Synthesis and biological evaluation of clovamide analogues as potent anti-neuroinflammatory agents in vitro and in vivo

Accepted Manuscript Synthesis and biological evaluation of clovamide analogues as potent antineuroinflammatory agents in vitro and in vivo Xiao-Long Hu, Jun Lin, Xian-Yu Lv, Jia-Hao Feng, Xiao-Qi Zhang, Hao Wang, WenCai Ye PII:

S0223-5234(18)30324-6

DOI:

10.1016/j.ejmech.2018.03.081

Reference:

EJMECH 10346

To appear in:

European Journal of Medicinal Chemistry

Received Date: 12 January 2018 Revised Date:

26 March 2018

Accepted Date: 30 March 2018

Please cite this article as: X.-L. Hu, J. Lin, X.-Y. Lv, J.-H. Feng, X.-Q. Zhang, H. Wang, W.-C. Ye, Synthesis and biological evaluation of clovamide analogues as potent anti-neuroinflammatory agents in vitro and in vivo, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.03.081. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Synthesis and biological evaluation of clovamide analogues as potent anti-neuroinflammatory agents in vitro and in vivo Xiao-Long Hua, Jun Lina, Xian-Yu Lva, Jia-Hao Fenga, Xiao-Qi Zhangb, Hao Wanga,*, and Wen-Cai Yeb a

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State Key Laboratory of Natural Medicines, Department of TCMs Pharmaceuticals, School of

Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 210009, People’s Republic of China.

Institute of Traditional Chinese Medicine & Natural Products, Jinan University, Guangzhou

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b

*

Corresponding author: Hao Wang

E-mail: [email protected]

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510632, People’s Republic of China.

Postal address: State Key Laboratory of Natural Medicines, Department of TCMs Pharmaceuticals,

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China Pharmaceutical University, Nanjing 210009, People’s Republic of China.

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ABSTRACT

A series of clovamide analogues, namely, 1a–13a and 1b–13b, was synthesized and evaluated for their anti-neuroinflammatory activities using BV-2 microglia cells. Among these compounds, six (1b, 4b–8b) showed NO inhibition with no or weak cytotoxicity (CC50＞100 µM), especially 4b, and showed an IC50 value of 2.67 µM. Enzyme activity and docking assay revealed that the six

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compounds, especially 4b, target inducible NO synthase (iNOS) and exhibit potent inhibitory effects on iNOS with IC50 values ranging from 1.01 µM to 29.23 µM. 4b significantly suppressed the expression of pro-inflammatory cytokines in lipopolysaccharide-stimulated cells. Notably, the oral administration of 4b remarkably improved dyskinesia, reduced the expression of glial fibrillary

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acidic protein (GFAP) － a marker of neuroinflammation, and increased tyrosine hydroxylasepositive cells in 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine-induced Parkinson’s disease (PD)

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mouse models. These observations demonstrated that 4b is an effective and promising candidate for PD therapy.

Keywords: clovamide analogues, neuroinflammation, nitric oxide, iNOS inhibitor, Parkinson’s

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disease

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1. Introduction

Inflammation is a complex process in the host defense system that involves numerous cellular and plasma regulators that restrict its action at a necessary time and place [1]. Moreover, nitric oxide (NO), an inflammatory mediator in living organisms, plays important roles in cardiovascular, immune, and neuronal systems [2, 3]. High-level NO production in pathological situations is an

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indicator of cytotoxicity during inflammation. Therefore, reducing NO production is a promising therapeutic strategy for the reduction of neuronal cell injury or death in various neuroinflammatory and neurodegenerative diseases. In mammals, NO is generated via the oxidation of L-arginine by NO synthase (NOS); furthermore, at least three isoforms of the NOS enzymes have been identified: endothelial NOS, neuronal NOS, and inducible NOS (iNOS) [4]. Suppressing the over-production

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of iNOS-mediated NO may be useful as a drug target for the treatment of inflammatory diseases,

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including neuroinflammatory diseases [5, 6].

Clovamide, which is also known as N-caffeoyl-L-dihydroxyphenyl-alanine, is an amide analog of rosmarinic acid that contains two distinctive functional moieties, namely, caffeic acid and L-dopa [7]. Clovamide displays anti-inflammatory effects via three different mechanisms, including proinflammatory cytokine release [8], inhibition of superoxide anion production [9], and NF-κB activation [10]. Most clovamide-related studies involve the use of clovamide or its derivatives that

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are directly obtained through separation from plants. Few studies conducted with synthesized analogues revealed their potent antioxidant activities in bulk lipids and moderate protection in emulsions [7, 11]. In Yoon’s study, a series of methyl ester of clovamide analogues was synthesized and was found to inhibit NO production in activated BV-2 microglial cells. However, clovamide

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derivatives with free carboxyl groups were not synthesized in the study [12]. Therefore, synthesizing additional clovamide analogues containing carboxyl groups is important in

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investigating their inhibitory effects on neuroinflammation. In the present study, 26 clovamide analogues with carboxylic acid or ester functionalities were designed and synthesized. Docking and iNOS assays were performed to confirm the inhibitory effects of clovamide analogues on iNOS activity. The most efficacious analogue, compound b4, significantly reduced pro-inflammatory cytokines in LPS-stimulated cells and alleviated the symptoms of 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine-induced Parkinson’s disease (PD) model.

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2. Results and discussion

2.1. Inhibition and cytotoxicity in BV-2 cells Lipopolysaccharide (LPS) is an important structural component of the outer membrane of gram-negative bacteria; furthermore, it is a well-studied immunostimulator that induces immune inflammatory responses, especially the expression of NO production and other pro-inflammatory

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cytokines [13]. In the current study, all analogues displayed improved NO inhibitory activity at a concentration of 10 µM (Fig. 1). Among the analogues, six compounds (1b and 4b–8b) showed potent inhibition rates (>50%). The IC50 values of these compounds (1b and 4b–8b) were 3.40 ± 0.98, 2.67 ± 0.48, 10.43 ± 1.77, 26.45 ± 2.38, 15.34 ± 2.12, and 4.41 ± 1.11 µM, respectively

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(Table 1). Notably, 4b exhibited the most potent inhibitory activity for NO production. Preliminary structure-activity relationship (SAR) analysis showed that analogues with carboxyl group showed

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better NO inhibitory activity than their corresponding esters (1b–13b vs. 1a–13a, respectively), and free phenolic hydroxyl-substituted analogues exhibited better NO inhibitory effects than those without phenolic hydroxyl groups. Compounds 1b and 4b–8b demonstrated no cytotoxicity on BV2 cells (CC50＞100 µM) (Table 1). On the basis of cellular viability and anti-inflammatory activity

2.2. Docking study of iNOS

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in vitro, compounds 1b and 4b–8b were further evaluated in the next experimental process.

To gain improved understanding on the potency of the studied compounds, we examined the interaction of all compounds with NO production-related kinases. The iNOS protein 3D structure

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obtained from the Protein Data Bank (PDB ID: 1r35) [14] showed the best matching scores with these compounds (data not shown). Consistent with the bioassay results of NO production, the

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docking results displayed that analogues possessing carboxylic acid and p-phenolic hydroxyl of phenylacrylic moiety functionalities exhibited the highest affinity among the other synthesized compounds (Table 1). Among the compounds, 4b showed maximum binding affinity with binding energy value (−48.34 kcal/mol) (Table 1). The carboxylic acid group of the amino acid residue of 4b interacted with amino acids TRP366 and GLY365 of the target protein forming two hydrogen bonds (H-bond) with bonding distances of 2.07 and 2.12 Å. The p-phenolic hydroxyl of phenylacrylic moiety in the same compound interacted with ARG382 to form another H-bond with bonding distances of 2.10 and 2.97 Å (Fig. 2). Thus, compound b4 is likely a potential iNOS inhibitor with perfect binding to the active site of murine iNOS. 2.3. Inhibitory effects of 1b and 4b–8b on iNOS activity 4

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To confirm the docking results, we performed the iNOS inhibition assay to evaluate the inhibitory effects of 1b and 4b–8b on iNOS activity. The six compounds showed potent iNOS inhibitory effects with IC50 values of 2.10 ± 0.97 (1b), 1.01 ± 0.65 (4b), 8.45 ± 1.56 (5b), 18.87 ± 2.98 (6b), 29.23 ± 3.11 (7b), and 2.98 ± 0.91 µM (8b) (Table 1). Interestingly, the inhibitory effects of these compounds on iNOS activity were positively correlated with their NO inhibition and docking scores. This finding is the first report of these clovamide analogues exhibiting potential as

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iNOS inhibitors, further explaining the action mechanisms of these compounds on neuroinflammation therapy. Thus, further biological evaluation of compound 4b as a potential lead is essential.

2.4. Effects of 4b on LPS-induced translocation of NF-κb p65 and inflammatory mediators in BV-2

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cells

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LPS can activate NF-κb to allow p65 translocation from cytoplasm to nucleus, further increasing the expression of the pro-inflammatory mediators COX-2, IL-1β, TNF-α, and iNOS [13]. Clovamide and its analogues can inhibit LPS-induced NF-κb activation. Therefore, Western blot analysis was employed to detect the effects of 4b on the expression of these related proteins. As shown in Fig. 3, LPS could significantly increase the expression levels of COX-2, IL-1β, TNF-α, and iNOS compared with control treatment. However, pretreatment with 1, 10, and 20 µM 4b could

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reverse the tendency in a dose-dependent manner. These results indicated that like clovamide, 4b can suppress NF-κb activation as well. Based on the above results, 4b may be involved in two action mechanisms, including the inhibition of NF-κb translocation, and iNOS activity. According to the mentioned results, we speculated that the iNOS inhibitory effect of these compounds is the

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main factor determining NO inhibition.

2.5. Effects of 4b on impairments in sensory-motor control and tyrosine hydroxylase (TH)

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expression in MPTP-intoxicated mice

NO over-production by iNOS in the brain has been implicated in the development of PD, which is characterized by the slow and progressive degeneration of dopaminergic neurons in the substantia nigra (SN). The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP) upregulates iNOS activity [15], leading to dopaminergic neurodegeneration in a murine model of PD. iNOS-deficient mice are almost completely protected from MPTP-induced toxicity, and treatment of adult mice with MPTP results in a pronounced reduction in locomotor activity [16]. Therefore, to verify the iNOS inhibition and anti-neuroinflammation of 4b in vivo, MPTP-induced PD mouse models were used to determine whether 4b can protect against PD. As shown in Fig. 4A, all mice were pretrained for 3 d to maintain themselves on the rod for 180 s at 25 rpm. The MPTP group showed significantly impaired performance compared with normal controls at 4, 24, 48, and 72 h 5

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after the last MPTP injection. However, oral administration of 4b at 10 or 20 mg/kg significantly alleviated the functional impairment observed in the MPTP-treated group at 4, 24, 48, and 72 h after MPTP injection (Fig. 4B). The early loss of TH activity, followed by a decline in TH protein levels was thought to contribute to dopamine deficiency. Our results showed that b4 can better increase TH-positive cells in substantia nigra (SN) and striatum (STR) compared with the MPTP (Fig. 4C).

PD. 2.6. Effects of 4b on MPTP-induced microglial activation

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The above results suggest 4b as a promising lead in the development of orally active therapies for

Astrocytes and microglia-mediated neuroinflammation may play important roles in Parkinson’s disease (PD). Furthermore, most studies have concluded that the activations of

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microglia and astrocytes are closely associated with neurotoxicity and neurodegeneration [17, 18]. In the current study, to further confirm the anti-neuroinflammation of 4b in vivo, we detected the

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expression of glial fibrillary acidic portein (GFAP), a marker for activated astrocytes, using immunofluorescence [19]. MPTP significantly increased the expression of GFAP in STR and SN, and pretreatment of 4b effectively blocked astroglial activation. These results further suggested that the anti-PD effects of 4b are involved in anti-neuroinflammation in vivo.

3. Conclusions

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A total of 26 clovamide analogues were designed and synthesized, and their antineuroinflammatory effects were evaluated using a model of LPS-induced BV-2 cells. Among these compounds, 1b and 4b–8b showed potent NO release inhibition at a concentration of 10 µM. Molecular docking studies, which were necessary to validate the results derived from the kinetic

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assay, revealed that these compounds are potential iNOS inhibitors. Notably, the optimal candidate compound, 4b, effectively reduced the level of pro-inflammatory mediators induced by LPS in

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vitro. Under in vivo conditions, oral treatment with 4b significantly relieved the behavior impairment, increased TH-positive cell number, and decreased the expression of GFAP, a neuroinflammation marker, in both the SN and STR of PD mice. All of these results highlight the potential of compound 4b as a promising lead in the development of orally active therapies for PD. 4. Experimental section 4.1 Chemistry 4.1.1 General Reagents and solvents were purchased from common commercial suppliers and were used without further purification. Reaction progress was monitored using analytical thin layer chromatography (TLC) on precoated silica gel GF254 (Qingdao Haiyang Chemical Plant, Qingdao, 6

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China) plates. Optical rotations were measured on a Rudolph Autopol IV polarimeter at room temperature. 1H and

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C NMR spectra were recorded in DMSO solutions using a Bruker AV-300

(300 MHz) spectrometer at 25 ℃. Chemical shifts are reported in δ (ppm) relative to internal Me4Si. J values are given in hertz, and spin multiplicities are expressed as s (singlet), d (doublet), t (triplet), q (quartet), or m (multiplet). Preparative HPLC was carried out on a Shimadzu LC-8A instrument equipped with an SPD-20A detector, and a YMC-pack Preparative HPLC column (C18,

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20 × 250 mm, 5 µm). HRESIMS data were acquired on an Agilent 6520 Q-TOF mass spectrometer (Agilent Technologies, USA). The purities of all compounds were confirmed to be higher than 95% through analytical HPLC performed with Agilent 1200 HPLC System.

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4.1.2 General procedure for the preparation of 1a-13a and 1b-13b

The analogues (1a-13a, 1b-13b) have been synthesized through previous reported methods [20], As shown in Scheme 1, the synthetic strategy involved a three-step sequence via methyl

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esterification of L-amino acid, amide condensation, and the hydrolysis of methyl ester. 210 µL SOCl2 was added in portions to 4 mL methanol at 0 ℃, then 1 mmol L-amino acid was added and the mixture was warmed to room temperature and stirred overnight. After the solvent was removed, 4

mL

Pyridine,

1.1

mmol

corresponding

substituted

acid

and

1.1

mmol

DCC

(dicyclohexylcarbodiimide) was added into the residue. The mixture was stirred for 72 h under N2

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to finish condensation. The reaction solution was added 20 mL 2 M H2SO4, and extracted with ethyl acetate (4 × 15 mL). The combined organic phase was dried over anhydrous Na2SO4 and finally evaporated in vacuum. The residue was purified by silica-gel chromatography using mixtures of PE/EtOAc as eluent to give 1a-13a. Compounds 1b-13b were prepared by hydrolysis of 1a-13a,

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respectively, which was treated by 3 mmol LiOH in THF/H2O (V/V 2:1) at 40 ℃ overnight. THF was removed in vacuum and the residual aqueous solution was acidified to pH 2 with 1N HCl. The

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aqueous phase was extracted four times with EtOAc to obtain the crude products. Finally, the residue was further purified by preparative HPLC to afford 1b-13b. At this stage, all compounds were fully analyzed and characterized by nuclear magnetic resonance (NMR), mass spectrometry (MS), and optical rotations before entering the biological assay. 4.1.3. N-[4'-Hydroxy-(E)-Cinnamoyl]-L-tyrosine methyl ester (1a) Light yellow powder in 57 % yield. [α]20D -17.7 (c 0.1, MeOH). 1H NMR (300 MHz, DMSO-d6) δ 9.80 (1H, s), 9.21 (1H, s), 8.33 (1H, d, J = 7.8 Hz), 7.38 (2H, d, J = 8.4 Hz), 7.29 (1H, d, J = 15.9 Hz), 7.01 (2H, d, J = 8.4 Hz), 6.79 (2H, d, J = 8.7 Hz), 6.65 (2H, d, J = 8.4 Hz), 6.47 (1H, d, J = 15.9 Hz), 4.50 (1H, m), 3.60 (3H, s), 2.95 (1H, dd, J = 5.4 Hz, 13.8 Hz), 2.83 (1H, dd, J = 9.0 Hz, 13.8 Hz), 13C NMR (75 MHz, DMSO-d6) δ 36.1, 51.7, 54.0, 115.0 (2C), 115.7, 117.8, 125.6, 127.1, 7

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129.2, 129.8, 139.4, 155.9, 158.9, 165.3, 172.2. HRMS: calcd. for C19H19NO5 [M + H]+ 342.1344, found 342.1338. 4.1.4. N-(E)-Cinnamoyl-L-tyrosine methyl ester (2a) Transparent crystal in 47 % yield. [α]20D -10.9 (c 0.1, MeOH). 1H NMR (300 MHz, DMSO-d6) δ 9.21 (1H, s), 8.48 (1H, d, J = 7.8 Hz), 7.56 (2H, d, J = 6.6 Hz), 7.28~7.43 (4H, m), 7.02 (2H, d, J

dd, J = 5.7 Hz, 13.8 Hz), 2.85 (1H, dd, J = 8.7 Hz, 13.8 Hz),

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= 8.4 Hz), 6.70 (1H, d, J = 15.6 Hz), 6.66 (2H, d, J = 8.1 Hz), 4.54 (1H, m), 3.62 (3H, s), 2.97 (1H, C NMR (75 MHz, DMSO-d6) δ

36.1, 51.8, 54.1, 115.1, 121.4, 127.1, 127.6, 128.9, 129.6, 129.9 (2C), 134.7, 139.4, 156.0, 164.9,

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172.2. HRMS: calcd. for C19H19O4 [M + H]+ 326.1395, found 326.1389. 4.1.5. N-[3'-Hydroxy-(E)-Cinnamoyl]-L-tyrosine methyl ester (3a)

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White powder in 40 % yield. [α]20D -21.3 (c 0.1, MeOH). 1H NMR (300 MHz, DMSO-d6) δ 9.55 (1H, s), 9.20 (1H, s), 8.46 (1H, d, J = 7.5 Hz), 7.29 (1H, d, J = 15.9 Hz), 7.19 (1H, t), 7.01 (2H, d, J = 8.4 Hz), 6.96 (1H, d, J = 7.5 Hz), 6.91 (1H, s), 6.77 (1H, dd, J = 1.5 Hz, 7.8 Hz), 6.65 (2H, d, J = 8.4 Hz), 6.59 (1H, d, J = 15.9 Hz), 4.51 (1H, m), 3.60 (3H, s), 2.95 (1H, dd, J = 5.4 Hz, 13.8 Hz), 2.82 (1H, dd, J = 9 Hz, 13.8 Hz),

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C NMR (75 MHz, DMSO-d6) δ 36.6, 52.3, 54.6,

114.2, 115.6 (2C), 117.3, 119.2, 121.7, 127.6, 130.4 (2C), 136.5, 140.1, 156.5, 158.2, 165.4, 172.7.

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HRMS: calcd. for C19H19NO5 [M + H]+ 342.1342, found 342.1337.

4.1.6. N-[4'-Hydroxy-3'-Methoxy-(E)-Cinnamoyl]-L-tyrosine methyl ester (4a) Light yellow powder in 64 % yield. [α]20D -14.6 (c 0.1, MeOH). 1H NMR (300 MHz, DMSO-

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d6) δ 9.42 (1H, s), 9.20 (1H, s), 8.28 (1H, d, J = 7.8 Hz), 7.29 (1H, d, J = 15.6 Hz), 7.12 (1H, s), 7.01 (2H, d, J = 8.1 Hz), 6.97 (1H, d, J = 8.1 Hz), 6.79 (1H, d, J = 8.1 Hz,), 6.66 (2H, d, J = 8.1

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Hz), 6.51 (1H, d, J = 15.9 Hz), 4.52 (1H, m), 3.81 (3H, s), 3.61 (3H, s), 2.95 (1H, dd, J = 5.4 Hz, 13.8 Hz), 2.83 (1H, dd, J = 9.0 Hz, 13.8 Hz),

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C NMR (75 MHz, DMSO-d6) δ 36.6, 52.2, 54.5,

56.1, 111.3, 115.5 (2C), 116.1, 118.6, 122.2, 126.7, 127.7, 130.4 (2C), 140.3, 148.3, 148.9, 156.5, 164.8, 172.8. HRMS: calcd. for C20H21NO6 [M + H]+ 372.1448, found 372.1443.

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4.6.7. N-[4'-Hydroxy-(E)-Cinnamoyl]-L-phenylalanine methyl ester (5a) Light yellow powder in 43 % yield. [α]20D -15.1 (c 0.1, MeOH). 1H NMR (300 MHz, DMSOd6) δ 9.83 (1H, s), 8.39 (1H, d, J = 8.0 Hz), 7.38 (2H, d, J = 8.5 Hz), 7.30 (1H, d, J = 15.5 Hz), 7.32 ~ 7.20 (5H, m), 6.79 (2H, d, J = 8.5 Hz), 4.61 (1H, m), 3.62 (3H, s), 3.08 (1H, dd, J = 5.5 Hz,14.0 Hz), 2.96 (1H, dd, J = 9.0 Hz, 14.0 Hz), 13C NMR (75 MHz, DMSO-d6) δ 37.3, 52.3, 54.2, 116.2 HRMS: calcd. for C19H19NO4 [M + Na]+ 348.1208, found 348.1201. 4.6.8. N-(E)-Cinnamoyl-L-phenylalanine methyl ester (6a)

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(2C), 118.2, 126.2, 127.0, 128.7 (2C), 129.5 (2C), 129.8 (2C), 137.7, 140.1, 159.5, 165.9, 172.8.

White powder in 60 % yield. [α]20D -22.4 (c 0.1, MeOH). 1H NMR (300 MHz, DMSO-d6) δ 8.53 (1H, d, J = 7.5 Hz), 7.55 (2H, d, J = 7.0 Hz), 7.42~7.36 (4H, m), 7.30~7.20 (5H, m), 6.68 (1H,

Hz, 14.0 Hz),

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d, J = 16.0 Hz), 4.62 (1H, m), 3.62 (3H, s), 3.09 (1H, dd, J = 5.5 Hz, 13.5 H), 2.97 (1H, dd, J = 9.5 C NMR (75 MHz, DMSO-d6) δ 37.3, 52.3, 54.2, 121.8, 127.0, 128.1 (2C), 128.7

(2C), 129.4 (2C), 129.5 (2C), 130.1, 135.2, 137.6, 140.4, 165.3, 172.5. HRMS: calcd. for

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C18H17NO4 [M + H]+ 310.1439, found 310.1433.

4.6.9. N-[3'-Hydroxy-(E)-Cinnamoyl]-L-phenylalanine methyl ester (7a) White powder in 63 % yield. [α]20D -20.7 (c 0.1, MeOH). 1H NMR (300 MHz, DMSO-d6) δ 9.54 (1H, s), 8.52 (1H, d, J = 7.5 Hz), 7.31 ~ 7.21 (6H, m), 7.20 (1H, t), 6.96 (1H, d, J = 7.5 Hz), 6.91 (1H, s), 6.78 (1H, dd, J = 1.5 Hz, 8.0 Hz), 6.59 (1H, d, J = 16.0 Hz), 4.61 (1H, m), 3.62 (3H, 13

C NMR (75 MHz,

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s), 3.08 (1H, dd, J = 5.5 Hz, 13.5 Hz), 2.96 (1H, dd, J = 9.5 Hz, 13.5 Hz),

DMSO-d6) δ 37.3, 52.3, 54.2, 114.2, 117.3, 119.2, 121.6, 127.0, 128.7, 129.5, 130.4, 136.4, 137.6, 140.2, 158.2, 165.4, 172.5. HRMS: calcd. for C19H19NO4 [M + H]+ 326.1391, found 326.1386. 4.6.10. N-[4'-hydroxy-3'-Methoxy-(E)-Cinnamoyl]-L-phenylalanine methyl ester (8a)

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White powder in 47 % yield. [α]20D -16.9 (c 0.1, MeOH). 1H NMR (300 MHz, DMSO-d6) δ 9.43 (1H, s), 8.37 (1H, d, J = 7.2 Hz), 7.32 ~ 7.23 (6H, m), 7.12 (1H, s), 6.98 (1H, d, J = 8.1 Hz),

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6.78 (1H, d, J = 8.1 Hz), 6.50 (1H, d, J = 15.6 Hz), 4.61 (1H, m), 3.80 (3H, s), 3.62 (3H, s), 3.08 (1H, dd, J = 4.8 Hz, 13.5 Hz), 2.95 (1H, dd, J = 9.6 Hz, 13.0 Hz), 13C NMR (75 MHz, DMSO-d6) δ 37.3, 52.3, 54.2, 56.0, 111.3, 116.1, 118.6, 122.3, 126.7, 127.0, 128.7, 129.5, 137.7, 140.4, 148.3, 148.9, 165.9, 172.7. HRMS: calcd. for C20H21NO5 [M + H]+ 356.1494, found 356.1489. 4.6.11. N-[4'-Hydroxy-(E)-Cinnamoyl]-L-Aspartic dimethyl ester (9a) White powder in 44 % yield. [α]20D -13.1 (c 0.1, MeOH). 1H NMR (300 MHz, DMSO-d6) δ 9.84 (1H, s), 8.43 (1H, d, J = 7.5 Hz), 7.40 (2H, d, J = 8.5 Hz), 7.35 (1H, d, J = 16.0 Hz), 6.78 (2H, d, J = 8.5 Hz), 6.47 (1H, d, J = 15.5 Hz), 4.76 (1H, m), 3.65 (3H, s), 3.62 (3H, s), 2.86 (1H, dd, J = 6.0 Hz, 16.5 Hz), 2.78 (1H, dd, J = 7.5 Hz, 16.5 Hz), 13C NMR (75 MHz, DMSO-d6) δ 36.3, 49.1, 52.1, 52.6, 116.2 (2C), 118.2, 126.2, 129.8, 140.3, 159.5, 165.8, 170.9, 171.7. HRMS calcd. for C15H17NO6 [M + H]+ 308.1131, found 308.1124. 9

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4.6.12. N-[3'-Hydroxy-(E)-Cinnamoyl]-L-Aspartic dimethyl ester (10a) Light yellow powder in 65 % yield. [α]20D -18.4 (c 0.1, MeOH). 1H NMR (300 MHz, DMSOd6) δ 9.54 (1H, s), 8.58 (1H, d, J = 8.1 Hz), 7.35 (1H, d, J = 15.6 Hz), 7.20 (1H, t), 6.98 (1H, d, J = 7.5 Hz), 6.94 (1H, s), 6.78 (1H, d, J = 7.8 Hz), 6.61 (1H, d, J = 15.6 Hz), 4.77 (1H, m), 3.65 (3H, s), 3.62 (3H, s), 2.86 (1H, dd, J = 6.0 Hz, 16.5 Hz), 2.78 (1H, dd, J = 7.5 Hz, 16.8 Hz), 13C NMR (75 MHz, DMSO-d6) δ 36.2, 49.2, 52.2, 52.7, 114.3, 117.4, 119.2, 121.5, 130.4, 136.4, 140.4, 158.2,

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165.4, 170.9, 171.6. HRMS: calcd. for C15H17NO6 [M + H]+ 308.1130, found 308.1124. 4.6.13. N-(E)-Cinnamoyl-L-glutamate dimethyl ester (11a)

White powder in 36 % yield. [α]20D -16.8 (c 0.1, MeOH). 1H NMR (300 MHz, DMSO-d6) δ 8.51 (1H, d, J = 7.5 Hz), 7.57 (2H, d, J = 7.0 Hz), 7.48 ~ 7.35 (4H, m), 6.69 (1H, d, J = 15.6 Hz), 13

C NMR (75

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MHz, DMSO-d6) δ 26.7, 30.1, 51.8, 51.9, 52.5, 121.7, 128.1 (2C), 129.4 (2C), 130.1, 135.2, 140.1,

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165.6, 172.6, 173.0. HRMS: calcd. for C16H19NO5 [M + H]+ 306.1339, found 306.1334. 4.6.14. N-[3'-Hydroxy-(E)-Cinnamoyl]-L-glutamate dimethyl ester (12a) White powder in 38 % yield. [α]20D -17.7 (c 0.1, MeOH). 1H NMR (300 MHz, DMSO-d6) δ 9.56 (1H, s), 8.48 (1H, d, J = 7.5 Hz), 7.35 (1H, d, J = 15.9 Hz), 7.21 (1H, t), 6.98 (1H, d, J = 7.5 Hz), 6.94 (1H, s), 6.79 (1H, d, J = 7.0 Hz), 6.61 (1H, d, J = 15.6 Hz), 4.10 (1H, m), 3.64 (3H, s), 3.59 (3H, s), 2.42 (2H, m), 2.04 (1H, m), 1.91 (1H, m). 13C NMR (75MHz, DMSO-d6) δ 26.7, 30.1,

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51.8, 51.9, 52.4, 114.2, 117.3, 119.2, 121.5, 130.4, 136.4, 140.2, 158.2, 165.6, 172.6, 173.0. HRMS: calcd. for C16H19NO6 [M + H]+ 322.1289, found 322.1283. 4.6.15. N-[4'-Hydroxy-3'-Methoxy-(E)-Cinnamoyl]-L-glutamate dimethyl ester (13a) Light yellow powder in 47 % yield. [α]20D -14.7 (c 0.1, MeOH). 1H NMR (300 MHz, DMSO-

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d6) δ 9.44 (1H, s), 8.35 (1H, d, J = 7.5 Hz), 7.34 (1H, d, J = 15.6 Hz), 7.14 (1H, s), 6.99 (1H, d, J = 7.8 Hz), 6.80 (1H, d, J = 8.1 Hz), 6.52 (1H, d, J = 15.6 Hz), 4.39 (1H, m), 3.81 (3H, s), 3.64 (3H, s), 13

C NMR (75 MHz, DMSO-d6) δ 26.7,

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3.59 (3H, s), 2.37 (2H, m), 2.05 (1H, m), 1.90 (1H, m).

30.1, 51.8, 51.9, 52.4, 56.0, 111.2, 116.1, 118.5, 122.2, 126.7, 140.4, 148.3, 148.9, 166.1, 172.7, 173.0. HRMS: calcd. for C17H21NO7 [M + H]+ 352.1395, found 352.1390. 4.1.16. N-[4'-Hydroxy-(E)-Cinnamoyl]-L-tyrosine (1b) Light yellow powder in 51 % yield. [α]20D -20.8 (c 0.1, MeOH). 1H NMR (300 MHz, DMSOd6) δ 7.89 (1H, d, J = 7.0 Hz), 7.36 (2H, d, J = 7.8 Hz), 7.25 (1H, d, J = 15.0 Hz), 7.00 (2H, d, J = 7.2 Hz), 6.79 (2H, d, J = 7.5Hz), 6.62 (2H, d, J = 7.2 Hz), 6.53 (1H, d, J = 15.0 Hz), 4.40 (1H, m), 3.02 (1H, dd, J = 1.5 Hz, 12.3 Hz), 2.80 (1H, dd, J = 9.0 Hz, 12.6 Hz), 13C NMR (75 MHz, DMSOd6) δ 36.1, 54.9, 114.8 (2C), 115.7 (2C), 118.8, 125.8, 128.5, 129.1, 130.0, 138.6, 155.6, 158.9, 164.9, 174.4. HRMS: calcd. for C18H17NO5 [M - H]- 326.1020, found 326.1025. 4.1.17. N-(E)-Cinnamoyl-L-tyrosine (2b) 10

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White powder in 42 % yield. [α]20D -17.1 (c 0.1, MeOH). 1H NMR (300 MHz, DMSO-d6) δ 7.91 (1H, d, J = 7.2 Hz), 7.53 (2H, d, J = 6.3 Hz), 7.44 ~ 7.31 (4H, m), 7.00 (2H, d, J = 7.8 Hz), 6.79 (1H, d, J = 15.6 Hz), 6.60 (2H, d, J = 7.8 Hz), 4.37 (1H, m), 3.06 (1H, dd, J = 2.4 Hz, 13 Hz), 2.83 (1H, dd, J = 7.8 Hz, 13 Hz), 13C NMR (75 MHz, DMSO-d6) δ 37.3, 56.0, 115.2, 123.4, 128.0 (2C), 129.3 (2C), 129.4, 129.7, 130.6 (2C), 135.2, 138.6, 156.0, 164.5, 172.3. HRMS: calcd. for C18H17NO4 [M - H]- 310.1076, found 310.1081.

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4.1.18. N-[3'-Hydroxy-(E)-Cinnamoyl]-L-tyrosine (3b)

Light yellow powder in 34 % yield. [α]20D -12.7 (c 0.1, MeOH). 1H NMR (300 MHz, DMSOd6) δ 8.30 (1H, d, J = 7.8 Hz), 7.27 (1H, d, J = 15.9 Hz), 7.19 (1H, t), 7.02 (2H, d, J = 8.4 Hz), 6.95 (1H, d, J = 8.1 Hz), 6.92 (1H, s), 6.78 (1H, dd, J = 1.8 Hz, 7.8Hz), 6.65 (2H, d, J = 8.1 Hz), 6.62 13

C NMR (75

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(1H, d, J = 15.9Hz), 4.45 (1H, m), 2.97 (1H, m), 2.82 (1H, dd, J = 9Hz, 13.5 Hz),

MHz, DMSO-d6) δ 36.1, 54.4, 114.2, 115.5 (2C), 117.2, 119.2, 122.1, 128.2, 130.3, 130.4 (2C),

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136.5, 139.7, 156.3, 158.2, 165.3, 173.8. HRMS calcd. for C18H17NO5 [M - H]- 326.1026, found 326.1031.

4.1.19. N-[4'-Hydroxy-3'-Methoxy-(E)-Cinnamoyl]-L-tyrosine (4b)

Light yellow powder in 59 % yield. [α]20D -19.7 (c 0.1, MeOH). 1H NMR (300 MHz, DMSOd6) δ 8.01 (1H, d, J = 7.5 Hz), 7.26 (1H, d, J = 15.5 Hz), 7.12 (1H, s), 7.01 (2H, d, J = 8 Hz), 6.97 (1H, d, J = 8 Hz), 6.78 (1H, d, J = 8 Hz), 6.65 (2H, d, J = 8 Hz), 6.55 (1H, d, J = 15.5 Hz), 4.44

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(1H, m), 3.80 (3H, s), 2.99 (1H, dd, J = 4.5 Hz, 14 Hz), 2.81 (1H, dd, J = 8.5 Hz, 14 Hz), 13C NMR (75 MHz, DMSO-d6) δ 36.7, 54.7, 56.0, 111.2, 115.4, 116.1, 119.2, 122.1, 126.8, 128.3, 130.5 (2C), 139.8, 148.3, 148.8, 156.3, 165.7, 173.9. HRMS: calcd. for C19H19NO6 [M - H]- 356.0950, found 356.1135.

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4.1.20. N-[4'-Hydroxy-(E)-Cinnamoyl]-L-pheylalanine (5b) White powder in 40 % yield. [α]20D -17.7 (c 0.1, MeOH). 1H NMR (300 MHz, DMSO-d6) δ

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7.62 (1H, d, J = 6.9 Hz), 7.36 (2H, d, J = 8.4 Hz), 7.23 (1H, d, J = 15.9 Hz), 7.22 ~ 7.10 (5H, m), 6.77 (2H, d, J = 8.4 Hz), 6.56 (1H, d, J = 15.9 Hz), 4.33 (1H, m), 3.16 (1H, dd, J = 4.5 Hz, 13.5 Hz), 2.95 (1H, dd, J = 6.9 Hz, 13.5 Hz), 13C NMR (75 MHz, DMSO-d6) δ 38.3, 55.9, 116.2 (2C), 119.8, 126.1, 126.4, 128.2 (2C), 129.6 (2C), 129.8 (2C), 138.7, 139.8, 159.4, 165.1, 174.5. HRMS: calcd. for C18H17NO4 [M - H]- 310.1076, found 310.1082. 4.1.21. N-(E)-Cinnamoyl-L-pheylalanine (6b) White powder in 55 % yield. [α]20D -13.8 (c 0.1, MeOH). 1H NMR (300 MHz, DMSO-d6) δ 8.16 (1H, d, J = 6.9 Hz), 7.54 (2H, d, J = 6.6 Hz), 7.44 ~7.31 (4H, m), 7.28 ~ 7.12 (5H, m), 6.75 (1H, d, J = 15.6 Hz), 4.49 (1H, m), 3.15 (1H, dd, J = 3.9 Hz, 13.5 Hz), 2.94 (1H, dd, J = 8.4 Hz, 13.5 Hz), 13C NMR (75 MHz, DMSO-d6) δ 37.7, 54.9, 122.8, 126.6, 128.0 (2C), 128.5 (2C), 29.3

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(2C), 29.7 (2C), 29.8, 135.4, 138.8, 139.1, 165.0, 173.9. HRMS: calcd. for C18H17NO3 [M - H]294.1127, found 294.1133. 4.1.22. N-[3'-Hydroxy-(E)-Cinnamoyl]-L-pheylalanine (7b) Light yellow powder in 56 % yield. [α]20D -14.7 (c 0.1, MeOH). 1H NMR (300 MHz, DMSOd6) δ 8.05 (1H, d, J = 6.9 Hz), 7.29 ~ 7.11 (7H, m), 6.95 (2H, m), 6.78 (1H, d, J = 8.1 Hz), 6.69 (1H, d, J =15.9 Hz), 4.44 (1H, m), 3.17 (1H, dd, J =4.8 Hz, 12.9 Hz), 2.93 (1H, dd, J =8.4 Hz, 13.5 13

C NMR (75 MHz, DMSO-d6) δ 37.3, 55.5, 114.3, 117.1, 119.0, 122.8, 126.4, 128.4 (2C),

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Hz),

29.5 (2C), 30.2, 136.7, 139.1, 139.3, 158.3, 164.9, 174.8. HRMS: calcd. for C18H17NO4 [M - H]310.1082, found 310.1087.

4.1.23. N-[4'-Hydroxy-3'-Methoxy-(E)-Cinnamoyl]-L-pheylalanine (8b)

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Light yellow powder in 42 % yield. [α]20D -15.2 (c 0.1, MeOH). 1H NMR (300 MHz, DMSOd6) δ 7.91 (1H, d, J = 7.5 Hz), 7.28 ~ 7.20 (6H, m), 7.12 (1H, s), 6.95 (1H, d, J = 8 Hz), 6.80 (1H, d, J = 8 Hz), 6.57 (1H, d, J = 15.5 H), 4.46 (1H, m), 3.79 (3H, s), 3.14 (1H, dd, J = 4.5 Hz, 14 Hz),

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2.94 (1H, dd, J = 8.5 Hz, 14 Hz), 13C NMR (75 MHz, DMSO-d6) δ 37.7, 55.0, 56.0, 111.1, 116.0, 119.2, 122.3, 126.6, 126.8, 128.5 (2C), 129.6 (2C), 138.7, 139.8, 148.3, 148.7, 165.8, 174.3. HRMS: calcd. for C19H19NO5 [M - H]- 340.1192, found 340.1197. 4.1.24. N-[4'-Hydroxy-(E)-Cinnamoyl]-L-aspartic acid (9b)

Light yellow powder in 38 % yield. [α]20D -14.1 (c 0.1, MeOH). 1H NMR (300 MHz, DMSO-

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d6) δ 12.62 (2H, s), 9.84 (1H, s), 8.25 (1H, d, J = 7.8 Hz), 7.40 (2H, d, J = 8.4 Hz, H-C2’, 6’), 7.34 (1H, d, J = 15.9 Hz, H-C7’), 6.79 (2H, d, J = 8.4 Hz), 6.52 (1H, d, J = 15.9 Hz), 4.64 (1H, m), 2.73 (1H, dd, J = 5.7 Hz, 16.5 Hz), 2.64 (1H, dd, J = 6.9 Hz, 16.5 Hz). 13C NMR (75 MHz, DMSO-d6) δ 36.8, 49.2, 116.2 (2C), 118.6, 126.3, 129.7 (2C), 39.9, 159.4, 165.7, 172.2, 173.0. HRMS: calcd. for

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C13H13NO6 [M - H]- 278.0670, found 278.0675.

4.1.25. N-[3'-Hydroxy-(E)-Cinnamoyl]-L-aspartic acid (10b)

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White powder in 32 % yield. [α]20D -20.4 (c 0.1, MeOH). 1H NMR (300MHz, DMSO-d6) δ 8.41 (1H, d, J = 7.8 Hz), 7.32 (1H, d, J = 15.9 Hz), 7.18 (1H, t), 6.96 (1H, d, J = 7.8 Hz), 6.92 (1H, s), 6.77 (1H, d, J = 7.8 Hz), 6.64 (1H, d, J = 15.9 Hz), 4.67 (1H, m), 2.78 (1H, m), 2.68 (1H, m), 13C NMR (300 MHz, DMSO-d6) δ 26.6, 51.5, 113.7, 116.4, 122.6, 130.0, 136.2, 139.5, 157.8, 165.1, 173.3, 173.7. HRMS: calcd. for C13H13NO6 [M - H]- 278.0668, found 278.0673. 4.1.26. N-(E)-Cinnamoyl-L-glutamic acid (11b) White powder in 30 % yield. [α]20D -21.6 (c 0.1, MeOH). 1H NMR (300 MHz, DMSO-d6) δ 8.41 (1H, d, J = 7.2 Hz), 7.57 (2H, d, J = 7.0 Hz), 7.47 ~7.37 (4H, m), 6.74 (1H, d, J = 15.9 Hz), 4.35 (1H, m), 2.35 (2H, m), 2.03 (1H, m), 1.85 (1H, m). 13C NMR (300 MHz, DMSO-d6) δ 26.9, 30.6, 51.8, 122.2, 128.0 (2C), 129.4 (2C), 130.0, 135.3, 139.6, 165.5, 173.7, 174.0. HRMS calcd. for C14H15NO5 [M - H]- 276.0878, found 276.0883. 12

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4.1.27. N-[3'-Hydroxy-(E)-Cinnamoyl]-L-glutamic acid (12b)

Light yellow powder in 34 % yield. [α]20D -23.7 (c 0.1, MeOH). 1H NMR (300 MHz, DMSOd6) δ 8.34 (1H, d, J = 7.8 Hz), 7.33 (1H, d, J = 15.9 Hz), 7.21 (1H, t), 6.98 (1H, d, J = 7.8 Hz), 6.94 (1H, s), 6.79 (1H, d, J = 8.1 Hz), 6.63 (1H, d, J = 15.9), 4.34 (1H, m), 2.30 (2H, m), 2.01 (1H, m), 1.85 (1H, m), 13C NMR (300 MHz, DMSO-d6) δ 26.6, 30.2, 51.5, 113.8, 116.8, 121.5, 130.0, 136.2, 139.5, 157.8, 165.1, 173.3, 173.7. HRMS: calcd. for C14H15NO6 [M - H]- 292.0827, found

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292.0832.

4.1.28. N-[4'-hydroxy-3'-Methoxy-(E)-Cinnamoyl]-L-glutamic acid (13b)

White powder in 43 % yield. [α]20D -18.1 (c 0.1, MeOH). 1H NMR (300 MHz, DMSO-d6) δ 9.43 (1H, s), 8.18 (1H, d, J=7.8 Hz), 7.34 (1H, d, J=15.9 Hz), 7.14 (1H, s), 7.00 (1H, d, J=8.1 Hz),

m), 1.84 (1H, m).

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6.80 (1H, d, J=8.1 Hz), 6.54 (1H, d, J=15.9 Hz), 4.33 (1H, m), 3.81 (3H, s), 2.31(2H, m), 2.03 (1H, C NMR (300 MHz, DMSO-d6) δ 26.6, 30.2, 55.6, 56.4, 110.8, 115.7, 118.5,

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121.7, 126.4, 139.4, 147.9, 148.5, 165.5, 173.3, 173.7. HRMS calcd. for C15H17NO7 [M - H]322.0929, found 322.0935.

4.2. Biological assays and experimental procedures 4.2.1 General

MTT, 0.25% trypsin-EDTA and dimethyl sulfoxide (DMSO) were purchased from Amresco (Solon, OH, USA). BCA proteins determining kit, NO determining kit, iNOS activity kit, and DAB

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staining kit were purchased from Beyotime (Shanghai, China). MPTP hydrochloride, paraformaldehyde (PFA) and Sodium azide were purchased from Sigma-Aldrich (St. Louis, MO, USA). RIPA Lysis Buffer was purchased from KeyGEN BioTECH (Nanjing, China). PVDF

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transfer membrane was purchased from Millipore Corp. (Bedford, MA, USA). Rabbit antibodies to IL-1β, iNOS, TNF-α, p65, COX-2, TH, GAPDH, and GFAP were purchased from Cell Signaling Technology (Beverly, MA, USA). Rabbit antibody to β-actin and Lamin B, and HRP-conjugated

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goat anti-rabbit (mouse) IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All remaining chemicals and reagents used in this experiment were the highest available analytic grade.

4.2.2. Cell culture

BV-2 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carls-bad, CA) supplemented with 10 % fetal bovine serum (Invitrogen), 100 units/mL penicillin, and 100 mg/mL streptomycin and incubated at 37 ℃ in a humidified atmosphere containing 5% CO2. 4.2.3. MTT assay

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BV-2 cells were seeded into 96-well plates with a density of 5 × 103 cells/well, and then were cultured for 12 hours. Then, 1-100 µM of all test compounds were added to the medium to incubate for another 24 hours. Afterwards, MTT was added to the culture medium to reach a final concentration of 0.5 mg/mL. After incubation at 37 ℃ for 4 h, the culture medium containing MTT was removed, and 150 µL DMSO was added into each well. The produced formazan was quantified by measuring the absorbance of the dye solution at 490 nm using a microtiter plate reader

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(TECAN M1000, Austria GmbH, Austria). 4.2.4. Assay for the inhibition of NO production

NO production was quantified by nitrite accumulation in the culture medium using the

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commercial kit (Beyotime Biotechnology, China). Briefly, BV-2 cells were pretreated with different concentrations (100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78 µM) compounds for 4 hours, and then stimulated with or without LPS (500 ng/mL) for 24 hours. The isolated supernatants were mixed

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with an equal volume of Griess reagent. NaNO2 was used to generate a standard curve, and nitrite production was determined by measuring the absorbance at 540 nm by a microplate reader (M1000, TECAN, Austria GmbH, Austria). The IC50 values of NO release inhibition were calculated by GraphPad Prism 7. 4.2.5. Assay for iNOS enzyme activity

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The iNOS enzyme activity was detected by a commercial kit (Beyotime Biotechnology, China). Briefly, after treated with LPS (500 ng/mL) and compounds 1b, 4b-8b (1-100 µM) for 4 hours at 37 ℃, the culture supernatant was removed and 100 µL of NOS assay buffer (1×) were

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added to each well. Then 100 µL of NOS assay reaction solution (50% NOS assay buffer, 39.8 % MilliQ water, 5 % L-Arginine solution, 5 % 0.1 mM NADPH, 0.2 % DAF-FMDA) was added to each well and incubated for 2 h at 37 ℃. Fluorescence was measured with a fluorescence plate

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reader (M1000, TECAN, Austria GmbH, Austria) at excitation of 485 nm and emission of 528 nm. The IC50 values of iNOS inhibition were determined by GraphPad Prism 7. 4.2.6. Western-blot analysis

BV-2 cells were seeded into 6-well plates with a density of 5 × 105 cells/well. After the treatments mentioned above, cells were collected and lysed in RIPA buffer with protease and phosphatase inhibitors. After centrifugation (4 ℃, 15 min, 10, 000 g), samples were prepared for Western blot analysis. For preparation of nuclear fraction, nuclear protein was extracted using the nuclear and cytoplasmic extraction kit (Beyotime Biotechnology, China) according to manufacturer’s instructions. Western blot was performed as previously described [21]. Equal amounts of protein extracts (30 µg) were subject to immunoblot analysis using GAPDH, IL-1β, 14

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iNOS, TNF-α, Lamin, NF-κB p65, and β-actin (all in 1:1000 dilution) antibodies followed by horseradish peroxidase-conjugated secondary antibodies (1:5000 dilution), and developed by ECL. Signal intensity was quantified using the software Image J2 in each group with β-actin or Lamin as internal control. The results were expressed as fold changes by normalizing the data to the control values.

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4.2.7. Animals, drug administration, and establishment of PD model Male C57BL/6 mice (7-8 weeks old, weighing approximately 20-22 g) were obtained from the Model Animal Research Center of Nanjing University. Animals were housed in a temperaturecontrolled (22-25 ℃) room with a 12:12-hrs light: dark cycle and had free access to food and water.

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Methylcellulose (0.5%) solution containing 1% Tween 80 was used to dissolve the 4b. Mice were randomly divided into five groups (8 mice/cage), including Group A, control; Group B, b4-only (10mg/kg); Group C, MPTP (20mg/kg); Group D, 4b (10 mg/kg) + MPTP (20 mg/kg); Group E, 4b

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(20 mg/kg) + MPTP (20 mg/kg), and allowed 3 days to acclimate before any treatments. 4b was pre-administered intragastrically (i.g.) at 10 mg/kg or 20 mg/kg body weight daily for 7 days. Group A and C animals were administered methylcellulose (0.5 %) solution containing 1 % Tween 80 before MPTP injection. After 7 days of pretreatment with 4b, four times of intraperitoneally (i.p.) injections of vehicle or MPTP (20 mg/kg) were administered to male C57BL/6 mice at 2-h intervals

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a day. All experiments were carried out according to the guidelines of the animal care and use committee at Animal Center of China Pharmaceutical University. 4.2.8. Rota-rod test

The test was similar to that described previously [22], and assessed using a rotary rod

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apparatus. All mice were pre-trained for 3 days prior to drug administration. The training consisted of three consecutive runs with a gradual increase in rpm up to a maximum 25 rpm until the mice

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were able to keep themselves without falling from the rotary rod for up to 180 s. After MPTP administration, mice were tested at 4, 24, 48, and 72 h. The average time of three tests was calculated for statistical analyses. 4.2.9. Tissue preparation

At 10 days after MPTP administration, mice were anesthetized with ether for 60 s, and then perfused intracardially with 0.9 % sodium chloride (PH = 7.4) and 4 % paraformaldehyde (PFA) in 0.1 M sodium dihydrogen phosphate containing 0.48 % sodium hydroxide (PH = 7.4). After fixative perfusion, brains were removed, placed in the same fixative solution at 4 ℃. overnight, and transferred to a 30 % sucrose solution till settle down. The cryoprotected brains were sectioned 15

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serially at 30 mm in the coronal plane using a freezing microtome (Microm, Walldorf, Germany), collected in Dulbecco’s PBS solution containing 0.1% sodium azide solution, and stored at 4 ℃. 4.2.10. Immunohistochemistry for TH and GFAP DAB immunostaining was similar to a previous study with some slight modifications [23]. Briefly, brain sections were treated with 0.6 % H2O2 in phosphate-buffered saline (PBS) for 20 min

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and then washed with Tris-buffered saline (TBS) for three times. After that, sections were blocked in 0.5 % Triton-x-100 in TBS and Triton X-100/3% horse serum (TBS-TS) for 60 min at room temperature, and incubated with primary antibody tyrosine hydroxylase (TH) and GFAP at 1:200 in TBS-TS at 4 ℃ for 48 h. Then after four times of washing with TBS, sections were incubated with

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secondary goat-rabbit IgG antibodies (1:1000, Bioworld) at room temperature for 2 h. After three times of washing with TBS, DAB solution was added to incubate for 5 min. Images were obtained with a DP72 digital camera (Olympus, Tokyo, Japan) and DP2-BSW microscope digital camera

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software (Olympus) and TH-positive cells were counted in entire extents of the SNpc. All cell counts were performed by the same informed investigator. 4.2.11. Docking assay

The iNOS was chosen as the target receptor. The 3D structure of the receptor was gained from

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Protein Data Bank (PDB ID: 1r35). Conformers of molecules were created by the aid of Omega and the up limit of conformer number for each molecule is set to 2000. Compound 1a-13a, 1b-13b were docked to the active site of iNOS by employing a protein-ligand docking program CDOCKER. Scoring functions, interaction energy, were used for exhaustive searching, solid body optimizing

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and interaction scoring. Fig. 2 was generated by the Discovery Studio 4.5 (DS 4.5) software.

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4.2.12. Statistical analysis

Data were represented as the mean ± S.E.M. Analysis of variance (ANOVA) with Tukey’s HSD-post hoc test procedure was used to determine the significances of differences between groups. The analysis was performed in Prism 7.0 software, and P < 0.05 was considered significant differences.

Conflict of interest The authors have no conflicts of interest.

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Acknowledgement

This work was financially supported by Jiangsu science and technology support program (BE2014710), and the National Natural Science Foundation of China (No. 81573309 and No.

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81473160).

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rat cardiomyoblasts H9c2 cell line, Food Funct, 5 (2014) 2542-2551. [10] H. Zeng, M. Locatelli, C. Bardelli, A. Amoruso, J.D. Coisson, F. Travaglia, M. Arlorio, S.

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Brunelleschi, Anti-inflammatory properties of clovamide and Theobroma cacao phenolic extracts in human monocytes: evaluation of respiratory burst, cytokine release, NF-kappaB activation, and PPARgamma modulation, J Agric Food Chem, 59 (2011) 5342-5350. [11] L. Alemanno, T. Ramos, A. Gargadenec, C. Andary, N. Ferriere, Localization and identification of phenolic compounds in Theobroma cacao L. somatic embryogenesis, Ann Bot-London, 92 (2003) 613-623. [12] J.Y. Park, B.W. Kim, H.U. Lee, D.K. Choi, S.H. Yoon, Synthesis of Clovamide Analogues That Inhibit NO Production in Activated BV-2 Microglial Cells, Biol Pharm Bull, 40 (2017) 1475-1482. [13] S. Aid, F. Bosetti, Targeting cyclooxygenases-1 and-2 in neuroinflammation: Therapeutic implications, Biochimie, 93 (2011) 46-51. 18

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[14] J.Q. He, L. Ma, Z. Wei, J. Zhu, F. Peng, M.F. Shao, L. Lei, L. He, M.H. Tang, L.H. He, Y.Z. Wu, L.J. Chen, Synthesis and biological evaluation of novel pyrazoline derivatives as potent anti-inflammatory agents, Bioorg Med Chem Lett, 25 (2015) 2429-2433. [15] G.T. Liberatore, V. Jackson-Lewis, S. Vukosavic, A.S. Mandir, M. Vila, W.G. McAuliffe, V.L. Dawson, T.M. Dawson, S. Przedborski, Inducible nitric oxide synthase stimulates dopaminergic neurodegeneration in the MPTP model of Parkinson disease, Nat Med, 5 (1999)

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[21] X.L. Hu, L.Y. Gao, Y.X. Niu, X. Tian, J. Wang, W.H. Meng, Q. Zhang, C. Cui, L. Han, Q.C. Zhao, Neuroprotection by Kukoamine A against oxidative stress may involve N-methyl-D-

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ACCEPTED MANUSCRIPT O R1

NH2

O

a

R1

OH

NH2 R3

OH

+ O

O

b

R2

R2

O

HN O

R3

c

R1

HN

O

OH

O

O

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Scheme 1

R3

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R1

R2

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Figure 1

Figure 2

20

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Figure 3

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Figure 4

21

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Figure 5

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22

ACCEPTED MANUSCRIPT Comp.

R1

1a-13aa R2 R3

Comp.

yield%

R1

1b-13bb R2

R3

yield%

OH

H

57

1b

OH

H

51

2a

H

H

47

2b

H

H

42

3a

H

OH

40

3b

4a

OH

OCH3

64

4b

5a

OH

H

43

5b

6a

H

H

7a

H

OH

8a

OH

OCH3

9a

OH

10a 11a

13a

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H

OH

34

OH

OCH3

59

OH

H

40

6b

H

H

55

63

7b

H

OH

56

47

8b

OH

OCH3

42

H

44

9b

OH

H

38

H

OH

65

10b

H

OH

32

H

H

36

11b

H

H

30

OH

38

12b

H

OH

34

OCH3

47

13b

OH

OCH3

43

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60

H

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12a

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1a

OH

Table 1 The chemical structure and reaction yield of each compounds.

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ACCEPTED MANUSCRIPT Compound

IC50 (NO), µM

IC50 (iNOS), µM

1b 4b

3.40±0.98 2.67±0.48

2.10±0.97 1.01±0.65

Docking score, kj/mol -46.34 -48.34

5b

10.43±1.77

8.45±1.56

-44.22

＞100

6b

26.45±2.38

18.87±2.98

-40.54

＞100

7b

15.34±2.12

29.23±3.11

-39.23

＞100

8b

4.41±1.11

2.98±0.91

-42.90

＞100

CC50, µM

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＞100 ＞100

Table 2 The inhibitory effects of 1b, 4b-8b on NO production, iNOS, and cell survival

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Note: The inhibition of the tested compounds on NO release, iNOS activity, cytotoxicity, and docking score. IC50: 50 % inhibitory concentration (means ± S.E.M. of three experiments). CC50: 50 % cell death concentration (means

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± S.E.M. of three experiments).

Graphical abstract

24

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Figure Legends

Scheme 1. (a) SOCl2, MeOH, 0 ℃ → R.T., overnight; (b) DCC, Pyridine, R.T., 72 h; (c) LiOH,

THF, H2O, 40 ℃, overnight.

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Figure 1. Synthetic compounds inhibited LPS-induced NO production in BV-2 cells. BV-2 cells

were pretreated with compounds (10 µM) for 4 h, and then stimulated with or without LPS (500 ng/mL) for 24 h. NO production was measured using nitrite and nitrate assay. Data were presented as Means ± S.E.M. (n=3).

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Figure 2. Docking study of 4b with iNOS. (A) 3D docking model of compound b4 with iNOS. (B)

2D model of 4b with iNOS, and the green segment represented H-bonding interactions (For

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interpretation of the color in this figure legend, the reader is recommended to see the web version of this article).

Figure 3. 4b inhibited LPS-induced inflammatory response in BV-2 cells. After pretreatment with b4 (1, 10, 20 µM), BV-2 cells were stimulated with LPS (500 ng/mL) for 24 h or 2 h. p65, IL-1β,

iNOS, TNF-α and COX-2 expression were detected by Western blot analysis. (A) Quantitative

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analysis of cyt-p65 expression, β-actin was used as loading control; (B) Quantitative analysis of Nup65 expression, Lamin B was used as loading control; (C) Quantitative analysis of IL-1β expression, β-actin was used as loading control; (D) Quantitative analysis of iNOS expression, βactin was used as loading control; (E) Quantitative analysis of TNF-α expression, β-actin was used

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as loading control; (F) Quantitative analysis of COX-2 expression, β-actin was used as loading control. #P < 0.05 or ##P < 0.01 compared with unstimulated cells, *P < 0.05 or **P < 0.01 compared

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with LPS-stimulated cells; Data were obtained by at least three independent experiments, and each was performed in duplicate.

Figure 4. The effects of 4b on motor function of mice, and TH expression. (A) Schematic of

experimental procedure; (B) The effects of 4b on rota-rod test; (C) Representative microphotographs of TH immunostaining of SN and Str. All the results were randomly obtained from three of the 8 mice/group. Figure 5. The effects of 4b on the expression of GFAP. After the 10th day of MPTP injection,

immunofluorescence histochemistry was performed. The representative microphotographs were the GFAP immunostaining of SN and Str. All the results were randomly obtained from three of the 8 mice/group. 25

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1. Twenty-six clovamide analogues were synthesized.

2. These clovamide analogues exhibited neuroinflammation in vitro. 3. Compounds b4 exhibited remarkable inhibitory effects on NO production and iNOS activity.

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4. Compounds b4 could improve the symptoms in a model of Parkinson’s disease.