5′-O-tritylinosine and analogues as allosteric inhibitors of human thymidine phosphorylase

Article abstract of DOI:10.1021/jm0605379

On the basis of our previous findings that 5′-O-tritylinosine (KIN59) behaves as an allosteric inhibitor of the angiogenic enzyme thymidine phosphorylase (TPase), we have undertaken the synthesis and enzymatic evaluation of a novel series of nucleoside an

Full text of DOI:10.1021/jm0605379

5562
J. Med. Chem. 2006, 49, 5562-5570
5′-O-Tritylinosine and Analogues as Allosteric Inhibitors of Human Thymidine Phosphorylase
Elena Casanova,† Ana-Isabel Herna´ndez,† Eva-Mar´ıa Priego,† Sandra Liekens,‡ Mar´ıa-Jose´ Camarasa,† Jan Balzarini,‡ and
Mar´ıa-Jesu´s Pe´rez-Pe´rez*,†
Instituto de Qu´ımica Me´dica (C.S.I.C.), Juan de la CierVa 3, E-28006 Madrid, Spain, and Rega Institute for Medical Research, Katholieke
UniVersiteit LeuVen, B-3000 LeuVen, Belgium
ReceiVed May 9, 2006
On the basis of our previous findings that 5′-O-tritylinosine (KIN59) behaves as an allosteric inhibitor of
the angiogenic enzyme thymidine phosphorylase (TPase), we have undertaken the synthesis and enzymatic
evaluation of a novel series of nucleoside analogues modified at positions 1, 2, or 6 of the purine ring and
at the 5′-position of the ribose moiety of the lead compound KIN59. SAR studies indicate that quite large
structural variations can be performed on KIN59 without compromising TPase inhibition. Thus, incorporation
of a cyclopropylmethyl or a cyclohexylmethyl group at position N1 of 5′-O-tritylinosine increases the inhibitory
activity against TPase 10-fold compared to KIN59. Moreover, the trityl group at the 5′-position of the
ribose seems to be crucial for TPase inhibition. The here reported results further substantiate that 5′-O-trityl
nucleosides represent a new class of TPase inhibitors that should be further explored in those biological
systems where TPase plays an instrumental role (i.e. angiogenesis).
Introduction
tightly interact with an allosteric site on the enzyme that is ∼15
Å distanct from the substrate active site.7,8
Nucleoside analogues constitute an important contribution to
the chemotherapy of cancer, viral diseases, and immunomodu-
lating therapies. Despite their long tradition as chemotherapeutic
agents, novel applications are still being explored and new
nucleoside structures reach the clinic. Some very recent
examples of nucleosides that have been approved for anticancer
or antiviral applications include nelarabine,1 entecavir,2 clo-
farabine,3 or azacitidine.4 In most cases, nucleosides behave as
prodrugs or bioprecursors that need to be activated to their
pharmacologically active metabolite by cellular and/or viral
enzymes. Still, there are an increasing number of compounds
with a nucleoside structure that do not require this activation
to become pharmacologically active. In some cases, these
nucleoside analogues bind at the substrate-binding site of the
target enzyme but are not recognized as substrates, and therefore,
they exert mostly a competitive inhibition with the natural
substrate. In a few other cases, the nucleoside analogues interact
with the target enzyme at a site different from the substrate-
binding site, and therefore, they behave as noncompetitive
inhibitors with respect to the natural substrate. This binding site,
which is topographically distinct, and quite often, distant, from
the substrate-binding site, can be designated as an allosteric
binding site.5 Allosteric inhibitors of enzymes may offer some
advantages when compared to compounds interacting at the
substrate-binding site. Since these allosteric ligands do not need
to be structurally related to the enzyme substrates, they may be
safer and more efficacious by overcoming selectivity issues and
substrate competition.5 Therefore, allosteric inhibitors are
considered as novel pharmacologically active compounds that
may increase our understanding of basic enzymatic or biological
processes involving the target enzymes.5,6 A typical example
of such allosteric inhibitors is represented by the non-nucleoside
reverse transcriptase inhibitors of the human immunodeficiency
virus (HIV), where a variety of apparently different structures
Since 1998, our research groups have been involved in the
discovery of novel inhibitors of the human enzyme thymidine
phosphorylase (TPase). TPase catalyses the reversible phos-
phorolysis of pyrimidine 2′-deoxynucleosides to 2-deoxyribose-
1-phosphate and their respective pyrimidine bases, including
the phosphorolysis of nucleoside analogues with important
antiviral or anticancer properties. Moreover, TPase, identified
also as the angiogenic platelet-derived endothelial cell growth
factor (PD-ECGF), stimulates endothelial cell migration in vitro
and angiogenesis in vivo and plays an important role in tumor
progression and metastasis.9-11 We have identified several series
of TPase inhibitors, including purine bases such as 7-deazax-
anthine (7-DX)12 or multisubstrate inhibitors such as TP6513-15
(Chart 1). More recently, we reported the inhibitory activity of
the purine riboside derivative KIN59 (5′-O-tritylinosine) (1)
against human and Escherichia coli recombinant TPase.16 In
contrast to previously described TPase inhibitors, KIN59
competes neither with the pyrimidine nucleoside nor with the
phosphate-binding site of the enzyme but noncompetitively
inhibits TPase when thymidine or phosphate is used as the
variable substrate.16 In addition, KIN59 is far more active than
our previously identified TPase inhibitors against TPase-induced
angiogenesis in an established angiogenesis assay (chicken
chorioallantoic membrane assay, CAM assay).16 It was dem-
onstrated that inosine, the parent nucleoside of KIN59, neither
inhibits the enzyme nor the angiogenic activity of TPase in the
CAM assay.16 This indicates that the antiangiogenic activity of
KIN59 requires the intact trityl-containing nucleoside. Therefore,
KIN59 is the prototype of a new family of nucleosides that
behave as allosteric inhibitors of TPase. Since the TPase
inhibitor KIN59 has such a marked effect on angiogenesis, we
speculated that KIN59 and analogues could help to explore a
novel allosteric binding site at TPase, different from the
thymidine and phosphate-binding sites that could play an
important role in TPase-triggered angiogenesis stimulation. In
this study, we performed modifications on the lead compound
* Corresponding author. Phone: 34 91 5622900, ext 411. Fax: 34 91
5644853. E-mail: mjperez@iqm.csic.es.
† Instituto de Qu´ımica Me´dica (C.S.I.C.).
‡ Katholieke Universiteit Leuven.
10.1021/jm0605379 CCC: $33.50 © 2006 American Chemical Society
Published on Web 08/12/2006

5′-O-Tritylinosine Analogues as TPase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18 5563
Chart 1. Structural Formulas of the TPase Inhibitors
7-Deazaxanthine (7DX), TP65, and KIN59
A more recent paper makes use of a (2-methoxyethoxy)methyl
(MEM) as the substituent of choice to perform this transforma-
tion from inosine to AICAR.23 Due to the high reactivity of the
MEMCl with alcohols, the 2′- and 3′-OH of 5′-O-tritylinosine
(1) were acetylated prior to the treatment with MEMCl. Thus,
reaction of 424 with MEMCl in dry CH2Cl2 in the presence of
i-Pr2NEt afforded the N1-MEM inosine derivative 5 in 72% yield
(Scheme 2). Treatment of 5 with refluxing 0.2 N NaOH for 24
h, as described,23 afforded 6 as the major compound (42%)
together with a smaller amount of the open analogue 7 (18%
yield) that still keeps the MEM moiety on the N4-carboxamide.
Only after longer reaction times was the 5′-O-trityl AICAR
derivative (8) detected. These results indicate that the opening
reaction of the pyrimidine ring is prior to the removal of the
MEM moiety, results that agree with the proposal of Shaw.22
Alternatively, treatment of 5 with refluxing 5 N NaOH for 6 h
afforded the 5′-O-trityl AICAR derivative 8 in 20% yield.
KIN59 to determine the structural requirements among this
series of purine nucleosides for TPase inhibition.
Next, we decided to replace the hypoxanthine moiety of the
lead compound 1 by other purines. In particular, we chose the
ribofuranosyl derivatives of 9H-purine (9) and 6-chloro-9H-
purine (10) (Scheme 3). Treatment of 9 with trityl chloride in
pyridine with a small amount of DMAP at 80 °C afforded the
5′-O-trityl nucleoside 11 in 35% yield. However, this procedure
failed when applied to the 6-chloropurin-9-yl riboside (10).
Alternatively, the 6-chloro-9-(5-O-trityl-â-D-ribofuranosyl)pu-
rine (12) was synthesized by reaction of 10 with TrCl in DMF
at 40 °C and in the presence of i-Pr2NEt, as described.25 Further
reaction of 12 with MeNH2 in MeOH afforded the 6-NHMe
purin-9-yl derivative (13) in 42% yield.
Chemistry
When considering structural modifications on our lead
compound 1, we have focused on positions 1 and 6 of the purine
base and the 5′-OH of the ribose moiety. Moreover, we have
cleaved the pyrimidine ring of the purine base by synthesizing
the corresponding AICAR (5-amino-(1-â-D-ribofuranosyl)imi-
dazole-4-carboxamide) derivatives, and we have also opened
the ribose ring of compound 1 by synthesizing the corresponding
2′,3′-seconucleoside.
On the basis of the known reactivity of position 1 of inosine,
the first series of targeted compounds were N1 substituted 5′-
O-tritylinosines that could be synthesized by the introduction
of different alkyl, allyl, or benzyl substituents at position N1 of
5′-O-tritylinosine (1). Thus, treatment of 116 with the corre-
sponding halide in dimethylacetamide (DMAC) and in the
presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)17 af-
forded the N1-substituted 5′-O-tritylinosines (2a-f) in good
yields (56-89%) (Scheme 1); only the (E)-3-methoxycarbonyl-
2-propenyl derivative 2g was obtained in a lower yield (32%).
As previously shown in different series of compounds, substitu-
tion occurs regioselectively at N1, while the O6-substituted
byproducts18 were, in our case, not detected.
It is well-known that N1-alkylinosines can be cleaved at the
pyrimidine ring by treatment with aqueous alkali to yield
5-amino-4-(N-alkylcarbamoyl)imidazole ribosides.19-21 Several
compounds from the above series were subjected to this
pyrimidine-opening reaction. Thus, reaction of 2a-d with 5 N
NaOH in refluxing EtOH afforded the 4-N-methyl, 4-N-propyl,
4-N-allyl, and 4-N-cyclopropylmethyl carbamoyl derivatives
3a-d in 46-53% yield (Scheme 1). In every case, the opening
of the pyrimidine ring is clear by the appearance in the 1H NMR
spectra of a broad singlet, corresponding to the 5-NH2 signal,
around 5.7 ppm and a triplet around 7.5 ppm that corresponds
to the 4-CONH, when the spectra were recorded in DMSO-d6.
It is also worth mentioning the upfield movement of the signal
corresponding to the anomeric proton H-1′ (∆δ) -0.4) when
comparing the open (3a-d) to the close analogues (2a-d).
We also considered of interest the synthesis of the free
4-CONH2 derivative that should correspond to the 5′-O-trityl-
AICAR (8). The synthesis of AICAR derivatives from inosine
by using p-toluenesulfonyl or methoxymethyl groups as N1-
substituents was described by Shaw.22 According to this report,
treatment of these N1-substituted inosines by aqueous alkali in
refluxing ethanol could simultaneously open the pyrimidine ring
and remove the N1-substituent to afford the AICAR compounds.
We also wanted to evaluate the impact of substituents
introduced on the trityl moiety on TPase inhibition (Scheme
4). For this purpose, the 5′-O-(4,4′-dimethoxytrityl)inosine (16)
was synthesized following described procedures.26 We also
prepared the 4-chlorotrityl derivative (17) by reaction of inosine
(14) with 4-chlorotrityl chloride. This reagent was synthesized
by treatment of the 4-chlorobenzophenone with phenylmagne-
sium bromide and further transformed into the desired chloride
by reaction of the resulting carbinol with acetyl chloride.27
Moreover, reaction of 2′-deoxyinosine (15) with trityl chloride
in pyridine, in the presence of DMAP, at 60 °C, afforded 5′-
trityl-2′deoxyinosine (18). It is worth mentioning that when the
reaction between inosine (14) and trityl chloride was performed
in DMF at 40 °C and in the presence of i-Pr2NEt, conditions
employed for the 5′-O-tritylation of 6-chloro-(1-â-D-ribofura-
nosyl)purine,25 the reaction on inosine did not occur at the 5′-
OH, as expected, but at position 1, yielding the N1-tritylinosine
derivative (19) as the major product (30% yield). To the best
of our knowledge there is only one previous report on the
introduction of a trityl moiety at N1 of inosine, although in this
report the 5′- and 3′-positions of 2′-deoxyinosine were protected
with acetyl groups prior to the introduction of the trityl moiety
at N1.28 The selective introduction of the trityl moiety at N1
under the experimental conditions above-described (in the
presence of the free OH groups of the ribose) has no precedent
and could be of value as a protective strategy.
To highlight the importance of the presence of the furanose
moiety in our lead structure 1, we have performed the synthesis
of the corresponding 2′,3′-seconucleoside (20). Thus, reaction
of 1 with sodium periodate followed by “in situ” reduction of
the resulting dialdehyde with sodium borohydride26,29 afforded
the target compound 20 in 60% yield (Scheme 5).
Different attempts have been made in our laboratory to
synthesize 5′-O-benzylinosine. The only reported procedure
involves reaction of 2′,3′-O-isopropylideneinosine with benzyl

5564 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18
CasanoVa et al.
Scheme 1. Synthesis of N1 Substituted 5′-O-Tritylinosines and Their Open Pyrimidine-Ring Analoguesa
a (a) R1-X, DBU, DMAC, Ar, rt; (b) 5 N NaOH, EtOH, ∆.
Scheme 2. Synthesis of 5′-O-Trityl AICARa
a (a) Ac2O, Py, rt; (b) MEMCl, i-Pr2NEt, CH2Cl2, 0 °C; (c) 0.2 N NaOH, ∆; (d) 5 N NaOH, ∆.
Scheme 3. Synthesis of 9-(5-O-Trityl-â-D-ribofuranosyl)purines
11, 12 and 13a
reactive and reacts with benzyl bromide under the same
experimental conditions as the 5′-OH.31 On the basis of these
results, we have undertaken the synthesis of the 1,5′-dibenzyli-
nosine (23). Thus, reaction of 2′,3′-O-isopropylideneinosine (21)
with benzyl bromide in DMF in the presence of NaH at room
temperature afforded the dibenzylated derivative (22) in 71%
yield (Scheme 6). In our hands, and in contrast to the described
method,31 it was important to perform this reaction at room
temperature to avoid undesired products. Deprotection of the
isopropylidene moiety by treatment with aqueous TFA afforded
2331 in 95% yield.
a (a) TrCl, DMAP, Py, 80 °C; (b) TrCl, i-Pr2NEt, DMF, 40 °C; (c)
MeNH2 in EtOH, 70 °C.
Biological Results and Discussion
bromide and potassium tert-butoxide in dioxane.30 In our hands,
this reaction afforded a mixture of 1- and 1,5′-dibenzyl products.
This is a reasonable result since position 1 of inosine is highly
The synthesized compounds have been evaluated for their
inhibitory activity against human TPase in the presence of 100
µM thymidine as the natural substrate. The 50% inhibitory

5′-O-Tritylinosine Analogues as TPase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18 5565
Table 1. Inhibitory Effect of the Synthesized Compounds on Human
TPase Enzymatic Activity
Scheme 4. Reaction of Inosine and 2′-Deoxyinosine with Trityl
Chloridesa
compd
IC50 (µM)a
compd
IC50 (µM)a
compd
IC50 (µM)a
1
30 ( 15
49 ( 4
3a
3b
3c
3d
5
6
7
8
137
11
12
16
17
18
20
23
45 ( 3
26
18 ( 3
4.5 ( 2.3
96
>200
>200
2a
2b
2c
2d
2e
2f
13 ( 3
23 ( 5
19
13 ( 2
11 ( 2
2.3 ( 0.4
4.4 ( 1.8
11 ( 1
>20
>100
>75
2g
24 ( 41
g40
a IC50: concentration at which the conversion of 100 µM thymidine to
thymine is inhibited by 50%.
TPase. In particular, incorporation of an alkyl (propyl, 2b),
alkenyl (propenyl, 2c), or a benzyl (2f) group at N1 of the lead
compound 1 increases the inhibitory activity of the new
compounds by 2.5-3-fold. Interestingly, those compounds that
incorporate a methylcycloalkyl substituent (2d, 2e) are at least
10-fold more inhibitory than 1. Moreover, the corresponding
AICAR analogues (3b, 3c) also show an inhibitory potency
similar to that of their parent inosine analogues (2b, 2c).
However, compounds with a MEM substituent at N1 either in
the inosine (6) or in the AICAR series (7) were devoid of
inhibitory activity against TPase up to 75-100 µM. The 5′-O-
trityl-AICAR 8 at the highest concentration tested (40 µM)
inhibited thymidine to thymine conversion at almost 50%.
a (a) DMTrCl or 4-CITrCl or TrCl, Py, DMAP, 60 or 80 °C; (b) TrCl,
i-Pr2NEt, DMF, 40 °C.
Scheme 5. Synthesis of 5′-O-Trityl-2′,3′-secoinosinea
Replacement of the hypoxanthine base of the lead compound
by other structurally related purines modified at position 6, like
in compounds 11 or 12, leads to compounds with activity similar
to that of the inosine derivative 1.
Concerning the 5′-position of the ribose moiety, the trityl
group of 1 can be replaced by a 4,4′-dimethoxytrityl (such as
in 16) or a 4-chlorotrityl (such as in 17) and the inhibitory
activity against TPase is preserved or even increased. However,
replacement of the 5′-O-trityl by a 5′-O-benzyl group results in
an inactive compound (compare compound 23 with the trityl
analogue 2f). These data further stress the crucial importance
of a trityl group at the 5′-position of these nucleoside derivatives
for TPase inhibition. Interestingly, the 5′-O-trityl-2′deoxyinosine
(18) also shows TPase inhibition although at higher concentra-
tions (IC50) 96 µM) than the ribose analogue 1. On the other
hand, the inactivity of the 5′-O-trityl-2′,3′-secoinosine derivative
20 compared to the intact nucleoside 1 indicates that not only
the 5′-O-trityl and the hypoxanthine moieties are important for
TPase inhibition but that the sugar scaffold of the nucleoside
seems to be instrumental to position the two substituents (the
5′-O-trityl and the hypoxanthine moiety) in the right way to
allow interaction with TPase. It should be mentioned, in this
respect, that we ascertained that the parent compound 1 and
several of its derivatives are fully stable in the presence of CEM
cell extracts and bovine serum, further attesting the important
role of the 5′-trityl group in the eventual biological activity of
the test compounds. Indeed there are an increasing number of
examples where a trityl moiety might be considered as a
pharmacophoric group, both in the nucleoside32-34 and in the
peptide field.35,36
a (a) NaIO4, dioxane:H2O, rt; (b) NaBH4, dioxane:H2O.
Scheme 6. Synthesis of 1,5′-O-Dibenzylinosinea
a (a) NaH, BnBr, DMF, rt; (b) 30% aqueous TFA.
Some of the most potent TPase inhibitors described in this
paper have also been tested against TPase-triggered angiogenesis
in the CAM assay.37 In particular, the 4-chlorotrityl derivative
17 has shown a marked inhibition of TPase-induced neovas-
cularization at 250 nmol.37 At a lower concentration (100 nmol),
compound 17 was still able to inhibit TPase-induced angiogen-
esis by 75% while, at the same concentration, the parent
compound 1 showed 32% inhibition.
concentrations (IC50 values), that is, the concentrations at which
these compounds inhibit thymidine to thymine conversion by
50%, are shown in Table 1. For comparative purposes, we may
indicate that 7-deazaxanthine (Chart 1), one of our previously
described TPase inhibitors that is competitive to the thymidine
substrate, shows an IC50 of 48 µM under similar experimental
conditions. Interestingly, several of the synthesized derivatives
are more inhibitory than the lead compound 1 against human

5566 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18
CasanoVa et al.
drofuran was dried by refluxing over sodium/benzophenone.
Anhydrous N,N′-dimethylformamide was purchased from Aldrich.
Anhydrous dimethylacetamide (DMAC) was purchased from Al-
drich. 2′,3′-O-Isopropylideneinosine was purchased from Aldrich.
General Procedure for the Preparation of N1-Substituted 5′-
O-Tritylinosines. To a solution of 5′-O-tritylinosine (1)16 (200 mg,
0.39 mmol) in DMAC (3 mL) were added the corresponding alkyl
or benzyl halide (0.51 mmol) and DBU (0.077 mL, 0.52 mmol).
The reaction mixture was stirred at room temperature under argon
overnight. The reaction was quenched by addition of a mixture of
ether:hexane 1:1 (8 mL) and the resulting suspension was placed
at -20 °C overnight. Solvents were decanted while still chilly, and
the resulting gum was evaporated under reduced pressure. The
resulting residue was partitioned into EtOAc (25 mL) and a
NaHCO3 solution (15 mL). The organic phase was further washed
with a NaHCO3 solution (2 × 10 mL) and finally with brine. The
organic phase was dried on anhydrous Na2SO4, filtered, and
evaporated. The residue was purified by CCTLC in the Chroma-
totron or by flash column chromatography as indicated for each
compound.
Conclusions
We have performed the synthesis and enzymatic evaluation
against human thymidine phosphorylase of a series of purine
nucleoside derivatives structurally related to the TPase lead
inhibitor 5′-O-tritylinosine. SAR studies indicate that quite large
structural variations can be performed on the lead structure 1
without compromising TPase inhibition. Modification of the N1
position of the hypoxanthine base by incorporating alkyl,
alkenyl, or cyclic alkyl substituents results in maintained or
increased TPase inhibition. In particular, those compounds that
incorporate a cyclopropylmethyl (2d) or cyclohexylmethyl (2e)
moiety are 10-fold more inhibitory against TPase than the parent
compound. Moreover, several 5-amino-4-(N-substituted-car-
bamoyl)imidazole ribosides (3b-d), derived from the cleavage
of the pyrimidine ring of the N1-substituted inosines, were
equally active against TPase as the parent 1. Also other purine
bases that lack a carbonyl (11) or have a chlorine (12) at position
6 retain activity. The trityl substituent at the 5′-position is crucial
for activity, since the corresponding 5′-O-benzyl analogue was
inactive. Moreover, the intact nucleoside structure in this family
of compounds is important to correctly position the nucleic base
and the 5′-O-trityl substituent for interaction with the target
enzyme, since the oxidative cleavage at positions 2′,3′ of the
ribose as in the 2′,3′-secoinosine derivative 20 renders an
inactive compound.
Attempts are now being made to cocrystallize 5′-O-trityli-
nosine with human TPase, but so far these attempts have not
been successful. Therefore, the allosteric binding site where 5′-
O-tritylinosine binds to TPase remains elusive. Alternatively,
the data reported in this paper, together with our recent report
that 5′-O-trityl derivatives of pyrimidines, exemplified by 1-(5′-
O-trityl-â-D-ribofuranosyl)thymine, also noncompetitively in-
hibit TPase,37 indicate that 5′-O-tritylnucleosides constitute a
novel class of TPase inhibitors. This family of compounds
accepts significant structural modifications without impairment
or even with a marked increase of their inhibitory activity against
TPase, a feature that is not unusual among allosteric inhibitors.
Some of the here described compounds, such as 17, have been
shown to be even more potent than the parent compound 1 in
biological assays (i.e., TPase-triggered angiogenesis), supporting
the interest of this class of nucleosides for further optimization.
1-Methyl-5′-O-tritylinosine (2a). Following the general proce-
dure for the preparation of N1-substituted 5′-O-tritylinosines,
reaction of 1 with methyl iodide (32 µL, 0.51 mmol) in the presence
of DBU afforded a residue that was purified in the Chromatotron
(CH2Cl2:MeOH, 10:1) to afford 159 mg (77% yield) of 2a as a
white solid: mp 181-184 °C (CH2Cl2:MeOH); MS (ES, positive
1
mode) m/z 525 (M + 1)+; H NMR (DMSO-d6) δ 3.24 (m, 2H,
H-5′), 3.49 (s, 3H, NCH3), 4.06 (q, J ) 5.1 Hz, 1H, H-4′), 4.20
(m, 1H, H-3′), 4.55 (m, 1H, H-2′), 5.25 (d, J ) 5.9 Hz, 1H, OH),
5.57 (d, J ) 5.9 Hz, 1H, OH), 5.88 (d, J ) 4.4 Hz, 1H, H-1′),
7.22-7.42 (m, 15 H, Ph), 8.20 (s, 1H, H-8), 8.32 (s, 1H, H-2).
Anal. (C30H28N4O5‚H2O) C, H, N.
1-Propyl-5′-O-tritylinosine (2b). Reaction of 1 with propyl
iodide (50 µl, 0.51 mmol), following the general procedure for the
preparation of N1-substituted 5′-O-tritylinosines, afforded a residue
that was purified by flash column chromatography (CH2Cl2:MeOH,
15:1) to yield 193 mg (89%) of 2b as a white solid: mp 115-118
°C (CH2Cl2:MeOH); MS (ES, positive mode) m/z 553 (M + 1)+;
1H NMR (DMSO-d6) δ 0.85 (t, J ) 7.0 Hz, 3H, CH3), 1.65 (m,
2H, CH2CH3), 3.20 (m, 2H, H-5′), 3.94 (t, J ) 7.0 Hz, 2H, NCH2),
4.05 (m, 1H, H-4′), 4.20 (m, 1H, H-3′), 4.55 (m, 1H, H-2′), 5.23
(d, J ) 5.7 Hz, 1H, OH), 5.57 (d, J ) 5.1 Hz, 1H, OH), 5.88 (d,
J ) 4.6 Hz, 1H, H-1′), 7.20-7.36 (m, 15 H, Ph), 8.20 (s, 1H, H-8),
8.32 (s, 1H, H-2). Anal. (C32H32N4O5‚1.5H2O) C, H, N.
1-Allyl-5′-O-tritylinosine (2c). Reaction of 1 with allyl bromide
(44 µl, 0.51 mmol), following the general procedure for the
preparation of N1-substituted 5′-O-tritylinosines, afforded a residue
that was purified by flash column chromatography (CH2Cl2:MeOH,
20:1) to yield 184 mg (85%) of 2c as a white solid: mp 93-94 °C
Experimental Section
1
(CH2Cl2:MeOH); MS (ES, positive mode) m/z 551 (M + 1)+; H
Chemical Procedures. Melting points were obtained on a
Reichert-Jung Kofler apparatus and are uncorrected. Microanalyses
were obtained with a Heraeus CHN-O-RAPID instrument. Elec-
trospray mass spectra were measured on a quadrupole mass
spectrometer equipped with an electrospray source (Hewlett-
Packard, LC/MS HP 1100). 1H and 13C NMR spectra were recorded
on a Varian Gemini operating at 200 MHz (1H) and 50 MHz (13C),
respectively, on a Varian INNOVA 300 operating at 299 MHz (1H)
and 75 MHz (13C), respectively, and Varian INNOVA-400 operating
at 399 MHz (1H) and 99 MHz (13C), respectively.
Analytical TLC was performed on silica gel 60 F254 (Merck)
precoated plates (0.2 mm). Spots were detected under UV light
(254 nm) and/or by charring with phosphomolybdic acid. Separa-
tions on silica gel were performed by preparative centrifugal circular
thin-layer chromatography (CCTLC) on a ChromatotronR (Kiesegel
60 PF254 gipshaltig (Merck)), with layer thickness of 1 or 2 mm
and flow rate of 4 or 8 mL/min, respectively. Flash column
chromatography was performed with silica gel 60 (230-400 mesh)
(Merck).
NMR (DMSO-d6) δ 3.20 (m, 2H, H-5′), 4.07 (m, 1H, H-4′), 4.22
(m, 1H, H-3′), 4.57 (m, 1H, H-2′), 4.63 (d, J ) 5.3 Hz, 2H, NCH2),
5.04-5.19 (m, 2H, CHdCH2), 5.23 (d, J ) 5.9 Hz, 1H, OH), 5.57
(d, J ) 5.7 Hz, 1H, OH), 5.89 (d, J ) 4.8 Hz, 1H, H-1′), 6.98 (m,
1H, CHdCH2), 7.20-7.43 (m, 15 H, Ph), 8.22 (s, 1H, H-8), 8.29
(s, 1H, H-2). Anal. (C32H30N4O5‚H2O) C, H, N.
1-(Cyclopropyl)methyl-5′-O-tritylinosine (2d). Reaction of 1
with (bromomethyl)cyclopropane (50 µL, 0.51 mmol), following
the general procedure for the preparation of N1-substituted 5′-O-
tritylinosines, afforded a residue that was purified by flash column
chromatography (CH2Cl2:MeOH, 20:1 to 10:1) to yield 180 mg
(81%) as a white solid: mp 120-123 °C (CH2Cl2:MeOH); MS
1
(ES, positive mode) m/z 565 (M + 1)+; H NMR (DMSO-d6) δ
0.39-0.48 (m, 4H, CH2), 1.23 (m, 1H, CH), 3.22 (m, 2H, H-5′),
3.86 (d, J ) 7.3 Hz, 2H, NCH2), 4.11 (m, 1H, H-4′), 4.21 (m, 1H,
H-3′), 4.56 (m, 1H, H-2′), 5.23 (d, J ) 5.9 Hz, 1H, OH), 5.58 (d,
J ) 5.9 Hz, 1H, OH), 5.89 (d, J ) 4.8 Hz, 1H, H-1′), 7.25-7.62
(m, 15 H, Ph), 8.21 (s, 1H, H-8), 8.35 (s, 1H, H-2). Anal.
(C33H32N4O5‚0.5H2O) C, H, N.
All experiments involving water-sensitive compounds were
conducted under scrupulously dry conditions. Triethylamine and
acetonitrile were dried by refluxing over calcium hydride. Tetrahy-
1-(Cyclohexyl)methyl-5′-O-tritylinosine (2e). Reaction of 1 with
(bromomethyl)cyclohexane (72 µL, 0.50 mmol) during 3 days,

5′-O-Tritylinosine Analogues as TPase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18 5567
following the general procedure for the preparation of N1-substituted
5′-O-tritylinosines, afforded a residue that was purified in the
Chromatotron (CH2Cl2:MeOH, 10:1) to yield 134 mg (56%) of 2e
as a white solid: mp 121-123 °C (CH2Cl2:MeOH); MS (ES,
positive mode) m/z 607 (M + 1)+; 1H NMR (CDCl3) δ 0.92-1.25
(m, 6H, C6H11), 1.55-1.70 (m, 5H, C6H11), 3.39 (m, 2H, H-5′),
3.43 (d, J ) 2.9 Hz, 1H, OH), 3.82 (d, J ) 7.3 Hz, 2H, NCH2),
4.35-4.44 (m, 2H, H-3′, H-4′), 4.78 (m, 1H, H-2′), 5.35 (d, J )
4.0 Hz, 1H, OH), 5.95 (d, J ) 5.9 Hz, 1H, H-1′), 7.18-7.38 (m,
15 H, Ph), 7.80 (s, 1H, H-8), 7.92 (s, 1H, H-2). Anal. (C36H38N4O5‚
1.5H2O) C, H, N.
5-Amino-1-(5-O-trityl-â-D-ribofuranosyl)imidazole-4-[(N-cy-
clopropyl)methyl]carboxamide (3d). Following a procedure analo-
gous to that described for the synthesis of 3a, compound 2d (100
mg, 0.18 mmol) reacted with 5 N NaOH to yield 3d (54 mg, 55%)
as a white solid: mp 92-93 °C (CH2Cl2:MeOH); MS (ES, positive
mode) m/z 555 (M + 1)+; 1H NMR (CDCl3) δ 0.24 (m, 2H, CH2),
0.51 (m, 2H, CH2), 0.95 (m, 1H, CH), 3.17-3.22 (m, 3H, H-5′,
OH), 3.46 (m, 2H, HNCH2), 4.12 (m, 1H, H-4′), 4.43 (m, 1H, H-3′),
4.58 (m, 1H, H-2′), 5.16 (br s, 2H, NH2), 5.31 (br s, 1H, OH), 5.42
(d, J ) 5.5 Hz, 1H, H-1′), 6.79 (t, J ) 5.0 Hz, 1H, NHCO), 6.90
(d, J ) 1.3 Hz, 1H, H-2), 7.21-7.39 (m, 15 H, Ph). Anal.
(C32H34N4O5‚H2O) C, H, N.
2′,3′-Di-O-acetyl-5′-O-trityl-1-[(2-methoxyethoxy)methyl]-
inosine (5). To a solution of 2′,3′-bis-O-acetyl-5′-O-tritylinosine
(4)24 (235 mg, 0.39 mmol) in dry CH2Cl2 (3 mL) at 0 °C were
added i-Pr2NEt (83 µL, 0.47 mmol) and 2-methoxyethoxymethyl
chloride (MEMCl) (54 µL, 0.47 mmol). The reaction mixture was
stirred at 0 °C for 1 h. The reaction was quenched by addition of
H2O (10 mL), stirred for an additional 20 min, and diluted with
CH2Cl2 (15 mL). The organic phase was decanted and the aqueous
phase was further extracted with CH2Cl2 (2 × 15 mL). The
combined organic phases were dried on MgSO4, filtered, and
evaporated. The final residue was purified by column chromatog-
raphy (CH2Cl2:acetone, 4:1) to yield 191 mg (72%) of 5 as white
solid: mp 68-69 °C (CH2Cl2:MeOH); MS (ES, positive mode)
m/z 683 (M + 1)+; 1H NMR (CDCl3) δ 2.07 (s, 3H, COCH3), 2.12
(s, 3H, COCH3), 3.34 (s, 3H, OCH3), 3.46-3.52 (m, 4H, OCH2-
CH2O), 3.79 (m, 2H, H-5′), 4.34 (m, 1H, H-4′), 5.53 (m, 2H,
NCH2O), 5.68 (m, 1H, H-3′), 6.06 (t, J ) 6.5 Hz, 1H, H-2′), 6.15
(d, J ) 6.5 Hz, 1H, H-1′), 7.21-7.44 (m, 15 H, Ph), 7.89 (s, 1H,
H-8), 7.99 (s, 1H, H-2).
1-[(2-Methoxyethoxy)methyl]-5′-O-tritylinosine (6). A suspen-
sion containing 5 (133 mg, 0.19 mmol) in 0.2 N NaOH (2 mL)
was refluxed for 6 h. Additional 0.2 N NaOH (2 mL) was added
and the reaction was refluxed overnight. Volatiles were removed,
and the residue was purified by flash column chromatography (CH2-
Cl2:MeOH, 15:1). The UV-positive fractions were further purified
by a SPE Silica cartridge eluting with mixtures CH2Cl2:MeOH,
100:1 to 60:1. The fastest moving fractions afforded 48 mg (42%)
of a white solid, identified as 6: mp 82-83 °C (CH2Cl2:MeOH);
MS (ES, positive mode) m/z 599 (M + 1)+; 1H NMR (DMSO-d6)
δ 3.11 (s, 3H, OCH3), 3.16 (m, 2H, H-5′), 3.33 (m, 2H, CH2OCH3),
3.56 (m, 2H, NCH2OCH2), 4.07 (m, 1H, H-4′), 4.21 (m, 1H, H-3′),
4.56 (m, 1H, H-2′), 5.26 (d, J ) 4.5 Hz, 1H, OH), 5.43 (d, J ) 6.8
Hz, 2H, NCH2O), 5.74 (br s, 1H, OH), 5.90 (d, J ) 4.6 Hz, 1H,
H-1′), 5.90 (br s, 2H, NH2), 7.21-7.37 (m, 15 H, Ph), 8.23 (s, 1H,
H-8), 8.38 (s, 1H, H-2). Anal. (C33H34N4O7‚0.5H2O) C, H, N.
The slowest moving fractions afforded 20 mg (18%) of a white
solid identified as 5-amino-1-(5-O-trityl-â-D-ribofuranosyl)imid-
azole-4-[N-(2-methoxyethoxy)methyl]carboxamide (7): mp 80-
81 °C; MS (ES, positive mode) m/z 589 (M + 1)+; 1H NMR
(DMSO-d6) δ 3.18 (d, J ) 3.7 Hz, 2H, H-5′), 3.21 (s, 3H, OCH3),
3.39 (m, 2H, CH2OCH3), 3.51 (m, 2H, NCH2OCH2), 4.02 (m, 1H,
H-4′), 4.10 (m, 1H, H-3′), 4.31 (m, 1H, H-2′), 4.32 (d, J ) 6.8 Hz,
2H, HNCH2), 5.20 (d, J ) 4.9 Hz, 1H, OH), 5.52-5.54 (m, 2H,
H-1′, OH), 5.90 (br s, 2H, NH2), 7.23-7.38 (m, 16 H, H-2, Ph),
8.24 (t, J ) 6.9 Hz, 1H, NHCO). Anal. (C32H36N4O7‚0.5H2O) C,
H, N.
1-Benzyl-5′-O-tritylinosine (2f). Reaction of 1 with benzyl
bromide (51 µL, 0.43 mmol), following the general procedure for
the preparation of N1-substituted 5′-O-tritylinosines, afforded a
residue that was purified in the Chromatotron (CH2Cl2:MeOH, 10:
1) to yield 156 mg (66%) of 2f as a white solid: mp 120-121 °C
1
(CH2Cl2:MeOH); MS (ES, positive mode) m/z 601 (M + 1)+; H
NMR (DMSO-d6) δ 3.19 (m, 2H, H-5′), 4.07 (m, 1H, H-4′), 4.22
(m, 1H, H-3′), 4.57 (m, 1H, H-2′), 5.22 (s, 2H, NCH2), 5.26 (d, J
) 5.8 Hz, 1H, OH), 5.61 (d, J ) 5.5 Hz, 1H, OH), 5.89 (d, J )
4.4 Hz, 1H, H-1′), 7.20-7.37 (m, 15 H, Ph), 8.23 (s, 1H, H-8),
8.52 (s, 1H, H-2). Anal. (C36H32N4O5‚H2O) C, H, N.
1-[(E)-3-Methoxycarbonyl-2-propenyl]-5′-O-tritylinosine (2 g).
Reaction of 1 with methyl-4-bromocrotonate (60 µL, 0.50 mmol)
following the general procedure for the preparation of N1-substituted
5′-O-tritylinosines, afforded a residue that was purified in the
Chromatotron (CH2Cl2:MeOH, 10:1) to yield 78 mg (32%) of 2g
as a white solid: mp 115-116 °C (CH2Cl2:MeOH); MS (ES,
1
positive mode) m/z 609 (M + 1)+; H NMR (CDCl3) δ 3.34 (m,
2H, H-5′), 3.65 (s, 3H, OCH3), 4.28 (m, 1H, H-4′), 4.36 (m, 1H,
H-3′), 4.69-4.72 (m, 3H, H-2′, NCH2), 5.00 (br s, 1H, OH), 5.76
(d, J ) 15.7 Hz, 1H, dCHCO), 5.91 (d, J ) 5.1 Hz, 1H, H-1′),
6.90 (m, 1H, NCH2CHd), 7.09-7.32 (m, 15 H, Ph), 7.75 (s, 1H,
H-8), 7.90 (s, 1H, H-2). Anal. (C34H32N4O7‚H2O) C, H, N.
5-Amino-1-(5-O-trityl-â-D-ribofuranosyl)imidazole-4-(N-
methyl)carboxamide (3a). A solution containing 2a (100 mg, 0.18
mmol) in EtOH (4 mL) and 5 N NaOH (2 mL) was refluxed for 3
h. Volatiles were removed, and the residue was purified by flash
column chromatography (CH2Cl2:MeOH:NH4OH, 10:1:0.5) to yield
49 mg (53%) of 3a as a white solid: mp 197-200 °C (CH2Cl2:
1
MeOH); MS (ES, positive mode) m/z 515 (M + 1)+; H NMR
(DMSO-d6) δ 2.66 (d, J ) 4.8 Hz, 2H, CH3), 3.16 (m, 2H, H-5′),
4.01 (m, 1H, H-4′), 4.09 (m, 1H, H-3′), 4.30 (m, 1H, H-2′), 5.24
(br s, 1H, OH), 5.50 (d, J ) 5.4 Hz, 1H, H-1′), 5.55 (br s, 1H,
OH), 5.75 (br s, 2H, NH2), 7.25-7.37 (m, 16 H, Ph, H-2), 7.42
(m, 1H, CONH). Anal. (C29H30N4O5‚0.5H2O) C, H, N.
5-Amino-1-(5-O-trityl-â-D-ribofuranosyl)imidazole-4-(N-pro-
pyl)carboxamide (3b). Following a procedure analogous to that
described for the synthesis of 3a, compound 2b (100 mg, 0.18
mmol) reacted with 5 N NaOH to yield 53 mg (52%) of 3b as a
white solid: mp 81-84 °C (CH2Cl2:MeOH); MS (ES, positive
mode) m/z 543 (M + 1)+; 1H NMR (DMSO-d6) δ 0.82 (t, J ) 7.6
Hz, 3H, CH3), 1.43 (m, J ) 7.6 Hz, 2H, CH3CH2CH2), 3.10 (q, J
) 7.6 Hz, 2H, HNCH2), 3.14 (d, J ) 4.0 Hz, 2H, H-5′), 4.00 (m,
1H, H-4′), 4.10 (dd, J ) 4.8 Hz, 1H, H-3′), 4.31 (dd, J ) 5.1 Hz,
1H, H-2′), 5.21 (d, J ) 5.5 Hz, 1H, OH), 5.47 (d, J ) 5.4 Hz, 1H,
H-1′), 5.48 (d, J ) 5.4 Hz, 1H, OH), 5.78 (br s, 2H, NH2), 7.26-
7.37 (m, 16H, Ph, H-2), 7.42 (t, J ) 6.0 Hz, 1H, CONH). Anal.
(C31H34N4O5‚2H2O) C, H, N.
5-Amino-1-(5-O-trityl-â-D-ribofuranosyl)imidazole-4-carbox-
amide (8). A suspension containing 5 (125 mg, 0.18 mmol) in 5 N
NaOH (2 mL) was refluxed for 6 h. Volatiles were removed and
the residue was purified by CCTLC in the Chromatotron (CH2Cl2:
MeOH, 15:1 to 10:1) to yield 22 mg (20%) of compound 8 as a
white solid: mp 114-116 °C (CH2Cl2:MeOH); MS (ES, positive
5-Amino-1-(5-O-trityl-â-D-ribofuranosyl)imidazole-4-(N-
allyl)carboxamide (3c). Following a procedure analogous to that
described for the synthesis of 3a, compound 2c (100 mg, 0.18
mmol) reacted with 5 N NaOH to yield 3c (45 mg, 46%) as a
solid: mp 93-94 °C (CH2Cl2:MeOH); MS (ES, positive mode)
1
mode) m/z 501 (M + 1)+; H NMR (DMSO-d6) δ 3.16 (m, 2H,
1
m/z 541 (M + 1)+; H NMR (DMSO-d6) δ 3.17 (d, J ) 3.6 Hz,
H-5′), 3.99 (m, 1H, H-4′), 4.09 (m, 1H, H-3′), 4.30 (m, 1H, H-2′),
5.20 (d, J ) 5.3 Hz, 1H, OH), 5.49-5.53 (m, 2H, H-1′, OH), 5.81
(br s, 2H, NH2), 6.64, 6.82 (br s, 2H, CONH2), 7.23-7.38 (m, 16
H, Ph, H-2). Anal. (C28H28N4O5‚2H2O) C, H, N.
General Procedure for the 5′-O-Tritylation of Ribonucleo-
sides. Prior to reaction, the corresponding ribonucleoside was
2H, H-5′), 3.78 (t, J ) 5.5 Hz, 2H, HNCH2), 4.01 (m, 1H, H-4′),
4.09 (m, 1H, H-3′), 4.30 (m, 1H, H-2′), 4.98-5.11 (m, 2H, CHd
CH2), 5.21 (d, J ) 5.3 Hz, 1H, OH), 5.51-5.54 (m, 2H, OH, H-1′),
5.78 (br s, 2H, NH2), 5.85 (m, 1H, CHdCH2), 7.23-7.38 (m, 16
H, Ph, H-2), 7.54 (t, J ) 6.0 Hz, 1H, CONH). Anal. (C31H32N4O5‚
H2O) C, H, N.

5568 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18
CasanoVa et al.
coevaporated twice with pyridine (5 mL). Then, to a suspension of
the nucleoside (1.0 mmol) in anhydrous pyridine (5 mL) were added
4-(dimethylamino)pyridine (DMAP) (5 mg, 0.04 mmol) and trityl
chloride (482 mg, 1.7 mmol). The reaction was heated at 80 °C
for 15 h. After reaching room temperature, EtOAc (100 mL) and
a NaHCO3 solution (20 mL) were added. The organic phase was
decanted and further washed with H2O (20 mL) and brine (20 mL).
Then it was dried on MgSO4, filtered, and evaporated to dryness.
The residue was purified as indicated for each compound.
1H, OH), 6.33 (t, J ) 6.4 Hz, 1H, H-1′), 7.19-7.41 (m, 15 H, Ph),
7.96 (s, 1H, H-8), 8.18 (s, 1H, H-2), 12.36 (br s, 1H, NH). Anal.
(C29H26N4O4‚0.5H2O) C, H, N.
1-Tritylinosine (19). To a suspension of inosine 14 (268 mg,
1.0 mmol) in DMF (4 mL) were added i-Pr2NEt (0.2 mL, 1.2 mmol)
and trityl chloride (558 mg, 2.0 mmol), and the mixture was stirred
at 40 °C for 18 h. The workup was identical to that of the general
procedure. The final residue was purified by CCTLC in the
Chromatotron (CH2Cl2:MeOH, 15:1) to afford 153 mg (30%) of
19 as a white solid: mp 123-125 °C; MS (ES, positive mode)
9-(5-O-Trityl-â-D-ribofuranosyl)purine (11) was prepared by
reaction of 9-(â-D-ribofuranosyl)purine (9) (252 mg, 1.0 mmol)
with trityl chloride, according to the general procedure. The final
residue was purified by flash column chromatography (CH2Cl2:
MeOH, 10:1) to yield 173 mg (35%) of 11 as a white solid: mp
94-97 °C (CH2Cl2:MeOH); MS (ES, positive mode) m/z 495 (M
+ 1)+; 1H NMR (CDCl3) δ 3.30 (dd, J ) 10.7, 3.2 Hz, 1H, H-5′),
3.49 (dd, J ) 10.7, 3.4 Hz, 1H, H-5′′), 4.46 (m, 2H, H-3′, H-4′),
4.90 (m, 1H, H-2′), 5.38 (br s, 1H, OH), 6.05 (d, J ) 5.9 Hz, 1H,
H-1′), 7.20-7.28 (m, 15H, Ph), 8.38 (s, 1H, H-8), 8.97 (s, 1H,
H-2), 9.22 (s, 1H, H-6). Anal. (C29H26N4O4‚H2O) C, H, N.
6-Chloro-9-(5-O-trityl-â-D-ribofuranosyl)purine (12). This
compound was prepared by a described procedure25 that consisted
of treatment of 6-chloro-9-(â-D-ribofuranosyl)purine (10) (287 mg,
1.0 mmol) in dry DMF (4 mL) and in the presence of i-Pr2NEt
(0.2 mL, 1.2 mmol) with trityl chloride (558 mg, 2.0 mmol) at 40
°C for 18 h. Purification of the crude mixture by CCTLC in the
Chromatotron (CH2Cl2:MeOH, 20:1) afforded 159 mg (30%) of
12 as a white solid: mp 94-96 °C (CH2Cl2:MeOH); MS (ES,
positive mode) m/z 551 (M + Na)+ with a Cl isotopic pattern; 1H
NMR (DMSO-d6) δ 3.23 (m, 2H, H-5′), 4.11 (q, J ) 4.2 Hz, 1H,
H-4′), 4.33 (m, 1H, H-3′), 4.72 (m, 1H, H-2′), 5.29 (d, J ) 6.1 Hz,
1H, OH), 5.65 (d, J ) 5.4 Hz, 1H, OH), 6.05 (d, J ) 3.9 Hz, 1H,
H-1′), 7.19-7.34 (m, 15 H, Ph), 8.71 (s, 1H, H-8), 8.81 (s, 1H,
H-2). Anal. (C29H25ClN4O4) C, H, N.
1
m/z 511 (M + 1)+; H NMR (DMSO-d6) δ 3.45-3.62 (m, 2H,
H-5′), 3.91 (m, 1H, H-4′), 4.11 (m, 1H, H-3′), 4.50 (m, 1H, H-2′),
5.02 (t, J ) 5.3 Hz, 1H, OH), 5.21 (d, J ) 4.9 Hz, 1H, OH), 5.48
(d, J ) 6.0 Hz, 1H, OH), 5.83 (d, J ) 5.7 Hz, 1H, H-1′), 7.23-
7.36 (m, 15 H, Ph), 8.32, 8.33 (2s, 2H, H-8, H-2). Anal.
(C29H26N4O5‚2H2O) C, H, N.
5′-O-Trityl-2′,3′-secoinosine (20). To a solution of 5′-O-
tritylinosine (1)16 (250 mg, 0.5 mmol) in 6 mL of dioxane/H2O
(5:1) was added a solution of NaIO4 (111 mg, 0.5 mmol) in H2O
(1 mL). After a few minutes, a suspension formed that was stirred
for 18 h. The mixture was diluted with dioxane (5 mL), and after
10 min of stirring, the solid was filtered and further washed with
dioxane (3 mL). The combined filtrates were cooled at 0 °C and
treated with NaBH4 (18 mg, 0.5 mmol). The resulting mixture was
stirred at room temperature for 2 h and then acetone (0.1 mL) was
added. After 5 min, the reaction was neutralized by addition of a
few drops of 10% AcOH. Volatiles were removed, and the residue
was partitioned between H2O and i-BuOH. The aqueous phase was
further extracted with i-BuOH. The combined organic phases were
dried on MgSO4, filtered, and evaporated. The final residue was
purified by CCTLC in the Chromatotron (CH2Cl2:MeOH, 6:1) to
afford 154 mg (60%) of 20 as an amorphous solid:. MS (ES, positive
1
mode) m/z 513 (M + 1)+; H NMR (DMSO-d6) δ 2.79 (m, 2H,
H-5′), 3.42 (m, 1H, H-3′), 3.78 (m, 1H, H-4′), 3.98 (m, 1H, H-2′),
4.78 (br s, 1H, OH), 5.21 (br s, 1H, OH), 5.47 (t, J ) 6.1 Hz, 1H,
H-1′), 7.07-7.26 (m, 15H, Ph), 8.06 (s, 1H, H-8), 8.28 (s, 1H,
H-2), 12.28 (br s, 1H, NH). Anal. (C29H28N4O5‚2H2O) C, H, N.
1-Benzyl-5′-O-benzyl-2′,3′-O-isopropylideneinosine (22). To a
solution of 2′,3′-O-isopropylideninosine (21) (200 mg, 0.63 mmol)
in dry DMF (4 mL) was added NaH (60% in mineral oil, 81 mg,
2.10 mmol). The reaction was stirred at room temperature for 1 h.
Then, benzyl bromide (0.17 mL, 1.41 mmol) was added and stirring
was continued for 1 h at room temperature. The reaction was
quenched by addition of a few drops of AcOH till the pH was 7-8.
Volatiles were removed, and the residue was purified by flash
column chromatography (CH2Cl2:MeOH, 99:1 to 97:3) to yield 221
mg (71%) of 22 as an oil: MS (ES, positive mode) m/z 489 (M +
6-Methylamino-9-(5-O-trityl-â-D-ribofuranosyl)purine (13).
To a solution of compound 12 (100 mg, 0.19 mmol) in EtOH (2
mL) was added MeNH2 in EtOH (33%) (4 mL). The mixture was
heated in a sealed tube at 70 °C for 4 h. Volatiles were removed,
and the residue was purified by CCTLC in the Chromatotron (CH2-
Cl2:MeOH, 15:1) to afford 42 mg (42%) of 13 as a white solid:
mp 218-220 °C (CH2Cl2:MeOH); MS (ES, positive mode) m/z
1
524 (M + 1)+; H NMR (DMSO-d6) δ 3.20 (m, 2H, H-5′), 3.94
(s, 3H, NHCH3), 4.04 (q, J ) 4.9 Hz, 1H, H-4′), 4.30 (m, 1H,
H-3′), 4.68 (m, 1H, H-2′), 5.20 (d, J ) 5.9 Hz, 1H, OH), 5.52 (d,
J ) 5.6 Hz, 1H, OH), 5.91 (d, J ) 4.4 Hz, 1H, H-1′), 7.18-7.36
(m, 15 H, Ph), 7.76 (br s, 1H, NH), 8.17 (s, 1H, H-8), 8.23 (s, 1H,
H-2). Anal. (C30H29N5O4‚H2O) C, H, N.
1
1)+; H NMR (CDCl3) δ 1.29 (s, 3H, CH3), 1.53 (s, 3H, CH3),
5′-O-[(4-Chlorophenyl)-1,1-(diphenyl)methyl]inosine (17). This
compound was prepared by reaction of inosine 14 (268 mg, 1.0
mmol) with 4-chlorotrityl chloride27 (313 mg, 1.0 mmol) according
to the general procedure for the 5′-O-tritylation of ribonucleosides.
Purification of the residue by flash column chromatography (CH2-
Cl2:MeOH, 20:1 to 10:1) afforded 174 mg (32%) of 17 as a white
solid: mp 154-157 °C (CH2Cl2:MeOH); MS (ES, positive mode)
m/z 545 (M + 1)+ showing Cl isotopic pattern; 1H NMR (DMSO-
d6) δ 3.22 (m, 2H, H-5′), 4.07 (q, J ) 5.1 Hz, 1H, H-4′), 4.23 (m,
1H, H-3′), 4.57 (m, 1H, H-2′), 5.24 (d, J ) 6.0 Hz, 1H, OH), 5.59
(d, J ) 5.8 Hz, 1H, OH), 5.91 (d, J ) 4.3 Hz, 1H, H-1′), 7.24-
7.38 (m, 14H, Ph), 8.00 (s, 1H, H-8), 8.22 (s, 1H, H-2), 12.40 (br
s, 1H, NH-1). Anal. (C29H25ClN4O5) C, H, N.
5′-O-Trityl-2′deoxyinosine (18). 2′-Deoxyinosine (15) (100 mg,
0.40 mmol) was coevaporated twice with anhydrous pyridine (1
mL) prior to reaction. Then it was dissolved in pyridine (2 mL),
and trityl chloride (188 mg, 0.67) and DMAP (2 mg, 0.02 mmol)
were added. The reaction was warmed at 60 °C for 4 h. Methanol
(1 mL) was added, and volatiles were removed. Purification of the
residue by flash column chromatography (CH2Cl2:MeOH, 30:1 to
10:1) afforded 70 mg (35%) of 18 as a white solid: mp 219-221
°C (CH2Cl2:MeOH); MS (ES, positive mode) m/z 495 (M + 1)+;
1H NMR (DMSO-d6) δ 2.32, 2.75 (m, 2H, H-2′), 3.15 (m, 2H,
H-5′), 3.95 (m, 1H, H-4′), 4.41 (m, 1H, H-3′), 5.38 (d, J ) 4.6 Hz,
3.56 (m, 2H, H-5′), 4.38 (d, J ) 1.8 Hz, 2H, CH2O), 4.44 (m, 1H,
H-4′), 4.83 (dd, J ) 6.0, 2.1 Hz, 1H, H-3′), 5.08 (dd, J ) 6.0, 2.4
Hz, 1H, H-2′), 5.16 (s, 2H, CH2N), 6.02 (d, J ) 2.4 Hz, 1H, H-1′),
7.11-7.28 (m, 10 H, Ph), 7.89 (s, 1H, H-8), 7.93 (s, 1H, H-2).
1-Benzyl-5′-O-benzylinosine (23).31 A solution containing 22
(100 mg, 0.2 mmol) in 30% aqueous TFA (2.2 mL) was stirred at
0 °C for 20 min and for 3 h at room temperature. Volatiles were
removed, and the residue was purified by CCTLC in the Chroma-
totron (CH2Cl2:MeOH, 20:1) to yield 87 mg (95%) of 23 as a white
solid: mp 65-68 °C (CH2Cl2:MeOH); m/z 449 (M + 1)+; 1H NMR
(CDCl3) δ 3.59-3.72 (m, 3H, H-5′, OH′), 4.32 (m, 1H, H-4′), 4.42
(m, 1H, H-3′), 4.46 (d, J ) 2.7 Hz, 2H, OCH2Ph), 4.61 (m, 1H,
H-2′), 5.08 (s, 2H, N CH2Ph), 5.28 (br s, 1H, OH), 5.95 (d, J )
5.4 Hz, 1H, H-1′), 7.18-7.30 (m, 10 H, Ph), 7.80 (s, 1H, H-8),
7.92 (s, 1H, H-2). Anal. (C24H24N4O5‚H2O) C, H, N.
TPase Enzyme Assays. The phosphorolysis of thymidine (dThd)
by human TPase was measured by HPLC analysis. The incubation
mixture (500 µL) contained 10 mM Tris-HCl (pH 7.6), 1 mM
EDTA, 2 mM potassium phosphate, 150 mM NaCl, 5% DMSO,
and 100 µM of dThd in the presence of 0.025 U TPase. Incubations
were performed at room temperature. At different time points (i.e.,
0, 20, 40, and 60 min), 100-µL fractions were withdrawn,
transferred to an Eppendorf tube thermoblock, and boiled at 95 °C

5′-O-Tritylinosine Analogues as TPase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18 5569
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HPLC analysis. The separation of Thy and dThd was performed
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Acknowledgment. We are grateful to Mrs. Ria Van Berwaer
for excellent technical assistance. E.C. acknowledges the
Conserjer´ıa de Educacio´n de la Comunidad de Madrid and the
Fondo Social Europeo (F.S.E.) for a predoctoral fellowship.
A.I.H. thanks the Spanish Ministerio de Educacio´n y Ciencia
for a predoctoral fellowship. S.L. holds a postdoctoral research
fellowship from the “Fonds voor Wetenschappelijk Onderzoek
(FWO)”. This work has been supported by grants of the Spanish
MEC (SAF2003-07219-C02-01) and the EU (QLRT-2001-
01004), the Centers of Excellence of the KULeuven (EF/05/
15), and the “Belgische Federatie tegen Kanker” (to S.L.).
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Supporting Information Available: Elemental analysis data
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material is available free of charge via the Internet at http://
pubs.acs.org.
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