Synthesis of antimitotic thioglycosides: In vitro and in vivo evaluation of their anticancer activity

Article abstract of DOI:10.1021/jm200961q

The synthesis and biological activity of oleylN-acetyl-α- and β-d-glucosaminides (1 and 2, respectively) and their thioglycosyl analogues (3 and 4, respectively) are reported. The compounds exhibited antimitotic activity on rat glioma (C6) and human lung carcinoma (A549) cell cultures in the micromolar range. Analysis of cell extracts using ultra performance liquid chromatography-mass spectrometry showed that the synthetic glycosides produce alterations in glycosphingolipid metabolism, with variable effect on the level of glucosylceramide depending on the configuration of the antimitotic used. In vivo experiments in nude mice bearing an implanted C6 glioma showed that the α-thioglycoside 3 reduced tumor volume, while the O-glycosyl derivative was inactive, highlighting the importance of using enzyme resistant glycosides.

Full text of DOI:10.1021/jm200961q

ARTICLE
pubs.acs.org/jmc
Synthesis of Antimitotic Thioglycosides: In Vitro and in Vivo
Evaluation of Their Anticancer Activity
†
Isabel García-Alvarez, Hugo Groult,‡,^ Josefina Casas,§ M. Asunciꢀon Barreda-Manso,†,||
ꢀ
Natalia Yanguas-Casꢀas,†,|| Manuel Nieto-Sampedro,†,|| Lorenzo Romero-Ramírez,*,†,|| and
Alfonso Fernꢀandez-Mayoralas*,‡
†Hospital Nacional de Paraplꢀejicos, SESCAM, Finca la Peraleda s/n, 45071 Toledo, Spain
‡Instituto de Química Orgꢀanica General, CSIC, Juan de la Cierva, 3, 28006 Madrid, Spain
§RUBAM Institut de Química Avanc-ada de Catalunya, CSIC, Jordi Girona, 18, 08034 Barcelona, Spain
||Instituto Cajal, CSIC, Avda. Doctor Arce 37, 28002 Madrid, Spain
S Supporting Information
b
ABSTRACT: The synthesis and biological activity of oleylN-acetyl-
α- and β-D-glucosaminides (1 and 2, respectively) and their thiogly-
cosyl analogues (3 and 4, respectively) are reported. The compounds
exhibited antimitotic activity on rat glioma (C6) and human lung
carcinoma (A549) cell cultures in the micromolar range. Analysis of
cell extracts using ultra performance liquid chromatographyÀmass
spectrometry showed that the synthetic glycosides produce altera-
tions in glycosphingolipid metabolism, with variable effect on the
level of glucosylceramide depending on the configuration of the antimitotic used. In vivo experiments in nude mice bearing an
implanted C6 glioma showed that the α-thioglycoside 3 reduced tumor volume, while the O-glycosyl derivative was inactive,
highlighting the importance of using enzyme resistant glycosides.
1. INTRODUCTION
Chart 1. Chemical Structures of Glycosides 1 and 2 and
Thio-glycosides 3 and 4
We have previously reported the synthesis of a family of glyco-
sides derived from N-acyl-glucosamine and their evaluation as
inhibitors of glioma and melanoma cell proliferation.1 The results
indicated that the activity of the compounds increased with a long
hydrocarbon chain at position C-1 of the glucosamine scaffold.
Among the compounds tested, oleyl glycoside 1 was the most
effective (Chart 1). A favorable effect of an oleyl moiety on the
antitumor activity was also observed in a family of alkylated
iminosugars.2 These findings suggest that the conjugation of a
sugar with an oleyl chain is beneficial for obtaining compounds
with good antitumor activity. Preliminary studies on the mechan-
ism of growth inhibition by 1 using proton magic angle spining
nuclear magnetic resonance (1H MAS NMR) suggested that the
glycoside may be altering the metabolism of lipids.3 Further
studies of its effect on the sphingolipidome of the A549 human
lung carcinoma cell line showed that 1 caused important changes
in glycosphingolipid and ganglioside content.4 These biomol-
ecules have important regulatory roles in tumor formation and
progression5,6 and are involved in processes of cell death and cell
proliferation.7,8
per day) by intratumor administration. After 8 days of treatment,
the glycoside neither slowed tumor growth nor caused any
reduction of tumor volume as compared to the controls. Never-
theless, in the case of the treatment with the highest dose of 1,
we observed the formation of a small bag or cyst, attached to the
surface of the tumor. The analysis of the intact cyst by 1H MAS
NMR spectroscopy showed that it was formed mainly by oleyl
alcohol, probably derived from the cleavage of 1. Because hydro-
lysis of 1 was not observed during experiments in cell culture,4
the cleavage of 1 could be the result of an inflammatory process
The convenient synthesis of 1, together with its antimitotic
activity on different cancer cell lines, makes this compound a
potential anticancer drug. With the aim to gain further insight in
this direction, recently, we performed preliminary in vivo experi-
ments in nude mice bearing an implanted C6 glioma (unpublished
work). Animals were treated with two doses of 1 (1 and 10 mg/kg
Received: July 20, 2011
Published: August 25, 2011
r
2011 American Chemical Society
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Scheme 1. Synthesis of Compounds 2À4a
Table 1. ID50 Values (μM)a for Compounds 1À4 on A549
and C6 Tumor Cells
1
2
3
4
A549
C6
25 ( 44
16 ( 4
50 ( 4
38 ( 3
35 ( 5
14 ( 1
30 ( 4
26 ( 2
a Results represent the mean ( SEM of two experiments in triplicate.
Cells were treated with serial dilutions of compounds (1À4) for 48 h.
Cell proliferation was measured by the MTT assay.
Scheme 2. Schematic Representation of Glycosphingolipid
Biosynthesis
5 with thiourea. Only the β-anomer was obtained, indicating that
the nucleophilic substitution at the anomeric position proceeded
through an SN2-like mechanism. Oleyl mesylate 8 was treated
with the isothiouronium salt 7 to give a mixture of α- and
β-thioglycosides 9 and 10 in a 1:1 ratio. This result is in contrast
with the general observation that glycosyl thiolates are config-
urationally stable;11 nevertheless, this isomerization allowed us to
obtain both anomeric thioglycosides through the same synthetic
route. Compounds 9 and 10 were separated by silica gel column
chromatography and subsequently treated with a solution of
sodium methoxide in methanol to give 3 and 4 (Scheme 1).
Compounds 2À4 together with 1 as a reference were eval-
uated for their ability to inhibit A549 (human adenocarcinoma)
and C6 (rat glioma) cell growth. Cell proliferation was measured
by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay, which is based on the conversion of the water-
soluble MTT into an insoluble formazan. The results, summarized
in Table 1, show that the new compounds 2À4 were as active as 1
against tumor cell lines with 50% inhibitory dose (ID50) values in
the same range than that of 1. Therefore, the change oftheanomeric
oxygen by a sulfur atom or the configuration at the anomeric posi-
tion have only a moderate influence on the inhibitory activity.
2.2. Effect on Glycosphingolipid Content. Glucosylcera-
mide (GlcCer) and lactosylceramide (LacCer) are the precursors
of glycosphingolipids, including gangliosides GM3 and GM2
(Scheme 2). Glycosphingolipids are involved in multiple cellular
processes5À8 and also in the formation of membrane lipid rafts.
Alterations in the metabolism of glycosphingolipids may result in
tumor growth inhibition.13,14 To compare the effect of com-
pounds 1À4 on the content of GlcCer, LacCer, and gangliosides
GM3 and GM2 of A549 cells, 1À4 (15 μM) were incubated with
the cells for 48 h, and the glycosphingolipids were then extracted
and analyzed by ultra performance liquid chromatographyÀmass
spectrometry (UPLC-MS), as described in the Experimental
Section. The results are shown in Figure 1.
a Reagents and conditions: (a) Oleyl alcohol, SnCl4, CH3CN, 55 ꢀC, 24 h
(48%). (b) 0.1 M NaOMe/MeOH, room temperature, 2 h (quantitative).
(c) Thiourea, acetone, 56 ꢀC, 30 min (73%). (d) Et3N, DMF, 60 ꢀC, 8 h
(60%). (e) 0.1 M NaOMe/MeOH, room temperature, 1 h (73% for 3,
77% for 4).
taking place in vivo after tumor implantation or compound injec-
tion. Activated macrophages can generate lysosomal enzymes,
including hexosaminidases with the capacity to cleave the glyco-
sidic bond of N-acetyl-glucosaminides.9,10
Thioglycosides are known to be resistant to glycosidase-
catalyzed hydrolysis.11 In view of the results obtained in vivo,
we decided to synthesize the corresponding thioanalogues
of 1 and its β-configured glycoside 2 (compounds 3 and 4,
respectively, Chart 1). In the present work, we describe the
synthesis of 2À4 and their inhibitory activity against C6 and
A549 tumor cell lines. The effect of the change of the oxygen by a
sulfur atom and the influence of the configuration at the
anomeric carbon on the ability to alter glycosphingolipid levels
in A549 cells have also been evaluated.
The stability of the thioanalogues to enzymatic hydrolysis has
been tested with the β-thioglycoside 4 against a commercially
available β-N-acetylhexosaminidase. Finally, we report the results
of testing the in vivo activity of the α-glycosides 1 and its
thioanalogue 3.
2. RESULTS AND DISCUSSION
2.1. Synthesis and Inhibitory Activity. The synthesis of 2
was carried out in two steps from α-D-glucosaminyl chloride
peracetate 5 (Scheme 1). Stereoselective glycosylation of 5 with
oleic alcohol using SnCl4 as an acid promotor gave the
β-glycopyranoside 6, which was submitted to O-deacetylation
to afford 2. For the synthesis of thioglycosides 3 and 4, we
employed the approach involving the reaction of alkyl halides or
mesylates with S-glycosyl isothiouronium salts.12 Thus, iso-
thiouronium derivative 7 was prepared by treatment of chloride
All compounds caused a similar effect on LacCer and gang-
lioside content, namely, a rise (40À60%) in LacCer and a marked
decrease in the levels of gangliosides GM3 and GM2. Consider-
ing the biosynthetic pathway of gangliosides (Scheme 2), these
results suggest that compounds 1À4 partially inhibited their
formation at the step of GM3 (the first ganglioside in the
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Figure 2. Enzymatic hydrolysis of compound 2 (circle) and 4 (square).
All experiments were performed in triplicate. Product formation, this is
GlcNAc formation, was calculated as % percentage and expressed as the
mean ( SEM.
Figure 1. Effect of compounds 1À4 (15 μM) on glycosphingolipid
content of A549 carcinoma cells. Glycosphingolipids: glucosilceramide
(GlcCer), lactosylceramide (LacCer), and gangliosides GM3 and GM2.
Data were normalized to the same number of cells (1 Â 106 cells). The
content in pmol per 1 Â 106 cells for the control was 150 ( 2, 13 ( 1,
608 ( 65, and 705 ( 7 for GlcCer, LacCer, GM3, and GM2, respectively.
The data are the means of two experiments performed by triplicate. The
diagram includes the ANOVA results for each treatment with respect to
control at a significance level of *p < 0.05, **p < 0.01, and ***p < 0.001.
Table 2. Detected Compounds by UPLC-MS in A549 Cells
after Treatment with Compounds 1À4 (15 μM)
compd
amounta
rt (min)b
exact massb (error in ppm)
1-treated cells
3.19
1
2916 ( 417
3.5 ( 0.5
472.3638 (À0.4)
634.4166 (1.3)
Gly-1c
3.01
2-treated cells
3.11
2
952 ( 69
320 ( 9
472.3638 (À1.9)
634.4166 (À1.4)
Figure 3. Evaluation of the antitumoral activity of compounds 1 and 3
in an orthotopical transplantation of the rat C6 glioma cells in the flank
of nude mice. The results are expressed as the mean ( SEM of at least six
animals per group. Intergroup differences for repeated measures were
evaluated statistically using two-way analyses of variance (ANOVA)
followed by posthoc Tukey test. Statistical significance of the treatments
related to control was assigned as *p < 0.05 and **p < 0.01.
Gly-2c
3.01
3-treated cells
3.34
3
3675 ( 409
NDd
488.3410 (1.0)
NDd
Gly-3c
NDd
4-treated cells
3.24
4
3333 ( 814
812 ( 178
488.3410 (1.3)
650.3938 (1.2)
Gly-4c
3.13
amounts in cells incubated with β-anomers 2 and 4 than with
α-anomer 1 (not detected in 3-treated cells, Table 2). On the basis
of their accurate mass measurement and their relative retention
times, these compounds have been putatively identified as oleyl
disaccharides derived from the glycosylation of 1, 2, and 4 with a
six-carbon glycosyl moiety (C6H10O5) (Gly-1, Gly-2, and Gly-4,
respectively, Table 2). The chemical resemblance between the
β-configured compounds 2 and 4 and GlcCer, which is also a
β-glycoside, makes us think that 2 and 4 have been galactosylated
by the enzyme that transformed GlcCer into LacCer (LacCer
synthase). If this were the case, the β-compounds would act as
“decoy acceptors” of LacCer synthase and would cause an accu-
mulation of GlcCer, as it is actually observed in the analysis of this
metabolite (Figure 1).
a Amount in pmol/1 Â 106 cells. b As indicated by UPLC-MS. c Com-
pounds Gly-1, Gly-2, Gly-3, and Gly-4 have been putatively identified as
disaccharides derived from glycosylation of 1, 2, 3, and 4, respectively
(see the text). d ND, not detected.
biosynthetic route), leading to lower ganglioside levels and the
accumulation of LacCer.
On the other hand, a different behavior between the glycosides
with α- and β-anomeric configuration was observed when anal-
yzing GlcCer content. While the α-compounds (1 and 3) reduced
the GlcCer content, the β-compounds (2 and 4) caused a rise
in the level of this metabolite (Figure 1). Differences between
anomers were also observed when the extracts were analyzed
for the presence of compounds 1À4 (Table 2). All of the com-
pounds were detected in cell extracts, which indicated that they
are efficiently taken up by cells. However, an unnatural metabolite
not present in control samples was detected in much higher
2.3. Hexosaminidase Hydrolysis Resistance. The stability of
the O- and S-glycosyl derivatives to glycosidases was assayed with
the β-configured compounds, taking advantage that N-acetyl-
β-D-glucosaminidase from Jack bean is commercially available. The
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Figure 4. Representative 1H MAS NMR spectra of C6 tumor tissue (T2 = 60 ms, ns = 256). (A) Tumor of control mice and (B) tumor of 3-treated mice
(10 mg/kg).
enzyme activity was determined using p-nitrophenyl β-D-glucopyr-
anoside as a substrate, by measuring the liberation of p-nitrophenol.
The assay was performed in the presence of 20% of isopropanol
to dissolve the glycosides. This cosolvent did not alter severely
the enzyme activity,15 as evidenced by the control reaction. More-
over, no glycoside product derived from transglycosidation of
isopropanol was observed. Compounds (2 or 4) were incubated
in the presence of the enzyme at pH 5 and 37 ꢀC for different
time periods. After 48 h of incubation, high-performance liquid
chromatography (HPLC) analysis showed that thioglycoside 4
remained unchanged, while 32% of 2 was hydrolyzed to N-acetyl-
D-glucosamine (GlcNAc) (Figure 2). This result confirmed the
higher resistance to enzymatic hydrolysis of thioglycosides as
compared to O-glycosyl derivatives, which makes these com-
pounds good candidates for in vivo assays.
pound 3 with the higher dose (10 mg/kg animal) reduce signifi-
cantly their size as compared to the controls. Conversely, there is no
significant effect in the size of the tumors treated with compound 1
or compound 3 with the lower concentration (1 mg/kg animal).
Moreover, 1H MAS NMR analysis of intact tumor tissues showed
higher cell death in the samples from treated animals than in the
controls (Figure 4). MAS NMR spectroscopy allows the simul-
taneous detection of many metabolites present in the sample of
interest and requires minimal or no sample preparation.16À18
Thus, in the case of mice treated with the higher dose (10 mg/kg)
of thioglycoside 3, the spectra showed a low intensity in the peak
centered at 3.0 ppm, which was assigned to creatine, as compared
to the control (Figure 4). Creatine is commonly used as an
internal concentration reference for in vivo 1H NMR because its
concentration correlates with the number of metabolically active
cells and is used as a measure of viable cell number.19 An increase
of choline-containing metabolites observed in the spectra of
treated mice may also be indicative of cell death processes.20 On
2.4. In Vivo Effect on Tumor Growth. The activity of com-
pound 1 and 3 on tumor growth was evaluated usingan orthotopic
model in female nude mice (Figure 3). Tumors treated with com-
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the other hand, the tumor samples of animals treated with 3
showed pronounced peaks corresponding to lactate and acet-
ate, metabolites that have been related to the anaerobic energy
metabolism of cancer cells.21 All together, the results from 1H
MAS NMR analysis suggest that a higher decrease in tumor
volume of treated mice could be obtained in treated mice if the
animals were kept alive longer, allowing macrophages to engulf
dead tissue.
chromatography (hexaneÀEtOAc, 1:1) to give 6 (396 mg, 48%). [α]D
À9.6ꢀ (c 1.1, MeOH). 1H NMR (400 MHz, CD3OD): δ 5.5À5.3 (m,
2H), 5.22 (dd, 1H, J = 10.5, 9.3 Hz), 4.98 (dd, 1H, J = 10.1, 9.5 Hz), 4.63
(d, 1H, J = 8.5 Hz), 4.28 (dd, 1H, J = 12.4, 4.8 Hz), 4.11 (dd, 1H, J = 12.3,
2.4 Hz), 3.9À3.7 (m, 2H), 3.6À3.4 (m, 2H), 2.1À2.0 (m, 13H), 1.90 (s,
3-H), 1.6À1.5 (m, 2H), 1.4À1.2 (m, 22 H), 0.90 (t, 3H, J = 6.9 Hz) ppm.
13C NMR (100 MHz, CD3OD): δ 173.2, 172.3, 171.9, 171.3, 130.9,
130.8, 102.1, 74.2, 72.8, 70.9, 70.2, 63.3, 55.5, 33.6, 33.1, 30.9, 30.8, 30.8,
30.8, 30.7, 30.6, 30.5, 30.3, 30.2, 28.2, 28.1, 27.1, 23.7, 22.8, 20.7, 20.6,
20.6, 14.5 ppm. HRMS (ESI) m/z calcd for C32H55NO9, 597.3897;
found, 598.3970 (M + H)+.
3. CONCLUSION
4.1.2. Oleyl 2-Acetamido-2-deoxy-β-D-glucopyraroside (2). Com-
pound 6 (800 mg, 1.30 mmol) was dissolved in MeOH (2 mL) and
treated with a 0.1 M solution of NaOMe (10 mL). The reaction was
stirred at room temperature for 2 h. After this time, the mixture was
neutralized with Amberlite IR-120 (H+ form), filtered off, and concen-
trated. The residue was purified by silica gel column chromatography
(EtOAcÀMeOH, 10:0 f 10:1) to give 2 (840 mg, quantitative) as a
On the basis of our previous studies on the antitumor activity
of glycoside 1, in the present work, we report on the synthesis and
biological activity of its β-anomer 2 and thioanalogues 3 and 4.
The new compounds also showed antiproliferative activity in
cancer cells and caused alterations in the levels of glycosphingo-
lipids of A549 cells. The anomeric configuration of the glycosides
was important for the effect on glucosylceramide content, which
was increased after treatment with the β-anomers, while with the
α-anomers a decrease in GlcCer content was observed as com-
pared to control. The different effect could be related to a glyco-
sylation of the β-anomers by glycosyltransferases involved in
GlcCer metabolism. In vivo experiments with thioglycoside 3 led
to a reduction of tumor volume, in contrast to the O-glycosyl
derivative 1 that did not cause any reduction of tumor volume.
The results show that α-thioglycoside 3 is an enzymatically stable
product that maintains antitumor activity, making it a good
candidate for further studies in cancer therapy.
1
white solid; mp 155À160 ꢀC; [α]D À9.0ꢀ (c 0.5, MeOH). H NMR
(400 MHz, CD3OD): δ 5.4À5.3 (m, 2H), 4.38 (d, 1H, J = 8.4 Hz),
3.9À3.8 (m, 2H), 3.69 (dd, 1H, J = 11.9, 5.5 Hz), 3.62 (dd, 1H, J = 10.3,
8.4), 3.5À3.4 (m, 1H), 3.34 (d, 1H, J = 9.6 Hz), 3.25 (ddd, 1H, J = 9.6,
5.5, 2.4 Hz), 2.1À1.9 (m, 7H), 1.5À1.4 (m, 2H), 1.3À1.2 (m, 26H),
0.88 (t, 3H, J = 7.0 Hz) ppm. 13C NMR (100 MHz, CD3OD): δ 173.4,
130.6, 130.5, 102.3, 79.1, 78.8, 78.5, 77.4, 75.7, 71.8, 70.5, 62.5, 57.1, 33,4,
32.8, 30.6, 30.6, 30.5, 30.5, 30.4, 30.40, 30.3, 30.3, 30.2, 30.9, 30.1, 30.1,
29.9, 28.0, 26.9, 23.5, 23.0, 14.4 ppm. HRMS (ESI) m/z calcd for
C26H49NO6, 471.3570; found, 472.3643 (M + H)+, 494.3461 (M + Na)+.
4.1.3. Oleyl Mesylate (8). Oleic alcohol (4 mL, 10.8 mmol) was dis-
solved in anhydrous CH2Cl2 (108 mL), and Et3N (4.5 mL, 32.4 mmol)
was added. Mesyl chloride (2.3 mL, 32.4 mmol) was added dropwise,
and the mixture was stirred at room temperature for 23 h. After the
completion of the mesylation (TLC: hexaneÀEtOAc, 2:1), the reaction
mixture was concentrated in vacuo and purified by silica gel column
chromatography (hexaneÀEtOAc, 3:1) to afford 8 (3.13 g, 84%) as a
yellow liquid. 1H NMR (300 MHz, CDCl3): δ 5.3À5.2 (m, 2H), 4.19
(t, 2H, J = 6.6 Hz), 3.0 (s, 3H), 2.0À1.9 (m, 4H), 1.9À1.6 (m, 2H),
1.3À1.1 (m, 22H), 0.85 (t, 3H, J = 6.6 Hz) ppm. 13C NMR (75 MHz,
CDCl3): δ 130.2, 129.9, 70.5, 37.5, 32.8, 32.1, 30.0, 29.9, 29.7, 29.5, 29.3,
29.3, 29.3, 29.2, 27.4, 27.4, 25.6, 22.9, 21.2, 14.4 ppm.
4. EXPERIMENTAL SECTION
4.1. Chemistry. General Synthetic Methods. All chemicals
were of reagent grade or higher and were purchased from commercial
suppliers or purified by standard techniques. Compounds 5 and 7 were
prepared following described procedures.22,23 Thin-layer chromatogra-
phy (TLC) was performed on aluminum sheets 60 F254 Merck silica gel,
and compounds were visualized by irradiation with UV light and/or by
treatment with a solution of Ce2MoO4 or 5% H2SO4 in EtOH, followed
by heating. Flash column chromatography was performed using thick-
walled columns, employing silica gel (Merck 60: 0.040À0.063 mm). The
eluent used is indicated, and solvent ratios refer to volume. Melting
points are not corrected and were measured with a Reicher Jung
Thermovar micromelting apparatus. Optical rotations were recorded
on a Perkin-Elmer 241 Polarimeter (λ = 589 nm, 1 dm cell). 1H NMR
spectra were registered at 400 or 300 MHz, and 13C NMR spectra were
obtained at 100 MHz on a Varian INOVA spectrometers, using CDCl3,
CD3OD, or D2O as the solvent at room temperature. Chemical shift
values are reported in parts per million (δ). Coupling constant values (J)
are reported in hertz (Hz), and spin multiplicities are indicated by the
following symbols: s (singlet), d (doublet), t (triplet), q (quartet), and m
(multiplet). High-resolution mass spectra (HRMS) were recorded on an
Agilent 6520 Accurate Mass Q-TOF spectrometer with an ESI source.
The purity of all compounds was g95% as determined by elemental
analyses using a Heraus CHN-O analyzer.
4.1.4. Oleyl 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-1-thio-α- and
β-D-Glucopyranoside (9 and 10). To a mixture of 723 (1.9 g, 4.30 mmol)
and Et3N (1.8 mL, 12.9 mmol) in anhydrous DMF (6 mL), 8 (2.2 g,
6.5 mmol) was added, under stirring at 60 ꢀC and under Ar atmosphere for
8 h. Thereaction mixture wasconcentrated, and the residue waspurified by
silica gel column chromatography (hexaneÀEtOAc 5:1 f 2:1) to give 9
(0.72 g 27%) as a yellow oil and 10 (0.89 g, 33%) as a white solid.
1
Compound 9: [α]D +112.4ꢀ (c 0.5, MeOH). H NMR (400 MHz,
CDCl3): δ 5.71 (d, 1H, J = 8.7 Hz), 5.39 (d, 1H, J = 5.4 Hz), 5.4À5.3
(m, 1H), 5.1À5.0 (m, 2H), 4.5À4.4 (m, 1H), 4.4À4.3 (m, 2H), 4.2À4.0
(m, 2H), 2.6À2.5 (m, 2H), 2.1À1.9 (m, 16H), 1.7À1.4 (m, 2H),
1.4À1.2 (m, 22H), 0.88 (t, 3H, J = 5.7 Hz) ppm. 13C NMR (75 MHz,
CD3OD): δ 171.6, 170.7, 169.8, 169.3, 130.0, 129.7, 84.6, 71.4, 68.3,
68.1, 62.8, 52.3, 36.5, 32.5, 31.9, 31.5, 29.7, 28.8, 27.2, 23.2, 22.7, 20.7,
14.6, 14.1 ppm. HRMS (ESI) m/z calcd for C32H55NO8S, 613.85;
found, 614.5 (M + H)+.
4.1.1. Oleyl 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyra-
noside (6). A solution of 522 (500 mg, 1.37 mmol) and oleic alcohol
(85%) (1.53 mL, 4.11 mmol) in anhydrous CH3CN (55 mL) containing
4 Å molecular sieves was stirred at room temperature for 10 min. Then,
SnCl4 (0.3 mL, 2.4 mmol) was added, and the reaction mixture was
stirred at 55 ꢀC for 24 h. After this time, the mixture was heated at 80 ꢀC
(under reflux) and stirred for 1 h. The reaction mixture was cooled at
room temperature, treated with Et3N (0.4 mL), filtered under Celite,
and concentrated in vacuo. The residue was purified by silica gel column
Compound 10: mp 132À143 ꢀC; [α]D: À13.1ꢀ (c 1.5, MeOH). 1H
NMR (400 MHz, CDCl3): δ 5.46 (d, 1H, J = 9.3 Hz), 5.4À5.3 (m, 2H),
5.2 5.0 (m, 2H), 4.57 (d, 1H, J = 10.5 Hz), 4.24 (dd, 1H, J = 4.9, 12.3 Hz),
4.2À4.0 (m, 2H), 4.1À4.0 (m, 1H), 3.7À3.6 (m, 1H), 2.7À2.6 (m, 2H),
2.0À1.9 (m, 16H), 1.7À1.5 (m, 2H), 1.4À1.2 (m, 22 H), 0.88 (t, 3H, J =
6.3 Hz) ppm. 13C NMR (100 MHz, CD3OD): δ 171.1, 170.7, 170.0,
169.3, 130.0, 129.8, 84.6, 76.7, 75.9, 73.8, 68.3, 62.30, 53.3, 32.6, 31.9,
30.1, 29.7, 29.6, 29.6, 29.5, 29.4, 29.3, 29.2, 29.1, 28.9, 27.2, 27.1, 23.3,
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22.7, 20.8, 20.7, 20.6, 14.1 ppm. HRMS (ESI) m/z calcd for C32H55N-
O8S, 613.85; found, 614.5 (M + H)+.
37 ꢀC/5% CO2. Then, cells were washed in PBS, collected by brief try-
psinization, and transferred to glass vials. An aliquot of the cells was taken
for cell counting, using a Countess automatic cell counter. Sphingolipid
extracts, spiked with internal standards (0.2 nmol), were prepared as
described24 and analyzed by liquid chromatographyÀmass spectro-
metry using a Waters Aquity UPLC system connected to a Waters
LCT Premier orthogonal accelerated time-of-flight mass spectrometer
(Waters, Milford, MA), operated in positive electrospray ionization
mode in the conditions previously reported.25
4.4. N-Acetyl-D-hexosaminidase Assay. 4.4.1. Chemicals.
N-Acetyl-β-D-hexosaminidase from Jack bean (1.2 mg protein/mL),
2-acetamido-2-deoxy-β-D-glucopiranose (GlcNAc), and p-nitrophenyl
2-acetamido-2-deoxy-β-D-glucopiranoside (pNP-GlcNAc) were purchased
from Sigma-Aldrich. Phosphate buffers were prepared by mixing appro-
priately concentrated aqueous sodium dihydrogenphosphate and dis-
odium hydrogen phosphate solutions to reach the required pH.
4.4.2. HPLC Analysis. All analyses were performed using a Jasco Pu-
2089 Plus, Dual Gradient Pump chromatograph, equipped with an
ultraviolet Jasco UV-2075 Plus detector and a 20 μL Rheodyne injector.
Data acquisition and processing were accomplished with the Jasco
ChromPass Chromatography Data System 1.8.6.1 software. A reverse
phase Lichrosorb C18 (5 μM, 4.6 mm  250 mm) column was used as
the stationary phase, using a C18 guard cartridge. GlcNAc separation
was carried out with a mobile phase of waterÀmethanol mixture,
following a gradient elution program: 95À5 (from 0 to 4 min) to
0À100 (from 8 to 20 min) to 95À5 (from 25 to 30 min). The flow rate
was 1.0 mL minÀ1, and the elution profile was monitored by recording
the UV absorbance at 205 nm. The amount of liberated GlcNAc was
calculated from a standard curve obtained using unlinked GlcNAc, with a
retention time of 2.8 min in 95% waterÀ5% methanol.
4.1.5. Oleyl 2-Acetamido-2-deoxy-1-thio-α-D-glucopyranoside (3).
Compound 9 (810 mg, 1.30 mmol) was dissolved in MeOH (2 mL) and
treated with a 0.1 M solution of NaOMe (10 mL), and the reaction
mixture was stirred at room temperature for 1 h. After this time, the
mixture was neutralized with Amberlite IR-120 (H+ form) and concen-
trated. The crude was purified by silica gel column chromatography
(EtOAcÀMeOH 10:1) to give 3 (0.47 g, 73%) as a white solid; mp
1
185À190 ꢀC; [α]D +132.0ꢀ (c 1.0, MeOH). H NMR (400 MHz,
CD3OD): δ 5.45 (d, 1H, J = 5.4 Hz), 5.4À5.3 (m, 2H), 4.00 (dd, 1H, J =
5.4, 11.1 Hz), 4.0À3.9 (m, 1H), 3.81 (dd, 1H, J = 2.0, 12.0 Hz), 3.70 (dd,
1H, J = 5.4, 12.0 Hz), 3.60 (t, 1H, J = 9.0 Hz), 3.3 (t, 1H, J = 9.3 Hz),
2.6À2.5 (m, 2H), 2.1À2.0 (m, 4H), 2.0 (s, 3H), 1.6À1.5 (m, 2H),
1.3À1.2 (m, 22H), 0.90 (t, 3H, J = 6.6 Hz) ppm. 13C NMR (100 MHz,
CD3OD): δ 173.6, 130.9, 130.8, 85.1, 74.2, 72.7, 72.6, 62.6, 55.9, 33.6, 33.1,
31.6, 30.9, 30.8, 30.6, 30.6, 30.5, 30.3, 29.9, 28.2, 28.1, 23.8, 22.6, 14.9 ppm.
MS (ESI) m/z calcd for C26H49NO5S, 487.5; found, 488.5 (M + H)+.
4.1.6. Oleyl 2-Acetamido-2-deoxy-1-thio-β-D-glucopyranoside (4).
Using the same procedure for the preparation of 3, starting from 10 (1.4 g,
2.23 mmol), 4 (0.84 g, 77%) was obtained as a white solid; mp 170À
175 ꢀC; [α]D À18.3ꢀ (c 1.1, MeOH). 1H NMR (400 MHz, CD3OD):
δ 5.4À5.3 (m, 2H), 4.47 (d, 1H, J = 10.3 Hz), 3.86 (dd, 1H, J = 2.2, 12.0
Hz), 3.73 (t, 1H, J = 10.1 Hz), 3.67 (dd, 1H, J = 5.7, 12.0 Hz), 3.43 (t, 1H,
J = 9.7 Hz), 3.3À3.2 (m, 2H), 2.7À2.6 (m, 2H), 2.1À2.0 (m, 4H), 2.00
(s, 3H), 1.6À1.5 (m, 2H), 1.4À1.2 (m, 22H), 0.90 (t, 3H, J = 6.6 Hz)
ppm. 13C NMR (100 MHz, CD3OD): δ 173.5, 130.9, 130.8, 85.7, 82.1,
77.4, 71.9, 62.9, 56.3, 33.6, 33.1, 30.9, 30.8, 30.8, 30.6, 30.5, 30.3, 30.2,
30.0, 28.2, 28.1, 23.8, 23.0, 14.5 ppm. HRMS (ESI) m/z calcd for
C26H49NO5S, 487.3341; found, 488.3415 (M + H)+.
4.2. In Vitro Activity of the Compounds: Inhibition of A549
and C6 Tumor Cell Proliferation. Human A549 line was cultured as
previously described.25 Rat glioma (C6 line) cells were maintained in
Dulbecco's modified Eagle's medium (DMEM) complete medium
(Sigma-Aldrich, St. Louis, MO), supplemented with fetal bovine serum
(FBS, 10%; GLinus, Madrid, Spain), glutamine (2 mM), penicillin (50
IU/mL), and streptomycin (50 mg/mL), at 37 ꢀC in a 5% CO2 humidified
atmosphere. Exponentially growing C6 cells were seeded on 96-well plates
(Beckton Dickinson, Le Pont de Claix, France), in complete DMEM
medium, at a density of 5 Â 104 cells/well. The cells were allowed to
attach with 5% of CO2 at 37 ꢀC for overnight, and then, the medium was
replaced by 100 μL of fresh medium. Stock solutions in EtOH of the
compounds 1À4 (100 mM) were finally dissolved in DMEM medium
containing EGF or basic FGF (10 ng/mL) for the treatments. The cells
were treated with serial dilutions of compounds (1À4) for 48 h. Cell
proliferation was evaluated with an MTT assay (Sigma-Aldrich), based
on the conversion of the water-soluble MTT into an insoluble formazan.
Briefly, after 48 h of compound treatment, the medium was replaced
by 100 μL of fresh DMEM without phenol red containing MTT
(5 mg/mL) solution, and cells were incubated for 3 h at 37 ꢀC with
5% CO2. After this time, the medium was removed, the precipitated
formazan was dissolved in 100 μL of DMSO, and the solution optical
density was measured at 595 nm in a Spectramax Plus (Molecular
Devices Corporation) for 96-well plates equipment. Absorption values
were referred to positive proliferation controls, cultured in DMEM
medium supplemented with EGF or basic FGF. Inhibition was expressed
as an ID50 value, the compound concentration that reduced maximal
proliferation by 50%.
4.4.3. Enzyme Assay. N-Acetyl-β-D-hexosaminidase activity was de-
termined using pNP-GlcNAc as substrate, by measuring the release of
p-nitrophenol (pNP) from pNP-GlcNAc. UV/Visible spectra were re-
corded at 405 nm on a SpectraMax Plus 384 spectrophotometer at
37 ꢀC. One unit was defined as that amount of enzyme that released
1 μmol p-nitrophenol minÀ1. Reaction mixtures consisted of 500 μL of
40 mM sodium phosphate buffer (pH 5) and 20% isopropanol, contain-
ing 1 mM of substrate (2 or 4) and 1 unit/ml of enzyme and were
incubated at 37 ꢀC (170 rpm in an orbital shaker). Fifty microliter
aliquots were collected at different time periods, varying between 0.25 and
48 h. The reaction was stopped by freezing at À80 ꢀC and then analyzed
by HPLC under the conditions described above. All experiments were
performed in triplicate. Product formation was calculated as % percentage
and expressed as the mean ( SEM (standard error of the mean).
4.5. Evaluation of the in Vivo Effect. The activity of compounds
1 and 3 on tumor growth was evaluated using an orthotopic model in
female nude mice (Foxn1nu/nu, 6À8 weeks old, Harlan Iberica, Barcelona,
Spain). Mice were maintained in the animalhouse of the Cajal Institute
(Madrid, Spain) with ad libitum food and water in a 12 h light/dark
cycle. Animals were handled complying with the European Union
guidelines for care and handling of experimental animals (86/609/
EEC), and the protocols were approved by the Cajal Institute animal
welfare committee. Mice were injected into a flank, sc, a suspension of 2 Â
106 cells of rat glioma C6. The tumor volume was calculated according to
the following formula: V (cm3) = (L Â W2 Â π)/6, where L is the length
and W is the width of the tumor, respectively. When tumors reached
250 mm3, the animals were treated with a daily intratumoral injection of
the compounds for 14 days. Stock solutions in DMSO of the compounds
(100 mM) were finally dissolved in PBS buffer containing fatty acid-free
BSA (5 mg/mL; Sigma-Aldrich, Steinheim, Germany) for the treatments.
Control animals received injections with the same solution of PBS buffer
with BSA and DMSO as other groups of animals treated with test
compounds. The animals were sacrificed by cervical dislocation after the
treatments, and the tumors were resected on ice until use. Animal weight
4.3. Liquid ChromatographyÀMass Spectrometry Analysis
of Glycosphingolipids (A549). The glycosphingolipids used as
internal standards were from Avanti Polar Lipids. A549 cells (2.5 Â
105 cells/mL) were seeded in 1 mL of medium with 10% FBS in 6-well
plates. Twenty-four hours later, media were replaced with fresh medium
containing test compound or vehicle solvent and cultured for 48 h at
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ARTICLE
was controlled to evaluate the general health state during treatments
(see the Supporting Information).
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4.6. 1H MAS NMR Spectroscopy. High-resolution proton MAS
NMR measurements were performed on a Bruker Avance 400 wide bore
(89 mm Ø) spectrometer (Bruker Instruments, Karlsruhe, Germany)
operating at 9.4 T (proton Larmor frecuency at 400.14 MHz). The
spectra were acquired at 20 ꢀC (293 K) using a Bruker double tuned
broad-band solid state CP/MAS probe head. After the in vivo experi-
ment, the animals were sacrificed, and the tumors were resected on ice.
The tumor samples were washed with deuterated phosphate-buffered
solution and placed into a 4 mm Ø zirconia rotor. The spectra were
acquired using a 90ꢀ pulse of 3.35 μs followed by a T2 filter of 60 ms. The
spinning rate was 3 kHz. The spectral width was 8.3 kHz, 16 k data
points, and the number of scans was 256 with a repetition rate of 5 s. The
residual water signal was suppressed by presaturation on the water
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chemical shifts were referenced to internal creatine (3.026 ppm).
’ ASSOCIATED CONTENT
S
Supporting Information. Glucosylceramide, lactosylcer-
b
amide, and ganglioside content in A549 cells as a function of the
acyl chain length and the number of insaturations; weight gain
curves of control and treated animals; and NMR spectra and
elemental analysis data of all new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
(14) Weiss, M.; Hettmer, S.; Smith, P.; Ladisch, S. Inhibition of
Melanoma Tumor Growth by a Novel Inhibitor of Glucosylceramide
Synthase. Cancer Res. 2003, 63, 3654–3658.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: +34 91 585 47 19. Fax: +34 91 585 47 54. E-mail: lromero@
cajal.csic.es (L.R.-R.). Tel: +34 91 562 29 00. Fax: +34 91 564 48 53.
E-mail: mayoralas@iqog.csic.es (A.F.-M.).
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co-solvent mixtures. Biotechnol. Tech. 1998, 12, 569–570.
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to biomedical studies. Prog. NMR Spectrosc. 2009, 55, 79–100.
(17) Sitter, B.; Bathen, T. F.; Tessem, M.-B.; Gribbestad, I. S. High
resolution magic angle spinning (HR MAS) MR spectroscopy in meta-
bolic characterization of human cancer. Prog. NMR Spectrosc. 2009,
54, 239–254.
Present Addresses
^Hospital Gregorio Mara~nꢀon, Maiquez, 9, 28007 Madrid, Spain.
’ ACKNOWLEDGMENT
ꢀ
(18) García-Alvarez, I.; Fernꢀandez-Mayoralas, A.; Garrido, L. Effect
The financial support provided by the Servicio de Salud de
Castilla La Mancha Community (SESCAM, PI2008/19), the
Ministry of of Science and Innovation (CTQ2007-67403/BQU
and CTQ2010-15418), the Comunidad de Madrid (S2009/
PPQ-1752), and Generalitat de Catalunya (Grant 2009SGR-
1072) is greatly appreciated. We thank Dr. Leoncio Garrido for
his help in obtaining MAS NMR spectra and Eva Dalmau for
excellent technical assistance.
of Drugs in Cells and Tissues by NMR Spectroscopy. Curr. Top. Med.
Chem. 2011, 11, 27–42.
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Nicholson, J. K.; Gr€ohn, O. H. J.; Kauppinen, R. A. High-resolution
magic-angle-spinning 1H NMR spectroscopy reveals different responses
in choline-containing metabolites upon gene therapy-induced pro-
grammed cell death in rat brain glioma. NMR Biomed. 2005, 18, 252–259.
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endogenous acidic metabolic products associated with carbohydrate
metabolism in tumor cells. Cell Biol. Toxicol. 2010, 26, 177–188.
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of 2-Amino-2-deoxy-1-thio-D-glucose. J. Org. Chem. 1962, 27, 1794–1800.
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Haynes, C. A.; Sullards, M. C.; Merril, A. H., Jr. Quantitative analysis of
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linear ion trap mass spectrometers. J. Lipid Res. 2009, 50, 1692–1707.
(25) Canals, D.; Mormeneo, D.; Fabriꢁas, G.; Llebaria, A.; Casas, J.;
Delgado, A. Synthesis and biological properties of Pachastrissamine
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17, 235–241.
’ ABBREVIATIONS USED
UPLC-MS, ultra performance liquid chromatographyÀmass
spectrometry; 1H MAS NMR, proton magic angle spining nuclear
magnetic resonance; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyltetrazolium bromide; ID50, 50% inhibitory dose
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