CN116444454A

CN116444454A - N-hydroxy amidine derivative, preparation method and application thereof, and tumor immunotherapy medicament

Info

Publication number: CN116444454A
Application number: CN202310712338.9A
Authority: CN
Inventors: 山广志; 周霞; 刘伊彤; 赵午莉; 赵聪; 左利民; 朱志玲; 李怡然; 赵学佳; 赵婷; 赵圣楠; 姜艺菲; 连晓芳; 孟庆国
Original assignee: Institute of Medicinal Biotechnology of CAMS
Current assignee: Institute of Medicinal Biotechnology of CAMS
Priority date: 2023-06-16
Filing date: 2023-06-16
Publication date: 2023-07-18
Anticipated expiration: 2043-06-16
Also published as: CN116444454B

Abstract

The invention provides an N-hydroxyamidine derivative, a preparation method and application thereof, and a tumor immunotherapy medicament, belonging to the technical field of medicines. The invention provides an N-hydroxyamidine derivative, which takes an N-hydroxyamidine inhibitor Epacadenostat in a phase III clinical test as a lead, reserves furazan ring, N-hydroxyamidino and benzene ring parts of active sites by researching the crystal structure of an Epacadenostat and IDO1 compound, carries out structural modification on side chain sulfamide, and designs and synthesizes a novel N-hydroxyamidine derivative with better IDO1 inhibition activity, thus being capable of being used for preparing tumor immunotherapy medicaments.

Description

N-hydroxy amidine derivative, preparation method and application thereof, and tumor immunotherapy medicament

Technical Field

The invention relates to the technical field of medicines, in particular to an N-hydroxyamidine derivative, a preparation method and application thereof, and a tumor immunotherapy medicament.

Background

Indoleamine 2,3-dioxygenase 1 (IDO 1) is a heme-containing enzyme that catalyzes the cleavage of the indole ring of tryptophan to N-formylkynurenine in the kynurenine metabolic pathway, a key rate-limiting enzyme that mediates this metabolic pathway. Recent studies have found IDO1 as an important target for the treatment of cancer and other diseases associated with the kynurenine metabolic pathway. The development of novel IDO1 inhibitors for the treatment of tumors associated with immune escape is of great theoretical interest.

Disclosure of Invention

The invention aims to provide an N-hydroxyamidine derivative, a preparation method and application thereof, and a tumor immunotherapy medicament.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an N-hydroxyamidine derivative, which has Sup>A structure shown in Sup>A formulSup>A I, sup>A formulSup>A II, sup>A formulSup>A III, sup>A formulSup>A IV-A, sup>A formulSup>A IV-7 or Sup>A formulSup>A IV-8:

a formula I; />A formula II; />Formula III;

formulSup>A IV-A; />Formula IV-7;

formula IV-8;

r in formula I ₁ Is that、/>、/>、/>、/>、/>、、/>、/>、/>、/>、/>、、/>、/>、/>、/>Or->；

R in formula II ₂ Is that、/>、/>、/>、/>Or->；

R in formula III ¹ is-Cl or-Br;

in the formulSup>A III and the formulSup>A IV-A, R ₃ is-H, -SCH ₃ or-OCH ₃ ，R ₄ is-H or-OCH ₃ ，R ₅ is-H or-OCH ₃ X is-O-or-NH-.

Preferably, the N-hydroxyamidine derivative is any one of the following compounds:

、/>、/>、

、/>、/>、、/>、/>、、/>、/>、、/>、/>、、/>、/>、、/>、/>、

。

the invention provides a preparation method of the N-hydroxyamidine derivative,

(i) The preparation method of the N-hydroxyamidine derivative with the structure shown in the formula I comprises the following steps:

mixing a substituted amine compound, an intermediate 6a and an organic solvent, and performing substitution reaction to obtain an intermediate 9;

mixing the intermediate 9, sodium hydroxide aqueous solution and organic solvent, and performing ring-opening reaction to obtain an N-hydroxyamidine derivative with a structure shown in a formula I;

The substituted amine compound is N-phenyl ethylenediamine, 2- (pyridin-2-yl) ethylamine, 4-fluorophenylethylamine, 3,4, 5-trifluorobenzylamine, 4-methylphenylethylamine, 4-bromophenylethylamine, 2- (4-trifluoromethylphenyl) ethylamine, 4-hydroxyphenylethylamine, 3-fluorophenylethylamine, 2-fluorophenylethylamine, 4-methylthiobenzylamine, 4-methoxybenzylamine, 3-chlorobenzylamine, 4-piperazinylbenzonitrile, 3-hydroxyphenylpiperazine, 1-benzoylpiperazine or 1-acetylpiperazine;

the structural formulas of the intermediate 6a and the intermediate 9 are shown in the following sequence:

；/>；

the R is ₁ As defined in formula I;

(ii) The preparation method of the N-hydroxyamidine derivative with the structure shown in the formula II comprises the following steps:

(ii-1) mixing a substituted formic acid compound, an intermediate 8, 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate, 1-hydroxybenzotriazole and triethylamine with an organic solvent, and carrying out condensation reaction to obtain an intermediate 10;

mixing the intermediate 10, sodium hydroxide aqueous solution and organic solvent, and performing ring-opening reaction to obtain an N-hydroxyamidine derivative with a structure shown in a formula II;

the substituted formic acid compound is 2- (4-morpholinyl) -5-nitrobenzoic acid, 2- (4-thiomorpholinyl) -5-nitrobenzoic acid, 2- (4-Boc-piperazinyl) -5-nitrobenzoic acid, 6-trifluoromethyl nicotinic acid or 2, 5-difluorobenzoic acid;

The structural formulas of the intermediate 8 and the intermediate 10 are shown in the following sequence:

；/>；

the R is ₂ As defined in formula II;

(ii-2) mixing the intermediate 10d with an organic solvent, and carrying out deprotection reaction under acidic conditions to obtain an intermediate 10e;

mixing the intermediate 10e, sodium hydroxide aqueous solution and organic solvent, and performing ring-opening reaction to obtain an N-hydroxyamidine derivative with a structure shown in a formula II;

the structural formulas of the intermediate 10d and the intermediate 10e are shown below in sequence:

；/>；

(iii) The preparation method of the N-hydroxyamidine derivative with the structure shown in the formula III comprises the following steps:

mixing the intermediate 6, the intermediate 13 and an organic solvent with a sodium hydroxide aqueous solution, and performing substitution-ring opening reaction to obtain an N-hydroxyamidine derivative with a structure shown in a formula III;

the structural formulas of the intermediate 6 and the intermediate 13 are shown below in sequence:

；/>；

the R is ¹ 、R ₃ 、R ₄ 、R ₅ And X is as defined in formula III;

(IV-1) Sup>A process for the preparation of an N-hydroxyamidine derivative having the structure represented by the formulSup>A IV-A, comprising the steps of:

mixing the intermediate 5a, the intermediate 14, potassium carbonate and an organic solvent, and carrying out substitution reaction to obtain an intermediate 15;

mixing the intermediate 15, sodium hydroxide aqueous solution and organic solvent, and performing ring-opening reaction to obtain an N-hydroxyamidine derivative with Sup>A structure shown in Sup>A formulSup>A IV-A;

The structural formulas of the intermediate 5a, the intermediate 14 and the intermediate 15 are shown in the following sequence:

；/>；/>；

the R is ₃ 、R ₄ 、R ₅ And X is as defined in formula IV; the R is ₇ is-Cl or-Br;

(IV-2) a process for producing an N-hydroxyamidine derivative having the structure represented by the formula IV-7, comprising the steps of:

mixing the intermediate 8, the intermediate 14a and the potassium carbonate with an organic solvent, and carrying out substitution reaction to obtain an intermediate 15g;

mixing 15g of the intermediate, sodium hydroxide aqueous solution and an organic solvent, and performing ring-opening reaction to obtain an N-hydroxyamidine derivative with a structure shown in a formula IV-7;

the structural formulas of the intermediate 14a and the intermediate 15g are shown below in sequence:

；/>；

(IV-3) a process for producing an N-hydroxyamidine derivative having a structure represented by the formula IV-8, comprising the steps of:

mixing compound CA-4, 2-bromoethanol, triphenylphosphine and diisopropyl azodicarboxylate with an organic solvent for substitution reaction to obtain an intermediate 16;

mixing the intermediate 16, the intermediate 8 and the potassium carbonate with an organic solvent, and performing substitution-ring opening reaction to obtain an N-hydroxyamidine derivative with a structure shown in a formula IV-8;

the structural formulas of the compound CA-4 and the intermediate 16 are shown in the following sequence:

；/>。

the invention provides application of the N-hydroxyamidine derivative or the pharmaceutically acceptable salt thereof in preparation of IDO1 inhibitors.

The invention provides application of the N-hydroxyamidine derivative or the pharmaceutically acceptable salt thereof in preparing tumor immunotherapy medicaments.

Preferably, the tumor comprises non-small cell lung cancer, breast cancer or glioblastoma.

The invention provides a tumor immunotherapy drug, which comprises an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is the N-hydroxyamidine derivative or the pharmaceutically acceptable salt thereof according to the technical scheme.

Preferably, the pharmaceutically acceptable carrier comprises one or more of water for injection, propylene glycol, mannitol, glycerol, stearic acid, sodium chloride, dextrin, glucose, starch, sucrose, lactose, sodium carboxymethyl starch, polyethylene glycol, alginic acid and polysorbate 80.

Preferably, the dosage form of the medicament comprises tablets, capsules, pills, granules, syrups, emulsions, suspensions, aerosols, injections or suppositories.

Preferably, the mode of administration of the drug includes intravenous, oral, inhalation or rectal administration.

The beneficial effects are that: according to the invention, an IDO1 inhibitor Epacadenostat in a phase III clinical test is taken as a lead, based on the crystal complex structure of Epacadenostat and IDO1 enzyme, the furazan ring, N-hydroxyamidino and benzene ring parts of the active sites are reserved, and the structure modification is carried out on side chain sulfamide, so that a novel N-hydroxyamidino derivative with better IDO1 inhibition activity is designed and synthesized. The inhibitory activity of the N-hydroxyamidine derivatives on IDO1 was measured at the enzyme activity level and at the cellular level in the test examples of the present invention, respectively, and the results showed that: compound I-10 (hIDO 1 IC) ₅₀ =0.1868±0.12 μm) and compound II-6 (hIDO 1 IC ₅₀ Enzyme inhibitory Activity = 0.08779 ±0.004 μm with epacoadostat (hIDO 1 IC) ₅₀ = 0.1787 ±0.01 μm); compound I-1 (HeLa EC ₅₀ Inhibitory Activity of IDO1 at cellular level with Epacadenostat (HeLa EC) =0.062.+ -. 0.004. Mu.M ₅₀ =0.068±0.009 μΜ) are equivalent; among them, the compound IV-7 (HeLa EC) has the strongest inhibitory activity on IDO1 at the cellular level ₅₀ = 0.00017 ±0.01 μm), is superior to epacoadostat (HeLa EC ₅₀ =0.068±0.009 μm). Comprehensive comparison of inhibitory Activity of N-hydroxyamidine derivatives of the present invention against IDO1 at both cellular and enzymatic levels, compounds I-1, I-2, II-5, II-6, IV-3, IV-6 and IV-7 are derivatives with better in vitro inhibitory potential for IDO1 activity. To investigate whether the cellular activity of the N-hydroxyamidine derivatives was caused by IDO1 inhibition or by cytotoxicity alone, the CC of the more active compounds I-1, I-2, II-5, II-6, IV-3, IV-6 and IV-7 on HeLa cells was also determined in the test examples according to the invention using a cell-based method ₅₀ And calculating a selection index SI (CC ₅₀ /EC ₅₀ ) Is 91.66-37423 (one)Is generally considered to select an index SI>30, indicating that the cellular activity is caused by IDO1 inhibition). In summary, the N-hydroxyamidine derivative provided by the invention has novel structure and good IDO1 inhibition activity, and can be used for preparing tumor immunotherapy medicaments.

Drawings

FIG. 1 is a graph showing the results of an assay for the activity of inhibiting IDO1 enzyme at the biochemical level of Epacadenostat;

FIG. 2 is a graph showing the results of an assay for the activity of inhibiting IDO1 enzyme at the level of Epacadenostat cells;

FIG. 3 is a graph showing the results of measurement of the activity of the compound I-1 in inhibiting IDO1 enzyme at biochemical level;

FIG. 4 is a graph showing the results of measurement of the inhibitory activity of IDO1 enzyme at the cellular level of Compound I-1;

FIG. 5 is a graph showing the results of measurement of the activity of the compound I-2 in inhibiting IDO1 enzyme at biochemical level;

FIG. 6 is a graph showing the results of measurement of the inhibitory activity of IDO1 enzyme at the cellular level of Compound I-2;

FIG. 7 is a graph showing the results of measurement of the activity of the compound IV-7 in inhibiting the IDO1 enzyme at biochemical level;

FIG. 8 is a graph showing the results of measurement of the inhibitory activity of IDO1 enzyme at the cellular level of Compound IV-7.

Detailed Description

a formula I; />A formula II; />Formula III;

formulSup>A IV-A; />Formula IV-7;

formula IV-8;

R in formula II ₂ Is that、/>、/>、/>、/>Or->；

R in formula III ¹ is-Cl or-Br;

In the present invention, the N-hydroxyamidine derivative is preferably any one of the following compounds:

；/>；/>；

；/>；；

；/>；/>；

。

the invention provides a preparation method of the N-hydroxyamidine derivative, which is described in detail below. In the present invention, unless otherwise specified, all materials are commercially available or prepared by methods well known to those skilled in the art.

In the invention, the preparation method of the N-hydroxyamidine derivative with the structure shown in the formula I comprises the following steps:

mixing a substituted amine compound, an intermediate 6a and an organic solvent, and performing a substitution reaction (denoted as a first substitution reaction) to obtain an intermediate 9;

mixing the intermediate 9, sodium hydroxide aqueous solution and organic solvent, and performing ring-opening reaction (marked as first ring-opening reaction) to obtain an N-hydroxyamidine derivative with a structure shown in a formula I;

；/>；

the R is ₁ As defined in formula I.

The present invention mixes a substituted amine compound, an intermediate 6a and an organic solvent, and performs a first substitution reaction to obtain an intermediate 9. In the present invention, the molar ratio of the substituted amine compound to the intermediate 6a is preferably (1.8 to 2.2): 1, more preferably 2:1. in the present invention, the organic solvent preferably includes Tetrahydrofuran (THF), and the ratio of the organic solvent to the substituted amine compound is preferably (8-12) mL: (0.43 to 1.06) mmol, more preferably 10 mL: (0.43-1.06) mmol. The mode of mixing the substituted amine compound, the intermediate 6a and the organic solvent is not particularly limited, and the mixture may be uniformly mixed. In the present invention, the temperature of the first substitution reaction is preferably 15 to 35 ℃, more preferably room temperature (25 ℃); the time is preferably 2.5 to 3.5 hours, more preferably 3 h. After the first substitution reaction, the present invention preferably concentrates the resulting product system under reduced pressure to afford intermediate 9, which is used directly in the next reaction.

After obtaining the intermediate 9, the invention mixes the intermediate 9, sodium hydroxide aqueous solution and organic solvent, and carries out a first ring-opening reaction to obtain the N-hydroxyamidine derivative with the structure shown in the formula I. In the invention, the concentration of the sodium hydroxide aqueous solution is preferably 1.5-2.5 mol/L, more preferably 2 mol/L; the organic solvent preferably comprises THF; based on the usage amount of the substituted amine compound, the usage amount ratio of the substituted amine compound, the sodium hydroxide aqueous solution and the organic solvent is preferably (0.43-1.06) mmol: (1.5-2.5) mL: (8-12) mL, more preferably (0.43-1.06) mmol:2 mL:10 And (3) mL. The method of mixing the intermediate 9, the aqueous sodium hydroxide solution and the organic solvent is not particularly limited, and the mixture may be uniformly mixed. In the invention, the temperature of the first ring-opening reaction is preferably 15-35 ℃, more preferably room temperature; the time is preferably 3.5 to 4.5 hours, more preferably 4 h. After the first ring-opening reaction, the obtained product system is dissolved by ethyl acetate, washed by water and saturated sodium chloride solution in sequence, dried by anhydrous sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure and then purified by silica gel column chromatography (the eluent is preferably petroleum ether: ethyl acetate=2:1 according to the volume ratio), so as to obtain the N-hydroxyamidine derivative with the structure shown in the formula I.

In the present invention, the preparation method of the N-hydroxyamidine derivative having the structure shown in the formula II is specifically classified into two cases, and is described in detail below.

First case: when the N-hydroxyamidine derivatives having the structure shown in the formula II are compounds II-1, II-2, II-3, II-4 and II-6, the preparation method comprises the following steps:

mixing a substituted formic acid compound, an intermediate 8, 2- (7-aza-benzotriazol) -N, N, N ', N' -tetramethyl urea Hexafluorophosphate (HATU), 1-hydroxybenzotriazole (HOBt) and triethylamine with an organic solvent for condensation reaction to obtain an intermediate 10;

mixing the intermediate 10, sodium hydroxide aqueous solution and organic solvent, and performing ring-opening reaction (denoted as second ring-opening reaction) to obtain an N-hydroxyamidine derivative with a structure shown in formula II;

；/>；

the R is ₂ As defined in formula II.

The invention mixes the substituted formic acid compound, the intermediate 8, HATU, HOBt and triethylamine with the organic solvent to carry out condensation reaction to obtain the intermediate 10. In the present invention, the molar ratio of the substituted formic acid compound, the intermediate 8, HATU, HOBt and triethylamine is preferably (0.51 to 1.27): (0.36 to 0.64): (1.00-1.50): (0.20 to 0.30): (3.50 to 4.00), more preferably (0.51 to 1.27): (0.36 to 0.64): 1.13:0.22:3.75. in the present invention, the organic solvent preferably includes Dichloromethane (DCM); the dosage ratio of the organic solvent to the substituted formic acid compound is preferably (35-45) mL: (0.51 to 1.27) mmol, more preferably 40 mL: (0.51-1.27) mmol. In the present invention, the substituted formic acid compound, HATU, HOBt, triethylamine and the organic solvent are preferably mixed to carry out an activation reaction, and then the obtained product system is mixed with the intermediate 8 to carry out a condensation reaction. In the invention, the temperature of the activation reaction is preferably 15-35 ℃, more preferably room temperature; the time is preferably 20 to 40 minutes, more preferably 30 minutes. In the invention, the temperature of the condensation reaction is preferably 15-35 ℃, more preferably room temperature; the time is preferably 5 to 7 hours, more preferably 6 h. After the condensation reaction, the obtained product system is preferably washed by water and saturated sodium chloride solution in sequence, then dried by anhydrous sodium sulfate, filtered, and filtrate is concentrated under reduced pressure to obtain an intermediate 10a which is directly used for the next reaction.

After the intermediate 10 is obtained, the intermediate 10, a sodium hydroxide aqueous solution and an organic solvent are mixed, and a second ring-opening reaction is carried out, so that the N-hydroxyamidine derivative with the structure shown in the formula II is obtained. In the present invention, the kind and amount of the aqueous sodium hydroxide solution and the organic solvent required for the second ring-opening reaction, the reaction conditions and the post-treatment method are preferably identical to those of the first ring-opening reaction, and are not described herein.

Second case: when the N-hydroxyamidine derivative having the structure shown in formula II is the compound II-5, the preparation method comprises the following steps:

mixing the intermediate 10d with an organic solvent, and carrying out deprotection reaction under acidic conditions to obtain an intermediate 10e;

mixing the intermediate 10e, sodium hydroxide aqueous solution and organic solvent, and performing ring-opening reaction (marked as third ring-opening reaction) to obtain an N-hydroxyamidine derivative with a structure shown in a formula II;

；/>。

the intermediate 10d is mixed with an organic solvent, and a deprotection reaction is performed under acidic conditions to obtain an intermediate 10e. In the present invention, the organic solvent preferably includes ethyl acetate. In the invention, the intermediate 10d, ethyl acetate and HCl ethyl acetate solution are preferably mixed for deprotection reaction; the concentration of HCl in the ethyl acetate solution of HCl is preferably 1.5-2.5 mol/L, more preferably 2 mol/L; the ratio of the amount of intermediate 10d, ethyl acetate to ethyl acetate solution of HCl is preferably 0.25 mmol: (35-45) mL: (15-25) mL, more preferably 0.25 mmol:40 mL:20 And (3) mL. In the invention, the temperature of the deprotection reaction is preferably 15-35 ℃, more preferably room temperature; the time is preferably 3 to 5 hours, more preferably 4 h. After the deprotection reaction, the obtained product system is preferably subjected to suction filtration, and a filter cake is washed by ethyl acetate and then dried under reduced pressure to obtain an intermediate 10e.

After the intermediate 10e is obtained, the intermediate 10e, a sodium hydroxide aqueous solution and an organic solvent are mixed, and a third ring-opening reaction is carried out, so that the N-hydroxyamidine derivative with the structure shown in the formula II is obtained. In the present invention, the kind and amount of the aqueous sodium hydroxide solution and the organic solvent required for the third ring-opening reaction, the reaction conditions and the post-treatment method are preferably identical to those of the first ring-opening reaction, and are not described herein.

In the present invention, a method for preparing an N-hydroxyamidine derivative having a structure represented by formula III includes the steps of:

mixing the intermediate 6, the intermediate 13 and an organic solvent with a sodium hydroxide aqueous solution, and performing a substitution-ring-opening reaction (denoted as a first substitution-ring-opening reaction) to obtain an N-hydroxyamidine derivative with a structure shown in a formula III;

；/>；

the R is ¹ 、R ₃ 、R ₄ 、R ₅ And X is as defined in formula III.

The invention mixes the intermediate 6, the intermediate 13, the organic solvent and the sodium hydroxide aqueous solution. In the present invention, the molar ratio of the intermediate 6 to the intermediate 13 is preferably 1: (0.8 to 1.2), more preferably 1:1. in the present invention, the organic solvent preferably includes THF; the concentration of the sodium hydroxide aqueous solution is preferably 1.5-2.5 mol/L, more preferably 2 mol/L; the dosage ratio of the intermediate 6, the sodium hydroxide aqueous solution and the organic solvent is preferably (0.40-0.61) mmol: (1.5-2.5) mL: (6-10) mL, more preferably (0.40-0.61) mmol:2 mL:8 mL. The method of mixing the intermediate 6, the intermediate 13, the organic solvent and the aqueous sodium hydroxide solution is not particularly limited, and may be uniformly mixed. In the invention, the temperature of the first substitution-ring opening reaction is preferably 15-35 ℃, more preferably room temperature; the time is preferably 3.5 to 4.5 hours, more preferably 4 h. After the first substitution-ring opening reaction, the obtained product system is preferably evaporated to dryness under reduced pressure, ethyl acetate is added into residues to be dissolved, organic matters are washed by water and saturated sodium chloride solution, then are dried by anhydrous sodium sulfate and filtered, and filtrate is subjected to silica gel column chromatography purification after reduced pressure concentration (the eluent is preferably petroleum ether: ethyl acetate=3:1 according to the volume ratio), so that the N-hydroxyamidine derivative with the structure shown in the formula III is obtained.

In the present invention, sup>A method for preparing an N-hydroxyamidine derivative having Sup>A structure represented by formulSup>A IV-A includes the steps of:

intermediate 5a, intermediate 14, potassium carbonate and an organic solvent are mixed and subjected to a substitution reaction (denoted as a second substitution reaction) to obtain intermediate 15;

mixing the intermediate 15, sodium hydroxide aqueous solution and organic solvent, and performing ring opening reaction (marked as fourth ring opening reaction) to obtain an N-hydroxyamidine derivative with Sup>A structure shown in formulSup>A IV-A;

；/>；/>；

the R is ₃ 、R ₄ 、R ₅ And X is as defined in formula IV; the R is ₇ is-Cl or-Br.

The intermediate 5a, the intermediate 14, potassium carbonate and an organic solvent are mixed to perform a second substitution reaction, thereby obtaining an intermediate 15. In the present invention, the molar ratio of the intermediate 5a to the intermediate 14 is preferably 1: (0.8 to 1.2), more preferably 1:1. in the present invention, the organic solvent preferably includes acetonitrile; the dosage ratio of the intermediate 5a, the potassium carbonate and the organic solvent is preferably (0.58-0.67) mmol: (1.00-1.50) mmol: (25-30) mL, more preferably (0.58-0.67) mmol:1.18 mmol:30 And (3) mL. The method of mixing the intermediate 5a, the intermediate 14, the potassium carbonate and the organic solvent is not particularly limited, and the respective components may be uniformly mixed. In the invention, the second substitution reaction is preferably performed under the reflux condition of the system, taking acetonitrile as an example of the organic solvent, and the temperature of the second substitution reaction is preferably 60 ℃; the second substitution reaction time is preferably 15 to 20 hours, more preferably 18 to h. After the second substitution reaction, the product system is preferably filtered to remove potassium carbonate, the filtrate is concentrated under reduced pressure, the remainder is dissolved by ethyl acetate, the organic phase is sequentially washed by water and saturated sodium chloride solution, then dried by anhydrous sodium sulfate, filtered, and the filtrate is purified by silica gel column chromatography (the eluent is preferably petroleum ether: ethyl acetate=1:2 according to the volume ratio) after being concentrated under reduced pressure to obtain an intermediate 15a which is directly used for the next reaction.

After the intermediate 15 is obtained, the intermediate 15, sup>A sodium hydroxide aqueous solution and an organic solvent are mixed, and Sup>A fourth cyclization reaction is carried out, so that the N-hydroxyamidine derivative with the structure shown in the formulSup>A IV-A is obtained. In the present invention, the kind and amount of the aqueous sodium hydroxide solution and the organic solvent required for the fourth ring opening reaction, the reaction conditions and the post-treatment method are preferably identical to the operation conditions related to the first ring opening reaction, and are not described herein.

In the present invention, a method for preparing an N-hydroxyamidine derivative having a structure represented by formula IV-7 includes the steps of:

intermediate 8, intermediate 14a, and potassium carbonate were mixed with an organic solvent, and substitution reaction was performed (denoted as a third substitution reaction), to obtain intermediate 15g;

mixing 15g of the intermediate, sodium hydroxide aqueous solution and an organic solvent, and performing ring-opening reaction (denoted as fifth ring-opening reaction) to obtain an N-hydroxyamidine derivative with a structure shown in formula IV-7;

；/>。

intermediate 8, intermediate 14a, potassium carbonate and an organic solvent are mixed and subjected to a third substitution reaction to obtain intermediate 15g. In the present invention, the types and amounts of potassium carbonate and organic solvent required for the third substitution reaction, the reaction conditions and the post-treatment method are preferably identical to those of the second substitution reaction, and are not described herein.

After 15g of intermediate is obtained, 15g of intermediate, sodium hydroxide aqueous solution and organic solvent are mixed, and a fifth ring-opening reaction is carried out, so that the N-hydroxyamidine derivative with the structure shown in the formula IV-7 is obtained. In the present invention, the kind and amount of the aqueous sodium hydroxide solution and the organic solvent required for the fifth ring-opening reaction, the reaction conditions and the post-treatment method are preferably the same as those of the first ring-opening reaction, and are not described herein.

In the present invention, a method for preparing an N-hydroxyamidine derivative having a structure represented by formula IV-8 includes the steps of:

mixing compound CA-4, 2-bromoethanol, triphenylphosphine and diisopropyl azodicarboxylate with an organic solvent, and performing a substitution reaction (marked as a fourth substitution reaction) to obtain an intermediate 16;

mixing the intermediate 16, the intermediate 8 and the potassium carbonate with an organic solvent, and performing a substitution-ring-opening reaction (denoted as a second substitution-ring-opening reaction) to obtain an N-hydroxyamidine derivative having a structure shown in formula IV-8;

；/>。

the invention mixes the compound CA-4, 2-bromoethanol, triphenylphosphine, diisopropyl azodicarboxylate and organic solvent to carry out the fourth substitution reaction to obtain the intermediate 16. In the present invention, the molar ratio of the compound CA-4, 2-bromoethanol, triphenylphosphine and diisopropyl azodicarboxylate is preferably 0.95: (1.0 to 1.5): (1.0 to 1.5): (1.0 to 1.5), more preferably 0.95:1.14:1.14:1.14. in the present invention, the organic solvent preferably includes toluene; the ratio of the organic solvent to the compound CA-4 is preferably 0.95 mmol: (8-12) mL, more preferably 0.95 mmol:10 And (3) mL. The method for mixing the compound CA-4, 2-bromoethanol, triphenylphosphine, diisopropyl azodicarboxylate and the organic solvent is not particularly limited, and the uniform mixing of the components can be ensured. In the present invention, the temperature of the fourth substitution reaction is preferably 15 to 35 ℃, more preferably room temperature; the time is preferably 20 to 28 hours, more preferably 24 to h. After the fourth substitution reaction, the solvent in the product system is preferably evaporated under reduced pressure, and the residue is separated and purified by silica gel column chromatography (the eluent used is preferably petroleum ether: ethyl acetate=4:1 by volume ratio).

After the intermediate 16 is obtained, the intermediate 16, the intermediate 8 and the potassium carbonate are mixed with an organic solvent to perform a second substitution-ring opening reaction, so that the N-hydroxyamidine derivative with the structure shown in the formula IV-8 is obtained. In the invention, the temperature of the second substitution-ring opening reaction is preferably 75-85 ℃, more preferably 80 ℃; the time is preferably 15 to 24 hours, more preferably 20 h. After the second substitution-ring opening reaction, the obtained product system is preferably filtered to remove potassium carbonate, the filtrate is decompressed and evaporated to dryness and then is separated and purified by silica gel column chromatography (the eluent is preferably dichloromethane: methanol=30:1 according to the volume ratio), so as to obtain the N-hydroxyamidine derivative with the structure shown in the formula III.

The process for preparing the reactants required for the preparation of the N-hydroxyamidine derivatives of the present invention is described below.

In the present invention, the reaction formula for preparing the intermediate 8 is as follows:

。

in the present invention, the preparation method of intermediate 2 preferably comprises the steps of:

mixing malononitrile, sodium nitrite, hydrochloric acid and water, and performing nitrosation reaction to obtain a nitrosation product system;

mixing the nitrosation product system with hydroxylamine hydrochloride aqueous solution, and carrying out rearrangement reaction to obtain a rearrangement product system;

And (3) regulating the pH value of the rearrangement product system to 9.5-10.5, and carrying out dehydration reaction to obtain an intermediate 2.

According to the invention, malononitrile, sodium nitrite and hydrochloric acid are mixed with water to perform nitrosation reaction, so as to obtain a nitrosation product system. In the invention, the concentration of the hydrochloric acid is preferably 1.8-2.2 mol/L, more preferably 2 mol/L; the water is preferably ultrapure water; the dosage ratio of the malononitrile, the sodium nitrite, the hydrochloric acid and the water is preferably 0.15 mol: (0.28 to 0.32) mol: (140-160) mL: (35-45) mL, more preferably 0.15 mol:0.3 mol:150 mL:40 And (3) mL. In the invention, malononitrile is preferably mixed with hydrochloric acid to obtain a malononitrile hydrochloric acid solution; mixing sodium nitrite with water to obtain sodium nitrite aqueous solution; and (3) dropwise adding the malononitrile hydrochloric acid solution into the sodium nitrite aqueous solution, and performing nitrosation reaction. In the invention, the temperature of the nitrosation reaction is preferably 15-35 ℃, more preferably room temperature; the nitrosation reaction time is preferably 10-15 hours, more preferably 12 h; the nitrosation reaction time is counted from the completion of the dropwise addition of the malononitrile hydrochloric acid solution.

After the nitrosation reaction, the present invention preferably cools the obtained nitrosation product system to 0 ℃ and mixes the nitrosation product system with hydroxylamine hydrochloride aqueous solution to perform rearrangement reaction to obtain a rearrangement product system. In the present invention, the molar ratio of the malononitrile to the hydroxylamine hydrochloride in the aqueous hydroxylamine hydrochloride solution is preferably 0.15 based on the amount of the malononitrile: (0.30 to 0.40), more preferably 0.15:0.34; the concentration of the hydroxylamine hydrochloride aqueous solution is preferably 8-9 mmol/mL, more preferably 8.5 mmol/mL. In the present invention, the temperature of the rearrangement reaction is preferably 0 ℃; the time is preferably 20 to 40 minutes, more preferably 30 minutes.

After the rearrangement reaction, the pH value of the obtained rearrangement product system is adjusted to 9.5-10.5 (preferably 10), and the intermediate 2 is obtained through dehydration reaction. In the present invention, the agent for adjusting the pH of the rearrangement product system is preferably an aqueous sodium hydroxide solution; the concentration of the aqueous sodium hydroxide solution is preferably 8 to 12 mol/L, more preferably 10 mol/L. In the present invention, the dehydration reaction preferably includes sequentially performing a first stage reaction and a second stage reaction; the temperature of the first-stage reaction is preferably 15-35 ℃, more preferably room temperature, and the time is preferably 1.5-2.5 h, more preferably 2 h; the second-stage reaction is preferably performed under the condition of system reflux (the temperature is preferably 100 ℃) for preferably 1.5-2.5 hours, and more preferably 2 h. After the dehydration reaction, the obtained product system is preferably placed at room temperature for 10-15 h (preferably 12 h), filtered, and the filter cake is washed with water and dried under reduced pressure to obtain an intermediate 2.

In the present invention, the preparation method of intermediate 3 preferably comprises the steps of:

intermediate 2, sodium nitrite, sodium chloride, hydrochloric acid, acetic acid and water are mixed for diazotization reaction to obtain intermediate 3.

In the invention, the concentration of the hydrochloric acid is preferably 5.5-6.5 mol/L, more preferably 6 mol/L; the dosage ratio of the intermediate 2, sodium nitrite, sodium chloride, hydrochloric acid, acetic acid and water is preferably 58.74 mmol: (55-60) mmol: (170-180) mmol: (25-30) mL: (130-140) mL, more preferably 58.74 mmol:58.0 mmol:177.8 mmol:29 mL:132 And (3) mL. According to the invention, preferably, sodium nitrite is mixed with part of water to obtain a sodium nitrite aqueous solution; mixing the intermediate 2 with the rest water, adding sodium chloride, hydrochloric acid and acetic acid into the obtained feed liquid, heating under reflux (preferably at 45 ℃) until the solution becomes clear and transparent, cooling to-10 to-5 ℃ (preferably at-8 ℃), and dropwise adding the sodium nitrite aqueous solution into the obtained feed liquid for diazotization reaction; the concentration of the sodium nitrite aqueous solution is preferably 3.5-4.5 mmol/mL, more preferably 4.1 mmol/mL. In the present invention, the temperature of the diazotization reaction is preferably 0 ℃; the diazotization reaction time is preferably 2.5-3.5 h, more preferably 3 h; the time of the diazotization reaction is counted from the end of the dropwise addition of the sodium nitrite aqueous solution. After the diazotization reaction, the obtained product system is preferably heated to room temperature and is placed for 2.5-3.5 h (3 h is preferred), the product system is filtered, a filter cake is washed by water, and the product system is dried under reduced pressure to obtain an intermediate 3.

In the present invention, the preparation method of intermediate 4 (specifically including intermediate 4a and intermediate 4 b) preferably includes the steps of:

mixing the intermediate 3, a halogenated aniline compound, sodium bicarbonate and an organic solvent for substitution reaction to obtain an intermediate 4; the halogenated aniline compound is 3-bromo-4-fluoroaniline or 3-chloro-4-fluoroaniline.

In the present invention, the organic solvent preferably includes Tetrahydrofuran (THF); the ratio of the intermediate 3, the halogenated aniline compound, sodium bicarbonate and the organic solvent is preferably 25 mmol: (25-30) mmol: (45-55) mmol: (45-55) mL, more preferably 25.00 mmol:26.95 mmol:50.00 mmol:50 And (3) mL. In the invention, the halogenated aniline compound is preferably mixed with part of the organic solvent to obtain halogenated aniline compound solution; mixing the intermediate 3, sodium bicarbonate and the rest organic solvent, heating to reflux (the temperature is preferably 45 ℃), and then dropwise adding the halogenated aniline compound solution for substitution reaction; the concentration of the halogenated aniline compound solution is preferably 3.0 to 3.5 mmol/mL, more preferably 3.4 mmol/mL. In the present invention, the substitution reaction is preferably carried out under reflux (preferably at 45 ℃ C.) conditions of the system; the time of the substitution reaction is preferably 2.5-3.5 h, more preferably 3 h; the time of the substitution reaction is counted from the completion of the dropwise addition of the halogenated aniline compound solution. After the substitution reaction, the obtained product system is preferably filtered while the system is hot, the filtrate is decompressed and concentrated, petroleum ether is added for pulping (the pulping time is preferably 2 h), suction filtration is carried out, and a filter cake is decompressed and dried, so that the intermediate 4 is obtained.

In the present invention, the preparation method of intermediate 5 (specifically including intermediate 5a and intermediate 5 b) preferably includes the steps of:

intermediate 4, N' -carbonyl diimidazole and an organic solvent are mixed for carrying out a hydroxamic protection reaction to obtain intermediate 5.

In the present invention, the organic solvent preferably includes ethyl acetate; the ratio of the intermediate 4, N' -carbonyldiimidazole to the organic solvent is preferably 12.66 mmol: (23-27) mmol: (75-85) mL, more preferably 12.66 mmol:25.32 mmol:80 And (3) mL. The method of mixing the intermediate 4, the N, N' -carbonyldiimidazole and the organic solvent is not particularly limited, and the components may be uniformly mixed. In the invention, the temperature of the hydroxamic protection reaction is preferably 15-35 ℃, more preferably room temperature; the time is preferably 3.5 to 4.5 hours, more preferably 4 h. After the hydroximic acid protection reaction, the obtained product system is preferably washed once by hydrochloric acid (the concentration is preferably 1 mol/L), water and saturated sodium chloride solution in sequence, then dried by anhydrous sodium sulfate, filtered, concentrated under reduced pressure, added with absolute ethyl alcohol for pulping (the pulping time is preferably 3 h), filtered, and dried under reduced pressure to obtain an intermediate 5.

In the present invention, the preparation method of intermediate 6 (specifically including intermediate 6a and intermediate 6 b) preferably includes the steps of:

intermediate 5, trifluoroacetic acid and aqueous hydrogen peroxide are mixed and subjected to oxidation reaction to obtain intermediate 6.

In the invention, the concentration of the hydrogen peroxide aqueous solution is preferably 28-30wt%; the ratio of the intermediate 5, trifluoroacetic acid to aqueous hydrogen peroxide is preferably 12.35 mmol: (85-95) mL: (25-35) mL, more preferably 12.35 mmol:90 mL:30 And (3) mL. The method of mixing the intermediate 5, trifluoroacetic acid and an aqueous hydrogen peroxide solution is not particularly limited, and the components may be uniformly mixed. In the invention, the temperature of the oxidation reaction is preferably 45-55 ℃, more preferably 45-50 ℃; the time is preferably 15 to 20 hours, more preferably 18 to h. After the oxidation reaction, the invention preferably places the obtained product system in an ice bath, and drops saturated Na ₂ SO ₃ The aqueous solution was filtered until a large amount of precipitation occurred, and the cake was washed with water and dried under reduced pressure to give intermediate 6.

In the present invention, the preparation method of intermediate 7 preferably comprises the steps of:

intermediate 6a, N-t-butoxycarbonyl-1, 2-ethylenediamine and an organic solvent were mixed and subjected to substitution reaction to obtain intermediate 7.

In the present invention, the organic solvent preferably includes tetrahydrofuran; the dosage ratio of the intermediate 6a, the N-tert-butoxycarbonyl-1, 2-ethylenediamine to the organic solvent is preferably 6.05 mmol: (11.70-12.30) mmol: (35-45) mL, more preferably 6.05 mmol:12.10 mmol:40 And (3) mL. The method of mixing the intermediate 6a, N-t-butoxycarbonyl-1, 2-ethylenediamine and the organic solvent is not particularly limited, and the components may be uniformly mixed. In the invention, the temperature of the substitution reaction is preferably 15-35 ℃, more preferably room temperature; the time is preferably 1.5 to 2.5 hours, more preferably 2 h. After the substitution reaction, the present invention preferably concentrates the resulting product system under reduced pressure to afford intermediate 7.

In the present invention, the preparation method of intermediate 8 preferably comprises the steps of:

intermediate 7, ethyl acetate and ethyl acetate solution of HCl were mixed and deprotected to afford intermediate 8.

In the invention, the concentration of the ethyl acetate solution of HCl is preferably 1.5-2.5 mol/L, more preferably 2 mol/L; the ratio of the amount of intermediate 7, ethyl acetate to ethyl acetate solution of HCl is preferably 3.76 mmol: (35-45) mL: (18-22) mL, more preferably 3.76 mmol:40 mL:20 And (3) mL. The method of mixing the intermediate 7, ethyl acetate and ethyl acetate solution of HCl is not particularly limited, and each component may be uniformly mixed. In the invention, the temperature of the deprotection reaction is preferably 15-35 ℃, more preferably room temperature; the time is preferably 3.5 to 4.5 hours, more preferably 4 h. After the deprotection reaction, the obtained product system is preferably subjected to suction filtration, and a filter cake is washed by ethyl acetate and then dried under reduced pressure to obtain an intermediate 8.

In the present invention, the reaction formula for preparing the intermediate 14 is as follows:

；；。

the R is ₃ 、R ₄ 、R ₅ And X is as defined in formula III; the R is ₆ Is that、/>Or->。

In the present invention, the preparation method of the intermediate 14 preferably includes the steps of:

mixing the intermediate 12, ethyl acetate and an ethyl acetate solution of HCl, and carrying out deprotection reaction to obtain an intermediate 13;

mixing the intermediate 13, triethylamine, an acylating reagent and an organic solvent, and carrying out substitution reaction to obtain an intermediate 14; the acylating agent is bromoacetyl bromide or chloroacetyl chloride.

Intermediate 12, ethyl acetate and HCl ethyl acetate solution are mixed for deprotection reaction to obtain intermediate 13. In the present invention, the reagents, material ratios, conditions for deprotection reaction and post-treatment methods used in preparing intermediate 14 are preferably identical to those used in preparing intermediate 8, and will not be described in detail.

After obtaining an intermediate 13, the invention mixes the intermediate 13, an acylating agent, triethylamine and an organic solvent for substitution reaction to obtain an intermediate 14; the acylating agent is bromoacetyl bromide or chloroacetyl chloride. In the present invention, the molar ratio of the intermediate 13, the acylating agent and the triethylamine is preferably (6.96 to 8.11): (10.44 to 12.22): (35 to 40), more preferably (6.96 to 8.11): (10.44 to 12.22): 38.76. in the present invention, the organic solvent is preferably dichloromethane, and the dosage ratio of the intermediate 13 to the organic solvent is preferably (6.96-8.11 mmol): (45-55 mL), more preferably (6.96-8.11) mmol:50 And (3) mL. In the invention, the intermediate 13 is preferably mixed with an organic solvent, triethylamine is added at 0 ℃ and reacts for 30-40 min at 0 ℃, and then an acylating reagent is added for substitution reaction. In the invention, the temperature of the substitution reaction is preferably 15-35 ℃, more preferably room temperature; the time is preferably 5 to 7 hours, more preferably 6 h. After the substitution reaction, the obtained product system is preferably evaporated to dryness under reduced pressure, the remainder is added with ethyl acetate for dissolution, the organic phase is sequentially washed with water and saturated sodium chloride solution, then dried by anhydrous sodium sulfate, filtered, and the filtrate is subjected to silica gel column chromatography purification after reduced pressure concentration (the eluent is preferably petroleum ether: ethyl acetate=1:1 according to the volume ratio), so as to obtain an intermediate 14.

The present invention is preferably prepared by different methods according to the kind of the X group in the intermediate 12, specifically, the intermediate 12 includes an intermediate 12-1 (i.e., x= -O-) and an intermediate 12-2 (i.e., x= -NH-), and the preparation methods of the intermediate 12-1 and the intermediate 12-2 are described in detail below, respectively.

In the present invention, the preparation method of the intermediate 12-1 preferably comprises the steps of:

mixing 2-fluoro-5-nitrobenzoic acid, an alcohol compound, triethylamine and HATU with an organic solvent for condensation reaction to obtain an intermediate 11-1; the structural formula of the alcohol compound is shown as follows:

；

the R is ₃ 、R ₄ And R is R ₅ As defined in formula III;

and mixing the intermediate 11-1, potassium carbonate and 1- (tert-butoxycarbonyl) piperazine with an organic solvent to perform substitution reaction to obtain an intermediate 12-1.

The invention mixes 2-fluoro-5-nitrobenzoic acid, alcohol compound, triethylamine and HATU with organic solvent to carry out condensation reaction to obtain intermediate 11-1. In the present invention, the molar ratio of the 2-fluoro-5-nitrobenzoic acid to the alcohol compound is preferably 1: (0.8 to 1.2), more preferably 1:1, a step of; the molar ratio of the 2-fluoro-5-nitrobenzoic acid, triethylamine and HATU is preferably (16.22-20.00): (90-110): (25-35), more preferably (16.22-20.00): 100:30. in the present invention, the organic solvent is preferably methylene chloride; the dosage ratio of the dichloromethane to the 2-fluoro-5-nitrobenzoic acid is preferably (45-55) mL: (16.22-20.00) mmol, more preferably 50mL: (16.22-20.00) mmol. The invention preferably mixes 2-fluoro-5-nitrobenzoic acid, triethylamine and HATU with organic solvent, and adds alcohol compound to carry out condensation reaction after fully dissolving. In the invention, the temperature of the condensation reaction is preferably 15-35 ℃, more preferably room temperature; the time is preferably 2.5 to 3.5 hours, more preferably 3 h. After the condensation reaction, the obtained product system is preferably concentrated under reduced pressure to remove the solvent, ethyl acetate is used for dissolving residues, the organic phase is sequentially washed by water and saturated sodium chloride solution, then the organic phase is dried by anhydrous sodium sulfate and filtered, and the filtrate is subjected to silica gel column chromatography purification (the eluent is preferably petroleum ether: ethyl acetate=3:1 according to the volume ratio) after the reduced pressure concentration, so as to obtain the intermediate 11-1.

After intermediate 11-1, the intermediate 11-1, potassium carbonate and 1- (tert-butoxycarbonyl) piperazine are mixed with an organic solvent for substitution reaction to obtain intermediate 12-1. In the invention, the molar ratio of the intermediate 11-1, 1- (tert-butoxycarbonyl) piperazine to potassium carbonate is preferably (5.48-6.56): (8.22-9.84): (12-13), more preferably (5.48-6.56): (8.22-9.84): 12.46. in the invention, the organic solvent is preferably N, N-dimethylformamide, and the dosage ratio of the organic solvent to the intermediate 11-1 is preferably (5.48-6.56) mmol: (10-20) mL, more preferably (5.48-6.56) mmol:15 And (3) mL. The method of mixing the intermediate 11-1, potassium carbonate, 1- (t-butoxycarbonyl) piperazine and the organic solvent is not particularly limited, and uniform mixing can be achieved. In the present invention, the substitution reaction is preferably carried out under reflux (temperature is preferably 80 ℃); the time for the substitution reaction is preferably 4 to 6 hours, more preferably 5 h. After the substitution reaction, the invention preferably filters the obtained product system to remove potassium carbonate, adds water into filtrate, extracts with ethyl acetate, washes organic phase by water and saturated sodium chloride solution, then dries with anhydrous sodium sulfate, filters, and carries out silica gel column chromatography purification (the eluent is preferably petroleum ether: ethyl acetate=4:1 according to volume ratio) after the filtrate is decompressed and concentrated, thus obtaining intermediate 12-1.

In the present invention, the preparation method of the intermediate 12-2 preferably comprises the steps of:

2-fluoro-5-nitrobenzoic acid, potassium carbonate and R ₆ Mixing H with an organic solvent to perform substitution reaction to obtain an intermediate 11-2;

mixing the intermediate 11-2, HOBt, HATU and triethylamine with an organic solvent for an activation reaction to obtain an activation product system;

mixing the activated product system with an amine compound, and performing condensation reaction to obtain an intermediate 12-2;

the structural formula of the amine compound is shown as follows:

；

the R is ₃ 、R ₄ And R is R ₅ As defined in formula III; the R is ₆ Is that、/>Or->。

The invention mixes 2-fluoro-5-nitrobenzoic acid, potassium carbonate and F reagent with organic solvent to carry out substitution reaction to obtain intermediate 11-2. In the invention, the molar ratio of the 2-fluoro-5-nitrobenzoic acid, the F reagent and the potassium carbonate is preferably (16.22-20): (30-48.66): (35 to 45), more preferably (16.22 to 20): (30-48.66): 40. in the invention, the organic solvent is preferably N, N-dimethylformamide, and the dosage ratio of the organic solvent to the 2-fluoro-5-nitrobenzoic acid is preferably (10-20) mL: (16.22-20) mmol, more preferably 15 mL: (16.22-20) mmol. The mode of mixing the 2-fluoro-5-nitrobenzoic acid, the potassium carbonate and the F reagent with the organic solvent is not particularly limited, and uniform mixing can be realized. In the present invention, the substitution reaction is preferably carried out under reflux (temperature is preferably 80 ℃); the time for the substitution reaction is preferably 4 to 6 hours, more preferably 5 h. After the substitution reaction, the invention preferably filters the obtained product system to remove potassium carbonate, adds water into the filtrate, adjusts the pH value to 4 by using 1 mol/L hydrochloric acid, then extracts by using ethyl acetate, and washes the organic phase by water and saturated sodium chloride solution, then dries by anhydrous sodium sulfate, filters, and carries out silica gel column chromatography purification (the eluent is preferably petroleum ether: ethyl acetate=1:1 according to the volume ratio) after the filtrate is decompressed and concentrated, thus obtaining the intermediate 11-2.

After the intermediate 11-2 is obtained, the intermediate 11-2, HOBt, HATU and triethylamine are mixed with an organic solvent for an activation reaction to obtain an activation product system. In the present invention, the molar ratio of the intermediates 11-2, HOBt, HATU to triethylamine is preferably 8.5: (2-3): (12-13): (40 to 45), more preferably 8.5:2.55:12.75:42.5. in the present invention, the organic solvent is preferably methylene chloride, and the ratio of the organic solvent to the intermediate 11-2 is preferably 8.50 mmol: (35-45) mL, more preferably 8.50 mmol:40 And (3) mL. The method for mixing the intermediate 11-2, HOBt, HATU, triethylamine and the organic solvent is not particularly limited, and uniform mixing can be realized. In the invention, the temperature of the activation reaction is preferably 15-35 ℃, more preferably room temperature; the time is preferably 25 to 35 minutes, more preferably 30 minutes. After the activation reaction, the invention does not need post-treatment, and the obtained activated product system is directly subjected to subsequent reaction.

After the activated product system is obtained, the activated product system is mixed with an amine compound to perform condensation reaction to obtain an intermediate 12-2. In the present invention, the molar ratio of the amine compound to the intermediate 11-2 is preferably 1: (0.8 to 1.2), more preferably 1:1. in the invention, the temperature of the condensation reaction is preferably 15-35 ℃, more preferably room temperature; the time is preferably 5 to 7 hours, more preferably 6 h. After the condensation reaction, the obtained product system is preferably washed by water and saturated sodium chloride solution in sequence, then dried by anhydrous sodium sulfate, filtered, and filtrate is concentrated under reduced pressure to obtain an intermediate 12-2.

In the present invention, the tumor preferably includes non-small cell lung cancer, breast cancer or glioblastoma.

The invention provides a tumor immunotherapy drug, which comprises an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is the N-hydroxyamidine derivative or the pharmaceutically acceptable salt thereof according to the technical scheme. In the invention, the content of the active ingredient in the medicament is preferably 0.1-99wt%, more preferably 1-99wt%, further preferably 10-90wt%, and even more preferably 30-85wt%. In the present invention, the pharmaceutically acceptable carrier preferably includes one or more of water for injection, propylene glycol, mannitol, glycerin, stearic acid, sodium chloride, dextrin, dextrose, starch, sucrose, lactose, sodium carboxymethyl starch, polyethylene glycol, alginic acid and polysorbate 80. In the present invention, the dosage form of the drug preferably includes tablets, capsules, pills, granules, syrups, emulsions, suspensions, aerosols, injections or suppositories. In the present invention, the administration mode of the drug preferably includes intravenous injection, oral administration, inhalation or rectal administration.

The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Preparation example 1

The synthesis procedure for intermediate 2 is as follows:

stirring and mixing sodium nitrite (20.70 g, 0.3 mol) and ultrapure water (40 mL) at room temperature to obtain a colorless transparent sodium nitrite aqueous solution; malononitrile (9.90 g, 0.15 mol) is pre-dissolved in hydrochloric acid with the concentration of 2 mol/L of 150 mL, then dropwise added into the sodium nitrite aqueous solution, stirred at room temperature for reaction of 12 h, then the obtained product system is cooled to 0 ℃, hydroxylamine hydrochloride (23.00 g, 340.00 mmol) aqueous solution (40 mL) is added for reaction at 0 ℃ for 30 min, then 10 mol/L of sodium hydroxide aqueous solution is used for regulating the pH value to 10, the mixture is moved to room temperature for reaction of 2 h, and then heated to reflux (100 ℃) for reaction of 2 h; after the reaction was completed, the resulting product system was left at room temperature for 12. 12 h, filtered, and the cake was washed with water and dried under reduced pressure to give 14.1. 14.1 g as a white solid as intermediate 2 in a yield of 65.7%.

Preparation example 2

The synthesis procedure for intermediate 3 is as follows:

intermediate 2 (8.40 g, 58.74 mmol) and water (118 mL) are stirred and mixed at room temperature to obtain a white turbid material liquid, then sodium chloride (10.40 g, 177.8 mmol), hydrochloric acid with the concentration of 29 mL of 6 mol/L and 60 mL acetic acid are added, the mixture is refluxed in an oil bath at 45 ℃ until the solution becomes clear and transparent, then cooled to-8 ℃, sodium nitrite (4.00 g, 58.0 mmol) is pre-dissolved in 14 mL water, and then the mixture is added dropwise into the reaction liquid cooled to-8 ℃ to react at 0 ℃ for 3 h; after the reaction was completed, the resulting product system was allowed to stand at room temperature for 3. 3 h, filtered, and the cake was washed with water and dried under reduced pressure to give 4.64. 4.64 g as a white solid as intermediate 3 in a yield of 48.7%.

Preparation example 3

The synthesis procedure for intermediate 4a is as follows:

intermediate 3 (4.05 g, 25.00 mmol), tetrahydrofuran (THF, 50 mL) and sodium bicarbonate (4.20 g, 50.00 mmol) were stirred and mixed in an oil bath at 45 ℃, 3-bromo-4-fluoroaniline (5.121 g, 26.95 mmol) was pre-dissolved in tetrahydrofuran at 8 mL, added dropwise to the above reaction solution at 45 ℃, and reacted for 3 h under reflux (45 ℃), after detection by tlc (petroleum ether: ethyl acetate=1:1, uv development); filtering the product system while the product system is hot, concentrating the filtrate under reduced pressure, adding 50 mL petroleum ether to pulp 2 h, carrying out suction filtration, and drying the filter cake under reduced pressure to obtain a white solid 6.23 g which is intermediate 4a, wherein the yield is 80%.

The procedure was followed for the synthesis of intermediate 4a, except that 3-chloro-4-fluoroaniline was used to prepare intermediate 4b in 78% yield.

Preparation example 4

The synthesis procedure for intermediate 5a is as follows:

80 mL ethyl acetate and intermediate 4a (4.00 g, 12.66 mmol) are stirred and mixed at room temperature, then N, N' -carbonyldiimidazole (4.10 g, 25.32 mmol) is added, the reaction is carried out at room temperature for 4 hours, and TLC detection is finished (the developing agent is petroleum ether: ethyl acetate=2:1, ultraviolet development is carried out according to the volume ratio); the obtained product system is washed once by 60 mL concentration hydrochloric acid, 60 mL water and 60 mL saturated sodium chloride solution in sequence, then dried by anhydrous sodium sulfate, filtered, concentrated under reduced pressure, added with 60 mL absolute ethanol to pulp 3 h, filtered, and dried under reduced pressure to obtain 3.67 g off-white solid which is intermediate 5a with a yield of 84.8%.

The procedure was followed for the synthesis of intermediate 5a, except that intermediate 5b was prepared in 76% yield using intermediate 4 b.

Preparation example 5

The synthesis procedure for intermediate 6a is as follows:

90 mL trifluoroacetic acid, an intermediate 5a (4.20 g, 12.35 mmol) and 30 mL hydrogen peroxide solution with a concentration of 30wt% are mixed, and reacted in an oil bath at 45 ℃ for 18 h, after TLC detection, the reaction is completed (the developing agent is petroleum ether: ethyl acetate=3:1, ultraviolet development is performed by volume ratio); the obtained product system is placed in ice bath, saturated Na is added dropwise ₂ SO ₃ The aqueous solution was filtered until a large amount of precipitate appeared, and the filter cake was washed with water and dried under reduced pressure to give 2.28. 2.28 g as a yellow solid as intermediate 6a in a yield of 49.7%.

The procedure was followed for the synthesis of intermediate 6a, except that intermediate 5b was used to prepare intermediate 6b in 51% yield.

Preparation example 6

The procedure for the synthesis of intermediate 7 is as follows:

40 mL of THF, intermediate 6a (2.24 g, 6.05 mmol) and N-t-butoxycarbonyl-1, 2-ethylenediamine (1.94 g, 12.10 mmol) were mixed and reacted under stirring at room temperature for 2 h to appear a large amount of off-white solid after TLC detection (petroleum ether: ethyl acetate=3:1 as developing agent, ultraviolet coloration); the resulting product system was concentrated under reduced pressure to give 2.34, g as an off-white solid in 79.9% yield as intermediate 7.

Preparation example 7

The synthesis procedure for intermediate 8 is as follows:

mixing 40 mL ethyl acetate, an intermediate 7 (1.82 g, 3.76 mmol) and 20 mL of HCl in ethyl acetate (HCl concentration is 2 mol/L), stirring at room temperature for reaction for 4 hours, and obtaining a large amount of off-white solid, wherein after TLC detection, the reaction is finished (the developing agent is petroleum ether: ethyl acetate=3:1, ultraviolet development is performed; the resulting product system was suction filtered, and the filter cake was washed with ethyl acetate and dried under reduced pressure to give 1.30. 1.30 g as an off-white solid in 89.7% yield as intermediate 8.

Preparation example 8

The procedure for the synthesis of intermediate 11a is as follows:

50 mL of DCM, 2-fluoro-5-nitrobenzoic acid (3.70 g,20.00 mmol), triethylamine (10.10 g,100.00 mmol) and HATU (11.40 g,30.00 mmol) are stirred at room temperature for 10 min to be fully dissolved, 4-methylthiobenzyl alcohol (3.08 g,20.00 mmol) is further added for reaction at room temperature for 3 h, TLC detection is completed (petroleum ether: ethyl acetate=4:1 as developing agent by volume ratio, ultraviolet development); the resulting product system was concentrated under reduced pressure to remove the solvent, the residue was dissolved with ethyl acetate, the organic phase was successively washed with water and saturated sodium chloride solution, then dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography on silica gel (in volume ratio, eluent: petroleum ether: ethyl acetate=3:1) to give 5.73. 5.73 g intermediate 11a as a yellow oily product in 89.2% yield.

The procedure was followed as for the synthesis of intermediate 11a, except that 2-fluoro-5-nitrobenzoic acid (3.00 g, 16.22 mmol) was used to react with 3,4, 5-trimethoxybenzyl alcohol (3.21 g, 16.22 mmol) to afford intermediate 11b as a yellow solid in 69.3% yield.

The procedure was followed as for the synthesis of intermediate 11a, except that 2-fluoro-5-nitrobenzoic acid (3.00 g, 16.22 mmol) was used to react with 4-methoxybenzyl alcohol (2.24 g, 16.22 mmol) to afford intermediate 11c as a yellow solid in 79.8% yield.

The procedure was followed as for the synthesis of intermediate 11a, except that 2-fluoro-5-nitrobenzoic acid (3.00 g, 16.22 mmol) was used to react with 3, 5-dimethoxybenzyl alcohol (2.72 g, 16.22 mmol) to give 4.40 g of intermediate 11d as a yellow solid in 81.0% yield.

The procedure for the synthesis of intermediate 11e is as follows:

15 mL of DMF, 2-fluoro-5-nitrobenzoic acid (3.70 g, 20.00 mmol), potassium carbonate (5.52 g, 40.00 mmol) and 1- (tert-butoxycarbonyl) piperazine (5.55 g, 30.00 mmol) were mixed and reacted under reflux (80 ℃) for 5 h, and TLC detection was completed (in volume ratio, the developing agent was petroleum ether: ethyl acetate=1:2, ultraviolet coloration); the resulting product system was filtered to remove potassium carbonate, 70 mL water was added to the filtrate, the pH was adjusted to 4 with 1 mol/L hydrochloric acid, then extracted with ethyl acetate, the organic phase was washed successively with water and saturated sodium chloride solution, then dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography on silica gel (in terms of volume ratio, eluent: petroleum ether: ethyl acetate=1:1), to give 6.67 g intermediate 11e as a yellow oily product in 95.0% yield.

The procedure was followed as for the synthesis of intermediate 11e, except for the reaction of 2-fluoro-5-nitrobenzoic acid (3.00 g, 16.22 mmol) with thiomorpholine (5.01 g, 48.66 mmol) to give 3.76 g intermediate 11f as an off-white solid in 86.4% yield.

The procedure was followed as for the synthesis of intermediate 11e, except that 2-fluoro-5-nitrobenzoic acid (3.00 g, 16.22 mmol) was used to react with morpholine (4.23 g, 48.66 mmol) to give 3.56 g of intermediate 11g as a yellow solid in 87.1% yield.

Preparation example 9

The procedure for the synthesis of intermediate 12a is as follows:

15 mL of DMF, intermediate 11a (2.00 g, 6.23 mmol), potassium carbonate (1.72 g, 12.46 mmol) and 1- (t-butoxycarbonyl) piperazine (1.75 g, 9.35 mmol) were mixed and reacted for 5 h under reflux (80 ℃ C.) conditions, TLC detection reaction was complete (petroleum ether: ethyl acetate=4:1, UV coloration: developing solvent by volume ratio); the resulting product system was filtered to remove potassium carbonate, 70 mL water was added to the filtrate, extracted with ethyl acetate, the organic phase was washed successively with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography on silica gel (in volume ratio, eluent: petroleum ether: ethyl acetate=4:1) to give intermediate 12a as a yellow oily product in 96.2% yield.

The procedure was followed as a synthesis of intermediate 12a, except for using intermediate 11b (2.00 g, 5.48 mmol) to react with 1-tert-butoxycarbonyl piperazine (1.53 g, 8.22 mmol) to give 2.63 g intermediate 12b as a yellow oily product in 90.3% yield.

The procedure was followed as a synthesis of intermediate 12a, except for using intermediate 11c (2.00 g, 6.56 mmol) to react with 1-tert-butoxycarbonyl piperazine (1.83 g, 9.84 mmol) to give 2.66 g intermediate 12c as a yellow oily product in 86.1% yield.

The procedure was followed as a synthesis of intermediate 12a, except for using intermediate 11d (2.00 g, 5.97 mmol) to react with 1-tert-butoxycarbonyl piperazine (1.08 g, 8.96 mmol) to give 2.70 g intermediate 12d as a yellow oily product in 90.3% yield.

The procedure for the synthesis of intermediate 12e is as follows:

40 mL Dichloromethane (DCM), intermediate 11e (3.00 g, 8.50 mmol), 1-hydroxybenzotriazole (HOBt, 344.25 mg, 2.55 mmol), 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (HATU, 4.85 g, 12.75 mmol) and triethylamine (4.30 g, 42.5 mmol) were mixed, reacted at room temperature for 0.5 h, then 4-methoxybenzylamine (1.17 g, 8.50 mmol) was added and reacted at room temperature for 6 h, TLC detection was complete (in volume ratio, the developer was petroleum ether: ethyl acetate=1:1, uv color development); the resulting product system was washed sequentially with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure to give 3.61. 3.61 g intermediate 12e as a yellow oily product in 90.2% yield.

The procedure was followed as a synthesis of intermediate 12e, except that intermediate 11f (3.00 g, 8.50 mmol) was used to react with 4-methylthiobenzylamine (1.30 g, 8.50 mmol) to afford intermediate 12f as a yellow oily product in 89.1% yield.

Preparation example 10

Operating following the synthetic procedure for intermediate 8, except using intermediate 12a (3.00 g, 6.16 mmol) to remove Boc, 2.03 g intermediate 13a was prepared as a yellow solid in 85.3% yield.

Operating following the synthetic procedure for intermediate 8, except using intermediate 12b (3.00 g, 5.65 mmol) to remove Boc, 1.95 g intermediate 13b was prepared as a yellow solid in 79.9% yield.

Operating following the synthetic procedure for intermediate 8, except using intermediate 12c (3.00 g, 6.37 mmol) to remove Boc, 1.96 g intermediate 13c was prepared as a yellow solid in 83.1% yield.

Operating following the synthetic procedure for intermediate 8, except using intermediate 12d (3.00 g, 5.99 mmol) to remove Boc, 2.04 g of intermediate 13d was prepared as a yellow solid in 85.0% yield.

Operating following the synthetic procedure for intermediate 8, except using intermediate 12e (3.00 g, 6.38 mmol) to remove Boc, 1.91 g intermediate 13e was prepared as a yellow solid in 80.9% yield.

Operating following the synthetic procedure for intermediate 8, except using intermediate 12f (3.00 g, 6.17 mmol) to remove Boc, 2.09 g intermediate 13f was prepared as a yellow solid in 87.8% yield.

PREPARATION EXAMPLE 11

The procedure for the synthesis of intermediate 14a is as follows:

50 mL of DCM was mixed with intermediate 13a (3.00 g, 7.75 mmol), triethylamine (3.91 g, 38.76 mmol) was added at 0deg.C and reacted at 0deg.C for 30 min, then bromoacetyl bromide (2.34 g, 11.63 mmol) was added, and the reaction was allowed to proceed to room temperature for 6 h, after which the TLC detection reaction was completed (in volume ratio, the developing agent was petroleum ether: ethyl acetate=1:2, UV-developed); the resulting product system was evaporated to dryness under reduced pressure, the residue was dissolved in ethyl acetate, the organic phase was washed with water and saturated sodium chloride solution, then dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether: ethyl acetate=1:1 as eluent by volume ratio) to give intermediate 14a as a yellow oily product in a yield of 74.9%.

The procedure was followed as a synthesis of intermediate 14a, except that intermediate 13b (3.00 g, 6.96 mmol) was used to react with chloroacetyl chloride (1.17 g, 10.44 mmol) to afford intermediate 14b as a yellow solid in 79.9% yield.

The procedure was followed as a synthesis of intermediate 14a, except that intermediate 13c (3.00 g, 8.09 mmol) was used to react with bromoacetyl bromide (2.44 g, 12.14 mmol) to afford intermediate 14c as a yellow oil in 83.4% yield.

The procedure was followed as a synthesis of intermediate 14a, except that intermediate 13d (3.00 g, 7.48 mmol) was used to react with bromoacetyl bromide (2.26 g, 11.22 mmol) to afford intermediate 14d as a yellow oil in 82.8% yield.

The procedure was followed as a synthesis of intermediate 14a, except that intermediate 13e (3.00 g, 8.11 mmol) was used to react with chloroacetyl chloride (1.36 g, 12.16 mmol) to afford intermediate 14e as a yellow oil in 77.9% yield.

The procedure was followed as a synthesis of intermediate 14a, except that intermediate 13f (3.00 g, 7.77 mmol) was used to react with chloroacetyl chloride (1.31 g, 11.66 mmol) to afford intermediate 14f as a yellow oil in 79.9% yield.

Example 1

The synthesis procedure for compound I-1 is as follows:

(1) 10 mL of THF, intermediate 6a (0.15 g, 0.40 mmol) and N-phenylethanediamine (0.11 g, 0.80 mmol) were mixed and reacted at room temperature for 3 h, after TLC detection was complete (in volume ratio, the developing agent was petroleum ether: ethyl acetate=4:1, ultraviolet coloration); concentrating the obtained product system under reduced pressure to obtain an intermediate 9a which is directly used for the next reaction;

(2) Mixing 10 mL of THF, an intermediate 9a and a 2 mol/L sodium hydroxide aqueous solution with the concentration of 2 mL, reacting for 4 hours at room temperature, and detecting the completion of the TLC detection reaction (according to the volume ratio, the developing agent is petroleum ether: ethyl acetate=3:1, and ultraviolet developing); the resulting product system was dissolved with 30 mL ethyl acetate, washed sequentially with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography on silica gel (petroleum ether: ethyl acetate=2:1 as eluent by volume) to give 115.00 mg as a tan solid, compound I-1, in 66.2% yield in two steps. m.p.131.8-132.7 ℃. ¹ H NMR (600 MHz, Chloroform-d) δ 7.25 – 7.17 (m, 3H), 7.01 (t, J = 8.4 Hz, 1H), 6.93 – 6.83 (m, 2H), 6.76 (t, J = 7.3 Hz,1H), 6.69 (d, J = 7.7 Hz, 2H), 5.92 (t, J = 5.3 Hz, 1H), 3.56 (q, J = 5.6 Hz, 2H), 3.48 (t, J = 5.7 Hz, 2H). ¹³ C NMR (151 MHz, Chloroform-d) δ 157.46, 155.83, 155.59, 147.87, 142.25, 138.35, 135.67, 129.47, 128.68, 124.38, 118.00, 116.15, 113.26, 108.70, 44.23, 42.70. HR-MS (ESI): calcd. for C ₁₇ H ₁₇ N ₆ O ₂ BrF, [M+H] ⁺ 435.05749; found: 435.05694。

Example 2

The procedure was followed as in example 1, except that intermediate 6a (0.15 g, 0.40 mmol) was used with 2- (pyridin-2-yl) ethylamine (97.60 mg, 0.80 mmol) to afford intermediate 9b; then, the intermediate 9b was used to prepare 112.00 mg compound I-2 as a milky solid in 66.7% yield. m.p.157.6-159.6 ℃. ¹ H NMR (600 MHz, DMSO-d ₆ ) δ 11.44 (s, 1H), 8.85 (s, 1H), 8.67 – 8.45 (m, 1H), 7.72 (td, J = 7.6, 1.8 Hz, 1H), 7.28 (d, J = 7.7 Hz, 1H), 7.26 – 7.22 (m, 1H), 7.18 (t, J = 8.8 Hz, 1H), 7.10 (dd, J = 6.0, 2.7 Hz, 1H), 6.75 (ddd, J = 8.8, 4.0, 2.8 Hz, 1H), 6.41 (t, J = 5.8 Hz, 1H), 3.59 (q, J = 6.9 Hz, 2H), 3.05 (t, J = 7.0 Hz, 2H). ¹³ C NMR (151 MHz, DMSO-d ₆ ) δ 159.39, 156.01, 155.00, 153.42, 149.59, 140.42, 139.72, 138.50, 137.10, 125.26, 123.68, 121.98, 116.41, 107.51, 44.06, 36.51. HR-MS (ESI): calcd. for C ₁₆ H ₁₅ N ₆ O ₂ BrF，[M+H] ⁺ 421.04184; found: 421.04214。

Example 3

The procedure was followed as in example 1, except that intermediate 6a (0.15 g, 0.40 mmol) and 4-fluorophenylethylamine (111.20 mg, 0.80 mmol) were used to give intermediate 9c; then using the intermediate 9c, 105.00, mg, compound I-3 was prepared as a yellow solid in 60.1% yield. m.p. 138.3-142.7 ℃. ¹ H NMR (600 MHz, Chloroform-d) δ 7.25 (s, 1H), 7.21 (dd, J = 5.8, 2.6 Hz, 1H), 7.19 – 7.15 (m, 2H), 7.04 – 6.97 (m, 3H), 6.90 (tt, J = 4.0, 2.1 Hz, 2H), 5.70 (t, J = 5.8 Hz, 1H), 3.55 (q, J = 7.0 Hz, 2H), 2.93 (t, J = 7.1 Hz, 2H). ¹³ C NMR (151 MHz, Chloroform-d) δ 162.52, 160.90, 157.47, 155.84, 155.30, 142.47, 138.29, 135.70, 134.38, 130.26, 128.70, 124.38, 116.17, 115.41, 108.72, 45.68, 34.23. HR-MS (ESI): calcd. for C ₁₇ H ₁₅ N ₅ O ₂ BrF ₂ ，[M+H] ⁺ 438.03717; found: 438. 03683。

Example 4

The procedure was followed as in example 1, except that intermediate 6a (0.15 g, 0.40 mmol) was used with 3,4, 5-trifluorobenzylamine (128.80 mg, 0.80 mmol) to give intermediate 9d; then compound I-4 was prepared 127.00 mg as a pale yellow solid in 69.2% yield using the intermediate 9 d. m.p. 128.0-130.7 ℃. ¹ H NMR (600 MHz, Chloroform-d) δ 7.27 – 7.24 (m, 2H), 7.04 (t, J = 8.4 Hz, 1H), 7.02 – 6.96 (m, 2H), 6.97 – 6.93 (m, 2H), 6.14 (t, J = 5.9 Hz, 1H), 4.44 (d, J = 6.1 Hz, 2H). ¹³ C NMR (151 MHz, Chloroform-d) δ 157.65, 156.02, 155.17, 152.18, 150.52, 142.60, 139.99, 138.26, 135.51, 134.09, 129.04, 124.72, 116.21, 111.56, 108.76, 47.34. HR-MS (ESI): calcd. for C ₁₆ H ₁₁ O ₂ N ₅ BrF ₄ ，[M+H] ⁺ 460.00268; found: 460.00388。

Example 5

The procedure was followed as in example 1, except that intermediate 6a (0.15 g, 0.40 mmol) was used with 4-methylphenylethylamine (108.00 mg, 0.80 mmol) to afford intermediate 9e; then using the intermediate 9e, 87.00 mg of compound I-5 was prepared as a yellow solid in 50.2% yield. m.p. 137.2-139.3 ℃. ¹ H NMR (600 MHz, Chloroform-d) δ 7.21 (dd, J = 5.8, 2.6 Hz, 1H), 7.16 – 7.10 (m, 4H), 7.07 (s, 1H), 7.02 (t, J = 8.4 Hz, 1H), 6.93 – 6.85 (m, 2H), 5.68 (t, J = 5.6 Hz, 1H), 3.56 (q, J = 7.0 Hz, 2H), 2.93 (t, J = 7.0 Hz, 2H), 2.34 (s, 3H). ¹³ C NMR (151 MHz, Chloroform-d) δ 157.43, 155.80, 155.36, 142.41, 138.32, 136.15, 135.75, 135.65, 129.28, 128.77, 128.62, 124.30, 116.14, 108.70, 45.68, 34.65, 21.06. HR-MS (ESI): calcd. for C ₁₈ H ₁₈ O ₂ N ₅ BrF，[M+H] ⁺ 434.06224; found: 434. 06165。

Example 6

The procedure was followed as in example 1, except that intermediate 6a (0.20 g, 0.53 mmol) and 4-bromophenylethylamine (212.00 mg, 1.06 mmol) were used to give intermediate 9f; then compound I-6 was prepared 158.00 mg using the intermediate 9f as an orange solid in 60.0% yield. m.p. 143.3-144.6 ℃. ¹ H NMR (600 MHz, Chloroform-d) δ 7.46 – 7.42 (m, 2H), 7.21 (dd, J = 5.8, 2.6 Hz, 1H), 7.15 (s, 1H), 7.09 (d, J = 8.3 Hz, 2H), 7.03 (t, J = 8.4 Hz, 1H), 6.93 – 6.87 (m, 2H), 5.68 (t, J = 5.8 Hz, 1H), 3.56 (q, J = 6.9 Hz, 2H), 2.92 (t, J = 7.0 Hz, 2H). ¹³ C NMR (151 MHz, Chloroform-d) δ 157.49, 155.86, 155.26, 142.50, 138.27, 137.77, 135.68, 131.69, 130.61, 128.74, 124.42, 120.43, 116.17, 108.72, 45.41, 34.42. HR-MS (ESI): calcd. for C ₁₇ H ₁₅ O ₂ N ₅ Br ⁸¹ BrF，[M+H] ⁺ 499.95506; found: 499. 95486。

Example 7

The procedure was followed as in example 1 except that intermediate 6a (0.20 g, 0.53 mmol) was used with 2- (4-trifluoromethylphenyl) ethylamine (200.34 mg, 1.06 mmol) to give intermediate 9g; then compound I-7 was prepared 173.00 mg as a pale yellow solid in 67.0% yield using 9g of the intermediate. m.p.124.6-126.0 ℃. ¹ H NMR (600 MHz, Chloroform-d) δ 7.57 (d, J = 8.1 Hz, 2H), 7.53 (s, 1H), 7.33 (d, J = 8.0 Hz, 2H), 7.21 (dd, J = 5.8, 2.7 Hz, 1H), 7.01 (t, J = 8.4 Hz, 1H), 6.95 – 6.83 (m, 2H), 5.77 (t, J = 5.9 Hz, 1H), 3.59 (q, J = 6.9 Hz, 2H), 3.02 (t, J = 7.1 Hz, 2H). ¹³ C NMR (151 MHz, Chloroform-d) δ 157.47, 155.85, 155.26, 142.93, 142.41, 138.33, 135.69, 129.18, 128.96, 128.71, 125.52, 125.14, 124.40, 123.34, 116.17, 108.71, 45.34, 34.81. HR-MS (ESI): calcd. for C ₁₈ H ₁₅ O ₂ N ₅ BrF ₄ ，[M+H] ⁺ 488.03398; found: 488.03333。

Example 8

The procedure was followed as in example 1, except that intermediate 6a (0.20 g, 0.53 mmol) and 4-hydroxyphenylethylamine (145.22 mg, 1.06 mmol) were used to give intermediate 9h; then compound I-8 was prepared 138.00 mg as an off-white solid in 59.9% yield using the intermediate 9 h. m.p. 178.6-178.8 ℃. ¹ H NMR (600 MHz, Methanol-d ₄ ) δ 7.17 – 6.97 (m, 4H), 6.87 – 6.72 (m, 3H), 3.49 (s, 2H), 2.85 (s, 2H). ¹³ C NMR (151 MHz, Methanol-d ₄ ) δ 155.50, 154.73, 140.67, 139.35, 137.54, 129.83, 129.37, 126.39, 123.53, 122.45, 121.68, 119.67, 115.45, 114.95, 107.41, 45.68, 33.71. HR-MS (ESI): calcd. for C ₁₇ H ₁₆ O ₃ N ₅ BrF，[M+H] ⁺ 436.04151; found: 436.04132。

Example 9

The procedure was followed as in example 1, except that intermediate 6a (0.20 g, 0.53 mmol) and 3-fluorophenylethylamine (147.34 mg, 1.06 mmol) were used to give intermediate 9i; then, the intermediate 9I was used to prepare 162.00 mg compound I-9 as a tan solid in 69.9% yield. m.p. 123.3-126.5 ℃. ¹ H NMR (600 MHz, Chloroform-d) δ 7.29 (ddd, J = 8.9, 7.6, 6.1 Hz, 1H), 7.22 (dd, J = 5.8, 2.6 Hz, 1H), 7.02 (dd, J = 16.1, 7.9 Hz, 3H), 6.98 – 6.93 (m, 2H), 6.93 – 6.86 (m, 2H), 5.69 (t, J = 5.6 Hz, 1H), 3.58 (q, J = 6.9 Hz, 2H), 2.97 (t, J = 7.0 Hz, 2H). ¹³ C NMR (151 MHz, Chloroform-d) δ 162.96, 156.69, 155.26, 142.54, 141.35, 138.26, 135.69, 130.14, 128.77, 124.56, 124.45, 116.11, 115.78, 113.52, 108.72, 45.31, 34.77. HR-MS (ESI): calcd. for C ₁₇ H ₁₅ O ₂ N ₅ BrF ₂ ，[M+H] ⁺ 438.03717; found: 438.03619。

Example 10

The procedure was followed as in example 1, except that intermediate 6a (0.20 g, 0.53 mmol) was used with 2-fluorophenylethylamine (147.34 mg, 1.06 mmol) to give intermediate 9j; then compound I-10 was prepared 127.00 mg as a white solid in 54.8% yield using the intermediate 9 j. m.p. 148.6-149.2 ℃. ¹ H NMR (600 MHz, Methanol-d ₄ ) δ 7.32 – 7.22 (m, 2H), 7.17 – 7.11 (m, 2H), 7.11 – 7.04 (m, 2H), 6.84 (ddd, J = 8.8, 4.1, 2.7 Hz, 1H), 3.56 (t, J = 7.1 Hz, 2H), 3.02 (t, J = 7.0 Hz, 2H). ¹³ C NMR (151 MHz, Methanol-d ₄ ) δ 161.35, 155.43, 155.25, 140.63, 139.35, 137.65, 130.95, 128.10, 126.36, 125.81, 123.98, 122.41, 115.33, 114.78, 107.41, 44.03, 27.86. HR-MS (ESI): calcd. for C ₁₇ H ₁₅ O ₂ N ₅ BrF ₂ ，[M+H] ⁺ 438.03717; found: 438.03677。

Example 11

The procedure is as in example 1, except that intermediate 6a (0.20 g, 0.53 mmol) is used in combination with4-methylthiobenzylamine (162.18 mg, 1.06 mmol) to afford intermediate 9k; then using the intermediate 9k, 120.00 g mg compound I-11 was prepared as a white solid in 50.2% yield. m.p. 100.4-100.8 ℃. ¹ H NMR (600 MHz, Chloroform-d) δ 7.52 (s, 1H), 7.26 (d, J = 8.2 Hz, 2H), 7.25 – 7.18 (m, 3H), 7.00 (t, J = 8.4 Hz, 1H), 6.94 – 6.86 (m, 2H), 5.96 (t, J = 5.7 Hz, 1H), 4.41 (d, J = 5.8 Hz, 2H), 2.46 (s, 3H). ¹³ C NMR (151 MHz, Chloroform-d) δ 157.45, 155.82, 155.33, 142.36, 138.31, 138.03, 135.69, 134.52, 128.68, 128.42, 126.88, 124.38, 116.16, 108.71, 48.07, 15.85. HR-MS (ESI): calcd. for C ₁₇ H ₁₅ O ₂ N ₅ ⁸¹ BrFNaS，[M+Na] ⁺ 475.99856; found: 475.99719。

Example 12

The procedure was followed as in example 1, except that intermediate 6a (0.20 g, 0.53 mmol) and 4-methoxybenzylamine (145.22 mg, 1.06 mmol) were used to give intermediate 9l; then compound I-12 was prepared 138.00 mg as a white solid in 59.9% yield using the intermediate 9 l. m.p. 130.1-130.7 ℃. ¹ H NMR (600 MHz, Chloroform-d) δ 7.32 (s, 1H), 7.29 (d, J = 8.3 Hz, 2H), 7.22 (dd, J = 5.6, 2.3 Hz, 1H), 7.02 (t, J = 8.4 Hz, 1H), 6.95 – 6.82 (m, 4H), 5.86 (t, J = 5.1 Hz, 1H), 4.41 (d, J = 5.6 Hz, 2H), 3.80 (s, 3H). ¹³ C NMR (151MHz, Chloroform-d) δ 159.21, 157.45, 155.82, 155.32, 142.43, 138.26, 135.73, 129.91, 129.30, 128.66, 124.34, 116.15, 114.15, 108.71, 55.36, 48.01. HR-MS (ESI): calcd. for C ₁₇ H ₁₅ O ₃ N ₅ BrFNa，[M+Na] ⁺ 458.02345; found: 458.02313。

Example 13

The procedure was followed as in example 1, except that intermediate 6a (0.20 g, 0.53 mmol) and 3-chlorophenyl ethylamine (164.30 mg, 1.06 mmol) were used to prepare intermediate 9m; then compound I-13 was prepared 144.00 mg as a yellow oil in 59.7% yield using the intermediate 9 m. ¹ H NMR (600 MHz, Chloroform-d) δ 7.72 (s, 1H), 7.25 – 7.18 (m, 4H), 7.10 – 7.07 (m, 1H), 6.99 (t, J = 8.4 Hz, 1H), 6.91 (s, 1H), 6.88 (ddd, J = 8.8, 4.1, 2.7 Hz, 1H), 5.80 (t, J = 5.9 Hz, 1H), 3.53 (td, J = 7.0, 5.9 Hz, 2H), 2.91 (t, J = 7.0 Hz, 2H). ¹³ C NMR (151 MHz, Chloroform-d) δ 157.42, 155.79, 155.26, 142.29, 140.80, 138.40, 135.73, 134.16, 130.03, 129.04, 128.65, 126.97, 124.36, 116.16, 108.69, 45.32, 34.67. HR-MS (ESI): calcd. for C ₁₇ H ₁₅ O ₂ N ₅ ⁸¹ BrClF，[M+H] ⁺ 456.00557; found: 456.00467。

Example 14

The procedure was followed as in example 1, except that intermediate 6a (0.20 g, 0.53 mmol) was used with 3-chlorobenzylamine (149.46 mg, 1.06 mmol) to give intermediate 9n; then compound I-14 was prepared 132.00 mg as a white solid in 56.7% yield using the intermediate 9 n. m.p.124.7-124.9 ℃. ¹ H NMR (600 MHz, Methanol-d ₄ ) δ 7.36 (t, J = 1.9 Hz, 1H), 7.30 (d, J = 7.6 Hz, 1H), 7.26 (ddt, J = 9.0, 7.3, 1.6 Hz, 2H), 7.15 (dd, J = 6.0, 2.7 Hz, 1H), 7.04 (t, J = 8.6 Hz, 1H), 6.85 (ddd, J = 8.8, 4.1, 2.7 Hz, 1H), 4.46 (s, 2H). ¹³ C NMR (151 MHz, Methanol-d ₄ ) δ 156.15, 155.46, 154.54, 140.80, 140.60, 139.45, 137.48, 134.03, 129.68, 127.10, 126.52, 125.49, 122.53, 115.41, 107.52, 47.00. HR-MS (ESI): calcd. for C ₁₆ H ₁₃ O ₂ N ₅ Br ³⁷ ClF，[M+H] ⁺ 441.98902; found: 441.98849。

Example 15

The procedure was followed as in example 1, except that intermediate 6a (0.18 g, 0.48 mmol) and 4-piperazinylbenzonitrile (179.52 mg, 0.96 mmol) were used to prepare intermediate 9o; then using the intermediate 9o, 130.00, mg, compound I-15 was prepared as a yellow solid in 55.8% yield. m.p. 189.5-195.3 ℃. ¹ H NMR (600 MHz, DMSO-d ₆ ) δ 7.63 – 7.56 (m, 3H), 7.16 (td, J = 7.5, 6.4, 4.4 Hz, 2H), 7.09 – 7.02 (m, 3H), 6.74 (ddd, J = 8.9, 4.1, 2.7 Hz, 1H), 3.47 (q, J = 5.0 Hz, 4H), 3.36 (dd, J = 6.8, 3.4 Hz, 4H). ¹³ C NMR (151 MHz, DMSO-d ₆ ) δ 158.33, 155.57, 153.97, 153.29, 143.04, 142.10, 141.07, 138.75, 137.60, 133.84, 126.02, 122.35, 122.22, 119.36, 117.07, 114.88, 107.93, 99.49, 47.23, 46.11. HR-MS (ESI): calcd. for C ₂₀ H ₁₈ O ₂ N ₇ BrF，[M+H] ⁺ 486.06839; found: 486.06766。

Example 16

The procedure was followed as in example 1, except that intermediate 6a (0.18 g, 0.48 mmol) and 3-hydroxyphenyl piperazine (170.88 mg, 0.96 mmol) were used to prepare intermediate 9p; then compound I-16 was prepared 114.00 mg as a milky oil in 49.8% yield using the intermediate 9 p. m.p. 74.8-75.0 ℃. ¹ H NMR (600 MHz, Methanol-d ₄ ) δ 7.07 (dd, J = 6.0, 2.7 Hz, 1H), 6.97 – 6.86 (m, 2H), 6.70 (ddd, J = 8.9, 4.1, 2.7 Hz, 1H), 6.36 (dd, J = 8.2, 2.4 Hz, 1H), 6.31 (t, J = 2.3 Hz, 1H), 6.24 (dd, J = 8.1, 2.2 Hz, 1H), 3.24 – 3.21 (m, 4H), 3.03 – 2.97 (m, 4H). ¹³ C NMR (151 MHz, Methanol-d ₄ ) δ 158.46, 157.87, 156.67, 155.05, 152.58, 141.26, 140.13, 136.35, 129.46, 127.14, 122.90, 115.95, 108.09, 107.41, 103.66, 48.70, 48.04. HR-MS (ESI): calcd. for C ₁₉ H ₁₉ O ₃ N ₆ ⁸¹ BrF，[M+H] ⁺ 479.06601; found: 479.06638。

Example 17

The procedure was followed as in example 1, except that intermediate 6a (0.16 g, 0.43 mmol) was used with 1-benzoylpiperazine (163.40 mg, 0.86 mmol) to give intermediate 9q; then using the intermediate 9q to prepare 90.00. 90.00 mg compound I-17 as a white solid in 42.9% yield. m.p. 106.8-107.1 ℃. ¹ H NMR (600 MHz, Chloroform-d) δ 9.03 (s, 1H), 7.46 – 7.39 (m, 5H), 7.36 (s, 1H), 7.12 (dd, J = 5.8, 2.7 Hz, 1H), 6.95 (dd, J = 8.8, 7.9 Hz, 1H), 6.72 (ddd, J = 8.8, 4.0, 2.7 Hz, 1H), 3.78 (s, 4H), 3.29 (s, 4H). ¹³ C NMR (151 MHz, Chloroform-d) δ 170.99, 158.24, 157.35, 155.72, 140.80, 140.27, 134.83, 134.75, 130.32, 128.73, 127.72, 127.13, 123.24, 116.77, 109.25, 48.47, 41.37. HR-MS (ESI): calcd. for C ₂₀ H ₁₉ O ₃ N ₆ BrF，[M+H] ⁺ 489.06806; found: 489.06802。

Example 18

The procedure was followed as in example 1, except that intermediate 6a (0.16 g, 0.43 mmol) and 1-acetylpiperazine (110.08 mg, 0.86 mmol) were used to give intermediate 9r; then using the intermediate 9q to prepare 95.00, mg compound I-18 as a white solid in 51.9% yield. m.p. 170.1-172.5 ℃. ¹ H NMR (600 MHz, Methanol-d ₄ ) δ 7.17 (dd, J = 5.9, 2.7 Hz, 1H), 7.06 (t, J = 8.6 Hz, 1H), 6.82 (ddd, J = 8.9, 4.1, 2.7 Hz, 1H), 3.62 – 3.57 (m, 2H), 3.57 – 3.53 (m, 2H), 3.29 – 3.25 (m, 2H), 3.21 (dd, J = 6.4, 4.1 Hz, 2H), 2.13 (s, 3H). ¹³ C NMR (151 MHz, Methanol-d ₄ ) δ 171.07, 158.26, 155.76, 141.24, 139.81, 136.43, 126.85, 122.77, 116.06, 107.98, 45.16, 40.42, 19.70. HR-MS (ESI): calcd. for C ₁₅ H ₁₇ O ₃ N ₆ BrF，[M+H] ⁺ 427.05241; found: 427.05276。

Example 19

The synthesis procedure for compound II-1 is as follows:

(1) 40 mL Dichloromethane (DCM), 2- (4-thiomorpholinyl) -5-nitrobenzoic acid (intermediate 11f, 0.20 g, 0.75 mmol), 1-hydroxybenzotriazole (HOBt, 29.70 mg, 0.22 mmol), 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (HATU, 429.40 mg, 1.13 mmol) and triethylamine (378.75 mg, 3.75 mmol) were mixed and reacted at room temperature for 0.5 h, intermediate 8 (146.3 mg, 0.38 mmol) was added and reacted at room temperature for 6 h, after TLC detection the reaction was complete (in volume ratio, the developer was petroleum ether: ethyl acetate=1:1, uv color); washing the obtained product system with water and saturated sodium chloride solution in turn, drying by anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure to obtain a yellow oily product which is a crude intermediate 10a and directly used for the next reaction;

(2) The procedure of step (2) of example 1 was followed except that intermediate 10a was used to prepare 200.00, 200.00 mg of compound II-1 as a yellow solid in 43.8% two-step yield. m.p.185.5-186.6 deg.c. ¹ H NMR (600 MHz, Chloroform-d) δ 9.67 (s, 1H), 8.62 (t, J = 5.9 Hz, 1H), 8.56 (d, J = 2.8 Hz, 1H), 8.09 (dd, J = 8.9, 2.8 Hz, 1H), 7.14 (dd, J = 5.9, 2.7 Hz, 1H), 7.11 (d, J = 9.0 Hz, 1H), 7.06 (s, 1H), 6.97 (t, J = 8.4 Hz, 1H), 6.86 (ddd, J = 8.8, 4.0, 2.7 Hz, 1H), 6.28 (t, J = 5.6 Hz, 1H), 3.80 (q, J = 5.8 Hz, 2H), 3.59 (q, J = 5.4 Hz, 2H), 3.39 – 3.29 (m, 4H), 2.82 – 2.73 (m, 4H). ¹³ C NMR (151 MHz, Chloroform-d) δ 166.78, 157.14, 156.58, 155.67, 155.51, 142.59, 141.91, 138.55, 135.94, 128.26, 127.56, 127.07, 126.97, 124.06, 120.42, 116.09, 108.52, 54.84, 44.56, 39.12, 27.76.HR-MS (ESI): calcd. for C ₂₂ H ₂₃ O ₅ N ₈ ⁸¹ BrFS，[M+H] ⁺ 611.06536; found: 611.06836。

Example 20

The procedure was followed as in example 19 except that 6-trifluoromethylnicotinic acid (0.20 g, 1.05 mmol) was used with intermediate 8 (242.55 mg, 0.63 mmol) to give intermediate 10b; the intermediate 10b was then used to prepare 240.00 mg compound II-2 as a white solid in 42.8% two-step yield. m.p. 186.1-187.8 ℃. ¹ H NMR (600 MHz, Methanol-d ₄ ) δ 9.09 (d, J = 2.1 Hz, 1H), 8.40 (dd, J = 8.2, 2.2 Hz, 1H), 7.93 (dd, J = 8.2, 0.8 Hz, 1H), 7.14 (dd, J = 6.0, 2.7 Hz, 1H), 7.03 (t, J = 8.7 Hz, 1H), 6.85 (ddd, J = 8.8, 4.1, 2.7 Hz, 1H), 3.71 (dd, J = 6.7, 5.1 Hz, 2H), 3.58 (dd, J = 6.6, 5.1 Hz, 2H). ¹³ C NMR (151 MHz, Methanol-d ₄ ) δ 165.97, 156.08, 155.77, 154.48, 149.40, 148.59, 140.75, 139.39, 137.60, 137.07, 133.12, 126.51, 122.56, 120.28, 115.32, 107.40, 43.38, 38.68. HR-MS (ESI): calcd. for C ₁₈ H ₁₅ O ₃ N ₇ BrF ₄ ，[M+H] ⁺ 534.03299; found: 534.03480。

Example 21

The procedure was followed as in example 19 except that 2- (4-morpholinyl) -5-nitrobenzoic acid (intermediate 11g, 0.20 g, 1.05 mmol) was used with intermediate 8 (242.55 mg, 0.63 mmol) to give intermediate 10c; then, the intermediate 10c was used to prepare 111.00 mg compound II-3 as a yellow solid in 39.0% two-step yield. m.p. 172.9-174.3 ℃. ¹ H NMR (600 MHz, DMSO-d ₆ ) δ 11.42 (s, 1H), 8.88 (s, 1H), 8.74 (t, J = 5.6 Hz, 1H), 8.21 – 7.97 (m, 2H), 7.16 (dd, J = 9.0, 7.6 Hz, 2H), 7.11 (dd, J = 6.0, 2.7 Hz, 1H), 6.78 (ddd, J = 9.0, 4.2, 2.8 Hz, 1H), 6.32 (t, J = 5.9 Hz, 1H), 3.68 (t, J = 4.6 Hz, 4H), 3.51 (q, J = 6.1 Hz, 2H), 3.44 (q, J = 6.2 Hz, 2H), 3.21 – 3.16 (m, 4H). ¹³ C NMR (151 MHz, DMSO-d ₆ ) δ 167.60, 156.17, 154.90, 153.43, 140.40, 139.73, 139.47, 138.49, 127.00, 126.68, 126.24, 125.33, 121.95, 118.07, 116.38, 107.50, 66.15, 51.05, 49.07, 44.04, 42.36, 38.67. HR-MS (ESI): calcd. for C ₂₂ H ₂₃ O ₆ N ₈ ⁸¹ BrF，[M+H] ⁺ 595.08820; found: 595.09027。

Example 22

The procedure was followed as in example 19 except that 2- (4-Boc-piperazinyl) -5-nitrobenzoic acid (intermediate 11e, 0.18 g, 0.51 mmol) was used with intermediate 8 (138.60 mg, 0.36 mmol) to give intermediate 10d; then, the intermediate 10d was used to prepare 176.00 mg compound II-4 as a yellow solid in a two-step yield of 49.9%. m.p. 106.6-110.7 ℃. ¹ H NMR (600 MHz, Chloroform-d) δ 9.83 (s, 1H), 8.54 (d, J = 2.8 Hz, 1H), 8.23 (t, J = 5.9 Hz, 1H), 8.14 (dd, J = 9.0, 2.8 Hz, 1H), 7.16 (dd, J = 5.9, 2.7 Hz, 1H), 7.07 (d, J = 9.0 Hz, 1H), 7.02 (s, 1H), 6.99 (t, J = 8.4 Hz, 1H), 6.87 (ddd, J = 8.8, 4.0, 2.7 Hz, 1H), 6.14 (t, J = 5.4 Hz, 1H), 3.85 – 3.79 (m, 2H), 3.68 – 3.54 (m, 6H), 3.13 (dd, J = 6.6, 3.6 Hz, 4H), 1.48 (s, 9H). ¹³ C NMR (151 MHz, Chloroform-d) δ 167.05, 157.22, 155.63, 155.23, 142.37, 142.05, 138.48, 136.01, 128.40, 127.46, 127.04, 126.86, 124.19, 119.01, 116.09, 108.57, 81.24, 51.99, 44.48, 39.05, 28.37. HR-MS (ESI): calcd. for C ₂₇ H ₃₀ O ₇ N ₉ BrF，[M-H] ^- 690.14301; found: 690.14156。

Example 23

The procedure was followed except that intermediate 10d (0.18 g, 0.25 mmol) was used to remove Boc to give intermediate 10e; then, the procedure of step (2) in example 19 was followed, except that compound II-5 was prepared 74.00 mg as a yellow solid in 50.0% two-step yield using the intermediate 10 e. m.p. 170.4-173.5 ℃. ¹ H NMR (600 MHz, Methanol-d ₄ ) δ 8.25 (d, J = 2.8 Hz, 1H), 8.12 (dd, J = 9.1, 2.8 Hz, 1H), 7.10 (d, J = 9.1 Hz, 1H), 7.01 (dd, J = 6.0, 2.7 Hz, 1H), 6.92 (t, J = 8.6 Hz, 1H), 6.74 (ddd, J = 8.9, 4.1, 2.7 Hz, 1H), 3.60 (dd, J = 6.7, 4.8 Hz, 2H), 3.46 (dd, J = 6.7, 4.8 Hz, 2H), 3.15 – 3.07 (m, 4H), 2.89 – 2.82 (m, 4H). ¹³ C NMR (151 MHz, Methanol-d ₄ ) δ 168.98, 156.04, 155.71, 155.28, 154.44, 140.75, 139.39, 137.65, 127.03, 126.43, 125.72, 122.52, 118.26, 115.32, 107.36, 51.52, 44.70, 43.84, 38.59. HR-MS (ESI): calcd. for C ₂₂ H ₂₄ O ₅ N ₉ ⁸¹ BrF，[M+H] ⁺ 594.10419; found: 594.10686。

Example 24

The procedure was followed as in example 19 except that 2, 5-difluorobenzoic acid (0.20 g, 1.27 mmol) was used with intermediate 8 (246.40 mg, 0.64 mmol) to give intermediate 10f; then, the intermediate 10f was used to prepare 254.00 mg compound II-6 as an off-white solid in 40.1% two-step yield. m.p. 163.8-165.9 ℃. ¹ H NMR (600 MHz, DMSO-d ₆ ) δ 11.46 (s, 1H), 8.88 (s, 1H), 8.54 (d, J = 2.1 Hz, 1H), 7.44 – 7.39 (m, 1H), 7.39 – 7.33 (m, 2H), 7.19 – 7.06 (m, 2H), 6.78 (ddd, J = 8.8, 4.2, 2.7 Hz, 1H), 6.32 (t, J = 6.0 Hz, 1H), 3.51 (q, J = 6.0 Hz, 2H), 3.43 (q, J = 6.1 Hz, 2H). ¹³ C NMR (151 MHz, DMSO-d ₆ ) δ 163.32, 159.02, 157.42, 156.54, 156.19, 155.01, 153.42, 140.46, 139.73, 138.51, 125.28, 121.91, 118.47, 116.74, 116.36, 107.52, 43.92, 38.61. HR-MS (ESI): calcd. for C ₁₈ H ₁₅ O ₃ N ₆ ⁸¹ BrF ₃ ，[M+H] ⁺ 501.03152; found: 501.03344。

Example 25

The synthesis procedure for compound III-1 is as follows:

8 mL of THF, intermediate 6a (151.00 mg, 0.40 mmol), intermediate 13a (154.80 mg, 0.40 mmol) and 2 mL of aqueous sodium hydroxide solution with a concentration of 2mol/L are mixed, reacted for 4 h at room temperature, and TLC detection is completed (petroleum ether: ethyl acetate=2:1 as developing agent, ultraviolet development is performed in volume ratio); the obtained product system was evaporated to dryness under reduced pressure, ethyl acetate was added to the residue to dissolve, the organic phase was washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (in terms of volume ratio, eluent: petroleum ether: ethyl acetate=3:1) to give 145.00 mg compound as yellow oil, compound III-1, yield 52.8%. ¹ H NMR (600 MHz, Chloroform-d) δ 8.83 (s, 1H), 8.60 (d, J = 2.8 Hz, 1H), 8.21 (dd, J = 9.1, 2.8 Hz, 1H), 7.43 (s, 1H), 7.39 (d, J = 8.3 Hz, 2H), 7.27 (d, J = 5.3 Hz, 2H), 7.09 (dd, J = 5.8, 2.7 Hz, 1H), 6.98 – 6.91 (m, 2H), 6.69 (ddd, J = 8.8, 4.0, 2.7 Hz, 1H), 5.31 (s, 2H), 3.32 – 3.26 (m, 4H), 3.14 – 3.08 (m, 4H), 2.48 (s, 3H). ¹³ C NMR (151 MHz, Chloroform-d) δ 165.76, 158.20, 157.48, 155.84, 155.72, 141.15, 140.29, 140.08, 139.31, 134.55, 132.04, 129.69, 128.17, 128.01, 126.44, 123.51, 121.47, 117.89, 116.88, 116.72, 109.32, 109.17, 67.12, 50.50, 47.89, 15.47. HR-MS (ESI): calcd. for C ₂₈ H ₂₆ O ₆ N ₇ ⁸¹ BrFS，[M+H] ⁺ 688.08067; found: 688.08392。

Example 26

Following the procedure in example 25, except using intermediate 6b (190.00 mg,0.58 mmol) and intermediate 13a (224.46 mg,0.58 mmol), compound III-2 was prepared 186.00 mg as a yellow oilThe yield of the product was 50.0%. ¹ H NMR (600 MHz, Chloroform-d) δ 8.61 (d, J = 2.8 Hz, 1H), 8.22 (dd, J = 9.1, 2.8 Hz, 2H), 7.40 (d, J = 8.3 Hz, 2H), 7.34 (s, 1H), 7.27 (d, J = 8.3 Hz, 2H), 7.02 – 6.92 (m, 3H), 6.66 (ddd, J = 8.8, 3.9, 2.7 Hz, 1H), 5.32 (s, 2H), 3.35 – 3.28 (m, 4H), 3.16 – 3.09 (m, 4H), 2.50 (s, 3H). ¹³ C NMR (150 MHz, Chloroform-d) δ 165.75, 158.20, 156.46, 155.70, 154.82, 141.15, 140.29, 140.06, 139.28, 134.30, 132.06, 129.70, 128.18, 128.00, 126.45, 125.17, 122.70, 121.54, 121.46, 121.41, 117.87, 117.03, 67.11, 50.50, 47.90, 15.48. HR-MS (APCI): calcd. for C ₂₈ H ₂₆ O ₆ N ₇ ClFS，[M+H] ⁺ 642.13323; found: 642.13336。

Example 27

The procedure is as in example 25, except that intermediate 6a (200.00 mg,0.54 mmol) and intermediate 13b (232.74 mg,0.54 mmol) are used to prepare 200.00 mg of compound III-3 as a yellow oil in 50.7% yield. ¹ H NMR (600 MHz, CDCl ₃ ) δ 8.62 (d, J = 2.8 Hz, 1H), 8.31 (s, 1H), 8.24 (dd, J = 9.1, 2.8 Hz, 1H), 7.31 (s, 1H), 7.08 (dd, J = 5.8, 2.7 Hz, 1H), 6.99 – 6.90 (m, 2H), 6.74 (s, 2H), 6.69 (ddd, J = 8.8, 3.9, 2.9 Hz, 1H), 5.30 (s, 2H), 3.88 (s, 9H), 3.40 – 3.30 (m, 4H), 3.17 – 3.06 (m, 4H). ¹³ C NMR (151 MHz, Chloroform-d) δ 165.86, 158.32, 157.21, 155.58, 155.48, 153.33, 140.93, 140.54, 140.05, 137.86, 135.01, 131.41, 128.02, 127.59, 123.13, 121.25, 117.64, 116.76, 116.61, 109.20, 109.05, 106.36, 67.60, 61.10, 56.36, 50.37, 48.15. HR-MS (APCI): calcd. For C ₃₀ H ₃₀ O ₉ N ₇ ⁸¹ BrF，[M+H] ⁺ 732.12465; found: 732.12634。

Example 28

The procedure is as in example 25, except that intermediate 6a (200.00 mg,0.54 mmol) and intermediate 13c (200.34 mg,0.54 mmol) are used to prepare 181.00 mg of compound III-4 as a yellow oil in 50.0% yield. ¹ H NMR (600 MHz, Chloroform-d) δ 8.79 – 8.61 (m, 1H), 8.57 (d, J = 2.8 Hz, 1H), 8.21 (dd, J = 9.1, 2.8 Hz, 1H), 7.41 (d, J = 8.6 Hz, 2H), 7.34 (s, 1H), 7.10 (dd, J = 5.8, 2.7 Hz, 1H), 6.98 – 6.90 (m, 4H), 6.70 (dt, J = 8.7, 3.6 Hz, 1H), 5.31 (s, 2H), 3.83 (s, 3H), 3.35 – 3.25 (m, 4H), 3.16 – 3.02 (m, 4H). ¹³ C NMR (151 MHz, Chloroform-d) δ 165.97, 159.72, 158.27, 157.45, 155.82, 155.56, 141.15, 140.28, 140.17, 134.60, 130.93, 127.98, 127.86, 127.80, 123.47, 121.87, 117.74, 116.87, 116.71, 114.30, 109.31, 109.16, 67.30, 55.62, 50.45, 47.93. HR-MS (ESI): calcd. for C ₂₈ H ₂₄ O ₇ N ₇ ⁸¹ BrF，[M-H] ^- 670.08787; found: 670.08765。

Example 29

The procedure is as in example 25 except that intermediate 6a (200.00 mg,0.54 mmol) and intermediate 13e (199.80 mg,0.54 mmol) are used to prepare 195.00 mg of compound III-5 as a yellow oil in 54.0% yield. ¹ H NMR (600 MHz, Chloroform-d) δ 9.00 (s, 1H), 8.74 (d, J = 2.8 Hz, 1H), 8.40 (t, J = 5.7 Hz, 1H), 8.19 (dd, J = 8.9, 2.8 Hz, 1H), 7.37 (s, 1H), 7.27 (d, J = 8.6 Hz, 2H), 7.16 – 7.07 (m, 2H), 7.02 – 6.90 (m, 1H), 6.85 (dd, J = 9.1, 2.3 Hz, 2H), 6.73 – 6.62 (m, 1H), 4.55 (d, J = 5.7 Hz, 2H), 3.75 (s, 3H), 3.01 (d, J = 5.6 Hz, 4H), 2.98 (d, J = 5.0 Hz, 4H). ¹³ C NMR (151 MHz, Chloroform-d) δ 164.84, 159.29, 157.98, 157.30, 155.66, 154.98, 143.40, 140.66, 140.26, 134.87, 129.96, 129.80, 128.26, 127.76, 127.19, 126.93, 123.24, 119.53, 116.74, 114.62, 109.13, 55.49, 51.49, 47.78, 43.52. HR-MS (ESI): calcd. for C ₂₈ H ₂₅ O ₆ N ₈ ⁸¹ BrF，[M-H] ^- 669.10385; found: 669.10364。

Example 30

The procedure is as in example 25 except that intermediate 6b (200.00 mg,0.61 mmol) is used with intermediate 13c (226.31 mg,0.61 mmol) to give 186.00 mg compound III-6 as a yellow oil in 48.8% yield. ¹ H NMR (600 MHz, Chloroform-d) δ 8.73 (s, 1H), 8.57 (d, J = 2.8 Hz, 1H), 8.20 (dd, J = 9.1, 2.8 Hz, 1H), 7.44 – 7.38 (m, 2H), 7.34 (s, 1H), 6.99 – 6.90 (m, 5H), 6.66 (ddd, J = 8.9, 3.9, 2.8 Hz, 1H), 5.30 (s, 2H), 3.83 (s, 3H), 3.34 – 3.25 (m, 4H), 3.15 – 3.04 (m, 4H). ¹³ C NMR (151 MHz, Chloroform-d) δ 165.95, 159.77, 158.24, 156.42, 155.58, 154.77, 141.09, 140.21, 134.37, 130.87, 127.89, 125.07, 122.59, 121.80, 121.52, 121.39, 117.76, 117.08, 116.93, 114.29, 67.31, 60.47, 55.58, 50.44, 47.90. HR-MS (ESI): calcd. for C ₂₈ H ₂₆ O ₇ N ₇ ClF，[M+H] ⁺ 626.15608; found: 626.15570。

Example 31

The procedure is as in example 25 except that intermediate 6a (200.00 mg,0.54 mmol) and intermediate 13d (216.54 mg,0.54 mmol) are used to prepare 174.00 mg of compound III-7 as a yellow oil in 46.1% yield. ¹ H NMR (600 MHz, Chloroform-d) δ 8.76 – 8.62 (m, 1H), 8.36 – 8.11 (m, 1H), 7.35 (d, J = 23.8 Hz, 1H), 7.12 (dd, J = 5.6, 2.7 Hz, 1H), 6.95 (ddt, J = 12.0, 8.6, 3.6 Hz, 2H), 6.76 – 6.67 (m, 1H), 6.66 – 6.55 (m, 2H), 6.50 – 6.42 (m, 1H), 5.29 (s, 2H), 3.79 (d, J = 2.8 Hz, 6H), 3.32 (s, 4H), 3.25 – 3.12 (m, 4H). ¹³ C NMR (151 MHz, Chloroform-d) δ 165.60, 160.99, 158.22, 157.45, 155.84, 141.15, 140.17, 137.50, 134.59, 128.33, 128.04, 127.92, 123.39, 121.29, 118.04, 116.79, 109.26, 106.60, 100.66, 67.34, 55.55, 50.56, 47.93. HR-MS (ESI): calcd. for C ₂₉ H ₂₈ O ₈ N ₇ BrF，[M+H] ⁺ 700.11613; found: 700.11578。

Example 32

The procedure is as in example 25, except that intermediate 6a (200.00 mg,0.54 mmol) and intermediate 13f (208.44 mg,0.54 mmol) are used to prepare 174.00 mg of compound III-8 as a yellow oil in 47.2% yield. ¹ H NMR (600 MHz, Chloroform-d) δ 8.78 (d, J = 2.8 Hz, 1H), 8.56 (s, 1H), 8.40 (t, J = 5.8 Hz, 1H), 8.22 (dd, J = 8.9, 2.8 Hz, 1H), 7.33 (s, 1H), 7.29 (d, J = 8.2 Hz, 2H), 7.20 (d, J = 8.2 Hz, 2H), 7.13 (d, J = 8.9 Hz, 1H), 7.09 (dd, J = 5.8, 2.7 Hz, 1H), 6.94 (t, J = 8.4 Hz, 1H), 6.67 (dt, J = 8.7, 3.6 Hz, 1H), 4.58 (d, J = 5.9 Hz, 2H), 3.11 – 3.04 (m, 4H), 3.04 – 2.96 (m, 4H), 2.45 (s, 3H). ¹³ C NMR (151 MHz, Chloroform-d) δ 164.93, 157.91, 154.90, 143.52, 140.73, 140.22, 138.35, 134.77, 134.61, 128.99, 128.28, 127.88, 127.28, 126.99, 126.84, 123.39, 119.51, 116.84, 116.68, 109.14, 60.43, 51.50, 47.90, 43.52, 15.47. HR-MS (ESI): calcd. for C ₂₈ H ₂₇ O ₅ N ₈ BrFS, [M+H] ⁺ , 685.09870; found: 685.10137。

Example 33

The synthesis procedure for compound IV-1 is as follows:

(1) 30 mL acetonitrile, intermediate 14a (300.00 mg, 0.59 mmol), intermediate 5a (201.78 mg, 0.59 mmol) and potassium carbonate (162.84 mg, 1.18 mmol) were mixed and reacted for 18 h under reflux (60 ℃) with TLC detection (petroleum ether: ethyl acetate=1:1 as developing agent by volume ratio, ultraviolet coloration); filtering the obtained product system to remove potassium carbonate, concentrating the filtrate under reduced pressure, dissolving residues with 30 mL ethyl acetate, washing the organic phase with water and saturated sodium chloride solution, drying the organic phase with anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure, and purifying the filtrate by silica gel column chromatography (the eluent is petroleum ether: ethyl acetate=1:2 according to the volume ratio), thereby obtaining an intermediate 15a which is directly used for the next reaction;

(2) The procedure of step (2) of example 1 was followed except that intermediate 15a was used to prepare 151.00 mg compound IV-1 as a yellow oil in 34.2% two-step yield. ¹ H NMR (600 MHz, Chloroform-d) δ 8.96 (s, 1H), 8.65 (d, J = 2.8 Hz, 1H), 8.24 (dd, J = 9.1, 2.8 Hz, 1H), 7.39 (d, J = 8.3 Hz, 2H), 7.27 (d, J = 8.3 Hz, 2H), 7.19 (dd, J = 5.8, 2.7 Hz, 1H), 7.02 – 6.92 (m, 3H), 6.91 – 6.84 (m, 2H), 5.32 (s, 2H), 4.13 (d, J = 4.4 Hz, 2H), 3.80 – 3.75 (m, 2H), 3.53 – 3.48 (m, 2H), 3.22 – 3.15 (m, 4H), 2.50 (s, 3H). ¹³ C NMR (151 MHz, Chloroform-d) δ 166.97, 165.46, 157.21, 155.71, 155.58, 154.88, 141.64, 140.49, 139.77, 138.69, 135.92, 135.90, 131.77, 129.48, 128.35, 128.25, 128.07, 126.38, 123.95, 121.25, 118.03, 116.13, 108.64, 67.21, 51.36, 50.72, 45.26, 43.96, 41.83, 15.54. HR-MS (ESI): calcd. for C ₃₀ H ₂₇ O ₇ N ₈ ⁸¹ BrFS，[M-H] ^- 743.08649; found: 743.08575。

Example 34

The procedure was followed as in example 33, except that intermediate 14c (300.00 mg,0.61 mmol) was used to prepare intermediate 15c from intermediate 5a (208.62 mg,0.61 mmol); then using the intermediate 15c, the preparation yielded 105.00, mg, compound IV-2 as a yellow oil in 23.6%. ¹ H NMR (600 MHz, Chloroform-d) δ 9.25 (s, 1H), 8.55 (d, J = 2.3 Hz, 1H), 8.18 – 8.08 (m, 1H), 7.33 (d, J = 8.4 Hz, 2H), 7.26 – 7.17 (m, 1H), 7.14 – 7.06 (m, 1H), 6.96 – 6.89 (m, 2H), 6.86 (t, J = 9.0 Hz, 3H), 6.83 – 6.76 (m, 3H), 5.23 (s, 2H), 4.03 (d, J = 4.1 Hz, 2H), 3.75 (s, 3H), 3.67 (s, 2H), 3.42 (s, 2H), 3.10 (s, 4H). ¹³ C NMR (151 MHz, Chloroform-d) δ 167.01, 165.66, 160.05, 159.23, 157.12, 155.54, 154.89, 141.55, 140.41, 138.74, 135.97, 132.95, 130.66, 128.77, 128.32, 128.04, 127.35, 123.82, 121.41, 117.91, 116.11, 114.05, 108.61, 67.41, 65.05, 55.38, 50.94, 45.24, 43.96, 41.84. HR-MS (ESI): calcd. for C ₃₀ H ₂₉ O ₈ N ₈ ⁸¹ BrF，[M+H] ⁺ 729.12498; found: 729.12463。

Example 35

The procedure was followed as in example 33, except that intermediate 14e (300.00 mg,0.67 mmol) was used to prepare intermediate 15e from intermediate 5a (229.14 mg,0.67 mmol); then using the intermediate 15e, 170.00 mg compound IV-3 was prepared as a yellow oil in 35.0% yield. ¹ H NMR (600 MHz, Chloroform-d) δ 8.80 (s, 1H), 8.75 (d, J = 2.8 Hz, 1H), 8.22 (dd, J = 8.9, 2.8 Hz, 1H), 8.16 (t, J = 5.6 Hz, 1H), 7.31 (d, J = 8.6 Hz, 2H), 7.19 (dd, J = 5.8, 2.7 Hz, 1H), 7.09 (d, J = 8.9 Hz, 1H), 7.00 (dd, J = 18.1, 9.8 Hz, 2H), 6.93 – 6.86 (m, 3H), 6.74 (t, J = 4.4 Hz, 1H), 4.59 (d, J = 5.6 Hz, 2H), 4.04 (d, J = 4.5 Hz, 2H), 3.78 (s, 3H), 3.57 (s, 2H), 3.22 (d, J = 4.7 Hz, 2H), 3.11 – 3.01 (m, 4H). ¹³ C NMR (151 MHz, Chloroform-d) δ 166.77, 164.95, 159.54, 157.26, 155.63, 154.86, 154.53, 143.36, 141.81, 138.63, 135.92, 135.89, 129.75, 128.34, 127.14, 126.92, 124.07, 124.02, 119.38, 116.15, 114.46, 108.66, 55.45, 52.04, 51.76, 45.21, 43.73, 43.62, 41.69. HR-MS (ESI): calcd. for C ₃₀ H ₂₉ O ₇ N ₉ BrFNa，[M+Na] ⁺ 748.12496; found: 748.12457。

Example 36

The procedure was followed as in example 33, except that intermediate 14b (300.00 mg,0.59 mmol) was used to prepare intermediate 15b from intermediate 5a (201.78 mg,0.59 mmol); then using intermediate 15b to afford 162.00 mg compound IV-4 as a yellow oil in 34.9% yield. ¹ H NMR (600 MHz, Chloroform-d) δ 8.72 (d, J = 2.4 Hz, 1H), 8.25 (d, J = 9.1 Hz, 1H), 7.18 (s, 1H), 6.97 (dd, J = 17.2, 7.4 Hz, 3H), 6.93 – 6.85 (m, 1H), 6.78 (s, 1H), 6.70 (s, 2H), 5.30 (s, 2H), 4.15 (d, J = 4.5 Hz, 2H), 3.89 (s, 6H), 3.86 (s, 3H), 3.80 (s, 2H), 3.60 (s, 2H), 3.31 – 3.18 (m, 4H). ¹³ C NMR (151 MHz, Chloroform-d) δ 167.03, 165.27, 155.85, 154.91, 153.50, 141.75, 140.41, 138.66, 138.41, 135.91, 130.78, 128.59, 128.21, 123.95, 120.88, 118.11, 116.13, 108.64, 105.89, 67.66, 60.95, 56.30, 51.23, 50.92, 45.31, 44.06, 41.86. HR-MS (ESI): calcd. for C ₃₂ H ₃₂ O ₁₀ N ₈ BrFNa，[M+Na] ⁺ 809.13010; found: 809.12854。

Example 37

The procedure was followed as in example 33, except that intermediate 14d (300.00 mg,0.58 mmol) was used to prepare intermediate 15d from intermediate 5a (198.36 mg,0.58 mmol); then prepared by using the intermediate 15d 158.00 mg of compound IV-5 as yellow oil in 35.9% yield. ¹ H NMR (600 MHz, Chloroform-d) δ 8.68 (d, J = 2.3 Hz, 1H), 8.32 – 8.17 (m, 1H), 7.23 – 7.12 (m, 1H), 7.05 – 6.92 (m, 3H), 6.92 – 6.82 (m, 1H), 6.83 – 6.75 (m, 1H), 6.60 (d, J = 1.8 Hz, 2H), 6.47 (s, 1H), 5.30 (s, 2H), 4.12 (d, J = 4.3 Hz, 2H), 3.81 (s, 6H), 3.79 (s, 2H), 3.61 – 3.45 (m, 2H), 3.21 (dd, J = 9.2, 4.4 Hz, 4H). ¹³ C NMR (151 MHz, Chloroform-d) δ 167.01, 165.46, 161.13, 155.69, 154.90, 141.73, 140.41, 138.68, 137.48, 135.94, 128.36, 128.19, 128.08, 123.87, 121.16, 117.95, 116.13, 108.63, 106.56, 100.20, 67.33, 55.51, 51.47, 50.58, 45.28, 43.98, 41.84. HR-MS (ESI): calcd. for C ₃₁ H ₃₁ O ₉ N ₈ ⁸¹ BrF，[M+H] ⁺ 759.13555; found: 759.13690。

Example 38

The procedure is as in example 33 except that intermediate 14f (300.00 mg,0.65 mmol) is used in combination with intermediate 5a (222.30 mg,0.65 mmol) to afford intermediate 15f; then, the intermediate 15f was used to prepare 164.00 mg compound IV-6 as a yellow oil product in 34.0% yield. ¹ H NMR (600 MHz, DMSO-d ₆ ) δ 11.70 (s, 1H), 9.13 (s, 1H), 8.89 (s, 1H), 8.20 (dd, J = 9.1, 2.8 Hz, 1H), 8.17 (d, J = 2.8 Hz, 1H), 7.34 (d, J = 8.3 Hz, 2H), 7.28 (d, J = 8.3 Hz, 2H), 7.23 – 7.15 (m, 2H), 7.13 (dd, J = 6.0, 2.7 Hz, 1H), 6.87 – 6.78 (m, 1H), 6.65 (t, J = 4.7 Hz, 1H), 4.44 (d, J = 5.9 Hz, 2H), 4.16 (d, J = 4.7 Hz, 2H), 3.55 (s, 2H), 3.45 (s, 2H), 3.23 (d, J = 26.8 Hz, 4H), 2.48 (s, 3H). ¹³ C NMR (151 MHz, DMSO-d ₆ ) δ 167.14, 166.86, 155.58, 154.95, 154.53, 153.36, 140.31, 140.00, 139.35, 138.66, 138.64, 137.23, 136.23, 129.02, 126.95, 126.61, 126.03, 125.17, 121.90, 118.13, 116.37, 107.46, 50.24, 49.99, 45.50, 43.58, 42.86, 41.42, 15.39. HR-MS (ESI): calcd. for C ₃₀ H ₂₀ O ₆ N ₉ ⁸¹ BrFS，[M+H] ⁺ 744.11812; found: 744.12000。

Example 39

The synthesis procedure for compound IV-7 is as follows:

the procedure is as in example 33 except that intermediate 14a (300.00 mg,0.59 mmol) is used with intermediate 8 (227.74 mg,0.59 mmol) to give intermediate 15g; then using 15g of the intermediate to prepare 180.00. 180.00 mg of compound IV-7 as yellow oil with a yield of 38.8%. ¹ H NMR (600 MHz, Chloroform-d) δ 8.58 (s, 1H), 8.16 (d, J = 8.8 Hz, 1H), 7.35 (d, J = 8.1 Hz, 2H), 7.24 (d, J = 8.1 Hz, 2H), 7.12 (s, 1H), 7.09 – 7.05 (m, 1H), 6.94 – 6.86 (m, 2H), 6.77 (d, J = 8.0 Hz, 1H), 6.52 (s, 1H), 5.28 (d, J = 14.3 Hz, 2H), 3.74 (s, 2H), 3.66 (s, 4H), 3.40 (s, 2H), 3.18 (s, 2H), 3.12 (d, J = 17.0 Hz, 4H), 2.46 (s, 3H). ¹³ C NMR (151 MHz, Chloroform-d) δ 165.45, 156.80, 155.56, 155.18, 141.25, 140.22, 139.55, 138.91, 136.13, 131.82, 129.45, 128.27, 128.03, 127.56, 126.39, 123.41, 120.88, 117.88, 116.14, 115.99, 108.58, 108.43, 67.17, 51.18, 50.39, 48.99, 47.19, 44.07, 42.57, 41.76, 15.52. HR-MS (ESI): calcd. for C ₃₂ H ₃₄ O ₇ N ₉ ⁸¹ BrFS，[M+H] ⁺ 788.14434; found: 788.14594。

Example 40

The synthesis procedure for compound IV-8 is as follows:

to toluene (10 mL) was added compound CA-4 (300 mg, 0.95 mmol), 2-bromoethanol (141 mg, 1.14 mmol), triphenylphosphine (299 mg, 1.14 mmol) and diisopropyl azodicarboxylate (230 mg, 1.14 mmol), and reacted at room temperature for 24 hours, after tlc detection (in volume ratio, developing solvent petroleum ether: ethyl acetate=3:1, uv color); evaporating the solvent in the obtained product system under reduced pressure, and separating and purifying the residue by silica gel column chromatography (petroleum ether: ethyl acetate=4:1 as eluent) to obtain a white oily product 215 mg which is an intermediate 16, wherein the yield is 54%;

Intermediate 16 (200 mg, 0.47 mmol), intermediate 8 (183 mg, 0.47 mmol) and potassium carbonate (129 mg, 0.94 mmol) were added to acetonitrile (30 mL),stirring at 80deg.C for 20 hr, and detecting by TLC (developing solvent is dichloromethane: methanol=10:1, ultraviolet developing; the obtained product system is filtered to remove potassium carbonate, the filtrate is evaporated to dryness under reduced pressure and then is separated and purified by silica gel column chromatography (the eluent is methylene dichloride: methanol=30:1 according to the volume ratio), and the off-white solid 90 mg is obtained as the compound IV-8, and the yield is 27%. m.p. 100.0-101.6 ℃. ¹ H NMR (600 MHz, Chloroform-d) δ 7.21 (dd, J = 5.9, 2.7 Hz, 1H), 7.13 (s, 1H), 6.98 (t, J = 8.4 Hz, 1H), 6.89 (td, J = 9.2, 8.7, 3.3 Hz, 2H), 6.84 (d, J = 1.9 Hz, 1H), 6.79 (d, J = 8.3 Hz, 1H), 6.50 (s, 2H), 6.46 (s, 2H), 6.06 (t, J = 5.8 Hz, 1H), 5.30 (s, 2H), 4.39 – 4.35 (m, 2H), 4.12 – 4.08 (m, 2H), 3.81 (s, 3H), 3.79 (s, 3H), 3.69 (s, 6H), 3.36 (q, J = 5.8 Hz, 2H), 2.96 (t, J = 5.9 Hz, 2H). ¹³ C NMR (151 MHz, Methanol-d ₄ ) δ 156.56, 155.85, 154.95, 152.86, 148.89, 147.41, 140.82, 138.95, 136.94, 133.40, 130.22, 129.34, 128.76, 127.44, 123.68, 122.71, 115.39, 114.55, 111.76, 107.37, 106.00, 103.39, 72.83, 67.59, 59.81, 55.11, 55.05, 41.77, 40.52, 38.17.HR-MS (ESI): calcd. for C ₃₁ H ₃₅ O ₇ N ₆ ⁸¹ BrF，[M+H] ⁺ 703.17087; found: 703.17194。

Test example 1

The N-hydroxyamidine derivatives prepared in examples 1 to 40 were tested for IDO1 inhibitory activity as follows:

(1) Biochemical level IDO1 inhibition Activity assay

In a standard reaction system of 100 mu L, the reagent mainly comprises 50 mM potassium dihydrogen phosphate buffer (pH 6.5), 10 mM ascorbic acid, 10 mu M methylene blue, 100 mu g/mL bovine liver catalase, 100 mu M L-Trp, 100 nM IDO1 and compounds to be tested with different concentrations. The 96-well plate was allowed to react at 37℃for 30 min, and 40. Mu.L of 40% (w/v) trichloroacetic acid was added to terminate the reaction. The samples were then incubated at 65℃for 15 min to promote kynurenine production. Finally 60. Mu.L of a solution of p-dimethylaminobenzaldehyde-glacial acetic acid (3.75%, w/v) was added. Measuring absorbance of the mixed solution at 490 and nm wavelength by using an enzyme-labeled instrument according to the following method The following formula calculates the IDO1 inhibition by compounds, wherein the concentration of the enzyme activity primary screening compound is 100. Mu.M, and compounds with inhibition greater than 50% are diluted and measured by a double dilution method, and IC is determined by nonlinear regression analysis using GraphPad Prism 9 ₅₀ Value:

。

(2) Determination of IDO1 inhibitory Activity at cellular level

HeLa cells were grown at 2X 10 ⁴ Density of wells/density of wells inoculated in 96 well plates at final volume of 200. Mu.L, placed at 37℃in 5% CO ₂ Culturing overnight in an incubator. The next day, the original medium was discarded, 200. Mu.L of cell culture medium containing 100 ng/mL of human interferon-gamma, 50. Mu. M L-Trp and different concentrations of test compound was added, and the mixture was placed at 37℃with 5% CO ₂ After 48℃and h incubation in an incubator, 140. Mu.L of the supernatant was mixed with 10. Mu.L of 40% (w/v) trichloroacetic acid and incubated at 65℃for 15 min. The mixture was then centrifuged at 2500 r/min for 10 min to remove the precipitate. Mixing 100 μl of supernatant with an equal volume of p-dimethylaminobenzaldehyde-glacial acetic acid solution (2%, w/v), incubating at room temperature for 10 min, reading absorbance at 490 nm wavelength, calculating IDO1 inhibition rate (formula same above), diluting with a double dilution method at a cell primary screening compound concentration of 10 μM, and measuring with GraphPad Prism 9 to calculate EC ₅₀ Values.

(3) Cytotoxicity assays

To investigate whether biological activity at the HeLa cell level was due to IDO1 inhibition, but not false positives due to cytotoxicity, cytotoxicity (CC) of a compound having both good biochemical and cellular level enzyme inhibitory activities was evaluated by MTT method ₅₀ ) (the positive control was 5-FU). HeLa cells at 5X 10 ³ Density of wells/well inoculated in 96 well plates at a final volume of 100. Mu.L, placed at 37℃in 5% CO ₂ Culturing overnight in an incubator. The next day, the original culture medium is discarded, 100 mu L of the medicine-containing culture medium is added into each hole, and the mixture is placed at 37 ℃ and 5% CO ₂ Culturing 48 h in a constant temperature incubator. Abandon the originalAnd adding 100 mu L (light-shielding operation) of 1 mg/mL MTT working solution into each hole of the culture medium, and culturing at the constant temperature of 37 ℃ for 3.5-4 hours. The reaction solution was discarded, 150. Mu.L of DMSO solution was added to each well, and the 96-well plate was placed on a micro-shaker and shaken for 5 min to ensure complete dissolution of the crystals. The absorbance at 490 nm was measured with a microplate reader. The relative cell viability was calculated according to the following formula, and drug CC was calculated by GraphPad Prism software ₅₀ Values and concentration-survival curves were plotted:

。

the measurement results are shown in tables 1 to 2 and FIGS. 1 to 8 (Epacadenostat is used as a positive control). Wherein, FIG. 1 and FIG. 2 are the measurement results of the inhibitory activity of Epacadostat against IDO1, FIG. 3 and FIG. 4 are the measurement results of the inhibitory activity of Compound I-1 against IDO1, FIG. 5 and FIG. 6 are the measurement results of the inhibitory activity of Compound I-2 against IDO1, and FIG. 7 and FIG. 8 are the measurement results of the inhibitory activity of Compound IV-7 against IDO 1.

TABLE 1 Activity test results of Compounds prepared in examples 1 to 24

Note that: ^a IC ₅₀ 、EC ₅₀ and CC ₅₀ The value of (2) is the average of at least three trials; "ND" represents undetected.

TABLE 2 Activity test results of the compounds prepared in examples 25 to 40

The activity evaluation experimental data of tables 1-2 and figures 1-8 show that the inhibitory activity of the compounds I-5, I-8 and I-13 on IDO1 is superior to that of the compounds I-11, I-12 and I-14; the compounds I-1 and I-2 are compared with the compounds I-3, I-5, I-6, I-7 and I-8, and the compounds newly introduced with substituent groups (electron-withdrawing substituent groups and electron-donating substituent groups) on the side chain benzene rings have 5-65 times of inhibition activity on IDO1 on the cellular level and have little difference in enzyme activity; meanwhile, piperazine is used for replacing methylene, and the obtained compounds I-15-I-18 have almost no inhibitory activity on IDO 1.

Comparative Compounds II-1, II-3, II-4, II-5, at the cellular level, inhibition of IDO1 by piperazine-substituted compounds on the side-chain benzene ring > morpholine-substituted compounds > thiomorpholine-substituted compounds > Boc piperazine-substituted compounds, heLa EC thereof ₅₀ The activity is not greatly different at the enzyme activity level between 0.199 and 8.65 mu M; comparing the compounds II-2 and II-3, changing benzene ring into pyridine, and making the activity of the compounds not greatly different; meanwhile, the C-2 heterocycle and C-5 nitro on the benzene ring are changed into fluorine, and the inhibitory activity of the compound II-6 on IDO1 is improved at the cellular level and the enzyme activity level.

In general, the compounds I-1, I-2, II-5, II-6, IV-3, IV-6 and IV-7 of the invention have a better IDO1 inhibitory activity at the cellular level, heLa EC ₅₀ Values below 200 nM. In order to investigate whether the cellular activity of the compounds is caused by IDO1 inhibition or by cytotoxicity alone, the invention also used cell-based methods to determine the CC of the more active compounds I-1, I-2, II-5, II-6, IV-3, IV-6 and IV-7 against HeLa cells ₅₀ And calculating a selection index SI (CC ₅₀ /EC ₅₀ ) Between 91.66 and 37423 (generally considered as the selection index SI>30, indicating that the cellular activity is caused by IDO1 inhibition). Of particular note is compound IV-7 (HeLa EC ₅₀ = 0.00017 ±0.01 μm), which is currently the compound of IDO1 inhibitors having the best inhibitory activity against IDO1 at the cellular level, heLa EC ₅₀ The value reaches the sub-nanomole level, has the value of further clinical development and application, and can be used as a drug lead molecule for further research and development.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. An N-hydroxyamidine derivative having Sup>A structure according to formulSup>A I, formulSup>A II, formulSup>A III, formulSup>A IV-Sup>A, formulSup>A IV-7 or formulSup>A IV-8:

a formula I; />A formula II; />Formula III;

formulSup>A IV-A; />Formula IV-7;

formula IV-8;

R in formula II ₂ Is that、/>、/>、/>、/>Or->；

R in formula III ¹ is-Cl or-Br;

2. The N-hydroxyamidine derivative of claim 1, wherein the N-hydroxyamidine derivative is any one of the following compounds:

、/>、/>、

。

3. a process for producing an N-hydroxyamidine derivative according to claim 1 or 2, wherein,

；/>；

the R is ₁ As defined in formula I;

；/>；

the R is ₂ As defined in formula II;

；/>；

the R is ¹ 、R ₃ 、R ₄ 、R ₅ And X is as defined in formula III;

；/>；/>；

；/>；

；/>。

4. use of an N-hydroxyamidine derivative according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, for the preparation of IDO1 inhibitors.

5. Use of an N-hydroxyamidine derivative of claim 1 or 2 or a pharmaceutically acceptable salt thereof in the manufacture of a tumor immunotherapeutic medicament.

6. The use of claim 5, wherein the tumor comprises non-small cell lung cancer, breast cancer or glioblastoma.

7. A tumor immunotherapeutic agent comprising an active ingredient which is the N-hydroxyamidine derivative according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

8. The medicament of claim 7, wherein the pharmaceutically acceptable carrier comprises one or more of water for injection, propylene glycol, mannitol, glycerol, stearic acid, sodium chloride, dextrin, dextrose, starch, sucrose, lactose, sodium hydroxymethylstarch, polyethylene glycol, alginic acid, and polysorbate 80.

9. The medicament according to claim 7 or 8, wherein the dosage form of the medicament comprises a tablet, capsule, pill, granule, syrup, emulsion, suspension, aerosol, injection or suppository.

10. The medicament of claim 9, wherein the mode of administration of the medicament comprises intravenous injection, oral administration, inhalation or rectal administration.