Synthesis and cytotoxic property of annonaceous acetogenin glycoconjugates

Article abstract of DOI:10.2147/DDDT.S259547

Background: Annonaceous acetogenins (ACGs) are secondary metabolites produced by the Annonaceae family and display potent anticancer activity against various cancer cell lines. Squamocin and bullatacin are two examples of ACGs that show promising antitumor activity; however, preclinical data are not sufficient partly due to their being highly lipophilic and poorly soluble in water. These compounds also display high toxicity to normal cells. Due to these disadvantageous properties, the therapeutic potential of squamocin and bullatacin as antitumor agents has not been fully evaluated. Methods: In order to enhance their water solubility and potentially improve their cancer targeting, squamocin and bullatacin were conjugated to a glucose or galactose to yield glycosylated derivatives by direct glycosylation or the Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) reaction (the click reaction). The synthesized compounds were evaluated for their anticancer property against HeLa, A549 and HepG2 cancer cell lines using MTT assay. Results: Nine glycosyl derivatives were synthesized and structurally characterized. Most of them show comparable in vitro cytotoxicity against HeLa, A549 and HepG2 cancer cell lines as their parent compounds squamocin and bullatacin. It appears that the type of sugar residue (glucose or galactose), the position at which the sugar residue is attached, and whether or not a linking spacer is present do not affect the potency of these derivatives much. The solubility of galactosylated squamocin 13 in phosphate buffer saline (PBS, pH = 7) is greatly improved (1.37 mg/mL) in comparison to squamocin (not detected in PBS). Conclusion: The conjugation of a glucose or galactose to squamocin and bullatacin yields glycosyl derivatives with similar level of anticancer activity in tested cell lines. Further studies are needed to demonstrate whether or not these compounds show reduced toxicity to normal cells and their therapeutic potential as antitumor agents.

Full text of DOI:10.2147/DDDT.S259547

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O R I G I N A L R E S E A R C H
Synthesis and Cytotoxic Property of Annonaceous
Acetogenin Glycoconjugates
This article was published in the following Dove Press journal:
Drug Design, Development and Therapy
Jing-Fang Shi1,2
Background: Annonaceous acetogenins (ACGs) are secondary metabolites produced by the
Ping Wu2
Annonaceae family and display potent anticancer activity against various cancer cell lines.
Xiao-Li Cheng2
Squamocin and bullatacin are two examples of ACGs that show promising antitumor
2
3
activity; however, preclinical data are not sufficient partly due to their being highly lipophilic
and poorly soluble in water. These compounds also display high toxicity to normal cells. Due
to these disadvantageous properties, the therapeutic potential of squamocin and bullatacin as
antitumor agents has not been fully evaluated.
Xiao-Yi Wei
Zi-Hua Jiang
1Guangdong Provincial Key Laboratory
for Crop Germplasm Resources
Methods: In order to enhance their water solubility and potentially improve their cancer
targeting, squamocin and bullatacin were conjugated to a glucose or galactose to yield
glycosylated derivatives by direct glycosylation or the Cu(I)-catalyzed azide-alkyne
1,3-dipolar cycloaddition (CuAAC) reaction (the click reaction). The synthesized compounds
were evaluated for their anticancer property against HeLa, A549 and HepG2 cancer cell lines
using MTT assay.
Preservation and Utilization, Agro-
Biological Gene Research Center,
Guangdong Academy of Agricultural
Sciences, Guangzhou 510640, People’s
2
Republic of China; Key Laboratory of
Plant Resources Conservation and
Sustainable Utilization, South China
Botanical Garden, Chinese Academy of
Sciences, Guangzhou 510650, People’s
Results: Nine glycosyl derivatives were synthesized and structurally characterized. Most of
them show comparable in vitro cytotoxicity against HeLa, A549 and HepG2 cancer cell lines
as their parent compounds squamocin and bullatacin. It appears that the type of sugar residue
(glucose or galactose), the position at which the sugar residue is attached, and whether or not
a linking spacer is present do not affect the potency of these derivatives much. The solubility
of galactosylated squamocin 13 in phosphate buffer saline (PBS, pH = 7) is greatly improved
(1.37 mg/mL) in comparison to squamocin (not detected in PBS).
3
Republic of China; Department of
Chemistry, Lakehead University, Thunder
Bay, Ontario P7B 5E1, Canada
Conclusion: The conjugation of a glucose or galactose to squamocin and bullatacin yields
glycosyl derivatives with similar level of anticancer activity in tested cell lines. Further
studies are needed to demonstrate whether or not these compounds show reduced toxicity to
normal cells and their therapeutic potential as antitumor agents.
Keywords: annonaceous acetogenins, squamocin, bullatacin, glycosylated, cytotoxicity,
anticancer, solubility
Correspondence: Zi-Hua Jiang
Department of Chemistry, Lakehead
University, 955 Oliver Road, Thunder Bay,
Ontario P7B 5E1, Canada
Tel +1-807-766-7171
Email zjiang@lakeheadu.ca
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
Xiao-Yi Wei
Key Laboratory of Plant Resources
Conservation and Sustainable Utilization,
South China Botanical Garden, Chinese
Academy of Sciences, Xingke Road 723,
Tianhe District, Guangzhou 510650,
People’s Republic of China
Tel +86-20-37252538
Email wxy@scbg.ac.cn
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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 bul-
latacin are conjugated to a glucose or galactose to improve
their solubility in water and potentially their cancer target-
ing. The synthesis and preliminary studies of the antic-
ancer activity of these new glycoconjugates are described.
Figure 1 Structures of squamocin and bullatacin.
adenocarcinoma multidrug-resistant cell line.13 In addi-
tion, 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.
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-dimethylaminopyri-
dine (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 trimethylsi-
lyl 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 4Å
molecular sieves. CH2Cl2 was refluxed with CaH2 for 4 h,
then distilled and immediately stored over activated 4Å
molecular sieves.
Conjugation of carbohydrates with drugs has become
increasingly important in bioorganic chemistry and chemi-
cal biology.16,17 Glycosylation of drug molecules has been
employed to modify their physico-chemical properties,
including polarity, solubility, and stability, without affect-
ing their biological activities.18,19 For example, glycosy-
lated porphyrins have been shown as promising
photosensitizers in photodynamic therapy (PDT) for can-
cer owing to their good solubility and specific membrane
interactions.20 Moreover, glycosylation has been widely
explored as a targeting strategy to selectively deliver antic-
ancer 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 facil-
itate the uptake of glucose. For example, glucose transpor-
ter-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
Optical rotations were obtained on a Perkin-Elmer 341
1
polarimeter with MeOH as solvent. The H 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.
(or another sugar that is also a glucose transporter sub- The splitting pattern abbreviations are as follows: s = singlet,
strate) may be taken up by cancer cells more rapidly via d = doublet, dd = double doublet, t = triplet, m = multiplet.
facilitation by glucose transporters and be more selective ESIMS data were obtained on an MDS SCIEX API 2000
toward cancer cells than normal cells. A number of antic- LC/MS instrument. HRESIMS data were obtained on
ancer agents such as doxorubicin,23 daunorubicin,24 a Bruker maXis Q-TOF mass spectrometer. Preparative
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Shi et al
HPLC was run on a Shimadzu LC-6A pump and a Shimadzu 2,3,4,6-tetra-O-acetyl-D-galactopyranose (1.756 g, 91%)
as a white solid.
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.
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 pro-
duct 3 (761 mg, 31%) as an amorphous solid.
Preparation of Galacosylated Squamocin
4 and 5
D-galactose (1 g, 5.56 mmol) was dissolved in dried pyr-
idine (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.
Squamocin (124 mg, 0.2 mmol), 3 (147 mg, 0.3 mmol)
were dissolved in dried CH2Cl2 (5 mL) at 0°C. 4Å mole-
cular 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.
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
o
at 40 C for 4 h (Scheme 1). The reaction was mon-
The deprotection was performed by treatment with Et3
N: 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
itored 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,
HPLC using 88% MeOH to afford 4 (2 mg, 1%, tR
=
filtered and evaporated under reduced pressure to afford 43.6 min) and 5 (2 mg, 1%, tR = 65.1 min).
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.
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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
Characterization Data of Compound 4
A yellowish waxy solid; [α]20 +4.6 (c 0.06, MeOH);
D
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).
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 fil-
trate 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 concen-
trated under reduced pressure. The crude products were
separated by Sephadex LH-20 size exclusion chromatogra-
phy 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
Characterization Data of Compound 5
A yellowish waxy solid; [α]20 −1.2 (c 0.07, MeOH);
D
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 metha-
nol (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
Scheme 2 Synthesis of glycosyl azides 8 and 9.
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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.
products. F-II was further separated by HPLC using
Characterization Data of Compound 12
MeCN-H2O (78:22, v/v) at a flow rate of 5 mL/min to afford A colorless waxy solid; [α]20 +16.0 (c 0.2, MeOH);
D
1H NMR (600 MHz, CDCl3): δ 7.82 (1H, s, H-6′), 7.00
11 (7 mg, tR = 61.47 min, 2.5%), 12 (9 mg, tR = 55.53 min,
(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).
3.3%), and 13 (20 mg, tR = 50.06 min, 7.2%).
Characterization Data of Compound 11
A colorless waxy solid; [α]20 +19.6 (c 0.2, MeOH);
D
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 13
A colorless waxy solid; [α]20 +15.6 (c 0.2, MeOH);
D
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′,
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11′, 12′), 3.75 (1H, m, H-24), 3.39 (1H, m, H-15), 2.68 12′), 3.58 (1H, m, H-11′), 3.51 (1H, m, H-28), 3.35
(2H, m, H-4′), 2.33 (2H, m, H-2′), 2.26 (2H, m, H-3), (1H, m, H-15), 2.68 (2H, m, H-4′), 2.35 (2H, m, H-2′),
1.85–2.03 (6H, m), 1.79 (2H, m), 1.44–1.61 (10H, m), 2.25 (2H, m, H-3), 1.94 (5H, m), 1.72 (2H, m), 1.52
1.41 (3H, d, J = 6.0 Hz, H-37), 1.20–1.37 (30H, m), 0.88 (11H, m), 1.41 (3H, d, J = 6.0 Hz, H-37), 1.19–1.37
(3H, t, J = 7.0 Hz, H-34); 13C NMR (150 MHz, CDCl3): δ (30H, m), 0.87 (3H, t, J = 7.0 Hz, H-34); 13C NMR (150
174.2 (C-1), 173.5 (C-1′), 149.2 (C-35, C-5′), 134.5 MHz, CDCl3): δ 174.1 (C-1), 173.6 (C-2′), 149.2 (C-35),
(C-2), 88.3 (C-7′), 84.0 (C-16), 83.2 (C-19), 83.0 134.4 (C-2), 129.0 (C-6′), 87.9 (C-7′), 83.6 (C-16), 82.6
(C-20), 82.8 (C-23), 77.8 (C-11′), 77.7 (C-36), 74.6 (C-20), 81.8 (C-19), 80.8 (C-23), 79.2 (C-11′), 77.6
(C-15), 74.3 (C-9′), 74.2 (C-28), 71.7 (C-24), 70.6 (C-36), 76.9 (C-9′), 75.1 (C-24), 74.4 (C-15), 72.7
(C-8′), 69.2 (C-10′), 61.7 (C-12′), 34.6 and 34.5 (C-27, (C-8′), 71.4 (C-28), 69.1 (C-10′), 61.1 (C-12′), 37.7 and
C-29), 34.0 (C-2′), 32.9 (C-14), 32.5 (C-25), 31.9 (C-32), 37.0 (C-27, C-29), 33.9 (C-2′), 33.1 (C-14), 32.0 (C-32),
29.9 (4C), 29.8 (2C), 29.7, 29.5, 29.4 (2C), 29.2 (2C), 31.3 (C-25), 29.9 (2C), 29.8 (3C), 29.7, 29.6, 29.5, 29.4,
28.7, 27.6, 25.8, 25.6, 25.4 (2C), 24.8 (C-4′), 24.7, 22.8 29.0, 28.6, 28.5, 27.6 (2C), 25.9, 25.7, 25.3, 24.8 (2C),
(C-33), 22.4 (C-26), 19.4 (C-37), 14.3 (C-34); HR- 24.5, 22.8 (C-33), 21.9 (C-26), 19.4 (C-37), 14.3 (C-34);
ESIMS m/z: 922.6017 [M
+
H]+ (calcd for HR-ESIMS m/z: 922.5996 [M H]+ (calcd for
+
C49H84N3O13, 922.5999).
C49H84N3O13, 922.5999).
Characterization Data of Compound 15
Preparation of Glucosylated Squamocin
Derivatives 14 and 15
A yellowish waxy solid; [α]20 +0.8 (c 1.1, MeOH);
D
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).
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 sus-
pended 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 chro-
matography 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, tR = 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, tR = 56.68 min, 11.7%).
Preparation of Galactosylated Bullatacin 17
Galactosylated bullatacin derivative was synthesized using
the same method as described for galactosylated squamocin
Characterization Data of Compound 14
A yellowish waxy solid; [α]20 +1.4 (c 1.1, MeOH); derivatives 11–13. Briefly, bullatacin (93 mg, 0.15 mmol),
D
1H NMR (600 MHz, CDCl3): δ 7.82 (1H, s, H-6′), 7.00 5-hexynoic acid (20 mg, 0.18 mmol) and DMAP (2 mg, 0.02
(1H, m, H-35), 5.59 (1H, d, J = 7.9 Hz, H-7′), 5.00 mmol) were dissolved in 3 mL dried CH2Cl2. DCC (62 mg,
(1H, m, H-36), 4.96 (1H, m, H-24), 4.01 (2H, m, H-23, 0.3 mmol) was added and the mixture was stirred under N2
8′), 3.82 (1H, m, H-20), 3.77 (6H, m, H-16, 19, 9′, 10′, atmosphere at room temperature for 16 h (Scheme 4). The
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resulting precipitate (DCU) was removed by filtration and the H-4, 9′, 11′, 12′), 3.73 (1H, m, H-24), 2.70 (2H, m, H-4′),
filtrate was concentrated under reduced pressure. The residue 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).
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 sus-
pended 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, tR = 65.24 min, 34.7%).
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 dis-
solved in 3 mL dried CH2Cl2. DCC (86 mg, 0.42 mmol)
was added and the mixture was stirred under N2 atmo-
sphere 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).
Characterization Data of Compound 17
A colorless waxy solid; [α]20 +20.3 (c 0.1, MeOH);
D
1H NMR (600 MHz, CDCl3): δ 7.83 (1H, s, H-6′), 7.20
The intermediate product 16B (115 mg) and β-
D-glucopyranosyl azide 9 (82 mg, 0.42 mmol) were sus-
(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 pended in 3 mL MeOH. Then, CuSO4·5H2O (15 mg, 0.06
(1H, m, H-16), 3.85 (3H, m, H-19, 20, 23), 3.81 (5H, m, mmol) and sodium ascorbate (24 mg, 0.12 mmol) in 1 mL
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.
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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, tR = 55.23 min, 28.7%).
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.
Characterization Data of Compound 18
A yellowish waxy solid; [α]20 +4.5 (c 1.1, MeOH);
Cell Viability Assay
D
A549, HeLa and HepG2 cells (5 × 104/well) were seeded in
flat-bottomed 96 well microplates. Then, squamocin/bullata-
cin and glycosylated squamocin/bullatacin derivatives dis-
solved 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 incu-
bator 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%.
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).
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
Aqueous Solubility of Squamocin and
Galactosyl Derivative 13
HPLC method with refractive index detector was the α,β-unsaturated γ-lactone moiety during the deprotec-
employed to measure the aqueous solubility of squamocin tion of benzyl groups on the sugar residue.29 We aim to
and its galactosyl derivative 13. Standard curve of com- prepare glycosylated acetogenin derivatives wherein the
pound 13 was found to be y=1.45*106x + 8.64*105 (R2 = acetogenin scaffold remains intact. The three hydroxyl
0.9970) by using six gradient concentrations (10, 5, 2.5, groups in squamocin and bullatacin (Figure 1) play less
1.25, 0.625, and 0.3125 mM) in methanol. Excess amount important roles in their anticancer activity13,28 and sugar
of squamocin/compound 13 was added to 1 mL of phos- residue(s) may be attached to these hydroxyl groups with-
phate-buffered saline (PBS, pH = 7.0) and sonicated for 5 out affecting their activities much.
min to provide saturated solution. The mixture was cen-
We first tried the trichloroacetimidate as the glycosyla-
trifuged (12000 rpm) for 2 min and the supernatant was tion donor. Galactose trichloroacetimidate (3) was readily
analyzed by HPLC for the content of squamocin/com- prepared from D-galactose in three steps (Scheme 1)
pound 13. The concentration of compound 13 was found according to literature procedure.30 Treatment of squamo-
to be 1.49 mM (1.37 mg/mL) in PBS (pH 7.0) at ambient cin with imidate 3 in the presence of trimethylsilyl trifluor-
temperature (around 28°C) while squamocin was not omethanesulfonate (TMSOTf) as the catalyst resulted in
detected in PBS (pH 7.0).
complicated product mixture comprising of mono- and di-
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glycosylated products (as indicated by mass spectrometry final products might have resulted in the loss of other minor
data). The glycosylation yield was also found to be very 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,
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 pro-
tection groups on the sugar, alternative methods of attach-
ing sugars to acetogenins are desirable.
1
and HR-ESIMS data. The selected H 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 hydro-
xyl 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 acyla-
tion of the hydroxyl group. When a hydroxyl group was
glycosylated, the carbon bearing that hydroxyl group exhib-
ited 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
The Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddi-
tion (CuAAC) reaction, popularly known as the “click reac-
tion”, 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 corre-
sponding commercially available per-acetylated sugar deriva-
tives 6 and 7 (Scheme 2). Squamocin was first treated with
5-hexynoic acid (1.2 or 1.5 eq.) in the presence of dicyclohex-
ylcarbodiimide (DCC) and 4-N,N-dimethylaminopyridine
(DMAP) to provide alkyne functionalized intermediate (10A
or 10B) after purification through Sephadex LH-20 gel per-
meation chromatography (Scheme 3). Treatment of 10A with
galactosyl azide 8 in the presence of CuSO4 and sodium
ascorbate afforded mono-galactosylated squamocin deriva-
tives 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-hex-
ynoic 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 purifica-
tion of the reaction intermediate products (16A/16B) and the
1
hydroxyl group (Table 2). In the case of H NMR data,
a significant downfield shift (>1.5 ppm) was observed for
the proton directly attached to the carbon bearing the hydro-
xyl group that was acylated while a smaller downfield shift
Table 1 Selected 1H NMR Data (600 MHz, CDCl3) for
Squamocin (1), Bullatacin (2) and Their Glycosylated Derivatives
Compound
δH (ppm)
H-4
H-15
H-24
H-28
Squamocin (1)a
3.33
4.03
3.39
4.83
3.39
3.39
3.35
3.37
3.38
4.86
3.39
3.76
3.74
3.63
3.59
5.00
3.75
4.96
3.78
3.83
3.73
3.83
3.52
3.57
3.63
3.52
3.53
4.88
3.51
4.86
4
5
11
12
13
14
15
Bullatacin (2)b
3.80
3.81
5.00
17
18
Notes: aData from Fujimoto et al.34 bData from Hui et al.35
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Table 2 Selected 13C NMR Data (150 MHz, CDCl3) for Squamocin (1), Bullatacin (2) and Their Glycosylated Derivatives
Compound
δC (ppm)
C-3
C-4
C-5
C-14
C-15
C-16
C-23
C-24
C-25
C-27
C-28
C-29
Squamocin (1)a
33.1
30.2
33.2
30.9
33.1
32.9
33.1
33.0
33.2
30.7
33.8
74.1
81.2
74.6
75.7
74.7
74.6
74.4
74.4
74.1
75.5
74.4
83.4
81.3
83.7
80.7
83.7
84.0
83.6
83.6
83.2
80.3
83.5
82.2
82.8
82.2
82.2
80.9
82.8
80.8
82.2
82.8
82.2
82.4
71.5
71.2
71.8
72.2
75.1
71.7
75.1
71.5
71.3
71.7
71.6
32.5
32.1
32.6
33.0
31.2
32.5
31.3
32.4
32.4
32.8
32.6
37.5c
37.7c
35.6c
37.8
71.6
71.7
81.4
71.9
71.4
74.2
74.1
73.9
37.2c
37.4c
34.1c
37.3
4
5
11
12
37.8
37.1
13
34.6
34.5
14
37.7c
34.4c
37.0c
34.3c
15
Bullatacin (2)b
33.2
33.3
29.7
69.9
70.0
72.3
37.3
37.5
33.3
17
18
Notes: aData from Fujimoto et al.34 bData from Hui et al.35 cAssignments interchangeable in the same row.
(0.1–0.7 ppm) resulted from O-galactosylation of that hydro- evaluated using MTT assay. The IC50 values of these
1
xyl group (Table 1). The H and 13C NMR spectra of all compounds were calculated based on three parallel
synthesized glycosyl derivatives of squamocin and bullatacin experiments and presented in Table 4. Overall, when
(4, 5, 11–15, 17 and 18) are provided in the Supplementary compared to squamocin (1) and bullatacin (2), the major-
Material of this article.
ity 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 com-
pounds 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, com-
pounds 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).
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 solubi-
lity 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 squa-
mocin significantly improved its solubility in water.
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 elec-
tron 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 pecu-
liar to the plasma membranes of cancer cells.37
Acetogenins cause cell cycle arrest at different phases
in different cancer cells and are strong apoptosis indu-
cers. Squamocin arrested T24 bladder cancer cells at the
G1 phase, enhanced caspase-3 activity, cleaved the
Biological Evaluation
The cytotoxicity of the synthesized glycoconjugate deri-
vatives against three tumor cell lines (Table 4) were
Table 3 Solubility of Squamocin and Its Derivative 13
Compound
Solubility (mg/mL)
Squamocin
Not detected
1.37
13
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Shi et al
Table 4 Cytotoxic Activity (IC50) of Glycosylated Derivatives of
Squamocin and Bullatacin
whether or not a linking spacer is present do not seem
to affect the potency much. The solubility of galactosy-
lated 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 com-
pounds exhibit reduced toxicity to normal cells, the
mechanism of action, and their therapeutic potential for
cancer.
Compound
IC50 (µM)
A549
HeLa
HepG2
Squamocin (1)
1.99 ± 0.49
6.62 ± 1.23
4.60 ± 0.69
3.88 ± 0.005
4.67 ± 0.69
2.99 ± 0.39
6.33 ± 0.96
1.80 ± 0.25
1.74 ± 0.34
1.46 ± 0.38
15.44 ± 2.93
0.93 ± 0.079
1.96 ± 0.23
2.24 ± 0.049
7.16 ± 1.45
3.24 ± 0.57
0.98 ± 0.13
1.56 ± 0.16
0.77± 0.0092
1.42 ± 0.14
13.77 ± 1.51
1.42 ± 0.12
1.70 ± 0.14
2.93 ± 0.44
2.82 ± 0.31
1.52 ± 0.40
1.97 ± 0.18
4.09 ± 0.68
1.68 ± 0.29
0.98 ± 0.042
2.48 ± 0.29
1.51 ± 0.09
1.13 ± 0.17
4
5
11
12
13
14
Acknowledgments
15
We thank Mr. Yunfei Yuan, South China Botanical
Garden, Chinese Academy of Sciences, for NMR spectro-
scopic measurements, and Ms. Aijun Sun, South China
Sea Institute of Oceanology, Chinese Academy of
Sciences, for HRESIMS measurements. This work was
supported by an NSFC grant (grant no. 31470423).
Bullatacin (2)
17
18
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 asso-
ciation 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 mito-
chondrial 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.
Disclosure
The authors declare no conflicts of interest.
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