Betulinic Acid and its Derivatives as Potential Antitumor Agents.

Betulinic Acid and its Derivatives as Potential Antitumor Agents Dong-Mei Zhang,1 ∗ Hong-Gui Xu,2 ∗ Lei Wang,1 Ying-Jie Li,1 Ping-Hua Sun,2 Xiao-Ming Wu,3 Guang-Ji Wang,3 Wei-Min Chen,2 and Wen-Cai Ye1 1 Guangdong

Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine and New Drugs Research, College of Pharmacy, Jinan University, Guangzhou 510632, P. R. China 2 Department of Medicinal Chemistry, College of Pharmacy, Jinan University, Guangzhou 510632, P. R. China 3 Institute of Pharmaceutical Research, College of Pharmacy, China Pharmaceutical University, Nanjing 210009, P. R. China Published online 2 June 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/med.21353

䉲 Abstract: Betulinic acid (BA) is a lupane-type pentacyclic triterpene, distributed ubiquitously throughout the plant kingdom. BA and its derivatives demonstrate multiple bioactivities, particularly an antitumor effect. This review critically describes the recent research on isolation, synthesis, and derivatization of BA and its natural analogs betulin and 23-hydroxybetulinic acid. The subsequent part of the review focuses on the current knowledge of antitumor properties, combination treatments, and pharmacological mechanisms of these compounds. A 3D-QSAR analysis of 62 BA derivatives against human ovarian cancer A2780 is also included to provide information concerning the structure–cytotoxicity relationships of these C 2015 Wiley Periodicals, Inc. Med. Res. Rev., 35, No. 6, 1127–1155, 2015 compounds. Key words: betulinic acid; derivatives; antitumor effect; structure–cytotoxicity relationship

1. INTRODUCTION Betulinic acid (3β-hydroxy-lup-20(29)-en-28-oic acid, BA, Fig. 1), a lupane-type pentacyclic triterpenoid, is widespread in many plants including Betula cordifolia,1 Coussarea paniculata,2 Tovomita krukovii,3 Physocarpus intermedium,4 Syncarpa glomulifera,5 Tetracera boiviniana,6 Zizyphus joazeiro,7 genus Uapaca (Euphorbiaceae),8 and Bacopa monniera.9 Betulin (3βlup-20(29)-en-3,28-diol, BN, Fig. 1), the reduction product of BA, is found in the bark of white-barked birch trees at levels up to 22%.10, 11 This could be the most convenient source for BA, which can be prepared by simple high yield reduction of BN.12 Another important analog, 23-hydroxybetulinic acid (3β, 23-dihydroxy-lup-20(29)-en-28-oic acid, 23-HBA, Fig. 1), was isolated first from Pulsatilla chinensis (Anemone chinensis).13 It has been ∗ These

authors have contributed equally to this work.

Correspondence to: Wen-Cai Ye, Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine and New Drugs Research, College of Pharmacy, Jinan University, Guangzhou 510632, P. R. China. E-mail: [email protected] and Wei-Min Chen, Department of Medicinal Chemistry, College of Pharmacy, Jinan University, Guangzhou 510632, P. R. China. E-mail: [email protected]. Medicinal Research Reviews, 35, No. 6, 1127–1155, 2015 C 2015 Wiley Periodicals, Inc.

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Figure 1.

Structures of betulinic acid (BA), betulin (BN), and 23-hydroxybetulinic acid (23-HBA).

demonstrated that BA and its natural occurring analogs possess various bioactivities, including antitumor, anti-HIV, antimalarial, antimicrobial, anti-inflammatory, antihelmentic, and antifeedant activities.14 Among these biological properties, the antitumor properties of BA and its analogs have attracted considerable attention worldwide since several of these compounds have shown quite promising results in the treatment of different types of cancer.15 To date, several simple and practical methods for the production of BA and its derivatives from a variety of botanical sources have been developed and applied. Another effective means of preparing BA is by synthesis from BN, which is very abundant in plants. Prior to 2002 there were only a few references in which BA could be synthesized from BN but the quantity of references has multiplied since 2003, reflecting greatly improved synthetic approaches to BA. In exploration of antitumor effects of BA derivatives over the past decade, a vast number of BA derivatives with various functional groups have been obtained, most of which were achieved by reactions at C-2, C-3, C-19, C-23, and C-28 or modifications of the skeleton. Furthermore, some of these derivatives have been developed as twin drugs or prodrugs. Since the discovery of BA, it has been demonstrated that over 20 different cancer cell lines are inhibited to some extent by BA or its derivatives. Many mechanisms, such as induction of apoptosis, antiangiogenesis, cell cycle arrest, inhibition of invasion and migration, multidrug resistance (MDR) reversal, immunoregulation, as well as autophagy induction have been proposed for this biological activity.16 Additionally, BA has also been combined with other chemotherapeutic drugs, ionizing radiation, or the death receptor ligand enhancing their anticancer efficacy.17 Data from the numerous derivatization of BA offer strong support for structure– cytotoxicity relationship studies of BA derivatives. 3D-QSAR analysis, usually utilizing comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA), has been demonstrated to be a reliable tool for analysis of structure–cytotoxicity relationships.18 Only one previous report, however, using a Topomer CoMFA method, has developed a 3D-QSAR analysis that provides a structure–cytotoxicity relationship study for 37 selected BA derivatives against human colon cancer HT29 cells.19 A 3D-QSAR analysis of 62 selected BA derivatives against human ovarian A2780 cancer cell lines, utilizing both CoMFA and CoMSIA, was expected to be useful to explore the essential structural features associated with the antitumor bioactivities of BA derivatives and is presented in this paper. Our aim in this review is to summarize the knowledge regarding chemistry, antitumor effects, and structure–cytotoxicity relationship of BA and its derivatives. We will emphasize information added to this topic since 2003. Medicinal Research Reviews DOI 10.1002/med

BETULINIC ACID AND ITS DERIVATIVES WITH ANTITUMOR ACTIVITIES

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2. CHEMISTRY OF BETULINIC ACID AND ITS DERIVATIVES A. Isolation and Synthesis BN was first observed by Lowitz in 1788,16 from the sublimation products formed when birch bark is heated. BA, the oxidation product of BN, was first described as an unknown compound extracted from Gratiola officinalis by Retzlaff in 1902.20 This compound was identified and named by Robertson et al. in 1939,21 while 23-HBA was first isolated by Huang et al. in 1962 from P. chinensis (Anemone chinensis).13 BN and BA can be isolated from many plant species,22 but 23-HBA is found only in genera such as Pulsatilla,23 Paeonia,24, 25 Rosmarinus,26 Oplopanax,27 and Glycyrrhiza.28 The procedure for isolation of BA and its derivatives always involves extraction with various organic solvents such as ethanol,29 methanol,30 n-hexane,25 ethyl acetate,31 or methanol/dichloromethane (1:1),26 followed by separation using chromatography on, for example, silica gel25, 29 or Sephadex LH-20.26 BA derivatives can be obtained by crystallization in methanol8 and are easily isolated from botanical sources as a consequence perhaps of their moderate polarity. Since many plant species contain BN at a relatively high level, researchers focused more on the extraction process than on the synthesis of BN. Several simple and practical approaches for isolating BN from birch bark have been published.31, 32 Improved yields and purity are achieved when BN is extracted from plants with ionic liquids.33 The quantities of natural occurring BA obtained from plants have proved to be insufficient and an effective and convenient way to deal with this problem is synthesis of BA from BN, which is available in satisfactory quantities from botanical sources (e.g., the outer bark of white-barked birch trees, in which BN constitutes as much as 22% of its weight).10, 11 In 2003, Cichewicz et al.22 summarized several methods for preparation of BA from BN, such as a two-step method, Jones oxidation of the C-3 and C-28 hydroxyls, followed by reduction of the resulting C-28 carboxyl acid by sodium borohydride. Another classic method was established in 1997, following a five-step strategy of protecting the C-3 and C-28 hydroxyls, then selectively removing the C-28 protective group, and finally oxidizing the C-28 hydroxyl and hydrolyzing the C-3 acetyl group.12 Recently, biotransformation of BN to BA by selected fungi with a maximum yield of 9.32% was reported.34, 35 To avoid the drawbacks of these approaches, including the formation of a mixture of epimers, low overall yields and small-scale preparations,34, 36 several novel and improved synthetic routes with which to prepare BA from BN have been established during the past decade. A representative summary of the novel and improved semisynthesis from BN to BA is provided in Scheme 1. In contrast to BA, less emphasis was placed on the semisynthesis of BN because of its large quantities in plants.37–39 The existing synthetic route included methylation of the C28 carboxylic acid by diazomethane, followed by reduction of the resulting ester by lithium aluminum hydride. To date, there have been no reports of the preparation of BA or BN from 23-HBA.

B. Derivatization Many researchers have contributed to the derivatization of BA and its natural analogs BN and 23-HBA, with the aim of investigating the structural features responsible for their antitumor activities and improving their pharmacokinetic properties.12,39,46,47, Dozens of derivatives prepared from BA, BN, and 23-HBA with representative functional groups were reported as antitumor agents between 2003 and 2014, with no systematic conclusions concerning these derivatives and the resulting antitumor effects. Therefore, a representative summary of these derivatizations reported during the past decade is provided in the following sections. The former parts will highlight those derivatives designed as twin drugs and prodrugs (see Schemes 2–4). Medicinal Research Reviews DOI 10.1002/med

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Scheme 1. Representative synthetic routes used for preparing BA from BN. (1) Method A was initiated by Pichette et al.40 in 2004 in a 54% overall yield. Oxidation of C-28 hydroxyl with high selectivity is the key step, using chromic acid (CrO3 ) adsorbed on solid supported reagent silicon dioxide (SiO2 ) in a mass ratio of 1:10. (2) Method B, a TEMPO-mediated electrochemical approach of two steps, was established in 2006.41 Then Csuk et al.36 improved it into a short one-step route, which is considered the simplest method for chemical synthesis of BA in laboratory and even for industrial production.42 (3) Barthel et al.43 developed Method C in 2008, giving a moderate overall yield of 64%. Trimethylsilyl choride (TMSCl) was used to protect C-3/C-28 hydroxyl before oxidation of C-28 hydroxyl. (4) Method D, employing ultrasound (20 kHz) when BN is dispersed in acetone before mixing with a solution of a Keggin structure 12-heteropoly acid, was an improved synthetic pathway with a satisfactory yield of 92%.44 (5) Method E was another three-step approach first established in 2013. This was an environmentally benign catalytic oxidation of BN to synthesize BA by avoiding the carcinogenic reactants such as CrO3 but using atmospheric oxygen. Being relatively effective and economic is another advantage of this method for its one-pot oxidation.45

Other derivatives obtained by conventional derivatization, including skeletal modification, single-site modification, as well as multisite modification, will be described in subsequent sections (see Schemes 5–10). Additionally, selected IC50 values of the involved derivatives and their precursors (BA/BN/23HBA), together with leading references, are provided. 1. Derivatives as Twin Drugs Two dimers of BA and BN (Scheme 2) as twin drugs have been reported with observed antitumor effects. BA dimer 3, an anhydride of BA at the C-28 carboxyl acid, was obtained as a side product during acetylation of BA by acetic anhydride.48 BN dimer 5 was synthesized from BN via a six-step reaction by Pettit et al. in 2014.49 An aza-Wittig-type reaction was applied in the Medicinal Research Reviews DOI 10.1002/med


Scheme 2.

Scheme 3.

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Synthesis of BA/BN dimers.

Hybridization of BA/BN and cytotoxic agents.

combination of the enamino derivative 4 in 27% yield. Compared to monomers, both dimers showed considerable loss of cytotoxicity toward 12 types of cancer cells. A limited number of hybrid molecules have been derived from BA/BN in combination with cytotoxic agents (Scheme 3). In contrast to the dimers described in the previous section, several of these hybrids have yielded promising results. BN and artesunic succinic acid (ASC) were successfully linked using the condensation agents dicyclohexylcarbodiimide (DCC) or 1-ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDCI). The easily Medicinal Research Reviews DOI 10.1002/med

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Scheme 4.

Synthesis of BA/BN prodrugs.

synthesized hybrids 6–8 showed enhanced inhibitory effects against multidrug-resistant leukemia cells CEM/ADR5000 (minimum IC50 = 11.9 μM) compared to BN and artemisinin.50 The BA-azidothymidine (AZT) hybrid 9 was coupled by click chemistry,51 and its antitumor effect toward KB and Hep-G2 was almost fourfold stronger than that of BA. Emmerich et al. introduced PtCl2 (cisplatin) fragment into BA using Pt(DMSO)2 Cl2 but found the inhibitory activities of hybrids 11 and 13 were weaker than their precursors 10 and 12.52 2. Derivatives as Prodrugs Followed prodrug monotherapy strategy, the in vitro noncytotoxic 28-O-β-D-glucuronide BA derivative 15 (Scheme 4) was first identified as a prodrug by Gauthier et al.53 Commercial methyl 2,3,4-tri-O-acetyl-1-bromo-D-glucopyranuronate first esterified C-28 carboxyl group, then the protecting groups were removed by potassium hydroxide. As expected, this prodrug could be enzymatically hydrolyzed and released 75% BA after 24 hr in vitro upon treatment with β-D-glucuronidase, an enzyme that occurred more common in necrotic tumor tissue than in normal tissue. Another BA prodrug 16 (Scheme 4) was synthesized by simply combining the C3 hydroxyl with the multiarm-polyethylene glycol (PEG) linker, using EDC as a condensation agent.54 This PEGylated derivative 16 possesses excellent water solubility of 160.2 mg/mL (750 folds higher than BA). Furthermore, both in vitro and in vivo tests in lung xenograft models showed that 16 to be a promising prodrug with a high therapeutic index. 3. Derivatives from Skeletal Modification Most of the earlier studies on skeletal modification of BA were focused on ring A (Scheme 5). An indole ring was introduced into the ring A of BA via the Fischer indole synthesis method.55 The resulting derivative 18 exhibited modest inhibitory properties toward eight different cancer cells with IC50 values 10 μM. Urban et al. have employed the diosphenol-like derivative 19 as an intermediate in the preparation of a series of ring A-seco BA derivatives, including the lactone derivative 20, the ring-opened derivative 21, and the ring-expanded derivative 22.48 However, most of these chemical modifications resulted in a loss of cytotoxicity, suggesting that ring A is not an appropriate region for modification in optimization studies of cytotoxicity. 4. Derivatives from Single-Site Derivatization A. Derivatives from Single-Site Derivatization at C-3, C-19, and C-23 Sites The C-3 hydroxyl, C-19 alkene, and C-23 hydroxyl of BA, BN, or 23-HBA are available for chemical modification to generate various functional groups (Scheme 6). Boc-protected Llysine was used to synthesize the C-3 L-lysine ester derivative 23 and the resulting enhanced activity is attributed to its improved water solubility.56 Derivatives 24–25 bearing 3α-alkyne and Medicinal Research Reviews DOI 10.1002/med


Scheme 5.

Scheme 6.

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Synthesis of BA derivatives from skeletal modification.

Representative derivatives from single-site derivatization at C-3, C-19, and C-23 sites.

3-sulfonate substituents could be prepared by a Mannich reaction and a sulfonylation reaction, respectively.57, 58 One of the approaches to creation of a C-3 amino group at BA (26) was to treat BA with CrO3 /NH4 OAc.59 The glucoside 27 was achieved in a four-step route and has slightly inhibitory effect on DLD-1 but no obvious cytotoxicity toward normal skin fibroblasts WS1 (IC50 > 75 μM).60 A microwave approach was first applied by Kommera et al. in the acetylation Medicinal Research Reviews DOI 10.1002/med

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Scheme 7.

Representative derivatives from single-site derivatization at C-28 site.

of the C-3 hydroxyl of BA, giving 28. 61 Other derivatives (29–32) bearing a hydrazone, ketone, oxime, and oxime ether at C-3 have been reported.62, 63 Baratto et al. synthesized derivatives 33–35 by treating BA first with oxidants such as osmic acid (OsO4 ), selenium dioxide (SeO2 ), or sodium periodate (NaIO4 ), and then functionalizing the resulting oxidation products.62 Epoxidation by m-chloroperbenzoic acid and cyclopropanation by carbenes have both been applied to the chemical modifications at this site, affording the epoxy alkane derivative 36 and the naphthenic derivative 37, respectively.58, 64 This double bond at C-19 position was also available for hydrogenation by Pd/H2 , giving 38.63 However, the IC50 values of 36–38 imply that the C-19 alkene substitute should not be modified. For the C-23 hydroxyl of 23-HBA, both oxidation and esterification resulted no loss of the desired bioactivity (see derivatives 39 and 40).65

B. Derivatives from Single-Site Derivatization at C-28 Site Numerous researchers have contributed to the preparation of BA/BN/23-HBA derivatives at the 28β-carboxylic acid or the 28β-hydroxymethyl. Several representative derivatives are illustrated in Scheme 7. Use of diazomethane and Lawesson reagent with BA delivered C-28 methyl ester derivative 41 and the thiocarboxylic acid derivative 42.58, 61 Transformation of BA into a quaternary ammonium salt as seen in derivative 43 was identified as a feasible approach to increase water solubility and cytotoxicity.66 For the 23-HBA derivative 44, amidation of the C-28 carboxyl acid group hindered the optimization of the cytotoxicity.67 Distinct from the highly cytotoxic 3-L-lysine BA derivative 23, the 28-L-glycine BN derivative 45 displayed unsatisfactory activity with IC50 value in excess of 50 μM to EPG85–257P, which may result from its poor water solubility.68 Other representative derivatives 46–48 bearing C-28 alkyne, lactone, and alkane substitutes were also prepared from BN and their activities against several cancer cell lines were determined.69–71 5. Derivatives from Multisites Derivatization A. Derivatives from Multisites Derivatization: Highlighted at C-2 Site The representative BA/BN derivatives highlighted on C-2 derivatization are presented in Scheme 8. Yu et al. synthesized a series of BA derivatives with triphenylphosphonium substituents by a multistep route and obtained 49, which shows considerably enhanced antitumor Medicinal Research Reviews DOI 10.1002/med


Scheme 8.

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Representative derivatives from multisites derivatization highlighted at C-2 site.

potency to mastocytoma P-815 (37-fold higher than BN).72 Derivatives 50 and 51, containing highlighted substitutes of a C-2 exo-alkene and exo-screw ring, respectively, could be easily synthesized by a Mannich reaction or an Aldol condensation.73 The other four derivatives 52–55 bearing specified groups such as C-2 bromide, azide, hydroxyl, and ketone, respectively, have been described by Pettit et al.49 The brominated derivative 52 was prepared by N-phenylN,N,N-trimethylammonium tribromide and a minor quantity of its 2β-bromine substitute diastereoisomer was observed in this reaction. Further reacting 52 with sodium azide (NaN3 ) and potassium carbonate (K2 CO3 ) gave rise to 53–55, respectively. Collectively, most of resulting IC50 values revealed that these chemical modifications support increased potency.

B. Derivatives from Multisites Derivatization: Highlighted at C-3 Site Scheme 9 presents several representatives highlighted on C-3 modification of BA. Derivatives 56–58 were synthesized by hydrogenation-oxidation and nucleophilic substitution with phenylhydrazine or hydroxylamine hydrochloride.74, 75 Based on their similar structural features and the corresponding IC50 values, it seems that the enamine (57) or hydroxylamine (58) substitution at C-3 is preferable to hydrazine (56). Another derivative, dithiophosphate (59), is a side product of the reaction with Lawesson reagent.58

C. Derivatives from Multisites Derivatization: Highlighted at C-19, C-23, and C-28 Sites Derivatives 60–66 in Scheme 10 are highlighted for they contain several special substituents at C-19/C-23/C-28 sites. In a study by Santos et al., the C-3/C-28 N-acyltriazole derivative Medicinal Research Reviews DOI 10.1002/med

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Scheme 9.

Representative derivatives from multisites derivatization highlighted at C-3 site.

60 was found to possess strong inhibitory activity against PC-3.76 Treating BA with benzene sulfonyl isocyanate under reflux produced the phenylsulfonyl isocyanate (61).58 The C-3/C-19 ketone derivative 62 can be prepared from BN via using the oxidants CrO3 , OsO4 , and NaIO4 in sequence.62 In accordance with the bioactivity of the C-3 monosaccharide derivative 27, decreased anticancer activity was observed after converting the C-28 free carboxyl acid into a monosaccharide (63) or branched trisaccharide (64).77 Distinct from the selective oxidation via pyridinum chlorochromate (PCC, see derivative 39), treatment of 23-HBA with Jones reagent led to oxidation of both C-3 and C-23 hydroxyls (see 23-HBA derivative 65).65

3. ANTITUMOR ACTIVITY OF BETULINIC ACID AND ITS DERIVATIVES A. Antitumor Activity In preliminary screening of antitumor activity of about 2500 plant extracts, BA attracted more attention because of its apparent activity against melanoma.78 Subsequent studies revealed that BA and its synthetic and naturally occurring derivatives were cytotoxic to an extensive variety of cancer cells including osteosarcoma, Ewing’s sarcoma, fibrosarcoma, embryonal neuroblastoma, glioma, leukemia, as well as several carcinomas, such as lung, colon, breast, prostate, hepatocellular, bladder, head and neck, stomach, pancreatic, cerebroma, ovarian, and cervical carcinoma.19, 55, 61, 76,79–83 The cytotoxicities of BA and BN toward cultured primary cancer cells from patient tumor tissues of ovarian carcinoma, cervical carcinoma, neuroblastoma, glioblastoma, and leukemia have also been reported.82,84–86 Beyond its potent cytotoxicity in vitro, BA also inhibits the growth of solid tumors in vivo in tumor-bearing nude mice. After treatment with BA, tumor size and weight are significantly Medicinal Research Reviews DOI 10.1002/med


Scheme 10.

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Representative derivatives from multisites derivatization highlighted at C-19, C-23, and C-28 sites.

decreased in MDA-MB-231 or the MCF-7 xenograft tumor model.87, 88 In addition, BA potently inhibits tumor growth in melanoma or pancreatic cancer xenografts.78, 89 Similar activity was observed when BA was injected into colon or prostate cancer cells implanted in mice; tumor weights decrease markedly but body weights and the histopathology of important organs fail to show significant changes, suggesting a favorable therapeutic prospect.90, 91 BA also obviously increased survival times of mice with ovarian cancer xenografts.92 A recent study showed that a BA liposome formulation inhibits growth of human lung and colon tumor xenografts without causing systemic toxicity.93 To enhance the solubility and bioavailability of BA, cyclodextrin (octakis-[6-deoxy-6-(2sulfanyl ethanesulfonic acid)]-γ -cyclodextrin) was used to form a hydrophilic complex with BA. This complex was found to reduce tumor volume and weight of a B164A5 murine melanoma in C57BL/6J mice.94 The 17-carboxylic acid ester derivatives of 23-HBA have potent growthinhibitory activity in mice bearing B16 melanoma or H22 liver tumors, and in this sense are similar to the clinical drugs cyclophosphamide and 5-fluorouracil.95 Importantly, BA is more cytotoxic to cancer cells than to normal cells. Many studies have demonstrated that normal cells such as human skin fibroblasts, peripheral blood lymphocytes, and melanocytes are more resistant to BA than cancer cells.96, 97 BA derivatives also showed Medicinal Research Reviews DOI 10.1002/med

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less toxicity to nontumoral Chang liver cells than to hepatocellular carcinoma HepG2 cells.98 BA can differentiate normal human keratinocytes into corneocytes while it induces apoptosis in skin cancer cells.96 A possible reason for this is that BA is mainly absorbed and distributed in murine tumors, to judge from pharmacokinetic studies.99, 100 Recently, Reiner et al.101 found that BA can potently suppress multiple deubiquitinases, enhance poly-ubiquitinated proteins, and decrease the level of oncoproteins, thereby triggering apoptosis in prostate cancer cells but not in normal prostate cells. This suggests that deubiquitinases might play a pivotal role in BA’s selective effects on cancer cells and normal cells. However, an understanding of the molecular mechanisms underlying this phenomenon remains an important issue for further study.

B. Combination Treatments Recent research has demonstrated that BA exerts synergistic effects with chemotherapeutic drugs, ionizing radiation, or the death receptor ligand TRAIL (TNF-related apoptosis-inducing ligand). As an effective supplement to vincristine, BA significantly reduces lung metastasis in a murine B16F10 melanoma model.102 Moreover, BA combined with different types of anticancer drugs, such as taxol, doxorubicin, etoposide, cisplatin, actinomycin D, and PI3K inhibitor GDC-0941, can decrease the survival of cancer cells by induction of mitochondrial apoptosis.17, 103 BA is also able to enhance the sensitivity of multidrug resistant colon cancer cells toward 5-fluorouracil, irinotecan, and oxaliplatin.104 In addition, BA in combination with other chemicals such as ginsenoside Rh2, epithelial growth factor receptor tyrosine kinase inhibitor PD153035, mithramycin A, or thalidomide increases cell death in various cancer cell lines.89, 105, 106 BA can be used to enhance the efficacy of hyperthermia in low pH adapted melanoma cells DB-1.107 Moreover, in combination with ionizing radiation BA provides an additive impact in human glioma cells.108 Furthermore, it sensitizes different cancer cell lines and primary cultured tumor cells to TRAIL-induced apoptosis.109 These findings indicate that BA and its derivatives have the potential to behave as a sensitizer, improving the clinical efficacy of cancer treatment.

C. Mechanisms of Action 1. Induction of Apoptosis Abundant studies had demonstrated that BA can trigger apoptosis in many kinds of cancer cells mainly through direct regulation of the mitochondrial apoptotic pathway, and supported by perturbations of the mitochondrial membrane potential, release of Smac and cytochrome c from mitochondria, increased activated forms of poly (ADP) ribose polymerase (PARP) and caspase 3.109, 110 Fulda et al. found that ectopic expression of antiapoptotic Bcl-2 or BclxL, counteracting apoptosis in BA-treated cells with a variety of changes associated with mitochondrial collapse, cytochrome c release, and increase of caspase activities, suggests that Bcl-2 or Bcl-xL is associated with these processes.111 However, Wick et al. reported that BA enhances Bax and Bcl-2 but does not affect Bcl-xL or Bcl-xS. Overexpression of Bcl-2 attenuates caspase cleavage, DNA fragmentation, and cell death induced by BA.112 It was also found that BA induces apoptosis, independent of Bax or Bak.113 Although these results are contradictory, they suggest that the role of Bcl-2 family members in the apoptosis induced by anticancer agents is complex and based on these above findings, it was concluded that presence of BA leads to mitochondrial apoptosis probably through direct opening of the permeability transition (PT) pore. Similar results have been found by Liu et al.114 Such an effect can be temporarily abrogated by antiapoptotic Bcl-2 family members.113 It was also found that p53 wild-type and p53 mutant Medicinal Research Reviews DOI 10.1002/med


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RMS cells are sensitive to BA treatment, assuming that the p53 pathway fails to take part in the regulation of BA-induced apoptosis in RMS cells.115 It was found that the signaling pathway of mitogen-activated protein kinases (MAPK) is associated with BA-induced apoptosis. After BA treatment, p38 and c-Jun NH2-terminal kinase (JNK) were activated but extracellular signal regulated kinases (ERK) were not phosphorylated in human melanoma UISO-Mel-1 cells. However, both SP600125 (a selective JNK inhibitor) and SB203580 (a specific inhibitor of p38) do not completely inhibit cellular apoptosis after BA treatment, suggesting that MAPK signaling pathway is involved in apoptosis but is not the sole determinant. Moreover, depolarization of the mitochondrial membrane potential was also observed. SB203580 and SP600125 can partly prevent membrane depolarization rapidly but SB203580 displays a more potently protective effect than SP600125, suggesting that p38, among three types of MAPKs, played the most prominent role in BA-induced apoptosis. Furthermore, preincubation with antioxidants such as glutathione, N-acetyl-L-cysteine, and vitamin E decreases the generation of reactive oxygen species (ROS) after BA treatment and blocks the process of apoptosis with a decrease in p-p38 and p-JNK as well as an increase in p-ERK. These data indicate that ROS acts as an upstream signaling molecule of the MAPK pathway in BA-treated cells but no known caspases such as Caspase-8, -9, or -3 are involved in the process.116 Specificity protein transcription factors, Sp1, Sp3, and Sp4, are upregulated in multiple types of cancer and relatively downregulated in human normal tissues, suggesting that they may be potential drug targets. BA induces degradation of Sp transcription factors in human prostate cancer LNCaP cells dependent on the proteasome pathway. Further investigation demonstrated BA-induced degradation of Sp proteins results in decrease of survivin and vascular endothelial growth factor (VEGF) by inhibition of the interaction between Sp and the promoters of survivin and VEGF. However, Sp protein levels were almost similar in lysates of liver from vehicle controls and from BA-treated mice, suggesting that degradation of Sp by BA may be specific for solid tumor or cancer cells that overexpress Sp when compared with normal tissue.91 This research group also found that BA suppressed not only the protein expressions but also promoters of Sp and Sp-regulated genes, such as cyclin D1, the p65 subunit of NF-κB, and EGFR in colon cancer cells. Downregulation of Sp induced by BA is associated with ROS-regulated disruption of miR-27a:ZBTB10 by proteasome-independent and proteasomedependent pathways, which are dependent on tumor type.90 Consistently, a previous report has also shown that the antitumor effect of BA is closely related to the microRNA-27a-ZBTB10Sp-axis.87 The signal transducer and activator of transcription (STAT) protein 3 activation has been shown to participate in the regulation of growth, survival, and invasion of multiple cancer cells. BA attenuates both the constitutive and inducible STAT3 phosphorylation, nuclear translocation, and its DNA binding in human various myeloma cells. It also downregulates constitutive activation of the upstream molecules of STAT3, JAK1, and JAK2, and pretreatment with sodium pervanadate or siRNA SHP-1 abolishes the decrease of STAT3 activation level induced by BA, indicating the involvement of a protein tyrosine phosphatase (PTP), especially SHP-1 (a nontransmembrane PTPase). BA also reduces the expression of STAT3-mediated genes, such as survivin, Cyclin D1, Bcl-2, and Bcl-xL, which is correlated with enhancement in caspase-3 and PARP activation as well as apoptosis. Furthermore, overexpression of STAT3 rescues apoptosis of cancer cells after BA treatment, implying the anticancer effect of BA may be mediated by STAT3 pathway.106 BA also effectively inhibits constitutive NF-κB activation in androgen-insensitive prostate cancer cells. BA abrogates NF-κB/p65-DNA binding and inhibits its translocation into the nucleus in PC3 cells by decrease of IKK activity and p-IκBα (Ser 32/36) followed by its degradation. These effects are correlated with an increase in Bax/Bcl-2 ratio and cleavage Medicinal Research Reviews DOI 10.1002/med

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of PARP, hallmarks of apoptosis induction. BA can also sensitize highly resistant PC-3 cells to TNFα-triggered apoptosis through suppression of TNFα-mediated NF-κB activation.117 However, another study reported that BA activates NF-κB signaling pathway in many kinds of tumor cells.118 It was further confirmed that BA was still effective in apoptosis of MDA-MB231 cells, which was not regulated by NF-κB.119 These contradictory findings suggested that the different regulatory effect of BA on NF-κB pathway may be dependent on cell type. Many BA derivatives with more potent inhibitory effects against a variety of cancer cells have been synthesized and also could induce apoptosis through the intrinsic apoptotic pathway.19, 120 Two methoxycarbonyl derivatives of BA, TP-295 and TP-296, could activate apoptosis in Bax/Bak–/– cells that lack two key components of the mitochondrial dependent apoptotic pathway, indicating that they can also lead to apoptosis via the extrinsic death receptor apoptotic pathway.121 It has been reported that BN also could induce apoptosis through the intrinsic apoptotic pathway.122 Further proteomic analysis demonstrated that BN triggers apoptosis by downregulation of isoform 1 of 3-hydroxyacyl-CoA dehydrogenase type 2, poly(rC)-binding protein 1, enoyl-CoA hydratase, and heat shock protein 90-α2, and upregulation of splicing factor arginine/serine-rich 1, malate dehydrogenase, and aconitate hydratase.123 2. Antiangiogenesis Although many studies have demonstrated that BA and its derivatives can induce an antiangiogenic response, the underlying mechanism is still not clear. The antiangiogenic effect of BA under hypoxia is mediated by prevention of formation of a tube-like structure in human umbilical vein endothelial cells (HUVECs) as well as reduction of secreted protein levels of VEGF and hypoxia-inducible factor-1α (HIF-1α) in PC-3 cells. Mechanistic studies showed that BA can inhibit hypoxia-induced STAT3 activation and the transcriptional activity of HIF-1α, and it also significantly disturbs the binding of STAT3 and HIF-1α to VEGF promoter. It was concluded that the STAT3/HIF-1α/VEGF signaling pathway plays a critical role in the antiangiogenesis of BA in PC-3 cells under hypoxia.124 Similarly, BA could attenuate pancreatic tumor angiogenesis through the Sp1/VEGF signaling pathway.89 It has also been found that BA inhibits prolidase activity to promote collagen degradation accompanied by decrease in levels of VEGF, HIF-1α, as well as integrins (α1 and α2) in human endometrial adenocarcinoma cells. This potential antiangiogenic mechanism might implicate proline and hydroxyproline that regulate ubiquitination and proteosomal degradation of HIF1α via Von Hippel–Lindau (VHL) tumor suppressor. Once HIF-1α is not degraded and exerts its transcription factor functions to enhance VEGF, angiogenesis can occur. Furthermore, α1 and α2 integrins markedly inhibit VEGF-driven angiogenesis. Therefore, BA suppresses prolidase activity, triggering the degradation of HIF-1α and consequent elimination of activation of VEGF expression, in which α1 and α2 integrins also take part, to inhibit angiogenesis.125 The new 3-O-acyl, 3-benzylidene, 3-hydrazone, 3-hydrazine, 17-carboxyacryloyl ester derivatives of BA also show antiangiogenic effects in vitro. These compounds efficiently inhibit the proliferation of endothelial cell and tube formation of ECV304 cells and some of them displayed better activity than BA in tube formation assay.126 Dehelean et al. found that BN reduces newly formed capillaries of the chicken embryo chorioallantoic membrane, suggesting that BN’s inhibition of angiogenesis is possibly associated with the elimination of the normal function of vascular endothelial cells.127 3. Cell Cycle Arrest A recent report showed that BA can block the cell cycle in the G2 /M phase in gastric adenocarcinoma cells through downregulation of Cyclin B1 and Hiwi in mRNA and protein expression Medicinal Research Reviews DOI 10.1002/med


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level.81 Similar results were found in breast cancer following phosphorylation of Cdc287 and in lung cancer after downregulation of cyclin A2, which was regulated by Sp1.128 Several novel BN derivatives synthesized by Rene et al. exhibit an excellent inhibitory effect on cell proliferation with a small number of cells arrested in G2 phase.57 BA can induce arrest of mice melanoma cells in the G0 /G1 phase in a dose-dependent manner.102 Recent research revealed that BA and its derivatives RS01, RS02, as well as RS03 have the ability to induce specific S phase cell cycle block in human hepatocellular carcinoma, cervical carcinoma, and leukemia. Furthermore, its derivatives are more significantly effective than those of BA in arresting S phase.98 It was found that BA-treated PC cells show a major cell cycle arrest in G1 /S phase.101 These findings indicate that cell cycle arrest induced by BA and its derivatives is associated with marked selectivity for tumor cells. 4. Inhibition of Invasion and Migration Gao et al. showed that BA has strong synergy with mithramycin A on inhibition of migration and invasion on pancreatic cancer cells at nontoxic concentrations. Their research results showed that combinations of BA and mithramycin A suppress Sp1 and uPAR in a dosedependent manner.89 Lei et al. first identified lamin B1 as a primary target of BA treatment in pancreatic cancer. Overexpression of lamin B1 contributes to the invasion and proliferation of pancreatic cancer cells, whereas suppression of lamin B1 by BA can partly attenuate the migration and invasion, suggesting that lamin B1 is a novel target that critically mediates the anticancer effects of BA.129 5. Multidrug Resistance Reversal Our group found that BBA, a derivative of 23-HBA, can significantly reverse the MDR of ABCB1 overexpressing cells by interacting directly with ABCB1 and greatly affecting the ABCB1 ATPase activity to block the transport function of ABCB1.130 Further investigation found that BBA shows potential ability to reverse ABCC10 (MRP7)-mediated MDR by inhibiting its efflux activity.131 Furthermore, two bipiperidinyl derivatives of 23-HBA, DABB (3,23-O-diacetyl-17-1,4’bipiperidinyl betulinic amide), and DHBB (3,23-O-dihydroxy-17-1,4’-bipiperidinyl betulinic amide) can also reverse MDR mediated by ATP-binding cassette transporter. We found that DABB or DHBB exhibits its potent ABCB1 ATPase suppression characteristics at nontoxic concentrations, rather than by altering mRNA and protein of ABCB1. These combined findings may support development of advance novel MDR modulators using derivatives of 23-HBA as an adjuvant antitumor chemotherapeutic agent.132 B5H7, a morpholine derivative of 23-HBA, could also contribute remarkably to the sensitivity of doxorubicin to ABCB1-overexpressing MDR cells. Further mechanistic investigations found that B5H7 does not influence the activity of ABCB1 ATPase or suppress the expression level of ABCB1. Though the exact mechanisms of B5H7 action remain unknown, it might be quite different to that of other 23-HBA derivatives such as BBA, DABB, and DHBB, and so expand our understanding of the reversal mechanisms of 23-HBA derivatives.133 6. Immunoregulation BA not only effectively inhibits tumor growth in mice, but also contributes to immunoregulation in vivo. Wang et al. found that BA can obviously improve the expression of multiple immunity-related cytokines such as IL-2, TNF-α, and CD4+ lymphocytes subsets, simultaneously upregulating the ratio of CD4+ /CD8+ , suggesting the antitumor effects of BA are involved in the body’s immune response.134 Medicinal Research Reviews DOI 10.1002/med

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7. Autophagy Induction BA potently induces autophagy as a survival mechanism in response to PT pore opening and mitochondrial damage. BA-induced autophagy, rather than autophagic cell death, prevents cells surviving. Furthermore, BA-induced cell death is not independent of necroptosis, autophagy, and caspases, indicating that an alternative manner of cell death next to apoptosis may be participating in BA-induced cell death.135 Yang et al. found that BA downregulates Beclin 1, but dose-dependently increases p62 and LC3-II indicating the inhibition of autophagic flux that contributes to apoptosis induction. Furthermore, BA combined with rapamycin, an autophagic inducer, causes the synergic accumulation of p62 and LC3-II and induction of apoptosis. Collectively, BA may have a different effect in the process of autophagy in different cancer cell lines.136 B10, a glycosylated derivative of BA, not only induces apoptosis, but also induces autophagy. B10 destabilizes lysosomes and releases lysosomal hydrolases, cathepsin B and Z, to the cytoplasm and induces cell death. Further mechanistic investigation confirmed that lysosomal permeabilization and apoptosis are dependent on ATG7, ATG5, and Beclin 1. Autophagic cell death is a vital part of B10-induced cell death, suggesting important implications of BA derivatives for apoptosis-resistant cancers.137

4. STRUCTURE–CYTOTOXICITY RELATIONSHIP ANALYSIS Generally, the result of classical SAR analysis is limited to 2D structure, few substitutions in a reference, and no graphical output for interpretation.138 To overcome such drawbacks, a clearer and more realistic method, 3D-QSAR analysis, has been applied more extensively over the past decade. The basic workflow of 3D-QSAR is illustrated in Figure 2. To the best of our knowledge, only one 3D-QSAR analysis of the antitumor bioactivity of BA and its derivatives has been reported.19 Hence, we next focused on the 3D-QSAR, aiming to reveal the essential structural features for increasing the antitumor bioactivities. In this section, a 3D-QSAR analysis was employed by CoMFA and CoMSIA for 62 selected BA derivatives against human ovarian cancer cell A2780, which has been reported by the same research group between 2010 and 2013.57, 70, 73,139–141

A. Materials and Methods for 3D-QSAR An overview of key information in this section is highlighted in Figure 3, while further details, including the compounds (S1–S62) involved in the next section, are provided in the Supporting Information.

B. Results and Discussion for 3D-QSAR 1. Graphical Interpretation of CoMFA In Figure 4A, the large green contour near R3 suggests that bulky groups at this position are beneficial to the bioactivity. That only 4 of the top 20 active compounds in this section possessed a small steric hydroxyl group at R3 is in agreement with the above observation. The yellow contour encompassing R2 reveals that small steric groups at this site are preferable for antitumor potency. Meanwhile, a bulky substituent (e.g., a five-membered ring) at the edge of R5 will hinder higher activity, as seen in compounds S40–S42. On the contrary, at the terminus of R5 there is a green contour, emphasizing the necessity of bulky substituents in this Medicinal Research Reviews DOI 10.1002/med


Figure 2.

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Flowchart of the 3D-QSAR analysis.

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Figure 3.

Overview of materials and methods employed in 3D-QSAR.

Figure 4. CoMFA STDEV*COEFF contour diagram combined with template S33. (A) Steric fields: green bulky groups favorable regions (80% contribution); yellow small steric groups favorable regions (20% contribution). (B) Electrostatic fields: blue electropositive groups favorable regions (80% contribution); red electronegative groups favorable regions (20% contribution).

area. Hence, the compound S3 exhibits better potency than compound S2, which possessed a diethylamino-carbonyl group and an ethoxycarbonyl group at the terminal of R5 , respectively. The electrostatic contour provided by CoMFA (Fig. 4B) shows a large blue contour enclosing R1 and suggesting that electropositive groups at this position should be beneficial to the inhibitory effect. However, electronegative groups are preferable at the position occupied by R2 . For the substituent at R4 , electropositive groups are unfavorable and this might explain why compounds S14–S26 bearing electropositive amino groups at the terminal of R4 have unsatisfactory activity. Two huge red contours around the R5 position emphasized the extreme importance of electronegative substituent. For example, S33 and S34, which bear electronegative substituents (alkynyl carbonyl group) at R5 , are the most active compounds. Medicinal Research Reviews DOI 10.1002/med


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Figure 5. CoMSIA STDEV*COEFF contour diagram combined with template S33. (A) Electrostatic fields: Blue electropositive groups favorable regions (90% contribution); red electronegative groups favorable regions (20% contribution). (B) Hydrogen bond donor fields: Cyan favorable regions (90% contribution); purple unfavorable regions (35% contribution). (C) Hydrogen bond acceptor fields: magenta favorable regions (80% contribution); white unfavorable regions (20% contribution).

2. Graphical Interpretation of CoMSIA The CoMSIA electrostatic contour diagram (Fig. 5A) will not be interpreted for its similarity to the CoMFA one. Three obvious purple contours located near R3 (Fig. 5B) demonstrated that hydrogen bond donor groups are not essential substituents at this site, which may explain why the compounds in the present study without a hydroxyl group at R3 usually displayed better activities. An obvious cyan contour around the R5 region implied the necessity of hydrogen bond donor groups at this position. Hence, compounds S47–S49 bearing a hydroxyl group at the edge of R5 displayed satisfactory bioactivities. Substituents with the hydrogen bond acceptor properties, at the magenta contour (Fig. 5C) enclosing R2 region, are essential, as can be seen in highly active compounds S31 and S32, which have a sulfoether or sulfone at R2 , respectively. Another magenta contour at the end of R5 revealed that hydrogen bond acceptor groups should produce better bioactivity, as seen in compounds S47–S49. Both one white and one magenta contour enclosing the beginning of R5 implies that the hydrogen bond acceptor substitutes may make no remarkable difference at this position.

C. Summary of Structure–Cytotoxicity Relationship Figure 6 shows the structure–cytotoxicity relationship of BA derivatives against human ovarian cancer cell A2780, based on the above 3D-QSAR study. An electropositive group at R1 ; a minor, electronegative, and hydrogen bond acceptor group at R2 ; bulky groups at the C-3βsite; bulky and electronegative groups at R4 ; minor, electronegative, and hydrogen bond donor groups at the beginning of C-28 side chain; and bulky, electronegative, and hydrogen bond acceptor groups at the terminus of C-28 side chain all would be beneficial to the antitumor potency. Medicinal Research Reviews DOI 10.1002/med

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Figure 6.

Structure–cytotoxicity relationship revealed by 3D-QSAR model.

5. PERSPECTIVES BA can be prepared simply from the more plentiful BN on a laboratory scale, and can be chemically modified easily. However, a practical industrial synthetic route had still not been developed and applied during the past decade. Most of the derivatizations were carried out by conventional organic synthesis methods, and were often costly in terms of reagents and energy. Consequently, further studies are required to focus on improvement of the synthetic approaches, such as applying microwave chemistry and combinatorial chemistry to become more efficient and environmentally friendly. BA and its derivatives attract more attention because of their broad-spectrum biological activities, especially in anticancer treatment. They not only can treat different cancers including multidrug resistant carcinoma with low toxicity in normal cells, but they also represent a novel class of autophagy inducers with complex mechanisms. Although BA derivatives are promising cancer therapeutic agents, the molecular targets and the precise mechanisms have yet not been identified. Thus, further study on the anticancer mechanisms of BA derivatives will provide a rationale for the design of optimal anticancer drug candidates, while molecular modification based on modern medicinal chemistry will support exploration of the precise mechanisms, which leads to the development in anticancer therapy. To conclude, much is yet to be done and the future of BA and its derivatives will mainly depend on the identification of the mechanisms responsible for their antitumor properties. The clearer these are, the larger the probability of their clinical application. 6. ABBREVIATIONS AIBN CDI DCC DMAP DMSO EDCI LDA m-CPBA PDC PTSA Py TBAB TEMPO TFA TMSOTf

= = = = = = = = = = = = = = =

2,2 -azobis(2-methylpropionitrile) 1,1 -carbonyldiimidazole dicyclohexylcarbodiimide 4-dimethylaminopyridine dimethylsulfoxide 1-ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride lithium diisopropylamide 3-chloroperoxybenzoic acid pyridinium dichromate p-toluene sulphonic acid pyridine tetrabutylammonium bromide 2,2,6,6-tetramethylpiperidinyl-1-oxide trifluoroacetic acid trimethylsilyl trifluoromethanesulfonate



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ACKNOWLEDGMENTS This work was financed by Science and Technology Program of China (2012ZX09103101053) and Guangzhou (2009A1-E011 and 2010U1-E00531), National Science Foundation of Guangdong Province (S2013050014183 and S2012010008130), Specialized Research Fund for the Doctoral Program of Higher Education (20124401110008), and Program for New Century Excellent Talents in University (D.-M.Z.).

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Dr. Dong-Mei Zhang graduated from the Chinese University of Hong Kong in 2007 with a Ph.D. degree investigating the anticancer activity and molecular mechanisms of natural products and their derivatives. Hong-Gui Xu obtained his B.Sc. degree at Guangdong Pharmaceutical University in 2013. He is currently pursuing a M.Sc. degree at Jinan University in the role of structural modification of naturally occurring antitumor products. Medicinal Research Reviews DOI 10.1002/med


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Dr. Lei Wang obtained his Ph.D. degree at China Pharmaceutical University in 2009 investigating naturally occurring antitumor products. Ying-Jie Li graduated from Jinan University in 2014 with an M.Phil. degree and now is pursuing a Ph.D. degree in investigating the anticancer activity of natural products and their derivatives. Dr. Ping-Hua Sun obtained his Ph.D. degree at Jinan University in 2010 investigating structural modification and SAR analysis of naturally occurring antitumor products. Dr. Xiao-Ming Wu obtained his Ph.D. degree at Kyushu University in 1993 investigating structural modification and SAR analysis of naturally occurring antitumor products. Dr. Guang-Ji Wang obtained his Ph.D. degree at University of Otago in 1995 investigating pharmacokinetics pharmacodynamics of antitumor agents. Dr. Wei-Min Chen received his Ph.D. degree from Sun Yat-sen University in 1998. Now his research interests focus on the structural modification of natural compounds and new drug discovery in the fields of anti-infection, anticancer, and antibacterial agents. Dr. Wen-Cai Ye obtained his Ph.D. degree at The Hong Kong University of Science and Technology in 2001. His Scientific focus is on the investigation of structurally unique and biologically active constituents from Chinese medicinal plants.

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web site: Table SI. Structures of the training and test set molecules Table SII. Summary of CoMFA-PLS and CoMSIA-PLS results (A2780 cells) Table SIII. Summary of the actual pIC50 s, predicted pIC50 s (Pred.), and their residuals (Res.) of the training and test set molecules Figure S1. Alignment of the compounds used in the training set. Figure S2. Graph of actual versus predicted pIC50 of the training set and the test set using CoMFA (left) and CoMSIA (right).


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