Synthesis and Cytotoxic Property of Annonaceous Acetogenin Glycoconjugates

Introduction

Annonaceous acetogenins (ACGs) have been isolated exclusively from species of the annonaceae family, exhibiting potent growth inhibitory activity against various cancer cell lines.^1–7 Bullatacin and squamocin (Figure 1) are two of the most cytotoxic acetogenins bearing adjacent tetrahydrofuran (THF) rings, which have been reported mainly in the seeds of these plants.^8–11 The anticancer potency of bullatacin has been reported to be much higher than some of the anticancer drugs currently used in the clinic. For example, bullatacin was found to be 10⁴–10⁵ times more potent than doxorubicin against both A549 and MCF-7 cell lines,¹² and 250 times more potent than doxorubicin against MCF-7/ADR, the human breast adenocarcinoma multidrug-resistant cell line.¹³ In addition, bullatacin was found to be 300 times more effective than taxol in treating L1210 murine leukemia in mouse model.¹⁴ Bullatacin and squamocin are promising anti-cancer agents worthy of further investigation. However, they display high toxicity toward normal cells as well¹⁰ and show poor solubility in water (less than 1 μg/mL).¹⁵ These properties prevent them from full evaluation of their therapeutic potential for cancer treatment. The preparation of derivatives for tumor-specific targeting by incorporation of different ligands could improve their activity and yield more suitable drugs.

Figure 1 Structures of squamocin and bullatacin.

Conjugation of carbohydrates with drugs has become increasingly important in bioorganic chemistry and chemical biology.^16,17 Glycosylation of drug molecules has been employed to modify their physico-chemical properties, including polarity, solubility, and stability, without affecting their biological activities.^18,19 For example, glycosylated porphyrins have been shown as promising photosensitizers in photodynamic therapy (PDT) for cancer owing to their good solubility and specific membrane interactions.²⁰ Moreover, glycosylation has been widely explored as a targeting strategy to selectively deliver anticancer drugs to cancer cells. This strategy is based on the findings that cancer cells require more glucose due to their rapid growth and altered metabolism than normal cells do, which results in the expression of higher levels of glucose transporters on plasma membrane of cancer cells to facilitate the uptake of glucose. For example, glucose transporter-1 (GLUT1), which also transports other hexoses such as galactose, mannose, and glucosamine,²¹ is expressed at a level 100–300 times higher in malignant cells than in normal cells.²² Anticancer agents conjugated to a glucose (or another sugar that is also a glucose transporter substrate) may be taken up by cancer cells more rapidly via facilitation by glucose transporters and be more selective toward cancer cells than normal cells. A number of anticancer agents such as doxorubicin,²³ daunorubicin,²⁴ paclitaxel and docetaxel,²⁵ 8-hydroxyquinoline and derivatives,²⁶ and benzodiazepins²⁷ have been redesigned to improve their cancer targeting and selectivity.

Previously, we reported a series of biotin-conjugated squamocin/bullatacin derivatives that showed tumor cell growth inhibitory activity with higher selectivity toward biotin receptor (+) tumor cells than their parent squamocin/bullatacin.²⁸ In this study, squamocin and bullatacin are conjugated to a glucose or galactose to improve their solubility in water and potentially their cancer targeting. The synthesis and preliminary studies of the anticancer activity of these new glycoconjugates are described.

Materials and Methods

Reagents and Instrumentation

Squamocin and bullatacin were obtained in our previous chemical investigation on the seeds of Annona squamosa.⁴² 2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl azide (6) and 2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl azide (7) were purchased from J&K scientific Ltd. (Beijing, China). Sodium ascorbate, 4-dimethylaminopyridine (DMAP) and N,N′–dicyclohexylcarbodiimide (DCC), copper sulfate pentahydrate, D-galactose, 5-hexynoic acid, acetic anhydride, trichloroacetonitrile, hydrazine acetate, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and trimethylsilyl trifluoromethanesulfonate (TMSOTf) were purchased from Aladdin Chemical Co., Ltd (Shanghai, China). Anhydrous sodium sulfate and N,N-dimethylformamide (DMF) were purchased from Guangzhou Reagent Factory (Guangzhou, China). Pyridine was refluxed with CaH₂ for 3 h, then distilled and immediately stored over activated 4Ǻ molecular sieves. CH₂Cl₂ was refluxed with CaH₂ for 4 h, then distilled and immediately stored over activated 4Ǻ molecular sieves.

Optical rotations were obtained on a Perkin-Elmer 341 polarimeter with MeOH as solvent. The ¹H and ¹³C NMR spectra were collected on a Bruker Avance-600 instrument at 600 (¹H) and 150 (¹³C) MHz. The 2D NMR (¹H-¹H COSY, HSQC, and HMBC) spectra were recorded on a Bruker Avance-600 instrument. Chemical shifts are reported as ppm (δ units) relative to tetramethylsilane (TMS) as an internal standard and the coupling constants as J in hertz. The splitting pattern abbreviations are as follows: s = singlet, d = doublet, dd = double doublet, t = triplet, m = multiplet. ESIMS data were obtained on an MDS SCIEX API 2000 LC/MS instrument. HRESIMS data were obtained on a Bruker maXis Q-TOF mass spectrometer. Preparative HPLC was run on a Shimadzu LC-6A pump and a Shimadzu RID-10A refractive index detector and all separations were carried out with an XTerra Prep MS C₁₈ column (19 × 300 mm, 10 μm) at a flow rate of 5 mL/min except where otherwise stated.

Preparation of Galacosylated Squamocin 4 and 5

D-galactose (1 g, 5.56 mmol) was dissolved in dried pyridine (10 mL). Acetic anhydride (5.33 mL, 55.6 mmol) was added and the mixture was stirred overnight at room temperature (Scheme 1). The solvent was removed under reduced pressure and then the residue was dissolved in EtOAc (100 mL). The EtOAc solution was extracted with 1 N HCl (2 x 50 mL), saturated aqueous NaHCO₃ (2 x 50 mL) and saturated aqueous NaCl solution (50 mL), respectively. The organic layer was dried over anhydrous Na₂SO₄, filtered and concentrated to provide D-galactose pentaacetate (2.186 g, 100%) as yellow syrup.

D-galactose pentaacetate (2.186 g, 5.56 mmol) was dissolved in DMF (16 mL). Hydrazine acetate (614 mg, 6.67 mmol) was added and then the mixture was stirred at 40 ^oC for 4 h (Scheme 1). The reaction was monitored by TLC (petroleum ether: acetone, 2:1). Upon completion, the solvent was removed under reduced pressure. The residue was dissolved in EtOAc and washed with saturated aqueous NaCl solution. The organic layer was then dried over anhydrous Na₂SO₄, filtered and evaporated under reduced pressure to afford 2,3,4,6-tetra-O-acetyl-D-galactopyranose (1.756 g, 91%) as a white solid.

2,3,4,6-Tetra-O-acetyl-D-galactopyranose (1.756 g, 5.05 mmol) was dissolved in dried CH₂Cl₂ (7 mL) under nitrogen atmosphere. Trichloroacetonitrile (7.2 mL, 50.5 mmol) and 1.8-diazabicyclo[5.4.0]undec-7-ene (DBU, 0.8 mL, 5.55 mmol) was slowly added, respectively, when the solution was cooled to 0°C. The mixture was stirred at 0°C for 7 h (Scheme 1) and monitored by TLC (petroleum ether: acetone, 2:1). When the reaction was completed, the mixture was filtered over celite, and was concentrated to give product 3 (761 mg, 31%) as an amorphous solid.

Squamocin (124 mg, 0.2 mmol), 3 (147 mg, 0.3 mmol) were dissolved in dried CH₂Cl₂ (5 mL) at 0°C. 4Ǻ molecular sieves (200 mg) and TMSOTf (0.02 mL, 0.1mmol) was added at nitrogen atmosphere and the mixture was stirred at 0°C for 30 min (Scheme 1). The reaction mixture was quenched with triethylamine and then concentrated under reduced pressure. The residue was dissolved in EtOAc and extracted with 1 N HCl, saturated aqueous NaHCO₃ and saturated aqueous NaCl solution. The organic layer was dried over anhydrous Na₂SO₄, filtered and concentrated to give crude product 245 mg.

The deprotection was performed by treatment with Et₃N: MeOH: H₂O (1:8:1) at 35°C for 30 h (Scheme 1). The reaction mixture was concentrated in vacuo and the crude product was separated by Sephadex LH-20 gel permeation chromatography using MeOH, followed by preparative HPLC using 88% MeOH to afford 4 (2 mg, 1%, t_R = 43.6 min) and 5 (2 mg, 1%, t_R = 65.1 min).

Characterization Data of Compound 4

A yellowish waxy solid; [α]²⁰_D +4.6 (c 0.06, MeOH); ¹H NMR (600 MHz, CDCl₃): 7.00 (1H, m, H-35), 5.00 (1H, m, H-36), 4.37 (1H, d, J = 7.6 Hz, H-1′), 4.03 (3H, m, H-15, 16, 4′), 3.86 (3H, m, H-20, 2′, 6′a), 3.80 (2H, m, H- 19, 6′b), 3.74 (1H, m, H-24), 3.62 (2H, m, H-23, 3′), 3.57 (1H, m, H-28), 3.53 (1H, m, H-5′), 2.26 (2H, t, J = 7.7 Hz, H-3), 1.95 (3H, m), 1.88 (1H, m), 1.82 (1H, m), 1.69 (1H, m), 1.45–1.58 (5H, m), 1.38–1.44 (4H, m), 1.41 (3H, d, J = 6 Hz, H-37), 1.20–1.37 (30H, m), 0.88 (3H, t, J = 6.9 Hz, H-34); ¹³C NMR (150 MHz, CDCl₃): 174.2 (C-1), 149.2 (C-35), 134.5 (C-2), 102.3 (C-1′), 82.8 (2C, C-16, C-19), 82.2 (C-20), 81.3 (C-23), 81.2 (C-15), 74.8 (C-5′), 77.5 (C-36), 73.7 (C-3′), 71.8 (C-28), 71.7 (C-2′), 71.2 (C-24), 69.2 (C-4′), 61.5 (C-6′), 37.7 and 37.4 (C-27, C-29), 32.1 (C-25), 31.6 (C-32), 30.2 (C-14), 30.0, 29.9 (3C), 29.8 (2C), 29.7, 29.5, 29.4, 29.3, 29.0, 28.9, 27.6, 26.0, 25.4, 25.1, 24.9, 22.9 (C-33), 21.7 (C-26), 19.4 (C-37), 14.3 (C-34); HR-ESIMS m/z: 785.5413 [M + H]⁺ (calcd for C₄₃H₇₇O₁₂, 785.5410).

Characterization Data of Compound 5

A yellowish waxy solid; [α]²⁰_D −1.2 (c 0.07, MeOH); ¹H NMR (600 MHz, CDCl₃): 7.00 (1H, m, H-35), 4.99 (1H, m, H-36), 4.30 (1H, d, J = 6.7 Hz, H-1′), 4.04 (1H, m, H-4′), 3.95 (2H, m, H-19, 20), 3.82 (5H, m, H-16, 23, 2′, 6′), 3.63 (3H, m, H-24, 28, 3′), 3.54 (1H, m, H-5′), 3.39 (1H, m, H-15), 2.28 (2H, t, J = 7.7 Hz, H-3), 1.93 (3H, m), 1.81 (1H, m), 1.72 (1H, m), 1.61 (1H, m), 1.45–1.56 (5H, m), 1.38–1.44 (4H, m), 1.40 (3H, d, J = 6 Hz, H-37), 1.20–1.35 (30H, m), 0.88 (3H, t, J = 6.9 Hz, H-34); ¹³C NMR (150 MHz, CDCl₃): 174.2 (C-1), 149.2 (C-35), 134.5 (C-2), 103.5 (C-1′), 83.6 (C-16), 83.1 (C-19), 82.7 (C-20), 82.2 (C-23), 81.4 (C-28), 77.7 (C-36), 74.6 (C-15), 74.4 (C-5′), 73.6 (C-3′), 71.8 (C-24, C-2′), 69.9 (C-4′), 61.4 (C-6′), 35.6 and 34.1 (C-27, C-29), 33.2 (C-14), 32.6 (C-25), 32.1 (C-32), 30.0, 29.9 (4C), 29.8, 29.7, 29.5, 29.4, 29.1, 29.0, 28.9, 27.6, 25.8, 25.7, 25.4 (2C), 22.9 (C-33), 21.7 (C-26), 19.4 (C-37), 14.4 (C-34); HR-ESIMS m/z: 785.5415 [M + H]⁺ (calcd for C₄₃H₇₇O₁₂, 785.5410).

Preparation of β-D-Galactopyranosyl Azide 8 and β-D-Glucopyranosyl Azide 9

Eighty microliters of sodium methoxide solution in methanol (5 M) was added to 2 mL dried MeOH, then 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl azide (186 mg, 0.5 mmol) was added to the solution and the mixture was stirred for 4 h at room temperature. 0.5 N HCl solution was then added to the reaction mixture until pH 6 (Scheme 2). The mixture was concentrated under reduced pressure and the residue re-dissolved in 0.5 mL MeOH and solid filtrated off. The filtrate was concentrated under reduced pressure to afford β-D-galactopyranosyl azide 8 (93 mg, 90.7%).

Eighty microliters of sodium methoxide was added to 2 mL dried MeOH, then 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl azide (261 mg, 0.7 mmol) was added to the solution (Scheme 2). Following the same procedure, as described for the preparation of 8, β-D-glucopyranosyl azide 9 (141 mg, 100%) was obtained.

Preparation of Galactosyl Squamocin Derivatives 11–13

Squamocin (187 mg, 0.3 mmol), 5-hexynoic acid (40 mg, 0.36 mmol) and DMAP (3 mg, 0.03 mmol) were dissolved in 3 mL dried CH₂Cl₂. DCC (123 mg, 0.6 mmol) was added and the mixture was stirred under N₂ atmosphere at room temperature for 16 h (Scheme 3). The resulting precipitate (DCU) was removed by filtration and the filtrate was concentrated under reduced pressure. The residue was dissolved in EtOAc and sequentially washed with 0.5% aqueous HCl solution, saturated aqueous NaHCO₃ solution and saturated aqueous NaCl. The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude products were separated by Sephadex LH-20 size exclusion chromatography using MeOH to give crude product 10A (204 mg).

The crude product 10A (204 mg) and β-D-galactopyranosyl azide 8 (93 mg) were suspended in MeOH. Then, CuSO₄·5H₂O (30 mg, 0.12 mmol) and sodium ascorbate (47 mg, 0.24 mmol) in 2 mL of distilled water were added. The suspension was stirred at 50°C for 16 h and monitored by TLC (CHCl₃: MeOH, 85:15) until the reaction was finished (Scheme 2). The suspension was concentrated under reduced pressure and then purified by silica gel column chromatography using CHCl₃-MeOH (97:3, and 93:7; v/v) to afford three fractions with F-II as the fraction of target products. F-II was further separated by HPLC using MeCN-H₂O (78:22, v/v) at a flow rate of 5 mL/min to afford 11 (7 mg, t_R = 61.47 min, 2.5%), 12 (9 mg, t_R = 55.53 min, 3.3%), and 13 (20 mg, t_R = 50.06 min, 7.2%).

Characterization Data of Compound 11

A colorless waxy solid; [α]²⁰_D +19.6 (c 0.2, MeOH); ¹H NMR (600 MHz, CDCl₃): δ 7.92 (1H, s, H-6′), 7.00 (1H, m, H-35), 5.55 (1H, d, J = 8.0 Hz, H-7′), 5.00 (1H, m, H-36), 4.83 (1H, m, H-15), 3.94 (1H, m, H-16), 4.29 (1H, m, H-8′), 4.14 (1H, m, H-10′), 3.80 (7H, m, H-19, 20, 23, 9′, 11′, 12′), 3.59 (1H, m, H-24), 3.52 (1H, m, H-28), 2.75 (2H, m, H-4′), 2.36 (2H, m, H-2′), 2.26 (2H, m, H-3), 1.85–2.02 (4H, m), 1.74 (2H, m), 1.48–1.59 (6H, m), 1.38–1.47 (6H, m), 1.41 (3H, d, J = 6.0 Hz, H-37), 1.16–1.35 (30H, m), 0.87 (3H, t, J = 6.6 Hz, H-34); ¹³C NMR (150 MHz, CDCl₃): δ 174.2 (C-1), 173.6 (C-1′), 149.2 (C-35), 147.2 (C-5′), 134.5 (C-2), 122.4 (C-6′), 88.3 (C-7′), 82.7 (C-20), 82.2 (C-23), 81.9 (C-19), 80.7 (C-16), 77.9 (C-11′), 77.7 (C-36), 75.7 (C-15), 74.0 (C-9′), 72.2 (C-24), 71.9 (C-28), 70.3 (C-8′), 69.0 (C-10′), 61.2 (C-12′), 37.8 and 37.3 (C-27, C-29), 33.8 (C-2′), 33.0 (C-25), 32.1 (C-32), 30.9 (C-14), 29.8 (3C), 29.7 (5C), 29.5, 29.4, 28.9, 28.6, 28.6, 27.6, 26.0, 25.5, 25.4 (2C), 24.7, 24.6 (C-4′), 22.9 (C-33), 22.4 (C-26), 19.4 (C-37), 14.3 (C-34); HR-ESIMS m/z: 922.6015 [M + H]⁺ (calcd for C₄₉H₈₄N₃O₁₃, 922.5999).

Characterization Data of Compound 12

A colorless waxy solid; [α]²⁰_D +16.0 (c 0.2, MeOH); ¹H NMR (600 MHz, CDCl₃): δ 7.82 (1H, s, H-6′), 7.00 (1H, m, H-35), 5.58 (1H, br s, H-7′), 5.00 (2H, m, H-36, H-24), 4.32 (1H, m, H-8′), 4.18 (1H, m, H-10′), 4.05 (1H, m, H-23), 3.85 (2H, m, H- 9′, 11′), 3.83 (1H, m, H-16), 3.80 (4H, m, H-19, 20, 12′), 3.53 (1H, m, H-28), 3.39 (1H, m, H-15), 2.67 (2H, m, H-4′), 2.35 (2H, m, H-2′), 2.26 (2H, m, H-3), 1.85–2.02 (5H, m), 1.76 (2H, m), 1.48–1.62 (5H, m), 1.37–1.47 (6H, m), 1.41 (3H, d, J = 6.0 Hz, H-37), 1.16–1.36 (30H, m), 0.87 (3H, t, J = 7.0 Hz, H-34); ¹³C NMR (150 MHz, CDCl₃): δ 174.3 (C-1), 174.2 (C-1′), 149.2 (C-35, C-5′), 134.5 (C-2), 88.3 (C-7′), 83.7 (C-16), 82.7 (C-20), 81.9 (C-19), 80.9 (C-23), 77.7 (C-11′, C-36), 75.1 (C-24), 74.7 (C-15), 73.9 (C-9′), 71.4 (C-28), 70.3 (C-8′), 69.3 (C-10′), 61.1 (C-12′), 37.8 and 37.1 (C-27, C-29), 33.9 (C-2′), 33.1 (C-14), 32.1 (C-32), 31.2 (C-25), 30.0, 29.9 (3C), 29.8 (2C), 29.7, 29.5, 29.4, 29.1, 28.8, 28.4, 27.6, 25.9, 25.7, 25.4 (2C), 24.9 (C-4′), 22.9, 22.9 (C-33), 22.0 (C-26), 19.4 (C-37), 14.4 (C-34); HR-ESIMS m/z: 922.6020 [M + H]⁺ (calcd for C₄₉H₈₄N₃O₁₃, 922.5999).

Characterization Data of Compound 13

A colorless waxy solid; [α]²⁰_D +15.6 (c 0.2, MeOH); ¹H NMR (600 MHz, CDCl₃): δ 7.85 (1H, s, H-6′), 7.00 (1H, m, H-35), 5.57 (1H, br s, H-7′), 5.00 (1H, m, H-36), 4.88 (1H, m, H-28), 4.28 (1H, m, H-8′), 4.16 (1H, m, H-10′), 3.91 (2H, m, H-19, 20), 3.83 (6H, m, H-16, 23, 9′, 11′, 12′), 3.75 (1H, m, H-24), 3.39 (1H, m, H-15), 2.68 (2H, m, H-4′), 2.33 (2H, m, H-2′), 2.26 (2H, m, H-3), 1.85–2.03 (6H, m), 1.79 (2H, m), 1.44–1.61 (10H, m), 1.41 (3H, d, J = 6.0 Hz, H-37), 1.20–1.37 (30H, m), 0.88 (3H, t, J = 7.0 Hz, H-34); ¹³C NMR (150 MHz, CDCl₃): δ 174.2 (C-1), 173.5 (C-1′), 149.2 (C-35, C-5′), 134.5 (C-2), 88.3 (C-7′), 84.0 (C-16), 83.2 (C-19), 83.0 (C-20), 82.8 (C-23), 77.8 (C-11′), 77.7 (C-36), 74.6 (C-15), 74.3 (C-9′), 74.2 (C-28), 71.7 (C-24), 70.6 (C-8′), 69.2 (C-10′), 61.7 (C-12′), 34.6 and 34.5 (C-27, C-29), 34.0 (C-2′), 32.9 (C-14), 32.5 (C-25), 31.9 (C-32), 29.9 (4C), 29.8 (2C), 29.7, 29.5, 29.4 (2C), 29.2 (2C), 28.7, 27.6, 25.8, 25.6, 25.4 (2C), 24.8 (C-4′), 24.7, 22.8 (C-33), 22.4 (C-26), 19.4 (C-37), 14.3 (C-34); HR-ESIMS m/z: 922.6017 [M + H]⁺ (calcd for C₄₉H₈₄N₃O₁₃, 922.5999).

Preparation of Glucosylated Squamocin Derivatives 14 and 15

Squamocin (249 mg, 0.4 mmol), 5-hexynoic acid (67 mg, 0.6 mmol) and DMAP (7 mg, 0.06 mmol) were dissolved in 5 mL dried CH₂Cl₂. DCC (247 mg, 1.2 mmol) was added and the mixture was stirred under N₂ atmosphere at room temperature for 16 h (Scheme 3). Following the same procedure as described for the preparation of 10A in the Experimental Section 3.4, intermediate product 10B (288 mg) was obtained.

The intermediate product 10B (288 mg) and β-D-glucopyranosyl azide 9 (82 mg, 0.4 mmol) were suspended in 4 mL MeOH. Then, CuSO₄·5H₂O (40 mg, 0.16 mmol) and sodium ascorbate (63 mg, 0.32 mmol) in 2 mL of distilled water were added. The suspension was stirred at 50°C for 16 h. The suspension was then concentrated in vacuo and the residue purified by silica gel column chromatography using CHCl₃-MeOH (97:3 and 93:7; v/v) to afford five fractions. F-II was further separated by HPLC using MeOH-H₂O (80:20, v/v) at a flow rate of 5 mL/min to afford 14 (34 mg, t_R = 75.74 min, 9.2%). F-III and F-IV were combined and further separated by HPLC using MeOH-H₂O (80:20, v/v) at a flow rate of 5 mL/min to afford 15 (43 mg, t_R = 56.68 min, 11.7%).

Characterization Data of Compound 14

A yellowish waxy solid; [α]²⁰_D +1.4 (c 1.1, MeOH); ¹H NMR (600 MHz, CDCl₃): δ 7.82 (1H, s, H-6′), 7.00 (1H, m, H-35), 5.59 (1H, d, J = 7.9 Hz, H-7′), 5.00 (1H, m, H-36), 4.96 (1H, m, H-24), 4.01 (2H, m, H-23, 8′), 3.82 (1H, m, H-20), 3.77 (6H, m, H-16, 19, 9′, 10′, 12′), 3.58 (1H, m, H-11′), 3.51 (1H, m, H-28), 3.35 (1H, m, H-15), 2.68 (2H, m, H-4′), 2.35 (2H, m, H-2′), 2.25 (2H, m, H-3), 1.94 (5H, m), 1.72 (2H, m), 1.52 (11H, m), 1.41 (3H, d, J = 6.0 Hz, H-37), 1.19–1.37 (30H, m), 0.87 (3H, t, J = 7.0 Hz, H-34); ¹³C NMR (150 MHz, CDCl₃): δ 174.1 (C-1), 173.6 (C-2′), 149.2 (C-35), 134.4 (C-2), 129.0 (C-6′), 87.9 (C-7′), 83.6 (C-16), 82.6 (C-20), 81.8 (C-19), 80.8 (C-23), 79.2 (C-11′), 77.6 (C-36), 76.9 (C-9′), 75.1 (C-24), 74.4 (C-15), 72.7 (C-8′), 71.4 (C-28), 69.1 (C-10′), 61.1 (C-12′), 37.7 and 37.0 (C-27, C-29), 33.9 (C-2′), 33.1 (C-14), 32.0 (C-32), 31.3 (C-25), 29.9 (2C), 29.8 (3C), 29.7, 29.6, 29.5, 29.4, 29.0, 28.6, 28.5, 27.6 (2C), 25.9, 25.7, 25.3, 24.8 (2C), 24.5, 22.8 (C-33), 21.9 (C-26), 19.4 (C-37), 14.3 (C-34); HR-ESIMS m/z: 922.5996 [M + H]⁺ (calcd for C₄₉H₈₄N₃O₁₃, 922.5999).

Characterization Data of Compound 15

A yellowish waxy solid; [α]²⁰_D +0.8 (c 1.1, MeOH); ¹H NMR (600 MHz, CDCl₃): δ 7.71 (1H, s, H-6′), 7.00 (1H, m, H-35), 5.57 (1H, d, J = 8.0 Hz, H-7′), 4.99 (1H, m, H-36), 4.86 (1H, m, H-28), 4.03 (1H, m, H-8′), 3.91 (2H, m, H-19, 20), 3.82 (3H, m, H-16, 12′), 3.78 (4H, m, H-23, 24, 9′, 10′), 3.61 (1H, m, H-11′), 3.37 (1H, m, H-15), 2.68 (2H, m, H-4′), 2.32 (2H, m, H-2′), 2.25 (2H, m, H-3), 1.93 (4H, m), 1.86 (1H, m), 1.77 (1H, m), 1.52 (12H, m), 1.40 (3H, d, J = 6.8 Hz, H-37), 1.19–1.35 (30H, m), 0.88 (3H, t, J = 7.0 Hz, H-34); ¹³C NMR (150 MHz, CDCl₃): δ 174.1 (C-1), 173.4 (C-2′), 149.1 (C-35), 146.9 (C-5′), 134.3 (C-2), 121.6 (C-6′), 87.8 (C-7′), 83.6 (C-16), 83.0 (C-19), 82.7 (C-20), 82.2 (C-23), 79.1 (C-11′), 77.6 (C-36), 76.9 (C-9′), 74.4 (C-15), 74.3 (C-28), 72.7 (C-8′), 71.5 (C-24), 69.0 (C-10′), 61.1 (C-12′), 34.4 and 34.3 (C-27, C-29), 33.9 (C-2′), 33.0 (C-14), 32.4 (C-25), 31.8 (C-32), 29.9, 29.8, 29.7 (2C), 29.6, 29.4, 29.3 (2C), 29.0, 29.0, 28.6, 27.5, 25.7, 25.4, 25.3, 24.9, 24.8, 24.6, 23.6, 22.7 (C-33), 22.2 (C-26), 19.3 (C-37), 14.2 (C-34); HR-ESIMS m/z: 922.5986 [M + H]⁺ (calcd for C₄₉H₈₄N₃O₁₃, 922.5999).

Preparation of Galactosylated Bullatacin 17

Galactosylated bullatacin derivative was synthesized using the same method as described for galactosylated squamocin derivatives 11–13. Briefly, bullatacin (93 mg, 0.15 mmol), 5-hexynoic acid (20 mg, 0.18 mmol) and DMAP (2 mg, 0.02 mmol) were dissolved in 3 mL dried CH₂Cl₂. DCC (62 mg, 0.3 mmol) was added and the mixture was stirred under N₂ atmosphere at room temperature for 16 h (Scheme 4). The resulting precipitate (DCU) was removed by filtration and the filtrate was concentrated under reduced pressure. The residue was dissolved in EtOAc and sequentially washed with 0.5% aqueous HCl solution, saturated aqueous NaHCO₃ solution and saturated aqueous NaCl. The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude products were separated by Sephadex LH-20 gel permeation chromatography using MeOH to provide intermediate product 16A (93 mg).

The intermediate product 16A (93 mg) and β-D-galactopyranosyl azide 8 (30 mg, 0.15 mmol) were suspended in 2 mL MeOH. Then, CuSO₄·5H₂O (15 mg, 0.06 mmol) and sodium ascorbate (47 mg, 0.24 mmol) in 1 mL of distilled water were added. The suspension was stirred at 50°C for 16 h and monitored by TLC (CHCl₃:MeOH, 85:15) until the reaction was finished (Scheme 2). The suspension was concentrated under reduced pressure and then purified by silica gel column chromatography using CHCl₃-MeOH (97:3, and 93:7; v/v) to afford three fractions with F-II as the fraction of target products. F-II was further separated by HPLC using MeCN-H₂O (77:23, v/v) at a flow rate of 5 mL/min to afford 17 (48 mg, t_R = 65.24 min, 34.7%).

Characterization Data of Compound 17

A colorless waxy solid; [α]²⁰_D +20.3 (c 0.1, MeOH); ¹H NMR (600 MHz, CDCl₃): δ 7.83 (1H, s, H-6′), 7.20 (1H, m, H-35), 5.49 (1H, m H-7′), 5.03 (1H, m, H-36), 4.86 (1H, m, H-15), 4.28 (1H, m, H-8′), 4.10 (1H, m, H-10′), 3.99 (1H, m, H-16), 3.85 (3H, m, H-19, 20, 23), 3.81 (5H, m, H-4, 9′, 11′, 12′), 3.73 (1H, m, H-24), 2.70 (2H, m, H-4′), 2.48 (1H, d, H-3a), 2.40 (1H, d, H-3b), 2.36 (2H, m, H-2′), 1.88–1.99 (6H, m), 1.77 (2H, m), 1.58 (2H, m), 1.51 (2H, m), 1.43 (3H, m), 1.39 (3H, d, J = 6.7 Hz, H-37), 1.20–1.33 (33H, m), 0.86 (3H, t, J = 7.0 Hz, H-34); ¹³C NMR (150 MHz, CDCl₃): δ 175.0 (C-1), 173.5 (C-2′), 152.3 (C-35), 147.1 (C-5′), 131.1 (C-2), 122.0 (C-6′), 88.4 (C-7′), 82.8 (C-19), 82.3 (C-20), 82.2 (C-23), 80.3 (C-16), 78.3 (C-11′), 77.9 (C-36), 74.1 (C-9′), 75.5 (C-15), 70.2 (C-8′), 70.0 (C-4), 71.7 (C-24), 69.0 (C-10′), 61.2 (C-12′), 37.5 (C-5), 33.8 (C-2′), 33.3 (C-3), 32.8 (C-25), 32.1 (C-32), 30.7 (C-14), 30.0, 29.8 (4C), 29.7 (2C), 29.6 (2C), 29.5 (2C), 28.8, 28.5, 28.2, 26.2, 25.7, 25.5, 24.9, 24.8, 24.5, 22.8 (C-33), 19.2 (C-37), 14.3 (C-34); HR-ESIMS m/z: 922.6013 [M + H]⁺ (calcd for C₄₉H₈₄N₃O₁₃, 922.5999).

Preparation of Glucosylated Bullatacin 18

Bullatacin (87 mg, 0.14 mmol), 5-hexynoic acid (22 mg, 0.21 mmol) and DMAP (2.4 mg, 0.02 mmol) were dissolved in 3 mL dried CH₂Cl₂. DCC (86 mg, 0.42 mmol) was added and the mixture was stirred under N₂ atmosphere at room temperature for 16 h (Scheme 4). The crude product was purified in a similar way as described for the preparation of 16A to provide the intermediate product 16B (115 mg).

The intermediate product 16B (115 mg) and β-D-glucopyranosyl azide 9 (82 mg, 0.42 mmol) were suspended in 3 mL MeOH. Then, CuSO₄·5H₂O (15 mg, 0.06 mmol) and sodium ascorbate (24 mg, 0.12 mmol) in 1 mL of distilled water were added. The suspension was stirred at 50°C for 16 h. The mixture was concentrated and then purified by silica gel column chromatography using CHCl₃-MeOH (97:3, and 93:7; v/v) to afford five fractions. The product containing fraction was further separated by HPLC using MeOH-H₂O (83:17, v/v) at a flow rate of 5 mL/min to afford 18 (37 mg, t_R = 55.23 min, 28.7%).

Characterization Data of Compound 18

A yellowish waxy solid; [α]²⁰_D +4.5 (c 1.1, MeOH); ¹H NMR (600 MHz, CDCl₃): δ 7.75 (1H, s, H-6′), 7.15 (1H, m, H-35), 5.58 (1H, d, J = 7.9 Hz, H-7′), 5.06 (1H, m, H-36), 5.00 (1H, m, H-4), 4.01 (1H, m, H-8′), 3.94 (1H, m, H-19), 3.90 (1H, m, H-20), 3.83 (5H, m, H-16, 23, 24, 12′), 3.74 (2H, m, H-9′, 10′), 3.59 (1H, m, H-11′), 3.39 (1H, m, H-15), 2.63 (2H, m, H-4′), 2.55 (1H, m, H-3a), 2.47 (1H, m, H-3b), 2.31 (2H, m, H-2′), 1.95 (3H, m), 1.88 (2H, m), 1.78 (1H, m), 1.41–1.61 (6H, m), 1.43 (3H, m), 1.32 (3H, d, J = 6.7 Hz, H-37), 1.20–1.33 (33H, m), 0.88 (3H, t, J = 7.0 Hz, H-34); ¹³C NMR (150 MHz, CDCl₃): δ 174.1 (C-1), 173.3 (C-2′), 152.2 (C-35), 147.0 (C-5′), 129.8 (C-2), 122.0 (C-6′), 87.8 (C-7′), 83.5 (C-16), 83.0 (C-20), 82.8 (C-19), 82.4 (C-23), 79.1 (C-11′), 78.1 (C-36), 76.9 (C-9′), 74.4 (C-15), 72.6 (C-8′), 72.3 (C-4), 71.6 (C-24), 69.1 (C-10′), 61.1 (C-12′), 34.1 (C-2′), 33.8 (C-14), 33.3 (C-5), 32.6 (C-25), 32.1 (C-32), 29.9, 29.8 (5C), 29.7 (2C), 29.6, 29.5 (3C), 29.1 (2C), 28.6, 26.3, 25.8, 25.4, 24.9, 24.6, 24.5, 22.9 (C-33), 19.1 (C-37), 14.3 (C-34); HR-ESIMS m/z: 922.6012 [M + H]⁺ (calcd for C₄₉H₈₄N₃O₁₃, 922.5999).

Aqueous Solubility of Squamocin and Galactosyl Derivative 13

HPLC method with refractive index detector was employed to measure the aqueous solubility of squamocin and its galactosyl derivative 13. Standard curve of compound 13 was found to be y=1.45*10⁶x + 8.64*10⁵ (R² = 0.9970) by using six gradient concentrations (10, 5, 2.5, 1.25, 0.625, and 0.3125 mM) in methanol. Excess amount of squamocin/compound 13 was added to 1 mL of phosphate-buffered saline (PBS, pH = 7.0) and sonicated for 5 min to provide saturated solution. The mixture was centrifuged (12000 rpm) for 2 min and the supernatant was analyzed by HPLC for the content of squamocin/compound 13. The concentration of compound 13 was found to be 1.49 mM (1.37 mg/mL) in PBS (pH 7.0) at ambient temperature (around 28°C) while squamocin was not detected in PBS (pH 7.0).

Cell Culture

A549, HeLa and HepG2 cell lines were obtained from Kunming Cell Bank, Chinese Academy of Sciences. The cell lines were cultured in RPMI1640 (Invitrogen) cell culture medium supplemented with 10% FBS and 1% penicillin and streptomycin. All cell lines were maintained in a humidified atmosphere of 5% CO₂ at 37°C.

Cell Viability Assay

A549, HeLa and HepG2 cells (5 × 10⁴/well) were seeded in flat-bottomed 96 well microplates. Then, squamocin/bullatacin and glycosylated squamocin/bullatacin derivatives dissolved in DMSO (10, 5, 2.5, 1.25, 0.625, 0.3125 μM as concentration gradient) were added to wells, respectively. The cells were incubated for 72h at 37°C in CO₂ gas incubator and then treated with 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (20 μL/well, 5 mg/mL). After another 4 h incubation, the medium was removed and 150 μL of DMSO was added to each well. Absorbance in each well, including the blanks, was measured at 570 nm in a microtiter plate reader after the samples were swirled gently. Experiments were repeated at least three times. Growth inhibition rate was calculated as follows: inhibition rate (%) =x{1 - [∆OD (compound) - ∆OD (blank)]/[∆OD (control) - ∆OD (blank)]}× 100%.

Results and Discussion

Synthesis and Structure Characterization

Queiroz et al reported the synthesis of several glycosyl derivatives of squamocin in which the squamocin scaffold was modified through either acetylation of the hydroxyl group(s) during the glycosylation reaction or reduction of the α,β-unsaturated γ-lactone moiety during the deprotection of benzyl groups on the sugar residue.²⁹ We aim to prepare glycosylated acetogenin derivatives wherein the acetogenin scaffold remains intact. The three hydroxyl groups in squamocin and bullatacin (Figure 1) play less important roles in their anticancer activity^13,28 and sugar residue(s) may be attached to these hydroxyl groups without affecting their activities much.

We first tried the trichloroacetimidate as the glycosylation donor. Galactose trichloroacetimidate (3) was readily prepared from D-galactose in three steps (Scheme 1) according to literature procedure.³⁰ Treatment of squamocin with imidate 3 in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) as the catalyst resulted in complicated product mixture comprising of mono- and di-glycosylated products (as indicated by mass spectrometry data). The glycosylation yield was also found to be very low partly due to the decomposition of the imidate. The glycosylated product was isolated and treated with Et₃N-MeOH-H₂O (1:8:1, v/v) to remove the acetyl groups on the sugar.³¹ Unfortunately, the γ-lactone ring of squamocin was not stable to allow for the complete removal of all acetyl groups. In the end, we managed to obtain two mono-glycosylated products 4 (glycosylated at C-15) and 5 (glycosylated at C-28) after purification by HPLC, albeit at only 1% yield. Due to the complications associated with glycosylation reaction and the removal of the acetyl protection groups on the sugar, alternative methods of attaching sugars to acetogenins are desirable.

The Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) reaction, popularly known as the “click reaction”, is an efficient method to covalently link two molecular entities together and has been widely used to prepare various glycoconjugates³² and carbohydrate macrocycles alike.³³ Here we use the CuAAC reaction to conjugate glucose or galactose with squamocin/bullatacin through an alkyne-functionalized linker. Thus, β-D-galactopyranosyl azide (8) and β-D-glucopyranosyl azide (9) were prepared from the corresponding commercially available per-acetylated sugar derivatives 6 and 7 (Scheme 2). Squamocin was first treated with 5-hexynoic acid (1.2 or 1.5 eq.) in the presence of dicyclohexylcarbodiimide (DCC) and 4-N,N-dimethylaminopyridine (DMAP) to provide alkyne functionalized intermediate (10A or 10B) after purification through Sephadex LH-20 gel permeation chromatography (Scheme 3). Treatment of 10A with galactosyl azide 8 in the presence of CuSO₄ and sodium ascorbate afforded mono-galactosylated squamocin derivatives 11–13. The 28-O-substituted product (13) was obtained in relatively higher yield, which coincided with our earlier observation that the 28-OH was most reactive among the three hydroxyl groups in squamocin.²⁸ Similarly, reaction of intermediate 10B with glucosyl azide 9 afforded mono-glucosylated squamocin derivatives 14 and 15. Following the same reaction sequence, bullatacin was first treated with 5-hexynoic acid (1.2 or 15 eq.) to give the ester intermediate (16A or 16B) which was then coupled with the glycosyl azide 8 or 9 to provide the mono-glycosylated bullatacin derivative 17 or 18 in 34.7% and 28.7% yield, respectively (Scheme 4). Compound 17 is 24-O-substituted while 18 4-O-substituted. This level of regio-selectivity is not expected for bullatacin.²⁸ Multiple chromatographic procedures involved in the purification of the reaction intermediate products (16A/16B) and the final products might have resulted in the loss of other minor products.

Typically, reaction products were purified by silica gel chromatography and preparative HPLC. All products were structurally confirmed by the analysis of 1D (¹H, ¹³C and DEPT) and 2D (COSY, HSQC and HMBC) NMR spectra, and HR-ESIMS data. The selected ¹H and ¹³C chemical shifts of the glycosylated derivatives were listed in Tables 1 and 2, respectively, and compared with those of squamocin or bullatacin. The changes in ¹H and ¹³C chemical shifts of these glycosylated derivatives with an ester linkage (11–15, 17 and 18) were similar with those observed for biotinylated acetogenins we reported earlier.²⁸ When a secondary hydroxyl group was acylated, the chemical shift of the carbon (C-4, C-15, C-24, and C-28) bearing an oxygen atom moved downfield (Table 2). Up to 3.4–3.6 ppm downfield shift was observed for C-24 in compound 12 and compound 14 while the shift for C-15 was smaller (1.6 ppm) in compound 11. On the other hand, the chemical shifts of the neighboring carbons (C-5, C-14, C-16, C-23, C-25, C-27, and C-29) moved upfield (in a magnitude of 1.1–4.1 ppm) upon acylation of the hydroxyl group. When a hydroxyl group was glycosylated, the carbon bearing that hydroxyl group exhibited a significant downfield shift (7.1 ppm for C-15 in 4 and 9.8 ppm for C-28 in 5). Similarly, upfield shifts (1.9–3.2 ppm) for the neighboring carbons (C-14, C-16, C-27, and C-29) were observed as a result of direct glycosylation of that hydroxyl group (Table 2). In the case of ¹H NMR data, a significant downfield shift (>1.5 ppm) was observed for the proton directly attached to the carbon bearing the hydroxyl group that was acylated while a smaller downfield shift (0.1–0.7 ppm) resulted from O-galactosylation of that hydroxyl group (Table 1). The ¹H and ¹³C NMR spectra of all synthesized glycosyl derivatives of squamocin and bullatacin (4, 5, 11–15, 17 and 18) are provided in the Supplementary Material of this article.

Table 1 Selected 1H NMR Data (600 MHz, CDCl3) for Squamocin (1), Bullatacin (2) and Their Glycosylated Derivatives

Table 2 Selected 13C NMR Data (150 MHz, CDCl3) for Squamocin (1), Bullatacin (2) and Their Glycosylated Derivatives

Solubility

Squamocin and bulattacin are poorly soluble in water, which is a major drawback in the further exploitation of their potential uses. The attachment of a sugar residue is expected to increase their water solubility. Squamocin derivative 13 bearing a galactose residue was suspended in the phosphate buffer saline (PBS, pH = 7.0) and the mixture sonicated for 5 min. The supernatant was analyzed by HPLC for the content of 13. The solubility of 13 in PBS was found to be 1.37 mg/mL (1.49 mM) at ambient temperature (around 28°C). On the other hand, the solubility of squamocin in PBS was close to zero because no squamocin was detected in the supernatant of the PBS saturated with squamocin (Table 3). The data clearly showed that the attachment of one sugar residue to squamocin significantly improved its solubility in water.

Table 3 Solubility of Squamocin and Its Derivative 13

Biological Evaluation

The cytotoxicity of the synthesized glycoconjugate derivatives against three tumor cell lines (Table 4) were evaluated using MTT assay. The IC₅₀ values of these compounds were calculated based on three parallel experiments and presented in Table 4. Overall, when compared to squamocin (1) and bullatacin (2), the majority of these derivatives show similar level of anticancer activity against all three tumor cell lines with their IC₅₀ values in µM range. Compound 15 bearing a glucose residue is slightly more active than squamocin. A few derivatives (eg, 11, 17 and 18) show up to 8–10 times higher IC₅₀ than squamocin or bullatacin against certain cell lines (HeLa or A549), suggesting that these compounds exhibit some selectivity toward certain type of cancer cells. The data also suggest that all glycosyl derivatives either via glycosidic linkage (4 and 5) or triazolyl-ester linkage retain the anticancer activity. Furthermore, the position at which the glycosyl residue is attached does not affect their potency much, eg, compounds 4 and 5 having similar level of potency. The glucosyl derivatives (14, 15 and 18) are similarly active as the galactosyl derivatives (12, 13 and 17).

Table 4 Cytotoxic Activity (IC50) of Glycosylated Derivatives of Squamocin and Bullatacin

Numerous studies have been conducted to elucidate the mechanism of action of Annonaceous acetogenins (ACGs) for their potent cytotoxic activity. ACGs are potent inhibitors of mitochondrial complex I in the electron transport system, with squamocin having the lowest IC₅₀ value reported for this class of compounds.³⁶ They are also powerful inhibitors of the NADH oxidases peculiar to the plasma membranes of cancer cells.³⁷ Acetogenins cause cell cycle arrest at different phases in different cancer cells and are strong apoptosis inducers. Squamocin arrested T24 bladder cancer cells at the G1 phase, enhanced caspase-3 activity, cleaved the functional protein of PARP and caused cell apoptosis.³⁸ Squamocin also inhibited the proliferation of chronic myeloid leukemia (K562) cells via G2/M arrest in association with the induction of p21, p27 and the reduction of Cdk1 and Cdc25C kinase activities.³⁹ Three other THF-containing acetogenins (squamostatin A, squamocin M and corossolone) also showed potent antiproliferative activity against human nasopharyngeal carcinoma (NPC) cell lines and induced G2/M phase arrest, mitochondrial damage and apoptosis, and increased cytosolic and mitochondrial Ca²⁺ in NPCs.⁴⁰ Furthermore, Dzhemilev et al recently showed that muricadienin, a linear acetogenin containing a 1Z,5Z-diene unit, induced apoptosis in the HEK293 kidney cancer cells and exhibited a moderate inhibitory activity against topoisomerases I and IIα.⁴¹ ACGs are versatile anticancer agents causing tumor cell death by different mechanisms. For these glycosylated acetogenins described here, more studies are required to demonstrate their cancer selectivity and mechanism(s) of action.

In conclusion, we have prepared nine glycosyl derivatives of squamocin and bullatacin and evaluated their growth inhibitory activity with three cancer cell lines, including A549, HeLa and HepG2. Most squamocin/bullatacin glycoconjugates show similar in vitro cytotoxicity against the tested cell lines as squamocin and bullatacin, respectively. Among the synthesized compounds, compound 15 displays slightly higher activity than squamocin while some other derivatives show up to 8–10 times lower activity in A549 or HeLa cell line than squamocin and bullatacin, respectively. The type of sugar residue (glucose or galactose), the position of attachment, and whether or not a linking spacer is present do not seem to affect the potency much. The solubility of galactosylated squamocin 13 in phosphate buffer saline (PBS, pH = 7) at ambient temperature is greatly improved (1.37 mg/mL) in comparison to squamocin (not detectable). Further studies are needed to show whether or not these compounds exhibit reduced toxicity to normal cells, the mechanism of action, and their therapeutic potential for cancer.

Scheme 1 Synthesis of galactosyl squamocin 4 and 5. Reagents and conditions: (a) Ac2O, pyridine, rt, N2, 16 h, 100%. (b) hydrazine acetate, DMF, 40 °C, 4 h, 91%. (c) trichloroacetonitrile, DBU, 0°C, 7 h, 31%. (d) 3, TMSOTf, CH2Cl2, rt, 16 h. (e) Et3N:MeOH:H2O (1:8:1, v/v), 35°C, 30 h.

Scheme 2 Synthesis of glycosyl azides 8 and 9.

Scheme 3 Synthesis of 11–15 by click chemistry. Reagents and conditions: (a) DCC, DMAP, CH2Cl2, rt, N2, 16 h. (b) CuSO4·5H2O, sodium ascorbate, MeOH/H2O, 50 °C, 16 h.

Scheme 4 Synthesis of 17 and 18 by click chemistry. Reagents and conditions: (a) DCC, DMAP, CH2Cl2, rt, N2, 16 h. (b) CuSO4·5H2O, sodium ascorbate, MeOH/H2O, 50 °C, 16 h.