Synthesis and biological evaluation of clovamide analogues with catechol functionality as potent Parkinson's disease agents in vitro and in vivo

Article abstract of DOI:10.1016/j.bmcl.2018.11.030

In this study, seven clovamide analogues (1–7) were designed and synthesized, and the neuroprotection of 1–7 as well as 8–15 (prepared in our previous work) against H₂O₂-induced oxidative stress was evaluated in SH-SY5Y cells. Results showed that 1–7 with catechol groups exhibited better neuroprotective effects than 8–15, and their EC₅₀ values ranged from 4.26 to 23.83 μM, especially 1, indicating that the moiety of catechol governed the activities of these compounds. Furthermore, oral administration of 1 (10 or 20 mg/kg) was demonstrated to possess anti-PD effect through alleviating apoptosis and oxidative stress in vitro and in vivo, and to up-regulate the expression of heme oxygenase-1 (HO-1) via PI3K/AKT/mTOR pathway. Finally, the pharmacokinetic (PK) assessment of 1 was determined in rats. These findings suggested that 1 might be an effective candidate for PD therapy.

Full text of DOI:10.1016/j.bmcl.2018.11.030

Accepted Manuscript
Synthesis and biological evaluation of clovamide analogues with catechol func-
tionality as potent Parkinson’s disease agents in vitro and in vivo
Jia-Hao Feng, Xiao-Long Hu, Xian-Yu Lv, Bao-Lin wang, Jun Lin, Xiao-Qi
Zhang, Wen-Cai Ye, Fei Xiong, Hao Wang
PII:
S0960-894X(18)30897-7
https://doi.org/10.1016/j.bmcl.2018.11.030
BMCL 26141
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
7 August 2018
23 October 2018
14 November 2018
Please cite this article as: Feng, J-H., Hu, X-L., Lv, X-Y., wang, B-L., Lin, J., Zhang, X-Q., Ye, W-C., Xiong, F.,
Wang, H., Synthesis and biological evaluation of clovamide analogues with catechol functionality as potent
Parkinson’s disease agents in vitro and in vivo, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://
doi.org/10.1016/j.bmcl.2018.11.030
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Synthesis and biological evaluation of clovamide analogues with
catechol functionality as potent Parkinson’s disease agents in vitro
and in vivo
Jia-Hao Fenga, Xiao-Long Hua, Xian-Yu Lva, Bao-Lin wanga, Jun Lina, Xiao-Qi Zhangb, Wen-Cai
Yeb, Fei Xiong*,c and Hao Wang*,a
a 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.
b Institute of Traditional Chinese Medicine & Natural Products, Jinan University, Guangzhou 510632,
People’s Republic of China.
c
State Key Laboratory of Bioelectronics, Jiangsu Laboratory for Biomaterials and Devices,
Southeast University, Nanjing 210009, People’s Republic of China.
*Corresponding author: Hao Wang, Professor
E-mail: wanghao@cpu.edu.cn
Postal address: Department of TCMs Pharmaceuticals, China Pharmaceutical University, Nanjing
210009, People’s Republic of China.
*Corresponding author: Fei Xiong, associate professor
E-mail: xiongfei@seu.edu.cn
Postal address: Jiangsu Laboratory for Biomaterials and Devices, Southeast University, Nanjing
210009 People’s Republic of China.
Abstract
1


In this study, seven clovamide analogues (1-7) were designed and synthesized, and the
neuroprotection of 1-7 as well as 8-15 (prepared in our previous work) against H2O2-induced
oxidative stress was evaluated in SH-SY5Y cells. Results showed that 1-7 with catechol groups
exhibited better neuroprotective effects than 8-15, and their EC50 values ranged from 4.26 to 23.83
μM, especially 1, indicating that the moiety of catechol governed the activities of these compounds.
Furthermore, oral administration of 1 (10 or 20 mg/kg) was demonstrated to possess anti-PD effect
through alleviating apoptosis and oxidative stress in vitro and in vivo, and to up-regulate the
expression of heme oxygenase-1 (HO-1) via PI3K/AKT/mTOR pathway. Finally, the
pharmacokinetic (PK) assessment of 1 was determined in rats. These findings suggested that 1 might
be an effective candidate for PD therapy.
Keywords: Clovamide analogue; Catechol functionality; Oxidative stress; Heme oxygenase-1;
Parkinson’s disease; Pharmacokinetic assessment
Parkinson’s disease (PD), the second most common age related neurodegenerative disease only
to Alzheimer's disease, is affecting 1% of the population above the age of 65, and the risk of PD is
2

continually increased in the elderly [1]. Although the exact etiology of PD is still unknown, the
pathogenesis is believed to be related with the progressive loss of dopamine-producing neurons in
the substantia nigra (SNpc), destroying the balance between excitatory neurotransmitter (acctyl
choline, ACh) and inhibitory neurotransmitter (dopamine, DA) [2]. Clinically, the symptoms of PD
included motor dysfunctions (resting tremor, postural instability, and muscle rigidity) and
non-motor-related symptoms (autonomic dysfunction, cognitive deficits, and sleep disorders) [3].
Since 1960’s, replacement or supplement of DA has always been the main therapeutic for PD. The
representative drugs are catechol-O-methyltransferase (COMT) inhibitors, monoamine oxidase type
B (MAO-B) inhibitors, and levodopa (L-DOPA) [4]. Although these drugs can greatly alleviate the
symptoms of PD, they cannot halt or reverse the disease progression induced by neuronal damage,
and long-term use of levodopa even accompanied by motor complications [5]. Therefore, it is
necessary to find out new therapeutic strategies that can protect neurons in SNpc to hamper PD
progression.
Oxidative stress, as the major inducer of nigral loss in both sporadic and genetic forms of PD,
represents the physiological response linked an imbalance between antioxidant defense systems and
reactive oxygen production, and plays a central role in the progression of the disorder [6]. Heme
oxygenase-1 (HO-1), also known as heat-shock protein 32 or inducible HO, is the rate-limiting
enzyme in heme catabolism with strong antioxidant activities. Emerging evidences support the role
for heme oxygenase-1 (HO-1) as important components of the cellular antioxidant and
cytoprotective defense [7, 8]. Recently, the anti-oxidant agents for PD treatment have received much
attention, and several anti-oxidants, such as Puerarin [9] and Desipramine [10], were found to
provide PD treatment involved in up-regulating the expression of HO-1, suggesting that HO-1 might
represent a promising and novel therapeutic strategy in treating Parkinsonism.
Clovamide, an amide analogue of rosmarinic acid, was first reported in red clover [11].
Evidences demonstrated that the protective effects of clovamides reducing oxidative stress-induced
damage to blood platelets, plasma and cardio myoblasts have been reported [12, 13]. However, there
are no significant data currently available on the neuroprotection against oxidative stress in vitro and
in vivo. In addition, catechol group was deemed as an important antioxidant pharmacophore in
various antioxidants, such as catecholamines and flavonoids [14], and clovamide analogues with
catechol group displayed significant effects of scavenging free radical [15]. Therefore, synthesizing
and evaluating the neuroprotection of clovamide analogues containing catechol functionality are
necessary in developing the neurodegenerative disease’s therapeutic. In order to evaluate whether the
catechol group plays an important role on the neuroprotective effects of clovamide analogues against
3

neuro-oxidative stress, seven representative clovamide analogues (1-7) with different position and
quantity of catechol groups were designed and synthesized, and the neuroprotective effects of 1-7 as
well as 8-15 without catechol functionality (prepared in our previous work [16]) against
H2O2-induced oxidative stress was evaluated in SH-SY5Y cells
Among them, 1-7 provide neuroprotection with EC50 values ranging from 4.26 μM to 23.83 μM,
while compounds 8-15 showed poor effects (Table 1), suggesting that catechol group might be the
important pharmacophore for their neuroprotective effects in this model, and their protective effects
are independent of the quantity of phenolic hydroxyl groups. Furthermore, the optimal one, N-[3',
4'-Dihydroxy-(E)-cinnamoyl]-L-phenylalanine methyl ester (1), was demonstrated to exhibit
significant neuroprotection involved in attenuating apoptosis and up-regulating the expression of
HO-1 via PI3K/AKT/mTOR pathway. In vivo, as far as we are aware, the anti-PD effects of 1
through apoptosis and oxidant pathways was firstly revealed in this study. Additionally,
ADME-related descriptors were calculated to predict the pharmacokinetic properties of the
compounds, and the pharmacokinetic (PK) assessment of 1 was determined in rats.
Scheme1. (a) SOCl2, MeOH, 0 °C→ R.T., 12 h; (b) HBTU, DIPEA, CH3CN, R.T., 2 h.
Table.1
Chemical structures and neuroprotective effects of compounds 1-15.
Compound
R1
H
R2
H
R3
OH
OH
H
R4
OH
OH
H
EC50, (μM)a
4.26 ± 1.37
11.26 ± 1.02
14.33 ± 1.77
15.58 ± 0.74
17.48 ± 1.53
19.07 ± 0.48
23.83 ± 2.16
N.D.b
1
2
3
4
5
6
7
8
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
OH
OH
H
OH
H
OH
OH
OH
OCH3
H
4

N.D.
N.D.
H
H
H
H
H
H
H
OH
OH
OH
H
H
OH
OCH3
H
H
H
9
10
N.D.
H
11
N.D.
OH
H
12
N.D.
H
H
13
N.D.
H
OH
OCH3
H
14
N.D.
H
OH
15
NACc
1.68 ± 0.41 (mM)
33.82 ± 3.22
Caffeic acidd
a
Note: EC50, 50% concentration of maximal effect (means ± S.E.M. of three experiments); b N.D. means the
EC50 was not detected because of the poor activity observe in the primary screening at 100 μM; c NAC
(N-Acetyl-L-cysteine) as the positive control (mM); d Caffeic acid: synthetic substrate of the compounds.
Initially, in order to confirm the optimal concentration of H2O2, cells were incubated with
various concentrations of H2O2 (100–1000 μM) for 24 h. As shown in Figure 1A, 600 μM H2O2
could decrease the cell viability to 52.66 ± 2.34 % of control. Therefore, 600 μM H2O2 was chosen
for the subsequent experiments. Among the analogues, seven compounds (1-7) exhibited potent
protection rates (>50%) at 100 μM, while others (8-15) showed poor protection rates (＜50%) at 100
μM ( Figure 1B). The EC50 values of these compounds 1-7 were 4.26 ± 1.37, 11.26 ± 1.02, 14.33 ±
1.77, 15.58 ± 0.74, 17.48 ± 1.53, 19.07 ± 0.48, 23.83 ± 2.16 μM, respectively. Above all, 1 exhibited
the most remarkable protective effect (EC50 = 4.26 ± 1.37 μM) (Table 1), and was considered as a
potential lead in further evaluation. Treatment alone with 1 at different concentrations (1-100 μM)
for 24 h didn’t affect the cell viability (Figure 1C). The effect of 1 against H2O2-induced cell death
was shown in Figure 1D, pretreatment with 1 at 1 and 10 μΜ could improve cell viability to 68.31 ±
2.09% and 88.99 ± 1.19% of control, respectively. The neurotoxicity of H2O2 and the
neuroprotective effect of 1 were further evaluated by lactate dehydrogenase (LDH) assay. As shown
in Figure 1E, the leakage of LDH in H2O2 group (339.93 ± 3.66 U/L) was much more than control
group (154.87 ± 1.41 U/L). In contrast, pretreatment with 1 (1, 10 μM) reduced the LDH release in a
dose-dependent manner (269.12 ± 2.47 and 180.66 ± 2.23 U/L, respectively). Moreover, 1 alone
treatment also has no influence on cell viability and LDH release. The neuroprotection of 1 was
further confirmed by observing the cell morphological under the inverted microscope (Figure 1F).
5

The above results confirmed that 1 at 1 and 10 μM could provide neuroprotection without
cytotoxicity.
Figure 1. Neuroprotection of 1 in SH-SY5Y cells. (A) Cytotoxicity of different concentrations of H2O2 in
SH-SY5Y cells. (B) Dose-dependent neuroprotection of pretreatment with 1 against H2O2-induced
cytotoxicity in SH-SY5Y cells. (C) Protective effect of the plasma membrane damage was analyzed by LDH
release. (D) Treated with 1 alone at 1, 10, 100 μΜ had no effect on cell viability. (E) The observation of the
cell morphological under the inverted microscope followed by above treatments. All the groups were treated
with less than 0.1% DMSO. The data were represented as mean ± S.E.M. of three independent experiments. #p
＜0.05 and ##p＜0.01 compared with control and **p＜0.01 compared with H2O2 treatment group.
As is known to all, reactive oxygen species (ROS), as the mitochondrial by-products in aerobic
respiration process, regulate many metabolic reactions [17]. However, the excessively increased of
ROS generation can induce oxidative stress and result in cell death via apoptosis in a wide variety of
cell types [18]. To confirm whether or not the protective effect of 1 via anti-oxidant, the level of
intracellular ROS was determined by dihydroethidium (DHE) fluorescence dye. Results showed that
the cells treated with H2O2 could increase the accumulation of intracellular ROS, which was about
4.0-fold relative to that of control cells. While pretreatment with 1 (1,10 μM) could significantly
decrease the ROS production in a dose-dependent manner, suggesting the anti-oxidative effects of it
6

via inducting ROS production (Figures 2A-C).
Mitochondria is a major site of ROS generation, the destruction of it integrity could cause the
increasing consumption of ATP and accumulating of intracellular ROS production [19]. Previous
study has confirmed that oxidative stress is involved in mitochondrial dysfunctions, as contributing
to open the mitochondrial permeability transition pore, resulting in depolarization of the
mitochondrial membrane potential (MMP) [20]. In this study, therefore, the effect of 1 reversed the
loss of MMP induced by H2O2 was detected using Rho 123 assay. Figures 2D-F showed that 1 at 1
and 10 μM could respectively increase the MMP to 79.43 ± 2.51% and 98.37 ± 1.87% compared to
H2O2 group (26.31 ± 3.32%) of control. These results suggested that 1 could prevent the loss of
MMP to provide neuroprotection, and we hypothesized that the ability of 1 to inhibit ROS generation
may attribute to the preservation of MMP loss. Moreover, under physiological conditions,
intracellular ROS levels are tightly controlled by a sophisticated cellular antioxidant system in
organism such as some antioxidant enzymes [21]. Superoxide dismutase (SOD), as one of the major
antioxidant enzymes, provides the most important anti-oxidant via catalyzing the dismutation
reaction of superoxide anion to H2O2. On the other hand, malondialdehyde (MDA), as an
end-product of lipid peroxidation, is regard as an important indicator of the oxidative damage.
Therefore, the anti-oxidative stress of 1 was further detected by the level of MDA and the activity of
SOD in SH-SY5Y cells. As shown in Figures 2G-H, being exposed to H2O2 could reduce the SOD
activity to 0.45 ± 0.047 units and increase the MDA level to 7.99 ± 0.55 μmol/g compared with
control cells (1.52 ± 0.028 units and 3.42 ± 0.037 μmol/g, respectively). However, pretreatment with
1 (1, 10 μΜ) could effectively improve the SOD activity and decrease the MDA level in a
dose-dependent manner, suggesting that 1 could mediate oxidant system to against oxidative stress
induced by H2O2 in SH-SY5Y cells.
7

8

Figure 2. Anti-oxidative effect of 1 in SH-SY5Y cells. The intracellular ROS production was detected by (A)
inverted fluorescence microscope and (B) flow cytometry. (C) Bar graphs showed intracellular ROS
production relative to the control group. The levels of ΔΨm was analyzed by (D) inverted fluorescence
microscope (E) and flow cytometry. (F) Bar graphs showed the quantitative analysis for MMP. Effects of 1 on
(G) the levels of MDA and (H) the activities of SOD in H2O2 treated cells. The data were represented as mean
± S.E.M. of three independent experiments. ##p＜0.01 compared with control and **p＜0.01 compared with
H2O2 treatment group.
Cell apoptosis induced by oxidative stress plays an important role in the pathological process of
neurodegenerative disorders [22]. To confirm the effects of 1 against the H2O2-induced apoptosis in
SH-SY5Y cells, Hoechst 33342 staining and Annexin V-PI double staining were performed in this
study. For the results of Hoechst 33342 staining, the nuclear fragmentation and DNA condensation
were occurred after treatment with H2O2. However, pretreatment with 1 could significantly alleviate
the typical characteristics of apoptosis (Figure 3A). For the results of AnnexinV-PI double staining,
the untreated cells showed slight total apoptosis (6.81 ± 0.73%) as well as only treated with 1 at 10
μM (total apoptotic of 4.14 ± 1.12%). Whereas treatment with 600 μM H2O2 markedly increased the
percentage of total apoptotic (84.17 ± 3.44%) and pretreatment with 1 at 1, 10 μM could decrease the
percentage to 24.13 ± 2.21% and 8.86 ± 1.63%, respectively (Figures 3B-C). These data proved the
anti-apoptotic activities of 1 against H2O2-induced neurotoxicity.
Cell apoptosis is primary to mitochondria dependent apoptotic pathways in neurodegenerative
diseases [23]. As the mitochondrial membrane-associated protein, the anti-apoptotic protein Bcl-2
located in the outer mitochondrial membrane with the anti-apoptotic function and the pro-apoptotic
protein Bax promotes cell apoptosis by translocating to the mitochondrial membrane. Cell survival in
the initiation phase of apoptosis mostly depends on the balance between the Bcl-2 and Bax. Thus,
Bcl-2/Bax ratio could be used to determine whether a cell has undergone apoptosis [24]. Moreover,
Bcl-2 has been reported to inhibit Bax expression in mitochondria, and inhibit subsequent activation
of the caspase-3 [25]. In this study, the expressions of these proteins were detected by western-blot
assay, β-actin as the internal standard. Results showed that the ratio of Bax/Bcl-2 and the expression
of cleaved-caspase-3 were significantly increased by treatment of H2O2 compared with control.
However, pretreatment with 1 (1,10 μM) could effectively reverse this tendency in a dose-dependent
manner. Treated with 1 alone had no obvious effects on the ratio of Bax/Bcl-2 and the expression of
cleaved-caspase-3 (Figures 3D–E). These results suggested that the anti-apoptosis activity of 1
might be mediated by modulating the expressions of apoptosis-related proteins. Additionally, the
9

absorption, distribution, metabolism, elimination (ADME) properties of 1-7 were performed well in
Discovery Studio 3.0 using ADME descriptors. Results indicated that the compounds have good
intestinal absorption, and the penetrating BBB ability of 1 might be better than other candidate
compounds (Supporting Information Table S1). Taken together, 1, the optimal one, was chosen to
further evaluate its anti-PD effects in vivo.
Figure 3. Effects of 1 on apoptotic and the expression of H2O2-induced apoptosis-related proteins in
SH-SY5Y cells. (A) Hoechst 33342 staining (B) Distribution of viable (lower left, Annexin V− PI−) necrotic
(upper left, Annexin V− PI+) late apoptotic (upper right, Annexin V+ PI+) and early apoptotic (lower right,
Annexin V+ PI−) (C) Bar graphs showed the quantitative results of AV−PI. (D) The original bands of Bax,
Bcl-2 and the quantitative analysis of Bcl-2/Bax ratio. (E) The original bands the quantitative analysis of
10

cleaved-caspase-3 expression. The data were represented as mean ± S.E.M. of three independent experiments.
##p＜0.01 compared with control and *p＜0.05 , **p＜0.01 and compared with H2O2 treatment group.
1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP) is an effective dopaminergic neurotoxin
in some mouse strains and has been widely used for inducing PD model of animal [26]. Evidences
indicate that MPTP’s deleterious cascade of events include mitochondrial respiration deficit,
oxidative stress, and activation of apoptotic genetic programs [27]. The above results revealed the
anti-apoptosis and anti-oxidative activities of 1 in H2O2-induced SH-SY5Y cells, implying 1 may
also has the anti-PD activity in MPTP-induced PD model. As mentioned above, the loss of
dopaminergic neurons in substantia nigra (SNpc) and striatum (Str) makes patients suffer from
different degrees of behavior disorders [3]. Therefore, in this study, the motor function of the MPTP
intoxicated mice were determined by Rota-rod test (Figure 4B). Results showed that the duration
time of mice on rotating-stick was significantly decreased compared to control at 4, 24, 48, and 72 h
after MPTP treatment. However, oral administration of 1 at 10 or 20 mg/kg markedly increased the
retention time compared with MPTP-treated group. Treatment with 1 alone had no influence of
motor function. Moreover, tyrosine hydroxylase (TH), as the rate-limiting step in the biosynthesis of
DA, is commonly regarded as a marker of dopaminergic neurons and also been used to evaluate the
degrees of PD [28]. Therefore, in this study, immunohistochemistry (IHC) analysis and western-blot
analysis were conducted for TH protein expression. As shown in Figures 4C-E, treatment with
MPTP could significantly reduce the TH expression both in SNpc and Str compared with control
group. However, the loss of TH protein was markedly prevented by 1 at 10 or 20 mg/kg. The above
results suggested that 1 could effectively alleviate the behavioral disorders and protect against
dopaminergic neuronal loss in MPTP-induced model.
11

Figure 4. Effects of 1 on motor function and TH-positive cells in the SNpc and Str of MPTP-treated 18 mice.
(A) Schematic of experimental procedure. (B) The results of 1 on Rota-rod test. At 10 days after MPTP
12

injection, DAB staining and western blots were performed. (C) The original bands and the quantitative
analysis of TH protein in SNpc. (D) The original bands and the quantitative analysis TH protein in Str; A
representative immunoblot was shown from three mice. (E) Representative microphotographs of TH
immunostaining of SNpc and Str. All the results were randomly obtained from three of the 3 mice/group and
#
*
the data were represented as mean ± S.D. (n = 6 mice/group). p＜0.05 compared with control and p＜
0.05,**p＜0.01 and compared with MPTP treatment group.
To further investigate the anti-PD activity of 1, we focused on the anti-apoptotic and
anti-oxidative effects of 1 in the brains. Consistent with the in vitro results, pretreatment of 1 at 10 or
20 mg/kg could significantly decrease the ratio of Bax/Bcl-2 and the expression of cleaved-caspase-3
both in SNpc and Str (Figures 5A-D). On the other hand, treatment with 1 at 10 or 20 mg/kg
dose-dependently increased SOD activity and decreased the level of MDA in the Str and SNpc of the
MPTP-induced PD mice (Figures 6A-D). Furthermore, pharmacokinetic (PK) assessment of the
optimum candidate compound 1 was determined in rats. When 1 was oral administrated (p.o., 25
mg/kg) and injected (i.v., 5 mg/kg), blood samples (100-200 μL) were collected from an orbit at 12
time points within 24 h (0.083, 0.168, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24 h) (Figure 7). The Tmax of 2 h
suggested that 1 was rapidly absorbed with short time of maximum observed concentration after
oral administration. The pharmacokinetic (PK) profiles of 1 is projected to have moderate blood
clearance (Cli.v.= 25.5 mL/min/kg), moderate volume of distribution (Vdss = 2.25 L/kg) and moderate
absolute bioavailability (F = 15.52 %) (Table 2) , These results highlight the potential of compound
1 as a promising lead in the development of orally active therapies for PD.
13

Figure 5. Effects of 1 on anti-oxidative and anti-apoptosis in vivo. Anti-apoptosis activity viewed by the
expression of apoptosis-related proteins. (A) The original bands of Bax, Bcl-2 and the quantitative analysis of
Bcl-2/Bax ratio in Str. (B) The original bands and the quantitative analysis of cleaved-caspease-3 expression
in Str. (C) The original bands of Bax, Bcl-2, and the expression of Bcl-2, Bax and the quantitative analysis of
Bcl-2/Bax ratio in SNpc (D) The original bands and the quantitative analysis of cleaved-caspease-3 expression
#
in SNpc. All data were represented as mean ± S.D. (n = 6 mice/group). p＜0.05, ##p＜0.01 compared with
control and *p＜0.05, **p＜0.01 and compared with MPTP treatment group.
14

Figure 6. Anti-oxidative activity in vivo viewed by MDA levels and SOD activity. (A) Bar graphs
represented the level of MDA in Str. (B) The activity of SOD in Str. (C) The level of MDA in SNpc. (D) The
#
##
activity of SOD in SNpc. The data were represented as mean ± S.D. (n = 6 mice/group). p＜0.05 and p＜
0.01 compared with control and *p＜0.05 and **p＜0.01 compared with MPTP treatment group;
Table 2
Pharmacokinetic parameters of 1
Parameters
p.o. (25 mg/kg)
i.v. (5 mg/kg)
Cl (mL/min/kg)
Vdss (L/kg)
AUC0-∞ (h·ng/mL)
T1/2 (h)
40.83 ± 8.45
5.98 ± 2.18
2495.56 ± 62.32
2.70 ± 1.16
2.00
25.5 ± 4.57
2.25 ± 1.46
3272.87 ± 133.41
0.85 ± 0.74
——
Tmax(h)
Cmax (ng/mL)
MRT (h)
807.95 ± 21.32
3.06 ± 0.92
——
1.45 ± 0.61
F (%)
15.25
Note：Dose: i.v., 5 mg/kg (5% DMSO +2% F68 + 93% saline)；p.o., 25 mg/kg, dissolved in methylcellulose
(0.5 %) solution containing 1% Tween 80. Cl: Plasma clearance; Vdss :Volume of distribution; AUC :Area
15

under curve; T1/2: Half-life; Tmax: time to reach peak plasma concentration; Cmax: peak plasma concentration;
MRT: Mean Retention Time; F: Absolute oral bioavailability.
Figure 7. Concentration-time curves in the PK study. Plasma concentration–time profile was evaluated
after i.v. (5 mg/kg) and p.o. (25 mg/kg) administration of compound 1 to male Sprague–Dawley rats (mean ±
SD, n = 3).
Normally, the level of HO-1 is low in neurons, it can be highly up-regulated during the
progression of Parkinson’s patients [29]. Accumulated evidences suggested that the up-regulation of
HO-1 by various stimuli may represent an adaptive and protective cellular response to alleviate the
damage caused by these stimuli [30]. Fu’s study has demonstrated that overexpression of HO-1
protected dopaminergic neurons against MPP+ induced neurotoxicity in midbrain neuron glia
co-cultures, indicating that HO-1 may be involved in the pathogenesis of Parkinsonism [31].
Therefore, we were interested in whether or not the neuroprotection of 1 was involved in modulating
HO-1 expression. In accordance with the postulation, treatment with 1 at 0.1 μM (111.39 ± 17.30%),
1 μM (154.79 ± 21.83%, p＜0.05) and 10 μM (205.82 ± 14.44%, p＜0.01) could dose-dependently
increase the HO-1 expression of control in SH-SY5Y cells. (Figure 8A). Moreover, H2O2 (600 μM),
as an oxidative stress stimuli, led to an increase of HO-1 expression (181.53 ± 24.67%, p＜0.05) of
control after 24 h treatment. However, pretreatment with 1 at 1 μM (273.55 ± 18.32%, p＜0.05) ) or
10 μM (324.80 ± 16.62%, p＜0.01) for 6 h, followed by H2O2 treatment for 24h, induced a more
significant increase of HO-1 expression compared with H2O2 treatment alone (Figure 8B). These
results suggested that 1 could specifically up-regulate the HO-1 expression in SH-SY5Y cells.
To further investigate the potential role of HO-1 in the 1-mediated neuroprotective activity,
cells were pretreated with or without specific HO-1 inhibitor, zinc protoporphyrin-IX (ZnPPIX 10
16

μM), and followed by 1 plus H2O2 treatment. As shown in Figure 8C, pretreatment with ZnPPIX
cloud significantly alleviate the protective effect of 1, indicating the induction of HO-1 is essential
for 1 to attenuate H2O2-induced cytotoxicity. On the other hand, an extremely high level of HO-1 can
result in more oxidative stress due to generating excessive free iron [32]. Our results showed that 1
could enhance the expression of HO-1 without cytotoxicity in SH-SY5Y cells, suggesting 1 was able
to up-regulate HO-1 appropriately.
Figure 8. Effect of 1 on the expression of HO-1 in SH-SY5Y cells. (A) The original bands and the
quantitative analysis of HO-1 expression in the cells which have been incubated with various concentrations
(0.1, 1 or 10 μM) of 1 for 24 h. (B) The original bands and the quantitative analysis of HO-1 expression in the
cells which was treated with 1 (1 or 10 μM) and H2O2 (600 μM). (C) Cells were pretreated with heme
oxygenase-1 inhibitor, ZnPPIX (10 μM) for 1h and 1 (10 μM) for 6 h, then cells were exposed to H2O2 (600
μM) for 24 h and the Cell viability was assessed by MTT assay. All the results are expressed as mean ± S.E.M.
#
*
**
from three-independent experiments. p＜0.05, ##p＜0.01 compared with control and p＜0.05, p＜0.01
compared with H2O2 treatment and &&p＜0.01 compared with 1 plus H2O2 group.
17

As mentioned in previous studies, various signaling pathways were related with up-regulation
of HO-1, including PI3K/AKT/mTOR [33, 34] and MAPKs [35]. Therefore, to examine the possible
signaling pathways involved in HO-1 induction, in this study, SH-SY5Y cells were respectively
pretreated with several inhibitors, including SP600125, SB203580, U0126, LY294002 and
Rapmycin for 1 h, and followed by stimulating with 1 and H2O2. As shown in Figure 9A,
pretreatment with the PI3K/AKT/mTOR inhibitors LY294002 and Rapmycin, respectively, could
significantly reduce the stimulated HO-1 expression by 1 plus H2O2. However, HO-1 expression not
affected by MEK1/2 inhibitor, JNK inhibitor or p38 MAPK inhibitor treatment. These results
suggested that the PI3K/AKT/mTOR pathway is required for the induction of HO-1 by 1 plus H2O2
in SH-SY5Y cells. Moreover, as well known, PI3K/AKT/mTOR pathway plays an important role in
cell proliferation and apoptosis in the brain [36]. To further test whether the pathway act as a
potential signal for neuroprotection of 1, we examined the effects of the pathway inhibition on the
extent of apoptosis in SH-SY5Y cells. As shown in Figure 9B, the neuroprotection of 1 was
significantly reduced by the pretreatment of PI3K/Akt/mTOR pathway inhibitors, LY294002 ( 60.53
± 2.04%, p＜0.01) and Rapmycin (65.94 ± 1.53, p＜0.01) compared with 1 plus H2O2 group (80.77
± 1.927 %). Furthermore, we investigated the effects of 1 on the expression of phospho-AKT
(p-AKT) and its downstream effector phospho-mTOR (p-mTOR) in SH-SY5Y cells. The time
course results as shown in Figures 9C-D, unstimulated cells appeared a relative low expression of
phosphorylated AKT and mTOR, while treatment with 1 in different times (5, 15, 30, 60, 120, 240,
360 min) could increase the phosphorylation of AKT and mTOR, and which both peaked at 30 min.
The above results supported that 1 regulates the expression of HO-1, at least in part, through the
PI3K/Akt/mTOR pathway, which mainly exert a neuroprotective effect in this experimental model.
18

Figure 9. Induction of HO-1 blocked by mTOR inhibition and PI3K inhibition. (A) SH-SY5Y cells were
preincubated for 1 h with either SB203580 (p38 inhibitor 10 μM), LY294002 (PI3K inhibitor 5 μM), U0126
(MEK1/2 inhibitor 10 μM), SP600125 (JNK inhibitor 5 μM) and Rapmycin (mTOR inhibitor 100 nM) and
then treated with 1 (10 μM) for 6 h before being exposed to 600 μM H2O2. The expression of HO-1 were
analyzed by Western blot and plotted as a graph after densitometric analysis; (B) Effect of blocking the
PI3K/AKT/mTOR pathways on the extent of apoptosis in SH-SY5Y cells. The cell viability was determined
by MTT. Effects of 1 on the expression of AKT and mTOR in SH-SY5Y cells. (C) Time courses of
phosphorylated AKT proteins, the original bands and the quantitative analysis of p-AKT/t-AKT ratio. (D)
Time courses of phosphorylated mTOR proteins, the original bands and the quantitative analysis of
p-mTOR/t-mTOR ratio. All the results are expressed as mean ± S.E.M. from three-independent experiments.
*
**
&
#p＜0.05, ##p＜0.01 compared with control and p＜0.05, p＜0.01 compared with H2O2 treatment and p＜
19

0.05, &&p＜0.01 compared with H2O2 plus 1 group.
In summary, seven compounds (1-7) with the catechol group showed the significant
neuroprotection against H2O2-induced SH-SY5Y cytotoxicity, compared to those of 8-15 without
catechol group, suggesting that catechol group might be the important pharmacophore for their
neuroprotective effects against oxidative damage, and their protective effects are independent of the
quantity of phenolic hydroxyl groups. Moreover, the optimal candidate, compound 1, provide
neuroprotection involved in reducing the lipid peroxidation and ROS generation, recovering
mitochondrial function, and mediating the expression of apoptosis-related proteins. The major
finding in our present study is the key role for HO-1 expression-related signals contributing to the
neuroprotective effect of 1. Furthermore, in vivo conditions, oral treatment with 1 could markedly
alleviate the behavior impairment and increase TH level both in SNpc and Str of MPTP-induced PD
mice. The pharmacokinetic (PK) assessment indicated that 1 has a normal oral bioavailability in the
rat. All these promising findings suggested that 1 might be a promising lead in the development of
orally active therapies for PD, and might provide a thought for the design of Parkinson's therapeutic
agents.
Acknowledgement
This work was financially supported by Jiangsu science and technology support program
(BE2014710), and National Natural Science Foundation of China (No. 81573309, 81473160) and
National Key R&D Program of China (No. 2017YFC1703802). Hao Wang*, Fei Xiong*, Wen-Cai
Ye, Jia-Hao Feng conceived and designed the study. Jia-Hao Feng and Xiao-Long Hu carried out
experiments, Xian-Yu Lv, Bao-Lin wang and Fei Xiong provided the technical assistance, Jia-Hao
Feng and Xiao-Long Hu wrote the first draft of the manuscript. All authors read and approved the
manuscript. The authors declare no competing financial interest.
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Highlights
1.Fiftten clovamide analogues were design and synthesized
2.Analogues containing catechol functionality exhibited neuroprotective effects against
H2O2-induced oxidative damage in vitro.
3.Compound 1 could up-regulate HO-1 expression via PI3K/AKT/mTOR pathway.
4. Oral administration of compound 1 improve the symptoms of Parkinson’s disease in
MPTP-induced mice model.
5. Pharmacokinetic (PK) assessment indicated that 1 has a normal oral bioavailability in the rat.
24