CN113416181B - Quinazoline derivatives and their uses - Google Patents

Abstract

The invention discloses a quinazoline derivative, a preparation method and application thereof, and belongs to the technical field of medicine. The present invention provides a compound of formula I or a pharmaceutically acceptable salt thereof. This series of compound molecules have inhibitory activity against ATR, and can be used to prepare ATR inhibitor drugs. Tumor activity; therefore, this series of compounds provides a new and effective choice for the preparation of ATR small molecule inhibitor drugs and drugs for the treatment and/or prevention of cancer, and has a good application prospect.

Description

Quinazoline derivatives and their uses

technical field

The invention belongs to the technical field of organic synthetic medicines, and particularly relates to quinazoline derivatives and a preparation method and application thereof.

Background technique

Ataxia telangiectasia and Rad3-related (ATR) is an atypical serine/threonine protein kinase that plays a key role in the DNA replication stress response. Both ATR and ataxia telangiectasia mutant kinase (ATM) and DNA-dependent protein kinase (DNA-PK) belong to the phosphatidylinositol 3-kinase-like kinase (PIKK) family and are important components of DNA damage response (DDR) In part; DDR has evolved to recognize, signal and facilitate the repair of damaged DNA. Unlike ATR, which responds to DNA replication stress, ATM and DNA-PK primarily respond to DNA double-strand breaks. Normally, the DDR coordinates the recognition, signaling, and repair of damaged DNA through the network, thereby ensuring genome stability. Numerous studies have shown that tumor cells with ATM mutations or loss of function, or other DDR deficiencies that promote replicative stress, are generally more dependent on ATR for survival. Based on the principle of synthetic lethality, this offers the possibility of using ATR inhibitors to kill cancer cells while sparing normal non-cancer cells. Therefore, the development of ATR kinase inhibitors for related cancer therapy has important application value.

SUMMARY OF THE INVENTION

The present invention develops a new class of quinazoline derivatives and a preparation method thereof. The quinazoline derivatives exhibit good antitumor activity on enzymes, cells and animals, and can be used to prepare ATR inhibitors.

The present invention first provides a compound represented by formula I or a pharmaceutically acceptable salt thereof:

The nitrogen heterocycle A is selected from: substituted or unsubstituted 4-10-membered azacycloalkyl, which in addition to N shown in formula I, also contains 0-2 heteroatoms, the heteroatoms are N, O or S; nitrogen In the heterocycle A, the substituents of the substituted 4-10-membered azacycloalkyl are selected from: C1-C8 alkyl, C1-C8 alkoxycarbonyl, amino, and sulfo;

R ₁ is selected from: H, a substituted or unsubstituted 6-10-membered aryl group, a substituted or unsubstituted 5-10-membered heteroaryl group, a substituted or unsubstituted pyridone, and a sulfonate group; in R ₁ , the The 5-10-membered heteroaryl group contains 1-3 heteroatoms, and the heteroatom is N, O or S; in R ₁ , the substituted 6-10-membered aryl group, the substituted 5-10-membered heteroaryl group, the substituted The substituents of the pyridone are selected from: C1-C8 alkyl, 5-10-membered cycloalkyl, cyano, amino, C1-C8 alkoxycarbonyl; in R ₁ , the 5-10-membered cycloalkyl contains 0- 2 heteroatoms, the heteroatoms are N, O or S;

R ₂ is selected from: H, a substituted or unsubstituted 6-10-membered aryl group, a substituted or unsubstituted 5-10-membered heteroaryl group, a substituted or unsubstituted pyridone, and a sulfonate group; in R ₂ , the The 5-10-membered heteroaryl group contains 1-3 heteroatoms, and the heteroatom is N, O or S; in R ₂ , the substituted 6-10-membered aryl group, the substituted 5-10-membered heteroaryl group, the substituted The substituents of the pyridone are selected from: C1-C8 alkyl, 5-10-membered cycloalkyl, cyano, amino, C1-C8 alkoxycarbonyl; in R ₂ , the 5-10-membered cycloalkyl contains 0 ~2 heteroatoms, where the heteroatoms are N, O, or S;

R ₁ and R ₂ are not H at the same time;

R₃中，所述5～10元杂芳基含有1～3杂原子，杂原子为N、O或S；R₃中，所述取代的6～10元芳基、取代的5～10元杂芳基的取代基选自：C1～C8烷基、氨基、C1～C8烷氧基、羟基、C1～C8烷氧羰基、卤素、5～10元环烷基；R₃中，所述5～10元环烷基含有0～2个杂原子，杂原子为N、O或S。R ₃ is selected from: substituted or unsubstituted 6-10-membered aryl, substituted or unsubstituted 5-10-membered heteroaryl,

In R ₃ , the 5- to 10-membered heteroaryl group contains 1-3 heteroatoms, and the heteroatom is N, O or S; in R ₃ , the substituted 6- to 10-membered aryl group, the substituted 5- to 10-membered aryl group, the substituted 5- to 10-membered aryl group The substituent of the heteroaryl group is selected from: C1-C8 alkyl, amino, C1-C8 alkoxy, hydroxyl, C1-C8 alkoxycarbonyl, halogen, 5-10-membered cycloalkyl; in R ₃ , the 5 ~10 membered cycloalkyl groups contain 0 to 2 heteroatoms, which are N, O or S.

Wherein, in the compound represented by the above formula I, the nitrogen heterocyclic ring A is selected from: substituted or unsubstituted 5-6 membered cycloalkyl, substituted or unsubstituted 7-8 membered bridged cycloalkyl, the In addition to N, it also contains 0-1 heteroatom, which is N, O or S; in nitrogen heterocycle A, the substituted 5-6 membered cycloalkyl, substituted 7-8 membered bridged cycloalkyl The substituent of is selected from: C1-C6 alkyl group, C1-C6 alkoxycarbonyl group, amino group, C1-C6 alkanesulfo group.

取代或未取代的

取代或未取代的

氮杂环A中，所述取代的吗啉、取代的哌嗪、取代的四氢吡咯、取代的六氢吡啶、取代的

取代的

取代的

的取代基选自：C1～C4烷基、C1～C4烷氧羰基、-NH₂、C1～C4烷磺基。Preferably, in the compound represented by the above formula I, nitrogen heterocycle A is selected from: substituted or unsubstituted morpholine, substituted or unsubstituted piperazine, substituted or unsubstituted tetrahydropyrrole, substituted or unsubstituted hexahydropyrrole Hydropyridine, substituted or unsubstituted

substituted or unsubstituted

substituted or unsubstituted

In the nitrogen heterocycle A, the substituted morpholine, substituted piperazine, substituted tetrahydropyrrole, substituted hexahydropyridine, substituted

replaced

replaced

The substituent of is selected from: C1-C4 alkyl group, C1-C4 alkoxycarbonyl group, -NH ₂ , C1-C4 alkanesulfo group.

R₄选自：H、C1～C4烷基、C1～C4烷氧羰基、-NH₂；R₅为C1～C4烷基。More preferably, in the compound represented by the above formula I, the nitrogen heterocycle A is selected from:

R ₄ is selected from: H, C1-C4 alkyl, C1-C4 alkoxycarbonyl, -NH ₂ ; R ₅ is C1-C4 alkyl.

Most preferably, in the compound represented by the above formula I, the nitrogen heterocycle A is selected from:

Wherein, in the compound represented by the above formula I, R ₁ is selected from: H, substituted or unsubstituted 6-membered aryl, substituted or unsubstituted 5- to 6-membered heteroaryl, substituted or unsubstituted pyridone, C1 ~C6 sulfonate group; in R ₁ , the 5- to 6-membered heteroaryl group contains 1-2 heteroatoms, and the heteroatom is N, O or S; in R ₁ , the substituted 6-membered aryl group, substituted The substituents of the 5-6-membered heteroaryl and substituted pyridone are selected from: C1-C6 alkyl, 5-6 membered cycloalkyl, cyano, amino, C1-C6 alkoxycarbonyl; in R ₁ , the The 5-6 membered cycloalkyl group contains 0-1 heteroatom, and the heteroatom is N, O or S.

Preferably, in the compound represented by the above formula I, R ₁ is selected from: H, substituted or unsubstituted phenyl, substituted or unsubstituted pyrazole, substituted or unsubstituted furan, substituted or unsubstituted thiophene, substituted or unsubstituted thiophene, or unsubstituted thiazole, substituted or unsubstituted oxazole, substituted or unsubstituted isoxazole, substituted or unsubstituted imidazole, substituted or unsubstituted pyrrole, substituted or unsubstituted pyridine, substituted or unsubstituted pyrimidine , substituted or unsubstituted pyridone, C1-C4 sulfonate group; in R ₁ , the substituted phenyl, substituted pyrazole, substituted furan, substituted thiophene, substituted thiazole, substituted oxazole, Substituted isoxazoles, substituted imidazoles, substituted pyrroles, substituted pyridines, substituted pyrimidines, substituted pyridones have substituents selected from: C1-C4 alkyl, 5-6 membered epoxyalkyl, cyano, Amino, C1-C4 alkoxycarbonyl.

R₆选自：C1～C4烷基、氰基、

R₇选自：C1～C4烷基、5～6元环氧烷基、C1～C4烷氧羰基；R₈选自：C1～C4烷基。More preferably, in the compound represented by the above formula I, R ₁ is selected from: H,

R ₆ is selected from: C1-C4 alkyl, cyano,

R ₇ is selected from: C1-C4 alkyl, 5- to 6-membered epoxy alkyl, C1-C4 alkoxycarbonyl; R ₈ is selected from: C1-C4 alkyl.

Most preferably, in the compound represented by the above formula I, R ₁ is selected from: H,

Wherein, in the compound represented by the above formula I, R ₂ is selected from: H, substituted or unsubstituted 6-membered aryl, substituted or unsubstituted 5- to 6-membered heteroaryl, substituted or unsubstituted pyridone, C1 ~C6 sulfonate group; in R ₂ , the 5- to 6-membered heteroaryl group contains 1-2 heteroatoms, and the heteroatom is N, O or S; in R ₂ , the substituted 6-membered aryl group, substituted The substituents of the 5-6-membered heteroaryl and substituted pyridone are selected from: C1-C6 alkyl, 5-6 membered cycloalkyl, cyano, amino, C1 _- C6 alkoxycarbonyl; The 5-6 membered cycloalkyl group contains 0-1 heteroatom, and the heteroatom is N, O or S.

Preferably, in the compound represented by the above formula I, R ₂ is selected from: H, substituted or unsubstituted phenyl, substituted or unsubstituted pyrazole, substituted or unsubstituted furan, substituted or unsubstituted thiophene, substituted or unsubstituted thiophene, or unsubstituted thiazole, substituted or unsubstituted oxazole, substituted or unsubstituted isoxazole, substituted or unsubstituted imidazole, substituted or unsubstituted pyrrole, substituted or unsubstituted pyridine, substituted or unsubstituted pyrimidine , substituted or unsubstituted pyridone, C1-C4 sulfonate group; in R ₂ , the substituted phenyl, substituted pyrazole, substituted furan, substituted thiophene, substituted thiazole, substituted oxazole, Substituted isoxazoles, substituted imidazoles, substituted pyrroles, substituted pyridines, substituted pyrimidines, substituted pyridones have substituents selected from: C1-C4 alkyl, 5-6 membered epoxyalkyl, cyano, Amino, C1-C4 alkoxycarbonyl.

R₆选自：C1～C4烷基、氰基、

R₇选自：C1～C4烷基、5～6元环氧烷基、C1～C4烷氧羰基；R₈选自：C1～C4烷基。More preferably, in the compound represented by the above formula I, R ₂ is selected from: H,

R ₆ is selected from: C1-C4 alkyl, cyano,

R ₇ is selected from: C1-C4 alkyl, 5- to 6-membered epoxy alkyl, C1-C4 alkoxycarbonyl; R ₈ is selected from: C1-C4 alkyl.

Most preferably, in the compound represented by the above formula I, R ₂ is selected from: H,

Wherein, in the compound represented by the above formula I, when R ₁ is selected from substituted or unsubstituted 6-10-membered aryl, substituted or unsubstituted 5-10-membered heteroaryl, substituted or unsubstituted pyridone, R ₂ is H; when R ₁ is selected from substituted or unsubstituted 6-10-membered aryl, substituted or unsubstituted 5-10-membered heteroaryl, substituted or unsubstituted pyridone, R ₂ is H; when When R ₁ is a sulfonate group, R ₂ is a sulfonate group.

Preferably, in the compound represented by the above formula I, R ₁ is selected from: substituted or unsubstituted 6- to 10-membered aryl, substituted or unsubstituted 5- to 10-membered heteroaryl, substituted or unsubstituted pyridone, Sulfonate group; R ₂ is selected from: H, sulfonate group, and R ₂ is a sulfonate group only when R ₁ is a sulfonate group.

所述取代的萘、取代的苯、取代的吲哚、取代氮杂吲哚、取代的吡唑、取代的苯并吡唑、取代的苯并咪唑的取代基选自：C1～C8烷基、氨基、C1～C6烷氧基、羟基、C1～C6烷氧羰基、卤素、5～6元环烷基；R₃中，所述5～6元环烷基含有0～1个杂原子，杂原子为N、O或S。Wherein, in the compound represented by the above formula I, R ₃ is selected from: substituted or unsubstituted naphthalene, substituted or unsubstituted benzene, substituted or unsubstituted indole, substituted or unsubstituted azaindole, substituted or unsubstituted Unsubstituted pyrazoles, substituted or unsubstituted benzopyrazoles, substituted or unsubstituted benzimidazoles,

The substituents of the substituted naphthalene, substituted benzene, substituted indole, substituted azaindole, substituted pyrazole, substituted benzopyrazole and substituted benzimidazole are selected from: C1-C8 alkyl, Amino, C1-C6 alkoxy, hydroxyl, C1-C6 alkoxycarbonyl, halogen, 5-6 membered cycloalkyl; in R ₃ , the 5-6 membered cycloalkyl contains 0 to 1 heteroatom, and the heteroatom contains 0 to 1 heteroatom. Atoms are N, O or S.

其中，X为C或N；R9为C1～C7烷基、C1～C4烷氧羰基、5～6元环烷基，所述5～6元环烷基含有0～1个杂原子，杂原子为N、O或S；R₁₀、R₁₁独立的选自：C1～C6烷基、C1～C6烷氧基、氨基、羟基、卤素；R₁₂选自：C1～C6烷基、C1～C6烷氧基、氨基、羟基、卤素，或者与芳环成5～6元脂肪环，5～6元脂肪环含有1～2个杂原子，杂原子为N或O。Preferably, in the compound represented by the above formula I, R ₃ is selected from:

Wherein, X is C or N; R9 is a C1-C7 alkyl group, a C1-C4 alkoxycarbonyl group, a 5-6 membered cycloalkyl group, and the 5-6 membered cycloalkyl group contains 0-1 heteroatom, and the heteroatom is N, O or S; R ₁₀ and R ₁₁ are independently selected from: C1-C6 alkyl, C1-C6 alkoxy, amino, hydroxyl, halogen; R ₁₂ is selected from: C1-C6 alkyl, C1-C6 Alkoxy, amino, hydroxyl, halogen, or form a 5-6-membered aliphatic ring with an aromatic ring, the 5-6-membered aliphatic ring contains 1-2 heteroatoms, and the heteroatom is N or O.

Most preferably, in the compound represented by the above formula I, R ₃ is selected from:

Preferably, the compound represented by the above formula I specifically includes the following structure:

The present invention also provides the use of the above compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment and/or prevention of anti-tumor.

Further, the present invention also provides the use of the compound or a pharmaceutically acceptable salt thereof in the preparation of ATR inhibitor drugs.

The present invention also provides a pharmaceutical composition, which is a preparation prepared by using the above compound or a pharmaceutically acceptable salt thereof as an active ingredient and adding auxiliary ingredients.

The present invention also provides the preparation method of the above-mentioned compound, and it comprises the following two synthetic routes:

Synthetic route one:

Reaction conditions: (a), K ₂ CO ₃ , DCM, 0°C, 1.5h; (b), R ₁ corresponds to boronic acid or boronic acid ester, Pd[P(C ₆ H ₅ ) ₃ ] ₄ , K ₂ CO ₃ , 1,4-dioxane/H ₂ O (V/V=50/1), 95℃, 12h; (c), Pd(dppf)Cl ₂ , K ₂ CO ₃ , 1,4-dioxo Hexacyclic/ _H2O (V/V=50/1), 105°C, 6h.

Synthetic route two:

Reaction conditions: (d), t-BuOK, THF, 0°C, 1.5h; (e), Pd[P(C ₆ H ₅ ) ₃ ] ₄ , K ₂ CO ₃ , 1,4-dioxane/ H ₂ O (V/V=50/1), 95°C, 12h; (f), Pd(dppf)Cl ₂ , K ₂ CO ₃ , 1,4-dioxane/H ₂ O (V/V =50/1), 105°C, 6h; (g), HCl (concentration 4M, solvent 1,4-dioxane), 1,4-dioxane/H ₂ O, 80°C, 1h; ( h), TsCl, DMAP, TEA, DMF, 80°C; (i), amine corresponding to R ₃ , 80°C.

Definition of Terms:

The compounds and derivatives provided by the present invention can be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstracts Service, Columbus, OH) nomenclature system.

The term "alkyl" is a straight or branched chain saturated hydrocarbon radical. Examples of C1-C8 alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4) , sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2- Butyl (C5), tert-amyl (C5) and n-hexyl (C6). Unless otherwise specified, each instance of alkyl is independently optionally substituted, ie, unsubstituted or substituted with one or more substituents. "Substitution" means that a hydrogen atom in a molecule is replaced by a different atom or molecule. In some embodiments, the C1-C8 alkyl group is a C1-C8 alkyl group substituted with halogen (fluorine, chlorine, bromine, iodine). In the case where the C1-C8 alkyl group is substituted by a substituent, the number of carbon atoms of the substituent is not counted.

The term "amino" refers to the group _-NH2 , -NHR or -NRR. Examples of amino groups include, but are not limited to, _-NH2 , N-methylamino, N-ethylaminomethyl, N,N-dimethylamino, N,N-dimethylamino, N-formamide, and N-acetamide. Unless otherwise specified, each instance of an amino group is independently optionally substituted, ie, unsubstituted or substituted with one or more substituents. In some embodiments, R and/or R, are C1-C8 alkyl, C1-C8 alkanoamide.

The cycloalkyl group mentioned in the present invention does not only refer to an alkyl group with only one ring, but also refers to a cycloalkyl group containing multiple rings, such as spirocycloalkyl group or bridged cycloalkyl group.

The term "pharmaceutically acceptable" means that a carrier, vehicle, diluent, adjuvant, and/or salt formed is generally chemically or physically compatible with the other ingredients that make up a pharmaceutical dosage form, and is physiologically Compatible with receptors.

The term "pharmaceutically acceptable salts" refers to the acid and/or base salts of the compounds of the present invention with inorganic and/or organic acids and bases, also including zwitterionic salts (inner salts), and also including quaternary ammonium salts, For example alkylammonium salts. These salts can be obtained directly in the final isolation and purification of the compounds. It can also be obtained by mixing the above-mentioned compound with a certain amount of acid or base as appropriate (eg, equivalent). These salts may be precipitated in solution and collected by filtration, recovered after evaporation of the solvent, or obtained by lyophilization after reaction in an aqueous medium. The salts described in the present invention can be hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate of the compound salt, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate.

Certain embodiments of the present invention include isotopically labeled compounds, which are the same as those listed herein, but in which one or more atoms are replaced by another atom whose atomic mass is Or mass number is different from the atomic mass or mass number commonly found in nature. Isotopes that may be incorporated into the compounds of the present invention include hydrogen, carbon, nitrogen, oxygen, sulfur, ie, 2H, 3H, 13C, 14C, 15N, 17O, 18O, 35S. Compounds of the present invention containing the above isotopes and/or other atomic isotopes, as well as pharmaceutically acceptable salts of such compounds, are intended to be included within the scope of the present invention. The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include, but are not limited to: oral, parenteral (intravenous, intramuscular or subcutaneous) and topical.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with (a) fillers or compatibilizers, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, For example, glycerol; (d) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, 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, hard Calcium fatty acid, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials well known in the art. They may contain opacifying agents, and the release of the active compound or compounds in such compositions may be in a certain part of the digestive tract in a delayed manner. Examples of embedding components that can be employed are polymeric substances and waxes. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances, and the like.

Besides these inert diluents, the compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances and the like.

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

Dosage forms for topical administration of the compounds of this invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required if necessary.

The pharmaceutically acceptable adjuvant of the present invention refers to the substance contained in the dosage form other than the active ingredient. The addition of the substance will not change the dominance of the above-mentioned pharmaceutical composition in the process of disease treatment, but only plays an auxiliary effect. These adjuvant effects are only the utilization of the known activity of the ingredient, which is the usual adjuvant treatment in the medical field. If the above-mentioned excipients are used in combination with the pharmaceutical composition of the present invention, it should still belong to the protection scope of the present invention.

The beneficial effects of the present invention are:

The present invention develops a new type of quinazoline derivatives, the series of compounds can be used to prepare ATR inhibitor drugs, and show strong cell anti-proliferation activity in various cell lines such as LoVo cells, and at the same time It also has good anti-tumor activity in animal experiments; therefore, the series of compounds provide a new effective choice for the preparation of ATR small molecule inhibitor drugs and drugs for treating and/or preventing cancer, and have good application prospects.

Description of drawings

Figure 1 is a graph showing the growth of subcutaneous tumors in the LoVo xenograft model of human colorectal adenocarcinoma after oral administration of the specified dose of compound 4-28 or solvent.

Figure 2 shows Western blot analysis of p-ATR, ATR, p-Chk1S ³⁴⁵ and Chk1 protein expression in subcutaneous tumor tissue after the indicated doses of compound 4-28 or solvent-treated LoVo xenograft model.

Detailed ways

Target Molecule Synthesis Example 1:

Preparation of intermediate (R)-4-(6-bromo-2-chloroquinazolin-4-yl)-3-methylmorpholine (2):

Weigh 6-bromo-2,4-dichloroquinazoline (1, 10.00g, 46.50mmol, 1.0eq) and (R)-3-methylmorpholine (5.64g, 55.81mmol, 1.2eq) and dissolve in 230mL In DCM, K ₂ CO ₃ (9.64 g, 69.76 mmol, 1.5 eq) was slowly added, and the reaction was carried out at room temperature for two hours. The reaction was monitored by TLC for completion. Post-processing: crude silica gel was directly added to the system, the sample was spin-dried, and the samples were separated by prep-CC (petroleum ether/ethyl acetate=4:1) to obtain 14.66 g of a white solid product with a yield of 92%.

1H NMR (400MHz, DMSO-d6) δ8.10 (d, J=1.7Hz, 1H), 7.96 (dd, J=8.9, 1.8Hz, 1H), 7.65 (d, J=8.9Hz, 1H), 4.67 (d, J=6.6Hz, 1H), 4.03 (d, J=13.3Hz, 1H), 3.90 (d, J=11.5Hz, 1H), 3.84–3.75 (m, 1H), 3.70 (s, 2H) , 3.55 (t, J=12.8Hz, 1H), 1.43 (d, J=6.8Hz, 3H).

13C NMR (101 MHz, DMSO-d6) δ 163.31, 156.10, 152.30, 137.10, 129.72, 128.17, 118.05, 116.08, 70.48, 66.62, 51.80, 44.70, 15.36.

Preparation of intermediate (R)-4-(6-R1-2-chloroquinazolin-4-yl)-3-methylmorpholine (3-X):

Weigh the intermediate (R)-4-(6-bromo-2-chloroquinazolin-4-yl)-3-methylmorpholine (2, 1.0eq) and the corresponding boronic acid or boronic acid ester (1.2eq) Dissolve in dioxane/water (v/v=50:1), then add K ₂ CO ₃ (2.0eq) and tetrakis(triphenylphosphine)palladium (0.15eq), under nitrogen protection, 95 ℃ The reaction was carried out for 12 h, and the reaction was monitored by TLC. Post-processing: add crude silica gel directly to the system, spin dry and mix samples, and separate by prep-CC (petroleum ether/ethyl acetate = 3:1-1:4) to obtain products 3a-z with a yield of 50-73 %.

Preparation of product (R)-4-(2-(1H-indol-4-yl)-6-R1-quinazolin-4-yl)-3-methylmorpholine (4-X):

Weigh out intermediate (R)-4-(6-R1-2-chloroquinazolin-4-yl)-3-methylmorpholine (3-X, 1.0eq) and 4-(4,4,5, 5-Tetramethyl-1,3,2-dioxaboran-2-yl)-1H-indole (2.0eq) was dissolved in 5mL dioxane/water (v/v=50:1) , and then K ₂ CO ₃ (0.10mmol, 2.0eq) and [1,1'-bis(diphenylphosphino)ferrocene]dichloride palladium (0.1eq) were added, and the reaction was carried out at 95°C under nitrogen protection. 6h, the reaction was monitored by TLC. Post-processing: crude silica gel was directly added to the system, spin-dried and mixed, and the product 4-X was obtained by prep-CC separation.

Target Molecule Synthesis Example 2:

Preparation of intermediate (R)-4-(6-bromo-2-chloroquinazolin-4-yl)-3-methylmorpholine (5):

To a solution of 6-bromo-2,4-dichloroquinazoline (1, 35.98 mmol) in THF (36 mL) at 0 °C was added t-BuOK (35.98 mmol, 1.0 eq). It was stirred at 0°C for 3 hours and the completion of the reaction was monitored by TLC. Work-up: The reaction solution was poured into aqueous H ₂ O/NH ₄ Cl (25 mL/25 mL) and extracted with ethyl acetate. The organic layer was dried ( _{Na2SO4 )} and filtered. The filtrate was concentrated and separated and purified by prep-CC (petroleum ether/ethyl acetate=4:1) to give off-white intermediate 5 in 90% yield.

1H NMR (400MHz, Chloroform-d) δ8.18 (dd, J=2.3, 0.5Hz, 1H), 7.85 (dd, J=8.9, 2.3Hz, 1H), 7.69-7.65 (m, 1H), 1.74 ( s, 9H).13C NMR (101 MHz, DMSO-d6) δ 166.49, 155.39, 150.69, 138.41, 129.36, 126.64, 120.53, 117.43, 86.30, 28.05.

Intermediates 5 to 6 were prepared using the preparation method 2-X to 3-X, and intermediates 6 to 7 were prepared using the preparation method 3-X to 4-X.

Preparation of intermediate 2-(1H-indol-4-yl)-6-(1-methyl-1H-pyrazol-5-yl)-4(3H)-quinazolone (8):

To the intermediate 4-(tert-butoxy)-2-(1H-indol-4-yl)-6-(1-methyl-1H-pyrazol-5-yl)quinazoline (7, 5.03 mmol ) in CH ₃ OH (50 mL) solution was added concentrated hydrochloric acid (10 mL) and placed at 50° C. for 3 h. Work-up: the reaction solution was filtered and the solid was washed with DCM. The yellow solid 8 was dried and used in the next step without further purification.

1H NMR (400MHz, DMSO-d6) δ 11.61 (s, 1H), 8.27 (d, J=2.0Hz, 1H), 8.08 (dd, J=8.5, 1.9Hz, 1H), 7.95 (d, J= 8.5Hz, 1H), 7.74–7.68 (m, 3H), 7.59 (t, J=2.6Hz, 1H), 7.56 (d, J=1.8Hz, 1H), 7.29 (t, J=7.8Hz, 1H) , 7.11(s, 1H), 6.61(d, J=1.8Hz, 1H), 3.95(s, 2H).

13C NMR(101MHz,DMSO-d6)δ161.51,156.50,141.84,138.51,136.96,135.52,129.24,128.22,126.33,125.92,125.27,121.42,121.29,121.11,120.95,116.63,107.11,107.08,102.20,38.18。

Preparation of product 4-R3-2-(1H-indol-4-yl)-6-(1-methyl-1H-pyrazol-5-yl)quinazoline (4-X):

To 2-(1H-indol-4-yl)-6-(1-methyl-1H-pyrazol-5-yl)-4(3H)-quinazolone (8, 0.88 mmol), DMAP (0.088 mmol, 0.1 eq), TEA (1.76 mmol, 2.0 eq) in DMF (3 mL) was added TsCl (0.97 mmol, 1.1 eq). Under nitrogen protection, the reaction was carried out at 80° C. for 1 hour, and then the corresponding amine (1.76 mmol, 2.0 eq) was added to react for 2 hours, and the completion of the reaction was monitored by TLC. Post-treatment: the reaction solution was concentrated and purified by pre-column chromatography (petroleum ether/ethyl acetate=1:1-0:1), the yield of compound 4-X was 10-35%.

Example molecular and NMR data:

Note: The synthesis of example molecules is selected from route one or route two synthesis.

Compound pharmacological activity evaluation

In Vitro Kinase Experiments of Quinazoline Derivatives

In vitro kinase assays were performed using the Kinase Profiler service provided by Eurofins. A mixture of the test compound 4-X and the kinase ATR/ATRIP was incubated with 50 nM of GST-CMyc-p53 and Mg-containing ATP buffer to initiate the reaction by Mg/ATP. After incubation at room temperature for 30 minutes, the reaction was terminated by adding stop buffer containing EDTA, and then adding detection buffer containing d2-tagged anti-GST monoclonal antibody and phosphorylated p53 Europium-tagged anti-phosphorylated monoclonal antibody. Put the reaction plate into the microplate reader, use the time-resolved fluorescence (HTRF) mode, and calculate the HTRF signal value according to the formula HTRF=(Em665nm/Em620nm)*10000.

Test results: The inhibition of ATR activity by compound 4-X was determined, and the results are shown in Table 2 (A means IC ₅₀ <100nM, B means 500nM>IC ₅₀ >100nM, C means 1000nM>IC ₅₀ >500nM)).

In Vitro Cell Experiments of Quinazoline Derivatives

(1) Cell culture consumables:

Cell culture medium: DMEM medium (Gibco company), RPMI-1640 (Gibco company); fetal bovine serum (FBS, Hyclone); penicillin solution and streptomycin solution (Invitrogen biological company)

Cells: HT-29 (human colorectal adenocarcinoma cells), LoVo (human colorectal adenocarcinoma cells), NCI-H23 (human non-small cell lung cancer cells), A549 (human lung cancer cells), HL60 (human myeloid leukemia cells) cells) were purchased from the American Type Species Conservation Center (ATCC).

Cell culture consumables: trypsin (Invitrogen Biological Company); 6-well plate, 24-well plate, 96-well plate, centrifuge tube, gun tip, etc. (Corning Company), 20, 100 mm cell culture dishes (WHB Company).

(2) Cell culture:

HT-29 and LoVo cells were cultured in DMEM medium (containing 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin); NCI-H23, A549, HL60 cells were cultured in RPMI1640 medium (containing 10% fetal bovine serum) bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin). All cells were simultaneously incubated in a humidified cell incubator at 37°C with 5% CO ₂ . During the cell culture process, trypsinize the cells for reasonable passage to ensure a suitable cell density. All experiments use cells in the logarithmic growth phase.

(3) MTT detection

A. Solution configuration

5mg/mL MTT solution: Dissolve MTT powder with physiological saline to prepare a 5mg/mL solution, filter with a 0.22 μm filter membrane, store at 4°C in the dark, and use up within 1 month.

20% acidic SDS solution: Weigh 80 g of SDS powder, add ultrapure water to 320 mL. Sonicate until dissolved, add 400 μL of concentrated hydrochloric acid, and finally make up to 400 mL with ultrapure water.

B. Adherent cell (HT-29, LoVo, A549) treatment

Cells in logarithmic growth phase were seeded in 96-well plates at 100 μL per well (the seeding numbers of LoVo, HT-29 and A549 cells were 3500, 2000 and 3000 cells/well, respectively), leaving blank group and cell control In the group, add 200 μL of normal saline for injection to the side wells, incubate the well plate overnight in a cell incubator, add 100 μL of medium containing different concentrations of compounds into the well plate inoculated with cells, and set 3 duplicate wells in each group. Incubate in a cell incubator for 72h.

C. Treatment of suspension cells (NCI-H23 and HL60)

100 μL of the medium containing different concentrations of compounds was added to the well plates seeded with cells, and then the cells in logarithmic growth phase were seeded in 96-well plates per 100 μL per well (the seeding numbers of NCI-H23 and HL60 cells were respectively). 8,000 and 10,000 cells/well), 3 duplicate wells were set in each group, a blank group and a cell control group were set aside, 200 μL of normal saline for injection was added to the side wells, and the cells were incubated in a cell incubator for 72 h.

D. color rendering

Add 20μL of 5mg/mL MTT staining solution to each well, incubate in the cell incubator for 2-4h, confirm that the cells are completely stained under the microscope, add 50μL of 20% acidic SDS solution, and continue to culture in the cell incubator overnight. Finally, the absorbance value was detected at 570 nm with a multi-function microplate reader.

E. Calculate _IC50

The absorbance value of each well was used to calculate the survival rate of cells under different concentrations of compounds. Taking the compound concentration as the abscissa and the survival rate as the ordinate, the IC ₅₀ was calculated by fitting with GraphPad Prism 5.0 software. Survival rate=100-100%×(control group-administration group)/(control group-blank group).

Lovo cell test results: The inhibition of ATR activity by compound 4-X was determined, and the results are shown in Table 2.

Table 2 Kinase activity and cellular activity data

Kinase selectivity assay for compounds 4-28

Table 3 Inhibition data of compounds 4-28 against 13 targets

In the kinase selectivity test, the present invention found that the compound 4-28 at a concentration of 10 μM showed excellent selectivity to 403 recombinant human protein kinase targets, and as can be seen from Table 3, the inhibition rate of 13 kinase targets was greater than 50 %. Further IC ₅₀ test showed that the kinase activity of compound 4-28 against 9 of the targets was within 10 μM, and the off-target activity was the ATR target, followed by the B-Raf target. Overall, compounds 4-28 showed at least 118-fold selectivity against 403 recombinant human protein kinases with excellent kinase selectivity.

LoVo tumor xenograft model experiments

Collect LoVo cells in logarithmic growth phase, wash the cells three times with serum-free DMEM-based medium, and finally adjust the density of the cell suspension to 1×108 cells/mL using serum-free medium. The number of 107 cells, ie, 100 μL of LoVo cell suspension, was subcutaneously injected into the right side of 6-week-old female Balb/C mice (5-week-old female Balb/c mice after 1 week of adaptive breeding). After the tumor had grown to 130-150 mm ³ , the mice were randomly divided into 3 groups with 6 mice in each group, and the body weight and tumor volume of the mice were measured, and this was the first day. Compounds 4-28 were orally administered to mice by oral gavage at 50 mg/kg and 100 mg/kg twice a day, respectively. The compounds were dissolved in 10% (v/v) DMSO, 40% (v/v) PEG 300, 5% (v/v) ) in Tween 80 and 45% (v/v) saline, and the control group was orally administered the same solvent at 200 μL per day. Mouse tumor volume was measured every 2-3 days and recorded. After 30 days of administration, the subcutaneous tumor tissue of the mice was taken, and the tumor tumor was weighed and photographed. Part of the tumor tissue was soaked in 4% paramethanol, while the heart, liver and spleen of the mice were taken out and soaked in 4% paramethanol for immunohistochemical detection. Calculation of subcutaneous tumor volume in mice: V (mm ³ )=0.5×a×b×b, a is the width of the tumor, b is the length of the tumor, and the unit is mm.

Relative tumor volume (RTV): RTV=Vt/V0, Vt is the tumor volume at the measurement time point, and V0 is the tumor volume at the initial administration.

Relative tumor proliferation rate: R=TRTV/CRTV×100%, TRTV is the relative tumor volume in the treatment group, and CRTV is the relative tumor volume in the treatment group. The drug was considered effective when R<60%.

As can be seen from Figure 1, in all treatment groups, 4-28 inhibited tumor growth in a dose-dependent manner, and at a dose of 100 mg/kg BID, the tumor inhibition rate was 72.8%.

To investigate the relationship between ATR inhibition and antitumor efficacy in vivo, western blot analysis was performed to measure ATR activity (p-ATR, p-Chk1S ³⁴⁵ ) in LoVo tumor xenograft models after treatment with different doses of 4-28. As shown in Figure 2, 4-28 significantly inhibited the phosphorylation of ATR and Chk1 compared with the control group. These results suggest that 4-28 inhibits LoVo xenograft tumor growth in vivo by inhibiting ATR activity.

Preliminary pharmacokinetic properties studies of compounds 4-28:

Table 4 Important pharmacokinetic parameters of compounds 4-28

Pharmacokinetic parameters Intravenous (5mg/kg) Oral administration (10mg/kg) Half-life t_1/2(h) 1.4 1.1 Maximum absorption time t_max(h) 0.1 1.7 Peak drug concentration C_max(ng/mL) 3396.3 895.3 Area under the drug-time curve AUC_(0-∞)ng/mL*h 3151.3 2719.6 Plasma clearance Cl (mL/min/kg) 26.5 — Steady state apparent volume of distribution Vss (L/kg) 2.03 — Bioavailability F — 43.1%

To evaluate the suitability of compound 4-28 for in vivo experiments, we tested the preliminary pharmacokinetic properties of compound 4-28. The preliminary pharmacokinetic properties of compounds 4-28 were determined by Shanghai Medicilon Biopharmaceutical Co., Ltd. Preliminary pharmacokinetic properties were determined by oral administration of 10 mg/kg and intravenous administration of 5 mg/kg to male Sprague-Dawley rats, and detailed pharmacokinetic data are listed in Table 4. In oral administration, elimination half-life (t1/2), peak value (Cmax), area under the concentration-time curve (AUC0-∞) and bioavailability (F) were 1.1h, 895.3ng/mL, 2719.6ng/mL *h, 43.1%. In intravenous injection, the elimination half-life (t1/2), peak value (Cmax), area under the concentration-time curve (AUC0-∞), plasma clearance (Cl) and apparent volume of distribution (Vss) were 1.4h, 3396.3ng/mL, 3151.3ng/mL*h, 26.5mL/min/kg and 2.03L/kg. The compound SKLB-197 has good bioavailability and fully supports oral administration, but its half-life is not too long, only 1.1 hours.

As a small molecule inhibitor of ATR, the compound of the present invention exhibits strong cell anti-proliferation activity in various cell lines such as LoVo cells, and also has good anti-tumor activity in animal experiments.

Claims (5)

1. The compound represented by formula I or a pharmaceutically acceptable salt thereof:

The nitrogen heterocycle A is selected from:

R1 is selected from _:

R ₂ is selected from: H,

_{R3 is} selected from:

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, comprising the following structure:

3. Use of the compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment and/or prevention of colorectal cancer. 4. Use of the compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof in the preparation of ATR inhibitor drugs. 5. A pharmaceutical composition, characterized in that: it is a preparation prepared by using the compound described in claim 1 or 2 or a pharmaceutically acceptable salt thereof as an active ingredient and adding auxiliary ingredients.

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