PYRIDOPYRIMIDINE-BASED COMPOUND AND APPLICATION THEREOF

The present disclosure provides a class of Pyridopyrimidine compounds having a structure shown in Formula (I) or their pharmaceutically acceptable salts, or stereoisomers or prodrug molecules and applications thereof. The compounds in the present disclosure can efficiently and selectively degrade AKT3 protein in cells without affecting AKT1/2, thereby significantly inhibiting tumor cell proliferation mediated by high expression of AKT3 protein. It can be used to prepare therapeutic drugs for cancer and other diseases related to abnormal expression of AKT3 protein.

BACKGROUND OF THE INVENTION

1. Technical Field

The present disclosure relates to pharmaceutical chemistry technology, in particular to a class of Pyridopyrimidine compounds and the applications thereof.

2. Background Art

AKT (AK mouse plus Transforming or Thymoma), also known as Protein kinase B (PKB), is a serine/threonine protein kinase with a molecular weight of approximately 57 kD. This family includes three subtypes: AKT1, AKT2, and AKT3. There are many similarities and differences in the functional and histological distribution of AKT subtypes, and their abnormal expression is closely related to the occurrence and development of various diseases. AKT1 widely exists in various tissues, including heart, liver, muscles, etc; AKT2 is mainly distributed in insulin sensitive tissues, such as skeletal muscle and adipose tissue; AKT3 is mainly distributed in tissues such as brain, heart, and kidneys. AKT1's expression increased in about 40% in breast cancer and ovarian cancer and more than 50% in prostate cancer; AKT2 is overexpressed in 40% of liver cancer and 57% of colorectal cancer; AKT2 deficiency will lead to hyperglycemia, Type 2 diabetes and glucose intake disorders; the overexpression of AKT1 and AKT2 is also associated with paclitaxel resistance in ovarian cancer; the overexpression of AKT3 exists in breast cancer, prostate cancer and some Osimertinib resistant non-small cell lung cancer. Therefore, selective regulation of AKT subtype proteins may play a positive role in the treatment of related diseases.

AKT is an important target for tumor treatment. At present, multiple kinase inhibitors of AKT have entered clinical trials and have shown good anti-tumor effects. However, these inhibitors lack selectivity towards various subtypes of AKT proteins, and undifferentiated inhibition may have certain clinical toxic side effects. In addition, similar to traditional targeted drugs, prolonged drug treatment may lead to patient resistance to AKT inhibitors, thereby weakening the efficacy. Moreover, AKT protein has both kinase and non-kinase functions, and simply inhibiting kinase function may be difficult to fully exert anti-tumor effects. Targeted degradation of the intact AKT3 protein can not only inhibit its kinase function, but also regulate its non-kinase function, thereby exerting a stronger anti-tumor effect.

Therefore, the development and synthesis of small molecule degrading agents which can selectively degrade AKT3 protein is of great significance for the development of therapeutic drugs for AKT3-mediated related diseases.

SUMMARY OF THE INVENTION

In response to the above-mentioned issues, the present disclosure provides a new class of Pyridopyrimidine compounds, which can selectively degrade AKT3 protein with high activity, inhibit the proliferation of various tumor cells, and can be used to treat diseases and tumors related to AKT3 protein.

The detailed technical solution is as follows:

Pyridopyrimidine compounds having a structure shown in Formula (I) or their pharmaceutically acceptable salts, or stereoisomers or prodrug molecules,

In some embodiments, E is selected from: H, cyclopropyl,

herein x is an integer from 0 to 3, and y is an integer from 0 to 3.

In some embodiments, L is selected from:

wherein n and m are independently integers from 0 to 14.

In some embodiments, L is selected from:

wherein n is an integer from 0 to 7, and m is an integer from 0 to 3.

In some embodiments, L is selected from:

or L is absent, wherein n is an integer from 2 to 7.

In some embodiments, Y is selected from: —CH2—, —CO—, —O—, or Y is absent; Z is selected from: —NHCO—, —NH—, or Z is absent.

wherein R is selected from: H

In some embodiments, R2is selected from:

In some embodiments, A is selected from:—NH—

B is absent or is selected from:

In some embodiments, R3is selected from:

The present disclosure also provides applications of the above-mentioned Pyridopyrimidine compounds or their pharmaceutically acceptable salts or stereoisomers or prodrug molecules, including the following technical solution:

An application of the above-mentioned Pyridopyrimidine compounds or their pharmaceutically acceptable salts or stereoisomers or prodrug molecules in the preparation of AKT3 protein degradation agent.

An application of the above-mentioned Pyridopyrimidine compounds or their pharmaceutically acceptable salts or stereoisomers or prodrug molecules in the preparation of drugs for preventing and/or treating diseases related to abnormal expression of AKT3 protein.

In some embodiments, the diseases related to abnormal expression of AKT3 protein include: tumor, cardiovascular disease, diabetes, hypertension, muscular dystrophy, Parkinson's disease and Alzheimer's disease.

An application of the above-mentioned Pyridopyrimidine compounds or their pharmaceutically acceptable salts or stereoisomers or prodrug molecules in the preparation of drugs for preventing and/or treating tumors or preventing postoperative recurrence of tumors.

The present disclosure also provides an AKT3 protein degrading agent, including the following technical solution:

An AKT3 protein degrading agent, wherein its active ingredient comprises the above-mentioned Pyridopyrimidine compounds or their pharmaceutically acceptable salts or stereoisomers or prodrug molecules.

The present disclosure also provides a pharmaceutical composition for treating and/or preventing tumors or preventing postoperative recurrence of tumors, including the following technical solution:

A pharmaceutical composition for treating and/or preventing tumors or preventing postoperative recurrence of tumors, is prepared from active ingredients and pharmaceutically acceptable carriers or excipients, wherein the active ingredients include the Pyridopyrimidine compounds or their pharmaceutically acceptable salts or stereoisomers or prodrug molecules.

The present disclosure provides a class of Pyridopyrimidine compounds or their pharmaceutically acceptable salts, or stereoisomers or prodrug molecules, which can efficiently and selectively degrade AKT3 protein in cells without affecting AKT1/2, thereby being able to significantly inhibit tumor cells proliferation mediated by high expression of AKT3, and can be used to prepare therapeutic drugs for diseases related to abnormal expression of AKT3 protein and various tumors.

DETAILED DESCRIPTION OF THE EMBODIMENTS

In the compounds of the present disclosure, when any variable (eg. R4, R5, etc.) occurs more than once in any component, its definition at each occurrence is independent from the definition at each other occurrences. Similarly, combinations of substituents and variables are permissible as long as the combination stabilizes the compound. Aline of self substituent to a ring system indicates that the indicated bond may be attached to any substitutable ring atom. If the ring system is polycyclic, it means that such bonds are only attached to any suitable carbon atoms adjacent to the ring. It is understood that an ordinary skilled in the art can select substituents and substitution patterns of the compounds of the present disclosure to provide compounds that are chemically stable and can be readily synthesized from the available starting materials by techniques in the art and by the methods described below. If a substituent itself is substituted by more than one group, it should be understood that these groups may be on the same carbon atom or on different carbon atoms, so long as the structure is stable.

The term “alkyl” in the present disclosure means saturated aliphatic hydrocarbon groups including branched and straight chain having the specified number of carbon atoms. For example, the definition of “C1-C6” in “C1-C6alkyl” includes groups having 1, 2, 3, 4, 5 or 6 carbon atoms arranged in a straight or branched chain. For example, “C1-C6alkyl” specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, hexyl.

The term “cycloalkyl” used in this article refers to a saturated or partially unsaturated monocyclic, bicyclic or polycyclic hydrocarbon group composed of carbon atoms. Double ring or multi ring includes spiral ring, fused ring, and bridge ring. For example, “cycloalkyl” includes but is not limited to the following groups: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,

The term “alkoxy” refers to a group with an —O-alkyl structure, such as —OCH3, —OCH2CH3, —OCH2CH2CH3, —O—CH2CH(CH3)2, —OCH2CH2CH2CH3, —O—CH(CH3)2, etc.

The term “heterocycloalkyl” is a saturated or partially unsaturated monocyclic, bicyclic or polycyclic substituent wherein one or more ring atoms are heteroatoms selected from N, O or S(O)m(wherein m is an integer from 0 to 2), and the remaining ring atoms are carbon, bicyclic or polycyclic including spiral cyclic, thick cyclic, and bridge cyclic, such as: morpholinyl, piperidinyl, tetrahydropyrrolyl, pyrrolidinyl, dihydroimidazolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydro oxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridyl, dihydropyrimidinyl, dihydropyrrolyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidine, tetrahydrofuranyl, tetrahydrothienyl,

and their N-oxides. The connection of heterocyclic substituents can be achieved through carbon atoms or through heteroatoms.

The term “heteroaryl” as used herein refers to an aromatic ring containing one or more heteroatoms selected from O, N or S. The aromatic ring can be monocyclic, bicyclic or polycyclic, include but not limited to: quinolinyl, pyrazolyl, pyrrolyl, thienyl, furanyl, pyridyl, pyrimidinyl, pyrazinyl, triazolyl, imidazolyl, oxazolyl, isoxazolyl, pyridazinyl, benzoyl Furanyl, benzothienyl, benzoxazole, indolyl, etc.; “heteroaryl” can also be understood to include the N-oxide derivatives of any nitrogen-containing heteroaryl groups. The attachment of heteroaryl substituents can be through carbon atoms or through heteroatoms.

The term “heteroaromatic ketone substituents” as used herein refers to an aromatic ring containing one or more cyclic carbonyl groups and one or more heteroatoms selected from O, N, or S, which can be monocyclic, bicyclic or polycyclic, such as but not limited to:

The attachment of heteroaromatic ketone substituents can be achieved by carbon atoms or heteroatoms.

The term “heterocyclic alkane ketone substituents” used in this article refers to a saturated or partially unsaturated single ring, double ring, or multi ring substituent group containing one or more cyclic carbonyl groups, wherein one or more ring atoms are heteroatoms selected from N, O, or S (O) m (where m is an integer of 0-2) heteroatoms, and the remaining ring atoms are carbon. Double rings or multi rings include spiral rings, fused rings, and bridge rings, such as but not limited to:

The attachment of heterocyclic alkane ketone substituents can be achieved by carbon atoms or heteroatoms.

As understood by the skilled in the art, “halogen” or “halo” as used herein means chlorine, fluorine, bromine and iodine.

The present disclosure provides a class of Pyridopyrimidine compounds having a structure shown in Formula (I),

The present disclosure includes free forms of compounds of Formula I, as well as pharmaceutically acceptable salts and stereoisomers thereof. Some specific exemplary compounds in the present disclosure are protonation salts of amine compounds. The term “free form” refers to the amine compound in non-salt form. The pharmaceutically acceptable salts include not only exemplary salts of the particular compounds described herein, but also typical pharmaceutically acceptable salts of free form of all compounds of Formula I. The free forms of specific salts of the compounds can be isolated using techniques known in the art. For example, the free form can be regenerated by treating the salt with appropriate dilute aqueous base, such as dilute aqueous NaOH, dilute aqueous potassium carbonate, dilute aqueous ammonia, and dilute aqueous sodium bicarbonate. The free forms differ somewhat from their respective salt forms in certain physical properties such as solubility in polar solvents, but for the purposes of the disclosure, such salts of acid or base are otherwise pharmaceutically equivalent to their respective free forms.

The pharmaceutically acceptable salts of the present disclosure can be synthesized from the compounds containing a basic or acidic moiety in the present disclosure by conventional chemical methods. Generally, salts of basic compounds can be prepared by ion exchanged chromatography or by reacting the free base with a stoichiometric or excess amount of inorganic or organic acid in the desired salt form in a suitable solvent or combination of solvents. Similarly, salts of acidic compounds can be formed by reaction with a suitable inorganic or organic base.

The preparation of the pharmaceutically acceptable salts described above and other typical pharmaceutically acceptable salts was described in more detail by Berg et al., in “Pharmaceutical Salts,” J. Pharm. Sci. 1977:66:1-19.

In one embodiment, the present disclosure provides a method of treating tumors, cardiovascular diseases, diabetes, hypertension, muscular dystrophy, Parkinson's disease, Alzheimer's disease and other diseases related to abnormal expression of AKT3 protein in humans or other mammals by using compounds of Formula I, as well as pharmaceutically acceptable salts and stereoisomers thereof.

In one embodiment, the compounds, as well as pharmaceutically acceptable salts and stereoisomers thereof in the present disclosure can be used for treating and/or preventing tumors, such as non-small cell lung cancer, malignant melanoma, prostate cancer, kidney cancer, liver cancer, bladder cancer, ovarian cancer, colon cancer, rectal cancer, breast cancer, cervical cancer, lung cancer, laryngeal cancer, nasopharyngeal cancer, pancreatic cancer, multiple myeloma, B lymphoma, leukemia, or preventing postoperative recurrence of tumors.

Drug Metabolites and Prodrugs:

The metabolites of the compounds of the present disclosure and their pharmaceutically acceptable salts, and prodrugs that can be converted into the structures of the compounds of the present disclosure or their pharmaceutically acceptable salts in vivo, also fall within the scope of protection defined by the claims of this application.

Pharmaceutical Composition

The present disclosure also provides a pharmaceutical composition, comprising active ingredients within a safe and effective dosage range, as well as pharmaceutically acceptable carriers or excipients.

The “active ingredient” mentioned in the present disclosure refers to the compounds of Formula I according to the present disclosure or their pharmaceutically acceptable salts, stereoisomers, or prodrug.

The “active ingredient” and pharmaceutical composition of the present disclosure can be used as AKT3 protein degradation agent, and can be used to prepare drugs to prevent and/or treat tumors, cardiovascular diseases, diabetes, hypertension, muscular dystrophy, Parkinson's disease, Alzheimer's disease, etc.

“Safe and effective dosage” refers to the amount of the active ingredients that are sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical compositions contain 1-2000 mg of active ingredients/formulation, and more preferably, 10-200 mg of active ingredients/formulation. Preferably, the ‘one dose’ is one tablet.

“Pharmaceutically acceptable carrier or excipient” refers to one or more compatible solid or liquid fillers or gel substances, which are suitable for human use, and must have sufficient purity and sufficiently low toxicity.

“Compatibility” here refers to that each component in the composition can be mixed with the active ingredients of the present disclosure and intermingled between each other without significantly reducing the efficacy of the active ingredients.

In another preferred embodiment, the compounds of Formula I of the present disclosure can form complexes with macromolecular compounds or macromolecule through nonbonding cooperation. In another preferred embodiment, the compound of Formula I of the present disclosure, as a small molecule, can also be connected with a macromolecular compound or a polymer through a chemical bond. The macromolecular compounds can be biological macromolecules such as polysaccharides, proteins, nucleic acids, peptides, etc.

There is no special restriction on application methods of the active ingredients or drug composition of the present disclosure, and typical administration methods include (but not limited to) oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), etc.

The solid dosage forms used for oral administration include capsules, tablets, pills, powders and granules.

In these solid dosage forms, the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients:(a) fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid;(b) adhesives, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and Arabic gum;(c) humectants, such as glycerin;(d) disintegrating agents, such as agar, calcium carbonate, potato starch or cassava starch, algic acid, some composite silicates, and sodium carbonate;(e) slow solvent, such as paraffin;(f) absorption accelerators, such as quaternary amine compounds;(g) wetting agents, such as cetyl alcohol and glyceryl monostearate;(h) adsorbents, such as kaolin; and(i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or their mixtures. In the case of capsules, tablets and pills, the dosage form may also include buffers.

The solid dosage form can also be prepared with coating and shell materials, such as casings and other materials known in the art. They may comprise an opaque agent. Furthermore, the active ingredients from such compositions may be released in certain part of the digestive tract in a delayed manner. Embodiments of embedding components that can be used are polymers and waxes.

Liquid dosage forms for oral administration include pharmaceutically acceptable lotion, solutions, suspensions, syrups or tinctures. In addition to the active ingredients, the liquid dosage form may include inert diluents commonly used in the art, such as water or other solvents, solubilizers, emulsifiers (such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide), and oil (especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances). In addition to these inert diluents, the composition may also include auxiliary agents, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and spices.

In addition to the active ingredients, the suspension may contain suspension agents, such as ethoxylated isooctadecanol, polyoxyethylene sorbitol and dehydrated sorbitol ester, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for re-dissolution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols, and their suitable mixtures.

The compound of the present disclosure can be administered separately or in combination with other therapeutic drugs.

When using a pharmaceutical composition, a safe and effective amount of the compound of the present disclosure is applied to mammals (such as humans) in need of treatment, wherein the dosage at the time of application is a pharmaceutically effective dosage. For an individual weighing 60 kg, the daily dosage is usually 1-2000 mg, preferably 20-500 mg. Of course, the specific dosage should also consider factors such as the route of administration and the patient's health status, etc., which are within the skill range of a skilled physician.

Drug Combinations

The compounds of Formula I may be used in combination with other drugs known to treat or improve similar conditions. In the case of combined administration, the administration mode and dosage of the original drug remain unchanged, while the compounds of Formula I are administered simultaneously or subsequently. When the compounds of Formula I are administered concomitantly with one or more other drugs, it is preferred to use a pharmaceutical composition containing one or more known drugs and the compounds of Formula I. Drug combination also includes administration of the compounds of Formula I with one or more other known drugs in overlapping time periods. When the compounds of Formula I are used in combination with one or more other drugs, the compounds of Formula I or known drugs may be administered at lower doses than that when they are administered alone.

The benefits of the present disclosure are:(1) A novel structure of Pyridopyrimidine compounds is provided.(2) This type of compounds can efficiently and highly selectively degrade AKT3 protein in cells without affecting AKT1/2. They can effectively inhibit the growth of various tumor cells and can be used to prepare anti-tumor drugs.

A further description about the present disclosure is given below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present disclosure, but not to limit its scope. The following embodiments, which have not specified specific conditions, are usually performed in accordance with conventional conditions, such as those described in Sambrook, et al., “Molecular Cloning: Laboratory Manual” (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturers. Unless otherwise specified, percentages and portions are calculated by weight.

Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those are familiar to the skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present disclosure. The preferred implementation methods and materials described herein are for demonstration purposes only.

The raw materials in the following embodiments can be obtained commercially, or prepared by methods known in the art, or according to the methods described herein.

The structure of the compound is determined by nuclear magnetic resonance (1H-NMR) and/or mass spectrometry (MS). NMR determination is carried out using the Bruker AV-400 nuclear magnetic resonance instrument, with deuterated chloroform (CDCl3) or deuterated dimethyl sulfoxide (DMSO-D6) as the solvent and TMS as the internal standard. The LCQAD-40000 mass spectrometer is used for the determination of MS. 200-300 mesh silica gel (produced by Qingdao Ocean Chemical Factory) is used for Column chromatography.

Compound 3a (2.57 g, 6.43 mmol) and crotonic acid (5.54 g, 64.3 mmol) were placed in a 250 mL two-necked round-bottom flask, and anhydrous tetrahydrofuran (40 mL) and N, N-diisopropylethylamine (11.2 mL) were added under argon protection. After stirring the mixture evenly, replaced with argon three times. Added Bis (cyanobenzene) palladium dichloride (II) (0.12 g, 5%) and tri (o-methylphenyl) phosphorus (96 mg, 5%) again, and replaced with argon three times. Then the temperature of the mixture was slowly raised to 70° C. After the complete reaction of the raw material 3a was detected by TLC, 1.5 mL of acetic anhydride was added. The reaction solution was heated to 80° C. and continued stirring for 8 hours. After the reaction was completed monitored by TLC, most of the organic solvent was evaporated under reduced pressure, and the residue was diluted with 100 mL of ethyl acetate. The organic layer was washed with 1N HCl (3×100 mL) and saturated sodium chloride solution (2×100 mL), and the separated organic phase was dried with anhydrous sodium sulfate, and then concentrated and purified through column chromatography (petroleum ether/ethyl acetate=2:1 elution) to obtain a white solid compound 4a (0.71 g, yield 30%). MS (ESI), m/z: 387.3 [M+H]+.1H NMR (400 MHz, DMSO-d6) δ 9.61 (s, 1H), 9.10 (s, 1H), 7.51-7.38 (m, 3H), 6.89 (dt, J=7.4, 1.7 Hz, 1H), 6.73 (d, J=1.4 Hz, 1H), 2.53 (d, J=1.3 Hz, 3H), 1.47 (s, 9H).

TFA (2 mL) was added into a DCM (10 mL) solution of compound 4a (0.50 g, 1.29 mmol), stirred at room temperature for 0.5 hours. After the reaction was complete, the organic solvent was removed by evaporating under reduced pressure. The obtained residue was dissolved in 10 mL of acetonitrile and the solution was sequentially added anhydrous potassium carbonate (0.36 g, 2.58 mmol) and acryloyl chloride (0.17 g, 1.94 mmol), continued stirring for 0.5 hours at room temperature. After the reaction was completed monitored by TLC, the organic solvent was evaporated and 50 mL of ice water was added. Continued stirring at room temperature for 1 hour and filtered under reduced pressure. The filter cake was washed with anhydrous acetone (2×25 mL). A white solid intermediate 5 (0.41 g, yield 930%) was obtained after the filter cake was dried. The crude intermediate could be used in the next reaction without purification. MS (ESI), m/z: 341.0 [M+H]+.1H NMR (400 MHz, DMSO-d6) δ ppm 10.69 (br, 1H), 9.11 (s, 1H), 7.78 (d, J=8.22 Hz, 1H), 7.71 (br, 1H), 7.49 (t, J=7.92 Hz, 1H), 7.00 (d, J=7.63 Hz, 1H), 6.74 (s, 1H), 6.56 (dd, J=10.27, 16.92 Hz, 1H), 6.26 (d, J=16.82 Hz, 1H), 5.76 (d, J=10.17 Hz, 1H), 2.55 (s, 3H).

5-Fluoro-2-nitroanisole 11 (3.00 g, 17.5 mmol), piperazine (7.53 g, 87.5 mmol), and anhydrous potassium carbonate (3.63 g, 26.3 mmol) were sequentially added to acetonitrile (50 mL), and the mixture was stirred at 80° C. for 6 hours. After the reaction was completed monitored by TLC, the organic solvent was evaporated under reduced pressure. The obtained residue was added to 20 mL of distilled water and stirred for 1 hour, then filtered under reduced pressure. The obtained filter cake was added to 40 mL of anhydrous ether, continued stirring for 1 hour, and filtered under reduced pressure. The filter cake was washed with iced anhydrous ether (3×20 mL). The dried filter cake was the relatively pure compound 12 (3.95 g, yield 95%). MS (ESI), m/z: 238.1 [M+H]+.1H NMR (400 MHz, DMSO-d6) δ 9.21 (s, 2H), 7.97-7.87 (m, 1H), 6.69-6.58 (m, 2H), 3.93 (s, 3H), 3.70-3.63 (m, 4H), 3.28-3.19 (m, 4H).

The methanol solution of compound 13 (2.98 g, 5 mmol) was added palladium carbon (0.55 g) and replaced with hydrogen gas three times. The reaction solution was stirred overnight at room temperature. After the reaction was complete, filtered under reduced pressure, the filtrate was evaporated to dryness. The obtained colorless oily liquid was compound 14 (2.8 g, yield 99%). Due to its high susceptibility to oxidation in the air, this compound did not require purification and could be directly used in the next step. MS (ESI), m/z: 567.4 [M+H]+

Embodiment 105: Effect of Compounds on AKT3 Protein Levels in H19750R Cells

Cell line: Non-small cell lung cancer H1975-OR cell line, which is an Osimertinib resistant H1975 cell line, and could be obtained through low dose gradual addition culture. Specific method: H1975 cells were spread in a 10 cm culture dish at a convergence rate of 60-70%, added 50 nM of Osimertinib to the culture medium. After the cell state stabilized, the concentration of Osimertinib was gradually double to 3 μM. After genetic detection, the obtained drug-resistant cell line (H1975-OR) showed significantly higher expression of AKT3 compared to the original H1975 cell line.

Conventional Western Blot (immunoblotting) was used for detection, as follows: a certain number of H1975-OR cells were planted on a 96 well plate, adherent cultured in an incubator overnight, and added a certain concentration of compound to act for 24 hours. 1×SDS lysis buffer with protease and phosphatase inhibitors was used to lyse cells. Cell lysates were separated by SDS-PAGE and transferred to PVDF membrane. Then, at room temperature, the PCDF membrane was taken out and immersed in a sealing solution (5% BSA TBS solution containing 0.5% Tween-20) for sealing treatment for 1 hour, and then incubated with a specific primary antibody at 4° C. overnight. The imprints were washed with TBST and incubated with horseradish peroxidase (HRP) labeled secondary antibodies at room temperature for 2 hours. Finally, ECL plus fluorescence detection reagent (Thermo Scientific, Waltham, MA) was used for protein development, and the Amersham Imager 600 system (GE, America) was used for photography. The detection results were processed using ImageJ software to obtain the grayscale value G (Density). The maximum degradation rate (Dmax) of the protein was calculated using the following formula: Dmax=1−(Gmax Gmin)/Gmax×100%, wherein Gmax=blank control group Density(target protein band)/Density(corresponding to GAPDH); Gmin was the Density(target protein band)/Density(corresponding to GAPDH)when the maximum degradation value of the target protein was observed in the compound treatment group. The obtained results were presented in Dmax (%), as shown in Table 1.

TABLE 1Ability of compounds to induce degradation ofAKT1, AKT2, and AKT3 proteins in H1975-OR cellsCompoundsDmax (%) at 1000 nMNo.AKT1AKT2AKT3ZX-HYT-0159.227.267.6ZX-HYT-020030.8ZX-HYT-0378.964.889.1ZX-HYT-040026.6ZX-HYT-0534.429.971.9ZX-HYT-0604762.9ZX-HYT-070069.2ZX-HYT-080066.8ZX-HYT-090075.7ZX-HYT-10025.357.1ZX-HYT-11015.391.1ZX-HYT-1256075.4ZX-HYT-13———ZX-HYT-14———ZX-HYT-15———ZX-HYT-16031.3770.3ZX-HYT-1746.445.976.4ZX-HYT-180030.6ZX-HYT-190028ZX-HYT-20———ZX-HYT-21006.8ZX-HYT-220025.02ZX-HYT-230041.38ZX-HYT-240051.5ZX-HYT-25———ZX-HYT-26———ZX-HYT-27030.1876.3ZX-HYT-28———ZX-HYT-2957.5136.4354.7ZX-HYT-3040.5337.1351.9ZX-HYT-31030.6455.5ZX-HYT-32024.2645.2ZX-HYT-33043.7355.3ZX-HYT-3455.467.5156.6ZX-HYT-3542.1739.1243.4ZX-HYT-360012.3ZX-HYT-370017.5ZX-HYT-380016.17ZX-HYT-3905155.3ZX-HYT-403230.753ZX-HYT-4357.530.537.9ZX-HYT-4422.423.640.1ZX-HYT-4532.817.629.1ZX-HYT-470067.45ZX-HYT-48034.2560.6ZX-HYT-49026.1361.6ZX-HYT-5930.324.738.9ZX-HYT-6013018.9ZX-HYT-6114.231.515.9ZX-HYT-6228.415.118.8

Embodiment 106: Effect of Compound ZX-HYT-11 on AKT1/2/3 Proteins in Other Tumor Cells

Tumor cells (111975, PC-9, 111299, and A549) was incubated with different concentrations of compound ZX-HYT-11 for 24 hours, and then analyzed the protein levels of AKT1/2/3 using immunoblotting method as described in Embodiment 105. The results showed (FIGURE) that compound ZX-HYT-11 could selectively degrade AKT3 protein in the aforementioned cells, without affecting AKT1/2, further demonstrating the effectiveness and universality of this compound in degrading AKT3 protein.

Embodiment 107: Inhibitory Activity of Compounds on Tumor Cells Proliferation

Tumor cells (see Table 2 to Table 5) were inoculated in 96-well plates in complete culture medium (2000-3000 cells/well). After overnight incubation, the compounds with different concentrations (0.000508 μM-10 μM) were used to treat the cells separately for 72 hours. CCK-8 (Cell Counting Kit 8, Dojindo Laboratories, Kumamoto, Japan) experiment was used to evaluate cell proliferation. GraphPad Prism 5.0 software (La Jolla, CA) was used to calculate the half-maximal inhibitory concentration (IC50) values by fitting the concentration response curve. Each IC50 value was represented as an average value±SD. The results were shown in Table 2 to Table 5.

TABLE 4The inhibitory activity of compounds on MDA-MB-231 and other tumor cells proliferationCompoundsIC50 (μM)No.MDA-MB-231MDA-MB-453BT-549Bel-7402Huh7HCC1937ZX-HYT-110.02935>10>10>100.6859>10ZX-HYT-19<0.001>100.0042090.041——ZX-HYT-500.1553>10>10>100.6317>10ZX-HYT-510.1449>100.6436>3>10>10ZX-HYT-521.9932.50.47750.5681.3520.002-0.005ZX-HYT-640.01515>100.14060.37—4ZX-HYT-650.1038>10>10>100.3351>10ZX-HYT-67——0.376—>10—ZX-HYT-68——0.03856—>10—ZX-HYT-69——2.996—>10—ZX-HYT-70——1.021—>10—ZX-HYT-71——1.251—>10—ZX-HYT-72——0.6261—3—ZX-HYT-89——0.02666—0.12—ZX-HYT-91——0.9371—3—

TABLE 5The inhibitory activity of compounds on A549 and other tumor cells proliferationIC50 (μM)Compounds No.A549HCT116K562MIAPACA-2PANC-1BXPC-3ZX-HYT-500.66820.011930.029340.0086450.013730.0421ZX-HYT-510.62850.055280.26260.051280.12480.3327ZX-HYT-520.83290.10360.25010.072090.12590.3033ZX-HYT-651.1730.072910.23410.051080.17860.447

The technical features of the Embodiments above can be combined arbitrarily. To simplify description, all possible combinations of the technical features of the Embodiments above are not described. However, as long as there is no contradiction in the combination of these technical features, they should be considered as falling within the scope of this specification.

The Embodiments above only express several implementations of the present disclosure. The descriptions of the Embodiments are relatively specific and detailed, but may not be construed as the limitation on the patent scope of the present disclosure. It should be noted that a person of ordinary skill in the art may make several variations and improvements without departing from the concept of the present disclosure. These variations and improvements all fall within the protection scope of the present disclosure. Therefore, the patent protection scope of the present disclosure shall be defined by the appended claims.