Antitumor activity of C-methyl-β-D-ribofuranosyladenine nucleoside ribonucleotide reductase inhibitors

Article abstract of DOI:10.1021/jm048944c

A series of adenosine derivatives substituted at the 1′-, 2′-, or 3′-position of the ribose ring with a methyl group was synthesized and evaluated for antitumor activity. From this study 3′-C-methyladenosine (3′-Me-Ado) emerged as the most active compound

Full text of DOI:10.1021/jm048944c

J. Med. Chem. 2005, 48, 4983-4989
4983
Antitumor Activity of C-Methyl-â-D-ribofuranosyladenine Nucleoside
Ribonucleotide Reductase Inhibitors
Palmarisa Franchetti,† Loredana Cappellacci,† Michela Pasqualini,† Riccardo Petrelli,† Patrizia Vita,†
Hiremagalur N. Jayaram,‡ Zsuzsanna Horvath,§ Thomas Szekeres,§ and Mario Grifantini*,†
Dipartimento di Scienze Chimiche, Universita` di Camerino, 62032 Camerino, Italy, Department of Biochemistry and Molecular
Biology, Indiana University School of Medicine and Richard Roudebush VA Medical Center, Indianapolis, Indiana 46202, and
Clinical Institute for Medical and Chemical Laboratory Diagnostics, School of Medicine, University of Vienna, Austria
Received December 31, 2004
A series of adenosine derivatives substituted at the 1′-, 2′-, or 3′-position of the ribose ring
with a methyl group was synthesized and evaluated for antitumor activity. From this study
3′-C-methyladenosine (3′-Me-Ado) emerged as the most active compound, showing activity
against human myelogenous leukemia K562, multidrug resistant human leukemia K562IU,
human promyelocytic leukemia HL-60, human colon carcinoma HT-29, and human breast
carcinoma MCF-7 cell lines with IC50 values ranging from 11 to 38 µM. Structure-activity
relationship studies showed that the structure of 3′-Me-Ado is crucial for the activity.
Substitution of a hydrogen atom of the N6-amino group with a small alkyl or cycloalkyl group,
the introduction of a chlorine atom in the 2-position of the purine ring, or the moving of the
methyl group from the 3′-position to other ribose positions brought about a decrease or loss of
antitumor activity. The antiproliferative activity of 3′-Me-Ado appears to be related to its ability
to deplete both intracellular purine and pyrimidine deoxynucleotides through ribonucleotide
reductase inhibition.
Introduction
tumor potential of these nucleoside analogues has not
been further explored, and on the basis of the knowledge
that adenosine derivatives such as 2-chloroadenosine
and 2-chloro-2′-deoxyadenosine are potent cytotoxic and
apoptotic agents,10 we have synthesized and tested as
antitumor agents a series of 1′-, 2′-, and 3′-C-methyl-
substituted adenosine and 2-chloroadenosine analogues
and N6-substituted derivatives (Chart 1).
Nucleoside analogues are a pharmacologically diverse
family that includes cytotoxic compounds, antiviral
agents, and immunosuppressive molecules. Consider-
able progress has been made in the search for novel
nucleoside structures with anticancer and/or antiviral
activity by modifications in the base or in the sugar
portion of the molecule. Several branched-chain sugar
pyrimidine nucleosides such as 1-(2-deoxy-2-methylene-
â-D-erythro-pentofuranosyl)cytosine,1 (E)-2′-deoxy-2′-
(fluoromethylene)cytidine,2 and other C-branched 2′-
deoxynucleosides have shown potent antitumor activity
both in vitro and in vivo. Among the â-D-ribo-pento-
furanosyl nucleosides, 1-(3-C-ethynyl-â-D-ribo-pento-
furanosyl)cytosine and its uracil congener have also
been reported to have a broad spectrum antitumor ac-
tivity.3 On the basis of these findings, we became in-
terested in the investigation of the antitumor activity
of purine nucleosides substituted at the carbon atoms
of the ribose ring with a methyl group. From a survey
of the literature it was found that all four possible
adeno-
Chemistry
The ribose-modified nucleoside analogues 3 and 5-9
were synthesized by catalyzed glycosylation of 6-chloro-
or 2,6-dichloropurine with the suitable protected sugars.
1′-C-Methyladenosine (1′-Me-Ado, 1), 2′-C-methylad-
enosine (2′-Me-Ado, 2), and 2-chloro-2′-C-methyladeno-
sine (2-Cl-2′-MeAdo, 4) were synthesized as previously
reported.4,5 2′-C-Methyladenosine derivatives 6 and 8
were obtained by ammination of 6-chloro-9H-[(2-C-
methyl)-2,3,5-tri-O-benzoyl-â-D-ribofuranosyl]purine (10)4
with aqueous methylamine or cyclopropylamine (Scheme
1).
The synthesis of 3′-C-methyl-derivatives was carried
out starting from 1,2,3-tri-O-acetyl-5-O-benzoyl-3-C-
methyl-D-ribofuranose (11, mixture of R and â anomers)
as described in Scheme 2. Compound 11 was synthe-
sized following the method described in the literature11a
with minor modifications (see the Supporting Informa-
tion).
sine derivatives containing a methyl group at the 1′-
C-, 2′-C-, 3′-C-, and 4′-C-position have been synthe-
sized.4-8 Of these, 2′- and 3′-C-methyladenosine showed
the ability to inhibit the growth of KB cells in culture,6
while 4′-C-methyladenosine proved to be noncytotoxic
up to a 100 µM concentration9 and 1′-C-methyladenosine
was never tested as a cytotoxic agent. Since the anti-
Coupling of 11 with 6-chloropurine (12) or 2,6-
dichloropurine (13) was performed by trimethylsilyl
trifluoromethanesulfonate (TMSiOTf) mediated N-gly-
cosylation in acetonitrile and in the presence of 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) to obtain the pro-
tected compounds 14 and 15, respectively, in high yields.
* To whom correspondence should be addressed. Phone: +39-0737-
402233. Fax +39-0737-637345. E-mail: mario.grifantini@unicam.it.
† Universita` di Camerino.
‡ Indiana University School of Medicine and Richard Roudebush VA
Medical Center.
§ University of Vienna.
10.1021/jm048944c CCC: $30.25 © 2005 American Chemical Society
Published on Web 06/28/2005

4984 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15
Franchetti et al.
Table 1. In Vitro Activities of Nucleosides 1-9 (IC50 in µM)a
against Human Myelogenous Leukemia K562, Multidrug
Resistant Human Leukemia K562IU, Human Colon Carcinoma
HT-29, and Human Breast Carcinoma MCF-7 Cell Lines
Chart 1
Compd
K562
K562IU
HT-29
MCF-7
1 (1′-Me-Ado)
53
>100
>100
38.3
>100
>100
>100
93.8
>100
>100
19.5
>100
>100
>100
>100
23.2
>100
>100
>100
74.3
>100
>100
85.6
>100
>100
>100
>100
17.5
>100
>100
>100
68.1
>100
>100
72.6
>100
93.4
2 (2′-Me-Ado)
16
3 (3′-Me-Ado)
18.2
>100
>100
>100
43.9
>100
73.0
15.4
>100
>100
4 (2-Cl-2′-Me-Ado)
5 (2-Cl-3′-Me-Ado)
6 (N6-Me-2′-Me-Ado)
7 (N6-Cp-2′-Me-Ado)
8 (N6-Cp-2′-Me-Ado)
9 (N6-Cp-3′-Me-Ado)
2-Cl-Ado
Scheme 1a
N6-Me-Ado
N6-Cp-Ado
a IC50 values represent the drug concentration required to
inhibit cancer cell replication by 50%. The compounds were tested
up to a concentration of 100 µM.
multidrug resistant human leukemia K562IU, human
colon carcinoma HT-29, and human breast carcinoma
MCF-7 cell lines, and the results are shown in Table 1.
2-Cl-Ado, N6-Me-Ado, and N6-Cp-Ado were used as
reference compounds.
Among the tested nucleosides, 3′-Me-Ado (3) showed
the best activity in all cell lines with IC50 values ranging
from 17.5 to 38.3 µM. When compared with 2-Cl-Ado,
compound 3 displayed a similar activity against both
human leukemia cell lines and exhibited more pro-
nounced cytotoxic properties against both human car-
cinoma HT-29 and MCF-7. Substitution of the N6-amino
group of 3 with a methylamino one (compound 7) brings
about a reduction of activity. However, the cytotoxicity
of 7 against both colon and breast carcinoma cell lines
was similar to that of 2-Cl-Ado.
The N6-substitution with a cyclopropylamino group
(compound 9) resulted in a decrease of the activity
against K562 cells and in the loss of the activity against
the other tumor cell lines, while the 2′-methyl analogue
8 was found to be not cytotoxic. 1′-Me-Ado (1) and 2′-
Me-Ado (2) showed a cell growth inhibitory activity only
against human leukemia K562. Finally, the lack of
cytotoxic activity of 2-chloro derivatives 4 and 5 indi-
cated that substitution at the 2-position with a chlorine
atom in 2′- and 3′-C-methyladenosine is not tolerated.
This is a surprising result, because a similar substitu-
tion in adenosine and 2′-deoxyadenosine (2-Cl-Ado and
a Reagents and conditions: (i) aqueous methylamine or cyclo-
propylamine, rt or ∆.
The synthesis of derivatives 14 and 15 resulted in a
stereoselective ribosylation (â/R, 99:1).
Nucleophilic displacement of 6-chlorine atom in pro-
tected compounds 14 and 15 with liquid ammonia,
aqueous methylamine, or cyclopropylamine gave the
deblocked nucleoside analogues 3, 5, 7, and 9. N6-
Methyladenosine (N6-Me-Ado), N6-cyclopropyladenosine
(N6-Cp-Ado), and 2-chloroadenosine (2-Cl-Ado) were also
synthesized as reported in the literature.12,13
Assignment of the â-anomeric configuration of nucleo-
sides 3, 5, 7, and 9 was performed by proton NOE data.
In particular, the â-anomeric configuration was deter-
mined by selective irradiation of the H-1′ signal that
increased the intensity of the H-4′ signal; this indicates
that H-1′ and H-4′ are located on the same face of the
ribosyl ring.14
Biological Evaluation
The synthesized nucleosides were evaluated for their
cytotoxicity against human myelogenous leukemia K562,
Scheme 2a
a Reagents and conditions: (i) TMSiOTf, CH3CN, DBU; (ii) liquid ammonia or aqueous RNH2, ∆.

Antitumor C-Methyl-â-D-ribofuranosyladenine
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15 4985
Figure 1. Inhibition of colony formation in CCL-228 cells by
2′-Me-Ado and 3′-Me-Ado after 7 days of incubation.
Figure 3. Concentration of dNTPs in HL-60 cells after
treatment with 2′-Me-Ado as measured by HPLC. Values
marked with an asterisk (*) are significantly different from
the control (P < 0.05).
Figure 2. Inhibition of colony formation in HT-29 cells by
2′-Me-Ado and 3′-Me-Ado after 7 days of incubation.
2-Cl-dAdo, respectively) gives rise to very potent cyto-
toxic and apoptotic compounds.10
Figure 4. Concentration of dNTPs in HL-60 cells after
treatment with 3′-Me-Ado as measured by HPLC. Values
marked with an asterisk (*) are significantly different from
the control (P < 0.05).
Adenosine-induced cell death has been claimed to
involve either activation of extracellular adenosine
receptors or intracellular actions. However, since 3′-Me-
Ado was found to be a very poor agonist at adenosine
receptors,15 activation of extracellular receptors prob-
ably did not play any significant role in the cytotoxic
effect of this nucleoside.
evaluated indirectly by measuring the level of intracel-
lular deoxyribonucleotide triphosphate pools in HL-60
cells by HPLC analysis before and after treatment with
2′- and 3′-Me-Ado.
We have further investigated the antitumor activity
of 2′-Me-Ado and 3′-Me-Ado by testing their cytotoxicity
against HL-60 human promyelocytic leukemia cells. In
this type of cell, the cytostatic effect of 3′-Me-Ado proved
to be slightly higher compared to that of the 2′-methyl
analogue after 3 days of incubation (IC50 ) 11 and 24
µM, respectively).
The clonogenic activity of 2′-Me-Ado and 3′-Me-Ado
in human colorectal adenocarcinoma CCL-228 and
human colon adenocarcinoma HT-29 cells was also
tested. In CCL-228 cells, 2′-Me-Ado was shown to be
slightly more effective, with an IC50 value of 24 µM after
7 days of incubation, as compared to the IC50 value of
3′-Me-Ado (32 µM), while in the HT-29 cell line both
nucleosides reached similar IC50 values (42 and 39 µM,
respectively) (results are shown in Figures 1 and 2).
The mechanism by which these nucleosides affect
human tumor cells could be related to the inhibition of
ribonucleotide reductase (RR). In fact, it was reported
that 2′- and 3′-Me-Ado, as 5′-diphosphates, behave as
mechanism-based inhibitors of ribonucleotide reductase
of Corynebacterium nephridii.11 To check this hypoth-
esis, the effect of these nucleosides on cellular RR was
Treatment of HL-60 cells with 2′-Me-Ado significantly
depleted intracellular dATP and dGTP pools (Figure 3).
Incubation for 24 h with 12.5, 25, and 50 µM 2′-Me-Ado
decreased dATP pools to 61%, 56%, and 35% of control
values, respectively. The corresponding values for dGTP
pools were 53%, 45%, and 23%. Changes in intracellular
dCTP and dTTP concentrations were not significant.
In contrast to 2′-Me-Ado, 3′-Me-Ado was able to
significantly decrease all dNTP concentrations (Figure
4). After treatment with 6.25 µM 3′-Me-Ado, dCTP,
dATP, and dGTP concentrations were decreased to 61%,
49%, and 15% of control values, respectively, whereas
intracellular dTTP concentrations showed an increase
to 130% of control values, perhaps due to reduction in
DNA synthesis in response to the restricted availability
of other deoxyribonucleoside triphosphates. Treatment
with 12.5 and 25 µM 3′-Me-Ado caused a marked
decrease in all dNTP concentrations. Intracellular dCTP
and dTTP pools experienced a decrease to 26%, 53%,
61%, and 58% of control values, respectively. These
values for dATP were 5% and 3%. Intracellular dGTP
pools were depleted beyond detectability by treatment
with 3′-Me-Ado.

4986 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15
Franchetti et al.
Figure 7. Incorporation of [14C]cytidine into DNA of HL-60
cells after treatment with 2′-Me-Ado. HL-60 cells were treated
with 2′-Me-Ado for 24 h and then pulsed with [14C]cytidine
for 30 min. Total DNA from the cells was extracted and
radioactivity was determined using a scintillation detector.
Data are means ( SEM of two measurements.
Figure 5. Concentration of dNTPs in HL-60 cells after
treatment with gemcitabine (dFdC) as measured by HPLC.
Values marked with an asterisk (*) are significantly different
from the control.
of [14C]cytidine into DNA elicited by 3′-Me-Ado confirms
the hypothesis that this nucleoside is able to inhibit the
enzyme.
It is known that the initial step in the mechanism of
action of RR is the homolytic cleavage of the 3′-carbon-
hydrogen bond mediated by a protein radical (Scheme
3).11,16 Such carbon-hydrogen bond cleavage is not
possible in the 3′-C-methyladenosine 5′-diphosphate (3′-
Me-ADP); thus, this ADP analogue cannot function as
a substrate of the enzyme but behaves as an inhibitor
of ADP reduction.11 3′-Me-Ado is the first example of a
â-D-ribonucleoside able to inhibit human RR functioning
as mechanism-based inhibitor of the enzyme.
The lower activity of the regioisomeric 2′-C-methyl-
adenosine might be due to its instability in the cell
culture medium. It is already known that this nucleo-
side is susceptible to enzymatic conversions by adeno-
sine deaminase and purine nucleoside phosphorylase.17
We have compared the stability of 2′-Me-Ado and 3′-
Me-Ado in MEM medium containing 10% fetal bovine
serum for 72 h at 37 °C. Under these conditions 2′-Me-
Ado proved to be quite unstable, with a half-life of 4.5
h, while the half-life of 3′-Me-Ado was 43.5 h. Other
factors that could negatively affect the activity of 2′-
Me-Ado are (a) inefficient intracellular metabolism to
the 5′-diphosphate derivative and (b) reduced inhibitory
activity of 2′-Me-ADP against human RR. Further
studies are underway to evaluate these hypotheses.
Figure 6. Incorporation of [14C]cytidine into DNA of HL-60
cells after treatment with 3′-Me-Ado. HL-60 cells were treated
with 3′-Me-Ado for 24 h and then pulsed with [14C]cytidine
for 30 min. Total DNA from the cells was extracted and
radioactivity was determined using a scintillation detector.
Data are means ( SEM of two measurements.
The effects of gemcitabine (dFdC), a clinically well-
established inhibitor of RR on intracellular dNTP pool
concentrations, were comparable to those of 3′-Me-Ado.
Treatment of HL-60 cells with 40 nM dFdC significantly
decreased intracellular dCTP and dATP pools to 6.1%
and 13.9% of control values, respectively, while dGTP
pools were depleted beyond detectability. In contrast,
dTTP pools experienced a significant increase to 192.6%
of control values, as shown in Figure 5.
These finding strengthen the hypothesis that the
cytotoxicity of these nucleosides is due to the inhibition
of RR. The higher activity of 3′-Me-Ado could be
explained by its greater ability to reduce the level of all
dNTPs, and in particular those of dGTP.
We have also determined the incorporation of [14C]-
cytidine into the DNA of HL-60 cells after incubation
with 3′-Me-Ado (Figure 6) and 2′-Me-Ado (Figure 7).
Cells were first preincubated with various concentra-
tions of these compounds. Incubation with 6.25 and 12.5
µM 3′-Me-Ado did not influence significantly the incor-
poration of [14C]cytidine into DNA. After treatment of
HL-60 cells with 25 and 50 µM 3′-Me-Ado for 24 h, [14C]-
cytidine incorporation into DNA was significantly de-
creased to 14.2% and 5% of control values, respectively.
In contrast, incubation with 2′-Me-Ado did not have a
significant effect on the incorporation of [14C]cytidine
into DNA, although values measured have shown a
meaningful increase.
Conclusion
In summary, a series of adenosine derivatives sub-
stituted at the 1′-, 2′-, or 3′-position of the ribose ring
with a methyl group have been synthesized and evalu-
ated as antitumor agents. 3′-C-Methyladenosine emerged
as the most active compound, showing activity against
human myelogenous leukemia K562, multidrug-resis-
tant human leukemia K562IU, human promyelocytic
leukemia HL-60, human colon carcinoma HT-29, and
human breast carcinoma MCF-7 cell lines with IC50
values in the micromolar range. Structure-activity
relationships studies have pointed out that the structure
of 3′-Me-Ado is crucial for the antitumor activity. Thus,
the substitution of a hydrogen atom of the amino group
in 6-position with a small alkyl or cycloalkyl group, the
introduction of a chlorine atom in the 2-position, or the
moving of the methyl group from the 3′-position to the
other positions of the ribose ring brings about a decrease
or loss of activity. The antitumor activity of 3′-Me-Ado
Since RR is the rate-limiting enzyme catalyzing de
novo DNA synthesis, the inhibition of the incorporation

Antitumor C-Methyl-â-D-ribofuranosyladenine
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15 4987
Scheme 3. Mechanism of Reduction of Ribonucleosides to 2′-Deoxyribonucleosides Catalyzed by Ribonucleotide
Reductase (RR) As Proposed by Stubbe and Ackles16 and Confirmed by Ong et al.11
tion by flash chromatography on silica gel (n-hexane/CHCl3,
95:5) gave 15 as a white solid (1.03 g, 78%). 1H NMR (CDCl3):
δ 1.84 (s, 3H, CH3), 2.17, 2.23 (2 s, 6H, 2 Ac), 4.56 (dd, J )
4.0, 12.5 Hz, 1H, H-5′), 4.85 (dd, J ) 3.5, 12.6 Hz, 1H, H-5′),
5.06 (t, J ) 3.7 Hz, 1H, H-4′), 5.94 (d, J ) 7.3 Hz, 1H, H-2′),
6.26 (d, J ) 7.3 Hz, 1H, H-1′), 7.45, 7.60, 8.03 (3m, 5H, arom.),
8.24 (s, 1H, H-8). Anal. (C22H20Cl2N4O7) C, H, N.
2-Chloro-9H-(3-C-methyl-â-D-ribofuranosyl)adenine (5).
The title compound was obtained as described for 3 starting
from 15 (reaction time 22 h). Chromatography on a silica gel
column (CHCl3/MeOH, 90:10) gave 5 as a white solid (yield
55%). 1H NMR (DMSO-d6): δ 1.29 (s, 3H, CH3), 3.50-3.71 (m,
2H, H-5′), 3.88 (t, J ) 3.1 Hz, 1H, H-4′), 4.38 (pseudo t, 1H,
H-2′), 4.87 (s, 1H, OH-3′), 5.20 (dd, J ) 4.4, 5.7 Hz, 1H, OH-
5′), 5.42 (d, J ) 7.0 Hz, 1H, OH-2′), 5.78 (d, J ) 8.1 Hz, 1H,
H-1′), 7.85 (br s, 2H, NH2), 8.40 (s, 1H, H-8). MS: m/z, 316 [M
+ H]+. Anal. (C11H14ClN5O4) C, H, N.
General Procedure for the Ammination of 10 and 14
into Compounds 6-9. To 6-chloro-9H-(2-C-methyl-2,3,5-tri-
O-benzoyl-â-D-ribofuranosyl)purine (10)4 or 6-chloro-9H-(3-C-
methyl-2,3-di-O-acetyl-5-O-benzoyl-â-D-ribofuranosyl)purine (14)
was added the suitable 40% aqueous amine (molar ratio 1:6),
and the mixture was stirred at room temperature or at 40 °C.
The solution was evaporated in vacuo and the residue was
purified by column chromatography.
appears to be related to its ability to deplete intracel-
lular purine and pyrimidine deoxynucleotides through
ribonucleotide reductase inhibition. Owing to the sig-
nificant antitumor activity of 3′-Me-Ado, its resistance
to the enzymatic degradation by adenosine deaminase,18
and its prolonged stability in fetal calf serum, additional
evaluation of the cytotoxic effects and in vivo activity
of this nucleoside in a panel of human tumors is
warranted.
Experimental Section
Chemistry. All reagent and solvents were purchased from
Aldrich Chemical Co. Thin-layer chromatography (TLC) was
run on silica gel 60 F254 plates; silica gel 60 (70-230 and 230-
400 mesh, Merck) for column chromatography was used.
Nuclear magnetic resonance spectra were recorded on a Varian
VXR-300 spectrometer with TMS as the internal standard for
1H NMR and external H3PO4 for 31P NMR. Chemical shift are
reported in part per million (δ) as s (singlet), d (doublet), t
(triplet), dd (double doublet), q (quartet), m (multiplet), or br
s (broad singlet). Stationary NOE experiments were run on
degassed solutions at 25 °C. Mass spectroscopy was carried
out on an HP 1100 series instrument. All measurements were
performed in the positive ion mode using atmospheric pressure
electrospray ionization (API-ESI).
N6-Methyl-9H-(2-C-methyl-â-D-ribofuranosyl)adenine
(6). Reaction of 10 with 40% aqueous methylamine at room
temperature for 3 h, followed by chromatography on a silica
gel column (CHCl3/MeOH, 90:10), gave 6 as a white solid (79%
yield). 1H NMR (DMSO-d6): δ 0.75 (s, 3H, CH3), 2.90 (br s,
3H, CH3NH), 3.60-3.90 (2m, 3H, H-4′, H-5′), 4.05 (m, 1H,
H-3′), 5.20 (m, 3H, OH), 5.95 (s, 1H, H-1′), 7.75 (br s, 1H, NH),
8.20 (s, 1H, H-2), 8.45 (s, 1H, H-8). MS: m/z 296.30 [M + H]+.
Anal. (C12H17N5O4) C, H, N.
6-Chloro-9H-(2,3-di-O-acetyl-5-O-benzoyl-3-C-methyl-
â-D-ribofuranosyl)purine (14). To a stirred mixture of 11
(800 mg, 2.03 mmol), 6-chloropurine (12) (340 mg, 2.25 mmol),
and DBU (0.86 mL, 6.08 mmol) in anhydrous acetonitrile (4
mL) at 0 °C was added TMSiOTf (1.47 mL, 8.11 mmol). After
3 h at 60 °C, the mixture was cooled at room temperature,
poured into aqueous NaHCO3 (1 M, 15 mL) and extracted with
CH2Cl2 (3 × 10 mL). The combined organic phase was dried
over Na2SO4 and evaporated to dryness. The residue was
purified by flash chromatography on silica gel (n-hexane/
N6-Cyclopropyl-9H-(2-C-methyl-â-D-ribofuranosyl)ad-
enine (8). Reaction of 10 with 40% aqueous cyclopropylamine
at 40 °C for 22 h, followed by chromatography on a silica gel
column (EtOAc/CHCl3/MeOH, 70:25:5), gave 8 as a white solid
(76% yield). 1H NMR (DMSO-d6): δ 0.50-0.75 (2m, 4H,
cyclopropyl), 0.75 (s, 3H, CH3), 3.0 (br s, 1H, CHNH), 3.65-
3.90 (m, 3H, H-4′, H-5′), 4.05 (pseudo t, 1H, H-3′), 5.20 (m,
3H, OH), 5.98 (s, 1H, H-1′), 7.98 (br s, 1H, NH), 8.25 (s, 1H,
H-2), 8.48 (s, 1H, H-8). MS: m/z 322.34 [M + H]+. Anal.
(C14H19N5O4) C, H, N.
1
EtOAc, 75:25) to give 14 as a white foam (720 mg, 72%). H
NMR (CDCl3): δ 1.82 (s, 3H, CH3), 2.05, 2.15 (2s, 6H, 2 Ac),
4.56 (dd, J ) 4.6, 12.6 Hz, 1H, H-5′), 4.82 (dd, J ) 3.3, 12.8
Hz, 1H, H-5′), 5.0 (t, J ) 3.8 Hz, 1H, H-4′), 6.10 (d, J ) 7.3
Hz, 1H, H-2′), 6.28 (d, J ) 7.3 Hz, 1H, H-1′), 7.50, 7.65, 8.05
(3m, 5H, arom.), 8.25 (s, 1H, H-2), 8.63 (s, 1H, H-8). Anal.
(C22H21ClN4O7) C, H, N.
9H-(3-C-Methyl-â-D-ribofuranosyl)adenine (3). A mix-
ture of 14 (700 mg, 1.43 mmol) and liquid ammonia was
reacted in a stainless steel bomb at 60 °C for 30 h. After
evaporation the residue was purified by flash chromatography
on silica gel eluting with CHCl3/MeOH (95:5) to give 3 as a
white solid (390 mg, 97%). 1H NMR (DMSO-d6): δ 1.30 (s, 3H,
CH3), 3.46-3.74 (m, 2H, H-5′), 3.88 (t, J ) 2.6 Hz, 1H, H-4′),
4.46 (pseudo t, 1H, H-2′), 4.85 (s, 1H, OH-3′), 5.42 (d, J ) 6.6
Hz, 1H, OH-2′), 5.82 (d, J ) 8.1 Hz, 1H, H-1′), 5.95 (dd, J )
3.5, 8.3 Hz, 1H, OH-5′), 7.42 (br s, 2H, NH2), 8.12 (s, 1H, H-2),
8.34 (s, 1H, H-8). MS: m/z 282 [M + H]+. Anal. (C11H15N5O4)
C, H, N.
N6-Methyl-9H-(3-C-methyl-â-D-ribofuranosyl)adenine
(7). Reaction of 14 with 40% aqueous methylamine at room
temperature for 1 h, followed by chromatography on a silica
gel column (CHCl3/MeOH/NH4OH, 92:7:1), gave 7 as a white
solid (72% yield). 1H NMR (DMSO-d6): δ 1.30 (s, 3H, CH3),
2.95 (br s, 3H, CH3NH), 3.60 (m, 2H, H-5′) 3.88 (t, J ) 2.6 Hz,
1H, H-4′), 4.45 (d, J ) 7.7 Hz, 1H, H-2′), 4.85 (s, 1H, OH-3′),
5.4 (br s, 1H, OH-2′), 5.83 (d, J ) 8.1 Hz, 1H, H-1′), 5.95 (dd,
J ) 3.3, 8.4 Hz, 1H, OH-5′), 7.90 (br s, 1H, NH), 8.20 (s, 1H,
H-2), 8.30 (s, 1H, H-8). MS: m/z 296.30 [M + H]+. Anal.
(C12H17N5O4) C, H, N.
2,6-Dichloro-9H-(2,3-di-O-acetyl-5-O-benzoyl-3-C-meth-
yl-â-D-ribofuranosyl)purine (15). The title compound was
prepared starting from 2,6-dichloropurine (13) (660 mg, 3.49
mmol) and 11 (1.0 g, 2.53 mmol) as described for 14. Purifica-
N6-Cyclopropyl-9H-(3-C-methyl-â-D-ribofuranosyl)ad-
enine (9). The title compound was obtained from 14 as
reported for 8, as a white solid (60% yield). 1H NMR (DMSO-
d6): δ 0.60-0.80 (2m, 4H, cyclopropyl), 1.30 (s, 3H, CH3), 3.07

4988 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15
Franchetti et al.
(br s, 1H, CHNH), 3.60 (m, 2H, H-5′), 3.88 (t, J ) 2.6 Hz, 1H,
H-4′), 4.45 (t, J ) 7.3 Hz, 1H, H-2′), 4.85 (s, 1H, OH-3′), 5.40
(d, J ) 6.6 Hz, 1H, OH-2′), 5.85 (d, J ) 8.1 Hz, 1H, H-1′), 5.92
(dd, J ) 3.5, 7.9 Hz, 1H, OH-5′), 8.08 (br s, 1H, NH), 8.22 (s,
1H, H-2), 8.35 (s, 1H, H-8). MS: m/z 322.34 [M + H]+. Anal.
(C14H19N5O4) C, H, N.
for 1 min. The lysate was rested on ice for 30 min and then
the proteins were separated by centrifugation at 15 000g for
10 min in an Eppendorf microcentrifuge. The supernatant was
removed and neutralized by adding 1.1 vol of Freon containing
0.5 M tri-n-octylamine. Aliquots (100 µL) of this sample were
periodated by adding 30 µL of 4 M methylamine solution and
10 µL of periodate solution (concentration 100 g/L). After
incubation at 37 °C for 30 min, the reaction was stopped by
adding 5 µL of rhamnose solution. The extracted dNTPs were
measured by using a Merck “La Chrom” HPLC system
equipped with an L-7200 autosampler, an L-7100 pump, an
L-7400 UV detector, and a D-7000 interface unit. Samples
were eluted with a 3.2 mol/L ammonium phosphate buffer, pH
3.8 containing 20 mol/L acetonitrile using a 4.6 × 250 mm
Partisil 10 SAX column. Separation was performed at constant
ambient temperature with a flow rate of 2 mL/min. The
concentration of dNTPs was calculated as a percent of the
control. The initial intracellular dNTP concentrations of
untreated control cells were determined by calibration to a
standard 100 µM dNTP solutions. Intracellular concentrations
of dNTPs in untreated control cells were 7.8, 46.1, 4.4, and
8.2 µM for dCTP, dTTP, dATP, and dGTP, respectively.
Incorporation of 14C-Labeled Cytidine into DNA. To
analyze the effect of 2′-Me-Ado and 3′-Me-Ado incubation on
the activity of DNA synthesis, an assay was performed as
previously described.21 HL-60 cells in the logarithmic phase
of growth (1 × 106 cells/mL) were incubated with various
concentrations of 2′-Me-Ado and 3′-Me-Ado for 24 h. After
incubation, cells were counted and pulsed with [14C]cytidine
(0.3125 µCi, 5 nM) for 30 min at 37 °C. Afterward, cells were
collected by centrifugation and washed with PBS. Total DNA
was extracted from 5 × 106 cells and the specific radioactivity
of the samples was determined using a Wallac 1414 liquid
scintillation counter (Perkin-Elmer, Boston, MA).
Biological Assays. Cells and Culture. The cell lines
human myelogenous leukemia K562, human promyelocytic
leukemia HL-60, human colon carcinoma HT-29, human
colorectal adenocarcinoma CCL-228, and human breast car-
cinoma MCF-7 were obtained from the American Type Culture
Collection (ATCC, Manasses, VA). Multidrug resistant K562
(K562IU) cells were developed by exposing sensitive K562 cells
to sublethal concentrations of tiazofurin over a period of 60
generations. K562IU exhibited resistance to tiazofurin, ben-
zamide riboside, and related nucleoside analogues. K562 and
K562IU cells were maintained in RPMI 1640 medium (Gibco/
Life Technologies, Gaithersburg, MD) containing 10% heat-
inactivated fetal bovine serum (FBS) (Atlanta Biologicals,
Atlanta, GA) and 10 000 U/L penicillin and 50 mg/L strepto-
mycin.19 HT-29 and MCF-7 cells were maintained in minimal
essential medium alpha (MEM) with Earl’s balanced salts, 10%
FBS, penicillin, and streptomycin as above. Logarithmically
growing HT-29 and MCF-7 cells were incubated with 0.05%
trypsin containing 1 mM EDTA at 37 °C for about 5 min until
cells were nonadherent and formed a single cell suspension.
Trypsin activity was neutralized by adding a 20-fold excess of
serum-containing medium. Cells were cultured at 37 °C in an
atmosphere of air and 5% CO2.
Antitumor Activity. Antitumor activity of the compounds
was determined by their cytotoxic action on cultured tumor
cells. Cytotoxicity assays were conducted by tetrazolium
reduction of MTS with PMS (CellTiter Assay, Promega,
Madison, WI). Logarithmically growing cells were plated in
0.1-mL aliquots in 96-well microtiter plates. Cells were plated
at an initial density of about 50 000 cells/mL and allowed to
acclimatize for 24 h. Cell suspensions were treated with
various dilutions of compounds in triplicate, mixed well, and
allowed to incubate for 48 h at 37 °C in an atmosphere of air
and 5% CO2. To the cell suspension was added 20 µL of
tetrazolium reagent, the mixture was incubated for 3 h at 37
°C in an atmosphere of air and 5% CO2, and absorbance at
490 nm was read by microplate reader. Control plates with
serial dilutions of cell types were counted as a control for the
assay. In all cases, controls indicated a linear response versus
cell number, R2 g 0.99.
Stability of 2′-Me-Ado and 3′-Me-Ado in Culture Me-
dium. Stability of 2′-Me-Ado and 3′-Me-Ado was evaluated by
incubation of the compounds (50 µM) in MEM medium
containing 10% fetal bovine serum (Sigma Chemical Co.) for
72 h at 37 °C. At various times, 200 µL of the medium was
removed and frozen. The collected samples were thawed and
passed through 0.22-µm syringe filters and then subjected to
HPLC. Samples were eluted with a linear gradient from water
to 100% methanol using a 4.6 × 250 mm Beckman Ultrasphere
5 µm C18 column. A HP1090 system was used. 2′-Me-Ado and
3′-Me-Ado were also analyzed without serum containing
medium and found to be stable.
Clonogenic Assay. The inhibition of colony formation by
2′-Me-Ado and 3′-Me-Ado was also investigated in HT-29 and
CCL-228 cells. A total of 103 cells/well were plated in 24-well
plates and allowed to attach overnight. The next day, the
medium was replaced by fresh medium containing various
concentrations of the drugs. The plates were incubated with
the drugs for 7 days, then the medium was removed and the
wells were stained with crystal violet solution. Colonies (>50
cells) were counted using a VWR Merck colony counter. All
experiments were run in triplicate.
Acknowledgment. This research was supported by
the Italian MIUR (PRIN 2002) and by the Project
Development Program, Research and Sponsored Pro-
grams, Indiana University, Indianapolis.
Supporting Information Available: Experimental de-
tails for the synthesis of compound 11 and analysis data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Analysis of Intracellular dNTP Pools by High Perfor-
mance Liquid Chromatography (HPLC). The effects of 2′-
Me-Ado and 3′-Me-Ado on intracellular dNTP pools were
analyzed in HL-60 cells. The effects of these nucleosides were
compared with that of a clinically well-established inhibitor
of ribonucleotide reductase (RR) on intracellular dNTP pool
concentrations, with dFdC as a reference substance. The
extraction of dNTPs from the cells was done by the method
described by Garrett et al.20 Cells (7 × 107) were seeded at a
concentration of 1 × 106/mL in 70 mL of complete medium
and were incubated with various concentrations of 2′-Me-Ado,
3′-Me-Ado, or dFdC for 24 h. After the incubation period, the
cell count was determined, and 7.5 × 107 cells were separated
for dNTP extraction. All steps of the extraction were carried
out on ice. The corrected number of cells was centrifuged at
600g for 5 min and then resuspended in 100 µL of phosphate-
buffered saline. In this suspension, cells were lysed by addition
of 10 µL of trichloroacetic acid, and the mixture was vortexed
References
(1) Niitsu, N.; Ishii, Y.; Matsuda, A.; Honma, Y. Induction of
differentiation of acute promyelocytic leukemia cells by a cytidine
deaminase-resistant analogue of 1-â-D-arabinofuranosylcytosine,
1-(2-deoxy-2-methylene-â-D-erythro-pentofuranosyl)-cytosine. Can-
cer Res. 2001, 61, 178-185.
(2) Sun, L. Q.; Li, Y. X.; Guillou, L.; Mirimanoff, R. O.; Coucke, P.
A. Antitumor and radiosensitizing effects of (E)-2′-deoxy-2′-
(fluoromethylene)cytidine, a novel inhibitor of ribonucleoside
diphosphate reductase, on human colon carcinoma xenografts
in nude mice. Cancer Res. 1997, 57, 4023-4028.
(3) Hattori, H.; Nozawa, E.; Iino, T.; Yoshimura, Y.; Shuto, S.;
Shimamoto, Y.; Nomura, M.; Fukushima, M.; Tanaka, M.;
Sasaki, T.; Matsuda, A. Nucleosides and nucleotides. 175.
Structural requirements of the sugar moiety for the antitumor
activities of new nucleoside antimetabolites, 1-(3-C-ethynyl-â-
D-ribo-pentofuranosyl)cytosine and -uracile. J. Med. Chem. 1998,
41, 2892-2902.

Antitumor C-Methyl-â-D-ribofuranosyladenine
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15 4989
(4) Franchetti, P.; Cappellacci, L.; Marchetti, S.; Trincavelli, L.;
Martini, C.; Mazzoni, M. R.; Lucacchini, A.; Grifantini, M. 2′-
C-Methyl analogues of selective adenosine receptor agonists:
Synthesis and binding studies. J. Med. Chem. 1998, 41, 1708-
1715.
(5) Cappellacci, L.; Barboni, G.; Palmieri, M.; Pasqualini, M.;
Grifantini, M.; Costa, B.; Martini, C.; Franchetti, P. Ribose-
modified nucleosides as ligands for adenosine receptors: Syn-
thesis, conformational analysis, and biological evaluation of 1′-
C-methyl adenosine analogues. J. Med. Chem. 2002, 45, 1196-
1202.
(6) Walton, E.; Jenkins, S. R.; Nutt, R. F.; Zimmerman, M.; Holly,
F. W. Branched-chain sugar nucleosides. New type of biological
active nucleoside. J. Am. Chem. Soc. 1966, 88, 4524-4525.
(7) Nutt, R. F.; Dickinson, M. J.; Holly, F. W.; Walton, E. Branched-
chain sugar nucleosides. III. 3′-C-Methyladenosine. J. Org.
Chem. 1968, 33, 1789-1795.
(8) Waga, T.; Nishizaki, T.; Miyakawa, I.; Ohrui, H.; Meguro, H.
Studies on sugar-modified nucleosides. Part I. Synthesis of 4′-
C-methylnucleosides. Biosci., Biotechnol., Biochem. 1993, 57,
1433-1438.
(9) Griffon, J.-F.; Dukhan, D.; Pierra, C.; Benzaria, S.; Loi, A. G.;
La Colla, P.; Sommadossi, J.-P.; Gosselin, G. 4′-C-Methyl-â-D-
ribofuranosyl purine and pyrimidine nucleosides revisited.
Nucleosides, Nucleotides Nucleic Acids 2003, 22, 707-709.
(10) Ceruti, S.; Franceschi, C.; Barbieri, D.; Malorni, W.; Camurri,
A.; Giammarioli, A. M.; Ambrosini, A.; Racagni, G.; Cattabeni,
F.; Abbracchio, M. P. Apoptosis induced by 2-chloro-adenosine
and 2-chloro-2′-deoxy-adenosine in a human astrocytoma cell
line: differential mechanisms and possible clinical relevance.
J. Neurosci. Res. 2000, 60, 388-400.
(11) (a) Ong, S. P.; Nelson, L. S.; Hogenkamp, H. P. Synthesis of 3′-
C-methyladenosine and 3′-C-methyluridine diphosphates and
their interaction with the ribonucleoside diphosphate reductase
from Corynebacterium nephridii. Biochemistry 1992, 31, 11210-
11215. (b) Ong, S. P.; McFarlan, S. C.; Hogenkamp, H. P. 2′-C-
Methyladenosine and 2′-C-methyluridine 5′-diphosphates are
mechanism-based inhibitors of ribonucleoside diphosphate re-
ductase from Corynebacterium nephridii. Biochemistry 1993, 32,
11397-11404.
(14) Rosemeyer, H.; Toth, G.; Seela, F. Assignment of anomeric
configuration of D-ribo-, arabino-, 2′-deoxyribo-, and 2′,3′-dideoxy-
ribonucleosides by NOE difference spectroscopy. Nucleosides
Nucleotides 1989, 8, 587-597.
(15) Cappellacci, L.; Franchetti, P.: Pasqualini, M.; Petrelli, R.; Vita,
P.; Lavecchia A.; Novellino, E.; Costa B.; Martini, C.; Klotz K.-
N.; Grifantini M. Synthesis, biological evaluation and molecular
modeling of ribose-modified adenosine analogues as adenosine
receptor agonists. J. Med. Chem. 2005, 48, 1555-1562.
(16) (a) Stubbe, J.; Ackles D. On the mechanism of ribonucleoside
diphosphate reductase from Escherichia coli. Evidence for 3′-
C-H bond cleavage. J. Biol. Chem. 1980, 265, 8027-8030.
(17) (a) Eldrup, A. B.; Allerson, C. R.; Bennett, C. F.; Bera, S.; Bhat,
B.; Bhat, N.; Bosserman, M. R.; Brooks, J.; Burlein, C.; Carrol,
S. S.; Cook, P. D.; Getty, K. L.; MacCoss, M.; MacMasters, D.
R.; Olsen, D. B.; Prakash, T. P.; Prhavc, M.; Song, Q.; Tomassini,
J. E.; Xia, J. Structure-activity relationship of purine ribonucleo-
sides for inhibition of hepatitis C virus RNA-dependent RNA
polymerase. J. Med. Chem. 2004, 47, 2283-2295. (b) Eldrup,
A. B.; Prhavc, M.; Brooks, J.; Bhat, B.; Prakash, T. P.; Song, Q.;
Bera, S.; Bhat, N.; Dande, P.; Cook, P. D.; Bennett, C. F.; Carroll,
S. S.; Ball, R. G.; Bosserman, M.; Burlein, C.; Colwell, L. F.; Fay,
J. F.; Flores, O. A.; Getty, K.; LaFemina, R. L.; Leone, J.;
MacCoss, M.; McMasters, D. R.; Tomassini, J. E.; Von Langen,
D.; Wolanski, B.; Olsen, D. B. Structure-activity relationship
of heterobase-modified 2′-C-methyl ribonucleosides as inhibitors
of hepatitis C virus RNA replication. J. Med. Chem. 2004, 47,
5284-5297.
(18) Kalinichenko, E. N.; Beigel’man, L. N.; Mikhailov, S. N.;
Mikhailopulo, I. A. Substrate specificity of adenosine deaminase.
The role of methyl groups at 2′, 3′, and 5′-carbon atoms of
adenosine. Bioorg. Khim. 1988, 14, 1157-1161.
(19) Yalowitz, J. A.; Pankiewicz, K.; Patterson, S. E.; Jayaram, H.
N. Cytotoxicity and cellular differentiation activity of methyl-
enebis(phosphonate) analogues of tiazofurin and mycophenolic
acid adenine dinucleotide in human cancer cell lines. Cancer Lett.
2002, 181, 31-38.
(20) Garrett, C.; Santi, D. V. A Rapid and sensitive high-pressure
liquid chromatography assay for deoxynucleoside triphosphates
in cell extracts. Anal. Biochem. 1979, 99, 268-273.
(12) Kusachi, S.; Thompson, R. D.; Bugni, W. J.; Yamada, N.; Olsson,
R. A. Dog coronary artery adenosine receptor: structure of the
N6-alkyl subregion. J. Med. Chem. 1985, 28, 1636-1643.
(13) Thompson, R. D.; Secunda, S.; Daly, J. W.; Olsson, R. A. Activity
of N6-substituted 2-chloroadenosine at A1 and A2 adenosine
receptors. J. Med. Chem. 1991, 34, 3388-3390.
(21) Szekeres, T.; Gharehbaghi, K.; Fritzer, M.; Woody, M.; Srivas-
tava, A.; van’t Riet, B.; Jayaram, H. N.; Elford, H. L. Biochemical
and antitumor activity of trimidox, a new inhibitor of ribonucle-
otide reductase. Cancer Chemother. Pharmacol. 1994, 34, 63-66.
JM048944C