Accepted Manuscript Synthesis and biological evaluation of betulonic acid derivatives as antitumor agents Sheng-Jie Yang, Ming-Chuan Liu, Qi Zhao, De-Yu Hu, Wei Xue, Song Yang PII:

S0223-5234(15)00255-X

DOI:

10.1016/j.ejmech.2015.04.006

Reference:

EJMECH 7821

To appear in:

European Journal of Medicinal Chemistry

Received Date: 29 August 2014 Revised Date:

1 April 2015

Accepted Date: 1 April 2015

Please cite this article as: S.-J. Yang, M.-C. Liu, Q. Zhao, D.-Y. Hu, W. Xue, S. Yang, Synthesis and biological evaluation of betulonic acid derivatives as antitumor agents, European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.04.006. 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 Graphical Abstract Twenty-five betulonic acid derivatives were designed and synthesized and found to possess good antiproliferative activities against five cell lines, among which compound 3k was found to induce

AC C

EP

TE D

M AN U

SC

RI PT

cell apoptosis on MGC-803 cells through the mitochondrial intrinsic pathway.

ACCEPTED MANUSCRIPT

Synthesis and biological evaluation of betulonic acid derivatives as antitumor agents Sheng-Jie Yanga,b,c¶, Ming-Chuan Liua,b¶, Qi Zhaoa, De-Yu Hua, Wei Xuea, and Song Yanga*

a

RI PT

State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key

Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University, Guiyang 550025, P.R. China

Research Institute, Sinphar Tian-Li Pharmaceutical Co., Ltd, Hangzhou, 311100, P.R. China

c

Research Institute of Traditional Chinese Medicine, Yangtze River Pharmaceutical Group Co.,

SC

b

¶

M AN U

Ltd, Taizhou, 225321, P.R. China

Both authors contributed equally to this work.

*Corresponding author. Ctr for R&D of Fine Chemicals, Guizhou University, Huaxi St., Guiyang, China 550025. Tel.: +86 851 829 2171; Fax: +86 851 829 2170.

TE D

E-mail address: [email protected]

Conflicts of interest: None.

EP

Abbreviation list: ADM: adriamycin

AC C

AO/EB: acridine orange/ethidium bromide BetA: betulonic acid 13

C NMR: 13C Nuclear Magnetic Resonance

DMF: N, N-dimethylformamide DMSO: dimethyl sulfoxide EDCI: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride HCPT: 10-hydroxyl camptothecine HOBt: 1-hydroxybenzotriazole

1

H NMR: Proton Nuclear Magnetic Resonance 1

ACCEPTED MANUSCRIPT IR: Infra-red MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

RI PT

TUNEL: terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling

Abstract: Structural modification was performed at the C-28 position of betulonic acid (BetA). Twenty-five BetA derivatives were synthesized, and evaluated for their antitumor activities against MGC-803, PC3, Bcap-37, A375, and MCF-7 human cancer cell lines by MTT assay. Among the

SC

derivatives, most of the derivatives had significant antiproliferative ability (IC50 < 19 µM). Compound 3k, the most active compound, showed IC50 values of 3.6, 5.6, 4.2, 7.8, and 5.2 µM on

M AN U

the five cancer cell lines respectively, and was selected to investigate cell apoptosis by subsequent florescence staining and flow cytometry analysis. The results revealed that compound 3k could induce apoptosis in MGC-803 cell lines, and the apoptosis ratios reached 28.33% after 36 h of treatment at 10 µM. In addition, the study of cancer cell apoptotic signaling pathway indicated that the apoptosis of MGC-803 cells induced by compound 3k could be through the mitochondrial

TE D

intrinsic pathway.

AC C

EP

Keywords: betulonic acid derivatives; synthesis; antitumor; apoptosis; mitochondrial pathway

2

ACCEPTED MANUSCRIPT 1. Introduction Cancer is the major cause of human deaths worldwide because of its high incidence and mortality [1,2]. In order to improve the survival and quality of life of cancer patients, the need of new therapies or therapeutic combinations is very urgent all over the world [3]. Natural products have

RI PT

played an important role in the antitumor drug development [4,5], and about 80% of clinically antitumor agents originate from natural products [6-9]. In recent years, interest in synthetic transformations of natural products for the purpose of preclinical development is a major objective of antitumor research programs [10]. Pentacylic triterpenes are one of the most abundant natural

SC

compounds found in plant kingdom [11-13].

Betulonic acid (BetA, 3-oxo-20(29)-lupen-28-oic acid), is the one of the major effective

M AN U

components of many traditional Chinese medicine [14], and it can be derived rather easily from betulin by oxidation with chromium trioxide [15], and betulin forms up to 30% of the dry weight of the extractive (the bark of birch trees) [16]. BetA and its derivatives have been found to possess several medicinal properties, such as anti-inflammatory [17,18], anti-melanoma [19] and anti-viral [20,21] activity. Furthermore, BetA was also found to be as an antitumor agent. Some studies have

TE D

shown that BetA could inhibit the growth of various types of human tumor cell lines, including SGC-7901, HepG-2, LNCaP, DU-145 and PC3 tumor cell lines [22,23]. The antitumor activity of BetA has attracted the attention of the pioneers who aim to develop novel antitumor agents. The

EP

structure-activity relationships (SAR) of other pentacyclic triterpene acids such as oleanolic acid (OA), ursolic acids (UA), and GA (glycyrrhetinic acid) had been established [24-26]. Interestingly,

AC C

modification of C-28 carboxylic acid group of UA into amides and amines analogous led to a rise in activity [27]. The amides of BetA with esters of amino acids and with aliphatic and heterocyclic amines were synthesized, and were revealed valuable biological activity [20,28]. BetA and other pentacyclic triterpenes shared similar observations of SAR [29]. In the previous work, our group have synthesized novel nitrogen-containing betulinic acid derivatives. And the pharmacological evaluations revealed the target compounds had potent antitumor activity compared with betulinic acid in addition to improved aqueous solubility [30]. In view of the mentioned facts above and an attempt to discover more potent and selective antitumor agents based on BetA scaffold, a series of new BetA derivatives were also synthesized. The new products were evaluated their antitumor activities against MGC-803, PC3, Bcap-37, 3

ACCEPTED MANUSCRIPT A375, and MCF-7 human cancer cell lines. Furthermore, the possible mechanism of MGC-803 cell growth inhibition by compound 3k was also investigated in the present study.

2. Results

RI PT

2.1. Chemistry The BetA was derived from betulin by oxidation with Jones regent. The syntheses of BetA derivatives are summarized in Schemes 1-2. The structure modification of BetA used as the leading compound was done at the position C-28. BetA was reacted with 1,2-dibromoethane,

SC

1,3-dibromopropane, or 1,4-dibromobutane in the presence of K2CO3 in DMF at room temperature to give the compounds 2a-2c in high yield. And then, the compounds 2a, 2b, or 2c were reacted

M AN U

with corresponding amines to yield the nitrogen-containing derivatives 3a-3l, respectively (Scheme 1). Compound 2a was treated with piperazine in DMF in the presence of K2CO3 at 80 oC, and reacted with aromatic or aliphatic carboxylic acids in the presence EDCI and HOBT to afford compounds 5a-5i (Scheme 2). All the compounds were fully characterized by various spectroscopic methods.

TE D

Scheme 1

Scheme 2

2.2. Biology

EP

The in vitro antitumor activity of BetA derivatives on five different human cancer cell lines: gastric carcinoma cell MGC-803, prostate carcinoma cell PC3, breast carcinoma cell Bcap-37,

AC C

malignant melanoma cell A375, and breast carcinoma cell MCF-7 cell lines were studied by MTT assay. HCPT (hydroxycamptothecine) and ADM (Adriamycin) were used as the positive controls. The negative control group was treated with culture medium containing 0.1% DMSO. All the compounds including BetA derivatives, ADM, and HCPT were dissolved in DMSO. Each experiment was repeated at least three times. The BetA derivatives showed dose dependent antitumor activity against the investigated cell lines. If IC50 value could not be reached at the highest concentration, then > 20 was given. The IC50 values are summarized in Table 1. Table 1 As shown in table 1, when the 28-COOH was only substituted with a dibromoalkane (compounds 2a-2c), the effect was significantly reduced, compared with the parent BetA. The previous study in 4

ACCEPTED MANUSCRIPT UA or BA derivatives showed that significant improvement of cell growth inhibition was not observed when the 28-COOH was just acetylated with a fatty alkyl group [30,31]. Similar results were seen in this study. Interestingly, the most active compounds (compound 3a-3l) with selected amino groups

RI PT

introduced were found to be two to eight times more active than BetA, IC50 values between 3 and 18 µM. And the activity of the compounds were increased as the length of the carbon chain was increased (4 > 3 > 2). The compound 3k, one of the most active compounds, had the IC50 values of 3.6, 5.6, 4.2, 7.8, and 5.2 µM on the five cancer cell lines, respectively.

SC

Among the derivatives substituted with acids, substituted benzoic acids (compounds 5a-5g) exhibited high and moderate activities, respectively, whereas substituted alkyl carboxylic acids

M AN U

(compounds 5h-5i) demonstrated weak antitumor activity. Compounds 5h-5i did not show significant antitumor activity as reported in the literature. One reason for low activity could be due the long fatty alkyl group. The results were similar with other pentacylic triterpenes [31]. In addition, selectivity of the active compounds (3i, 3j, 3k, 5a, 5e, and 5f) was also assessed on normal cell line NIH3T3. The selectivity index was calculated by IC50 value in normal cells

TE D

divided by IC50 value in cancer cells, and the results are summarized in Table 2. It was observed that these compounds were 2 to 9 times more selective towards cancer cells than normal cells. That is to say that these compounds did not show significant cytotoxic effect on normal cells.

EP

Table 2

2.3. Preliminary investigation of the apoptosis-inducing effect of title compound 3k

AC C

To determine whether the growth inhibitory activity of the selected compound (compound 3k) were related to the induction of apoptosis, the morphological character changes of MGC-803 cells were investigated using the AO/EB, Hoechst 33258 staining under fluorescence microscopy. The morphologic changes in the cell after treatment with compound 3k were assessed by fluorescene microscopy after staining with AO/EB. AO is a vital dye and will stain both live and dead cells, but EB will stain only cells that have lost membrane integrity. The stained cells revealed four different types under a fluorescence microscope: living cells (normal green nucleus), early apoptotic (bright green nucleus with condensed or fragmented chromatin), late apoptotic (orange-stained nuclei with chromatin condensation or fragmentation), and necrotic cells 5

ACCEPTED MANUSCRIPT (uniformly orange-stained cell nuclei) [32]. With HCPT as positive control at 10 µM for 48 h, the compound 3k at 5 µM against MGC-803 cells from 12 to 48 h was detected via AO/EB staining. As can be seen in Figure 1A, the cells treated with compound 3k from 12 to 48 h had changed. Yellow and orange dots in MGC-803 cells showed early and late apoptotic cells, and the

RI PT

appearance of little red cells indicated that compound 3k was low cytotoxicity. Therefore, it can be concluded that compound 3k could induce apoptosis without any significant cytotoxicity.

Hoechst 33258 staining was also used to investigate the apoptosis induction on cells. Live cells with uniformly light blue nuclei were treated with Hoechst 33258 and observed under a

SC

fluorescence microscope [33]. Hoechst 33258 staining showed apoptosis in all four types of cells, which was characterized by cytoplasmic and nuclear shrinkage, chromatin condensation and

M AN U

apoptosis body [34]. With HCPT as positive control at 10 µM for 48 h, the compound 3k at 5 µM against MGC-803 cells from 12 to 48 h was detected via Hoechst 33258 staining. As can be seen in Figure 1B, the cells of the negative group (DMSO) were normal blue. However, the cells of HCPT group appeared compact condensed, and crescent-shaped. The cells exhibited strong blue fluorescence, revealing the typical apoptosis characteristics. The cells treated with compound 3k

TE D

from 12 to 48 h had changed. The cell nuclei appeared to be highly condensed and crescent-shaped; indicating that compound 3k could induce apoptosis against MGC-803 cell lines. These results were identical with the previous AO/EB double staining.

EP

In addition, TUNEL is a popular method for identifying apoptotic cells by detection of DNA fragmentation [33]. The cells were observed that where brown precipitate was the result of

AC C

positive apoptosis. With HCPT as positive control at 10 µM for 48 h, the compound 3k at 5 µM against MGC-803 cells from 12 to 48 h was detected via TUNEL assay. As can be seen in Figure 1C, the cells of the negative group (DMSO) did not appear as brown precipitates, whereas the other groups, namely, HCPT, appeared as brown precipitates. The cells treated with compound 3k from 12 to 48 h had changed. Therefore, it can be further concluded that compound 3k induced apoptosis against MGC-803 cells. The results were identical with the previous experiment. Figure 1 The apoptosis ratios induced by compound 3k in tumor cells were quantitatively assessed by FCM. In early apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the external cellular 6

ACCEPTED MANUSCRIPT environment. Annexin V is a 35-36 kDa Ca2+ dependent phospholipid-binding protein that has a high affinity for PS, and is used to detect early apoptotic cells. PI (Propidine Iodide) was a red fluorescent dye and stained cells that had lost membrane integrity. So, the different periods of apoptotic cells could be distinguished when Annexin V matched with PI: the upper left quadrant

RI PT

contains the necrotic cells (Annexin-/PI+); the upper right quadrant contains late apoptotic cells (Annexin-/PI+); the lower left quadrant contains the intact cells (Annexin-/PI+); the lower right quadrant contains the early apoptotic cells (Annexin-/PI+) [35]. As shown in Figure 2, with HCPT as positive control, compound 3k (10 µM) could induce apoptosis of MGC-803 cells, and highest

SC

apoptosis ratios, 28.33% for compound 3k, were obtained after 36 h of treatment at a concentration of 10 µM. Furthermore, as shown in Figure 3 the apoptosis of MGC-803 cells which

M AN U

treated with compound 3k increased gradually in a time-dependent manner. Figure 2

Figure 3

2.4. The study of cancer cell apoptotic signaling pathway

p53 can initiate apoptosis, the programmed cell death, if DNA damage proves to be irreparable,

TE D

and it is able to reduce bcl-2 and promote Bax expression [36]. Thus, cytochrome c, which is a component of the electron transport chain in mitochondria, will be released [37]. This release of cytochrome c in turn activates caspase 9, a cysteine protease. And then, caspase 9 can activate

EP

caspase 3 to induce cell apoptosis. As shown in Figure 4A, the expression of p53 and Bax, was significantly promoted [38]. At the same time, the treatment of compound 3k also resulted in a

AC C

significant activation of caspase 3/9 (Figure 4B). These findings indicated that compound 3k could induce apoptosis of MGC-803 cells through the mitochondrial intrinsic pathway, involving suppression of the expressions of p53, Bax, caspase 9 and caspase 3. Figure 4

3. Conclusions In conclusion, we have described the synthesis of BetA derivatives and their in vitro antitumor activity on MGC-803, PC3, Bcap-37, A375, and MCF-7 human cell lines. The pharmacological results showed that most of the compounds displayed moderate to high levels of antitumor activity, against the five cancer cell lines and that most exhibited more potent inhibitory activity compared 7

ACCEPTED MANUSCRIPT with the parent BetA. Meanwhile, the results also indicated that (1) Significant improvement of the cell growth inhibition was achieved when amino groups were introduced at the C-28 position. (2) Substituted benzoic acids (compounds 5a-5g) displayed moderate to high inhibitory activity, whereas substituted alkyl carboxylic acids (compounds 5h-5i) demonstrated weak antitumor

RI PT

activity. (3) When the 28-COOH was only substituted with a dibromoalkane (compounds 2a-2c), the effect was significantly reduced.

Moreover, the compound 3k, one of the most active compounds, had the IC50 values of 3.6, 5.6, 4.2, 7.8, and 5.2 µM on the five cancer cell lines respectively, and it was selected to analyze the

SC

mechanism by subsequent florescence staining and flow cytometry analysis. The preliminary mechanism indicated that compound 3k could induce apoptosis in MGC-803 cell lines, and the

M AN U

apoptosis ratio reached 28.33% after 36 h of treatment at 10 µM, higher than the ratios observed for the positive control HCPT. Anything else, compound 3k can induce apoptosis of MGC-803 cells through the mitochondrial intrinsic pathway, involving suppression of the expressions of p53, Bax, caspase 9 and caspase 3. These suggest that BetA derivatives could possess a strong potential for development as a preventive and therapeutic agent against human carcinomas. Further study is

TE D

needed to investigate the active BetA derivatives and the underlying mechanism. And this study also provides a very powerful incentive for further research on the chemical modification and

EP

structure-activity relationships of BetA and other triterpenoid acids.

4. Experimental

AC C

4.1. General

Betulin with more than 98% purity was purchased from Zhejiang Tiancao Biotech Co., Ltd. Reagents of analytical grade were obtained from Yuda Chemistry Co., Ltd., and used without further purification unless otherwise noted. Infrared spectra were recorded on a Bruker VECTOR22 spectrometer in KBr disks.

1

H-NMR and

13

C-NMR were recorded using a

JEOL-ECX500 spectrometer at 22 °C, with tetramethylsilane as the internal standard and CDCl3 as the solvent. Column chromatography was performed using silica gel (200–300 meshes) (Qingdao Marine Chemistry Co., Qingdao, China)

4.2. Synthesis 8

ACCEPTED MANUSCRIPT 4.2.1. General procedure for compounds (3a-3l). BetA (1 mmol) and K2CO3 (2 mmol) were added to DMF (25 mL) and stirred at room temperature for 10 min, after which 1,2-dibromoethane, 1,3-dibromopropane, or 1,4-dibromobutane (4 mmol) was added. After being stirred for another 24 h, the reaction mixture was poured onto 100 mL of

RI PT

distilled water and partitioned with DCM (3×25 mL). The organic layer was washed with saturated sodium chloride, dried over Na2SO4 and purified via silica gel column chromatography with petroleum ether/ethyl acetate to obtain compounds 2a-2c. The compounds 2a, 2b or 2c (1 mmol) and K2CO3 (2 mmol) were assed to DMF (10 mL) and stirred at room temperature for 10

SC

min, after saturated nitrogen heterocyclic rings (2 mmol) was added. After being stirred for 24 h, the reaction mixture was poured into the 100 mL distilled water and partitioned with DCM (3×30

M AN U

mL). The organic layer was washed with saturated sodium chloride, dried over Na2SO4, and purified via silica gel column chromatography with CHCl3/MeOH (10:1, v/v) to obtain compounds 3a-3l.

4.2.1.1. (4-(piperidin-1-yl)butyl) 3-oxo-20(29)-lupen-28-oate (3k)

TE D

According to the general procedure, compound 2a was treated with 1,4-dibromobutane, and then purified on silica gel column using petroleum ether/ethyl acetate (15:1, v/v) to get compound 3k, Rf [petroleum ether/ ethyl acetate (3:1, v/v)] = 0.24.

EP

Yield: 72.8%; pale yellow oil; IR (KBr) : νmax 3441, 2938, 2877, 1689, 1640, 880 cm-1; 1H NMR (CDCl3, 500 MHz) δ: 4.67 (1H, s, Hb-29), 4.54 (1H, s, Ha-29), 4.04 (2H, m, CH2), 2.94 (3H, m,

AC C

H-2, 19), 2.44 (4H, m, CH2), 2.38 (2H, m, CH2), 1.63 (3H, s, CH3), 1.58 (2H, m, CH2), 1.41 (4H, m, CH2), 1.01 (3H, s, CH3), 0.96 (3H, s, CH3), 0.91 (3H, s, CH3), 0.89 (3H, s, CH3), 0.86 (3H, s, CH3); 13C NMR (CDCl3, 125 MHz) δ: 218.2 (C-3), 176.1 (C-28), 150.5 (C-20), 109.7 (C-29), 63.7 (CH2), 58.6 (CH2), 56.5 (C-17), 54.9 (C-5), 54.3 (CH2), 49.9 (C-9), 49.4 (C-18), 47.4 (C-4), 47.0 (C-19), 42.5 (C-14), 40.6 (C-8), 39.7 (C-1), 38.4 (C-13), 37.0 (C-22), 36.9 (C-10), 34.2 (C-2), 33.7 (C-7), 32.1 (C-16), 30.6 (C-21), 29.7 (C-15), 26.8 (C-23), 26.7 (CH2), 25.6 (C-12), 25.4 (CH2), 24.1 (CH2), 22.9 (CH2), 21.5 (C-11), 21.1 (C-24), 19.7 (C-6), 19.4 (C-30), 16.0 (C-26), 15.9 (C-25), 14.7 (C-27).

4.2.2. General procedure for compounds (5a-5i). 9

ACCEPTED MANUSCRIPT Compound 2a (1 mmol) and K2CO3 (2 mmol) were added to DMF (15 mL) and stirred at room temperature for 10 min, and then piperazine (5 mmol) was dripped into the mixture, which was stirred at 80 °C for 5 h. After cooling to room temperature, the reaction mixture was poured onto 100 mL of distilled water and partitioned with DCM (3×20 mL). The organic layer was washed

RI PT

with saturated sodium chloride, dried over Na2SO4 and purified via silica gel column chromatography with CHCl3/MeOH (10:1, v/v) to obtain compound 4. Compound 4 (1 mmol), amine compounds (1.2 mmol), EDCI (1.2 mmol) and HOBt (1.2 mmol) were added to DCM (25 mL) containing Et3N (0.5 mmol); the mixture was then stirred at room temperature for 6-12 h.

SC

After the reaction was completed, the mixture was poured onto 100 mL of distilled water and partitioned with ethyl acetate (3×50 mL). The target compounds were purified on a flash column

M AN U

with chloroform/methanol (20:1, v/v) to yield compounds 5a-5i.

4.2.2.1. (2-(4-benzoylpiperazin-1-yl)ethyl) 3-oxo-20(29)-lupen-28-oate (5a) According to the general procedure, compound 4 was treated with aromatic acids and then purified on silica gel column using chloroform/methanol (10:1, v/v) to obtain compound 5a, Rf

TE D

[chloroform/methanol (5:1, v/v)] = 0.28.

Yield: 71.1%; colorless oil; IR (KBr): νmax 3444, 2931, 2869, 1713, 1644, 881, 755, 701 cm-1; 1H NMR (CDCl3, 500 MHz) δ: 7.37 (5H, s, PhH), 4.69 (1H, s, Hb-29), 4.57 (1H, s, Ha-29), 4.19 (2H,

EP

t, J = 5 Hz, CH2), 3.43 (2H, brs, H in piperazine), 2.97 (3H, m, H-2, 19), 2.64 (2H, t, J = 6.5 Hz, CH2), 2.46 (2H, m, H in piperazine), 2.38 (2H, m, H in piperazine), 1.67 (3H, s, CH3), 1.04 (3H, s, 13

C NMR

AC C

CH3), 0.99 (3H, s, CH3), 0.94 (3H, s, CH3), 0.92 (3H, s, CH3), 0.89 (3H, s, CH3);

(CDCl3, 125 MHz) δ: 218.2 (C-3), 175.9 (C-28), 170.4 (C=O), 150.5 (C-20), 135.8 (C), 129.8 (CH), 128.6 (CH), 127.1 (CH), 109.8 (C-29), 60.8 (CH2), 56.8 (CH2), 55.6 (C-17), 55.0 (C-5), 53.7 (CH2), 53.1 (CH2), 49.9 (C-9), 49.3 (C-18), 47.8 (CH2), 47.4 (C-4), 47.0 (C-19), 42.5 (C-14), 42.2 (CH2), 40.7 (C-8), 39.7 (C-1), 38.4 (C-13), 36.9 (C-22), 36.9 (C-10), 34.2 (C-2), 33.7 (C-7), 32.1 (C-16), 30.7 (C-21), 29.7 (C-15), 26.6 (C-23), 25.5 (C-12), 21.5 (C-11), 21.1 (C-24), 19.7 (C-6), 19.4 (C-30), 16.0 (C-26), 15.9 (C-25), 14.7 (C-27).

4.3. Cell lines and culture MGC-803, PC3, Bcap-37, A375, and MCF-7 cell lines were obtained from the Institute of 10

ACCEPTED MANUSCRIPT Biochemistry and Cell Biology, China Academy of Science. MGC-803 is gastric cancer cell line, PC3 is prostate cancer cell line, A375 is malignant melanoma cell line, Bcap-37, and MCF-7 are breast cancer cell lines. The entire cancer cell lines were maintained in the RPMI 1640 medium. They were supplemented with 10% heat-inactivated fetal bovine serum (FBS) in a humidified

RI PT

atmosphere of 5% CO2 at 37 °C. All cell lines were maintained at 37 °C in a humidified 5% carbon dioxide and 95% air incubator.

4.4. MTT assays

SC

All compounds were dissolved in DMSO and subsequently diluted in the culture medium before treatment of the cultured cells. When the cells were 80-90% confluent, they were harvested by

M AN U

treatment with a solution containing 0.25% trypsin, thoroughly washed and resuspended in supplemented growth medium. Cells (2×103/well) were seeded in 96-well plates overnight. The tested compounds at different concentrations were added into wells and cells were treated at 37 oC for 72 h. In parallel, the cells treated with 0.1% DMSO served as negative control and ADM (Adriamycin) as positive control. Then MTT (5 mg/ml, in PBS) was added into each well and

TE D

cultured for another 4 h. The MTT-formazan formed by metabolically viable cells was dissolved in 100 µL of SDS for 12 h. The absorbance was then measured at 595 nm with a microplate reader, which is directly proportional to the number of living cells in culture. The percentage cytotoxicity

AC C

curves [39].

EP

was calculated using the formula. IC50 values were calculated according to the dose-dependent

4.5. AO/EB staining

The cells (5×104 cell/mL in a volume of 0.6 mL) were seeded in 6-well tissue culture plates. Following incubation, the medium was removed and replaced with fresh medium plus 10% FBS and then supplemented with compounds. After the treatment period, cells were washed with 1×PBS once, and the cover slip with monolayer cells was inverted on the glass slide with 20 µL of AO/EB stain (100 µg/mL) [40]. The fluorescence was read using an IX71SIF-3 fluorescence microscope.

4.6. Hoechst 33258 staining 11

ACCEPTED MANUSCRIPT The cells (5×104 cell/mL in a volume of 0.6 mL) were seeded in 6-well tissue culture plates. Following incubation, the medium was removed and replaced with fresh medium plus 10% FBS and then supplemented with compounds (5 µmol/L). After the treatment period, cells were washed with 1×PBS twice, and the cells were fixed in 4% paraformaldehyde for 10 min. The cells were

RI PT

washed twice with PBS, and were consequently stained with 0.5 mL of Hoechst 33258 staining solution for 5 min. Then the wells were washed twice, (3min/once) with 1×PBS after staining. Later, coverslips having cells were covered the glass slide, detected by IX71SIF-3 fluorescence

SC

microscope at 350 nm excitation and 460 nm emissions [41].

4.7. TUNEL assay

M AN U

The cells (5×104 cell/mL in a volume of 0.6 mL) were seeded in 6-well tissue culture plates. Following incubation, the medium was removed and replaced with fresh medium plus 10% FBS and then supplemented with compounds. TUNEL assays were performed using a colorimetric TUNEL apoptosis assay kit according to the manufacturer’s instructions. (1) After the treatment period, cells were washed with 1×PBS and fixed in 4% paraformaldehyde for 40 min. The cells

TE D

were washed once with PBS, and were consequently permeabilized with immunol staining wash buffer for 2 min on ice. (2) The cells were rewashed once with PBS, and were consequently incubated in 0.3% H2O2 in methanol at room temperature for 20 min to inactivate the endogenous

EP

peroxidases, after which the cells were washed thrice with PBS. (3) The cells were incubated with 2 µL of TdT-enzyme and 48 µL of Biotin-dUTP per specimen for 60 min at 37 °C. The cells were

AC C

terminated for 10 min, and were consequently incubated with streptavidin-HRP (50 µL per specimen) conjugate diluted at 1:50 in sample diluent for 30 min. (4) The cells were washed three times with PBS, and were consequently incubated with diaminobenzidine solution (200 µL per specimen) for 10 min. At last, the cells were rewashed twice with PBS, and were consequently imaged under an XDS-1B inverted biological microscope [42].

4.8. Flow cytometry analysis Prepared MGC-803 cells (1×106/mL) were washed twice with cold PBS and then re-suspended gently in 500 µL binding buffer. Thereafter, cells were stained in 5 µL Annexin V-FITC and shaked well. Finally, 5 µL PI was added to these cells and incubated for 20 min in a dark place, 12

ACCEPTED MANUSCRIPT analyzed by FACS Calibur, Becton Dickinson.

4.9. Western blotting analysis Cells were collected after treatment with compound 3k at 2.5, 5, and 10 µM for 12 h, respectively.

RI PT

Western blotting analysis was performed as previously described [43], using the following antibodies at dilutions of 1: 500 to 1:1000: anti-p53, anti-Bax, and anti-β actin.

5.0. Caspase 3 enzyme assay

SC

Cells were collected after treatment with compound 3k at 2.5, 5, and 10 µM for 12 h, respectively. Prepared MGC-803 cells (1×106/mL, 5ml) were washed twice with cold PBS. Then, 100 µL of

M AN U

lysis buffer was added to the cells for 25 min on ice and centrifuged at 18000 g for 15 min. 80 µL of reaction buffer and 10 µL of Ac-DEVED-pNA were added to 10 µL of supernatant liquid. After incubating at 37 oC for 2-3 h in darkness, the absorbance was measured at 405 nm, with the lysis buffer and reaction buffer as control.

TE D

5.1. Caspase 9 enzyme assay

Cells were collected after treatment with compound 3k at 2.5, 5, and 10 µM for 12 h, respectively. Prepared MGC-803 cells (1×106/mL, 5ml) were washed twice with cold PBS. Then, 100 µL of lysis

EP

buffer was added to the cells for 25 min on ice and centrifuged at 16000 g for 15 min. 80 µL of reaction buffer and 10 µL of Ac-LEHD-pNA were added to 10 µL of supernatant liquid. After

AC C

incubating at 37 oC for 2-3 h in darkness, the absorbance was measured at 405 nm, with the lysis buffer and reaction buffer as control.

5.2. Statistical Analysis

All statistical analyses were performed using SPSS 10.0, and the data were analyzed using one-way ANOVA. The mean separations were performed using the least significant difference method. Each experiment was performed in triplicate, and all experiments were run thrice and yielded similar results. Measurements from all the replicates were combined, and the treatment effects were analyzed.

13

ACCEPTED MANUSCRIPT Acknowledgments The authors wish to thank the National Nature Science Foundation of China (Grant Nos. 21172048, 21372052), Key Technologies R&D Program (No. 2011BAE06B02), the Research Project of Chinese Ministry of Education (No. 213033A), and Guizhou Key S&T Project (Nos.

RI PT

[2012]6012, [2011]3016, [2010]3052) for the financial support.

References

[1] G. Danaei, S.V. Hoorn, A.D. Lopez, C.J. Murray, M. Ezzati, Causes of cancer in the world:

SC

Comparative risk assessment of nine behavioural and environmental risk factors, Lancet. 366 (2005) 1784-1793.

M AN U

[2] C.D. Mathers, D. Loncar, Projections of global mortality and burden of disease from 2002 to 2030, Plos Med. 3 (2006) 2011-2029.

[3] K. Sikora, S. Advani, V. Koroltchouk, I. Magrath, L. Levy, H. Pinedo, G. Schwartsmann, M. Tattersall, S. Yan, Essential drugs for cancer therapy: A World Health Organization consultation, Ann. Oncol. 10 (1999) 385-390.

191-198.

TE D

[4] S. Dev, Impact of natural products in modern drug development, India J. Exp. Biol. 48 (2010)

[5] A.D. Kinghorn, Y.W Chin, S.M. Swanson, Discovery of natural product anticancer agents

EP

from biodiverse organisms, Curr. Opin. Drug Discov. Devel. 12 (2009) 189-196. [6] M. Shoeb, Anticancer agents from medicinal plants, Bangl J. Pharmacol. 1 (2006) 35-41.

AC C

[7] D.R.A. Mans, A.B. Da Rocha, G. Schwartsmann, Anti-cancer drug discovery and development in Brazil: Targeted plant collection as a rational strategy to acquire candidate anti-cancer compounds, Oncologist 5 (2000) 185-198.

[8] D.J. Newman, G.M. Gragg, K.M. Snader, The influence of natural products upon drug discovery, Nat. Prod. Rep. 17 (2000) 215-234.

[9] T.W. Hodges, C.F. Hossain, Y.P. Kim, Y.D. Zhou, D.G. Nagle, Molecular-targeted antitumor agents: The saururus cernuus dineolignans manassantin B and 4-O-demethylmanassantin B are potent inhibitors of hypoxia-activated HIF-1, J. Nat. Prod. 67 (2004) 767-771. [10] D.G. Nagle, Y.D. Zhou, F.D. Mora, K.A. Mohammed, Y.P. Kim, Mechanism targeted discovery of antitumor marine natural products, Curr. Med. Chem. 11 (2004) 1725-1756. 14

ACCEPTED MANUSCRIPT [11] Z, Skrzypek, H. Wysokińska, Sterols and triterpenes in cell culture of Hyssopus officinalis L., Z Naturforsch C 58 (2003) 308-312. [12] S. Jäger, H. Trojan, T. Kopp, M.N. Laszczyk, A. Scheffler, Pentacyclic triterpene distribution in various plants - rich sources for a new group of multi-potent plant extracts, Molecules 14

RI PT

(2009) 2016-2031. [13] A.A.L. Gunatilaka, Triterpenoids and steroids of Sri Lankan plants: A review of occurrence and chemistry, J. Natl. Sci. Coun. Sri Lanka 14 (1986) 1-54.

[14] K.H. Lee, S. Morris-Natschke, K. Qian, Y. Dong, X. Yang, T. Zhou, E. Belding, S.F. Wu, K.

SC

Wada, T. Akiyama, Recent progress of research on herbal products used in traditional Chinese medicine: The herbs belonging to the divine husbandman’s herbal foundation canon,

M AN U

J. Tradit. Complem. Med. 2 (2012) 6-26.

[15] A.V. Symon, N.N. Veselova, A.P. Kaplun, N.K. Vlasenkova, G.A. Fedorova, A.I. Liutik, G.K. Gerasimova, V.I. Shvrts, Synthesis and antitumor activity of cyclopropane derivatives of betulinic and betulonic acids, Russ. J. Bioorg. Chem. 31 (2005) 320-325. [16] P. Silhár, S. Alakurtti, K. Čapková, F. Xiaochuan, C.B. Shoemaker, J. Yli-kauhaluoma, K.D.

TE D

Janda, Synthesis and evaluation of library of betulin derivatives against the botulinum neurotoxin A protease, Bioorg. Med. Chem. Lett. 21 (2011) 2229-2231. [17] A.I. Govdi, I.V. Sorokina, T.G. Tolstikova, S.F. Vasilevsky, G.A. Tolstikov, Synthesis and

397-402.

EP

biological activity of novel acetylene betulonic acid derivatives, Chem. Sust. Dev. 18 (2010)

AC C

[18] S.F. Vasilevsky, A.I. Govdi, E.E. Shults, M.M. Shakirov, I.V. Sorokina, T.G. Tolstikova, D.S. Baev, G.A. Tolstikov, I.V. Alabugin, Efficient synthesis of the first betulonic acid-acetylene hybrids and their hepatoprotective and anti-inflammatory activity, Bioorg. Med. Chem. 17 (2009) 5164-5169.

[19] D.Z.L. Bastos, I.C. Pimentel, D.A. de Jesus, B.H. de Oliveira, Biotransformation of betulinic and betulonic acids by fungi, Phytochemistry 68 (2007) 834-839. [20] O.B. Flekhter, E.I. Boreko, L.R. Nigmatullina, E.V. Tret'yakova, N.I. Pavlova, L.A. Baltina, S.N. Nikolaeva, O.V. Savinova, V.F. Eremin, F.Z. Galin, G.A. Tolstikov, Synthesis and antiviral activity of betulonic acid amides and conjugates with amino acids, Russ. J. bioorg. Chem. 30 (2004) 80-88. 15

ACCEPTED MANUSCRIPT [21] P. Coric, S. Turcaud, F. Souquet, L. Briant, B. Gay, J. Royer, N. Chazal, S. Bouaziz, Synthesis and biological evaluation of a new derivative of bevirimat that targets the Gag CA-SP1 cleavage site, Eur. J. Med. Chem. 62 (2013) 453-465. [22] X. Zhang, H. Li, Y. Jin, G. Fang, Effects of betulonic acid on SGC-7901, HepG-2 and mice of

RI PT

bearing S180 tumor cells, Nat. Prod. Res. Dev. 21 (2009) 766-770. [23] S. Yang, Q. Zhao, H. Xiang, M. Liu, Q. Zhang, W. Xue, B. Song, S. Yang, Antiproliferative activity and apoptosis-inducing mechanism of constituents from Toona sinensis on human cancer cells, Cancer Cell Int. 13 (2013) 12.

SC

[24] R.C. Santos, J.A. Salvador, S. Marín, M. Cascante, J.N. Moreira, T.C. Dinis, Synthesis and structure-activity relationship study of novel cytotoxic carbamate and N-acylheterocyclic

M AN U

bearing derivatives of betulin and betulinic acid, Bioorg. Med. Chem. 18 (2010) 4385-4396. [25] 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) 460-466.

[26] S. Schwarz, B. Siewert, N.M. Xavier, A.R. Jesus, A.P. Rauter, R. Csuk, A "natural" approach:

TE D

synthesis and cytoxicity of monodesmosidic glycyrrhetinic acid glycosides, Eur. J. Med. Chem. 72 (2014) 78-83.

[27] K.K. Bai, Z. Yu, F.L. Chen, F. Li, W.Y. Li, Y.H. Guo, Synthesis and evaluation of ursolic acid

EP

derivatives as potent cytotoxic agents, Bioorg. Med. Chem. Lett. 22 (2012) 2488-2493. [28] O.B. Flekhter, E.I. Boreko, E.V. Tret'iakova, N.I. Pavlova, L.A. Baltina, S.N. Nikolaeva, O.V.

AC C

Savinova, V.F. Eremin, F.Z. Galin, G.A. Tolstikov, Synthesis and antiviral activity of amides and conjugates of betulonic acid with amino acids, Russ. J. bioorg. Chem. 30 (2004) 89-98.

[29] Y. Liu, W.X. Lu, M.C. Yan, Y. Yu, T. Ikejima, M.S. Cheng, Synthesis and tumor cytotoxicity of novel amide derivatives of β-hederin, Molecules 15 (2010) 7871-7883.

[30] S. Yang, N. Liang, H. Li, W. Xue, D. Hu, L. Jin, Q. Zhao, S. Yang, Design, synthesis and biological evaluation of novel betulinic acid derivatives, Chem. Cent. J. 6 (2012) 141. [31] J.W. Shao, Y.C. Dai, J.P. Xue, J.C. Wang, F.P. Lin, Y.H. Guo, In vitro and in vivo anticancer activity evaluation of ursolic acid derivatives, Eur. J. Med. Chem. 46 (2011) 2652-2661. [32] F. Attari, H. Sepehri, L. Delphi, B. Goliaei, Apoptotic and necrotic effects of pectic acid on rat pituitary GH3/B6 tumor cells, Iran. Biomed. J. 13 (2009) 229-236. 16

ACCEPTED MANUSCRIPT [33] M.C. Liu, S.J. Yang, L.H. Jin, D.Y. Hu, Z.B. Wu, S. Yang, Chemical constituents of the ethyl acetate extract of Belamcanda chinensis (L.) DC Roots and their antitumor activities, Molecules 17 (2012) 6156-6169. [34] Y.L. Wu, B. Sun, X.J. Zhang, S.N. Wang, H.Y. He, M.M. Qiao, J. Zhong, J.Y. Xu, Growth

RI PT

inhibition and apoptosis induction of sulindac on human gastric cancer cells. World J. Gastroenterol. 7 (2001) 796-800.

[35] B. Schutte, R. Nuydens, H. Geerts, F. Ramaekers, Annexin V binding assay as a tool to measure apoptosis in differentiated neuronal cells, J. Neurosci. Meth. 86 (1998) 63-69.

apoptosis, Cancer Sci. Ther. 4 (2012) 368-370.

SC

[36] R. Kumariya, A. K. Barui, S. Singh, p53-Cells’ inbuilt mechanism to inhibit cancer through

M AN U

[37] J. Cai, J. Yang, D.P. Jones, Mitochondrial control of apoptosis: the role of cytochrome c, Biochim. Biophys. Acta. 1366 (1998) 139-149.

[38] X. Jiang, X. Wang, Cytochrome c-mediated apoptosis, Annu. Rev. Biochem. 73 (2004), 87-106

[39] P. Lan, J. Wang, D.M. Zhang, C.Shu, H.H. Cao, P. H. Sun, X.M. Wu, W.C. Ye, W.M. Chen,

TE D

Synthesis and antiproliferative evaluation of 23-hydroxybetulinic acid derivatives, Eur. J. Med. Chem. 46 (2011) 2490-2502.

[40] D. Ribble, N.B. Goldstein, D.A. Norris, Y.G. Shellman, A simple technique for quantifying

EP

apoptosis in 96-well plates, BMC Biotechnol. 5 (2005) 12. [41] G.Y. Wang, J.W. Zhang, Q.H. Lü, R.Z. Xu, Q.H. Dong, Berbamine induces apoptosis in

AC C

human hepatoma cell line SMMC7721 by loss in mitochondrial transmembrane potential and caspase activation, J. Zhejiang Univ. Sci. B 8 (2007) 248-255..

[42] X. Xu, X. Gao, L. Jin, P.S. Bhadury, K. Yuan, D. Hu, B. Song, S. Yang, Antiproliferation and cell apoptosis inducing bioactivities of constituents from Dysosma versipellis in PC3 and Bcap-37 cell lines, Cell Div. 6 (2011) 14.

[43] J.Q. Yu, Y. Yin, J.C. Lei, X.Q. Zhang, W. Chen, C.L. Ding, S. Wud, X.Y. He, Y.W. Liu, G. L. Zou, Activation of apoptosis by ethyl acetate fraction of ethanol extract of Dianthus superbus in HepG2 cell line, Cancer Epidemiol. 36 (2012) e40-e45.

17

ACCEPTED MANUSCRIPT Table 1 Antitumor activity in a panel of various human cancer cell lines in vitro Figure 1. Apoptosis induction studies of compound 3k. (A) AO\EB staining. (B) Hoechst 33258 staining. (C) TUNEL assay. Figure 2. The apoptosis ratios, including the early (B4) and late apoptosis ratios (B2).

RI PT

Figure 3. Annexin V/PI dual staining of MGC-803 cell lines. A: negative control; B, C, and D: cells treated with HCPT (10 µM) as positive control; E, F, and G: cells treated with compound 3k (10 µM).

Figure 4. Expression levels of caspases, p53, and Bax. A: western blot analysis of p53 and Bax in

SC

MGC-803 cells; B: activation of caspase 3/9 in MGC-803 cells.

Scheme 1. Regents and conditions: (i) BrCH2CH2Br, BrCH2CH2CH2Br, or BrCH2(CH2)2CH2Br,

M AN U

K2CO3, DMF, r.t.; (ii) amine, K2CO3, DMF, r.t.

Scheme 2. Reagents and conditions: (i) piperazine, K2CO3, DMF, 80 °C; (ii) EDCI, HOBT,

AC C

EP

TE D

aromatic carboxylic acids, DCM, r.t..

18

ACCEPTED MANUSCRIPT Table 1 Antitumor activity in a panel of various human cancer cell lines in vitro IC50 in µM for cancer cell lines MGC-803

PC3

Bcap-37

MCF-7

1

17.7 ± 0.3

13.9 ± 0.7

25.7 ± 0.4

28.9 ± 0.7

18.2 ± 0.3

2a

> 20

> 20

> 20

> 20

> 20

2b

> 20

> 20

> 20

> 20

> 20

2c

> 20

> 20

> 20

> 20

> 20

3a

11.7 ± 1.1

10.1 ± 0.7

15.6 ± 0.8

14.3 ± 1.4

12.8 ± 0.3

3b

10.8 ± 0.1

12.3 ± 0.3

10.2 ± 0.9

9.7 ± 0.3

9.4 ± 0.4

3c

12.5 ± 1.5

18.8 ± 0.2

16.9 ± 1.3

17.1 ± 0.1

16.0 ± 0.2

3d

9.4 ± 0.3

11.3 ± 0.7

8.8 ± 0.4

10.5 ± 0.6

10.1 ± 0.7

3e

6.8 ± 0.4

7.0 ± 0.6

6.1 ± 0.3

3f

7.4 ± 0.1

7.6 ± 0.8

9.2 ± 0.4

3g

6.3 ± 0.3

5.7 ± 0.3

9.5 ± 0.4

3h

5.6 ± 0.3

6.6 ± 0.8

3i

3.8 ± 0.1

3j

RI PT

A375

SC

Compound

7.3 ± 0.6

8.3 ± 0.4

8.4 ± 0.3

9.5 ± 0.6

6.6 ± 0.4

7.2 ± 0.8

8.6 ± 0.4

9.3 ± 0.2

4.4 ± 0.8

5.2 ± 0.2

9.2 ± 0.7

7.9 ± 0.5

4.0 ± 0.1

6.7 ± 0.4

4.7 ± 0.8

8.5 ± 0.7

5.4 ± 0.2

3k

3.6 ± 0.1

5.6 ± 0.3

4.2 ± 0.1

7.8 ± 0.4

5.2 ± 0.5

3l

5.6 ± 0.3

7.2 ± 0.7

5.2 ± 0.5

6.8 ± 0.5

8.5 ± 0.6

5a

5.3 ± 0.3

4.1 ± 0.6

7.4 ± 0.4

6.3 ± 0.5

4.7 ± 0.4

5b

4.9 ± 0.2

6.8 ± 0.5

6.4 ± 0.1

7.6 ± 0.3

5.4 ± 0.2

5c

5.3 ± 0.2

9.3 ± 0.2

7.5 ± 0.4

8.4 ± 0.2

8.1 ± 0.1

5d

13.4 ± 0.1

11.0 ± 0.6

9.4 ± 0.5

9.3 ± 0.4

7.3 ± 0.4

5e

4.5 ± 0.3

8.7 ± 0.8

6.4 ± 0.4

4.5 ± 0.9

5.4 ± 1.0

5f

4.4 ± 0.2

4.6 ± 0.7

3.9 ± 0.8

7.2 ± 0.4

3.7 ± 0.7

5g

16.6 ± 0.4

14.1 ± 0.5

> 20

17.4 ± 0.1

10.8 ± 1.2

5h

> 20

> 20

> 20

> 20

17.6 ± 0.1

5i

13.4 ± 0.9

> 20

18.7 ± 1.2

> 20

15.4 ± 0.7

HCPT

29.1 ± 2.6

34.5 ± 1.5

28.1 ± 1.0

27.8 ± 1.2

48.2 ± 0.4

ADM

0.7 ± 0.2

0.6 ± 0.1

1.2 ± 0.2

1.0 ± 0.6

1.35 ± 0.1

AC C

EP

TE D

M AN U

9.3 ± 0.2

19

ACCEPTED MANUSCRIPT Table 2 Selectivity index of the compounds towards tumor cells MGC-803

PC3

Bcap-37

A375

MCF-7

3i

6.15

5.32

4.50

2.54

2.96

3j

7.18

4.28

6.11

3.38

5.31

3k

7.38

4.75

6.33

3.41

5.11

5a

7.62

9.85

5.46

6.41

8.60

5e

4.69

2.43

3.30

4.69

3.91

5f

4.20

4.02

4.74

2.57

AC C

EP

TE D

M AN U

SC

RI PT

Compound

20

5.00

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

staining. (C) TUNEL assay.

M AN U

Figure 1. Apoptosis induction studies of compound 3k. (A) AO\EB staining. (B) Hoechst 33258

21

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Figure 2. The apoptosis ratios, including the early (B4) and late apoptosis ratios (B2).

22

SC

RI PT

ACCEPTED MANUSCRIPT

Fi

Figure 3. Annexin V/PI dual staining of MGC-803 cell lines. A: negative control; B, C, and D:

M AN U

cells treated with HCPT (10 µM) as positive control; E, F, and G: cells treated with compound 3k

AC C

EP

TE D

(10 µM).

23

RI PT

ACCEPTED MANUSCRIPT

SC

Figure 4. Expression levels of caspases, p53, and Bax. A: western blot analysis of p53 and Bax in

AC C

EP

TE D

M AN U

MGC-803 cells; B: activation of caspase 3/9 in MGC-803 cells.

24

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Scheme 1. Regents and conditions: (i) BrCH2CH2Br, BrCH2CH2CH2Br, or BrCH2(CH2)2CH2Br,

AC C

EP

TE D

K2CO3, DMF, r.t.; (ii) amine, K2CO3, DMF, r.t.

25

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Scheme 2. Reagents and conditions: (i) piperazine, K2CO3, DMF, 80 °C; (ii) EDCI, HOBT,

AC C

EP

TE D

aromatic carboxylic acids, DCM, r.t..

26

ACCEPTED MANUSCRIPT Highlights 1. Two series of betulonic acid derivatives were designed and synthesized. 2. The new compounds showed good antiproliferative activities against tumor cells. 3. Further mechanism study indicates that the new compounds can induce cell apoptosis through

AC C

EP

TE D

M AN U

SC

RI PT

the mitochondrial intrinsic pathway.

ACCEPTED MANUSCRIPT

Supporting Information Synthesis and biological evaluation of betulonic acid derivatives as antitumor agents

a

RI PT

Sheng-Jie Yanga,b,c¶, Ming-Chuan Liua,b¶, Qi Zhaoa, De-Yu Hua, Wei Xuea, and Song Yanga*

State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key

Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou

SC

University, Guiyang 550025, P.R. China

Research Institute, Sinphar Tian-Li Pharmaceutical Co., Ltd, Hangzhou, 311100, P.R. China

c

Research Institute of Traditional Chinese Medicine, Yangtze River Pharmaceutical Group Co.,

Ltd, Taizhou, 225321, P.R. China

¶

M AN U

b

Both authors contributed equally to this work.

*Corresponding author. Ctr for R&D of Fine Chemicals, Guizhou University, Huaxi St., Guiyang,

TE D

China 550025. Tel.: +86 851 829 2171; Fax: +86 851 829 2170. E-mail address: [email protected]

Data of other compounds

EP

Compound (1): Yield: 81.8%; white powder; mp 291-293 oC; IR (KBr, cm-1): νmax 3440, 2945, 1689, 1680; 1H NMR (CDCl3, 500 MHz) δ: 4.71 (1H, s, H-29), 4.58 (1H, s, H-29), 3.01 (3H, m,

AC C

H-2, 19), 1.71 (3H, s, CH3), 1.10 (3H, s, CH3), 0.99 (3H, s, CH3), 0.95 (3H, s, CH3), 0.93 (3H, s, CH3); 13C NMR (CDCl3, 125 MHz) δ: 219.5 (C-3), 181.8 (C-28), 150.4 (C-20), 109.7 (C-29), 55.9 (C-17), 54.8 (C-5), 49.8 (C-9), 49.5 (C-18), 47.4 (C-4), 47.0 (C-19), 42.5 (C-14), 40.6 (C-8), 39.7 (C-1), 38.6 (C-13), 37.2 (C-22), 36.8 (C-10), 34.2 (C-2), 33.7 (C-7), 32.2 (C-16), 30.7 (C-21), 29.7 (C-15), 26.8 (C-23), 25.6 (C-12), 21.5 (C-11),21.3 (C-24), 19.5 (C-6), 19.4 (C-30), 16.1 (C-26), 15.8 (C-25), 14.5 (C-27). The above data were identical to the literature data [1].

Compound (2a): Yield: 76.9%; colorless oil; IR (KBr, cm-1): νmax 3444, 2955, 2870, 1640, 1428, 881; 1H NMR (CDCl3, 500 MHz) δ: 4.72 (1H, s, Hb-29), 4.60 (1H, s, Ha-29),4.24 (2H, t, J = 7.5 1

ACCEPTED MANUSCRIPT Hz, CH2), 3.69 (2H, t, J = 10.5 Hz, CH2), 2.99 (3H, m, H-2, 19), 1.67 (3H, s, CH3), 1.06 (3H, s, CH3), 1.01 (3H, s, CH3), 0.97 (3H, s, CH3), 0.95 (3H, s, CH3), 0.91(3H, s, CH3); 13C NMR (CDCl3, 125 MHz) δ: 218.2 (C-3), 175.9 (C-28), 150.4 (C-20), 109.8 (C-29), 62.1 (CH2), 55.6 (C-17), 55.1 (C-5), 49.9 (C-9), 49.4 (C-18), 47.4 (C-4), 46.9 (C-19), 42.5 (C-14), 40.7 (C-8), 39.7 (C-1), 38.4

RI PT

(C-13), 36.9 (C-22), 36.9 (C-10), 34.2 (C-2), 33.7 (C-7), 32.1 (C-16), 30.7 (C-21), 29.7 (CH2), 29.7 (C-15), 26.8 (C-23), 25.6 (C-12), 21.5 (C-11),21.1 (C-24), 19.7 (C-6), 19.5 (C-30), 16.0 (C-26), 15.9 (C-25), 14.7 (C-27)

SC

Compound (2b): Yield: 71.3%; colorless oil; IR (KBr, cm-1): νmax 3446, 3069, 2850, 1705, 1453, 884; 1H NMR (CDCl3, 500 MHz) δ: 4.70 (1H, s, Hb-29), 4.57 (1H, s, Ha-29), 4.20 (4H, m, CH2),

M AN U

3.46 (2H, t, J = 13 Hz, CH2), 2.97 (3H, m, H-2, 19), 1.65 (3H, s, CH3), 1.03 (3H, s, CH3), 0.98 (3H, s, CH3), 0.94 (3H, s, CH3), 0.93 (3H, s, CH3), 0.89 (3H, s, CH3); 13C NMR (CDCl3, 125 MHz) δ: 218.2 (C-3), 175.9 (C-28), 150.4 (C-20), 109.8 (C-29), 61.6 (CH2), 56.6 (C-17), 55.0 (C-5), 49.9 (C-9), 49.4 (C-18), 47.4 (C-4), 47.0 (C-19), 42.5 (C-14), 40.7 (C-8), 39.7 (C-1), 38.4 (C-13), 37.0 (C-22), 36.9 (C-10), 34.2 (C-2), 33.7 (C-7), 32.2 (C-16), 31.7 (C-21), 30.6 (CH2), 29.7 (C-15),

TE D

29.6 (CH2), 26.7 (C-23), 25.5 (C-12), 21.5 (C-11), 21.1 (C-24), 19.7 (C-6), 19.5 (C-30), 16.1 (C-26), 15.9 (C-25), 14.7 (C-27).

Compound (2c): Yield: 74.2%; colorless oil; IR (KBr, cm-1): νmax 3449, 2933, 2872, 1695, 1458,

EP

882; 1H NMR (CDCl3, 500 MHz) δ: 4.70 (1H, s, Hb-29), 4.57 (1H, s, Ha-29), 4.12 (2H, t, J = 7.5

AC C

Hz, CH2), 3.51 (2H, t, J = 11.5 Hz, CH2), 2.99 (3H, m, H-2, 19), 1.75 (4H, m, CH2), 1.66 (3H, s, CH3), 1.02 (3H, s, CH3), 0.97 (3H, s, CH3), 0.94 (3H, s, CH3), 0.91 (3H, s, CH3), 0.88 (3H, s, CH3); 13C NMR (CDCl3, 125 MHz) δ: 218.2 (C-3), 175.9 (C-28), 150.5 (C-20), 110.1 (C-29), 62.6 (CH2), 56.6 (C-17), 55.0 (C-5), 49.9 (C-9), 49.4 (C-18), 47.3 (C-4), 47.2 (C-19), 42.5 (C-14), 40.5 (C-8), 39.9 (C-1), 38.4 (C-13), 37.0 (C-22), 36.8 (C-10), 34.1 (C-2), 33.7 (C-7), 32.3 (C-16), 31.7 (C-21), 30.2 (CH2), 29.7 (C-15), 29.6 (CH2), 27.4 (CH2), 26.7 (C-23), 25.7 (C-12), 21.5 (C-11), 21.0 (C-24), 19.7 (C-6), 19.5 (C-30), 16.2 (C-26), 15.9 (C-25), 14.7 (C-27).

Compound (3a): Yield: 67.5%; pale yellow oil; IR (KBr, cm-1): νmax 3444, 2955, 1706, 1635, 1368, 881; 1H NMR (CDCl3, 500 MHz) δ: 4.71 (1H, s, Hb-29), 4.59 (1H, s, Ha-29), 4.20 (2H, t, J = 11.5 2

ACCEPTED MANUSCRIPT Hz, CH2), 1.65 (3H, s, CH3), 1.03 (3H, s, CH3), 1.01 (6H, t, J = 5.0 Hz, CH3), 0.99 (3H, s, CH3), 0.95 (3H, s, CH3), 0.91 (3H, s, CH3), 0.88 (3H, s, CH3);

13

C NMR (CDCl3, 125 MHz) δ: 218.6

(C-3), 175.5 (C-28), 150.6 (C-20), 109.9 (C-29), 62.6 (CH2), 56.5 (C-17), 55.0 (C-5), 52.4 (CH2), 49.9 (C-9), 49.2 (C-18), 48.5 (CH2), 47.4 (C-4), 47.0 (C-19), 42.5 (C-14), 40.9 (C-8), 39.7 (C-1),

RI PT

38.3 (C-13), 37.1 (C-22), 36.9 (C-10), 34.5 (C-2), 33.7 (C-7), 31.1 (C-16), 30.7 (C-21), 29.5 (C-15), 26.7 (C-23), 25.6 (C-12), 21.6 (C-11), 21.1 (C-24), 19.9 (C-6), 19.5 (C-30), 16.0 (C-26), 15.9 (C-25), 14.7 (C-27), 10.3 (CH3).

SC

Compound (3b): Yield: 72.4%; pale yellow oil; IR (KBr, cm-1): νmax 3442, 3066, 2945, 2870, 1705, 1641, 883; 1H NMR (CDCl3, 500 MHz) δ: 4.71 (1H, s, Hb-29), 4.58 (1H, s, Ha-29), 4.24 (2H, t, J

M AN U

= 10 Hz, CH2), 3.01 (3H, m, H-2, 19), 2.74 (2H, t, J = 12.5 Hz, CH2), 2.57 (4H, m, CH2), 1.77 (4H, m, CH2), 1.67 (3H, s, CH3), 1.05 (3H, s, CH3), 1.01 (3H, s, CH3), 0.95 (3H, s, CH3), 0.93 (3H, s, CH3), 0.90 (3H, s, CH3); 13C NMR (CDCl3, 125 MHz) δ: 218.3 (C-3), 176.0 (C-28), 150.7 (C-20), 109.7 (C-29), 62.9 (CH2), 56.5 (CH2), 55.0 (C-17), 54.6 (CH2), 54.5 (C-5), 50.0 (C-9), 49.4 (C-18), 47.4 (C-4), 46.9 (C-19), 42.5 (C-14), 40.7 (C-8), 39.7 (C-1), 38.4 (C-13), 37.0 (C-22), 36.9 (C-10),

TE D

34.2 (C-2), 33.7 (C-7), 32.1 (C-16), 31.0 (C-21), 29.7 (C-15), 26.7 (C-23), 25.6 (C-12), 23.6 (CH2), 21.5 (C-11), 21.1 (C-24), 19.7 (C-6), 19.5 (C-30), 16.0 (C-26), 15.9 (C-25), 14.7 (C-27).

Compound (3c): Yield: 78.5%; pale yellow oil; IR (KBr, cm-1): νmax 3445, 3081, 2939, 2861, 1704,

EP

1456, 881; 1H NMR (CDCl3, 500 MHz) δ: 4.71 (1H, s, Hb-29), 4.58 (1H, s, Ha-29), 4.23 (2H, t, J

AC C

= 12 Hz, CH2), 2.99 (3H, m, H-2, 19), 2.62 (2H, t, J = 12 Hz, CH2), 2.46 (4H, m, CH2), 1.68 (3H, s, CH3), 1.58 (6H, m, CH2), 1.04 (3H, s, CH3), 1.02 (3H, s, CH3), 0.99 (3H, s, CH3), 0.93 (3H, s, CH3), 0.90 (3H, s, CH3); 13C NMR (CDCl3, 125 MHz) δ: 218.2 (C-3), 175.9 (C-28), 150.6 (C-20), 109.7 (C-29), 61.3 (CH2), 57.3 (CH2), 56.6 (C-17), 55.0 (C-5), 54.7 (CH2), 49.9 (C-9), 49.4 (C-18), 47.4 (C-4), 46.9 (C-19), 42.5 (C-14), 40.7 (C-8), 39.7 (C-1), 38.4 (C-13), 37.0 (C-22), 36.9 (C-10), 34.2 (C-2), 33.7 (C-7), 32.1 (C-16), 30.7 (C-21), 29.7 (C-15), 26.7 (C-23), 25.8 (CH2), 25.6 (C-12), 24.1 (CH2), 21.5 (C-11), 21.1 (C-24), 19.7 (C-6), 19.5 (C-30), 16.0 (C-26), 15.9 (C-25), 14.7 (C-27).

Compound (3d): Yield: 73.1%; pale yellow oil; IR (KBr, cm-1): νmax 3450, 2939, 1701, 1640, 1463, 3

ACCEPTED MANUSCRIPT 883; 1H NMR (CDCl3, 500 MHz) δ: 4.71 (1H, s, Hb-29), 4.58 (1H, s, Ha-29), 4.22 (2H, t, J = 12 Hz, CH2), 3.69 (4H, m, J = 9.5 Hz, CH2), 2.99 (3H, m, H-2, 19), 2.62 (2H, t, J = 11.5 Hz, CH2), 2.49 (4H, m, CH2), 1.67 (3H, s, CH3), 1.05 (3H, s, CH3), 1.03 (3H, s, CH3), 0.96 (3H, s, CH3), 0.94 (3H, s, CH3), 0.90 (3H, s, CH3); 13C NMR (CDCl3, 125 MHz) δ: 218.3 (C-3), 175.0 (C-28),

RI PT

150.6 (C-20), 109.7 (C-29), 67.1 (CH2), 60.8 (CH2), 57.3 (CH2), 56.6 (C-17), 55.0 (C-5), 53.8 (CH2), 49.9 (C-9), 49.4 (C-18), 47.4 (C-4), 47.0 (C-19), 42.5 (C-14), 40.7 (C-8), 39.7 (C-1), 38.4 (C-13), 37.0 (C-22), 36.9 (C-10), 34.2 (C-2), 33.7 (C-7), 32.1 (C-16), 30.7 (C-21), 29.7 (C-15), 26.7 (C-23), 25.6 (C-12), 21.5 (C-11), 21.1 (C-24), 19.7 (C-6), 19.4 (C-30), 16.0 (C-26), 15.9

SC

(C-25), 14.7 (C-27).

M AN U

Compound (3e): Yield: 69.6%; pale yellow oil; IR (KBr, cm-1): νmax 3446, 2944, 2868, 1711, 1635, 1363, 880; 1H NMR (CDCl3, 500 MHz) δ: 4.71 (1H, s, Hb-29), 4.59 (1H, s, Ha-29), 4.15 (2H, t, J = 12 Hz, CH2), 2.95 (3H, m, H-2, 19), 2.47 (2H, m, CH2), 1.67 (3H, s, CH3), 1.05 (3H, s, CH3), 1.03 (3H, s, CH3), 1.00 (6H, t, J = 4.5 Hz, CH3), 0.96 (3H, s, CH3), 0.93 (3H, s, CH3), 0.90 (3H, s, CH3); 13C NMR (CDCl3, 125 MHz) δ: 218.2 (C-3), 175.9 (C-28), 150.3 (C-20), 109.9 (C-29), 61.3

TE D

(CH2), 56.6 (C-17), 54.7 (C-5), 49.9 (C-9), 49.3 (C-18), 48.8 (CH2), 47.4 (C-4), 46.9 (C-19), 46.5 (CH2), 42.5 (C-14), 40.7 (C-8), 39.7 (C-1), 38.4 (C-13), 37.1 (C-22), 36.9 (C-10), 34.2 (C-2), 33.7 (C-7), 32.1 (C-16), 30.7 (C-21), 29.7 (C-15), 26.7 (C-23), 25.5 (C-12), 24.0 (CH2), 21.5 (C-11),

EP

21.1 (C-24), 19.7 (C-6), 19.4 (C-30), 16.0 (C-26), 15.9 (C-25), 14.7 (C-27), 9.22 (CH2).

Compound (3f): Yield: 70.8%; pale yellow oil; IR (KBr, cm-1): νmax 3438, 2947, 2833, 1712, 1458,

AC C

886; 1H NMR (CDCl3, 500 MHz) δ: 4.66 (1H, s, Hb-29), 4.54 (1H, s, Ha-29), 4.09 (2H, d, J = 12.5 Hz, CH2), 3.02 (3H, m, H-2, 19), 2.41 (4H, m, CH2), 1.60 (3H, s, CH3), 0.99 (3H, s, CH3), 0.94 (3H, s, CH3), 0.90 (3H, s, CH3), 0.87 (3H, s, CH3), 0.85 (3H, s, CH3); 13C NMR (CDCl3, 125 MHz) δ: 218.3 (C-3), 175.8 (C-28), 150.2 (C-20), 109.9 (C-29), 60.9 (CH2), 56.5 (CH2), 54.9 (C-17), 53.6 (CH2), 52.7 (C-5), 49.9 (C-9), 49.3 (C-18), 47.3 (C-4), 46.9 (C-19), 42.5 (C-14), 40.7 (C-8), 39.6 (C-1), 38.3 (C-13), 36.9 (C-22), 36.9 (C-10), 34.2 (C-2), 33.6 (C-7), 31.9 (C-16), 30.5 (C-21), 29.7 (C-15), 26.7 (C-23), 25.6 (CH2), 25.5 (C-12), 23.4 (CH2), 21.5 (C-11),21.1 (C-24), 19.7 (C-6), 19.4 (C-30), 16.0 (C-26), 15.9 (C-25), 14.7 (C-27).

4

ACCEPTED MANUSCRIPT Compound (3g): Yield: 75.8%; pale yellow oil; IR (KBr, cm-1): νmax 3458, 3067, 2942, 1704, 1641, 883; 1H NMR (CDCl3, 500 MHz) δ: 4.67 (1H, s, Hb-29), 4.55 (1H, s, Ha-29), 4.07 (2H, m, CH2), 3.46 (2H, t, J = 13 Hz, CH2), 2.95 (3H, m, H-2, 19), 2.44 (2H, m, CH2), 2.35 (4H, m, CH2), 1.63 (3H, s, CH3), 1.54 (6H, m, CH2), 1.03 (3H, s, CH3), 0.96 (3H, s, CH3), 0.92 (3H, s, CH3), 0.90 (3H, 13

C NMR (CDCl3, 125 MHz) δ: 218.1 (C-3), 176.1 (C-28), 150.6

RI PT

s, CH3), 0.87 (3H, s, CH3);

(C-20), 109.7 (C-29), 62.6 (CH2), 56.5 (CH2), 56.1 (C-17), 55.0 (CH2), 54.7 (C-5), 49.9 (C-9), 49.3 (C-18), 47.4 (C-4), 47.0 (C-19), 42.5 (C-14), 40.7 (C-8), 39.7 (C-1), 38.4 (C-13), 37.0 (C-22), 36.9 (C-10), 34.2 (C-2), 33.7 (C-7), 32.1 (C-16), 30.7 (C-21), 29.7 (C-15), 26.7 (C-23), 26.4 (CH2),

SC

26.0 (CH2), 25.6 (C-12), 24.5 (CH2), 21.5 (C-11),21.1 (C-24), 19.7 (C-6), 19.4 (C-30), 16.0 (C-26),

M AN U

15.9 (C-25), 14.7 (C-27).

Compound (3h): Yield: 74.7%; pale yellow oil; IR (KBr, cm-1): νmax 3439, 3066, 2949, 2869, 1702, 1451, 884; 1H NMR (CDCl3, 500 MHz) δ: 4.68 (1H, s, Hb-29), 4.55 (1H, s, Ha-29), 4.11 (2H, m, CH2), 3.68 (4H, t, J = 9 Hz, CH2), 2.98 (3H, m, H-2, 19), 2.40 (4H, m, CH2), 2.21 (2H, m, CH2), 1.66 (3H, s, CH3), 1.56 (2H, m, CH2), 1.052 (3H, s, CH3), 0.97 (3H, s, CH3), 0.93 (3H, s, CH3),

TE D

0.90 (3H, s, CH3), 0.87 (3H, s, CH3); 13C NMR (CDCl3, 125 MHz) δ: 218.1 (C-3), 176.1 (C-28), 150.5 (C-20), 109.8 (C-29), 67.0 (CH2), 62.2 (CH2), 56.5 (C-17), 55.6 (CH2), 55.0 (C-5), 53.7 (CH2), 49.9 (C-9), 49.3 (C-18), 47.4 (C-4), 47.0 (C-19), 42.5 (C-14), 40.7 (C-8), 39.7 (C-1), 38.4

EP

(C-13), 37.0 (C-22), 36.9 (C-10), 34.2 (C-2), 33.7 (C-7), 32.1 (C-16), 30.7 (C-21), 29.7 (C-15), 26.6 (C-23), 25.9 (CH2), 25.6 (C-12), 21.5 (C-11), 21.1 (C-24), 19.7 (C-6), 19.4 (C-30), 16.0

AC C

(C-26), 15.9 (C-25), 14.7 (C-27).

Compound (3i): Yield: 72.1%; pale yellow oil; IR (KBr, cm-1): νmax 3445, 2928, 2860, 1690, 1455, 885; 1H NMR (CDCl3, 500 MHz) δ: 4.70 (1H, s, Hb-29), 4.57 (1H, s, Ha-29), 4.26 (2H, d, J =10 Hz, CH2), 2.99 (3H, m, H-2, 19), 2.68 (4H, m, CH2), 2.54 (2H, m, CH2), 1.65 (3H, s, CH3), 1.03 (3H, s, CH3), 1.02 (3H, s, CH3), 1.01 (6H, m, J = 5 Hz, CH3), 0.99 (3H, s, CH3), 0.98 (3H, s, CH3), 0.94 (3H, s, CH3), 0.89 (3H, s, CH3); 13C NMR (CDCl3, 125 MHz) δ: 218.2 (C-3), 176.1 (C-28), 150.6 (C-20), 109.7 (C-29), 62.1 (CH2), 56.5 (C-17), 55.0 (C-5), 51.3 (CH2), 49.9 (C-9), 49.4 (C-18), 47.6 (C-4), 47.4 (C-19), 46.9 (CH2), 42.5 (C-14), 40.7 (C-8), 39.7 (C-1), 38.4 (C-13), 37.0 (C-22), 36.9 (C-10), 34.2 (C-2), 33.7 (C-7), 32.1 (C-16), 30.6 (C-21), 29.7 (C-15), 26.7 (CH2), 5

ACCEPTED MANUSCRIPT 26.7 (C-23), 25.6 (C-12), 23.8 (CH2), 21.5 (C-11), 21.1 (C-24), 19.7 (C-6), 19.4 (C-30), 16.0 (C-26), 15.9 (C-25), 14.7 (C-27), 12.0 (CH3).

Compound (3j): Yield: 74.4%; pale yellow oil; IR (KBr, cm-1): νmax 3454, 2928, 2854, 1704, 1364,

RI PT

881; 1H NMR (CDCl3, 500 MHz) δ: 4.69 (1H, s, Hb-29), 4.57 (1H, s, Ha-29), 4.08 (2H, d, J = 12.5 Hz, CH2), 2.96 (3H, m, H-2, 19), 2.46 (4H, m, CH2), 2.37 (2H, m, CH2), 1.78 (4H, m, CH2), 1.65 (3H, s, CH3), 1.03 (3H, s, CH3), 1.01 (3H, s, CH3), 0.98 (3H, s, CH3), 0.94 (3H, s, CH3), 0.89 (3H, s, CH3); 13C NMR (CDCl3, 125 MHz) δ: 218.3 (C-3), 176.1 (C-28), 150.4 (C-20), 109.8 (C-29),

SC

62.7 (CH2), 56.5 (CH2), 55.2 (C-17), 55.0 (C-5), 49.9 (C-9), 49.4 (C-18), 47.4 (C-4), 47.0 (C-19), 42.5 (C-14), 40.7 (C-8), 39.7 (C-1), 38.4 (C-13), 37.0 (C-22), 36.9 (C-10), 34.2 (C-2), 33.7 (C-7),

M AN U

32.1 (C-16), 30.6 (C-21), 29.7 (C-15), 26.7 (C-23), 26.4 (CH2), 25.6 (C-12), 23.4 (CH2), 22.9 (CH2), 21.5 (C-11), 21.1 (C-24), 19.7 (C-6), 19.4 (C-30), 16.0 (C-26), 15.9 (C-25), 14.7 (C-27).

Compound (3l): Yield: 73.5%; pale yellow oil; IR (KBr, cm-1): νmax 3484, 3112, 2944, 1713, 1459, 885; 1H NMR (CDCl3, 500 MHz) δ: 4.69 (1H, s, Hb-29), 4.56 (1H, s, Ha-29), 4.07 (2H, m, CH2),

TE D

3.67 (4H, t, J = 7.5 Hz, CH2), 2.96 (3H, m, H-2, 19), 2.44 (4H, m, CH2), 2.33 (2H, m, CH2), 2.21 (4H, m, CH2), 1.64 (3H, s, CH3), 1.02 (3H, s, CH3), 0.97 (3H, s, CH3), 0.93 (3H, s, CH3), 0.90 (3H, s, CH3), 0.88 (3H, s, CH3);

13

C NMR (CDCl3, 125 MHz) δ: 218.2 (C-3), 176.1 (C-28), 150.5

EP

(C-20), 109.7 (C-29), 66.7 (CH2), 63.8 (CH2), 58.6 (CH2), 56.5 (C-17), 55.0 (C-5), 53.7 (CH2), 49.9 (C-9), 49.4 (C-18), 47.4 (C-4), 47.0 (C-19), 42.5 (C-14), 40.7 (C-8), 39.7 (C-1), 38.4 (C-13),

AC C

37.0 (C-22), 36.9 (C-10), 34.2 (C-2), 33.7 (C-7), 32.1 (C-16), 30.6 (C-21), 29.7 (C-15), 26.8 (C-23), 26.7 (CH2), 25.6 (C-12), 23.1 (CH2), 21.5 (C-11), 21.1 (C-24), 19.7 (C-6), 19.4 (C-30), 16.0 (C-26), 15.9 (C-25), 14.7 (C-27).

Compound (5b): Yield: 68.5%; colorless oil; IR (KBr, cm-1): νmax 3451, 2944, 2881, 1699, 1461, 880, 761; 1H NMR (CDCl3, 500 MHz) δ: 7.39 (1H, m, PhH), 7.12 (3H, m, PhH), 4.70 (1H, s, Hb-29), 4.59 (1H, s, Ha-29), 4.22 (2H, d, J = 9.5 Hz, CH2), 3.76 (2H, brs, H in piperazine), 3.38 (2H, brs, H in piperazine), 2.98 (3H, m, H-2, 19), 2.65 (2H, m, CH2), 2.58 (2H, brs, H in piperazine), 2.47 (2H, m, H in piperazine), 1.67 (3H, s, CH3), 1.06 (3H, s, CH3), 1.00 (3H, s, CH3), 0.96 (3H, s, CH3), 0.93 (3H, s, CH3), 0.90 (3H, s, CH3); 6

13

C NMR (CDCl3, 125 MHz) δ: 218.3

ACCEPTED MANUSCRIPT (C-3), 175.9 (C-28), 168.8 (C=O), 163.6 (C), 150.5 (C-20), 137.8 (C), 130.5 (CH), 122.9 (CH), 116.9 (CH), 114.5 (CH), 109.8 (C-29), 60.8 (CH2), 56.8 (CH2), 56.6 (C-17), 55.0 (C-5), 53.6 (CH2), 53.0 (CH2), 49.9 (C-9), 49.3 (C-18), 47.7 (CH2), 47.4 (C-4), 47.0 (C-19), 42.5 (C-14), 42.3 (CH2), 40.7 (C-8), 39.7 (C-1), 38.3 (C-13), 37.1 (C-22), 36.9 (C-10), 34.2 (C-2), 33.7 (C-7), 32.1

19.4 (C-30), 16.0 (C-26), 15.9 (C-25), 14.7 (C-27).

RI PT

(C-16), 30.6 (C-21), 29.7 (C-15), 26.7 (C-23), 25.6 (C-12), 21.5 (C-11), 21.1 (C-24), 19.7 (C-6),

Compound (5c): Yield: 64.7%; colorless oil; IR (KBr, cm-1): νmax 3457, 2944, 2870, 1705, 1641,

SC

1366, 881, 744; 1H NMR (CDCl3, 500 MHz) δ: 7.29 (2H, m, PhH), 7.02 (1H, t, J = 7.5 Hz, PhH), 4.69 (1H, s, Hb-29), 4.58 (1H, s, Ha-29), 4.19 (2H, t, J = 8 Hz, CH2), 3.81 (2H, brs, H in

M AN U

piperazine), 3.25 (2H, brs, H in piperazine), 2.97 (3H, m, H-2, 19), 2.65 (2H, t, J = 11 Hz, CH2), 2.58 (2H, brs, H in piperazine), 2.46 (2H, m, H in piperazine), 1.66 (3H, s, CH3), 1.04 (3H, s, CH3), 0.99 (3H, s, CH3), 0.94 (3H, s, CH3), 0.92 (3H, s, CH3), 0.89 (3H, s, CH3);

13

C NMR

(CDCl3, 125 MHz) δ: 218.3 (C-3), 175.9 (C-28), 161.9 (C=O), 157.7 (C), 150.5 (C-20), 131.9 (C), 130.9 (C), 135.6 (CH), 124.5 (CH), 114.6 (CH), 109.8 (C-29), 60.8 (CH2), 56.8 (CH2), 56.6

TE D

(C-17), 55.0 (C-5), 53.4 (CH2), 52.8 (CH2), 49.9 (C-9), 49.3 (C-18), 47.4 (CH2), 47.0 (C-4), 46.6 (C-19), 42.5 (C-14), 41.8 (CH2), 40.7 (C-8), 39.7 (C-1), 38.3 (C-13), 37.1 (C-22), 36.9 (C-10), 34.2 (C-2), 33.7 (C-7), 32.1 (C-16), 30.6 (C-21), 29.7 (C-15), 26.7 (C-23), 25.6 (C-12), 21.5

EP

(C-11), 21.1 (C-24), 19.7 (C-6), 19.4 (C-30), 16.0 (C-26), 16.0 (C-25), 14.7 (C-27).

Compound (5d): Yield: 61.8%; colorless oil; IR (KBr, cm-1): νmax 3450, 2924, 2876, 1690, 1458,

AC C

883, 746; 1H NMR (CDCl3, 500 MHz) δ: 7.66 (1H, d, J = 15.5 Hz, PhH), 7.50 (2H, d, J = 6 Hz, PhH), 7.35 (2H, m, PhH), 6.87 (1H, d, J = 15 Hz, CH), 5.22 (1H, s, CH), 4.70 (1H, s, Hb-29), 4.58 (1H, s, Ha-29), 4.20 (2H, t, J = 10.5 Hz, CH2), 3.72 (2H, brs, H in piperazine), 2.99 (3H, m, H-2, 19), 2.64 (2H, m, CH2), 2.53 (2H, m, H in piperazine), 2.44 (2H, m, H in piperazine), 1.67 (3H, s, CH3), 1.03 (3H, s, CH3), 0.99 (3H, s, CH3), 0.95 (3H, s, CH3), 0.93 (3H, s, CH3), 0.89 (3H, s, CH3);

13

C NMR (CDCl3, 125 MHz) δ: 218.2 (C-3), 175.9 (C-28), 165.4 (C=O), 150.5 (C-20),

142.9 (CH), 135.3 (C), 129.7 (CH), 128.9 (CH), 127.8 (CH), 117.1 (CH), 109.8 (C-29), 60.9 (CH2), 56.8 (CH2), 55.6 (C-17), 55.0 (C-5), 53.5 (CH2), 52.9 (CH2), 49.9 (C-9), 49.4 (C-18), 47.4 (C-4), 47.0 (C-19), 46.0 (CH2), 42.5 (C-14), 42.2 (CH2), 40.7 (C-8), 39.7 (C-1), 38.4 (C-13), 37.0 7

ACCEPTED MANUSCRIPT (C-22), 36.9 (C-10), 34.2 (C-2), 33.7 (C-7), 32.1 (C-16), 30.7 (C-21), 29.7 (C-15), 26.7 (C-23), 25.6 (C-12), 21.5 (C-11), 21.1 (C-24), 19.7 (C-6), 19.4 (C-30), 16.0 (C-26), 15.9 (C-25), 14.7 (C-27).

RI PT

Compound (5e): Yield: 62.1%; colorless oil; IR (KBr, cm-1): νmax 3446, 2931, 1710, 1640, 887, 768, 711; 1H NMR (CDCl3, 500 MHz) δ: 7.19 (4H, m, PhH), 4.69 (1H, s, Hb-29), 4.57 (1H, s, Ha-29), 4.19 (2H, t, J = 9.5 Hz, CH2), 3.79 (2H, brs, H in piperazine), 2.97 (3H, m, H-2, 19), 2.63 (2H, t, J = 9 Hz, CH2), 2.46 (2H, m, H in piperazine), 2.38 (2H, m, H in piperazine), 1.67 (3H, s,

CH3);

SC

CH3), 1.04 (3H, s, CH3), 1.01 (3H, s, CH3), 0.94 (3H, s, CH3), 0.92 (3H, s, CH3), 0.89 (3H, s, 13

C NMR (CDCl3, 125 MHz) δ: 218.2 (C-3), 175.9 (C-28), 170.0 (C=O), 150.5 (C-20),

M AN U

136.1 (C), 134.2 (C), 130.5 (CH), 128.9 (CH), 126.0 (CH), 125.8 (CH), 109.8 (C-29), 60.8 (CH2), 56.8 (CH2), 55.6 (C-17), 55.0 (C-5), 53.7 (CH2), 53.1 (CH2), 49.9 (C-9), 49.3 (C-18), 47.4 (CH2), 47.0 (C-4), 46.9 (C-19), 42.5 (C-14), 41.5 (CH2), 40.7 (C-8), 39.7 (C-1), 38.4 (C-13), 37.0 (C-22), 36.9 (C-10), 34.2 (C-2), 33.7 (C-7), 32.1 (C-16), 30.6 (C-21), 29.7 (C-15), 26.7 (C-23), 25.6 (C-12), 21.5 (C-11), 21.1 (C-24), 19.7 (C-6), 19.4 (CH3), 19.1 (C-30), 16.0 (C-26), 15.9 (C-25),

TE D

14.7 (C-27).

Compound (5f): Yield: 66.7%; colorless oil; IR (KBr, cm-1): νmax 3444, 2955, 1681, 1643, 1377,

EP

885, 755, 661; 1H NMR (CDCl3, 500 MHz) δ: 7.38 (1H, m, PhH), 6.94 (1H, t, J = 7.5 Hz, PhH), 6.82 (1H, J = 12 Hz, PhH), 4.70 (1H, s, Hb-29), 4.58 (1H, s, Ha-29), 4.20 (2H, d, J = 10.5 Hz,

AC C

CH2), 3.77 (2H, brs, H in piperazine), 3.30 (2H, brs, H in piperazine), 2.97 (3H, m, H-2, 19), 2.64 (2H, m, CH2), 2.58 (2H, m, H in piperazine), 2.47 (2H, m, H in piperazine), 1.66 (3H, s, CH3), 1.05 (3H, s, CH3), 1.00 (3H, s, CH3), 0.95 (3H, s, CH3), 0.93 (3H, s, CH3), 0.90 (3H, s, CH3); 13C NMR (CDCl3, 125 MHz) δ: 218.2 (C-3), 175.9 (C-28), 164.4 (C=O), 162.6 (C), 159.8 (C), 150.5 (C-20), 130.7 (CH), 120.4 (C), 112.4 (CH), 109.8 (C-29), 104.8 (CH), 60.8 (CH2), 56.7 (CH2), 56.5 (C-17), 55.0 (C-5), 53.4 (CH2), 52.9 (CH2), 49.9 (C-9), 49.4 (C-18), 47.4 (CH2), 47.2 (C-4), 47.0 (C-19), 42.5 (C-14), 42.2 (CH2), 40.7 (C-8), 39.7 (C-1), 38.3 (C-13), 37.1 (C-22), 36.9 (C-10), 34.2 (C-2), 33.7 (C-7), 32.1 (C-16), 30.6 (C-21), 29.7 (C-15), 26.7 (C-23), 25.6 (C-12), 21.5 (C-11), 21.1 (C-24), 19.7 (C-6), 19.4 (C-30), 16.0 (C-26), 15.9 (C-25), 14.7 (C-27).

8

ACCEPTED MANUSCRIPT Compound (5g): Yield: 63.7%; colorless oil; pale yellow oil; IR (KBr, cm-1): νmax 3446, 2942, 2877, 1702, 1642, 885, 749; 1H NMR (CDCl3, 500 MHz) δ: 7.31 (1H, t, J = 7.5 Hz, PhH), 7.24 (2H, m, PhH), 4.69 (1H, s, Hb-29), 4.57 (1H, s, Ha-29), 4.17 (2H, m, CH2), 3.81 (2H, m, H in piperazine), 3.22 (2H, t, J = 9.5 Hz, H in piperazine), 2.96 (3H, m, H-2, 19), 2.64 (2H, t, J = 11 Hz,

RI PT

CH2), 2.60 (2H, t, J = 10.5 Hz, H in piperazine), 2.48 (2H, t, J = 13 Hz, H in piperazine), 1.67 (3H, s, CH3), 1.04 (3H, s, CH3), 0.99 (3H, s, CH3), 0.94 (3H, s, CH3), 0.92 (3H, s, CH3), 0.88 (3H, s, CH3);

13

C NMR (CDCl3, 125 MHz) δ: 218.3 (C-3), 175.9 (C-28), 163.7 (C=O), 150.5 (C-20),

135.0 (C), 131.8 (C), 130.5 (CH), 128.1 (CH), 109.8 (C-29), 60.8 (CH2), 56.8 (CH2), 56.5 (C-17),

SC

55.9 (C-5), 53.3 (CH2), 52.7 (CH2), 49.9 (C-9), 49.3 (C-18), 47.4 (CH2), 47.0 (C-4), 46.3 (C-19), 42.5 (C-14), 41.6 (CH2), 40.7 (C-8), 39.7 (C-1), 38.3 (C-13), 37.1 (C-22), 36.9 (C-10), 34.2 (C-2),

M AN U

33.7 (C-7), 32.1 (C-16), 30.6 (C-21), 29.7 (C-15), 26.7 (C-23), 25.6 (C-12), 21.5 (C-11), 21.1 (C-24), 19.7 (C-6), 19.4 (C-30), 16.0 (C-26), 16.0 (C-25), 14.7 (C-27).

Compound (5h): Yield: 70.8%; colorless oil; IR (KBr, cm-1): νmax 3447, 2941, 2866, 1703, 1640, 882; 1H NMR (CDCl3, 500 MHz) δ: 4.69 (1H, s, Hb-29), 4.58 (1H, s, Ha-29), 4.20 (2H, t, J = 11.5

TE D

Hz, CH2), 3.58 (2H, brs, H in piperazine), 3.55 (2H, t, J = 10.5 Hz, H in piperazine), 2.98 (3H, m, H-2, 19), 2.63 (2H, m, CH2), 2.49 (2H, m, H in piperazine), 2.37 (2H, m, H in piperazine), 1.59 (3H, s, CH3), 1.03 (3H, s, CH3), 0.99 (3H, s, CH3), 0.95 (3H, s, CH3), 0.93 (3H, s, CH3), 0.86(3H,

EP

s, CH3), 0.83 (3H, t, J = 13.5 Hz, CH3); 13C NMR (CDCl3, 125 MHz) δ: 218.2 (C-3), 175.9 (C-28), 171.8 (C=O), 150.5 (C-20), 109.8 (C-29), 60.9 (CH2), 56.8 (CH2), 56.5 (C-17), 55.0 (C-5), 53.5

AC C

(CH2), 53.1 (CH2), 49.9 (C-9), 49.4 (C-18), 47.4 (C-4), 47.0 (C-19), 45.7 (CH2), 42.5 (C-14), 41.6 (CH2), 40.7 (C-8), 39.7 (C-1), 38.4 (C-13), 37.0 (C-22), 36.9 (C-10), 34.2 (C-2), 33.7 (C-7), 33.4 (CH2), 32.1 (C-16), 31.9 (CH2), 30.7 (C-21), 29.7 (C-15), 29.6 (CH2), 29.5 (CH2), 29.3 (CH2), 26.7 (C-23), 25.6 (C-12), 25.4 (CH2), 22.7 (CH2), 21.5 (C-11),21.1 (C-24), 19.7 (C-6), 19.4 (C-30), 16.0 (C-26), 15.9 (C-25), 14.7 (C-27), 14.2 (CH3).

Compound (5i): Yield: 68.6%; colorless oil; IR (KBr, cm-1): νmax 3445, 2947, 2866, 1704, 1667, 885; 1H NMR (CDCl3, 500 MHz) δ: 4.69 (1H, s, Hb-29), 4.57 (1H, s, Ha-29), 4.20 (2H, t, J = 11.5 Hz, CH2), 3.64 (2H, brs, H in piperazine), 3.52 (2H, brs, H in piperazine), 2.98 (3H, m, H-2, 19), 2.61 (2H, t, J=10.9 Hz, CH2), 2.46 (2H, brs, H in piperazine), 2.37 (2H, m, H in piperazine), 1.65 9

ACCEPTED MANUSCRIPT (3H, s, CH3), 1.03 (3H, s, CH3), 0.99 (3H, s, CH3), 0.95 (3H, s, CH3), 0.93 (3H, s, CH3), 0.89 (3H, s, CH3), 0.83 (6H, m, CH3);

13

C NMR (CDCl3, 125 MHz) δ: 218.2 (C-3), 175.9 (C-28), 174.7

(C=O), 150.5 (C-20), 109.8 (C-29), 60.9 (CH2), 56.8 (CH2), 56.5 (C-17), 55.0 (C-5), 53.8 (CH2), 53.5 (CH2), 49.9 (C-9), 49.4 (C-18), 47.4 (C-4), 47.0 (C-19), 45.7 (CH2), 42.5 (C-14), 42.4 (CH2),

RI PT

41.7 (CH2), 40.7 (C-8), 39.7 (C-1), 38.4 (C-13), 37.0 (C-22), 36.9 (C-10), 34.2 (C-2), 33.7 (C-7), 32.5 (C-16), 32.1 (CH2), 30.7 (C-21), 29.9 (C-15), 29.7 (CH2), 26.7 (C-23), 26.0 (CH2), 25.6 (C-12), 22.9 (CH2), 21.5 (C-11), 21.1 (C-24), 19.7 (C-6), 19.4 (C-30), 16.0 (C-26), 15.9 (C-25),

SC

14.7 (C-27), 14.1 (CH3), 12.2 (CH3).

References

M AN U

[1] H.J. Zhang, G.T. Tan, V.D. Hoang, N.V. Hung, N.M. Cuong, D.D. Soejarto, J.M. Pezzuto, H.H. Fong, Natural anti-HIV agents. Part IV. Anti-HIV constituents from Vatica cinerea, J.

AC C

EP

TE D

Nat. Prod. 66 (2003) 263-268.

10

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

The copies of 1H NMR and 13C NMR of selected compounds

11

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

12

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

13

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

14

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

15

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

16

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

17

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

18

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

19

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

20