CN111233834B

CN111233834B - FAK-targeting compound and markers thereof, and preparation methods and applications of FAK-targeting compound and markers

Info

Publication number: CN111233834B
Application number: CN202010157272.8A
Authority: CN
Inventors: 张华北; 齐月恒
Original assignee: Beijing Normal University
Current assignee: Beijing Normal University
Priority date: 2020-03-09
Filing date: 2020-03-09
Publication date: 2021-08-31
Anticipated expiration: 2040-03-09
Also published as: CN111233834A

Abstract

The invention provides a FAK-targeting compound, which has a structure shown in the following formula (I), wherein R₁is-F, -Cl, -Br, -CH₃or-OCH₃；R₂、R₃、R₅Are methoxy or-H respectively; r₄Is composed of

Or

R₆is-H, -CH₂CH₂OH or-CH₂CH₂F. The invention also provides a class of radiolabeled compounds targeting FAK. The compound has excellent in-vitro FAK kinase activity inhibition effect; the radioactive marker has ideal biological distribution in S180 tumor-bearing mice. The invention also provides a preparation method of the compound and a radioactive marker thereof and application of the compound and the radioactive marker thereof in preparing tumor drugs.

Description

FAK-targeting compound and markers thereof, and preparation methods and applications of FAK-targeting compound and markers

Technical Field

The invention relates to the field of compounds, in particular to a FAK (Focal Adhesion Kinase) targeted compound and an F-18 marker thereof, and a preparation method and application thereof.

Background

Focal Adhesion Kinase (FAK) is an non-receptor tyrosine Kinase and plays a crucial role in various links such as tumor generation, development and metastasis. FAK is highly or over-expressed in most tumor cell types, and thus, FAK is a potential tumor diagnosis target.

It is also necessary to develop radiopharmaceuticals with high specificity and high sensitivity to tumors. Over the past three decades, a number of radiopharmaceuticals have been designed and developed to image and identify the unique biochemical properties of tumor tissue. The high expression phenomenon of FAK in tumors can also be used as early diagnosis, treatment and prognosis evaluation of tumors on the level of radiopharmaceuticals.

In the prior art, some FAK small molecule inhibitors have been in clinical study on the general pharmaceutical level, as listed in table 1 below:

TABLE 1 FAK Small molecule inhibitors currently in the discovery and study stage

However, the tumor inhibition activity of these FAK small molecule inhibitors still needs to be improved. Therefore, it is necessary to develop a new FAK-targeting tumor growth inhibitor, and a good foundation is provided for the FAK-targeting drugs on the radiopharmaceutical layer.

Disclosure of Invention

The primary objects of the present invention are: a novel FAK-targeting compound is provided, which can be used as an effective tumor growth inhibitor molecule and also can be used as an inhibitor molecule of a rare disease tumor body such as pleural mesothelioma.

Another object of the present invention is to: the FAK-targeting compound provides a class of radioactive labels which can be used as an excellent tumor imaging agent for early diagnosis of tumors.

Yet another object of the present invention is to: methods of making the FAK-targeting compound and the radiolabel are presented.

Yet another object of the present invention is to: the FAK-targeting compound and the application of the radioactive marker in preparing early tumor treatment or diagnosis medicines are provided.

The above object of the present invention is achieved by the following technical solutions:

firstly, a compound targeting FAK is provided, and the structure of the compound is shown as the following formula (I):

wherein R is₁is-F, -Cl, -Br, -CH₃or-OCH₃；R₂、R₃、R₅Are respectively-OCH₃or-H; r₄Is composed of

Or

R₆is-H, -CH₂CH₂OH or-CH₂CH₂F。

In a preferred embodiment of the present invention, in the structure of the FAK targeting compound represented by formula (I), R₂is-OCH₃，R₃And R₅Are all H, R₄Is composed of

Namely, the FAK targeting compound has the structure shown in the following formula (I-I):

wherein R is₁is-F, -Cl, -Br, -CH₃or-OCH₃；R₆is-H, -CH₂CH₂OH or-CH₂CH₂F。

More preferably, the FAK targeting compound represented by the formula (I-I) is specifically selected from any one of the following compounds:

compound 27aa, having the structure:

compound 27ba, structure:

compound 27ac, having the structure:

compound 27ae, having the structure:

compound 28ab, having the structure:

compound 28bb, structure:

compound 29, having the structure:

in another preferred embodiment of the present invention, in the structure of the FAK targeting compound represented by formula (I), R₂Is a compound of the formula-H,

wherein R is₁is-F, -Cl, -Br, -CH₃or-OCH₃(ii) a More preferably-Cl or-Br.

More preferably, the FAK targeting compound represented by the formula (I-II) is specifically selected from any one of the following compounds:

compound 31ab, having the structure:

compound 31bb, structure:

compound 31bc, having the structure:

compound 31bd, structure:

alternatively, the first and second electrodes may be,

compound 31be, having the structure:

the invention also provides a compound targeting FAK, which has the structure shown in the following formula (II):

wherein R is₇is-Br or-CF₃，R₈Is composed of

Or

R₉is-OH or-F.

More preferred compounds of formula (II) according to the present invention are specifically selected from any one of the following compounds:

compound 39, having the structure:

compound 43, having the structure:

alternatively, the first and second electrodes may be,

compound 44, having the structure:

on the basis, the invention further provides a radioactive nuclide F-18 labeled compound targeting FAK, which has a structure shown in the following formula (IV), wherein R is₁is-Cl or-Br:

the invention further provides another radionuclide F-18 labeled FAK targeting compound, the structure of which is shown in the following formula (V), wherein R is₁is-CH₃or-Br:

the invention further provides another radionuclide F-18 labeled FAK targeting compound, the structure of which is shown in the following formula (VI), wherein R₇is-Br or-CF₃，R₈Is composed of

Or

The invention further provides a method for preparing the FAK targeting compound and the marker thereof.

When the compound with the structure shown as the formula (I-I) is prepared, when R is₆is-CH₂CH₂OH or-CH₂CH₂When F, the synthetic route is generalThe method is recorded as the first method as follows:

the preparation method comprises the following steps:

A1) stirring 2-chloro-3-nitropyridine (compound 1) and methylsulfonylmethylamine (compound 2) with equal mass in an organic solvent under an alkaline condition at 0 ℃ for reaction for 1-2h to obtain a compound 3;

A2) hydrogenating and reducing the nitro group of the compound 3 obtained in A1) into amino group under the condition of pressure catalysis at room temperature to obtain a compound 4;

A3) stirring and reacting the compound 4 obtained in the step A2) and a compound 5(2,4, 5-trichloropyrimidine, 5-bromo-2, 4-dichloropyrimidine, 5-fluoro-2, 4-dichloropyrimidine or 5-methoxy-2, 4-dichloropyrimidine) for 4-6h at the temperature of 80-90 ℃ under an alkaline condition to obtain a compound 6;

A4) introducing a piperazine structure into 4-fluoro-2-methoxynitrobenzene (compound 9), and hydrogenating under a pressurized catalytic condition to obtain a compound 17;

A5) heating the compound 6 obtained in the step A3), the compound 17 obtained in the step A4) and p-toluenesulfonic acid in an organic solution to 100-120 ℃, stirring and reacting for 4-6h, and extracting with ethyl acetate to obtain the compound with the structure shown in the formula (II).

In a preferred embodiment of the present invention, the introduction of the piperazine structure into the 4-fluoro-2-methoxynitrobenzene (compound 9) in step a4) of the first method step can be specifically accomplished by the following steps:

A41) stirring 4-fluoro-2-methoxynitrobenzene (compound 9) and 1- (2-hydroxyethyl) piperazine (compound 10) in an acetonitrile solution and triethylamine at room temperature overnight until solid is separated out, and separating to obtain solid, namely compound 11;

A42) hydrogenating and reducing the nitro group of the compound 11 obtained in the step A41) into amino group under the condition of pressurized catalysis to obtain a compound 12;

the synthetic route is as follows:

alternatively, the first and second electrodes may be,

A43) heating 4-fluoro-2-methoxynitrobenzene (compound 9) and piperazine (compound 13) in a1, 4-dioxane solution to 110-;

A44) stirring the compound 14 obtained in the step A43) and 1-bromo-2-fluoroethane in an acetonitrile solution and triethylamine at the temperature of 80-90 ℃ for reacting for 2-3h, and extracting to obtain a compound 15;

A45) hydrogenating the nitro group of the compound 15 obtained in the step A44) under the pressure catalysis condition to reduce the nitro group into amino group to obtain a compound 16.

The synthetic route is as follows:

when the compound with the structure shown in the formula (I-II) is prepared, the synthetic route of the invention is generally as follows, and is marked as a second method:

the preparation method comprises the following steps:

B1) stirring 3,4, 5-trimethoxybenzoic acid (compound 18) in a mixed solution of nitric acid and acetic acid at 40 ℃ for reacting for 3-4h to obtain a solid compound 19;

B2) stirring the compound 19 obtained in the step B1) and boron trichloride in an organic solvent at 40 ℃ for reacting for 4-5h, and extracting to obtain a solid compound 20;

B3) stirring the compound 20 obtained in the step B2) and 2-bromoethanol or bromofluoroethane in an organic solvent at a molar ratio of 1: 1.5-2 under an alkaline condition at 70-80 ℃ for 2-3h to obtain a compound 21;

B4) hydrogenating and reducing the nitro group of the compound 21 obtained in the step B3) into amino group under the condition of pressurized catalysis to obtain a compound 22;

B5) the compound 22 obtained in the step B4) and the compound 6 obtained in the step A3) of the method react with p-toluenesulfonic acid in an organic solution by heating to 100 ℃ and 120 ℃ for stirring for 4-6h, and the compound with the structure shown in the formula (I-II) is obtained by extraction with ethyl acetate.

The invention further provides a method for preparing the targeting FAK compound shown as the formula (II) when R is₇is-Br and

the preparation method comprises the following steps:

C1) stirring and reacting 2-amino-N-methylbenzamide and 5-bromo-2, 4-dichloropyrimidine in an organic solvent at 80-90 ℃ for 4-6h under an alkaline condition to obtain a compound 8;

C2) heating compound 8 obtained from p-aminophenylethanol (compound 38) and C1) and p-toluenesulfonic acid in an organic solution to 100-120 ℃, stirring and reacting for 4-6h, and extracting with ethyl acetate to obtain a compound 39;

C3) adding triethylamine and p-toluenesulfonyl chloride into the compound 39 obtained in C2) at 0 ℃ in a dichloromethane solvent, and stirring and reacting for 2-3h at room temperature to obtain a compound 40;

C4) reacting the compound 40 obtained in C3) with tetrabutylammonium fluoride in a tetrahydrofuran solvent at 80-90 ℃ for 1-2h by stirring, and extracting with ethyl acetate to obtain a compound 43.

The invention further provides a method for preparing the targeting FAK compound shown as the formula (II) when R is₇is-CF₃And said R is₈Is composed of

Then, the synthesis route is as follows:

the preparation method comprises the following steps:

D1) adding diisopropylethylamine and 3-aminomethyl benzenesulfonyl hydrochloride (compound 36) into tetrahydrofuran solution of 2, 4-dichloro-5-trifluoromethylpyrimidine (compound 35), and stirring at 0 deg.C for overnight reaction to obtain compound 37;

D2) heating the compound 37 obtained in the step D1) with sulfanilamide alcohol (compound 38) and p-toluenesulfonic acid in an organic solution to 100-;

D3) adding triethylamine and p-toluenesulfonyl chloride into the compound 41 obtained in the step D2) at 0 ℃ in a dichloromethane solvent, and stirring and reacting for 2-3h at room temperature to obtain a compound 42;

D4) reacting the compound 42 obtained in D3) with tetrabutylammonium fluoride in a tetrahydrofuran solvent at 80-90 ℃ for 1-2h under stirring, and extracting with ethyl acetate to obtain a compound 44.

The invention still further provides a process for preparing a radiolabeled targeted FAK compound of formula (IV) when R₁When the molecular weight is-Cl or-Br, the method is marked as method five, and the synthetic route is as follows:

the method comprises the following specific steps:

E1) preparation of labeled precursor Compounds:

heating 4-fluoro-2-methoxynitrobenzene (compound 9) and piperazine (compound 13) in a1, 4-dioxane solution to 110-; catalytically hydrogenating the nitro group of compound 14 to provide compound 23; the compound 23 and the compound 6 obtained in the step A3) of the reference method and p-toluenesulfonic acid are heated to 100 ℃ and 120 ℃ in an organic solution and stirred for reaction for 4-6h, and the labeled precursor compound 29 is obtained by extraction with ethyl acetate;

E2) radiolabeling:

using Kryptofix 2.2.2./, [ 2 ]¹⁸F]KF/K₂CO₃Complexing said labelled precursor compound 29 obtained in step E1)¹⁸F-nucleophilic substitution fluorination to obtain the F-18 labeled targeting FAK compound with the structure shown in the formula (IV).

The invention further provides a method for preparing the radiolabeled target FA shown in the formula (V)A process for preparing a compound of K, wherein R₁is-CH₃or-Br, as method six, the synthetic route is as follows:

the method comprises the following specific steps:

F1) preparation of labeled precursor Compounds:

compound 20 was prepared according to method two steps B1) -B2); catalytically hydrogenating the nitro group of compound 20 to provide compound 31; heating the compound 31 and the compound 6 prepared in the step A3) of the reference method and p-toluenesulfonic acid in an organic solution to 100-120 ℃, stirring and reacting for 4-6h, and extracting with ethyl acetate to obtain a labeled precursor compound 32;

F2) radiolabeling:

using Kryptofix 2.2.2./, [ 2 ]¹⁸F]KF/K₂CO₃Complexing said labelled precursor compound 32 obtained in step F1)¹⁸F-nucleophilic substitution fluorination to obtain the F-18 labeled targeting FAK compound with the structure shown in the formula (V).

The invention still further provides a process for preparing a radiolabeled targeted FAK compound of formula (VI) when R is₇is-Br, and R₈Is composed of

Then, the synthesis route is as follows:

the method comprises the following specific steps:

G1) preparation of labeled precursor Compounds:

preparation according to the method of method three steps C1) -C3) to give the tagged precursor compound 40;

G2) radiolabeling:

using Kryptofix 2.2.2./, [ 2 ]¹⁸F]KF/K₂CO₃Compound step by stepStep G1) of obtaining said labelled precursor compound 40¹⁸F-nucleophilic substitution fluorination to obtain the F-18 labeled targeting FAK compound with the structure shown in the formula (VI).

The invention still further provides a method for preparing a radiolabeled targeted FAK compound of formula (VI), wherein R₇is-CF₃And R is₈Is composed of

Then, the method is written as method eight, and the synthetic route is as follows:

the method comprises the following specific steps:

H1) preparation of labeled precursor Compounds:

preparation of tagged precursor compound 42 according to method three steps D1) -D3);

H2) radiolabeling:

using Kryptofix 2.2.2./, [ 2 ]¹⁸F]KF/K₂CO₃Complexing said labelled precursor Compound 42 obtained in step H1)¹⁸F-nucleophilic substitution fluorination to obtain the F-18 labeled targeting FAK compound with the structure shown in the formula (VI).

The FAK-targeting compound has excellent in-vitro FAK kinase activity inhibition effect; the radioactive marker has ideal biological distribution in S180 tumor-bearing mice.

The radioactive labeled targeting FAK compound prepared by the method has the radiochemical purity of more than 98 percent after conventional separation and purification, and shows good matching with a corresponding standard substance without radioactive labeling within the range of tolerance error and good in-vitro stability as a result of coinjection with the corresponding standard substance without radioactive labeling.

The invention further provides application of the FAK-targeting compound in preparation of tumor treatment medicines, wherein the tumor treatment medicines comprise tumor cell growth inhibition medicines and the like.

The invention further provides application of the radiolabeled targeting FAK compound in preparation of an imaging agent for early diagnosis of tumors.

Drawings

FIG. 1 is a radioligand [ 2 ]¹⁸F]28ab and F-19 standard thereof¹⁹F]HPLC co-injection analysis chromatogram of 28 ab.

FIG. 2 is a radioligand [ 2 ]¹⁸F]31bb and its F-19 standard substance [ alpha ]¹⁹F]HPLC co-injection analysis chromatogram of 31 bb.

FIG. 3 is a radioligand [ 2 ]¹⁸F]31bd and F-19 standard product thereof¹⁹F]HPLC co-injection analysis chromatogram of 31 bd.

FIG. 4 is a radioligand [ 2 ]¹⁸F]43 and F-19 Standard product thereof¹⁹F]43 HPLC co-injection analysis chromatogram.

FIG. 5 is a radioligand [ 2 ]¹⁸F]44 and its F-19 standard product¹⁹F]HPLC co-injection analysis chromatogram of 44.

FIG. 6 shows a compound [ 2 ]¹⁹F]28ab antitumor activity in S180 mouse model.

FIG. 7 shows a compound [ 2 ]¹⁹F]28ab mouse weight change in the S180 mouse model.

Detailed Description

In order to further explain the technical solution of the present invention in detail, the present invention will be further explained below by way of specific examples, but the technical solution of the present invention is not limited to the examples.

Example 1 organic Synthesis of Compound 27aa

A solution of Compound 2(20g, 183.4mmol) in DMF (300mL) was cooled to 0 ℃ on an ice bath, sodium hydride (8.8g, 220mmol, 60% in mineral oil, 1.2equiv) was added portionwise, stirring was carried out at 0 ℃ for 1h, 2-chloro-3-nitropyridine (Compound 1) (20g, 126.5mmol, 0.7equiv) was added, the temperature was slowly raised to room temperature, and stirring was continued for 5 h. After the reaction was complete, DMF was poured into 1L of water and then extracted with ethyl acetate (300mL x 3 times). The ethyl acetate layers were combined, dried over anhydrous sodium sulfate, filtered, concentrated by rotary evaporation, and separated by silica gel column chromatography (petroleum ether/ethyl acetate: 10/1 to 3/1) to give compound 3(22.2g, a tan solid, yield 75.9%); compound 3 is structurally characterized as follows:

¹H NMR(400MHz,CDCl₃,δppm):8.67(dd,J＝1.6Hz,4.7Hz,1H),8.26-8.23(m,1H),7.47-7.44(m,1H),3.42(s,3H),3.07(s,3H).

¹³C NMR(400MHz,CDCl₃,δppm):151.46,146.30,143.23,133.87,123.03,37.38,36.35.

ESI-HRMS m/z calcd for C₇H₁₀N₃O₄S⁺232.0387,found 232.0381[M+H]⁺.

a solution of compound 3(10g,43.3mmol,1equiv) in methanol (200mL) was added to the autoclave at room temperature, 10% palladium on carbon (1g,0.1equiv) was added, hydrogen was introduced to 10bar, and the reaction was carried out at room temperature for 2 hours. Palladium on carbon was filtered through celite, and the filtrate was concentrated by rotary evaporation to give compound 4(8.6g, a violet black solid, yield 99.0%); compound 4 was structurally characterized as follows:

¹H NMR(400MHz,CDCl₃,δppm):7.86(dd,J＝2.4Hz,3.7Hz,1H),7.11-7.06(m,2H),4.27(s,2H),3.23(s,3H),3.08(s,3H).

¹³C NMR(400MHz,CDCl₃,δppm):140.85,140.14,137.80,124.45,124.03,36.93,35.37.

ESI-HRMS m/z calcd for C₇H₁₂N₃O₂S⁺202.0645,found 202.0650[M+H]⁺.

to a solution of compound 4(5g,24.8mmol,1equiv) in DMF (100mL) was added 2,4, 5-trichloropyrimidine (5.4g,29.6mmol,1.2equiv), K₂CO₃(5.1g,37.2mmol,1.5equiv), the mixture was stirred at 90 ℃ for 5 h. After the reaction was completed, the DMF was poured into 500mL of water and then extracted with ethyl acetate (100mL × 3 times). The ethyl acetate layers were combined, dried over anhydrous sodium sulfate, filtered, concentrated by rotary evaporation, and separated by silica gel column chromatography (petroleum ether/ethyl acetate: 10/1 to 3/1) to give compound 6a (7.1g, a tan solid, yield 82.5%); compound 6a was structurally characterized as follows:

¹H NMR(400MHz,CDCl₃,δppm):8.79(d,J＝8.2Hz,1H),8.70(s,1H),8.28(d,J＝4.4Hz,2H),7.44-7.41(m,1H),3.31(s,3H),3.04(s,3H).

¹³C NMR(400MHz,CDCl₃,δppm):157.53,156.16,154.93,144.43,143.69,132.11,130.87,124.13,114.84,37.53,34.76.

ESI-MS:m/z calcd for C₁₁H₁₀Cl₂N₅O₂S^-345.99,found 346.70[M-H]^-.

adding 1- (2-hydroxyethyl) piperazine (compound 10) (9.1g,70.0mmol,1.2equiv) to a solution of 4-fluoro-2-methoxynitrobenzene (compound 9) (10g,58.4mmol,1equiv) in acetonitrile (200mL) at room temperature, the solution being turbid, adding triethylamine (14.8g,146.0mmol,2.5equiv), continuing stirring until the starting material is completely dissolved, the solution being orange and transparent, stirring overnight at room temperature, precipitating a solid, and collecting the solid by filtration to obtain compound 11(12.8g, yellow solid, yield 78.0%); compound 11 was structurally characterized as follows:

¹H NMR(400MHz,CDCl₃,δppm):7.94(d,J＝9.3Hz,1H),6.39(dd,J＝2.2Hz,9.3Hz,1H),6.28(d,J＝2.0Hz,1H),3.90(s,3H),3.66(t,J＝5.2Hz,2H),3.40(t,J＝4.9Hz,4H),2.64(t,J＝5.0Hz,4H),2.60(t,J＝5.3Hz,2H).

¹³C NMR(400MHz,CDCl₃,δppm):155.93,155.22,129.17,128.39,105.13,96.71,59.03,57.56,55.85,52.05,46.74.

ESI-HRMS m/z calcd for C₁₃H₂₀N₃O₄ ⁺282.1448,found 282.1451[M+H]⁺.

a solution of compound 11(10g,35.5mmol,1equiv) in methanol (200mL) was added to the autoclave at room temperature, 10% palladium on carbon (1g,0.1equiv) was added, hydrogen was introduced to 10bar, and the reaction was carried out at room temperature for 2 hours. Palladium on carbon was filtered through celite, and the filtrate was concentrated by rotary evaporation to give compound 12(8.8g, a violet black solid, yield 99.0%); compound 12 was structurally characterized as follows:

¹H NMR(400MHz,CDCl₃,δppm):6.66(d,J＝8.3Hz,1H),6.51(d,J＝2.2Hz,1H),6.42(dd,J＝2.3Hz,8.3Hz,1H),3.83(s,3H),3.67(t,J＝5.2Hz,2H),3.09(t,J＝4.7Hz,4H),2.70(t,J＝4.9Hz,4H),2.60(t,J＝5.3Hz,2H).

¹³C NMR(400MHz,CDCl₃,δppm):147.76,144.61,129.96,115.2,109.20,102.07,59.02,57.47,55.18,52.80,51.01.

ESI-HRMS m/z calcd for C₁₃H₂₂N₃O₂ ⁺252.1707,found 252.1705[M+H]⁺.

to a solution of compound 6a (1g,2.8mmol,1equiv) in DMF (50mL) was added compound 12(1.1g,4.2mmol,1.5equiv), and p-toluenesulfonic acid (0.6g,3.4mmol,1.2equiv), and the mixture was heated to 110 ℃ and stirred for 5 h. After completion of the reaction, the reaction mixture was poured into 200mL of water, followed by extraction with ethyl acetate (50mL × 3 times). The ethyl acetate layers were combined, dried over anhydrous sodium sulfate, filtered, and the concentrated filtrate was rotary-evaporated, and separated by silica gel column chromatography (dichloromethane/methanol ═ 20/1 to 5/1) to give compound 27aa (0.72g, yellow solid, yield 46.2%); compound 27aa was structurally characterized as follows:

¹H NMR(400MHz,CDCl₃,δppm):8.80(dd,J＝1.2Hz,9.4Hz,1H),8.33(s,1H),8.18(dd,J＝1.4Hz,4.4Hz,1H),8.07(s,1H),7.92(d,J＝8.6Hz,1H),7.29-7.26(m,2H),6.52(d,J＝2.2Hz,1H),6.46(dd,J＝2.2Hz,8.7Hz,1H),3.84(s,3H),3.71(t,J＝5.2Hz,2H),3.28(s,3H),3.19(t,J＝4.5Hz,4H),3.04(s,3H),2.76(t,J＝4.7Hz,4H),2.67(t,J＝5.3Hz,2H),

¹³C NMR(400MHz,CDCl₃,δppm):157.58,155.00,154.51,149.49,147.20,143.99,142.39,133.06,130.77,123.63,121.63,120.90,107.59,105.30,100.27,59.09,57.40,55.31,52.64,49.63,37.39,34.90.

ESI-HRMS m/z calcd for C₂₄H₃₂ClN₈O₄S⁺563.1950,found 563.1941[M+H]⁺.

EXAMPLE 2 organic Synthesis of Compound 27ab

Compound 4 was prepared according to the procedure of example 1 and reacted with compound 4 using 5-bromo-2, 4-dichloropyrimidine instead of 2,4, 5-trichloropyrimidine to give compound 6b having a structure similar to compound 6 a;

compound 12 was prepared according to the procedure of example 1, and compound 12 was reacted with compound 6b instead of compound 6a to give compound 27ab (0.51g, pale green solid, yield 33.1%) structurally similar to compound 27 aa. Compound 27ab structure is characterized as follows:

¹H NMR(400MHz,CDCl₃,δppm):8.80(d,J＝6.3Hz,1H),8.35(s,1H),8.19-8.17(m,2H),7.94(d,J＝5.8Hz,1H),7.29-7.27(m,1H),7.19(s,1H),6.53(d,J＝1.6Hz,1H),6.47(dd,J＝1.6Hz,5.8Hz,1H),3.85(s,3H),3.69(t,J＝3.4Hz,2H),3.29(s,3H),3.20(t,J＝3.1Hz,4H),3.05(s,3H),2.75(t,J＝2.8Hz,4H),2.66(t,J＝3.4Hz,2H).

¹³C NMR(400MHz,CDCl₃,δppm):158.66,157.92,156.26,150.01,147.67,144.65,142.91,133.84,131.36,124.15,122.34,121.42,108.35,100.95,94.85,59.72,57.82,55.91,55.84,53.22,50.22,37.88,35.55.

ESI-HRMS m/z calcd for C₂₄H₃₂BrN₈O₄S⁺607.1445,found 607.1446[M+H]⁺.

EXAMPLE 3 organic Synthesis of Compound 27ac

Compound 4 was prepared according to the procedure of example 1, and compound 4 was reacted with 5-fluoro-2, 4-dichloropyrimidine instead of 2,4, 5-trichloropyrimidine to give compound 6c having a structure similar to compound 6 a.

Compound 12 was reacted with compound 6c instead of compound 6b according to the procedure of example 2 to give compound 27ca (0.98g, yellow solid, yield 59.6%) having a structure similar to that of compound 27 ab. Compound 27ac structure is characterized as follows:

¹H NMR(400MHz,CDCl₃,δppm):8.85(d,J＝8.2Hz,1H),8.19(dd,J＝1.2Hz,4.4Hz,1H),8.00-7.96(m,3H),7.32-7.29(m,1H),7.19(s,1H),6.54(d,J＝2.2Hz,1H),6.50(dd,J＝2.0Hz,8.7Hz,1H),3.86(s,3H),3.75(t,J＝4.8Hz,2H),3.28(s,3H),3.23(s,4H),3.04(s,3H),2.82(s,4H),2.72(s,2H).

¹³C NMR(400MHz,CDCl₃,δppm):155.35,149.20,146.75,143.76,142.29,142.00,141.16,140.97,132.96,130.34,123.76,122.44,120.18,107.92,100.54,59.25,57.26,55.36,52.73,49.71,37.56,34.86.

ESI-HRMS m/z calcd for C₂₄H₃₂FN₈O₄S⁺547.2246,found 547.2248[M+H]⁺.

EXAMPLE 4 organic Synthesis of Compound 27ae

Compound 4 was prepared according to the procedure of example 1 and reacted with compound 4 using 5-methoxy-2, 4-dichloropyrimidine instead of 2,4, 5-trichloropyrimidine to give compound 6e having a structure similar to compound 6 a.

Compound 12 was reacted with compound 6e instead of compound 6b according to the procedure of example 2 to give compound 27ae (0.96g, tan solid, yield 59.6%) having a structure similar to that of compound 27 ab. Compound 27ae was structurally characterized as follows:

¹H NMR(400MHz,CDCl₃,δppm):8.99(dd,J＝1.4Hz,8.3Hz,1H),8.28(s,1H),8.14(dd,J＝1.5Hz,4.5Hz,1H),8.09(d,J＝8.6Hz,1H),7.76(s,1H),7.31-7.28(m,1H),7.11(s,1H),6.55-6.50(m,2H),3.91(s,3H),3.87(s,3H),3.75(t,J＝5.0Hz,2H),3.28(s,3H),3.25-3.22(m,4H),3.07(s,3H),2.85-2.83(m,4H),2.74(t,J＝5.1Hz,2H).

¹³C NMR(400MHz,CDCl₃,δppm):153.78,151.34,148.99,146.16,143.42,141.51 136.5,135.05,133.68,129.65 123.91,123.41,119.75,108.28,100.84,59.35,57.24,57.17,57.17,55.43,52.82,49.88,37.54,35.12.

ESI-HRMS m/z calcd for C₂₅H₃₄N₈O₅S⁺559.2446,found 559.2443[M+H]⁺.

EXAMPLE 5 organic Synthesis of Compound 28ab

To a solution of 4-fluoro-2-methoxynitrobenzene (compound 9) (10g,58.4mmol,1equiv) in 1, 4-dioxane (300mL) was added piperazine (compound 13) (6.0g,70.0mmol,1.2equiv), the temperature was raised to 120 ℃ and the mixture was stirred for 2 h. The reaction was concentrated by rotary evaporation and chromatographed on silica gel (dichloromethane/methanol 20/1 to 5/1) to give compound 14(12.1g, yellow solid, 87.6% yield); compound 14 was structurally characterized as follows:

¹H NMR(400MHz,DMSO-d₆,δppm):7.90(d,J＝8.9Hz,1H),6.64-6.61(m,2H),6.28(d,J＝2.0Hz,1H),3.90(s,3H),3.65(t,J＝4.8Hz,4H),3.22(t,J＝5.0Hz,4H).

¹³C NMR(400MHz,DMSO-d₆,δppm):155.84,154.80,129.42,128.21,105.96,98.36,56.65,44.01,42.50.

ESI-HRMS m/z calcd for C₁₁H₁₆N₃O₃ ⁺238.1186,found 238.1192[M+H]⁺.

to a solution of compound 14(10g,42.2mmol,1equiv) in acetonitrile (200mL) was added 1-bromo-2-fluoroethane (7.9g,63.2mmol,1.5equiv) and triethylamine (6.4g,63.3mmol,1.5equiv) at room temperature, and stirring was continued until the starting material was completely dissolved, and the temperature was raised to 90 ℃ and stirred for 3 hours. After completion of the reaction, the reaction mixture was poured into 500mL of water, followed by extraction with ethyl acetate (300mL × 3 times). The ethyl acetate layers were combined, dried over anhydrous sodium sulfate, filtered, and the concentrated filtrate was rotary-evaporated, and separated by silica gel column chromatography (dichloromethane/methanol ═ 20/1 to 5/1) to give compound 15(11.0g, yellow solid, yield 92.1%); compound 15 is structurally characterized as follows:

¹H NMR(400MHz,DMSO-d₆,δppm):7.89(d,J＝9.4Hz,1H),6.61(dd,J＝2.2Hz,9.4Hz,1H),6.53(d,J＝1.8Hz,1H),4.66(t,J＝4.4Hz,1H),4.54(t,J＝4.4Hz,1H),3.91(s,3H),3.45(s,4H),2.73-2.59(m,6H).

¹³C NMR(400MHz,DMSO-d₆,δppm):156.14,155.58,128.32,105.46,97.19,82.77,81.13,57.58,57.38,56.45,52.63,46.52.

ESI-HRMS m/z calcd for C₁₃H₁₉FN₃O₃ ⁺284.1405,found 284.1412[M+H]⁺.

a solution of compound 15(10g,35.3mmol,1equiv) in methanol (200mL) was added to the autoclave at room temperature, 10% palladium on carbon (1g,0.1equiv) was added, hydrogen was introduced to 10bar, and the reaction was carried out at room temperature for 2 hours. Palladium on carbon was filtered through celite, and the filtrate was concentrated by rotary evaporation to give compound 16(8.8g, a violet black solid, yield 99.0%); compound 16 was structurally characterized as follows:

¹H NMR(400MHz,CDCl₃,δppm):6.63(d,J＝8.3Hz,1H),6.51(d,J＝2.4Hz,1H),6.41(dd,J＝2.2Hz,8.3Hz,1H),4.66(t,J＝4.8Hz,1H),4.55(t,J＝4.8Hz,1H),3.81(s,3H),3.09(t,J＝4.9Hz,4H),2.79-2.68(m,6H).

¹³C NMR(400MHz,CDCl₃,δppm):147.67,144.56,129.90,115.12,109.12,102.01,82.43,80.77,57.95,57.75,55.10,53.37,50.80.

ESI-MS m/z calcd for C₁₃H₂₁FN₃O⁺254.16,found 254.38[M+H]⁺.

to compound 6a prepared by the method described in example 1, compound 16(1.1g,4.2mmol,1.5equiv), and p-toluenesulfonic acid (0.6g,3.4mmol,1.2equiv) were added to a solution of compound 6a (1g,2.8mmol,1equiv) in DMF (50mL), and the mixture was stirred at 110 ℃ for 5 hours. After completion of the reaction, the reaction mixture was poured into 200mL of water, followed by extraction with ethyl acetate (50mL × 3 times). The ethyl acetate layers were combined, dried over anhydrous sodium sulfate, filtered, and the concentrated filtrate was rotary-evaporated, and separated by silica gel column chromatography (dichloromethane/methanol ═ 20/1 to 5/1) to give compound 28ab (1.1g, yellow solid, yield 71.1%). Compound 28ab structure was characterized as follows:

¹H NMR(400MHz,CDCl₃,δppm):8.82(dd,J＝1.3Hz,8.2Hz,1H),8.35(s,1H),8.20(dd,J＝1.5Hz,4.5Hz,1H),8.10(s,1H),7.95(d,J＝8.7Hz,1H),7.30-7.27(m,1H),7.20(s,1H),6.55(d,J＝2.3Hz,1H),6.50-6.47(m,1H),4.71(t,J＝4.6Hz,1H),4.59(t,J＝4.6Hz,1H),3.86(s,3H),3.30(s,3H),3.22(t,J＝4.6Hz,4H),3.06(s,3H),2.76-2.71(m,6H).

¹³C NMR(400MHz,CDCl₃,δppm):157.69,155.10,154.62,149.58,147.46,144.09,142.39,133.19,130.87,123.61,121.65,121.02,107.64,100.37,99.71,82.51,82.47,57.99,57.80,55.36,53.27,49.81,37.43,34.99.

ESI-HRMS m/z calcd for C₂₄H₃₁ClFN₈O₃S⁺565.1907,found 565.1911[M+H]⁺.

EXAMPLE 6 organic Synthesis of Compound 28bb

Compound 6b was prepared according to the procedure of example 2 and compound 16 was prepared according to the procedure of example 5, except that compound 6b was used instead of compound 6a to react with compound 16, to give compound 28bb (1.0g, yellow-green solid, 63.5% yield) structurally close to compound 28ab, which was characterized as follows:

¹H NMR(400MHz,CDCl₃,δppm):8.79(dd,J＝1.1Hz,8.2Hz,1H),8.37(s,1H),8.19-8.18(m,2H),7.90(s,1H),7.28-7.26(m,2H),6.54(d,J＝2.3Hz,1H),6.48(dd,J＝2.3Hz,8.7Hz,1H),4.73(t,J＝4.6Hz,1H),4.61(t,J＝4.6Hz,1H),3.85(s,3H),3.29(s,3H),3.24(t,J＝4.5Hz,4H),3.06(s,3H),2.81-2.78(m,6H).

¹³C NMR(400MHz,CDCl₃,δppm):158.19,157.27,155.78,149.76,147.38,144.11,142.44,133.29,130.85,123.67,121.64,121.29,107.76,100.45,94.26,82.19,80.52,57.91,57.71,55.39,53.20,49.60,37.38,35.06.

ESI-MS m/z calcd for C₂₄H₃₁BrFN₈O₃S⁺609.14,found 609.17[M+H]⁺.

example 7 organic Synthesis of Compound 31bb

3,4, 5-Trimethoxybenzoic acid (Compound 18) (20.00g,94.3mmol,1equiv) was added to a mixture of nitric acid (100mL) and acetic acid (200mL) at 0 ℃. The temperature is increased to 40 ℃, and the mixture is stirred for 4 hours. The reaction solution was poured into ice water (500mL), a white solid precipitated, and the solid was collected by filtration to give compound 19(13.6g, yield 68.1%, dark red solid); compound 19 was structurally characterized as follows:

¹H NMR(400MHz,CDCl₃,δppm):7.52(s,2H),3.948(s,3H),3.944(s,6H).

¹³C NMR(400MHz,CDCl₃,δppm):152.60,143.50,143.15,101.07,60.86,56.21.

to a solution of compound 19(10.00g,46.9mmol,1equiv) in anhydrous dichloromethane (200mL) was added dropwise boron trichloride (1.0mol/L dichloromethane solution) (93.8mL,93.8mmol,2.0equiv) at 0 ℃. Slowly heating to 40 ℃, and stirring for 5 h. After completion of the reaction, the reaction mixture was poured into 500mL of water, followed by extraction with ethyl acetate (300mL × 3 times). The ethyl acetate layers were combined, dried over anhydrous sodium sulfate, filtered, and the concentrated filtrate was rotary-evaporated, and subjected to silica gel column chromatography (dichloromethane/methanol ═ 20/1 to 10/1) to give compound 20(3.0g, yellow solid)Bulk, yield 32.8%); compound 20 is structurally characterized as follows:¹H NMR(400MHz,CDCl₃,δppm):7.53(s,2H),3.95(s,6H).

to a solution of compound 20(2g,10.0mmol,1equiv) in acetonitrile (50mL) was added 1-bromo-2-fluoroethane (1.9g,15.0mmol,1.5equiv), and triethylamine (1.5g,15.0mmol,1.5equiv) at room temperature, and the mixture was heated to 80 ℃ and stirred for 2 h. After completion of the reaction, the reaction mixture was poured into 500mL of water, followed by extraction with ethyl acetate (200mL × 3 times). The ethyl acetate layers were combined, dried over anhydrous sodium sulfate, filtered, and the concentrated filtrate was rotary-evaporated, and separated by silica gel column chromatography (dichloromethane/methanol ═ 20/1 to 10/1) to give compound 25(2.2g, yellow solid, yield 90.0%); compound 25 is structurally characterized as follows:

¹H NMR(400MHz,CDCl₃,δppm):7.52(s,2H),4.77(t,J＝4.0Hz,1H),4.21(t,J＝4.0Hz,1H),4.38(t,J＝4.2Hz,1H),4.31(t,J＝4.1Hz,1H),3.93(s,6H).

¹³C NMR(400MHz,CDCl₃,δppm):152.72,143.50,142.05,101.01,83.26,81.57,72.03,71.83,56.26

a solution of compound 25(2g,8.1mmol,1equiv) in methanol (50mL) was added to the autoclave at room temperature, 10% palladium on carbon (0.2g,0.1equiv) was added, hydrogen was introduced to 10bar, and the reaction was carried out at room temperature for 2 hours. Palladium on carbon was filtered through celite, and the filtrate was concentrated by rotary evaporation to give compound 26(1.6g, a violet black solid, 92.0% yield); compound 26 is structurally characterized as follows:

¹H NMR(400MHz,CDCl₃,δppm):5.93(s,2H),4.73(t,J＝4.2Hz,1H),4.61(t,J＝4.2Hz,1H),4.18(t,J＝4.2Hz,1H),4.11(t,J＝4.4Hz,1H),3.80(s,6H).

¹³C NMR(400MHz,CDCl₃,δppm):153.71,142.88,129.04,92.51,83.36,71.95，71.75，55.73.

ESI-HRMS m/z calcd for C₁₀H₁₅FNO₃ ⁺216.1030,found 216.1034[M+H]⁺.

referring to preparation of compound 6b by the method of example 2, compound 26(0.9g,4.2mmol,1.5equiv), and p-toluenesulfonic acid (0.6g,3.4mmol,1.2equiv) were added to a solution of compound 6b (1g,2.8mmol,1equiv) in DMF (50mL), and the mixture was stirred at 110 ℃ for 5 h. After completion of the reaction, the reaction mixture was poured into 200mL of water, followed by extraction with ethyl acetate (50mL × 3 times). The ethyl acetate layers were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by rotary evaporation, and separated by silica gel column chromatography (dichloromethane/methanol ═ 20/1 to 5/1) to give compound 31bb (1.3g, tan solid, yield 88.3%); compound 31bb was structurally characterized as follows:

¹H NMR(400MHz,CDCl₃,δppm):8.80(d,J＝7.9Hz,1H),8.42(s,1H),8.16(d,J＝3.4Hz,1H),8.11(s,1H),7.31(s,1H),7.26-7.22(m,1H),6.74(s,2H),4.76(t,J＝4.0Hz,1H),4.65(t,J＝4.0Hz,1H),4.26(t,J＝4.1Hz,1H),4.19(t,J＝4.1Hz,1H),3.75(s,6H),3.28(s,3H),3.06(s,3H).

¹³C NMR(400MHz,CDCl₃,δppm):157.57,155.12,154.31,153.11,143.94,142.50,135.17,133.02,132.49,130.17,123.86,106.17,98.63,83.39,81.71,71.92,71.72,55.92,37.49,34.93.

ESI-HRMS m/z calcd for C₂₁H₂₅ClFN₆O₅S⁺527.1274,found 527.1270[M+H]⁺.

example 8 Synthesis of Compound 31ab

Compound 6a was obtained according to the procedure of example 1, and compound 6a was reacted with compound 26 instead of compound 6b according to the procedure of example 7 to give compound 31ab (1.3g, tan solid, yield 88.3%); the structure of compound 31ab is characterized as follows:

EXAMPLE 9 Synthesis of Compound 31bc

Compound 6c was obtained according to the procedure of example 3, and compound 31bc (0.4g, tan solid, 28.0% yield) structurally close to compound 31bb was obtained by reacting compound 26 with compound 6c instead of compound 6b according to the procedure of example 7, and its structure was characterized as follows:

¹H NMR(400MHz,CDCl₃,δppm):8.82(d,J＝8.1Hz,1H),8.18(d,J＝3.3Hz,1H),8.07(s,1H),8.01(d,J＝1.6Hz,1H),7.30-7.27(m,1H),7.06(s,1H),6.76(s,2H),4.77(t,J＝4.0Hz,1H),4.65(t,J＝4.0Hz,1H),4.26(t,J＝4.2Hz,1H),4.19(t,J＝4.1Hz,1H),3.78(s,6H),3.29(s,3H),3.05(s,3H).

¹³C NMR(400MHz,CDCl₃,δppm):153.50,149.85,149.75,144.16,142.90,135.79,133.12,132.65,130.28,124.26,98.49,83.75,82.06,72.28,72.08,56.30,37.98,35.19.

ESI-HRMS m/z calcd for C₂₁H₂₅F₂N₆O₅S⁺511.1570,found 511.1578[M+H]⁺.

EXAMPLE 10 Synthesis of Compound 31be

Compound 6e was prepared according to the procedure of example 4; referring to the procedure of example 7, compound 6e instead of compound 6b was reacted with compound 26 to give compound 31be (0.7g, tan solid, 50.0% yield) structurally close to compound 31bb, which was characterized as follows:

¹H NMR(400MHz,CDCl₃,δppm):8.97(d,J＝8.2Hz,1H),8.38(s,1H),8.12(d,J＝3.6Hz,1H),7.71(s,1H),7.38(s,1H),7.27-7.24(m,1H),6.79(s,2H),4.76(t,J＝4.0Hz,1H),4.64(t,J＝4.0Hz,1H),4.25(t,J＝4.2Hz,1H),4.18(t,J＝4.2Hz,1H),3.92(s,3H),3.78(s,6H),3.28(s,3H),3.07(s,3H).

¹³C NMR(400MHz,CDCl₃,δppm):153.71,153.46,151.82,143.77,142.08,136.57,135.58,135.32,133.76,132.07,129.60,124.36,97.98,83.75,82.06,72.28,72.08,57.38,56.28,37.90,35.38.

ESI-HRMS m/z calcd for C₂₂H₂₈FN₆O₆S⁺523.1770,found 523.1777[M+H]⁺.

EXAMPLE 11 Synthesis of Compound 31bd

Preparing a compound 4 according to the method of example 1, and reacting the compound 4 with 5-methyl-2, 4-dichloropyrimidine instead of 2,4, 5-trichloropyrimidine to obtain a compound 6d which has a structure similar to that of the compound 6 a; referring to the procedure of example 7, compound 6d was reacted with compound 26 instead of compound 6b to give compound 31bd (0.4g, tan solid, yield 28.0%) having a structure close to that of compound 31 bb. The structure is characterized as follows:

¹H NMR(400MHz,CDCl₃,δppm):8.87(dd,J＝0.9Hz,8.3Hz,1H),8.13(dd,J＝1.3Hz,4.4Hz,1H),7.96(s,1H),7.80(s,1H),7.28-7.22(m,2H),6.78(s,2H),4.76(t,J＝4.0Hz,1H),4.64(t,J＝4.0Hz,1H),4.25(t,J＝4.2Hz,1H),4.17(t,J＝4.2Hz,1H),3.74(s,6H),3.28(s,3H),3.04(s,3H),2.17(s,3H).

¹³C NMR(400MHz,CDCl₃,δppm):158.35,158.13,155.67,153.06,143.45,141.82,135.97,133.76,131.87,130.23,123.89,106.74,98.04,83.38,81.69,71.91,71.71,55.86,37.3,34.71,12.76.

ESI-HRMS m/z calcd for C₂₂H₂₈FN₆O₅S⁺507.1820,found 507.1817[M+H]⁺.

EXAMPLE 12 Synthesis of Compound 43

To a solution of 2-amino-N-methylbenzamide (Compound 7) (10g,66.6mmol,1equiv) in DMF (200mL) was added 5-bromo-2, 4-dichloropyrimidine (Compound 5b) (14.5g,79.7mmol,1.2equiv), K₂CO₃(13.8g,100.0mmol,1.5equiv), the mixture was stirred at 90 ℃ for 5 h. A yellow solid precipitated from the reaction solution, and the reaction solution was poured into a mixed solution of acetonitrile/water (v: v ═ 1:1,500mL), stirred, filtered, and the filter cake was washed with acetonitrile, and the filter cake was collected as the product compound 8b (15.9g, yellow solid, yield 80.7%); the structure is characterized as follows:

¹H NMR(400MHz,DMSO-d₆,δppm):12.16(s,1H),8.83(s,1H),8.51(d,J＝8.3Hz,1H),8.45(s,1H),7.79(d,J＝7.8Hz,1H),7.60(t,J＝7.7Hz,1H),7.22(t,J＝7.5Hz,1H),2.80(d,J＝4.4Hz,3H).

¹³C NMR(400MHz,DMSO-d₆,δppm):169.17,157.15,156.68,155.83,138.79,132.29,128.60,123.62,121.69,121.36,115.37,26.80.

ESI-HRMS m/z calcd for C₁₂H₁₁C_l2N₄O⁺297.0304,found 297.0310[M+H]⁺.

to a solution of p-aminophenethyl alcohol (compound 38) (5.0g,36.5mmol,1equiv) in DMF (200mL) were added compound 8b (10.0g,29.5mmol,0.8equiv) and p-toluenesulfonic acid (2.0g,11.6mmol,0.3equiv), and the reaction was stirred at 100 ℃ for 5 h. After completion of the reaction, the reaction mixture was poured into 200mL of water, followed by extraction with ethyl acetate (50mL × 3 times). The ethyl acetate layers were combined, dried over anhydrous sodium sulfate, filtered, and the concentrated filtrate was rotary-evaporated, and separated by silica gel column chromatography (dichloromethane/methanol ═ 20/1 to 5/1) to give compound 39(9.3g, white solid, yield 57.7%); the structure is characterized as follows:

¹H NMR(400MHz,DMSO-d₆,δppm):11.30(s,1H),9.32(s,1H),8.70(d,J＝4.5Hz,1H),8.63(d,J＝8.6Hz,1H),8.22(s,1H),7.70(d,J＝7.8Hz,1H),7.51(d,J＝8.4Hz,2H),7.46(m,1H),7.12(m,1H),7.07(d,J＝8.4Hz,2H),4.58(t,J＝5.2Hz,1H),3.55(dd,J＝7.2Hz,12.3Hz,2H),2.77(d,J＝4.5Hz,3H),2.64(t,J＝7.1Hz,2H).

¹³C NMR(400MHz,DMSO-d₆,δppm):169.34,158.76,158.04,156.32,139.75,138.56,133.42,131.80,129.27,128.45,122.52,122.28,121.70,120.30,94.52,62.96,26.84.

ESI-HRMS m/z calcd for C₂₀H₂₁BrN₅O₂ ⁺442.0873,found 442.0869[M+H]⁺.

to a solution of compound 39(1g,2.2mmol,1equiv) in dichloromethane (50mL) at 0 ℃ was added triethylamine (10g,4.9mmol,2.2equiv), and the mixture was stirred at 0 ℃ for 10 minutes, and a solution of p-toluenesulfonyl chloride (1.0g,5.2mmol,2.3equiv) in dichloromethane (5mL) was slowly added dropwise, and after the addition was completed, the mixture was warmed to room temperature and stirred for 2 hours. The reaction solution was concentrated by rotary evaporation and subjected to silica gel column chromatography (dichloromethane/methanol: 20/1 to 10/1) to obtain compound 40(1.1g, yellow solid, yield 84.6%). The structure is characterized as follows:

¹H NMR(400MHz,DMSO-d₆,δppm):11.34(s,1H),9.39(s,1H),8.70(d,J＝2.9Hz,1H),8.61(d,J＝3.3Hz,1H),8.24(s,1H),7.70(d,J＝5.2Hz,1H),7.64(d,J＝5.5Hz,2H),7.49(d,J＝5.4Hz,2H),7.44(t,J＝5.0Hz,1H),7.36(d,J＝5.4Hz,2H),7.11(t,J＝5.0Hz,1H),7.00(d,J＝5.5Hz,2H),4.17(t,J＝4.3Hz,2H),2.80(m,5H),2.33(s,3H).

¹³C NMR(400MHz,DMSO-d₆,δppm):169.06,157.23,145.91,138.37,136.19,131.90,130.64,129.70,128.65,128.52,127.99,126.03,124.12,122.87,122.55,94.45,62.77,38.99,26.88,21.31.

ESI-HRMS m/z calcd for C₂₇H₂₇BrN₅O₄S⁺596.0962 found to be free of [ M + H [ ]]⁺.

A solution of compound 40(0.5g,0.8mmol,1equiv) in tetrahydrofuran (50mL) and a solution of tetrabutylammonium fluoride in 1M tetrahydrofuran (4.0mL,4.0mmol,5equiv) were added to the digestion tank, and the reaction was stirred at 85 ℃ for 1h after the tank was closed. After completion of the reaction, the reaction mixture was poured into 200mL of water, followed by extraction with ethyl acetate (50mL × 3 times). The ethyl acetate layers were combined, dried over anhydrous sodium sulfate, filtered, and the concentrated filtrate was rotary-evaporated, and separated by silica gel column chromatography (dichloromethane/methanol ═ 20/1 to 5/1) to give compound 43(0.3g, yellow-green solid, yield 85.4%); the structure is characterized as follows:

¹H NMR(400MHz,DMSO-d₆,δppm):11.32(s,1H),9.37(s,1H),8.70(d,J＝2.8Hz,1H),8.62(d,J＝4.5Hz,1H),8.23(s,1H),7.70(d,J＝4.8Hz,1H),7.55(d,J＝5.5Hz,2H),7.45(t,J＝5.1Hz,1H),7.12(m,3H),4.62(t,J＝4.3Hz,1H),4.54(t,J＝4.3Hz,1H),2.91(t,J＝4.3Hz,1H),2.87(t,J＝4.3Hz,1H),2.77(d,J＝3.0Hz,2H).

¹³C NMR(400MHz,DMSO-d₆,δppm):169.33,158.70,158.05,156.32,139.71,139.10,131.80,130.18,129.41,128.46,122.57,122.28,121.71,120.34,94.67,85.43,83.78,36.15,35.95,26.84.

ESI-HRMS m/z calcd for C₂₀H₂₀BrFN₅O⁺444.0830,found 444.0834[M+H]⁺.

EXAMPLE 13 Synthesis of Compound 44

To a solution of 2, 4-dichloro-5-trifluoromethylpyrimidine (compound 35) (1g,4.6mmol,1equiv) in THF (50mL) at 0 deg.C was added 3-aminomethylbenzenesulfonyl hydrochloride (compound 36) (1.1g,5.9mmol,1.3equiv), diisopropylethylamine (1.8g,13.8mmol,3equiv), and the suspension was stirred at 0 deg.C overnight. The reaction was concentrated by rotary evaporation, the residue was dissolved in dichloromethane (100mL) and filtered with stirring, the filter cake was washed with dichloromethane (100mL), water (100mL) and the filter cake was collected as product 37(0.6g, white solid, 37.5% yield);

¹H NMR(400MHz,DMSO-d₆,δppm):8.60(t,J＝3.8Hz,1H),8.41(s,1H),7.87(s,1H),7.79(d,J＝4.9Hz,1H),7.62(m,2H),4.68(d,J＝3.9Hz,2H),3.15(s,3H).

¹³C NMR(400MHz,DMSO-d₆,δppm):168.09,163.86,161.34,146.40,145.40,137.94,135.08,131.36,131.18,110.93,110.61,49.17,49.04.

ESI-HRMS m/z calcd for C₁₃H₁₂ClF₃N₃O₂S⁺366.0285,found 366.0281[M+H]⁺.

to a solution of p-aminophenethyl alcohol (compound 38) (5.0g,36.5mmol,1equiv) in DMF (200mL) were added compound 37(10.0g,27.3mmol,0.7equiv) and p-toluenesulfonic acid (2.0g,11.6mmol,0.3equiv), and the reaction was stirred at 100 ℃ for 5 h. After completion of the reaction, the reaction mixture was poured into 200mL of water, followed by extraction with ethyl acetate (50mL × 3 times). The ethyl acetate layers were combined, dried over anhydrous sodium sulfate, filtered, and the concentrated filtrate was rotary-evaporated, and separated by silica gel column chromatography (dichloromethane/methanol ═ 20/1 to 5/1) to give compound 41(7.4g, white solid, yield 43.6%); the structure is characterized as follows:

¹H NMR(400MHz,CD₃OD,δppm):8.10(s,1H),7.78(d,J＝5.3Hz,2H),7.49(m,2H),7.19(s,2H),7.11(s,2H),4.48(s,2H),3.73(t,J＝4.6Hz,2H),3.00(s,3H),2.79(t,J＝4.6Hz,2H).

ESI-HRMS m/z calcd for C₂₁H₂₂F₃N₄O₃S⁺467.1359,found 467.1365[M+H]⁺.

to a solution of compound 41(1g,2.1mmol,1equiv) in dichloromethane (50mL) at 0 ℃ was added triethylamine (0.5g,4.9mmol,2.3equiv), and the mixture was stirred at 0 ℃ for 10 minutes, and a solution of p-toluenesulfonyl chloride (1.0g,5.2mmol,2.4equiv) in dichloromethane (5mL) was slowly added dropwise, and after completion of the addition, the mixture was warmed to room temperature and stirred for 2 hours. The reaction solution was concentrated by rotary evaporation and subjected to silica gel column chromatography (dichloromethane/methanol: 20/1 to 10/1) to obtain compound 42(1.0g, white solid, yield 77.8%). The structure is characterized as follows:

¹H NMR(400MHz,DMSO-d₆,δppm):9.54(s,1H),8.21(s,1H),7.88(s,2H),7.77(d,J＝4.8Hz,1H),7.66(d,J＝7.2Hz,2H),7.59(s,2H),7.46(d,J＝6.8Hz,1H),7.38(d,J＝6.0Hz,2H),7.09(d,J＝6.2Hz,1H),6.97(d,J＝7.2Hz,1H),4.72(s,2H),4.19(t,J＝6.5Hz,2H),3.08(s,3H),2.80(t,J＝7.6Hz,2H),2.35(s,3H).

¹³C NMR(400MHz,DMSO-d₆,δppm):158.20,146.04,141.43,140.79,138.27,136.19,135.69,132.46,130.05,129.59,128.62,126.10,126.02,125.72,125.68,121.50,119.25,62.66,44.36,43.98,38.94,21.31.

ESI-HRMS m/z calcd for C₂₈H₂₈F₃N₄O₅S₂ ⁺621.1448,found 621.1451[M+H]⁺.

a solution of compound 42(0.5g,0.8mmol,1equiv) in tetrahydrofuran (50mL) and a solution of tetrabutylammonium fluoride in 1M tetrahydrofuran (4.0mL,4.0mmol,5equiv) were added to the digestion tank, and the reaction was stirred at 85 ℃ for 1h after the tank was closed. After completion of the reaction, the reaction mixture was poured into 200mL of water, followed by extraction with ethyl acetate (50mL × 3 times). The ethyl acetate layers were combined, dried over anhydrous sodium sulfate, filtered, and the concentrated filtrate was rotary-evaporated, and separated by silica gel column chromatography (dichloromethane/methanol ═ 20/1 to 5/1) to give compound 44(0.3g, white solid, yield 81.5%); the structure is characterized as follows:

¹H NMR(400MHz,CDCl₃,δppm):8.17(s,1H),7.91(s,1H),7.85(d,J＝5.0Hz,1H),7.57(m,2H),7.38(d,J＝5.2Hz,2H),7.15(d,J＝5.4Hz,2H),4.82(d,J＝3.8Hz,2H),4.65(t,J＝4.0Hz,1H),4.58(d,J＝4.0Hz,1H),2.99(m,5H).

¹³C NMR(400MHz,CDCl₃,δppm):59.13,158.76,144.23,141.10,139.80,136.59,133.03,132.98,132.39,129.97,129.50,126.57,126.38,120.78,84.95,83.27,59.32,44.44,36.46,36.26.

ESI-MS m/z calcd for C₂₁H₂₁F₄N₄O₂S⁺469.13,found 469.33[M+H]⁺.

example 14 Synthesis of Label precursor Compound 29, Compound 32, Compound 33

Referring to example 3, compound 14 was prepared, and a solution of compound 14(10g,42.1mmol,1equiv) in methanol (200mL) was added to an autoclave at room temperature, 10% palladium on carbon (1g,0.1equiv) was added, hydrogen was introduced to 10bar, and the reaction was carried out at room temperature for 2 hours. Palladium on carbon was filtered through celite, and the filtrate was concentrated by rotary evaporation to give compound 17(8.6g, a violet black solid, yield 99.0%); compound 23 is structurally characterized as follows:

¹H NMR(400MHz,CDCl₃,δppm):6.61(d,J＝7.8Hz,1H),6.43(s,1H),6.35(s,1H),3.78(s,3H),3.27(s,4H),3.19(s,4H).

¹³C NMR(400MHz,CDCl₃,δppm):147.65,143.13,131.07,115.00,110.24,102.78,55.09,48.35,43.35.

ESI-HRMS m/z calcd for C₁₁H₁₈N₃O⁺208.1444,found 208.1451[M+H]⁺.

compound 6a was prepared by the method described in example 1, and to a solution of Compound 6a (1g,2.8mmol,1equiv) in DMF (50mL) were added compound 23(0.9g,4.2mmol,1.5equiv), and p-toluenesulfonic acid (0.6g,3.4mmol,1.2equiv), and the mixture was heated to 110 ℃ and stirred for 5 h. After completion of the reaction, the reaction mixture was poured into 200mL of water, followed by extraction with ethyl acetate (50mL × 3 times). The ethyl acetate layers were combined, dried over anhydrous sodium sulfate, filtered, and the concentrated filtrate was rotary-evaporated, and separated by silica gel column chromatography (dichloromethane/methanol ═ 20/1 to 5/1) to give compound 29(1.3g, pale yellow solid, yield 90.5%). Compound 29 is structurally characterized as follows:

¹H NMR(400MHz,CDCl₃,δppm):8.79(d,7.8Hz,1H),8.37(s,1H),8.22(d,J＝3.6Hz,1H),8.11(s,1H),8.04(d,J＝8.7Hz,1H),7.77(d,J＝7.7Hz,1H),7.31-7.29(m,1H),7.22(d,J＝6.7Hz,1H),6.51(s,1H),6.47(d,J＝8.8Hz,1H),3.86(s,3H),3.40-3.39(m,8H),3.30(s,3H),3.05(s,3H).

¹³C NMR(400MHz,CDCl₃,δppm):157.38,155.15,154.54,149.17,145.86,144.27,142.64,133.06,130.95,125.47,123.61,120.24,109.05,105.77,101.44,55.47,47.90,43.60,37.39,34.94.

ESI-HRMS m/z calcd for C₂₂H₂₈ClN₈O₃S⁺519.1688,found 519.1683[M+H]⁺.

compound 20 was prepared according to the method of example 7 by adding a solution of compound 20(2g,10.0mmol,1equiv) in methanol (100mL) to an autoclave at room temperature, adding 10% palladium on carbon (0.2g,0.1equiv), introducing hydrogen to 10bar, and reacting at room temperature for 2 hours. Palladium-carbon is filtered by diatomite, filtrate is evaporated and concentrated in a rotary mode to obtain a crude product compound 31(1.6g, purple black solid, yield 95.0%), the crude product is unstable, the next reaction is directly carried out, and mass spectrum low resolution confirmation is only carried out in the step;

ESI-MS m/z calcd for C₈H₁₂NO₃ ⁺170.08,found 170.08[M+H]⁺.

compound 6b was prepared according to the method of example 2, and to a solution of Compound 6b (1g,2.5mmol,1equiv) in DMF (50mL) were added compound 31(0.6g,3.8mmol,1.5equiv), and p-toluenesulfonic acid (0.5g,3.0mmol,1.2equiv), and the mixture was heated to 110 ℃ and stirred for 5 h. After completion of the reaction, the reaction mixture was poured into 200mL of water, followed by extraction with ethyl acetate (50mL × 3 times). The ethyl acetate layers were combined, dried over anhydrous sodium sulfate, filtered, and the concentrated filtrate was rotary-evaporated, and separated by silica gel column chromatography (dichloromethane/methanol ═ 20/1 to 5/1) to give compound 32(0.42g, a reddish brown solid, yield 32.3%); compound 32 was structurally characterized as follows:

¹H NMR(400MHz,CDCl₃,δppm):8.72(s,1H),8.66(d,J＝5.2Hz,1H),8.20(dd,J＝1.0Hz,4.2Hz,1H),8.10(s,1H),7.22(dd,J＝3.1Hz,5.5Hz,1H),6.68(s,2H),3.79(s,6H),3.29(s,3H),3.05(s,3H).

¹³C NMR(400MHz,CDCl₃,δppm):157.07,156.62,147.07,144.64,143.64,132.89,132.41,130.84,129.99,129.76,124.19,100.93,94.50,56.50,37.79,35.28.

ESI-HRMS m/z calcd for C₁₉H₂₂BrN₆O₅S⁺525.0550,found 525.0541[M+H]⁺.

compound 20 was prepared according to the method of example 7, and a solution of Compound 20(2g,10.0mmol,1equiv) in methanol (100mL) was added to a autoclave at room temperature, 10% palladium on carbon (0.2g,0.1equiv) was added, hydrogen was introduced to 10bar, and the reaction was carried out at room temperature for 2 hours. Palladium-carbon is filtered by diatomite, filtrate is evaporated and concentrated in a rotary mode to obtain a crude product 30(1.6g, purple black solid, yield 95.0%), the crude product is unstable, the next reaction is directly carried out, and mass spectrum low resolution confirmation is only carried out in the step;

ESI-MS m/z calcd for C₈H₁₂NO₃ ⁺170.08,found 170.08[M+H]⁺.

referring to preparation of compound 6d in example 11, compound 30(0.7g,4.5mmol,1.5equiv), and p-toluenesulfonic acid (0.6g,3.6mmol,1.2equiv) were added to a solution of compound 6d (1g,3.0mmol,1equiv) in DMF (50mL), and the mixture was stirred at 110 ℃ for 5 h. After completion of the reaction, the reaction mixture was poured into 200mL of water, followed by extraction with ethyl acetate (50mL × 3 times). The ethyl acetate layers were combined, dried over anhydrous sodium sulfate, filtered, and the concentrated filtrate was rotary-evaporated, and separated by silica gel column chromatography (dichloromethane/methanol ═ 20/1 to 5/1) to give compound 33(0.59g, pale yellow solid, yield 42.7%); the structure is characterized as follows:

¹H NMR(400MHz,CDCl₃,δppm):8.86(d,J＝5.5Hz,1H),8.11(d,J＝3.0Hz,1H),7.93(s,1H),7.79(s,1H),7.23(dd,J＝3.0Hz,5.4Hz,1H),6.96(s,1H),6.76(s,2H),3.80(s,6H),3.28(s,3H),3.04(s,3H),2.17(s,3H).

¹³C NMR(400MHz,CDCl₃,δppm):158.89,158.75,155.92,147.05,143.67,142.03,134.21,131.83,131.12,130.44,124.23,106.72,99.62,77.42,77.30,77.10,76.78,56.39,37.99,35.10,13.08.

ESI-MS m/z calcd for C₂₀H₂₅N₆O₅S⁺461.16,found 461.19[M+H]⁺.

example 15 the Compound [ 2 ]¹⁸F]28ab、[¹⁸F]31bb and [ 2 ]¹⁸F]F-18 labeling and isolation purification of 31bd

15mg of Kryptofix 222 was dissolved in 0.7mL of anhydrous acetonitrile, 1mg of K₂CO₃Dissolving in 0.3mL water, and mixing to obtain 1.0mL Kryptofix 222/K₂CO₃An eluent; capturing on QMA column with the eluate¹⁸F^-Leaching into a reaction flask, and adding N at 100 DEG C₂Blowing the solvent in the reaction bottle to dry, then adding 0.5mL of anhydrous acetonitrile into the solvent, blowing the solvent to dry again, and repeating the process for three times to ensure that the water in the reaction bottle is fully removed; a solution of the labeled precursor 1, 2-bis-methylphenoxyethane (4mg) in anhydrous acetonitrile (0.3mL) was quickly added to the above reaction flask, sealed, and reacted at 110 ℃ for 10 min. After the reaction, the mixture was subjected to radio-HPLC separation and purification using acetonitrile/water at a ratio of 1:1 as the mobile phase at a flow rate of 4mL/min and a wavelength of 254nm on a C18 reversed-phase semi-preparative column (Agela Technologies, 5 μm,

10X 250 mm). Product 2-, [ 2 ]¹⁸F]The retention time of fluoroethyl tosylate was 12 minutes.

2- [18F ] of the product of the previous step]HPLC receiver of fluoroethyl tosylate was collected in a 100mL sterile vial and 50mL water was added. The diluted product solution was loaded onto a Sep-Pak C-18 column, N₂The Sep-Pak C-18 column was air dried, the product on the Sep-Pak C-18 column was eluted with 3mL of diethyl ether into a sterile vial and dried at 40 ℃ with nitrogen flow. A solution of the labeling precursor (compound 29, compound 32 or compound 33 prepared in example 6) in 4mg of acetonitrile (0.4mL) was added thereto, and anhydrous potassium carbonate (1mg) was further added thereto, followed by uniform mixing and reaction at 120 ℃ for 20 minutes. The column was purified by semi-preparative column (agela technologies, 5 μm,

10X 250mm) to obtain a F-18 labeled radioactive compound [ alpha ], [ beta ] -n ] or a salt thereof¹⁸F]28ab、[¹⁸F]31bb and [ 2 ]¹⁸F]31bd。

As shown in FIG. 1, the product [ alpha ], [ product ]¹⁸F]The liquid phase retention time of 28ab is 10.7 minutes, and¹⁹F]the liquid phase co-injection analysis of 28ab confirmed the radioligand [ 2 ]¹⁸F]28ab accuracy.

As shown in FIG. 2, the product [ 2 ]¹⁸F]The liquid phase retention time of 31bb is 17.1 minutes and¹⁹F]the liquid phase coinjection analysis of 31bb confirmed the radioligand [ 2 ], [¹⁸F]Accuracy of 31 bb.

As shown in FIG. 3, the product [ 2 ]¹⁸F]31bd has a liquid phase retention time of 14.3 minutes and¹⁹F]the liquid phase coinjection analysis of 31bd confirmed the radioligand [ 2 ]¹⁸F]Accuracy of 31 bd.

Example 16 the Compound¹⁸F]43 and [ 2 ]¹⁸F]44F-18 labeling and isolation purification

15mg of Kryptofix 222 was dissolved in 0.7mL of anhydrous acetonitrile, 1mg of K₂CO₃Dissolving in 0.3mL water, and mixing to obtain 1.0mL Kryptofix 222/K₂CO₃An eluent; capturing on QMA column with the eluate¹⁸F^-Leaching into a reaction flask, and adding N at 100 DEG C₂Blowing the solvent in the reaction bottle to dry, then adding 0.5mL of anhydrous acetonitrile into the solvent, blowing the solvent to dry again, and repeating the process for three times to ensure that the water in the reaction bottle is fully removed; a solution (0.3mL) of the labeled precursor compound 40(2mg) prepared according to the method of example 12 (or the labeled precursor compound 42(2mg) prepared according to the method of example 13) in anhydrous acetonitrile was quickly added to the above reaction flask, sealed, and reacted at 120 ℃ for 20 min. After the reaction was completed, distilled water (10mL) was added to quench the reaction, and the reaction solution was drawn up with a syringe and passed through a Sep-Pak C18 solid phase extraction column, N, which was activated in advance₂And (3) air-drying the Sep-Pak C18 solid-phase extraction column, then eluting the reaction product from the C18 small column by using 1mL of acetonitrile, and collecting eluent. The column was purified by semi-preparative column (Agela Technologies, 5 μm,

10X 250mm) to obtain a F-18 labeled radioactive compound [ alpha ], [ beta ] -n ] or a salt thereof¹⁸F]43 or [ alpha ], [ beta ], [ alpha ], [ beta ]¹⁸F]44。

As shown in FIG. 4, the product [ 2 ]¹⁸F]43 has a liquid phase retention time of 9.2 minutes and is as defined in [1 ], [¹⁹F]43 liquid phase coinjection analysis confirmed the radioligand [ alpha ], [ beta ] -cyclodextrin¹⁸F]43 accuracy.

As shown in FIG. 5, the product [ 2 ]¹⁸F]44 has a liquid phase retention time of 8.3 minutes and is as defined in [ 2 ]¹⁹F]44 liquid phase coinjection analysis confirmed the radioligand [ alpha ], [ beta ] -cyclodextrin¹⁸F]44 accuracy.

Examples of the experiments

1. In-vitro FAK enzyme activity inhibition experiment and selective inhibition experiment of different kinase activities of standard compound

The 15 compounds prepared in the preceding examples were used as standards to be evaluated in this experiment.

We use

(Homogeneous Time-Resolved Fluorescence ) kinEASE^TMTK kit and truncated human FAK (PTK2) [376 and 1052(end) amino acids of access number NP-722560.1]The enzyme was subjected to FAK enzyme activity inhibition experiments.

Optimization of enzyme concentration, enzyme reaction time and ATP concentration was first performed prior to the official test.

For the in vitro inhibition experiment of FAK enzyme activity, the optimized conditions are as follows: mu.L of kinase buffer solution of FAK (final concentration of the enzyme reaction step: 0.11 ng/. mu.L (10. mu.L)) in 2. mu.L of kinase buffer, 2. mu.L of saturated TK substrate-biotin solution (final concentration of the enzyme reaction step: 1. mu.M (10. mu.L)), and 2. mu.L of ATP in kinase buffer solution (final concentration of the enzyme reaction step: 13.8. mu.M (10. mu.L)) at room temperature for 50 minutes;

for selective inhibition experiments of different kinase activities, the optimized conditions were: mu.L of kinase buffer solution of kinase 2. mu.L (final concentration of the enzyme reaction step is 0.037 ng/. mu.L (10. mu.L)), 2. mu.L of saturated TK substrate-biotin solution (final concentration of the enzyme reaction step is 1. mu.M (10. mu.L)), together with 2. mu.L of ATP kinase buffer solution (final concentration of the enzyme reaction step is 3. mu.M (10. mu.L) for PYK2, JAK2, EGFR and PDGFR. beta.), final concentration of the enzyme reaction step is 1.6. mu.M (10. mu.L) for IGF-1R, and 1.5. mu.M (10. mu.L) for InsR), at room temperature for a specified time (30 minutes, 40 minutes, 50 minutes, 40 minutes, and 50 minutes for PYK2, JAK2, IGF-1R, InsR, EGFR and PDGFR. beta., respectively).

During the next enzymatic reaction step, 4. mu.L of the serially diluted small molecule compound to be tested (each standard compound described above), 2. mu.L of the TK substrate-biotin solution and 2. mu.L of the FAK kinase solution were first incubated, and then 2. mu.L of the ATP solution was added to start the enzymatic reaction. Next, enzyme buffer (from)

kinEASE^TMTK kit), 5mM MgCl₂1mM DTT (dithiothreitol) and 25nM SEB (staphyloccal enterotoxin B) solution.

The detection reagent (5. mu.L of Eu) is added³⁺Cryptate-labeled TK-antibody and 5. mu.L of streptavidin-XL 665) were dissolved in the detection buffer (in the presence of EDTA), stirred and added to the reaction system. After 1 hour of the above incubation, the TR-FRET (time-resolved fluorescence energy transfer) signal, which is proportional to the phosphorylation level of the kinase, is detected with a microplate reader (BMG FS type). For each small molecule compound (corresponding F-19 standard molecule or drug intermediate molecule) tested, its IC for inhibition of FAK enzyme activity was determined by making a sigmoidal dose-response curve using GraphPad Prism Software (GraphPad Software, San Diego, Calif., USA)₅₀(median inhibitory concentration) value.

In experiments with inhibition of FAK kinase activity in vitro, all standard compounds were evaluated using the HTRF method.

The specific detection results of the standard compound of the invention are shown in table 1.

TABLE 1 results of in vitro inhibition of FAK enzyme Activity by Standard Compounds

As can be seen from table 1 above, the compounds 27aa, 27ab, 28bb and 31bb of the present invention have significant inhibitory effects on the in vitro FAK enzyme activity. The excellent FAK in-vitro kinase activity inhibition capacity of the compounds lays a solid experimental foundation for the compounds to show excellent tumor growth inhibition effect after being prepared into tumor treatment medicines in future, and provides a more sufficient experimental basis for the compounds to be developed into radioactive medicines for early diagnosis and research of tumors in future.

Biodistribution experiment of F-18 radiopharmaceuticals in S180 tumor-bearing mice

2.1 animal model establishment

Under aseptic condition, disinfecting oxter skin of normal female Kunming mouse (18-22g) with alcohol, sucking well-grown S180 ascites, diluting with normal saline (2-3 times), inoculating to oxter subcutaneous tissue of mouse, and inoculating cell number (1-5) x 10⁶. A size that is as long as the tumor size to a diameter of 0.5-0.8cm (about one week is required) can be used for the biological fractionAnd (5) performing a cloth experiment to obtain the S180 tumor mouse.

2.2 in vivo distribution experiments in animal radioactivity

The product of example 15-16 purified by HPLC¹⁸F]28ab、[¹⁸F]31bb、[¹⁸F]31bd、[¹⁸F]43 and [ 2 ]¹⁸F]44(10 μ Ci, dissolved in 0.1mL of physiological saline, containing 5% DMSO) was injected into S180 tumor mice by tail vein injection (18-22g, female, n ═ 5), the mice were sacrificed at 5min, 15min, 30min, 60min, 120min, decapitation, bleeding, brain, heart, liver, spleen, lung, kidney, muscle, bone, intestine, stomach, S180 tumor and tail were dissected, the wet weight of each organ was weighed and measured with γ -counter, uptake per tissue was finally expressed as% ID/g,% ID/g ═ ID/g ÷ 1%, where ID/g ═ radioactivity count of tissue (counts) ÷ tissue mass (mg), 1% ═ average of 1% ID per phase-tail radioactivity count/100.

2.3 discussion of results

TABLE 2. Compound [ 2 ]¹⁸F]Distribution of 28ab in female Kunming S180 tumor mice (18-22g)

The data in the table are the mean ± standard deviation of five measurements;

TABLE 3. Compound [ 2 ]¹⁸F]31bb distribution in female Kunming S180 tumor mice (18-22g)

The data in the table are the mean ± standard deviation of five measurements;

TABLE 4. Compound [ 2 ]¹⁸F]31bd in female Kunming S180 tumor mouse(18-22g) distribution in vivo

The data in the table are the mean ± standard deviation of five measurements.

TABLE 5. Compound [ 2 ]¹⁸F]43 distribution in female Kunming S180 tumor mice (18-22g)

The data in the table are the mean ± standard deviation of five measurements.

TABLE 6. Compound [ 2 ]¹⁸F]44 distribution in female Kunming S180 tumor mice (18-22g)

The data in the table are the mean ± standard deviation of five measurements.

As shown in tables 2-6, the results of the preliminary experiments indicate that the F-18 marker of the present invention shows a trend of increasing tumor uptake in S180 tumor-bearing mice relative to other organs in terms of biodistribution. And the absorption value of the tumor is higher than that of most other organs. The radioactive marker of the invention has ideal biological distribution in S180 tumor-bearing mice, and also provides more sufficient experimental basis for the early diagnosis and research of tumors by developing radioactive drugs for the compounds in the future.

3. Compound [ 2 ]¹⁹F]Pharmacodynamic study of 28ab in S180 murine tumor mouse model

3.1. Establishment of animal model

Under aseptic condition, disinfecting oxter skin of normal female Kunming mouse (18-22g) with alcohol, sucking well-grown S180 ascites, diluting with normal saline (2-3 times), inoculating to oxter subcutaneous tissue of mouse, and inoculating cell number (1-5) x 10⁶。

3.2 animal groups

The test is provided with a negative control group, a positive control group and a treatment group. Wherein the treatment group is provided with a high dosage group, a middle dosage group and a low dosage group. The number of animals per group was 6. The animal models were randomly grouped the next day after establishment.

3.3 dosing regimens

3.4 drug configuration and route of administration

Compound [ 2 ]¹⁹F]28ab was dissolved in 5% Gelucire Glycerol laurate in sterile aqueous solution. The administration was performed orally with a gavage apparatus.

3.5 results of the experiment

Dosing was started after randomization, tumor size was measured and mouse body weight was weighed every 1-2 days. The tumor is approximated as a sphere and the Tumor Volume (TV) is calculated as: (4/3) π r ^ 3. From the measurement results, Relative Tumor Volume (RTV) was calculated, which is Vt/V0. Where V0 is the tumor volume measured at the time of caged administration (i.e., d0) and Vt is the tumor volume at each measurement. The evaluation index of the antitumor activity is relative tumor proliferation rate T/C (%): T/C% ═ TRTV/CRTV × 100%. TRTV: treatment group RTV; CRTV: negative control group RTV. The test period was 18 days.

Table 7. the Compound [ alpha ], [¹⁹F]Pharmacodynamic experiment result of 28ab in S180 murine tumor mouse model

From the results shown in Table 7 above, it is understood that the compound [ 2 ]¹⁹F]28ab has better tumor inhibition activity. Meanwhile, as shown in FIG. 6, the term "2¹⁹F]The three dose groups of 28ab had tumor growth-inhibiting effects, with inhibition rates TGI (%) of 65.3%, 74.5%, 68.2% at the end of the day 18 trial, respectively. The inhibition rate was highest in the medium dose group (25.0mg/kg QOD), and the tumor growth was at the lowest growth rate all the time compared to the other groups, and there was also a case where the tumor volume was decreased. As shown in FIG. 7, the weight of the mice in the dose group (25.0mg/kg QOD) was not significantly reduced in view of the rate of increase in body weight, and was in a state of steady increase, indicating that it had less toxic and side effects in vivo and a higher inhibitory rate.

Claims

1. A method for preparing a radionuclide F-18 labeled FAK-targeting compound,

the structure of the radionuclide F-18 labeled FAK targeting compound is shown as the following formula (IV), wherein R₁is-Cl or-Br:

the preparation method is as follows, wherein R₁is-Cl or-Br:

the method comprises the following specific steps:

E1) preparation of labeled precursor Compounds:

heating 4-fluoro-2-methoxynitrobenzene (compound 9) and piperazine (compound 13) in a1, 4-dioxane solution to 110-; catalytically hydrogenating the nitro group of compound 14 to provide compound 23; heating the compound 23, the compound 6 and p-toluenesulfonic acid in an organic solution to 100-120 ℃, stirring and reacting for 4-6h, and extracting with ethyl acetate to obtain a labeled precursor compound 29;

the compound 6 is synthesized by the following route, wherein R₁is-Cl or-Br:

the method comprises the following specific steps:

A1) stirring 2-chloro-3-nitropyridine (compound 1) and methylsulfonylmethylamine with equal mass in an organic solvent at 0 ℃ to room temperature under an alkaline condition for reaction for 1-2h to obtain a compound 3;

A3) stirring and reacting the compound 4 obtained in the step A2) with the compound 5 at 80-90 ℃ for 4-6h under an alkaline condition to obtain a compound 6; the compound 5 is selected from 2,4, 5-trichloropyrimidine, 5-bromo-2, 4-dichloropyrimidine, 5-fluoro-2, 4-dichloropyrimidine or 5-methoxy-2, 4-dichloropyrimidine;

E2) radiolabeling:

2. The method according to claim 1, characterized in that step a1) comprises in particular the steps of: cooling 300mL DMF solution containing 183.4mmol of methylsulfonylmethylamine to 0 ℃ in ice bath, adding 220mmol of sodium hydride in batches, stirring at 0 ℃ for 1h, adding 126.5mmol of 2-chloro-3-nitropyridine, slowly heating to room temperature, and continuing to stir for 5 h; after the reaction is finished, pouring DMF into 1L of water, and then extracting for 3 times by using 300mL of ethyl acetate; the ethyl acetate layers were combined, dried over anhydrous sodium sulfate, filtered, concentrated by rotary evaporation, and separated by silica gel column chromatography using petroleum ether/ethyl acetate (10/1-3/1) to give compound 3.

3. The method according to claim 2, characterized in that step a2) comprises in particular the steps of: adding 43.3mmol of compound 3 in 200mL of methanol solution in a high-pressure reaction kettle at room temperature, adding 0.1equiv of 10% palladium-carbon, introducing hydrogen to 10bar, and reacting for 2h at room temperature; palladium carbon is filtered by diatomite, and the filtrate is evaporated and concentrated to obtain a compound 4.

4. The method according to claim 3, characterized in that step A3) comprises in particular the steps of: to a solution of 24.8mmol of Compound 4 in 100mL of DMF was added 29.6mmol of 2,4, 5-trichloropyrimidine and 37.2mmol of K₂CO₃Stirring the mixture for 5 hours at 90 ℃; after the reaction is finished, cooling to room temperature, pouring DMF into 500mL of water, and then extracting for 3 times by using 300mL of ethyl acetate; the ethyl acetate layers were combined, dried over anhydrous sodium sulfate, filtered, concentrated by rotary evaporation, and separated by silica gel column chromatography using petroleum ether/ethyl acetate 10/1 to 3/1 to give compound 6 a.

5. The process according to claim 1, wherein the preparation of compound 14 in step E1) comprises in particular the following steps: adding 70.0mmol of piperazine (compound 13) into a solution of 58.4mmol of 4-fluoro-2-methoxynitrobenzene (compound 9) in 300mL of 1, 4-dioxane, heating to 120 ℃, and stirring for 2 h; the reaction mixture was concentrated by rotary evaporation and separated by silica gel column chromatography using dichloromethane/methanol 20/1 to 5/1, whereby compound 14 was obtained.

6. The process according to any one of claims 4 or 5, characterized in that the preparation of compound 29 in step E1) comprises in particular the following steps: adding 4.2mmol of compound 23 and p-3.4 mmol of toluenesulfonic acid into 50mL of DMF solution of 2.8mmol of compound 6a, heating to 110 ℃, and stirring for 5 h; after the reaction is finished, pouring the reaction solution into 200mL of water, and then extracting for 3 times by using 50mL of ethyl acetate; the ethyl acetate layers were combined, dried over anhydrous sodium sulfate, filtered, and the concentrated filtrate was rotary-evaporated, and separated by silica gel column chromatography using dichloromethane/methanol from 20/1 to 5/1, to give compound 29.

7. The method according to any one of claims 1 or 6, characterized in that step E2) comprises in particular the steps of:

15mg of Kryptofix 222 was dissolved in 0.7mL of anhydrous acetonitrile, 1mg of K₂CO₃Dissolving in 0.3mL water, and mixing to obtain 1.0mL Kryptofix 222/K₂CO₃An eluent; capturing on QMA column with the eluate¹⁸F^-Leaching into a reaction flask, and adding N at 100 DEG C₂Blowing the solvent in the reaction bottle to dry, then adding 0.5mL of anhydrous acetonitrile into the solvent, blowing the solvent to dry again, and repeating the process for three times to ensure that the water in the reaction bottle is fully removed; rapidly adding 0.3mL of anhydrous acetonitrile solution of 4mg of 1, 2-bis (methylphenoxyethane) into the reaction bottle, sealing, and reacting for 10min at 110 ℃; after the reaction is finished, acetonitrile and water are used as mobile phases with the ratio of 1:1 to carry out radio-HPLC separation and purification, the flow rate is 4mL/min, the wavelength is 254nm, a C18 reversed phase semi-preparation column is adopted, and the product is 2-, [ product ] 2- ]¹⁸F]The retention time of fluoroethyl tosylate was 12 minutes;

2- [18F ] of the product of the previous step]HPLC receiver of fluoroethyl tosylate was collected in a 100mL sterile vial and 50mL water was added; the diluted product solution was loaded onto a Sep-Pak C-18 column, N₂Air flow drying the Sep-Pak C-18 small column, eluting a product on the Sep-Pak C-18 column into a sterile vial by using 3mL of diethyl ether, and drying by using nitrogen flow at 40 ℃; adding 0.4mL acetonitrile solution of 4mg of compound 29, adding 1mg of anhydrous potassium carbonate, uniformly mixing, and reacting for 20 minutes at 120 ℃; the product was liquid phase separated through a semi-preparative column to give the F-18 labelled radioactive compound.