ROS-responsive and multifunctional anti-Alzheimer prodrugs: Tacrine-ibuprofen hybrids via a phenyl boronate linker

Graphical abstract

ROS-Responsive and Multifunctional Anti-Alzheimer Prodrugs:

Tacrine-Ibuprofen Hybrids via a Phenyl Boronate Linker

a,b,1

a,b

a,b *

Zhikun Liu , Bin Zhang , Shengjin Xia , Lei Fang , Shaohua Gou

A series of ROS-responsive and multi-functional anti-Alzheimer compounds

were designed and synthesized. The representative compound 22 possessed

pronounced cholinesterase-inhibiting, anti-inflammatory and neuron-protective effects

in vitro, and perfectly ameliorated the cognitive damage of AD in vivo. The study

offers a promising strategy for developing new anti-Alzheimer drugs.

ROS-Responsive and Multifunctional Anti-Alzheimer Prodrugs:

Tacrine-Ibuprofen Hybrids via a Phenyl Boronate Linker

a,b,1

a,b

a,b *

Zhikun Liu

, Bin Zhang ,Shengjin Xia , Lei Fang , Shaohua Gou

a

Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research, Southeast

University, Nanjing, 211189, China

b

Pharmaceutical Research Center and School of Chemistry and Chemical

Engineering, Southeast University, Nanjing 211189, China

ABSTRACT Current drugs available in clinic for Alzheimer’s disease (AD)

treatment can only alleviate disease symptoms without clearly curing or delaying the

process of AD. And some AD drugs failed in Phase III clinical trials are only focused

on targeting amyloid-β (Aβ). Therefore, an alternative strategy in AD drug design is

meaningful to be involved in the multiple pathogenic factors which can affect each

other at multiple levels. Herein, we report a series of ROS-responsive prodrugs based

on multi-target-directed ligands (MTDLs) approach, which can specifically release

tacrine derivatives and ibuprofen under oxidation of ROS and show

acetylcholinesterase (AChE)-inhibiting, neuron-protective and anti-inflammatory

effects in extracellular or intracellular assays. Related biological study illustrated that

compound 22 was able to permeate blood-brain-barrier (BBB) showing little

hepatotoxicity in comparison to tacrine. Besides, 22 hinted a therapeutic clue in

AD-treatment by regulating proinflammatory factors (IL-1β and TNF-α) and

apoptosis related proteins (Bax, Bcl-2 and cleaved caspase-3). Further spatial memory

assays in Aβ-induced AD model showed that 22 enhanced the ability of learning and

memory. Our study proves that the strategy of ROS-responsive prodrugs has promise

for AD treatments in future and offers a way for AD drug development.

1

These authors contributed equally.

*

Corresponding authors

E-mail: lei.fang@seu.edu.cn (L. Fang); 2219265800@qq.com (S. Gou)

Keywords: ROS stimulation, Prodrug, Multifunctional agent, Alzheimer

1

. INTRODUCTION

Alzheimer's disease (AD), one of the most common forms of dementia, is an

age-related neurodegenerative disease characterized by progressive cognitive decline,

[

1,2]

behavior disorders and degenerative brain disease

. It is estimated that about 50

million people have been affected by AD in 2018 and the number is expected to reach

1

2

52 million costing more than $2.0 trillion for treatment in the United States along by

[3,4]

050

. Without doubt, the sustaining and high-speed growth of AD patients will

bring about huge social and economic burden, revealing that treatment of AD is of

great significance.

Much attention has been paid to AD, but no efficient therapeutic treatment is

developed at present because of complex etiology of this neurodegenerative disease

[

5,6]

and its multifactorial progression . Several hypotheses based on AD’s pathogenic

factors have been proposed, including the production and aggregation of amyloid-β

(Aβ), accumulation of hyperphosphorylated τ-proteins, oxidative stress, decrease of

[

7,8]

synaptic cholinergic level and inflammation response

. Upon these hypotheses,

only a few of acetylcholinesterase inhibitors (AChEIs shown in Figure 1) including

tacrine (withdrawn from the market for hepatotoxicity), donepezil, rivastigmine,

galantamine and the N-methyl-D-aspartate receptor (NMDAR) antagonist

(memantine), were obtained for AD treatment. However, these drugs can only

alleviate the progress of AD by improving memory and cognitive function to a certain

degree instead of definitively curing or stopping this disease. Therefore, development

of novel approach to treat AD is much necessary and required. In view of the complex

pathogenesis, present research and development on AD drugs are mainly focused on

multitarget-directed ligands (MTDLs) approach by efficiently ameliorating memory

[

9-13]

and modifying cognitive decline

.

Indeed, a number of drug candidates have undergone clinical trials and most of

them aim to reduce the production of toxic aggregated Aβ or promote the

[

14-17]

depolymerization or clearance of aggregation Aβ proteins to protect neurons

.

However, with further understanding of mechanisms about AD, oxidative stress is

found to happen earlier than other pathogenesis and then affect each other at multiple

[

18-20]

levels

. When dopamine (DA) is oxidized by oxygen (O ) or upon the formation

2

of metal ion-binding Aβ protein, a large number of reactive oxygen species (ROS)

•

−

(

e.g. superoxide anion radical (O ) and hydrogen peroxide (H O ) generate and

2 2 2

subsequently induce more pathogenesis such as oxidative damage to biomolecules

(lipids, proteins, and DNA), mitochondrial dysfunction and final disorders of neuronal

[

21-23]

homeostasis in the brain

. Moreover, prominent activation of inflammatory

process (inflammatory cytokines, IL-1β, TNF-α) induced by Aβ may drive AD

pathology or become ongoing disease process, though neuroinflammation exerts both

[

24-26]

toxic and protective effects in AD process

. Alternatively, in order to reduce the

toxic side effects induced by anti-inflammatory drugs and increase neuron-protective

activities, much attention has also been paid on microenvironment-responsive prodrug

[

25, 27a]

strategies

.

In recent years, quite a number of H O -specific probes or H O -responsive

2

prodrugs for antitumor study have been designed and synthesized using arylboronic

[

27].

acid or ester as the protecting group

As a ROS-responsive unit, phenylboronate

group can be self-immolative upon exposure to H O , which has been widely used in

2

[

28-30]

both oxidation-responsive polymers and prodrugs

. Tacrine, the first AChEI

approved for AD treatment, has been withdrawn from clinical use, but we believe that

it is still of great significance for further investigation on tacrine derivatives to reduce

[

6, 31,32]

hepatotoxicity

. Inkielewicz-Stepniak and co-workers have explained the great

therapeutic potential of tacrine in Alzheimer’s disease via multifunctional ligands

[32c]

modification

. In addition, nonsteroidal anti-inflammatory drugs (NSAIDs) like

ibuprofen or aspirin were reported to reduce inflammatory markers and reverse spatial

memory deficits in APPsw transgenic mice or improve memory and learning, and

[

25,33,34]

decrease stress-related behaviors in FAD5X/Ppara-null mice

. Therefore, the

great therapeutic potential effects of tacrine and ibuprofen motivate us to develop a

novel agent to simultaneously tackle the cognitive impairment caused by Alzheimer's

disease. Much different from ‘one molecule, one target’ paradigm, MTDLs approach

usually offers more than one functional biological activities, which is suitable for

complex pathogenesis of AD. Besides, MTDLs approach can also offer an ample

variety of derivatives in which key structure or linker length can modulate their

biological properties. Moreover, considering the overproduction of ROS in brain of

AD and specific characteristics of local inflammation, we have constructed

self-immolative dendritic prodrugs consisting of two ligands, ibuprofen and tacrine

derivatives, based on the MTDLs with a concept mentioned above.

As illustrated in Figure 2, we pursue to develop ROS-responsive prodrugs based

on AD’s pathological characteristics, including oxidative stress decreased synaptic

cholinergic level and inflammation response. With this aim in mind, we expect these

prodrugs release those two ligands at specific pathological oxidation environment of

AD upon the stimulation of ROS. In order to achieve our designing goals, quite a

number of biological assays have been performed including intracellular/extracellular

tests and therapeutic effects in AD models in vivo. Herein we present such a

promising strategy for the design of AD drugs as well as significant results we have

obtained.

2

. RESULTS AND DISCUSSION

2

.1. Chemistry

Preparation of tacrine derivatives is shown in Scheme S1, in which intermediates

[

31,32a]

8

-11 were synthesized according to the previous reports

. Synthetic way of our

target compounds 19-22 is displayed in Scheme 1. Compound 13 was obtained

through modifying with two protection groups of TBDMS on 12, using imidazole as

an absorb acid agent in anhydrous N,N-dimethylformamide (DMF). Then, the

nucleophilic

substitution

-bromomethylphenylboronic acid pinacol ester produced compound 14. Intermediate

5 was obtained from deprotection reaction of 14 under the condition of catalytic

reaction

between

compound

13

and

4

1

amount of PTSA in methanol at room temperature. Single TBDMS-covered 16 was

separated with silica gel chromatography. Compound 18 was made from deprotection

of TBDMS-covered 17 followed by condensation reaction with ibuprofen.

Compounds 19-22 were obtained by modifying two ligands of 18 and tacrine

derivatives 8-11, respectively, in the presence of N,N'-carbonyldiimidazole (CDI) in

DMF.

2

.2. Cytotoxicity assay

Cytotoxicity of the target compounds (19-22) against SH-SY5Y cells was

measured by MTT method. As shown in Figure 3A, no obvious cytotoxicity was

induced by 19-22 in the concentration ranged from 7.81 to 31.25 μM. Interestingly, 19

(with a two-methylene linker) and 21 (with a four-methylene linker) exhibit moderate

cytotoxicity to SH-SY5Y cells at 31.25 μM, whereas both of them have negligible

cytotoxicity at 7.81 μM and 15.62 μM. Moreover, 20 (with a three-methylene linker)

and 22 (with a six-methylene linker) have better cellular adaptability, especially for

compound 22. Judging from the results mentioned above, 22 exhibited negligible

cytotoxicity to SH-SY5Y cells even at higher concentration of 31.25 μM as

comparison to that of control group (Figure 3A), whereas 19-21 induced little to high

cytotoxicity to some degree under this concentration. Therefore, we decided to select

2

2 to investigate the following biological activities in alleviating Alzheimer’s disease.

As reducing the damage from oxidative stress in cells is believed to be feasible to

[34b-34d]

protect the nerve cells

, we subsequently investigated neuroprotective effects of

compounds 19-22 against H O . Briefly, SH-SY5Y cells were pre-incubated with

2

o

1

9-22 followed by the addition of H O . After another 24 h co-incubation at 37 C,

2 2

cell viability of SH-SY5Y cells was obtained. The results in Figure 3B indicate that

these compounds at the concentration of 5 μM show moderate to high neuroprotective

activity against H O and the values are all more than 50 % but significantly increase

2

to more than 80 % when the concentration was elevated to 20 µM (exception for 21),

exerting stronger neuroprotective activities than that of H O -treated group (cell

2

viability was about 20 %). However, ibuprofen, a component of the target compound,

does not show apparent detoxification against H O . On the other hand, the combined

2

group of ibuprofen and 11, two components of 22, seems not to rescue the cells

against cytotoxicity induced by H O even at high concentration of 20 µM. Taken all

2

the results together, 22 is the most potential compound in protecting neurons. Thus,

compound 22 is selected for further biological experiments.

Encouraged by this, we further investigate the H O -scavenging capability of our

2

target compounds. The consuming H O after 24 h co-incubation with 22 was

2

quantified via QuantiChrom™ Peroxide Assay Kit. As illustrated in Figure S1A, the

amount of scavenging H O is significantly elevated in a liner relationship as the

2

increasing dose of 22. Trolox, a positive control, also shows strong H O -cosuming

2

capacity (Figure S1A). In contrast, 11 and ibuprofen only show negligible

H O -eliminating capability as compared with 22 and Trolox. All the illustrated

2

results mentioned above revealed that compound 22 can protect neurons from

apoptosis against H O and be beneficial for decrease of oxidation stress of

2

Alzheimer.

2

.3. The release of ibuprofen and tacrine derivative 11

The target compound, especially compound 22, induces no cytotoxicity to

SH-SY5Y cells (Figure 3A) but seems to be more potential in protecting neurons

from apoptosis against the ROS (Figure 3B). Motivated by this, we evaluated the

stability of compound 22 under the oxidation of H O . A mixed solution of 22 and

2

o

H O at 37 C was used to detect the stability of 22 by reverse phase high

2

performance liquid chromatography (RP-HPLC). For the purpose of matching the

peaks generating from 22, solutions of ibuprofen, 11 and 12 at certain concentrations

were prepared. As shown in Figure S1, the peak attributed to 22 in RP-HPLC

appeared at about 12.381 min (Figure S1F). However, when treated with H O , the

2

peak of 22 gradually decreased with the generation of other three peaks matching the

position of compound 11, compound 12 and ibuprofen, respectively. This

phenomenon indicated that 22 acted as a ROS-responsive prodrug. Moreover, the

reaction rate did not accelerate a lot during the first 5 hours until the addition of

another portion of H O , hinting that amount of H O was scavenged with

2

self-immolation of 22 (Figure S1F).

The results in Figure S1F indicated that 22 degrades into the fragments of 11, 12

and ibuprofen gradually within 10 h upon stimulation with H O . Therefore, we

2

speculated that the envisaged collapse mechanism of 22 under oxidative condition

was as follows (Scheme S3): Firstly, phenylboronic ester reacted with H O to

2

generate phenyl oxide structure. Subsequently, the electron rearrangement around the

aromatic structure and finally caused rapidly release of the two ligands. In order to

1

substantiate this speculation, the process was also monitored with H-NMR

characterization. As shown in Figure S2, initially, there existed no peaks from

4

.10-4.60 ppm where were methylene protons of benzyl alcohols. However, when

o

mixed with H O (6.0 equiv.) at 37 C, methylene protons of 22 (a, b) gradually

2

decreased along with another increased peaks at 4.50 and 4.36 ppm (a′, b′, Figure S2).

These two peaks (a′, b′) belong to the methylene protons of phenylboronate and cresol

units, indicating the collapse of 22 under the oxidative condition of H O .

2

Interestingly, the pinacol unmasking reaction was not detected may due to the fast

1

degradation of this intermediate. Moreover, the significant changes in H-NMR peaks

of e′-f′ and c also proved the hydrolysis of the benzyl ester bonds and produce of

fragments 11 and ibuprofen under the oxidative condition of H O . In sum,

2

considering the results in Figure S1F, we can conclude that our target compound 22

served as a ROS-responsive prodrug and obtained great potential in treatment of

Alzheimer’s disease.

2

.4. Blood-brain barrier (BBB) permeation and cellular uptake and assays

[

35a]

Brain permeation abilities are initially required for development of AD drugs

.

Therefore, in order to investigate the ability of 22 to penetrate the BBB we

constructed a widely adopted BBB model by growing a dense layer of bEnd.3 cells on

[

35b]

the upper chamber of a transwell

. Briefly, bEnd.3 cells are cultured and allowed

to adhere in monolayer in DMEM (10% FBS) and transendothelial electrical

resistance (TEER) values of cells are monitored until the TEER values reach about

2

1

80 Ω

•

cm and used for BBB assays. The schematic of BBB model is given in Figure

S3A while change of TEER values during the monolayer culture is showed in Figure

S3B. As illustrated in Figure S3C, TEER values are decreased in bEnd.3 cells

monomers upon the addition of 22 while little change in TEER value occurs in

DMSO-treated group. The decreased TEER at the first 15 min is induced by solvent,

[

36a]

which was reported previously

. Further immunofluorescence staining indicates

that 22 induces discontinued intervals or even full absence to tight-junction related

protein (ZO-1) between bEnd.3 monomer cells (Figure S3D), which reveals that 22

regulates tight junction molecules to assist 22 to permeate BBB for delivering drug to

brain. Furthermore, we also tested the content of 22 in the medium and intracellular

abilities of lower chamber via BBB model by HPLC. As illustrated in Figure S4B, 22

easily passes through monomer cells in significant dose-dependent manner and can be

easily ingested by SH-SY5Y cells in lower chamber (Figure S4C), indicating better

BBB permeation abilities and intracellular properties. In sum, all of the results

indicate that 22 efficiently permeates BBB via regulation expression of ZO-1 between

bEnd.3 cells and easily entered SH-SY5Y cells.

For further research of visual experiment in BBB permeation assays and

intracellular uptake assays, compound 25 with a fluorescent coumarin moiety at the

[

36, 37]

position of 11 was constructed according to previous reports

(Scheme S2).

Intensely florescent images in SH-SY5Y cells in the BBB model indicate that 25 can

efficiently cross through the monolayer barrier and be easily ingested by SH-SY5Y

cells (Figure S4D). In order to investigate the uptake by SH-SY5Y and BV-2 cells, 25

were co-incubated with SY-SY5Y or BV-2 cells, respectively, and cellular uptake

results were obtained from laser confocal microscope. The intensely florescent color

in Figure 4 indicates that 25 has entered either SY-SY5Y or BV-2 cells efficiently.

Considering the great BBB permeation properties and efficient intracellular uptake of

2

2 and 25 in SHSY5Y cells we conclude that 22 is in great potential for treatment of

Alzheimer’s disease.

2

.5. The AChE inhibition study

The results in Figure S1F illustrated that our target compounds act as

ROS-responsive prodrugs upon stimulation with H O . Therefore, we forecast that

2

AChE inhibition activity of our target compounds may vary greatly before and after

treatment with H O . In order to confirm our hypothesis, AChE (from electric eel,

2

eeAChE) inhibition assays were performed using tacrine and untreated-compounds

[

34, 38]

1

9-22 as controls with Ellman’s methods

. The decreased enzymatic activity

results induced by 19-22 or H O -sitimulated 19-22 are shown in Figure 5. It is noted

2

in Figure 5A that compounds 19-22 have moderate or no obvious AChE inhibitory

activity even at 10 μM. Besides, the inhibitory rate of AChE increased from 28.25%

19) to 47.25% (22), probably due to the extension of carbon linkers that enhanced the

(

interaction between AChE and the tacrine group. However, after co-incubation with

H O , the AChE inhibitory effects of the target compounds were enhanced apparently

2

with the increasing alkyl chains. As shown in Figure 5B, H O -treated 19-22 exhibit

2

AChE inhibitory activity with IC values in range of 39.16 - 608.1 nM, of which the

5

0

activity of compound 22 containing a six methylene linker tacrine group is the

strongest with a value comparable to that of tacrine (IC value was 21.63 nM) and 11

5

0

(

IC value was 54.58 nM). It is noticed that the length of the alkyl chain has a close

50

relationship with the AChE activity of the resulting compounds. Motivated by AChE

inhibitory activity of 22, we chose the compound 22 for the following biological

study.

2

.6. Molecular docking

The AChE inhibition study proved that our target compounds 19-22 showed little

inhibitory effects on AChE, whereas they turned out to be efficient AChE imbibition

agents upon exposure to H O . Generally, AChE was reported as dumbbell-shape with

2

two active sites: the catalytic active site (CAS) at the bottom and the peripheral active

[

31]

site (PAS) at the lip . To investigate the difference before and after treatment with

H O , we performed the molecular docking studies of compounds 11 and 22. The

2

crystal structure of torpedo californica AChE (2CKM) was obtained from RSCB

protein data bank, and the structure of 22 was constructed in Chem 3D 14.0 with

energy minimized in Sybyl-X 2.0. The results obtained from Sybyl-X 2.0

surflex-dock model were shown in Figure 6. The binding model illustrated that the

tacrine fragment of 11 bound to the bottom of AChE, is surrounded with Asp 72, Val

7

1, Tyr 70, Trp 84, Glu 199, Met 83, Gly 118 and Pro 86, exhibiting a potent π-π

interaction between the heterocycle moiety and Trp 84; while the aliphatic alkyl chain

is surrounded by Ser 200, Trp 84, Gly 441, His 440 and Try 442 (Figure 6A and 6B).

The formation of H-bond between aliphatic alkyl chain of 11 and residues Tyr 442

and Ser 200 may contribute to the inhibitory effect on AChE as well. However, the

binding model in Figure 6C and 6D suggested that 22 only occupies the mouth of the

gorge and was only surrounded with residues Gly 335, Phe 331, His 440, Asp 72, Val

1, Try 70, Asp 276 and Trp 279 without significant intermolecular interactions with

7

residues around the PAS. Taken all these docking results together, the apparent

difference of the binding models between 22 or 11 and AChE may explain why 22

shows little AChE inhibition activity excepting for stimulating with H O .

2

.7. Inflammatory cytokines assay by qRT-PCR and LPS-induced ROS accumulation

assays

Proinflammatory cytokines (such as TNF-α and IL-β) secreted by microglia play

[39-41]

an important role in the pathogenesis and development of cognitive impairment

.

Therefore, BV-2 cells were co-incubated with 22 for 6 h followed with treatment of

LPS for another 24 h. The total RNA was isolated, purified, quantified and reversely

[

25,39a]

transcripted to cDNA according to the previous report by q-PCR

. The results in

Figure 7 showed that, under stimulation of LPS at concentration of 1 μg/mL and 3

μg/mL, expression of IL-1β was significantly increased to 2.22- and 3.68-fold, while

that of TNF-α was significantly increased to 2.51- and 3.86-fold when compared to

those of the control. This clearly indicates that LPS is capable of inducing

inflammation in BV-2 cells. However, when co-incubated with 22 (5 μM, 10 μM) and

LPS (3 μg/mL) for another 24 h, the expression of IL-1β in BV-2 cells decreased to

1

.36- and 0.86-fold in a concentration-dependent manner while the expression of

TNF-α decreased to 1.60- and 1.20-fold as comparison to control group (Figure 7).

Moreover, the expression level of both IL-1β and TNF-α was decreased to the same

level as that of untreated group, revealing that compound 22 acquired the

anti-inflammatory activity by regulating relative proinflammatory cytokines (TNF-α

and IL-β). Similarly, ibuprofen also showed the same anti-inflammatory effects by

maintaining the normal level of the expression of TNF-α and IL-1β.

The results in Figure 7 indicated that 22 modulates LPS-induced IL-1β and

TNF-α to the normal levels in LPS-stimulated BV-2 cells as comparison with that of

untreated cells, which may raise a question that whether the anti-inflammation effects

come from 22 itself or from the released ibuprofen from 22 under the stimulation of

LPS. No doubtfully, LPS elevated the intracellular ROS accumulation, which

[

39b]

thereafter modulates the expression of neurotoxic factors in microglia

. Besides,

disruption of the production or accumulation of intracellular ROS provides a method

[

39c]

to investigate the proinflammatory signals in cells

. Therefore, in order to

investigate this mechanism of anti-inflammation of 22, intracellular ROS

accumulation in BV-2 cells were studied. As shown in Figure S5, the intracellular

ROS levels in BV-2 cells treated with LPS (3 μg/mL) were significantly elevated

(Figure S5A2-S5C2), labeling as more cells were stained in strong green-florescence

as comparison with untreated-cells (Figure S5A1-S5C1). However, when

co-incubated with 22 (10 µM) and LPS (3 μg/mL) for another 18 h, ROS levels were

apparently decreased, illustrating that 22 regulated oxidative stress induced by LPS

(Figure S5A3-S5C3). Interestingly, the remarkable ROS accumulation in the cells

treated with combined group of ibuprofen (10 µM) and 11 (10 µM) indicated that both

ibuprofen and 11 did not detoxify against LPS by eliminating the ROS accumulation

(Figure S5A4-S5C4), though the cells were pretreated with the two components of 22

for 6 h. Moreover, BV-cells of 6 h pretreatment of LPS (3 μg/mL) and another 18 h

co-incubation with 22 (10 µM) were also in normal ROS levels condition when

compared with that of untreated cells (Figure S5A5- S5C5), revealing that 22 also

showed neuroprotective activity in a model of oxidative stress in BV-2 cells. Taken

the anti-inflammatory of 22 in LPS-treated cells in consideration, we believed that 22

rescued cells against cytotoxicity induced by LPS not only depending on the collapse

of 22 in oxidative condition but also on its ROS-eliminating capability.

2

.8. Apoptosis detection

On the basis of the promising neurons protection activity of 22 against H O , we

2

subsequently performed apoptosis assays by FACS to investigate whether the

neuroprotection was attributed to apoptosis inhibition. Firstly, the apoptosis of

SH-SY5Y cells induced by 22 was studied. As shown in Figure 8A-8D, 22 did not

exert obvious cytotoxicity against the cells after 24 h co-incubation with a cell

viability value over 90%, consistent with the result observed in Figure 3. In order to

evaluate the neuroprotective effect of 22 against H O , SH-SY5Y cells were incubated

2

with 22 (5 μM, 10 μM) for 6 h followed by another 24 h treatment of H O (100 μM).

2

Similarly, cells exposed to equal concentration H O were used as positive control.

2

The apoptotic results are given in Figure 8E-8H. Notably, the percentage of living

cells decreased from 93.60% (DMSO-treated group) to 41.7% (H O -treated group).

2

In comparison with H O , SH-SY5Y cells treated with 22 exhibited high percentage

2

of living cells from 73.70% of 22 (5 μM) to 86.60% of 22 (10 μM). Significantly, the

potency of 22 in protecting neurons from H O induced cytotoxicity presented in a

2

dose-dependent manner. In sum, these findings verify the results as obtained in

Figure 3 and the neuroprotective activity of compound 22.

2

.9. Hepatotoxicity study

Tacrine was the first AChEI approved for AD treatment, but it has been

withdrawn from the clinical use due to its hepatotoxicity. However, this drug

[

31,

molecule still deserves to be investigated for non-hepatotoxic tacrine derivatives

4

0-44]

.

It is learned that the hepatotoxicity induced by tacrine mainly resulted in

elevating the levels liver transaminase, decreasing albumin concentration and

[

31, 42-44]

inducing ROS in hepatocyte

. In order to investigate whether our target

compound can decrease the hepatotoxicity, compound 22 was used to for ROS

accumulation tests with tacrine as positive control. As shown in Figure 9, in

comparison to the control group, tacrine caused significant hepatotoxicity as

illustrated with more HepG2 cells in green florescence. Interestingly, less amount of

HepG2 cells in green florescence were detected when cells were treated with 22 (25

or 50 μM) for 24 h. Generally, the higher the accumulation of ROS was present, the

[

45,46]

more serious damage was induced to the cells

. Taken all of the results mentioned

above, compound 22 caused lower hepatotoxicity at a concentration of 25 or 50 μM

than tacrine which induced serious hepatotoxicity at 50 μM.

2

.10. Cell morphology assays under H O

2 2

In order to investigate the change of cell morphology, calcein AM/PI double

staining probes were used. SH-SY5Y cells were pre-incubated with 22 (10 μM) for 6

o

h at 37 C, using DMSO as control group. Subsequently, the cells in 22-treated group

and untreated group were incubated with H O (100 μM) for another 24 h and stained

2

with calcein AM/PI double staining probes. As shown in Figure 10, the cells without

any treatment are in spindle outline and mostly stained in green florescence (Figure

0A1), while little cells are stained in red florescence (Figure 10B1). However, after

treated with H O most cells become oval or irregular nucleus in apoptosis or death

1

2

state and are stained in red florescence by the PI probe (Figure 10A2-10C2).

Moreover, after pre-treatment with 22 (Figure 10B3), less cells with red florescence

are found as compared with H O -treated group (Figure 10A2-10C2), illustrating that

2

2 shows perfect neuroprotective activities against the apoptosis induced by H O .

2 2

Besides, the neuroprotective effects also confirmed the better anti-apoptosis activity

of 22 as evaluated in Figure 8.

2

.11. Western blotting assays

Given the great potential neuroprotective activities of 22 revealed above, we

subsequently explore the mechanism of its neuroprotective effects against cytotoxicity

induced by H O via western blotting technique. SH-SY5Y cells were pre-incubated

2

with DMSO, 22(5 μM, 10 μM), ibuprofen (10 μM) and ferulic acid (10 μM) for 6 h

and subsequently treated with H O (100 μM) for another 24 h. The total cytosolic

2

protein of SH-SY5Y cells was collected and subjected to western blotting assay with

β-actin as internal reference. As shown in Figure 11, levels of relative apoptosis

related proteins of Bcl-2, Bax and cleaved-caspase 3 in H O -treated group are

2

significantly changed in comparison to those of untreated-group, which clearly

revealed that H O can induce apoptosis through mitochondrial apoptosis pathway.

2

However, when cells co-incubated with 22 and H O , the levels of Bax and

2

cleaved-caspase 3 were decreased while Bcl-2 was up-regulated in contrast to those of

H O -treated group, indicating that 22 exerted neural protective effects by regulating

2

the expression of apoptotic protein and anti-apoptotic protein. Moreover, this

neuroprotective ability of 22 was in a concentration-dependent manner in regulating

related apoptotic proteins. As expected, the positive control (ferulic acid-treated cells)

also exerted the same neuroprotective effects by regulating the expression of relative

proteins.

2

.12. Morris Water Maze (MWM) assay

Lastly, we carried out morris water maze (MWM) test to investigate the

therapeutic effect of 22 on the learning and memory of AD model rats induced by

intrahippocampal aggregated Aβ25-35 (2 μL, 5 μg/μL). After another a week recovery

from the surgery, the rats were treated daily intraperitoneal injection of tacrine (1.0

µmol/100g b.wt., 0.2 mg/100g, qd), 22 (1.0 µmol/100g b.wt., 0.9 mg/100g, qd), using

equal amount of normal saline for control group. As shown in Figure 12, the escape

latency of rats in different groups had no significant difference of approximately 65 s.

During the following four days, rats of tacrine-treated group and 22-treated group

displayed a significant decrease in escape latency, especially for short searching

distance of tacrine-treated and 22-treated groups on the day five (Figure 12B).

Moreover, the shorter searching path of rats in 22-treated group revealed that 22

gained better memory improvement than tacrine. Not surprisingly, the escape latency

of the model group changed little in comparison to that of the first day. These results

apparently proved that 22 can ameliorate cognitive deficit induced by Aβ polymers.

Furthermore, the results in Figure 12C indicated that rats in the control group

swam aimlessly in the tank, whereas rats in tacrine-treated and 22-treated groups

preferentially swam in the northeast of the target quadrant. Similarly, rats in

2

2-treated group took more time (2.2 seconds) in the targeting platform than that of

the control or tacrine-treated rats (1.3 seconds), demonstrating that 22 has potential in

ameliorating cognitive deficit and improving spatial memory when compared with

tacrine-treated group. More excitingly, the more times (4.2) of 22-treated rats crossed

the platform than those of the control (0.4) and tacrine-treated rats (2.4) disclosed that

2

2 showed significantly therapeutic effect in ameliorating cognitive deficit and

improving spatial memory of AD model rats.

3

. CONCLUSION

In conclusion, a series of ROS-responsive prodrugs have been designed and

synthesized based on the clinic pathological characteristics of ROS overproduction,

inflammation and decrease of choline transmitters in brain. The prodrugs were

constructed and catered to our design with MTDLs strategies as a potential and novel

symptomatic- and disease-improving approach in AD treatments. The cytotoxicity

tests showed that the resulting compounds themselves had little cytotoxic activity

toward SH-SY5Y cells, but exerted excellent neuron-protective effects in the presence

of H O . Besides, little inhibitory activity on AChE was detected even at 10 μM,

2

whereas H O -treated 19-22 showed AChE inhibitory activity with IC values from

2

50

3

9.16 to 608.1 nM. Further examination indicated that upon treatment with H O , 22

2 2

can release 11 and ibuprofen, and show strong neuroprotective by inhibiting

proinflammatory cytokines (TNF-α and IL-1β) in LPS-treated microglial cells (BV-2)

and regulating apoptosis related proteins (Bax, Bcl-2 and cleaved caspase-3). In

addition, 22 showed neglected hepatotoxicity in HepG2 cells by inducing less

intracellular ROS than tacrine, demonstrating that this compound has better

biocompatibility. More importantly, compound 22 exhibited significantly therapeutic

effect in ameliorating cognitive deficit and improving spatial memory of Aβ-induced

AD model rats. Taken together, our research on these ROS-responsive prodrugs has

promise for AD treatments in future and offers a way for developing new AD drugs.

4

. EXPERIMENTAL SECTION

4

.1. Materials and Instruments

All the reagents and solvents were reagent grade with a minimum purity of 98%

purchased from Aladdin, Energy Chemical or Sinopharm Chemical Reagent Co., Ltd

and were used without further purification. All reactions were carried out under

1

nitrogen in anhydrous conditions, unless otherwise indicated. The H-NMR and

1

3

C-NMR were recorded with Bruker 600 MHz spectrometer in DMSO-d or CDCl₃

6

with TMS as the internal standard. Chemical shifts were obtained in parts per million

(

ppm) and spin multiplicities in NMR spectra were abbreviated as: s (singlet), d

doublet), t (triplet), m (multiplet) or br (broad). All of the intermediates and target

(

products were purified by recrystallization or silica gel chromatography. Silica gel

200-300 mesh) was purchased from Qingdao Haiyang Chemical Co. Ltd. and was

(

used without further treatment. Reactions were monitored by UV detector using thin

player chromatography (TLC) on silica gel plates (Yantai Jiangyou Silica Gel

Development Co., Ltd.). High-resolution mass spectrometry (HR-MS) were recorded

by an Agilent 6224 ESI/TOF MS instrument. All target compounds with purity of

9

5% or above were used in the following biological studies. The purity testes were

made with RP-HPLC (waters, e2695 system) equipped with ODS column (250×4.6

mm, 5 μm) and UV/Vis detector (2489 system, using wavelength of 254 nm), running

4

5:55 acetonitrile: water (V/V) (0.05% trifluoroacetic acid in water) at a flow rate of 1

mL/min. ROS tracker (KGAF018), mitochondrial membrane potential test kit

KGA604), and Annexin-V/PI double staining kit as well as the cells used were

(

purchased from KeyGEN BioTECH Corp. Primary antibodies of Bax, Bcl-2 and

cleaved capase-3 were obtained from Imgenex, USA. Secondary antibodies labeled

with horseradish peroxidase with a dilution of 1:2000 were from Santa Cruz

Biotechnologies. Primary antibody of anti-ZO-1 (ab216880) and secondary goat

anti-rabbit IgG H&L (ab150077) were from Abcam. QuantiChrom™ Peroxide Assay

Kit was purchased from BioAssay Systems, ROS tracker (DCFH-DA, S0033S,

Beyotime).

4

.2. Synthesis of Intermediates

,6-bis(((tert-butyldimethylsilyl)oxy)methyl)-4-methylphenol (13). To a solution

2

of compound 12 (4.0 g, 23.7 mmol) and imidazole (3.7 g, 54.5 mmol) in DMF (30

mL), TBDMSCl (7.9 g, 52.3 mmol) was added dropwise at 0 ℃ and kept stirring for 2

h at the same temperature. After the completion of the reaction, EA (300 mL) was

added to dissolve the residue. The resulting solution was washed with sat. saline

solution three times. The organic layer was dried with anhydrous Na SO and

2

4

concentrated in vacuum to give crude oil, which was isolated on silica gel

1

chromatography to give compound 13. (colorless oil, 8.4 g). Yield 90.0%. H NMR

(

600 MHz, CDCl ) δ 8.03 (s, 1H, Ar-OH), 6.91 (s, 2H, Ar-H), 4.83 (s, 4H, Ar-CH ),

3

2

.26 (s, 3H, Ar-H), 0.95 (s, 18H, C(CH ) ), 0.13 (s, 12H, Si(CH ) ).

3

3 2

(((5-methyl-2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)-1,3-p

henylene)bis(methylene))bis(oxy))bis(tert-butyldimethylsilane) (14). A solution of

compound 13 (4.2 g, 10.6 mmol) in DMF (15 mL) was cooled to 0 °C, then K CO

2

3

(2.8 g, 20.2 mmol) was added and stirred for 10 min at the same temperature before

the addition of 2-(4-bromobenzyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (3.0 g).

After the completion of the reaction monitored by TLC, EA (300 mL) was added to

dissolve the residue that was washed with sat. saline solution three times. The organic

layer was dried with anhydrous Na SO and concentrated in vacuum to give crude

2

4

colorless oil, which was purified by silica gel chromatography to obtain compound 14.

1

(

colorless oil, 4.8 g). Yield 75.1%. H NMR (600 MHz, CDCl ) δ 7.85 (d, J = 8.0 Hz,

3

2

4

H, Ar-H), 7.43 (d, J = 8.0 Hz, 2H, Ar-H), 7.18 (s, 2H, Ar-H), 4.90 (s, 2H, Ar-CH₂),

.71 (s, 4H, Ar-CH ), 2.35 (s, 3H, Ar-CH ), 1.37 (s, 12H, OC(CH ) ), 0.93 (s, 18H,

2

3

3 2

-

C(CH ) ), 0.08 (s, 12H, -Si(CH ) ).

3 3 3 2

(

5-methyl-2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)-1,3-ph

enylene)dimethanol (15). To a solution of compound 14 (1.1 g, 1.86 mmol) in MeOH

15 mL) was added PTSA (35.4 mg). The mixture was stirred at room temperature for

0 min, then the solvent was removed under reduced pressure after the completion of

(

3

the reaction. EA (150 mL) was added to dissolve the residue that was washed with sat.

saline solution three times. The organic layer was dried with anhydrous Na SO and

2

4

concentrated in vacuum. The residue was isolated by silica gel chromatography to

1

give compound 15 (colorless oil, 640.0 mg). Yield 89.5%. H NMR (600 MHz,

CDCl ) δ 7.82 (d, J = 7.9 Hz, 2H, Ar-H), 7.40 (d, J = 7.9 Hz, 2H, Ar-H), 7.13 (s, 2H,

3

Ar-H), 4.90 (s, 2H, Ar-CH ), 4.62 (s, 4H, Ar-CH ), 2.30 (s, 3H, Ar-CH ), 1.35 (s, 12H,

2

3

OC(CH ) )).

3

2

(

3-(((tert-butyldimethylsilyl)oxy)methyl)-5-methyl-2-((4-(4,4,5,5-tetramethyl-1,3,

-dioxaborolan-2-yl)benzyl)oxy)phenyl)methanol (16). To a solution of compound 15

1.5 g, 3.9 mmol) and imidazole (398.2 g, 5.9 mmol) in DMF (15 mL) was added

2

(

TBDMSCl (764.0 g, 5.1 mmol) dropwise at 0 °C. The mixture was stirred overnight

at room temperature. After the completion of the reaction monitored by TLC, EA

(150 mL) was added to dissolve the residue that was washed with sat. saline solution

three times. The organic layer was dried with anhydrous Na SO and concentrated in

2

4

vacuum to give crude oil, which was isolated on silica gel chromatography to obtain

1

compound 16. (colorless oil, 964.0 mg). Yield 49.6%. H NMR (600 MHz, CDCl ) δ

3

7

.97 (s, 1H, ), 7.85 (d, J = 7.9 Hz, 2H, Ar-H), 7.43 (d, J = 7.9 Hz, 2H, Ar-H), 7.22 (d,

J = 1.5 Hz, 1H, Ar-H), 7.10 (d, J = 1.7 Hz, 1H, Ar-H), 4.93 (s, 2H, Ar-CH ), 4.72 (s,

2

0

H, Ar-CH ), 4.63 (s, 2H, Ar-CH ), 2.33 (s, 3H, Ar-CH ), 1.36 (s, 12H, -OC(CH ) ),

2 2 3 3 2

.93 (s, 9H, -C(CH ) ), 0.09 (s, 6H, -Si(CH ) ).

3

3 2

3-(((tert-butyldimethylsilyl)oxy)methyl)-5-methyl-2-((4-(4,4,5,5-tetramethyl-1,3,2

-dioxaborolan-2-yl)benzyl)oxy)benzyl 2-(4-isobutylphenyl)propanoate (17).

A

solution of ibuprofen (1.48 g, 7.2 mmol) and DCC (1.9 g, 9.4 mmol) in anhydrous

DCM (25 mL) was stirred at room temperature for 20 min. Then, compound 16 (3.2 g,

6

.6 mmol) was added dropwise at the same temperature and stirred overnight. After

the completion of the reaction, the deposit was filtered out and the organic layer was

concentrated under reduced pressure. The residue was further isolated by silica gel

chromatography with the eluent of PE and EA (30:1-25:1) to obtain compound 17.

1

(

colorless oil, 3.4 g). Yield 75.6%. H NMR (600 MHz, CDCl ) δ 7.81 (d, J = 7.7 Hz,

3

2

H, Ar-H), 7.34 (d, J = 7.7 Hz, 2H, Ar-H), 7.23 (s, 1H, Ar-H), 7.17 (d, J = 8.0 Hz, 2H,

Ar-H), 7.04 (d, J = 8.0 Hz, 2H, Ar-H), 6.93 (s, 1H, Ar-H), 5.17-5.12 (m, 2H,

Ar-CH -O), 4.75 (s, 2H, Ar-CH -O), 4.65 (s, 2H, Ar-CH -O), 3.72 (q, J = 7.1 Hz, 1H,

2

Ar-CH-CO), 2.40 (d, J = 7.2 Hz, 2H, Ar-CH -CH), 2.27 (s, 3H, Ar-CH ), 1.83-1.77

2

3

(

m, 1H, -CH(CH ) ), 1.49 (d, J = 7.2 Hz, 3H, CH(CH ) ), 1.37 (s, 12H, -OC(CH ) ),

3 2 3 2 3 2

0

.92 (s, 9H, -C(CH ) ), 0.88 (s, 3H, -CH(CH ) ), 0.87 (s, 3H, -CH(CH ) ), 0.06 (s, 6H,

3 3 3 2 3 2

-

Si(CH ) ).

3 2

3

-(hydroxymethyl)-5-methyl-2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)

benzyl)oxy)benzyl 2-(4-isobutylphenyl)propanoate (18). To a solution of compound

7 (3.7 g, 5.5 mmol) in MeOH (25 mL) PTSA (104.0 mg) was added. The mixture

1

was stirred at room temperature for 30 min, the solvent was removed under reduced

pressure and EA (150 mL) was added. The resulting solution was washed three times

with sat. saline solution. The organic layer was dried with anhydrous Na SO and

2

4

concentrated in vacuum. The residue was isolated by silica gel chromatography to

1

give compound 18. (colorless oil, 1.9 g). Yield 63.0%. H NMR (600 MHz, CDCl )

3

δ 8.24 (d, J = 9.1 Hz, 2H, Ar-H), 7.81 (d, J = 7.9 Hz, 2H, Ar-H), 7.36 (d, J = 7.9 Hz,

2

1

5

H, Ar-H), 7.26 (d, J = 2.3 Hz, 1H, Ar-H), 7.25 (d, J = 2.3 Hz, 1H, Ar-H), 7.20 (d, J =

.8 Hz, 1H, Ar-H), 7.06 (d, J = 1.8 Hz, 1H, Ar-H), 5.23 (s, 2H, Ar-CH -O), 5.23 –

2

.14 (m, 2H, Ar-CH -O), 4.84 (s, 2H, Ar-CH -O), 3.74 (q, J = 7.1 Hz, 1H,

2

Ar-CH-CO), 2.38 (d, J = 7.2 Hz, 2H, Ar-CH -CH), 2.27 (s, 3H, Ar-CH ), 1.83 – 1.75

2

3

(

m, 1H, -CH(CH ) ), 1.50 (d, J = 7.2 Hz, 3H, -CH(CH )), 1.36 (s, 12H, -OC(CH ) ),

3 2 3 3 2

0

4

.87 (s, 3H, -CH(CH ) ), 0.86 (s, 3H, -CH(CH ) ).

3 2 3 2

.3. General preparations of compounds 19-22

To a solution of compound 18 (300 mg, 0.52 mmol) in DMF (5 mL) CDI (92.8

mg, 0.57 mmol) was added. The mixture was stirred at room temperature for 2 h, then

compound 8/9/10/11 (0.68 mmol) was added. After the resulting solution was kept

stirring for another 2 h, DCM (150 mL) was added. The organic phase was washed

three times with sat. saline solution, and then dried over anhydrous Na SO and

2

4

concentrated in vacuum. The residue was isolated by silica gel chromatography with

the eluent of DCM and MeOH (40:1-10:1) to generate target compounds 19-22.

5-methyl-3-((((2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)carbamoyl)oxy)me

thyl)-2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)benzyl2-(4-isobut

1

ylphenyl)propanoate (19). (pale yellow solid, 150 mg). Yield 34.3 %. H NMR (600

MHz, CDCl ) δ 14.40 (s, 1H, -NH-), 8.23 (d, J = 8.5 Hz, 1H, Ar-H), 8.11 (d, J = 8.5

3

Hz, 1H, Ar-H), 7.76 (d, J = 7.9 Hz, 2H, Ar-H), 7.35 (d, J = 7.9 Hz, 2H, Ar-H), 7.20 –

7

6

.15 (m, 2H, Ar-H), 7.14 (d, J = 8.0 Hz, 2H, Ar-H), 7.02 (d, J = 8.0 Hz, 3H, Ar-H),

.94 (s, 1H, Ar-H), 6.64 (s, 1H, -NH-), 5.15 (s, 2H, Ar-CH -O), 5.15 – 5.08 (m, 2H,

2

Ar-CH -O), 4.80 (s, 2H, Ar-CH -O), 4.07 (s, 2H, NHCH -), 3.71-3.67 (m, 3H,

2

Ar-CH-CO, NHCH -), 3.15 (s, 2H, -CH -), 2.45 (s, 2H, -CH -), 2.38 (d, J = 7.2 Hz,

2

H, Ar-CH -CH), 2.19 (s, 3H, Ar-CH ), 1.81-1.73 (m, 5H, -CH(CH ) , -CH -), 1.46

2 3 3 2 2

(

d, J = 7.2 Hz, 3H, -CH(CH )), 1.33 (s, 12H, -OC(CH ) ), 0.86 (s, 3H, -CH(CH ) ),

3 3 2 3 2

1

3

0

1

.85 (s, 3H, -CH(CH ) ); C NMR (150 MHz, CDCl ) δ 174.44, 158.65, 155.54,

3 2 3

53.10, 150.65, 140.62, 139.88, 138.28, 137.41, 134.96(C×2，Ar-H，overlapped

signals), 134.20, 131.72, 130.87, 130.61, 129.51, 129.47, 129.40, 129.34(C×2，Ar-H，

overlapped signals ), 127.19(C×2，Ar-H，overlapped signals), 126.89(C×2, Ar-H，

overlapped signals), 124.69, 124.40, 120.04, 115.42, 110.91, 83.87(C×2 ，

CH -C-(CH ) , overlapped signals), 62.21, 61.51, 50.66, 45.12, 44.97, 40.71, 30.13,

3

3 2

2

1

9.70, 28.33, 24.88(C×4, -CH ,overlapped signals ), 23.87, 22.39, 21.87, 20.79, 20.60,

3

+

8.35, 14.11；HR-MS(ESI) calcd for [M+H] = 840.4759, found 840.4827.

5-methyl-3-((((3-((1,2,3,4-tetrahydroacridin-9-yl)amino)propyl)carbamoyl)oxy)

methyl)-2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)benzyl2-(4-iso

1

butylphenyl)propanoate (20). (pale yellow solid, 134.4 mg). Yield 30.3 %. H NMR

(

600 MHz, CDCl3) δ 14.69 (s, 1H, -NH-), 8.45 (d, J = 8.5 Hz, 1H, Ar-H), 8.16 (d, J =

.5 Hz, 1H, Ar-H), 7.77 (d, J = 7.8 Hz, 2H, Ar-H), 7.61 (t, J = 7.8 Hz, 1H, Ar-H), 7.38

t, J = 7.8 Hz, 1H, Ar-H), 7.34 (d, J = 7.7 Hz, 2H, Ar-H), 7.32 (s, 1H, Ar-H), 7.15 (d,

J = 7.9 Hz, 2H, Ar-H), 7.03 (d, J = 7.9 Hz, 2H, Ar-H), 6.94 (s, 1H, Ar-H), 5.78 (s, 1H,

NH-), 5.16 – 5.09 (m, 4H, Ar-CH -O), 4.79 (s, 2H, Ar-CH -O), 3.88 (s, 2H,

8

(

-

2

NHCH -), 3.72-3.68 (m, 1H, Ar-CH-CO), 3.37-3.34 (m, 2H, -NHCH -), 3.27-3.24 (m,

2

H, -CH -), 2.66 (s, 2H, -CH -), 2.38 (d, J = 7.2 Hz, 2H, Ar-CH -CH), 2.20 (s, 3H,

2 2 2

Ar-CH ), 1.84-1.76 (m, 7H, -CH(CH ) , -CH -), 1.47 (d, J = 7.1 Hz, 3H, -CH(CH )),

3

3 2

2

3

13

1

.32 (s, 12H, -OC(CH ) ), 0.86 (s, 3H, -CH(CH ) ), 0.85 (s, 3H, -CH(CH ) ); C

3 2 3 2 3 2

NMR (150 MHz, CDCl ) δ 174.46, 157.75, 155.80, 153.29, 151.17, 140.62, 139.97,

3

1

38.61, 137.45, 134.97(C×2，Ar-H, overlapped signals), 134.08, 132.14, 130.80,

30.79, 129.61, 129.42, 129.38, 129.35(C×2，Ar-H, overlapped signals), 127.21(C×2，

1

Ar-H, overlapped signals), 126.89(C×2，Ar-H, overlapped signals), 125.21, 123.93,

1

4

2

20.79, 115.76, 111.29, 83.86(C×2, CH -C-(CH ) , overlapped signals), 62.13, 61.54,

3 3 2

5.15, 44.98, 43.70, 37.28, 31.24, 30.13, 28.46, 24.87(C×4, -CH , overlapped signals),

3

4.17, 22.39, 22.38, 22.04, 20.79, 20.66, 18.37, 14.13; HR-MS(ESI) calcd for [M

+

H] = 854.4916, found 854.5190.

5-methyl-3-((((4-((1,2,3,4-tetrahydroacridin-9-yl)amino)butyl)carbamoyl)oxy)me

thyl)-2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)benzyl2-(4-isobut

1

ylphenyl)propanoate (21). (pale yellow solid, 141.0 mg). Yield 31.2 %. H NMR (600

MHz, DMSO) δ 13.89 (s, 1H, -NH-), 8.37 (d, J = 8.5 Hz, 1H, Ar-H), 7.94 (d, J = 8.5

Hz, 1H, Ar-H), 7.83 (t, J = 7.7 Hz, 1H, Ar-H), 7.73 (s, 1H, Ar-H), 7.66 (d, J = 7.8 Hz,

2

5

6

H, Ar-H), 7.54 (t, J = 7.7 Hz, 1H, Ar-H), 7.35 (d, J = 7.8 Hz, 2H, Ar-H), 7.28 (t, J =

.6 Hz, 1H, Ar-H), 7.14 (d, J = 8.1 Hz, 2H, Ar-H), 7.04 (d, J = 8.0 Hz, 2H, Ar-H),

.92 (s, 1H, -NH-), 5.09 (d, J = 12.6 Hz, 1H, Ar-CH -O), 5.04 (d, J = 12.5 Hz, 1H,

2

Ar-CH -O), 4.97 (s, 2H, Ar-CH -O), 4.76 (s, 2H, Ar-CH -O), 3.84-3.82 (m, 2H,

2

NHCH -), 3.79 – 3.75 (m, 1H, Ar-CH-CO), 3.01-2.98 (m, 4H, -NHCH -, -CH -), 2.64

2

(s, 2H, -CH -), 2.35 (d, J = 7.1 Hz, 2H, Ar-CH -CH), 2.16 (s, 3H, Ar-CH ), 1.81-1.80

2 2 3

(

m, 4H, -CH ), 1.77-1.68 (m, 3H, -CH -), 1.47 – 1.43 (m, 2H, -CH -), 1.36 (d, J = 7.1

2

Hz, 3H, -CH(CH )), 1.30 (s, 12H, -OC(CH ) ), 0.81 (s, 3H, -CH(CH ) ), 0.81 (s, 3H,

3

3 2

1

3

-

CH(CH ) ). C NMR (150 MHz, CDCl ) δ 174.48, 156.62, 155.50, 153.22, 151.17,

3 2 3

1

40.62, 139.92, 138.67, 137.43, 134.97(C×2，Ar-H, overlapped signals), 134.06,

32.20, 130.79, 130.68, 129.77, 129.34(C×3，Ar-H, overlapped signals), 127.20(C×3，

Ar-H, overlapped signals ), 127.00(C×2，Ar-H, overlapped signals), 125.12, 124.30,

1

6

2

20.71, 115.72, 110.84, 83.86(C×2, CH -C-(CH ) , overlapped signals), 61.70,

3 3 2

1.55(C×2, Ar-CH -O, overlapped signals), 45.14, 44.97, 40.20, 31.58, 30.13, 28.37,

2

7.96, 27.04, 24.87(C×4, -CH , overlapped signals), 22.38(C×2,-CH -, overlapped

3

2

+

signals), 21.89, 20.79, 20.59, 18.35, 14.14; HR-MS(ESI) calcd for [M +H] =

68.5072, found 868.5058.

-methyl-3-((((6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)carbamoyl)oxy)m

8

5

ethyl)-2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)benzyl2-(4-isobu

1

tylphenyl)propanoate (22). (pale yellow solid, 116.4 mg). Yield 25.0 %. H NMR

(

600 MHz, CDCl ) δ 15.10 (s, 1H, -NH-), 8.53 (d, J = 8.5 Hz, 1H, Ar-H), 8.18 (d, J =

3

8

7

.5 Hz, 1H, Ar-H), 7.78 (d, J = 7.7 Hz, 2H, Ar-H), 7.66 (t, J = 7.7 Hz, 1H, Ar-H),

.41 (t, J = 7.7 Hz, 1H, Ar-H), 7.33 (d, J = 7.7 Hz, 2H, Ar-H), 7.14 (d, J = 7.9 Hz, 2H,

Ar-H), 7.12 (s, 1H, Ar-H), 7.03 (d, J = 7.9 Hz, 2H, Ar-H), 6.94 (s, 1H, Ar-H), 5.99 (s,

1

2

3

2

1

H, -NH-), 5.16 – 5.05 (m, 4H, Ar-CH -O), 4.89 (t, J = 5.7 Hz, 1H, NH-CH ), 4.77 (s,

2 2

H, Ar-CH -O), 3.90-3.87 (m, 2H, -NH-CH -), 3.70 (q, J = 7.1 Hz, 1H, Ar-CH-CO),

2

.29 (t, J = 6.1 Hz, 2H), 3.19-3.15 (m, 2H, -CH -), 2.58 (t, J = 5.8 Hz, 2H, -CH -),

2

.38 (d, J = 7.2 Hz, 2H, Ar-CH -CH), 2.21 (s, 3H, Ar-CH ), 1.80-1.80 (m, 2H, -CH -),

2

3

2

.84 – 1.77 (m, 6H, -CH -), 1.47-1.46 (m, J = 7.2 Hz, 7H, -CH -), 1.34 (s, 12H,

2

13

-

OC(CH ) ), 0.86 (s, 3H, -CH(CH ) ), 0.85 (s, 3H, -CH(CH ) ); C NMR (150 MHz,

3 2 3 2 3 2

CDCl ) δ 174.44, 156.48, 155.46, 153.38, 151.30, 140.60, 139.94, 138.85, 137.47,

3

1

34.98(C×2，Ar-H), 134.05, 132.24, 130.97, 130.81, 129.79(C×2，Ar-H), 129.40,

1

29.34(C×2，Ar-H), 127.21(C×2，Ar-H), 126.98(C×2，Ar-H), 125.12, 124.27, 120.95,

15.78, 110.74, 83.85(C×2, CH -C-(CH ) , overlapped signals), 61.73, 61.58(C×2,

3

3 2

Ar-CH -O-, overlapped signals), 45.15, 44.99, 40.59, 30.91, 30.12, 29.81, 28.38,

2

1

6.03, 26.00, 24.88(C×4,-CH ,), 23.80, 22.40, 22.38, 21.93, 20.78, 20.65, 18.37,

3

+

4.13; HR-MS(ESI) calcd for [M +H] = 896.5585, found 896.5562.

6-((2-oxo-2H-chromen-7-yl)oxy)hexanoic acid (24). To a solution of compound

2

3 (1.0 g, 6.17 mmol) in DMF (10 mL), K CO (3.4 g, 24.8 mmol), 6-bromohexanoic

2

3

acid (2.4 g, 12.34 mmol) and catalytic amount of KI were added. The resulting

o

solution was stirred at 55 C for 6 h, then the solvent was removed under reduced

pressure. The residue was isolated on silica gel with the eluent of DCM and MeOH

1

(

80:1-50:1) to produce compound 24. Pale yellow solid (1.3 g). Yield 76.5%. H

NMR (600 MHz, DMSO) δ 12.00 (s, 1H, -COOH), 7.98 (d, J = 9.5 Hz, 1H, Ar-H),

.61 (d, J = 8.6 Hz, 1H, Ar-H), 6.97 (d, J = 2.4 Hz, 1H, Ar-H), 6.93 (dd, J = 8.6, 2.4

Hz, 1H, Ar-H), 6.27 (d, J = 9.5 Hz, 1H, Ar-H), 4.06 (t, J = 6.5 Hz, 2H, Ar-O-CH -),

7

2

.23 (t, J = 7.4 Hz, 2H, -CH -COOH), 1.76 – 1.71 (m, 2H, -CH -), 1.6-1.54 (m, 2H,

2 2

-

CH -), 1.45-1.39 (m, 2H, -CH -).

2 2

3-(((2-(4-isobutylphenyl)propanoyl)oxy)methyl)-5-methyl-2-((4-(4,4,5,5-tetramet

hyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)benzyl6-((2-oxo-2H-chromen-7-yl)oxy)hexan

oate (25). DCC (174.5 g, 0.84 mmol) was added to a solution of compound 24 (180.0

mg, 0.65 mmol) in DCM (15 mL). The mixing solution was stirred for 10 min until

white solids precipitated. Then, compound 17 (372.1 mg, 0.65 mmol) and catalytic

amount of DMAP was added to the solution which was stirred for another 2 h. After

removal of the precipitate, the product was isolated with silica gel chromatography

with the eluent of EA and PE (10:1-5:1) as compound 25 (150 mg, white solid). Yield

1

2

7.8 %. H NMR (600 MHz, CDCl ) δ 7.80 (d, J = 8.0 Hz, 2H, Ar-H), 7.62 (d, J = 3.9

3

Hz, 1H, Ar-H), 7.35 – 7.34 (m, 3H, Ar-H), 7.16 (d, J = 8.0 Hz, 2H, Ar-H), 7.13 (d, J

1.9 Hz, 1H, Ar-H), 7.04 (d, J = 8.0 Hz, 2H, Ar-H), 6.98 (d, J = 1.9 Hz, 1H, Ar-H),

.81-6.79 (m, 1H, Ar-H), 6.78 (d, J = 2.3 Hz, 1H, Ar-H), 6.24 (d, J = 9.4 Hz, 1H,

Ar-H), 5.17 – 5.11 (m, 2H, Ar-CH -O), 5.11 (s, 2H, Ar-CH -O), 4.80 (s, 2H,

=

6

2

Ar-CH -O), 3.97 (t, J = 6.4 Hz, 2H, -O-CH - ), 3.71 (q, J = 7.1 Hz, 1H, Ar-CH(CH )),

2

3

2

.40 (d, J = 7.2 Hz, 2H, Ar-CH -CH(CH ) ), 2.34 (t, J = 7.5 Hz, 2H, OOCCH ), 2.24

2 3 2 2

(

s, 3H, Ar-CH -), 1.83-1.76 (m, 3H, -CH -, -CH(CH ) ), 1.71-1.66 (m, 2H, -CH -),

3 2 3 2 2

1

.51 – 1.46 (m, 5H, -CH -, Ar-CH(CH )), 1.35 (s, 12H, -OC(CH ) ), 0.87 (d, J = 0.9

2 3 3 2

1

3

Hz, 3H, -CH(CH ) ), 0.86 (d, J = 0.9 Hz, 3H, -CH(CH ) ). C NMR (150 MHz,

3

2

3 2

CDCl ) δ 174.45, 173.26, 162.31, 161.32, 155.91, 153.72, 143.48, 140.61, 139.95,

3

1

37.52, 135.03(C×2, Ar-H, overlapped signals), 134.11, 131.29, 131.16, 129.53,

29.36(C×2, Ar-H, overlapped signals), 129.27, 128.74(C×2, Ar-H, overlapped

1

signals), 127.24(C×2, Ar-H, overlapped signals), 126.88(C×2, Ar-H, overlapped

signals), 112.98, 112.94, 112.45, 101.37, 83.86(C×2, CH3-C(CH ) , overlapped

3

2

signals), 76.99, 68.28, 61.63, 61.51, 45.19, 45.01, 34.13, 30.15, 28.67, 25.57,

2

4.90(C×4, -CH , overlapped signals), 24.58, 22.42, 22.40, 20.78, 18.39.

3

4

.4. Cell Culture

SH-SY5Y, BV-2, and b.End.3 cells were maintained in RPMI 1640 or

Dulbecco’s Modified Eagle Medium (DMEM, Life Technologies) supplemented with

0 % fetal bovine serum (FBS, Gibco), penicillin (100 U/mL) and streptomycin (100

μg/mL) solution. All cells were grown at 37 °C in 5% CO atmosphere and 95% air.

1

2

4

.5. Cytotoxicity assay

The cytotoxicity was measured with MTT methods. SH-SY5Y cells were seeded

5

into 96-well plates at a density of 1×10 cells/mL and allowed to adhere for overnight.

Then compounds of 19-22 and tacrine at different concentrations in DMSO were

added to the well and co-incubated for 24 h, 48 h or 72 h at an atmosphere of 5 %

CO and 95 % air. Subsequently, MTT (5 mg/mL) was added for an extra 4 h of

2

incubation. The medium was replaced with DMSO to dissolve formazan precipitate

and the absorbance of OD was recorded with enzyme-labeling instrument at a

wavelength of 570 nm. The results of cell viabilities were analyzed with SPSS

software for three independent experiments. Neuroprotective assays of 19-22 were

carried out followed the procedures mentioned above. Briefly, SH-SY5Y cells were

pre-incubated with 19-22 (5, 10, 20 μM), ibuprofen (5, 10, 20 μM) and combined

group (ibuprofen and 11, 5, 10, 20 μM) for 6 h followed with another 24h treatment of

H O . The results obtained through MTT assays were shown as mean ± SD of three

2

independent experiments.

4

.6. Release of ibuprofen and tacrine derivative 11

A solution of 22 (40 μmmol/L) was prepared in purified water and acetonitrile

(

50 % : 50 %, V/V), then the solution of 22 and H O (3.0 equiv.) were co-incubated

2 2

o

at 37 C in dark. The release process was recorded and analyzed with reversed phase

high performance liquid chromatography (RP-HPLC, waters, e2695 system; 2489

UV/Vis detector). If the process slowed down, extra portion of H O (3.0 equiv.) was

2

added until the completion of release. Similarly, other solutions of ibuprofen (40

μmmol/L), 11 (10 μmmol/L), and 12 (40 μmmol/L) were prepared in purified water

and acetonitrile (50%:50%, V/V). All of the tests were detected on ODS column

(250×4.6 mm, 5 μm) at different time points with an eluent of acetonitrile/water

(10:90-100:0, 16 min; 100:0, 20 min; 50:50, V/V) (0.05 % trifluoroacetic acid in

water). The volume of sample injection was 10 μL and the flow rate was 1 mL/min

o

with column oven of 35 C. All solutions prefiltered with 0.45 µm filter were detected

using UV detector under 254 nm.

4

.7. In vitro H O -eliminating capability

2 2

Various concentrations of trolox (1,5,10, 20, 30 µM), 22 (1,5,10, 20, 30 µM),

ibuprofen (1,5,10, 20, 30 µM) and 11 (1,5,10, 20, 30 µM) were incubated in 4 mL

o

PBS (7.4) containing 200 µM H O at 37 C for 24 h. The remaining H O in various

2

reaction solutions were detected via QuantiChrom Peroxide Assay Kit (DIOX-250,

BioAssay Systems) while the scavenged H O was calculated by graph pad prism 8.0.

2

The relationship between consuming H O and concentrations of various compounds

2

were graphed via origin pro 2016.

4

.8. Blood-brain barrier (BBB) permeation assays and cellular uptake

As for the blood-brain barrier permeation assays, b.End.3 cells at a density of

5

2

×10 cells/cm were seeded into the upper chamber of transwell while SH-SY5Y

5

2

cells (1×10 cells/cm ) were seeded into the lower chamber of transwell. All of the

cells were cultured in monolayer. Trans-Endothelial electrical resistance (TEER)

values of cells in upper chamber were monitored with voltammeter (Millipore) and

2

used for BBB assays until the TEER values reached more than 180 Ω cm . Thereafter,

•

2

2 and 25 (10 μM) were added to the upper chamber, respectively and incubated for 2

o

h at 37 C. The concentration of 22 in the lower culture medium was analyzed with

HPLC. After another 6 h co-incubation, SH-SY5Y cells were collected, lysed with

RIPA lysis buffer and cytoplasm was collected for HPLC detection. The concentration

of 22 in SH-SY5Y cells calculated with Graph Pad Prism 8.0 and showed as one of

three independent experiment. TEER= (R -R )*A, R and R represent the total

t

b

t

b

resistance and background resistance, respectively, and A is the transwell area (0.33

2

cm ). As for 25, the cells in lower chamber were washed twice with cold PBS and

gathered for images with laser confocal microscope.

As for uptake of 25, SH-SY5Y or BV-2 cells were seeded onto six-well plates

5

o

(

1×10 cells/mL) and allowed to adhere at 37 C for overnight. After 80 % confluence,

o

the cells were treated with compound 25 (10 μM) for 12 h at 37 C. Subsequently, the

cells were washed twice with PBS, recorded and analyzed with laser scanning

confocal microscope. (Ex. 405 nm, Em. 488 nm).

4

.9. Immunofluorescence assays of ZO-1

5

BEnd.3 (5×10 cells/mL) cells were seeded into glass bottom cell culture dishes

till the formation of monomer cells. Then the cells were treated with DMSO or 22 for

5 min. After that, the cells were washed with cold PBS, fixed in paraformaldehyde

4%) and permeabilized with 0.5% Triton X-100 in PBS. Then, the cells were labeled

4

(

with primary antibodies (ab216880, abcam) followed with Alexa-Fluoro488-labeled

goat anti-rabbit secondary antibody. The nuclei were stained with DAPI, while images

were recorded and analyzed via laser confocal microscope. The results were shown as

one of three independent experiments.

4

.10. LPS-induced ROS accumulation assays

BV-2 cells were seeded onto six-well plates (1×10 cells/mL) and allowed to

adhere overnight. After 80 % confluence, the cells were pretreated with compound 22

10 μM), ibuprofen+11 (10 µM + 10 µM) for 6 h followed with another 18 h

5

(

co-treatment with LPS (3 μg/mL). Similarly, untreated cells (DMSO) and LPS (3

μg/mL)-treated cells were used as controls. BV-2 cells pretreated with LPS (3 μg/mL)

for 6 h followed with another 18 h co-incubation with 22 (10 µM) was also used as

o

control. After 24 h co-treatment at 37 C, the cells were trypsinized, collected for

DCFH-DA staining at dark for 20 min and imaged via laser confocal microscope with

the excitation wavelength and emission wavelength of 488 nm and 530 nm. Results

were displayed as one of three independent experiments.

4

.11. AChE inhibition study

The AChE inhibition was investigated with colorimetric method according to the

[32,34]

research previously reported by Ellman et al

. The details of materials were as

follows: AChE (EC3.1.1.7, Type VI−S, Electric Eel); (5,5'-Dithiobis-(2-nitrobenzoic

acid)) DTNB (pH=8.0 Gluco Biotechnology Co., Ltd, Nanjing); acetylthiocholine

chloride (ATC, Sigma Aldrich); tacrine (Sigma Aldrich). Tacrine and target

-9

-5

compounds were dissolved in DMSO and diluted to 10 -10 M with 0.1 M

NaH PO /Na HPO buffer (pH 7.4). As for the measurement of AChE assay of target

2

4

2

4

compounds under H O . Compounds 19-22 was dissolved in DMSO and diluted with

2

PBS buffer to 40 mM followed with the addition of H O (6.0 equiv.) and

2

o

co-incubated for 10 h at 37 C in dark. The release solution was diluted with PBS

buffer to a final concentration range and used for the following assays.

For measurement, the AChE was diluted to 2.0 units/mL in 2 mL aliquots. After

5

min co-incubation of contained PBS (1 mL), AChE (10 μL), test compound solution

o

(

10 μL) and DTNB (0.01 M, 30 μL) at 25 C, the reaction was started with

o

acetylthiocholine chloride (0.01 M, 30 μL) and incubated for 1 min at 37 C. The

absorbance of various solution was recorded with enzyme-labeled instrument at 410

nm for three independent experiments. Similarly, pure water was tested as control.

The IC values were calculated with Graph Pad Prism 6.0.

5

0

4

.12. Molecular docking studies

The moleular docking were performed with Sybyl-X 2.0 (Sybyl-X 2.0 was

available from state key laboratory for the chemistry and molecular engineering of

medicinal resources of guangxi normal university). The structures used in this

stdudies were constructed with chem 3D pro 14.0 followed by ennergy minimized

with tripos force field, using optimization gradient of 0.001 kcal/mol·Å and a

maximum optimal step size of 1000. The crystal structure of torpedo californica

AChE (PDB ID: 2CKM) was obtained from the RCSB protein data bank and

modified with Sybyl-X 2.0. For preparing receptor protein, the water molecules were

removed and the missing hydrogen atoms were added. The docking results were

obtained in Sybyl-X 2.0 Surflex-Dock model. Besides, the docking parameters were

kept as default values.

4

.13. The detection of inflammatory factors in BV-2 cells by real-time quantitative

PCR (qRT-PCR)

The effects of 22 on expression of inflammatory factors induced by H O were

2

detected by real-time quantitative PCR (qRT-PCR). BV-2 cells were seeded onto

5

o

six-well plates (1×10 cells/mL) and allowed to adhere at 37 C for overnight. When

cells were 80% confluence, the cells were pre-treated with or without 22 (5 μM, 10

μM) for 6 h and treated with H O (100 μM) for another 24 h at the same condition.

2

The total RNA isolated with TRIzol reagent (Invitrogen Co., Carlsbad, CA), purified

with chloroform and isopropanol and quantified with the NanoDrop 2000c

spectrophotometer (ThermoFisher Scientific, Madrid, Spain) at 260/280 wavelength.

After reverse transcription cDNA by a High Capacity RNA-to-cDNA kit (TOYOBO,

Japan), real-time quantitative PCR was performed with 10 μL of final reaction volume

including SYBR green I master mix. The method of amplification was carried out

[

47]

according to the previous report

and the results were analyzed with ΔΔCt method

[

48]

.

The primer sequences were used as follows: IL-1β R-AGC TCT CCA CCT CAA

TGG AC; TNF-α R-CTG TAG CCC ACG TCG TAG; GAPDH R-CAC TGA GGA

CCA GGT TGT CT

4

.14. Apoptosis detection

The neuroprotective effects of compound 96 were studied using apoptosis assay.

5

SH-SY5Y cells were seeded onto six-well plates (1×10 cells/mL) and allowed to

o

adhere at 37 C for overnight. The cells were pretreated with control (DMSO),

o

compound 96 (5 μM and 10 μM) for 6 h at 37 C. Then the cells were treated H O

2

(100 μM) and incubated for another 24 h at the same condition. Then both the

adherent and floating cells were collected, centrifuged (2000 rpm for 5 min) and

washed twice with cold PBS. Lastly, the cells were resuspended with 500 μL binding

buffer, stained with Annexin V-FITC (5 μL) and propidium iodide (5 μL) for 15 min

at room temperature and analyzed with flow cytometry (Cell Quest; BD Biosciences).

4

.15. Hepatotoxicity Studies

In order to investigate whether our target copunds obtained decreased

hepatotoxicity in coparison to tacrine. ROS accumulation assays induced by 22 or

5

tacrine were measured. Briefly, Hep G2 were seeded onto six-well plates (1×10

o

cells/mL) and allowed to adhere at 37 C for overnight. Subsequently, the cells were

treated with control (DMSO), compound 22 (25 μM and 50 μM) and tacrine (50 μM)

o

at 37 C for another 24 h. Thereafter, the cells were washed twice with cold PBS,

stained with ROS tracker followed by washing with PBS. The accumulation of ROS

was recorded and analyzed with laser confocal microscope (Ex. 490 nm/Em. 535 nm).

4

.16. Cell morphology assay

The cell morphology assay was detected with cytotoxicity assay kit (KGAF001,

5

KeyGEN BioTECH). SH-SY5Y cells were seeded onto six-well plates (1×10

o

cells/mL) and allowed to adhere at 37 C for overnight. Then the cells were treated

o

with control (DMSO), compound 22 (5 μM and 10 μM) for 6 h at 37 C. Subsequently,

the cells were treated with H O (100 μM) and incubated for another 24 h at the same

2

condition. Lastly, the cells were stained with calcein AM/PI probe prepared according

to the illustration of the manufacture for 30 min in dark at room temperature. The

cells were washed twice with cold PBS and analyzed with laser confocal microscope.

4

.17. Western blotting assay

In order to investigate the expression of related protein in the mitochondrial

apoptosis pathway, the western blotting study was performed. SH-SY5Y cells were

5

seeded into six-well plates at a density of 1×10 cells/mL and allowed to adhere at 37

o

C for overnight. Thereafter, the cells were pre-incubated with different conditions,

including 22 (5 μM and 10 μM), control (DMSO) for 6 h. Then the cells were exposed

to H O (100 μM) (one group of control was not treated) and incubated for another 24

2

Article Doi

ROS-responsive and multifunctional anti-Alzheimer prodrugs: Tacrine-ibuprofen hybrids via a phenyl boronate linker

DOI: 10.1016/j.ejmech.2020.112997

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.ejmech.2020.112997

Full text of DOI:10.1016/j.ejmech.2020.112997

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