Bioorganic & Medicinal Chemistry Letters 24 (2014) 4749–4753
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Synthesis and acetylcholinesterase inhibitory activity of Mannich base derivatives flavokawain B Hao-ran Liu, Xue-qin Huang, Ding-hui Lou, Xian-jun Liu, Wu-kun Liu, Qiu-an Wang ⇑ College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China
a r t i c l e
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Article history: Received 23 April 2014 Revised 25 July 2014 Accepted 30 July 2014 Available online 27 August 2014 Keywords: Flavokawain B Chalcone Mannich bases Synthesis AChE inhibitory activity
a b s t r a c t A novel series of flavokawain B derivatives, chalcone Mannich bases (4–10) were designed, synthesized, characterized, and evaluated for the inhibition activity against acetylcholinesterase (AChE). Biological results revealed that four compounds displayed potent activities against AChE with IC50 values below 20 lM. Moreover, the most promising compound 8 was 2-fold more active than rivastigmine, a wellknown AChE inhibitor. The log P values of 4–10 were around 2 which indicated that they were sufficiently lipophilic to pass blood brain barriers in vivo. Enzyme kinetic study suggested that the inhibition mechanism of compound 8 was a mixed-type inhibition. Meanwhile, the molecular docking showed that this compound can both bind with the catalytic site and the periphery of AChE. Ó 2014 Elsevier Ltd. All rights reserved.
Alzheimer’s disease (AD), the most common cause of dementia, is a progressive neurodegenerative disorder characterized by deterioration of memory and cognition in elder patients. The main cause of the loss of cognitive functions in AD patients is a continuous decline of the cholinergic neurotransmission in cortical and other regions of the human brain. Cholinergic neurotransmission is mediated by the neurotransmitter acetylcholine (ACh), which is released and carried out the effect followed by rapidly hydrolysis via acetylcholinesterase (AChE).1,2 So AChE plays an important role in central and peripheral nervous systems for its main function to regulate the impulse transmission at cholinergic synapses. Meanwhile, recent studies have identified that AChE could also play a key role in accelerating the assembly of b-amyloid into amyloid fibrils which are characteristically found in the brain cells of AD patients.3,4 One of the major therapeutic strategies adopted for primarily symptomatic AD is based on the cholinergic hypothesis.5 The widely used treatment is to inhibiting AChE to increase the ACh level in brain, which remains the most effective therapeutic approach against AD for several decades.6 Currently, four AChE inhibitors have been approved by the European and US regulatory authorities: tacrine, donepezil, galanthamine and rivastigmine (Fig. 1). They are important agents for the treatment of AD, but Abbreviations: AD, Alzheimer’s disease; AChE, acetylcholinesterases; ACh, acetylcholine; PAS, peripheral anionic sites; Log P, octanol/water partition; CNS, central nervous system; BBB, blood brain barrier. ⇑ Corresponding author. Tel.: +86 0731 83860540. E-mail address:
[email protected] (Q.-a. Wang). http://dx.doi.org/10.1016/j.bmcl.2014.07.087 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
accumulatively some side effects or demerits such as hepatotoxicity, periphery side effect, short half life, or gastrointestinal tract excitement cast a shadow in the clinic application.7 Therefore, the search for novel AChE inhibitors is still of great interest. Recently, it becomes a trend to discover cholinesterase inhibitors from natural products because of their slight side effects. Chalcones, an open-chain flavonoid, exhibit diverse biological activities.8 Flavokawain B (3), 20 -hydroxy-40 ,60 -dimethoxy chalcone, is isolated from the roots of kava and has been shown to exhibit an interesting spectrum of pharmacological effects.9,10 Besides its remarkable anti-proliferative activity against different cancer cell lines,11,12 flavokawain B also exhibited anti-inflammatory, anti-hepatitis, hepatoprotective and antinociceptive effects.13,14 However, the natural resource of flavokawain B (3) is limited due to the low content in the plants, which negatively influenced its further bioactivity evaluation. Therefore, in our investigation flavokawain B (3) was first total synthesized from phloroglucinol by the Houben–Hoesch acetylization, regioselective O-methylation and Claisen–Schmidt condensation with benzaldehyde. According to the structure of acetylcholine AChE substrate and some listed AChE inhibitors illustrated in Figure 1, a novel series of flavokawain B (3) derivatives, chalcone Mannich bases (4–10), were designed and synthesized. Mannich base is possible to improve the properties of original compounds, such as bioactivity.15,16 The title compounds were synthesized according to the route shown in Scheme 1. 2,4,6-Trihydroxyacetophenone (1) was acetylated by using the Houben–Hoesch reaction of phloroglucinol
4750
H.-r. Liu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4749–4753 Table 1 Half-inhibitory concentration against AChE and log P values for compounds 4–10
N
18.8 ± 1.6
1.61
5
N
15.1 ± 0.89
1.69
6
N
54.8 ± 2.21
1.77
7
N
16.1 ± 1.13
1.74
8
N
4.15 ± 0.28
1.75
9
N
O
265 ± 18.6
1.63
N
N
Rivastigmine*
according to the reported method.17 Taking advantage of hydrogen bonding between the carbonyl and an ortho phenolic-OH group, regioselective O-methylation of compound 1 with dimethyl sulfate and anhydrous potassium carbonate in dry acetone gained compound 2 in 68% yield. Chalcone flavokawain B (3) was prepared by the Claisen–Schmidt condensation of compound 2 with 1 equiv of benzaldehyde in the presence of KOH in a mixture of water and EtOH (1:4). Mannich reaction of compound 3 with HCHO and different amines in 2-propanol gave novel chalcone Mannich base derivatives 4–10 in 70–80% yield. The classical conditions of Mannich reaction for the hydroxyl compounds are based on the substrate, amine and formaldehyde ratio in alcohol with prolonged heating. In our experiment, flavokawain B, formaldehyde and amines in 1:1:1 ratio, respectively, were refluxed and stirred in isopropanol for 2–3 h to afford the C-aminomethylated derivatives. The regioselectivity of the reaction occurred preferentially at C-30 position of B ring of chalcone backbone. The compounds 4–10 were characterized by MS, 1H NMR and IR spectra. In the 1H NMR spectra of 4–10, the signal for H-30 was disappeared, and a signal at d 4.1–4.5 indicated the presence of an aminomethyl group at C-8 of flavokawain B. For a compound with potential effect to treat AD, the ability to penetrate the blood brain barrier (BBB) is vital. Although the factors that affect the penetration of a drug from the systemic circulation into the central nervous system (CNS) were complicated, logarithm 1-octanol/water partition coefficient (log P) was thought
HO
HO
OH
OH
OH 1
NR1 R2 d
O
OH
O
1.58 —
O
OH
c O
O
O
O
2
4 NR 1R 2=
N
6 NR 1R 2=
O
f lavokawain B (3)
CH 3
5 NR 1R 2=
N
N
7 NR 1R 2=
N
8 NR 1R 2=
N
9 NR 1R 2=
N
10 NR 1 R2 =
N
CH 3
OH
O
12.4 ± 0.83 10.54 ± 0.86
as an important physical chemistry parameter to valuate or predict a compound’s ability to cross BBB, and widely used in medicinal chemistry investigation. The log P of 4–10 were determined by the classical shake-flask method18 using reverse phase high performance liquid chromatography (RP-HPLC). Hansch19 presumed that the log P with optimum CNS penetration was around 2 ± 0.7. As showed in Table 1, the log P values of 4–10 were 1.61, 1.69, 1.77, 1.74, 1.75, 1.63 and 1.58, respectively, which indicated 4–10 were sufficiently lipophilic to pass blood brain barriers in vivo. All the synthetic compounds were tested for the acetylcholinesterases (AChE) inhibitory activities in vitro according to the modified Ellman’s method.20 Rivastigmine, a well-known AChE inhibitor, was used as the positive control. The inhibit ratio against AChE was calculated according to the absorbance of the product in the AChE-catalyzed reaction and the results were shown in Table 1. Preliminary investigation demonstrated that flavokawain B (3) showed poor inhibiting effect against AChE (IC50 >104), but most of its derivatives revealed potent AChE inhibitory activities. Furthermore, the inhibition was found to be influenced markedly by aminomethylene group at C-30 position. Among them, compound 8, a flavokawain B derivative contained piperidine group, was found to have the most potent inhibitory activity with IC50 value of 4.15 ± 0.28 lM, which was 2-fold of inhibitory effect compared with rivastigmine (IC50 = 10.54 ± 0.86 lM, lit.21: 9.12 lM). The dimethylamine, diethylamine, dipropylamine, pyrrolidine and
b
phloroglucinol
—
a IC50 values of compounds represent the concentration that caused 50% enzyme activity loss. b The partition coefficients of in the octanol/buffer solution at pH 7.4 were determined by the classical shake-flask method. * Used for positive control for IC50.
O
OH
a
Log Pb
4
10
Figure 1. The chemistry structure of acetylcholine (ACh) and the listed AChE inhibitors.
IC50a (lM) ± SD
–NR1R2
Compound
O
N
Scheme 1. The synthetic route of flavokawain B (3) and its Mannich base derivatives 4–10. Reagents and conditions: (a) (1) CH3CN, ZnCl2, Et2O, HCl, 0 °C; (2) concentrated HCl, H2O, reflux 2 h; (b) (CH3)2SO4, K2CO3, acetone, reflux 2 h; (c) (1) KOH, CH3OH, benzaldehyde; (2) HCl (aq); (d) concentrated HCl, HCHO, HNR1R2, isopropanol, reflux 3 h.
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H.-r. Liu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4749–4753 Table 2 Kinetic parameters of AChE inhibited by compound 8 Compound 8/lM
Michaelis–Menten equation
Km/mM
vmax/D Amin1
Ki/lM
Ki0 /lM
0 2.2 4.4
1/v = 87.80/[S]+23.44 1/v = 145.26/[S]+34.74 1/v = 218.32/[S]+47.13
3.749 4.183 4.632
0.0427 0.0288 0.0212
2.75
4.58
N-methyl piperazine-contained flavokawain B also showed potent AChE inhibitory activities. However, compound 9, a hydrophilic morpholine ring-contained flavokawain B, showed significant reduced AChE inhibitory activity, which might suggest that the hydrophobic interactions in this binding region of the enzymes is required for intensive inhibition.22 Compound 8 was selected for further kinetic measurement for its most potent inhibitory against AChE among these compounds. The linear Lineweaver–Burk equation of the Michaelis–Menten was applied to evaluate the inhibition profile.23 The graphical analysis of the steady-state inhibition data of compound 8 was shown in Figure 2. These results showed that increasing the concentration of compound 8 resulted in different slopes and intercepts. According to Table 2, Km but not vmax increased with increasing concentration of compound 8, which presented a mixed-type inhibition. It seemed that compound 8 can bind both with the free enzyme and the enzyme–substrate complex with different bind equilibrium constants. The inhibition constants for the free enzyme (E), Ki, and that one for enzyme-substrate (ES) complex, Ki0 , are obtained from the replots of the slope and intercept versus concentration of compound 8, respectively, which are both linear in Figure 2. The Ki and Ki0 value are 2.75 lM and 4.58 lM, respectively, which indicates that compound 8 revealed a mixed inhibiting effect against AChE includes competitive and uncompetitive profile. Moreover, competitive inhibition is easier than uncompetitive inhibition for compound 8.24
To elucidate further the binding mechanism of compound 8 with AChE which X-ray crystal structure (PDB code: 1EVE) was obtained from protein data bank, we performed a docking study on compound 8 by utilizing the Molecular Operating Environment (MOE). It is well known that two distinct binding sites exist in the active pocket of AChE peripheral binding site (PAS) and catalytic active site (CAS), located at the entrance and the bottom of the active-site gorge, respectively. These sites are characterized by two tryptophan residues, Trp 84 at the active site and Trp 279 at the mouth of the gorge (PAS).25 Compound 8 manifested binding mode with AChE in Figure 3. The chalcone moiety adopted an appropriate orientation for its binding to PAS via the p–p stacking interactions with Trp 279 and Tyr 334, and their ring-to-ring distance was between 4.48 and 4.44 Å. Moreover, the conformation of the side chain conformed to the shape of the mid-gorge, and in the bottom of the gorge, the charged nitrogen of piperidine ring was observed to bind to the CAS via a cation–p interaction between Trp 84 and Phe 330. These results suggested that it can both bind with the catalytic site and the periphery of AChE. In conclusion, flavokawain B was totally synthesized for the first time from phloroglucinol by the Houben–Hoesch acetylization, regioselective O-methylation and Claisen–Schmidt condensation with benzaldehyde. The Mannich base derivatives of flavokawain B were designed, synthesized, and subjected to biological evaluated as the AChE inhibitors. All derivatives exhibited AChE inhibitory activity compared with flavokawain B in the preliminary
Figure 2. (A) Lineweaver–Burk plot for the inhibition of AChE by compound 8; (B) the replot of the intercept versus concentration of compound 8; (C) the replot of the slope versus concentration of compound 8.
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H.-r. Liu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4749–4753
Figure 3. Molecular modeling of compound 8 with AChE generated by MOE.
bioassay, which indicates that the base group seemed necessary to inhibit AChE. Among them, compound 8 revealed the most potent inhibitory against AChE. The kinetic assay of compound 8 suggested that it can bind both with free enzyme and the enzyme–substrate complex. The molecular modeling study further indicated that this compound can bind both with the CAS and PAS of AChE. Overall, Compound 8 might serve as an inhibition agent against AChE for the treatment of AD. Additional investigations on the biological activity as well as an extended structure–activity relationship study are in progress and will be part of a forthcoming Letter. Acknowledgments The present investigation was supported by the Grant of ‘‘The Natural Science Foundation of China (No. 21342015)’’, ‘‘The Project of Science and Technology of Hu’nan Province (No. 2012SK3183)’’ and ‘‘The Research Funds for younger investigators of Hu’nan University (No. HNU2013066)’’.
Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014. 07.087.
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