3, 4-dihydropyrazino [1, 2-b] indazole-1 (2H)-ketone derivative as well as preparation method and application thereof

Abstract

The invention relates to a 3, 4-dihydropyrazino [1, 2-b] indazole-1 (2H)-ketone derivative with a structure as shown in a formula 6, or a stereoisomer or pharmaceutically acceptable salt thereof, and a preparation method and application of the 3, 4-dihydropyrazino [1, 2-b] indazole-1 (2H)-ketone derivative. The 3, 4-dihydropyrazino [1, 2-b] indazole-1 (2H)-ketone derivative can significantly inhibit release of nitric oxide (NO) induced by LPS, can significantly inhibit gene expression of a proinflammatory cytokine IL-6, has a good anti-inflammatory effect, has the advantages of low cytotoxicity and good safety, is expected to become a potential drug for treating inflammation-related diseases, and has a good application prospect. And an aza-ring framework which can be used for drug development is enriched.

Description

Technical Field

[0001] The present invention belongs to the field of organic synthesis and pharmaceutical technology, and relates to the synthesis of anti-inflammatory drugs, and specifically to a class of 3,4-dihydropyrazino[1,2-b]indazole-1(2H)-one derivatives, a preparation method thereof, and an application thereof in anti-inflammatory treatment. Background Art

[0002] Inflammation is a protective response of the body aimed at maintaining tissue homeostasis by eliminating pathogenic microbial infection, irritants, or tissue damage. Based on its pathological characteristics and duration, inflammation can be divided into acute inflammation and chronic inflammation. Acute inflammation is an immediate response to infection or injury and usually lasts from a few days to a week. Chronic inflammation, on the other hand, is persistent and manifests as low-grade inflammation and immune dysregulation. Inflammation is like a double-edged sword with complex and multifaceted effects. When inflammation is out of balance, it becomes a pathological state and is associated with a variety of diseases, such as metabolic disorders, neurodegenerative diseases, autoimmune diseases, inflammatory bowel disease, etc. Therefore, the development of inhibitors for the treatment of inflammation and its related diseases is of great significance.

[0003] Ring systems significantly influence the shape, volume, electrostatic properties, and bioactivity of compounds. These bioactive scaffolds have been widely used in drug discovery. Among them, nitrogen-containing heterocycles have garnered significant attention due to their diverse bioactivities and wide applications. These heterocycles are crucial components in the development of novel drugs, pesticides, and advanced materials. Therefore, it is imperative to develop novel, efficient synthetic methods to synthesize more novel nitrogen-containing heterocyclic scaffolds for the development of novel small-molecule anti-inflammatory drugs. Summary of the Invention

[0004] Based on this, the purpose of the present invention is to provide a method for synthesizing 3,4-dihydropyrazino[1,2-b]indazole-1(2H)-one derivatives having a novel nitrogen-containing heterocyclic skeleton structure, and to provide novel nitrogen-containing heterocyclic lead compounds with good anti-inflammatory effects.

[0005] The technical solutions for achieving the above-mentioned purpose include the following.

[0006] In the first aspect, the present invention provides a 3,4-dihydropyrazino[1,2-b]indazol-1(2H)-one derivative having a structure shown in Formula 6, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0007]

[0008] Wherein, n is selected from: 1, 2, 3, 4;

[0009] R1 is selected from the group consisting of: hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy;

[0010] Each R2 is independently selected from: hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy;

[0011] R3 is selected from the group consisting of: hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy;

[0012] R4 is selected from: C1-C6 alkyl, C3-C8 cycloalkyl, C6-C 10 Aryl-substituted C1-C6 alkyl, one or more R1-substituted or unsubstituted C6-C 10 Aryl.

[0013] In some embodiments, R1 is selected from the group consisting of hydrogen, fluorine, chlorine, bromine, methyl, ethyl, propyl, methoxy, ethoxy, and propoxy.

[0014] In some embodiments, each R2 is independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, methyl, ethyl, propyl, methoxy, ethoxy, and propoxy.

[0015] In some embodiments, R3 is selected from the group consisting of hydrogen, fluorine, chlorine, bromine, methyl, ethyl, propyl, methoxy, ethoxy, and propoxy.

[0016] In some embodiments, R4 is selected from: C1-C4 alkyl, C5-C6 cycloalkyl, C1-C2 alkyl substituted with phenyl, and one or more R1 substituted or unsubstituted phenyl.

[0017] In some embodiments, R4 is selected from the group consisting of: methyl, ethyl, propyl, n-butyl, tert-butyl, isobutyl, cyclohexyl, cyclopentyl, benzyl, and m-xylyl.

[0018] In some embodiments, the 3,4-dihydropyrazino[1,2-b]indazol-1(2H)-one derivative is selected from the following compounds:

[0019]

[0020] In a second aspect, the present invention provides a method for preparing the 3,4-dihydropyrazino[1,2-b]indazol-1(2H)-one derivative, comprising the following steps:

[0021] (1) Compound 1, Compound 2, Compound 3 react with Compound 4 to obtain Compound 5;

[0022] (2) The obtained compound 5 undergoes further intramolecular cyclization reaction under the action of a base to obtain compound 6;

[0023] The reaction formula is as follows:

[0024]

[0025] wherein n, R1, R2, R3, R4 are as described above.

[0026] In some embodiments, the solvent for the reaction in step (1) is methanol.

[0027] In some embodiments, the solvent for the reaction in step (2) is selected from at least one of methanol, N,N-dimethylformamide, tetrahydrofuran, 1,4-dioxane, dichloroethane, isopropanol and acetonitrile, more preferably methanol.

[0028] In some embodiments, the base in step (2) is selected from at least one of sodium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, triethylamine, diazabicyclo[5.4.0]undec-7-ene and 4-dimethylaminopyridine.

[0029] In some embodiments, the base in step (2) is potassium carbonate and / or sodium carbonate.

[0030] In some embodiments, the molar ratio of compound 1, compound 2, compound 3, compound 4 to base is 1:0.8-1.2:0.8-1.2:0.8-1.2:1.8-2.2.

[0031] In some embodiments, the ratio of solvent to compound 1 in step (1) is 1.8 mL-2.2 mL:1.0 mmol.

[0032] In some embodiments, the ratio of solvent to compound 1 in step (2) is 8 mL-12 mL:1.0 mmol.

[0033] In some embodiments, the reactions in step (1) and step (2) are both carried out under air atmosphere.

[0034] In some embodiments, the temperature for the reaction in step (1) is 20°C-40°C, and the time is 6 hours-16 hours, preferably 10 hours-12 hours.

[0035] In some embodiments, the temperature for the reaction in step (2) is 20°C-40°C, and the time is 4 hours-12 hours, preferably 6 hours-9 hours.

[0036] In some embodiments, the method for preparing the 3,4-dihydropyrazino[1,2-b]indazol-1(2H)-one derivative comprises the following steps:

[0037] (1) under air atmosphere, mixing and stirring the compound 1, methanol and the compound 2 for 8 minutes-10 minutes, adding the compound 3 and the compound 4, and stirring the resulting mixture at 20°C-40°C for 6 hours-12 hours;

[0038] (2) adding more methanol to the reaction mixture obtained in step (1), and then adding the base, and continuing to stir the resulting mixture at 20° C.-40° C. for 4 hours-12 hours, and performing post-treatment and purification to obtain the 3,4-dihydropyrazino[1,2-b]indazol-1(2H)-one derivative;

[0039] In step (1), the ratio of methanol to compound 1 is 1.8 mL-2.2 mL: 1.0 mmol;

[0040] In step (2), methanol is added to compound 1 in a ratio of 8 mL-12 mL: 1.0 mmol.

[0041] In a third aspect, the present invention provides the use of the 3,4-dihydropyrazino[1,2-b]indazol-1(2H)-one derivative or its stereoisomer or its pharmaceutically acceptable salt in the preparation of anti-inflammatory drugs.

[0042] In a fourth aspect, the present invention provides an anti-inflammatory drug prepared from an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient comprises the 3,4-dihydropyrazino[1,2-b]indazole-1(2H)-one derivative described in the present invention or a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0043] The present invention has the following beneficial effects:

[0044] This invention synthesizes a class of 3,4-dihydropyrazino[1,2-b]indazol-1(2H)-one derivatives with novel azatricyclic rings through a multi-component, one-pot reaction. These compounds significantly inhibit LPS-induced nitric oxide (NO) release and gene expression of the pro-inflammatory cytokine IL-6, exhibiting strong anti-inflammatory effects, low cytotoxicity, and good safety. They are expected to become potential drugs for the treatment of inflammatory diseases, enriching the nitrogen heterocyclic skeletons available for drug development.

[0045] The synthetic method for preparing 3,4-dihydropyrazino[1,2-b]indazol-1(2H)-one derivatives provided by the present invention has the following advantages: high yield and a wide range of substrate applicability; no additives or catalysts are required, and the reaction can be carried out solely under the action of a base; the reaction is carried out at room temperature and in an air atmosphere, requiring no special protection, resulting in simple operation, low energy consumption, and low production cost; the two-step reaction can be carried out in one pot, and the intermediate compound does not require post-treatment or separation and purification, further simplifying the operation and reducing labor and material costs. These advantages are conducive to its industrialized and large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1The figures show the inhibitory effects of compounds 6e and 6r on LPS-induced NO release; A is the dose-response curve of compound 6e in the NO release assay; B is the NO release concentration of RAW264.7 cells stimulated with LPS for 1 hour and then treated with compound 6e for 24 hours; C is the NO release concentration of RAW 264.7 cells stimulated with LPS for 1 hour and then treated with compound 6r for 24 hours.

[0047] Figure 2 The inhibitory effect of compound 6e on IL-6 gene expression was analyzed by qPCR.

[0048] Figure 3 The cytotoxicity of compounds 6e and 6r on THP-1 cells. DETAILED DESCRIPTION

[0049] To facilitate understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the present disclosure more thorough and comprehensive.

[0050] In the following examples, the experimental methods without specific conditions are generally carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0052] Furthermore, as used herein, the term "or" is inclusive and equivalent to the term "and / or," unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on other factors not described, unless the context clearly dictates otherwise. Furthermore, throughout this specification, the meanings of "a," "an," and "the" include plural referents. The meaning of "in" includes "in" and "on."

[0053] The present invention is further described in detail below with reference to specific embodiments.

[0054] Example 1 Synthesis of Compound 6a under different conditions

[0055]

[0056] Compound 1a (1.0mmol, 1.0 equivalent), methanol (2mL) and compound 2a (1.0mmol, 1.0 equivalent) are added in reaction tube successively, and stirred 10 minutes under air atmosphere. Subsequently, compound 3a (1.0mmol, 1.0 equivalent) and compound 4a (1.0mmol, 1.0 equivalent) are added respectively. The mixture is stirred 12 hours under room temperature (25 ℃) and air atmosphere, and the reaction process is monitored by thin layer chromatography (TLC). When no compound 4a is detected, the reaction solution is spin-dried, and the residue is purified by silica gel column chromatography, and ethyl acetate / hexane (0-30%) is used to carry out gradient elution to obtain compound 5a, yield 86%.

[0057] Obtained compound 5a is dissolved in 10mL of the solvent shown in Table 1, and then the base (2.0mmol, 2.0 equivalents) shown in Table 1 is added. The reaction mixture is stirred under air atmosphere and the temperature shown in Table 1 for a certain time (specific time is as shown in Table 1), and monitored by TLC. If compound 5a is not detected, the reaction mixture is poured into water and extracted with ethyl acetate. The ethyl acetate phase is dried over anhydrous sodium sulfate and concentrated. The residue is purified by silica gel column chromatography using ethyl acetate / hexane (0-30%) for gradient elution to ultimately obtain target compound 6a, and the yield is as shown in Table 1.

[0058] Table 1 Reaction conditions and yields of intramolecular cyclization reaction

[0059]

[0060]

[0061] As shown in Table 1, compound 5a was treated with different bases in DMF (N,N-dimethylformamide). The yield of compound 6a varied depending on the base. The yield of compound 6a (80%) was the highest when sodium carbonate was used as the base. Using sodium carbonate as the base, the yield of compound 6a varied in different solvents, with methanol providing the highest yield (91%). Further increasing the reaction temperature and extending the reaction time did not improve the product yield, and the reaction tended to decrease.

[0062] Example 2 One-pot synthesis of compound 6a

[0063]

[0064] Compound 1a (1.0 mmol, 1.0 equiv), methanol (2 mL) and compound 2a (1.0 mmol, 1.0 equiv) were added sequentially into a reaction tube and stirred for 10 minutes under air atmosphere. Subsequently, compound 3a (1.0 mmol, 1.0 equiv) and compound 4a (1.0 mmol, 1.0 equiv) were added respectively. The mixture was stirred for 12 hours at room temperature (25 °C) under air atmosphere and the reaction progress was monitored by thin layer chromatography (TLC). When no compound 4a was detected, more methanol (total solvent amount was about 10 mL) was added into the bottle followed by sodium carbonate (2.0 mmol, 2.0 equiv). The reaction mixture was continued to stir for 6 hours at room temperature (25 °C) under air atmosphere and monitored by TLC. If compound 5a was not detected, the reaction mixture was poured into water and extracted with ethyl acetate. The ethyl acetate phase was dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography using a gradient elution of ethyl acetate / hexane (0-30%) to give the target product 6a with a total yield of 73% for two steps.

[0065] Preparation of compounds 6a-6r of Example 3

[0066]

[0067] Compounds 6b-6g were prepared according to the same procedure as in Example 2 by replacing compound 2a in Example 2 with compounds 2b-2g respectively.

[0068] Compounds 6h-6k were prepared according to the same procedure as in Example 2 by replacing compound 1a in Example 2 with compounds 1h-1k respectively.

[0069] Compounds 61-6m were prepared according to the same procedure as in Example 2 by replacing compound 4a in Example 2 with compounds 41-4m respectively.

[0070] Compounds 6p-6r were prepared according to the same procedure as in Example 2 by replacing compound 3a in Example 2 with compounds 3p-3r respectively.

[0071] The structural formulas of compounds 2b-2g are as follows respectively:

[0072]

[0073] The structural formulas of compounds 1h-1k are as follows respectively:

[0074]

[0075] The structural formulas of compounds 41-4o are as follows respectively:

[0076]

[0077] The structural formulas of compound 3p-compound 3r are as follows:

[0078]

[0079] The structural formulas and yields of compounds 6a-6r are as follows:

[0080]

[0081] The structural characterization data of compounds 6a-6s are as follows:

[0082] 4-Benzyl-N-(tert-butyl)-1-oxo-2-phenyl-1,2,3,4-tetrahydropyrazino[1,2-b]indazole-3-carboxamide (Compound 6a)

[0083] 1 H-NMR (400MHz, CDCl3) δ8.20(d,J=8.3Hz,1H),7.84(d,J=8.6Hz,1H),7.44(dt,J=15.8,7.9Hz,3H),7.39-7.30(m,4H),7.25-7.17(m,3H),7.04(dd,J =6.1,2.5Hz,2H),5.80(s,1H),5.66(dd,J=10.7,3.9Hz,1H),4.40(s,1H), 3.52(dd,J=13.8,4.4Hz,1H), 3.18(dd,J=13.8,10.9Hz,1H), 1.10(s,9H). 13 C-NMR (101MHz, CDCl3) δ167.4,156.8,148.9,140.5,135.1,129.6,129.2,129.1,127.7 ,127.1,127.1,125.4,124.3,123.8,122.5,121.0,118.5,65.0,62.5,52.3,40.5,28.4.

[0084] 4-Benzyl-N-(tert-butyl)-1-oxo-2-(4-chlorophenyl)-1,2,3,4-tetrahydropyrazino[1,2-b]indazole-3-carboxamide (Compound 6b)

[0085] 1H-NMR (400 MHz, CDC13) δ 8.18 (d, J = 8.3 Hz, 1H), 7.83 (d, J = 8.7 Hz, 1H), 7.48-7.39 (m, 3H), 7.38-7.32 (m, 1H), 7.29 (s, 1H), 7.26 (dd, J = 5.9, 3.4 Hz, 4H), 7.01 (dd, J = 6.2, 2.6 Hz, 2H), 5.71 (s, 1H), 5.62 (dd, J = 10.3, 4.4 Hz, 1H), 4.35 (s, 1H), 3.51 (dd, J = 13.9, 4.5 Hz, 1H), 3.18 (dd, J = 13.8, 10.5 Hz, 1H), 1.10 (s, 9H). 13 C-NMR (101 MHz, CDC13) δ 167.1, 156.8, 148.9, 138.9, 135.0, 132.6, 129.7, 129.3, 129.0, 127.8, 127.2, 125.7, 125.5, 123.7, 122.5, 121.0, 118.5, 64.9, 62.6, 52.4, 40.6, 28.4.

[0086] 4-benzyl-N-(tert-butyl)-1-oxo-2-(4-bromophenyl)-1,2,3,4-tetrahydropyrazino[1,2-b]indazole-3-carboxamide (Compound 6c)

[0087] 1 H-NMR (400 MHz, CDC13) δ 8.17 (d, J = 8.3 Hz, 1H), 7.82 (d, J = 8.7 Hz, 1H), 7.59-7.52 (m, 2H), 7.45-7.38 (m, 1H), 7.34 (dd, J = 12.9, 4.8 Hz, 2H), 7.26-7.16 (m, 5H), 6.60-6.45 (m, 1H), 5.85 (s, 1H), 5.60 (ddd, J = 12.6, 9.2, 4.1 Hz, 1H), 4.38 (d, J = 1.1 Hz, 1H), 3.49 (dd, J = 13.9, 4.6 Hz, 1H), 3.19 (dd, J = 13.8, 10.1 Hz, 1H), 1.10 (s, 9H). 13 C-NMR (101 MHz, CDC13) δ 167.1, 157.0, 148.8, 139.4, 135.0, 132.6, 132.0, 129.3, 129.1, 127.8, 127.2, 126.2, 125.5, 123.9, 122.4, 121.0, 120.5, 118.4, 116.8, 64.8, 62.6, 52.3, 40.7, 28.4.

[0088] 4-benzyl-N-(tert-butyl)-l-oxo-2-(2-bromophenyl)-l,2,3,4-tetrahydropyrazino[l,2- b]indazole-3-carboxamide (Compound 6d)

[0089] 1 H-NMR (400 MHz, CDC13) δ 8.21 (d, J = 8.3 Hz, 1H), 7.87 (d, J = 8.6 Hz, 1H), 7.52 (t, J = 1.9 Hz, 1H), 7.50-7.43 (m, 2H), 7.41-7.31 (m, 2H), 7.30-7.26 (m, 4H), 7.03 (dd, J = 6.3, 2.9 Hz, 2H), 5.75-5.61 (m, 2H), 4.38 (d, J = 1.1 Hz, 1H), 3.53 (dd, J = 13.9, 4.5 Hz, 1H), 3.22 (dd, J = 13.9, 10.3 Hz, 1H), 1.14 (s, 9H). 13 C-NMR (101 MHz, CDC13) δ 167.0, 156.6, 148.9, 141.6, 134.9, 130.7, 130.1, 129.3, 129.0, 127.8, 127.8, 127.2, 125.6, 123.7, 122.9, 122.7, 122.5, 121.0, 118.5, 64.9, 62.4, 52.4, 40.7, 28.4.

[0090] 4-benzyl-N-(tert-butyl)-l-oxo-2-(5-bromo-2-chlorophenyl)-l,2,3,4-tetrahydropyrazino[l,2- b]indazole-3-carboxamide (Compound 6e)

[0091] 1 H-NMR (400 MHz, CDC13) δ 8.22 (d, J = 8.3 Hz, 1H), 7.82 (d, J = 8.6 Hz, 1H), 7.58 (s, 1H), 7.51-7.40 (m, 3H), 7.35 (dd, J = 13.1, 8.4 Hz, 6H), 5.28 (dd, J = 13.6, 9.4 Hz, 2H), 4.12 (s, 1H), 3.71-3.52 (m, 2H), 1.21 (s, 9H). 13 C-NMR (101 MHz, CDC13) δ 167.1, 156.8, 148.7, 138.9, 135.6, 132.7, 131.9, 131.8, 129.4, 129.2, 127.8, 127.2, 125.4, 123.8, 122.4, 121.4, 121.1, 118.1, 64.3, 63.8, 52.5, 40.5, 28.4.

[0092] 4-Benzyl-N-(tert-butyl)-1-oxo-2-(4-methoxyphenyl)-1,2,3,4-tetrahydropyrazino[1,2-b]indazole-3-carboxamide (Compound 6f)

[0093] 1 H-NMR (400MHz, CDCl3) δ8.22(d,J=8.3Hz,1H),7.85(d,J=8.7Hz,1H),7.47-7.24(m,7H),7.11(dd,J=7.0,2.1Hz,2H),7.04-6.91(m,2H),5. 77(s,1H),5.70-5.58(m,1H),4.36(d,J=1.1Hz,1H),3.87(s,3H),3.54(dd,J=13.9,4.5Hz,1H),3.22(dd,J=13.8,10.8Hz,1H),1.13(s,9H). 13 C-NMR (101MHz, CDCl3) δ167.5,158.4,157.0,148.8,135.2,133.1,129.2,129.1,127.7,12 7.0,126.0,125.3,123.9,122.4,121.0,118.4,114.8,65.2,62.5,55.6,52.2,40.5,28.4.

[0094] 4-Benzyl-N-(tert-butyl)-1-oxo-2-(2-bromo-5-methylphenyl)-1,2,3,4-tetrahydropyrazino[1,2-b]indazole-3-carboxamide (Compound 6g)

[0095] 1 H-NMR (400MHz, CDCl3) δ8.14(d,J=8.3Hz,1H),7.70(d,J=8.7Hz,1H),7.49(d,J=8.2Hz,1H),7.35-7.10(m,8H),6.98(dd,J=8.2,1 .3Hz,1H),5.23(s,1H),5.21-5.10(m,1H),4.04(d,J=0.9Hz,1H),3.58(ddd,J=18.7,13.8,7.6Hz,2H),2.19(s,3H),1.08(s,9H). 13C-NMR (101 MHz, CDC13) δ 167.6, 157.1, 148.7, 139.3, 138.8, 136.0, 133.5, 130.8, 129.5, 129.4, 129.2, 127.6, 127.0, 125.1, 124.1, 122.3, 121.5, 119.5, 118.0, 64.6, 63.8, 52.2, 40.5, 28.3, 21.0.

[0096] N-(tert-butyl)-4-(4-methoxybenzyl)-l-oxo-2-phenyl-l,2,3,4-tetrahydropyrazino[l,2- b]indazole-3-carboxamide (Compound 6h)

[0097] 1 H-NMR (400 MHz, CDC13) δ 8.20 (d, J = 8.3 Hz, 1H), 7.84 (d, J = 8.6 Hz, 1H), 7.51 - 7.30 (m, 7H), 6.95 (d, J = 8.5 Hz, 2H), 6.76 (d, J = 8.5 Hz, 2H), 5.81 (s, 1H), 5.60 (dd, J = 10.7, 4.0 Hz, 1H), 4.41 (s, 1H), 3.76 (s, 3H), 3.47 (dd, J = 13.9, 4.4 Hz, 1H), 3.11 (dd, J = 13.8, 11.0 Hz, 1H), 1.11 (s, 9H). 13 C-NMR (101 MHz, CDC13) δ 167.6, 157.1, 148.7, 139.3, 138.8, 136.0, 133.5, 130.8, 129.5, 129.4, 129.2, 127.6, 127.0, 125.1, 124.1, 122.3, 121.5, 119.5, 118.0, 64.6, 63.8, 52.2, 40.5, 28.3, 21.0.

[0098] N-(tert-butyl)-4-(4-methoxybenzyl)-l-oxo-2-phenyl-l,2,3,4-tetrahydropyrazino[l,2- b]indazole-3-carboxamide (Compound 6h)

[0099] 1H-NMR (400MHz, CDCl3) δ8.19(d,J=8.3Hz,1H),7.82(d,J=8.6Hz,1H),7.44(dt,J=19.0,7.9Hz,3H),7.39-7.30(m,4H),6.97-6.85(m ,4H),5.91(s,1H),5.63(dd,J=9.8,4.6Hz,1H),4.41(d,J=0.9Hz,1H),3.46(dd,J=14.0,4.8Hz,1H),3.21-3.13(m,1H),1.10(s,9H). 13 C-NMR (101MHz, CDCl3) δ167.3, 163.4, 160.9, 156.8, 148.8, 140.4, 130.8 (d, J = 3.3Hz), 130.7 (d, J = 8.1Hz), 129 .7,127.1(d,J=3.9Hz),125.5,124.2,123.8,122.4,120.9,118.5,116.2,116.0,65.1,62.4,52.3,39.7,28.4.

[0100] N-(tert-Butyl)-4-(4-methylbenzyl)-1-oxo-2-phenyl-1,2,3,4-tetrahydropyrazino[1,2-b]indazole-3-carboxamide (Compound 6j)

[0101] 1 H-NMR (400MHz, CDCl3) δ8.26-8.19(m,1H),7.86(d,J=8.6Hz,1H),7.52-7.32(m,7H),7.06(d,J=7.8Hz,2H),6.96(d,J=7.9Hz,2H),5.81(s,1H),5 .64(ddd,J=10.8,4.4,1.2Hz,1H),4.43(d,J=1.3Hz,1H),3.51(dd,J=13.8,4.4Hz,1H),3.15(dd,J=13.8,10.9Hz,1H),2.31(s,3H),1.13(s,9H). 13 C-NMR (101MHz, CDCl3) δ167.5,156.8,148.9,140.5,137.4,132.0,129.9,129.6,129. 0,127.0,125.4,124.3,123.8122.5,121.0118.5,64.9,62.6,52.2,40.1,28.4,21.1.

[0102] N-(tert-Butyl)-4-(4-bromobenzyl)-1-oxo-2-phenyl-1,2,3,4-tetrahydropyrazino[1,2-b]indazole-3-carboxamide (Compound 6k)

[0103] 1 H-NMR (400MHz, CDCl3) δ8.22(d,J=8.3Hz,1H),7.85(d,J=8.6Hz,1H),7.53-7.42(m,3H),7.41-7.32(m,6H),6.89(d,J=8.3Hz,2H),5.88(s ,1H),5.67(ddd,J=10.2,4.7,1.1Hz,1H),4.40(d,J=1.2Hz,1H),3.47(dd,J=13.9,4.8Hz,1H),3.18(dd,J=13.9,10.3Hz,1H),1.13(s,9H). 13 C-NMR (101MHz, CDCl3) δ167.2,156.6,148.9,140.4,134.0,132.3,130.7,129.7,127.1 ,127.1,125.6,124.0,123.8,122.5,121.8,120.9,118.5,65.1,62.1,52.4,39.9,28.4.

[0104] 4-Benzyl-N-butyl-1-oxo-2-phenyl-1,2,3,4-tetrahydropyrazino[1,2-b]indazole-3-carboxamide (Compound 61)

[0105] 1 H-NMR(400MHz, CDCl3)δ8.13(d,J=8.4Hz,1H),7.83(d,J=8.7Hz,1H),7.48-7.2 8(m,7H),7.25-7.16(m,3H),7.03-6.91(m,2H),6.27(t,J=5.7Hz,1H),5.72(dd, J=10.6,4.5Hz,1H),4.53(s,1H),3.51(dd,J=13.8,4.5Hz,1H),3.23-3.05(m,3H ),1.29-1.13(m,2H),0.94(ddd,J=11.7,7.2,3.8Hz,2H),0.62(d,J=7.3Hz,3H). 13C-NMR (101 MHz, CDC13) δ 167.2, 156.9, 148.9, 140.5, 135.0, 129.6, 129.2, 129.1, 127.6, 127.1, 127.0, 125.4, 124.2, 124.0, 122.5, 121.0, 118.4, 64.7, 62.5, 49.0, 40.5, 32.5, 32.3, 25.2, 24.5, 24.3.

[0106] 4-benzyl-N-cyclohexyl-1-oxo-2-phenyl-1,2,3,4-tetrahydropyrazino[1,2-b]indazole-3- carboxamide (Compound (6m)

[0107] 1 H-NMR (400 MHz, CDC13) δ 8.16 (d, J = 8.4 Hz, 1H), 7.82 (d, J = 8.7 Hz, 1H), 7.49 - 7.27 (m, 7H), 7.23 - 7.13 (m, 3H), 6.96 (dd, J = 7.4, 1.6 Hz, 2H), 6.09 (d, J = 8.2 Hz, 1H), 5.77 - 5.61 (m, 1H), 4.51 (d, J = 1.1 Hz, 1H), 3.69 - 3.53 (m, 1H), 3.49 (dd, J = 13.8, 4.6 Hz, 1H), 3.19 (dd, J = 13.8, 10.4 Hz, 1H), 1.83 - 1.64 (m, 1H), 1.55 (dd, J = 9.4, 4.1 Hz, 1H), 1.49 - 1.34 (m, 3H), 1.30 - 1.14 (m, 1H), 1.13 - 0.93 (m, 3H), 0.85 - 0.71 (m, 1H). 13 C-NMR (101 MHz, CDC13) δ 167.2, 156.9, 148.9, 140.5, 135.0, 129.6, 129.2, 129.1, 127.6, 127.1, 127.0, 125.4, 124.2, 124.0, 122.5, 121.0, 118.4, 64.7, 62.5, 49.0, 40.5, 32.5, 32.3, 25.2, 24.5, 24.3.

[0108] 4-benzyl-N-cyclohexyl-1-oxo-2-phenyl-1,2,3,4-tetrahydropyrazino[1,2-b]indazole-3- carboxamide (Compound (6m)

[0109] 1H-NMR (400 MHz, CDC13) δ 8.11 (d, J = 8.3 Hz, 1H), 7.87 (d, J = 8.6 Hz, 1H), 7.59 - 7.28 (m, 8H), 7.26 (s, 1H), 7.22 (s, 3H), 7.12 - 7.05 (m, 1H), 7.03 - 6.93 (m, 3H), 6.73 (d, J = 7.2 Hz, 2H), 6.60 (s, 1H), 5.75 (d, J = 6.7 Hz, 1H), 4.58 (s, 1H), 4.41 (dd, J = 14.6, 6.0 Hz, 1H), 4.23 (dd, J = 14.7, 4.8 Hz, 1H), 3.54 (dd, J = 13.8, 3.3 Hz, 1H), 3.26 - 3.12 (m, 1H). 13 C-NMR (101 MHz, CDC13) δ 168.4, 156.8, 148.9, 140.4, 136.8, 134.9, 129.6, 129.2, 129.1, 128.5, 127.7, 127.6, 127.1, 127.0, 125.6, 123.9, 122.7, 121.0, 118.5, 64.8, 62.6, 43.9, 40.4.

[0110] 4-benzyl-N-(2,6-dimethylphenyl)-l-oxo-2-phenyl-l,2,3,4-tetrahydropyrazino[l,2-b]indazole-3-carboxamide (Compound 6o)

[0111] 1 H-NMR (400 MHz, CDC13) δ 8.20 (d, J = 8.2 Hz, 1H), 7.87 (d, J = 8.5 Hz, 1H), 7.64 - 7.48 (m, 5H), 7.48 - 7.43 (m, 1H), 7.38 (t, J = 7.2 Hz, 2H), 7.26 (t, J = 7.4 Hz, 3H), 7.09 - 6.97 (m, 3H), 6.91 (d, J = 7.4 Hz, 2H), 5.85 (dd, J = 10.0, 4.3 Hz, 1H), 4.80 (s, 1H), 3.59 (dd, J = 13.7, 4.4 Hz, 1H), 3.38 - 3.20 (m, 1H), 1.67 (s, 6H). 13C-NMR (101MHz, CDCl3) δ167.0,156.8,148.9,140.4,135.1,134.9,132.4,129.7,129.2,129.2,128 .3,127.9,127.7,127.2,127.0,125.8,123.9,123.7,122.9,120.7,118.5,65.2,62.9,40.5,17.8.

[0112] 4-Benzyl-N-(tert-butyl)-8-chloro-1-oxo-2-phenyl-1,2,3,4-tetrahydropyrazino[1,2-b]indazole-3-carboxamide (Compound 6p)

[0113] 1 H-NMR (400MHz, CDCl3) δ8.15(d,J=8.9Hz,1H),7.84(d,J=1.1Hz,1H),7.53-7.45(m,2H) ,7.36(dd,J=10.5,4.4Hz,3H),7.31(dd,J=8.9,1.7Hz,1H),7.27(dd,J=6.0,3.4Hz,3H) ,7.06(dd,J=6.5,2.8Hz,2H),5.74(s,1H),5.64(ddd,J=10.5,4.6,1.2Hz,1H),4.41(d, J=1.3Hz,1H),3.53(dd,J=13.8,4.6Hz,1H),3.22(dd,J=13.8,10.6Hz,1H),1.14(s,9H). 13 C-NMR (101MHz, CDCl3) δ167.2,156.5,149.0,140.3,134.9,133.1,129.7,129.2,12 9.1,127.8,127.3,126.8,124.4,122.2,120.7,117.5,65.0,62.6,52.3,40.4,28.4.

[0114] 4-Benzyl-9-bromo-N-(tert-butyl)-1-oxo-2-phenyl-1,2,3,4-tetrahydropyrazino[1,2-b]indazole-3-carboxamide (Compound 6q)

[0115] 1H-NMR (400 MHz, CDC13) δ 8.34-8.30 (m, 1H), 7.66-7.60 (m, 1H), 7.44-7.37 (m, 3H), 7.27 (dd, J = 10.7, 4.3 Hz, 3H), 7.18-7.15 (m, 3H), 6.95 (dd, J = 6.5, 2.8 Hz, 2H), 5.69 (s, 1H), 5.58 (ddd, J = 10.6, 4.6, 1.2 Hz, 1H), 4.33 (d, J = 1.3 Hz, 1H), 3.43 (dd, J = 13.8, 4.6 Hz, 1H), 3.11 (dd, J = 13.8, 10.7 Hz, 1H), 1.05 (s, 9H). 13 C-NMR (101 MHz, CDC13) δ 167.1, 156.4, 147.3, 140.3, 134.9, 130.9, 129.7, 129.2, 129.1, 127.8, 127.2, 124.2, 123.4, 123.4, 120.1, 119.5, 64.9, 62.5, 52.4, 40.4, 28.4.

[0116] 4-benzyl-N-(tert-butyl)-9-methyl-1-oxo-2-phenyl-1,2,3,4-tetrahydropyrazino[1,2-b]indazole-3-carboxamide (Compound 6r)

[0117] 1 H-NMR (400 MHz, CDC13) δ 7.98 (s, 1H), 7.75 (d, J = 8.9 Hz, 1H), 7.48 (dd, J = 10.6, 5.1 Hz, 2H), 7.41-7.31 (m, 3H), 7.30-7.22 (m, 4H), 7.04 (dd, J = 6.6, 2.8 Hz, 2H), 5.85 (s, 1H), 5.67 (ddd, J = 10.7, 4.5, 1.3 Hz, 1H), 4.42 (d, J = 1.3 Hz, 1H), 3.52 (dd, J = 13.8, 4.5 Hz, 1H), 3.18 (dd, J = 13.8, 10.7 Hz, 1H), 2.52 (s, 3H), 1.13 (s, 9H). 13 C-NMR (101 MHz, CDC13) δ 167.5, 156.9, 147.8, 140.6, 135.5, 135.2, 129.8, 129.6, 129.2, 129.1, 127.6, 126.9, 124.2, 122.9, 119.3, 118.1, 65.0, 62.3, 52.2, 40.5, 28.4, 22.0.

[0118] Example 4 Anti-inflammatory effect of test compounds in LPS-stimulated RAW264.7 cell model

[0119] Pattern recognition receptors (PRRs) on immune cells, especially TLR4, are able to recognize LPS and other inflammatory stimuli, triggering the production of various inflammatory mediators including nitric oxide (NO), interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Among them, NO is a key inflammatory mediator, and excessive NO can cause cell damage and possibly promote the progression of diseases such as sepsis. This example tests the effect of compounds 6e and 6r on LPS-induced nitric oxide release. The specific operation is as follows:

[0120] RAW 264.7 cells were seeded into 96-well plates at a density of 5.0 x 10 3 cells per well and incubated overnight. The next day, the culture medium was removed, and the cells were treated with 100 μL of LPS (1 μg / mL) for 1 hour before adding the test compounds for continued treatment for 24 hours. This experiment set up a blank group (RAW264.7 cells, DMSO), a model group (RAW 264.7 cells, DMSO, 1 μg / mL LPS), and a drug group (RAW264.7 cells, test compound, 1 μg / mL LPS). The drug group was treated with different concentrations of test compounds (0.46875 μM, 0.9375 μM, 1.875 μM, 3.75 μM, 7.5 μM, 15 μM, 30 μM, and 60 μM), respectively. The nitric oxide concentration in the culture supernatant was determined using a nitric oxide detection kit (S0021S, Biyun Tian Biological Technology). The standard solution was prepared by diluting the test sample solution to the following concentrations: 1.875 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM. 50 μL of the standard and sample were added to the 96-well plate. Subsequently, 50 μL of Griess Reagent I and 50 μL of Griess Reagent II were added to each well. The absorbance (OD value) was measured at 540 nm using a microplate reader. The NO concentration in the sample was determined according to the standard curve.

[0121] The results, as shown in Figure 1 , showed that compounds 6e and 6r exhibited concentration-dependent inhibition of LPS-induced nitric oxide release. Among them, compound 6e had higher inhibitory activity, with a half-inhibitory concentration value (IC 50 ) of 8.55 ± 0.32 μM.

[0122] Example 5 Inhibitory activity of compound 6e on inflammatory cytokine transcription level

[0123] This example tested the effect of compound 6e at 12.5 μM, 25 μM, and 50 μM concentrations on the transcription level of the inflammatory cytokine IL-6 gene in THP-1 cells by quantitative polymerase chain reaction (qPCR). The specific procedures are as follows:

[0124] THP-1 cells in the logarithmic growth phase were seeded in 96-well plates and treated with 100 ng / mL PMA overnight to differentiate into THP-Ms. The next day, the culture medium was replaced and THP-Ms cells were treated with different concentrations of test compounds (12.5 μM, 25 μM and 50 μM) for 18 hours and stimulated with LPS (1 μg / mL) for 6 hours. Total RNA was extracted from THP-Ms using RNAiso Plus (TaKaRa, 9109). The purity and concentration of RNA were determined using a Thermo Scientific NanoDrop spectrophotometer. PrimeScript TM Reverse transcription was performed using the RT Master Mix Kit (Takara, RR036A) to synthesize cDNA from RNA. TaqPro Universal SYBR qPCR MasterMix (Vazyme, Q712-03) and Real-time quantitative PCR (qPCR) was performed using the 480II Real-time PCR System (Roche). The expression level of the relevant gene was calculated as 2-ΔCT. GAPDH was used as an internal reference gene to normalize the expression of the target gene. The target gene primer sequences are as follows:

[0125] IL-6-F 5'-GTAGCCGCCCCACACAGA-3';

[0126] IL-6-R 5'-CATGTCTCCTTTCTCAGGGCTG-3';

[0127] GAPDH-F 5'-AAGGTCCGGAGTCAACGGATTT-3';

[0128] GAPDH-R 5'-AGATGATGACCCTTTTGGCTC-3'.

[0129] The results are as follows Figure 2 As shown: Compound 6e significantly reduced the transcription level of IL-6 in a concentration-dependent manner.

[0130] Example 6 Cytotoxicity Test

[0131] This example tests the cytotoxicity of the compound by CCK-8 assay: THP-1 cells in the logarithmic growth phase were seeded into 96-well plates and stimulated with 100 ng / mL PMA overnight to differentiate into THP-Ms. After removing the old culture medium, the experimental group was added with fresh culture medium containing different concentrations (3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM) of the compound, and the control group was added with fresh culture medium containing the maximum concentration of DMSO in the experimental group. The cells were incubated at 37°C for 24 hours. After the incubation was completed, 10 μL of CCK-8 reagent was added to each well and incubated for another 2 hours at 37°C and 5% CO2. The absorbance (OD value) of each well was measured at a wavelength of 450 nm using a microplate reader (Tecan, Spark). Cell viability was calculated as follows: Cell viability (%) = (OD (化合物处理) -OD 背景 ) / (OD (DMSO处理) -OD 背景 )×100%.

[0132] The results are as follows Figure 3 As shown in the results, compound 6e showed no toxicity in THP-Ms cells, while compound 6r slightly inhibited the proliferation of THP-Ms cells at high concentrations. Overall, the half-inhibitory concentrations of compounds 6e and 6r on cell proliferation exceeded 100 μM, indicating that they are essentially non-toxic to THP-Ms cells.

[0133] From the above experimental data, it can be seen that the 3,4-dihydropyrazino[1,2-b]indazol-1(2H)-one derivatives of the present invention show significant anti-inflammatory effects and have the advantages of low cytotoxicity and good safety, and are expected to become potential drugs for the treatment of inflammation-related diseases.

[0134] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A 3,4-dihydropyrazino[1,2-b]indazol-1(2H)-one derivative having the structure shown in Formula 6, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, in, n is selected from: 1, 2, 3, 4; R1 is selected from the group consisting of: hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy; Each R2 is independently selected from: hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy; R3 is selected from the group consisting of: hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy; R4 is selected from: C1-C6 alkyl, C3-C8 cycloalkyl, C6-C 10 Aryl-substituted C1-C6 alkyl, one or more R1-substituted or unsubstituted C6-C 10 Aryl.

2. The 3,4-dihydropyrazino[1,2-b]indazol-1(2H)-one derivative according to claim 1, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that: R1 is selected from the group consisting of hydrogen, fluorine, chlorine, bromine, methyl, ethyl, propyl, methoxy, ethoxy, and propoxy; and / or, each R2 is independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, methyl, ethyl, propyl, methoxy, ethoxy, and propoxy; and / or, R3 is selected from the group consisting of: hydrogen, fluorine, chlorine, bromine, methyl, ethyl, propyl, methoxy, ethoxy, propoxy; And / or, R4 is selected from the group consisting of: methyl, ethyl, propyl, n-butyl, tert-butyl, isobutyl, cyclohexyl, cyclopentyl, benzyl, and m-xylyl.

3. The 3,4-dihydropyrazino[1,2-b]indazol-1(2H)-one derivative according to claim 1, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein the 3,4-dihydropyrazino[1,2-b]indazol-1(2H)-one derivative is selected from the following compounds:

4. A method for preparing the 3,4-dihydropyrazino[1,2-b]indazol-1(2H)-one derivative according to any one of claims 1 to 3, characterized in that: The steps include: (1) Compound 1, Compound 2, Compound 3 react with Compound 4 to obtain Compound 5; (2) The obtained compound 5 undergoes further intramolecular cyclization reaction under the action of a base to obtain compound 6; The reaction formula is as follows: wherein n, R1, R2, R3, and R4 are as described in any one of claims 1 to 3, respectively.

5. The method for preparing a 3,4-dihydropyrazino[1,2-b]indazol-1(2H)-one derivative according to claim 4, characterized in that: The solvent for the reaction in step (1) is methanol; and / or, the solvent for the reaction in step (2) is selected from at least one of methanol, N,N-dimethylformamide, tetrahydrofuran, 1,4-dioxane, dichloroethane, isopropanol and acetonitrile, preferably methanol; And / or, the base in step (2) is selected from at least one of sodium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, triethylamine, diazabicyclo[5.4.0]undec-7-ene and 4-dimethylaminopyridine, preferably potassium carbonate and / or sodium carbonate.

6. The method for preparing a 3,4-dihydropyrazino[1,2-b]indazol-1(2H)-one derivative according to claim 4 or 5, characterized in that: The molar ratio of compound 1, compound 2, compound 3, compound 4 and base is 1:0.8-1.2:0.8-1.2:0.8-1.2:1.8-2.2; And / or, the ratio of the solvent to compound 1 in step (1) is 1.8 mL-2.2 mL: 1.0 mmol; And / or, the ratio of the solvent in step (2) to compound 1 is 8 mL-12 mL: 1.0 mmol.

7. The method for preparing a 3,4-dihydropyrazino[1,2-b]indazol-1(2H)-one derivative according to claim 4 or 5, characterized in that: The reactions in step (1) and step (2) are both carried out under air atmosphere; And / or, the reaction temperature in step (1) is 20°C-40°C, and the reaction time is 6 hours-16 hours, preferably 10 hours-12 hours; And / or, the reaction temperature in step (2) is 20° C.-40° C., and the reaction time is 4 hours-12 hours, preferably 6 hours-9 hours.

8. The method for preparing a 3,4-dihydropyrazino[1,2-b]indazol-1(2H)-one derivative according to claim 4, characterized in that: The steps include: (1) In an air atmosphere, the compound 1, methanol, and the compound 2 were mixed and stirred for 8-10 minutes, and the compound 3 and the compound 4 were added. The resulting mixture was stirred and reacted at 20°C-40°C for 6-12 hours; (2) adding more methanol to the reaction mixture obtained in step (1), and then adding the base, and continuing to stir the resulting mixture at 20° C.-40° C. for 4 hours-12 hours, and performing post-treatment and purification to obtain the 3,4-dihydropyrazino[1,2-b]indazol-1(2H)-one derivative; In step (1), the ratio of methanol to compound 1 is 1.8 mL-2.2 mL: 1.0 mmol; In step (2), methanol is added to compound 1 in a ratio of 8 mL-12 mL: 1.0 mmol.

9. Use of the 3,4-dihydropyrazino[1,2-b]indazol-1(2H)-one derivative according to any one of claims 1 to 3, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof in the preparation of an anti-inflammatory drug.

10. An anti-inflammatory drug, characterized in that The invention is prepared from an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient comprises the 3,4-dihydropyrazino[1,2-b]indazol-1(2H)-one derivative according to any one of claims 1 to 3, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

Images