Bioorganic & Medicinal Chemistry 23 (2015) 1437–1446

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Synthesis and antitumor activity of novel per-butyrylated glycosides of podophyllotoxin and its derivatives Cheng-Ting Zi a,b, Dan Yang b, Fa-Wu Dong b, Gen-Tao Li b, Yan Li b, Zhong-Tao Ding a, Jun Zhou b, Zi-Hua Jiang c,⇑, Jiang-Miao Hu b,⇑ a b c

Key Laboratory of Medicinal Chemistry for Natural Resource, Yunnan University, Kunming 650091, China State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China Department of Chemistry, Lakehead University, 955 Oliver Road, Thunder Bay, ON P7B 5E1, Canada

a r t i c l e

i n f o

Article history: Received 25 November 2014 Revised 31 January 2015 Accepted 11 February 2015 Available online 24 February 2015 Keywords: Podophyllotoxin Epipodophyllotoxin 40 -Demethylepipodophyllotoxin Butyrylated glycosides Antitumor Cytotoxic Synthesis

a b s t r a c t A series of perbutyrylated glycosides of podophyllotoxin and its derivatives were synthesized and evaluated for their antitumor activity in vitro. Most of them exhibit cytotoxic activity against a panel of five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, SW480) using MTT assays. Among the synthesized compounds, epipodophyllotoxin a-D-galactopyranoside 8b, epipodophyllotoxin a-D-arabinopyranoside 8e, and podophyllotoxin b-D-glucopyranoside 11a show the highest potency of anticancer activity with their IC50 values ranging from 0.14 to 1.69 lM. Structure activity relationship analysis indicates that the type of glycosidic linkage, the configuration at C-4 of the podophyllotoxin scaffold, and the substitution at 40 -position (OH vs OCH3) can all have significant effect on the potency of their anticancer activity. Several compounds are more active than the control drugs Etoposide and Cisplatin, suggesting their potential as anticancer agents for further development. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Podophyllotoxin (1, Fig. 1), extracted from the roots and rhizomes of podophyllum species such as Podohyllum hexandrum and Podophyllum peltatum,1 shows strong cytotoxic activity against various cancer cell lines. Due to its complicated side effects such as nausea, vomiting and damage of normal tissues, attempts to use podophyllotoxin in the treatment of human neoplasia have been mostly unsuccessful.2 Several podophyllotoxin derivatives such as Etoposide (2, Fig. 1) and Etopophos (3, Fig. 1) have been developed for clinical use as antineoplastic agents. Etoposide and Etopophos function as inhibitors of the enzyme DNA-topoisomerase II by stabilizing a cleavable complex in which the DNA is cleaved and covalently linked to the enzyme.3 Previous structure–activity relationship (SAR) studies have shown that intact A ring, the trans-lactone and 40 -demethyl moieties are essential to maintain the anticancer activity as topoisomerase II inhibitors.5–7 More recently, diverse podophyllotoxin analogues have been synthesized in an

⇑ Corresponding authors. Tel.: +1 807 766 7171; fax: +1 807 346 7775 (Z.-H.J.); tel.: +86 871 6522 3264; fax: +86 871 6522 3261 (J.-M.H.). E-mail addresses: [email protected] (Z.-H. Jiang), [email protected] (J.-M. Hu). http://dx.doi.org/10.1016/j.bmc.2015.02.021 0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

effort to discover novel anticancer agents with improved therapeutic efficacy and to overcome drug resistance.4,8–11 Preparation of glycoconjugates of small molecule anticancer drugs has become an attractive strategy in recent years in order to improve drug efficacy and pharmacokinetics, and reduce side effects.12–14 Both Etoposide (2) and Etopophos (3), the two anticancer drugs widely prescribed in the clinic, are derivatives of b-D-glucopyranoside of 40 -demethylepipodophyllotoxin. Besides b-D-glucopyranosides, other types of podophyllotoxin glycosides are quite limited.15–20 For example, a-D-glucoside, a-D-galactoside, and D-mannoside are still not known for podophyllotoxin and its derivatives. Thus, in the present study we plan to synthesize a series of glycoconjugates of podophyllotoxin, epipodophyllotoxin, and 40 -demethylepipodophyllotoxin combined with a number of different sugars. Short chain fatty acid (SCFA)-derivatized 2-deoxy-2-acetamidoD-mannose (ManNAc) analogs such as O-butyrylated ones were originally designed to increase the lipophilicity and cellular uptake of ManNAc in modifying cell surface carbohydrates in living cells. Some of the butyrylated ManNAc analogs also showed considerable anti-cancer effects including the induction of apoptosis, inhibition of NF-jB, and suppression of proinvasive oncogenes.21–23 Earlier studies suggested that the anticancer activity of these butyrylated ManNAc analogs may be partly contributed by the

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C.-T. Zi et al. / Bioorg. Med. Chem. 23 (2015) 1437–1446 OBut O O O A O

5

OH 3 2

1 8

O

But = CH 3CH 2CH2 CO O

N

3

N

O

12

N

O

O

O

O

O O

E H3 CO

O

O

D O

1'

O ButO

OH

11

4

ButO ButO

O

HO

OCH3 4' OCH3

H3 CO

O

OCH3 OR H 3CO

2 R = H Etoposide 3 R = P(O)(OH)2 Etopophos

1 Podophyllotoxin

OCH 3 OH 4

Figure 1. Structures of podophyllotoxin (1) and its semisynthetic derivatives (2–4).

butyrate species released by these compounds inside the cell.22 Butyrate is a well known histone deacetylase (HDAC) inhibitor and its anticancer effect shows promising therapeutic potential.24 Previously, we reported the synthesis and anticancer property of a group of glucosylated podophyllotoxin derivatives linked via 4b-triazole rings (e.g., 4 in Fig. 1).25 We found that derivatives with per-butyrylated glucose residues generally displayed higher anticancer activity than their counterparts carrying free glucose residues or per-acetylated glucose residues. Here in this paper, we report the synthesis and in vitro anticancer activity of a series of perbutyrylated glycosides of podophyllotoxin and its derivatives generated from D-glucose, D-galactose, D-mannose, L-rhamnose, Darabinose, maltose and lactose.

Direct glycosylation of podophyllotoxin (1) with 7a–7g under the treatment of trifluoroboron etherate (BF3Et2O)28,29 resulted in the formation of glycosylated epipodophyllotoxin 8a–8g in good yield (Scheme 2). In each case, the reaction stereoselectively produced the a-glycoside as the major product; other minor products were detected on TLC but not purified for structure determination. Similarly, the reaction of 40 -demethylepipodophyllotoxin 532 with 7a–7g produced the corresponding a-glycoside 9a–9g in a stereoselective manner in good yield (Scheme 2). As illustrated in Scheme 2, the glycosylation of both podophyllotoxin 1 and 40 -demethylepipodophyllotoxin 5 with 7a–7g all resulted in the formation of the major products (8a–8g and 9a– 9g) having an a-glycosidic linkage and a b-configuration at C-4 of the aglycone. The glycosylation reaction was supposed to undergo through a benzylic carbocation intermediate.28,29 As an example, the reaction mechanism of 7c with podophyllotoxin (1) is depicted in Scheme 3. Under the treatment of BF3OEt2, podophyllotoxin (1) is converted to a secondary benzylic carbocation (13) which is attacked by the sugar alcohol 7c. Compound 7c exists as an anomeric mixture (ab ratio = 10:1) with 7c–a being the major nucleophile present, leading to the formation of the a-glycoside as the major glycosylated product. The nucleophilic attack of 13 by 7c–a occurs preferably from the top face (b-face) due to the presence of the bulky aryl group at the lower face at C-1, resulting in the formation of glycoside 8c as the major product which has a b-configuration at C-4 of the aglycone. Nucleophilic attack of 13 by the minor b-anomer 7c–b would yield the b-glycoside 8c–I while the attack from the lower face (a-face) at C-4 of the aglycone by either 7c–b or 7c–a would produce 8c–II or 8c–III, respectively. Glycosides 8c–I, 8c–II, and 8c–III probably all formed in this reaction, albeit at a much lower rate, as indicated by the presence of multiple minor products detected on TLC.

2. Results and discussion 2.1. Chemical synthesis There have been a few methods reported for constructing the glycosidic linkages of podophyllotoxin glycosides and their analogues by using glycosyl iodide donors,26 and silyl glycoside donors.27 Here we are adopting the previously reported strategy by reacting the aglycone with the anomeric OH group of sugars.28,29 O-Butyryl protected sugar building blocks with a free anomeric OH group 7a–7g were prepared from the corresponding free sugars: D-glucose (6a), D-galactose (6b), D-mannose (6c), Lrhamnose (6d), D-arabinose (6e), maltose (6f) and lactose (6g) (Scheme 1). Butyrylation with butyric anhydride through iodinepromoted Lewis acid-catalyzation followed by treatment with 25% ammonia solution in acetonitrile afforded 7a–7g as an anomeric mixture in 56–68% yield.30,31

O HO

O

i, ii

ButO

OH

OH

O But

ButO

OBut ButO

O

O

ButO ButO

ButO ButO

ButO ButO

But = CH 3CH2 CH 2CO

7a - 7g

6a - 6g

ButO 7b

OH

7a

OH

OH

OBut O

O

ButO ButO

7c

OBut

OH 7d

OBut

ButO ButO

OBut O

7e

ButO ButO OH

O ButO

OBut

ButO O ButO 7f

OBut

O

O ButO

OBut

ButO

ButO

O

O ButO

ButO

OH

OH

7g

Scheme 1. Reagents and conditions: (i) (CH3CH2CH2CO)2O, I2, 0–5 °C, 1 h, 100%; (ii) NH3, CH3CN, rt, overnight, 56–68%.

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C.-T. Zi et al. / Bioorg. Med. Chem. 23 (2015) 1437–1446 R1

R1 O

O

i

O

ButO

O O

O

OH

O

O

O

O

7a - 7g H 3CO

H 3CO

O CH 3

O CH 3 OR

OR 1 R1 = α -OH, R = CH 3 5 R1 = β -OH, R = H R1 =

OBut

OBut

ButO ButO

ButO a

OBut O

ButO ButO ButO

O

O

ButO ButO

8a - 8g R = CH3 9a - 9g R = H

O

ButO ButO

ButO b

OBut d

c

O But

ButO ButO

OBut O

O

ButO ButO

e

OBut

O

O

O ButO

OBut

ButO

OBut ButO

ButO ButO

O

O ButO

ButO

ButO g

f But = CH 3CH2 CH 2CO

Scheme 2. Reagents and conditions: (i) BF3Et2O, CH2Cl2, 78 °C to rt, 57–72%.

OBut O

ButO ButO ButO

ButO ButO ButO

OH

7c-α OH

7c- β H OH O Ar 1

Ar =

O

ButO ButO ButO

OBut O O

-H

O

O Ar 12

H3 CO

ButO ButO ButO

attack from top face

BF3

BF3 .Et2 O

O

OBut O

O

O

Ar

O

OBut O

ButO ButO ButO

O

ButO ButO ButO

OBut O O

OBut O O

OCH3 OCH 3

But = CH 3CH2 CH2 CO

O

O Ar

O

Ar 8c

13

O

Ar

O

O

Ar 8c-ΙΙΙ

8c-II

8c-Ι

O

Scheme 3. Plausible mechanism for the formation of 8c as the major glycosylation product.

Since the glycosylation of podophyllotoxin with anomeric free sugar alcohol (7a–7g) only provided a-glycosides of epipodophyllotoxin with a b-configuration at C4, we decided to use trichloroacetimidates as glycosylation donors to access podophyllotoxin b-glycosides. Thus, trichloroacetimidates 10a/10b were readily prepared from their corresponding 1-OH compound 7a/ 7b by treatment with trichloroacetonitrile (CCl3CN) and the catalytic amount of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (Scheme 4).33 Glycosylation reaction of imidates 10a/10b with podophyllotoxin (1) in the presence of BF3Et2O as the catalyst at 78 °C to room temperature furnished the corresponding podophyllotoxin b-glycosides 11a/11b in 60% and 68% yield, respectively (Scheme 5). All the synthesized compounds were characterized by 1H NMR, 13 C NMR, ESI–MS, and HRESI–MS. The characteristic 1H NMR and 13 C NMR data of compounds 8a–8g, 9a–9g, 11a, and 11b are shown

OBut 7a/7b

i

ButO ButO

ButO

ButO

OBut O

O NH O

10a

CCl3

ButO ButO 10b

NH O CCl3

But = CH3 CH 2 CH 2CO

Scheme 4. Reagents and conditions: (i) CCl3CN, DBU, CH2Cl2, 0 °C, 67% for 10a and 85% for 10b.

in Table 1. In the 1H NMR spectra, the proton at C-4 of the aglycone occurs at d 4.90–4.59 ppm, usually with a coupling constant J3,4 40 5.32 0.60 >40 >40 16.87 4.89 2.71 2.54 26.49 >40 9.59 0.61 3.15 0.31 1.17

>40 0.62 >40 15.21 0.78 >40 >40 16.82 3.78 3.84 2.68 17.10 >40 20.24 0.83 18.09 8.12 6.43

>40 0.61 >40 7.62 0.61 >40 >40 16.04 5.70 3.10 3.52 23.15 >40 18.64 1.12 17.81 11.92 9.24

>40 1.27 >40 13.48 1.42 >40 >40 39.13 5.67 7.50 4.71 29.32 >40 >40 1.69 22.11 32.82 15.86

>40 1.65 >40 16.83 1.11 >40 >40 38.71 10.65 4.85 5.05 >40 >40 >40 1.26 26.52 17.11 13.42

Some 40 -demethylepipodophyllotoxin glycosides (9a, 9c and 9d) show higher activity than their corresponding epipodophyllotoxin glycosides (8a, 8c and 8d) while other 40 -demethylepipodophyllotoxin glycosides (9b and 9e) show lower activity than their counterparts (8b and 8e). These data indicate that the 40 -demethylation of epipodophyllotoxin glycosides of the present study may lead to significant increase or decrease of anticancer activity. For example, 9e is up to 44 times less active against HL60 cancer cells than 8e, while 9c is at least 15 times more active against HL-60 cancer cells than 8c. Interestingly, b-D-glucopyranoside 11a with the normal podophyllotoxin as the aglycone is highly potent with IC50 values in the range of 0.61–1.69 lM against all five cancer cell lines, which is much more active than epipodophyllotoxin a-D-glucopyranoside 8a with IC50 values >40 lM against all five cancer cell lines. However, this trend of increase in anticancer activity is not observed for D-galactosederived glycosides between compounds 11b and 8b. In fact, podophyllotoxin b-D-galactopyranoside 11b has significantly lower activity than epipodophyllotoxin a-D-galactopyranoside 8b. These data suggest that the type of glycosidic linkage (a vs b) as well as the configuration at C-4 of the podophyllotoxin scaffold (a vs b) may have significant effect on the anticancer potency of these glycosides. 3. Conclusions In summary, a series of novel per-butyrylated glycosides of podophyllotoxin and its derivatives have been synthesized and assessed for their anticancer activity against a panel of five human cancer cell lines including HL-60 (leukemia), SMMC-7721 (hepatoma), A-549 (lung cancer), MCF-7 (breast cancer), and SW480 (colon cancer). Most compounds exhibit good cytotoxicity against all five cancer cell lines evaluated and several of them are more active than the control drugs Etoposide and Cisplatin. Epipodophyllotoxin a-D-galactopyranoside 8b, epipodophyllotoxin a-D-arabinopyranoside 8e, and podophyllotoxin b-D-glucopyranoside 11a are the most active compounds of this study with IC50 values ranging from 0.14 to 1.69 lM. Structure activity relationship analysis reveals that the type of glycosidic linkage, the configuration at C-4 of podophyllotoxin scaffold, and the substitution at 40 -position (OH vs OCH3) can all have significant effect on the potency of their anticancer activity. Our results suggest that some of these compounds have potential for further development as anticancer agents.

D-Glucose, D-galactose, maltose and lactose were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). D-Mannose was purchased from Acros Organics (New Jersey, USA). L-Rhamnose and D-arabinose were obtained from Aladdin Chemical Co., Ltd (Guangzhou, China). Podophyllotoxin was obtained from Shanghai Yuanye Bio-Technology Co., Ltd (Shanghai, China). Melting points were uncorrected. MS data were obtained in the ESI mode on API Qstar Pulsar instrument. HRMS data were obtained in the ESI mode on LCMS-IT-TOF (Shimadzu, Kyoto, Japan). NMR spectra were acquired on Bruker AV-400 or DRX500 or Bruker AVANCE I-600 (Bruker BioSpin GmbH, Rheinstetten, Germany) instruments, using tetramethylsilane (TMS) as an internal standard. Column chromatography (CC) was performed on flash silica gel (200–300 mesh; Qingdao Makall Group Co., Ltd; Qingdao; China). All reactions were monitored using thin-layer chromatography (TLC) on silica gel plates.

4.2. General procedure for the synthesis of compounds 7a–7g The sugar 6a–6g (10.0 mmol) was suspended in butyric anhydride (7.5 mL, 46 mmol) and stirred at 0–5 °C. Iodine (75 mg) was added and the stirring was continued for 1 h. The reaction mixture was diluted with CH2Cl2 (30 mL) and washed successively with aqueous saturated sodium thiosulphate (Na2S2O3) and aqueous saturated sodium bicarbonate (NaHCO3) solutions. The organic layer was then dried with sodium sulphate (Na2SO4) and concentrated in vacuo to give the per-butyrylated product. The per-butyrylated product was dissolved in acetonitrile (20 mL) and 25% ammonia solution (0.4 mL, 20 mmol) was added dropwise slowly. The mixture was stirred stirring at room temperature for 6 h. The solvent was evaporated and the residue was purified by column chromatography (silica gel, petroleum ether 60–90 °C:ethyl acetate = 4:1) to afford the product 7a–7g (56–68%). 4.2.1. 2,3,4,6-Tetra-O-butyryl-a/b-D-glucopyranose (7a) Yield: 60%. a/b ratio = 3/1. 1H NMR (CDCl3, 400 MHz) d 5.54 (t, 3/4H, J = 10.0 Hz, C3-Ha), 5.42 (d, 3/4H, J = 4.0 Hz, C1-Ha), 5.25 (t, 1/4H, J = 10.0 Hz, C3-Hb), 5.11–5.06 (m, 1H, C4-H), 4.85 (m, 1H, C2-H), 4.70 (d, 1/4H, J = 8.0 Hz, C1-Hb), 4.24–4.17 (m, 1H), 4.15– 4.12 (m, 2H, C6-CH2), 3.70 (br s, 1H, OH), 2.30–2.18 (m, 8H, 4  COCH2), 1.64–1.52 (m, 8H, 4  CH2CH3); 0.91–0.86 (m, 12H, 4  CH2CH3); 13C NMR (CDCl3, 100 MHz) d 173.5 (C@O), 172.8 (C@O), 172.7 (C@O), 172.1 (C@O), 95.6 (C-1b), 90.2 (C-1a), 73.0, 72.2, 71.7, 71.0, 69.4, 68.1, 67.3, 61.7 (C-6), 36.0 (COCH2), 35.8 (COCH2), 35.8 (COCH2), 35.6 (COCH2), 18.3 (CH2CH3), 18.2 (CH2CH3), 18.2 (CH2CH3), 18.1 (CH2CH3), 13.6 (CH2CH3), 13.5 (CH2CH3), 13.5 (CH2CH3), 13.5 (CH2CH3); ESIMS: m/z 483 [M+Na]+. 4.2.2. 2,3,4,6-Tetra-O-butyryl-a/b-D-galactopyranose (7b) Yield: 65%. a/b ratio = 2/1. 1H NMR (CDCl3, 400 MHz) d 5.46– 5.40 (m, 2H, C3-H, C4-H), 5.39 (d, 2/3H, J = 3.2 Hz, C1-Ha), 5.12 (m, 1H, C2-H), 5.06 (d, 1/3H, J = 6.1 Hz, C1-Hb), 4.69–4.67 (m, 1H), 4.44 (t, 1H, J = 6.8 Hz), 4.05–4.03 (m, 2H), 3.70 (br s, 1H, OH), 2.34–2.15 (m, 8H, 4  COCH2), 1.65–1.55 (m, 8H, 4  CH2CH3), 0.94–0.86 (m, 12H, 4  CH2CH3); 13C NMR (CDCl3, 100 MHz) d 173.7 (C@O), 173.3 (C@O), 173.2 (C@O), 173.1 (C@O), 172.8 (C@O), 172.7 (C@O), 172.6 (C@O), 172.5 (C@O), 95.9 (C-1b), 90.7 (C-1a), 70.9, 70.7, 70.2, 68.2, 67.9, 67.1, 66.8, 66.1, 61.4 (C-6a), 61.1 (C-6b), 36.0 (COCH2), 35.9 (COCH2), 35.8 (COCH2), 35.7 (COCH2), 18.4 (CH2CH3), 18.3 (CH2CH3), 18.2 (CH2CH3), 18.0

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C.-T. Zi et al. / Bioorg. Med. Chem. 23 (2015) 1437–1446

(CH2CH3), 13.5 (CH2CH3), 13.5 (CH2CH3), 13.5 (CH2CH3), 13.4 (CH2CH3); ESIMS: m/z 483 [M+Na]+. 4.2.3. 2,3,4,6-Tetra-O-butyryl-a/b-D-mannopyranose (7c) Yield: 56%. a/b ratio = 10/1. 1H NMR (CDCl3, 400 MHz) for the a-isomer: d 5.39–5.32 (m, 2H, C3-H, C4-H), 5.23 (dd, 1H, J = 1.6 Hz, 4.0 Hz, C2-H), 5.16 (d, 1H, J = 1.6 Hz, C1-H), 4.19–4.13 (m, 3H, C5-H, C6-CH2), 3.88 (br s, 1H, OH), 2.34–2.15 (m, 8H, 4  COCH2), 1.67–1.50 (m, 8H, 4  CH2CH3), 0.93–0.84 (m, 12H, 4  CH2CH3); 13C NMR (CDCl3, 100 MHz) d 173.6 (C@O), 172.8 (C@O), 172.7 (C@O), 172.3 (C@O), 92.8 (C-1b), 92.1 (C-1a), 72.3, 71.0, 69.9, 69.8, 68.8, 68.3, 65.6, 65.1, 62.1, 36.0 (COCH2), 35.9 (COCH2), 35.9 (COCH2), 35.7 (COCH2), 18.4 (CH2CH3), 18.3 (CH2CH3), 18.0 (CH2CH3), 18.0 (CH2CH3), 13.6 (CH2CH3), 13.5 (CH2CH3), 13.5 (CH2CH3), 13.4 (CH2CH3); ESIMS: m/z 483 [M+Na]+; ESIMS: m/z 483 [M+Na]+.

0

(m, 7/5H, C1 -H, C1-H), 5.21 (t, 2/5H, J = 10.0 Hz, C3-H), 5.10–5.04 0 0 (m, 1H, C3 -H), 4.92 (dd, 1H, J = 4.0 Hz, 10.0 Hz, C2 -H), 4.75 (m, 2 1 1H, C -H), 4.67 (d, 3/5H, J = 8.0 Hz, C -Hb), 4.49–4.41 (m, 2H), 4.11–4.07 (m, 2H), 3.84–3.71 (m, 2H), 2.35–2.09 (m, 14H, 7  COCH2) 1.68–1.48 (m, 14H, 7  CH2CH3), 0.95–0.84 (m, 21H, 7  CH2CH3); 13C NMR (CDCl3, 100 MHz) d 173.1 (C@O), 173.0 (C@O), 172.9 (C@O), 172.8 (C@O), 172.7 (C@O), 172.6 (C@O), 172.5 (C@O), 172.2 (C@O), 171.6 (C@O), 171.5 (C@O), 100.8 (C10 ), 95.3 (C-1b), 90.0 (C-1a), 75.8, 72.6, 71.1, 70.9, 70.7, 68.8, 68.7, 68.2, 66.4, 61.4 (C-60 ), 60.7 (C-6), 36.0 (COCH2), 35.9 (COCH2), 35.9 (COCH2), 35.8 (COCH2), 35.8 (COCH2), 35.7 (COCH2), 37.5 (COCH2), 18.4 (CH2CH3), 18.4 (CH2CH3), 18.3 (CH2CH3), 18.2 (CH2CH3), 18.1 (CH2CH3), 18.1 (CH2CH3), 17.9 (CH2CH3), 13.6 (CH2CH3), 13.5 (CH2CH3), 13.5 (CH2CH3), 13.5 (CH2CH3), 13.5 (CH2CH3), 13.5 (CH2CH3), 13.5 (CH2CH3); ESIMS: m/z 855 [M+Na]+. 4.3. General method for the synthesis of compounds 8a–8g

4.2.4. 2,3,4-Tri-O-butyryl-a/b-L-rhamnopyranose (7d) Yield: 57%. a/b ratio = 9/1. 1H NMR (CDCl3, 400 MHz) d 5.32 (dd, 1H, J = 4.0 Hz, 10.0 Hz, C3-H), 5.22–5.21 (m, 1H, C4-H), 5.09–5.07 (m, 1H, C2-H), 5.04 (s, 9/10H, C1-Ha), 4.98 (m, 1H, C3-H), 4.12– 4.07 (m, 1H), 2.35–2.11 (m, 6H, 3  COCH2), 1.65–1.48 (m, 6H, 3  CH2CH3), 1.15 (d, 3H, J = 8.0 Hz, C6-CH3), 0.94–0.82 (m, 9H, 3  CH2CH3); 13C NMR (CDCl3, 100 MHz) d 173.5 (C@O), 173.0 (C@O), 172.8 (C@O), 172.7 (C@O), 12.6 (C@O), 92.6 (C-1b), 92.0 (C-1a), 71.0, 70.8, 70.5, 70.2, 70.0, 68.8, 66.2, 36.0 (COCH2), 35.9 (COCH2), 35.8 (COCH2), 18.4 (CH2CH3), 18.3 (CH2CH3), 18.0 (CH2CH3), 17.3 (C-6), 13.5 (CH2CH3), 13.4 (CH2CH3), 13.4 (CH2CH3); ESIMS: m/z 397 [M+Na]+. 4.2.5. 2,3,4-Tri-O-butyryl-a/b-D-arabinopyranose (7e) Yield: 62%. a/b ratio = 5/4. 1H NMR (CDCl3, 400 MHz) d 5.44– 5.39 (m, 2H, C3-H, C4-H), 5.33 (d, 4/9H, J = 1.6 Hz, C1-Hb), 5.18 (m, 1H, C2-H), 5.08 (d, 5/9H, J = 4.0 Hz, C1-Ha), 4.20–4.17 (m, 1H), 3.67 (dd, 1H, J = 1.7 Hz, 10.0 Hz), 2.35–2.19 (m, 6H, 3  COCH2), 1.68–1.55 (m, 6H, 3  CH2CH3), 0.95–0.89 (m, 9H, 3  CH2CH3); 13 C NMR (CDCl3, 100 MHz) d 173.8 (C@O), 173.1 (C@O), 173.0 (C@O), 172.9 (C@O), 172.6 (C@O), 172.5 (C@O), 96.1 (C-1b), 90.9 (C-1a), 71.0, 69.9, 68.8, 68.4, 67.7, 66.7, 64.2, 60.3, 36.1 (COCH2), 36.0 (COCH2), 35.9 (COCH2), 18.4 (CH2CH3), 18.3 (CH2CH3), 18.1 (CH2CH3), 13.5 (CH2CH3), 13.5 (CH2CH3), 13.4 (CH2CH3); ESIMS: m/z 383 [M+Na]+. 4.2.6. 20 ,30 ,40 ,60 -Tetra-O-butyryl-a-D-glucopyranosyl-(10 ? 4)2,3,6-tri-O-butyryl-a/b-D-glucopyranose (7f) Yield: 59%. a/b ratio = 3/2. 1H NMR (CDCl3, 400 MHz) d 5.54 (t, 0 0 1H, J = 10.0 Hz, C3 -H), 5.35–5.32 (m, 2H, C4 -H, C4-H), 5.31 (d, 1H, 10 J = 4.0 Hz, C -H), 5.28 (d, 3/5H, J = 4.0 Hz, C1-Ha), 5.04 (t, 1H, 0 J = 10.0 Hz, C3-H), 4.80 (m, 1H, C2 -H), 4.64 (dd, 1H, J = 4.0 Hz, 2 10.0 Hz, C -H), 4.47 (d, 2/5H, J = 8.0 Hz, C1-Hb), 4.18–4.16 (m, 3H), 3.99–3.90 (m, 3H), 2.30–2.20 (m, 14H, 7  COCH2), 1.63– 1.48 (m, 14H, 7  CH2CH3), 0.90–0.82 (m, 21H, 7  CH2CH3); 13C NMR (CDCl3, 100 MHz) d 173.2 (C@O), 173.1 (C@O), 172.9 (C@O), 172.8 (C@O), 172.6 (C@O), 172.1 (C@O), 172.8 (C@O), 95.2 (C-10 ), 95.1 (C-1b), 89.6 (C-1a), 74.6, 73.8, 72.1, 72.0, 71.7, 71.4, 69.6, 68.9, 68.3, 68.2, 67.4, 62.3 (C-60 ), 61.0 (C-6), 35.7 (COCH2), 35.7 (COCH2), 35.6 (COCH2), 35.6 (COCH2), 35.5 (COCH2), 35.7 (COCH2), 35.5 (COCH2), 18.1 (CH2CH3), 18.0 (CH2CH3), 18.0 (CH2CH3), 18.0 (CH2CH3), 17.9 (CH2CH3), 17.9 (CH2CH3), 17.9 (CH2CH3), 13.4 (CH2CH3), 13.4 (CH2CH3), 13.4 (CH2CH3), 13.3 (CH2CH3), 13.3 (CH2CH3), 13.3 (CH2CH3), 13.3 (CH2CH3); ESIMS: m/z 855 [M+Na]+. 4.2.7. 20 ,30 ,40 ,60 -Tetra-O-butyryl-b-D-galactopyranosyl-(10 ? 4)2,3,6-tri-O-butyryl-a/b-D-glucopyranose (7g) Yield: 60%. a/b ratio = 3/2. 1H NMR (CDCl3, 400 MHz) d 5.49 (t, 0 3/5H, J = 10.0 Hz, C3-H), 5.34–5.32 (m, 2H, C4 -H, C4-H), 5.31–5.30

To the mixture of sugar alcohol 7a–7g (0.2 mmol), podophyllotoxin 1 (82.8 mg, 0.2 mmol) in CH2Cl2 (3 mL) was added dropwise a solution of BF3Et2O (25 lL, 0.02 mmol) in CH2Cl2 (1 mL) at 78 °C. After another 1 h of stirring at room temperature, Et3N (0.1 mL) was added to the mixture, and AcOH (0.1 mL) was added. The solvent was evaporated and the residue was purified by column chromatography (silica gel, petroleum ether 60–90 °C: ethyl acetate = 9:1 ? 4:1) to afford 8a–8g (61–72%). 4.3.1. 4-O-(200 ,300 ,400 ,600 -Tetra-O-butyryl-a-D-glucopyranosyl)epipodophyllotoxin (8a) Yield 68%; mp 145–148 °C; [a]21.8 +83.4 (c 0.14, CHCl3); 1H NMR D (CDCl3, 400 MHz) d 6.94 (s, 1H, C5-H), 6.52 (s, 1H, C8-H), 6.42 (s, 2H, 0 0 00 C2 ,C6 -H), 5.98–5.96 (m, 2H, OCH2O), 5.42 (t, 1H, J = 8.0 Hz, C3 -H), 100 400 5.37 (d, 1H, J = 4.0 Hz, C -H), 5.11–5.08 (m, 1H, C -H), 4.83 (dd, 00 1H, J = 4.0 Hz, 8.0 Hz, C2 -H), 4.74 (d, 1H, J = 4.0 Hz, C4-H), 4.38 (d, 1 1H, J = 4.0 Hz, C -H), 4.34–4.29 (m, 1H), 4.22 (dd, 1H, J = 2.0 Hz, 8.0 Hz), 4.12 (dd, 1H, J = 4.0 Hz, 10.0 Hz), 3.93–3.91 (m, 1H), 3.85 0 0 0 (s, 3H, C4 -OCH3), 3.81 (s, 6H, C3 ,C5 -OCH3), 3.65–3.63 (m, 1H), 2 3.35 (dd, 1H, J = 4.0 Hz, 10.0 Hz, C -H), 3.13–3.08 (m, 1H, C3-H), 2.38–2.19 (m, 8H, 4  COCH2), 1.63–1.53 (m, 8H, 4  CH2CH3), 0.92–0.89 (m, 12H, 4  CH2CH3); 13C NMR (CDCl3, 100 MHz) d 177.8 (C-12), 173.3 (C@O), 173.1 (C@O), 172.3 (C@O), 172.0 (C@O), 153.5 (C-30 , C-50 ), 150.0 (C-6), 146.7 (C-7), 137.5 (C-40 ), 136.9 (C-10 ), 131.5 (C-10), 128.4 (C-9), 109.7 (C-8), 106.8 (C-5), 105.3 (C-20 , C-60 ), 101.4 (OCH2O), 95.8 (C-100 ), 75.8 (C-4), 71.1, 69.5, 68.3, 67.8 (C-11), 67.5, 60.9 (40 -OCH3), 60.7 (C-600 ), 56.2 (30 ,50 -OCH3), 45.0 (C-2), 44.2 (C-1), 38.1 (C-3), 35.9 (COCH2), 35.8 (COCH2), 35.7 (COCH2), 35.6 (COCH2), 18.2 (CH2CH3), 18.2 (CH2CH3), 18.1 (CH2CH3), 18.1 (CH2CH3), 13.6 (CH2CH3), 13.6 (CH2CH3), 13.6 (CH2CH3), 13.6 (CH2CH3); ESIMS: m/z 879 [M+Na]+, HRESIMS: calcd for C44H56O17Na [M+Na]+ 879.3410, found 879.3312. 4.3.2. 4-O-(200 ,300 ,400 ,600 -Tetra-O-butyryl-a-D-galactopyranosyl)epipodophyllotoxin (8b) Yield 70%; mp 109–110 °C; [a]22.2 +19.1 (c 0.25, CHCl3); 1H NMR D (CDCl3, 500 MHz) d 6.97 (s, 1H, C5-H), 6.54 (s, 1H, C8-H), 6.24 (s, 2H, 0 0 00 C2 , C6 -H), 5.98 (d, 2H, J = 8.0 Hz, OCH2O), 5.42–5.41 (m, 1H, C4 -H), 00 5.32 (dd, 1H, J = 4.0 Hz, 8.0 Hz, C3 -H), 5.24 (dd, 1H, J = 4.0 Hz, 00 00 8.0 Hz, C2 -H), 5.20 (d, 1H, J = 4.0 Hz, C1 -H), 4.78 (d, 1H, 4 1 J = 2.7 Hz, C -H), 4.65 (d, 1H, J = 4.0 Hz, C -H), 4.26–4.24 (m, 1H), 0 0 4.14–4.04 (m, 3H), 3.79 (s, 3H, C4 -OCH3), 3.73 (s, 6H, C3 , 50 2 C -OCH3), 3.46 (dd, 1H, J = 4.0 Hz, 10.0 Hz, C -H), 2.96–2.88 (m, 1H, C3-H), 2.38–2.27 (m, 8H, 4  COCH2), 1.70–1.54 (m, 8H, 4  CH2CH3), 0.95–0.90 (m, 12H, 4  CH2CH3); 13C NMR (CDCl3, 100 MHz) d 174.4 (C-12), 173.0 (C@O), 172.8 (C@O), 172.6 (C@O), 172.5 (C@O), 152.6 (C-30 , C-50 ), 148.7 (C-6), 147.2 (C-7), 137.2 (C-40 ), 135.0 (C-10 ), 132.6 (C-10), 128.4 (C-9), 110.5 (C-8), 109.7

C.-T. Zi et al. / Bioorg. Med. Chem. 23 (2015) 1437–1446

(C-5), 108.2 (C-20 , C-60 ), 101.6 (OCH2O), 98.3 (C-100 ), 75.5 (C-4), 67.7, 67.7, 67.2, 67.2, 66.3 (C-11), 61.3 (C-600 ), 60.7 (40 -OCH3), 56.2 (30 ,50 -OCH3), 43.7 (C-2), 40.5 (C-1), 38.3 (C-3), 35.9 (COCH2), 35.8 (COCH2), 35.8 (COCH2), 35.6 (COCH2), 18.5 (CH2CH3), 18.2 (CH2CH3), 18.2 (CH2CH3), 18.0 (CH2CH3), 13.6 (CH2CH3), 13.6 (CH2CH3), 13.5 (CH2CH3), 13.5 (CH2CH3); ESIMS: m/z 879 [M+Na]+, HRESIMS: calcd for C44H56O17Na [M+Na]+ 879.3410, found 879.3332. 4.3.3. 4-O-(200 ,300 ,400 ,600 -Tetra-O-butyryl-a-D-mannopyranosyl)epipodophyllotoxin (8c) Yield 65%; mp 102–104 °C; [a]21.6 27.7 (c 0.16, CHCl3); 1H D NMR (CDCl3, 500 MHz) d 6.93 (s, 1H, C5-H), 6.54 (s, 1H, C8-H), 0 0 6.24 (s, 2H, C2 ,C6 -H), 6.00–5.98 (m, 2H, OCH2O), 5.36 (t, 1H, 00 400 J = 8.0 Hz, C -H), 5.29 (dd, 1H, J = 4.0 Hz, 8.0 Hz, C3 -H), 5.15– 200 100 5.14 (m, 1H, C -H), 4.95 (s, 1H, C -H), 4.82 (d, 1H, J = 2.9 Hz, C4-H), 4.64 (d, 1H, J = 4.0 Hz, C1-H), 4.40 (t, 1H, J = 8.0 Hz), 4.27– 4.20 (m, 2H), 4.15–1.12 (m, 1H), 4.96–4.92 (m, 1H), 3.79 (s, 3H, 0 0 0 C4 -OCH3), 3.73 (s, 6H, C3 ,C5 -OCH3), 3.33 (dd, 1H, J = 4.0 Hz, 2 10.0 Hz, C -H), 2.93–2.86 (m, 1H, C3-H), 2.45–2.19 (m, 8H, 4  COCH2), 1.73–1.55 (m, 8H, 4  CH2CH3), 0.99–0.89 (m, 12H, 4  CH2CH3); 13C NMR (CDCl3, 100 MHz) d 174.2 (C-12), 173.3 (C@O), 172.9 (C@O), 172.4 (C@O), 172.1 (C@O), 152.5 (C-30 , C-50 ), 148.7 (C-6), 147.3 (C-7), 137.3 (C-40 ), 134.8 (C-10 ), 132.7 (C-10), 128.3 (C-9), 110.5 (C-8), 109.7 (C-5), 108.3 (C-20 , C-60 ), 101.6 (OCH2O), 98.6 (C-100 ), 76.1 (C-4), 69.7, 69.6, 68.1, 67.0 (C-11), 65.6, 61.3 (C-600 ), 60.7 (40 -OCH3), 56.3 (30 ,50 -OCH3), 43.7 (C-2), 40.7 (C-1), 38.1 (C-3), 36.0 (COCH2), 35.8 (COCH2), 35.8 (COCH2), 35.8 (COCH2), 18.4 (CH2CH3), 18.3 (CH2CH3), 18.2 (CH2CH3), 18.1 (CH2CH3), 13.7 (CH2CH3), 13.5 (CH2CH3), 13.5 (CH2CH3), 13.5 (CH2CH3); ESIMS: m/z 879 [M+Na]+, HRESIMS: calcd for C44H56O17Na [M+Na]+ 879.3410, found 879.3346. 4.3.4. 4-O-(200 ,300 ,400 -Tri-O-butyryl-a-L-rhamnopyranosyl)epipodophyllotoxin (8d) Yield 63%; mp 195–197 °C; [a]21.7 81.2 (c 0.11, CHCl3); 1H D NMR (CDCl3, 500 MHz) d 6.92 (s, 1H, C5-H), 6.60 (s, 1H, C8-H), 0 0 6.24 (s, 2H, C2 , C6 -H), 6.02–6.01 (m, 2H, OCH2O), 5.23–5.21 (m, 00 300 200 2H, C -H, C -H), 5.17 (t, 1H, J = 8.0 Hz, C4 -H), 4.93 (d, 1H, 100 4 J = 1.1 Hz, C -H), 4.84 (d, 1H, J = 2.6 Hz, C -H), 4.66 (d, 1H, 0 J = 5.0 Hz, C1-H), 4.42–4.39 (m, 2H), 3.82 (s, 3H, C4 -OCH3), 3.75 30 50 (s, 6H, C ,C -OCH3), 3.47 (dd, 1H, J = 5.0 Hz, 10.0 Hz, C2-H), 3.02– 2.95 (m, 1H, C3-H), 2.44–2.18 (m, 6H, 3  COCH2), 1.71–1.57 (m, 6H, 4  CH2CH3), 1.00–0.92 (m, 9H, 3  CH2CH3); 13C NMR (CDCl3, 100 MHz) d 174.4 (C-12), 172.6 (C@O), 172.5 (C@O), 172.4 (C@O), 152.5 (C-30 ,C-50 ), 148.9 (C-6), 146.7 (C-7), 137.2 (C-40 ), 135.4 (C10 ), 133.5 (C-10), 125.7 (C-9), 110.5 (C-8), 110.3 (C-5), 108.2 (C20 ,C-60 ), 101.6 (OCH2O), 93.5 (C-100 ), 70.3 (C-4), 69.2, 69.0, 67.6, 66.9 (C-11), 60.7 (40 -OCH3), 56.2 (30 ,50 -OCH3), 43.9 (C-2), 40.7 (C1), 37.5 (C-3), 36.0 (COCH2), 36.0 (COCH2), 35.8 (COCH2), 18.4 (CH200 CH3), 18.4 (CH2CH3), 18.0 (CH2CH3), 17.7 (C6 -CH3), 13.6 (CH2CH3), 13.6 (CH2CH3), 13.6 (CH2CH3); ESIMS: m/z 793 [M+Na]+, HRESIMS: calcd for C40H50O15Na [M+Na]+ 793.3042, found 793.2980. 4.3.5. 4-O-(200 ,300 ,400 -Tri-O-butyryl-a-D-arabinopyranosyl)epipodophyllotoxin (8e) Yield 62%; mp 96–98 °C; [a]21.7 20.1 (c 0.19, CHCl3); 1H NMR D (CDCl3, 500 MHz) d 7.03 (s, 1H, C5-H), 6.48 (s, 1H, C8-H), 6.25 (s, 0 0 00 2H, C2 , C6 -H), 5.99–5.96 (m, 2H, OCH2O), 5.29 (s, 1H, C1 -H), 400 300 5.25–5.23 (m, 1H, C -H), 5.06 (dd, 1H, J = 5.0 Hz, 10.0 Hz, C -H), 00 4.88 (d, 1H, J = 5.0 Hz, C2 -H), 4.59 (d, 1H, J = 4.0 Hz, C4-H), 4.48 (d, 1H, J = 5.0 Hz, C1-H), 4.35–4.33 (m, 2H), 4.10 (dd, 1H, 0 0 0 J = 5.0 Hz, 10.0 Hz), 3.79 (s, 3H, C4 -OCH3), 3.75 (s, 6H, C3 , C5 2 OCH3), 3.73–3.72 (m, 1H), 3.20 (dd, 1H, J = 5.0 Hz, 10.0 Hz, C -H),

1443

2.91–2.84 (m, 1H, C3-H), 2.38–2.17 (m, 6H, 3  COCH2), 2.69– 1.53 (m, 6H, 3  CH2CH3), 0.96–0.88 (m, 9H, 3  CH2CH3); 13C NMR (CDCl3, 100 MHz) d 174.4 (C-12), 173.0 (C@O), 172.6 (C@O), 171.7 (C@O), 152.5 (C-30 ,C-50 ), 148.5 (C-6), 147.2 (C-7), 137.1 (C-40 ), 134.8 (C-10 ), 132.4 (C-10), 128.6 (C-9), 110.6 (C-8), 109.8 (C-5), 108.1 (C-20 , C-60 ), 101.4 (OCH2O), 100.9 (C-100 ), 73.9 (C-4), 70.1, 69.1, 67.4, 66.6, 63.7 (C-11), 60.8 (C-500 ), 60.7 (40 -OCH3), 56.5 (30 ,50 -OCH3), 43.7 (C-2), 40.8 (C-1), 37.8 (C-3), 36.0 (COCH2), 35.9 (COCH2), 35.8 (COCH2), 18.4 (CH2CH3), 18.4 (CH2CH3), 18.3 (CH2CH3), 13.6 (CH2CH3), 13.5 (CH2CH3), 13.5 (CH2CH3); ESIMS: m/z 779 [M+Na]+, HRESIMS: calcd for C39H48O15Na [M+Na]+ 779.2885, found 779.2813. 4.3.6. 4-O-[(2000 ,3000 ,4000 ,6000 -Tetra-O-butyryl-a-D-glucopyranosyl(1000 ? 400 )-200 ,300 ,600 -tri-O-butyryl-a-D-glucopyranosyl)]epipodophyllotoxin (8f) Yield 61%; mp 89–90 °C; [a]21.7 5.3 (c 0.14, CHCl3); 1H NMR D (CDCl3, 600 MHz) d 6.77 (s, 1H, C5-H), 6.48 (s, 1H, C8-H), 6.15 (s, 0 0 2H, C2 , C6 -H), 5.95 (d, 2H, J = 12.0 Hz, OCH2O), 5.34–5.31 (m, 2H, 000 4000 400 C -H, C -H), 5.21 (t, 1H, J = 10.0 Hz, C3 -H), 5.04 (t, 1H, 300 J = 10.0 Hz, C -H), 4.84 (d, 1H, J = 2.4 Hz, C4-H), 4.81 (d, 1H, 000 00 J = 4.0 Hz, C1 -H), 4.79 (d, 1H, J = 4.0 Hz, C1 -H), 4.68–4.65 (m, 2H, 000 00 C2 -H, C2 -H), 4.49 (d, 1H, J = 4.8 Hz, C1-H), 4.27–4.16 (m, 3H), 4.11 (dd, 1H, J = 6.0 Hz, 10.0 Hz), 3.99–3.90 (m, 3H), 3.73 (s, 3H, 0 0 0 C4 -OCH3), 3.66 (s, 6H, C3 ,C5 -OCH3), 3.62–3.60 (m, 1H), 3.07 (dd, 2 1H, J = 6.0 Hz, 10.0 Hz, C -H), 2.84–2.78 (m, 1H, C3-H), 2.38–2.32 (m, 14H, 7  COCH2), 1.65–1.28 (m, 14H, 7  CH2CH3), 0.92–0.70 (m, 21H, 7  CH2CH3); 13C NMR (CDCl3, 150 MHz) d 174.9 (C-12), 173.4 (C@O), 173.2 (C@O), 173.2 (C@O), 172.8 (C@O), 172.7 (C@O), 172.2 (C@O), 172.2 (C@O), 152.8 (C-30 , C-50 ), 148.2 (C-6), 147.6 (C-7), 137.3 (C-40 ), 135.2 (C-10 ), 133.1 (C-10), 127.8 (C-9), 110.0 (C-8), 109.3 (C-5), 108.3 (C-20 , C-60 ), 101.9 (OCH2O), 98.1 (C-1000 ), 95.6 (C-100 ), 74.8 (C-4), 73.6, 72.7, 72.0, 71.7, 70.0, 69.2, 68.9, 67.9 (C-11), 67.7, 62.1 (C-600 ), 61.4 (C-6000 ), 61.0 (40 -OCH3), 56.4 (30 ,50 -OCH3), 44.0 (C-2), 41.2 (C-1), 37.7 (C-3), 36.3 (COCH2), 36.1 (COCH2), 36.1 (COCH2), 36.0 (COCH2), 36.0 (COCH2), 35.9 (COCH2), 35.9 (COCH2), 18.6 (CH2CH3), 18.5 (CH2CH3), 18.5 (CH2CH3), 18.4 (CH2CH3), 18.3 (CH2CH3), 18.3 (CH2CH3), 18.2 (CH2CH3), 13.9 (CH2CH3), 13.9 (CH2CH3), 13.9 (CH2CH3), 13.9 (CH2CH3), 13.8 (CH2CH3), 13.8 (CH2CH3), 13.7 (CH2CH3); ESIMS: m/z 1251 [M+Na]+, HRESIMS: calcd for C62H84O25Na [M+H]+ 1251.5194, found 1251.5092. 4.3.7. 4-O-[(2000 ,3000 ,4000 ,6000 -Tetra-O-butyryl-b-D-galactopyranosyl(1000 ? 400 )-200 ,300 ,600 -tri-O-butyryl-a-D-glucopyranosyl)]epipodophyllotoxin (8g) Yield 72%; mp 90–92 °C; [a]21.7 40.8 (c 0.16, CHCl3); 1H NMR D 5 (CDCl3, 500 MHz) d 6.79 (s, 1H, C -H), 6.53 (s, 1H, C8-H), 6.21 (s, 0 0 2H, C2 , C6 -H), 5.99–5.96 (m, 2H, OCH2O), 5.37 (d, 1H, J = 3.0 Hz, 00 100 C -H), 5.21 (t, 1H, J = 10.0 Hz, C3 -H), 5.11 (dd, 1H, J = 8.0 Hz, 000 2000 10.0 Hz, C -H), 4.97 (dd, 1H, J = 4.0 Hz, 10.0 Hz, C3 -H), 4.89 (dd, 200 1H, J = 4.0 Hz, 10.0 Hz, C -H), 4.86 (d, 1H. J = 4.0 Hz, C4-H), 4.67– 000 00 4.65 (m, 2H, C4 -H, C4 -H), 4.55 (d, 1H, J = 5.0 Hz, C1-H), 4.48 (d, 1000 1H, J = 8.0 Hz, C -H), 4.32 (t, 1H, J = 10.0 Hz), 4.21 (t, 1H, 0 J = 8.0 Hz), 4.10–4.05 (m, 3H), 3.89–3.86 (m, 1H), 3.78 (s, 3H, C4 30 50 OCH3), 3.71 (s, 6H, C ,C -OCH3), 3.59–3.56 (m, 1H), 3.13 (dd, 1H, J = 5.0 Hz, 10.0 Hz, C2-H), 2.89–2.80 (m, 1H, C3-H), 2.40–1.99 (m, 14H, 7  COCH2), 1.71–1.36 (m, 14H, 7  CH2CH3), 0.98–0.76 (m, 21H, 7  CH2CH3); 13C NMR (CDCl3, 100 MHz) d 174.6 (C-12), 172.9 (C@O), 172.9 (C@O), 172.6 (C@O), 172.5 (C@O), 172.3 (C@O), 171.8 (C@O), 171.6 (C@O), 152.5 (C-30 , C-50 ), 148.7 (C-6), 146.9 (C-7), 137.1 (C-40 ), 134.9 (C-10 ), 132.9 (C-10), 127.5 (C-9), 110.8 (C-8), 109.0 (C-5), 108.1 (C-20 , C-60 ), 101.6 (OCH2O), 100.9 (C-1000 ), 98.8 (C-100 ), 75.7 (C-4), 73.4, 73.0, 71.6, 71.0, 70.9, 70.8,

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68.2, 67.7 (C-11), 66.4, 61.1 (C-600 ), 60.7 (40 -OCH3), 60.6 (C-6000 ), 56.2 (30 ,50 -OCH3), 43.8 (C-2), 40.9 (C-1), 37.5 (C-3), 35.9 (COCH2), 35.8 (COCH2), 35.8 (COCH2), 35.8 (COCH2), 35.7 (COCH2), 35.7 (COCH2), 35.7 (COCH2), 18.4 (CH2CH3), 18.4 (CH2CH3), 18.3 (CH2CH3), 18.2 (CH2CH3), 18.1 (CH2CH3), 18.1 (CH2CH3), 17.9 (CH2CH3), 13.7 (CH2CH3), 13.6 (CH2CH3), 13.6 (CH2CH3), 13.5 (CH2CH3), 13.5 (CH2CH3), 13.5 (CH2CH3), 13.4 (CH2CH3); ESIMS: m/z 1273 [M+HCOO], HRESIMS: calcd for C62H84O25 HCOO [M+HCOO] 1273.5284, found 1273.5008. 4.4. General method for the synthesis of compounds 9a–9g To the mixture of sugar alcohol 7a–7g (0.2 mmol), 40 -demethylepipodophyllotoxin 5 (80.0 mg, 0.2 mmol) in CH2Cl2 (3 mL) was added dropwise a solution of BF3Et2O (25 lL, 0.02 mmol) in CH2Cl2 (1 mL) at 78 °C. After another 1 h of stirring at room temperature, Et3N (0.1 mL) was added to the mixture, and AcOH (0.1 mL) was added. The solvent was evaporated and the residue was purified by column chromatography (silica gel, petroleum ether 60–90 °C:ethyl acetate = 9:1 ? 4:1) to afford 9a–9g (57–70%). 4.4.1. 4-O-(200 ,300 ,400 ,600 -Tetra-O-butyryl-a-D-glucopyranosyl)-40 demeththylepipodophyllotoxin (9a) Yield 70%; mp 128–130 °C; [a]22.0 1.9 (c 0.17, CHCl3); 1H NMR D 5 (CDCl3, 400 MHz) d 6.94 (s, 1H, C -H), 6.56 (s, 1H, C8-H), 6.45 (s, 2H, 0 0 00 C2 , C6 -H), 5.98–5.97 (m, 2H, OCH2O), 5.42 (t, 1H, J = 10.0 Hz, C3 100 400 H), 5.36 (d, 1H, J = 4.0 Hz, C -H), 5.08 (t, 1H, J = 10.0 Hz, C -H), 00 4.83 (dd, 1H, J = 4.0 Hz, 10.0 Hz, C2 -H), 4.74 (d, 1H, J = 4.0 Hz, C41 H), 4.42 (d, 1H, J = 4.0 Hz, C -H), 4.34–4.30 (m, 1H), 4.22–4.19 (m, 0 0 1H), 4.10 (dd, 1H, J = 4.0 Hz, 12.0 Hz), 3.76 (s, 6H, C3 ,C5 -OCH3), 2 3.64–3.62 (m, 1H), 3.34 (dd, 1H, J = 4.0 Hz, 10.0 Hz, C -H), 3.14– 3.09 (m, 1H, C3-H), 2.60–2.18 (m, 8H, 4  COCH2), 1.82–1.53 (m, 8H, 4  CH2CH3), 1.04–0.89 (m, 12H, 4  CH2CH3); 13C NMR (CDCl3, 100 MHz) d 174.3 (C-12), 173.3 (C@O), 172.6 (C@O), 172.6 (C@O), 171.9 (C@O), 148.7 (C-6), 147.0 (C-7), 146.4 (C-30 , C-50 ), 132.9 (C40 ), 130.4 (C-10 ), 128.3 (C-10), 127.3 (C-9), 110.9 (C-8), 109.0 (C5), 107.9 (C-20 , C-60 ), 101.6 (OCH2O), 98.7 (C-100 ), 72.1 (C-4), 70.9, 70.5, 69.4, 68.3 (C-11), 66.3, 61.7 (C-600 ), 56.5 (30 ,50 -OCH3), 43.6 (C-2), 44.7 (C-1), 38.2 (C-3), 35.9 (COCH2), 35.9 (COCH2), 35.8 (COCH2), 35.7 (COCH2), 18.2 (CH2CH3), 18.2 (CH2CH3), 18.2 (CH2CH3), 18.1 (CH2CH3), 13.6 (CH2CH3), 13.6 (CH2CH3), 13.5 (CH2CH3), 13.4 (CH2CH3); ESIMS: m/z 865 [M+Na]+, HRESIMS: calcd for C43H54O17Na [M+Na]+ 865.3253, found 865.3183. 4.4.2. 4-O-(200 ,300 ,400 ,600 -Tetra-O-butyryl-a-D-galactopyranosyl)-40 demeththylepipodophyllotoxin (9b) Yield 69%; mp 129–130 °C; [a]22.4 +26.7 (c 0.17, CHCl3); 1H NMR D 5 (CDCl3, 400 MHz) d 6.90 (s, 1H, C -H), 6.48 (s, 1H, C8-H), 6.19 (s, 2H, 0 0 C2 , C6 -H), 5.93–5.89 (m, 2H, OCH2O), 5.42 (br d, 1H, J = 2.0 Hz, 00 400 C -H), 5.34 (dd, 1H, J = 4.0 Hz, 10.0 Hz, C3 -H), 5.23 (dd, 1H, 00 200 J = 4.0 Hz, 10.0 Hz, C -H), 5.20 (d, 1H, J = 4.0 Hz, C1 -H), 4.78 (d, 4 1 1H, J = 2.8 Hz, C -H), 4.64 (d, 1H, J = 5.2 Hz, C -H), 4.23–4.20 (m, 0 0 2H), 4.15–4.01 (m, 3H), 3.70 (s, 6H, C3 ,C5 -OCH3), 3.37 (dd, 1H, J = 4.0 Hz, 10.0 Hz, C2-H), 2.88–2.80 (m, 1H, C3-H), 2.34–2.07 (m, 8H, 4  COCH2), 1.64–1.47 (m, 8H, 4  CH2CH3), 0.91–0.83 (m, 12H, 4  CH2CH3); 13C NMR (CDCl3, 100 MHz) d 174.5 (C-12), 173.0 (C@O), 172.8 (C@O), 172.6 (C@O), 172.5 (C@O), 148.8 (C6), 147.2 (C-7), 146.5 (C-30 , C-50 ), 134.2 (C-40 ), 132.8 (C-10 ), 130.5 (C-10), 128.4 (C-9), 110.5 (C-8), 109.7 (C-5), 108.0 (C-20 , C-60 ), 101.6 (OCH2O), 98.3 (C-100 ), 75.6 (C-4), 67.8, 67.2, 66.3 (C-11), 61.3 (C-600 ), 56.5 (30 ,50 -OCH3), 43.6 (C-2), 40.7 (C-1), 38.3 (C-3), 35.9 (COCH2), 35.9 (COCH2), 35.8 (COCH2), 35.7 (COCH2), 18.5 (CH2CH3), 18.3 (CH2CH3), 18.2 (CH2CH3), 18.0 (CH2CH3), 13.6 (CH2CH3), 13.6 (CH2CH3), 13.6 (CH2CH3), 13.6 (CH2CH3); ESIMS: m/z 881

[M+K]+, HRESIMS: calcd for C43H54O17K [M+K]+ 881.2993, found 881.2880. 4.4.3. 4-O-(200 ,300 ,400 ,600 -Tetra-O-butyryl-a-D-mannopyranosyl)-40 demethylepipodophyllotoxin (9c) Yield 57%; mp 176–178 °C; [a]21.6 27.8 (c 0.14, CHCl3); 1H D NMR (CDCl3, 500 MHz) d 6.95 (s, 1H, C5-H), 6.56 (s, 1H, C8-H), 0 0 6.27 (s, 2H, C2 , C6 -H), 6.01 (s, 2H, OCH2O), 5.46 (br s, 1H, OH), 00 5.38 (t, 1H, J = 10.0 Hz, C4 -H), 5.29 (dd, 1H, J = 4.0 Hz, 10.0 Hz, 00 00 00 C3 -H), 5.17–5.16 (m, 1H, C2 -H), 4.96 (s, 1H, C1 -H), 4.84 (d, 1H, 4 1 J = 3.0 Hz, C -H), 4.64 (d, 1H, J = 5.0 Hz, C -H), 4.40 (t, 1H, J = 8.0 Hz), 4.29–4.21 (m, 2H), 4.16 (dd, 1H, J = 5.0 Hz, 15.0 Hz), 0 0 3.98–3.94 (m, 1H), 3.75 (s, 6H, C3 , C5 -OCH3), 3.34 (dd, 1H, 2 J = 5.0 Hz, 10.0 Hz, C -H), 2.94–2.87 (m, 1H, C3-H), 2.47–2.20 (m, 8H, 4  COCH2), 1.75–1.57 (m, 8H, 4  CH2CH3), 1.01–0.91 (m, 12H, 4  CH2CH3); 13C NMR (CDCl3, 125 MHz) d 174.3 (C-12), 173.3 (C@O), 172.9 (C@O), 172.5 (C@O), 172.1 (C@O), 148.7 (C6), 147.3 (C-7), 146.4 (C-30 , C-50 ), 134.2 (C-40 ), 133.0 (C-10 ), 130.3 (C-10), 128.3 (C-9), 110.4 (C-8), 109.7 (C-5), 108.0 (C-20 , C-60 ), 101.6 (OCH2O), 98.7 (C-100 ), 76.1 (C-4), 69.7, 69.6, 68.2, 67.0 (C11), 65.6, 62.1 (C-600 ), 56.5 (30 ,50 -OCH3), 43.6 (C-2), 40.9 (C-1), 38.0 (C-3), 36.0 (COCH2), 35.9 (COCH2), 35.9 (COCH2), 35.8 (COCH2), 18.4 (CH2CH3), 18.3 (CH2CH3), 18.3 (CH2CH3), 18.2 (CH2CH3), 13.7 (CH2CH3), 13.6 (CH2CH3), 13.6 (CH2CH3), 13.6 (CH2CH3); ESIMS: m/z 865 [M+Na]+, HRESIMS: calcd for C43H54O17Na [M+Na]+ 865.3253, found 865.3169. 4.4.4. 4-O-(200 ,300 ,400 -Tri-O-butyryl-a-L-rhamnopyranosyl)-40 demethylepipodophyllotoxin (9d) Yield 62%; mp 207–208 °C; [a]21.6 67.6 (c 0.22, CHCl3); 1H D NMR (CDCl3, 500 MHz) d 6.88 (s, 1H, C5-H), 6.57 (s, 1H, C8-H), 0 0 6.22 (s, 2H, C2 , C6 -H), 5.99–5.97 (m, 2H, OCH2O), 5.42 (br s, 1H, 00 00 OH), 5.20–5.18 (m, 2H, C3 -H, C2 -H), 5.14 (t, 1H, J = 10.0 Hz, 400 100 C -H), 4.90 (d, 1H, J = 1.0 Hz, C -H), 4.81 (d, 1H, J = 2.9 Hz, C4-H), 4.62 (d, 1H, J = 5.0 Hz, C1-H), 4.37–4.35 (m, 2H, C11-CH2), 3.75 (s, 0 0 6H, C3 , C5 -OCH3), 3.42 (dd, 1H, J = 5.0 Hz, 10.0 Hz, C2-H), 2.98– 2.91 (m, 1H, C3-H), 2.43–2.15 (m, 6H, 3  COCH2), 1.68–1.54 (m, 00 6H, 3  CH2CH3), 1.32 (d, 3H, J = 10.0 Hz, C6 -CH3), 0.97–0.89 (m, 13 9H, 3  CH2CH3); C NMR (CDCl3, 125 MHz) d 174.5 (C-12), 0 0 172.6 (C@O), 172.5 (C@O), 172.4 (C@O), 152.5 (C-3 , C-5 ), 148.9 0 (C-6), 146.4 (C-7), 134.1 (C-4 ), 133.7 (C-10), 130.9 (C-9), 125.8 (C-10 ), 111.5 (C-8), 110.3 (C-5), 108.0 (C-20 , C-60 ), 101.6 (OCH2O), 93.6 (C-100 ), 70.4 (C-4), 69.3 (C-11), 69.2, 69.1, 67.6, 66.9, 56.5 (30 ,50 -OCH3), 43.8 (C-2), 40.8 (C-1), 37.5 (C-3), 36.1 (COCH2), 36.0 (COCH2), 35.8 (COCH2), 18.4 (CH2CH3), 18.4 (CH2CH3), 18.0 (CH200 CH3), 17.7 (C6 -CH3), 13.6 (CH2CH3), 13.6 (CH2CH3), 13.6 (CH2CH3); ESIMS: m/z 779 [M+Na]+, HRESIMS: calcd for C39H48O15Na [M+Na]+ 779.2885, found 779.2819. 4.4.5. 4-O-(200 ,300 ,400 -Tri-O-butyryl-a-D-arabinopyranosyl)-40 demethylepipodophyllotoxin (9e) Yield 60%; mp 218–220 °C; [a]21.6 44.6 (c 0.18, CHCl3); 1H D NMR (CDCl3, 500 MHz) d 7.06 (s, 1H, C5-H), 6.51 (s, 1H, C8-H), 0 0 6.28 (s, 2H, C2 , C6 -H), 6.02–5.98 (m, 2H, OCH2O), 5.44 (br s, 1H, 00 00 OH), 5.31 (s, 1H, C1 -H), 5.27 (dd, 1H, J = 8.0 Hz, 10.0 Hz, C4 -H), 300 5.09 (dd, 1H, J = 4.0 Hz, 10.0 Hz, C -H), 4.89 (d, 1H, J = 4.0 Hz, 00 C2 -H), 4.61 (d, 1H, J = 5.0 Hz, C4-H), 4.51 (d, 1H, J = 5.0 Hz, C1-H), 4.36–4.34 (m, 2H), 4.12 (dd, 1H, J = 3.0 Hz, 10.0 Hz), 3.78 (s, 6H, 0 0 C3 ,C5 -OCH3), 3.72–3.69 (m, 1H), 3.21 (dd, 1H, J = 5.0 Hz, 10.0 Hz, 2 C -H), 2.93–2.86 (m, 1H, C3-H), 2.39–2.21 (m, 6H, 3  COCH2), 1.73–1.45 (m, 6H, 3  CH2CH3), 0.99–0.90 (m, 9H, 3  CH2CH3); 13 C NMR (CDCl3, 125 MHz) d 174.4 (C-12), 173.0 (C@O), 172.6 (C@O), 171.7 (C@O), 148.5 (C-6), 147.2 (C-7), 146.4 (C-30 , C-50 ), 134.1 (C-40 ), 132.6 (C-10 ), 130.3 (C-10), 128.6 (C-9), 110.6 (C-8), 109.9 (C-20 , C-60 ), 107.9 (C-5), 101.4 (OCH2O), 101.0 (C-100 ), 74.0 (C-4), 70.1, 69.3, 67.5, 66.6 (C-500 ), 63.7 (C-11), 56.4 (30 ,50 -OCH3),

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43.6 (C-2), 40.9 (C-1), 37.8 (C-3), 36.1 (COCH2), 35.9 (COCH2), 35.9 (COCH2), 18.5 (CH2CH3), 18.3 (CH2CH3), 18.1 (CH2CH3), 13.5 (CH2CH3), 13.5 (CH2CH3), 13.5 (CH2CH3); ESIMS: m/z 765 [M+Na]+, HRESIMS: calcd for C38H46O15Na [M+Na]+ 765.2729, found 765.2648. 4.4.6. 4-O-[(2000 ,3000 ,4000 ,6000 -Tetra-O-butyryl-a-D-glucopyranosyl0 (1000 ? 400 )-200 ,300 ,600 -tri-O-butyryl-a-D-glucopyranosyl)]-4 demethylepipodophyllotoxin (9f) Yield 57%; mp 92–94 °C; [a]21.7 +2.2 (c 0.23, CHCl3); 1H NMR D 5 (CDCl3, 500 MHz) d 6.84 (s, 1H, C -H), 6.54 (s, 1H, C8-H), 6.24 (s, 0 0 2H, C2 , C6 -H), 6.01–5.99 (m, 2H, OCH2O), 5.42–5.38 (m, 2H, 000 4000 400 C -H, C -H), 5.27 (t, 1H, J = 10.0 Hz, C3 -H), 5.19–5.08 (m, 1H, 000 300 4 C -H), 4.90 (d, 1H, J = 2.5 Hz, C -H), 4.88 (d, 1H, J = 4.0 Hz, C1 100 H), 4.86 (d, 1H, J = 3.0 Hz, C -H), 4.73–4.71 (m, 2H), 4.55 (d, 1H, J = 5.0 Hz, C1-H), 4.33–4.17 (m, 4H), 4.08–4.06 (m, 3H), 3.76 (s, 0 0 6H, C3 ,C5 -OCH3), 3.68–3.67 (m, 1H), 3.12 (dd, 1H, J = 5.0 Hz, 10.0 Hz, C2-H), 2.89–2.83 (m, 1H, C3-H), 2.43–1.98 (m, 14H, 7  COCH2), 1.73–1.35 (m, 14H, 7  CH2CH3), 0.99–0.77 (m, 21H, 7  CH2CH3); 13C NMR (CDCl3, 125 MHz) d 174.6 (C-12), 173.1 (C@O), 172.9 (C@O), 172.9 (C@O), 172.5 (C@O), 172.4 (C@O), 171.9 (C@O), 171.9 (C@O), 148.7 (C-6), 146.9 (C-7), 146.4 (C-30 , C-50 ), 134.1 (C-40 ), 133.1 (C-10 ), 130.4 (C-10), 127.5 (C-9), 113.1 (C-8), 109.1 (C-5), 108.0 (C-20 , C-60 ), 101.6 (OCH2O), 98.5 (C-1000 ), 00 95.5 (C-1 ), 74.6 (C-4), 73.4, 72.6, 72.1, 71.6, 69.8, 69.0, 68.8, 67.7 00 (C-11), 67.6, 62.0 (C-6 ), 61.3 (C-6000 ), 56.5 (30 ,50 -OCH3), 43.7 (C-2), 41.1 (C-1), 37.5 (C-3), 36.1 (COCH2), 35.9 (COCH2), 35.9 (COCH2), 35.9 (COCH2), 35.8 (COCH2), 35.7 (COCH2), 35.7 (COCH2), 18.4 (CH2CH3), 18.2 (CH2CH3), 18.2 (CH2CH3), 18.2 (CH2CH3), 18.1 (CH2CH3), 18.0 (CH2CH3), 18.0 (CH2CH3), 13.6 (CH2CH3), 13.6 (CH2CH3), 13.6 (CH2CH3), 13.6 (CH2CH3), 13.6 (CH2CH3), 13.6 (CH2CH3), 13.4 (CH2CH3); ESIMS: m/z 680 [M+Na]+, HRESIMS: calcd for C31H35N3O13H [M+H]+ 658.2243, found 658.2223. 4.4.7. 4-O-[(2000 ,3000 ,4000 ,6000 -Tetra-O-butyryl-b-D-galactopyranosyl0 (1000 ? 400 )-200 ,300 ,600 -tri-O-butyryl-a-D-glucopyranosyl)]-4 demethylepipodophyllotoxin (9g) Yield 64%; mp 92–93 °C; [a]21.9 46.4 (c 0.21, CHCl3); 1H NMR D (CDCl3, 400 MHz) d 6.80 (s, 1H, C5-H), 6.54 (s, 1H, C8-H), 6.23 (s, 0 0 2H, C2 , C6 -H), 6.00–5.97 (m, 2H, OCH2O), 5.42 (br s, 1H, OH), 00 00 5.38 (d, 1H, J = 3.1 Hz, C1 -H), 5.22 (t, 1H, J = 10.0 Hz, C3 -H), 3000 200 5.15–5.11 (m, 1H, C -H), 4.99 (dd, 1H, J = 3.1 Hz, 10.0 Hz, C -H), 000 4.92–4.89 (m, 1H, C2 -H), 4.86 (d, 1H, J = 4.0 Hz, C4-H), 4.69–4.66 000 (m, 2H), 4.55 (d, 1H, J = 5.0 Hz, C1-H), 4.48 (d, 1H, J = 8.0 Hz, C1 H), 4.34–4.30 (m, 1H), 4.22–4.19 (m, 1H), 4.11–4.03 (m, 3H), 0 0 3.90–3.86 (m, 1H), 3.76 (s, 6H, C3 ,C5 -OCH3), 3.60–3.58 (m, 1H), 2 3.13 (dd, 1H, J = 5.0 Hz, 10.0 Hz, C -H), 2.88–2.82 (m, 1H, C3-H), 2.42–2.01 (m, 14H, 7  COCH2), 1.73–1.38 (m, 14H, 7  CH2CH3), 0.99–0.78 (m, 21H, 7  CH2CH3); 13C NMR (CDCl3, 100 MHz) d 174.6 (C-12), 172.9 (C@O), 172.9 (C@O), 172.6 (C@O), 172.5 (C@O), 172.3 (C@O), 171.9 (C@O), 171.6 (C@O), 148.7 (C-6), 146.9 (C-7), 146.4 (C-30 , C-50 ), 134.1 (C-40 ), 133.1 (C-10 ), 130.5 (C10), 127.5 (C-9), 110.8 (C-8), 109.0 (C-5), 107.8 (C-20 , C-60 ), 101.6 (OCH2O), 100.9 (C-1000 ), 98.8 (C-100 ), 75.7 (C-4), 73.5, 72.6, 72.6, 71.0, 70.9, 70.8, 68.8, 67.7 (C-11), 66.4, 62.0 (C-600 ), 61.3 (C-6000 ), 56.5 (30 ,50 -OCH3), 43.6 (C-2), 41.0 (C-1), 37.5 (C-3), 35.9 (COCH2), 35.8 (COCH2), 35.8 (COCH2), 35.7 (COCH2), 35.7 (COCH2), 35.7 (COCH2), 35.7 (COCH2), 18.5 (CH2CH3), 18.4 (CH2CH3), 18.3 (CH2CH3), 18.2 (CH2CH3), 18.1 (CH2CH3), 18.1 (CH2CH3), 17.9 (CH2CH3), 13.7 (CH2CH3), 13.6 (CH2CH3), 13.6 (CH2CH3), 13.5 (CH2CH3), 13.5 (CH2CH3), 13.4 (CH2CH3), 13.4 (CH2CH3); ESIMS: m/z 1237 [M+Na]+, HRESIMS: calcd for C61H82O25Na [M+Na]+ 1237.5032, found 1237.5043.

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4.5. General procedure for the synthesis of compounds 10a and 10b A mixture of sugar alcohol 7a or 7b (5.0 mmol) and CCl3CN (20.0 mmol) in CH2Cl2 (40 mL) was added DBU (10.0 mmol) at 0 °C. The mixture was stirred for 2 h until TLC showed the reaction to be complete. The solvent was evaporated and the residue was purified by column chromatography (silica gel, petroleum ether 60–90 °C:ethyl acetate = 9:1) to afford mainly the a-trichloroacetimidate. 4.5.1. 2,3,4,6-Tetra-O-butyryl-a-D-glucopyranosyl-trichloroimidate (10a) Yield: 67%. 1H NMR (CDCl3, 400 MHz) d 8.65 (s, 1H, NH), 6.52 (d, 1H, J = 4.0 Hz, C1-H), 5.57 (t, 1H, J = 10.0 Hz, C3-H), 5.18 (t, 1H, J = 10.0 Hz, C4-H), 5.11 (dd, 1H, J = 4.0 Hz, 10.0 Hz, C2-H), 4.22– 4.15 (m, 2H), 4.12–4.09 (m, 1H), 2.29–2.17 (m, 8H, 4  COCH2), 1.63–1.52 (m, 8H, 4  CH2CH3), 0.89–0.85 (m, 12H, 4  CH2CH3); 13 C NMR (CDCl3, 100 MHz) d 173.0 (C@O), 172.4 (C@O), 172.3 (C@O), 171.9 (C@O), 160.6 (CONH), 92.9 (C-1), 87.4 (CCl3), 70.2, 69.5, 69.4, 67.3, 61.1 (C-6), 35.8 (COCH2), 35.8 (COCH2), 35.7 (COCH2), 35.6 (COCH2), 18.2 (CH2CH3), 18.2 (CH2CH3), 18.2 (CH2CH3), 18.1 (CH2CH3), 13.6 (CH2CH3), 13.5 (CH2CH3), 13.5 (CH2CH3), 13.5 (CH2CH3). 4.5.2. 2,3,4,6-Tetra-O-butyryl-a-D-galactopyranosyltrichloroimidate (10b) Yield: 85%. 1H NMR (CDCl3, 400 MHz) d 6.57 (d, 1H, J = 4.0 Hz, C1-H), 5.57–5.56 (m, 1H, C4-H), 5.46 (dd, 1H, J = 4.0 Hz, 10.0 Hz, C3-H), 5.37 (dd, 1H, J = 4.0 Hz, 10.0 Hz, C2-H), 4.44–4.42 (m, 1H), 4.14–4.04 (m, 2H), 2.40–2.19 (m, 8H, 4  COCH2), 1.70–1.56 (m, 8H, 4  CH2CH3), 0.97–0.88 (m, 12H, 4  CH2CH3); 13C NMR (CDCl3, 100 MHz) d 172.9 (C@O), 172.6 (C@O), 172.6 (C@O), 172.4 (C@O), 160.8 (CONH), 93.5 (C-1), 88.0 (CCl3), 69.1, 67.3, 67.2, 66.7, 61.1 (C-6), 35.9 (COCH2), 35.8 (COCH2), 35.8 (COCH2), 35.7 (COCH2), 18.2 (CH2CH3), 18.1 (CH2CH3), 18.0 (CH2CH3), 17.9 (CH2CH3), 13.6 (CH2CH3), 13.5 (CH2CH3), 13.5 (CH2CH3), 13.5 (CH2CH3). 4.6. General method for the synthesis of compounds 11a and 11b To the mixture of compound 10a or 10b (0.2 mmol), podophyllotoxin 1 (82.8 mg, 0.2 mmol) in dichloromethane (3 mL) was added dropwise a solution of BF3Et2O (25 lL, 0.02 mmol) in dichloromethane (1 mL) at 78 °C. After another 1 h of stirring at room temperature, Et3N (0.1 mL) was added to the mixture, and AcOH (0.1 mL) was added. The solvent was evaporated and the residue was purified by column chromatography (silica gel, petroleum ether 60–90 °C:ethyl acetate = 9:1 ? 4:1) to afford 11a (63%) or 11b (74%). 4.6.1. 4-O-(200 ,300 ,400 ,600 -Tetra-O-butyryl-b-D-glucopyranosyl)podophyllotoxin (11a) Yield 63%; mp 123–125 °C; [a]22.0 67.4 (c 0.21, CHCl3); 1H D NMR (CDCl3, 400 MHz) d 6.99 (s, 1H, C5-H), 6.50 (s, 1H, C8-H), 0 0 6.36 (s, 2H, C2 , C6 -H), 6.02–5.99 (m, 2H, OCH2O), 5.19 (t, 1H, 00 00 00 J = 10.0 Hz, C3 -H), 5.10–5.01 (m, 2H, C4 -H, C2 -H), 4.93 (d, 1H, 4 100 J = 8.0 Hz, C -H), 4.60 (d, 1H, J = 8.0 Hz, C -H), 4.57 (d, 1H, J = 4.0 Hz, C1-H), 4.55–4.52 (m, 1H), 4.10–4.03 (m, 3H), 3.79 (s, 0 0 0 3H, C4 -OCH3), 3.75 (s, 6H, C3 ,C5 -OCH3), 3.63–3.59 (m, 1H), 2.93– 3 2.85 (m, 1H, C -H), 2.79 (dd, 1H, J = 4.0 Hz, 10.0 Hz, C2-H), 2.33– 1.95 (m, 8H, 4  COCH2), 1.61–1.45 (m, 8H, 4  CH2CH3), 0.90– 0.85 (m, 12H, 4  CH2CH3); 13C NMR (CDCl3, 100 MHz) d 173.9 (C-12), 173.1 (C@O), 172.7 (C@O), 172.0 (C@O), 171.8 (C@O),

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152.7 (C-30 , C-50 ), 148.2 (C-6), 147.6 (C-7), 137.4 (C-40 ), 135.3 (C-10 ), 132.5 (C-10), 129.3 (C-9), 109.7 (C-8), 108.5 (C-20 , C-60 ), 107.7 (C5), 101.6 (OCH2O), 98.1 (C-100 ), 79.5 (C-4), 71.4, 71.3, 71.2, 71.1, 68.3 (C-11), 61.9 (C-600 ), 60.7 (40 -OCH3), 56.4 (30 ,50 -OCH3), 45.7 (C-2), 44.0 (C-1), 38.4 (C-3), 35.9 (COCH2), 35.9 (COCH2), 35.8 (COCH2), 35.4 (COCH2), 18.2 (CH2CH3), 18.2 (CH2CH3), 18.2 (CH2CH3), 18.1 (CH2CH3), 13.6 (CH2CH3), 13.6 (CH2CH3), 13.6 (CH2CH3), 13.5 (CH2CH3); ESIMS: m/z 879 [M+Na]+, HRESIMS: calcd for C44H56O17Na [M+Na]+ 8798.3410, found 879.3320.

Acknowledgments This work was financially supported by the Fund of State Key Laboratory of Phytochemistry and Plant Resource in West China (P2010-KF07). The authors thank the staff of analytical group of the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, for measurements of all spectra. References and notes

4.6.2. 4-O-(200 ,300 ,400 ,600 -Tetra-O-butyryl-b-D-galactopyranosyl)podophyllotoxin (11b) Yield 74%; mp 109–110 °C; [a]22.2 +19.0 (c 0.18, CHCl3); 1H NMR D 5 (CDCl3, 400 MHz) d 6.86 (s, 1H, C -H), 6.46 (s, 1H, C8-H), 6.39 (s, 2H, 0 0 00 C2 , C6 -H), 5.97–5.86 (m, 2H, OCH2O), 5.31–5.30 (m, 1H, C4 -H), 200 5.23 (dd, 1H, J = 8.0 Hz, 10.0 Hz, C -H), 4.95 (dd, 1H, J = 4.0 Hz, 00 00 10.0 Hz, C3 -H), 4.89 (d, 1H, J = 8.0 Hz, C1 -H), 4.87 (d, 1H, J = 8.0 Hz, C4-H), 4.47 (dd, 1H, J = 3.0 Hz, 10.0 Hz), 4.40–4.36 (m, 1H), 4.28–4.26 (m, 1H), 4.19 (d, 1H, J = 5.2 Hz, C1-H), 4.14–4.12 0 0 0 (m, 2H), 3.86 (s, 3H, C4 -OCH3), 3.81 (s, 6H, C3 , C5 -OCH3), 3.31 2 (dd, 1H, J = 4.0 Hz, 10.0 Hz, C -H), 3.16–3.10 (m, 1H, C3-H), 2.41– 2.16 (m, 8H, 4  COCH2), 1.71–1.52 (m, 8H, 4  CH2CH3), 0.99– 0.88 (m, 12H, 4  CH2CH3); 13C NMR (CDCl3, 100 MHz) d 178.2 (C-12), 173.1 (C@O), 172.8 (C@O), 172.6 (C@O), 171.7 (C@O), 153.5 (C-30 , C-50 ), 148.3 (C-6), 146.7 (C-7), 137.5 (C-40 ), 137.1 (C-10 ), 132.2 (C-10), 127.0 (C-9), 109.6 (C-8), 107.6 (C-5), 105.6 (C-20 , C-60 ), 101.4 (OCH2O), 98.7 (C-100 ), 75.5 (C-4), 71.2, 70.7, 68.4, 68.3 (C-11), 67.0, 61.6 (C-600 ), 61.9 (40 -OCH3), 56.2 (30 ,50 OCH3), 45.0 (C-2), 44.0 (C-1), 39.1 (C-3), 36.0 (COCH2), 35.9 (COCH2), 35.8 (COCH2), 35.7 (COCH2), 18.6 (CH2CH3), 18.3 (CH2CH3), 18.3 (CH2CH3), 18.0 (CH2CH3), 13.7 (CH2CH3), 13.6 (CH2CH3), 13.5 (CH2CH3), 13.5 (CH2CH3); ESIMS: m/z 879 [M+Na]+, HRESIMS: calcd for C44H56O17Na [M+Na]+ 879.3410, found 879.3322. 4.7. Cell culture and cytotoxicity assay The following human tumor cell lines were used: HL-60, SMMC7721, A-549, MCF-7, and SW480. All the cells were cultured in RMPI-1640 or DMEM medium (Hyclone, Logan, UT, USA), supplemented with 10% fetal bovine serum (Hyclone) at 37 °C in a humidified atmosphere with 5% CO2. Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO, USA). Briefly, adherent cells (100 lL) were seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition, both with an initial density of 1  105 cells/mL in 100 lL of medium. Each tumor cell line was exposed to the test compound at various concentrations in triplicate for 48 h. After the incubation, MTT (100 lg) was added to each well, and the incubation continued for 4 h at 37 °C. The cells lysed with SDS (200 lL) after removal of 100 lL of medium. The optical density of lysate was measured at 595 nm in a 96-well microtiter plate reader (Bio-Rad 680). The IC50 value of each compound was calculated by Reed and Muench’s method.

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