CN113480543B

CN113480543B - 2,6, 8-polysubstituted imidazo [1,2-a ] pyrazine and synthetic method and application thereof

Info

Publication number: CN113480543B
Application number: CN202110765383.1A
Authority: CN
Inventors: 王鑫; 邓凯元
Original assignee: Wuxi No 2 Peoples Hospital
Current assignee: Wuxi No 2 Peoples Hospital
Priority date: 2021-07-07
Filing date: 2021-07-07
Publication date: 2022-05-17
Anticipated expiration: 2041-07-07
Also published as: CN113480543A; WO2023279938A1

Abstract

The invention discloses a 2,6, 8-polysubstituted imidazo [1,2-a ] as shown in formula (I)]Pyrazine or a pharmaceutically acceptable salt thereof. The invention also discloses the 2,6, 8-polysubstituted imidazo [1,2-a ]]Pyrazine or pharmaceutically acceptable salts thereof are used as TYK2 inhibitors and are applied to preparation of medicines for preventing and treating tumor or inflammatory diseases. The invention also provides the 2,6, 8-polysubstituted imidazo [1,2-a ]]A method for synthesizing pyrazine or pharmaceutically acceptable salts thereof.

Description

2,6, 8-polysubstituted imidazo [1,2-a ] pyrazine and synthetic method and application thereof

Technical Field

The invention belongs to the field of pharmaceutical synthesis chemical industry, and relates to 2,6, 8-polysubstituted imidazo [1,2-a ] pyrazine and a synthesis method and application thereof, in particular to a 2,6, 8-polysubstituted imidazo [1,2-a ] pyrazine compound serving as a TYK2 inhibitor and a preparation method and application thereof.

Background

Tyrosine kinase 2(Tyrosine kinase 2, TYK2) is a member of a non-receptor protein Tyrosine kinase JAK family, is an intersection point of various signal transduction pathways, and plays an important role in normal tumor cell proliferation, differentiation and apoptosis. Downstream protein STATs can be recruited by activated JAK, and then phosphorylated by JAK to form homodimers or heterodimers, enter cell nucleus, and are combined with the promoter of downstream genes to regulate the transcription of the downstream genes. The JAK-STAT signaling pathway is closely related to the development and progression of tumors, exhibiting over-activation in many malignancies.

In recent years, more and more studies have shown that TYK2 is closely related to many tumors. In Anaplastic Large Cell Lymphoma (ALCL), TYK2 is highly expressed and promotes the growth of ALCL cells through abnormal activation of TYK2-STAT1/3-Bcl2 pathway, and ALCL apoptosis can be promoted through the use of TYK2 inhibitor. The ubiquity of TYK2 high expression was found in samples of patients with Malignant Peripheral Nerve Sheath Tumors (MPNSTs), cell death was significantly increased by lower TYK2 in both murine and human MPNST cells, while aberrant activation of the TYK2-STAT1/3-Bcl2 pathway was also detected in MPNST. In animal in vivo studies, it was found that the tumor growth of TYK2(-/-) mice was rapidly grown and the metastatic ability was significantly stronger than that of TYK2(+/+) mice when 4T1 breast cancer cells were injected into TYK2(-/-) mice and TYK2(+/+) mice, respectively. Therefore, based on the biological function of TYK2 and the extensive research results in tumors, TYK2 can be concluded to have an important regulatory role in the development of tumors, and TYK2 is a very potential tumor therapy target.

In conclusion, the expression level of TYK2 is abnormal in various tumor patient samples, which is closely related to the occurrence of tumors. Although there are reported TYK2 inhibitors that have entered clinical trials, there are no TYK2 inhibitors that have been used in tumor therapy. The TYK2 is a potential drug target for anti-tumor therapy, and a targeted inhibitor of the TYK2 is very potential to be developed for anti-tumor drugs, so that the development of TYK2 inhibitors for tumor therapy is urgently needed.

Disclosure of Invention

The 2,6, 8-polysubstituted imidazo [1,2-a ] pyrazine compound provided by the invention is a TYK2 inhibitor, and can be used for treating various tumors, including various malignant tumors such as lymphoma, ovarian cancer, gastric cancer, breast cancer, lung cancer and the like.

The 2,6, 8-polysubstituted imidazo [1,2-a ] pyrazine and pharmaceutically acceptable salts thereof provided by the invention have a structural general formula shown as (I):

wherein:

R₁is selected from

Wherein E, Y, A, Q are each independently selected from C, N, O, S, R in (1)₄Selected from hydrogen radicals, amino radicals, methylamino radicals, dimethylamino radicals, hydroxyl radicals, halogen atoms, cyano radicals, hydrocarbon radicals, cycloalkyl radicals, heteroatomic cycloalkyl radicals, aryl or heteroaromatic radicals, substituted aryl radicals or substituted heteroaromatic radicals,

In (2), L is selected from

R₅Selected from hydrogen radicals, aryl or heteroaromatic rings, substituted aryl or substituted heteroaromatic rings;

R₂selected from cycloalkyl or substituted cycloalkyl, heteroatomic cycloalkyl or substituted heteroatomic cycloalkyl or

When R is₂When the structure is selected from the structure in (3), M, X, Z, T are independently selected from C, N, R₆Selected from hydrogen, amino, substituted amino, hydroxyl, halogen atom, cycloalkyl or cycloalkyl containing substituent, heteroatom cycloalkyl or heteroatom cycloalkyl containing substituent, cyano, alkyl, aryl or heteroaromatic ring, substituted aryl or substituted heteroaromatic ring;

R₃selected from the group consisting of hydrogen atoms,

Preferably, R₁Is selected from

Wherein E, Y, A, Q are each independently selected from C, N, R in (1)₄Selected from amino, cyano, in (2) L is selected from

R₅Is selected from

R₂Selected from cyclopropane or

Wherein M, X, Z, T are each independently selected from C, N, R₆Selected from halogen atoms,

R₃Selected from the group consisting of hydrogen atoms,

Further preferably, R₁Is selected from

R₅Is selected from

R₂Selected from cyclopropane or

Wherein M, X, Z, T are each independently selected from C, N, R₆Selected from fluorine atoms,

R₃Selected from the group consisting of hydrogen atoms,

The invention also provides a pharmaceutical composition, which comprises the compound shown in the formula (I) or the pharmaceutically acceptable salt thereof and one or more medicinal carriers and/or diluents, and is prepared into a clinical medicinal preparation. Wherein, the pharmaceutical composition is prepared into tablets, capsules, liquid preparations or the like. The compounds of the present invention may be combined with pharmaceutical carriers or excipients (e.g., pharmaceutically acceptable carriers and excipients) to form pharmaceutical formulations according to conventional pharmaceutical formulation techniques. The 2,6, 8-polysubstituted imidazo [1,2-a ] pyrazine compounds may be incorporated as the active ingredient in any of the usual oral dosage forms including tablets, capsules and liquid preparations (e.g., elixirs and suspensions) containing coloring, flavoring, stabilizing and taste-masking substances. For mixed oral dosage forms, the 2,6, 8-polysubstituted imidazo [1,2-a ] pyrazine compounds as active ingredients may be mixed with various common tablet materials (e.g., starch, calcium carbonate, lactose, sucrose, and dicalcium phosphate) to facilitate tableting and encapsulation, and the 2,6, 8-polysubstituted imidazo [1,2-a ] pyrazine compounds may be dissolved or suspended in a pharmaceutically acceptable sterile liquid carrier such as sterile water, sterile organic solvents, or a mixture of both. The liquid carrier may be a carrier suitable for injection, such as physiological saline, propylene glycol or aqueous polyethylene glycol solution. In other cases, it may also be prepared by dispersing the micronised active ingredient in an aqueous solution of starch or sodium carboxymethylcellulose or in a suitable oil, for example arachis oil. Liquid pharmaceutical formulations (referred to as sterile solutions or suspensions) can be used for intravenous, intramuscular, intraperitoneal or subcutaneous injection. The pharmaceutical composition may also comprise one or more inorganic or organic, solid or liquid pharmaceutically acceptable carriers or excipients. The term "pharmaceutically acceptable" refers to additives or compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction (e.g., dizziness, etc.) when administered to an animal such as a mammal (e.g., a human). Pharmaceutical carriers and excipients may include, but are not limited to, diluents, such as lactose, glucose, mannose, and/or glycerol; a lubricant; polyethylene glycol; binders, such as magnesium aluminum silicate, starch, gelatin, methylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone; and, if desired, disintegrating agents, such as starch, agar, alginic acid or a salt thereof such as sodium alginate; and/or adsorbents, colorants, preservatives, stabilizers, flavorants and sweeteners.

The invention also provides application of the compound shown in the formula (I) or pharmaceutically acceptable salt thereof or the pharmaceutical composition in preparation of the TYK2 inhibitor.

The invention also provides application of the compound shown in the formula (I) or pharmaceutically acceptable salt thereof or the pharmaceutical composition in preparing medicines for preventing and treating tumor or inflammatory diseases. The tumor comprises various malignant tumors such as lymphoma, ovarian cancer, gastric cancer, breast cancer, lung cancer and the like.

The compound or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition is used for inhibiting the proliferation, growth, infiltration and migration of tumor cells, or promoting the apoptosis of the tumor cells.

The invention also provides a preparation method of the compound shown in the formula (I) or the pharmaceutically acceptable salt thereof: obtaining 2-substituted-6, 8-dibromo-imidazo [1,2-a ] pyrazine through ring closure of 2-amino-3, 5-dibromo-pyrazine, then obtaining 2, 6-polysubstituted-8-dibromo-imidazo [1,2-a ] pyrazine through substitution reaction, and finally obtaining the target compound 2,6, 8-polysubstituted imidazo [1,2-a ] pyrazine through scouring reaction:

the compound disclosed by the invention has the beneficial effects that a cell activity determination experiment and a TYK2 enzyme activity determination experiment are carried out on the compound, and the experiment result shows that the compound disclosed by the invention has good application in preventing and treating cancer or inflammation related diseases.

Detailed Description

The following detailed description of the preferred embodiments of the present invention is provided to enable those skilled in the art to more readily understand the advantages and features of the present invention, and to clearly and unequivocally define the scope of the present invention.

Various aspects and features of the disclosure are described further below.

Abbreviations used herein are generally well known to those skilled in the art or may be readily understood based on basic knowledge.

The starting materials employed in the preparation of the compounds of the invention are known, can be prepared according to known methods or are commercially available.

The invention also relates to novel intermediates and/or starting materials. The same or similar reaction conditions and novel intermediates as those mentioned in the examples are particularly preferred.

Both intermediates and final products can be worked up and/or purified according to conventional methods including pH adjustment, extraction, filtration, drying, concentration, chromatography, trituration, crystallization, and the like.

In addition, the compounds of the present invention may be prepared by various methods known in the art or variations of the methods described herein.

The following examples are intended to illustrate the invention and are not intended to limit it in any way.

Example 1

Preparation of 3-cyano-N- (3- (8- ((4-morpholinophenyl) amino) imidazo [1,2-a ] pyrazin-6-yl) phenyl) benzamide

Step 1.1: preparation of 4- (4-nitrophenyl) morpholine (1b)

P-fluoronitrobenzene (35.44mmol) and excess morpholine (205mmol) were stirred at 80 ℃ for 5 hours in a 100mL single-neck flask. After the reaction was complete, the reaction mixture was slowly poured into 1000mL of ice water, if anyThe yellow solid precipitated, was filtered and dried to give a yellow powdery solid with a yield of 99%.¹H NMR(400 MHz,CDCl₃)δ8.14(d,J＝9.4Hz,2H),6.84(d,J＝9.4Hz,2H),3.90–3.83(m, 4H),3.37(dd,J＝5.9,4.0Hz,4H).¹³C NMR(101MHz,CDCl₃)δ155.0,138.9, 125.9,112.6,66.4,47.1.

Step 1.2: preparation of 4-morpholinoaniline (1c)

Under the protection of argon, 4- (4-nitrophenyl) morpholine (19mmol) is dissolved in 60mL of ethanol in a two-neck bottle, 10% palladium-carbon and hydrazine hydrate are sequentially added, and the mixture is heated to 75 ℃ to react for 8 hours. After the reaction is finished, filtering by using kieselguhr, evaporating the organic solvent under reduced pressure, and carrying out column chromatography separation to obtain the target compound with the yield of 95%.¹H NMR(400MHz,DMSO-d₆)δ6.68(d,J＝8.8Hz,2H),6.51(d,J＝8.8Hz,2H),4.58 (s,2H),3.73–3.65(m,4H),2.88–2.85(m,4H)；¹³C NMR(101MHz,DMSO-d₆)δ 142.8,142.8,118.0,115.2,66.8,51.1.

Step 1.3: preparation of 6-bromo-N- (4-morpholinophenyl) imidazo [1,2-a ] pyrazin-8-amine (1d)

4-Morpholine aniline (5.6mmol) was dissolved in 60mL of isopropanol solution, and DIPEA (8.4mmol) and 6, 8-dibromo-imidazole [1,2-a ] were added in this order]Pyrazine (6.72mmol) was heated to 85 ℃ and reacted for 8 hours. After the reaction, cooled to room temperature, the organic solvent was removed under reduced pressure, the residue was dissolved in ethyl acetate, washed with water and then with saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered, and separated by column chromatography silica gel to obtain a white solid with a yield of 85%.¹H NMR(400MHz,DMSO-d₆)δ9.81(s, 1H),8.18(d,J＝5.7Hz,1H),7.91(d,J＝3.1Hz,1H),7.80(d,J＝9.3Hz,2H),7.60 (d,J＝2.4Hz,1H),6.92(d,J＝8.3Hz,2H),3.73(d,J＝3.1Hz,4H),3.06(dt,J＝ 8.3,4.6Hz,4H)；¹³C NMR(101MHz,DMSO-d₆)δ147.58,144.9,132.9,132.1, 131.8,122.0,121.3,116.7,115.7,111.2,66.6,49.4.

Step 1.4: preparation of 6- (3-aminophenyl) -N- (4-morpholinophenyl) imidazo [1,2-a ] pyrazin-8-amine (1e)

Under argon flow, 6-bromo-N- (4-morpholinophenyl) imidazole [1,2-a ] is added into a two-neck bottle in sequence]Pyrazine-8-amine (1mmol), amino boronic acid ester (1.2mmol), PdCl₂(dppf) (0.1mmol) and 1M aqueous sodium carbonate solution (1.5mmol) were heated to 100 ℃ and reacted overnight. After the reaction was completed, it was cooled to room temperature, filtered through celite, and the organic solvent was distilled off. Dissolving in ethyl acetate, washing with water and saturated sodium chloride solution, and drying the organic phase with anhydrous sodium sulfate. Separating with column chromatography silica gel to obtain pure product with yield of 54%.¹H NMR(400MHz,DMSO-d₆)δ9.45(s,1H),8.39(s,1H),8.05(d,J＝8.6Hz,2H), 7.98(s,1H),7.61(s,1H),7.22(d,J＝2.2Hz,1H),7.12(d,J＝4.7Hz,2H),6.99(d, J＝8.6Hz,2H),6.59(dt,J＝6.1,2.7Hz,1H),5.42–5.13(m,2H),3.76–3.72(m, 4H),3.07(t,J＝4.7Hz,4H)；¹³C NMR(101MHz,DMSO-d₆)δ149.4,146.8,145.1, 138.4,137.6,133.2,132.6,132.6,129.6,121.2,116.7,116.0,114.3,113.8,111.9, 108.3,66.6,49.6.

Step 1.5: preparation of 3-cyano-N- (3- (8- ((4-morpholinophenyl) amino) imidazo [1,2-a ] pyrazin-6-yl) phenyl) benzamide (1)

Sequentially adding the compound 6- (3-aminophenyl) -N- (4-morpholinophenyl) imidazole [1,2-a ] into a single-mouth bottle at normal temperature]Pyrazine-8-amine (0.5mmol), 3-cyanobenzoic acid (0.7mmol), EDCI (0.9mmol), HOBt (0.9mmol), DIEA (1.2mmol) and stirred overnight. After TLC monitoring reaction, the reaction solution was diluted with ethyl acetate,the reaction mixture was washed with water and saturated brine in this order, and the organic phase was dried over anhydrous sodium sulfate. And separating by column chromatography silica gel to obtain the pure product with the yield of 53 percent.¹H NMR(400MHz, DMSO-d₆)δ10.61(s,1H),9.58(s,1H),8.70(s,1H),8.56(s,1H),8.49(s,1H), 8.34(d,J＝8.0Hz,1H),8.10(dd,J＝8.3,5.8Hz,3H),8.03(s,1H),7.81–7.72 (m,2H),7.67(d,J＝9.9Hz,2H),7.48(t,J＝7.9Hz,1H),7.03(d,J＝8.6Hz, 2H),3.74(t,J＝4.6Hz,4H),3.07(t,J＝4.8Hz,4H).¹³C NMR(101MHz, DMSO-d₆)δ164.04,146.79,145.13,139.69,138.12,136.70,136.31,135.51, 133.08,132.96,132.43,131.88,130.31,129.45,121.52,121.48,120.55,118.83, 118.73,117.02,116.07,112.04,66.60,49.62.

Example 2

Preparation of 6- (5-aminopyridin-3-yl) -N- (4-morpholinophenyl) imidazo [1,2-a ] pyrazin-8-amine (2)

Under argon flow, 6-bromo-N- (4-morpholinophenyl) imidazole [1,2-a ] is added into a two-neck bottle in sequence]Pyrazine-8-amine (1.5mmol), 5-aminopyridine-3-boronic acid pinacol ester (1.8mmol), PdCl₂(dppf) (0.15mmol) and 1M aqueous sodium carbonate solution (3mmol) were heated to 100 ℃ and reacted overnight. After the reaction, the reaction mixture was cooled to room temperature, filtered through celite, the organic solvent was removed by rotary evaporation, and the mixture was dissolved in ethyl acetate, washed with water and saturated brine in this order, and the organic phase was dried over anhydrous sodium sulfate. Separating with column chromatography silica gel to obtain pure product with yield of 73%.¹H NMR(400MHz,DMSO-d₆)δ9.51(s,1H),8.51(s, 1H),8.35(s,1H),8.07–7.91(m,4H),7.63(s,1H),7.48(t,J＝2.3Hz,1H),6.99(d, J＝8.5Hz,2H),5.51(s,2H),3.75(t,J＝4.8Hz,4H),3.08(t,J＝4.8Hz,4H).；¹³C NMR(101MHz,DMSO-d₆)δ146.51,145.00,135.80,134.65,134.45,133.85, 132.97,132.46,132.25,132.14,120.97,116.79,116.44,115.48,108.41,66.17,49.11.

Example 3

Preparation of N- (5- (8- ((4-morpholinophenyl) amino) imidazo [1,2-a ] pyrazin-6-yl) -pyridin-3-yl) -3- (trifluoromethyl) benzamide (3)

Sequentially adding the compound 6- (3-aminophenyl) -N- (4-morpholinophenyl) imidazole [1,2-a ] into a single-mouth bottle at normal temperature]Pyrazine-8-amine (0.26mmol), 3-trifluoromethylbenzoic acid (0.28mmol), EDCI (0.39mmol), HOBt (0.39mmol) and DIEA (0.52mmol) were stirred at room temperature overnight. After completion of the TLC monitoring reaction, the reaction mixture was diluted with ethyl acetate, washed with water and saturated brine in this order, and the organic phase was dried over anhydrous sodium sulfate. Separating with column chromatography silica gel to obtain pure product with yield of 66%.¹H NMR(400 MHz,DMSO-d₆)δ10.83(s,1H),9.64(s,1H),9.04(s,1H),8.95(s,1H),8.85(d, J＝2.4Hz,1H),8.71(s,1H),8.44–8.32(m,2H),8.11–7.99(m,4H),7.83(t,J ＝7.8Hz,1H),7.66(s,1H),7.02(d,J＝8.6Hz,2H),3.77–3.69(m,4H),3.11– 3.02(m,4H).¹³C NMR(101MHz,DMSO-d₆)δ164.48,146.58,145.12,141.87, 141.08,135.71,135.23,133.55,132.88,132.47,132.41,132.21,131.93,129.89, 125.37,124.59,124.46,124.42,121.29,116.71,115.52,108.97,66.17,49.56, 49.08.

Example 4

Preparation of N- (5- (8- (cyclopropylamino) imidazo [1,2-a ] pyrazin-6-yl) -pyridin-3-yl) -3-fluorobenzamide (4)

Step 4.1: preparation of 6-bromo-N-cyclopropylimidazo [1,2-a ] pyrazin-8-amine (4b)

Adding 6, 8-dibromo-imidazole [1,2-a ] into a 100mL two-mouth bottle]Pyrazine (9.1mmol) was dissolved in 20mL of isopropanol, cyclopropylamine (13.6mmol) and DIPEA (13.6mmol) were added in that order, heated to 80 ℃ and reactedShould be 3 hours. After the reaction is finished, cooling to room temperature, removing the organic solvent under reduced pressure, adding ethyl acetate into the residue to dissolve the residue, precipitating yellow solid, and filtering to obtain a pure product with the yield of 92%.¹H NMR(400MHz, DMSO-d₆)δ8.13(d,J＝4.3Hz,1H),8.04(s,1H),7.83(s,1H),7.49(s,1H), 2.91(tt,J＝8.1,3.8Hz,1H),0.75–0.63(m,4H).¹³C NMR(101MHz,DMSO-d₆) δ148.47,132.03,131.47,122.16,115.81,109.40,23.85,6.16.

Step 4.2: preparation of 6- (5-aminopyridin-3-yl) -N-cyclopropylimidazo [1,2-a ] pyrazin-8-amine (4c)

Under argon flow, 6-bromo-N-cyclopropyl imidazole [1,2-a ] is added into a two-neck bottle in sequence]Pyrazine-8-amine (2.1mmol), 5-aminopyridine-3-boronic acid pinacol ester (2.5mmol), PdCl₂(dppf) (0.2mmol) and 1M aqueous sodium carbonate solution (4.2mmol) were heated to 100 ℃ and reacted overnight. After the reaction was completed, it was cooled to room temperature, filtered through celite, and the organic solvent was distilled off. Dissolving in ethyl acetate, washing with water and saturated sodium chloride solution, and drying the organic phase with anhydrous sodium sulfate. Separating with column chromatography silica gel to obtain pure product with yield of 81%.¹H NMR(400MHz,DMSO-d₆)δ8.37(s,2H),8.03–7.84(m,2H),7.75 (d,J＝4.2Hz,1H),7.56–7.49(m,2H),5.41(s,2H),3.07(tq,J＝7.7,4.0Hz,1H), 0.77(dh,J＝8.1,3.9Hz,2H),0.69(hept,J＝4.1Hz,2H).¹³C NMR(101MHz, DMSO-d₆)δ148.72,135.72,135.03,134.62,131.87,116.99,115.87,113.79,111.51, 109.58,107.07,23.68,6.23.

Step 4.3: preparation of N- (5- (8- (cyclopropylamino) imidazo [1,2-a ] pyrazin-6-yl) -pyridin-3-yl) -3-fluorobenzamide (4)

Sequentially adding the compound 6- (5-aminopyridin-3-yl) -N-cyclopropyl imidazole into a single-mouth bottle at normal temperatureAzole [1,2-a ]]Pyrazine-8-amine (0.4mmol), 3-fluorobenzoic acid (0.4mmol), EDCI (0.6mmol), HOBt (0.6mmol) and DIPEA (0.6mmol) were stirred at room temperature overnight. After completion of the TLC monitoring reaction, the reaction mixture was diluted with ethyl acetate, washed with water and saturated brine in this order, and the organic phase was dried over anhydrous sodium sulfate. Separating with column chromatography silica gel to obtain pure product with yield of 28%.¹H NMR(400MHz, DMSO-d₆)δ10.63(s,1H),8.96(d,J＝2.0Hz,1H),8.91(d,J＝2.4Hz,1H), 8.83(t,J＝2.3Hz,1H),8.53(s,1H),7.94(s,1H),7.85(dd,J＝16.8,7.9Hz, 3H),7.63(td,J＝8.0,5.8Hz,1H),7.54(s,1H),7.49(td,J＝8.6,2.7Hz,1H), 3.11(tq,J＝7.6,4.0Hz,1H),0.80(dt,J＝6.7,3.2Hz,2H),0.72(q,J＝4.1Hz, 2H).¹³C NMR(101MHz,DMSO-d₆)δ164.68,163.16,160.73,148.88,142.14, 141.21,135.55,134.05,133.09,132.10,132.08,130.76,130.68,124.47,124.05, 124.02,118.71,116.14,114.75,114.53,107.69,23.73,6.26.

Example 5

Preparation of N- (3- (8-cyclopropylamino) imidazo [1,2-a ] pyrazin-6-yl) phenyl) -1H-1,2, 4-triazole-3-carboxamide (5)

Step 5.preparation of 16- (3-aminophenyl) -N-cyclopropylimidazo [1,2-a ] pyrazin-8-amine (5a)

Under the protection of argon, 6-bromine-N-cyclopropyl imidazole [1,2-a ] is added into a two-mouth bottle in sequence]Pyrazine-8-amine (4.0mmol), 3-aminophenylboronic acid pinacol ester (4.8mmol), PdCl₂(dppf) (0.4mmol) and 1M aqueous sodium carbonate (8.0mmol) were heated to 100 ℃ and reacted overnight. After the reaction was completed, it was cooled to room temperature, filtered through celite, and the organic solvent was distilled off. Dissolving in ethyl acetate, washing with water and saturated sodium chloride solution, and drying the organic phase with anhydrous sodium sulfate. Separating with column chromatography silica gel to obtain pure product with yield of 69%.¹H NMR(400MHz,DMSO-d₆)δ8.24(s,1H),7.88(s,1H),7.64(d,J＝4.2Hz, 1H),7.49(s,1H),7.26(t,J＝1.9Hz,1H),7.09(dt,J＝15.3,7.7Hz,2H),6.56(dt,J ＝7.8,1.6Hz,1H),5.12(s,2H),3.08(tq,J＝7.7,4.0Hz,1H),0.77(td,J＝7.1,4.6 Hz,2H),0.68(q,J＝4.0Hz,2H).¹³C NMR(101MHz,DMSO)δ148.71,148.39, 138.04,137.42,132.03,131.71,128.88,115.69,113.66,113.43,111.54,106.46, 23.68,6.24.

Step 5.2 preparation of N- (3- (8-cyclopropylamino) imidazo [1,2-a ] pyrazin-6-yl) phenyl) -1H-1,2, 4-triazole-3-carboxamide (5)

Sequentially adding the compound 6- (3-aminophenyl) -N-cyclopropyl imidazole [1,2-a ] into a single-mouth bottle at normal temperature]Pyrazine-8-amine (0.4mmol), 1H-1,2, 4-triazole-3-carboxylic acid (0.5mmol), EDCI (0.7mmol), HOBt (0.7mmol) and DIPEA (1.02mmol), and stirring at room temperature overnight. After completion of the TLC monitoring reaction, the reaction mixture was diluted with ethyl acetate, washed with water and saturated brine in this order, and the organic phase was dried over anhydrous sodium sulfate. Separating with column chromatography silica gel to obtain pure product with yield of 35%.¹H NMR(400MHz, DMSO-d₆)δ14.81(s,1H),10.56(s,1H),8.59(d,J＝34.5Hz,2H),8.38(s,1H), 7.93(s,1H),7.82–7.72(m,3H),7.52(s,1H),7.43(t,J＝7.9Hz,1H),3.13(tq, J＝7.9,4.1Hz,1H),0.80(dq,J＝6.9,4.3Hz,2H),0.70(p,J＝4.4Hz,2H).¹³C NMR(101MHz,DMSO)δ148.57,146.87,138.48,138.01,136.56,132.05, 131.91,131.43,128.75,121.44,120.21,118.18,115.94,112.74,106.98,23.71, 6.26.

Example 6

Preparation of N- (3- (8-cyclopropylamino) imidazo [1,2-a ] pyrazin-6-yl) phenyl) -3- (methylthio) benzamide (6)

Sequentially adding the compound 6- (3-amino) into a single-mouth bottle at normal temperaturePhenyl) -N-cyclopropylimidazole [1,2-a]Pyrazine-8-amine (0.6mmol), 3- (methylthio) benzoic acid (0.9mmol), EDCI (1.2mmol), HOBt (1.2mmol) and DIPEA (1.6mmol) were stirred at room temperature overnight. After completion of the TLC monitoring reaction, the reaction mixture was diluted with ethyl acetate, washed with water and saturated brine in this order, and the organic phase was dried over anhydrous sodium sulfate. Separating with column chromatography silica gel to obtain pure product with yield of 72%.¹H NMR(400MHz, DMSO-d₆)δ10.41(s,1H),8.49–8.42(m,1H),8.38(s,1H),7.97–7.91(m, 1H),7.84(s,1H),7.76(q,J＝5.7,3.9Hz,4H),7.54–7.42(m,4H),3.13(tt,J＝ 7.5,3.7Hz,1H),2.56(s,3H),0.80(dh,J＝7.8,3.5Hz,2H),0.71(q,J＝3.9Hz, 2H).¹³C NMR(101MHz,DMSO)δ165.18,148.59,139.28,138.74,137.97, 136.63,135.67,132.06,131.89,128.99,128.72,124.71,124.19,121.24,120.23, 120.19,118.08,115.95,106.93,23.73,14.62,6.28.

Example 7

Preparation of N- (3- (8-cyclopropylamino) imidazo [1,2-a ] pyrazin-6-yl) phenyl) -3- (methylthio) benzamide (7)

At 0 ℃, adding N- (3- (8-cyclopropylamino) imidazole [1,2-a ]]Pyrazin-6-yl) phenyl) -3- (methylthio) benzamide (0.4mmol) was dissolved in dichloromethane and excess m-chloroperoxybenzoic acid (1.6mmol) was added with stirring. After the reaction, a sodium sulfite saturated solution was added, liquid separation was performed, the organic phase was dried over anhydrous sodium sulfate, and column analysis separation was performed to obtain a white solid with a yield of 65%.¹H NMR(400MHz,DMSO-d₆)δ 10.64(s,1H),8.51(t,J＝1.8Hz,1H),8.45(t,J＝2.0Hz,1H),8.39(s,1H),8.33 (dt,J＝7.8,1.4Hz,1H),8.15(dt,J＝7.7,1.4Hz,1H),7.94(d,J＝1.1Hz,1H), 7.85(t,J＝7.8Hz,1H),7.80–7.74(m,3H),7.52(d,J＝1.1Hz,1H),7.46(t,J ＝7.9Hz,1H),3.13(tt,J＝7.2,3.9Hz,1H),0.80(td,J＝7.1,4.7Hz,2H),0.73– 0.67(m,2H).¹³C NMR(101MHz,DMSO)δ164.09,148.61,141.13,139.01, 138.07,136.53,135.99,132.75,132.06,131.93,129.86,129.83,128.85,126.20, 121.48,120.35,118.23,115.99,107.00,43.50,23.74,6.29.

Example 8

Preparation of 6- (5-aminopyridin-3-yl) -N- (4- ((1R,4R) -5-isopropyl-2, 5-diazabicyclo [2.2.1] heptan-2-yl) phenyl) imidazo [1,2-a ] pyrazin-8-amine (8)

Under argon flow, 6-bromo-N- (4- ((1R,4R) -5-isopropyl-2, 5-diazabicyclo [2.2.1] was added sequentially to a two-necked flask]Heptane-2-yl) phenyl) imidazole [1,2-a]Pyrazine-8-amine (1.0mmol), 5-aminopyridine-3-boronic acid pinacol ester (1.2mmol), PdCl₂(dppf) (0.1mmol) and 1M aqueous sodium carbonate solution (2.0mmol) were heated to 100 ℃ and reacted overnight. After the reaction was completed, it was cooled to room temperature, filtered through celite, and the organic solvent was distilled off. Dissolving in ethyl acetate, washing with water and saturated sodium chloride solution, and drying the organic phase with anhydrous sodium sulfate. Separating with column chromatography silica gel to obtain pure product with yield of 30%.¹H NMR(400 MHz,DMSO-d₆)δ9.43(s,1H),8.47(s,1H),8.34(s,1H),7.98–7.92(m,4H),7.62 (s,1H),7.45(d,J＝2.7Hz,1H),6.68(d,J＝8.5Hz,2H),5.48(s,2H),4.40(s,1H), 3.21(s,5H),2.89–2.59(m,2H),1.97(s,1H),1.12–1.01(m,6H).¹³C NMR(101 MHz,DMSO)δ145.05,144.73,142.50,142.32,135.96,134.81,134.68,133.01, 132.21,132.17,121.61,116.71,116.45,112.85,108.16,59.81,58.55,57.98,56.40, 55.02,20.82,14.13.

Example 9

Preparation of 6- (3-aminophenyl) -N- (5-fluoropyrimidin-2-yl) imidazo [1,2-a ] pyrazin-8-amine (9)

Step 9.16 preparation of bromo-N- (5-fluoropyrimidin-2-yl) imidazo [1,2-a ] pyrazin-8-amine (9b)

Under argon flow, 6, 8-dibromo-imidazole [1,2-a ] is sequentially added into a two-mouth bottle]Pyrazine (3.6mmol), 2-amino-5-fluoro-pyridine (5.4mmol), Pd₂(dba)₃(0.36mmol), Xantphos (0.72mmol) and cesium carbonate (7.2mmol) were heated to 100 ℃ for reaction overnight. After the reaction was complete, the reaction mixture was cooled to room temperature, filtered through celite, and the organic solvent was evaporated. Dissolving in ethyl acetate, washing with water and saturated sodium chloride solution, and drying the organic phase with anhydrous sodium sulfate. Separating with column chromatography silica gel to obtain pure product with yield of 45%.¹H NMR(400MHz,DMSO-d₆)δ9.33(s,1H),8.41(s,1H),8.38(d,J＝3.1Hz,1H), 8.33(dd,J＝9.2,4.1Hz,1H),8.01(d,J＝1.1Hz,1H),7.87(td,J＝8.7,3.1Hz,1H), 7.68(d,J＝1.1Hz,1H).¹³C NMR(101MHz,DMSO)δ147.95,143.08,135.76, 135.51,133.42,131.35,125.58,125.38,119.71,116.84,114.32,113.32.

Step 9.26 preparation of (3-aminophenyl) -N- (5-fluoropyrimidin-2-yl) imidazo [1,2-a ] pyrazin-8-amine (9)

Under argon flow, 6-bromo-N- (5-fluoropyrimidin-2-yl) imidazole [1,2-a ] is added into a two-neck bottle in sequence]Pyrazine-8-amine (2.0mmol), 3-aminophenylboronic acid pinacol ester (2.4mmol), PdCl₂(dppf) (0.2mmol) and 1M aqueous sodium carbonate solution (4.0mmol) were heated to 100 ℃ and reacted overnight. After the reaction was completed, it was cooled to room temperature, filtered through celite, and the organic solvent was distilled off. Dissolving in ethyl acetate, washing with water and saturated sodium chloride solution, and drying the organic phase with anhydrous sodium sulfate. Separating with column chromatography silica gel to obtain pure product with yield of 45%.¹H NMR(400MHz,DMSO-d₆)δ8.96(s,1H),8.74(dd,J＝9.2, 4.0Hz,1H),8.58(s,1H),8.37(d,J＝3.1Hz,1H),8.06(d,J＝1.1Hz,1H),7.89(td, J＝8.7,3.1Hz,1H),7.68(d,J＝1.1Hz,1H),7.24(t,J＝1.9Hz,1H),7.16–7.09(m, 2H),6.61(dt,J＝7.5,1.9Hz,1H),5.27(s,2H).¹³C NMR(101MHz,DMSO)δ 156.42,153.97,149.05,148.66,143.08,137.27,136.70,135.63,135.38,133.07, 131.69,129.31,125.61,125.42,116.85,113.97,113.74,113.70,113.21,111.42, 109.89.

Example 10

Preparation of 3-cyano-N- (3- (8- ((5-fluoropyrimidin-2-yl) amino) imidazo [1,2-a ] pyrazin-6-yl) phenyl) benzamide

At normal temperature, the compound 6- (3-aminophenyl) -N- (5-fluoropyrimidin-2-yl) imidazole [1,2-a ] is added into a single-mouth bottle in sequence]Pyrazine-8-amine (0.5mmol), 3-cyanobenzoic acid (0.7mmol), EDCI (0.9mmol), HOBt (0.9mmol), and DIPEA (1.2mmol) were stirred at room temperature overnight. After completion of the TLC monitoring reaction, the reaction mixture was diluted with ethyl acetate, washed with water and saturated brine in this order, and the organic phase was dried over anhydrous sodium sulfate. Separating with column chromatography silica gel to obtain pure product with yield of 77%.¹H NMR(400 MHz,DMSO-d₆)δ10.54(s,1H),8.96(s,1H),8.86(dd,J＝9.2,4.1Hz,1H), 8.73(s,1H),8.68(s,1H),8.47(s,1H),8.33(dd,J＝9.5,5.4Hz,2H),8.08(d,J ＝8.9Hz,2H),7.83(ddd,J＝21.8,10.9,5.5Hz,2H),7.67(td,J＝11.3,9.2,4.8 Hz,3H),7.46(t,J＝7.9Hz,1H).¹³C NMR(101MHz,DMSO)δ163.78,156.46, 154.01,148.55,143.15,139.40,137.13,135.96,135.70,135.56,135.31,135.02, 133.14,132.58,131.70,131.35,129.87,129.05,125.54,125.35,120.91,119.96, 118.39,117.84,117.04,114.08,111.55,110.31.

Example 11

Preparation of 6- (3-cyanophenyl) -N-methyl-8- ((4-morpholinophenyl) amino) imidazo [1,2-a ] pyrazine-2-benzamide (11)

Step 11.preparation of 16, 8-dibromoimidazo [1,2-a ] pyrazine-2-carboxylic acid ethyl ester (11b)

In a 100mL single-necked flask, 2-amino-3, 5-dibromopyrazine (20mmol) and ethyl 3-bromopyruvate (24mmol) were dissolved in 40mL of ethylene glycol dimethyl ether and stirred at 100 ℃ overnight. After the reaction, the reaction mixture was added with ether, filtered and dried to obtain a brown solid with a yield of 47%.¹H NMR(400MHz, CDCl₃)δ8.31(dd,J＝7.2,1.7Hz,2H),4.48(qd,J＝7.1,1.6Hz,2H),1.43(td,J＝ 7.1,1.7Hz,3H).¹³C NMR(101MHz,CDCl₃)δ161.81,139.73,138.31,135.93, 121.68,119.55,119.32,62.14,14.46.

Step 11.2: preparation of ethyl 6-bromo-8- ((4-morpholinophenyl) amino) imidazo [1,2-a ] pyrazine-2-carboxylate (11c)

Dissolving 4-morpholinoaniline (7.2mmol) in 30mL acetonitrile solution, sequentially adding DIPEA (16.2mmol) and 6, 8-dibromoimidazole [1,2-a ]]Ethyl pyrazine-2-carboxylate (6.0mmol) was heated to 85 ℃ and reacted overnight. After the reaction, the reaction mixture was cooled to room temperature, the organic solvent was removed under reduced pressure, the residue was dissolved in ethyl acetate, washed with water and then with saturated brine in this order, the organic phase was dried over anhydrous sodium sulfate, filtered, and separated by column chromatography silica gel to obtain a yellow solid with a yield of 39%.¹H NMR(400MHz,CDCl₃) δ8.13(s,1H),8.00(s,1H),7.70(d,J＝8.6Hz,2H),7.60(s,1H),6.92(d,J＝ 8.5Hz,2H),4.44(q,J＝7.1Hz,2H),3.85(t,J＝4.5Hz,5H),3.12(t,J＝4.6Hz, 4H),1.41(d,J＝14.1Hz,3H).¹³C NMR(101MHz,CDCl₃)δ162.31,148.02, 144.66,136.04,132.43,130.76,123.83,121.06,118.96,116.32,110.01,66.94, 61.57,49.68,14.47.

Step 11.3: preparation of 6-bromo-N-methyl-8- ((4-morpholinophenyl) amino) imidazo [1,2-a ] pyrazine-2-carboxamide (11d)

Mixing 6-bromo-8- ((4-morpholinophenyl) amino) imidazole [1,2-a]Pyrazine-2-carboxylic acid ethyl ester (2.35mmol) was dissolved in 10mL of tetrahydrofuran solution, and 12mL of 30% methylamine aqueous solution was added and stirred at room temperature overnight. After the reaction was completed, filtration was carried out to obtain a pale yellow solid with a yield of 85%.¹H NMR(400MHz, CDCl₃)δ8.01(s,1H),7.79(s,1H),7.74(d,J＝8.5Hz,2H),7.67(s,1H),7.23(d,J ＝5.6Hz,1H),7.00(s,2H),3.90(s,4H),3.17(t,J＝4.8Hz,4H),3.05(d,J＝5.0Hz, 3H).¹³C NMR(101MHz,CDCl3)δ162.84,149.54,144.59,137.06,133.61,130.59, 123.82,121.06,119.08,116.31,110.76,77.48,77.16,76.84,66.92,61.67,26.07.

Step 11.4: preparation of 6- (3-cyanophenyl) -N-methyl-8- ((4-morpholinophenyl) amino) imidazo [1,2-a ] pyrazine-2-benzamide (11)

Under argon flow, 6-bromo-N-methyl-8- ((4-morpholinophenyl) amino) imidazole [1,2-a ] is added into a two-neck bottle in sequence]Pyrazine-2-carboxamide (0.23mmol), 3-cyanophenylboronic acid pinacol ester (0.27mmol), PdCl₂(dppf) (0.02mmol) and 1M aqueous sodium carbonate (0.46mmol) were heated to 100 ℃ and reacted overnight. After the reaction was completed, it was cooled to room temperature, filtered through celite, and the organic solvent was distilled off. Dissolving in ethyl acetate, washing with water and saturated sodium chloride solution, and drying the organic phase with anhydrous sodium sulfate. Separating with column chromatography silica gel to obtain pure product with yield of 42%.¹H NMR(400MHz,DMSO-d₆)δ9.41(s, 1H),8.72(s,1H),8.35(d,J＝1.8Hz,1H),8.32–8.28(m,2H),8.14(q,J＝4.8Hz, 1H),7.92–7.88(m,2H),7.85(dt,J＝7.7,1.4Hz,1H),7.71(t,J＝7.8Hz,1H),7.02 –6.97(m,2H),3.77–3.73(m,4H),3.12–3.07(m,4H),2.86(d,J＝4.8Hz,3H). ¹³C NMR(101MHz,DMSO-d₆)δ161.85,147.01,145.19,139.32,138.14,135.12, 131.70,131.62,131.55,130.22,130.13,128.96,121.36,118.83,117.14,115.38, 111.88,109.47,66.17,48.95,25.60.

Example 12

Biological assay

TYK2 enzyme Activity assay

A384-well microplate TR-FRET (time resolved fluorescence energy transfer) endpoint assay was used for the TYK2 kinase activity assay. Same assay for IC of Small molecule inhibitors₅₀And (4) measuring. In general, the kinase reaction is carried out in a volume of 25. mu.L of a reaction solution containing: mu.L of compound (in 10% DMSO), 20. mu.L of a mixture of TYK2, polypeptide, and ATP in assay buffer (25mM HEPES, pH 7.5, 0.001% Brij-35, 0.01% Triton,0.5 mM EGTA). All reactions were performed in 384-well white flat-bottom optical density plates (Perkin Elmer) at room temperature for 60min, then 25. mu.L of stop reaction solution (100mM HEPES, pH 7.5, 0.015% Brij-35, 0.2% Coating Reagent #3,50mM EDTA) was added. Read on an Evison Multilabel reader (Perkin Elmer, Envision 2102- & 0010).

TABLE 1

Example numbering	Inhibition against TYK2 at 1μM(％)
		1	76％(IC₅₀＝0.2μM)
2	55％
		3	3％
4	10％
		5	8％
6	6％
		7	3％

Cell activity assay

And (3) inoculating cells in logarithmic growth phase into a 96-well plate according to the density of 3000 cells per 100 mu L, adding 100 mu L of compounds to be detected with different concentrations after the cells adhere to the wall, and taking 6-8 concentration gradients. Each group was provided with five parallel wells, and a control group was provided. After 72 hours of co-incubation of compound and tumor cells, 10. mu.L of CCK-8 solution was added per well. After incubation for 1-2 hours in a cell incubator, absorbance (OD value) of each well was measured with an enzyme-linked immunosorbent assay, and inhibition rate was calculated: inhibition (IR%) (1-TOD/COD) x 100%, TOD: administration group OD mean value; COD: OD mean of solvent control group.

TABLE 2

The above embodiments are merely illustrative of the technical concept and features of the present invention, and the present invention is not limited thereto, and any equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of the present invention.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, characterized by the following:

。

2. a pharmaceutical composition comprising a compound of claim 1 or a pharmaceutically acceptable salt thereof together with one or more pharmaceutically acceptable carriers and/or diluents, in a clinically acceptable pharmaceutical formulation.

3. The pharmaceutical composition of claim 2, wherein the pharmaceutical formulation is selected from the group consisting of a tablet, a capsule, and a liquid formulation.

4. Use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 2, in the preparation of an inhibitor of TYK 2.

5. The use of the compound or pharmaceutically acceptable salt thereof according to claim 1, or the pharmaceutical composition according to claim 2 for the preparation of a medicament for the prevention and treatment of neoplastic or inflammatory diseases.

6. The use of claim 5, wherein said compound or pharmaceutically acceptable salt thereof, or said pharmaceutical composition is used to inhibit proliferation, growth, infiltration and migration of tumor cells, or to promote apoptosis of tumor cells.