Novel isopentenyladenosine analogues: Synthesis, characterization, and evaluation of antiproliferative activity on bladder carcinoma cells

Article abstract of DOI:10.1080/15257770903155550

Isopentenyladenosine (iPA), a member of the cytokinin family of plant hormones, exerts a marked antiproliferative activity on some leukemic and epithelial cancer cell lines. To characterize the molecular moieties required for the in vitro antitumor activity of the molecule and to obtain cytostatic iPA derivatives potentially useful as chemotherapeutic agents, N9-acyclic analogues have been synthesized using regioselective Mitsunobu reaction and characterized by elemental analyses, ¹H and ¹³C NMR. These compounds were analyzed for their activity on human bladder cancer cell lines. In this study, we report that iPA inhibited the proliferation but not the migration of human bladder cancer cells, while the newly synthesized analogues revealed no significant cytostatic activity apart from the compound with a saturated double bond of the isopentenyl chain. These results indicate that the integrity of the ribose ring is required for the cytostatic activity of iPA.

Full text of DOI:10.1080/15257770903155550

Nucleosides, Nucleotides and Nucleic Acids, 28:736–751, 2009
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770903155550
NOVEL ISOPENTENYLADENOSINE ANALOGUES: SYNTHESIS,
CHARACTERIZATION, AND EVALUATION OF ANTIPROLIFERATIVE
ACTIVITY ON BLADDER CARCINOMA CELLS
Roberta Ottria, Silvana Casati, Jeanette A. M. Maier, Massimo Mariotti, and
Pierangela Ciuffreda
Dipartimento di Scienze Precliniche LITA Vialba-Universita` degli Studi di Milano,
Milano, Italy
Isopentenyladenosine (iPA), a member of the cytokinin family of plant hormones, exerts a
2
marked antiproliferative activity on some leukemic and epithelial cancer cell lines. To characterize
the molecular moieties required for the in vitro antitumor activity of the molecule and to obtain
cytostatic iPA derivatives potentially useful as chemotherapeutic agents, N9-acyclic analogues have
been synthesized using regioselective Mitsunobu reaction and characterized by elemental analyses,
1H and 13C NMR. These compounds were analyzed for their activity on human bladder cancer cell
lines. In this study, we report that iPA inhibited the proliferation but not the migration of human
bladder cancer cells, while the newly synthesized analogues revealed no significant cytostatic activity
apart from the compound with a saturated double bond of the isopentenyl chain. These results
indicate that the integrity of the ribose ring is required for the cytostatic activity of iPA.
Keywords Isopentenyladenosine; Acyclic nucleosides; Antitumor agents; Bladder car-
cinoma cells
INTRODUCTION
Cytokinins are an important class of plant growth hormones which
regulate cell division, differentiation and gene expression.[1] The only
known cytokinin existing in animal cells is isopentenyladenosine (iPA, 1,
Figure 1), a modified nucleoside derived from mevalonate. It has been
detected in the cytosol of many eukaryotic and prokaryotic cells as a free
compound, bound to tRNAs or to a 26 kDa protein.[2] At the moment,
Received 12 May 2009; accepted 9 June 2009.
This work was financially supported by Universita` degli Studi di Milano (Fondi FIRST).
Address correspondence to Pierangela Ciuffreda, Dipartimento di Scienze Precliniche
LITA Vialba-Universita` degli Studi di Milano-Via G.B. Grassi,74-20157, Milano, Italy. E-mail:
pierangela.ciuffreda@unimi.it
736

Novel Isopentenyladenosine Analogues
737
FIGURE 1 The structures of the modified iPA analogous.
however, the biological role of iPA in mammalian cells is rather obscure.[3,4]
It has been demonstrated to inhibit protein prenylation and compete for
nucleoside transport. In addition, as a component of mammalian serine
tRNA, it is conceivable that iPA participates to regulate RNA and protein
synthesis.
Spinola et al.[5] have recently shown that iPA exerts potent in vitro
antitumor activity on human epithelial cancer cells. This inhibition of cell
growth is paralleled by the upregulation of genes involved in arresting cell
cycle, as demonstrated by gene expression profile of iPA-treated cancer
cells.[6] However, iPA has only slight effect on tumor growth in rodents;[7]
this is in agreement with the results obtained in a pilot clinical trial.[8] This
lack of effect in vivo might be due to the short plasma half-life of iPA, in
analogy to other nucleosides.
With the aim of synthesizing compounds endowed with antiproliferative
activity both in vitro and in vivo, we developed structural modifications of
iPA. We focused our interest on the modifications of the N6-isopentenyl
moiety as well as on the modifications of the ribosidic moiety.
Initially, the modification of the N6-isopentenyl moiety with N6-isopentyl
moiety has been considered (isopentyladenosine 2). Successively, our atten-
tion was devoted to the ribosidic portion of iPA, since the β-N-glycosidic
bond can be cleaved by enzymatic phosphorolysis in vivo. In this respect,
in order to obtain more stable compounds, the β-N-glycosidic bond (N-C-
O) was replaced with a stronger N-C-C bond. Therefore, the synthesis of
the acyclic nucleosides 3–6, analogues of the antiviral drug penciclovir,[9]

738
R. Ottria et al.
was considered. The acyclic chain may be indeed considered as resulting
from the suppression of a methylene group with respect to a carbohydrate
moiety.[10]
Here, we describe the synthesis and biological activity of compound
2–6. Substituent at position 6 (isopentyl instead of isopentenyl) of adenine
and the ribose moiety modification at position 9 were chosen to verify
whether these modifications could affect the antiproliferative activity of
human bladder cancer cells. Bladder cancer is the second most common
malignancy of the genitourinary tract, and the second most common cause
of death of all genitourinary tumors. Of all types of cancer, bladder cancer
has an unusually high propensity for recurring after therapy, regardless of
treatment with surgery, chemotherapy, or immunotherapy. The availability
of novel molecules to treat this neoplasia is therefore a crucial topic. This
is the reason why we evaluated the activity of iPA and its derivatives on two
different human bladder cancer cell lines. We found that iPA was very potent
in arresting cell proliferation, while it did not inhibit cell migration, an early
pivotal event in cancer invasion. Newly synthesized iPA derivatives, however,
were not cytostatic apart from the compound 2 with a saturated double bond
of the isopentenyl chain.
RESULTS AND DISCUSSION
Chemistry
Monosubstituted N6-isopentyladenosine 2 was prepared in a one-step
conversion from 6-chloropurine riboside by nucleophilic substitution with
isopentylamine.
For the synthesis of nucleoside analogues in which a modified base
was linked to the modified sugar moiety, the N-alkylation of nucle-
obases has great significance since it is a direct route in the nucleosides
synthesis.[11–13] The N-alkylation of purine, or pyrimidine nucleobases, is
usually achieved using different sources of carbon electrophiles includ-
ing: alkyl halides,[14–16] alkyl tosylates,[17] alkyl mesylates,[18] epoxides,[19]
Michael acceptors,[20] and allylic esters catalyzed by Pd(0).[21] Finally Mit-
sunobu conditions were also applied[22–26] due to the advantageous strategy
of the direct synthesis of acyclic nucleosides from alcohols.
The first attempt to obtain 6-chloro-9-[(2,2-dimethyl-1,3-dioxan-4-
yl)methyl]purine (9) (Scheme 1), key intermediate of compounds 3 and
4, was the reaction of the chloride 7a with 6-chloropurine (8) and sodium
hydride in DMF. Unfortunately, in these conditions, the reaction gave a
mixture of undesired by-product 7-isomer and desired product 9-isomer 9
in low yields (8 and 15%, respectively).
Therefore to obtain compound 9, we tried to introduce the nucleic
base by substitution with alcohol 7b under Mitsunobu conditions. The

Novel Isopentenyladenosine Analogues
739
SCHEME 1 Reagents and conditions: a) DIAD, THF, Ph3P, room temperature, 15 hours; b) RNH2,
K2CO3, DMF, reflux, 3 hours; c) HCl/MeOH, room temperature, 2 hours.
expected compound 9 was obtained in reasonable yield (64%). It should be
noticed that only the N-9 regioisomer formed without another substitution
product. The side of N-alkylation was assigned by 1H and 13C NMR spectra
analysis.
1
Figure 2 shows relevant H/13C HMBC correlations for the identifica-
tion of compounds 9 as the N-9 regioisomers, in particular the diagnostic
HMBC correlations between CH2 or CH protons of the acyclic group with
1
H8, H5, and H2. In the H NMR spectrum of 9 the signal of proton H2
was unequivocally assigned by detecting the three-bond interaction with C4
and C6 (HMBC), whereas the signal of H8 was coupled by three-bond scalar
interaction with C4 and C5.
The stereochemistry of 9 was also elucidated by NOE correlations
observed between H2 and CH and CH2 of the acyclic moiety (Figure 3).
Monosubstituted N6-isopentenyl derivative 10 and N6-isopentyl deriva-
tive 11 were prepared in a one step conversion from 9 by nucleophilic
substitution with isopentenylamine and isopentylamine in almost quan-
titative yields. These compounds were successively deprotected with

740
R. Ottria et al.
FIGURE 2 Relevant 1H/13C HMBC correlations for identification of compounds 9 and 15 as the N-9
regioisomers.
hydrochloric acid in methanol at room temperature to obtain the target
compounds 3 and 4 in 67 and 79% yields, respectively. The overall chemical
yield from 8 was 43% for compound 3 and 50% for 4.
The synthesis of the 1,3-protected triol 13, essential for the achievement
of compounds 5 and 6, was performed modifying the procedures reported
in literature (Scheme 2).[9]
The commercially available starting material triethyl-1,1,2-
ethanetricarboxylate (12) was converted in 2-(hydroxymethyl)butane-
1,4-diol (13) through a reduction reaction with an excess of sodium
borohydride in tert-butyl alcohol in 90% yield. The crude product
contained a polar contaminant so it was convenient to purify it by column
FIGURE 3 NOE correlations in compounds 9 and 15.

Novel Isopentenyladenosine Analogues
741
SCHEME 2 Reagents and conditions: a) NaBH4, tBuOH, MeOH reflux for 60 minutes and room
temperature overnight; b) 2,2-dimethoxypropane, H2SO4, acetone, room temperature, 19 hours; c)
DIAD, THF, Ph3P, room temperature, 15 hours; d) RNH2, K2CO3, DMF, reflux, 3 hours; e) HCl/MeOH,
room temperature, 2 hours.
chromatography. Lithium aluminum hydride (LAH) attempt to reduce
the triester, resulted in a mixture of two unidentified components.
The selective 1,3-protection of the triol 13 was completed by the
introduction of the isopropylidene group through the reaction of 13
with 2,2-dimethoxypropane catalyzed by sulfuric acid. The reaction took
19 hours to produce 5-(2-hydroxyethyl)-2,2-dimethyl-1,3-dioxane (14) in
57% yield. A small amount of 7-membered ring by-product, very close
in TLC to the desired 6-membered ring product 14, also formed. It was
therefore necessary a particularly careful column chromatography to purify
compound 14.
The alcohol 14 was treated with 6-chloropurine (8), triphenylphosphine
and DIAD under Mitsunobu conditions (Scheme 2). The expected com-
pound 15 was obtained in reasonable yield (72%). It should be noticed that
only the N-9 regioisomer formed. The side of N-alkylation was assigned by
detecting the HMBC and NOESY interactions as analogously reported for
the compound 9 (Figure 2 and Figure 3).
Monosubstituted N6-isopentenyl derivative 16 and N6-isopentyl deriva-
tive 17 were prepared in a one step conversion from 15 by nucleophilic

742
R. Ottria et al.
substitution with isopentenylamine and isopentylamine in nearly quantita-
tive yields. These compounds were then deprotected with hydrochloric acid
in methanol at room temperature, to obtain the target compounds 5 (67%
yield) and 6 (70% yield). The overall chemical yield from 8 was 48% for
compound 5 and 50% for 6.
Effects of IPA Analogues on Human Bladder Cancer Cell Lines
Two different cell lines derived from human transitional cell bladder
carcinomas were selected for these studies: non tumorigenic T24 and
tumorigenic J82. We first evaluated the effects of iPA on the proliferation
of these cells. After 72 hours in the presence of different concentrations of
iPA, the cells were trypsinized and counted. We found that iPA inhibits cell
growth in a dose dependent fashion (Figure 4a).
To test whether this inhibition was reversible or not, after 12 hours ex-
posure to iPA (10 µM), we removed the compound, washed the monolayer
and incubated the cells in their culture medium for additional 60 hours.
Figure 4b shows that iPA inhibitory effect was irreversible both in T24 and
in J82 cells. We then compared iPA and its analogues 2–6 for their capability
to inhibit cell growth. Because the concentration of iPA which exerted the
maximal effect is 10 µM, the cells were exposed to 10 µM of each molecule
for 72 hours. Apart from compound 2, which showed a partial inhibitory
activity, all the analogues had no effect on cell growth both in J82 and in
T24 cells (Figure 4c). Consistently, whereas iPA completely inhibited type
clonogenity after 10 days of treatment, compound 2 reduced the number of
colonies and the other molecules had no effect on the clonogenicity of T24
and J82 cells (Figure 5).
We also evaluated the effects of iPA on cell migration, which is funda-
mental for invasion and metastasis. T24 cells were treated for 8 hours with
iPA and then exposed to EGF (10 ng/mL) for 14 additional hours. iPA and
its derivatives exerted no effect on basal or EGF-stimulated migration in T24
cells (Figure 6). Similar results were obtained in J82 cells (not shown).
CONCLUSION
Carcinoma of the bladder is the fifth most frequent human cancer,
primarily affecting people over the age of 50 years, and accounts for
approximately 3% of all cancer-related deaths.[27] It might aggressively
invade the bladder wall and originate metastases. These tumors frequently
recur after excision; consequently, it is relevant to search for novel molecules
that could prevent their recurrence and/or inhibit their progression. Our
results demonstrate that iPA is a potent irreversible inhibitor of bladder
cancer cell growth, while it does not exert any activity on cell migration,
which is necessary to invade the surrounding tissues and, eventually, to

Novel Isopentenyladenosine Analogues
743
FIGURE 4 Effects of iPA and its derivatives on bladder cancer cell growth. a) T24 and J82 cells were
cultured in the presence or in the absence of different concentrations of iPA. Seventy-two hours later,
the cells were trypsinized and counted as described in the methods. Results are expressed as the mean
standard deviation. b) Twenty-four hours after seeding, the cells were exposed to iPA (10 µM) for
12 hours. Then iPA was removed and the cells incubated for an additional 60 hours in their culture
medium before being counted as described above. c) T24 and J82 cells were cultured in the presence or
in the absence of iPA and its derivatives (10 µM). The experiment was performed as described in A. ∗p
= 0.0036, ∗∗p = 0.015, #p = 0.0044, ##p = 0.0041.
generate metastases. Interestingly, iPA was reproducibly unable to modulate
cell motility of several other cancer cell lines. On these bases, we propose
that iPA interferes with the complex regulation of cell proliferation without
affecting the signalling pathways necessary for cell migration.

744
R. Ottria et al.
FIGURE 5 Effects of iPA and its derivatives on the clonogenic activity. T24 and J82 cells were seeded
at very low density, treated with iPA and derivatives for 72 hours and maintained thereafter in culture
medium alone until the end of the experiment. The cells were stained and photographed (magnification
40 ×).
In spite of its inhibitory effects in vitro, iPA is not active in vivo,[7]
probably because it is rapidly inactivated. For this reason, iPA derivatives
were synthesized and tested for their activity on bladder cancer cell lines.
Various iPA analogues, modified at position 9, were synthesized by direct
coupling using Mitsunobu reaction that provided the expected compounds
in an efficient way, leading only to the N-9 adenine analogue. These deriva-
tives were evaluated for their in vitro effects, but no significant effect was
detected both on cell migration and on cell growth. In particular, when the
lateral chain was modified, a less active compound was obtained (compound

Novel Isopentenyladenosine Analogues
745
FIGURE 6 Effects of iPA and its derivatives on bladder cancer cell migration. Wound assay was
performed on confluent T24 cells. The cells were treated with EGF (100 ng/ml) and the different
molecules for 16 hours. The wound area was calculated by a specific software and expressed using an
arbitrary value scale.
2), whereas the substitution of the ribose moiety gave completely inactive
compounds (3–6).
We conclude that the antiproliferative action of iPA seems to depend
on two factors, namely, the ribose moiety and the substitution of amine
group in position 6. Further studies are necessary to identify the chemical
modifications which might preserve the biological activity also in vivo.
EXPERIMENTAL
General
Melting points were determined with a Stuart Scientific SMP3 melting
point apparatus and left uncorrected. Optical rotations were measured
on a 241 polarimeter (sodium D line at 25◦C; Perkin Elmer, Waltham,
MA, USA). NMR spectra were done on a Bruker (Rheinstetten, Germany)
AVANCE 500 spectrometer equipped with a 5mm broadband reverse probe
1
with field z-gradient operating at 500.13 and 125.76 MHz for H and 13C,
respectively. 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 δ (ppm) scale. The central peak of
CDCl3 signals (7.26 ppm for 1H and 77.7 ppm for 13C) and of CD3OD signals
1
(3.31 ppm for H and 49.1 ppm for 13C) were used as internal reference
standard. (NMR descriptions; s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet, and bs, broad singlet). Mass spectra were recorded on a Finnigan
LCQdeca (TermoQuest) in ESI negative ion mode, kV 5.00, 220◦C, 15 V.
Only significant m/z peaks, with their percentage of relative intensity in
parentheses are reported.
Reactions progress was monitored by analytical thin-layer chromatog-
raphy (TLC) on pre-coated glass plates (silica gel 60 F254-plate; Merck,
Darmstaat, Germany) and the products were visualized by UV light.

746
R. Ottria et al.
Elemental analyses were obtained on all intermediates and are within
0.4% of theoretical values.
(S)-(+)-2,2-Dimethyl-1,3-dioxolane-4-methanol (7b), dimethylallylbro-
mide, 1-amino-3-methylbutane and 6-chloropurine riboside, the other
reagents and all solvents 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.
3,3-dimethylallylamine was synthesized starting from 3,3-dimethyl allyl-
bromide according to literature method.[28]
Syntheses
General Procedure for Mitsunobu Reaction
Diisopropyl azodicarboxylate (DIAD, 1.2 mmol) was added dropwise
over 3 hours to a suspension of 6-chloropurine (8) (1 mmol), triph-
enylphosphine (1.2 mmol) and suitably protected alcohol (1.2 mmol) in
dry acetonitrile (20 mL), under argon atmosphere at 0◦C. The resulting
solution was stirred at room temperature overnight to yield a light orange
solution. Evaporation of the solvent gave a residue that was submitted
to column chromatography on silica gel (CH2Cl2/MeOH) to afford the
corresponding acyclonucleoside.
General Procedure for 6-Chlorine Substitution and Acyclic Chain Deprotection
K2CO3 (4.5 mmol) and the appropriate amine (4.5 mmol) were added
to a solution of 6-chloropurine derivative (1.5 mmol) in DMF (20 mL). The
mixture was refluxed for 3 hours, cooled to room temperature, filtered
on celite, and the solvent was removed under vacuum. The crude residue
was stirred in HCl (1.0 M in MeOH, 2.2 mL) at room temperature for 2
hours. The solution was neutralized by addition of DOWEX 50WX8–200.
The basic resin was filtered off, the solvent removed and the residue purified
by column chromatography on silica gel.
6-(3-Methylbutyl)-9-(β-D-ribofuranosyl)purine (2)
Compound 2 was prepared following the above described procedure
starting from 6-chloropurine riboside and 1-amino-3-methylbutane. Rf =
0.48 (CH2Cl2/MeOH, 90:10); white solid 68% yield; m.p. 152–154◦C; [α]20
D
-58.7 (c 1, MeOH) [lit.[29] m.p. 154.5–156◦C, [α]20D -42 (c 1.03, EtOH)]; 1H
NMR (CD3OD) δ = 1.00 (d, J = 6.6 Hz, 6H, 2 × CH3), 1.60 (dt, J = 6.6 Hz,
J = 6.6 Hz, 2H, β-CH2), 1.76 (dq, J = 6.6 Hz, J = 6.6 Hz, 1H, γ -CH), 3.63
(bs, 2H, α-CH2), 3.76 (dd, J = 2.5 Hz, J = 12.6 Hz, 1H, H5ꢁa), 3.91 (dd,
J = 2.5 Hz, J = 12.6 Hz, 1H, H5ꢁb), 4.19 (ddd, J = 2.5 Hz, J = 2.5 Hz, J =
2.5 Hz, 1H, H4ꢁ), 4.34 (dd, J = 2.5 Hz, J = 5.2 Hz, 1H, H3ꢁ), 4.76 (dd, J =
5.2 Hz, J = 6.3 Hz, 1H, H2ꢁ), 5.97 (d, J = 6.3 Hz, 1H, H1ꢁ), 8.23 (s, 1H,

Novel Isopentenyladenosine Analogues
747
H2), 8.25 (s, 1H, H8); 13C NMR δ = 19.98 (2 × CH3), 24.06 (γ -CH), 36.50
(α-CH2), 37.00 (β-CH2), 60.62 (C5ꢁ), 69.81 (C4ꢁ), 72.55 (C2ꢁ), 85.32 (C3ꢁ),
88.41(C1ꢁ), 120.33 (C5), 138.47 (C8), 147.66 (C4), 150.66 (C2), 153.45
(C6); ESIMS m/z 336 (M-1, 30%), 673 (2M-1, 100%).
6-Chloro-9-[(2,2-dimethyl-1,3-dioxolan-4-yl)methyl]purine (9)
Compound 9 was prepared following the above described procedure
starting from 6-chloropurine (8) and (S)-(+)-2,2-dimethyl-1,3-dioxolane-4-
methanol (7b). Rf = 0.39 (CH2Cl2/MeOH, 98:2); amorphous foam; 64%
yield; m.p. 98–100◦C; 1H NMR (CDCl3) δ = 1.33 (s, 3H, CH3), 1.38 (s, 3H,
CH3), 3.72 (dd, J = 5.5 Hz, J = 9.0 Hz, 1H, CH2-O), 4.15 (dd, J = 6.2 Hz,
J = 9.0 Hz, 1H, CH2-O), 4.37 (dd, J = 6.9 Hz, J = 15.2 Hz, 1H, N-CH2), 4.46
(dd, J = 3.5 Hz, J = 15.2 Hz, 1H, N-CH2), 4.51 (dddd, J = 3.5 Hz, J = 5.5
Hz, J = 6.2 Hz, J = 6.9 Hz, 1H, CH-O), 8.27 (s, 1H, H8), 8.74 (s, 1H, H2);
13C NMR δ = 24.92 (CH3), 26.63 (CH3), 46.19 (N-CH2), 66.30 (CH2-O),
73.55 (CH-O), 110.38 (C(CH3)2), 131.28 (C5), 146.32 (C8), 151.12 (C4),
151.28 (C6), 151.96 (C2); Anal. calcd for C11H13ClN4O2: C, 49.17; H, 4.88;
Cl, 13.19; N, 20.85; O, 11.9. Found: C, 49.29; H, 4.92; Cl, 13.22; N, 20.65.
6-(3-Methyl-2-butenylamino)-9-(propane-2,3-diol-1-yl)purine (3)
Compound 3 was prepared following the above described procedure
starting from 6-chloro-9-[(2,2-dimethyl-1,3-dioxan-4-yl)methyl]purine (9)
and 3,3-dimethylallylamine to give 6-(3-methyl-but-2-enylamino)-9-[(2,2-
dimethyl-1,3-dioxan-4-yl)-methyl]purine (10) that was deprotected to give
the desired compound 3. Rf = 0.21 (CH2Cl2/MeOH, 90:10); white solid;
1
67% yield; m.p. 154–156◦C; [α]20 -23.2 (c 1, MeOH); H NMR (CDCl3)
D
δ = 1.77 (s, 3H, CH3), 1.79 (s, 3H, CH3), 3.49 (dd, J = 5.5 Hz, J = 11.8 Hz,
1H, CH2-OH), 3.57 (dd, J = 4.2 Hz, J = 11.8 Hz, 1H, CH2-OH), 4.09 (dddd,
J = 4.2 Hz, J = 4.2 Hz, J = 5.5 Hz, J = 5.5 Hz, 1H, CH-OH), 4.23 (bs, 2H,
α-CH2), 4.34 (dd, J = 4.2 Hz, J = 14.5 Hz, 1H, N-CH2), 4.39 (dd, J = 5.5 Hz,
J = 14.5 Hz, 1H, N-CH2), 5.40 (t, J = 7.0 Hz, 1H, β-CH), 5.81 (bs, 1H, NH),
7.74 (s, 1H, H8), 8.38 (s, 1H, H2); 13C NMR δ = 18.00 (CH3), 25.66 (CH3),
38.75 (α-CH2), 46.78 (N-CH2), 62.77 (CH2-OH), 70.62 (CH-OH), 119.96
(β-CH), 136.85 (γ -C), 137.00 (C5), 140.63 (C8), 151.00 (C4), 152.98 (C2),
154.78 (C6); ESIMS m/e 276 (M-1, 100%), 553 (2M-1, 31%); Anal. calcd for
C16H23N5O2: C, 60.55; H, 7.30; N, 22.07; O, 10.08. Found: C, 60.43; H, 7.38;
N, 22.17.
6-(3-Methyl-butylamino)-9-(propane-2,3-diol-1-yl)purine (4)
Compound 4 was prepared following the above described proce-
dure starting from 6-chloro-9-[(2,2-dimethyl-1,3-dioxan-4-yl)methyl]purine
(9) and 1-amino-3-methylbutane to give 6-(3-methyl-butylamino)-9-[(2,2-
dimethyl-1,3-dioxan-4-yl)-methyl]purine (11) that was deprotected to the
desired compound 4. Rf = 0.23 (CH2Cl2/MeOH, 90:10); white solid; 69%

748
R. Ottria et al.
yield; m.p. 98–100◦C; [α]20 -20.0 (c 1, MeOH); 1H NMR (CDCl3) δ = 0.98
D
(d, J = 6.6 Hz, 6H, 2 × CH3), 1.60 (dt, J = 6.6 Hz, J = 6.6 Hz, 2H, β-CH2),
1.76 (dq, J = 6.6 Hz, J = 6.6 Hz, 1H, γ -CH), 3.50 (dd, J = 6.2 Hz, J =
11.8 Hz, 1H, CH2-OH), 3.58 (dd, J = 4.2 Hz, J = 11.8 Hz, 1H, CH2-OH),
3.66 (bs, 2H, α-CH2), 4.09 (dddd, J = 4.2 Hz, J = 4.2 Hz, J = 5.5 Hz, J =
6.2 Hz, 1H, CH-OH), 4.32–4.40 (AB part of ABX system, 2H, N-CH2), 6.05
(bs, 1H, NH), 7.75 (s, 1H, H8), 8.35 (s, 1H, H2); 13C NMR δ = 22.50 (2 ×
CH3), 25.78 (γ -CH), 38.43 (α-CH2), 39.00 (β-CH2), 46.79 (N-CH2), 62.76
(CH2-OH), 70.57 (CH-OH), 139.23 (C5), 140.60 (C8), 151.98 (C4), 153.01
(C2), 154.88 (C6); ESIMS m/z 278 (M-1, 100%), 557 (2M-1, 52%); Anal.
calcd for C16H25N5O2: C, 60,17; H, 7.89; N, 21.93; O, 10.02. Found: C, 60,09;
H, 7.76; N, 22.06.
2-(Hydroxymethyl)butane-1,4-diol (13)[9]
Triethyl-1,1,2-ethanetricarboxylate (12) (4.65 mL, 20.3 mmol) and
NaBH4 (2.0 g, 53 mmol) were dissolved in tert-butylalcohol (40 mL). The
mixture was heated to reflux and then MeOH (2.5 mL) was added in
portions. The mixture was refluxed for 1 additional hour, then cooled
to room temperature and stirred overnight. HCl (5M, 3 mL) was added
carefully until the solution was neutral. A large amount of white solid
formed. The mixture was filtered and the residue was washed with EtOH
(30 mL), after removal of the solvent the residue was purified by column
chromatography on silica gel (CH2Cl2/MeOH, 95:5) to give a colorless oil
13 (2.17 g, 91%); Rf = 0.35 (CH2Cl2/MeOH, 95:5); 1H NMR (DMSO) δ =
1.39 (q, J = 6.6 Hz, 2H, CH-CH2), 1.54–1.58 (m, 1H, CH-CH2), 3.33–3.39
(m, 2H, ax-CH), 4.42–4.45 (m, 2H, eq-CH).
5-(2-Hydroxyethyl)-2,2-dimethyl-1,3-dioxane (14)[9]
Sulfuric acid 96% (0.2 mL) was added to
a
solution of
2-(hydroxymethyl)butane-1,4-diol (13) (1 g, 8.4 mmol) and 2,2-
dimethoxypropane (2 mL, 16.3 mmol) in acetone (5 mL) and the mixture
was stirred overnight at room temperature. Ca(OH)2 was added until the
solution was neutral, the mixture was filtered, and the solvent removed
under vacuum. The residue was purified by column chromatography on
silica gel (CH2Cl2/MeOH, 95:5) to give 14 as a colorless oil (0.450 g, 33%);
Rf = 0.35 (CH2Cl2/MeOH, 95:5); 1H NMR (CDCl3) δ = 1.44 (s, 3H, CH3),
1.46 (s, 3H, CH3), 1.58 (dt, J = 6.2 Hz, J = 6.2 Hz, 2H, CH-CH2), 1.95–2.00
(m, 2H, CH-CH2), 3.66 (dd, J = 8.2 Hz, J = 12.2 Hz, 2H, ax-CH), 3.74 (t,
J = 6.3 Hz, 2H, CH2OH), 3.96 (dd, J = 4.6, J = 12.2 Hz, 2H, eq-CH).
6-Chloro-9-[(2,2-dimethyl-1,3-dioxan-5-yl)ethyl]purine (15)
Compound 15 was prepared following the above described procedure
starting from 6-chloropurine (8) and 5-(2-hydroxyethyl)-2,2-dimethyl-1,3-
dioxane (14). Rf = 0.4 (CH2Cl2/MeOH, 95:5); white solid; 72% yield; m.p.

Novel Isopentenyladenosine Analogues
749
1
146–148◦C; [lit.[19] m.p. 148–150◦C]; H NMR (CDCl3) δ = 1.45 (s, 6H, 2
× CH3), 1.70–1.76 (m, 1H, CH-CH2), 2.06 (dt, J = 6.9 Hz, J = 6.9 Hz, 2H,
CH-CH2), 3.70 (dd, J = 6.9 Hz, J = 11.8 Hz, 2H, CH2-O), 4.01 (dd, J = 4.2
Hz, J = 11.8 Hz, 2H, CH2-O), 4.38 (t, J = 6.9 Hz, 2H, N-CH2), 8.16 (s, 1H,
H2), 8.78 (s, 1H, H8).
6-(3-Methyl-2-butenylamino)-9-[4-hydroxy-3-(hydroxymethyl)but-1-yl]purine
(5)
Compound 5 was prepared following the above described proce-
dure starting from 6-chloro-9-[(2,2-dimethyl-1,3-dioxan-5-yl)ethyl]purine
(15) and 3,3-dimethylallylamine to give 6-(3-methyl-but-2-enylamino)-9-
[(2,2-dimethyl-1,3-dioxan-5-yl)ethyl]purine (16) that was deprotected to the
desired compound 5. Rf = 0.18 (CH2Cl2/MeOH, 90:10); white solid; 67%
yield; m.p. 104–106◦C; [α]20D -00.0 (c 1, MeOH); 1H NMR (CDCl3) δ = 1.75
(s, 3H, CH3), 1.76 (m, overlapped, 1H, CH-CH2-OH); 1.77 (s, 3H, CH3),
2.01 (dt, J = 6.9 Hz, J = 6.9 Hz, 2H, CH-CH2), 3.74–3.80 (m, 4H, 2 ×
CH2-OH), 4.22 (bs, 2H, α-CH2), 4.34 (t, J = 6.9 Hz, 2H, N-CH2), 5.38 (t, J =
7.0 Hz, 1H, β-CH), 5.93 (bs, 1H, NH), 7.76 (s, 1H, H8), 8.37 (s, 1H, H2); 13C
NMR δ = 18.02 (CH3), 25.69 (CH3), 29.33 CH-CH2), 38.70 (α-CH2), 39.87
(CH-CH2-OH), 42.03 (N-CH2), 64.35 (CH2-OH), 120.05 (β-CH), 136.95
(γ -C), 137.00 (C5), 139.60 (C8), 152.13 (C4), 153.01 (C2), 154.70 (C6);
ESIMS m/z 304 (M-1, 100%), 609 (2M-1, 50%); Anal. calcd for C15H23N5O2:
C, 59.00; H, 7.59; N, 22.93; O, 10.48. Found: C, 58.88; H, 7.42; N, 22.98.
6-(3-Methyl-butylamino)-9-[4-hydroxy-3-(hydroxymethyl)but-1-yl]purine (6)
Compound 6 was prepared following the above described proce-
dure starting from 6-chloro-9-[(2,2-dimethyl-1,3-dioxan-5-yl)ethyl]purine
(15) and 1-amino-3-methylbutane to give 6-(3-methyl-butylamino)-9-[(2,2-
dimethyl-1,3-dioxan-5-yl)ethyl] purine (17) that was deprotected to the
desired compound 6. Rf = 0.22 (CH2Cl2/MeOH, 90:10); white solid; 70%
1
yield; m.p. 96–98◦C; αD 0.00 (c = 1 in MeOH); H NMR (CDCl3) δ =
0.99 (d, J = 6.6 Hz, 6H, 2 × CH3), 1.60 (dt, J = 6.6 Hz, J = 6.6 Hz, 2H,
β-CH2), 1.77 (dq, J = 6.6 Hz, J = 6.6 Hz, 1H, γ -CH), 2.04 (dt, J = 6.9
Hz, J = 6.9 Hz, 2H, CH-CH2), 3.68 (bs, 2H, α-CH2), 3.76–3.83 (m, 4H, 2
× CH2-OH), 4.36 (t, J = 6.9 Hz, 2H, N-CH2), 5.86 (bs, 1H, NH), 7.76 (s,
1H, H8), 8.38 (s, 1H, H2); 13C NMR δ = 22.52 (2 × CH3), 25.77 (γ -CH),
29.38 (CH-CH2), 38.53 (β-CH2), 38.98 (α-CH2), 39.74 (CH-CH2-OH), 41.96
(N-CH2), 64.68 (CH2-OH), 119.80 (C5), 139.51 (C8), 148.67 (C4), 153.08
(C2), 155.04 (C6); ESIMS m/z 306 (M-1, 100%), 613 (2M-1, 72%); Anal.
calcd for C15H25N5O2: C, 58.61; H, 8.20; N, 22.78; O, 10.41. Found: C, 58.46;
H, 8.12; N, 22.88.

750
R. Ottria et al.
Cell Culture, Proliferation, and Migration
T24 and J82 cells were cultured in DMEM containing 10% calf serum.
All the reagents for cell culture were from Gibco Invitrogen Co. (Scotland,
UK). Stock solutions of iPA (1) and compounds 2–6 were prepared in
DMSO and kept at −20◦C. Appropriate dilutions of the compounds were
freshly prepared just prior the assays. The controls were additioned with the
final concentrations of DMSO.
Cell migration was determined using an in vitro model of wound repair
as previously described.[30] Briefly, the cells were grown in 24-well plates
to confluence. After an overnight of starvation in DMEM containing 0.1%
serum, the monolayer was wounded, EGF (100 ng/ml) and the different
molecules (10 µM) were added for an additional 16 hours. The wound area
was calculated by the ImageJ software and expressed using an arbitrary value
scale. For proliferation assays, the cells were seeded at low density in growth
medium.[30] Sixteen hours after plating, iPA and its derivatives were added.
Seventy-two hours later, the cells were trypsinized, stained with a trypan blue
solution (0,4%) and the viable cells were counted using a Burker chamber.
In some experiments iPA was added for 12 hours, washed, and the cells were
maintained in culture medium for 60 hours. For clonogenic assays, T24 and
J82 cells were plated at a density of 500 cells/well in 6 well-plates and treated
with iPA and its analogues (10 µM) for 10 days. Plates were then rinsed with
PBS and stained with crystal violet.
The experiments were performed 5 times in triplicates. Data are shown
as the mean
standard deviation. Statistical significance was determined
using the student’s t test.
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