Design, Synthesis of N-phenethyl Cinnamide Derivatives and ... - MDPI

1 downloads 0 Views 981KB Size Report
Oct 16, 2018 - inhibitors, including tacrine, donepezil, rivastigmine, galanthamine, and huperzine A, and the. N-methyl-D-aspartate (NMDA) receptor ...
molecules Article

Design, Synthesis of N-phenethyl Cinnamide Derivatives and Their Biological Activities for the Treatment of Alzheimer’s Disease: Antioxidant, Beta-amyloid Disaggregating and Rescue Effects on Memory Loss Tian Chai 1 , Xiao-Bo Zhao 2 , Wei-Feng Wang 2 , Yin Qiang 1 , Xiao-Yun Zhang 1, * and Jun-Li Yang 2, * 1 2

*

School of Pharmacy, Lanzhou University, Lanzhou 730000, China; [email protected] (T.C.); [email protected] (Y.Q.) CAS Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics (LICP), Chinese Academy of Sciences (CAS), Lanzhou 730000, China; [email protected] (X.-B.Z.); [email protected] (W.-F.W.) Correspondence: [email protected] (X.-Y.Z.); [email protected] (J.-L.Y.); Tel.: +86-931-8915686 (X.-Y.Z.); +86-931-4968385 (J.-L.Y.)

Received: 1 September 2018; Accepted: 10 October 2018; Published: 16 October 2018

 

Abstract: Gx-50 is a bioactive compound for the treatment of Alzheimer’s disease (AD) found in Sichuan pepper (Zanthoxylum bungeanum). In order to find a stronger anti-AD lead compound, 20 gx-50 (1–20) analogs have been designed and synthesized, and their molecular structures were determined based on nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis, as well as comparison with literature data. Compounds 1–20 were evaluated for their anti-AD potential by using DPPH radical scavenging assay for considering their anti-oxidant activity, thioflavin T (ThT) fluorescence assay for considering the inhibitory or disaggregate potency of Aβ, and transgenic Drosophila model assay for evaluating their rescue effect on memory loss. Finally, compound 13 was determined as a promising anti-AD candidate. Keywords: Alzheimer’s disease; gx-50; DPPH assay; ThT assay; transgenic Drosophila model assay

1. Introduction Alzheimer’s disease (AD) is a neurodegenerative disorder that mainly occurs in the elderly. It is characterized by intelligence decline and memory loss, as well as changes in emotions and personality [1]. There are approximately 36 million AD patients worldwide, and this figure will double every 20 years [2]. Since the 1990s, a great deal of financial support has been invested to explore the molecular pathogenesis of AD, which had provided robust support to develop effective pharmacological treatments [3]. Currently, the clinically used AD drugs mainly include cholinesterase inhibitors, including tacrine, donepezil, rivastigmine, galanthamine, and huperzine A, and the N-methyl-D-aspartate (NMDA) receptor antagonist, that is memantine. These drugs could alleviate cognitive symptoms in this disease. Unfortunately, there is no effective means to cure, or only slow, the progression of this disease [4,5]. The pathogenesis of AD has not yet been fully understood. Therefore, a variety of AD hypotheses have been proposed, such as the amyloid cascade hypothesis, the Tau phosphorylation hypothesis, the neurovascular hypothesis, the oxidative stress hypothesis, and the immune hypothesis [4]. One of Molecules 2018, 23, 2663; doi:10.3390/molecules23102663

www.mdpi.com/journal/molecules

Molecules 2018, 23, 2663

2 of 11

the major pathological hallmarks of AD is the aggregation of amyloid plaques in the brain. Its essence are the fibrils of the amyloid-β peptide (Aβ) [6,7]. Oxidative stress also plays an important role in the development and progress of AD, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay is one of the most common methods to evaluate the anti-oxidative activity of compounds [8,9]. Moreover, transgenic Drosophila that can express AD-related proteins has been successfully used to screen the potential anti-AD compounds [10]. N-[2-(3,4-Dimethoxyphenyl)ethyl]-3-phenyl-acrylamide (gx-50), a potential drug candidate for the treatment of AD, was isolated from Sichuan pepper (Zanthoxylum bungeanum). Gx-50 could penetrate the blood brain barrier (BBB) and enter the brain tissue to improve the cognitive function of dementia mice, and meanwhile could reduce the Aβ plaques in brain tissues [11–13]. In order to find a much stronger drug candidate for the treatment of AD, 20 analogues of gx-50 were designed and synthesized in this study. Consequently, the DPPH assay and thioflavin T (ThT) assay were employed to evaluate their anti-oxidative activity and their inhibition and disaggregation on Aβ aggregation. Finally, Pavlov’s olfactory memory test was used to evaluate their rescue effects on memory loss as analyzed on the behavior of AD model flies. Herein, we will report the design, synthesis, and anti-AD bioactivity of these gx-50 derivatives. 2. Results and Discussion 2.1. Chemistry The synthesis of the gx-50 derivatives is described in Schemes 1 and 2. All compounds were synthesized by following previously reported methods with little modifications [14]. Specifically, the cinnamic acid was synthesized firstly by the substituted aromatic aldehyde with malonic acid through the Knoevenagel reaction. Then in the presence of 3-(ethyliminomethylideneamino)-N,Ndimethylpropan-1-amine hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP), the cinnamic acids with different substitutions reacted with 4-methoxyphenethylamine, 2-phenylethylamine, 3,4-dimethoxyphenethylamine, and 3-methoxyphenethylamine, respectively. Finally, one molecule of water was removed at room temperature to produce the corresponding target compounds 1–20. Among them, compounds 12, 16, and 20 have never been reported by searching the SciFinder database. Regarding the known compounds, their chemical structures were determined and confirmed by NMR and MS data analysis as well as literature comparison. Some of them were reported to show potent biological activities. For example, compounds 1, 9, and 13 exhibited indution of apoptosis in U-937 cells at 100 µM [15]. Compound 3 and 11 had an antihyperglycemic effect as inhibition percentage of 20.1% and 30.7% in sucrose-loaded model (SLM) at a dosage of 100 mg/kg-body weight [16,17]. Compound 5 inhibited platelet aggregation with the IC50 value of 2.6 µM [18]. Compound 10 and 11 showed anti-inflammatory activity with 50% NO inhibition concentration as 14.08 µM and 15.08 µM, respectively [19,20]. Compound 10 and 14 revealed 5-lipoxygenase inhibitory activity with the IC50 value of 0.12 µM and 1 µM, respectively [21]. Compound 15 could inhibit tyrosinase as the IC50 value of 0.6 mM [22,23]. In this study, all compounds 1–20 were evaluated for their anti-AD bioactivity via DPPH assay, ThT test, and Pavlov’s olfactory memory test for the first time.

Molecules 2018, 2018, 23, 23, x 2663 Molecules FOR PEER REVIEW Molecules 2018, 23, x FOR PEER REVIEW

of 11 11 33 of 3 of 11

Scheme 1. Synthesis of compounds 1–16. Reagents and conditions: (a) anhydrous pyridine, Scheme 1.1.Synthesis of compounds 1–16.1–16. Reagents and conditions: (a) anhydrous pyridine, piperidine, Synthesis of compounds Reagents and conditions: (a) anhydrous pyridine, piperidine, malonic acid, ◦oil bath, 90 °C, 4 h; (b) cinnamic acid, amine, EDCI, DMAP, DMF, room malonic acid, oil bath, 90 oil C, bath, 4 h; (b) acid, amine, acid, EDCI,amine, DMAP, DMF,DMAP, room temperature, piperidine, malonic acid, 90 cinnamic °C, 4 h; (b) cinnamic EDCI, DMF, room temperature, overnight. overnight. overnight. temperature,

of compounds 17–20. Reagents and conditions: (a) anhydrous pyridine, piperidine, Scheme 2.2.Synthesis Synthesis of compounds 17–20. Reagents and conditions: (a) anhydrous pyridine, Scheme 2. Synthesis of ◦compounds 17–20. Reagents and conditions: (a) anhydrous pyridine, malonic acid, oil bath, 90 oil C, bath, 4 h; (b) acid, amine, acid, EDCI,amine, DMAP, DMF,DMAP, room temperature, piperidine, malonic acid, 90 cinnamic °C, 4 h; (b) cinnamic EDCI, DMF, room piperidine, malonic acid, oil bath, 90 °C, 4 h; (b) cinnamic acid, amine, EDCI, DMAP, DMF, room overnight. overnight. temperature, temperature, overnight.

2.2. Pharmacology Pharmacology 2.2. 2.2. Pharmacology 2.2.1. Determination of Anti-Oxidant Activity Based on DPPH Assay 2.2.1. Determination of Anti-Oxidant Activity Based on DPPH Assay 2.2.1. Determination of Anti-Oxidant Activity Based on DPPH Assay The oxidative damage to neurons is closely related to the pathogenesis of AD [24]. In this article, The oxidative damage to neurons is closely related to the pathogenesis of AD [24]. In this article, The oxidativeactivity damage neurons is1–20 closely to the of AD [24]. InCthis the anti-oxidative of to compounds wasrelated evaluated by pathogenesis DPPH assay with Vitamin (Vc)article, as the the anti-oxidative activity of compounds 1–20 was evaluated by DPPH assay with Vitamin C (Vc) as the anti-oxidative ofradical compounds 1–20 ability was evaluated by DPPH Vitamin C (Vc) as positive control [25].activity The free scavenging was evaluated basedassay on thewith UV absorbance change the positive control [25]. The free radical scavenging ability was evaluated based on the UV the positive control [25]. The free radical scavenging ability was evaluated based on the UV of the solution measured at 517 nm using a microplate reader [26]. As shown in Table 1, the compound absorbance change of the solution measured at 517 nm using a microplate reader [26]. As shown in absorbance of the solution measured at 517 nm using a microplate reader [26]. As shown in concentrationchange and their anti-oxidative activity exhibited a favorable concentration-dependent relationship. Table 1, the compound concentration and their anti-oxidative activity exhibited a favorable Table compound concentrationrelationship, and their compounds anti-oxidative exhibited a favorable In order1,tothe discuss their structure-activity 1–20activity were classified into three groups concentration-dependent relationship. In order to discuss their structure-activity relationship, concentration-dependent relationship. In order discuss their structure-activity relationship, as follows. The first group contains compounds 1–8, to which only contained methoxy substituents. These compounds 1–20 were classified into three groups as follows. The first group contains compounds compounds showed 1–20 were classified three groups as follows. The group first group contains compounds weak DPPHinto scavenging activity. The second contains compounds 9–16, 1–8, which only contained methoxy substituents. These compounds showed weak DPPH scavenging 1–8, which only contained of methoxy substituents. TheseThese compounds showed weak DPPH scavenging which had the substituents hydroxyls and methoxyls. compounds exhibited comparable DPPH activity. The second group contains compounds 9–16, which had the substituents of hydroxyls and activity. Theactivity, secondcompared group contains compounds 9–16, which had the substituents of hydroxyls and scavenging to the positive control. Moreover, compounds 13–16 with two hydroxyls methoxyls. These compounds exhibited comparable DPPH scavenging activity, compared to the methoxyls. These compounds exhibited comparable DPPH scavenging activity, compared the showed much stronger activity than compounds 9–12 with just one hydroxyl substituent. The third to group positive control. Moreover, compounds 13–16 with two hydroxyls showed much stronger activity positive control. Moreover, compounds 13–16 with two hydroxyls showed much stronger activity contains compounds 17–20, which contained 3,4-(methylenedioxy)cinnamic acid moiety. These compounds than compounds 9–12 with just one hydroxyl substituent. The third group contains compounds 17– than compounds 9–12 withscavenging just one hydroxyl third group contains 17–3 showed the weakest DPPH activity.substituent. In addition, The the DPPH scavenging ratecompounds of compound 20, which contained 3,4-(methylenedioxy)cinnamic acid moiety. These compounds showed the 20, which 3,4-(methylenedioxy)cinnamic acid stronger moiety. DPPH These scavenging compoundsability showed the (gx-50) was contained less than 10%, and its derivatives 9–16 exhibited ranging weakest DPPH scavenging activity. In addition, the DPPH scavenging rate of compound 3 (gx-50) weakest activity. In addition, the scavenging rate be of acompound 3 (gx-50) from 16.69DPPH ± 0.46scavenging to 60.85 ± 0.37. As discussed above, theDPPH phenolic hydroxyl may functional group for was less than 10%, and its derivatives 9–16 exhibited stronger DPPH scavenging ability ranging from was less than 10%, andofitsgx-50 derivatives 9–16 This exhibited stronger DPPH scavenging ability ranging from the anti-oxidant ability derivatives. summarized structure-activity relationship is consistent 16.69 ± 0.46 to 60.85 ± 0.37. As discussed above, the phenolic hydroxyl may be a functional group for 16.69the ± 0.46 to 60.85reported ± 0.37. As discussed[27]. above, the phenolic hydroxyl may be a functional group for with previously conclusion the anti-oxidant ability of gx-50 derivatives. This summarized structure-activity relationship is the anti-oxidant ability of gx-50 derivatives. This summarized structure-activity relationship is consistent with the previously reported conclusion [27]. consistent with the previously reported conclusion [27].

Molecules 2018, 23, 2663

4 of 11

Table 1. The DPPH assay result for compounds 1–20. Inhibition Ratio (%)

Compound

R1

R2

R3

R4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Vc

H H H H H H H H OCH3 OCH3 OCH3 OCH3 OH OH OH OH OCH3 H OCH3 H

H H H H OCH3 OCH3 OCH3 OCH3 OH OH OH OH OH OH OH OH H H OCH3 OCH3

OCH3 H OCH3 H OCH3 H OCH3 H OCH3 H OCH3 H OCH3 H OCH3 H -

H H OCH3 OCH3 H H OCH3 OCH3 H H OCH3 OCH3 H H OCH3 OCH3 -

50 µM

100 µM

200 µM

2.97 ± 1.67 3.1 ± 0.30 2.92 ± 1.7 0.99 ± 0.55 4.78 ± 2.8 1.08 ± 0.70 3.61 ± 0.89 0.06 ± 2.63 30.21 ± 1.52 16.69 ± 0.46 17.43 ± 0.46 28.74 ± 0.78 54.34 ± 1.44 54.21 ± 1.21 57.85 ±0.15 58.86 ±0.43 2.13 ± 0.32 0.63 ± 0.27 1.44 ± 1.68 1.57 ± 0.13 41.42 ± 2.83

4.35 ± 2.29 6.69 ± 0.37 5.03 ± 2.01 2.91 ± 1.24 9.78 ± 0.61 5.11 ± 2.06 5.58 ± 0.78 2.81 ± 2.78 36.28 ± 3.43 34.06 ± 2.57 40.28 ± 0.21 46.93 ± 0.42 56.16 ± 1.78 55.01 ± 0.35 59.96 ± 0.79 60.85 ± 0.37 4.60 ± 0.33 1.99 ± 1.50 1.39 ± 0.85 4.09 ± 0.31 72.00 ± 1.15

4.93 ± 2.13 9.20 ± 0.67 6.18 ± 0.65 3.13 ± 0.59 11.22 ± 0.43 12.91 ± 1.57 9.58 ± 1.39 3.42 ± 0.78 46.57 ± 0.86 44.07 ± 2.20 50.19 ± 1.90 51.83 ± 1.42 56.37 ± 2.86 54.17 ± 2.62 58.33 ± 2.71 57.68 ± 0.54 4.82 ± 0.36 2.27 ± 1.52 2.02 ± 0.52 4.53 ± 0.83 78.88 ± 0.80

Values are expressed as the means ± SD of at least three independent experiments.

2.2.2. Inhibition of Cu2+ -Induced Aβ Aggregation and Disaggregation The inhibitory activities of compounds 1–20 on copper-mediated Aβ1–42 aggregation and disaggregation were evaluated by using ThT assay [28,29] with resveratrol and curcumin as positive controls. As shown in Table 2, the inhibitory and disaggregate potency of compounds 1–4 were less than 5%. Specifically, the inhibitory and disaggregate potency of compound 3 (gx-50) were 1.73 ± 2.15% and 2.39 ± 1.35%, respectively. However, when R2 becomes methoxyl group, as in compounds 5–8, the inhibitory and disaggregate potency obviously increased except for compound 8. Therefore, R2 may be an active position that could increase inhibitory or disaggregate potency of the test compounds. The inhibitory and disaggregate potency of compounds 9–12 were less than 10%. Compounds 13–16 exhibited equal or better inhibitory and disaggregate potency than curcumin and resveratrol. Compounds 17–20 had little inhibitory or disaggregate potency. The most active compound was compound 15, with 61.85 ± 1.70% and 64.44 ± 0.76%, respectively. As discussed, the catechol part could be the bioactive part for the gx-50 derivatives to inhibit Aβ aggregation and disaggregate Aβ aggregate. Table 2. Thioflavin T (ThT) assay results for compounds 1–20. Compound

R1

R2

R3

R4

Aβ Aggregation (inhib. %) a

Disaggregation (%) b

1 2 3 4 5 6 7 8 9 10 11 12

H H H H H H H H OCH3 OCH3 OCH3 OCH3

H H H H OCH3 OCH3 OCH3 OCH3 OH OH OH OH

OCH3 H OCH3 H OCH3 H OCH3 H OCH3 H OCH3 H

H H OCH3 OCH3 H H OCH3 OCH3 H H OCH3 OCH3

4.83 ± 8.10 0.14 ± 1.49 1.73 ± 2.15 2.28 ± 3.24 14.47 ± 4.15 12.26 ± 7.37 7.87 ± 2.90 3.33 ± 1.26 2.72 ± 3.65 5.94 ± 3.13 4.37 ± 3.20 2.78 ± 4.27

4.40 ± 2.11 0.27 ± 0.94 2.39 ± 1.35 3.85 ± 2.22 11.92 ± 2.32 11.19 ± 1.70 9.04 ± 1.99 4.49 ± 0.49 1.05 ± 1.04 4.16 ± 1.65 2.04 ± 0.71 0.33 ± 0.45

Molecules 2018, 23, 2663 Molecules 2018, 23, x FOR PEER REVIEW

13 14 Compound 15 16 13 14 17 15 18 16 19 17 20 18 curcumin 19 20 resveratrol a

OH OH R1 OH OH OH OH3 OCH OH H OH OCH3 OCH3 H H OCH3 H

OH OH R2 OH OH OH OH H OH H OH OCH3 H OCH H3 OCH3 OCH3

5 of 11 5 of 11

HTable 2. Cont. 56.43 ± 1.72 OCH3 H H 60.68 ± 1.76 R33 OCHR3 4 Aβ Aggregation (inhib. %) a 61.85 ± 1.70 OCH 3 38.47 ±± 2.37 H OCH3 OCHH 56.43 1.72 60.68 1.76 −H − H 14.34 ±± 2.37 OCH 61.85 ± 1.70 3 − −OCH3 3.47 ± 7.57 H OCH3 38.47 ± 2.37 − − 13.06 ± 4.58 − − 14.34 ± 2.37 −− − − 12.93 3.66 3.47±± 7.57 48.85 ±± 1.20 − − 13.06 4.58 − − 12.93 3.66 60.49 ±± 2.71

52.76 ± 0.33 57.28 ± 3.61 Disaggregation 64.44 ± 0.76(%) b 51.51 ± 1.42 52.76 ± 0.33 57.28 ± 3.61 15.50 ± 0.07 64.44 0.76 7.74 ±±2.00 51.51 ± 1.42 18.68 ± 1.17 15.50 ± 0.07 0.61 ± 0.38 7.74 ± 2.00 43.83 ± 1.22 18.68 ± 1.17 0.61 ±± 0.38 51.27 3.31

curcumin 48.85 ± 1.20 43.83 ± 1.22 b The disaggregation percentage of Cu2+The inhibition percentage Cu2+-induced Aβ1−42 aggregation.60.49 resveratrol ± 2.71 51.27 ± 3.31

Aβ1–42 aggregates. Assays were carried out in the presence of 25 μM inhibitor and 25 μM Aβ1– ainduced The inhibition percentage Cu2+ -induced Aβ1–42 aggregation. b The disaggregation percentage of Cu2+ -induced

42. Values are expressed as the means ± SD of at least three independent experiments. Aβ 1–42 aggregates. Assays were carried out in the presence of 25 µM inhibitor and 25 µM Aβ1–42 . Values are expressed as the means ± SD of at least three independent experiments.

2.2.3. Pavlovian Olfactory Aversive Immediate Memory Test 2.2.3. Pavlovian Olfactory Aversive Immediate Memory Test The Pavlovian olfactory memory experiment was used to observe the rescue effect of The Pavlovian olfactory memory experiment used toand observe rescue effectcompounds of compounds on compounds on memory loss. As analyzed from was the DPPH ThTthe assay results, 3 and memory loss.selected As analyzed from thewhether DPPH and ThT assayrescue results,the compounds and of 13–16 selectedAD to 13–16 were to explore they could memory 3loss the were Drosophila explore they 3could the memory loss of the AD model. Compounds 3 and 13–16 model. whether Compounds and rescue 13–16 were administered at aDrosophila concentration of 10 μM. As shown in Figure were at aAD concentration of 10 µM. As (P35*H29.3) shown in Figure PI valueless of the negative 1, theadministered PI value of the negative control group was 1, 27the (required thanAD 45). The PI control group (P35*H29.3) was 27 (required less(P35*2U) than 45).and Thethe PI difference between genetic control difference between the genetic control group AD negative groupthe was 28 (required group thethere AD negative group was 28 (required biggerthe than 15). Also, (MEM) there was significant bigger(P35*2U) than 15).and Also, was a significant difference between memantine 100a μM group difference between (MEM) 100 µM and the ADhad group (p < 0.01). It demonstrated and the AD group the (p