Synthesis, pharmacological evaluation and mechanistic study of scutellarin methyl ester -4′-dipeptide conjugates for the treatment of hypoxic-ischemic encephalopathy (HIE) in rat pups

B i o o r g an i c C h e m i s tr y 1 0 1 (2 020 )1 03980

Contents lists available at ScienceDirect

Bioorganic Chemistry

journal homepage: www.elsevier.com/locate/bioorg

Synthesis, pharmacological evaluation and mechanistic study of scutellarin

methyl ester -4′-dipeptide conjugates for the treatment of hypoxic-ischemic

encephalopathy (HIE) in rat pups

Tao Li^a,b,d,1, Dirong Wu^a,b,d,1, Yang Yang^e, Tao Xiao^a,b,d, Yilin Han^a,b,d, Jing Li^a,b,d, Ting Liu^a,b,c

,

⁎

Li Li^a,b, Zeqin Dai^a,b, Yongjun Li^a,c,d, Xiaozhong Fu^a,b,d,

^aState Key Laboratory of Functions and Applications of Medicinal Plants, Guizhou Medical University, Guiyang 550004, Guizhou, China

^bSchool of Pharmacy, Guizhou Medical University, Guiyang 550004, China

^cGuizhou Provincial Key Laboratory of Pharmaceutics, Guizhou Medical University, Guiyang 550004, China

^dNational Engineering Research Center of Miao's Medicines & Engineering Research Center for the Development and Application of Ethnic Medicine and TCM, Ministry of

Education, Guiyang 550004, Guizhou, China

^eThe Second People's Hospital of Jiangyou City, Jiangyou City 621701, Sichuan Province, China

A R T I C L E I N F O

A B S T R A C T

Keywords:

A series of novel scutellarin methyl ester-4′-dipeptide conjugates exhibiting active transport characteristics and

protection against pathological damage caused by hypoxic-ischemic encephalopathy (HIE) were successfully

designed and synthesized. The physiochemical properties of the obtained compounds, as well as the Caco-2 cell-

based permeability and uptake into hPepT1-MDCK cells were evaluated using various analytical methods.

Scutellarin methyl ester-4′-Val-homo-Leu dipeptide (5k) was determined as the optimal candidate with a high

Hypoxic-ischemic encephalopathy

Scutellarin

Oligopeptide transporter 1

Dipeptide conjugates

Pathomorphological damage

apparent permeability coeﬃcient (P_{app A to B}) of 1.95

0.24 × 10⁻⁶cm/s, low ER (P_{app BL to AP}/P_{app AP to BL}) of

0.52 in Caco-2 cells, and high uptake of 25.47 μmol/mg/min in hPepT1-MDCK cells. Comprehensive mechanistic

studies demonstrated that pre-treatment of PC12 cells with 5k resulted in more potent anti-oxidative activity,

which was manifested by a signiﬁcant decrease in the malondialdehyde (MDA) and reactive oxygen species

(ROS) levels, attenuation of the H₂O₂-induced apoptotic cell accumulation in the sub-G1 peak, and improvement

in the expression of the relevant apoptotic proteins (Bcl-2, Bax, and cleave-caspase-3). Moreover, evaluation of in

vivo neuroprotective characteristics in hypoxic-ischemic rat pups revealed that 5k signiﬁcantly reduced in-

farction and alleviated the related pathomorphological damage. The compound was also shown to ameliorate

the neurological deﬁcit at 48 h as well as to decrease the brain tissue loss at 4 weeks. Conjugate 5k was de-

monstrated to reduce the amyloid precursor protein (APP) and β-site APP-converting enzyme-1 (BACE-1) ex-

pression. Pharmacokinetic characterization of 5k indicated favorable druggability and pharmacokinetic prop-

erties. The conducted docking studies revealed optimal binding of 5k to PepT1. Hydrogen bonding as well as

cation-π interactions with the corresponding amino acid residues in the target active site were clearly observed.

The obtained results suggest 5k as a potential candidate for anti-HIE therapy, which merits further investigation.

1. Introduction

development of immature brain and subsequently leads to long-term

disabilities, such as epilepsy, mental retardation, cerebral palsy, and

Brain diseases, including Alzheimer’s disease (AD), vascular de-

mentia (VD), and Parkinson’s disease (PD) are accompanied by cogni-

tive impairments, such as deﬁcits in learning, memory, language, per-

ception, and intellect [1]. Previous clinical and animal experiments

have revealed that hypoxic-ischemic encephalopathy (HIE) is a major

cause of morbidity and mortality in infants [2]. HIE aﬀects the proper

behavioral diﬃculties [2–3]. Moreover, HIE causes delayed cell death

via excitotoxicity, inﬂammation, and oxidative stress [4–7]. Thus, de-

velopment of drugs able to improve brain damage and cognitive im-

pairment caused by HIE is essential for the prevention and treatment of

broad vascular cognitive impairment and dementia [8].

Scutellarin (4′,5,6-trihydroxyﬂavone-7-glucuronide, Scu), (Fig. 1) is

^⁎Corresponding author at: School of Pharmacy, Guizhou Medical University, Guiyang 550004, China.

E-mail address: xiaozhong_fu@sina.com (X. Fu).

¹Tao Li and Dirong Wu contributed equally to this work, they are the co-ﬁrst authors.

https://doi.org/10.1016/j.bioorg.2020.103980

Received 11 February 2020; Received in revised form 13 April 2020; Accepted 29 May 2020

A v a i l a b l e o n l i n e 0 1 J un e 2 020

T. Li, et al.

BioorganicChemistry101(2020)103980

Fig. 1. Designed target compounds scutellarin methyl ester-4′-dipeptide conjugates.

an active ingredient extracted from traditional Chinese medicine Erigeron

breviscapus (Vant) Hand–Mazz, which has been clinically used in China to

treat acute cerebral infarction and paralysis induced by cerebrovascular

diseases, including hypertension, cerebral thrombosis, and cerebral he-

morrhage since 1984 [9–10]. So far, various studies have identiﬁed the

anti-oxidative eﬀects of scutellarin against oxidative stress caused by

hydrogen peroxide, glutamate, and hypoxia [10–13]. There is also

growing evidence, which suggests that scutellarin exhibits neuroprotec-

tive eﬀects against focal cerebral ischemia induced by middle cerebral

artery occlusion (MCAO). These results can be attributed to the inhibitory

eﬀects of this compound on microglial activation in neuroinﬂammation,

as well as to anti-oxidative, anti-inﬂammatory, and anti-apoptotic activ-

ities [14–17]. More recently, it has been revealed that scutellarin can also

ameliorate learning and memory impairment under chronic cerebral

hypoperfusion induced by permanent bilateral common carotid artery

occlusion (pBCAO). This observation is suggested to be a consequence of

the reduction in the Aβ formation by inhibition of the amyloid precursor

protein (APP) and β-site APP-converting enzyme-1 (BACE-1) production

[18]. Although the above ﬁndings demonstrate that scutellarin exhibits

potent protective activities against brain injury and a remarkable ther-

apeutic potential, so far only few attempts have been made to explore

utilization of scutellarin for the treatment of pathological damage in a HIE

model. Accordingly, it is essential to explore whether scutellarin displays

therapeutic eﬀects in the treatment of HIE and to evaluate its underlying

mechanism. The challenges of using scutellarin clinically include an un-

favorable ADME proﬁle, i.e., poor solubility in both water and lipids, low

blood–brain barrier (BBB) permeability, very low absolute bioavailability,

and metabolic instability [19–23].

Over the past decade, the human intestinal oligopeptide transporter

1 (PepT1), has been widely explored as a target for increasing the oral

bioavailability of poorly absorbed drugs [24,25]. PepT1 is a promising

and valuable target protein, responsible for the uptake of dipeptides

and tripeptides. Several dipeptide-drug conjugate were synthesized,

such as Gly-Val-Acyclovir, Val-Trp-Acyclovir [26], Val-Ala-Oleanolic

Acid [27], Pro-Ile-Zidovudine [28] and they appeared to combine both

chemical stability and good aﬃnity for the PepT1 transporter, the

peptide carrier strategy applied to improve oral absorption and bioa-

vailability via targeting the PepT1 transporter.

In recent years, to overcome disadvantages of scutellarin, a prodrug

strategy has been used to modify the structure of Scu. The approach

involves introduction of long aliphatic chain; ethyl, benzyl, N,N-dia-

lkylglycolamide ester and N,N-dialkylacetyl amino moieties to the

glucuronic acid carboxyl group and the 4′-hydroxyl functionality of the

compound [29–31], and previous research also revealed that the glu-

curonic acid carboxyl moiety and the 4′-hydroxyl group play important

roles in the structural modiﬁcation of Scu. Although the developed Scu

2

T. Li, et al.

BioorganicChemistry101(2020)103980

derivatives displayed good protective activity against oxidative damage

and optimal absorption characteristics in vitro, scutellarin analogs ex-

hibiting both improved active transportation and enhanced protective

properties against the HIE impairment have not been described.

Based upon above research conclusions, in the present study, mo-

tivated by our continuing interest in exploring novel agents for the

treatment of HIE, we ﬁrstly envisaged that scutellarin methyl ester-4′-

dipeptide conjugates derived from scutellarin can be used as neuro-

protective agents. We envisioned that the novel conjugates would dis-

play desirable protective activities against the HIE impairment and

exhibit optimal active transport characteristics (Fig. 1). Herein, we re-

port our recent ﬁndings on the design, synthesis, and biological eva-

luation of scutellarin methyl ester-4′-dipeptide conjugates. The pre-

pared analogs displayed improved physiochemical properties, Caco-2

cell permeability, enhanced hPepT1-mediated transport properties,

superior anti-oxidative activities in vitro and neuroprotective char-

acteristics in vivo, as well as improved pharmacokinetic properties.

Amino acid tert-butyl ester hydrochlorides, such as alanine (Ala)

and valine (Val) (1a-b) were dissolved in anhydrous CH₂C1₂.

Subsequently, a solution of triphosgene [bis(trichloromethyl) carbo-

nate] (BTC) in CH₂C1₂was added to the initial solution at −10 °C.

Following stirring at −10 °C for 2 h, the solution was washed with a

cold 0.1 M aqueous solution of hydrochloric acid, dried, and con-

centrated in vacuo to obtain -amino acid ester isocyanates 2a-b quan-

titatively as light yellow oils.^LScutellarin methyl ester was then coupled

with 2a-b in the presence of Et₃N in anhydrous DMF at 50 °C for 10 h

under N₂atmosphere. Subsequently, the solvent was evaporated in

vacuo and the resulting residue was puriﬁed using routine ﬂash column

chromatography

on

silica

gel

(eluent:

chloroform/me-

thanol = 15:1–20:1 v/v) to obtain compounds 3a-b. Intermediates 3a-

b were then deprotected using triﬂuoroacetic acid (TFA) at 0 °C for

5–6 h to aﬀord 4a-b. The scutellarin methyl ester-dipeptide conjugates

(5a-l) were synthesized by condensation of compounds 4a-b with

-

L

amino acid tert-butyl ester hydrochlorides (e.g. Val, Phe, Glu, Asp, Leu,

Ile). The reactions were carried out in the presence of peptide-coupling

reagents, such as EDC or HBTU/DIPEA in anhydrous DMF at 0 °C for

0.5 h, and then at room temperature for 24 h. Finally, the deprotection

reactions were carried out utilizing TFA according to the procedures

described above to generate the desired target compounds. The struc-

tures of analogs 5a-l were conﬁrmed using ¹H/¹³C nuclear magnetic

resonance (NMR) spectroscopy, electrospray ionization-mass

2. Results and discussion

2.1. Chemical synthesis

The procedure for the preparation of scutellarin methyl ester-4′-

dipeptide conjugates (5a–l) is outlined in Scheme 1.

Scheme 1. Reagents and conditions: (a) bis(trichloromethyl) carbonate (BTC), pyr, DCM, −10 °C, 2 h, 1 M HCl, 90–95%; (b) anhydrous THF/DMF, Et₃N, 50 °C,

40–55%, 12 h; (c) TFA, 0 °C, 5–6 h, 29.9–44.3%; (d) amino acid tert-butyl ester, EDC, HBTU/DIPEA, DMF, 0 °C for 0.5 h, then room temperature for 24 h; (e) TFA,

DCM, 0 °C, 5–6 h, yields for last two steps were 23.2–57.9%.

3

T. Li, et al.

BioorganicChemistry101(2020)103980

Table 1

Table 2

The physicochemical properties for scutellarin methyl ester carbamate deriva-

tives.

Apparent permeability coeﬃcients (P_app) of scutellarin methyl ester carbamate

derivatives in Caco-2 cells.

Compds Aqueous

Fold

Stability in

Stability in simulated

intestinal ﬂuid

(t_1/2h)

Compounds

P_app

×10⁻⁶

P_app

(cm/^Bs)^t^a^o

× 10⁻⁶

ER (P_app

P_app)

to B

/

A

to

B

A

B

to

(cm/^As)^a

b

solubility

increase

Plasma

A

(μg/mL)

(t_1/2min)

Scutellarin^d

0.14

0.31

0.75

0.77

1.36

1.09

1.27

1.09

1.29

1.15

1.95

0.71

0.24^c

0.15

0.11

0.27

0.13

0.24

0.19

0.27

0.19

0.37

0.24

0.15

0.27

0.67

1.17

0.86

1.02

1.57

0.89

1.55

0.85

1.77

1.02

1.16

0.15

0.09

0.23

0.22

0.17

0.31

0.12

0.27

0.08

0.25

0.18

0.21

1.93

2.16

1.56

1.12

0.75

1.44

0.70

1.42

0.66

1.54

0.52

1.63

Scu

5a

5b

5c

5d

5e

5f

8.52

1.0

–

5a

5b

5c

5d

5f

126.94

167.84

247.93

313.53

40.04

14.9

19.7

29.1

36.8

4.7

10

35

45

35

10

45

40

45

> 4

> 2

> 4

> 2

> 4

5g

5h

5i

351.87

179.77

189.14

149.10

253.04

332.91

427.70

41.3

21.1

22.2

17.5

29.7

39.1

50.2

5g

5h

5i

5j

5k

5l

5j

5k

5l

a

P_{app A to B}: transport of the compound from apical to basolateral; P_{app B to A}

transport of the compound from basolateral to apical

:

b

c

d

ER (P_{app B to A}/P_{app A to B}): the ratio of P_{app B to A}to P_{app A to B}

spectrometry (MS-ESI) as well as high resolution mass spectrometry

(HRMS-ESI) analyses. Spectral data were in accordance with the as-

signed structures and all of the characterization data are provided in the

experimental section.

Data are mean

SD (n = 3)

The concentration of the test compounds was at 2.0 × 10⁻⁴M for scu-

tellarin and 1.5 × 10⁻⁴M for compounds 5a-l. The incubation time was up to

120 min

2.2. Biological evaluation

2.2.1. Physicochemical property studies

The study of the physicochemical properties of the obtained com-

pounds (Table 1) revealed that analogs containing a free carboxyl

functionality (5c–d and 5i-j) or a larger steric hindrance alkyl moiety

(5e–f and 5k-l) in their carbamate group were signiﬁcantly more stable

(t_1/235–45 min) in the plasma. Evaluation in artiﬁcial intestinal ﬂuid

showed that the dipeptide compounds 5a-l exhibited good stability (t_1/

> 4 h) in this environment. These outcomes are attributed to the

2

increasing steric hindrance of the carbamate strand and the formation

of intermolecular hydrogen bonds, which block the approach of en-

zymes to the carbamate linkage. With the exception of compound 5e,

aqueous solubility of all target compounds was signiﬁcantly higher

(126.94–427.70 μg/mL) than that of scutellarin (8.52 μg/mL). Parti-

cularly noteworthy are the solubilities of compounds 5f (351.87 μg/mL)

and 5l (427.70 μg/mL), which were 41–50 times higher than the so-

lubility of scutellarin. Moreover, metabolic studies using liver micro-

somes demonstrated that analogs 5a–l were ﬁrst converted into scu-

tellarin methyl ester by a hydrolase enzyme before being transformed

into scutellarin.

Fig. 2. The apical-to-basolateral apparent permeability coeﬃcients (P_app

AP to

_BL) of scutellarin methyl ester 4′-L-dipeptide carbamate conjugates 5a-5l in

Caco-2 cells (mean

SD, n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001

vs. scutellarin.

2.2.2. Caco-2 cell-based permeability assay

Val carbamate moieties in compounds 5g-l signiﬁcantly enhanced P_app

_{AP to BL}, indicating that the Val carbamate dipeptide conjugates exhibit

greater potential for future applications. The obtained outcomes also

demonstrated that analogs 5d, 5g, 5i, and 5k display signiﬁcantly

higher Caco-2 cell permeability and lower ER values than scutellarin;

thus, mechanistic evaluation of the uptake of these compounds would

be valuable.

The Caco-2 cell permeability assay (Table 2) demonstrated that

except for compound 5a with a higher ratio of P_{app BL to AP}/P_{app AP to BL}

(ER 2.16) than the ratio for scutellarin (ER 1.93), the ratios of P_{app BL to}

_AP/P_app

(ER) for compounds 5b-5l were within the range of

AP to BL

0.52–1.63. These values are lower than the ER of scutellarin, suggesting

that the eﬄux eﬀect of scutellarin is higher than that of compounds 5b-

5l. Among them, ER values of 5b, 5c, 5f, 5h, 5j, and 5l were higher

than 1.0, while ER values of 5d, 5g, 5i, and 5k were lower than 1.0.

These outcomes indicate greater permeability in the basolateral plate

(BL) to apical plate (AP) direction for compounds 5b, 5c, 5f, 5h, 5j, and

5l than for compounds 5d, 5g, 5i, and 5k. Additionally, compared with

scutellarin, Caco-2 cell permeability of 5d, 5g, 5i, 5j, and 5k con-

siderably increased, while their P_{app AP to BL}values were 9.7, 9.1, 9.2,

2.2.3. Uptake of target compounds 5d, 5g, 5i, and 5k into hPepT1-Madin-

Darby Canine Kidney (MDCK) cells

To evaluate the functionality of the cell system, protein expression

of PepT1 in stably transfected hPepT1-MDCK cell lines was initially

established according to a previously reported method [32]. Im-

portantly, the identiﬁed results were consistent with the data available

in the literature [33]. Fig. 3 demonstrates that the uptake rate in the

hPepT1-MDCK cells for all tested prodrugs (5d, 5g, 5i, 5k) signiﬁcantly

increased following the attachment of the dipeptides to the scutellarin

methyl ester; however, in MDCK cells, the uptake rate for all

8.2, and 13.9 times higher than the P_app

value of scutellarin.

AP to BL

Among the prepared compounds, 5k exhibits the highest P_{app AP to BL}

value (1.95

0.24 × 10⁻⁶cm/s) and the lowest ER value (0.52)

(Fig. 2). The initial SAR evaluation revealed that except for 5l, sub-

stitution of Ala carbamate in compounds 5a-f with the corresponding

4

T. Li, et al.

BioorganicChemistry101(2020)103980

2.2.5. In vitro anti-oxidative activities of compound 5k

Considering that compound 5k exhibited the highest permeability

in Caco-2 cells and highest uptake in hPepT1-MDCK cells, we subse-

quently examined its anti-oxidative activity in vitro and studied the

corresponding mechanisms.

2.2.5.1. Eﬀects of compound 5k on LDH and MDA in PC12

cells. Compared to Scu, pretreatment with analog 5k at 10 and

20 μM, followed by treatment with H₂O₂(1000 μM) attenuated the

LDH activity by approximately 40% and 50%, respectively (P < 0.05

and

P < 0.01 vs. Scu; Fig. 5A). Moreover, compared to Scu,

pretreatment with 5k at 2, 10, and 20 μM resulted in a decrease in

the level of MDA in a concentration dependent manner. Particularly, at

20 μM, 5k reduced the MDA level by approximately 30% (P < 0.05 vs.

Scu; Fig. 5B).

Fig. 3. The intake of scutellarin and target compounds (5d, 5g, 5i, 5k) in the

hPepT1-MDCK and MDCK cell lines. Data are shown as mean

(***p < 0.001).

SD

2.2.5.2. Eﬀects of 5k on the DNA condensation, intracellular ROS

production, and MMP loss in PC12 cells. Hoechst 33,342 staining

conﬁrmed that nuclear DNA condensation and nuclear fragmentation

occurred following treatment with 1000 μM of H₂O₂. Pretreatment with

Scu and 5k inhibited these apoptotic features (Fig. 6A). Microscopic

observation revealed that in the control group, the PC12 cells exhibited

regular, rounded nuclei, as whereas nuclear pyknosis, dense staining,

and a large number of apoptotic bodies indicative of apoptosis were

seen after the cells were treated with H₂O₂(Fig. 5A, arrows). Based on

the analysis of the blank group, the ROS production in the H₂O₂

compounds remained practically unchanged. Moreover, we noted that

the uptake rates of Scu dipeptide derivatives 5d, 5g, 5i, 5k were sig-

niﬁcantly higher than that of Scu (***p < 0.001) in the hPepT1-MDCK

cells. Among all prodrugs, 5k (Scu-Val-Leu) exhibited the highest up-

take rate of 25.74 μmol/mg/min, which was 11.2-fold higher than that

of Scu. These observations strongly suggest that the introduction of the

Val-Leu dipeptide into scutellarin methyl ester improves its uptake in

the hPepT1-transfected MDCK cells and that analog 5k is an optimal

prodrug candidate for further evaluation.

treatment group signiﬁcantly increased to 195.22

Nevertheless, pretreatment with Scu (20 μM) reduced the ROS

production to 179.06 16.68% (P < 0.05 vs·H₂O₂group), while

pretreatment with 5k (20 μM) further reduced the ROS production to

11.23%.

2.2.4. Molecular docking studies of compound 5k with PepT1

To provide insight into the molecular mechanism of oligopeptide

transporter 1 (PepT1), a computational docking experiment was carried

out. The binding modes of compound 5k in stereoview are illustrated in

Fig. 4. The prodrug molecules were docked into the protein binding

pocket using the “Protein Preparation Wizard 2017″ workﬂow in the

Schrödinger suite and the Force Field OPLS3 was utilized to optimize

the protein, including addition of missing hydrogen atoms to form

disulﬁde bonds. Employing the precision analysis of Schrödinger XP,

the lowest energy structure was calculated and selected as the optimal

conformational structure. The possible binding modes of compound 5k

with PepT1 were explored by docking the analog into the PepT1

binding cleft.

104.60

9.79% (P < 0.01 vs. Scu; Fig. 6B). Furthermore, treatment

of the blank group with 1,000 µM of H2O2 considerably decreased the

mean ﬂuorescence intensity of the MMP probe (Rho123) to

28.73

2.53% (^###

p < 0.001 vs. blank; Fig. 6C-D). However,

pretreatment with 20 µM of 5k noticeably increased the mean

ﬂuorescence intensity of Rho123 to 87.38

11.57% (P < 0.05 vs.

Scu; Fig. 6C-D). These observations suggested that compound 5k

displays signiﬁcant anti-apoptotic eﬀects against H2O2-induced

apoptosis in PC12 cells.

2.2.5.3. Cell apoptosis assay. We subsequently conducted identiﬁcation

of early and late apoptotic cells by Annexin-V/PI staining. As shown in

Fig. 7A-B, compared with the blank group, the treatment of PC12 cells

with 1000 μM of H₂O₂for 6 h signiﬁcantly increased the apoptosis rate

As it can be seen in Fig. 4(A), the dipeptide moiety of 5k extends

into the central cavity of binding pocket and displays an analogous

binding position to the transported dipeptide substrate. Fig. 4(B) shows

the ﬁt of 5k into active site of the transporter and a number of inter-

actions exist between 5k and amino acid residues of its action target.

Fig. 4(C) further illustrates the binding relationship between 5k and

amino acid residues of its target. For instance, the oxygen atom of

terminal leucine carboxycarbonyl and hydrogen atom of amide bond in

the dipeptide structure of the compound 5k form two hydrogen bonds

with Ser404 and Ser149 residues, respectively. Moreover, the hydroxyl

group at the 6 position and the oxygen atom of glycoside bond at the 7

position on the A ring of 5k form three hydrogen bonds with Tyr68,

Tyr30 and Arg26 residues of binding target, respectively. There are two

additional hydrogen bonds between the 3 and 4-position hydroxyl

groups of glucuronic acid and Asn156 and Glu400 residues of binding

target, while the A ring of 5k forms cation-π interaction with the

Lys126 residue, respectively. The binding results are shown in Fig. 4A-C

and it can be seen that compound 5k approaches the PepT1 binding

to 34.8

3.81%. Furthermore, compared with the H₂O₂group,

pretreatment with Scu or 5k resulted in a reduction in the apoptosis

rate. Especially, compared with the Scu group (22.1 2.62%) at

20 µM, pretreatment with 10 µM and 20 µM of 5k considerably reduced

the apoptosis rate to 12.3 1.21% and 11.7 0.57%, respectively

(P < 0.05 and P < 0.05 vs. Scu; Fig. 7A-B). To further evaluate

whether H₂O₂induced the cell death via apoptotic pathways, the

percentage of apoptotic cells was measured utilizing ﬂow cytometry.

The presence of the sub-G1 peak in the ﬂow cytometry detection is

considered as an indicator of cell apoptosis. Our results demonstrated

that treatment of cells with 1000 μM of H₂O₂for 6 h notably induced

the sub-G1 peak, indicating an apoptotic cell accumulation of

approximately 41.26%. Additionally, scutellarin and 5k signiﬁcantly

attenuated the H₂O₂-induced apoptotic cell accumulation in the sub-G1

peak (Fig. 7C). It is noteworthy that analog 5k (20 μM) attenuated the

H₂O₂-induced apoptotic cell accumulation in the sub-G1 peak to just

5.93% (P < 0.05 vs. Scu; Fig. 7C). Overall, our results imply that

compound 5k reduces apoptosis triggered by H₂O₂in PC12 cells.

channel. It appears that compounds containing -carbamate dipeptide

L

groups exhibit more optimal binding activities as a result of their hy-

drophilic and hydrophobic properties. These observations conﬁrmed

the validity of our strategy for designing scutellarin methyl ester-4′-

dipeptide conjugates as substrates for oligopeptide transporter 1 to

enhance the transport eﬃciency.

2.2.5.4. Eﬀect of 5k on the expression of cleaved caspase-3, Bcl-2, and Bax

levels in H₂O₂-induced PC12 cells. Among the proteins regulating

apoptosis, pro-apoptotic proteins belonging to the Bcl-2 family are

5

T. Li, et al.

BioorganicChemistry101(2020)103980

Fig. 4. Binding of compound 5k in PepT1 (PDB ID: 4d2c): View of the optimal conformation of prodrugs binding in the cave-like binding pocket (A), the interaction

modes of prodrugs in stereoview (B) and 2D display (C). The homology model structure of PepT1 (PDB ID: 4d2c) is displayed as a solid ribbon. Compound 5k is

shown in the substrate-binding pocket in the stick mode. The hydrogen bonds and cation-π interaction are shown as purple arrows and red ray, respectively. (For

interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

recognized to play an essential role. Bax, a pro-apoptotic protein,

accelerates cell death, whereas Bcl-2, an anti-apoptotic protein,

suppresses cell death. Thus, the Bcl-2/Bax signaling pathway is key in

the process of apoptosis [34,35]. In the present study, the treatment

with compound 5k upregulated the expression of the anti-apoptotic

protein Bcl-2 (Fig. 8A). These changes caused an increase in the Bcl-2/

Bax ratio. Based on the obtained results, it could be reasonably

speculated that 5k attenuates apoptosis. To further support our

hypothesis, we investigated the cleave-caspase-3 activity (Fig. 8A).

The analysis revealed that Scu and 5k exhibit anti-apoptotic activities.

6

T. Li, et al.

BioorganicChemistry101(2020)103980

Fig. 5. Eﬀects of 5k on LDH and MDA.

The cells were incubated with the in-

dicated concentrations of 5k for 24 h,

then stimulated with H₂O₂(1000 μM)

for 6 h. (A) The release of LDH. (B) The

MDA level. The data are represented as

mean

SEM (n = 3) **p < 0.01,

***p < 0.001 vs. blank, ^#p < 0.05,

^##p

<

0.01 vs·H₂O₂, ^&p

< 0.05,

&&

p < 0.01, ^&&&p < 0.001 vs. Scu.

Fig. 6. Eﬀects of 5k on H₂O₂-induced nuclear condensation, ROS, and MMP in PC12 cells. Cells were pretreated with 5k (2, 10, 20 μM) for 24 h and then exposed to

H₂O₂(1000 μM) for 6 h. (A) Eﬀect of 5k on MMP loss in H₂O₂-induced PC12 cells. (B) Eﬀects of 5k against H₂O₂-induced ROS production in PC12 cells. (C)

Representative ﬂuorescence images were obtained following Hoechst 33,342 staining in PC12 cells (200 × ). (D) The quantitative analysis of the mean ﬂuorescence

&&

intensity of 5k. The data are represented as mean

p < 0.01 vs. Scu.

SEM (n = 3). **p < 0.01, ***p < 0.001 vs. blank, ^#p < 0.05, ^###p < 0.001 vs·H₂O₂, ^&p < 0.05,

Moreover, compared with Scu (20 µM), at the same concentration, 5k

signiﬁcantly increased the Bcl-2/Bax ratio (P < 0.05 vs. Scu; Fig. 8C)

5k reduced the infarct volume compared to vehicle at 48 h post HIE.

Moreover, a high dose (4.4 mg/kg) proved to be most eﬀective,

reducing the infarcted area by approximately 43% compared with the

Scu group (2.8 mg/kg), suggesting that 5k drastically reduced the

infarct volume (P < 0.05 vs. Scu; Fig. 9A-B). As shown in Fig. 9C,

hematoxylin-eosin (HE) staining provided a direct observation method

for the analysis of the morphological characteristics of the damaged

area. The investigation of the protective eﬀects of compound 5k on the

hippocampus of the HIE rat pups revealed that in the sham group, the

hippocampal pyramidal cells were abundant, compact, and normal.

and attenuated the cleave-caspase-3 activation (P

< 0.01 vs. Scu;

Fig. 8B), which was accordance with the previous observations.

2.2.6. In vivo neuroprotective characteristics of compound 5k

2.2.6.1. Eﬀects of compound 5k on infarct volume, brain tissue

histopathology and neurological deﬁcits at 48 h post HIE. The 2,3,5-

triphenyltetrazolium

hydrochloride

(TTC)

staining

results

demonstrated that two doses (1.3 mg and 4.4 mg/kg) of compound

7

T. Li, et al.

BioorganicChemistry101(2020)103980

Fig. 7. The eﬀects of 5k on apoptosis. The cells were incubated with indicated concentrations of 5k for 24 h, then stimulated with H₂O₂(1000 μM) for 6 h. (A) PC12

cells were stained with Annexin-V/PI for FACS-based quantiﬁcation of apoptotic cells. (B) Quantitative analysis of the apoptotic ratio. (C) The cell cycle. The data are

represented as mean

SEM (n = 3), ^##p < 0.01 vs. Blank, **p < 0.01 vs·H₂O₂, ^&p < 0.05 vs. Scu.

Conversely, in the vehicle group, the hippocampal pyramidal cells were

entirely necrosed and nuclear pyknosis, deep staining as well as

fragmentation and dissolution were noted. In addition, when the HIE

rat pups were pretreated with 5k and scutellarin, the related

pathomorphological damage was reduced to varying degrees. In these

lesions, the group treated with compound 5k (4.4 mg/kg) was

signiﬁcantly lighter than the Scu (2.8 mg/kg) group, and only a small

number of pyramidal cells were necrotic. In addition, as shown in

Fig. 9D, at a high dose (4.4 mg/kg), 5k greatly ameliorated the

neurological deﬁcits compared with the scutellarin (4.4 mg/kg)

sham-operated groups as well as scutellarin-treated HIE group).

Moreover, the obtained results show that 5k considerably reduced the

infarct area and ameliorated the neurological deﬁcit following cerebral

HIE to diﬀerent degrees. Hence, the data indicated that the prodrug of

5k has a more potent protective eﬀect on the ischemic injury induced

by cerebral HIE in rat pups than the parent drug scutellarin.

2.2.6.2. Eﬀects of compound 5k on long-term brain morphology at 4 weeks

post HIE. Representative images of the brain morphology and the

microphotographs with Nissl-stained brain sections at 4 weeks post

HIE are illustrated in Fig. 10A. Nissl's staining showed that the 5k

pretreated HIE group (p

< 0.05, compared with the vehicle- and

8

T. Li, et al.

BioorganicChemistry101(2020)103980

Fig. 8. Eﬀects of 5k on the downregulation of the Bcl-2/Bax signaling pathway induced by H₂O₂. (A) At 48 h following the H₂O₂treatment, the protein expression

levels of Bcl-2, Bax, and cleave-caspase-3 in the PC12 cells were evaluated by western blotting analysis. (B) and (C) Semi-quantiﬁed results. The outcomes are

presented as the mean

standard error of the mean and are representative of three independent experiments. ***P < 0.001 vs. blank, ^###p < 0.001 vs·H₂O₂

group, ^&p < 0.05, ^&&p < 0.01 vs. Scu group.

treatment group attenuated brain tissue loss at 4 weeks post HIE. In

exhibited signiﬁcant suppression in the expression levels of APP and

BACE-1 in the brain tissue (p < 0.01) compared to the Scu (2.8 mg/kg)

group (Fig. 11A–C).

addition, 5k treatment signiﬁcantly reduced the brain tissue loss area at

4

weeks post HIE compared to the vehicle (Fig. 10B). These

observations imply that the modiﬁcation of scutellarin using the

dipeptide carbamate strategy signiﬁcantly enhanced the protective

activity of the target compounds against HIE-induced brain tissue

loss. The above results may be a consequence of eﬃcient transport

through active transport carriers, such as ^L-type amino acid transporter

1 (LAT1) or peptide transporter 2 (PepT2), which are expressed at the

BBB and in the membrane of the brain parenchymal cells. As a result,

the concentration of the drug in the brain as well as its neuroprotective

eﬀects were signiﬁcantly enhanced.

2.2.7. Pharmacokinetic studies on 5k

Following the injection of the tested compounds, the concentrations

of scutellarin and prodrug 5k in the blood were evaluated and the

pharmacokinetic parameters were calculated. As shown in Table 3 and

Fig. 12, compound 5k exhibited a better pharmacokinetic proﬁle than

scutellarin, with signiﬁcantly longer mean retention time (MRT:

17.328

1.907 min) and half-life (t_1/2: 41.044

25.736 min) than

1.983 min; t_1/2

those of scutellarin (MRT: 9.647

:

7.589

0.833 min). These results suggest that 5k slowly and

2.2.6.3. Compound 5k reduces APP and BACE-1 expression in the brain

tissues of HIE- suckling rat. Amyloid precursor protein (APP) is widely

known as the precursor molecule for the generation of Aβ, while the β-

site APP-converting enzyme-1 (BACE-1) is a β-secretase, which cleaves

the N-terminal of APP to generate Aβ [36]. It has previously been

reported that BACE-1 expression is upregulated in the brain tissue of AD

patients and that excessive expression of this protein can cause the

onset of the disease [37–38]. Based on western blot analysis, compared

to the sham group, the HIE group showed signiﬁcant upregulation in

the expression of APP and BACE-1 in the brain tissue (p < 0.01 and

p < 0.001, respectively) (Fig. 11A–C). The expression levels of APP

and BACE-1 in the 5k (1.3 mg /kg) group was not much diﬀerent from

those in the Scu group (Fig. 9A-C). Moreover, the 5k (4.4 mg/kg) group

smoothly releases the parent drug, allowing reduced metabolic oppor-

tunities for scutellarin, and subsequently prolonging the action time of

the drug. Moreover, the AUC

value of 5k (66.896

5.113 mg/

42.171 mg/

0.810 l/kg)

0.020 l/kg).

(0-t)

L.min) was lower than that of scutellarin (363.568

L.min), while volume of distribution of 5k (V_d: 5.876

was signiﬁcantly higher than that of scutellarin (0.301

These observations could be explained by the fact that due to its 4′-

dipeptide conjugate structure, 5k is transported by active transport

carriers, such as _L-type amino acid transporter 1 (LAT1) or peptide

transporter 2 (PepT2), which are expressed at the BBB and in the

membrane of the brain parenchymal cells; therefore, 5k is suspected to

display the tendency for targeting the brain. Consequently, following

the transporter-mediated delivery of 5k across the BBB and cellular

9

T. Li, et al.

BioorganicChemistry101(2020)103980

Fig. 9. Eﬀects of compound 5k on infarct volume and short-term neurological deﬁcits at 48 h post HIE. Representative TTC stained brain sections (A) and TTC

analysis (B) at 48 h post HIE with 5k treatment signiﬁcantly reduced the infarct volume. (C) Eﬀects of compound 5k on the HE staining in the HIE rat pups at 48 h. a:

sham group. b: vehicle group. c: 2.8 mg/kg Scu. d: 1.3 mg/kg of 5k. e: 4.4 mg/kg of 5k. (D) Neurological score at 48 h post HIE with 5k treatment showed an

improvement in the outcome. Data are presented as mean

SD. ***P < 0.001 vs. sham, ^#P < 0.05 vs. vehicle, ^&P < 0.05 vs. Scu, n = 6 per group. The

diﬀerences between groups were evaluated by one-way ANOVA followed by Tukey's test.

membrane barrier, the parent drug will be released at its target site

inside the brain parenchymal cells and as a result may lead to a de-

crease in the concentration of 5k in the blood. We therefore postulate

that the presence of the 4′-dipeptide fragments beneﬁts “drug-like”

molecular physicochemical properties, resulting in improved prodrug

absorption, as demonstrated in both in vitro and in vivo assays.

conducted in vivo studies. Overall, our results suggest that the scu-

tellarin methyl ester-4′-dipeptide conjugates strategy may be used as an

eﬀective method for the design of new scutellarin prodrugs with en-

hanced therapeutic eﬀects, improved absorptive potential in vitro and

encouraging pharmacokinetic proﬁles. Further work on the struc-

ture–activity relationships (SAR) of these compounds is currently being

carried out in our laboratory.

3. Conclusions

4. Experimental procedures

In the current study, based on our previous research, we designed

and synthesized a series of scutellarin methyl ester-4′-dipeptide con-

jugates. The obtained compounds were evaluated as potential anti-HIE

agents. Among them, compound 5k exhibited the most optimal P_{app A to}

_B, the lowest ER values in Caco-2 cells, and the highest uptake value in

hPepT1-MDCK cells. Moreover, the mechanistic studies demonstrated

that pre-treatment of the PC12 cells with 5k resulted in more potent

protective activity against oxidative damage by improving the relevant

in vitro detection indicators. Analog 5k displayed remarkable ther-

apeutic eﬀects in the HIE rat pups model. A reduction in the infarct

area, decrease in the related pathomorphological damage, amelioration

of the neurological deﬁcits, attenuation of the brain tissue loss as well

as reduction in the APP and BACE-1 expression were observed. Finally,

the favorable pharmacokinetic proﬁle of 5k was also conﬁrmed in the

4.1. Chemical synthesis

Scutellarin (Scu, purity > 95%, HPLC) was provided by Feng shang

jian Pharmaceutical Co. Ltd. (Yunnan, China). Scutellarin methyl ester

was prepared according to the available literature procedures (Lu et al.,

2010). All other reagents were 97–99% pure and purchased from

Sinopharm Chemical Reagent Co., Ltd. (SCRC). ¹H/¹³C nuclear mag-

netic resonance (NMR) spectra were recorded on a Varian Mercury 400

spectrometer. Tetramethylsilane was used as the reference and the J

values are given in Hz. Low-resolution mass spectra were obtained

utilizing an ACQUITY TQD (triple quadrupole) low-resolution mass

spectrometer (Waters, America). High-resolution mass spectra were

obtained using the microTOF-Q II ESI-Q-ToF LC/MS/MS apparatus

10

T. Li, et al.

BioorganicChemistry101(2020)103980

Fig. 10. Representative brain morphology images of Nissl-stained brain sections at 4 weeks post HIE. (A) Treatment with 5k signiﬁcantly reduced the percentage of

tissue loss. (B) Signiﬁcant reduction of the brain tissue loss area following treatment with 5k compared to vehicle. Data are presented as mean

SD, **P < 0.01 vs.

sham, ^#P < 0.05 vs. vehicle, ^&P < 0.05 vs. Scu, n = 6 per group. The diﬀerences between groups were evaluated by one-way ANOVA, followed by Tukey's test.

(Bruker Daltonics). Flash chromatography was carried out on silica gel

(200–300 mesh), and chromatographic solvent ratios are expressed as

volume:volume. All anhydrous solvents were distilled over CaH₂or Na/

benzophenone prior to use.

4.1.1. Procedure for the preparation of scutellarin methyl ester-4′-dipeptide

conjugates

A 250 mL, three-necked, round-bottomed ﬂask, ﬁtted with a ni-

trogen inlet adapter, was charged with 2.5 mmol of -amino acid tert-

L

Fig. 11. Eﬀects of scutellarin (Scu) and 5k on APP and BACE-1 expression in the brain tissues. (A) Representative western blots illustrating diﬀerences in the band

intensities of APP and BACE-1. (B) Scu and 5k signiﬁcantly attenuated the upregulation of the APP expression in the brain. (C) Scu and 5k considerably decreased the

upregulation of BACE-1 expression in the brain. Data are presented as mean

SEM (n = 6 in each group; ^**p < 0.01, ^***p < 0.001 vs. sham, ^##p < 0.01,

^#p < 0.05 vs. vehicle, ^&p < 0.05 vs. Scu group).

11

T. Li, et al.

BioorganicChemistry101(2020)103980

Table 3

reaction mixture was stirred at 0 °C for 10 min prior to the addition of _L-

amino acid tert-butyl ester hydrochloride (e.g., Val, Phe, Glu, Asp, Leu,

Ile). Subsequently, the reaction mixture was stirred at room tempera-

ture for 12 h·H₂O (10 mL) and CH₂Cl₂(10 mL) were then added and the

mixture was extracted three times with CH₂Cl₂(3 × 30 mL). The or-

ganic extracts were dried over anhydrous MgSO₄, ﬁltered, and evapo-

rated in vacuo. The resulting residue was puriﬁed by column chroma-

tography on silica gel (eluent: chloroform/methanol, 35:1 v/v),

followed by a deprotection with TFA/CH₂Cl₂, 10:1 (v/v) at 0 °C. The

crude product was dried in vacuo to aﬀord compound 5a-l as a light

yellowish-green solid. The yields were given for the last two steps of the

reaction.

Pharmacokinetic parameters of scutellarin and 5k after i.v. administration to

rats (n = 6, Mean

Parameter

SD).

Unit

Scutellarin

363.56

5k

AUC

μg/mL.min

min

42.17

66.89

17.32

41.04

5.87

5.11

1.90

25.73

MRT ^0-120

9.64

7.58

0.30

1.98

0.83

0.020

0-120

t_1/2

V_d

min

L/kg

0.81

4.1.2. ((4-(5,6-dihydroxy-4-oxo-7-(((2S,3S,4R,5R,6S)-3,4,5-trihydroxy-

6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)-4H-chromen-2-yl)

phenoxy)carbonyl)-^L-alanyl-L-valine (5a)

Yield 42.1%, light yellowish green solid. ¹H NMR (400 MHz,

DMSO‑d₆) δ 10.45 (s, 1H, 6-OH), 8.10 (d, J = 8.7 Hz, 1H, NHCH

(CH₃)₂), 8.01 (d, J = 6.9 Hz, 1H, NHCHCH₃), 7.92 (d, J = 8.7 Hz, 2H,

C_2′6′–2H), 7.06 (s, 1H, C₃-H), 6.92 (d, J = 8.7 Hz, 2H,C_3′5′–2H), 6.87 (s,

1H, C₈-H), 5.29 (d,

J = 7.0 Hz, 1H, C_′′-H), 4.25–4.05 (m,

3H,C_{2′′3′′4′′}–OH), 3.62 (s, 3H, COOCH₃), 3.34 (dt, J = 16.7, 8.7 Hz, 3H,

C_{2′′3′′4′′}–3H), 2.01 (dq, J = 13.1, 7.0, 6.6 Hz, 1H, CH(CH₃)₂), 1.25 (d,

J = 7.0 Hz, 3H, CHCH₃), 0.82 (d, J = 4.9 Hz, 6H, CH(CH₃)₂). ¹³C NMR

(101 MHz, DMSO‑d₆) δ 182.75, 173.53, 173.42, 169.74, 165.12,

162.00, 155.77, 153.99, 153.59, 153.17, 129.18(2C), 124.05, 121.52,

116.56(2C), 106.04, 103.40, 99.77, 93.91, 75.70, 73.22, 71.87, 57.48,

55.44, 52.56, 50.54, 30.42, 19.55, 18.48, 18.34. HRESIMS m/z (pos):

691.1982 C₃₁H₃₄N₂O₁₆(calcd. 691.1987).

Fig. 12. Concentration proﬁles of scutellarin and 5k in plasma following i.v.

4.1.3. ((4-(5,6-dihydroxy-4-oxo-7-(((2S,3S,4R,5R,6S)-3,4,5-trihydroxy-

6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)-4H-chromen-2-yl)

phenoxy)carbonyl)-^L-alanyl-L-phenylalanine (5b)

administration.

Yield 45.9%, light yellowish green solid. ¹H NMR (400 MHz,

DMSO‑d₆) δ 10.46 (s, 1H, 6-OH), 8.27 (d, J = 7.8 Hz, 1H, -NHCH₂Ph),

7.96 (s, 1H, NHCHCH₃), 7.93 (d, J = 8.7 Hz, 2H, C_2′6′–2H), 7.46 – 7.07

(m, 6H, Ph-H), 7.06 (s, 1H, C₃-H), 6.92 (d, J = 8.8 Hz, 2H, C_3′5′–2H),

6.88 (s, 1H, C₈-H), 5.52 (d, J = 5.2 Hz, 1H,C_1′-H), 5.42 – 5.13 (m, 3H,

butyl ester hydrochloride, 50 mL of anhydrous CH₂Cl₂, and 0.8 mL

(0.121 mol) of pyridine. The resulting solution was cooled at −10 °C

for 30 min prior to the addition of the triphosgene [bis(trichloromethyl)

carbonate] (BTC) solution (1.68 mmol in 3.0 mL of CH₂Cl₂) using a

syringe over 30 s. The resulting light yellow solution was stirred at

−10 °C for 2 h. The reaction mixture was then washed twice with

50 mL of a cold 0.1 M aqueous solution of HCl, ca. 30 mL of crushed ice

and 20 mL of cold saturated aqueous solution of NaCl. The organic

layers were separated, dried over anhydrous MgSO₄, ﬁltered, and

concentrated in vacuo to obtain _L-amino acid ester isocyanate (2a–b)

quantitatively as a light yellow oil.

C

_2′3′4′–OH), 4.42 (q, J = 7.8 Hz, 1H, CHCH₂Ph), 4.18 (d, J = 9.3 Hz,

1H, C_5′-H), 4.09 (p, J = 7.1 Hz, 1H,CHCH₃), 3.63 (s, 3H, COOCH₃),

3.04 – 2.83 (m, 2H,CH₂Ph), 1.22 (d, J = 7.1 Hz, 3H,CH₃). ¹³C NMR

(101 MHz, DMSO‑d₆) δ 182.76, 173.12 (d, J = 13.0 Hz), 169.74,

165.13, 162.01, 155.81, 153.75 (d, J = 43.3 Hz), 153.53, 153.15,

137.73, 129.78 (2C), 129.20, 128.74 (2C), 127.01, 124.11, 121.52 ,

116.57 (2C), 106.06, 103.42, 99.98, 94.03, 75.76, 73.22 (2C), 71.86,

55.47, 53.97, 52.57, 50.59, 37.22, 28.01, 18.77. HRESIMS m/z (pos):

739.1982 C₃₅H₃₄N₂O₁₆(calcd. 739.1987).

Under N₂atmosphere, scutellarin methyl ester (770 mg, 1.6 mmol)

was dissolved in 15 mL of anhydrous N,N-dimethylformamide (DMF).

Subsequently, a solution of compounds 2a–b (2.43 mmol in 10 mL

anhydrous THF) and Et₃N (34 μL, 0.24 mmol) was added to the mix-

ture, and the reaction mixture was stirred at 50 °C for 10 h.

Subsequently, the solvent was evaporated in vacuo, and the residue was

puriﬁed by routine ﬂash column chromatography on silica gel (eluent:

chloroform/methanol = 15:1–20:1 v/v) to aﬀord scutellarin methyl

ester-4′-_L-amino acid carbamate conjugates 3a–b.

4.1.4. ((4-(5,6-dihydroxy-4-oxo-7-(((2S,3S,4R,5R,6S)-3,4,5-trihydroxy-

6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)-4H-chromen-2-yl)

phenoxy)carbonyl)-^L-alanyl-L-glutamic acid (5c)

Yield 35.0%, light yellowish green solid. ¹H NMR (400 MHz,

DMSO‑d₆) ¹H NMR (400 MHz, DMSO‑d₆) δ8.22 (d, J = 7.9 Hz, 1H,

Compounds 3a–b (0.22 mmol) were dissolved in triﬂuoroacetic acid

(TFA) (3 mL) and the solution was stirred at 0 °C for 5–6 h. The reaction

was monitored by thin layer chromatography (TLC) until completion.

TFA was then evaporated in vacuo, and the resulting residue was wa-

shed with 10 mL chloroform and petroleum ether respectively using the

centrifuge subside method to provide compounds 4a–b.

–NHCH₂COOH), 8.01 (d, J = 7.2 Hz, 1H, NHCHCH₃), 7.92(d,

J = 8.8 Hz, 2H, C_2′6′–2H), 7.06 (s, 1H, C₃-H), 6.92 (m, 2H, C_3′5′–2H),

6.86 (s, 1H, C₈-H), 5.29 (d, J = 6.8 Hz, 1H, C_1′-H), 4.24 – 4.14 (m, C_5′-

H, NHCHCHCH₃), 4.07 (p, J = 7.1 Hz, 1H), 3.62 (s, 3H, COOCH₃),2.28

– 2.20 (m, 2H, CH₂CH₂COOH), 1.93 (tq, J = 16.1, 7.9, 7.4 Hz, 3H),

1.25 (d, J = 7.2 Hz, 3H, CH₃). ¹³C NMR (101 MHz, DMSO‑d₆) δ 182.75,

174.37, 173.66, 173.20, 169.73, 165.12, 161.99, 155.79, 154.00,

153.62, 153.16, 129.19(2C), 124.08, 121.56, 116.57(2C), 106.08,

103.44, 99.96, 94.00, 75.75, 73.22, 71.84, 52.56, 51.74, 50.64, 49.13,

30.65, 26.98, 18.50. HRESIMS m/z (pos): 721.1728 C₃₁H₃₂N₂O₁₈

(calcd. 721.1726).

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide

hydrochloride

(EDC)

(0.254

mmol),

N,N-Diisopropylethylamine (DIPEA)

(0.305 mmol), and O-benzotriazole- N,N,N',N'-tetramethyluronium-

hexaﬂuorophosphate (HBTU) (0.508 mmol) were added to a stirred

solution of compounds 4a–b (0.254 mmol) in DMF (20 mL) at 0 °C. The

12

T. Li, et al.

BioorganicChemistry101(2020)103980

4.1.5. ((4-(5,6-dihydroxy-4-oxo-7-(((2S,3S,4R,5R,6S)-3,4,5-trihydroxy-

6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)-4H-chromen-2-yl)

phenoxy)carbonyl)-^L-alanyl-L-aspartic acid (5d)

94.03, 78.97, 75.77, 73.30, 71.80, 60.65, 52.51, 47.99, 31.29, 19.61,

18.79, 17.60. HRESIMS m/z (pos): 691.1990 C₃₁H₃₄N₂O₁₆(calcd.

691.1987).

Yield 23.2%, light yellowish green solid. ¹H NMR (400 MHz,

DMSO‑d₆)

δ

10.42 (s, 1H, 6-OH), 8.28 (d,

J

=

8.1 Hz,

4.1.9. (S)-2-((S)-2-(((4-(5,6-dihydroxy-4-oxo-7-(((2S,3S,4R,5R,6S)-

3,4,5-trihydroxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)-4H-

chromen-2-yl)phenoxy)carbonyl)amino)-3-methylbutanamido)-3-

phenylpropanoic acid (5h)

1H,–NHCH₂COOH), 7.96 (d, J = 8.0 Hz, 1H, NHCHCH₃), 7.93 (d,

J = 8.9 Hz, 2H, C_2′6′–2H), 7.05 (s, 1H, C₃-H), 6.92 (d, J = 8.8 Hz, 2H,

C

_3′5′–2H), 6.86 (s, 1H, C₈-H), 5.29 (d, J = 6.9 Hz, 1H, C_1′′-H), 4.54 (q,

J = 6.2 Hz, 1H, CHCH₂COOH), 4.18 (d, J = 9.4 Hz, 1H, C_5′′-H), 4.12 –

4.06 (m, 1H, CHCH₃), 3.63 (s, 3H, COOCH₃), 2.71 – 2.53 (m, 2H,

CH₂COOH), 1.26 (d, J = 7.0 Hz, 3H, CH₃). ¹³C NMR (101 MHz,

DMSO‑d₆) δ 182.74, 172.91, 172.78, 172.17, 169.69, 165.14, 162.01,

155.81, 153.98, 153.59, 153.16, 129.19(2C), 124.15, 121.55,

116.58(2C), 106.09, 103.45, 100.10, 94.07, 75.79(2C), 73.28, 71.83,

52.55, 50.60, 49.00, 36.52, 18.74. HRESIMS m/z (pos): 707.1570

Yield 40.5%, light yellowish green solid. ¹H NMR (400 MHz,

DMSO‑d₆) δ 10.46 (s, 1H, 6-OH), 8.35 (d, J = 7.3 Hz, 1H, -NHCH₂Ph),

7.93 (d, J = 8.4 Hz, 2H, C_2′6′–2H), 7.72 (d, J = 8.7 Hz, 1H, NHCHCH₃),

7.22 (d, J = 5.6 Hz, 4H, Ph-H), 7.16 (d, J = 6.1 Hz, 1H, Ph₄-H), 7.06 (s,

1H, C₃-H), 6.92 (d, J = 8.4 Hz, 2H, C_3′5′–2H), 6.88 (s, 1H, C₈-H), 5.50

(d, J = 5.7 Hz, 1H,), 5.30 (d, J = 5.9 Hz, 1H, C_1′′-H), 4.43 (q,

J = 7.5 Hz, 1H, CHCH₂Ph), 4.17 (d, J = 8.8 Hz, 1H, C_5′′-H), 3.90 (t,

J = 7.9 Hz, 1H, CHCH₃), 3.62 (s, 3H, COOCH₃), 2.93 (ddd, J = 44.6,

13.9, 7.4 Hz, 2H, CH₂Ph), 1.94 (dq, J = 12.2, 5.9, 5.5 Hz, 1H, CH

(CH₃)₂), 0.85 (d, J = 6.4 Hz, 6H, CH(CH₃)₂). ¹³C NMR (101 MHz,

DMSO‑d₆) δ 182.78, 173.28, 171.56, 169.67, 165.13, 162.02, 155.96,

153.94, 153.22, 137.82, 129.69(2C), 129.20, 128.75(4C), 127.01,

124.23, 121.53, 116.57(2C), 106.07, 103.41, 100.13, 94.07, 75.83,

75.75, 73.26, 71.80, 60.62, 54.01, 52.56, 37.30, 32.07, 19.61, 18.61.

HRESIMS m/z (pos): 767.2300 C₃₇H₃₈N₂O₁₆(calcd. 767.2300).

C

₃₀H₃₀N₂O₁₈(calcd. 707.1572).

4.1.6. ((4-(5,6-dihydroxy-4-oxo-7-(((2S,3S,4R,5R,6S)-3,4,5-trihydroxy-

6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)-4H-chromen-2-yl)

phenoxy)carbonyl)-^L-alanyl-L-leucine(5e)

Yield 46.0%, light yellowish green solid. ¹H NMR (400 MHz,

Methanol‑d₄) δ 7.82 (d, J = 6.7 Hz, 2H, C_2′6′–2H), 6.89 (d, J = 8.7 Hz,

2H, C_3′5′–2H), 6.62 (s, 1H, C₈-H), 5.21 (d, J = 7.3 Hz, 1H, C_1′′-H), 4.43

(dd, J = 10.0, 5.0 Hz, 1H, CHCH₂CH(CH₃)₂), 4.19 (q, J = 7.0, 5.4 Hz,

2H, CHCH₃, C_5′′–OH), 3.74 (s, 3H, COOCH₃), 3.68 – 3.52 (m, 4H,

C_{2′′,3′′,4′′,5′′}-4H), 1.71 (dq, J = 13.0, 6.7 Hz, 1H, CH(CH₃)₂), 1.65 – 1.55

(m, 2H, CH₂CH(CH₃)₂), 1.43 (d, J = 7.1 Hz, 3H, CHCH₃), 0.91 (dd,

J = 11.5, 6.5 Hz, 6H, CH(CH₃)₂. ¹³C NMR (101 MHz, Methanol‑d₄) δ

182.83, 174.98, 174.15, 169.48, 165.69, 161.67, 155.49, 154.52,

154.23, 152.99, 128.36(2C), 123.94, 121.58, 115.72(2C), 106.08,

102.57, 100.22, 93.85, 75.46, 75.40, 72.98, 71.46, 51.65, 51.05, 50.75,

40.34, 24.62, 22.12, 20.68, 16.80. HRESIMS m/z (pos): 705.2148

4.1.10. (S)-2-((S)-2-(((4-(5,6-dihydroxy-4-oxo-7-(((2S,3S,4R,5R,6S)-

3,4,5-trihydroxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)-4H-

chromen-2-yl)phenoxy)carbonyl)amino)-3-methylbutanamido)

pentanedioic acid (5i)

Yield 36.4%, light yellowish green solid. ¹H NMR (400 MHz,

DMSO‑d₆) δ 8.30 (d, J = 7.4 Hz, 1H, –NHCH₂COOH), 7.89 (d,

J = 8.8 Hz, 2H, C_2′6′–2H), 7.82 (d, J = 8.6 Hz, 1H, NHCHCH₃), 7.03 (s,

1H, C₃-H), 6.90 (d, J = 8.8 Hz, 2H, C_3′5′–2H), 6.81 (s, 1H, C₈-H), 5.28

(d, J = 6.8 Hz, C_1′′-H), 4.24 – 4.18 (m, 1H, NHCHCOOH), 4.16 (d,

J = 9.1 Hz, 1H, NHCHCHCH₃), 3.61 (s, 3H, COOCH₃), 3.36–3.30 (m,

3H, C_{2′′3′′4′′}–3H), 2.32 – 2.18 (m, 2H, CH₂CH₂COOH), 2.01 – 1.87 (m,

2H, CH₂CH₂COOH), 1.78 (ddd, J = 11.5, 9.2, 6.4 Hz, 1H, CH(CH₃)₂),

0.91 (t, J = 7.2 Hz, 6H, CH(CH₃)₂). ¹³C NMR (101 MHz,) δ 182.74,

174.33, 173.60, 171.79, 169.66, 165.12, 161.97, 155.88, 154.01,

153.92, 153.17, 129.19(2C), 124.16, 121.52, 116.56(2C), 106.06,

103.40, 100.03, 94.00, 75.74, 73.23, 71.78, 60.64, 52.56, 51.78, 31.28,

30.56, 29.53, 26.73, 19.63, 18.74. HRESIMS m/z (pos): 749.2036

C

₃₂H₃₆N₂O₁₆(calcd. 705.2143)

4.1.7. ((4-(5,6-dihydroxy-4-oxo-7-(((2S,3S,4R,5R,6S)-3,4,5-trihydroxy-

6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)-4H-chromen-2-yl)

phenoxy)carbonyl)-^L-alanyl-L-alloisoleucine (5f)

Yield 44.8%, light yellowish green solid. ¹H NMR (400 MHz,

DMSO‑d₆) δ 10.43 (s, 1H, 6-OH), 8.09 – 8.03 (d, 1H, NHCHCOOH), 8.00

(d, J = 7.1 Hz, 1H, NHCHCH₃), 7.93 (d, J = 8.8 Hz, 2H, C_2′6′–2H), 7.06

(s, 1H, C₃-H), 6.92 (d, J = 8.8 Hz, 2H, C_3′5′–2H), 6.87 (s, 1H, C₈-H),

5.29 (d, J = 7.1 Hz, 1H, C_1′′-H), 4.24 – 4.08 (m, 3H, C_5′′-H, NHCHCH₃,

NHCHCOOH), 3.62 (s, 3H, COOCH₃), 1.73 (dp, J = 13.1, 6.7 Hz, 1H,

CH(CH₃)CH₂CH₃), 1.40 – 1.29 (m, 1H, CH(CH₃)CH₂CH₃), 1.24 (d,

J = 7.1 Hz, 3H, CHCH₃), 1.20 – 1.05 (m, 1H, CH(CH₃)CH₂CH₃), 0.79 (t,

J = 7.4 Hz, 6H, CH(CH₃)CH₂CH₃). ¹³C NMR (101 MHz, DMSO‑d₆) δ

182.75, 173.42, 173.35, 169.74, 165.12, 162.00, 155.78, 153.99,

153.58, 153.18, 129.19(2C), 124.08, 121.54, 116.57(2C), 106.04,

103.43, 99.79, 93.95, 75.73, 73.22, 71.87, 56.82, 55.46, 52.56, 50.51,

36.94, 25.04, 18.50, 16.01, 11.98. HRESIMS m/z (pos): 705.2151

C₃₂H₃₆N₂O₁₆(calcd. 705.2143).

C₃₃H₃₇N₂O₁₈(calcd. 749.2041).

4.1.11. (S)-2-((S)-2-(((4-(5,6-dihydroxy-4-oxo-7-(((2S,3S,4R,5R,6S)-

3,4,5-trihydroxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)-4H-

chromen-2-yl)phenoxy)carbonyl)amino)-3-methylbutanamido)succinic

acid (5j)

Yield 32.0%, light yellowish green solid. ¹H NMR (400 MHz,

DMSO‑d₆) δ 8.39 (d, J = 7.0 Hz, 1H, NHCH₂COOH), 7.93 (d,

J = 8.7 Hz, 2H, C_2′6′–2H), 7.79 (d, J = 8.0 Hz, 1H, NHCHCH₃), 7.06 (s,

1H, C₃-H), 6.92 (d, J = 8.7 Hz, 2H, C_3′5′–2H), 6.87 (s, 1H, C₈-H), 5.29

(d, J = 6.5 Hz, 1H, C_1′′-H), 4.53 (q, J = 6.6 Hz, 1H, CHCH₂COOH),

4.16 (d, J = 9.1 Hz, 1H, C_5′′-H), 4.12 – 3.94 (m, 1H, CHCH(CH₃)₂), 3.91

(t, 1H, J = 7.7 Hz, C_3″–OH), 3.62 (s, 3H, COOCH₃), 3.41 – 3.22 (m, 3H,

4.1.8. ((4-(5,6-dihydroxy-4-oxo-7-(((2S,3S,4R,5R,6S)-3,4,5-trihydroxy-

6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)-4H-chromen-2-yl)

phenoxy)carbonyl)-^L-valylalan-ine(5g)

C

_{2′′3′′4′′}–3H), 2.61 (qd, J = 16.8, 6.3 Hz, 2H, CH₂COOH), 1.97 (dq,

Yield 44.8%, light yellowish green solid. ¹H NMR (400 MHz,

DMSO‑d₆) δ 8.33 (d, J = 7.0 Hz, 1H, NHCHCH₃), 7.92 (d, J = 8.8 Hz,

2H, C_2′6′–2H), 7.77 (d, J = 8.1 Hz, 1H, NHCH(CH₃)₂), 7.05 (s, 1H,C₃-

H), 6.92 (d, J = 8.8 Hz, 2H,C_3′5′–2H), 6.86 (s, 1H, C₈-H), 5.29 (d,

J = 6.9 Hz, 1H, C_1′′-H), 4.22 (q, J = 7.1 Hz, 1H, CHCH₃), 4.17 (d,

J = 9.2 Hz, 1H, C_3′′–OH), 3.90 – 3.84 (m, 1H, CH(CH₃)₂), 3.63 (s, 3H,

COOCH₃), 3.34 (dt, J = 17.1, 8.5 Hz, 3H, C_{2′′3′′4′′}–3H), 1.98 (dq,

J = 13.1, 6.4 Hz, 1H, CH(CH₃)₂), 1.25 (d, J = 7.2 Hz, 3H,CHCH₃), 0.99

– 0.85 (m, 6H, CH(CH₃)₂). ¹³C NMR (101 MHz,) δ 182.75, 174.49,

171.40, 169.63, 165.14, 161.99, 155.90, 153.95, 153.19, 129.16(2C),

124.21, 121.54, 117.06, 116.55, 114.19, 106.08, 103.42, 100.19,

J = 20.1, 6.4 Hz, 1H, CH(CH₃)₂), 0.90 (t, J = 7.4 Hz, 6H,CH(CH₃)₂).

¹³C NMR (101 MHz, DMSO‑d₆) δ 182.74, 172.79, 172.17, 171.33,

169.64, 165.11, 161.98, 155.90, 154.01, 153.92, 153.17, 129.18(2C),

124.21, 121.50, 116.55(2C), 106.05, 103.38, 100.15, 94.04, 75.77(2C),

73.24, 71.76, 60.53, 52.54, 49.02, 36.47, 31.55, 19.59, 18.54.

HRESIMS m/z (pos): 735.1887 C₃₂H₃₄N₂O₁₈(calcd. 735.1885).

4.1.12. ((4-(5,6-dihydroxy-4-oxo-7-(((2S,3S,4R,5R,6S)-3,4,5-

trihydroxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)-4H-

chromen-2-yl)phenoxy)carbonyl)-^L-valyl-L-leucine (5k)

Yield 57.9%, light yellowish green solid. ¹H NMR (400 MHz,) δ 8.21

13

T. Li, et al.

BioorganicChemistry101(2020)103980

(d, J = 7.4 Hz, 1H, NHCHCOOH), 7.89 (d, J = 8.8 Hz, 2H, C_2′6′–2H),

7.77 (d, J = 8.7 Hz, 1H, NHCH), 7.03 (s, 1H, C₃-H), 6.90 (d, J = 8.8 Hz,

2H, C_3′5′–2H), 6.82 (s, 1H, C₈-H), 5.28 (d, J = 6.5 Hz, 1H, C_1′-H), 4.22 –

4.17 (m, 1H, CHCH₂CH(CH₃)₂), 4.16 (d, J = 9.1 Hz, 1H, CHCH₃), 3.93

– 3.84 (m, 1H, NHCH-CH(CH₃)₂), 3.61 (s, 3H, COOCH₃), 3.33 (dq,

J = 14.4, 7.8, 6.4 Hz, 3H, C_{2′′3′′4′′}–3H), 1.99 (dq, J = 13.6, 7.0 Hz, 1H,

CH(CH₃)₂), 1.68 – 1.55 (m, 1H, CHCH₂CH(CH₃)₂), 1.54 – 1.33 (m, 2H,

CHCH₂CH(CH₃)₂), 0.90 (t, J = 7.3 Hz, 6H, CH(CH₃)₂), 0.83 (d,

J = 6.5 Hz, 3H, CHCH₂CHCH₃), 0.78 (d, J = 6.5 Hz, 3H, CHCH₂CH-

CH₃). ¹³C NMR (101 MHz, DMSO‑d₆): δ 182.76, 174.48, 171.58,

169.67, 165.11, 161.99, 155.90, 153.92(2C), 153.21, 129.18(2C),

124.25, 121.52, 116.56(2C), 106.04, 103.40, 99.98, 94.02, 75.84,

75.73, 73.23, 71.80, 60.65, 52.55, 50.86, 31.33, 29.55, 24.71, 23.40,

21.89, 19.66, 18.70. HRESIMS m/z (pos): 733.2458 C₃₄H₄₁N₂O₁₆

(calcd. 733.2456).

120 °C; desolvation temperature, 350 °C; cone gas ﬂow, 50 l/h; deso-

lvation gas ﬂow, 650 l/h; collision gas ﬂow, 0.16 mL/min; capillary

voltage, 3.0 kV. The multi reaction monitor (MRM) mode was used. In

the positive ion mode (ESI⁺), the conﬁrmation ion pairs were (m/z)

691.1 → 287(5a), 739.1 → 287 (5b), 721.1 → 287 (5c), 707.1 → 287

(5d), 705.2 → 287 (5e), 705.2 → 287 (5f), 691.1 → 287 (5g), 767.2 →

287 (5 h), 749.2 → 287 (5i), 735.1 → 287 (5j), 733.2 → 287 (5k),

733.2 → 287(5l). The cone voltage and collision voltage were in the

range of 30–40 V. The UPLC-MS/MS results are summarized in Table 1.

4.3. Biological evaluation

Melatonin, dimethyl sulfoxide (DMSO), Lipofectamine^TM2000

transfection reagent, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-

trazolium bromide (MTT) were purchased from Sigma-Aldrich (St.

Louis, MO, USA). The Hoechst 33,342 nuclear staining assay kit and

Annexin V/propidium iodide (PI) apoptosis detection kit were obtained

from the Beyotime Institute of Biotechnology (BD Pharmingen^TM, USA).

Anti-β-actin (abs132001) was purchased from Absin Bioscience Inc

(Shanghai, China). Anti-caspase 3 (ab32351), anti-Bcl-2 (ab32124), and

anti-Bax (ab32503) were obtained from Abcam (Cambridge, MA, USA).

For the in vitro studies, compounds 5a-l and scutellarin were dissolved

in DMSO and formulated into 25 mM stock solutions.

4.1.13. ((4-(5,6-dihydroxy-4-oxo-7-(((2S,3S,4R,5R,6S)-3,4,5-

trihydroxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)-4H-

chromen-2-yl)phenoxy)carbonyl)-^L-valyl-L-alloisoleucine (5l)

Yield 52.1%, light yellowish green solid. ¹H NMR (400 MHz,

DMSO‑d₆) δ 10.43 (s, 1H, 6-OH), 8.09 (d, J = 7.4 Hz, 1H, NHCHC-

OOH), 7.93 (d, J = 8.3 Hz, 2H, C_2′6′–2H), 7.80 (d, J = 8.1 Hz, 1H,

NHCH-CH₃), 7.07 (s, 1H, C₃-H), 6.92 (d, J = 8.5 Hz, 2H, C_3′5′–2H), 6.88

(s, 1H, C₈-H), 5.31 – 5.27 (m, 1H, C_1′′-H), 4.18 – 4.10 (m, 2H, CHCH₂CH

(CH₃)₂, CHCH₃), 3.97 (t, J = 7.5 Hz, 1H, C_3″-H), 3.62 (s, 3H, COOCH₃),

1.98 (dt, J = 12.8, 6.0 Hz, 1H, CH(CH₃)₂), 1.73 – 1.72 (m, 1H, CH

(CH₃)–CH₂CH₃), 1.38 (dt, J = 10.5, 5.2 Hz, 1H, CH(CH₃)CHCH₃), 1.21

– 1.12 (m, 1H, CH(CH₃)CHCH₃), 0.90 – 0.89 (m, 6H, CH(CH₃)₂), 0.82 –

0.79 (m, 6H, CH(CH₃)–CH₂CH₃). ¹³C NMR (101 MHz, DMSO‑d₆) δ

182.77, 173.31, 171.86, 169.67, 165.16, 162.00, 155.93, 153.97,

153.25, 129.20(2C), 124.27, 121.57, 116.58(2C), 106.08, 103.44,

100.03, 94.04, 75.84, 75.76, 73.28, 71.81, 60.56, 56.94, 55.44, 52.54,

36.75, 31.34, 25.22, 19.64, 18.73, 15.99, 11.90. HRESIMS m/z (pos):

733.2468 C₃₄H₄₁N₂O₁₆(calcd. 733.2456).

4.3.1. Caco-2 cell permeability assay

4.3.1.1. Caco-2 cell culture. The Caco-2 cells were obtained from the

Shanghai Institute of Material Medica (SIMM) and seeded onto

Millicell^TMCaco-2 plate at a density of 1.0 × 10⁵cells/cm². The

Caco-2 cells were cultured in Dulbecco’s Modiﬁed Eagle’s Medium

(DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL

penicillin and streptomycin, 3.7 g/L NaHCO₃, and 1% L-glutamine

(Gln). The cells were grown at 37 °C in a humidiﬁed incubator with an

atmosphere of 5% CO₂.

4.3.1.2. Caco-2 cell-based permeability assay. Caco-2 cells were seeded

at a density of approximately 1.0 × 10⁵cells/cm²on a 6-well

Millicell^TMplate and left to grow for 21 days until reaching

conﬂuence and diﬀerentiation. The integrity and transportation

ability of the Caco-2 cell monolayer were examined by measuring the

transepithelial electrical resistance (TEER) utilizing an epithelial

voltohmmeter (Millicell-ERS electrical resistance system, Millipore,

Bedford, MA). Inserts with TEER values ≥ 500 Ω cm²in the culture

medium were selected for the transport experiments. On the 21st day,

the Caco-2 cell monolayer was washed three times with a warm HBSS

medium (pH = 7.4) and equilibrated in the same buﬀer. To determine

the rate of the drug transport in the apical to basolateral direction,

4.2. Physiochemical property studies

For in vitro stability evaluation, 1 mg of the target compounds 5a–l

was dissolved in 20 μL DMSO to obtain stock solutions. Distilled water

was then added to the above solutions to achieve a ﬁnal concentration

of 0.2 mg/mL. Subsequently, 200 μL of the diluted solution was added

to 2 mL of plasma and intestinal ﬂuid. The resulting solutions were

maintained at 37

0.5 °C in screw-capped vials in a water bath. The

samples were withdrawn at appropriate time intervals, and analyzed by

ultra-performance liquid chromatography-tandem mass spectrometry

(UPLC–MS/MS). For the solubility test, 50 μL of distilled water was

added to 2–4 mg of compounds 5a–l. The solutions were ultrasonicated

and ﬁltered through a microporous membrane ﬁlter (0.45 μm). The

saturated solutions were then diluted with distilled water to the cor-

responding multiple and the solubility for each compound was calcu-

lated using standard curves.

0.4 mL of target compounds 5a-l with a concentration of 1.5 × 10⁻⁴

M

and scutellarin with a concentration of 2.0 × 10⁻⁴M were added to the

apical plate (AP), and the transport basolateral plate (BL) was ﬁlled

with 0.6 mL of the HBSS buﬀer. Furthermore, to determine the

transport rates in the basolateral to apical direction, 0.6 mL of the

target compounds 5a-l with a concentration of 1.5 × 10⁻⁴M and

scutellarin with a concentration of 2.0 × 10⁻⁴M were added to the BL

plate, and the ﬁlter wells (apical compartment, AP) were ﬁlled with

0.4 mL of the HBSS buﬀer. To establish the target compounds 5a-l and

scutellarin, 50 μL of the HBSS solution was taken from the AP or BL side

and 150 μL of methanol was added to dilute the solution. The solution

was subsequently centrifuged at 15,000 r/min for 10 min. A 10 μL

aliquot of the supernatant solution was used for the UPLC–MS/MS assay

according to the method described for the evaluation of the

physiochemical properties. The obtained results are summarized in

Table 2 and Fig. 1.

4.2.1. UPLC–MS/MS analysis conditions

UPLC analyses were performed utilizing a Waters ACQUITY UPLC

instrument. The samples were separated on a BEH C l8 column

(2.1 mm × 50 mm, 1.7 μm). The mobile phase consisted of acetonitrile

containing 0.1% formic acid (A) and water containing 0.1% formic acid

(B). The elution gradient was as follows: 10% A (0–2 min), 90% A

(2–3 min), and 10% A (3 min). The mobile phase ﬂow rate was set at

0.35 mL/min and the column temperature was 45 °C. The injection

volume was 1 μL.

Mass spectrometry: All of the mass spectrometry experiments were

carried out using a Waters ACQUITY TQD (triple quadrupole) spec-

trometer equipped with a Z-spray ESI source connected to the ACQUITY

UPLC system. The acquisition parameters included collision gas, argon

(Ar); nebulizing and drying gas, nitrogen (N₂); source temperature,

4.3.2. Cell culture and stable transfection

The Madin Darby Canine Kidney (MDCK) cells were obtained from

the American Type Culture Collection (Manassas, VA, USA) and grown

14

T. Li, et al.

BioorganicChemistry101(2020)103980

in DMEM. The BCA protein quantitation reagent was purchased from

Beijing Solarbio Science & Technology Co., Ltd. Lipofectamine^TM2000

was obtained from Invitrogen (Carlsbad, CA), while the anti-IgG anti-

body was purchased from Santa Cruz Biotechnology. The pcDNA3.1-

hPepT1 plasmid was constructed by Beijing Bomeid Gene Technology

Co., Ltd.

ﬂuorescence microscope. The condensation and fragmentation of nuclei

was considered to be representative of apoptosis.

For the detection of intracellular reactive oxygen species (ROS), the

cells were incubated with 10 μmol/L DCFH-DA at 37 °C for 30 min.

Following washing twice with PBS, the cells were harvested and sus-

pended in the same buﬀer. The ﬂuorescence intensity was measured

using a ﬂow cytometer (BD).

According to D'Mello et al. [35], the transfection protocol involved

transfection of the MDCK cells with the pcDNA3.1-hPepT1 plasmid

using Lipofectamine. Brieﬂy, cells were seeded in 6-well plates at a

density of 3 × 10⁵cells/well and incubated at 37 °C. After 24 h, the

volume/mass ratio of Lipofectamine^TM2000:pcDNA3.1-hPepT1 (2:1)

was transfected into the MDCK cells. After 48 h, G418 was added at the

ﬁnal concentration of 800 μg/ml and the cells were incubated for

2 weeks until antibiotic-resistant colonies were observed for the stably

hPepT1-overexpressed mono-clone MDCK cells. Subsequently, hPepT1-

MDCK stably transfected cells and MDCK were incubated with the ty-

pical substrate Gly-Sar (200 µM) at 37 °C for 5–50 min to identify the

substrate uptake conditions.

4.3.4.2. Measurement of the mitochondrial membrane potential

(ΔΨm). The mitochondrial membrane potential (ΔΨm) was

determined using the Rhodamine 123 staining kit. The PC12 cells

were grown in a 6-well plate at a density of 8 × 10⁴cells/well. The

cells were then washed twice with PBS and incubated with 10 μg/ml

Rhodamine 123 staining solution at 37 °C for 30 min in darkness. After

washing away the unbound dye, the cells were ﬁxed and observed

under a ﬂuorescence microscope.

4.3.4.3. Annexin V/PI staining assay. Apoptotic cells were analyzed

utilizing the Annexin

V

(ﬂuorescein isothiocyanate (FITC)-

4.3.2.1. Western blotting of the PepT1 expression. The above two kinds of

cells were collected and lysed by radio immunoprecipitation assay

(RIPA) buﬀer containing 1 mM phenylmethylsulfonyl ﬂuoride (PMSF).

The protein concentration was measured utilizing a BCA assay kit

(Solarbio), the samples were separated by SDS-PAGE gel

electrophoresis and transferred onto a polyvinylidene ﬂuoride (PVDF)

membrane. The employed primary antibodies included the anti-IgG

antibody obtained from Santa Cruz Biotechnology.

conjugated)/PI apoptosis kit (BD Pharmingen^TM

, USA) by ﬂow

cytometry. The cells were subcultured in a 6-well plate at a density of

1 × 10⁵cells/well. Following treatment with varying concentrations of

scutellarin and 5k (2, 10, and 20 μM) for 24 h, the cells were harvested

and resuspended in DMEM. Subsequently, the cells were incubated with

5 μL of Annexin V-FITC and 5 μL of PI for 15 min in the dark. After

incubation, 400 μL of the buﬀer was added to each sample and the

sample were analyzed using a ﬂow cytometer (BD FACSCantoⅡ). The

percentage of cells residing in the lower right region of the scatter plot

(early apoptotic cells) of Annexin V-FITC was calculated for

comparison.

4.3.3. Uptake of target compounds in the hPepT1 –MDCK cells

Based on the above analysis, the uptake mechanism for target

compounds 5d, 5g, 5i, and 5k in the hPepT1-MDCK cells was evaluated

at 30 min. Subsequently, the RIPA buﬀer (250 μL) was added to lyse the

cells. The cell lysis solution was then centrifuged at 12,000g for 20 min

at 4 °C and an aliquot of the supernatant (20 μL) was withdrawn for the

determination of the protein levels using the BCA protein assay kit. In

the second experiment, methanol (800 μL) was added to the cell lysis

solution (200 μL) and the mixed solution was centrifuged twice at

12,000g for 20 min at 4 °C to precipitate the proteins. An aliquot of the

supernatant solution (150 μL) was assayed using the UPLC-MS/MS

method established for the evaluation of the physiochemical properties

described above. All of the results are illustrated in Fig. 6 (A-C).

4.3.4.4. Cell cycle study. Based on the cell viability assay results, the

PC12 cells were seeded into 6-well plates at a density of 8 × 10⁴cells/

well and treated with 2, 10, and 20 μM of compound 5k and 20 μM of

Scu for 24 h. Following this treatment, the cells were digested with

trypsin and centrifuged for 5 min (1500g). One mL of ice-cold PBS was

then added to wash the centrifuged cells twice before ﬁxing the cells

with formalin for 30 min. Subsequently, the cellular membrane was

broken using 0.5%Triton-X100 for 20 min at room temperature. The

cells were then washed twice with PBS and collected by centrifugation.

Propidium iodide (0.5 mL) was added to each tube to stain the cells,

which were incubated in the dark at 37 °C for 30 min. The samples were

then immediately analyzed with a ﬂow cytometer (BD canto II plus).

4.3.4. The lactate dehydrogenase (LDH) and malondialdehyde (MDA)

assay

The PC12 cell line was obtained from Shanghai Cell Bank of the

Chinese Academy of Sciences and was maintained at 37 °C in a medium

supplemented with 5% heat-inactivated horse serum, 10% FBS, 100 U/

mL penicillin, and 100 U/mL streptomycin.

4.3.4.5. Western blot analysis. The PC12 cells were subcultured in a 6-

well plate at

2

×

10⁵cells/well and treated with varying

concentrations of Scu and 5k (2, 10, and 20 μM) for 24 h. The cells

were harvested, washed twice with cold PBS, and then lysed in the RIPA

extraction buﬀer containing 1 mM PMSF for 30 min on ice. The lysates

were centrifuged at 12,000 rpm for 20 min at 4 °C. The supernatants

were subsequently collected, and the protein concentrations were

determined utilizing the Bio-rad Dc protein assay (Bio-rad

Laboratories, CA, USA) using bovine serum albumin as the standard.

Following SDS-PAGE, the protein samples were transferred onto a PVDF

membrane. The employed primary antibodies included anti-caspase 3

(ab32351), anti-Bcl-2 (ab32124), anti-Bax (ab32503), and anti-β-actin

(abs132001).

After the cells were exposed to a 1000 µM solution of H₂O₂in the

presence of scutellarin for 24 h, the medium was collected, and the

amount of LDH released by the cells was determined using an assay kit

according to the manufacturer’s protocol. The absorbance of the sam-

ples was read at 450 nm. The concentration of free MDA in the PC12

cells, i.e., the product formed due to peroxidation of the cell membrane,

was assessed using an MDA kit according to the manufacturer’s in-

structions. In brief, the assay was based on the reaction of MDA with

thiobarbituric acid (TBA), forming stable thiobarbituric acid-reactive

substances (TBARS), which absorb at 530 nm. The lipid peroxide level

was expressed as nmol of MDA per mg of protein.

4.3.4.6. Molecular docking assay. The Schrodinger software was used

for the molecular docking studies. The crystal structure of PepT1 (PDB

ID: 4d2c) was obtained from the Protein Data Bank. Proteins were

prepared using the Protein Preparation Wizard based on the force ﬁeld

OPLS (optimized potentials for liquid simulations) 2005. Compound 5k

was prepared using the LigPrep module to transform to the three-

dimensional structure. The calculation was performed based on the

Schrodinger extra precision (XP) mode and the structure with the

4.3.4.1. Cytotoxicity assay with Hoechst 33,342. The PC12 cells were

grown in 6-well plates at a density of 8 × 10⁴cells/well. The cells were

treated with varying concentrations of scutellarin and 5k (2, 10, and

20 μM) for 24 h. Following this treatment, the cells were stained with

Hoechst 33,342 for 10 min at 37 °C. The cells were then washed three

times with PBS and the ﬂuorescence images were acquired utilizing a

15

T. Li, et al.

BioorganicChemistry101(2020)103980

lowest energy was chosen as the ﬁnal result. The details of the docking

procedure were as follows: 1) Protein preparation: The PepT1 (PDB ID:

4d2c) protein was used as the docking template. The protein was

processed and optimized using the Protein Preparation Wizard module

of the Schrodinger software. 2) Ligand preparation: The ligand

structure was drawn in ChemDraw, and the three dimensional

structure of the molecule was transformed utilizing the LigPrep

module to generate diﬀerent conformations. 3) Molecular docking:

The XP mode in the Schrodinger software was employed for molecular

docking. Based on the binding mode of compound 5k and the proteins,

the docking conformation with the lowest docking energy was deemed

as the ﬁnal result.

spontaneously.

4.3.5.7. Western blot analysis. After 48 h of HIE, six rat pups from each

group were sacriﬁced and the brains on the same side of the

embolization was immediately dissected and used for protein

extraction. The rat pups’ brains were homogenized in the RIPA buﬀer

containing 1% protease and phosphatase inhibitor. The total proteins

were collected from the supernatant after centrifugation at 12,000g for

15 min at 4 °C. Protein concentrations were measured using the

Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA).

The samples were separated by the SDS-PAGE gel electrophoresis and

transferred onto a PVDF membrane. The employed primary antibodies

included rabbit anti-Amyloid Precursor Protein (ab32136) and anti-

BACE1 (ab108394). The data was analyzed by the Quantity one

software.

4.3.5. In vivo neuroprotective characteristics evaluation

4.3.5.1. Animals. Four male Sprague-Dawley (SD) rats and 10–12

female rats were purchased from Tianqin Biotechnology Co., Ltd.,

Changsha City, Hunan Province.

undenatured SD rat pups were used.

A

total of 300 seven-day-old

4.4. Pharmacokinetic evaluation of scutellarin and its prodrug 5k

The male SD rats (200 ~ 220 g) were divided into two groups, each

with six rats, and fasted for 12 h prior to intravenous administration of

scutellarin and 5k at a dose of 10 mg/kg and 16 mg/kg, respectively.

Blood samples (0.5 mL) were collected from suborbital vein into he-

parinized 1.5 mL centrifuge tubes at 0, 2, 5, 10, 30, 45, 60, and 120 min

following drug administration. All of the blood samples were cen-

trifuged at 15000g for 10 min after which the plasma samples were

collected. The plasma concentrations of scutellarin and its prodrug 5k

were measured using the LC-MS/MS method (injection volume of 2 μL)

described above for the evaluation of the physiochemical properties.

The PK parameters were calculated utilizing DAS (version 2.0).

4.3.5.2. Hypoxic-ischemic encephalopathy (HIE) model. The HIE model

of neonatal rats was established based on the experimental scheme of

the rodent model established by Rice et al. [39]. Rat pups produced by

mating between male and female SD rats were anesthetized with 2–4%

isoﬂurane and their right carotid arteries were surgically ligated.

Subsequently, the rat pups were returned to 37 °C for 1 h before

being placed in a hypoxic chamber (8% oxygen-92% nitrogen) for

120 min. Pups treated by the sham operation were generated by

ligating the ipsilateral carotid artery, but not subjecting them to

hypoxic conditions.

4.3.5.3. Drug administration method. The rat pups were divided into ﬁve

groups, i.e., the sham group (n = 6), HIE + vehicle group (n = 6),

HIE + Scu (2.8 mg/kg) group (n = 6), HIE + 5k (0.84 mg/kg) group

(n = 6), and HIE + 5k (2.8 mg/kg) group (n = 6). The pups were

given an intraperitoneal injection of the tested drugs from the sixth day

after birth. They were then treated with the HIE regimen, and the drugs

were given for another 48 h by using the same route of administration.

4.5. Statistical analysis

Data are shown as the mean

standard deviation (SD). Statistical

analysis was performed using one-way ANOVA or Student's t-test. A P-

value of

< 0.05 was considered to be statistically signiﬁcant.

GraphPad Prism was employed to perform the statistical analyses and

generate the graphs.

4.3.5.4. TTC staining results. Cerebral infarct size was measured by

2,3,5-triphenyltetrazolium chloride (TTC) staining 48 h after HIE [40].

Brieﬂy, the rats were decapitated after neurological evaluation, and the

brains were removed quickly before being sliced into six uniform

coronal sections (2 mm thickness each). The sections were stained with

2% TTC (Sigma-Aldrich) at room temperature for 15 min and then ﬁxed

in a 4% formaldehyde solution. The normal brain tissue was stained

red, while the infarct areas were pale. The posterior surface of each

slice was photographed and analyzed utilizing the ImageJ software. The

Declaration of Competing Interest

None.

Acknowledgements

This work was supported by the grants from National Natural

Science Foundation of China (NSFC Nos. 81260473 and 81460523),

Projects of Guizhou Science and Technology Department (No. 2012-

3013), and Excellent Youth Scientiﬁc Talents Foundation of Guizhou

Province (No. 2013-45).

infarct area was calculated as

contralateral hemisphere.

a percentage of the area of the

4.3.5.5. HE and Nissl staining results. After HIE rat pups were killed by

deep anesthesia, the brains were perfused with heparinized physiologic

saline, followed by 4% paraformaldehyde. The brains were removed

and postﬁxed in 4% paraformaldehyde for 24 h before being embedded

in paraﬃn. For measuring HE, the staining sections were cut and

stained with hematoxylin and eosin 48 h after HIE, while for evaluating

Nissl, the staining sections were stained with toluidine blue staining

solution 4 weeks after HIE.

Appendix A. Supplementary material

Supplementary data to this article can be found online at https://

doi.org/10.1016/j.bioorg.2020.103980.

References

[1] M. Fotuhi, V. Hachinski, P.J. Whitehouse, Changing perspectives regarding late-life

dementia, Nat. Rev. Neurol. 5 (12) (2009) 649–658 .

[2] R.C. Vannucci, J.R. Connor, D.T. Mauger, et al., Rat model of perinatal hypoxic-

ischemic brain damage, Neurosci. Res. 55 (2) (1999) 158–163 .

[3] S. Adhikari, K.S. Rao, Neurodevelopmental outcome of term infants with perinatal

asphyxia with hypoxic ischemic encephalopathy stage II, Brain Dev. 39 (2) (2017)

107–111 .

4.3.5.6. Neurological deﬁcit score. The neurological deﬁcit score of each

rat pup 48 h after HIE was evaluated by an assessor, who was blinded to

the treatment protocols. The neurological deﬁcit was determined

according to a ﬁve-point scale established by Bederson et al. [41], as

follows: 0 = no neurological symptoms; 1 = unable to fully extend the

front paw on the contralateral side; 2 = circling to the contralateral

side; 3 = falling to the contralateral side; and 4 = unable to walk

[4] C. McLean, D. Ferriero, Mechanisms of hypoxic-ischemic injury in the term infant,

Semin. Perinatol. 28 (6) (2004) 425–432 .

[5] J.M. Lorenz, D.E. Wooliever, J.R. Jetton, et al., A quantitative review of mortality

and developmental disability in extremely premature newborns, Arch. Pediatr.

16

T. Li, et al.

BioorganicChemistry101(2020)103980

Adolesc. Med. 152 (5) (1998) 425–435 .

[24] F. Cao, J. Jia, Z. Yin, et al., Ethylene glycol-linked amino acid diester prodrugs of

oleanolic acid for PepT1-mediated transport: synthesis, intestinal permeability and

pharmacokinetics, Mol. Pharm. 9 (8) (2012) 2127–2135 .

[6] D. Doycheva, G. Shih, H. Chen, et al., Granulocytecolony stimulating in combina-

tion with stem cell factor confers greater neuroprotection after hypoxic-ischemic

brain damage in the neonatal rats than a solitary treatment, Transl. Stroke Res. 4 (2)

(2013) 171–178 .

[25] Y. Gong, X. Wu, T. Wang, et al., Targeting PEPT1: A novel strategy to improve the

antitumor efcacy of Doxorubicin in human hepatocellular carcinoma therapy,

Oncotarget 8 (25) (2017) 40454–40468 .

[7] J. Shetty, Neonatal seizures in hypoxic-ischemic encephalopathy–risks and beneﬁts

of anticonvulsant therapy, Dev. Med. Child Neurol. 57 (2015) 40–43 .

[8] B.J. Dixon, C. Reis, W.M. Ho, et al., Neuroprotective strategies after neonatal hy-

poxic ischemic encephalopathy, Int. J. Mol. Sci. 16 (9) (2015) 22368–22401 .

[9] L. Wang, Q. Ma, Clinical beneﬁts and pharmacology of scutellarin: a comprehensive

review, Pharmacol. Ther. 190 (2018) 105–127 .

[26] B.S. Anand, J. Patel, A.K. Mitra, Interactions of the dipeptide ester prodrugs of

acyclovir with the intestinal oligopeptide transporter: competitive inhibition of

glycylsarcosine transport in human intestinal cell line-Caco-2, J. Pharmacol. Exp.

Ther. 304 (2) (2003) 781–791 .

[27] L. Fang, M. Wang, S. Gou, et al., Combination of amino acid/dipeptide with nitric

oxide donating oleanolic acid derivatives as PepT1 targeting antitumor prodrugs, J.

Med. Chem. 57 (3) (2014) 1116–1120 .

[10] J. Zhu, L. Chen, Y. Qi, et al., Protective eﬀects of Hand.- Mazz. (EBHM) extract in

retinal neurodegeneration models, Mol. Vis. 24(2018) 315–325.

[11] H. Liu, X. Yang, R. Tang, et al., Eﬀect of scutellarin on nitric oxide production in

early stages of neuron damage induced by hydrogen peroxide, Pharmacol. Res. 51

(3) (2005) 205–210 .

[28] L.H. Zhang, L. Zhang, T. Luo, Synthesis and evaluation of a dipeptide drug con-

jugate library as substrates for PEPT1, ACS Comb. Sci. 14 (2012) 108–114 .

[29] G. Ni, Y. Tang, M. Li, et al., Synthesis of Scutellarein Derivatives with a Long

Aliphatic Chain and Their Biological Evaluation against Human Cancer Cells.

Molecules. 23(2)(2018) pii: E310.

[12] H. Hong, G.Q. Liu, Scutellarin attenuates oxidative glutamate toxicity in PC12 cells,

Planta Med. 70 (5) (2004) 427–431 .

[13] L.L. Guo, Z.Z. Guan, Y.L. Wang, Scutellarin protects against Abeta-induced learning

and memory deﬁcits in rats: involvement of nicotinic acetylcholine receptors and

cholinesterase, Acta Pharmacol. Sin. 32 (12) (2011) 1446–1453 .

[14] H.F. Zhang, X.M. Hu, L.X. Wang, et al., Protective eﬀects of scutellarin against

cerebral ischemia in rats: Evidence for inhibition of the apoptosis-inducing factor

pathway, Planta Med. 75 (2) (2009) 121–126 .

[30] F. Cao, J.X. Guo, Q.N. Ping, et al., Prodrugs of scutellarin: ethyl, benzyl and N, N-

diethylglycolamide ester synthesis, physicochemical properties, intestinal metabo-

lism and oral bioavailability in the rats, Eur. J. Pharm. Sci. 29 (5) (2006) 385–393 .

[31] Z.X. Dong, Z.H. Shi, N.G. Li, et al., Design, Synthesis, and Biological Evaluation of

Scutellarein Derivatives Based on Scutellarin Metabolic Mechanism In Vivo. Chem.

Biol. Drug Des. 87(6)(2016)946-957.

[15] L. Qian, M. Shen, H. Tang, et al., Synthesis and protective eﬀect of scutellarein on

focal cerebral ischemia/reperfusion in rats, Molecules 17 (9) (2012) 10667–10674 .

[16] Y. Yuan, H. Zha, P. Rangarajan, et al., Anti-inﬂammatory eﬀects of Edaravone and

Scutellarin in activated microglia in experimentally induced ischemia injury in rats

and in BV-2 microglia, BMC Neurosci. 15 (2014) 125 .

[32] C. Chu, C.T. Okamoto, S.F. Hamm-Alvarez, et al., Stable transfection of MDCK cells

with epitope-tagged human PepT1, Pharm. Res. 21 (11) (2004) 1970–1973 .

[33] Y. Fujisawa, R. Tateoka, T. Nara, et al., The extracellular pH dependency of

transport activity by human oligopeptide transporter 1 (hPEPT1) expressed stably

in Chinese hamster ovary (CHO) cells: a reason for the bell-shaped activity versus

pH, Biol. Pharm. Bull. 29 (5) (2006) 997–1005 .

[17] L.L. Guo, Z.Z. Guan, Y. Huang, et al., The neurotoxicity of beta-amyloid peptide

toward rat brain is associated with enhanced oxidative stress, inﬂammation and

apoptosis, all of which can be attenuated by scutellarin, Exp. Toxicol. Pathol. 65 (5)

(2013) 579–584 .

[34] Y. Wang, R. Wang, Y. Wang, et al., Ginkgo biloba extract mitigates liver fbrosis and

apoptosis by regulating p38 MAPK, NF-κ B/I κ Bα, and Bcl-2/Bax signaling, Drug Des.

Devel. Ther. 9 (2015) 6303–6317 .

[18] J.W. Shin, K.J. Kweon, D.K. Kim, et al., Scutellarin Ameliorates Learning and

Memory Deﬁcit via Suppressing β-Amyloid Formation and Microglial Activation in

Rats with Chronic Cerebral Hypoperfusion, Am. J. Chin. Med. 46 (6) (2018)

1203–1223 .

[35] S.R. D'Mello, C.Y. Kuan, R.A. Flavell, et al., Caspase-3 is required for apoptosis-

associated DNA fragmentation but not for cell death in neurons deprived of po-

tassium. J. Neurosci. Res. 59(1)(2000)24-31.

[36] R. Vassar, B.D. Bennett, S. Babu-Khan, et al., Beta-secretase cleavage of Alzheimer's

amyloid precursor protein by the transmembrane aspartic protease BACE, Science.

286 (5440) (1999) 735–741 .

[19] Y.J. Gong, J.X. Wang, Y. Zhang, et al., Intestinal lymphatic transport of breviscapine

orally administered in rat. Acta. Pharm. Sin. 46(10)(2011)1262-1267.

[20] X. Chen, L. Cui, X. Duan, et al., Pharmacokinetics and metabolism of the ﬂavonoid

scutellarin in humans after a single oral administration, Drug Metab Dispos 34 (8)

(2006) 1345–1352 .

[37] X. Sun, G. He, H. Qing, et al., Hypoxia facilitates Alzheimer's disease pathogenesis

by up-regulating BACE1 gene expression, Proc. Natl. Acad. Sci. USA 103 (49)

(2006) 18727–18732 .

[21] X.M. Hu, M.M. Zhou, X.M. Hu, et al., Neuroprotective eﬀects of scutellarin on rat

neuronal damage induced by cerebral ischemia/reperfusion, Acta Pharmacol. Sin.

26 (12) (2005) 1454–1459 .

[38] J.A. Bonds, A. Shetti, A. Bheri, et al., Depletion of Caveolin-1 in Type-2 Diabetes

Model Induces Alzheimer's disease Pathology Precursors. J. Neurosci. 39(43)(2019)

8576-8583.

[22] Y. Wang, H. Ao, Z. Qian, et al., Intestinal transport of scutellarein and scutellarin

and ﬁrst-pass metabolism by UDPglucuronosyltransferase-mediated glucuronida-

tion of scutellarein and hydrolysis of scutellarin, Xenobiotica 41 (7) (2011)

538–548 .

[39] J.E. Rice 3rd, R.C. Vannucci, J.B. Brierley, The inﬂuence of immaturity on hypoxic-

ischemic brain damage in the rat, Ann Neurol. 9 (2) (1981) 131–141 .

[40] J.B. Bederson, L.H. Pitts, M. Tsuji, et al., Rat middle cerebral artery occlusion:

evaluation of the model and development of a neurologic examination. Stroke.

17(3)(1986) 472-476.

[23] W. Cong, L. Shen, D. Xu, et al., Solid dispersion tablets of breviscapine with poly-

vinylpyrrolidone K30 for improved dissolution and bioavailability to commercial

breviscapine tablets in beagle dogs, Eur. J. Drug Metab. Pharmacokinet. 39 (3)

(2014) 203–210 .

[41] L. Ye, Z. Feng, D. Doycheva, et al., CpG-ODN exerts a neuroprotective eﬀect via the

TLR9/pAMPK signaling pathway by activation of autophagy in a neonatal HIE rat

model, Exp. Neurol. 301 (2018) 70–80 .

17

Article Doi

Synthesis, pharmacological evaluation and mechanistic study of scutellarin methyl ester -4′-dipeptide conjugates for the treatment of hypoxic-ischemic encephalopathy (HIE) in rat pups

DOI: 10.1016/j.bioorg.2020.103980

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.bioorg.2020.103980

Full text of DOI:10.1016/j.bioorg.2020.103980

Products guided by the article

R&D Labs maybe for 1432474-27-5

Relevant to this article

Hot Product