Synthesis and biological evaluation of novel H6 analogues as drug resistance reversal agents

Accepted Manuscript Synthesis and biological evaluation of novel H6 analogues as drug resistance reversal agents Xiao Wang, Qian-wen Ren, Xian-xuan Liu, Yan-ting Yang, Bing-hua Wang, Rong Zhai, Jia Grace Qi, Hong-bo Wang, Yi Bi PII:

S0223-5234(18)30902-4

DOI:

10.1016/j.ejmech.2018.10.033

Reference:

EJMECH 10819

To appear in:

European Journal of Medicinal Chemistry

Received Date: 8 July 2018 Revised Date:

10 October 2018

Accepted Date: 13 October 2018

Please cite this article as: X. Wang, Q.-w. Ren, X.-x. Liu, Y.-t. Yang, B.-h. Wang, R. Zhai, J.G. Qi, H.-b. Wang, Y. Bi, Synthesis and biological evaluation of novel H6 analogues as drug resistance reversal agents, European Journal of Medicinal Chemistry (2018), doi: https://doi.org/10.1016/ j.ejmech.2018.10.033. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Synthesis and biological evaluation of novel H6 analogues as drug resistance reversal agents Xiao Wang1,#, Qian-wen Ren1,#, Xian-xuan Liu1, Yan-ting Yang1,2, Bing-hua Wang1, Rong Zhai1, Jia Grace Qi1, Hong-bo Wang1,*, Yi Bi1,* 1

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

2

State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and

Peking Union Medical College, Beijing, 100050, China * Correspondence: [email protected] (H.W.), [email protected] (Y. B.) These authors contributed equally to this work

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

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Graphical abstract

ACCEPTED MANUSCRIPT

Synthesis and biological evaluation of novel H6 analogues as drug resistance reversal agents Xiao Wang1,#, Qian-wen Ren1,#, Xian-xuan Liu1, Yan-ting Yang1,2, Bing-hua Wang1, Rong Zhai1, Jia Grace Qi1, Jing-wei Tian1,*, Hong-bo Wang1,*, Yi Bi1,* School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation

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Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, 264005, China 2

State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and

Peking Union Medical College, Beijing, 100050, China

*Correspondence: [email protected] (J.T.), [email protected] (H.W.), [email protected] (Y. B.) These authors contributed equally to this work

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ABSTRACT

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Hederagenin is a naturally occurring pentacyclic triterpenoids compound with multiple pharmacological activities. We recently showed that H6, a synthetic derivative of hederagenin, could enhance the anticancer activity of paclitaxel in drug-resistant cells in vitro and in vivo, but showed poor solubility. With the aim of improving the drug resistant reversal activity of H6, here we designed and synthesized a series of novel H6 analogues. Our results showed that compound 10 at the concentration of 5 µM significantly enhanced the cytotoxicity of paclitaxel to drug-resistant KBV cells and sensitized cells to paclitaxel in arresting cells in G2/M phase and inducing apoptosis. We found that compound 10 might block the drug efflux of P-gp via stimulating P-gp ATPase activity. Importantly, compound 10 enhanced the efficacy of paclitaxel against KBV cancer cell-derived xenograft tumors. Finally, we summarized a preliminary structure-activity relationship of hederagenin by the drug resistant reversal activity

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of H6 analogues in vitro and compound 10 and H6 in vivo. This study highlights the importance of nitrogen-containing derivatives of hederagenin C-28 in the development of novel drug resistance reversal agents. Keywords: H6 analogues

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Synthesis Drug resistance reverse activity P-glycoprotein

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1． ．Introduction

According to cancer statistics, more than 50% of malignancies are resistant to traditional chemical drugs, and the

development of multidrug resistance (MDR) is one of the main reasons for the failure of chemotherapy [1, 2]. One of the earliest identified MDR mechanisms is the overexpression of P-glycoprotein (P-gp). It extracts and expels substrates from the membrane to promote multidrug resistance [3, 4]. Several therapeutic agents have been developed for the treatment of MDR, however their application is limited due to toxic and other side effects [5, 6]. Therefore, the identification of novel MDR reversal agents such as P-gp inhibitors with low toxicity, high efficiency and specificity is urgently needed to improve the efficacy of clinical application. Because natural products have the characteristics with rich source and a variety of pharmacological activities, natural products have been a useful source for potential MDR reversal agents, such as tetrandrine, alisol B, ursolic acid, betulinic acid and oleanolic acid [7-10]. Pentacyclic triterpenoids are mainly classified into oleanane-type, ursane-type, lupine-type and friedelane-type depending on the structural skeleton of the alkane [11]. Nabekura et al. reported that ursolic acid could increase the cellular accumulation of daunorubicin in KB-C2 cells in a dose-dependent manner and possibly inhibit P-gp function by stimulating P-gp ATPase activity [9]. Fernandes et al. reported that betulinic acid and oleanolic acid (Fig. 1) could inhibit the

ACCEPTED MANUSCRIPT growth of Lucena 1, a vincristine-resistant K562 cell line, indicating a possible action of betulinic acid and oleanolic acid in modulating P-gp. Fernandes et al. also studied the activity of triterpenes on Lucena 1 in the absence of vincristine and found that betulinic acid and oleanolic acid impacted Lucena 1 growth, indicating their anti-MDR properties [10]. Oleanolic acid and hederagenin (H) are both pentacyclic triterpene compounds, and hederagenin differs from oleanolic acid at the 23-hydroxyl group. Our research group has studied hederagenin derivatives as potential drug resistance reversal agents. H (Fig. 1) and its derivatives have a variety of pharmacological activities [12-21]. Our previous studies [17] showed that H6 (Fig. 1), a H derivative, combined with paclitaxel at 10 µM showed an IC50 value of 2.4 nΜ against drug-resistant KBV cells. We

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found that H6 could activate P-gp ATPase, resulting in the inability of drug-resistant cells to exclude drugs from the body. Thus, H6 could enhance the antitumor activity of paclitaxel in KBV cells, and the drug reversal effects were stronger than that of an equal dose of verapamil (IC50 = 4.9 nM). In addition, in vivo experiments revealed that xenograft nude mice showed a slight drop in weight and tumor weight was decreased to 42% after the combination of paclitaxel (30 mg/kg) and H6 (10 mg/kg). The results indicated that H6 had good drug resistant reversal activity in vivo, however, had poor solubility due to benzyl at C-28.

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Based on these studies, here we designed and synthesized a series of novel H6 analogues with the aim of improving its

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activity in reversing drug resistance of cancer cells.

Fig. 1. Chemical structure of ursolic acid, betulinic acid, oleanolic acid, H and H6. 2. Results and discussion

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2.1. Chemistry

H has several hot spots such as C-3 hydroxyl, C-23 hydroxyl, ring-C double bond and C-28 carboxylic acid, which provide enough space for chemical transformations. We used H, derived from Hedera nepalensis var. sinensis, as our starting material.

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Based on our previous research, H6 was selected as the lead compound, following structural modification of 23-hydroxyl and 28-carboxyl on ring-A fused pyrazine (see Scheme 1). Compound 20 were prepared by oxidation of H. Compounds 21–23 were prepared (90.0% yield) by a reaction of H with unsaturated polynitrogen compounds in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) in dichloromethane (DCM) (see Scheme 2). Compounds 24–25 were prepared (90.0% yield) by a reaction of compound 20 with unsaturated polynitrogen compounds. Compound 26–29 were prepared by polymerization of H. Several H6 analogues were designed and synthesized, including carboxylic acid derivatives (1–9, 13-20), nitrogen-containing derivatives (10–12), unsaturated polynitrogen derivatives (21–25) and dimer derivatives (26–29).

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Scheme 1. Synthetic route of target compounds 1-19. (a) anhydrides, DMAP, DCM, r.t.; (b) 10% Pd/C, H2, CH3OH, r.t.; (c) Ac2O, pyridine, DCM, r.t.; (d) (COCl)2, DCM, 0 °C - r.t.; (e) amino acid hydrochloride, triethylamine or succinimide, r.t.; (f) 10%

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NaOH, THF/CH3OH or THF/H2O, r.t..

Scheme 2. Synthetic route of target compounds 20-29. (a) BrBn, DMF, K2CO3, 50 °C; (b) TBSCl, DMAP, DCM, r.t.; (c) PCC, DCM, r.t.; (d) 30% H2O2, HCO2H, DCM, r.t., 24 h; (e) 10% HCl, acetone, r.t.; (f) 10% Pd/C, H2, CH3OH, r.t.; (g) EDCI, RH,

ACCEPTED MANUSCRIPT DCM, r.t.; (h) bromoalkane, K2CO3, DMF, reflux. The group at C-28 of intermediate X4c is N-hydroxysuccinimide, and the side chains of compounds 7 and 12 at C-28 are O-amidobutyric acid and O-amidobutyric acid methyl ester, respectively. We speculated that the amide bonds of N-hydroxysuccinimide in these compounds were alcoholylated and hydrolyzed, respectively. Similarly, fragmentation of the amide of C-28 position of intermediate X4g in solvent tetrahydrofuran and ethanoamine (3:2) leads to alcoholysis to generate compound 11. Compound 10 differs from compounds 11, 12 in that when C-23 is hydrolyzed, C-28 does not change and remains the C-28 position of X4f.

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In the 1H NMR spectra, the formation of ring-C oxidation confirmed by the signal disappear around δ = 5.38 corresponding to H-12 of the ring-C. Several H6 analogues were confirmed by NMR and HR-MS and were not reported previously through a SciFinder search. 2.2. Biological screening

The drug reversal activity and cytotoxic activity of all the synthesized H6 analogues were tested using MTT assays.

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Verapamil, LY335979, AK110701 and compound H6 were used as reference standards (Table 1-2).

As shown in Table 1, the compounds had no effect on KBV cell growth in the absence of paclitaxel. However, some compounds with paclitaxel showed good cytotoxic activity. Compound 10 showed the highest drug resistance reversal activity in

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KBV cells, which was higher than that of the standard H6. Compounds 11 and 23 showed inhibitory effects through their drug resistance reversal activity in KBV cells comparable to that of verapamil. Compound 12 showed moderate drug resistance reversal activity. The common denominator of compounds 10, 11, 12 and 23 was the presence of a nitrogen at C-28. Other synthesized compounds showed less drug resistance reversal activity; the common denominator of these compounds was the presence of terminal carboxyl groups.

The results showed that 10 as well as LY335979 and AK110701, dramatically decreased the IC50 values of paclitaxel in the drug-resistant cells at concentrations of 5 µM and 10 µM (Table 2-3). Compound 10, with the highest drug resistance reversal

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activity, sensitized KBV cells to paclitaxel in a dose-dependent and time-dependent manner from 5 µM and 10 µM and showed higher inhibitory effects from its drug resistance reversal activity than verapamil at 5 µM and 10 µM. We thus selected compound 10 for examination of its underlying mechanism of action and as a candidate compound. Table 1 Effects of novel H6 analogues (5 µM) on the cytotoxic activity of paclitaxel (100 nM) against KBV cells. KBV Cell Survival Rate (%)

Without Paclitaxel

KBV Cell Survival Rate (%) Cpd. (5 µM)

With Paclitaxel (100 nM)

Without Paclitaxel 121±17

98±19

-

14

91±11

Verapamil

23±4

-

15

94±38

94±38

H6

18±4

-

16

80±0

91±13

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Paclitaxel

With Paclitaxel (100 nM)

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Cpd. (5 µM)

H

100±7

-

17

81±7

90±12

1

85±10

116±7

18

68±10

76±18

2

100±20

95±2

19

76±20

102±26

3

102±8

96±3

20

104±11

102±9

4

61±9

89±5

21

115±14

94±4

5

101±19

96±2

22

111±13

97±8

6

129±8

94±7

23

23±3

103±5

7

112±16

89±0

24

127±14

110±6

8

264±9

98±9

25

112±11

98±6

9

114±11

83±6

26

37±3

136±8

10

10±1

97±2

27

45±3

107±5

11

24±1

116±6

28

44±5

92±8

29

42±12

123±11

12

32±2

86±3

13

76±15

130±12

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IC50

PTX

RF

398.34±0.58

Vera

10 µM

6.15±1.21

64.82

5 µM

12.72±0.74

31.32

10 µM

1.59±0.59

250.77

5 µM

12.26±0.11

32.50

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10

LY335979 10 µM

3.49±0.97

5 µM

5.24±0.07

10 µM

4.46±1.56

114.11 75.95

AK110701

89.41

24h

48h

IC50

RF

IC50 1396.57

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Table 3 The effects of 10 on the cytotoxic activity of paclitaxel with different incubation time.

72h

RF

IC50

RF

-

396.33

-

-

3837.57

-

10

5 µM

49.77±6.75

77.10

21.18±1.00

65.92

14.13±3.16

28.05

10 µM

34.95±9.98

109.79

17.22±1.52

81.11

4.92±1.13

80.59

2.3 The relationship of hydrophilicity and activity

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As shown in Table 4, the lgP of compound 10 was -1.25, which was lower than that of H6 (-0.54) due to the introduction of nitrogen-containing groups at C-28. The lgP of several compounds between -1.25 and -0.54, but these compounds were not active. In addition, the lgP of compounds 8, 9 and 12 were -1.07, -0.78 and -1.24 respectively, but only compound 12 was active

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compound. The results displayed that there were no direct correlation between hydrophilicity and activity. However, an active compound possessed good hydrophilicity that it was beneficial to its druggability.

lgP

Compound

lgP

Compound

lgP

Compound

lgP

H6

-0.54

7

-0.78

14

0.38

22

-0.75

1

-0.66

8

-1.07

15

-0.20

23

-0.40

2

-0.33

9

-0.78

16

-0.66

24

0.14

10

-1.25

17

-0.39

25

0.10

3 4 5 6

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Table 4 Lipo-hydro partition coefficient of compounds.

-0.28 0.24

11

0.36

18

-0.03

-0.43

12

-1.24

19

-0.11

-0.32

13

0.08

21

-0.23

2.4 Structure-activity relationships Based on the drug resistance reversal activity and lgP of H6 analogues, plausible structure-activity relationships (SARs) could be reasoned out and are shown in Fig. 2: drug resistance reversal activity of H derivatives based on ring-A fused pyrazine were improved; when the hydroxyl at C-3 and the double bond at C-12 were converted to ketones, activity was decreased; when the hydroxyl at C-23 and the carboxyl at C-28 were the terminal carboxyl, activity was decreased; when the carboxyl at C-28 was converted to the terminal amino, activity was improved; when carboxyl and hydroxyl groups were both introduced into C-28 site, activity was decreased; when nitrogen groups were substituted at C-28, activity was improved.

ACCEPTED MANUSCRIPT Compound 20 is obtained through oxidation of H and its activity is lower than H. Specifically, when the hydroxyl at C-3 and the double bond at C-12 are converted to ketones, activity is decreased. Comparing compounds 23, H6 and 10, we can infer that ring-A fused pyrazine as well as nitrogen-containing groups at C-28 may increase activity in KBV cells. The results indicate that introduced groups are the main factors affecting activity. And the compound is not active when it contains carboxyl group. Importantly, the compound with introduction of nitrogen-containing groups have good activity. On the basis of the presence of nitrogen-containing groups, the compound of possessed good hydrophilicity that it was beneficial to its druggability. It guides the

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design of subsequent derivatives with nitrogen-containing groups at C-28.

2.5 Compound 10 inhibits the efflux function of P-gp

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Fig. 2. Preliminary structure-activity relationships of H6 analogues

The P-gp substrate Rhodamine123 was used as a biomarker to explore the effect of compound 10 on the efflux function of P-gp. As shown in Fig. 3, treatment of either compound 10 or verapamil increased the accumulation of Rhodamine123 in KBV cells in a dose-dependent manner, which indicated the resistance reversal activity of compound 10 might occur by blocking the

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efflux of paclitaxel via P-gp in drug resistant cancer cells.

Fig. 3. Effects of compound 10 on the Rhodamine123 efflux in KBV cells.

KBV cells were incubated with Rhodamine123 in the presence of compound 10 or verapamil as indicated for 30 min, and then the cells were harvested for analysis by flow cytometry. All data were expressed as mean ± SD (n = 3). *p < 0.05, compared with Rho only group. 2.6 Compound 10 sensitizes KBV cells to paclitaxel in arresting cells in the G2/M phase and undergoing cell apoptosis We next explored the effect of compound 10 on the activity of paclitaxel in the regulation of cell cycle distribution and cell apoptosis by flow cytometry. As shown in Fig. 4, no obvious effect was observed after 24 h with paclitaxel treatment on the cell cycle and cell apoptosis in KBV cells. However, co-treatment with compound 10 at the concentration of 10 µM was sufficient to arrest KBV cells in the G2/M phase and induce apoptosis, which was indicated by the increased ratio of cells in the sub-G1 phase

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(Fig. 4).

Fig. 4. Effects of compound 10 on the activity of paclitaxel in the regulation of cell cycle distribution and cell apoptosis.

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Cells were seeded into 6-well plates and treated with indicated compounds at the concentration of 10 µM for 24 h. Cells were then subjected to flow cytometry assay to determine the cell cycle (A) and cell cycle distributions were determined (B). All data were expressed as mean ± SD (n = 3). *p < 0.05, compared with control and paclitaxel groups. 2.7 Compound 10 enhances antitumor activity of paclitaxel against KBV xenograft tumors in nude mice To explore the effect of compound 10 on the antitumor activity of paclitaxel in vivo, we established xenograft tumors in nude mice using KBV cells. Consistent with the in vitro findings, the xenograft tumors were resistant to paclitaxel treatment (Fig. 5). While compound 10 alone did not display antitumor activity, co-treatment with compound 10 at a dose of 50 mg/kg

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significantly increased the tumor inhibitory activity of paclitaxel without increasing extra toxicity (Fig. 5). The results showed that the derivative 10 displayed similar activity to the parent compound H6. H6 also did not exert activity on its own. Importantly, compound 10 was treated by oral gavage administration, and it has significant improvement than H6 in administration. One

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possible reason for this phenomenon might be that the group of compound 10 at C-28 has improved water solubility.

Fig. 5. Effects of compound 10 on the antitumor activity of paclitaxel against KBV xenograft tumors in nude mice. Nude mice were inoculated with KBV cells and treated with indicated agents at a dose of 50 mg/kg. At the end of treatment, the mice were euthanized and the tumors were removed and weighed. All data were expressed as mean ± SD (n = 4 or 5). *p < 0.01, compared with control group and paclitaxel group. 2.8 Compound 10 increases P-gp ATPase activity To test the effect of compound 10 on P-gp ATPase activity, the P-gp-Glo™ Assay was performed using a SpectraMax M5 microplate reader, with verapamil as the positive control. As shown in Fig. 6, treatment with compound 10 at the concentration

ACCEPTED MANUSCRIPT of 0.5 mM could produce a significant increase in luminescence, similar to verapamil, indicating that compound 10 could

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stimulate P-gp ATPase activity.

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Fig. 6. Effects of compound 10 on the ATPase activity of P-gp.

Purified P-gp was incubated with the indicated compounds, and luminescence was detected. All data were expressed as means ± SD (n = 3). *p < 0.05, compared with basal group.

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2.9 Molecular docking simulation of H6 analogues compound 10

The P-gp protein consist two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). The reported P-gp inhibitors could either bind to drug-binding pocket in TMD as a competitive inhibitor, or block ATP-binding in NBDs as a non-competitive inhibitor [22]. In order to explore the mode of interaction between 10 H6 analogues and P-gp, molecular docking simulation was performed. Docking results of H6 analogues were shown in Table 5. Obviously 10, 11, H6 and verapamil bound in the same amino acid residue Gln990 in P-gp (Fig. 7). Docking score of compound 10 was 5.3814 which had a high binding affinity, and its hydrogen bonds was formed by the interaction with the oxygen atom of the carbonyl moiety of compound 10 and the backbone NH of Gln990. Moreover, verapamil and H6 both had interaction with amino acid residue

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Gln990 as same as 10, and also possessed good docking scores (5.0900 and 4.5567). The results of molecular docking indicated that compound 10 showed strong interactions with P-gp. Compound 11 formed two hydrogen bonds with Gln990 (-CO···OC-Gln990) and Met986 (-NH···O-Met986). Compound 12 has moderate drug resistance reversal activity and has good docking (6.1239) [23]. From the result of docking, we found that there was an similar order of docking scores with cytotoxic

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activity values of 4 ligands (compounds 10, 11, 12 and H6).

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Fig. 7. Molecular modeling of compound 10 (A), verapamil (B) ,or H6 (C) or compound 11 (D) binding to homology P-gp. Table 5 Scores of docking models of P-gp and H6 analogues. Total Score

Crash

Verapamil

5.0900

-1.1839

1.2168

H6

4.5567

-0.5309

0.6352

1

5.1724

-0.9444

2.4301

2

3.5344

-1.3556

2.6441

3

3.3447

-1.0000

2.1327

4

2.7140

-1.5921

1.1482

5

2.9936

-0.8359

0.0001

6

3.4169

-1.7483

1.1204

7

3.1603

-1.9444

0.2631

8

3.2809

-0.9505

1.0920

9

4.2720

-2.6642

1.9220

5.3814

-1.2070

0.7134

4.8007

-2.2485

2.0765

6.1239

-1.1839

1.2166

4.6983

-1.5922

0.0610

3.7152

-0.7493

1.1648

15

3.8768

-1.5045

2.2221

16

3.1672

-1.7391

0.0086

17

4.6682

-1.5418

3.3170

18

3.2296

-1.9107

1.7733

19

2.5480

-2.9521

1.7628

20

2.4160

-0.6622

2.0988

21

3.0566

-0.9229

1.1216

22

4.5005

-2.1061

1.4586

23

1.7598

-0.9374

2.0484

24

3.3518

-0.4261

1.0326

25

3.8533

-2.4209

1.0907

26

2.0758

-4.9001

0.9525

27

2.1232

-1.0440

0.9202

28

4.5172

-2.1061

1.4586

29

2.8683

-2.2171

0.6216

11 12 13

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Polar

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3. Conclusions A series of novel carboxylic acid derivatives, unsaturated polynitrogen derivatives, nitrogen-containing derivatives and dimer derivatives of H was synthesized by esterification, amidation and hydrolysis reaction. All the synthesized H6 analogues were screened for their drug resistance reversal activity and cytotoxic activity in KBV cells using the MTT assay. A plausible SARs has been summarized from the data. Notably, compound 10 was found to inhibit the efflux function of P-gp via activating P-gp ATPase activity. Compound 10 sensitized KBV cells to paclitaxel in arresting the cells in the G2/M phase and inducing cell

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apoptosis. Most importantly, compound 10 enhanced the efficacy of paclitaxel against KBV cancer cell-derived xenograft tumors with acceptable toxicity. Further intensive modifications at C-28 and ring-A studies are currently being performed in our research group, and the results will be announced in due course. 4. Experimental section

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4.1. Chemistry

Most chemicals and solvents were obtained from companies and were used that further pure or dried when necessary. Thin layer chromatography (TLC) analysis was carried out to determine the reaction progress and a 10% ethanol sulfate solution was

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used for detection of the spots. The 1H and 13C NMR spectra were recorded with Bruker av400 or JNM-ECZS 400 instruments using CDCl3 or CD3OD as solvent in the indicated solvents (TMS). High-resolution mass spectra were recorded using an Agilent QTOF 6520 or 6530. Melting points were determined with a SGW®X-4 micro melting point determination apparatus and was uncorrected. Concentration were determined with a Agilent 1100 Series. 4.1.1. General procedure for the synthesis of 1-9, 13-20

The key intermediate H6 was obtained from the raw H according to the previous experimental method [16]. To a solution of H6 and 4-dimethylaminopyridine (DMAP) in dry pyridine, different anhydride was added. The key intermediates X1a-X1c were obtained through action at room temperature (r.t.), then obtained 1-3 through catalytic reduction of hydrogen. (see Scheme 1) The

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compound 4 was obtained through catalytic reduction of hydrogen H6. (see Scheme 1) To a solution of H6 in dry pyridine, acetic anhydride was slowly added. The reaction mixture was stirred at r.t. for 5 h, then obtained the key intermediate X3 through catalytic reduction of hydrogen, and oxalyl chloride was added to a solution of X3 in dry dichloromethane (8.0 mL) in 0 °C. After, Acyl Chloride, the appropriate branched chain and dry DCM were obtained X4a-X4e and X4h-X4n. To a solution of

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X4a-X4e and X4h-X4n in tetrahydrofuran with methanol (MA) or water, 10% sodium hydroxide was slowly added. Compounds 5-9 were obtained at room temperature for 10 h by hydrolysis reaction. (see Scheme 2) The intermediate X7 was prepared from the reaction of a solution of the intermediate X6 in DMF with formic acid and 30% hydrogen peroxide (H2O2), then compound X7 in absolute acetone was stirred with 10% hydrogen chloride (10% HCl) to

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removal of C-23 protective groups at r.t.. A mixture of X8 and 10% Pd/C in methanol was stirred at r.t. under H2. (see Scheme 2) 4.1.1.1. The synthesis of X1a-X1c

2-(Benzyl olean[3,2-b]pyrazine-12-en-28-oate-23-oxy)-oxobenzoic acid (X1a) To a solution of H6 (100.0 mg, 0.2 mmol) in dry DCM (8.0 mL) was added DMAP (41.0 mg, 0.3 mmol) and o-phthalic

anhydride (251.8 mg, 1.5 mmol), the mixture was refluxed at r.t. for 8 h. The mixture was concentrated and diluted with ethyl acetate, washed with water, and brine in sequence, dried anhydrous sodium sulfate (Na2SO4), and concentrated. The white solid product X1a was obtained by column chromatography (chloroform/methanol, 40:1-30:1) (95.8 mg, 75.7%). The compounds X1b and X1c were maked and purified with the same as synthesis of X1a. 4.1.1.2. 23-acetoxy-olean[3,2-b]pyrazine-12-en-28-acid (X3) To a H6 (2.0 g, 3.4 mmol) in 10.0 ml of pyridine (30.0 mL), Ac2O (16.0 mL) was slowly added while stirring. The mixture was then stirred 8 h at r.t.. The mixture was diluted with ethyl acetate, washed with water, and brine in sequence, dried anhydrous

ACCEPTED MANUSCRIPT sodium sulfate (Na2SO4), and concentrated. The pure product 1 was obtained by column chromatography (petroleum ether/ethyl acetate, 20:1) and was a white solid (1.9 g, 89.0% yield). A mixture of X2 (1.9 g, 3.0 mmol) and 10% Pd/C (0.6 g, 6.0 mmol) in methanol (30.0 mL) was stirred at r.t. under H2 for 6 h. The mixture was filtered and concentrated. The white solid product X3 was obtained by column chromatography (petroleum ether/ethyl acetate, 8:1) (1.5 g, 92.3% yield). 4.1.1.3. 2-(Benzyl olean[3,2-b]pyrazine-12-en-28-acid-23-oxy)-oxobenzoic acid (1)

RI PT

The compound 1 was synthesized with the synthesis of X3 as a white solid. Yield: 81.4% (after chromatograph with chloroform/methanol, 40:1-30:1). m.p. 166.8-167.2 °C. 1H-NMR (400 MHz, CDCl3) δ: 8.48 (s, 1H, H-pyrazine), 8.28 (s, 1H, H-pyrazine), 7.76 (d, J = 6.9 Hz, 1H, H-Ar), 7.53-7.38 (m, 2H, 2×H-Ar), 7.31 (s, 1H, H-Ar), 5.38 (s, 1H, H-12), 4.56 (d, J = 10.7 Hz, 2H, H-23), 2.99 (t, J = 8.3 Hz, 1H, H-1a), 2.89 (d, J = 15.7 Hz, 1H, H-18), 2.58 (d, J = 16.3 Hz, 1H, H-1b), 1.33 (s, 3H, CH3), 1.17 (s, 3H, CH3), 0.94 (d, J = 6.9 Hz, 6H, 2×CH3), 0.92 (s, 3H, CH3), 0.88 (s, 3H, CH3); 13C-NMR (100 MHz, CDCl3) δ: 183.71, 169.92, 167.72, 156.59, 151.78, 143.59, 142.78, 140.87, 132.34, 131.14, 131.15, 130.85, 129.50, 128.45, 122.24, 71.77, 46.91,

SC

46.60, 45.81, 45.72, 45.59, 43.30, 41.83, 41.11, 39.14, 36.54, 33.81, 33.02, 32.36, 31.70, 30.64, 27.63, 25.69, 23.51, 23.31, 22.97, 19.90, 19.80, 16.70, 15.90; HR-MS (ESI) m/z: calcd. for C40H50N2O6 [M+H]+: 655.3747, found: 655.3731.

M AN U

4.1.1.4. 2-(Benzyl olean[3,2-b]pyrazine-12-en-28-acid-23-oxy)-oxocyclohexanecarboxylic acid (2)

The compound 2 was synthesized with the synthesis of X3 as a white solid. Yield: 95.5% (after chromatograph with chloroform/methanol, 40:1-30:1). m.p. 150.8-153.1 °C. 1H-NMR (400 MHz, CDCl3) δ: 8.46 (s, 1H, H-pyrazine), 8.30 (dd, J = 7.6, 2.3 Hz, 1H, H-pyrazine), 5.38 (s, 1H, H-12), 4.32 (ddd, J = 30.6, 26.9, 10.6 Hz, 2H, H-23), 2.99 (dd, J = 16.6, 3.8 Hz, 1H, -CH-), 2.88 (d, J = 10.3 Hz, 1H, H-18), 2.73 (s, 1H, -CH-), 2.08 (d, J = 18.6 Hz, 4H, 2×-CH2-), 1.26 (d, J = 1.5 Hz, 3H, CH3), 1.18 (s, 3H, CH3), 0.95 (s, 3H, CH3), 0.93 (s, 6H, 2×CH3), 0.87 (s, 3H, CH3); 13C-NMR (100 MHz, CDCl3) δ: 183.64, 178.18, 177.81, 172.96, 155.86, 151.25, 143.12, 142.25, 121.81, 70.14, 59.96, 46.11, 45.28, 45.02, 42.66, 41.69, 41.36, 40.62, 38.68, 36.04, 33.34, 32.59, 31.85, 31.34, 30.19, 27.09, 26.25, 25.16, 22.85, 22.47, 19.28, 19.20, 16.13, 15.48, 15.34, 13.70; HR-MS

TE D

(ESI) m/z: calcd. for C36H56N2O6 [M+H]+: 661.4217, found: 661.4195. 4.1.1.5. 4-(Benzyl olean[3,2-b]pyrazine-12-en-28-acid-23-oxy)-4-oxo-butyric acid (3) The compound 3 was synthesized with the synthesis of X3 as a white solid. Yield: 85.0% (after chromatograph with chloroform/methanol, 40:1-30:1). m.p. 143.6-145.8 °C. 1H-NMR (400 MHz, CDCl3) δ: 8.48 (d, J = 2.2 Hz, 1H, H-pyrazine), 8.29

EP

(d, J = 2.4 Hz, 1H, H-pyrazine), 5.38 (s, 1H, H-12), 4.32 (s, 2H, H-23), 2.96 (s, 1H, H-1a), 2.88 (dd, J = 13.5, 3.5 Hz, 1H, H-18), 2.74-2.51 (m, 1H, H-1b), 2.50-2.31 (m, 4H, 2×-CH2-), 1.27 (s, 3H, CH3), 1.21 (s, 3H, CH3), 0.95 (s, 3H, CH3), 0.92 (s, 6H, 2×CH3), 0.87 (s, 3H, CH3); 13C-NMR (100 MHz, CDCl3) δ: 183.98, 176.08, 171.61, 156.44, 151.74, 143.57, 142.71, 140.98, 122.27, 70.72,

AC C

46.97, 46.60, 45.87, 45.78, 45.62, 43.20, 41.79, 41.10, 39.13, 36.47, 33.78, 33.01, 32.37, 31.71, 30.66, 28.84, 28.58, 27.59, 25.70, 23.54, 23.31, 22.91, 19.74, 16.64, 15.84, 14.16; HR-MS (ESI) m/z: calcd. for C36H50N2O6 [M+H]+: 607.3747, found: 607.3730. 4.1.1.6. Benzyl 23-hydroxy-olean[3,2-b]pyrazine-12-en-28-acid (4) The compound 4 was obtained through catalytic reduction of hydrogen H6 as a white solid. Yield: 89.2% (after

chromatograph with petroleum ether/ethyl acetate, 6:1). m.p. 231.0-232.5 °C. 1H-NMR (400 MHz, CD3OD) δ: 8.50 (d, J = 2.1 Hz, 1H, H-pyrazine), 8.27 (d, J = 2.5 Hz, 1H, H-pyrazine), 5.36 (s, 1H, H-12), 3.85 (d, J = 10.6 Hz, 1H, H-23a), 3.60 (d, J = 10.6 Hz, 1H, H-23b), 2.96 (s, 1H, H-1a), 2.92 (s, 1H, H-18), 2.61 (s, 1H, H-1b), 1.26 (s, 3H, CH3), 1.20 (s, 3H, CH3), 0.98 (s, 3H, CH3), 0.94 (s, 6H, 2×CH3), 0.93 (s, 3H, CH3); 13C-NMR (100 MHz, CD3OD) δ: 182.27, 160.00, 154.46, 145.82, 144.39, 142.60, 123.96, 70.75, 49.01, 48.21, 47.72, 47.43, 46.61, 46.56, 43.71, 43.40, 40.99, 38.06, 35.46, 34.31, 34.10, 33.56, 32.15, 29.36, 26.82, 25.06, 24.63, 24.49, 21.30, 20.69, 17.96, 16.85; HR-MS (ESI) m/z: calcd. for C32H46N2O3 [M+H]+: 507.3581, found: 507.3566. 4.1.1.7. The synthesis of X4a-X4e N-glycine 23-acetoxy-olean[3,2-b]pyrazine-12-en-28-oate (X4a)

ACCEPTED MANUSCRIPT To a X3 (50.0 mg, 0.1 mmol) in 6.0 mL of dry DCM (6.0 mL) at 0 °C for 10 min, oxalyl chloride (77.0 µL) was slowly added while stirring. The mixture was stirred 1 h at r.t. and was concentrated, that a mixture of it, glycine methyl ester hydrochloride (11.4 mg, 0.2 mmol) and triethylamine (38.0 µL) in dry DCM (6.0 mL) was stirred at r.t. for 2 h, then the mixture was diluted with ethyl acetate, washed with water, and brine in sequence, dried anhydrous sodium sulfate (Na2SO4), and concentrated. The white solid product 3a was obtained by column chromatography (petroleum ether/ethyl acetate, 8:1) (48.0 mg, 85.0% yield). The compound X4b-X4e and X4h-X4n were maked and purified with the same as synthesis of X4a. 4.1.1.8. N-glycine 23-hydroxy-olean[3,2-b]pyrazine-12-en-28-oate (5)

RI PT

To a solution of X4a (40.0 mg, 1.0 mmol) in tetrahydrofuran with methanol (3:2) (5.0 mL), 10% sodium hydroxide (0.1 mL) was slowly added. The reaction mixture was stirred at room temperature for 5 h. The organic solution was diluted with ethyl acetate (20.0 mL) and washed with 5% HCl, water and brine successively, dried over anhydrous sodium sulfate. Yield: 84.3% (after chromatograph with chloroform/methanol, 60:1) as a white solid. m.p. 191.1-194.3 °C. 1H-NMR (400 MHz, CDCl3) δ: 8.46, 8.21 (each 1 H, ds, J = 2.3 Hz, H-pyrazine), 6.92 (s, 1H, NH), 5.64 (s, 1H, H-12), 3.99 (m, 2H, NCH2), 3.78 (d, J = 10.6 Hz,

SC

1H, H-23a), 3.49 (d, J = 10.6 Hz, 1H, H-23b), 2.73-2.59 (m, 1H, H-18), 1.26 (s, 3H, CH3), 1.23 (s, 3H, CH3), 0.93 (s, 3H, CH3), 0.91 (s, 3H, CH3), 0.88 (s, 3H, CH3), 0.83 (s, 3H, CH3); 13C-NMR (100 MHz, CDCl3) δ: 178.65, 171.80, 159.46, 151.96, 144.00, 142.50, 139.88, 123.76, 70.37, 46.81, 46.73, 46.35, 46.24, 45.61, 43.79, 42.74, 42.34, 42.05, 39.41, 36.08, 34.07, 33.01, 31.94,

M AN U

31.66, 30.74, 27.26, 25.49, 23.99, 23.82, 23.46, 19.95, 19.39, 16.28, 15.82; HR-MS (ESI) m/z: calcd. for C34H49N3O4 [M+H]+: 564.3801, found: 564.3788.

4.1.1.9. N-phenylalanine 23-hydroxy-olean[3,2-b]pyrazine-12-en-28-oate (6)

The compound 6 was synthesized with the synthesis of 5 as a white solid. Yield: 79.3% (after chromatograph with chloroform/methanol, 100:1). m.p. 195.5-197.9 °C. 1H-NMR (400 MHz, CDCl3) δ: 8.44 (d, J = 18.1 Hz, 1H, H-pyrazine), 8.29 (d, J = 31.2 Hz, 1H, H-pyrazine), 7.28-7.14 (m, 5H, 5×H-Ar), 6.66 (d, J = 5.9 Hz, 1H, NH), 5.24 (s, 1H, H-12), 4.80 (dd, J = 10.6, 5.3 Hz, 1H, NCH), 3.79 (d, J = 10.5 Hz, 1H, H-23a), 3.49 (d, J = 10.5 Hz, 1H, H-23b), 3.22 (d, J = 5.4 Hz, 1H, CHAr),

0.73 (s, 3H, CH3);

13

TE D

3.16 (d, J = 4.7 Hz, 1H, CHAr), 1.24 (s, 3H, CH3), 1.20 (s, 3H, CH3), 0.93 (s, 3H, CH3), 0.88 (s, 3H, CH3), 0.81 (s, 3H, CH3), C-NMR (100 MHz, CDCl3) δ: 178.19, 172.34, 158.80, 151.34, 142.63, 141.97, 139.67, 135.86, 129.26,

128.80, 128.21, 127.77, 126.50, 123.36, 69.89, 52.74, 46.34, 46.19, 45.94, 45.12, 43.27, 41.89, 41.84, 38.87, 36.75, 35.88, 33.68, 32.50, 31.94, 31.36, 30.20, 29.22, 26.83, 24.88, 23.38, 22.97, 22.81, 19.46, 18.91, 15.78, 15.72; HR-MS (ESI) m/z: calcd. for C41H55N3O4 [M+H]+: 654.4271, found: 654.4259.

EP

4.1.1.10. O-amidobutyric acid 23-hydroxy-olean[3,2-b]pyrazine-12-en-28-oate (7) The compound 7 was synthesized with the synthesis of 5 as a white solid. Yield: 58.0% (after chromatograph with chloroform/methanol, 80:1). m.p. 149.0-155.7 °C. 1H-NMR (400 MHz, CDCl3) δ: 9.68 (s, 1H, NH), 8.39, 8.34 (each 1 H, ds, J =

AC C

2.3 Hz, H-pyrazine), 5.37 (s, 1H, H-12), 3.78 (d, J = 10.6 Hz, 1H, H-23a), 3.47 (d, J = 10.5 Hz, 1H, H-23b), 2.88 (d, J = 13.1 Hz, 1H, H-18), 2.68 (d, J = 6.2 Hz, 2H, COCH2), 2.47 (d, J = 16.9 Hz, 2H, COOCH2), 1.27 (s, 3H, CH3), 1.15 (s, 3H, CH3), 0.91 (s, 3H, CH3), 0.88 (s, 6H, 2×CH3), 0.76 (s, 3H, CH3);

13

C-NMR (100 MHz, CDCl3) δ: 176.07, 158.18, 151.77, 142.86, 141.81,

141.56, 122.86, 77.19, 70.37, 47.21, 46.87, 46.81, 45.58, 43.34, 41.91, 41.33, 39.20, 36.41, 33.66, 32.93, 32.34, 31.76, 30.58, 29.66, 28.74, 28.33, 27.68, 27.57, 25.63, 23.51, 23.32, 22.96, 20.01, 19.40, 16.59, 16.11; HR-MS (ESI) m/z: calcd. for C36H51N3O6 [M+H]+: 622.3856, found: 622.3844. 4.1.1.11. N-alanine 23-hydroxy-olean[3,2-b]pyrazine-12-en-28-oate (8) The compound 8 was synthesized with the synthesis of 5 as a white solid. Yield: 78.5% (after chromatograph with chloroform/methanol, 70:1). m.p. 197.5-203.5 °C. 1H-NMR (400 MHz, CDCl3) δ: 8.47 (d, J = 2.4 Hz, 1H, H-pyrazine), 8.22 (d, J = 2.6 Hz, 1H, H-pyrazine), 7.03 (d, J = 4.9 Hz, 1H, NH), 5.62 (s, 1H, H-12), 4.41 (dd, J = 6.9, 5.1 Hz, 1H, NCH), 3.77 (d, J = 10.6 Hz, 1H, H-23a), 3.47 (d, J = 10.6 Hz, 1H, H-23b), 2.63 (d, J = 15.8 Hz, 1H, H-18), 1.45 (d, 3H, CH3) 1.24 (s, 3H, CH3), 1.21 (s, 3H, CH3), 0.94 (s, 3H, CH3), 0.92 (s, 3H, CH3), 0.85 (s, 3H, CH3), 0.78 (s, 3H, CH3); 13C-NMR (100 MHz, CDCl3) δ: 178.09, 174.80, 159.40, 151.81, 143.59, 142.49, 139.86, 123.63, 70.33, 48.39, 46.61, 46.56, 46.46, 46.32, 45.59, 43.72, 42.48,

ACCEPTED MANUSCRIPT 42.22, 39.39, 36.07, 34.00, 33.03, 32.39, 31.66, 30.73, 27.37, 25.99, 23.86, 23.53, 23.47, 19.92, 19.34, 18.98, 16.23, 15.80; HR-MS (ESI) m/z: calcd. for C35H51N3O4 [M+H]+: 578.3958, found: 578.3950. 4.1.1.12. N-valine 23-hydroxy-olean[3,2-b]pyrazine-12-en-28-oate (9) The compound 9 was synthesized with the synthesis of 5 as a white solid. Yield: 92.3% (after chromatograph with chloroform/methanol, 70:1). m.p. 158.4-164.1 °C. 1H-NMR (400 MHz, CDCl3) δ: 8.47 (d, J = 2.2 Hz, 1H, H-pyrazine), 8.36 (t, J = 6.9 Hz, 1H, H-pyrazine), 6.71 (d, J = 7.0 Hz, 1H, NH), 5.60 (s, 1H, H-12), 4.48 (dt, J = 15.8, 7.9 Hz, 1H, NCH), 3.79 (d, J = 10.6

RI PT

Hz, 1H, H-23a), 3.49 (d, J = 10.6 Hz, 1H, H-23b), 2.70 (t, J = 16.2 Hz, 1H, H-18), 2.53 (d, J = 17.0 Hz, 1H, -CH-), 1.25 (s, 3H, CH3), 1.22 (s, 3H, CH3), 0.99 (d, J = 6.9 Hz, 3H, CH3), 0.95 (t, J = 9.7 Hz, 9H, 3×CH3), 0.84 (s, 3H, CH3), 0.79 (s, 3H, CH3); 13

C-NMR (100 MHz, CDCl3) δ: 178.08, 173.50, 158.92, 151.44, 143.51, 142.41, 140.46, 123.31, 70.26, 57.07, 46.80, 46.63, 46.51,

46.36, 45.53, 43.60, 42.47, 42.16, 39.31, 36.21, 34.00, 33.09, 32.96, 31.73, 31.64, 30.72, 27.37, 25.80, 23.66, 23.56, 23.21, 19.91, 19.36, 18.73, 18.18, 16.25, 16.11; HR-MS (ESI) m/z: calcd. for C37H55N3O4 [M+H]+: 606.4271, found: 606.4260.

SC

4.1.1.13. N-leucine 23-hydroxy-olean[3,2-b]pyrazine-12-en-28-oate (13)

The compound 13 was synthesized with the synthesis of 5 as a white solid. Yield: 63.5% (after chromatograph with chloroform/methanol, 70:1). m.p. 189.1-192.2 °C. 1H-NMR (400 MHz, CDCl3) δ: 8.43 (d, J = 2.2 Hz, 1H, H-pyrazine), 8.30 (d, J

M AN U

= 2.5 Hz, 1H, H-pyrazine), 6.70 (d, J = 5.6 Hz, 1H, NH), 5.56 (s, 1H, H-12), 4.51 (s, 1H, NCH), 3.77 (d, J = 10.6 Hz, 1H, H-23a), 3.47 (d, J = 10.6 Hz, 1H, H-23b), 2.64 (d, J = 10.1 Hz, 1H, H-18), 2.16 (s, 2H, -CH2-), 1.24 (s, 3H, CH3), 1.23 (s, 3H, CH3), 1.20 (s, 3H, CH3), 0.95 (s, 3H, CH3), 0.93 (s, 6H, 2×CH3), 0.92 (s, 3H, CH3), 0.85 (s, 3H, CH3); 13C-NMR (100 MHz, CDCl3) δ: 158.97, 151.77, 143.83, 142.43, 140.74, 123.43, 70.46, 46.91, 46.76, 46.64, 45.72, 43.73, 42.52, 42.35, 42.12, 39.45, 36.35, 34.16, 33.11, 32.76, 31.80, 30.82, 29.77, 29.43, 27.45, 25.71, 25.08, 23.79, 23.72, 23.64, 22.94, 22.78, 22.76, 20.10, 19.51, 16.39, 16.28, 14.19; HR-MS (ESI) m/z: calcd. for C38H57N3O4 [M+H]+: 620.4422, found: 620.4421. 4.1.1.14. N-proline 23-hydroxy-olean[3,2-b]pyrazine-12-en-28-oate (14)

TE D

The compound 14 was synthesized with the synthesis of 5 as a white solid. Yield: 68.7% (after chromatograph with chloroform/methanol, 70:1). m.p. 164.8-166.9 °C. 1H-NMR (400 MHz, CDCl3) δ: 8.38-8.31 (m, 2H, H-pyrazine), 5.35 (s, 1H, H-12), 4.57 (s, 1H, NCH), 3.78 (d, J = 10.6 Hz, 2H, NCH2), 3.50 (d, J = 10.6 Hz, 1H, H-23a), 3.47 (d, J = 10.6 Hz, 1H, H-23b), 3.17 (d, J = 11.5 Hz, 1H, H-18), 2.16 (s, 2H, -CH2-), 1.30 (s, 3H, CH3), 1.19 (s, 3H, CH3), 0.94 (s, 3H, CH3), 0.92 (s, 3H, CH3), 0.90 (s, 3H, CH3), 0.81 (s, 3H, CH3); 13C-NMR (100 MHz, CDCl3) δ: 157.95, 152.08, 144.10, 144.06, 142.22, 141.81, 141.73,

EP

122.39, 70.70, 48.43, 48.10, 47.65, 47.24, 45.91, 45.84, 43.38, 42.71, 42.31, 36.65, 33.71, 33.18, 32.04, 30.60, 29.77, 29.61, 27.79, 26.42, 26.10, 24.04, 23.38, 22.89, 21.18, 20.21, 19.43, 16.68, 16.16, 14.20; HR-MS (ESI) m/z: calcd. for C37H53N3O4 [M+H]+:

AC C

604.4109, found: 604.4106.

4.1.1.15. N-methionione 23-hydroxy-olean[3,2-b]pyrazine-12-en-28-oate (15) The compound 15 was synthesized with the synthesis of 5 as a white solid. Yield: 76.9% (after chromatograph with

chloroform/methanol, 50:1). m.p. 188.3-189.7 °C. 1H-NMR (400 MHz, CDCl3) δ: 8.46 (s, 1H, H-pyrazine), 8.24 (d, 1H, H-pyrazine), 6.96 (d, J = 5.6 Hz, 1H, NH), 5.59 (s, 1H, H-12), 4.55 (s, 1H, NCH), 3.78 (d, J = 10.6 Hz, 1H, H-23a), 3.48 (d, J = 10.5 Hz, 1H, H-23b), 2.64 (m, 1H, H-18), 2.48 (d, J = 17.1 Hz, 2H, SCH2), 2.08 (s, 3H, CH3), 1.24 (s, 3H, CH3), 1.23 (s, 2H, -CH2-), 1.21 (s, 3H, CH3), 0.95 (s, 3H, CH3), 0.93 (s, 3H, CH3), 0.84 (s, 3H, CH3), 0.78 (s, 3H, CH3); 13C-NMR (100 MHz, CDCl3) δ: 178.39, 151.84, 143.61, 142.60, 140.14, 123.73, 70.42, 46.76, 46.65, 45.72, 43.86, 42.53, 42.34, 39.50, 36.26, 34.15, 33.10, 32.89, 32.17, 31.99, 31.83, 30.85, 30.06, 29.76, 29.43, 27.52, 25.73, 23.91, 23.67, 23.54, 22.76, 20.06, 19.50, 16.35, 16.11, 15.61, 14.20; HR-MS (ESI) m/z: calcd. for C37H55N3O4S [M+H]+: 638.3986, found: 638.3982. 4.1.1.16. N-glutamic 23-hydroxy-olean[3,2-b]pyrazine-12-en-28-oate (16) The compound 16 was synthesized with the synthesis of 5 as a white solid. The organic solution was concentrated, it was diluted with ethyl acetate (20.0 mL) and washed with water. Then water phase PH was adjusted to 5. The water was diluted with

ACCEPTED MANUSCRIPT ethyl acetate and dried over anhydrous sodium sulfate. Yield: 88.3% (after chromatograph with chloroform/methanol, 50:1). m.p. 152.4-153.3 °C. 1H-NMR (400 MHz, CD3OD) δ: 8.43 (m, 1H, H-pyrazine), 8.24 (d, J = 2.5 Hz, 1H, H-pyrazine), 5.44 (s, 1H, H-12), 4.39 (m, 1H, NCH), 3.81 (d, J = 10.6 Hz, 1H, H-23a), 3.53 (m, 1H, H-23b), 2.89 (d, J = 17.0 Hz, 1H, H-18), 2.42 (m, 2H, -CH2-), 2.18 (dd, J = 14.3, 6.5 Hz, 2H, -CH2-), 1.23 (s, 3H, CH3), 1.16 (s, 2H, -CH2-), 0.94 (s, 3H, CH3), 0.91 (s, 3H, CH3), 0.88 (s, 3H, CH3), 0.84 (s, 3H, CH3), 0.78 (s, 3H, CH3); 13C-NMR (100 MHz, CD3OD) δ: 178.97, 175.16, 173.87, 158.16, 152.61, 152.60, 143.51, 142.57, 140.77, 122.97, 68.92, 60.22, 52.14, 46.40, 46.19, 45.59, 44.75, 41.98, 41.64, 39.28, 36.22, 36.20, 33.84, 30.31,

4.1.1.17. N-aspartic 23-hydroxy-olean[3,2-b]pyrazine-12-en-28-oate (17)

RI PT

29.88, 27.29, 26.45, 24.96, 23.27, 22.80, 22.72, 19.57, 19.43, 18.88, 16.16, 15.07, 13.17; HR-MS (ESI) m/z: calcd. for C37H53N3O6 [M+H]+: 636.4007, found: 636.4000.

The compound 17 was synthesized with the synthesis of 16 as a white solid. Yield: 89.4% (after chromatograph with chloroform/methanol, 50:1). m.p. 177.1-179.9 °C. 1H-NMR (400 MHz, CD3OD) δ: 8.46 (d, J = 2.5 Hz, 1H, H-pyrazine), 8.23 (d, J = 2.5 Hz, 1H, H-pyrazine), 5.46 (m, 1H, H-12), 4.55 (t, J = 5.1 Hz, 1H, NCH), 3.81 (d, J = 10.6 Hz, 1H, H-23a), 3.53 (m, 1H,

SC

H-23b), 2.88 (d, J = 5.0 Hz, 1H, H-18), 2.84 (d, J = 5.2 Hz, 1H), 2.79 (dd, J = 12.2, 3.8 Hz, 1H), 1.23 (s, 3H, CH3), 1.15 (s, 3H, CH3), 0.93 (s, 3H, CH3), 0.90 (s, 3H, CH3), 0.88 (s, 3H, CH3), 0.87 (s, 3H, CH3); 13C-NMR (100 MHz, CD3OD) δ: 178.50, 167.96, 158.17, 152.58, 143.35, 142.57, 140.75, 132.24, 128.56, 123.17, 68.92, 65.33, 49.40, 48.37, 46.55, 45.60, 44.71, 42.06, 41.78,

M AN U

41.66, 39.33, 35.60, 33.84, 32.24, 31.70, 30.40, 27.20, 24.94, 23.30, 22.91, 19.50, 18.94, 18.86, 16.09, 15.12, 12.77; HR-MS (ESI) m/z: calcd. for C36H51N3O6 [M+H]+: 622.3851, found: 622.3845.

4.1.1.18. N-tyrosine 23-hydroxy-olean[3,2-b]pyrazine-12-en-28-oate (18)

The compound 18 was synthesized with the synthesis of 16 as a white solid. Yield: 90.1% (after chromatograph with chloroform/methanol, 50:1). m.p. 231.6-232.8 °C. 1H-NMR (400 MHz, CD3OD) δ: 8.45 (d, J = 2.1 Hz, 1H, H-pyrazine), 8.22 (d, J = 2.5 Hz, 1H, H-pyrazine), 7.01 (d, J = 8.4 Hz, 2H, H-Ar), 6.67 (d, J = 8.4 Hz, 2H, H-Ar),5.29 (s, 1H, H-12), 4.44 (s, 1H, NCH), 3.80 (d, J = 10.7 Hz, 1H, H-23a), 3.58 (d, J = 8.2 Hz, H-23b), 3.13 (dd, J = 13.8, 4.4 Hz, 1H), 2.82 (d, J = 5.0 Hz, 1H, H-18), 2.62

TE D

(d, J = 16.2 Hz, 1H), 1.16 (s, 6H, 2×CH3), 0.88 (s, 6H, 2×CH3), 0.83 (s, 3H, CH3), 0.55 (s, 3H, CH3);

13

C-NMR (100 MHz,

CD3OD) δ: 178.62, 158.20, 156.19, 152.64, 143.55, 142.52, 140.73, 130.19, 127.74, 122.83, 114.92, 68.92, 46.24, 46.20, 45.59, 44.76, 44.72, 44.71, 44.66, 41.84, 41.50, 39.11, 36.14, 35.82, 33.87, 32.37, 32.17, 31.74, 31.30, 30.25, 29.13, 27.12, 24.88, 24.86, 23.18, 22.92, 22.63, 19.40, 18.88, 15.51, 15.02; HR-MS (ESI) m/z: calcd. for C41H55N3O5 [M+H]+: 670.4214, found: 670.4208.

EP

4.1.1.19. N-serine 23-hydroxy-olean[3,2-b]pyrazine-12-en-28-oate (19) The compound 19 was synthesized with the synthesis of 16 as a white solid. Yield: 50.2% (after chromatograph with chloroform/methanol, 50:1). m.p. 151.3-152.2 °C. 1H-NMR (400 MHz, CDCl3) δ: 8.51-8.46 (m, 1H, H-pyrazine), 8.24 (s, 1H,

AC C

H-pyrazine), 7.31 (s, 1H, NH), 5.63 (s, 1H, H-12), 4.47 (d, J = 3.4 Hz, 1H), 4.03 (dd, J = 11.2, 2.8 Hz, 1H), 3.79 (d, J = 10.6 Hz, 1H, H-23a), 3.46 (d, J = 10.6 Hz, 1H, H-23b), 2.67 (d, J = 12.9 Hz, 1H, H-18), 1.24 (s, 6H, 2×CH3), 0.96 (s, 3H, CH3), 0.94 (s, 3H, CH3), 0.87 (s, 3H, CH3), 0.80 (s, 3H, CH3);

13

C-NMR (100 MHz, CDCl3) δ: 180.95, 159.48, 151.84, 143.40, 142.66, 140.04,

124.05, 70.43, 64.33, 56.14, 46.68, 45.65, 43.93, 42.63, 39.55, 36.26, 34.10, 32.99, 32.71, 32.00, 31.75, 30.82, 29.77, 27.47, 25.40, 23.85, 23.72, 23.63, 22.77, 20.01, 19.46, 16.51, 16.37, 15.96, 14.19; HR-MS (ESI) m/z: calcd. for C35H51N3O5 [M+H]+: 594.3901, found: 594.3896.

4.1.1.20. Benzyl 3-oxo-23-t-butyldimethylsilyloxy-olean-12-oxo-28-oate (X7) To a solution of X5 (500.0 mg, 0.7 mmol) in dry DCM (8.0 mL) was added formic acid (20.0 mL) and 30% H2O2 (20.0 mL), the mixture was refluxed at r.t. for 28 h. The mixture was concentrated and diluted with ethyl acetate, washed with hypertonic sodium bicarbonate solution and brine in sequence, dried anhydrous sodium sulfate (Na2SO4), and concentrated. The white solid product X7 was obtained by column chromatography (petroleum ether/ethyl acetate, 40:1) (377.7 mg, 73.9% yield).

ACCEPTED MANUSCRIPT 4.1.1.21. 3-oxo-23-hydroxy-olean-12-oxo-28-acid (20) The compound X7 (0.4 mmol) in absolute acetone was stirred with 10% HCl (2.0 ml) to removal of C-23 protective groups at r.t.. The white solid product benzyl 3-oxo-23-hydroxy-olean-12-oxo-28-oate (X8) was obtained by column chromatography (petroleum ether/ethyl acetate, 5:1) (89.5% yield). The compound 20 was obtained through catalytic reduction of hydrogen X8. The white solid product 20 was purified by column chromatography (chloroform/methanol, 150:1). Yield: 91.3%. m.p. 122.8-124.1 °C; 1H-NMR (400 MHz, CDCl3) δ: 3.68 (d, J = 11.4 Hz, 1H, H-23a), 3.37 (d, J = 11.4 Hz, 1H, H-23b), 2.74 (d, J =

13

RI PT

13.5 Hz, 1H, H-18), 1.06 (s, 3H,CH3), 1.03 (s, 3H,CH3), 0.97 (s, 3H, CH3), 0.95 (s, 3H, CH3), 0.93 (s, 3H, CH3), 0.88 (s, 3H, CH3); C-NMR (100 MHz, CDCl3) δ: 218.20, 210.97, 184.00, 176.98, 66.41, 52.45, 51.83, 48.91, 48.41, 47.08, 41.96, 38.46, 38.12,

36.44, 36.00, 34.96, 34.34, 33.28, 32.98, 31.72, 31.03, 30.57, 27.50, 23.04, 22.51, 20.47, 18.98, 16.66, 16.21, 14.93; HR-MS (ESI) m/z: calcd. for C30H46O5 [M+H]+: 487.3424, found: 487.3413. 4.1.2. General procedure for the synthesis of 10-12

SC

4.1.2.1. O-1’-isopropanol-3’-dimethylamino group 23-acetoxy -olean[3,2-b]pyrazine-12-en-28-oate (X4f)

The compound X4f was synthesized with the synthesis of X4a as a yellow oil. Yield: 83.9% (after chromatograph with

M AN U

chloroform/methanol, 30:1). The compound X4g was synthesized with the synthesis of X4f.

4.1.2.2. O-1’-isopropanol-3’-dimethylamino group 23-hydroxy-olean[3,2-b]pyrazine-12-en-28-oate (10) The compound 10 was obtained from X4f according to the deprotection reaction. Yield: 93.5% (after chromatogra ph with chloroform/methanol, 25:1) as a white solid. m.p. 121.1-123.2 °C. 1H-NMR (400 MHz, CDCl3) δ: 8.34 (d, J = 2.3 Hz, 1H, H-pyrazine), 8.31 (d, J = 2.5 Hz, 1H, H-pyrazine), 5.34 (t, J = 3.3 Hz, 1H, H-12), 4.07 (t, J = 6.1 H z, 2H, OCH2), 3.76 (d, J = 10.6 Hz, 1H, H-23a), 3.45 (d, J = 10.6 Hz, 1H, H-23b), 2.85 (d, J = 14.4 Hz, 1H, H-1 8), 2.76 (d, J = 11.0 Hz, 2H, NCH2), 2.57 (s, 6H, 2×NCH3), 1.29 (s, 3H, CH3), 1.15 (s, 3H, CH3), 0.90 (s, 3H, CH 3),

0.90 (s, 3H, CH3), 0.88 (s, 3H, CH3), 0.78 (s, 3H, CH3).

13

C-NMR (100 MHz, CDCl3) δ: 177.51, 157.81, 151.96,

TE D

143.72, 142.11, 141.61, 122.16, 70.56, 61.47, 55.62, 47.63, 46.99, 46.79, 45.68, 45.60, 43.75, 43.28, 41.92, 41.40, 39.1 8, 36.46, 33.76, 32.99, 32.39, 31.84, 30.64, 29.64, 27.54, 25.67, 25.03, 23.50, 23.31, 22.99, 20.07, 19.36, 16.79, 16.04. HR-MS (ESI) m/z: calcd. for C37H57N3O3 [M+H]+: 592.4473, found: 592.4478. 4.1.2.3. O-monoethanolamine 23-hydroxy-olean[3,2-b]pyrazine-12-en-28-oate (11)

EP

To a solution of X4g in tetrahydrofuran with ethanolamine (3:2), 10% sodium hydroxide was slowly added. The compound 11 was obtained according to the synthesis conditions of the compound 5. Yield: 56.2% (after chromatograph with chloroform/methanol, 600:1) as a white solid. m.p. 102.4-105.1 °C. 1H-NMR (400 MHz, CDCl3) δ: 8.38 (dd, J = 8.0, 2.3 Hz, 2H,

AC C

H-pyrazine), 5.40 (t, J = 3.4 Hz, 1H, H-12), 4.12 (dt, J = 24.4, 5.8 Hz, 2H, OCH2), 3.80 (d, J = 10.6 Hz, 1H, H-23a), 3.50 (d, J = 10.6 Hz, 1H, H-23b), 3.01 (d, J = 16.6 Hz, 2H, NCH2), 2.93 (d, J = 13.8 Hz, 1H, H-18), 2.64 (s, 2H, NH2), 1.34 (s, 3H, CH3), 1.21 (s, 3H, CH3), 0.96 (s, 6H, 2×CH3), 0.93 (s, 3H, CH3), 0.86 (s, 3H, CH3); 13C-NMR (100 MHz, CDCl3) δ: 177.16, 157.38, 151.56, 143.47, 141.69, 141.12, 121.68, 70.17, 47.26, 46.68, 46.46, 45.29, 45.22, 42.82, 41.53, 41.10, 40.56, 38.78, 36.03, 33.38, 32.56, 32.02, 31.45, 30.22, 29.19, 27.13, 25.24, 23.09, 22.91, 22.60, 19.66, 18.88, 16.34, 15.61; HR-MS (ESI) m/z: calcd. for C34H51N3O3 [M+H]+: 550.4003, found: 550.4005. 4.1.2.4. O-amidobutyric acid methyl ester 23-hydroxy-olean[3,2-b]pyrazine-12-en-28-oate (12) The compound 12 was obtained from X4f according to the deprotection reaction. Yield: 50.2% (after chromatograph with petroleum ether/ethyl acetate, 5:1) as a white solid. m.p. 128.3-129.8 °C. 1H-NMR (400 MHz, CDCl3) δ: 9.07 (s, 1H, NH), 8.35, 8.33 (ds, J = 2.3 Hz, 2H, H-pyrazine), 5.39 (s, 1H, H-12), 3.77 (d, J = 10.5 Hz, 1H, H-23a), 3.68 (s, 3H, OCH3), 3.46 (d, J = 10.5 Hz, 2H, H-23b), 3.93-2.86 (m, 1H, H-18), 2.68 (t, J = 6.7 Hz, 2H, NHCOCH2), 2.59-2.43 (m, 2H, OCOCH2), 1.30 (s, 3H, CH3), 1.17 (s, 3H, CH3), 0.92 (s, 6H, 2×CH3), 0.89 (s, 3H, CH3), 0.80 (s, 3H, CH3); 13C-NMR (100 MHz, CDCl3) δ: 172.62, 157.39, 151.52, 142.42, 141.69, 141.14, 122.39, 117.42, 70.16, 51.58, 47.24, 46.67, 46.33, 45.18, 45.11, 42.78, 41.46, 40.87, 38.77, 36.01,

ACCEPTED MANUSCRIPT 33.21, 32.47, 31.85, 31.36, 30.13, 29.20, 28.56, 28.29, 27.49, 27.32, 27.13, 25.18, 23.06, 22.89, 22.47, 19.66, 18.89; HR-MS (ESI) m/z: calcd. for C37H53N3O6 [M+H]+: 636.4013, found: 636.3998. 4.1.3. General procedure for the synthesis of 21-25 H (0.1 mmol) and EDCI (0.6 mmol) were added to a solution of 1-hydroxy-7-azabenzotriazole (HOAT), 6-chloro-1-hydroxybenzotriazole or imidazole (1.2 mmol) in dry dichloromethane (8.0 mL). The mixture was stirred at r.t. for 3 h. The solvent was evaporated under reduced pressure to afford the crude product . It was purified by column chromatography

same as synthesis of 21. (see Scheme 2) 4.1.3.1. 7’-azabenzotriazol-1’-yl-(3β,4α)-3,23-dihydroxyolean-12-en-28-oate (21)

RI PT

(chloroform/methanol, 150:1) to afford 21-23. The compound 24, 25 of 20 derivatives were synthesisd and purified with the

Yield: 95.5% (after chromatograph with chloroform/methanol, 150:1) as a white solid. m.p. 239.0-240.9 °C; 1H-NMR (400 MHz, CDCl3) δ: 8.66 (d, J = 4.2 Hz, 1H, H-Ar), 8.35 (d, J = 8.2 Hz, 1H, H-Ar), 7.37 (dd, J = 8.3, 4.5 Hz, 1H, H-Ar), 5.30 (s, 1H, H-12), 3.71 (s, 1H, H-23a), 3.62 (s, 1H, H-3), 3.42 (s, 1H, H-23b), 2.93 (d, J = 6.0 Hz, 1H, H-18), 2.87 (d, J = 8.1 Hz, 2H, H-11),

SC

1.18 (s, 3H, CH3), 0.95 (s, 3H, CH3), 0.93 (s, 3H, CH3), 0.91 (s, 3H, CH3), 0.86 (s, 3H, CH3), 0.81 (s, 3H, CH3); 13C-NMR (100 MHz, CDCl3) δ: 172.86, 151.11, 141.59, 140.31, 134.49, 128.74, 123.17, 120.22, 62.69, 49.36, 47.09, 46.99, 45.07, 41.43, 41.13, 38.97, 37.74, 36.39, 33.19, 32.45, 32.07, 31.67, 30.13, 27.58, 26.12, 25.27, 23.04, 22.94, 22.79, 18.01, 16.45, 15.27, 13.44, 11.00,

M AN U

10.89; HR-MS (ESI) m/z: calcd. for C35H50N4O4 [M+H]+: 591.3910, found: 591.3901.

4.1.3.2. 6’-Chloro-benzotriazol-1’-yl-(3β,4α)-3,23-dihydroxyolean-12-en-28-oate (22)

Yield: 91.7% (after chromatograph with chloroform/methanol, 150:1) as a white solid. m.p. 149.8-153.9 °C. 1H-NMR (400 MHz, CDCl3) δ: 7.94 (d, J = 9.3 Hz, 1H, H-Ar), 7.34 (m, 2H, 2×H-Ar), 5.39 (t, J = 3.2 Hz, 1H, H-12), 3.71 (d, J =10.2 Hz, 1H, H-23a), 3.62 (t, J =7.6 Hz, 1H, H-3), 3.41 (d, J = 10.2 Hz, 1H, H-23b), 2.96 (dd, J =13.5, 4.0 Hz, 1H, H-18), 1.19 (s, 3H, CH3), 0.98 (s, 3H, CH3), 0.95 (s, 3H, CH3), 0.94 (s, 3H, CH3), 0.87 (s, 3H, CH3), 0.81 (s, 3H, CH3); 13C-NMR (100 MHz, CDCl3) δ: 172.99, 141.72, 141.60, 134.60, 128.86, 125.53, 123.50, 121.00, 107.71, 76.24, 71.45, 49.29, 47.13, 47.07, 44.91, 41.46, 41.32,

TE D

41.28, 39.04, 37.72, 36.44, 33.08, 32.44, 32.17, 31.84, 30.19, 27.41, 26.19, 25.36, 23.06, 22.99, 22.63, 17.96, 16.92, 15.23, 11.00; HR-MS (ESI) m/z: calcd. for C36H50ClN3O4 [M+H]+: 624.3568, found: 624.3565. 4.1.3.3. Imidazol-1’-yl-(3β,4α)-3,23-dihydroxyolean-12-en-28-oate (23) Yield: 61.8% (after chromatograph with chloroform/methanol, 150:1) as a white solid. m.p. 116.5-125.8 °C. 1H-NMR (400

EP

MHz, CDCl3) δ: 8.29 (s, 1H, H-Ar), 7.60-7.54 (m, 1H, H-Ar), 7.04 (d, J = 16.9 Hz, 1H, H-Ar), 5.33 (d, J = 22.8 Hz, 1H, H-12), 3.69 (t, J = 11.3 Hz, 1H, H-23a), 3.64 (t, J = 7.8 Hz, 1H, H-3), 3.40 (d, J = 10.3 Hz, 1H, H-23b), 3.06 (d, J = 10.8 Hz, 1H, H-18), 1.16 (s, 3H, CH3), 0.97 (s, 3H, CH3), 0.96 (s, 3H, CH3), 0.96 (s, 3H, CH3), 0.88 (s, 3H, CH3), 0.69 (s, 3H, CH3); 13C-NMR (100

AC C

MHz, CDCl3) δ: 174.21, 142.11, 136.62, 130.46, 129.12, 128.36, 122.69, 117.03, 71.40, 49.36, 47.06, 45.33, 42.04, 41.36, 41.25, 38.69, 37.64, 36.40, 33.40, 32.38, 31.86, 30.84, 29.93, 27.13, 26.11, 25.40, 23.32, 22.99, 22.90, 17.86, 16.23, 15.23,10.99; HR-MS (ESI) m/z: calcd. for C33H50N2O3 [M+H]+: 523.3900, found: 523.3895. 4.1.3.4.O-benzotriazol-1’-yl-3-oxo-23-hydroxy-olean-12-oxo-28-oate (24) Yield: 92.0%. m.p. 126.7-130.5 °C; 1H-NMR (400 MHz, CDCl3) δ: 8.07 (d, J = 8.4 Hz, 1H, H-Ar), 7.55 (t, J = 7.6 Hz, 1H,

H-Ar), 7.43 (t, J = 8.0 Hz, 1H, H-Ar), 7.36 (d, J = 8.3 Hz, 1H, H-Ar), 5.32 (d, J = 19.0 Hz, 1H, H-12), 3.72 (d, J = 11.4 Hz, 1H, H-23a), 3.41 (d, J = 11.4 Hz, 1H, H-23b), , 2.98 (d, J = 13.3 Hz, 1H, H-18), 2.90 (d, J = 4.3 Hz, 1H, H-13), 1.18 (s, 3H, CH3), 1.11 (s, 3H, CH3), 1.05 (s, 3H, CH3), 1.03 (s, 3H, CH3), 1.01 (s, 3H, CH3), 0.98 (s, 3H, CH3); 13C-NMR (100 MHz, CDCl3) δ: 218.88, 211.30, 175.16, 144.51, 129.68, 129.61, 125.77, 121.57, 108.87, 67.39, 53.53, 52.74, 50.06, 49.50, 48.92, 43.18, 42.46, 39.82, 39.27, 37.47, 36.74, 35.94, 35.16, 34.16, 34.07, 33.32, 32.09, 30.63, 28.73, 24.04, 23.81, 21.51, 19.96, 17.69, 17.29, 15.97; HR-MS (ESI) m/z: calcd. for C36H49N3O5 [M+H]+: 604.3751, found: 604.3747.

ACCEPTED MANUSCRIPT 4.1.3.5.O-6’-Chloro-benzotriazol-1’-yl-3-oxo-23-hydroxy-olean-12-oxo-28-oate (25) Yield: 90.2%. m.p. 128.2-130.5 °C; 1H-NMR (400 MHz, CDCl3) δ: 8.00 (d, J = 8.9 Hz, 1H, H-Ar), 7.39 (dd, J = 8.9, 1.8 Hz, 1H, H-Ar), 7.34 (d, J = 1.5 Hz, 1H, H-Ar), 3.72 (d, J = 5.4 Hz, 1H, H-23a), 3.40 (d, J = 11.4 Hz, 1H, H-23b), 2.97 (d, J = 13.4 Hz, 1H, H-18), 2.86 (d, J = 4.3 Hz, 1H, H-13), 1.17 (s, 3H, CH3), 1.11 (s, 3H, CH3), 1.05 (s, 3H, CH3), 1.03 (s, 3H, CH3), 1.00 (s, 3H, CH3), 0.98 (s, 3H, CH3), 13C-NMR (100 MHz, CDCl3) δ: 218.86, 211.24, 175.02, 143.06, 136.30, 130.19, 127.14, 122.98, 108.79, 67.38, 53.53, 52.82, 50.02, 49.46, 48.97, 43.18, 42.46, 39.50, 39.25, 37.47, 36.73, 36.93, 36.04, 34.13, 34.00, 33.36, 32.08, 31.53,

638.3357. 4.1.4. General procedure for the synthesis of 26-29 4.1.4.1. Ethyl-(3β,4α)-3,23-dihydroxyolean-12-en-28-oate (26)

RI PT

28.68, 24.04, 23.81, 21.53, 19.94, 17.69, 17.27, 15.96; HR-MS (ESI) m/z: calcd. for C36H48ClN3O5 [M+H]+: 638.3361, found:

To a solution of H (50.0 mg, 0.1 mmol) in DMF (6.0 mL) was added potassium carbonate (10.1 mg, 0.1 mmol) and ethylene

SC

dibromide (4.8 µl), the mixture was refluxed at 50 °C for 12 h. The mixture was concentrated and diluted with ethyl acetate, washed with water and brine in sequence, dried Na2SO4, and concentrated. The white solid product 26 was obtained by column chromatography (chloroform/methanol, 140:1-100:1) (35.3 mg, 66.2%). m.p. 220.7-223.1 °C. 1H-NMR (400 MHz, CDCl3) δ: 5.30

M AN U

(t, J = 3.3 Hz, 2H, 2×H-12), 4.20 (dd, J = 20.5, 9.0 Hz, 4H, 2×OCH2), 3.73 (d, J = 10.3 Hz, 2H, 2×H-23a), 3.65 (t, J = 7.8 Hz, 2H, 2×H-3), 3.43 (d, J = 10.3 Hz, 2H, 2×H-23b), 2.87 (d, J = 13.5 Hz, 2H, 2×H-18), 2.62 (s, 4H, 2×H-11), 1.14 (s, 6H, 2×CH3), 0.97 (s, 6H, 2×CH3), 0.93 (s, 6H, 2×CH3), 0.92 (s, 6H, 2×CH3), 0.90 (s, 6H, 2×CH3), 0.75 (s, 6H, 2×CH3); 13C-NMR (100 MHz, CDCl3) δ: 177.01, 143.06, 121.96, 76.46, 71.70, 61.70, 49.36, 47.10, 46.20, 45.32, 41.25, 41.23, 40.77, 38.83, 37.61, 36.44, 33.36, 32.61, 32.02, 31.93, 30.21, 27.29, 26.16, 26.40, 23.12, 22.92, 22.54, 18.01, 16.46, 15.23, 10.96; HR-MS (ESI) m/z: calcd. for C62H98O8 [M+Na]+: 993.7154, found: 993.7151.

4.1.4.2. Propyl -(3β,4α)-3,23-dihydroxyolean-12-en-28-oate (27)

TE D

The compound 27 was synthesized with the synthesis of 26 as a white solid. Yield: 67.0% (after chromatograph with chloroform/methanol, 140:1-100:1). m.p. 210.1-213.5 °C; 1H-NMR (400 MHz, CDCl3) δ: 5.29 (s, 2H, 2×H-12), 4.10 (dd, J = 8.9, 6.1 Hz, 4H, 2×OCH2), 3.72 (d, J = 10.2 Hz, 2H, 2×H-23a), 3.64 (t, J = 7.7 Hz, 2H, 2×H-3), 3.42 (d, J = 10.2 Hz, 2H, 2×H-23b), 2.87 (s, 2H, 2×H-18), 1.14 (s, 6H, 2×CH3), 0.95 (s, 6H, 2×CH3), 0.93 (s, 6H, 2×CH3), 0.91 (s, 6H, 2×CH3), 0.90 (s, 6H, 2×CH3), 0.73 (s, 6H, 2×CH3); 13C-NMR (100 MHz, CDCl3) δ: 177.09, 143.20, 121.91, 76.44, 71.64, 60.39, 49.33, 47.09, 46.24, 45.34,

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41.25, 41.24, 40.83, 38.82, 37.62, 36.43, 33.37, 32.61, 32.06, 32.00, 30.20, 27.68, 27.18, 26.15, 25.42, 23.15, 22.92, 22.50, 18.02, 16.62, 15.24, 10.96; HR-MS (ESI) m/z: calcd. for C63H100O8 [M+Na]+: 1007.7310, found: 1007.7307.

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4.1.4.3. N-butyl -(3β,4α)-3,23-dihydroxyolean-12-en-28-oate (28) The compound 28 was synthesized with the synthesis of 26 as a white solid. Yield: 65.3% (after chromatograph with

chloroform/methanol, 140:1-100:1). m.p. 205.2-206.9 °C; 1H-NMR (400 MHz, CDCl3) δ: 5.29 (t, J = 3.2 Hz, 2H, 2×H-12), 4.05 (s, 4H, 2×OCH2), 3.80–3.70 (m, 2H, 2×H-23a), 3.69-3.61 (m, 2H, 2×H-3), 3.43 (d, J = 10.3 Hz, 2H, 2×H-23b), 2.87 (dd, J = 13.7, 3.9 Hz, 2H, 2×H-18), 1.14 (s, 6H, 2×CH3), 0.96 (s, 6H, 2×CH3), 0.94 (s, 6H, 2×CH3), 0.92 (s, 6H, 2×CH3), 0.90 (s, 6H, 2×CH3), 0.74 (s, 6H, 2×CH3); 13C-NMR (100 MHz, CDCl3) δ: 177.21, 143.27, 121.84, 76.43, 71.63, 63.26, 49.32, 47.09, 46.22, 45.37, 41.27, 41.25, 40.84, 38.84, 37.62, 36.43, 33.40, 32.62, 32.08, 32.00, 30.21, 27.18, 26.17, 25.41, 25.00, 23.14, 22.93, 22.53, 18.03, 16.59, 15.23, 10.96; HR-MS (ESI) m/z: calcd. for C64H102O8 [M+Na]+: 1021.7467, found: 1021.7460. 4.1.4.4. N-Pentyl-(3β,4α)-3,23-dihydroxyolean-12-en-28-oate (29) The compound 29 was synthesized with the synthesis of 26 as a white solid. Yield: 61.4% (after chromatograph with chloroform/methanol, 140:1-100:1). m.p. 223.8-226.1 °C; 1H-NMR (400 MHz, CDCl3) δ: 5.29 (t, J = 3.2 Hz, 2H, 2×H-12), 4.03 (t, J = 6.1 Hz, 4H, 2×OCH2), 3.73 (d, J = 10.3 Hz, 2H, 2×H-23a), 3.65 (t, J = 7.8 Hz, 2H, 2×H-3), 3.43 (d, J = 10.4 Hz, 2H, 2×H-23b), 2.88 (dd, J = 13.7, 3.9 Hz, 2H, 2×H-18), 1.14 (s, 6H, 2×CH3), 0.96 (s, 6H, 2×CH3), 0.94 (s, 6H, 2×CH3), 0.92 (s, 6H,

ACCEPTED MANUSCRIPT 2×CH3), 0.90 (s, 6H, 2×CH3), 0.74 (s, 6H, 2×CH3); 13C-NMR (100 MHz, CDCl3) δ: 177.22, 143.28, 121.82, 76.42, 71.59, 63.51, 49.32, 47.11, 46.18, 45.39, 41.27, 41.25, 40.84, 38.84, 37.63, 36.42, 33.41, 32.62, 32.10, 31.99, 30.22, 27.81, 27.17, 26.19, 25.42, 23.14, 22.93, 22.56, 22.30, 18.03, 16.58, 15.22, 10.94; HR-MS (ESI) m/z: calcd. for C65H104O8 [M+H]+: 1013.7809, found: 1013.7804. 4.2 Lipo-hydro partition coefficient A high performance liquid chromatography method (HPLC) was applied to determine the lipo-hydro partition coefficient of some compounds in n-octanol-buffer solution systems. A buffer of pH 6.8 was placed at room temperature. The above solution

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was used as the aqueous phase, and n-octanol was used as the oil phase. In this study, it was taken in a ratio of 1:1. The mixture was shaken for 4 h to evenly distribute. The concentration of the compound in the two phases was measured [24]. 4.3. Biology 4.3.1. Cell culture and treatment

The cancer cell line KBV was kindly provided by Dr. Xiaoguang Chen from Institute of Materia Medica, Chinese Academy

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of Medical Sciences. All the cells were cultured in RPMI-1640 (Hyclone) with 10% heat-inactivated fetal calf serum (Life Technology), and 100 U/ml penicillin and l00 g/ml streptomycin, in an atmosphere of 5% CO2 at 37°C. Cells in logarithmic

4.3.2. Cell proliferation assays

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growth phase were used for further experiments.

The MTT assay was conducted to detect the cell viability following our previously protocol [25]. Briefly, the cells were seeded into 96-well plates and treated with the tested articles at the desired concentration for 72 h or indicated time period. MTT solution was then added into the wells and incubated, and then DMSO was added and the optical density was measured at 570 nm, in which the cell survival rate were calculated.

4.3.3. Intracellular Rhodamine123 accumulation assay

The effect of compound 10 on the Rhodamine123 accumulation was measured by flow cytometry analysis following our

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previously protocol [17]. Briefly, KBV cells were treated with or without compound 10 for 2 h at the concentration indicated, and then the cells were incubated with 10 µM Rhodamine123 for 30 min. Subsequently, the cells were harvested and washed three times prior to analysis by flow cytometry. The mean fluorescence intensity was then automatically calculated from the fluorescence intensity of 10,000 cells by the flow cytometry machine (BD, C6, USA).

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4.3.4. Cell cycle distribution analysis

A flow cytometry assay was used to analyze the cell cycle distribution as previously reported [26]. Briefly, the cells were plated into 6-well plates and incubated overnight. After treatment with the test articles for 24 h, the cells were harvested and

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fixed in ethanol solution (70%) overnight at -20 °C, and then washed with PBS and stained with PI solution (20 mg/mL PI and 20 mg/mL RNaseA in PBS) for 30 min.The cell fluorescence was measured using flow cytometry machine (BD, C6, USA) and the cell cycle distribution was analyzed. 4.3.5 ATPase activity assay

The P-gp-Glo™ assay kit (Promega, USA) was used to detect the effect of compound 10 on the ATPase activity following

our published protocol [17]. Briefly, the diluted P-gp protein was added into the wells containing 10, or the positive control, verapamil. After incubation at 37 °C for about 5 min, MgATP were added and mixed, and then incubated for 40 min at 37 °C. The reactions were stopped and the luminescence initiated by adding ATP Detection Reagent. Solutions were mixed, and then plates were incubated at r.t. for 20 min. Luminescence was read using a SpectraMax M5 multifunctional microplate reader (Molecular Devices, USA). 4.3.6 Xenograft Studies Nude mice (6−8 weeks old, BALB/c, male) were used to establish the xenograft tumors following our published Protocol [27]. Briefly, KBV cells (5 × 106) were implanted in the dorsal region of recipient mice by means of subcutaneous injection.

ACCEPTED MANUSCRIPT Once a tumor had reached around 100-200 mm3 in size, the mice were randomized into four groups as control, paclitaxel (30 mg/kg), 10 (50 mg/kg), and paclitaxel (30 mg/kg) plus 10 (50 mg/kg) with five mice per group. The animals were administrated by intraperitoneal injection twice a week for the paclitaxel, and the 10 was given via gavage. Tumor growth were measured every three days during the treatment. At the end of the treatment, mice were sacrificed and tumors were removed and weighed. The use of animals was approved by the Animal Experimentation Ethics Committee of Yantai University (protocol number 20180321) in accordance with the guidelines for ethical conduct in the care and use of animals. 4.4. Molecular modeling

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The model of the three compounds (10, H6 and verapamil) and P-gp was studied by Surflex-dock (SYBYL Software, Version X2.0.). The P-gp structure of three-dimensional for docking was derived from published homology model of P-gp [28]. 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 [29]. The three compounds were optimized by Minimize module. The force field was Tripos force field. Gasteiger-Hückel charge was loaded, and the other parameters were the default values.

The authors confirm that this article content has no conflict of interest. Acknowledgements

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Conflict of interest

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This work was partially supported by NSFC (81773563 and 81728020), Key Research Project of Shandong Province (2017GSF18177), Natural Science Foundation of Shandong Province (ZR2018LH025). We thank Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript. References

[1] Y.Q. Wu, W.R. Fang, Y.M. Li, Mechanisms and reversing drugs of cancer multidrug resistance, Pharm. & Clin. Res. 2016(1):43-47. [2] C.F. Higgins. Multiple molecular mechanisms for multidrug resistance transporters, Nature, 2007, 446(7137):749. [3] S.G. Aller, J. Yu, A. Ward, Y. Weng, S. Chittaboina, structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding, Science,

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2009, 323(323):1718-1722. [4] X.Q. Zhao, J. D. Xie, X.G. Chen, H.M. Sim, X. Zhang, Y.J. Liang, S. Singh, T.T. Talele, Y.L. Sun, S.V. Ambudkar, Z.S. Chen, L.W. Fu, Neratinib (HKI-272) reverses ABCB 1-mediated chemotherapeutic drug resistance in vitro,in vivo and ex vivo, Molecular Pharmacology Fast Forward. 2012.

[5] G. Szakács, J.K. Paterson, J.A. Ludwig, Targeting multidrug resistance in cancer, Nat. Rev. Drug Discov. 2006, 5(3):219-234.

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[6] T. Saeki, T. Nomizu, M. Toi, Y. Ito, S. Noguchi, T. Kobayashi, Dofequidar fumarate (MS-209) in combination with cyclophosphamide, doxorubicin, and fluorouracil for patients with advanced or recurrent breast cance, J. Clin. Oncol, 2007, 25(4):411-7. [7] L. Fu, Y. Liang, L. Deng, Y. Ding, L.M. Chen, Y.L. Ye, X.P. Yang, Q.C. Pan, Characterization of tetrandrine, a potent inhibitor of

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P-glycoprotein-mediated multidrug resistance, Cancer Chemoth. & Pharm. 2004, 53(4):349-356. [8] C. Wang, J.X. Zhang, X.L. Shen, C.K. Wan, A.K.W. Tse, W.F. Fong, Anfernee Kai-Wing Tse, Reversal of P-glycoprotein-mediated multidrug resistance by Alisol B 23-acetate, Biochem. Pharmacol. 2004, 68(5):843-855.

[9] T. Nabekura, T. Yamaki, T. Hiroi, K. Ueno, S. Kitagawa, Inhibition of anticancer drug efflux transporter P-glycoprotein by rosemary phytochemicals, Pharmacol. Res. 2010, 61(3):259-263.

[10] J. Fernandes, R.O. Castilho, M.R.D. Costa, K.W. Souza, M.A.C. Kaplan, C.R. Gattass, Pentacyclic triterpenes from Chrysobalanaceae species: cytotoxicity on multidrug resistant and sensitive leukemia cell lines, Cancer Lett. 2003, 190(2):165-169. [11] X.X. Liu, Y.T. Yang, X. Wang, K.Y. Wang, J.Q. Liu, L. Lei, X.M. Luo, R.Zhai, F.H. Fu, H.B. Wang, Y. Bi, Design, synthesis and biological evaluation of novel α-hederagenin derivatives with anticancer activity, Eur. J. Med. Chem. 2017, 141:427-439. [12] C.W. Lee, S.M. Park, R.J. Zhao, C. Lee, W.J. Chun, Y. Son, S.H. Kim, J.Y. Jung, K.H.Jegal, I.J. Cho, S.K. Ku, Y.W. Kim, S.A. Ju, Hederagenin, a major component of Clematis mandshurica Ruprecht root, attenuates inflammatory responses in RAW 264.7 cells and in mice, Int. Immunopharmacol. 2015, 29(2):528-537. [13] F.S.T. Ndjateu, R.B.N. Tsafack, B.K. Nganou, M.D. Awouafack, H.K. Wabo, M. Tene, P. Tane, J.N. Eloff, Antimicrobial and antioxidant

ACCEPTED MANUSCRIPT activities of extracts and ten compounds from three Cameroonian medicinal plants: Dissotis perkinsiae, (Melastomaceae), Adenocarpus mannii, (Fabaceae) and Barteria fistulosa, (Passifloraceae), S. Afr. J. Bot. 2014, 91(3):37-42. [14] G.J. Kim, D.S. Song, H.S. Yoo, K.H. Chung, K.J. Lee, J.H. An, Hederagenin Supplementation Alleviates the Pro-Inflammatory and Apoptotic Response to Alcohol in Rats, Nutrients. 2017, 9(1):41. [15] B.F. Liang, F. Huang, H.T. Wang, G.H. Wang, X. Yuan, M.Z. Zhang, H.B. Guo, Y.F. Cheng, J.P. Xu, Involvement of norepinephrine and serotonin system in antidepressant-like effects of hederagenin in the rat model of unpredictable chronic mild stress-induced depression, Pharm. Biol. 2015, 53(3):368-77.

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[16] S.H. Lu, J.H. Guan, Y.L. Huang, Y.W. Pan, W. Yang, H. Lan, Experimental study of antiatherosclerosis effects with hederagenin in rats, Evid.-Based Compl. Alt. Medicine,2015,(2015-10-19), 2015, 2015(34):456354.

[17] 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, reverses multidrug resistance in vitro and in vivo, Toxicol. & Appl. Pharm. 2018, 341:98.

[18] U.V. Mallavadhani, N.R. Vanga, M.K. Jeengar, V.G.M. Naidu, Synthesis of novel ring-A fused hybrids of oleanolic acid with capabilities to

SC

arrest cell cycle and induce apoptosis in breast cancer cells, Eur. J. Med. Chem. 2014, 74(3):398-404.

[19] D. Rodríguez-Hernández, A.J. Demuner, L.C. Barbosa, C. Rene, H. Lucie, Hederagenin as a triterpene template for the development of new antitumor compounds, Eur. J. Med. Chem. 2015, 105:57.

agents, Eur. J. Med. Chem. 2016, 115:257-267.

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[20] D. Rodríguez-Hernández, A.J. Demuner, L.C. Barbosa, H. Lucie, C. Rene, Novel hederagenin-triazolyl derivatives as potential anti-cancer

[21] 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. W, H.M. Lei, Design, synthesis, and cytotoxic analysis of novel hederagenin–pyrazine derivatives based on partial least squares discriminant analysis, Int. J. Mol. Sci. 2018, 19:994.

[22] Y.K. Zhang, H.Y. Zhang, G.N. Zhang, Y.J. Wang, R.J. Kathawala, R. Si1, B.A. Patel, J.X. Xu, Z.S. Chen, Semi-synthetic ocotillol analogues as selective ABCB1-mediated drug resistance reversal agents, Oncotarget. 2015, 6(27):24277.

[23] G.F. Zha , J. Leng, N. Darshini, T. Shubhavathi, H.K. Vivek, A. M. Asiri, H.M. Marwani, K.P. Rakesh, N. Mallesha, H.L. Qin, Synthesis, SAR

2017, 27(14):3148-3155.

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and molecular docking studies of benzo[d]thiazole-hydrazones as potential antibacterial and antifungal agents, Bioorg. & Med. Chem. Lett.

[24] X.Y Rao, S Yin, G.S Zhang, X.J Luo, H Jian, Y.L Feng, S.L Yang, Determination of equilibrium solubility and n-octanol / water partition coefficient of pulchinenosiden D by HPLC, Chin Pharmacol J. 2014, 39(9):1593-1596. [25] D Zhang, Q Xu, N Wang, Y.T Yang, J.Q Liu, G.H Yu, X Yang, H Xu, H.B Wang, A complex micellar system co-delivering curcumin with

EP

doxorubicin against cardiotoxicity and tumor growth, Int. J. Nanomedicine. 2018, 13:4549-4561. [26] 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. 2017, 7(1):52-58.

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[27] Y.T Ma, Y.T Yang, P Cai, D.Y Sun, PA Sánchez-Murcia, X.Y Zhang, W.Q Jia, L Lei, M.Q Guo, F Gago, H.B Wang, W.S Fang, A Series of Enthalpically Optimized Docetaxel Analogues Exhibiting Enhanced Antitumor Activity and Water Solubility, J Nat Prod. 2018, 81(3):524-533.

[28] J. Li, K.F. Jaimes, S.G. Aller, Refined structures of mouse P-glycoprotein, Protein Sci. 2013, 23(1):34-46. [29] A.C. Wallace, R.A. Laskowski, J.M. Thornton, LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions, Protein Eng. 1995, 8(2):127-134.

ACCEPTED MANUSCRIPT Synthesis and biological evaluation of novel H6 analogues as drug resistance reversal agents Xiao Wang1,#, Qian-wen Ren1,#, Xian-xuan Liu1, Yan-ting Yang1,2, Bing-hua Wang1, Rong Zhai1, Jia Grace Qi1, Hong-bo Wang1,*, Yi Bi1,* 1

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

State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and

Peking Union Medical College, Beijing, 100050, China * Correspondence: [email protected] (H.W.), [email protected] (Y. B.) #

These authors contributed equally to this work

Highlights： ： Synthesis of 29 novel H6 analogues.

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2

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

paclitaxel to the KBV cells.

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Compound 10 at the concentration of 5 µM significantly enhanced the cytotoxicity of

Compound 10 might block the drug efflux of P-gp via stimulating P-gp ATPase activity. Compound 10 enhanced the efficacy of paclitaxel against KBV cancer cell-derived xenograft tumors.

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It was the first time to summed up the preliminary structure-activity relationship of

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hederagenin of the reverse activity.