Design, synthesis, and biological evaluation of hederagenin derivatives with improved aqueous solubility and tumor resistance reversal activity

European Journal of Medicinal Chemistry 2 1 1 (2021) 1 13107

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Research paper

Design, synthesis, and biological evaluation of hederagenin derivatives

with improved aqueous solubility and tumor resistance reversal

activity

Binghua Wang ¹, Shuqi Liu ¹, Wentao Huang, Mengxin Ma, Xiaoqian Chen, Wenxuan Zeng,

Kaicheng Liang, Hongbo Wang^*, Yi Bi^**, Xiaopeng Li

School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation

Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, 264005, PR China

a r t i c l e i n f o

a b s t r a c t

Article history:

Multidrug resistance (MDR) has become a major obstacle to malignancies treatment by chemothera-

peutic drugs, therefore, it is important to develop MDR reversal agents with high activity. We have

previously found that the hederagenin (HD) derivative HBQ showed good tumor MDR reversal activity

in vitro and in vivo but had poor solubility. In this study, to enhance the aqueous solubility and tumor

MDR reversal activity of HBQ, three series of HD derivatives were designed and synthesized. Nitrogen-

containing heterocyclic-substituted, PEGylated, and ring-A substituted derivatives signiﬁcantly reversed

the MDR phenotype of KBV (multidrug-resistant oral epidermoid carcinoma) cells toward paclitaxel at a

Received 22 September 2020

Received in revised form

5 December 2020

Accepted 14 December 2020

Available online 17 December 2020

Keywords:

Hederagenin

concentration of 10 mM in MTT assays. The PEGylated derivatives 10ce10e had increased aqueous sol-

ubility compared with HBQ by 18e657 fold, while maintaining tumor MDR reversal activity. The most

in vitro active compound 10c possessed good chemical stability to an esterase over 24 h and enhanced

the sensitivity of KBV cells to paclitaxel and vincristine with IC₅₀values of 4.58 and 0.79 nM, respectively.

Mechanism studies indicated that compound 10c increased the accumulation of P-glycoprotein (P-gp)

substrates rhodamine 123 and Flutax1 in KBV cells and MCF-7T (paclitaxel-resistant breast carcinoma)

cells, that is to say, compound 10c exerted the reversal effect of tumor MDR by inhibiting the efﬂux

function of P-gp. Finally, the structureeactivity relationships were further investigated by analyzing the

relationship between structure and tumor MDR reversal activity of HD derivatives. This study highlights

the potential of PEGylated HD derivatives such as compound 10c for the development of tumor MDR

reversal agents and provides information for the further improvement of the aqueous solubility and

tumor MDR reversal activity of HD derivatives in the future.

PEGylated derivatives

Synthesis

Tumor resistance reversal activity

Aqueous solubility

P-glycoprotein

1. Introduction

particularly important to overcome the MDR phenotype, and the

development of MDR reversal agents has become the focus of

Tumor multidrug resistance (MDR) phenotype refers to the

cross-resistance of tumor cells to antitumor drugs with different

structures and mechanisms of action, which is the main reason for

the failure of tumor chemotherapy [1,2]. The majority of antitumor

drugs have a small difference between the dose to achieve a ther-

apeutic effect and the dose that produce toxicity. Therefore, it is

current tumor treatment [3e5]. The overexpression of P-glyco-

protein (P-gp) is considered to be one of the major mechanisms

that induce MDR. P-gp is a member of the family of ATP binding

cassette (ABC) transporters [6,7], and it uses ATP hydrolysis to drive

the efﬂux of antitumor drugs, thus decreasing the efﬁcacy of

chemotherapy and leading to MDR [8,9]. Therefore, inhibition of P-

gp is a potential method to overcome MDR, but it has only achieved

limited success in clinical research [10]. As a ﬁrst-generation P-gp

inhibitor, verapamil (Fig. 1) signiﬁcantly enhances the sensitivity of

drug-resistant tumor cells to chemotherapeutic drugs, such as

paclitaxel, but has considerable cardiac toxicity. Second and third-

generation inhibitors such as dofequidar (MS-209, Fig. 1),

* Corresponding author.

** Corresponding author.

E-mail addresses: hongbowangyt@gmail.com (H. Wang), beeyee_413@163.com

(Y. Bi).

1

These authors have equally contributed to the work.

https://doi.org/10.1016/j.ejmech.2020.1 13107

B. Wang, S. Liu, W. Huang et al.

European Journal of Medicinal Chemistry 211 (2021) 113107

Fig. 1. Chemical structures of verapamil, dofequidar, zosuquidar and tariquidar.

zosuquidar (LY335979, Fig. 1) and tariquidar (XR9576, Fig. 1), are

limited in clinical application because of inherent toxicity or poor

potency [1 1,12]. Therefore, the search for novel tumor MDR reversal

agents with strong activity and low toxicity has become important

in the ﬁeld of malignant tumor treatment.

Hederagenin (HD, Fig. 2), ﬁrst found in the leaves of English ivy

in 1849, is an oleanane-type pentacyclic triterpene, which has been

found to have a variety of pharmacological effects, including anti-

tumor [20], anti-inﬂammatory [21], antidepressant [22], and anti-

neurodegenerative [23] effects. In particular, many researchers

have modiﬁed the structure of HD, and some of the derivatives

could enhance the antitumor activity of HD [24e27]. Our group ﬁrst

discovered that HD derivatives have tumor MDR reversal activity.

The derivative HBQ (Fig. 2), obtained by structural modiﬁcation of

HD, had good tumor MDR reversal activity both in vitro and in vivo

[28]. HBQ had no obvious cytotoxicity in vitro when used alone at a

Compared with the previous three generations of P-gp in-

hibitors, natural products have high chemical diversity and low

toxicity, which are considered to be a reliable source of potential P-

gp inhibitors and termed “the fourth-generation inhibitors” [13,14].

Many different structural types of natural products have been

shown to have tumor MDR reversal activity with less toxicity,

including ﬂavonoids [13,15], alkaloids [14,15], terpenes [15,16], and

coumarins [15]. Most notably, several triterpenoids with various

structures could effectively inhibit the function of P-gp in different

drug-resistant cells [1 7]. Nabekura et al. reported that glycyr-

rhetinic acid (Fig. 2) inhibited the function of P-gp in KB-C2

(multidrug-resistant human epidermal carcinoma) cells, and MDR

associated protein 1 (MRP1) in KB/MRP (human MRP1 gene-

transfected multidrug-resistant epidermal carcinoma) cells, which

enhanced the sensitivity of KB-C2 cells to vincristine and KB/MRP

cells to doxorubicin [18]. Ren et al. reported that the 24R-ocotillol-

type amide derivative OR1 (Fig. 2) signiﬁcantly enhanced the

sensitivity of drug-resistant tumor cells to paclitaxel in vitro and

in vivo and inhibited the efﬂux of the P-gp substrate rhodamine 123

in KBV (multidrug-resistant oral epidermoid carcinoma) cells by

increasing P-gp ATPase activity [1 7]. However, most of the natural

products with good activity had poor aqueous solubility and low

bioavailability, which has limited their applications [19]. Increasing

the aqueous solubility of natural products is one of the important

ways to improve the druggability of compounds.

concentration of 10

proliferative activity of paclitaxel in drug-resistant KBV cells with

an IC₅₀value of 2.39 0.74 nM when combined with 100 nM

mM, and signiﬁcantly increased the anti-

paclitaxel. In addition, the tumor growth inhibition rate was 41.88%

when HBQ (10 mg/kg) was combined with paclitaxel (30 mg/kg) in

a drug-resistant KBV cell xenograft nude mice model. HBQ can

reverse MDR by stimulating the ATPase activity of P-gp and then

competing with the chemotherapeutic drugs to bind to P-gp.

However, the poor solubility of HBQ has limited its further use. To

improve the aqueous solubility of HBQ, we synthesized a series of

HBQ derivatives, of which HBQ-5 (Fig. 2), is a representative de-

rivative. However, HBQ-5 had poor activity in vivo compared with

HBQ and limited solubility [29]. Therefore, further structural

modiﬁcation to improve the druggability of HBQ, while maintain-

ing or enhancing the activity, was investigated.

To overcome the poor aqueous solubility of small-molecule

drugs, polyethylene glycol (PEG, Fig. 3) has received extensive

attention in the structural modiﬁcation of small-molecule drugs in

recent years [30,31]. PEG comprises a repeating ethylene glycol

subunit structure and has good biocompatibility and high aqueous

solubility. PEGylation has been shown to improve the aqueous

solubility and pharmacokinetic properties of antitumor drugs such

as adriamycin, paclitaxel, and camptothecin [30,32,33]. NKTR-102

(Fig. 3), obtained by linking irinotecan to a four-arm PEG mole-

cule, has an extended half-life and increased area under the con-

centration time-curve (AUC) compared with irinotecan, and is in

phase III clinical trials for metastatic breast cancer [34]. Medina-

O’Donnell et al. have designed and synthesized a series of PEGy-

lated derivatives of maslinic acid (MA). The aqueous solubility of

the derivatives was higher than that of MA, and increased with

length of PEG chains, the compound MA-8 (Fig. 3) had 477-fold

higher solubility than MA [35]. Thus, PEGylation may be an effec-

tive method to improve the aqueous solubility of HBQ derivatives.

Therefore, in this study, to enhance the aqueous solubility and

tumor MDR reversal activity of HBQ, and to further explore the

structureeactivity relationships (SARs), we designed and synthe-

sized several series of HD derivatives. The designed strategies are

Fig. 2. Chemical structures of glycyrrhetinic acid, OR1, HD, HBQ and HBQ-5.

2

B. Wang, S. Liu, W. Huang et al.

European Journal of Medicinal Chemistry 211 (2021) 113107

Fig. 3. Chemical structures of PEG, NKTR-102, and MA-8.

shown in Fig. 4. First, several PEG molecules with different chain

lengths were introduced at the C-23 and C-28 positions of HBQ to

explore the inﬂuence of different chain lengths of PEG and different

modiﬁcation positions on the solubility and activity of the target

compounds. In addition, the presence of nitrogen-containing het-

erocycles such as piperazinyl, substituted piperazinyl, and mor-

pholinyl groups has been shown to enhance the tumor MDR

reversal activity of compounds [12,36], and previous SAR studies in

our group have indicated that the activity was improved when C-28

was substituted with a nitrogen-containing group. Therefore, we

designed and synthesized derivatives with nitrogen-containing

heterocycles substituted at C-28 in an attempt to improve the ac-

tivity of HBQ. Moreover, based on the good activity of HBQ, in

which ring-A is a fused pyrazine, the ring-A fused pyrazole de-

rivatives were designed by using the principles of bioisosterism

[37,38]. In addition, to further enrich the SARs, ring-A fused methyl

thiazole analogues were designed. Herein, we report the synthesis

of these derivatives and the evaluation of their tumor MDR reversal

activities in vitro. In addition, the aqueous solubility, in vitro sta-

bility, and mechanism of action of compound 10c were also

investigated.

previous synthetic method [28]. Compounds 6aed were prepared

by an amide reaction with different cyclic amines with NH moiety

after deprotection of the C-28 carboxyl of HBQ. The C-28 carboxyl

and C-23 hydroxyl of HBQ were reacted with ethyl bromoacetate

and succinic anhydride, respectively, to obtain intermediates 8 and

3, which were designed to reduce steric hindrance in the next

modiﬁcation with PEG. Intermediates 8 and 3 were reacted with

PEG with different chain lengths in the presence of 4-

dimethylaminopyridine (DMAP) and 1-(3-dimethylaminopropyl)-

3-ethylcarbodiimide hydrochloride (EDCI) in anhydrous dichloro-

methane (DCM) to obtain target compounds 9aee and 10aee,

respectively. In addition, using HD as the starting material, the C-28

carboxyl was protected by a benzyl group and the C-23 hydroxyl

was protected by a tert-butyldimethylsilyl (TBDMS) group in turn,

and then the C-3 hydroxyl was oxidized by pyridinium chlor-

ochromate (PCC) to obtain intermediate 13. Intermediate 13 was

reacted with ethylenediamine in morpholine at 150 ^ꢀC to furnish

14. Compound 18 was synthesized by oxidation of intermediate

14 at the C-12 position with 30% H₂O₂and deprotection of the C-23

hydroxyl. Intermediate 15 was substituted by bromine at the C-2

position and then reacted with thioacetamide in ethyl alcohol ab-

solute at 95 ^ꢀC to produce compound 21a. Intermediate 15 was

deprotected in the presence of Pd/C and then the methyl thiazole

ring was fused to ring-A to produce compound 21b. Intermediate

13 was reacted with ethyl formate under potassium tert-butanol

catalysis to obtain intermediate 16. Intermediate 16 was reacted

with hydrazine hydrate in ethyl alcohol absolute in a heated reﬂux

system to give intermediate 23. Compounds 24a and 24b were

prepared by deprotection of the C-23 hydroxyl and deprotection of

the C-28 carboxyl of intermediate 23 in turn.

2. Results and discussion

2.1. Synthetic chemistry

The synthetic methods for the preparation HBQ derivatives

6aed, 9aee and 10aee obtained by structural modiﬁcation at the

C-23 and C-28 positions of HBQ are shown in Scheme 1, and the

synthetic methods for the preparation HBQ analogues 18, 21aeb

and 24aeb based on ring-A substitutions are shown in Scheme 2.

The lead compound HBQ was obtained according to our

The NMR spectra of compounds 9aee and 10aee were similar to

the lead compound HBQ, the proton signals of the PEG group in

Fig. 4. The designed strategies for the HD derivatives.

3

B. Wang, S. Liu, W. Huang et al.

European Journal of Medicinal Chemistry 211 (2021) 113107

aa

Scheme 1. Synthesis of HD derivatives 6a-d, 9a-e, 10a-e Reagents and conditions: (a) Ac₂O, pyridine, r.t.; (b) 10% Pd/C, H₂, CH₃OH, r.t.; (c) (COCl)₂, DCM, 0 ^ꢀC to r.t.; (d) cyclic

amines with NH moiety, triethylamine, DCM, r.t.; (e) NaOH, CH₃OH/H₂O or CH₃OH/THF, r.t.; (f) ethyl bromoacetate, K₂CO₃, DMF, 65 ^ꢀC; (g) PEG, DMAP, EDCI, DCM, r.t.; (h) succinic

anhydride, DMAP, DCM, r.t.

Scheme 2. Synthesis of HD derivatives 18, 21a-b, 24a-b ^bbReagents and conditions: (a) benzyl bromide, DMF, K₂CO₃, 50 ^ꢀC; (b) tert-butyldimethylsilyl chloride, DMAP, DCM, r.t.; (c)

PCC, DCM, r.t.; (d) S, ethylenediamine, morpholine, reﬂux; (e) 30% H₂O₂, HCO₂H, DCM, r.t.; (f) 10% HCl, acetone/CH₃OH, r.t.; (g) pyridinium bromide perbromide, CHCl₃, r.t.; (h)

thioacetamide, ethyl alcohol absolute, 95 ^ꢀC; (i) 10% Pd/C, H₂, CH₃OH, r.t.; (j) potassium t-butoxide, ethyl formate, DCM, r.t.; (k) hydrazine hydrate, EtOH, reﬂux.

each compound being the main difference. The ¹H NMR spectra of

PEG had signals at 3.6e3.4 ppm from the protons of

dOdCH₂dCH₂d (the PEG chain) and the ¹³C NMR spectra had

signals at 72e68 ppm from the carbons in the methylene groups.

d

4

B. Wang, S. Liu, W. Huang et al.

European Journal of Medicinal Chemistry 211 (2021) 113107

The structures of all the HD derivatives were conﬁrmed by ¹H NMR,

788.88

7.74

m

g/mL, respectively. The results showed that the

¹³C NMR, and HR-MS.

aqueous solubility of compounds 10ae10e was signiﬁcantly

improved by PEGylation, and the solubility increased with length of

PEG chains, which may be because of the increase in hydrogen

bond acceptors. Thus, PEGylation is an effective method to main-

tain activity and improve the aqueous solubility of compounds and

the improved solubility of compound 10c should enable further

research and application of this compound.

2.2. Biological screening

The cytotoxicity and tumor MDR reversal activity toward KBV

cells of the synthesized HD derivatives were investigated with the

MTT assay [39], and the results are summarized in Table 1 and

Fig. S1.

No compounds showed appreciable cytotoxicity at a concen-

2.4. In vitro stability of compound 10c

tration of 10 mM, which was reﬂected in the high survival rate of

KBV cells in the absence of paclitaxel. Further, verapamil (a classic

P-gp inhibitor) and compound HBQ were used as reference stan-

dards to evaluate the tumor MDR reversal activity of the synthe-

sized compounds in combination with paclitaxel. Compounds 6a,

6d, 10c, 10d, and 10e showed superior or equal activity compared

The excellent tumor resistance reversal activity in vitro and

signiﬁcantly improved aqueous solubility of compound 10c indi-

cated that the compound was worthy of further research. There-

fore, we ﬁrst investigated its stability in vitro to provide a basis for

subsequent mechanism studies. The esterases in mammals are

widely distributed in the plasma, organs, and tissues and play an

indispensable role in drug metabolism. Porcine liver esterase (PLE),

a widely accepted model of esterase activity [40,41], was selected to

be added to PBS buffer (pH 7.4, 37 ^ꢀC) to study the stability of

compound 10c in a simulated physiological environment. Mean-

while, benorylate [Fig. 5(C)], a recognized cleavable ester [42], was

selected as a control to attest the viability of this test. The stability

results are displayed in Fig. 5(B, D). Compound 10c was highly

stable under simulated physiological conditions as indicated by

presence of 98.35 0.06%, 81.84 0.03%, and 78.92 0.03% of the

starting material following 4, 12, and 24 h of incubation, respec-

tively. Compound 10c, which is not a prodrug, possessed good

chemical stability to an esterase and may remain stable in the cir-

culation over 24 h, which would facilitate its stable transportation

and tumor resistance reversal activity. Benorylate was hydrolyzed

quickly under the same conditions, and only 14.10 0.23% of the

starting material existed after half an hour, which proved the

viability of this stability determination method.

with the lead compound HBQ at a concentration of 10 mM, dis-

played in bold font in Table 1. Among them, the lowest survival rate

(11.97%) of KBV cells occurred when compound 10c was combined

with paclitaxel, which indicated that compound 10c had the

highest tumor MDR reversal activity. Moreover, compound 10c

increased the sensitivity of KBV cells to paclitaxel in a dose-

dependent manner at concentrations of 2, 5 and 10 mM (Fig. S2).

The above results showed that, as expected, HBQ derivatives with

nitrogen-containing heterocycles or PEG modiﬁcation could

ameliorate the activity of HBQ. Most of the other derivatives also

produced a lower survival rate for KBV cells when combined with

paclitaxel, for example, the activity of compounds 6b, 6c, 9c, 9d, 18,

and 24a was stronger than verapamil but weaker than HBQ.

2.3. Aqueous solubility determination of PEGylated derivatives

The poor aqueous solubility of HBQ is the major obstacle for

further research. It is important to improve the aqueous solubility

through speciﬁc structural modiﬁcation methods while ensuring

excellent activity. Therefore, we paid particular attention to the

aqueous solubility of the most active derivative 10c and the

aqueous solubility of the series of PEGylated derivatives 10ae10e

was measured to explore the speciﬁc effects of PEGylation on the

solubility. The aqueous solubility results are shown in Fig. 5(A).

As expected, compared with HBQ, the aqueous solubility of the

PEGylated derivatives was signiﬁcantly improved. The aqueous

2.5. Effect of compound 10c on enhancing the sensitivity of KBV and

MCF-7T cells toward paclitaxel and vincristine

The good solubility and stability of compound 10c enlightened

our in-depth activity evaluation and mechanism study. The effect of

compound 10c on the sensitivity of KBV and MCF-7T (paclitaxel-

resistant breast carcinoma) cells toward paclitaxel and vincristine

was investigated by MTT assay and compared with the lead HBQ,

verapamil, and tariquidar (a third-generation P-gp inhibitor). The

MDR reversal potency and reversal fold (RF) values of compound

10c are summarized in Table 2.

solubility of compound 10c was 21.59

approximately 20-fold higher than that of HBQ (1.20 0.37

0.97

m

g/mL, which was

g/mL).

m

Because of the strong solubilizing ability of PEG molecules with a

large molecular weight, compounds 10d and 10e showed much

higher solubility than HBQ, with values of 546.18

6.49 and

Compound 10c could signiﬁcantly enhance the sensitivity of

Table 1

Effect of HD derivatives (10

m

M) on the cytotoxicity of paclitaxel (100 nM) toward KBV cells^a.

Cpd. (10 M)

m

KBV Cell Survival Rate (%)

With Paclitaxel (100 nM)

Cpd. (10

m

M)

KBV Cell Survival Rate (%)

With Paclitaxel (100 nM)

Without Paclitaxel

Paclitaxel

Verapamil

HD

HBQ

HBQ-5

6a

6b

6c

6d

9a

103.48 2.88

25.03 0.50

107.17 4.15

18.17 2.36

12.65 0.79

16.37 0.50

25.27 4.57

23.51 3.37

16.67 1.27

26.15 0.63

28.76 1.00

25.88 0.43

e

9d

9e

25.16 1.47

39.13 2.53

79.47 6.19

43.54 0.95

11.97 0.06

15.38 2.42

15.93 0.32

20.77 0.41

27.85 0.51

61.78 2.97

21.95 0.50

99.31 5.52

76.03 7.41

73.26 6.01

72.66 9.42

82.53 2.94

90.29 13.37

70.58 2.71

69.69 2.69

129.31 11.52

98.59 6.19

96.01 3.34

83.13 3.27

108.54 13.47

e

10a

10b

10c

10d

10e

18

21a

21b

24a

24b

e

67.51 1.21

110.02 3.15

59.43 8.02

124.41 17.98

120.48 16.62

81.65 1.54

80.47 0.97

75.43 4.31

9b

9c

a

The cell survival rates are presented as the mean SD of three independent experiments.

5

B. Wang, S. Liu, W. Huang et al.

European Journal of Medicinal Chemistry 211 (2021) 113107

Fig. 5. (A) Aqueous solubility values of HBQ and the PEGylated derivatives 10ae10e in deionized water at 37 ^ꢀC. Data are represented as the mean SD (n ¼ 3). (B) In vitro stability

of compound 10c in the following environment: 0.01 M PBS at pH 7.4 with 20 units PLE (n ¼ 3, mean SD). (C) Chemical structure of benorylate. (D) In vitro stability of benorylate in

the following environment: 0.01 M PBS at pH 7.4 with 57 units PLE (n ¼ 3, mean SD).

**p < 0.01, compared with HBQ.

Table 2

Effect of compound 10c, HBQ, verapamil, and tariquidar on the cytotoxicity of paclitaxel and vincristine.

KBV

MCF-7T

IC₅₀^a(nM)

RF^b

IC₅₀^a(nM)

RF^b

Paclitaxel (nM)

e

778.24 52.42

21.84 2.13

4.58 0.39**

59.17 6.49

15.80 2.10

36.77 2.57

23.08 2.01

7.57 0.52

1.00

22.73 3.05

5.42 0.23

0.89 0.24

12.99 1.95

1.48 0.51

16.49 1.47

8.51 1.83

3.26 1.26

0.26 0.12

1.00

4.19

25.50

1.75

15.40

1.38

2.67

6.97

86.28

þ5

m

M 10c

M HBQ

M Verapamil

M Tariquidar

35.63

169.96

13.15

49.25

21.16

33.72

102.80

174.60

þ10

m

þ5

m

þ10

m

þ5

m

þ10

m

þ5

m

þ10

m

4.46 0.35

Vincristine (nM)

e

435.85 19.09

4.02 0.23

0.79 0.30**

16.58 3.05

2.23 0.10

4.59 0.10

2.97 0.24

3.34 0.24

2.05 0.21

1.00

9.45 8.98

5.31 0.71

0.04 0.03

6.85 0.54

0.03 0.02

10.17 0.98

5.29 1.23

0.71 0.21

0.22 0.15

1.00

1.78

269.55

1.38

309.98

0.93

1.79

13.23

43.35

þ5

m

M 10c

M HBQ

M Verapamil

M Tariquidar

108.38

550.59

26.28

195.27

94.86

146.72

130.50

212.45

þ10

m

þ5

m

þ10

m

þ5

m

þ10

m

þ5

m

þ10

m

**p < 0.01, compared with control group.

a

IC₅₀values are represented as the mean SD (n ¼ 3).

b

RF value was calculated using the formula: RF ¼ IC₅₀of chemotherapeutic drug alone/IC₅₀of chemotherapeutic drug in the presence of reversal agent.

KBV cells to the chemotherapeutic drugs paclitaxel and vincristine.

and 550.59, respectively, compared with the chemotherapeutic

drugs alone. The sensitization effect of compound 10c on chemo-

therapeutic drugs was stronger than either the lead compound

HBQ or the standard compound verapamil, and was almost

Compound 10c at a concentration of 10

mM decreased the IC₅₀

values (4.58 and 0.79 nM when combined with paclitaxel and

vincristine, respectively) and the RF values were as high as 169.96

6

B. Wang, S. Liu, W. Huang et al.

European Journal of Medicinal Chemistry 211 (2021) 113107

equivalent to tariquidar, and showed a dose-dependent effect at

Figs. S3(A and B) that compound 10c inhibited the P-gp-mediated

efﬂux of rhodamine 123 in a dose-dependent manner, which was

reﬂected in the increased accumulation of rhodamine 123 in KBV

and MCF-7T cells. The effect was stronger than the positive controls

HBQ and verapamil. Flutax1, a paclitaxel derivative that emits

ﬂuorescence upon binding to microtubules, is used as a P-gp sub-

strate to characterize the function of P-gp [44]. As shown in Fig. 6(C

and D) and Figs. S3(C and D), treatment with compound 10c

increased the accumulation of Flutax1 in KBV cells at a concen-

concentrations of 5 and 10

mM. Similarly, compound 10c also

enhanced the sensitivity of chemotherapy drugs to MCF-7T cells to

varying degrees. These results indicated that compound 10c could

enhance the antitumor activity of chemotherapeutic drugs toward

drug-resistant tumor cells.

2.6. Effect of compound 10c on the efﬂux function of P-gp

tration of 5 mM and in MCF-7T cells at a concentration of 10 mM,

Enhanced sensitivity of drug-resistant tumor cells to chemo-

therapeutic drugs may be achieved by increasing the accumulation

of the chemotherapeutic drugs in the cells. Therefore, to investigate

whether compound 10c exerts a tumor resistance reversal effect by

inhibiting the drug efﬂux function of P-gp, the effect on the efﬂux of

rhodamine 123 and Flutax1 from KBV and MCF-7T cells was

studied.

thereby enhancing the antitumor effect of paclitaxel. The above

results suggested that compound 10c exerted a reversal effect on

tumor MDR by effectively inhibiting the efﬂux function of P-gp.

2.7. Effect of compound 10c on paclitaxel-induced cell cycle arrest

and cell apoptosis in KBV cells

Rhodamine123 is a well-known P-gp substrate that is used as a

biomarker to evaluate the effect of MDR reversal agents on the

efﬂux function of P-gp [43]. It can be seen in Fig. 6(A and B) and

To investigate the effect of compound 10c on paclitaxel-induced

cell cycle arrest and apoptosis in KBV cells, the KBV cell cycle

Fig. 6. Effect of compound 10c on efﬂux function of P-gp in KBV and MCF-7T cells. (A) Effect of compound 10c on the accumulation of rhodamine 123 in KBV cells. KBV cells were

incubated with rhodamine 123 in the presence or absence of test compounds for 30 min for analysis by ﬂow cytometry. (B) Effect of compound 10c on the accumulation of

rhodamine 123 in MCF-7T cells. MCF-7T cells were incubated with rhodamine 123 in the presence or absence of test compounds for 30 min for analysis by ﬂow cytometry. (C) Effect

of compound 10c on the accumulation of Flutax1 in KBV cells. KBV cells were incubated with Flutax1 in the presence or absence of the test compounds for 30 min and then

harvested for analysis by ﬂow cytometry. (D) Effect of compound 10c on the accumulation of Flutax1 in MCF-7T cells. MCF-7T cells were incubated with Flutax1 in the presence or

absence of the test compounds for 30 min and then harvested for analysis by ﬂow cytometry. All data are presented as the mean SD (n ¼ 3), *p < 0.05, compared with the Rh123

group (A, B) and Flutax1 group (C, D). Rh123: rhodamine 123; Vrp: verapamil.

7

B. Wang, S. Liu, W. Huang et al.

European Journal of Medicinal Chemistry 211 (2021) 113107

Table 3

distribution was analyzed by ﬂow cytometry using propidium io-

dide (PI) staining. As shown in Fig. 7, compound 10c signiﬁcantly

increased the rate of apoptosis in a dose-dependent manner. This

effect was manifested as an increase in the proportion of KBV cells

in the sub-G1 phase, which was increased from 2.1% to 48.9% after

treatment with 10 mM compound 10c in combination with pacli-

taxel. However, compound 10c had little effect on the paclitaxel-

induced cell cycle arrest of the G2/M phase.

Scores of the docking models of HD derivatives with a homology model of human P-

gp.

Compound

Total_Score

Crash

Polar

Verapamil

HBQ

6a

6b

6c

6d

9a

9b

9c

6.6301

4.6288

4.2866

2.614

ꢁ1.9269

ꢁ8.3403

ꢁ10.4029

ꢁ11.1818

ꢁ4.0402

ꢁ7.7473

ꢁ9.2299

ꢁ10.4232

ꢁ1.6426

ꢁ6.1117

ꢁ10.6122

ꢁ12.8774

ꢁ9.5701

ꢁ6.1117

ꢁ2.9517

ꢁ11.9120

ꢁ5.2249

ꢁ5.6353

ꢁ7.4755

ꢁ9.0318

ꢁ7.5693

0.0907

1.0207

0.0006

0.9548

1.0882

0.0001

1.1056

0.1304

1.9870

2.0635

2.7504

0.0061

0.9939

2.0635

1.4684

0.9938

0.0007

1.0246

0

3.4525

2.8164

5.9506

5.3933

4.6424

9.0604

4.2427

5.9765

5.1472

7.0268

11.5472

4.3699

4.0631

3.5854

0.8395

2.7847

2.0442

2.8. Molecular docking simulations

We used SYBYL software to conduct a molecular docking study

to explore the speciﬁc inﬂuence of the structure of the synthesized

compounds on their binding with P-gp. QZ-SSS with more drug-

binding residues in a pair of stereoisomers in the literature

[45,46] was selected as the reference molecule, and its binding

pocket was used for molecular docking research. The docking

scores of 19 compounds with a homology model of human P-gp are

shown in Table 3. Most PEGylated derivatives showed higher

docking scores than HBQ, except for compounds 9e and 10e. The

docking positions of representative compounds 9d and 10c in the

P-gp binding pocket are shown in Fig. 8(A and B). The oxygen atom

of the carbonyl moiety of compound 9d interacted with the amino

9d

9e

10a

10b

10c

10d

10e

18

21a

21b

24a

24b

0

1.3406

Fig. 7. Effect of HBQ and compound 10c on paclitaxel activity in cell cycle distribution and apoptosis in KBV cells. (A): cell cycle distribution; (B): cell apoptosis; (C): HBQ and

compound 10c both enhanced paclitaxel-induced apoptosis, and compound 10c demonstrated a greater ability to enhance cell apoptosis. All data are presented as the mean SD

(n ¼ 3), **p < 0.01, compared with control group. Ptx: paclitaxel; Vrp: verapamil.

8

B. Wang, S. Liu, W. Huang et al.

European Journal of Medicinal Chemistry 211 (2021) 113107

Fig. 8. Molecular docking mode of compounds 9d (A), 10c (B), HBQ (C), and 6a (D) with a homology model of human P-gp. The compounds and amino acid residues are shown in

stick format and hydrogen bonds are shown as dotted red lines. The ﬁgures were generated using PyMOL. (For interpretation of the colors in this ﬁgure, the reader is referred to the

Web version of this article.).

acid residue GLN990 to form two hydrogen bonds (2.1 and 2.7 Å),

which was similar to the binding interactions of HBQ [Fig. 8(C)]. In

addition, the hydroxyl group at C-23 and the end of the PEG moiety

were also bridged to SER993, ASN721, and SER766 by three

hydrogen-bonding interactions, which further stabilized the bind-

ing in the pocket of P-gp. In contrast, the oxygen atom in the PEG

side chain formed a hydrogen bond with the amino acid residue

GLN725 in compound 10c. However, through ﬂexible changes in

the docking position, compound 10c could also be deeply

embedded in the active pocket of P-gp. Amino acid residues

GLN990, ASN721 and GLN725 mentioned above are identical to the

binding residues of QZ-SSS. In addition, the derivatives 6aed with

nitrogen-containing heterocycles substituted at C-28 had relatively

low docking scores. For example, the docking position of compound

6a [Fig. 8(D)] was closer to the top of the P-gp protein, and the

compound did not penetrate as deeply as compound 10c into the P-

gp active pocket, which may be the reason why the activity of

compound 6a was lower than that of compound 10c. In summary,

these results indicated that PEGylated derivatives may reverse tu-

mor MDR by acting on P-gp and that this docking method could

provide direction for the future structural modiﬁcation of HD de-

rivatives with improved tumor MDR reversal activity.

unsaturated nitrogen-containing heterocycle, activity was

improved and the activity was further improved when the C-28

carboxyl was substituted by a benzyl group.

The tumor MDR reversal activity of compound 6d was improved

compared with compound HBQ, because of the N-ethyl piperazine

substituted at C-28; however, it had less tumor MDR reversal ac-

tivity than compound HBQ-5 with 3-dimethylamino-1-propanol

substituted at C-28, which indicated that open-chain nitrogen

groups make a greater contribution to the activity than the N-ethyl

piperazine group. Compounds 10c, 10d, and 10e had higher tumor

MDR reversal activity compared with compound HBQ, which is

likely because of the introduction of PEG to increase the aqueous

solubility. Compounds 21a and 24a displayed activity equal to

verapamil at the same dose, and better activity than compounds

21b and 24b, respectively. These results show, under the premise

that the C-28 carboxyl is protected by benzyl, that the activity is

improved when ring-A is a fused unsaturated nitrogen-containing

heterocycle. The existing SAR studies will be used to guide the

design and synthesis of novel tumor MDR reversal agents with high

activity, and more structural modiﬁcations of HD should continue

to be carried out to further improve knowledge of the SAR.

3. Conclusions

2.9. Structureeactivity relationships

In summary, we have designed and synthesized several series of

nitrogen-containing heterocyclic-substituted, PEGylated, and ring-

According to the tumor MDR reversal activity evaluation of the

synthesized HD derivatives and the SARs summarized in our pre-

vious study [29], the SARs were reﬁned, as displayed in bold font in

Fig. 9. When saturated nitrogen-containing heterocycles were

substituted at C-28, activity was improved; however, open-chain

nitrogen containing groups were better than the saturated

nitrogen-containing heterocycles. When ring-A was a fused pyr-

azine and the C-28 carboxyl and the C-23 hydroxyl were

substituted by PEG, activity was improved. When ring-A was a

fused pyrazine and the double bond at C-12 was converted to a

A

substituted derivatives of hederagenin through amidation,

esteriﬁcation, and cyclization reactions, respectively. The MTTassay

was used to evaluate the tumor MDR reversal activity of the de-

rivatives as drug resistance reversal agents for tumor therapy.

Almost all of the derivatives had a good reversal effect on drug-

resistant KBV cells, with an effect stronger than, or equivalent to,

verapamil. In addition, knowledge of the SARs of the HD derivatives

were further improved. These structural modiﬁcation methods will

provide an important reference for the development of potential

MDR reversal agents in the future. Notably, the aqueous solubility

ketone, activity was improved. When ring-A was

a fused

9

B. Wang, S. Liu, W. Huang et al.

European Journal of Medicinal Chemistry 211 (2021) 113107

Fig. 9. Supplementary SARs of HD derivatives. The new SARs based on the results of this research are displayed in bold font.

of some of the PEGylated derivatives was 12e657 fold higher than

the lead compound HBQ, and the aqueous solubility increased with

length of PEG chains. Therefore, PEGylation is an effective method

to improve aqueous solubility while maintaining tumor MDR

reversal activity.

solid compounds were tested using a Micro melting point deter-

mination apparatus SGW®X-4 (Shanghai INESA Physico-Optical

Instrument Co., Ltd, CHN).

4.1.1. General procedure for the synthesis of 5a~5d

Compound 10c was selected to evaluate the stability and the

mechanism of the tumor MDR reversal effect. The results showed

that compound 10c possessed good chemical stability to an

esterase over 24 h. Additionally, compound 10c could signiﬁcantly

enhance the sensitivity of KBV cells to paclitaxel and vincristine,

with IC₅₀values of 4.58 and 0.79 nM, respectively. The combination

of compound 10c and paclitaxel signiﬁcantly increased the

apoptosis rate of KBV cells. The results of ﬂow cytometry showed

that treatment with compound 10c increased the accumulation of

rhodamine 123 and Flutax1 in KBV and MCF-7T cells at concen-

The lead compound HBQ was obtained according to our previ-

ous synthetic method [28]. HBQ (2.0 g, 3.3 mmol) was dissolved in

pyridine (30.0 mL) and acetic anhydride (15.9 mL) was added. The

reaction was reacted with stirring at room temperature for 8 h. The

mixture was diluted with ethyl acetate, washed with water and

saturated brine in sequence, dried over Na₂SO₄and concentrated.

The residue was puriﬁed by silica gel column chromatography (V

_{petroleum ether}: V

89.0% yield).

¼ 20:1) to give a white solid 1 (1.5 g,

ethyl acetate

1 (1.9 g, 3.0 mmol) was dissolved in methanol (30.0 mL) and 10%

Pd/C (0.6 g, 6.0 mmol) was added. The reaction was reacted with

stirring under H₂at room temperature for 6 h. The solution was

collected by vacuum ﬁltration. Then the mixture was concentrated

trations of 5 and 10

mM, which indicated that compound 10c

exerted the reversal of tumor resistance by effectively inhibiting

the efﬂux function of P-gp. Moreover, compound 10c showed a

higher docking score than HBQ in the molecular docking study.

Therefore, the above results strongly suggest that the in vivo ac-

tivity, distribution, and metabolism of compound 10c should be

investigated, and these results will be published in due course.

and puriﬁed by silica gel column chromatography (V _{petroleum ether}

:

V

¼ 6:1) to give a white solid 4 (1.5 g, 92.3% yield).

ethyl acetate

4 (90.0 mg, 0.2 mmol) was dissolved in anhydrous DCM (6 mL)

with stirring for 10 min at 0 ^ꢀC and then oxalyl chloride (160.0

mL,

1.9 mmol) was added with stirring for 1 h. Remaining oxalyl chlo-

ride was removed by vacuum distillation, and the residue was

dissolved in anhydrous DCM (6 mL) again, morpholine (160.0 mL,

4. Experimental section

1.8 mmol) was added. The reaction was reacted with stirring at

room temperature for 2 h. The mixture was diluted with ethyl ac-

etate, washed with water and saturated brine in sequence, dried

over Na₂SO₄and concentrated. The residue was puriﬁed by silica

gel column chromatography (V _{petroleum ether}: V _{ethyl acetate}¼ 8:1) to

give a white solid 5a (85.1 mg, 83.9% yield). The compounds 5b~5d

were made and puriﬁed with the same as synthesis of 5a.

4.1. Chemistry

All solvents and reagents were purchased from commercial

sources, which were dried further by standard methods when

necessary. All reactions were stirred magnetically, and the reaction

progress was monitored by thin layer chromatography (TLC) anal-

ysis on silica gel HSGF254 and the spots visualized by using 10%

ethanol sulfate solution and UV ﬂuorescence with a wavelength of

254 nm. All the compounds we synthesized were puriﬁed by col-

umn chromatography on silica gel (200e300 mesh). ¹H (400 MHz)

NMR spectra and ¹³C (100 MHz) NMR spectra were recorded on

Bruker av400 (Bruker, GER) or JNM-ECZ400S (JEOL Ltd., JPN) in-

strument in CDCl₃, CD₃OD or DMSO‑d₆with TMS as internal stan-

4.1.2. N-morpholine 23-hydroxy-olean[3,2-b]pyrazine-12-en-28-

amide (6a)

5a (50 mg, 0.1 mmol) was dissolved in a component solvent (V

_{tetrahydrofuran}: V _methanol¼ 3:2), and 10% sodium hydroxide solution

(246.0 mL) was dripped into the solution with stirring, reacted at

dard. All chemical shifts were expressed in

d

values parts per

room temperature for 5 h. The mixture was concentrated and then

diluted with ethyl acetate. The solution was adjusted to acidic by

using 5% hydrochloric acid solution. The organic phase was washed

with water and saturated brine in sequence, dried over Na₂SO₄,

concentrated and puriﬁed by silica gel column chromatography (V

_{petroleum ether}: V _{ethyl acetate}¼ 8:1) to give a white solid 6a (42.1 mg,

million (ppm) and the coupling constants (J) were given in Hertz

(Hz). Took 1 mg each of all compounds and dissolved in DMSO, then

diluted to 100 mg/mL with methanol to determine high resolution

mass spectra (HR-MS). The HR-MS were taken on Agilent QTOF

6520 (Agilent Technologies Co. Ltd., USA), or Q ExactiveTM Orbitrap

MS system (Thermo Scientiﬁc, USA). Melting points (m.p.) of all

90.3% yield). m.p. 147.1e149.3 ^ꢀC. ¹H NMR (400 MHz, CDCl₃)

d 8.34

10

B. Wang, S. Liu, W. Huang et al.

European Journal of Medicinal Chemistry 211 (2021) 113107

(d, J ¼ 8.0 Hz, 2H, H-pyrazine), 5.34 (t, J ¼ 3.1 Hz, 1H, H-12), 3.78 (d,

J ¼ 10.6 Hz, 1H, H-23a), 3.75e3.54 (m, 8H, 4 ꢂ NCH₂), 3.47 (d,

J ¼ 10.6 Hz, 1H, H-23b), 3.10 (d, J ¼ 11.2 Hz, 1H, H-18), 2.99 (d,

J ¼ 16.6 Hz, 1H, H-1a), 2.49 (d, J ¼ 16.7 Hz, 1H, H-1b), 1.30 (s, 3H,

CH₃),1.18 (s, 3H, CH₃), 0.93 (s, 6H, 2 ꢂ CH₃), 0.89 (s, 3H, CH₃), 0.83 (s,

603.4633, found: 603.4623.

4.1.6. General procedure for the synthesis of 8

According to the synthesis method of 4, HBQ was deprotected to

give crude sodium 2, which could be used directly for the next step

without further puriﬁcation.

3H, CH₃); ¹³C NMR (100 MHz, CDCl₃)

d 176.98, 159.36, 153.51,

146.03, 143.43, 143.00, 122.81, 71.98, 68.34, 61.79, 49.01, 48.86,

48.51, 47.70, 47.47, 47.28, 45.06, 44.75, 43.48, 40.44, 37.79, 35.38,

34.43, 33.43, 31.79, 31.29, 31.07, 29.34, 27.25, 25.43, 24.72, 24.10,

21.53, 20.77, 17.98, 17.49; HR-MS (ESI) m/z: calcd. for C₃₆H₅₃N₃O₃

[MþH]^þ: 576.4160, found: 576.4161.

Ethyl Bromoacetate (965.4 mL, 8.7 mmol) and potassium car-

bonate (467.9 mg, 3.4 mmol) were added to a solution of 2

(816.0 mg, 1.6 mmol) in DMF (20 mL). The mixture was stirred for

3 h at 65 ^ꢀC. Then the mixture was diluted with ethyl acetate,

washed with water and saturated brine in sequence, dried over

Na₂SO₄and concentrated to give crude sodium 7, which could be

used directly for the next step without further puriﬁcation.

7 (1023.0 mg, 1.7 mmol) was dissolved in a component solvent

4.1.3. N- piperidine 23-hydroxy-olean[3,2-b]pyrazine-12-en-28-

amide (6b)

According to the synthesis method of 6a, 5b was deprotected to

give a white solid 6b (11.0 mg, 88.9% yield) after puriﬁed by silica

(V _methanol: V

¼ 4:1), and sodium hydroxide (552.6 mg,

water

13.8 mmol) was added. The mixture was stirred for 1 h at room

temperature. The mixture was concentrated and then diluted with

ethyl acetate, washed with 5% hydrochloric acid solution, water and

saturated brine in sequence, dried over Na₂SO₄and concentrated.

The residue was puriﬁed by silica gel column chromatography (V

_chloroform: V _methanol¼ 100:1) to give a white solid 8 (879.7 mg, 81.9%

yield).

gel column chromatography (V _DCM: V

¼ 60:1). m.p.

methanol

145.2e149.6 ^ꢀC. ¹H NMR (400 MHz, CDCl₃)

d

8.36 (q, J ¼ 2.4 Hz, 2H,

H-pyrazine), 5.35 (t, J ¼ 3.6 Hz,1H, H-12), 3.79 (d, J ¼ 10.6 Hz,1H, H-

23a), 3.67e3.50 (m, 4H, 2 ꢂ NCH₂), 3.48 (d, J ¼ 10.5 Hz, 1H, H-23b),

3.14 (dd, J ¼ 14.6, 3.0 Hz, 1H, H-18), 3.01 (d, J ¼ 16.6 Hz, 1H, H-1a),

2.50 (d, J ¼ 16.6 Hz, 1H, H-1b), 1.33 (s, 3H, CH₃), 1.19 (s, 3H, CH₃),

0.95 (s, 3H, CH₃), 0.94 (s, 3H, CH₃), 0.90 (s, 3H, CH₃), 0.87 (s, 3H,

CH₃); ¹³C NMR (100 MHz, CDCl₃)

d

174.79, 158.09, 152.25, 145.13,

4.1.7. O-(23-hydroxy-olean[3,2-b]pyrazine-12-en-28-acyl)-

142.13, 141.61, 121.19, 70.76, 53.50, 47.75, 47.51, 47.36, 46.69, 46.61,

46.04, 43.84, 43.38, 42.17, 39.12, 36.69, 34.21, 33.17, 32.18, 30.50,

29.85, 29.77, 28.11, 26.23, 25.90, 24.92, 24.18, 23.43, 22.76, 20.30,

19.43,16.74,16.20; HR-MS (ESI) m/z: calcd. for C₃₇H₅₅N₃O₂[MþH]^þ:

574.4367, found: 574.4365.

acetyldiethylene glycol (9a)

Diethylene glycol (108.0

mL, 1.4 mmol), DMAP (150.1 mg,

1.2 mmol) and EDCI (148.8 mg, 0.8 mmol) were added to a solution

of 8 (73 mg, 0.1 mmol) in dry DCM (5 mL). The mixture was stirred

for 10 h at room temperature. Then the mixture was concentrated

and dissolved in methanol, and the solution was dialyzed (MWCO

500 Da) in deionized water for 28 h and changed the dialysis liquid

four times interval of 7 h. Dialysis substance was concentrated by

vacuum distillation. The residue was puriﬁed by silica gel column

4.1.4. N-(4ʼ-methyl) piperazine 23-hydroxy-olean[3,2-b]pyrazine-

12-en-28-amide (6c)

According to the synthesis method of 6a, 5c was deprotected to

give a white solid 6c (45.0 mg, 92.0% yield) after puriﬁed by silica

chromatography (V _DCM: V

¼ 185:1) to give a yellow

methanol

gel column chromatography (V _DCM: V

¼ 80:1). m.p.

transparent syrup 9a (47.4 mg, 56.2% yield). ¹H NMR (400 MHz,

CDCl₃)

methanol

138.5e144.2 ^ꢀC. ¹H NMR (400 MHz, CDCl₃)

d

8.32 (d, J ¼ 8.4 Hz, 2H,

d

8.37 (d, J ¼ 2.1 Hz, 1H, H-pyrazine), 8.35 (d, J ¼ 2.5 Hz, 1H,

H-pyrazine), 5.32 (t, J ¼ 3.4 Hz, 1H, H-12), 3.76 (d, J ¼ 10.5 Hz,1H, H-

23a), 3.68e3.61 (m, 4H, 2 ꢂ NCH₂a), 3.46 (d, J ¼ 10.5 Hz, 1H, H-23b),

3.09 (d, J ¼ 13.4 Hz, 1H, H-18), 2.97 (d, J ¼ 16.6 Hz,1H, H-1a), 2.47 (d,

J ¼ 16.6 Hz, 1H, H-1b), 2.42e2.32 (m, 4H, 2 ꢂ NCH₂b), 2.29 (s, 3H,

NCH₃), 1.29 (s, 3H, CH₃), 1.16 (s, 3H, CH₃), 0.91 (s, 6H, 2 ꢂ CH₃), 0.88

H-pyrazine), 5.38 (t, J ¼ 3.5 Hz,1H, H-12), 4.65 (d, J ¼ 15.8 Hz,1H, H-

23a), 4.54 (d, J ¼ 15.8 Hz, 1H, H-23b), 4.38e4.28 (m, 2H, CH₂O PEG

group), 3.79 (d, J ¼ 10.6 Hz, 1H, CH₂a), 3.75e3.57 (m, 6H, CH₂O PEG

group), 3.49 (d, J ¼ 10.6 Hz,1H, CH₂b), 3.01 (d, J ¼ 16.6 Hz,1H, H-1a),

2.92 (d, J ¼ 13.2 Hz, 1H, H-18), 2.51 (d, J ¼ 16.6 Hz, 1H, H-1b), 1.33 (s,

3H, CH₃), 1.20 (s, 3H, CH₃), 0.95 (s, 6H, 2 ꢂ CH₃), 0.92 (s, 3H, CH₃),

(s, 3H, CH₃), 0.81 (s, 3H, CH₃); ¹³C NMR (100 MHz, CDCl₃)

d 175.08,

158.00, 152.26, 144.84, 142.22, 141.63, 121.40, 70.71, 55.22, 47.78,

47.54, 47.27, 46.45, 45.99, 45.97, 45.23, 43.78, 43.40, 42.15, 39.13,

36.67, 34.12, 33.13, 32.15, 31.00, 30.48, 29.99, 29.34, 27.99, 25.92,

24.12, 23.41, 22.78, 20.28,19.44,16.71,16.18; HR-MS (ESI) m/z: calcd.

for C₃₇H₅₆N₄O₂[MþH]^þ: 589.4476, found: 589.4470.

0.83 (s, 3H, CH₃); ¹³C NMR (100 MHz, CDCl₃)

d 177.20, 168.02,

157.93, 152.09, 143.47, 142.14, 141.62, 122.35, 72.45, 70.65, 68.81,

64.01, 61.70, 60.45, 47.74, 47.15, 46.86, 45.97, 45.73, 43.33, 41.98,

41.36, 39.26, 36.53, 33.90, 33.05, 32.16, 31.91, 30.69, 27.60, 25.69,

23.60, 23.40, 23.11, 20.18,19.40,16.63,16.14; HR-MS (ESI) m/z: calcd.

for C₃₈H₅₆N₂O₇[MþH]^þ: 653.4161, found: 653.4156.

4.1.5. N-(4ʼ-ethyl) piperazine 23-hydroxy-olean[3,2-b]pyrazine-12-

en-28-amide (6d)

According to the synthesis method of 6a, 5d was deprotected to

give a white solid 6d (38.0 mg, 88.5% yield) after puriﬁed by silica

4.1.8. O-(23-hydroxy-olean[3,2-b]pyrazine-12-en-28-acyl)-

acetyltriethylene glycol (9b)

According to the synthesis method of 9a, 8 was reacted with

triethylene glycol to give a yellow transparent syrup 9b (70.1 mg,

56.8% yield) after dialyzed (MWCO 500 Da) and puriﬁed by silica gel

gel column chromatography (V _DCM: V

¼ 80:1). m.p.

methanol

133.5e139.1 ^ꢀC. ¹H NMR (400 MHz, CDCl₃)

d

8.36 (d, J ¼ 2.5 Hz, 1H,

H-pyrazine), 8.34 (d, J ¼ 2.5 Hz, 1H, H-pyrazine), 5.35 (t, J ¼ 3.5 Hz,

1H, H-12), 3.80 (d, J ¼ 10.5 Hz, 1H, H-23a), 3.71e3.63 (m, 4H,

2 ꢂ NCH₂a), 3.49 (d, J ¼ 10.5 Hz, 1H, H-23b), 3.16e3.10 (m, 1H, H-

18), 3.00 (d, J ¼ 16.6 Hz, 1H, H-1a), 2.50 (d, J ¼ 16.7 Hz, 1H, H-1b),

2.46e2.37 (m, 6H, 3 ꢂ NCH₂b), 1.32 (s, 3H, CH₃), 1.19 (s, 3H, CH₃),

1.10 (t, J ¼ 7.2 Hz, 3H, CH₃), 0.94 (s, 6H, 2 ꢂ CH₃), 0.91 (s, 3H, CH₃),

column chromatography (V _DCM: V

¼ 135:1). ¹H NMR

methanol

(400 MHz, CDCl₃)

d

8.37 (d, J ¼ 2.2 Hz, 1H, H-pyrazine), 8.35 (d,

J ¼ 2.5 Hz, 1H, H-pyrazine), 5.38 (t, J ¼ 3.5 Hz, 1H, H-12), 4.67 (d,

J ¼ 15.8 Hz, 1H, H-23a), 4.55 (d, J ¼ 15.8 Hz, 1H, H-23b), 4.34e4.29

(m, 2H, CH₂O PEG group), 3.80 (d, J ¼ 10.6 Hz, 1H, CH₂a), 3.72e3.59

(m, 10H, CH₂O PEG group), 3.49 (d, J ¼ 10.5 Hz, 1H, CH₂b), 3.01 (d,

J ¼ 16.6 Hz, 1H, H-1a), 2.92 (dd, J ¼ 13.8, 4.1 Hz, 1H, H-18), 2.51 (d,

J ¼ 16.6 Hz, 1H, H-1b), 1.33 (s, 3H, CH₃), 1.20 (s, 3H, CH₃), 0.95 (s, 6H,

2 ꢂ CH₃), 0.92 (s, 3H, CH₃), 0.83 (s, 3H, CH₃); ¹³C NMR (100 MHz,

0.85 (s, 3H, CH₃); ¹³C NMR (100 MHz, CDCl₃)

d 174.92,157.93,152.21,

144.81, 142.11, 141.56, 121.29, 70.59, 53.00, 52.29, 47.70, 47.44, 47.13,

46.38, 45.91, 45.26, 43.69, 43.36, 42.08, 39.05, 36.59, 34.05, 33.07,

32.08, 30.41, 29.91, 27.91, 25.86, 24.06, 23.34, 22.70, 20.19, 19.38,

16.66, 16.10, 11.87; HR-MS (ESI) m/z: calcd. for C₃₈H₅₈N₄O₂[MþH]^þ:

CDCl₃)

d 177.05,168.03,157.92, 152.09, 143.52, 142.14,141.60, 122.31,

72.50, 70.65, 70.61, 70.31, 68.87, 64.07, 61.75, 60.33, 47.75, 47.16,

11

B. Wang, S. Liu, W. Huang et al.

European Journal of Medicinal Chemistry 211 (2021) 113107

46.83, 45.90, 45.74, 43.33, 41.99, 41.37, 39.26, 36.53, 33.91, 33.05,

32.18, 31.90, 30.69, 27.59, 25.68, 23.60, 23.39, 23.12, 20.18, 19.39,

16.62, 16.13; HR-MS (ESI) m/z: calcd. for C₄₀H₆₀N₂O₈[MþH]^þ:

697.4423, found: 697.4420.

C

₆₀H₁₀₀N₂O₁₈[MþH]^þ: 1137.7044, found: 1137.7046.

4.1.12. General procedure for the synthesis of 3

To a solution of HBQ (443 mg, 0.7 mmol) in anhydrous DCM

(25 mL) was added succinic anhydride (742.8 mg, 7.4 mmol) and

DMAP (453.4 mg, 3.7 mmol). After stirring for 2 h at room tem-

perature, the mixture was diluted with DCM, washed with water

and saturated brine in sequence, dried over Na₂SO₄and concen-

trated to give crude sodium 3, which could be used directly for the

next step without further puriﬁcation.

4.1.9. O-(23-hydroxy-olean[3,2-b]pyrazine-12-en-28-acyl)-

acetyltetraethylene glycol (9c)

According to the synthesis method of 9a, 8 was reacted with

tetraethylene glycol to give a yellow transparent syrup 9c (76.1 mg,

58.0% yield) after dialyzed (MWCO 500 Da) and puriﬁed by silica gel

column chromatography (V _DCM: V

¼ 100:1). ¹H NMR

methanol

(400 MHz, CDCl₃)

d

8.37 (s, 1H, H-pyrazine), 8.35 (s, 1H, H-pyr-

4.1.13. 4-(Benzyl olean[3,2-b]pyrazine-12-en-28-oate-23-oxy)-4-

azine), 5.38 (t, J ¼ 3.4 Hz, 1H, H-12), 4.66 (d, J ¼ 15.8 Hz, 1H, H-23a),

4.55 (d, J ¼ 15.9 Hz, 1H, H-23b), 4.33e4.28 (m, 2H, CH₂O PEG

group), 3.79 (d, J ¼ 10.6 Hz, 1H, CH₂a), 3.71e3.59 (m, 14H, CH₂O PEG

group), 3.49 (d, J ¼ 10.6 Hz,1H, CH₂b), 3.01 (d, J ¼ 16.5 Hz,1H, H-1a),

2.92 (dd, J ¼ 13.8, 4.0 Hz, 1H, H-18), 2.51 (d, J ¼ 16.6 Hz, 1H, H-1b),

1.33 (s, 3H, CH₃), 1.19 (s, 3H, CH₃), 0.95 (s, 6H, 2 ꢂ CH₃), 0.92 (s, 3H,

oxo-butyryldiethylene glycol (10a)

Diethylene glycol (101.7 mL, 1.2 mmol) was dissolved in anhy-

drous DCM (8 mL), and 3 (90 mg, 0.1 mmol), DMAP (149.9 mg,

1.2 mmol), EDCI (148.5 mg, 0.8 mmol) were added in turn. The

mixture was stirred for 10 h at room temperature. Then the mixture

was concentrated and dissolved in methanol, and the solution was

dialyzed (MWCO 500 Da) in deionized water for 28 h and changed

the dialysis liquid four times interval of 7 h. Dialysis substance was

concentrated by vacuum distillation. The residue was puriﬁed by

CH₃), 0.83 (s, 3H, CH₃); ¹³C NMR (100 MHz, CDCl₃)

d 177.06, 168.07,

157.97, 152.15, 143.59, 142.19, 141.66, 122.35, 72.57, 70.70, 70.69,

70.62, 70.56, 70.38, 68.95, 64.22, 61.78, 60.38, 47.80, 47.19, 46.87,

45.94, 45.79, 43.39, 42.04, 41.41, 39.31, 36.58, 33.96, 33.09, 32.21,

31.95, 30.74, 27.65, 25.73, 23.66, 23.44, 23.17, 20.23, 19.45, 16.67,

16.18; HR-MS (ESI) m/z: calcd. for C₄₂H₆₄N₂O₉[MþH]^þ: 741.4685,

found: 741.4682.

silica gel column chromatography (V _DCM: V

¼ 250:1) to

methanol

give a yellow transparent syrup 10a (47.0 mg, 45.8% yield). ¹H NMR

(400 MHz, CDCl₃) 8.42 (s, 1H, H-pyrazine), 8.29 (s, 1H, H-pyr-

d

azine), 7.38e7.28 (m, 5H, 5 ꢂ HeAr), 5.37 (t, J ¼ 3.5 Hz, 1H, H-12),

5.14e5.03 (m, 2H, CH₂Ar), 4.32 (d, J ¼ 10.5 Hz, 1H, H-23a), 4.27 (d,

J ¼ 10.5 Hz, 1H, H-23b), 4.20e4.16 (m, 2H, CH₂O PEG group),

3.77e3.57 (m, 6H, CH₂O PEG group), 2.99e2.93 (m, 2H, H-1a, H-18),

2.51 (d, J ¼ 16.3 Hz, 1H, H-1b), 2.46e2.33 (m, 4H, 2 ꢂ CH₂), 1.28 (s,

3H, CH₃), 1.19 (s, 3H, CH₃), 0.94 (s, 3H, CH₃), 0.91 (s, 3H, CH₃), 0.88 (s,

4.1.10. O-(23-hydroxy-olean[3,2-b]pyrazine-12-en-28-acyl)-

acetylpolyethylene glycol 400 (9d)

According to the synthesis method of 9a, 8 was reacted with

polyethylene glycol 400 to give a yellow transparent syrup 9d

(68.3 mg, 40.1% yield) after dialyzed (MWCO 500 Da) and puriﬁed

by silica gel column chromatography (V _DCM: V _methanol¼ 80:1). ¹H

3H, CH₃), 0.69 (s, 3H, CH₃); ¹³C NMR (100 MHz, CDCl₃)

d 177.43,

171.91, 171.82, 156.19, 152.31, 143.72, 142.39, 141.65, 136.41, 128.43,

128.03, 127.94, 122.29, 72.53, 71.01, 68.93, 65.97, 63.72, 61.71, 47.66,

46.81, 46.11, 45.85, 45.66, 43.18, 41.91, 41.56, 39.20, 36.58, 33.89,

33.10, 32.35, 31.82, 30.72, 29.70, 29.32, 29.02, 28.81, 27.63, 25.75,

23.61, 23.35, 23.10, 19.85, 19.79, 16.64, 15.74; HR-MS (ESI) m/z:

calcd. for C₄₇H₆₄N₂O₈[MþH]^þ: 785.4735, found: 785.4723.

NMR (400 MHz, CDCl₃)

d

8.38 (d, J ¼ 2.1 Hz,1H, H-pyrazine), 8.35 (d,

J ¼ 2.4 Hz, 1H, H-pyrazine), 5.38 (t, J ¼ 3.5 Hz, 1H, H-12), 4.66 (d,

J ¼ 15.8 Hz, 1H, H-23a), 4.54 (d, J ¼ 15.8 Hz, 1H, H-23b), 4.32e4.27

(m, 2H, CH₂O PEG group), 3.80 (d, J ¼ 10.6 Hz, 1H, CH₂a), 3.74e3.57

(m, 32H, CH₂O PEG group), 3.49 (d, J ¼ 10.6 Hz, 1H, CH₂b), 3.01 (d,

J ¼ 16.6 Hz, 1H, H-1a), 2.91 (dd, J ¼ 14.3, 3.4 Hz, 1H, H-18), 2.51 (d,

J ¼ 16.6 Hz, 1H, H-1b), 1.33 (s, 3H, CH₃), 1.20 (s, 3H, CH₃), 0.95 (s, 6H,

2 ꢂ CH₃), 0.92 (s, 3H, CH₃), 0.83 (s, 3H, CH₃); ¹³C NMR (100 MHz,

4.1.14. 4-(Benzyl olean[3,2-b]pyrazine-12-en-28-oate-23-oxy)-4-

oxo-butyryltriethylene glycol (10b)

According to the synthesis method of 10a, 3 was reacted with

triethylene glycol to give a yellow transparent syrup 10b (46.0 mg,

47.7% yield) after dialyzed (MWCO 500 Da) and puriﬁed by silica gel

CDCl₃)

d

176.99, 168.01, 152.07, 143.55, 142.11, 141.63, 122.28, 99.93,

72.62, 70.68e70.24, 68.88, 64.23, 61.67, 60.34, 47.72, 47.17, 46.83,

45.91, 45.75, 43.33, 41.99, 41.38, 39.26, 36.54, 33.92, 33.06, 32.19,

31.92, 30.70, 27.60, 25.68, 23.62, 23.40, 23.13, 20.18, 19.40, 16.63,

16.15; HR-MS (ESI) m/z: calcd. for C₅₂H₈₄N₂O₁₄[MþH]^þ: 961.5996,

found: 961.5984.

column chromatography (V _DCM: V

¼ 150:1). ¹H NMR

methanol

(400 MHz, CDCl₃)

d

8.42 (d, J ¼ 2.1 Hz, 1H, H-pyrazine), 8.29 (d,

J ¼ 2.3 Hz, 1H, H-pyrazine), 7.37e7.28 (m, 5H, 5 ꢂ HeAr), 5.37 (t,

J ¼ 3.4 Hz, 1H, H-12), 5.13e5.04 (m, 2H, CH₂Ar), 4.32 (d, J ¼ 10.5 Hz,

1H, H-23a), 4.27 (d, J ¼ 10.5 Hz, 1H, H-23b), 4.21e4.15 (m, 2H, CH₂O

PEG group), 3.74e3.59 (m, 10H, CH₂O PEG group), 3.02e2.90 (m,

2H, H-1a, H-18), 2.52 (d, J ¼ 13.8 Hz, 1H, H-1b), 2.49e2.31 (m, 4H,

2 ꢂ CH₂), 1.27 (s, 3H, CH₃), 1.19 (s, 3H, CH₃), 0.94 (s, 3H, CH₃), 0.91 (s,

3H, CH₃), 0.88 (s, 3H, CH₃), 0.69 (s, 3H, CH₃); ¹³C NMR (100 MHz,

4.1.11. O-(23-hydroxy-olean[3,2-b]pyrazine-12-en-28-acyl)-

acetylpolyethylene glycol 600 (9e)

According to the synthesis method of 9a, 8 was reacted with

polyethylene glycol 600 to give a yellow transparent syrup 9e

(106.5 mg, 45.2% yield) after dialyzed (MWCO 1000 Da) and puri-

ﬁed by silica gel column chromatography (V _DCM: V _methanol¼ 75:1).

CDCl₃) d 177.43, 171.97, 171.89, 156.15, 152.34, 143.70, 142.38, 141.73,

¹H NMR (400 MHz, CDCl₃)

d

8.38 (s, 2H, H-pyrazine), 5.38 (t,

136.41, 128.43, 128.02, 127.94, 122.30, 72.53, 70.98, 70.58, 70.34,

69.02, 65.97, 63.67, 61.74, 47.67, 46.81, 46.14, 45.85, 45.65, 43.19,

41.91, 41.55, 39.19, 36.58, 33.89, 33.10, 32.35, 31.82, 30.72, 29.69,

29.36, 28.94, 28.79, 27.62, 25.75, 23.61, 23.35, 23.10, 19.86, 19.79,

16.64, 15.73; HR-MS (ESI) m/z: calcd. for C₄₉H₆₈N₂O₉[MþH]^þ:

829.4998, found: 829.4988.

J ¼ 3.5 Hz, 1H, H-12), 4.66 (d, J ¼ 15.8 Hz, 1H, H-23a), 4.54 (d,

J ¼ 15.8 Hz,1H, H-23b), 4.31e4.29 (m, 2H, CH₂O PEG group), 3.80 (d,

J ¼ 10.4 Hz,1H, CH₂a), 3.74e3.59 (m, 50H, CH₂O PEG group), 3.49 (d,

J ¼ 10.5 Hz, 1H, CH₂b), 3.01 (d, J ¼ 17.1 Hz, 1H, H-1a), 2.92 (dd,

J ¼ 13.5, 4.3 Hz, 1H, H-18), 2.51 (d, J ¼ 15.0 Hz, 1H, H-1b), 1.33 (s, 3H,

CH₃), 1.20 (s, 3H, CH₃), 0.95 (s, 6H, 2 ꢂ CH₃), 0.92 (s, 3H, CH₃), 0.83

(s, 3H, CH₃); ¹³C NMR (100 MHz, CDCl₃)

d

176.99, 168.00, 152.08,

4.1.15. 4-(Benzyl olean[3,2-b]pyrazine-12-en-28-oate-23-oxy)-4-

oxo-butyryltetraethylene glycol (10c)

According to the synthesis method of 10a, 3 was reacted with

tetraethylene glycol to give a yellow transparent syrup 10c

(46.3 mg, 45.6% yield) after dialyzed (MWCO 500 Da) and puriﬁed

143.54, 142.08, 141.66, 122.29, 72.67, 70.76e70.24, 68.87, 64.22,

61.66, 60.33, 47.73, 47.13, 46.82, 45.91, 45.74, 43.35, 41.99, 41.37,

39.26, 36.53, 33.91, 33.05, 32.17, 31.90, 30.69, 27.60, 25.68, 23.61,

23.40, 23.12, 20.18, 19.40, 16.62, 16.14; HR-MS (ESI) m/z: calcd. for

12

B. Wang, S. Liu, W. Huang et al.

European Journal of Medicinal Chemistry 211 (2021) 113107

by silica gel column chromatography (V _DCM: V _methanol¼ 150:1). ¹H

previous experimental method [24].

NMR (400 MHz, CDCl₃)

d

8.41 (d, J ¼ 2.4 Hz,1H, H-pyrazine), 8.29 (d,

J ¼ 2.5 Hz, 1H, H-pyrazine), 7.36e7.30 (m, 5H, 5 ꢂ HeAr), 5.37 (t,

J ¼ 3.5 Hz, 1H, H-12), 5.13e5.04 (m, 2H, CH₂Ar), 4.32 (d, J ¼ 10.5 Hz,

1H, H-23a), 4.27 (d, J ¼ 10.5 Hz, 1H, H-23b), 4.21e4.16 (m, 2H, CH₂O

PEG group), 3.74e3.59 (m, 14H, CH₂O PEG group), 2.99e2.93 (m,

2H, H-1a, H-18), 2.54 (d, J ¼ 16.4 Hz, 1H, H-1b), 2.49e2.34 (m, 4H,

2 ꢂ CH₂), 1.27 (s, 3H, CH₃), 1.19 (s, 3H, CH₃), 0.94 (s, 3H, CH₃), 0.91 (s,

3H, CH₃), 0.88 (s, 3H, CH₃), 0.69 (s, 3H, CH₃); ¹³C NMR (100 MHz,

4.1.19. General procedure for the synthesis of 17

13 (7.3 g, 10.8 mmol) and sulphur (10.4 mg, 324.3 mmol) were

dissolved in dry morpholine (250.0 mL) and ethylene diamine

(14.4 mL, 215.6 mmol) was added. The reaction was reﬂuxed at

150 ^ꢀC for 2 h. The mixture was diluted with ethyl acetate, washed

with water and saturated brine in sequence, dried over Na₂SO₄and

concentrated. The residue was puriﬁed by silica gel column chro-

CDCl₃)

d

177.44, 171.99, 171.92, 156.19, 152.31, 143.70, 142.41, 141.69,

matography (V _{petroleum ether}: V

solid 14 (4.3 g, 56.2% yield).

To a solution of 14 (90.0 mg, 0.1 mmol) in anhydrous DCM (6 mL)

was added methanoic acid (312.0 L) and 30% H₂O₂(128 L), the

reaction was reacted with stirring at room temperature for 28 h.

The reaction solution was washed to neutral with saturated sodium

bicarbonate solution and washed twice with saturated brine, dried

over Na₂SO₄and concentrated. The residue was puriﬁed by silica

gel column chromatography (V _{petroleum ether}: V _{ethyl acetate}¼ 8:1) to

give a white solid 17 (72.8 mg, 61.0% yield).

¼ 25:1) to give a white

ethyl acetate

136.41, 128.43, 128.02, 127.94, 122.30, 72.57, 70.97, 70.64, 70.55,

70.53, 70.33, 69.06, 65.97, 63.77, 61.74, 47.65, 46.82, 46.14, 45.85,

45.66, 43.20, 41.91, 41.56, 39.20, 36.58, 33.90, 33.10, 32.36, 31.82,

30.73, 29.70, 28.93, 28.79, 27.63, 27.42, 25.76, 23.62, 23.36, 23.11,

19.86, 19.80, 16.65, 15.74; HR-MS (ESI) m/z: calcd. for C₅₁H₇₂N₂O₁₀

[MþH]^þ: 873.5260, found: 873.5223.

m

4.1.16. 4-(Benzyl olean[3,2-b]pyrazine-12-en-28-oate-23-oxy)-4-

oxo-butyrylpolyethylene glycol 400 (10d)

According to the synthesis method of 10a, 3 was reacted with

polyethylene glycol 400 to give a yellow transparent syrup 10d

(76.8 mg, 40.2% yield) after dialyzed (MWCO 500 Da) and puriﬁed

by silica gel column chromatography (V _DCM: V _methanol¼ 80:1). ¹H

4.1.20. Benzyl 23-hydroxy-olean[3,2-b]pyrazine-12-oxo-28-oate

(18)

17 (60 mg, 0.1 mmol) was dissolved in acetone (5 mL) and 10%

hydrochloric acid solution (282.0 mL) was added with stirring for

10 h at room temperature. The mixture was diluted with ethyl ac-

etate, washed with water to neutral and then washed with satu-

rated brine, dried over Na₂SO₄and concentrated. The residue was

puriﬁed by silica gel column chromatography (V _{petroleum ether}: V

NMR (400 MHz, CDCl₃)

d

8.41 (d, J ¼ 2.2 Hz,1H, H-pyrazine), 8.29 (d,

J ¼ 2.3 Hz, 1H, H-pyrazine), 7.37e7.29 (m, 5H, 5 ꢂ HeAr), 5.38 (t,

J ¼ 3.5 Hz, 1H, H-12), 5.12e5.04 (m, 2H, CH₂Ar), 4.33 (d, J ¼ 10.5 Hz,

1H, H-23a), 4.27 (d, J ¼ 10.5 Hz, 1H, H-23b), 4.20e4.14 (m, 2H, CH₂O

PEG group), 3.73e3.60 (m, 32H, CH₂O PEG group), 2.98e2.93 (m,

2H, H-1a, H-18), 2.51 (d, J ¼ 16.9 Hz, 1H, H-1b), 2.54e2.45 (m, 4H,

2 ꢂ CH₂), 1.27 (s, 3H, CH₃), 1.19 (s, 3H, CH₃), 0.94 (s, 3H, CH₃), 0.91 (s,

3H, CH₃), 0.88 (s, 3H, CH₃), 0.69 (s, 3H, CH₃); ¹³C NMR (100 MHz,

¼ 6:1) to give a white solid 18 (41.5 mg, 82.1%). m.p.

ethyl acetate

159.6e161.9 ^ꢀC. ¹H NMR (400 MHz, CDCl₃)

d

8.29 (d, J ¼ 3.9 Hz, 1H,

H-pyrazine), 8.02 (d, J ¼ 4.0 Hz, 1H, H-pyrazine), 7.39e7.30 (m, 5H,

5 ꢂ HeAr), 5.21, 5.09 (each 1H, d, J ¼ 12.2 Hz, 1H, CH₂Ar), 3.80 (d,

J ¼ 10.7 Hz, 1H, H-23a), 3.46 (d, J ¼ 10.7 Hz, 1H, H-23b), 3.24 (d,

J ¼ 18.2 Hz, 1H, H-13), 2.85 (dd, J ¼ 13.4, 3.2 Hz, 1H, H-1a), 2.51 (d,

J ¼ 4.2 Hz, 1H, H-18), 2.45 (dd, J ¼ 16.3, 4.5 Hz, 1H, H-1b), 1.32 (s, 3H,

CH₃),1.01 (s, 3H, CH₃), 0.95 (s, 3H, CH₃), 0.92 (s, 3H, CH₃), 0.81 (s, 3H,

CDCl₃) d 177.43, 171.97, 171.89, 156.10, 152.36, 143.70, 142.36, 141.78,

136.40, 128.43, 128.02, 127.94, 122.30, 72.63, 70.97e70.27, 69.03,

65.96, 63.80, 61.69, 47.71, 46.81, 46.14, 45.84, 45.65, 43.18, 41.90,

41.55, 39.19, 36.58, 33.89, 33.10, 32.35, 31.82, 30.72, 29.69, 28.98,

28.90, 28.78, 27.62, 25.75, 23.61, 23.34, 23.10, 19.86, 19.80, 16.64,

15.73; HR-MS (ESI) m/z: calcd. for C₆₁H₉₂N₂O₁₅[MþH]^þ: 1093.6571,

found: 1093.6556.

CH₃), 0.68 (s, 3H, CH₃); ¹³C NMR (100 MHz, CDCl₃)

d 210.24, 177.39,

161.44, 143.30, 142.61, 136.23, 131.48, 128.44, 128.12, 69.81, 65.92,

53.39, 51.68, 48.31, 47.10, 45.16, 44.04, 42.08, 40.97, 38.35, 37.96,

36.14, 35.40, 34.38, 33.32, 32.73, 31.98, 30.62, 30.30, 27.28, 25.59,

23.08, 22.58, 20.31, 19.70, 18.72, 16.36, 15.05; HR-MS (ESI) m/z:

calcd. for C₃₉H₅₂N₂O₄[MþH]^þ: 613.4000, found: 613.3998.

4.1.17. 4-(Benzyl olean[3,2-b]pyrazine-12-en-28-oate-23-oxy)-4-

oxo-butyrylpolyethylene glycol 600 (10e)

According to the synthesis method of 10a, 3 was reacted with

polyethylene glycol 600 to give a yellow transparent syrup 10e

(68.8 mg, 41.4% yield) after dialyzed (MWCO 1000 Da) and puriﬁed

by silica gel column chromatography (V _DCM: V _methanol¼ 60:1). ¹H

4.1.21. General procedure for the synthesis of 19

According to the synthesis method of 18, 13 was deprotected to

give crude sodium 15, which could be used directly for the next step

without further puriﬁcation.

15 (458.0 mg, 0.8 mmol) was dissolved in chloroform (50.0 mL)

and pyridinium bromide perbromide (259.0 mg, 0.8 mmol) was

added with stirring for 4 h at room temperature. The mixture was

diluted with DCM, washed with water and saturated brine in

sequence, dried over Na₂SO₄and concentrated to give crude so-

dium 19, which could be used directly for the next step without

further puriﬁcation.

NMR (400 MHz, CDCl₃)

d

8.41 (d, J ¼ 2.3 Hz,1H, H-pyrazine), 8.29 (d,

J ¼ 2.4 Hz, 1H, H-pyrazine), 7.37e7.31 (m, 5H, 5 ꢂ HeAr), 5.38 (t,

J ¼ 3.5 Hz, 1H, H-12), 5.13e5.04 (m, 2H, CH₂Ar), 4.33 (d, J ¼ 10.5 Hz,

1H, H-23a), 4.27 (d, J ¼ 10.5 Hz, 1H, H-23b), 4.19e4.15 (m, 2H, CH₂O

PEG group), 3.73e3.60 (m, 50H, CH₂O PEG group), 2.99e2.93 (m,

2H, H-1a, H-18), 2.52 (d, J ¼ 16.5 Hz, 1H, H-1b), 2.50e2.32 (m, 4H,

2 ꢂ CH₂), 1.27 (s, 3H, CH₃), 1.19 (s, 3H, CH₃), 0.94 (s, 3H, CH₃), 0.91 (s,

3H, CH₃), 0.88 (s, 3H, CH₃), 0.69 (s, 3H, CH₃); ¹³C NMR (100 MHz,

CDCl₃) d 177.44, 171.98, 171.89, 156.14, 152.34, 143.70, 142.38, 141.74,

136.41, 128.43, 128.02, 127.94, 122.30, 72.64, 70.97e70.28, 69.03,

65.96, 63.80, 61.70, 47.69, 46.81, 46.14, 45.84, 45.65, 43.19, 41.90,

41.55, 39.19, 36.58, 33.90, 33.10, 32.35, 31.82, 30.72, 29.69, 28.91,

28.78, 27.62, 25.76, 23.61, 23.35, 23.11, 19.86, 19.80, 18.43, 16.64,

4.1.22. Benzyl 23-hydroxy-olean[3,2-d]-(2-methyl)thiazole-12-en-

28-oate (21a)

19 (32.0 mg, 50.0

mmol) was dissolved in ethyl alcohol absolute

15.73; HR-MS (ESI) m/z: calcd. for

C

₆₉H₁₀₈N₂O₁₉[MþH]^þ:

(20.0 mL). Thioacetamide (38.0 mg, 50.0 mmol) was dissolved in

1269.7619, found: 1269.7604.

ethyl alcohol absolute (20.0 mL) and the solution was dropped into

the above solution at a rate of 10 drops per half hour. The reaction

was reﬂuxed at 95 ^ꢀC for 48 h. Ethyl alcohol absolute was removed

by vacuum distillation, and the residue was dissolved in DCM,

washed with 10% hydrochloric acid solution, saturated sodium bi-

carbonate solution, water and saturated brine in sequence, dried

4.1.18. General procedure for the synthesis of 13

The synthesis of the key intermediate 13 used HD as the starting

compound with three steps of protection of C-28 carboxyl, pro-

tection of C-23 hydroxyl, oxidation of C-3 hydroxyl, according to the

13

B. Wang, S. Liu, W. Huang et al.

European Journal of Medicinal Chemistry 211 (2021) 113107

over Na₂SO₄and concentrated. The residue was puriﬁed by silica

gel column chromatography (V _{petroleum ether}: V _{ethyl acetate}¼ 10:1) to

give a white solid 21a (24.0 mg, 62.8% yield). m.p.109.2e113.1 ^ꢀC. ¹H

gel column chromatography (V _{petroleum ether}: V

¼ 2:1).

ethyl acetate

7.38 (d, J ¼ 4.0 Hz,

m.p. 132.7e135.7 ^ꢀC. ¹H NMR (400 MHz, CDCl₃)

d

5H, 5 ꢂ HeAr), 7.25 (s, 1H, H-pyrazole), 5.39 (t, J ¼ 3.3 Hz,1H, H-12),

5.12 (q, J ¼ 12.5 Hz, 2H, CH₂Ar), 3.73 (d, J ¼ 10.5 Hz, 1H, H-23a), 3.40

(d, J ¼ 10.5 Hz, 1H, H-23b), 2.97 (d, J ¼ 13.9 Hz, 1H, H-18), 2.60 (d,

J ¼ 14.9 Hz, 1H, H-13), 1.24 (s, 3H, CH₃), 1.18 (s, 3H, CH₃), 0.96 (s, 3H,

CH₃), 0.94 (s, 3H, CH₃), 0.90 (s, 3H, CH₃), 0.71 (s, 3H, CH₃); ¹³C NMR

NMR (400 MHz, CDCl₃)

d

7.34 (s, 5H, 5 ꢂ HeAr), 5.34 (t, J ¼ 3.5 Hz,

1H, H-12), 5.14e5.03 (m, 2H, CH₂Ar), 3.74 (d, J ¼ 10.3 Hz,1H, H-23a),

3.35 (d, J ¼ 10.3 Hz, 1H, H-23b), 2.93 (d, J ¼ 16.2 Hz, 1H, H-1a), 2.70

(d, J ¼ 15.8 Hz, 1H, H-18), 2.63 (s, 3H, CH₃), 2.24 (d, J ¼ 16.3 Hz, 1H,

H-1b), 1.25 (s, 3H, CH₃), 1.14 (s, 3H, CH₃), 0.97 (s, 3H, CH₃), 0.93 (s,

3H, CH₃), 0.90 (s, 3H, CH₃), 0.68 (s, 3H, CH₃); ¹³C NMR (100 MHz,

(100 MHz, CDCl₃)

d 177.49, 149.56, 143.59, 136.39, 131.13, 128.42,

128.01, 127.93, 122.46, 114.08, 70.33, 66.96, 47.48, 46.80, 46.04,

45.96, 41.89, 41.49, 39.28, 38.97, 38.23, 35.84, 33.87, 33.11, 32.34,

31.94, 30.71, 29.70, 29.32, 27.66, 25.71, 23.62, 23.38, 23.08, 19.79,

19.05, 16.66, 15.46; HR-MS (ESI) m/z: calcd. for C₃₈H₅₂N₂O₃

[MþH]^þ: 585.4051, found: 585.4040.

CDCl₃) d 177.42,143.81, 136.41, 128.43, 128.04,127.95,127.58, 122.13,

71.05, 65.98, 46.90, 46.79, 46.17, 45.87, 41.92, 41.52, 41.46, 39.37,

38.48, 38.35, 33.89, 33.10, 32.34, 31.91, 30.72, 29.70, 28.21, 27.79,

27.63, 25.72, 24.94, 23.62, 23.35, 23.08, 19.79, 19.15, 17.68, 16.65,

15.92; HR-MS (ESI) m/z: calcd. for C₃₉H₅₃NO₃S [MþH]^þ: 616.3819,

found: 616.3816.

4.1.27. 23-hydroxy-olean[3,2-c]pyrazole ꢁ12-en-28-oic acid (24b)

According to the synthesis method of 4, 24a was deprotected to

give a white solid 24b (180.5 mg, 87.0% yield) after puriﬁed by silica

4.1.23. General procedure for the synthesis of 22

According to the synthesis method of 4, 15 was deprotected to

give crude sodium 20, which could be used directly for the next

step without further puriﬁcation.

gel column chromatography (V _chloroform: V

¼ 6:1). m.p.

methanol

122.8e124.1 ^ꢀC. ¹H NMR (400 MHz, CD₄O)

d 7.91 (s, 1H, NH), 7.21 (s,

According to the synthesis method of 19, 20 was reacted with

pyridinium bromide perbromide to give crude sodium 22, which

could be used directly for the next step without further puriﬁcation.

1H, H-pyrazole), 5.32 (t, J ¼ 3.3 Hz, 1H, H-12), 3.57 (dd, J ¼ 53.5,

10.9 Hz, 2H, H-23a), 2.90 (d, J ¼ 13.7 Hz, 1H, H-18), 2.59 (d,

J ¼ 14.7 Hz, 1H, H-13), 1.21 (s, 3H, CH₃), 1.12 (s, 3H, CH₃), 0.96 (s, 3H,

CH₃), 0.92 (s, 3H, CH₃), 0.90 (s, 6H, 2 ꢂ CH₃); ¹³C NMR (100 MHz,

4.1.24. 23-hydroxy-olean[3,2-d]-(2-methyl)thiazole-12-en-28-oic

acid (21b)

According to the synthesis method of 21a, 22 was reacted with

thioacetamide to give a white solid 21b (167.0 mg, 64.6% yield) after

puriﬁed by silica gel column chromatography (V _{petroleum ether}: V

CD₄O) d 180.37, 147.13, 143.63, 132.20, 122.26, 114.26, 68.63, 53.34,

46.22, 46.00, 45.87, 41.65, 41.37, 39.09, 38.72, 38.00, 35.55, 33.47,

32.34, 32.14, 31.69, 30.17, 27.45, 24.81, 23.07, 22.65,22.52, 18.93,

18.18, 16.07, 14.52; HR-MS (ESI) m/z: calcd. for C₃₁H₄₆N₂O₃[MþH]^þ:

495.3581, found: 495.3579.

¼ 1:1). m.p. 121.4e125.2 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

ethyl acetate

d

5.31 (t, J ¼ 3.5 Hz, 1H, H-12), 3.73 (d, J ¼ 10.9 Hz, 1H, H-23a), 3.52

(d, J ¼ 10.9 Hz, 1H, H-23b), 2.91e2.85 (m, 1H, H-1a), 2.73 (d,

J ¼ 15.6 Hz, 1H, H-18), 2.63 (s, 3H, CH₃), 2.31 (d, J ¼ 16.0 Hz, 1H, H-

1b), 1.21 (s, 3H, CH₃), 1.09 (s, 3H, CH₃), 0.98 (s, 3H, CH₃), 0.95 (s, 3H,

CH₃), 0.91 (s, 3H, CH₃), 0.90 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-

4.2. Aqueous solubility determination

An excess of each compound was added to 4 mL of deionized

water. The suspensions were placed and shaken in a constant

temperature oscillator for 24 h at 37 ^ꢀC, and then the mixtures were

centrifuged at 14,000 rpm for 5 min. The supernatants were ﬁltered

with 0.45 mm microporous membrane and diluted properly, then

the concentration of compounds was determined by UVeVis

D₆)

d 179.10, 161.32, 154.16, 144.29, 127.78, 122.04, 46.23, 46.05,

46.01, 44.30, 42.48, 42.06, 41.46, 38.34, 33.88, 33.36, 32.60, 31.97,

31.68, 30.94, 30.36, 29.23, 27.78, 25.99, 23.88, 23.35, 23.18, 19.48,

19.30, 18.78, 17.15, 16.04; HR-MS (ESI) m/z: calcd. for C₃₂H₄₇NO₃S

[MþH]^þ: 526.3350, found: 526.3350.

spectrophotometry.

4.1.25. General procedure for the synthesis of 23

To a solution of 13 (300.0 mg, 0.4 mmol) in anhydrous DCM

(20.0 mL) was added potassium t-butoxide (151.0 mg, 1.3 mmol),

the reaction was reacted with stirring at room temperature for

2e3 h. Ethyl formate was added when the solution turned brown

and the reaction continued overnight. DCM was removed by vac-

uum distillation, and the residue was dissolved in ethyl acetate,

washed with saturated brine, dried over Na₂SO₄and concentrated

to give crude sodium 16, which could be used directly for the next

step without further puriﬁcation.

To a solution of 16 (334.0 mg, 0.5 mmol) in ethyl alcohol abso-

lute (20.0 mL) was added hydrazine hydrate (0.2 mL, 3.9 mmol), the

reaction was reﬂuxed at 80 ^ꢀC for 4 h. Then water (10.0 mL) was

added and ethanol was removed by vacuum distillation, and the

residue was dissolved in ethyl acetate, washed with water and

saturated brine in sequence, dried over Na₂SO₄and concentrated.

The residue was puriﬁed by silica gel column chromatography (V

_{petroleum ether}: V _{ethyl acetate}¼ 5:1) to give a white solid 23 (329.0 mg,

69.9% yield).

4.3. Stability studies

For stability studies, compound 10c (9 mg, 0.010 mmol) was

dissolved in 0.01 M PBS (6 mL, pH 7.4, containing 0.5% Tween 80)

and PLE (20 units) was added. The PBS solution was placed and

shaken in a constant temperature oscillator at 37 ^ꢀC. At scheduled

time intervals, 1 mL solutions were removed respectively, diluted

for 10 times and centrifuged at 14,000 rpm for 5 min. The super-

natants were analyzed for compound 10c by HPLC (LC-20A, Shi-

madzu, JPN) with UVeVis detector (SPD-20A) using a ZORBAX SB-

C18 column (4.6 ꢂ 150 mm, 5

m

m) at 37 ^ꢀC. An isocratic elution

method of 95% methanol/water (containing 0.1% triﬂuoroacetic

acid) was applied at a ﬂow rate of 1 mL/min. The concentration of

compound 10c was detected by UV at 306 nm. A reference ab-

sorption graph was made from dilution series of the compounds in

methanol. The percentage of compound 10c remaining at each time

point relative to the 0 h sample was then calculated from HPLC

concentration. Benorylate (9 mg, 0.029 mmol) was dissolved in

0.01 M PBS (17 mL, pH 7.4, containing 0.5% Tween 80) and PLE (57

units) was added. The methods of sample processing and data

determination were the same as above, excepted that the isocratic

elution method of 56% methanol/water was applied at a ﬂow rate of

1 mL/min.

4.1.26. Benzyl 23-hydroxy-olean[3,2-c]pyrazole ꢁ12-en-28-oate

(24a)

According to the synthesis method of 18, 23 was deprotected to

give a white solid 24a (225.7 mg, 82.0% yield) after puriﬁed by silica

14

B. Wang, S. Liu, W. Huang et al.

European Journal of Medicinal Chemistry 211 (2021) 113107

4.4. Biology

the Tripos force ﬁeld, and the Gasteiger-Hückel charge was loaded.

After protein preparation and molecular optimization, other pa-

rameters used system default values to complete the molecular

docking process. The docking results were visualized using the

PyMOL software (http://www.pymol.org).

4.4.1. Cell culture

The MDR cancer cell lines KBV and MCF-7T were kindly pro-

vided by Dr. Xiaoguang Chen from Institute of Materia Medica,

Chinese Academy of Medical Sciences. Both cells were cultured in

DMEM medium (Dulbecco’s Modiﬁed Eagle Medium) with 10%

heat-inactivated fetal calf serum, 100 U/mL penicillin and 0.1 mg/

mL streptomycin. All cells were incubated at 37 ^ꢀC in a humidiﬁed

atmosphere containing 5% CO₂. Moreover, 10 nM paclitaxel was

termly added to KBV and MCF-7T medium to keep the drug resis-

tance phenotype. Cells were harvested in the exponential growth

phase for further assays.

4.5. Statistical analysis

Date were presented as mean SD. The statistical signiﬁcance of

differences between groups was evaluated by the Student’s t-test

and indicated with *p < 0.05, **p < 0.01.

Declaration of competing interest

4.4.2. Cytotoxicity and MDR reversal assay

The authors declare that they have no known competing

ﬁnancial interests or personal relationships that could have

appeared to inﬂuence the work reported in this paper.

The viability of cells was tested by the MTT assays as reported

previously [47]. In short, cells were seeded in 96-well plates and

treated with the tested articles at the desired concentration for

72 h, then cells were treated with MTT solution for about 2 h. After

the medium of 96-well plates was discarded, DMSO was added,

then the absorbance was measured at 570 nm to calculate the

survival rate. The RF which was the index for MDR-reversal activity

deﬁned as the ratio between the IC₅₀value presented by the drugs

alone and drugs combined with the test compounds.

Acknowledgment

This work was partially supported by National Natural Science

Foundation of China (81773563), The Science and Technology

Support Program for Youth Innovation in Universities of Shandong

(2020KJM003), Natural Science Foundation of Shandong Province

(ZR2018LH025), Graduate Innovation Foundation of Yantai Uni-

versity, GIFYTU (YDZD2033), Top Talents Program for One Case One

Discussion of Shandong Province. We thank Victoria Muir, PhD,

from Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for

editing the English text of a draft of this manuscript.

4.4.3. Cell cycle distribution analysis

A ﬂow cytometry assay was conducted to analyze the cell cycle

distribution as previously reported [48]. Brieﬂy, cells were seeded

in 6-well plates, then added the tested compounds to the culture

medium for the next day. Cells were harvested after the indicated

time, ﬁxed in 80% ethanol overnight at ꢁ20 ^ꢀC, and then washed

with PBS and stained with PI solution (20 mg/mL PI and 20 mg/mL

RNase in PBS) for 30 min. The cell ﬂuorescence was measured by a

ﬂow cytometry machine (BD, C6, USA).

Appendix A. Supplementary data

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2020.1 13107.

4.4.4. Intracellular rhodamine 123 accumulation assay

References

The effects of HBQ and compound 10c on the Rhodamine123

accumulation were measured by ﬂow cytometry following our

previous protocol [28]. In short, KBV and MCF-7T cells were both

treated with or without HBQ and compound 10c for 2 h at indicated

[1] I.S. Mohammad, W. He, L.F. Yin, Understanding of human ATP binding cassette

superfamily and novel multidrug resistance modulators to overcome MDR,

Biomed. Pharmacother. 100 (2018) 335e348.

[2] Q. Wu, Z.P. Yang, Y.Z. Nie, Y.Q. Shi, D.M. Fan, Multi-drug resistance in cancer

chemotherapeutics: mechanisms and lab approaches, Canc. Lett. 347 (2014)

159e166.

concentrations, then both cells were treated with 10 mM Rhoda-

mine123 for 30 min. Subsequently, the cells were harvested and

washed with PBS for three times, then detected by ﬂow cytometry

machine (BD, C6, USA). 10,000 cells were detected to calculate the

mean ﬂuorescence intensity.

ꢀ

[3] Y.G. Assaraf, A. Brozovic, A.C. Gonçalves, D. Jurkovicova, A. Line,

M. Machuqueiro, S. Saponara, A.B. Sarmento-Ribeiro, C.P.R. Xavier,

M.H. Vasconcelos, The multi-factorial nature of clinical multidrug resistance in

cancer, Drug Resist. Updates 46 (2019) 100645.

ꢁ ꢂ

[4] S. Dallavalle, V. Dobricic, L. Lazzarato, E. Gazzano, M. Machuqueiro, I. Pajeva,

I. Tsakovska, N. Zidar, R. Fruttero, Improvement of conventional anti-cancer

drugs as new tools against multidrug resistant tumors, Drug Resist. Updates

50 (2020) 100682.

4.4.5. Flutax1 ﬂuorescence staining assay

Flutax1 is a ﬂuorescent paclitaxel derivative that binds to the

paclitaxel microtubule binding site with high afﬁnity. The KBV and

MCF-7T cells were treated with or without compound 10c for 3 h,

[5] H. Zhang, H.W. Xu, C.R. Ashby, Y.G. Assaraf, Z.S. Chen, H.M. Liu, Chemical

molecular-based approach to overcome multidrug resistance in cancer by

targeting P-glycoprotein (P-gp), Med. Res. Rev. (2020) 1e31.

[6] E.E. Chufan, H.M. Sim, S.V. Ambudkar, Molecular basis of the polyspeci ﬁ city of

P-glycoprotein (ABCB1): recent biochemical and structural studies, Adv. Canc.

Res. 125 (2015) 71e96.

[7] R. Silva, V. Vilas-Boas, H. Carmo, R.J. Dinis-Oliveira, F. Carvalho, M.L. Bastos,

F. Remiao, Modulation of P-glycoprotein ef ﬂ ux pump: induction and activa-

tion as a therapeutic strategy, Pharmacol. Ther. 149 (2015) 1e123.

[8] G. Szakacs, J.K. Paterson, J.A. Ludwig, C. Booth-Genthe, M.M. Gottesman, Tar-

then incubated with 1 mM Flutax1 for 1 h. The cells were harvested

and washed with PBS for three times, then detected by ﬂow

cytometry machine (BD, C6, USA). 10,000 cells were detected to

calculate the mean ﬂuorescence intensity.

4.4.6. Molecular docking

geting multidrug resistance in cancer, Nat. Rev. Drug Discov.

5 (2006)

The binding mode of compounds and a homology model of

human P-gp was constructed and studied via surﬂex-dock module

in software SYBYL-X2.1.1 (Tripos Associates Inc., St. Louis, USA). The

three-dimensional structure model of human P-gp protein was

derived from the literature [45,46], the binding site of QZ-SSS

derived from the crystal structure of mouse P-gp (PDB ID:4M1M)

was recognized as the active site of the homology model of human

P-gp [45]. The Powell energy gradient method was used for the 3D

structure optimization process of all compounds, the force ﬁeld was

219e234.

[9] N. Kopcho, G. Chang, E.A. Komives, Dynamics of ABC transporter P-glyco-

protein in three conformational states, Sci. Rep. 9 (2019) 15092.

[10] A.K. Nanayakkara, C.A. Follit, G. Chen, N.S. Williams, P.D. Vogel, J.G. Wise,

Targeted inhibitors of P-glycoprotein increase chemotherapeutic-induced

mortality of multidrug resistant tumor cells, Sci. Rep. 8 (2018) 967.

[11] R.J. Kathawala, P. Gupta, C.R. Ashby, Z.S. Chen, The modulation of ABC

transporter-mediated multidrug resistance in cancer: a review of the past

decade, Drug Resist. Updates 18 (2015) 1e17.

[12] H.H. Yin, J.J. Dong, Y.C. Cai, X.M. Shi, H. Wang, G.X. Liu, Y. Tang, J.W. Liu, Lei Ma,

Design, synthesis and biological evaluation of chalcones as reversers of P-

15

B. Wang, S. Liu, W. Huang et al.

European Journal of Medicinal Chemistry 211 (2021) 113107

glycoprotein-mediated multidrug resistance, Eur. J. Med. Chem. 180 (2019)

350e366.

[31] J. Hodon, L. Borkova, J. Pokorny, A. Kazakova, M. Urban, Design and synthesis

of pentacyclic triterpene conjugates and their use in medicinal research, Eur. J.

Med. Chem. 182 (2019) 111653.

[32] A. Kolate, D. Baradia, S. Patil, I. Vhora, G. Kore, A. Misra, PEGda versatile

conjugating ligand for drugs and drug delivery systems, J. Contr. Release 192

(2014) 67e81.

[33] S.A. Senevirathne, K.E. Washington, M.C. Biewer, M.C. Stefan, PEG based anti-

cancer drug conjugated prodrug micelles for the delivery of anti-cancer

agents, J. Mater. Chem. B 4 (2016) 360e370.

[13] J. Yu, P. Zhou, J. Asenso, X.D. Yang, C. Wang, W. Wei, Advances in plant-based

inhibitors of P-glycoprotein, J. Enzym. Inhib. Med. Chem. 31 (2016) 867e881.

ꢂ

[14] A. Paterna, A. Kincses, G. Spengler, S. Mulhovo, J. Molnar, M. Ferreira, Dreg-

amine and tabernaemontanine derivatives as ABCB1 modulators on resistant

cancer cells, Eur. J. Med. Chem. 128 (2017) 247e257.

[15] A. Kumar, V. Jaitak, Natural products as multidrug resistance modulators in

cancer, Eur. J. Med. Chem. 176 (2019) 268e291.

[16] B.M.F. Gonçalves, D.S.P. Cardoso, M.J.U. Ferreira, Overcoming multidrug

resistance: ﬂ avonoid and terpenoid nitrogen-containing derivatives as ABC

transporter modulators, Molecules 25 (2020) 3364.

[17] Q.W. Ren, G.Q. Yang, M.Q. Guo, J.W. Guo, Y. Li, J. Lu, Q. Yang, H.H. Tang, Y. Li,

X.J. Fang, Y.X. Sun, J.G. Qi, J.W. Tian, H.B. Wang, Design, synthesis, and dis-

covery of ocotillol-type amide derivatives as orally available modulators of P-

glycoprotein-mediated multidrug resistance, Eur. J. Med. Chem. 161 (2019)

118e130.

[34] D. Tripathy, S.M. Tolaney, A.D. Seidman, C.K. Anders, N. Ibrahim, H.S. Rugo,

C. Twelves, V. Dieras, V. Muller, M. Tagliaferri, A.L. Hannah, J. Cortes, ATTAIN:

phase III study of etirinotecan pegol versus treatment of physician ’s choice in

patients with metastatic breast cancer and brain metastases, Future Oncol. 15

(2019) 2211e2225.

[35] M. Medina-O ’Donnell, F. Rivas, F.J. Reyes-Zurita, A. Martinez, F. Galisteo-

ꢂ

~

Gonzalez, J.A. Lupianez, A. Parra, Synthesis and in vitro antiproliferative

evaluation of PEGylated triterpene acids, Fitoterapia 120 (2017) 25e40.

[36] S.M. Schmitt, K. Stefan, M. Wiese, Pyrrolopyrimidine-derivatives as novel in-

hibitors of multidrug resistance- associated protein 1 (MRP1, ABCC1), J. Med.

Chem. 59 (2016) 3018e3033.

[18] T. Nabekura, T. Yamaki, K. Ueno, S. Kitagawa, Inhibition of P-glycoprotein and

multidrug resistance protein 1 by dietary phytochemicals, Canc. Chemother.

Pharmacol. 62 (2008) 867e873.

[19] M. Zhou, R.H. Zhang, M. Wang, G.B. Xu, S.G. Liao, Prodrugs of triterpenoids and

their derivatives, Eur. J. Med. Chem. 131 (2017) 222e236.

[37] G.A. Patani, E.J. LaVoie, Bioisosterism: A rational approach in drug design,

Chem. Rev. 96 (1996) 3147e3176.

[20] J. Zeng, T. Huang, M. Xue, J.X. Chen, L.L. Feng, R.F. Du, Y. Feng, Current

knowledge and development of hederagenin as a promising medicinal agent:

a comprehensive review, RSC Adv. 8 (2018) 24188e24202.

[21] C.W. Lee, S.M. Park, R.J. Zhao, C. Lee, W. Chun, Y. Son, S.H. Kim, J.Y. Jung,

K.H. Jegal, I.J. Cho, S.K. Ku, Y.W. Kim, S.A. Ju, S.C. Kim, W.G. An, Hederagenin, a

major component of Clematis mandshurica Ruprecht root, attenuates in-

ﬂ ammatory responses in RAW 264.7 cells and in mice, Int. Immunopharm. 29

(2015) 528e537.

[38] N.A. Meanwell, Synopsis of some recent tactical application of bioisosteres in

drug design, J. Med. Chem. 54 (2011) 2529e2591.

[39] T. Mosmann, Rapid colorimetric assay for cellular growth and survival:

application to proliferation and cytotoxicity assays, J. Immunol. Methods 65

(1983) 55e63.

[40] Y. Ding, Y.Y. Zhou, H. Chen, D.D. Geng, D.Y. Wu, J. Hong, W.B. Shen, T.J. Hang,

C. Zhang, The performance of thiol-terminated PEG-paclitaxel-conjugated

gold nanoparticles, Biomaterials 34 (2013) 10217e10227.

[41] L. Roux, S. Priet, N. Payrot, C. Weck, M. Fournier, F. Zoulim, J. Balzarini,

B. Canard, K. Alvarez, Ester prodrugs of acyclic nucleoside thiophosphonates

compared to phosphonates: synthesis, antiviral activity and decomposition

study, Eur. J. Med. Chem. 63 (2013) 869e881.

[22] D. Zhou, H. Jin, H.B. Lin, X.M. Yang, Y.F. Cheng, F.J. Deng, J.P. Xu, Antidepressant

effect of the extracts from fructus akebiae, Pharmacol., Biochem. Behav. 94

(2010) 488e495.

[23] A.G. Wu, W. Zeng, V.K.W. Wong, Y.Z. Zhu, B.Y.K. Law, Hederagenin and

a -

hederin promote degradation of proteins in neurodegenerative diseases and

improve motor de ﬁ cits in MPTP-mice, Pharmacol. Res. 115 (2017) 25e44.

[24] X.X. Liu, Y.T. Yang, X. Wang, K.Y. Wang, J.Q. Liu, L. Lei, X.M. Luo, R. Zhai, F.H. Fu,

[42] A. Robertson, J.P. Glynn, A.K. Watson, The absorption and metabolism in man

of 4-Acetamidophenyl-2-acetoxybenzoate (Benorylate), Xenobiotica 2 (1972)

339e347.

H.B. Wang, Y. Bi, Design, synthesis and biological evaluation of novel

hederagenin derivatives with anticancer activity, Eur. J. Med. Chem. 141

(2017) 427e439.

a

-

[43] Z. Darzynkiewicz, F. Traganos, L. Staiano-Coico, J. Kapuscinski, M.R. Melamed,

Interactions of rhodamine 123 with living cells studied by ﬂ ow cytometry,

Canc. Res. 42 (1982) 799e806.

[25] D. Rodriguez-Hernandez, A.J. Demuner, L.C.A. Barbosa, R. Csuk, L. Heller,

Hederagenin as a triterpene template for the development of new antitumor

compounds, Eur. J. Med. Chem. 105 (2015) 57e62.

[26] K. Fang, X.H. Zhang, Y.T. Han, G.R. Wu, D.S. Cai, N.N. Xue, W.B. Guo, Y.Q. Yang,

M. Chen, X.Y. Zhang, H. Wang, T. Ma, P.L. Wang, H.M. Lei, Design, synthesis,

and cytotoxic analysis of novel hederageninepyrazine derivatives based on

partial least squares discriminant analysis, Int. J. Mol. Sci. 19 (2018) 2994.

[27] D. Rodríguez-Hernandez, L.C.A. Barbosa, A.J. Demuner, J.P.A. Martins,

L. Fischer, R. Csuk, Hederagenin amide derivatives as potential anti-

proliferative agents, Eur. J. Med. Chem. 168 (2019) 436e446.

[44] J.F. Díaz, R. Strobe, Y. Engelborghs, A.A. Souto, J.M. Andreu, Molecular recog-

nition of taxol by microtubules. Kinetics and thermodynamics of binding of

ﬂ uorescent taxol derivatives to an exposed site, J. Biol. Chem. 275 (2000)

26265e26276.

[45] J. Li, K.F. Jaimes, S.G. Aller, Re ﬁ ned structures of mouse P-glycoprotein, Protein

Sci. 23 (2013) 34e46.

[46] S.G. Aller, J. Yu, A. Ward, Y. Weng, S. Chittaboina, R.P. Zhuo, P.M. Harrell,

Y.T. Trinh, Q.H. Zhang, I.L. Urbatsch, G. Chang, Structure of P-Glycoprotein

reveals a molecular basis for poly-speci ﬁ c drug binding, Science 323 (2009)

1718e1722.

[28] Y.T. Yang, D.K. Guan, L. Leia, J. Lua, J.Q. Liu, G.Q. Yang, C.H. Yan, R. Zhai,

J.W. Tian, Y. Bi, F.H. Fua, H.B. Wang, H6, a novel hederagenin derivative, re-

verses multidrug resistance in vitro and in vivo, Toxicol. Appl. Pharmacol. 341

(2018) 98e105.

[47] F.F. Li, Z.L. Liu, H.Y. Sun, C.M. Li, W.Y. Wang, L. Ye, C.H. Yan, J.W. Tian,

H.B. Wang, PCC0208017, a novel small-molecule inhibitor of MARK3/MARK4,

suppresses glioma progression in vitro and in vivo , Acta Pharm. Sin. B 10

(2020) 289e300.

[29] X. Wang, Q.W. Ren, X.X. Liu, Y.T. Yang, B.H. Wang, R. Zhai, J.G. Qi, J.W. Tian,

H.B. Wang, Y. Bi, Synthesis and biological evaluation of novel H6 analogues as

drug resistance reversal agents, Eur. J. Med. Chem. 161 (2019) 364e377.

[30] W.J. Li, P. Zhan, E.D. Clercq, H.X. Lou, X.Y. Liu, Current drug research on

PEGylation with small molecular agents, Prog. Polym. Sci. 38 (2013) 421e444.

[48] G.Y. Lv, D.J. Sun, J.W. Zhang, X.X. Xie, X.Q. Wu, W.S. Fang, J.W. Tian, C.H. Yan,

H.B. Wang, F.H. Fu, Lx2-32c, a novel semi-synthetic taxane, exerts antitumor

activity against prostate cancer cells in vitro and in vivo , Acta Pharm. Sin. B 7

(2017) 52e58.

16

Article Doi

Design, synthesis, and biological evaluation of hederagenin derivatives with improved aqueous solubility and tumor resistance reversal activity

DOI: 10.1016/j.ejmech.2020.113107

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.ejmech.2020.113107

Full text of DOI:10.1016/j.ejmech.2020.113107

Products guided by the article

R&D Labs maybe for 1025795-44-1

Relevant to this article

Hot Product