treatment of EGFR-mediated diseases.

12. The use according to claim 11, characterized in that, The diseases mentioned include tumors.

13. The use according to claim 12, characterized in that, The tumor is selected from at least one of lung cancer, breast cancer, colorectal cancer, kidney cancer, prostate cancer, and pancreatic cancer.

14. The use according to claim 11, characterized in that, The EGFR in the EGFR-mediated disease is selected from at least one of EGFR wild-type and EGFR mutant.

15. The use according to claim 12, characterized in that, The EGFR mutant is selected from at least one of the following: EGFR L858R mutant, EGFR Exon19 Del mutant, EGFR T790M mutant, EGFR C797S mutant, EGFR T790M / C797S complex mutant, and EGFR Del19 / T790M / C797S complex mutant.

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