Synthesis and cytostatic activity of purine nucleosides derivatives of allofuranose

Article abstract of DOI:10.1016/j.ejmech.2010.09.046

Several new purine nucleosides derivatives of allofuranose were prepared according to Vorbrüggen method, starting from 1,2,5,6-di-O-isopropylidene- α-d-allofuranose and using 1,2,3,5,6-pentaacetoxy-β-d-allofuranose as key intermediate. The synthesized all

Full text of DOI:10.1016/j.ejmech.2010.09.046

European Journal of Medicinal Chemistry 45 (2010) 6114e6119
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and cytostatic activity of purine nucleosides derivatives of allofuranose
**
*
Pedro Besada , Tamara Costas, Marta Teijeira, Carmen Terán
Department of Organic Chemistry, University of Vigo, 36310, Vigo, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 July 2010
Received in revised form
15 September 2010
Accepted 20 September 2010
Available online 25 September 2010
Several new purine nucleosides derivatives of allofuranose were prepared according to Vorbrüggen
method, starting from 1,2,5,6-di-O-isopropylidene- -allofuranose and using 1,2,3,5,6-pentaacetoxy-
-allofuranose as key intermediate. The synthesized allofuranosyl nucleosides, as well as some acetyl
derivatives, were evaluated for their cytotoxicity in vitro in three human cancer cell lines (MCF-7, Hela-
229 and HL-60). Among the studied compounds the 9-(2,3,5,6-tetra-O-acetyl- -allofuranosyl)-
a
-
D
b-
D
b-D
2,6-dichloropurine (9) was the most potent one on the three cell lines evaluated, being its activity against
HL-60 cells similar to cisplatin.
Keywords:
Allofuranose
Cancer
Ó 2010 Elsevier Masson SAS. All rights reserved.
Cytostatic activity
Hexofuranosyl nucleosides
Purine
1. Introduction
Most of purine derivatives described before show an amino
group or similar (alkoxy, chloro) at 6th position of the heterocyclic
Purine nucleoside analogues play an important role as cytostatic
agents. Thus, cladribine (1a) and fludarabine (1b) are used in
oncology for the treatment of both hematological malignant
diseases and some solid tumors [1] (Fig. 1). In the last years
a number of new purine nucleosides have been developed. Some of
these compounds, such as nelarabine (1c) or clofarabine (1d), are
currently under investigation in clinical studies [2]. For instance,
clofarabine is a new deoxyadenosine analogue that has demon-
strated single-agent anticancer activity in pediatric and also in
adult acute leukaemias [3].
Purine nucleoside analogues, in general, act as antimetabolites
after their metabolic phosphorylation to the corresponding 50-
triphosphates, compete with physiological nucleosides and interact
with a number of cellular targets to induce cytotoxicity. All purine
nucleoside analogues develop a similar mechanism of cytotoxicity
both in proliferating and quiescent cells, such as inhibition of DNA
synthesis and repair, and accumulation of DNA strand breaks, being
apoptosis the end-point of their action [4,5]. However, significant
differences exist not only in the target enzymes but also in the
drugetarget interactions of these antimetabolites, which could
explain their different biological activities [4].
base which could be essential to establish hydrogen bonds with
polymerases and other key enzymes of nucleic acid metabolism [6].
However, purine nucleosides bearing C-substituents at C-6 (aryl,
alkyl, benzyl, or heteroaryl) also possess significant cytostatic
effects [6,7]. In addition, substitution at C-2 of the purine ring by
halogen seems to be favorable [8]. The sugar moieties have been
also modified for the development of new purine nucleosides, in
particular 20 and 30 positions. A number of 20 or 30 modified
nucleosides have shown potent cytostatic activity, such as 30-C-
methyl derivatives [9], 20-O-alkenyl analogues [10] as well as
20-deoxy derivatives and nucleosides with arabino configuration,
that in many cases have proven to be inhibitors of multienzymes
[11]. On the other hand, structural changes at 40 position of the
sugar moiety have been studied with minor profusion although
several 40-modified nucleoside analogues have been synthesized
and some of them have shown significant biological properties
[12e14].
Regarding this aspect, it is necessary to emphasize the potential
interest of hexofuranosyl nucleosides because of their great struc-
tural similitude with the ribosyl nucleosides. The main difference
between both types of analogues is the presence at 40 of an ethylene
glycol group replacing the habitual hydroxymethyl group [15].
Some hexofuranosyl nucleosides have been previously synthesized
and biologically evaluated as antiviral agents [16], however there
are not recent studies about the potential cytostatic activity of
this class of derivatives [17]. A reason for the lack of cytotoxicity
* Corresponding author. Tel.: þ34 986 812276; fax: þ34 986 812262.
** Corresponding author.
E-mail addresses: pbes@uvigo.es (P. Besada), mcteran@uvigo.es (C. Terán).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.09.046

P. Besada et al. / European Journal of Medicinal Chemistry 45 (2010) 6114e6119
6115
NH2
N
R6
Cl
N
N
N
N
N
R2
N
HO
HO
R2'
O
N
O
N
N
Cl
N
R2
H
N
RO
RO
OH
O
OH
H
H
1a
1b R6= NH2, R2 = F, R2' = OH
1c R6 = OCH3, R2 = NH2, R2' = OH
1d R6= NH2, R2 = Cl, R2' = F
OR OR
2a R = R2 = H,
9
R = Ac, R2 = Cl
Fig. 1. Nucleoside cytostatic drugs.
Fig. 3. Protoneproton interactions observed in the NOE spectrums of compounds 2a
and 9.
data could be the fact that they are currently considered as con-
formationally restricted acyclic nucleosides [18], and structurally
related to an important group of antiviral agents.
Taking into account that nucleoside analogues are among the
most complex and promising cytostatic agents and as part of our
research program in this field [19], we are describing in this work
the synthesis and cytostatic activity of several purine nucleosides
derivatives of allofuranose of structure 2 (Fig. 2). The selection of
substituents at the purine nucleus was based on the analogy to
some of the most active compounds known.
nucleophilic reagents. Thus, treatment of 9 with NH3 0.5 M in
dioxane at r.t. provided the aminopurine derivative peracetylated 11.
On the other hand, chemoselective removal of O-acetyl groups in
compound 9 was achieved using dibutyltin oxide (DBTO) [24]. When
9 was refluxed for 48 h with an excess of DBTO in toluene the
chloropurine analogue 2b was obtained as mainly product (49%)
together with the 20,30-diacetyl derivative 10 (32%). Both
compounds can be easily separated by flash chromatography.
In addition, treatment of nucleosides 7 and 9 with 25% aqueous
ammonia at reflux resulted in substitution of the chloro at C-6
together with deacetylation providing the aminopurine analogues
2c [15] and 2d. Similarly, reaction of 7 and 9 with an excess of
methylamine hydrochloride and triethylamine in MeOH gave the
nucleosides 2e and 2f in 78% and 62% yields respectively.
The 6-phenyl nucleoside 2g was prepared in good yield in two
steps, a SuzukieMiyaura type coupling of 7 with phenylboronic
acid using anhydrous K2CO3 and Pd(Ph3P)4 in toluene [6] to give
the 20,30,50,60-tetra-O-acetyl-6-phenyl nucleoside 8 in 62% yield,
followed by deacetylation with methanolic ammonia at room
temperature.
2. Results and discussion
2.1. Chemistry
The allofuranosyl nucleosides 2aeh were prepared according to
Vorbrüggen method [20] using 1,2,3,5,6-pentaacetoxy-
anose (6 ) as key intermediate.
The allofuranose derivative 6
the commercially available 1,2,5,6-di-O-isopropylidene-
b-D-allofur-
b
b
was obtained in good yield from
-allo-
a-D
furanose (3) in four steps that included a sequence of two selective
O-isopropylidene deprotections [21] each one followed of corres-
ponding acetylation (Scheme 1). Thus, compound 6 was obtained
Finally, the treatment of 9 at room temperature with K2CO3 in
a mixture of dichloromethane-ethanol gave the 2-chloro-6-ethoxy
derivative 2h.
as a mixture of
a- and b-anomers (a:b ratio 1:3.5), which were
The stereochemistry of two series of derivatives was further
established by NOE correlation experiments. Thus, in the NOE
spectrum of 2a, as well as in the NOE spectrum of 9, strong NOE
effects were observed between the anomeric proton H-10 and H-40,
separated by column chromatography [22].
The acetyl protected nucleoside 7 was obtained in 41% yield by
coupling between furanose 6b and the adequate purine, following
Vorbrüggen conditions, as shown in Scheme 2. Thus, 6-chlo-
which indicate that both nucleosides are b-isomers (Fig. 3). More-
ropurine was silylated in situ using the conventional methodology
over, the NOE effects observed for both compounds between H-8
and H-10 provide evidence about the N-9 substitution of the purine
ring [25].
[23] and then was condensed with 6b. The reaction was performed
under reflux in acetonitrile with the presence of trimethylsilyl
triflate (TMSOTf). Two critical factors for the success of this reaction
were the solvent and Lewis acid, since the use of dichloromethane
under similar reaction conditions resulted in a complicated mixture
of products from which the nucleoside 7 was isolated in only a 17%
yield. Moreover, when SnCl2 was used instead of TMSOTf
compound 7 was not obtained.
2.2. Cytostatic activity
The synthesized allofuranosyl nucleosides 2aeh, as well as the
acetyl derivatives 7e11, were evaluated for their cytotoxicity in vitro
in three human cancer cell lines: breast cancer (MCF-7), cervix
Similarly, The acetyl protected nucleoside 9 was obtained in
a
74% yield when furanose 6b was treated with silylated
2,6-dichloropurine. Deacetylation of 7 was performed with metha-
nolic ammonia at 0 ꢀC to provide compound 2a in 73% yield.
However, ammonia was not suitable for selective deacetylation of
9 because of the high reactivity of the chlorine atom at C-6 with
O
O
HO
HO
AcO
AcO
a
b
O
O
O
O
O
O
OH O
OH O
AcO
O
3
4
5
R6
N
2a R6 = Cl, R2 = H
2b R6 = Cl, R2 = Cl
2c R6 = NH2, R2 = H
2d R6 = NH2, R2 = Cl
2e R6 = NHCH3, R2 = H
2f R6= NHCH3, R2 = Cl
2g R6 = Ph, R2 = H
2h R6 = OEt, R2 = Cl
HO
HO
N
N
AcO
AcO
AcO
AcO
N
OAc
c
O
O
O
R2
OAc
AcO
AcO
OAc
OAc
OH OH
6α
6β
Scheme 1. Reagents and conditions: (a) 80% aq. AcOH, r.t., 92%; (b) Ac2O, Py, r.t., 96%;
Fig. 2. Purine nucleosides derivatives of allofuranose synthesized.
(c) (i) 80% aq. TFA, 0 ꢀC, (ii) Ac2O, Py, DMAP, r.t., 84%.

6116
P. Besada et al. / European Journal of Medicinal Chemistry 45 (2010) 6114e6119
NHR6
N
N
N
N
HO
HO
Cl
Cl
N
O
N
N
N
AcO
AcO
c
2c R6 = H
2e R6 = CH3
Ph
N
RO
RO
N
H
HO
N
OH
OAc
a
N
O
O
N
N
AcO
OAc
N
RO
RO
RO OR
7 R = Ac
2a R = H
d
6
Cl
N
N
b
O
N
N
a
NHR6
N
RO OR
R = Ac
2g R = H
N
H
N
N
Cl
RO
RO
8
e
Cl
N
N
Cl
O
N
N
N
11 R = Ac R6 = H
2d R = R6 = H
2f R = H R6 = CH3
RO
RO
g or c
RO
OR
Cl
O
OEt
R'O
OR'
N
N
N
h
HO
HO
9 R = R' = Ac
2b + 10
f
N
Cl
O
2b R = R' = H
10 R=H R' = Ac
HO OH
2h
Scheme 2. Reagents and conditions: (a) HMDS, (NH4)2SO4, TMSOTf, CH3CN, reflux, 41% (7), 74% (9); (b) NH3 2 M MeOH, 0 ꢀC, 73%; (c) NH4OH 25%, CH3CN, reflux, 65% (2c), 60% (2d);
CH3NH2$HCl, Et3N, MeOH, 78% (2e), 62% (2f); (d) PhB(OH)2, K2CO3, Pd(PPh3)4, toluene, 100 ꢀC, 62%; (e) NH3 2 M MeOH, r.t., 90%; (f) DBTO, toluene, 100 ꢀC, 49% (2b), 32% (10); (g) NH3
0.5 M, dioxane, r.t., 70% (11); (h) K2CO3, CH2Cl2-EtOH, r.t., 50%.
cancer (Hela-229) and promyelocytic leukaemia (HL-60) using
cisplatin as the reference compound and the results are included in
Table 1.
Among all the acetyl-free nucleosides tested (compounds 2aeh)
only the 2,6-dichloropurine derivative 2b showed moderate cytos-
tatic activity against MCF-7 and Hela-229 cells with similar IC50
key intermediate. Cytotoxicity studies revealed the 2,6-dichlor-
opurine derivative 2b as the most interesting of the target
compounds, these results are in accordance with previous studies
for the related compounds [8,26]. Compound 2b showed lower
activity than their acetyl derivatives 9 and 10. Thus, the tetra-
O-acetyl derivative 9 was the most potent one on the three cell lines
evaluated, being its activity against HL-60 cells similar to cisplatin.
The cytostatic activity of these chloropurine derivatives appears to
be modulated by inclusion of acetyl groups in the sugar moiety.
Acetyl groups habitually increase the lipophilicity of drugs sup-
porting their cell penetration. This could also explain the activity
shown by the acetyl protected nucleoside 7.
values (54.2 and 47.1
cell line at concentrations below of 100
derivatives (compounds 9e10) showed good activity against the
three tested cell lines with IC50 values ranging from 16.1 to 45.0 M.
mM respectively), being inactive against HL-60
mM. However, their acetyl
m
On the other hand, the acetylated nucleoside 7 that contains
a chlorine atom at C-6 displayed significant cytostatic activity that
was selective against HL-60 cells (IC50 79.3 mM).
In conclusion, a new series of purine nucleosides derivatives of
allofuranose were prepared in good yield according to Vorbrüggen
3. Experimental section
method and using 1,2,3,5,6-pentaacetoxy-b-D-allofuranose (6b) as
3.1. Chemistry
All starting materials and common laboratory chemicals were
purchased from commercial sources and used without further
purification. All solvents were distilled and dried according to
standard procedures.1H and 13C NMR spectra were recorded on
a Bruker ARX400 and Bruker Avance DPX400 instruments, using
Table 1
Cytostatic activity for the nucleosides 2a-h and for the acetyl derivatives 7-11.
Compound
IC50 (m
M)a
MCF-7
Hela-229
HL-60
TMS as internal standard [chemical shifts (d) in ppm, J in Hz].
2a
2b
2c
2d
2e
2f
2g
2h
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
Assignment of the signals was performed by NOE, COSY, DEPT,
HMQC experiments. High resolution mass spectra were recorded
using a Bruker microTOF focus spectrometer. Silica gel (Merck 60,
230e400 mesh) was used for flash chromatography (FC). Prepa-
rative thin layer chromatography was performed on Analtech silica
54.2 (ꢁ 3.7)
47.1 (ꢁ 2.2)
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
gel GF 1000
mm plates. Analytical TLC was performed on plates
> 100
> 100
7
8
> 100
> 100
> 100
> 100
79.3 (ꢁ 5.1)
precoated with silica gel (Merck 60 F254, 0.25 mm).
> 100
9
10
11
22.4 (ꢁ 0.8)
36.3 (ꢁ 1.0)
> 100
24.2 (ꢁ 1.1)
36.3 (ꢁ 1.2)
> 100
16.1 (ꢁ 0.9)
45.0 (ꢁ 0.7)
> 100
3.2. Preparation of 9-(2,3,5,6-tetra-O-acetyl-b-D-allofuranosyl)-
6-chloropurine (7)
Cisplatin
9.2 (ꢁ 0.6)
0.9 (ꢁ 0.2)
14.8 (ꢁ 1.2)
a
A mixture of 6-chloropurine (198 mg, 1.28 mmol), HMDS (2 mL)
Values are means of three experiments, standard deviation is given in
parentheses.
and (NH4)2SO4 (5 mg) was refluxed for 2 h and then the solvent

P. Besada et al. / European Journal of Medicinal Chemistry 45 (2010) 6114e6119
6117
was removed in vacuo. To a mixture of the silylated 6-chloropurine
in CH3CN (4 mL) a solution of compound 6 (191 mg, 0.49 mmol) in
H-20), 5.65 (dd, J ¼ 5.4, 2.1 Hz, 1H, H-30), 5.56 (d, J ¼ 5.1 Hz, 1H,
OH-50), 4.78 (t, J ¼ 5.5 Hz, 1H, OH-60), 4.32e4.30 (m, 1H, H-40),
3.82e3.86 (m, 1H, H-50), 3.43e3.39 (m, 2H, H-60), 2.14 (s, 3H, CH3),
b
CH3CN (3 mL) and TMSOTf (0.13 mL, 0.73 mmol) was added. The
reaction mixture was stirred at room temperature for 1 h and then
refluxed for 15 h, followed by quenching with saturated aq. NaHCO3
(10 mL). The product was extracted with EtOAc (3 ꢂ 10 mL). The
organic layer was dried over Na2SO4, and concentrated to dryness.
The residue was purified by column chromatography on silica gel
(hexaneeethyl acetate, 1:1) to afford 7 (96 mg, 41%) as a colorless
oil. Rf ¼ 0.15 (hexaneeethyl acetate, 1:1); 1H NMR (400 MHz,
1.97 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3):
d
¼ 169.4, 169.1, 153.1,
151.3, 150.1, 146.1, 131.0, 85.1, 84.3, 73.2, 71.1, 70.2, 62.1; HRMS-ESI:
m/z [M
þ
H]þ calcd for C15H17Cl2N4O7: 435.04688, found:
435.04665. Compound 2b: Rf ¼ 0.28 (CH2Cl2-MeOH, 9:1); 1H NMR
(400 MHz, DMSO-d6):
d
¼ 8.97 (s, 1H, H-8), 5.95 (d, J ¼ 6.2 Hz, 1H,
H-10), 5.55 (d, J ¼ 6.0 Hz, 1H, OH-20), 5.25e5.23 (m, 2H, OH-30, OH-
50), 4.65 (t, 1H, J ¼ 5.6 Hz, OH-60), 4.56e4.52 (m, 1H, H-20),
4.26e4.22 (m, 1H, H-30), 4.02e3.99 (m, 1H, H-40), 3.77e3.73 (m, 1H,
H-50), 3.44e3.40 (m, 2H, H-60); 13C NMR (100 MHz, DMSO-d6):
CDCl3):
d
¼ 8.76 (s, 1H, H-purine), 8.18 (s, 1H, H-purine), 6.13
(d, J ¼ 6.4 Hz, 1H, H-10), 6.12e6.07 (m, 1H, H-20), 5.80 (dd, J ¼ 5.3,
3.6 Hz, 1H, H-30), 5.49e5.45 (m, 1H, H-50), 4.44 (dd, J ¼ 12.2, 4.0 Hz,
1H, 1H-60), 4.39 (dd, J ¼ 5.3, 3.6 Hz, 1H, H-40), 4.10 (dd, J ¼ 12.2,
5.2 Hz, 1H, 1H-60), 2.17 (s, 3H, CH3), 2.13 (s, 3H, CH3), 2.06 (s, 3H,
d
¼ 153.2, 151.2, 149.9, 146.4, 130.9, 87.4, 86.0, 74.2, 71.3, 69.3, 62.4;
HRMS-ESI: m/z [M þ Na]þ calcd for C11H12Cl2N4NaO5: 373.16350,
found: 373.16383.
CH3), 2.03 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3):
169.3, 169.1, 152.3, 151.8, 151.2, 144.0, 132.5, 86.6, 81.5, 71.9, 70.5,
70.0, 61.8, 20.9, 20.6, 20.5, 20.3; HRMS-ESI: m/z [M þ H]þ calcd for
C19H22ClN4O9: 485.10698, found: 485.10883.
d
¼ 170.4, 169.8,
3.6. Preparation of 9-b-D-allofuranosyladenine (2c)
To a solution of compound 7 (38 mg, 0.08 mmol) in CH3CN
(1 mL) was added 25% NH4OH (6 mL). The reaction mixture was
stirred at 100 ꢀC in a sealed tube for 13 h. After removal of the
solvent, the residue was purified by preparative thin layer chro-
matography on silica gel (CH2Cl2-MeOH, 8:2) to give 2c (15 mg,
65%) as a white solid. Rf ¼ 0.30 (CH2Cl2-MeOH, 8:2). 1H NMR
3.3. Preparation of 9-(2,3,5,6-tetra-O-acetyl-b-D-allofuranosyl)-
2,6-dichloropurine (9)
Obtained from 2,6-dichloropurine (271 mg, 1.39 mmol), 6
b
(211 mg, 0.54 mmol) and TMSOTf (0.15 mL, 0.81 mmol) following the
same procedure as for preparation of compound 7. The residue was
purified by column chromatography on silica gel (hexaneeethyl
acetate, 1.5:1) to afford 9 (208 mg, 74%) as a colorless oil. Rf ¼ 0.24
(DMSO-d6):
d
¼ 8.22 (s, 1H, H-purine), 8.01 (s, 1H, H-purine), 7.25
(s, 2H, NH2), 5.73 (d, J ¼ 7.3 Hz, 1H, H-10), 5.73e5.70 (m, 1H, OH-50),
5.27 (d, J ¼ 6.6 Hz, 1H, OH-20), 5.04 (d, J ¼ 3.8 Hz, 1H, OH-30),
4.57e4.51 (m, 2H, H-20, OH-60), 4.08e4.06 (m, 1H, H-30), 3.92e3.90
(m, 1H, H-40), 3.60e3.58 (m, 1H, H-50), 3.31e3.28 (m, 2H, H-60). 13C
(hexaneeethyl acetate, 1:1); 1H NMR (400 MHz, CDCl3):
d
¼ 8.20 (s,
1H, H-8), 6.13 (d, J ¼ 6.5 Hz, 1H, H-10), 5.88e5.85 (m, 1H, H-20), 5.73
(dd, J ¼ 5.6, 3.3 Hz, 1H, H-30), 5.47e5.44 (m, 1H, H-50), 4.46 (dd,
J ¼ 12.3, 3.8 Hz,1H,1H-60), 4.38 (dd, J ¼ 5.6, 3.3 Hz,1H, H-40), 4.12 (dd,
J ¼ 12.3, 5.2 Hz, 1H, 1H-60), 2.17 (s, 3H, CH3), 2.13 (s, 3H, CH3), 2.06
NMR (DMSO-d6):
d
¼ 156.6, 152.7, 149.4, 140.6, 119.9, 87.9, 87.1, 73.7,
72.4, 69.9, 62.8. HRMS (ESI) m/z [M þ H]þ calcd for C11H16N5O5:
298.11460; found: 298.11598.
(s, 3H, CH3), 2.04 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3):
d
¼ 170.4,
3.7. Preparation of 9-b-D
-allofuranosyl-N6-methyladenine (2e)
169.8, 169.3, 169.2, 153.5, 152.6, 152.5, 144.0, 131.5, 86.2, 81.6, 72.3,
70.4, 69.9, 61.8, 20.8, 20.6, 20.5, 20.3; HRMS-ESI: m/z [M þ H]þ calcd
for C19H21Cl2N4O9: 519.06801, found: 519.07004.
To a solution of compound 7 (20 mg, 0.04 mmol) in MeOH
(1 mL) was added methylamine hydrochloride (139 mg, 2.06 mmol)
and Et3N (0.43 mL). The reaction mixture was stirred at 65 ꢀC in
a sealed tube for 24 h. After removal of the solvent, the residue was
purified by preparative thin layer chromatography on silica gel
(CH2Cl2-MeOH, 8:2) to give 2e (10 mg, 78%) as a white solid.
3.4. Preparation of 9-b-D-allofuranosyl-6-chloropurine (2a)
To a solution of compound 7 (15 mg, 0.03 mmol) in MeOH
(0.5 mL) was added NH3 2 M in MeOH (3 mL) and the reaction
mixture was stirred at 0 ꢀC for 4 h. After removal of the solvent, the
residue was purified by preparative thin layer chromatography on
silica gel (CH2Cl2-MeOH, 9:1) to give 2a (7 mg, 73%) as a white solid.
Rf ¼ 0.31 (CH2Cl2-MeOH, 8:2). 1H NMR (DMSO-d6):
d
¼ 8.32 (s, 1H,
H-purine), 8.22 (s, 1H, H-purine), 7.85 (s, 1H, NH), 5.84 (d, J ¼ 7.4 Hz,
1H, H-10), 5.84e5.82 (m, 1H, OH-50), 5.36 (d, J ¼ 6.7 Hz, 1H, OH-20),
5.12 (d, J ¼ 3.8 Hz, 1H, OH-30), 4.67e4.61 (m, 2H, OH-60, H-20),
4.20e4.16 (m, 1H, H-30), 4.03e4.01 (m, 1H, H-40), 3.74e3.70 (m, 1H,
H-50), 3.43e3.41 (m, 2H, H-60), 2.96 (s, 3H, CH3). 13C NMR (DMSO-
Rf ¼ 0.15 (CH2Cl2-MeOH, 9:1). 1H NMR (DMSO-d6):
d
¼ 8.94 (s, 1H,
H-purine), 8.81 (s, 1H, H-purine), 6.02 (d, J ¼ 6.5 Hz, 1H, H-10), 5.50
(d, J ¼ 6.1 Hz, 1H, OH-20), 5.25 (d, J ¼ 4.9 Hz, 1H, OH-50), 5.21
(d, J ¼ 4.6 Hz, 1H, OH-30), 4.63e4.60 (m, 2H, H-20, OH-60), 4.28e4.25
(m, 1H, H-30), 4.03e4.01 (m, 1H, H-40), 3.80e3.70 (m, 1H, H-50),
d6):
d
¼ 155.1, 152.2, 147.9, 139.9, 120.0, 87.5, 86.6, 73.3, 71.9, 69.5,
62.3, 26.9. HRMS (ESI) m/z [M þ H]þ calcd for C12H18N5O5:
312.13025; found: 312.13079.
3.40e3.36 (m, 2H, H-60). 13C NMR (DMSO-d6):
d
¼ 151.7 (2C), 149.3,
145.8, 131.3, 87.4, 86.1, 74.0, 71.4, 69.4, 62.4. HRMS (ESI) m/z
[M þ H]þ calcd for C11H14ClN4O5: 317.06472; found: 317.06466.
3.8. Preparation of 9-b-D-allofuranosyl-2-chloroadenine (2d)
Obtained from 9 (27 mg, 0.05 mmol) following the same
procedure as for preparation of compound 2c from 7. The residue
was purified by preparative thin layer chromatography on silica gel
(CH2Cl2-MeOH, 8:2) to afford 2d (10 mg, 60%) as a white solid.
3.5. Preparation of 9-(2,3-di-O-acetyl-
b
-D-allofuranosyl)-2,6-
dichloropurine (10) and 9-
(2b)
b-D-allofuranosyl-2,6-dichloropurine
Rf ¼ 0.36 (CH2Cl2-MeOH, 8:2). 1H NMR (DMSO-d6):
d
¼ 8.37 (s, 1H,
A mixture of 9 (30 mg, 0.06 mmol) and Bu2SnO (124 mg,
0.49 mmol) in toluene (8 mL) was refluxed for 48 h and then the
solvent was removed in vacuo. The residue was purified by column
chromatography on silica gel (CH2Cl2-MeOH, 95:5) to afford 10
(8 mg, 32%) and 2b (10 mg, 49%) both as a colorless oils. Compound
10: Rf ¼ 0.68 (CH2Cl2-MeOH, 9:1);1H NMR (400 MHz, DMSO-d6):
H-8), 7.85 (s, 2H, NH2), 5.79 (d, J ¼ 7.2 Hz, 1H, H-10), 5.41e5.37
(m, 1H, OH-20), 5.23e5.21 (m, 1H, OH-50), 5.16e5.14 (m, 1H, OH-30),
4.61e4.59 (m, 1H, OH-60), 4.55e4.53 (m, 1H, H-20), 4.20e4.18
(m, 1H, H-30), 3.99e3.97 (m, 1H, H-40), 3.72e3.71 (m, 1H, H-50),
3.42e3.40 (m, 2H, H-60). 13C NMR (DMSO-d6):
d
¼ 156.7, 152.9,
150.3, 140.1, 118.1, 86.6, 86.1, 73.6, 71.6, 69.5, 62.3. HRMS (ESI) m/z
d
¼ 8.94 (s, 1H, H-8), 6.24 (d, J ¼ 6.8 Hz, 1H, H-10), 5.85e5.83 (m, 1H,
[M þ H]þ calcd for C11H15ClN5O5: 332.07562; found: 332.07477.

6118
P. Besada et al. / European Journal of Medicinal Chemistry 45 (2010) 6114e6119
3.9. Preparation of 9-
(2f)
b
-
D
-allofuranosyl-2-chloro-N6-methyladenine
(s, 3H, CH3). 13C NMR (CDCl3):
152.7, 152.0, 143.0, 135.2, 131.8, 131.2, 129.8, 128.7, 86.2, 81.3, 71.9,
70.6, 70.2, 61.9, 20.9, 20.7, 20.6, 20.3. HRMS (ESI) m/z [M þ H]þ calcd
for C25H27N4O9: 527.17725; found: 527.17671.
d
¼ 170.4, 169.8, 169.4, 169.2, 155.6,
Obtained from 9 (22 mg, 0.04 mmol) following the same
procedure as for preparation of compound 2e from 7. The residue
was purified by column chromatography on silica gel (CH2Cl2-
MeOH, 93:7) to afford 2f (9 mg, 62%) as a white solid. Rf ¼ 0.38
3.13. Preparation of 9-(2,3,5,6-tetra-O-acetyl-b-D-allofuranosyl)-2-
chloroadenine (11)
(CH2Cl2-MeOH, 8:2). 1H NMR (DMSO-d6):
d
¼ 8.37 (s, 1H, H-8), 8.31
(s, 1H, NH), 5.79 (d, J ¼ 7.0 Hz, 1H, H-10), 5.40 (d, J ¼ 6.6 Hz, 1H, OH-
20), 5.23 (d, J ¼ 4.7 Hz,1H, OH-50), 5.15 (d, J ¼ 4.5 Hz,1H, OH-30), 4.60
(t, J ¼ 5.6 Hz, 1H, OH-60), 4.56e4.52 (m, 1H, H-20), 4.21e4.17 (m, 1H,
H-30), 3.99e3.97 (m, 1H, H-40), 3.72e3.68 (m, 1H, H-50), 3.44e3.40
(m, 2H, H-60), 2.93 (d, J ¼ 4.4 Hz, 3H, CH3). 13C NMR (DMSO-d6):
A solution of compound 9 (10 mg, 0.02 mmol) in NH3 0.5 M in
dioxane (3 mL) was stirred at room temperature for 48 h. After
removal of the solvent, the residue was purified by preparative thin
layer chromatography on silica gel (CH2Cl2-MeOH, 96:4) to give 11
(7 mg, 70%) as a white solid. Rf ¼ 0.65 (CH2Cl2-MeOH, 9:1). 1H NMR
d
¼ 155.5, 153.2, 149.2, 139.9, 118.7, 86.7, 86.1, 73.6, 71.6, 69.5, 62.4,
(DMSO-d6):
d
¼ 8.39 (s,1H, H-8), 7.93 (s, 2H, NH2), 6.15 (d, J ¼ 6.1 Hz,
27.1. HRMS (ESI) m/z [M þ H]þ calcd for C12H17ClN5O5: 346.09127;
1H, H-10), 5.94e5.90 (m, 1H, H-20), 5.68e5.64 (m, 1H, H-30),
5.44e5.40 (m, 1H, H-50), 4.34e4.29 (m, 2H, H-40, 1H-60), 4.08 (dd,
J ¼ 12.3, 5.6 Hz, 1H, 1H-60), 2.13 (s, 3H, CH3), 2.04 (s, 3H, CH3), 2.03
found: 346.09224.
3.10. Preparation of 9-b-D-allofuranosyl-6-phenylpurine (2g)
(s, 3H, CH3), 1.99 (s, 3H, CH3). 13C NMR (DMSO-d6):
d
¼ 169.9, 169.5,
169.2, 169.1, 156.8, 153.2, 150.0, 140.6, 118.3, 85.1, 79.8, 71.3, 69.8,
69.4, 61.4, 20.5, 20.4, 20.3, 20.1. HRMS (ESI) m/z [M þ H]þ calcd for
C19H23ClN5O9: 500.11788; found: 500.11888.
To a solution of compound 8 (15 mg, 0.03 mmol) in MeOH
(0.5 mL) was added NH3 2 M in MeOH (2 mL) and the reaction
mixture was stirred at room temperature for 4 h. After removal of
the solvent, the residue was purified by column chromatography on
silica gel (CH2Cl2-MeOH, 9:1) to give 2g (9 mg, 90%) as a white solid.
3.14. Cytotoxicity assays in vitro
Rf ¼ 0.34 (CH2Cl2-MeOH, 9:1). 1H NMR (DMSO-d6):
d
¼ 9.01 (s, 1H,
Cytotoxicity effects on MCF-7, Hela-229 and HL-60 cells were
determined by means of a colorimetric microculture assay, 3-
(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide
(MTT) assay [27e29]. Briefly, cells were seeded in 96-well plates
(104 cells per well for MCF-7 and HL-60 cell lines, and 4 ꢂ 103 for
Hela-229 cell line), incubated for 24 h in the culture medium and
treated at 37 ꢀC for 96 h (MCF-7), 24 h (HL-60) or 48 h (Hela-229)
with varying doses of evaluated compounds and the reference
drug, cisplatin, dissolved in DMSO. Three wells were used for each
of the variants tested. Aliquots of MTT solution in phosphate-
H-purine), 8.92 (s, 1H, H-purine), 8.83e8.81 (m, 2H, H-phenyl),
7.63e7.60 (m, 3H, H-phenyl), 6.07 (d, J ¼ 6.7 Hz, 1H, H-10), 5.52
(d, J ¼ 6.3 Hz, 1H, OH-20), 5.35 (d, J ¼ 4.8 Hz, 1H, OH-50), 5.23
(d, J ¼ 4.5 Hz, 1H, OH-30), 4.71e4.69 (m, 1H, H-20), 4.65 (t, J ¼ 5.6 Hz,
1H, OH-60), 4.28e4.26 (m, 1H, H-30), 4.04e4.02 (m, 1H, H-40),
3.79e3.75 (m, 1H, H-50), 3.45e3.41 (m, 2H, H-60). 13C NMR (DMSO-
d6):
d
¼ 153.1, 152.3, 151.9, 145.1, 135.2, 131.2, 130.9, 129.4, 128.7,
87.0, 86.1, 73.8, 71.6, 69.5, 62.5. HRMS (ESI) m/z [M þ H]þ calcd for
C17H19N4O5: 359.13500; found: 359.13476.
buffered saline (10 mL) were added to each well and incubated for
3.11. Preparation of 9-b-D-allofuranosyl-2-chloro-6-ethoxypurine
4 h. The color formed was quantified by a spectrophotometric plate
reader (Tecan Ultra evolution) at 595 nm wavelength. In all
experiments, DMSO controls were included. The percentage cell
viability was calculated by dividing the average absorbance of cells
treated with a compound by that of the control. 50% Inhibitory
concentrations (IC50) were calculated from concentration-effect
curves by using GraphPad Prism software, version 2.01. Correlation
coefficients (r2) were higher than 0.998 for all compounds tested.
(2h)
A mixture of compound 9 (15 mg, 0.03 mmol), K2CO3 (7 mg,
0.05 mmol), CH2Cl2 (1 mL) and EtOH (3 mL) was stirred at room
temperature for 4 h. After removal of the solvent, the residue was
purified by preparative thin layer chromatography on silica gel
(CH2Cl2-MeOH, 98:2) to give 2h (5 mg, 50%) as a white solid.
Rf ¼ 0.26 (CH2Cl2-MeOH, 9:1). 1H NMR (DMSO-d6):
d
¼ 8.19 (s, 1H,
H-8), 5.84 (d, J ¼ 5.9 Hz, 1H, H-10), 4.62 (q, J ¼ 7.0 Hz, 2H, CH2),
4.60e4.56 (m, 1H, H-20), 4.41e4.39 (m, 1H, H-30), 4.20e4.18 (m, 1H,
H-40), 4.02e3.98 (m, 1H, H-50), 3.48e3.44 (m, 2H, H-60), 1.46
(t, J ¼ 7.0 Hz, 3H, CH3). HRMS (ESI) m/z [M þ H]þ calcd for
C13H18ClN4O6: 361.09094; found: 361.09161.
Acknowledgements
We acknowledge the Xunta de Galicia (PGIDIT07PXIB, INCI-
TE08ENA314019ES and INCITE09E1R314094ES) and the Uni-
versidade de Vigo for the financial support. P.B. thanks the Xunta de
Galicia for an Isidro Parga Pondal contract.
3.12. Preparation of 9-(2,3,5,6-tetra-O-acetyl-b-D-allofuranosyl)-6-
phenylpurine (8)
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