Literature DB >> 24232736

Synthesis and anticancer activity of glucosylated podophyllotoxin derivatives linked via 4β-triazole rings.

Cheng-Ting Zi¹, Feng-Qing Xu, Gen-Tao Li, Yan Li, Zhong-Tao Ding, Jun Zhou, Zi-Hua Jiang, Jiang-Miao Hu.

Abstract

A series of 4β-triazole-linked glucose podophyllotoxin conjugates have been designed and synthesized by employing a click chemistry approach. All the compounds were evaluated for their anticancer activity against a panel of five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, SW480) using MTT assays. Most of these triazole derivatives have good anticancer activity. Among them, compound 35 showed the highest potency against all five cancer cell lines tested, with IC₅₀ values ranging from 0.59 to 2.90 μM, which is significantly more active than the drug etoposide currently in clinical use. Structure-activity relationship analysis reveals that the acyl substitution on the glucose residue, the length of oligoethylene glycol linker, and the 4'-demethylation of podophyllotoxin scaffold can significantly affect the potency of the anticancer activity. Most notably, derivatives with a perbutyrylated glucose residue show much higher activity than their counterparts with either a free glucose or a peracetylated glucose residue.

Entities: CellLine Chemical Disease Gene Species

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Year: 2013 PMID： 24232736 PMCID： PMC6270044 DOI： 10.3390/molecules181113992

Source DB: PubMed Journal: Molecules ISSN： 1420-3049 Impact factor: 4.411

1. Introduction

Podophyllotoxin (1, Figure 1), which is a lignan mainly isolated from Podophyllum peltatum and Podophyllum hexandrum [1,2], shows strong cytotoxic activity against various cancer cell lines by inhibiting tubulin polymerization and preventing microtubule formation. 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 [3]. The unique cyclolignan scaffold of 1 has however drawn a lot of attention for the discovery and development of new anticancer agents. Extensive structural modifications, particularly at the C-4 and C-4' position of podophyllotoxin have led to the development of many semisynthetic derivatives of podophyllotoxin [4,5,6]. Among them, five semisynthetic derivatives, etoposide (2), teniposide (3), etopophos (4), GL-331 (5) and TOP-53 (6) (Figure 1) are currently used in the chemotherapy for a variety of cancers, including small-cell lung cancer, non-Hodgkin’s lymphoma, leukemia, Kaposi’s sarcoma, neurobslastoma and soft tissue sarcoma. These derivatives display binding activity to DNA topoisomerase II during the late S and early G2 cell cycle stages and are potent inhibitors of the enzyme [7,8,9,10,11,12,13,14]. Their anticancer activity proceeds through a mechanism of action entirely different from that of their parent compound podophyllotoxin (1). Etoposide (2), teniposide (3), and etopophos (4) are three semisynthetic glucosidic cyclic acetals of 1, and in particular, etoposide (2) is considered to be one of the most successful pharmaceuticals derived from plants. Both GL-331 (5) and TOP-53 (6) are more active than etoposide (2) and are currently under clinical investigation [12].

Figure 1

Structures of podophyllotoxin (1) and its semisynthetic derivatives.

Structures of podophyllotoxin (1) and its semisynthetic derivatives. Recently, novel podophyllotoxin hybrids obtained by covalently linking another biologically active molecule to podophyllotoxin have been reported. For example, thiocolchicine-podophyllotoxin conjugates were reported to have improved solubility and anticancer activity [15]. In addition, a series of conjugates of podophyllotoxin with 5-fluorouracil (5-FU) were reported to have better cytotoxic activity than VP-16 [16]. Structure-activity relationship (SAR) studies [17] have demonstrated that C-4 is the molecular area tolerable to significant structural diversification. Chemotherapeutic agents such as the podophyllotoxin derivatives 2–4 are often associated with undesirable side effects and the development of multi-drug resistance by cancer cells. Thus, structural modification of podophyllotoxin for developing new antitumor drugs with increased selectivity and reduced toxicity is highly desirable. In recent years, the altered glucose metabolism in cancer cells has been explored for targeted cancer therapy [18]. Glucose is the main source of metabolic energy of animal cells, generating ATP through glycolysis and oxidative phosphorylation. Cancer cells are well known to display an enhanced uptake and consumption of glucose, which is metabolized primarily through the fermentative pathway instead of tricarboxylic acid cycle and oxidative phosphorylation in the mitochondria of normal cells [19]. The transport of glucose across the plasma membrane into the cytosol is mediated by a family of glucose transporters (GLUTs) [20,21]. Due to their enhanced glucose consumption, cancer cells generally express higher levels of GLUTs than normal cells [22]. For example, glucose transporter class 1 (GLUT1) has been found to be overexpressed in a variety of both solid and hematological malignancies such as large B-cell lymphoma, colorectal carcinomas, hepatocellular carcinoma, head and neck cancer, gastrointestinal stromal tumor (GIST), prostate carcinoma, thyroid carcinoma, renal cell cancer, lung cancer, pancreatic cancer, sarcomas and laryngeal carcinomas [19]. Thus, in this study we planned to covalently link a glucose residue to podopyllotoxin so the resulting cytotoxic agents may be preferably taken up by cancer cells through the mediation of GLUTs. Recently, the click reaction has been widely used to covalently link two molecular fragments in creating a wide variety of drug-like molecules [23,24]. Typically, a terminal alkyne and an azide undergo a copper-catalyzed [3+2]-cycloaddition to generate a substituted 4β-triazole ring [25,26]. Podophyllotoxin derivatives containing the featured 4β-triazole ring have also been reported as potential DNA topoisomerase-II inhibitors [27], including a few compounds bearing a sugar residue [28]. Through click reactions we have now synthesized a series of glucose-podophyllotoxin conjugates in order to systematically study the effect of: (a) the length of the linker; (b) the substituent on the glucose; (c) the configuration of the anomeric carbon of glucose residue; and (d) the substituent on the 4-position of the E-ring of the podophyllotoxin scaffold on the anticancer activity of such conjugates. Herein we report the synthesis, the preliminary anticancer activity and the structure activity relationship of these conjugates.

2. Results and Discussion

2.1. Chemical Synthesis

The preparation of terminal-alkynes is shown in Scheme 1. Compounds 10 and 11 were prepared in 70% yield by treatment of triethylene glycol and hexaethylene glycol with sodium hydride and propargyl bromide as previous described [29,30]. Fisher glycosylation of D-glucose with propargyl alcohols 9–11 in the presence of H2SO4-silica as a catalyst [31] provided the corresponding glycosides 12–17 as α/β mixtures. The α-isomer was usually obtained as the major isomer, and the α/β ratio was typically 6:1. The preparation of compounds 12 [32], 13 [32] and 15 [33] using a similar method has been reported in the literature. The major α-glycosides 12, 14 and 16 were subjected to peracetylation and perbutyrylation with acetic anhydride and butyric anhydride in the presence of pyridine, to give the corresponding peracetylated (compounds 18–20) and perbutyrylated products (compounds 21–23), respectively, in 92%–98% yield. The preparation of compound 18 has been described in literature [34].

Scheme 1

Synthesis of glucosylated terminal alkynes.

Synthesis of glucosylated terminal alkynes. Reagents and Reaction Conditions: (a) propargyl bromide, sodium hydride, THF, reflux; (b) cat. H2SO4-silica, D-glucose; (c) acetic anhydride or n-butyric anhydride, pyridine, 0 °C–rt. To introduce the azido functionality for the click-reaction, podophyllotoxin was readily converted to 4β-azido-4-deoxypodophyllotoxin (7) and 4β-azido-4-deoxy-4'-demethypodophyllotoxin (8) according to known procedures [35,36]. The glycosylated terminal alkynes 12–23 were allowed to react with azide 7 or 8 in the presence of copper (II) acetate and sodium ascorbate to yield a series of 4β-triazole-linked glucose-podophyllotoxin conjugates (Scheme 2, Table 1).

Scheme 2

Click-chemistry strategy for the synthesis of 4β-triazole-linked glucose podophyllotoxin conjugates.

Table 1

A series of 4β-triazole-linked glucose-podophyllotoxin conjugates.

Compounds	n	R	R₁	1'''–configuration	Yield% ^a
24	0	CH₃	H	α	98
25	0	CH₃	CH₃CO	α	92
26	0	CH₃	C₃H₇CO	α	98
27	0	H	H	α	98
28	0	H	CH₃CO	α	94
29	0	H	C₃H₇CO	α	96
30	3	CH₃	H	α	91
31	3	CH₃	CH₃CO	α	94
32	3	CH₃	C₃H₇CO	α	90
33	3	H	H	α	93
34	3	H	CH₃CO	α	94
35	3	H	C₃H₇CO	α	92
36	6	CH₃	H	α	91
37	6	CH₃	CH₃CO	α	91
38	6	CH₃	C₃H₇CO	α	91
39	6	H	H	α	93
40	6	H	CH₃CO	α	90
41	6	H	C₃H₇CO	α	97
42	0	H	H	β	97
43	3	H	H	β	92
44	6	H	H	β	94

a Total isolated yield (%) was calculated after column chromatography.

Click-chemistry strategy for the synthesis of 4β-triazole-linked glucose podophyllotoxin conjugates. A series of 4β-triazole-linked glucose-podophyllotoxin conjugates. a Total isolated yield (%) was calculated after column chromatography. All the products were characterized by 1H-NMR, 13C-NMR, ESI-MS, and HRESI-MS. In the 1H-NMR spectra, the proton at C-4 of 4β-triazole-substituted compounds appears as a doublet at δ 5.9–6.3 ppm, usually with a coupling constant J3,4 < 5.0 Hz, indicating a cis-relationship between H-3 and H-4. The formation of the triazole ring was confirmed by the resonance of its C5"-H signal (δ 7.8–8.2 ppm) in the aromatic region in the 1H-NMR spectra, which was further supported by two characteristic carbon signals at around 145 ppm and 126 ppm in the 13C-NMR spectra. The coupling constant of the anomeric proton of the glucose residue (J1"',2"') is typically <4.0 Hz for the α-linkage and >7.6 Hz for the β-linkage. In addition, the anomeric carbon of an α-glucoside always has a lower chemical shift than the corresponding β-glucoside in the 13C-NMR spectra. Poor water-solubility is a common problem in developing podophyllotoxin derivatives for therapeutic use. For the glucose-podophyllotoxin conjugates described in this study, compounds with a peracetylated or perbutyrylated glucose residue are slightly soluble in water. In general, the conjugates with a free glucose residue are soluble in water and methanol, while those with a peracetylated or a perbutyrylated glucose residue are more soluble in chloroform. For example, at room temperature compound 29 with a perbutyrylated glucose residue has a solubility of 1.7 mg/mL in water while compound 30 with a free glucose residue has a solubility of 13.3 mg/mL in water. Overall, the inclusion of the triazole-ring and the multiple triethylene glycol units improved the aqueous solubility of these compounds, and in the case when a free glucose residue is present, the compound becomes fairly soluble in water.

2.2. Evaluation of Biological Activity

All the 4β-triazole-linked glucose-podophyllotoxin conjugates 24–44 were tested for their anticancer activity against five human cancer cell lines, including HL-60 (leukemia), SMMC-7721 (hepatoma), A-549 (lung cancer), MCF-7 (breast cancer), and SW480 (colon cancer). Etoposide and cisplatin were taken as control drugs and the anticancer activity data are presented in Table 2. Our first observation is that compounds having a free glucose residue (compounds 24, 27, 30, 33, 36, 39, and 42–44) mostly show weak activity (all having IC50 > 40 μM, except 30), while several derivatives containing a peracetylated glucose residue (compounds 28, 31 and 37) show improved activity.

Table 2

In vitro anticancer activity (IC50, μM) of 4β-triazole-linked glucose-podophyllotoxin conjugates 24–44.

Entry	IC₅₀ (μM)
Entry	HL-60	SMMC-7721	A-549	MCF-7	SW480
24	>40	>40	>40	>40	>40
25	>40	>40	>40	>40	>40
26	>40	>40	>40	>40	>40
27	>40	>40	>40	>40	>40
28	6.77	27.17	>40	34.54	>40
29	0.80	3.03	4.05	3.90	4.36
30	13.13	19.12	20.17	22.33	28.58
31	12.12	38.33	33.73	29.41	>40
32	2.05	3.38	5.55	12.49	12.59
33	>40	>40	>40	>40	>40
34	>40	>40	>40	>40	>40
35	0.59	0.99	1.38	2.90	1.50
36	>40	>40	>40	>40	>40
37	12.82	26.31	31.84	38.69	40.00
38	1.66	3.79	4.55	5.20	6.54
39	>40	>40	>40	>40	>40
40	>40	>40	>40	>40	>40
41	15.07	17.65	26.68	28.77	28.05
42	>40	>40	>40	>40	>40
43	>40	>40	>40	>40	>40
44	>40	>40	>40	>40	>40
Etoposide (2)	0.31	8.12	11.92	32.82	17.11
Cisplatin	1.17	6.43	9.24	15.86	13.42

In vitro anticancer activity (IC50, μM) of 4β-triazole-linked glucose-podophyllotoxin conjugates 24–44. A group of compounds that display relatively high potency are those bearing the perbutyrylated glucose residue (compounds 29, 32, 35 and 38). In most cases, derivatives with a perbutyrylated sugar residue are more active than those with a peracetylated glucose residue (29 vs. 28, 32 vs. 31, 35 vs. 34, 38 vs. 37, and 40 vs. 41), which in turn are more active than those with a free glucose residue (28 vs. 27 and 37 vs. 36). The length of the linking spacer between the glucose moiety and the 1,2,3-triazole residue does not exhibit a uniform effect on the cytotoxic potency of these compounds. For example, among the set of compounds with a perbutyrylated sugar moiety and a methoxyl group at the C-4'-position (compounds 26, 32 and 38), compound 38 with the longest linking spacer (six ethylene glycol repeating units) is the most active compound against four out of five cancer cell lines tested, followed by 32 which has a shorter linker (three ethylene glycol repeating units), and 26 which has no linking spacer in-between is the least active. However, among the set of compounds with a perbutyrylated sugar moiety and a hydroxyl group at the C-4'-position (compounds 29, 35 and 41), compound 41 with the longest linking spacer is the least active while 35 with the shorter linker is the most active against all five cancer cell lines. Methylation of 4'-OH group in the E ring can lead to either increased or decreased activity, as indicated by the IC50 values of the pairs of compounds between 25/31/37 vs. 28/34/40 and 26/32/38 vs. 29/35/41. The effect of glycosidic linkage on the cytotoxicity potency of these compounds can’t be concluded from this study because the exact IC50 values for the three pairs of α/β isomers (27/33/39 vs. 42/43/44) have not been determined (all having IC50 > 40 μM against all cancer cells tested). Previously, Reddy and co-workers [28] reported the cytotoxic activity of several 4β-triazole-linked sugar-podophyllotoxin conjugates. The cytotoxic potency of compound 42 against A-549 cells reported in their paper was quite similar to our data, however, our findings that peracetylation and perbutyrylation of the glucose residue lead to increased activity are different from their observation [28]. Among all the synthesized compounds, several compounds (i.e., 32, 35 and 38) display significantly higher activity than etoposide (2), and in general derivatives containing a perbutyrylated D-glucose moiety are more active than other derivatives. n-Butyrate, a naturally occurring short-chain fatty acid, is a well known histone deacetylase (HDAC) inhibitor [37]. In recent years, HDAC inhibition has attracted much attention for the development of anticancer drugs because HDAC inhibitors are able to disrupt cell cycle progression or selectively induce apoptosis via depression of certain genes [38,39,40,41]. Thus, the butyrate species liberated from the hydrolysis of these butyrylated derivatives may have contributed to the apparently higher potency of these compounds. Further studies are needed in order to confirm the potential role of the butyryl substituents on the sugar residue.

3. Experimental

3.1. General

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 DRX-500 or Bruker AVANCE Ш-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.

3.2. General Procedure for Fisher Glycosylation Catalyzed with H2SO4-on-Silica Gel (Preparation of 12–17)

d-Glucose (5 mmol) was suspended in propargyl alcohol (25 mmol) and stirred at 65 °C. H2SO4-silica (25 mg) was added and stirring was continued until all solids had dissolved (~2.5 h). After cooling to room temperature, the reaction mixture was transferred to a short silica gel column and eluted using CHCl3/CH3OH = 9:1. The preparation of 12, 13 and 15 using the same method has been reported in the literature [32,33]. 2-[2-[2-(2-Propyn-1-yloxy)ethoxy]ethoxy-α- (14). 1H-NMR (CD3OD, 400 MHz): δ 4.78 (d, 1H, J = 3.7 Hz, C1-H), 4.14 (d, 2H, J = 2.4 Hz, CH2-C≡C), 3.74 (d, 2H, J = 1.8 Hz), 3.61 (t, 6H, J = 1.1 Hz), 3.55 (t, 6H, J = 3.7 Hz), 3.33–3.34 (m, 4H), 3.25 (s, 4H), 2.82 (t, 1H, J = 2.3 Hz, C≡CH); 13C-NMR (CD3OD, 100 MHz): δ 100.2 (C-1), 80.6 (C≡CH), 76.1 (C≡CH), 75.1, 73.6, 71.7, 71.4, 71.3, 70.0, 68.1, 62.6 (C-6), 59.0 (CH2-C≡C); ESIMS: m/z 373 [M+Na]+. 3,6,9,12,15,18-Hexaoxaheneicos-20-yn-1-yl-α- (16). 1H-NMR (CD3OD, 400 MHz): δ 4.82 (d, 1H, J = 3.6 Hz, C1-H), 4.18 (d, 2H, J = 2.0 Hz, CH2-C≡C), 3.78 (d, 1H, J = 11.4 Hz), 3.69 (t, 12H, J = 5.1 Hz), 3.64 (t, 12H, J = 2.0 Hz), 3.38–3.34 (m, 4H), 2.87 (s, 4H), 2.87 (t, 1H, J = 1.9 Hz, C≡CH); 13C-NMR (CD3OD, 100 MHz): δ 100.3 (C-1), 80.6 (C≡CH), 76.0 (C≡CH), 75.1, 73.6, 71.8, 71.5, 71.3, 70.1, 68.1, 62.6 (C-6), 59.0 (CH2-C≡C); ESIMS: m/z 505 [M+Na]+. 3,6,9,12,15,18-Hexaoxaheneicos-20-yn-1-yl-β- (17). 1H-NMR (CD3OD, 400 MHz): δ 4.31 (d, 1H, J = 7.8 Hz, C1-H), 4.21 (d, 2H, J = 2.4 Hz, CH2-C≡C), 3.86 (d, 1H, J = 11.6 Hz), 3.68 (m, 12H), 3.66 (m, 12H), 3.21 (s, 4H), 2.88 (t, 1H, J = 2.4 Hz, C≡CH); 13C-NMR (CD3OD, 100 MHz): δ 104.4 (C-1), 80.6 (C≡CH), 77.9, 77.8, 75.9 (C≡CH), 71.6, 71.5, 71.5, 71.5, 71.3, 70.1, 70.0, 62.8, 59.0 (CH2-C≡C); ESIMS: m/z 505 [M+Na]+.

3.3. General Procedure for Acetylation and Butyrylation of the Glucoside (Preparation of 18–23)

To a solution of a propargyl glycosides 12–17 (1 mmol) in pyridine (4.0 mL) at 0 °C, acetic anhydride (or butyryl anhydride) (4.0 mL) was added. The reaction mixture was stirred overnight until the starting material disappeared as indicated by TLC. The reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (3 × 20 mL). The organic layer was washed with 10% aqueous hydrochloric acid (20 mL) and brine (20 mL). The organic layer was dried over magnesium sulfate and evaporated to give a residue, which was chromatographed on silica gel with petroleum ether/acetone = 4:1→2:1 to give the peracetylated or perbutyrylated product. The preparation of 18 using the same method has been reported in the literature [34]. 2-[2-[2-(2-Propyn-1-yloxy)ethoxy]ethoxy-per-O-acetyl-α- (19). Yield: 96%. 1H-NMR (CD3OD, 400 MHz): δ 5.43 (t, 1H, J = 9.9 Hz, C3-H), 5.12 (d, 1H, J = 2.9 Hz, C1-H), 5.03 (t, 1H, J = 9.8 Hz, C4-H), 4.82 (d, 1H, J = 2.9 Hz, C2-H), 4.28–4.24 (m, 3H, C6-CHa, CH2-C≡C), 4.19–4.09 (m, 2H, C6-CHb, C5-H), 3.67 (m, 6H), 3.65 (m, 6H), 2.85 (s, 1H, C≡CH), 2.06 (s, 3H, COCH3), 2.05 (s, 3H, COCH3), 2.02 (s, 3H, COCH3), 1.99 (s, 3H, COCH3); 13C-NMR (CD3OD, 100 MHz): δ 172.3 (C=O), 171.6 (C=O), 171.6 (C=O), 171.3 (C=O), 97.0 (C-1), 80.7 (C≡CH), 76.0 (C≡CH), 72.1, 71.7, 71.6, 71.5, 71.4, 71.2, 70.1, 70.0, 68.7, 68.6, 63.2 63.2 (C-6), 59.0 (CH2-C≡C), 20.7 (COCH3), 20.7 (COCH3), 20.7 (COCH3), 20.7 (COCH3); ESIMS: m/z 541 [M+Na]+. 3,6,9,12,15,18-Hexaoxaheneicos-20-yn-1-yl-per-O-butyryl-α- (20). Yield: 96%. 1H-NMR (CD3OD, 400 MHz): δ 5.43 (t, 1H, J = 9.8 Hz, C3-H), 5.12 (d, 1H, J = 3.5 Hz, C1-H), 5.03 (t, 1H, J = 9.7 Hz, C4-H), 4.82 (dd, 1H, J = 3.7 Hz, 10.3 Hz, C2-H), 4.27–4.24 (m, 3H, C6-CHa, CH2-C≡C), 4.17–4.12 (m, 2H, C6-CHb, C5-C), 3.65 (m, 12H), 3.63 (m, 12H), 2.86 (s, 1H, C≡CH), 2.06 (s, 3H, COCH3), 2.05 (s, 3H, COCH3), 2.02 (s, 3H, COCH3), 2.00 (s, 3H, COCH3); 13C-NMR (CD3OD, 100 MHz): δ 172.3 (C=O), 171.7 (C=O), 171.7 (C=O), 171.3 (C=O), 97.1 (C-1), 80.7 (C≡CH), 76.0 (C≡CH), 72.1, 71.7, 71.6, 72.6, 72.5, 71.4, 71.2, 70.1, 70.0, 63.2 (C-6), 59.0 (CH2-C≡C), 20.7 (COCH3), 20.7 (COCH3), 20.7 (COCH3), 20.7 (COCH3) ; ESIMS: m/z 673 [M+Na]+. 2-Propyn-1-yl-per-O-butyryl-α- (21). Yield: 92%. 1H-NMR (CD3OD, 400 MHz): δ 5.45 (t, 1H, J = 9.9 Hz, C3-H), 5.29 (d, 1H, J = 3.6 Hz, C1-H), 5.12 (t, 1H, J = 9.8 Hz, C4-H), 4.90 (dd, 1H, J = 3.7 Hz, 10.3 Hz, C2-H), 4.35–4.24 (m, 3H, C6-CHa, CH2-C≡C), 4.13–4.09 (m, 2H, C6-CHb, C5-H), 2.91 (s, 1H, C≡CH), 2.34–2.24 (m, 2H, COCH2), 2.34-2.24 (m, 2H, COCH2), 2.34–2.24 (m, 2H, COCH2), 2.34–2.24 (m, 2H, COCH2), 1.66–1.56 (m, 2H, CH2CH3), 1.66–1.56 (m, 2H, CH2CH3), 1.66–1.56 (m, 2H, CH2CH3), 1.66–1.56 (m, 2H, CH2CH3), 0.96 (t, 3H, J = 7.4 Hz, CH2CH3), 0.94 (t, 3H, J = 7.4 Hz, CH2CH3), 0.92 (t, 3H, J = 7.4 Hz, CH2CH3), 0.90 (t, 3H, J = 7.4 Hz, CH2CH3); 13C-NMR (CD3OD, 100 MHz): δ 174.6 (C=O), 174.0 (C=O), 173.9 (C=O), 173.5 (C=O), 95.6 (C-1), 79.5 (C≡CH), 77.1 (C≡CH), 71.8, 71.6, 71.0, 69.5, 69.3, 62.9 (C-6), 56.0 (CH2-C≡C), 36.9 (CH2C=O), 36.8 (CH2C=O), 36.8 (CH2C=O), 36.8 (CH2C=O), 19.5 (CH2CH3), 19.4 (CH2CH3), 19.4 (CH2CH3), 19.3 (CH2CH3), 14.1 (CH2CH3), 14.1 (CH2CH3), 14.1 (CH2CH3), 14.1 (CH2CH3); ESIMS: m/z 522 [M+Na]+. 2-[2-[2-(2-Propyn-1-yloxy)ethoxy]ethoxy-per-O-butyryl-α- (22). Yield: 92%. 1H-NMR (CD3OD, 400 MHz): δ 5.48 (d, 1H, J = 9.9 Hz, C3-H), 5.12 (d, 1H, J = 3.2 Hz, C1-H), 5.10 (t, 1H, J = 9.7 Hz, C4-H), 4.82 (d, 1H, J = 3.1 Hz, C2-H), 4.25–4.18 (m, 3H, C6-CHa, CH2-C≡C), 4.15–4.10 (m, 2H, C6-CHb, C5-H), 3.68 (m, 6H), 3.65 (m, 6H), 2.85 (s, 1H, C≡CH), 2.35-2.20 (m, 2H, COCH2), 2.35–2.20 (m, 2H, COCH2), 2.35–2.20 (m, 2H, COCH2), 2.35–2.20 (m, 2H, COCH2), 1.65–1.56 (m, 2H, CH2CH3), 1.65–1.56 (m, 2H, CH2CH3), 1.65–1.56 (m, 2H, CH2CH3), 1.65–1.56 (m, 2H, CH2CH3), 0.96 (t, 3H, J = 3.2 Hz, CH2CH3), 0.94 (t, 3H, J = 3.2 Hz, CH2CH3), 0.92 (t, 3H, J = 3.2 Hz, CH2CH3), 0.90 (t, 3H, J = 3.2 Hz, CH2CH3); 13C-NMR (CD3OD, 100 MHz): δ 174.7 (C=O), 174.0 (C=O), 173.9 (C=O), 173.6 (C=O), 97.1 (C-1), 80.6 (C≡CH), 76.0 (C≡CH), 72.1, 71.7, 71.6, 71.5, 71.2, 71.2, 70.1, 69.6, 68.7, 62.9 (C-6), 59.0 (CH2-C≡C), 36.9 (CH2C=O), 36.8 (CH2C=O), 36.8 (CH2C=O), 36.7 (CH2C=O), 19.4 (CH2CH3), 19.4 (CH2CH3), 19.3 (CH2CH3), 19.3 (CH2CH3), 14.0 (CH2CH3), 14.0 (CH2CH3), 14.0 (CH2CH3), 14.0 (CH2CH3); ESIMS: m/z 653 [M+Na]+. 3,6,9,12,15,18-Hexaoxaheneicos-20-yn-1-yl-per-O-butyryl-α- (23). Yield: 92%. 1H-NMR (CD3OD, 400 MHz): δ 5.45 (t, 1H, J = 9.8 Hz, C3-H), 5.12 (d, 1H, J = 3.5 Hz, C1-H), 5.07 (t, 1H, J = 9.7 Hz, C4-H), 4.85 (dd, 1H, J = 11.6 Hz, 3.6 Hz, C2-H), 4.21–4.19 (m, 3H, C6-CHa, CH2-C≡C), 4.19–4.13 (m, 2H, C6-CHb, C5-H), 3.65 (m, 12H), 3.65 (m, 12H), 3.63 (m, 12H), 2.86 (s, 1H, C≡CH), 2.33–2.22 (m, 2H, COCH2), 2.33–2.22 (m, 2H, COCH2), 2.33–2.22 (m, 2H, COCH2), 2.33–2.22 (m, 2H, COCH2), 1.65–1.56 (m, 2H, CH2CH3), 1.65–1.56 (m, 2H, CH2CH3), 1.65–1.56 (m, 2H, CH2CH3), 1.65–1.56 (m, 2H, CH2CH3), 0.96 (t, 3H, J = 3.8 Hz, CH2CH3), 0.94 (t, 3H, J = 3.8 Hz, CH2CH3), 0.92 (t, 3H, J = 3.8 Hz, CH2CH3), 0.91 (t, 3H, J = 3.8 Hz, CH2CH3); 13C-NMR (CD3OD, 100 MHz): δ 174.7 (C=O), 174.0 (C=O), 173.9 (C=O), 173.6 (C=O), 97.1 (C-1), 80.6 (C≡CH), 76.0 (C≡CH), 72.1, 71.7, 71.6, 71.6, 71.6, 71.6, 71.4, 71.3, 71.2, 70.1, 69.6, 68.7, 63.0 (C-6), 59.0 (CH2-C≡C), 36.9 (CH2C=O), 36.8 (CH2C=O), 36.7 (CH2C=O),. 36.7 (CH2C=O), 19.4 (CH2CH3), 19.3 (CH2CH3), 19.3 (CH2CH3), 19.3 (CH2CH3), 14.0 (CH2CH3), 14.0 (CH2CH3), 14.0 (CH2CH3), 14.0 (CH2CH3); ESIMS: m/z 785 [M+Na]+.

3.4. Click Chemistry-General Procedure

To a solution of a terminal-alkyne 12–23 (0.25 mmol) and 4β-azido-podophyllotoxin analogue (7 or 8, 0.25 mmol) in t−BuOH/H2O (1:2, 1.0 mL) and THF (1.0 mL) at room temperature were added copper (II) acetate (4.6 mg, 0.025 mmol) and sodium ascorbate (1.0 M in H2O, 0.1 mL). The reaction mixture was stirred at room temperature for 31 h until the starting material disappeared as indicated by TLC. Then, the mixture was diluted with water (30 mL) and extracted with ethyl acetate (3 × 20 mL), and the combined organic layer was dried over sodium sulfate. The solvent was evaporated and the residue was purified by column chromatography to afford the cycloaddition product. 4β-[4-(α- (24). White amorphous powder, yield 98% (after chromatography with CHCl3/CH3OH, 9:1); mp 168 °C; [α]D24.1: +14.2 (c 0.22, CH3OH); 1H-NMR (CD3OD, 500 MHz): δ 7.84 (s, 1H, C5''-H), 6.65 (s, 1H, C5-H), 6.58 (s, 1H, C8-H), 6.41 (s, 2H, C2', C6'-H), 6.22 (d, 1H, J = 4.7 Hz, C4-H), 5.93 (d, 2H, J = 7.2 Hz, OCH2O), 4.90 (d, 1H, J = 2.8 Hz, C1'''-H), 4.76 (d, 1H, J = 4.0 Hz, C1-H), 4.66 (s, 2H, C6''-CH2); 4.63–4.61 (m, 1H), 4.38–4.34 (m, 1H), 3.85–3.82 (m, 2H), 3.80 (s, 2H, C6'''-CH2), 3.72 (s, 6H, C3'', C5''-OCH3), 3.70 (s, 3H, C4''-OCH3), 3.68–3.58 (m, 2H), 3.45-3.14 (m, 4H); 13C-NMR (CD3OD, 125 MHz): δ 175.8 (C-12), 153.9 (C-3', C-5'), 150.5 (C-7), 149.2 (C-6), 145.6 (C-4''), 138.2 (C-1'), 136.7 (C-9), 134.7 (C-10), 126.8 (C-4'), 125.9 (C-5''), 111.1 (C-5), 109.8 (C-8), 109.3 (C-2', C-6'), 103.3 (OCH2O), 99.7 (C-1'''), 74.9 (C-5'''), 73.9 (C-3'''), 73.3 (C-2'''), 71.7 (C-4'''), 68.9 (C-11), 62.6 (C-6''), 61.5 (C-6'''), 61.1 (4'-OCH3), 59.8 (C-2), 56.6 (3', 5'-OCH3), 44.8 (C-4), 42.4 (C-1), 38.5 (C-3); ESIMS: m/z 680 [M+Na]+, HRESIMS: calcd for C31H35N3O13H [M+H]+ 658.2243, found 658.2223. 4β-{4-[1-(2,3,4,6-Tetra-O-acetyl-α- (25).White amorphous powder, yield 92% (after chromatography with petroleum ether/acetone, 1:1); mp 137 °C; [α]D24.6: +16.3 (c 0.28, CH3OH); 1H-NMR (CD3OD, 400 MHz): δ 7.87 (s, 1H, C5''-H), 6.70 (s, 1H, C5-H), 6.59 (s, 1H, C8-H), 6.41 (s, 2H, C2', C6'-H), 6.25 (d, 1H, J = 4.7 Hz, C4-H), 5.95 (d, 2H, J = 8.3 Hz, OCH2O), 5.38 (t, 1H, J = 9.7 Hz, C3'''-H), 5.18 (d, 1H, J = 3.6 Hz, C1'''-H), 5.03 (t, 1H, J = 8.0 Hz, C4'''-H), 4.79–4.67 (m, 5H, C6''-CH2, C1-H, C2'''-H, C5'''-H), 4.39–4.35 (m, 1H), 4.26–4.20 (m, 1H), 4.11–4.01 (m, 2H), 3.42 (dd, 1H, J = 4.0 Hz, 16.0 Hz, C2-H), 3.21–3.16 (m, 1H, C3-H), 2.04 (s, 3H, COCH3), 1.99 (s, 3H, COCH3), 1.97 (s, 3H, COCH3), 1.95 (s, 3H, COCH3); 13C-NMR (CD3OD, 100 MHz): δ 175.8 (C-12), 172.3 (C=O), 171.7 (C=O), 171.5 (C=O), 171.3 (C=O), 153.9 (C-3', C-5'), 150.5 (C-7), 149.3 (C-4''), 144.5 (C-6), 138.3 (C-1'), 136.7 (C-9), 134.7 (C-10), 126.9 (C-4'), 126.4 (C-5''), 111.2 (C-5), 109.9 (C-8), 109.4 (C-2', C-6'), 103.3 (OCH2O), 95.6 (C-1'''), 72.2 (C-5'''), 71.2 (C-3'''), 69.8 (C-2'''), 68.9 (C-11), 68.8 (C-4'''), 63.1 (C-6''), 61.3 (C-6'''), 61.1 (4'-OCH3), 59.8 (C-2), 56.6 (3', 5'-OCH3), 44.9 (C-4), 42.5 (C-1), 38.5 (C-3), 20.7 (COCH3), 20.7 (COCH3), 20.7 (COCH3), 20.7 (COCH3); ESIMS: m/z 848 [M+Na]+, HRESIMS: calcd for C39H43N3O17Na [M+Na]+ 848.2485, found 848.2472. 4β-{4-[1-(2,3,4,6-Tetra-O-butyryl-α- (26). White amorphous powder, yield 98% (after chromatography with petroleum ether/acetone, 1:1); mp 101 °C; [α]D25.1: −41.3 (c 0.23, CH3OH); 1H-NMR (CD3OD, 400 MHz): δ 7.74 (s, 1H, C5''-H), 6.67 (s, 1H, C5-H), 6.59 (s, 1H, C8-H), 6.41 (s, 2H, C2', C6'-H), 6.24 (d, 1H, J = 4.5 Hz, C4-H), 5.95 (d, 2H, J = 5.6 Hz, OCH2O), 5.26 (t, 1H, J = 9.4 Hz, C3'''-H), 5.08 (t, 1H, J = 12.0 Hz, C4'''-H), 4.93–4.73 (m, 6H, C1'''-H, C6''-CH2, C1-H, C2'''-H, C5'''-H), 4.33-3.89 (m, 4H), 3.72 (s, 6H, C3'', C5''-OCH3), 3.70 (s, 3H, C4''-OCH3), 3.40 (dd, 1H, J = 4.0 Hz, 16.0 Hz, C2-H), 3.14–3.10 (m, 1H, C3-H), 2.31–2.13 (m, 2H, COCH2), 2.31–2.13 (m, 2H, COCH2), 2.31–2.13 (m, 2H, COCH2), 2.31–2.13 (m, 2H, COCH2), 1.63–1.47 (m, 2H, CH2CH3), 1.63–1.47 (m, 2H, CH2CH3), 1.63–1.47 (m, 2H, CH2CH3), 1.63-1.47 (m, 2H, CH2CH3), 0.92 (t, 3H, J = 4.1 Hz, CH2CH3), 0.90 (t, 3H, J = 4.1 Hz, CH2CH3), 0.87 (t, 3H, J = 4.1 Hz, CH2CH3), 0.83 (t, 3H, J = 4.1 Hz, CH2CH3); 13C-NMR (CD3OD, 100 MHz): δ 175.6 (C-12), 174.6 (C=O), 173.8 (C=O), 173.5 (C=O), 173.5 (C=O), 153.9 (C-3', C-5'), 150.5 (C-7), 149.2 (C-4''), 145.3 (C-6), 138.3 (C-1'), 136.6 (C-9), 134.7 (C-10), 126.9 (C-4'), 125.9 (C-5''), 111.2 (C-5), 109.8 (C-8), 109.4 (C-2', C-6'), 103.3 (OCH2O), 101.1 (C-1'''), 73.8 (C-5'''), 73.1 (C-3'''), 72.4 (C-2'''), 69.4 (C-4'''), 68.8 (C-11), 63.3 (C-6''), 62.7 (C-6'''), 61.1 (4'-OCH3), 59.8 (C-2), 56.6 (3', 5'-OCH3), 44.9 (C-4), 42.5 (C-1), 38.5 (C-3), 36.8 (COCH2), 36.7 (COCH2), 36.7 (COCH2), 36.7 (COCH2), 19.4 (CH2CH3), 19.3 (CH2CH3), 19.3 (CH2CH3), 19.2 (CH2CH3), 14.0 (CH2CH3), 14.0 (CH2CH3), 14.0 (CH2CH3), 14.0 (CH2CH3); ESIMS: m/z 960 [M+Na]+, HRESIMS: calcd for C47H59N3O17H [M+H]+ 938.3917, found 938.3877. 4β-[4-(α- (27). White amorphous powder, yield 98% (after chromatography with CHCl3/CH3OH, 9:1); mp 227 °C; [α]D24.9: +1.0 (c 0.30, CH3OH); 1H-NMR (CD3OD, 400 MHz): δ 7.87 (s, 1H, C5''-H), 6.68 (s, 1H, C5-H), 6.64 (s, 1H, C8-H), 6.38 (s, 2H, C2', C6'-H), 6.26 (d, 1H, J = 4.8 Hz, C4-H), 5.97 (d, 2H, J = 9.7 Hz, OCH2O), 4.84–4.42 (m, 6H, C1'''-H, C1-H, C6''-CH2, C11-CH2), 3.83–3.78 (m, 3H, C3'''-H, C6'''-CH2), 3.75 (s, 6H, C3'', C5''-OCH3), 3.67–3.60 (m, 3H), 3.43–3.34 (m, 1H, C2-H), 3.26–3.18 (m, 1H, C3-H); 13C-NMR (CD3OD, 100 MHz): δ 176.5 (C-12), 150.7 (C-7), 149.6 (C-6), 148.1 (C-3', C-5'), 145.7 (C-4''), 135.9 (C-1'), 135.2 (C-9), 131.5 (C-10), 126.8 (C-4'), 126.2 (C-5''), 111.3 (C-5), 109.2 (C-8), 109.9 (C-2', C-6'), 103.4 (OCH2O), 99.8 (C-1'''), 74.9 (C-5'''), 74.0 (C-3'''), 73.4 (C-2'''), 71.7 (C-4'''), 69.2 (C-11), 62.7 (C-6''), 61.5 (C-6'''), 60.1 (C-2), 56.9 (3', 5'-OCH3), 44.8 (C-4), 42.8 (C-1), 38.6 (C-3); ESIMS: m/z 667 [M+Na]+, HRESIMS: calcd for C30H34N3O13Na [M+Na]+ 667.1984, found 667.1961. 4β-{4-[1-(2,3,4,6-Tetra-O-acetyl-α- (28). White amorphous powder, yield 94% (after chromatography with petroleum ether/acetone, 1:1); mp 128 °C; [α]D24.8: +27.0 (c 0.14, CH3OH); 1H-NMR (CD3OD, 400 MHz): δ 8.24 (s, 1H, C5''-H), 6.60 (s, 1H, C5-H), 6.26 (s, 1H, C8-H), 6.58 (s, 2H, C2', C6'-H), 6.02–5.91 (m, 3H, C4-H, OCH2O), 5.42 (t, 1H, J = 9.9 Hz, C3'''-H), 5.24 (d, 1H, J = 3.5 Hz, C1'''-H), 5.04 (t, 1H, J = 9.4 Hz, C4'''-H), 4.85 (s, 2H, C6''-CH2), 4.81–4.76 (m, 2H, C2'''-H, C5'''-H), 4.67 (d, 1H, J = 4.2 Hz, C1-H), 4.26–4.00 (m, 4H, C6'''-CH2, C11-CH2), 3.78 (s, 6H, C3'', C5''-OCH3), 3.57-3.46 (m, 1H, C3-H), 3.25 (dd, 1H, J = 4.0 Hz, 16.0 Hz, C2-H), 2.03 (s, 3H, COCH3), 2.00 (s, 3H, COCH3), 1.99 (s, 3H, COCH3), 1.98 (s, 3H, COCH3); 13C-NMR (CD3OD, 100 MHz): δ 176.0 (C-12), 172.3 (C=O), 171.7 (C=O), 171.5 (C=O), 171.2 (C=O), 149.7 (C-7), 149.1(C-6), 148.6 (C-3', C-5'), 144.7 (C-4''), 135.5 (C-1'), 134.3 (C-9), 131.6 (C-10), 129.1 (C-4'), 126.2 (C-5''), 110.9 (C-5), 109.2 (C-2', C-6'), 107.2 (C-8), 103.1 (OCH2O), 96.2 (C-1'''), 72.1 (C-5'''), 71.3 (C-3'''), 71.3 (C-11), 69.8 (C-2'''), 68.9 (C-4'''), 63.9 (C-2), 62.9 (C-6''), 61.7 (C-6'''), 56.8 (3', 5'-OCH3), 46.6 (C-4), 45.1 (C-1), 39.9 (C-3), 20.6 (COCH3), 20.6 (COCH3), 20.6 (COCH3), 20.5 (COCH3); ESIMS: m/z 834 [M+Na]+, HRESIMS: calcd for C38H41N3O17Na [M+H]+ 812.2509, found 812.2480. 4β-{4-[1-(2,3,4,6-Tetra-O-butyryl-α- (29). White amorphous powder, yield 96% (after chromatography with petroleum ether/acetone, 1:1); mp 102 °C; [α]D24.7: +25.9 (c 0.29, CH3OH); 1H-NMR (CD3OD, 400 MHz): δ 8.21 (s, 1H, C5''-H), 6.57 (s, 3H, C2', C6'-H, C5-H), 6.18 (s, 1H, C8-H), 5.91 (d, 2H, J = 6.0 Hz, OCH2O), 5.93–5.88 (m, 3H, OCH2O, C4-H), 5.47 (t, 1H, J = 9.7 Hz, C3'''-H), 5.25 (d, 1H, J = 3.0 Hz, C1'''-H), 5.11 (t, 1H, J = 9.7 Hz, C4'''-H), 4.84 (s, 2H, C6''-CH2), 4.81 (d, 1H, J = 4.0 Hz, C1-H), 4.77–4.61 (m, 2H), 4.23–4.03 (m, 4H, C6'''-CH2, C11-CH2), 3.55–3.44 (m, 1H, C3-H), 3.06 (d, 1H, J = 4.0 Hz, 16.0 Hz, C2-H), 2.29–2.14 (m, 2H, COCH2), 2.29-2.14 (m, 2H, COCH2), 2.29–2.14 (m, 2H, COCH2), 2.29–2.14 (m, 2H, COCH2), 1.62–1.47 (m, 2H, CH2CH3), 1.62–1.47 (m, 2H, CH2CH3), 1.62–1.47 (m, 2H, CH2CH3), 1.62–1.47 (m, 2H, CH2CH3), 0.90 (t, 3H, J = 7.8 Hz, CH2CH3), 0.88 (t, 3H, J = 7.8 Hz, CH2CH3), 0.86 (t, 3H, J = 7.8 Hz, CH2CH3), 0.81 (t, 3H, J = 7.8 Hz, CH2CH3); 13C-NMR (CD3OD, 100 MHz): δ 175.8 (C-12), 174.7 (C=O), 174.0 (C=O), 173.9 (C=O), 173.5 (C=O), 149.5 (C-7), 149.0 (C-6), 148.5 (C-3', C-5'), 144.6 (C-4''), 135.4 (C-1'), 134.3 (C-9), 131.7 (C-10), 129.0 (C-4'), 126.2 (C-5''), 111.0 (C-5), 109.2 (C-2', C-6'), 107.3 (C-8), 103.1 (OCH2O), 96.3 (C-1'''), 72.0 (C-5'''), 71.1 (C-3'''), 69.5 (C-2'''), 69.1 (C-4'''), 71.3 (C-11), 63.8 (C-2), 62.8 (C-6''), 61.7 (C-6'''), 56.9 (3', 5'-OCH3), 46.4 (C-4), 45.0 (C-1), 39.8 (C-3), 36.9 (COCH2), 36.9 (COCH2), 36.8 (COCH2), 36.7 (COCH2), 19.4 (CH2CH3), 19.3 (CH2CH3), 19.3 (CH2CH3), 19.3 (CH2CH3), 14.1 (CH2CH3), 14.1 (CH2CH3), 14.1 (CH2CH3), 14.1 (CH2CH3); ESIMS: m/z 946 [M+Na]+, HRESIMS: calcd for C46H57N3O17H [M+H]+ 924.3761, found 924.3722. 4β-{4-[1-(α- (30). White amorphous powder, yield 91% (after chromatography with CHCl3/CH3OH, 9:1); [α]D25.0: +0.5 (c 0.15, CH3OH); 1H-NMR (CD3OD, 600 MHz): δ 7.84 (s, 1H, C5''-H), 6.70 (s, 1H, C5-H), 6.63 (s, 1H, C8-H), 6.41 (s, 2H, C2', C6'-CH), 6.27 (d, 1H, J = 3.2 Hz, C4-H), 5.98 (d, 2H, J = 4.8 Hz, OCH2O), 4.82 (d, 1H, J = 3.5 Hz, C1'''-H), 4.81 (d, 1H, J = 4.0 Hz, C1-H), 4.62 (s, 2H, C6''-CH2), 3.79–3.86 (m, 4H), 3.74 (s, 6H, C3'', C5''-OCH3), 3.72 (s, 3H, C4''-OCH3), 3.69–3.67 (m, 6H), 3.65–3.61 (m, 6H), 3.47–3.44 (m, 2H), 3.38–3.36 (m, 2H), 3.30–3.27 (m, 1H, C3-H), 3.17–3.14 (m, 1H, C2-H); 13C-NMR (CD3OD, 150 MHz): δ 176.0 (C-12), 154.1 (C-3', C-5'), 150.7 (C-7), 149.5 (C-6), 146.1 (C-4''), 138.4 (C-1'), 136.9 (C-9), 134.9 (C-10), 127.1 (C-4'), 126.1 (C-5''), 111.3 (C-5), 110.0 (C-8), 109.4 (C-2', C-6'), 103.5 (OCH2O), 100.5 (C-1'''), 75.3 (C-5'''), 73.8 (C-3'''), 73.8 (C-2'''), 71.9 (C-4'''), 71.6, 71.6, 71.6, 71.5, 71.1 (C-11), 69.1, 68.3, 65.1 (C-6''), 62.8 (C-6'''), 61.2 (4'-OCH3), 59.9 (C-2), 56.8 (3', 5'-OCH3), 45.1 (C-4), 42.7 (C-1), 38.7 (C-3); ESIMS: m/z 812 [M+Na]+, HRESIMS: calcd for C37H47N3O16Na [M+Na]+ 812.2849, found 812.2822. 4β-{4-[1-(2,3,4,6-Tetra-O-acetyl-α- (31). White amorphous powder, yield 94% (after chromatography with petroleum ether/acetone, 1:1); [α]D25.0: +14.3 (c 0.20, CH3OH); 1H-NMR (CD3OD, 400 MHz): δ 8.18 (s, 1H, C5''-H), 6.62 (s, 2H, C2', C6'-CH), 6.40 (s, 1H, C5-H), 6.25 (s, 1H, C8-H), 5.97–5.92 (m, 3H, C4-H OCH2O), 5.43 (t, 1H, J = 12.0 Hz, C3'''-H), 5.10 (d, 1H, J = 3.1 Hz, C1'''-H), 5.02 (t, 1H, J = 12.0 Hz, C4'''-H), 4.84–4.76 (m, 2H), 4.61 (s, 2H, C6''-CH2), 4.25–4.06 (m, 4H, C6'''-CH2, C11-CH2), 3.78 (s, 6H, C3'', C5''-OCH3), 3.72 (s, 3H, C4''-OCH3), 3.71–3.60 (m, 12H), 3.25 (dd, 1H, J = 4.0 Hz, 16.0 Hz, C2-H), 3.19–3.14 (m, 1H, C3-H), 2.02 (s, 3H, COCH3), 1.99 (s, 3H, COCH3), 1.98 (s, 3H, COCH3), 1.96 (s, 3H, COCH3); 13C-NMR (CD3OD, 100 MHz): δ 175.8 (C-12), 172.3 (C=O), 171.6 (C=O), 171.6 (C=O), 171.3 (C=O), 153.9 (C-3', C-5'), 150.5 (C-4''), 149.7 (C-7), 149.3 (C-6), 136.9 (C-1'), 134.7 (C-9), 133.9 (C-10),127.0 (C-4'), 125.6 (C-5''), 110.9 (C-5), 109.9 (C-8), 109.4 (C-2', C-6'), 103.2 (OCH2O), 97.1 (C-1'''), 72.1 (C-5'''), 71.7, 71.7, 71.6, 71.6, 71.5, 71.5, 71.2, 71.0, 71.0, 69.9 (C-2'''), 68.7 (C-11), 68.5 (C-4'''), 64.6 (C-6''), 63.2 (C-6'''), 61.1 (4'-OCH3), 56.7 (C-2), 56.6 (3', 5'-OCH3), 45.2 (C-4), 45.5 (C-1), 39.9 (C-3), 20.7 (COCH3), 20.7 (COCH3), 20.7 (COCH3), 20.7 (COCH3); ESIMS: m/z 980 [M+Na]+, HRESIMS: calcd for C45H55N3O20H [M+H]+ 958.3452, found 958.3403. 4β-{4-[1-(2,3,4,6-Tetra-O-butyryl-α- (32). White amorphous powder, yield 90% (after chromatography with petroleum ether/acetone, 1:1); [α]D24.0: +19.3 (c 0.14, CH3OH); 1H-NMR (CD3OD, 400 MHz): δ 8.17 (s, 1H, C5''-H), 6.61 (s, 2H, C2', C6'-H), 6.40 (s, 1H, C5-H), 6.25 (s, 1H, C8-H), 5.97–5.90 (m, 3H, C4-H, OCH2O), 5.48 (t, 1H, J = 12.0 Hz, C3'''-H), 5.11–5.05 (m, 2H, C1'''-H, C4'''-H), 4.87–4.83 (m, 2H), 4.76 (d, 1H, J = 4.0 Hz, C1-H), 4.60 (s, 2H, C6''-CH2), 4.23–4.08 (m, 4H, C6'''-CH2, C11-CH2), 3.71 (s, 6H, C3'', C5''-OCH3), 3.70 (s, 3H, C4''-OCH3), 3.70–3.59 (m, 12H), 3.22–3.12 (m, 2H, C2-H, C3-H), 2.31–2.19 (m, 2H, COCH2), 2.31–2.19 (m, 2H, COCH2), 2.31–2.19 (m, 2H, COCH2), 2.31–2.19 (m, 2H, COCH2), 1.63–1.53 (m, 2H, CH2CH3), 1.63–1.53 (m, 2H, CH2CH3), 1.63-1.53 (m, 2H, CH2CH3), 1.63-1.53 (m, 2H, CH2CH3), 0.92 (t, 3H, J = 4.0 Hz, CH2CH3), 0.90 (t, 3H, J = 4.0 Hz, CH2CH3), 0.88 (t, 3H, J = 4.0 Hz, CH2CH3), 0.86 (t, 3H, J = 4.0 Hz, CH2CH3); 13C-NMR (CD3OD, 100 MHz): δ 175.7 (C-12), 174.7 (C=O), 174.1 (C=O), 174.0 (C=O), 173.6 (C=O), 153.9 (C-3', C-5'), 150.5 (C-4''), 149.6 (C-7), 146.2 (C-6), 137.9 (C-9), 136.9 (C-10), 133.9 (C-1'), 127.0 (C-4'), 125.6 (C-5''), 110.9 (C-5), 109.8 (C-8), 109.4 (C-2', C-6'), 103.2 (OCH2O), 97.1 (C-1'''), 72.1 (C-5'''), 71.7, 71.7, 71.6, 71.6, 71.6, 71.2 (C-3'''), 69.6 (C-2'''), 68.7 (C-4'''), 68.6 (C-11), 65.2 (C-6''), 63.0 (C-6'''), 61.1 (4'-OCH3), 56.7 (C-2), 56.7 (3', 5'-OCH3), 45.2 (C-4), 42.5 (C-1), 39.9 (C-3), 36.8 (COCH2), 36.8 (COCH2), 36.8 (COCH2), 36.8 (COCH2), 19.4 (CH2CH3), 19.3 (CH2CH3), 19.3 (CH2CH3), 19.3 (CH2CH3), 14.1 (CH2CH3), 14.1 (CH2CH3), 14.1 (CH2CH3), 14.1 (CH2CH3); ESIMS: m/z 1092 [M+Na]+, HRESIMS: calcd for C53H71N3O20H [M+H]+ 1070.4704, found 1070.4658. 4β-{4-[1-(α- (33). White amorphous powder, yield 93% (after chromatography with CHCl3/CH3OH, 9:1); [α]D25.3: −37.2 (c 0.11, CH3OH); 1H-NMR (CD3OD, 600 MHz): δ 7.83 (s, 1H, C5''-H), 6.69 (s, 1H, C5-H), 6.66 (s, 1H, C8-H), 6.38 (s, 2H, C2', C6'-H), 6.27 (d, 1H, J = 3.2 Hz, C4-H), 5.99 (d, 2H, J = 5.6 Hz, OCH2O), 4.82 (d, 1H, J = 4.0 Hz, C1'''-H), 4.78 (d, 1H, J = 4.0 Hz, C1-H), 4.63 (s, 2H, C6''-CH2), 3.86–3.79 (m, 4H, C6'''-CH2, C11-CH2), 3.75 (s, 6H, C3'', C5''-OCH3), 3.69–3.61 (m, 12H), 3.44–3.40 (m, 2H), 3.38–3.36 (m, 2H), 3.30–3.26 (m, 2H), 3.18–3.14 (m, 2H, C2-H, C3-H); 13C-NMR (CD3OD, 150 MHz): δ 176.2 (C-12), 150.7 (C-7), 149.4 (C-4''), 148.8 (C-3', C-5'), 146.1 (C-6), 136.1 (C-1'), 135.3 (C-9), 131.5 (C-10), 127.1 (C-4'), 126.1 (C-5''), 111.4 (C-5), 109.9 (C-8), 109.4 (C-2', C-6'), 103.4 (OCH2O), 100.5 (C-1'''), 75.3 (C-5'''), 73.8 (C-3'''), 73.8 (C-2'''), 72.0 (C-4'''), 71.7, 71.6, 71.6, 71.5 (C-11), 71.1, 69.1, 68.3, 65.1 (C-6''), 62.8 (C-6'''), 60.0 (C-2), 56.9 (3', 5'-OCH3), 44.9 (C-4), 42.8 (C-1), 38.7 (C-3); ESIMS: m/z 798 [M+Na]+, HRESIMS: calcd for C36H45N3O16Na [M+Na]+ 798.2692, found 798.2669. 4β-{4-[1-(2,3,4,6-Tetra-O-acetyl-α- (34). White amorphous powder, yield 94% (after chromatography with petroleum ether/acetone, 1:1); [α]D23.8: +14.4 (c 0.23, CH3OH); 1H-NMR (CD3OD, 400 MHz): δ 8.18 (s, 1H, C5''-H), 6.61 (s, 1H, C5-H), 6.58 (s, 2H, C2', C6'-H), 6.25 (s, 1H, C8-H), 6.03–5.93 (m, 3H, C4-H, OCH2O), 5.42 (d, 1H, J = 8.0 Hz, C3'''-H), 5.10 (d, 1H, J = 2.8 Hz, C1'''-H), 5.02 (t, 1H, J = 8.0 Hz, C4'''-H), 4.83–4.81 (m, 2H), 4.70–4.67 (m, 3H, C6''-CH2, C1-H), 4.25–4.07 (m, 4H, C6'''-CH2, C11-CH2), 3.79 (s, 6H, C3'', C5''-OCH3), 3.72–3.63 (m, 12H), 3.57–3.45 (m, 1H, C3-H), 3.26 (dd, 1H, J = 4.0 Hz, 16.0 Hz, C2-H), 2.02 (s, 3H, COCH3), 2.01 (s, 3H, COCH3), 1.99 (s, 3H, COCH3), 1.96 (s, 3H, COCH3); 13C-NMR (CD3OD, 100 MHz): δ 176.0 (C-12), 172.4 (C=O), 171.8 (C=O), 171.7 (C=O), 171.7 (C=O), 149.7 (C-7), 149.1 (C-6), 148.6 (C-3', C-5'), 145.8 (C-4''), 135.6 (C-1'), 134.3 (C-9), 131.6 (C-10), 129.2 (C-4'), 125.6 (C-5''), 110.9 (C-5), 109.2 (C-2', C-6'), 107.2 (C-8), 103.1 (OCH2O), 97.1 (C-1'''), 72.1 (C-5'''), 71.7, 71.6, 71.6,71.5 (C-3'''), 71.2, 71.0, 70.0 (C-2'''), 68.7 (C-11), 68.5 (C-4'''), 65.1 (C-6''), 63.9 (C-2), 63.2 (C-6'''), 56.8 (3', 5'-OCH3), 46.6 (C-4), 45.1 (C-1), 39.9 (C-3), 20.7 (COCH3), 20.6 (COCH3), 20.6 (COCH3), 20.6 (COCH3); ESIMS: m/z 966 [M+Na]+, HRESIMS: calcd for C44H53N3O20H [M+H]+ 944.3295, found 944.3249. 4β-{4-[1-(2,3,4,6-Tetra-O-butyryl-α- (35). White amorphous powder, yield 92% (after chromatography with petroleum ether/acetone, 1:1); [α]D25.0: +22.4 (c 0.24, CH3OH); 1H-NMR (CD3OD, 400 MHz): δ 8.18 (s, 1H, C5''-H), 6.61 (s, 1H, C5-H), 6.59 (s, 2H, C2'-H, C6'-H), 6.26 (s, 1H, C8-H), 6.04–5.94 (m, 3H, C4-H, OCH2O), 5.47 (t, 1H, J = 12.0 Hz, C3'''-H), 5.11 (d, 2H, J = 3.7 Hz, C1'''-H), 5.06 (t, 1H, J = 8.0 Hz, C4'''-H), 4.85–4.81 (m, 2H), 4.71–4.69 (m, 3H, C6''-CH2, C1-H), 4.23–4.09 (m, 4H, C6'''-CH2, C11-CH2), 3.80 (s, 6H, C3'', C5''-OCH3), 3.72–3.64 (m, 12H), 3.55–3.49 (m, 1H, C3-H), 3.28–3.26 (m, 1H, C2-H), 2.32–2.19 (m, 2H, COCH2), 2.32–2.19 (m, 2H, COCH2), 2.32–2.19 (m, 2H, COCH2), 2.32–2.19 (m, 2H, COCH2), 1.62–1.53 (m, 2H, CH2CH3), 1.62–1.53 (m, 2H, CH2CH3), 1.62–1.53 (m, 2H, CH2CH3), 1.62–1.53 (m, 2H, CH2CH3), 0.93 (t, 3H, J = 4.0 Hz, CH2CH3), 0.91 (t, 3H, J = 4.0 Hz, CH2CH3), 0.89 (t, 3H, J = 4.0 Hz, CH2CH3), 0.87 (t, 3H, J = 4.0 Hz, CH2CH3); 13C-NMR (CD3OD, 100 MHz): δ 176.0 (C-12), 174.8 (C=O), 174.1 (C=O), 174.1 (C=O), 173.6 (C=O), 149.7 (C-7), 149.1 (C-6), 148.6 (C-3', C-5'), 134.3 (C-1'), 131.5 (C-9), 131.5 (C-10), 127.2 (C-4'), 125.5 (C-5''), 110.9 (C-5), 109.2 (C-2', C-6'), 107.2 (C-8), 103.1 (OCH2O), 97.1 (C-1'''), 72.1 (C-5'''), 71.7, 71.6, 71.6, 71.3, 71.2 (C-3'''), 71.0, 69.6 (C-2'''), 68.7 (C-4'''), 68.7 (C-11), 65.1 (C-6''), 63.9 (C-2), 62.9 (C-6'''), 56.8 (3', 5'-OCH3), 46.6 (C-4), 45.2 (C-1), 39.9 (C-3), 36.9 (COCH2), 36.8 (COCH2), 36.7 (COCH2), 36.7 (COCH2), 19.4 (CH2CH3), 19.3 (CH2CH3), 19.3 (CH2CH3), 19.2 (CH2CH3), 14.0 (CH2CH3), 14.0 (CH2CH3), 14.0 (CH2CH3), 14.0 (CH2CH3); ESIMS: m/z 1078 [M+Na]+, HRESIMS: calcd for C52H69N3O20H [M+H]+ 1056.4547, found 1056.4484. 4β-{4-[1-(α- (36). White amorphous powder, yield 91% (after chromatography with CHCl3/CH3OH, 9:1); [α]D24.3: −2.5 (c 0.17, CH3OH); 1H-NMR (CD3OD, 400 MHz): δ 7.82 (s, 1H, C5''-H), 6.69 (s, 1H, C5-H), 6.64 (s, 1H, C8-H), 6.41 (s, 2H, C2', C6'-H), 6.26 (d, 1H, J = 4.8 Hz, C4-H), 5.98 (d, 2H, J = 5.5 Hz, OCH2O), 4.82–4.80 (m, 2H, C1'''-H, C1-H), 4.62 (s, 2H, C6''-CH2), 3.87–3.78 (m, 4H, C6'''-CH2, C11-CH2), 3.74 (s, 6H, C3'', C5''-OCH3), 3.72 (s, 3H, C4''-OCH3), 3.68–3.60 (m, 24H), 3.47–3.42 (m, 2H), 3.38–3.35 (m, 2H), 3.28–3.25 (m, 1H), 3.18–3.13 (m, 1H); 13C-NMR (CD3OD, 100 MHz): δ 175.8 (C-12), 153.9 (C-3', C-5'), 150.6 (C-7), 149.3 (C-6), 148.1 (C-4''), 138.3 (C-1'), 136.7 (C-9), 134.7 (C-10), 126.9 (C-4'), 125.9 (C-5''), 111.2 (C-5), 109.8 (C-8), 109.3 (C-2', C-6'), 103.3 (OCH2O), 100.3 (C-1'''), 75.2 (C-5'''), 73.7 (C-3'''), 73.7 (C-2'''), 71.8 (C-4'''), 71.5, 71.5, 71.5, 71.3, 70.9 (C-11), 68.9, 68.1, 65.0 (C-6''), 62.7 (C-6'''), 61.1 (4'-OCH3), 59.8 (C-2), 56.7 (3', 5'-OCH3), 44.9 (C-4), 42.5 (C-1), 38.6 (C-3); ESIMS: m/z 944 [M+Na]+, HRESIMS: calcd for C43H59N3O19Na [M+Na]+ 944.3635, found 944.3622. 4β-{4-[1-(2,3,4,6-Tetra-O-acetyl-α- (37). White amorphous powder, yield 91% (after chromatography with petroleum ether/acetone, 1:1); [α]D23.9: −24.9 (c 0.22, CH3OH); 1H-NMR (CD3OD, 400 MHz): δ 8.21 (s, 1H, C5′′-H), 6.62, (s, 2H, C2′, C6′-H), 6.60 (s, 1H, C5-H), 6.25 (s, 1H, C8-H), 6.05–5.94 (m, 3H, C4-H, OCH2O), 5.43 (t, 1H, J = 12.0 Hz, C3'''-H), 5.11 (d, 1H, J = 3.5 Hz, C1'''-H), 5.02 (t, 1H, J = 12.0 Hz, C4'''-H), 4.85–4.80 (m, 3H), 4.70 (s, 2H, C6''-CH2), 4.26–4.03 (m, 4H, C6'''-CH2, C11-CH2), 3.79 (s, 6H, C3'', C5''-OCH3), 3.73 (s, 3H, C4''-OCH3), 3.65–3.59 (m, 24H), 3.55-3.45 (m, 2H, C2-H, C3-H), 2.04 (s, 3H, COCH3), 2.03 (s, 3H, COCH3), 2.00 (s, 3H, COCH3), 1.97 (s, 3H, COCH3); 13C-NMR (CD3OD, 100 MHz): δ 175.8 (C-12), 172.3 (C=O), 171.7 (C=O), 171.7 (C=O), 171.3 (C=O), 153.9 (C-3', C-5'), 153.9 (C-7), 149.7 (C-6), 149.2 (C-4''), 137.9 (C-1'), 137.0 (C-9), 133.9 (C-10), 129.3 (C-4'), 125.7 (C-5''), 110.9 (C-5), 109.3 (C-2', C-6'), 107.2 (C-8), 103.2 (OCH2O), 97.1 (C-1'''), 72.1 (C-5'''), 71.7, 71.6, 71.5, 71.5, 71.2 (C-3'''), 71.0 (C-2'''), 70.0, 68.7 (C-4'''), 68.5 (C-11), 65.1 (C-6''), 63.8 (C-2), 63.2 (C-6'''), 61.1 (4'-OCH3), 56.7 (3', 5'-OCH3), 45.5 (C-4), 45.3 (C-1), 39.9 (C-3), 20.7 (COCH3), 20.6 (COCH3), 20.6 (COCH3), 20.6 (COCH3); ESIMS: m/z 1090 [M+H]+, HRESIMS: calcd for C51H67N3O23H [M+H]+ 1090.4238, found 1090.4177. 4β-{4-[1-(2,3,4,6-Tetra-O-butyryl-α- (38). White amorphous powder, yield 91% (after chromatography with petroleum ether/acetone, 1:1); [α]D23.7: +17.4 (c 0.20, CH3OH); 1H-NMR (CD3OD, 400 MHz): δ 8.20 (s, 1H, C5''-H), 6.62 (s, 2H, C2', C6'-H), 6.59 (s, 1H, C5-H), 6.25 (s, 1H, C8-H), 6.04–5.93 (m, 3H, C4-H, OCH2O), 5.48 (t, 1H, J = 8.0 Hz, C3'''-H), 5.11–5.04 (m, 2H, C1'''-H, C4'''-H), 4.85–4.81 (m, 2H), 4.72–4.70 (m, 3H, C6''-CH2, C1-H), 4.23–4.09 (m, 4H, C6'''-CH2, C11-CH2), 3.79 (s, 6H, C3'', C5''-OCH3), 3.72 (s, 3H, C4''-OCH3), 3.65–3.61 (m, 24H), 3.27–3.26 (m, 1H), 3.18–3.13 (m, 1H), 2.33–2.20 (m, 2H, COCH2), 2.33–2.20 (m, 2H, COCH2), 2.33–2.20 (m, 2H, COCH2), 2.33-2.20 (m, 2H, COCH2), 1.65–1.53 (m, 2H, CH2CH3), 1.65–1.53 (m, 2H, CH2CH3), 1.65–1.53 (m, 2H, CH2CH3), 1.65–1.53 (m, 2H, CH2CH3), 0.94 (t, 3H, J = 4.0 Hz, CH2CH3), 0.91 (t, 3H, J = 4.0 Hz, CH2CH3), 0.90 (t, 3H, J = 4.0 Hz, CH2CH3), 0.88 (t, 3H, J = 4.0 Hz, CH2CH3); 13C-NMR (CD3OD, 100 MHz): δ 175.8 (C-12), 174.7 (C=O), 174.1 (C=O), 174.0 (C=O), 173.6 (C=O), 153.9 (C-3', C-5'), 149.7 (C-7), 149.2 (C-6), 146.3 (C-4''), 137.9 (C-1'), 136.9 (C-9), 133.9 (C-10), 129.3 (C-4'), 125.8 (C-5''), 110.9 (C-5), 109.4 (C-2', C-6'), 107.3 (C-8), 103.2 (OCH2O), 97.1 (C-1'''), 72.1 (C-5'''), 71.7, 71.6, 71.5, 71.2, 71.0 (C-3'''), 69.6 (C-2'''), 68.7 (C-11), 68.6 (C-4'''), 65.2 (C-6''), 63.8 (C-2), 63.0 (C-6'''), 61.1 (4'-OCH3), 56.7 (3', 5'-OCH3), 46.5 (C-4), 45.2 (C-1), 39.9 (C-3), 36.9 (COCH2), 36.8 (COCH2), 36.7 (COCH2), 36.7 (COCH2), 19.4 (CH2CH3), 19.3 (CH2CH3), 19.3 (CH2CH3), 19.3 (CH2CH3), 14.0 (CH2CH3), 14.0 (CH2CH3), 14.0 (CH2CH3), 14.0 (CH2CH3); ESIMS: m/z 1224 [M+Na]+, HRESIMS: calcd for C59H83N3O23H [M+H]+ 1202.5490, found 1202.5423. 4β-{4-[1-(α- (39). White amorphous powder, yield 93% (after chromatography with CHCl3/CH3OH, 9:1); mp 158 °C; [α]D25.4: −4.0 (c 0.11, CH3OH); 1H-NMR (CD3OD, 600 MHz): δ 7.82 (s, 1H, C5''-H), 6.69 (s, 1H, C5-H), 6.66 (s, 1H, C8-H), 6.38 (s, 2H, C2', C6'-H), 6.27 (d, 1H, J = 3.6 Hz, C4-H), 5.98 (d, 2H, J = 5.6 Hz, OCH2O), 4.82 (d, 1H, J = 2.4 Hz, C1'''-H), 4.78 (d, 1H, J = 3.6 Hz, C1-H), 4.64 (s, 2H, C6''-CH2), 3.85–3.80 (m, 4H, C6'''-CH2, C11-CH2), 3.75 (s, 6H, C3'', C5''-OCH3), 3.69–3.60 (m, 24H), 3.42–3.40 (m, 2H), 3.38–3.36 (m, 1H), 3.18–3.14 (m ,2H, C2-H, C3-H); 13C-NMR (CD3OD, 150 MHz): δ 176.2 (C-12), 150.7 (C-7), 149.4 (C-6), 148.8 (C-3', C-5'), 135.2 (C-4''), 132.5 (C-1'), 131.5 (C-9), 130.0 (C-10), 127.1 (C-4'), 126.1 (C-5''), 111.4 (C-5), 109.9 (C-8), 109.4 (C-2', C-6'), 103.4 (OCH2O), 100.4 (C-1'''), 75.3 (C-5'''), 73.9 (C-3'''), 73.9 (C-2'''), 71.9 (C-4'''), 71.7, 71.7, 71.6, 71.5, 71.1, 70.9 (C-11), 69.2, 68.2, 65.1 (C-6''), 62.3 (C-6'''), 60.0 (C-2), 56.9 (3', 5'-OCH3), 44.9 (C-4), 42.9 (C-1), 68.7 (C-3); ESIMS: m/z 930 [M+Na]+, HRESIMS: calcd for C42H57N3O19Na [M+Na]+ 930.3478, found 930.3462. 4β-{4-[1-(2,3,4,6-Tetra-O-acetyl-α- (40). White amorphous powder, yield 90% (after chromatography with petroleum ether/acetone, 1:1); [α]D25.0: +25.1 (c 0.23, CH3OH); 1H-NMR (CD3OD, 400 MHz): δ 8.18 (s, 1H, C5''-H), 6.60 (s, 1H, C5-H), 3.58 (s, 2H, C2', C6'-H), 6.23 (s, 1H, C8-H), 6.01–5.92 (m, 3H, C4-H, OCH2O), 5.43 (t, 1H, J = 12.0 Hz, C3'''-H), 5.11 (d, 1H, J = 4.0 Hz, C1'''-H), 5.02 (t, 1H, J = 12.0 Hz, C4'''-H), 4.69 (s, 2H, C6''-CH2), 4.65 (d, 1H, J = 4.0 Hz, C1-H), 4.30–4.07 (m, 4H, C6'''-CH2, C11-CH2), 3.78 (s, 6H, C3'', C5''-OCH3), 3.69–3.59 (m, 24H), 3.44–3.41 (m, 1H, C3-H), 3.21 (dd, 1H, J = 4.0 Hz, 12.0 Hz, C4-H), 2.04 (s, 3H, COCH3), 2.03 (s, 3H, COCH3), 2.00 (s, 3H, COCH3), 1.97 (s, 3H, COCH3); 13C-NMR (CD3OD, 100 MHz): δ 174.4 (C-12), 170.8 (C=O), 170.2 (C=O), 170.2 (C=O), 169.8 (C=O), 148.1 (C-7), 147.6 (C-6), 147.1 (C-3', C-5'), 144.6 (C-4''), 134.0 (C-1'), 132.7 (C-9), 130.1 (C-10), 128.3 (C-4'), 127.7 (C-5''), 109.4 (C-5), 107.6 (C-2', C-6'), 105.7 (C-8), 101.6 (OCH2O), 95.5 (C-1'''), 72.1, 71.6, 71.6, 71.5, 71.2, 71.0 (C-5'''), 70.0 (C-3'''), 68.4 (C-2'''), 67.2 (C-11), 66.9 (C-4'''), 63.5 (C-6''), 62.3 (C-2), 61.6 (C-6'''), 55.3 (3', 5'-OCH3), 45.0 (C-4), 43.6 (C-1), 38.3 (C-3), 20.7 (COCH3), 20.7 (COCH3), 20.7 (COCH3), 20.7 (COCH3); ESIMS: m/z 1098 [M+Na]+, HRESIMS: calcd for C50H65N3O23H [M+H]+ 1076.4082, found 1076.4031. 4β-{4-[1-(2,3,4,6-Tetra-O-butyryl-α- (41). White amorphous powder, yield 97% (after chromatography with petroleum ether/acetone, 1:1); [α]D24.6: −3.1 (c 0.29, CH3OH); 1H-NMR (CD3OD, 400 MHz): δ 8.18 (s, 1H, C5''-H), 6.62 (s, 1H, C5-H), 6.59 (s, 2H, C2', C6'-H), 6.25 (s, 1H, C8-H), 5.95–5.84 (m, 3H, C4-H, OCH2O), 5.37 (t, 1H, J = 8.0 Hz, C3'''-H), 5.02 (d, 2H, J = 4.0 Hz, C1'''-H), 4.97 (t, 1H, J = 12.0 Hz, C4'''-H), 4.61–4.59 (m, 3H, C6''-CH2, C1-H), 4.13–4.00 (m, 4H, C6'''-CH2, C11-CH2), 3.80 (s, 6H, C3'', C5''-OCH3), 3.53–3.51 (m, 24H), 3.45–3.39 (m, 2H, C2-H, C3-H), 2.24–2.10 (m, 8H), 1.56–1.43 (m, 8H), 0.85 (t, 3H, J = 4.0 Hz, CH2CH3), 0.82 (t, 3H, J = 4.0 Hz, CH2CH3), 0.80 (t, 3H, J = 4.0 Hz, CH2CH3), 0.79 (t, 3H, J = 4.0 Hz, CH2CH3); 13C-NMR (CD3OD, 100 MHz): δ 176.0 (C-12), 174.8 (C=O), 174.0 (C=O), 174.0 (C=O), 173.6 (C=O), 149.7 (C-7), 149.1 (C-6), 148.6 (C-3', C-5'), 146.2 (C-4''), 135.6 (C-1'), 134.3 (C-9), 131.6 (C-10), 129.2 (C-4'), 125.6 (C-5''), 110.0 (C-5), 109.1 (C-2', C-6'), 107.1 (C-8), 103.0 (OCH2O), 97.1 (C-1'''), 72.1 (C-5'''), 71.7, 71.6, 71.5, 71.2 (C-3'''), 69.6 (C-2'''), 68.6 (C-4'''), 68.6 (C-11), 65.1 (C-6''), 63.8 (C-2), 62.9 (C-6'''), 56.8 (3', 5'-OCH3), 46.7 (C-4), 45.1 (C-1), 39.9 (C-3), 36.8 (COCH2), 36.7 (COCH2), 36.7 (COCH2), 36.7 (COCH2), 19.4 (CH2CH3), 19.3 (CH2CH3), 19.3 (CH2CH3), 19.3 (CH2CH3), 14.0 (CH2CH3), 14.0 (CH2CH3), 14.0 (CH2CH3), 14.0 (CH2CH3); ESIMS: m/z 1210 [M+Na]+, HRESIMS: calcd for C58H81N3O23H [M+H]+ 1188.5334, found 1188.5298. 4β-[4-(α- (42). White amorphous powder, yield 97% (after chromatography with CHCl3/CH3OH, 9:1); mp 148 °C;1H-NMR (CD3OD, 400 MHz): δ 8.22 (s, 1H, C5''-H), 6.61 (s, 1H, C5-H), 6.59 (s, 2H, C2', C6'-H), 6.25 (s, 1H, C8-H), 6.03–5.93 (m, 3H, C4-H, OCH2O), 5.03 (d, 1H, J = 12.0 Hz, C1'''-H), 4.85 (s, 2H, C6''-CH2), 4.70 (d, 1H, J = 4.3 Hz, C1-H), 4.43 (d, 1H, J = 4.0 Hz), 4.24–4.16 (m, 2H, C6'''-CH2), 3.91 (d, 1H, J = 12.0 Hz), 3.79 (s, 6H, C3'', C5''-OCH3), 3.68 (dd, 1H, J = 4.0 Hz, 12.0 Hz, C2-H), 3.57–3.46 (m, 1H, C3-H), 3.39–3.35 (m, 2H), 3.27–3.21 (m, 2H); 13C-NMR (CD3OD, 100 MHz): δ 176.1 (C-12), 149.7 (C-7), 149.2 (C-6), 148.6 (C-3', C-5'), 145.9 (C-4''), 135.6 (C-1'), 134.2 (C-9), 131.6 (C-10), 129.2 (C-4'), 125.8 (C-5''), 110.9 (C-5), 109.2 (C-2', C-6'), 107.2 (C-8), 103.8 (C-1'''), 103.1 (OCH2O), 78.1 (C-5'''), 77.9 (C-3'''), 75.0 (C-2'''), 71.6 (C-4'''), 71.3 (C-11), 63.9 (C-2), 63.2 (C-6''), 62.7 (C-6'''), 56.8 (3', 5'-OCH3), 46.6 (C-4), 45.1 (C-1), 39.9 (C-3); ESIMS: m/z 666 [M+Na]+, HRESIMS: calcd for C30H33N3O13Na [M+Na]+ 666.1906, found 666.1900. 4β-{4-[1-(β- (43). White amorphous powder, yield 92% (after chromatography with CHCl3/CH3OH, 9:1); mp 104–106 °C; [α]D25.1: −40.3 (c 0.18, CH3OH); 1H-NMR (CD3OD, 400 MHz): δ 7.82 (s, 1H, C5''-H), 6.68 (s, 1H, C5-H), 6.64 (s, 1H, C8-H), 6.38 (s, 2H, C2', C6'-H), 6.25 (d, 2H, J = 4.8 Hz, C4-H), 5.97 (d, 2H, J = 5.0 Hz, OCH2O), 4.78 (d, 1H, J = 4.0 Hz, C1-H), 4.62 (s, 2H, C6''-CH2), 4.31 (d, 1H, J = 8.0 Hz, C1'''-H), 4.02–3.98 (m, 2H), 3.87–3.79 (m, 2H), 3.74 (s, 6H, C3'', C5''-OCH3), 3.70–3.61 (m, 12H), 3.43–3.39 (m, 2H), 3.28–3.26 (m, 2H), 3.21–3.13 (m, 2H); 13C-NMR (CD3OD, 100 MHz): δ 176.0 (C-12), 150.5 (C-7), 149.2 (C-6) , 148.7 (C-3', C-5'), 145.9 (C-4''), 135.1 (C-9), 131.3 (C-10), 131.3 (C-1'), 126.9 (C-4'), 125.9 (C-5''), 111.2 (C-5), 109.7 (C-8), 109.3 (C-2', C-6'), 104.4 (OCH2O), 103.2 (C-1'''), 77.9 (C-5'''), 77.9 (C-3'''), 75.1 (C-2'''), 71.6 (C-4'''), 71.4, 71.4, 70.9 (C-11), 69.6, 68.9, 64.9 (C-6''), 62.7 (C-6'''), 59.8 (C-2), 56.7 (3', 5'-OCH3), 44.7 (C-4), 42.7 (C-1), 38.5 (C-3); ESIMS: m/z 798 [M+Na]+, HRESIMS: calcd for C36H45N3O13Na [M+Na]+ 798.2692, found 798.2669. 4β-{4-[1-(β- (44). White amorphous powder, yield 94% (after chromatography with CHCl3/CH3OH, 9:1); [α]D24.9: −37.7 (c 0.23, CH3OH); 1H-NMR (CD3OD, 400 MHz): δ 7.81 (s, 1H, C5''-H), 6.67 (s, 1H, C5-H), 6.63 (s, 1H, C8-H), 6.38 (s, 2H, C2', C6'-H), 6.24 (d, 1H, J = 4.7 Hz, C4-H), 5.97 (d, 2H, J = 5.3 Hz, OCH2O), 4.77 (d, 1H, J = 4.0 Hz, C1-H), 4.64 (s, 2H, C6''-CH2), 4.35 (d, 1H, J = 8.0 Hz, C1'''-H), 4.04–4.00 (m, 2H), 3.88–3.78 (m, 2H), 3.75 (s, 6H, C3'', C5''-OCH3), 3.68–3.63 (m, 24H), 3.48–3.39 (m, 2H), 3.31–3.29 (m, 2H), 3.25–3.21 (m, 1H), 3.17–3.11 (m, 1H); 13C-NMR (CD3OD, 100 MHz,): δ 176.0 (C-12), 150.5 (C-7), 149.2 (C-6), 148.7 (C-3', C-5'), 146.0 (C-4''), 136.2 (C-1'), 135.1 (C-9), 131.3 (C-10), 126.9 (C-4'), 125.9 (C-5''), 111.2 (C-5), 109.7 (C-8), 109.3 (C-2', C-6'), 104.4 (OCH2O), 103.2 (C-1'''), 77.9 (C-5'''), 77.9 (C-3'''), 75.1 (C-2'''), 71.6 (C-4'''), 71.1, 71.0, 70.9, 70.9 (C-11), 69.6, 68.9, 65.0 (C-6''), 62.7 (C-6'''), 59.8 (C-2), 56.8 (3', 5'-OCH3), 44.7 (C-4), 42.7 (C-1), 38.5 (C-3); ESIMS: m/z 931 [M+Na]+, HRESIMS: calcd for C42H57N3O19Na [M+Na]+ 930.3478, found 930.3465.

3.5. Cell Culture and Cytotoxicity Assay

The following human tumor cell lines were used: HL-60, SMMC-7721, 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 μL) 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 μL 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 μg) was added to each well, and the incubation continued for 4 h at 37 °C. The cells lysed with SDS (200 μL) after removal of 100 μL 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.

4. Conclusions

In conclusion, we have used an effective and facile Fisher glycosylation strategy to prepare glucose-bearing terminal-alkynes with a catalyst of H2SO4-silica. Then, all glycosides were subjected to peracetylation or perbutyrylation, and the resulting glycosylated terminal alkynes underwent click-reactions with azide derivatives of podophyllotoxin to yield a series of 4β-triazole-linked glucose-podophyllotoxin conjugates in high yields. All conjugated derivatives were screened for 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). All these derivatives display different level of anticancer activity which can be affected by the nature of substituents on the glucose residue, the length of the linking spacer between the sugar and the triazole ring, and the substituent on the 4'-position of the E-ring of podophyllotoxin scaffold. Derivatives with a perbutyrylated glucose residue generally display higher anticancer activity than other derivatives. The two most active compounds 29 and 35, both having a perbutyrylated glucose residue and a 4'-OH on the E ring, are significantly more active than etopodide or cisplatin. Further investigation of these compounds in in vivo tumor models is necessary in order to evaluate their therapeutic potential for cancer treatment.

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