WO2012075849A1

WO2012075849A1 - 4',5-dihydroxy-7-[4-(n, n-diethylamino) butoxy] isoflavones, and preparation method and application thereof

Info

Publication number: WO2012075849A1
Application number: PCT/CN2011/080037
Authority: WO
Inventors: 谭仁祥; 史大华; 吴俊华; 宴志强; 张丽娜; 王玉蓉
Original assignee: 南京大学
Priority date: 2010-12-06
Filing date: 2011-09-22
Publication date: 2012-06-14
Also published as: CN102127046B; CN102127046A

Abstract

Disclosed is a genistein derivative, 4',5-dihydroxy-7-[4-(N, N-diethylamino) butoxy] isoflavones, and a preparation method and application thereof. The 4',5-dihydroxy-7-[4-(N, N-diethylamino) butoxy] isoflavones is acquired via chemical modification of genistein, has acetylcholinesterase inhibiting activity, and estrogenic activity, and offers protection for nerve cells. As a compound having multiple active target sites, the compound can be used for the preparation of an anti-Alzheimer disease medicament.

Description

4',5-dihydroxy-7-[4-(N,N-diethylamino)butoxy]isoflavone and preparation method and application thereof

The invention relates to a genistein derivative and a preparation method and application thereof, in particular to 4',5-dihydroxy- _7- [4-(N,N-diethylamino)butoxy]isoflavone And its preparation method and application. Background technique

Alzheimer's disease is a common senile neurodegenerative disorder with a high incidence and has become one of the diseases that threaten the lives and health of the elderly in modern society. Typical pathological changes in the disease include amyloid deposition to form senile plaques, neurofibrillary tangles, basal forebrain cholinergic dysfunction, and extensive neuronal loss and synaptic morphology in the cortex, hippocampus (see: Roberson ED, Mucke L) 100 years and counting: prospects for defeating Alzheimer's disease. Science 2006;314 (5800): 781-4).

The study found that the lesions in the brain endothelium and hippocampus of patients with Alzheimer's disease were more obvious, and the brain basal ganglia cholinergic neurons were most destroyed. Therefore, by increasing the content of acetylcholine, or acting on choline receptors, the function of the central cholinergic system can be enhanced and improved, thereby achieving the goal of promoting mental healing (Baskin DS, Browning JL, Pirozzolo FJ, Korporaal S, Baskin JA, Appel SH. Brain choline acetyltransferase and mental function in Alzheimer disease. Archives of Neurology 1999;56(9):1121-3). Acetylcholinesterase inhibitors are the first and most mature class of drugs developed for the treatment of Alzheimer's disease clinically based on this theory. ( Taylor P. Development of acetylcholinesterase inhibitors in the therapy of Alzheimer's disease. Neurology 1998; 51 (Supplement 1), S30-S35), this class of drugs is effective in patients with mild to moderate Alzheimer's disease, which can partially improve their cognitive function and other symptoms. A variety of acetylcholinesterase inhibitors have been used in the treatment of Alzheimer's disease.

However, the efficacy of acetylcholinesterase inhibitors depends on the completeness of cholinergic neurons and is not suitable for severe patients. At the same time, this class of drugs can only increase the content of acetylcholine, and can not prevent the progressive degeneration of central cholinergic neurons. As the disease progresses, the central cholinergic neurons die progressively, and the acetylcholinesterase inhibitor drugs The effect will gradually decrease. Therefore, the search for a new type of acetylcholinesterase inhibitor that inhibits acetylcholinesterase activity and prevents the death of central cholinergic neurons is the key to finding a therapeutic drug for Alzheimer's disease.

Amyloid accumulation causes the formation of senile plaques, promotes the activation of neurotoxic pathways by inflammatory reactions, leads to dysfunction and death of nerve cells, and is one of the main causes of senile plaques, causing neuronal degeneration ( Riviere C, Richard T, Vitrac X, Merillon JM, Vails J, Monti JP. New polyphenols active on Beta-amyloid aggregation. Bioorganic and Medicinal Chemistry Letters 2008; 18(2): 828-31). Stone research shows that estrogen can inhibit amyloid-induced neuronal cell death and has a good neuroprotective effect. However, estrogen causes hyperplasia and carcinogenesis of estrogen-related non-neuronal cells such as breast cells and endometrial cells in the treatment of Alzheimer's disease, and thus is limited in clinical applications (Bang OY, Hong HS, Kim DH, Kim) H, Boo JH, Huh K, et al. Neuroprotective effect of genistein against beta amyloid-induced neurotoxicity. Neurobiology of Disease 2004; 16(1): 21 -8).

Genistein (4',5,7-trihydroxyisoflavone) is the main biologically active substance of soy isoflavones, which has a strong estrogenic activity. Studies have shown that genistein can inhibit amyloid-induced neuronal cell death without causing normal cell carcinogenesis like estrogen, and has a good application prospect in the treatment of Alzheimer's disease. However, this compound does not have acetylcholinesterase inhibitory activity, and its application is limited.

Considering that the development of Alzheimer's disease is caused by many factors, "multi-target-directed drug design strategy" (ie, a compound molecule can interact with multiple targets in the development of Alzheimer's disease). Design concept) was introduced into the development of therapeutic drugs for Alzheimer's disease (Bolognesi ML, Cavalli A, Valgimigli L, Bartolini

M, Rosini M, Andrisano V, et al. Multi-target-directed drug design strategy: from a dual binding site acetylcholinesterase inhibitor to a trifunctional compound against Alzheimer's disease.

Journal of Medicinal Chemistry 2007;50(26):6446-9 Alzheimer's disease, which can simultaneously act on multiple targets, has become a trend in drug development for Alzheimer's disease. The chemical modification of genistein to obtain a multi-target compound having both estrogenic activity and acetylcholinesterase inhibitory activity has a good application prospect in the treatment of Alzheimer's disease. Summary of the invention

The object of the present invention is to provide a genistein derivative having both acetylcholinesterase inhibitory activity and estrogenic activity, specifically 4',5-dihydroxy-7-[4-(N,N-diethylamino) Butoxy]isoflavone, and a process for the preparation of the same and for the preparation of a medicament for the treatment of Alzheimer's disease.

The present invention employs the following technique: A genistein derivative having the structure represented by formula (I):

The genistein derivative 4',5-dihydroxy-7-[4-(N,N-diethylamino)butoxy]isoflavone can be synthesized as follows, genistein and 1, 4 -Dibromobutane reaction to produce the intermediate 4',5-dihydroxy-7-(4-bromobutoxy)isoflavone, The obtained intermediate product is reacted with diethylamine to obtain a target genistein derivative.

The method specifically includes the following steps:

1) Preparation of 4',5-dihydroxy-7-(4-bromobutoxy)isoflavone

Genistein (0.27 g, l mmol), 1, 4-dibromobutane (5.4 g, 25 mrnol), K ₂ C0 ₃ (0.07 g, 0.5 mmol) dissolved in 60 mL of anhydrous DMF, 40 ° Ultrasonic reaction at C-50 ° C for 1-2 hours. After completion of the reaction, the reaction mixture was cooled to room temperature, and the insoluble material was filtered. The filtrate was evaporated under reduced pressure to give a pale yellow solid.

2) Preparation of 4', 5-dihydroxy-7-[4-(N, N-diethylamino)butoxy]isoflavone

The compound 4',5-dihydroxy-7-(4-bromobutoxy)isoflavone (0.40 g, 1 mmol) was dissolved in 5 mL of anhydrous DMF, and diethylamine C 0.37 g was slowly added dropwise. 5 mmol), stirring at 80 ° C - 90 ° C for 1-2 hours, the reaction product was slowly poured into a 100 mL ice water mixture, allowed to stand overnight, the solid was filtered off, and acetone was recrystallized.

By studying the biological activity of the compound, it was found that the compound has a strong acetylcholinesterase inhibitory action, an estrogenic activity comparable to the parent compound genistein, and a protective effect against amyloid-induced neuronal damage. Therefore, the compound can be used as a multi-target anti-Alzheimer's disease compound for the preparation of a medicament for the prevention and treatment of Alzheimer's disease.

The invention also relates to the use of the genistein derivative described in the manufacture of a medicament for the treatment of Alzheimer's disease.

4',5-Dihydroxy-7-[4-(N,N-diethylamino)butoxy]isoflavone as a multi-targeted anti-Alzheimer's disease compound capable of simultaneously having acetylcholine Esterase inhibitory activity, estrogenic activity, and neuronal cell protection have a good therapeutic effect on Alzheimer's disease in the preparation of a medicament for treating Alzheimer's disease.

The genistein derivative 4',5-dihydroxy-7-[4-(N,N-diethylamino)butoxy]isoflavone obtained by chemically modifying genistein according to the invention has both acetylcholine ester Enzyme inhibitory activity and estrogen activity, which have protective effects on amyloid-induced neuronal injury, and can be used as a multi-targeted anti-Alzheimer's disease compound for the preparation of drugs for the treatment of Alzheimer's disease. in.

The invention is further illustrated by the following examples, but the scope of the invention is not limited by the specific examples, but is defined by the claims.

detailed description

Example 1: 4',5-Dihydroxy-7-[4-(N,N-diethylamino)butoxy]isoflavone) and preparation thereof Preparation of the genistein derivative 4',5-dihydroxy-7-[4-(N,N-diethylamino)butoxy]isoflavone, comprising the following steps:

Preparation of 1,4',5-dihydroxy-7-(4-bromobutoxy)isoflavone

Genistein (0.27 g, 1 mmol), 1, 4-dibromobutane (5.4 g, 25 mmol), K ₂ C0 ₃ (0.07 g, 0.5 mmol) dissolved in 60 mL of anhydrous DMF, 40 ° The ultrasonic reaction was carried out for 1.5 hours under C. After completion of the reaction, the reaction mixture was cooled to room temperature. After the residue was filtered, the filtrate was evaporated to give a pale yellow solid, which crystallised from acetone to give pale yellow needle crystals, yield 86%, m.

¹ H NMR (DMSO-de): 1.83 (m, 2H), 1.94 (m, 2H), 3.60 (t, J = 6.2 Hz, 2H), 4.12 (t, J = 6.4 Hz, 2H), 6.40 (d , J = 2.0 Hz, 1H), 6.65 (d, J = 2.0 Hz, 1H), 6.80 (d, J = 8.5 Hz, 2H), 7.38 (d, J = 8.5 Hz, 2H), 8.40 (s, 1H ), 9.65 (s, 1H), 12.95 (s, 1H). ESI-MS C ₁₉ H ₁₇ Br0 ₅ [M+H] ⁺ 405. Anal. Calcd for C19H17 Br0 ₅ : C, 56.31; H, 4.23. : C, 56.28; H, 4.27.

Preparation of 2, 4', 5-dihydroxy-7-[4-(N, N-diethylamino)butoxy]isoflavone

The compound 4',5-dihydroxy-7-(4-bromobutoxy)isoflavone (0.40 g, 1 mmol) was dissolved in 5 mL of anhydrous DMF, and diethylamine (0.37 g, 5) was slowly added dropwise. Ment), stirring at 80 ° C - 90 ° C for 1 hour, the reaction product was slowly poured into 100 mL of ice water mixture, allowed to stand overnight, the solid was filtered off, acetone was recrystallized, yield 82%, melting point 170-171 ° C .

1 H NMR (DMSO-de ): 0.94 (t, J = 7.2 Hz, 6H), 1.52 (m, 2H), 1.72 (m, 2H), 2.40 (m, 2H), 3.33 (t, J = 7.0 Hz , 4H), 4.10 (t, J = 6.5 Hz, 2H), 6.39 (d, J = 2.0 Hz, 1H), 6.64 (d, J = 2.0 Hz, 1H), 6.82 (d, J = 8.5 Hz, 2H ), 7.39 (d, J = 8.5 Hz, 2H), 8.40 (s, 1H), 9.60 (s, 1H), 12.93 (s, 1H). ESI-MS

C ₂₃ H ₂₇ N0 ₅ [M+H] ⁺ 398. Anal. calc. for C ₂₃ H ₂₇ N0 ₅ : C, 69.50; H, 6.85; N, 3.52. Found: C, 69.60; H, 6.79; N, 3.58.

Example 2: Genistein derivative acetylcholinesterase inhibitory activity of 4',5-dihydroxy-7-[4-(N,N-diethylamino)butoxy]isoflavone

1,4',5-Dihydroxy-7-[4-(N,N-diethylamino)butoxy]isoflavone inhibits electroacupuncture acetylcholinesterase

The enzymatic reaction was carried out in a phosphate buffer of 0.1 M pH 8.0 at 25 ° C for a total reaction volume of 200 μL containing 3.33 mM 5,5 '-dithio-linked O-nitrobenzoic acid) 20 μ , 0.35 U /mL acetylcholinesterase solution 20 and 5.3 mM thioacetylcholine solution 20 μL·, test the compound solution 10 μL·, and measure at 412 nm for 5 min with a microplate reader (Sunrise, Tecan, Austria). The sample was repeated three times. The optical density value of the unfilled compound pores was taken as 100%, and the optical density of the pores of the compound was compared, and the percentage of reduction was the enzyme inhibition rate. The compound is tested for at least 5 concentration gradients against acetylcholinesterase The inhibitory activity was plotted by the logarithm of the compound concentration versus the inhibition rate of IC _5Q (concentration of the compound at 50% inhibition of enzyme activity).

The present invention tested the acetylcholinesterase inhibitory activity of 4',5-dihydroxy-7-[4-(N,N-diethylamino)butoxy]isoflavone. The compound has significant acetylcholinesterase inhibitory activity, and its IC _5Q value for inhibiting acetylcholinesterase is 0.17 μΜ, which is comparable to the positive control tacrine (IC _5Q value 0.17 μΜ), and the parent compound genistein at the 100 μΜ There was no significant acetylcholinesterase inhibitory activity at the concentration.

2,4',5-Dihydroxy-7-[4-(indolyl-diethylamino)butoxy]isoflavone inhibits electrokinetics of acetylcholinesterase

The activity of AChE at 0 μΜ, ΙΟ μΜ, 20 μΜ and 40 μΜ at substrate concentrations of 50 μΜ, 100 μΜ, 200 μΜ, 300 μΜ and 400 μΜ was determined at 25 °C. The measured data was plotted using the Lineweaver-Burk method, and the inhibition type of the enzyme was observed. The slope of the Lineweaver-Burk plot is plotted for the concentration of the compound (the dissociation constant of the compound in combination with the enzyme). Double reciprocal plots show that the compound is a non-competitive inhibitor of acetylcholinesterase. The Ki value of the compound inhibiting acetylcholinesterase was 0.23 ± 0.01 μΜ (η = 6).

3. Computer simulation of the interaction of 4',5-dihydroxy-7-[4-(indolyl-diethylamino)butoxy]isoflavone with acetylcholinesterase The crystal structure of electroporation AChE was downloaded from the PDB database. (PDB numbers are 1C2B, respectively), and energy optimization is performed using Amber9 software. The three-dimensional structure of the compound was constructed and optimized using Chemoffice 3D software. Molecular docking with AUTODOCK 4.0 software. Molecular docking revealed that the compound binds to the distal anion binding site of acetylcholinesterase, the 4-OH of the compound forms a hydrogen bond with S289 at the distal anion binding site, and the other moiety can interact with the acetylcholine ester via hydrophobic interactions. The distal anion binding site of the enzyme interacts with the W82, Y120, W282, R292, Y333 F334, Y337, H443 residues in the vicinity.

Example 3: Estrogen activity of genistein derivative 4 ',5-dihydroxy-7-[4-(indolyl-diethylamino)butoxy]isoflavone

1,4 ',5-Dihydroxy-7-[4-(indolyl-diethylamino)butoxy]isoflavone promotes estrogen-dependent proliferation of MCF-7 cells

MCF-7 cells at a concentration of 1.5X 10 ⁴ seeded in 96-well plates. After the cells were cultured for 24 h, the original medium was aspirated, and the 1640 medium containing 5% activated carbon/dextran-treated fetal bovine serum was replaced. Continue to culture for 48 h to deplete the estrogen in the cells. The test was divided into a blank control group, a drug treatment group, and a positive control group. The blank group was replaced with 1640 medium containing 5% activated carbon/dextran for fetal bovine serum; the drug treatment group was replaced with 100 μΜ, 50 μΜ, 10 μΜ, 1 μΜ, 0.1 μΜ, 0.01 μΜ, 0.001 μΜ, 0.0001 μΜ And 0.00001 μΜ 4 ',5-dihydroxy-7-[4-(Ν,Ν-diethylamino) 1640 medium of butoxylated] isoflavones (containing 5% activated carbon/dextran for fetal bovine serum); the positive control group was changed to a concentration of 100 μΜ, 50 μΜ, 10 μΜ, 1 μΜ, 0.1 μΜ, 0.01 μΜ, 0.001 μΜ, 0.0001 μΜ and 0.00001 μΜ genistein in 1640 medium (containing 5% activated carbon/dextran for fetal bovine serum). After 96 h of culture, MTT assayed cell viability. Cell proliferation rate (%) = ΔΟϋ drug treatment / ΔΟϋ blank control χ 100. The experimental results are shown in Table 1.

Table 1 4 ',5-Dihydroxy-7-[4-(N,N-diethylamino)butoxy]isoflavone promotes proliferation of MCF-7 cells

(n=6), cell viability (%)

As can be seen from Table 1, when the compound concentration is lower than Ι μΜ, both the compound and the genistein can promote the proliferation of estrogen-dependent MCF-7 cells, i.e., have estrogen activity. At ΙμΜ, the compound and genistein have a stronger effect on the growth of MCF-7 cells, and the proliferation of MCF-7 cells is basically equivalent. However, above this concentration, the number of viable cells is reduced due to the cytotoxic effect of the compound and genistein on MCF-7 cells. At compound concentrations of 0.01 and 0.001 μΜ, the survival rate of MCF-7 cells treated with this compound was slightly higher than that of genistein treated cells, and the estrogenic activity of the compound was presumably stronger than that of its parent compound genistein.

2. Computer simulation of the interaction of 4',5-dihydroxy-7-[4-(indolyl-diethylamino)butoxy]isoflavone with estrogen receptor

ERc (the crystal structure of the sum is downloaded from the PDB database (PDB numbers 2I0J and 2I0G respectively) and energy optimized with Amber9 software. 4 ' ,5-Dihydroxy-7-[4-(N,N-II The three-dimensional structure of amino)butoxy]isoflavones was constructed and optimized using Chemoffice 3D software. Molecular docking was performed using AUTODOCK 4.0 software. Molecular docking revealed that the compound interacts with ERc (and both. -8.18 kcal/mol is much higher than its Binding energy of ER^ (-11.66 kcal/mol). The dissociation constants of this compound and ERc (and ER^ were 1.01 μΜ and 2.86 ηΜ, respectively. Therefore, the compound has a binding ability to ER^ which is about 350 times stronger and has a higher selectivity to ER^ (Table 2). Table 2 Binding energy and value of 4 ',5-dihydroxy-7-[4-(N,N-diethylamino)butoxy]isoflavone interaction with ERcc

Parameter ER ER^

Binding Energy -8.18 kcal/mol -11.66 kcal/mol

1.01 μΜ 2.86 nM

The 4'-OH of the compound forms a hydrogen bond with Ν483 of the ΕΚβ receptor, and the ruthenium atom on the 7-0 substituent forms a hydrogen bond with Ε305 of the ΕΚβ acceptor. The anthracene ring of this compound and the aliphatic chain attached to 7-0 were inserted into a lipophilic pocket formed by M340, L339, L343, F356 and L298 by hydrophobic interaction. The benzene ring of the B ring is bonded to L476 and M295 by a hydrophobic interaction.

The interaction of this compound with ERcc is weaker than that of ΕΚβ. The compound is embedded in the hydrophobic pocket of ERa consisting of M343, L346, F404, M421, 1424, L523 by hydrophobic interaction, but has no strong hydrogen bonding interaction.

Example 4 _: Protective effect of 4',5-dihydroxy-7-[4-(N,N-diethylamino)butoxy]isoflavone on amyloid-induced SH-SY5Y cell injury

SH-SY5Y cells were seeded in multi-well plates at lx10 ⁴ /mL, and cultured for 24 h, and then replaced with DMEM medium containing 10 μ of retinoic acid (RA). The medium was changed every two days to treat the day and induce cell differentiation. The cells differentiated were gently washed twice with D-Hank's solution, and the test was divided into a blank control group, a model group, a drug treatment group, and a positive control group. The blank control group was replaced with phenol red 1640 medium containing 10% activated carbon/dextran for fetal bovine serum; the model group was added with a final concentration of 15 μΜ amyloid; the drug treatment group was added with different concentrations of 4 ', 5- Dihydroxy-7-[4-(N,N-diethylamino)butoxy]isoflavone was added for 15 h before adding 15 μΜ amyloid; positive control group was added with estrogen 2 nM or genistein 1 nM Amyloid was added after 3 h. Cell viability was measured by MTT assay after 72 h of cell culture in each treatment group. The experimental results are shown in Table 3.

Table 3 Protective effect of 4',5-dihydroxy-7-[4-(N,N-diethylamino)butoxy]isoflavone on β-amyloid-induced SH-SY5Y cell injury

Group cell survival rate (%) blank control 100 ± 5.89 Model group 7.64 ± 3.85

4',5-dihydroxy-7-[4-(N,N-diethylamino)butoxy]isoflavone (ΙΟΟΟηΜ) 90.98±4.48

4',5-dihydroxy-7-[4-(N,N-diethylamino)butoxy]isoflavone (ΙΟΟηΜ) 82.97±6.52

4',5-dihydroxy-7-[4-(N,N-diethylamino)butoxy]isoflavone (ΙΟηΜ) 82.42±6.01

4',5-Dihydroxy-7-[4-(N,N-diethylamino)butoxy]isoflavone (InM) 94.21 ±8.59

4',5-dihydroxy-7-[4-(N,N-diethylamino)butoxy]isoflavone (0.1 nM) 85.74±4.19

4',5-Dihydroxy-7-[4-(N,N-diethylamino)butoxy]isoflavone (0.01 nM) 85.31 ±3.20 genistein (0.001 nM) 85.50±2.92 estrogen (InM) 93.85±4.21 It can be seen from Table 3 that after treatment of differentiated SH-SY5Y cells with 15 μΜ amyloid for 72 h, the cell survival rate decreased to 75.6% of the blank control. Treatment of cells with 1000 nM, 1 nM and 0.1 nM significantly improved cell viability. The survival rate of SH-SY5Y cells in the treated group was also increased, but there was no significant difference compared with the model group. InM concentration of this compound has the strongest cytoprotective effect (cell survival rate 94.2%), better than 2 nM estrogen and the same concentration of the parent compound genistein (cell survival rate of 93.9% and 85.5%, respectively)

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Claims

1 - a genistein derivative 4',5-dihydroxy-7-[4-(N,N-diethylamino)butoxy] having the structure represented by formula (I),

2. A process for the preparation of a genistein derivative according to claim 1, characterized in that the genistein is reacted with, 4-dibromobutanoxime to obtain an intermediate product 4',5-dihydroxy-7. - (4-Bromobutoxy)isoflavone, the obtained intermediate product is reacted with diethylamine to give 4',5-dihydroxy-7-[4-(N,N-diethylamino)butoxy] Isoflavones.

The method for producing a genistein derivative according to claim 2, which comprises the following steps

1) Preparation of 4',5-dihydroxy-7-(4-bromobutoxy)isoflavone

Genistein 0.27 g, 1, 4-dibromobutane 5.4 g, K ₂ CO ₃ 0.07 g dissolved in 60 mL of anhydrous DMF, ultrasonically reacted at 40 ° C - 50 ° C for 1-2 hours; reaction completed After the reaction mixture was cooled to room temperature, the insoluble matter was filtered off, and the filtrate was evaporated under reduced pressure to give a pale yellow solid, which crystallised from acetone to give pale yellow needle crystals;

2) Preparation of 4', 5-dihydroxy-7-[4-(N, N-diethylamino)butoxy]isoflavone

The compound 4',5-dihydroxy-7-(4-bromobutoxy)isoflavone 0.40 g obtained in the step 1) was dissolved in 5 mL of anhydrous DMF, and diethylamine 0.37 g, 80° was slowly added dropwise. After stirring at C-90 ° C for 1-2 hours, the reaction product was slowly poured into a 100 mL ice-water mixture, allowed to stand overnight, and the solid was filtered, and then crystallised from acetone.

4. Use of a genistein derivative according to claim 1 for the preparation of a medicament for the treatment of Alzheimer's disease.

The use of the genistein derivative according to claim 4 for the preparation of a medicament for treating Alzheimer's disease, characterized in that the genistein derivative has acetylcholinesterase inhibitory activity, estrogenic activity and nerve Cytoprotection.