Design, synthesis and evaluation of genistein-polyamine conjugates as multi-functional anti-Alzheimer agents.

A series of genistein-polyamine conjugates (4a-4h) were designed, synthesized and evaluated as multi-functional anti-Alzheimer agents. The results showed that these compounds had significant cholinesterases (ChEs) inhibitory activity. Compound 4b exhibited the strongest inhibition to acetylcholinesterase (AChE) with an IC50 value of 2.75 μmol/L, which was better than that of rivastigmine (5.60 μmol/L). Lineweaver-Burk plot and molecular modeling study showed that compound 4b targeted both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. Besides, compound 4b showed potent metal-chelating ability. In addition, it was found that 4a-4h did not affect HepG-2 cell viability at the concentration of 10 μmol/L.

Introduction

Alzheimer׳s disease (AD), the most common form of neurodegenerative senile dementia, is associated with selective loss of cholinergic neurons and reduced level of acetylcholine neurotransmitter. It is characterized by memory deficit and progressive impairment of cognitive functions. AD affects millions of elderly people, and the number of patients is expected to increase in the next 20 years. Many factors have been found to be implicated in AD, such as low levels of acetylcholine, β-amyloid deposits, oxidative damage and metal ions, which seem to play significant roles in the disease. Current treatment of AD focuses on increasing cholinergic neurotransmission in the brain by inhibiting cholinesterases (ChEs) with medicines including tacrine, donepezil, rivastigmine and galantamine. Unfortunately, the potential effectiveness offered by the above inhibitors is often limited by their side effects. For example, clinical studies have shown that tacrine has hepatotoxic liability. Due to the multi-pathogenesis of AD, one of the current strategies is to develop novel anti-AD agents with multiple potencies.

Genistein is biosynthetically the simplest isoflavonoid compound of the Leguminosae. It expresses a wide range of biological activities, such as antioxidant, anti-cancer and antimicrobial7, 8, 9. In recent years, it was reported that genistein showed neuroprotective effect and ameliorated learning and memory deficits in the AD rat model10, 11. Besides, a number of genistein derivatives have been reported as anti-AD agents in the past years (Fig. 1)12, 13. These results indicate that genistein could be used as leading compound for the treatment of AD.

Figure 1

Chemical structures of genistein and genistein derivatives.

Polyamines are aliphatic molecules with amine groups distributed along their structures. They have always been the concern of medicinal chemists as a universal template. Our group has been involved in the development of polyamine conjugates as potential drugs for many years16, 17, 18, 19. It was found that quinoline-polyamine conjugates exhibited potent ChEs inhibition activity and polyamine occupied the gorge of AChE. Therefore, in the present study, in order to enhance the pharmacological potential of genistein, a series of genistein conjugates modified with polyamine were designed, synthesized as anti-Alzheimer agents.

Results and discussion

Synthesis of target compounds 4a–4h

The synthetic routes to target compounds are summarized in Scheme 1. The starting material genistein 1 was treated with ethyl 2-chloroacetate in acetone to give intermediate 2, which was heated with K2CO3 in water and then mixed with HCl yielding compound 3. Then compound 3 converted to the intermediates by reaction with amines or Boc protected polyamines in DMF. At last, the Boc groups subsequently were removed using HCl (4 mol/L) at room temperature, producing target compounds 4a–4h as hydrochloride salts. All the structures of the target compounds were confirmed by 1H NMR, ESI-MS and elemental analysis.

Scheme 1

Reaction conditions and reagents: (a) ethyl 2-chloroacetate, K2CO3, I2, acetone, reflux, 6 h; (b) DMSO, K2CO3, H2O, 85 °C, 10 h; (c) 10% HCl, rt, overnight; (d) EDC, NHS, amines, DMF, rt, overnight; (e) 4 mol/L HCl, EtOH, rt, overnight.

Enzyme inhibition assays

All the newly synthesized compounds (4a–4h) were screened against AChE and BChE in vitro according to the modified Ellman method. Rivastigmine was used as control. The ChEs inhibition results were listed as the inhibition ratio at a tested concentration of 50 µmol/L (Table 1). We also tested the IC50 value of compounds 4b and 4h (Table 2).

Table 1

Inhibitory activity of target compounds for AChE and BChE.

Compound	R	X	Inhibition ratios for AChEa (%)	Inhibition ratios for BChEa (%)
4a		0	7.88±3.58	1.19±0.59
4b		1	90.40±1.23	26.05±2.52
4c		1	37.02±2.07	13.77±1.05
4d		1	34.49±2.76	9.02±0.23
4e		2	19.28±1.87	11.72±0.82
4f		2	10.19±0.44	9.52±1.48
4g		2	16.54±1.36	17.88±2.83
4h		2	51.04±0.55	39.20±5.64
Rivastigmine	–	–	86.45±0.71	94.60±3.19

Inhibition ratios for AChE and BChE in the presence of 50 μmol/L compound (mean±SEM of two experiments), AChE from Electric Eel, BChE from equine serum.

Table 2

IC50 of some target compounds for AChE and BChE.

Compound	IC50 AChEa (μmol/L)	IC50 BChEa (μmol/L)
4b	2.75±0.28	>50
4h	46.59±3.87	>50
Genistein	>100	>100
Rivastigmine	5.60±1.50	1.65±0.05

IC50 (μmol/L), 50% inhibitory concentration (mean±SEM of three experiments) of AChE or BChE.

The results showed that all of the target compounds possessed ChEs inhibition activity, and compound 4b exhibited the strongest inhibition to AChE with an IC50 value of 2.75 μmol/L which was better than rivastigmine (5.60 μmol/L), compound 4h also showed good activity with IC50 values of 46.59 μmol/L. Genistein, the parent molecule, inhibited the AChE activity to less than 50% at the concentration of 100 μmol/L (Table 2). It indicated that conjugation polyamines with genistein could increase the inhibition activity of AChE. Besides, it seemed that AChE inhibitory potency of conjugates was closely related to the length and the end group of the polyamine chain. Compounds (4b–4d) modified by diamine were more active than compounds conjugated with monoamine or triamine.

In the assay of BChE inhibtion studies, compound 4h showed the most potent inhibition for BChE with an inhibition rate of 39.20% at the concentration of 50 μmol/L. These compounds showed quite weaker inhibitory effect than AChE. The result indicated that these genistein derivatives might favor the binding to AChE, which was in agreement with the literature reported.

Kinetic characterization of AChE inhibition

The inhibition type of AChE was investigated by graphical analysis of steady state inhibition data (Fig. 2A) using compound 4b as a typical example. The Lineweaver–Burk plots describing 4b inhibition showed both increasing slopes and increasing intercepts with higher inhibitor concentration, indicating a mixed-type inhibition. These results revealed that compound 4b bounded to both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE, which is also in agreement with the results of our molecular modeling studies.

Figure 2

Lineweaver–Burk plots (A) and the docking model (B) for compound 4b with TcAChE.

Molecular modeling

To investigate the interaction mode of compound 4b with TcAChE (protein data bank (PDB) code:1ZGB) molecular modeling was carried out by AUTODOCK 4.0 package with PyMOL program (Fig. 2B)21, 22. The docking result demonstrated that compound 4b exhibited multiple binding modes with AChE. In the 4b-TcAChE complex, compound 4b occupied the entire enzymatic CAS, mid-gorge and PAS. The charged nitrogen made a cation–π interaction with the Trp84. At the midgorge recognition site, the chromone moiety displayed classic π–π stacking with the phenyl ring of Try334, with the ring-to-ring distance being 4.4 Å and 4.7 Å, respectively. At the PAS, the benzene of genistein moiety stacked against the Trp279 through π–π interaction with the distance of 4.4 Å. The result showed that compound 4b was able to bind both CAS and PAS of AChE which was in agreement with the result of kinetic study.

Metal-chelating study

The abnormally high levels of biometals in affected areas of the brain catalyze the formation of reactive oxygen species, which further aggravates oxidative stress contributing to β-amyloid formation. These effects have rendered metal chelators as very promising drugs for AD. Thus, the chelation abilities of compound 4b towards biometal Fe3+, Cu2+ and Zn2+ in water were studied by UV-Vis spectrometry. The results in Fig. 3 shows that the absorbance spectra of 4b exhibited an apparent increase after the addition of Fe3+ or Zn2+, and a red-shift in the maximum absorption from 259 nm to 268 nm occurred. This result indicated that there was an interaction between compound 4b and Fe3+ or Zn2+ ion due to complex formation. However, slight changes were observed in the UV spectrum of 4b after adding Cu2+, indicating the poor chelating ability of compound 4b for Cu2+ ion.

Figure 3

UV–Vis spectrum of compound 4b (20 μmol/L) alone or in the presence of 20 μmol/L Fe3+, Cu2+ and Zn2+.

MTT assay of cell viability

The toxicity of synthesized compounds was determined in HepG-2. Results indicated that the most potent two inhibitors, 4b and 4h, showed no obvious effect on cell viability at concentration of 10 μmol/L, as shown in Table 3. Compared with tacrine, they had lower toxicity on cell viability.

Table 3

MTT assay of HepG-2 cell viability.

Compound	Inhibition ratiosa (%)
4a	6.40
4b	1.53
4c	14.57
4d	1.17
4e	8.51
4f	11.74
4g	16.72
4h	16.30
Tacrine	57.35

Inhibition ratios for HepG-2 cell viability in the presence of 10 μmol/L compound, each sample is the mean of three independent experiments.

Conclusions

In conclusion, a series of novel genistein–polyamine conjugates (4a–4h) were designed, synthesized and evaluated for cholinesterase inhibition, metal-chelating activity and human hepatoma cell viability. Results indicated that these compounds had significant ChEs inhibitory activity. Compound 4b exhibited the strongest inhibition to AChE with an IC50 value of 2.75 μmol/L. Lineweaver–Burk plot and molecular modeling study showed that compound 4b targeted both the CAS and PAS of AChE. Besides, compound 4b showed potent metal chelating ability. In addition, these compounds showed low cytotoxicity by MTT assay in vitro. Compound 4b may be considered to be a novel multi-functional low-toxic drug candidate for the treatment of AD.

Experimental

Materials

Acetylcholinesterase (AChE, E.C. 3.1.1.7, from Electric Eel), butyrylcholinesterase (BChE, E.C. 3.1.1.8, from equine serum), 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), butyrylthiocholine chloride (BTC), and acetylthiocholine chloride (ATC) were purchased from Sigma-Aldrich and rivastigmine hydrochloride standard was purchased from Sunve Pharmaceutical Co., Ltd. (Shanghai, China). 1H NMR spectra were recorded using TMS as the internal standard in DMSO or D2O with a Bruker AV-400 spectrometer at 400 MHz. MS spectra were recorded on a Shimadzu LCMS-2010A instrument with an ESI mass selective detector. Elemental analyses were performed on a Gmbe VarioEL Elemental Instrument. Flash column chromatography was performed with silica gel (200–300 mesh) purchased from Qingdao Haiyang Chemical Co., Ltd.

Chemistry

Synthesis of intermediate 2

Genistein (2.70 g, 10 mmol), ethyl 2-chloroacetate (1.47 g, 12 mmol), anhydrous K2CO3 (0.69 g, 5 mmol) and catalytic amount KI (0.05 g) were added in anhydrous acetone (100 mL), and the mixture was refluxed for 6 h. The solution was filtered and the filter cake was recrystallized from EtOH to give light yellow solid 2.60 g; Yield 73%; MS (ESI) m/z 357.1 [M+H]+.

Synthesis of intermediate 3

Compound 2 (0.71 g, 2 mmol), 5% Na2CO3 and DMSO (40 mL) were heated at 85 °C for 10 h. Then the reaction mixture was poured into 10% HCl (300 mL), kept overnight and filtered. The filter cake was recrystallized from EtOH to give light brown solid 0.46 g; Yield 70%.

Synthesis of target compounds 4a–4h

Compound 3 (0.36 g, 1 mmol), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC, 0.29 g, 1.5 mmol) and N-hydroxysuccinimide (NHS, 0.17 g, 1.5 mmol) were stirred at room temperature for 1.5 h, and then amines or Boc protected amines (1.2 mmol) were added. The mixture was stirred at room temperature overnight. Then the solvent was poured into water and extracted with ethyl acetate (20 mL×3). The solution was dried over anhydrous Na2SO4 and concentrated. The intermediates were purified by flash chromatography with chloroform/methanol/ammonia (20:1:0.5, v/v/v) elution.

The intermediates were dissolved in EtOH (10 mL) and stirred at 0 °C for 10 min. Then 4 mol/L HCl (diluted with EtOH) was added dropwise at 0 °C. The reaction mixture was stirred at room temperature overnight. The solution typically gave a white solid precipitate. The precipitate was collected and washed several times with absolute ethanol and ether, and dried under vacuum to give pure target compounds 4a–4h.

N-butyl-2-(5-hydroxy-3-(4-hydroxyphenyl)-4-oxo-4H-chromen-7-yloxy)acetamide (): White solid; m.p. 180–183 °C; Yield 63%. 1H NMR (400 MHz, DMSO-d6): δ 12.95 (s, 1H), 9.63 (s, 1H), 8.43 (s, 1H), 8.16 (s, 1H), 7.44–7.36 (m, 2H), 6.87–6.79 (m, 2H), 6.66 (d, J=2.3 Hz, 1H), 6.45 (d, J=2.3 Hz, 1H), 4.61 (s, 2H), 3.13 (dd, 2H, J=13.0, 6.8 Hz), 1.46–1.37 (m, 2H), 1.26 (dd, J=15.1, 7.5 Hz), 0.86 (t, J=7.3 Hz). ESI-MS m/z: 384.4 [M+H]+. Anal. Calcd. for C21H21NO6·0.1C2H5OH: C, 65.52; H, 5.76; N, 3.60, Found C, 65.44; H, 5.82; N, 3.43.

N-(2-(dimethylamino)ethyl)-2-(5-hydroxy-3-(4-hydroxyphenyl)-4-oxo-4H-chromen-7-yloxy)acetamide hydrochloride (): White solid; m.p. 143–145 °C; Yield 49%. 1H NMR (400 MHz, D2O): δ 7.99–7.89 (m, 1H), 7.22 (s, 2H), 6.81 (s, 2H), 6.41–6.34 (m, 1H), 6.22 (s, 1H), 4.36 (s, 2H), 3.52 (s, 2H), 3.19 (s, 2H), 2.58 (s, 6H). ESI-MS m/z: 399.4 [M+H-HCl]+. Anal. Calcd. for C21H21N2O6·HCl·0.4H2O: C, 57.05; H, 5.43; N, 6.34, Found C, 57.03; H, 5.52; N, 6.73.

N-(3-aminopropyl)-2-(5-hydroxy-3-(4-hydroxyphenyl)-4-oxo-4H-chromen-7-yloxy)acetamide hydrochloride (): White solid; m.p. 187–189 °C; Yield 50%. 1H NMR (400 MHz, D2O): δ 7.98 (s, 1H), 7.27 (d, 2H, J=8.5 Hz), 6.87 (d, 2H, J=8.4 Hz), 6.38 (s, 1H), 6.23 (d, 1H, J=2.0 Hz), 4.31 (s, 2H), 3.30 (t, 2H, J=6.8 Hz), 3.00–2.93 (m, 2H), 1.90–1.82 (m, 2H). ESI-MS m/z: 385.4 [M+H-HCl]+. Anal. Calcd. for C20H20N2O6·HCl·3.5H2O: C, 49.64; H, 5.83; N, 5.79, Found C, 49.69; H, 5.60; N, 5.58.

N-(4-aminobutyl)-2-(5-hydroxy-3-(4-hydroxyphenyl)-4-oxo-4H-chromen-7-yloxy)acetamide hydrochloride (): White solid; m.p. 214–216 °C; Yield 48%: 1H NMR (400 MHz, D2O): δ 7.83 (s, 1H), 7.14 (s, 2H), 6.76 (d, 2H, J=7.9 Hz), 6.21 (s, 1H), 6.08 (s, 1H), 4.14 (s, 2H), 3.12 (t, 2H, J=6.8 Hz), 2.95–2.85 (m, 2H), 1.50 (dd, 4H, J=31.9, 7.1 Hz). ESI-MS m/z: 399.4 [M+H-HCl]+. Anal. Calcd. for C21H22N2O6·HCl·2.5H2O: C, 52.56; H, 5.88; N, 5.84, Found C, 52.64; H, 5.51; N, 5.57.

2-(5-hydroxy-3-(4-hydroxyphenyl)-4-oxo-4H-chromen-7-yloxy)-N-(4-(piperazin-1-yl)butyl)acetamide hydrochloride (): White solid; m.p. 215–217 °C; Yield 46%: 1H NMR (400 MHz, D2O): δ 7.95 (s, 1H), 7.23 (d, 2H, J=8.4 Hz), 6.81 (d, 2H, J=8.5 Hz), 6.36 (s, 1H), 6.19 (s, 1H), 4.30 (s, 2H), 3.47 (s, 8H), 3.14 (dd, 4H, J=14.9, 7.6 Hz), 1.62 (s, 2H), 1.50–1.40 (m, 2H). ESI-MS m/z: 468.5 [M+H-2HCl]+. Anal. Calcd. for C25H29N3O6·2HCl·0.4H2O: C, 54.83; H, 5.85; N, 7.67, Found C, 54.49; H, 5.78; N, 7.63.

N-(3-(3-aminopropylamino)propyl)-2-(5-hydroxy-3-(4-hydroxyphenyl)-4-oxo-4H-chromen-7-yloxy)acetamide hydrochloride (): White solid; m.p. 147–149 °C; Yield 37%: 1H NMR (400 MHz, D2O): δ 7.96 (s, 1H), 7.24 (d, 2H, J=7.8 Hz), 6.82 (d, 2H, J=8.0 Hz), 6.37 (s, 1H), 6.20 (s, 1H), 4.31 (s, 2H), 3.24 (t, 2H, J=6.6 Hz), 3.06–2.92 (m, 6H), 1.97 (dt, J=15.8, 7.9 Hz), 1.86–1.77 (m, 2H). ESI-MS m/z: 442.5 [M+H-2HCl]+. Anal. Calcd. for C23H27N3O6·2HCl·1.2H2O: C, 51.54; H, 5.90; N, 7.84, Found C, 51.86; H, 5.92; N, 7.45.

N-(4-(3-aminopropylamino)butyl)-2-(5-hydroxy-3-(4-hydroxyphenyl)-4-oxo-4H-chromen-7-yloxy)acetamide hydrochloride (): QWhite solid; m.p. 176–178 °C; Yield 35%: 1H NMR (400 MHz, D2O): δ 7.86 (s, 1H), 7.15 (d, 2H, J=8.6 Hz), 6.74 (d, 2H, J=8.6 Hz), 6.23 (d, 1H, J=2.0 Hz), 6.08 (d, 1H, J=2.2 Hz,), 4.13 (s, 2H), 3.08 (t, 2H, J=6.9 Hz), 3.01–2.90 (m, 6H), 1.92 (dd, 2H, J=10.5, 5.3 Hz), 1.52 (d, 2H, J=6.3 Hz), 1.45–1.37 (m, 2H). ESI-MS m/z: 456.5 [M+H-2HCl]+. Anal. Calcd. for C24H29N3O6·2HCl·4H2O: C, 48.00; H, 6.55; N, 7.00, Found C, 48.01; H, 6.81; N, 7.38.

N-(4-(4-aminobutylamino)butyl)-2-(5-hydroxy-3-(4-hydroxyphenyl)-4-oxo-4H-chromen-7-yloxy)acetamide hydrochloride (): White solid; m.p. 187–189 °C; Yield 37%: 1H NMR (400 MHz, D2O): δ 7.62 (s, 1H), 7.04 (d, 2H), 6.71 (d, 2H, J=7.8 Hz), 5.97 (s, 2H), 3.98 (s, 2H), 3.13 (s, 2H), 3.04–2.95 (m, 6H), 1.72 (s, 4H), 1.63 (s, 2H), 1.49 (d, 2H, J=7.0 Hz). ESI-MS m/z: 470.5 [M+H-2HCl]+. Anal. Calcd. for C25H31N3O6·2HCl·2.5H2O: C, 51.11; H, 6.52; N, 7.15, Found C, 51.03; H, 6.29; N, 6.93.

All the assays were under 0.1 mol/L KH2PO4/K2HPO4 buffer, pH 8.0, using a Shimadzu 2450 Spectrophotometer. Enzyme solutions were prepared to give 2.0 units/mL in 2 mL aliquots. The assay medium contained phosphate buffer (pH 8.0), 50 µL of 0.01 mol/L DTNB, 10 µL of enzyme, and 50 µL of 0.01 mol/L substrate (ATC). The substrate was added to the assay medium which contained enzyme, buffer and DTNB with inhibitor (0, 5, 10, 20, 35, and 50 µmol/L) after 15 min of incubation time. The activity was determined by measuring the increase in absorbance at 412 nm at 1 min intervals at 37 °C. Calculations were performed according to the method of the equation in Ellman et al.. The in vitro BChE assay used the similar method described above. The concentration of compound that effected 50% inhibition of ChEs activities (IC50) was calculated by nonlinear regression of the inhibition ratio–concentration curve, using Origin 7.5 program.

Kinetic characterization of AChE was performed using a reported method. Six different concentrations of substrate were mixed in the 1 mL 0.1 mol/L KH2PO4/K2HPO4 buffer (pH 8.0), containing 50 µL of DTNB, 10 µL AChE and 50 µL substrate. Test compound was added into the assay solution and pre-incubated with the enzyme at 37 °C for 15 min, followed by the addition of substrate. Kinetic characterization of the hydrolysis of ATC catalyzed by AChE was done spectrometrically at 412 nm. A parallel control with no inhibitor in the mixture, allowed adjusting activities to be measured at various times.

The crystal structure of the torpedo AChE (code ID: 1ZGB) was obtained in the PDB after eliminating the inhibitor and water molecules. The 3D structure of compound 4b was prepared as similar as previously described.

Docking studies were carried out using the AUTODOCK 4.0 program using ADT. The enzyme structure was used as an input for the AUTOGRID program. AUTOGRID performed a precalculated atomic affinity grid maps for each atom type in the ligand plus an electrostatics map and a separate desolvation map present in the substrate molecule. All maps were calculated with 0.375 Å spacing between grid points. The center of the grid box was placed at the bottom of the active site gorge (AChE [2.781 64.383 67.971]). The dimensions of the active site box were set at 50 Å×46 Å×46 Å.

Flexible ligand docking was performed for the compounds. Docking calculations were carried out using the Lamarckian genetic algorithm (LGA) and all parameters were the same for each docking.

The chelating studies were made in water using a UV-Vis spectrophotometer (SHIMADZC UV-2450PC). The absorption spectrum of compound 4b (20 μmol/L), alone or in the presence of FeCl3, CuSO4 or ZnCl2 (20 μmol/L), was recorded with wavelength ranging from 200 to 500 nm after incubating for 30 min at room temperature. The final volume of reaction mixture was 1 mL, and the final concentrations of tested compound and metals were 20 µmol/L.

MTT assay of HepG-2 cell viability

Cells were cultured at 37 °C under a 5% CO2 atmosphere. The antiproliferative ability of compounds was evaluated in HepG-2 cells by the conversion of MTT to a purple formazan precipitate as previously described. Cells were seeded into 96-well plates at 5×103 cells/well. After 12 h, 10 μmol/L of compounds was subsequently added and incubated for 48 h. The inhibition rate was calculated from plotted results using untreated cells as 100%.