Synthesis of a new N1-pentyl analogue of cyclic inosine diphosphate ribose (cIDPR) as a stable potential mimic of cyclic ADP ribose (cADPR)

Article abstract of DOI:10.1002/1099-0690(200212)2002:24<4234::AID-EJOC4234>3.0.CO;2-8

The new analogue 7 of cADPR (1), a cyclic nucleotide bis(phosphate) involved in Ca²⁺ metabolism, was prepared starting from 2′,-3′-isopropylideneinosine (8) which was alkylated at N-1, leading to the intermediate 11. Bis(phosphorylation) of 11 through two alternative procedures, followed by phosphate deprotection steps, afforded derivatives 15 and 16, the substrates for the intramolecular pyrophosphate bond formation. Both 15 and 16 were converted into derivative 17 in high yields, which was finally deprotected to give the target compound 7. Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2002.

Full text of DOI:10.1002/1099-0690(200212)2002:24<4234::AID-EJOC4234>3.0.CO;2-8

FULL PAPER
Synthesis of a New N1-Pentyl Analogue of Cyclic Inosine Diphosphate Ribose
(cIDPR) as a Stable Potential Mimic of Cyclic ADP Ribose (cADPR)
Aldo Galeone,[a] Luciano Mayol,*[a] Giorgia Oliviero,[a] Gennaro Piccialli,[a] and
Michela Varra[a]
Keywords: cADPR analogues / cIDPR / Nucleotides
The new analogue 7 of cADPR (1), a cyclic nucleotide bi-
s(phosphate) involved in Ca2+ metabolism, was prepared
16, the substrates for the intramolecular pyrophosphate bond
formation. Both 15 and 16 were converted into derivative 17
starting from 2Ј,3Ј-isopropylideneinosine (8) which was al- in high yields, which was finally deprotected to give the tar-
kylated at N-1, leading to the intermediate 11. Bis(phosphor- get compound 7.
ylation) of 11 through two alternative procedures, followed ( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
by phosphate deprotection steps, afforded derivatives 15 and
Germany, 2002)
Introduction
Cyclic-ADP ribose (cADPR, 1, Scheme 1), a naturally
occurring compound related to NADϩ, has been shown to
be a general mediator involved in Ca2ϩ signalling and in-
tracellular mobilization in various cells.[1] This cyclic nucle-
otide is characterized by a very labile glycosidic bond to N-
1 which is rapidly hydrolyzed both enzymatically, by cADP
hydrolase, and nonenzymatically, to give ADP-ribose even
in a neutral aqueous solution.[2,3] This biological and chem-
ical instability deeply hinders further studies on cADPR
aimed at elucidating its physiological role, particularly as
far as the regulation of Ca2ϩ mobilization in the cells is
concerned. Hence, stable, yet active, cADPR analogues give
rise to great interest in this field.
Several enzymes involved in the metabolism of cADPR
have been described, among which is the ubiquitous ADP-
ribosyl cyclase, first discovered in sea urchin eggs and par-
ticularly abundant in Aplysia california.[4] The Aplysia cy-
clase shows a significant catalytic activity in the cyclization
of NADϩ and NADPϩ to cADPR and has been utilized to
produce new analogues of the natural metabolite from their
linear counterparts.[5Ϫ8] However, the enzyme specificity se-
verely limits the applicability of enzymatic or chemo-en-
zymatic procedures. Further analogues have been synthe-
sized and their biological properties investigated.[9Ϫ12] Some
of these analogues are by far more stable to hydrolysis than
Scheme 1
the natural metabolite and exhibit interesting biological ac- of one or two atoms of the original metabolite, thus pre-
tivity. This is the case, for example, in compounds 2Ϫ5 venting substantial conformational modifications. Particu-
where the structural changes are limited to the substitution larly in compound 5 (cIDPcR), a stable mimic of cADPR
synthesized by Matsuda and co-workers,[10] the adenine
base is replaced by the structurally related hypoxanthine
whereas a carbocyclic moiety is linked at N-1. In the same
paper, a general method for the chemical synthesis of new
cADPR analogues is supplied through an extensive study
[a]
Dipartimento di Chimica delle Sostanze Naturali,
Via D. Montesano 49, 80131 Napoli, Italy
Fax: (internat.) ϩ 39-081/678552
E-mail: mayoll@unina.it
4234
 2002 WILEY-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim 1434Ϫ193X/02/1224Ϫ4234 $ 20.00ϩ.50/0 Eur. J. Org. Chem. 2002, 4234Ϫ4238

A New N1-Pentyl Analogue of Cyclic Inosine Diphosphate Ribose
FULL PAPER
of the pyrophosphate bond formation involved in the cyc-
lization step.
Recently, we reported the synthesis of a new product,
6,[13] with more severe structural changes than the previ-
ously reported cADPR analogues. This compound, de-
signed to investigate the role of the N-9-attached ribosyl
moiety in the mechanism of Caϩ2 intracellular modulation,
displays a carbaribosyl and a butyl moiety at N-1 and N-9
of a hypoxanthine base, respectively. We wish to report here
the synthesis of a new cIDPR congener 7 having a pentyl
chain at N-1 of a hypoxanthine base, which is expected to
be resistant to both enzymatic and chemical hydrolysis as
similar N1-alkylated analogues.[14]
Results and Discussion
The synthetic route adopted for 7 (Scheme 2) shows three
main steps: i) introduction of the 5-hydroxypentyl chain on
N-1 of the protected inosine 9 which leads to 11; ii) bis(pho-
sphorylation) of derivative 11 through two alternative pro-
cedures to give 12 or 14, the direct precursors of 15 and
16, respectively; iii) cyclization of derivatives 15 or 16 by
pyrophosphate bond formation. For the N1-alkylation of
inosine we utilized the already described procedure based
on the reaction of N1-(2,4-dinitrophenyl)inosine with al-
kylamine.[15] Thus, acetylation of 2Ј,3Ј-isopropylideneinos-
ine (8) with Ac2O in pyridine led to the 5Ј-O-acetyl derivat-
ive 9[16] which was, in turn, converted into the N1-(2,4-di-
nitrophenyl) derivative 10 by reaction with 2,4-dinitrochlo-
robenzene and K2CO3 in DMF (90% yields). Compound
Scheme 2. Reagents and conditions: a) Ac2O, pyridine, room temp.,
6 h; b) 1-chloro-2,4-dinitrobenzene, K2CO3, DMF, 80 °C, 2.5 h; c)
5-aminopentan-1-ol, 80 °C, 6 h; d) (PhNH)2POCl (excess), pyrid-
ine, room temp., ca. 12 h; e) (PhNH)2POCl, pyridine, room temp.,
ca. 12 h; f) S,SЈ-diphenyl dithiophosphate, 1H-tetrazole, TPSCl,
pyridine, N2, room temp., 8 h; g) isoamyl nitrite, pyridine/AcOH/
˚
Ac2O, room temp., 8 h; h) 15, I2, pyridine, 3-A molecular sieves,
room temp., 15 h; i) 16, EDC, MPD, room temp.; 60 h; l) aq.
HCO2H, room temp., 3.5 h
10 was obtained as a 1:1 mixture of atropisomers at the yield). Both 15 and 16, the substrates for the intramolecular
N-1 position.[15] Treatment of 10 with 5-aminopentan-1-ol condensation, were purified by HPLC on a C18 reversed-
afforded the N1-(5-hydroxypentyl)inosine derivative 11 phase column and characterized as triethylammonium salts
(85% yield), with the concomitant aminolysis of the 5Ј-O- by NMR and MS data. Particularly, the proton-decoupled
acetyl protecting group. The formation of the N1-alkylated 31P NMR spectrum of 15 showed two singlets at δ ϭ 2.3
derivative 11 proceeds through a rearrangement of the pyri- and 18.9 ppm attributed to the phosphate and phenylthi-
midine ring, induced by nucleophilic attack of the amine at ophosphate groups, respectively. As expected, in the 31P
C-2, whose electrophilicity was enhanced by the presence NMR spectrum of 16 two close signals at δ ϭ 0.4 and
of the 2,4-dinitrophenyl group at N-1. Compound 11, after Ϫ0.1 ppm were present, pertinent to the phosphomonoes-
purification, underwent two distinct phosphorylation pro- ter functions.
cedures leading to the bis(phosphate) derivatives 12 and 14.
Two alternative intramolecular condensation reactions
Thus, reaction of 11 with a 1.2 molar excess of N,NЈ-di- were performed to cyclize derivatives 15 and 16. In the for-
phenylphosphorodiamidic chloride[17] afforded a mixture of mer method 15, dissolved in pyridine, was added over 15 h
monophosphate and bis(phosphate) derivatives 13 and 12 to I2, dissolved in the same solvent, in the presence of activ-
[21]
˚
in a 9:1 ratio, which were separated by silica gel chromato- ated molecular sieves (3 A). HPLC purification on a C18
graphy. Treatment of 13 with S,SЈ-diphenyl dithiophosphate reversed-phase column furnished the cyclic product 17 (60%
as a cyclohexylammonium salt,[18] in the presence of 2,4,6- isolated yield). In the latter case the intramolecular con-
triisopropylbenzenesulfonyl chloride (TPSCl) and 1H-tetra- densation reaction of the two phosphate groups of 16 was
zole[19] gave the protected bis(phosphate) 14 (80% yield). performed by addition of a slight excess of 1-[3-(dimethyl-
Alternatively, 11 treated with a 3.5 molar excess of amino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) in
(PhNH)2POCl furnished the bis(phosphate) 12 (85% yield). N-methylpyrrolidone (MPD) and allowing the mixture to
Deprotection of the phosphate groups in 14 was achieved stand at 50 °C for 60 h. The desired cyclic product 17 was
by sequential treatments with isoamyl nitrite in pyridine/ obtained in 80% yield after HPLC purification.
[19]
AcOH/Ac2O (2:1:1, v/v)[20] and H3PO2
in pyridine af-
In the light of the literature data, the above results are
fording compound 15 (75% yield). The complete deprotec- rather surprising. Although a number of well-established
tion of both phosphate groups in 12, achieved by treatment procedures have been described for the formation of a pyro-
with isoamyl nitrite in pyridine/AcOH/Ac2O, led to 16 (85% phosphate bridge, most of the available data refer to inter-
Eur. J. Org. Chem. 2002, 4234Ϫ4238
4235

A. Galeone, L. Mayol, G. Oliviero, G. Piccialli, M. Varra
FULL PAPER
references. 31P NMR spectra were recorded with a Bruker WM 400
spectrometer (85% H3PO4 as an external standard). NMR signals
were assigned to the pertinent nuclei through two-dimensional 1H-
1H and 1H-13C COSY experiments. Mass spectra were registered
with a Finnigan MAT instrument. General reagents and solvents
were purchased from Sigma-Aldrich-Fluka. UV measurements
were carried out with a Jasco V-530 UV spectrophotometer. HPLC
molecular reactions with only a few papers dealing with
cyclization processes involving an intramolecular condensa-
tion of two phosphate groups. From these data, it was ap-
parent that the following factors play a major role in regu-
lating the course of the reaction: i) the size of the cycle; ii)
the conformation adopted by the linear precursor; iii) the
electrostatic repulsion between the two phosphate species to
be condensed. Studies on the synthesis of compounds 4 and
5 seem to indicate the electrostatic repulsion as the domin-
purifications were performed on
a Nucleosil C18 column
(MachereyϪNagel, 250/10, 7 µm). The following abbreviations
were used throughout the text: TPSCl ϭ 2,4,6-triisopropylbenzene-
ant factor in determining the cyclization yields. In fact, very sulfonyl chloride, EDC ϭ 1-[3-(dimethylamino)propyl]-3-ethylcar-
bodiimide hydrochloride, MPD ϭ N-methylpyrrolidone, DMF ϭ
N,N-dimethylformamide, TEAA triethylammonium acetate,
low (23%) or not detectable yields are reported for the pre-
paration of 5 through the cyclization with EDC of the lin-
ear precursors possessing favorable or nonfavorable con-
formational requisites (syn or anti conformation around the
glycosidic bond, respectively). On the other hand, very high
yields were observed if the cyclization was performed min-
imizing the charge repulsion between the two phosphates,
independently from the conformation. On the contrary, our
finding suggests that the electrostatic repulsion is not so
crucial, at least in the cyclization of linear precursors char-
acterized by high conformational flexibility, as in the case
of 15 and 16. In fact, in the procedure adopted for com-
pound 15, neutral, reactive metaphosphate species are gen-
erated, whereas cyclization of 16 involves two charged phos-
phomonoester functions, one of them activated by EDC.
Nevertheless, a higher yield for the cyclization is observed
for 16 than for 15.
ϭ
TEAB ϭ triethylammonium bicarbonate, DNP ϭ 2,4-dinitro-
phenyl, s’s ϭ singlets, d’s ϭ doublets, m’s ϭ multiplets, q’s ϭ quad-
ruplets.
5Ј-O-Acetyl-2Ј,3Ј-O-isopropylideneinosine (9): Commercially avail-
able 2Ј,3Ј-O-isopropylideneinosine (8) (1.0 g, 3.25 mmol) was
treated with Ac2O/pyridine solution (6:4 v/v, 10 mL). After 6 h at
room temperature, the dried mixture was dissolved in CHCl3 and
washed with water. The dried organic layer gave 9[16] (1.08 g, 95%)
as a white solid.
5Ј-O-Acetyl-1-(2,4-dinitrophenyl)-2Ј,3Ј-O-isopropylideneinosine (10):
A mixture of 9 (1.0 g, 2.86 mmol), 1-chloro-2,4-dinitrobenzene
(1.15 g, 5.72 mmol) and K2CO3 (790 mg, 5.72 mmol) was stirred in
anhydrous DMF (8.0 mL) at 80 °C for 2.5 h. After cooling, the
mixture was filtered and the solid was washed with CHCl3. The
filtrate and washings, concentrated to dryness under reduced pres-
sure, were purified on a silica gel column eluted with an increasing
amount of CH3OH in CHCl3 (from 0 to 5%) to give 10 (1.33 g,
90%) as a 1:1 mixture of atropisomers at the N-1Ϫphenyl bond as
a pale yellow amorphous solid. M.p. 204Ϫ207 °C (from CH3OH).
1H NMR (CDCl3): δ ϭ 9.07 (s, 1 H, 3-H DNP), 8.65 (d, J ϭ
9.0 Hz, 1 H, 5-H DNP), 8.04, 8.03, 7.98, 7.95 (s’s, 0.5 H each, 2-H
and 8-H), 7.72 and 7.70 (d’s, 0.5 H, each J ϭ 9.0 Hz, 6-H DNP),
6.12 and 6.14 (s’s, 0.5 H each, 1Ј-H), 4.95 (m, 1 H, 3Ј-H), 4.56 and
4.51 (m’s, 0.5 H each, 4Ј-H), 4.34 and 4.26 (m’s, 1 H each, 5Ј-H2),
2.27 (m, 1 H, 2Ј-H), 2.05 (s, 3 H, CH3CO) 1.63 and 1.40 (s’s, 3 H
each, isopropylidene) ppm. 13C NMR (CDCl3): δ ϭ 170.1, 158.0,
152.8, 151.4, 149.9, 144.9, 143.0, 142.2, 129.1, 126.7, 121.9, 121.8,
114.7, 91.3, 84.9, 84.2, 81.3, 63.8, 27.0, 25.1, 20.4 ppm. ESI-MS:
calcd. for C21H20N6O10 516.12, found 517 [M ϩ H]ϩ. UV (CHCl3):
λmax ϭ 249 nm.
The structure of 17 was confirmed by 1H, 13C, 31P NMR
and ESI-MS data. Particularly, the 31P NMR spectrum
showed two broad singlets at δ ϭ Ϫ10.4 and Ϫ11.5 ppm,
which are diagnostic for a pyrophosphate moiety.
Final removal of the isopropylidene protecting group,
performed by treating 17 with aqueous HCO2H at room
temperature for 3.5 h, furnished 7 (85%, after purification).
Product 7 was purified by HPLC on a C18 reversed-phase
column and its structure was confirmed by 1H and 31P
NMR and MS data.
In conclusion, we have reported the high-yield synthesis
of a new stable analogue 7 of the cIDPR carrying a pentyl
chain at N-1 of inosine, which substitutes the ribosyl moi-
ety. In the synthesis of this compound, characterized by
high conformational flexibility, we tested two condensation
procedures on the linear bis(phosphate) derivatives 15 and
16 to obtain the closure of a 19-membered ring, by a pyro-
phosphate bond formation. The surprisingly and unpreced-
ented high cyclization yields observed using EDC as a con-
densing agent of the two phosphomonoester functions of
1-(5-Hydroxypentyl)-Substituted Compound 11: A solution of 10
(1.0 g, 1.94 mmol) in dry DMF (8.0 mL) was treated with 5-amino-
pentan-1-ol (2.0 g, 19.4 mmol) at 80 °C for 6 h whilst stirring. The
resulting solution, dried under reduced pressure, was purified on a
silica gel column eluted with an increasing amount of CH3OH in
CHCl3. The fractions eluted with 15% CH3OH furnished 11
the linear precursor 16, indicates this reaction as an efficient (0.65 g, 85%) as a white amorphous solid which could not be in-
1
duced to crystallize. H NMR (CDCl3): δ ϭ 7.99 and 7.87 (s, 1 H
each, 2-H and 8-H), 5.85 (d, J ϭ 4.5 Hz, 1 H, 1Ј-H), 5.07 and 5.04
(m’s, 1 H each, 2Ј-H and 3Ј-H), 4.49 (br. s, 1 H, 4Ј-H), 4.05 (t, J ϭ
7.2 Hz, 2 H, CH2N), 3.94 (d, J ϭ 12.4 Hz, 1 H, 5Ј-Ha), 3.77 (m, 1
H, 5Ј-Hb), 3.62 (t, J ϭ 6.3 Hz, 2 H, CH2O), 1.80 (m, 2 H, pentyl
chain methylene), 1.61 (s, 3 H, isopropylidene), 1.57 and 1.43 (m’s,
2 H each, pentyl chain methylene groups), 1.35 (s, 3 H, isopropylid-
ene) ppm.13C NMR (CDCl3): δ ϭ 158.2, 149.7, 148.6, 141.3, 125.3,
115.3, 92.3, 88.5, 85.9, 82.9, 63.4, 62.9, 48.0, 33.2, 30.5, 27.6, 25.6,
24.3 ppm. ESI-MS: calcd. for C18H26N4O6 394.18, found 395 [M
ϩ H]ϩ. UV (CH3OH): λmax ϭ 247, 251, shoulder 268 nm.
and easy method to obtain large rings characterized by a
high conformational mobility.
Experimental Section
General Methods: 1H and 13C NMR spectra were recorded in
CDCl3 with a Bruker WM 500 spectrometer. Residual proton and
carbon signals of the solvent (CDCl3: δ ϭ 7.24 ppm and 77.5;
CD3OD: δ ϭ and 77.5; D2O: δ ϭ 4.80 ppm) were used as internal
4236
Eur. J. Org. Chem. 2002, 4234Ϫ4238

A New N1-Pentyl Analogue of Cyclic Inosine Diphosphate Ribose
5Ј-O-(Dianilinophosphoryl)-1-[5-O-(dianilinophosphoryl)pentyl]-
FULL PAPER
2Ј,3Ј-O-Isopropylidene-5Ј-O-[(phenylthio)phosphoryl]-1-[5-O-
2Ј,3Ј-O-isopropylideneinosine
(12):
(PhNH)2POCl
(1.18 g,
(phosphoryl)pentyl]inosine (15):
A
mixture of 14 (60 mg,
4.43 mmol) was added to 11 (500 mg, 1.27 mmol), dissolved in dry
pyridine (4.0 mL), and the solution was stirred at room temper-
ature overnight. The resulting mixture, dried under reduced pres-
sure, was chromatographed on a silica gel column eluted with an
0.068 mmol) and isoamyl nitrite (200 µL, 3.3 mmol) in pyridine/
AcOH/Ac2O (2:1:1, v/v, 1.5 mL) was stirred at room temperature
for 8 h. The mixture, dried under reduced pressure (at Ͻ 50 °C),
was dissolved in a mixture of H3PO2 (70 µL, 4.3 mmol), Et3N (292
increasing amount of CH3OH in CHCl3. The fractions eluted with µL, 2.1 mmol) and pyridine (5.5 mL) and the resulting solution was
5% CH3OH furnished 12 (0.92 g, 85%) as a pale yellow amorphous stirred at room temperature for 11 h. The mixture was dried under
solid which could not be induced to crystallize. 1H NMR (CDCl3):
δ ϭ 7.81 and 7.51 (s’s, 1 H each, 2-H and 8-H), 7.15Ϫ6.25 (complex
signals, 20 H, phenyl groups), 6.97 (br. s, 1 H, 1Ј-H), 6.66 and 6.53
(d’s, 2 H each, J ϭ 7.3, 7.3 Hz, NH), 5.21 (m, 1 H, 2Ј-H), 5.24 (m,
reduced pressure (at Ͻ 50 °C) and the residue, dissolved in H2O,
was partitioned between CHCl3 and H2O. Pyridine (11.0 mL) was
added to the aqueous layer and the resulting solution was dried
under reduced pressure (at Ͻ 50 °C). The resulting solid residue
1 H, 3Ј-H), 4.51 (m, 1 H, 4Ј-H), 4.06 (complex signals 4 H, CH2N was dissolved in a TEAA buffer (0.1 , pH ϭ 7.0) and chromato-
and 5Ј-H2), 1.66, 1.55 and 1.29 (m’s, 2 H each, pentyl chain methyl-
graphed on a C18 reversed-phase HPLC column, eluted with a lin-
ear gradient from 0 to 30% of CH3CN in a TEAA buffer (0.1 ,
ene groups), 1.60 and 1.31 (s’s, 3 H each, isopropylidene) ppm. 13
C
NMR (CDCl3): δ ϭ 156.6, 147.7, 146.7, 139.8, 140.2, 140.0, 129.5, pH ϭ 7.0) in 30 min, flow 2.0 mL/min. The product with retention
129.3, 125.6, 122.1, 121.9, 118.2, 118.1, 114.6, 91.9, 86.1, 84.8, 82.4,
65.7, 64.8, 46.9, 29.5, 28.7, 27.3, 25.4, 22.8 ppm. 31P NMR
(CDCl3): δ ϭ 6.9, 6.4 ppm. ESI-MS: calcd. for C42H48N8O8P2
time 12.2 min was concentrated under reduced pressure and excess
of TEAA was removed by C18 reversed-phase HPLC column eluted
with 30% of aqueous CH3CN to give 15 (0.04 g, 75%) as a mono(-
854.31, found 855 [M ϩ H]ϩ. UV (CH3OH): λmax ϭ 276, 250 nm. triethylammonium) salt as a white amorphous solid. M.p. Ͼ 230
1
°C (dec., from CH3OH). H NMR (D2O): δ ϭ 8.32 and 8.22 (s, 1
1-[5-O-(Dianilinophosphoryl)pentyl]-2Ј,3Ј-O-isopropylideneinosine
(13): (PhNH)2POCl (405 mg, 1.52 mmol) was added to 11 (500 mg,
1.27 mmol), dissolved in dry pyridine (5.0 mL), and the solution
was stirred at room temperature overnight. The resulting mixture
H each, 2-H and 8-H), 7.25Ϫ7.05 (complex signals, 5 H, PhS), 6.31
(br. s, 1 H, 1Ј-H), 5.48 (m, 1 H, 2Ј-H), 5.11 (m, 1 H, 3Ј-H), 4.69
(m, 1 H, 4Ј-H), 4.25 and 4.13 (m’s, 1 H each, CH2O), 4.05 (m, 2
H, 5Ј-H2), 3.85 (m, 2 H, CH2N), 3.21 (q, J ϭ 7.2 Hz, 6 H, CH2
was dried under reduced pressure and chromatographed on a silica
triethylammonium), 1.74Ϫ1.63 (m’s, 4 H, pentyl chain methylene
gel column eluted with an increasing amount of CH3OH in CHCl3.
groups), 1.44 and 1.41 (s’s, 3 H each, isopropylidene), 1.35 (m, 2
The fraction eluted with 15% of CH3OH furnished 13 (0.55 g, 70%)
H, pentyl chain methylene), 1.17 (t, 9 H, J ϭ 7.2 Hz, CH3 triethyl-
as a yellow oil and 12 (0.08 g, 8%) was also recovered from the
ammonium) ppm. 13C NMR (D2O): δ ϭ 158.0, 149.7, 147.5, 140.2,
1
column. H NMR (CDCl3): δ ϭ 8.00 and 7.88 (s’s, 1 H each, 2-H
134.4, 129.7, 127.2, 126.1, 125.0, 115.1, 92.3, 86.4, 85.5, 82.6, 66.9,
and 8-H), 7.15Ϫ6.87 (complex signals, 10 H, phenyl groups), 6.18
65.7, 59.8, 47.7, 31.4, 30.4, 27.5, 25.8, 24.0, 8.8 ppm. 31P NMR
(br. s, 2 H, NH), 5.88 (d, J ϭ 4.0 Hz, 1 H, 1Ј-H), 5.08 (dd, J ϭ 4.0
(D2O): δ ϭ 18.9, 2.3 ppm. ESI-MS: calcd. for C24H32N4O11P2S
and ϭ 5.1 Hz, 1 H, 2Ј-H) 5.03 (dd, J ϭ 5.1, 5.1 Hz, 1 H, 3Ј-H),
646.13, found 647 [M ϩ H]ϩ. UV (H2O): λmax ϭ 244, shoulder
268 nm.
4.48 (br. s, 1 H, 4Ј-H), 4.09 (m, 2 H, CH2O), 3.90 (complex signal,
3 H, 5Ј-Ha and CH2N), 3.76 (d, J ϭ 12.0 Hz, 1 H, 5Ј-Hb), 1.68 (m,
4 H, pentyl chain methylene groups), 1.61 (s, 3 H, isopropylidene),
2Ј,3Ј-O-Isopropylidene-5Ј-O-phosphoryl-1-[5-O-(phosphoryl)-
1.39 (m, 2 H, pentyl chain methylene), 1.35 (s, 3 H, isopropylidene)
pentyl]inosine (16): A mixture of 12 (800 mg, 0.94 mmol) and
ppm. 13C NMR (CDCl3): δ ϭ 155.5, 147.5, 146.2, 140.1, 139.8,
isoamyl nitrite (1.2 mL, 6.1 mmol) in pyridine/AcOH/Ac2O (2:1:1,
129.5, 126.4, 122.1, 118.5, 114.2, 93.7, 86.3, 84.0, 81.6, 66.1, 63.3,
v/v, 15 mL) was stirred at room temperature for 8 h. The mixture
47.0, 29.8, 29.1, 27.8, 25.5, 22.8 ppm. 31P NMR (CDCl3): δ ϭ
was dried under reduced pressure and the residue, dissolved in a
6.2 ppm. ESI-MS: calcd. for C30H37N6O7P 624.25, found 625 [M
TEAA buffer (0.1 , pH ϭ 7.0), was purified by a C18 reversed-
ϩ H]ϩ. UV (CH3OH): λmax ϭ 275, 250 nm.
phase HPLC column, eluted with a linear gradient from 0 to 50%
1-[5-O-(Dianilinophosphoryl)pentyl]-2Ј,3Ј-O-isopropylidene-5Ј-O-
[bis(phenylthio)phosphoryl]inosine (14): S,SЈ-Diphenyl dithi-
ophosphate (cyclohexylammonium salt, 350 mg, 0.92 mmol) and
TPSCl (695 mg, 2.3 mmol) were added to a solution of 13 (500 mg,
0.81 mmol) in dry pyridine (5.0 mL) and the mixture was stirred
for 8 h at room temperature under N2. The mixture was dried un-
der reduced pressure and the residue was purified on a silica gel
column by eluting with an increasing amount of CH3OH in CHCl3.
The fraction eluted with 5% of CH3OH furnished 14 (0.57 g, 80%)
of CH3CN in a TEAA buffer (0.1 , pH ϭ 7.0) in 45 min. The
product with retention time 22.9 min was concentrated and excess
of TEAA was removed by C18 reversed-phase HPLC column eluted
with 30% aqueous CH3CN to give 16 (0.52 g, 85%) as a mono(trie-
thylammonium) salt as a white amorphous solid. M.p. Ͼ 230 °C
1
(dec., from CH3CH2OH). H NMR (D2O): δ ϭ 8.37 and 8.36 (s,
1 H each, 2-H and 8-H), 6.27 (br. s, 1 H, 1Ј-H), 5.40 (m, 1 H, 2Ј-
H), 5.17 (m, 1 H, 3Ј-H), 4.61 (m, 1 H, 4Ј-H), 4.13 and 4.09 (m’s, 2
H each, 5Ј-H2 and CH2O), 3.83 (m, 2 H, CH2N), 3.19 (q, J ϭ
7.1 Hz, 6 H, CH2 triethylammonium), 1.80 and 1.68 (m’s, 4 H,
pentyl chain methylene groups), 1.65 and 1.43 (s’s, 3 H each, isop-
ropylidene), 1.40 (m, 2 H, pentyl chain methylene), 1.22 (t, J ϭ
7.1 Hz, 9 H, CH3 triethylammonium) ppm. 13C NMR (D2O): δ ϭ
158.7, 149.4, 148.1, 140.8, 124.0, 115.4, 94.7, 91.3, 85.8, 81.8, 65.8,
65.0, 47.5, 42.6, 29.8, 28.7, 26.1, 24.3, 22.1, 9.2 ppm. 31P NMR
(D2O): δ ϭ 0.4, Ϫ0.1 ppm. ESI-MS: calcd. for C18H28N4O12P2
554.12, found 553 [M Ϫ H]Ϫ. UV (H2O): λmax ϭ 250 nm.
1
a yellow oil. H NMR (CD3OD): δ ϭ 7.87 and 7.70 (s’s, 1 H each,
2-H and 8-H), 7.50Ϫ7.18 (complex signals, 10 H, PhN), 7.16Ϫ6.79
(complex signals, 10 H, PhS), 6.10 (m, 2 H, 2NH), 6.01 (br. s, 1 H,
1Ј-H), 5.09 (m, 1 H, 2Ј-H), 4.89 (m, 1 H, 3Ј-H), 4.38 (m, 3 H, 4Ј-
H and CH2O), 4.08 (m, 2 H, 5Ј-H2), 3.90 (m, 2 H, CH2N), 1.68
(m, 4 H, pentyl chain methylene groups), 1.58 (s, 3 H, isopropyl-
idene), 1.35 (m, 2 H, pentyl chain methylene), 1.30 (s, 3 H, isoprop-
ylidene) ppm. 13C NMR (CD3OD): δ ϭ 156.5, 147.5, 147.0, 140.1,
144.1, 136.2, 129.2, 128.1, 127.9, 125.8, 124.4, 122.0, 118.0, 114.9,
91.4, 85.2, 84.9, 81.9, 66.5, 65.5, 46.8, 29.8, 29.0, 27.6, 25.2, 2Ј,3Ј-Isopropylidene-Substituted Cyclopyrophosphate Compound 17
22.5 ppm. 31P NMR (CD3OD): δ ϭ 49.8, 5.9 ppm. ESI-MS: calcd.
from 15: A solution of 15 (20 mg, 0.027 mmol) in pyridine
for C42H46N6O8P2S2 888.23, found 889 [M ϩ H]ϩ. UV (CH3OH): (20.0 mL) was added slowly over 15 h, using a syringe pump, to a
˚
λmax ϭ 270, 256, 235 nm.
mixture of I2 (125 mg, 0.475 mmol) and dried 3-A molecular sieves
Eur. J. Org. Chem. 2002, 4234Ϫ4238
4237

A. Galeone, L. Mayol, G. Oliviero, G. Piccialli, M. Varra
FULL PAPER
(1.2 g) in pyridine (20 mL) at room temperature in the dark. After
filtering and washing, the combined filtrate and washings were con-
centrated to dryness. The residue was dissolved in a TEAA buffer
NMR (D2O): δ ϭ 158.4, 148.6, 147.5, 142.1, 125.2, 90.4, 84.1, 72.1,
70.5, 66.1, 65.4, 59.0, 47.0, 29.1, 26.3, 21.5, 8.7 ppm. 31P NMR
(D2O): δ ϭ Ϫ10.2, Ϫ11.1 ppm. ESI-MS: calcd. for C15H22N4O11P2
(0.1 , pH ϭ 7.0) and purified by a C18 reversed-phase HPLC 496.08, found 495 [M Ϫ H]Ϫ. UV (H2O): λmax ϭ 249 nm (10100),
column eluted with a linear gradient (from 0 to 70% in 70 min) of
CH3CN in a TEAA buffer (0.1 , pH ϭ 7.0) flow 2.0 mL/min. The
fractions with retention time 28.8 min were concentrated to dryness
and excess of TEAA was removed by C18 reversed-phase HPLC
column eluted with 40% of aqueous CH3CN to give 17 (0.01 g,
60%) as a bis(triethylammonium) salt as a white amorphous solid
which could not be induced to crystallize. 1H NMR (D2O): δ ϭ
8.43 and 8.22 (s, 1 H each, 2-H and 8-H), 6.37 (br. s, 1 H, 1Ј-H),
5.82 (m, 1 H, 2Ј-H), 5.46 (m, 1 H, 3Ј-H), 4.56 (m, 1 H, 4Ј-H), 4.08
and 3.93 (m’s, 2 H each, CH2O and CH2N), 3.77 (m, 2 H, 5Ј-H2),
3.20 (q’s, 12 H, J ϭ 7.2 Hz, CH2 triethylammonium), 1.91 (m, 2
H, pentyl chain methylene), 1.65 (s, 3 H, isopropylidene), 1.58 (m,
2 H, pentyl chain methylene), 1.47 (s, 3 H, isopropylidene), 1.35
(m, 2 H, pentyl chain methylene), 1.23 (t’s, 18 H, J ϭ 7.2 Hz, CH3
triethylammonium) ppm. 13C NMR (D2O): δ ϭ 158.0, 148.8, 147.3,
142.2, 124.2, 114.4, 91.2, 87.3, 83.8, 82.1, 65.9, 65.3, 47.3, 46.7,
29.0, 26.5, 25.8, 24.2, 21.6, 8.1 ppm. 31P NMR (D2O): δ ϭ 10.4,
Ϫ11.5 ppm. ESI-MS: calcd. for C18H26N4O11P2 536.11, found 537
[M ϩ H]ϩ. UV (H2O): λmax ϭ 249 nm.
shoulder 266 (5400).
Acknowledgments
This work was supported by the Italian M.U.R.S.T. (PRIN 2001)
and Regione Campania (L. 41). The authors are grateful to ‘‘Cen-
tro Ricerche Interdipartimentale di Analisi Strumentale’’,
C.R.I.A.S., for supplying NMR and MS facilities.
[1]
H. C. Lee, Annu. Rev. Pharmacol. Toxicol. 2001, 41, 317Ϫ345.
[2]
A. Galione, Science 1993, 259, 325Ϫ326.
[3]
A. H. Guse, Cell Signalling 1999, 11, 309Ϫ316.
R. Aarhus, M. R. Graeff, D. M Dickey, T. F. Walseth, H. C.
[4]
Lee, J. Biol. Chem. 1995, 270, 30327Ϫ30333.
R. Zocchi, L. Franco, L. Guida, U. Benfatti, A. Bargellesi, E.
[5]
Malavasi, H. C. Lee, A. De Flora, Biochem. Biophys. Res. Com-
mun. 1993, 196, 1459Ϫ1465.
K. Inageda, K. Takahashi, K. Tokita, H. Nishina, Y. Kanaho,
[6]
I. Kukimoto, K. Kontani, S. Hoshino, T. J. Katada, Biochem-
istry (Tokio) 1995, 117, 125Ϫ131.
L. Guida, L. Franco, R. Zocchi, S. Buzzone, U. Benfatti, G.
[7]
Damonte, H. C. Lee, J. Biol. Chem. 1997, 272, 12945Ϫ12951.
Product 17 from 16: EDC (151 mg, 0.79 mmol) was added to a
solution of 16 (100 mg, 0.15 mmol) in MDP (20 mL) and the mix-
ture stirred at room temperature for 60 h. The solvents were evap-
orated under reduced pressure and the residue was dissolved in
20 mL of a TEAA buffer (0.1 , pH ϭ 7.0). The solution was
purified and desalted by a C18 reversed-phase HPLC column, as
described before for the same compound to give 17 (0.09 g, 80%)
as a bis(triethylammonium) salt. Spectroscopic data are identical
to product 17 obtained from 15.
[8]
Y. Fukushi, I. Kato, S. Takasawa, T. Sasaki, B. H. Ong, M.
Sato, A. Ohsaga, K. Sato, H. Okamoto, Y. Maruyama, J. Biol.
Chem. 2001, 276, 649Ϫ655.
S. Shuto, M. Shirato, Y. Sumita, Y. Ueno, A. Matsuda, J. Org.
Chem. 1998, 52, 1986Ϫ1994.
M. Fukuoka, S. Shuto, N. Minakawa, Y. Ueno, A. Matsuda,
J. Org. Chem. 2000, 65, 5238Ϫ5248.
S. Shuto, M. Fukuoka, A. Manikowsky, Y. Ueno, T. Nakano,
R. Kuroda, H. Kuroda, A. Matsuda, J. Am. Chem. Soc. 2001,
123, 8750Ϫ8759.
[9]
[10]
[11]
[12]
T. F. Walseth, H. C. Lee, Biochim. Biophys. Acta 1993, 1178,
235Ϫ242.
A. Galeone, L. Mayol, G. Oliviero, G. Piccialli, M. Varra, Tet-
Cyclopyrophosphate Compound 7:
A solution of 17 (56 mg,
0.076 mmol) in aqueous HCO2H (60%, 2.0 mL) was stirred at room
temperature for 3.5 h. After the solvent was evaporated under re-
duced pressure, the residue, dissolved in H2O, was purified by a
C18 reversed-phase HPLC column, eluted with a linear gradient
(from 0 to 40% in 80 min) of CH3CN in a TEAB buffer (0.1 ,
pH ϭ 7.0), flow 2.0 mL/min. The fractions with retention time
24.4 min were concentrated under reduced pressure and excess of
TEAB was coevaporated with H2O. The residue was freeze-dried
to give 7 (0.05 g, 85%) as a bis(triethylammonium) salt as a white
amorphous solid. M.p. Ͼ 230 °C (dec., from CH3CH2OH). 1H
NMR (D2O): δ ϭ 8.39 and 8.21 (s, 1 H each, 2-H and 8-H), 6.04
(br. s, 1 H, 1Ј-H), 5.38 (m, 1 H, 2Ј-H), 4.50 (m, 1 H, 3Ј-H), 4.31
(m, 1 H, 4Ј-H), 3.96 and 3.84 (m’s, 2 H each, CH2O and CH2N),
3.76 (m, 2 H, 5Ј-H2), 3.20 (q’s, J ϭ 7.0 Hz, 12 H, CH2 triethyl-
ammonium), 1.85, 1.58 and 1.32 (m’s, 2 H each, methylene groups),
1.21 (t’s, J ϭ 7.0 Hz, 18 H, CH3 triethylammonium) ppm. 13C
[13]
rahedron 2002, 58, 363Ϫ368.
[14]
F. Zhang, Q. Gu, C. J. Sih, Bioorg. Med. Chem. 1999, 7,
653Ϫ664.
[15]
L. De Napoli, A. Messere, D. Montesarchio, G. Piccialli, M.
Varra, J. Chem. Soc., Perkin Trans. 1 1997, 2079Ϫ2082.
[16]
K. H. Scheit, Angew. Chem. Internat. Edit. 1967, 6, 180Ϫ181.
[17]
J. Smrt, Tetrahedron Lett. 1973, 47, 4727Ϫ4728.
[18]
T. Hata, K. Yamaguchi, S. Honda, I. Nakagawa, Chem. Lett.
1978, 507Ϫ508.
[19]
M. Sekine, J. Matsuzaki, T. Hata, Tetrahedron Lett. 1981, 22,
3209Ϫ3212.
[20]
M. Sekine, T. Hata, Tetrahedron Lett. 1983, 24, 5741Ϫ5744.
Y. Nakagawa, S. Koni, S. Ohtani, T. Hata, Synthesis 1980,
[21]
556Ϫ557.
Received July 9, 2002
[O02372]
4238
Eur. J. Org. Chem. 2002, 4234Ϫ4238