Design, synthesis, and anti-tobacco mosaic virus (TMV) activity of glycoconjugates of phenanthroindolizidines alkaloids

Abstract

Glycoconjugates of phenanthroindolizidine alkaloids targeting tobacco mosaic virus (TMV) RNA were designed, synthesized, and evaluated for their antiviral activity against TMV for the first time. The glycoconjugation of $(S)$-6-O-desmethylantofine (2) and 14-hydroxyltylophorines (3–6) was accomplished in three ways (O-glycosylation manner, using carbamoyloxy as linker arm, and using 1,2,3-triazole as linker arm) with three different sugar units (glucose, galactose, and mannose). The glycoconjugates showed improved water solubility and molecule polarity compared with phenanthroindolizidine alkaloids. The bioassay results showed that C6 was a suitable position for glycoconjugation and O-glycosylation can increase the antiviral activity of phenanthroindolizidine alkaloids indicating that the introduction of sugar units can improve the antiviral activity profile of glycoconjugates. Two O-glycosides of $(S)$-6-O-desmethylantofine, (13aS)-6-O-$\upbeta$-d-galactopyranosyl-2,3-dimethoxyphenanthro [9,10-b]-11-indolizidinone (10) and (13aS)-6-O-$\upbeta$-d-mannopyranosyl-2,3-dimethoxyphenanthro [9,10-b]-11-indolizidinone (11) displayed significant higher activity than commercial ningnanmycin, and thus could be considered for novel therapy against plant virus infection.

Introduction

Plant viruses are pathogenic to plants and they affect more than 700 plants up to now [1]. For example, tobacco mosaic virus (TMV) infect tobacco, tomato, pepper, and cucumbers [2]. Plant viruses and their hosts have an intricate relationship that complicates the strategic designs to control plant viruses. Because of the economic loss caused by TMV and the low antiviral activities ($<$60 %) of the existing commercial antiviral agents such as Ribavirin and Ningnanmycin [3], many efforts have focused towards the development of novel and more effective antiviral agents. However, the development of antiviral agents is restricted due to that the study of molecular mechanism between antiviral agents and virus is quite limited [4, 5].

Phenanthroindolizidine alkaloids (Fig. 1) exhibited several interesting pharmacological properties including notable anticancer and antiviral activity [6–8]. We found that $(R)$-antofine (1, Fig. 1) isolated from cyanchum komarovii showed excellent antiviral activity against TMV [9]. Many phenanthroindolizidine alkaloids were screened to evaluate their antiviral activity against TMV and many of them were found to exhibit higher antiviral activity than Ribavirin and Ningnanmycin both in vitro and in vivo [10–12].

Fig. 1

Chemical structure of phenanthroindolizidine alkaloids

However, to date, phenanthroindolizidine alkaloids have not been successfully developed as anticancer or antiviral agents as they have several drawbacks including poor water solubility, low in vivo activity, and central nervous system (CNS) toxicity [13, 14]. Suffness et al. [14] pointed out that more polar analogs, which cannot clear the blood brain barrier, could have less side effects. As we know, glycoconjugation is widely used to improve the parent compound’s stability and water solubility [15, 16], and reduce toxicity of some natural products [17]. Glycoconjugation could certainly improve the water solubility and molecule polarity of phenanthroindolizidine alkaloid due to the added hydroxyl groups contained in the sugar structure.

Glycoconjugation is usually a key modification of plant natural products during their biosynthesis, and is also one of the major factors that determine their bioactivity and bioavailability [18, 19]. Glycosylated natural products have served as reliable platforms for the development of many existing front-line drugs and glycoconjugation is a powerful tool to enhance the pharmacodymanics and/or pharmacokinetics of small molecule-based therapeutics, including natural products [20–22]. In nature, glycoconjugation is performed by more than 80 families of glycosyltransferases (GT). For the unglycosylated natural products, chemical glycoconjugation can serve as a platform for rational structural modification and, particularly, for more effective exploitation of chemical structure diversity offered by nature [23].

Xi et al. indicated that the phenanthroindolizidine alkaloid skeleton of antofine was important for virus assembly inhibition, while the substituent groups on the phenanthrene ring had little effect on drug–RNA interaction [24, 25]. Consequently, glycoconjugation on the phenanthrene ring and indolizidine ring would not affect the interaction between the phenanthroindolizidine alkaloids and TMV RNA. Glycoconjugates of phenanthroindolizidine alkaloids targeting TMV RNA were designed and synthesized for the first time, and evaluated for their antiviral activity against TMV.

Results and discussion

Chemistry

Optimization of glycoconjugates is involved in modification of aglycones and sugar units since both entities influence target recognition, selectivity, and pharmacology. $(S)$-6-$O$-desmethylantofine (2, Fig. 1) and 14-hydroxyltylophorines (3–6, Fig. 1) were chosen as the aglycons and glucose, galactose, and mannose were chosen as the sugar units. As changing glycosylation patterns is also an effective strategy to influence the biological activity [26], three glycoconjugation approaches were used: (1) $O$-glycosylation, (2) introduction of a carbamoyloxy group as linker arm, and (3) introduction of a 1,2,3-triazole group as linker arm. All of the newly synthesized glycoconjugates were evaluated for their antiviral activities against TMV.

Many $O$-glycosylation methods have been disclosed since the emergence of the Koenigs–Knorr procedure [27, 28]. After trying several glycosyl donors, it was deteremined that trichloroacetimidate derivatives [29] were most suitable for the glycoconjugation of $(S)$-6-$O$-desmethylantofine (2) and 14-hydroxyltylophorine (4) (Scheme 1). 2,3,4,6-Tetra-$O$-acetyl-$\upalpha$-d-glucopyranosyl trichloroacetimidate (31) [30], 2,3,4,6-tetra-$O$-acetyl-$\upalpha$-d-galactopyranosyl trichloroacetimidate (32) [31] and 2,3,4,6-tetra-$O$-acetyl-$\upalpha$-d-mannopyranosyl trichloroacetimidate (33) [30] were synthesized according to the literature. As shown in Scheme 1, treatment of $(S)$-6-$O$-desmethylantofine (2) with different $\upalpha$-trichloroacetimidates (31, 32, 33) at $-40\, ^{\circ }\hbox {C}$ in $\hbox {CH}_{2}\hbox {Cl}_{2}$ in the presence of catalytic amounts of the Lewis acid $\hbox {BF}_{3}\cdot \hbox {Et}_{2}\hbox {O}$, and subsequent removal of the protecting groups (acetate) using $\hbox {CH}_{3}\hbox {ONa}$, gave $\upbeta$-glycosides 9–11 in high yield (78–81 %, 2 steps). However, the yield of O-glycosylation of 14-hydroxyltylophorine (4) under these reaction conditions is low (28 %). The reason is that 14-hydroxyltylophorine (4) and trichloroacetimidate (31) were both unstable, especially in acid medium. The desired 14-hydroxyltylophorine glycoside 8 was obtained through deprotection of compound 7.

Scheme 1

O-Glycosylation of phenanthroindolizidine alkaloids

Scheme 2

Glycoconjugation of phenanthroindolizidine alkaloids using 1,2,3-triazole as a linker arm

Scheme 3

Glycoconjugation of phenanthroindolizidine alkaloids using carbamoyloxy as a linker arm

In order to assess if the distance between the phenanthroindolizidine alkaloid and the sugar unit has any effect in the biological activity of the conjugates, a linker arm was introduced. Meldal et al. [32] and Sharpless et al. [33] found that the classical 1,3-dipolar cycloaddition of azides and terminal alkynes can be catalyzed by $\hbox {Cu}^{\mathrm{I}}$ salts. Now, $\hbox {Cu}^{\mathrm{I}}$-catalyzed Huisgen azide-alkyne 1,3-dipolar cycloaddition reaction to give a 1,4-di-substituted 1,2,3-triazole unit in high yield and chemoselectivity is usually used as a powerful method for glycoconjugation [34, 35]. As shown in Scheme 2, treatment of 2 and 3 with 3-bromopropyne can introduce terminal alkyne to the oxygen on C6 and C14 to afford 12 and 13. Then, 12 and 13 were treated with 2,3,4,6-tetra-$O$-acetyl-$\upbeta$-d-glucopyranosyl azide (34), 2,3,4,6-tetra-$O$-acetyl-$\upbeta$-d-galactopyranosyl azide (35) [36] and 2,3,4,6-tetra-$O$-acetyl-$\beta$-d-mannopyranosyl azide (36) [37] under Sharpless reaction conditions ($\hbox {CuSO}_{4}$, sodium ascorbate) [38] to afford the corresponding glycoconjugates 14–16 and 20–22 in high yields (89–96 %). Finally, removal of the acetate protecting groups afforded the desired the $N$-glycosyl phenanthroindolizidine alkaloid triazoles 17–19 and 23–25.

Recently, sugar isocyanates were used in glycoconjugation reaction [38, 39]. Since sugar isocyanates are highly reactive species they could be used to react with the hydroxyl group at C6 or C14 of phenanthroindolizidine alkaloids to afford glycoconjugates with a carbamoyloxy as a linker arm. 1,3,4,6-Tetra-$O$-acetyl-2-deoxy-2-isocyanato-$\upbeta$-d-glucopyranose (37) was prepared according to the literature [38]. (S)-6-O-desmethylantofine (2), and the four 14-hydroxyltylophorines 3–6 were treated with 37 in the presence of triethylamine to afford the corresponding glycoconjugates 26–30 in high yields (83–92 %) (Scheme 3).

Biological activities and SARs

(S)-6-O-desmethylantofine (2), 14-hydroxyltylophorines 3–6 and their glycoconjugates 8–11, 14, 17–19 and 23–30 were evaluated for their antiviral activities against tobacco mosaic virus (TMV) (Table 1). Since we have already assessed the antiviral activity of compounds 3 and 5 against TMV [12], we used these two compounds as well as commercially available plant virucide Ningnanmycin as controls.

Table 1 In vitro and in vivo (protection effect, inactivation effect, and curative effect) antiviral profile of phenanthroindolizidine alkaloids and their glycoconjugates against TMV

Our results indicate that all of the compounds generally exhibit the same activity fluctuations in vitro and in vivo. Comparing (S)-6-O-desmethylantofine (2) with its O-glycosides 9–11 it can be observed that galactoside 10 and mannoside 11 showed much higher antiviral activity than 2 indicating the positive impact the sugar unit has on the antiviral activity of these compounds while glucoside 9 shows the same antiviral activity of 2, which indicates that the activity of the glycoconjugate has great relationship with the sugar unit. O-glycoside 11 exhibits much higher antiviral activity than 11a, which suggests that the protection group of the sugar is unfavorable to the activity of the glycoconjugate. Comparing the 14-hydroxyltylophorine 4 with its O-glycoside 9, glucoside 9 exhibits much lower antiviral activity. Comparing (S)-6-O-desmethylantofine (2) with its glycoconjugates 14 and 17–19, it can be observed that they show the same antiviral activity level, except for 17 which shows a slightly lower in vitro activity, indicating that introducing sugar unit with 1,2,3-triazole as a linker arm could maintain the antiviral activity of (S)-6-O-desmethylantofine. Compounds 14 and 17 showed the same activity, which indicates protection group do not affect the activity of (S)-6-O-desmethylantofine glycoconjugates with 1,2,3-triazole as a linker arm. Compounds 23–25 show lower antiviral activity than DCB-3503 (3), which suggests that 1,2,3-triazole is not suitable to be used as the linker arm between the sugar and the hydroxyl at C14. The glycoconjugate 26 shows lower antiviral activity than (S)-6-O-desmethylantofine (2) and 27–30 show much lower antiviral activity than 14-hydroxyltylophorines 3–6, respectively. In addition, glycoconjugates 26–30 also show lower activity than other glycoconjugates, which indicates carbamoyloxy is not suitable to be used as the linker arm between the sugar and phenanthroindolizidine alkaloid.

In general, antiviral activity results indicate that all 14-hydroxyltylophorine-based glycoconjugates show lower activity than 14-hydroxyltylophorines. For (S)-6-O-desmethylantofine (2), its O-glycosides 9–11 exhibit higher activity and the sugar unit affected the activity significantly. However, there is little change in the antiviral activity when the sugar unit is connected to the C6 of (S)-6-O-desmethylantofine using 1,2,3-triazole as a linker arm. The distance between the sugar unit and the (S)-6-O-desmethylantofine in the glycoconjugates 14 and 17–19 is longer than the O-glycosides 9–11, maybe too long that the sugar has little effect on activity. The glycoconjugates 26–30 show lower antiviral activity, and 11a shows lower activity than 11, which indicates that the hydroxyl on the sugar was important for phenanthroindolizidine alkaloids glycoconjugates to maintain high activity. Among the synthesized glycoconjugates, 10 and 11 are found to exhibit excellent antiviral activities which are better than Ningnanmycin.

Experimental section

Chemistry

$^{1}\hbox {H NMR}$ spectra were obtained at 400 MHz using a Bruker AC-P 400. Chemical shift values ($\updelta )$ are given in ppm and are downfield from internal tetramethylsilane. Abbreviations used for the NMR signals are s(singlet), d(doublet), t(triplet), m(multiplet), and br(broad). High-resolution mass spectra (HRMS) were recorded on FT-ICR MS (Ionspec, 7.0 T). Melting points were determined on an X-4 binocular microscope melting point apparatus (Beijing Tech Instruments Co., Beijing, China) and were uncorrected. Reagents were purchased from commercial sources and were used as received. All anhydrous solvent were dried and purified by standard techniques before use.

Synthesis of (13aR,14S)-14-O-$\beta$-d-glucopyranosyl-2,3,6,7-tetramethoxyphenanthro[9,10-b]-11-indolizidinone (8)

To a solution of 14-hydroxyltylophorine 4 (0.47 g, 1.15 mmol) in $\hbox {CH}_{2}\hbox {Cl}_{2}$ was added $4\,{{\AA }}$ molecular sieves under argon, and then the mixture was stirred at room temperature for 0.5 h. After cooling to $-40\,^{\circ }\hbox {C},\,\hbox {BF}_{3} \cdot \hbox {Et}_{2}\hbox {O}$ (0.2 mL) was added and the mixture was stirred for another 0.5 h. 2,3,4,6-Tetra-$O$-acetyl-$\upalpha$-d-glucopyranosyl trichloroacetimidate (0.75 g, 1.20 mmol) in $\hbox {CH}_{2}\hbox {Cl}_{2}$ (15 mL) was added slowly and the mixture was stirred for 4 h at $-40\, ^{\circ }\hbox {C}$. The reaction was then quenched with water, and the organic layer separated. The water phase was extracted with $\hbox {CH}_{2}\hbox {Cl}_{2}$ (20 mL $\times$ 3). The combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by column chromatography (EtOAc, and then $\hbox {CH}_{2}\hbox {Cl}_{2}{-}\hbox {MeOH}$, 30:1, v/v) to give 7 (0.24 g, 28 %) as a light-yellow solid. As 7 was unstable, it was added immediately to a solution of MeONa (0.012 g, 0.23 mmol) in MeOH (30 mL). The solution was stirred at room temperature for 1 h and concentrated to remove MeOH. Cold water (15 mL) was added, and the mixture was stirred and filtered to give product 8. The filtrate was extracted with $\hbox {CH}_{2}\hbox {Cl}_{2}{-}\hbox {MeOH}$ (7:1) and then concentrated to give additional desired product. The combined product was dried to afford 8 (0.18g, 95 %) as a white solid (Scheme 1). $\hbox {Mp}\,211-213\,^{\circ }\hbox {C};\,^{1}\hbox {H NMR}\,(400\,\hbox {MHz},\, \hbox {DMSO-}d_{6})$: $\delta$ $8.00\,(\hbox {s}, 1\hbox {H}),$ $7.95\,(\hbox {s}, 1\hbox {H}), 7.93\,(\hbox {s}, \,1\hbox {H})$, $7.22\,(\hbox {s}, 1\hbox {H})$, 5.76 $(\hbox {d},\,J \!=\! 6.4\,\hbox {Hz}, 1\hbox {H})$, 4.97 $(\hbox {t}, J \!=\! 6.0\, \hbox {Hz}, 2\hbox {H})$, 4.93 $(\hbox {d}, J = 4.8\,\hbox {Hz}, 1\hbox {H})$, 4.77 $(\hbox {t}, J \!=\! 6.0\,\hbox {Hz}, 1\hbox {H})$, 4.60 $(\hbox {d}, J \!=\! 7.2\,\hbox {Hz}, 1\hbox {H})$, 4.52 $(d, J \!=\!$ 15.2 $\hbox {Hz}, 1\hbox {H}),$ 4.03 $(\hbox {s}, 3\hbox {H})$, 4.01 $(\hbox {s}, 3\hbox {H})$, 3.93 $(\hbox {s},3\hbox {H})$, 3.91 $(\hbox {s}, 3\hbox {H}), 3.78-3.83\,(\hbox {m}, 1\hbox {H}),$ 3.56 $(\hbox {d}, J \!=\! 14.8 \hbox {Hz}, 1\hbox {H})$, 3.45 ${-}$ 3.48 $(\hbox {m}, 1\hbox {H}), 3.01{-}3.07\,(\hbox {m}, 1\hbox {H}),$ 2.91${-}$2.96 $(\hbox {m}, 1\hbox {H}),$ 2.37 ${-}$ 3.46 $(\hbox {m}, 2\hbox {H})$, 1.92${-}$2.01 $(\hbox {m}, 1\hbox {H})$, 1.79$-$1.86 $(\hbox {m}, 2\hbox {H}). ^{13}\hbox {C NMR}\,(100\,\hbox {MHz}, \hbox {DMSO-}d_{6})$: $\delta$ 155.6, 149.3, 148.4, 129.6, 126.4, 126.2, 126.0, 124.1, 124.0, 123.0, 116.8, 108.3, 104.2, 101.8, 75.9, 73.5, 70.4, 68.5, 60.9, 59.9, 55.7, 55.4, 54.5, 53.3, 33.1, 31.0, 21.2. $\hbox {HRMS}\,(\hbox {ESI}): \,\hbox {calcd}.$ $\hbox {for}\,\hbox {C}_{30}\,\hbox {H}_{38}\hbox {NO}_{10}$ $[\hbox {M{+}H}]^{+}572.2490;$ found 572.2485.

Synthesis of the glycoconjugates 9–11 and 11a

The glycoconjugates 9–11 and 11a were synthesized using the same procedure with the preparation of 8. (S)-6-O-desmethylantofine (2) (0.40 g, 1.15 mmol) was treated with 2,3,4,6-tetra-$O$-acetyl-$\upalpha$-d-glucopyranosyl trichloroacetimidate, 2,3,4,6-tetra-$O$-acetyl-$\upalpha$-d-galactopyranosyl trichloroacetimidate and 2,3,4,6-tetra-$O$-acetyl-$\upalpha$-d-mannopyranosyl trichloroacetimidate to give glycoconjugates 9–11 and 11a.

(13aS)-6-O-$\beta$-d-Glucopyranosyl-2,3-dimethoxyphenanthro[9,10-b]-11-indolizidinone (9)

$\hbox {Mp}\,256{-}258\,^{\circ }\hbox {C}$; $^{1}\hbox {H NMR}\,(400\,\hbox {MHz}, \hbox {DMSO-}d_{6})$: $\delta$ 8.25 $(\hbox {s}, 1\hbox {H}), 8.03\,(\hbox {s}, 1\hbox {H}),$ 7.83 $(\hbox {d}, J\!=\! 9.2\,\hbox {Hz}, 1\hbox {H}),$ 7.33 $(\hbox {s}, 1\hbox {H}),$ 7.32 $(\hbox {d}, J= 9.2\,\hbox {Hz}, 1\hbox {H}),$ 5.40 $(\hbox {s}, 1\hbox {H}),$ 5.18 $(\hbox {s}, 1\hbox {H}),$ 5.06 ${-}$ 5.13 $(\hbox {m}, 2\hbox {H}),$ 4.71 ${-}$ 4.78 $(\hbox {m}, 1\hbox {H}),$ 4.57 $(\hbox {d}, J= 12.8\,\hbox {Hz}, 1\hbox {H}),$ 4.01 $(\hbox {s}, 3\hbox {H}),$ 3.95 $(\hbox {s}, 3\hbox {H}),$ 3.74 ${-}$ 3.81 $(\hbox {m}, 1\hbox {H}),$ 3.46 ${-}$ 3.55 $(\hbox {m}, 3\hbox {H}),$ 3.23 ${-}$ 3.26 $(\hbox {m}, 1\hbox {H}),$ 3.13 ${-}$ 3.23 $(\hbox {m}, 1\hbox {H}),$ 2.28 ${-}$ 2.42$(\hbox {m}, 2\hbox {H}),$ 2.14 ${-}$ 2.21 $(\hbox {m}, 1\hbox {H}),$ 1.77 ${-}$ 1.89 $(\hbox {m}, 2\hbox {H}),$ 1.59 ${-}$ 1.68$(\hbox {m}, 1\hbox {H}).\,^{13}\hbox {C}$ $\hbox {NMR}\,(100\,\hbox {MHz}, \hbox {DMSO-}d_{6})$: $\delta$ 155.5, 149.2, 148.3, 129.5, 126.3, 126.2, 126.0, 124.1, 124.0, 123.0, 116.7, 108.1, 104.1, 104.1, 101.1, 77.3, 76.7, 73.4, 70.0, 60.9, 59.8, 55.6, 55.4, 54.4, 53.3, 33.0, 30.9, 21.2. $\hbox {HRMS}\,(\hbox {ESI}):$ calcd. for $\hbox {C}_{28}\hbox {H}_{34}\hbox {NO}_{8}\,[\hbox {M{+}H}]^{+}512.2279;$ found 512.2281.

(13aS)-6-O-$\beta$-d-Galactopyranosyl-2,3-dimethoxyphenanthro[9,10-b]-11-indolizidinone (10)

$\hbox {Mp}$ 249 ${-}$ 251 $^{\circ }\hbox {C}$; $^{1}\hbox {H NMR}\,(400\,\hbox {MHz}, \hbox {DMSO-}d_{6})$: $\delta$ 8.28 $(\hbox {s}, 1\hbox {H}),$ 8.03 $(\hbox {s}, 1\hbox {H}),$ 7.84 $(\hbox {d}, J =$ 8.8 $\hbox {Hz}, 1\hbox {H})$, 7.35 $(\hbox {s}, 1\hbox {H}),$ 7.32 $(\hbox {d}, J=$ 8.8 $\hbox {Hz}, 1\hbox {H}),$ 5.24 $(\hbox {d}, J =$ 4.4 $\hbox {Hz}, 1\hbox {H}),$ 5.07 $(\hbox {d}, J =$ 4.0 $\hbox {Hz},\,1\hbox {H}),$ 4.96 $(\hbox {d}, J= 5.6$ $\hbox {Hz}, 1\hbox {H})$, 4.82 $(\hbox {t}, J=$ 5.2 $\hbox {Hz}, 1\hbox {H}),$ 4.61 $(\hbox {d}, J=$ 4.0 $\hbox {Hz}, 1\hbox {H}),$ 4.02 $(\hbox {s}, 3\hbox {H}),$ 3.96 $(\hbox {s}, 3\hbox {H}),$ 3.75 ${-}$ 3.78 $(\hbox {m}, 2\hbox {H}),$ 3.67 ${-}$ 3.70 $(\hbox {m}, 1\hbox {H}),$ 3.58 ${-}$ 3.61 $(\hbox {m}, 2\hbox {H}),$ 3.41 ${-}$ 3.50 $(\hbox {m}, 3\hbox {H}),$ 2.82 ${-}$ 2.93 $(\hbox {m}, 1\hbox {H}),$ 2.22 ${-}$ 2.24 $(\hbox {m}, 1\hbox {H}),$ 1.85 ${-}$ 1.95 $(\hbox {m}, 2\hbox {H}), 1.68 {-} 1.85 (\hbox {m}, 1\hbox {H}).{^{13}}\hbox {C}\, \hbox {NMR}\,(100\,\hbox {MHz}, \hbox {DMSO-}d_{6})$: $\delta$ 155.6, 149.3, 148.4, 129.6, 126.4, 126.2, 126.0, 124.1, 124.0, 123.0, 116.8, 108.3, 104.2, 101.8, 75.9, 73.5, 70.4, 68.5, 60.9, 59.9, 55.7, 55.4, 54.5, 53.3, 33.1, 31.0, 21.2. $\hbox {HRMS}\,(\hbox {ESI})$: calcd. for $\hbox {C}_{28}\hbox {H}_{34}\hbox {NO}_{8}$ $[\hbox {M{+}H}]^{+}\,512.2279;$ found 512.2284.

(13aS)-6-O-$\beta$-d-Mannopyranosyl-2,3-dimethoxyphenanthro[9,10-b]-11-indolizidinone (11)

$\hbox {Mp} 258{-}260\,^{\circ }\hbox {C}$; $^{1}\hbox {H NMR}$ (400 $\hbox {MHz}, \hbox {DMSO-}d_{6})$: $\delta$ 8.36 $(\hbox {s}, 1\hbox {H}),$ 8.06 $(\hbox {s}, 1\hbox {H}),$ 7.82 $(\hbox {d}, J=$ 9.2 $\hbox {Hz}, 1\hbox {H}),$ 7.38 $(\hbox {d}, J=$ 8.8 $\hbox {Hz}, 1\hbox {H})$, 7.31 $(\hbox {s}, 1\hbox {H}),$ 5.58 $(\hbox {s}, 1\hbox {H}),$ 5.11 $(\hbox {br}, 1\hbox {H}),$ 4.84 ${-}$ 4.99 $(\hbox {m}, 2\hbox {H}),$ 4.53 ${-}$ 4.63 $(\hbox {m}, 2\hbox {H}),$ 4.02 $(\hbox {s}, 3\hbox {H}),$ 3.96 $(\hbox {s}, 1\hbox {H}),$ 3.94 $(\hbox {s}, 3\hbox {H}),$ 3.82 $(\hbox {d}, J=$ 6.4 $\hbox {Hz}, 1\hbox {H}),$ 3.68 $(\hbox {d}, J=$ 10.4 $\hbox {Hz}, 1\hbox {H}),$ 3.48 ${-}$ 3.56 $(\hbox {m}, 2\hbox {H}),$ 2.71 ${-}$ 2.77 $(\hbox {m}, 1\hbox {H}),$ 2.22 ${-}$ 2.37$(\hbox {m}, 2\hbox {H}),$ 2.06 ${-}$ 2.18 $(\hbox {m}, 1\hbox {H}),$ 1.75 ${-}$ 1.88 $(\hbox {m},\,2\hbox {H}),$ 1.59 ${-}$ 1.64 $(\hbox {m}, 1\hbox {H}).$ $^{13}\hbox {C} \,\hbox {NMR}\,(100\,\hbox {MHz}, \hbox {DMSO-}d_{6})$: $\delta$ 154.4, 149.1, 148.2, 129.5, 126.1, 126.1, 126.0, 124.2, 123.9, 122.9, 116.8, 109.7, 104.2, 103.9, 99.4, 74.8, 70.6, 70.1, 66.8, 61.0, 59.6, 55.6, 55.2, 54.3, 53.1, 32.9, 30.7, 21.0. $\hbox {HRMS}\,(\hbox {ESI}):$ calcd. for $\hbox {C}_{28}\hbox {H}_{34}\hbox {NO}_{8}$ $[\hbox {M{+}H}]^{+}\,512.2279;$ found 512.2275.

(13aS)-6-O-(2,3,4,6-Tetra-O-acetyl-$\beta$-d-Mannopyranosyl)-2,3-dimethoxyphenanthro[9,10-b]-11-indolizidinone (11a)

$\hbox {Mp}\,186{-}188\,^{\circ }\hbox {C}$; $^{1}\hbox {H NMR}\,(400\,\hbox {MHz}, \hbox {DMSO-}d_{6})$: $\delta$ 8.14 $(\hbox {s}, 1\hbox {H}),$ 7.88 $(\hbox {s}, 1\hbox {H}),$ 7.71 $(\hbox {d}, J=$ 9.2 $\hbox {Hz}, 1\hbox {H}),$ 7.33 $(\hbox {d}, J=$ 8.0 $\hbox {Hz}, 1\hbox {H})$, 7.21 $(\hbox {s}, 1\hbox {H}),$ 5.73 $(\hbox {s}, 1\hbox {H})$, 5.66 $(\hbox {d}, J=$ 9.6 $\hbox {Hz}, 1\hbox {H}),$ 5.56 $(\hbox {s}, 1\hbox {H})$, 4.42 $(\hbox {t}, J=$ 10.0 $\hbox {Hz}, 1\hbox {H}),$ 4.93 ${-}$ 4.96 $(\hbox {m}, 1\hbox {H})$, 4.30 ${-}$ 4.34 $(\hbox {m}, 1\hbox {H})$, 4.18 ${-}$ 4.21 $(\hbox {m}, 1\hbox {H}),$ 4.16 $(\hbox {s}, 3\hbox {H})$, 4.09 ${-}$ 4.12 $(\hbox {m}, 2\hbox {H})$, 4.04 $(\hbox {s}, 3\hbox {H})$, 3.39 ${-}$ 3.42 $(\hbox {m}, 1\hbox {H})$, 3.15 ${-}$ 3.24 $(\hbox {m}, 2\hbox {H})$, 2.36 ${-}$ 2.41 $(\hbox {m}, 1\hbox {H})$, 2.25 $(\hbox {s}, 3\hbox {H})$, 2.15 ${-}$ 2.17 $(\hbox {m}, 1\hbox {H})$, 2.08 $(\hbox {s}, 6\hbox {H})$, 1.95 ${-}$ 2.03 $(\hbox {m}, 3\hbox {H})$, 1.91 $(\hbox {s}, 3\hbox {H}).\,\hbox {HRMS}\,(\hbox {ESI})$: calcd. for $\hbox {C}_{36}\hbox {H}_{41}\hbox {NO}_{12}$ $[\hbox {M{+}H}]^{+}680.2702;$ found 680.2700.

Synthesis of (13aS)-2,3-dimethoxy-6-propargyloxyphenanthro[9,10-b]-11-indolizidinone (12)

To a solution of ($S)$-6-$O$-desmethylantofine (0.81 g, 2.33 mmol) in N,N-dimethylformamide (15 mL) was added $\hbox {Cs}_{2}\hbox {CO}_{3}$ $(0.91\, \hbox {g}, 2.79\,\hbox {mmol})$. The mixture was stirred at room temperature for 0.5 h and then brought to $0\,^{\circ }\hbox {C}$, then a solution of propargyl bromide (0.33 g, 2.79 mmol) in N,N-dimethylformamide (10 mL) was added slowly. The mixture was warmed to room temperature and stirred until the reaction was complete. EtOAc (30 mL) and water (30 mL) were added and the solution was separated, then the water phase was extracted with EtOAc (20 mL $\times$ 2). The combined organic phase was dried over sodium sulfate, filtered, and evaporated to give crude product. This material was then purified by column chromatography (EtOAc as eluate) to give 12 (0.92 g, 85 %) as a white solid (Scheme 2). $\hbox {Mp}\,195{-}197\,^{\circ }\hbox {C}$; $^{1}\hbox {H NMR}\,(400\,\hbox {MHz}, \hbox {CDCl}_{3})$: $\delta$ $8.04\,(\hbox {br}, 1\,\hbox {H}),$ $7.91\,(\hbox {s}, 1\,\hbox {H})$, $7.83\,(\hbox {d}, J = 9.2\,\hbox {Hz},1 \hbox {H})$, $7.31\,(\hbox {s}, 1\,\hbox {H})$, $7.24{-}7.26\,(\hbox {m}, 1\,\hbox {H})$, $4.90\,(\hbox {s}, 2\,\hbox {H})$, 4.69 $(\hbox {d}, J =$ 14.8 $\hbox {Hz}, 1\,\hbox {H})$, 4.10 $(\hbox {s}, 3\,\hbox {H})$, 4.06 $(\hbox {s}, 3\,\hbox {H})$, 3.69 $(\hbox {d}, J =$ 14.4 $\hbox {Hz}, 1\,\hbox {H})$, 3.47 $(\hbox {t}, J =$ 8.0 $\hbox {Hz}, 1\,\hbox {H})$, 3.35 $(\hbox {d}, J =$ 15.6, $1\,\hbox {H})$, 2.87 ${-}$ 2.93 $(\hbox {m}, 1\,\hbox {H})$, 2.59 $(\hbox {s}, 1\,\hbox {H}$), 2.44 ${-}$ 2.52 $(\hbox {m}, 2\,\hbox {H})$, 2.20 ${-}$ 2.29 $(\hbox {m}, 1\,\hbox {H})$, 2.00 ${-}$ 2.06 $(\hbox {m}, 1\,\hbox {H})$, 1.90 ${-}$ 1.97 $(\hbox {m}, 1\,\hbox {H})$, 1.74 ${-}$ 1.82 $(\hbox {m}, 1\, \hbox {H})$. $^{13}\hbox {C NMR}$ (100 $\hbox {MHz}, \hbox {CDCl}_{3})$: $\delta$ 155.4, 149.5, 148.4, 130.1, 127.1, 126.6, 126.1, 124.7, 124.3, 123.5, 115.1, 106.6, 104.0, 103.8, 78.7, 75.8, 60.2, 56.3, 56.0, 55.9, 55.1, 53.9, 33.8, 31.3, 21.6. $\hbox {HRMS}\,(\hbox {ESI})$: calcd. for $\hbox {C}_{25}\hbox {H}_{26}\hbox {NO}_{3}$ $[\hbox {M{+}H}]^{+}388.1907;$ found 388.1909.

Synthesis of (13aS,14S)-2,3,6,7-tetramethoxy-14-propargyloxyphenanthro[9,10-b]-11-indolizidinone (13)

To a solution of DCB-3503 (3) (0.83 g, 2.03 mmol) in N,N-dimethylformamide (60 mL) was added NaH (0.49 g, 20.29 mmol). The mixture was stirred at $40{-}45\,^{\circ }\hbox {C}$ for 1 h. Propargyl bromide (1.19 g, 10.15 mmol) was added at room temperature and the mixture was stirred overnight. Ethyl acetate and water were added to the mixture slowly. The water phase was extracted with ethyl acetate and the combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by column chromatography (EtOAc/petroleum ether, 1:1, v/v) to give 0.73 g (80 %) of13 as a white solid (Scheme 2). $\hbox {Mp} \,205{-}207\,^{\circ }\hbox {C}$; $^{1}\hbox {H NMR}\,(400\,\hbox {MHz}, \hbox {CDCl}_{3})$: $\delta$ $7.87\,(\hbox {s},1\,\hbox {H})$, $7.84\,(\hbox {s}, 1\,\hbox {H})$, $7.82\,(\hbox {s},1\,\hbox {H})$, $7.22\,(\hbox {s}, 1\,\hbox {H})$, $5.29\,(\hbox {s}, 1\,\hbox {H})$, 4.68 $(\hbox {d}, J=$ 15.2 $\hbox {Hz}, 1\,\hbox {H})$, 4.61 $(\hbox {d},\,J=$ 15.2 $\hbox {Hz}, 1\,\hbox {H})$, 4.13 $(\hbox {s}, 6\,\hbox {H})$, 4.08 $(\hbox {s}, 3\,\hbox {H}$), 4.06 $(\hbox {s}, 3\,\hbox {H})$, 3.81 $(\hbox {d}, J=$ 15.6 $\hbox {Hz}, 1\,\hbox {H}$), 3.53 $(\hbox {d}, J=$ 15.2 $\hbox {Hz}, 1\,\hbox {H})$, 3.43 $(\hbox {t}, J=$ 8.0, $1\,\hbox {H}$), 3.54 ${-}$ 3.58 $(\hbox {m}, 1\,\hbox {H})$, 3.33 ${-}$ 3.43 $(\hbox {m}, 2\,\hbox {H})$, 2.31 $(\hbox {s}, 1\,\hbox {H})$, 1.92 ${-}$ 2.05 $(\hbox {m}, 3\,\hbox {H})$. $^{13}\hbox {C NMR}\,(100\,\hbox {MHz}, \hbox {CDCl}_{3})$: $\delta$ 149.5, 149.3, 149.0, 130.5, 126.3, 125.0, 124.1, 124.0, 123.6, 105.3, 103.7, 103.4, 103.1, 81.6, 73.3, 71.2, 65.4, 56.2, 56.1, 55.5, 54.5, 54.4, 24.6, 22.0. $\hbox {HRMS}\,(\hbox {ESI})$: calcd. for $\hbox {C}_{27}\hbox {H}_{30}\hbox {NO}_{5}$ $[\hbox {M{+}H}]^{+}448.2118;$ found 448.2124.

Synthesis of glycoconjugates 17–19 and 23–25

To a mixture of compound 12 or 13 (0.27 mmol) and $\beta$-1-azido-2,3,4,6-tetra-$O$-acetyl-d-glucose azide, $\beta$-1-azido-2,3,4,6-tetra-$O$-acetyl-d-glucose azide or $\beta$-1-azido-2,3,4,6-tetra-$O$-acetyl-d-glucose azide (0.11 g, 0.29 mmol) in $\hbox {CH}_{2}\hbox {Cl}_{2}$ (20 mL) and $\hbox {H}_{2}\hbox {O}$ (20 mL) were added $\hbox {CuSO}_{4}\cdot 5\hbox {H}_{2}\hbox {O}$ (0.02 g, 0.081 mmol), ascorbic acid sodium (0.05 g, 0.27 mmol) at room temperature. The mixture was stirred at reflux for 2 h and cooled to room temperature. The water phase was extracted with $\hbox {CH}_{2}\hbox {Cl}_{2}$. The combined organic phase was washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by column chromatography to give 14–16 (EtOAc, and then $\hbox {CH}_{2}\hbox {Cl}_{2}{-}\hbox {MeOH},25{:}1, \hbox {v/v})$ or 20–22 (EtOAc/petroleum ether, 1:1, v/v, and then EtOAc) as a light-yellow solid. Then the compound 14–16 or 20–22 was added to a solution of MeONa (2.7 mg, 0.05 mmol) in MeOH (10 mL). The solution was stirred at room temperature for 1 h and concentrated to remove MeOH. Cold water (5 mL) was added, and the mixture was stirred and filtered to give the glycoconjugates 17–19 or 23–25. The filtrate was extracted with $\hbox {CH}_{2}\hbox {Cl}_{2}{-}\hbox {MeOH}\,(6{:}1)$ and then concentrated to give additional product. The combined product 17–19 or 23–25 was dried to afford a white solid (Scheme 2).

(13aS)-6-(1-(2,3,4,6-Tetra-O-acetyl-$\beta$-d-glucopyranosyl)-1H-1,2,3-triazol-4-yl)methoxy-2,3-dimethoxyphenanthro[9,10-b]indolizidine (14)

$\hbox {Mp}\,204{-}206\,^{\circ }\hbox {C}$;$^{1}\hbox {H NMR}\,(400\,\hbox {MHz}, \hbox {CDCl}_{3})$: $\delta$ 8.05 $(\hbox {d}, J=$ 1.6 $\hbox {Hz}, 1\,\hbox {H})$, 7.94 $(\hbox {s}, 1\,\hbox {H}),$ 7.92 $(\hbox {s}, 1\,\hbox {H}$), 7.81 $(\hbox {d},J=$ 9.2 $\hbox {Hz}, 1\,\hbox {H})$, 7.30 $(\hbox {s}, 1\,\hbox {H})$, 7.24 ${-}$ 7.26 $(\hbox {m}, 1\,\hbox {H})$, 5.87 $(\hbox {d},J=$ 8.8 $\hbox {Hz}, 1\,\hbox {H})$, 5.44 $(\hbox {s}, 2\,\hbox {H})$, 5.37 ${-}$ 5.44 $(\hbox {m}, 1\,\hbox {H})$, 5.23 $(\hbox {t}, J=$ 9.2 $\hbox {Hz}, 1\,\hbox {H})$, 4.68 $(\hbox {d},J=$ 14.8 $\hbox {Hz}, 1\,\hbox {H})$, 4.29 $(\hbox {dd}, J=$ 12.8, 5.2 $\hbox {Hz}, 1\,\hbox {H})$, 4.14 $(\hbox {s}, 3\,\hbox {H})$, 4.13 $(\hbox {d},J=$ 12.8 $\hbox {Hz}, 1 \,\hbox {H}),$ 4.06 $(\hbox {s}, 3\,\hbox {H})$, 3.96 ${-}$ 3.99 $(\hbox {m}, 1\,\hbox {H})$, 3.68 $(\hbox {d}, J=$ 14.8 $\hbox {Hz}, 1 \,\hbox {H})$, 3.44 ${-}$ 3.48 $(\hbox {m}, 1\,\hbox {H})$, 3.34 $(\hbox {dd}, J=$ 15.2, 2.4 $\hbox {Hz}, 1\,\hbox {H})$, 2.85 ${-}$ 2.92 $(\hbox {m}, 1\,\hbox {H})$, 2.41 ${-}$ 2.52 $(\hbox {m}, 2\,\hbox {H})$, 2.20 ${-}$ 2.28 $(\hbox {m}, 1\,\hbox {H})$, 2.06 $(\hbox {s}, 7\,\hbox {H})$, 2.01 $(\hbox {s}, 3\,\hbox {H})$, 1.89 ${-}$ 1.94 $(\hbox {m}, 1\,\hbox {H})$, 1.72 ${-}$ 1.78 $(\hbox {m}, 2\,\hbox {H})$, 1.67 $(\hbox {s}, 3\,\hbox {H})$. $^{13}\hbox {C NMR}\,(100\,\hbox {MHz}, \hbox {CDCl}_{3})$: $\delta$ 170.5, 169.9, 169.3, 168.9, 155.9, 149.4, 148.5, 145.2, 130.1, 127.0, 126.6, 125.9, 124.4, 124.3, 123.6, 121.3, 115.6, 106.0, 103.9, 103.8, 85.8, 75.2, 72.6, 70.2, 67.7, 62.2, 61.5, 60.2, 56.0, 55.9, 55.1, 53.9, 33.8, 31.3, 21.6, 20.7, 20.5, 20.5, 19.9. $\hbox {HRMS}\,(\hbox {ESI})$: calcd. for $\hbox {C}_{39}\hbox {H}_{45}\hbox {N}_{4}\hbox {O}_{12}$ $[\hbox {M{+}H}]^{+}761.3028;$ found 761.3033.

(13aS)-6-(1-(2,3,4,6-Tetra-O-acetyl-$\beta$-d-galactopyranosyl)-1H-1,2,3-triazol-4-yl)methoxy-2,3-dimethoxyphenanthro[9,10-b]indolizidine (15)

$\hbox {Mp}\,200{-}202\,^{\circ }\hbox {C};$ $^{1}\hbox {H NMR}\,(400\,\hbox {MHz}, \hbox {CDCl}_{3})$: $\delta$ 8.06 ${-}$ 8.08 $(\hbox {m}, 1\,\hbox {H}),$ 8.03 $(\hbox {s}, 1\,\hbox {H})$, 7.93 $(\hbox {s}, 1\,\hbox {H}),$ 7.81 $(\hbox {d},J=$ 9.2 $\hbox {Hz}, 1\,\hbox {H})$, 7.29 $(\hbox {s}, 1\,\hbox {H})$, 7.25 ${-}$ 7.28 $(\hbox {m}, 1\,\hbox {H})$, 5.84 $(\hbox {d}, J=$ 9.2 $\hbox {Hz}, 1\,\hbox {H})$, 5.53 ${-}$ 5.59 $(\hbox {m}, 2\,\hbox {H})$, 5.45 $(\hbox {s}, 2\,\hbox {H}),$ 5.21 ${-}$ 5.25 $(\hbox {m}, 1\,\hbox {H}),$ 4.69 $(\hbox {d}, J=$ 15.2$\hbox {Hz}, 1\,\hbox {H})$, 4.17 ${-}$ 4.22 $(\hbox {m}, 1\,\hbox {H})$, 4.15 $(\hbox {s}, 3\,\hbox {H})$, 4.10 ${-}$ 4.13 $(\hbox {m}, 1\,\hbox {H})$, 4.06 $(\hbox {s}, 3\,\hbox {H})$, 3.70 $(\hbox {d}, J=$ 14.8 $\hbox {Hz}, 1\,\hbox {H})$, 3.47 $(\hbox {t},J=$ 6.8 $\hbox {Hz}, 1\,\hbox {H})$, 3.34 $(\hbox {dd}, J=$ 16.0 $\hbox {Hz},$ 2.8 $\hbox {Hz}, 1\,\hbox {H})$, 2.87 ${-}$ 2.95 $(\hbox {m}, 1\,\hbox {H})$, 2.43 ${-}$ 2.50 $(\hbox {m}, 1\,\hbox {H})$, 2.23 ${-}$ 2.26 $(\hbox {m}, 1 \,\hbox {H})$, 2.21 $(\hbox {s}, 3\,\hbox {H}),$ 2.03 ${-}$ 2.07 $(\hbox {m}, 1\,\hbox {H})$, 2.03 $(\hbox {s}, 3\,\hbox {H})$, 1.99 $(\hbox {s}, 3 \,\hbox {H})$, 1.90 ${-}$ 1.95 $(\hbox {m}, 1\,\hbox {H})$, 1.78 ${-}$ 1.83 $(\hbox {m}, 1\,\hbox {H})$, 1.69 ${-}$ 1.75 $(\hbox {m}, 3\,\hbox {H})$. $^{13}\hbox {C NMR}$ (100 $\hbox {MHz},\,\hbox {CDCl}_{3})$: $\delta$ 170.3, 170.0, 169.8, 169.0, 156.0, 149.4, 148.5, 145.1, 130.1, 126.9, 125.8, 124.3, 123.6, 121.4, 115.7, 115.5, 106.1, 106.0, 103.9, 103.8, 86.3, 77.2, 74.1, 70.7, 67.8, 66.8, 62.2, 61.2, 60.2, 56.0, 55.9, 55.0, 33.6, 31.2, 21.6, 20.6, 20.5, 20.1, 20.0. $\hbox {HRMS}\,(\hbox {ESI})$: calcd. for $\hbox {C}_{39}\hbox {H}_{45}\hbox {N}_{4}\hbox {O}_{12}$ $[\hbox {M{+}H}]^{+}$ 761.3028; found 761.3027.

(13aS)-6-(1-(2,3,4,6-Tetra-O-acetyl-$\beta$-d-mannopyranosyl)-1H-1,2,3-triazol-4-yl)methoxy-2,3-dimethoxyphenanthro[9,10-b]indolizidine (16)

$\hbox {Mp}\,197{-}199\,^{\circ }\hbox {C}$ ;$^{1}\hbox {H NMR}\,(400\,\hbox {MHz},\,\hbox {CDCl}_{3})$: $\delta$ $8.05\,(\hbox {d}, J =$ 12.0 $\hbox {Hz}, 1\,\hbox {H})$, 7.88 $-$ 7.91 $(\hbox {m}, 2\,\hbox {H}),$ 7.76 $(\hbox {d},J=$ 12.8 $\hbox {Hz}, 1\,\hbox {H})$, 7.27 $(\hbox {s}, 1\,\hbox {H})$, 7.20 ${-}$ 7.22 $(\hbox {m}, 1\,\hbox {H})$, 6.16 $(\hbox {s}, 1\,\hbox {H})$, 5.73 $(\hbox {s}, 1\,\hbox {H})$, 5.45 $(\hbox {s}, 2\,\hbox {H})$, 5.26 ${-}$ 5.36 $(\hbox {m}, 2\,\hbox {H})$, 4.75 $(\hbox {d}, J=$ 14.4 $\hbox {Hz}, 1\,\hbox {H})$, 4.32 $(\hbox {dd},J=$ 12.4 $\hbox {Hz},$ 2.0 $\hbox {Hz}, 1\,\hbox {H})$, 4.20 $(\hbox {d}, J=$ 12.4 $\hbox {Hz}, 1\,\hbox {H})$, 4.13 $(\hbox {s}, 3\,\hbox {H})$, 4.06 $(\hbox {s}, 3\,\hbox {H})$, 3.94 ${-}$ 3.98 $(\hbox {m}, 1\,\hbox {H})$, 3.77 ${-}$ 3.86 $(\hbox {m}, 1\,\hbox {H})$, 3.50 ${-}$ 3.61 $(\hbox {m}, 1\,\hbox {H})$, 3.67 $(\hbox {d}, J=$ 15.2 $\hbox {Hz}, 1\,\hbox {H})$, 3.00 ${-}$ 3.06$(\hbox {m}, 1\,\hbox {H})$, 2.55 ${-}$ 2.76 $(\hbox {m}, 2\,\hbox {H})$, 2.25 ${-}$ 2.33 $(\hbox {m}, 1\,\hbox {H})$, 2.08 $(\hbox {br}, 7\,\hbox {H})$, 1.97 $(\hbox {s}, 4\,\hbox {H})$, 1.69 ${-}$ 1.85 $(\hbox {m}, 4\,\hbox {H})$. $^{13}\hbox {C NMR}\,(100\,\hbox {MHz}, \hbox {CDCl}_{3})$: $\delta$ 170.5, 169.8, 169.6, 168.9, 156.1, 149.6, 148.7, 144.5, 130.3, 124.2, 124.1, 123.7, 121.9, 121.8, 115.7, 115.5, 106.1, 106.0, 103.9, 84.9, 77.2, 75.8, 70.7, 70.6, 68.8, 68.7, 64.9, 62.3, 62.3, 62.2, 56.1, 55.9, 30.9, 21.5, 20.7, 20.7, 20.5, 20.1. $\hbox {HRMS}\,(\hbox {ESI})$: calcd. for $\hbox {C}_{39}\hbox {H}_{45}\hbox {N}_{4}\hbox {O}_{12}$ $[\hbox {M{+}H}]^{+}$ 761.3028; found 761.3027.

(13aS,14S)-14-(1-(2,3,4,6-Tetra-O-acetyl-$\beta$-d-glucopyranosyl)-1H-1,2,3-triazol-4-yl)methoxy-2,3,6,7-tetramethoxy phenanthro[9,10-b]-11-indolizidinone (20)

$\hbox {Mp}\,118{-}120\,^{\circ }\hbox {C}$; $^{1}\hbox {H NMR}$ (400 $\hbox {MHz},\,\hbox {CDCl}_{3})$: $\delta$ 7.84 $(\hbox {s}, 1\,\hbox {H})$, 7.82 $(\hbox {s}, 1\,\hbox {H})$, 7.80 $(\hbox {s}, 1\,\hbox {H})$, 7.37 $(\hbox {s}, 1 \,\hbox {H})$, 7.22 $(\hbox {s}, 1\,\hbox {H})$, 5.73 $(\hbox {d}, J =$ 8.4 $\hbox {Hz}, 1\,\hbox {H})$, 5.29 ${-}$ 5.32 $(\hbox {m}, 2\,\hbox {H})$, 5.26 $(\hbox {s}, 1\,\hbox {H})$, 5.08 ${-}$ 5.17 $(\hbox {m}, 2\,\hbox {H})$, 4.70 $(\hbox {d}, J =$ 15.2 $\hbox {Hz}, 1\,\hbox {H}$), 4.21 ${-}$ 4.27$(\hbox {m}, 2\,\hbox {H})$, 4.12 $(\hbox {s}, 6\,\hbox {H})$, 4.05 $(\hbox {s}, 3\,\hbox {H}$), 3.95 $(\hbox {s}, 3\,\hbox {H})$, 3.86 ${-}$ 3.89 $(\hbox {m}, 1\,\hbox {H})$, 3.41 ${-}$ 3.54 $(\hbox {m}, 2\,\hbox {H})$, 2.55 $(\hbox {br}, 1\,\hbox {H})$, 2.33 ${-}$ 2.41 $(\hbox {m}, 2\,\hbox {H})$, 2.07 $(\hbox {s}, 3\,\hbox {H})$, 2.04 $(\hbox {s}, 3\,\hbox {H})$, 2.00 ${-}$ 2.02 $(\hbox {m}, 2\,\hbox {H})$, 1.99 $(\hbox {s}, 3\,\hbox {H}),$ 1.90 ${-}$ 1.97 $(\hbox {m}, 2\,\hbox {H})$. $^{13}\hbox {C NMR}$ (100 $\hbox {MHz},\, \hbox {DMSO-}d_{6})$: $\delta$ 170.6, 167.0, 169.4, 168.8, 149.4, 149.2, 148.9, 148.8, 147.3, 130.4, 126.1, 124.9, 124.3, 124.0, 123.5, 120.3, 105.2, 103.7, 103.4, 103.1, 85.6, 75.0, 72.8, 71.6, 70.2, 67.8, 65.9, 65.4, 61.7, 60.3, 56.1, 56.1, 55.4, 54.4, 24.6, 22.0, 20.7, 20.6, 20.6, 20.2. $\hbox {HRMS}\,(\hbox {ESI})$: calcd. for $\hbox {C}_{41}\hbox {H}_{49}\hbox {N}_{4}\hbox {O}_{14}$ $[\hbox {M{+}H}]^{+}$ 821.3240; found 821.3237.

(13aS,14S)-14-(1-(2,3,4,6-Tetra-O-acetyl-$\beta$-d-galactopyranosyl)-1H-1,2,3-triazol-4-yl)methoxy-2,3,6,7-tetramethoxy phenanthro[9,10-b]-11-indolizidinone (21)

$\hbox {Mp}\,117{-}119\,^{\circ }\hbox {C}$; $^{1}\hbox {H NMR}\,(400\,\hbox {MHz},\,\hbox {CDCl}_{3})$: $\delta$ 7.88 $(\hbox {s}, 1\,\hbox {H}),$ 7.86 $(\hbox {s}, 1\,\hbox {H})$, 7.84 $(\hbox {s}, 1\,\hbox {H}),$ 7.45 $(\hbox {s}, 1 \,\hbox {H})$, 7.26 $(\hbox {s}, 1\,\hbox {H})$, 5.75 $(\hbox {d}, J =$ 9.2 $\hbox {Hz}, 1\,\hbox {H})$, 5.50 $(\hbox {d}, J =$ 3.2 $\hbox {Hz}, 1\,\hbox {H})$, 5.43 $(\hbox {t}, J =$ 10.0 $\hbox {Hz}, 1\,\hbox {H})$, 5.33 $(\hbox {s}, 1\,\hbox {H})$, 5.18 $(\hbox {dd}, J =$ 10.4 $\hbox {Hz}$, 3.2 $\hbox {Hz}, 1\,\hbox {H})$, 5.13 $(\hbox {d}, J =$ 12.0 $\hbox {Hz}, 1\,\hbox {H})$, 4.75 $(\hbox {d}, J =$ 14.8 $\hbox {Hz}, 1\,\hbox {H})$, 4.31 $(\hbox {d}, J =$ 12.4 $\hbox {Hz}, 1\,\hbox {H})$, 4.16 ${-}$ 4.20 $(\hbox {m}, 2\,\hbox {H})$, 4.15 $(\hbox {s}, 3\,\hbox {H})$, 4.13 $(\hbox {s}, 3\,\hbox {H})$, 4.10 $(\hbox {br}, 1\,\hbox {H}),$ 4.08 $(\hbox {s}, 3\,\hbox {H})$, 3.98 $(\hbox {s}, 3\,\hbox {H})$, 3.59 $(\hbox {d}, J =$ 15.2 $\hbox {Hz}, 1\,\hbox {H})$, 3.47 $(\hbox {t}, J =$ 7.6 $\hbox {Hz}, 1\,\hbox {H})$, 2.62 ${-}$ 2.65 $(\hbox {m}, 1\,\hbox {H})$, 2.36 ${-}$ 2.49 $(\hbox {m}, 2\,\hbox {H})$, 2.20 $(\hbox {s}, 3\,\hbox {H})$, 2.08 ${-}$ 2.11 $(\hbox {m}, 1\,\hbox {H})$, 2.05 $(\hbox {s}, 3\,\hbox {H})$, 2.02 ${-}$ 2.04 $(\hbox {m}, 1\,\hbox {H})$, 1.98 $(\hbox {s}, 3\,\hbox {H})$, 1.92 ${-}$ 1.95 $(\hbox {m}, 1\,\hbox {H}$), 1.81 $(\hbox {s}, 3\,\hbox {H})$. $^{13}\hbox {C NMR}\,(100\,\hbox {MHz},\, \hbox {CDCl}_{3})$: $\delta$ 170.4, 170.1, 169.9, 169.0, 149.4, 149.2, 148.9, 148.8, 147.3, 130.5, 126.2, 124.9, 124.3, 124.0, 123.6, 120.5, 105.3, 103.7, 103.4, 103.2, 86.2, 73.9, 71.7, 71.0, 67.8, 66.9, 65.4, 61.2, 60.3, 56.2, 56.1, 55.5, 54.4, 24.6, 22.0, 20.7, 20.7, 20.6, 20.3. $\hbox {HRMS}\,(\hbox {ESI})$: calcd. for $\hbox {C}_{41}\hbox {H}_{49}\hbox {N}_{4}\hbox {O}_{14}$ $[\hbox {M{+}H}]^{+}$ 821.3240; found 821.3236.

(13aS,14S)-14-(1-(2,3,4,6-Tetra-O-acetyl-$\beta$-d-mannopyranosyl)-1H-1,2,3-triazol-4-yl)methoxy-2,3,6,7-tetramethoxy-phenanthro[9,10-b]-11-indolizidinone (22)

$\hbox {Mp}$ 139 ${-}$ 141 $^{\circ }\hbox {C}$; $^{1}\hbox {H NMR}$ (400 $\hbox {MHz},\,\hbox {CDCl}_{3})$: $\delta$ 7.86 $(\hbox {s}, 2\,\hbox {H})$, 7.84 $(\hbox {s}, 1\,\hbox {H})$, 7.38 $(\hbox {s}, 1\,\hbox {H})$, 6.05 $(\hbox {s}, 1 \,\hbox {H})$, 5.60 $(\hbox {s}, 1\,\hbox {H})$, 5.34 $(\hbox {s}, 1\,\hbox {H})$, 5.26 $(\hbox {s}, 1\,\hbox {H})$, 5.28 $(\hbox {t}, J =$ 9.6 $\hbox {Hz},1\,\hbox {H})$, 5.20 $(\hbox {d},J =$ 9.6 $\hbox {Hz}, 1\,\hbox {H})$, 5.09 $(\hbox {d}, J =$ 12.0 $\hbox {Hz}, 1\,\hbox {H})$, 4.74 $(\hbox {d}, J =$ 15.2 $\hbox {Hz}, 1\,\hbox {H})$, 4.34 $(\hbox {d}, J =$ 12.8 $\hbox {Hz}, 1\,\hbox {H})$, 4.27 ${-}$ 4.30 $(\hbox {m}, 1\,\hbox {H})$, 4.19 $(\hbox {d}, J =$ 12.8 $\hbox {Hz}, 1\,\hbox {H})$, 4.15 $(\hbox {s}, 3\,\hbox {H})$, 4.14 $(\hbox {s}, 3\,\hbox {H})$, 4.08 $(\hbox {s}, 3\,\hbox {H})$, 3.99 $(\hbox {s}, 3\,\hbox {H})$, 3.88 ${-}$ 3.92 $(\hbox {m}, 1\,\hbox {H})$, 3.58 $(\hbox {m}, d, J =$ 15.2 $\hbox {Hz}, 1\,\hbox {H})$, 3.46 ${-}$ 3.49 $(\hbox {m}, 2\,\hbox {H})$, 2.61 ${-}$ 2.65 $(\hbox {m}, 1\,\hbox {H})$, 2.36 ${-}$ 2.42 $(\hbox {m}, 2\,\hbox {H})$, 2.09 $(\hbox {s}, 6\,\hbox {H})$, 1.98 ${-}$ 2.06 $(\hbox {m}, 3\,\hbox {H})$, 1.96 $(\hbox {s}, 3\,\hbox {H})$, 1.79 $(\hbox {s}, 3\,\hbox {H})$. $^{13}\hbox {C NMR}\,(100\,\hbox {MHz},\,\hbox {DMSO-}d_{6})$: $\delta$ 170.7, 169.8, 169.7, 169.1, 149.5, 149.1, 149.0, 148.9, 146.9, 130.6, 126.1, 124.8, 124.3, 124.0, 123.6, 121.2, 105.3, 103.7, 103.3, 103.0, 84.7, 75.7, 71.7, 70.9, 68.9, 65.3, 65.1, 62.4, 60.4, 56.2, 56.1, 56.1, 55.5, 54.4, 24.6, 22.0, 20.8, 20.8, 20.6, 20.3. $\hbox {HRMS}\,(\hbox {ESI})$: calcd. for $\hbox {C}_{41}\hbox {H}_{49}\hbox {N}_{4}\hbox {O}_{14}$ $[\hbox {M{+}H}]^{+}$ 821.3240; found 821.3236.

(13aS)-6-(1-($\beta$-d-Glucopyranosyl)-1H-1,2,3-triazol-4-yl)methoxy-2,3-dimethoxyphenanthro[9,10-b]indolizidine (17)

$\hbox {Mp}$ 259 ${-}$ 261 $^{\circ }\hbox {C}$; $^{1}\hbox {H NMR}$ (400 $\hbox {MHz},\,\hbox {DMSO-}d_{6})$: $\delta$ 8.54 $(\hbox {s}, 1\,\hbox {H}),$ 8.34 $(\hbox {s}, 1\,\hbox {H})$, 8.16 $(\hbox {s}, 1\,\hbox {H})$, 7.85 $(\hbox {d}, J=$ 8.8 $\hbox {Hz}, 1\,\hbox {H})$, 7.36 $(\hbox {br}, 2\,\hbox {H})$, 5.59 $(\hbox {d}, J=$ 9.2 $\hbox {Hz}, 1\,\hbox {H})$, 5.45 $(\hbox {d}, J=$ 9.2 $\hbox {Hz}, 1\,\hbox {H})$, 5.44 $(\hbox {s}, 2\,\hbox {H})$, 5.34 $(\hbox {d}, J=$ 4.4 $\hbox {Hz}, 1\,\hbox {H})$, 5.21 $(\hbox {d}, J=$ 5.2 $\hbox {Hz}, 1\,\hbox {H})$, 4.78 ${-}$ 4.81 $(\hbox {m}, 1\,\hbox {H})$, 4.66 $(\hbox {br}, 1\,\hbox {H})$, 4.05 $(\hbox {s}, 3 \,\hbox {H})$, 3.97 $(\hbox {s}, 3\,\hbox {H})$, 3.78 ${-}$ 3.84 $(\hbox {m}, 1\,\hbox {H})$, 3.69 ${-}$ 3.72 $(\hbox {m}, 1\,\hbox {H})$, 3.41 ${-}$ 3.52 $(\hbox {m}, 4\,\hbox {H})$, 3.27 ${-}$ 3.28 $(\hbox {m}, 1\,\hbox {H})$, 2.94 ${-}$ 2.98 $(\hbox {m}, 1\,\hbox {H})$, 2.29 $(\hbox {br}, 1\,\hbox {H})$, 1.97 $(\hbox {br}, 1\,\hbox {H})$, 1.75 ${-}$ 1.79 $(\hbox {m}, 1\,\hbox {H})$. $^{13}\hbox {C NMR}$ $(100 \,\hbox {MHz},\hbox {DMSO-}d_{6})$: $\delta$ 157.6, 150.6, 149.9, 143.9, 131.1, 127.0, 126.2, 125.5, 125.2, 124.4, 124.2, 117.0, 107.3, 105.8, 105.4, 88.7, 81.2, 78.2, 73.3, 70.7, 62.6, 61.9, 61.5, 57.1, 56.6, 54.8, 49.8, 31.2, 22.0. $\hbox {HRMS}\,(\hbox {ESI})$: calcd. for $\hbox {C}_{31}\hbox {H}_{37}\hbox {N}_{4}\hbox {O}_{8}$ $[\hbox {M{+}H}]^{+}$ 593.2606; found 593.2604.

(13aS)-6-(1-($\beta$-d-Galactopyranosyl)-1H-1,2,3-triazol-4-yl)methoxy-2,3-dimethoxyphenanthro[9,10-b]indolizidine (18)

$\hbox {Mp}$ 234 ${-}$ 236 $^{\circ }\hbox {C}$; $^{1}\hbox {H NMR}$ (400 $\hbox {MHz}, \hbox {DMSO-}d_{6})$: $\delta$ 8.46 $(\hbox {s}, 1\,\hbox {H})$, 8.30 $(\hbox {s}, 1\,\hbox {H})$, 8.14 $(\hbox {s}, 1\,\hbox {H})$, 7.83 $(\hbox {d},J=$ 8.8 $\hbox {Hz}, 1\,\hbox {H}),$ 7.33 $(\hbox {br}, 2\,\hbox {H})$, 5.54 $(\hbox {d}, J=$ 8.8 $\hbox {Hz}, 1\,\hbox {H})$, 5.43 $(\hbox {s}, 2\,\hbox {H})$, 5.33 $(\hbox {d}, J=$ 2.4 $\hbox {Hz}, 1\,\hbox {H})$, 4.72 $(\hbox {br}, 2\,\hbox {H})$, 4.56 $(\hbox {d}, J =$ 15.2 $\hbox {Hz}, 1\,\hbox {H})$, 4.07 ${-}$ 4.14 $(\hbox {m}, 1\,\hbox {H})$, 4.05 $(\hbox {s}, 3\,\hbox {H})$, 3.95 $(\hbox {s}, 3\,\hbox {H}),$ 3.74 ${-}$ 3.78 $(\hbox {s}, 2\,\hbox {H})$, 3.54 $(\hbox {br}, 4\,\hbox {H}, \hbox {OH})$, 3.31 $(\hbox {br}, 2\,\hbox {H})$, 2.72 ${-}$ 2.79 $(\hbox {m}, 1\,\hbox {H})$, 2.30 ${-}$ 2.33 $(\hbox {m}, 2\,\hbox {H})$, 2.14 ${-}$ 2.15 $(\hbox {m}, 1\,\hbox {H})$, 1.84 $(\hbox {br}, 2\,\hbox {H})$, 1.62 ${-}$ 1.66 $(\hbox {m},$ $2\,\hbox {H})$. $^{13}\hbox {C NMR}\,(100\,\hbox {MHz}, \hbox {DMSO-}d_{6})$: $\delta$ 156.1, 149.3, 148.3, 142.9, 129.7, 126.3, 125.6, 124.2, 123.6, 123.5, 123.0, 115.6, 105.9, 104.6, 104.1, 88.1, 78.4, 73.7, 69.3, 68.4, 61.3, 60.4, 59.8, 55.8, 55.4, 54.4, 53.3, 33.0, 30.9, 21.2. $\hbox {HRMS}\,(\hbox {ESI})$: calcd. for $\hbox {C}_{31}\hbox {H}_{37}\hbox {N}_{4}\hbox {O}_{8}$ $[\hbox {M{+}H}]^{+}$ 593.2606; found 593.2605.

(13aS)-6-(1-($\beta$-d-Mannopyranosyl)-1H-1,2,3-triazol-4-yl)methoxy-2,3-dimethoxyphenanthro[9,10-b]indolizidine (19)

$\hbox {Mp}$ 226 ${-}$ 228 $^{\circ }\hbox {C}$; $^{1}\hbox {H NMR}$ (400 $\hbox {MHz}, \hbox {DMSO-}d_{6})$: $\delta$ 8.42 $(\hbox {s}, 1\,\hbox {H})$, 8.30 $(\hbox {s}, 1\,\hbox {H})$, 8.13 $(\hbox {s}, 1\,\hbox {H})$, 7.82 $(\hbox {d}, J =$ 9.2 $\hbox {Hz}, 1\,\hbox {H})$, 7.29 ${-}$ 7.32 $(\hbox {m}, 2\,\hbox {H})$, 6.06 $(\hbox {s}, 1\,\hbox {H})$, 5.43 $(\hbox {s}, 2\,\hbox {H})$, 5.08 ${-}$ 5.15 $(\hbox {m}, 1\,\hbox {H})$, 4.56 $(\hbox {d}, J=$ 15.2 $\hbox {Hz}, 1\,\hbox {H})$, 4.04 $(\hbox {s}, 3\,\hbox {H})$, 3.94 $(\hbox {s}, 3\,\hbox {H})$, 3.91 $(\hbox {s}, 1\,\hbox {H})$, 3.75 $(\hbox {d}, J=$ 11.6 $\hbox {Hz}, 1\,\hbox {H})$, 3.61 ${-}$ 3.64 $(\hbox {m}, 1\,\hbox {H})$, 3.41 ${-}$ 3.53 $(\hbox {m}, 4\,\hbox {H})$, 3.31 $(\hbox {br}, 1\,\hbox {H})$, 3.72 ${-}$ 3.78 $(\hbox {m}, 1\,\hbox {H})$, 2.27 ${-}$ 2.35 $(\hbox {m}, 2\,\hbox {H})$, 2.13 ${-}$ 2.15 $(\hbox {m}, 1\,\hbox {H})$, 1.98 ${-}$ 1.89 $(\hbox {m}, 2 \,\hbox {H})$, 1.58 ${-}$ 1.68 $(\hbox {m}, 1\,\hbox {H})$. $^{13}\hbox {C NMR}$ (100 $\hbox {MHz}, \hbox {DMSO-}d_{6})$: $\delta$ 156.0, 149.2, 148.2, 142.3, 129.6, 126.2, 125.5, 124.3, 124.1, 123.4, 122.9, 115.6, 105.8, 104.5, 104.0, 85.9, 80.3, 73.0, 70.4, 66.1, 61.3, 61.0, 59.7, 55.7, 55.3, 54.3, 53.2, 33.0, 30.8, 21.1. $\hbox {HRMS}\,(\hbox {ESI})$: calcd. for $\hbox {C}_{31}\hbox {H}_{37}\hbox {N}_{4}\hbox {O}_{8}$ $[\hbox {M{+}H}]^{+}$ 593.2606; found 593.2604.

(13aS,14S)-14-(1-($\beta$-d-Glucopyranosyl)-1H-1,2,3-triazol-4-yl)methoxy-2,3,6,7-tetramethoxy phenanthro[9,10-b]-11-indolizidinone (23)

$\hbox {Mp}$ 172 ${-}$ 174 $^{\circ }\hbox {C}$; $^{1}\hbox {H NMR}$ (400 $\hbox {MHz}, \hbox {DMSO-}d_{6})$: $\delta$ 8.04 $(\hbox {s}, 1\,\hbox {H})$, 8.03 $(\hbox {s}, 1\,\hbox {H})$, 8.02 $(\hbox {s}, 1\,\hbox {H})$, 7.74 $(\hbox {s}, 1 \,\hbox {H})$, 7.26 $(\hbox {s}, 1\,\hbox {H})$, 5.46 $(\hbox {d}, J =$ 9.2 $\hbox {Hz}, 1\,\hbox {H})$, 5.30 $(\hbox {d}, J =$ 3.6 $\hbox {Hz}, 1\,\hbox {H})$, 5.25 $(\hbox {d}, J =$ 4.4 $\hbox {Hz}, 1\,\hbox {H})$, 5.11 ${-}$ 5.14 $(\hbox {m}, 1\,\hbox {H})$, 4.96 $(\hbox {d}, J =$ 11.6 $\hbox {Hz}, 1\,\hbox {H})$, 4.72 $(\hbox {d}, J =$ 7.6 $\hbox {Hz}, 1\,\hbox {H})$, 4.58 $(\hbox {m}, 1\,\hbox {H})$, 4.05 $(\hbox {s}, 6\,\hbox {H})$, 3.94 $(\hbox {s}, 3\,\hbox {H})$, 3.87 $(\hbox {s}, 3\,\hbox {H})$, 3.66 ${-}$ 3.69 $(\hbox {m}, 2\,H)$, 3.38 ${-}$ 3.44 $(\hbox {m}, 3\,\hbox {H})$, 3.19 ${-}$ 3.23 $(\hbox {m}, 1\,\hbox {H})$, 2.35 ${-}$ 2.37 $(\hbox {m}, 1\,\hbox {H})$, 2.23 ${-}$ 2.38 $(\hbox {m}, 2\,\hbox {H})$, 1.86 ${-}$ 1.93 $(\hbox {m}, 3\,\hbox {H})$. $^{13}\hbox {C NMR}$ (100 $\hbox {MHz}$, $\hbox {DMSO-}d_{6})$: $\delta$ 149.2, 148.7, 148.6, 144.9, 129.9, 125.4, 124.3, 123.8, 123.5, 122.8, 122.4, 105.2, 104.1, 103.9, 87.4, 79.9, 77.0, 72.0, 70.5, 69.6, 64.9, 60.8, 59.8, 55.9, 55.9, 55.5, 55.3, 54.8, 53.7, 24.1, 21.4. $\hbox {HRMS}\,(\hbox {ESI})$: calcd. for $\hbox {C}_{33}\hbox {H}_{41}\hbox {N}_{4}\hbox {O}_{10}$ $[\hbox {M{+}H}]^{+}$ 653.2817; found 653.2812.

(13aS,14S)-14-(1-($\beta$-d-Galactopyranosyl)-1H-1,2,3-triazol-4-yl)methoxy-2,3,6,7-tetramethoxy phenanthro[9,10-b]-11-indolizidinone (24)

$\hbox {Mp}$ 177 ${-}$ 179 $^{\circ }\hbox {C}$; $^{1}\hbox {H NMR}$ (400 $\hbox {MHz},\hbox {DMSO-}d_{6})$: $\delta$ 8.06 $(\hbox {s}, 1\,\hbox {H})$, 8.05 $(\hbox {s}, 1\,\hbox {H})$, 8.03 $(\hbox {s}, 1 \,\hbox {H})$, 7.80 $(\hbox {s}, 1\,\hbox {H})$, 7.31 $(\hbox {s}, 1 \,\hbox {H})$, 5.41 $(\hbox {d}, J =$ 9.2 $\hbox {Hz}, 1\,\hbox {H})$, 5.23 $(\hbox {s}, 1\,\hbox {H})$, 5.01 $(\hbox {d}, J =$ 11.6 $\hbox {Hz}, 1\,\hbox {H})$, 4.78 $(\hbox {d}, J =$ 16.0 $\hbox {Hz}, 1\,\hbox {H})$, 3.35 ${-}$ 3.39 $(\hbox {m}, 1\,\hbox {H})$, 4.05 $(\hbox {s}, 6\,\hbox {H})$, 3.96 $(\hbox {s}, 3 \,\hbox {H})$, 3.89 $(\hbox {s}, 3\,\hbox {H})$, 3.74 $(\hbox {s}, 1\, \hbox {H})$, 3.65 ${-}$ 3.67 $(\hbox {m}, 1\,\hbox {H})$, 3.47 ${-}$ 3.53 $(\hbox {m}, 4\,\hbox {H})$, 3.43 ${-}$ 3.45 $(\hbox {m}, 3\,\hbox {H})$, 2.29 ${-}$ 2.36 $(\hbox {m}, 2\,\hbox {H})$, 1.88 ${-}$ 1.99 $(\hbox {m}, 3\,\hbox {H})$. $^{13}\hbox {C NMR}\,(100\,\hbox {MHz}, \hbox {DMSO-}d_{6})$: $\delta$ 149.2, 148.6, 145.0, 129.9, 125.5, 124.3, 123.9, 123.5, 122.8, 122.0, 105.2, 104.1, 104.0, 88.0, 79.2, 78.3, 73.7, 70.5, 69.3, 68.5, 65.0, 60.4, 60.0, 55.9, 55.5, 55.3, 54.8, 53.7, 24.1, 21.4. $\hbox {HRMS}\,(\hbox {ESI})$: calcd. for $\hbox {C}_{33}\hbox {H}_{41}\hbox {N}_{4}\hbox {O}_{10}$ $[\hbox {M{+}H}]^{+}$ 653.2817; found 653.2816.

(13aS,14S)-14-(1-($\beta$-d-Mannopyranosyl)-1H-1,2,3-triazol-4-yl)methoxy-2,3,6,7-tetramethoxy phenanthro[9,10-b]-11-indolizidinone (25)

$\hbox {Mp}$ 171 ${-}$ 173 $^{\circ }\hbox {C}$; $^{1}\hbox {H NMR}$ (400 $\hbox {MHz}, \hbox {DMSO-}d_{6})$: $\delta$ 8.05 $(\hbox {s}, 1\,\hbox {H})$, 8.03 $(\hbox {s}, 1\,\hbox {H})$, 7.97 $(\hbox {s}, 1 \,\hbox {H})$, 7.28 $(\hbox {s}, 1\,\hbox {H})$, 5.92 $(\hbox {s}, 1 \,\hbox {H})$, 5.20 ${-}$ 5.22 $(\hbox {m}, 2\,\hbox {H})$, 4.94 ${-}$ 5.06 $(\hbox {m}, 3\,\hbox {H})$, 4.75 $(\hbox {d}, J =$ 15.6 $\hbox {Hz}, 1 \,\hbox {H})$, 4.58 ${-}$ 4.59 $(\hbox {m}, 1\,\hbox {H})$, 4.05 $(\hbox {s}, 7\,\hbox {H})$, 3.95 $(\hbox {s}, 3\,\hbox {H})$, 3.88 $(\hbox {s}, 3\,\hbox {H})$, 3.81 $(\hbox {br}, 1\,\hbox {H})$, 3.70 ${-}$ 3.74 $(\hbox {m}, 1\,\hbox {H})$, 3.57 $(\hbox {br}, 1\,\hbox {H})$, 3.45 ${-}$ 3.47 $(\hbox {m}, 2\,\hbox {H})$, 2.37 ${-}$ 2.44 $(\hbox {m}, 1\,\hbox {H})$, 2.26 ${-}$ 2.29 $(\hbox {m}, 2\,\hbox {H})$, 1.80 ${-}$ 1.99 $(\hbox {m}, 3\,\hbox {H})$. $^{13}\hbox {C NMR}$ (100 $\hbox {MHz}, \hbox {DMSO-}d_{6})$: $\delta$ 149.2, 148.6, 144.6, 129.9, 125.4, 124.3, 123.8 123.5, 122.8, 122.7, 105.1, 104.1, 103.9, 85.8, 80.3, 73.2, 70.5, 70.4, 66.2, 64.9, 61.1, 60.0, 55.9, 55.9, 55.5, 55.3, 54.8, 53.7, 29.6, 24.1, 21.4. $\hbox {HRMS}\,(\hbox {ESI})$: calcd. for $\hbox {C}_{33}\hbox {H}_{41}\hbox {N}_{4}\hbox {O}_{10}$ $[\hbox {M{+}H}]^{+}$ 653.2817; found 653.2812.

Synthesis of (13aS)-6-(1,3,4,6-tetra-O-acetyl-$\beta$-d-glucopyranosyl-2-deoxy-2-amino)methanoyl-2,3-dimethoxyphenanthro[9,10-b]-11-indolizidinone (26)

To a solution of ($S)$-6-$O$-desmethylantofine (2) (0.18 g, 0.52 mmol) in CH$_{2}$Cl$_{2}$ (40 mL) was added 1,3,4,6-tetra-$O$-acetyl-2-deoxy-2-isocyanato-$\upbeta$-d-glucopyranose (G) (0.27 g, 0.62 mmol) and Et$_{3}$N (0.4 mL). The mixture was stirred at room temperature for 3 h, and then concentrated. The crude product was purified by column chromatography (EtOAc, and then $\hbox {CH}_{2}\hbox {Cl}_{2}{-}\hbox {MeOH}$, 10:1, v/v) to give 26 (0.35 g, 92 %) as a light-yellow solid (Scheme 3). $\hbox {Mp}\,196{-}198\,^{\circ }\hbox {C}$; $^{1}\hbox {H NMR}\,(400\,\hbox {MHz},\hbox {CDCl}_{3})$: $\delta$ $8.16\,(\hbox {s}, 1\hbox {H})$, $7.86\,(\hbox {s}, 1\hbox {H})$, $7.84\,(\hbox {d}, J= 13.2\,\hbox {Hz}, 1\hbox {H})$, $7.31\,(\hbox {s}, 1\hbox {H})$, $7.28\,(\hbox {d}, J= 13.2 \,\hbox {Hz}, 1\hbox {H})$, $5.87\,(\hbox {d}, J= 8.4\,\hbox {Hz}, 1\hbox {H})$, $5.43\,(\hbox {d}, J= 8.4\,\hbox {Hz}, 1\hbox {H})$, $5.33{-}5.38\,(\hbox {m}, 1\hbox {H})$, $5.17\,(\hbox {t}, J= 9.6\,\hbox {Hz}, 1\hbox {H}),$ 4.67 $(\hbox {d}, J =$ 14.8 $\hbox {Hz}, 1\hbox {H})$, 4.28 ${-}$ 4.32 $(\hbox {m}, 1\hbox {H})$, 4.13 $(\hbox {s}, 1\hbox {H})$, 4.10 $(\hbox {s}, 3\hbox {H})$, 4.06 $(\hbox {s}, 3\hbox {H})$, 3.96 ${-}$ 4.02 $(\hbox {m}, 1\hbox {H})$, 3.82 ${-}$ 3.84 $(\hbox {m}, 1\hbox {H})$, 3.68 $(\hbox {d}, J=$ 14.8 $\hbox {Hz}, 1\hbox {H})$, 3.44 ${-}$ 3.49 $(\hbox {m}, 1\hbox {H})$, 3.33 ${-}$ 3.37 $(\hbox {m}, 1\hbox {H})$, 2.89 ${-}$ 2.95 $(\hbox {m}, 1\hbox {H})$, 2.42 ${-}$ 2.53 $(\hbox {m}, 2\hbox {H})$, 2.23 ${-}$ 2.25 $(\hbox {m}, 1\hbox {H})$, 2.19 $(\hbox {s}, 3\hbox {H})$, 2.14 $(\hbox {s}, 3\hbox {H})$, 2.10 $(\hbox {s}, 3\hbox {H})$, 2.06 $(\hbox {s}, 3\hbox {H})$, 1.91 ${-}$ 2.02 $(\hbox {m}, 2\hbox {H})$, 1.76 ${-}$ 1.82 $(\hbox {m}, 1\hbox {H})$. $^{13}\hbox {C NMR}\,(100\,\hbox {MHz}, \hbox {CDCl}_{3})$: $\delta$ 171.0, 170.7, 169.4, 154.4, 149.6, 148.6, 129.8, 127.8, 127.1, 126.9, 126.5, 124.0, 123.7, 119.7, 114.1, 103.9, 92.5, 72.8, 72.4, 67.9, 65.8, 61.6, 60.1, 55.9, 55.9, 55.4, 55.0, 53.8, 33.8, 31.3, 29.7, 21.6, 21.0, 20.8, 20.7, 20.6, 15.3. $\hbox {HRMS}\,(\hbox {ESI})$: calcd. for $\hbox {C}_{37}\hbox {H}_{43}\hbox {N}_{2}\hbox {O}_{13}$ $[\hbox {M{+}H}]^{+}$ 723.2760; found 723.2765.

Synthesis of the glycoconjugate 27–30

To a solution of 14-hydroxyltylophorine (3–6) (0.18 g, 0.44 mmol) in $\hbox {CH}_{2}\hbox {Cl}_{2}$ (40 mL) was added 1,3,4,6-tetra-$O$-acetyl-2-deoxy-2-isocyanato-$\upbeta$-d-glucopyranose (G) (0.76 g, 1.76 mmol) and $\hbox {Et}_{3}\hbox {N}$ (0.4 mL). The mixture was heated to reflux under stirring under $\hbox {N}_{2}$ for 3 h and concentrated. The crude product was purified by column chromatography (EtOAc/petroleum ether, 10:1, v/v) to give 27–30 as a light-yellow solid.

(13aS,14S)-14-(1,3,4,6-Tetra-O-acetyl-$\beta$-d-glucopyranosyl-2-deoxy-2-amino)methanoyl-2,3,6,7-tetramethoxyphenanthro[9,10-b]-11-indolizidinone (27)

$\hbox {Mp}$ 200 ${-}$ 202 $^{\circ }\hbox {C}$; $^{1}\hbox {H NMR}$ (400 $\hbox {MHz}, \hbox {CDCl}_{3})$: $\delta$ 7.80 $(\hbox {s}, 1\hbox {H})$, 7.78 $(\hbox {s}, 1\hbox {H})$, 7.52 $(\hbox {s}, 1\hbox {H})$, 7.06 $(\hbox {s}, 1\hbox {H})$, 6.57 $(\hbox {s}, 1\hbox {H})$, 5.72 $(\hbox {d}, J=$ 8.4 $\hbox {Hz}, 1\hbox {H})$, 5.09 ${-}$ 5.17 $(\hbox {m}, 2\hbox {H})$, 4.64 ${-}$ 4.73 $(\hbox {m}, 1\hbox {H})$, 4.28 $(\hbox {dd}, J=$ 13.4 $\hbox {Hz}, 2.4\,\hbox {Hz}, 1\hbox {H})$, 4.16 $(\hbox {br}, 1\hbox {H})$, 4.13 $(\hbox {s}, 3\hbox {H})$, 4.10 $(\hbox {s}, 3\hbox {H})$, 4.04 ${-}$ 4.06 $(\hbox {m}, 1\hbox {H})$, 3.98 $(\hbox {br}, 6\,\hbox {H})$, 3.82 ${-}$ 3.84 $(\hbox {m}, 1\hbox {H})$, 3.63 ${-}$ 3.67 $(\hbox {m}, 1\hbox {H})$, 3.50 ${-}$ 3.57 $(\hbox {m}, 1\hbox {H})$, 2.74 ${-}$ 2.78 $(\hbox {m}, 1\hbox {H})$, 2.46 ${-}$ 2.56 $(\hbox {m}, 1\hbox {H})$, 2.14 $(\hbox {s}, 3\hbox {H})$, 2.10 $(\hbox {br}, 4\hbox {H})$, 2.03 $(\hbox {br}, 2\hbox {H})$, 2.00 $(\hbox {s}, 3\hbox {H})$, 1.87 ${-}$ 1.90 $(\hbox {m}, 2\hbox {H})$, 1.77 $(\hbox {s}, 3\hbox {H})$. $^{13}\hbox {C NMR}$ (100 $\hbox {MHz}, \hbox {CDCl}_{3})$: $\delta$ 170.8, 170.5, 169.6, 169.2, 156.7, 149.7, 149.4, 149.0, 148.9, 125.2, 125.1, 124.2, 124.1, 123.3, 104.1, 103.6, 103.4, 103.3, 92.9, 72.8, 71.78, 68.3, 63.9, 61.9, 61.8, 61.8, 56.2, 56.2, 56.1, 55.0, 54.7, 53.6, 24.7, 21.5, 20.9, 20.7, 20.1. $\hbox {HRMS}\,(\hbox {ESI})$: calcd. for $\hbox {C}_{39}\hbox {H}_{47}\hbox {N}_{2}\hbox {O}_{15}$ $[\hbox {M{+}H}]^{+}783.2971;$ found 783.2979.

(13aR,14S)-14-(1,3,4,6-Tetra-O-acetyl-$\beta$-d-glucopyranosyl-2-deoxy-2-amino)methanoyl-2,3,6,7-tetramethoxyphenanthro[9,10-b]-11-indolizidinone (28)

$\hbox {Mp}$ 205 ${-}$ 207 $^{\circ }\hbox {C}$; $^{1}\hbox {H NMR}$ (400 $\hbox {MHz}, \hbox {CDCl}_{3})$: $\delta$ 7.77 $(\hbox {s}, 1\hbox {H})$, 7.68 $(\hbox {s}, 1\hbox {H})$, 7.21 $(\hbox {s}, 1\hbox {H})$, 6.97 $(\hbox {s}, 1\hbox {H})$, 6.55 $(\hbox {d}, J=$ 7.2 $\hbox {Hz}, 1\hbox {H})$, 5.62 $(\hbox {d}, J=$ 8.8 $\hbox {Hz}, 1\hbox {H})$, 5.09 ${-}$ 5.11 $(\hbox {m}, 2\hbox {H})$, 4.38 $(\hbox {d}, J=$ 14.8 $\hbox {Hz}, 1\hbox {H})$, 4.24 $(\hbox {dd}, J=$ 14.8 $\hbox {Hz},$ 4.4 $\hbox {Hz}, 1\hbox {H})$, 4.13 $(\hbox {s}, 3\hbox {H})$, 4.08 ${-}$ 4.10 $(\hbox {m}, 2\hbox {H})$, 4.05 $(\hbox {s}, 3\hbox {H})$, 3.98 $(\hbox {s}, 3\hbox {H})$, 3.81 $(\hbox {s}, 3\hbox {H})$, 3.72 ${-}$ 3.76 $(\hbox {m}, 1\hbox {H})$, 3.47 ${-}$ 3.52 $(\hbox {m}, 1\hbox {H})$, 3.30 ${-}$ 3.34 $(\hbox {m}, 1\hbox {H})$, 2.46 ${-}$ 2.56 $(\hbox {m}, 2\hbox {H})$, 2.20 $(\hbox {s}, 3\hbox {H})$, 2.12 ${-}$ 2.17 $(\hbox {m}, 1\hbox {H})$, 2.09 $(\hbox {s}, 3\hbox {H})$, 2.01 $(\hbox {s}, 3\hbox {H})$, 1.89 ${-}$ 1.98 $(\hbox {m}, 3\hbox {H})$, 1.83 $(\hbox {s}, 3\hbox {H})$. $^{13}\hbox {C NMR}$ (100 $\hbox {MHz}, \hbox {CDCl}_{3})$: $\delta$ 170.5, 170.4, 169.7, 169.4, 156.7, 148.6, 148.1, 147.8, 147.7, 129.4, 125.1, 124.3, 124.0, 123.6, 122.6, 103.9, 103.3, 103.0, 102.7, 93.6, 75.1, 72.7, 71.3, 69.0, 65.9, 65.2, 62.4, 55.8, 55.6, 55.0, 54.3, 54.1, 53.1, 29.7, 21.9, 21.1, 20.7, 20.6, 19.8. $\hbox {HRMS}\,(\hbox {ESI})$: calcd. for $\hbox {C}_{39}\hbox {H}_{47}\hbox {N}_{2}\hbox {O}_{15}$ $[\hbox {M{+}H}]^{+}$ 783.2971; found 783.2969.

(13aR,14R)-14-(1,3,4,6-Tetra-O-acetyl-$\beta$-d-glucopyranosyl-2-deoxy-2-amino)methanoyl-2,3,6,7-tetramethoxyphenanthro[9,10-b]-11-indolizidinone (29)

$\hbox {Mp}$ 205 ${-}$ 207 $^{\circ }\hbox {C}$; $^{1}\hbox {H NMR}$ (400 $\hbox {MHz}, \hbox {CDCl}_{3})$: $\delta$ 7.73 $(\hbox {s}, 1\hbox {H})$, 7.64 $(\hbox {s}, 1\hbox {H}),$ 7.34 $(\hbox {s}, 1\hbox {H})$, 6.85 $(\hbox {s}, 1\hbox {H})$, 6.51 $(\hbox {s}, 1\hbox {H})$, 5.48 ${-}$ 5.59 $(\hbox {m}, 2\hbox {H})$, 5.16 $(\hbox {t}, J=$ 9.6 $\hbox {Hz}, 1\hbox {H})$, 5.06 $(\hbox {t}, J=$ 9.6 $\hbox {Hz}, 1\hbox {H})$, 4.38 $(\hbox {d}, J=$ 11.6 $\hbox {Hz}, 1\hbox {H})$, 4.20 ${-}$ 4.27 $(\hbox {m}, 1\hbox {H})$, 4.14 $(\hbox {s}, 3\hbox {H})$, 4.08 ${-}$ 4.13 $(\hbox {m}, 1\hbox {H})$, 4.05 $(\hbox {s}, 3\hbox {H})$, 3.91 $(\hbox {s}, 3\hbox {H})$, 3.83 ${-}$ 3.86 $(\hbox {m}, 1\hbox {H})$, 3.75 $(\hbox {s}, 3\hbox {H})$, 3.38 ${-}$ 3.39 $(\hbox {m}, 1\hbox {H})$, 2.73 $(\hbox {br}, 1\hbox {H})$, 2.37 $(\hbox {d}, J=$ 8.0 $\hbox {Hz}, 1\hbox {H})$, 2.11 $(\hbox {s}, 3\hbox {H})$, 2.07 $(\hbox {s}, 3\hbox {H})$, 2.04 $(\hbox {br}, 1\hbox {H})$, 2.00 $(\hbox {s}, 3\hbox {H})$, 2.00 $(\hbox {s}, 3\hbox {H})$, 1.91 $(\hbox {br}, 2\hbox {H})$, 1.75 $(\hbox {s}, 2\hbox {H})$. $^{13}\hbox {C NMR}$ (100 $\hbox {MHz}, \hbox {CDCl}_{3})$: $\delta$ 170.8, 170.4, 169.5, 169.2, 157.1, 149.3, 148.9, 148.7, 148.5, 128.9, 125.2, 124.9, 124.2, 124.0, 123.3, 104.0, 103.5, 103.3, 103.2, 92.2, 73.7, 72.9, 68.0, 63.4, 61.6, 60.5, 56.1, 56.0, 55.8, 55.1, 54.1, 53.7,24.8, 21.6,21.2, 20.9,20.7, 20.3. $\hbox {HRMS}\,(\hbox {ESI})$: calcd. for $\hbox {C}_{39}\hbox {H}_{47}\hbox {N}_{2}\hbox {O}_{15}$ $[\hbox {M{+}H}]^{+}$ 783.2971; found 783.2979.

(13aS,14R)-14-(1,3,4,6-Tetra-O-acetyl-$\beta$-d-glucopyranosyl-2-deoxy-2-amino)methanoyl-2,3,6,7-tetramethoxyphenanthro[9,10-b]-11-indolizidinone (30)

$\hbox {Mp}$ 206 ${-}$ 208 $^{\circ }\hbox {C}$; $^{1}\hbox {H NMR}$ (400 $\hbox {MHz}, \hbox {CDCl}_{3})$: $\delta$ 7.61 $(\hbox {s}, 1\hbox {H})$, 7.40 $(\hbox {s}, 1\hbox {H})$, 6.83 $(\hbox {s}, 1\hbox {H})$, 6.44 $(\hbox {s}, 1\hbox {H})$, 6.43 $(\hbox {d}, J=$ 7.2 $\hbox {Hz}, 1\hbox {H})$, 5.66 $(\hbox {d}, J=$ 8.8 $\hbox {Hz}, 1\hbox {H})$, 5.17 $(\hbox {t}, J =$ 9.6 $\hbox {Hz}, 1\hbox {H})$, 5.05 $(\hbox {t}, J=$ 10.0 $\hbox {Hz}, 1\hbox {H})$, 4.44 ${-}$ 4.48 $(\hbox {m}, 1\hbox {H})$, 4.16 ${-}$ 4.22 $(\hbox {m}, 2\hbox {H})$, 4.16 $(\hbox {s}, 3\hbox {H})$, 4.03 ${-}$ 4.08 $(\hbox {m}, 3\hbox {H})$, 3.99 $(\hbox {s}, 3\hbox {H})$, 3.86 ${-}$ 3.89 $(\hbox {m}, 1\hbox {H})$, 3.76 $(\hbox {s}, 3\hbox {H})$, 3.39 $(\hbox {s}, 3\hbox {H})$, 3.16 $(\hbox {br}, 1\hbox {H})$, 2.60 ${-}$ 2.61 $(\hbox {m}, 1\hbox {H})$, 2.29 ${-}$ 2.34 $(\hbox {m}, 1\hbox {H})$, 2.19 $(\hbox {s}, 3\hbox {H})$, 2.08 ${-}$ 2.14 $(\hbox {m}, 2\hbox {H})$, 2.06 $(\hbox {s}, 3\hbox {H})$, 1.99 $(\hbox {s}, 3\hbox {H})$, 1.88 $(\hbox {s}, 3\hbox {H})$, 1.80 ${-}$ 1.87 $(\hbox {m}, 4\hbox {H})$. $^{13}\hbox {C NMR}$ (100 $\hbox {MHz}, \hbox {CDCl}_{3})$: $\delta$ 170.9, 170.6, 169.3, 169.3, 156.8, 148.8, 148.4, 147.9, 147.9, 125.2, 124.4, 124.1, 123.8, 122.8, 104.2, 103.3, 103.1, 102.8, 91.8, 74.4, 72.9, 67.9, 65.2, 61.4, 56.0, 55.9, 55.8, 55.0, 54.5, 54.0, 29.5, 22.1, 21.0, 20.8, 20.7, 20.3. $\hbox {HRMS}\,(\hbox {ESI})$: calcd. for $\hbox {C}_{39}\hbox {H}_{47}\hbox {N}_{2}\hbox {O}_{15}$ $[\hbox {M{+}H}]^{+}$ 783.2971; found 783.2960.

Antiviral biological assay

The purification procedure of TMV, the method to test the antiviral activity of compounds against TMV in vitro and the method to test the protective effect, the inactivation effect and the curative effect of compounds against TMV in vivo were described in the literature [22].

Antiviral activity of compounds against TMV in vitro

Fresh leaf of the 5–6 growth stage of tobacco (Nicotiana tabacum var. Xanthi nc) inoculated by the juice-leaf rubbing method (concentration of TMV is 5.88 $\times$ $10^{-2}\,\upmu \hbox {g}/\hbox {mL}$) was cut into halves along the main vein. The halves were immersed into the solution of $500\,\upmu \hbox {g}/\hbox {mL}\,(\hbox {or}\,100\,\upmu \hbox {g}/\hbox {mL})$ of the compounds and double-distilled water for 20 min and then cultured at $25 \,^{\circ }\hbox {C}$ for 72 h. Each compound was tested three times.

Protective effect of compounds against TMV in vivo

A solution of the test compound was smeared on the left side, and the solvent served as a control on the right side for growing N. tabacum var. Xanthi nc leaves of the same ages. The leaves were then inoculated with the virus after 12 h. A brush was dipped in TMV of 6 $\times$ $10^{-3}\,\hbox {mg/mL}$ to inoculate the leaves, which were previously scattered with silicon carbide. The leaves were then washed with water and rubbed softly along the nervature once or twice. The number of local lesion appearing 3–4 days after inoculation were counted. There are three replicates for each compound. The deviation of values was $\pm$5 %.

Inactivation effect of compounds against TMV in vivo

The virus was inhibited by mixing with a solution of a test compound at the same volume for 30 min. The mixture was then inoculated on the left side of the leaves of N. tabacum var. Xanthi nc, whereas the right side of the leaves was inoculated with the mixture of solvent and the virus for control. The local lesion numbers were recorded 3–4 days after inoculation. There are three replicates for each compound. The deviation of values was $\pm$5 %.

Curative effect of compounds against TMV in vivo

Growing leaves of N. tabacum var. Xanthi nc of the same ages were selected. TMV (concentration of 6.0 $\times$ $10^{-3}\,\hbox {mg mL}^{-1})$ was dipped and inoculated on the whole leaves. Then, the leaves were washed with water and dried. The compound solution was smeared on the left side, and the solvent was smeared on the right side for control. The local lesion numbers were then counted and recorded 3–4 days after inoculation. There are three replicates for each compound. The deviation of values was $\pm$5 %.

The in vitro and in vivo inhibition rates of the compound were calculated according to the following formula (“av” means average, and controls were not treated with compound):

inhibition rate % $=$ [(av local lesion number of control $-$ av local lesion number of drug treated)/av local lesion number of control] $\times$100 %

Conclusion

The first glycoconjugation of phenanthroindolizidine alkaloids was accomplished and their glycoconjugates evaluated for their antiviral activities against TMV. The observed antiviral activity suggests that C6 of phenanthroindolizidine alkaloids was a suitable position for glycoconjugation. Introducing sugar unit through O-glycosylation to C6 could increase the antiviral activity of phenanthroindolizidine alkaloids. When the distance between sugar unit and phenanthroindolizidine alkaloid was longer than O-glycosides or the hydroxyl groups on sugar unit were protected, the antiviral activities decreased dramatically. In addition, all the deprotected ($S)$-6-$O$-desmethylantofine glycoconjugates exhibited higher water solubility ($>$4 mg/mL). O-glycosylation could be regarded as a powerful tool to enhance the antiviral activity and pharmacology of phenanthroindolizidine alkaloids. (13aS)-6-O-$\upbeta$-d-galactopyranosyl-2,3-dimethoxyphenanthro[9,10-b]-11-indolizidinone (10) and (13aS)-6-O-$\upbeta$-d-mannopyranosyl-2,3-dimethoxyphenanthro [9,10-b]-11-indolizidinone (11) could be considered for further use as antiviral agents against TMV in plants.