Total synthesis, chemical modification and structure-activity relationship of bufadienolides

Journal Pre-proof Total synthesis, chemical modification and structure-activity relationship of bufadienolides Yue- Zhong, Chao- Zhao, Wen-Yu Wu, Tian-Yuan Fan, Nian-Guang Li, Min- Chen, Jin-Ao Duan, Zhi-Hao Shi PII:

S0223-5234(20)30005-2

DOI:

https://doi.org/10.1016/j.ejmech.2020.112038

Reference:

EJMECH 112038

To appear in:

European Journal of Medicinal Chemistry

Received Date: 15 November 2019 Revised Date:

13 December 2019

Accepted Date: 3 January 2020

Please cite this article as: Y.- Zhong, C.- Zhao, W.-Y. Wu, T.-Y. Fan, N.-G. Li, M.- Chen, J.-A. Duan, Z.-H. Shi, Total synthesis, chemical modification and structure-activity relationship of bufadienolides, European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2020.112038. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Masson SAS.

Graphical Abstract

Total synthesis, chemical modification and structure-activity relationship of bufadienolides

Yue-Zhonga, Chao-Zhaoa, Wen-Yu Wuc, Tian-Yuan Fana, Nian-Guang Lia,*, Min-Chena, Jin-Ao Duana, Zhi-Hao Shib,* a

National and Local Collaborative Engineering Center of Chinese Medicinal Resources

Industrialization and Formulae Innovative Medicine, Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210023, China b c

Department of Organic Chemistry, China Pharmaceutical University, Nanjing 211198, China

Department of Nuclear Medicine, Nanjing First Hospital, Nanjing Medical University, Nanjing

210006, China ∗Corresponding author. Tel.: +86-25-85811916; fax: +86-25-85811916; e-mail: [email protected] ∗Corresponding author. Tel.: +86-25-86185172; fax: +86-25-86185182; e-mail: [email protected]

O O

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O H H

AcO

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AcO

HO

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O O

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H O H

AcO

O

H HO

O

OAc O

H

O O AcO

O

H H O H HO

OH O

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OH

H HO H

OH

O

HO H HO

HO

O O

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HO

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CO2H O

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H H HO

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Total synthesis, chemical modification and structure-activity relationship of bufadienolides Yue-Zhonga, Chao-Zhaoa, Wen-Yu Wuc, Tian-Yuan Fana, Nian-Guang Lia,*, Min-Chena, Jin-Ao Duana, Zhi-Hao Shib,* a

National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine, Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210023, China b Department of Organic Chemistry, China Pharmaceutical University, Nanjing 211198, China c Department of Nuclear Medicine, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, China ∗Corresponding author. Tel.: +86-25-85811916; fax: +86-25-85811916; e-mail: [email protected] ∗Corresponding author. Tel.: +86-25-86185172; fax: +86-25-86185182; [email protected]

e-mail:

Abstract Bufadienolides are a type of natural cardiac steroids and originally isolated from the Traditional Chinese Medicine Chan’Su, they have been used for the treatment of heart disease in traditional remedies as well as in modern medicinal therapy with potent anti-tumor activities. Due to their unique molecular structures with unsaturated six-membered lactones attached to the steroid core, bufadienolides have received great attention in the synthetic organic community. This review presents total synthetic efforts to some representative bufadienolides, chemical modification of bufadienolides will also be given to discuss their structure-activity relationship in anti-tumor. Key words: Chan’Su, bufadienolide, cardiac steroids, heart disease, anti-tumor 1. Introduction 2. Total synthesis of bufadienolides 2.1 Starting materials 2.2 Total synthesis of bufalin 2.3 Total synthesis of α-isobufalin from digitoxigenin 2.4 Total synthesis of α'-isobufalin from the usual steroidal ketone 2.5 Total synthesis of γ-isobufalin from testosterone 2.6 Total synthesis of β-isoresibufogenin from digitoxigenin 2.7 Total synthesis of α-isobufalin, α-isoresibufogenin and β-isoresibufogenin from testosterone 2.8 Total synthesis of scillarenin 2.9 Total synthesis of telocinobufagin from digitoxigenin 2.10 Synthesis of cinobufagin from bufotalin 2.11 Synthesis of bufadienolide from dehydroepiandrosterone 2.12 Synthesis of bufadienolide from deoxycholic acid 2.13 Synthesis of bufadienolide from 3β-acetoxy-5α-pregnan-21-al 2.14 Synthesis of bufadienolide from 3β-acetoxy-5-androsten-17-one 2.15 Synthesis of 3β-hydroxy-5β,14α-bufa-20,22-dienolide from deoxycorticosterone 2.16 Transformation of bufalin into other members of the bufadienolides 1

2.17 Other methods for the construction of pyrone ring 3. Chemical structural modification of bufadienolides 4. Structure-activity relationship of bufadienolides 5. Concluding remarks 6. Acknowledgements 7. References 1. Introduction The bufadienolides are a wide-spread group of heart-active steroids occurring in the Ch’an Su (in the free state or as conjugates), which is the secretion of toads as well as in certain plants as glycosides [1]. As one of the sources of bufadienolides, the Ch’an Su preparation has been traditionally used as a cardiotonic, diuretic, anti-tumor and local anaesthetic agent. Bufadienolides along with adrenaline and indole alkaloids (some of which show psychotropic activity) [2,3] can be obtained from Ch’an Su. Bufalin 1 (Figure 1), telocinobufagin 2 and 14,15β-epoxides 3–5, shown in Figure 1, are the main bufadienolide constituents of Ch’an Su. These substances all contain α-pyrone ring at the 17β position, as well as a 14β-hydroxy group (e.g., bufalin 1) or a 14β,15β-oxido group (e.g., resibufogenin 3).

Figure 1. The chemical structures of some represented bufadienolides. Many bufadienolides isolated from plant sources of several species or prepared by the biotransformation of relatively easily accessible bufadienolides have been examined with respect to their cytotoxicity. These works have recently been extensively reviewed [4-7]. For example, bufadienolide glycosides 6 and 7 (Figure 1), have been isolated from rhizomes of Helleborus orientalis and their cytotoxic activity against selected tumor cells has been evaluated [8]. Both rhamnosides 6 and 7 showed high specific cytotoxicity towards human squamous carcinoma cells (HSC-2) and human melanoma cells (A375), and a weak effect on parental human pulp cells (HPC). From comparison of 6 with its aglycone, it has been concluded that the sugar moiety markedly contributes to the tumor specificity. The most noticeable role of bufadienolides was the inhibition towards Na+/K+-ATPase, leading to its cardiotonic effects [9,10]. Besides, anti-tumor activities were found in bufadienolides, which were manifested in the induction of apoptosis, autophagy and cell cycle arrest of cancer cells by inhibiting the PI3K/AKT/mTOR pathway, as well as blocked tumor invasion and metastasis via the down-regulation of β-catenin [11,12,13]. Despite attractive biological activities of bufadienolides, most of these natural products were isolated from medicinal plants and secretion of toads with unsatisfactory contents and yields [1,14]. Considering the limited resources, the total synthesis of bufadienolides has been widely studied in recent years. A review article describing natural and synthetic 2H-pyran-2-ones provided an overview of the natural bufadienolides reported up to 2005 [15]. A methodological analysis of synthetic efforts to 2

bufadienolide aglycones has been reviewed, and special emphasis was given to cross-coupling reactions applied for the attachment of lactone subunits at sterically very hindered positions of the steroid core [1]. This review presents total synthetic efforts to some representative bufadienolides, chemical modification of bufadienolides will also be given to discuss their structure-activity relationship in anti-tumor. 2. Total synthesis of bufadienolides Approach to the synthesis of natural bufadienolides such as bufalin 1 and resibufogenin 3 has a history of over 50 years. The main difficulty encountered was to build both α-pyrone ring and labile 14β-hydroxy (or epoxide) together at the D-ring keeping thermodynamically unstable configurations [16]. In 1969, Sondheimer announced first formal total synthesis of 1 and 3, ingeniously solving this problem [14]. Shortly afterward, Hoechst Farbwerke’s work had developed attractive and methodologically different routes as exemplified by the synthesis of scillarenin [17,18]. Meanwhile, Pettit and his co-workers also had made valuable contributions to the bufadienolide chemistry including the conversion of digitoxigenin (cardenolide) to some bufadienolides [19,20]. The α-pyrone ripe in bufadienolide synthetic studies had been elaborated either directly by cyclization of unsaturated esters of type 8 [21], or from dihydro precursors of types 10 [22], 12 [23] and 13 [24] (Figure 2).

Figure 2. The chemical synthetic routes to the α-pyrone ripe in bufadienolide. 2.1. Starting materials Several commercially available steroids were used as starting materials for bufadienolide synthesis (Figure 3). Digitoxigenin 14, a steroid derived from digitalis, was the most common used starting material and could also be used in certain cardiac drug treatments [25,26]. Dehydroepiandrosterone 15 and its acetate 16 were the basic starting materials for the partial synthesis of bufadienolides [21,23]. Testosterone 17 and 3β-(benzyloxy)-5β-androst-15-en-17-one 18 were other convenient options among androstane (C19) derivatives [27,28]. Various pregnane derivatives were used as the 21-carbon atom scaffold for cardenolide synthesis, including 3β-acetoxy-5α-pregnan-21-al 19 and 5β-pregn-14-en-3β-ol-20-one acetate 20 [29,30]. A pregnane nucleus with higher degree of functionalization might be obtained from corticosteroids, such as deoxycorticosterone 21, 15α-hydroxycortexone 22 or 14α-hydroxycortexolone 23 [14,17,31,32]. Bile acids represented another group of common steroids starting materials. These included cholanic acid 24 and deoxycholic acid 25 [33,34]. 14-dehydrobufalin 26 and 3β-acetoxy-14-dehydrobufalin 27 were involved in the synthesis of bufadienolides as well 3

[19,35,36]. Moreover, bufotalin 28, which was isolated from Ch’an Su, could be transformed into cinobufagin 5 while bufalin 1 was employed as relay to afford bufotalin 28, cinobufagin 5 and other bufadienolides [37,38].

Figure 3. Structures of major starting materials for bufadienolide synthesis. 2.2 Total synthesis of bufalin Synthesis of bufalin from 14α-hydroxycortexolone In 1969, Sondheimer et al. reported the total synthesis of bufalin 1 and resibufogenin 3, starting from 14α-hydroxycortexolone 23, which was available in quantity as a by-product in the commercial microbiological hydroxylation of cortexolone 29 to cortisol [14] (Scheme 1). Side-chain degradation with sodium bismuthate followed by catalytic hydrogenation of 30 led mainly to 14α-hydroxy-5β-androstane-3,17-dione 31, which was reduced at C-3 with sodium borohydride (NaBH4) yielded 59% (based on 30) of the 3α-ol 32 and 22% of the 3β-ol 33. The major product 32 was next reconverted to a pregnane through reaction with lithium ethoxyacetylide (from n-butyllithium and ethoxyacetylene), followed by rearrangement of the 17-ethoxy acetylenic carbinol in dioxane with 2N sulfuric acid. The resulting unsaturated ester 34 on saponification with potassium carbonate gave the acid 35. The chemical reduction of the double bond in 35 using a large excess of potassium in liquid ammonia afforded the saturated acid 36, which was treated with ethereal diazomethane to obtain the methyl ester 37. Acetylation of 37 yielded the noncrystalline acetate 38, dehydration of 38 in pyridine with phosphorus oxychloride gave ca. 55% of the ∆14 compound 39, as well as ca. 30% of the ∆8(14) isomer. Saponification of 39 with potassium carbonate led to the ∆14-hydroxy acid 40. The carboxylic acid function in 40 was reduced to an aldehyde through reacting with an excess of N,N′-carbonyldiimidazole, followed by reduction of the resulting 3,21-bis derivative 41 at C-21 with excess lithium tri-t-butoxyaluminum hydride. Hydrolysis of the product 42 at C-3 with dilute sulfuric acid gave the noncrystalline hydroxy aldehyde 43. Treatment with boiling methanol in the presence of p-toluenesulfonic acid (p-TSA) led to the dimethyl acetal 44, which was acetylated to 45. The acetal 45 was subjected to the Vilsmeier-Haack reaction by adding a reagent prepared from equal volumes of phosphorus oxychloride and dimethylformamide (DMF). Preparative thin layer chromatography then gave 60% of the 'cis' isomer of the enol ether 46 and 19% of the 'trans' isomer. Hydrolysis of the vinylogous ester 'cis'-46 with sodium hydroxide (NaOH) led to a mixture of the enolized β-dialdehydes 47 and 48, which was subjected to Reformatsky reaction by heating with methyl bromoacetate and zinc. Preparative thin layer chromatography resulted in the α-pyrone 49. Saponification of 49 with hydrochloric acid yielded the 3α-ol, which was converted to the p-toluenesulfonate and then heated in DMF. The resulting 3β formate was saponified by shaking in ether with alkaline alumina to the 3β-ol, which was then acetylated. Treatment of 27 in aqueous acetone with N-bromosuccinimide (NBS) and subsequent chromatography on basic 4

alumina gave 45% of resibufogenin acetate 50. Saponification by absorption in ether on basic alumina led to 3 in high yields. Finally, reduction of 3 with an excess of lithium aluminum hydride in ether afforded 50% of natural bufalin 1. Although this was the first reported route of bufalin synthesis, the final product was obtained in less than 1% yield in more than 28 steps of transformation, which was not suitable for large-scale industrial production.

Scheme 1. Synthesis of bufalin from 14α-hydroxycortexolone. Synthesis of bufalin from digitoxigenin In 1970, Petti’s group reported the total synthesis of resibufogenin 3 and bufalin 1 from digitoxigenin 14 [25] (Scheme 2). This synthesis represented a new approach to the bufadienolides and constituted the first synthetic route from a cardenolide to a bufadienolide. Digitoxigenin 14 prepared by hydrolysis of digitoxin 51 was acetylated to 52 and then dehydrated to 14-dehydrodigitoxigenin acetate 53. Upon treatment with sodium methoxide in methanol (followed by acidification) cardenolide 53 was transformed, presumably via aldehyde 54, to lactol 55. Methanolysis of lactone 55 using methanol containing p-TSA readily afforded acetal methyl ester 56. The protection was achieved by first acetylating alcohol 56 and then treatment with ethanedithiol containing 70% perchloric acid afforded carboxylic acid 58. Homologation of acid 58 via acid chloride 59 and diazo ketone 60 led to carboxylic acid 61. Cleavage of the ethylene thioacetal was accomplished using a mercuric oxide-mercuric chloride-aqueous acetic acid procedure to afford 62. Enol cyclization of aldehyde 62 was achieved using p-TSA as catalyst to afford 63. Dehydrogenation of enol lactone 63 using the sulfur procedure afforded the product 14-dehydrobufalin acetate 27. Treatment of 14-dehydrobufalin acetate 27 with m-chloroperbenzoic acid (m-CPBA) prepared resibufogenin acetate 51. Selective saponification of acetate 50 using basic alumina afforded the product resibufogenin 3, which was completely identical with a natural specimen. Selective reduction of resibufogenin was employed to obtain bufalin 1. The reaction conditions of this synthetic route were quite mild and easy to operate. However, the expensive starting materials and lower yields limited the promotion of this synthetic route. 5

O

O O

O O

O CO2H CHO

CH3ONa

H 14 R=H 51 R=3(D-digitoxose) 52 R=COCH 3

S

H 58 R=COCH 3, R1=OH oxalyl chloride benzene 59 R=COCH 3, R1=Cl diazomethane ether 60 R=COCH 3, R1=CHN 2

HO

H 53

54

H

CO2H

Ag2O, Na 2S2O3, K2CO3 dioxane

RO

55

O

COR1 S

RO

CH3CO2

ethanedithiol 70% HClO 4 H

56R=H Ac2O, pyridine 57 R=COCH 3

O

O

O

O

O

O

O

R p-TSA dry benzene

OCH3

PTSA MeOH

CH3OH

OH RO

CO2CH3 OCH3

OH

m-CPBA CHCl3

S, CS2

LiAlH4 ether OH

O

CH3C O2

H

S

AcOH HgCl2 HgO

S

61 R=

CH3C O2

H 63

CH3C O2

H 27

RO

H 50 R=COCH 3

HO

H 1

activated alumina ether 3 R=H

62 R=-CHO

Scheme 2. Synthesis of bufalin from digitoxigenin. In 1982, Tsai and Wiesner converted digitoxigenin to bufalin [39] (Scheme 3). The starting material was the dibenzyl aldehyde 64 easily prepared from digitoxigenin 14 [26]. Acetalization with benzene and p-TSA yielded 92% of the only ∆14,15-acetal 65. The acetal 65 was irradiated with a 100 W high pressure mercury lamp at ‒70°C in CH2Cl2 in the presence of meso-tetraphenylporphine (0.4%) while oxygen was bubbled through the solution. The resulting endoperoxide solution was treated with a large excess of dimethylsulfide, and the solvent was evaporated to dryness. The residue was taken up in aqueous tetrahydrofuran (THF), 2N NaOH and reduced with an excess of NaBH4 to yield 82% of the pure oily acetal 66. Compound 66 was heated under reflux in a mixture of THF and 3N HCl to afford the crude hemiacetal 67, which was then heated under reflux with silver carbonate on Celite in benzene to give 75% of the hydroxy lactone 68. The hydroxy lactone 68 reacted with mesyl chloride (MsCl) and trimethylamine (TMA), and then heated under reflux with 1,5-diazabicyclo[4.3.0]non-5-ene (DBN) in benzene to afford the α-pyrone 69 in a yield of 85%. The introduction of the C14-β hydroxy group was performed as described previously [40]. The benzyl derivative 70 was isolated in a yield of 70%. Finally, debenzylation with Pd(OH)2/C as described previously yielded 70% of semi-synthetic bufalin 1 [26]. Compared with Pettit’s work, the yield of this route had been improved, but the reaction conditions were relatively harsh due to the requirement of specific wavelengths under lighting conditions.

Scheme 3. Synthesis of bufalin from digitoxigenin. Transformation of 14-dehydrobufalin to bufalin 6

In 1973, Pettit’s group simplified the transformation of 14-dehydrobufalin 26 via resibufogenin 3 to bufalin 1 by employing a halohydrin approach [35] (Scheme 4). The reaction of N-iodosuccinimide and either NBS or N-bromoacetamide (NBA) transformed 14-dehydrobufalin 26 to, respectively, iodohydrin 71 and bromohydrin 72. Hydrogenolysis of halohydrins 71 and 72 using Urushibara nickel A or Randy nickel readily afforded bufalin 1 in good yields. However, the shortcomings of expensive starting materials and long routes had not been improved.

Scheme 4. Transformation of 14-dehydrobufalin to bufalin. Synthesis of bufalin from steroid 5β-pregn-14-en-3β-ol-20-one acetate In 1977, Yoshii et al. investigated more effective short-step synthesis of bufalin 1 and resibufogenin 3 from steroid 5β-pregn-14-en-3β-ol-20-one acetate 20 [30] (Scheme 5). The aldehyde 73 was obtained by perchloric acid catalyzed condensation of 20 with trimethyl orthoformate [41]. It was then reacted with weakly acidic methanol to produce a mixture of β-methoxyvinyl ketone 74 and the dimethylacetal 75, which were transformed to a single product 74 through brief treatment with potassium tert-butoxide. The reaction of 74 with large excess of dimethylsulfonium methylide followed by treatment of the crude product with methanolic hydrogen chloride afforded methoxydihydropyrane 76. Hydrolysis of the methyl ether group of acetylated 76 was affected with buffered hydrochloric acid. Jones oxidation of the resulting pyranol 77 produced buf-20(22)-enolide 78. The reaction of 78 with slight excess of aqueous NBA afforded unstable 15β-bromo-14β-hydrin, which was readily transformed to 14β,15β-epoxide 79 on alumina chromatography. Addition of bromide to 79 in the presence of an acetate buffer and subsequent dehydrobromination with DBU provided resibufogenin acetate 50. Hydrolytic removal of the acetate group gave resibufogenin 3, whose conversion to bufalin 1 was affected by reductive cleavage of the epoxide ring [25]. This route possessed mild reaction conditions, while expensive starting materials and low yields limited its development to a large extent. O OCH3 O CH3

H3CO

O

O

75

CH3OH, THF, pyridine hydrobromide AcO

AcO

20

OCH3

AcO t-BuOK, t-BuOH

O

O

73 74

OCH3 (1) KH, Me2SCH2, THF, (CH3)3SI (2) HCl, MeOH

AcO OH O

O Jones reagent, O acetone

HO

O

70% HClO4, NBA, H2O, acetone

77

AcO

78

AcO

76

O

O

10% AcOK-AcOH, O Br2, AcOH, ether

O

O AcO

HCl, KCl, THF

O AcO

79

50

O

O

O

O HO

H 3

OH HO

H 1

Scheme 5. Synthesis of bufalin from steroid 5β-pregn-14-en-3β-ol-20-one acetate. 7

Synthesis of bufalin from the usual steroidal ketone In 1983, Wiesner’s group applied their furan strategy to approach bufalin 1 [24,42]. The first four steps were identical with the already described preparation of β-isoresibufogenin [28] (Scheme 6). The 15'β-hydroxy group of compound 84 was eliminated with MsCl in pyridine to yield the crystalline ∆14′,15′-derivative 65. 65 was irradiated with a 100 W high-pressure Hg-lamp at −70°C in CH2Cl2 in the presence of 5,10,15,20-tetraphenylporphyrin (TPP) while O2 was bubbled through the solution. The peroxide bond of the resulting endoperoxide 85 was cleaved with a large excess of Me2S and the crude unsaturated keto aldehyde 86 was immediately reduced with an excess of NaBH4 to afford the oily product 66. Compound 66 was heated under reflux with dilute HCl in THF. Hydrolysis of the ethylene glycol acetal and hemiacetal formation yielded the diastereoisomeric mixture 67, which was oxidized with Ag2CO3/Celite in refluxing benzene to afford the unsaturated hydroxylactone 68. Compound 68 was dissolved in CH2Cl2 and mesylated with MsCl and triethylamine (Et3N). The crude mesylate was then subjected to elimination by heating with DBN in benzene to produce the elimination product 69. The introduction of the 14'β-hydroxy group was performed as described previously by the modified method of Engel to afford the product 3-O-benzylbufalin 70 [28,42]. Finally, debenzylation of 70 with Pd(OH)2/C yielded the synthetic bufalin 1. The specific wavelength in this route was hard to control and the use of strong base n-butyl lithium was dangerous, thus it was difficult to synthesize bufalin 1 in a large scale.

Scheme 6. Synthesis of bufalin from the usual steroidal ketone. Synthesis of bufalin from deoxycholic acid In 1986, Welzel’s group reported the synthesis of bufalin from deoxycholic acid 25 [43] (Scheme 7). Deoxycholic acid 25 was firstly transformed into 87 in 4 steps as already described [44]. Swern oxidation of 87 gave 88. The ZnCl2-catalyzed reaction of 88 with the tert-butyldimethylsilyl ketene acetal derived from tert-butyl methylsulfanylacetate provided the corresponding Michael adducts 89. Reaction of 89 with potassium fluoride in THF-methanol led to the cleavage product 90. Refluxing a benzene solution of 90 in the presence of p-TSA provided 91 (1:1 mixture of 23-isomers). Selective irradiation of 91 into the CO band followed by immediate reduction with LiAlH(OtBu)3 afforded 92. Introduction of the missing double bond, mesylation and subsequent solvolysis of 93 under Masamune’s conditions gave 94 [45]. The bufalin 1 was formed by ester hydrolysis of 94. The starting material deoxycholic acid was cheaper than digitoxigenin and this synthetic route greatly reduced the economic cost. However, the reaction conditions were quite harsh as multi-step low temperature reactions and the ultraviolet lamp irradiation reaction were covered in this route, which limited large-scale applications.

8

Scheme 7. Synthesis of bufalin from deoxycholic acid. 2.3 Total synthesis of α-isobufalin from digitoxigenin In 1982, Wiesner’s group disclosed a simple conversion of digitoxigenin 14 to α-isobufalin 103 [26] (Scheme 8). The furan derivative 95 was benzylated with sodium hydride, 18-crown-6 ether and benzyl bromide in refluxing dioxane to afford the dibenzyl derivative 96. An ether solution of compound 96 was firstly treated at ‒70°C with n-butyl lithium and then with DMF under ice cooling. The formyl derivatives 97 and 64 were obtained in a ratio 3:2. Compound 97 was reduced quantitatively with LiAlH4 in ether to the oily alcohol 98, which was then oxidized in the presence of sodium acetate with m-CPBA in CH2Cl2 to afford the mixture of epimeric hemiacetals 99. The hemiacetal 99 was oxidized in CH2Cl2 with CrO3-diPy and the relatively unstable ketolactone was immediately reduced with an excess of Zn(BH4)2 in ether to produce the oily epimeric hydroxylactones 100. 100 was firstly mesylated with MsCl and TMA in CH2Cl2 and the resulting derivative 101 was then heated with DBN in benzene to afford the oily homogeneous dibenzyl α-isobufalin 102. The dibenzyl derivative 102 was heated under reflux in an ethanol-benzene mixture (2:1) with Pd(OH)2/C and cyclohexene to afford the product α-isobufalin 103.

Scheme 8. Total synthesis of α-isobufalin from digitoxigenin. 2.4 Total synthesis of α'-isobufalin from the usual steroidal ketone In 1983, Wiesner’s group applied their furan strategy to approach natural α'-isobufalin 114 [24,42] (Scheme 9). The starting materials were the Li-derivative 80, prepared by direct lithiation of the ethylene glycol acetal of furfural and the usual steroidal ketone 18. The synthesis proceeded uneventfully (→104→105→106) to compound 107, which was oxidized by NBS and then reduced by NaBH4 to the diastereoisorneric mixture 108. The acid-catalyzed conversion of 108 afforded 109. Oxidation of the pyranose 109 with Ag2CO3/Celite gave the crystalline 9

hydroxy-lactone 110. The elimination of the two hydroxy groups of compound 110 was performed in two steps. A monomesylation followed by heating of the crude mesylate with lithium bromide (LiBr) in DMF yielded the α-pyrone 111, and the remaining hydroxy group was eliminated with SOCl2 and pyridine to afford 112. The introduction of the 14'β-hydroxy group and the debenzylation of the resulting compound 113 was accomplished by the same methods as before.

Scheme 9. Total synthesis of α'-isobufalin from the usual steroidal ketone. 2.5 Total synthesis of γ-isobufalin from testosterone Biological activity evaluations of bufadienolides indicated that the derivatives which were connected to the steroid moiety by the β-carbon of the enone system of the six-membered ring showed both potency and margin of safety superior to those natural cardioactive steroid glycosides. Thus, in 1988, Wiesner’s group synthesized γ-isobufalin 127 from testosterone 17 using α-furans as masking groups of a carboxylic acid [27] (Scheme 10). The γ-pyrone ring in γ-isobufalin 127 still retained the right setup for the enone system, but it possessed electronic and chemical properties (i.e., dipole moment, pKa) different from those of the isomeric α-pyrones. Testosterone 17 was converted to the α,β-unsaturated ketone 18, which was then alkylated in dry ether at 0°C with a preformed solution of α-furyl lithium (furan and n-butyl lithium in dry ether at ‒78°C) [42]. The crude tertiary alcohol was acetylated with acetic anhydride in pyridine to compound 116, which was subjected to allylic rearrangement in refluxing aqueous acetone in the presence of calcium carbonate to yield the allylic alcohol 117. The crude product 117 was hydrogenated with palladium on calcium carbonate (Pd/CaCO3) in ethanol to produce the 15β-hydroxy compound 118. The 15β-hydroxy group was acetylated with acetic anhydride in pyridine to yield compound 119. Treatment of compound 119 with NBS in dioxane/water followed by KMnO4/NaIO4 oxidation in the same solvent mixture in the presence of potassium carbonate yielded the carboxylic acid 121 via the keto-aldehyde 120 [24]. The carboxylic acid 121, upon treatment with N,N′-carbonyldiimidazole in dry chloroform at 50°C, yielded quantitatively the carbonyl imizadole derivate 122 which was immediately reacted with a preformed solution of 4-methoxy-3-buten-2-one potassium enolate in dry THF at ‒78°C produced the expected γ-pyrone 123 [46]. Hydrolysis of the acetyl group of compound 123 with aqueous HCl in refluxing methanol yielded the secondary alcohol 124 which was regioselectively dehydrated with thionyl chloride in pyridine at ‒15°C to yield the unsaturated compound 125. Treatment of the unsaturated compound 125 with NBS in the presence of aqueous HClO4 in acetone/water followed immediately by debromination with a large excess of Raney nickel in the presence of acetic acid in dichloromethane/water at room temperature yielded the 3-benzyl-γ-isobufalin 126. Debenxylation of compound 126 in refluxing ethanol/benzene with cyclohexene over Pd(OH)2 10

yielded γ-isobufalin 127.

Scheme 10. Total synthesis of γ-isobufalin from testosterone. 2.6 Total synthesis of β-isoresibufogenin from digitoxigenin In 1982, Wiesner’s group disclosed a simple conversion of digitoxigenin 14 to β-isoresibufogenin 133 [26] (Scheme 11). Reduction of 64 with LiAlH4 and oxidation of the resulting primary alcohol with m-CPBA yielded the mixture of epimeric acetals 128. Oxidation and reduction of 128 gave a mixture of the epimeric hydroxylactones 129. Finally, mesylation and elimination of the mesyloxy group gave dibenzyl β-isobufalin 130. Debenzylation of compound 130 yielded 6% β-isobufalin 131 and 80% of the ∆14,15-derivative 132. When 132 was treated with NBS and Raney nickel, intermediate bromohydrin eliminated hydrobromic acid and yielded β-isoresibufogenin 133.

Scheme 11. Total synthesis of β-isoresibufogenin from digitoxigenin. 2.7 Total synthesis of α-isobufalin, α-isoresibufogenin and β-isoresibufogenin from testosterone In 1982, Wiesner et al. synthesized α-isobufalin 103, α-isoresibufogenin 152 and β-isoresibufogenin 133 by a method which features a novel oxidative furan to pyrone transformation [28]. The steroid starting material was the α,β-unsaturated ketone 18, which could be readily prepared from testosterone [42].

11

Scheme 12. Total synthesis of α-isobufalin and α-isoresibufogenin from testosterone. Compound 134 was first converted to the lithium derivative 135 by treatment with butyllithium (Scheme 12), and 135 was immediately reacted with the steroid 18. The obtained tertiary alcohol 136 was then acetylated, and intermediate 137 was subjected to an allylic rearrangement in refluxing aqueous acetone in the presence of calcium carbonate to afford the product 138. Hydrogenation of 138 with Pd/CaCO3 in ethanol gave the 17β-derivative 139, and the subsequent deprotection and NaBH4 reduction of the aldehyde group (intermediates 140 and 141) proceeded quantitatively. The oxidative conversion of furan to pyrone succeeded by treatment of the alcohol 141 with m-CPBA, the epimeric hemiacetals 143 was obtained presumably by rearrangement of the primary oxidation product 142. The hemiacetal hydroxy group of 143 was firstly blocked by a selective monoacetylation with sodium acetate/acetic anhydride in benzene, and the epimeric monoacetates 144 were obtained. A fully regioselective elimination of the 15β-hydroxy group with thionyl chloride in pyridine yielded the ∆14,15-derivative 145, which was then saponified to the hemiacetal 146. The hemiacetal hydroxy group was oxidized with the chromic acid dipyridine complex and the quite unstable ketolactone was formed, which was immediately reduced with zinc borohydride in ether to afford the epimeric hydroxylactones 147. The alcohols 147 were converted to the corresponding methanesulfonates 148 with MsCl and Et3N in CH2Cl2, and 148 was heated under reflux with DBN in benzene to afford the ∆14,15-pyrone 149. The ∆14,15-derivative 149 was treated with NBS in aqueous acetone, and the intermediate bromohydrin was debrominated by stirring with Raney nickel to afford the 3-O-benzyl-α-isobufalin 150. Compound 150 was heated under reflux in benzenelethanol with Pd(OH)2/C and cyclohexene to yield the final product α-isobufalin 103. Compound 149 reacted with NBS and basic alumina to obtain the epoxide 151, and then debenzylation by the Hanessian technique yielded 84% of α-isoresibufogenin 152 [47].

12

Scheme 13. Total synthesis of β-isoresibufogenin from testosterone. For the synthesis of β-isoresibufogenin 133, the starting materials were the protected steroid ketone 18 and the reagent 80 obtained by lithiation of the bromoacetal 153 (Scheme 13). The synthesis proceeded in a precisely analogous manner. A major difference in reactivity was the ∆14,15-derivative 162. The bromohydrin obtained from this material by NBS in acetone and Raney nickel gave exclusively 3-O-benzyl-β-isoresibufogenin 163. Debenzylation of 163 with Pd/C approximately yielded equal quantities of the crystalline β-isoresibufogenin 133 and its dihydro derivative 164. 2.8 Total synthesis of scillarenin Synthesis of scillarenin from 15α-hydroxycortexone Stache et al. synthesized scillarenin 178 from 15α-hydroxycortexone 22 [17,32] (Scheme 14). The 21 position in 22 was functionalized by Cu(OAc)2 oxidation in aqueous MeOH to afford 165, which was followed by acetal formation and the C14-hydroxy group elimination using TsCl to produce 168. After protection of the A/B ring enone moiety, an addition of dimethylsulfonium methylide to the side chain carbonyl group of 169 was carried out and the intermediate epoxide 170 was subsequently transformed into bromohydrin 171. The bromohydrin moiety was converted to epoxide 172 and the Horner–Emmons–Wadsworth olefination of 172 afforded the epoxy ester 173, which was smoothly rearranged into aldehyde 174. Acid-catalyzed ring closure of ester aldehyde 174 afforded the α-pyrone derivative 175. Using hypobromous acid, the obtained 14-anhydro-scillarenone 175 was converted into 15α-l4β-bromohydrin 176, whose 15α-bromine atom was subsequently eliminated hydrogenolytically and the obtained scillarenone 177 reduced to 178 with LiAl(tert-C4H9O)3H. Reduction of 177 with lithium borohydride (LiBH4)/pyridine produced 5α-bufalin 179.

13

Scheme 14. Synthesis of scillarenin from 15α-hydroxycortexone. Synthesis of scillarenin from bufalin In 1972, Pettit’s group reported a total synthesis of the plant bufadienolide scillarenin 178 employing bufalin as relay [20] (Scheme 15). 14-dehydrobufalin 26 was treated with N-iodosuccinimide in acetone-water and the crude iodohydrin 71 was reduced with Urushibara nickel A to complete a new synthesis of bufalin 1. Next, bufalin was easily oxidized (chromium trioxide in acetic acid) to bufalone 180, which was brominated at C-4 using NBS in carbon tetrachloride. The α-bromo ketone was dehydrohalogenated by refluxing in collidine and scillarenone 177 was obtained. Reduction of scillarenone to scillarenin 178 was easily realized by lithium tri-tert-butoxyaluminum hydride or LiBH4.

Scheme 15. Synthesis of scillarenin from bufalin. In 1974, Pettit’s group utilized bufalin 1 as relay in a new synthetic route to scillarenin 178 [48] (Scheme 16). Important steps in the synthesis of scillarenin included bromination and dehydrohalogenation of bufalone 180 to yield scillarenone 177. Selective chromic acid oxidation (Sarett) of bufalin 1 provided bufalone 180, and treatment of ketone 180 with bromine in DMF or acetic acid gave the corresponding 4-bromo derivative 181, which was subjected directly to dehydrobromination with LiBr in DMF or lithium chloride in dimethylacetamide (DMAc) to afford scillarenone 177. Reduction of ketone 177 to scillarenin 178 using lithium tri-tert-butoxyaluminum hydride in THF completed a new and formal total synthesis of this plant bufadienolide.

Scheme 16. Synthesis of scillarenin from bufalin. 14

2.9 Total synthesis of telocinobufagin from digitoxigenin

Scheme 17. Total synthesis of telocinobufagin from digitoxigenin. With the synthesis of bufalin 1 and scillarenin 178 from digitoxigenin 14 in hand [25,35,36,49], Pettit’s group completed the synthesis of telocinobufagin 2 in 1974 [50] (Scheme 17). Oxidation of olefin 178 with m-CPBA yielded β-epoxide 182. Oxidation of alcohol 182 by either chromium trioxide-pyridine or NBA provided ketone 183 in good yields. Reaction of epoxy ketone 183 with chromium (II) acetate in alcohol afforded telocinobufagone 184. Treating 3-ketone 184 with either Urushibara nickel A or W-2 Raney nickel in alcohol provided telocinobufagin 2. 2.10 Synthesis of cinobufagin from bufotalin

Scheme 18. Synthesis of cinobufagin from bufotalin. In 1972, Pettit reported a partial synthesis of cinobufagin 5 in the following route [37] (Scheme 18). Bufotalin 28, which was isolated from the toad venom preparation Ch’an Su, was acetylated to afford bufotalin acetate 185. Dehydration of 185 in pyridine with thionyl chloride yielded olefin 186. Hypobromous acid prepared in situ from NBA or NBS was added to 14-dehydrobufotalin acetate 186, and the resulting bromohydrin 187 was treated with basic alumina or pyridine to afford cinobufagin acetate 188. Finally, acid hydrolysis of cinobufagin acetate using Dowex-50 W-X8 gave cinobufagin 5. 2.11 Synthesis of bufadienolide from dehydroepiandrosterone In 1971, Pettit’s group developed a synthetic method for converting the readily available dehydroepiandrosterone 15 to 3β-acetoxy-5β,14α-bufa-20,22-dienolide 203 [23] (Scheme 19). Treatment of 15 with acetic-formic anhydride provided formate 190. Ketone 190 was condensed with the carbanion derived from diethyl cyanomethylphosphonate to afford olefin 191. The selective reduction of sidechain olefin 191 using Pd/CaCO3 as catalyst afforded nitrile 192, oppenauer oxidation of 192 led to ketone 193. In the present case 5% Pd/CaCO3 in acetonitrile, hydrogenation of 193 resulted in 34% of the 5α (194, positive Cotton effect) and 55% of the 5β 15

isomer (195, negative Cotton effect). Reaction of ketone 195 with chloroiridic acid and trimethyl phosphite resulted in axial 3β alcohol 196. The nitrile 196 was hydrolyzed and acetylated to provide carboxylic acid 197. Carboxylic acid 197 was converted to acid chloride 198 and reduced to aldehyde 199 by a Rosenmund reaction. The aldehyde 199 was converted via the piperidine enamine derivative and subsequently reacted with methyl acrylate to afford methyl ester 200. Selective saponification of methyl ester 200 followed by enol lactonization with p-TSA in refluxing benzene gave bufenolide 202. Dehydrogenation of bufenolide 202 to the required bufadienolide 203 was performed using molten sulfur. Although this route was not lengthy, the yield was very low especially in the last two steps (only 14%), which limited the application of this reaction in a certain extent. O

RO

O P N O O NaH, THF O HCO

15 R=H aceticformic anhydride, pyridine 190 R=CHO

191

CH2R (1) piperidine, toluene (2) CH2=CHCOOCH3, CH3CN

(1) KOH, MeOH-H2O (2) Ac2O, pyridine

H 196

O

192

CH2CN H2, 5% Pd/CaCO3 CH3CN

O

193 O

CH3CO2 HO

O HCO

CH2CN

chloroiridic acid P(OCH3)3 2-propanol

CH2CN aluminum isopropoxide xylene-cyclohexanone

CHCN H2, 5% Pd/CaCO 3 THF

H 197 R=CO2H oxalyl chloride benzene

CO2R

CH2CN

H 194 5 195 5

O O

O

CHO TsOH, benzene

S, 212-217 oC

H CH3CO2

198 R=COCl H2, 5% Pd/BaSO 4 toluene 199 R=CHO

H 200 R=CH3 Na2CO3, CH3OH/THF 201 R=H

CH3CO2

H 202

CH3CO2

H 203

Scheme 19. Synthesis of bufadienolide from dehydroepiandrosterone. 2.12 Synthesis of bufadienolide from deoxycholic acid In 1987, Welzel’s group reported a new procedure for the conversion of deoxycholic acid 25 to bufadienolide 210 [34] (Scheme 20). This novel strategy was centered around a new method for the conversion of 14α-H into 14β-hydroxy steroids using photochemical isomerixation. Deoxycholic acid 25 was transformed into 87 by (a) Iwasaki degradation, (b) Mitsunobu reaction, (c) photooxygenation-reduction, and (d) MnO2 oxidation, as already described [44]. Swern oxidation of 87 gave 88. The unsaturated aldehyde 88 was then reacted with the anion derived from tert-butyl ester 204 to give 206 (mixture of diastereoisomers). Refluxing a benzene solution of 206 (mixture of diastereoisomers) in the presence of p-TSA provided 208. Similarly, the direct cyclization of 207 (mixture of stereoisomers) furnished 209. To complete the synthesis of 210, the bufenolides 208 were separately oxidized with sodium metaperiodate in methanol-water, and the reaction mixtures were then heated in sealed tubes to give 210 in 52% and 47% yield, respectively. Similarily, a mixture of 209 was oxidized at ‒78°C with m-CPBA in CH2Cl2 solution and warming to room temperature caused already elimination to furnish 210 in 84% yield. However, this reaction sequence cannot be performed on a substrate with a pyrone ring, because the bufadienolide 2-pyrone has a UV absorption at the same wavelength as a ketone. Therefore, it was necessary to introduce the second double bond of the pyrone ring after the photolysis step.

16

Scheme 20. Synthesis of bufadienolide from deoxycholic acid. In 1989, Welzel’s group observed a pronounced solvent effect in the ZnCl2-catalyzed reaction of 88 with the unsubstituted ketene acetal 211, which was very fast even at ‒78°C in dimethoxyethane solution, providing 212. 212 was treated with tetrabutylammonium fluoride (TBAF) in THF to afford 213 as the main product [49]. Ester hydrolysis and acid-catalyzed enol lactone formation of 213 afforded 214 (Scheme 21). O O

AcO

88

H H

211

O

OSiMe 2tBu CO2Me

OMe OSitBuMe2

O

O TBAF, THF

AcO

O

AcO 212

CO2Me (1) Na2CO3, THF, CH3OH (2) p-TSA, benzene

O O

AcO 213

214

Scheme 21. Synthesis of bufadienolide from deoxycholic acid. 2.13 Synthesis of bufadienolide from 3β-acetoxy-5α-pregnan-21-al In 1969, Engel et al. published a bufadienolide synthesis which consisted of the Michael addition of diethyl malonate to a 20-methylene 21-aldehyde and the transformation of the resulting aldehydo diester into an enol lactone, which was then dehydrogenated to the α-pyrone [51] (Scheme 22). However, the yields of the last step were not very satisfactory.

Scheme 22. Synthesis of bufadienolide from 3β-acetoxy-5α-pregnan-21-al. In 1978, Engel et al. therefore prepared derivatives of the 20-methylene 21-aldehyde which had served for their first bufadienolide synthesis, in which the methylene group carried a leaving group (structure 220) [29]. The dimethyl acetal 219 of aldehyde 19 was subjected to a Vilsmeier-Haack reaction. This led to a mixture of the geometrically isomeric methoxymethylene aldehydes 220 and 221. Addition of ethyl lithioacetate to the 20-methoxymethylene 21-aldehyde 220 produced 222, and then treated with hydrochloric acid to give a mixture of the (Z)-and (E)-aldehydo cholenic esters 223 and 224. The major Z-isomer 223 reacted with hydrochloric acid in boiling ethanol, followed by reacetylation afforded the bufadienolide 218 (Scheme 23). This route not only saved the cost of synthesis, but also improved the yield, which was a great progress to scientific researchers.

Scheme 23. Synthesis of bufadienolide from 3β-acetoxy-5α-pregnan-21-al.

17

2.14 Synthesis of bufadienolide from 3β-acetoxy-5-androsten-17-one In 1983, Wicha’s group synthesized pentadienolide 203 from 3β-acetoxy-5-androsten-17-one via common intermediates [21] (Scheme 24). The starting material of acetoxy ketone 16 was transformed in four steps into the protected diketone 225. The carbonyl group in compound 225 was reduced with lithium tri-sec-butylborohydride (L-Selectride) in hexane-THF solution to the axial hydroxyl group, then the ketal group was hydrolyzed with p-TSA in acetone, and the crude hydroxy ketone was acetylated to afford compound 226. Condensation of ketone 226 with ethyl cyanoacetate in boiling toluene, in the presence of ammonium acetate, gave rise to the cyano ester 227. The condensation product was reduced with an excess of NaBH4 in methanol to the saturated alcohol 228, which was presented as a mixture of stereoisomers. The hydroxy group in compound 228 was then protected as its tetrahydropyranyl (THP) ether and the derivative 229 was subjected to reduction with an excess of diisobutylaluminum hydride (DIBAL) in toluene at ‒78°C. The obtained key intermediate 230 was first acetylated and then treated with an ylide prepared from triethyl phosphonoacetate and sodium hydride in THF to yield the condensation product 231. The protective THP group in compound 231 was removed and the alcohol was oxidized with pyridinium chlorochromate (PCC) to give the α,β-unsaturated aldehyde 232. Compound 232 was heated in benzene solution in the presence of a catalytic amount of p-TSA to afford pentadienolide 203.

Scheme 24. Synthesis of bufadienolide from 3β-acetoxy-5-androsten-17-one. 2.15 Synthesis of 3β-hydroxy-5β,14α-bufa-20,22-dienolide from deoxycorticosterone

Scheme 25. Synthesis of 3β-hydroxy-5β,14α-bufa-20,22-dienolide from deoxycorticosterone. In 1983, Watt’s group developed an unsaturated homoenolate anion equivalent 237 to furnish an intermediate 238, which was cyclized to α-pyrone in acceptable yield [31] (Scheme 25). Conversion of the C-21 hydroxyl group in 21 to a methyl ether and stereoselective reduction of 18

21-methoxypregn-4-ene-3,20-dione 233 using palladium on carbon in pyridine furnished 21-methoxy-5β-pregnane-3,20-dione 234. Further stereo- and regioselective reduction of the C-3 carbonyl group in 234 employed Henbest’s iridium chloride-trimethyl phosphite procedure to obtain the 3β- and 3α-alcohols 236 and 235 in a 91:9 ratio. The anion of 1-(phenylthio)-1-(trimethylsilyl)-2-propene 237 condensed with 3β-hydroxy-21-methoxy-5β-pregnan-20-one 236 furnished the adduct 238. The metalation of 238 used sec-butyllithium in a 10% hexamethylphosphoramide-THF solution containing 12-crown-4 at ‒78°C and subsequent exposure of the trianion to oxygen gave the thiol ester 239. Exposure of 239 to hydrobromic acid in refluxing acetonitrile involved the cyclization of the thiol ester and converted 239 to 240. The route was simple and convenient, but the functional groups were less tolerant. 2.16 Transformation of bufalin into other members of the bufadienolides Synthesis of isobufalin from bufalin In 1970, Pettit’s group synthesized isobufalin from bufalin [52] (Scheme 26). Upon treatment with potassium hydroxide (KOH) in methanol, bufalin 1 readily afforded isobufalin methyl ester 241. Acetylation of alcohol 241 gave 3β-acetoxyisobufalin methyl ester 242. Saponification of methyl ester 242 with NaOH in ethanol gave isobufalin 243.

Scheme 26. Synthesis of isobufalin from bufalin. Synthesis of bufotalin, cinobufagin, bufalitoxin, and bufotoxin from bufalin In order to provide a solution to the structural problem presented by bufotoxin and obtain sufficient amount of the substance for antineoplastic and other biological evaluation, Pettit’s group undertook (beginning in 1957) a series of interrelated synthetic investigations which have culminated in the formal total syntheses of toad venom constituents bufotalin 28, cinobufagin 5, bufalitoxin 245, and bufotoxin 249 [38] (Scheme 27). Bufalin 1 was employed as relay and converted to 14-dehydrobufalin 3-acetate 27. Selective oxidation of olefin 27 with a chromium trioxide-pyridine reagent afforded 16-ketone 246, which was previously transformed to bufotalin 28 [53] and thence to cinobufagin 5 [37]. Condensation of bufalin 1 with suberic anhydride followed by a mixed carbonic anhydride reaction sequence using arginine monohydrochloride yielded bufalitoxin 245, and an analogous route from bufotalin 28 led to bufotoxin 249.

19

Scheme 27. Synthesis of bufotalin, cinobufagin, bufalitoxin, and bufotoxin from bufalin. Synthesis of Bufotalien, 15α-Hydroxybufalin, and Resibufogenin from 14-dehydrobufalin In 1971, Pettit’s group synthesized bufotalien 251, 15α-hydroxybufalin 254 and 3β-acetoxyresibufogenin 50 from 3β-acetoxy-14-dehydrobufalin 27 [36] (Scheme 28). An extensive attempt to convert olefin 27 to diene 250 by means of sulfur dehydrogenation proved impractical, while mild treatment of 27 with NBS followed by pyridine-catalyzed dehydrohalogenation afforded 3β-acetoxybufotalien 250 successfully. Selective saponification of acetate 250 to bufotalien 251 was achieved using alumina. m-CPBA oxidation of 27 formed 14α,15α-epoxide 253, the mild aqueous sulfuric acid catalyzed opening of epoxide 253 afforded 15α-hydroxybufalin 254. Further diol 254 was treated with methanesulfonyl chloride, which provided a useful route to 3β-acetoxyresibufogenin 50. When 27 was treated with NBA in dioxane-water containing perchloric acid, bromohydrin 252 was obtained. When the crude bromohydrin was chromatographed on basic alumina, 3β-acetoxyresibufogenin 50 was obtained.

Scheme 28. Synthesis of bufotalien, 15α-hydroxybufalin, and resibufogenin from 14-dehydrobufalin. Synthesis of 15β-hydroxybufalin from 14-dehydrobufalin Since 15β-hydroxybufalin 257 may be a component of other toad venoms and possess potentially useful biological properties [54], in 1975, Pettit’s group developed a new synthesis of this substance based on their prior route to 15β-hydroxy digitoxigenin [19,55] (Scheme 29). Treatment of 14-dehydrobufalin 26 or its 3-acetate derivative 27 with iodine and silver acetate led to 15β-acetate 255 and 256. Next, an acid-catalyzed hydrolysis procedure was utilized to convert 20

the acetate derivatives 255 and 256 to 15β-hydroxybufalin 257.

Scheme 29. Synthesis of 15β-hydroxybufalin from 14-dehydrobufalin. 2.17 Other methods for the construction of pyrone ring Synthesis of 5β,14α-bufa-20,22-dienolide from cholanic acid In 1970, Sarel’s group reported the synthesis of 5β,14α-bufa-20,22-dienolide from a cholanic acid side-chain 24 which already contains the required carbon chain [33] (Scheme 30). Lactone 258 was prepared in a three-step synthesis from 24 and then converted into the methyl 20-hydroxycholanate 259 [56]. 259 was exposed to the action of POCl3 in dry pyridine at room temperature, the product consists of a mixture of 260 (cis and trans, 70%), 22% of 261-262 and 8% of 263. The oxidative cyclisation step 260→265 was achieved by applying the NBS reaction to the chol-20(22)-enic acid 264 rather than its ester 260, which afforded (57% yield) the hitherto unknown 5β,14α-bufa-20(22)-enolide 265. Finally, 265 was exposed to the action of 2,3-dichloro-5,6-dicyanoquinone (DDQ) in boiling dioxan containing p-TSA and the dehydrogenation was specific at C-21 and C-23, which provided 5β,14α-bufa-20,22-dienolide 266 in almost quantitative yield. Since the six-membered lactone ring was a key step in the synthesis of bufadienolides, this route represented an alternative synthetic pattern for the construction α-pyrone ring from a cholanic acid side-chain.

Scheme 30. Synthesis of 5β,14α-bufa-20,22-dienolide from cholanic acid. Synthesis of bufadienolide by palladium-catalysed coupling Since the first successful bufadienolide synthesis [14], many ingenious methods for the elaboration of steroidal 2H-pyran-2-ones have been described. These methods have all been based on a linear strategy, in which a suitable five-carbon chain is first built up at C-17, followed with cyclization and dehydrogenation (if necessary) to give the desired 2H-pyran-2-one. In 1996, Liu 21

and Meinwald reported a convergent strategy that an intact 2H-pyran-2-one ring joined directly to a variety of substrates, including steroids [57] (Scheme 31). It involved the palladium-catalysed coupling of 5-trimethylstannyl-2H-pyran-2-one 270 (prepared from the readily available 5-bromo-2H-pyran-2-one) with a 14β-hydroxy-16-ene-17-ol triflate steroid derivative 273 to yield a bufadienolide analogue 274 studied by Stille [58]. 274 was then deprotected prior to catalytic hydrogenation of the C16‒C17 double bond to give the bufadienolide 276. This methodology avoided the lengthy and inefficient conversion of functional groups and was conducive to expand more possible lead compounds. Nevertheless, lithium reagents, high temperature and oxygen free conditions were needed in the coupling, which constituted an obstacle to industrialization.

Scheme 31. Synthesis of bufadienolide by palladium-catalysed coupling. However, the use of highly toxic tin reagents was a serious concern when the possibility of in vivo testing was taken into account. Even though the desired steroids were subjected to several purification steps after the Stille coupling [58], organotin residues were particularly difficult to remove completely. Gravett et al. investigated the preparation of a suitable 2-pyrone boronate ester and studied the Suzuki–Miyaura coupling reaction of this compound to steroid vinyl triflates [59] (Scheme 32). Free radical dibromination of 2-pyrone 277 followed by base elimination of HBr afforded 5-bromo-2-pyrone 268. The coupling of pinacolborane 279 to 268 in the presence of catalytic PdCl2(PPh3)2 and NEt3 in toluene at reflux temperature led to the desired 2-pyrone-5-boronate 280. Coupling reactions between aryl boronates and triflates was successful with the use of PdCl2(dppf) catalyst and an inorganic base with K3PO4. Hydrogenation of the C16‒C17 double bond in 282 to give bufadienolides was achieved straightforwardly using the conditions reported by Meinwald and Liu [57]. This Pd(0)-mediated cross-coupling of boronates to a variety of steroid vinyl triflates was considered as an efficient method for preparing bufadienolides without involving toxic tin reagents, thus avoiding the risk of contamination of products with tin residues.

Scheme 32. Synthesis of bufadienolide by palladium-catalysed coupling. 3. Chemical structural modification of bufadienolides Currently, many experimental and clinical studies had suggested that bufadienolides had 22

significant anti-tumor activities towards different types of human cancers, such as breast cancer, nonsmall-cell lung cancer, liver cancer and gastric cancer [60]. However, bufadienolides as Na+/K+-ATPase inhibitors exhibited significant cardiotoxicity, which limited their clinical usage and drug development [61]. In recent years, numerous analogues of bufalin, cinobufagin and resibufogenin had been prepared by chemical synthesis or biological transformation [62,63] and the cytotoxicities of these bufadienolide analogues had been evaluated in vitro [64,65]. This article only reviewed the chemical structural modification of bufadienolides, the biological transformation of bufadienolides could be seen in a recent review [6]. 3.1 Chemical structural modification of resibufogenin Resibufogenin 3 was prepared from skin secretions of toads of the Bufo genus (venenum Bufonis), and it was reported to be cardiotoxic with a high mortality rate when used in the United States [66]. In 2012, Rice’s group achieved a greatly improved synthesis of 283 from 3 by acetic formic anhydride in pyridine and oxidation respectively [67] (Scheme 33). The (+)-20R,21R-compound 283 had been found to have high affinity as an IL-6 receptor antagonist, and over-activation of the IL-6 receptor was involved in the onset, development and poor prognosis of cancer [68,69].

Scheme 33. Chemical structural modification of resibufogenin. 3.2 Chemical structural modification of proscillaridin. The cardiac glycoside proscillaridin had been widely used in the treatment of congestive heart failure [70]. However, this drug showed very narrow concentration of positive inotropic effect (PIE) development and occasionally caused arrhythmia [71]. In 1982, Stache’s group obtained the 14,15β-epoxy-3β-hydroxy-l4β-bufa-4,20,22-trienolide 3-α-L-rhamnopyranoside 285 from proscillaridin A 284 by first protecting the sugar hydroxy groups as acetates, and then dehydrating the 14β-hydroxy group to the 14(15) double bond. Conversion of 14(15) double bond to the bromohydrin or iodohydrin led subsequently to the 14,15β-epoxy analog [72] (Scheme 34). At the isolated guinea pig’s heart, four times higher doses of 285 had the same positive inotropic activity as 284, and the same dose of 285 had the same potency as digoxin. The in vivo positive inotrope of 285 was similar to 284 and stronger than digoxin. Besides, 285 displayed inhibitory effect of its glycosides on the Na+/K+-ATPase from in vitro bovine brains.

23

Scheme 34. Chemical structural modification of proscillaridin. In 1987, Sakakibara et al. reported extensive chemical modifications of 284 and found that C22-C23 hydrogenated proscillaridin, 3β-[(6-deoxy-α-L-mannopyranosyl)oxy]-14β-hydroxybufa-4,20-dienolide 286, had a greatly expanded concentration range of PIE development on papillary muscles isolated from guinea-pig hearts [73,74] (Scheme 35). When 286 at the dose of 11.9 mg/kg, an equivalent dose of 4.4 times the pD2 values (The concentrations at which half maximum PIE just developed), was administrated intravenously by bolus injection into guinea pigs, no arrhythmias were observed on the electrocardiogram (ECG).

Scheme 35. Chemical structural modification of proscillaridin. In 1988, Sakakibara’s group modified proscillaridins with a higher margin of safety [75] (Scheme 36). Although the activities of proscillaridin derivatives 287, 288, 289 and 290 were less potent than that of 284, they showed slightly expanded concentration ranges of PIE development on guinea-pig papillary muscle preparations.

Scheme 36. Chemical structural modification of proscillaridin. In 1991, Sakakibara’s group investigated the Diels-Alder reactions of proscillaridin 284 with some dienophiles [76] (Scheme 37). Among the proscillaridin derivatives, compound 291 and 292 moderately inhibited Na+/K+-ATPase activity. Furthermore, the concentration range of 292, whose PIE on guinea-pig papillary muscle preparations increased from 5% to 95% of maximum, was broader than that of 284, exhibiting concentration dependency over a greater range. O

O O

COOCH3

O COOCH 3

OH OH O O CH3 OH OH

284

OH OH O O CH3

291

OH OH

OH OH O O CH3

292

OH OH

Scheme 37. Chemical structural modification of proscillaridin. In 1992, Sakakibara’s group synthesized a para-substituted benzylalcohol 293, methylbenzamides 294 and 296, and ethylbenzamides 295 and 297 of proscillaridin 284 [77] (Scheme 38). Although the biological activities of these derivatives were less potent than that of 284, they inhibited the activity of Na+/K+-ATPase almost as potently as naturally occurring cardiac glycosides such as digoxin and digitoxin.

24

Scheme 38. Chemical structural modification of proscillaridin. In order to reduce the vascular contracting effect of proscillaridin 284, in 1991, Sakakibara’s group prepared all kinds of its nitrates by utilizing an isopropylidene function effectively as a protective group [78] (Scheme 39). The PIE and Na+/K+-ATPase inhibitory activities of mononitrates (302,303,304) and dinitrates (299,300,301) were a little less potent than 284, but those of trinitrate 298 were much reduced. Every nitrate did not exhibit a vascular contracting effect but a relaxing effect. Among them, the vascular relaxing effects of 2′,3′-dinitrate 299 and 2′,4′-dinitrate 300 were more potent than those of the other nitrates.

Scheme 39. Chemical structural modification of proscillaridin. The guanidyl group of arginine had been reported to generate nitric oxide (NO) in vivo which acted as the endothelium-derived relaxing factor (EDRF) [79]. In 1994, Sakakibara’s group prepared new bufotoxin homologues 305 with various lengths of alkyl chain including longer ones than a suberoyl group at C-3 of the steroid nucleus from proscillaridin 284 via its genuine aglycone, scillarenin 178 [80] (Scheme 40). The bufotoxin homologues 305 showed only slight contraction of vascular muscle followed by a small relaxation, in addition to the high Na+/K+-ATPase inhibitory activity and PIE comparable to those of clinically used ouabain, digoxin and digitoxin.

Scheme 40. Chemical structural modification of proscillaridin. 3.3 Chemical structural modification of bufalin Bufalin was one of the most potent bufadienolides against cancer cells, such as human prostate carcinoma cells (PC3, DU145) [81], human leukemia cells (U937, HL60) [82,83] and human cervical carcinoma cells (HeLa) [84], with IC50 values of 10‒9 to 10‒8 mol/L. Furthermore, bufalin could inhibit the growth of human HepG2 cell-transplanted tumor in nude mice and prolong the survival of host significantly [85]. The mechanism of the anti-cancer effects of bufalin was still subject to study. Similar to other cardiac glycosides, bufalin could bind and inhibit the Na+/K+-ATPase [86,87], which increased the intracellular concentration of Ca2+ [(Ca2+) intra] and 25

then led to cardiotonic effects. Moreover, the effects of bufalin on Na+/K+ pump induced endoplasmic reticulum stress in cancer cells expressing Na+/K+-ATPase subunits and eventually caused cell death [88]. From the 1990s, a lot of bufalin analogues had been prepared by isolation, and chemical or biological transformation, and these compounds were evaluated in vitro and in vivo for their cytotoxicities [3,62,67,89,90]. The essential structural requirements of bufalin for increasing cytotoxicities had been indentified: a steroidal C/D cis ring juncturing with a C14β-hydroxyl group and a C17β-2-pyrone ring [91]. Unfortunately, these efforts had provided few new and more active bufalin derivatives. Therefore, new ideas and approaches were needed to extend investigations of the use of these bufadienolides as anticancer agents. Considering that structurally simpler polar substituents could be advantageously grafted on C3 of bufalin to increase activity with simultaneously increased solubility. N-Heterocycles were found in a variety of biologically active compounds and were easily to form water-soluble salts with organic or inorganic acids. In 2013, Hu’s group designed and synthesized a series of nitrogen-contained carbocycle esters [92] (Scheme 41). The structure–activity relationships of this new series revealed that C3 moiety had important influence on cytotoxic activity. On two cell lines, the bufalin-3-piperidinyl-4-carboxylate compound 306 (IC50 values on HeLa and A549 cell lines were 0.76 nM and 0.34 nM, respectively) displayed significant cytotoxic potency compared to the parent compound bufalin.

Scheme 41. Chemical structural modification of bufalin. Unfortunately, compound 306 was inactive in vivo. Considering that the ester was prone to metabolism in vivo because ester groups are susceptible to hydrolysis by esterases in the blood, Hu’s group designed bufalin-3-yl piperazine-1-carbamate by bioisosteric replacement, and the activity test indicated that compound 307 could maintain cytotoxic potency (IC50 (HeLa) = 0.77 nM) [93]. In order to find new compounds with better cytotoxic potency, Hu’s group designed and synthesized a series of bufalin-3-yl nitrogen-containing-carbamate derivatives in 2016, and evaluated for their proliferation inhibition activities. Among them, compound 308 (BF211) had a strong cytotoxic effect on various tumor cells with IC50 = 0.30–10.78 nM. Furthermore, BF211 significantly inhibited tumor growth by 100% at a dose of 2 mg/kg by i.v., or 4 mg/kg by i.g. (Scheme 41). Fibroblast activation protein α (FAPα) was a tumor-specific protease, which was observed on the surface of carcinoma-associated fibroblasts (CAFs) in the stroma of >90% solid tumors while generally absent from adjacent normal tissues and nonmalignant tumors [94]. FAPα, a type II integral membrane serine protease, was distinguished from other proteases in the dipeptidyl deptidase (DPP) subfamily, as its activity was restricted to substrates containing the N-terminal benzyloxycarbonyl blocked Gly-Pro (Z-GP) [95]. The specific proteolytic activity and highly tumor-restricted distribution of FAPα made it a very attractive target for the design of anticancer prodrugs. In 2017, Hu’s group used a FAPα-based prodrug strategy to synthesize a dipeptide 26

(Z-Gly-Pro)-conjugated BF211 prodrug named BF211-03 (309) to exclude the potential cardiotoxicity of BF211 [96] (Scheme 41). 309 was hydrolyzed by recombinant human FAPα (rhFAPα) and cleaved by homogenates of human colon cancer HCT-116 or human gastric cancer MGC-803 xenografts. In contrast, 309 showed good stability in plasma and in the homogenates of FAPα-negative normal tissues, such as heart and kidney. In HCT-116 tumor-bearing nude mice, doubling the dose of 309, compared with BF211, caused less weight loss and similar inhibitory effects on tumor growth. The results suggested that 309 was converted to active BF211 in tumor tissues and exhibited anti-tumor activities in tumor-bearing nude mice. FAPα-targeted 309 displayed tumor selectivity and might be a useful targeting agent to improve the safety profile of cytotoxic natural products in cancer therapy. Androgen receptor (AR) belonged to the steroid nuclear receptor super-family [97]. It was activated by endogenous androgens as testosterone and 5a-dihydrotestosterone (DHT) or exogenous compounds, and regulated genes for male differentiation and development. However, high levels of AR expression might lead to severe diseases like prostate cancer (PCa). Recently, AR was found to play a critical role in PCa since approximately 80–90% of PCa were androgen dependent at initial diagnosis [98]. Thus, it had become an attractive target for the treatment of PCa. Close examination of the structure of 1 revealed the similarity to those of steroidal AR antagonists, such as VN/85-1 [99]. Both of them possessed a steroidal skeleton with an unsaturated substitution at C-17. However, the steroid moiety of bufalin was saturated, which was in contrast to the presence of at least one double bond in the steroidal AR antagonists. Ye’s group hypothesized that introduction of a double bond in bufalin would increase the interactions with AR [100]. So, they synthesized compounds 310 and 26. The AR competitive binding assay indicated that the IC50 values of the three compounds were 1.9, >50 and >50 µM (relative binding affinity) respectively (Scheme 42). The active 310 was further tested for the cytotoxic activities on the androgen dependent PCa cells LNCaP and androgen independent cells PC3 with taxol served as the positive control. It was found that 310 showed more potent inhibitory activity against LNCaP cells with an IC50 value of 6.8 µM than PC3 cells with an IC50 value of 16.4 µM. The inhibitory activity of 310 on Na+/K+-ATPase was also explored. It was found that 310 showed much weaker inhibition with an IC50 value of 5.6 µM than bufalin (1, IC50 value of 0.022 µM), indicating two hundred folds decrease of inhibition on Na+/K+-ATPase. Because the lactone moiety was the most important for targeting the Na+/K+-ATPase, Jiang’s group hypothesised that the modification in the lactone moiety would lead to changed potency [101]. They synthesized two bufalin derivatives, bufadienolactam 311 and secobufalinamide 312, by treating bufalin with ammonium acetate in DMF solution (Scheme 42). Compound 311 and 312 expressed strong inhibitory activities against androgen-dependent PCa cells (IC50 values about 10 µM), but only weak inhibition on Na+/K+-ATPase (Ki about 70 µM), indicating that they might be potential anti-PCa agents without severe cardiac toxicity.

27

Scheme 42. Chemical structural modification of bufalin. In 2014, Jiang’s group carried out the simultaneous modifications on the lactone moiety and 14-hydoxyl group, and prepared ∆14,15-anhydro-24-thiocarbonylbufalin 313 by the reaction of natural product bufalin with Lawesson reagent [102] (Scheme 42). The inhibition on Na+/K+-ATPase property of 313 and the parent compound bufalin were compared. Compound 313 showed weak inhibition on Na+/K+-ATPase with IC50 value of 35.8 µM, while the parent compound bufalin showed potent inhibition with IC50 value of 0.011 µM, indicating that replacement of the carbonyl group with a thiocarbonyl group and dehydration at C-14 significantly decreased the inhibition on the Na+/K+-ATPase. Tumor-specific protease-activated prodrugs were a promising approach to reduction of the toxicity of anticancer agents [103]. In this prodrug strategy, coupling of the peptides to the toxic anticancer agents was accomplished to generate a low or nontoxic prodrug, which was a specific substrate of enzymes known to be expressed in tumor tissues. This prodrug could be activated by tumor specific proteases to kill tumor cells, while it was inactive in normal tissues. In 2016, Qin’s group employed esterase-sensitive β-thioester bond to link water soluble and biocompatible PEG with the 3α-hydroxyl group of BUF to fabricate PEGylated polymeric prodrug of BUF, PEGS-BUF 314, whose water solubility was good with unaffected anticancer activity [104] (Scheme 43). PEGS-BUF exhibited little drug release at neutral conditions without esterase. While in the presence of esterase, β-thioester bonds hydrolysed quickly, leading to fast drug release. The water solubility and the stability of PEGS-BUF improved dramatically compared with that of small molecular BUF. Besides, PEGS-BUF exhibited comparable anticancer performance in comparison with that of free BUF both in vitro and in vivo. O O

O

H H HO

H C3H7 O S

O

H

HO

H O

113

HO H

HO

H

Esterase

H

Active BUF

O OH

PEGS-BUF 314

H 1

O

O

O PEG

OH 113

HO

O C3H7 O S

O

OH O -COOH

Scheme 43. Chemical structural modification of bufalin. 3.4 FAPα-activated tripeptide arenobufagin prodrug Motivated by reducing or avoiding the cardiac toxicity of bufadienolides, in 2017, Ye’s group designed and synthesized the FAPα activated tripeptide arenobufagin prodrugs with the 28

purpose of improving the safety of arenobufagin 315 [105] (Scheme 44). 316 exhibited the best hydrolytic efficiency by rhFAPα and was activated in tumors. The LD50 of 316 was 6.5-fold higher than that of arenobufagin. There were not apparent changes in echocardiography, pathological section of cardiac muscle, and the lactate dehydrogenase activities (LDH) in 316 treatment tumor-bearing mice, even when the dose reached 3 times the amount of parent drug arenobufagin that was used. Compound 316 also exhibited significant antitumor activity in vitro and in vivo. The improved safety profile and favorable anticancer properties of 316 warranted further studies of the potential clinical implications.

Scheme 44. FAPα-activated tripeptide arenobufagin prodrug. 4. Structure–activity relationship of bufadienolides Bufadienolides were an important group of polyhydroxy C-24 steroids and their glycosides. They were characterised by the presence of a six-membered lactone (α-pyrone) ring located at position C-17β. Since the isolation of the first bufadienolide, scillaren A, from Egyptian squill in 1933 [106], this family of compounds had attracted the attention of scientists to isolate numerous bufadienolides with various chemical structures and diverse biological activities. The structures and conformations of bufadienolides played an important role in determining their Na+/K+-ATPase inhibitory potency [107], it had been concluded that the sugar moiety markedly contributed to the tumor specificity [8]. The 5β,14β-androstane-3β,14β-diol containing the 17β-lactone was the optimal steroid nucleus for bufadienolides to induce the Na+/K+-ATPase inhibition. Conversion of C14-C15 double bond to the 14,15β-epoxy ring, and hydrogenation the C22-C23 double bond could decrease the positive inotropic activity [72,74]. The C3 moiety was of great significance for cytotoxic activity (BF211), where the introduction of carbamate groups annihilated a variety of tumor cells [92,93]. The modification of the lactone moiety into lactam ring showed strong inhibitory activities against androgen-dependent PCa cells while only weak inhibition on Na+/K+-ATPase [101]. Replacement of the carbonyl group with a thiocarbonyl group in lactone ring and dehydration at C-14 significantly could also decrease the inhibition on the Na+/K+-ATPase effect [102]. Furthermore, the tumor-specific protease-activated prodrugs were a promising approach to reduction of the toxicity of bufadienolides. FAPα-targeted prodrugs displayed tumor selectivity and might be useful as a targeting agent to improve the safety profile of bufadienolides for use in cancer therapy [96,104,105]. 5. Concluding remarks As a kind of cardiac glycosides, bufadienolides showed inhibitory activity against Na+/K+ pump [86,87]. In addition to its effects on cardiomyocytes, bufadienolides could kill tumor cells dependent on Na+/K+-ATPase [88]. In order to reduce the toxicity or cardiotoxicity of bufadienolides, some bufadienolide derivatives were designed and synthesized as anticancer agents through a tumor-specific protease-activated prodrugs approach. This strategy allowed the 29

low or nontoxic prodrug to be a specific substrate of enzymes known to be expressed in tumor tissues, which could be activated by tumor specific proteases to kill tumor cells, while it was inactive in normal tissues. Daniel and co-authors reported selective antitumor activity towards malignant T lymphoblasts induced by hellebrin derivatives that lacked cardioactive groups in contrast to normal peripheral blood mononuclear cells [108]. This study suggested the possibility of obtaining potent anticancer bufadienolides with low or even no cardiotoxic effects. There has been a longstanding interest in developing efficient methodologies for the synthesis of cardiotonic steroids to make available structural analogues with an improved therapeutic index. Behind such efforts a realistic background seems to exist. In the rats, two different types of cardiac glycoside receptors mediating positive inotropy and toxicity have been identified. If two types of receptors could also be distinguished in human heart, more specific and saver drugs could be developed hopefully [109]. This approach provides new hope for the bufadienolides to reduce their cardiotoxicity. 6. Acknowledgement This work was financially supported by National Natural Science Foundation of China (81973171), Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, Project Funded by the Flagship Major Development of Jiangsu Higher Education Institutions (PPZY2015A070), Key Laboratory of Therapeutic Material of Chinese Medicine, Jiangsu Province, State Key Laboratory Cultivation Base for TCM Quality and Efficacy, Nanjing University of Chinese Medicine. 7. References [1] M. Michalak, K. Michalak, J. Wicha, The synthesis of cardenolide and bufadienolide aglycones, and related steroids bearing a heterocyclic subunit, Nat. Prod. Rep. 34 (2017) 361−410. [2] P. Zhang, Z. Cui, Y. Liu, D. Wang, N. Liu, M. Yoshikawa, Quality evaluation of traditional Chinese drug toad venom from different origins through a simultaneous determination of bufogenins and indole alkaloids by HPLC, Chem. Pharm. Bull. 53 (2005) 1582−1586. [3] T. Nogawa, Y. Kamano, A. Yamashita, G.R. Pettit, Isolation and structure of five new cancer cell growth inhibitory bufadienolides from the Chinese traditional drug Ch'an Su, J. Nat. Prod. 64 (2001) 1148−1152. [4] L. Krenn, B. Kopp, Bufadienolides from animal and plant sources, Phytochemistry 48 (1998) 1−29. [5] P.S. Steyn, F.R. van Heerden, Bufadienolides of plant and animal origin, Nat. Prod. Rep. 15 (1998) 397−413. [6] H. Gao, R. Popescu, B. Kopp, Z. Wang, Bufadienolides and their antitumor activity, Nat. Prod. Rep. 28 (2011) 953−969. [7] A. Kamboj, A. Rathour, M. Kaur, Bufadienolides and their medicinal utility: A review, Int. J. Pharm. Pharm. Sci. 5 (2013) 20−27. [8] K. Watanabe, Y. Mimaki, H. Sakagami, Y. Sashida, Bufadienolide and spirostanol glycosides from the rhizomes of helleborusorientalis, J. Nat. Prod. 66 (2003) 236−241. [9] T. Mijatovic, R. Kiss, Cardiotonic steroids-mediated Na+/K+-ATPase targeting could circumvent various chemoresistance pathways, Planta. Med. 79 (2013) 189–198. 30

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Bufadienolides, originally isolated from Chan’Su, are a type of natural heart-active steroids that have traditionally been used to treat heart disease. The bufadienolides are widely considered as effective agents for cardiac, diuretic, anti-tumor and local anesthesia.

The unique molecular chemical structures and numerous biological activities of the bufadienolides attracted the interest of scientists. Related researches of bufadienolides have been summarized to provide references for the future research on novel bufadienolides with better biological activities and reduced toxicity.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: