N6-Alkyladenosines: Synthesis and evaluation of in vitro anticancer activity

Article abstract of DOI:10.1016/j.bmc.2010.09.030

A series of adenosine analogues differently substituted in N ⁶-position were synthesized to continue our studies on the relationships between structure and biological activity of iPA. The structures of the compounds were confirmed by standard studies of ¹H NMR, MS and elemental analysis. These molecules were then evaluated for their anti-proliferative activity on bladder cancer cells. We found that some of these compounds possess anti-proliferative activity but have no effect on cell invasion and metalloprotease activity.

Full text of DOI:10.1016/j.bmc.2010.09.030

Bioorganic & Medicinal Chemistry 18 (2010) 8396–8402
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
N6-Alkyladenosines: Synthesis and evaluation of in vitro anticancer activity
⇑
Roberta Ottria, Silvana Casati, Erika Baldoli, Jeanette A. M. Maier, Pierangela Ciuffreda
Dipartimento di Scienze Cliniche ‘Luigi Sacco’, Via G. B. Grassi 74, Università degli Studi di Milano, 20157 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
A series of adenosine analogues differently substituted in N6-position were synthesized to continue our
studies on the relationships between structure and biological activity of iPA. The structures of the com-
pounds were confirmed by standard studies of 1H NMR, MS and elemental analysis. These molecules
were then evaluated for their anti-proliferative activity on bladder cancer cells. We found that some of
these compounds possess anti-proliferative activity but have no effect on cell invasion and metallopro-
tease activity.
Article history:
Received 24 June 2010
Revised 6 September 2010
Accepted 14 September 2010
Available online 18 September 2010
Keywords:
Isopentenyladenosine
Cytokinins
Ó 2010 Published by Elsevier Ltd.
Modified nucleosides
Antitumor agents
Bladder carcinoma cells
1. Introduction
biological activity of iPA,8,9 we focused our attention on the isopen-
tenyl chain and synthesised a series of adenosine analogues differ-
Cytokinins, an important group of plant growth regulatory
substance, are N6-substituted adenine derivatives.1 They occur
endogenously as free bases, nucleosides or nucleotides. Cytokinins
are often present in a very low concentration and regulate many
processes in plants such as bud formation and release, leaf expan-
sion, promotion of seed germination and chloroplast formation.2
Cytokinins induce callus, a cluster of differentiated plant cells that
proliferate indefinitely in a disorganized manner similar to human
cancer cells, to re-differentiate into adventitious buds.3 Since there
are some similarities in the biological phenotypes of cancer and
callus cells, cytokinins might also affect the growth and differenti-
ation of human cancer cells. The only known cytokinin existing in
animal cells is isopentenyladenosine (iPA, 1a, Fig. 1), a modified
nucleoside with a pentaatomic isopentenyl chain that binds the
nitrogen at the position 6 of the purinic base. iPA has been detected
in the cytosol of many eukaryotic and prokaryotic cells as a free
compound or bound to tRNA.4 It has been demonstrated that iPA
exerts a potent anti-proliferative activity on cultured tumour epi-
thelial cells,5 but it has only a slight effect on tumour growth in ro-
dents.6 Discouraging results have also been obtained in a pilot
clinical trial.7 The discrepancy between in vitro and in vivo results
might be due to the rapid catabolism in vivo, or to the short plasma
half-life of iPA, in analogy to other nucleosides. We therefore tried
to investigate which structural modifications might yield iPA ana-
logues which maintained their biological activity in vivo. To con-
tinue our studies concerning the relation between structure and
ently substituted in N6-position (Fig. 1). In this study, we prepared
a group of adenosine derivatives with modification in N6-chain and
we tested them on T24 cells, a cell line established from a human
urinary bladder cancer patient, in order to verify whether our
R
HN
N
N
N
HO
N
O
HO
OH
1
c
a
d
b
e
f
OH
OH
i
l
g
j
h
k
N
OH
n
o
m
⇑
Corresponding author. Tel.: +39 0250319695; fax: +39 0250319694.
E-mail address: pierangela.ciuffreda@unimi.it (P. Ciuffreda).
Figure 1. Structures of the modified iPA analogues.
0968-0896/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2010.09.030

R. Ottria et al. / Bioorg. Med. Chem. 18 (2010) 8396–8402
8397
and 1n inhibited the formation of clones (Fig. 3). Since tumor cell
invasion of basement membranes represents one of the critical
steps in the metastatic process, we also investigated whether iPA
or its active derivatives modulated cell invasion through basement
membrane-like matrigel barriers. In particular, matrigel has been
extensively used as an in vitro surrogate for the in vivo process
of tumor invasion through basement membranes.15 Figure 4a
shows that iPA and its derivatives 1b, 1e, 1i, 1k and 1n did not ex-
ert any effect on T24 cell invasion on matrigel. Because increased
matrix metalloprotease (MMP) activity has been correlated with
the metastatic potential of many cancers,16 we also analyzed
MMP activity by zymography on media collected from T24 cells
treated with iPA and its derivatives 1b, 1e, 1i, 1k and 1n. A clear
band of gelatinolytic activity, sized to 88 kDa corresponding to
activated MMP-9, was detected. However, none of the molecules
tested altered MMP activity of T24 cells (Fig. 4b). Accordingly, these
molecules did not inhibit T24 cell migration in an in vitro model of
wound repair (data not shown).
Cl
N
NHR
N
N
N
N
N
N
N
HO
HO
O
O
a
HO
OH
HO
OH
1b-o
Scheme 1. Synthesis of iPA analogues 1b–o. Reagents and conditions: (a) appro-
priate amine, EtOH/Et3N, 80 °C, 3 h.
modification could affect the anti-proliferative activity of iPA on
cell cultures. Bladder cancer is the second most common malig-
nancy of the genitourinary tract. Multifocality and frequent recur-
rence are the typical characteristics of this tumor.10 Therefore,
novel molecules are needed to treat this neoplasia.
2. Results and discussion
2.1. Chemistry
3. Conclusions
Bladder cancer is the fourth most common cancer in men and the
five-years relative survival rates are as low as 6% if it is diagnosed at a
late stage.17 Although chemotherapy has improved the treatment of
advanced tumors, the associated side effects induced by lack of spec-
ificity to tumor cells remain a challenging problem.
As part of our continuing efforts to develop iPA analogues
endowed with in vivo anti-proliferative activity we turned our
attention to compounds characterised by a different substituent
in N6 position (Fig. 1). We synthesised a series of compounds with
a saturated linear or ramified chain (1b–e), one or more hydroxyl
or amino groups on the N6-substituent (1f–h), an unsaturated
chain (1i), a cyclic substituent (1j–m) and an aromatic ring (1n–o).
Monosubstituted N6-alkyladenosines were prepared by con-
Tumor cell lines represent valuable preclinical models to deci-
pher underlying biology and identify potential therapy targets
and pharmacologically useful compounds. We have utilized blad-
der carcinoma T24 cells which have provided important insights
into bladder tumour progression events and metastatic dissemina-
tion. In these cells we have previously shown that modifications of
N6-position of iPA impact on the cytostatic activity, while modifi-
cations of sugar moiety or the purine base generate inactive mole-
cules.8,9 The anti-proliferative activity is optimal in N6-substituted
adenosine analogues with side chains containing at least three car-
bon atoms. In addition, the activity is enhanced by the presence in
the chain of an unsaturation site and the presence of an hetero-
atom in the side chain causes a lack in the anti-proliferative
activity.
The ability of tumor cells to invade is one of the hallmarks of the
metastatic phenotype. We found that iPA 1a and its derivatives
with anti-proliferative activity do not impact on the invasive phe-
notype of bladder cancer cells. Indeed, no differences between un-
treated or treated T24 cells were detected using matrigel invasion
assay, which shows a strong correlation between the ability of
tumor cells to invade in vitro and their invasive behavior in vivo.15
In addition, because MMPs represent the most prominent family of
proteases associated with invasion and metastasis,16 it is notewor-
thy that all the compounds tested were unable to modulate MMP
activity in T24 cells. We conclude that iPA and derivatives target
DNA replication18 while they do not interfere with the signalling
pathways involved in modulating cell invasion.
densing 6-chloropurine riboside (6-chloro-9-b-D-ribofuranosyl-
9H-purine) with the corresponding amines by nucleophilic substi-
tution in absolute ethanol, using triethylamine as acid acceptors
(Scheme 1).11,12 The compounds were isolated and purified by
crystallization. The only exception was 6-(cyclobutylmethylami-
no)-adenosine (1n). In this case, cyclobutylmethylbromide was
used to alkylate adenosine, and the N1-alkylated intermediate
was rearranged under Dimroth13,14 conditions to yield 1k.
The structure of all prepared compounds 1b–o, were confirmed
from MS, 1D and 2D NMR spectroscopic data as well as by elemen-
tal (C, H, N) analyses (see Section 4.3) which allowed the correct
identification and determined the purity of all compounds.
2.2. Biological assays
Starting from results obtained from dose/response curves per-
formed with iPA,8 we selected 10
lM as the concentration to uti-
lize to compare iPA (1a) and its analogues 1b–o for their ability
to inhibit cell proliferation, clonogenity and invasion. All the com-
pounds were dissolved in DMSO and subsequently diluted in the
culture medium before treatment of T24 bladder carcinoma cells,
which have been used before to test different iPA analogues.8,9
To screen all the synthesized compounds for their anti-prolifera-
tive capacity, microtiter tetrazolium (MTT) assay was performed.
Figure 2a demonstrates a significant anti-proliferative capacity
by iPA derivatives 1b, 1e, 1i, 1k and 1n. In particular, the activity
of compound 1n is comparable to iPA. These derivatives were
tested on another bladder carcinoma cell line, that is, J82, and
similar results were obtained (Fig. 2b). Derivatives 1b, 1e, 1i, 1k
and 1n showed anti-proliferative activity also on colon carcinoma
CaCo2 and breast carcinoma MDAMB231 cells (data not shown).
The molecules which resulted active by MTT assay were further
investigated by T24 cell counting after trypsinization and con-
firmed to inhibit cell growth (Fig. 2c). Accordingly, clonogenic as-
says on T24 cells demonstrated that compounds 1b, 1e, 1i, 1k
4. Materials and methods
4.1. General procedures
Melting points were determined with a Stuart Scientific SMP3
melting point apparatus. Optical rotations were measured on a
Perkin–Elmer 241 polarimeter (sodium D line at 25 °C). NMR spec-
tra were done on a Bruker AVANCE 500 spectrometer equipped
with a 5 mm broadband reverse probe with field z-gradient operat-
ing at 500.13 MHz for 1H. All NMR spectra were recorded at 298 K
in CD3OD (isotopic enrichment 99.95%) solution and the chemical

8398
R. Ottria et al. / Bioorg. Med. Chem. 18 (2010) 8396–8402
a
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
*
*
*
**
**
**
CTRL
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
b
c
3.0
2.5
2.0
1.5
1.0
0.5
0.0
*
*
**
**
**
**
CTRL
1a
1b
1e
1i
1k
1n
120000
100000
80000
60000
40000
20000
0
**
**
**
**
**
**
CTRL
1a
1b
1e
1i
1k
1n
Figure 2. MTT and proliferation assay on bladder carcinoma cells treated with iPA and derivatives. (a) MTT quantification on T24 cells exposed to 10
l
M of iPA or synthesized
derivatives for 72 h. (b) MTT quantification on J82 cells exposed to 10
were trypsinized and counted after 72 h of treatment.
lM of iPA or some of its derivatives for 72 h. (c) T24 cells treated with 10 lM iPA or selected derivatives
shifts were reported on a d (ppm) scale. The central peak 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, multiplet, and br s, broad peak.
Mass spectra were recorded on a Finnigan LCQ deca (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.
The progress of the reaction was monitored by analytical thin-
layer chromatography (TLC) on pre-coated glass plates (Silica Gel
60 F254-plate-Merck, Darmstadt, Germany) and the products were
visualized by UV light. Elemental analyses were obtained for all
intermediates and are within 0.4% of theoretical values.
Purity of all compounds (P99%) was verified by thin-layer chro-
matography, NMR and Mass Spectrometry measurements.
Sigma–Aldrich (St. Louis, MO, USA). Organic solvents were dried
in the presence of appropriate drying agents and were stored over
suitable molecular sieves.
4.3. Synthesis of N6-alkyladenosines
To a solution of 6-chloropurine riboside (1.5 mmol) in absolute
EtOH (10 mL), Et3N, (4.5 mmol) and the appropriate amine
(4.5 mmol) were added. The mixture was stirred at 80 °C for 3 h,
cooled to room temperature and the solvent was removed under
vacuum to leave syrupy residue. The addition of dry Et2O precipi-
tated Et3NHCl, which was filtered off. The crude residue after evap-
oration was crystallised from MeOH.
4.3.1. N6-Isopentyladenosine 1b
Compound 1b was prepared following the above described pro-
cedure starting from 6-chloropurine riboside and 3-methylbutyl-
amine. Rf = 0.48 (CH2Cl2/MeOH, 90:10); white solid 78% yield;
4.2. Chemicals
Adenosine, 6-chloropurine riboside, all available starting
amines, the other reagents and all solvents were purchased from
mp 156–157 °C; ½a D20
ꢀ
ꢁ57.8 (c 1, MeOH) [lit.19 mp 154.5–156 °C,
½
a 2D0
ꢀ
ꢁ42.0 (c 1.03, EtOH)]; 1H NMR (CD3OD) d = 1.0 (d, 6H,

R. Ottria et al. / Bioorg. Med. Chem. 18 (2010) 8396–8402
8399
Figure 3. Clonogenic assay on T24 cells treated with iPA and derivatives. T24 cells were seeded at very low density and treated with iPA and derivatives. After a week, the
cells were stained and photographed.
a
14000
12000
10000
8000
6000
4000
2000
0
CTRL
1a
1b
1e
1i
1k
1n
b
CTRL
1a
1b
1e
1i
1k
1n
- MMP9
Figure 4. Invasion assay on matrigel and MMP secretion in T24 cells treated with iPA and derivatives. (a) T24 cells were cultured in transwell chambers with Matrigel-coated
membranes. Invasion was evaluated as described in Section 4. (b) Zymography was performed on media collected form T24 cells exposed to iPA and derivatives as described
in Section 4.
J = 6.6 Hz, CH3 ꢂ 2), 1.61 (dt, 2H, J = 7.0 Hz, J = 7.3 Hz, CH2CH2CH),
1.76 (tq, 1H, J = 6.6, J = 7.0, CH2CH2CH), 3.63 (br s, 2H, CH2CH2CH),
3.76 (dd, 1H, J = 2.4 Hz, J = 12.6 Hz, H50a), 3.91 (dd, 1H, J = 2.4 Hz,
J = 12.6 Hz, H50b), 4.19 (ddd, 1H, J = 2.4 Hz, J = 2.4 Hz, J = 2.4 Hz,
H40), 4.34 (dd, 1H, J = 2.4 Hz, J = 5.2 Hz, H30), 4.77 (dd, 1H,
J = 5.2 Hz, J = 6.3 Hz, H20), 5.97 (d, 1H, J = 6.3 Hz, H10), 8.23 (br s,
1H, H2), 8.25 (s, 1H, H8); ESIMS m/z 336 (Mꢁ1, 30%), 673
(2Mꢁ1, 100%).
NMR (CD3OD) d = 1.33 (d, 6H, J = 6.5 Hz, CH3 ꢂ 2), 3.51 (q, 1H,
J = 6.5 Hz, NHCH), 3.74 (dd, 1H, J = 2.2 Hz, J = 12.2 Hz, H50a), 3.90
(dd, 1H, J = 2.2 Hz, J = 12.2 Hz, H50b), 4.19 (ddd, 1H, J = 2.2 Hz,
J = 2.2 Hz, J = 2.5 Hz, H40), 4.33 (dd, 1H, J = 2.5 Hz, J = 5.9 Hz, H30),
4.76 (dd, 1H, J = 5.9 Hz, J = 6.3 Hz, H20), 5.91 (d, 1H, J = 6.3 Hz,
H10), 8.23 (br s, 1H, H2), 8.27 (s, 1H, H8); ESIMS m/z 308 (Mꢁ1,
70%), 617 (2Mꢁ1, 100%).
4.3.3. N6-Isobutyladenosine 1d
4.3.2. N6-Isopropyladenosine 1c
Compound 1d was prepared following the above described pro-
cedure starting from 6-chloropurine riboside and 2-methyl-pro-
pylamine. Rf = 0.60 (CH2Cl2/MeOH, 90:10); white solid 84% yield;
Compound 1c was prepared following the above described pro-
cedure starting from 6-chloropurine riboside and 1-methyl-ethyl-
amine. Rf = 0.54 (CH2Cl2/MeOH, 90:10); white solid 86% yield;
mp 166–167 °C; ½a D20
ꢀ
ꢁ64.0 (c 1, MeOH); 1H NMR (CD3OD)
mp 172–174 °C; ½a D20
ꢀ
ꢁ21.6 (c 1, MeOH); [lit.20 mp 157 °C]; 1H
d = 1.02 (d, 6H, J = 6.5 Hz, CH3 ꢂ 2), 2.01 (tq, 1H, J = 6.5, J = 7.0,

8400
R. Ottria et al. / Bioorg. Med. Chem. 18 (2010) 8396–8402
NHCH2CH), 3.44 (br s, 2H, NHCH2CH), 3.76 (dd, 1H, J = 2.2 Hz,
J = 12.2 Hz, H50a), 3.90 (dd, 1H, J = 2.2 Hz, J = 12.2 Hz, H50b), 4.19
(ddd, 1H, J = 2.2 Hz, J = 2.2 Hz, J = 2.5 Hz, H40), 4.34 (dd, 1H,
J = 2.5 Hz, J = 5.2 Hz, H30), 4.76 (dd, 1H, J = 5.2 Hz, J = 6.5 Hz, H20),
5.97 (d, 1H, J = 6.5 Hz, H10), 8.23 (br s, 1H, H2), 8.27 (s, 1H, H8);
ESIMS m/z 322 (Mꢁ1, 15%), 645 (2Mꢁ1, 100%). Anal. Calcd for
3.90 (dd, 1H, J = 1.5 Hz, J = 12.2 Hz, H50b), 4.18 (ddd, 1H,
J = <1.0 Hz, J = 1.2 Hz, J = 1.5 Hz, H40), 4.31 (dd, 1H, J = <1.0 Hz,
J = 5.2 Hz, 1H, H30), 4.77 (dd, 1H, J = 5.2 Hz, J = 6.5 Hz, H20), 5.93
(d, 1H, J = 6.5 Hz, H10), 8.23 (br s, 1H, H2), 8.26 (s, 1H, H8), 8.57
(br s, 1H, NH); ESIMS m/z 351 (Mꢁ1, 60%), 703 (2Mꢁ1, 100%). Anal.
Calcd for C15H24N6O4: C, 51.13; H, 6.86; N, 23.85; O, 18.16. Found:
C, 51.22; H, 6.78; N, 23.74.
C14H21N5O4: C, 52.00; H, 6.55; N, 21.66; O, 19.79. Found: C,
51.98; H, 6.62; N, 21.49.
4.3.8. N6-Allyladenosine 1i
Compound 1i was prepared following the above described pro-
cedure starting from 6-chloropurine riboside and allylamine.
Rf = 0.40 (CH2Cl2/MeOH, 90:10); white solid 75% yield; mp 166–
4.3.4. N6-Butyladenosine 1e
Compound 1e was prepared following the above described pro-
cedure starting from 6-chloropurine riboside and n-butylamine.
Rf = 0.52 (CH2Cl2/MeOH, 90:10); white solid 76% yield; mp 176–
167 °C; ½a 2D0
ꢀ
ꢁ49.6 (c 1, MeOH); [lit.11 mp 166–167 °C; ½a 2D5
ꢁ101
ꢀ
177 °C; ½a 2D0
ꢀ
ꢁ62.6 (c 1, MeOH); [lit.21 mp 171–173 °C, ½a 2D0
ꢀ
(c 0.094, EtOH)]; 1H NMR (CD3OD) d 3.77 (dd, 1H, J = 1.2 Hz,
J = 12.2 Hz, H50a), 3.91 (dd, 1H, J = 1.5 Hz, J = 12.2 Hz, H50b), 4.20
(ddd, 1H, J = <1.0 Hz, J = 1.2 Hz, J = 1.5 Hz, H40), 4.25 (br s, 2H,
NCH2CH), 4.35 (dd, 1H, J = <1.0 Hz, J = 5.2 Hz, 1H, H30), 4.77 (dd,
1H, J = 5.2 Hz, J = 6.4 Hz, H20), 5.18 (d, 1H, J = 10.3 Hz, CH@CHH),
5.29 (d, 1H, J = 16.8 Hz, CH@CHH), 5.95–6.10 (m, 1H, CH2CH@),
5.99 (d, 1H, J = 6.4 Hz, H10), 8.24 (s, 1H, H2), 8.29 (s, 1H, H8); ESIMS
m/z 306 (Mꢁ1, 40%), 613 (2Mꢁ1, 100%).
ꢁ36.4 (c 1, MeOH)] 1H NMR (CD3OD) d = 1.00 (t, 3H, J = 7.7 Hz,
CH3), 1.49 (qt, 2H, J = 6.8 Hz, J = 7.7 Hz, NHCH2CH2CH2CH3), 1.69
(dt, 2H, J = 6.8 Hz, J = 7.0 Hz, NHCH2CH2CH2CH3), 3.60 (br m, 2H,
NHCH2CH2CH2CH3), 3.76 (dd, 1H, J = 1.2 Hz, J = 12.2 Hz, H50a),
3.90 (dd, 1H, J = 1.5 Hz, J = 12.2 Hz, H50b), 4.19 (ddd, 1H,
J = <1.0 Hz, J = 1.2 Hz, J = 1.5 Hz, H40), 4.33 (dd, 1H, J = <1.0 Hz,
J = 5.1 Hz, H30), 4.76 (dd, 1H, J = 5.1 Hz, J = 6.4 Hz, H20), 5.97
(d, 1H, J = 6.4 Hz, H10), 8.23 (br s, 1H, H2), 8.26 (s, 1H, H8); ESIMS
m/z 322 (Mꢁ1, 30%), 645 (2Mꢁ1, 100%).
4.3.9. N6-Cyclopropylmethyladenosine 1j
Compound 1j was prepared following the above described pro-
cedure starting from 6-chloropurine riboside and cyclopropylm-
ethylamine Rf = 0.42 (CH2Cl2/MeOH, 90:10); white solid 80%
4.3.5. N6-(3-Hydroxy-2-methylpropyl)-adenosine 1f
Compound 1f was prepared following the above described pro-
cedure starting from 6-chloropurine riboside and (+)-2-amino-1-
butanol. Rf = 0.42 (CH2Cl2/MeOH, 90:10); white solid 74% yield;
yield; mp 173–175 °C; ½a D20
ꢀ
ꢁ66.4 (c 1, MeOH); [lit.12 mp 176 °C;
25
½
a
ꢀ Dꢀ ꢁ64.5 (c 0.217, EtOH)]; 1H NMR (CD3OD) d = 0.35 (dd, 2H,
½
mp 192–194 °C; ½a D20
ꢀ
ꢁ76.8 (c 1, MeOH); 1H NMR (CD3OD)
J = 5.8 Hz, J = 7.7 Hz, CHHCHCHH), 0.58 (dd, 2H, J = 5.1 Hz,
J = 5.8 Hz, CHHCHCHH), 1.17–1.22 (m, 1H, NCH2CH), 3.47 (br s,
2H, NCH2CH@), 3.77 (dd, 1H, J = 1.2 Hz, J = 12.2 Hz, H50a), 3.90
(dd, 1H, J = 1.5 Hz, J = 12.2 Hz, H50b), 4.19 (ddd, 1H, J = <1.0 Hz,
J = 1.2 Hz, J = 1.5 Hz, H40), 4.34 (dd, 1H, J = <1.0 Hz, J = 5.2 Hz, 1H,
H30), 4.76 (dd, 1H, J = 5.2 Hz, J = 6.4 Hz, H20), 5.97 (d, 1H,
J = 6.4 Hz, H10), 8.23 (br s, 1H, H2), 8.28 (s, 1H, H8), 8.57 (br s,
1H, NH); ESIMS m/z 320 (Mꢁ1, 38%), 641 (2Mꢁ1, 100%).
d = 1.01 (dd, 3H, J = 7.1 Hz, J = 7.1 Hz, CH2CH3), 1.65 (ddq, 1H,
J = 7.1 Hz, J = 7.1 Hz, J = 14.8 Hz, CHHCH3), 1.81 (ddq, 1H,
J = 5.8 Hz, J = 7.1 Hz, J = 14.8 Hz, CHHCH3), 3.70 (d, 2H, J = 4.5 Hz,
CH2OH), 3.76 (dd, 1H, J = 1.2 Hz, J = 12.2 Hz, H50a), 3.90 (dd, 1H,
J = 1.5 Hz, J = 12.2 Hz, H50b), 4.19 (ddd, 1H, J = <1.0 Hz, J = 1.2 Hz,
J = 1.5 Hz, H40), 4.31 (br s, 1H, NHCH), 4.34 (dd, 1H, J = <1.0 Hz,
J = 5.2 Hz, 1H, H30), 4.76 (dd, 1H, J = 5.2 Hz, J = 6.5 Hz, H20), 5.98
(d, 1H, J = 6.5 Hz, H10), 8.04 (br s, 1H, NH), 8.23 (br s, 1H, H2),
8.27 (s, 1H, H8); ESIMS m/z 338 (Mꢁ1, 35%), 677 (2Mꢁ1, 100%).
Anal. Calcd for C14H21N5O5: C, 49.55; H, 6.24; N, 20.64; O, 23.57.
Found: C, 49.48; H, 6.39; N, 20.61.
4.3.10. N6-Cyclobutylmethyladenosine 1k
BaCO3 (2.4 mmol) and cyclobutylmethylbromide (2.25 mmol)
were added to
a solution of adenosine (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 mixture 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 sol-
vent was removed and the residue purified by column chromatog-
raphy on silica gel (eluent CH2Cl2/MeOH, 98:2). Rf = 0.40 (CH2Cl2/
4.3.6. N6-2,3-Dihydroxy-propyladenosine 1g
Compound 1g was prepared following the above described pro-
cedure starting from 6-chloropurine riboside and (ꢁ)-3-amino-1,2-
propanediol. Rf = 0.16 (CH2Cl2/MeOH, 90:10); white solid 74%
yield; mp 196–198 °C; ½a D20
ꢀ
ꢁ48.6 (c 1, MeOH); 1H NMR (CD3OD)
d = 3.22 (dd, 1H, J = 7.1 Hz, J = 13.5 Hz, NHCHH), 3.43 (dd, 1H,
J = 4.5 Hz, J = 13.5 Hz, NHCHH), 3.59 (d, 2H, J = 5.2 Hz, CH2OH),
3.71 (ddt, 1H, J = 4.5 Hz, J = 5.2 Hz, J = 7.1 Hz, CH2CHOHCH2OH),
3.79 (dd, 1H, J = 1.2 Hz, J = 12.2 Hz, H50a), 3.87 (dd, 1H, J = 1.5 Hz,
J = 12.2 Hz, H50b), 4.16 (ddd, 1H, J = <1.0 Hz, J = 1.2 Hz, J = 1.5 Hz,
H40), 4.31 (dd, 1H, J = <1.0 Hz, J = 5.2 Hz, 1H, H30), 4.73 (dd, 1H,
J = 5.2 Hz, J = 6.5 Hz, H20), 5.93 (d, 1H, J = 6.5 Hz, H10), 8.08 (br s,
1H, NH), 8.23 (br s, 1H, H2), 8.28 (s, 1H, H8); ESIMS m/z 340
(Mꢁ1, 15%), 681 (2Mꢁ1, 100%). Anal. Calcd for C13H19N5O6: C,
45.75; H, 5.61; N, 20.52; O, 28.13. Found: : C, 45.62; H, 5.68; N,
20.64.
MeOH, 95:5); white solid 55% yield; mp 181–183 °C; ½a D20
ꢁ42.1
ꢀ
(c 1, MeOH); 1H NMR (CD3OD) d = 1.81–1.88 (m, 2H, CHHCHCHH),
1.91–2.00 (m, 2H, CHCH2CH2CH2), 2.09–2.17 (m, 2H, CHHCHCHH),
2.67–2.75 (m, 1H, NCH2CH), 3.63 (br s, 2H, NCH2CH), 3.76 (dd, 1H,
J = 1.2 Hz, J = 12.2 Hz, H50a), 3.90 (dd, 1H, J = 1.5 Hz, J = 12.2 Hz,
H50b), 4.19 (ddd, 1H, J = <1.0 Hz, J = 1.2 Hz, J = 1.5 Hz, H40), 4.33
(dd, 1H, J = <1.0 Hz, J = 5.2 Hz, 1H, H30), 4.76 (dd, 1H, J = 5.2 Hz,
J = 6.4 Hz, H20), 5.97 (d, 1H, J = 6.4 Hz, H10), 8.23 (br s, 1H, H2),
8.25 (br s, 1H, NH), 8.27 (s, 1H, H8); ESIMS m/z 334 (Mꢁ1, 25%),
669 (2Mꢁ1, 100%). Anal. Calcd for C15H21N5O4: C, 53.72; H, 6.31;
N, 20.88; O, 19.08. Found: C, 53.62; H, 6.40; N, 20.96.
4.3.7. N6-30-(Dimethylamino)propyladenosine 1h
Compound 1h was prepared following the above described pro-
cedure starting from 6-chloropurine riboside and 3-dimethyl-
amino-propylamine. Rf = 0.32 (CH2Cl2/MeOH, 90:10); white solid
4.3.11. N6-Cyclopentyladenosine 1l
75% yield; mp 176–178 °C; ½a D20
ꢀ
ꢁ33.2 (c 1, MeOH); 1H NMR
Compound 1l was prepared following the above described pro-
cedure starting from 6-chloropurine riboside and cyclopentyl-
amine Rf = 0.58 (CH2Cl2/MeOH, 98:2); white solid 79% yield; mp
(CD3OD) d = 1.89 (m, 2H, NCH2CH2CH2N(CH3)2), 2.26 (s, 6H,
N(CH3)2), 2.46 (m, 2H, NCH2CH2CH2N(CH3)2), 3.63 (br s, 2H,
NCH2CH2CH2N(CH3)2), 3.77 (dd, 1H, J = 1.2 Hz, J = 12.2 Hz, H50a),
120–122 °C [lit.22 mp 120–122 °C]; ½a 2D0
ꢀ
ꢁ35.1 (c 1, MeOH); 1H

R. Ottria et al. / Bioorg. Med. Chem. 18 (2010) 8396–8402
8401
NMR (CD3OD) d = 1.60–1.74 (m, 4H, 2 ꢂ CH2), 1.79–1.86 (m, 2H,
CH2), 2.08–2.11 (m, 2H, CH2), 3.33 (br s, 1H, NCHCH2), 3.76 (dd,
1H, J = 1.5 Hz, J = 12.2 Hz, H50a), 3.90 (dd, 1H, J = 1.5 Hz,
J = 12.2 Hz, H50b), 4.19 (ddd, 1H, J = <1.0 Hz, J = 1.5 Hz, J = 1.5 Hz,
H40), 4.34 (dd, 1H, J = <1.0 Hz, J = 5.0 Hz, 1H, H30), 4.75 (dd, 1H,
J = 5.0 Hz, J = 6.6 Hz, H20), 5.97 (d, 1H, J = 6.6 Hz, H10), 8.23 (br s,
1H, H2), 8.24 (s, 1H, H8); ESIMS m/z 334 (Mꢁ1, 13%), 669
(2Mꢁ1, 100%).
studies, the different molecules were used at a concentration
10
M.8 Briefly, 1 h before the end of treatment the medium was
l
replaced with medium containing 0.5 mg/ml of 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyltetrasodium bromide (MTT) (Sigma,
Oakville, Ontario, Canada). At the end of the incubation, media
were removed and formazan crystals generated by the cellular
reduction activity were dissolved in DMSO. Absorbance was mea-
sured at 575 nm and data are expressed as the ratio of absorbance
of treated cells versus initial MTT absorbance, corresponding to the
level of MTT reduced to formazan crystals by cells in the initial day
of treatment.9
For proliferation assays, the cells were seeded at low density
(2000/cm2) in 6-well plates and allowed to attach for 16 h before
being treated with different compounds.8,9 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.
4.3.12. N6-Cyclohexyladenosine 1m
Compound 1m was prepared following the above described
procedure starting from 6-chloropurine riboside and cyclohexyl-
amine Rf = 0.34 (CH2Cl2/MeOH, 95:5); white solid 80% yield; mp
25
184–186 °C; ½a 2D0
ꢀ
ꢁ60.4 (c 1, MeOH); [lit.12 mp 185 °C; ½
a
ꢀ Dꢀ
½
ꢁ59 (c 0.312, EtOH);]; 1H NMR (CD3OD) d = 0.87–0.93 (m, 2H,
CH2), 1.25–1.54 (m, 4H, 2 ꢂ CH2), 1.81–1.86 (m, 2H, CH2), 2.04–
2.08 (m, 2H, CH2), 3.76 (dd, 1H, J = 1.2 Hz, J = 12.2 Hz, H50a), 3.90
(dd, 1H, J = 1.4 Hz, J = 12.2 Hz, H50b), 4.10–4.14 (br s, 1H, NCHCH2),
4.19 (ddd, 1H, J = <1.0 Hz, J = 1.2 Hz, J = 1.4 Hz, H40), 4.34 (dd, 1H,
J = <1.0 Hz, J = 5.2 Hz, 1H, H30), 4.76 (dd, 1H, J = 5.2 Hz, J = 6.5 Hz,
H20), 5.54 (br s, 1H, NH), 5.96 (d, 1H, J = 6.5 Hz, H10), 8.22 (br s,
1H, H2), 8.27 (s, 1H, H8); ESIMS m/z 348 (Mꢁ1, 22%), 697
(2Mꢁ1, 100%).
4.4.3. Clonogenic assay
T24 cells (5000 per well) were seeded into 6-well plates and
cultured in the presence of iPA or its derivatives (10 lM). The med-
ium was replaced every 2 or 3 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.
4.3.13. N6-Benzyladenosine 1n
Compound 1n was prepared following the above described pro-
cedure starting from 6-chloropurine riboside and benzylamine
Rf = 0.63 (CH2Cl2/MeOH, 90:10); white solid 76% yield; mp 168–
4.4.4. Matrigel invasion assay and MMP activity
For the Matrigel invasion assays, 105 cells in serum-free culture
medium were plated in the top transwell chamber (24-well insert;
169 °C [lit.23 mp 167–169 °C]; ½a 2D0
ꢀ
ꢁ68.4 (c 0.5, MeOH); 1H NMR
pore size 8 lm; Greiner bio-one, Germany) with Matrigel-coated
(CD3OD) d = 3.76 (dd, 1H, J = 1.2 Hz, J = 12.2 Hz, H50a), 3.81 (br s,
2H, NCH2), 3.91 (dd, 1H, J = 1.4 Hz, J = 12.2 Hz, H50b), 4.19 (ddd,
1H, J = <1.0 Hz, J = 1.2 Hz, J = 1.4 Hz, H40), 4.34 (dd, 1H, J = <1.0 Hz,
J = 5.2 Hz, 1H, H30), 4.77 (dd, 1H, J = 5.2 Hz, J = 6.5 Hz, H20), 5.51
(br s, 1H, NH), 5.89 (d, 1H, J = 6.5 Hz, H10), 7.25–7.40 (m, 5H, aro-
matics), 8.26 (br s, 1H, H2), 8.28 (s, 1H, H8); ESIMS m/z 356
(Mꢁ1, 41%), 713 (2Mꢁ1, 100%).
membrane (Becton Dickinson, Franklin Lakes, NJ). Medium supple-
mented with 10% FBS and Epidermal Growth Factor (50 ng/ml) was
added to each well of the plate to act as a chemoattractant in the
lower chamber.25 After 24 h, cells on the lower surface of the mem-
brane and cells attached to the surface of the lower chamber were
trypsinised and counted. These experiments were conducted in
triplicate and performed three times.
For zymography, conditioned media were incubated at 4 °C
overnight with gelatin–Sepharose and resolved on an 8% polyacryl-
amide gels co-polymerized with 1 mg/ml gelatin type B (Sigma Al-
drich) under non-reducing conditions without heating. Gels were
then washed twice for 30 min in 2.5% Triton X-100 at room tem-
perature and incubated overnight in collagenase buffer
(50 mmol/L Tris–HCl, pH 7.5, 10 mmol/L CaCl2, 150 mmol/L NaCl)
at 37 °C. Gels were stained in Coomassie Blue R 250 (Bio-Rad,
Milano, Italy) in a mixture of methanol–acetic acid–water (4:1:5)
for 1 h and destained in the same solution without the dye. The
experiment has been performed three times and one representa-
tive zymography is shown.
4.3.14. N6-2-Phenylethyladenosine 1o
Compound 1o was prepared following the above described pro-
cedure starting from 6-chloropurine riboside and 2-phenylethyl-
amine Rf = 0.65 (CH2Cl2/MeOH, 90:10); white solid 85% yield; mp
170–171 °C; [lit.24 mp 166–168 °C]; ½a 2D0
ꢀ
ꢁ28.0 (c 0.5, MeOH); 1H
NMR (CD3OD) d = 3.49–3.53 (m, 2H, NCH2CH2), 3.77 (dd, 1H,
J = 1.2 Hz, J = 12.2 Hz, H50a), 3.86 (br s, 2H, NCH2), 3.91 (dd, 1H,
J = 1.4 Hz, J = 12.2 Hz, H50b), 4.19 (ddd, 1H, J = <1.0 Hz, J = 1.2 Hz,
J = 1.4 Hz, H40), 4.34 (dd, 1H, J = <1.0 Hz, J = 5.0 Hz, 1H, H30), 4.77
(dd, 1H, J = 5.0 Hz, J = 6.6 Hz, H20), , 5.87 (d, 1H, J = 6.6 Hz, H10),
7.21–7.30 (m, 5H, aromatics), 8.24 (br s, 1H, H2), 8.26 (s, 1H,
H8); ESIMS m/z 370 (Mꢁ1, 39%), 741 (2Mꢁ1, 100%).
4.4.5. Statistical analysis
4.4. Biological assays
All the experiments were performed at least three times in trip-
licates and data are shown as the mean standard deviation. Sta-
tistical significance was determined using the Student’s T test. In
4.4.1. Cell culture
*
**
T24 and J82 cells were grown in D-MEM or RPMI, respectively,
the figures: p <0.05; p <0.01.
supplemented with 10% fetal bovine serum (FBS), 1 mM L-gluta-
mine and 1 mM penicillin/streptomycin. The cells were cultured
in 5% CO2 at 37 °C. All the reagents for cell culture were from Gibco.
Stock solutions of iPA and various compounds were prepared in
DMSO and kept at ꢁ20 °C. Appropriate dilutions of the compounds
were freshly prepared in culture medium just prior the assays. The
controls were added with the final concentrations of DMSO
(0.01%).
Acknowledgement
This work has been partially supported by Università degli Studi
di Milano (Fondi FIRST).
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