CN112375042A

CN112375042A - Trimethoxy styryl six-membered ring and pyrazolopyrimidine compound, preparation and application thereof

Info

Publication number: CN112375042A
Application number: CN202011163292.2A
Authority: CN
Inventors: 程文丹; 于浩然; 王琛; 田大胜; 荆珏华; 尚贤波; 徐春归; 石静波; 舒海洋
Original assignee: Anhui Medical University
Current assignee: Anhui Medical University
Priority date: 2020-10-27
Filing date: 2020-10-27
Publication date: 2021-02-19

Abstract

The invention discloses a trimethoxy styryl six-membered ring and pyrazolopyrimidine compound, a preparation and application thereof, and relates to the technical field of anti-inflammatory compounds.

Description

Trimethoxy styryl six-membered ring and pyrazolopyrimidine compound, preparation and application thereof

The technical field is as follows:

the invention relates to the technical field of anti-inflammatory compounds, and particularly relates to a trimethoxy styryl six-membered ring and pyrazolopyrimidine compound, a preparation, and preparation and application thereof.

Background art:

the stilbene compound generally refers to a compound having a stilbene parent structure in which two benzene rings and a vinyl group are linked. As a monomer of a natural astragalus compound, the compound can be divided into cis form and trans form. Compared with the trans-structure of glucoside or polyglycoside, the natural plant has obvious effects of resisting oxidation, tumors, inflammation, cardiovascular diseases, immunity, aging, radiation and the like. The most representative are Resveratrol (Resveratrol), diethylstilbestrol (diethylstilbestrol), pterostilbene (pterostilbene), Ospemifene (Ospemifene) and the like, and the corresponding structural formulas are respectively as follows:

wherein, the resveratrol (3, 4', 5-trihydroxy stilbene) has important biological functions of resisting senile dementia, cancer, bacteria, oxidation, blood fat reduction and the like; diethylstilbestrol (Diethylstilbestrol) is a non-steroidal estrogen substance which is artificially synthesized and can generate all pharmacological and therapeutic effects same as natural estradiol; ospemifene (Ospemifene) is one of the active metabolites of toremifene, and is also a novel selective estrogen receptor modulator, having both estrogenic and antiestrogenic effects, exhibiting tissue selectivity. Due to the continuous update of new drug targets, the pharmaceutical chemistry of the compounds is rapidly advanced, a new field of styrene compounds is formed, and the compounds are promising compounds. Styrene compounds have attracted great attention from domestic and foreign scholars due to their unique effects.

In summary, stilbene-based framework modifications are of potential interest. The pyrimidine derivative is a structural framework with wide biological activity, and the thienopyrimidine derivative which is obtained by fusing the two heterocyclic frameworks through a heterocyclic ring has wide biological activity.

Pyrimidines are one of the attractive fused groups. Many pharmacological literature studies report that the core structure contains a thienopyrimidine skeleton, such as buspirone hydrochloride (BuspiHydro-chloride) which is developed and marketed in 1985 by Meadohnson of the United states has good anxiolytic application, and can be used as a 5-HTA partial agonist to treat acute and chronic anxiety disorders. The product has the characteristics of high selectivity, definite curative effect, small addiction and the like. Nimustine hydrochloride which is developed and marketed in 1980 by Japan flood medicine Kabushiki Kaisha is an antitumor drug with good curative effect, and is used for relieving diseases such as brain tumor, digestive tract tumor (gastric cancer, liver cancer and colorectal cancer), lung cancer, malignant lymphoma and leukemia.

It is because of this broad range of biological activities that medicinal chemists have synthesized a large number of heterocyclic split pyrimidine derivatives. Several fused pyrimidine derivatives such as pyrazolopyrimidine, pyrrolopyrimidine, thiazolopyrimidine and imidazopyrimidine known so far have shown good antibacterial activity; in the previous research, the pyrazolopyrimidine compound has a good inhibition effect on the expression of an inflammatory factor NO induced by an adjuvant, and has a remarkable inhibition effect on the activity of iNOS enzyme.

Based on that the structural modification of the pyrimidine skeleton has important potential medicinal value, the styryl pyrimidine compound is synthesized, the efficient anti-inflammatory drug is provided, the inhibition effect of the styryl pyrimidine compound on the activity of iNOS enzyme of articular chondrocytes is researched, and the styryl pyrimidine compound has great significance for constructing a novel iNOS inhibitor drug for treating osteoarthritis.

The invention content is as follows:

the invention aims to solve the technical problem of synthesizing a series of novel trimethoxy styryl six-membered ring and pyrazolopyrimidine compounds for developing efficient anti-inflammatory drugs.

The technical problem to be solved by the invention is realized by adopting the following technical scheme:

a trimethoxy styryl six-membered ring compound comprises a compound with a structure shown in a formula I:

wherein, the compound contains three structures (1): x ═ H, Y ═ CH; (2): x ═ Cl, Y ═ CH; (3): x ═ OH, Y ═ N;

R₁and R₂Is selected from any one of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl and phenyl substituted phenyl.

The preparation method of the trimethoxy styryl six-membered ring compound comprises the following steps of preparing a compound 2 from a compound 1 and an amine derivative under the conditions of isopropanol and triethylamine, and reacting the compound 2 with trimethoxybenzaldehyde under the condition of hydrochloric acid to obtain the trimethoxy styryl six-membered ring compound, wherein the reaction process is as follows:

the invention also provides a pyrazolopyrimidine compound, which comprises compounds with structures shown as a formula II-a and a formula II-b:

wherein the content of the first and second substances,

R₄and R₅Any one selected from hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, morpholinyl, substituted morpholinyl and phenyl-substituted phenyl;

R₆any one selected from hydrogen, alkyl, substituted alkyl and halogen;

z is C, N or O;

n is 1 or 2;

linker is CH₂-CH₂Or CH ═ CH.

The preparation method of the pyrazolopyrimidine compound comprises the steps of reacting a compound 3 with an acrylic acid derivative to obtain a compound 4, cyclizing the compound 4 under the condition of sodium methoxide to obtain a compound 5, and performing chlorination reaction on the intermediate 5 by using excessive phosphorus oxychloride to obtain a key intermediate 6; and (3) carrying out substitution reaction on the intermediate 6 and an amine derivative to obtain the pyrazolopyrimidine compound, wherein the reaction process is as follows:

the amine derivative is selected from any one of isopropylamine, cyclopropylamine, morpholine, thiomorpholine, 4-chlorophenylethylamine, 4-fluorophenylethylamine, 4-fluorobenzylamine and 2-chlorophenylethylamine.

The structural formula of the trimethoxystyryl six-membered ring and pyrazolopyrimidine compound is as follows:

the invention also provides application of the trimethoxy styryl six-membered ring and pyrazolopyrimidine compound in preparation of anti-inflammatory drugs.

The invention also provides a pharmaceutical preparation taking the trimethoxy styryl six-membered ring and pyrazolopyrimidine compounds as active compounds.

The dose of the active compound in the pharmaceutical preparation is 0.01-500 mg/kg. Preferably, the dose of active compound in the pharmaceutical preparation is 250 mg/kg.

The pharmaceutical preparation is prepared into tablets, capsules, pastilles, injections, suspending agents, suppositories or ointments by adding pharmaceutically acceptable auxiliary materials to the active compound.

The auxiliary materials comprise an excipient and a carrier, wherein the excipient is one or more of calcium carbonate, calcium phosphate, saccharides, starch, cellulose derivatives, gelatin, vegetable oil and polyethylene glycol, and the carrier is one or more of a diluent, a disintegrating agent, an adhesive and a lubricant.

The invention has the beneficial effects that:

(1) the trimethoxy styryl six-membered ring and pyrazolopyrimidine compounds belong to novel compounds, are synthesized for the first time and are subjected to structural representation;

(2) the preparation method of the trimethoxystyryl six-membered ring and pyrazolopyrimidine compounds is simple and convenient to operate, and the compounds can be quickly synthesized;

(3) researches show that the trimethoxy styryl six-membered ring and pyrazolopyrimidine compounds have good anti-inflammatory activity, show good application prospect in treatment of various acute and chronic inflammations, and show good application potential in treatment of acute inflammations such as sepsis and chronic inflammations represented by type II diabetes, Alzheimer's disease and atherosclerosis.

The specific implementation mode is as follows:

in order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.

Example 1

(E) Preparation of (E) -N-isopropyl-2- (3,4, 5-trimethoxy styryl) pyrimidin-4-amine (structure shown in formula I-1):

preparation of intermediate 2-1:

compound 1-1(200mg, 1.34mmol), isopropylamine (0.115mL, 1.34mmol) and TEA (0.556mL, 4.02mmol) were dissolved in isopropanol (15mL) and refluxed for 6 h. After the reaction was completed, the solvent was removed to obtain intermediate 2-1.

Preparation of Compound I-1:

compound intermediate 2-1(77mg, 0.51mmol) and 3,4, 5-trimethoxybenzaldehyde (100mg, 0.51mmol) were added to hydrochloric acid (6mL) and refluxed for 6 h. After completion of the reaction, the solvent was removed under reduced pressure, and the residue was purified by column chromatography to give compound I-1. And (3) structural data characterization:¹H NMR(400MHz,DMSO-d₆)δ8.02(s,1H),7.70(d,J＝15.9Hz,1H),7.18(d,J＝7.5Hz,1H),6.98(s,2H),6.97(d,J＝15.9Hz,1H),6.27(d,J＝5.9Hz,1H),4.27(s,1H),3.84(s,6H),3.68(s,3H),1.18(d,J＝6.5Hz,6H).HR-MS(ESI):calcd for C₁₈H₂₄N₃O₃[M+H]⁺,330.1812；found 330.1814.

example 2

(E) Preparation of (E) -N-phenethyl-2- (3,4, 5-trimethoxystyryl) pyrimidin-4-amine (structure shown in formula I-2):

the same synthesis method as that of the compound I-1 is adopted. And (3) structural data characterization:¹H NMR(400MHz,DMSO-d₆)δ8.05(s,1H),7.74(d,J＝15.9Hz,1H),7.42(s,1H),7.34–7.28(m,4H),7.22(s,1H),7.03(d,J＝15.9Hz,1H),6.98(s,2H),6.33(d,J＝5.8Hz,1H),3.85(s,6H),3.69(s,3H),3.61(m,2H),2.89(t,J＝7.5Hz,2H).¹³C NMR(101MHz,DMSO-d₆)δ163.81,162.02,154.37,153.57(2C),140.11,138.57,136.35,132.09,129.24(2C),128.82(2C),128.71,126.60,105.17(2C),104.52,60.54,56.39(2C),41.96,35.30.HR-MS(ESI):calcd for C₂₃H₂₆N₃O₃[M+H]⁺,392.1969；found392.1962.

example 3

(E) Preparation of (E) -N- (4-fluorophenethyl) -2- (3,4, 5-trimethoxy styryl) pyrimidin-4-amine (structure shown in formula I-3):

the same synthesis method as that of the compound I-1 is adopted. And (3) structural data characterization:¹H NMR(400MHz,DMSO-d₆)δ8.04(s,1H),7.72(d,J＝15.9Hz,1H),7.40(s,1H),7.33(dd,J＝8.5,5.7Hz,2H),7.13(t,J＝8.9Hz,2H),7.01(d,J＝15.9Hz,1H),6.98(s,2H),6.32(d,J＝6.0Hz,1H),3.84(s,6H),3.68(s,3H),3.59(m,2H),2.88(t,J＝7.3Hz,2H).¹³C NMR(101MHz,DMSO-d₆)δ163.73,162.53,161.15(d,J＝205.03Hz),154.23,153.55(2C),138.58,136.31,136.09,132.06,130.84(d,J＝8.08Hz,2C),128.42,115.27(d,J＝21.21Hz,2C),104.93(2C),104.46,60.31,56.14(2C),41.90,34.38.HR-MS(ESI):calcd for C₂₃H₂₅FN₃O₃[M+H]⁺,410.1874；found 410.1876.

example 4

(E) Preparation of (E) -N- (4-chlorophenylethyl) -2- (3,4, 5-trimethoxystyryl) pyrimidin-4-amine (the structure is shown as formula I-4):

the same synthesis method as that of the compound I-1 is adopted. And (3) structural data characterization:¹H NMR(400MHz,DMSO-d6)δ8.05(s,1H),7.72(d,J＝15.9Hz,1H),7.45–7.23(m,5H),7.01(d,J＝15.9Hz,1H),6.97(s,2H),6.32(d,J＝5.9Hz,1H),3.85(s,6H),3.69(s,3H),3.59(s,2H),2.88(t,J＝7.2Hz,2H).¹³C NMR(101MHz,DMSO)δ163.81,162.30,154.54,153.57(2C),139.15,138.65,136.36,132.09(2C),131.23,131.14(2C),129.22,128.81,128.69(2C),105.23(2C),60.54,56.42(2C),41.59,34.54.HR-MS(ESI):calcd for C²³H²⁴ClN³O³[M+H]⁺,426.1585；found 426.1579.

example 5

(E) Preparation of-6-chloro-N-isopropyl-2- (3,4, 5-trimethoxystyryl) pyrimidin-4-amine (structure shown in formula I-5):

preparation of intermediate 2-2:

compound 1-2(217mg, 1.34mmol), isopropylamine (0.115mL, 1.34mmol) and TEA (0.556mL, 4.02mmol) were dissolved in isopropanol (15mL) and refluxed for 6 h. After the reaction was completed, the solvent was removed to obtain intermediate 2-2.

Preparation of Compound I-5:

compound intermediate 2-2(95mg, 0.51mmol) and 3,4, 5-trimethoxybenzaldehyde (100mg, 0.51mmol) were added to hydrochloric acid (6mL) and refluxed for 6 h. After completion of the reaction, the solvent was removed under reduced pressure, and the residue was purified by column chromatography to give compound I-5. And (3) structural data characterization:¹H NMR(400MHz,DMSO-d₆)δ7.72(d,J＝15.8Hz,1H),7.53(d,J＝7.9Hz,1H),7.03(s,2H),6.99(d,J＝15.8Hz,1H),6.30(s,1H),4.30(s,1H),3.84(s,6H),3.69(s,3H),1.18(d,J＝6.6Hz,6H).¹³C NMR(101MHz,DMSO-d₆)δ164.59,162.98,157.45,153.56(2C),138.91,138.17,131.59,127.13,105.52(2C),101.52,60.53,56.42(2C),42.10,22.81(2C).HR-MS(ESI):calcd for C₁₈H₂₃ClN₃O₃[M+H]⁺,364.1422；found 364.1422.

example 6

(E) Preparation of-6-chloro-N-phenethyl-2- (3,4, 5-trimethoxystyryl) pyrimidin-4-amine (structure shown in formula I-6):

the same synthesis method as that of the compound I-5 is adopted. And (3) structural data characterization:¹H NMR(400MHz,DMSO-d₆)δ7.75(d,J＝16.0Hz,1H),7.73(s,1H),7.36–7.26(m,4H),7.27–7.17(m,1H),7.03(s,2H),7.00(d,J＝16.7Hz,1H),6.37(s,1H),3.85(s,6H),3.69(s,3H),3.65(m,2H),2.88(t,J＝7.3Hz,2H).¹³C NMR(101MHz,DMSO-d₆)δ164.61,163.68,157.50,153.58(2C),139.87,138.99,138.28,131.57,129.26(2C),128.82(2C),127.11,126.65,105.57,101.65,60.54(2C),56.44,42.24,35.23.HR-MS(ESI):calcd for C₂₃H₂₅ClN₃O₃[M+H]⁺,426.1579；found 426.1578.

example 7

(E) Preparation of (E) -6-chloro-N- (4-fluorophenethyl) -2- (3,4, 5-trimethoxy styryl) pyrimidin-4-amine (structure shown in formula I-7):

the same synthesis method as that of the compound I-5 is adopted. And (3) structural data characterization:¹H NMR(400MHz,DMSO-d₆)δ7.73(d,J＝16.5Hz,1H),7.71(s,1H),7.36–7.27(m,2H),7.18–7.07(m,2H),7.03(s,2H),6.99(s,1H),6.36(s,1H),3.84(s,6H),3.69(s,3H),3.67–3.59(m,2H),2.86(t,J＝7.3Hz,2H).¹³C NMR(101MHz,DMSO-d₆)δ164.59,163.66,161.36(d,J＝242.40Hz),157.49,153.57(2C),138.96,138.29,136.02,131.56,131.06(d,J＝8.08Hz,2C),127.09,115.46(d,J＝20.20Hz,2C),105.55(2C),101.64,60.54,56.42(2C),42.20,34.34.HR-MS(ESI):calcd for C₂₃H₂₄ClFN₃O₃[M+H]⁺,444.1485；found 444.1504.

example 8

(E) Preparation of (E) -4- (isopropylamino) -6- (3,4, 5-trimethoxystyryl) -1,3, 5-triazin-2-ol (structure shown in formula I-8):

preparation of intermediates 2-3:

compounds 1-3(218mg, 1.34mmol), isopropylamine (0.115mL, 1.34mmol) and TEA (0.556mL, 4.02mmol) were dissolved in isopropanol (15mL) and refluxed for 6 h. After the reaction was completed, the solvent was removed to obtain intermediate 2-3.

Preparation of Compound I-8:

compound intermediates 2-3(95mg, 0.51mmol) and 3,4, 5-trimethoxybenzaldehyde (100mg, 0.51mmol) were added to hydrochloric acid (6mL) and refluxed for 6 h. After completion of the reaction, the solvent was removed under reduced pressure, and the residue was purified by column chromatography to give compound I-8.

And (3) structural data characterization:¹H NMR(400MHz,DMSO-d₆)δ11.43(s,1H),δ7.84(d,J＝16.0Hz,1H),7.66(d,J＝8.1Hz,1H),7.00(s,1H),6.90(s,2H),6.71(d,J＝15.9Hz,1H),4.03(m,1H),3.84(s,6H),3.71(s,3H),1.13(d,J＝6.6Hz,6H).¹³C NMR(101MHz,DMSO-d₆)δ164.64,162.03,156.38,153.67(2C),141.61,140.05,130.33,119.10,105.90(2C),60.63,56.44(2C),42.22,22.47(2C).HR-MS(ESI):calcd for C₁₇H₂₃N₄O₄[M+H]⁺,347.1714；found 347.1747.

example 9

(E) Preparation of (E) -4- (phenylethylamino) -6- (3,4, 5-trimethoxystyryl) -1,3, 5-triazin-2-ol (structure shown in formula I-9):

the same synthesis method as that of the compound I-8 is adopted. And (3) structural data characterization:¹H NMR(400MHz,DMSO-d₆)δ7.93–7.76(m,2H),7.36–7.16(m,5H),6.96(d,J＝22.8Hz,2H),6.72(d,J＝16.0Hz,1H),4.17–4.07(m,1H),3.84(s,6H),3.71(s,3H),3.55–3.43(m,2H),2.84(t,J＝7.4Hz,2H).HR-MS(ESI):calcd for C₂₂H₂₅N₄O₄[M+H]⁺,409.1870；found 409.1864.

example 10

(E) Preparation of-4- ((4-fluorophenethyl) amino) -6- (3,4, 5-trimethoxystyryl) -1,3, 5-triazin-2-ol (structure shown in formula I-10):

the same synthesis method as that of the compound I-8 is adopted. And (3) structural data characterization:¹H NMR(400MHz,DMSO-d₆)δ11.51(s,1H),7.94–7.79(m,2H),7.32–7.26(m,2H),7.17–7.06(m,2H),6.95(d,J＝16.0Hz,1H),6.92(s,1H),6.72(d,J＝16.0Hz,1H),3.84(s,6H),3.71(s,3H),3.51–3.41(m,2H),2.83(t,J＝7.3Hz,2H).¹³C NMR(101MHz,DMSO-d₆)δ165.49,162.26,160.30(d,J＝242.40Hz),156.31,153.64(2C),141.77,140.02,136.02(d,J＝3.03Hz),130.90(d,J＝8.08Hz,2C),130.32,119.17,115.46(d,J＝21.21Hz,2C),105.92(2C),60.62,56.42(2C),42.21,33.98.HR-MS(ESI):calcd for C₂₂H₂₄FN₄O₄[M+H]⁺,427.1776；found 427.1779.

example 11

(E) Preparation of (E) -4- (cyclopropylamino) -6- (3,4, 5-trimethoxystyryl) -1,3, 5-triazin-2-ol (structure shown in formula I-11):

the same synthesis method as that of the compound I-8 is adopted. And (3) structural data characterization:¹H NMR(400MHz,DMSO-d₆)δ11.53(s,1H),7.94(s,1H),7.82(d,J＝15.9Hz,1H),6.99(s,1H),6.90(s,2H),6.71(d,J＝15.9Hz,1H),3.83(s,6H),3.71(s,3H),2.78(m,1H),0.69(m,2H),0.53(m,2H).HR-MS(ESI):calcd for C₁₇H₂₁N₄O₄[M+H]+,345.1557；found 345.1534.

example 12

(E) Preparation of-4- ((4-chlorophenethyl) amino) -6- (3,4, 5-trimethoxystyryl) -1,3, 5-triazin-2-ol (structure shown in formula I-12):

the same synthesis method as that of the compound I-8 is adopted. And (3) structural data characterization:¹H NMR(400MHz,DMSO-d₆)δ11.51(s,1H),7.83(d,J＝16.0Hz,2H),7.34–7.21(m,5H),6.95(d,J＝16.1Hz,1H),6.92(s,1H),6.71(d,J＝16.1Hz,1H),3.83(s,6H),3.71(s,3H),3.46(q,J＝6.8Hz,2H),2.84(t,J＝7.1Hz,2H).¹³C NMR(101MHz,DMSO-d₆)δ164.19,161.24,158.94,153.64(2C),141.77,140.01,138.95,131.21,131.04(2C),130.31,128.70(2C),119.07,105.91(2C),60.62,56.42(2C),41.97,34.09.HR-MS(ESI):calcd for C₂₂H₂₄ClN₄O₄[M+H]+,443.1481；found 443.1477.

example 13

(E) Preparation of-N-cyclopropyl-1-methyl-3-propyl-5- (2- (3,5, 6-trimethylpyrazin-2-yl) vinyl) -1H-pyrazolo [4,3-d ] pyrimidin-7-amine (structure shown as formula II-a-1):

preparation of intermediate 4-1:

(E) -3- (3,5, 6-trimethylpyrazin-2-yl) acrylic acid (0.96g, 5mmol), EDCI (1.434g, 7.5mmol) and HOBT (1.103g, 7.5mmol) were dissolved in dry DCM (10mL) and the mixture was stirred at 25 ℃ for 30 min. Compound 3(1.001g, 5.5mmol) and TEA (2.073mL, 15mmol) were added and the solution was stirred at room temperature overnight. The reaction mixture was evaporated, the precipitate was filtered, washed with water and dried to give compound 4-1.

Intermediate 5-1 preparation

Compound 4-1(1.715g, 5mmol) and sodium methoxide (30%, 10mL) were dissolved in methanol (20mL) and refluxed for 10 h. The mixture was concentrated under vacuum. The residue was acidified to pH 7 with 2N HCl. The precipitate was filtered, washed with water, and dried to give compound 5-1.

Intermediate 6-1 preparation

Compound 5-1(1.692g, 5mmol) was dissolved in phosphorus oxychloride (POCl)₃10mL) and stirred at 100 ℃ for 10 h. After completion of the reaction, the mixture was concentrated in vacuo to afford the product as a dark oil. Cooling the residue with cold H₂O washed and extracted with DCM (30 mL. times.3). The combined organic layers were washed with saturated NaHCO₃The solution was washed with saturated saline solution and then with anhydrous Na₂SO₄Drying and concentrating to obtain the key intermediate 6-1.

Preparation of intermediate II-a-1

Intermediate 6-1(100mg, 0.281mmol), cyclopropylamine (0.021mL, 0.30mmol) and TEA (0.117mL, 0.843mmol) were dissolved in isopropanol (15mL) and refluxed for 6 h. After completion of the reaction, the solvent was removed, and the residue was purified by column chromatography to give compound II-a-1. And (3) structural data characterization:¹H NMR(400MHz,CDCl₃)δ8.70(s,1H),8.29(d,J＝14.9Hz,1H),7.79(d,J＝14.9Hz,1H),4.25(s,3H),3.28–3.28(m,1H),2.90(t,J＝7.5Hz,2H),2.61(s,3H),2.54–2.47(m,6H),1.75–1.66(m,2H),1.03–0.89(m,7H).¹³C NMR(101MHz,DMSO-d₆)δ156.59,150.81,150.71,149.65,147.63,144.46,144.32,142.48,134.04,129.39,121.45,39.47,27.69,24.49,22.08,21.94,21.90,20.82,14.36,7.05(2C).HR-MS(ESI):calcd for C₂₁H₂₈N₇[M+H]⁺,378.2041；found 378.2043.

example 14

(E) Preparation of-N-isopropyl-1-methyl-3-propyl-5- (2- (3,5, 6-trimethylpyrazin-2-yl) vinyl) -1H-pyrazolo [4,3-d ] pyrimidin-7-amine (the structure is shown as formula II-a-2):

the same synthesis method as that for compound II-a-1 was adopted. And (3) structural data characterization:¹H NMR(400MHz,CDCl₃)δ8.38(s,1H),8.23(d,J＝14.9Hz,1H),7.79(d,J＝14.9Hz,1H),4.86–4.77(m,1H),4.31(s,3H),2.91(t,J＝7.5Hz,2H),2.63(s,3H),2.52(d,6H),1.75–1.66(m,2H),1.43(d,J＝6.6Hz,6H),0.96(t,J＝7.3Hz,3H).¹³C NMR(101MHz,DMSO-d₆)δ156.55,150.76,149.61,149.13,147.56,144.45,144.32,142.47,134.23,129.02,121.33,42.80,39.49,27.68,22.21(2C),22.09,21.91,21.88,20.82,14.35.HR-MS(ESI):calcd for C₂₁H₃₀N₇[M+H]⁺,380.2557；found 380.2559.

example 15

(E) Preparation of-1-methyl-N-phenethyl-3-propyl-5- (2- (3,5, 6-trimethylpyrazin-2-yl) vinyl) -1H-pyrazolo [4,3-d ] pyrimidin-7-amine (the structure is shown as formula II-a-3):

the same synthesis method as that for compound II-a-1 was adopted. And (3) structural data characterization:¹H NMR(400MHz,DMSO-d₆)δ8.80(s,1H),8.14(d,J＝14.9Hz,1H),7.71(d,J＝14.9Hz,1H),7.32(dd,J＝13.0,5.6Hz,4H),7.23(t,J＝7.0Hz,1H),4.26(s,3H),3.98-3.92(m,2H),3.08-3.00(m,2H),2.85(t,J＝7.5Hz,2H),2.61(s,3H),2.51(s,6H),1.75-1.66(m,2H),0.95(t,J＝7.3Hz,3H).¹³C NMR(101MHz,DMSO-d₆+Pyridine-d₅)δ156.44,150.85,149.58,149.54,147.64,144.30,144.17,141.48,140.07,133.68,129.59,129.09(2C),128.85(2C),126.62,121.42,42.72,39.58,35.23,27.74,22.16,21.83(2C),20.82,14.27.HR-MS(ESI):calcd for C₂₆H₃₂N₇[M+H]⁺,442.2714；found442.2713.

example 16

(E) Preparation of (E) -N- (3-chlorophenylethyl) -1-methyl-3-propyl-5- (2- (3,5, 6-trimethylpyrazin-2-yl) vinyl) -1H-pyrazolo [4,3-d ] pyrimidin-7-amine (the structure is shown in formula II-a-4):

the same synthesis method as that for compound II-a-1 was adopted. And (3) structural data characterization:¹H NMR(400MHz,DMSO-d₆)δ7.92(d,J＝15.2Hz,1H),7.55(d,J＝15.2Hz,1H),7.49(s,1H),7.42–7.25(m,4H),4.16(s,3H),3.87–3.81(m,2H),3.08–3.00(m,2H),2.80(t,J＝7.5Hz,2H),2.56(s,3H),2.49(s,3H),2.46(s,3H),1.79–1.71(m,2H),0.95(t,J＝7.3Hz,3H).¹³C NMR(101MHz,DMSO-d₆+Pyridine-d₅)δ156.67,150.71,149.53,149.50,147.60,144.49,144.43,142.69,142.31,134.22,133.64,130.55,129.25,128.97,127.84,126.61,121.43,42.18,39.50,34.79,27.76,22.15,21.79(2C),20.88,14.26.HR-MS(ESI):calcd for C₂₆H₃₁ClN₇[M+H]⁺,476.2324；found 476.2321.

example 17

(E) -N-) 4-fluorobenzyl) -1-methyl-3-propyl-5- (2- (3,5, 6-trimethylpyrazin-2-yl) vinyl) -1H-pyrazolo [4,3-d ] pyrimidin-7-amine (the structure is shown as formula II-a-5) is prepared:

the same synthesis method as that for compound II-a-1 was adopted. And (3) structural data characterization:¹H NMR(400MHz,DMSO-d₆)δ7.93(t,J＝5.9Hz,1H),7.76(d,J＝15.2Hz,1H),7.55(dd,J＝8.4,5.6Hz,2H),7.47(d,J＝15.2Hz,1H),7.15(t,J＝8.9Hz,2H),4.79(d,J＝5.7Hz,2H),4.25(s,3H),2.80(t,J＝7.5Hz,2H),2.53(s,3H),2.47(s,3H),2.45(s,3H),1.80–1.71(m,2H),0.94(t,J＝7.3Hz,3H).¹³C NMR(101MHz,DMSO-d₆)δ162.50(d,J＝242.40Hz),156.46,150.81,149.60,149.13,147.56,144.47,144.25,142.47,136.65(d,J＝3.03Hz),134.04,129.60(d,J＝8.08Hz,2C),129.15,121.23,115.36(d,J＝21.21Hz,2C),43.61,39.63,27.69,22.12,21.91,21.88,20.86,14.36.HR-MS(ESI):calcd for C₂₅H₂₉FN₇[M+H]⁺,446.2463；found 446.2458.

example 18

(E) Preparation of (E) -N-cyclopropyl-1-methyl-3-propyl-5- (2- (pyridin-3-yl) vinyl) -1H-pyrazolo [4,3-d ] pyrimidin-7-amine (the structure is shown in formula II-a-6):

preparation of intermediate 4-2:

(E) pyridine acrylic acid (0.745g, 5mmol), EDCI (1.434g, 7.5mmol) and HOBT (1.103g, 7.5mmol) were dissolved in dry DCM (10mL) and the mixture was stirred at 25 ℃ for 30 min. Compound 3(1.001g, 5.5mmol) and TEA (2.073mL, 15mmol) were added and the solution was stirred at room temperature overnight. The reaction mixture was evaporated, the precipitate was filtered, washed with water and dried to give compound 4-2.

Intermediate 5-2 preparation

Compound 4-2(1.567g, 5mmol) and sodium methoxide (30%, 10mL) were dissolved in methanol (20mL) and refluxed for 10 h. The mixture was concentrated under vacuum. The residue was acidified to pH 7 with 2N HCl. The precipitate was filtered, washed with water and dried to give compound 5-2.

Preparation of intermediate 6-2

Compound 5-2(1.477g, 5mmol) was dissolved in phosphorus oxychloride (POCl)₃10mL) and stirred at 100 ℃ for 10 h. After completion of the reaction, the mixture was concentrated in vacuo to afford the product as a dark oil. Cooling the residue with cold H₂O washed and extracted with DCM (30 mL. times.3). The combined organic layers were washed with saturated NaHCO₃The solution was washed with saturated saline solution and then with anhydrous Na₂SO₄Drying and concentrating to obtain the key intermediate 6-2.

Preparation of intermediate II-a-6

Intermediate 6-2(88mg, 0.281mmol), cyclopropylamine (0.021mL, 0.30mmol) and TEA (0.117mL, 0.843mmol) were dissolved in isopropanol (15mL) and refluxed for 6 h. After completion of the reaction, the solvent was removed, and the residue was purified by column chromatography to give compound II-a-6. And (3) structural data characterization:¹H NMR(400MHz,CDCl₃)δ9.17(s,1H),8.82(d,J＝5.2Hz,1H),8.60(d,J＝7.9Hz,2H),8.20(d,J＝15.8Hz,1H),7.93(dd,J＝8.1,5.3Hz,1H),7.81(d,J＝15.9Hz,1H),4.26(s,3H),3.42-3.36(m,1H),2.94(t,J＝7.5Hz,2H),1.77-1.68(m,2H),1.07-0.87(m,7H).¹³C NMR(101MHz,DMSO-d₆)δ156.61,150.74,149.49,149.37,144.34,142.34,133.88,132.52,131.55,131.41,124.35,121.45,39.48,27.69,24.54,22.16,14.36,7.01(2C).HR-MS(ESI):calcd for C₁₉H₂₃N₆[M+H]⁺,335.1979；found 335.1977.

example 19

(E) Preparation of (E) -N-isopropyl-1-methyl-3-propyl-5- (2- (pyridin-3-yl) vinyl) -1H-pyrazolo [4,3-d ] pyrimidin-7-amine (the structure is shown in formula II-a-7):

the same synthetic method as that for compound II-a-6 was adopted. And (3) structural data characterization:¹H NMR(400MHz,DMSO-d₆)δ8.83(d,J＝2.2Hz,1H),8.49(dd,J＝4.7,1.6Hz,1H),8.16(dt,J＝8.1,2.0Hz,1H),7.73(d,J＝16.0Hz,1H),7.40(dd,J＝8.0,4.7Hz,1H),7.26(d,J＝16.0Hz,1H),6.69(d,J＝7.7Hz,1H),4.71-4.66(m,1H),4.19(s,3H),2.80(t,J＝7.5Hz,2H),1.80-1.70(m,2H),1.34(d,J＝6.6Hz,6H),0.94(t,J＝7.4Hz,3H).¹³C NMR(101MHz,DMSO-d₆)δ156.60,149.47,149.33,149.15,144.33,142.38,133.89,132.52,131.73,131.05,124.33,121.32,42.50,39.51,27.69,22.36,22.17(2C),14.35.HR-MS(ESI):calcd for C₁₉H₂₅N₆[M+H]⁺,337.2135；found 337.2133.

example 20

(E) Preparation of (E) -1-methyl-N-phenethyl-3-propyl-5- (2- (pyridin-3-yl) vinyl) -1H-pyrazolo [4,3-d ] pyrimidin-7-amine (the structure is shown in formula II-a-8):

the same synthetic method as that for compound II-a-6 was adopted. And (3) structural data characterization:¹H NMR(400MHz,CDCl₃)δ8.82(d,J＝2.4Hz,1H),8.53(d,J＝4.3Hz,1H),7.97(d,J＝8.0Hz,1H),7.88(d,J＝15.9Hz,1H),7.44–7.35(m,2H),7.37–7.26(m,5H),5.15(t,J＝5.6Hz,1H),4.06–4.01(m,2H),4.03(s,3H),3.11(t,J＝6.7Hz,2H),2.95(t,J＝7.7Hz,2H),1.89-1.82(m,2H),1.04(t,J＝7.4Hz,3H).¹³C NMR(101MHz,DMSO-d₆)δ156.61,149.48,149.45,149.21,144.37,142.36,140.13,133.81,132.50,131.74,131.08,129.16(2C),128.86(2C),126.59,124.34,121.31,42.57,39.45,35.17,27.70,22.09,14.34.HR-MS(ESI):calcd for C2₄H₂₇N₆[M+H]⁺,399.2292；found 399.2291.

example 21

(E) Preparation of (E) -N- (3-chlorophenylethyl) -1-methyl-3-propyl-5- (2- (pyridin-3-yl) vinyl) -1H-pyrazolo [4,3-d ] pyrimidin-7-amine (having the structure shown in formula II-a-9):

the same synthetic method as that for compound II-a-6 was adopted. And (3) structural data characterization:¹H NMR(400MHz,CDCl₃)δ8.82(s,1H),8.53(d,J＝3.9Hz,1H),7.98(d,J＝7.9Hz,1H),7.87(d,J＝15.9Hz,1H),7.36–7.28(m,5H),7.19(d,J＝7.0Hz,1H),5.20(t,J＝5.8Hz,1H),4.11(s,3H),4.02(m,2H),3.10(t,J＝6.9Hz,2H),2.95(t,J＝7.7Hz,2H),1.88–1.86(m,2H),1.04(t,J＝7.4Hz,3H).¹³C NMR(101MHz,DMSO-d₆)δ156.59,149.53,149.39,149.34,144.37,142.77,142.36,133.83,133.45,132.48,131.64,131.17,130.67,129.09,128.04,126.61,124.35,121.28,42.15,39.47,34.75,27.71,22.16,14.38.HR-MS(ESI):calcd for C₂₄H₂₆ClN₆[M+H]⁺,433.1902；found 433.1905.

example 22

(E) Preparation of (E) -N- (4-fluorobenzyl) -1-methyl-3-propyl-5- (2- (pyridin-3-yl) vinyl) -1H-pyrazolo [4,3-d ] pyrimidin-7-amine (having the structure shown in formula II-a-10):

the same synthetic method as that for compound II-a-6 was adopted. And (3) structural data characterization:¹H NMR(400MHz,CDCl₃)δ8.78(d,J＝2.2Hz,1H),8.52(dd,J＝4.8,1.6Hz,1H),7.93(dt,J＝7.9,2.0Hz,1H),7.80(d,J＝15.9Hz,1H),7.49–7.40(m,2H),7.35–7.27(m,2H),7.13–7.05(m,2H),5.48(t,J＝5.5Hz,1H),4.94(d,J＝5.4Hz,2H),4.24(s,3H),3.01–2.92(m,2H),1.91–1.83(m,2H),1.04(t,J＝7.4Hz,3H).¹³C NMR(101MHz,DMSO-d₆)δ161.55(d,J＝243.41Hz),156.52,149.52,149.34,149.16,144.36,142.43,136.69(d,J＝3.03Hz),133.84,132.48,131.48,131.40,129.87(d,J＝8.08Hz,2C),124.35,121.24,115.35(d,J＝21.21Hz,2C),43.54,39.63,27.70,22.18,14.36.HR-MS(ESI):calcd for C₂₃H₂₄FN₆[M+H]⁺,403.2041；found 403.2042.

example 23

(E) Preparation of (E) -3- (2- (7- (cyclopropylamino) -1-methyl-3-propyl-1H-pyrazolo [4,3-d ] pyrimidin-5-yl) vinyl) pyridine 1-oxide (having the structure shown in formula II-a-11):

preparation of Compound II-a-11:

compound II-a-6(100mg, 0.297mmol) was dissolved in acetone (6mL) and water (3 mL). NaHCO is added within 1 hour₃(749mg, 8.918mmol) and KHSO₅(912mg, 2.97mmol) was added 3 times. The mixture was stirred at room temperature for 4 h. After the reaction was completed, the solvent was removed. The residue was purified by column chromatography to give compound II-a-11. And (3) structural data characterization:¹H NMR(600MHz,DMSO-d₆)δ8.59(s,1H),8.14(d,J＝6.5Hz,1H),7.75(d,J＝8.2Hz,1H),7.71(d,J＝15.7Hz,1H),7.44–7.39(m,1H),7.33(d,J＝15.9Hz,1H),7.28(d,J＝3.0Hz,1H),4.15(s,3H),3.13–3.06(m,1H),2.80(t,J＝7.5Hz,2H),1.81–1.70(m,2H),0.94(t,J＝7.3Hz,3H),0.92–0.88(m,2H),0.73–0.66(m,2H).¹³C NMR(101MHz,DMSO-d₆)δ156.18,150.74,144.46,142.30,138.21,137.93,136.35,133.73,129.49,126.86,123.39,121.48,39.52,27.69,24.58,22.14,14.36,6.98(2C).HR-MS(ESI):calcd for C₁₉H₂₃N₆O[M+H]⁺,351.1928；found 351.1931.

example 24

(E) Preparation of 3- (2- (7- (isopropylamino) -1-methyl-3-propyl-1H-pyrazolo [4,3-d ] pyrimidin-5-yl) vinyl) pyridine 1-oxide (having the structure shown in formula II-a-12):

the same synthesis method as that for compound II-a-11 was adopted. And (3) structural data characterization:¹H NMR(400MHz,DMSO-d₆)δ8.59(t,J＝1.7Hz,1H),8.14(dd,J＝6.3,1.0Hz,1H),7.75(d,J＝8.4Hz,1H),7.63(d,J＝15.9Hz,1H),7.41(dd,J＝8.0,6.4Hz,1H),7.30(d,J＝16.0Hz,1H),6.72(d,J＝7.8Hz,1H),4.72–4.64(m,1H),4.20(s,3H),2.79(t,J＝7.5Hz,2H),1.79–1.70(m,2H),1.33(d,J＝6.5Hz,6H),0.93(t,J＝7.3Hz,3H).¹³C NMR(101MHz,DMSO-d₆)δ156.18,149.14,144.44,142.30,138.18,137.90,136.37,133.85,129.19,126.85,123.47,121.34,42.47,39.50,27.68,22.39(2C),22.15,14.35.HR-MS(ESI):calcd for C₁₉H₂₅N₆O[M+H]⁺,353.2084；found 353.2083.

example 25

(E) Preparation of (E) -3- (2- (1-methyl-7- (phenethylamino) -3-propyl-1H-pyrazolo [4,3-d ] pyrimidin-5-yl) vinyl) pyridine 1-oxide (having the structure shown in formula II-a-13):

the same synthesis method as that for compound II-a-11 was adopted. And (3) structural data characterization:¹H NMR(400MHz,DMSO-d₆)δ8.56(s,1H),8.16(d,J＝6.3Hz,1H),7.72(d,J＝8.1Hz,1H),7.66(d,J＝15.9Hz,1H),7.46–7.37(m,2H),7.39–7.29(m,5H),7.27–7.16(m,1H),4.16(s,3H),3.88–3.78(m,2H),3.06–2.98(m,2H),2.79(t,J＝7.6Hz,2H),1.82–1.68(m,2H),0.94(t,J＝7.4Hz,3H).¹³C NMR(101MHz,DMSO-d₆)δ156.20,149.46,144.47,142.25,140.13,138.23,137.75,136.33,133.87,129.25(2C),129.21,128.87(2C),126.93,126.61,123.50,121.32,42.49,39.51,35.17,27.70,22.14,14.38.HR-MS(ESI):calcd for C₂₄H₂₇N₆O[M+H]⁺,415.2241；found 415.2239.

example 26

(E) Preparation of (E) -3- (2- (7- ((3-chlorophenethyl) amino) -1-methyl-3-propyl-1H-pyrazolo [4,3-d ] pyrimidin-5-yl) vinyl) pyridine 1-oxide (having the structure shown in formula II-a-14):

the same synthesis method as that for compound II-a-11 was adopted. And (3) structural data characterization:¹H NMR(600MHz,DMSO-d₆)δ8.57(s,1H),8.14(d,J＝6.5Hz,1H),7.73(d,J＝8.0Hz,1H),7.65(d,J＝15.9Hz,1H),7.42(m,2H),7.39–7.23(m,5H),4.15(s,3H),3.88–3.85(m,2H),3.03(t,J＝7.4Hz,2H),2.79(t,J＝7.6Hz,2H),1.75(m,2H),0.94(t,J＝7.3Hz,3H).¹³C NMR(101MHz,DMSO-d₆)δ156.16,149.41,144.48,142.74,142.30,138.21,137.81,136.34,133.80,133.43,130.56,129.23,129.16,128.06,126.85,126.55,123.39,121.29,42.00,39.47,34.81,27.70,22.14,14.37.HR-MS(ESI):calcd for C₂₄H₂₆ClN₆O[M+H]⁺,449.1851；found 449.1856.

example 27

(E) Preparation of (E) -3- (2- (7- ((4-fluorobenzyl) amino) -1-methyl-3-propyl-1H-pyrazolo [4,3-d ] pyrimidin-5-yl) vinyl) pyridine 1-oxide (having the structure shown in formula II-a-15):

the same synthesis method as that for compound II-a-11 was adopted. And (3) structural data characterization:¹H NMR(400MHz,DMSO-d₆)δ8.57(t,J＝1.7Hz,1H),8.14(dd,J＝6.3,1.0Hz,1H),7.94(t,J＝5.9Hz,1H),7.71(d,J＝8.2Hz,1H),7.63–7.51(m,3H),7.41(dd,J＝8.0,6.4Hz,1H),7.27(d,J＝16.0Hz,1H),7.20–7.11(m,2H),4.82(d,J＝5.7Hz,2H),4.24(s,3H),2.79(t,J＝7.5Hz,2H),1.79–1.69(m,2H),0.93(t,J＝7.4Hz,3H).¹³C NMR(101MHz,DMSO-d₆)δ161.57(d,J＝243.41Hz),156.07,149.18,144.49,142.37,138.23,137.81,136.63(d,J＝3.03Hz),136.30,133.61,130.01(d,J＝8.08Hz,2C),129.41,126.90,123.48,121.27,115.32(d,J＝21.21Hz,2C),43.42,39.64,27.68,22.15,14.35.HR-MS(ESI):calcd for C₂₃H₂₄FN₆O[M+H]⁺,419.1990；found 419.1989.

example 28

Preparation of N-cyclopropyl-1-methyl-3-propyl-5- (2- (pyridin-3-yl) ethyl) -1H-pyrazolo [4,3-d ] pyrimidin-7-amine (the structure is shown in formula II-a-16):

a mixture of compound II-a-6(100mg, 0.297mmol) and 5% Pd/C (3.14mg, 0.0297mmol) in THF (6mL) was stirred under hydrogen (2atm) at room temperature for 10 h. After completion of the reaction, the mixture was filtered, concentrated under vacuum, and the residue was purified by column chromatography to give compound II-a-16. And (3) structural data characterization:¹H NMR(600MHz,DMSO-d₆)δ8.44(d,J＝2.2Hz,1H),8.34(dd,J＝4.7,1.6Hz,1H),7.65(d,J＝7.8,1H),7.25(dd,J＝7.8,4.7Hz,1H),7.12(d,J＝2.9Hz,1H),4.09(s,3H),3.14(t,J＝7.3Hz,2H),3.06(t,J＝7.7Hz,2H),2.96–2.94(m,1H),2.73(t,J＝7.5Hz,2H),1.73–1.66(m,2H),0.89(t,J＝7.3Hz,3H),0.83–0.75(m,2H),0.65–0.61(m,2H).¹³C NMR(101MHz,DMSO-d₆)δ161.14,151.00,150.18,147.37,143.46,142.14,137.74,136.33,123.70,121.25,39.78,39.34,31.13,27.63,24.45,22.09,14.27,6.99(2C).HR-MS(ESI):calcd for C₁₉H₂₅N₆[M+H]⁺,337.2135；found 337.2130.

example 29

Preparation of N-isopropyl-1-methyl-3-propyl-5- (2- (pyridin-3-yl) ethyl) -1H-pyrazolo [4,3-d ] pyrimidin-7-amine (the structure is shown in formula II-a-17):

the same synthetic method as that for compound II-a-16 was adopted. And (3) structural data characterization:¹H NMR(400MHz,DMSO-d₆)δ8.41(d,J＝1.4Hz,1H),8.34(dd,J＝4.8,1.7Hz,1H),7.64(dt,J＝7.8,2.0Hz,1H),7.27–7.23(m,1H),6.58(d,J＝7.8Hz,1H),4.53–4.41(m,1H),4.14(s,3H),3.09(t,J＝6.8Hz,2H),3.05–2.97(m,2H),2.73(t,J＝7.5Hz,2H),1.74–1.65(m,2H),1.26(d,J＝6.6Hz,6H),0.89(t,J＝7.4Hz,3H).¹³C NMR(101MHz,DMSO-d₆)δ161.17,150.09,149.43,147.40,143.48,142.21,137.67,136.29,123.73,121.09,42.35,39.78,39.40,31.26,27.64,22.34(2C),22.10,14.30.HR-MS(ESI):calcd for C₁₉H₂₇N₆[M+H]⁺,339.2292；found 339.2293.

example 30

Preparation of 1-methyl-N-phenethyl-3-propyl-5- (2- (pyridin-3-yl) ethyl) -1H-pyrazolo [4,3-d ] pyrimidin-7-amine (the structure is shown in formula II-a-18):

the same synthetic method as that for compound II-a-16 was adopted. And (3) structural data characterization:¹H NMR(400MHz,DMSO-d₆)δ8.43(s,1H),8.34(d,J＝3.1Hz,1H),7.64(d,J＝7.8Hz,1H),7.36–7.24(m,5H),7.25–7.20(m,2H),4.11(s,3H),3.76–3.66(m,2H),3.13(t,J＝6.7Hz,2H),3.04(t,J＝6.7Hz,2H),2.98–2.90(m,2H),2.74(t,J＝7.5Hz,2H),1.77–1.61(m,2H),0.90(t,J＝7.4Hz,3H).¹³C NMR(101MHz,DMSO-d₆)δ161.23,150.09,149.72,147.45,143.50,142.17,140.13,137.70,136.29,129.15(2C),128.87(2C),126.59,123.76,121.07,42.50,39.53,39.37,35.19,31.33,27.67,22.10,14.34.HR-MS(ESI):calcd for C₂₄H₂₉N₆[M+H]⁺,401.2448；found 401.2450.

example 31

Preparation of N- (3-chlorophenylethyl) -1-methyl-3-propyl-5- (2- (pyridin-3-yl) ethyl) -1H-pyrazolo [4,3-d ] pyrimidin-7-amine (the structure is shown in formula II-a-19):

the same synthetic method as that for compound II-a-16 was adopted. And (3) structural data characterization:¹H NMR(600MHz,DMSO-d₆)δ8.40(d,J＝2.3Hz,1H),8.32(dd,J＝4.8,1.6Hz,1H),7.61(dt,J＝7.8,2.1Hz,1H),7.35–7.26(m,2H),7.25–7.19(m,4H),4.08(s,3H),3.73–3.67(m,2H),3.09(t,J＝7.7Hz,2H),3.05–2.98(m,2H),2.92(t,J＝7.3Hz,2H),2.71(t,J＝7.5Hz,2H),1.68(m,2H),0.88(t,J＝7.4Hz,3H).¹³C NMR(101MHz,DMSO-d₆)δ161.21,150.09,149.68,147.44,143.53,142.78,142.20,136.28,133.40,130.63,129.15,129.05,127.97,126.55,123.74,121.05,42.02,39.53,39.35,34.71,31.37,27.66,22.09,14.34.HR-MS(ESI):calcd for C₂₄H₂₈ClN₆[M+H]⁺,435.2058；found 435.2060.

example 32

Preparation of N- (4-fluorobenzyl) -1-methyl-3-propyl-5- (2- (pyridin-3-yl) ethyl) -1H-pyrazolo [4,3-d ] pyrimidin-7-amine (the structure is shown in formula II-a-20):

the same synthetic method as that for compound II-a-16 was adopted. And (3) structural data characterization:¹H NMR(400MHz,DMSO-d₆)δ8.32(dd,J＝4.8,1.7Hz,1H),8.28(dd,J＝2.3,0.9Hz,1H),7.81(t,J＝5.9Hz,1H),7.51–7.42(m,3H),7.21–7.17(m,1H),7.16–7.09(m,2H),4.70(d,J＝5.8Hz,2H),4.19(s,3H),3.01–2.92(m,4H),2.72(t,J＝7.5Hz,2H),1.73–1.63(m,2H),0.89(t,J＝7.4Hz,3H).¹³C NMR(101MHz,DMSO-d₆)δ161.54(d,J＝243.41Hz),161.02,150.04,149.51,147.37,143.51,142.24,137.59,136.59(d,J＝3.03Hz),136.23,129.85(d,J＝8.08Hz,2C),123.66,121.02,115.29(d,J＝21.21Hz,2C),43.30,39.89,39.53,31.11,27.64,22.10,14.31.HR-MS(ESI):calcd for C₂₃H₂₆FN₆[M+H]⁺,405.2197；found 405.2196.

example 33

(E) Preparation of-4- (1-methyl-3-propyl-5- (2- (3,5, 6-trimethylpyrazin-2-yl) vinyl) -1H-pyrazolo [4,3-d ] pyrimidin-7-yl) morpholine (structure shown as formula II-b-1):

the same synthesis method as that for compound II-a-1 was adopted. And (3) structural data characterization:¹H NMR(400MHz,DMSO-d₆)δ8.18(d,J＝15.0Hz,1H),7.81(d,J＝15.0Hz,1H),4.12(s,3H),3.99–3.96(m,4H),3.83–3.81(m,4H),2.96(t,J＝7.5Hz,2H),2.65(s,3H),2.52(d,6H),1.78–1.69(m,2H),0.98(t,J＝7.3Hz,3H).¹³C NMR(101MHz,DMSO-d₆+Pyridine-d₅)δ156.08,153.63,150.95,149.60,147.76,146.06,144.67,144.16,133.35,129.82,123.96,66.12(2C),49.98(2C),39.21,27.76,21.97,21.83,21.80,20.82,14.29.HR-MS(ESI):calcd for C₂₂H₃₀N₇O[M+H]⁺,408.2506；found 408.2510.

example 34

(E) Preparation of-4- (1-methyl-3-propyl-5- (2- (3,5, 6-trimethylpyrazin-2-yl) vinyl) -1H-pyrazolo [4,3-d ] pyrimidin-7-ylthiomorpholine 1, 1-dioxide (structure shown in formula II-b-2):

the same synthesis method as that for compound II-a-1 was adopted. And (3) structural data characterization:¹H NMR(400MHz,DMSO-d₆)δ8.07(d,J＝15.1Hz,1H),7.74(d,J＝15.1Hz,1H),4.18-4.15(m,4H),4.14(s,3H),3.47-3.73(m,4H),2.93(t,J＝7.5Hz,2H),2.64(s,3H),2.52(d,6H),1.82-1.73(m,2H),0.97(t,J＝7.3Hz,3H).¹³C NMR(101MHz,DMSO-d₆)δ155.78,152.55,151.12,149.69,147.84,146.02,145.09,144.00,132.97,129.95,123.66,50.70(2C),48.10(2C),39.38,27.70,21.97,21.92(2C),20.89,14.38.HR-MS(ESI):calcd for C₂₂H₃₀N₇O₂S[M+H]⁺,456.2176；found 456.2178.

example 35

(E) Preparation of (E) -4- (1-methyl-3-propyl-5- (2- (pyridin-3-yl) vinyl) -1H-pyrazolo [4,3-d ] pyrimidin-7-yl) morpholine (having the structure shown in formula II-b-3):

the same synthetic method as that for compound II-a-6 was adopted. And (3) structural data characterization:¹H NMR(400MHz,CDCl₃)δ9.14(s,1H),8.73(d,J＝3.9Hz,1H),8.67(d,J＝8.2Hz,1H),7.94(d,J＝16.0Hz,1H),7.84(dd,J＝8.1,5.3Hz,1H),7.59(d,J＝16.0Hz,1H),4.10(s,3H),3.85–3.82(m,4H),3.67–3.66(m,4H),2.89(t,J＝7.5Hz,2H),1.83-1.74(m,2H),0.97(t,J＝7.3Hz,3H).¹³C NMR(101MHz,DMSO-d₆)δ156.06,153.68,149.63,149.40,145.94,144.56,133.93,132.32,131.94,130.72,124.31,123.93,66.15(2C),50.00(2C),39.27,27.75,22.02,14.38.HR-MS(ESI):calcd for C₂₀H₂₅N₆O[M+H]⁺,365.2084；found 365.2088.

example 36

(E) Preparation of (E) -4- (1-methyl-3-propyl-5- (2- (pyridin-3-yl) vinyl) -1H-pyrazolo [4,3-d ] pyrimidin-7-yl) thiomorpholine 1, 1-dioxide (having the structure shown in formula II-b-4):

the same synthetic method as that for compound II-a-6 was adopted. And (3) structural data characterization:¹H NMR(400MHz,DMSO-d₆)δ8.88(d,J＝2.2Hz,1H),8.52(dd,J＝4.8,1.6Hz,1H),8.21(dt,J＝8.0,2.0Hz,1H),7.82(d,J＝16.0Hz,1H),7.46-7.43(m,1H),7.40(d,J＝16.0Hz,1H),4.12(s,3H),4.05-4.02(m,4H),3.44–3.41(m,4H),2.88(t,J＝7.5Hz,2H),1.84-1.75(m,2H),0.97(t,J＝7.4Hz,3H).¹³C NMR(101MHz,DMSO-d₆)δ155.90,152.63,149.76,149.54,145.89,144.99,134.04,132.27,132.24,130.48,124.37,123.69,50.76(2C),48.12(2C),39.41,27.72,22.04,14.39.HR-MS(ESI):calcd for C₂₀H₂₅N₆O₂S[M+H]⁺,413.1754；found 413.1779.

example 37

(E) Preparation of (E) -3- (2- (1-methyl-7-morpholino-3-propyl-1H-pyrazolo [4,3-d ] pyrimidin-5-yl) vinyl) pyridine 1-oxide (having the structure shown in formula II-b-5):

the same synthesis method as that for compound II-a-11 was adopted. And (3) structural data characterization:¹H NMR(400MHz,CDCl₃)δ8.43(s,1H),8.17(d,J＝6.3Hz,1H),7.68(d,J＝15.9Hz,1H),7.57(d,J＝8.0Hz,1H),7.37(d,J＝15.9Hz,1H),7.32(dd,J＝8.1,6.4Hz,1H),4.14(s,3H),4.00–3.93(m,4H),3.63(t,J＝4.7Hz,4H),3.05–2.97(m,2H),1.94–1.85(m,2H),1.06(t,J＝7.4Hz,3H).¹³C NMR(101MHz,DMSO-d₆)δ155.66,153.62,146.02,144.50,138.37,138.04,136.17,132.86,130.14,126.89,123.95,123.42,66.14(2C),49.96(2C),39.40,27.75,22.01,14.39.HR-MS(ESI):calcd for C₂₀H₂₅N₆O₂[M+H]⁺,381.2034；found 381.2038.

example 38

(E) Preparation of (E) -3- (2- (7- (1, 1-thiomorpholino) -1-methyl-3-propyl-1H-pyrazolo [4,3-d ] pyrimidin-5-yl) vinyl) pyridine 1-oxide (having the structure shown in formula II-b-6):

the same synthesis method as that for compound II-a-11 was adopted. And (3) structural data characterization:¹H NMR(400MHz,DMSO-d₆)δ8.64(t,J＝1.8Hz,1H),8.15(dd,J＝6.3,1.0Hz,1H),7.79(d,J＝8.8Hz,1H),7.71(d,J＝16.0Hz,1H),7.47–7.40(m,2H),4.11(s,3H),4.05–4.03(m,4H),3.43–3.38(m,4H),2.87(t,J＝7.5Hz,2H),1.83–1.74(m,2H),0.96(t,J＝7.4Hz,3H).¹³C NMR(101MHz,DMSO-d₆)δ155.30,152.61,146.10,144.65,138.40,137.91,136.14,132.53,130.19,127.16,125.23,123.71,50.69(2C),47.99(2C),39.01,27.54,22.01,14.26.HR-MS(ESI):calcd for C₂₀H₂₅N₆O₃S[M+H]⁺,429.1703；found 429.1703.

example 39

Preparation of 4- (1-methyl-3-propyl-5- (2- (pyridin-3-yl) ethyl) -1H-pyrazolo [4,3-d ] pyrimidin-7-yl) morpholine (the structure is shown in formula II-b-7):

the same synthetic method as that for compound II-a-16 was adopted. And (3) structural data characterization:¹H NMR(400MHz,CDCl₃)δ8.50(d,J＝2.3Hz,1H),8.44(dd,J＝4.9,1.6Hz,1H),7.68–7.65(m,1H),7.25(dd,J＝7.4,5.2Hz,1H),4.08(s,3H),3.92–3.86(m,4H),3.55–3.48(m,4H),3.32–3.18(m,4H),3.02–2.92(m,2H),1.89–1.80(m,2H),1.02(t,J＝7.3Hz,3H).¹³C NMR(101MHz,CDCl₃)δ160.99,153.75,149.74,146.91,146.83,144.90,137.39,136.33,124.20,123.34,66.38(2C),49.88(2C),39.54,38.43,31.64,27.79,22.16,14.05.HR-MS(ESI):calcd for C₂₀H₂₇N₆O[M+H]⁺,367.2242；found 367.2236.

example 40

Preparation of 4- (1-methyl-3-propyl-5- (2- (pyridin-3-yl) ethyl) -1H-pyrazolo [4,3-d ] pyrimidin-7-yl) thiomorpholine 1, 1-dioxide (having the structure shown in formula II-b-8):

the same synthetic method as that for compound II-a-16 was adopted. And (3) structural data characterization:¹H NMR(400MHz,CDCl₃)δ8.46(dd,J＝4.9,1.6Hz,1H),8.42(d,J＝2.2Hz,1H),7.68(dt,J＝7.8,2.0Hz,1H),7.34–7.27(m,1H),4.11–4.02(m,4H),4.08(s,3H),3.32–3.29(m,2H),3.24–3.22(m,2H),3.17(t,J＝5.3Hz,4H),3.01–2.93(m,2H),1.90–1.81(m,2H),1.04(t,J＝7.4Hz,3H).¹³C NMR(101MHz,CDCl₃)δ160.80,151.88,149.32,147.31,146.88,145.91,137.19,136.66,123.66,123.56,50.54(2C),47.76(2C),39.42,38.43,31.71,27.76,22.09,14.06.HR-MS(ESI):calcd for C₂₀H₂₇N₆O₂S[M+H]⁺,415.1911；found 415.1915.

EXAMPLE 41

The trimethoxystyryl six-membered ring and pyrazolopyrimidine compounds prepared in the above examples are used as active compounds, and the active compounds are prepared into one or more dosage forms of tablets, capsules, troches, injections, suspensions, suppositories, and ointments according to a conventional preparation method of pharmaceutical preparations. Wherein, the preparation comprises the active compound and pharmaceutically acceptable auxiliary materials, and the auxiliary materials are carriers or excipients.

Wherein the dosage of the active compound in the pharmaceutical preparation is 0.01-500 mg/kg.

Wherein the excipient is calcium carbonate, calcium phosphate, saccharide, starch, cellulose derivative, gelatin, vegetable oil or polyethylene glycol; the carrier is diluent, disintegrant, binder or lubricant.

Anti-inflammatory activity verification experiment:

in order to verify that the trimethoxystyryl six-membered ring and pyrazolopyrimidine compounds disclosed by the invention have anti-inflammatory activity, the trimethoxystyryl six-membered ring compounds prepared in the above examples are selected for an activity experiment.

1. The experimental principle is as follows: the test adopts an NO content determination method, nitrate is reduced into nitrite by nitrate reductase, and then the nitrite is detected by Griess Reagent, so that the total NO content is calculated.

2. The experimental procedure was as follows:

(1) collecting cells in logarithmic growth phase, and counting by using a cell counting plate; cell dilution with medium to a concentration of 1.4 x 10⁵The cells are inoculated in a 24-well plate, the volume of each well is 500 mu L, and the cells are cultured overnight;

(2) the 24-well plate is divided into three groups: blank (cells only), model (cells + LPS), experimental (cells + LPS + compounds);

(3) changing the culture medium after overnight, directly changing 500 μ L fresh culture medium for blank group and model group, and adding compound solution 500 μ L containing different concentrations (40, 20, 10, 5, 2.5 μ M) into each well of experimental group;

(4) adding LPS after 1h for stimulation to make the final concentration of LPS be 1 μ g/mL (generally, high-concentration LPS can be diluted with a small amount of culture medium and then dripped);

(5) observing cell supernatant after 24h, detecting by using a nitric oxide detection kit produced in Biyun day, and calculating the concentration of NO in the cell supernatant;

(6) configuring a standard curve: diluting the standard substance with fresh culture medium to concentration of 1, 5, 10, 20, 40, 60, 100 μ M;

(7) adding the standard substance and the cell supernatant into a 96-well plate according to 50 mu L of each well;

(8) adding 50 mu L of Griess Reagent at room temperature into each hole, and adding 50 mu L of Griess Reagent II into each hole;

(9) absorbance was measured at 540nm using a microplate reader, and the NO content was calculated by introducing a standard.

(10) The above experiments were repeated three times, and the results were analyzed and plotted using SPSS17.0 statistical analysis software.

3. The experimental results are as follows:

the results of the experiment are shown in table 1:

table 1: data statistics for anti-inflammatory Activity of the corresponding numbered Compounds of each example

Note: IC (integrated circuit)₅₀The unit of value is: mu mol/L.

The relation between the balanced toxicity and the anti-inflammatory activity is taken as guidance to design and synthesize novel molecules with effective drug-like anti-inflammatory activity, and part of compounds show strong NO generation and iNOS inhibition activity and have lower cytotoxicity to normal stem cell of human. The log P values for oral drugs that permeate by passive diffusion are best in the 0-3 range, which is the log P for these compounds.

As can be seen from table 1, the trimethoxystyryl six-membered ring and pyrazolopyrimidine compounds provided in this example show good anti-inflammatory activity in a RAW 264.7 cell model induced by LPS, which indicates that they may have good application prospects in treatment of various acute and chronic inflammations, and show good application potentials in treatment of acute inflammations such as sepsis and rheumatoid arthritis, and chronic inflammations represented by type ii diabetes, alzheimer's disease, and atherosclerosis.

The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. A trimethoxy styryl six-membered ring compound is characterized in that: a compound comprising the structure shown in formula I:

2. The method for preparing a trimethoxystyryl six-membered ring compound according to claim 1, wherein: preparing a compound 2 from the compound 1 and an amine derivative under the conditions of isopropanol and triethylamine, and reacting the compound 2 with trimethoxybenzaldehyde under the condition of hydrochloric acid to obtain a trimethoxystyryl six-membered ring compound, wherein the reaction equation is as follows:

3. a pyrazolopyrimidine compound characterized in that: comprising a compound having the structure shown in formula II-a and formula II-b:

wherein the content of the first and second substances,

R₆any one selected from hydrogen, alkyl, substituted alkyl and halogen;

z is C, N or O;

n is 1 or 2;

linker is CH₂-CH₂Or CH ═ CH.

4. A process for producing a pyrazolopyrimidine compound as claimed in claim 3, characterized in that: reacting the compound 3 with an acrylic acid derivative to obtain a compound 4, cyclizing the compound 4 under the condition of sodium methoxide to obtain a compound 5, and performing chlorination reaction on the intermediate 5 by using excessive phosphorus oxychloride to obtain a key intermediate 6; and (3) carrying out substitution reaction on the intermediate 6 and an amine derivative to obtain the pyrazolopyrimidine compound, wherein the reaction process is as follows:

5. the production method according to claim 2 or 4, characterized in that: the amine derivative is selected from any one of isopropylamine, cyclopropylamine, morpholine, thiomorpholine, 4-chlorophenylethylamine, 4-fluorophenylethylamine, 4-fluorobenzylamine and 2-chlorophenylethylamine.

6. The use of trimethoxystyryl six-membered ring compound according to claim 1 for the preparation of anti-inflammatory drugs.

7. Use of a pyrazolopyrimidine compound according to claim 3 in the preparation of an anti-inflammatory drug.

8. A pharmaceutical preparation comprising the trimethoxystyryl six-membered ring compound according to claim 1 as an active compound.

9. A pharmaceutical preparation comprising the pyrazolopyrimidine compound according to claim 3 as an active compound.

10. The pharmaceutical formulation according to claim 8 or 9, characterized in that: the pharmaceutical preparation is prepared into tablets, capsules, pastilles, injections, suspending agents, suppositories or ointments by adding pharmaceutically acceptable auxiliary materials to the active compound; the auxiliary materials comprise an excipient and a carrier, wherein the excipient is one or more of calcium carbonate, calcium phosphate, saccharides, starch, cellulose derivatives, gelatin, vegetable oil and polyethylene glycol, and the carrier is one or more of a diluent, a disintegrating agent, an adhesive and a lubricant.