A solid-phase approach to the synthesis of N-1-alkyl analogues of cyclic inosine-diphosphate-ribose (cIDPR)

Article abstract of DOI:10.1016/j.tet.2010.01.013

We report here an efficient solid-phase synthesis of N-1-alkyl-substituted analogues of cyclic inosine-diphosphate-ribose (cIDPR), a mimic of cyclic ADP-ribose (cADPR). Our synthetic strategy makes use of a polystyrene support to which inosine was bonded through a 2′,3′-acetal linkage. Insertion of a ω-hydroxy-polymethylene chain of variable length on N-1, followed by conversion into N-1-alkylinosine-bis-phosphate derivatives and cyclization, allowed to obtain analogues of cIDPR of various ring size. The cyclization step was carried out both in solid-phase and in solution by pyrophosphate bond formation. The effect of the N-1-polymethylene chain length on the cyclization yields as well as the reaction conditions, which led to the solid-phase pyrophosphate bond formation, were thoroughly investigated.

Full text of DOI:10.1016/j.tet.2010.01.013

Tetrahedron 66 (2010) 1931–1936
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
A solid-phase approach to the synthesis of N-1-alkyl analogues of cyclic
inosine-diphosphate-ribose (cIDPR)
Giorgia Oliviero a, Stefano D’Errico a, Nicola Borbone a, Jussara Amato a, Vincenzo Piccialli b,
,
a
Michela Varra a, Gennaro Piccialli a , Luciano Mayol
*
a
`
Dipartimento di Chimica delle Sostanze Naturali, Universita degli Studi di Napoli ‘‘Federico II’’, Via D. Montesano 49, I-80131 Napoli, Italy
b
`
Dipartimento di Chimica Organica e Biochimica, Universita degli Studi di Napoli ‘‘Federico II’’, Via Cynthia 4, I-80126 Napoli, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 May 2009
Received in revised form 18 November 2009
Accepted 4 January 2010
Available online 11 January 2010
We report here an efficient solid-phase synthesis of N-1-alkyl-substituted analogues of cyclic inosine-
diphosphate-ribose (cIDPR), a mimic of cyclic ADP-ribose (cADPR). Our synthetic strategy makes use of
a polystyrene support to which inosine was bonded through a 20,30-acetal linkage. Insertion of a
u-hy-
droxy-polymethylene chain of variable length on N-1, followed by conversion into N-1-alkylinosine-bis-
phosphate derivatives and cyclization, allowed to obtain analogues of cIDPR of various ring size. The
cyclization step was carried out both in solid-phase and in solution by pyrophosphate bond formation.
The effect of the N-1-polymethylene chain length on the cyclization yields as well as the reaction
conditions, which led to the solid-phase pyrophosphate bond formation, were thoroughly investigated.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
permeabilized human Jurkat T lymphocytes.4 The chemical synthesis
proposed for the great part of these analogues, makes use of mul-
Cyclic ADP-ribose (cADPR, 1, Fig. 1) is a metabolite involved in
Ca2þ signalling in various cell types. It is biosynthesized from NADþ
and regulates the calcium mobilization from intracellular stores in
a wide variety of biological systems via ryanodine receptors.1 New
cADPR analogues, exhibiting both biological properties as agonists or
antagonists and resistance to the enzymatic and chemical hydrolysis,
are required to investigate cADPR-mediated Ca2þ signalling. In order
to elucidate the structure–activity relationship, a number of ana-
logues of cADPR have been synthesized using enzymatic, chemo-
enzymatic and chemical approaches and pharmacologically tested.2
8-Br-cIDPR (2, Fig. 1), has been recently described as an agonist of
cADPR/Ca2þ signalling system.3 A series of N-1-substituted,4 N-9-
substituted5 and C-8-substituted3 cIDPR (3, Fig. 1) have been syn-
thesized and some of them have been shown to retain an interesting
Ca2þ-related biological activity. Particular attention has been de-
voted to the N-1-substitutions. In particular, the synthesis of N-1-
carborybosyl,6 N-1-glycosyl,7 and N-1-ethoxymethyl5a,7,8 analogues
have been reported. In some cases, these modifications have also
been combined with a C-8 substitution (i.e., 8-Cl, 8-Br, 8-N3). Among
the synthesized compounds, the N-1-ethoxymethyl-cIDPR (cIDPRE,
4, Fig. 1), containing a structurally simplified N-1 molecular moiety,
has shown a strong potency in inducing Ca2þ release in intact and
tistep sequences carried out in solution which require purification of
the products after each reaction step. In an effort to facilitate the
chemical synthesis of these substances as well as to enlarge the
number of accessible structurally diverse analogues, we report here
a new and general solid-phase approach to the synthesis of N-1-alkyl
analogues of cIDPR (18a–d, Scheme 1).
2. Results and discussion
Our synthetic approach uses the inosine-binding solid support 7
as key intermediate. This was prepared in almost quantitative yield
by reaction of the 4-(hydroxymethyl)-20,30-benzylidene-50-
TBDPS-inosine
6
with the commercially available poly-
styrenemonomethoxytrityl chloride resin (MMTCl, 1.3 mmol/g). In
this way inosine was bound to the trityl resin by a linker having two
acid labile functions (a benzyl–trityl ether and a 20,30-acetal link-
age) that are selectively cleavable by treatment with anhydrous
acids or aqueous acids, respectively. Carboxy-functionalized 20,30-
benzylidene acetal linkers have previously been used to attach
nucleosides to amino-loaded solid supports through amide bond
formation.9 Intermediate 6 was in turn prepared in 85% yield by
reaction of 50-protected inosine 5 with 4-(hydroxymethyl)-benz-
aldehyde dimethyl acetal in the presence of catalytic amounts of
p-toluensulfonic acid (PTSA). This reaction proved highly diaster-
eoselective giving the stereoisomer possessing the R configuration
at the benzylidene acetal carbon (dr>96%) as ascertained by a 2D-
* Corresponding author. Tel.: þ39 081678541; fax: þ39 081678552.
E-mail address: picciall@unina.it (G. Piccialli).
0040-4020/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.01.013

1932
G. Oliviero et al. / Tetrahedron 66 (2010) 1931–1936
OH
HO
OH
HO
O
O
O
NH2
O
N
O
N
N
O
N
N
N
O
O
O
O
O
O
O
X
N
N
N
N
N
N
P
P
P
HO
HO
HO
O
O
O
O
O
O
O
O
P
P
P
O
O
OH
OH
OH
OH
OH
HO
4 cIDPRE
OH
HO
HO
1 (cADPR)
2 X = Br (8-Br-cIDPR)
3 X = H (cIDPR)
Figure 1. Structures of cADPR, cIDPR, 8-Br-cIDPR and cIDPRE.
Scheme 1. Reagents and conditions: (i) 4-(hydroxymethyl)-benzaldehyde dimethyl acetal (2.7 equiv), PTSA (0.3 equiv), DCM, 7 h, reflux; (ii) MMTCl resin, 6 (1.5 equiv), pyridine
(1.5 mL/250 mg of resin), DMAP (0.2 equiv), 24 h, rt; (iii) (a) NH4F (55 equiv), MeOH, reflux, 12 h, (b) Ac2O in pyridine, 30 min, rt; (iv) DNCB (7.5 equiv), K2CO3 (7.5 equiv), DMF, 2 h,
80 ꢀC.; (v) OH–(CH2)2,4–6–NH2 (66 equiv), DMF, 8 h, 50 ꢀC; (vi) TFA 2% in DCM, 8 min, rt; (vii) (a) (CEO)2PN(iPr)2 (24 equiv), tetrazole (48 equiv) in THF/DCM, 15 h, rt; (b) tert-
butylhydroperoxide (69 equiv), decane/THF, 3 h, rt; (viii) conc. NH4OH, 15 h, 55 ꢀC; (ix) EDC (6.4 equiv for 13a–d or 5.0 equiv for 16a–d), DMF, 72 h, rt; (x) TFA/H2O/DCM, 15 min, rt;
(xi) Ac2O in pyridine, 15 h, rt; (xii) conc. NH4OH, 5 h, rt.
NOESY experiment that showed intense correlations between the
acetal proton and the ribose H-20 and H-30 protons. In previous
papers we demonstrated that N-1-(2,4-dinitrophenyl)-inosine can
be easily converted into N-1-alkylated inosine by reaction with
primary alkylamines, both in solution10–12 or 50-bound to a poly-
meric support.13 In particular, reaction of the C-2 purine carbon
with amino nucleophiles leads to the opening of the six-membered
ring through the cleavage of the N1-C2 bond. The successive fast
ring re-closure, favored by loss of 2,4-dinitroaniline, furnishes the
N-1-alkylinosine derivatives in high yields. Based on these pre-
cedents, N-1-(2,4-dinitrophenyl)-inosine-containing support 9 was
synthesized in high yields (94%) by reaction of 7 with 2,4-
dinitrochlorobenzene (DNCB) in the presence of K2CO3. It is to be
noted that a 50 acetyl replaces the TBDMS protecting group in 8.
This protecting group modification (NH4F treatment of 7 followed
by acetylation) was suggested by the observation that the presence
of a 50-O-acetyl on the sugar furnishes higher yields of the dini-
trophenylderivative 9 during the conversion of 8 to 9. This trans-
formation demonstrated that the above process works well on
a solid support too, allowing the successful replacement of the
time-consuming step required for the preparation of N-1-(2,4-
dinitrophenyl)-inosine in solution before its loading onto the solid
support.13 Next, reaction of 9 with some
(HO–(CH2)n–NH2, n¼2,4–6, a–d, Table 1) furnished the N-1-
u-hydroxyalkylamines
Table 1
Products and reaction yields
u
-hydroxyalkylamines
11a–d
15a–d
18a–d (from 13)
18a–d (from 16)
18a–d
yielda (%)
yielda (%)
yieldb (%)
yieldc;
yielda (%)
a OH–(CH2)2–NH2
b OH–(CH2)4–NH2
c OH–(CH2)5–NH2
d OH–(CH2)6–NH2
91
92
92
90
78
78
77
74
20
13
10
10
89
84
85
84
74
70
70
68
a
Overall yield starting from support 7.
Calculated on the amount of 13 (0.063 mmol/g) converted in 18.
Calculated on the amount of 16 converted in 18.
b
c

G. Oliviero et al. / Tetrahedron 66 (2010) 1931–1936
1933
u
-hydroxyalkyl-inosine supports 10a–d (90–92% yield) by the
were subjected, without further purification, to the pyrophos-
phate bond formation by addition of EDC in DMF to give the
cyclic diphosphate derivatives 17a–d in high yields (Table 1).
Finally removal of the acetyl groups, performed by aqueous
conc. ammonia treatment, furnished the target products 18a–
d the structures of which were confirmed by 1H, 31P NMR and
MS data. It is to be noted that the cyclization yields (84–89%,
Table 1) are scarcely affected by the N-1-polymethylene chain
lengths though the best yield was obtained with 18a including
the shortest, bis-methylene, chain. In addition, we also observed
that almost the same cyclization yields were obtained when the
reaction was carried out on the hydroxymethyl-acetylated 20,30-
benzylidene derivatives of 15a–d (not shown in Scheme 1) re-
leased from supports 13a–d by anhydrous acidic treatment
followed by standard acetylation of the p-CH2OH group on the
benzene ring. This second pathway demonstrated that a 20,30-
bonded bulky group does not hamper the cyclization reaction
and, furthermore, furnishes an alternative synthetic procedure,
which uses an acid-labile 20,30-protecting group.
concomitant aminolysis of the 50-O-acetyl group. It is to be noted
that when 3-amino-propanol was used, the resin assumed an en-
during gel-like consistency that prevented its further manipulation
and therefore the synthesis of the expected N-1-(3-hydrox-
ypropyl)-inosine. The reaction yield, as well as the structures of
10a–d, were ascertained analyzing the HPLC purified nucleoside
material 11a–d released from a weighted amount of resin 10a–d by
treatment with a 2% TFA solution in dry CH2Cl2 (DCM) taking into
account that the starting support 7 had a nucleoside functionali-
zation of 0.72 mmol/g. Then, inosine derivatives 10a–d were
converted into the bis-protected diphosphate derivatives 12a–d
that, after phosphate deprotection, gave diphosphate derivatives
13a–d suitable for cyclization by intramolecular pyrophosphate
bond formation as detailed below.
The intramolecular cyclization is a crucial step in the chemical
synthesis of cADPR, cIDPR and their derivatives, that requires the
closure of a 18-membered cycle. The most employed method uses
the condensation between a phosphomonoester and a S-phenyl
phosphodiester group, promoted by AgNO3 that converts the
phosphorothioate into a reactive metaphosphate species.6 However,
this procedure, that minimizes the charge repulsion between the
two phosphates, requires two distinct phosphorylation reactions and
suitable protection/deprotection steps. In previous work, we dem-
onstrated that in the 20,30-protected diphosphate 15c, though char-
acterized by a high conformational flexibility, the two charged
phosphate functions could be efficiently condensed in dilute con-
ditions in the presence of 1-[3-(dimethylamino)propyl]-3-eth-
3. Conclusions
The herein reported solid-phase approach, besides the synthesis
of several cIDPR analogues, furnishes a general and fast method-
ology for: (i) the synthesis of N-1 nucleoside analogues; (ii) the
synthesis of their bis-phosphorylated derivatives; (iii) the intra-
molecular cyclization of bis-phosphate species by pyrophosphate
bond formation. Particular attention has been devoted to the latter
reaction that was carried out both in solid-phase and in solution.
While the solid-phase cyclizations mostly lead to polymeric spe-
cies, due to the formation of intermolecular pyrophosphate bonds,
very high yields of the target cyclic products were obtained by
performing the final cyclization step in solution.
ylcarbodiimide hydrochloride (EDC), generating
a
19-
membered ring.11 Thus, following this approach, the reaction
of 10a–d with bis-cyanoethyl-N,N0-diisopropylphosphoramidite14
(CEO)2PN(iPr)2 in THF, in the presence of tetrazole, furnished
the bis-protected diphosphate derivatives 12a–d (95–98% yield)
after phosphorus oxidation with tert-butylhydroperoxide. The
complete deprotection of both phosphates, achieved by treating
12a–d with conc. aqueous ammonia, furnished supports 13a–
d in almost quantitative yields. The solid-phase phosphorylation
yields, as well as the efficiency of ammonia deprotection of the
phosphotriester groups, were evaluated analyzing the N-1-alkyl-
inosine diphosphate derivatives 15a–d (Table 1) released from
13a–d by aqueous acid treatments (TFA/DCM/H2O). The struc-
tures of 15a–d were confirmed by spectroscopic data after HPLC
purifications. Next, the intramolecular cyclization of supports
13a–d by pyrophosphate bond formation was addressed. We
observed that the previously employed conditions for the cy-
clization of 15c in solution (EDC, 5.0 equiv, N-methylpyrrolidone,
60 h, rt) when applied to solid-supports 13a–d led to very low
yields of cyclic products 18a–d (5–15%) even after prolonged
reaction time (various EDC ratios, solvents and temperatures
were tested). In particular, it was observed that high amounts
(60–70%) of polymeric species were obtained, notwithstanding
the pseudo-diluted conditions characterizing the solid-phase
cyclization. The presence of these species, demonstrated by
HPLC and MS analyses, can be explained by hypothesizing that
the formation of intermolecular pyrophosphate bonds may oc-
cur due to the high functionalization of the support and the
consequent low distance between the reactive inosine di-
phosphate moieties. On the other hand, only a modest in-
creased yield of the cyclic products was obtained (12–22%)
when the cyclization was carried out using an ad hoc synthe-
4. Experimental section
4.1. General
4-Methoxytrityl chloride resin (MMT-Cl, 1% divinylbenzene,
200–400 mesh,1.3 mmol gꢁ1 substitution) was purchased from CBL
Patras, Greece. Anhydrous solvents were used for reactions. All the
other reagents were obtained from commercial sources and were
used without further purification. The reactions on solid phase
were performed using glass columns (10 mm diameter, 100 mm
length) with fused-in sintered glass-disc PO (bore of plug 2.5 mm),
which were shaken on an orbital shaker, or in round bottomed
flasks, when the process required high temperatures. The 1H and
13C NMR spectra were performed on a Varian Mercury Plus
400 MHz using SiMe4 as an internal standard and CD3OD and D2O
as solvents; chemical shifts are reported in parts per million (d)
relative to residual solvent signals: CD2HOD 3.31, HDO 4.80 for 1H
NMR and CD2HOD 49.0 for 13C NMR. 31P-NMR were performed on
a Varian Unity Inova 500 MHz using H3PO4 85% as an external
standard. RP-HPLC analyses and purifications were carried out on
a Jasco UP-2075 Plus pump using a 4.8ꢂ150 mm C-18 reverse-
phase column (particle size 5 mm) eluted with a linear gradient of
CH3CN in H2O (from 0 to 100% in 60 min, flow 1.0 mL minꢁ1, system
A) or with a linear gradient of CH3CN in 0.1 M TEAB (pH 7.0, from
0 to 100% in 120 min, flow 1.0 mL minꢁ1, system B) equipped with
a Jasco UV-2075 Plus UV detector. The UV spectra were recorded on
a Jasco V-530 UV spectrophotometer. IR spectra were recorded on
a Jasco FTIR 430 spectrometer. The High Resolution MS were
recorded on a Bruker APEX II FT-ICR mass spectrometer using
electron spray ionization (ESI) technique. Column chromatography
was performed on silica gel (Merck, Kieselgel 60, 0.063–0.200 mm).
Analytical TLC analyses were performed using F254 silica gel plates
sized support
7 characterized by a severe lowering of the
support functionalization (up to 0.063 mmol/g). Therefore, we
decided to perform the final cyclization step in solution. To this
end, the diphosphate derivatives 15a–d were converted quan-
titatively into the 20,30-di acetyl derivatives 16a–d by reaction
with acetic anhydride in pyridine solution. These compounds

1934
G. Oliviero et al. / Tetrahedron 66 (2010) 1931–1936
(0.2 mm, Merck). TLC spots were detected under UV light (254 nm).
Cationic exchange chromatographies were performed on Dowex
50 W X8 (Naþ form, 50–100 mesh, Fluka).
DMF, was left in contact with the appropriate u-hydroxyalkylamine
(a–d, 5.0 mmol, Table 1) in DMF (1.5 mL) under shaking for 8 h at
50 ꢀC. After filtration and washings with DMF (3ꢂ5 mL), DMF/
MeOH (1:1, v/v, 3ꢂ5 mL) and MeOH (3ꢂ5 mL), supports 10a–d
were dried under reduced pressure. The yields of N-1-alkyl-ino-
sines 11a–d (90–92%, from 9) were calculated by quantization of
the products obtained after HPLC purification (system A, see
General) of the crude nucleoside material released from a weighed
amount of resin by treatment with 2% (v/v) TFA in dry DCM for
8 min at rt followed by DMF washings (collected). 1H NMR spectra
confirmed the purity of the products.
4.1.1. 50-O-TBDPS-20,30-O-(4-hydroxymethyl-(1R)-benzyliden)-in-
osine 6. A mixture of 5 (1.0 g, 3.7 mmol), PTSA (0.19 g, 0.99 mmol),
and 4-(hydroxymethyl)benzaldehyde dimethyl acetal (9.9 mmol,
1.8 g) was suspended in dry DCM (15 mL) and the solution refluxed
for 7 h. The reaction was monitored by TLC (AcOEt/MeOH¼95:5).
After cooling, the solvent was removed under reduced pressure and
the residue was applied on a silica gel column eluted with in-
creasing amounts of MeOH in AcOEt (from 0 to 5%) to afford pure 6
(1.6 g, 70%) as an amorphous white solid; IR nmax (neat) 3500
4.2.5. Solid-phase phosphorylation of supports 10a–d; (supports
12a–d and 13a–d). In a typical experiment, solid support 10a
(0.10 g, 0.070 mmol), swollen in dry THF, was treated with
(CEO)2PN(iPr)2 (0.46 g, 1.7 mmol) and tetrazole (0.24 g, 3.4 mmol)
in dry THF/DCM (9:1, v/v,1.5 mL) and the whole was shaken for 15 h
at rt. The obtained solid support was filtered and washed with THF
(3ꢂ5 mL), THF/MeOH (1:1, v/v, 3ꢂ5 mL) and MeOH (3ꢂ5 mL) and
finally dried in vacuo. Treatment with tert-butylhydroperoxide (5–
6 M in decane, 0.85 mL, 4.8 mmol) in THF (1.5 mL) for 3 h at rt,
followed by washings with THF (3ꢂ5 mL), THF/MeOH (1:1, v/v,
3ꢂ5 mL), and MeOH (3ꢂ5 mL), gave 12a. Successively, support 12a
(0.10 g) was left in to contact with conc. NH4OH for 15 h at 55 ꢀC.
After filtration and washings with H2O (3ꢂ5 mL), H2O/MeOH (1:1,
v/v, 3ꢂ5 mL) and MeOH (3ꢂ5 mL), the resulting support 13a was
dried under reduced pressure. The solid-phase phosphorylation
yields, as well as the efficiency of ammonia deprotection, were
evaluated by analyzing the nucleotide material released from
weighed amounts of 13a–d by treatment with TFA/H2O/DCM
(3:2:95, v/v/v, 15 min, rt). HPLC purifications (system B, see Gen-
eral) furnished the N-1-alkyl-inosine-diphosphate derivatives 15a–
d the structures of which were confirmed by spectroscopic data
(overall yields from 7 in Table 1). 1H NMR and MS data, performed
after cationic exchange and lyophilization (see General), confirmed
the purity and the structure of the products.
(broad, OH), 1690 (carbonyl) cmꢁ1 1H NMR (400 MHz, CD3OD) dH
;
8.20, 7.80 (1H each, s’s, H-8 and H-2), 7.61–7.24 (14H, complex
signals, aromatic of TBDPS), 6.35 (1H, d, J¼1.7 Hz, H-10), 6.01 (1H, s,
CH-benzylidene), 5.52 (1H, dd, J¼6.6, 2.0 Hz, H-20), 5.13 (1H, dd,
J¼6.7, 3.0 Hz, H-30), 4.65 (2H, s, CH2-benzylidene), 4.54 (1H, m,
H-40), 3.87 (1H, dd, J¼11.2, 5.1 Hz, Ha-50), 3.81 (1H, dd, J¼11.2,
5.1 Hz, Hb-50), 0.99 (9H, s, t-But); 13C NMR (100 MHz, CD3OD, as-
signments by a HSQC experiment) dC 20.0 (C-Si), 27.3 (3ꢂCH3, t-
But), 64.8 (CH2Ph), 65.5 (C-50), 83.8 (C-40), 86.4 (C-20), 89.1 (C-30),
91.9 (C-10), 108.8 (CH acetal), 126.2 (C-5), 127.9 (C-2 and C-6 ben-
zylidene),128.2 (C-3 and C-5 benzylidene),128.7,128.8, 130.9,131.0,
134.0, 134.3, 135.7 (aromatic carbons), 136.7 (C-1 benzylidene),
136.7 (aromatic), 141.3 (C-8), 144.7 (C-4 benzylidene), 146.5 (C-2),
149.2 (C-4), 158.8 (C-6); m/z (HRESIMS) 625.2497 ([MþH]þ,
C34H37N4O6Si, requires, 625.2482).
4.2. General procedure for the scaffold synthesis
4.2.1. Loading of 6 on MMT-Cl resin. Solid support 7. A mixture of 6
(1.0 g, 1.6 mmol) and DMAP (0.23 mmol, 0.028 g) co-evaporated
with dry pyridine (3ꢂ1.5 mL) and dissolved in dry pyridine (6 mL)
were added to the MMT-Cl resin (0.9 g, 1.1 mmol) and the whole
was gently shaken for 24 h at rt. The obtained support 7 (inosine
loading 0.72 mmol/g) was filtered and washed with DCM (3ꢂ5 mL),
DCM/MeOH (1:1, v/v, 3ꢂ5 mL) and MeOH (3ꢂ5 mL), and finally
dried under reduce pressure. The reaction yield (96%) was calcu-
lated both measuring the weight increment of the support and
analyzing and quantizing the nucleoside material detached from
the solid support by treatment with 2% TFA in DCM (v/v, 8 min, rt).
4.2.6. Pyrophosphate bond formation on supports 13a–d. In a typi-
cal experiment, support 13a (0.050 g, 0.031 mmol), swollen in dry
DMF, was treated with EDC (0.039 g, 0.20 mmol) and the mixture
was shaken for 72 h at rt. After filtration and washings with DMF
(3ꢂ5 mL), DMF/MeOH (1:1, v/v, 3ꢂ5 mL) and MeOH (3ꢂ5 mL),
support 14a was dried under reduced pressure. The yield of cyclic
compound 18a (22%, from 13a, Table 1) was calculated by UV
quantification of the product obtained after HPLC purification
(system B, see General) of the crude nucleotide material released
from a weighed amount of resin 14a by treatment with TFA/H2O/
DCM (3:2:95, v/v/v, 15 min) at rt. 1H NMR and MS data, performed
after cationic exchange and lyophilization (see General), confirmed
the purity and the structure of the product 18a.
4.2.2. Solid support 8. To the solid support 7 (1.0 g, 0.72 mmol),
suspended in dry MeOH (20 mL), was added NH4F (40 mmol, 1.5 g)
and the mixture was refluxed for 12 h. The resin was filtered and
washed with MeOH (3ꢂ5 mL), DCM/MeOH (1:1, v/v, 3ꢂ5 mL) and
DCM (3ꢂ5 mL) and finally dried in vacuo. The 50-hydroxy-inosine
support was then acetylated by treatment with a solution of acetic
anhydride in pyridine (2:8, v/v, 3 mL, 30 min, rt). After washing
with pyridine (3ꢂ5 mL) and DCM (3ꢂ5 mL), support 8 was dried
under reduced pressure.
4.2.7. Synthesis of compounds 16a–d. N-1-alkyl-inosine di-
phosphate 15a–d (25 mg), were acetylated by reaction with a so-
lution of Ac2O in pyridine (2:8, v/v, 1 mL, 15 h) to give pure 16a–d in
almost quantitative yield. 1H NMR spectra, performed after cationic
exchange and lyophilization (see General) confirmed the structure
of products.
4.2.3. Solid support 9. To the solid support 8 (1.0 g, 0.87 mmol),
suspended in dry DMF (20 mL), K2CO3 (0.9 g, 6.5 mmol) and DNCB
(1.9 g, 6.5 mmol) were added and the mixture kept at 80 ꢀC for 2 h.
After filtration, the support was washed with DMF (3ꢂ5 mL), DMF/
H2O (1:1, v/v, 3ꢂ5 mL), H2O (3ꢂ5 mL), H2O/MeOH (1:1, v/v, 3ꢂ5 mL)
and MeOH (3ꢂ5 mL) and then dried under reduced pressure to give
support 9 (0.76 mmol/g). The reaction yield (94%) was evaluated by
detaching 1-(2,4-dinitrophenyl)inosine13 under acid hydrolytic
conditions (TFA/H2O/DCM (3:2:95, v/v/v, 15 min, rt)).
4.2.8. Pyrophosphate bond formation in solution; cyclic compounds
18a–d. In
a typical experiment, to compound 16a (10 mg,
0.018 mmol), dissolved in dry DMF (1.5 mL), EDC (17.2 mg,
0.090 mmol) was added and the solution was shaken for 72 at rt.
The DMF was evaporated under reduced pressure and the crude
mixture, dissolved in water, was purified by HPLC (system B, see
General) to give 17a (8.6 mg, 89%). The product was treated with
4.2.4. Treatment of solid support 9 with
u-hydroxyalkylamines a–d;
(supports 10a–d). Solid support 9 (0.10 g, 0.076 mmol), swollen in

G. Oliviero et al. / Tetrahedron 66 (2010) 1931–1936
1935
conc. NH4OH for 5 h at rt and then dried under reduced pressure to
give 18a in almost quantitative yield.
The structure of 18a, converted in sodium salt by cationic
exchange chromatography (see General), was confirmed by 1H and
31P NMR spectroscopy and HRESIMS data.
3.87 (2H each, m’s, CH2O and Ha,b-50), 1.87 (2H, m, CH2), 1.67 (2H, m,
CH2); 31P-NMR (202 MHz, D2O) dP 0.51, ꢁ0.20 (s’s); m/z (HRESIMS)
499.0642 ([MꢁH]ꢁ, C14H21N4O12P2, requires 499.0631).
4.2.15. 1-N-(5-O-Phosphorylpentyl)-50-O-phosphorylinosine
15c. Amorphous solid, sodium salt; IR nmax (neat) 3600–2600
(broad, OH’s), 1678 (strong, carbonyl), 1056, 915 (both strong,
phosphates) cmꢁ1; 1H NMR (400 MHz, D2O) dH 8.59, 8.42 (1H each,
s’s, H-8 and H-2), 6.14 (1H, d, J¼5.2 Hz, H-10), 4.76 (1H, m, H-20), 4.52
(1H, m, H-30), 4.38 (1H, m, H-40), 4.16 (2H, m, CH2N); 4.04, 3.81 (2H
each, m’s, CH2O and Ha,b-50),1.81 (2H, m, CH2),1.59 (2H, m, CH2),1.44
(2H, m, CH2); 31P-NMR (202 MHz, D2O) dP 0.42, ꢁ0.16 (s’s); m/z
(HRESIMS) 513.0829 ([MꢁH]ꢁ, C15H23N4O12P2, requires 513.0788).
4.2.9. 1-N-(2-Hydroxyethyl)-20,30-O-(4-hydroxymethyl-(1R)-benzyli-
den)inosine 11a. Colorless oil; IR nmax (neat) 3600–2600 (broad,
OH’s), 1670 (strong, carbonyl) cmꢁ1; 1H NMR (400 MHz, CD3OD) dH
8.26, 8.23 (1H each, s’s, H-8 and H-2), 7.40 (2H, d, J¼8.0 Hz, aro-
matic), 7.29 (2H, d, J¼8.0, aromatic), 6.23 (1H, d, J¼2.9 Hz, H-10),
6.13 (1H, s, CHPh), 5.31 (1H, dd, J¼6.4, 2.9 Hz, H-20), 5.07 (1H, dd,
J¼6.4, 4.0 Hz, H-30), 4.53 (2H, s, CH2Ph), 4.31 (1H, m, H-40), 4.11
(2H, t, J¼4.8 Hz, CH2N), 3.75–3.72 (4H, m, CH2O, Ha,b-50), m/z
(HRESIMS) 431.1555 ([MþH]þ, C20H23N4O7, requires, 431.1567).
4.2.16. 1-N-(6-O-Phosphorylhexyl)-50-O-phosphorylinosine
15d. Amorphous solid, sodium salt; IR nmax (neat) 3600–2600
(broad, OH’s), 1677 (strong, carbonyl), 1056, 915 (both strong,
phosphates) cmꢁ1; 1H NMR (400 MHz, D2O) dH 8.52, 8.40 (1H each,
s’s, H-8 and H-2), 6.10 (1H, d, J¼5.2 Hz, H-10), 4.73 (1H, m, H-20),
4.52 (1H, m, H-30), 4.39 (1H, m, H-40), 4.19 (2H, t, J¼6.2 Hz, CH2N),
4.06, 3.82 (m’s, 2H each, CH2O and Ha,b-50), 1.82 (2H, m, CH2), 1.62
(2H, m, CH2), 1.41 (4H, m’s, 2ꢂCH2); 31P-NMR (202 MHz, D2O) dP
0.46, ꢁ0.17 (s’s); m/z (HRESIMS) 527.0932 ([MꢁH]ꢁ, C16H25N4O12P2,
requires 527.0944).
4.2.10. 1-N-(4-Hydroxybutyl)-20,30-O-(4-hydroxymethyl-(1R)-benzy-
liden)inosine 11b. Colorless oil; IR nmax (neat) 3600–2600 (broad,
OH’s), 1672 (strong, carbonyl) cmꢁ1; 1H NMR (400 MHz, CD3OD) dH
8.26 (2H, br s, H-8 and H-2), 7.47 (2H, d, J¼8.1 Hz, aromatic), 7.33
(2H, d, J¼8.1 Hz, aromatic), 6.24 (1H, d, J¼2.9 Hz, H-10), 5.93 (1H, s,
CHPh), 5.31 (1H, dd, J¼6.2, 2.8 Hz, H-20), 5.02 (1H, dd, J¼6.5, 2.3 Hz,
H-30), 4.55 (2H, s, CH2Ph), 4.42 (1H, m, H-40), 4.04 (2H, t, J¼7.3 Hz,
CH2N), 3.67 (m, ABX system, J¼10.4, 4.2 Hz, 2H, Ha,b-50), 3.49 (2H, t,
J¼6.4 Hz, CH2O), 1.75 (2H, m, CH2), 1.48 (2H, m, CH2); m/z (HRE-
SIMS) 459.1842 ([MþH]þ, C22H27N4O7, requires, 459.1880).
4.2.17. 1-N-(2-O-Phosphorylethyl)-20,30-di-O-acetyl-50-O-phosphor-
ylinosine 16a. Amorphous solid, sodium salt; IR nmax (neat)
3600–2600 (broad), 1742 (strong, carbonyl acetate), 1687 (strong,
4.2.11. 1-N-(5-Hydroxypentyl)-20,30-O-(4-hydroxymethyl-(1R)-ben-
zyliden)inosine 11c. Amorphous solid; IR nmax (neat) 3600–2600
carbonyl), 1215 (acetate), 1056, 915 (both strong, phosphates) cmꢁ1
;
(broad, OH’s), 1670 (strong, carbonyl) cmꢁ1
;
1H NMR (400 MHz,
1H NMR (400 MHz, D2O) dH 8.52, 8.41, (s’s, 1H each, H-8 and H-2),
6.31 (1H, d, J¼5.2 Hz, H-10), 5.77 (1H, m, H-20), 5.65 (1H, m, H-30),
4.60 (1H, m, H-40), 4.26 (2H, m, CH2N), 4.12, 3.93 (2H each, m’s, CH2O
and Ha,b-50), 2.20 (3H, s, CH3), 2.11 (3H, s, CH3); 31P-NMR (202 MHz,
D2O) dP 0.46, ꢁ0.15 (s’s); m/z (HRESIMS) 555.0567 ([MꢁH]ꢁ,
C16H21N4O14P2, requires 555.0529).
CD3OD) dH 8.35, 8.34 (s’s, 1H each, H-8 and H-2), 7.57 (2H, d,
J¼8.1 Hz, aromatic), 7.43 (2H, d, J¼8.1 Hz, aromatic), 6.33 (1H, d,
J¼2.93 Hz, H-10), 6.03 (1H, s, CHPh), 5.41 (1H, dd, J¼6.4, 2.9 Hz,
H-20), 5.12 (1H, dd, J¼6.4, 2.5 Hz, H-30), 4.65 (2H, s, CH2Ph), 4.51 (1H,
m, H-40), 4.11 (2H, t, J¼7.2 Hz, CH2N), 3.77 (m, ABX system, J¼10.4,
4.1 Hz, 2H, Ha,b-50), 3.55 (2H, t, J¼6.3 Hz, CH2O), 1.80 (2H, m, CH2),
1.59 (2H, m, CH2), 1.43 (2H, m, CH2); m/z (HRESIMS) 473.2054
([MþH]þ, C23H29N4O7, requires, 473.2036).
4.2.18. 1-N-(4-O-Phosphorylbutyl)-20,30-di-O-acetyl-50-O-phosphor-
ylinosine 16b. Amorphous solid, sodium salt; IR nmax (neat) 3600–
2600 (broad), 1740 (strong, carbonyl acetate), 1687 (strong, carbonyl),
4.2.12. 1-N-(6-Hydroxyhexyl)-20,30-O-(4-hydroxymethyl-(1R)-benzy-
1215 (acetate), 1056, 915 (both strong, phosphates) cmꢁ1 1H NMR
;
liden)inosine 11d. Amorphous solid; IR nmax (neat) 3600–2600
(400 MHz, D2O) dH 8.45, 8.41 (1H each, s’s, H-8 and H-2), 6.36 (1H, d,
J¼5.6 Hz, H-10), 5.79 (1H, m, H-20), 5.65 (1H, m, H-30), 4.61 (1H, m, H-40),
4.27 (2H, m, CH2N), 4.15, 3.98 (2H each, m’s, CH2O and Ha,b-50), 2.18 (3H,
s, CH3), 2.09 (3H, s, CH3), 1.87 (2H, m, CH2), 1.68 (2H, m, CH2); 31P-NMR
(202 MHz, D2O) dP 0.48, ꢁ0.16 (s’s); m/z (HRESIMS) 583.0883 ([MꢁH]ꢁ,
C18H25N4O14P2, requires 583.0842).
(broad, OH’s), 1670 (strong, carbonyl) cmꢁ1 1H NMR (400 MHz,
;
CD3OD) dH 8.26 (br s, 2H each, H-8 and H-2), 7.42 (2H, d, J¼8.0 Hz,
aromatic), 7.31 (2H, d, J¼8.0 Hz, aromatic), 6.24 (1H, d, J¼2.8 Hz,
H-10), 6.14 (1H, s, CHPh), 5.33 (1H, m, H-20), 5.08 (1H, dd, J¼6.4,
4.1 Hz, H-30), 4.55 (2H, s, CH2Ph), 4.33 (1H, m, H-40), 4.03 (2H, t,
J¼7.5 Hz, CH2N), 3.77–3.65 (2H, m, Ha,b-50), 3.46 (2H, t, J¼6.4 Hz,
CH2O), 1.71 (2H, m, CH2), 1.46 (2H, m, CH2), 1.34 (4H, m, 2ꢂCH2); m/
z (HRESIMS) 487.2234 ([MþH]þ, C24H31N4O7, requires, 487.2193).
4.2.19. 1-N-(5-O-Phosphorylpentyl)-20,30-di-O-acetyl-50-O-phos-
phorylinosine 16c. Amorphous solid, sodium salt; IR nmax (neat)
3600–2600 (broad), 1740 (strong, carbonyl acetate), 1687 (strong,
4.2.13. 1-N-(2-O-Phosphorylethyl)-50-O-phosphorylinosine
15a. Amorphous solid, sodium salt; IR nmax (neat) 3600–2600
(broad, OH’s), 1678 (strong, carbonyl), 1056, 915 (both strong,
carbonyl),1215 (acetate),1056, 915 (both strong, phosphates) cmꢁ1
;
1H NMR (400 MHz, D2O) dH 8.55, 8.41 (1H each, s’s, H-8 and H-2),
6.33 (1H, d, J¼5.2 Hz, H-10), 5.76 (1H, m, H-20), 5.63 (1H, m, H-30),
4.60 (1H, m, H-40), 4.28 (2H, m, CH2N), 4.16, 4.00 (2H each, m’s,
CH2O and Ha,b-50), 2.16 (3H, s, CH3), 2.08 (3H, s, CH3), 1.86 (2H, m,
CH2), 1.61 (2H, m, CH2), 1.44 (2H, m, CH2); 31P-NMR (202 MHz, D2O)
dP 0.39, ꢁ0.18 (s’s); m/z (HRESIMS) 597.1024 ([MꢁH]ꢁ,
C19H27N4O14P2, requires 597.0999).
phosphates) cmꢁ1 1H NMR (400 MHz, D2O) dH 8.55, 8.46, (s’s, 1H
;
each, H-8 and H-2), 6.16 (1H, d, J¼4.8 Hz, H-10), 4.78 (1H, covered by
solvent signal, m, H-20), 4.45 (1H, m, H-30), 4.32 (3H, m, CH2N and
H-40), 4.04 (4H, m, CH2O and Ha,b-50); 31P-NMR (202 MHz, D2O) dP
0.40, ꢁ0.11 (s’s); m/z (HRESIMS) 471.0359 ([MꢁH]ꢁ, C12H17N4O12P2,
requires 471.0318).
4.2.20. 1-N-(6-O-Phosphorylhexyl)-20,30-di-O-acetyl-50-O-phosphor-
ylinosine 16d. Amorphous solid, sodium salt; IR nmax (neat) 3600–
2600 (broad), 1742 (strong, carbonyl acetate), 1687 (strong, car-
bonyl),1215 (acetate),1056, 915 (both strong, phosphates) cmꢁ1; 1H
NMR (400 MHz, D2O) dH 8.54, 8.41(1H each s’s, H-8 and H-2), 6.29
(1H, d, J¼5.4 Hz, H-10), 5.80 (1H, m, H-20), 5.63 (1H, m, H-30), 4.59
(1H, m, H-40), 4.26 (2H, m, CH2N), 4.16, 3.98 (2H each, m’s, CH2O and
4.2.14. 1-N-(4-O-Phosphorylbutyl)-50-O-phosphorylinosine
15b. Amorphous solid, sodium salt; IR nmax (neat) 3600–2600
(broad, OH’s), 1677 (strong, carbonyl), 1056, 915 (both strong,
phosphates) cmꢁ1; 1H NMR (400 MHz, D2O) dH 8.44, 8.40 (1H each,
s’s, H-8 and H-2), 6.10 (1H, d, J¼5.1 Hz, H-10), 4.74 (1H, m, H-20), 4.47
(1H, m, H-30), 4.35 (1H, m, H-40), 4.16 (2H, t, J¼6.3 Hz, CH2N), 4.08,

1936
G. Oliviero et al. / Tetrahedron 66 (2010) 1931–1936
Ha,b-50), 2.16 (3H, s, CH3), 2.06 (3H, s, CH3), 1.86 (2H, m, CH2), 1.65
(2H, m, CH2),1.36 (4H, m, 2ꢂCH2); 31P-NMR (202 MHz, D2O) dP 0.45,
ꢁ0.21 (s’s); m/z (HRESIMS) 611.1116 ([MꢁH]ꢁ, C20H29N4O14P2, re-
quires 611.1155).
carbonyl), 1227, 1120, 1052, 935 (all strong, pyrophosphate) cmꢁ1; 1H
NMR (400 MHz, D2O) dH 8.55, 8.45 (1H each, s’s H-2 and H-8), 6.16
(1H, d, J¼5.1 Hz, H-10), 5.43 (1H, m, H-20), 4.74 (1H, m, H-30), 4.53
(1H, m, Ha-50), 4.41 (1H, m, H-40), 4.20 (2H, m, CH2N), 4.05 (1H, m,
Hb-50), 3.83 (2H, m, CH2O), 1.80,1.71, (2H each, m’s, 2ꢂCH2), 1.41 (4H,
m, 2ꢂCH2); 31P NMR (202 MHz, D2O) dP ꢁ9.6, ꢁ10.4; m/z (HRESIMS)
509.0860 ([MꢁH]ꢁ, C16H23N4O11P2, requires 509.0839).
4.2.21. 1-N-Ethyl-cyclic inosine-diphosphate 18a. Amorphous solid,
sodium salt; IR nmax (neat) 3600–2600 (broad, OH’s), 1678 (strong,
carbonyl), 1227, 1120, 1052, 935 (all strong, pyrophosphate) cmꢁ1
;
1H NMR (400 MHz, D2O) dH 8.50, 8.40 (1H each, s’s, H-8 and H-2),
6.13 (1H, d, J¼5.1 Hz, H-10), 5.34 (1H, m, H-20), 4.42 (1H, m, Ha-50),
4.78 (1H, m, H-30), 4.30 (1H, m, H-40), 4.27–4.13 (2H, m, CH2N), 4.05
(1H, m, Hb-50), 3.66 (2H, m, CH2O); 31P NMR (202 MHz, D2O) dP
ꢁ9.4, ꢁ10.5; m/z (HRESIMS) 453.0225 ([MꢁH]ꢁ, C12H15N4O11P2,
requires 453.0213).
References and notes
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J. Org. Chem. 2000, 65, 5238–5248.
4.2.22. 1-N-Butyl-cyclic inosine–diphosphate 18b. Amorphous solid,
sodium salt; IR nmax (neat) 3600–2600 (broad, OH’s), 1680 (strong,
carbonyl),1227,1120,1052, 935 (all strong, pyrophosphate) cmꢁ1; 1H
NMR (400 MHz, D2O) dH 8.43, 8.19 (1H each, s’s, H-8 and H-2), 6.03
(1H, d, J¼3.4 Hz, H-10), 5.41 (1H, m, H-20), 4.75, (1H, m, H-30), 4.39
(1H, m, Ha-50), 4.32 (1H, m, H-40), 4.24–4.10 (2H, m, CH2N), 4.03 (1H,
m, Hb-50), 3.62 (2H, m, CH2O), 1.80, 1.50 (2H each, m’s, 2ꢂCH2); 31P
NMR (202 MHz, D2O) dP ꢁ9.7, ꢁ10.4; m/z (HRESIMS) 481.0521
([MꢁH]ꢁ, C14H19N4O11P2, requires 481.0526).
7. Huang, L.-J.; Zhao, Y. Y.; Yuan, L.; Min, J.-M.; Zhang, L.-H. J. Med. Chem. 2002, 45,
5340–5352.
8. Huang, L.-J.; Zhao, Y. Y.; Yuan, L.; Min, J.-M.; Zhang, L.-H. Bioorg. Med. Chem.
2002, 12, 887–889.
9. (a) Epple, R.; Kudirka, R.; Greenberg, W. A. J. Comb. Chem. 2003, 5, 292–310; (b)
Bozzoli, A.; Kazmierski, W.; Kennedy, G.; Pasquarello, A.; Pecunioso, A. Bioorg.
Med. Chem. Lett. 2000, 10, 2759–2763; (c) Palom, Y.; Alazzouzi, E.; Gordillo, F.;
Grandas, A.; Pedroso, E. Tetrahedron Lett. 1993, 34, 2195–2198.
10. De Napoli, L.; Messere, A.; Montesarchio, D.; Piccialli, G. J. Org. Chem. 1995, 60,
2251–2253.
11. Galeone, A.; Mayol, L.; Oliviero, G.; Piccialli, G.; Varra, M. Eur. J. Org. Chem. 2002,
4234–4238.
12. Terrazas, M.; Ariza, X.; Farra`s, J.; Vilarrasa, J. Chem. Commun. 2005, 3968–3970.
13. Oliviero, G.; Amato, J.; Borbone, N.; D’Errico, S.; Piccialli, G.; Mayol, L. Tetrahe-
dron Lett. 2007, 48, 397–400.
4.2.23. 1-N-Pentyl-cyclic inosine-diphosphate 18c. Amorphous solid,
sodium salt; IR nmax (neat) 3600–2600 (broad, OH’s), 1680 (strong,
carbonyl),1227,1120,1052, 935 (all strong, pyrophosphate) cmꢁ1; 1H
NMR (400 MHz, D2O) dH 8.42, 8.22 (1H each, s’s, H-8 and H-2), 6.05
(1H, d, J¼3.5 Hz, H-10), 5.40 (1H, m, H-20), 4.76 (1H, m, H-30), 4.50
(1H, m, Ha-50), 4.38 (1H, m, H-40), 4.24 (2H, m, CH2N), 4.03 (1H, m,
Hb-50), 3.82 (2H, m, CH2O), 1.84, 1.58, 1.30 (2H each, m’s, 3ꢂCH2). 31P
NMR (202 MHz, D2O) dP ꢁ9.5, ꢁ10.5; m/z (HRESIMS) 495.0654
([MꢁH]ꢁ, C15H21N4O11P2, requires 495.0682).
4.2.24. 1-N-Hexyl-cyclic inosine-diphosphate 18d. Amorphous solid,
sodium salt; IR nmax (neat) 3600–2600 (broad, OH’s), 1679 (strong,
14. Uhlmann, E.; Engels, J. Tetrahedron Lett. 1996, 27, 1023–1026.