CN116568689A

CN116568689A - Polycyclic pyrimidine derivative as SOS1 inhibitor, and preparation method and application thereof

Info

Publication number: CN116568689A
Application number: CN202280007413.5A
Authority: CN
Inventors: 郑苏欣; 谢成英; 陆晓杰; 郑明月; 乔刚
Original assignee: Suzhou Almai Biotechnology Co ltd
Current assignee: Suzhou Almai Biotechnology Co ltd
Priority date: 2021-02-09
Filing date: 2022-01-14
Publication date: 2023-08-08
Also published as: WO2022170917A1

Abstract

The invention belongs to the technical field of polycyclic pyrimidine derivatives, and particularly relates to a polycyclic pyrimidine derivative serving as an SOS1 inhibitor, and a preparation method and application thereof. The structure of the polycyclic pyrimidine derivative is shown as a formula (I). The invention provides a brand-new polycyclic pyrimidine derivative which has the functions of obviously inhibiting RAS signal paths and treating cancers, wherein the cancers are pancreatic cancer, colorectal cancer, lung cancer, hepatocellular carcinoma, renal cancer, gastric cancer, cholangiocarcinoma and the like.

Description

Polycyclic pyrimidine derivative as SOS1 inhibitor, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of polycyclic pyrimidine derivatives, and particularly relates to a polycyclic pyrimidine derivative serving as an SOS1 inhibitor, and a preparation method and application thereof.

Background

RAS family proteins belong to a small GTPase comprising three subfamilies of KRAS, NRAS and HRAS. Mutated RAS gene is an important oncogene, and the discovery of RAS gene mutation is found in 20-30% of human tumors, particularly pancreatic, colorectal and lung cancers. The various isoforms of RAS proteins have a balance of GTP-bound active and GDP-bound inactive states, GTPase-activated proteins (GTPase-activating proteins, GAPs) may facilitate the conversion of GTP to GDP, thereby converting RAS proteins to inactive states, and guanine nucleotide exchange factors (guanine nucleotide exchange factor, GEFs) may facilitate the release of GDP and the binding of GTP, thereby converting RAS proteins to active states. Activation of RAS proteins promotes proliferation of cells, apoptosis escape, metabolic recombination and the like through signal pathways of RAS-RAF-MEK-ERK and RAS-PI3K-PDK1-AKT, thereby promoting occurrence and development of tumors.

SOS1 (son of sevenless 1) is a key guanine nucleotide exchange factor (GEF) capable of binding to RAS proteins,promote the binding of RAS protein and GTP, and turn RAS protein to active state. Recent studies have found that SOS1 inhibitors not only inhibit RAS mutant cell growth, but also produce synergistic effects with MEK inhibitors, producing significant inhibition of KRAS-driven tumors ^1-2 . The development of SOS1 inhibitors has become a focus of research and SOS1 inhibitors of different structural types have been reported in several patents, such as WO2018172250, WO2019201848, WO2018115380, WO2019122129, WO2020173935, WO2020180768 and WO2020180770, etc.

However, there is still uncertainty in the effectiveness, safety or selectivity of these compounds and test drugs disclosed in the prior art, and thus there is a need to study and develop new selective SOS1 inhibitors.

Reference is made to:

1、Hillig et al.Discovery of poetent SOS1 inhibitors that block RAS activation via disruption of the RAS-SOS1 interaction.PNAS.116,2251-2560(2019).

2、Hofmann et al.BI-3406,a potent and selective SOS1::KRAS interaction inhibitor,is effective in KRAS-driven cancers through combined MEK inhibition.Cancer Discov.CD-20-0142(2020).

disclosure of Invention

In order to solve the above problems of the prior art, the present invention aims to provide a polycyclic pyrimidine derivative, a pharmaceutically acceptable salt thereof, a tautomer thereof or a stereoisomer thereof, so as to screen out a compound serving as an SOS1 inhibitor, which has excellent performance in terms of performance such as effectiveness, safety and selectivity.

It is another object of the present invention to provide a process for the preparation of said derivatives, pharmaceutically acceptable salts thereof, tautomers thereof or stereoisomers thereof.

In order to achieve the aim of the invention, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a polycyclic pyrimidine derivative, a pharmaceutically acceptable salt thereof, a tautomer thereof, or a stereoisomer thereof, wherein the structure of the polycyclic pyrimidine derivative is shown in formula (I):

wherein: r is R ¹ Selected from hydrogen or C ₁ -C ₃ An alkyl group; preferably hydrogen or methyl;

A ¹ selected from N or C-R ¹¹ ；

R ¹¹ Selected from H, C ₁ -C ₃ Alkyl or C ₁ -C ₃ A haloalkyl group;

A ² selected from N or C-R ² ；

R ² Selected from-OR ²¹ Or cyano;

R ²¹ selected from H, C ₁ -C ₃ Alkyl, 3-7 membered cycloalkyl, 4-7 membered heterocyclyl, wherein C ₁ -C ₃ Alkyl, 3-7 membered cycloalkyl, 4-7 membered heterocyclyl are optionally substituted with 1-3R ²² Substituted;

R ²² selected from halogen, C ₁ -C ₄ Alkyl, cyano, hydroxy;

l may be absent or selected from O, NH or NCH ₃ ；

R ³ Selected from 3-7 membered cycloalkyl, 4-7 membered heterocyclyl, 6-10 membered fused heterocyclyl, 6-10 membered bridged heterocyclyl, 6-10 membered spiroheterocyclyl, wherein 3-7 membered cycloalkyl, 4-7 membered heterocyclyl, 6-10 membered fused heterocyclyl, 6-10 membered bridged heterocyclyl, 6-10 membered spiroheterocyclyl is optionally substituted with 1-3R ³¹ Substituted;

R ³¹ Selected from C ₁ -C ₃ Alkyl, C ₁ -C ₃ Haloalkyl, hydroxy, halogen, cyano, -NR ^a R ^b 、C ₁ -C ₃ Alkoxy, =o, -NHCOR ³² or-COR ³² ；

R ³² Selected from C ₁ -C ₃ Alkyl, C ₁ -C ₃ Haloalkyl, 3-7 membered cycloalkyl or 4-7 membered heterocyclyl;

R ⁴ is-CH ₃ ；

AR is selected from 6-10 membered aryl or 5-10 membered heteroaryl, wherein aryl or heteroaryl is optionally substituted with 1-4R ⁵ Substituted;

R ⁵ selected from halogen, C ₁ -C ₄ Alkyl, C ₁ -C ₄ Haloalkyl, hydroxy-C ₁ -C ₄ Alkyl, hydroxy-C ₁ -C ₄ Haloalkyl, 3-6 membered cycloalkyl, 4-7 membered heterocyclyl, -OR ^a 、-NR ^a R ^b ；

R ^a Selected from H, C ₁ -C ₄ Alkyl, C ₁ -C ₄ Haloalkyl, 3-6 membered cycloalkyl or 4-7 membered heterocyclyl;

R ^b selected from H, C ₁ -C ₄ Alkyl, C ₁ -C ₄ Haloalkyl, 3-6 membered cycloalkyl or 4-7 membered heterocyclyl;

in the above definition, the following conditions cannot occur simultaneously:

1)A ¹ is C-R ¹¹ ；

2)A ² Is C-OR ²¹ ；

3) L is O or absent;

the hetero atoms in the heterocyclic group and the heteroaryl group in the formula (I) are 1-3 and are selected from one or more of oxygen, nitrogen and sulfur.

Preferably, the structure of the polycyclic pyrimidine derivative is shown as a formula (II):

wherein R is ¹ 、L、R ³ And R is ⁵ Has the same limit range as the above; n=1-4 (e.g., n=1, n=2, n=3, n=4);

preferably, for compounds of formula (II), the phenyl group is optionally substituted with 1 to 4R ⁵ Substituted when said R ⁵ When the number of R is 2-4, R is ⁵ May be the same or different;

and/or when said R ⁵ Is C ₁ -C ₄ In the case of haloalkyl, said R ⁵ 1-3, when 2-3R's are present ⁵ When said R is ⁵ May be the same or different;

and/or when said R ⁵ Is C ₁ -C ₄ When haloalkyl, halogen atom is fluorine or chlorine;

and/or when said R ⁵ In the case of halogen, R is ⁵ 1-3, when 2-3R's are present ⁵ When said R is ⁵ May be the same or different;

and/or when said R ⁵ When halogen, the halogen atom is fluorine or chlorine;

and/or when said R ⁵ is-NR ^a R ^b When said R is ⁵ 1-3, when 2-3R's are present ⁵ When said R is ⁵ May be the same or different;

and/or when said R ⁵ is-NR ^a R ^b When said R is ^a And R is ^b May be the same or different.

Preferably, the structure of the polycyclic pyrimidine derivative is shown as a formula (III):

wherein R is ¹ 、R ²¹ 、R ³ And R is ⁵ Has the same limit range as the above; n=1-4 (e.g., n=1, n=2, n=3, n=4);

preferably, for the compound of formula (III), R ³ Is optionally covered with 1-3R ³¹ When the substituted 4-7 membered heterocyclic group is a 5-6 membered heterocyclic group, the 4-7 membered heterocyclic group is a 5-6 membered heterocyclic group;

and/or R ³ Is optionally covered with 1-3R ³¹ When substituted 4-7 membered heterocyclyl, the heterocyclyl contains 1-2 heteroatoms;

And/or, the R ³ Is optionally covered with 1-3R ³¹ When the substituted 4-7 membered heterocyclic group is, the heteroatom of the heterocyclic group is nitrogen and/or oxygen;

and/or, the R ³ Is optionally covered with 1-3R ³¹ When the number of the hetero atoms of the heterocyclic group is two, the two hetero atoms are the same or different;

and/or, the R ²¹ Selected from H or optionally substituted with 1-3R ²² Substituted C ₁ -C ₃ Alkyl, said R ²² Selected from halogen, C ₁ -C ₄ Alkyl, cyano, hydroxy;

wherein R is ³¹ And R is ²² Has the same limiting scope as described above.

Preferably, the structure of the polycyclic pyrimidine derivative is shown as a formula (IV):

preferablyFor compounds of formula (IV), R when L is absent or O ³ Is optionally covered with 1-3R ³¹ Substituted 3-7 membered cycloalkyl or 4-7 membered heterocyclyl;

and/or when said R ³ Is optionally covered with 1-3R ³¹ When the substituted 4-7 membered heterocyclic group is a heterocyclic group, a heteroatom in the heterocyclic group is nitrogen and/or oxygen;

and/or when said R ³ Is optionally covered with 1-3R ³¹ When the substituted 4-7 membered heterocyclic group is, 1-2 hetero atoms are used;

wherein R is ³¹ Has the same limiting scope as described above.

Preferably, the structure of the polycyclic pyrimidine derivative is shown as a formula (V):

wherein R is ¹ 、A ² 、L、R ³ And R is ⁵ Has the same limit range as the above; n=1-4 (e.g., n=1, n=2, n=3, n=4).

Further preferably, the polycyclic pyrimidine derivative is selected from any one of the following structures:

typical compounds of the invention include, but are not limited to, compounds in the following table:

in a second aspect, the present invention provides a method for preparing a polycyclic pyrimidine derivative, a pharmaceutically acceptable salt thereof, a tautomer thereof, or a stereoisomer thereof according to the first aspect, which is selected from one of four schemes:

scheme one

The preparation method of the compound shown in the general formula (I) or the stereoisomer, the tautomer or the medicinal salt thereof comprises the following steps:

preparation of key intermediate (I-A), method one:

step one, performing metal catalytic cross coupling on a compound of a general formula (I-1) to obtain a compound of a general formula (I-2);

secondly, reacting the compound shown in the general formula (I-2) under the condition of a catalyst to obtain a chiral sulfimide compound shown in the general formula (I-3);

thirdly, reducing the compound of the general formula (I-3) by a metal reducing agent to obtain a chiral compound of the general formula (I-4);

Fourthly, the compound of the general formula (I-4) is cracked into the chiral benzylamine compound of the general formula (I-A) under the acid condition.

Preparation of key intermediate (I-A), method two:

the first step, alcohol hydroxyl of the compound of the general formula (I-5) is oxidized to obtain aldehyde compound of the general formula (I-6);

secondly, carrying out format reaction on the compound shown in the general formula (I-6) to obtain a compound shown in the general formula (I-7);

thirdly, oxidizing the alcoholic hydroxyl group of the compound of the general formula (I-7) to obtain a chiral compound of the general formula (I-2);

fourth to sixth steps are the same as above.

Wherein AR, R ⁴ Has the same limiting scope as described above.

Preparation of key intermediate (I-B), method one:

firstly, oxidizing a compound methyl of a general formula (I-8) to obtain an acid compound of a general formula (I-9);

secondly, carrying out esterification reaction on the compound shown in the general formula (I-9) to obtain a compound shown in the general formula (I-10);

thirdly, reacting the compound shown in the general formula (I-10) with the compound shown in the general formula (I-11) under alkaline conditions to obtain a compound shown in the general formula (I-12);

fourthly, reducing the nitro group of the compound of the general formula (I-12) to obtain a compound of the general formula (I-13);

and fifthly, the compound of the general formula (I-13) and nitrile are subjected to amidine formation under an acidic condition, and then the ring closure is carried out to obtain the compound of the general formula (I-B).

Wherein A is ¹ 、A ² 、R ¹ 、R ³ And L has the same defined range as above; a is that ¹ Preferably C; a is that ² Preferably N; l is preferably absent; r is R ¹ Methyl is preferred.

Preparation of key intermediate (I-B), method II:

firstly, carrying out acyl chlorination and ammonia water reaction on a compound shown in a general formula (I-9) to obtain an amide compound shown in a general formula (I-14);

secondly, reacting the compound of the general formula (I-14) with the compound of the general formula (I-11) under alkaline conditions to obtain a compound of the general formula (I-15);

thirdly, reducing the nitro group of the compound of the general formula (I-15) to obtain a compound of the general formula (I-16);

fourth, the compound of the formula (I-16) is reacted with triethyl orthoacetate to give the compound (R) of the formula (I-B) ¹ =methyl); the compound of the formula (I-16) is reacted with triethyl orthoformate to give the compound (R) of the formula (I-B) ¹ ＝H)。

Wherein A is ¹ 、A ² 、R ¹ 、R ³ And L has the same defined range as above; a is that ¹ Preferably C; a is that ² Preferably N; l is preferably absent or O; r is R ¹ Methyl is preferred.

Preparation of key intermediate (I-C), method one:

in the first step, the compound of the general formula (I-B) is subjected to halogenation reaction to obtain the compound of the general formula (I-C).

Wherein A is ¹ Preferably C, A ² Preferably N, L is preferably absent or O, R ¹ Methyl is preferred.

Preparation of key intermediate (I-C), method two:

The first step, a compound of a general formula (I-11) and tert-butyl bromoacetate undergo substitution reaction under alkaline conditions to obtain a compound of a general formula (I-17);

secondly, removing tert-butyl from the compound of the general formula (I-17) under an acidic condition to obtain a compound of the general formula (I-18);

thirdly, carrying out esterification reaction on the compound of the general formula (I-18) and alcohol under an acidic condition to obtain a compound of the general formula (I-19);

fourthly, reacting the compound of the general formula (I-20) with ammonia water to obtain a compound of the general formula (I-21);

fifthly, carrying out halogenation reaction on the compound of the general formula (I-21) to obtain a compound of the general formula (I-22);

sixthly, carrying out Suzuki reaction on the compound of the general formula (I-22) and vinyl potassium trifluoroborate under alkaline conditions in the presence of a metal catalyst and a ligand to obtain a compound of the general formula (I-23);

seventh, oxidizing double bonds in the compound of the general formula (I-23) to obtain a compound of the general formula (I-24);

eighth, reacting the compound of the general formula (I-24) with the compound of the general formula (I-19) under alkaline conditions to obtain a compound of the general formula (I-25);

and ninth, hydroxyl substitution of the compound of the general formula (I-25) is carried out to obtain the compound of the general formula (I-C).

Wherein A is ¹ ，A ² ，R ¹ ，R ³ And L has the same defined range as above; a is that ¹ Preferably N; a is that ² Preferably CR ² (R ² Selected from-OR ²¹ ) The method comprises the steps of carrying out a first treatment on the surface of the L is preferably NH, R ¹ Methyl is preferred.

Preparation of general formula (I):

in the first step, the compound of the general formula (I-B) and the compound of the general formula (I-A) are reacted under condensation conditions to obtain the compound of the general formula (I).

Wherein A is ¹ 、A ² 、AR、R ¹ 、R ³ 、R ⁴ And L has the same defined range as described above.

Scheme II

in the first step, the compound of the general formula (I-C) and the compound of the general formula (I-A) are reacted under alkaline conditions to obtain the compound of the general formula (I).

Wherein A is ¹ 、A ² 、AR、R ¹ 、R ³ 、R ⁴ And L is as defined in formula (I).

Scheme III

firstly, demethylating the compound of the general formula (I-26) under alkaline conditions to obtain a compound of the general formula (I-27);

secondly, carrying out esterification reaction on the compound shown in the general formula (I-27) to obtain a compound shown in the general formula (I-28);

thirdly, protecting groups on hydroxyl groups of the compound of the general formula (I-28) to obtain a compound of the general formula (I-29);

Fourthly, reducing the nitro group of the compound of the general formula (I-29) to obtain a compound of the general formula (I-30);

fifthly, forming amidine with nitrile under the acidic condition of the compound shown in the general formula (I-30) and then closing the ring to obtain the compound shown in the general formula (I-31);

step six, the hydroxyl of the compound of the general formula (I-31) is halogenated to obtain the compound of the general formula (I-32);

seventh, reacting the compound of the general formula (I-32) with the compound of the general formula (I-A) under alkaline conditions to obtain a compound of the general formula (I-33);

eighth step, deprotecting the compound of formula (I-33) under acidic conditions to obtain a compound of formula (I-34);

ninth, reacting the compound of the general formula (I-34) with trifluoromethanesulfonic anhydride under alkaline conditions to obtain a compound of the general formula (I-35);

tenth, the compound of formula (I-35) and the compound of formula (I-11) are reacted under basic conditions in the presence of a metal catalyst and a ligand to give the compound of formula (I).

Wherein PG is a protecting group, preferably benzyl; a is that ¹ 、A ² 、AR、R ¹ 、R ³ 、R ⁴ And L has the same defined range as above; l is preferably NH; a is that ¹ Preferably C; a is that ² Preferably CR ² (R ² preferably-OR ²¹ )。

Scheme IV

Firstly, oxidizing a compound methyl of a general formula (I-36) to obtain an acid compound of the general formula (I-37);

secondly, performing nitration reaction on the compound of the general formula (I-37) to obtain a compound of the general formula (I-38);

thirdly, reacting the compound shown in the general formula (I-38) with the compound shown in the general formula (I-11) under alkaline conditions to obtain a compound shown in the general formula (I-39);

fourthly, the compound of the general formula (I-39) is subjected to acyl chlorination and ammonia water reaction to obtain an amide compound of the general formula (I-40);

fifthly, reducing the nitro group of the compound of the general formula (I-40) to obtain a compound of the general formula (I-41);

sixth, the compound of formula (I-41) is reacted with triethyl formate to give the compound (R) of formula (I-42) ¹ ＝H)；

Seventh, the compound of the general formula (I-42) is subjected to halogenation reaction to obtain a compound of the general formula (I-43);

eighth, reacting the compound of the general formula (I-43) with the compound of the general formula (I-A) under alkaline conditions to obtain a compound of the general formula (I-44);

and a ninth step, reacting the compound of the general formula (I-44) with zinc cyanide in the presence of a metal catalyst to obtain the compound of the general formula (I).

Wherein X is ¹ 、X ² And X ³ Is halogen; x is X ¹ Preferably fluorine; x is X ² Bromine is preferred; x is X ³ Preferably chlorine; a is that ¹ 、A ² 、AR、R ¹ 、R ³ 、R ⁴ And L has the same defined range as above; a is that ¹ Preferably C; a is that ² Is CR (CR) ² (R ² Preferably cyano); l is preferably O; r is R ¹ Methyl is preferred.

In the case of the above-mentioned preparation method,

the reagent for providing alkaline conditions is selected from organic bases or inorganic bases, wherein the organic bases are one or more of triethylamine, N-diisopropylethylamine, N-butyllithium, lithium diisopropylamide, lithium bistrimethylsilylamino, sodium tert-butoxide, sodium methoxide and potassium tert-butoxide, and the inorganic bases are one or more of sodium hydride, potassium phosphate, sodium carbonate, potassium acetate, cesium carbonate, sodium hydroxide, potassium hydroxide, sodium bicarbonate and lithium hydroxide;

the reagent providing the acidic condition is one or more of hydrogen chloride, 1, 4-dioxane solution of hydrogen chloride, methanol solution of hydrogen chloride, trifluoroacetic acid, formic acid, acetic acid, hydrochloric acid, sulfuric acid, methanesulfonic acid, nitric acid and phosphoric acid;

the metal catalyst is palladium/carbon, raney nickel, tetra-triphenylphosphine palladium, palladium dichloride, palladium acetate, and [1,1' -bis (diphenylphosphino) ferrocene]Palladium dichloride (Pd (dppf) Cl) ₂ ) [1,1' -bis (diphenylphosphino) ferrocene]Palladium dichloride dichloromethane complex, ditriphenylphosphine palladium dichloride (Pd (PPh) ₃ )Cl ₂ ) And tris (dibenzylideneacetone) dipalladium (Pd) ₂ (dba) ₃ ) One or more of the following;

The ligand is one or more of 2-dicyclohexylphosphine-2, 6' -dimethoxybiphenyl (SPhos), 4, 5-bis-diphenylphosphine-9, 9-dimethylxanthene (Xantphos), 2-dicyclohexylphosphine-2, 4, 6-triisopropylbiphenyl (XPhos), 2-dicyclohexylphosphino-2 ' - (N, N-dimethylamine) -biphenyl (DavePhos), 1' -bis (diphenylphosphine) ferrocene (Dppf) and 1,1' -binaphthyl-2, 2' -bis-diphenylphosphine (BINAP), preferably 1,1' -binaphthyl-2, 2' -bis-diphenylphosphine (BINAP);

the reducing agent is one or more of sodium borohydride, potassium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride and lithium aluminum hydride;

the oxidant is one or more of potassium permanganate, manganese dioxide, potassium dichromate, sodium dichromate and potassium osmium;

the above reaction is preferably carried out in a solvent selected from one or more of N, N-dimethylformamide, N-methylpyrrolidone, dimethylsulfoxide, 1, 4-dioxane, water, tetrahydrofuran, methylene chloride, 1, 2-dichloroethane, methanol, ethanol, toluene, petroleum ether, ethyl acetate, N-hexane and acetone.

In a third aspect, the present invention provides a pharmaceutical composition comprising a polycyclic pyrimidine derivative according to the first aspect, a pharmaceutically acceptable salt thereof, a tautomer thereof, or a stereoisomer thereof; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient.

In the present invention, the term "comprising" means that the various ingredients may be applied together in a mixture or composition of the present invention. Thus, the terms "consisting essentially of and" consisting of are encompassed by the term "containing.

In the present invention, a "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio.

In the present invention, a "pharmaceutically acceptable carrier" is a pharmaceutically acceptable solvent, suspending agent or excipient for delivering the active substance of the present invention or a physiologically acceptable salt thereof to an animal or human. The carrier may be a liquid or a solid.

In the present invention, the pharmaceutical composition contains a safe and effective amount (e.g., 0.001 to 99.9 parts by weight, more preferably, 0.01 to 99 parts by weight, still more preferably, 0.1 to 90 parts by weight) of the compound represented by the formula (I) or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier or excipient, wherein the total weight of the composition is 100 parts by weight.

Alternatively, the pharmaceutical composition of the present invention contains 0.001 to 99.9wt%, more preferably 0.01 to 99 wt%, still more preferably 0.1 to 90 wt% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof based on the total weight; and a pharmaceutically acceptable carrier or excipient, wherein the total weight of the composition is 100 wt%.

In another preferred embodiment, the preferred ratio of the compound of formula (I) to the pharmaceutically acceptable carrier, excipient or slow release agent is such that formula (I) is present as active ingredient in an amount of more than 65% by weight, the remainder being present in an amount of 0.5 to 40% by weight, or more preferably 1 to 20% by weight, or most preferably 1 to 10% by weight.

The pharmaceutical compositions of the present invention comprise in unit dose form 0.05mg to 500mg, preferably 0.5mg to 200mg, more preferably 0.1mg to 100mg of the compound of formula (I), enantiomer, racemate, pharmaceutically acceptable salt or mixture thereof per dose.

When the pharmaceutical composition contains an additional pharmaceutically active ingredient for treating or preventing cancer, the amount of the active ingredient may be generally the conventional amount or less in the art.

The pharmaceutical compositions of the invention may be in a variety of forms, such as tablets, capsules, powders, syrups, solutions, suspensions, aerosols and the like, wherein the compounds of formula (I) may be presented in a suitable solid or liquid carrier or diluent. The pharmaceutical compositions of the present invention may also be stored in a suitable injectable or instillation disinfection device. The pharmaceutical composition may also contain odorants, flavoring agents, etc.

The compounds of formula (I) or pharmaceutical compositions comprising the compounds of formula (I) of the present invention may be administered to mammals, including humans, clinically by oral, nasal, dermal, pulmonary or gastrointestinal routes of administration. The preferred route of administration is oral. The preferred daily dosage is 0.5mg-200mg/kg body weight, administered once or in divided doses. Regardless of the method of administration, the optimal dosage for an individual will depend on the particular treatment. Typically starting from a small dose, the dose is gradually increased until the most appropriate dose is found.

The effective dose of the active ingredient used may vary with the compound used, the mode of administration and the severity of the condition to be treated. However, generally, satisfactory results are obtained when the compounds of the invention are administered at a dose of about 1-300mg/kg animal body weight per day, preferably at 1-3 divided doses per day, or in a sustained release form. For most large mammals, the total daily dose is about 5-1000mg, preferably about 10-500mg. Dosage forms suitable for oral administration comprise about 1-200mg of the active compound intimately mixed with a solid or liquid pharmaceutically acceptable carrier. This dosage regimen can be adjusted to provide the optimal therapeutic response. For example, separate doses may be administered several times per day, or the dose may be proportionally reduced, as dictated by the urgent need for the treatment of the condition.

The compounds or pharmaceutically acceptable salts thereof and compositions thereof may be administered orally as well as intravenously, intramuscularly or subcutaneously. Preferred pharmaceutical compositions are solid compositions, especially tablets and solid filled or liquid filled capsules, from the standpoint of ease of preparation and administration. Oral administration of the pharmaceutical composition is preferred.

The solid support comprises: starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose and kaolin, while the liquid carrier includes: sterile water, polyethylene glycols, non-ionic surfactants and edible oils (e.g., corn, peanut and sesame oils) as are appropriate to the nature of the active ingredient and the particular mode of administration desired. Adjuvants commonly used in the preparation of pharmaceutical compositions may also advantageously be included, for example flavouring agents, pigments, preservatives and antioxidants such as vitamin E, vitamin C, BHT and BHA.

The active compound or a pharmaceutically acceptable salt thereof and compositions thereof may also be administered parenterally or intraperitoneally. Solutions or suspensions of these active compounds (as the free base or pharmaceutically acceptable salt) may also be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquids, polyethylene glycols and mixtures thereof in oils. Under normal conditions of storage and use, these formulations contain preservatives to prevent microbial growth.

Pharmaceutical forms suitable for injection include: sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, these forms must be sterile and must be fluid to facilitate the discharge of the fluid from the syringe. Must be stable under the conditions of manufacture and storage and must be able to prevent the contaminating effects of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, alcohols (such as glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

The compounds of formula (I) or pharmaceutically acceptable salts and compositions thereof may also be administered in combination with other active ingredients or drugs for the treatment or prophylaxis of chronic pain disorders. When two or more drugs are administered in combination, there is generally an effect superior to the effect of the two drugs administered separately.

In a fourth aspect, the present invention provides the use of a polycyclic pyrimidine derivative according to the first aspect, a pharmaceutically acceptable salt thereof, a tautomer thereof, a stereoisomer thereof, or a pharmaceutical composition according to the third aspect, alone or in combination with a MEK signaling pathway inhibitor, in the manufacture of a medicament for the treatment of cancer or in the manufacture of a SOS1 inhibitor; preferably, the cancer includes, but is not limited to, astrocyte cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, hepatocellular cancer, laryngeal cancer, pancreatic cancer, lung cancer, oral cancer, ovarian cancer, prostate cancer, thyroid cancer, sarcoma, renal cancer, and cholangiocarcinoma; further preferably, the cancer includes, but is not limited to: pancreatic cancer, colorectal cancer, lung cancer, hepatocellular carcinoma, renal cancer, gastric cancer, and cholangiocarcinoma. And/or the MEK signaling pathway inhibitor such as RAF, KRAS, MEK or ERK1/2 inhibitor.

Preferably, the dosage of the polycyclic pyrimidine derivative according to the first aspect, the pharmaceutically acceptable salt thereof, the tautomer thereof, the stereoisomer thereof or the pharmaceutical composition according to the third aspect is in the range of 10-100mg/kg; and/or the dosage range of the MEK inhibitor is selected from 0.1-0.2mg/kg; preferably, the dosage range of the polycyclic pyrimidine derivative, the pharmaceutically acceptable salt thereof, the tautomer thereof, the stereoisomer thereof or the pharmaceutical composition is 25-50mg/kg; and/or the dosage range of the MEK inhibitor is selected from 0.125mg/kg; and/or the MEK inhibitor is Trametinib.

The use of the fourth aspect, wherein the cancer is a RAS family related cancer; preferably, the cancer is a KRAS, HRAS or NRAS G12C-related cancer, a KRAS, HRAS or NRAS G12D-related cancer, a KRAS, HRAS or NRAS G12A-related cancer, a KRAS, HRAS or NRAS G12V-related cancer, a KRAS, HRAS or NRAS G13D-related cancer, a KRAS, HRAS or nrg 13C-related cancer, a KRAS, HRAS or NRAS Q61X-related cancer, a KRAS, HRAS or NRAS a 146T-related cancer, a KRAS, HRAS or NRAS a 146V-related cancer or a KRAS, HRAS or NRAS a 146P-related cancer.

In another aspect, there is provided a method of treating cancer comprising providing to a cancer patient a therapeutically effective amount of formula (I), a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt thereof. In one embodiment, the cancer is a RAS family-related cancer.

The compositions and methods provided herein are useful for treating a variety of cancers, including prostate, breast, brain, skin, cervical cancer, testicular cancer, and the like. More specifically, cancers treatable by the compositions and methods of the invention include, but are not limited to, tumor types such as astrocyte, breast, cervical, colorectal, endometrial, esophageal, stomach, head-neck, hepatocellular, laryngeal, lung, oral, ovarian, prostate and thyroid cancers and sarcomas. More specifically, these compounds are useful in the treatment of: and (3) heart: sarcomas (hemangiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma, and teratoma; lung: bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondrioma, mesothelioma; gastrointestinal tract: esophageal (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), gastric (carcinoma, lymphoma, leiomyosarcoma), pancreatic (ductal adenocarcinoma, insulinoma, glucagon, gastrinoma, carcinoid, VIPoma), small intestine (adenocarcinoma, lymphoma, carcinoid, kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large intestine (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); urogenital tract: kidney (adenocarcinoma, nephroblastoma (nephroblastoma), lymphoma, leukemia), bladder and urinary tract (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); liver: liver cancer (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; biliary tract: gall bladder cancer, ampulla cancer, bile duct cancer; bone: osteosarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, ewing's sarcoma, malignant lymphoma (reticuloma), multiple myeloma, malignant giant cell tumor chordoma, bone chronicity tumor (osteochondrosarcoma), benign tumor, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumor; the nervous system: skull (bone tumor, hemangioma, granuloma, xanthoma, osteomyelitis), meninges (meningioma, glioma), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germ cell tumor (pineal tumor), glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, congenital tumor), spinal fibroma, meningioma, glioma, sarcoma; gynaecology: uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-cancerous cervical dysplasia), ovary (ovarian carcinoma (serous cystic adenocarcinoma, myxocyst adenocarcinoma, unclassified carcinoma), granulosa sheath cell carcinoma, backing cell carcinoma, germ cell carcinoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, cystoid sarcoma (embryonal rhabdomyosarcoma), fallopian tube (carcinoma), hematology (acute and chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), hodgkin's disease, non-hodgkin's lymphoma (malignant lymphoma), skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, kaposi's sarcoma, nevus, lipoma, hemangioma, skin fibroma, lupus erythematosus, psoriasis, and adrenal gland neuroblastoma).

In one embodiment, the cancer is a RAS family-related cancer, such as KRAS, NRAS, or HRAS-related cancer. In certain embodiments, the RAS family-related cancer is non-small cell lung cancer or pancreatic cancer. In one embodiment, the cancer is a cancer associated with SOS 1. In certain embodiments, the SOS 1-associated cancer is lung adenocarcinoma, embryonal rhabdomyosarcoma, support cell testicular tumor, and skin granulosa cell tumor.

Preferably, the cancer is pancreatic cancer, colorectal cancer, lung cancer, hepatocellular cancer, renal cancer, gastric cancer, or cholangiocarcinoma.

Interpretation of the terms

Unless stated to the contrary, some of the terms used in the specification and claims of the present invention are defined as follows:

as used herein, "KRAS G12C" refers to a mutant form of a mammalian KRAS protein that contains an amino acid that replaces glycine with a cysteine at amino acid position 12.

The determination of amino acid codons and residue positions for human KRAS is based on UniProtKB/SwissProt P01116: amino acid sequence of variant p.Gly12 Cys.

"KRAS G12D" refers to a mutant of a mammalian KRAS protein that contains an amino acid that replaces glycine at position 12 with aspartic acid. The amino acid codon and residue positions of human KRAS are according to UniProtKB/Swi-prot P01116: variant P.Gly12 Asp.

"KRAS G12V" refers to a mutant of a mammalian KRAS protein that contains an amino acid valine substituted for glycine at position 12. The amino acid codon and residue positions of human KRAS are according to UniProtKB/Swi-prot P01116: variant P.Gly12 Val.

"KRAS G12S" refers to a mutant of a mammalian KRAS protein comprising an amino acid having a serine substituted for glycine at amino acid position 12. The amino acid codon and residue positions of human KRAS are according to UniProtKB/SwissProt P01116: the amino acid sequence identified by variant p.Gly12Ser.

As used herein, "KRAS G12A" refers to a mutant form of a mammalian KRAS protein that contains an amino acid with alanine in place of glycine at amino acid position 12. The amino acid codon and residue positions of human KRAS are according to UniProtKB/SwissProt P01116: amino acid sequence identified by variant p.Gly12 Ala.

"KRAS G13D" refers to a mutant of a mammalian KRAS protein comprising an amino acid with an aspartic acid substituted for glycine at amino acid position 13. The determination of amino acid codons and residue positions for human KRAS is based on UniProtKB/SwissProt P01116: the amino acid sequence determined by variant p.gly 13 Asp.

"KRAS G13C" refers to a mutant of a mammalian KRAS protein that contains an amino acid with a cysteine at amino acid 13 in place of glycine. The amino acid codon and residue positions of human KRAS are according to UniProtKB/SwissProt-Prot P01116: the amino acid sequence of the variant P.Gly 13Cys is determined.

As used herein, "KRAS Q61L" refers to a mutant form of a mammalian KRAS protein that contains the amino acid leucine in place of glutamine at amino acid 61. The determination of amino acid codons and residue positions of human KRAS is based on the amino acid sequence of UniProtKB/SwissProt P01116: variant p.Gln61Leu.

As used herein, "KRAS A146T" refers to a mutant form of a mammalian KRAS protein that contains an amino acid that replaces alanine with threonine at amino acid position 146. The amino acid codon and residue positions of human KRAS are according to UniProtKB/SwissProt P01116: the amino acid sequence identified by variant p.al46thr.

As used herein, "KRAS A146V" refers to a mutant form of a mammalian KRAS protein that contains an amino acid with valine at amino acid position 146 in place of alanine. The amino acid codon and residue positions of human KRAS are according to UniProtKB/SwissProt P01116: amino acid sequence identified by variant p.al46Val.

As used herein, "KRAS A146P" refers to a mutant form of a mammalian KRAS protein that contains an amino acid with proline at amino acid position 146 in place of alanine. The amino acid codon and residue positions of human KRAS are according to UniProtKB/SwissProt P01116: the amino acid sequence identified by variant p.Al46Pro.

As used herein, "HRAS G12C" refers to a mutant form of mammalian HRAS protein which contains an amino acid which replaces glycine with a cysteine at amino acid position 12. According to UniProtKB/SwissProt P01112: amino acid sequences identified by variants p.Gly12 Cys, amino acid codons and residue positions of human HRAs were determined.

"HRAS G12D" refers to a mutant of a mammalian HRAS protein containing the amino acid substitution amino acid position 12 of aspartic acid-substituted glycine, the determination of the amino acid codon and residue position of human HRAS being based on UniProtKB/SwissProt P01112: amino acid sequence of variant p.gly 12 Asp.

"HRAS G12S" refers to a mutant of a mammalian HRAS protein containing an amino acid with serine substituted for glycine at 12. Amino acid codon and residue positions of human HRAS are according to UniProtKB/SwissProt P01112: variant P.Gly12Ser.

"HRAS G12A" refers to a mutant of a mammalian HRAS protein, the amino acid glycine being replaced with alanine at protein 12. The amino acid codon and residue positions of human KRAS are according to UniProtKB/SwissProt P01112: amino acid sequence identified by variant p.gly 12 Ala.

"HRAS G13D" refers to a mutant of mammalian HRAS protein, which contains an amino acid with aspartic acid substituted for glycine at position 13. Amino acid codon and residue positions of human HRAS are according to UniProtKB/Swi-prot P01112: variant p.gly 13 Asp.

"HRAS G13C" is a mutant of a mammalian HRAS protein, which contains a cysteine at amino acid 13 in place of the glycine amino acid. Determination of amino acid codon and residue positions for human HRAS is based on UniProtKB/SwissProt-Prot P01112: amino acid sequence of variant p.gly 13 Cys.

As used herein, "HRAS Q61L" refers to a mutant form of a mammalian HRAS protein comprising the amino acid leucine in place of glutamine at amino acid position 61, with amino acid position 61. The amino acid codons and residue positions of human HRAs were determined from the amino acid sequence identified by UniProtKB/SwissProt P01112 variant p.gln61leu.

As used herein, "HRAS A146T" refers to a mutant form of mammalian HRAS protein which contains an amino acid threonine at amino acid position 146 in place of alanine. Amino acid codon and residue positions of human NRAS are according to UniProtKB/SwissProt P01112: the amino acid sequence identified by variant p.Al46Thr.

As used herein, "HRAS A146V" refers to a mutant form of mammalian HRAS protein which contains the amino acid valine at amino acid position 146 in place of alanine. One amino acid replaces alanine at amino acid position 146. Amino acid codon and residue positions of human NRAS are according to UniProtKB/SwissProt P01112: the amino acid sequence identified by variant p.al46Val.

As used herein, "HRAS A146P" refers to a mutant form of mammalian HRAS protein which contains an amino acid with proline at amino acid position 146 in place of alanine. Amino acid codon and residue positions of human NRAS are according to UniProtKB/SwissProt P01112: variant p.Al46Pro identified amino acid sequence determination.

"NRAS G12C" refers to a mutant form of a mammalian NRAS protein that contains an amino acid that has a cysteine at amino acid 12 that replaces glycine. Amino acid codon and residue positions of human NRAS are according to UniProtKB/SwissProt-Prot P01111: variant p.Gly12cys.

"NRAS G12D" refers to a mutant of a mammalian NRAS protein that contains an amino acid that replaces glycine at position 12 with aspartic acid. Amino acid codon and residue positions of human NRAS are according to UniProtKB/Swi-prot P01111: variant P.Gly12 Asp.

"NRAS G12S" refers to a mutant of a mammalian NRAS protein that contains an amino acid that replaces glycine with serine at amino acid 12. Amino acid codon and residue positions of human NRAS are according to UniProtKB/SwissProt P01111: the amino acid sequence identified by variant p.Gly12Ser.

"NRAS G12A" refers to a mutant form of a mammalian NRAS protein that contains an amino acid that replaces glycine with alanine at amino acid 12. The determination of amino acid codons and residue positions for human KRAS is based on UniProtKB/SwissProt P01111: amino acid sequence of variant p.Gly12 Ala.

As used herein, "NRAS G13D" refers to a mutant form of a mammalian NRAS protein that contains an amino acid substitution of aspartic acid at amino acid 13 for glycine at the amino acid position. The determination of amino acid codons and residue positions for human NRAS is based on UniProtKB/SwissProt P01111: amino acid sequence determined by variant p.Gly13 Asp.

"HNras G13C" refers to a mutant form of a mammalian NRAS protein that contains an amino acid with a cysteine substituted for glycine at amino acid 13. The determination of amino acid codons and residue positions for human NRAS is based on UniProtKB/SwissProt P01111: amino acid sequence of variant p.gly 13 Cys.

As used herein, "HRAS Q61L" refers to a mutant form of a mammalian HRAS protein comprising the amino acid leucine in place of glutamine at amino acid 61, which has an amino acid position of 61. The amino acid codons and residue positions of human HRAs were determined from the amino acid sequence identified by UniProtKB/SwissProt P01112 variant p.gln61leu.

As used herein, "NRAS A146T" refers to a mutant form of a mammalian NRAS protein that contains an amino acid threonine at amino acid 146 instead of alanine. Amino acid codon and residue positions of human NRAS are according to UniProtKB/SwissProt P01111: the amino acid sequence identified by variant p.al46thr.

As used herein, "NRAS A146V" refers to a mutant form of a mammalian NRAS protein that contains an amino acid valine for alanine at amino acid 146. Amino acid codon and residue positions of human NRAS are according to UniProtKB/SwissProt P01111: the amino acid sequence identified by variant p.al46Val.

As used herein, "NRAS a146P" refers to a mutant form of a mammalian NRAS protein that contains an amino acid with proline at amino acid 146 in place of alanine. Amino acid codon and residue positions of human NRAS are according to UniProtKB/SwissProt P01111: the amino acid sequence identified by variant p.Al46Pro.

As used herein, "RAS family member" or "RAS family" refers to KRAS, HRAS, NRAS and its activating mutants, including at the G12, G13, Q61 and A146 positions.

As used herein, a 'RAS family related disease or disorder' refers to a disease or disorder associated with, mediated by or having an activating RAS mutation, such as RAS at the G12, G13, Q61 or a146 position. Non-limiting examples of RAS family related diseases or disorders are KRAS, HRAS or NRAS G12C related cancer, KRAS, HRAS or NRAS G12D related cancer, KRAS, HRAS or NRAS G12V related cancer, KRAS, HRAS or NRAS G12A related cancer, KRAS, HRAS or NRAS G13D related cancer, KRAS, HRAS or NRAS G13C related cancer, KRAS, HRAS or NRAS Q61L related cancer, KRAS, HRAS or NRAS a146T related cancer, KRAS, HRAS or NRAS a146V related cancer or KRAS, HRAS or nr146P related cancer.

As used herein, an "SOS 1-related disease or disorder" refers to a disease or disorder associated with, mediated by, or having an activated SOS1 mutation. Examples of activating SOS1 mutations include SOS1N233S and SOS1N233Y mutations.

As used herein, "SOS1N233S" refers to a mutant form of a mammalian SOS1 protein that contains an amino acid substitution of serine for glutamine at amino acid position 233. The determination of amino acid codon and residue positions of human SOS1 is based on UniProtKB/SwissProt Q07889: amino acid sequence identified as variant p.Gln2338er.

As used herein, "SOS1N233Y" refers to a mutant form of a mammalian SOS1 protein in which the amino acid containing tyrosine substituted glutamine has been substituted for amino acid 233. The determination of amino acid codon and residue positions of human SOS1 is based on UniProtKB/SwissProt Q07889: amino acid sequence identified by variant p.gln2335yr.

As used herein, "SOS1 inhibitor" refers to a compound of the present invention, as described herein, represented by formula (I). These compounds negatively inhibit all or part of the interaction of SOS1 with RAS family mutants or SOS1 activating mutations, thereby reducing and/or modulating the nucleotide exchange activity of RAS family member-SOS 1 complexes.

As used herein, "KRAS inhibitor" refers to inhibitors known in the art for various KRAS mutations. Inhibitors such as KRAS G12C, KRAS G12D, KRAS G12V may be targeted.

As used herein, an "MEK signaling pathway" inhibitor refers to inhibitors known in the art for RAF, MEK, and ERK 1/2.

"NF-1/NF-2 related disease or disorder" refers to a disease or disorder caused by a loss-of-function mutation in a neurocellulase (NF-1) gene or a neurocellulase 2 (NF-2) gene.

"loss-of-function mutation" as used herein refers to any point mutation, splice site mutation, fusion, nonsense mutation (amino acid mutation to stop codon), intra-frame mutation or frame transfer mutation, including insertions and deletions, as well as homozygous deletion of the gene encoding the protein in the cell of interest or cancer cell, resulting in partial or complete loss of presence, activity and/or function of the encoded protein.

"alkyl" refers to a saturated aliphatic hydrocarbon group comprising 1 to 20 carbon atoms, or 1 to 10 carbon atoms, or 1 to 6 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms, or 1 to 2 carbon atoms, saturated straight or branched chain monovalent hydrocarbon groups, wherein the alkyl groups may be independently optionally substituted with one or more substituents described herein. Examples of alkyl groups further include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl, and the like. The alkyl group may be optionally substituted or unsubstituted.

"alkenyl" refers to a straight or branched monovalent hydrocarbon radical of 2 to 12 carbon atoms, or 2 to 8 carbon atoms, or 2 to 6 carbon atoms, or 2 to 4 carbon atoms, wherein at least one C-C is sp ² Double bonds, in which the alkenyl groups may independently optionally be substituted by 1 or more substituents as described in the present inventionIncluding, but not limited to, vinyl, allyl, and allyl groups, and the like. Alkenyl groups may be optionally substituted or unsubstituted.

"cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring comprising 3 to 20 carbon atoms, preferably comprising 3 to 12 carbon atoms, more preferably comprising 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like; polycyclic cycloalkyl groups include spiro, fused and bridged cycloalkyl groups. Cycloalkyl groups may be optionally substituted or unsubstituted.

"spirocycloalkyl" refers to a 5 to 18 membered, two or more cyclic structure, and monocyclic polycyclic groups sharing one carbon atom (called spiro atom) with each other, containing 1 or more double bonds within the ring, but no ring has a completely conjugated pi-electron aromatic system. Preferably 6 to 14 membered, more preferably 7 to 10 membered. The spirocycloalkyl group is classified into a single spiro group, a double spiro group or a multiple spirocycloalkyl group according to the number of common spiro atoms between rings, preferably single spiro group and double spirocycloalkyl group, preferably 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered. Non-limiting examples of "spirocycloalkyl" include, but are not limited to:

"fused ring alkyl" refers to an all-carbon polycyclic group containing two or more cyclic structures sharing a pair of carbon atoms with each other, one or more of the rings may contain one or more double bonds, but none of the rings has a fully conjugated pi-electron aromatic system, preferably 6 to 12 members, more preferably 7 to 10 members. The number of constituent rings may be classified as a bicyclic, tricyclic, tetracyclic or polycyclic fused ring alkyl group, preferably a bicyclic or tricyclic, more preferably a 5-membered/5-membered or 5-membered/6-membered bicycloalkyl group. Non-limiting examples of "fused ring alkyl" include, but are not limited to:

"bridged cycloalkyl" means an aromatic system having 5 to 18 members, containing two or more cyclic structures, sharing two all-carbon polycyclic groups with one another that are not directly attached to a carbon atom, one or more of the rings may contain one or more double bonds, but none of the rings has a fully conjugated pi electron, preferably 6 to 12 members, more preferably 7 to 10 members. Cycloalkyl groups which may be classified as bicyclic, tricyclic, tetracyclic or polycyclic bridged according to the number of constituent rings are preferably bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of "bridged cycloalkyl" include, but are not limited to:

The cycloalkyl ring may be fused to an aryl, heteroaryl or heterocyclyl ring, wherein the ring attached to the parent structure is cycloalkyl, non-limiting examples include indanyl, tetrahydronaphthyl, benzocycloheptyl, and the like.

"heterocyclyl", "heterocycle" or "heterocyclic" are used interchangeably herein and refer to a saturated or partially unsaturated, monocyclic, bicyclic or tricyclic, non-aromatic heterocyclic group containing 3 to 12 ring atoms, at least one of which is a heteroatom such as oxygen, nitrogen, sulfur, and the like. Preferably having a 5 to 7 membered mono-or 7 to 10 membered bi-or tri-ring, which may contain 1,2 or 3 atoms selected from nitrogen, oxygen and/or sulphur. Examples of "heterocyclyl" include, but are not limited to, morpholinyl, oxetanyl, thiomorpholinyl, tetrahydropyranyl, 1-dioxo-thiomorpholinyl, piperidinyl, 2-oxo-piperidinyl, pyrrolidinyl, 2-oxo-pyrrolidinyl, piperazin-2-one, 8-oxa-3-aza-bicyclo [3.2.1] octyl, and piperazinyl. The heterocyclyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring attached to the parent structure is heterocyclyl. The heterocyclyl group may be optionally substituted or unsubstituted.

"spiroheterocyclyl" refers to a 5-to 18-membered, two or more cyclic structure, polycyclic group having single rings sharing one atom with each other, containing 1 or more double bonds within the ring, but no ring having a fully conjugated pi-electron aromatic system in which one or more ring atoms are selected from nitrogen, oxygen, sulfur or S (O) _m The remaining ring atoms are carbon, m=1 or 2. Preferably 6 to 14 membered, more preferably 7 to 10 membered. The spiroheterocyclyl groups are classified into a single spiroheterocyclyl group, a double spiroheterocyclyl group or a multiple spiroheterocyclyl group according to the number of common spiro atoms between rings, and preferably a single spiroheterocyclyl group and a double spiroheterocyclyl group. More preferably a 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered single spiro heterocyclic group. Non-limiting examples of "spiroheterocyclyl" include, but are not limited to:

"fused heterocyclyl" refers to an all-carbon polycyclic group containing two or more cyclic structures sharing a common pair of atoms with each other, one or more of the rings may contain one or more double bonds, but none of the rings has a fully conjugated pi-electron aromatic system in which one or more of the ring atoms is selected from nitrogen, oxygen, sulfur or S (O) _m The remaining ring atoms are carbon, m=1 or 2. Preferably 6 to 14 membered, more preferably 7 to 10 membered. The number of constituent rings may be classified as a bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic group, preferably a bicyclic or tricyclic, more preferably a 5-membered/5-membered or 5-membered/6-membered bicyclic fused heterocyclic group. Non-limiting examples of "fused heterocyclyl" include, but are not limited to:

"bridged heterocyclyl" means 5 to 18 membered, containingPolycyclic groups having two or more cyclic structures sharing two atoms not directly attached to each other, one or more of the rings may contain one or more double bonds, but none of the rings has a fully conjugated pi-electron aromatic system in which one or more of the ring atoms is selected from nitrogen, oxygen, sulfur, or S (O) _m The remaining ring atoms are carbon, m=1 or 2. Preferably 6 to 14 membered, more preferably 7 to 10 membered. Heterocyclic groups which may be classified as bicyclic, tricyclic, tetracyclic or polycyclic bridged according to the number of constituent rings are preferably bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of "bridged heterocyclyl" include, but are not limited to:

"aryl" refers to a carbocyclic aromatic system containing one or two rings, wherein the rings may be linked together in a fused manner. The term "aryl" includes aromatic groups such as phenyl, naphthyl, tetrahydronaphthyl. Preferably aryl is C ₆ -C ₁₀ Aryl, more preferably aryl is phenyl and naphthyl, most preferably phenyl. Aryl groups may be substituted or unsubstituted. The "aryl" may be fused to a heteroaryl, heterocyclyl, or cycloalkyl group, wherein the aryl ring is attached to the parent structure, non-limiting examples include, but are not limited to:

"heteroaryl" refers to an aromatic 5-to 6-membered monocyclic or 9-to 10-membered bicyclic ring, which may contain 1 to 4 atoms selected from nitrogen, oxygen and/or sulfur. Examples of "heteroaryl" include, but are not limited to, furyl, pyridyl, 2-oxo-1, 2-dihydropyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, thienyl, isoxazolyl, oxazolyl, oxadiazolyl, imidazolyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, 1,2, 3-thiadiazolyl, benzodioxolyl, benzimidazolyl, indolyl, isoindolyl, 1, 3-dioxo-isoindolyl, quinolinyl, indazolyl, benzisothiazolyl, benzoxazolyl and benzisoxazolyl. Heteroaryl groups may be optionally substituted or unsubstituted. The heteroaryl ring may be fused to an aryl, heterocyclyl, or cycloalkyl ring, wherein the ring attached to the parent structure is a heteroaryl ring, non-limiting examples include, but are not limited to:

"alkoxy" refers to a group of (alkyl-O-). Wherein alkyl is as defined herein. C (C) ₁ -C ₆ Is preferably selected. Examples include, but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy and the like.

"haloalkyl" refers to an alkyl group having one or more halo substituents, wherein the alkyl group has the meaning as described herein. Examples of haloalkyl include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, perfluoroethyl, 1-dichloroethyl, 1, 2-dichloropropyl, and the like.

"hydroxy" refers to an-OH group.

"halogen" means fluorine, chlorine, bromine and iodine, preferably fluorine, chlorine and bromine.

"amino" means-NH ₂ 。

"cyano" refers to-CN.

"nitro" means-NO ₂ 。

"benzyl" means-CH ₂ -phenyl.

"carboxy" means-C (O) OH.

"acetyl" means-C (O) CH ₃ Or Ac.

"carboxylate" refers to-C (O) O (alkyl) or (cycloalkyl), wherein alkyl, cycloalkyl are as defined above.

"optional" means that the event described may, but need not, occur. For example, "AR is optionally 1 to multiple R ^c Substitution the description includes that the AR group may be substituted with 1 to a plurality of R ^c Substituted or not by R ^c Substitution case.

"substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms in the group are independently substituted with a corresponding number of substituents. It goes without saying that substituents are only in their possible chemical positions, and that the person skilled in the art is able to determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, amino or hydroxyl groups having free hydrogen may be unstable when bound to carbon atoms having unsaturated (e.g., olefinic) bonds.

"substituted" or "substituted" as used herein, unless otherwise indicated, means that the group may be substituted with one or more groups selected from the group consisting of: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, alkenyl, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, amino, haloalkyl, hydroxyalkyl, carboxyl, carboxylate, =o, -C (O) R ^b 、-OC(O)R ^b 、-NR ^b R ^b 、-C(O)NR ^b R ^b 、-NR ^b C(O)R ^b 、-S(O)NR ^b R ^b or-S (O) ₂ NR ^b R ^b Wherein R is ^b The definition of (C) is as described in the general formula (I).

As used herein, the term "subject," "individual," or "patient" is used interchangeably to refer to any animal, including mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, primates, and humans. In some embodiments, the patient is a human. In some embodiments, the subject has experienced and/or displayed a disease or at least one symptom of a disease to be treated and/or prevented. In some embodiments, the subject has been determined or diagnosed as a cancer having a KRAS G12 or G13 mutation (e.g., as determined by an FDA-approved regulatory agency, such as an FDA-approved, assay, or kit). In some embodiments, the subject has a tumor positive for a KRAS G12C mutation, a KRAS G12D mutation, a KRAS G12S mutation, a KRAS G12V mutation, a KRAS G12A mutation, a KRAS G13D mutation, or a KRAS G13C mutation (e.g., as determined by regulatory agency-approved assays or kits). The subject may be a tumor patient with a KRAS G12C mutation, a KRAS G12D mutation, a KRAS G12V mutation, a KRAS G12A mutation, a KRAS G13D mutation, or a KRAS G13C mutation (e.g., approved regulatory bodies-such as FDA approved, analytical, or kit). The subject may be a subject whose tumor has a KRAS G12C mutation, a KRAS G12D mutation, a KRAS G12V mutation, a KRAS G12A mutation, a KRAS G13D mutation, or a KRAS G13C mutation (e.g., the tumor is determined by an FDA-approved regulatory agency, kit, or assay). In some embodiments, the subject is suspected of having a KRAS G12 or G13 gene-related cancer. In some embodiments, the subject has a clinical record indicating that the subject has a tumor with a KRAS G12C mutation (and optionally a clinical record indicating that the subject should be treated with any of the compositions provided herein).

The term "pediatric patient" as used herein refers to a patient less than 16 years of age at the time of diagnosis or treatment. The term "child" can also be divided into the following subclasses: neonates (from birth to first month of birth); infants (1 month to two years); children (2 to 12 years); teenagers (12 to 21 years old (up to but not including 22 years of birth)). Berhman RE, kliegman R, arvin AM, nelson we. Nelson textbook, 15 th edition. Philadelphia: w.b. samanders company, 1996; rudolph AM, et al. Pediatric version 21 of rudoffer. New York: mcGrow-Hill, 2002; and Avery MD, first LR. Pediatric medicine, second edition. Balm: williams & Wilkins;1994.

as used herein, an "effective amount" of a compound refers to an amount sufficient to down-regulate or inhibit SOS 1 enzymatic activity.

As used herein, a "therapeutically effective dose" of a compound refers to an amount sufficient to ameliorate or somehow reduce symptoms, stop or reverse progression of a disease, or down regulate or inhibit SOS 1 activity. Such doses may be administered as a single dose or may be administered according to a regimen so as to be effective.

As used herein, "treating" refers to ameliorating or otherwise altering the condition, disorder, or symptom or pathology of a disease in a patient in any manner.

As used herein, "ameliorating a symptom of a particular disease by use of a particular compound or pharmaceutical composition" refers to any reduction, whether permanent or temporary, persistent or temporary, attributable to or associated with the use of the composition.

The definition and use of stereochemistry in the present invention is generally referred to in the following documents:

S.P. Parker, ed., mcGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hillbook Company, new York; and Eliel, e.and Wilen, s., "Stereochemistry of Organic Compounds", john Wiley & Sons, inc., new York,1994. The compounds of the invention may contain asymmetric or chiral centers and thus exist as different stereoisomers. All stereoisomeric forms of the compounds of the invention, including, but in no way limited to, diastereomers, enantiomers, atropisomers, and mixtures thereof, such as racemic mixtures, form part of the present invention. Diastereomers can be separated into the individual diastereomers by chromatography, crystallization, distillation, or sublimation, based on their physical-chemical differences. Enantiomers may be converted into diastereomeric mixtures by separation by reaction with an appropriate optically active compound (e.g., a chiral auxiliary such as a chiral alcohol or Mosher's acid chloride), separating the diastereomers, and converting the individual diastereomers to the corresponding pure enantiomers. The intermediates and compounds of the invention may also exist in different tautomeric forms and all such forms are encompassed within the scope of the invention. Many organic compounds exist in optically active form, i.e. they have the ability to rotate the plane of plane polarized light. In describing optically active compounds, the prefix D, L or R, S is used to denote the absolute configuration of the chiral center of the molecule. The prefix d, l or (+), (-) is used to name the sign of the compound plane polarization rotation, where (-) or l means that the compound is left-handed and the prefix (+) or d means that the compound is right-handed. The atoms or groups of atoms of these stereoisomers are connected in the same order but in different steric structures. The particular stereoisomer may be an enantiomer, and the mixture of isomers is commonly referred to as an enantiomeric mixture. 50: mixtures of enantiomers of 50 are referred to as racemic mixtures or racemates, which may result in no stereoselectivity or stereospecificity during chemical reactions. The terms "racemic mixture" and "racemate" refer to a mixture of two enantiomers in equimolar amounts, lacking optical activity.

"tautomer" or "tautomeric form" refers to isomers of structures of different energies that can be interconverted by a low energy barrier. For example, proton tautomers (i.e., proton-shifted tautomers) include tautomerism by proton shift, such as keto-enol and imine-enamine isomerisation. Valency (valence) tautomers include tautomers that reorganize into bond electrons. Unless otherwise indicated, the structural formulae described herein include all isomeric forms (e.g., enantiomers, diastereomers, and geometric isomers): for example, R, S configuration containing asymmetric centers, the (Z), (E) isomers of double bonds, and the conformational isomers of (Z), (E). Thus, individual stereochemical isomers of the compounds of the invention, or enantiomers, diastereomers, or mixtures of geometric isomers thereof, are all within the scope of the invention.

By "pharmaceutically acceptable salts" is meant salts of the compounds of the present invention which are safe and effective when used in the human or animal body. Salts of the compounds may be obtained by dissolving the corresponding addition salts in pure solution or in a suitable inert solvent with sufficient amounts of base or acid. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic ammonia, magnesium salts, and the like, and pharmaceutically acceptable acid addition salts include inorganic and organic acid salts including hydrochloric acid, hydrobromic acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, monohydrogen sulfate, acetic acid, maleic acid, malonic acid, succinic acid, fumaric acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, methanesulfonic acid, and the like (see Berge et al, "Pharmaceutical Salts", journal of Pharmaceutical Science 66:1-19 (1977)).

The invention provides an SOS1 inhibitor with a novel structure, and test results show that the polycyclic pyrimidine derivative has excellent SOS1 inhibition activity and excellent safety and selectivity, and can be used for preparing medicines for treating cancers, especially pancreatic cancer, colorectal cancer, lung cancer, hepatocellular carcinoma, renal cancer, gastric cancer, cholangiocarcinoma and other diseases.

Drawings

FIG. 1 is a graph showing the effect of compounds according to the present invention on KRAS/ERK1/2 signaling pathways of K-562 cells.

FIG. 2 is a graph showing in vivo anti-tumor effects of SOS1 inhibitors according to the present invention on KRAS G12D mutant PANC-1 pancreatic xenograft tumor nude mice model alone.

FIG. 3 is a graph showing the effect of SOS1 inhibitors according to the present invention on the body weight of mice in a model of KRAS G12D mutant PANC-1 pancreatic xenograft tumor nude mice alone.

Figure 4 is a graph of in vivo anti-tumor effects of SOS1 inhibitors and Trametinib (Trametinib) according to the invention, alone or in combination, on KRAS G12C mutant MIAPaCa-2 pancreatic xenograft tumor nude mouse models.

Figure 5 is a graph of the effect of SOS1 inhibitor and Trametinib (Trametinib) according to the invention, alone or in combination, on the body weight of mice from a KRAS G12C mutant MIAPaCa-2 pancreatic xenograft tumor nude mouse model.

Detailed Description

The method of the present invention will be described by way of specific examples, so that the technical solution of the present invention can be understood and grasped more easily, but the present invention is not limited thereto. In the following examples ¹ The H NMR spectrum was determined with a Bruker instrument (400 MHz) and the chemical shifts were expressed in ppm. Using tetramethyl siliconInternal standard (0.00 ppm). ¹ H NMR representation method: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, br=broadened, dd=doublet of doublet, dt=doublet of triplet. If coupling constants are provided, they are in Hz.

The mass spectrum is measured by an LC/MS instrument, and the ionization mode is ESI.

High performance liquid chromatograph model: agilent 1260, siemens flying U3000; chromatographic column model: waters xbridge C18 (4.6. Times.150 mm,3.5 μm); mobile phase: ACN, B Water (0.1% H) ₃ PO ₄ ) The method comprises the steps of carrying out a first treatment on the surface of the Flow rate: 1.0mL/min; gradient: 5%A for 1min,increase to 20%A within 4min,increase to 80%A within 8min,80%A for 2min,back to 5%A within 0.1min; wavelength: 220nm; column incubator: 35 ℃.

The thin layer chromatography silica gel plate is a smoke table yellow sea HSGF254 or Qingdao GF254 silica gel plate, the specification of the silica gel plate used by the Thin Layer Chromatography (TLC) is 0.2mm-0.3mm, and the specification of the thin layer chromatography separation and purification product is 0.4mm-0.5mm.

Column chromatography generally uses tobacco stage yellow sea silica gel 200-300 mesh silica gel as carrier.

In the following examples, unless otherwise indicated, all temperatures are in degrees celsius and unless otherwise indicated, various starting materials and reagents are either commercially available or synthesized according to known methods, and are used without further purification, and unless otherwise indicated, commercially available manufacturers include, but are not limited to, the national pharmaceutical community, the carbofuran technologies, the tencel (Shanghai) chemical industry development limited, the Shanghai Pico pharmaceutical technologies limited, the Shanghai Michelson chemical technologies limited, and the like.

CD ₃ OD: deuterated methanol

CDCl ₃ : deuterated chloroform

DMSO-d ₆ : deuterated dimethyl sulfoxide

Pd ₂ (dba) ₃ : tris (dibenzylideneacetone) dipalladium

Pd(dppf)Cl ₂ : [1,1' -bis (diphenylphosphino) ferrocene]Palladium dichloride

XantPhos:4, 5-bis-diphenylphosphine-9, 9-dimethylxanthene

XPhos: 2-dicyclohexylphosphorus-2, 4, 6-triisopropylbiphenyl

HATU:2- (7-Oxybenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate

TLC: thin layer chromatography

HPLC: high performance liquid chromatography

purity: purity of

And (3) the following steps: and

the hydrogen atmosphere is defined as the reaction flask being connected to a hydrogen balloon of about 1L volume.

The examples are not particularly described, and the solution in the reaction is an aqueous solution.

The examples are not specifically described, and the reaction temperature is room temperature and is 20℃to 30 ℃.

The monitoring of the progress of the reaction in the examples employed Thin Layer Chromatography (TLC), the developing reagent used for the reaction, the system of eluent for column chromatography employed for purifying the compound or the developing reagent system of thin layer chromatography included: a: petroleum ether and ethyl acetate systems; b: methylene chloride and methanol systems; c: n-hexane: ethyl acetate; the volume ratio of the solvent is different according to the polarity of the compound, and can be adjusted by adding a small amount of acidic or alkaline reagent, such as acetic acid or triethylamine.

Preparation of intermediates

Intermediate 1

(R) -1- (3-nitro-5- (trifluoromethyl) phenyl) ethanamine IN-1

First step 1- (3-nitro-5- (trifluoromethyl) phenyl) ethan-1-one IN-1b

3-bromo-5-nitrobenzotrifluoride IN-1a (2.0 g,7.41 mmol), tributyl (1-ethoxyethylene) tin (3.5 g,9.69 mmol) and Pd (PPh) ₃ ) ₂ Cl ₂ (520 mg,0.74 mmol) was added sequentially to toluene (25 mL), heated to 100deg.C under nitrogen, and reacted overnight, TLC showed completion of the reaction. The reaction solution was cooled to room temperature, hydrochloric acid (15 ml,3 n) was added, stirring was performed for 30 minutes, celite was added, the filtrate was separated, the aqueous phase was extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by silica gel column chromatography to give the title compound IN-1b (1.25 g, yield 72%) as a yellow oil.

¹ H NMR(400MHz,CDCl ₃ )δ8.94(s,1H),8.68(s,1H),8.53(s,1H),2.75(s,3H).

Second step (R, Z) -2-methyl-N- (1- (3-nitro-5- (trifluoromethyl) phenyl) ethylene) propane-2-sulfinamide IN-1c

A mixture of compound IN-1b (1.25 g,5.36 mmol), (R) - (+) -tert-butylsulfinamide (974 mg,8.04 mmol) and tetraethyl titanate (10 mL,47.70 mmol) was heated to 80℃for 3 hours and the TLC showed little starting material remaining. The reaction solution was cooled to room temperature, poured into ice water (60 mL), extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was chromatographed on silica gel to give the title compound IN-1c (1.01 g, yield 56%) as a yellow oil.

Third step (R) -2-methyl-N- ((R) -1- (3-nitro-5- (trifluoromethyl) phenyl) ethyl) propane-2-sulfinamide IN-1d

Compound IN-1c (260 mg,0.77 mmol) was dissolved IN tetrahydrofuran (2.5 mL) and water (0.05 mL), cooled to-60℃and sodium borohydride (74 mg,1.95 mmol) was added IN portions, and after addition, stirring was continued for 1 hour at-60℃until TLC showed complete reaction. The reaction solution was quenched with water, extracted with ethyl acetate, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by silica gel column chromatography to give the title compound IN-1d (150 mg, yield 58%) as a white solid.

¹ H NMR(400MHz,CDCl ₃ )δ8.43-8.42(m,2H),7.95(s,1H),4.75-4.69(m,1H),3.55(d,J＝4.4Hz,1H),1.61(d,J＝6.8Hz,3H),1.25(s,9H).

Fourth step (R) -1- (3-nitro-5- (trifluoromethyl) phenyl) ethanamine IN-1

Compound IN-1d (164 mg,0.48 mmol) was dissolved IN tetrahydrofuran (3 mL), concentrated hydrochloric acid (0.5 mL) was added dropwise, and after the addition, the reaction was completed by TLC at room temperature for 1 hour. The reaction was then added dropwise to a saturated aqueous sodium carbonate solution to adjust ph=8, extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound IN-1 (105 mg, yield 92%) as a yellow oil.

LC-MS:m/z＝235.1[M+H] ⁺

Intermediate 2

(R) -3- (1-aminoethyl) -5- (difluoromethyl) -4-fluoroaniline IN-2

First step 1-bromo-3-difluoromethyl-2-fluorobenzene IN-2b

2-fluoro-3-bromobenzaldehyde IN-2a (10.0 g,49.26 mmol) was dissolved IN dichloromethane (200 mL), cooled to 0deg.C, diethylaminosulfur trifluoride (15.9 g,98.64 mmol) was slowly added dropwise, and after the dropwise addition, the mixture was slowly warmed to room temperature and stirred for 1 hour, and TLC showed complete reaction of the starting materials. The reaction solution was quenched by pouring into saturated aqueous sodium bicarbonate, extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by silica gel column chromatography to give the title compound IN-2b (8.1 g, yield 73%) as a yellow liquid.

¹ H NMR(400MHz,CDCl ₃ )δ7.68(t,J＝7.2Hz,1H),7.55(t,J＝7.2Hz,1H),7.14(t,J＝8.0Hz,1H),6.89(t,J＝54.8Hz,1H).

Second step 1- (3- (difluoromethyl) -2-fluoroacetophenone IN-2c

Compound IN-2b (8.1 g,36.00 mmol) was dissolved IN 1, 4-dioxane (80 mL), triethylamine (9.1 g,89.93 mmol) and tributyl (1-ethoxyethylene) tin (15.6 g,43.20 mmol) were added sequentially at room temperature, nitrogen was bubbled for 15 min, pd (PPh) was added ₃ ) ₂ Cl ₂ (250 mg,0.36 mmol), nitrogen substitution multiple times, heating to 100deg.C and stirring for 1 hr, TLC showed completion of the reaction. The reaction mixture was cooled to room temperature, and diluted hydrochloric acid (14.4 mL, 72.0) was addedmmol, 5M), stirred at room temperature for 1 hour, TLC indicated complete reaction of starting material. The reaction solution was diluted with water, extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by silica gel column chromatography to give the title compound IN-2c (5.6 g, yield 83%) as a yellow liquid.

¹ H NMR(400MHz,CDCl ₃ )δ8.00(t,J＝7.2Hz,1H),7.79(t,J＝6.8Hz,1H),7.34(t,J＝7.6Hz,1H),6.94(t,J＝54.8Hz,1H),2.67(d,J＝5.2Hz,3H).

Third step 1- (3- (difluoromethyl) -2-fluoro-5-nitrophenyl) ethan-1-one IN-2d

Potassium nitrate (45.6 g,0.45 mmol) was dissolved IN concentrated sulfuric acid (100 mL), stirred at room temperature for 30 min, cooled to about 0deg.C, and compound IN-2c (8.5 g,45.18 mmol) was slowly added dropwise, stirred at 0deg.C for 5 min, and TLC showed complete reaction. The reaction solution was slowly added to ice water, extracted with ethyl acetate, and the organic phases were combined, washed with saturated aqueous sodium bicarbonate, saturated brine, concentrated, and the crude product was purified by silica gel column to give the title compound IN-2d (9.0 g, yield 86%).

¹ H NMR(400MHz,CDCl ₃ )δ8.88-8.86(m,1H),8.66-8.64(m,1H),7.12-6.85(m,1H),2.73(d,J＝4.8Hz,3H).

Fourth step (R, Z) -N- (1- (3- (difluoromethyl) -2-fluoro-5-nitrophenyl) ethylene) -2-methylpropane-2-sulfinamide IN-2e

Compound IN-2d (9.0 g,38.60 mmol) was dispersed IN tetraethyltitanate (90 mL) and (R) - (+) -tert-butylsulfinamide (7.0 g,57.76 mmol) was added at room temperature and slowly warmed to 100deg.C and stirred for 1 hour, TLC indicated complete reaction of starting materials. The reaction solution was cooled to room temperature, poured into vigorously stirred ice water, stirred continuously for 10 minutes, filtered, the filter cake was repeatedly washed with ethyl acetate, the filtrate extracted with ethyl acetate, the organic phases were combined, saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound IN-2e (9.0 g, crude) as a yellow oil, which was used directly IN the next step.

LC-MS:m/z＝337.1[M+H] ⁺

Fifth step (R) -N- (1- (3- (difluoromethyl) -2-fluoro-5-nitrophenyl) ethyl) -2-methylpropane-2-sulfinamide IN-2f

Compound IN-2e (9.0 g, crude) was dissolved IN tetrahydrofuran (150 mL) and water (2 mL), cooled to about-60℃and sodium borohydride (3.1 g,81.95 mmol) was added IN portions, after which the reaction was continued at room temperature for 2 hours with slow warming, and TLC indicated that the starting material was consumed. The reaction solution was poured into ice water to quench, extracted with ethyl acetate, and the organic phases were combined, washed with water, saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by silica gel column to give the title compound IN-2f (2.8 g, two-step yield 21%) as a brown oil.

LC-MS:m/z＝339.1[M+H] ⁺

Sixth step (R) -1- (3- (difluoromethyl) -2-fluoro-5-nitrophenyl) ethan-1-amine IN-2g

Compound IN-2f (2.8 g,8.28 mmol) was dissolved IN tetrahydrofuran (30 mL), concentrated hydrochloric acid (1.4 mL,16.52 mmol) was added and stirred at room temperature for 1 hour, TLC showed starting material to be consumed. The reaction solution was cooled to room temperature, saturated aqueous sodium hydrogencarbonate solution was then added to adjust the pH to alkaline, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate and concentrated to give a brown liquid, which solidified upon standing to give the title compound IN-2g (1.8 g, yield 95%).

LC-MS:m/z＝235.1[M+H] ⁺

¹ H NMR(400MHz,DMSO-d ₆ )δ8.76-8.74(m,1H),8.41-8.39(m,1H),7.52-7.25(m,1H),4.42(q,J＝6.4Hz,1H),2.48-2.24(m,2H),1.35(d,J＝6.8Hz,3H).

Seventh step (R) -3- (1-aminoethyl) -5- (difluoromethyl) -4-fluoroaniline 2e

Compound IN-2g (300 mg,1.28 mmol) was dissolved IN ethanol and water (6 mL/2 mL), reduced iron powder (356 mg,6.41 mmol) and ammonium chloride (346 mg,6.69 mmol) were added at room temperature, and the reaction was heated to 90℃for 2 hours, and TLC detection showed complete reaction. The reaction solution was cooled to room temperature, filtered through celite, and the filtrate was concentrated to give the title compound IN-2 (350 mg, crude) which was used directly IN the next step.

Intermediate 3

(R) -1- (3- (difluoromethyl) -2-fluorophenyl) ethanamine IN-3

First step (R, Z) -N- (1- (3- (difluoromethyl) -2-fluorophenyl) ethylene) -2-methylpropane-2-sulfinamide IN-3a

Compound IN-2c (5.6 g,29.76 mmol) was dissolved IN tetraethyltitanate (50 mL) and (R) - (+) -tert-butylsulfinamide (10.8 g,89.11 mmol) was added at room temperature and slowly warmed to 100deg.C and stirred for 1 hour, TLC indicated complete reaction of starting materials. The reaction solution was cooled to room temperature, poured into vigorously stirred ice water, stirred continuously for 10 minutes, filtered, the filter cake was repeatedly washed with ethyl acetate, the filtrate extracted with ethyl acetate, the organic phases were combined, saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound IN-3a (9.5 g, crude) as a yellow oil, which was used directly IN the next step.

LC-MS:m/z＝292.1[M+H] ⁺

Second step (R) -N- ((R) -1- (3- (difluoromethyl) -2-fluorophenyl) ethyl) -2-methylpropane-2-sulfinamide IN-3b

Compound IN-3a (9.5 g, crude) was dissolved IN tetrahydrofuran (100 mL) and water (2 mL), cooled to about-60℃and sodium borohydride (3.7 g,97.80 mmol) was added IN portions, after which the reaction was continued at room temperature for 2 hours with slow warming to TLC indicating depletion of the starting material. The reaction solution was quenched by pouring ice water, extracted with ethyl acetate, and the organic phases were combined, washed with water, saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by silica gel column chromatography to give the title compound IN-3b (2.9 g, two-step yield 33%) as a yellow oil.

LC-MS:m/z＝294.1[M+H] ⁺

Third step (R) -1- (3- (difluoromethyl) -2-fluorophenylethylamine IN-3

Compound IN-3b (2.9 g,9.88 mmol) was dissolved IN tetrahydrofuran (50 mL) and concentrated hydrochloric acid (720 mg,19.8mmol, 12N) was added at room temperature, and the mixture was stirred for 1 hour at 80℃until TLC indicated that the starting material was consumed. The reaction solution was cooled to room temperature, saturated aqueous sodium hydrogencarbonate solution was then added to adjust the pH to alkaline, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound IN-3 (2.0 g, crude) as a brown liquid, which was used directly IN the next step.

LC-MS:m/z＝190.1[M+H] ⁺

¹ H NMR(400MHz,CDCl ₃ )δ7.58(t,J＝7.4Hz,1H),7.47(t,J＝6.8Hz,1H),7.23(t,J＝7.6Hz,1H),6.90(t,J＝55.2Hz,1H),4.45(q,J＝6.8Hz,1H),1.80(s,2H),1.43(d,J＝6.4Hz,3H).

Intermediate 4

(S) -2- ((tetrahydrofuran-3-yl) oxy) acetic acid ethyl ester IN-4

First step (S) -tetrahydrofuran-3-yl-4-nitrobenzoate IN-4b

(R) - (-) -3-hydroxytetrahydrofuran IN-4a (10.0 g,0.113 mol) was dissolved IN tetrahydrofuran (130 mL) under nitrogen, nitrobenzoic acid (18.9 g,0.113 mol) and triphenylphosphine (35.6 g,0.136 mol) were added at room temperature, the temperature was lowered to 0℃and a solution of diisopropyl azodicarboxylate (27.8 g,0.137 mol) IN tetrahydrofuran (20 mL) was added dropwise, and the reaction was completed at room temperature by TLC, indicating that the starting material was reacted for 2 hours. The reaction mixture was extracted with water and ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by silica gel column chromatography to give the title compound IN-4b (29.8 g, crude product, containing a small amount of phosphorus oxychloride) as a white solid.

Second step (S) -3-hydroxytetrahydrofuran IN-4c

Compound IN-4b (29.8 g, crude) was dissolved IN ethanol (300 mL) and sodium carbonate (25.0 g,0.236 mol) was added and heated to 85℃for 2 hours, and TLC showed complete reaction of starting material. The reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated, and the crude product was chromatographed on silica gel to give the title compound IN-4c (8.1 g, 81% IN two steps).

Third step (S) -2- ((tetrahydrofuran-3-yl) oxy) acetic acid tert-butyl ester IN-4d

Compound IN-4c (3.0 g,34.05 mmol) was dissolved IN toluene (40 mL), tetrabutylammonium bromide (3.0 g,9.30 mmol) and aqueous sodium hydroxide (30 mL,360mmol,12 mol/L) were added, tert-butyl bromoacetate (9.9 g,50.75 mmol) was added with stirring at room temperature, and stirring overnight at 20℃was complete, and TLC showed complete reaction. The reaction mixture was extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was chromatographed on a silica gel column to give the title compound IN-4d (5.5 g, yield 80%) as a yellow oil.

¹ H NMR(400MHz,CDCl ₃ )δ4.22-4.25(m,1H),3.97(s,2H),3.80-3.95(m,4H),1.96-2.09(m,2H),1.48(s,9H).

Fourth step (S) -2- ((tetrahydrofuran-3-yl) oxy) acetic acid IN-4e

Compound IN-4d (5.5 g,27.19 mmol) was dissolved IN dichloromethane (15 mL) and trifluoroacetic acid (30 mL) was added and heated to 45℃for 2 hours, and TLC showed substantial completion. The reaction solution was concentrated (dichloromethane with 3 times) to give the title compound IN-4e (13.5 g, crude) as a pale yellow oil, which was used directly IN the next step.

Fifth step (S) -2- ((tetrahydrofuran-3-yl) oxy) ethyl acetate IN-4

Compound IN-4e (13.5 g, crude) was dissolved IN ethanol (100 mL), concentrated sulfuric acid (30 mL) was added at room temperature, and the mixture was heated to 85℃for reaction for 5 hours. The reaction solution was cooled to room temperature, poured into brine ice, extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated, and the crude product was chromatographed on silica gel to give the title compound IN-4 (2.1 g, two-step yield 44%) as a yellowish oil.

¹ H NMR(400MHz,CDCl ₃ )δ4.29-4.18(m,3H),4.08(s,2H),3.96-3.79(m,4H),2.09-1.96(m,2H),1.29(t,J＝7.2Hz,3H).

Intermediate 5

R) - (3- (1-aminoethyl) -5- (difluoromethyl) -4-fluorophenyl) carbamic acid tert-butyl ester IN-5

First step 3-bromo-2-fluoro-5-nitrobenzoic acid IN-5b

Concentrated sulfuric acid (60 mL) was cooled to 0deg.C, 2-fluoro-5-nitrobenzoic acid IN-5a (10.0 g,54.0 mmol) was added, dibromohydantoin (9.30 g,32.4 mmol) was added after dissolution, and the reaction was allowed to proceed to room temperature overnight, and TLC indicated complete reaction of starting materials. The reaction solution was poured into ice water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound IN-5b (15.0 g, crude) as a yellow solid, which was used directly IN the next step.

Second step (3-bromo-2-fluoro-5-nitrophenyl) methanol IN-5c

Compound IN-5b (15.0 g, crude) was dissolved IN tetrahydrofuran (100 mL), cooled to 0deg.C under nitrogen protection, borane tetrahydrofuran solution (85.3 mL,85.3mmol, 10M) was added dropwise, and the reaction was allowed to warm to room temperature overnight, with complete TLC. The reaction solution was cooled to 0℃and quenched with methanol dropwise until no gas was generated, concentrated, and the residue was dissolved IN ethyl acetate, washed with water, saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound IN-5c (14.5 g, crude) as a pale yellow solid, which was used directly IN the next step.

Third step (5-amino-3-bromo-2-fluorophenyl) methanol IN-5d

Compound IN-5c (14.5 g, crude) and stannous chloride (44.0 g,232 mmol) were dissolved IN ethyl acetate (200 mL), cooled to 0deg.C, concentrated hydrochloric acid (9.70 mL,116mmol, 12M) was added dropwise, and the mixture was stirred for 3 hours at room temperature after natural warming, and TLC detection was complete. The reaction solution was quenched with ice water, adjusted ph=14 with aqueous sodium hydroxide (2N), extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound IN-5d (13.5 g, crude) as a pale yellow solid, which was used directly IN the next step.

LC-MS:m/z＝220.0[M+H] ⁺

Fourth step (3-bromo-4-fluoro-5- (hydroxymethyl) phenyl) carbamic acid tert-butyl ester IN-5e

Compound IN-5d (12.8 g, crude) was dissolved IN N, N-dimethylformamide (100 mL), di-tert-butyl dicarbonate (25.3 g,116 mmol) was added at room temperature, and the mixture was stirred for 3 hours at 60℃and the reaction was complete by TLC. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated to give the title compound IN-5e (18.6 g, crude product) as a pale yellow solid, which was used directly IN the next step.

LC-MS:m/z＝318.0[M-H] ^-

Fifth step (3-bromo-4-fluoro-5-formylphenyl) carbamic acid tert-butyl ester IN-5f

Compound IN-5e (18.6 g, crude) was dissolved IN dichloromethane (200 mL), cooled to 0deg.C, and was reacted at room temperature for 2 hours with the addition of dess-Martin reagent (36.9 g,87.0 mmol) and TLC checked for complete reaction of starting material. The reaction solution was cooled to 0℃and quenched with saturated aqueous sodium bicarbonate, extracted with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated to give the title compound IN-5f (18.4 g, crude) as a yellow solid, which was used directly IN the next step.

Sixth step (3-bromo-5- (difluoromethyl) -4-fluorophenyl) carbamic acid tert-butyl ester IN-5g

Compound IN-5f (18.4 g, crude product) was dissolved IN dichloromethane (200 mL), cooled to 0deg.C, diethylaminosulfur trifluoride (19.6 g,122 mmol) was added dropwise, the reaction was continued for 1 hour at 0deg.C, and then warmed to room temperature for 2 hours, and TLC detection was complete. The reaction mixture was quenched with water, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product purified by silica gel column chromatography to give the title compound IN-5g (19.2 g, crude product) as a yellow solid, which was used directly IN the next reaction.

Seventh step (3-acetyl-5- (difluoromethyl) -4-fluorophenyl) carbamic acid tert-butyl ester IN-5h

The compound IN-5g (19.2 g, crude product), tributyl (1-ethoxyethylene) tin (20.1 g,56.5 mmol) and ditriphenylphosphine palladium dichloride (793 mg,1.13 mmol) were added sequentially to toluene (100 mL), warmed to 100deg.C under nitrogen protection and stirred overnight, and TLC detected the disappearance of starting material. The reaction was cooled to room temperature, diluted hydrochloric acid (50 mL, 2N) was added and stirred for 3 hours, and TLC showed complete reaction. The reaction mixture was extracted with water and ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by silica gel column chromatography to give the title compound IN-5h as a yellow solid (9.1 g, yield 52% over seven steps).

Eighth step (R, Z) - (3- (1- ((tert-butylsulfinyl) imino) ethyl) -5- (difluoromethyl) -4-fluorophenyl) carbamic acid tert-butyl ester IN-5i

Compound IN-5h (5.50 g,18.1 mmol) and t-butylsulfinamide (3.30 g,27.2 mmol) were dissolved IN tetrahydrofuran (80 mL), isopropyl titanate (10.3 g,36.3 mmol) was added at room temperature, warmed to 80℃and stirred overnight, and TLC detection was complete. The reaction solution was cooled to room temperature, quenched with water, filtered to remove insoluble materials, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by silica gel column chromatography to give the title compound IN-5i (4.60 g, yield 62%) as a yellow solid.

Ninth step (3- ((R) -1- (((R) -tert-butylsulfinyl) amino) ethyl) -5- (difluoromethyl) -4-fluorophenyl) carbamic acid tert-butyl ester IN-5j

Compound IN-5i (4.60 g,11.3 mmol) was dissolved IN tetrahydrofuran (50 mL) and water (3 mL), cooled to-20deg.C, sodium borohydride (850 mg,22.6 mmol) was added, and the mixture was stirred for 1 hour at room temperature by natural heating, and the reaction was complete by TLC. The reaction solution was quenched with water, extracted with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by silica gel column chromatography to give the title compound IN-5j (1.92 g, yield 42%) as a yellow oil.

Tenth step (R) - (3- (1-aminoethyl) -5- (difluoromethyl) -4-fluorophenyl) carbamic acid tert-butyl ester IN-5

Compound IN-5j (1.92 g,4.70 mmol) was dissolved IN tetrahydrofuran (20 mL), concentrated hydrochloric acid (0.80 mL,9.40mmol, 12N) was added and stirred at room temperature for 1 hour, and TLC detection was complete. The reaction solution was quenched with water, neutralized with saturated aqueous sodium bicarbonate, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the crude title compound IN-5 (1.41 g, yield 99%) as a yellow oil by purification on a silica gel column.

LC-MS:m/z＝305.1[M+H] ⁺

¹ H NMR(400MHz,CDCl ₃ )δ7.64-7.57(m,1H),7.54-7.46(m,1H),6.83(t,J＝54.8Hz,1H),6.80(br,1H),4.43(q,J＝6.8Hz,1H),2.51(s,2H),1.50(s,9H),1.43(d,J＝6.8Hz,3H).

Intermediate 6

(R) - (3- (1-aminoethyl) -5- (difluoromethyl) -4-fluorophenyl) carbamic acid isopropyl ester IN-6

First step 1- (5-amino-3- (difluoromethyl) -2-fluorophenyl) ethan-1-one IN-6a

Compound IN-5h (3.0 g,9.89 mmol) was dissolved IN dichloromethane (30 mL) and trifluoroacetic acid (10 mL) was added and reacted at room temperature for 3 h, and the reaction was complete by TLC. The reaction solution was concentrated, and the residue was neutralized with saturated aqueous sodium carbonate, extracted with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound IN-6a (2.5 g, crude) as a yellow oil, which was used directly IN the next step.

Second step benzyl (3-acetyl-5- (difluoromethyl) -4-fluorophenyl) carbamate IN-6b

Compound IN-6a (2.5 g, crude) was dissolved IN tetrahydrofuran (20 mL) and water (8 mL), sodium carbonate (5.2 g,49.06 mmol) was added, benzyl chloroformate (2.0 g,11.72 mmol) was added dropwise, and the reaction was completed by TLC at room temperature for 4 hours. The reaction mixture was extracted with water and ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by silica gel column chromatography to give the title compound IN-6b (2.2 g, 67% yield IN two steps) as a yellow solid.

LC-MS:m/z＝393.1[M+H] ⁺

Third step (R, Z) - (3- (1- ((tert-butylsulfinyl) imino) ethyl) -5- (difluoromethyl) -4-fluorophenyl) carbamic acid isopropyl ester IN-6c

Compound IN-6b (2.2 g,6.52 mmol) and (R) - (+) -tert-butylsulfinamide (870 mg,7.18 mmol) were dissolved IN tetrahydrofuran (20 mL), tetraisopropyl titanate (9.2 g,32.37 mmol) was added at room temperature, heated to 70℃and reacted overnight, and TLC detection was essentially complete. The reaction solution was cooled to room temperature, ethyl acetate and water were then added thereto, stirring vigorously for half an hour, filtering, ethyl acetate extraction of the filtrate, washing with saturated brine, drying over anhydrous sodium sulfate, concentration, and purification of the crude product by silica gel column chromatography gave the title compound IN-6c (1.4 g, yield 55%) as a pale yellow solid.

Fourth step (3- ((R) -1- (((R) -tert-butylsulfinyl) amino) ethyl) -5- (difluoromethyl) -4-fluorophenyl) carbamic acid isopropyl ester IN-6d

Compound IN-6c (1.4 g,3.57 mmol) was dispersed IN tetrahydrofuran (15 mL) and water (1.5 mL), cooled to-60℃and sodium borohydride (6755 mg,17.85 mmol) was added IN portions, and after addition, the reaction was slowly warmed to room temperature for 2 hours and was complete by TLC. The reaction solution was quenched by pouring into ice water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the crude product as a pale yellow solid of the title compound IN-6d (710 mg, yield 50%) by column chromatography on silica gel.

Fifth step (R) - (3- (1-aminoethyl) -5- (difluoromethyl) -4-fluorophenyl) carbamic acid isopropyl ester IN-6

Compound IN-6d (710 mg,1.80 mmol) was dissolved IN tetrahydrofuran (10 mL), concentrated hydrochloric acid (0.2 mL,2.36mmol, 12N) was added and reacted at room temperature for 2 hours, and TLC detection was complete. The reaction solution was neutralized by adding saturated aqueous sodium carbonate, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by silica gel column chromatography to give the title compound IN-6 (495 mg, yield 95%) as a white solid.

LC-MS:m/z＝291.1[M+H] ⁺

¹ H NMR(400MHz,CDCl ₃ )δ7.68-7.53(m,2H),7.41-7.30(m,1H),6.83(t,J＝54.8Hz,1H),5.03-4.93(m,1H),4.47(q,J＝6.4Hz,1H),3.59(br,2H),1.45(d,J＝6.8Hz,3H),1.28(d,J＝6.4Hz,6H).

Example 1

N ⁴ - ((R) -1- (3-amino-5- (trifluoromethyl) phenyl) ethyl) -7-methoxy-2-methyl-N ⁶ - ((S) -tetrahydrofuran-3-yl) quinazoline-4, 6-diamine 1

First step 5-hydroxy-4-methoxy-2-nitrobenzoic acid 1b

2-nitro-4, 5-dimethoxybenzoic acid 1a (15.0 g,66.03 mmol) was dissolved in aqueous sodium hydroxide (60 mL, 6M) and the reaction was stirred for 3 hours at 100deg.C and the reaction was complete by TLC. The reaction solution was cooled to room temperature, pH was adjusted to 2-3 with hydrochloric acid, extracted with ethyl acetate, washed with brine, and concentrated after organic phase drying to give the title compound 1b (14.1 g, crude) as a yellow solid, which was used directly in the next step.

¹ H NMR(400MHz,DMSO-d ₆ )δ13.44(s,1H),10.76(s,1H),7.56(s,1H),7.08(s,1H),3.90(s,3H).

Second step 5-hydroxy-4-methoxy-2-nitrobenzoic acid methyl ester 1c

Compound 1b (14.1 g, crude product) was dissolved in methanol (150 mL), thionyl chloride (23.6 g,198.37 mmol) was added dropwise, and the reaction was stirred for 28 hours at 80℃and was detected to be complete by TLC. The reaction solution was cooled to room temperature, concentrated, and the residue was dissolved in ethyl acetate, washed with water, saturated brine, and dried in organic phase, and concentrated to give the title compound 1c (14.1 g, crude product) as a yellow solid, which was used directly in the next step.

Third step 5- (benzyloxy) -4-methoxy-2-nitrobenzoic acid methyl ester 1d

Compound 1c (14.1 g, crude) and benzyl bromide (12.7 g,74.26 mmol) were dissolved in dimethyl sulfoxide (100 mL), cesium carbonate (40.4 g,123.99 mmol) was added, stirred overnight at room temperature, and TLC was complete. The reaction was taken up in water, extracted with ethyl acetate, washed with brine, and the organic phase was dried and concentrated to give the title compound 1d (18.4 g, crude) as a yellow oil, which was used directly in the next step.

Fourth step 2-amino-5- (benzyloxy) -4-methoxybenzoic acid methyl ester 1e

Compound 1d (18.4 g, crude product) was dissolved in ethanol (200 mL) and water (40 mL), iron powder (16.2 g,0.29 mol) and ammonium chloride (15.5 g,0.29 mol) were added, and the reaction mixture was stirred for 2 hours at 90℃and was checked by TLC to be complete. The reaction solution was filtered through celite while it was hot, the filtrate was concentrated, the residue was dissolved in ethyl acetate, washed with brine, and the organic phase was dried and concentrated, and the residue was purified by silica gel column chromatography to give the title compound 1e (14.6 g, four-step yield 77%) as a yellow oil.

Fifth step 6- (benzyloxy) -7-methoxy-2-methyl-quinazolin-4 (3H) -one 1f

Compound 1e (1.0 g,3.48 mmol) was dissolved in acetonitrile (10 mL), 4M hydrochloric acid/1, 4-dioxane solution (20 mL, 4M) was added, and the reaction was warmed to 90℃and stirred overnight. The reaction solution was cooled to room temperature, and the precipitated precipitate was filtered to collect a solid. The solid was dissolved in water, the pH was adjusted to 8 with saturated aqueous sodium bicarbonate, the solid was precipitated, the solid was collected by filtration, and the solid was dried to give the title compound 1f (930 mg, crude) as a yellow solid, which was used directly in the next step.

LC-MS:m/z＝297.1[M+H] ⁺

Sixth step 6- (benzyloxy) -4-chloro-7-methoxy-2-methyl quinazoline 1g

Compound 1f (930 mg, crude) was dissolved in phosphorus oxychloride (20 mL), N-diisopropylethylamine (1.2 g,9.29 mmol) was added, and the reaction was stirred for 8 hours at 100℃until completion by TLC. The reaction solution was cooled to room temperature, concentrated, and the residue was dissolved in methylene chloride, quenched by dropwise addition of cooled aqueous sodium bicarbonate, washed with brine, and the organic phase was concentrated and purified by column chromatography to give the title compound as a brown solid (1 g, 400mg, two-step yield 36%).

LC-MS:m/z＝315.1[M+H] ⁺

Seventh step (R) -6- (benzyloxy) -7-methoxy-2-methyl-N- (1- (3-nitro-5- (trifluoromethyl) phenyl) ethyl) quinazolin-4-amine for 1h

1g (400 mg,1.27 mmol) of compound and IN-1 (328 mg,1.40 mmol) were dissolved IN dimethyl sulfoxide (10 mL), N-diisopropylethylamine (493 mg,3.81 mmol) was added, the reaction was heated to 80℃and stirred overnight, and the reaction was complete by TLC. The reaction was cooled to room temperature, water was then added, extracted with ethyl acetate, washed with brine, and the organic phase was dried and concentrated to give the title compound as a yellow solid for 1h (700 mg, crude) which was used directly in the next step.

Eighth step (R) -7-methoxy-2-methyl-4- ((1- (3-nitro-5- (trifluoromethyl) phenyl) ethyl) amino) quinazolin-6-ol 1i

The compound 1h (700 mg, crude) was dissolved in trifluoroacetic acid (6 mL) and the reaction was stirred for 2 hours at 80℃with complete reaction by TLC. The reaction was cooled to room temperature, concentrated, the residue was dissolved in dichloromethane, washed with water, and the organic phase was dried and concentrated to give the title compound 1i (577 mg, crude) as a yellow solid, which was used directly in the next step.

LC-MS:m/z＝423.2[M+H] ⁺

Ninth step (R) -7-methoxy-2-methyl-4- ((1- (3-nitro-5- (trifluoromethyl) phenyl) ethyl) amino) quinazolin-6-yl trifluoromethanesulfonate 1j

Compound 1i (577 mg, crude) was dissolved in dichloromethane (20 mL), pyridine (216 mg,2.73 mmol) and trifluoromethanesulfonic anhydride (578 mg,2.05 mmol) were added, and the reaction was stirred at room temperature for 3 hours, and the reaction was complete by TLC. The reaction mixture was extracted with dichloromethane, washed with brine, and the organic phase was concentrated and purified by silica gel column chromatography to give the title compound 1j (460 mg, three steps yield 65%) as a yellow oil.

LC-MS:m/z＝555.2[M+H] ⁺

Tenth step 7-methoxy-2-methyl-N ⁴ - ((R) -1- (3-nitro-5- (trifluoromethyl) phenyl) ethyl) -N ⁶ - ((S) -tetrahydrofuran-3-yl) quinazoline-4, 6-diamine 5l

Compound 1j (130 mg,0.23 mmol) and (S) -3-aminotetrahydrofuran 1k (61 mg,0.70 mmol) were dissolved in toluene (20 mL) and Pd was added ₂ (dba) ₃ (43 mg,0.047 mmol), BINAP (59 mg,0.095 mmol) and cesium carbonate (152 mg,0.47 mmol), the reaction was warmed to 100℃under nitrogen and stirred for 5 hours, and TLC detected the basic reaction of the starting materials. The reaction solution was cooled to room temperature, water was then added, extraction was performed with ethyl acetate, and the organic phase was dried and concentrated, and the residue was purified by silica gel column chromatography to give the title compound 1l (56 mg, yield 49%) as a yellow solid.

LC-MS:m/z＝492.2[M+H] ⁺

Eleventh step N ⁴ - ((R) -1- (3-amino-5- (trifluoromethyl) phenyl) ethyl) -7-methoxy-2-methyl-N ⁶ - ((S) -tetrahydrofuran-3-yl) quinazoline-4, 6-diamine 1

Compound 1k (56 mg,0.11 mmol) was dissolved in ethanol (6 mL) and water (1 mL), iron powder (32 mg,0.57 mmol) and ammonium chloride (30 mg,0.56 mmol) were added, and the reaction mixture was warmed to 90℃and stirred for 2 hours, and the reaction was complete by TLC. The reaction solution was filtered through celite while it was hot, the filtrate was concentrated, and the residue was purified by Prep-TLC to give the title compound 1 (26 mg, yield 49%) as a pale yellow solid.

LC-MS:m/z＝462.3[M+H] ⁺

¹ H NMR(400MHz,CD ₃ OD)δ7.20(s,1H),6.98(s,1H),6.97(s,1H),6.94(s,1H),6.80(s,1H),5.66(q,J＝7.2Hz,1H),4.35-4.29(m,1H),4.07-3.96(m,5H),3.91-3.85(m,1H),3.75-3.72(m,1H),2.47(s,3H),2.44-2.35(m,1H),1.99-1.92(m,1H),1.65(d,J＝7.2Hz,3H).(96.49％purity by HPLC)

Example 2

N- ((R) -1- (3-amino-5- (trifluoromethyl) phenyl) ethyl) -7-methoxy-2-methyl-6- (((S) -tetrahydrofurane-3-yl) oxy) pyrido [2,3-d ] pyrimidin-4-amine 2

First step 6-chloro-2-methyl-4-pyrimidinamine 2b

4, 6-dichloro-2-methylpyrimidine 2a (15.2 g,93.25 mmol) was added to an aqueous ammonia solution (150 mL, 25%) and the reaction was carried out at 65℃for 3 hours, with TLC indicating completion of the reaction of the starting material. The reaction solution was concentrated to give the title compound 2b (11.9 g, crude) as a white solid, which was used directly in the next step.

LC-MS:m/z＝144.1[M+H] ⁺

Second step 6-chloro-5-iodo-2-methyl-4-pyrimidinamine 2c

Compound 2b (11.9 g, crude) was added to acetic acid (120 mL) and N-iodosuccinimide (18.64 g,82.85 mmol) was added in portions at room temperature and heated to 70℃for 4 hours, and TLC showed complete reaction of starting material. The reaction solution was cooled to room temperature, saturated aqueous sodium hydrogencarbonate solution was then added to adjust to neutrality, the organic phases were combined, washed with saturated aqueous sodium hydrogencarbonate solution, aqueous sodium sulfite solution, saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound 2c (11.82 g, crude product) as a white solid, which was used directly in the next step.

LC-MS:m/z＝270.0[M+H] ⁺

Third step 6-chloro-2-methyl-5-vinyl-4-pyrimidinamine 2d

Compound 6c (18.0 g, crude product) was dissolved in ethylene glycol dimethyl ether (180 mL), and potassium vinyltrifluoroborate (17.89 g,0.13 mol), potassium phosphate (35.9 g,0.17 mol) and Pd (dppf) Cl were added sequentially at room temperature ₂ Dichloromethane complex (1.64 g,2.01 mmol), nitrogen displacement 3 times, temperature up to 80 ℃ for 3 hours, TLC indicated complete reaction of starting material. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with ethyl acetate, and the organic phases were combined, saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was subjected to silica gel column chromatography to give the title compound 2d (6.1 g, three-step yield 25%) as a white solid.

Fourth step 4-amino-6-chloro-2-methylpyrimidine-5-carbaldehyde 2e

Compound 2d (5.00 g,29.48 mmol) was dissolved in acetone and water (120 mL/30 mL) and sodium periodate (14.32 g,66.95 mmol) and aqueous potassium osmium sulfate (catalytic amount) were added and reacted at room temperature for 2 hours, TLC indicated complete reaction of starting materials. The reaction mixture was extracted with water and ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was chromatographed on silica gel to give the title compound 2e (2.5 g, yield 49%) as an off-white solid.

LC-MS:m/z＝172.1[M+H] ⁺

Fifth step (S) -4-chloro-2-methyl-6- ((tetrahydrofuran-3-yl) oxy) pyrido [2,3-d ] pyrimidin-7 (8H) -one 2f

Compound 2e (2.50 g,14.57 mmol) was dissolved IN tetrahydrofuran (500 mL) and intermediate IN-4 (3.30 g,18.94 mmol) was added at room temperature, cooled to-60℃and lithium diisopropylamide (22 mL,43.86mmol, 2.0M) was added dropwise and reacted at-60℃for 1 hour, slowly warmed to room temperature overnight. The reaction solution was quenched by addition of citric acid, extracted with ethyl acetate, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was chromatographed on a silica gel column to give the title compound 2f (1.0 g, yield 24%) as a pale yellow solid.

LC-MS:m/z＝282.1[M+H] ⁺

Sixth step (S) -4-chloro-7-methoxy-2-methyl-6- ((tetrahydrofuran-3-yl) oxy) pyrido [2,3-d ] pyrimidine 2g

Compound 2f (70 mg,0.25 mmol) was dissolved in chloroform (5 mL), silver carbonate (102 mg,0.37 mmol) and methyl iodide (704 mg,4.97 mmol) were added at room temperature, heated to 65℃and reacted overnight, and TLC showed some starting material remained. The reaction was cooled to room temperature, filtered, the filter cake washed with dichloromethane, the filtrate concentrated and the crude purified by Prep-TLC to give the title compound 2g (56 mg, 77% yield) as a white solid.

LC-MS:m/z＝296.1[M+H] ⁺

¹ H NMR(400MHz,CDCl ₃ )δ7.34(s,1H),5.06-5.01(m,1H),4.17(s,3H),4.17-3.97(m,3H),3.93-3.87(m,1H),2.77(s,3H),2.37-2.26(m,1H),2.23-2.15(m,1H).

Seventh step 7-methoxy-2-methyl-N- ((R) -1- (3-nitro-5- (trifluoromethyl) phenyl) ethyl) -6- (((S) -tetrahydrofurane-3-yl) oxy) pyrido [2,3-d ] pyrimidin-4-amine for 2h

2g (44 mg,0.15 mmol) of the compound was dissolved IN dimethyl sulfoxide (3 mL), N-diisopropylethylamine (58 mg,0.45 mmol) and compound IN-1 (38 mg,0.16 mmol) were added at room temperature, and the reaction was heated to 100℃for 10 hours, and TLC showed the starting material was complete. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with ethyl acetate, and the organic phases were combined, saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by Prep-TLC to give the title compound as a yellow solid for 2h (55 mg, yield 75%).

LC-MS:m/z＝494.2[M+H] ⁺

Eighth step N- ((R) -1- (3-amino-5- (trifluoromethyl) phenyl) ethyl) -7-methoxy-2-methyl-6- (((S) -tetrahydrofurane-3-yl) oxy) pyrido [2,3-d ] pyrimidin-4-amine 2

Compound 2h (55 mg,0.11 mmol) was dissolved in ethanol (10 mL) water (3 mL), reduced iron powder (31 mg,0.56 mmol) and ammonium chloride (28 mg,0.52 mmol) were added at room temperature and heated to 90℃for 3 h, and TLC showed complete reaction of starting material. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with ethyl acetate, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by Prep-TLC to give the title compound 2 (35 mg, yield 67%) as a white solid.

LC-MS:m/z＝464.3[M+H] ⁺

¹ H NMR(400MHz,CD ₃ OD)δ8.05(s,1H),7.01-6.95(m,2H),6.81(s,1H),5.68(q,J＝7.2Hz,1H),5.20-5.15(m,1H),4.11(s,3H),4.06-3.95(m,3H),3.94-3.90(m,1H),2.55(s,3H),2.41-2.31(m,1H),2.23-2.16(m,1H),1.68(d,J＝7.2Hz,3H).(99.74％purity by HPLC)

Example 3

N- ((R) -1- (5-amino-3- (difluoromethyl) -2-fluorophenyl) ethyl) -2-methyl-6- (((S) -tetrahydrofurane-3-yl) oxy) pyrido [3,4-d ] pyrimidin-4-amine 3

First step 2-chloro-5-nitroisonicotinic acid 3b

2-chloro-5-nitro-4-methylpyridine 3a (20 g,115.9 mmol) was dissolved in concentrated sulfuric acid (150 mL), chromium trioxide (28.97 g,289.7 mmol) was added in portions at room temperature and reacted overnight at room temperature, TLC showed completion of the reaction. The reaction solution was poured into ice water, stirred for 1 hour, filtered, and the filter cake was washed with water and dried to give the title compound 3b (21.6 g, crude) as a solid as an off-white solid, which was used directly in the next step.

Second step 2-chloro-5-nitroisonicotinamide 3c

Compound 3b (10.0 g,49.37 mmol) was heated to reflux in thionyl chloride (100 mL) for 3 h and TLC was complete. The reaction mixture was cooled to room temperature, concentrated, and the residue was dissolved in tetrahydrofuran (50 mL), slowly added dropwise to aqueous ammonia (100 mL) cooled to about 0deg.C, and reacted at 0deg.C for 0.5 hours after the addition, and the reaction was complete by TLC. The reaction solution was extracted with water and ethyl acetate, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound 3c (8.5 g, crude) as a yellow solid, which was used directly in the next step.

Third step (S) -5-nitro-2- ((tetrahydrofuran-3-yl) oxy) isonicotinamide 3d

Compound 3c (3.0 g, crude) was dissolved IN acetonitrile (30 mL), cesium carbonate (9.69 g,29.74 mmol) and S-3-hydroxytetrahydrofuran IN-4c (3.27 g,37.11 mmol) were added at room temperature, the temperature was raised to 30℃and reacted overnight, and TLC showed little starting material was unreacted. The reaction mixture was extracted with water and ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by silica gel column chromatography to give the title compound 3d as a yellow solid (1.67 g, three-step yield 35%).

LC-MS:m/z＝254.1[M+H] ⁺

Fourth step (S) -5-amino-2- ((tetrahydrofuran-3-yl) oxy) isonicotinamide 3e

Compound 3d (1.67 g,6.60 mmol) was dissolved in ethanol (30 mL) and water (10 mL), reduced iron powder (1.84 g,32.95 mmol) and ammonium chloride (1.69 g,31.59 mmol) were added at room temperature, and the reaction was heated to 90℃for 3 hours, and TLC showed complete reaction of starting material. The reaction solution was cooled to room temperature, filtered through celite, and the cake was washed with ethanol, the filtrate was concentrated, water was added, extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound 3e (1.38 g, crude) as a yellow solid, which was used directly in the next step.

LC-MS:m/z＝224.2[M+H] ⁺

Fifth step (S) -2-methyl-6- ((tetrahydrofuran-3-yl) oxy) pyrido [3,4-d ] pyrimidin-4-ol 3f

Compound 3e (300 mg, crude) was dissolved in triethyl orthoacetate (6 mL), and reacted at 120℃for 20 hours by TLC, indicating completion of the reaction of the starting material. The reaction was cooled to room temperature, filtered, and the filter cake washed with methyl tert-butyl ether and dried to give the title compound 3f (170 mg, crude) as a white solid, which was used directly in the next step.

LC-MS:m/z＝248.1[M+H] ⁺

Sixth step (S) -4-chloro-2-methyl-6- ((tetrahydrofuran-3-yl) oxy) pyrido [3,4-d ] pyrimidine 3g

Compound 3f (170 mg, crude) was suspended in 1, 4-dioxane (10 mL), N-diisopropylethylamine (355 mg,2.75 mmol) and phosphorus oxychloride (426 mg,2.75 mmol) were added at room temperature and reacted for 4 hours with heating to 100deg.C, TLC indicated complete reaction of starting materials. The reaction solution was cooled to 0℃and quenched with saturated aqueous sodium bicarbonate solution, extracted with ethyl acetate, the organic phases combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by silica gel column chromatography to give the title compound 3g as a pale yellow solid (150 mg, three-step yield 29%).

Seventh step N- ((R) -1- (5-amino-3- (difluoromethyl) -2-fluorophenyl) ethyl) -2-methyl-6- (((S) -tetrahydrofurane-3-yl) oxy) pyrido [3,4-d ] pyrimidin-4-amine 3

3g (65 mg,0.26 mmol) of the compound was dissolved IN dimethyl sulfoxide (3 mL), N-diisopropylethylamine (100 mg,0.77 mmol) and intermediate IN-2 (53 mg,0.26 mmol) were added at room temperature, and the reaction was heated to 80℃for 1.5 hours, and TLC showed complete reaction of the starting material. The reaction solution was cooled to room temperature, water was added, extraction was performed with ethyl acetate, the organic phases were combined, saturated brine was washed, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by Prep-TLC to give a yellow solid, which was purified by Prep-HPLC to give the title compound 3 (21 mg, yield 19%) as a pale yellow solid.

LC-MS:m/z＝434.2[M+H] ⁺

¹ H NMR(400MHz,CD ₃ OD)δ8.66(s,1H),7.58(s,1H),7.02-6.81(m,2H),6.78-5.68(m,1H),5.73(q,J＝7.2Hz,1H),5.65-5.657(m,1H),4.08-3.87(m,4H),2.43(s,3H),2.39-2.27(m,1H),2.22-2.13(m,1H),1.63(d,J＝7.2Hz,3H).(99.46％purity by HPLC)

Example 4

4- (((R) -1- (3-amino-5- (trifluoromethyl) phenyl) ethyl) amino) -2-methyl-6- (((S) -tetrahydrofurane-3-yl) oxy) quinazolin-7-carbonitrile 4

First step 4-bromo-3-fluorobenzoic acid 4b

3-fluoro-4-bromotoluene 4a (5.0 g,26.45 mmol) was dissolved in pyridine (20 mL) and water (20 mL), heated to 90℃and potassium permanganate (16.7 g,105.68 mmol) was added in portions over 1 hour and the reaction was continued for 2 hours with basic completion as detected by TLC. The reaction solution was filtered through celite, the filtrate was cooled, adjusted to ph=3 with concentrated hydrochloric acid, extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound 4b (3.2 g, crude) as a white solid, which was used directly in the next step.

Second step 4-bromo-5-fluoro-2-nitrobenzoic acid 4c

Compound 4b (3.2 g, crude) was dissolved in concentrated sulfuric acid (30 mL), potassium nitrate (1.7 g,16.82 mmol) was added in portions, and the reaction was completed by TLC at room temperature for 2 hours. The reaction solution was extracted with water and ethyl acetate, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound 4c (3.8 g, crude) as a pale yellow solid, which was used directly in the next step.

Third step (S) -4-bromo-2-nitro-5- ((tetrahydrofuran-3-yl) oxy) benzoic acid 4d

Compound 4c (3.8 g, crude) and compound IN-4c (3.8 g,43.13 mmol) were dissolved IN N, N-diisopropylethylamine (30 mL) and cesium carbonate (14.0 g,42.97 mmol) was added at room temperature and heated to 100deg.C for 4 hours and the basic reaction was complete as detected by TLC. The reaction solution was cooled to room temperature, diluted hydrochloric acid (3N) was adjusted to ph=3, extracted with ethyl acetate, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound 4d (5.2 g, crude) as a yellow solid, which was used directly in the next step.

LC-MS:m/z＝330.0[M-H] ^-

Fourth step (S) -4-bromo-2-nitro-5- ((tetrahydrofuran-3-yl) oxy) benzamide 4e

Compound 4d (5.2 g, crude) was dissolved in thionyl chloride (30 mL) and heated to 80deg.C for 3 hours. The reaction mixture was cooled to room temperature, concentrated, and the residue was dissolved in tetrahydrofuran (50 mL), added dropwise to aqueous ammonia (80 mL) cooled to about 0deg.C, and reacted at 0deg.C for 0.5 hours after the addition, and TLC was found to be complete. The reaction solution was diluted with water, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by silica gel column chromatography to give the title compound 4e (3.6 g, four-step yield 41%) as an off-white solid.

LC-MS:m/z＝331.1[M+H] ⁺

Fifth step (S) -2-amino-4-bromo-5- ((tetrahydrofuran-3-yl) oxy) benzamide 4f

Compound 4e (1.0 g,3.02 mmol) was dissolved in ethanol (20 mL) and water (4 mL), iron powder (676 mg,12.10 mmol) and ammonium chloride (806 mg,15.11 mmol) were added at room temperature, heated to 80℃and reacted for 3 hours, and the completion of the reaction was detected by LCMS. The reaction solution was cooled to room temperature, filtered through celite, and the filtrate was concentrated, extracted with water, ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound 4f (830 mg, crude) as a yellow solid, which was used directly in the next step.

LC-MS:m/z＝301.1[M+H] ⁺

Sixth step (S) -7-bromo-2-methyl-6- ((tetrahydrofuran-3-yl) oxy) quinazolin-4 (3H) -one 4g

Compound 4f (830 mg, crude) was dissolved in triethyl orthoformate (15 mL), heated to 150deg.C and reacted for 5 hours, and the reaction was complete by TLC. The reaction solution was cooled to room temperature, and a solid was precipitated and filtered to give a crude solid which was purified by silica gel column chromatography to give 4g (490 mg, 50% yield in two steps) of the title compound as an off-white solid.

LC-MS:m/z＝325.0[M+H] ⁺

Seventh step (S) -7-bromo-4-chloro-2-methyl-6- ((tetrahydrofuran-3-yl) oxy) quinazoline for 4h

4g (490 mg,1.51 mmol) of the compound was dispersed in 1, 4-dioxane (5 mL), pyridine (73 mg,0.92 mmol) and phosphorus oxychloride (143 mg,0.93 mmol) were added at room temperature, and the mixture was heated to 100℃for 2 hours to complete the reaction by TLC. The reaction solution was cooled to room temperature, water was then added thereto, neutralized with sodium hydrogencarbonate, extracted with ethyl acetate, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound as a yellow solid for 4h (510 mg, crude product) which was used directly in the next step.

Eighth step 7-bromo-2-methyl-N- ((R) -1- (3-nitro-5- (trifluoromethyl) phenyl) ethyl) -6- (((S) -tetrahydrofuranyl-3-yl) oxy) quinazolin-4-amine 4i

Compound 4h (80 mg, crude) and intermediate IN-1 (41 mg,0.18 mmol) were dissolved IN dimethyl sulfoxide (2 mL), N-diisopropylethylamine (46 mg,0.36 mmol) was added at room temperature, heated to 100deg.C and reacted for 2 hours, and TLC detection was complete. The reaction solution was cooled to room temperature, water was added, extraction was performed with ethyl acetate, the organic phases were combined, saturated brine was washed, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by Prep-TLC to give the title compound 4i as a yellow solid (70 mg, 74% yield in two steps).

LC-MS:m/z＝541.1[M+H] ⁺

Ninth step 2-methyl-4- (((R) -1- (3-nitro-5- (trifluoromethyl) phenyl) ethyl) amino) -6- (((S) -tetrahydrofuran-3-yl) oxy) quinazolin-7-carbonitrile 4j

Compound 4i (70 mg,0.13 mmol) was dissolved in N, N-diisopropylethylamine (3 mL), zinc cyanide (30 mg,0.26 mmol) and tetrakis triphenylphosphine palladium (30 mg,0.026 mmol) were added at room temperature, nitrogen was displaced 3 times, heated to 120℃and reacted overnight, and LCMS detected the starting material portion remaining. The reaction solution was cooled to room temperature, water was added, extraction was performed with ethyl acetate, the organic phases were combined, saturated brine was washed, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by Prep-TLC to give the title compound 4j (50 mg, crude product) as a yellow solid mixture, which was used directly in the next step.

Tenth step 4- (((R) -1- (3-amino-5- (trifluoromethyl) phenyl) ethyl) amino) -2-methyl-6- (((S) -tetrahydrofuran-3-yl) oxy) quinazolin-7-carbonitrile 4

Compound 4j (50 mg, crude) was dissolved in ethanol (5 mL) and water (1 mL), ammonium chloride (30 mg,0.56 mmol) and iron powder (30 mg,0.54 mmol) were added sequentially at room temperature, and the reaction was heated to 80℃for 3 hours, and the reaction was complete by TLC. The reaction solution was cooled to room temperature, filtered through celite, the filtrate was concentrated, the residue was extracted with water, ethyl acetate, the organic phases were combined, saturated brine was dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by Prep-TLC to give the title compound 4 (8 mg, 14% yield in two steps) as a yellow solid.

LC-MS:m/z＝458.2[M+H] ⁺

¹ H NMR(400MHz,CD ₃ OD)δ7.91(s,1H),7.86(s,1H),7.01-6.95(m,2H),6.81(s,1H),5.65(q,J＝7.2Hz,1H),5.33-5.27(m,1H),4.19-4.00(m,3H),3.99-3.93(m,1H),2.48(s,3H),2.43-2.31(m,1H),2.28-2.19(m,1H),1.67(d,J＝7.2Hz,3H).(96.53％purity by HPLC)

Example 5

N- ((R) -1- (3- (difluoromethyl) -2-fluorophenyl) ethyl) -2-methyl-6- ((S) -2-methylpiperazin-1-yl) pyrido [3,4-d ] pyrimidin-4-amine 5

First step (S) -4- (4-carbamoyl-5-nitropyridin-2-yl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester 5a

Compound 3c (2.0 g,9.92 mmol) was dissolved in dimethyl sulfoxide (10 mL), and (S) -4-N-t-butoxycarbonyl-2-methylpiperazine (2.2 g,10.98 mmol) and N, N-diisopropylethylamine (2.6 g,20.12 mmol) were added at room temperature and reacted at 80℃for 5 hours, followed by completion of the reaction by TLC. The reaction solution was cooled to room temperature, water was added, a solid was precipitated, and the filtrate was filtered, and the cake was washed and dried to give the title compound 5a (4.2 g, crude product) as a yellow solid, which was used directly in the next step.

LC-MS:m/z＝388.2[M+Na] ⁺

Second step (S) -4- (5-amino-4-carbamoylpyridin-2-yl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester 5b

Compound 5a (4.2 g, crude product) was dissolved in ethanol (80 mL) and water (15 mL), and ammonium chloride (2.6 g,48.61 mmol) and reduced iron powder (2.2 g,39.39 mmol) were added at room temperature and heated to 80℃for 3 hours to react essentially completely as detected by TLC. The reaction solution was filtered while it was hot through celite, the filtrate was concentrated, the residue was added with water, extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound 5b (3.5 g, crude) as a yellow solid, which was used directly in the next step.

LC-MS:m/z＝336.3[M+H] ⁺

(S) -4- (4-hydroxy-2-methylpyrido [3,4-d ] pyrimidin-6-yl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester 5c

Compound 5b (3.5 g, crude) and p-toluenesulfonic acid (30 mg,0.17 mmol) were dissolved in triethyl orthoacetate (30 mL), heated to 150deg.C and reacted for 5 hours, and TLC detected the completion of the reaction. The reaction solution was cooled to room temperature, diluted hydrochloric acid (20 ml,1 n) was added, stirring was performed at room temperature for 0.5 hour, the organic phase was discarded by extraction with ethyl acetate, the aqueous phase was neutralized with sodium carbonate, the ethyl acetate extracts, the organic phases were combined, saturated brine was washed, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by silica gel column chromatography to give the title compound 5c (1.7 g, three-step yield 48%).

(S) -4- (4-chloro-2-methylpyrido [3,4-d ] pyrimidin-6-yl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester 5d

Compound 5c (1.0 g,2.78 mmol) was dissolved in 1, 4-dioxane (10 mL), triethylamine (845 mg,8.35 mmol) was added at room temperature, phosphorus oxychloride (1.28 g,8.35 mmol) was added dropwise, and the reaction was carried out for 2 hours after the dropwise addition, heated to 100deg.C, and TLC detected that the starting material was essentially complete. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with ethyl acetate, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound 5d (1.0 g, crude product) as brown, which was used in the next step.

LC-MS:m/z＝378.3[M+H] ⁺

Fifth step (S) -4- (4- (((R) -1- (3- (difluoromethyl) -2-fluorophenyl) ethyl) amino) -2-methylpyrido [3,4-d ] pyrimidin-6-yl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester 5e

Compound 5d (123 mg, crude) and intermediate IN-3 (50 mg,0.26 mmol) were dissolved IN dimethyl sulfoxide (2 mL), N-diisopropylethylamine (67 mg,0.52 mmol) was added at room temperature, heated to 90℃and reacted for 2 hours, and the reaction was complete by TLC. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with ethyl acetate, and the organic phases were combined, saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by Prep-TLC to give the title compound 5e (120 mg, two-step yield 66%) as a yellow solid.

LC-MS:m/z＝531.3[M+H] ⁺

Sixth step N- ((R) -1- (3- (difluoromethyl) -2-fluorophenyl) ethyl) -2-methyl-6- ((S) -2-methylpiperazin-1-yl) pyrido [3,4-d ] pyrimidin-4-amine 5

Compound 5e (120 mg,0.23 mmol) was dissolved in dichloromethane (2 mL), trifluoroacetic acid (2 mL) was added and reacted at room temperature for 2 hours, and the reaction was complete by TLC. The reaction solution was concentrated, neutralized with saturated sodium bicarbonate solution, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by Prep-TLC to give the title compound 5 (90 mg, yield 91%) as a yellow solid.

LC-MS:m/z＝431.3[M+H] ⁺

¹ H NMR(400MHz,CD ₃ OD)δ8.67(s,1H),7.59(t,J＝7.2Hz,1H),7.47(t,J＝7.2Hz,1H),7.30(s,1H),7.22(t,J＝7.6Hz,1H),7.14-6.87(m,1H),5.81(q,J＝7.2Hz,1H),4.71-4.57(m,1H),4.00(dd,J＝12.8,2.4Hz,1H),3.18-3.03(m,3H),3.01-2.95(m,1H),2.92-2.80(m,1H),2.38(s,3H),1.69(d,J＝7.2Hz,3H),1.24(d,J＝6.8Hz,3H).(99.64％purity by HPLC)

Example 6

N- ((R) -1- (3- (difluoromethyl) -2-fluorophenyl) ethyl) -6- ((S) -2, 4-dimethylpiperazin-1-yl) pyrido [3,4-d ] pyrimidin-4-amine 6

First step (S) -4- (4-hydroxypyrido [3,4-d ] pyrimidin-6-yl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester 6a

Compound 5b (510 mg,1.52 mmol) was dissolved in triethyl orthoformate (15 mL), p-toluenesulfonic acid (10 mg,0.058 mmol) was added at room temperature, and the reaction was heated to 150℃for 3 hours, and TLC showed completion of the reaction. The reaction was diluted with hydrochloric acid (1N), extracted with ethyl acetate, the organic phase was discarded, the aqueous phase was neutralized with sodium carbonate, extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude Prep-TLC purified to give the title compound 6a as a yellowish brown solid (330 mg, yield 63%).

Second step (S) -4- (4-Chloropyrimido [3,4-d ] pyrimidin-6-yl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester 6b

Compound 6a (330 mg,0.96 mmol) was dissolved in 1, 4-dioxane (5 mL), pyridine (440 mg,5.56 mmol) was added at room temperature, phosphorus oxychloride (377 mg,2.46 mmol) was added dropwise, and the reaction was completed by TLC after heating to 100deg.C for 2 hours. The reaction solution was cooled to room temperature, diluted with dilute hydrochloric acid (10 ml,1 n) and extracted with ethyl acetate, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound 6b (350 mg, crude) as a brown solid, which was used directly in the next step.

Third step (S) -4- (4- (((R) -1- (3- (difluoromethyl) -2-fluorophenyl) ethyl) amino) pyrido [3,4-d ] pyrimidin-6-yl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester 6c

Compound 6b (130 mg, crude) and intermediate IN-3 (31 mg,0.16 mmol) were dissolved IN dimethyl sulfoxide (2 mL), N-diisopropylethylamine (42 mg,0.32 mmol) was added at room temperature, heated to 100deg.C and reacted for 2 hours, TLC showed starting material remaining, additional intermediate IN-3 (31 mg,016 mmol) was added and the reaction was continued for 2 hours, TLC detection was complete. The reaction was cooled to room temperature, water was added, a solid was precipitated, and the filter cake was purified by Prep-TLC to give the title compound 6c (20 mg, yield 12%) as a yellow solid.

LC-MS:m/z＝517.3[M+H] ⁺

Fourth step N- ((R) -1- (3- (difluoromethyl) -2-fluorophenyl) ethyl) -6- ((S) -2-methylpiperazin-1-yl) pyrido [3,4-d ] pyrimidin-4-amine 6d

Compound 6c (20 mg,0.039 mmol) was dissolved in dichloromethane (2 mL), trifluoroacetic acid (0.5 mL) was added at room temperature, and the reaction was heated to 35℃for 2 hours, and the reaction was complete by TLC. The reaction solution was cooled to room temperature, saturated aqueous sodium bicarbonate was then added to adjust to basicity, the mixture was extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound 6d (20 mg, crude) as a yellow solid, which was used directly in the next step.

Fifth step N- ((R) -1- (3- (difluoromethyl) -2-fluorophenyl) ethyl) -6- ((S) -2, 4-dimethylpiperazin-1-yl) pyrido [3,4-d ] pyrimidin-4-amine 6

Compound 6d (20 mg, crude) was dissolved in methanol (5 mL), aqueous formaldehyde (20 mg,0.25mmol, 37%) and palladium on carbon (10 mg, 10%) were added at room temperature and the reaction was warmed to 30℃under hydrogen atmosphere for 3 hours, and TLC showed complete reaction. The reaction was cooled to room temperature, filtered, the filter cake washed, the filtrate concentrated, and the crude product purified by Prep-TLC to give the title compound 6 (10 mg, 59% in two steps) as a yellow solid.

LC-MS:m/z＝431.3[M+H] ⁺

¹ H NMR(400MHz,CD ₃ OD)δ8.80(s,1H),8.28(s,1H),7.59(t,J＝7.6Hz,1H),7.49-7.47(m,2H),7.24(t,J＝8.0Hz,1H),7.14-6.89(m,1H),5.81(q,J＝6.8Hz,1H),5.06-4.97(m,1H),4.55-4.42(m,1H),3.60-3.34(m,4H),3.13-3.03(m,1H),2.89(s,3H),1.71(d,J＝6.8Hz,3H),1.36(d,J＝7.2Hz,3H).(98.88％purity by HPLC)

Example 7

N ⁴ - ((R) -1- (5-amino-3- (difluoromethyl) -2-fluorophenyl) ethyl) -2-methyl-N ⁶ - ((S) -tetrahydrofuran-3-yl) pyrido [3,4-d]Pyrimidine-4, 6-diamine 7

First step 2-chloro-5-nitroisonicotinic acid methyl ester 7a

Compound 3b (43.50 g,0.21 mol) was dissolved in N, N-dimethylformamide (300 mL), potassium carbonate (60.00 g,0.43 mol) was added at room temperature, methyl iodide (45.91 g,0.32 mol) was added dropwise, and the reaction was completed at room temperature for 2 hours by TLC. The reaction mixture was extracted with water and ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by silica gel column chromatography to give the title compound 7a (46.1 g, yield 99%) as a yellow solid.

LC-MS:m/z＝217.1[M+H] ⁺

Second step (S) -5-nitro-2- ((tetrahydrofuran-3-yl) amino) isonicotinic acid methyl ester 7b

Compound 7a (1.92 g,8.87 mmol) was dissolved in tetrahydrofuran (30 mL), cesium carbonate (4.33 g,13.29 mmol) and (S) -3-aminotetrahydrofuran 1k (850 mg,9.76 mmol) were added at room temperature, the temperature was raised to 30℃and reacted overnight, and TLC showed that a small amount of starting material was unreacted. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with ethyl acetate, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound 7b (2.28 g, crude product) as a yellow solid, which was used directly in the next step.

LC-MS:m/z＝268.1[M+H] ⁺

Third step (S) -5-amino-2- ((tetrahydrofuran-3-yl) amino) isonicotinic acid methyl ester 7c

Compound 7b (2.28 g, crude) was dissolved in methanol (30 mL), palladium on carbon (400 mg, 10%) was added and the reaction was allowed to proceed to 30℃for 5 hours under hydrogen atmosphere, and TLC showed complete reaction of starting material. The reaction solution was cooled to room temperature, filtered through celite, and the cake was washed with methanol, and the filtrate was concentrated, and the crude product was purified by silica gel column chromatography to give the title compound 7c as a yellowish brown solid (1.67 g, 80% in two steps).

LC-MS:m/z＝238.2[M+H] ⁺

Fourth step (S) -2-methyl-6- ((tetrahydrofuran-3-yl) amino) pyrido [3,4-d ] pyrimidin-4-ol 7d

Compound 7c (700 mg,2.95 mmol) was dissolved in acetonitrile (30 mL), cooled to about 0deg.C, and reacted overnight with hydrogen chloride gas at 80℃over 2 hours, with TLC indicating complete reaction of the starting material. The reaction solution was cooled to room temperature, sodium hydrogencarbonate was added to adjust to basicity, filtration, cake water washing, methanol washing, cake addition to toluene, and concentration gave the title compound 7d (650 mg, crude) as a yellow solid, which was used directly in the next step.

LC-MS:m/z＝247.1[M+H] ⁺

Fifth step (S) -4-chloro-2-methyl-N- (tetrahydrofuran-3-yl) pyrido [3,4-d ] pyrimidin-6-amine 7e

Compound 7d (500 mg, crude) was suspended in 1, 4-dioxane (20 mL), N-diisopropylethylamine (1.05 g,8.12 mmol) and phosphorus oxychloride (1.24 g,8.09 mmol) were added at room temperature and reacted for 4 hours with heating to 90℃and TLC indicated that the starting material was complete. The reaction solution was cooled to 0 ℃, quenched by dropwise addition of saturated aqueous sodium bicarbonate, extracted with ethyl acetate, the organic phases combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound 7e (200 mg, crude) as a pale yellow solid, which was used directly in the next step.

LC-MS:m/z＝265.1[M+H] ⁺

Sixth step N ⁴ - ((R) -1- (5-amino-3- (difluoromethyl) -2-fluorophenyl) ethyl) -2-methyl-N ⁶ - ((S) -tetrahydrofuran-3-yl) pyrido [3,4-d]Pyrimidine-4, 6-diamine 7

Compound 7e (78 mg, crude) was dissolved IN dimethyl sulfoxide (3 mL), N-diisopropylethylamine (95 mg,0.74 mmol) and intermediate IN-2 (60 mg,0.29 mmol) were added at room temperature, and the reaction was warmed to 80℃for 8 hours, and TLC showed complete reaction of the starting material. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with ethyl acetate, and the organic phases were combined, saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by Prep-HPLC to give the title compound 7 (6 mg, three-step yield 6%) as a pale yellow solid.

LC-MS:m/z＝433.3[M+H] ⁺

¹ H NMR(400MHz,CD ₃ OD)δ8.59(s,1H),7.06(s,1H),7.05-6.85(m,2H),6.80-6.77(m,1H),5.75(q,J＝7.2Hz,1H),4.45-4.37(m,1H),4.10-3.98(m,2H),3.94-3.87(m,1H),3.73(dd,J＝3.6,8.8Hz,1H),2.41(s,3H),2.41-2.33(m,1H),2.02-1.93(m,1H),1.67(d,J＝7.2Hz,3H).(97.91％purity by HPLC)

Example 8

N ⁴ - ((R) -1- (3-amino-5- (trifluoromethyl) phenyl) ethyl) -7-methoxy-2-methyl-N ⁶ - ((S) -1-methylpyrrolidin-3-yl) quinazoline-4, 6-diamine 8

First step 2-amino-5-bromo-4-methoxybenzoic acid 8b

2-amino-4-methoxybenzoic acid 8a (15.0 g,89.7 mmol) was dissolved in N, N-dimethylformamide (100 mL), cooled to 0℃and N-bromosuccinimide (17.6 g,98.7 mmol) was added thereto, and the reaction was allowed to stand at room temperature for 2 hours by TLC to complete the reaction. The reaction solution was added with water, adjusted to ph=3 with dilute hydrochloric acid (1N), extracted with ethyl acetate, washed with an organic phase saturated aqueous sodium sulfite solution, dried over anhydrous sodium sulfate, and concentrated to give the title compound 8b (22.1 g, crude product) as a pale yellow solid, which was used directly in the next step.

Second step 2-amino-5-bromo-4-methoxybenzoic acid methyl ester 8c

Compound 8b (22.1 g, crude product) was dissolved in methanol (200 mL), concentrated sulfuric acid (10 mL) was added dropwise at room temperature, and the addition was completed, and stirred overnight at 80℃under TLC to detect that the starting material was essentially complete. The reaction solution was cooled to room temperature, quenched with saturated aqueous sodium bicarbonate, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound 8c (19.5 g, 84% yield in two steps) as a pale yellow solid.

Third step 6-bromo-7-methoxy-2-methylquinazolin-4 (3H) -one 8d

Compound 8c (19.5 g,75.0 mmol) was dissolved in acetonitrile (150 mL), methanesulfonic acid (40 mL) was added at room temperature, and the mixture was stirred overnight at 90℃and the reaction was complete as determined by TLC. The reaction solution was concentrated, diluted with water, and ph=8-9 was adjusted with aqueous sodium hydroxide (2N), and a solid was precipitated, filtered, and the cake was washed with water and dried to give the title compound 8d (10.5 g, yield 52%) as an off-white solid.

Fourth step 6-bromo-4-chloro-7-methoxy-2-methyl quinazoline 8e

Compound 8d (1.00 g,3.72 mmol) was dissolved in phosphorus oxychloride (6 mL), N-dimethylformamide (0.1 mL) was added at room temperature, and the reaction was warmed to 90℃for 6 hours, and the reaction was complete by TLC. The reaction solution was cooled to room temperature, poured into ice water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by silica gel column chromatography to give the title compound 8e (535 mg, yield 50%) as a pale yellow solid.

LC-MS:m/z＝287.0[M+H] ⁺

Fifth step (R) -6-bromo-7-methoxy-2-methyl-N- (1- (3-nitro-5- (trifluoromethyl) phenyl) ethyl) quinazolin-4-amine 8f

Compound 8e (500 mg,1.74 mmol) and intermediate IN-1 (407 mg,1.74 mmol) were dissolved IN dimethyl sulfoxide (20 mL), N-diisopropylethylamine (674 mg,5.22 mmol) was added at room temperature, and the mixture was stirred overnight at 80℃until the reaction was complete by TLC. The reaction solution was cooled to room temperature, quenched with water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound 8f (810 mg, crude) as a yellow solid, which was used directly in the next step.

LC-MS:m/z＝485.1[M+H] ⁺

Sixth step N ⁶ - ((S) -1-Benzylpyrrolidin-3-yl) -7-methoxy-2-methyl-N ⁴ - ((R) -1- (3-nitro-5- (trifluoromethyl) phenyl) ethyl) quinazoline-4, 6-diamine for 8h

Compound 8f (430 mg,0.89 mmol) and 8g (312 mg,1.77 mmol) of (S) -1-benzyl-3-aminopyrrolidine were dissolved in toluene (20 mL), tris (dibenzylideneacetone) dipalladium (162 mg,0.18 mmol), 1 '-binaphthyl-2, 2' -bisdiphenylphosphine (220 mg,0.35 mmol) and cesium carbonate (577 mg,1.77 mmol) were added at room temperature, and the mixture was stirred overnight at 100℃under nitrogen and the reaction of the starting materials was complete by TLC. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by silica gel column chromatography to give the title compound as a yellow solid for 8h (290 mg, yield 56.4%).

Seventh step N ⁴ - ((R) -1- (3-amino-5- (trifluoromethyl) phenyl) ethyl) -N ⁶ - ((S) -1-Benzylpyrrolidin-3-yl) -7-methoxy-2-methylquinazoline-4, 6-diamine 8i

Compound 8h (290 mg,0.50 mmol) was dissolved in ethanol (20 mL) and water (4 mL), iron powder (140 mg,2.50 mmol) and ammonium chloride (134 mg,2.50 mmol) were added at room temperature, and the mixture was stirred for 2 hours at 90℃and the reaction was complete by TLC. The reaction solution was filtered through celite while it was still hot, the filtrate was concentrated, the residue was dissolved in ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound 8i (275 mg, crude) as a yellow oil, which was used directly in the next step.

Eighth step (3- ((R) -1- ((6- (((S) -1-benzyl pyrrolidin-3-yl) amino) -7-methoxy-2-methyl-quinazolin-4-yl) amino) ethyl) -5- (trifluoromethyl) phenyl) carbamic acid tert-butyl ester 8j

Compound 8i (275 mg, crude) was dissolved in tetrahydrofuran (32 mL), sodium carbonate (159 mg,1.50 mmol) and di-tert-butyl dicarbonate (218 mg,1.00 mmol) were added, reacted overnight at room temperature and the basic reaction was complete by TLC. The reaction mixture was extracted with water and ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by Prep-TLC to give the title compound 8j (70 mg, 22% yield in two steps) as a pale yellow solid.

LC-MS:m/z＝651.3[M+H] ⁺

Ninth step (3- ((R) -1- ((7-methoxy-2-methyl-6- (((S) -pyrrolidin-3-yl) amino) quinazolin-4-yl) amino) ethyl) -5- (trifluoromethyl) phenyl) carbamic acid tert-butyl ester 8k

Compound 8j (70 mg,0.11 mmol) was dissolved in ethanol (10 mL), palladium on carbon (20 mg, 10%) was added and reacted at room temperature under hydrogen atmosphere for 2 hours, and TLC detected the completion of the starting material reaction. The reaction solution was filtered through celite, and the filtrate was concentrated to give the title compound 8k (50 mg, crude) as a pale yellow oil, which was used directly in the next step.

Tenth step (3- ((R) -1- ((7-methoxy-2-methyl-6- (((S) -1-methylpyrrolidin-3-yl) amino) quinazolin-4-yl) amino) ethyl) -5- (trifluoromethyl) phenyl) carbamic acid tert-butyl ester 8l

Compound 8k (50 mg, crude) and aqueous formaldehyde (26 mg,0.32mmol, 37%) were dissolved in ethanol (10 mL), palladium on carbon (20 mg, 10%) was added and reacted at room temperature under hydrogen atmosphere for 3 hours, and TLC detected complete reaction of the starting materials. The reaction was filtered, the filtrate was concentrated, and the crude product was purified by Prep-TLC to give 8l (37 mg, 60% yield in two steps) of the title compound as a pale yellow solid.

Eleventh step N ⁴ - ((R) -1- (3-amino-5- (trifluoromethyl) phenyl) ethyl) -7-methoxy-2-methyl-N ⁶ - ((S) -1-methylpyrrolidin-3-yl) quinazoline-4, 6-diamine 8

Compound 8l (37 mg,0.06 mmol) was dissolved in methanol (1 mL), and methanol hydrochloride solution (3 mL, 4M) was added and reacted at room temperature for 2 hours, and TLC was used to detect completion of the starting material reaction. The reaction was neutralized with methanolic ammonia (4M), the insoluble matter was removed by filtration, the filtrate was concentrated, and the crude product was purified by Prep-TLC to give the title compound 8 (20 mg, yield 66%) as a pale yellow solid.

LC-MS:m/z＝475.3[M+H] ⁺

¹ H NMR(400MHz,CD ₃ OD)δ7.11(s,1H),6.99(s,1H),6.97(s,1H),6.94(s,1H),6.80(s,1H),5.64(q,J＝6.8Hz,1H),4.32-4.25(m,1H),3.98(s,3H),3.00-2.86(m,2H),2.70-2.66(m,1H),2.63-2.55(m,1H),2.55-2.47(m,1H),2.45(s,6H),1.82-1.74(m,1H),1.64(d,J＝6.8Hz,3H).

Example 9

4- (((R) -1- (3-amino-5- (trifluoromethyl) phenyl) ethyl) amino) -6- (((S) -tetrahydrofurane-3-yl) oxy) quinazolin-7-carbonitrile 9

First step (S) -7-bromo-6- ((tetrahydrofuran-3-yl) oxy) quinazolin-4 (3H) -one 9a

Compound 4f (1.0 g,3.32 mmol) and p-toluenesulfonic acid (20 mg,0.12 mmol) were reacted in triethylorthoformate (10 mL) heated to 150℃for 6 hours, and TLC showed complete reaction of starting materials. The reaction solution was cooled to room temperature, and a solid was precipitated, filtered, and the cake was washed and dried to give the title compound 9a (540 mg, yield 52%) as an off-white solid.

LC-MS:m/z＝311.0[M+H] ⁺

Second step (S) -7-bromo-4-chloro-6- ((tetrahydrofuran-3-yl) oxy) quinazoline 9b

Compound 9a (300 mg,0.96 mmol) was dispersed in 1, 4-dioxane (10 mL), pyridine (228 mg,2.88 mmol) and phosphorus oxychloride (442 mg,2.88 mmol) were added at room temperature, and the reaction was heated to 90℃for 2 hours, and TLC detected complete reaction of the starting materials. The reaction solution was cooled to room temperature, concentrated, and the residue was neutralized with saturated aqueous sodium bicarbonate, extracted with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by Prep-TLC to give the title compound 9b (73 mg, yield 23%) as a yellow solid.

Third step 7-bromo-N- ((R) -1- (3-nitro-5- (trifluoromethyl) phenyl) ethyl) -6- (((S) -tetrahydrofurane-3-yl) oxy) quinazolin-4-amine 9c

Compound 9b (73 mg,0.22 mmol) and intermediate IN-1 (52 mg,0.22 mmol) were dissolved IN dimethyl sulfoxide (1 mL), N-diisopropylethylamine (57 mg,0.44 mmol) was added at room temperature, and the reaction was heated to 100deg.C for 2 hours, and TLC detection was essentially complete. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by Prep-TLC to give the title compound 9c (85 mg, yield 73%) as a yellow solid.

LC-MS:m/z＝527.1[M+H] ⁺

Fourth step 4- (((R) -1- (3-nitro-5- (trifluoromethyl) phenyl) ethyl) amino) -6- (((S) -tetrahydrofurane-3-yl) oxy) quinazoline-7-carbonitrile 9d

Compound 9c (85 mg,0.16 mmol) was dissolved in N, N-dimethylacetamide (3 mL), and tetrakis triphenylphosphine palladium (18 mg,0.016 mmol) and zinc cyanide (47 mg,0.40 mmol) were added at room temperature and heated to 140℃under nitrogen for 7 hours, and TLC detected complete reaction of the starting materials. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by Prep-TLC to give the title compound 9d (54 mg, yield 71%) as a yellow solid.

LC-MS:m/z＝474.2[M+H] ⁺

Fifth step 4- (((R) -1- (3-amino-5- (trifluoromethyl) phenyl) ethyl) amino) -6- (((S) -tetrahydrofurane-3-yl) oxy) quinazoline-7-carbonitrile 9

Compound 9d (54 mg,0.11 mmol) was dissolved in ethanol (5 mL) and water (1 mL), ammonium chloride (29 mg,0.55 mmol) and reduced iron powder (25 mg,0.44 mmol) were added at room temperature, and the reaction was heated to 90℃for 2 hours, and the TLC detection of the starting materials was essentially complete. The reaction solution was cooled to room temperature, filtered through celite, the filtrate was concentrated, extracted with water, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by Prep-TLC to give the title compound 9 (32 mg, yield 63%) as a yellow solid.

LC-MS:m/z＝444.2[M+H] ⁺

¹ H NMR(400MHz,CD ₃ OD)δ8.40(s,1H),8.02(s,1H),7.90(s,1H),6.96(s,2H),6.81(s,1H),5.60(q,J＝6.8Hz,1H),5.37-5.31(m,1H),4.19-4.01(m,3H),3.99-3.94(m,1H),2.48-2.32(m,1H),2.32-2.20(m,1H),1.67(d,J＝6.8Hz,3H).

Example 10

N- ((R) -1- (3- (difluoromethyl) -2-fluorophenyl) ethyl) -2-methyl-6- ((R) -1-methylpiperidin-3-yl) pyrido [3,4-d ] pyrimidin-4-amine 10-1

N- ((R) -1- (3- (difluoromethyl) -2-fluorophenyl) ethyl) -2-methyl-6- ((S) -1-methylpiperidin-3-yl) pyrido [3,4-d ] pyrimidin-4-amine 10-2

First step 5-amino-2-chloropyridine-4-carboxamide 10a

Compound 3c (1.0 g,4.96 mmol) was dissolved in ethanol (20 mL) and water (4 mL), ammonium chloride (1.3 g,24.08 mmol) and reduced iron powder (1.1 g,19.84 mmol) were added at room temperature, and the reaction was heated to 90℃for 3 hours, and the TLC detected complete reaction of the starting materials. The reaction solution was cooled to room temperature, filtered through celite, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound 10a (680 mg, crude) as a yellowish brown solid, which was used directly in the next step.

Second step 5-amino-4-carbamoyl-5 ',6' -dihydro- [2,3' -bipyridine ] -1' (2 ' H) -carboxylic acid tert-butyl ester 10c

Compound 10a (680 mg, crude) was dissolved in 1, 4-dioxane (8 mL) water (2 mL), 5- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -3, 6-dihydropyridine-1 (2H) -carboxylic acid tert-butyl ester 10b (1.5 g,4.93 mmol), sodium carbonate (803 mg,7.58 mmol) and [1,1' -bis (diphenylphosphine) ferrocene ] palladium dichloride dichloromethane complex (50 mg,0.061 mmol) were added sequentially at room temperature and heated to 80℃under nitrogen to react for 2 hours under TLC to detect the starting material was essentially complete. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with ethyl acetate, washing was performed with saturated brine, drying was performed with anhydrous sodium sulfate, and the residue was purified by silica gel column chromatography to give the title compound 10c (710 mg, 45% yield in two steps) as a yellow solid.

LC-MS:m/z＝319.2[M+H] ⁺

Third step 3- (5-amino-4-carbamoylpyridin-2-yl) piperidine-1-carboxylic acid tert-butyl ester 10d

Compound 10c (710 mg,2.23 mmol) was dissolved in ethanol (20 mL) and ethyl acetate (10 mL), palladium on carbon (100 mg, 10%) was added and the reaction was heated to 50℃under a hydrogen atmosphere for 5 hours, and the reaction was complete as detected by LC-MS. The reaction solution was cooled to room temperature, filtered through celite, and the filtrate was concentrated to give the title compound 10d (680 mg, crude) as a yellow solid, which was used directly in the next step.

LC-MS:m/z＝321.3[M+H] ⁺

Fourth step 3- (4-hydroxy-2-methylpyrido [3,4-d ] pyrimidin-6-yl) piperidine-1-carboxylic acid tert-butyl ester 10e

Compound 10d (680 mg, crude) was dissolved in triethyl orthoacetate (6 mL), p-toluenesulfonic acid (10 mg) was added, and the mixture was heated to 150℃for 6 hours, and TLC showed completion of the reaction of starting material. The reaction solution was cooled to room temperature, a small amount of diluted acid (1N) was then added thereto and stirred for 0.5 hour, extraction was performed with ethyl acetate, washing was performed with saturated brine, drying was performed with anhydrous sodium sulfate, and concentration was performed, and the residue was purified by silica gel column chromatography to give the title compound 10e (188 mg, 24% in two steps) as a brown solid.

Fifth step 3- (4-chloro-2-methylpyrido [3,4-d ] pyrimidin-6-yl) piperidine-1-carboxylic acid tert-butyl ester 10f

Compound 10e (160 mg,0.44 mmol) was dissolved in 1, 4-dioxane (5 mL), phosphorus oxychloride (409 mg,2.6 mmol) and N, N-diisopropylethylamine (344 mg,2.6 mmol) were added at room temperature, and the reaction was heated to 90℃for 1.5 hours, and TLC was checked for complete reaction of the starting materials. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with ethyl acetate, washing was performed with saturated brine, drying was performed with anhydrous sodium sulfate, and the residue was purified by silica gel column chromatography to give the title compound 10f (95 mg, yield 57%) as a yellow solid.

Sixth step (R) -tert-butyl 3- (4- (((R) -1- (3- (difluoromethyl) -2-fluorophenyl) ethyl) amino) -2-methylpyrido [3,4-d ] pyrimidin-6-yl) piperidine-1-carboxylate 10g-1& (S) -3- (4- (((R) -1- (3- (difluoromethyl) -2-fluorophenyl) ethyl) amino) -2-methylpyrido [3,4-d ] pyrimidin-6-yl) piperidine-1-carboxylate 10g-2

Compound 10f (75 mg,0.20 mmol) and intermediate IN-3 (46 mg,0.24 mmol) were dissolved IN dimethyl sulfoxide (1.5 mL), N-diisopropylethylamine (80 mg,0.60 mmol) was added at room temperature, and the reaction was heated to 95℃for 3 hours and was complete by TLC. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with ethyl acetate, washing was performed with saturated brine, drying was performed with anhydrous sodium sulfate, and the residue was purified by Prep-TLC to give the title compound as a white solid (10 g, 100mg, yield 94%). Chiral resolution of 10g of compound (cellophane AD-H,30×250mm,5 μm,30mL/min, ethanol: hexane=5:95) gave 10g-1 (peak No. 1, RT 7.79 min) (30 mg, yield 28%) as a white solid and 10g-2 (peak No. 2, RT 16.10 min) (48 mg, yield 45%). The configuration and the property of the compound need to be further detected, and 10g-1 and 10g-2 are tentatively set to the above configurations.

LC-MS:m/z＝516.3[M+H] ⁺

Seventh step N- ((R) -1- (3- (difluoromethyl) -2-fluorophenyl) ethyl) -2-methyl-6- ((R) -piperidin-3-yl) pyrido [3,4-d ] pyrimidin-4-amine 10h-1&N- ((R) -1- (3- (difluoromethyl) -2-fluorophenyl) ethyl) -2-methyl-6- ((S) -piperidin-3-yl) pyrido [3,4-d ] pyrimidin-4-amine 10h-2

Compound 10g-1 (30 mg,0.058 mmol) was dissolved in dichloromethane (1 mL), trifluoroacetic acid (1 mL) was added and stirred at room temperature for 1 hour, and TLC detection was complete. The reaction solution was concentrated, water was added, saturated aqueous sodium carbonate was neutralized, ethyl acetate was extracted 2 times, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound (10 h-1) (25 mg, crude) as a yellow solid, which was used directly in the next step.

Compound 10g-2 (48 mg,0.093 mmol) was dissolved in dichloromethane (1 mL), trifluoroacetic acid (1 mL) was added and stirred at room temperature for 1 hour, and TLC detection was complete. The reaction solution was concentrated, water was added, saturated aqueous sodium carbonate was neutralized, ethyl acetate was extracted 2 times, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound (10 h-2) (30 mg, crude) as a yellow solid, which was used directly in the next step.

The configuration and the property of the compound need to be further detected, and the tentative 10h-1 and 10h-2 are the configurations.

Eighth step N- ((R) -1- (3- (difluoromethyl) -2-fluorophenyl) ethyl) -2-methyl-6- ((R) -1-methylpiperidin-3-yl) pyrido [3,4-d ] pyrimidin-4-amine 10-1&N- ((R) -1- (3- (difluoromethyl) -2-fluorophenyl) ethyl) -2-methyl-6- ((S) -1-methylpiperidin-3-yl) pyrido [3,4-d ] pyrimidin-4-amine 10-2

The compound 10h-1 (25 mg, crude) was dissolved in ethanol (2 mL), aqueous formaldehyde (4 drops, 37%) and palladium on carbon (10 mg, 10%) were added and reacted under hydrogen atmosphere at 35 ℃ for 3 hours, and TLC detection showed complete reaction. The reaction was cooled to room temperature, filtered through celite, and the filtrate concentrated, and the residue was purified by Prep-TLC to give the title compound 10-1 (8 mg, 32% in two steps) as a white solid.

The compound 10h-2 (30 mg, crude) was dissolved in ethanol (2 mL), aqueous formaldehyde (4 drops, 37%) and palladium on carbon (10 mg, 10%) were added and reacted under hydrogen atmosphere at 35 ℃ for 3 hours, and TLC detection showed complete reaction. The reaction was cooled to room temperature, filtered through celite, and the filtrate was concentrated, and the residue was purified by Prep-TLC to give the title compound 10-2 (21 mg, 53% yield in two steps) as a white solid.

The configuration and the property of the compound need to be further detected, and the tentative 10-1 and 10-2 are the configurations.

10-1：

LC-MS:m/z＝430.2[M+H] ⁺

¹ H NMR(400MHz,CD ₃ OD)δ8.96(s,1H),8.26(s,1H),7.66(t,J＝7.2Hz,1H),7.49(t,J＝6.8Hz,1H),7.24(t,J＝7.6Hz,1H),7.00(t,J＝54.8Hz,1H),5.86(q,J＝7.2Hz,1H),3.84-3.35(m,4H),3.24-3.03(m,1H),2.96(s,3H),2.48(s,3H),2.30-1.84(m,4H),1.72(d,J＝7.2Hz,3H).

10-2：

LC-MS:m/z＝430.2[M+H] ⁺

¹ H NMR(400MHz,CD ₃ OD)δ8.92(s,1H),8.13(s,1H),7.62(t,J＝7.2Hz,1H),7.49(t,J＝6.8Hz,1H),7.24(t,J＝7.6Hz,1H),7.00(t,J＝54.8Hz,1H),5.86(q,J＝7.2Hz,1H),3.28-3.18(m,2H),3.12-3.04(m,1H),2.65-2.55(m,1H),2.50(s,3H),2.46(s,3H),2.40-2.28(m,1H),2.16-2.06(m,1H),1.98-1.74(m,3H),1.72(d,J＝7.2Hz,3H).

Example 11

(R) -4- ((1- (3-amino-5- (trifluoromethyl) phenyl) ethyl) amino) -6- (4-methylpiperazin-1-yl) quinazolin-7-carbonitrile 11

First step 4-bromo-5-fluoro-2-nitrobenzoic acid methyl ester 11a

Compound 4c (2.6 g,9.85 mmol) was dissolved in N, N-dimethylformamide (10 mL), methyl iodide (2.8 g,19.73 mmol) and potassium carbonate (4.0 g,28.94 mmol) were added, reacted overnight at room temperature, and the reaction was complete by TLC. The reaction mixture was extracted with water (50 mL), ethyl acetate (100 mL), and the organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and concentrated to give the title compound 11a (3.0 g, crude) as a pale yellow solid, which was used directly in the next step.

Second step 4-bromo-5- (4-methylpiperazin-1-yl) -2-nitrobenzoic acid methyl ester 11b

Compound 11a (3.0 g, crude) was dissolved in acetonitrile (20 mL), N-methylmorpholine (1.6 g,15.98 mmol) and potassium carbonate (2.0 g,14.47 mmol) were added at room temperature, heated to 80℃and reacted overnight, and the reaction was complete by TLC. The reaction mixture was extracted with water (50 mL), ethyl acetate (100 mL), and the organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and concentrated to give the title compound 11b (2.5 g, crude) as a pale yellow oil, which was used directly in the next step.

Third step methyl 2-amino-4-bromo-5- (4-methylpiperazin-1-yl) benzoate 11c

Compound 11b (2.5 g, crude) was dissolved in ethanol (20 mL), iron powder (1.5 g,26.86 mmol), ammonium chloride (1.5 g,28.04 mmol) and water (2 mL) were added at room temperature, and the reaction was heated to 80℃for 2 hours, and the reaction was complete by TLC. The reaction solution was filtered through celite while it was still hot, the filtrate was diluted with water, extracted with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by silica gel column chromatography to give the title compound 11c as a yellowish brown solid (1.5 g, three steps yield 47%).

LC-MS:m/z＝328.1[M+H] ⁺

Fourth step 7-bromo-6- (4-methylpiperazin-1-yl) quinazolin-4-ol 11d

Compound 11c (1.5 g,4.57 mmol) was dissolved in ethylene glycol monomethyl ether (10 mL), formamidine acetate (3.0 g,28.82 mmol) was added at room temperature, heated to 120deg.C and reacted overnight, and TLC detected completion of the reaction. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with methylene chloride, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by silica gel column chromatography to give the title compound 11d (1.3 g, yield 87%) as a pale yellow solid.

LC-MS:m/z＝323.1[M+H] ⁺

Fifth step 7-bromo-4-chloro-6- (4-methylpiperazin-1-yl) quinazoline 11e

Compound 11d (200 mg,0.62 mmol) was dissolved in 1, 4-dioxane (10 mL), N-diisopropylethylamine (2 mL) and phosphorus oxychloride (1 mL) were added at room temperature, heated to 90℃and reacted overnight, and TLC detected the completion of the reaction. The reaction solution was cooled to room temperature, concentrated, added with water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound 11e (84 mg, crude) as a yellowish brown solid, which was used directly in the next step.

Sixth step (R) -7-bromo-6- (4-methylpiperazin-1-yl) -N- (1- (3-nitro-5- (trifluoromethyl) phenyl) ethyl) quinazolin-4-amine 11f

Compound 11e (84 mg, crude product) was dissolved IN dimethyl sulfoxide (2 mL), N-diisopropylethylamine (70 mg,0.54 mmol) and intermediate IN-1 (73 mg,0.31 mmol) were added sequentially at room temperature, and the reaction was warmed to 100℃for 2 hours and was complete by TLC. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated to give the title compound 11f (130 mg, crude product) as a yellowish brown solid, which was used directly in the next step.

Seventh step (R) -N- (1- (3-amino-5- (trifluoromethyl) phenyl) ethyl) -7-bromo-6- (4-methylpiperazin-1-yl) quinazolin-4-amine 11g

Compound 11f (130 mg, crude) was dissolved in ethanol (5 mL), iron powder (81 mg,1.45 mmol), ammonium chloride (77 mg,1.44 mmol) and water (1 mL) were added at room temperature, and the reaction was heated to 90℃for 2 hours, and the completion of the reaction was detected by TLC. The reaction solution was filtered with celite while it was hot, the filtrate was diluted with water, extracted with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by Prep-TLC to give the title compound as a pale yellow solid 11g (30 mg, 10% yield in three steps).

LC-MS:m/z＝509.1[M+H] ⁺

Eighth step (R) -4- ((1- (3-amino-5- (trifluoromethyl) phenyl) ethyl) amino) -6- (4-methylpiperazin-1-yl) quinazoline-7-carbonitrile 11

11g (30 mg,0.059 mmol) of the compound was dissolved in N, N-dimethylacetamide (5 mL), zinc cyanide (30 mg,0.26 mmol) and tris (dibenzylideneacetone) dipalladium (10 mg,0.01 mmol) were added at room temperature, and the reaction was heated to 120℃under nitrogen protection for 2 hours and was complete by TLC detection. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was obtained as a pale yellow solid title compound 11 (25 mg, yield 93%) by Prep-HPLC.

LC-MS:m/z＝456.2[M+H] ⁺

¹ HNMR(400MHz,CD ₃ OD)δ8.39(s,1H),8.01(s,1H),7.98(s,1H),6.99-6.94(m,2H),6.80(s,1H),5.58(q,J＝7.2Hz,1H),3.38-3.34(m,4H),2.84-2.75(m,4H),2.45(s,3H),1.67(d,J＝7.2Hz,3H).

Example 12

4- (((R) -1- (5-amino-3- (difluoromethyl) -2-fluorophenyl) ethyl) amino) -2-methyl-6- (((S) -tetrahydrofuran-3-yl) oxy) quinazolin-7-carbonitrile 12

First step (3- ((R) -1- ((7-bromo-2-methyl-6- (((S) -tetrahydrofuran-3-yl) oxy) quinazolin-4-yl) amino) ethyl) -5- (difluoromethyl) -4-fluorophenyl) carbamic acid tert-butyl ester 12a

Compound 4h (110 mg, crude) and intermediate IN-5 (60 mg,0.20 mmol) were dissolved IN dimethyl sulfoxide (2 mL), N-diisopropylethylamine (52 mg,0.40 mmol) was added at room temperature and heated to 100deg.C for 2 hours, and TLC detection of basic reaction was complete. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by Prep-TLC to give the title compound 12a (63 mg, 34% in two steps) as a yellow solid.

LC-MS:m/z＝611.2[M+H] ⁺

Second step (3- ((R) -1- ((7-cyano-2-methyl-6- (((S) -tetrahydrofuran-3-yl) oxy) quinazolin-4-yl) amino) ethyl) -5- (difluoromethyl) -4-fluorophenyl) carbamic acid tert-butyl ester 12b

Compound 12a (63 mg,0.10 mmol) was dissolved in N, N-dimethylacetamide (3 mL), and tetrakis triphenylphosphine palladium (18 mg,0.016 mmol) and zinc cyanide (29 mg,0.25 mmol) were added at room temperature and heated to 140℃under nitrogen for 7 hours, and the reaction was complete by TLC. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated to give the title compound 12b (73 mg, crude product) as a yellow solid, which was used directly in the next step.

Step three 4- (((R) -1- (5-amino-3- (difluoromethyl) -2-fluorophenyl) ethyl) amino) -2-methyl-6- (((S) -tetrahydrofurane-3-yl) oxy) quinazoline-7-carbonitrile 12

Compound 12b (73 mg, crude) was dissolved in dichloromethane (3 mL), trifluoroacetic acid (3 mL) was added and reacted at room temperature for 4 hours, and TLC detection was complete. The reaction was adjusted to basicity by dropwise addition of saturated aqueous sodium bicarbonate, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the crude product as a pale yellow solid, title compound 12 (31 mg, 66% in two steps) by Prep-TLC purification.

LC-MS:m/z＝458.2[M+H] ⁺

¹ H NMR(400MHz,CD ₃ OD)δ7.93(s,1H),7.91(s,1H),7.03-6.72(m,3H),5.76(q,J＝6.8Hz,1H),5.37-5.31(m,1H),4.19-4.01(m,3H),3.99-3.94(m,1H),2.45(s,3H),2.43-2.35(m,1H),2.32-2.22(m,1H),1.67(d,J＝6.8Hz,3H).

Example 13

N ⁴ - ((R) -1- (5-amino-3- (difluoromethyl) -2-fluorophenyl) ethyl) -7-methoxy-2-methyl-N ⁶ - ((S) -tetrahydrofuran-3-yl) quinazoline-4, 6-diamine 13

First step (R) - (3- (1- ((6-bromo-7-methoxy-2-methylquinazolin-4-yl) amino) ethyl) -5- (difluoromethyl) -4-fluorophenyl) carbamic acid tert-butyl ester 13a

Compound 8e (600 mg,2.09 mmol) and intermediate IN-5 (635 mg,2.09 mmol) were dissolved IN dimethyl sulfoxide (15 mL), N-diisopropylethylamine (81mg, 6.27 mmol) was added at room temperature, and the mixture was stirred overnight at 80℃until the reaction was complete by TLC. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by silica gel column chromatography to give the title compound 13a (1.01 g, yield 87%) as a pale yellow solid.

LC-MS:m/z＝555.1[M+H] ⁺

Second step (3- (difluoromethyl) -4-fluoro-5- ((R) -1- ((7-methoxy-2-methyl-6- (((S) -tetrahydrofuranyl-3-yl) amino) quinazolin) -4-yl) amino) ethyl) phenyl) carbamic acid tert-butyl ester 13b

Compound 13a (100 mg,0.18 mmol) and (S) -3-aminotetrahydrofuran 1k (32 mg,0.36 mmol) were dissolved in toluene (15 mL), tris (dibenzylideneacetone) dipalladium (33.0 mg,0.036 mmol), 1 '-binaphthyl-2, 2' -bisdiphenylphosphine (45 mg,0.072 mmol) and cesium carbonate (117 mg,0.36 mmol) were added, and the mixture was stirred for 10 hours at 100℃under nitrogen protection, and the reaction was essentially complete by TLC. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated, and the crude product was purified by silica gel column chromatography to give the title compound 13b (70 mg, yield 69%) as a yellow solid.

LC-MS:m/z＝562.3[M+H] ⁺

Third step N ⁴ - ((R) -1- (5-amino-3- (difluoromethyl) -2-fluorophenyl) ethyl) -7-methoxy-2-methyl-N ⁶ - ((S) -tetrahydrofuran-3-yl) quinazoline-4, 6-diamine 13

Compound 13b (50.0 mg,0.09 mmol) was dissolved in dichloromethane (2 mL), trifluoroacetic acid (2 mL) was added and reacted at room temperature for 2 hours, and TLC was found to be essentially complete. The reaction solution was concentrated, the residue was neutralized with saturated aqueous sodium bicarbonate, extracted with dichloromethane, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by Prep-TLC to give the title compound 13 (20 mg, yield 49%) as a pale yellow solid.

LC-MS:m/z＝462.2[M+H] ⁺

¹ H NMR(400MHz,DMSO-d ₆ )δ7.20(s,1H),7.03-6.85(m,3H),6.80-6.75(m,1H),5.78(q,J＝6.8Hz,1H),4.41-4.32(m,1H),4.09-3.85(m,6H),3.75-3.72(m,1H),2.48-2.32(m,4H),2.01-1.93(m,1H),1.64(d,J＝7.2Hz,3H).

Example 14

N- ((R) -1- (5-amino-3- (difluoromethyl) -2-fluorophenyl) ethyl) -6- ((R) -3, 4-dimethylpiperazin-1-yl) -2-methylpyridine [3,4-d ] pyrimidin-4-amine 14

First step (R) -1, 2-dimethylpiperazine 14b

(R) -3, 4-Dimethylpiperazine-1-carboxylic acid tert-butyl ester 14a (1.5 g,7.00 mmol) was dissolved in dichloromethane (10 mL), trifluoroacetic acid (10 mL) was added and reacted at room temperature for 3 hours, and TLC detection was complete. The reaction was concentrated to give the title compound 14b (1.5 g, crude) as a yellow oil, which was used directly in the next step.

Second step (R) -3, 4-dimethylpiperazine-1-carboxylic acid benzyl ester 14c

Compound 14b (1.5 g, crude) was dispersed in tetrahydrofuran (20 mL) and water (10 mL), sodium carbonate (6.9 g,65.78 mmol) was added, stirred for 10 minutes, benzyl chloroformate (930 mg,13.15 mmol) in tetrahydrofuran (5 mL) was added dropwise, and the reaction was completed by TLC at room temperature for 2 hours. The reaction solution was filtered, the filtrate was extracted with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by silica gel column chromatography to give the title compound 14c (1.2 g, two-step yield 70%) as a transparent oil.

LC-MS:m/z＝249.2[M+H] ⁺

Third step (R) -1, 2-dimethylpiperazine 14d

Compound 14c (1.2 g,4.80 mmol) was dissolved in tetrahydrofuran (15 mL), palladium on carbon (120 mg, 10%) was added and the reaction was heated to 50℃under hydrogen atmosphere for 12 hours, and the reaction was complete by TLC. The reaction was cooled to room temperature, filtered over celite, and the filter cake washed with ethyl acetate, and the filtrate was concentrated to give the title compound 14d (620 mg, crude) as a yellow oil, which was used directly in the next step.

Fourth step (R) -2- (3, 4-dimethylpiperazin-1-yl) -5-nitroisonicotinic acid methyl ester 14e

Compound 14d (132 mg, crude) and methyl 2-chloro-5-nitroisonicotinate compound 7a (250 mg,1.15 mmol) were dissolved in dimethyl sulfoxide (2 mL), N-diisopropylethylamine (447 mg,34.7 mmol) was added at room temperature, and the reaction was heated to 90℃for 2 hours and was complete by TLC. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with ethyl acetate, and the organic phase was washed with saturated brine 3 times, dried over anhydrous sodium sulfate and concentrated to give the title compound 14e (270 mg, crude product) as a brown solid, which was used directly in the next step.

LC-MS:m/z＝295.2[M+H] ⁺

Fifth step (R) -5-amino-2- (3, 4-dimethylpiperazin-1-yl) isonicotinic acid methyl ester 14f

Compound 14e (270 mg, crude product) was dispersed in ethanol/water (5 mL/1.5 mL), reduced iron powder (258 mg,4.6 mmol) and ammonium chloride (258 mg,4.6 mmol) were added at room temperature, heated to 100deg.C and reacted for 3 hours, and TLC detection was complete. The reaction solution was filtered through celite while it was hot, the cake was washed with ethanol, the filtrate was concentrated, water was added, extraction was performed with ethyl acetate, the organic phase was washed with saturated brine for 3 times, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by silica gel column chromatography to give the title compound 14f as a yellow solid (230 mg, three-step yield 75%).

Sixth step (R) -6- (3, 4-dimethylpiperazin-1-yl) -2-methylpyrido [3,4-d ] pyrimidin-4-ol 14g

Compound 14f (130 mg,0.49 mmopl) was dissolved in acetonitrile (10 mL), cooled to 0℃and then purged with hydrochloric acid for 2 hours, heated to 80℃and reacted for 2 hours, whereupon TLC indicated complete reaction of the starting materials. The reaction was cooled to room temperature, concentrated, neutralized by adding methanolic ammonia (15 mL, 4M) at low temperature, concentrated, and the crude product purified by Prep-TLC to give 14g (120 mg, 95% yield) of the title compound as a yellow solid.

LC-MS:m/z＝274.2[M+H] ⁺

Seventh step (R) -4-chloro-6- (3, 4-dimethylpiperazin-1-yl) -2-methylpyrido [3,4-d ] pyrimidine for 14h

14g (120 mg,0.43 mmol) of the compound was dissolved in 1, 4-dioxane (2 mL), N-diisopropylethylamine (113 mg,0.87 mmol) was added at room temperature, cooled to 0℃and phosphorus oxychloride (134 mg,0.87 mmol) was added dropwise thereto, and after the addition, the mixture was heated to 90℃and reacted for 2 hours, and TLC was found to be complete. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with ethyl acetate, and the organic phase was washed with saturated brine 3 times, dried over anhydrous sodium sulfate and concentrated to give the title compound as a yellow solid 14h (126 mg, crude product) which was used directly in the next step.

Eighth step N- ((R) -1- (5-amino-3- (difluoromethyl) -2-fluorophenyl) ethyl) -6- ((R) -3, 4-dimethylpiperazin-1-yl) -2-methylpyridin [3,4-d ] pyrimidin-4-amine 14

Compound 14h (126 mg, crude) and intermediate IN-2 (97 mg,0.47 mmol) were dissolved IN dimethyl sulfoxide (2 mL), N-diisopropylethylamine (167 mg,1.29 mmol) was added at room temperature, heated to 95℃and reacted for 2 h, and the reaction was complete by TLC. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by Prep-TLC to give the title compound 14 (28 mg, 14% in two steps) as a yellow solid.

LC-MS:m/z＝460.3[M+H] ⁺

¹ H NMR(400MHz,CD ₃ OD)δ8.70(s,1H),7.57(s,1H),7.00-6.93(m,1H),6.88(t,J＝55.2Hz,1H),6.75-6.73(m,1H),5.76(q,J＝7.2Hz,1H),4.53-4.37(m,2H),3.46(d,J＝12.4Hz,1H),3.38-3.32(m,1H),3.19-3.00(m,3H),2.81(s,3H),2.45(s,3H),1.67(d,J＝6.8Hz,3H),1.43(d,J＝6.0Hz,3H).

Example 15

N- ((R) -1- (5-amino-3- (difluoromethyl) -2-fluorophenyl) ethyl) -6- ((S) -2, 4-dimethylpiperazin-1-yl) -2-methylpyridine [3,4-d ] pyrimidin-4-amine 15

First step (S) -4- (4- (((R) -1- (5-amino-3-difluoromethyl) -2-fluorophenyl) ethyl) amino) -2-methylpyrido [3,4-d ] pyrimidin-6-yl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester 15a

Compound 5d (91 mg,0.24 mmol) was dissolved IN dimethyl sulfoxide (2 mL), intermediate IN-2 (50 mg,0.24 mmol) and N, N-diisopropylethylamine (62 mg,0.48 mmol) were added at room temperature, and the reaction was warmed to 100deg.C for 2 hours and completed by TLC. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by Prep-TLC to give the title compound 15a (50 mg, yield 38%) as a yellow solid.

Second step (S) -4- (4- (((R) -1- (5-acetamido-3-difluoromethyl) -2-fluorophenyl) ethyl) amino) -2-methylpyrido [3,4-d ] pyrimidin-6-yl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester 15b

Compound 15a (50 mg,0.092 mmol) was dissolved in dichloromethane (5 mL), pyridine (22 mg,0.28 mmol) and acetic anhydride (14 mg,0.14 mmol) were added at room temperature, and the temperature was raised to 30℃for 4 hours, and the reaction was complete by TLC. The reaction solution was cooled to room temperature, concentrated, water was then added thereto, extraction was performed with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound 15b (85 mg, crude product) as a yellow solid, which was used directly in the next step.

LC-MS:m/z＝588.3[M+H] ⁺

Third step N- (3- (difluoromethyl) -4-fluoro-5- ((R) -1- (2-methyl-6- ((S) -2-methylpiperazin-1-yl) pyrido [3,4-d ] pyrimidin-4-yl) amino) ethyl) phenyl) acetamide 15c

Compound 15b (85 mg, crude) was dissolved in dichloromethane (2 mL), trifluoroacetic acid (1 mL) was added at room temperature, and the reaction was warmed to 30℃for 2 hours, and the reaction was complete by TLC. The reaction solution was cooled to room temperature, concentrated, and the residue was neutralized with saturated sodium bicarbonate solution, extracted with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound 15c (55 mg, crude product) as a yellow solid, which was used directly in the next step.

LC-MS:m/z＝488.3[M+H] ⁺

Fourth step N- (3- (difluoromethyl) -5- ((R) -1- ((6- ((S) -2, 4-dimethylpiperazin-1-yl) -2-methylpyrido [3,4-d ] pyrimidin-4-yl) amino) ethyl) -4-fluorophenyl) acetamide 15d

Compound 15c (55 mg, crude) was dissolved in ethanol (5 mL), and aqueous formaldehyde (30 mg,0.37mmol, 37%) and palladium on charcoal (10 mg, 10%) were added and reacted at room temperature for 2 hours, and TLC detection was complete. The reaction solution was filtered through celite, and the filtrate was concentrated to give the title compound 15d (66 mg, crude) as a yellow solid, which was used directly in the next step.

Fifth step N- ((R) -1- (5-amino-3- (difluoromethyl) -2-fluorophenyl) ethyl) -6- ((S) -2, 4-dimethylpiperazin-1-yl) -2-methylpyridin [3,4-d ] pyrimidin-4-amine 15

Compound 15d (66 mg, crude) was dispersed in ethanol (2 mL) and aqueous sodium hydroxide (2.0 mL,8.00mmol, 4M) and heated to 90deg.C for 4 hours, and TLC showed the starting material to be essentially complete. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by Prep-TLC to give the title compound 15 (12 mg, four-step yield 29%) as a yellow solid.

LC-MS:m/z＝460.3[M+H] ⁺

¹ H NMR(400MHz,CD ₃ OD)δ8.60(s,1H),7.33(s,1H),7.00-6.55(m,3H),5.65(q,J＝6.8Hz,1H),4.77-4.67(m,2H),4.14(d,J＝14.2Hz,1H),3.18-3.02(m,2H),2.75-2.64(m,1H),2.58-2.47(m,4H),2.34(s,3H),1.56(d,J＝7.2Hz,3H),1.20(d,J＝6.8Hz,3H).

Example 16

N- ((R) -1- (5-amino-3- (difluoromethyl) -2-fluorophenyl) ethyl) -2-methyl-6- ((1S, 4S) -5-methyl-2, 5-diazabicyclo [2.2.1] heptan-2-yl) pyrido [3,4-d ] pyrimidin-4-amine 16

First step (1S, 4S) -5- (4-carbamoyl-5-nitropyridin-2-yl) -2, 5-diazabicyclo [2.2.1] heptane-2-carboxylic acid tert-butyl ester 16b

Compound 3c (500 mg,2.50 mmol) and (1S, 4S) -2, 5-diazabicyclo [2.2.1] heptane-2-carboxylic acid tert-butyl ester 16a (500 mg,2.50 mmol) were dissolved in dimethyl sulfoxide, N-diisopropylethylamine (967 mg,7.50 mmol) was added at room temperature, and the reaction was heated to 95℃for 2 hours and was complete by TLC. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by silica gel column chromatography to give the title compound 16b (1.0 g, yield 94%) as a brown solid.

Second step (1S, 4S) -5- (5-amino-4-carbamoylpyridin-2-yl) -2, 5-diazabicyclo [2.2.1] heptane-2-carboxylic acid tert-butyl ester 16c

Compound 16b (1.0 g,2.7 mmol) was dissolved in ethanol (20 mL) and water (5 mL), reduced iron powder (770 mg,13.7 mmol) and ammonium chloride (770 mg,13.7 mmol) were added at room temperature, and the reaction was heated to 100deg.C for 2 hours, and the reaction was complete by TLC. The reaction solution was filtered hot through celite, the filter cake was washed, the filtrate was concentrated, water was added, extraction was performed 3 times with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound 16c (950 mg, crude) as a brown solid, which was used directly in the next step.

Third step (1S, 4S) -5- (2-methyl-4-oxo-3, 4-dihydropyrido [3,4-d ] pyrimidin-6-yl) -2, 5-diazabicyclo [2.2.1] heptane-2-carboxylic acid tert-butyl ester 16d

Compound 16c (950 mg, crude) was dissolved in triethyl orthoacetate (9 mL), p-toluenesulfonic acid (30 mg,0.17 mmol) was added at room temperature, and the mixture was heated to 150℃for 2 hours, and TLC showed complete reaction of starting materials. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed 3 times with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by silica gel column chromatography to give the title compound 16d (570 mg, 58% yield in two steps) as a brown solid

LC-MS:m/z＝358.2[M+H] ⁺

Fourth step (1S, 4S) -5- (4-chloro-2-methylpyrido [3,4-d ] pyrimidin-6-yl) -2, 5-diazabicyclo [2.2.1] heptane-2-carboxylic acid tert-butyl ester 16e

Compound 16d (200 mg,0.56 mmol) was dissolved in 1, 4-dioxane (3 mL), phosphorus oxychloride (257 mg,1.6 mmol) and N, N-diisopropylethylamine (217 mg,1.6 mmol) were added at room temperature, and the reaction was heated to 90℃for 1.5 hours, and TLC was checked for complete reaction of the starting materials. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, whereby the crude product was purified by silica gel column chromatography to give the title compound 16e (122 mg, yield 58%) as a yellow solid.

Fifth step (1S, 4S) -5- (4- (((R) -1- (3- (difluoromethyl) -2-fluoro-5- ((isopropoxycarbonyl) amino) phenyl) ethyl) amino) -2-methylpyrido [3,4-d ] pyrimidin-6-yl) -2, 5-diazabicyclo [2.2.1] heptane-2-carboxylic acid tert-butyl ester 16f

Compound 16e (122 mg,0.32 mmol) was dissolved IN dimethyl sulfoxide (2 mL), intermediate IN-6 (93 mg,0.32 mmol) and N, N-diisopropylethylamine (42 mg,0.96 mmol) were added at room temperature, and the reaction was heated to 95℃for 1.5 h and was complete by TLC. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated to give the title compound 16f (150 mg, crude product) as a yellow solid, which was used directly in the next step.

Step six (3- ((R) -1- ((6- ((1S, 4S) -2, 5-diazabicyclo [2.2.1] heptan-2-yl) -2-methylpyrido [3,4-d ] pyrimidin-4-yl) amino) ethyl) -5- (difluoromethyl) -4-fluorophenyl) carbamic acid isopropyl ester 16g

Compound 16f (150 mg, crude) was dissolved in dichloromethane (5 mL), trifluoroacetic acid (2.5 mL) was added and reacted at room temperature for 2 hours, and TLC detection was complete. The reaction solution was concentrated, quenched with ice water, made alkaline with saturated aqueous sodium bicarbonate, extracted with ethyl acetate, washed with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, and concentrated to give 16g (137 mg, crude) of the title compound as a yellow solid, which was used directly in the next step.

Seventh step (3- (difluoromethyl) -4-fluoro-5- ((R) -1- ((2-methyl-6- ((1S, 4S) -5-methyl-2, 5-diazabicyclo [2.2.1] heptan-2-yl) pyrido [3,4-d ] pyrimidin-4-yl) amino) ethyl) phenyl) carbamic acid isopropyl ester 16h

16g (137 mg, crude) of the compound was dissolved in ethanol (3 mL), and formaldehyde solution (167 mg,0.20mmol, 37%) and palladium on carbon (10 mg, 10%) were added and heated to 30℃under hydrogen atmosphere to react for 2 hours, and the reaction was complete by TLC. The reaction was cooled to room temperature, filtered through celite, and the filter cake was washed, and the filtrate was concentrated to give the title compound as a yellow solid for 16h (137 mg, crude) which was used directly in the next step.

LC-MS:m/z＝544.3[M+H] ⁺

Eighth step N- ((R) -1- (5-amino-3- (difluoromethyl) -2-fluorophenyl) ethyl) -2-methyl-6- ((1S, 4S) -5-methyl-2, 5-diazabicyclo [2.2.1] heptan-2-yl) pyrido [3,4-d ] pyrimidin-4-amine 16

Compound 16h (137 mg, crude) was dissolved in ethanol (2 mL), aqueous sodium hydroxide (2 mL,6mmol, 3M) was added at room temperature, and the reaction was heated to 95℃for 3 hours, and the reaction was complete by TLC. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed 3 times with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by Prep-TLC to give the title compound 16 (45 mg, four-step yield 30%) as a yellow solid.

LC-MS:m/z＝458.2[M+H] ⁺

¹ H NMR(400MHz,CD ₃ OD)δ8.55(s,1H),7.07(s,1H),6.95-6.66(m,3H),5.67(q,J＝7.2Hz,1H),4.77(s,1H),3.77(s,1H),3.60(d,J＝10.4Hz,1H),3.51(dd,J＝10.4,2.0Hz,1H),2.98-2.88(m,2H),2.50(s,3H),2.34(s,3H),2.08(d,J＝10.4Hz,1H),1.97(d,J＝10.0Hz,1H),1.59(d,J＝7.2Hz,3H).

Example 17

N- ((R) -1- (3-amino-5- (trifluoromethyl) phenyl) ethyl) -6- ((S) -2, 4-dimethylpiperazin-1-yl) pyrido [3,4-d ] pyrimidin-4-amine 17

First step (S) -4- (4- (((R) -1- (3-amino-5- (trifluoromethyl) phenyl) ethyl) amino) pyrido [3,4-d ] pyrimidin-6-yl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester 17a

Compound 6b (63 mg,0.17 mmol) and intermediate IN-1 (41 mg,0.17 mmol) were dissolved IN dimethyl sulfoxide (1 mL), N-diisopropylethylamine (67 mg,0.52 mmol) was added at room temperature, and the reaction was heated to 100deg.C for 2 hours and was complete by TLC. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with ethyl acetate, saturated brine was performed, dried over anhydrous sodium sulfate, and concentrated to give the title compound 17a (130 mg, crude product) as a yellow solid, which was used directly in the next step.

Second step (S) -4- (4- (((R) -1- (3-amino-5- (trifluoromethyl) phenyl) ethyl) amino) pyrido [3,4-d ] pyrimidin-6-yl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester 17b

Compound 17a (130 mg, crude product) was dispersed in ethanol/water (10 mL/2 mL), reduced iron powder (64 mg,1.15 mmol) and ammonium chloride (64 mg,1.15 mmol) were added at room temperature, and the reaction was heated to 100℃for 3 hours, and the completion of the reaction was detected by TLC. The reaction solution was filtered through celite while it was hot, the cake was washed, the filtrate was concentrated, extracted with water, ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by Prep-TLC to give the title compound 17b (87 mg, 94% in two steps) as a yellow solid.

LC-MS:m/z＝532.3[M+H] ⁺

Third step (S) -4- (4- (((R) -1- (3-acetamido-5- (trifluoromethyl) phenyl) ethyl) amino) pyrido [3,4-d ] pyrimidin-6-yl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester 17c

Compound 17b (87 mg,0.16 mmol) was dissolved in dichloromethane (3 mL), N-diisopropylethylamine (42 mg,0.32 mmol) and acetic anhydride (20 mg,0.19 mmol) were added at room temperature, heated to 30℃and reacted for 4 hours, and the reaction was complete by TLC. The reaction solution was cooled to room temperature, concentrated, added with water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound 17c (100 mg, crude) as a yellow solid, which was used directly in the next reaction.

Fourth step N- (3- ((R) -1- ((6- ((S) -2-methylpiperazin-1-yl) pyrido [3,4-d ] pyrimidin-4-yl) amino) ethyl) -5- (trifluoromethyl) phenyl) acetamide 17d

Compound 17c (100 mg, crude) was dissolved in dichloromethane (3 mL), trifluoroacetic acid (0.5 mL) was added at room temperature, and the reaction was heated to 30℃for 3 hours, and the reaction was complete by TLC. The reaction solution was concentrated, water was added, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by Prep-TLC to give the title compound 17d (77 mg, 99% in two steps) as a yellow solid.

Fifth step N- (3- ((R) -1- ((6- ((S) -2, 4-dimethylpiperazin-1-yl) pyrido [3,4-d ] pyrimidin-4-yl) amino) ethyl) -5- (trifluoromethyl) phenyl) acetamide 17e

Compound 17d (77 mg,0.16 mmol) was dissolved in ethanol (3 mL), aqueous formaldehyde (55 mg,0.67mmol, 37%) and palladium on carbon (10 mg, 10%) were added at room temperature and heated to 35℃for 3 hours, and TLC detection was complete. The reaction was cooled to room temperature, filtered through celite, and the filtrate was concentrated to give the title compound 17e (78 mg, crude) as a yellow oil, which was used directly in the next step.

LC-MS:m/z＝488.3[M+H] ⁺

Sixth step N- ((R) -1- (3-amino-5- (trifluoromethyl) phenyl) ethyl) -6- ((S) -2, 4-dimethylpiperazin-1-yl) pyrido [3,4-d ] pyrimidin-4-amine 17

Compound 17e (78.0 mg, crude) was dissolved in ethanol (2 mL), aqueous sodium hydroxide (2.0 mL,8.0mmol, 4M) was added at room temperature, and the reaction was heated to 75deg.C overnight and was complete by TLC. The reaction solution was concentrated, extracted with ethyl acetate, washed with saturated brine 3 times, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by Prep-TLC to give the title compound 17 (10 mg, 13% yield in two steps) as a yellow solid.

LC-MS:m/z＝446.2[M+H] ⁺

¹ H NMR(400MHz,CD ₃ OD)δ8.74(s,1H),8.24(s,1H),7.30(s,1H),6.95(s,2H),6.81(s,1H),5.54(q,J＝6.8Hz,1H),4.80-7.83(m,1H),4.12(d,J＝12.8Hz,1H),3.29-3.20(m,1H),3.03(d,J＝11.2Hz,1H),2.93(d,J＝11.2Hz,1H),2.44(d,J＝8.0Hz,1H),2.39(s,3H),2.30-2.20(m,1H),1.65(d,J＝6.8Hz,3H),1.26(d,J＝6.8Hz,3H).

Example 18

N- ((R) -1- (5-amino-3- (difluoromethyl) -2-fluorophenyl) ethyl) -2-methyl-6- (6-methyl-3, 6-diazabicyclo [3.1.1] heptan-3-yl) pyrido [3,4-d ] pyrimidin-4-amine 18

First step 3- (4-carbamoyl-5-nitropyridin-2-yl) -3, 6-diazabicyclo [3.1.1] heptane-6-carboxylic acid tert-butyl ester 18b

Compound 3c (1.0 g,4.96 mmol) was dissolved in dimethyl sulfoxide (3 mL), 3, 6-diazabicyclo [3.1.1] heptane-6-carboxylic acid tert-butyl ester 18a (990 mg,4.99 mmol) and N, N-diisopropylethylamine (1.9 g,14.70 mmol) were added at room temperature and reacted to 90℃for 3 hours, and the reaction was complete by TLC. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with ethyl acetate, and the organic phase was saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by silica gel column chromatography to give the title compound 18b (1.7 g, yield 94%) as a yellow solid.

LC-MS:m/z＝364.2[M+H] ⁺

Second step 3- (5-amino-4-carbamoyl pyridin-2-yl) -3, 6-diazabicyclo [3.1.1] heptane-6-carboxylic acid tert-butyl ester 18c

Compound 18b (1.7 g,4.68 mmol) was dispersed in ethanol (20 mL) and water (5 mL), reduced iron powder (1.5 g,26.86 mmol) and ammonium chloride (1.4 g,26.17 mmol) were added at room temperature, and the reaction was heated to 90℃for 3 hours, and the reaction was complete by TLC. The reaction solution was filtered while it was still hot, the filter cake was washed with ethanol, the filtrate was concentrated, water was added, ethyl acetate was extracted 3 times, the organic phases were combined, saturated brine was dried over anhydrous sodium sulfate, and concentrated, and the crude product was chromatographed on a silica gel column to give the title compound 18c (1.4 g, yield 90%) as a yellow solid.

LC-MS:m/z＝334.2[M+H] ⁺

Third step 3- (2-methyl-4-oxo-3, 4-dihydropyrido [3,4-d ] pyrimidin-6-yl) -3, 6-diazabicyclo [3.1.1] heptane-6-carboxylic acid tert-butyl ester 18d

Compound 18c (1.4 g,4.20 mmol) was dispersed in triethyl orthoacetate (16 mL), p-toluenesulfonic acid (60 mg,0.35 mmol) was added at room temperature, heated to 150℃and reacted for 3 hours, and the reaction was complete by TLC. The reaction solution was cooled to room temperature, diluted hydrochloric acid (10 ml,1 n) was added and stirred for 10 minutes, the organic phase was discarded, the aqueous phase was neutralized with saturated aqueous sodium carbonate, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was chromatographed on a silica gel column to give the title compound 18d (805 mg, yield 54%).

Fourth step 3- (4-chloro-2-methylpyrido [3,4-d ] pyrimidin-6-yl) -3, 6-diazabicyclo [3.1.1] heptane-6-carboxylic acid tert-butyl ester 18e

Compound 18d (100 mg,0.29 mmol) was dissolved in 1, 4-dioxane (3 mL), N-diisopropylethylamine (108 mg,0.84 mmol) was added, the temperature was reduced to 0℃and phosphorus oxychloride (128 mg,0.83 mmol) was added dropwise, and the reaction was completed by TLC after heating to 90℃for 3 hours. The reaction solution was cooled to room temperature, ice water was added, extraction was performed with ethyl acetate, saturated brine was performed, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by Prep-TLC to give the title compound 18e (54 mg, yield 50%) as a yellow solid.

Fifth step 3- (4- (((R) -1- (3- (difluoromethyl) -2-fluoro-5- ((isopropoxycarbonyl) amino) phenyl) ethyl) amino) -2-methylpyrido [3,4-d ] pyrimidin-6-yl) -3, 6-diazabicyclo [3.1.1] heptane-6-carboxylic acid tert-butyl ester 18f

Compound 18e (54 mg,0.14 mmol) was dissolved IN dimethyl sulfoxide (2 mL), intermediate IN-6 (42 mg,0.14 mmol) and N, N-diisopropylethylamine (67 mg,0.52 mmol) were added at room temperature, and the reaction was heated to 95℃for 1.5 hours and was complete by TLC. The reaction solution was cooled to room temperature, water was then added thereto, extraction was performed with ethyl acetate, saturated brine was performed, dried over anhydrous sodium sulfate, and concentrated to give the title compound 18f (100 mg, crude product) as a green solid, which was used directly in the next step.

LC-MS:m/z＝630.3[M+H] ⁺

Step six (3- ((1R) -1- ((6- (3, 6-diazabicyclo [3.1.1] heptan-3-yl) -2-methylpyrido [3,4-d ] pyrimidin-4-yl) amino) ethyl) -5- (difluoromethyl) -4-fluorophenyl) carbamic acid isopropyl ester 18g

Compound 18f (100 mg, crude) was dissolved in dichloromethane (5 mL), trifluoroacetic acid (2 mL) was added and reacted at room temperature for 2 hours, and TLC detection was complete. The reaction solution was concentrated, water was then added thereto, a saturated aqueous sodium hydrogencarbonate solution was added to adjust the pH to neutrality, extraction was performed with ethyl acetate, saturated brine was performed, and dried over anhydrous sodium sulfate, and 18g (90 mg, crude product) of the title compound was concentrated to obtain a yellow solid, which was directly used in the next step.

LC-MS:m/z＝530.3[M+H] ⁺

Seventh step (3- (difluoromethyl) -4-fluoro-5- ((1R) -1- ((2-methyl-6- (6-methyl-3, 6-diazabicyclo [3.1.1] heptan-3-yl) pyrido [3,4-d ] pyrimidin-4-yl) amino) ethyl) phenyl) carbamic acid isopropyl ester 18h

18g (90 mg, crude) of the compound was dissolved in ethanol (3 mL), and formaldehyde solution (55 mg,0.68mmol, 37%) and palladium on carbon (10 mg, 10%) were added and heated to 30℃under hydrogen atmosphere to react for 2 hours, and TLC detection was complete. The reaction was cooled to room temperature, filtered through celite, and the filtrate was concentrated, and the crude product was purified by Prep-TLC to give the title compound as a white solid for 18h (30 mg, four steps yield 19%).

LC-MS:m/z＝544.3[M+H] ⁺

Eighth step N- ((R) -1- (5-amino-3- (difluoromethyl) -2-fluorophenyl) ethyl) -2-methyl-6- (6-methyl-3, 6-diazabicyclo [3.1.1] heptan-3-yl) pyrido [3,4-d ] pyrimidin-4-amine 18

Compound 18h (30 mg,0.055 mmol) was dissolved in ethanol (2 ml), aqueous sodium hydroxide (2 mL,6.00mmol, 3N) was added at room temperature, and the reaction was heated to 95℃for 3 h, and the reaction was complete by TLC. The reaction solution was cooled to room temperature, water was then added, extraction was performed 3 times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by Prep-TLC to give the title compound 18 (10 mg, yield 40%) as a yellow solid.

LC-MS:m/z＝458.3[M+H] ⁺

¹ H NMR(400MHz,CD ₃ OD)δ8.73(s,1H),7.33(s,1H),7.03-6.92(m,1H),6.88(t,J＝55.2Hz,1H),6.80-6.72(m,1H),5.77(q,J＝6.8Hz,1H),4.51-3.85(m,6H),3.25-3.10(m,1H),2.97-2.78(m,1H),2.56-2.46(m,2H),2.44(s,3H),2.11-1.93(s,1H),1.68(d,J＝7.2Hz,3H).

Test example 1 Compounds for inhibition of K-562 cell proliferation ₅₀ Measurement

Human chronic myelogenous leukemia cell K-562 (CCL-243) used in the present invention was purchased from American Type Culture Collection (ATCC). Cells in RPMI 1640 medium with 10% Fetal Bovine Serum (FBS) and 1% diabody at 37 ℃,5% co ₂ Is grown in the environment of (a).

The inhibition of proliferation of K-562 cells cultured in vitro by the compounds was determined by the following method:

1) Cell inoculation: inoculating K-562 cells in good logarithmic phase into 96-well plate at 20000 cells/well and 90 μl, inoculating at 37deg.C with 5% CO ₂ Culturing for 24 hours under the condition.

2) Adding the medicine: the compound to be tested was diluted in a gradient in complete medium and 10 μl of diluted compound was added to 90 μl of cells to give final concentrations of 10000, 3000, 1000, 300, 100, 30, 10, 3, 1nM, with corresponding vehicle controls.Placing at 37deg.C and 5% CO ₂ The cells were cultured in a cell incubator for 96 hours.

3) And (3) detection: after adding 10. Mu.L of 5mg/mL MTT working solution (ABCONE, M9609) per well and allowing to act at 37℃for 4 hours, a triple solution (10% SDS,0.5% isopropyl alcohol, 0.1mol/L HCL) was added until the cell lysate was completely dissolved, and OD570 and OD690 values were read using a TECAN SPARK microplate reader.

4) And (3) calculating: the cell growth inhibition was calculated by the following formula:

inhibition = (control wells _{OD570nm-OD690nm} -an administration orifice _{OD570nm-OD690nm} ) Control well _{OD570nm-OD690nm} ×100％

Calculation of IC from compound concentration and corresponding inhibition ratio using Graphpad prism 5.0 software ₅₀ Values. The test results are shown in Table 1.

TABLE 1 IC for inhibition of proliferation of K-562 cells by the compounds of the present invention ₅₀ (nM)

Numbering of compounds	K-562	Numbering of compounds	K-562
BI-3406	35.2	10-1	303.8
1	36.4	10-2	147.8
2	111.0	11	40.7
3	138.9	12	210.25
4	203.4	13	22.5
5	89.5	14	35.6
6	129.1	15	19.0
7	92.4	16	143.7
8	153.4	17	32.7
9	105.7	18	10.0

Conclusion: the compounds of the examples of the present invention have proliferation inhibitory effect on K-562 cells, and the activity of a plurality of compounds is equivalent to that of BI-3402.

Test example 2 Effect of Compounds on the level of ERK1/2 phosphorylation of the KRAS downstream Signal molecule in K-562 cells

The effect of the compounds of the invention on ERK1/2 phosphorylation levels in K-562 cells was detected by the following method:

1) Cell inoculation: taking K-562 cells with good logarithmic growth phase at 1×10 ⁶ The cells/wells were inoculated into six well plates and incubated overnight at 37℃under 5% CO 2.

2) Adding the medicine: the compounds to be tested were added to the cells after gradient dilution in complete medium to give final concentrations of 1000, 100, 10, 1nM. Placing at 37deg.C and 5% CO ₂ The cells were cultured in a cell incubator for 24 hours.

3) Protein sample preparation: cell suspensions were collected, centrifuged at 500g for 5 min, the supernatant was discarded, washed 3 times with PBS, and 100. Mu.L of cells were lysed with 1 XSDS gel loading buffer (50 mM Tris-HCl (pH 6.8), 100mM DTT,2%SDS,10% glycerol, 0.1% bromophenol blue). Cell lysates were denatured by heating at 100℃for 10 min.

4) Western blot: carrying out SDS-PAGE electrophoresis on a protein sample, transferring the protein to a PVDF membrane by a wet transfer system after the electrophoresis is finished, placing the PVDF membrane in a sealing solution (5% skimmed milk powder is diluted in TBS/T) for sealing for 1 hour at room temperature, and then resisting reaction I and II; after washing the membrane, color development was performed with Immobilon Western HRP Substrate luminal reagent reagent and a Western Blot imager (Tanon, 4600) was used to photograph. The following are the antibody information used: p-ERK1/2 (CST: 4370); ERK1/2 (CST: 9102); beta-tubulin (CST: 2146).

The effect of compounds on ERK1/2 phosphorylation in K-562 cells is shown in FIG. 1.

Conclusion: the compound provided by the embodiment of the invention has obvious inhibition effect on the phosphorylation of the ERK1/2 of the K-562 cells, and the inhibition activity is concentration gradient dependent.

Test example 3 Compounds for SOS1 inhibitory Activity

The experimental steps are as follows:

treatment of compounds

The compound was formulated at 400-fold final concentration, e.g., 5uM for detection, and at 400-fold concentration, i.e., 2mM. The compound was diluted in gradient to the set number of concentration spots with an automated microwell pipette.

Transfer of compounds to 384 well plate reaction plates

The diluted compounds were transferred from Echo 384 well plates to 384 well reaction plates using an ultrasonic nanoliter liquid treatment system, and both negative and positive controls transferred 50nL of 100% dmso.

Preparation and transfer of 4-fold Tag1-SOS1 solution

A4-fold Tag1-SOS1 solution was prepared with the reagent provided in the kit, transferred into 5ul to 384 well reaction plates, and for negative control wells, transferred 5ul of reagent instead of enzyme solution, and centrifuged at 1000rpm for 1 min.

Preparing 4 times Tag2-KRAS G12C solution

A4-fold Tag2-KRAS G12C solution was prepared with the reagent provided in the kit, transferred to a 5ul 384 well reaction plate, and centrifuged at 1000rpm for 1 minute.

Transferring 2 times of detection solution

2-fold Anti-Tag1-Tb3+ and Anti-Tag2-XL665 solutions were prepared using the Detection Buffer provided in the kit, transferred to 10ul to 384 well reaction plates, centrifuged at 1000rpm for 1 min and incubated at room temperature for 60 min.

Reading the number

The data fluorescence signal values were read with a microplate reader Envision (Ex 665/Em 615).

Inhibition calculation and IC50 fitting

Values were copied from the plate reader, where maximum refers to the reading of the positive control and minimum refers to the reading of the negative control. Inhibition ratio (%) = (maximum value-sample value)/(maximum value-minimum value) ×100%.

Data were imported into MS Excel and IC50 values were fitted with XLFIT Excel add-in version 5.4.0.8;

inhibitory Activity of Compounds of Table 2 on SOS1

Numbering of compounds	IC50(nM)	Numbering of compounds	IC50(nM)	Numbering of compounds	IC50(nM)
1	5.7	2	6.7	3	10.1
5	6.6	6	7.5	8	5.4
9	8.5	10-2	13.0	11	11
12	6.5	13	4.0	14	5.6
15	4.7	16	5.4	17	5.1
18	4.2

Fitting formula: y=bottom+ (Top-Bottom)/(1+ (IC 50/X)/(HillSlope)

Test example 4 Compounds for liver microsomal stability experiments on mice and humans

The experimental steps are as follows:

(1) Taking out liver microsomes (20 mg protein/mL) from a refrigerator at-80 ℃, placing the liver microsomes on a water bath constant temperature oscillator at 37 ℃ for pre-incubation for 3min, and melting for later use.

(2) According to the above "constitution of the experimental incubation system", a mixed solution of the incubation system (without beta-NADPH) was prepared.

(3) Preparing 100 mu M test compound working solution for later use.

(4) Control group (without β -NADPH): and (3) respectively taking 25 mu L of PB solution into 75 mu L of the mixed solution of the incubation system (2), swirling for 30s, uniformly mixing, and carrying out reaction with the total volume of 100 mu L, and repeating. Incubation was performed in a 37℃water bath thermostatted shaker and timing was started with sampling time points of 0min and 60min.

(5) Sample group: mu.L of beta-NADPH solution (4 mM) was added to 75. Mu.L of the reaction system (2), and the mixture was vortexed for 30s, mixed well, and the total volume of the reaction was 100. Mu.L, and the reaction was repeated. Incubation was performed in a 37℃water bath thermostated shaker and timing was started with sampling times of 0min,5min,15min,30min,60min.

(6) The sample tube was removed at each time point and 300. Mu.L of cold stop reagent (containing internal standard) was added to stop the reaction.

(7) Vortex and centrifuge.

(8) 150 μl of the supernatant was added with 150 μl of water, vortexed and mixed well, and analyzed by LC-MS/MS.

Data analysis

Half-life (t 1/2) and Clearance (CL) were calculated using the following first order kinetic formulas

Ct＝C0*e-kt

Ct＝(1/2)*C0

t1/2＝ln2/k＝0.693/k

CL＝Vd*k

Vd=1/protein content in liver microsomes

CLint (lever) =clint (mic) ×liver weight to weight ratio×liver microsomal protein concentration per gram of liver

The parameters in the formula are shown in Table 3:

table 3 list of parameters

The experimental results are shown in table 4:

microsomal stability of the compounds of Table 4 in different species

In the human liver microsome stability experiment, the stability of the compound 13 is obviously better than that of BI-3406; in the mouse liver microsome stability experiments, the stability of compounds 16, 17 was significantly better than BI-3406. In summary, liver microsomal stability of the various species of the compounds of the examples was significantly better than BI-3406.

Test example 5 rat pharmacokinetic Properties of Compounds

The compound of example 1 (10 mg/kg) was orally administered to overnight fasted SD rats (male, n=3/time point). Rats were fed a further 2h after dosing. Blood samples were collected before and 1.5h, 4h and 8h after the administration, respectively, and centrifuged (4500 rpm) at 4℃for 10min to obtain serum. Livers were harvested 1.5h, 4h and 8h before and after dosing, respectively. Liver samples were then flash frozen and stored at-80 ℃. mu.L of MeOH/ACN (1:1, v/v) was added to 10. Mu.L of serum to precipitate the mixture, which was then vortexed for 1min and centrifuged (11000 rpm) for 5min to obtain a supernatant. mu.L of the supernatant was dissolved in 20. Mu.L of ACN/H2O (1:1, v/v) and analyzed by ultra performance liquid chromatography. The liver samples were homogenized by adding 10 times the weight of MeOH/ACN (1:1, v/v) and then homogenizing with a homogenizer at 50Hz for 120 s. The homogenate was centrifuged (11000 rpm) for 5 minutes and the supernatant collected. Then 20. Mu.L of the supernatant was redissolved in 20. Mu.L of ACN/H2O (1:1, v/v) and analyzed by ultra high performance liquid chromatography. The results are shown in Table 5:

Rat pharmacokinetic profile of the compounds of Table 5

As can be seen from table 5, compound 1 has a better plasma exposure and a better half-life in rats.

Test example 6 Compounds for in vivo anti-tumor Activity

Human pancreatic cancer cells MIAPaCa-2 or PANC-1 (1.0X107 cells/mouse) were subcutaneously injected on the right side of female BALB/c mice (6-8 weeks). Mice were dosed by gavage, compound 1 (Cpd 1) (50 mg/kg), trametinb (0.125 mg/kg, bid), and combinations of compound 1 and trametinb (combination), twice a day (bid), and continuously. Mice were monitored daily and caliper measurements were started when tumors became visible. The tumor volume was calculated by measuring two perpendicular diameters using the following formula: (L x W2)/2, wherein L and W refer to the length and width of the tumor diameter. When the average tumor volume reached 100mm3, mice were grouped (D0, n=5/group) and given the compound. Tumor volume and mouse body weight were measured 1 time every 3 days during dosing. The results are shown in FIGS. 2-5 below.

Conclusion: experimental results show that the compound 1 of the embodiment of the invention has good anti-tumor activity on different mutant tumor models of KRAS under single and combined dosages. In the two anti-tumor models, the compound 1 of the example has no obvious influence on the weight of mice under the single and combined dosages, and shows better safety. The compound 1 of the example has a certain inhibition activity on PANC-1 (KRAS G12D) nude mice transplanted tumor, and the tumor growth inhibition rate is 40.6 percent (P < 0.01); the compound 1 of the example has a certain inhibition activity on MIAPaCa-2 (KRAS G12C) nude mice transplanted tumor, the tumor growth inhibition rate is 65.4 percent (P < 0.01), the tumor inhibition rate of a group combined with MEK inhibitor Trametinib is 105.6 percent, the tumor occurrence of all mice in the group combined is retracted, and the activity is obviously enhanced compared with that of a single group (P < 0.001).

The applicant states that the present invention is illustrated by the above examples as a polycyclic pyrimidine derivative as SOS1 inhibitor, and its preparation method and application, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must be practiced by the above examples. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.

In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Claims

A polycyclic pyrimidine derivative, a pharmaceutically acceptable salt thereof, a tautomer thereof or a stereoisomer thereof, which is characterized in that the structure of the polycyclic pyrimidine derivative is shown as a formula (I):

wherein: r is R ¹ Selected from hydrogen or C ₁ -C ₃ An alkyl group; preferably hydrogen or methyl;

A ¹ selected from N or C-R ¹¹ ；

R ¹¹ Selected from H, C ₁ -C ₃ Alkyl or C ₁ -C ₃ A haloalkyl group;

A ² selected from N or C-R ² ；

R ² Selected from-OR ²¹ Or cyano;

R ²¹ selected from H, C ₁ -C ₃ Alkyl, 3-7 membered cycloalkyl, 4-7 membered heterocyclyl, wherein C ₁ -C ₃ Alkyl, 3-7 membered cycloalkyl, 4-7 membered heterocyclyl are optionally substituted with 1-3R ²² Substituted;

R ²² selected from halogen, C ₁ -C ₄ Alkyl, cyano, hydroxy;

l is absent or selected from O, NH or NCH ₃ ；

R ³ Selected from 3-7 membered cycloalkyl, 4-7 membered heterocyclyl, 6-10 membered fused heterocyclyl, 6-10 membered bridged heterocyclyl, 6-10 membered spiroheterocyclyl, wherein 3-7 membered cycloalkyl, 4-7 membered heterocyclyl, 6-10 membered fused heterocyclyl, 6-10 membered bridged heterocyclyl, 6-10 membered spiroheterocyclyl is optionally substituted with 1-3R ³¹ Substituted;

R ³¹ selected from C ₁ -C ₃ Alkyl, C ₁ -C ₃ Haloalkyl, hydroxy, halogen, cyano, -NR ^a R ^b 、C ₁ -C ₃ Alkoxy, =o, -NHCOR ³² or-COR ³² ；

R ³² Selected from C ₁ -C ₃ Alkyl, C ₁ -C ₃ Haloalkyl, 3-7 membered cycloalkyl or 4-7 membered heterocyclyl;

R ⁴ is-CH ₃ ；

AR is selected from 6-10 membered aryl or 5-10 membered heteroaryl, wherein aryl or heteroaryl is optionally substituted with 1-4R ⁵ Substituted;

R ⁵ selected from halogen, C ₁ -C ₄ Alkyl, C ₁ -C ₄ Haloalkyl, hydroxy-C ₁ -C ₄ Alkyl, hydroxy-C ₁ -C ₄ Haloalkyl, 3-6 membered cycloalkyl, 4-7 membered heterocyclyl, -OR ^a 、-NR ^a R ^b ；

R ^a Selected from H, C ₁ -C ₄ Alkyl, C ₁ -C ₄ Haloalkyl, 3-6 membered cycloalkyl or 4-7 membered heterocyclyl;

R ^b selected from H, C ₁ -C ₄ Alkyl, C ₁ -C ₄ Haloalkyl, 3-6 membered cycloalkyl or 4-7 membered heterocyclyl;

in the above definition, the following conditions cannot occur simultaneously:

1)A ¹ is C-R ¹¹ ；

2)A ² Is C-OR ²¹ ；

3) L is O or absent;

the hetero atoms in the heterocyclic group and the heteroaryl group in the formula (I) are 1-3 and are selected from one or more of oxygen, nitrogen and sulfur.
The polycyclic pyrimidine derivative, a pharmaceutically acceptable salt thereof, a tautomer thereof, or a stereoisomer thereof according to claim 1, wherein the polycyclic pyrimidine derivative has a structure represented by formula (ii):

wherein R is ¹ 、L、R ³ And R is ⁵ Having the same limitations as claim 1; n=1 to 4;

preferably, for compounds of formula (II), the phenyl group is optionally substituted with 1 to 4R ⁵ Substituted when said R ⁵ When the number of R is 2-4, R is ⁵ May be the same or different;

and/or when said R ⁵ Is C ₁ -C ₄ In the case of haloalkyl, said R ⁵ 1-3, when 2-3R's are present ⁵ When said R is ⁵ May be the same or different;

And/or when said R ⁵ Is C ₁ -C ₄ When haloalkyl, halogen atom is fluorine or chlorine;

and/or when said R ⁵ In the case of halogen, R is ⁵ 1-3, when 2-3R's are present ⁵ When said R is ⁵ May be the same or different;

and/or when said R ⁵ When halogen, the halogen atom is fluorine or chlorine;

and/or when said R ⁵ is-NR ^a R ^b When said R is ⁵ 1-3, when storedAt 2-3R ⁵ When said R is ⁵ May be the same or different;

and/or when said R ⁵ is-NR ^a R ^b When said R is ^a And R is ^b May be the same or different.
The polycyclic pyrimidine derivative, a pharmaceutically acceptable salt thereof, a tautomer thereof, or a stereoisomer thereof according to claim 1, wherein the polycyclic pyrimidine derivative has a structure represented by formula (III):

wherein R is ¹ 、R ²¹ 、R ³ And R is ⁵ Having the same limitations as claim 1; n=1 to 4;

preferably, for the compound of formula (III), R ³ Is optionally covered with 1-3R ³¹ When the substituted 4-7 membered heterocyclic group is a 5-6 membered heterocyclic group, the 4-7 membered heterocyclic group is a 5-6 membered heterocyclic group;

and/or R ³ Is optionally covered with 1-3R ³¹ When substituted 4-7 membered heterocyclyl, the heterocyclyl contains 1-2 heteroatoms;

and/or, the R ³ Is optionally covered with 1-3R ³¹ When the substituted 4-7 membered heterocyclic group is, the heteroatom of the heterocyclic group is nitrogen and/or oxygen;

And/or, the R ³ Is optionally covered with 1-3R ³¹ When the number of the hetero atoms of the heterocyclic group is two, the two hetero atoms are the same or different;

and/or, the R ²¹ Selected from H or optionally substituted with 1-3R ²² Substituted C ₁ -C ₃ Alkyl, said R ²² Selected from halogen, C ₁ -C ₄ Alkyl, cyano, hydroxy;

wherein R is ³¹ And R is ²² Has the same limitations as claim 1.
The polycyclic pyrimidine derivative, a pharmaceutically acceptable salt thereof, a tautomer thereof, or a stereoisomer thereof according to claim 1, wherein the polycyclic pyrimidine derivative has a structure represented by formula (IV):

wherein R is ¹ 、L、R ³ And R is ⁵ Having the same limitations as claim 1; n=1 to 4;

preferably, for compounds of formula (IV), R when L is absent or O ³ Is optionally covered with 1-3R ³¹ Substituted 3-7 membered cycloalkyl or 4-7 membered heterocyclyl;

and/or when said R ³ Is optionally covered with 1-3R ³¹ When the substituted 4-7 membered heterocyclic group is, the hetero atom in the heterocyclic group is nitrogen and/or oxygen;

and/or when said R ³ Is optionally covered with 1-3R ³¹ When the substituted 4-7 membered heterocyclic group is, 1-2 hetero atoms are used;

wherein R is ³¹ Has the same limitations as claim 1.
The polycyclic pyrimidine derivative, a pharmaceutically acceptable salt thereof, a tautomer thereof, or a stereoisomer thereof according to claim 1, wherein the polycyclic pyrimidine derivative has a structure represented by formula (V):

wherein R is ¹ 、A ² 、L、R ³ And R is ⁵ Having the same limitations as claim 1; n=1-4.
The polycyclic pyrimidine derivative, a pharmaceutically acceptable salt thereof, a tautomer thereof, or a stereoisomer thereof according to claim 1, wherein the polycyclic pyrimidine derivative is selected from any one of the following structures:
a process for the preparation of the polycyclic pyrimidine derivative, a pharmaceutically acceptable salt thereof, a tautomer thereof or a stereoisomer thereof according to any one of claims 1 to 6, which is selected from one of four processes:

a first method,

Wherein A is ¹ 、A ² 、AR、R ¹ 、R ³ 、R ⁴ And L has the same definition as claim 1;

a second method,

Wherein A is ¹ 、A ² 、AR、R ¹ 、R ³ 、R ⁴ And L has the same definition as claim 1;

method III,

Wherein A is ¹ 、A ² 、AR、R ¹ 、R ³ 、R ⁴ And L has the same definition as claim 1; l is preferably NH; a is that ¹ Preferably C; a is that ² Preferably CR ² ，R ² preferably-OR ² ；

A fourth method,

Wherein X is ¹ 、X ² And X ³ Is halogen; x is X ¹ Preferably fluorine; x is X ² Bromine is preferred; x is X ³ Preferably chlorine; a is that ¹ 、A ² 、AR、R ¹ 、R ³ 、R ⁴ And L has the same definition as claim 1; a is that ¹ Preferably C; a is that ² Is CR (CR) ² ，R ² Preferably cyano; l is preferably O; r is R ¹ Methyl is preferred.
A pharmaceutical composition comprising a polycyclic pyrimidine derivative according to any one of claims 1 to 6, a pharmaceutically acceptable salt thereof, a tautomer thereof, or a stereoisomer thereof;

preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient.
Use of a polycyclic pyrimidine derivative, a pharmaceutically acceptable salt thereof, a tautomer thereof, a stereoisomer thereof, or a pharmaceutical composition according to claim 8 alone or in combination with a MEK signaling pathway inhibitor in the manufacture of a medicament for the treatment of cancer or in the manufacture of a SOS1 inhibitor according to any one of claims 1-6; preferably, the cancer includes, but is not limited to, astrocyte cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, hepatocellular cancer, laryngeal cancer, pancreatic cancer, lung cancer, oral cancer, ovarian cancer, prostate cancer, thyroid cancer, sarcoma, renal cancer, and cholangiocarcinoma; further preferably, the cancer includes, but is not limited to: pancreatic cancer, colorectal cancer, lung cancer, hepatocellular carcinoma, renal cancer, gastric cancer, and cholangiocarcinoma. And/or the MEK signaling pathway inhibitor such as RAF, KRAS, MEK or ERK1/2 inhibitor.
The use of claim 9, wherein the dosage of the polycyclic pyrimidine derivative, pharmaceutically acceptable salt thereof, tautomer thereof, stereoisomer thereof, or pharmaceutical composition is in the range of 10-100mg/kg; and/or the dosage range of the MEK inhibitor is selected from 0.1-0.2mg/kg; preferably, the dosage range of the polycyclic pyrimidine derivative, the pharmaceutically acceptable salt thereof, the tautomer thereof, the stereoisomer thereof or the pharmaceutical composition is 25-50mg/kg; and/or the dosage range of the MEK inhibitor is selected from 0.125mg/kg; and/or the MEK inhibitor is Trametinib.
The use of claim 9, wherein the cancer is a RAS family-related cancer; preferably, the cancer is a KRAS, HRAS or NRAS G12C-related cancer, a KRAS, HRAS or NRAS G12D-related cancer, a KRAS, HRAS or NRAS G12A-related cancer, a KRAS, HRAS or NRAS G12V-related cancer, a KRAS, HRAS or NRAS G13D-related cancer, a KRAS, HRAS or nrg 13C-related cancer, a KRAS, HRAS or NRAS Q61X-related cancer, a KRAS, HRAS or NRAS a 146T-related cancer, a KRAS, HRAS or NRAS a 146V-related cancer or a KRAS, HRAS or NRAS a 146P-related cancer.
A method for treating and/or preventing cancer comprising administering to a human a therapeutically effective amount of the polycyclic pyrimidine derivative according to any one of claims 1-6, a pharmaceutically acceptable salt thereof, a tautomer thereof, a stereoisomer thereof, or a pharmaceutical composition according to claim 8.