Synthesis and evaluation of in vitro anticancer activity of some novel isopentenyladenosine derivatives

Article abstract of DOI:10.1016/j.bmc.2010.04.093

The present study describes the synthesis, the characterization and the evaluation of some derivatives of N⁶-isopentenyladenosine on T24 human bladder carcinoma cells. In particular we have modified the hydroxyl groups in the ribose moiety, the position of the isopentenyl chain in the purine ring and the base moiety. The structures of the compounds were confirmed by standard studies of NMR, MS and elemental analysis. We here show that only two derivatives, 1-(3-methyl-2-butenylamino)-9-(3′-deoxy-β-d-ribofuranosyl)-purine hydrobromide and 2-amino-6-(3-methyl-2- butenylamino)-9-(β-d-ribofuranosyl)-purine, inhibit the growth of T24 cells, although to a lower extent than N⁶-isopentenyladenosine. We conclude that the integrity of ribosidic and purine moiety and the N⁶ position of the chain are essential for maintaining the antiproliferative activity.

Full text of DOI:10.1016/j.bmc.2010.04.093

Bioorganic & Medicinal Chemistry 18 (2010) 4249–4254
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of in vitro anticancer activity of some novel
isopentenyladenosine derivatives
Roberta Ottria a, Silvana Casati a, Ada Manzocchi b, Erika Baldoli a, Massimo Mariotti a,
Jeanette A. M. Maier a, Pierangela Ciuffreda a,
*
a Dipartimento di Scienze Cliniche ‘Luigi Sacco’, Via G. B. Grassi 74, Università degli Studi di Milano, 20157 Milano, Italy
b Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Via Saldini 50, Università degli Studi di Milano, 20133 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The present study describes the synthesis, the characterization and the evaluation of some derivatives of
N6-isopentenyladenosine on T24 human bladder carcinoma cells. In particular we have modified the
hydroxyl groups in the ribose moiety, the position of the isopentenyl chain in the purine ring and the base
moiety. The structures of the compounds were confirmed by standard studies of NMR, MS and elemental
Received 25 March 2010
Revised 27 April 2010
Accepted 28 April 2010
Available online 20 May 2010
analysis. We here show that only two derivatives, 1-(3-methyl-2-butenylamino)-9-(30-deoxy-b-
D-ribo-
furanosyl)-purine hydrobromide and 2-amino-6-(3-methyl-2- butenylamino)-9-(b-
D-ribofuranosyl)-pur-
Keywords:
ine, inhibit the growth of T24 cells, although to a lower extent than N6-isopentenyladenosine. We
conclude that the integrity of ribosidic and purine moiety and the N6 position of the chain are essential
for maintaining the antiproliferative activity.
Isopentenyladenosine
Modified nucleosides
Antitumor agents
Bladder carcinoma cells
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Bladder cancer is the fourth most common cancer among men and
the ninth most common cancer among women.8 Its treatment re-
N6-Isopentenyladenosine (iPA, 1a) is a naturally occurring
nucleoside present in yeast and mammalian t-RNA.1,2 In vitro iPA
(1a) was found to inhibit cell multiplication on a variety of human
epithelial cancers.3 Recently, iPA has been shown to act by inhibit-
ing DNA synthesis, causing a cell cycle arrest that correlates with a
decrease in the levels of cyclin E, cyclin A and cyclin D1 and with a
concomitant increase in the levels of cyclin-dependent kinase
inhibitor and p27kip1. Moreover, iPA induces apoptosis through
the downregulation of antiapoptotic products and caspase-3
activation.4
mains a challenge despite significant improvements in preventing
disease progression and improving survival, because bladder can-
cer is characterized by a high rate of tumour recurrence and poten-
tial progression, regardless of treatment with surgery,
chemotherapy, or immunotherapy. We have previously demon-
strated that iPA inhibited the proliferation of human bladder can-
cer cells in a dose-dependent fashion.7
One of our goals is to individuate structural modifications nec-
essary to obtain iPA analogues which maintain biological activity
in vivo. Indeed, we recently reported the synthesis of iPA analogues
with modifications at N9-position which were investigated for
their biological activity in vitro.7 We here report the synthesis of
iPA derivatives modified on ribose and base moiety evaluated for
their biological activity on bladder carcinoma cells.
However, in vivo testing of iPA in the conventional animal tu-
mour screening revealed a modest antitumoral activity which
might be due to the short plasma half-life of iPA, in analogy to
other nucleosides.5,6
To continue our studies concerning the relation between struc-
ture and biological activity,7 as well as to obtain compounds en-
dowed with antiproliferative activity both in vitro and in vivo,
we have synthesized a number of iPA analogues modified in the su-
gar portion (compounds 1b–f), in the alkylation position (com-
pounds 2b–f), in the purine base (compounds 3a–c) or with a
stable base-sugar linkage (compounds 4a–b). We have studied
these molecules for their antiproliferative activity on T24 cells, a
cell line established from a human urinary bladder cancer patient.
2. Results and discussion
2.1. Chemistry
To investigate the structure-anticancer in vitro activity relation-
ship of the lead compound iPA (1a) we modified the sugar portion
(compounds 1b–f), the alkylation position (compounds 2b–f) and
the purine base (compounds 3a–c) (Fig. 1).
Since our aim is to maintain the antiproliferative activity of iPA
in vivo, we were also interested in synthesizing compounds resis-
tant to the action of purine nucleoside phosphorylase (PNP), one of
* Corresponding author. Tel.: +39 0250319695; fax: +39 0250319694.
E-mail address: pierangela.ciuffreda@unimi.it (P. Ciuffreda).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.093

4250
R. Ottria et al. / Bioorg. Med. Chem. 18 (2010) 4249–4254
HN
N
NH2
N
O
N
HN
N
N
N
N
N
N
N
N
N
N
N
N
Br
HO
R1
R1
R1
X
N
O
O
O
O
HO OH
R2 R3
1
R2 R3
R2 R3
3
4
2
a: R1=R2=R3=OH
a: X=Cl
a: R1=R2=R3=OH
b: R1=R2=OH; R3=H
c: R1=R3=OH; R2=H
d: R2=R3=OH; R1=H
e: R1=OH; R2=R3=H
b: X=NH2
b: R1=R2=OH; R3=H
c: R1=OH; R2=R3=H
f: R3=β-OH; R1=R2=OH
Figure 1. Structures of the modified iPA analogues.
the key enzymes in the purine-metabolism.9 Since the substitution
of the hydrogen atom at the 2-position of the purine ring renders
the molecules resistant to metabolic enzymes10 we have also syn-
thesized compounds 4a–b.
2b–f, 3a–c and 4a–b to compare their capability to inhibit cell pro-
liferation and clonogenicity.
Initially, the anti-proliferative capacity of the different com-
pounds was assessed on quiescent and proliferating T24 cells by
the microtiter tetrazolium assay (MTT) as described in the meth-
ods. Our results show the anti-proliferative activity of iPA both in
quiescent (Fig. 2a) and proliferating (Fig. 2b) T24 cells and the lack
of any effect of its derivatives 1b, 1d, 1f, 2b, 2e, 2f, 3a–c and 4a.
Among all synthesized iPA derivatives, compound 4b showed a
slight, albeit statistically significant, inhibitory effect on proliferat-
ing cells. Interestingly, 4b significantly reduced quiescent cell
number, probably because of a cytotoxic effect as previously de-
scribed for iPA.7 It is noteworthy that, when added to the medium
of quiescent T24 cells, compound 2c showed a selective capacity to
reduce cell number, while it did not affect the growth of the cells in
10% FBS. We found that 30-deoxy-adenosine had no effects on T24
cells in the two culture conditions tested (data not shown).
The molecules that had shown some inhibitory effects, namely
2c and 4b, were further investigated by proliferation assays both
Initially, it was necessary to obtain the key intermediates 30-
deoxyadenosine, 50-deoxyadenosine, and 20,30-dideoxyadenosine
that were synthesized according to the procedures reported in
the literature11–13 and were identified by their physical and spec-
troscopic data. On the contrary, the intermediates adenine-9-b-D-
arabinofuranoside, adenine-2-chloro-9-b-
D
-ribofuranoside and
adenine-2-amino-9-b-
D-ribofuranoside
were
commercially
available.
All monosubstituted N6-isopentenyl-deoxyadenosine deriva-
tives 1b–e, were prepared by N1-alkylation of the opportune sub-
strate
with
3,3-dimethylallylbromide.
The
N1-alkylated
derivatives 2b–e were submitted to Dimroth rearrangement to
N6-isomers.14,15 We observed that this alkylation-rearrangement
procedure invariably contained a small amount of the starting
nucleoside that was removed by column chromatography. Analo-
gous reactions using adenine 9-b-D-arabinofuranoside gave at first
the N1-alkylated derivative 2f and then the corresponding N6-iso-
pentenyl derivative 1f.
20,30-Dideoxyinosine and 20-deoxyinosine, key intermediates to
obtain compounds 3b and 3c, respectively, were obtained by enzy-
matic deamination of 20-deoxyadenosine and 20,30-dideoxyadeno-
sine with adenosine deaminase.16 The one step alkylation
reaction gave the desired products 3b and 3c. Isopentenyl-deriva-
tives 3a, 4a and 4b were obtained by one step alkylation reaction
which yield only N6-alkylated compounds starting from the com-
3
a
starvation medium
2.5
2
1.5
*
1
*
*
0.5
mercially available inosine-9-b-
chloro-9-b- -ribofuranoside and
ribofuranoside.
D
-ribofuranoside, adenine-2-
0
D
adenine-2-amino-9-b-
D-
The structure of the synthetic intermediates and of the deriva-
tives 1b–f, 2b–f, 3a–c and 4a–b was confirmed from MS, 1D and
2D NMR spectroscopic data and elemental (C, H, N) analyses which
allowed the correct identification and determined the purity of all
compounds (see Section 5).
complete growth medium
3.5
b
3
2.5
2
*
The correct site of N-alkylation was elucidated by NOE experi-
ments. NOE correlation between H–N6 and
a-CH2 and b-CH of
1.5
1
the isopentenyl moiety could be detected in N6-alkylated deriva-
tives 1b–f, while it is absent in N1-alkylated derivatives 2b–f, thus
demonstrating the binding of the isopentenyl chain in N6-position.
*
0.5
0
Moreover a
-CH2 in side chain resonances of N6-alkylated com-
pounds 1b–f, is more shielded than
a
-CH2 of N1-alkylated com-
pounds 2b–f.
Figure 2. MTT assay on T24 cells treated with iPA and derivatives. (a) MTT
quantification on T24 cells exposed to starvation medium and treated with 10
of iPA or synthesized derivatives. (b) MTT quantification on T24 cells exposed to
2.2. Biological assays
l
M
On the basis of results obtained from dose/response curve per-
formed with iPA,7 we utilized 10
M of iPA and its analogues 1b–f,
complete growth medium and treated with 10
l
M of iPA or synthesized derivatives.
*
l
p <0.05.

R. Ottria et al. / Bioorg. Med. Chem. 18 (2010) 4249–4254
4251
80
70
60
50
40
30
20
10
0
starvation medium
complete growth medium
CTRL
1a
2c
4b
Figure 3. Proliferation assay on T24 cells treated with iPA and derivatives. The cells
were treated with 10
treatment. p <0.05.
lM iPA or selected derivatives and counted after 72 h of
*
on quiescent and proliferating T24 cells. Figure 3 shows counted
cells after 72 h of treatment, only 4b had a slight inhibitory effect
on cell cultured in complete growth medium while compounds
2c and 4b markedly reduced the number of quiescent cells.
We then performed clonogenic assays in the presence of iPA
and all its derivatives (10 lM). After a week, colonies were fixed
with crystal violet and the plates were photographed. We con-
firmed that iPA inhibited the formation of clones, while, in agree-
ment with the aforementioned results, apart from
a slight
reduction of number and dimension of clones in cells treated with
4b, all the compounds did not exert any effect on the formation of
clones (Fig. 4).
We also evaluated the effects of iPA and synthesized analogues
on cell migration, which is fundamental for invasion and metasta-
sis. T24 cells were treated for 8 h with iPA or analogues and then
exposed to EGF (10 ng/mL) for additional 14 h. iPA and its deriva-
tives exerted no effect on basal or EGF-stimulated migration in
T24 cells. Figure 5 shows that, in spite of their antiproliferative
activity, iPA, compound 2c and 4b do not inhibit cell migration.
3. Conclusion
In summary, we have synthesized a series of novel iPA deriva-
tives and we have determined their antiproliferative activity after
modification of the hydroxyl groups in the ribose moiety, of the
alkylation position and of the base moiety.
Figure 4. Clonogenic assay on T24 cells treated with iPA and derivatives. T24 cells
were seeded at very low density, treated with iPA and derivatives for 72 h and
maintained thereafter in culture medium alone until the end of the experiment. The
cells were stained and photographed (magnification 40ꢀ).
All the modifications drastically inhibited iPA biological activity.
On these bases, we can conclude that the iPA ribofuranosidic and
base moiety are pivotal for iPA antiproliferative activity in vitro.
Among all the synthesized derivatives only compound 4b, charac-
terized by the presence of an amino group in position 2, showed a
slight inhibitory effect on proliferating cells and a more evident ac-
tion on quiescent cells. Therefore, 4b is the only compound which
mimics iPA, even though with a lower efficiency. We hypothesize
that the presence of the amino group in 2-position in 4b renders
this molecule more bioavailable than the others. Interestingly,
compound 2c, which has the isopentenyl chain in N1-position,
selectively acts on quiescent cells. The differential action of com-
pound 2c on quiescent and proliferating cells is puzzling. That a
molecule selectively acts on quiescent cells might be relevant in
primary or metastatic tumors, where part of the cells are dormient
and might reactivate after many years, as often reported in the case
of bladder carcinoma. However, at the moment we can not provide
any explanation for these different effects. More studies are neces-
sary to understand the mechanisms involved.
250
CTRL
1a
2c
4b
200
150
100
50
0
Basal
EGF
Figure 5. Effects of iPA, 2c and 4b on T24 bladder cancer cell migration. The cells
were treated with EGF (100 ng/ml) and the different molecules (or an equal amount
of DMSO as a control—CTRL-) for 16 h. The wound area was calculated by a specific
software and expressed using an arbitrary value scale.
It is noteworthy that iPA and its derivatives exert no effect on
cell migration, which is fundamental in invasion and metastasis.

4252
R. Ottria et al. / Bioorg. Med. Chem. 18 (2010) 4249–4254
We argue that iPA and its derivatives do not interfere with the
complex signalling pathways necessary for cell migration.
In conclusion, our results demonstrate that the integrity of
ribosidic and purine moiety and the N6-position of the isopentenyl
chain are essential for maintaining the antiproliferative activity. In-
deed, it is sufficient to modify the stereochemistry of 20-hydroxy
group in the ribose moiety (compound 1f) or to substitute a nitro-
gen with an oxygen atom in the 6-position of the base (compound
3a) to completely abolish the activity.
The results of this investigation prompt us to continue the
development and testing of novel derivatives of iPA with the aim
of individuating novel biologically active compounds which main-
tain their activity also in vivo.
MeOH); 1H NMR (CD3OD) d = 1.86 (s, 3H, CH3), 1.88 (s, 3H, CH3),
2.51 (ddd, J = 3.5 Hz, J = 6.3 Hz, J = 13.2 Hz, 1H, H20a), 2.77 (ddd,
J = 6.3 Hz, J = 6.3 Hz, J = 13.2 Hz, 1H, H20b), 3.76 (dd, J = 4.2 Hz,
J = 12.2 Hz, 1H, H50a), 3.82 (dd, J = 3.5 Hz, J = 12.2 Hz, 1H, H50b),
4.07 (ddd, J = 2.8 Hz, J = 3.5 Hz, J = 4.2 Hz, 1H, H40), 4.59 (ddd,
J = 2.8 Hz, J = 3.5 Hz, J = 6.3 Hz, 1H, H30), 4.87 (d, J = 6.6 Hz, 2H,
a-
CH2), 5.44 (t, J = 6.6 Hz, 1H, b-CH), 6.48 (dd, J = 6.3 Hz, J = 6.3 Hz,
1H, H10), 8.40 (s, 1H, H2), 8.53 (s, 1H, H8); MS (EI): m/z 320
(MꢂHBr+1, 100%). Anal. Calcd for C15H22BrN5O3: C, 45.01; H,
5.54; Br, 19.96; N, 17.50; O, 11.99. Found: C, 45.12; H, 5.44; Br,
19.82; N, 17.41.
4.2.1.2. 6-(3-Methyl-2-butenylamino)-9-(20-deoxy-b-
D-ribofur-
anosyl)-purine (1b).
White solid; 86% yield; Rf = 0.63
(CH2Cl2/MeOH, 90:10); mp 108–109 °C; ½a D20
ꢂ19.2 (c 1, MeOH)
ꢁ
4. Experimental
4.1. General
{lit.17 mp 106–109 °C; ½a 2D0
ꢁ
ꢂ19.5 (c 1, MeOH)}; 1H NMR (CD3OD)
d = 1.77 (s, 6H, 2 ꢀ CH3), 2.42 (ddd, J = 3.5 Hz, J = 8.2 Hz,
J = 13.4 Hz, 1H, H20a), 2.82 (ddd, J = 5.9 Hz, J = 6.3 Hz, J = 13.4 Hz,
1H, H20b), 3.76 (dd, J = 3.1 Hz, J = 12.3 Hz, 1H, H50a), 3.86 (dd,
J = 2.8 Hz, J = 12.3 Hz, 1H, H50b), 4.09 (ddd, J = 2.1 Hz, J = 2.8 Hz,
Melting points were determined with a Stuart Scientific SMP3
melting point apparatus and left uncorrected. Optical rotations
were measured on a Perkin–Elmer 241 polarimeter (sodium D line
at 25 °C). NMR spectra were done on a Bruker AVANCE 500 spec-
trometer equipped with a 5 mm broadband reverse probe with
field z-gradient operating at 500.13 MHz for 1H. All NMR spectra
were recorded at 298 K in CDCl3 (isotopic enrichment 99.95%) or
CD3OD (isotopic enrichment 99.95%) solution and the chemical
shifts were reported on a d (ppm) scale. The central peak of CDCl3
signals (7.26 ppm) and of CD3OD signals (3.31 ppm) were used as
internal reference standard. The splitting pattern abbreviations
are as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multi-
plet, and br, broad singlet.
J = 3.1 Hz, 1H, H40), 4.17 (br, 2H,
a-CH2), 4.59 (ddd, J = 2.1 Hz,
J = 3.5 Hz, J = 5.9 Hz, 1H, H30), 5.39 (t, J = 6.9 Hz, 1H, b-CH), 6.43
(dd, J = 6.3 Hz, J = 8.2 Hz, 1H, H10), 8.22 (s, 1H, H2), 8.26 (s, 1H,
H8); MS (EI): m/z 320 (M+1, 100%), 343 (M+23, 32%).
4.2.2. 6-(3-Methyl-2-butenylamino)-9-(30-deoxy-b-
D-ribofurano-
syl)-purine (1c)
4.2.2.1. 1-(3-Methyl-2-butenylamino)-9-(30-deoxy-b-
D-ribofurano-
syl)-purine hydrobromide (2c).
White solid; 55% yield;
Rf = 0.28 (CH2Cl2/MeOH, 90:10); mp 177–179 °C; ½a D20
ꢂ44.7 (c 1,
ꢁ
MeOH); 1H NMR (CD3OD) d = 1.86 (s, 3H, CH3), 1.88 (s, 3H, CH3),
2.05 (ddd, J = 2.7 Hz, J = 6.2 Hz, Hz, J = 13.8 Hz, 1H, H30a), 2.36
(ddd, J = 6.2 Hz, J = 9.7, J = 13.8 Hz, 1H, H30b), 3.71 (dd, J = 3.4 Hz,
J = 12.2 Hz, 1H, H50a), 3.94 (dd, J = 2.8 Hz, J = 12.2 Hz, 1H, H50b),
4.57 (dddd, J = 2.8 Hz, J = 3.4 Hz, J = 6.2 Hz, J = 9.7 Hz, 1H, H40),
4.59 (ddd, J = 1.4 Hz, J = 2.7 Hz, J = 6.2 Hz, 1H, H20), 4.87 (d,
Mass spectra were recorded on a Finnigan LCQdeca (Thermo-
Quest) in ESI negative ion mode, kV 5.00, 220 °C, 15 V. Only signif-
icant m/z peaks, with their percentage of relative intensity in
parentheses are reported.
Reactions progress was monitored by analytical thin-layer chro-
matography (TLC) on pre-coated glass plates (Silica Gel 60 F254-
plate-Merck, Darmstaat, Germany) and the products were visual-
ized by UV light. Elemental analyses were obtained for all interme-
diates and are within 0.4% of theoretical values.
J = 6.6 Hz, 2H,
a-CH2), 5.44 (t, J = 6.6 Hz, 1H, b-CH), 6.04 (d,
J = 1.4 Hz, 1H, H10), 8.37 (s, 1H, H2), 8.60 (s, 1H, H8); MS (EI): m/z
320 (MꢂHBr+1, 100%), 343 (MꢂHBr+1+(23), 80%). Anal. Calcd for
C15H22BrN5O3: C, 45.01; H, 5.54; Br, 19.96; N, 17.50; O, 11.99.
Found: C, 45.15; H, 5.48; Br, 20.02; N, 17.60.
Adenosine, 20-deoxyadenosine, inosine, adenine 9-b-
furanoside, adenine-2-chloro-9-b- -ribofuranoside and adenine-
2-amino 9-b- -ribofuranoside, the other reagents and all solvents
D-arabino-
D
D
4.2.2.2. 6-(3-Methyl-2-butenylamino)-9-(30-deoxy-b-
D-ribofurano-
were purchased from Sigma–Aldrich (St. Louis, MO, USA). Organic
solvents were dried in the presence of appropriate drying agents
and were stored over suitable molecular sieves.
syl)-purine (1c).
White solid; 88% yield; Rf = 0.78 (CH2Cl2/
MeOH, 90:10); mp 148–149 °C; ½a D20
ꢁ
ꢂ42.8 (c 1, MeOH); 1H NMR
(CD3OD) d = 1.79 (s, 6H, 2 ꢀ CH3), 2.09 (ddd, J = 3.8 Hz, J = 6.6 Hz,
J = 13.2 Hz, 1H, H30a), 2.39 (ddd, J = 6.2 Hz, J = 9.4 Hz, J = 13.2 Hz,
1H, H30b), 3.68 (dd, J = 3.5 Hz, J = 12.2 Hz, 1H, H50a), 3.94 (dd,
4.2. General procedure for N6-alkylation
J = 2.4 Hz, J = 12.2 Hz, 1H, H50b), 4.19 (br, 2H,
a-CH2), 4.54 (dddd,
BaCO3 (2.4 mmol) and 3,3-dimethylallylbromide (2.25 mmol)
were added to a solution of nucleoside derivative (1.5 mmol) in
DMF (20 mL). The mixture was stirred at room temperature for
24 h while protected from light and moisture. TLC indicated that
N1-alkylation was about 90% complete. The moisture was filtered
using a Celite pad and washed with DMF. The combined filtrate
was evaporated to a small volume and purified by column chroma-
tography on silica gel. The N1-alkylated derivative was treated with
Me2NH–MeOH (1 M, 4.5 mL) at room temperature for 16 h. The
solvent was removed and the residue purified by column chroma-
tography on silica gel.
J = 2.4 Hz, J = 3.5 Hz, J = 6.6 Hz, J = 9.4 Hz, 1H, H40), 4.72 (ddd,
J = 2.8 Hz, J = 3.8 Hz, J = 6.2 Hz, 1H, H20), 5.41 (t, J = 6.9 Hz, 1H, b-
CH), 5.96 (d, J = 2.8 Hz, 1H, H10), 8.26 (s, 1H, H2), 8.37 (s, 1H,
H8); MS (EI): m/z 320 (M+1, 20%), 389 (M+1+(3 ꢀ 23), 100%). Anal.
Calcd for C15H21N5O3: C, 56.41; H, 6.63; N, 21.93; O, 15.03. Found C,
56.28; H, 6.86; N, 21.79.
4.2.3. 6-(3-Methyl-2-butenylamino)-9-(50-deoxy-b-
D-ribofurano-
syl)-purine (1d)
4.2.3.1. 1-(3-Methyl-2-butenylamino)-9-(50-deoxy-b-
D-ribofurano-
syl)-purine hydrobromide (2d).
White solid; 48% yield;
Rf = 0.36 (CH2Cl2/MeOH, 90:10); mp 96–98 °C; ½a D20
ꢂ23.2 (c 1,
ꢁ
4.2.1. 6-(3-Methyl-2-butenylamino)-9-(20-deoxy-b-
D-ribofurano-
1
MeOH); H NMR (CD3OD) d = 41.45 (d, J = 6.5 Hz, 3H, H50) 1.83 (s,
3H, CH3), 1.87 (s, 3H, CH3), 4.09 (dd, J = 4.5 Hz, J = 5.2 Hz, 1H,
H30), 4.16 (dq, J = 5.2 Hz, J = 6.5 Hz, 1H, H40), 4.68 (dd, J = 4.5,
syl)-purine (1b)
4.2.1.1. 1-(3-Methyl-2-butenylamino)-9-(20-deoxy-b-
D
-ribofur-
anosyl)-purine hydrobromide (2b).
White solid; 62% yield;
J = 5.2, 1H, H20), 4.90 (d, J = 6.5 Hz, 2H,
a-CH2), 5.40 (t, J = 6.5 Hz,
Rf = 0.25 (CH2Cl2/MeOH, 90:10); mp 104–106 °C; ½a D20
ꢂ19.8 (c 1,
ꢁ
1H, b-CH), 6.04 (d, J = 4.5 Hz, 1H, H10), 8.46 (s, 1H, H2), 8.51 (s,

R. Ottria et al. / Bioorg. Med. Chem. 18 (2010) 4249–4254
4253
1H, H8); MS (EI): m/z 343 (MꢂHBr+1+23, 100%). Anal. Calcd for
4.2.6. 6-(3-Methyl-2-butenyloxy)-9-(b-
(3a)
D-ribofuranosyl)-purine
C15H22BrN5O3: C, 45.01; H, 5.54; Br, 19.96; N, 17.50; O, 11.99.
Found: C, 44.98; H, 5.39; N, 17.61.
White solid; 46% yield; Rf = 0.37 (CH2Cl2/MeOH, 95:5); mp 56–
58 °C; ½a 2D0
ꢁ
ꢂ37.4 (c 1, MeOH); 1H NMR (CD3OD) d = 1.79 (s, 3H,
4.2.3.2. 6-(3-Methyl-2-butenylamino)-9-(50-deoxy-b-
D-ribofurano-
CH3), 1.87 (s, 3H, CH3), 3.77 (dd, J = 3.1 Hz, J = 12.2 Hz, 1H, H50a),
3.88 (dd, J = 2.8 Hz, J = 12.2 Hz, 1H, H50b), 4.15 (ddd, J = 2.8 Hz,
J = 3.1 Hz, J = 3.8 Hz, 1H, H40), 4.34 (dd, J = 3.8 Hz, J = 5.6 Hz, 1H,
H30), 4.64 (dd, J = 5.6 Hz, J = 5.6 Hz, 1H, H20), 4.72 (d, J = 6.9 Hz,
syl)-purine (1d).
White solid; 77% yield; Rf = 0.67 (CH2Cl2/
MeOH, 90:10); mp 124–126 °C {lit.15 mp 96–97 °C}; ½a 2D0
ꢂ37.7;
ꢁ
1H NMR (CD3OD) d = 1.45 (d, J = 6.5 Hz, 3H, CH3), 1.79 (s, 6H,
2 ꢀ CH3), 4.09 (dd, J = 4.5 Hz, J = 5.2 Hz, 1H, H30), 4.14 (dq,
2H,
a-CH2), 5.36 (t, J = 6.9 Hz, 1H, b-CH), 6.02 (d, J = 5.6 Hz, 1H,
J = 5.2 Hz, J = 6.5 Hz, 1H, H40), 4.20 (br, 2H,
a-CH2), 4.70 (dd,
H10), 8.29 (s, 1H, H2), 8.33 (s, 1H, H8); MS (EI): m/z 359 (M+23,
47%), 695 (2M+23, 100%). Anal. Calcd for C15H20N4O5: C, 53.56;
H, 5.99; N, 16.66; O, 23.78. Found C, 53.42; H, 5.88; N, 16.49.
J = 4.5 Hz, J = 5.2 Hz, 1H, H20), 5.41 (t, J = 6.9 Hz, 1H, b-CH), 5.97
(d, J = 4.5 Hz, 1H, H10), 8.20 (s, 1H, H8), 8.27 (s, 1H, H2); MS (EI):
m/z 388 (M+(3 ꢀ 23), 100%).
4.2.7. 6-(3-Methyl-2-butenyloxy)-9-(20-deoxy-b-
D-ribofuranosyl)-
purine (3b)
4.2.4. 6-(3-Methyl-2-butenylamino)-9-(20,30-dideoxy-b-
D-ribofur-
anosyl)-purine (1e)
White solid; 52% yield; Rf = 0.21 (CH2Cl2/MeOH, 90:10); mp
4.2.4.1. 1-(3-Methyl-2-butenylamino)-9-(20,30-dideoxy-b-
D
-ribo-
126–128 °C; ½a 2D0
ꢁ
ꢂ4.0 (c 1, MeOH); 1H NMR (CD3OD) d = 1.79 (s,
furanosyl)-purine hydrobromide (2e).
White solid; 45%
3H, CH3), 1.87 (s, 3H, CH3), 2.46 (ddd, J = 3.2 Hz, J = 6.3 Hz,
J = 13.2 Hz, 1H, H20a), 2.76 (ddd, J = 6.3 Hz, J = 6.3 Hz, J = 13.2 Hz,
1H, H20b), 3.75 (dd, J = 3.5 Hz, J = 12.2 Hz, 1H, H50a), 3.82 (dd,
J = 3.5 Hz, J = 12.2 Hz, 1H, H50b), 4.05 (ddd, J = 3.5 Hz, J = 3.5 Hz,
J = 3.5 Hz, 1H, H40), 4.58 (ddd, J = 3.2 Hz, J = 3.5 Hz, J = 6.3 Hz, 1H,
yield; Rf = 0.22 (CH2Cl2/MeOH, 90:10); mp 100–102 °C;
½ ꢁ
a 2D0
ꢂ5.73 (c 1, MeOH); 1H NMR (CD3OD) d = 1.84 (s, 3H, CH3), 1.87
(s, 3H, CH3), 2.12–2.24 (m, 2H, H30), 2.48–2.56 (m, 2H, H20), 3.69
(dd, J = 4.5 Hz, J = 12.1 Hz, 1H, H50a), 3.86 (dd, J = 3.1 Hz,
J = 12.1 Hz, 1H, H50b), 4.26–4.30 (m, 1H, H40), 4.81 (d, J = 6.9 Hz,
H30), 4.72 (d, J = 6.6 Hz, 2H,
a-CH2), 5.38 (t, J = 6.6 Hz, 1H, b-CH),
2H,
a
-CH2), 5.43 (t, J = 6.9 Hz, 1H, b-CH), 6.32 (dd, J = 6.6 Hz,
6.44 (dd, J = 6.3 Hz, J = 6.3 Hz, 1H, H10), 8.28 (s, 1H, H2), 8.32 (s,
1H, H8); MS (EI): m/z 344 (M+1+23, 28%), 665 (2 ꢀ (M+1)+23,
100%). Anal. Calcd for C15H20N4O4: C: 56.24, H: 6.29, N: 17.49, O:
19.98. Found C, 56.12; H, 6.10; N, 17.39.
J = 3.1 Hz, 1H, H10), 8.27 (s, 1H, H2), 8.49 (s, 1H, H8); MS (EI): m/z
304 (MꢂHBr+1, 100%). Anal. Calcd for C15H22BrN5O2: C, 46.88; H,
5.77; Br, 20.79; N, 18.23; O, 8.33. Found: C, 46.72; H, 5.70; Br,
20.82; N, 18.38.
4.2.8. 6-(3-Methyl-2-butenyloxy)-9-(20,30-deoxy-b-
purine (3c)
D-ribofuranosyl)-
4.2.4.2. 6-(3-Methyl-2-butenylamino)-9-(20,30-dideoxy-b-
D-ribo-
furanosyl)-purine (1e).
White solid; 82% yield; Rf = 0.74
White solid; 68% yield; Rf = 0.53 (CH2Cl2/MeOH, 90:10); mp
(CH2Cl2/MeOH, 90:10); mp 105–107 °C; ½a D20
ꢁ
ꢂ12.6 (c 1, MeOH);
110–112 °C; ½a 2D0
ꢁ
ꢂ4.08 (c 1, MeOH); 1H NMR (CD3OD) d = 1.79
1H NMR (CD3OD) d = 1.79 (s, 6H, 2 ꢀ CH3), 2.12–2.24 (m, 2H,
H30), 2.48–2.58 (m, 2H, H20), 3.68 (dd, J = 3.8 Hz, J = 12.2 Hz, 1H,
(s, 3H, CH3), 1.87 (s, 3H, CH3), 2.07–2.20 (m, 2H, H30), 2.47–2.61
(m, 2H, H20), 3.69 (dd, J = 4.5 Hz, J = 11.8 Hz, 1H, H50a), 3.85 (dd,
J = 3.2 Hz, J = 11.8 Hz, 1H, H50b), 4.24–4.29 (m, 1H, H40), 4.72 (d,
H50a), 3.89 (dd, J = 2.8 Hz, J = 12.2 Hz, 1H, H50b), 4.19 (br, 2H,
a-
CH2), 4.26–4.31 (m, 1H, H40), 5.40 (t, J = 6.9 Hz, 1H, b-CH), 6.30
(dd, J = 4.5 Hz, J = 5.9 Hz, 1H, H10), 8.25 (s, 1H, H2), 8.36 (s, 1H,
H8); MS (EI): m/z 304 (M+1, 100%). Anal. Calcd for C15H21N5O2: C,
59.39; H, 6.98; N, 23.09; O, 10.55. Found C, 59.26; H, 6.88; N, 23.14.
J = 6.9 Hz, 2H, a-CH2), 5.36 (t, J = 6.9 Hz, 1H, b-CH), 6.31 (dd,
J = 3.5 Hz, J = 6.3 Hz, 1H, H10), 8.24 (s, 1H, H2), 8.27 (s, 1H, H8);
MS (EI): m/z 336 (Mꢂ1, 30%), 673 (2Mꢂ1, 100%). Anal. Calcd for
C15H20N4O3: C, 59.20; H, 6.62; N, 18.41; O, 15.77. Found C, 59.12;
H, 6.48; N, 18.26.
4.2.5. 6-(3-Methyl-2-butenylamino)-9-b-D-arabinofuranosyl)-
purine (1f)
4.2.9. 2-Chloro-6-(3-methyl-2- butenylamino)-9-(b-D-
ribofuranosyl)-purine (4a)
4.2.5.1. 1-(3-Methyl-2-butenylamino)-9-b-
D
-arabinofuranosyl)-
White solid; 34% yield; Rf = 0.27 (CH2Cl2/MeOH, 95:5); mp 148–
purine hydrobromide (2f).
White solid; 52% yield; Rf = 0.28
150 °C; ½a 2D0
ꢁ
ꢂ6.9 (c 1, MeOH); 1H NMR (CD3OD) d = 1.74 (s, 6H,
(CH2Cl2/MeOH, 90:10); mp 147–148; ½a D20
ꢁ
+2.2 (c 1, MeOH); 1H
2 ꢀ CH3), 3.75 (dd, J = 3.1 Hz, J = 12.2 Hz, 1H, H50a), 3.86 (dd,
NMR (CD3OD) d = 1.87 (s, 3H, CH3), 1.90 (s, 3H, CH3), 3.87 (dd,
J = 3.4 Hz, J = 11.9 Hz, 1H, H50a), 3.91 (dd, J = 4.5 Hz, J = 11.9 Hz,
1H, H50b), 4.02 (ddd, J = 3.4 Hz, J = 4.5 Hz, J = 4.5 Hz, 1H, H40), 4.31
(dd, J = 4.5 Hz, J = 4.5 Hz, 1H, H30), 4.35 (dd, J = 4.5 Hz, J = 4.9 Hz,
J = 2.4 Hz, J = 12.2 Hz, 1H, H50b), 3.95 (d, J = 7.0 Hz, 2H,
a-CH2),
4.14 (ddd, J = 2.4 Hz, J = 3.1 Hz, J = 3.1 Hz, 1H, H40), 4.33 (dd,
J = 3.1 Hz, J = 5.4 Hz, 1H, H30), 4.79 (dd, J = 5.4 Hz, J = 6.7 Hz, 1H,
H20), 5.33 (t, J = 7.0 Hz, 1H, b-CH), 5.85 (d, J = 6.7 Hz, 1H, H10),
8.00 (s, 1H, H8); MS (EI): m/z 370 (M+1, 100%). Anal. Calcd for
C15H20ClN5O4: C, 48.72; H, 5.45; Cl, 9.59; N, 18.94;O, 7.31. Found
C, 48.59; H, 5.39; N 19.02.
1H, H20), 4.96 (d, J = 6.8 Hz, 2H,
a-CH2), 5.45 (tt, J = 1.5 Hz,
J = 6.8 Hz, 1H, b-CH), 6.46 (d, J = 4.9 Hz, 1H, H10), 8.55 (s, 1H, H2),
8.26 (s, 1H, H8); MS (EI) m/z 336 (MꢂHBr+1, 100%). Anal. Calcd
for C15H22BrN5O4: C, 43.28; H, 5.33; Br, 19.20; N, 16.82; O, 15.37.
Found: C, 43.12; H, 5.44; Br, 19.12; N, 16.91.
4.2.10. 2-Amino-6-(3-methyl-2-butenylamino)-9-(b-D-ribofurano-
syl)-purine (4b)
4.2.5.2. 6-(3-Methyl-2-butenylamino)-9-b-D-arabinofuranosyl)-
White solid; 48% yield; Rf = 0.7 (CH2Cl2/MeOH, 95:5); mp 210–
212 °C; ½a 2D0
ꢁ
ꢂ2.9 (c 1, MeOH); 1H NMR (CD3OD) d = 1.82 (s, 3H,
purine (1f).
White solid; 84% yield; Rf = 0.65 (CH2Cl2/MeOH,
90:10); mp 180–182 °C; ½a D20
ꢁ
2.5 (c 1, MeOH) {lit.15 mp 161.5–
CH3), 1.89 (s, 3H, CH3), 3.72 (d, J = 7.0 Hz, 2H,
a-CH2), 3.77 (dd,
162 °C; ½a 2D0
ꢁ
1.9 (c 1, MeOH)}; 1H NMR (CD3OD) d = 1.79 (s, 6H,
J = 3.1 Hz, J = 12.2 Hz, 1H, H50a), 3.91 (dd, J = 2.4 Hz, J = 12.2 Hz,
1H, H50b), 4.17 (ddd, J = 2.4 Hz, J = 3.1 Hz, J = 3.1 Hz, 1H, H40),
4.34 (dd, J = 3.1 Hz, J = 5.2 Hz, 1H, H30), 4.69 (dd, J = 5.2 Hz,
J = 6.0 Hz, 1H, H20), 5.34 (t, J = 7.0 Hz, 1H, b-CH), 5.94 (d,
J = 6.0 Hz, 1H, H10), 8.31 (s, 1H, H8); MS (EI): m/z 351 (M+1,
100%). Anal. Calcd for C15H22N6O4: C, 51.42; H, 6.33; N, 23.99; O,
18.27. Found: C, 51.32; H, 6.38, N, 24.01.
2 ꢀ CH3), 3.86 (dd, J = 4.5 Hz, J = 11.9 Hz, 1H, H50a), 3.92 (dd,
J = 3.5 Hz, J = 11.9 Hz, 1H, H50b), 4.00 (ddd, J = 3.5 Hz, J = 4.5 Hz,
J = 4.9 Hz, 1H, H40), 4.20 (d, J = 6.9 Hz, 2H,
a-CH2), 4.28–4.32 (m,
2H, H20, H30), 5.41 (t, J = 6.9 Hz, 1H, b-CH), 6.40 (d, J = 4.9 Hz, 1H,
H10), 8.25 (s, 1H, H2), 8.32 (s, 1H, H8); MS (EI): m/z 320 (M+1,
100%), 343 (M+23, 52%).

4254
R. Ottria et al. / Bioorg. Med. Chem. 18 (2010) 4249–4254
4.3. Biological assays
incubated with EGF (100 ng/mL) to stimulate cell migration and
the different molecules (10 M) for additional 16 h. This initial
l
4.3.1. Cell culture
T24 (human bladder carcinoma), were grown in D-MEM med-
ium supplemented with 10% fetal bovine serum (FBS), 1 mM L-glu-
wounding and the movement of the cells in the scratched area
were photographically monitored using an Axiovert Zeiss 200
microscope with a 10ꢀ (0.25 numerical aperture) objective linked
to camera. The 16 h-time interval has been chosen because it is
shorter than cell doubling time in these conditions. Four different
fields from each sample were analysed using the ImageJ software
for quantitative estimations of the number of cells that have mi-
grated to the wounded area. The values are expressed as the rela-
tive cell migration compared to control condition.
tamine and 1 mM penicillin/streptomycin (complete growth
medium). In some experiments cells were cultured in D-MEM with
0.1% FBS (starvation medium) for 48 h to induce quiescence. This
treatment significantly reduced cell growth and 3H-thymidine
incorporation (not shown). The cells were cultured in 5% CO2 at
37 °C. All the reagents for cell culture were from Gibco. Stock solu-
tions of iPA and various compounds were prepared in DMSO and
kept at ꢂ20 °C. Appropriate dilutions of the compounds were
freshly prepared in culture medium just prior the assays. The con-
trols were added with the final concentrations of DMSO (0.01%).
4.3.5. Statistical analysis
All the experiments were performed at least 3 times in tripli-
cates and data are shown as the means standard deviation. Sta-
tistical significance was determined using the Student’s T test. In
*
4.3.2. Cell proliferation
the figures: p <0.05.
For proliferation assays, the cells were seeded at low density
(2000/cm2) in 6-well plates in growth medium. Cells were allowed
to attach for 16 h before being exposed to starvation or complete
growth medium. After 48 h, the cells were treated with different
compounds. On the basis of previous studies, the different mole-
Acknowledgement
This work has been supported by Università degli Studi di
Milano (Fondi FIRST).
cules were used at a concentration 10 l
M.7 After 72 h, the cells
were harvested with trypsin-EDTA and stained with a trypan blue
solution. The viable cells were counted using Burker chamber.
In another set of experiments, the different molecules were
tested on proliferating or quiescent cells seeded at low density
(2000/cm2) by the MTT reduction assay. This method is based on
the reduction of MTT by mitochondrial dehydrogenase of intact
cells to a purple formazan product. Briefly, 16 h after seeding, the
cells were exposed either to a starvation medium or to fresh com-
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.04.093.
References and notes
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added for three additional days.
lM) were
Before (1 h) the end of treatment the medium was replaced
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yl)-2,5-diphenyltetrasodium bromide (Sigma, Oakville, Ontario,
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4.3.3. Clonogenic assay
5000 T24 cells per well were seeded into 6-well plates and cul-
tured in D-MEM containing 10% FBS supplemented with the com-
pound of interest (10 lM). The medium was replaced every two
days and supplemented with fresh compounds. After culture for
a week, colonies were fixed in 0.5% crystal violet in methanol
and extensively washed. The plates were then photographed.
4.3.4. Migration assay
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Cell migration was determined using an in vitro model of
wound repair.18 Briefly, the cells were grown in 24-well plates to
confluence. After 24 h starvation in medium with 0.1% FBS, the
monolayer was wounded by scratching the surface as uniformly
as possible with a pipette tip and then cells were simultaneously
18. Castiglioni, S.; Mariotti, M.; Maier, J. A. M. Cancer Lett. 2009, 273, 155.