CN112480134B - Pair of isomers, preparation method and application thereof - Google Patents

Abstract

The invention discloses a pair of isomers, a preparation method and application thereof, wherein the chemical structures of the pair of isomers are shown as formulas (I) and (II):

（I）

(II) the preparation method is as follows: mixing (E) -2- (2- ((Z) -1,3, 3-trimethylindole-2-ethylidene) furan [2,3-b]Mixing quinoline-3, 4 (2H, 9H) -diketone, diethyl sulfate, anhydrous potassium carbonate and an organic solvent, heating for reaction, concentrating an organic phase, and separating to obtain a product. The invention adopts a one-pot method to prepare two compounds, wherein the organic micromolecule (I) can realize positioning and fluorescent response to intracellular lipid drops and has the advantages of high sensitivity, quick response, high specificity and the like; the organic small molecule (II) has fluorescent response to the viscosity of the solution, can be used for detecting the viscosity of a system, has stronger acetylcholinesterase inhibition activity, and can be used for treating related diseases.

Description

Pair of isomers, preparation method and application thereof

Technical Field

The invention belongs to the fields of pharmaceutical chemical industry and biological detection. In particular to a pair of isomers, a preparation method and application thereof.

Background

The core of the lipid droplet is composed of neutral fat, mainly including triglyceride and cholesterol ester, and a monolayer of phospholipid molecules and various proteins are coated outside the core. The phospholipid molecules are mainly phosphatidylcholine and phosphatidylethanolamine, and secondly phosphatidylinositol. Lipid droplets interact with other organelles and may play important roles in lipid metabolism and storage, membrane transport, protein degradation, and signal transduction. In addition, studies have shown that various metabolic diseases, such as obesity, fatty liver, cardiovascular diseases and diabetes, neutral lipid storage diseases are associated.

Commercial lipid drop fluorescent probes such as Nile red, Bodipy and the like have the defects of low specificity for identifying lipid drops, small Stokes shift, poor light stability, background noise and the like. In order to overcome the defects, the design of the lipid droplet probe which is simple to prepare, high in sensitivity, high in selectivity and high in response speed has important significance for the distribution and function research of lipid droplets in a living body.

Viscosity, which may also be referred to as viscosity, refers to the resistance that a fluid exhibits to flow. When a fluid (gas or liquid) flows, one part flows over the other part, and is subjected to resistance, which is the internal friction of the fluid. To enable fluid flow, tangential forces are applied in the direction of fluid flow to counter the resistance. The traditional viscosity probe has the defects of low fluorescence intensity, poor stability, short fluorescence life and the like after being applied to materials, so that the viscosity probe with high sensitivity, high stability, high response speed and long fluorescence life has certain significance for viscosity detection.

Acetylcholinesterase (abbreviated as AChE) is a key enzyme in biological nerve conduction, and is mainly present in synaptic clefts of cholinergic nerve cells, and the enzyme can degrade neurotransmitter acetylcholine, terminate the excitation of acetylcholine on postsynaptic membranes, and ensure the normal transmission of nerve signals in organisms. Alzheimer's disease is commonly called senile dementia, is a very common disease in the elderly population, and at present, AChE inhibitors such as donepezil, rivastigmine and galantamine are mainly used as clinical treatment medicines, and the medicines can be used for preparing the hydrolysis of AChE on acetylcholine and improving the level of neurotransmitter acetylcholine in the brain of a patient, so that the memory and cognitive functions of the patient can be improved. There is another cholinesterase which has a structure very similar to that of AChE, called butyrylcholinesterase (abbreviated as BChE), and can hydrolyze butyrylcholine, but it is mainly present in the peripheral system. Many of the compounds reported in the literature have no selectivity for AChE and BChE inhibition and therefore have toxic side effects on the peripheral system. The development of new compounds that selectively inhibit AChE is of great interest for the treatment of alzheimer's disease.

Disclosure of Invention

The primary object of the present invention is to provide a pair of isomers having lipid droplet localization, viscosity detection or acetylcholinesterase inhibitory activity. Another object of the present invention is to provide a process for the preparation of the above isomers. The invention also aims to provide application of the isomer in lipid drop detection, viscosity and acetylcholinesterase activity inhibition.

Based on the above purpose, the invention is realized by the following technical scheme:

a pair of isomers capable of detecting lipid droplets, detecting viscosity and inhibiting acetylcholinesterase, wherein the chemical structures of the isomers are shown as formulas (I) and (II):

（I） （II）

the preparation method of the isomer comprises the following preparation steps: mixing (E) -2- (2- ((Z) -1,3, 3-trimethylindole-2-ethylidene) furan [2,3-b ] quinoline-3, 4 (2H, 9H) -diketone, diethyl sulfate, anhydrous carbonate and an organic solvent, heating for reaction, filtering to obtain a filtrate, washing a filter cake with chloroform, drying a combined solution by distillation, adding ice water, extracting with chloroform, drying an organic phase, concentrating the organic phase, and separating by column chromatography to obtain the isomer, wherein the anhydrous carbonate is anhydrous potassium carbonate or anhydrous sodium carbonate.

The organic solvent is methanol, ethanol, dichloromethane, chloroform, acetonitrile, acetone, ethyl acetate or N, N-dimethylformamide.

The molar ratio of (E) -2- (2- ((Z) -1,3, 3-trimethylindole-2-ethylidene) furan [2,3-b ] quinoline-3, 4 (2H, 9H) -diketone to diethyl sulfate to anhydrous carbonate is 1 (1-2) to (1-3).

Isomers of (A), (B), (C), and a)I) Eluting with petroleum ether/ethyl acetate =2:1 (volume ratio) during column chromatography, and eluting isomer (II) with CHCl₃MeOH =200:1 (volume ratio) was eluted, isomer (I) was eluted first, and isomer (II) was eluted second.

The heating temperature is 40-80 ℃.

The compound shown in the formula (I) is applied to preparing a probe for detecting lipid droplets in biological cells, and the cells are SH-SY5Y or Hela cells.

The application of the compound shown in the formula (II) in preparing a viscosity detection agent, wherein the viscosity refers to the viscosity in a glycerol-water system.

The compound shown in the formula (II) is applied to a high polymer material as a fluorescent probe, and preferably, the high polymer material is epoxy resin.

The application of the compound shown in the formula (II) in preparing acetylcholinesterase inhibitors.

The preparation method and the product of the invention have the following advantages:

1. the method has simple reaction steps, can obtain two compounds by only one step, and has simple and convenient post-treatment method and high yield.

2. The isomer (I) of the present invention can enter cells and be used for lipid droplet detection with rapid response and little background interference.

3. The isomer (II) of the invention can not enter cells, can be used for detecting the viscosity of solution, and has high sensitivity and stable fluorescence property. After the polymer material is prepared, the loss of fluorescence intensity is less than 5 percent after the polymer material is continuously irradiated for 72 hours at 365 nm.

4. The AChE inhibitory activity of the isomer (II) is 27 times that of the commercial medicine Rivastigmine, and the inhibition selectivity of the isomer (II) on AChE is far higher than that of BChE.

Drawings

FIG. 1 is a scheme for the synthesis of isomers (I) and (II) of the present invention;

FIG. 2 shows the isomer (I) according to the invention¹A HNMR map;

FIG. 3 is an illustration of the present inventionProcess for the preparation of isomer (I)¹³A CNMR map;

FIG. 4 shows the isomer (II) according to the present invention¹A HNMR map;

FIG. 5 shows the isomer (II) according to the present invention¹³A CNMR map;

FIG. 6 fluorescence response image of isomer (I) obtained in example 1 to SH-SY5Y and Hela intracellular lipid droplets, using the commercial dye Nile Red for comparison;

FIG. 7 is a graph showing fluorescence spectra of isomer (II) obtained in example 1 in water-glycerol systems at different ratios;

FIG. 8 is a graph showing the fold change in fluorescence of isomer (II) obtained in example 1 in response to the ratio of glycerol in a water-glycerol system;

FIG. 9 is a graph showing the change in fluorescence of the isomer (II) obtained in example 1 after it is applied to a crystal drop (epoxy resin) and continuously irradiated with a 365 nm ultraviolet lamp for 72 hours;

FIG. 10 is a graph showing the change in the intensity of the maximum fluorescence emission wavelength (521 nm) of the isomer (II) obtained in example 1 after it is applied to a crystal drop (epoxy resin) and continuously irradiated with a 365 nm ultraviolet lamp for 72 hours;

FIG. 11 is the average time for the isomer (II) obtained in example 1 to find a plateau in the mouse water maze experiment using rivastigmine as a control.

Detailed Description

The present invention will be further described with reference to the following examples and drawings, but the embodiments of the present invention are not limited thereto.

The nuclear magnetic spectrum is measured by a Bruker AV-300 nuclear magnetic resonance spectrometer, deuterated DMSO is used as a solvent, the fluorescence spectrum adopts an Agilent fluorescence spectrophotometer, and the ultraviolet spectrum adopts a Beijing Leiberttaco instrument, Inc. UV9100A ultraviolet spectrophotometer. Other parameters may be set with reference to conventional instrumentation.

Example 1

Isomers (I) and (II), as shown in fig. 1, were synthesized as follows:

the reaction mixture of (E) -2- (2- ((Z) -1,3, 3-trimethylindol-2-ethylene) furan [2,3-b]Quinoline-3, 4 (2H, 9H) -dione (384 mg, 1.0 mmol) and anhydrous K₂CO₃(207 mg, 1.5 mmol) was added to 30mL of anhydrous DMF, and 10 mL of a solution of diethyl sulfate (231 mg, 1.5 mmol) in DMF was added dropwise from a constant pressure dropping funnel with stirring. After the dropwise addition, the temperature is raised to 60 ℃ and the reaction is stirred for 30 minutes. After the reaction was complete, the filtrate was filtered and the filter cake was washed three times with 10 mL of chloroform each time. The solution was combined and evaporated to dryness, 30mL of ice water was added, and the mixture was extracted three times with 30mL each time of chloroform. The organic layers were combined, dried over anhydrous sodium sulfate, evaporated to dryness and purified by column chromatography. Elution with petroleum ether/ethyl acetate =2:1 (vol.%) gave 210 mg of isomer (I) in 51% yield. The nuclear magnetic spectra of the obtained product are shown in fig. 2 and fig. 3, respectively. The product identification data is as follows:

¹HNMR (300 MHz, CDCl₃) δ 8.37 (d, J = 8.4 Hz, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.78 (t, J = 7.2 Hz, 1H), 7.49 – 7.38 (m, 2H), 7.32 (s, 1H), 7.29 (s, 1H), 7.04 (t, J = 7.2 Hz, 1H), 6.83 (d, J = 7.2 Hz, 1H), 5.95 (d, J = 13.2 Hz, 1H), 5.26 (q, J = 7.2 Hz, 2H), 3.37 (s, 3H), 1.73 (s, 6H), 1.62 (t, J = 7.2 Hz, 3H). ¹³CNMR (75 MHz, CDCl₃) δ 175.32, 165.50, 164.41, 161.89, 148.42, 143.05, 140.81, 138.42, 131.60, 126.93, 126.49, 124.13, 123.14, 120.70, 120.53, 119.04, 112.84, 106.31, 101.07, 88.91, 71.84, 45.81, 28.67, 27.40, 27.40, 14.59.

continued use of CHCl₃MeOH =200:1 (volume ratio) gave 128 mg of isomer (II) in 31% yield. The nuclear magnetic spectra of the obtained product are shown in fig. 4 and 5, respectively. The product identification data is as follows:

¹HNMR (300 MHz, CD₂Cl₂) δ 8.30 (d, J = 7.2 Hz, 1H), 7.66 – 7.56 (m, 1H), 7.45 (d, J = 8.1 Hz, 1H), 7.28 (t, J = 7.2 Hz, 1H), 7.14 (td, J = 9.0, 8.1, 4.8 Hz, 3H), 6.89 (t, J = 7.4 Hz, 1H), 6.72 (d, J = 8.1 Hz, 1H), 5.50 (d, J = 13.2 Hz, 1H), 4.35 (q, J = 7.2 Hz, 2H), 3.22 (s, 3H), 1.56 (s, 6H), 1.43 (t, J = 7.2 Hz, 3H). ¹³CNMR (75 MHz, CD₂Cl₂) δ 176.14, 172.35, 165.80, 165.76, 144.29, 141.43, 139.49, 137.70, 132.91, 128.03, 127.76, 127.49, 124.35, 121.83, 121.57, 115.37, 112.63, 107.41, 102.94, 87.28, 46.96, 39.24, 29.64, 28.33, 28.33, 13.44.

example 2

The isomers (I) and (II) of this example were synthesized as follows:

mixing (E) -2- (2- ((Z) -1,3, 3-trimethylindole-2-ethylidene) furan [2,3-b]Quinoline-3, 4 (2H, 9H) -dione (384 mg, 1.0 mmol) and anhydrous Na₂CO₃(318 mg, 3.0 mmol) was added to 30mL of acetonitrile, 10 mL of an acetonitrile solution containing diethyl sulfate (231 mg, 1.5 mmol) was added dropwise with stirring, and after completion of the addition, the temperature was raised to 70 ℃ to stir and react for 30 minutes. After the reaction was completed, the reaction solution was filtered, and the cake was washed with chloroform three times by 20 mL. The filtrates were combined, the solvent was evaporated to dryness, 30mL of ice water was added, extraction was carried out three times with 30mL each time, the organic layers were combined, dried over anhydrous sodium sulfate, evaporated to dryness and purified by column chromatography. The eluent was petroleum ether/ethyl acetate =2:1 (volume ratio), giving isomer (I) 196 mg, 48% yield. The nuclear magnetic data are the same as for isomer (I) in example 1.

Continued use of CHCl₃MeOH =200:1 (vol) gave 150 mg of isomer (II) in 36% yield. The nuclear magnetic data are identical to isomer (II) in example 1.

And (3) performance testing:

(1) imaging of intracellular lipid droplets by isomer (I)

The isomer (I) prepared in example 1 was dissolved in DMSO to prepare a 10 mM stock solution. SH-SY5Y or Hela cells were seeded into sterilized 35 mm imaging plates at a density of 2.0X 10⁴One dish in an incubator (temperature 37 ℃, 5% volume fraction CO)₂) After the cells are attached, compound (I) and the commercial dye Nile Red are added to the cells at a final concentration of 5. mu.M isomer (I) and 10. mu.M Nile Red. After half an hourThe medium was discarded, the cells were washed 3 times with PBS buffer, and fluorescence imaging was performed (excitation wavelength of Compound (I) was 488 nm, yellow channel 550-600 nm; excitation wavelength of Nile Red was 473 nm, red channel 570-650 nm) with the results shown in FIG. 6. Wherein A is an imaging picture of the isomer (I) synthesized by the invention in SH-SY5Y cells; b is an imaging picture of the commercial dye nile red in SH-SY5Y cells; c is an overlay of A and B; d is an imaging picture of the isomer (I) in Hela cells; e is an imaging picture of nile red in Hela cells; f is an overlay of D and E; as can be seen in fig. 6, compound (I) is consistent with the imaging position of nile red, indicating that isomer (I) is localized to lipid droplets in the cell. Compound (I) at 5. mu.M has a similar brightness to Nile Red at 10. mu.M, indicating that the fluorescent properties of isomer (I) of the present invention are superior to that of the commercial dye Nile Red.

(2) Fluorescence response of isomer (II) to solution viscosity

The isomer (II) prepared in example 1 was dissolved in DMSO to prepare a 10 mM stock solution. Respectively taking 5 mL of glycerol and H with different volume ratios₂The mixed solution of O (0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% by volume of glycerin, respectively) was added to the stock solution of isomer (II) (final concentration of 10 μ M), and the fluorescence spectrum of each system was measured by fluorescence spectrophotometry, as shown in fig. 7, with an excitation wavelength of 504 nm. As can be seen from FIG. 7, as the proportion of glycerol increases (the viscosity of the system gradually increases), the fluorescence intensity at 560 nm increases, and the enhancement factor and the proportion of glycerol form a good curve relationship (FIG. 8), which indicates that the isomer (II) has a fluorescence response to the viscosity of the solution, and thus has a potential application value for detecting the viscosity of the system.

(3) Application of isomer (II) in high polymer material

Adding the stock solution of the isomer (II) into crystal dripping glue (commercially available epoxy resin AB glue, uniformly mixed according to the specification proportion), fully stirring, curing the mixture, and irradiating the cured block material under a 365 nm ultraviolet lamp to emit bright green fluorescence. The irradiation with the ultraviolet lamp was continued to examine the stability of the fluorescence of the material, as shown in fig. 9 and 10. As can be seen from FIG. 10, after the continuous irradiation of the material for 72 hours under a 365 nm ultraviolet lamp, the fluorescence intensity attenuation of the material at the maximum emission wavelength of 521 nm is less than 5%, which indicates that the stability of (II) in the glue dripping is better.

(4) In vitro inhibitory Activity of isomer (II) on AChE

TABLE 1 inhibitory Activity of Compounds on AChE and BChE

The inhibitory activity of isomer (II) on AChE was tested using Ellman ultraviolet spectroscopy. The stock solution of (II) above was taken and tested by dilution to five different concentrations (0.1 μ M, 1 μ M, 10 μ M, 100 μ M, 200 μ M) with phosphate buffer (0.1 mol/L, pH = 8.0) with rivastigmine as a positive control drug. By plotting the concentration of compound against the inhibition of AChE, the median Inhibitory Concentration (IC) can be calculated₅₀Values), the results are shown in table 1. IC of Isomer (II)₅₀Is only 0.24 mu M and is 27/1 of the marketed drug Rivastigmine, which shows that the inhibition activity of (II) on AChE is much higher than that of Rivastigmine. Meanwhile, the inhibitory activity of the compound on BChE is tested, and the IC of the compound on BChE is found out₅₀The value was 120.35. mu.M, indicating that (II) is able to selectively inhibit AChE. IC of isomer (I) for two cholinesterases₅₀The activity is more than 50 mu M, and the inhibition activity is weaker.

(5) Effect of isomer (II) on improving memory impairment in mice

The mouse is continuously trained for four days by using a scopolamine induced mouse memory impairment model and adopting a Morris water maze method, and the model mouse has longer time for finding a platform in the water maze than a normal mouse, which indicates that the model building is successful. In the experiment, compound (II) and rivastigmine were administered to model mice at the same dose (gavage, 20 μmol/kg body weight, physiological saline) for four consecutive days, and the results were shown in fig. 11 on the fourth day. As can be seen from fig. 11, the mice given compound (II) found a shorter mean time to develop a hydrothorax platform than the rivastigmine group, indicating that compound (II) has a stronger effect in improving memory cognitive function than rivastigmine.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which are equivalent to the above embodiments without departing from the spirit and principle of the present invention are also included in the protection scope of the present invention.

Claims (10)

1. A pair of isomers, wherein the chemical structures of said isomers are represented by formulas (I) and (II):

（I） （II）

2. the process for producing an isomer of claim 1, which comprises the following production steps: mixing (E) -2- (2- ((Z) -1,3, 3-trimethylindole-2-ethylidene) furan [2,3-b ] quinoline-3, 4 (2H, 9H) -dione, diethyl sulfate, anhydrous carbonate and an organic solvent, heating, stirring, reacting, concentrating an organic phase, and separating and purifying to obtain the compounds shown in the formulas (I) and (II), wherein the anhydrous carbonate is anhydrous potassium carbonate or anhydrous sodium carbonate.

3. The method for preparing an isomer of claim 2, wherein the organic solvent is selected from methanol, ethanol, dichloromethane, chloroform, acetonitrile, acetone, ethyl acetate and N, N-dimethylformamide, and the molar ratio of (E) -2- (2- ((Z) -1,3, 3-trimethylindole-2-ethylene) furan [2,3-b ] quinoline-3, 4 (2H, 9H) -dione, diethyl sulfate and anhydrous carbonate is 1 (1-2) to (1-3).

4. The method for producing an isomer according to claim 2, characterized in that: the heating temperature is 40-80 ℃, the separation and purification is column chromatography and/or recrystallization, and the used separation solvent is selected from a single solvent or a mixed solvent in any proportion of methanol, ethanol, ethyl acetate, acetone, dichloromethane, chloroform, petroleum ether and n-hexane.

5. Use of a compound of formula (I) according to claim 1 for the preparation of a probe for the detection of lipid droplets in biological cells.

6. The use according to claim 5, wherein said cells are SH-SY5Y or Hela cells.

7. Use of a compound of formula (II) according to claim 1 in the preparation of a viscosity detector.

8. Use according to claim 7, wherein viscosity is in a glycerol-water system.

9. Use of a compound of formula (II) according to claim 1 as a fluorescent probe in an epoxy resin.

10. Use of a compound of formula (II) according to claim 1 for the preparation of an acetylcholinesterase inhibitor.

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