Accepted Manuscript: European Journal of Medicinal Chemistry

Accepted Manuscript
Structure-activity relationship studies of (E)-3,4-dihydroxystyryl alkyl sulfones as novel

neuroprotective agents based on improved antioxidant, anti-inflammatory activities
and BBB permeability
Ying Chen, Bolin Wu, Yameng Hao, Yunqi Liu, Zhili Zhang, Chao Tian, Xianling Ning,
Ying Guo, Junyi Liu, Xiaowei Wang
PII: S0223-5234(19)30262-4
DOI: https://doi.org/10.1016/j.ejmech.2019.03.044
Reference: EJMECH 11214
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 January 2019

Revised Date: 25 February 2019
Accepted Date: 17 March 2019
Please cite this article as: Y. Chen, B. Wu, Y. Hao, Y. Liu, Z. Zhang, C. Tian, X. Ning, Y. Guo,
J. Liu, X. Wang, Structure-activity relationship studies of (E)-3,4-dihydroxystyryl alkyl sulfones
as novel neuroprotective agents based on improved antioxidant, anti-inflammatory activities and
BBB permeability, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.03.044.
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Structure-activity Relationship Studies of (E)-3,4-dihydroxystyryl
Alkyl Sulfones as Novel Neuroprotective Agents Based on Improved
Antioxidant, Anti-inflammatory Activities and BBB Permeability

Ying Chen a, Bolin Wu a, Yameng Hao a, Yunqi Liu a, Zhili Zhang a, Chao Tian a,
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Xianling Ning b, Ying Guo a, Junyi Liu a, c, **, Xiaowei Wang a, *
a
Department of Chemical Biology, School of Pharmaceutical Sciences, Peking
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University, Beijing 100191, China
b
Institute of Systems Biomedicine, School of Basic Medical Sciences, Beijing Key
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Laboratory of Tumor Systems Biology, Peking University Health Science Center,
Beijing 100191, China
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c
State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing
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100191, China
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ABSTRACT
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(E)-3,4-dihydroxystyryl alkyl sulfones, as new analogues of neurodegenerative agents,

were designed and synthesized. The biological results demonstrated that most of the
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target compounds preserved antioxidant and anti-inflammatory potency in scavenging

reactive free radicals, protecting neuronal cells against neurotoxins such as H2O2,
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6-hydroxydopamine and inhibiting lipopolysaccharide (LPS)-induced over production

of NO. Among these compounds, 6.22 with cyclopentyl propyl exhibited prominent
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antioxidant activity at low concentration (2.5 µM) in H2O2 model (cell viability =
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94.5%). In addition, 6.22 (IC50 = 1.6 µM) displayed better anti-inflammatory activity
than that of lead compound 1 (IC50 = 13.4 µM). In view of the outstanding
*
Corresponding author.
**
Corresponding author. Department of Chemical Biology, School of Pharmaceutical Sciences, Peking
University, Beijing 100191, China. Phone/fax: +86-10-82805203.
E-mail: jyliu@bjmu.edu.cn. (Junyi Liu), E-mail: xiaoweiwang@bjmu.edu.cn. (Xiaowei Wang)

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performance of 6.22, the apoptotic rates of H2O2-damaged PC12 cells were detected
by Annexin V-FITC/ PI assay. 6.22 showed higher potency in inhibition of apoptosis
than 1 at low concentration (2.5 µM), consisting with the antioxidant and
anti-inflammatory models. Furthermore, with the predicted CNS (+) blood-brain
barrier (BBB) permeability (Pe = 6.84 × 10−6 cm s−1), low cytotoxicity and favorable
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physiochemical properties based on calculation, compound 6.22 can be further
developed as a potential multifunctional neuroprotective agent.
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Keywords
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Neurodegenerative diseases, Antioxidant, Annexin V-PI apoptosis stain,
Anti-inflammatory, BBB permeability
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1. Introduction
Neurodegenerative diseases (NDs), including Alzheimer’s (AD), Parkinson’s (PD),
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Huntington’s (HD) disease, traumatic brain injury (TBI) and amyotrophic lateral
sclerosis (ALS), are progressive age-related disorders of the central nervous system,
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resulting in progressively and irreversibly deteriorated nervous system and greatly

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reduced life quality [1]. Mechanisms of neurodegenerative disorders are complex, but
some common elements, such as oxidative stress, inflammation, apoptosis and
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mitochondrial injury are considered to play important roles in the pathogenesis of

these diseases [2]. Recently, the antioxidative and anti-inflammatory strategies have
shown promise in neurodegenerative diseases treatment. Oxidative stress induced by
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excessive reactive oxygen species (ROS) leads to DNA injury and lipid peroxidation
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in neural cells and it is implicated in the pathogenesis of neurodegenerative diseases

[3]. Inflammatory reaction is also considered to be an important contributor to NDs,
which is related to the generation of nitric oxide (NO) and superoxide [4]. NO is a
crucial cell signaling molecule involved in many physiological functions, which is
also related to the pathogenesis of inflammatory process [5]. Excessive NO can
mediate inflammatory response and react with superoxide anion to form the reactive
nitrogen species (RNS) like potent oxidant peroxynitrite (ONOO−), which causes lipid
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peroxidation (LPO) [6], DNA damage [7] and mitochondria dysfunction [8] and
therefore leads to neuronal necrosis or apoptosis [9].
Accordingly, searching small molecule that can prevent the death of neurons by
antioxidative and anti-inflammatory will make great contribution to the effective
therapy method for NDs. In the previous study, our group has designed and
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synthesized several effective neuroprotective agents such as (E)-3,4-dihydroxystyryl
aralkyl sulfonamides, ketones, sulfones and sulfoxides [2,10,11]. Among them, the
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(E)-3,4-dihydroxystyryl aralkyl sulfone analogues displayed potent effects against
oxidative damage and anti-neuro-inflammatory properties in neuronal cells. However,
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these analogues have low solubility and permeability, which reduce their
bioavailability. Moreover, a successful drug for central nervous system (CNS)
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diseases requires not only good pharmacological activities but also sufficient
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permeation to across the blood brain barrier (BBB) into the CNS [12]. The entry of
active molecules into the CNS is efficiently controlled by the BBB, which is formed
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by microvessel endothelial cells [13]. One of the useful strategies to facilitate the
molecules’ BBB permeation is to increase the passive diffusion through structure
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modification of drug molecules [14,15]. Herein, we focused on developing drug

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candidates with good neuroprotection and BBB permeability in this study.

As CAPE analogues, (E)-3,4-dihydroxystyryl aralkyl sulfones showed antioxidant
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activity due to the catechol ring group as an effective free radical scavenger.
Furthermore, these analogues are hydrogen donors and could inhibit the generation of
reactive oxygen species (ROS) [14] such as hydroxyl free radical (•OH), superoxide
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anion (O2−) and lipid peroxide free radicals (ROO•) [15,16,17]. Meanwhile, the
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double bond of the side chain would stabilize the phenolic radical. Therefore,
(E)-3,4-dihydroxystyryl group is reserved in the designed compounds. As reported,
molecules’ solubility and permeability are impacted by lipophilicity [18], which could
affect the bioavailability of compounds [19]. To investigate the effect of lipophilicity
and distribution coefficient, the phenyl group was substituted with various alkyl side
chains. In addition, the cycloalkyl and heterocyclic alkyl moieties were introduced to
these molecules to examine the influence of flexibility, spatial effects and molecular
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conformation on permeability and neuroprotective activities. We also designed

several analogues bearing terminal alkenyl or hydroxyl group on the alkyl side chain
to reveal the conformation and polarity effects on antioxidant and anti-inflammatory
activities. In this report, we focused on the modification of compound 1 with alkyl
moieties to improve the antioxidative and anti-inflammatory activities and
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permeability, as illustrated in Fig. 1. For the potential compound, its effect on
apoptosis were detected by Annexin V-FITC/PI staining assay. Physiochemical
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properties (log P, log BB, TPSA and CNS MPO) were calculated to predict if the
compounds can cross the BBB and, PAMPA−BBB assay was conducted on the
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(E)-3,4-dihydroxystyryl alkyl sulfones to verify the calculated values.
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Fig 1. Structures of 1 and target compounds.

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2. Chemistry
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The synthetic routes of (E)-4-(2-((3-phenylpropyl)sulfonyl)vinyl)benzene-1,2-diol

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analogues are illustrated in Scheme 1 [2]. Most of the alkyl bromides are
commercially available except 3.18−3.26, which were synthesized from substituted
alcohols. As shown in Scheme 1, the reactions between substituted alcohols
2.18−2.26 and PBr3 in anhydrous THF are effective to give alkyl bromides 3.18−3.21,
while others were activated with p-toluenesulfonic chloride (TsCl) in the presence of
Et3N and DMAP to afford corresponding p-toluene sulfonates 3.22−3.26.
Subsequently, the alkyl sulfanyl acetic acids 4.1−4.21 were obtained with high yields
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(approximately 80%) from mercaptoacetic acid in methanol catalyzed by NaOH at

room temperature. However, sulfanyl acetic acids 4.22−4.26 were provided under
reflux condition. After the oxidation work-up with H2O2 (30% aqueous solution) at
room temperature, intermediates 5.1−5.26 were obtained and condensed with
3,4-dihydroxybenzaldehyde by using pyrrolidine and acetic acid as catalysts in
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refluxed THF to afford target compounds 6.1−6.26.
The pyrrolidinyl derivative 6.25 was synthesized from 1-Boc-pyrrolidine-2-
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methanol. Followed by removal of the t-butyloxy-carbonyl (Boc) protecting group by
treatment with hydrogen chloride in methanol, 6.27 was prepared with good yield
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(Scheme 2). The target compounds, with E geometry, were characterized with 1H
NMR, 13C NMR and HRMS.
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Scheme 1. Synthetic route of compounds 6.1−6.26. Reagents and conditions: (a) PBr3, THF,
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0oC-rt, 90-95%; (b) TsCl, Et3N, DMAP, 0oC-rt, 90-100%; (c) HSCH2COOH, NaOH, MeOH, rt,
72-80%; (d) HSCH2COOH, NaOH, MeOH, reflux, 80-90%; (e) 30% H2O2, acetic acid, rt, 70-87%;
(f) pyrrolidine, acetic acid, THF, reflux, 49-72%.

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Scheme 2. Synthetic route of compound 6.27. Reagents and conditions: (g) concentrated
hydrochloric acid, MeOH, rt, 70%.
3. Biological Evaluation
The neuroprotective properties of target compounds were assessed by several
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experimental pharmacological models in vitro. The antioxidant and anti-inflammatory
properties were evaluated in PC12 cells and BV2 microglial cells, respectively. The
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apoptotic rate of H2O2-damaged PC12 cells was detected by Annexin V-FITC/PI
staining assay. In addition, a parallel artificial membrane permeation assay
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(PAMPA−BBB) and calculated physiochemical properties were performed to predict
BBB permeability of the target compounds. The results were discussed as follows.
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3.1 Free Radical Scavenging Ability (DPPH).
Antioxidants can react with reactive species such as DPPH, a stable free radical
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existing in vitro, which could be used to screen the capability of compounds for
scavenging reactive free radicals [20,21]. The free radical scavenging abilities were
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monitored by fluorescence spectrophotometer at 514 nm according to the published

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method [11]. The results suggested that most of target compounds, except 6.12 and
6.27, exhibited satisfactory radical scavenging activities with EC50 ranging from 5.9
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to 11.4 µM, as shown in Table 1. Especially, compounds 6.2–6.6, 6.9–6.11, 6.13–6.15,

6.19, 6.20, 6.22, 6.23 and 6.25 (EC50 = 5.9 to 7.9 µM) showed stronger free radical
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scavenging capacities in comparison to 1 (EC50 = 11.5 µM). The results confirmed

that the replacement of the phenyl groups by the cycloalkyl and alkyl chains would
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provide positive effects on radical scavenging activity. In addition, 6.27 (EC50 = 13.8
µM) and 6.24 (EC50 = 10.5 µM), bearing pyrrolidinyl and tetrahydrofuranyl,
respectively, showed weaker free radical quenching abilities compared with the
corresponding cyclopentyl substituted compound 6.18 (EC50 = 9.4 µM). Introducing
heteroatom to the cycloalkyl would decrease the activity of scavenging free radicals.
Meanwhile, 6.19 (EC50 = 7.5 µM) with cyclohexyl showed better scavenging activity
than that of 6.16−6.18 (EC50 = 10.5, 8.8 and 9.4 µM) with cyclopropyl, cyclobutyl and
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cyclopentyl substituents, indicating that the flexibility and conformation of rings

would significantly affect the free radical quenching ability.
Table 1
Free Radical Scavenging Abilities of target compounds by the DPPH Methoda.
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Compd. n R EC50 (µM) Compd. n R EC50 (µM)
6.1 2 C2H5 8.1 ± 0.2*** 6.16 0 cyclopropyl 10.5 ± 0.9
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6.2 2 C3H7 7.3 ± 0.8*** 6.17 0 cyclobutyl 8.8 ± 0.4**
6.3 2 C4H9 7.1 ± 0.1*** 6.18 0 cyclopentyl 9.4 ± 0.6
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6.4 2 C5H11 6.4 ± 0.2*** 6.19 0 cyclohexyl 7.5 ± 0.4***
6.5 2 C6H13 7.7 ± 0.1*** 6.20 1 cyclopentyl 6.8 ± 0.1***
6.6 2 C7H15
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6.8 ± 0.3*** 6.21 1 cyclohexyl 9.8 ± 0.3
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6.7 2 C8H17 11.4 ± 0.7 6.22 2 cyclopentyl 6.3 ± 0.7***
6.8 2 C9H19 8.5 ± 0.6** 6.23 2 cyclohexyl 7.9 ± 1.1***

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6.9 2 C4H8OH 5.9 ± 0.1*** 6.24 0 tetrahydrofuranyl 10.5 ± 0.3
6.10 2 C5H10OH 6.7 ± 0.1*** 6.25 0 N-BOC pyrrolidinyl 7.1 ± 0.1***

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6.11 2 C6H12OH 7.5 ± 0.3*** 6.26 0 tetrahydropyranyl 10.8 ± 0.1

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6.12 2 C8H16OH 22.6 ± 1.4*** 6.27 0 pyrrolidinyl 13.8 ± 0.4
6.13 2 C2H4CH=CH2 7.1 ± 0.8*** 1b 11.5 ± 0.1

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6.14 2 C3H6CH=CH2 6.4 ± 0.1*** CAPE 14.6 ± 1.1
6.15 2 C4H8CH=CH2 6.9 ± 0.1***

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The results are expressed as means ± SEM (n=3) Significance levels *p < 0.05, **p < 0.01, ***p < 0.001 as compared with the
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respective 1.
a
Data are expressed as the mean ± SD, n = 3.
b
1 as the positive control compound
3.2 Neuronal Protection Effect against Damage Induced by H2O2.

In order to further study the antioxidant property of target compounds,
neuroprotection against oxidative stress was assayed in dopaminergic PC12 cells.
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Oxidative stress models caused by reactive oxygen species (ROS) were used to assess
the therapeutic potential for NDs. Hydrogen peroxide (H2O2) can generate free
radicals and lead to damaging of lipid, protein, RNA and DNA [22] as well as
neurons apoptosis. Therefore, it is widely used as an inducer of oxidative stress in
many cellular models [23]. Here, the antioxidant activity of target compounds were
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tested on PC12 cells against oxidative injuries stimulated by H2O2 [24]. The
protective effect against H2O2 can be detected by the cell viability through the MTS
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assay. Most target compounds showed protective effects and especially, compounds
6.1, 6.2, 6.13–6.15, 6.19, 6.22 and 6.23 displayed approximately 20% higher activities
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than 1 and 30% higher than CAPE at 5 µM, as shown in Fig. 2. It is remarkable that
the cycloalkyl- substituted compounds 6.16–6.19 showed significant differences on
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antioxidant activity with IC50 values of 6.61, 4.81, 2.19 and 1.85 µM, respectively.
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Moreover, cyclohexyl substituted compound 6.19 showed 3-fold higher activity than
cyclopropyl substituted compound 6.16 as shown in Table 2. Compounds 6.22 (IC50 =
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1.28 µM) and 6.23 (IC50 = 1.00 µM) showed slightly better antioxidant properties than
6.18−6.21 (IC50 = 2.19, 1.85, 1.96, 2.00 µM), which may suggest that the length of the
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linker has minor effect on antioxidant activity. Moreover, 6.22 displayed dramatically
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higher activities than 1 at 2.5 and 5 µM as shown in Fig. 2b. However, when hetero
atoms were introduced to the alkyl substituents, the antioxidant abilities were
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significantly decreased. For example, the IC50 values of 6.24 (IC50 = 27.10 µM) and
6.27 (IC50 is not detected), bearing tetrahydrofuranyl and pyrrolidinyl, respectively,
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were lower than that of the corresponding cyclopentyl substituted compound 6.18
(IC50 = 2.19 µM). Similar results were observed on the alkyl side chain compounds.
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6.9−6.12 (IC50 = 9.91, 7.91, 7.91, 24.10 µM) with terminal hydroxyl side chain
showed weaker antioxidant properties than that of other alkyl substituted compounds
6.1−6.8 and 6.13−6.15. It is notable that compounds 6.9−6.12, 6.24 and 6.27
exhibited significant free radical quenching abilities in DPPH model while weaker
antioxidant activities in H2O2 model on PC12 cells. This result indicated that the
radical scavenging ability is not the only factor to affect the protection from oxidative
stress on PC12 cells. The possible explanation would be that the introduction of
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heteroatoms on alkyl substituents reduced the lipophilicity of the compounds and

affected the passively diffuse across cell membranes.
2a
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*** *** *** *** 2.5 µM
100 *** 5 µM
10 µM
cell viability (%)
** *
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*
50 *
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0
E
10
11
12
13
14
1
9
l
C 2
ro
2O
6.
6.
6.
6.
6.
6.
6.
6.
6.
P
6.
6.
6.
6.
6.
nt
A
H
co
H2O2
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2b
*** *** *** *** *** 2.5 µM
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***
100 *** 5 µM
10 µM
cell viability (%)
** *
50
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0
D
H l
2
PE
1
15
16
17
18
19
20
21
22
23
24
25
26
27
ro
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6.
6.
6.
6.
6.
6.
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6.
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co
H2O2
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Fig. 2. Protective effects of target compounds 6.1−6.27, 1 and CAPE against H2O2-induced
apoptosis in PC12 cells (2a and 2b). PC12 cells were incubated with compounds (2.5, 5, and 10
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µM) for 3 h; H2O2 (400 µM) was added, and incubation continued for 3 h. Cell viability was
determined by the MTS assay. The viability of untreated cells is defined as 100%. Data are
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expressed as the mean ± SD, n = 3. Significance levels *p < 0.05, **p < 0.01, ***p < 0.001 as
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compared with the H2O2-treated group.
Table 2
H2O2-induced injury half-maximal inhibitory concentration (IC50) of target compounds 6.1−6.27,
1 and CAPE in PC12 cellsa.
Compd. IC50 (µM) Compd. IC50 (µM)

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6.1 1.83 ± 0.05 6.16 6.61 ± 0.34***
6.2 1.30 ± 0.06*** 6.17 4.81 ± 0.14***
6.3 1.33 ± 0.09** 6.18 2.19 ± 0.07**
6.4 1.45 ± 0.06*** 6.19 1.85 ± 0.07
6.5 1.93 ± 0.01** 6.20 1.96 ± 0.03**
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6.6 2.21 ± 0.03*** 6.21 2.00 ± 0.02**
6.7 2.34 ± 0.05*** 6.22 1.28 ± 0.08***
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6.8 2.27 ± 0.05*** 6.23 1.00 ± 0.01***
6.9 9.91 ± 0.19*** 6.24 27.10 ± 0.34***
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6.10 7.91 ± 0.41*** 6.25 7.20 ± 0.09***
6.11 7.91 ± 0.10*** 6.26 3.34 ± 0.03***
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6.12 24.10 ± 2.09*** 6.27 ND
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6.13 1.62 ± 0.04*** 1 1.86 ± 0.01
6.14 1.35 ± 0.04*** CAPE 2.00 ± 0.03

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6.15 1.90 ± 0.01*
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respective 1.
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a
Data are expressed as the mean ± SD, n = 3. ND: not detected.
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3.3 Neuroprotective effect on PC12 cells injury induced by 6-OHDA

6-OHDA can also induce neurons apoptosis via uncoupling mitochondrial
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oxidative phosphorylation, resulting in energy deprivation and ROS accumulation

[25], which could destruct mitochondrial membrane integrity and inducing
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mitochondrial dysfunction by oxidative stress reduce the cellular resistance [26].

Therefore, 6-OHDA-induced toxicity in PC12 cells model was used to evaluate the
antioxidative effects of target compounds with the MTT method [27]. As shown in
Fig. 3, most of target compounds, except 6.16, 6.24, 6.25 and 6.27, exhibited
significantly protective effects against damage induced by 6-OHDA at 10 µM. Most
of the compounds with alkyl side chain, cyclohexyl and cyclopentyl were found with
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better antioxidant activity than 1 and CAPE at 2.5 µM. Overall, activities of 6.1−6.15
with alkyl chains are higher than those of 6.16−6.27 with cycloalkyl. It was also
observed that compounds 6.9−6.12 and 6.24−6.27 bearing terminal hydroxy and
heterocycle, respectively, showed weaker protection effects, which is consistent with
the results from H2O2 model. Compound 6.19 with cyclohexyl showed much better
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activity than 6.16, 6.17 and 6.18 with cyclopropyl, cyclobutyl and cyclopentyl. These
results confirmed that the conformation and lipophilicity of the compounds affect the
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antioxidant effects in cellular models. It was noteworthy that in contrast with results
from H2O2 model, compound 6.22 did not show prominent protection effect against
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6-OHDA. The different results from two cellular models are likely ascribed to the
different mechanisms of the oxidants.
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3a
*** 2.5 µM
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100 *** *** ***
5 µM
10 µM
cell viability (%)
***
*** ***
50
** ** ***
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0
D
A
PE
1
1
2
3
4
5
6
7
8
9
10
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13
14
O l
6- tro
6.
6.
6.
6.
6.
6.
6.
6.
6.
D
6.
6.
6.
6.
6.
A
H
n
C
co
6-OHDA
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3b
100 *** *** *** *** *** 2.5 µM

5 µM
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10 µM
cell viability (%)
*** ***
50
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0
PE
1
15
16
17
18
19
20
21
22
23
24
25
26
27
A
6- rol
D
6.
6.
6.
6.
6.
6.
6.
6.
6.
6.
6.
6.
6.
nt
A
H
C
O
co
6-OHDA
Fig. 3. Protective effects of all target compounds at 2.5, 5, 10 µM against 6-OHDA-induced
apoptosis in PC12 cells (3a and 3b). PC12 cells were incubated with compounds (2.5, 5, and 10
µM) for 3 h; 6-OHDA (250 µM) was added, and incubation continued for 24 h. Cell viability was
determined by the MTT assay. The viability of untreated cells is defined as 100%. Data are
expressed as the mean ± SD, n = 3. Significance levels *p < 0.05, **p < 0.01, ***p < 0.001 as
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compared with the 6-OHDA-treated group.
3.4 In vitro 15-LOX inhibition assay

15-lipoxygenase (15-LOX) is one of the key mediators in neurodegenerative
disease[28,29], associating with oxidative stress and inflammatory[30]. Compound
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6.22, which exhibited efficient antioxidant activity, was also subjected to in vitro
lipoxygenase inhibition assay using LOX assay kit (catalog no.760700; Cayman
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Chemicals Inc., Ann Arbor, MI, USA). The non-selective LOX inhibitor,
nordihydroguaiaretic acid (NDGA), was used as positive control for comparison. The
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results were shown in Table 3.
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Table 3.
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In vitro 15-LOX enzyme inhibitory activities of compounds 6.22, 1 and NDGA a.
Compd. 15-LOX IC50 (µM)
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6.22 102.9 ± 9.2***
1 140.2 ± 8.2***
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NDGA 2.1 ± 0.7

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respective 1.
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a
Data are expressed as the mean ± SD, n = 3
As shown in table 3, the inhibitory activities of 6.22 (IC50 = 102.9µM) and 1 (IC50
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= 140.2µM) were less than that of NDGA, which indicated the antioxidant activity of
6.22 may due to other mechanisms than 15-LOX inhibitory.
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3.5 Inhibiting Nitric Oxide Production in LPS-Stimulated BV2 Microglial Cells.

The excessive production of pro-inflammatory mediators, such as nitric oxide
(NO), plays important roles in neurodegenerative diseases. Activated microglial cells
can release an inflammatory mediator, nitric oxide, the overproduction of which
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results in neural cell death in neurodegenerative disorders [31] because the excessive
NO is detrimental due to the inflammatory response amplification [32]. Therefore, the
anti-inflammatory properties of target compounds was evaluated by investigating the
NO production suppression in lipopolysaccharide (LPS)-induced stimulated BV2
microglial cells with the Griess assay [33]. Compounds with alkyl side chains,
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cyclopentyl and cyclohexyl significantly inhibited the NO production as shown in
Table 4. Among them, 6.19 (IC50 = 6.7 µM) exhibited stronger inhibitory effect than
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6.16−6.18 (IC50 = 44.1, 20.8, 15.8 µM) bearing cyclopropyl, cyclobutyl and
cyclopentyl substituents. This phenomenon is consistent with the fact that the
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flexibility and conformation of the ring can largely affect the anti-inflammatory
activity. Surprisingly, compound 6.22 (IC50 = 1.6 µM) with cyclopentyl propyl
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substituent exhibited slightly higher activity than 6.20 (IC50 = 2.5 µM) with
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cyclopentyl ethyl and approximately 10-fold higher activity than 6.18 (IC50 = 15.8 µM)
with cyclopentyl methyl. This may indicate that the linker length had significant
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effect on anti-inflammatory activity. Especially, 6.22 is approximately 8-fold more

active than lead compound 1 (IC50 = 13.4 µM) and 4-fold more active than CAPE
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(IC50 = 6.0 µM) (Fig. 4). In addition, 6.27 (IC50 = 103.0 µM) and 6.24 (IC50 = 54.6
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µM), bearing pyrrolidinyl and tetrahydrofuranyl, respectively, showed weaker

anti-inflammatory activities than the corresponding cyclopentyl substituted compound
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6.18 (IC50 = 15.8 µM). It is notable that when 6.27 (IC50 = 103.0 µM) is protected by
t-butyloxy carbonyl (6.25, IC50 = 7.0 µM), the anti-inflammatory activity increased
rapidly. This result is consistent with the antioxidant models and verifies that the low
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lipophilicity is detrimental to activities in cellular models. Meanwhile, cell viabilities

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are more than 90% with target compounds in all concentrations determined by the
MTS assay in this experiment, which indicates that the NO production inhibition is
not from the cytotoxicity of the target compounds. The background level of NO
production was determined from BV2 cells after 24h without LPS stimulation.
Table 4
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Inhibitory Activities against LPS-induced NO production in BV2 microglial cells of compounds
6.1−6.27, 1 and CAPE a.
Compd. IC50 (µM) Compd. IC50 (µM)
6.1 3.3 ± 0.4*** 6.16 33.5 ± 0.1***
6.2 3.8 ± 0.1*** 6.17 20.8 ± 0.8***
PT
6.3 8.5 ± 0.2*** 6.18 15.8 ± 0.9
6.4 7.6 ± 0.9*** 6.19 6.7 ± 0.6***
RI
6.5 7.4 ± 0.4*** 6.20 2.5 ± 0.3***
6.6 7.3 ± 0.8*** 6.21 11.3 ± 0.2
SC
6.7 6.9 ± 0.6*** 6.22 1.6 ± 0.2***
6.8 6.6 ± 0.4*** 6.23 9.2 ± 0.2**
U
6.9 14.3 ± 0.6 6.24 54.6 ± 1.2***
AN
6.10 12.2 ± 0.8 6.25 7.0 ± 1.3***
6.11 11.0 ± 0.5 6.26 6.9 ± 0.4***

M
6.12 13.3 ± 0.5 6.27 103.0 ± 1.6***
6.13 11.2 ± 0.2 1 13.4 ± 0.3

D
6.14 8.7± 0.3*** CAPE 6.0 ± 0.1

TE
6.15 8.3 ± 0.1***
respective 1.
EP
a
Data are expressed as the mean ± SD, n = 3
C
*** **
100
***
6.22
NO scavenging rate（ %）
*** 1
AC
***
50
**
0
0 10 20 30 40
concentration
Fig. 4. Inhibitory Activities against LPS-induced NO production in BV2 microglial cells of
compounds 6.22 and 1. BV2 cells were incubated with compounds (0.625, 1.25, 2.5, 5, 10, 20 and
−1
40 µM) for 3 h; LPS (0.1 µg mL ) was added, and incubation continued for 24 h. The
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supernatants of the culture media were mixed with Griess reagent in a new 96-well plate. The
absorbance was detected at 540 nm. The results are expressed as means ± SEM (n=3) Significance
levels *p < 0.05, **p < 0.01, ***p < 0.001 as compared with the respective 1.
PT
3.5 Cytotoxicity
The cytotoxicity of all compounds towards PC12 and BV2 cells were determined
RI
by the MTT assay. As shown in Table 5, most of the tested compounds displayed low
toxicity for PC12 and BV2 cells at 100 µM, except 6.6−6.8. When the number of
SC
carbon atoms in alkyl chain exceed 9, the cytotoxicity increases rapidly. Comfortingly,
almost all of the cycloalkyl-substituted compounds exhibited lower cytotoxicity than
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1. It can be seen from toxcity results of 6.3 and 6.19 that when the carbon atom
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number of the substituents is equal, the toxicity of cycloalkyl compound is weaker
than that of the alkyl chain compound, especially towards BV2 cells.
M
Table 5
Cytotoxicity of target compounds on PC12 and BV2 cells at 100 µMa.

D
PC12 Viability BV2 PC12 BV2

Compd. Compd.
TE
(%) Viability (%) Viability (%) Viability (%)
6.1 57.4 ± 1.8*** 70.1 ± 3.4 6.16 92.9 ± 2.3 87.8 ± 4.8*
EP
6.2 63.7 ± 2.2*** 56.2 ± 1.5** 6.17 80.2 ± 1.6 83.8 ± 7.1
6.3 85.7 ± 0.2 52.5 ± 2.9** 6.18 85.4 ± 0.8 72.7 ± 3.4
C
6.4 73.6 ± 0.9*** 63.6 ± 6.9 6.19 87.0 ± 1.5 74.7 ± 3.1
6.5 70.6 ± 1.5*** 66.6 ± 6.1 6.20 77.7 ± 3.3 76.6 ± 6.0
AC
6.6 59.7 ± 0.7*** 34.1 ± 3.3*** 6.21 94.9 ± 0.9 62.6 ± 2.2
6.7 52.1 ± 2.3*** 23.2 ± 0.8*** 6.22 70.5 ± 0.6*** 72.4 ± 4.0
6.8 28.2 ± 2.3*** 25.2 ± 1.1*** 6.23 91.3 ± 0.3 77.7 ± 2.3
6.9 91.8 ± 3.6 72.0 ± 6.5 6.24 98.0 ± 1.8** 93.9 ± 3.3**
6.10 87.3 ± 1.4 49.5 ± 4.9** 6.25 52.5 ± 2.8*** 68.7 ± 2.2
6.11 95.2 ± 1.5 54.4 ± 2.0** 6.26 88.3 ± 4.0 78.8 ± 2.6
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6.12 100.2 ± 0.6*** 50.4 ± 5.9** 6.27 55.9 ± 1.9*** 76.4 ± 3.8
6.13 67.5 ± 6.1*** 58.9 ± 0.5 1 86.1 ± 2.5 72.0 ± 2.8
6.14 66.9 ± 3.3** 73.4 ± 5.0 CAPE 85.1 ± 1.6 69.3 ± 2.7
6.15 74.7 ± 3.1 71.9 ± 5.3
Cells (1 × 105 cells mL−1, 100 µL per well) were plated in 96-well plates for 24h and then treated with varying concentrations of
PT
compounds for 24h, and the cell viability was determined by the MTT assay. The viability of untreated cells is defined as 100%.
Cell viabilities were obtained by the following formula: ODtreated / ODcontrol*100%. aData are expressed as the mean ± SD, n = 3.
RI
respective 1.
U SC
3.6 Effect of compounds 6.22 and 1 on H2O2-Induced apoptosis in PC12 cells.
AN
Based on results above on antioxidant and anti-inflammatory activities, 6.22
shows the optimal multifunctional neuroprotection activity. The protection effect of
M
compound 6.22 on apoptosis was further verified by test on the H2O2-damaged PC12
cells, which were double-labeled with Annexin V-FITC (AV-FITC) and propidium
D
iodide (PI). The populations of apoptotic PC12 cells preincubated with or without
TE
compounds 6.22 and 1 were determined using fluorescence microscope [34] (Fig. 5).
Most cells in the control group were viable. Exposure to H2O2 significantly increased
EP
the number of early (AV-FITC) and late (AV-FITC and PI) apoptotic cells (green
fluorescence and green + red fluorescence, respectively) of PC12 cells. When cells
C
were preincubated with two concentrations (2.5 and 10 µM) of compounds 6.22 and 1,
AC
the percentage of apoptotic cells decreased in different degrees. Preincubated with

compound 6.22 protected the PC12 cells against H2O2 oxidation and the percentage of
apoptotic cells decreased significantly at 2.5 µM, which was much better than that of
1. When cells were preincubated with 6.22 and 1 at 10 µM , the late apoptotic cells
were considerably less and the early apoptotic rate was similar to that of the control
group. These results suggest that the neuroprotective activity of compounds 6.22 and
1 is concentration dependent and 6.22 has better antioxidant activity than 1 at low
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concentration.
Control H2O2 6.22 (2.5 µM) 6.22 (10 µM) 1 (2.5 µM) 1 (10 µM)
brightfield
PT
Annexin V
RI
SC
PI
Annexin V+PI
U
AN
M
Fig.5. Cells were counted for co-staining of Annexin V-FITC (green filter) and PI (red filter).
Cells are shown for brightfield, Annexin V-FITC, PI and merged of AV-FITC and PI. Cells
D
stained for Annexin V are green, cells stained for PI are red, and cells stained for both are merged
TE
and shown as yellow. Images were captured using a 20× objective.

EP
3.7 In Vitro Evaluation of the BBB Permeability.
The blood-brain barrier (BBB) is a major impediment to the development of

C
central nervous system (CNS) drugs. Compounds with high potency but poor
AC
permeation through the BBB show low therapeutic value. Therefore, good BBB
permeability is an important property for the CNS drug candidates [35]. The parallel
artificial membrane permeation assay for BBB (PAMPA-BBB) that is used to predict
the capacity to cross the blood-brain barrier (BBB) was first developed by Di et al
[36]. In this assay, porcine polar brain lipid in dodecane was used to simulate the
BBB membrane [13]. The permeability (Pe) of target compounds was detected with
verapamil and hydrocortisone as positive and negative controls, respectively [2]. It is
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well known that the compound with good BBB permeation (CNS+) should show Pe >
4.0 × 10−6 cm s−1. Otherwise, the BBB permeation is low (CNS−) [37] with Pe < 2.0 ×
10−6 cm s−1. If 2.0 × 10−6 cm s−1 < Pe < 4.0 × 10−6 cm s−1, the BBB permeation is
uncertain (CNS±). As shown in Table 6, Pe values of the positive and negative
controls, verapamil and hydrocortisone, are similar with the results of Di Li
PT
(verapamil Pe = 16 × 10−6 cm s−1, hydrocortisone Pe = 1.9 × 10−6 cm s−1) [36], which
confirmed the reliability of the assay. The results demonstrated that most target
RI
compounds exhibit a predicted CNS (+) with Pe values up to 1.12 × 10−5 cm s−1,
which are better than CAPE (Pe = 1.91 × 10−6 cm s−1). The influence of the alkyl
SC
chain length on the BBB permeability is significant, which was confirmed by the
increasing Pe (10−6 cm s−1) values for compounds 6.4−6.7 and 6.9−6.12 (6.4−6.7: 5.23,
U
9.49, 8.68 and 11.20; 6.9−6.12: 2.21, 3.21, 4.73 and 6.76). Meanwhile, compounds
AN
6.24 (Pe = 4.47 × 10−6 cm s−1) and 6.27 (Pe = 2.70 × 10−6 cm s−1), with pyrrolidinyl
and tetrahydrofuranyl substituent, showed weaker permeability than 6.18 (Pe = 6.94 ×
M
10−6 cm s−1) with cyclopentyl, indicating that the introduction of heteroatoms

dramatically decreased the BBB permeability. Similar phenomenon was observed on
D
the alkyl side chain compounds. The Pe value of N-Boc (6.25, Pe = 9.43 × 10−6 cm s−1)
protected 6.27 (Pe = 2.70 × 10−6 cm s−1) is increased rapidly. In addition, compounds
TE
6.18 and 6.19 (Pe = 6.94 and 5.78 × 10−6 cm s−1) with cyclopentyl and cyclohexyl
showed better permeability than that of 6.16 and 6.17 (Pe = 3.93 and 5.16 × 10−6 cm
EP
s−1) with cyclopropyl, cyclobutyl substituents.

C
AC
Table 6
Effective permeability (Pe) of target compounds and control drugs in the PAMPA−BBB assaya.
Compd. Pe (10−6 cm s−1) prediction Compd. Pe (10−6 cm s−1) prediction
6.1 4.66 ± 0.26 CNS+ 6.17 5.16 ± 0.78 CNS+
6.2 8.33 ± 0.18 CNS+ 6.18 6.94 ± 0.32 CNS+
6.3 5.65 ± 0.39 CNS+ 6.19 5.78 ± 0.53 CNS+

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6.4 5.23 ±0.07 CNS+ 6.20 8.21 ± 0.13 CNS+
6.5 9.49 ±0.12 CNS+ 6.21 6.44 ± 0.32 CNS+
6.6 8.68 ± 0.68 CNS+ 6.22 6.84 ± 0.25 CNS+
6.7 11.20 ± 0.35 CNS+ 6.23 5.20 ± 0.05 CNS+
6.8 8.86 ± 0.20 CNS+ 6.24 4.47 ± 0.15 CNS+
PT
6.9 2.21 ± 0.24 CNS± 6.25 9.43 ± 0.05 CNS+
6.10 3.21 ± 0.14 CNS± 6.26 4.30 ± 0.11 CNS+
RI
6.11 4.73 ± 0.48 CNS+ 6.27 2.70 ± 0.07 CNS-
6.12 6.76 ± 0.10 CNS+ 1 5.29 ± 0.60 CNS+
SC
6.13 4.53 ± 0.08 CNS+ Hydrocortisone 1.80 ± 0.05 CNS-
6.14 3.54 ± 0.01 CNS± Verapamil 16.9 ± 0.11 CNS+
U
6.15 3.53 ± 0.40 CNS± CAPE 1.91 ± 0.08 CNS-
AN
6.16 3.93 ± 0.31 CNS±
a
Data are expressed as the mean ± SD, n = 3. PBS was used as the solvent.
M
3.8 The Calculation of Physiochemical Properties.

D
To further study the predicted BBB permeability in the novel compounds and gain
TE
insight into their CNS drug likeness, CNS multiparameter optimization (CNS MPO)
algorith from Pfizer was used [38]. The CNS MPO desirability tool consisted of six
EP
fundamental physicochemical properties: lipophilicity, calculated partition coefficient

(Clog P); calculated distribution coefficient at pH 7.4 (Clog D); molecular weight
C
(MW); topological polar surface area (TPSA); number of hydrogen-bond donors

(HBDs) and most basic center (pKa) [39] predicted by ChemAxon [40] or
AC
Molinspiration Software (Table S2 in the Supporting Information). Lipophilicity is a

measure of the polar attraction between the drug and cell membrane, and it affects the
diffusivity of drugs [41]. The log P value is a descriptor of lipophilicity which is an
important index to evaluate the permeability to cross the BBB for neuroprotective
agents. Log BB is also a common measurement of the degree of BBB penetration [42]
which is determined by log P and TPSA [43]. Some researches predict that
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compounds with CNS MPO desirability scores greater than 4 [44] (in a scale from 0
to 6), log BB values greater than −1 [45], Log P values in the range of 2−4 [46] and
TPSA values less than 76 Å [47] are expected to penetrate the brain and exhibit better
ADME attributes. Substitution of the phenyl with the cycloalkyl and alkyl chain
would change the dipole moment, polarity and lipophilicity of molecules since the
PT
sp3-hybridized carbon exists lower electronegativity than sp2-hybridized carbon.
Therefore, the log P, TPSA, log BB and CNS MPO values of designed compounds
RI
were calculated.
As shown in Table 7, all compounds except 6.25 and 6.27 have log BB values
SC
greater than −1 and most of the compounds possessing prominent activities have
favorable CNS MPO scores, thus would be predicted to penetrate the brain. The
U
replacement of phenylpropyl by nonyl and cyclohexane propyl greatly increases the
AN
log P and log BB values (1 log P = 3.04, log BB = −0.50; 6.5 log P = 4.13, log BB =
−0.34; 6.23 log P = 3.51 log BB = −0.43), which means that compounds with alkyl
M
substituent have better BBB permeability. Besides, increasing the length of the alkyl
chain augments the log P and log BB values but introducing the hydroxy lowers them.
D
Compounds 6.27 (log P = −0.16, log BB = −1.17) and 6.24 (log P = 1.00, log BB =
TE
−0.95) bearing pyrrolidinyl and tetrahydrofuranyl, respectively, exhibited weaker

permeability than the corresponding cyclopentyl substituted compound 6.18 (log P =
EP
2.25, log BB = −0.62). Almost all the compounds except 6.9−6.12 and 6.24−6.27
have TPSA values of 74.60 Å, smaller than 76 Å. Compound 6.22 with efficient
antioxidative and anti-inflammatory activities also show desirable CNS MPO score of
C
4.36, log P value of 3.06, log BB value of −0.50 and TPSA value of 74.60 Å, which
AC
indicates compound 6.22 can penetrate the brain efficiently and possess appropriate
drug-like properties. The predicted ability of compounds to cross the blood−brain
barrier is consistent with Pe values of PAMPA-BBB model.
Table 7
Summary of calculated physiochemical properties for 6.1−6.27 and 1.

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Compd. Log P TPSA Log BBa CNS MPO Compd. Log P TPSA Log BBa CNS MPO
6.1 2.35 74.60 −0.61 4.75 6.15 3.82 74.60 −0.38 3.60
6.2 2.79 74.60 −0.54 4.52 6.16 1.36 74.60 −0.76 4.92
6.3 3.24 74.60 −0.47 4.18 6.17 1.80 74.60 −0.69 4.92
6.4 3.68 74.60 −0.41 3.74 6.18 2.25 74.60 −0.62 4.80
PT
6.5 4.13 74.60 −0.34 3.35 6.19 2.69 74.60 −0.56 4.57
6.6 4.57 74.60 −0.27 3.13 6.20 2.62 74.60 −0.57 4.61
RI
6.7 5.02 74.60 −0.20 2.92 6.21 3.06 74.60 −0.50 4.36
6.8 5.46 74.60 −0.14 2.86 6.22 3.06 74.60 −0.50 4.36
SC
6.9 1.80 94.83 −0.99 4.42 6.23 3.51 74.60 −0.43 3.91
6.10 2.25 94.83 −0.92 4.30 6.24 1.00 83.83 −0.95 4.92
U
6.11 2.69 94.83 −0.86 4.08 6.25 1.96 104.14 −1.10 4.28
AN
6.12 3.58 94.83 −0.72 3.27 6.26 1.44 83.83 −0.88 4.92
6.13 2.93 74.60 −0.52 4.45 6.27 −0.16 86.62 −1.17 4.65
M
6.14 3.38 74.60 −0.45 4.04 1 3.04 74.60 −0.50 4.38
a
[log BB = −0.0148 × TPSA + 0.152 × log P + 0.139] was used for calculations of log BB values.
D
TE
4. Conclusion
In this study, we have designed and synthesized a new series of
EP
(E)-3,4-dihydroxystyryl alkyl sulfones as potent antioxidant and anti-inflammatory

compounds with improved neuroprotective activity and the BBB permeability. Alkyl
C
substituents, including different length of alkyl side chains, cycloalkyl, heterocyclic

alkyl and the alkyl side chains bearing terminal alkenyl or hydroxyl group, were
AC
introduced to replace the phenyl counterpart. From the biological results, we

discovered that the target compounds with alkyl moieties not only retained their
antioxidant activities in H2O2 and 6-OHDA-induced PC12 cells injury models and
anti-inflammatory activities in LPS-Stimulated BV2 cells model, but also exhibited
enhanced permeability compared to lead compound 1. It is notable that the
cyclopentyl propyl substituted compound 6.22 (IC50 = 1.6 µM) shows better inhibition
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on NO production than lead compound 1 (IC50 = 13.4 µM), which indicates that the
length of the linker and the conformation of cycloalkyl play important roles in
anti-inflammatory activities. Moreover, the antioxidant activity of 6.22 in H2O2 model
is stronger than 1 especially at low concentrations (2.5 and 5 µM). Further research on
H2O2-induced apoptosis in PC12 cells suggests that 6.22 showed better protective
PT
effect on early and late apoptosis than the lead compound at 2.5 µM as evidenced
from the Annexin-V-FITC/PI assay. Compound 6.22 also has favorable log BB values
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greater than −1, CNS MPO values greater than 4 and Pe (10−6 cm s−1) values of 6.84,
which suggested that 6.22 would penetrate the BBB efficiently. Taken together,
SC
(E)-3,4-dihydroxystyryl alkyl sulfones can be considered as potential multifunctional
neuroprotective agents due to the enhanced anti-inflammatory activity, BBB
U
permeability, excellent antioxidant activity and low cytotoxicity. What is more, the
AN
relationships between structure−activity and structure−BBB permeability were
recognized by evaluation of the neuroprotective agents modified with different
M
substituents.
D
5. Experimental section
TE
5.1 Chemistry
All solutions were carried out in vacuo with Heidolph rotary evaporator. 1H and
EP
13
C NMR spectra recorded on a Bruker Avance III 400 MHz NMR spectrometer with
tetramethylsilane (TMS, Me4Si) as an internal standard. CDCl3 and DMSO-d6 were
C
used as the solvents for NMR experiments. HRMS spectra were recorded on a Bruker
AC
Apex IV FTMS spectrometer. All the target compounds were analyzed by HPLC
(Agilent 1260 infinity HPLC system) to determine their purity. All assayed
compounds displayed a purity ≥ 95% (Table S2 in the Supporting Information). All
reactions were detected by Thin-layer chromatography (TLC) using the silica Gel
GF254 plates. The spots were visualized under UV light irradiation or by iodine vapor
staining. Melting points were determined on SGW X-4 type melting point apparatus
and were uncorrected. Silica gel (500-600 mesh) was employed for vacuum column
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chromatography separations. Unless otherwise stated, all reagents were purchased

from commercial sources. If necessary, they were purified and dried by standard
methods.
5.1.1 General Procedure for the Synthesis of Alkyl bromide 3.18−3.21.
PT
The PBr3 (1.5 equiv) was added to a solution of alkyl alcohol (1.0 equiv) in
anhydrous tetrahydrofuran (5.0 mL) at 0 °C and then the ice bath was removed and
RI
the reaction mixture was further stirred at room temperature for 5h. Water (30.0 mL)
was added and then extracted with EtOAc, the combined organic extracts were dried
SC
with Na2SO4, and the solvent was evaporated in vacuo to get title compound, suitable
for use in the next step without further purification.
U
AN
5.1.2 General Procedure for tosylation of alcohols 3.22−3.26.
To a solution of alcohol (5.0 equiv) and N, N-dimethylamino-pyridine (DMAP,
M
1.0 equiv) in CH2Cl2 was slowly added a solution of p-toluenesulfonic chloride (TsCl)
(5.5 equiv) in CH2Cl2 at 0 °C, then the Et3N (10.0 equiv) was added. The reaction
D
mixture was stirred at 0 °C for 1 h, and then warmed to room temperature for another
TE
3 h. After diluted with water, the reaction mixture was extracted with EtOAc.
EP
5.1.3 General Procedure for the Synthesis of alkylsulfanyl-acetic Acids 4.1−4.26.

The alkyl bromide (1.0 equiv) was added to a solution of mercaptoacetic acid (1.0
equiv) in methanol (10.0 mL) and the resulting solution stirred. Then a solution of
C
NaOH (2.0 equiv) in methanol (5.0 mL) was added slowly, and the final mixture was
AC
stirred at room temperature until absence of the alkyl bromide (checked by TLC). The
reaction mixture was concentrated in vacuo, diluted with H2O, and neutralized with 1
N HCl. Then the obtained reaction mixture was extracted with EtOAc. The combined
organic fractions were washed with brine, dried with Na2SO4, and concentrated in
vacuo. Purification of the crude residue by column chromatography (petroleum ether/
EtOAc) afforded the title compound.
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5.1.4 General Procedure for the Synthesis of Alkylsulfonyl-acetic Acids 5.1−5.26.

The alkylsulfonyl-acetic acids (1.0 equiv) was added to a solution of 30% H2O2
aqueous solution (1.0 mL) in acetic acid (3.0 mL) and the resulting solution stirred at
room temperature until absence of the alkylsulfonyl-acetic acids (checked by TLC).
PT
The reaction mixture was diluted with H2O and extracted with EtOAc. The combined
organic fractions were washed with brine, dried with Na2SO4, and concentrated in
RI
vacuo. Purification of the crude residue by column chromatography (petroleum ether/
EtOAc) afforded the title compound.
U SC
5.1.5 General procedure for the Synthesis of (E)-3,4-Dihydroxystyryl Alkyl Sulfones
AN
6.1−6.26.
3,4-dihydroxybenzaldehyde (1.0 equiv), pyrrolidine (1.1 equiv), and acetic acid
M
(1.1 equiv) were added to a solution of the (alkylsulfinyl)-acetic acid (2.0 equiv)
solution in THF (15 mL) and the resulting solution heated to reflux until absence of
D
the 3,4-Dihydroxybenzaldehyde (checked by TLC). The reaction solution was

TE
evaporated under vacuum, diluted with H2O, and extracted with EtOAc. The
combined organic fractions were washed with brine, dried with (Na2SO4), and
EP
concentrated in vacuo. Purification of the crude residue by column chromatography

(petroleum ether/ EtOAc) afforded the title compounds.
C
5.1.5.1 (E)-4-(2-(pentylsulfonyl)vinyl)benzene-1,2-diol (6.1). white solid (58%); mp

AC
102–104°C; IR (KBr, cm-1): 3476, 3282 (O-H), 2956 (-CH3), 1607 (C=C aromatic),
1290, 1122 (SO2); 1H NMR (400 MHz, DMSO-d6) δ 9.71 (s, 1H), 9.19 (s, 1H), 7.27
(d, J = 15.4 Hz, 1H), 7.09–6.97 (m, 3H), 6.78 (d, J = 8.1 Hz, 1H), 3.15–3.04 (m, 2H),
1.67–1.59 (m, 2H), 1.38–1.24 (m, 4H), 0.85 (t, J = 7.0 Hz, 3H). 13C NMR (100 MHz,
DMSO-d6) δ 149.26 , 146.09 , 143.60 , 124.32 , 122.70 , 122.38 , 116.16 , 115.69 ,
54.37 , 30.22 , 22.37 , 22.09 , 14.15. HR-MS (ES−) m/z 269.0850 [M − H]−, found
269.0848 [M − H]−.
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5.1.5.2 (E)-4-(2-(hexylsulfonyl)vinyl)benzene-1,2-diol (6.2). white solid (62%); mp

127–129 °C; IR (KBr, cm-1): 3484, 3255 (O-H), 2929 (-CH2-), 2867 (-CH3), 1607
(C=C aromatic), 1280, 1122 (SO2); 1H NMR (400 MHz, DMSO-d6) δ 9.70 (s, 1H),
9.18 (s, 1H), 7.27 (d, J = 15.4 Hz, 1H), 7.11–6.96 (m, 3H), 6.79 (d, J = 8.1 Hz, 1H),
PT
3.17–3.05 (m, 2H), 1.70–1.57 (m, 2H), 1.44–1.19 (m, 6H), 0.85 (t, J = 6.7 Hz, 3H).
13
C NMR (100 MHz, DMSO-d6) δ 149.26, 146.10, 143.59, 124.34, 122.74, 122.36,
RI
116.17, 115.71, 54.43, 31.16, 27.73, 22.65, 22.28, 14.29. MS (ES−) m/z 283.10 [M −
H]−.
SC
5.1.5.3 (E)-4-(2-(heptylsulfonyl)vinyl)benzene-1,2-diol (6.3). white solid (56%); mp
U
115–116°C; IR (KBr, cm-1): 3494, 3246 (O-H), 2929, 2850 (-CH2-), 1595 (C=C
AN
aromatic), 1276, 1113 (SO2); 1H NMR (400 MHz, DMSO-d6) δ 9.71 (s, 1H), 9.18 (s,
1H), 7.27 (d, J = 15.4 Hz, 1H), 7.13–6.96 (m, 3H), 6.79 (d, J = 8.1 Hz, 1H), 3.18–3.04
M
13
(m, 2H), 1.66–1.59 (m, 2H), 1.44–1.15 (m, 8H), 0.84 (t, J = 6.3 Hz, 3H). C NMR
(100 MHz, DMSO-d6) δ 149.09, 146.09, 143.59, 124.33, 122.72, 122.37, 116.16,
D
115.70, 54.41, 31.44, 28.62, 28.02, 22.69, 22.43, 14.36. HR-MS (ES–) m/z 297.1168
[M − H]−, found 297.1161 [M − H]−.
TE
EP
5.1.5.4 (E)-4-(2-(octylsulfonyl)vinyl)benzene-1,2-diol (6.4). white solid (62%); mp

100°C; IR (KBr, cm-1): 3491, 3326 (O-H), 2920, 2850 (-CH2-), 1598 (C=C aromatic),
C
(d, J = 15.4 Hz, 1H), 7.16–6.95 (m, 3H), 6.79 (d, J = 8.1 Hz, 1H), 3.20–3.04 (m, 2H),
AC
13
1.66–1.59 (m, 2H), 1.41–1.33 (m, 2H), 1.23 (s, 8H), 0.84 (t, J = 6.2 Hz, 3H). C
NMR (100 MHz, DMSO-d6) δ 149.25, 146.09, 143.59,124.33, 122.72, 122.36, 116.15,
115.69, 54.41, 31.60, 28.92, 28.87, 28.06, 22.68, 22.50, 14.39. MS (ES−) m/z 311.13
[M − H]−.
5.1.5.5 (E)-4-(2-(nonylsulfonyl)vinyl)benzene-1,2-diol (6.5). white solid (64%); mp

100–102°C; IR (KBr, cm-1): 3483, 3325 (O-H), 2932, 2850 (-CH2-), 1596 (C=C
ACCEPTED MANUSCRIPT
1H), 7.26 (d, J = 15.4 Hz, 1H), 7.13–6.96 (m, 3H), 6.79 (d, J = 8.1 Hz, 1H), 3.14–3.05
(m, 2H), 1.66–1.59 (m, 2H), 1.35 (d, J = 6.5 Hz, 2H), 1.23 (s, 10H), 0.85 (t, J = 6.2 Hz,
13
3H). C NMR (100 MHz, DMSO-d6) δ 149.25, 146.09, 143.59, 124.33, 122.71,
122.35, 116.15, 115.69, 54.41, 31.69, 29.17, 29.03, 28.95, 28.04, 22.68, 22.53, 14.40.
PT
MS (ES−) m/z 325.15 [M − H]−.
RI
5.1.5.6 (E)-4-(2-(decylsulfonyl)vinyl)benzene-1,2-diol (6.6). white solid (49%); mp
SC
1
1300, 1115 (SO2); H NMR (400 MHz, DMSO-d6) δ 9.75 (s, 1H), 9.22 (s, 1H), 7.28
(d, J = 15.4 Hz, 1H), 7.13–7.00 (m, 3H), 6.81 (d, J = 8.2 Hz, 1H), 3.17–3.07 (m, 2H),
U
13
1.69–1.59 (m, 2H), 1.25 (s, 14H), 0.87 (t, J = 6.7 Hz, 3H). C NMR (100 MHz,
AN
DMSO-d6) δ 149.27, 146.11, 143.60, 124.34, 122.74, 122.36, 116.16, 115.71, 54.42,
31.74, 29.34, 29.22, 29.12, 28.95, 28.05, 22.68, 22.55, 14.42. HR-MS (ES−) m/z
M
339.1633 [M − H]−, found 339.1630 [M − H]−.

D
5.1.5.7 (E)-4-(2-(undecylsulfonyl)vinyl)benzene-1,2-diol (6.7). white solid (57%); mp

TE
EP
(d, J = 15.4 Hz, 1H), 7.12–6.93 (m, 3H), 6.79 (d, J = 8.1 Hz, 1H), 3.25–2.95 (m, 2H),
1.70–1.56 (m, 2H), 1.35 (d, J = 6.8 Hz, 2H), 1.23 (s, 14H), 0.85 (t, J = 6.4 Hz, 3H).
13
C NMR (100 MHz, DMSO-d6) δ 149.25, 146.09, 143.59, 124.33, 122.72, 122.35,
C
116.15, 115.69, 54.44, 31.74, 29.41, 29.38, 29.21, 29.15, 28.95, 28.04, 22.68, 22.55,
AC
14.42. HR-MS (ES–) m/z 353.1786 [M − H]−, found 353.1787 [M − H]−.
5.1.5.8 (E)-4-(2-(dodecylsulfonyl)vinyl)benzene-1,2-diol (6.8). white solid (72%); mp

100–101°C; IR (KBr, cm-1): 3544, 3360 (O-H), 2920, 2850 (-CH2-), 1608 (C=C
1H), 7.27 (d, J = 15.4 Hz, 1H), 7.09–6.97 (m, 3H), 6.79 (d, J = 8.1 Hz, 1H), 3.15–3.04
(m, 2H), 1.66–1.59 (m, 2H), 1.41–1.31 (m, 2H), 1.23 (s, 16H), 0.86 (t, J = 6.8 Hz, 3H).
ACCEPTED MANUSCRIPT
13
C NMR (100 MHz, DMSO-d6) δ 149.26, 146.10, 143.59, 124.34, 122.74, 122.35,
116.16, 115.71, 54.43, 31.74, 29.45, 29.37, 29.21, 29.15, 28.95, 28.05, 22.67, 22.55,
14.41. HR-MS (ES−) m/z 367.1938 [M − H]−, found 367.1943 [M − H]−.
5.1.5.9 (E)-4-(2-((7-hydroxyheptyl)sulfonyl)vinyl)benzene-1,2-diol (6.9). white solid
PT
(60%); mp 107–109°C; IR (KBr, cm-1): 3543, 3365 (O-H), 2921, 2848 (-CH2-), 1606
RI
9.19 (s, 1H), 7.27 (d, J = 15.4 Hz, 1H), 7.13–6.94 (m, 3H), 6.79 (d, J = 8.1 Hz, 1H),
4.32 (s, 1H), 3.38 (d, J = 5.6 Hz, 2H), 3.10 (s, 2H), 1.67–1.59 (m, 2H), 1.48–1.21 (m,
SC
13
9H). C NMR (100 MHz, DMSO-d6) δ 149.25, 146.09, 143.58, 124.33, 122.73,
122.37, 116.17, 115.70, 61.09, 54.48, 32.85, 28.87, 28.13, 25.70, 22.65. HR-MS (ES−)
U
m/z 313.1111 [M − H]−, found 313.1110 [M − H]−.
AN
5.1.5.10 (E)-4-(2-((8-hydroxyoctyl)sulfonyl)vinyl)benzene-1,2-diol (6.10). white solid
M
(65%); mp 99–100 °C; IR (KBr, cm-1): 3454, 3491 (O-H), 2931, 2853 (-CH2-), 1602
D
9.19 (s, 1H), 7.27 (d, J = 15.3 Hz, 1H), 7.13–6.95 (m, 3H), 6.79 (d, J = 8.1 Hz, 1H),
TE
4.32 (s, 1H), 3.37 (s, 2H), 3.17–3.04 (m, 2H), 1.69–1.57 (m, 2H), 1.42–1.32 (m, 4H),
13
1.25 (s, 6H). C NMR (100 MHz, DMSO-d6) δ 149.25, 146.09, 143.59, 124.33,
EP
122.73, 122.37, 116.16, 115.70, 61.14, 54.42, 32.93, 29.13, 29.02, 28.05, 25.85, 22.68.
MS (ES−) m/z 327.10 [M − H]−.
C
5.1.5.11 (E)-4-(2-((9-hydroxynonyl)sulfonyl)vinyl)benzene-1,2-diol (6.11). white solid

AC
(59%); mp 104–104 °C; IR (KBr, cm-1): 3491, 3172 (O-H), 2932, 2852 (-CH2-), 1609
9.21 (s, 1H), 7.26 (d, J = 15.4 Hz, 1H), 7.09–6.97 (m, 3H), 6.79 (d, J = 7.9 Hz, 1H),
4.33 (s, 1H), 3.15–3.06 (m, 2H), 1.66–1.59 (m, 2H), 1.43–1.32 (m, 5H), 1.24 (s, 9H).
13
C NMR (100 MHz, DMSO-d6) δ 149.26, 146.09, 143.61, 124.32, 122.70, 122.38,
116.16, 115.67, 61.17, 54.42, 32.96, 29.27, 28.93, 28.05, 25.90, 22.68. MS (ES−) m/z
341.14 [M − H]−.
ACCEPTED MANUSCRIPT
5.1.5.12 (E)-4-(2-((11-hydroxyundecyl)sulfonyl)vinyl)benzene-1,2-diol (6.12). white

solid (57%); mp 100–102°C ; IR (KBr, cm-1): 3490, 3175 (O-H), 2924, 2850 (-CH2-),
1606 (C=C aromatic), 1287, 1115 (SO2); 1H NMR (400 MHz, DMSO-d6) δ 9.72 (s,
1H), 9.19 (s, 1H), 7.26 (d, J = 15.4 Hz, 1H), 7.10–6.97 (m, 3H), 6.78 (d, J = 8.1 Hz,
PT
13
1H), 4.32 (s, 1H), 3.14–3.06 (m, 2H), 1.63–1.60 (m, 2H), 1.41 –1.16 (m, 18H). C
NMR (100 MHz, DMSO-d6) δ 149.25, 146.09, 143.59, 124.32, 122.70, 122.37,
RI
116.14, 115.68, 61.17, 54.39, 33.00, 29.51, 29.41, 29.36, 29.23, 28.97, 28.06, 25.96,
22.68. HR-MS (ES–) m/z 369.1730 [M − H]−, found 369.1736 [M − H]−.
SC
5.1.5.13 (E)-4-(2-(hept-6-en-1-ylsulfonyl)vinyl)benzene-1,2-diol (6.13). white solid
U
(69%); mp 89–92 °C; IR (KBr, cm-1): 3492, 3438 (O-H), 2933, 2860 (-CH2-), 1597
AN
9.19 (s, 1H), 7.27 (d, J = 15.4 Hz, 1H), 7.11 –6.97 (m, 3H), 6.79 (d, J = 8.1 Hz, 1H),
M
5.83–5.72 (m, 1H), 5.03–4.90 (m, 2H), 3.17–3.04 (m, 2H), 2.00 (d, J = 6.4 Hz, 2H),
13
1.70–1.58 (m, 2H), 1.44–1.31 (m, 5H). C NMR (100 MHz, DMSO-d6) δ 146.09,
D
143.61, 138.98, 122.70, 122.38, 116.16, 115.70, 115.30, 54.36, 33.34, 28.17, 27.58,
22.56. MS (ES−) m/z 295.10 [M − H]−.
TE
EP
5.1.5.14 (E)-4-(2-(oct-7-en-1-ylsulfonyl)vinyl)benzene-1,2-diol (6.14). white solid

(57%); mp78–80 °C; IR (KBr, cm-1): 3472, 3274 (O-H), 2931, 2853 (-CH2-), 1602
C
9.17 (s, 1H), 7.27 (d, J = 15.4 Hz, 1H), 7.11 –6.96 (m, 3H), 6.79 (d, J = 8.1 Hz, 1H),
AC
5.82–5.73 (m, 1H), 5.04–4.88 (m, 2H), 3.16–3.06 (m, 2H), 2.00 (q, J = 6.6 Hz, 2H),
13
1.65–1.61 (m, 2H), 1.42–1.21 (m, 7H). C NMR (100 MHz, DMSO-d6) δ 146.10,
143.61, 139.16, 122.73, 122.36, 116.17, 115.72, 115.17, 54.42, 33.48, 28.43, 27.87,
22.64. MS (ES−) m/z 309.12 [M − H]−.
5.1.5.15 (E)-4-(2-(non-8-en-1-ylsulfonyl)vinyl)benzene-1,2-diol (6.15). white solid

(60%); mp 84 °C; IR (KBr, cm-1): 3490, 3326 (O-H), 2930, 2851 (-CH2-), 1596 (C=C
ACCEPTED MANUSCRIPT
1H), 7.27 (d, J = 15.4 Hz, 1H), 7.13 –6.95 (m, 3H), 6.79 (d, J = 8.1 Hz, 1H), 5.86–
5.69 (m, 1H), 5.06–4.87 (m, 2H), 3.19–3.03 (m, 2H), 2.02–1.97 (m, 2H), 1.73–1.54
13
(m, 2H), 1.38–1.24 (m, 8H). C NMR (100 MHz, DMSO-d6) δ 149.26, 146.10,
143.61, 139.22, 122.72, 122.36, 116.16, 115.70, 115.11, 54.41, 33.56, 28.75, 28.66,
PT
28.58, 28.00, 22.66. HR-MS (ES−) m/z 323.1318 [M − H]−, found 323.1317 [M −
H]−.
RI
5.1.5.16 (E)-4-(2-((cyclopropylmethyl)sulfonyl)vinyl)benzene-1,2-diol (6.16). white
SC
solid (46%); mp 169–160°C; IR (KBr, cm-1): 3505, 3305 (O-H), 1602 (C=C aromatic),
U
(d, J = 15.3 Hz, 1H), 7.08 (d, J = 2.5 Hz, 1H), 7.06–6.95 (m, 2H), 6.79 (d, J = 8.1 Hz,
AN
1H), 3.08 (d, J = 7.3 Hz, 2H), 0.98 (t, J = 7.0 Hz, 1H), 0.56 (d, J = 7.7 Hz, 2H), 0.31
13
(d, J = 4.9 Hz, 2H). C NMR (100 MHz, DMSO-d6) δ 149.23, 146.11, 143.61,
M
124.45, 123.05, 122.31, 116.19, 115.59, 59.30, 5.02, 4.50. MS (ES−) m/z 253.05 [M −
H]−.
D
TE
5.1.5.17 (E)-4-(2-((cyclobutylmethyl)sulfonyl)vinyl)benzene-1,2-diol (6.17). white

solid (56%); mp 122–123°C; IR (KBr, cm-1): 3365, 3053 (O-H), 2969 (-CH2-), 1595
EP
9.21 (s, 1H), 7.24 (d, J = 15.4 Hz, 1H), 7.11–6.89 (m, 3H), 6.79 (d, J = 7.8 Hz, 1H),
3.41 (s, 1H), 3.23 (d, J = 6.7 Hz, 2H), 2.77–2.59 (m, 1H), 2.05 (d, J = 7.1 Hz, 2H),
C
1.95–1.65 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 149.25, 146.09, 143.49, 124.39,
AC
123.17, 122.36, 116.18, 115.63, 60.33, 29.90, 28.30, 19.22. MS (ES−) m/z 267.09 [M
− H]−.
5.1.5.18 (E)-4-(2-((cyclopentylmethyl)sulfonyl)vinyl)benzene-1,2-diol (6.18). white

solid (56%); mp 162–163°C; IR (KBr, cm-1): 3456, 3344 (O-H), 2958, 2869 (-CH2-),
1H), 9.20 (s, 1H), 7.27 (d, J = 15.4 Hz, 1H), 7.19–6.95 (m, 3H), 6.79 (d, J = 8.1 Hz,
ACCEPTED MANUSCRIPT
1H), 3.17 (d, J = 6.7 Hz, 2H), 2.22–2.20 (m, 1H), 1.84 (d, J = 6.1 Hz, 2H), 1.67–1.41
13
(m, 4H), 1.29–1.24 (m, 2H). C NMR (100 MHz, DMSO-d6) δ 149.24, 146.10,
143.27, 124.38, 123.49, 122.36, 116.17, 115.64, 60.02, 34.43, 32.51, 24.74. HR-MS
(ES–) m/z 281.0847 [M − H]−, found 281.0848 [M − H]−.
PT
5.1.5.19 (E)-4-(2-((cyclohexylmethyl)sulfonyl)vinyl)benzene-1,2-diol (6.19). white
solid (69%); mp 122–125°C; IR (KBr, cm-1): 3504, 3287 (O-H), 2931, 2853 (-CH2-),
RI
1H), 9.19 (s, 1H), 7.27 (d, J = 15.6 Hz, 1H), 7.12–6.96 (m, 3H), 6.79 (d, J = 7.9 Hz,
SC
1H), 3.03 (d, J = 5.0 Hz, 2H), 1.85 (d, J = 12.0 Hz, 3H), 1.65–1.56 (m, 3H), 1.31–0.97
13
(m, 5H). C NMR (100 MHz, DMSO-d6) δ 149.24, 146.10, 142.97, 124.37, 123.85,
U
122.34, 116.19, 115.65, 60.84, 32.77, 32.73, 25.94, 25.72. MS (ES−) m/z 295.10 [M −
AN
H]−.
M
5.1.5.20 (E)-4-(2-((2-cyclopentylethyl)sulfonyl)vinyl)benzene-1,2-diol (6.20). white

solid (49%); mp 123–125 °C; IR (KBr, cm-1): 3478, 3268 (O-H), 2951, 2855 (-CH2-),
D
TE
1H), 9.19 (s, 1H), 7.27 (d, J = 15.3 Hz, 1H), 7.07-7.01 (m, 3H), 6.79 (d, J = 8.1 Hz,
1H), 3.18–3.06 (m, 2H), 1.88–1.43 (m, 9H), 1.15–1.00 (m, 2H). 13C NMR (100 MHz,
EP
DMSO-d6) δ 149.26, 146.09, 143.59, 124.33, 122.68, 122.39, 116.16, 115.69, 53.87,
38.71, 32.28, 28.56, 25.11. HR-MS (ES–) m/z 295.1006 [M − H]−, found 295.1004 [M
− H]−.
C
AC
5.1.5.21 (E)-4-(2-((2-cyclohexylethyl)sulfonyl)vinyl)benzene-1,2-diol (6.21). white

solid (72%); mp 140–141°C; IR (KBr, cm-1): 3354 (O-H), 2929, 2850 (-CH2-), 1593
9.20 (s, 1H), 7.26 (d, J = 15.4 Hz, 1H), 7.10–6.97 (m, 3H), 6.79 (d, J = 8.1 Hz, 1H),
3.18–3.04 (m, 2H), 1.69–1.49 (m, 7H), 1.31 (s, 1H), 1.22–1.04 (m, 3H), 0.87 (q, J =
13
11.6 Hz, 2H). C NMR (100 MHz, DMSO-d6) δ 149.26, 146.09, 143.57, 124.33,
122.69, 122.39, 116.16, 115.66, 52.46, 36.36, 32.77, 29.79, 26.37, 26.08. HR-MS
ACCEPTED MANUSCRIPT
(ES–) m/z 309.1163 [M − H]−, found 309.1161 [M − H]−.
5.1.5.22 (E)-4-(2-((3-cyclopentylpropyl)sulfonyl)vinyl)benzene-1,2-diol (6.22). white

solid (51%); mp 127–128 °C; IR (KBr, cm-1): 3494, 3253 (O-H), 2942, 2866 (-CH2-),
PT
1H), 9.19 (s, 1H), 7.27 (d, J = 15.4 Hz, 1H), 7.12–6.98 (m, 3H), 6.79 (d, J = 8.1 Hz,
1H), 3.15–3.06 (m, 2H), 1.77–1.34 (m, 11H), 1.05-0.99 (m, 2H). 13C NMR (100 MHz,
RI
DMSO-d6) δ 149.26, 146.10, 143.60, 124.34, 122.72, 122.34, 116.16, 115.69, 54.56,
SC
− H]−.
U
5.1.5.23 (E)-4-(2-((3-cyclohexylpropyl)sulfonyl)vinyl)benzene-1,2-diol (6.23). white
AN
solid (69%); mp 122-123°C; IR (KBr, cm-1): 3484, 3313 (O-H), 2931, 2852 (-CH2-),
M
1H), 9.19 (s, 1H), 7.26 (d, J = 15.4 Hz, 1H), 7.12 – 6.96 (m, 3H), 6.79 (d, J = 8.1 Hz,
1H), 3.08 (t, J = 8.0 Hz, 2H), 1.64 (d, J = 12.3 Hz, 7H), 1.23-1.08 (m, 6H), 0.85 (t, J =
D
13
11.5 Hz, 2H). C NMR (100 MHz, DMSO-d6) δ 149.25, 146.09, 143.62, 124.35,
TE
122.72, 122.37, 116.17, 115.71, 54.64, 40.63, 37.02, 35.66, 33.07, 26.56, 26.17.
HR-MS (ES–) m/z 281.0847 [M − H]− , found 281.0848 [M − H]−.
EP
5.1.5.24 (E)-4-(2-(((tetrahydrofuran-2-yl)methyl)sulfonyl)vinyl)benzene-1,2-diol
(6.24). white solid (49%); mp 100–101°C; IR (KBr, cm-1): 3378, 3194 (O-H), 2973,
C
2877 (-CH2-), 1598 (C=C aromatic), 1292, 1122 (SO2); 1H NMR (400 MHz,
AC
DMSO-d6) δ 9.73 (s, 1H), 9.21 (s, 1H), 7.39–6.66 (m, 5H), 4.37–4.05 (m, 1H), 3.75–
13
3.62 (m, 2H), 3.38 (d, J = 14.3 Hz, 2H), 2.18–1.45 (m, 4H). C NMR (100 MHz,
DMSO-d6) δ 149.27, 146.13, 142.94, 124.32, 123.85, 122.39, 116.23, 115.48, 73.32,
− H]−.
5.1.5.25 tert-butyl (E)-2-(((3,4-dihydroxystyryl)sulfonyl)methyl)pyrrolidine-1-

ACCEPTED MANUSCRIPT
carboxylate (6.25). white solid (65%); mp 79–80°C; IR (KBr, cm-1): 3370, 3053
(O-H), 2973, 2877 (-CH3), 1607 (C=C aromatic), 1298, 1113 (SO2); 1H NMR (400
MHz, DMSO-d6) δ 9.73 (s, 1H), 9.19 (s, 1H), 7.30 (d, J = 15.4 Hz, 1H), 7.14–6.99 (m,
3H), 6.79 (d, J = 8.1 Hz, 1H), 4.12–4.01 (m, 1H), 3.33–3.13 (m, 4H), 2.08–1.96 (m,
2H), 1.92–1.85 (m, 1H), 1.79–1.74 (m, 1H), 1.36 (d, J = 14.0 Hz, 9H). 13C NMR (100
PT
MHz, DMSO-d6) δ 153.26, 149.38, 146.07, 144.08, 124.24, 122.57, 116.13, 115.86,
79.45, 57.83, 52.43, 46.09, 30.88, 28.55, 22.48. MS (ES−) m/z 382.41 [M − H]−.
RI
5.1.5.26 (E)-4-(2-(((tetrahydro-2H-pyran-2-yl)methyl)sulfonyl)vinyl)benzene-1,2-diol
SC
(6.26). white solid (51%); mp 144–148 °C; IR (KBr, cm-1): 3396, 3070 (O-H), 2929,
2850 (-CH2-), 1598 (C=C aromatic), 1290, 1095 (SO2); 1H NMR (400 MHz,
U
DMSO-d6) δ 9.67 (s, 1H), 9.21 (s, 1H), 7.25 (d, J = 15.4 Hz, 1H), 7.06–6.90 (m, 3H),
AN
6.78 (d, J = 8.1 Hz, 1H), 3.89–3.81 (m, 1H), 3.77–3.72 (m, 1H), 3.40–3.26 (m, 1H),
3.30–3.24 (m, 2H), 1.75 (d, J = 10.9 Hz, 1H), 1.67 (d, J = 12.0 Hz, 1H), 1.52–1.39 (m,
M
13
3H), 1.34–1.25 (m, 1H). C NMR (100 MHz, DMSO-d6) δ 149.20, 146.10, 142.54,
124.28, 122.31, 116.21, 115.46, 72.62, 67.95, 60.69, 31.26, 25.54, 23.00. MS (ES−)
D
m/z 297.41 [M − H]−.

TE
5.1.5.27 (E)-4-(2-((pyrrolidin-2-ylmethyl)sulfonyl)vinyl)benzene-1,2-diol (6.27). To a

EP
solution of 6.25 (1 equiv) in MeOH (5mL), concentrated hydrochloric acid (catalytic

amount) was added and the resulting solution was stirred for 1 h at room temperature.
The solvent was evaporated under vacuum and the mixture was diluted with H2O and
C
extracted with ethyl acetate. The combined organic fractions were washed with brine,
AC
dried with sodium sulfate, and concentrated in vacuo. Purification of the crude
product by column chromatography (petroleum ether /EtOAc) afforded the target
compound 6.27 as a white solid (90%); mp 227–229°C; IR (KBr, cm-1): 3370, 3149
(O-H), 2982, 2735 (-CH2-), 1607 (C=C aromatic), 1289, 1121 (SO2); 1H NMR (400
MHz, DMSO-d6) δ 9.90 (s, 1H), 9.45 (s, 1H), 9.35 (s, 2H), 7.42 (d, J = 15.4 Hz, 1H),
7.16–7.04 (m, 3H), 6.84 (d, J = 8.2 Hz, 1H), 3.85–3.67 (m, 3H), 3.18 (s, 2H), 2.24 –
2.14 (m, 1H), 1.99–1.62 (m, 3H). 13C NMR (100 MHz, DMSO-d6) δ 149.74, 146.16,
ACCEPTED MANUSCRIPT
145.59, 124.03, 122.79, 121.53, 116.27, 116.08, 55.70, 54.04, 45.42, 30.70, 23.23.
HR-MS (ES–) m/z 282.0800 [M − H]−, found 282.0800 [M − H]−.
5.2 Biological Assays.

All tests were undertaken on three replicates and the results were averaged. IC50
PT
values were determined for each compound as the best fit non-linear regression values
of the log [inhibitor] vs. response curve using Excel. Statistical significance was
RI
estimated by Analysis of Variance (ANOVA) followed by Tukey post hoc
comparison or T-test (IC50 of H2O2 models) using GraphPad Prism software. P-value
SC
< 0.05 was considered significant.
U
5.2.1 DPPH Free Radical Scavenging Ability.
AN
The free radical scavenging ability was performed using stable free radicals
named 1,1'-diphenyl-2-picrylhydrazyl (DPPH) [48,49]. DPPH was purchased from
M
Sigma. 100 mL of the test solutions (different concentrations of different compounds)

and 100 mL of the DPPH solution (0.2 mM) were mixed in 96-well microplates
D
separately, and 100 mL of ethanol and DPPH was mixed as the control. The mixture
TE
was incubated at room temperature for 1h. The capacity of each compound to reduce
free radicals was detected by measuring the absorbance at 517 nm. The scavenging
EP
ability was compared with the well-known antioxidant vitamin C. The percentage of
reduction was obtained from the following formula: [1-(Ac– Ab)/A0] ×100, where Ac is
the absorbance of the test sample, Ab is the absorbance of sample in ethanol and A0 is
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the absorbance of the control.

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5.2.2 Protection of PC12 Cells against H2O2- and 6-OHDA-Induced Cell Injury
The PC12 cells were purchased from the Shanghai Institute of Cell Biology,
Chinese Academy of Sciences. Fetal bovine serum (FBS) was obtained from Hyclone,
penicillin streptomycin (PS) leagene was purchased from, trypsin−EDTA was
purchased from Life, and high-glucose DMEM were purchased from Hyclone. MTT,
MTS and DMSO were obtained from Amresco. H2O2 and 6-OHDA were purchased
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from Sigma. To evaluate properties of the synthesized compounds, the

[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium, inner salt; (MTS)] and the [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl
-2-H-tetrazolium bromide (MTT)] assays were used. PC12 cells (2 × 105 cells mL−1,
100 µL per well) were plated in 96-well microplates for 1 day and then treated with
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various concentrations of test compounds for 3 h. Afterward, 20 µL of H2O2 (diluted
with medium to a final concentration of 400 µM) or 6-OHDA (diluted with medium to
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a final concentration of 250 µM) solution was added. After 3 and 24 h, respectively,
the cell viability was determined by MTS and MTT assay. The absorbance was
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detected at 490 nm and 570 nm.
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5.2.3 In vitro 15-lipoxygenase (LOX) inhibition assay
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The ability of the test compounds to inhibit soyabean 15-LOX was determined using
Cayman lipoxygenase inhibitor screening assay kit Catalog No. (760700) supplied by
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Cayman chemicals, Ann Arbor, MI, USA. The preparation of reagents and testing
procedures for determining IC50 values of the tested compounds were carried out
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following the manufacturer's instructions as reported methods[50]. In this method, 90

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µL of soybean LOX was pipetted into a 96-well flat-bottomed plate. Next, 10 µL of

test compound at concentrations (1.25µM, 2.5 µM, 5.0 µM, 10 µM, 20µM, 25µM,
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50µM, 100µM) dissolved in assay buffer were added to each well. The reaction was
initiated by adding 10 µL substrate (arachidonic acid or linoleic acid) and the plate
was placed on a shaker for at least ten minutes. Finally, 100 µL of chromogen was
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added to each well to stop enzyme catalysis and develop the reaction. The absorbance
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of the solution was measured at λ 490-500nm.
5.2.4 Inhibiting NO Production of LPS-Stimulated BV2 Microglial Cells.

BV2 microglial cells were purchased from the Institute of Basic Medicine,
Chinese Academy of Medical Sciences. LPS was purchased from Sigma. BV2
microglial cells (3 × 105 cells mL−1, 100 µL per well) were plated in 96-well
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microplates for 1 day and then treated with various concentrations of test compounds
diluted with medium for 3 h. Afterward, the cells were challenged with 20 µL of LPS
(diluted with medium to a final concentration of 100 ng mL−1), followed by additional
culturing for 24 h. The supernatants of the culture media were mix with Griess
reagent in a new 96-well plate. The absorbance was detected at 540 nm. The
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percentage of inhibition of NO was obtained from the following formula:
−
In % = × 100%
−
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Where In (%) is the inhibition rate of NO. Rl is the release amount of only the
LPS-treated group, R0 is the release amount of the normal control, and Rc is the
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release amount of the test compound and LPS-treated group.
5.2.5 Cytotoxicity screening.

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PC12 and BV2 cells were incubated in high-glucose DMEM supplemented with10%
FBS and 1% PS in a humidified atmosphere of 5% CO2 at 37 °C. PC12 cells (1 × 105
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cells mL−1, 100 µL per well) were plated in 96-well plates for 1 day followed by
incubation with the drugs for 24 h. The cytotoxicity of drugs was determined by the
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MTS assay according to published procedure [25]. BV2 cells (2 × 105 cells mL−1, 100
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µL per well) were plated in 96-well plates for 1 day followed by incubation with the
drugs for 24 h. The cytotoxicity of drugs was determined by the MTT assay according
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to published procedure.
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5.2.6 Annexin-V/FITC assay

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Cells were assayed by the Annexin-V-FITC Apoptosis Detection Kit (keyGEN

bioTECH, China) according to the manufacturer's instructions. Briefly, PC12 cells
(1.5 × 105 cells mL−1, 3 mL per well) were plated in 35mm culture dish for 1 day and
then treated with compound 6.22 or 1 at 2.5 µM and 10 µM for 3h. Afterward, H2O2
(diluted with medium to a final concentration of 400 µM) solution was added. After
3h, PC12 cells were washed twice with ice-cold PBS, and stained with a mixture of 5
µL Annexin-V-FITC, 5 µL PI and 1000 µL Binding Buffer for 5 min in the dark at 25
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o
C, and analyzed by fluorescence microscope.
5.2.7 In Vitro Blood−Brain Barrier Permeability Assay.

Brain penetration of target compounds was evaluated by parallel artificial
membrane permeation assay (PAMPA) [36,37,51], The Verapamil, and
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hydrocortisone were purchased from Sigma, phosphate buffer saline (PBS), DMSO
(for biology), and dodecane (analytical standard,) were obtained from Acros and Alfa
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Aesar, The porcine polar brain (PBL) lipid was purchased from Avanti Polar Lipids.
The acceptor plate (catalog no. MAIPN4550) and the donor plate (catalogno.
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MATRNPS50) were both purchased from Millipore. The hydrophobic polyvinylidene
fluoride (PDVF) membrane units were obtained from Symta. Test compounds were
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dissolved in DMSO to make a 5 mM stock solution. Ten microliters (10 µL) of this
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compound stock solution were diluted with PBS to get secondary stock solution (final
concentration 25 mg mL−1). 300 µL of the secondary stock solution were added to the
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donor wells. The filter membrane was coated with 4 µL of porcine polar brain lipid
solution which was dissolved in dodecane at 20 mg mL−1. The acceptor well was
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filled with 150 µL of PBS. The acceptor filter plate was carefully put on the donor
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plate to form a “sandwich” which was consisted of the donor with tested compounds
on the bottom, artificial lipid membrane in the middle, and the aqueous acceptor on
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the top. The ‘sandwich’ was left undisturbed for 18h at room temperature. The
concentrations of tested compounds in the acceptor, the donor, and the reference
solutions were determined by a UV plate reader. Reference solutions were prepared
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by mixing the sample secondary stock solution (300 µL ) and 150 µL PBS. The Pe
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was obtained by the following equation [2,51]:
=− ln 1 −
+
Where Vd (mL) = volume of the donor compartment, Va (mL) = volume of the
acceptor compartment, Ca = the concentration of the acceptor well, Cr = the
concentration of the reference solution, s (cm2) = membrane area, and t (s) =
incubation time.
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Notes
The authors declare no competing financial interest.
Acknowledgment
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This study was supported by the National Natural Science Foundation of China
(Grants 20972011, 21042009, and 21172014) and Ministry of Science and
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Technology of China (Grant 2009ZX09301-010).
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Abbreviations used
CAPE, caffeic acid phenethyl ester; AD, Alzheimer’s disease; PD, Parkinson’s
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disease; HD, Huntington’s disease; ALS, amyotrophic lateral sclerosis; CNS MPO,
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Central Nervous System Multiparameter Optimization; TPSA, topological polar
surface area; log BB, brain-to-blood concentration ratio; HBDs, hydrogen-bond
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donors; iNOS, inducible nitric oxide synthase; IC50, half-maximal inhibitory

concentration; BBB, blood−brain barrier; PAMPA, parallel artificial membrane
D
permeability assay; PAMPA-BBB, parallel artificial membrane permeation assay for

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the blood−brain barrier; DPPH, 1,1-diphenyl-2-picrylhydrazyl; NO, nitric oxide; LPS,

lipopolysaccharide; PC12, rat pheochromocytoma cells; H2O2, hydrogen peroxide;
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6-OHDA, 6-hydroxydopamine; ROS, reactive oxygen species; DA, dopaminergic;

CNS, central nervous system; TLC, thin layer chromatography; HR-MS,
high-resolution mass spectrometry; HPLC, high-performance liquid chromatography
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Appendix A. Supplementary data
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Highlights
1. (E)-3,4-dihydroxystyryl alkyl sulfones were synthesized as neuroprotective agents.
2. 6.22 is a new compound with clopentyl propyl substituent.
3. 6.22 showed 8-fold higher inhibitory effects on NO production than lead compound 1.
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4. 6.22 inhibited PC12 apoptosis at 2.5 µM were confirmed by Annexin-V-FITC/PI assay.
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5. The CNS-MPO was first used to evaluate the BBB permeability of CAPE derivatives.
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Accepted Manuscript

Structure-activity relationship studies of (E)-3,4-dihydroxystyryl alkyl sulfones as novel

To appear in: European Journal of Medicinal Chemistry

Received Date: 1 January 2019

Structure-activity Relationship Studies of (E)-3,4-dihydroxystyryl

Alkyl Sulfones as Novel Neuroprotective Agents Based on Improved

Antioxidant, Anti-inflammatory Activities and BBB Permeability

(E)-3,4-dihydroxystyryl alkyl sulfones, as new analogues of neurodegenerative agents,

target compounds preserved antioxidant and anti-inflammatory potency in scavenging

6-hydroxydopamine and inhibiting lipopolysaccharide (LPS)-induced over production

University, Beijing 100191, China. Phone/fax: +86-10-82805203.

E-mail: jyliu@bjmu.edu.cn. (Junyi Liu), E-mail: xiaoweiwang@bjmu.edu.cn. (Xiaowei Wang)

resulting in progressively and irreversibly deteriorated nervous system and greatly

mitochondrial injury are considered to play important roles in the pathogenesis of

in neural cells and it is implicated in the pathogenesis of neurodegenerative diseases

modification of drug molecules [14,15]. Herein, we focused on developing drug

candidates with good neuroprotection and BBB permeability in this study.

conformation on permeability and neuroprotective activities. We also designed

Fig 1. Structures of 1 and target compounds.

The synthetic routes of (E)-4-(2-((3-phenylpropyl)sulfonyl)vinyl)benzene-1,2-diol

(approximately 80%) from mercaptoacetic acid in methanol catalyzed by NaOH at

(f) pyrrolidine, acetic acid, THF, reflux, 49-72%.

hydrochloric acid, MeOH, rt, 70%.

monitored by fluorescence spectrophotometer at 514 nm according to the published

to 11.4 µM, as shown in Table 1. Especially, compounds 6.2–6.6, 6.9–6.11, 6.13–6.15,

scavenging capacities in comparison to 1 (EC50 = 11.5 µM). The results confirmed

cyclopentyl substituents, indicating that the flexibility and conformation of rings

Free Radical Scavenging Abilities of target compounds by the DPPH Methoda.

6.1 2 C2H5 8.1 ± 0.2*** 6.16 0 cyclopropyl 10.5 ± 0.9

6.3 2 C4H9 7.1 ± 0.1*** 6.18 0 cyclopentyl 9.4 ± 0.6

6.5 2 C6H13 7.7 ± 0.1*** 6.20 1 cyclopentyl 6.8 ± 0.1***

6.8 2 C9H19 8.5 ± 0.6** 6.23 2 cyclohexyl 7.9 ± 1.1***

6.9 2 C4H8OH 5.9 ± 0.1*** 6.24 0 tetrahydrofuranyl 10.5 ± 0.3

6.10 2 C5H10OH 6.7 ± 0.1*** 6.25 0 N-BOC pyrrolidinyl 7.1 ± 0.1***

6.11 2 C6H12OH 7.5 ± 0.3*** 6.26 0 tetrahydropyranyl 10.8 ± 0.1

6.12 2 C8H16OH 22.6 ± 1.4*** 6.27 0 pyrrolidinyl 13.8 ± 0.4

6.13 2 C2H4CH=CH2 7.1 ± 0.8*** 1b 11.5 ± 0.1

6.14 2 C3H6CH=CH2 6.4 ± 0.1*** CAPE 14.6 ± 1.1

6.15 2 C4H8CH=CH2 6.9 ± 0.1***

3.2 Neuronal Protection Effect against Damage Induced by H2O2.

heteroatoms on alkyl substituents reduced the lipophilicity of the compounds and

compared with the H2O2-treated group.

H2O2-induced injury half-maximal inhibitory concentration (IC50) of target compounds 6.1−6.27,

1 and CAPE in PC12 cellsa.

Compd. IC50 (µM) Compd. IC50 (µM)

6.1 1.83 ± 0.05 6.16 6.61 ± 0.34***

6.2 1.30 ± 0.06*** 6.17 4.81 ± 0.14***

6.3 1.33 ± 0.09** 6.18 2.19 ± 0.07**

6.4 1.45 ± 0.06*** 6.19 1.85 ± 0.07

6.5 1.93 ± 0.01** 6.20 1.96 ± 0.03**

6.7 2.34 ± 0.05*** 6.22 1.28 ± 0.08***

6.9 9.91 ± 0.19*** 6.24 27.10 ± 0.34***

6.11 7.91 ± 0.10*** 6.26 3.34 ± 0.03***

6.14 1.35 ± 0.04*** CAPE 2.00 ± 0.03

6.15 1.90 ± 0.01*

3.3 Neuroprotective effect on PC12 cells injury induced by 6-OHDA

oxidative phosphorylation, resulting in energy deprivation and ROS accumulation

mitochondrial dysfunction by oxidative stress reduce the cellular resistance [26].

100 *** *** *** *** *** 2.5 µM

Fig. 3. Protective effects of all target compounds at 2.5, 5, 10 µM against 6-OHDA-induced

compared with the 6-OHDA-treated group.

3.4 In vitro 15-LOX inhibition assay

6.22 102.9 ± 9.2***

6.5 2 C6H13 7.7 ± 0.1* 6.20 1 cyclopentyl 6.8 ± 0.1*

6.8 2 C9H19 8.5 ± 0.6 6.23 2 cyclohexyl 7.9 ± 1.1*

6.10 2 C5H10OH 6.7 ± 0.1* 6.25 0 N-BOC pyrrolidinyl 7.1 ± 0.1*

6.2 1.30 ± 0.06* 6.17 4.81 ± 0.14*

6.3 1.33 ± 0.09 6.18 2.19 ± 0.07

6.5 1.93 ± 0.01 6.20 1.96 ± 0.03

6.7 2.34 ± 0.05* 6.22 1.28 ± 0.08*

6.9 9.91 ± 0.19* 6.24 27.10 ± 0.34*

6.11 7.91 ± 0.10* 6.26 3.34 ± 0.03*

100 * * * * *** 2.5 µM

6.1 3.3 ± 0.4* 6.16 33.5 ± 0.1*

6.2 3.8 ± 0.1* 6.17 20.8 ± 0.8*

6.4 7.6 ± 0.9* 6.19 6.7 ± 0.6*

6.8 6.6 ± 0.4* 6.23 9.2 ± 0.2