CN108424382B - 3,3' -disubstituted bipyridine derivative and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a 3,3 '-disubstituted bipyridine derivative, a preparation method thereof and application thereof in preparing anti-Alzheimer's disease drugs. The chemical formula of the 3,3' -disubstituted bipyridine derivative is shown as a formula I and a formula II. Wherein R is-H, halogen, -O (CH)2)mCH3Amino substituted with 1 or more methyl or ethyl groups; r1is-O (CH)2)mCH3，‑OH；R2An aliphatic hydrocarbon of C1 to C5, an amino group substituted with 1 or more methyl groups or ethyl groups,
，
. The invention also discloses a preparation method and application of the derivative. The 3,3' -disubstituted bipyridine derivative has the functions of inhibiting A beta aggregation, metal complexing ability and inhibiting metal ion-induced A beta aggregation activityHas low toxicity and high safety. Therefore, the 3,3' -disubstituted bipyridine derivative has wide application space in preparing the anti-Alzheimer disease medicine.
Description
Technical Field
The invention relates to the technical field of medicines and chemical engineering, in particular to a 3,3' -disubstituted bipyridine derivative and a preparation method and application thereof.
Background
Alzheimer's Disease (AD), also known as senile dementia, is a chronic, progressive degenerative disease of the central nervous system. In recent years, with the development of society and the improvement of living standard of people, the world population is aging, so the incidence rate of AD is rising year by year, and the AD becomes one of the main diseases seriously threatening the life health and life quality of the elderly. To date, the pathogenesis of AD is unclear, and various factors such as gene defects, metabolic disorders, free radical damage, immune neuroinflammation, neuronal apoptosis, and environmental toxins may be involved. In response to these factors, many hypotheses have been proposed, and it is now well accepted that the amyloid cascade hypothesis considers that aggregation of a β protein is the main cause of brain neurodegeneration. Wherein A β is a 39-43 residue peptide cleaved from the C-terminus of the amyloid precursor protein, and the most abundant A β is40And Abeta42，Aβ42Is more neurotoxic. According to the amyloid cascade hypothesis, an increase in A β in synaptic cleft may lead to the formation of toxic oligomersIn turn, these toxic oligomers repolymerize to form insoluble aggregates and fibrils, ultimately leading to deposition of amyloid. Excessive deposition of a β can lead to a series of inflammatory responses, which in turn lead to loss of neurons and decline of cognitive function. However, the self-aggregating properties of a β are not sufficient to account for the accumulation of peptides in specific brain regions of AD patients. In addition, it has been found that the concentration of metal ions, such as copper ions and iron ions, is abnormally high in senile plaques of AD patients. These metal ions can affect the synthesis, degradation and clearance of a β. And the A beta is metal ion binding protein, has metal ion binding sites, can be combined with metal ions to form an insoluble metal-A beta compound, enhances the neurotoxicity of the A beta, and catalyzes and accelerates the generation of Reactive Oxygen Species (ROS) in vivo, thereby causing oxidative damage and inducing apoptosis. These studies indicate that the appearance of amyloid pathology is associated with the deregulation of these metal ions. Therefore, based on the role of metal-a β in AD, it has been proposed to disrupt metal-a β interactions by means of metal-complexing therapy, thereby reducing the neurotoxicity of metal-a β and restoring the metal ion balance in the brain.
In summary, in order to further study the relationship between metal ions and a β and find better potential drugs for treating AD, designing and synthesizing metal-a β -based metal chelators, i.e., small molecule compounds with activity of inhibiting a β aggregation, metal ion complexation, and inhibition of metal ion-induced a β aggregation, may be an effective strategy for treating AD.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a novel 3,3' -disubstituted bipyridine derivative.
The invention also aims to provide a preparation method of the derivative and application of the compound in preparing medicaments for preventing and/or treating Alzheimer disease, cerebrovascular dementia or myasthenia gravis.
3,3' -bipyridyldiamine is a preferred Cu2+And Zn2+The literature reports that the bipyridine compound has a certain neuroprotective effect in cells and has an anti-oxidative stress effect.Therefore, in order to enhance the effect of the bipyridyl compound on inhibiting A beta aggregation, the patent introduces an aromatic group which is easy to react with A beta on a bipyridyl mother nucleus, designs and synthesizes a series of novel 3,3' -disubstituted bipyridyl derivatives, and proves that the compounds have the activities of inhibiting A beta aggregation, metal ion complexation and inhibiting metal ion-induced A beta aggregation.
The technical purpose of the invention is realized by the following technical scheme:
the invention provides a 3,3 '-disubstituted bipyridine derivative, the structural formula of the 3,3' -disubstituted bipyridine derivative is shown as the formula (I) and the formula (II),
wherein R is-H, hydroxy, halogen, -O (CH)2)mCH3Amino substituted with 1 or more methyl or ethyl groups; r1is-O (CH)2)mCH3-OH; n is any integer of 0-3; m is any integer of 0-3; r2Is C1-C5 aliphatic hydrocarbon, amino substituted by 1 or more methyl or ethyl,
Preferably, the structural formula of the 3,3' -disubstituted bipyridine derivative is as follows:
the invention also provides a preparation method of the 3,3' -disubstituted bipyridine derivative, which comprises the following steps:
s1. the
Mixing with activated copper powder, dissolving in proper solvent, and reflux reacting under nitrogen protection for several hours to obtain compound
S2. the
Heating the mixture and stannous chloride dihydrate in concentrated hydrochloric acid to perform reduction reaction to obtain a compound
S3, mixing
Carrying out condensation reaction with different carboxylic acids to obtain target products
Wherein R is-H, halogen, -O (CH)2)mCH3Amino substituted with 1 or more methyl or ethyl groups; r1is-O (CH)2)mCH3-OH; n is any integer of 0-3; m is any integer of 0-3; r2Is C1-C5 aliphatic hydrocarbon, amino substituted by 1 or more methyl or ethyl,
Preferably, the target product is purified by column chromatography or recrystallization.
The invention also provides application of the 3,3 '-disubstituted bipyridine derivative, and in particular relates to application of the 3,3' -disubstituted bipyridine derivative in preparing a medicament for preventing and/or treating Alzheimer disease, cerebrovascular dementia or myasthenia gravis.
The preparation form of the medicine for preventing and/or treating Alzheimer disease, cerebrovascular dementia or myasthenia gravis is tablets, pills, capsules, injection, suspending agents or emulsion.
The 3,3' -disubstituted bipyridine derivative provided by the invention has better inhibitory activity on the self-aggregation of A beta, and the inhibitory rate on the self-aggregation of A beta is more than 65% under the test concentration of 20 mu M, wherein the compound 3d shows the strongest inhibitory activity and reaches 93.3%; ultraviolet-visible spectrophotometry tests the chelation of the compound 3d with the strongest inhibitory activity and metal ions to find that the compound 3d can better chelate Cu2+And Fe2+(ii) a And can inhibit metal ion-induced A beta aggregation by chelating metal ions, which can reduce Cu content by 96.9%2+Induced Ass aggregation, reduced by 75.5% by Fe2+Induced aggregation of a β. The toxicity of the compound of the patent on SH-SY5Y cells is tested by an MTT method, which shows that the compound of the patent has lower cytotoxicity, wherein the compound 3d has IC (integrated Circuit) on SH-SY5Y cells50The value is 143.8 mu M, and the safety is high. Therefore, the 3,3' -disubstituted bipyridine derivative can be used as a bifunctional reagent based on the effect of metal-Abeta to prepare the medicine for resisting Alzheimer disease.
Compared with the prior art, the invention has the following beneficial effects:
the compound has good activity of inhibiting A beta aggregation, shows good chelation with copper ions, and can inhibit Cu by chelating metal ions2+Induced aggregation of a β, which provides a material basis for further studies of the participation of metal ions in the pathological processes of AD. The derivative has the advantages of simple preparation method, few steps, low raw material cost, obvious inhibition effect on the A beta aggregation of the Alzheimer disease and the A beta aggregation induced by metal ions, low cytotoxicity and high safety, can be prepared into the anti-Alzheimer disease medicine based on the metal-A beta effect, and has high medical value and wide market prospect.
Drawings
FIG. 1: ultraviolet spectrum of the compound 3d with metal ions.
FIG. 2: compounds 3c to 3e inhibit metal ion-induced Ass aggregation
Detailed Description
The technical solution of the present invention is further illustrated by the following specific examples and the accompanying drawings.
Reagents, equipment and methods employed in the present invention are reagents, equipment and methods conventionally commercially available in the art and conventionally used methods, unless otherwise specified.
Example 1:
synthesis of N, N ' - [ (2,2' -bipyridyl) -3,3' -diyl ] bis (4-chlorobenzamide) (Compound 3a)
0.20g of 4-chlorobenzoic acid and 0.14mL of N, N-Diisopropylethylamine (DIEA) were added to a round-bottomed flask containing 8mL of DMF, and the mixture was stirred to completely dissolve the solid, and cooled to 0-5 ℃ with an ice bath. Adding 0.21g of 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU), stirring for 5min, adding 0.10g of 3,3 '-diamino-2, 2' -bipyridine, stirring in an ice bath for 10min, naturally heating to room temperature for reaction, tracking by TLC, pouring the reaction solution into 20mL of ice water after the reaction is finished, and stirring to separate out a light yellow solid. And (5) carrying out suction filtration, washing and drying on the solid. The crude product was purified by silica gel chromatography to give 3a as a pale yellow powder in 81% yield.1H NMR(400MHz,CDCl3):δ14.35(s,2H),9.39(s,2H),8.42(s,2H),7.65(d,J＝8.0Hz,4H),7.47(d,J＝8.0Hz,4H),7.02(s,2H).ESI-MS m/z:464.3[M+H]+。
Example 2 Synthesis of Compound 3b
The procedure is as in example one, except that benzoic acid is used instead of 4-chlorobenzoic acid and the crude product is purified by silica gel column chromatography to give 3b as a pale yellow solid in 84% yield.1H NMR(400MHz,CDCl3)δ14.56(s,2H),9.43(d,J＝7.9Hz,2H),8.42(s,2H),8.08(d,J＝5.7Hz,4H),7.57(d,J＝7.0Hz,6H),7.48-7.45(m,2H)..ESI-MS m/z:395.5[M+H]+。
Example 3 Synthesis of Compound 3c
The procedure is as in example one, except that 3, 4-dimethoxybenzoic acid is used instead of 4-chlorobenzoic acid and the crude product is purified by silica gel chromatography to give 3c as a white solid in 85% yield.1H NMR(400MHz,CDCl3):δ14.35(s,2H),9.39(s,2H),8.42(s,2H),7.64(s,4H),7.45(s,2H),7.02(s,2H),3.99(s,12H).ESI-MS m/z:515.5[M+H]+。
Example 4 Synthesis of Compound 3d
The procedure is as in example one, except that 4-dimethylaminobenzoic acid is used instead of 4-chlorobenzoic acid and the crude product is purified by silica gel chromatography to give a pale yellow solid 3d in 84% yield.1H NMR(400MHz,CDCl3):δ8.72(s,2H),8.48-8.39(m,2H),8.14-8.12(m,4H),7.45-7.42(m,2H),6.76(d,J＝3.9Hz,4H),3.13(s,12H).ESI-MS m/z:481.6[M+H]+。
EXAMPLE 5 Synthesis of Compound 3e
The procedure is as in example one, except that 2-hydroxybenzoic acid is used instead of 4-chlorobenzoic acid and the crude product is purified by silica gel chromatography to give 3e as a pale yellow solid in 72% yield.1H NMR(400MHz,CDCl3):δ14.72(s,2H),12.31(s,2H),9.02(s,2H),8.41(s,2H),8.08(s,2H),7.95-7.91(m,2H),7.49-7.43(m,2H),7.38-7.32(m,2H),7.02(s,2H).ESI-MS m/z:427.4[M+H]+。
Example 6 Synthesis of Compound 3f
The procedure is as in example one, except that 4-chlorobenzoic acid is replaced by phenylacetic acid and the crude product is passed through a silica gel layerThe product was purified by precipitation to give 3f as a white solid in 83% yield.1H NMR(400MHz,CDCl3):δ13.21(s,2H),9.04(d,J＝8.3Hz,2H),8.28(s,2H),7.48(s,2H),7.39-7.36(m,6H),7.03(d,J＝9.8Hz,4H),3.79(s,4H).ESI-MS m/z:423.5[M+H]+。
Example 7 Synthesis of Compound 3g
The procedure is as in example one, except that 4-methoxybenzoic acid is used instead of 4-chlorobenzoic acid and the crude product is purified by silica gel chromatography to give 3g of a pale yellow solid in 85% yield.1H NMR(400MHz,CDCl3):δ14.53(s,2H),9.28(s,2H),8.33(s,2H),8.05(s,4H),7.14(s,4H),7.01(s,2H),3.90(s,6H).ESI-MS m/z:455.5[M+H]+。
Example 8 Synthesis of Compound 3h
The procedure is as in example one, except that 2, 2-dimethylpropionic acid is used instead of 4-chlorobenzoic acid and the crude product is purified by silica gel chromatography to give a white solid in 3h, 87% yield.1H NMR(400MHz,CDCl3):δ13.17(s,2H),9.15(d,J＝8.4Hz,2H),8.34(d,J＝4.0Hz,2H),7.42-7.37(m,2H),1.34(s,18H).ESI-MS m/z:355.3[M+H]+。
Example 9 Synthesis of Compound 3i
The procedure is as in example one, except that acetic acid is used instead of 4-chlorobenzoic acid and the crude product is purified by silica gel chromatography to give 3i as a pale yellow solid in 86% yield.1HNMR(400MHz,CDCl3):δ12.98(s,2H),9.08(d,J＝8.6Hz,2H),8.35(d,J＝4.5Hz,2H),7.40(dd,J＝8.5,4.6Hz,2H),2.24(s,6H).ESI-MS m/z:271.4[M+H]+。
Example 10 Synthesis of Compound 3j
The procedure is as in example one, except that 3-diethylaminopropionic acid is used instead of 4-chlorobenzoic acid and the crude product is purified by chromatography on silica gel to give 3i as a pale yellow solid in 80% yield.1HNMR(400MHz,CDCl3):δ12.86(s,2H),9.04(d,J＝8.5Hz,2H),8.34(t,J＝4.5Hz,2H),7.36(dd,J＝8.4,4.6Hz,2H),2.89(t,J＝7.2Hz,4H),2.60-2.52(m,12H),1.02(t,J＝7.1Hz,12H).ESI-MS m/z:441.7[M+H]+。
Example 11 Synthesis of Compound 3k
The procedure is as in example one, except that 3- (4-methylpiperazin-1-yl) -propionic acid is used instead of 4-chlorobenzoic acid and the crude product is purified by chromatography on silica gel to give 3k as a pale yellow solid in 78% yield.1H NMR(400MHz,CDCl3):δ12.91(s,2H),9.01(dd,J＝8.5,1.4Hz,2H),8.36(dd,J＝4.5,1.4Hz,2H),7.38(dd,J＝8.5,4.6Hz,2H),2.80(d,J＝7.2Hz,4H),2.64(d,J＝7.2Hz,4H),2.57(s,8H),2.43(s,8H),2.28(s,6H).ESI-MS m/z:495.9[M+H]+。
Example 12 Synthesis of Compound 3l
The procedure is as in example one, except that 3- (pyrrol-1-yl) -propionic acid is used instead of 4-chlorobenzoic acid and the crude product is purified by silica gel chromatography to give 3l of a pale yellow solid in 82% yield.1H NMR(400MHz,CDCl3):δ12.82(s,2H),8.99(d,J＝8.4Hz,2H),8.37(d,J＝4.4Hz,2H),7.38(dd,J＝8.0,4.6Hz,2H),2.89(t,J＝7.1Hz,4H),2.67(t,J＝8.2Hz,4H),2.55(s,8H),1.74(s,8H).ESI-MS m/z:437.7[M+H]+。
Example 13: the 3,3' -disubstituted bipyridine derivative has the inhibiting effect on the self-aggregation of Abeta
The compounds prepared in examples 1 to 12 were selected and the Α β self-aggregation inhibitory activity was determined by the ThT method.
1. Preparing a solution:
(1)20mM pH 7.4 Phosphate Buffer Solution (PBS): 3.618g of Na are weighed out2HPO4And 0.6027g KH2PO4And adding 100mL of ultrapure water, after the solid is dissolved, using the ultrapure water to fix the volume to 200mL, and adjusting the pH value of the solution to 7.4.
(2)Aβ1-42Protein solution: 1mg of protein was dissolved in 100. mu.L of 1% NH4The OH solution with a concentration of 2300 μ M was stored in a refrigerator at-80 deg.C for further use. The solution was diluted to 40. mu.M with PBS buffer.
(3)50mM glycine-NaOH buffer: 0.938g of glycine was weighed, dissolved in 250mL of ultrapure water, adjusted to pH 8.50 with 1mol/L NaOH solution and stored in a refrigerator at 4 ℃.
(4)5 μ M Thioflavin T solution (now ready for use): weighing sulfur T powder 2.2mg, dissolving in 689 μ L glycine-NaOH buffer solution with pH 8.5, ultrasonic treating to dissolve the solid completely, and standing in dark place.
(5) Preparation of compound solution: an appropriate amount of the compound was accurately weighed with a precision analytical balance, diluted with DMSO to a clear solution of 10mM in concentration, and diluted with phosphate buffer to the test concentration at the time of use.
2. And (3) testing the inhibitory activity:
respectively taking 10 mu L of 40 mu M Abeta1-42The protein was mixed with 10. mu.L of a compound at a concentration of 40. mu.M, and incubated in a thermostat at 37 ℃ for 48 h. Blank control was 10. mu.L of 40. mu.M A.beta.1-42Mixing the protein with 10 mu L of phosphate buffer solution with pH 7.4 for incubation; the positive control is Abeta1-42Incubating the protein and the resveratrol together. 72hThereafter, the incubation solution was transferred to a black 96-well plate, and 180. mu.L of a 5. mu.M thioflavin T solution was added thereto, and the mixture was allowed to stand still in the dark at room temperature for 5 min. Finally, measuring the fluorescence absorption value by using a multifunctional microplate reader, wherein the excitation wavelength is 450nm, the absorption wavelength is 485nm, and A beta in a negative control test is used1-42The fluorescence intensity of binding to Thioflavin T was used as a control to determine the A.beta.pair of compounds1-42Inhibition of protein aggregation. The results are shown in Table 1. The results show that most of the compounds described in this patent are directed to A β1-42Has stronger inhibition effect, the inhibition rate is more than 65 percent under the concentration of 20 mu M, wherein the compound 3d shows the strongest inhibition activity which reaches 93.3 percent. Therefore, the 3,3' -disubstituted bipyridine derivative has a development prospect and can be used for preparing the medicine for resisting the Alzheimer disease.
TABLE 13, 3' -disubstituted bipyridine derivatives vs. Abeta1-42Inhibitory Activity of self-aggregation
| Compound (I) | Aβ1-42Inhibition of self-aggregation | Compound (I) | Aβ1-42Inhibition of self-aggregation |
| 3a | 85.9±1.2 | 3h | 68.1±1.7 |
| 3b | 88.5±1.5 | 3i | 64.9±1.3 |
| 3c | 90.2±1.4 | 3j | 83.5±1.3 |
| 3d | 93.3±0.8 | 3k | 74.8±1.5 |
| 3e | 91.5±1.1 | 3l | 77.6±1.1 |
| 3f | 88.1±1.8 | Resveratrol | 79.2±1.5 |
| 3g | 89.3±1.5 |
Example 14 Metal Complex experiment of 3,3' -disubstituted bipyridine derivative 3d described in this patent
The compound prepared in example 4 was selected and the metal complexing ability of the compound was determined by the UV-vis method.
1. Preparing a solution:
(1) compound 3d solution: a certain amount of the compound was weighed up to 1mM with absolute ethanol.
(2) Metal ion solution: balanceTaking a certain amount of ZnSO4,CuSO4And FeSO4By dd H2O10 mM, then diluted to 1mM with absolute ethanol.
2. Compound 3d with ZnSO4,CuSO4And FeSO4Function of
3 pieces of 1.5mL pear tubes were added with 20. mu.L of 1mM 3d compound solution, and then 20. mu.L of 1mM ZnSO4,CuSO4And FeSO4And finally, adding absolute ethyl alcohol into the solution to ensure that the total volume is 1000 mu L, and the final concentration of the metal ions and the 3d are both 20 mu M. Blank control 20. mu.L of 1mM compound solution supplemented with absolute ethanol to the same concentration as the compound in the sample. Mixing, standing at room temperature for 30min, pouring into quartz dish, and scanning absorption curve with ultraviolet-visible spectrometer. The test temperature is room temperature, the test range is 200-600nm, the wavelength interval is 1nm, the scanning speed is 200nm/min, each sample is tested for three times, and the average value is taken. The results are shown in FIG. 1. The result shows that the compound 3d has stronger metal complexing ability and has strong Cu complexing ability2+，Fe2+Has better selectivity. Therefore, the 3,3' -disubstituted bipyridine derivative can complex excessive copper ions in vivo and can be used for preparing the anti-Alzheimer drugs.
Example 15: the 3,3' -disubstituted bipyridine derivatives 3c to 3e described in the patent have an inhibitory effect on metal ion-induced A beta aggregation
The compounds prepared in examples 3 to 5 were selected and the metal ion-induced Α β aggregation inhibitory activity was determined by the ThT method.
1. Preparing a solution:
(1)20 μmol/L pH 6.6HEPES (150 μmol/L NaCl) solution preparation: HEPES 2.38mg and NaCl 4.38mg were weighed, purified water was added thereto to make a volume of 500mL, and the pH was adjusted to 6.6 with a small amount of NaOH solution.
(2)75μM Cu2+Solution preparation: from 10mM CuSO4mu.L of the solution was taken out and diluted to 1mL with 20. mu. mol/L of a pH 6.6HEPES (150. mu. mol/L NaCl) solution.
(3)75μM Fe2+Solution preparation: from 10mM FeSO47.5. mu.L of the solution was taken and treated with 20. mu. mol/L of HEPES pH 6.6 (M) (150. mu. mol/L NaCl) solution to 1 mL.
(4)75 μ M compound formulation: from 10mM compound stock solution 7.5. mu.L, with 20. mu. mol/L pH 6.6HEPES (150. mu. mol/L NaCl) solution diluted to 1 mL.
(5)75μM Aβ1-42Solution preparation: a tube of stock solution (10. mu.L, 1000. mu.M) was taken and added with 123. mu.L of 20. mu. mol/L HEPES pH 6.6 (150. mu. mol/L NaCl) to a concentration of 75. mu.M. Subpackaging 10 μ L of each tube.
2. Sample preparation
Test samples: taking 2 mu L of subpackaged Abeta 1-422 μ L of Cu at a concentration of 75 μ M was added2+Or Fe2+Incubating at room temperature for 2 min; then add 2. mu.L of compound. Mix well and incubate at 37 ℃ for 24 h.
Control sample 1: taking 2 mu L of subpackaged Abeta 1-422 μ L of Cu at a concentration of 75 μ M was added2+Then, 2. mu.L of 20. mu. mol/L of pH 6.6HEPES (150. mu. mol/L NaCl) solution was added thereto, and the mixture was mixed and incubated at 37 ℃ for 24 hours.
Control sample 2: taking 2 mu L of subpackaged Abeta 1-422 μ L of 75 μ M Fe was added2+Then, 2. mu.L of 20. mu. mol/L of pH 6.6HEPES (150. mu. mol/L NaCl) solution was added thereto, and the mixture was mixed and incubated at 37 ℃ for 24 hours.
Control sample 3: taking 2 mu L of subpackaged Abeta1-42mu.L of a solution of 20. mu. mol/L of pH 6.6HEPES (150. mu. mol/L NaCl) was added thereto, and the mixture was mixed and incubated at 37 ℃ for 24 hours.
3. Activity test for inhibition of metal ion-induced a β aggregation:
reference is made to the test method for the self-aggregation inhibitory activity of a β in example 13. The results are shown in FIG. 2. The results show that Cu2+And Fe2 +Can promote A beta1-42In which Cu is present2+Inducing aggregation compared with Fe2+Is remarkable. When the compound is added for incubation, the fluorescence value of the sample is greatly reduced. Wherein the compound 3d has obvious effect, and can reduce 96.9% of Cu2+Induced Abeta1-42Aggregation, reduction by 75.5% of Fe2+Induced Abeta1-42Aggregation, and this inhibitory effect is better than resveratrol (85.3% reduction by Cu)2+Induced Abeta1-42Aggregation, reduction by 65.7% from Fe2+Induced Abeta1-42Aggregate), indicating that 3d can chelate Cu2+And Fe2+Thereby stopping Cu2+And Fe2+Induced aggregation of a β. Wherein, the compound 3d is to Cu2+The inhibitory activity of the induced Abeta is higher than that of Fe2+Inhibitory activity of induced a β aggregation.
EXAMPLE 16 study of the toxicity of the Compounds described in this patent on nerve cells
The compounds prepared in examples 1-12 were selected and tested for SH-SY5Y cytotoxicity by MTT method.
1. Solution preparation
(1) DMEM medium: the dry powder medium was dissolved in 300mL of ultrapure water using a 1000mL beaker, the inner face of the package was rinsed twice with 300mL of ultrapure water, the solutions were combined, and the solution was completely dissolved by magnetic stirring. 3.7g of sodium bicarbonate and 2.38g of HEPES were added and the mixture was magnetically stirred to complete the dissolution. The pH was adjusted to 7.5 with 10M sodium hydroxide under stirring, sterilized by filtration through a 0.22. mu.M filter in a super clean bench, and stored in a refrigerator at 4 ℃. Antibiotics (final concentration penicillin 100U/mL, streptomycin 100. mu.g/mL) and serum (10%) were added at the time of use.
(2) PBS buffer solution: accurately weighing 8g of NaCl and 0.2g of KH2PO4And 2.88g Na2HPO4·12H2And O, diluting to 1L with ultrapure water, sterilizing at 120 ℃ for 20min under high pressure, and storing in a refrigerator at 4 ℃.
(3) MTT solution: 5g/L of the extract is prepared by PBS solution, filtered and sterilized by a 0.22 mu M filter membrane, and stored in a refrigerator at 4 ℃ in a dark place.
2. Culture of SH-SY5Y nerve cells
Taking the nerve cell strain SH-SY5Y, using DMEM medium containing 10% fetal calf serum, 100U/mL penicillin and 100 mu g/mL streptomycin, at 37 ℃, with saturated humidity and 5% CO in the environment2And culturing in 95% air incubator for 2-3 days.
3. Nerve cell toxicity assay
(1) Cells in logarithmic growth phase were taken, digested with 0.25% pancreatin, washed twice with PBS, resuspended in complete medium and counted under microscope using cell counting plateAnd adjusting the cell concentration to 5X 104One cell per mL, inoculated in a 96-well cell culture plate, 100. mu.L/well, cultured for 24h to allow the cells to adhere to the wall.
(2) The original medium was aspirated, and compound solutions of different concentrations diluted with the medium were added, 100. mu.L per well, with 5 replicate wells. Adding culture medium instead of compound into blank and control group, and standing at 37 deg.C and 5% CO2The incubator is used for 48 hours. The medium containing 5mg/mL MTT was added to the sample and control groups 4h before termination of the experiment, 100. mu.L/well, and incubation was continued for 4 h.
(3) The supernatant was discarded, 100. mu.L of DMSO was added to each well, the resultant formazan was dissolved sufficiently by shaking, and the absorbance (OD value) of each well was measured on a full-wavelength microplate reader at a wavelength of 570 nm. Cell survival rate (%) as ═ OD (OD) in each sampleSample (I)-ODBlank space)/(ODControl-ODBlank space) X is 100%; the cell inhibition ratio (%) of each sample was 100% -the cell survival ratio (%) of each sample was plotted by the inhibition ratio versus the concentration, and the concentration at which the inhibition ratio was 50% was the compound IC50The value is obtained. The results are shown in Table 2. The results show the IC of the compounds of this patent on SH-SY5Y cells50Values were all greater than 90 μ M, showing lower neuro-cytotoxicity. Therefore, the compound disclosed by the patent is relatively safe, and provides guarantee for further development of new drugs.
TABLE 2MTT method for testing the toxicity of compounds on SH-SY5Y cells
And (4) conclusion: the data in the table 1 show that all tested 3,3' -disubstituted bipyridine derivatives have good inhibitory activity on the self-aggregation of A beta, and the inhibitory rate on the self-aggregation of A beta is more than 65% under the tested concentration of 20 mu M, wherein the compound 3d shows the strongest inhibitory activity and reaches 93.3%; ultraviolet-visible spectrophotometry tests show that the compound 3d has strong chelated Cu2+And Fe2+And Cu can be suppressed2+And Fe2+Induced A beta aggregation, which can reduce Cu by 96.9%2+Induced A beta aggregation, reduction75.5% of Fe2+Induced a β aggregation; the toxicity of the compound to SH-SY5Y cells tested by MTT method indicates that the compound disclosed by the patent has lower cytotoxicity, wherein the IC of the compound 3d to SH-SY5Y cells50The value is 143.8 mu M, and the safety is high. Therefore, the 3,3' -disubstituted bipyridine derivative can be used as a multifunctional reagent based on the effect of metal-Abeta to prepare the medicament for resisting the Alzheimer disease.
Claims (7)
1. A3, 3 '-disubstituted bipyridine derivative is characterized in that the structural formula of the 3,3' -disubstituted bipyridine derivative is shown as a formula I and a formula II,
wherein R is-H, hydroxy, halogen, -O (CH)2)mCH3Amino substituted with 1 or more methyl or ethyl groups; r1is-O (CH)2)mCH3-OH; n is any integer of 0-3; m is any integer of 0-3; r2is-C (CH)3)3Amino substituted by 1 or more methyl or ethyl groups,
2. The 3,3' -disubstituted bipyridine derivative of claim 1, wherein R is-H, -Cl, -OCH3，-N(CH3)2；R1is-OCH3-OH; n is any integer of 0-3; r2is-C (CH)3)3，
3. The 3,3 '-disubstituted bipyridine derivative of claim 1, wherein the structural formula of the 3,3' -disubstituted bipyridine derivative is as follows:
4. a process for preparing a 3,3' -disubstituted bipyridine derivative of claim 1, comprising the steps of:
s1. the
Mixing with activated copper powder, dissolving in solvent, and reflux reacting under nitrogen protection to obtain compound
S2. the
Heating the mixture and stannous chloride dihydrate in concentrated hydrochloric acid to perform reduction reaction to obtain a compound
S3, mixing
Carrying out condensation reaction with different carboxylic acids to obtain target products
Wherein R is-H, -Cl, -OCH3，-N(CH3)2；R1is-OCH3-OH; n is 0 or 1; r2is-C (CH)3)3，
5. The method according to claim 4, wherein the target product is purified by column chromatography or recrystallization.
6. Use of the 3,3 '-disubstituted bipyridine derivative according to claim 1 for the preparation of a medicament for the prevention and/or treatment of alzheimer's disease, cerebrovascular dementia or myasthenia gravis.
7. The use according to claim 6, wherein the medicament for preventing and/or treating Alzheimer's disease, cerebrovascular dementia or myasthenia gravis is in the form of a tablet, pill, capsule, injection, suspension or emulsion.
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