A Novel Potent Anticancer Compound Optimized from a Natural

Article pubs.acs.org/jmc

A Novel Potent Anticancer Compound Optimized from a Natural Oridonin Scaffold Induces Apoptosis and Cell Cycle Arrest through the Mitochondrial Pathway Shengtao Xu,† Hong Yao,† Shanshan Luo,‡ Yun-Kai Zhang,§ Dong-Hua Yang,§ Dahong Li,†,⊥ Guangyu Wang,† Mei Hu,† Yangyi Qiu,† Xiaoming Wu,† Hequan Yao,*,† Weijia Xie,† Zhe-Sheng Chen,*,§ and Jinyi Xu*,† †

State Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, P. R. China ‡ Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai 201203, P. R. China § College of Pharmacy and Health Sciences, St. John’s University, 8000 Utopia Parkway, Queens, New York 11439, United States ⊥ Key Laboratory of Structure-Based Drug Design and Discovery of Ministry of Education and School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 103 Wen Hua Road, Shenyang 110016, China S Supporting Information *

ABSTRACT: The cytotoxicity of the natural ent-kaurene diterpenoid, oridonin, has been extensively studied. However, the application of oridonin for cancer therapy was hampered primarily by its moderate potency. In this study, a series of oridonin A-ring modified analogues, and their derivatives bearing various substituents on 14-OH position, were designed, synthesized, and evaluated for anticancer efficacy. Some of the derivatives were significantly more potent than oridonin against both drug-sensitive and drug-resistant cancer cells. The most potent compound, 13p, was 200-fold more efficacious than oridonin in MCF-7 cancer cells. Furthermore, 13p induced apoptosis and cell cycle arrest at the G2/M phase. A decrease in mitochondrial membrane potential and an increase in Bax/Bcl-2 ratio, accompanied by activated caspase-3 cleavage, were observed in MCF-7 cells after treatment with 13p, suggesting that the mitochondrial pathway was involved in the 13p-mediated apoptosis. Moreover, 13p significantly inhibited tumor growth in mouse xenograft models and had no observable toxic effect.

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cancer,14 cervical carcinoma,15 hepatocellular carcinoma,16 and other tumors.17 Unfortunately, although oridonin was determined to have safe, unique, and extensive antitumor activity, the use of oridonin for cancer therapy was hampered by its moderate potency and complex oxygenated diterpenoid scaffold. Hunderds of derivatives of oridonin have been synthesized to enhance its antitumor potency.18−20 However, it should be noted that only a few derivatives have significantly improved antitumor efficacy.21,22 Developing novel oridonin analogues with potent antitumor efficacy through effective modifications is imperative. Therefore, it is important to optimize the scaffold of oridonin for the further development of novel derivatives with enhanced potency and acceptable safety to obtain promising anticancer compounds. According to the previous SARs studies with oridonin, the Bring (Figure 1) is inert due to the low reactivity of the 7-

INTRODUCTION Plants are an important source of new drugs, especially in the discovery of anticancer compounds. Over 60% of all clinically used anticancer drugs have their origin from natural sources.1 However, natural product based drug discovery is greatly impeded by the structural complexity of natural products or their moderate efficacies. An effective solution to these problems is to optimize natural product scaffolds based on structure−activity relationships (SARs), which could produce novel analogues with improved efficacies.2,3 Oridonin (1, Figure 1), an ent-kaurene diterpenoid isolated from genus Isodon (Rabdosia), was first identified in 1967 as an antitumor compound.4 Recently, oridonin has attracted attention due to its extensive pharmacological and physiological effects, such as prevention of hepatic fibrosis 5−7 and Alzheimer’s disease,8 as well as its antimicrobial,9 antiinflammatory,10 and antitumor efficacy. A number of researches have shown that oridonin has antitumor efficacy for human breast cancer,11 gallbladder cancer,12 leukemia,13 gastric © 2017 American Chemical Society

Received: November 9, 2016 Published: February 6, 2017 1449

DOI: 10.1021/acs.jmedchem.6b01652 J. Med. Chem. 2017, 60, 1449−1468

Journal of Medicinal Chemistry

Article

A-ring was still insufficient due to the densely functionalized and stereochemistry-rich framework of oridonin. Currently, more than 700 novel ent-kauranoids have been identified from the genus Isodon, and we found that some entkaurene diterpenoids had similar structures to oridonin (Figure 1, 2−9). The structural differences between these diterpenoids and oridonin were mainly on the A-ring.24 It is noteworthy that some of these ent-diterpenoids have similar or even enhanced anticancer activity compared to oridonin. For example, lasiokaurin (5), an acetylated oridonin, has similar anticancer efficacy as oridonin.25 Eriocalyxin B (4) and longikaurin A (7) have greater antiproliferative efficacy than oridonin.26,27 These results indicated that modification of the A-ring of oridonin is feasible, and it might be an effective way to increase the potency of oridonin. Furthermore, during the course of our work, a few novel oridonin analogues bearing a thiazole-fused A-ring and dienone were reported, and some compounds had enhanced anticancer efficacy.28,29 Therefore, we attempted to find a new A-ring modified analogue of oridonin as a scaffold to develop novel oridonin derivatives with increased efficacy, using pharmacophore exploration and optimization around the Aring of oridonin.30−32 Previous studies showed that eriocalyxin B (4) significantly inhibited the proliferation of cancer cells.26 The additional enone moiety in the A-ring of eriocalyxin B led us to insert an enone or other pharmacophores, like epoxy and halogen, into the A-ring of oridonin to enhance its antiproliferative efficacy.33 Structurally, oridonin is a highly oxygenated diterpenoid bearing four hydroxy groups, and the structure of longikaurin A (7), which was more potent than oridonin, indicated that eliminating the 1-hydroxy group of oridonin might be an effective way to increase the potency of oridonin. Previously, we have reported that the esterfication of the 14-hydroxy group

Figure 1. Structures of oridonin and representative naturally existing oridonin analogues.

hydroxy group and the hydrogen bond of 6-hydroxy group with the 15-carbonyl group. The esterfication of hydroxy group on the C-ring is an efficient way to enhance the antiproliferative activity of oridonin. The α,β-unsaturated ketone in the D-ring is the active moiety of oridonin, and reduction or opening will significantly reduce the antiproliferative efficacy of oridonin. Regarding the A-ring of oridonin, our previous work indicated that oxidation or acetylation of the 1-hydroxy group of oridonin maintained antiproliferative efficacy.23 However, the SAR of the

Scheme 1. Synthesis of Oridonin A-Ring Modified Derivatives 10−19 and Their Derivatives 13a−t and 15a−ca

Reagents and conditions: (a) TsOH, 2,2-dimethoxypropaneaceton, reflux, 10 min, 95%; (b) MsCl, TEA, 0 °C, 2 h, 80%; (c) LiBr, Li2CO3, DMF, 110 °C, 0.5 h, 75%; (d) 10%HCl, THF (v/v, 1:1), rt, 0.5 h, 90%; (e) SeO2, 1,4-dioxane, 100 °C, 56 h, 20%; (f) Jones reagent, acetone, 0 °C, 20 min, 78%; (g) m-CPBA, DCM, rt, 24 h, 71%; (h) corresponding acids, DMAP, EDCI, CH2Cl2, rt, 2−72 h, 34−93%; (i) Ac2O, TEA, DCM, rt, 3 h, 89%; (j) corresponding acids, DMAP, EDCI, CH2Cl2, rt, 5−16 h, 73−88%; (k) 10% HCl, THF, rt, 30 min, 82%; (l) 5% H2SO4, THF, rt, 1.5 h, 65%. a

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Table 1. Antiproliferative Efficacy of Oridonin (1) and Its A-Ring Modified Derivatives (12−19) in Four Human Cancer Cell Linesa IC50 (μM)b compd. 1 12 13 14 15 16 17 18 19

MGC-803 9.06 3.61 1.22 61.10 5.58 1.42 3.09 3.18 5.74

± ± ± ± ± ± ± ± ±

0.88 0.27 0.09 5.27 0.67 0.12 0.48 0.28 0.64

Bel-7402 5.41 3.66 1.59 25.49 3.43 1.32 2.07 1.69 5.77

± ± ± ± ± ± ± ± ±

0.34 0.28 0.12 1.98 0.43 0.14 0.29 0.18 0.46

MCF-7 17.89 1.49 1.99 303.02 11.13 1.58 4.13 3.43 5.37

± ± ± ± ± ± ± ± ±

1.90 0.17 0.129 21.09 1.05 0.12 0.33 0.21 0.54

K562 4.33 7.98 0.22 41.12 2.79 1.66 1.88 2.30 1.59

± ± ± ± ± ± ± ± ±

0.35 0.89 0.02 3.19 0.29 0.18 0.19 0.17 0.13

a

MGC-803: human gastric cancer cells; Bel-7402: human hepatocellular carcinoma cells; MCF-7: human breast cancer cells; K562: human leukemia cells. bMTT methods; cells were incubated with indicated compounds for 72 h (means ± SD, n = 3).

The Antiproliferative Efficacy of Oridonin Analogues on Human Cancer Cells. The in vitro antiproliferative efficacy of the oridonin A-ring modified analogues 11−17 was determined by the MTT assay using human hepatocellular carcinoma Bel-7402 and compared with the reference compound, oridoin (1). Except for compound 14, which had a 3-hydroxy moiety, all of the newly synthesized analogues were significantly more potent than oridonin in inhibiting cancer cell proliferation at 1 μM (Supporting Information Figure S1). These results led us to further evaluate the biological functions of these compounds. As shown in Table 1, eight analogues, including 12−19, and the parent compound oridonin (1), were evaluated against four human cancer cell lines. Among the eight tested compounds, with the exception of compound 14, all of the analogues were significantly more potent against the four cancer cell lines than oridonin. Interestingly, the introduction of a 3β-OH into the A-ring caused a 5−17-fold loss in antiproliferative efficacy relative to oridonin in the cancer cell lines. However, after the acetylation of the 3-hydroxy group, the acetylated compound 17 was equipotent to oridonin. The 1ene, 13, was the most potent compound in this series, with IC50 values ranging from 0.220 to 1.997 μM in the cancer cell lines. The comparison between the structures of the 1-ene (13) and oridonin (1) indicated that eliminating the 1-hydroxy of oridonin was critical for increasing the antiproliferative efficacy, especially in K562 cells, where compound 13 was 20-fold more potent than oridonin, with an IC50 value of 0.220 μM. The above results indicate that a polar moiety is deleterious for the antiproliferative potency of oridonin, and the elimination of 1hydroxy is an effective way to increase the efficacy of oridonin. Furthermore, the installation of additional pharmacophores to the A-ring of oridonin, like α,β-unsaturated ketone or epoxy moiety, did not significantly contribute to the increase of its antiproliferative efficacy. Compound 15, bearing an active 1ene-3-ketone moiety in the A-ring, was only about 2-fold more efficacious than oridonin in the cancer cell lines, while epoxy, 19, was slightly more potent against MCF-7 and K562 cells than oridonin. However, compound 18 exhibited potent anticancer efficacy against the MGC-803, Bel-7402, MCF-7, and K562 cancer cell lines, with IC50 values of 3.18, 1.69, 3.43, and 2.30 μM, respectively. Relative to oridonin, a 1.9- to 5.2fold increase in the potency of compound 18 suggested that the introduction of halogens to the A-ring of oridonin might be responsible for the enhanced antiproliferactive efficacy. Previously, we reported that the 14-O-derivatives of oridonin had significant efficacy against several human cancer cell lines

significantly increased the antiproliferative efficacy of oridonin and its analogues.23,31 Thus, we further designed and synthesized a series of derivatives and conducted the SARs analysis of the substituents on the 14-OH of newly synthesized oridonin analogues. Herein, we report the synthesis of oridonin A-ring modified analogues using a semisynthetic approach and the evaluation of their in vitro and in vivo antitumor efficacy. In addition, the mechanism of action of the representative compound 13p was also investigated.

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RESULTS AND DISCUSSION

Chemical Synthesis. The design and synthesis of oridonin A-ring modified analogues were successfully processed by preparing the key intermediate 12, which was obtained following the procedures reported in Scheme 1. In order to obtain 1-subsituted mesylate 11, protection of the 7,14dihydroxy of commercially available oridonin (1) with 2,2dimethoxypropane was performed to afford compound 10 in 95% yield. The 1-hydroxy of compound 10 was then selectively activated by MsCl, which was subsequently subjected to an elimination reaction in the presence of Li2CO3 and LiBr in DMF at 110 °C to provide the key intermediate 1-ene 12 with a 75% yield. The removal of the acetonide group in 12 with 10% HCl aqueous solution produced 13 with a 90% yield. Subsequently, 13 could be modified as a lead compound by coupling ester moieties to its 14-hydroxy position to generate new derivatives (13a−t) in 34−93% yields. The addition of selenium dioxide to 13 in refluxing 1,4-dioxane stereoselectively produced the 3β-hydroxyl analogue 14 with a poor yield, which underwent acetylation to yield a diacetyl, 17. The oxidation of 14 with the Jones reagent in an ice−water bath selectively oxidized the 3-hydroxy group, producing 1-ene-3-ketone 15 in 78% yield, which was regarded as an analogue bearing another active α, β-unsaturated ketone. Subsequently, the introduction of different substituents on the 4-hydroxy position of 15 provided new derivatives (15a−c) in 73−88% yields. In order to append the epoxy group, another active pharmacophore into the skeleton of oridonin, the key synthon 12 was subjected to epoxidation through m-CPBA to give intermediate 16 with a 71% yield.34 The removal of the protecting group with 5% H2SO4 produced the desired analogue 19 bearing a C-1,2epoxy group in 65% yield. The introduction of a halogen and the removal of the protecting group were finished in one step with 10% HCl, producing compound 18 in 82% yield. 1451



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Table 2. Antiproliferative Efficacy of Ester Derivatives of 13 and 15 (13a−h and 15a−c) in Four Human Cancer Cell Linesa

IC50 (μM)a compd. 5-Fu 1 13 13a 13b 13c 13d 13e 13f 13g 13h 15 15a 15b 15c

MGC-803 3.12 9.06 1.22 1.82 3.60 6.19 2.71 1.75 2.46 1.02 2.47 5.58 5.19 3.76 3.61

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.21 0.87 0.09 0.09 0.42 0.53 0.22 0.12 0.18 0.07 0.28 0.67 0.37 0.29 0.36

Bel-7402 2.25 5.41 1.59 1.67 4.06 6.00 4.34 1.85 3.18 0.45 1.77 3.43 3.61 3.30 2.93

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.14 0.34 0.12 0.12 0.38 0.79 0.33 0.18 0.36 0.02 0.16 0.43 0.27 0.48 0.24

MCF-7 1.75 17.89 1.99 2.57 5.83 2.49 2.44 1.73 2.66 0.18 1.37 11.13 9.41 5.15 3.84

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.11 1.90 0.12 0.26 0.47 0.19 0.28 0.14 0.22 0.01 0.19 1.05 1.04 0.39 0.27

K562 3.54 4.33 0.22 1.67 3.22 1.74 3.00 2.70 2.96 1.27 0.58 2.79 4.64 2.94 2.61

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.27 0.35 0.02 0.09 0.47 0.05 0.25 0.17 0.38 0.07 0.02 0.29 0.36 0.33 0.16

a


both in vitro and in vivo.23 Based on the above preliminary results, we continued to modify the potent analogues 13 and 15 by introducing different types of substituents to the 14-hydroxy position, including alkyl-, aryl-, and heterocycle-substituted esters. Therefore, compounds 13a−h and 15a−c were synthesized and subsequently evaluated against four human cancer cell lines, and the results are shown in Table 2. Although the antiproliferative efficacy of derivatives 13a−h were greater than that of oridonin, when compared with the unsubstituted 13, compounds 13a−d bearing alkyl groups were 1.1- to 14.7fold less potent against the human cancer cell lines. The derivatives with aromatic substitutions (13e−h) had potencies comparable to or even greater than that of 13 with IC50 values ranging from 0.18 to 3.18 μM. Notably, compound 13g, with a 3-methylphenyl substituent, had significant efficacy against the MGC-803, Bel-7402, MCF-7, and K562 cancer cell lines, with IC50 values of 1.02, 0.45, 0.18, and 1.27 μM, respectively. Unfortunately, the introduction of aromatic groups to the 14hydroxy of compound 15 did not significantly increase its antiproliferative efficacy. The above results suggested that compound 13 could be a potential lead compound for further optimization. Due to the

fact that the aryl substitution on the 14-OH of compound 13 contributed to an enhanced antiproliferative efficacy, a series of 14-aryl derivatives were synthesized based on the obtained SARs. Since compound 13g, bearing a 3-methylphenyl moiety, exhibited potent activity, compounds 13i−j were prepared by introducing different methylphenyl substituents to the 14-OH position. Also, compounds 13p−t, containing trans-cinnamic acid moieties, were synthesized based on 13h. Besides, the substituents examined included different substituted phenyls bearing an electron-releasing group (OMe), electron-withdrawing group (NO2), and halogens (F, Cl, Br) at different positions. Furthermore, the antiproliferative efficacy of these compounds was evaluated. As shown in Table 3, SAR analysis indicated that both steric and electronic factors can explain the different potencies of these compounds. The presence of substituents at the ortho-position(s) (compounds 13i and 13k) produced a significant loss in antiproliferative efficacy, exemplified by compound 13k, where the presence of two omethoxy moieties resulted in a 6- to 31-fold decrease in the antiproliferative efficacy compared to 13. By comparing 13m to 13n, we concluded that strong electron-withdrawing groups may be unsuitable, as replacing the halogen (13n) with a nitro 1452



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Table 3. Antiproliferative Efficacy of Ester Derivatives of 13 (13i−t) in Four Human Cancer Cell Linesa

IC50 (μM)a compd.

MGC-803 1.22 3.78 6.94 21.22 1.00 6.39 2.01 3.65 1.03 5.11 2.91 2.46 3.91

13 13i 13j 13k 13l 13m 13n 13o 13p 13q 13r 13s 13t

± ± ± ± ± ± ± ± ± ± ± ± ±

Bel-7402

0.09 0.28 0.77 3.12 0.08 0.78 0.21 0.34 0.07 0.34 0.28 0.21 0.47

1.59 2.33 7.11 45.33 1.58 5.14 1.06 4.16 1.03 2.58 2.42 2.22 2.44

± ± ± ± ± ± ± ± ± ± ± ± ±

MCF-7

0.12 0.12 0.99 5.87 0.12 0.78 0.09 0.32 0.06 0.29 0.25 0.17 0.14

1.99 3.58 6.14 12.46 2.11 4.47 0.14 2.80 0.08 2.51 0.95 0.82 2.36

± ± ± ± ± ± ± ± ± ± ± ± ±

K562

0.12 0.48 0.48 1.90 0.49 0.59 0.08 0.37 0.01 0.14 0.05 0.04 0.21

0.22 1.68 4.19 6.85 1.83 1.70 0.33 2.23 0.29 1.34 0.88 0.43 0.55

± ± ± ± ± ± ± ± ± ± ± ± ±

0.02 0.13 0.27 0.66 0.13 0.13 0.04 0.21 0.02 0.09 0.05 0.02 0.04

a


Table 4. IC50a Values (μM) of Representative Compounds in the Drug-Resistant and Drug-Sensitive Cancer Cellsb IC50 (μM) compd. 1 13 13g 13n 13p 13s 15 18 19 cisplatin

KB-3−1 13.24 5.16 6.21 5.82 5.70 8.31 16.69 6.54 20.96 2.62

± ± ± ± ± ± ± ± ± ±

0.96 0.43 0.75 0.87 0.41 0.83 2.65 0.73 2.87 0.32

KB/CP4 10.80 2.98 1.01 3.24 1.56 4.61 16.11 5.92 7.74 9.41

± ± ± ± ± ± ± ± ± ±

0.87 0.35 0.08 0.26 0.23 0.61 2.19 0.73 0.32 0.86

NCI-H460 20.51 2.69 2.44 5.31 2.62 5.31 19.22 8.80 8.81 −

± ± ± ± ± ± ± ± ±

2.54 0.26 0.32 0.64 0.35 0.62 2.84 0.76 0.64

NCI-H460/MX20 25.86 11.08 14.18 4.73 13.61 5.53 54.02 48.99 11.18 −

± ± ± ± ± ± ± ± ±

3.12 1.43 0.94 0.52 1.75 0.74 7.56 5.86 1.32

Bel-7404 17.92 3.91 6.56 5.35 5.94 6.06 16.21 6.96 13.73 2.89

± ± ± ± ± ± ± ± ± ±

Bel-7404/CP20

2.05 0.24 0.54 0.74 0.53 0.98 1.32 0.84 0.87 0.21

17.17 4.98 3.42 6.05 4.73 6.87 15.10 5.98 21.61 22.77

± ± ± ± ± ± ± ± ± ±

1.94 0.53 0.25 0.82 0.52 0.71 1.76 0.54 2.54 2.53

a MTT methods; cells were incubated with indicated compounds for 48 h (means ± SD, n = 3). bMTT cytotoxicity assay was assessed in pairs of parental and drug-resistant cancer cell lines: KB-3-1: human cervix carcinoma; KB/CP4: cisplatin-resistant human cervix carcinoma; NCI-H460: human lung carcinoma; NCI-H460/MX20: mitoxantrone-resistant human lung carcinoma; Bel-7404: human hepatocellular carcinoma; Bel-7404/ CP20: cisplatin-resistant human hepatocellular carcinoma.

than electronic factors, accounted for the different potency of these compounds. Furthermore, the antiproliferative data obtained with 13h and 13p−t revealed that the size of transcinnamic acid moiety was crucial. Indeed, compound 13p, which has no substituent on phenyl group, had the most potent anticancer activity against the MGC-803, Bel-7402, MCF-7, and

(13m) led to a decrease in antiproliferative efficacy against all cancer cell lines, and compound 13g, bearing a 3-methyl phenyl substituent, also exhibited potent cytotoxicities against the cancer cell lines. However, the introduction of another electron-releasing o-methyl group (13i and 13j) resulted in decreased antiproliferative efficacy, suggesting that steric, rather 1453



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Figure 2. Colony formation of MCF-7 cells inhibited by compound 13p. (a) MCF-7 cells were incubated with varying concentrations of 13p (0, 75, 150, 300 nM) and stained with crystal violet. (b) The micrographic differences between the colonies. Images were taken of stained single colonies observed under a microscope. (c) Bar chart showing the decreased proportion of the colonies after incubation with 13p. One colony was defined to be an aggregate of >50 cells. Data are shown as the mean ± SD of three independent experiments, **p < 0.01 and ***p < 0.001 vs control group (untreated cells).

comparable or even higher selectivity than oridonin (Supporting Information Table S1). Particularly, compound 13g seemed to be more selective than oridonin, with an SI (selective index, IC50 of normal cells/IC50 of tumor cells) value of 14.58. Although the SI value of compound 13p was not very high (4.07), it was chosen for further biological studies because it had significantly lower IC50 against MCF-7 cells compared to the other compounds. Compound 13p Inhibited Tumor Cell Colony Formation. Colony formation assay is used to test cells for their ability to undergo unlimited division and form colonies.37 We investigated the antiproliferative efficacy of compound 13p by performing a colony formation assay. As shown in Figure 2, the exposure of MCF-7 cells to compound 13p resulted in a significant suppression of colony formation. Cells incubated with 13p formed fewer and smaller colonies in a concentrationdependent manner compared to the control group. At 75 nM, the 13p incubated cells showed a significant decrease in the number and the size of colonies, and colony formation was almost completely suppressed with 300 nM of 13p. These results demonstrated that 13p inhibited the growth of MCF-7 cells and the inhibitory effect of 13p lasted for a significant period of time. Compound 13p Induced Cell Cycle Arrest with a Change in the Expression of cdc2/cyclin B and pcdc25c. Most antitumor compounds inhibit cell proliferation through induction of cell cycle arrest.38 To determine whether our analogues affect cancer cell proliferation by inhibiting cell cycle progression, DNA-based cell cycle analysis was performed using flow cytometry. As illustrated in Figure 3, incubation with 13p blocked the cell cycle at the G2/M phase. Compared to the control cells incubated with DMSO, the incubation of MCF-7 cells with increasing concentrations of 13p (0.125, 0.25, 0.5, 1 μM) increased the percentage of cells in the G2/M phase from 13.05% to 34.23%, whereas the percentage of cells in S and G1 phases decreased concomitantly. We further investigated whether 13p blocks other cancer cells at the G2/M phase in the same way as in MCF-7 cells. As shown in Figure 3b, compound 13p induced the accumulation of Bel-7402 cells

K562 cancer cell lines, with IC50 values of 1.03, 1.03, 0.08, and 0.29 μM, respectively. Most notably, compound 13p was about 200-fold more potent than oridonin (1) against MCF-7 cells (IC50 values of 0.08 and 17.89 μM, respectively), which deserved further investigation. The cellular membrane permeabilities of active compounds 13p and 13 were then examined to evaluate the impact of the cinnamic acid moiety on the antitumor activity of 13p. The results showed that 13p possessed better cellular membrane permeability, suggesting that the cinnamic acid moiety may provide some advantages for the cellular delivery of 13p (Supporting Information Table S2). Effects of Compounds on Multidrug-Resistant Cancer Cells. Multidrug resistance (MDR) is one of the major reasons for the failure of cancer chemotherapy.35 To investigate whether our oridonin analogues are efficacious against drugresistant cancer cells, the representative compounds 13, 13g, 13n, 13p, 13s, 15, 18, and 19 were tested for their antiproliferative efficacy in the drug-resistant and parentalsensitive cells using the MTT assay. As shown in Table 4, oridonin had moderate anticancer efficacy against both parental and drug-resistant cells. The antiproliferative efficacy of the synthesized compounds was more potent in drug-resistant KB/ CP4 cells than other cells, with the drug-resistant fold below 1.0 (Supporting Information Figure S2). Particularly, compounds 13g and 13p exhibited potent antiproliferative efficacy on drugresistant KB/CP4 cells, with IC50 values of 1.01 and 1.56 μM, respectively, which were significantly lower than that of the drug-sensitive KB-3-1 cells. Compounds 13g and 13p Selectively Inhibited Cancer Cell Growth in Vitro. Nonselective cytotoxicity is one of the main factors that limits the clinical use of anticancer drugs.36 To obtain insight into the cytotoxic potential of these new compounds on normal human cells, the effect of compounds 13, 13g, 13n, 13p, 15, 19, and oridonin (1) were evaluated in human liver cancer cell line Bel-7402 and normal liver cell line L-O2. Oridonin was approximately 6-fold more selectivity in inhibiting the growth of Bel-7402 cells vs LO2 cells. In addition, all the tested analogues exhibited 1454



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Figure 3. Compound 13p induced G2/M arrest in MCF-7 and Bel-7402 cancer cells. (a, b) MCF-7 cells were incubated with DMSO and varying concentrations of 13p [0.125, 0.25, 0.5, 1 μM, (a)] for 48 h, and Bel-7402 cells were incubated with DMSO and varying concentrations of 13p [0.5, 1, 2, 4 μM, (b)] for 48 h. Cells were harvested and stained with PI and then analyzed by flow cytometry. The percentages of cells in different phases of cell cycle were analyzed by ModFit 4.1. (c, d) Histograms display the percentage of cell cycle distribution. (e) Western blotting analysis on the effect of 13p on the G2/M regulatory proteins. The cells were harvested and lysed for the detection of p-cdc25c, cdc2, and cyclin B1. (f) Histograms display the density ratios of p-cdc25c, cdc2, and cyclin B1 to β-actin. Data are represented as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001; control compared with 13p treated cells.

in the G2/M phase at 0.5 μM and above. The effect of 13p on G2/M arrest was less pronounced in Bel-7402 cells as compared with MCF-7 cells. In addition, flow cytometric analysis was also performed to examine the effects of compound 13g on the cell cycle progression of MCF-7 cells. Similar cell cycle arrest at the G2/M phase was also observed (Supporting Information Figure S3). The above results suggested that these analogues induce cancer cell cycle arrest at the G2/M phase. To obtain insight into the mechanism of 13p in MCF-7 cell cycle arrest, Western Blotting assay was performed to determine the expression of cell cycle regulatory proteins. It

has been reported that the decreased expression of the cdc2/ cyclin B complex inhibits cell cycle progression from the G2 to the M phase. Cdc25c is a major phosphatase that dephosphorylates cdc2 and activates cdc2/cyclin B upon entry of the cells into mitosis.39 As shown in Figure 3, 13p induced G2/M phase arrest in MCF-7 cells, decreased cdc2 and cyclin B protein levels, and increased the expression of pcdc25c in a concentration-dependent manner. These data suggested that the 13p-induced G2/M arrest may be correlated with a change of expression of cdc2/cyclin B and p-cdc25c. Compound 13p Induced Cancer Cell Apoptosis. Cell viability is most often defined based on the integrity of the cell 1455



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Figure 4. Compound 13p induced apoptosis of MCF-7 cells. (a, b) LDH assay of selected compounds on Bel-7402 and MCF-7 cells. Cytotoxicity was assayed by LDH assay from culture media of indicated cells that were incubated with different concentrations (10, 20, 50 μM) of compounds for 24 h. The data are expressed as percentage of unstimulated cells. (c) Cell morphological alterations and nuclear changes associated with MCF-7 cells after incubation with 13p were assessed by staining with Hoechst 33342 and visualized by fluorescence microscopy, bars denote 20 μM. (d) Compound 13p induced apoptosis in MCF-7 cells. MCF-7 cells were incubated with varying concentrations of 13p (0, 0.125, 0.25, 0.5, 1 μM). After 24 h of incubation, cells were collected and stained with Annexin V/PI, followed by flow cytometric analysis. (e) Histograms display the percentage of cell distribution. Data are represented as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 vs control group.

apoptosis. Therefore, we used Hoechst 33342 staining to assess nuclear changes in cells. As shown in Figure 4c, MCF-7 cells incubated with 13p for 36 h displayed significant changes in cell morphology, such as chromatin condensation, indicating cell apoptosis. To further confirm whether 13p could induce apoptosis in MCF-7 cells, vehicle- or 13p-incubated MCF-7 cells were stained with Annexin V and PI. As shown in Figure 4d, incubation with 13p induced a concentration-dependent (0.125, 0.25, 0.5, 1 μM) increase (2.9−67.6%) in both the early

membrane. The measurement of the leakage of cell membrane components, such as lactate dehydrogenase (LDH), from the cytoplasm into the culture medium has been widely accepted as a valid method to estimate the number of nonviable cells.40 When MCF-7 and Bel-7402 cells were incubated with varying concentrations of selected compounds for 24 h, no LDH was detected in the culture media (Figure 4a, b). This result indicated that the inhibition of cell viability by these compounds was not by cytolysis, but most likely due to cell 1456



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Figure 5. Accumulation of ROS production in MCF-7 cells induced by the loss of MMP. (a) Fluorescence photomicrograph of changes in the MMP after incubated with 13p for 36 h in MCF-7. 13p (0.25, 0.5, 1 μM) significantly changed the fluorescence intensity from red to green in treated cells, bars denote 50 μM. (b) Independent experiments with 10,000 cells per group reveal a decrease in the photometric ratio of red to green fluorescence after incubation with 13p. Data are represented as mean ± SD of three independent experiments. (c) The generation of ROS was measured by using the ROS-detecting fluorescent dye DCF-DA in combination with FACScan flow cytometry. (d) The corresponding histograms of FACScan flow cytometry is shown. *p < 0.05, **p < 0.01, ***p < 0.001.

time, the ratio of fluorescent intensity of red to green was determined and used as an indicator of cell viability. As shown in Figure 5b, compared with the control cells, incubation with 1 μM of 13p decreased the ratio from 1.433 to 0.385. These findings suggested that compound 13p reduced mitochondrial membrane potential and induced mitochondrial dysfunction in MCF-7 cancer cells. ROS are considered to play an important role in apoptosis in various types of cells, and mitochondrial membrane depolarization is associated with mitochondrial production of ROS.42 Therefore, we used the fluorescent probe 2′,7′-dichlorofluoresceindiacetate (DCF-DA) to monitor intracellular ROS levels in the presence and absence of 13p. We found that 13p induced intracellular ROS generation. The ratio of DCF positive cells was increased from 3.77% in cells incubated with DMSO to 24.31% in cells incubated with 1 μM of 13p. Moreover, the increased ROS was almost completely inhibited by pre-incubation with 2.5 mM of the ROS scavenger, N-acetly

and late stage apoptosis of MCF-7 cells. Overall, these results demonstrated that 13p induced apoptosis in MCF-7 cancer cells. Compound 13p Induced Mitochondrial Depolarization and Reactive Oxygen Species (ROS) Generation. It is well-known that apoptosis is a programmed process, and there are two major apoptotic pathways: the death-receptor-induced extrinsic pathway and the mitochondria-apoptosome-mediated apoptotic intrinsic pathway. Mitochondria play an important role in regulating cellular functions, and mitochondrial dysfunction has been proposed to be involved in many pathological processes.41 In order to determine whether 13pinduced apoptosis was involved in a disruption of mitochondrial membrane integrity, the fluorescent probe JC-1 was used to measure the mitochondrial membrane potential (MMP). As shown in Figure 5a, incubation of MCF-7 cells with 13p induced the dissipation of MMP in a concentration-dependent manner, as indicated by a decrease in red fluorescence emission and an increase in green fluorescence emission. At the same 1457



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Figure 6. MCF-7 cells were incubated with various concentrations of 13p (0, 0.125, 0.25, 0.5, 1 μM) for 48 h. (a) The expressions of pro-caspase-3, caspase-3, caspase-9, Bcl-2, Bax, Bad, p53, p-ERK, and cytotochrome c were determined by Western blotting using specific antibodies. β-actin was used as internal control. (b) The density ratio of proteins to β-actin was shown as relative expression. (c) Histograms display the density ratios of caspase 3 and pro-caspase 3 to β-actin. Data are represented as mean ± SD of three independent experiments. ***p < 0.001, control compared with 13p treated cells.

caspase-3 in tumor cells.45 Therefore, experiments were conducted to determine if caspase-3 is involved in 13p-induced apoptosis. As shown in Figure 6a, 13p produced a concentration-dependent cleavage of pro-caspase-3 and activation of caspase-3, which occurred sequentially in MCF-7 cells. As shown in Figure 6c, the inactive pro-caspase-3 protein was activated, and the ratio of pro-caspase-3 to caspase-3 was significantly decreased as the concentration of 13p was increased. This result is congruent with the mitochondrial depolarization results described above. Furthermore, a significant increase of caspase-9 was observed, which interacted with caspase-3 and activated apoptosis. These results suggested that mitochondrial death pathway was involved in 13p-induced apoptosis. The summary of the proposed mitochondrial pathway for 13p-induced apoptosis in MCF-7 cells is outlined in Figure 7. Determination of the Antitumor Effect of 13p in Vivo. To evaluate the in vivo antitumor activity of compound 13p, a mouse breast cancer xenograft model was established by subcutaneous inoculation of MCF-7 cells into nude mice. The tumor size and body weights of the mice were monitored and recorded every 2 days. Cyclophosphamide was selected as a positive control. The reduction in tumor weight reached 64.64% at a dose of 20 mg/kg/day (iv) of cyclophosphamide at

cysteine (NAC) (Figure 5d). These results indicated that ROSmediated apoptosis in MCF-7 cells incubated with 13p. Compound 13p Regulates the Expression of Apoptosis-Related Proteins. The above results showed that 13pinduced apoptosis was closely related to the mitochondrial pathway. The Bcl-2 family has been identified as essential proteins in controlling the mitochondrial pathway.43 This family includes proapoptotic (e.g., Bax, Bad) and antiapoptotic proteins (e.g., Bcl-2, Bcl-xL). Bax acts on the mitochondria to increase mitochondria permeability, resulting in the release of certain cellular components, such as cytochrome c. To confirm whether such a mechanism is involved in apoptosis induced by 13p, the expression of Bax, Bad, Bcl-2, and cytotochrome c was determined by Western blot analysis. As shown in Figure 6a, the expression of Bax, Bad, and cytotochrome c began to increase after incubation with 13p for 48 h, while Bcl-2 expression was significantly decreased at concentrations ≥0.5 μM (Figure 6b). Cytochrome c normally functions via its association with other molecules to form a caspase-activating complex, which plays a key role in the caspase-dependent apoptotic pathway.44 Caspase-3 is one of the most important “executioner” caspases, and it cleaves many important cellular substrates. Many studies have shown that natural products can affect the expression of 1458



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Figure 7. Schematic diagram shown the proposed mitochondrial pathway for 13p-induced apoptosis in MCF-7 cells.

28 days after initiation of treatment as compared to vehicle. Compound 13p significantly decreased the tumor volume and reduced tumor weight by 69.77% at a dose of 20 mg/kg/day (iv), which was significantly greater than that of the positive control, cyclophosphamide (Figure 8a). Notably, none of the compounds significantly affected body weight, and they had no overt toxicity under the current treatment paradigm (Figure 8c). Furthermore, as shown in the Figure 9, H&E staining of the heart, liver, kidney, lung, and spleen collected at the end of the study also suggested no observable major organ-related toxicities. In addition, it was found that 13p also produced significant antitumor efficacy in a mouse liver cancer xenograft model (Supporting Information Figure S4). Overall, these data indicated that compound 13p was efficacious in inhibiting the growth of cancer in vivo and deserved further evaluation.

14-position, was 200-fold more potent than the parent molecule oridonin against MCF-7 cancer cells. In addition, the molecular mechanism study of 13p on the suppression of cancer cell growth revealed that compound 13p caused cancer cells arrest at the G2/M phase of the cell cycle, and it significantly increased cell apoptosis, which was illustrated by chromatin condensation, externalization of phosphatidylserine, and loss of MMP. Mitochondria-dependent apoptosis, which is upstream of caspase activation, is regulated by members of the Bcl-2 family. In the present study, Western blotting analysis showed a decrease in the level of antiapoptotic protein (Bcl-2) and an increase in the proapoptotic proteins (Bax, Bid) in MCF-7 cells. The permeabilization of the mitochondrial membrane facilitates the release of cytotochrome c from the mitochondria, which activates caspase-9 to initiate the protease cascade. Overall, our results indicated that the mitochondria pathway is involved in the apoptosis induced by 13p. More importantly, the antitumor efficacy of 13p was verified in both breast and liver cancer xenograft mouse models with no notable toxicity. Compound 13p significantly suppressed the tumor volume and reduced tumor weight by 69.77% at a dose of 20 mg/kg/day (iv) in an MCF-7 breast cancer xenograft nude mice model, which was greater than that of the positive control, cyclophosphamide (64.64%). Collectively, our results demonstrated that the newly developed oridonin analogue 13p showed significant antitumor efficacy both in vitro and in vivo and has the potential to be further developed into a promising antitumor compound.

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CONCLUSIONS In summary, the structural modification of the natural product oridonin around the A-ring led to the discovery of a series of diterpenoids with a novel scaffold. Some of the oridonin derivatives displayed significantly enhanced antiproliferative efficacy in a panel of human cancer cell lines. The analysis of the SARs indicated that the A-ring of oridonin was suitable for modification to improve its antitumor efficacy. The hydroxy group at C-1 position was not required for efficacy. Instead, this group could be removed to generate the potent 1-ene derivative, 13. The antiproliferative efficacy of the compounds derived from compound 13 was highly dependent on the structural modifications of its 14-position. The most potent compound, 13p, bearing a trans-cinnamic acid moiety on the 1459



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Figure 8. 13p inhibits breast cancer xenograft growth in vivo. (a) MCF-7 cells were subcutaneously inoculated into the right flank of nude mice. The mice were randomly divided into 5 groups with 6 mice in each group. Mice were treated intravenously with 13p (20 mg/kg), 13 (20 mg/kg), oridonin (20 mg/kg), cyclophosphamide (20 mg/kg), and DMSO (dissolved in sodium chloride, control) every day for 4 weeks. The resulting tumors were excised from the animals after treatment. (b) Visible tumor formation and photographs of representative tumors removed from mice at 28 days after initiation of treatment. (c) Body weight changes of mice during treatment. (d) 13p treatment resulted in significantly lower tumor weight compared with controls. ***p < 0.001.

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30.4, 29.2, 25.8, 22.4, 19.6; MS (ESI) m/z: 405.2 [M + H]+, 439.4 [M + Cl]−. ent-(1α-O-Methylsulfonyl)-6β-hydroxy-7,14-isopropylideneketal15-oxo-7,20-epoxy-16-kaurene (11). Three mL triethylamine was added to a solution of compound 10 (2.10 g, 5.19 mmol) in 20 mL of anhydrous CH2Cl2 at 0 °C. Methylsulfonyl chloride (2 mL) was added dropwise to the solution within 1 h, and then the mixture was allowed to stir for another 1 h. The mixture was quenched with water, extracted with dichloromethane, dried over anhydrous Na2SO4, filtered, and evaporated to yield a crude product, which was purified by column chromatography (MeOH/CH2Cl2 1:100, v/v) to obtain pure compound 11 (2.01 g, 80%) as a white solid. 1H NMR (CDCl3, 300 MHz): δ (ppm) 6.17 (s, 1H), 5.79 (d, J = 12.0 Hz, 1H), 5.58 (s, 1H), 4.75 (d, J = 1.2 Hz, 1H), 4.60 (m, 1H), 4.14 (2H, s), 3.93 (m, 1H), 3.07 (d, J = 9.3 Hz, 1H), 2.99 (s, 3H), 2.51 (m, 1H), 2.07 (m, 1H), 1.89 (m, 2H), 1.76 (m, 3H), 1.59 (s, 3H), 1.50 (m, 1H), 1.42 (m, 2H), 1.33 (s, 3H), 1.19 (s, 3H), 1.18 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm) 204.7, 149.9, 120.0, 100.6, 84.2, 72.5, 69.4, 61.9, 59.0, 52.3, 49.5, 40.2, 39.7, 37.8, 32.7, 32.4, 29.7, 29.6, 25.9, 24.9, 21.9, 18.3; MS (ESI) m/z: 483.2 [M + H]+; HR-MS (ESI) m/z: calcd for C24H34NaO8S [M + Na]+ 505.1867, found 505.1873. ent-6β-Hydroxy-7,14-isopropylideneketal-15-oxo-7,20-epoxy-1alkene-16-kaurene (12). Lithium carbonate (3.08 g, 41.65 mmol) and lithium bromide (3.62 g, 41.65 mmol) were added to a solution of compound 11 (2.01 g, 4.17 mmol) in 20 mL anhydrous dimethylformamide (DMF). The mixture was stirred violently at 110 °C for 1 h and then cooled to room temperature. After the inorganic precipitate was filtered off, the reaction mixture was diluted with 150 mL CH2Cl2 and then washed with water (20 mL× 3) and brine (20 mL× 3). The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated to yield a crude product, which was purified by column chromatography using CH2Cl2 to obtain pure compound 12 (1.21 g, 75%). 1H NMR (CDCl3, 300 MHz): δ (ppm) 6.09 (s, 1H), 5.68 (m, 1H), 5.49 (s, 1H), 5.31 (d, J = 12.0 Hz, 1H),

EXPERIMENTAL SECTION

Chemistry. General Methods. All commercially available reagents were used without further purification. Anhydrous solvents were dried through routine protocols. Flash column chromatography was carried out on 200−300 mesh silica gel (Qingdao Haiyang Chemical, China). Reactions were monitored by thin-layer chromatography (TLC) on 0.25 mm silicagel plates (GF254) and visualized under UV light. 1H NMR and 13C NMR spectra were recorded with a Bruker AV-300 spectrometer (Bruker Company, Germany) in the indicated solvents (CDCl3 or DMSO-d6, TMS as internal standard): the values of the chemical shifts are expressed in δ values (ppm) and the coupling constants (J) in Hz. Low- and high-resolution mass spectra (LRMS and HRMS) were measured on Finnigan MAT 95 spectrometer (Finnigan, Germany). The purity (≥95%) of the compounds was verified by the HPLC (Aglilent Technologies 1260 Infinity) study performed on an Aglilent C18 column (4.6 × 150 mm, 5 μm) using a mixture of solvent methanol/water at the flow rate of 1.0 mL/min and peak detection at 240 nm under UV. ent-1α,6β-Dihydroxy-7,14-isopropylideneketal-15-oxo-7,20epoxy-16-kaurene (10). Compound 1 (2 g, 5.49 mmol) was dissolved in anhydrous acetone (30 mL). TsOH and 3 mL 2,2-dimethoxypropane were added to this solution. The mixture was stirred at 56 °C for 30 min, then diluted with water, and extracted with dichloromethane. The extract was washed with saturated NaHCO3 solution and brine, dried over anhydrous Na2SO4, filtered, and evaporated to afford compound 10 (2.10 g, 95%) as a white powder. 1 H NMR (CDCl3, 300 MHz): δ (ppm) 6.15 (s, 1H), 5.78 (d, J = 8.1 Hz, 1H), 5.56 (s, 1H), 4.80 (d, J = 1.2 Hz, 1H), 4.24, 4.04 (dd, JA = JB = 10.2 Hz, each 1H), 3.90 (m, 1H), 3.47 (m, 1H), 3.06 (d, J = 9.0 Hz, 1H), 2.50 (m, 1H), 2.08 (m, 1H), 1.91 (m, 2H), 1.73 (m, 3H), 1.68 (m, 2H), 1.67 (s, 3H), 1.44 (m, 1H), 1.37 (s, 3H), 1.28 (s, 3H), 1.14 (s, 3H); 13C NMR (DMSO-d6, 75 MHz): δ (ppm) 206.3, 151.5, 119.7, 100.1, 94.6, 72.7, 72.4, 70.0, 62.8, 58.8, 56.3, 50.5, 40.5, 38.7, 33.4, 1460



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Figure 9. No obviously organ-related toxicity was observed after in vivo evaluation of the antitumor effect of 13p. Animals were sacrificed, and the organs were fixed in formalin overnight and processed for paraffin embedding. The paraffin-embedded blocks were sectioned and stained by hematoxylin and eosin. 5.11 (dd, J = 10.2, 2.5 Hz), 4.75 (s, 1H), 3.91, 3.73 (dd, JA = JB = 9.9 Hz, each1H), 3.81 (m, 1H), 3.00 (d, J = 6.0 Hz, 1H), 2.46 (m, 1H), 1.85 (m, 1H), 1.67 (m, 4H), 1.58 (s, 3H), 1.52 (m, 2H), 1.28 (s, 3H), 1.11 (s, 3H), 0.98 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm) 203.8, 150.3, 129.8, 123.7, 119.8, 86.1, 71.5, 69.6, 64.3, 57.5, 48.6, 40.7, 39.8, 37.7, 31.7, 30.6, 29.8, 29.7, 25.0, 21.6, 16.8; MS (ESI) m/z: 387.2 [M + H]+; HR-MS (ESI) m/z: calcd for C23H30NaO5 [M + Na]+ 409.1985, found 409.1989. ent-6β,7β,14β-Trihydroxy-15-oxo-7,20-epoxy-1-alkene-16-kaurene (13). Compound 12 (1.21 g, 3.13 mmol) was added to 20 mL of 10% HCl/THF (1:1), and the solution was stirred at room temperature for 1 h. Then the mixture was diluted with water and extracted with dichloromethane. The extract was washed with brine, dried over anhydrous Na2SO4, filtered, and evaporated to give compound 13 (0.98 g, 90%) as a white powder. 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 7.14 (s, 1H), 6.03 (s, 1H), 5.96 (s, 1H), 5.74 (m, 1H), 5.63 (s, 1H), 5.52 (d, J = 10.8 Hz, 1H), 5.22 (dd, J = 10.5, 2.4 Hz, 1H), 4.78 (s, 1H), 3.83, 3.72 (dd, JA = JB = 9.9 Hz, each1H), 3.58 (m, 1H), 2.91 (d, J = 9.3 Hz, 1H), 2.42 (m, 1H), 1.88 (m, 2H), 1.80 (m, 1H), 1.62 (m, 1H), 1.52 (m, 1H), 1.47 (m, 1H), 1.38 (m, 1H), 1.05 (s, 3H), 0.96 (s, 3H); 13C NMR (DMSO-d6, 75 MHz): δ (ppm) 207.0, 151.5, 129.4, 124.9, 120.4, 97.4, 72.3, 72.1, 64.2, 61.4, 58.3, 51.6, 42.6, 40.6, 37.8, 31.9, 30.7, 29.6, 21.6, 17.0; MS (ESI) m/z: 347.2 [M + H]+; HR-MS (ESI) m/z: calcd for C20H26NaO5 [M + Na]+ 369.1672, found 369.1679. ent-6β,7β-Dihydroxy-(14β-O-acetyl)-15-oxo-7,20-epoxy-1-alkene-16-kaurene (13a). Compound 13 (50 mg, 0.14 mmol) was

dissolved in dichloromethane, and dimethylaminopyridine (catalytic amount), acetic anhydride (0.05 mL), and triethylamine (0.1 mL) were added. The mixture was stirred at room temperature for 2 h, diluted with water, and extracted with dichloromethane. The extract was washed with saturated NaHCO3 solution and brine, dried over anhydrous Na2SO4, filtered, and evaporated to afford a crude product, which was purified by column chromatography (MeOH/CH2Cl2 1:120, v/v) to obtain compound 13a (46 mg, 82%). 1H NMR (CDCl3, 300 MHz): δ (ppm) 6.20 (s, 1H), 5.87 (s, 1H), 5.77 (m, 1H), 5.58 (d, J = 11.7 Hz, 1H), 5.56 (s, 1H), 5.14 (dd, J = 10.2, 2.4 Hz, 1H), 4.21 (s, 1H), 3.97, 3.87 (dd, JA = JB = 10.2 Hz, each1H), 3.78 (m, 1H), 3.13 (d, J = 9.3 Hz, 1H), 2.61 (m, 1H), 2.03 (s, 3H), 1.94 (m, 4H), 1.63 (m, 2H), 1.52 (m, 1H), 1.16 (s, 3H), 1.06 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm) 204.5, 169.0, 149.2, 130.4, 123.7, 120.7, 96.0, 74.7, 73.0, 64.9, 61.3, 57.6, 52.8, 40.9, 40.5, 38.0, 31.8, 30.2, 28.8, 21.2, 20.9, 17.3; MS (ESI) m/z: 389.2 [M + H]+; HR-MS (ESI) m/z: calcd for C22H28NaO6 [M + Na]+ 411.1778, found 411.1781. ent-6β,7β-Dihydroxy-(14β-O-cyclopentoyl)-15-oxo-7,20-epoxy-1alkene-16-kaurene (13b). Compound 13 (60 mg, 0.17 mmol) was dissolved in dichloromethane, and then 1-(3-(dimethylamino)propyl)3-ethylcarbodiimide hydrochloride (EDCI, 36 mg, 0.19 mmol) DMAP and cyclohexanecarboxylic acid (24 mg, 0.19 mmol) were added. The reaction mixture was stirred at room temperature for about 8 h. Then the mixture was washed with 10% HCl. The organic layer was washed with brine and dried over anhydrous Na 2 SO 4 . After flash chromatography (MeOH/CH2Cl2 1:200, v/v), 13b was obtained as white solid (58 mg, 74%). 1H NMR (CDCl3, 300 MHz): δ (ppm) 6.12 1461



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JB = 11.1 Hz, each1H), 3.79 (m, 1H), 3.27 (d, J = 9.6 Hz, 1H), 2.67 (m, 1H), 2.36 (s, 3H), 1.98 (m, 4H), 1.67 (m, 2H), 1.56 (m, 1H), 1.17 (s, 3H), 1.03 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm) 204.7, 165.0, 149.3, 137.9, 133.7, 130.4, 129.6, 129.0, 127.9, 126.4, 123.7, 120.8, 96.0, 75.4, 73.1, 65.0, 61.5, 57.7, 52.9, 41.0, 40.5, 38.0, 31.8, 30.2, 29.8, 21.2, 20.8, 17.4; MS (ESI) m/z: 465.2 [M + H]+; HR-MS (ESI) m/z: calcd for C28H32NaO6 [M + Na]+ 487.2091, found 487.2100. ent-6β,7β-Dihydroxy-(14β-O-m-nitrylphenylvinoyl)-15-oxo-7,20epoxy-1-alkene-16-kaurene (13h). Following the procedure described for preparation of compound 13b, compound 13h was prepared from compound 13 as a white solid (Yield 63%). 1H NMR (CDCl3, 300 MHz): δ (ppm) 8.25 (s, 1H), 8.15 (dd, J = 8.1, 1.5 Hz, 1H), 7.72 (d, J = 7.8 Hz, 1H), 7.59, 6.39 (dd, JA = JB = 15.9 Hz, each1H), 7.51 (t, J = 7.8 Hz, 1H), 6.16 (s, 1H), 5.97 (s, 1H), 5.72 (m, 1H), 5.52 (s, 1H), 5.51 (m, 1H), 5.12 (dd, J = 10.2, 2.1 Hz, 1H), 3.90, 3.80 (dd, JA = JB = 10.2 Hz, each1H), 3.69 (d, J = 8.4 Hz, 1H), 3.11 (d, J = 9.3 Hz, 1H), 2.57 (m, 1H), 1.91 (m, 3H), 1.80 (m, 1H), 1.59 (m, 2H), 1.48 (d, J = 8.1 Hz, 1H), 1.07 (s, 3H), 0.95 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm) 204.6, 164.4, 149.2, 148.1, 142.2, 135.4, 133.2, 130.4, 129.5, 124.2, 123.7, 122.1, 121.0, 120.4, 96.0, 74.7, 73.2, 64.9, 61.5, 57.4, 52.8, 41.1, 40.4, 38.0, 31.8, 30.2, 29.8, 21.2, 17.4; MS (ESI) m/z: 522.2 [M + H]+; HR-MS (ESI) m/z: calcd for C29H31NNaO8 [M + Na]+ 544.1942, found 544.1943. ent-6β,7β-Dihydroxy-(14β-O-(2,3-dimethylbenzoyl))-15-oxo7,20-epoxy-1-alkene-16-kaurene (13i). Following the procedure described for preparation of compound 13b, compound 13i was prepared from compound 13 as a white solid (Yield 47%). 1H NMR (CDCl3, 300 MHz): δ (ppm) 7.43 (d, J = 7.8 Hz, 1H), 7.18 (d, J = 7.8 Hz, 1H), 6.99 (t, J = 7.8 Hz, 1H), 6.16 (s, 1H), 5.99 (s, 1H), 5.73 (m, 1H), 5.56 (d, J = 11.1 Hz, 1H), 5.50 (s, 1H), 5.15 (dd, J = 10.2, 2.4 Hz, 1H), 4.23 (br, 1H), 3.92, 3.85 (dd, JA = JB = 11.1 Hz, each1H), 3.73 (m, 1H), 3.20 (d, J = 9.3 Hz, 1H), 2.61 (m, 1H), 2.34 (s, 3H), 2.21 (s, 3H), 1.87 (m, 3H), 1.80 (m, 1H), 1.58 (m, 2H), 1.47 (d, J = 8.1 Hz, 1H), 1.08 (s, 3H), 0.95 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm) 204.6, 166.3, 149.4, 137.5, 133.2, 130.4, 129.4, 127.5, 124.9, 123.7, 120.8, 96.0, 75.3, 72.9, 65.0, 61.4, 57.7, 52.8, 41.0, 40.5, 38.0, 31.8, 30.3, 29.8, 29.2, 21.2, 20.0, 17.4, 16.1; MS (ESI) m/z: 479.3 [M + H]+; HR-MS (ESI) m/z: calcd for C29H34NaO6 [M + Na]+ 501.2248, found 501.2254. ent-6β,7β-Dihydroxy-(14β-O-(2,5-dimethylbenzoyl))-15-oxo7,20-epoxy-1-alkene-16-kaurene (13j). Following the procedure described for preparation of compound 13b, compound 13j was prepared from compound 13 as a white solid (Yield 64%). 1H NMR (CDCl3, 300 MHz): δ (ppm) 7.47 (s, 1H), 7.10 (d, J = 7.8 Hz, 1H), 7.01 (d, J = 7.8 Hz, 1H), 6.17 (s, 1H), 6.00 (s, 1H), 5.74 (m, 1H), 5.60 (m, 1H), 5.51 (s, 1H), 5.15 (dd, J = 10.2, 2.4 Hz, 1H), 4.19 (br, 1H), 3.92, 3.85 (dd, JA = JB = 11.1 Hz, each1H), 3.73 (m, 1H), 3.19 (d, J = 9.3 Hz, 1H), 2.69 (m, 1H), 2.41 (s, 3H), 2.21 (s, 3H), 1.86 (m, 3H), 1.79 (m, 1H), 1.59 (m, 2H), 1.48 (d, J = 8.1 Hz, 1H), 1.09 (s, 3H), 0.96 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm) 204.7, 165.7, 149.4, 136.9, 135.1, 132.8, 131.2, 130.4, 130.3, 123.7, 120.8, 96.0, 75.2, 73.0, 65.0, 61.4, 57.7, 52.8, 41.1, 40.5, 38.0, 31.8, 30.2, 29.8, 29.2, 21.2, 20.7, 20.4, 17.4; MS (ESI) m/z: 479.2 [M + H]+; HR-MS (ESI) m/z: calcd for C29H34NaO6 [M + Na]+ 501.2248, found 501.2242. ent-6β,7β-Dihydroxy-(14β-O-(2,6-dimethxoylbenzoyl))-15-oxo7,20-epoxy-1-alkene-16-kaurene (13k). Following the procedure described for preparation of compound 13b, compound 13k was prepared from compound 13 as a white solid (Yield 75%). 1H NMR (CDCl3, 300 MHz): δ (ppm) 7.24 (t, J = 8.4 Hz, 1H), 6.50 (d, J = 8.4 Hz, 2H), 5.92 (s, 1H), 5.72 (m, 1H), 5.57 (d, J = 11.4 Hz, 1H), 5.29 (s, 1H), 5.21 (s, 1H), 5.17 (dd, J = 10.2, 2.4 Hz, 1H), 4.82 (s, 1H), 4.06, 3.90 (dd, JA = JB = 11.1 Hz, each1H), 3.73 (m, 1H), 3.66 (s, 6H), 2.91 (d, J = 9.6 Hz, 1H), 2.41 (m, 1H), 1.88 (m, 3H), 1.72 (m, 2H), 1.55 (m, 1H), 1.08 (s, 3H), 1.06 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm) 204.7, 165.0, 149.3, 132.9, 130.7, 130.4, 129.6, 128.4, 127.8, 123.8, 120.9, 96.0, 74.8, 73.1, 65.0, 61.5, 58.2, 57.6, 52.8, 41.1, 40.5, 38.0, 31.7, 30.3, 29.2, 20.9, 17.4; MS (ESI) m/z: 511.2 [M + H]+; HR-MS (ESI) m/z: calcd for C29H34NaO8 [M + Na]+ 533.2146, found 533.2140.

(s, 1H), 5.75 (m, 2H), 5.63 (m, 1H), 5.47 (s, 1H), 5.14 (dd, J = 10.2, 2.4 Hz, 1H), 4.44 (br, 1H), 3.90, 3.80 (dd, JA = JB = 10.2 Hz, each1H), 3.76 (m, 1H), 3.03 (d, J = 9.3 Hz, 1H), 2.52 (m, 1H), 2.17 (m, 1H), 1.86 (m, 6H), 1.61 (m, 4H), 1.45 (m, 1H), 1.21 (m, 6H), 1.08 (s, 3H), 0.96 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm) 204.6, 174.0, 149.2, 130.4, 123.6, 120.5, 96.1, 75.0, 72.7, 65.0, 61.2, 57.8, 52.7, 42.7, 42.3, 40.9, 40.5, 38.0, 31.8, 30.2, 29.6, 28.3, 28.0, 21.2, 17.3; MS (ESI) m/z: 457.3 [M + H]+; HR-MS (ESI) m/z: calcd for C27H36NaO6 [M + Na]+ 479.2404, found 479.2411. ent-6β,7β-Dihydroxy-(14β-O-valeryl)-15-oxo-7,20-epoxy-1-alkene-16-kaurene (13c). Following the procedure described for preparation of compound 13b, compound 13c was prepared from compound 13 as a white solid (Yield 93%). 1H NMR (CDCl3, 300 MHz): δ (ppm) 6.13 (s, 1H), 5.78 (s, 1H), 5.73 (m, 1H), 5.52 (s, 1H), 5.14 (dd, J = 10.5, 2.4 Hz, 1H), 4.50 (br, 1H), 3.92, 3.81 (dd, JA = JB = 10.2 Hz, each1H), 3.73 (d, J = 9.1 Hz, 1H), 3.06 (d, J = 9.6 Hz, 1H), 2.52 (m, 1H), 2.20 (m, 2H), 1.86 (m, 4H), 1.50 (m, 6H), 1.28 (m, 4H), 1.08 (s, 3H), 0.96 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm) 204.7, 171.9, 149.2, 130.3, 123.6, 120.7, 96.0, 74.7, 72.8, 64.9, 61.2, 57.7, 52.7, 40.9, 40.5, 37.9, 33.8, 31.8, 30.2, 29.7, 26.3, 26.1, 21.6, 17.2, 13.2; MS (ESI) m/z: 431.2 [M + H]+; HR-MS (ESI) m/z: calcd for C25H34NaO6 [M + Na]+ 453.2248, found 453.2246. ent-6β,7β-Dihydroxy-(14β-O-octanoyl)-15-oxo-7,20-epoxy-1-alkene-16-kaurene (13d). Following the procedure described for preparation of compound 13b, compound 13d was prepared from compound 13 as a white solid (Yield 83%). 1H NMR (CDCl3, 300 MHz): δ (ppm) 6.13 (s, 1H), 5.77 (s, 1H), 5.75 (m, 1H), 5.62 (d, J = 11.4 Hz, 1H), 5.48 (s, 1H), 5.22 (dd, J = 10.5, 2.4 Hz, 1H), 4.34 (s, 1H), 3.90, 3.81 (dd, JA = JB = 10.2 Hz, each1H), 3.73 (m, 1H), 3.06 (d, J = 9.6 Hz, 1H), 2.52 (m, 1H), 2.19 (t, J = 7.5 Hz, 2H), 1.85 (m, 4H), 1.52 (m, 5H), 1.20 (m, 11H), 1.08 (s, 3H), 0.96 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm) 204.6, 171.8, 149.2, 130.3, 123.6, 120.6, 96.0, 74.9, 72.8, 64.9, 61.2, 57.7, 52.7, 40.9, 40.5, 37.9, 34.1, 31.8, 31.1, 30.2, 29.7, 28.4, 28.3, 24.1, 22.0, 21.2, 17.3, 13.5; MS (ESI) m/z: 473.3 [M + H]+; HR-MS (ESI) m/z: calcd for C28H40NaO6 [M + Na]+ 495.2717, found 495.2722. ent-6β,7β-Dihydroxy-(14β-O-3-chloro-4-pyridoyl)-15-oxo-7,20epoxy-1-alkene-16-kaurene (13e). Following the procedure described for preparation of compound 13b, compound 13e was prepared from compound 13 as a white solid (Yield 59%). 1H NMR (CDCl3, 300 MHz): δ (ppm) 8.32 (m, 1H), 8.10 (m, 1H), 7.08 (m, 1H), 6.14 (m, 2H), 5.74 (m, 1H), 5.65 (m, 1H), 5.54 (s, 1H), 5.18 (d, J = 10.2 Hz, 1H), 4.43 (m, 1H), 3.88 (m, 2H), 3.71 (m, 1H), 3.17 (d, J = 9.3 Hz, 1H), 2.57 (m, 1H), 1.88 (m, 4H), 1.61 (m, 1H), 1.46 (m, 1H), 1.18 (m, 1H), 1.06 (s, 3H), 0.94 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm) 204.5, 162.6, 151.1, 149.4, 140.4, 130.4, 123.8, 121.7, 121.0, 95.9, 74.7, 73.5, 64.9, 61.6, 57.1, 52.8, 41.2, 40.4, 38.1, 31.8, 30.3, 29.8, 21.2, 17.3; MS (ESI) m/z: 486.2 [M + H]+; HR-MS (ESI) m/z: calcd for C26H28ClNNaO6 [M + Na]+ 508.1497, found 508.1502. ent-6β,7β-Dihydroxy-(14β-O-o-trifluoromethylbenzoyl)-15-oxo7,20-epoxy-1-alkene-16-kaurene (13f). Following the procedure described for preparation of compound 13b, compound 13f was prepared from compound 13 as a white solid (Yield 83%). 1H NMR (CDCl3, 300 MHz): δ (ppm) 7.70 (m, 1H), 7.70 (m, 1H), 7.38 (m, 2H), 6.13 (m, 2H), 5.76 (m, 1H), 5.60 (m, 1H), 5.52 (s, 1H), 5.16 (dd, J = 10.2, 2.4 Hz, 1H), 4.19 (br, 1H), 3.94, 3.84 (dd, JA = JB = 10.2 Hz, each 1H), 3.72 (m, 1H), 3.16 (d, J = 9.3 Hz, 1H), 2.58 (m, 1H), 1.88 (m, 4H), 1.61 (m, 1H), 1.47 (m, 1H), 1.18 (m, 1H), 1.08 (s, 3H), 0.95 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm) 204.9, 149.8, 131.9, 130.9, 130.4, 126.3, 124.3, 121.3, 96.4, 75.6, 73.8, 65.5, 62.0, 57.7, 53.4, 41.4, 41.0, 38.6, 32.3, 30.8, 30.3, 29.7, 21.6, 17.9; MS (ESI) m/z: [M + H]+; HR-MS (ESI) m/z: calcd for C28H29F3NaO6 [M + Na]+ 541.1808, found 541.1813. ent-6β,7β-Dihydroxy-(14β-O-m-methylbenzoyl)-15-oxo-7,20epoxy-1-alkene-16-kaurene (13g). Following the procedure described for preparation of compound 13b, compound 13g was prepared from compound 13 as a white solid (Yield 74%). 1H NMR (CDCl3, 300 MHz): δ (ppm) 7.71 (m, 2H), 7.30 (m, 2H), 6.24 (s, 1H), 6.07 (s, 1H), 5.82 (m, 1H), 5.63 (d, J = 11.4 Hz, 1H), 5.56 (s, 1H), 5.21 (dd, J = 10.2, 2.4 Hz, 1H), 4.21 (s, 1H), 3.98, 3.91 (dd, JA = 1462



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ent-6β,7β-Dihydroxy-(14β-O-(2,3,4,5-tetrafluorobenzoyl))-15oxo-7,20-epoxy-1-alkene-16-kaurene (13l). Following the procedure described for preparation of compound 13b, compound 13l was prepared from compound 13 as a white solid (Yield 52%). 1H NMR (CDCl3, 300 MHz): δ (ppm) 7.52 (m, 1H), 6.17 (s, 1H), 6.13 (s, 1H), 5.74 (m, 1H), 5.53 (d, J = 12.0 Hz, 1H), 5.54 (s, 1H), 5.15 (dd, J = 10.5, 2.4 Hz, 1H), 3.98 (s, 1H), 3.92, 3.84 (dd, JA = JB = 11.1 Hz, each1H), 3.67 (m, 1H), 3.13 (d, J = 9.3 Hz, 1H), 2.55 (m, 1H), 1.88 (m, 4H), 1.61 (m, 1H), 1.46 (m, 1H), 1.18 (m, 1H), 1.07 (s, 3H), 0.95 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm) 204.2, 149.1, 130.4, 123.7, 121.0, 113.1, 112.9, 95.8, 75.0, 73.5, 65.0, 61.7, 57.1, 52.9, 41.2, 40.7, 38.1, 31.8, 30.2, 29.8, 29.2, 21.1, 17.5; MS (ESI) m/z: 523.2 [M + H]+; HR-MS (ESI) m/z: calcd for C27H26F4NaO6 [M + Na]+ 545.1558, found 545.1566. ent-6β,7β-Dihydroxy-(14β-O-p-nitrylbenzoyl)-15-oxo-7,20epoxy-1-alkene-16-kaurene (13m). Following the procedure described for preparation of compound 13b, compound 13m was prepared from compound 13 as a white solid (Yield 83%). 1H NMR (CDCl3, 300 MHz): δ (ppm) 8.07 (m, 4H), 6.18 (s, 1H), 6.11 (s, 1H), 5.75 (m, 2H), 5.56 (s, 1H), 5.17 (dd, J = 10.2, 2.4 Hz, 1H), 4.61 (br, 1H), 3.90, 3.83 (dd, JA = JB = 11.1 Hz, each1H), 3.69 (m, 1H), 3.17 (d, J = 9.3 Hz, 1H), 2.67 (m, 1H), 1.86 (m, 3H), 1.79 (m, 1H), 1.62 (m, 2H), 1.43 (d, J = 8.1 Hz, 1H), 1.04 (s, 3H), 0.93 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm) 204.8, 163.5, 149.8, 149.1, 135.4, 130.5, 130.4, 123.7, 122.8, 121.2, 95.8, 74.8, 73.4, 64.9, 61.6, 57.2, 52.7, 41.2, 40.4, 38.0, 31.8, 30.3, 29.8, 29.2, 21.1, 17.4; MS (ESI) m/z: 496.3 [M + H]+; HR-MS (ESI) m/z: calcd for C27H29NNaO8 [M + Na]+ 518.1785, found 518.1781. ent-6β,7β-Dihydroxy-(14β-O-p-bromobenzoyl)-15-oxo-7,20epoxy-1-alkene-16-kaurene (13n). Following the procedure described for preparation of compound 13b, compound 13n was prepared from compound 13 as a white solid (Yield 78%). 1H NMR (CDCl3, 300 MHz): δ (ppm) 7.73 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 8.4 Hz, 2H), 6.16 (s, 1H), 6.03 (s, 1H), 5.76 (m, 1H), 5.60 (br, 1H), 5.51 (s, 1H), 5.15 (dd, J = 10.2, 2.4 Hz, 1H), 4.10 (br, 1H), 3.89, 3.81 (dd, JA = JB = 11.1 Hz, each1H), 3.69 (m, 1H), 3.15 (d, J = 9.3 Hz, 1H), 2.58 (m, 1H), 1.88 (m, 3H), 1.77 (m, 1H), 1.62 (m, 2H), 1.43 (d, J = 8.1 Hz, 1H), 1.06 (s, 3H), 0.93 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm) 204.7, 164.3, 149.3, 131.2, 130.9, 130.4, 128.5, 127.7, 122.8, 121.0, 95.9, 74.8, 73.3, 64.9, 61.5, 57.4, 52.8, 41.1, 40.5, 38.0, 31.8, 30.3, 29.8, 29.2, 21.2, 17.4; MS (ESI) m/z: 529.1 [M + H]+; HR-MS (ESI) m/z: calcd for C27H29BrNaO6 [M + Na]+ 551.1040, found 551.1035. ent-6β,7β-Dihydroxy-(14β-O-m-chlorobenzoyl)-15-oxo-7,20epoxy-1-alkene-16-kaurene (13o). Following the procedure described for preparation of compound 13b, compound 13o was prepared from compound 13 as a white solid (Yield 62%). 1H NMR (CDCl3, 300 MHz): δ (ppm) 7.92 (s, 1H), 7.82 (d, J = 7.8 Hz, 1H), 7.48 (d, J = 7.8 Hz, 1H), 7.31 (t, J = 7.8 Hz, 1H), 6.24 (s, 1H), 6.13 (s, 1H), 5.80 (m, 1H), 5.67 (m, 1H), 5.58 (s, 1H), 5.22 (dd, J = 10.2, 2.4 Hz, 1H), 4.22 (br, 1H), 3.97, 3.91 (dd, JA = JB = 11.1 Hz, each1H), 3.76 (m, 1H), 3.21 (d, J = 9.3 Hz, 1H), 2.64 (m, 1H), 1.97 (m, 3H), 1.86 (m, 1H), 1.68 (m, 2H), 1.54 (m, 1H), 1.13 (s, 3H), 1.00 (s, 3H); 13 C NMR (CDCl3, 75 MHz): δ (ppm) 204.7, 163.9, 149.3, 132.6, 130.4, 129.3, 129.2, 127.5, 123.8, 120.9, 95.9, 74.8, 73.3, 64.9, 61.6, 57.3, 52.8, 41.2, 40.5, 38.0, 31.8, 30.2, 29.8, 29.2, 21.1, 17.4; MS (ESI) m/z: 485.2 [M + H]+; HR-MS (ESI) m/z: calcd for C27H29ClNaO6 [M + Na]+ 507.1545, found 507.1552. ent-6β,7β-Dihydroxy-(14β-O-phenylvinoyl)-15-oxo-7,20-epoxy-1alkene-16-kaurene (13p). Following the procedure described for preparation of compound 13b, compound 13p was prepared from compound 13 as a white solid (Yield 73%). 1H NMR (CDCl3, 300 MHz): δ (ppm) 7.56, 6.27 (dd, JA = JB = 16.0 Hz, each1H), 7.40 (m, 2H), 7.29 (m, 3H), 6.15 (s, 1H), 5.89 (s, 1H), 5.72 (m, 1H), 5.55 (d, J = 11.4 Hz, 1H), 5.49 (m, 1H), 5.13 (dd, J = 10.2, 2.2 Hz, 1H), 4.24 (s, 1H), 3.90, 3.82 (dd, JA = JB = 10.2 Hz, each1H), 3.78 (m, 1H), 3.15 (d, J = 9.3 Hz, 1H), 2.58 (m, 1H), 1.88 (m, 3H), 1.78 (m, 1H), 1.58 (m, 1H), 1.47 (m, 1H), 1.18 (m, 1H), 1.09 (s, 3H), 0.96 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm) 204.6, 164.9, 149.2, 145.7, 133.5, 130.3, 130.1, 128.4, 127.8, 123.7, 120.8, 116.7, 96.1, 75.2, 72.9, 64.9,

61.4, 57.8, 52.8, 41.0, 40.5, 38.0, 31.8, 30.2, 29.8, 21.2, 17.3; MS (ESI) m/z: 477.2 [M + H]+; HR-MS (ESI) m/z: calcd for C29H32NaO6 [M + Na]+ 499.2091, found 499.2093. ent-6β,7β-Dihydroxy-(14β-O-p-fluorophenylvinoyl)-15-oxo-7,20epoxy-1-alkene-16-kaurene (13q). Following the procedure described for preparation of compound 13b, compound 13q was prepared from compound 13 as a white solid (Yield 63%). 1H NMR (CDCl3, 300 MHz): δ (ppm) 7.61, 6.27 (dd, JA = JB = 16.2 Hz, each1H), 7.48 (m, 2H), 7.06 (t, J = 7.8 Hz, 2H), 6.23 (s, 1H), 5.98 (s, 1H), 5.81 (m, 1H), 5.67 (m, 1H), 5.58 (s, 1H), 5.22 (dd, J = 10.2, 2.4 Hz, 1H), 4.30 (br, 1H), 3.98, 3.95 (dd, JA = JB = 10.2 Hz, each1H), 3.82 (m, 1H), 3.22 (d, J = 9.0 Hz, 1H), 2.63 (m, 1H), 1.93 (m, 3H), 1.86 (m, 1H), 1.67 (m, 2H), 1.53 (d, J = 8.1 Hz, 1H), 1.17 (s, 3H), 1.04 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm) 204.7, 164.9, 161.9, 149.2, 144.3, 130.4, 129.8, 129.7, 123.7, 120.9, 116.5, 115.7, 115.4, 96.1, 75.1, 72.9, 65.0, 61.4, 57.7, 52.8, 41.0, 40.5, 38.0, 31.8, 30.2, 29.8, 29.2, 21.2, 17.3; MS (ESI) m/z: 495.2 [M + H]+; HR-MS (ESI) m/z: calcd for C29H31FNaO6 [M + Na]+ 517.1997, found 517.1997. ent-6β,7β-Dihydroxy-(14β-O-p-methoxylphenylvinoyl)-15-oxo7,20-epoxy-1-alkene-16-kaurene (13r). Following the procedure described for preparation of compound 13b, compound 13r was prepared from compound 13 as a white solid (Yield 52%). 1H NMR (CDCl3, 300 MHz): δ (ppm) 7.60, 6.22 (dd, JA = JB = 15.9 Hz, each1H), 7.44 (d, J = 7.8 Hz, 2H), 6.88 (d, J = 7.8 Hz, 2H), 6.22 (s, 1H), 5.93 (s, 1H), 5.85 (m, 1H), 5.78 (m, 1H), 5.56 (s, 1H), 5.21 (dd, J = 10.2, 2.4 Hz, 1H), 4.44 (br, 1H), 4.00, 3.90 (dd, JA = JB = 10.2 Hz, each 1H), 3.86 (m, 1H), 3.83 (s, 3H), 3.23 (d, J = 9.0 Hz, 1H), 2.64 (m, 1H), 1.97 (m, 3H), 1.88 (m, 1H), 1.66 (m, 2H), 1.53 (d, J = 8.1 Hz, 1H), 1.17 (s, 3H), 1.04 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm) 204.7, 165.2, 161.2, 149.2, 145.6, 130.3, 129.6, 126.2, 123.7, 120.9, 113.9, 113.0, 96.1, 75.3, 72.8, 65.0, 61.3, 57.9, 54.9, 52.7, 40.9, 40.5, 38.0, 31.8, 30.2, 29.7, 29.1, 21.2, 17.3; MS (ESI) m/z: 507.2 [M + H]+; HR-MS (ESI) m/z: calcd for C30H34NaO7 [M + Na]+ 529.2197, found 529.2192. ent-6β,7β-Dihydroxy-(14β-O-o-methoxylphenylvinoyl)-15-oxo7,20-epoxy-1-alkene-16-kaurene (13s). Following the procedure described for preparation of compound 13b, compound 13s was prepared from compound 13 as a white solid (Yield 34%). 1H NMR (CDCl3, 300 MHz): δ (ppm) 7.85, 6.39 (dd, JA = JB = 15.9 Hz, each1H), 7.35 (dd, J = 7.8, 1.5 Hz, 1H), 7.27 (t, J = 7.8 Hz, 1H), 6.83 (m, 2H), 6.15 (s, 1H), 5.84 (s, 1H), 5.74 (m, 1H), 5.58 (br, 1H), 5.48 (s, 1H), 5.17 (dd, J = 10.2, 2.1 Hz, 1H), 4.30 (br, 1H), 3.91, 3.79 (dd, JA = JB = 10.2 Hz, each1H), 3.84 (m, 1H), 3.82 (s, 3H), 3.18 (d, J = 9.0 Hz, 1H), 2.58 (m, 1H), 1.90 (m, 3H), 1.80 (m, 1H), 1.59 (m, 2H), 1.47 (d, J = 8.1 Hz, 1H), 1.10 (s, 3H), 0.97 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm) 204.7, 165.5, 158.2, 149.1, 142.0, 131.5, 130.3, 129.3, 123.6, 122.3, 120.8, 120.2, 116.9, 110.6, 96.2, 75.7, 72.7, 65.0, 61.2, 58.0, 55.0, 52.8, 40.8, 40.6, 38.0, 31.9, 30.2, 29.7, 21.2, 17.3; MS (ESI) m/z: 507.3 [M + H]+; HR-MS (ESI) m/z: calcd for C30H34NaO7 [M + Na]+ 529.2197, found 529.2196. ent-6β,7β-Dihydroxy-(14β-O-(3,4,5-trimethoxylphenylvinoyl))15-oxo-7,20-epoxy-1-alkene-16-kaurene (13t). Following the procedure described for preparation of compound 13b, compound 13t was prepared from compound 13 as a white solid (Yield 58%). 1H NMR (CDCl3, 300 MHz): δ (ppm) 7.47, 6.17 (dd, JA = JB = 15.9 Hz, each1H), 6.65 (s, 2H), 6.16 (s, 1H), 5.88 (s, 1H), 5.73 (m, 1H), 5.53 (br, 1H), 5.51 (s, 1H), 5.13 (dd, J = 10.2, 2.1 Hz, 1H), 4.30 (br, 1H), 3.91, 3.79 (dd, JA = JB = 10.2 Hz, each1H), 3.84 (m, 1H), 3.82 (s, 3H), 3.18 (d, J = 9.0 Hz, 1H), 2.58 (m, 1H), 1.90 (m, 3H), 1.80 (m, 1H), 1.59 (m, 2H), 1.47 (d, J = 8.1 Hz, 1H), 1.10 (s, 3H), 0.97 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm) 204.7, 164.8, 152.9, 149.1, 145.7, 130.4, 128.9, 123.6, 121.0, 115.7, 104.9, 96.2, 75.5, 72.9, 65.0, 61.3, 60.5, 57.8, 55.7, 52.8, 40.9, 40.5, 38.0, 31.8, 30.3, 29.8, 29.2, 21.2, 17.3; MS (ESI) m/z: 567.3 [M + H]+; HR-MS (ESI) m/z: calcd for C32H38NaO9 [M + Na]+ 589.2408, found 589.2402. ent-3β,6β,7β,14β-Tetrahydroxy-15-oxo-7,20-epoxy-1-alkene-16kaurene (14). Selenium dioxide (480 mg, 4.33 mmol) was added to a solution of compound 13 (300 mg, 0.87 mmol) in 15 mL dioxane, and the reaction was stirred at 110 °C for 48 h. The resulting mixture was 1463



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diluted with 50 mL dichloromethane and filtered through Celite, and the solvents were removed under reduced pressure. The residue was purified by column chromatography (MeOH/CH2Cl2 1:80, v/v) to obtain pure compound 14 as a light yellow solid (62.2 mg, 20%). 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 7.16 (s, 1H), 6.04 (s, 1H), 5.96 (s, 1H), 5.87 (m, 1H), 5.64 (s, 1H), 5.44 (d, J = 11.1 Hz, 1H), 5.28 (d, J = 10.2 Hz, 1H), 4.78 (s, 1H), 4.56 (d, J = 6.9 Hz, 1H), 3.83, 3.67 (dd, JA = JB = 9.9 Hz, each1H), 3.60 (m, 1H), 2.92 (d, J = 9.0 Hz, 1H), 2.43 (m, 1H), 1.81 (m, 2H), 1.73 (d, J = 8.4 Hz, 1H), 1.51 (m, 1H), 1.45 (m, 1H), 1.23 (s, 1H), 1.02 (s, 3H), 0.87 (s, 3H); 13C NMR (DMSO-d6, 75 MHz): δ (ppm) 206.8, 151.6, 131.8, 127.0, 120.4, 97.5, 72.0, 72.0, 70.9, 64.1, 61.3, 51.1, 50.7, 42.6, 37.8, 36.5, 29.7, 26.0, 21.8, 16.9; MS (ESI) m/z: 363.2 [M + H]+; HR-MS (ESI) m/z: calcd for C20H26NaO6 [M + Na]+ 385.1622, found 385.1625. The β-orientation of 3-OH was confirmed according to the literature report.34 ent-6β,7β,14β-Trihydroxy-3,15-dioxo-7,20-epoxy-1-alkene-16kaurene (15). Compound 14 (100 mg, 0.27 mmol) was dissolved in 15 mL acetone. To this solution, Jones reagent was added dropwise until a red color persisted. The mixture was stirred at 0 °C for 15 min, and isopropanol was added. Then the mixture was diluted with water and extracted with dichloromethane. The extract was washed with brine, dried over anhydrous Na2SO4, filtered, and evaporated to give a crude product, which was purified by column chromatography (MeOH/CH2Cl2 1:120, v/v) to obtain compound 15 (78 mg, 78%) as a white powder. 1H NMR (CDCl3, 300 MHz): δ (ppm) 7.19 (s, 1H), 6.48 (d, J = 10.2 Hz, 1H), 6.12 (s, 1H), 6.04 (s, 1H), 5.91, 5.74 (dd, JA = JB = 10.5 Hz, each1H), 5.66 (s, 1H), 4.80 (s, 1H), 4.09, 3.90 (dd, JA = JB = 9.9 Hz, each1H), 3.73 (m, 1H), 2.97 (d, J = 9.9 Hz, 1H), 2.44 (m, 1H), 1.94 (m, 2H), 1.87 (m, 1H), 1.55 (m, 2H), 1.15 (s, 3H), 1.13 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm) 207.1, 202.6, 151.0, 145.0, 128.6, 120.8, 97.4, 72.2, 71.3, 63.7, 61.1, 56.5, 50.2, 43.8, 42.6, 38.6, 29.2, 23.7, 21.6, 16.7; MS (ESI) m/z: 361.2 [M + H]+; HRMS (ESI) m/z: calcd for C20H25O6 [M + H]+ 361.1646, found 361.1649. ent-6β,7β-Dihydroxy-3,15-dioxo-(14β-O-acetyl)-7,20-epoxy-1-alkene-16-kaurene (15a). Following the procedure described for preparation of compound 13a, compound 15a was prepared from compound 15 as a white solid (Yield 73%). 1H NMR (CDCl3, 300 MHz): δ (ppm) 6.32, 6.02 (dd, JA = JB = 10.5 Hz, each1H), 6.24 (s, 1H), 5.87 (s, 1H), 5.76 (d, J = 11.1 Hz, 1H), 5.61 (s, 1H), 4.29 (s, 1H), 4.13, 4.10 (dd, JA = JB = 9.9 Hz, each1H), 3.93 (m, 1H), 3.19 (d, J = 9.6 Hz, 1H), 2.67 (m, 1H), 2.09 (m, 1H), 2.06 (s, 3H), 1.94 (m, 1H), 1.85 (m, 1H), 1.58 (m, 2H), 1.34 (s, 3H), 1.28 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm) 212.2, 201.0, 148.5, 141.9, 129.8, 121.6, 92.7, 74.7, 71.8, 70.4, 64.3, 56.1, 52.3, 51.7, 40.7, 29.8, 24.8, 23.1, 21.3, 20.9, 17.2; MS (ESI) m/z: 403.2 [M + H]+; HR-MS (ESI) m/z: calcd for C22H26NaO7 [M + Na]+ 425.1571, found 425.1576. ent-6β,7β-Dihydroxy-3,15-dioxo-(14β-O-3-chloro-4-pyridoyl)7,20-epoxy-1-alkene-16-kaurene (15b). Following the procedure described for preparation of compound 13b, compound 15b was prepared from compound 15 as a white solid (Yield 79%). 1H NMR (CDCl3, 300 MHz): δ (ppm) 8.48 (dd, J = 4.8, 1.8 Hz, 1H), 8.17 (dd, J = 7.8, 1.8 Hz, 1H), 7.28 (m, 1H), 6.27 (s, 1H), 6.24 (s, 1H), 6.35, 6.04 (dd, JA = JB = 10.5 Hz, each1H), 5.73 (d, J = 12.0 Hz, 1H), 5.66 (s, 1H), 4.17 (m, 2H), 3.92 (m, 2H), 3.30 (m, 1H), 2.74 (m, 1H), 2.09 (m, 2H), 1.96 (m, 1H), 1.79 (m, 2H), 1.59 (s, 3H), 1.33 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm) 212.2, 204.3, 151.5, 141.9, 140.4, 129.9, 121.9, 91.0, 74.7, 72.4, 64.3, 55.4, 51.9, 44.0, 41.0, 38.8, 29.4, 23.1, 21.4, 17.4; MS (ESI) m/z: 500.2 [M + H]+; HR-MS (ESI) m/z: calcd for C26H26ClNNaO7 [M + Na]+ 522.1290, found 522.1287. ent-6β,7β-Dihydroxy-3,15-dioxo-(14β-O-o-trifluoromethylbenzoyl)-7,20-epoxy-1-alkene-16-kaurene (15c). Following the procedure described for preparation of compound 13b, compound 15c was prepared from compound 15 as a white solid (Yield 88%). 1H NMR (CDCl3, 300 MHz): δ (ppm) 7.69 (m, 1H), 7.56 (m, 1H), 7.40 (m, 2H), 6.13 (s, 2H), 5.74 (m, 1H), 5.61 (m, 1H), 5.53 (s, 1H), 5.17 (dd, J = 10.5, 2.4 Hz, 1H), 4.24 (br, 1H), 3.93, 3.84 (dd, JA = JB = 10.2 Hz, each1H), 3.72 (m, 1H), 3.17 (d, J = 9.0 Hz, 1H), 2.58 (m, 1H), 1.90 (m, 3H), 1.61 (m, 1H), 1.45 (m, 1H), 1.08 (s, 3H), 0.96 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm) 204.9, 165.1, 149.8, 131.9, 130.9,

130.4, 126.3, 124.3, 121.3, 96.4, 75.6, 73.8, 65.5, 62.0, 57.7, 53.5, 41.4, 40.9, 38.6, 32.3, 30.8, 30.3, 21.6, 17.9; MS (ESI) m/z: 533.2 [M + H]+; HR-MS (ESI) m/z: calcd for C28H27F3NaO7 [M + Na]+ 555.1601, found 555.1606. ent-1β,2β-Epoxy-6β-hydroxy-7,14-isopropylideneketal-15-oxo7,20-epoxy-16-kaurene (16). Metachloroperbenzoic acid (m-CPBA, 134 mg, 0.78 mmol) was added to a solution of 12 (200 mg, 0.52 mmol) in 30 mL dichloromethane. The mixture was stirred at room temperature for 48 h, and then the mixture was diluted with water and extracted with dichloromethane. The extract was washed with brine, dried over anhydrous Na2SO4, filtered, and evaporated to give a crude product, which was purified by column chromatography (MeOH/ CH2Cl2 1:200, v/v) to obtain compound 16 (148 mg, 71%) as a white powder. 1H NMR (CDCl3, 300 MHz): δ (ppm) 6.18 (s, 1H), 5.58 (s, 1H), 5.49 (d, J = 11.7 Hz, 1H), 4.83 (s, 1H), 4.11, 3.99 (dd, JA = JB = 9.6 Hz, each1H), 3.79 (m, 1H), 3.24 (m, 1H), 3.07 (d, J = 6.0 Hz, 1H), 2.58 (d, J = 3.9 Hz, 1H), 2.51 (m, 1H), 2.04 (m, 1H), 1.85 (m, 2H), 1.67 (m, 4H), 1.61 (s, 3H), 1.29 (s, 3H), 1.19 (s, 3H), 1.17 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm) 203.9, 149.8, 120.2, 100.9, 94.8, 71.4, 69.5, 64.4, 53.9, 52.7, 51.9, 45.1, 39.9, 39.7, 35.5, 33.2, 29.8, 29.6, 29.5, 25.0, 23.0, 16.1; MS (ESI) m/z: 403.2 [M + H]+; HR-MS (ESI) m/z: calcd for C23H30NaO6 [M + Na]+ 425.1935, found 425.1938. ent-(3β,14β-O-Acetyl)-6β,7β-drihydroxy-15-oxo-7,20-epoxy-1-alkene-16-kaurene (17). Compound 14 (50 mg, 0.14 mmol) was dissolved in dichloromethane, and dimethylaminopyridine (DMAP, catalytic amount), acetic anhydride (0.1 mL), and triethylamine (0.2 mL) were added. The mixture was stirred at room temperature for 2.5 h, diluted with water, and extracted with dichloromethane. The extract was washed with saturated NaHCO3 solution and brine, dried over anhydrous Na2SO4, filtered, and evaporated to afford a crude product, which was purified by column chromatography (MeOH/CH2Cl2 1:150, v/v) to obtain compound 17 (55 mg, 89%). 1H NMR (CDCl3, 300 MHz): δ (ppm) 6.17 (s, 1H), 5.93 (m, 1H), 5.79 (s, 1H), 5.56 (d, J = 11.4 Hz, 1H), 5.53 (s, 1H), 5.42 (d, J = 10.2 Hz, 1H), 4.76 (d, J = 10.2 Hz, 1H), 4.21 (s, 1H), 3.87 (s, 2H), 3.76 (m, 1H), 3.09 (d, J = 9.6 Hz, 1H), 2.53 (m, 1H), 2.23 (s, 1H), 1.99 (s, 3H), 1.97 (s, 3H), 1.91 (m, 1H), 1.81 (m, 2H), 1.57 (m, 1H), 1.07 (s, 3H), 1.00 (s, 3H); 13 C NMR (CDCl3, 75 MHz): δ (ppm) 204.5, 170.1, 169.0, 148.9, 129.6, 128.1, 121.1, 96.0, 74.8, 72.9, 72.4, 64.6, 52.4, 52.0, 40.8, 38.0, 35.3, 24.8, 20.9, 20.8, 20.6, 17.3; MS (ESI) m/z: 447.2 [M + H]+; HRMS (ESI) m/z: calcd for C24H30NaO8 [M + Na]+ 469.1833, found 469.1845. ent-1β,6β,7β,14β-Tetrahydroxy-2α- chloro-15-dioxo-7,20-epoxy16-kaurene (18). Compound 16 (100 mg, 0.25 mmol) was added to 10 mL of 10% HCl/THF (1:1), and the solution was stirred at room temperature for 2 h. Then the mixture was diluted with water and extracted with dichloromethane. The extract was washed with brine, dried over anhydrous Na2SO4, filtered, and evaporated to give compound 18 (82 mg, 82%) as a white powder. 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 6.91 (s, 1H), 6.09 (s, 1H), 6.06 (d, J = 10.2 Hz, 1H), 6.02 (s, 1H), 5.62 (s, 1H), 5.52 (d, J = 4.8 Hz, 1H), 4.77 (s, 1H), 4.45, 3.91 (dd, JA = JB = 10.2 Hz, each1H), 4.20 (m, 1H), 3.63 (m, 1H), 2.95 (d, J = 9.3 Hz, 1H), 2.42 (m, 1H), 2.15 (m, 1H), 1.88 (m, 2H), 1.80 (m, 1H), 1.62 (m, 1H), 1.52 (m, 1H), 1.47 (m, 1H), 1.38 (m, 1H), 1.05 (s, 3H), 0.96 (s, 3H); 13C NMR (DMSO-d6, 75 MHz): δ (ppm) 208.3, 151.6, 120.0, 96.7, 72.6, 72.3, 68.1, 64.2, 60.9, 59.2, 53.6, 47.3, 42.6, 39.6, 39.8, 34.2, 32.7, 29.5, 25.2, 15.1; MS (ESI) m/z: 399.1 [M + H]+; HR-MS (ESI) m/z: calcd for C20H27ClNaO6 [M + Na]+ 421.1388, found 421.1395. The stereostructure of compound was determined by NOESY experiment. ent-1β,2β-Epoxy-6β,7β,14β-trihydroxy-15-oxo-7,20-epoxy-16kaurene (19). Compound 16 (100 mg, 0.25 mmol) was added to 10 mL of 10% H2SO4/THF (1:1), and the solution was stirred at room temperature for 0.5 h. Then the mixture was diluted with water and extracted with dichloromethane. The extract was washed with brine, dried over anhydrous Na2SO4, filtered, and evaporated to give compound 19 (58 mg, 65%) as a white powder. 1H NMR (CDCl3, 300 MHz): δ (ppm) 6.18 (m, 2H), 5.62 (s, 1H), 5.53 (s, 1H), 4.83 (s, 1H), 4.56 (s, 1H), 4.07, 3.98 (dd, JA = JB = 9.6 Hz, each1H), 3.63 (m, 1464



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1H), 3.18 (m, 1H), 2.98 (d, J = 9.0 Hz, 1H), 2.51 (d, J = 3.6 Hz, 1H), 2.46 (m, 1H), 2.02 (m, 1H), 1.81 (m, 1H), 1.77 (m, 1H), 1.67 (m, 3H), 1.47 (s, 1H), 0.97 (s, 3H), 0.95 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm) 205.9, 150.4, 121.2, 97.1, 73.0, 71.7, 65.0, 61.2, 53.4, 52.8, 52.1, 48.4, 42.3, 39.5, 36.0, 31.9, 29.5, 29.1, 22.8, 16.3; MS (ESI) m/z: 363.2 [M + H]+; HR-MS (ESI) m/z: calcd for C20H26NaO6 [M + Na]+ 385.1622, found 385.1628. Biology. Materials. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), propidium iodide (PI), 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1), 2′,7′-dichlorofluorescein diacetate (DCF-DA), and crystal violet were purchased from Sigma Chemical Co. (St. Louis, MO, USA). CytoTox-ONE Homogeneous Membrane Integrity Assay Kit was purchased from Promega (Madison, WI, USA). The Annexin V-FITC apoptosis detection kit was purchased from BD Parmingen (San Diego, CA, USA). Primary antibodies against caspase-3, caspase-9, pro-caspase-3, Bax, Bad, Bcl-2, p-ERK, P53, cytotochrome c, p-cdc25c, cdc2, cyclin B1, and β-actin were purchased from Beyotime (Jiangsu, China), and fluorescent secondary antibodies (goat-antirabbit or goatantimouse) were purchased from Life Technologies (Grand Island, NY, USA). All cell culture reagents including non-essential amino acids (NEAA) and the BCA protein assay kit were purchased from Life Technologies (Grand Island, NY, USA). Cell Lines and Cell Culture. Human gastric cancer cells (MGC-803) and human breast cancer cells (MCF-7) were grown in DMEM medium (Life Technologies, USA), and 1× NEAA (Life Technologies, USA) was used for the culture of MCF-7 cells. Human hepatocellular carcinoma cells (Bel-7402), human leukemia cells (K562), human cervix carcinoma cells (KB-3-1), human lung carcinoma cells (NCIH460), and human hepatocellular carcinoma cells (Bel-7404) were grown in RPMI 1640 medium (Life Technologies, USA). The medium for all cell lines were supplemented with 10% fetal bovine serum (Life Technologies, USA), 100 μg/mL streptomycin (Life Technologies, USA), and 100 U/mL penicillin (Life Technologies, USA) and maintained at 37 °C in a humidified atmosphere with 5% CO2. Determination of Anticancer Activity in Vitro. The overall growth of human cancer cell lines was determined using the colorimetric MTT assay. Briefly, the cell lines were incubated at 37 °C in a humidified 5% CO2 incubator for 24 h in 96-microwell plates prior to the experiments. MGC-803, MCF-7, Bel-7402, and K562 cells were seeded at a density of 3500 cells/well, KB-3-1, NCI-H460, and Bel7404 cells at 4000 cells/well, and KB/CP4, NCI-H460/MX20, and Bel-7404/CP20 cell at 7000 cells/well. After the removal of medium, 100 μL of fresh medium containing the test compound at different concentrations was added to each well and incubated at 37 °C for 48 or 72 h. The percentage of DMSO in the medium was not exceeded 0.25%. The number of living cells after 72 h (or 48 h) of culture in the presence (or absence: control) of the various compounds is directly proportional to the intensity of the blue, which is quantitatively measured by spectrophotometry (Biorad, Nazareth, Belgium) at a 570 nm wavelength. The experiment was performed in quadruplicate and repeated three times. Caco2 Permeability Assay. Caco2 cells between passages 20 and 40 were cultured at 37 °C. After 21 days of culture, the integrity of the cell monolayer was verified by measuring the trans-epithelial electrical resistance (TEER). Only cell monolayers with TEER exceeding 200 Ω cm2 were selected for the measurement of drug transport from the apical side to the basolateral side (A to B). The monolayer of cells was treated in triplicate with 100 μM of 13 or 13p in the well with the appropriate pH (pH 6.8 for apical side and pH 7.4 for basolateral side) for 30 min. Samples were collected from the donor and receiver sides at 0 and 5 min of incubation. The concentrations of the drug were determined by HPLC (Phenomenex Gemini C18, 4.6 × 150 mm, 5 μm, samples were detected using a wavelength of 240 nm with a mobile phase of 80:20 (v/v) methanol:water at a flow rate of 1 mL/ min).46 Colony Formation Assay. MCF-7 cells (3 × 102 per well) were plated in 30 mm plates. After an overnight incubation, the cells were treated with various concentrations of 13p dissolved in DMSO. Some cells were treated with vehicle (DMSO) only as negative control. After

24 h, the medium was removed, and cells were allowed to grow for 10 days. Then, the cells were fixed with methanol for 15 min and then stained with 0.1% crystal violet for 15 min. After washing away the crystal violet, the plates were photographed and counted, and one colony was defined to be an aggregate of >50 cells. At least three independent experiments were performed for each assay. Cell Cycle Analysis. Five ×104 cells were seeded into six-well plates and incubated overnight. Cells were then treated with various concentrations of compounds for 48 h. The cells were harvested, washed with cold PBS, and then fixed with 70% ethanol in PBS at −20 °C for 12 h. Subsequently, the cells were resuspended in PBS containing 100 ug/mL RNase and 50 ug/mL PI and incubated at 37 °C for 30 min. Cell cycle distribution of nuclear DNA was determined by flow cytometry on an FC500 cytometer (Beckman Coulter). Cell Cytotoxicity. To measure cell cytotoxicity, MCF-7 and Bel7402 cells were seeded at a density of ∼5 × 103 cells per well in 96well plates (Coring) for 24 h, followed by incubation of indicated compounds for 36 h. Then the medium was collected and measured for cytotoxicity using LDH assay Kit (Promega) according to manufacturer’s instruction. Assay plate is allowed to equilibrate to ambient temperature, and CytoTox-ONE reagent is added to each well and incubated for 10 min. Then, a stop solution is added, and the fluorescent signal is measured with an excitation wavelength of 560 nm and an emission wavelength of 590 nm. Hoechst 33342 Staining. Approximately 5 × 104 cells per well were plated in six-well plates, and the cells were then incubated with 0, 0.125, 0.25, 0.5, 1 μM 13p for 36 h. After incubation, cells were washed with PBS, fixed in 4% paraformaldehyde for 30 min, and then stained with 20 μg/mL Hoechst 33342 for 15 min at room temperature in the dark. Cells were then assessed by fluorescence microscopy for morphological changes after 13p treatment. JC-1 Assay To Determine MMP. The MMP was determined with the dual-emission mitochondrial dye JC-1. After treatment with 0, 0.25, 0.5, 1 μM 13p for 36 h, MCF-7 cells were loaded with 10 μg/mL JC-1 dye for 30 min at 37 °C and then washed for 5 min in PBS buffer. After incubation, samples were immediately assessed for red and green fluorescence by a microplate reader or fluorescence microscopy. The fluorescent signal of monomers is measured with an excitation wavelength of 490 nm and an emission wavelength of 535 nm. The fluorescent signal of aggregates is detected with an excitation wavelength of 525 nm and an emission wavelength of 600 nm. All the experiments were performed in triplicate. Measurement of Intracellular ROS Generation. Intracellular ROS production was detected by using the peroxide-sensitive fluorescent probe DCF-DA. In brief, after treatment with 0, 0.125, 0.25, 0.5, 1 μM 13p for 36 h, the cells were incubated with 10 mM DCF-DA at 37 °C for 15 min. The intracellular ROS mediated oxidation of DCF-DA to the fluorescent compound 2′,7′-dichlorofluorescein (DCF). Then cells were harvested, and the pellets were suspended in 1 mL PBS. Samples were analyzed at an excitation wavelength of 480 nm and an emission wavelength of 525 nm by flow cytometry on a FC500 cytometer (Beckman Coulter). Flow Cytometry Analysis for Cell Apoptosis. MCF-7 cells (1 × 105/well) were cultured in complete medium in six-well plates for 24 h and treated in triplicate with different concentrations of 13p for 24 h. The control cells were treated with vehicle (1‰ DMSO in complete medium). The cells were harvested, washed, and stained with PI and FITC-Annexin-V in the dark for 15 min using the FITC-AnnexinV Kit (BD Parmingen, San Diego, CA, USA). The percentages of apoptotic cells were determined by flow cytometry on an FC500 cytometer (Beckman Coulter). Western Blot Analysis. MCF-7 cells were incubated in the presence of 13p and collected after 48 h. Cells were centrifuged and washed two times with ice cold phosphate buffered saline. The pellet was then resuspended in lysis buffer. After the cells were lysed on ice for 20 min, lysates were centrifuged at 13,000 g at 4 °C for 15 min. The protein concentration in the supernatant was determined using the BCA protein assay reagents. Equal amounts of protein (20 μg) were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (8−12% acrylamide gels) and transferred to 1465



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PVDF Hybond-P membrane. Membranes were blocked for 1 h at room temperature. Membranes were then incubated with primary antibodies against caspase-3, caspase-9, pro-caspase-3, Bax, Bad, Bcl-2, p-ERK, P53, cytotochrome c, p-cdc25c, cdc2, cyclin B1, and β-actin, with gentle rotation overnight at 4 °C. Membranes were next incubated with fluorescent secondary antibodies for 60 min. In Vivo Tumor Xenograft Study. Five-week-old male Institute for Cancer Research (ICR) mice and six-week-old male BALB/c nude mice were purchased from Shanghai SLAC Laboratory Animals Co. Ltd. A total of 1 × 106 H22 or MCF-7 cells were subcutaneously inoculated into the right flank of ICR mice or BALB/c mice, respectively, according to protocols of tumor transplant research. After 7 d of tumor growth, mice were weighed and divided into five groups of eight or six animals at random. The groups with oridonin, 13, and 13p were administered intravenously 20 mg/kg in a vehicle of 10% DMF/2% poloxamer/88% saline, respectively. The positive control group was treated with cyclophosphamide (20 mg/kg) through intravenous injection. The negative control group received 0.9% normal saline through intravenous injection. Treatments were performed at a frequency of intravenous injection one dose per day for a total 21 or 28 consecutive days. Body weights and tumor volumes were measured every 2 days. After the treatments, all of the mice were sacrificed and weighed. The following formula was used to determine tumor volumes: tumor volume = L × W2/2, where L is the length and W is the width. Ratio of inhibition of tumor (%) = (1 − average tumor weight of treated group/average tumor weight of control group) × 100%. All procedures were performed following institutional approval in accordance with the NIH Guide for the Care and Use of Laboratory Animals. H&E Staining. Mouse organs (heart, liver, spleen, lung, kidney) were isolated and fixed in 4% paraformaldehyde and embedded in paraffin using tissue embedding machine. The tissues were sectioned in the vertical plane into 5 μm-thick and stained with H&E. Briefly, sections were prepared orderly by dewaxing, stainingm and dehydration. After staining in Harris hematoxylin solution, sections were stained in eosin-phloxine solution for 1 min and then dehydrated and mounted with neutral resin. The tissue morphology was observed under a microscope.

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ACKNOWLEDGMENTS

We thank Dr. Charles R. Ashby, Jr. (College of Pharmacy and Health Sciences, St. John’s University, New York) for editing the manuscript.

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ABBREVIATIONS USED Ac2O, acetic anhydride; DCM, dichloromethane; DMAP, dimethylaminopyridine; EDCI, 1-(3-(dimethylamino)propyl)3-ethylcarbodiimide hydrochloride; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; DCF-DA, 2′,7′-dichlorofluorescein diacetate; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine; LDH, lactate dehydrogenase; m-CPBA, meta-chloroperoxybenzoic acid; MDR, multidrug resistance; MMP, mitochondrial membrane potential; MsCl, mesyl chloride; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-Htetrazolium bromide; NMR, nuclear magnetic resonance; NAC, N-acetly cysteine; PI, propidium iodide; PS, phosphatidylserine; ROS, reactive oxygen species; rt, room temperature; SAR, structure−activity relationships; SI, selective index; TEA, triethylamine; THF, tetrahydrofuran; TLC, thin-layer chromatography; TsOH, p-toluenesulfonic acid

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b01652.

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Article

Biological investigations and NMR spectra (PDF) Compound data (CSV)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +86-25-83271042. *E-mail: [email protected]. Phone: 1-718-990-1432. *E-mail: [email protected]. Phone: +86-25-83271299. ORCID

Hequan Yao: 0000-0003-4865-820X Jinyi Xu: 0000-0002-1961-0402 Funding

This work is supported by the National Natural Science Foundation of China (nos. 81373280, 81673306), the Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (no. SKLNMZZCX201404), and China Postdoctoral Science Foundation (no. 2015M581903). Notes

The authors declare no competing financial interest. 1466



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