Antiproliferation and apoptosis induced by C-glycosides in human leukemia cancer cells

Article abstract of DOI:10.1016/j.bmcl.2006.05.079

A large series of alkyl C-glycosides was synthesized from d-glucal or d-galactal. These compounds were screened against the human promyelocytic leukemia cell line (HL60), showing significant activity and apoptosis. Up to 13 C-glucopyranosides, but no C-ga

Full text of DOI:10.1016/j.bmcl.2006.05.079

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4223–4227
Antiproliferation and apoptosis induced by C-glycosides
in human leukemia cancer cells
a
a
b
b,c
´
´
´
Carlos A. Sanhueza, Carlos Mayato, Marıa Garcıa-Chicano, Raquel Dıaz-Penate,
˜
a
a,
*
´
´
Rosa L. Dorta and Jesus T. Vazquez
aInstituto Universitario de Bio-Orga´nica ‘Antonio Gonza´lez’, Universidad de La Laguna, Avda, Astrofı´sico Fco,
Sa´nchez 2, 38206 La Laguna, Tenerife, Spain
bUnidad de Investigacio´n del Hospital de Gran Canaria Dr. Negrı´n, Bco. de la Ballena S/N, 35020 Las Palmas de Gran Canaria, Spain
cInstituto Canario de Investigacio´n del Ca´ncer (ICIC), Bco. de la Ballena S/N, 35020 Las Palmas de Gran Canaria, Spain
Received 25 April 2006; revised 24 May 2006; accepted 24 May 2006
Available online 9 June 2006
Abstract—A large series of alkyl C-glycosides was synthesized from D-glucal or D-galactal. These compounds were screened against
the human promyelocytic leukemia cell line (HL60), showing significant activity and apoptosis. Up to 13 C-glucopyranosides, but
no C-galacto- or C-mannopyranosides, exhibited inhibitory concentrations (IC50 values) below 20 lM, five of them in the range
4–8 lM. Preliminary structure–activity relationships were established.
Ó 2006 Elsevier Ltd. All rights reserved.
Cell surface glycoproteins are known to be active in
numerous important disease states.1 Their carbohydrate
epitopes have been shown to play a central role in a vari-
ety of significant biological events, including inflamma-
tion, metastasis, immune response, and bacterial and
viral infection. This has recently stimulated the develop-
ment of effective therapeutic strategies based upon
recognition of these cell surface carbohydrates. One ap-
proach lies in the replacement of the exocyclic oxygen
with a carbon, to afford C-glycosidic analogs, due to
their increased resistance to degradation by glycosi-
dases. This has led to a wealth of synthetic approaches2
and consequently biological data on C-glycosides have
started to emerge.3
ties,8 including antitumor and antiviral action, the aim
of the present study was to carry out the synthesis of
easily obtained simple C-glycosides and check their
cytotoxic activities.
Among the different methods for the preparation of C-
glycosides,2 the addition of carbon nucleophiles to acti-
vated glycal epoxides has been widely used.9 Therefore,
the C-glycoside derivatives under study were prepared in
three steps: (i) O-alkylation of D-glucal or D-galactal; (ii)
epoxidation using dimethyldioxirane in CH2Cl2, accord-
ing to Danishefsky’s protocol;10 and (iii) epoxide
opening with Grignard reagents or diorganocuprates
(Scheme 1).11
In addition, comparative biological studies have re-
vealed that C-glycosides retain the biological properties
of natural O-glycosides.4
Thus, addition of numerous Grignard reagents or diorg-
anocuprates to the 1,2-anhydrosugar led to a mixture of
a- and b-C-glycoside derivatives, the a/b ratio being var-
iable and in most cases favoring the b-isomer. In some
cases, further simple structural modifications were
Since glycosylarenes5 (or C-aryl glycosides), 4-keto
unsaturated C-glycosides,6 C-glycoglycerolipids,7 and
unsaturated C-glycosides with an attached ring system
bonded at C-4 and C-6 exhibit diverse biological activi-
α
β
O
O
O
R'
R'
RO
RO
1. DMDO
RO
RO
1
1
+
2. R'MgX or
R'2CuMgX
RO
RO
OH
OH
Keywords: C-Glycosides; Leukemia (HL60); Apoptosis; Cancer.
*
OR
OR
OR
Corresponding author. Tel.: +34 922318581; fax: +34 922318571;
e-mail: jtruvaz@ull.es
Scheme 1. Synthesis of C-gluco- and C-galactopyranosides.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.05.079

4224
C. A. Sanhueza et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4223–4227
performed by means of standard chemical transforma-
tions. The stereochemistry of the synthesized C-glyco-
20 lM (Scheme 2), except compound 12, possess an
underivatized hydroxyl group at C-2 with an equa-
torial configuration. Compounds with an acetyl,
benzyl, 2-deoxy, etc., group at position 2 showed
a lowering or total loss of activity (Schemes 3
and 4).
1
sides (Scheme 1) was established by analyzing the H
NMR J1,2 value (b-configuration around 9.5 Hz, a-con-
figuration around 5.8 Hz). Sometimes it was confirmed
by means of the T-ROESY experiments, by observing
the clear crosspeaks involving the pseudo-anomeric pro-
ton H1.
Furthermore, the ketone 15, obtained by oxidation of
the alcohol 1 by treatment with dimethylsulfoxide/ace-
tic anhydride, as well as the b-C-mannopyranoside 35,
derived from 15 by reduction with NaBH4 in CH2Cl2/
MeOH (1:1),13 led to a lowering or total loss of activ-
ity. The mannopyranosides 36–39, similarly obtained
from their corresponding glucopyranosides (6–8 and
17) did not show activity either. Therefore, the equato-
rial disposition of the hydroxyl group at position 2,
glucose configuration, seems to be crucial for the inhib-
itory process.
The cytotoxic activity of the whole series was mea-
sured as growth inhibition or decreased viability on
the human promyelocytic leukemia cell line HL60.
Table 1 shows the compounds with a cytotoxic effect,
with their 50% inhibitory concentrations (IC50 values)
below 50 lM. These data were determined by the
MTT assay12 and calculated from at least three inde-
pendent experiments. Schemes 2 and 3 show the
structures of those compounds exhibiting IC50 values
below 20 lM and in the range 20–50 lM,
respectively.
O
O
O
BnO
BnO
BnO
BnO
BnO
BnO
1
1
The C-glucopyranosides 1–13 inhibited growth below
20 lM. Compounds 3, 4, 5, and 7 showed IC50 values
around 8.5 lM, while compound 9, the most active
one, exhibited an IC50 of 4.1 lM (Scheme 2). A moder-
ate or low activity was observed for compounds 14–23
(Scheme 3), whereas compounds 24–64, having an IC50
higher than 50 lM, were not active.
OH
OH
OH
OBn
OBn
OBn
35
36, C1 β
37, C1 α
38, C1 β
39, C1 α
To test the importance of the configuration at C-4, the
galactopyranoside derivatives 40–43 were analyzed.
Their lack of cytotoxic activity confirms the importance
of having the glucose configuration, as in the C-
mannopyranosides.
Some preliminary structure–activity relationships can
be established. Thus, the present study reveals the
importance of the underivatized hydroxyl group at
C-2 for cytotoxic activity. All compounds having
antiproliferative activity against HL60 cell line below
O
O
BnO
BnO
BnO
BnO
1
OH
OH
OBn
OBn
40, C1 β
41, C1 α
42, C1 β
43, C1 α
Table 1. Effects of the C-glycosides on the growth of the HL-60 cell
line
Compound
IC50
SD
To test the role of the benzyl groups linked to the
hydroxyl groups at C-3, C-4, and C-6 in cytotoxic
activity, all possible deprotected derivatives of the ac-
tive compound 3 (compounds 21–23 and 44–47) were
synthesized. They were satisfactorily obtained from
compound 3 by partial hydrogenolysis with H2 and
1
15.1
11.8
8.7
2.5
3.4
1.5
2.5
5.0
3.9
1.2
4.0
1.1
1.4
0.7
3.4
2.7
0.4
5.7
7.0
3.0
2.9
4.1
3.2
3.7
3.9
1.5
—
2
3
4
8.4
8.2
5
6
11.7
8.5
Pd–C as catalyst, when
allowed.
a 90% conversion was
7
8
13.9
4.1
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24–64
17.2
12.1
12.5
19.3
26.9
31.8
26.3
28.2
39.3
38.5
46.4
20.2
35.3
26.6
n.a.a
44, R1 = Bn, R2 = H, R3 = H
45, R1 = H, R2 = Bn, R3 = H
46, R1 = H, R2 = H, R3 = Bn
47, R1 = H, R2 = H, R3 = H
O
R3O
R2O
OH
OR1
As seen in Table 1, only those compounds retaining two
out of three O-benzyl groups (21–23) showed activity,
somewhat lower than their parent compound 3. On
the basis of their IC50 values, the contribution to the
inhibitory effect of 3 on the growth of HL60 was higher
when an O-benzyl group was located at C-4 rather than
at C-3, and at C-3 higher than C-6. Meanwhile, those
having one O-benzyl group or none (44–47) showed
no significant cytotoxic activity. This highlights the con-
a n.a., not active.

C. A. Sanhueza et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4223–4227
4225
O
O
O
O
O
O
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
OH
OH
OH
OH
OH
OH
OBn
OBn
OBn
OBn
OBn
OBn
1
6
2
3
4
5
O
O
O
O
O
BnO
BnO
BnO
BnO
BnO
BnO
O
OH
BnO
BnO
BnO
BnO
1
1
O
OH
OH
OH
OH
OBn
OBn
12
OBn
13
OBn
OBn
7, C1
8, C1
9, C1
10, C1
11
Scheme 2. Structures of C-glycosides with antiproliferative activity against HL60 cell line below 20 lM.
O
O
O
nO
nO
H3CO
H3CO
O
O
BnO
BnO
BnO
BnO
O
BnO
BnO
nO
OH
OCH3
48
OH
nO
OH
51, n = 14
OAc
O
O
O
n
OH
OBn
OBn
49, n = 3
50, n = 14
n
OBn
14
15
16
O
O
BnO
BnO
O
BnO
BnO
BnO
BnO
Regarding the C-aglycon, the data reveal a preference
for the b equatorial configuration at C-1 to achieve a
growth inhibiting effect. Most of the a axial epimers at
C-1 showed no activity or a lower activity compared
with their b stereoisomer, except for the neopentyl ster-
eoisomers, where the a isomer is cytotoxic (11), and its b
is not (58).
OH
O
OBn
OBn
OBn
19
17
18
O
BnO
BnO
R3O
Cl
Cl
R2O
OH
OR1
OBn
20
21, R1 = Bn, R2 = Bn, R3 = H
22, R1 = Bn, R2 = H, R3 = Bn
23, R1 = H, R2 = Bn, R3 = Bn
The relationships observed between the cytotoxicity and
the length of the saturated chain of the C-aglycon are
really striking. While the methyl, ethyl, and propyl
3,4,6-tri-O-benzyl b-C-glucopyranosides 4–6 exhibited
cytotoxicity, the corresponding n-butyl (56) and n-pentyl
(57) did not. Regarding their axial epimers at C-1, only
the propyl a-C-glucopyranoside 17 showed some cyto-
toxic activity. Compounds having a branched aglycon,
for example, 7–11, also inhibited cell growth; the b
stereoisomers 7 and 9 to a greater extent than their a
isomers 8 and 10. The neopentyl derivative 11 has the
opposite behavior, as already mentioned.
Scheme 3. Structures of C-glycosides with antiproliferative activity
against HL60 cell line between 20 and 50 lM.
venience of having the three above-mentioned hydroxyl
groups protected. Furthermore, when these aromatic
groups are changed for methyl, n-butyl, or pentadecyl
(48–51), no cytotoxic activity was observed. Conse-
quently, the benzyl groups at C-3, C-4, and C-6, besides
increasing cell permeability, may favor an aromatic
interaction with the receptor, resulting in increased
binding affinity.14
In addition, an IC50 data comparison between C-glyco-
sides with unsaturated aglycons and their saturated ana-
logs was performed. Thus, compounds 1, 2, and 3
O
O
O
O
BnO
BnO
BnO
BnO
BnO
BnO
RO
RO
OAc
OR
OR
OR
OBn
OBn
OBn
OR
24
25, R = H
26, R = Ac
27, R = H
28, R = Ac
29, R = H
30, R = Ac
O
O
O
O
BnO
BnO
BnO
BnO
BnO
BnO
OAc
OAc
OAc
OBn
AcO
AcO
OAc
OBn
OBn
OBn
OAc
31
32
33
34
Scheme 4. Structures of C-glycosides with no antiproliferative activity against HL60.

4226
C. A. Sanhueza et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4223–4227
showed higher IC50 values than compounds 6, 5, and 9,
respectively, revealing more potent cytotoxic activity for
C-glycosides with saturated aglycons (Scheme 5).
a
c
1
To test if these C-glucosides induce apoptosis in HL60
leukemia cells, some active compounds were analyzed.
The cells were cultured in absence (C) or presence of
20 lM of the selected compounds for 24 h, then fixed
with PFA, stained with DAPI, and nuclei were visual-
ized using fluorescence microscopy (Fig. 1). The percent-
age of apoptotic cells was determined by quantitative
fluorescence microscopy and the results of a representa-
tive experiment are shown. Each point represents aver-
age SE of duplicate determinations.
5
12
These data indicate that 3,4,6-tri-O-benzyl b-C-gluco-
sides 1, 5, 7, and 12 induce apoptosis in HL60 cells,
the most potent being compound 12.
b
2.0
1.5
1.0
0.5
In summary, a large series of C-glycosides have been
synthesized and screened against the human promyelo-
cytic leukemia cell line (HL60). Several C-glucopyrano-
sides showed promising antiproliferative and apoptotic
activity against HL60 cells. The preliminary structure–
activity study revealed some structural features related
with the potency of the cytotoxic activity. A b equatorial
configuration at C-1, an underivatized equatorial
hydroxyl group at C-2, and O-benzyl groups at C-3,
C-4, and C-6 led to the highest antiproliferative activity.
However, compound 12 having an acetonide group was
the most apoptotic derivative.
C
1
4
5
7
9
10
11
12
Figure 1. (a) Fluorescence microscopy of untreated (C) or treated
(compounds 1, 5, and 12) HL60 cells. (b) Percentage of apoptotic cells
determined by quantitative fluorescence microscopy.
medical significance, further chemical and biochemical
studies are currently under way.
Since compounds which regulate apoptosis and over-
come apoptosis deficiency of cancer cells are of high
Acknowledgments
This work was partially financed by the Universidad de
La Laguna and the Instituto de Salud Carlos III (ISCiii,
RTICCC C03/10). C.A.S. and C.M. thank the Univers-
O
O
O
BnO
BnO
BnO
BnO
BnO
BnO
n
OH
OH
OH
´
idad de La Laguna as well as the Direccion General de
Universidades e Investigacion (Gobierno de Canarias)
OBn
OBn
OBn
´
53, n = 1
54, n = 3
52
55
´
for a fellowship. We also thank J. M. Garcıa Castellano
´
and J. M. Padron for their collaboration.
O
O
O
BnO
BnO
BnO
BnO
BnO
BnO
n
OH
OH
OH
References and notes
OBn
OBn
OBn
56, n = 3
57, n = 4
58
59
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O
O
O
O
BnO
BnO
BnO
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O
OH
OH
OH
OBn
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OH
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60
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62
O
O
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63
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against HL60.

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