CN117865965B - Pyrrolopyrimidine-4-amine and pyrazolopyrimidine-4-amine derivatives and preparation methods and applications thereof - Google Patents

Abstract

The present invention belongs to the technical field of chemical synthesis, and specifically relates to pyrrolopyrimidine-4-amine and pyrazolopyrimidine-4-amine derivatives, preparation methods and applications thereof, and the structural formula thereof is shown in I. The present invention is based on Syringaresinol, a diepoxylignan active compound previously discovered in corn roots, and replaces its parent nucleus skeleton with a bioisostere through a skeleton transition strategy, thereby designing and synthesizing two new pyrrolopyrimidine-4-amine and pyrazolopyrimidine-4-amine derivatives, which are used for the treatment of nervous system diseases caused by oxidative stress injury, excitatory injury and inflammatory injury.

Description

Pyrrolopyrimidine-4-amine and pyrazolopyrimidine-4-amine derivatives, preparation method and application thereof

Technical Field

The invention belongs to the technical field of chemical synthesis, and particularly relates to pyrrolopyrimidine-4-amine and pyrazolopyrimidine-4-amine derivatives, and a preparation method and application thereof.

Background

Oxidative stress injury, excitatory injury and inflammatory injury are the main pathogenesis of neurological diseases leading to stroke, neurodegenerative diseases such as parkinsonism, alzheimer's disease, huntington's dance disorders and the like, as well as psychotic diseases such as depression and the like. Because of the complex pathogenesis of the above diseases, a satisfactory clinical effect cannot be obtained by controlling only one factor. Thus, interventions based on multifactorial, multi-target and multi-signaling pathways can achieve satisfactory clinical efficacy. There has been a great deal of progress made by researchers in the discovery of cerebral protectants that can simultaneously inhibit oxidative stress, excitatory injury, and inflammatory injury. However, the huge therapeutic demands of clinical patients still cannot be met, and development of new brain protecting agents is needed in order to solve the problem of lack of medicines for treating the clinical diseases.

Disclosure of Invention

Aiming at the problems in the background technology, the invention provides a pyrrolopyrimidine-4-amine derivative, a synthesis method and application. The compounds of the invention have greatly improved antioxidant, anti-inflammatory and anti-excitatory injury activities, and have remarkable progress, and the design ideas of the pyrrolopyrimidine-4-amine and pyrazolopyrimidine-4-amine derivatives are shown in figure 1.

In order to achieve the above purpose, the invention adopts the following technical scheme: pyrrolo-pyrimidine-4-amine derivatives, wherein the structural formula of the pyrrolo-pyrimidine-4-amine derivatives is shown as I

Wherein X is C or N; r1 groups are aryl methyl, aryl ethyl ketone or alicyclic heterocyclic substituted ethyl ketone substituent groups; the R2 group is a condensed aromatic heterocyclic substituent.

Further, the R1 group is

R2 is a radical

Further, the structural formulas of the pyrrolopyrimidine-4-amine and pyrazolopyrimidine-4-amine derivatives are as follows:

a second object of the present invention is to provide a process for the synthesis of pyrrolopyrimidin-4-amine and pyrazolopyrimidin-4-amine derivatives, comprising the steps of:

s1, synthesizing an intermediate C1-C19 by taking a compound A1 or A2 and a compound B as substrates, wherein the reaction route is as follows:

the method comprises the following steps: 4.24mmol of compound B is added into a solution of 3.26mmol of compound A1 or A2, 6.52mmol of potassium carbonate and 30ml of acetonitrile, the mixture is heated and refluxed for 3 hours, the mixture is cooled to room temperature, the potassium carbonate is removed by filtration, the solvent is removed in vacuum, petroleum ether and ethyl acetate are used as eluent, and the product is separated and purified by silica gel column chromatography, thus obtaining intermediate compound C1-C19.

S2, synthesizing E1-E24 by using intermediates C1-C19 obtained in S1 and a compound D, wherein the reaction route is as follows:

The method comprises the following steps: under the protection of argon, 0.82mmol of compound C and 1.23mmol of compound D are dissolved in 20ml of absolute ethanol, then 0.13ml 4M HCl in Dioxane is added, the temperature is raised to 95 ℃ for reaction for 4 hours, TLC monitors the completion of the reaction, the reaction is cooled to room temperature, the solvent is removed in vacuum, dichloromethane and methanol are used as eluent, and the product is separated and purified by silica gel column chromatography to obtain the target compounds E1-E24.

The beneficial technical effects of the invention are as follows:

Based on a double epoxy lignin active compound discovered in the early stage in corn roots, the invention carries out bioisostere replacement on the parent nucleus skeleton thereof through skeleton transition, designs and synthesizes a series of novel pyrrolopyrimidine-4-amine and pyrazolopyrimidine-4-amine derivatives, and is used for treating nervous system diseases caused by oxidative stress injury, excitatory injury and inflammatory injury.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.

FIG. 1 is an in vitro anti-inflammatory activity of Compound E24 of example 3 of the present invention. (A) Effect of E24 pretreatment on BV2 cells after LPS treatment. (B) E24 inhibits the release of NO. (C) E24 inhibits mRNA levels of iNOS. (D) E24 inhibits the mRNA level of COX-2. (E) E24 inhibits the mRNA level of TNF- α. (F) E24 inhibits mRNA levels of IL-1β. (G) E24 inhibits the mRNA level of IL-6.

FIG. 2 is the in vivo anti-neuroinflammatory activity of compound E24 of example 3 of the invention, (A) the effect of E24 treatment on the level of Nib's body in the hippocampal region of brain tissue of C57B6/J mice, scale: 100 μm. (B) E24 effects on mRNA levels of iNOS, IL-6, COX2, IL-1β and TNFα. (C-D) Effect of E24 on IL-6 and TNFα levels in mouse brain tissue (D) Effect of E24 on TLR-NFkB signaling pathway-associated protein expression. * P < 0.05, P < 0.01, P < 0.001, P < 0.0001, ns indicates no significant difference.

FIG. 3 is the effect of compound E24 (20 mg/kg) of example 3 of the present invention on the depressive-like behaviour of mice. (A) E24 improved the saccharophilic bias of CUMS mice. (B-C) E24 improved resting time in CUMS mice tail-suspension experiments and forced swim experiments. (D-F) E24 improved the overall distance traveled and prolonged the time in the middle zone in CUMS mouse mine experiments.

FIG. 4 shows the inhibitory activity of Compound E24 of example 3 of the present invention on cerebral damage caused by ischemic stroke. Compound E24 significantly reduced infarct size in brain tissue of MCAO model mice.

Detailed Description

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

Example 1: preparation of intermediate compound C1:

3, 5-Dimethoxybromobenzyl (B1) (947.51 mg,4.24 mmol) was added to a solution of 4-chloro-1H-pyrazolo [3,4-d ] pyrimidine (A1) (503.86 mg,3.26 mmol) and potassium carbonate (901.13 mg,6.52 mmol) in acetonitrile (30 ml), and the reaction was refluxed at elevated temperature for 3 hours (completion of TLC monitoring). Cooling to room temperature, filtering to remove potassium carbonate, removing solvent in vacuo, and subjecting the product to silica gel column chromatography using petroleum ether: ethyl acetate=10:1 as eluent to give 4-chloro-1- (4-chloro-2-fluorobenzyl) -1H-pyrazolo [3,4-d ] pyrimidine (C1) as a white solid (894.22 mg,2.71mmol, 83% yield), ESI-MS: m/z 298.1[ m+h ] ⁺.

The preparation of intermediate compounds C2-C19 is identical to C1:

the compound C2 is obtained, and the structure is as follows: Namely, 4-chloro-1- (2-chloro-3-fluorobenzyl) -1H-pyrazolo [3,4-d ] pyrimidine is obtained as a white solid in a yield of 81% and ESI-MS: m/z 298.1[ M+H ] ⁺.

The compound C3 is obtained, and the structure is as follows: Namely, 4-chloro-1- (3, 5-dichlorobenzyl) -1H-pyrazolo [3,4-d ] pyrimidine is obtained as a white solid with a yield of 84% and an ESI-MS: m/z 314.1[ M+H ] ⁺.

The compound C4 is obtained, and the structure is as follows: namely, 4-chloro-1- (4-fluorobenzyl) -1H-pyrazolo [3,4-d ] pyrimidine as a white solid in 87% yield as ESI-MS: m/z 263.7[ M+H ] ⁺.

The compound C5 is obtained, and the structure is as follows: Namely, 4-chloro-7- (3, 5-dimethoxy benzyl) -7H-pyrrole [2,3-d ] pyrimidine, which is white solid with the yield of 94 percent, ESI-MS is m/z 304.8[ M+H ] ⁺;

The compound C6 is obtained, and the structure is as follows: Namely, 4-chloro-7- (4-methoxybenzyl) -7H-pyrrolo [2,3-d ] pyrimidine, a white solid, with a yield of 89%, ESI-MS: m/z 274.4[ M+H ] ⁺.

The compound C7 is obtained, and the structure is as follows: namely, 4-chloro-7- (3-methoxybenzyl) -7H-pyrrolo [2,3-d ] pyrimidine, a white solid with a yield of 87%, ESI-MS: m/z 274.3[ M+H ] ⁺.

The compound C8 is obtained, and the structure is as follows: Namely, 7 benzyl-4-chloro-7H-pyrrolo [2,3-d ] pyrimidine, a white solid, with a yield of 81%, ESI-MS: m/z 244.8[ M+H ] ⁺.

The compound C9 is obtained, and the structure is as follows: Namely, 2- (4-chloro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) -1-acetophenone-1-one, the yield was 71% and ESI-MS: m/z 272.8[ M+H ] ⁺.

The compound C10 is obtained, and the structure is as follows: i.e., 4-chloro-7- (2-chloro-4-fluorobenzyl) -7H-pyrrolo [2,3-d ] pyrimidine, in 92% yield as a white solid, ESI-MS: m/z 297.1[ M+H ] ⁺.

The compound C11 is obtained, and the structure is as follows: namely, 4-chloro-7- (4-chlorobenzyl) -7H-pyrrolo [2,3-d ] pyrimidine, a white solid, with a yield of 78%, ESI-MS: m/z 279.1[ M+H ] ⁺.

The compound C12 is obtained, and the structure is as follows: I.e., 8- ((4-chloro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) methyl) quinoline, as a white solid in 74% yield, ESI-MS: m/z 295.8[ M+H ] ⁺.

The compound C13 is obtained, and the structure is as follows: Namely, 4-chloro-7- (2, 5-difluorobenzyl) -7H-pyrrolo [2,3-d ] pyrimidine, a white solid with a yield of 85%, ESI-MS: m/z 280.7[ M+H ] ⁺.

The compound C14 is obtained, and the structure is as follows: I.e., 4-chloro-7- (3-chloro-5-fluorobenzyl) -7H-pyrrolo [2,3-d ] pyrimidine, in 87% yield as a white solid, ESI-MS: m/z 297.1[ M+H ] ⁺.

The compound C15 is obtained, and the structure is as follows: Namely, the white solid of 4-chloro-7- (4-chloro-3-fluorobenzyl) -7H-pyrrolo [2,3-d ] pyrimidine has a yield of 81%, and ESI-MS: m/z 297.5[ M+H ] ⁺.

The compound C16 is obtained, and the structure is as follows: Namely, the yield of the 4-chloro-7- (2, 4-difluorobenzyl) -7H-pyrrolo [2,3-d ] pyrimidine is 86%, and the yield of ESI-MS is m/z 280.7[ M+H ] ⁺.

The compound C17 is obtained, and the structure is as follows: Namely 7- (benzo [ d ] [1,3] dioxole-5-methyl) -4-chloro-7H-pyrrolo [2,3-d ] pyrimidine, a white solid, in 76% yield, ESI-MS: m/z 288.7[ M+H ] ⁺.

The compound C18 is obtained, and the structure is as follows: Namely, 2- (4-chloro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) -1-morpholinoethyl-1-one, the yield was 74% and ESI-MS: m/z 281.7[ M+H ] ⁺.

The compound C19 is obtained, and the structure is as follows: namely, 4-chloro-7- (4-fluorobenzyl) -7H-pyrrolo [2,3-d ] pyrimidine, a white solid, in 87% yield, ESI-MS: m/z 262.8[ M+H ] ⁺.

Example 2: synthesis of pyrrolopyrimidin-4-amine and pyrazolopyrimidin-4-amine derivatives:

A total of 24 target compounds E1-E24 were obtained by synthesis of pyrrolopyrimidin-4-amine and pyrazolopyrimidin-4-amine derivatives; the 24 target compounds are all novel compounds and are synthesized by ¹ H-NMR spectrum, ¹³ C-NMR spectrum and HR-MS, and the synthesis method, physical properties, melting point and spectrum data of the 24 target compounds are as follows:

(1) Synthesis of the target compounds E1-E24:

4-chloro-1- (4-chloro-2-fluorobenzyl) -1H-pyrazolo [3,4-D ] pyrimidine (C1) (243.63 mg,0.82 mmol) and 5-aminobenzimidazolone (D1) (183.45 mg,1.23 mmol) were dissolved in absolute ethanol under argon, then 0.13ml 4M HCl in Dioxane was added and the temperature was raised to 95℃for 4 hours. TLC monitored completion of the reaction, cooled to room temperature, solvent removed in vacuo, and the product purified by silica gel column chromatography with eluent: dichloromethane: methanol=30:1 to give 5- ((1- (3-chloro-5-fluorobenzyl) -1H-pyrazolo [3,4-d ] pyrimidin-4-yl) amino) 1, 3-dihydro-2H-benzo [ d ] imidazol-2-one (E1) as a white solid (299.16 mg,0.73mmol, 89% yield).

Target Compound E2-E24 1, 3-dihydro-2H-benzo [ d ] imidazol-2-one (E1) according to step 5- ((1- (3-chloro-5-fluorobenzyl) -1H-pyrazolo [3,4-d ] pyrimidin-4-yl) amino) as described in E1

White solid, 89% yield, melting point ：342-345℃.¹H NMR(600MHz,DMSO)δ10.65(s,1H),10.60(s,1H),10.02(s,1H),8.72–8.05(m,2H),7.60(s,1H),7.35(dt,J＝8.8,2.2Hz,1H),7.22(s,1H),7.16–7.10(m,1H),7.07–7.00(m,1H),6.94(d,J＝8.3Hz,1H),5.58(s,2H).¹³C NMR(151MHz,DMSO)δ161.83(162.66,161.01,d,J＝248.7Hz),155.36,155.31,154.98,153.19,141.13(141.16,141.10,d,J＝8.3Hz),133.82(133.86,133.78,d,J＝10.9Hz),132.34,131.82,129.69,126.62,123.39(123.40,123.38,d,J＝3.1Hz),115.08,114.73(114.82,114.65,d,J＝25.0Hz),113.06(113.14,112.99,d,J＝22.2Hz),108.00,103.84,100.46,48.60.¹⁹F NMR(565MHz,DMSO)δ-110.27.HRMS(ESI):calcd for C₁₉H₁₃N₇OClFNa[M+Na]⁺m/z,432.0746;found,432.0742.

5- ((1- (2-Chloro-3-fluorobenzyl) -1H-pyrazolo [3,4-d ] pyrimidin-4-yl) amino) 1, 3-dihydro-2H-benzo [ d ] imidazol-2-one (E2)

White solid, 89% yield, melting point ：378-381℃.¹H NMR(600MHz,DMSO)δ10.66(s,1H),10.61(s,1H),10.02(s,1H),8.72–8.07(m,2H),7.62(s,1H),7.41–7.16(m,3H),6.95(d,J＝8.3Hz,1H),6.82(d,J＝7.7Hz,1H),5.65(s,2H).¹³C NMR(151MHz,DMSO)δ157.26(158.07,156.44,d,J＝246.6Hz),155.30,155.20,154.91,153.35,136.57,132.33,131.79,129.70,127.96(127.98,127.93,d,J＝8.1Hz),126.62,124.86(124.87,124.85,d,J＝3.2Hz),118.76(118.82,118.70,d,J＝18.1Hz),115.46(115.53,115.39,d,J＝21.2Hz),115.05,108.00,103.80,100.35,46.79(46.80,46.78,d,J＝3.2Hz).¹⁹F NMR(565MHz,DMSO)δ-114.96.HRMS(ESI):calcd for C₁₉H₁₃N₇OClFNa[M+Na]⁺m/z,432.0746;found,432.0741.

5- ((1- (3, 5-Dichlorobenzyl) -1H-pyrazolo [3,4-d ] pyrimidin-4-yl) amino) 1, 3-dihydro-2H-benzo [ d ] imidazol-2-one (E3)

White solid, 91% yield, melting point ：357-360℃.¹H NMR(600MHz,DMSO)δ10.66(s,1H),10.61(s,1H),10.03(s,1H),8.72–8.00(m,2H),7.91–7.52(m,1H),7.52–7.48(m,1H),7.25(d,J＝1.9Hz,2H),7.23(s,1H),6.95(d,J＝8.3Hz,1H),5.56(s,2H).¹³C NMR(151MHz,DMSO)δ155.32,155.27,154.93,153.15,140.79,133.87,132.35,131.77,129.68,126.87,126.61,125.99,115.02,107.95,103.79,100.44,48.47.HRMS(ESI):calcd for C₁₉H₁₃N₇OCl₂Na[M+Na]⁺m/z,448.0451;found,448.0443.

5- ((1- (4-Fluorobenzyl) -1H-pyrazolo [3,4-d ] pyrimidin-4-yl) amino) 1, 3-dihydro-2H-benzo [ d ] imidazol-2-one (E4)

White solid, 89% yield, melting point ：339-342℃.¹H NMR(600MHz,DMSO)δ10.65(s,1H),10.60(s,1H),10.01(s,1H),8.70–8.03(m,2H),7.58(s,1H),7.30-7.37(m,1H),7.28(s,1H),7.14–7.08(m,1H),7.14–7.05(m,2H),6.92(d,J＝8.3Hz,1H),5.54(s,2H).¹³C NMR(151MHz,DMSO)δ161.56(161.96,160.35,d,J＝243.3Hz),155.37,154.09,151.01,149.31,134.04(134.10,134.08,d,J＝3.1Hz),134.31,129.81,129.12(129.15,129.09,d,J＝8.3Hz),125.12,125.03,114.99(115.06,114.92,d,J＝21.3Hz),113.29,107.56,104.31,100.53,47.79.¹⁹F NMR(565MHz,DMSO)δ-107.23.HRMS(ESI):calcd for C₁₉H₁₆N₇OF[M+H]⁺m/z,377.1244;found,376.1243.

Synthesis of 5- ((7- (3, 5-dimethoxybenzyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -1, 3-dihydro-2H-benzo [ d ] imidazol-2-one (E5)

White solid, yield 81%, melting point ：255-258℃.¹H NMR(600MHz,DMSO)δ10.57(s,1H),10.49(s,1H),9.26(s,1H),8.27(s,1H),7.65(d,J＝2.0Hz,1H),7.32(d,J＝3.5Hz,1H),7.25(dd,J＝8.4,2.0Hz,1H),6.89(d,J＝8.3Hz,1H),6.74(d,J＝3.5Hz,1H),6.39(t,J＝2.3Hz,1H),6.37(d,J＝2.2Hz,2H),5.30(s,2H),3.68(s,6H).¹³C NMR(151MHz,DMSO)δ160.64,155.65,153.97,151.11,149.62,140.55,133.60,129.69,125.38,125.14,113.81,108.22,105.51,103.42,102.67,98.93,98.73,55.18,47.17.HRMS(ESI):calcd for C₂₂H₂₁N₆O₃[M+H]⁺m/z,417.1670;found,417.1658.

5- ((7- (4-Methoxybenzyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -1, 3-dihydro-2H-benzo [ d ] imidazol-2-one (E6)

White solid, 84% yield, melting point ：305-308℃.¹H NMR(600MHz,DMSO)δ10.57(s,1H),10.49(s,1H),9.23(s,1H),8.28(s,1H),7.67(d,J＝2.1Hz,1H),7.29(d,J＝3.5Hz,1H),7.26(dd,J＝8.4,2.1Hz,1H),7.24–7.19(m,2H),6.89(d,J＝8.3Hz,1H),6.87–6.84(m,2H),6.73(d,J＝3.4Hz,1H),5.30(s,2H),3.70(s,3H).¹³CNMR(151MHz,DMSO)δ158.62,155.60,153.90,151.02,149.46,133.63,130.16,129.66,128.90,125.29,124.78,113.92,113.67,108.14,103.44,102.54,98.82,55.06,46.62.HRMS(ESI):calcd for C₂₁H₁₈N₆O₂[M+H]⁺m/z,387.1564;found,387.1552.

5- ((7- (3-Methoxybenzyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -1, 3-dihydro-2H-benzo [ d ] imidazol-2-one (E7)

White solid, 82% yield, melting point ：268-271℃.¹H NMR(600MHz,DMSO)δ10.57(s,1H),10.50(s,1H),9.25(s,1H),8.28(s,1H),7.67(d,J＝2.0Hz,1H),7.32(d,J＝3.5Hz,1H),7.26(dd,J＝8.4,2.1Hz,1H),7.24–7.18(m,1H),6.89(d,J＝8.3Hz,1H),6.84–6.81(m,2H),6.79–6.75(m,1H),6.76(d,J＝3.4Hz,1H),5.35(s,2H),3.70(s,3H).¹³C NMR(151MHz,DMSO)δ159.36,155.60,153.94,151.10,149.59,139.78,133.60,129.69,129.67,125.32,125.02,119.40,113.68,113.25,112.52,108.15,103.41,102.56,98.91,55.00,47.05.HRMS(ESI):calcd for C₂₁H₁₈N₆O₂[M+H]⁺m/z,387.1564;found,387.1552.

5- ((7-Benzyl-7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -1, 3-dihydro-2H-benzo [ d ] imidazol-2-one (E8)

White solid, yield 87%, melting point ：315-318℃.¹H NMR(600MHz,DMSO)δ10.58(s,1H),10.50(s,1H),9.26(s,1H),8.28(s,1H),7.67(d,J＝2.1Hz,1H),7.34–7.28(m,3H),7.28–7.20(m,4H),6.90(d,J＝8.3Hz,1H),6.76(d,J＝3.6Hz,1H),5.38(s,2H).¹³C NMR(151MHz,DMSO)δ155.61,153.95,151.11,149.59,138.23,133.60,129.67,128.55,127.39,127.30,125.33,124.98,113.71,108.16,103.43,102.58,98.94,47.14.HRMS(ESI):calcd for C₂₀H₁₇N₆O[M+H]⁺m/z,357.1458;found,357.1448;C₂₀H₁₆N₆ONa[M+Na]⁺m/z,379.1278;found,379.1265.

5- ((7- (2-Oxo-2-phenylethyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) 1, 3-dihydro-2H-benzo [ d ] imidazol-2-one (E9)

White solid, 71% yield, melting point ：340-343℃.¹H NMR(600MHz,DMSO)δ10.58(s,1H),10.50(s,1H),9.29(s,1H),8.20(s,1H),8.13–8.08(m,2H),7.75–7.70(m,1H),7.66(d,J＝2.0Hz,1H),7.63-7.58(m,2H),7.30–7.22(m,2H),6.90(d,J＝8.3Hz,1H),6.80–6.73(m,1H),5.83(s,2H).¹³C NMR(151MHz,DMSO)δ193.81,155.64,153.93,150.87,150.10,134.52,134.04,133.63,129.71,129.03,128.05,126.22,125.38,113.83,108.23,103.41,102.69,98.66,50.70.HRMS(ESI):calcd for C₂₁H₁₆N₆O₂Na[M+Na]⁺m/z,407.1227;found,407.1225.

5- ((7- (2-Chloro-4-fluorobenzyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -1, 3-dihydro-2H-benzo [ d ] imidazol-2-one (E10)

White solid, yield 85%, melting point ：329-332℃.¹H NMR(600MHz,DMSO)δ10.57(s,1H),10.50(s,1H),9.30(s,1H),8.25(s,1H),7.66(d,J＝2.0Hz,1H),7.50(dd,J＝8.7,2.7Hz,1H),7.31–7.24(m,2H),7.18–7.11(m,1H),6.90(d,J＝8.3Hz,1H),6.86(dd,J＝8.7,6.1Hz,1H),6.83–6.77(m,1H),5.45(s,2H).¹³C NMR(151MHz,DMSO)δ161.25(162.07,160.43,d,J＝247.5Hz),155.60,154.01,151.25,149.72,133.52,132.52(132.55,132.48,d,J＝10.6Hz),131.77(131.78,131.76,d,J＝3.5Hz),130.31(130.34,130.28,d,J＝9.2Hz),129.67,125.38,124.99,116.75(116.84,116.67,d,J＝25.4Hz),114.67(114.74,114.60,d,J＝21.2Hz),113.74,108.17,103.47,102.61,99.29,44.53.¹⁹F NMR(565MHz,DMSO)δ-112.69.HRMS(ESI):calcd for C₂₀H₁₅N₆OClF[M+H]⁺m/z,409.0974;found,409.0963.

5- ((7- (4-Chlorobenzyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -1, 3-dihydro-2H-benzo [ d ] imidazol-2-one (E11)

White solid, 86% yield, melting point ：278-281℃.¹H NMR(600MHz,DMSO)δ10.57(s,1H),10.50(s,1H),9.27(s,1H),8.26(s,1H),7.64(d,J＝2.0Hz,1H),7.40–7.35(m,2H),7.33(d,J＝3.5Hz,1H),7.27–7.21(m,3H),6.89(d,J＝8.3Hz,1H),6.75(d,J＝3.4Hz,1H),5.37(s,2H).¹³C NMR(151MHz,DMSO)δ155.65,154.02,151.21,149.58,137.29,133.57,132.08,129.70,129.24,128.59,125.41,124.99,113.86,108.24,103.49,102.72,99.13,46.52.HRMS(ESI):calcd for C₂₀H₁₆N₆OCl[M+H]⁺m/z,391.1069;found,391.1055.

7- (3, 5-Dimethoxybenzyl) -N- (1H-indazol-6-yl) -7H-pyrrolo [2,3-d ] pyrimidin-4-amine (E12)

White solid, yield 85%, melting point ：305-308℃.¹H NMR(600MHz,DMSO)δ12.88(s,1H),9.51(s,1H),8.51(s,1H),8.40(s,1H),7.97(s,1H),7.68(d,J＝8.6Hz,1H),7.43–7.38(m,2H),6.91(d,J＝3.5Hz,1H),6.39(s,3H),5.33(s,2H),3.68(s,6H).¹³C NMR(151MHz,DMSO)δ160.67,153.75,150.99,149.76,140.68,140.48,138.56,133.34,125.67,120.28,118.60,115.55,105.54,104.09,99.64,98.92,98.78,55.19,47.24.HRMS(ESI):calcd for C₂₂H₂₁N₆O₂[M+H]⁺m/z,401.1721;found,401.1711;C₂₂H₂₀N₆O₂Na[M+Na]⁺m/z,423.1540;found,423.1527.

N- (7- (3, 5-Dimethoxybenzyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) benzo [ d ] thiazol-5-amine (E13)

White solid, yield 83%, melting point ：189-192℃.¹HNMR(600MHz,DMSO)δ9.63(s,1H),9.36(s,1H),8.83(d,J＝2.0Hz,1H),8.40(s,1H),8.08(d,J＝8.7Hz,1H),7.91(dd,J＝8.7,2.1Hz,1H),7.41(d,J＝3.5Hz,1H),6.89(d,J＝3.5Hz,1H),6.39(s,3H),5.34(s,2H),3.68(s,6H).¹³C NMR(151MHz,DMSO)δ160.66,156.67,153.80,153.66,151.01,149.83,140.47,139.00,126.88,125.77,121.98,119.45,113.80,105.53,103.99,98.83,98.79,55.19,47.25.HRMS(ESI):calcd for C₂₂H₂₀N₅O₂S[M+H]⁺m/z,418.1332;found,418.1321.

N- (benzo [ d ] [1,3] dioxol-5-yl) -7- (3, 5-dimethoxybenzyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-amine (E14)

White solid, 89% yield, melting point ：156-159℃.¹HNMR(600MHz,DMSO)δ9.29(s,1H),8.28(s,1H),7.56(d,J＝2.1Hz,1H),7.33(d,J＝3.5Hz,1H),7.18(dd,J＝8.4,2.1Hz,1H),6.89(d,J＝8.3Hz,1H),6.75(d,J＝3.5Hz,1H),6.40–6.38(m,1H),6.38–6.36(m 2H),6.00(s,2H),5.30(s,2H),3.67(s,6H).¹³C NMR(151MHz,DMSO)δ160.65,153.84,151.08,149.71,147.01,142.58,140.53,134.47,125.34,113.67,107.93,105.51,103.49,103.32,100.94,98.82,98.74,55.19,47.19.HRMS(ESI):calcd for C₂₂H₂₁N₄O₄[M+H]⁺m/z,405.1557;found,405.1546.

5- ((7- (Quinolin-8-ylmethyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -1, 3-dihydro-2H-benzo [ d ] imidazol-2-one (E15)

White solid, 65% yield, melting point ：288-291℃.¹H NMR(600MHz,DMSO)δ10.58(s,1H),10.51(s,1H),9.28(s,1H),9.03(dd,J＝4.2,1.8Hz,1H),8.41(dd,J＝8.3,1.8Hz,1H),8.24(s,1H),7.90(dd,J＝8.2,1.4Hz,1H),7.70–7.65(m,1H),7.62(dd,J＝8.3,4.2Hz,1H),7.49–7.45(m,1H),7.40(d,J＝3.5Hz,1H),7.27(dd,J＝8.4,2.0Hz,1H),7.05(dd,J＝7.1,1.4Hz,1H),6.90(d,J＝8.3Hz,1H),6.79(d,J＝3.4Hz,1H),6.03(s,2H).¹³C NMR(151MHz,DMSO)δ155.69,154.07,151.16,150.22,149.91,145.16,136.57,135.88,133.68,129.72,127.88,127.67,127.15,126.42,125.77,125.39,121.87,113.85,108.27,103.55,102.71,98.87,43.82.HRMS(ESI):calcd for C₂₃H₁₈N₇O[M+H]⁺m/z,408.1567;found,408.1556;C₂₃H₁₇N₇ONa[M+Na]⁺m/z,430.1387;found,430.1373.

5- ((7- (2, 5-Difluorobenzyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -1, 3-dihydro-2H-benzo [ d ] imidazol-2-one (E16)

White solid, 84% yield, melting point ：355-558℃.¹H NMR(600MHz,DMSO)δ10.58(s,1H),10.51(s,1H),9.32(s,1H),8.27(s,1H),7.66(d,J＝2.0Hz,1H),7.33–7.25(m,3H),7.21–7.15(m,1H),6.89(d,J＝8.3Hz,1H),6.85–6.79(m,2H),5.44(s,2H).¹³C NMR(151MHz,DMSO)δ158.03(158.84,158.82,157.24,157.23,dd,J＝240.4,1.8Hz),155.91(156.72,156.70,155.12,155.10,dd,J＝241.8,2.3Hz),155.59,153.98,151.25,149.61,133.50,129.66,126.89(126.97,126.92,126.86,126.80,dd,J＝17.7,7.7Hz),125.38,124.92,117.07(117.18,117.12,117.02,116.96,dd,J＝24.2,8.7Hz),116.05(116.15,116.10,115.99,115.94,dd,J＝23.7,8.4Hz),115.84(115.94,115.91,115.77,115.74,dd,J＝24.7,4.2Hz),113.76,108.15,103.43,102.62,99.34,41.09(41.10,41.07,d,J＝3.7Hz).¹⁹F NMR(565MHz,DMSO)δ-118.31,-118.34,-123.56,-123.59.HRMS(ESI):calcd for C₂₀H₁₅N₆OF₂[M+H]⁺m/z,393.1270;found,393.1258.

6- ((7- (2-Chloro-4-fluorobenzyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) benzo [ d ] oxazol-2 (3H) -one (E17)

White solid, 91% yield, melting point ：262-265℃.¹HNMR(600MHz,DMSO)δ11.59(s,1H),9.49(s,1H),8.31(s,1H),7.93(d,J＝2.2Hz,1H),7.50(dd,J＝8.7,2.7Hz,1H),7.42(dd,J＝8.7,2.2Hz,1H),7.32(d,J＝3.5Hz,1H),7.24(d,J＝8.7Hz,1H),7.17–7.12(m,1H),6.91–6.86(m,2H),5.46(s,2H).¹³C NMR(151MHz,DMSO)δ161.27(162.09,160.45,d,J＝247.5Hz),154.81,153.65,151.05,149.79,138.64,136.43,132.56(132.59,132.52,d,J＝10.6Hz),131.68(131.69,131.67,d,J＝3.2Hz),130.35(130.38,130.32,d,J＝9.1Hz),130.27,125.42,116.77(116.85,116.68,d,J＝25.1Hz),114.68(114.75,114.61,d,J＝21.2Hz),113.75,109.29,103.76,102.67,99.22,44.57.¹⁹F NMR(565MHz,DMSO)δ-112.64.HRMS(ESI):calcd for C₂₀H₁₄N₅O₂ClF[M+H]⁺m/z,410.0815;found,410.0812.

N- (benzo [ d ] [1,3] dioxol-5-yl) -7- (2-chloro-4-fluorobenzyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-amine (E18)

White solid, 93% yield, melting point ：196-199℃.¹HNMR(600MHz,DMSO)δ9.33(s,1H),8.27(s,1H),7.57(d,J＝2.1Hz,1H),7.50(dd,J＝8.8,2.7Hz,1H),7.29(d,J＝3.5Hz,1H),7.18(dd,J＝8.4,2.2Hz,1H),7.16–7.12(m,1H),6.90(d,J＝8.3Hz,1H),6.86(dd,J＝8.7,6.1Hz,1H),6.81(d,J＝3.5Hz,1H),6.00(s,2H),5.45(s,2H).¹³C NMR(151MHz,DMSO)δ161.25(162.07,160.43,d,J＝247.4Hz),153.85,151.18,149.78,146.96,142.58,134.35,132.52(132.55,132.48,d,J＝10.6Hz),131.72(131.73,131.71,d,J＝3.3Hz),130.30(130.33,130.27,d,J＝9.2Hz),125.16,116.74(116.83,116.66,d,J＝25.2Hz),114.66(114.73,114.59,d,J＝21.2Hz),113.66,107.87,103.50,103.31,100.90,99.16,44.53.¹⁹F NMR(565MHz,DMSO)δ-112.68.HRMS(ESI):calcd for C₂₀H₁₅N₄O₂ClF[M+H]⁺m/z,397.0862;found,397.0859.

5- ((7- (3-Chloro-5-fluorobenzyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -1, 3-dihydro-2H-benzo [ d ] imidazol-2-one (E19)

White solid, 89% yield, melting point ：329-332℃.¹H NMR(600MHz,DMSO)δ10.58(s,1H),10.50(s,1H),9.30(s,1H),8.28(s,1H),7.65(s,1H),7.38(d,J＝3.5Hz,1H),7.36–7.30(m,1H),7.26(dd,J＝8.4,2.0Hz,1H),7.13(s,1H),7.05(d,J＝9.3Hz,1H),6.90(d,J＝8.3Hz,1H),6.78(s,1H),5.41(s,2H).¹³C NMR(151MHz,DMSO)δ162.15(162.97,161.33,d,J＝248.3Hz),155.61,154.02,151.32,149.56,142.74(142.77,142.72,d,J＝8.1Hz),134.07(134.11,134.03,d,J＝11.0Hz),133.49,129.68,125.42,124.97,123.46(123.47,123.45,d,J＝3.2Hz),115.05(115.14,114.97,d,J＝25.3Hz),113.80,113.21(113.28,113.14,d,J＝22.0Hz),108.17,103.49,102.66,99.33,46.25.¹⁹F NMR(565MHz,DMSO)δ-110.36.HRMS(ESI):calcd for C₂₀H₁₅N₆ClF[M+H]⁺m/z,409.0977;found,409.0971.

5- ((7- (4-Chloro-3-fluorobenzyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -1, 3-dihydro-2H-benzo [ d ] imidazol-2-one (E20)

White solid, yield 85%, melting point ：318-321℃.¹H NMR(600MHz,DMSO)δ10.58(s,1H),10.50(s,1H),9.30(s,1H),8.27(s,1H),7.65(d,J＝2.0Hz,1H),7.54–7.51(m,1H),7.36(d,J＝3.5Hz,1H),7.28(dd,J＝10.2,2.0Hz,1H),7.25(dd,J＝8.4,2.1Hz,1H),7.04(dd,J＝8.2,2.3Hz,1H),6.89(d,J＝8.4Hz,1H),6.77(d,J＝3.5Hz,1H),5.40(s,2H).¹³C NMR(151MHz,DMSO)δ157.07(157.89,156.25,d,J＝247.0Hz),155.60,153.96,151.20,149.53,139.97(140.00,139.95,d,J＝6.3Hz),133.47,130.84,129.67,125.41,124.93,124.53(124.54,124.52,d,J＝3.2Hz),118.40(118.46,118.34,d,J＝17.5Hz),115.74(115.81,115.67,d,J＝21.2Hz),113.80,108.17,103.48,102.66,99.26,46.21.¹⁹F NMR(565MHz,DMSO)δ-116.02.HRMS(ESI):calcd for C₂₀H₁₅N₆ClF[M+H]⁺m/z,409.0977;found,409.0968.

5- ((7- (2, 4-Difluorobenzyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -1, 3-dihydro-2H-benzo [ d ] imidazol-2-one (E21)

White solid, 86% yield, melting point ：330-233℃.¹H NMR(600MHz,DMSO)δ10.57(s,1H),10.50(s,1H),9.27(s,1H),8.27(s,1H),7.66(d,J＝2.2Hz,1H),7.28–7.26(m,2H),7.25(d,J＝2.2Hz,1H),7.18–7.12(m,1H),7.05–7.00(m,1H),6.89(d,J＝8.3Hz,1H),6.77(d,J＝3.5Hz,1H),5.41(s,2H).¹³C NMR(151MHz,DMSO)δ161.78(162.64,162.56,161.01,160.93,dd,J＝246.2,12.2Hz),159.89(160.75,160.67,159.11,159.03,dd,J＝248.1,12.4Hz),155.60,153.97,151.20,149.61,133.54,131.01(131.06,131.02,130.99,130.96,dd,J＝9.9,5.6Hz),129.67,125.37,124.86,121.27(121.33,121.31,121.23,121.21,dd,J＝15.0,3.7Hz),113.74,111.67(111.76,111.73,111.61,111.59,dd,J＝21.5,3.7Hz),108.16,104.01(104.18,104.01,103.84,t,J＝25.7Hz),103.43,102.61,99.19,40.76(40.77,40.75,d,J＝3.6Hz).¹⁹FNMR(565MHz,DMSO)δ-110.80,-110.82,-113.73,-113.75.HRMS(ESI):calcd for C₂₀H₁₅N₆OF₂[M+H]⁺m/z,393.1270;found,393.1268.

5- ((7-Benzo [ d ] [1,3] dioxol-5-methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -1, 3-dihydro-2H-benzo [ d ] imidazol-2-one (E22)

White solid, yield 81%, melting point ：324-327℃.1H NMR(600MHz,DMSO)δ10.57(s,1H),10.50(s,1H),9.24(s,1H),8.29(s,1H),7.67(d,J＝2.0Hz,1H),7.31(d,J＝3.5Hz,1H),7.26(dd,J＝8.4,2.0Hz,1H),6.89(d,J＝8.3Hz,1H),6.87(d,J＝1.7Hz,1H),6.84(d,J＝7.9Hz,1H),6.77(dd,J＝8.0,1.7Hz,1H),6.73(d,J＝3.5Hz,1H),5.96(s,2H),5.27(s,2H).13C NMR(151MHz,DMSO)δ155.62,153.95,151.10,149.47,147.35,146.59,133.63,131.99,129.68,125.32,124.80,121.00,113.71,108.25,108.17,108.11,103.46,102.58,101.01,98.88,46.94.HRMS(ESI):calcd for C21H17N6O3[M+H]+m/z,401.1357;found,401.1356.

5- ((7- (2-Morpholinyl-2-oxoethyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) 1, 3-dihydro-2H-benzo [ d ] imidazol-2-one (E23)

White solid, 75% yield, melting point ：322-325℃.¹H NMR(600MHz,DMSO)δ10.57(s,1H),10.50(s,1H),9.22(s,1H),8.22(s,1H),7.66(d,J＝2.0Hz,1H),7.26(dd,J＝8.4,2.0Hz,1H),7.16(d,J＝3.5Hz,1H),6.89(d,J＝8.3Hz,1H),6.70(d,J＝3.5Hz,1H),5.12(s,2H),3.65(t,J＝4.7Hz,2H),3.58(q,J＝4.6Hz,4H),3.43(t,J＝4.8Hz,2H).¹³C NMR(151MHz,DMSO)δ165.96,155.63,153.89,150.77,150.07,133.69,129.69,126.31,125.31,113.75,108.19,103.29,102.61,98.36,66.05,65.98,45.15,44.70,41.85.HRMS(ESI):calcd for C₁₉H₁₉N₇O₃Na[M+Na]⁺m/z,416.1442;found,401.1441.

5- ((7- (4-Fluorobenzyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -1, 3-dihydro-2H-benzo [ d ] imidazol-2-one (E24)

White solid, 86% yield, melting point ：321-324℃.¹H NMR(600MHz,DMSO)δ10.58(s,1H),10.50(s,1H),9.26(s,1H),8.27(s,1H),7.64(d,J＝2.0Hz,1H),7.32(d,J＝3.5Hz,1H),7.32–7.26(m,3H),7.25(dd,J＝8.4,2.0Hz,1H),7.17–7.10(m,2H),6.89(d,J＝8.3Hz,1H),6.74(d,J＝3.5Hz,1H),5.36(s,2H).¹³C NMR(151MHz,DMSO)δ161.53(162.34,160.73,d,J＝243.3Hz),155.68,154.04,151.21,149.56,134.50(134.51,134.49,d,J＝3.1Hz),133.61,129.71,129.53(129.56,129.50,d,J＝8.3Hz),125.42,124.93,115.4(115.47,115.33,d,J＝21.3Hz),113.89,108.26,103.51,102.74,99.07,46.49.¹⁹F NMR(565MHz,DMSO)δ-115.14.HRMS(ESI):calcd for C₂₀H₁₆N₆OF[M+H]⁺m/z,375.1364;found,375.1353.

Test of Activity of the Compounds of example 3

We performed antioxidant, anti-excitatory injury and anti-inflammatory activity tests on the compounds synthesized as described above. First, the cytotoxicity of the above compounds was examined, and as shown in Table 1, after SH-SY5Y cells were treated with 10. Mu.M of the compounds for 24 hours, no significant cytotoxicity was observed in all the compounds, and the effect on cell viability was small (the viability was over 85%). We therefore tested the above 24 compounds for antioxidant, anti-inflammatory and anti-excitatory injury activity.

Cytotoxic effects of the compounds of Table 6 on SH-SY5Y cells

Since the above compounds have better safety, we further examined the antioxidant and anti-excitatory injury activity of these compounds. As shown in Table 2, the above compounds all exhibited antioxidant activity to varying degrees. Wherein the antioxidant activity of the compound E24 is optimal, the cell survival rate is 80.57%, and the neuroprotection rate is 63.78%. Cell viability was 78.54% and 79.83% for compound E1 and E4 treated groups, respectively, neuroprotection was 60.00% and 62.01%, respectively, with antioxidant activity very close to that of E24. In addition, the survival rates of compounds E7, E8, E11, E12, E21 and E22 were all over 60%, and also showed more pronounced antioxidant activity compared to the H ₂O₂ -treated group (survival rate of only 46.35%). The cell viability of the treatment groups of compounds E6, E14, E19 and E23 exceeded 55%, indicating that these three compounds have a certain antioxidant activity.

Table 2 Activity of related Compounds against H ₂O₂ -induced oxidative damage

We continued to test the compounds for anti-excitatory activity. As shown in Table 3, the above compounds all showed various degrees of anti-excitatory injury activity, with the anti-excitatory injury activity of compound E24 being optimal, the cell survival rate being 85.26%, and the neuroprotection rate being 72.44%. Cell viability was 71.54% and 82.33% for compound E1 and E4 treated groups, and neuroprotection was 62.39% and 69.10% respectively, suggesting that these two compounds also have strong anti-excitatory injury activity. In addition, the cell viability of the compound E12, E13, E15, E20, E21, E22 and E23 treated groups was more than 60%, and also showed a more pronounced anti-excitatory injury activity compared to the NMDA treated group (viability only 42.81%). The cell viability of the treated groups of compounds E3, E8, E10, E11, E14, E17 and E18 exceeded 50%, indicating that these three compounds also have some anti-excitatory injury activity.

TABLE 3 anti-NMDA induced excitatory injury Activity of related Compounds ^a

^a The control group had a survival rate of 100% and the NMDA treated group had a survival rate of 42.81%.

Neuroinflammation is considered to be a major factor in brain injury. Usually characterized by microglial activation and collateral brain damage and caused by a strong inflammatory response. We therefore selected BV2 as a mouse-derived microglial cell line that can be used as an in vitro model to study neurodegenerative diseases and related cellular conditions and processes, such as neuroinflammation. In addition, BV2 cells are considered an alternative model system for primary microglia. We therefore selected BV2 cell model for examining the neuroinflammatory inhibitory activity of the compounds. As shown in Table 9 and Table 10, the above compounds showed various degrees of anti-inflammatory activity, wherein the anti-inflammatory activity and safety of Compound E24 were optimal, the cell viability was 95.30% at 10. Mu.M concentration, the inhibition of NO release was 95.01%, and the neuroprotection was 90.77%. Whereas the NO inhibition rate of the positive drug galanthamine is only 55.73%. Wherein, the inhibition ratio of the NO release of the compounds E3, E7, E8, E9, E11, E12 and E14 is more than 70%, the inhibition ratio of the NO release of the compounds E1 and E6 is more than 60%, the inhibition ratio of the NO release of the compounds E17, E21, E22 and E23 is more than 50%, and the compounds all show stronger anti-inflammatory activity.

Cytotoxic effects of the Table 4 Compounds (10. Mu.M) on BV2 cells

Gal positive control was galanthamine.

Table 5 anti-inflammatory Activity of related Compounds ^a

^a Gal is positive control drug galanthamine with survival rate 55.73. The survival rate of the control group is 100%.

As shown by the activity test results, the E24 not only has better safety, but also has better anti-oxidation, anti-inflammatory and anti-excitatory injury activities, wherein the anti-inflammatory activity is most remarkable. Thus E24 was selected as a representative compound for subsequent in vivo and in vitro efficacy studies.

First, we studied the anti-neuroinflammatory activity of E24 on the LSP-induced BV2 cell model. Morphological observations showed that the BV2 cell bodies after LPS treatment were rounded, the antenna was reduced, and an activated morphology was shown. Whereas the E24 treated BV2 cells, the cell bodies were smaller, showing a morphology similar to that of the control group (FIG. 1A). The results in FIG. 1B show that the NO release inhibition IC ₅₀ of E24 is 768.6 + -33 nM. As shown in FIGS. 1C-1G, we next performed LPS treatment on BV2 cells after 12h of E24 pretreatment, and found that E24 significantly reduced the mRNA levels of LPS-induced inflammatory factors iNOS, COX-2, TNF- α, IL-1β and IL-6. However, the positive drug galantamine only reduced the mRNA levels of COX-2 after treatment. Next, we constructed an in vivo neuroinflammation model by injecting LPS into the abdominal cavity of the mice, and examined the in vivo efficacy. Through the nikov staining experiments, we found that the number of nikov bodies in the CA3 region and DG region of the LPS group was significantly reduced compared to the mice in the blank group, while the number of nikov bodies in these two regions was significantly increased in the mice given E24 pretreatment. This suggests that E24 was able to reduce neuronal damage due to neuroinflammation (fig. 2A). mRNA levels of COX-2, iNOS, TNF- α, IL-6 and IL-1β were detected in the mouse cerebral cortex using RT-PCR, and it was shown that E24 was able to significantly reduce transcription of these inflammatory factors (FIG. 2B). The levels of IL-6 and TNF- α in the mouse brain were measured by ELISA, and the results showed that E24 significantly inhibited the LPS-induced rise of IL-6 and TNF- α in the mouse brain (FIG. 2C). Finally, we examined the TLR-NF- κb signaling pathway protein expression levels. As shown in fig. 2D, E24 dose-dependently inhibited protein expression of TLR4 compared to the LPS group. In addition, E24 was able to significantly inhibit the phosphorylation levels of NF- κB and IκB.

Studies have shown that oxidative stress, inflammation and excitatory lesions also play a critical role in depression. In view of the fact that E24 has antioxidant, anti-inflammatory and anti-excitatory injury activities of different degrees at the same time, a mouse CUMS animal model is also constructed, and the influence of E24 (20 mg/kg) on mouse depression-like behaviors is examined. As shown in fig. 3, E24 was able to significantly improve the depressive-like behavior of the CUMS mice, mainly in that E24 significantly improved the saccharophilic preference of the CUMS mice, the stationary time of Forced Swimming (FST) and tail suspension experiments (TST), and the total distance of movement and time in the middle region in the mine experiments (OFT). The results show that E24 has good antidepressant activity.

In view of the key role of oxidative stress, inflammation and excitatory injury in brain injury caused by stroke, we also constructed a rat MCAO model, investigating the effect of E24 (20 mg/kg) on ischemia reperfusion injury in rats. As shown in fig. 4, the TTC experimental results indicate that the cerebral infarction area of rats of the MCAO model group is significantly larger than that of rats of the control group. And the E24 treated can obviously improve the cerebral infarction area of MCAO rats, which suggests that the MCAO rats have good activity of resisting cerebral ischemia reperfusion injury.

Finally, what should be said is: the above embodiments are only for illustrating the technical aspects of the present invention, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention, which is intended to be encompassed by the claims.

Claims (6)

1. The pyrrolopyrimidine-4-amine and pyrazolopyrimidine-4-amine derivatives are characterized in that the structural formulas of the pyrrolopyrimidine-4-amine and pyrazolopyrimidine-4-amine derivatives are shown as I

（I）

Wherein X is C or N;

the R1 group is: 、、

、、、

、、、

、、、

、、、

、 Or (b) ；

The R2 is、、、

Or (b)。

2. The pyrrolopyrimidin-4-amine and pyrazolopyrimidin-4-amine derivatives according to claim 1, characterized in that the pyrrolopyrimidin-4-amine and pyrazolopyrimidin-4-amine derivatives have the structural formula:

Or (b)

。

3. A process for the synthesis of pyrrolopyrimidin-4-amine and pyrazolopyrimidin-4-amine derivatives according to any one of claims 1-2, characterized in that the steps comprise:

s1, synthesizing an intermediate C1-C19 by taking a compound A1 or A2 and a compound B as substrates, wherein the reaction route is as follows:

；

the structures of intermediates C1-C19 are as follows:

；

s2, synthesizing E1-E4 and E6-E24 by using intermediates C1-C19 obtained in S1 and a compound D, wherein the reaction route is as follows:

the structures of the target compounds E1-E24 are as follows:

。

4. A method according to claim 3, wherein the specific process of step S1 is: adding 4.24 mmol of compound B into 3.26 mmol of compound A1 or A2 and 6.52 mmol of potassium carbonate and 30 ml of acetonitrile solution, heating and refluxing for 3 hours, cooling to room temperature, filtering to remove potassium carbonate, removing solvent in vacuum, using petroleum ether and ethyl acetate as eluent, and separating and purifying the product by silica gel column chromatography to obtain an intermediate compound C1-C19.

5. A method according to claim 3, wherein the specific process of step S2 is: under the protection of argon, 0.82 mmol compound C and 1.23 mmol compound D are dissolved in 20ml absolute ethanol, then 4 mol per liter of 1, 4-dioxane hydrochloride solution of 0.13 ml is added, the temperature is raised to 95 ℃ for reaction for 4 hours, TLC monitors the completion of the reaction, the reaction is cooled to room temperature, the solvent is removed in vacuum, dichloromethane and methanol are used as eluent, and the product is separated and purified by silica gel column chromatography to obtain the target compounds E1-E4, E6-E24.

6. Use of pyrrolopyrimidin-4-amine and pyrazolopyrimidin-4-amine derivatives according to any one of claims 1-2 for the preparation of a medicament for the prevention and/or treatment of neurological diseases caused by oxidative stress injury, excitatory injury and/or inflammatory injury.