Design and synthesis of glycoside inhibitors of glioma and melanoma growth

Article abstract of DOI:10.1021/jm0611556

An N-acetylglucosaminide derivative with a pentaerythritol substituent at position C-6 was previously synthesized and shown to inhibit neural tumor growth. Now, we report the preparation of a series of new synthetic compounds introducing systematic change

Full text of DOI:10.1021/jm0611556

364
J. Med. Chem. 2007, 50, 364-373
Design and Synthesis of Glycoside Inhibitors of Glioma and Melanoma Growth
Isabel Garc´ıa-AÄ lvarez,† Guillermo Corrales,† Ernesto Doncel-Pe´rez,‡,§ Ana Mun˜oz,| Manuel Nieto-Sampedro,*,‡,§ and
Alfonso Ferna´ndez-Mayoralas*,†
Instituto de Qu´ımica Orga´nica General, CSIC, Juan de la CierVa 3, 28006 Madrid, Spain, Hospital Nacional de Paraple´jicos, SESCAM, 45071
Toledo, Spain, Instituto Cajal, CSIC, AVda. Doctor Arce 37, 28002 Madrid, Spain, and Laboratorios FAES FARMA, SA, Ma´ximo Aguirre 14,
Leioa, Vizcaya
ReceiVed October 5, 2006
An N-acetylglucosaminide derivative with a pentaerythritol substituent at position C-6 was previously
synthesized and shown to inhibit neural tumor growth. Now, we report the preparation of a series of new
synthetic compounds introducing systematic changes in the nature, polarity, and size of the sugar substituents.
The antimitotic activity of the new compounds was tested on cultured rat (C6) and human (U-373) glioma
lines and on a human melanoma line (A-375). The antimitotic and antitumoral activity of the new compounds
on glioma cell lines increased up to 2 orders of magnitude with respect to the parent compound or was
abolished, permitting a detailed structure-function analysis of the new antitumorals. One of the glycosides
inhibited melanoma division with an ID50 below the micromolar range.
Chart 1. Glycoside 1 Inhibited the Division of Human Glioma
Introduction
U-373 Cells in Culture10
Glioblastoma and melanoma are two types of tumors with a
high proliferative potential and poor prognosis. Only half of
the patients receiving standard treatment for brain tumor in the
United States survived 1 year after diagnosis.1 Melanoma
survival rate often depends on the severity of the melanoma
and whether or not metastases have occurred. Brain metastases
are a common feature of malignant melanoma, making the
prognosis of melanoma patients exceedingly poor.2 Therefore,
the development of new drugs able to control the proliferation
of glioblastoma and melanoma cells is essential for the treatment
of the diseases.
We have described the presence in brain extracts of inhibitors
of astroblast and astrocytoma division.3-5 The inhibitor had
glycidic epitopes immunologically related to those of the
epidermal growth factor receptor and of blood groups A, H, or
Lewix X.6 On the basis of these observations we synthesized a
family of oligosaccharides with Lewis X-type structure and
tested their activities as inhibitors of normal and transformed
neural cells division.7 The tetrasaccharide R-D-GalNAc(1,3)-â-
D-Gal(1,4)[R-L-Fuc(1,3)]-D-GlcNAc inhibited the proliferation
of rat C6 glioma cells in culture and the growth of brain tumors
formed after intracerebral transplantation of C6 cells.8 From the
results obtained with the oligosaccharide tested, the practical
synthesis of a second generation of differently substituted mono-
and disaccharides was carried out.9,10 We showed that an octyl
N-acetylglucosaminide with a pentaerythritol chain at position
6 (compound 1, Chart 1) inhibited the growth of a neuroecto-
dermic tumor implanted in rats.10 Compound 1 inhibited the
division of human glioma U-373 cells in culture, although with
a modest ID50 value (43 ( 14 µM). We report here the synthesis
of a new series of derivatives with improved antimitotic activity,
by systematic modifications of the substituents at positions 1,
2, 3, and 6 of the glucosamine backbone, and their effects on
inhibition of proliferation on rat (C6) and human (U-373) glioma
and human melanoma (A-375).
Results and Discussion
Chemistry. By acylation of the amine group of D-glu-
cosamine with acetic anhydride, pent-4-enoyl chloride, or oleoyl
chloride, the corresponding N-acylglucosamines (2-4, Scheme
1) were obtained. Subsequent Fischer-type glycosidation with
a variety of alcohols furnished the R-glycosides 5-13.
Compounds with a pentaerythritol chain were synthesized
from 10 and 11 by the sequence of reactions shown in Scheme
2. Thus, selective tritylation at HO-6 followed by benzylation
at HO-3 and HO-4 and detritylation afforded alcohols 14
and 15. Alkylation of 14 and 15 with cyclic sulfate 16 gave
17 and 18, which were submitted to hydrogenolysis of benzyl
and benzylidene groups to give sulfates 19 and 20. Finally,
acid hydrolysis of the sulfate group in 19 and 20 furnished 21
and 22.
In a similar manner, alcohols 14 and 15 were treated with
(R/S)-2,3-isopropylideneglyceryl tosylate to give 23 and 24
(Scheme 3), as a 1:1 mixture of epimers at C-2 of the glyceryl
chain. After acid hydrolysis and hydrogenolysis, compounds
27 and 28 were obtained. We also worked with pure epimers
(R)-24 and (S)-24 obtained from (R/S)-24 after column chro-
matography, which after deprotection led to (R)-28 and (S)-28.
The R configuration at C-2 of the glyceryl chain in (R)-24 could
be deduced by the alkylation of 14 with the pure enantiomer
(R)-2,3-isopropylideneglyceryl tosylate, which gave a compound
identical to (R)-24.
* To whom correspondence should be addressed. A.F.-M.: phone, 34
91 562 29 00; fax, 34 91 564 48 53; e-mail, mayoralas@iqog.csic.es.
M.N.-S.: phone, 34 91 585 47 20; fax, 34 91 585 47 54; e-mail,
mns@cajal.csic.es
† Instituto de Qu´ımica Orga´nica General, CSIC.
‡ Instituto Cajal, CSIC.
§ Hospital Nacional de Paraple´jicos, SESCAM.
| Laboratorios FAES FARMA, SA.
10.1021/jm0611556 CCC: $37.00 © 2007 American Chemical Society
Published on Web 12/22/2006

Glycoside Inhibitors of Glioma and Melanoma Growth
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 365
Scheme 3a
Scheme 1a
Reagents and conditions: (a) Na, MeOH or 1 N NaOH, (b) BF3‚OEt2,
130-165 °C.
Scheme 2a
Reagents and conditions: (a) NaH, DMF, (R/S)-2,2-dimethyl-1,3-
dioxolan-4-ylmethyl p-toluenesulfonate, 90 °C, (b) trifluoroacetic acid,
MeOH, rt, (c) 10% Pd/C, H2, MeOH.
derivatives 36-39, respectively, which were reacted with SO3-
pyridine complex leading, after neutralization with KOH, to the
targets 40-43 in excellent yields. It is worth noting that the
hydrolysis of the isopropylidene group took place during the
sulfation reaction probably through an intramolecular catalysis
by the recently introduced bisulfate group. Similarly, compounds
with a sulfate group at C-6 were synthesized by sulfation of
the acetals 44, 45, 46, obtained by reaction of glycosides 10,
12, and 13 with butane-2,3-dione. Acid hydrolysis of 47, 48,
and 49 led to 50, 51, and 52, respectively.
Biological Activity. The antimitotic activity of compounds
with substitutions at positions C-1 and C-2 of the glucosamine
backbone was tested on rat (C6) and human (U-373) glioma
and human melanoma (A-375) in cell culture. The results,
summarized in Table 1, show that compounds bearing a phenyl
ring at position C-1 (5-7, 10) or C-2 (29) were inactive in all
cell types. Much more encouraging were the inhibition results
of compounds with aliphatic chains at those positions, although
their activity depended on the chain length of the substituent
and the absence or presence of a double bond in the chain. Thus,
the octyl glycoside 8 was inactive in all cell types, the
hexadecanoyl derivative 9 was inhibitory for C6 and A-375 cell
lines, and the oleyl glycoside 12 showed the best antimitotic
activity of this family. Compound 13 with an N-oleoyl substitu-
tion at C-2 exhibited also good inhibitory activity, although with
higher ID50 values than 12.
Table 2 summarizes the antimitotic activities of the glycosides
substituted with a hydroxylated alkyl group at position C-6 of
the sugar. As previously discussed, an alkyl group was preferred
to a phenyl ring as indicated by the ID50 values of the related
pairs of compounds 20/19, 22/21, and 28/27. Interestingly, the
presence of a sulfate group at the pentaerythritol substituent in
19 and 20 improved efficacy with respect to the nonsulfated
derivatives 21 and 22. Similarly, the compound substituted with
a glyceryl chain 28 showed better antimitotic activity than the
Reagents and conditions: (a) (Ph)3CCl, Py, 75 °C, (b) NaH, THF, Bu4NI,
BnBr, 80 °C, (c) p-TsOH, 1:2 CH2Cl2-MeOH, rt, (d) NaH, THF-DMF,
16, 90 °C, (e) 10% Pd/C, H2, MeOH, (f) 1 M H2SO4, 3:1 dioxane-MeOH.
Modifications at C-2 were also achieved by acyl exchange
at the amido group of some of the synthesized glycosides using
different procedures. Thus, the treatment11 of 8 with Ba(OH)2
gave the amine, which after benzoylation afforded the benzoyl
glucosamine derivative 29 (Scheme 4), although in low overall
yield (45%). Alternatively, the pent-4-enoyl group in compound
26 was removed by treatment12 with iodine to give amine 30,
which was reacted with palmitoyl chloride to give 31, also in
low overall yield (30%), which after deprotection gave 32. Better
results were obtained in the N-transacylation reaction13 on 33,
previously prepared by peracetylation of 1, by treatment with
oleoyl chloride and DMAP under reflux in pyridine, affording
34 in 58% yield. Deprotection step on 34 led to 35.
Compounds bearing a sulfate group at C-3 position were
obtained as follows (Scheme 5). Glycosides 10-13 were treated
with 2,2-dimethoxypropane to give the 4,6-O-isopropylidene

366 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Garc´ıa-AÄ lVarez et al.
Scheme 4a
Scheme 5a
Reagents and conditions: (a) 2,2-dimethoxypropane, p-TsOH, DMF, rt,
(b) SO3-pyridine complex, Py, rt, (c) butane-2,3-dione, camphorsulfonic
acid, triethylorthoformate, EtOH, 60 °C, (d) SO3-pyridine complex, Py,
rt, (e) 2:1 acetic acid-H2O, 70 °C.
The mechanism of growth inhibition by the new compounds
is still unknown. It has been recently shown that substituted
aryl glycopyranosides cause selective inhibition of the DNA
synthesis that precedes induction of apoptosis and growth
inhibition of human glioblastoma cells.14 However, our
results show that the glycosides containing a phenyl ring were
inactive against the cell lines tested. An alternative mode of
action for the new glycosides to exert their effects could be by
altering the biosynthesis of tumor-associated gangliosides.15 This
type of glycosphingolipid has been shown to have crucial
regulatory roles in tumor onset and progression.16 The initial
step of all glucosylceramide (GlcCer)-based sphingolipids,
including gangliosides, is the coupling of ceramide and glucose
catalyzed by the enzyme glucosylceramide synthase. In this
context, two imino-sugars substituted with an alkyl substitu-
ent17,18 (NB-DNJ17 and OGT-237818) inhibit melanoma tumor
growth by inhibiting glucosylceramide synthase, and NB-DNJ
has also been proposed for brain cancer treatment.19 The
chemical structures of our compounds resemble that of GlcCer
and, therefore, they could be operating by inhibiting GlcCer
formation or, alternatively, by competing with GlcCer for the
galactosyltransferase that synthesizes lactosylceramide. On the
other hand, imino sugars derived from functionalized pyrro-
lidines that inhibit R-mannosidase activity have been shown to
have inhibitory properties for human glioblastoma and mela-
noma cells.20
Reagents and conditions: (a) Ba(OH)2, 1:2 H2O-MeOH, 90 °C, (b)
benzoic anhydride, Et3N, MeOH, rt, (c) I2, 1:1 H2O-THF, sodium
thiosulfate, (d) palmitoyl chloride, Py, CH2Cl2, rt, (e) 10% Pd/C, H2, THF,
rt, (f) oleoyl chloride, Py, 4-(dimethylamino)pyridine, reflux, (g) 1 M
NaOMe-MeOH, rt.
parent with a pentaerythritol group 22. The configuration of
the chiral center of the glyceryl chain has little effect on the
activity against U-373 and A-375 cell lines, since the epimers
(R)-28 or (S)-28 showed similar ID50 values.
The influence of a sulfate group at position C-3 or C-6 of
the pyranoid ring on the antimitotic activity was next studied
(Table 3). No significant differences in efficacy were observed
between the regioisomeric pairs 40/50, 42/51, and 43/52.
However, when comparing the activity of the oleyl glycoside
12 (Table 1) with the sulfated analogues 42 and 51, it was
observed that while the negatively charged group was detri-
mental for the antimitotic activity against C6 and U-373 glioma
cells, it led to an increase in activity on A-375 melanoma cell
cultures. It is worth noting that 51 was effective on the
melanoma cell line at a concentration below micromolar.
Normal stromal cells derived from rat bone marrow were less
sensitive to inhibition than glioma and melanoma cells after
exposure to the best inhibitors, 12 and 51. The growth of stromal
cells was inhibited at slightly higher concentration of 12
(ID50 ) 10 µM) and at higher concentration of 51 (ID50 ) 40
µM) than glioma and melanoma cells (see Tables 1 and 3,
respectively). Therefore, some concentration-dependent selectiv-
ity toward tumoral cells versus nontumoral cells was shown by
the glycosides.
In conclusion, we have evaluated a series of new substituted
N-acyl-R-D-glucoamininide derivatives as inhibitors of pro-
liferation of glioma and melanoma cell lines. The results
obtained indicate that the most inhibitory molecules have an
oleyl group or a long alkyl chain at position C-1 of the

Glycoside Inhibitors of Glioma and Melanoma Growth
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 367
Table 1. Inhibition of Rat Glioma (C6), Human Glioma (U-373), and Human Melanoma (A-375) Cell Cultures by Compounds 5-13, 29
ID50 (µM)
U-373
compd
R1
R2
C6
A-375
5
6
7
8
9
10
11
12
13
29
CH2Ph
CH3
CH3
CH3
CH3
CH3
(CH2)2CHdCH2
(CH2)2CHdCH2
CH3
>50
>50
>50
(CH2)2Ph
(CH2)4Ph
(CH2)7CH3
(CH2)15CH3
(CH2)2Ph
(CH2)15CH3
(CH2)8CHdCH(CH2)7CH3
(CH2)7CH3
(CH2)7CH3
>50
>50
>50
>50
>50
>50
>50
>50
>50
13.0 ( 3.7
>50
>50
29.3 ( 5.2
>50
>50
>50
>50
>50
a
1.3 ( 0.8
12.0 ( 4.6
>50
4.6 ( 2.2
13.3 ( 4.1
>50
4.8 ( 2.7
6.0 ( 1.0
>50
a
(CH2)7CHdCH(CH2)7CH3
Ph
a The central double bond is cis-configured.
Table 2. Influence of a Hydroxylated Alkyl Chain at Position C-6 of the Glycoside
ID50 (µM)
compd
R1
R2
C6
U-373
A-375
19
20
21
22
27
28
(R)-28
(S)-28
32
(CH2)2Ph
(CH2)15CH3
(CH2)2Ph
(CH2)15CH3
(CH2)2Ph
(CH2)15CH3
(CH2)15CH3
(CH2)15CH3
(CH2)15CH3
(CH2)7CH3
(CH2)3CH3
(CH2)3CH3
(CH2)3CH3
(CH2)3CH3
(CH2)3CH3
(CH2)3CH3
(CH2)3CH3
(CH2)3CH3
(CH2)14CH3
8.6 ( 5.6
11.0 ( 6.0
>50
6.7 ( 0.7
3.2 ( 0.2
>50
9.3 ( 1.7
2.3 ( 0.3
>50
18.3 ( 7.2
>50
27.3 ( 3.7
>50
17.0 ( 2.0
>50
NDa
4.6 ( 0.3
4.9 ( 0.6
5.6 ( 1.7
3.3 ( 0.8
20.6 ( 5.2
3.0 ( 0.5
2.2 ( 0.2
3.4 ( 0.3
9.3 ( 0.6
13.3 ( 3.3
NDa
NDa
2.6 ( 1.2
11.9 ( 4.6
b
35
(CH2)7CHdCH(CH2)7CH3
a ND means not determined. b The central double bond is cis-configured.
Table 3. Influence of a Sulfate Group at Position C-3 or C-6 of the Glycoside
ID50 (µM)
U-373
>50
33.0 ( 3.5
9.6 ( 1.2
15.6 ( 4.3
>50
13.0 ( 3.0
12.6 ( 7.7
compd
R1
R2
C6
A-375
40
41
42
43
50
51
52
(CH2)2Ph
(CH2)15CH3
(CH2)2CHdCH2
(CH2)2CHdCH2
CH3
(CH2)7CHdCH(CH2)7CH3
(CH2)2CHdCH2
CH3
>50
>50
22.6 ( 3.7
12.3 ( 0.3
5.4 ( 2.5
>50
7.2 ( 6.3
2.1 ( 0.6
16.0 ( 2.0
>50
a
a
(CH2)8CHdCH(CH2)7CH3
(CH2)7CH3
a
a
(CH2)2Ph
(CH2)8CHdCH(CH2)7CH3
(CH2)7CH3
13.0 ( 3.5
5.6 ( 1.7
0.6 ( 0.3
15.6 ( 2.8
(CH2)7CHdCH(CH2)7CH3
a The central double bond is cis-configured.
glucosamine backbone. Additionally, the activity can be modu-
lated, making the compounds more inhibitory on some of the
cell lines tested, by introducing a hydroxylated alkyl chain or a
sulfate group at position C-6. The present work has provided
some compounds exhibiting ID50 values below micromolar
concentration.

368 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Garc´ıa-AÄ lVarez et al.
172.42, 139.27, 128.84, 128.25, 126.10, 97.28, 72.64, 71.73, 71.09,
68.47, 61.49, 54.30, 35.66, 21.56. MS (ES) m/z (calcd 325.2): 326.3
(M + 1), 327.2 (M + 2). Anal. (C16H23NO6) C, H, N.
Experimental Section
Chemistry. General Methods. Chemicals were purchased puriss
p.A. from commercial suppliers or purified by standard techniques.
Solvents were distilled over drying agents: dimethylformamide,
BaO; dichloromethane, CaH2; tetrahydrofuran, sodium/benzophe-
none ketyl; acetonitrile, CaH2; and pyridine, BaO. Thin-layer
chromatography (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 chroma-
tography was performed using thick-walled columns, employing
silica gel (Merck 60, 0.040-0.063 mm). The eluent used is
indicated, and solvent ratios refer to volume. Melting points are
not corrected. Optical rotations were recorded on a Perkin-Elmer
Phenylbutyl 2-N-Acetyl-2-amino-2-deoxy-r-D-glucopyrano-
side (7). To a mixture of 2 (500 mg, 2.26 mmol) and 4-phenyl-1-
butanol (1.5 mL, 9.73 mmol) was added BF3‚OEt2 (50 µL) under
Ar. The mixture was stirred at 165 °C for 10 min, cooled at room
temperature, and purified by column chromatography (CH2Cl2-
methanol, 10:1) to provide 7 (380 mg, 47%) as a white solid. Mp:
161-162 °C. [R]D: +135° (c 1.04, MeOH). 1H NMR (300 MHz,
CD3OD): δ 7.2-7.1 (m, 5H), 4.73 (d, 1H, J ) 3.4 Hz), 3.8-3.5
(m, 6H), 3.4-3.3 (m, 2H), 2.60 (t, 2H, J ) 7.3 Hz), 1.90 (s, 3H),
1.7-1.6 (m, 4H). 13C NMR (75 MHz, CD3OD): δ 172.48,
142.49, 128.29, 128.18, 125.62, 97.29, 72.60, 71.64, 71.25,
67.59, 61.60, 54.40, 35.46, 28.95, 28.95, 28.08, 21.51. MS (ES)
m/z (calcd 353.2): 354.2 (M + 1), 355.2 (M + 2). Anal. (C18H27-
NO6) C, H, N.
Octyl 2-N-Acetyl-2-amino-2-deoxy-r-D-glucopyranoside (8).
To a solution of 2 (10 g, 45.2 mmol) in nitromethane (350 mL)
were added octanol (21.3 mL, 135 mmol) and BF3‚OEt2 (0.91 mL)
under Ar. The mixture was heated at 100 °C for 2 h and then cooled
at room temperature until a precipitate appeared. The solid was
filtered off, the filtrate was concentrated, and the residue was
purified by column chromatography (EtOAc-methanol, 10:1f6:
1) to give 8 (3.43 g, 40%). [R]D: +142° (c 1.05, MeOH). 1H NMR
(200 MHz, CD3OD): δ 4.74 (d, 1H, J ) 3.5 Hz), 3.9-3.5 (m,
8H), 3.4-3.3 (m, 5H), 1.94 (s, 3H), 1.6-1.5 (m, 2H), 1.4-1.3 (m,
12H), 0.87 (t, 3H, J ) 6.6 Hz). 13C NMR (50 MHz, CD3OD): δ
172.42, 97.27, 72.57, 71.64, 71.24, 61.58, 54.40, 48.46, 31.85,
29.37, 29.30, 29.27, 26.13, 22.55, 21.49, 13.29. MS (ES) m/z
(calcd 333.2): 334.2 (M + 1), 334.2 (M + 2). Anal. (C16H31NO6)
C, H, N.
1
241 Polarimeter (λ ) 589 nm, 1-dm cell). H NMR spectra were
registered at 400, 300, or 200 MHz, and 13C NMR were obtained
at 100, 75, or 50 MHz using CDCl3, CD3OD, D2O, or DMSO as
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:
s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet). Mass
spectroscopy spectra were registered on a HP series 1100 MSD
spectrometer.
2-Amino-2-deoxy-2-N-(pent-4-enoyl)-r,â-D-glucopyranose (3).
To a solution of Na (1.95 g, 0.08 mmol) in methanol (40 mL) was
added D-glucosamine hydrochloride (18.45 g, 85.5 mmol). The
mixture was slowly stirred for 3 min and the solid was filtered off.
The filtrate was treated with 4-pentenoic anhydride (15 mL, 82.1
mmol) for 3 min and the solid was filtered and washed with Et2O-
methanol (1:1) to give 3 (17.69 g, 79%).
2-Amino-2-deoxy-2-N-oleoyl-r,â-D-glucopyranose (4). Oleyl
chloride was added dropwise to a cooled solution (-10 °C) of
D-glucosamine hydrochloride (10 g, 50 mmol) in 1 N NaOH (51
mL). The temperature was allowed to gradually rise to room
temperature with stirring for 2 h. After this time, the mixture was
filtered off and the filtrate was washed with ethanol and ether. The
solid was crystallized from ethanol to give 4 (15.4 g, 70%) as a
solid. Mp: 159-161 °C. 1H NMR (300 MHz, DMSO): δ 7.63 (d,
<1H, J ) 8.1 Hz), 7.48 (d, <1H, J ) 7.8 Hz), 6.44 (d, <1H, J )
6.6 Hz), 6.37 (d, <1H, J ) 4.6 Hz), 5.4-5.3 (m, 2H), 4.53 (dd,
1H, J ) 5.4 Hz, J ) 6.6 Hz), 4.41 (t, 1H, J ) 5.4 Hz), 3.7-3.2
(m, 4H), 3.1-3.0 (m, 1H), 2.1-1.9 (m, 7H), 1.5-1.4 (m, 2H), 1.3-
1.1 (m, 20H), 0.84 (t, 3H, J ) 6.3 Hz). 13C NMR (75 MHz, CD3-
OD): δ 130.37, 130.33, 130.29, 91.32, 72.72, 71.90, 71.17, 61.88,
54.93,39.36, 35.98, 31.95, 29.86, 29.78, 29,51, 29.46, 29.35, 29.35,
29.27, 29.16, 27.33, 27.26, 25.99, 22.76, 14.61. MS (ES) m/z (calcd
443.3): 444.4 (M + 1).
Hexadecanoyl 2-N-Acetyl-2-amino-2-deoxy-r-D-glucopyra-
noside (9). A mixture of 2 (1.0 g, 4.52 mmol) and 1-hexadecanol
(5.48 g, 22.60 mmol) was heated at 160 °C under Ar and BF3‚
OEt2 (100 µL) was added. The reaction was stirred at 160 °C for
15 min, cooled at room temperature, and diluted with CH2Cl2 (25
mL). Hexane was added, and the precipitate was collected and
crystallized from Et2O to give 9 (271.9 mg, 14%) as a white solid.
1
Mp: 159-161 °C. [R]D: +108° (c 1.01, MeOH). H NMR (200
MHz, CD3OD): δ 4.86 (d, 1H, J ) 3.5 Hz), 4.0-3.6 (m, 8H),
3.5-3.4 (m, 3H), 2.06 (s, 3H), 1.7-1.6 (m, 2H), 1.5-1.4 (m, 26H),
0.98 (t, 3H, J ) 6.2 Hz). 13C NMR (50 MHz, CD3OD): δ 173.49,
98.30, 73.57, 72.66, 72.22, 68.84, 62.60, 55.41, 32.99, 30.72,
30.53, 30.48, 30.40, 27.23, 23.66, 22.61, 14.65. MS (ES) m/z
(calcd 445.3): 446.5 (M + 1), 447.5 (M + 2). Anal. (C24H47NO6)
C, H, N.
Phenylethyl 2-Amino-2-deoxy-2-N-(pent-4-enoyl)-r-D-glu-
copyranoside (10). A mixture of 3 (7.5 g, 28.7 mmol), 2-phenyle-
thanol (20.6 mL, 168 mmol), and BF3·OEt2 (0.75 mL) was heated
at 100 °C under Ar for 10 min, cooled at room temperature, and
purified by column chromatography (CH2Cl2-methanol, 10:1) to
give 10 (5.5 g, 52%). [R]D: +131° (c 1.05, MeOH). 1H NMR (300
MHz, CD3OD): δ 7.3-7.1 (m, 5H), 5.8-5.7 (m, 1H), 5.0-4.9
(m, 2H), 4.70 (d, 1H, J ) 3.4 Hz), 3.9-3.5 (m, 6H), 3.4-3.3 (m,
2H), 2.9-2.8 (m, 2H), 2.3-2.1 (m, 4H). 13C NMR (75 MHz, CD3-
OD): δ 174.94, 139.25, 137.19, 128.33, 128.27, 126.13, 114.61,
97.38, 72.65, 71.69, 71.09, 68.59, 61.46, 54.20, 35.72, 35.10, 29.65.
MS (ES) m/z (calcd 365.2): (M + 1), (M + 2). Anal. (C19H27-
NO6) C, H, N.
Benzyl 2-N-Acetyl-2-amino-2-deoxy-r-D-glucopyanoside (5).
To a mixture of 2 (752 mg, 3.39 mmol) and benzyl alcohol (2.80
mL, 27.04 mmol) was added BF3‚OEt2 (75 µL) under inert
atmosphere. The mixture was stirred at 130 °C for 40 min, cooled
at room temperature, and purified by column chromatography (CH2-
Cl2-methanol, 10:0f10:1) to give 5 (402 mg, 38%). [R]D: +203°
1
(c 1.12, MeOH). H NMR (300 MHz, CD3OD): δ 7.4-7.3 (m,
5H), 5.44 (d, 1H, J ) 2.4 Hz), 4.8-4.7 (m, 2H), 4.70 (dd, 1H, J
) 2.2 Hz, J ) 12.2 Hz), 4.45 (dd, 1H, J ) 2.4 Hz, J ) 12.2 Hz),
3.9-3.8 (m, 2H), 3.7-3.6 (m, 3H), 3.4-3.3 (m, 1H), 1.90 (s, 3H).
13C NMR (75 MHz, CD3OD): δ 172.45, 137.85, 128.24, 128.09,
127.69, 96.40, 72.91, 71.55, 71.26, 69.02, 61.58, 54.26, 21.43. MS
(ES) m/z (calcd 311.2): 312.2 (M + 1), 313.2 (M + 2). Anal.
(C15H21NO6) C, H, N.
Hexadecanoyl 2-Amino-2-deoxy-2-N-(pent-4-enoyl)-r-D-glu-
copyranoside (11). A mixture of 3 (7 g, 28.7 mmol), hexadecanol
(32.47, 140 mmol), and BF3·OEt2 (0.64 mL) was heated at 120 °C
under Ar for 30 min, cooled at room temperature, and purified by
column chromatography (CH2Cl2-methanol, 1:0f10:1) to give 11
(4.40 g, 34%) as a white solid after recrystallization from methanol.
Phenylethyl 2-N-Acetyl-2-amino-2-deoxy-r-D-glucopyrano-
side (6). To a mixture of 2 (500 mg, 2.26 mmol) and 2-phenyle-
thanol (1.35 g, 22.60 mmol) was added BF3‚OEt2 (50 µL). The
mixture was heated at 165 °C under Ar for 5 min, cooled at room
temperature, and purified by column chromatography (CH2Cl2-
methanol, 10:1) to give 6 (360 mg, 49%). [R]D: +153° (c 1.16,
1
Mp: 160-166 °C. [R]D: +99° (c 1.03, MeOH). H NMR (400
1
MeOH). H NMR (200 MHz, CD3OD): δ 7.3-7.2 (m, 5H), 4.74
MHz, CD3OD): δ 6.0-5.8 (m, 1H), 5.1-4.8 (m, 2H), 4.79 (d,
1H, J ) 3.9 Hz), 3.9-3.6 (m, 4H), 3.4-3.3 (m, 4H), 2.4-2.3 (m,
4H), 1.7-1.6 (m, 2H), 1.4-1.3 (m, 26H), 0.92 (t, 3H, J ) 5.9
(d, 1H, J ) 3.7 Hz), 3.9-3.5 (m, 6H), 3.4-3.3 (m, 2H), 2.88 (t,
2H, J ) 6.6 Hz), 1.89 (s, 3H). 13C NMR (50 MHz, CD3OD): δ

Glycoside Inhibitors of Glioma and Melanoma Growth
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 369
Hz). MS (ES) m/z (calcd 485.3): 486.3 (M + 1). Anal. (C27H51-
NO6) C, H, N.
(m, 5H), 3.8-3.6 (m, 8H), 2.4-2.1 (m, 4H), 1.6-1.5 (m, 2H), 1.3-
1.2 (m, 26H), 0.9-0.8 (t, 3H, J ) 6.1 Hz).
Oleyl 2-Amino-2-N-acetyl-2-deoxy-r-D-glucopyranoside (12).
To a mixture of 2 (1 g, 4.52 mmol) and 85% oleyl alcohol (6 mL)
was added BF3·OEt2 (0.91 mL, 7.2 mmol). The reaction mixture
was stirred at 100 °C under Ar for 2 h, cooled at room temperature,
and concentrated under reduce pressure. The crude was purified
by column chromatography (EtOAc-methanol, 10:0f10:1) to give
12 (200 mg, 10%). [R]D: +103.1° (c 0.95, MeOH). 1H NMR (300
MHz, CD3OD): δ 5.4-5.3 (m, 2H), 4.74 (d, 1H, J ) 3.5 Hz),
3.9-3.8 (m, 2H), 3.7-3.5 (m, 4H), 3.4-3.3 (m, 2H), 2.0-1.9 (m,
4H), 1.94 (s, 3H), 1.6-1.5 (m, 2H), 1.3-1.2 (m, 22H), 0.86 (t,
3H, J ) 7.2 Hz). 13C NMR (75 MHz, CD3OD): δ 172.41, 129.70,
129.66, 97.29, 72.55, 71.65, 71.22, 67.79, 61.56, 54.39, 32.63,
32.07, 31.92, 31.86, 31.81, 31.71, 31.62, 31.45, 31.41, 31.36, 31.30,
29.19, 28.33, 22.56, 21.49, 13.33. MS (ES) m/z (calcd 471.4): 472.4
(M + 1), 473.4 (M + 2). Anal. (C26H49NO6) C, H, N.
Phenylethyl 2-Amino-3,4-di-O-benzyl-2-deoxy-6-O-(2-(p-meth-
oxybenzilidene)-3-hydroxy-3-O-(oxosulfonyl)propyl)-2-N-(pent-
4-enoyl)-r-D-glucopyranoside (17). To a solution of 14 (850 mg,
1.56 mmol) in a mixture of THF (34 mL) and DMF (3 mL) was
added NaH (77 mg, 3.21 mmol) under Ar, with stirring for 10 min.
When H2 evolution had ceased, 16 (761 mg, 2.41 mmol) was added.
The reaction mixture was stirred at 90 °C for 80 min and then
cooled at room temperature. The excess of NaH was quenched by
addition of methanol (0.5 mL), and then the solvent was removed
and the residue purified by column chromatography (EtOAc-
methanol, 1:0f10:1) to give 17 (1.31, 95%). [R]D: +61.34° (c
1.00, MeOH). 1H NMR (300 MHz, CD3OD): δ 7.88 (s, 1H), 7.3-
7.2 (m, 15H), 7.1-7.0 (m, 2H), 6.77 (d, 2H, J ) 8.8 Hz), 5.8-5.6
(m, 1H), 5.2-5.1 (m, 1H), 4.9-4.5 (m, 12 H), 4.27 (s, 2H), 4.0-
3.9 (m, 2H), 3.8-3.7 (m, 1H), 3.6-3.2 (m, 6H), 2.9-2.8 (m, 3H),
2.7-2.6 (m, 2H), 2.2-2.1 (m, 4H).
Phenylethyl 2-Amino-2-deoxy-6-O-[2,2-bis(hydroxymethyl)-
3-hydroxy-3-O-(oxosulfonyl)propyl)]-2-N-penthanoyl-r-D-glu-
copyranoside (19). 17 (310 mg, 0.35 mmol) was dissolved in
methanol (14 mL), and 10% Pd/C (150 mg) was added. The
suspension was stirred under H2 for 3 h. After this time, the solution
was filtered through Celite and washed with EtOAc. Removal of
the solvent gave 19 (180 mg, 87%). [R]D: +77° (c 1.39, MeOH).
1H NMR (200 MHz, CD3OD): δ 7.3-7.2 (m, 5H), 4.74 (d, 1H, J
) 3.5 Hz), 4.1-4.4 (m, 2H), 3.9-3.8 (m, 2H), 3.6-3.3 (m, 12H),
2.89 (t, 2H, J ) 7.1 Hz), 2.18 (t, 2H, J ) 7.9 Hz), 1.6-1.5 (m,
2H), 1.4-1.2 (m, 2H), 0.91 (t, 3H, J ) 7.5 Hz). 13C NMR (75
MHz, CD3OD): δ 176.80, 140.42, 130.00, 129.41, 127.27, 98.48,
72.81, 72.59, 72.10, 71.68, 71.50, 69.88, 68.15, 62.47, 62.38, 55.13,
46.57, 36.83, 36.74, 29.06, 23.31, 14.19. MS (ES) m/z (calcd
587.2): 605.2, 604.2. Anal. (C24H38NNaO12S) C, H, N, S.
Octyl 2-Amino-2-deoxy-2-N-(oleoyl)-r-D-glucopyranoside (13).
To a mixture of 4 (500 mg, 1.13 mmol) and octanol (21.3 mL, 135
mmol) was added BF3·OEt2 (30 µL). The reaction mixture was
heated at 110 °C under Ar for 5 min, cooled at room temperature,
and purified by column chromatography (EtOAc-methanol, 1:0f10:
1
1) to give 13 (308 g, 50%). [R]D: +85.1° (c 1.37, MeOH). H
NMR (300 MHz, CD3OD): δ 5.4-5.3 (m, 2H), 4.78 (d, 1H, J )
3.4 Hz), 3.9-3.5 (m, 5H), 3.4-3.3 (m, 3H), 2.3-2.2 (m, 2H), 2.1-
2.0 (m, 4H), 1.7-1.5 (m, 4H), 1.3-1.2 (m, 30H), 0.9-0.8 (m, 6H).
13C NMR (75 MHz, CD3OD): δ 178.61, 130.90, 130.81, 98.44,
73.78, 72.70, 72.47, 69.02, 62.78, 55.53, 37.10, 33.11, 33.10, 30.92,
30.88, 30.83, 30.67, 30.54, 30.50, 30.47, 30.39, 30.36, 30.29, 28.23,
28.20, 27.43, 27.16, 23.79, 23.70, 14.52, 14.50. MS (ES) m/z (calcd
555.4): 556.5 (M + 1). Anal. (C32H61NO6) C, H, N.
Phenylethyl 2-Amino-3,4-di-O-benzyl-2-deoxy-2-N-(pent-4-
enoyl)-r-D-glucopyranoside (14). A solution of 10 (1.20 g, 3.31
mmol) in pyridine (7.5 mL) was heated at 75 °C under Ar and
triphenylmethyl chloride (2.77 g, 9.93 mmol) was added. The
reaction mixture was stirred at this temperature for 30 min and
then cooled at room temperature. Evaporation of the solvent and
purification by silica gel column chromatography (EtOAc-
methanol, 1:0f10:1) gave a residue (2 g) which was dissolved in
anhydrous THF (18 mL) and cooled at 0 °C. Then, NaH (253 mg,
10.56 mmol) was added under Ar and the temperature was allowed
to gradually rise to room temperature with stirring over 15 min.
When H2 evolution had ceased, tetrabutylamonium iodide (0.60 g,
1.62 mmol) and benzyl bromide (0.87 mL, 6.92 mmol) were added.
The reaction mixture was stirred at 80 °C for 2 h and then cooled
at room temperature. The excess of NaH was removed by addition
of methanol (0.5 mL). Then, CH2Cl2 (100 mL) was added and the
mixture was washed with water (40 mL × 3). The organic layer
was separated, dried (Na2SO4), filtered, and concentrated, affording
a brown crude. The crude product without purification was dissolved
in a mixture of CH2Cl2-methanol (1:2, 40 mL), and p-toluene-
sulfonic acid (160 mg, 0.84 mmol) was added. The reaction mixture
was stirred at room temperature for 3 h and then neutralized with
triethylamine, and the solvent was removed under reduced pressure.
The residue was purified by column chromatography (EtOAc-
hexane, 1:1) to give 14 (64%, three steps) as a white solid after
recrystallization from EtOAc (minimum volume) and hexane. [R]D:
+73.1° (c 1.00, MeOH). 1H NMR (300 MHz, CD3OD): δ 7.6-
7.2 (m, 15H), 5.8-5.7 (m, 1H), 4.8-4.6 (m, 2H), 5.0-4.9 (m, 5H),
4.10 (dd, 1H, J ) 10.5 Hz, J ) 3.9 Hz), 3.9-3.5 (m, 6H), 3.5-3.4
(m, 1H), 2.92 (t, 2H, J ) 6.3 Hz), 2.3-2.2 (m, 4H). 13C NMR (75
MHz, CD3OD): δ 175.32, 140.45, 140.09, 139.81, 138.27, 130.07,
129.48, 129.37, 129.33, 128.82, 128.69, 128.60, 127.35, 115.83,
98.67, 81.47, 79.57, 75.82, 75.75, 73.16, 69.66, 62.02, 54.60, 36.88,
36.35, 30.79.
Hexadecanoyl 2-Amino-2-deoxy-6-O-[3-hydroxy-2,2-bis(hy-
droxymethyl)-3-O-(oxosulfonyl)propyl]-2-N-penthanoyl-r-D-glu-
copyranoside (20). Compound 15 was reacted under similar
conditions as described for 14, to give 18 (87%). 18 (550 mg, 0.55
mmol) was dissolved in a mixture of methanol-EtOAc (8:1, 45
mL), and 10% Pd/C (230 mg) was added. The suspension was then
stirred under H2 for 3 h. After this time, the solution was filtered
through Celite and washed with EtOAc. Removal of the solvent
and purification of the residue by column chromatography (CH2-
Cl2-methanol, 5:1) gave 20 (280 mg, 72%). [R]D: +78.6° (c 1.03,
1
MeOH). H NMR (200 MHz, CD3OD): δ 4.77 (d, 1H, J ) 3.5
Hz), 4.06 (s, 2H), 3.90 (dd, 1H, J ) 3.5 Hz, J ) 10.6 Hz), 3.8-
3.4 (m, 11H), 2.26 (t, 2H, J ) 7.5 Hz), 1.7-1.2 (m, 32H), 1.0-
0.8 (m, 6H). MS (ES) m/z (calcd 707.3): 724.3, 725.3. Anal.
(C32H62NNaO12S) C, H, N, S.
Phenylethyl 2-N-Penthanoyl-2-amino-2-deoxy-6-O-(2,2-bis-
(hydroxymethyl)-3-hydroxypropyl)-r-D-glucopyranoside (21).
19 (180 mg, 0.31 mmol) was dissolved in a mixture of dioxane-
methanol (3:1, 12 mL) and a solution of 1 M H2SO4 (30 µL) was
added. The reaction mixture was stirred for 7 h. After this time,
the mixture was neutralized by addition of saturated NaHCO3
solution. Removal of the solvent under reduced pressure and
purification of the residue by column chromatography (CH2Cl2-
methanol, 5:1) gave 21 (95 mg, 66%). [R]D: +89° (c 0.82, MeOH).
1H NMR (400 MHz, D2O): δ 7.2-7.1 (m, 5H), 4.74 (d, 1H, J )
3.5 Hz), 3.8-3.2 (m, 16H), 2.8-2.7 (t, 2H, J ) 5.9 Hz), 2.1-2.0
(t, 2H, J ) 7.7 Hz), 1.4-1.3 (m, 2H), 1.2-1.0 (m, 2H), 0.72 (t,
2H, J ) 7.1 Hz). 13C NMR (50 MHz, CD3OD): δ 176.65, 140.42,
130.05 (2C), 129.47 (2C), 127.34, 98.58, 72.66 (2C), 72.40, 72.34,
71.90, 69.96, 63.20 (2C), 55.35, 46.99, 36.94, 36.77, 29.12, 23.38,
14.24. MS (ES) m/z (calcd 485.3): 486.3 (M + 1). Anal. (C24H39-
NO9) C, H, N.
Hexadecanoyl 2-Amino-3,4-di-O-benzyl-2-deoxy-2-N-(pent-
4-enoyl)-r-D-glucopyranoside (15). Compound 11 was reacted
under similar conditions as described for 10, to give 15 (73%).
[R]D: +49.9° (c 1.00, MeOH). H NMR (300 MHz, CD3OD): δ
7.4-7.3 (m, 10H), 5.8-5.7 (m, 1H), 5.4-5.3 (m, 2H), 5.1-4.7
Hexadecanoyl 2-Amino-2-deoxy-2-N-penthanoyl-6-O-(2,2-bis-
(hydroxymethyl)-3-hydroxypropyl)-r-D-glucopyranoside (22).
20 (147 mg, 0.21 mmol) was dissolved in a mixture of dioxane-
methanol (1:1, 4 mL), and a solution of 1 M H2SO4 (30 µL) was
added. The reaction mixture was stirred for 20 h. After this time,
1

370 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Garc´ıa-AÄ lVarez et al.
the solution was neutralized by addition of saturated NaHCO3
solution. Removal of the solvent and purification of the residue by
column chromatography (CH2Cl2-methanol, 10:1f5:1) gave 22
+82° (c 1, MeOH). 1H NMR (300 MHz, CD3OD): δ 4.97 (d, 1H,
J ) 3.7 Hz), 4.05 (dd, 1H, J ) 11.0 Hz, J ) 3.5 Hz), 4.0-3.8 (m,
6H), 3.8-3.5 (m, 6H), 2.44 (t, 2H, J ) 6.2 Hz), 1.8-1.7 (m, 4H),
1.6-1.4 (m, 28H), 1.2-1.0 (m, 6H). MS (ES) m/z (calcd 561.4):
562.3 (M + 1), 563.3 (M + 2). Anal. (C30H59NO8) C, H, N.
Compound (R)-28 and (S)-28 were prepared from (R)-26 and
(S)-26, respectively, using the same procedure as described above
for the preparation of (R/S)-28 from (R/S)-26.
1
(126 mg, 100%). [R]D: +108° (c 1.01, MeOH). H NMR (200
MHz, CD3OD): δ 4.77 (d, 1H, J ) 3.5 Hz), 3.86 (dd, 1H, J ) 3.5
Hz, J ) 10.6 Hz), 3.8-3.3 (m, 13H), 2.24 (t, 2H, J ) 7.5 Hz),
1.7-1.2 (m, 32H), 1.1-0.9 (m, 6H). MS (ES) m/z (calcd 605.5):
606.3 (M + 1), 607.5 (M + 2). Anal. (C32H63NO9) C, H, N.
Phenylethyl 2-Amino-3,4-di-O-benzyl-2-deoxy-6-O-(2,2-dim-
ethyl-1,3-dioxolan-4(R/S)-ylmethyl)-2-N-(pent-4-enoyl)-r-D-glu-
copyranoside ((R/S)-23). To a solution of 14 (100 mg, 0.18 mmol)
in anhydrous DMF (1.53 mL) was added NaH (18 mg, 0.75 mmol)
under Ar, with stirring for 10 min. When H2 evolution had ceased,
(R/S)-2,2-dimethyl-1,3-dioxolan-4-ylmethyl p-toluenesulfonate (761
mg, 2.41 mmol) was added. The reaction mixture was stirred at 90
°C for 80 min and then cooled at room temperature. The excess of
NaH was quenched by addition of methanol (0.5 mL). Removal of
the solvent and purification of the residue by column chromatog-
(S)-Hexadecanoyl 2-Amino-2-deoxy-6-O-(2,3-dihydroxypro-
pyl)-2-N-pentanoyl-r-D-glucopyranoside ((S)-28). Yield: 100%.
1
[R]D: +50° (c 0.93, MeOH). H NMR (300 MHz, CD3OD): δ
4.97 (d, 1H, J ) 3.7 Hz), 4.05 (dd, 1H, J ) 11.0 Hz, J ) 3.5 Hz),
4.0-3.8 (m, 6H), 3.8-3.5 (m, 6H), 2.44 (t, 2H, J ) 6.2 Hz), 1.8-
1.7 (m, 4H), 1.6-1.4 (m, 28H), 1.2-1.0 (m, 6H). MS (ES) m/z
(calcd 561.4): 562.3 (M + 1), 563.3 (M + 2). Anal. (C30H59NO8)
C, H, N.
(R)-Hexadecanoyl 2-Amino-2-deoxy-6-O-(2,3-dihydroxypro-
pyl)-2-N-pentanoyl-r-D-glucopyranoside ((R)-28). Yield: 100%.
1
raphy (EtOAc-methanol, 2:1) gave (R/S)-23 (85 mg, 70%). H
1
Mp 91-97 °C. [R]D: +82° (c 1, MeOH). H NMR (300 MHz,
NMR (300 MHz, CDCl3): δ 7.4-7.2 (m, 13H), 7.2-7.1 (m, 2H),
5.8-5.7 (m, 1H), 5.0-4.6 (m, 7H), 4.4-3.9 (m, 3H), 3.8-3.4 (m,
10H), 2.9-2.8 (m, 2H), 2.2-1.9 (m, 4H), 1.58 (s, 6H).
CD3OD): δ 4.97 (d, 1H, J ) 3.7 Hz), 4.05 (dd, 1H, J ) 11.0 Hz,
J ) 3.5 Hz), 4.0-3.8 (m, 6H), 3.8-3.5 (m, 6H), 2.44 (t, 2H, J )
6.2 Hz), 1.8-1.7 (m, 4H), 1.6-1.4 (m, 28H), 1.2-1.0 (m, 6H).
MS (ES) m/z (calcd 561.4): 562.3 (M + 1), 563.3 (M + 2). Anal.
(C30H59NO8) C, H, N.
Hexadecanoyl 2-Amino-3,4-di-O-benzyl-2-deoxy-6-O-(2,2-
dimethyl-1,3-dioxolan-4(R/S)-ylmethyl)-2-N-(pent-4-enoyl)-r-D-
glucopyranoside ((R/S)-24). To a solution of 15 (1 g, 1.50 mmol)
in anhydrous DMF (12 mL) was added NaH (200 mg, 8.33 mmol),
stirring under Ar for 10 min. When H2 evolution had ceased, (R/
S)-2,2-dimethyl-1,3-dioxolan-4-ylmethyl p-toluenesulfonate (1.3 g,
4.54 mmol) was added. The reaction mixture was stirred at 90 °C
for 30 min and then cooled at room temperature. The excess of
NaH was quenched by addition of methanol (0.5 mL), the solvent
was removed, and the residue was purified by column chromatog-
raphy (hexane-EtOAc, 3:2) to give (S)-24 (160 mg), (R)-24 (150
mg), and (R/S)-24 (260 mg). 1H NMR (200 MHz, CDCl3): δ 7.3-
7.1 (m, 10H), 5.8-5.7 (m, 1H), 5.4-4.5 (m, 8H), 3.5-3.3 (m, 12H),
2.3-2.1 (m, 4H), 1.6-0.9 (m, 37H).
Octyl 2-Amino-2-N-benzoyl-2-deoxy-r-D-glucopyranoside (29).
A solution of 8 (400 g, 1.2 mmol) in H2O-methanol (1:2, 6 mL)
was heated at 90 °C and Ba(OH)2 (568 mg, 1.80 mmol) was added.
The mixture was stirred at 90 °C for 37 h. After this time, it was
cooled at room temperature and neutralized by bubbling CO2 until
pH 7. The mixture was filtered off and the filtrate was concentrated
to provide a yellow crude product (750 mg), which was dissolved
in methanol (9 mL) and treated with triethylamine (166 µL, 1.2
mmol) and benzoic anhydride (299 mg, 1.32 mmol) with stirring
at room temperature for 3 h. Removal of the solvent, purification
by column chromatography (CH2Cl2-methanol, 6:1), and crystal-
lization from methanol gave 29 (211 mg, 45% the two steps). [R]D:
1
Hexadecanoyl 2-Amino-3,4-di-O-benzyl-2-deoxy-6-O-(2(R/
S),3-dihydroxypropyl)-2-N-(pent-4-enoyl)-r-D-glucopyrano-
side ((R/S)-26). To a solution of (R/S)-24 (520 mg, 0.67 mmol) in
methanol (5 mL) was added trifluoroacetic acid (7.4 µL, 0.1 mmol).
The reaction mixture was stirred at room temperature for 1 h. After
this time the solvent was evaporated and the residue (580 mg) was
purified by column chromatography (hexane-EtOAc, 1:1f0:1) to
+116° (c 1.09, MeOH). H NMR (300 MHz, CD3OD): δ 7.9-
7.8 (m, 2H), 7.5-7.4 (m, 3H), 4.95 (d, 1H, J ) 3.4 Hz), 4.1-4.0
(m, 1H), 3.9-3.6 (m, 5H), 3.5-3.4 (m, 2H), 1.6-1.5 (m, 2H), 1.4-
1.1 (m, 10 H), 0.86 (t, 3H, J ) 7.1 Hz). 13C NMR (75 MHz, CD3-
OD): δ 170.12, 133.92, 130.70, 129.49, 129.40, 98.33, 73.78, 72.61,
72.42, 68.93, 62.77, 58.28, 32.92, 30.48, 30.37, 27.34, 27.31, 23.65,
14.42. MS (ES) m/z (calcd 395.2): 396.3 (M + 1), 397.2 (M + 2).
Anal. (C21H53NO6) C, H, N.
1
give (R/S)-26 (389 mg, 79%). H NMR (200 MHz, CD3OD): δ
7.5-7.3 (m, 10H), 5.9-5.8 (m, 1H), 5.2-4.7 (m, 9H), 4.3-4.2
(m, 1H), 3.9-3.4 (m, 10H), 2.5-2.3 (m, 4H), 1.7-1.3 (m, 28H),
0.92 (t, 3H, J ) 6.7 Hz).
Hexadecanoyl 2-Amino-3,4-di-O-benzyl-2-deoxy-2-N-(hexa-
decanoyl)-6-O-(2(R/S),3-dihexadecanoylpropyl)-r-D-glucopyra-
noside ((R/S)-31). A solution of (R/S)-26 (389 mg, 0.52 mmol) in
H2O-THF (1:1, 3.6 mL) was treated with I2 (532 mg, 2.10 mmol),
with stirring at room temperature for 15 min. After this time, sodium
thiosulfate (774 mg, 3.12 mmol) was added and the mixture was
concentrated. The residue was purified by column chromatography
(hexane-EtOAc-methanol, 1:3:0f0:1:0f0:10:1) (202 mg), dis-
solved in anhydrous CH2Cl2 (3 mL), and treated with anhydrous
pyridine (25 µL, 0.30 mmol) and palmitoyl chloride (190 µL, 0.60
mmol). The mixture was stirred under Ar for 1.5 h. Then, the
solvent was evaporated and the residue (360 mg) was purified by
column chromatography (hexane-EtOAc 2:1) to give (R/S)-31 (193
mg, 30%). 1H NMR (200 MHz, CDCl3): δ 7.4-7.3 (m, 10H), 5.85
(d, 1H, J ) 8.8 Hz), 5.30 (s, 4H), 4.77 (d, 1H, J ) 4.3 Hz), 4.2-
4.0 (m, 3H), 3.8-3.4 (m, 9H), 2.4-2.2 (m, 6H), 1.6-1.2 (m, 106H),
0.9-0.8 (m, 12H). MS (ES) m/z (calcd 1372.1): 1372.9 (M), 1373.9
(M + 1), 1374.8 (M + 2).
Hexadecanoyl 2-Amino-2-deoxy-2-N-(hexadecanoyl)-6-O-
(2(R/S),3-dihydroxypropyl)-r-D-glucopyranoside ((R/S)-32). (R/
S)-31 (182 mg, 0.20 mmol) was dissolved in THF (2 mL), and
10% Pd/C (91 mg) was added. The suspension was then stirred
under H2 for 2.5 h. After this time, the mixture was filtered through
Celite. Removal of the solvent afforded a residue (240 mg, 0.10
mmol) that was dissolved in 0.1 M NaOMe in methanol (10 mL),
with stirring at r room temperature for 1 h. After this time the
Phenylethyl 2-Amino-2-deoxy-6-O-(2(R/S),3-dihydroxypro-
pyl)-2-N-penthanoyl-r-D-glucopyranoside ((R/S)-27). To a solu-
tion of (R/S)-23 (82 mg, 0.12 mmol) in methanol (2 mL) was
added trifluoroacetic acid (5 µL, 0.07 mmol). The reaction mixture
was stirred at room temperature for 2 h. After this time, the
solvent was evaporated, the residue was purified by column
chromatography (hexane-EtOAc, 1:1) (78 mg) and dissolved in
methanol (10 mL), and 10% Pd/C (35 mg) was added, with
stirring under H2 for 3 h. After this time, the solution was filtered
through Celite and the filtrate concentrated to give (R/S)-27 (41
1
mg, 74%). [R]D: +97.5° (c 1.02, MeOH). H NMR (300 MHz,
CD3OD): δ 7.4-7.1 (m, 5H), 4.77 (d, 1H, J ) 3.8 Hz), 3.9-3.3
(m, 13H), 2.89 (t, 2H, J ) 7.0 Hz), 2.17 (t, 2H, J ) 8.1 Hz), 1.6-
1.5 (m, 2H), 1.4-1.3 (m, 2H), 0.92 (t, 3H, J ) 7.9 Hz). MS (ES)
m/z (calcd 441.2): 442.3 (M + 1), 443.3 (M + 2). Anal. (C22H35-
NO8) C, H, N.
Hexadecanoyl 2-Amino-2-deoxy-6-O-(2(R/S),3-dihydroxypro-
pyl)-2-N-pentanoyl-r-D-glucopyranoside ((R/S)-28). To a solution
of (R/S)-26 (260 mg, 0.33 mmol) in methanol (18 mL) were added
trifluoroacetic acid (13 µL) and 10% Pd/C (97 mg). The suspension
was then stirred under H2 for 2.5 h. After this time, the solution
was filtered through Celite, and the filtrate concentrated to give
(R/S)-28 (190 mg, 100%) as a white solid. Mp: 91-97 °C. [R]D:

Glycoside Inhibitors of Glioma and Melanoma Growth
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 371
mixture was neutralized with Amberlist (IR-120), filtered off, and
concentrated. The residue was purified by column chromatography
(CH2Cl2-methanol, 7:0f7:1) to give (R/S)-32 (115 mg, 79%). 1H
NMR (300 MHz, CD3OD): δ 4.63 (d, 1H, J ) 3.7 Hz), 3.78 (dd,
1H, J ) 4.0 Hz, J ) 10.3 Hz), 3.7-3.6 (m, 4H), 3.5-3.2 (m, 8H),
2.1-2.0 (m, 2H), 1.5-1.4 (m, 4H), 1.2-1.0 (m, 50H), 0.7-0.6
(m, 6H). 13C NMR (100 MHz, CDCl3-CD3OD, 10:1): δ 128.65,
97.61, 73.15, 72.89, 72.19, 71.30, 71.13, 70.82, 70.37, 68.45, 63.52,
36.76, 32.17, 29.95, 29.82, 29.74, 29.66, 29.60, 29.50, 29.46, 26.47,
26.05, 22.91, 22.90, 11.22, 11.20. MS (ES) m/z (calcd 715.6): 716.5
(M + 1). Anal. (C41H81NO8) C, H, N.
33.06, 30.77, 30.74, 30.61, 30.51, 30.45, 27.34, 23.72, 14.47. MS
(ES) m/z (calcd 603.0): 604.0 (M + 1). Anal. (C27H50KNO9S) C,
H, N, S.
Oleoyl 2-N-Acetamide-2-deoxy-3-O-(oxosulfonyl)-r-D-glu-
copyranoside (42). 12 (76 mg, 0.16 mmol) was reacted under
similar conditions as described for 10 to give 42 (82.1 mg, 82%).
1
[R]D: +39.4° (c 0.89, MeOH). H NMR (300 MHz, CD3OD): δ
5.4-5.3 (m, 2H), 4.48 (d, 1H, J ) 3.2 Hz), 4.48 (dd, 1H, J ) 10.7
Hz, J ) 11.0 Hz), 3.96 (dd, 1H, J ) 3.7 Hz, J ) 10.7 Hz), 3.8-
3.5 (m, 4H), 3.4-3.3 (m, 2H), 2.0-1.9 (m, 7H), 1.6-1.5 (m, 2H),
1.3-1.2 (m, 22H), 0.90 (t, 3H, J ) 7.1 Hz). 13C NMR (75 MHz,
CD3OD): δ 173.69, 130.84, 130.81, 98.32, 80.03, 73.64, 70.85,
69.15, 62.26, 54.10, 33.57, 33.02, 30.86, 30.80, 30.74, 30.65,
30.48, 30.40, 30.35, 30.29, 28.13, 27.28, 23.70, 22.87, 14.46. MS
(ES) m/z (calcd 589.3): 590.2 (M + 1). Anal. (C26H48KNO9S) C,
H, N, S.
Octyl 2-Amino-2-deoxy-6-O-[2,2-bis(hydroxymethyl)-3-hy-
droxypropyl]-2-N-oleoyl-r-D-glucopyranoside (35). 1 (210.2 mg,
0.46 mmol) was dissolved in anhydrous pyridine (2 mL), and
acetic anhydride (2 mL) was added. The reaction mixture was
stirred at room temperature for 12 h, concentrated, and purified by
column chromatography (EtOAc-hexane, 2:1) to give a white solid
(313 mg), which was dissolved in anhydrous pyridine (5 mL) and
treated with 85% oleyl chloride (0.47 mL, 1.40 mmol) and
dimethylaminopyridine (4.5 mg, 0.03 mmol). The mixture was
refluxed for 2 h, concentrated, and purified by column chroma-
tography (hexane-EtOAc, 2:1f1:1) to give a white solid (241 mg),
which was dissolved in methanol (30 mL) and treated with a
solution of NaOMe in MeOH (1M, 8 mL), with stirring at
room temperature for 2 h. After this time, the solution was
neutralized with Amberlist (IR-120), filtered, and concentrated. The
residue was purified by column chromatography (CH2Cl2-
methanol, 10:1) to give 35 (172 mg, 54%). [R]D: +69.7° (c 0.96,
Octyl 2-N-Oleoyl-2-amino-2-deoxy-3-O-(oxosulfonyl)-r-D-glu-
copyranoside (43). 13 (145 mg, 0.26 mmol) was reacted under
similar conditions as described for 10 to give 43 (77.2 mg, 42%).
1
[R]D: +43.6° (c 2.01, MeOH). H NMR (200 MHz, CD3OD): δ
5.4-5.3 (m, 2H), 4.87 (d, 1H, J ) 3.7 Hz), 4.6-4.4 (m, 1H), 3.9-
3.3 (m, 7H), 2.3-2.2 (m, 6H), 1.6-1.1 (m, 34H), 1.0-0.8 (m, 6H).
13C NMR (50 MHz, CD3OD): δ 176.55, 130.88, 130.80, 98.18,
79.78, 73.80, 70.91, 69.15, 62.40, 54.26, 32.29, 33.14, 33.10, 30.96,
30.91, 30.68, 30.60, 30.64, 30.59, 30.51, 30.49, 30.37, 30.30, 28.24,
26.20, 27.46, 26.98, 23.84, 23.80, 14.54, 14.50. MS (ES) m/z (calcd
673.4): 674.3 (M + 1). Anal. (C32H60KNO9S) C, H, N, S.
Oleoyl 2-N-Acetamide-2-deoxy-3,4-O-(2,3-diethoxybut-2,3-
diyl)-6-O-(oxosulfonyl)-r-D-glucopyranoside (48). A solution of
12 (100 mg, 0.21 mmol) in ethanol (1.5 mL) was treated with
butane-2,3-dione (41 µL, 0.47 mmol), camphorsulfonic acid (10
mg, 0.04 mmol), and triethylorthoformiate (0.23 mL, 1.4 mmol)
under Ar. The mixture was stirred for 3.5 h at 60 °C. After cooling,
the mixture was neutralized with triethylamine, concentrated, and
purified by column chromatography (hexane-EtOAc, 1:1f0:1) to
give a solid (98 mg), which was dissolved in anhydrous pyridine
(5 mL) and then treated with SO3-pyridine complex (509 mg, 3.20
mmol), with stirring at room temperature under Ar for 1 h. After
this time, the mixture was concentrated, and the residue was
dissolved in methanol-water (2:1, 7 mL), neutralized with a 0.5
M KOH solution, and concentrated. The residue was purified by
column chromatography (CH2Cl2-methanol, 6:1) to give 48 (95
mg, 61%). 1H NMR (200 MHz, CD3OD): δ 5.4-5.3 (m, 2H), 4.83
(d, 1H, J ) 3.4 Hz), 4.6-4.4 (m, 2H), 4.3-3.4 (m, 8H), 2.2-2.0
(m, 7H), 1.6-1.2 (m, 36H), 1.0-0.9 (m, 3H).
1
MeOH). H NMR (300 MHz, CD3OD): δ 5.4-5.3 (m, 2H), 4.78
(d, 1H, J ) 4.0 Hz), 3.86 (dd, 1H, J ) 4.1 Hz, J ) 10.6 Hz),
3.7-3.3 (m, 15H), 2.23 (t, 2H, J ) 7.0 Hz), 2.1-1.9 (m, 4H),
1.6-1.5 (m, 4H), 1.3-1.2 (m, 30H), 0.9-0.8 (m, 6H). MS (ES)
m/z (calcd 673.5): 674.5 (M + 1), 675.5 (M + 2). Anal. (C37H71-
NO9) C, H, N.
Phenylethyl 2-Amino-2-deoxy-2-N-(pen-4-enoyl)-3-O-(oxosul-
fonyl)-r-D-glucopyranoside (40). 10 (150 mg, 0.41 mmol) was
dissolved in anhydrous DMF (2.5 mL) and treated with 2,2-
dimethoxypropane (0.46 mL, 2.05 mmol) and p-toluensulphonic
acid (10 mg). The mixture was stirred at room temperature for 2 h,
neutralized with triethylamine, and concentrated to give a residue
(166 mg) that was dissolved in anhydrous pyridine (11 mL) and
then treated with the SO3-pyridine complex (1.3 g, 8.20 mmol),
with stirring at room temperature under Ar for 1.5 h. After this
time, the mixture was concentrated, and the residue was dissolved
in methanol-water (2:1, 5 mL), neutralized with a 0.5 M KOH
solution, and concentrated. The residue was extracted with metha-
nol, concentrated, and purified by column chromatography (CH2-
Cl2-methanol, 6:1) to give 40 (178.8 mg, 90%). [R]D: +82.4° (c
1.19, MeOH). 1H NMR (300 MHz, CD3OD): δ 7.4-7.1 (m, 5H),
5.9-5.7 (m, 1H), 5.05 (dd, 1H, J ) 2.0 Hz, J ) 16.9 Hz), 4.97
(dd, 1H, J ) 2.0 Hz, J ) 10.0 Hz), 4.87 (d, 1H, J ) 3.9 Hz), 4.48
(dd, 1H, J ) 10.7 Hz, J ) 10.7 Hz), 4.0-3.9 (m, 2H), 3.8-3.4
(m, 4H), 3.20 (dd, 1H, J ) 7.3 Hz, J ) 15.1 Hz), 2.91 (t, 2H, J )
6.8 Hz), 2.3-2.2 (m, 2H), 1.31 (t, 2H, J ) 7.3 Hz). 13C NMR (75
MHz, CD3OD): δ 175.62, 140.43, 138.32, 130.14, 129.46, 127.34,
115.78, 98.32, 79.81, 73.89, 70.79, 69.92, 62.26, 54.05, 36.90,
36.42, 30.67. MS (ES) m/z (calcd 483.1): 484.1 (M + 1). Anal.
(C19H26KNO9S) C, H, N, S.
Oleoyl 2-N-Acetyl-2-deoxy-6-O-(oxosulfonyl)-r-D-glucopyra-
noside (50). A solution of 10 (150 mg, 0.41 mmol) in ethanol (3
mL) was treated with butane-2,3-dione (82 µL, 0.93 mmol),
camphorsulfonic acid (20 mg, 0.09 mmol), and triethylorthoformate
(0.46 mL, 2.8 mmol) under Ar. The mixture was stirred for 3 h at
60 °C. After cooling, the mixture was neutralized with triethylamine
and concentrated to give a residue (507 mg) that was dissolved in
anhydrous pyridine (10 mL) and treated with SO3-pyridine
complex (1.3 g, 8.20 mmol), with stirring at room temperature under
Ar for 1 h. After this time, the mixture was concentrated, and the
residue was dissolved in methanol-water (2:1, 7 mL), neutralized
with a 0.5 M KOH solution, and concentrated. The residue was
extracted with methanol, concentrated, and purified by column
chromatography (CH2Cl2-methanol, 6:1) to give a solid. The solid
was dissolved in a mixture of acetic acid-water (2:1, 20 mL) and
stirred at 70 °C for 3 h. The mixture was concentrated and the
residue was purified by column chromatography (CH2Cl2-
methanol, 10:1f4:1) to give 50 (80 mg, 41%, three steps). [R]D:
Hexadecanoyl 2-Amino-2-deoxy-2-N-oleoyl-3-O-(oxosulfonyl)-
r-D-glucopyranoside (41). 11 (60 mg, 0.12 mmol) was reacted
under similar conditions as described for 10 to give 41 (47 mg,
1
1
63%). [R]D: +62.3° (c 1.28, MeOH). H NMR (300 MHz, CD3-
+89.5° (c 1.10, MeOH). H NMR (300 MHz, CD3OD): δ 7.3-
OD): δ 5.9-5.8 (m, 1H), 5.08 (dd, 1H, J ) 1.9 Hz, J ) 17.0 Hz),
4.98 (dd, 1H, J ) 1.9 Hz, J ) 10.2 Hz), 4.90 (d, 1H, J ) 3.6),
4.50 (dd, 1H, J ) 8.3 Hz, J ) 10.9 Hz), 3.95 (dd, 1H, J ) 3.6 Hz,
J ) 10.7 Hz), 3.8-3.6 (m, 5H), 3.4-3.3 (m, 1H), 2.4-2.3 (m,
4H), 1.7-1.6 (m, 2H), 1.4-1.1 (m, 26H), 0.90 (t, 3H, J ) 7.0
Hz). 13C NMR (75 MHz, CD3OD): δ 175.84, 138.23, 115.80,
98.23, 79.85, 73.65, 70.81, 69.10, 62.26, 55.27, 54.07, 36.44,
7.2 (m, 5H), 5.8-5.7 (m, 1H), 5.0-4.9 (m, 2H), 4.67 (d, 1H, J )
3.9 Hz), 4.1-4.0 (m, 2H), 3.9-3.8 (m, 1H), 3.8-3.6 (m, 2H), 3.5-
3.4 (m, 1H), 3.4-3.3 (m, 2H), 2.83 (t, 2H, J ) 5.9 Hz) 2.2-2.1
(m, 4H). MS (ES) m/z (calcd 483.1): 484.0 (M + 1). Anal. (C19H26-
KNO9S) C, H, N, S.
Oleoyl 2-N-Acetyl-2-amino-2-deoxy-6-O-(oxosulfonyl)-r-D-
glucopyranoside (51). 48 (79 mg, 0.11 mmol) was dissolved in a

372 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Garc´ıa-AÄ lVarez et al.
mixture of acetic acid-water (2:1, 10 mL) and stirred at 65 °C for
3 h. The mixture was concentrated and the residue was purified by
column chromatography (CH2Cl2-methanol, 5:1f4:1) to give 51
(42 mg, 66%). [R]D: +69.0° (c 1.18, MeOH). 1H NMR (300 MHz,
CD3OD): δ 5.4-5.3 (m, 2H), 4.76 (d, 1H, J ) 3.4 Hz), 4.26 (dd,
1H, J ) 2.4 Hz, J ) 11.0 Hz), 4.18 (dd, 1H, J ) 5.6 Hz, J ) 10.7
Hz), 3.89 (dd, 1H, J ) 3.7 Hz, J ) 10.7 Hz), 3.8-3.6 (m, 4H),
3.4-3.3 (m, 2H), 2.0-1.9 (m, 7H), 1.6-1.5 (m, 2H), 1.3-1.2 (m,
22H), 0.90 (t, 3H, J ) 6.8 Hz). MS (ES) m/z (calcd 589.3): 590.3
(M + 1). Anal. (C26H48KNO9S) C, H, N, S.
processing, Microsoft Excel for Windows XP and GraphPad Prism
4 programs were used.
Acknowledgment. Financial support provided by FAES
FARMA S.A., the Ministry of Educacio´n y Ciencia (Grant
CTQ2004-03523/BQU), and the CSIC (PIF, 200580F0062) is
greatly appreciated. E.D.-P. is supported by the Servicio de
Salud de Castilla La Mancha Community (SESCAM). We thank
to Maria Angeles Oliva for her technical assistance in cell
culture assays.
Octyl 2-Amino-2-deoxy-2-N-oleoyl-6-O-(oxosulfonyl)-r-D-glu-
copyranoside (52). 13 (250 mg, 0.45 mmol) in ethanol (3.2 mL)
was treated with butane-2,3-dione (87 µL, 0.99 mmol), camphor-
sulfonic acid (21 mg, 0.09 mmol), and triethylorthoformate (0.49
mL, 3.0 mmol) under Ar. The mixture was stirred for 3 h at 70 °C.
After cooling, the mixture was neutralized with triethylamine and
concentrated. The crude was purified by column chromatography
(hexane-EtOAc, 2:1) to give a residue (121 mg) that was dissolved
in anhydrous pyridine (5 mL) and treated with SO3-pyridine
complex (552 mg, 3.46 mmol), with stirring at room temperature
under Ar for 1 h. After this time, the mixture was concentrated,
and the residue was dissolved in methanol-water (2:1, 7 mL),
neutralized with a 0.5 M KOH solution, and concentrated. The
residue was purified by column chromatography (CH2Cl2-
methanol, 8:1) to give a solid (116 mg) that was dissolved in a
mixture of acetic acid-water (2:1, 20 mL) and stirred at 70 °C for
3 h. The mixture was concentrated and the residue was purified by
column chromatography (CH2Cl2-methanol, 10:1f4:1) to give 52
(80 mg, 13%, three steps). [R]D: +70.3° (c 0.86, MeOH). 1H NMR
(400 MHz, CD3OD): δ 5.4-5.3 (m, 2H), 4.76 (d, 1H, J ) 3.9
Hz), 4.26 (dd, 1H, J ) 2.4 Hz, J ) 10.8), 4.18 (dd, 1H, J ) 5.7
Hz, J ) 10.8 Hz), 3.88 (dd, 1H, J ) 3.9 Hz, J ) 10.8 Hz), 3.8-
3.7 (m, 3H), 3.4-3.3 (m, 2H), 2.3-2.2 (m, 2H), 2.0-1.9 (m, 4H),
1.6-1.5 (m, 4H), 1.3-1.2 (m, 30H), 0.9-0.8 (m, 6H). 13C NMR
(100 MHz, CD3OD): δ 176.62, 131.43, 130.37, 98.36, 72.43, 72.14,
71.72, 69.05, 68.30, 55.36, 37.04, 33.11, 33.07, 30.90, 30.85, 30.69,
30.62, 30.61, 30.56, 30.49, 30.46, 30.36, 30.34, 28.18, 28.14, 27.41,
27.16, 23.80, 23.75, 14.50, 14.48. MS (ES) m/z (calcd 673.3): 673.4
(M + 1), 673.5 (M + 2). Anal. (C32H60KNO9S) C, H, N, S.
Cell Lines. C6 rat glioma cells21 and U-373 MG human glioma
cells22 were seeded on plastic flasks and maintained in DMEM
culture medium, supplemented with 10% fetal calf serum (FCS), 2
mM glutamine, 50 IU/mL penicillin, and 50 mg/mL streptomycin
(DM-10S) and maintained in a humidified atmosphere of 5% CO2,
at 37 °C.
Supporting Information Available: Elemental analysis data
of target compounds 5-13, 19-22, 27-29, 32, 35, 40-43, and
50-52. This material is available free of charge via the Internet at
http://pubs.acs.org.
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Cell Proliferation Assays. For proliferation assays the C6, U373,
or A375 cell lines were seeded in DM-10S at 1 × 104 cells/well
and allowed to attach for 6 h. After the cells were attached to the
substrate, the medium was changed to serum-free DMEM, and the
cells were incubated for 36 h. Then the medium was replaced by
DMEM medium plus 1% FCS, containing the test inhibitors, in
triplicate. The cultures were incubated for 24 h and washed with
serum-free DMEM medium. For [3H]thymidine (Amersham) in-
corporation assay, a 6 h-pulse (0.5 µCi/well) was given; nonincor-
porated radioactivity was washed away, the cells were lysed with
0.5% SDS solution, diluted in scillation fluid, and the plates were
measured in a WALLAC liquid scintillation counter (1450 Micro-
beta Trilux model; Turku, Finland). Inhibition was calculated using
the formula:
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% inhibition ) 100 - 100[(X - B)/(A - B)]
where A is [3H]thymidine dpm incorporated corresponding to cells
maintained in DM-10S (high mitosis control); B is the dpm
incorporated from cells in serum-free medium (low mitosis control),
and X corresponds to dpm incorporated in cells treated with test
inhibitors.6 Dose-response plots of percent inhibition versus
concentration were obtained from triplicate samples and adjusted
to sigmoidal curves, from which values of the 50% inhibitory
concentration (ID50) were calculated. The ID50 values plus standard
error of the mean (SEM) are shown in the tables. For data
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