European Journal of Medicinal Chemistry 95 (2015) 240e248

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Original article

Synthesis of heterocycle-modified betulinic acid derivatives as antitumor agents Hai-Wei Cui a, 1, Yuan He b, 1, Jinhua Wang b, Wei Gao a, Ting Liu c, Min Qin b, Xue Wang b, Cheng Gao a, Yan Wang a, Ming-Yao Liu b, Zhengfang Yi b, *, Wen-Wei Qiu a, * a

Department of Chemistry, School of Chemistry and Molecular Engineering, East China Normal University, 500 Dongchuan Road, Shanghai 200241, China Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China c Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China Normal University, North Zhongshan Road, Shanghai 200062, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 February 2015 Received in revised form 11 March 2015 Accepted 20 March 2015 Available online 21 March 2015

A series of novel heterocycle-modified betulinic acid (BA) derivatives were synthesized and investigated for their activity against the growth of eight non-drug resistant and one multidrug-resistant tumor cell line using a sulforhodamine B (SRB) assay. The most active compound 17 showed an average IC50 1.19 mM, which was about 20 times more potent than the lead compound BA. It is amazing that for most synthetic saturated N-heterocycle derivatives, MCF-7/ADR was the most sensitive tumor cells, especially 17 showed the most potent antitumor activity (IC50 ¼ 0.33 mM) on this multidrug-resistant tumor cell line, that was 117 times more potent than BA. Most of the tested compounds displayed less toxic on human fibroblasts (HAF) in comparison with the tumor cell lines. The cytometry and transwell migration assays were used to test the ability of 17 to induce apoptosis and inhibit metastasis on tumor cell lines respectively. © 2015 Published by Elsevier Masson SAS.

Keywords: Triterpenoids Betulinic acid Antitumor Apoptosis Migration

1. Introduction Betulinic acid (BA, Scheme 1) is a type of pentacyclic triterpene acid that can be found in several species of plants, principally the white birch [1]. It has a variety of biological activities, such as antiHIV [2], anti-inflammatory, antimalarial, antimicrobial, and especially antitumor activity [3e5]. BA was initially known for its highly selective antitumor activity against the human melanoma cells [6]. Subsequent studies revealed that this natural product had a broad inhibitory effects in various cancerous tumors, including neuroblastoma, medulloblastoma, Ewing's sarcoma [7], leukemia [8], brain-tumors [9], gliomas [10], colon carcinoma [11], lung, breast, prostate and cervical cancers [12]. The inhibitory mechanisms of BA in various cancerous cells remains to be unraveled detailedly, which have been reported to involve the inhibition of nuclear factor kappa B (NF-kB) [13], topoisomerases I and II [14], and cholesterol

* Corresponding authors. E-mail addresses: [email protected] (Z. Yi), [email protected] (W.-W. Qiu). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ejmech.2015.03.048 0223-5234/© 2015 Published by Elsevier Masson SAS.

acyltransferases (ACAT-1 and ACAT-2) [15], activation of caspases and DNA fragmentation [16], and induction of apoptosis in a CD-95 and p53 independent manners [17,18]. Previous studies suggested that introduction of the nitrogencontaining heterocyclic rings to the pentacyclic triterpenoids can significantly improve the biological activities [19e23]. Thus, in order to search for agents with high antitumor activity and selectivity, a series of BA derivatives were synthesized by introducing nitrogen-containing heterocycles at C-3 position with ester, and especially amide linkages (amides are more stable than esters in metabolism). Their inhibitory activities against the growth of eight non-drug resistant tumor cell lines and one multidrug-resistant breast cancer cell line MCF-7/ADR were evaluated using an SRB assay. BA and its derivatives were screened by flow cytometry to determine their apoptotic behaviors, and their inhibitory effects on tumor cell migration were also tested.

2. Chemistry A series of heterocycle-modified BA derivatives with amide or ester linkage were synthesized according to the pathways

H.-W. Cui et al. / European Journal of Medicinal Chemistry 95 (2015) 240e248

COOH

HO

b

c

R

H2N

O Betulonic acid

Betulinic acid

COOH

COOH

COOH

a

1

N H

2

R=

3

R=

N

O 14 R = O 15 R =

5

H N

6

R=

N

Boc N

O

N

NBoc O

10 R =

R=

NBoc

N

O

H N

8

Boc N

9 R= O

N H

R= O

4 R=

d

7

N N

O COOH R

O

O

O

7, 8, 9, 10, 11, 12, 13

241

NBoc

O

R= Cl

11 R =

N

n O

O 16 R =

12 R =

NH

NBoc

n O

O

13 R =

17 R =

n=1

n=2 NBoc

n

n=3

NH O 18 R =

NH

n

n=1

O 19 R =

NH

n

n=2 NH

O 20 R =

n

n=3

Scheme 1. Synthesis of betulinic acid derivatives 2e6 and 14e20. Reagents and conditions: (a) IBX, DMSO, rt, 6 h, 90%; (b) NaCNBH3, CH3COONH4, CH3OH, rt, 12 h, 82%; (c) CDI, DMAP, CH2Cl2, rt, 6 h, 73e83% for 2e13; (d) boron trifluoride etherate, Et2O, rt, 10 min, 75e86% for 14e20.

described in Schemes 1 and 2. The heterocycle-modified BA derivatives with amide linkage were synthesized as shown in Scheme 1. Oxidation of BA with IBX gave betulonic acid. The important intermediate 1 was obtained

by Borch reduction with sodium cyanoborohydride and ammonium acetate in methanol [24]. Unfortunately, the yields of preparing compounds 2e6 and intermediates 7e13 by condensation of 1 with heterocyclic carboxylic acids under various condensation

COOH

COOH

a R

HO

Betulinic acid

COOH

b R

O

O 21 R =

NBoc

O

O 23 R =

O

NH O

22 R =

24 R = NBoc

NH

Scheme 2. Synthesis of betulinic acid derivatives 23 and 24. Reagents and conditions: (a) DCC, DMAP, CH2Cl2, rt, 4 h, 79% for 21 and 75% for 22; (b) boron trifluoride etherate, Et2O, rt, 10 min, 81% for 23 and 80% for 24.

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agents, such as N,N'-dicyclohexylcarbodiimide (DCC), 1-Ethyl-3(3-dimethylaminopropyl)carbodiimide (EDCI) and N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) were poor. Therefore a model system was employed to find the optimal condensation condition (Table 1). After examining several different conditions, we found the N, N0 -Carbonyldiimidazole (CDI) was the optimal condensation agent and provided compounds 2e6 and intermediates 7e13 with significant improvements in yield (Entry 2 and 9, Table 1 and Scheme 1). Finally, the Boc group was deprotected in the presence of mild boron trifluoride etherate and produced compounds 14e20 successfully. The heterocyclemodified BA derivatives with ester linkage were also synthesized as shown in Scheme 2. Intermediates 21 and 22 were synthesized by condensation of BA with 1-Boc-3-piperidinecarboxylic acid and 1-Boc-4-piperidinecarboxylic acid under DCC respectively. Then compounds 23 and 24 were obtained by deprotection of the Boc group in a manner similar to that of 14e20.

3. Biological evaluation The synthetic BA derivatives 2e6, 14e20, and 23e24 were evaluated for their in vitro antiproliferative activity in an SRB assay against human colon carcinoma cell lines (HCT-116 and HT-29), human prostate cancer cell lines (PC-3 and DU-145), human breast cancer cell lines (MDA-MB-231, MCF-7 and T47D), murine breast cancer cell line (4T1) and multidrug-resistant human breast cancer cell line (MCF-7/ADR). These derivatives showed potent antiproliferative activity against above-mentioned tumor cell lines, except the aromatic heterocyclic compounds 2e6. The compound possessed the most potent antitumor activity on tumor cell lines was selected for testing the apoptotic behavior by flow cytometry assay and the inhibitory effect on tumor cell migration by transwell migration assay.

4. Result and discussion 4.1. Antiproliferative activity To evaluate the anticancer potency of these synthetic heterocycle-modified BA derivatives, the antiproliferative activity of 2e6, 14e20, and 23e24 was first screened in the SRB assay in various tumor cell lines (Table 2). The results showed that aromatic N-heterocycles such as pyrazine-2-carboxamide (2), 5methylpyrazine-2-carboxamide (3), nicotinamide (4), isonicotinamide (5) and 2-chloronicotinamide (6) in position C-3 were not favorable substituted groups to improve antitumor activity, which resulted in a lower inhibition of cell growth compared with lead compound BA. Thus they were excluded from further investigations. Most derivatives bearing a saturated N-heterocycle in position C-3 possessed more potent antiproliferative activity than BA, except the 2-pyrrolidinecarboxamide (14) and 2piperidinecarboxamide (15) substituted compounds. For saturated N-heterocycle substituents, the six-membered piperidinecarboxamide derivatives (16e20 and 23e24) had much more activity than five-membered 2-pyrrolidinecarboxamide compound (14). The position of NH group in piperidinecarboxamide was also important, the sequence of antitumor activity was 4-position (17) > 3-position (16) > 2-position (15). The most potent compound 17 showed the average IC50 1.19 mM, which was 20 folds higher activity than BA. It is amazing that for most saturated Nheterocycle derivatives (16, 17, 20, 23 and 24), the multidrugresistant MCF-7/ADR was the most sensitive tumor cell line. In particular, compound 17 showed the most potent antitumor activity (IC50 ¼ 0.33 mM) on MCF-7/ADR, that was 117 folds more potent than BA (IC50 ¼ 38.5 mM). Perhaps the appropriate distance between A-ring of BA to the NH group of heterocycles was important for antiproliferative activity, compounds 18 (n ¼ 1), 19 (n ¼ 2), and 20 (n ¼ 3) were synthesized based on 17, which had more

Table 1 Optimization of the condensation conditions of compound 1 with heterocyclic carboxylic acids.

COOH

COOH

Heterocyclic carboxylic acids (2.0 eq) Condensation agent (2.0 eq)

H2N

R

Additive (2.0 eq), Solvent, rt

N H O

1 (1.0 eq) 5R= N

(Entry 1 and 2)

O 10 R =

(Entry 3-9) NBoc

Entry

Condensation agent

Additive

Solvent

Yield (%)b

1 2 3 4 5 6 7 8 9

EDCI CDI EDCI EDCI EDCI EDCI DCC EEDQ CDI

DMAP, HOBT DMAP DMAP DMAP DMAP, HOBT DMAP, HOBT DMAP

CH2Cl2 CH2Cl2 CH2Cl2 DMF CH2Cl2 DMF CH2Cl2 CH3CN/tBuOHa CH2Cl2

54 78 43 49 52 57 41 39 83

The bold type was used to emphasize the good results. a The ratio of CH3CN/tBuOH was 4:1 (v: v). b Isolated yield.

DMAP

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243

Table 2 IC50 values of BA and its derivatives against the growth of various cancer cell lines. IC50 (uM)

Compound

BA 2 3 4 5 6 14 15 16 17 18 19 20 23 24 ADR DOC

HCT-116

HT-29

PC-3

DU-145

MDA-MB-231

4T1

T47D

MCF-7

MCF-7/ADR

Average

22.13 >25 >25 >25 >25 >25 17.53 11.42 1.28 1.11 2.67 6.86 5.61 4.60 1.40 e e

17.32 >25 >25 >25 >25 >25 18.77 20.07 1.19 0.97 2.46 2.39 4.38 4.89 1.32 e e

27.51 >25 >25 >25 >25 >25 20.31 >25 1.30 1.11 2.55 3.42 5.23 8.09 2.18 e e

36.16 >25 >25 >25 >25 >25 10.96 7.31 3.16 2.45 3.72 4.39 7.70 11.29 2.69 e e

34.57 >25 >25 >25 >25 >25 12.83 10.59 1.94 1.01 3.17 3.27 3.44 4.97 1.60 e e

14.04 >25 >25 >25 >25 16.13 8.03 7.87 2.18 1.77 5.15 2.99 6.01 7.65 3.74 e e

15.56 >25 >25 >25 >25 17.72 10.97 6.49 2.24 1.40 2.37 2.59 5.26 9.73 3.05 e e

22.61 >25 >25 >25 >25 >25 15.25 4.81 0.94 0.54 3.43 2.90 6.49 4.42 2.41 0.009 0.005

38.50 e e e e e e e 0.39 0.33 3.00 4.42 0.93 1.60 0.59 9.26 5.18

23.74 >25 >25 >25 >25 e 14.33 e 1.62 1.19 3.17 3.69 5.01 6.36 2.11 e e

From SRB assay after 96 h of treatment; the values are averaged from at least 3 independent experiments; variation ± 10%; data not tested or calculated. The bold type was used to emphasize the good results.

4.3. Apoptosis

Table 3 IC50 values of BA and its derivatives against the growth of HAF. Compound

IC50(UM)

BA 16 17 18 19

34.5 3.18 4.45 9.21 6.89

± ± ± ± ±

3.54 0.05 0.41 0.45 0.84

SIa

Compound

IC50(uM)

0.9  2.2 1.0  8.2 1.8  13.5 1.8  3.9 1.0  2.7

20 23 24 ADR

9.15 5.42 5.70 0.016

± ± ± ±

0.13 0.98 0.02 0.006

SIa 1.2  9.8 0.5  3.4 1.5  9.7 e

From SRB assay after 96 h of treatment; the values are averaged from at least 3 independent experiments. a The selectivity indexes (SI) were calculated by IC50 values in HAF divided by IC50 values in cancer cell lines; data not calculated.

distance and afford less potency. The results also revealed that the amide linkage was better than the ester linkage, the average IC50 values (Table 2) of amides (16 and 17) were 1.8e3.9 times more potent than corresponding esters (23 and 24). Considering that amides are more stable than esters in metabolism, these synthetic amides derivatives are more promising candidates for antitumor agents than these esters derivatives. Adriamycin (ADR) and docetaxel (DOC) are selected as positive controls and they are both resisted by MCF-7/ADR significantly [25,26] and the results are consistent with this. 4.2. Selectivity BA and compounds (16e20, 23 and 24) with potent antiproliferative activity were chosen for selectivity test on a human fibroblast (HAF) cell line using the SRB assay. The selectivity indexes (SI) were calculated by IC50 values in HAF divided by IC50 values in cancer cell lines. The results (Table 3) revealed that most of the tested compounds were less toxic on human fibroblasts in comparison with the tumor cells. The most active compound 17 showed 1.8 to 13.5 times more selective towards cancer cells than human fibroblasts respectively. For the multidrug-resistant MCF-7/ADR, compound 17 was the most selective, and the selectivity index was 13.5. However the control compound ADR showed significant cytotoxicity (IC50 ¼ 0.016 mM) on HAF and possessed poor selectivity (0.0017 times) towards MCF-7/ADR cells (IC50 ¼ 9.26 mM) than human fibroblasts.

Compound 17, BA and control compound DOC were investigated their potential to induce apoptosis for 24 h using flow cytometry assay (Fig. 1). In comparison with untreated controls, 17 (5 mM) treatment of MDA-MB-231 cells generated apoptosis in 31.43% of cells (5.56% early apoptosis and 25.87% late apoptosis), which was more potent than 80 mM BA (29.52% apoptosis, contained 1.94% early apoptosis and 27.58% late apoptosis). For MCF-7 and its corresponding MCF-7/ADR cells, BA (80 mM) and DOC (1 mM) showed much more apoptosis to MCF-7 than MCF-7/ADR cells, especially DOC (16.24% apoptosis for MCF-7 and 2.96% apoptosis for MCF-7/ADR) which possessed 5.5 times more selectivity. To our delight, compound 17 almost displayed same apoptosis ability for MCF-7 (20.32% apoptosis) and multidrug-resistant MCF-7/ADR (19.16% apoptosis) cells. 4.4. Migration Migration is an important step during the metastasis of cancer. To determine anti-migration ability of compound 17 and BA, transwell migration assay was performed (Fig. 2). The results revealed that BA inhibited the migration of MDA-MB-231 cancer cells obviously at various concentrations (10, 25 and 50 mM), compared with the control group. Interestingly, the migration of MDA-MB-231 cells was significantly inhibited by compound 17 and almost completely suppressed by this compound at medium concentration (10 mM). In addition, the inhibitory effect on migration of 17 at 2.5 mM was similar to that of BA at 50 mM, it means that 17 was about 20 times more potent than BA on antimetastatic activity of MDA-MB-231 cancer cells. 5. Conclusion Herein we synthesized a series of novel heterocycle-modified BA derivatives with amide or ester linkages at the C-3 position. These derivatives were screened for antiproliferative activity in various cancer cell lines. The aromatic N-heterocycles modified derivatives showed a decreased inhibition of cell growth compared with BA. However, introduction of saturated N-heterocycles was quite promising. Most of the piperidinecarboxamide derivatives showed significantly improved IC50 values, especially 17. The

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Fig. 1. Compound 17, BA and DOC induced apoptosis of MCF-7, MDA-MB-231 and MCF-7/ADR cancer cells in flow cytometry assay compared with control groups. After treatment of various concentrations of 17, BA or DOC for 24 h, cancer cells with the supernatant were collected, centrifuged, and resuspended with PBS. After centrifugation, cells were resuspended with binding buffer and stained with Annexin V-FITC and PI for 0.5 h. Analysis was performed by flow cytometry, and the percentage displayed the apoptosis cells of all cells. Cells in the lower right quadrant indicate Annexin-positive/Annexin-negative early apoptotic cells. Cells in the upper right quadrant indicate Annexin-positive/PI-positive late apoptotic cells. The right quadrants of each diagram (Annexinþ/IP and Annexinþ/IPþ) represent apoptotic cells for each compound treatment.

appropriate distance between A-ring of BA to the NH group of piperidinecarboxylic acids was important for antiproliferative activity, increased the distance only afforded less potency. The amide linkage was better than the ester linkage to improve the antitumor activity. In addition, most of the piperidinecarboxamide derivatives were more sensitive towards the multidrug-resistant MCF-7/ADR than other non-drug resistant tumor cells, especially compound 17. Most of the tested compounds were less toxic on human fibroblasts in comparison with the tumor cell lines. Moreover, as demonstrated by flow cytometry assay, tumor cells death trigged by 17 resulted from apoptotic processes and this compound exhibited potent antimetastatic activity on the tested cancer cell line by transwell migration assay. In conclusion, we report BA heterocyclic derivatives as a series of new chemical entities for the first time. Especially 17, which displayed potent antitumor and antimetastatic activity, could be used as a promising lead for further development.

suppliers and used without further purification unless otherwise stated. When needed, the reactions were carried out in oven-dried glassware. Column chromatography was performed on silica gel (QinDao, 200e300 mesh) using the indicated eluents. Thin-layer chromatography was carried out on silica gel plates (QinDao) with a layer thickness of 0.25 mm. Melting points were determined using the MEL-TEMP 3.0 apparatus and uncorrected. 1H (400 and 500 MHz) and 13C (100 MHz) NMR spectra were recorded on Bruker AM-400 and Bruker AV-500 spectrometer with CDCl3 or DMSO-d6 as solvent and tetramethylsilane (TMS) as the internal standard. All chemical shift values were reported in units of d (ppm). The following abbreviations were used to indicate the peak multiplicity: s ¼ singlet; d ¼ doublet; t ¼ triplet; m ¼ multiplet; br ¼ broad. High-resolution mass data were obtained on a Bruker microOTOFQ II spectrometer.

6. Experimental section

To a solution of betulinic acid (5.0 g, 10.9 mmol) in DMSO (50 mL), IBX (6.2 g, 22.0 mmol) was added. The reaction mixture was stirred for 6 h at room temperature and then ice water (100 mL) and AcOEt (50 mL) were added. The mixture was filtered and the filtrate was extracted with AcOEt (30 mL  3). The

6.1. General All reagents and chemicals were purchased from commercial

6.2. Synthesis of compound 1

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Fig. 2. Compounds 17 and BA inhibit migration of breast cancer MDA-MB-231 in a transwell migration model. 8  104 MDA-MB-231 cells in 200 mL serum-free medium were seeded in the top-chamber, and 700 mL of medium with 10% fetal bovine serum (FBS) was added in the bottom-chambers. Different concentrations of 17 and BA were added in both chambers. The cancer cells were allowed to migrate for 12 h. The stained migrated purple cells in the images were photographed. Migrated cells were quantified by manual counting. The means and error bars representing 95% confidence intervals from three independent experiments are presented. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

combined organic extract was washed with brine (30 mL  2), dried over anhydrous Na2SO4 and concentrated to give betulonic acid (4.5 g, 90%) as a white solid. To a solution of betulonic acid (4.5 g, 9.9 mmol) and CH3COONH4 (7.6 g, 99 mmol)in CH3OH (100 mL), NaCNBH3 (935 mg,14.9 mmol) was added and the reaction mixture was stirred for 12 h at room temperature. The reaction mixture was concentrated, H2O (100 mL) was added. The pH of the mixture was adjusted to 9 with NaOH solution (1 M), and filtered to give crude product. The crude product was purified by silica gel column chromatography (CH2Cl2/MeOH, 10/1, v/v) to give compound 1 (3.7 g, 82%) as a white solid. 6.3. General procedure for the preparation of compounds 213 To a solution of heterocyclic carboxylic acid (0.88 mmol) in dry CH2Cl2 (10 mL), CDI (142 mg, 0.88 mmol) was added. After stirring for 2 h at room temperature, compound 1 (200 mg, 0.44 mmol) and DMAP (108 mg, 0.88 mmol) were added and the reaction mixture was stirred for another 8 h. The reaction mixture was concentrated, H2O (30 mL) was added, and then the aqueous phase was extracted with AcOEt (20 mL  3). The combined organic extract was washed with brine, dried over anhydrous Na2SO4 and concentrated. The residue was purified by silica gel column chromatography (CH2Cl2/ MeOH, 20/1, v/v) to give the desired product. 6.3.1. Compound 2 White solid; yield: 76%; mp: 343e346  C. 1H NMR (400 MHz, CDCl3) d 9.27 (s, 1H), 8.75 (s, 2H), 4.92‒4.83 (m, 1H), 4.75 (s, 1H), 4.62 (s, 1H), 3.05e2.98 (m, 1H), 2.30e2.18 (m, 2H), 2.05e1.94 (m, 2H), 1.87e1.73 (m, 3H), 1.71 (s, 3H), 1.66e1.60 (m, 1H), 1.58e1.48 (m, 2H), 1.48e1.37 (m, 6H), 1.34 (s, 1H), 1.30e1.19 (m, 3H), 1.13e1.06 (m, 2H), 1.01 (s, 3H), 1.00 (s, 3H), 0.96 (s, 3H), 0.94 (s, 3H), 0.91 (s, 3H); 13 C NMR (100 MHz, CDCl3) d 182.14, 162.50, 150.52, 147.09, 144.81,

144.51, 142.59, 109.82, 57.01, 56.48, 56.11, 50.49, 49.30, 47.04, 42.52, 40.73, 39.16, 38.46, 38.19, 37.21, 37.17, 34.28, 32.27, 30.66, 29.78, 28.65, 25.59, 25.52, 20.89, 19.42, 18.63, 16.52, 16.15, 16.13, 14.77. HRMS (ESI): calcd for C35H52N3O3 [M þ H]þ 562.4003; found 562.3998.

6.3.2. Compound 3 White solid; yield: 81%; mp: 339e342  C. 1H NMR (400 MHz, CDCl3) d 9.13 (s, 1H), 8.61 (s, 1H), 4.88e4.82 (m, 1H), 4.75 (s, 1H), 4.62 (s, 1H), 3.04e2.98 (m, 1H), 2.66 (s, 3H), 2.31e2.18 (m, 2H), 2.05e1.95 (m, 2H), 1.85e1.76 (m, 3H), 1.70 (s, 3H), 1.63 (t, J ¼ 11.2 Hz, 1H), 1.54e1.51 (m, 2H), 1.49e1.38 (m, 7H), 1.34 (s, 1H), 1.13e1.04 (m, 2H), 1.00 (s, 6H), 0.96 (s, 3H), 0.92 (s, 3H), 0.91 (s, 3H); 13C NMR (100 MHz, pyridine-d5) d 179.31, 163.59, 163.32, 157.72, 151.75, 144.08, 143.18, 110.41, 66.25, 57.08, 55.50, 54.86, 52.47, 51.19, 50.16, 48.23, 43.28, 41.47, 39.94, 39.02, 38.04, 35.06, 33.29, 31.64, 30.66, 29.32, 26.26, 24.41, 23.59, 22.02, 21.57, 19.90, 19.31, 16.80, 15.98, 15.35. HRMS (ESI): calcd for C36H53N3NaO3 [M þ Na]þ 598.3979; found 598.4012.

6.3.3. Compound 4 White solid; yield: 77%; mp: 331e335  C. 1H NMR (400 MHz, DMSO-d6) d 12.03 (s, 1H), 8.96 (s, 1H), 8.68 (d, J ¼ 4.7 Hz, 1H), 8.15 (d, J ¼ 7.9 Hz, 1H), 8.07 (d, J ¼ 9.3 Hz, 1H), 7.48 (dd, J ¼ 7.6, 5.0 Hz, 1H), 4.70 (s, 1H), 4.59 (s, 1H), 3.79e3.73 (m, 1H), 2.98e2.92 (m, 1H), 2.24 (t, J ¼ 10.9 Hz, 1H), 2.12 (d, J ¼ 8.4 Hz, 1H), 1.87e1.74 (m, 3H), 1.66 (s, 3H), 0.97 (s, 3H), 0.89 (s, 3H), 0.82 (s, 3H), 0.81 (s, 6H); 13C NMR (100 MHz, DMSO-d6) d 177.33, 164.88, 151.60, 150.38, 148.49, 135.23, 130.72, 123.40, 109.71, 56.59, 55.67, 55.52, 49.94, 48.63, 46.72, 42.11, 40.33, 38.51, 37.70, 36.82, 36.45, 33.92, 31.81, 30.20, 29.29, 28.74, 25.18, 24.78, 20.55, 19.02, 18.29, 17.01, 15.95, 15.80, 14.42. HRMS (ESI): calcd for C36H53N2O3 [M þ H]þ 561.4051; found 561.4086.

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6.3.4. Compound 5 White solid; yield: 78%; mp: 348e352  C. 1H NMR (400 MHz, DMSO-d6) d 12.03 (s, 1H), 8.69 (d, J ¼ 4.9 Hz, 2H), 8.13 (d, J ¼ 9.4 Hz, 1H), 7.72 (d, J ¼ 5.0 Hz, 2H), 4.70 (s, 1H), 4.57 (s, 1H), 3.76 (dd, J ¼ 15.2, 6.3 Hz, 1H), 3.17 (s, 1H), 2.98e2.92 (m, 1H), 2.24 (t, J ¼ 10.6 Hz, 1H), 2.13e2.11 (m, 1H), 1.88e1.75 (m, 3H), 1.66 (s, 3H), 1.20e1.08 (m, 2H), 0.97 (s, 3H), 0.89 (s, 3H), 0.82 (s, 3H), 0.80 (s, 6H); 13 C NMR (100 MHz, DMSO-d6) d 177.26, 164.76, 150.33, 150.02, 142.17, 121.51, 109.65, 56.67, 55.58, 55.46, 49.86, 48.57, 46.66, 42.06, 40.28, 38.49, 37.64, 36.76, 36.39, 33.85, 31.75, 30.15, 29.24, 28.68, 25.12, 24.65, 20.49, 18.97, 18.22, 16.95, 15.90, 15.74, 14.37. HRMS (ESI): calcd for C36H53N2O3 [M þ H]þ 561.4051; found 561.4098. 6.3.5. Compound 6 White solid; yield: 68%; mp: 250e252  C. 1H NMR (400 MHz, DMSO-d6) d 12.06 (s, 1H), 8.44 (dd, J ¼ 4.8, 1.8 Hz, 1H), 8.22 (d, J ¼ 9.5 Hz, 1H), 7.82 (dd, J ¼ 7.5, 1.8 Hz, 1H), 7.47 (dd, J ¼ 7.5, 4.8 Hz, 1H), 4.69 (s, 1H), 4.57 (s, 1H), 3.69e3.62 (m, 1H), 2.99e2.91 (m, 1H), 2.23 (t, J ¼ 10.5 Hz, 1H), 2.12 (d, J ¼ 7.1 Hz, 1H), 1.86e1.78 (m, 2H), 1.66 (s, 3H), 1.63e1.60 (m, 2H), 1.56e1.48 (m, 3H), 1.44e1.33 (m, 11H), 1.24 (s, 1H), 1.09 (t, J ¼ 7.0 Hz, 3H), 0.97 (s, 3H), 0.91 (s, 3H), 0.88 (s, 3H), 0.79 (s, 3H), 0.74 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d 177.22, 164.72, 150.30, 146.30, 137.89, 133.94, 128.86, 122.99, 109.61, 56.58, 55.59, 55.40, 49.78, 48.51, 46.59, 42.00, 38.20, 37.58, 36.66, 36.31, 33.77, 31.67, 30.06, 29.18, 28.51, 25.05, 20.43, 18.91, 18.10, 16.72, 15.83, 15.69, 14.33. HRMS (ESI): calcd for C36H51ClN2O3 [MH]- 593.3515; found 593.3510. 6.3.6. Compound 7 White solid; yield: 77%. 1H NMR (400 MHz, CDCl3) d 4.73 (s, 1H), 4.59 (s, 1H), 4.30 (d, J ¼ 5.0 Hz, 1H), 3.63 (dd, J ¼ 9.6, 7.0 Hz, 1H), 3.42 (s, 2H), 3.06e2.94 (m, 1H), 2.32e2.10 (m, 4H), 1.68 (s, 3H), 1.45 (s, 9H), 0.96 (s, 3H), 0.91 (s, 3H), 0.87 (s, 3H), 0.80 (s, 3H), 0.73 (s, 3H). 6.3.7. Compound 8 White solid; yield: 72%. 1H NMR (400 MHz, CDCl3) d 4.73 (s, 2H), 4.60 (s, 1H), 4.21e3.89 (m, 1H), 3.81e3.46 (m, 1H), 3.10e2.90 (m, 1H), 2.76 (d, J ¼ 13.4 Hz, 1H), 2.45e2.08 (m, 4H), 1.68 (s, 3H), 1.46 (s, 9H), 0.97 (s, 3H), 0.92 (s, 3H), 0.85 (s, 3H), 0.79 (s, 3H), 0.70 (s, 3H). 6.3.8. Compound 9 White solid; yield: 75%. 1H NMR (500 MHz, CDCl3) d 4.72 (s, 1H), 4.59 (s, 1H), 4.18e4.97 (m, 3H), 3.65 (s, 1H), 2.99e2.78 (m, 3H), 2.51e2.43 (m, 1H), 2.30e2.16 (m, 2H), 1.68 (s, 3H), 1.45 (s, 9H), 0.96 (s, 3H), 0.92 (s, 3H), 0.83 (s, 3H), 0.80 (s, 3H), 0.73 (s, 3H). 6.3.9. Compound 10 White solid; yield: 83%. 1H NMR (400 MHz, CDCl3) d 4.73 (s, 1H), 4.60 (s, 1H), 4.21e4.06 (m, 3H), 3.65 (s, 1H), 2.99 (d, J ¼ 10.2 Hz, 1H), 2.76 (d, J ¼ 11.6 Hz, 2H), 2.35e2.14 (m, 4H), 1.68 (s, 3H), 1.44 (s, 9H), 0.97 (s, 3H), 0.92 (s, 3H), 0.83 (s, 3H), 0.80 (s, 3H), 0.73 (s, 3H).

6.3.12. Compound 13 White solid; yield: 74%. 1H NMR (500 MHz, CDCl3) d 4.72 (s, 1H), 4.61 (s, 1H), 4.05 (d, J ¼ 12.2 Hz, 2H), 3.65 (t, J ¼ 10.0 Hz, 1H), 3.00 (t, J ¼ 10.2 Hz, 1H), 2.65 (t, J ¼ 11.9 Hz, 2H), 2.33e2.10 (m, 5H), 2.07e1.86 (m, 3H), 1.68 (s, 3H), 1.44 (s, 9H), 0.96 (s, 6H), 0.85 (s, 3H), 0.79 (s, 3H), 0.73 (s, 3H). 6.4. General procedure for the preparation of compounds 1420 One of compounds 7e13 (0.4 mmol) was dissolved in anhydrous diethyl ether (20 mL) and boron trifluoride etherate (5 mL) was added. The reaction mixture was stirred for 2 h at room temperature and then quenched with saturated aqueous NaHCO3 (20 mL). The mixture was extracted with AcOEt (20 mL  3). The combined organic extract was washed with brine, dried over anhydrous Na2SO4 and concentrated. The residue was purified by silica gel chromatography (CH2Cl2/MeOH, 10/1, v/v) to give the desired product. 6.4.1. Compound 14 White solid; yield: 75%; mp: 349e352  C. 1H NMR (400 MHz, DMSO-d6) d 12.05 (s, 1H), 8.06 (d, J ¼ 9.4 Hz, 1H), 4.67 (d, J ¼ 16.9 Hz, 1H), 4.56 (s, 1H), 4.13 (t, J ¼ 7.6 Hz, 1H), 3.58e3.47 (m, 1H), 3.29e3.16 (m, 2H), 2.98e2.91 (m, 1H), 2.35e2.20 (m, 2H), 2.12 (d, J ¼ 9.0 Hz, 1H), 1.94e1.73 (m, 5H), 1.65 (s, 3H), 1.17 (t, J ¼ 7.1 Hz, 3H), 0.95 (s, 3H), 0.88 (s, 3H), 0.79 (s, 3H), 0.73 (s, 3H), 0.72 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d 177.26, 167.55, 150.34, 109.66, 59.78, 56.48, 55.46, 55.31, 49.81, 48.57, 46.65, 45.73, 42.07, 40.26, 38.19, 37.62, 36.69, 36.40, 33.79, 31.75, 30.16, 30.08, 29.25, 28.38, 27.42, 25.10, 24.80, 23.56, 20.78, 18.99, 18.18, 16.63, 15.91, 15.73, 14.35. HRMS (ESI): calcd for C35H57N2O3 [M þ H]þ 553.4364; found 553.4378. 6.4.2. Compound 15 White solid; yield: 82%; mp: 338e341  C. 1H NMR (400 MHz, DMSO-d6) d 8.01 (d, J ¼ 9.5 Hz, 1H), 4.69 (s, 1H), 4.56 (s, 1H), 3.71e3.62 (m, 1H), 3.59e3.49 (m, 1H), 3.20 (d, J ¼ 12.3 Hz, 1H), 2.99e2.86 (m, 2H), 2.22 (dd, J ¼ 16.8, 7.1 Hz, 1H), 2.09 (dd, J ¼ 25.0, 9.8 Hz, 2H), 1.65 (s, 3H), 0.95 (s, 3H), 0.87 (s, 3H), 0.79 (s, 3H), 0.73 (s, 3H), 0.71 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d 177.31, 168.16, 150.36, 109.70, 65.02, 59.83, 57.18, 56.06, 55.49, 49.84, 48.59, 46.68, 43.30, 42.08, 40.28, 38.22, 37.63, 36.72, 33.82, 31.78, 30.18, 29.28, 28.29, 27.79, 25.13, 24.84, 21.83, 21.34, 20.50, 19.02, 18.23, 16.67, 15.95, 15.75, 15.24, 14.36. HRMS (ESI): calcd for C36H59N2O3 [M þ H]þ 567.4520; found 567.4524.

6.3.10. Compound 11 White solid; yield: 82%. 1H NMR (500 MHz, CDCl3) d 4.72 (s, 1H), 4.60 (s, 1H), 4.06 (d, J ¼ 12.2 Hz, 2H), 3.71e3.59 (m, 1H), 3.00 (t, J ¼ 9.8 Hz, 1H), 2.69 (t, J ¼ 11.6 Hz, 2H), 2.30e2.16 (m, 2H), 1.68 (s, 3H), 1.43 (s, 9H), 0.96 (s, 3H), 0.95 (s, 3H), 0.84 (s, 3H), 0.79 (s, 3H), 0.73 (s, 3H).

6.4.3. Compound 16 White solid; yield: 78%; mp: 334e336  C. 1H NMR (400 MHz, DMSO-d6) d 7.61e7.50 (m, 1H), 4.67 (s, 1H), 4.54 (s, 1H), 3.46e3.40 (m, 1H), 3.03e2.94 (m, 1H), 2.86 (dd, J ¼ 24.6, 11.7 Hz, 2H), 2.38e2.24 (m, 2H), 2.12 (d, J ¼ 11.6 Hz, 1H), 1.64 (s, 3H), 0.93 (s, 3H), 0.86 (s, 3H), 0.77 (s, 3H), 0.71 (s, 3H), 0.69 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d 177.30, 172.66, 150.38, 109.69, 55.65, 55.54, 55.50, 49.93, 48.62, 46.69, 42.82, 42.75, 42.08, 40.30, 38.02, 37.67, 36.76, 36.43, 36.00, 33.89, 31.79, 30.19, 29.28, 28.45, 25.88, 25.16, 25.04, 23.13, 20.50, 19.01, 18.25, 16.73, 15.94, 15.84, 15.77, 14.41. HRMS (ESI): calcd for C36H59N2O3 [M þ H]þ 567.4520; found 567.4524.

6.3.11. Compound 12 White solid; yield: 78%. 1H NMR (500 MHz, CDCl3) d 4.73 (s, 1H), 4.61 (s, 1H), 4.07 (d, J ¼ 12.9 Hz, 2H), 3.65 (t, J ¼ 8.8 Hz, 1H), 3.08e2.92 (m, 1H), 2.65 (t, J ¼ 12.3 Hz, 2H), 2.32e2.10 (m, 4H), 1.69 (s, 3H), 1.44 (s, 9H), 0.97 (s, 3H), 0.96 (s, 3H), 0.85 (s, 3H), 0.80 (s, 3H), 0.74 (s, 3H).

6.4.4. Compound 17 White solid; yield: 83%; mp: 352e355  C. 1H NMR (400 MHz, DMSO-d6) d 7.43 (d, J ¼ 9.4 Hz, 1H), 4.65 (s, 1H), 4.53 (s, 1H), 3.49 (s, 1H), 3.23 (d, J ¼ 10.7 Hz, 1H), 3.08 (d, J ¼ 9.7 Hz, 2H), 2.59 (d, J ¼ 11.6 Hz, 1H), 2.42 (d, J ¼ 13.2 Hz, 2H), 2.24 (s, 1H), 2.14 (d, J ¼ 10.7 Hz, 1H), 2.09e2.01 (m, 1H), 1.63 (s, 3H), 0.93 (s, 3H), 0.86 (s,

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3H), 0.77 (s, 3H), 0.72 (s, 3H), 0.72 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d 177.27, 168.71, 150.35, 109.66, 55.72, 55.47, 49.93, 48.59, 46.66, 42.05, 40.27, 37.80, 37.65, 36.73, 36.40, 33.88, 31.76, 30.16, 29.24, 28.41, 25.12, 22.83, 20.48, 18.98, 18.24, 16.68, 15.94, 15.75, 14.37. HRMS (ESI): calcd for C36H59N2O3 [M þ H]þ 567.4520; found 567.4565. 6.4.5. Compound 18 White solid; yield: 81%; mp: 331e333  C. 1H NMR (400 MHz, DMSO-d6) d 7.41 (d, J ¼ 9.0 Hz, 1H), 4.66 (s, 1H), 4.54 (s, 1H), 3.49e3.44 (m, 1H), 3.03 (d, J ¼ 10.3 Hz, 3H), 2.14 (d, J ¼ 8.0 Hz, 2H), 2.03 (d, J ¼ 6.5 Hz, 1H), 1.64 (s, 3H), 0.94 (s, 3H), 0.87 (s, 3H), 0.78 (s, 3H), 0.73 (s, 3H), 0.69 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d 177.31, 171.58, 150.38, 109.70, 55.71, 55.49, 49.92, 48.60, 46.68, 43.47, 42.07, 40.29, 37.91, 37.66, 36.75, 36.41, 35.63, 35.13, 33.88, 32.75, 31.77, 30.17, 29.27, 28.50, 25.16, 22.56, 20.49, 19.01, 18.26, 16.78, 15.95, 15.77, 14.40. HRMS (ESI): calcd for C37H59N2O3 [MH]579.4531; found 579.4525. 6.4.6. Compound 19 White solid; yield: 86%; mp: 323e326  C. 1H NMR (400 MHz, DMSO-d6) d 7.42 (d, J ¼ 9.4 Hz, 1H), 4.64 (s, 1H), 4.52 (s, 1H), 3.48 (s, 1H), 3.22 (d, J ¼ 10.7 Hz, 1H), 3.07 (d, J ¼ 9.7 Hz, 2H), 2.58 (d, J ¼ 11.6 Hz, 1H), 2.41 (d, J ¼ 13.2 Hz, 2H), 2.23 (s, 1H), 2.13 (d, J ¼ 10.7 Hz, 1H), 2.09e2.01 (m, 1H), 1.62 (s, 3H), 0.92 (s, 3H), 0.85 (s, 3H), 0.76 (s, 3H), 0.71 (s, 3H), 0.71 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d 177.23, 171.54, 150.26, 109.60, 55.59, 55.49, 55.38, 54.81, 49.81, 48.48, 46.57, 43.25, 41.95, 40.17, 37.77, 37.55, 36.63, 36.30, 33.76, 32.63, 31.68, 30.05, 29.15, 28.38, 28.23, 28.14, 25.03, 20.70, 20.37, 18.88, 18.13, 16.65, 15.83, 15.65, 14.28. HRMS (ESI): calcd for C38H63N2O3 [M þ H]þ 595.4833; found 595.4867. 6.4.7. Compound 20 White solid; yield: 77%; mp: 318e322  C. 1H NMR (400 MHz, DMSO-d6) d 7.40 (d, J ¼ 9.0 Hz, 1H), 4.65 (s, 1H), 4.53 (s, 1H), 3.43 (d, J ¼ 19.6 Hz, 1H), 3.02 (d, J ¼ 10.3 Hz, 3H), 2.20e2.07 (m, 2H), 2.06e1.96 (m, 1H), 1.63 (s, 3H), 0.93 (s, 3H), 0.86 (s, 3H), 0.77 (s, 3H), 0.71 s, 3H), 0.68 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d 177.36, 171.61, 150.40, 109.76, 55.71, 55.51, 49.92, 48.59, 46.71, 43.48, 42.09, 40.30, 37.94, 37.67, 36.76, 36.43, 35.64, 35.17, 33.88, 32.78, 31.78, 30.17, 29.29, 28.53, 28.50, 25.23, 25.16, 22.60, 20.50, 19.02, 18.28, 16.81, 15.98, 15.79, 14.42. HRMS (ESI): calcd for C39H65N2O3 [M þ H]þ 609.4990; found 609.4947. 6.5. General procedure for the preparation of compounds 21and 22 To a solution of N-Boc-piperidine carboxylic acid (1.32 mmol) in dry CH2Cl2 (10 mL), betulinic acid (300 mg, 0.66 mmol), DCC (272 mg, 1.32 mmol) and DMAP (161 mg, 1.32 mmol) were added. The reaction mixture was stirred for 4 h at room temperature, and then concentrated. H2O (30 mL) was added to the residue, and then the aqueous phase was extracted with AcOEt (30 mL  3). The combined organic extract was washed with brine, dried over anhydrous Na2SO4 and concentrated. The residue was purified by silica gel column chromatography (CH2Cl2/MeOH, 10/1, v/v) to give the desired product. 6.5.1. Compound 21 White solid; yield: 79%. 1H NMR (400 MHz, CDCl3) d 4.74 (s, 1H), 4.61 (s, 1H), 4.51e4.44 (m, 1H), 4.23e4.14 (m, 1H), 3.93 (d, J ¼ 12.8 Hz, 1H), 3.02e2.89 (m, 2H), 2.77 (t, J ¼ 12.4 Hz, 1H), 2.43 (t, J ¼ 10.7 Hz, 1H), 2.27 (d, J ¼ 12.6 Hz, 1H), 2.23e2.16 (m, 1H), 1.97 (dd, J ¼ 13.3, 5.5 Hz, 2H), 1.69 (s, 3H), 1.63e1.57 (m, 4H), 1.45 (s, 9H), 1.30e1.25 (m, 2H), 1.19 (d, J ¼ 13.3 Hz, 1H), 0.97 (s, 3H), 0.94 (s, 3H), 0.85 (s, 6H), 0.83 (s, 3H).

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6.5.2. Compound 22 White solid; yield: 75%. 1H NMR (400 MHz, CDCl3) d 4.74 (s, 1H), 4.61 (s, 1H), 4.52e4.45 (m, 1H), 3.99 (d, J ¼ 13.3 Hz, 2H), 3.05e2.96 (m, 1H), 2.85 (td, J ¼ 11.3, 3.6 Hz, 2H), 2.43 (td, J ¼ 10.7, 5.4 Hz, 1H), 2.31e2.14 (m, 2H), 2.03e1.92 (m, 2H), 1.87 (t, J ¼ 10.7 Hz, 2H), 1.69 (s, 3H), 1.64 (s, 2H), 1.63e1.56 (m, 4H), 1.45 (s, 9H), 1.38 (s, 3H), 1.30e1.25 (m, 2H), 1.18 (d, J ¼ 13.4 Hz, 1H), 0.97 (s, 3H), 0.94 (s, 3H), 0.85 (s, 3H), 0.83 (s, 6H). 6.6. General procedure for the preparation of compounds 23 and 24 By a similar procedure described for 1420, 23 and 24 was obtained. 6.6.1. Compound 23 White solid; yield: 81%; mp: 319e321  C. 1H NMR (400 MHz, DMSO-d6) d 4.69 (s, 1H), 4.56 (s, 1H), 4.41 (dd, J ¼ 11.0, 4.5 Hz, 1H), 3.25 (d, J ¼ 12.6 Hz, 2H), 2.94 (d, J ¼ 8.8 Hz, 3H), 2.68 (s, 1H), 2.23 (s, 1H), 2.13 (s, 1H), 1.99 (t, J ¼ 13.1 Hz, 2H), 1.80 (d, J ¼ 6.8 Hz, 1H), 1.65 (s, 3H), 0.95 (s, 3H), 0.88 (s, 3H), 0.81 (s, 6H), 0.79 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d 177.17, 172.63, 150.27, 109.60, 80.26, 55.37, 54.53, 49.64, 48.51, 46.58, 42.43, 42.39, 42.01, 40.22, 38.00, 37.63, 37.54, 37.49, 36.61, 36.30, 33.69, 31.66, 30.07, 29.18, 27.63, 25.01, 24.75, 24.52, 23.25, 20.45, 18.91, 17.68, 16.42, 15.83, 15.65, 14.33. HRMS (ESI): calcd for C36H56NO4 [MH]- 566.4215; found 566.4195. 6.6.2. Compound 24 White solid; yield: 80%; mp: 313e317  C. 1H NMR (400 MHz, DMSO-d6) d 4.68 (s, 1H), 4.55 (s, 1H), 4.40 (dd, J ¼ 11.0, 4.5 Hz, 1H), 3.24 (d, J ¼ 12.6 Hz, 2H), 2.93 (d, J ¼ 8.8 Hz, 3H), 2.67 (t, J ¼ 11.0 Hz, 1H), 1.79 (d, J ¼ 6.8 Hz, 1H), 1.64 (s, 3H), 0.94 (s, 3H), 0.87 (s, 3H), 0.80 (s, 6H), 0.78 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d 177.30, 171.41, 150.38, 109.68, 80.65, 55.47, 54.93, 54.58, 52.87, 49.82, 49.69, 48.59, 46.67, 44.92, 43.97, 43.04, 42.09, 40.30, 38.52, 37.59, 36.68, 36.39, 31.78, 31.57, 30.15, 29.26, 27.73, 25.38, 25.12, 24.50, 23.34, 21.41, 20.52, 18.99, 17.75, 16.51, 15.89, 15.74, 14.41. HRMS (ESI): calcd for C36H58NO4 [M þ H]þ 568.4366; found 568.4413. 6.7. Biological assay 6.7.1. SRB assay The cell viability was determined by SRB (Sigma Aldrich, St. Louis, MO, USA) assay [27]. Stock solutions (20 mM) of the test compounds were dissolved in DMSO. After seeded into 96-well plates at an appropriate cell density for 24 h, cells were treated with different concentrations of compounds and the same amount of DMSO (as control). After incubation for 96 h, the cells were fixed with 25 mL 50% trichloroacetic acid (TCA) for 2 h at 4  C. The plates were washed with deionized water for five times and allowed to dry using hair dryer. The cells were stained with 50 mL of 0.4% (W/V) SRB solution for 10 min then washed with 100 mL 1% acetic acid for five times to remove the unbound dye. After dying, the bound dye was dissolved with 100 mL of 10 mM Tris base solution for determination of optical densities at the wavelength of 515 nm in a SpectraMAX 190 Microplate Reader. The IC50 was calculated using GraphPad software (San Diego, CA, USA). 6.7.2. Flow cytometry assay Apoptosis cells of three human breast cancer cell lines MDAMB-231, MCF-7 and MCF-7/ADR with multidrug resistance induced by BA, 17 or Docetaxel were monitored with Annexin VFITC Apoptosis Detection Kit I (BD Bioscience, San Diego, CA, USA) [28]. Briefly, cells were seeded into 6-well plates at an appropriate cell density. After incubation for 24 h, cells were treated different

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concentrations of compounds for 24 h. Then the cells were collected, centrifuged, and resuspended with cold PBS. After centrifugation, cells were resuspended with 100 mL binding buffer and incubated with 5 mL Annexin V and 5 mL PI for 30 min at room temperature in dark condition. Then 400 mL binding buffer was added and cells were analyzed immediately by flow cytometry (FACS Calibur, BD biosciences). 6.7.3. Transwell migration assay The inhibitory effect on migration of human breast cancer cell line MDA-MB-231 was assessed by transwell migration assay [29]. Briefly, 8  104 MDA-MB-231 cells in 200 mL serum-free medium were added in the upper chamber, and 700 mL of medium with 10% FBS was added at the bottom. Different concentrations of BA and 17 were added in both chambers. Cells were allowed to migrate for 12 h. Non-migrated cells on the upper surface of the membrane were gently scraped away with a cotton swab. The migrated cells were fixed in 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for 5 min. Migrated cells in 5 randomly selected fields were counted under an inverted microscope (Olympus). The percentage of migrated cells inhibited by compounds was normalized to untreated control cell migration.

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

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Acknowledgment [19]

This work was supported by Shanghai Science and Technology Council (Grant 12ZR1408500) and National Natural Science Foundation of China (81272463, 81472788).

[20]

Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2015.03.048.

[21]

[22]

References [1] M.M. O'Connel, M.D. Bently, C.S. Cambell, Betulin and lupeol in bark from four white-barked birches, Phytochemistry 27 (1998) 2175e2176. [2] F. Hashimoto, Y. Kashiwada, L.M. Cosentino, C.H. Chen, P.E. Garrett, K.H. Lee, Anti-AIDS agentseXXVII. Synthesis and anti-HIV activity of betulinic acid and dihydrobetulinic acid derivatives, Bioorg. Med. Chem. 15 (1997) 2133e2143. [3] P. Yogeeswari, D. Sriram, Betulinic acid and its derivatives: a review on their biological properties, Curr. Med. Chem. 12 (2005) 657e666. [4] D. Emmerich, K. Vanchanagiri, L.C. Baratto, H. Schmidt, R. Paschke, Synthesis and studies of anticancer properties of lupane-type triterpenoid derivatives containing a cisplatin fragment, Eur. J. Med. Chem. 75 (2014) 460e466. [5] R. Csuk, Betulinic acid and its derivatives: a patent review (2008e2013), Expert Opin. Ther. Pat. 24 (2014) 913e923. [6] E. Pisha, H. Chai, I.S. Lee, T.E. Chagwedera, N.R. Farnsworth, G.A. Cordell, C.W.W. Beecher, H.H.S. Fong, A.D. Kinghorn, D.M. Brown, M.C. Wani, M.E. Wall, T.J. Hieken, T.K. Dasgupta, J.M. Pezzuto, Discovery of betulinic acid as a selective inhibitor of human-melanoma that functions by induction of apoptosis, Nat. Med. 1 (1995) 1046e1051. [7] S. Fulda, I. Jeremias, T. Pietsch, K.M. Debatin, Betulinic acid: a new chemotherapeutic agent in the treatment of neuroectodermal tumors, Klin. Padiatr 211 (1999) 319e322. [8] H. Ehrhardt, S. Fulda, M. Führer, K.M. Debatin, I. Jeremias, Betulinic acidinduced apoptosis in leukemia cells, Leukemia 18 (2004) 1406e1412. [9] S. Fulda, I. Jeremias, H.H. Steiner, T. Pietsch, K.M. Debatin, Betulinic acid: a new

[23]

[24] [25] [26]

[27]

[28]

[29]

cytotoxic agent against malignant brain-tumor cells, Int. J. Cancer 82 (1999) 435e441. W. Wick, C. Grimmel, B. Wagenknecht, J. Dichgans, M. Weller, Betulinic acidinduced apoptosis in glioma cells: a sequential requirement for new protein synthesis, formation of reactive oxygen species, and caspase processing, J. Pharmacol. Exp. Ther. 289 (1999) 1306e1312. S. Chintharlapalli, S. Papineni, P. Lei, S. Pathi, S. Safe, Betulinic acid inhibits colon cancer cell and tumor growth and induces proteasome-dependent and -independent downregulation of specificity proteins (Sp) transcription factors, BMC Cancer 11 (2011) 371. J.H. Kessler, F.B. Mullauer, G.M. de Roo, J.P. Medema, Broad in vitro efficacy of plant-derived betulinic acid against cell lines derived from the most prevalent human cancer types, Cancer Lett. 251 (2007) 132e145. T. Rabi, S. Shukla, S. Gupta, Betulinic acid suppresses constitutive and TNFalpha-induced NF-kappaB activation and induces apoptosis in human prostate carcinoma PC-3 cells, Mol. Carcinog. 47 (2008) 964e973. T. Syrovets, B. Büchele, E. Gedig, J.R. Slupsky, T. Simmet, Acetyl-boswellic acids are novel catalytic inhibitors of human topoisomerases I and IIalpha, Mol, Pharmacol 58 (2000) 71e81. W.S. Lee, K.R. Im, Y.D. Park, N.D. Sung, T.S. Jeong, Human ACAT-1 and ACAT-2 inhibitory activities of pentacyclic triterpenes from the leaves of lycopus lucidus TURCZ, Biol. Pharm. Bull. 29 (2006) 382e384. S. Fulda, C. Scaffidi, S.A. Susin, P.H. Krammer, G. Kroemer, M.E. Peter, K.M. Debatin, Activation of mitochondria and release of mitochondrial apoptogenic factors by betulinic acid, J. Biol. Chem. 273 (1998) 33942e33948. M. Raisova, A.M. Hossini, J. Eberle, C. Riebeling, T. Wieder, I. Sturm, P.T. Daniel, C.E. Orfanos, C.C. Geilen, The Bax/Bcl-2 ratio determines the susceptibility of human melanoma cells to CD95/Fas-mediated apoptosis,, J. Invest. Dermatol 117 (2001) 333e340. V. Zuco, R. Supino, S.C. Righetti, L. Cleris, E. Marchesi, C. Gambacorti-Passerini, F. Formelli, Selective cytotoxicity of betulinic acid on tumor cell lines, but not on normal cells, Cancer Lett. 175 (2002) 17e25. W.W. Qiu, Q. Shen, F. Yang, B. Wang, H. Zou, J.Y. Li, J. Li, J. Tang, Synthesis and biological evaluation of heterocyclic ring-substituted maslinic acid derivatives as novel inhibitors of protein tyrosine phosphatase 1B, Bioorg. Med. Chem. Lett. 19 (2009) 6618e6622. J. Xu, Z. Li, J. Luo, F. Yang, T. Liu, M. Liu, W.W. Qiu, J. Tang, Synthesis and biological evaluation of heterocyclic ring-fused betulinic acid derivatives as novel inhibitors of osteoclast differentiation and bone resorption, J. Med. Chem. 55 (2012) 3122e3134. M. Urban, M. Vlk, P. Dzubak, M. Hajduch, J. Sarek, Cytotoxic heterocyclic triterpenoids derived from betulin and betulinic acid, Bioorg. Med. Chem. 20 (2012) 3666e3674. T.A. Dang Thi, N.T. Kim Tuyet, C. Pham The, H. Thanh Nguyen, C. Ba Thi, T. Doan Duy, M. D'hooghe, T. Van Nguyen, Synthesis and cytotoxic evaluation of novel ester-triazole-linked triterpenoid-AZT conjugates, Bioorg. Med. Chem. Lett. 24 (2014) 5190e5194. C. Gao, F.J. Dai, H.W. Cui, S.H. Peng, Y. He, X. Wang, Z.F. Yi, W.W. Qiu, Synthesis of novel heterocyclic ring-fused 18b-glycyrrhetinic acid derivatives with antitumor and antimetastatic activity, Chem. Biol. Drug. Des. 84 (2014) 223e233. R.D. Goff, J.S. Thorson, Enhancing the divergent activities of betulinic acid via neoglycosylation, Org. Lett. 11 (2009) 461e464. Y. Mi, L. Lou, ZD6474 reverses multidrug resistance by directly inhibiting the function of P-glycoprotein, Br. J. Cancer 97 (2007) 934e940. K. Honma, K. Iwao-Koizumi, F. Takeshita, Y. Yamamoto, T. Yoshida, K. Nishio, S. Nagahara, K. Kato, T. Ochiya, RPN2 gene confers docetaxel resistance in breast cancer, Nat. Med. 14 (2008) 939e948. €hl, Synthesis and biological activity of R. Csuk, S. Schwarz, R. Kluge, D. Stro some antitumor active derivatives from glycyrrhetinic acid, Eur. J. Med. Chem. 45 (2010) 5718e5723. C. Fang, J. Zhang, D. Qi, X. Fan, J. Luo, L. Liu, Q. Tan, Evodiamine induces G2/M arrest and apoptosis via mitochondrial and endoplasmic reticulum pathways in H446 and H1688 human small-cell lung cancer cells, PLoS One 9 (2014) e115204. J. Wu, L. Yu, F. Yang, J. Li, P. Wang, W. Zhou, L. Qin, Y. Li, J. Luo, Z. Yi, M. Liu, Y. Chen, Optimization of 2-(3-(arylalkyl amino carbonyl) phenyl)-3-(2methoxyphenyl)-4-thiazolidinone derivatives as potent antitumor growth and metastasis agents, Eur. J. Med. Chem. 80 (2014) 340e351.

Synthesis of heterocycle-modified betulinic acid derivatives as antitumor agents.

A series of novel heterocycle-modified betulinic acid (BA) derivatives were synthesized and investigated for their activity against the growth of eigh...
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